Syed Marjuk

Biomass Conversion and Biorefinery https://doi.org/10.1007/s--w ORIGINAL ARTICLE A novel technology towards the high‑density and continuous production of the marine copepod, Pseudodiaptomus annandalei (Sewell, 1919) Perumal Santhanam1 · Mohammed Syed Marjuk1 · Shanmugam Gunabal1 · Palani Sridhar1 · Piliyan Raju1 · Selvaraj Ananth1,2 · Ravichandran Nandakumar1 · Moorthy Kaviyarasan1 · Ayyanar Shenbaga Devi1 · Selvakumaran Jeyanthi1,3 · Meril Divya1,4 · Nagarajan Krishnaveni1 · Ayyasamy Gowthami1 · Pachiappan Perumal1 Received: 3 February 2023 / Revised: 10 May 2023 / Accepted: 13 May 2023 © The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature 2023 Abstract The copepods are nutritionally superior and important live-feed organisms in aquaculture. However, attaining the high density and continuous production of copepods to fulfil the requirements of fish larval production is still a challenge, as compared to other live feeds such as artemia and rotifer. The lack of effective technology in the live feed industry failed to produce a mass density of copepods. Pseudodiaptomus annandalei is a common tropical calanoid copepod species. Hence, the present study attempted the high-density and continuous production of P. annandalei through various optimization technologies, namely, selective and induced breeding by environmental induction, hormone induction, and microbial induction. The copepod P. annandalei was tested with various selective breeding methods, i.e. (a) 18°C cold selective breeding (18°C CSB), (b) 26°C normal selective breeding (26°C NSB), and (c) non-selective breeding (control). This study reveals, compared to 18°C CSB and control copepods, the 26°C NSB copepod P. annandalei produced a significant population of 17181.6 ± 371.2 ind/l using RASC. Across the five generations, the mean nauplii production genetic gain (∆G) for G5 was 29.6% with calculated heritability ­(h2) of 0.30. For the continuous production of copepod, P. annandalei was optimized with suitable environmental parameters. In hormone induction, 20 μg/L bisphenol A and 40 μg/L 17β-estradiol produced high NPR, but the survival rate (SR) decreased while using these hormones. In the meantime, probiotic induction gave a positive result in terms of NPR. The maximum NPR in P. annandalei was induced by Bacillus subtilis at a concentration of ­106 CFU/mL, and probiotic induction also improved the copepod SR. Our findings provide the first concrete proof that P. annandalei would be a candidate species with favourable outcomes for selective breeding—improving its reproductive capacity. Keywords Copepod · High density · P. annandalei · Selective breeding · Hormones · Probiotics Perumal Santhanam and Mohammed Syed Marjuk contributed equally to this work. Highlights 1. The P. annandalei copepod at 26°C NSB yield was significant through RASC, with a total population of 17181.6 ± 371.2 ind/l in 2 months. Whereas, 18°C CSB, and the control yielded a population of 10237.6 ± 225.8 and 12030.6 ± 364.4 ind/l. 2. The genetic gain (∆G) for G5 increased by 31.9% and 29.6% at 18°C and 26°C, respectively. But the mean nauplii production of the select line in G5 at 18°C was 53.6 ± 2.1 nauplii/female only, whereas the 26°C produced 78.6 ± 1.8 nauplii/female. 3. In hormone induction, high NPR was achieved in bisphenol A at a concentration of 20 μg/L (83 ± 2.8 nauplii/female) and 17β-estradiol at 40 μg/L (75.3 ± 1.4 nauplii/female). But long-term hormone effect needs to be studied because it severely affects the survival rate of the copepod. 4. The current work demonstrates B. subtilis probiotic induction can improve copepod productivity and which provides excellent results at a concentration of ­106 CFU/mL (74.3 ± 1.8 nauplii/female) and also increased the survival rate of copepod when compared to control I. galbana. 