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• Published: 13 January 2020

Fish Feed Quality Is a Key Factor in Impacting Aquaculture Water Environment: Evidence from Incubator Experiments

• Wenwen Kong 1 ,
• Suiliang Huang 1 ,
• Zhenjiang Yang 1 ,
• Feifei Shi 1 ,
• Yibei Feng 1 &
• Zobia Khatoon 1  

Scientific Reports volume  10 , Article number:  187 ( 2020 ) Cite this article

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The effect of fish feed quality has gained increasing attention to alleviate the harmful environmental impacts induced by intensive aquaculture. In current research, we have conducted an incubator experiment to highlight the effect of fish feed quality on aquaculture water environment. Fish feed from three manufactures with two different dosages (0.1000 g, 0.2000 g) was added to the culture medium with and without Microcystis aeruginosa . Treatments with Microcystis aeruginosa were named as MHT, MHP and MZT; while the treatments without Microcystis aeruginosa named as HT, HP and ZT. Microcystis aeruginosa densities and nutrients concentrations were measured in the study. Results have shown that fish feed quality (manufactures) has a great effect on nutrients concentrations in the absence of Microcystis aeruginosa ( P  < 0.05). Meanwhile, fish feed can stimulate Microcystis aeruginosa growth that is also influenced by fish feed quality excluding lag phase (0~12 day) significantly in general ( P  < 0.05). The maximum Microcystis aeruginosa density ( N max ) is 1221.5, 984.5, 581.0, 2265.9, 2056.8 and 1766.6 1 × 10 4 cells mL −1 for MHT 0.1 g, MHP 0.1 g, MZT 0.1 g, MHT 0.2 g, MHP 0.2 g and MZT 0.2 g, respectively. In treatments with algae, fish feed quality affect total phosphorus (TP) concentrations (except the difference between MHT and MHP) and total nitrogen (TN) concentrations significantly ( P  < 0.05). For most of consumed nutrients, the obvious differences among all treatments were observed excluding lag phase in general ( P  < 0.05), which suggest that the nutrient utilization is also dependent on fish feed quality. Keeping in mind the above facts it is concluded that fish feed quality is a key factor in impacting aquaculture water environment.

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Introduction

Aquaculture is one of the fastest growing food producing sectors around the world. Global production of aquaculture increased from 4.17 × 10 7 tonnes in 2000 to 8.0 × 10 7 tonnes in 2016, and the annual growing rate reached 5.2% during this period 1 . Freshwater aquaculture is probably the most important form of aquaculture for the time being, and fish is by far the dominating product in freshwater aquaculture 2 , 3 . In fact, aquaculture production heavily depends on the external aquafeeds or nutrients supply to the aquaculture system 4 . Aquafeeds production has been widely recognized as one of the fastest expanding agricultural industries in the world 5 , and the annual growth rate of aquafeeds production reached 17% in China 6 . In 2018, total output of global aquafeeds was 40.1 million tonnes, of which Asia-Pacific’s aquafeeds production reached 28.5 million tonnes 7 . In practice, fish feed is the most important kind of aquafeeds with China being the top 1 in the world production of the fish feed 8 .

Currently, the rapid development and low entry barriers for China’s feed industry have led to the emergence of aquafeeds enterprises with insufficient conditions 9 . Meanwhile, production of carp and other omnivorous species is intensifying in China, and commercial aquafeeds enterprises are also being developed to serve these industries 10 , 11 , 12 . In 2017, there have been 6469 feeds manufactures in China, and 3145 feeds manufactures’ output are lower than 1 million tonnes 13 . Due to different production levels of fish feed producers, the fish feed qualities significantly differ both imaginably and practically. In Soong et al .’s 14 study, although all grouper fish feed meal produced by 30 manufacturers can be used to feed grouper fish, the nutritional indicators and quality of these feed meals are not the same.

Quality analysis of fish feed is mostly founded on the growth rate of fish 15 , 16 , quality benefit of fish 15 , 17 , 18 , feed coefficient 19 and so on 14 , 20 . In order to healthily promote the development of fisheries, some standards in US, Europe and China for fish feed have been formulated, such as “Nutrient Requirements of Fish and Shrimp” published by American National Research Council 21 and regulation EC NO.767/2009 issued by European Parliament and Council 22 . In China, the Ministry of Agriculture has issued 21 standards for aquafeeds industry, and AQSIQ (General Administration of Quality Supervision, Inspection and Quarantine of the People’s Republic of China) and the National Standards Management Committee have issued 7 national standards for aquafeeds 23 . In these standards, nutritional indicators are primarily protein, crude fat, crude ash, calcium, phosphorus, lysine and so on. Moreover, these indicators are mainly following the “lower limit rule” rather than the specific contents, and actually they are still incomplete for fish feed and detailed ingredients of fish feed. Thus, fish feed quality from different producers may be still different even if they all meet the standards.

Despite huge potential benefits of aquaculture development, there are always concerns about its environmental impacts 24 . Recently, concerns on both fish feed quality and effects of fish feed on the aquaculture water environment have been elevated to a new level 25 , 26 , 27 , 28 , 29 , 30 , 31 , and the environmental effect of cage farms is believed to be a critical part for sustainable aquaculture 32 . Wasted fish feed in Ballester-Moltó et al .’s assays were estimated in the range of between 8.52% and 52.20% in aquaculture water bodies 29 , 30 . Edwards also believed that when harvesting fish, only about1/3 of the nutrients in the feed were removed, and 2/3 of nutrients were voided by fish during the growth process 27 . In general, 57% of the total feed nitrogen (N) and 76% of the total feed phosphorus (P) for fish lost to the environment, respectively 33 . However, most discussions about the effects of different fish feed on nutrients (N and P) enrichment did not consider the difference of fish feed quality 27 , 32 , 34 . In fact, uneaten fish feed or fish excretion from aquaculture activities release major macronutrients like nitrogen and phosphorous 33 , the released nutrients concentrations from uneaten fish feed of different quality or manufactures are expected to be different.

Meanwhile, nitrogen and phosphorus released from aquafeeds are not only the basic ingredients incorporated in feed to achieve good growth of aquatic animals (e.g. fish, shrimp), but also required for algal growth in water bodies 28 , 32 , 35 , 36 . In fish ponds of feeding common carp ( Cyprinus carpio ), 57~71% N and 44~58% P came from the fish feed, and they can be accumulated in fish, plankton and benthic organisms 37 . Nutrients produced through aquaculture activities are rapidly assimilated by phytoplankton, and this results in low concentration of inorganic nutrients in the water column 38 . Li et al .’ study noted that the massive growing phytoplankton absorb DIN and DIP effectively 39 .

In addition, aquaculture activities not only stimulate algal growth but also affect phytoplankton communities 40 , 41 along with deteriorating water quality and adversely influencing human health aquaculture activities 42 . Wu et al .’s results showed that the release of N and P from fish feed stimulated algae growth 43 . Algae densities increased with increasing fish feed dosages in moderate nutrients concentrations from fish feed 43 . In Huang et al .’s study, enclosures with fish feed have higher algae biomass than those without fish feed, and blue-green algae dominated phytoplankton communities in enclosures with fish feed 28 . With the rapid growth of marine aquaculture activities in the coastal areas of Weihai, China, cellular abundance of diatoms and dinoflagellates increased between 2006 and 2014 39 . Affected by organic enrichment and sediment resuspension by shrimp, a shift in species dominance from Diatoms and Dinoflagellates to green algae was observed in shrimp aquaculture ponds in Hugues et al .’s study 44 . In sum, although fish feed qualities were also not considered in Rahman et al . 37 , Wu et al . 43 and Huang et al .’s 28 studies, the effect of different fish feed quality on algae growth is worthy to be studied.

Polyculture of Chinese carp, using large amount of commercial compound freshwater fish feed, has been recognized as a traditional way of increasing nutrient utilization in freshwater bodies. Additionally, in many freshwater lakes in China, Microcystis aeruginosa ( M. aeruginosa ) is a common cyanobacterium of harmful algal blooms 45 . In light of the above facts, compound freshwater fish feed from three different manufactures were selected to investigate effects of uneaten fish feed with different qualities on aquaculture water environment, including the characteristics of nutrients release, the effects of different fish feed on M. aeruginosa growth and nutrients utilization by M. aeruginosa through incubator experiment.

Materials and Methods

Experimental materials.

M. aeruginosa (cyanobacterium) was obtained from the Freshwater Algae Culture Collection of the Institution of Hydrobiology (FACHB-905), which belongs to Chinese Academy of Sciences. The algae were cultivated in an illumination.

Commercial adult fish feeds, named HT, HP and ZT, are selected according to their popularity in aquafeeds market, which are used for polyculture in freshwater bodies such as lakes, reservoirs, ponds and so on. In other words, they are more or less fish-friendly feeds. HT is produced by Huaian Tongwei Company Limited, and this company is a large-scale feed enterprise invested and built by Tongwei in 2001. Huaian Tongwei Company Limited mainly produces aquafeeds as well as animal feeds, and the aquafeeds are widely used in the mainland of China. HP is widely used in Hebei province, China, which is produced by Hebei Panda Feed Company Limited. The company is incorporated in 2013 which mainly produces aquafeeds along with animal feeds. ZT is produced by Zhongshan City Taishan Feed Company Limited incorporated in 2004 and is widely used in Guangdong province, China. In 2010, feed sales of Zhongshan City Taishan Feed Company were 170000 tonnes, of which 90000 tonnes were for aquafeeds. Retail price of HT, HP and ZT feed are 6.2, 7.5 and 7.7 yuan kg −1 when we bought online for experiment, respectively, and the price is free of transportation. We think the higher retail price of ZT fish feed is caused by transportation costs. These fish feeds were crushed and sieved through Taylor pore size of 0.85 mm before use. HT, HP and ZT fish feed contains TP with 13.41, 12.15 and 11.37 g kg −1 respectively, while contains TN with 49.70, 45.85 and 38.75 g kg −1 respectively by analysis 43 , 46 . Nutritional indicators of these fish feeds disclosed by their respective manufacturers are shown in Table  1 . These indicators are different with the same usage of fish feed and we believe that the quality of fish feeds is also different.

Algal pre-culture

M. aeruginosa were cultured in M-II culture medium for 15 days before the experiment. The M-II culture medium was prepared in deionized water with 100 mg L −1 NaNO 3 , 10 mg L −1 K 2 HPO 4 , 75 mg L −1 MgSO 4  × 7H 2 O, 40 mg L −1 CaCl 2  × 2H 2 O, 20 mg L −1 Na 2 CO 3 , 6 mg L −1 Fe·citrate × H 2 O and 1 mg L −1 Na 2 EDTA × 2H 2 O. The initial pH value was adjusted to approximately 8.0 with 0.5 mol L −1 NaOH and 0.5 mol L −1 HCl. The operational temperature and light intensity were 28 °C and 3000 lx for the experiment undertaken under light conditions. In comparison, the corresponding values during the period of darkness were 20 °C and 0 lx. The cycle of light and darkness comprised 12 h of light and 12 h of darkness.

The medium containing algae was collected and then centrifuged for 15 min at a speed of 3000 r min −1 . After removal of the supernatant, the algae were rinsed with 15 mg L −1 NaHCO 3 solution and then centrifuged again. After repeating the above procedure twice, the algae obtained via this procedure were cultured in M-II medium without nitrogen or phosphorus, the process was defined as starvation cultivation. Three days later, the algae would deplete the intracellular polyphosphate stores 43 .

Experimental methods

Effects of different fish feed on nutrients release and algae growths were assessed using batch incubation experiments. In the experiment, 400 mL sterilized M-II culture medium without nitrogen and phosphorus was used, and weights of 0.1000 g and 0.2000 g of the three different fish feed (from different manufactures) were added into the media served as P and N sources with 1 L flask. Treatments without algae containing 0.1 g fish feed were named “HT 0.1 g”, “HP 0.1 g”, “ZT 0.1 g”, and containing 0.2 g fish feed named “HT 0.2 g”, “HP 0.2 g” and “ZT 0.2 g”, respectively. Meanwhile, treatments with algae containing 0.1 g fish feed were named “MHT 0.1 g”, “MHP 0.1 g”, “MZT 0.1 g”, and containing 0.2 g fish feed named “MHT 0.2 g”, “MHP 0.2 g” and “MZT 0.2 g” conforming to the treatments’ name, respectively. Duplicates were prepared. Flasks were shaken and their positions were changed at random three times a day. The initial algae density was 1.0 × 10 5 cells mL −1 .

During the experimental period (37 days), algal cell densities were counted every two days using a haemacytometer under a microscope 43 , 47 . Counting was performed three times per sample. Water sampling started 1 day after algae addition, and total phosphorus (TP), total dissolved phosphorus (TDP), total particulate phosphorus (TPP = TP-TDP), orthophosphate (PO 4 3− -P), total nitrogen (TN), total dissolved nitrogen (TDN), total particulate nitrogen (TPN = TN-TDN) and ammonia (NH 4 + -N) were also measured every two days. Concentrations of PO 4 3− -P, TDP and TP were determined via the persulphate digestion and ammonium molybdate spectrophotometric method 48 . NH 4 + -N was analyzed using the phenol-hypochlorite method 48 . TN and TDN were analyzed using the procedure of alkaline potassium persulfate digestion with ultra-violet light spectroscopy 49 .

M. aeruginosa growth kinetics

Algal growth can be well described by (original) Logistic function 50 , 51 , 52 , 53 , 54 . However, this function does not satisfy the initial conditions of algal growth. A modified Logistic function was proposed by Huang et al . 49 and it is as follows:

where N (1 × 10 4 cells mL −1 ) is the algae density at any time, N max is the maximum algae density (1 × 10 4 cells mL −1 ), r (d −1 ) is the intrinsic growth rate, N 0 is the algae density at 0 day, and N 0 is 10 × 10 4 cells mL −1 in the present study, t (d) is time and a (−) is a constant. N max , a and r can be obtained by fitting Eq. ( 1 ) to experimental data.

Growth rates \({\mu ^{\prime} }_{c}\) (1 × 10 4 cells (mL·d) −1 ) can be derived from modified Logistic function in Huang et al .’s 49 study as follows:

The growth rate reached its maximal value \({\mu ^{\prime} }_{cmax}=\frac{r{N}_{max}}{4}\) (1 × 10 4 cells (mL·d) −1 ) when N equals to half of N max 49 , 51 , 53 , 55 .

The formula of the specific growth rate from the modified Logistic function as shown in Eq. ( 3 ), describing variations of specific growth rates with time is also better than that derived from Logistic function 49 :

where \({\mu }_{c}\) (d −1 ) is defined as the computed specific growth rate.

Statistical analysis

Experimental data was analyzed statistically by using Origin 8.6 and SPSS 19.0. Logistic model was examined for their fit to the experimental data using Origin 8.6. Origin 8.6 or SPSS 19.0 is used to determine correlation coefficients between the measured and predicted variables as well as between M. aeruginosa densities and nutrients concentrations. The statistical analysis is applied to identify the significant differences among groups with different fish feed by analysis of variance (ANOVA) with SPSS 19.0. Moreover, standard deviation was calculated and data was expressed in terms of means + SD of the two replicates.

Results and Discussion

Effects of different fish feed on nutrients concentrations without algae, effects of different fish feed on phosphorus concentrations.

Phosphorus is chemical compound found in fish feed 33 , its labile form (PO 4 3− -P) is a major form of released phosphorus from fish feed 43 . From Fig.  1 , TP, PO 4 3− -P and TDP concentrations in treatments with HT, HP and ZT increase gradually in the first 10 days and then enter into a stable phase. Meanwhile, released concentrations of TDP and PO 4 3− -P from fish feed reached 85.39~90.00% and 75.23~89.91% of their corresponding maximal values at the first sampling day (or 24 hours). Akhan and Gedik’s research results also indicated that release of nutrients from fish feed occurred rapidly, they believed that uneaten fish feed should be removed quickly to avoid nutrient enrichment 32 .

Variations of TP, TDP, TPP and PO 4 3− -P concentrations with time in groups without algae (HT, fish feed of HT; HP, fish feed of HP; ZT, fish feed of ZT). Data shown is the mean ± SD of two independent measurements.

Under same fish feed dosage, TP (TDP or PO 4 3− -P) concentrations in treatments with HT and HP feed are 1.33~1.66 times higher than those of ZT, which is not consistent with their nutritional indicator of TP (in Table  1 ). This may be because the TP indicator in these feeds just follows “the lower limit rule”. Calculated results shows that average TP concentrations are 1.97, 1.96 and 1.28 mg L −1 , average TDP concentrations are 1.75, 1.74 and 1.06 mg L −1 , average PO 4 3− -P concentrations are 1.60, 1.59 and 0.91 mg L −1 for HT 0.1 g, HP 0.1 g and ZT 0.1 g respectively, and these concentrations also doubles in treatments with 0.2 g correspondingly. This also implies that both HT and HP feed have much larger capacities in releasing phosphorus nutrients than ZT feed. In addition, significant analysis shows that there is a noteworthy difference in releasing phosphorus nutrients between HT and ZT and between HP and ZT ( P  < 0.001), while there is no significant difference between HT and HP ( P  > 0.05). Significant analysis also shows that fish feed dosage affects TP, TDP and PO 4 3− -P concentrations quite significantly ( P  < 0.001), which conforms to Wu et al .’s results 43 .

In Fig.  1(b,d) , variations of TPP concentrations with time are quite different from those of TDP. In general, TPP concentrations in HT, HP and ZT groups are quite low and close to each other with the same dosage of fish feed, and all increase firstly and then decrease slightly. Fish feed quality does not have a significant effect on TPP concentrations in general ( P  > 0.05).

Effects of different fish feed on nitrogen concentrations

Uneaten fish feed is probably the major input of nitrogen to the aquatic environment 35 , 56 , 57 , 58 , and the nitrogen cycle in aquaculture ecosystem begins with the introduction of protein in fish feed and NH 4 + -N is a by-product of protein catabolism 26 . From Fig.  2 , compared with the released process of phosphorus nutrients from HT, HP and ZT fish feed, nitrogen concentrations rise comparatively very slowly and the time to reach nitrogen nutrients equilibrium concentrations is much longer. TN, TDN and NH 4 + -N concentrations increase gradually in about 15 days, and then reach equilibrium in the following days. In addition, it is clearly observed from Figs.  1 and 2 that TN equilibrium concentrations are higher than TP equilibrium concentrations (1.40~5.04 mg L −1 ) in the present experiment. Fernandes et al . also observed that leaching loads of fish feed for the bluefin tuna were slightly high for nitrogen as 26 kg N tonne −1 , but significantly low for phosphorus as 4 kg P tonne −1   25 .

Variations of TN, TDN, TPN and NH 4 + -N concentrations with time in groups without algae (HT, fish feed of HT; HP, fish feed of HP; ZT, fish feed of ZT). Data shown is the mean ± SD of two independent measurements.