1 Introduction In recent years, there has been an increase in global protein demand [1, 2]. The average edible fish required per person has increased from 9.0 kg to 20.5 kg in 2018. Between 1986 and 1995, aquaculture production increased to 82.1 million tonnes, up from 14.9 million tonnes [3]. Aquaculture is considered to be a most excellent option to meet the growing demand for fish protein [4–6]. But the major bottleneck in marine fish larviculture is mass larval mortality owing to insufficient nutrition during the first feeding stage [7]. Copepods act as a promising crustacean as live feed; they have small naupliar stages, showing a high concentration of essential fatty acids and nutrients, and they possess a unique swimming motion that encourages the fish fry to eat [8–10]. Extended author information available on the last page of the article 13 Vol.:-) Biomass Conversion and Biorefinery While calanoid copepods are considered to be an effective live feed for marine fish larviculture [8, 11, 12], the calanoid copepod, Pseudodiaptomus annandalei, is an excellent model organism that is used as a live feed for aquatic animals in the aquafeed business, and it is one of the most frequent copepods seen in the wild [13–16]. P. annandalei is numerous and can adjust to changes in salinity, temperature, nutritional concentration, oxygen levels, and suspended particles and, thus, a potential one for outdoor culture [14, 17–19]. To date, highdensity copepod culture has been difficult when compared to other live feeds such as artemia and rotifers [20–23]. The continuous production of copepods is also complicated, and maintaining stock culture is also a significant problem here [24, 25]. Given the above constraint, we need to develop new techniques and methods for the high-density and continuous production of copepods. Therefore, presently the following methods have been tried for the tropical calanoid copepod P. annandalei in high-density and continuous production by selective breeding [21, 26] and induced breeding through hormone induction [27], microbial induction [28], and environmental induction by changing and optimizing the different parameters, viz., salinity, temperature, pH, light intensity, photo-period or light regime, and dissolved oxygen. 2 Material and methods 2.1 Microalgal culture In this study, the experimental copepod P. annandalei was fed with the microalga Isochrysis galbana. The microalga strain, I. galbana was collected, isolated, and maintained at the Marine Planktonology and Aquaculture Laboratory (MPAL), Department of Marine Science, Bharathidasan University, Tiruchirappalli, Tamil Nadu, India. Algal stock and mass cultures were kept in 1-L conical flasks and 20-L carboys indoors at a temperature of 23 ± 2°C with a cycle of 12:12 light: dark hours. Fluorescent tubes were used as a light source, with a light intensity of 2000 lux. For algal cultivation, filtered and sterilized seawater 25 PSU enriched with Conway’s medium was utilized (Walne, 1970). For mass cultivation, I. galbana was cultivated in a 1000-L FRP tank. 2.2 Copepod culture The copepod species, P. annandalei (KY982586), was cultured and maintained at the MPAL laboratory; The strain was initially collected using 158-μm mesh size plankton nets (0.35-m diameter opening) from Nagore (Lat. 10°49′37.804″ N; Long. 79°51′49.060″ E), Southeast Coast of India, and has been cultured, isolated, identified and the 13 stock was maintained in our laboratory without interruption since 2018. The copepods were transferred to the laboratory and given gentle aeration with aerators. The copepod samples were then coarsely filtered to exclude the nauplii and other larval forms using the superimposed sieves. The males and females of P. annandalei were separated from the diluted samples and the mother stock cultured in 1-L glass beakers with 700 mL of saline water. The copepods were then mass cultured in 100-L fibre-reinforced plastic (FRP) tanks filled with saltwater filtered through a 1-μm filter bag to remove the additional impurities from the water. The salinity and temperature of seawater were adjusted to 26 PSU and 26 ± 2°C, respectively 8 pH, 1000 lux light intensity, 12:12 light-and-dark photoperiod, and 4 mg/L dissolved oxygen. The I. galbana microalga was fed to P. annandalei every 2 days at 3×104 cells/ml concentration. The faecal pellets and detritus were siphoned out once in 4 days, and total water was exchanged at 15-day intervals. 2.3 Research design The research was conducted to develop high density and continuous production of marine copepod P. annandalei using various methods like (a) selective breeding normal 26°C and cold selective breeding 18°C, (b) environmental induction, (c) hormone induction, and (d) microbial induction. Each experiment consisted of triplicates (n=3). All the experiments were conducted under indoor conditions and maintained the same parameters as for stock culture (except different experiments were under different parameters depending on the study). For each experiment with copepod, we took care to acclimate all copepods to the experimental conditions for at least 24 h prior to conducting any experiments to minimize any potential effects of acclimation on our results. Copepods and developmental stages were enumerated by an inverted microscope (Micros Austria SUNDEW MCX1600, India, with Tuscan Discovery CH30 3.0 MP Camera). The experiment was performed in the Marine Planktonology and Aquaculture Laboratory (MPAL), Department of Marine Science, Bharathidasan University, located in Tiruchirappalli, Tamil Nadu, India. 2.3.1 Recirculation aquaculture system for copepods (RASC) The working principle of Recirculation Aquaculture System for Copepods (RASC) to get high-density production of copepods is shown in Fig. 1. In brief, freshwater or saltwater (according to need) was pumped to 5000-L and 3000-L capacity storage tanks (ST). The raw water was pumped from the ST using the pump (P) and filtered through a series of filters such as sand filter (S), biological filter (B), Biomass Conversion and Biorefinery membrane filter (M), and UV filter (UV), and filtered water and was stored in reservoir tank (RT). From the RT, the filtered water was supplied to all 6 copepod culture tanks (CC) and microalga culture tanks (MC). A known volume (5 L) of needful species of microalga was obtained from indoor stock culture facility and was inoculated in 1000-L capacity FRP tanks filled with filtered water and fertilized with commercial fertilizers such as ammonium sulphate, urea, and super phosphate in the ratio of 10:1:1 gm/L of water. The microalgal culture was developed prior to copepod inoculation. A known numbers of copepods (>1000 ind/l); both including male and female or egg carrying female (based on experiment) was inoculated in CC tanks. For the selective breeding, selection of male and female was done according to Alajmi & Zeng [21]. The aeration for both copepods and microalgal tanks was supplied through the PVC pipeline and silicon tube attached with the air stones by using industrial air compressor (AC). The cultured microalga was supplied as feed for copepods using an algal dosing pump (ADP). The water quality parameters such as temperature, salinity, pH, and dissolved oxygen in the culture tanks were maintained using a probe connected with automatic controller (ATC). The available wastewater in the CC tanks was drained through the wastewater pipeline and collected at recirculation tank (RCT). From the RCT, the wastewater was pumped by using pump (P) and passed through the sand filter (S), biological filter (B), and degasser cum skimmer (D) where the vigorous aeration was supplied by using the blower (BL) to remove the foam and ammonia. Finally, the purified water from degasser cum skimmer (D) was recirculated to all the culture tanks. The different experiment copepods, such as 26°C non-selective breeding (control), 18°C cold selective breeding (18°C CSB), and 26°C normal selective breeding (26°C NSB) adults, were maintained in the separate tanks such as CC1, CC2, and CC3, respectively. The cultured copepods were harvested by draining the water through one end of the outlet fitted with mechanical filters made up of plankton sieve holders. The wastewater seeped from the culture tanks during harvest was drained through another end of the outlet after one crop is over, and the wastewater was discharged through the drainage canal (Fig. 1). Overall, the RASC was designed to provide a stable and controlled environment for the copepods, allowing precise manipulation of environmental variables such as temperature, salinity, pH, photoperiod, photo regimes, and water quality. While RASC for copepod culture is uncommon, it has several advantages over other culture methods, including the ability to maintain consistent water quality and temperature and to reduce water usage and waste discharge. Fig. 1  The schematic diagram of Recirculation Aquaculture System for Copepods (RASC) for high-density copepod culture 13 Biomass Conversion and Biorefinery 2.3.2 Selective breeding A consistent selective breeding approach, slightly modified from Alajmi and Zeng [29] was adopted, for high density RASC culture system, for raising the copepod P. annandalei at the different temperatures of 26°C [29] and 18°C [26]. In this study, the selection was based on the rate of nauplii production of P. annandalei (Fig. 2). For