As shown in Fig.  2 , released TN, TDN and NH 4 + -N concentrations from different fish feed are significantly different ( P  < 0.05): TN, TDN or NH 4 + -N concentrations with HT are the most, next with HP and the smallest with ZT; and actually these nutrients concentrations in the whole experimental period from HT fish feed are 1.17~1.52 times and 1.23~1.37 times the concentrations of HP and ZT, respectively for the same fish feed dosage. Average TN concentrations are 9.85, 7.20 and 5.36 mg L −1 , average TDN concentrations are 7.93, 5.76 and 4.73 mg L −1 , and average NH 4 + -N equilibrium concentrations are 6.63, 4.15 and 2.98 mg L −1 for HT 0.1 g, HP 0.1 g and ZT 0.1 g respectively, and corresponding concentrations with 0.2 g fish feed are almost twice their respective concentrations of treatments with 0.1 g fish feed. In reality, as shown in Table  1 , ZT fish feed also contains the lowest crude protein, which may be due to the reason that ZT fish feed releases the smallest amount of nitrogen. In addition, similar to variations of TPP with time, TPN concentrations in Fig.  2(b,d) also fluctuate in low concentrations in all treatments during the whole period. Meanwhile, TPN concentrations are significantly different among the three different fish feed ( P  < 0.05).

As shown in Figs.  1 and 2 , although the nutrients concentrations are significantly different in most experimental runs among HT 0.1 g, HP 0.1 g, ZT 0.1 g, HT 0.2 g, HP 0.2 g and ZT 0.2 g ( P  < 0.05), the nutrients’ proportions, namely, TDP:TP, PO 4 3− -P:TP, TPP:TP, TDN:TN, NH 4 + -N:TN and TPN:TN, are quite close after all nutrients concentrations reach their equilibrium concentrations, as shown in Table  2 , for example, TDP is 84.48~91.95%, 88.80~94.90% and 80.91~90.93% of TP for HT, HP and ZT respectively. From the results in Table  2 , the ratio of PO 4 3− -P to TP and NH 4 + -N to TN are obviously lower than those of TDP to TP and TDN to TN respectively because PO 4 3− -P and NH 4 + -N are only one part of them, respectively. Proportions of PO 4 3− -P and NH 4 + -N are in good agreement with Wu et al .’s results, and PO 4 3− -P and NH 4 + -N have high proportions of TP and TN 43 , respectively. Butz and Ven-Cappell 59 and Kibria et al . 35 also believe that fish feed contained major phosphorus fraction in a labile form, namely, the total phosphorus in fish feed, the more the water-soluble phosphorus. Thus, according to released P (TP, TDP and PO 4 3− -P) and N (TN, TDN and NH 4 + -N) concentrations, we believed that HT contains the most nutrients, HP is next while ZT is the lowest in a comprehensive view. It is consistent with crude protein indicators of fish feed in general, ZT fish feed has the lowest crude protein level at 20%. Thus, based on trade-offs among feed price, feed efficiency, feed cost, feed quality, environmental impacts and so forth in aquaculture operations, we could improve protein bioavailability and design reasonable ratio of protein to energy to save protein and reduce nutrients emission.

Effects of different fish feed on M. aeruginosa growth

Effects of different fish feed on m. aeruginosa densities.

Fish feed contributes to abundant nutrient loads as discussed in the above, and it can effectively promote the growth of phytoplankton 28 , 43 , 60 . From Fig.  3 , in the first few days of the experiment, algal cell densities increase very slowly due to their acclimation in fish feed medium with abundant nutrients in the medium. As time goes, M. aeruginosa cell densities increase very fast in the exponential phase (12~25 days) followed by a stable phase.

The growth of M. aeruginosa (MHT, M. aeruginosa  + fish feed of HT; MHP, M. aeruginosa  + fish feed of HP; MZT, M. aeruginosa  + fish feed of ZT). Data shown is the mean ± SD of two independent measurements.

Not only fish feed dosage but also their quality affects algae growth greatly, and the algae densities’ rankings in Fig.  3 are in agreement with those rankings of nutrients concentrations generally. The order of algae densities from the three different fish feed is MHT 0.2 g (MHT 0.1 g) > MHP 0.2 g (MHP 0.1 g) > MZT 0.2 g (MZT 0.1 g) during the whole experimental period (Fig.  3 ), and the corresponding measured maximum algae density is 2526.1 (1278.9), 2042.0 (1016.4) and 1757.2 (595.2) 1 × 10 4 cells mL −1 , respectively. Two kinds of significant difference analysis of algae densities are conducted, namely, including and excluding lag phase, which indicates that the algae densities of MZT are significant different from those of MHT and MHP when excluding lag phase ( P  < 0.05), while they are not significantly different when including lag phase ( P  > 0.05), and this may be because the algae density is low and close to each other during the lag phase among the three different fish feed. In addition, fish feed dosage also has a significant effect on algae densities ( P  < 0.05).

Eutrophication is a major environmental problem induced by aquaculture activities, and algae densities reflect the level of eutrophication. Generally speaking, the lower the algae densities simulated by fish feed, the better the water quality is. Algae densities are coherent with released nutrients concentrations from fish feed and also consistent with nutritional indicators of fish feed in general. Thus, the above results imply that in order to protect aquaculture water environment, “environmentally friendly feed” are needed to both stimulate fish growth greatly and to lessen their effects on the water environment effectually in a balanced way. Meanwhile, new method is greatly needed to decrease the uneaten fish feed when throwing feed to fish manually and the uneaten fish feed also should be removed quickly before it releases nutrients to water.

In our study, both Fig.  3 and Table  3 show that the modified Logistic function can describe M. aeruginosa growth with good accuracy ( R 2  = 0.984~0.999) in agreement with the reported results 49 . Consistent with measured algae densities, \({N}_{max}\) and \({N}_{ave}\) (time-averaged algae density) of different fish feed are also in the order of MHT > MHP > MZT with the same fish feed dosage, and \({N}_{max}\) and \({N}_{ave}\) also increase with increasing dosages of fish feed. Specifically, the fitted N max are 2557.32, 2044.95, 1753.91, 1232.98, 979.49 and 593.59 1 × 10 4 cells (mL·d) −1 for MHT 0.2 g, MHP 0.2 g, MZT 0.2 g, MHT 0.1 g, MHP 0.1 g and MZT 0.1 g respectively, as shown in Table  3 .

Effects of different fish feed on the growth rate of M. aeruginosa

As shown in Fig.  4(a,b) , both measured and computed growth rates in different groups all increase monotonously with time before they reached their maximal values, and then all decrease monotonously, which is consistent with Huang et al .’s study 49 . From Fig.  4(a,b) and correlation analysis, the computed growth rates agree reasonably well with measured ones with correlation coefficients ( R ) of 0.911, 0.954, 0.825, 0.970, 0.970 and 0.975 for MHT 0.1 g, MHP 0.1 g, MZT 0.1 g, MHT 0.2 g, MHP 0.2 g and MZT 0.2 g respectively, and all correlations are significant ( P  < 0.001). Although the analysis of significant difference shows that the fish feed quality does not have significant effects on growth rate ( P  > 0.05), maximal calculated growth rates ( \({\mu }_{cmax}^{^{\prime} }\) ) and averaged calculated specific growth rates of MHT are obviously the most, next those of MHP while those of MZT the smallest, as shown in Table  3 .

Variations of growth rates and specific growth rates of M. aeruginosa in fish feed with time (MHT, M. aeruginosa  + fish feed of HT; MHP, M. aeruginosa  + fish feed of HP; MZT, M. aeruginosa  + fish feed of ZT). Data shown are average value of two independent measurements.

Effects of different fish feed on the specific growth rate of M. aeruginosa

Correlation analysis between measured and computed specific growth rates is conducted, the correlation coefficients ( R ) between measured and computed specific growth rates in all groups range from 0.713 to 0.841 ( P  = 0.002~0.037) except in group MZT 0.1 g with R  = 0.579 and P  = 0.188. This indicates that Eq. ( 3 ) is reasonably well in describing specific growth rates of algae generally. In Fig.  4(c,d) , the computed specific growth rates increase firstly, then decrease in general. In addition, both measured and computed specific growth rates among different qualities’ fish feed are quite close with the same fish feed dosage, significant difference analysis also shows that fish feed quality does not influence the specific growth rates significantly( P  > 0.05). This is because the specific growth rate is defined as the growth rate relative to (divided by) the algae density (as described in Eq. ( 3 )).

Interaction of different fish feed and M. aeruginosa growth on nutrients concentrations

As discussed in 2.1, different quality of fish feeds has markedly different influence on released nutrients concentrations in general, that further affect algae growth. Wu et al . believe that in the presence of both algae and fish feed, nutrients releases were mainly controlled by fish feed dosage and algae utilization 43 . In the present study, not only fish feed dosage and algae utilization but also fish feed quality is taken into account to study the interaction of different fish feed and M. aeruginosa growth on nutrients concentrations.

Interaction of different fish feed and M. aeruginosa growth on phosphorus concentrations

Figure  5 shows variations of TP, TDP, TPP and PO 4 3− -P concentrations with time in treatments with algae. From Fig.  5(a,b) , some fluctuations of TP concentrations in treatments with algae were observed during the whole experimental period, and TP concentrations is not related to algae growth ( R  = −0.213~0.461, P  = 0.072~0.928). Variations of PO 4 3− -P concentrations with time are similar to those of TDP, and both concentrations decrease gradually to minimal values, which have negative relationships with M. aeruginosa growth ( R  = −0.965~−0.623, P  < 0.010 for PO 4 3− -P; R  = −0.975~−0.539, P  < 0.031 for TDP).The above variations of PO 4 3− -P and TDP with time in the present study are consistent with Zhou et al .’s 16 and Wu et al .’s 43 studies.

Variations of TP, TDP, TPP and PO 4 3− -P concentrations with time (MHT, M. aeruginosa  + fish feed of HT; MHP, M. aeruginosa  + fish feed of HP; MZT, M. aeruginosa  + 0.1 g fish feed of ZT). Data shown is the mean ± SD of two independent measurements.

The bioavailability of phosphorus depends on the phosphorus speciation, and algae take up phosphorus predominantly in the form of free orthophosphate 35 , 61 . Zhou et al .’s results also show that the dissolved reactive phosphorus (mainly PO 4 3− -P) could be assimilated by algae at a higher velocity than other phosphorus forms 17 . As shown in Fig.  5 , with the same fish feed dosage, any forms of P (TP, TDP and PO 4 3− -P) concentrations in MHT and MHP are close to each other, which are higher than those of MZT. There are significant differences only between MHT and MZT as well as between MHP and MZT for TP concentrations and also there is a significant difference between MHP and MZT for TDP concentrations ( P  < 0.05). However, if we compare maximal and averaged TP, TDP, PO 4 3− -P concentrations in the three different fish feed, they are actually quite different, and the most appears in MHT, and MHP is next while MZT is the smallest in general.

As shown in Fig.  5(b,d) , TPP concentrations increase rapidly in the first 13 days then increase slowly in the following days. This is mainly related to initially released large quantities of phosphorus nutrients and uptake of PO 4 3− -P nutrients by algae. In Huang et al .’s 28 study, TPP concentrations are closely related to the algae biomass, namely, variations of TPP concentrations with time are similar to those of algae biomass. Correlation analysis in the present study also shows that there are positive correlations between TPP concentrations and algae densities in most groups ( R  = 0.710~−0.917, P  < 0.002) expect group MZT 0.2 ( R  = 0.349, P  = 0.192). This is because TPP concentrations do not increase and even decrease since day 11 in group MZT 0.2. Meanwhile, consistent with algae density, the order of TPP concentrations is also MHT 0.2 g (MHT 0.1 g) > MHP 0.2 g (MHP 0.1 g) > MZT 0.2 g (MZT 0.1 g), and the corresponding average TPP concentrations is 1.94 (0.89), 1.78 (0.70) and 1.47 (0.52) mg L −1 . However, quality or dosage has no significant effect on TPP concentrations in general ( P  > 0.05), which maybe because the difference of algae density among different quality of fish feeds are not significant especially during lag phase.

In addition, it is needed to point out that TP includes both extracellular P and intracellular P in treatments with algae, thus variations of TP concentrations with time in treatments with and without algae should be similar. However, we noted that, influenced by algae utilization and algae deposition, TP concentrations in groups with algae fluctuate and are lower than those in group without algae 43 , 62 .

Interaction of different fish feed and M. aeruginosa on nitrogen concentrations

From Fig.  6(a,c) , TN concentrations in treatments with algae increase gradually in the first 15 days and then keep stable in the following days, the variations are consistent with those in treatments without algae. Meanwhile, algae densities are also related to TN concentrations released from fish feed in general ( R  = 0.616~0.908, P  < 0.011), while the correlation coefficients are low in group MZT 0.2 with R  = 0.357 ( P  = 0.175). Fish feed quality has significant influence on TN concentrations ( P  < 0.05), and the order of TN concentrations in groups is MHT > MHP > MZT in Fig.  6 . Maximal TN concentrations are 11.00, 7.56 and 6.09 mg L −1 , the average values are 9.10, 6.09 and 4.57 mg L −1 for MHT 0.1 g, MHP 0.1 g and MZT 0.1 g respectively. Meanwhile the corresponding TN concentrations almost double in treatments with 0.2 g fish feed in general.

Variations of TN, TDN, TPN and NH 4 + -N concentrations with time (MHT, M. aeruginosa  + fish feed of HT; MHP, M. aeruginosa  + fish feed of HP; MZT, M. aeruginosa  + fish feed of ZT). Data shown is the mean ± SD of two independent measurements.

NH 4 + -N is the main form of TDN also being the preferred form of nitrogen for algae growth 63 . From Fig.  6 , both TDN and NH 4 + -N concentrations in treatments with algae increase to their maximal values firstly which is mainly affected by the release of TDN and NH 4 + -N from fish feed, then decrease in the following days affected by algal nutrients utilization generally. In general, correlation analysis indicates that there are negative relationships between algae densities and TDN concentrations ( R  = −0.887~−0.369, P  = 0.001~0.159) and between algae densities and NH 4 + -N concentrations ( R  = −0.867~−0.504, P  < 0.046). Different from the results in treatments without algae fish feed quality observes no significant effect on TDN and NH 4 + -N concentrations among MHT, MHP and MZT ( P  > 0.05), except that there is significant difference of TDN concentrations between MHT and MZT. Whereas, maximal and average values also show that MHT contains most TDN and NH 4 + -N concentrations, MHP next while MZT contains the lowest. Actually, NH 4 + -N concentrations have dropped to almost 0 mg L −1 in treatments with 0.1 g fish feed in the later period of algae growth and to 0.33~0.38 mg L −1 in treatments with 0.2 g fish feed (Fig.  6(a,c) ).

In Fig.  6(b,d) , TPN concentrations increase gradually in the first 20 days and then reach stable concentrations with time going in MHT, MHP and MZT. Consistent with TPP, TPN concentrations also have positive correlation with algae densities during the whole experimental period ( R  = 0.744~0.920, P  < 0.001). Also, the order of TPN concentrations at the same time among different treatments is MHT 0.2 g (MHT 0.1 g) > MHP 0.2 g (MHP 0.1 g) > MZT 0.2 g (MZT 0.1 g), and the corresponding average TPN concentrations are 10.95 (6.77), 9.30 (4.50) and 7.19 (3.61) mg L −1 . However, fish feed quality has no significant influence on TPN concentrations among all treatments with algae ( P  > 0.05), and this may be also because fish feed has no significant influence on algae densities when including the data in the lag phase ( P  > 0.05, n  = 16).

Due to the effect of algae growth, the fractional composition in treatments with algae, as shown in Figs.  5 and 6 and Table  2 , is different from that without algae, as shown in Figs.  1 , 2 and Table  2 . For example, due to the algae utilization, the ratio of TDN:TN is 8.88~12.64%, 9.12~17.48% and 6.22~17.80% for MHT, MHP and MZT respectively (in Table  2 ), which are largely lower than those of HT, HP and ZT mainly because of selective uptake of nutrients by algae.

Effectsof different fish feed on nutrients utilization by M. aeruginosa

Nutrients releases from HT, HP and ZT fish feed are different as discussed in 2.1, which further affect algae growth and nutrients utilization. In order to study the interaction between different fish feed and M. aeruginosa growth, nutrients utilization by algae is also explored. In Huang et al .’s 49 and Goudar et al .’s 50 studies, Logistic function is also used to simulate nutrients consumption versus incubation time and as follows:

in which t is the incubation time (d), ΔC (i.e. △ TDP, ΔPO 4 3− -P, ΔTDN and ΔNH 4 + -N) is consumed nutrient concentrations (difference of nutrients concentrations between without and with algae) at time t (mg L −1 ), \(\Delta {C}_{max}\) is the maximum consumed nutrient concentrations, \({r}_{\bigtriangleup C}\) is the consumed rate constant (d −1 ) and \({a}_{\Delta C}\) is a constant.

As shown in Fig.  7 , △ TDP, △ PO 4 3− -P, △ TDN and △ NH 4 + -N concentrations increase rapidly until it reaches their respective maximal consumed concentrations, then they remain stable. From Fig.  7 and Table  4 , Eq.( 4 ) can well describe variations of △ TDP, △ PO 4 3− -P, △ TDN and △ NH 4 + -N concentrations with time ( \({R}^{2}\)  = 0.89~0.99), which is consistent with Kong et al .’s 55 and Huang et al .’s 49 study. In Table  4 , it can also be founded that maximal calculated consumed TDP, PO 4 3− -P, TDN and NH 4 + -N concentrations ( \(\Delta {C}_{max}\) ) and averaged measured consumed concentrations ( \(\Delta {C}_{ave}\) ) in different treatments are in the order of MHT 0.2 g > MHP 0.2 g > MZT 0.2 g > MHT 0.1 g > MHP 0.1 g > MZT 0.1 g, for example, the corresponding △ C max of TDP is 3.85, 3.33, 1.99, 1.39, 1.38 and 0.75 mg L −1 , respectively, this conforms to measured results. \(\Delta {C}_{max}\) increases with increasing maximum density of M. aeruginosa ( \({N}_{max}\) ), which indicates that more algae need more nutrients to grow (Fig.  7 ). Correlation analysis also shows that there is a positive correlation between algae density and consumed TDP, PO 4 3− -P, TDN as well as NH 4 + -N concentrations with \({R}^{2}\)  = 0.738~0.949, \({R}^{2}\)  = 0.840~0.955, \({R}^{2}\)  = 0.816~0.949, \({R}^{2}\)  = 0.879~0.977, respectively. Meanwhile, fish feed quality has statistically significant effect on nutrient utilization if excluding the lag phase in general ( P  < 0.05) but no significant effect if including the lag phase ( P  > 0.05), and this is also because the algae density is close during the lag phase with different fish feed. In sum, the result implies that the nutrient utilization is dependent not only on the fish feed dosage but also on their quality.

Variations of consumed TDP, PO 4 3− -P, TDN and NH 4 + -N concentrations with time (MHT, M. aeruginosa  + fish feed of HT; MHP, M. aeruginosa  + fish feed of HP; MZT, M. aeruginosa  + fish feed of ZT). Data shown is the mean ± SD of two independent measurements.

In Tijani et al .’s study, both nitrogen and phosphorus utilization display a significant increase during the first 2~21 days, then enter a stationary phase on the 21st day and the utilization has an initial 48 h lag phase 64 . However, in the present study, as shown in Fig.  7 , algae have consumed 0~1.5 mg L −1 of P and 0~7.5 mg L −1 of N in the lag phase of algae growth, and the nutrients utilization do not show clearly a lag phase even if the algae densities are low. This may be because the algae in Tijani et al .’s 64 experiment do not experience the starvation just before their experiments.

Conclusions

Three selected commercial compound fish feeds, HT, HP and ZT demonstrate different effects on released nutrients concentrations and M. aeruginosa growth because of their different qualities.