selective breeding, 50 active pairs of adult P. annandalei were randomly selected from the laboratory stock cultures to produce the base population (G0). From the base population, 150 pre-matured female P. annandalei at copepodite stage V (CV) were individually isolated in Petri dishes and observed for sexual maturation. The animals from 16th day of the culture or 2 days after reaching the CV stage were considered as sexually mature and further confirmed by the moulting of the animal according to the standard protocol [30]. An adult male was partnered with mature adult female. Each pair was maintained in a 50-ml glass beaker submerged in filtered saltwater at a temperature of 26°C normal selective breeding (26°C NSB) in one group and another 18°C cold selective breeding (18°C CSB), 25 ± 2 PSU salinity, 8–8.5 pH, 4 mg/l dissolved oxygen, 1000 lux light intensity, and 12:12 h of light: dark photoperiod, and the copepods were fed with microalga I. galbana, 2 days once 3×10 4 cells/ml concentration. A 90-μm mesh was inserted at the bottom so that only the nauplii were permitted through the bottom mesh. A Bogorov counting chamber and a stereomicroscope were used to count the nauplii that accumulated at the bottom of the beakers. Throughout the experiment, copepods were given I. galbana. The family selection was used to develop a copepod strain with a higher nauplii production. A pilot-scale experiment was conducted to determine the nauplii production pattern of P. annandalei. The study identified the highest nauplii output period based on the high nauplii production rate (NPR) over the lifespan of the copepod. As per the study findings, the days between day 3 and day 12 were determined as the optimal days for taking nauplii counts. Previous studies have also reported higher nauplii output during the early stages of copepod culture [31]. However, it is important to keep in mind that the optimal days for nauplii counting may vary depending on the specific experimental conditions and research objectives. Therefore, it is always recommended to conduct pilot studies to determine the optimal 13 days for nauplii counting under the specific experimental conditions of each study. To track total nauplii production, nauplii generated by each G0 female were collected on a 25-μm mesh and recorded every day until the nauplii production stopped. These nauplii were cultivated until they matured. The nauplii were taken from each G0 pair to guarantee that a sufficient number of offspring were collected to start the next generation. The G1 was developed by selecting the progeny of the top 30% of G0 females with the highest nauplii production on the selected days as parents and an equivalent number of male individuals from these top 30% of females. The control copepod was formed by randomly pairing male and female individuals from G0’s offspring. By mating newly matured adults, fifty pairs were produced for each control and experiment. To eliminate inbreeding, according to Nomura and Yonezawa study [32], a circular mating design was used, which allows males from each group to be transferred to the neighbouring group in a rotational fashion for mating. The same selection criteria were used in all five generations of selection. A control line was kept alongside each succeeding generation with random mating without bias for a particular trait. After the final G5, the top 30% was inoculated into a 1-L beaker for further culture. After obtaining an adequate number (copepods >1000 ind/L) of adult copepods, their progenies were transported into a 100-L FRP tank for mass production of P. annandalei. Then the cultures were transferred to a 2000-L FRP tank controlled by RASC (Recirculation Aquaculture System for Copepod). After reaching the adequate density of copepod P. annandalei (days 20 to 28 from inoculation to the RASC), the total population consisting of nauplii, copepodite, and adults were counted by the Bogorov counting chamber under a stereomicroscope by random sampling of 1 L from each 2000-L RASC tank. The salinity, pH, oxygen, photoperiod, and light intensity were kept at the same optimal levels as that of the stock culture. Calculation of response to selection (R) genetic gain (∆G) and realized heritability (­ h 2 ) The difference in mean phenotype (nauplii production) between the progeny generation and the previous generation determined the response (R). The selection differential (S) was determined using the mean phenotypic difference between the chosen parents and the overall parent population. The slope of the generation representing the regression Biomass Conversion and Biorefinery Fig. 2  Illustrative diagram explaining the selective breeding of copepod P. annandalei by 26°C NSB method and 18°C CSB line plotted against the cumulative selection differential was used to estimate the realized heritability ­( h 2 ) [33]. Genetic gain (∆G) achieved throughout every two successive generations of selection were calculated according to the following equation: 13 Biomass Conversion and Biorefinery ΔGn = (Sn − Cn) where S is the select line’s mean phenotypic value, C is the control line’s mean phenotypic value, and n is the generation number. 2.3.3 Environmental induction and optimization Responses of the P. annandalei to various environmental factors such as temperature (15, 18, 21, 23, 26, 29, 32, and 35°C), salinity (15, 20, 25, 30, 35, and 40 PSU), pH (6.5, 7, 7.5, 8, 8.5, and 9 pH), light intensity (500, 1000, 1500, 2000, 2500, and 3000 lux), photoperiod (12:12, 14:10, 10:14, 08:16, and 16:08 light-and-dark hours), and dissolved oxygen (1, 2, 3, 4, 5 mg/L) were observed (in order to increase NPR and to optimize culture parameters. For each experiment, a pair of P. annandalei male and female CV stages were isolated from a stock culture with the help of a stereomicroscope and transferred into a 50-ml glass beaker. The nauplii were counted on selected days (days 3 to 12). Conditions similar to stock maintenance were employed except for parameter-specific experiments. 2.3.4 Hormonal induction The induced breeding method was followed by adopting the method of [27], with slight alterations. In brief, the estrogenic chemicals such as bisphenol A (CAS 80-05-7; >97) and 17β-estradiol (CAS 50-28-2; ≥98) were purchased from Sigma-Aldrich. Stock solutions were prepared using dimethyl sulfoxide (DMSO; CAS 67-68-5; ≥99.9%) (SigmaAldrich) as carrier solvent. The solutions were stirred and stored at 4°C in the dark until used. A ten-fold dilution series in 100% DMSO was made from each stock solution. Ten microliters of the dilution series was mixed with 100 mL seawater to make a nominal concentration. In the solvent control, bisphenol A, and 17β-estradiol treatments, the final concentration of DMSO was 0.0001% (v/v). Two pairs of CV stage P. annandalei were inoculated into the beaker filled with filtered seawater. The induced nauplii production experiment was started with the test chemicals bisphenol A and 17β-estradiol with concentrations much less than acute toxic test values, viz., 10, 20, 30, 40, and 50 μg/L. Prior studies [27, 34–36] on various copepod species, have reported higher effective concentration (EC) and lethal concentration (LC) values (>0.5 mg/L) for bisphenol-A and 17β-estradiol. So, in comparison, this study used a very less concentration of hormone to avoid toxicity to the animal. The number of nauplii released by the copepod P. annandalei was counted 13 using the Bogorov counting chamber under the stereo microscope on selected days (days 3 to 12). 2.3.5 Microbial induction In brief, the probiotic bacterium Bacillus subtilis was cultured in nutrient broth (HiMedia, Mumbai, India) and incubated at 37°C overnight. The next day, the culture was centrifuged at 5000g for 10 min at 4°C. The supernatants were removed, and the cell pellets were washed three times before being resuspended in a sterile saline solution (0.9 % NaCl). The spread plate method was used to determine the concentration of bacterium by colony-forming unit (CFU/ mL). The bacterium was quantified and stored in suspension form at 4°C for use in feed preparation. The prepared probiotic bacteria cells were inoculated in a 50-ml beaker containing two pairs of P. annandalei Copepodite V (CV) and were maintained in the same environmental condition as stock. One treatment contained I. galbana alga at a concentration of 30,000 cells/mL; the actual concentration added was adjusted daily by determining cell density with the particle counter (control). The probiotic bacterium B. subtilis was adjusted to the concentration of 1­ 05, ­106, ­107, and 1­ 08 CFU/mL for (treatment). The copepod nauplii were counted for NPR on selected days (days 3 to 12). 