In treatments without M. aeruginosa (HT, HP, ZT), released P (TP, TDP, PO 4 3− P) and N(TN, TDN, NH 4 + -N) concentrations from different fish feeds are significantly different in general ( P  < 0.05), while there is no significant difference between HT and HP for released P concentrations ( P  > 0.05).

In treatments with M. aeruginosa (MHT, MHP and MZT), fish feed quality affects TP and TN concentrations significantly in general ( P  < 0.05). In addition, for most forms of consumed nutrients concentrations, the differences among all treatments excluding the lag phase are significant in most comparisons ( P  < 0.05), which suggests that the nutrient utilization is dependent on not only fish feed dosage but also fish feed quality. Maximum M. aeruginosa densities and growth rates in different fish feeds are also quite different, their orders are MHT > MHP > MZT with the same dosage.

In our study we experimentally studied the environmental effect of fish feed through incubator experiments without fish as a first try. Our preliminary results demonstrated that fish feed quality should be considered in terms of water environment protection.

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Acknowledgements

The study is financially supported by the National Natural Science Foundation of China (11672139, 5181101344, and 4181101396), the Natural Science Foundation of Tianjin (18YFZCSF00510) and China-Poland Science and Technology Cooperation Committee Regular Meeting Exchange Program (37-14).The authors are grateful to the editors and the anonymous reviewers for their insightful comments and suggestions, which improved the paper greatly.

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S.L. Huang, W.W. Kong and F.F. Shi conceived the research and designed the experiments; W.W. Kong, Z.J. Yang, F.F. Shi and Y.B. Feng performed the experiments; S.L. Huang, W.W. Kong, and F.F. Shi analyzed the data; W.W. Kong and S.L. Huang wrote the article; Z. Khatoon edited the manuscript; S.L. Huang supervised and edited the manuscript. All authors read and approved the final manuscript. All authors agree to authorship and submission of the manuscript for peer review.

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Kong, W., Huang, S., Yang, Z. et al. Fish Feed Quality Is a Key Factor in Impacting Aquaculture Water Environment: Evidence from Incubator Experiments. Sci Rep 10 , 187 (2020). https://doi.org/10.1038/s41598-019-57063-w

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Fish Pathology Research and Diagnosis in Aquaculture of Farmed Fish; a Proteomics Perspective

Márcio moreira.

1 CCMAR—Centre of Marine Sciences, University of Algarve, Campus de Gambelas, 8005-139 Faro, Portugal; tp.glau@arieromjm (M.M.); tp.glau@amarhcsd (D.S.); tp.glau@ednesera (A.P.F.); tp.glau@arieuqrecam (M.C.); tp.glau@osoparsc (C.R.d.M.); tp.glau@ohlirracvr (R.C.)

2 University of Algarve, Campus de Gambelas, 8005-139 Faro, Portugal

3 IPMA—Portuguese Institute for the Sea and Atmosphere, EPPO—Aquaculture Research Station, Av. Parque Natural da Ria Formosa s/n, 8700-194 Olhão, Portugal

Denise Schrama

Ana paula farinha, marco cerqueira, cláudia raposo de magalhães, raquel carrilho, pedro rodrigues, associated data.

Not applicable.

Simple Summary

The objective of this review is to provide readers with a state-of-the-art description of the main factors affecting farmed fish pathologies and its diagnoses. A special focus is given to the use proteomics technologies as a tool in the evaluation of pathogens and host-pathogen interactions and its impact in disease characterization and control.

One of the main constraints in aquaculture production is farmed fish vulnerability to diseases due to husbandry practices or external factors like pollution, climate changes, or even the alterations in the dynamic of product transactions in this industry. It is though important to better understand and characterize the intervenients in the process of a disease outbreak as these lead to huge economical losses in aquaculture industries. High-throughput technologies like proteomics can be an important characterization tool especially in pathogen identification and the virulence mechanisms related to host-pathogen interactions on disease research and diagnostics that will help to control, prevent, and treat diseases in farmed fish. Proteomics important role is also maximized by its holistic approach to understanding pathogenesis processes and fish responses to external factors like stress or temperature making it one of the most promising tools for fish pathology research.

1. Introduction

The demand for animal protein for human consumption is rising as a result of an exponential increase in the world population. Aquaculture is becoming an increasingly important source of protein available for human consumption since is an industry capable of providing solutions to feed a rapidly growing human population and reduce poverty in many countries [ 1 , 2 , 3 ]. To achieve that, the scale of aquaculture production and the range of farmed species has increased dramatically over the last two decades [ 4 ]. Live production always comprises a risk for loss due to infectious diseases [ 5 ], with farmed fish, due to husbandry practices in aquaculture, being more vulnerable than wild fish to diseases from a wide range of bacterial, viral, parasitic and fungal infections [ 6 ]. Also, the tendency to higher density production systems, the perturbations in ecological systems balance related to pollution and climatic changes, and the expected increase in international transactions of aquaculture products and their derivatives contributed to alterations on the dynamics of interaction between organisms, infectious agents, and people. This influences pathogen rates of replication and proliferation, leading to a broader geographic distribution of pathogenic agents and an increase in species affected by disease outbreaks [ 7 , 8 ]. This makes disease outbreaks an important constraint to this industry, with a significant impact on the quality, safety and volume of the fish produced throughout the world [ 9 , 10 , 11 , 12 ], that can lead to market access exclusion and major economic loss or costs to the producer [ 8 , 13 , 14 ].

For several authors, disease outbreaks in aquaculture are the result of a complex network of interactions on aquatic systems between the produced organism, several environmental and zootechnical aspects, and possible pathogenic agents, that present a series of unique challenges in aquatic organism’s health [ 15 , 16 , 17 , 18 , 19 ], as represented in Figure 1 .

Aquaculture disease diagram, indicating the main factors for the evaluation of pathogen, and host-pathogen interactions intervening in fish disease outbreaks (adapted from [ 19 , 20 ]).

To address infectious pathologies in farmed fish, approaches like epidemiological studies on main areas of aquatic animal health as transboundary and emerging aquatic animal diseases, animal health surveillance and biosecurity program development should be performed. These are crucial to disease prevalence monitorization, early detection of emerging exotic and new diseases and quality management improvement of aquaculture operations [ 15 , 18 , 19 , 21 ].

Nevertheless, to obtain proper epidemiological models, animal health surveillance and biosecurity programs must integrate environmental information and information from different areas like pathogenesis, disease diagnosis, disease resistance, physiological response to pathogens, pathogen characterization, host immune system responses characterization, disease biomarkers and organism response to disease treatment products [ 22 , 23 ].

The amount of data from different origins and an increase in the reported frequency and severity of marine diseases demands that new diagnostic tools should be implemented for a more rapid and effective diagnosis [ 24 , 25 , 26 ]. Thus, several scientific advances in aquatic health continue to close the gap to veterinary medicine, and new optical, analytical chemistry, molecular biology [ 27 ], and Omics techniques are becoming a reality that offers a vast array of benefits to the aquaculture industry [ 12 , 28 ]. Proteomics techniques are one of those new tools, and one of the most interesting approaches for health management, epidemiology, and fish disease research [ 3 , 22 , 23 , 29 , 30 ]. Proteomics refers to the methodology that addresses the study of the entire complement of proteins expressed in a specific state of an organism or a cell population [ 31 , 32 ]. The proteome, or the full protein complement of the genome, is a highly structured entity, where proteins exert their cellular functions with specificity in time and location, in physical or functional association with other proteins or biomolecules [ 33 , 34 ]. High-throughput proteomics methods based on mass spectrometry (MS) allow the measurement of multiple properties for thousands of proteins, including their abundance, tissue distribution, sub-cellular localization, post-translational modifications and protein-protein interactions [ 34 ]. Proteomics-based approaches can therefore offer unique insights into fish cellular regulation in response to pathogens and during disease progression, besides enabling fast and sensitive pathogen detection and identification.

In this manuscript, detailed information regarding the use of proteomics in several disease aspects, with a special focus on the role of stress and welfare in disease, and the importance of pathogen identification and host-pathogen interactions on disease diagnostics and characterization, will be provided.

2. Fish Health, Stress and Welfare

Despite being the most consumed animal, fish are seldom afforded the same level of concern regarding their welfare as other vertebrates. The scientific research around fish welfare is at an early stage compared with other land animals produced for human consumption [ 35 ]. In part, this lack of consideration is due to the gap between public perception of their intelligence and the scientific evidence [ 36 ], along with the absence of a unified definition of the concept [ 37 ]. Nevertheless, most definitions consider mainly a feelings-based and a function-based approach [ 38 ]. The first gives regard to the emotional-like state of the animal, while good welfare is defined as the absence of negative feelings and the presence of positive feelings [ 39 ]. The second definition is more focused on the biological, physiological and health perspective of the animal, while good welfare is defined as the fish’s ability to cope and adapt to its environment while maintaining homeostasis [ 40 ]. Although the fish’s health state offers objective criteria as part of a welfare assessment, it does not provide the complete picture. Good health is essential to ensure good welfare, however, it does not necessarily indicate that the fish is in a good welfare state [ 37 ]. On the other hand, poor health i.e., the reduced ability of the animal to normal functioning, to cope with stressful conditions and to prevent disease, generally implies/leads to a bad welfare status in a variety of contexts. For example, deceased fish, as a consequence of disease, constitute a source of infection and compromise water quality [ 41 ]. Additionally, chemical treatments for specific outbreaks can also trigger some level of disturbance on the fish [ 42 , 43 ]. Importantly, a healthy animal in an optimal welfare environment can also be suddenly struck by an acute infection reducing its welfare. For instance, in the case of fish produced in cages, pathogens are naturally embedded in the environment [ 44 ]. In most cases, it is often the lousy welfare status itself, due to poor husbandry conditions, which translates into impaired health. Thus, in summary, health and welfare are intimately linked, and poor welfare can be interpreted both as a cause and a consequence of poor health. This section focuses on health as a cornerstone for fish welfare assessment and the effects of stressors on disease resistance, reviewing the most recent approaches employed to study the relationship between certain diseases/pathologies and welfare.

In aquaculture, inappropriate husbandry conditions, or even standard farming practices, are everyday stressors in culture systems [ 45 ]. The allostatic load imposed on the animals can reduce functioning immune mechanisms, consequently favoring diseases and threatening fish welfare ( Figure 2 ). For instance, drastic changes in water temperature (from 27 °C to either 19–23 °C or 31–35 °C) decreased the immune response and resistance to pathogens in Mozambique tilapia ( Oreochromis mossambicus ) [ 46 ]. More recently, using a transcriptomics approach, the rearing density in Nile tilapia ( Oreochromis niloticus ) was shown to significantly impact on the susceptibility to the oomycete Saprolegnia parasitica [ 47 ]. However, the association between husbandry-induced stress and disease is not that straightforward. For example, acute stressors have been reported to enhance [ 48 , 49 , 50 , 51 ] or decrease [ 52 , 53 ] some innate immune responses in fish. On the contrary, chronic stressors have mainly been indicated as immunosuppressors [ 54 , 55 , 56 , 57 , 58 ]. From a productivity perspective, the health of the fish is often interpreted as “absence of disease”, since from either an ethical or an economic point of view, any disease state is unacceptable for the industry [ 44 ]. Therefore, disease prevention and eradication are crucial aspects of a fish farm to ensure the production’s sustainability. Providing optimal welfare conditions, monitoring the health parameters routinely and alleviating stress are necessary steps towards this goal.

Interaction between welfare, allostatic load, disease susceptibility and the repetitive/chronic stressful experiences appraised by the fish. Stressful stimuli may induce either adaptive (eustress) or maladaptive allostasis (distress). If the stressor persists, recovery to the original homeostatic state (homeostatic set point) may be incomplete. In this case, a newly defined set point for future adaptation is settled (allostatic setpoint). As a result, the welfare status is decreased with time and stress experienced. The cumulative burden of adaptation (allostatic load) is thus constituted by the beneficial stressful events that the fish can cope with, while the allostatic overload represents the state when stress overcomes the organism’s natural regulatory capacity, which may induce a state of no-recovery. At this step, primary barrier function is severely impaired increasing disease susceptibility, which may cause illness and ultimately death.

Stress is considered a state of threatened homeostasis [ 59 ], which is re-established by a complex network of changes in the physiological systems (allostasis) [ 60 ]. As in all other vertebrates, in the face of a perceived stressor, fish launch a widespread reaction, the so-called physiological stress response, which allows the individual to adjust and cope with the predictable and unpredictable changes in its surroundings (eustress) [ 61 ]. As a primary response, cortisol and catecholamines are released into the bloodstream, which will induce a series of downstream reactions [ 62 ]. In fact, stress is not necessarily detrimental nor immediately equates compromised welfare. Instead, in the short term, it is an essential adaptation to ensure the best chances of survival [ 37 ]. However, when reaching an allostatic overload, usually as a result of a prolonged, repeated and/or unavoidable stressor, maladaptive effects such as impaired growth and/or reproductive and immune functions, arise ( Figure 2 ) [ 63 , 64 ]. In this case, these are largely associated with diminished welfare and may jeopardize fish health and survival (distress) [ 65 ]. The questions raised here are the cost of this acclimation and why stress increases diseases’ susceptibility in fish. First, in terms of energetic costs, the adaptive physiological response needed to counteract the disrupted homeostasis requires a significant amount of energy. This means that if part of the fish’s energy is allocated to face the challenge, then fewer resources will be available for other energy-demanding biological functions, such as some mechanisms of the defense repertoire: the epithelial barriers and the immune system [ 44 ]. In terms of immune responses, several mechanisms are immediately activated to respond directly to the challenge. These include an increase of inflammatory markers, the release of hormones and the expression of acute-phase proteins [ 66 ]. Even if a fish has managed to adapt to the stressor for a certain period, these energy stores will eventually be depleted if the stressor persists. Consequently, the total consumption of energy reserves gives rise to the allostatic overload, and the fish may no longer be able to adapt, which can lead to immunosuppression, disease, and in the case of more severe disturbances, even death ( Figure 2 ) [ 63 ]. Moreover, several studies also demonstrated the impact of stressful husbandry conditions on the functioning of the epithelial barriers, i.e., the mucus and the epidermal surfaces of the skin, gills and intestine, which constitute the primary lines of defense against pathogens and harmful substances, showing that injury of these barriers, inevitably leads to impaired disease resistance [ 67 ]. Changes in these barriers have been reported in Atlantic salmon ( Salmo salar ), Atlantic cod ( Gadus morhua ) and rainbow trout ( Oncorhynchus mykiss ) subjected to different acute stressors [ 68 , 69 ]. Moreover, in Atlantic salmon reared under low dissolved oxygen levels, impaired intestinal barrier function was also observed [ 70 ]. These disturbances have mainly been associated with high cortisol levels, though various other hormones, such as catecholamines, endogenous opioids, pituitary hormones, and serotonin, intervene here [ 71 ]. Indeed, it is known that cortisol plays an immunomodulatory role, inhibiting specific constituents of the immune system and enhancing others, such as induction of apoptosis, change of differentiation patterns, inhibition of cytokine release and inhibition of immunocyte migration [ 72 , 73 , 74 , 75 ]. Nevertheless, the cortisol response may vary among different species and even among individuals (coping styles) [ 76 ] and be affected by several other parameters (e.g., domestication level, age, nutritional state, stressor severity, among others) [ 53 , 77 , 78 , 79 ], which may obscure the relationship between stress and immune status. A detailed description of how the endocrine-immune response is mounted and the mechanisms behind these immunoregulatory changes is out of the scope of this review, for this, the authors refer to recent publications [ 66 , 80 ].

Deepening our scientific knowledge on the mechanisms relating to stress, fish health and welfare, is paramount for the sustainable aquaculture industry. In recent years, more advanced high-throughput technologies, as the case of proteomics, started to be successfully employed in aquaculture research, including for the study of fish diseases and welfare, providing a holistic understanding of the molecular events underlying the physiological stress response and valuable insights on the differential proteins involved in inflammatory processes and immune responses [ 30 , 58 , 81 ]. Proteomic studies on fish target mainly the liver, however, blood plasma and mucus are taking crescent importance, mainly from an immunological point of view, as skin mucus is one of the primary barriers of defense in fish [ 82 , 83 , 84 , 85 , 86 ] and plasma acts as a mirror/reporter of physiological or pathological conditions [ 87 , 88 ]. Important applications of proteomics in this field concern the study of the effects of certain diseases and parasites on the proteins’ abundance and modifications and the investigation of the host-pathogen interactions [ 88 , 89 , 90 , 91 , 92 , 93 , 94 , 95 ]. For example, joint studies evaluating changes in the proteome of fish challenged with a specific pathogen after exposure to a rearing stressor are scarce. However, the existing proteomic studies demonstrating aquaculture and environmental stressors clearly modulating the fish’s immune function [ 58 , 96 , 97 ] reveal that these technologies are already promising sensitive approaches to study this relationship.

3. Disease Diagnostics

To properly diagnose pathology in aquaculture, we must consider disease as a problem with multiple levels of increasing biological complexity, ranging from environmental to the cell, genome and proteome level ( Figure 3 ) [ 26 , 27 ].

Disease diagnosis concentric ring, representing layers of disease diagnoses as environment, community, organism, tissue, and omics as a tool to interpret cell/tissue responses (adapted from [ 26 ]).

New areas like Proteomics can be an important complement to more classical approaches like pathogen identification, disease symptomatology and histopathological analysis to achieve a good disease diagnosis in aquaculture [ 22 , 23 , 27 , 29 , 30 ]. In Proteomics, regardless the complexity of the analysed protein mixtures that can range from hundreds, to several thousands of proteins, the major goal is the accurate identification of the highest number of proteins as possible in those mixtures [ 32 ]. In gel-based approaches, proteins are first separated by one (1-DE)—or two-dimensional gel electrophoresis (2-DE) and then identified by mass spectrometry, whereas in gel-free approaches (or MS-based) protein mixtures remain in solution prior to protein identification. In each case, protein samples may be digested to peptides by a sequence-specific enzyme, typically trypsin, in a so-called peptide-based “bottom-up” proteomics approach, to distinguish it from the analysis of entire proteins in “top-down” proteomics. Peptide samples can then be separated and analysed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), usually employing electrospray ionization (ESI) as the method to convert the peptides to gas phase ions for MS analysis. Alternatively, peptide samples can be analysed by matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry. The method of choice will always depend on the main research objective, costs and expertise, with MALDI-TOF MS based strategies being most suited for microbial identification and diagnosis, as a rapid, sensitive and economical in terms of both labour and costs [ 98 ]. On the other hand, LC-MS/MS is most suited for large-scale, systematic characterization of proteomes, e.g., involved in host-pathogen interactions, allowing multiplex sample analysis and quantitation. In the following sections we will discuss in more detail main applications of proteomics in pathogen characterization and in host-pathogen interactions.

3.1. Pathogen Identification

Pathogen identification is a key area in disease diagnosis and management. Classical, immunological and molecular methods have been routinely and extensively used to address this area [ 26 ]. However, in the last ten years proteomics has emerged as a powerful tool for pathogen identification, strain typing and epidemiological studies [ 98 ], as can be observed in Table 1 .

Resume of some of the proteomic techniques applied to pathogen identification, characterization, and virulence.

Proteomic techniques abbreviations—1-DE: One-dimensional Electrophoresis; 2-DE: Two-dimensional Electrophoresis; SDS-PAGE: Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis; iTRAQ: Isobaric Tag for Relative and Absolute Quantitation; MALDI-TOF-MS: Matrix-Assisted Laser Desorption and Ionization Time-of-Flight Mass Spectrometry; MALDI-TOF/TOF-MS: Matrix-Assisted Laser Desorption and Ionization (Time-of-Flight) 2 Mass Spectrometry; MALDI-TOF/TOF-MS/MS: Matrix-Assisted Laser Desorption and Ionization (Time-of-Flight) 2 tandem Mass Spectrometry; LC-MALDI-TOF/TOF-MS/MS: Automated Liquid Chromatography Matrix-Assisted Laser Desorption and Ionization (Time-of-Flight) 2 tandem Mass Spectrometry; LC-MS/MS: Liquid Chromatography tandem Mass Spectrometry; ESI MS/MS: Electrospray Ionization tandem Mass Spectrometry; LC- ESI-Q-TOF MS/MS: Liquid Chromatography Electrospray Ionization Quadrupole Time-of-Flight tandem Mass Spectrometry; LC-nano ESI-Q-TOF MS/MS: Liquid Chromatography and Nano-Electrospray Ionization Quadrupole Time-of-Flight tandem Mass Spectrometry; LC-ESI-MS/MS: Liquid Chromatography Electrospray Ionization tandem Mass Spectrometry; nLC-ESI-MS/MS: Nano-scale Liquid Chromatography Electrospray Ionization tandem Mass Spectrometry; NanoUPLC-HDMS E : Ultra-Performance Liquid Chromatography with High Definition tandem Mass Spectrometry.

This powerful tool can be used for pathogen identification as a complement to other molecular genetic techniques, being Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) the main technique used for this purpose [ 98 ]. Is also very useful for virulence factors characterization and life cycle characterization of pathogens [ 125 , 126 ].

3.2. Symptomatology

Pathogens have different impacts on fish since the severity of infection depends on diverse factors, such as the host species, fish age and physiological state, environmental conditions, and disease stage [ 127 , 128 ].

Generally, diseases can be expressed in different stages and can develop from an acute to a chronic disease or the reverse way. This is the case of the infectious salmon anaemia (ISA) in Atlantic salmon outbreaks, with initial low mortality, causing minor alterations in the fish (e.g., anaemia). This chronic stage can go unnoticed if diagnostic measures are not performed. Acute disease stages with high mortality may occur sporadically, increasing the severity of the disease (e.g., ascitis and haemorrhages). Furthermore, ISA chronic infection develops in the autumn, while the acute stage is observed more in the spring [ 129 ].

Besides infections with virus, bacteria, parasites and fungus, fish can be exposed to secondary infections that can aggravate their health status and increase mortality rate [ 130 ]. Observation of clinical signs (external or internally) and behaviour alterations can help to detect a pathogen presence in fish. However, the signs exhibited in response to a disease can be non-specific of that disease and very similar between different pathogen infections ( Table 2 ). Moreover, the fish might show few or none of these signs. After these observations, gross and microscopic pathology can be used to confirm some pathogens yet is often necessary the use of more specific types of diagnosis for the identification [ 19 ].

Symptomatology of important diseases caused by virus, bacteria, and parasites.

As shown in Table 2 , even if disease symptomatology is extremely used in disease characterization, it is difficult to distinguish between several diseases with similar symptomatology. Taking this into account, several researchers suggested that host-pathogen interaction are more reproducible and more reliable indicators for disease diagnostics [ 21 , 81 , 168 ].

4. Tools for the Study of Host-Pathogen Interactions

4.1. the “holobiome” approach: metagenomics and metaproteomics.

The host-pathogen interactions are extremely complex and can be established at multiple levels, ranging from molecular, cellular and physiological, to populations and ecosystems levels [ 169 ]. The host-pathogen interaction starts when the host organism is challenged by a pathogenic agent e.g., virus, bacteria, prion, fungus, viroid, or parasite, thus triggering a biological response; the pathogen, in turn, develops a back-fighting response [ 170 , 171 ]. This interaction implies induction of gene expression and protein synthesis on both sides, and an infectious process may develop in the host, leading ultimately to death, if the host response or defense system fails to combat the pathogenic challenge [ 171 ]. However, a wider perspective on host-pathogen interactions may be undertaken [ 172 ], spanning these interactions to the associated microbial populations e.g., the host microbiome, known as the “holobiome” approach [ 173 ]. Indeed, it has been demonstrated that the microbiota may play a critical role in the immune response of organisms [ 174 ]. The “holobiome” approach on the study of fish host-pathogen interactions i.e., between the fish host, its microbiome, the pathogen, and other environmental microorganisms, has been pointed as a critical aspect for further development of rational strategies aiming at fish disease prevention and resistance [ 172 ]. Moreover, this holistic knowledge of fish host-pathogen interactions could contribute to promote sustainability in aquaculture, by reducing the use of antibiotics, responsible for a negative environmental impact of this industry [ 172 ].

Metagenomics and metaproteomics are among the most powerful and emerging high-throughput tools in marine/ocean environments to disclose the genome and proteome, of the associated microbial communities [ 172 , 175 , 176 , 177 ]. These methodologies are still scarce on aquaculture research, although it might be extremely useful in the study of microbial populations inherent to the farmed fish surrounding environment. Furthermore, metagenomics and metaproteomics approaches enable the characterization of the microbiota associated to fish skin mucous or fish gut, thus unravelling key genes or proteins in the immune function, that may act as the whole biosystem through complex networks during fish host-pathogen interactions. An additional and major benefit of these tools is the possibility of accessing to unculturable species, the vast majority of disease-related microbes in aquaculture, whose identity and function would otherwise remain unknown [ 172 ].

4.2. Omics-Based Strategies and Protein-Protein Interaction (PPI) Networks

The knowledge on the genes/proteins and metabolites involved in host-pathogen interactions during infectious events has assisted to considerable advances in the last years, due to the implementation of high-throughput technologies like RNA-sequencing (RNA-Seq) [ 178 ] mass-spectrometry based proteomics [ 126 ] and metabolomics [ 179 ]. On the other hand, the combination of omics-based approaches with in vivo studies, addressing interactions from the single cell to the whole animal level, by using zebrafish ( Danio rerio ) larvae as infection models [ 180 , 181 ], constituted a step forward in the understanding of the cellular mechanisms that occur during fish-pathogen interactions.

The large-scale proteome characterization from both pathogen(s) and fish host, either in health or disease conditions, allowed to identify proteins with a major role in disease defense mechanisms (recently reviewed by [ 126 ]), whose regulatory complexity might be represented by protein-protein interaction (PPI) networks. The integration of proteomics with other omics-based approaches may be used to model networks capable of predicting the interaction dynamics between cellular bio-components involved in fish-pathogen immune responses (e.g., DNA, RNA, protein, metabolite) to foster new therapeutic strategies in aquaculture [ 27 , 179 ]. It can be stated that proteins as the main key players and building blocks across all life forms, since they catalyze and control virtually all cellular processes [ 33 ], hence occupy a central role in host-pathogen interactions. PPIs networks, either determined at experimental level e.g., through interactome proteomic approaches [ 182 ] or predicted by computational methodologies, are gaining increasing popularity and becoming one of the most useful tools in the understanding of pathogenesis [ 183 ]. PPI networks may offer unique insights into host-pathogen and pathogen co-infection interactions, by identifying effective health/disease biomarkers, thus accelerating the implementation of prevention measures, treatment of fish diseases and vaccination development [ 183 ]. PPI network analysis will be no doubt, one of the most powerful and cost-effective tools to assist in fish disease management in the aquaculture sector.

In sum, there is a significant number of emerging tools to address fish host-pathogen interactions that can help in the control, prevention, and treatment of diseases in farmed fish, becoming evident that these interactions are extremely complex, requiring integrated, complementary, and holistic approaches to be fully understood.

Proteomics is also highly used to understand the fish immune response, surviving strategies of the pathogen and interactions between fish and pathogen [ 126 ]. As this technique can show differential expression of identified proteins in various stages of fish development, and different conditions of feeding, stress and disease [ 184 ], it provides a holistic overview of several functions of the fish metabolism [ 185 ]. Differential expression of proteins affected by any pathogen might be studied using gel-based (1 or 2-DE) or gel-free applications (LC-MS/MS) [ 186 ]. An overview of some proteomic studies with fish pathogens is shown in Table 3 . In the case of viruses, several proteins have been modified although differences depend on the type of virus. Spleen tissue of infected zebrafish and turbot ( Scophtalmus maximus ) with Megalocytivirus showed that cytoskeletal and cellular signal transduction proteins were modified in both species [ 89 , 187 ]. Pancreas disease caused by salmonid alphavirus in Atlantic salmon showed that humoral components of the serum were affected during the first weeks after infection [ 94 ]. Proteins involved in the glycolytic pathway and cytoskeleton were modified during viral haemorrhagic septicaemia rhabdovirus in zebrafish [ 188 ]. Host defences against spring viremia of carp virus use mainly proteins like vitellogenin and grass carp reovirus induced protein Gig2 [ 189 ]. These proteins seem to have a potential antiviral activity. Red blood cells in teleost can respond to pathogens and trigger an immune response against the viral septicaemia haemorrhagic virus [ 190 ]. As a defensive mechanism against cyprinid herpesvirus-2 several proteins like herpes simplex infection pathway, p53 signalling pathways and phagosome pathway were induced [ 191 ]. Against bacterial infections, the immune system of teleost fish is triggered, as shown by both the induced acute phase and immune responses in the liver or spleen, respectively of rainbow trout against Aeromonas salmonicida [ 192 , 193 ]. Or by the enhanced immune response against Aeromonas hydrophila in common carp ( Cyprinus carpio ) and zebrafish [ 91 , 194 ]. More specific, proteins involved in the cellular stress response were modified in channel catfish ( Ictalurus punctatus ) after a challenge with Edwardsiella ictaluri [ 195 ]. Enteric redmouth disease in salmonids resulted in several differentially expressed proteins in head kidney and liver samples of rainbow trout like antioxidants, lysozyme, metalloproteinase, cytoskeleton and c-type lectin receptor proteins [ 95 ]. Up-regulated proteins involved in peptidase and hydrolase activity, lysosome and metabolic pathways were identified in intestinal mucosal samples [ 196 ]. Detected on the first defence barrier of fish, the skin mucus showed differentially expressed proteins of the immune system of Atlantic cod with vibriosis [ 83 ]. Also, by proteins like heat-shock proteins, cathepsins and complement components it is shown that the immune response is up-regulated against Streptococcus parauberis in olive flounder ( Paralichthys olivaceus ) [ 197 ]. Mitochondrial enzymes also showed altered expression upon Moraxella sp. infection in kidney tissues of gilthead seabream ( Sparus aurata ) [ 198 ]. Infections with the ciliated parasite Ichthyophthirius multifiliis results in increased mucus secretion in fish. Proteomics of mucus in infested common carp with I. multifiliis showed an up-regulation of immune-related and signal transduction proteins in the first defence barrier of fish [ 199 ]. Infestations of Atlantic salmon with the ectoparasite Lepeophtheirus salmonis were studied on fish mucus and detected an increase in proteins involved in proteolysis [ 82 ]. When looking into the plasma of infested gilthead seabream with Amyloodinium ocellatum , differences were found in proteins involved in the acute-phase response, inflammation, homeostasis and wound healing but, in this case, the innate immunological system was not activated [ 88 ]. Another ectoparasite that affects Atlantic salmon is the amoeba Neoparamoeba perurans , causing amoebic gill disease. Proteomic analysis showed that proteins involved in the cell cycle regulation, inflammation pathway, oxidative metabolism and immunity were affected [ 200 , 201 ].

Summary of some modified proteins identified by proteomics in fish infectious diseases.

Although some examples were given in Table 3 , more studies were performed as each tissue/organ in fish represents a specific barrier against pathogens, and several of them have been used in proteomic studies. Like the shotgun proteomic approach of serum proteins from turbot infected by Edwardsiella tarda , showing that immunoglobulins and complement component proteins were important antimicrobial proteins [ 202 ]. Or the study on Infections by Mycrocystis aeruginosa infections on medaka ( Oryzias latipes ) fish, that showed differences in liver proteins such as stress response, lipid metabolism and developmental processes [ 203 ].

Proteomics may also be used to analyse the pathogen in vitro, which is shown by the reduced expression of proteins related to the tricarboxylic acid cycle and chemotaxis when chlortetracycline antibiotic was used against A. hydrophila [ 204 ]. Virulence mechanisms of bacteria can be studied using proteomics for the visualization of up and down-regulated proteins in virulent and avirulent strains. In the case of E. tarda proteins, like antigenic protein Et 46, bifunctional polymyxin resistance protein and iron-cofactored superoxide dismutase type I were identified [ 92 ]. And in the case of Y. ruckeri proteins like anti-sigma regulatory factor, arginine deiminase, and superoxide dismutase Cu-Zu were identified [ 107 ]. It is known that different conditions like temperature may affect a facultative pathogen like Pseudomonas plecoglossicida which showed upregulation of the pyoverdine protein at 18 °C, which is important for bacterial multiplication [ 205 ]. The iron metal is essential for bacteria [ 27 ], as shown in Vibrio spp., which was able to trap iron [ 125 ], and by Aeromonas salmonicida [ 206 ]. The outer membrane proteins, important for virulence by Y. ruckeri on Atlantic salmon and rainbow trout were identified in different isolates [ 109 ]. As parasites go through various life stages different proteins are needed in each one of them. Proteomics was applied to identify these proteins in I. multifiliis and showed proteins involved in biological processes, cellular components, molecular functions, binding and catalytic activity [ 207 ]. And in the case of Anisakis simplex proteins like pseudocoelomic globin, endochitinase 1 and paramyosin were identified in L3 developmental stage [ 208 ].

To understand the interaction between a pathogen and its host proteomics seems to be a good tool. As mentioned before the outer membrane proteins are important for pathogenicity. The immunity of fish might be reduced as proteins of bacteria are capable of interacting with extracellular proteins [ 209 ]. In the case of Gram-negative bacteria, outer membrane proteins seem to be able to survive inside the fish [ 210 ] and can present resistance to antimicrobial peptides [ 211 ]. Another technique used to identify pathogenic proteins was immunoproteomics. Immunized sera from rohu ( Labeo rohita ) and grass carp ( Ctenopharyngodon idella ) was used to identify outer membrane proteins from E. tarda [ 212 ] and Flavobacterium columnare [ 213 ]. Several outer membrane proteins were identified by immunized sera of Nile Tilapia with Francisella noatunensis subsp. orientalis [ 211 ].

5. Conclusions

Overall, we can look at proteomics as a very promising tool for fish pathology research and diagnostic, allowing a more holistic approach to pathogenesis processes, giving important information on pathogen identification and virulence mechanisms characterization and in host-pathogen interactions, enlightening new stress response routes and previously unknown physiological host responses.

However, the use of proteomics in fish aquaculture is still in its early days and limited to some sequenced organisms. Further progress in defining aquacultural proteomes and large-scale datasets from diseased fish and fish pathogens will boost the use of proteomic techniques in aquaculture, that will lead to new and exciting discoveries on this field.

But one of the most promising and interesting areas and one that we believe being the future trend in further understanding the fish response to pathogens, is the study of the interaction holobiome-host-pathogen, with a strong potential for new and more detailed and integrated knowledge of fish pathogenesis.

Author Contributions

M.M.: Conceptualization, Methodology, Formal analysis, Investigation, writing—Original Draft, writing—Review & Editing Visualization. D.S.: Writing—Original Draft, Writing—Review & Editing. A.P.F. and M.C.: Writing—Original Draft. C.R.d.M.: Writing—Original Draft, Visualization. R.C.: Writing—Original Draft, Writing—Review & Editing. P.R.: Project Administration, Funding Acquisition, Conceptualization, Writing—Review & Editing, Supervision. All authors have read and agreed to the published version of the manuscript.

This work received national funding through the Foundation for Science and Technology (FCT) through project UID/Multi/04326/2020 and projects WELFISH (Refª 16-02-05-FMP-12, “Establishment of Welfare Biomarkers in farmed fish using a proteomics approach”), ALLYFISH (Refª 16-02-01-FMP-0014, “Development of a farmed fish with reduced allergenic potential”) and project SAÚDE & AQUA (MAR-02.05.01-FEAMP-0009), both financed by Mar2020, in the framework of the program Portugal 2020. Márcio Moreira, Cláudia Raposo de Magalhães, and Denise Schrama acknowledge FCT PhD fellowships SFRH/BD/118601/2016, SFRH/BD/138884/2018, and SFRH/BD/136319/2018, respectively.

Institutional Review Board Statement

Informed consent statement.

Informed consent was obtained from all subjects involved in the study.

Data Availability Statement

Conflicts of interest.

The authors declare no conflict of interest and the funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

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• Open access
• Published: 27 August 2020

Recent studies on probiotics as beneficial mediator in aquaculture: a review

• Kazi Nurul Hasan 1 &
• Goutam Banerjee 2  

The Journal of Basic and Applied Zoology volume  81 , Article number:  53 ( 2020 ) Cite this article

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The diseases in fish and other economic aquatic species is a great concern, and every year it causes a huge loss in aquaculture sectors. The use of probiotics might be a good option to reduce the disease risk and to enhance the productivity.

We have gathered information from various important research and review articles related to fish diseases, probiotics, and gut microbial community. We have tried our level best to represent the up-to-date information in a concise manner.

In this present review, we have demonstrated the various beneficial aspects of probiotics in aquaculture sectors. Probiotics are considered as novel functional agents that have potential implications in influencing the gut microbiota of any aquatic organism. Researchers have already documented that probiotics play a wide spectrum functions (such as decrease diseases and stress, enhance immunity, modulate gut microbiota, helps in nutrition, improve water quality, etc.) in host body. Furthermore, the beneficial effects of probiotics contribute to increase feed value and growth of the animal, and improve spawning and hatching rate in aquaculture system. Here, we have discussed each and every functions of probiotics and tried to correlate with the previous knowledge.

The reports regarding the efficacy of probiotics and its detailed mechanism of action are scarce. Till date, several probiotics have been reported; however, their commercial use has not been implicated. Most of the studies are based on laboratory environment and thus the potentiality may vary when these probiotics will be used in natural environments (pond and lakes).

Aquaculture is the fastest growing food industry in several countries like China, India, Norway, etc. According to Food and Agriculture Organization (FAO), the aquaculture production reached 106 million tonnes with an estimated cost of USD 163 $ in the year 2017 with a growth rate of 6.6. The production/captured of finfish was recorded to be highest in Asian countries, followed by Americans countries, Europe, and Africa. Aquatic animals maintain a close relationship with their external environment, which enhance the risk of diseases susceptibility (Banerjee & Ray, 2017 ). Furthermore, high stocking density, water pollution, insecticides containing agricultural drainage water, and unscientific feeding enhance the risk of bacterial, fungal, and viral diseases in cultured animals (Banerjee & Ray, 2017 ). In intensive culture system, disease outbreak is a major difficulty that decreases the profit in food industries, as well as hampers the socio-economic condition of the country (Bondad-Reantaso et al., 2005 ).