2.3.6 Survival rate (SR) The survival rate of copepod P. annandalei was investigated for hormone induction and microbial induction. Experiments i, were done in triplicates using 10 active adults of P. annandalei for each experiment. For hormone induction, the copepods were treated with bisphenol A and 17β-estradiol with which concentration produces high NPR, vis., bisphenol-A 20 μg/L, and 17β-estradiol was 40 μg/L. For microbial induction, the probiotic bacterium B. subtilis was taken at ­106 CFU/mL, and I. galabana ­104 cells/ml was taken and induced to the copepod. The Kaplan-Meier survival curve method was used for analysis SR [37]. 2.4 Statistical analysis SPSS statistic software version 26 was used for statistical studies. To assess the significant differences between treatments and controls, the data were analysed using ANOVA followed by the Tukey post hoc test. For the survival study, the Kaplan–Meier survival analysis (Log-rank and Gehan-Breslow-Wilcoxon tests) method was used to compare and estimate adult copepod’s survival duration (days). All graphs were plotted with the help of GraphPad prism version 8. Biomass Conversion and Biorefinery 3 Results 3.1 Selective breeding During selective breeding, the copepod P. annandalei was cultured two different methods viz, non-selective breeding (control) and selective breeding. The selective breeding was done in two different temperatures viz., 26°C and 18°C. The copepods were eventually mass cultured using the Recirculation Aquaculture System for Copepods (RASC) to validate the increase in population. As a result (Table 1), copepods reared through 26°C NSB produced a high-density population of 17181.6 ± 371.2 ind./l, consisting of 10430 ± 351.5 nauplii, 3985.3 ± 77.4 copepodites, and 2766.3 ± 103.4 adults, followed by non-selective breeding (control) with a total population of 12030.6 ± 364.4 ind./l, consisting of 7260.3 ± 189.2 nauplii, 3014 ± 136.5 copepodites, and 1756.3 ± 68.4 adults, and at 18°C CSB, the yield was low when compared to the control and at 26°C NSB a total population of 10237.6 ± 225.8 ind/l, comprising 6638 ± 201.3 nauplii, 2414 ± 259.4 copepodites, and 1185.6 ± 155.5 adults. A total population between the non-selective breeding (control), 26°C NSB, and 18°C CSB are extremely significant (P < 0.001). Also, 26°C NSB was extremely significant when compared to 18°C CSB (P < 0.001). But there was no statistically significant difference in nauplii, copepodite, and adult production between control and 18°C CSB (P > 0.05). 3.1.1 Genetic gain The results show (Table 2) that at 26°C, the mean nauplii production before selection (G0) was 59.6 ± 2 nauplii/ female (days 3 to 12 pooled). The 26°C select line copepod nauplii output has been significantly enhanced as a result of selective breeding. In 26°C NSB G5 P. annandalei, nauplii production was increased by 78.6 ±1.8 nauplii/female which was a 29.6% genetic gain as compared to the control. In contrast, the initial nauplii production (G0) of 18°C CSB was 31.6 ± 2 nauplii/female (days 3–12 pooled), which was then increased to 53.6 ± 2.1 nauplii/female, by representing Table 1  The total population, nauplii, copepodites, and adults (ind/L) of P. annandalei through non-selective breeding (control 26°C), selective breeding 26°C NSB and 18°C CSB a 31.9% genetic gain over the 18°C control. At 26°C NSB, nauplii production was significantly increased (P<0.001) when compared to the 18°C CSB and both controls. Overall, after five generations of selection at 26°C and 18°C, the percentage of pooled nauplii production for days 3 and 12 was increased from 16.1% (G1) to 29.6% (G5) in 26°C NSB and 18.4% (G1) to 31.9% (G5) in 18°C CSB. Based on the observed selection responses, the heritability (h2) for nauplii production in P. annandalei at 18°C was 0.64 ± 0.05 and at 26°C was 0.30 ± 0.05 (Fig. 3). 3.2 Environmental induction Environmental induction was used to gauge the NPR response of copepod P. annandalei. This will help to release the nauplii immediately to the water column. The highest rate of NPR was achieved at 26°C yielded 67 ± 4.5 nauplii/female, salinity at 20 PSU gave 69 ± 3 nauplii/female, oxygen shock at 4 mg/L produced 70 ± 7.5 nauplii/female, pH shock at 8 pH yielded 68.6 ± 3.5 nauplii/female, light intensity at 1000 lux produced 68 ± 3 nauplii/female, and photoperiod at 10:14 dark: light hours produced 71.3 ± 2 nauplii/female. All these parameters were taken into consideration for the optimization of P. annandalei. So, the parameters optimized were; 26°C temperature, 20 PSU salinity, 4 mg/L oxygen, 8 pH, 1000 lux light intensity, and 10:14 light: dark hours photoperiod, for better survival and high NPR. Lower NPR of P. annandalei was recorded at temperature 15°C cold and 35°C hot, 15 PSU salinity, 1 mg/L oxygen, 6.5 pH, 3000 lux light intensity, and 16:08 light: dark hours photoperiod (Fig. 4). 