The use of antibiotics in aquaculture as a preventive measure associated with the evolution and spread of several resistant human pathogens like Aeromonas sp., Escherichia tarda , Escherichia coli , Vibrio vulnificus , Vibrio parahaemolyticus , Vibrio cholerae , and many more (Allameh et al., 2016 ; Brogden et al., 2014 ). In a review, Lakshmi, Viswanath, and Sai Gopal ( 2013 ) have provided the information regarding the resistance development in aquatic pathogens under long-term antibiotic pressure (Lakshmi et al., 2013 ). Thus, the uses of certain antibiotics in aquaculture industries have been restricted in several countries like the USA and Canada. So, the use of probiotics along with dietary supplementation is a very fruitful strategy to combat pathogenic agents through a variety of mechanisms as an alternative driving force of antibiotic treatment (Bandyopadhyay et al., 2015 ; Wu, Jiang, Ling, & Wang, 2015 ). The term ‘probiotic’ came from Greek words ‘ pro ’ (= favor) and ‘ bios ’ (= life) which are live organisms (usually bacteria or yeast or combination of both) and taken with food to confer beneficial effects to host in various ways (Fuller, 1989 ). The concept of probiotics, in the field of aquaculture, is fundamentally different from those which are used in terrestrial organisms depending upon certain critical influencing factors. It is now well established that probiotics play a vital role in maintaining the gut health by modulation of microbial community structure (Nayak, 2010 ). The microbes also proliferate independently of the host animal in response to diseases (Bondad-Reantaso et al., 2005 ; Irianto & Austin, 2002 ). The first experimental attempt of the probiotic application in aquaculture was made by Kozasa ( 1986 ), considering the beneficial effects of probiotics on humans and poultry (Kozasa, 1986 ). The rapid evolution of probiotics in aquaculture is well established due to the adverse effects of widely used antibiotics and broad spectrum chemicals which kill most of the beneficial bacteria along with the pathogenic bacteria to the aquatic species (Lakshmi et al., 2013 ). Additionally, probiotics also work through different mechanisms in aquaculture system to eliminate the organic wastes and pollutants, as a result of incorporation of ‘bioremediation’ and ‘biocontrol’ when dealing with the environmental problems. In this context, probiotics can play an effective role in aquaculture production by providing greater non-specific disease protection as well as pollution free water sources (Nandi, Banerjee, Dan, Ghosh, & Ray, 2018 ; Panigrahi, Kiron, Satoh, & Watanabe, 2010 ). The goal of this review is to summarize and evaluate the current information on the efficacy and mechanism of action of probiotics for the enumeration in a complex microbial community in aquaculture.

Application methods of probiotics

Based on the mode of action, probiotics can be divided into two broad categories: (a) gut probiotics: which are administrated orally along with food to improve the gut associated beneficial microbial flora (Table 1 ) and, (b) water probiotics: these types of agents proliferate in water medium and exclude the pathogenic bacteria from the specific medium by consuming all available nutrients, resulting in elimination of the pathogenic bacteria through starvation (Table 2 ).

Candidates as probiotics

Recently, the application of probiotics is a very popular practice in aquaculture sectors and it is mainly isolated from fish gut. Among several bacterial candidates, lactic acid bacteria (LAB), Bifidobacterium , and Streptococcus (Giri et al., 2013 ) gain more popularity. Despite the fact that implication of probiotics is relatively a very new approach but it has gained attention due to their potential activity in controlling different physiological activities of aquatic organisms. Thereafter, many probiotics such as Aeromonas media , Bacillus subtilis , Lactobacillus helveticus , Enterococcus faecium , Carnobacterium inhibens , etc. are considered to be significantly effective at present. However, Gram-negative facultative symbiotic anaerobes such as Vibrio , Pseudomonas , Plesiomonas , and Aeromonas were also reported to be potential probiotic candidates present in the gastro-intestinal tract (GIT) of fish and shellfish (Lakshmi et al., 2013 ; Verschuere, Rombaut, Sorgeloos, & Verstraete, 2000 ). Apart from these discussed laboratory-based probiotics, various experimentally approved commercial probiotics are also available in the market which is also effective in aquaculture (Table 3 ).

Screening of probiotics

Although, probiotics have been used in aquaculture due to their broad spectrum biological activities but the selection methods of inappropriate microorganisms lead to failure of many related researches. Screening of probiotics is the first and foremost crucial step that has to be achieved through a step by step fundamental scientific research. Till date, several probiotic candidates have been reported by different research groups; however, their use is restricted in laboratory scale. A full-scale trial of these probiotics is important to commercialize these products in the market. In order to select the potential probiotics, knowledge about the mechanisms of its action is essential (Pandiyan et al., 2013 ). It is widely accepted that a probiotic must contain some definite features in order to aid the correct establishment of effective agents (Priyodip, Prakash, & Balaji, 2017 ; Thakur, Rokana, & Panwar, 2016 ). The selection criteria of probiotic include the following: (a) it should be harmless to the host; (b) it must be non-invasive, and non-carcinogenic; (c) it should reach effectively at the host’s target site; (d) it should contain plasmid without antibiotic and virulence resistance genes; (e) it should be colonized for a stable time period and replicate within the host; and (f) it should actually work in host model system as opposed to in vitro findings.

However, the probiotic screening to date is concentrated on the search for active agents against a pathogen which induce the interruption in the aquatic environment. In in vitro screening for potential probiotics, most of the researchers employ identification of inhibitory or antagonistic activity (Kesarcodi-Watson, Kaspar, Lategan, & Gibson, 2008 ; Sahu, Swarnakumar, Sivakumar, Thangaradjou, & Kannan, 2008 ). To screen for inhibitory substances in vitro, four methods are commonly applied; the double layer method, the well diffusion method, the cross-streak method, and the disc diffusion method. The basic principle of all these methods is based on the fact that a bacterium (producer) produces an extracellular substance which is inhibitory to itself, or another bacterial strain (indicator) (Kesarcodi-Watson et al., 2008 ; Priyodip et al., 2017 ). The methods used in aquaculture include some major steps: (a) a background knowledge about the application of probiotics; (b) attainment of alleged probiotics; (c) both in vivo and in vitro assessment of their pathogenicity; and (d) a long-term practical evaluation of the treated probiotics. Recently, a number of fast and sensitive molecular tools are also used for selection and evaluation of probiotics includes ERIC-PCR and PCR-DGGE/TGGE techniques, FISH, and 16S rRNA gene sequencing (Qi, Zhang, Boon, & Bossier, 2009 ; Wu et al., 2015 ) (Fig. 1 ).

Demonstrates the flow chart of different screening methods and selection criteria of probiotics in aquaculture

Beneficial effects and mode of action of probiotic in aquaculture

The risk of disease enhancement in aquaculture industries fosters the probiotic research for developing sustainable aquaculture. With the increased public concern on the use of antibiotics, it is not surprising to increase a rapid growth of the probiotic for aquaculture. Food and Agricultural Organization (FAO) has now recommended the application of probiotics for the improvement of aquatic environmental quality by reducing the mortality (Subasinghe, 2005 ), or by increasing the resistance against putative pathogens of host (Irianto & Austin, 2002 ). The beneficial effects are temporal on occasion, depending on the time of application (Verschuere et al., 2000 ). The effectiveness and mode of actions of many probiotics used recently in aquaculture are summarized in Table 4 .

Maintenance of water quality

Probiotics help to improve water quality due to their ability to participate in the turnover of organic nutrients in aquaculture (Wang & Wang, 2008 ; Wang, Zheng, Liao, Huang, & Sun, 2007 ). Organic enrichment and nitrogenous wastes, including ammonium and ammonia (NH 3 ), are a serious concern in aquaculture, for example in pond rearing of catfish (Sahu et al., 2008 ). To date, the information regarding the maintenance of the balance of NH 3 /NO 2 /NO 3 in pond by probiotic candidates is limited (Wang et al., 2007 ) (Fig. 2 ). There is a strong tendency to combine different photosynthetic bacteria, Bacillus , nitrifiers, and denitrifiers together; therefore, probiotics are often labeled as multifunctional and can be applied to various species under diverse culture conditions (Wang & Wang, 2008 ). Apart from these, probiotics are more efficient in transforming the organic matter to CO 2 (Fig. 2 ); therefore, it is suggested to maintain their high levels in production ponds to reduce the organic carbon load and to enhance the water quality and fish health.

Diagrammatic presentation of the summary of information gathered from the studies on different aquatic organisms to explain the possible role of probiotics in aquaculture system. Probiotics improve (indicated by upwards arrow) body weight, digestion rate, surface area of microvilli, antioxidative enzymes, stress tolerance, immune response, fecundity, fertilization of the host, as well as water quality of the aquatic environment. It also downregulates (indicated by downwards arrow) pathogenic bacteria and their enteric colonization, viral activity, cortisol level in host organisms. But the effects of probiotics on the reproductive axis of aquatic animals are still lacking (indicated by question mark). Ag antigen, CAT catalase, DC dendritic cell, GnIH gonadotropin-inhibiting hormone, GnRH gonadotropin-releasing hormone, GPx glutathione peroxidase, GtH gonadotropin, Ig immunoglobulin, IGF - I insulin-like growth factor-I, IHNV infectious hematopoietic necrosis virus, MAMPs microbial-associated molecular patterns, PRR pathogen pattern recognition receptors, SOD superoxide dismutase

Augmentation of growth and survival rate

Probiotic is also used to promote the growth of different cultivated species in aquaculture. In Javanese carp ( Puntius gonionotus ), Enterococcus faecalis causes significant weight gain when supplemented at 10 7 and 10 9 cfu g −1 diet compared to the control group of carp (Allameh et al., 2016 ). The microorganisms are able to colonize within the GIT due to their higher multiplication rate than the rate of expulsion after the administration over a long period of time. Probiotics are added constantly to fish cultures to maintain the health by enhancing the expression of several immunological factors, and to reduce the pathogen load to the gut mucus layer by occupying the physical space (Banerjee & Ray, 2017 ). Furthermore, probiotic candidate also play a vital role in nutrient enhancement in host. Hamdan et al. ( 2016 ) have reported the enhancement of crude lipid, total protein, and body weight in Nile tilapia ( Oreochromis niloticus ) fed with probiotic strain of Lactobacillus sp. (Hamdan et al., 2016 ). This also depends on factors such as water quality, hydrobionts species, enzyme levels, and genetic resistance. Tan, Chan, Lee, and Goh ( 2016 ) have also reported that growth and survival rate increase in Xiphophorus helleri , Xiphophorus maculates , and Poecilia reticulate fed with probiotic supplemented food containing Bacillus subtilis and Streptomyces sp. (Tan et al., 2016 ).

Improvement in nutrient utilization

Probiotic microorganisms have beneficial effects in GIT of aquatic animals in the digestion of dietary nutrients as well as in production of energy. The most commonly used probiotic preparations in this purpose are the lactic acid bacteria (Ringø et al., 2018 ). It is found in large numbers in the gut of healthy animals and, in the words of Food and Drug Administration (FDA), is generally regarded as safe (GRAS status) (Giri et al., 2013 ). However, this increased nutrient digestibility are due to the elevated level of digestive enzymes (protease, amylase, cellulose, phytase, etc.) produced by the probiotic altered gut-associated microbial community in the host (Banerjee, Nandi, & Ray, 2017 ; Burr & Gatlin, 2005 ; Ghosh, Banerjee, Moon, Khan, & Dutta, 2017 ). For example, few bacteria (viz. Rhodobacter sphaeroides and Bacillus sp.) participate effectively in the digestion processes by activating protease, lipase, amylase, and cellulase enzymes significantly in white shrimp ( Litopenaeus vannamei ) (Wang & Wang, 2008 ) and in bivalves (Sahu et al., 2008 ). Additionally, a few recent studies have shown that probiotics may also stimulate the nutrient absorption by increasing the surface area of the host GIT, based on quantitative changes in histological measurements of the area of intestinal fold, enterochromaffin cells, and microvillus (Zhou, Buentello, & Gatlin, 2010 ) (Fig. 2 ). It is also suggested that Lactobacillus brevis and Bacillus subtilis are capable of producing higher amount of enzyme phytase (up to 1,354,906.6 U/mL) which helps to utilize the plant product phytate, chemically known as myo -inositol hexaphosphate (Priyodip et al., 2017 ). Till date, several bacterial candidates ( Pseudomonas sp., Brevibacterium sp., Microbacterium sp., Agrobacterium sp., and Staphylococcus sp.) have been reported to contribute in nutritional and metabolism physiology in Arctic charr ( Salvelinus alpines ) (Ringø, Dimitroglou, Hoseinifar, & Davies, 2014 ). Different bacterial strains in the form of probiotics also contribute significantly by modulating gut microbial population of the host organisms especially by synthesizing the fatty acids, minerals, vitamins, and essential amino acids (Nayak, 2010 ; Newaj-Fyzul, Al-Harbi, & Austin, 2014 ).

Effects on phytoplankton

Probiotic bacteria play vital role in controlling algal growth, particularly of red tide plankton (Qi et al., 2009 ). Bacteria antagonistic toward algae will be undesirable in green water larval rearing technique in hatchery where unicellular algae are cultured, but will be advantageous when undesired algae species are developed in the culture pond.

Bacteriostatic effects of probiotics

Probiotic bacterial populations may release a variety of chemical substances that have a bactericidal or bacteriostatic effect on both Gram-negative and Gram-positive bacteria. These inhibitory substances belong to different origin such as proteinaceous substance (lysozyme and different kind of proteases), chemical (hydrogen peroxides), and iron-chelating compound like sideropheres (Giri et al., 2013 ). LAB produces a compound—bacteriocins that can alter inter-population relationships by influencing the outcome of competition for chemicals, or energy (Kesarcodi-Watson et al., 2008 ; Ringø et al., 2018 ). These inhibitory substances play an important role in pathogen inhibition and proliferation, and thereby reduce the pathogen load. The information about the inhibitory substances produced by probiotic bacteria are given in Table 5 .

Stimulation of decolonization of pathogenic bacteria

One possible mechanism for preventing colonization by pathogens is physical competition for attachment sites on the gut mucosal layer in host. It is known that the ability to adhere to mucus and wall surfaces is necessary for bacteria to become established in fish intestines (Cruz, Ibáñez, Hermosillo, & Saad, 2012 ; Roeselers et al., 2011 ). Since bacterial adhesion to tissue surface is important during the initial stages of pathogenic infection, competition for adhesion receptors with pathogens might be the first probiotic effect (Chabrillón, Arijo, Díaz-Rosales, Balebonz, & Moriñigo, 2006 ). In general, probiotic microorganisms possess mucus binding proteins which help in the acceleration of the binding process. In an investigation, Mackenzie et al. ( 2010 ) have reported the differential expression pattern of a key receptor mub in different strains of Lactobacillus , and have compared their binding efficacy in the gut mucosa (Mackenzie et al., 2010 ).

Augmentation in the immune system

Probiotics play the beneficial role as immunostimulatory to assist in the protection of aquatic cultured species by reducing the impact of diseases and entrance of pathogens (Dawood & Koshio, 2016 ; Hai, 2015 ). Thus, its use as an immunostimulants is very practical approach to improve the success of the aquaculture. Many authors have confirmed the use of probiotics to elevate immune response, disease resistance, and reduce malformations in carp species (Wu et al., 2015 ). The possible mechanism of its action is cellular as well as humoral immune responses, and expression of IL-1b, TNFα, and lysozyme-C are increased when fish are fed with Aeromonas veronii , Vibrio lentus , and Flavobacterium sasangense enriched diet (Dawood & Koshio, 2016 ). Myeloperoxidase, lysozyme, complement component C3, albumin and immunoglobulin levels, respiratory burst activity, and phagocytic activity by blood leucocytes are improved in several fish species (Chi et al., 2014 ; Giri et al., 2013 ). An experimental report have supported that probiotics supplemented at 10 CFU/g diet for 2 weeks act as an immunomodulator by binding its MAMPs (microbial associated molecular patterns) to pathogen pattern recognition receptors (PRRs) on immunogenic cells like dendritic cells, macrophages, which trigger intracellular signaling cascade, resulting in the release of specific cytokines and interleukins by the activated T cells to exert anti-viral, pro- or anti-inflammatory exercise effects (Akhter, Wu, Memon, & Mohsin, 2015 ; Balcázar et al., 2006 ) (Fig. 2 ). Unfortunately, the specific role of probiotic supplementation on the immunological parameter expression is still not clearly understood.

Effects on viral pathogens

Though, data indicate that virus inactivation can occur by some extracts from different probiotic bacterial strains in aquaculture but the exact mechanism by which it exerts its action is not known. It is well established that probiotic candidates like Pseudomonas sp. and Vibrios sp. are very effective against ‘infectious hematopoietic necrosis virus’ (IHNV) (Sahu et al., 2008 ). Furthermore, Paralychthys olivaceus fed with Sporolac ( Lactobacillus sp.) supplemented food develop resistance against lymphocystis disease virus (LCDV) (Harikrishnan, Balasundaram, & Heo, 2010 ). Similar experiments have also proved the enhanced virus resistance power in grouper fish fed with probiotic strain of Bacillus subtilis E20 (Liu, Chiu, Wang, & Cheng, 2012 ).

Effects on reproduction

The use of probiotics on disease resistance ability is well documented, but research on the effects and action mechanism of probiotics on the reproductive performance of aquatic animals are lacking (Fig. 2 ). Very few studies have attempted to demonstrate the role of probiotic supplementation on reproductive performance in aquaculture (Abasali & Mohammad, 2011 ; Ghosh, Sinha, & Sahu, 2007 ), using various strains like B . subtilis , Lactobacillus acidophilus , Lactobacillus casei . It is well documented that probiotics influence reproduction in different factors like fertilization, gonadosomatic index, fecundity, and production of fry from the females (Abasali & Mohammad, 2011 ). Recent study also reported that probiotics increase the daily numbers of ovulated eggs compared to control levels with higher hatching rate and faster embryonic development in zebrafish (Gioacchini et al., 2013 ). However, rigorous experiments still need to be established for the utilization of probiotics to increase the production rate of aquatic animals.

Other functions

Very few recent investigations also highlight the effects of probiotics on some major physiological processes in aquatic organisms. In European seabass, it helps to increase the body weight by stimulating the mRNA transcription of insulin-like growth factor (IGF)-I (Carnevali, Sun, Merrifield, Zhou, & Picchietti, 2014 ). Additionally, it is now profoundly accepted that probiotics reduce the concentration of the stress hormone cortisol and activate the expression of antioxidative enzymes (superoxide dismutase, catalase, and glutathione peroxidase) to increase the stress tolerance (Zolotukhin, Prazdnova, & Chistyakov, 2018 ) (Fig. 2 ), which are essential for better reproductive performance in aquatic organisms (Hasan, Moniruzzaman, & Maitra, 2014 ; Hasan, Pal, & Maitra, 2020 ).

The mode of probiotic application can be in several ways: (i) addition to the artificial diet and culture water, and (ii) bathing and addition via live food. Furthermore, understanding the mode of action along with proper application methods may be the key for probiotics use in aquaculture. Although the exact mode of action is yet to be revealed, it often exert host as well as strain-specific differences in their activities. However, the use of probiotics is gaining potential scientific and commercial interest in aquaculture at global basis (Banerjee & Ray, 2017 ; Carnevali et al., 2014 ; Hoseinifar, Ringø, Masouleh, & Esteban, 2016 ).

Probiotics and different types of food in aquaculture

The use of balanced probiotic containing feed is a common practice in commercial aquaculture sectors which provides several beneficiary effects to farmer and consumers in term of improved growth performance, flesh quality, production rate, fish immunity, protein quantity, carcass quality, intestinal health, and reduced malformations (Hai, 2015 ; Ige, 2013 ). However, a huge number of farmers from developing and low-income countries still rely on natural feeds (usually phytoplankton and zooplankton) for fish farming to reduce the production cost, but it reduces the production rate, flesh quality, and enhances mortality, and thus ultimately affects the income. Several researchers have proved that probiotic feeding in fish from their first stage of life (larvae) is profitable due to diseases load is low in later stage (Table 6 ), but the delivery of probiotics during early stage is quite difficult. The protection of hatchling or larvae is the most challenging issue in aquaculture. So, the manipulation of microbiota by inoculating probiotic strain and their uses is a promising alternative. However, in later stage, probiotic-enriched formulated artificial balanced diet is good for fish health and the application of it is very easy. Moreover, farmers have to be careful of three main constraints (Vadstein et al., 2018 ; Vine, Leukes, & Kaiser, 2006 ) viz., (a) leaching of feed which reduces the availability of probiotic to the host. Thus, dose standardization and regular monitoring is required. (b) Probiotic candidate confers beneficial effects to the host only when it is active or live under different appropriate environmental conditions, so farmers have to be concern about these facts. (c) Nature of various ponds differ depending on the physicochemical parameters and natural feeds (zooplankton and phytoplankton). So, application, types, and dose of probiotics will be varied accordingly.

Probiotics and fish gut microbial community

Gut environment provides a favorable niche for indigenous microorganism by providing space, attachment sites, and nutrition. Balanced microbial communities are very important for maintaining gut health (Banerjee & Ray, 2017 ; Giatsis et al., 2016 ). During disease condition, the natural microbial communities in the gut are disrupted, which creates several health-related problems. Fish lives in such a condition which is surrounded by a huge population of pathogenic bacteria, fungi, and deadly virus (Egerton, Culloty, Whooley, Stanton, & Ross, 2018 ). Restoration of gut microbial communities through dietary probiotic supplementation is an effective method to improve fish health (Han et al., 2015 ). However, selection of probiotics varies greatly from one fish species to another to properly maintain the good to bad ratio of bacteria in the gut mucosal surface. Till date several bacterial candidates have been tested for probiotic potential; however, few candidates of Bacillus sp., Micrococcus sp., Enterococcus sp., Phaeobacter sp., Shewanella sp., lactic acid bacteria, and Pseudomonas sp. have gained popularity in manipulating gut flora in fish (Lobo et al., 2014 ; Merrifield et al., 2010 a, b ). In an investigation, Asaduzzaman and co-workers have reported the beneficiary effects of three probiotics ( Shewanella sp. AFG21, Bacillus sp. AHG22, and Alcaligenes sp. AFG22) in Tor tambroides which are able to shift the microbial composition toward good bacterial populations (Asaduzzaman et al., 2018 ). Several researchers reported that probiotic significantly induced many fold gut microbiota to produce several metabolites including volatile short-chain fatty acids (VSCFs), which play a vital role in maintaining gut health in fish (Fig. 3 ) (Allameh, Ringø, Yusoff, Daud, & Ideris, 2017 ; Asaduzzaman et al., 2018 ; Burr & Gatlin, 2005 ). Researchers also reported that probiotic modulation of gut microbiota is not restricted to fish age and maturation, as probiotics confer beneficial effects to all age group ranging from larvae to adult (Merrifield & Carnevali, 2014 ). A previous study reported that probiotic supplemented diet in rainbow trout was very effective to enhance the population of beneficial bacterium Bacillus subtilis (Newaj-Fyzul et al., 2007 ). They also reported that colonization of B . subtilis on the gut epithelial surface conferred protection (boost immunity, reduced oxidative stress, increased serum lysozyme concentration, and enhance phagocytic activity of specialized cell) against pathogenic strain of Aeromonas sp. The finding of Newaj-Fyzul and co-workers (Newaj-Fyzul et al., 2007 ) was further supported by the study conducted by Bagheri, Hedayati, Yavari, Alizade, and Farzanfar ( 2008 ), who used commercial probiotic product (Bioplus) containing a mixture of B . subtilis and Bacillus licheniformis. In the same direction, an investigation conducted in four fish species ( Poecilia sphenops , Xiphophorus maculates , Poecilia reticulate , and Xiphophorus helleri ) fed with B . subtilis containing diet and reported the population enhancement of B . subtilis on the intestinal mucosal surface (Ghosh, Sinha, & Sahu, 2008 ). Recently, the effects of two probiotic strains Bacillus subtilis and Rhodococcus sp. have evaluated on gut microbiota of Oreochromis niloticus (Martínez Kathia et al., 2018 ). The results of their study clearly indicated a significant shifting of gut microbial community (increasing percentage of proteobacteria and bacteroidetes) in probiotic fed fish compared to control. Furthermore, study also reported that bacteria belongs to phyla proteobacteria are important members as they are involved in mineralization of organic compounds and nutrient recycling process in fish (Cardona et al., 2016 ). However, the gut microbiota restoration capability of two probiotics also tested in diseased black molly ( Poecilia sphenops ) treated with antibiotics (Schmidt, Gomez-Chiarri, Roy, Smith, & Amaral-Zettler, 2017 ). Results of their study indicated that both the probiotic candidates ( Phaeobacter inhibens S4Sm and Bacillus pumilus RI06-95Sm) were able to restore the microbial community back to the normal. Among several probiotic strains, lactobacillus groups as probiotics in aquaculture have been studied extensively. It is well established that lactobacilli has high colonization property and thus retain for a longer time on the gut epithelial surface, and confer greater beneficial effects to host and gut microbiota (Merrifield & Carnevali, 2014 ). Researches on germ-free fish model indicated that probiotic along with environmental factors have high impact on gut microbiota modulation in term of antibody production, stress release, and resistance colonization (Kelly & Salinas, 2017 ). The microbial manipulating property of probiotic on gut mucosal surface depends on several external/environmental (water quality, temperature and pH) and internal (fish age, binding strength of the probiotic, duration of probiotic supplement diet, delivery system, etc.) factors. Alteration in any of these factors may hamper the probiotic efficiency. The cross talk between host and microbe on the gut epithelial surface is a complex phenomenon and is responsible to maintain a healthy environment. Restoration of gut microbiota in patient using fecal microbial therapy (microbiota collected from healthy individual) to solve several diseases is common practice in human (Aas, Gessert, & Bakken, 2003 ). The probiotic research in mammal including human is at peak level; however, such depth of research is still lacking in the case of aquaculture.

Effects of probiotics on metabolites production by gut microbial flora

Probiotics and mucosal immunity

Apart from systemic immunity, fish possess a well-defined mucosal immunity which is very important for protection and survival. Till date, the mucosal immunity in fish has been studied mostly in teleost fish (Lazado & Caipang, 2014 ). Mucosa-associated lymphoid tissues (MALT) in teleosts can be divided into three broad categories: skin-associated lymphoid tissue (SALT), gut-associated lymphoid tissue (GALT), and gill-associated lymphoid tissue (GIALT). However, lymphoid tissue [nasopharynx-associated lymphoid tissue (NALT)] has recently been discovered by Salinas ( 2015 ). Immunomodulation by probiotic bacteria is a vital process which confers strength to fish for combating with surrounding pathogen in the water, as well as inside the body. The mucosal secretion in fish contain a wide spectrum of anti-microbial peptides (AMPs) such as AJN-10 (Liang, Guan, Huang, & Xu, 2011 ), Gaduscidin-1 and -2 (Browne, Feng, Booth, & Rise, 2011 ), Piscidin 3 (Dezfuli, Giari, Lui, Lorenzoni, & Noga, 2011 ), and YFGAP (Seo, Lee, Go, Park, & Park, 2012 ), which have direct role in pathogen inhibition (Fuochi et al., 2017 ; Gallo & Nakatsuji, 2011 ; Gomez, Sunyer, & Salinas, 2013 ). Skin mucus layer act as a first defence barrier in fish, as it is in direct contact with water. Among the lymphoid tissues, GALT is the most important one and interestingly in fish it lacks Peyer’s patches like mammal. However, GALT contains the other important components (plasma cells, macrophages, lymphocytes, etc.), which are necessary for defense (Lazado & Caipang, 2014 ). It was reported that probiotic modulate the mucosal immunity in fish by increasing the population (10–30%) of granulocytes and lymphocytes cells which is related to cell mediated mucosal defence (Lazado & Caipang, 2014 ). Furthermore, an investigation on GALT of seabream ( Sparus aurata ) also reported that oral administration of a mixture of probiotic strains ( Lactobacillus plantarum and Lactobaccillus fructivorans ) enhanced the production of antibody and G7 + granulocytes cells (Picchietti et al., 2007 ). In general, plasma cells of fish produce three types of antibodies: IgM, IgD, and IgZ. The action of IgT/IgZ is thought to be associated with the gut mucosal immunity in fish (Salinas, Zhang, & Oriol Sunyer, 2011 ). Whereas, IgM is a general immunoglobulin responsible for combating invaded pathogen and the level of this antibody is elevated in the gut mucus in fish fed with probiotic supplemented diet (Salinas et al., 2008 ). Probiotic administration also enhanced the population of IgM producing B cell in gut lamina propria in juvenile fish (Abelli, Randelli, Carnevali, & Picchietti, 2009 ). Similarly, effect of probiotic on gut integrity and gut mucosal immunity in rainbow trout ( Oncorhynchus mykiss ) fingerling have also tested (Gisbert, Castillo, Skalli, Andree, & Badiola, 2013 ). Result of various studies also confirmed that Bacillus cereus confers significant beneficial effects on gut by increasing villi height (average 14.5%), villi area (average 28.6%), villi weight, as well as by enhancing the leucocytes infiltration and goblet cell number (1.63 ± 0.03 in respect to control 1.22 ± 0.05 per 100 μm of intestinal epithelium) (Asaduzzaman et al., 2018 ; Gisbert et al., 2013 ). Nowadays, the research on fish mucosal immunity gain a huge popularity and several researchers are involved in this field (Table 7 ). Immunization/vaccination is an effective method in disease resistance, but its use is still limited in aquaculture sectors (Liu et al., 2019 ). It is believed that vaccination of fish to boost the gut mucosal immunity is more effective rather than systemic immunity. Though, probiotics are very effective in protection against a wide range of pathogens, but the use of mucosal vaccines is the first choice as it lengthens the protection period (Munang'andu, Mutoloki, & Evensen, 2015 ).

Conclusion and future perspectives

The current researches improvise and optimize the utilization of probiotics in aquaculture industry. Notably, the future application also looks bright due to the ever-increasing demand of probiotics for aquacultured animals. Further investigations will demonstrate the techniques to screen host specific probiotic strains from aquaculture rearing system to manage significantly its quality and functional properties. Furthermore, research should also focus on studying the effects and mechanism of action of probiotics on the reproductive performance and gonadal development of aquatic organisms in an industrial scale hatchery system. Probiotic bacteria confer a broad spectrum of beneficiary effects to host, but still there are certain limitations. For example, antimicrobial compounds or bacteriocins produced by probiotic candidates against pathogenic bacteria are not species specific. Thus, strain improvement is necessary to enhance the efficiency of probiotic bacteria. There are several molecular biology techniques such as recombinant technology, mutagenesis, etc. that are available to improve the genetic makeup of the probiotic strain. However, application of these techniques is limited to probiotic candidates used for aquaculture. Future investigation must be done to solve these serious issues and to prepare effective probiotics.

Availability of data and materials

Data and relevant information were obtained from NCBI-PUBMED central, Scopus, MEDLINE and Google scholar.

Abbreviations

Anti-microbial peptides

Colony forming unit

Food and Agriculture Organization

Food and Drug Administration

Gut-associated lymphoid tissue

Gill-associated lymphoid tissue

Gastro-intestinal tract

Insulin-like growth factor

Infectious hematopoietic necrosis virus

Interleukin

Lactic acid bacteria

Lymphocystis disease virus

Mucosa-associated lymphoid tissues

Microbial-associated molecular patterns

Nasopharynx-associated lymphoid tissue

Polymerase chain reaction

Pathogen pattern recognition receptors

Skin-associated lymphoid tissue

Tumour necrosis factor alpha

Volatile short-chain fatty acids

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We are very much thankful to Department of Zoology, Sidho-Kanho-Birsha University, Purulia, India for providing necessary supports. The authors are thankful to Mr. Akash Acharyya for his academic support.

This work was funded by University Grants Commission (UGC) [F.30- 448/2018(BSR)] to KNH and DBT-Boost Programme, Govt of West Bengal, India.

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• Aquaculture
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Role and relevance of fish cell lines in advanced in vitro research

• Published: 11 January 2022
• Volume 49 , pages 2393–2411, ( 2022 )

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• M. Goswami   ORCID: orcid.org/0000-0001-6863-7647 1 ,
• B. S. Yashwanth 1 ,
• Vance Trudeau 2 &
• W. S. Lakra 3  

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Introduction

Cell line derived from fish has been established as a promising tool for studying many key issues of aquaculture covering fish growth, disease, reproduction, genetics, and biotechnology. In addition, fish cell lines are very useful in vitro models for toxicological, pathological, and immunological studies. The easier maintenance of fish cell lines in flexible temperature regimes and hypoxic conditions make them preferable in vitro tools over mammalian cell lines. Great excitement has been observed in establishing and characterizing new fish cell lines representing diverse fish species and tissue types. The well-characterized and authenticated cell lines are of utmost essential as these represent cellular functions very similar to in vivo state of an organism otherwise it would affect the reproducibility of scientific research.

The fish cell lines have exhibited encouraging results in several key aspects of in vitro research in aquaculture including virology, nutrition and metabolism, production of vaccines, and transgenic fish production. The review paper reports the cell lines developed from fish, their characterization, and biobanking along with their potential applications and challenges in in vitro research.

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Fish cell line: depositories, web resources and future applications

Murali S. Kumar, Vijay Kumar Singh, … Kuldeep Kumar Lal

Applications of Fish Cell Cultures

ABCD of Zebrafish Culture

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The Development of chemically defined cell culture medium like Leibovitz -15 (L-15) and the development of antibiotics with gradual improvisation of cell culture techniques eventually made the generation of cultured cells for deriving continuous cell lines. In addition to being an important biomedical tool like any other cell line, cell cultures prepared from fish, shellfish and seaweeds can provide a significant contribution to the growth of aquaculture. The scientific knowledge gained through the cell culture system can be utilized for manipulating the whole organism to enhance its usefulness for aquaculture. Their cell line could be useful for providing basic insights into growth, reproduction, and health, creating opportunities for manipulation and thus the cell lines could be used as sources of biochemical products in place of the whole organism [ 1 ]. Cell-based aquaculture systems using cell cultures could be a game-changing practice to produce seafood and other aqua food across multiple species for meeting the demand of the burgeoning world population [ 2 ]. A cell-based aqua food production system utilizing cells in place of whole fish could also lead to greater preservation of the aquatic environments. This practice has to meet the regulatory framework/guidelines developed by the FDA [ 3 ] for the safety of food produced using such animal cell culture technology.

Fish cell culture offers several advantages over mammalian cell culture in terms of adaptation to a broad range of temperature, higher tolerance to hypoxia, easier maintenance of cell culture for longer periods. Cell lines from fish have been increasingly established from different aquaculture species and they are being used in in vitro research related to aquaculture and other interdisciplinary areas. However, there are emerging issues regarding standardization of cell line nomenclature, characterization of cell lines following SOP/recommended guidelines, and conservation of cell lines in separate biobanks across the world-which we review below is of utmost essential to maintain scientific reproducibility in cell-based biological research using fish cell lines. The key areas of aquaculture like fish health, disease diagnosis, safety, and nutritional aspects challenging aquaculture production can be studied using fish cell lines without scarifying whole live fish (Fig.  1 ). The scientific knowledge generated using fish cell lines would be immensely useful for quality fish production in a sustainable manner. Cell lines would facilitate in vitro research for developing climate-resilient and sustainable aquaculture systems to minimize the key challenges and provide nutritional security to the burgeoning world population.

Implications of fish cell line in aquaculture

Global status of fish cell lines

An increasing trend has been observed for the development of fish cell lines from a wider range of tissues covering both tropical and temperate water since the first establishment of the RTG-2 cell line in 1962 [ 4 ]. Bairoch enlisted 883 fish cell lines out of 104,421 cell lines from > 590 species in Cellulosaurus; a knowledge resource on cell lines [ 5 ]. In general, cell lines have been developed globally using different types of fish tissue samples including gill, caudal fin, eye, liver, and kidney. Fish cell lines have also been established using tissue samples like intestine [ 66 ], brain [ 95 ], vertebra [ 105 ], and snout [ 121 ]. Spontaneous differentiation is one of the most challenging for the development of embryonic stem cell culture from fish and this is the main cause behind a very limited number of stem cell lines. Few embryonic fish stem cell lines were developed from sea bream Sparus aurata [ 6 ], sea perch Lateolabrax [ 7 ], sea bass; Lates calcarifer [ 8 ], Catla catla [ 9 ], Labeo rohita [ 10 ]. A feeder-free cell culture system used for the development of Embryonic Stem (ES) cell lines from medaka and zebrafish has boosted fish stem cell research by replacing the use of feeder layers to inhibit spontaneous differentiation in fish stem cell culture [ 11 ].

Cell line characterization

The numbers of cell lines developed from fish have been increasing rapidly which raises the concern for accurate authentication and characterization of fish cell lines to provide reproducible scientific data. The comprehensive guidelines for using cell lines highlight various aspects of cell culture, issues of misidentification, contamination with microbes along with recommendations to overcome these problems [ 12 ]. Although these guidelines are meant for scientists in the UK, the basic principles remain the same for international implications. Research and development using cell lines need detailed knowledge on the purity and originality of the cell line [ 13 ]. The characterized cell lines are indispensable as they facilitate the researchers to perform in vitro research and standard guidelines are available for their characterization. However, many fish cell lines don’t meet uniform international standards. The Food and Drug Administration has described the steps to be considered while characterizing a cell line used to produce biological products [ 14 ]. Such standard protocol for the characterization and authentication of fish cell lines should be practiced throughout the world. Standard protocols for authentication of cell line have been reported [ 15 ] wherein standard methods like cytochrome c oxidase subunit1 (CO1) barcode, Short Tandem Repeat (STR) profiling, karyotyping, Single Nucleotide Polymorphisms (SNP) profiling, use of species-specific primers, whole-genome sequencing (WGS), etc. are described as ideal approaches for authentication and maintenance of quality cell lines. Several STR databases of cell lines are maintained by ATCC, DSMZ across the world. CLASTR: The Cellosaurus STR similarity search tool is now in the public domain for comparing STR profiles of the cell lines [ 16 ]. Cross-contamination also causes a disastrous feature of the cell line as the cell line losses its originality and hence cross-contamination needs to be avoided by following standard operating procedure (SOP). Development of a framework for cell line annotation linked to STR and SNP profiles in the form of a catalog of synonymous cell lines to avoid or detect cross-contamination [ 17 ].

Misidentification of cell lines leads to irreproducible data and hence proper authentication of cell lines using molecular markers is essential. It was obligatory to provide DNA-based certification of the cell line developed [ 18 ]. Mitochondrial DNA genes like 16S rRNA and CO1 are used for the authentication of cell lines. Cox I gene has been used as a molecular identification system for animal species which is popularly referred to as ‘‘DNA barcoding’’ [ 19 ]. The cox I gene was used as DNA barcodes for the authentication of 67 cell lines [ 20 ]. Similarly, many fish cell lines have been DNA barcoded using cox I [ 21 , 22 , 23 ]. Cell line repositories like DSMZ, ATCC use DNA barcoding as a standard method for cell line identification. Protein expression signature has also been used for the identification of cell lines derived from fish [ 24 , 25 ].

Cell lines developed from fish are mostly applied in basic, biomedical and toxicological research in addition to their potential applications in aquaculture. Several key issues in aquaculture can be addressed by cell culture technology and they are reviewed below.

Fish health management

The fish disease has been considered as one of the most critical challenges for sustainable aquaculture production due to the economic loss and widespread use of antibiotics and other compounds causing great risk to the aquatic environment. Fish cell culture has great potential to provide tools and strategies for disease control in aquaculture. In vitro models that use cell culture methods and experimental systems facilitate a deeper understanding of the complex interactions underlying disease outbreaks and its advancement in which the interactions between aggressors and the host can be dissected [ 26 ]. Fish cell lines have potential applications in understanding disease mechanisms, developing assays for disease diagnosis, developing drugs and vaccines for the control of the fish disease. The export trade of seafood depends upon the quality and health status of the seafood. The fish cell line model has been considered useful for detecting viral pathogens and strategies need to be implemented accordingly for the health protection of major aquaculture species. Zoonotic disease associated with fish is another concern where consumption of unhealthy fish might be a risk to a human being. Associated in vitro assays would be useful in detecting such harmful pathogens and allergens so that the quality of seafood can be augmented. In vitro methods using cell cultures for addressing health issues in molluscs and crustaceans are equally important. Department of Biotechnology, Govt. of India has funded a national programme on the isolation and characterization of finfish and shellfish viruses using cell lines in India. In vitro approach using permanent cell lines needs to be validated for fish and shellfish disease surveillance and health certification. Transboundary movements of live aquatic animals have greatly increased concern for spreading disease in the aquaculture system.

The viral disease used to cause devastating loss to the aquaculture industry. The entire world has witnessed the deadliest effect of the spread of the virus Covid-19. The isolation of the novel Covid-19 using the animal model has begun and the successful isolation would be useful in understanding the biology and evolution of the Covid-19 in developing drugs, vaccines, and rapid diagnosis kits. Isolation of viruses using fish cell lines is one of the most sensitive techniques for the discernment of the important pathogens causing viral disease in many fish and other species. Hence, the development of control measures to halt the spread of the viral disease depends on the unitisation of fish cell lines for such purposes. A comprehensive list of fish cell lines used in virus susceptibility studies is given in Table 1 . Research on the avoidance of infectious fish disease in aquaculture necessitates a cell culture- based approach for understanding the underlying disease mechanism. Fish cell culture-based isolation and propagation of virus has provided momentum to virological studies and facilitated research on viral diseases in important aquaculture species. Propagation of viruses in a cell culture system is one of the bases of a virus surveillance system using cell culture. Ariel et al. developed standard methods to reduce false negatives in cell culture-based surveillance systems in testing fish cell line susceptibility for the viruses [ 27 ].

Highly specific cell lines are used for investigating unique virus which otherwise doesn’t propagate in any normal cell line. The susceptibility of the fish cell lines to the virus varies with species as well as tissue from where the cell line is developed . This raises the importance of the development of species-specific and tissue-specific cell lines from various important aquaculture species. Some fish cell lines like bluegill fry (BF-2), chinook salmon embryo (CHSE-214), epithelioma papulosum cyprinid (EPC), fathead minnow (FHM), rainbow trout gonad (RTG-2), and SAF-1 have shown susceptibility to some of the most commonly available viruses like Infectious pancreatic necrosis (IPN), VHSV, IHNV, IPNV, SVC, koi herpesvirus (KHV) and Channel catfish virus (CCV) that have severely affected several aquaculture species [ 28 , 29 ]. MEF-8C1 cloned cells obtained from the MEF cell line from mandarin fish suitably propagated megalocytiviruses that cause major problems in finfish aquaculture in China [ 30 ]. A transgenic fish cell line RTG-P1 was applied to estimate viremia of Salmonid alphavirus (SAV) which causes a serious viral disease i.e. Salmon Pancreas Disease (SPD) in Atlantic salmon farming [ 31 ]. SISK and SISS cell lines developed from the kidney and spleen of Lates calcarifer respectively and SIGE cell line developed from the eye of Epinephelus coioides showed their ability to propagate a nodavirus strain [ 32 ]. Yashwanth et al. reported the susceptibility of the OCF cell line to NNV [ 23 ]. SSN-1 cell line supported the replication of snakehead fish vesiculovirus (SHVV) which causes great economic loss in fish culture in East Asian countries [ 33 ]. Understanding the transmission of viral infection between the two most important aquaculture species mandarin fish and snakehead fish, it would be useful to develop control measures to prevent the spreading of such viral diseases. Fish cell cultures or cell lines could be used for investigating viral pathogenesis and host–pathogen interactions.

In the past, such in vitro methods were used for some bacterial pathogens like mycobacterial host–pathogen interactions using the carp monocytic cell line CLC (carp leukocyte culture) [ 34 ]. Recently, Cardiac Primary Cultures (SCPCs) from Atlantic salmon pre-hatch embryos were used to investigate viral host–pathogen interactions and pathogenesis [ 35 ]. A blend of cell culture and molecular biology methods will provide deeper insights into host–pathogen interactions. Although advanced antibody-based techniques are being developed in disease control in aquaculture, fish cell culture continues to be an indispensable technique for isolation and characterization of the pathogenic virus and intracellular bacteria and studying their pathogenicity [ 26 ]. These fish cell lines are going to play a crucial role in virus isolation and understanding viral pathogenicity and thereby controlling these viral diseases to enhance sustainable aquaculture production.

Pathological & immunological studies

Several fish health-related issues can be studied in vitro using fish cell lines. The most prominent is the application of fish cell lines in disease diagnostics and immunological studies. Some intracellular bacterial fish pathogens like Rickettsiae spp and Renibacterium salmoninarum have been detected in fish using cell cultures [ 36 , 37 ]. The in vitro investigation using fish cell line (CHSE-214) has improved the knowledge of the infection process by  Yersinia ruckeri  in salmonid fish as well as the interaction between the pathogen and host cells [ 38 ].

Cell cultures are promising in vitro tools in studying the host defense mechanism and thereby help in exploiting the immunological information for the health protection of fish and shellfish used in aquaculture. Fish leukocyte cell lines and macrophages developed from many aquaculture species like carp, catfish have been used for immunological studies. Several monocyte-like cell lines have also been developed using peripheral blood leukocytes of channel catfish [ 39 ]. Cell lines developed from gut, skin, and gill are promising in vitro tools for studying the defense mechanism in fish. The immunological potentials of DNA vaccines, synthetic peptides and immunostimulants, and other products can be tested using these fish cell lines. A continuous blood cell line developed from peripheral blood mononuclear cells of Cyprinus carpio was useful in understanding the fundamental aspects of fish immunology [ 40 ]. Fish macrophage cell lines are found to be very useful in numerous research applications including immunological studies. Two macrophage cell lines i.e., CTM and CCM [ 41 ] developed from Catla catla could be useful in investigating the importance of these cell lines in the differentiation and maturation of thymocytes and other fish immunological studies. SHK-1 macrophage-like cell line developed from Atlantic salmon showed the reaction to monoclonal antibodies against Atlantic salmon peripheral blood leukocytes and the cell line was able to phagocytose bacteria [ 42 ]. Macrophage-like cell line RTS11 developed from rainbow trout was used as a promising tool for investigating immune cell-specific responses in vitro [ 43 , 44 ].

Saprolegniales are considered the most important fungi causing disease in freshwater fish. The cytological response of their piscine hosts is not precisely understood. RTS11 cell line developed from rainbow trout was used to check the response of macrophage to water molds Achlya and Saprolegni [ 45 ]. Fish cell lines are a very useful aid in understanding pathogenicity arising due to nutritional issues. Such studies were carried out using fish cell lines to investigate the proinflammatory mechanism underlying the relationship between dietary PUFA and cardiac lesions using a cell line developed from chum salmon [ 46 ].

gene-editing and genetic engineering

Genetically edited fish cell lines have enormous biotechnological and clinical applications. CRISPR (Clustered Regularly Interspaced palindromic repeats-Cas9 (CRISPR associated) has revolutionized gene editing. Generation of improved fish cell lines using CRISPR-Cas9 technology would facilitate aquaculture biotechnological research including fish disease studies. Genetically edited cell lines using genome editing technology would be useful to enhance the transfection efficiency of fish cell lines and utilize those cell lines for the efficient production of viruses for vaccine development. This technique has been mostly used for gene editing in mammalian cell lines whereas the use of gene editing for fish cell lines is in the infancy stage. The use of CRISPR-Cas9 based gene editing method has been reported earlier in fish but an efficient method for gene editing was developed in a fish cell line CHSE developed from Chinook salmon Oncorhynchus tshawytscha for the first time [ 47 ]. The cell line was genetically engineered to overexpress different forms of CHSE cell line. Although various attempts have been made, a convincing fish knock-out in vitro model has not yet been developed.

A stable trout head kidney cell line was transfected with a variety of plasmids expressing cytokines Interleukin-6 macrophage colony-stimulating factor (MSCF). Rainbow trout head kidney cell line and RTG-2 stable cell lines were engineered in developed conditioned media to express interleukin (IL-2), IL-6, and macrophage colony-stimulating factor (MCSF) [ 48 ]. Greasy grouper Epinephelus tauvina liver cell line GL-av was genetically modified to assess the effectiveness of the anti-apoptotic protein Bcl-xL [ 49 ]. Fish cell lines have also important applications in in vitro ploidy manipulation. Polyploidization was successfully obtained in a crucian carp induced by a chemical compound and developed an autotetraploid cell line [ 50 ].

Genetically engineered fish cell lines have enormous potentials to be used in fish health, genetics, and biotechnological research. The establishment of a stable cell line is the need of the hour for functional genomics studies for fish genetics and health. With the progress of gene delivery methods, the number of stable genetically modified fish cell lines has increased. Not much effort has been made for the functional characterization of immortal fish cell lines towards developing genetically engineered methodologies [ 51 ]. Genetical modification of goldfish cell line, Chinook salmon Oncorhynchus tshawytscha embryo CHSE cell line, rainbow trout Oncorhynchus mykiss hepatoma RTH cell line have provided interesting information for fish disease and immunological research [ 52 , 53 ]. A transformed EPC (Epithelioma Papulosum Cyprini) cell line under the control of the tilapia HSP70 promoter expressed a green fluorescent protein (GFP)-luciferase fusion gene in response to cellular stress [ 54 ]. The fish cell lines can be used for studying stressors concerning infectious fish disease in addition to their usage in investigating environmental stressors concerning climate change. A novel in vitro system was developed using genetically modified Chinook salmon embryonic (CHSE)-TOF cell line to measure the sensitivity of some important virus-like Infectious Haematopoietic Necrosis Virus (IHNV), Infectious Pancreatic Necrosis Virus (IPNV), Salmon Alphavirus (SAV), and Epizootic Haematopoietic Necrosis Virus (EHNV) [ 55 ].

Transgenic studies and reproductive biotechnology

Gene targeting and transfer of the genes for transgenic fish production become easier with the advancement of cell culture techniques. Transgenic zebrafish produced applying primary cultures of genetically modified zebrafish male germ cells has paved the way for the development of transgenic lines in model organisms or other animal systems [ 56 ]. Genetically modified myogenic cell culture was developed from a transgenic trout ( Oncorhynchus mykiss ) having a construct containing the GFP reporter gene driven by a fast myosin light chain 2 (MlC2f) promoter [ 57 ]. The transgenic line can be produced by utilizing the primordial germ cell (PGC) cultures. Vasa marker facilitates isolation and characterization of targeted PGCs for germline-specific expression in fish. Tanaka et al. developed a transgenic line of medaka using GFP expressed germ cells [ 58 ]. Successful transplantation of germ cells in fish demonstrated the possibility of surrogate broodstock production in the aquaculture system. Intraperitoneal transplantation of PGCs was used to produce seedlings in rainbow trout for the first time [ 59 ]. The progress in stem cell culture and their subsequent applications in vitro basic research, as well as aquaculture biotechnology, will transform the fisheries sector for achieving the blue revolution. Spermatogonial stem cells transplantation offers many scopes for a successful captive breeding programme for aquaculture species. A spermatogonial cell line (SG3) developed from the mature testis of medaka was capable of producing sperm [ 60 ]. The production of fertile medaka fish using ES cells proved the possibility of generating nuclear transplants using fish embryonic cells [ 61 ]. More research needs to be carried out in aquaculture species utilising these ES cells. Gene transfer through embryonic stem (ES) cell is a promising tool for the production of transgenic animals [ 62 ]. Yoshizaki et al. successfully developed a stem cell-mediated gene transfer method to produce transgenic rainbow trout [ 63 ]. ES cells along with PGCs and nuclear transfer strategy make the transformation efficient for transgenic fish production.

Fish cell lines as in vitro models

Fish cell lines have enormous potentials to be used model systems for studying fish disease, immunology, biotechnology, nutrition, and toxicity testing of chemicals and therapeutic agents used in aquaculture as they are ideal substitutes for the whole organism which involves increasing questionable ethical issues. In vitro model has been used for investigating the viral replication and genetics and the production of experimental vaccines to be used in aquaculture. Organ culture developed from tilapia, eel, and trout pituitary glands was used as in vitro model for the production of the growth hormone prolactin [ 64 ]. Fish cell lines were found to complement in vivo development studies and recognize the involvement of signaling pathways in the developmental processes [ 65 ].

Cell cultures developed from fish can be effectively used as model systems to investigate nutrient assimilation and metabolism in fish but rarely such a culture system has been used to study that aspect of fish nutrition. This also raises the need for the development and characterization of the intestinal cell culture systems to support such studies. Cell line developed from the fish intestine is useful in understanding the effect of functional feed ingredients like probiotics and dietary exposure to chemicals in the aquatic system. Kawano et al. reported the use of the intestinal rainbow trout epithelial cell line (RTgutGC) to elucidate the metabolism of environmentally relevant contaminants in the intestinal tract of fish [ 66 ]. Recently, RTgutGC was used as an in vitro model for understanding the functional immunity system of the fish gut as well as the effects of functional feed ingredients in the gut cells [ 67 ].

Langan et al. investigated the function of spheroid size in the metabolism of propranolol using an RTgutGC cell line as a 3D fish intestinal model [ 68 ]. The cell line of the intestinal epithelial region rainbow trout acts as a barrier to study cellular mechanism of immune function, physiological and pathological response, nutrient uptake, and toxicants [ 118 ]. The above RTgutGC cells were compared with new cell lines from the proximal and distal intestine of rainbow trout such as RTpi-MI & RTdi-MI and these cells formed a polarized barrier, which was not permeable to larger molecules and absorbed glucose and proline [ 119 ]. The RTgill-W1 cells were used as in vitro model for accessing acute toxicity of select chemicals associated with Whole Effluent Toxicity (WET) testing in both marine and freshwater conditions [ 120 ]. A physiologically realistic model system- fish-gut-on-chip was developed by combining intestinal cell culture from rainbow trout ( Oncorhynchus mykiss ) with microchip technology and microfluidic engineering to study its barrier function towards the environment i.e. food & water and to monitor the function in real-time [ 69 ]. In vitro models are extremely important to study collagen synthesis and secretion in humans and other higher vertebrates. Very few models have been established to investigate collagen synthesis and secretion in fish. Lee and Bols reviewed the potential applications of fish cell lines to study collagen as in vitro model for evaluation of physic-chemical factors controlling synthesis, secretion, and deposition of collagen [ 70 ].

• Cell-based aquaculture

Aquaculture has been growing very fast and facing several challenges to meet the rising demand ensuring the safety and quality of fishery products. The concept of producing cell-based seafood has been emerging as a new approach to producing alternate animal protein. This alternative approach of animal protein production from fish would address several key challenges faced by the conventional aquaculture systems and declining marine capture fisheries. This alternative fish production system will reduce pressure on natural resources and the environment. Accordingly, the entire world is moving towards climate-resilient production systems and in vitro meat production has emerged as an area of cutting edge and priority research. The successful launch of the in vitro hamburger in 2013 has accelerated the research focus on cell-based meats [ 71 ]. The ease of growing fish cells at a lower temperature compared to mammalian cells may give cost benefits to the production of cellular fish meat as compared to cellular animal meat. Tissue engineering blend with modern aquaculture techniques can be explored to utilize marine cell culture as an attractive opportunity for the production of in vitro fish meat. Fish muscle cell culture can be used for in vitro fish meat production by exploiting their salient physiological properties like tolerance to a hypoxic-conditions, high buffering capacity, and lower temperature [ 2 ]. Fish muscle cell cultures are more adaptable to in vitro conditions than mammalian ones and hence in vitro meat production will be more feasible with fish muscle cell cultures. More concerted efforts and investigations are required to generate information on fish and shellfish muscle cell culture systems to suit in vitro fish meat production systems. The fastest possible path to produce cellular fish meat should start with zebrafish for research and development purposes [ 72 ].

The importance of genetic modification and closed aquaculture system paves the way for the innovative concept of cell-based fish production i.e. cellular aquaculture [ 73 ]. American space organization NASA had supported the first research program on in vitro edible muscle protein production from goldfish for space travelers during long-term manned space exploration [ 74 ]. A better understanding of the myogenesis involved in the muscle cell and tissue culture would be essential to trap the benefits of muscle cell culture in promoting cellular aquaculture. In vitro models like C2C12 cell lines have been utilized in understanding molecular mechanisms underlying muscle growth and differentiation in mammals [ 2 ]. Such studies are in the infancy stage in teleost due to the unavailability of equivalent permanent muscle cell lines except for a few fish muscle cell lines [ 75 , 76 , 77 , 77 ]. Most of them are not from aquaculture species except muscle cell lines developed from Wallago attu [ 21 ], olive flounder  Paralichthys olivaceus [ 78 ] and some myosatellite cells developed from the primary culture of muscle-derived from carp [ 79 ] and rainbow trout [ 80 ]. Prospects of cell-based aquacultures will rely on the development of appropriate cell lines, optimization of growth media, and other factors, mass production of cells. Some institutes like Good Food Institute; New York, USA have taken initiatives to develop cell-based seafood.

The cell-based molecular mechanism studies will provide basic research data for cell-based fish production. Some investigations on harvested native muscle tissues from fresh water and marine fish provide interesting insights into the potentials of developing a muscle cell culture system [ 81 , 82 ]. The genome editing by CRISPR/Cas9 system is a promising tool that attains targeted gene editing with high efficiency, without the requirement of integrating an exogenous gene. Its potential is yet to be exploited much in aquaculture using fish cell lines. CRISPR/Cas9 system has been used to get higher skeletal muscle/ muscular growth in aquaculture species like red sea bream; Pagrus major [ 83 ] and channel catfish; Ictalurus punctatus [ 84 ]. Clean meat farm is a million-dollar industry but academic research lags to propagate clean meat production [ 85 ]. Academic research focusing on the development of muscle cell culture systems, standardization media, and bioreactor facilities for large-scale cell production would be required to accelerate in vitro fish meat production and bring it to market.

Vaccine and other products developed from fish cell culture

Global aquaculture particularly shrimp farming used to suffer a major economic loss every year due to the occurrence of viral diseases. The development of vaccines has great relevance to the aquaculture industry to mitigate viral diseases. Purified viruses are likely to be the first health product for use as vaccines obtained from piscine cell cultures [ 1 ]. Several viral vaccines have been produced with improved techniques for their delivery at affordable prices [ 86 ]. Several fish cell lines have been tested for virus replication towards vaccine development. There is a need for scaling up the efforts towards the development of effective vaccines.

Cell-culture-based technology can be used as a robust and reliable alternative for the production of vaccines. The development of the vaccine and its potency testing requires a large number of live fish. Fish cell culture can be used as an alternative to whole live fish for the production and testing efficacy of fish vaccines. Cell lines like Vero, Madin Darby canine kidney (MDCK), chicken embryo fibroblasts (CEFs) have been mainly used for viral vaccine production [ 87 ]. Cell lines developed from humans, monkeys, hamsters, dogs, and chickens have so far been used for the development of vaccines. The studies for the development of viral vaccines using fish cell lines are very much limited. Oh et al. reported that the formalin-inactivated RSIV vaccine was developed from the viruses propagated in Grunt Fin (GF) cells [ 88 ]. Several inactivated or attenuated fish viral vaccines have been developed for iridovirus and NNV protection [ 89 , 90 ], and some of them have been commercialized [ 91 ]. However, few cell lines are available to replicate megalocytivirus, betanodavirus, herpesvirus, and aquareovirus for vaccine production, and hence more efforts are warranted to develop specific cell lines for the proliferation of these viruses. Fish cell cultures have great applications in modern vaccine technology including recombinant, DNA/RNA particle vaccines. Only a few fish cell lines have been used in viral propagation leading to vaccine development and diagnostics, and many are under trial for vaccine production. Rainbow trout pronephros cells as in vitro model could be used to screen fish DNA vaccine [ 92 ]. The anti-VP5 polyclonal antibody was able to neutralize Grass carp reovirus (GCRV) through in vitro micro neutralizing assay in a grass carp cell line CIK [ 93 ]. This would be important towards the development of a vaccine to prevent the infection of GCRV in grass carp which causes great damage to grass carp production in China.

The deficiency of treatment options and limited availability of vaccine poses a challenge for control of viral disease control in aquaculture. In this regard, JL122, a broad-spectrum antiviral agent oxazolidine compounds, was proven to inhibit transmission of IHNV, VHSV, and SVCV in the EPC cell line [ 94 ]. Another small molecule LJ001, lipophilic thiazolidine derivative also showed broad-spectrum antiviral properties for inhibition of IHNV infection in the EPC cell line. These hold promise as an immersion treatment option for the outbreak of aquatic rhabdoviral infection. The complex interaction between Infectious kidney and spleen necrosis (ISKNV) and its host Chinese Perch Brain (CPB) cells generated new information for understanding viral pathogenesis and developing antiviral treatment strategies [ 95 ].

Fish cell lines exhibit less transfection efficiency, unlike mammalian cell lines. Low transfected cell lines are not useful for the production of recombinant protein and other products. In the case of mammalian cell lines, a higher range of transfection efficiency of mammalian cell lines with the aid of the right combination of cell type and method was achieved up to 100% [ 96 ]. However, the same methods applied to fish cell-cultured at lower temperatures (5–15 °C) provided the low transfection efficiency which is often below 10% [ 97 ] whereas the transfection efficiency of the head kidney cell line was improved from 11.6% to 90.8% using Amaxa's cell line nucleofector solution T and program T-20 [ 98 ]. Hence, alternative reagents or methods should be explored to enhance transfection efficiency in fish cell lines. In addition to vaccine production, fish cell lines should also be explored for the production of human pharmaceutical proteins. The ability of the fish cells to grow at as lows as 4 0 C could be exploited in this regard. Transformed fish cell line Epithelioma papulosum cyrpini cells (EPC) were used to stably express and secret recombinant pleurocidin (Ple), a linear cationic peptide of 25 amino acids uninterruptedly for more than 2 years [ 99 ]. Fibroblast cell plays an important role in increasing collagen synthesis, collagen secretion under the stimulatory influence of ascorbic acid. Some cell lines developed from fish have been reported to be an ideal in vitro source for the synthesis of collagen [ 100 ]. Cytokines such as interferon could be considered for their potential therapeutic potential to fill the gap of shortage of fish therapeutics [ 1 ]. Fish interferon was partially purified in small quantity from the rainbow trout gonadal cell line, RTG-2. Transfected RTG-2 cell line expressed Interleukin Cytokines (Interleukin (IL)-2, IL-6, and Macrophage Colony Stimulating Factor (MCSF) and the transfected cell line was used to produce conditioned media-rich in these cytokines [ 48 ].

Toxicological and environmental monitoring studies

Different inorganic and organic aquatic pollutants influence the quality and health status of farmed fish and shellfish. Proper investigation to know the ill effects of the aquatic pollutants on farmed fish and shellfish is the need of the hour to improve the marketing of quality seafood. The cell lines have been used as alternative tools to replace the use of whole live fish due to a significant correlations observed between in vitro and in vivo data. Cell lines have been applied as a rapid and economic in vitro tool for screening toxicity of chemicals and environmental samples [ 1 , 93 ]. Fish cell lines have important applications in studying the effects of different aquatic pollutants on the metabolism of aquatic biological systems and hence there is a potential application of fish cell lines in environmental monitoring. Fish cell lines have been adopted as an in vitro tool for ecotoxicological evaluation of chemicals by many international regulatory bodies like Registration, Evaluation, Authorisation, and Restriction of Chemicals (REACH) in Europe, Food, and Drug Administration (FDA) in the USA. Fish cell cultures facilitated in vitro investigation to find toxic effects of polycyclic aromatic hydrocarbons and aflatoxins in farmed fish [ 101 ]. Primary cultures have been used in the case of toxicological investigation in invertebrates as permanent invertebrate cell lines are not available. Considerable progress has to be made for the development of invertebrate cell lines to facilitate in vitro investigations in farmed shelf fish and mollusc. In addition to the aquatic pollutants, toxic and residual effects of antimicrobial drugs used in aquaculture need to be investigated where fish cell culture can be utilized to replace the whole live fish model. In vitro studies established a correlation between in vitro immunosuppression and the interference of various antimicrobial drugs [ 102 ]. In this regard, in vitro investigation will provide more insights to increase the awareness of global antimicrobial resistance (AMR) initiated World Health Organization.

Fish nutrition and metabolism

Fish cell lines have the potentials to be used in fish feed formation using alternative ingredients as they provide an excellent in vitro model to study nutrient absorption and assimilation. To facilitate such studies, more intestinal fish culture systems need to be developed. Due to the lack of targeted research tools, the current understanding of the underlying effects of feed ingredients on fish nutrition is limited. The application of appropriate fish cell lines would facilitate further research on the basic functions of the digestive tract and the effects of functional feed ingredients on various aspects of fish nutrition [ 67 ]. The vital role of cell lines in biological experimentations is to reduce animals, with major three R rules such as reduction, replacement, and refinement [ 103 ]. That enhances the interest of researchers to utilize the in vitro model to study cellular environmental conditions of living biological components. The primary cultures of adipocytes or hepatocytes and myoblasts were significantly used to study molecular mechanisms related to fish nutrition [ 104 , 105 , 106 , 107 , 108 ]. This approach provides significant progress to a limited extent because the primary cultures failed to allow the functional genomic analysis to study the specific gene functions.

Morin et al., 2020 studied the role of RTH-149, RT hepatoma-derived cell line to address nutrition-related queries based on major pathways such as macroautophagy (autophagy), general control nonderepressible 2 (GCN2), and mechanistic Target of Rapamycin (mTOR) pathway that regulate cell homeostasis through amino acids to study the nutrient-sensing signalling. These pathways had attention concerning rainbow trout nutrition, which strongly relies on the supply of amino acid and assessing (1) their capacity to be repressed or induced by starvation, (2) their specific regulation by amino acid availabilities, and (3) their related kinetics. They demonstrate that the starvation can be sensed by RTH-149 cells, which then induce the activation of GCN2 and drive the expression of ISR-related genes in an amino acid-dependent manner. The high concentration of HF (1000 nM) upregulates chop but represses the induction of other ISR-related genes. This result corroborates previous findings from different species demonstrating that Chop overexpression contributes to a negative feedback loop responsible for attenuating the starvation-induced GCN2 response. They also demonstrated RT specificities for amino acid dependencies, time response, and the activation levels of their downstream targets [ 109 ]. They concluded that RT cell lines could be an alternative in vivo to analyze nutrition-related queries in Rainbow trout and other carnivorous fish using dietary proteins that provide most of energetic metabolism.

The regulations of atg4, lc3b, and sqstm1 observed in RTH-149 cells were previously described in a mouse cell line to be induced following starvation in a GCN2/ATF4-dependent manner [ 110 ]. The starvation-induced autophagy kinetics measured in RTH-149 cells matches with the cells of starved mouse embryonic fibroblast (MEF) [ 111 ]. That indicates the amino acid sensing and mTOR activation in RTH-149 cells follow the mechanisms shared between trout, human, and mice cell lines [ 112 , 113 ] and that has been conserved throughout evolution.

Several fish cell lines have been used as in vitro models to study elongation and desaturation of different PUFA. These in vitro models were also useful for unveiling the pro- inflammatory mechanisms underlying the relationship between dietary PUFA and cardiac lesions in salmon [ 46 ], the effect of fatty acid diet on fish inflammatory responses [ 122 , 123 ]. To study fish nutrition and metabolism, cell lines provide a great interest near future, especially advanced methods, such as CRISPR/cas9 and that may certainly work with new feed formulations for the development of sustainable aquaculture.

With global biodiversity rapidly declining, the need to preserve and conserve biological specimens becomes crucial. Cryopreservation techniques have long been used in agriculture for conservation. Proper freezing of cells can generate a bank of genetic material that can remain viable for hundreds or even thousands of years in the future, with the potential not just to act as reference specimens, but the capacity to regenerate live individuals of a species. While classic cryopreservation methods result in frozen sperm, which would need a fresh egg or frozen embryos–which poses challenges for proper freezing–new technology allows for the production of viable offspring from spermatogonial stem cells of fish.

The rapidly increasing number of fish cell lines raises the need for their long-term storage and conservation in different locations. Fish cell lines are not only the source material for in vitro research but also critical for the conservation of fish germplasm. Integrated efforts protect animal populations within their natural habitat (in situ conservation) and outside their natural environments ( ex-situ conservation). Similarly, ancillary conservation facilities like repositories of serum, DNA, and cell lines have been supporting basic and applied research [ 114 ]. Bio-banking is emerging as one of the most efficient approaches to provide security at the highest level against the loss of diversity of species [ 115 ]. Caulfield and Murdoch have critically reviewed various social and technical issues of biobank globally including public perception, biorights, privacy, technology, and commercialization [ 116 ]. The standard operating procedure should be followed for the long-term conservation of fish cell lines. The stability of the cell lines and recovery rate of the fish cell lines should be assessed using different replicates in the freezing medium at different passages to minimize the loss of cells. Working and master stocks of the fish cell lines also need to be maintained separately in the cell line repository. An automated controlled-rate programmable freezer would be ideal to provide reproducible cryopreservation with an optimized freezing program as per the cell’s requirement [ 117 ]. A simpler device like cryocan filled with liquid nitrogen may also be used for the storage of cell lines. Researchers can access fully characterized and quality-controlled cell lines from a repository without spending time to develop as per their requirement and at a minimal cost. The repository acts as an “insurance” to secure the loss of cell lines developed by a single laboratory. There should be many standby repositories at different places to avoid loss of the cell lines in case of any catastrophic event. Hence, the cell line repository facilitates promote the propagation of in vitro research as well as the conservation of fish germplasm.

The leading cell line repositories in the world like American Type Culture Collection (ATCC), European Collections of Cell Cultures (ECACC), German Collection of Microorganisms and Cell Cultures (DSMZ) have been providing characterized and authenticated cell line to researchers across the world. Details of cell lines maintained in the repositories worldwide are given in Table 2 . Some of the cell lines in the repository suffer from misidentification and contamination due to multiple transfers between laboratories. A certificate citing STR profile for each line is essential to guarantee authentic and contamination-free cell lines [ 12 ]. A very encouraging progress in the development of cell lines from different fish species including aquaculture species has been observed in India during the last decade. DBT, Govt. of India New Delhi, India has been instrumental in funding various projects in the development and characterization of fish cell lines in India which resulted in a rapid increase in the number of fish cell lines. This raises the need of establishing fish cell line repositories at a national level for the conservation of fish cell lines in secured places. The authors (M Goswami and W S Lakra) have developed a state-of-the-art facility for the development and storage of cell lines at ICAR-National Bureau of Fish Genetic Resources (NBFGR), Lucknow, India. Recognizing the expertise and research contributions of the authors and other colleagues in the country in the fish cell culture area, DBT funded a megaproject to establish a National Repository of Fish Cell lines (NRFC) at NBFGR, Lucknow. This National Repository of Fish Cell Lines (NRFC) has been in operation at NBFGR, Lucknow, since 2010 which is serving as a National Referral Centre of fish cell lines for research use in the country and abroad. More than 50 fish cell lines from 24 different fish species are being maintained and cryopreserved in the NRFC (Table 3 ). The facility provides services for deposition, characterization, cryopreservation, and distribution of fish cell lines to the scientific community in India. Many cell lines have been supplied to domestic researchers for their research experiments. This cell line repository would play a critical in contributing to the global biobank as many international scientific communities have expressed interest in sharing fish cell lines from India for collaborative in vitro research.

The growing interests in conducting in vitro research using fish cell lines have necessitated intensification of efforts to maintain constant quality and authenticate cell lines using standard protocol throughout their in vitro life. Fish cell culture has been increasingly used in modern biological research. However, fish cell culture research confronts many challenges like misidentification and contamination. As aquaculture continues to grow worldwide, the application of the fish cell lines in addressing fish disease, genetics, and biotechnological interventions will also increase many folds. Although the total number of fish cell lines has been increasing development and characterization of cell lines from crustaceans and other important marine and aquaculture species are still elusive. Preliminary efforts have been made for the development of stem cell cultures from fish but this area needs more focus to explore their use in modern aquaculture and biotechnology. The potentials of fish cell lines in developing vaccines for aquaculture and other derivable products from cells have yet not been explored fully. Hence, scaling up the fish cell culture systems is essential to grab the opportunities of using fish cell cultures in cell-based aquaculture .

There is a need for global networking and collaborations towards applications of fish cell lines for carrying out advanced in vitro research in fisheries and aqua, blue economy, human health, and environmental management. With technological interventions, fish cell lines could be explored to produce several new products. The information provided by the authors in this paper will add new knowledge to the global database of the fish cell lines besides their potential application in the advancement of aquaculture biotechnology and fisheries science research.

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Acknowledgements

Director, ICAR-National Bureau of Fish Genetic Resources, Lucknow; Director, ICAR-Central Institute of Fisheries Education, Mumbai are thankfully acknowledged for providing the facilities. The authors gratefully acknowledge the Department of Biotechnology, Govt. of India, New Delhi for financial support.

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M. Goswami & B. S. Yashwanth

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All the authors contributed to the preparation of this manuscript. MG, and YBS were responsible for the literature search and the first draft of this article. VT and WSL were responsible for language polishing and further editing the manuscript. All authors read and approved the final manuscript. Dr. MG Covered a major aspects of fish cell line for in vitro research like, the current status of fish cell line, novel characterization methods for fish cell line, biobanking, role of cell line in fish health management including the pathological and immunological studies, gene editing of fish cell line and the major contributor of the establishment of NRCF, India. YBS Contributed in the studies on vaccine and other products developed from fish cell culture and the studies on cell-based aquaculture. VT Contributed in the studies of fish cell line in transgenic studies and reproductive biology. WSL Contributed in the studies of fish cell line in toxicological research and environmental monitoring.

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Goswami, M., Yashwanth, B.S., Trudeau, V. et al. Role and relevance of fish cell lines in advanced in vitro research. Mol Biol Rep 49 , 2393–2411 (2022). https://doi.org/10.1007/s11033-021-06997-4

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Received : 27 July 2021

Accepted : 19 November 2021

Published : 11 January 2022

Issue Date : March 2022

DOI : https://doi.org/10.1007/s11033-021-06997-4

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Rabbitfish (Siganus guttatus) culture in floating net cage with different stocking densities

Rachman Syah 1 , Makmur 1 , B R Tampangallo 1 , M C Undu 2 , A I J Asaad 1 and Asda Laining 1

Published under licence by IOP Publishing Ltd IOP Conference Series: Earth and Environmental Science , Volume 564 , The 3rd International Symposium Marine and Fisheries (ISMF) 2020 5 – 6 June 2020, South Sulawesi, Indonesia Citation Rachman Syah et al 2020 IOP Conf. Ser.: Earth Environ. Sci. 564 012022 DOI 10.1088/1755-1315/564/1/012022

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1 Research Institute for Coastal Aquaculture and Fisheries Extention, Jalan Makmur Dg. Sitakka No.129, Maros-90512, Indonesia

2 Polytechnic of Marine and Fisheries of Jembrana, Village of Pengambengan, District of Negara, Regency of Jembrana, Bali-82218, Indonesia

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Rabbitfish ( Siganus guttatus ) is a schooling species which potential for being farmed in high density, however the information about stocking density of this species remain rare. This study was aimed to evaluate growth, survival rate, FCR, stress level and osmoregulation of Rabbitfish under different stocking densities. The tested fishes were the second generation (G2) of Rabbitfish that produced by hatchery outdoor of Research Institute of Coastal Aquaculture and Fisheries Extension, Maros, Indonesia. There were two stages of this study: the first stage was fingerling production. The seeds of Rabbitfish with average of length 6.2±0.8 cm and body weight 4.7±1.9 g/ind were reared in total of 12 units of 1 x 1 x 1 m 3 floating net cage for 90 days. The stocking densities were 50, 100, 150 and 200 ind/m 3 . The second stage of this study was fish growing, where the tested fishes were cultivated with stocking density of 100, 150 and 200 ind/m 3 in the same size of net as the first stage. The result of first stage showed that stocking density did not significantly affect growth rate of Rabbitfish fingerling. However, survival rate at stocking density of 50 (99.33±1.15%) and 100 ind/m 3 (98,33±0,58%) were higher compared to stocking densities 150 and 200 ind/m 3 (94.89±1.39 and 93.50±2.65%, respectively). The result of second stage showed that stocking density of 100 ind/m 3 resulted significant growth, survival rate and FCR compared to that observed on 150 and 200 ind/m 3 . The fish stocked with density of 100 ind/m 3 had length (18.6±0.3 cm), weigh (121.8±9.8 g/ind.), survival rate (92.0±2.6%) and FCR (4,41±0,43). Whereas, the result for fish stocked with 150 ind/m 3 were 17.6±0.3 cm, 107.1±7.0 gram, 86.2±7.1 % and 5.15±0.59, respectively, and 200 ind/m 3 were 16.9±0.2 cm, 96.5±5.9 gram, 82.2±2.3 % and 5,64±0,6, respectively. High stocking density might trigger stress on Rabbitfish and lower blood osmolality found on stocking density of 150 ind/m 3 (490.00±59.77 mOsm/kg) and 200 ind/m 3 (469.00±23.30 mOsm/kg) compared to that observed on density of 100 ind/m 3 (501.67±23.50 mosm/kg). During hypo-osmotic condition, the osmoregulation was not regulated by stocking density as indicated by blood osmotic performance levels <1, which was 0.37 mOsm/kg (100 ind/m 3 ) and 0.33 mOsm/kg (for 150 and 200 ind/m 3 ).

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