3.3 Hormone induction The copepod P. annandalei was tested with the hormones, bisphenol A and 17β-estradiol for continuous production of copepod. Bisphenol-A significantly (P<0.001; Fig. 5) increased NPR of the copepod P. annandalei, with the maximum NPR 83 ± 2.8 nauplii/ Stages Control (non-selective breeding) 26°C 18°C CSB 26°C NSB Nauplii (ind/l) Copepodite (ind/l) Adult (ind/l) Total population (ind/l) 7260.3 ± 189.2b 3014 ± 136.5b 1756.3 ± 68.4b 12030.6 ± 364.4b 6638 ± 201.3b(ns) 2414 ± 259.4b(ns) 1185.6 ± 155.5b(ns) 10237.6 ± 225.8b(***) 10430 ± 351.5a(***) 3985.3 ± 77.4a(*) 2766.3 ± 103.4a(*) 17181.6 ± 371.2a(***) Values are expressed as mean ± SE (n=3). Different letters in the same row are indicated significant differences within copepod stages. Different superscripts in brackets are expressed 18°C and 26°C selective breeding; population are compared with control (non-significant (ns), P<0.033(*), P<0.001(***)) analysed by two-way ANOVA (F (6.24) =33.10, P<0.001) 13 Biomass Conversion and Biorefinery Table 2  Mean nauplii production (days 3 to 12 pooled); nauplii/female, cumulative genetic gain, and response to selection (%) of selected line across five generations of P. annandalei females Temperature Generation Selected population (S) Selected parents 26°C G0 G1 G2 G3 G4 G5 G0 G1 G2 G3 G4 G5 59.6 ± 2 62.3 ± 0.8a 62 ± 1.1a 66.6 ± 1.8a 70.6 ± 2.8a 78.6 ± 1.8a 31.6 ± 2 36.3 ± 1.4a 38.3 ± 1.4a 44.6 ± 0.6a 49.3 ± 2.4a 53.6 ± 2.1a 61.6 ± 1.2 65.6 ± 3.1 72.6 ± 2 75.3 ± 5.7 81 ± 1.5 90.3 ± 2.7 38.3 ± 5 40.6 ± 1.4 45.3 ± 1.7 51.3 ± 1.4 58 ± 1.5 61.3 ± 1.3 18°C Control line (C) b R (Sn-Cn) ∆G (%) 53.6 ± 1.2 54 ± 2.5b 57.6 ± 2.9b 56 ± 1.1b 60.6 ± 1.2b - - 30.6 ± 1.4 b 32 ± 1.7 b 36.3 ± 0.8 b 38.6 ± 1.7 b 40.6 ± 1.4 b - - *Selection differential- Values are expressed as mean ± SE. Values in the same row with different superscripts are significantly different (P < 0.001) *Selection differential was calculated by weighing each parent by the number of its offspring female attained at a hormone concentration of 20 μg/L bisphenol-A. The minimal nauplii production was 37.6 ± 2 nauplii/female at a dosage of 50 μg/L, and 65.3 ± 3.1 and 50 ± 1.7 nauplii/female NPR were achieved at 10 and 40 μg/L of bisphenol A, respectively. Meanwhile, NPR was found to be higher at 75.3 ± 1.4 nauplii/female in the 40 μg/L dose and 65.6 ± 1.4, 60.3 ± 2.7 nauplii/female in the 30 μg/L, and 50 μg/L doses of 17β-estradiol, respectively. In 10 and 20 μg/L 17β-estradiol was not significantly produced NPR 51 ± 1.5, 56 ±1.5 nauplii/female. Overall bisphenol A 20 μg/L and 17β-estradiol 40 μg/L concentration induced better nauplii production of copepod P. annandalei, for continuous production. The SR of P. annandalei (Fig. 6), in hormones bisphenol A (20 μg/L) and 17β-estradiol (40 μg/L) was investigated. The survival percentage of the control was high compared to the hormones (test), throughout the experiment. Both, control and treatment had a 100% survival till day 3. From day 4, a steady decrease in SR was observed in both the hormone treatments. The control SR was stable from day 6 to day 12(82%) but, dropped to 55% on days 13 to 15. Despite the drop, SR of control was always higher than the hormone treatments. The treatment of bisphenol A dropped the survival rate rapidly, day 5 to 6 83.3%, day 7 to 8 74.07%, day 9 to 11 63.49%, day 12 and 13 47.61%, and finally, day 14 to day 15 have 21.8%. In 17β-estradiol from day 6 to 7 has 82.5%, on day 8 to 9 72.18%, on day 10 60.15%, on 11 to 12 48.12%, and on the last day 13 to 15 survival percentage declined to 32.08%. 13 3.4 Microbial induction The microbial induction was done by the using probiotic bacterium B. subtilis as sole diet to the copepod P. annandalei. The microbial induction had significant (P<0.001; Fig. 7) effect on nauplii production of the copepod. The highest NPR was attained at 1­ 06 CFU/mL NPR 74.3 ± 1.8 nauplii/female. The remaining B. subtilis concentrations of ­105, ­107, and ­104 CFU/mL produced 68.6 ±1.4, 65 ± 2, and 57.6 ±1.8 nauplii/female. ­103 CFU/mL showed least NPR (47 ± 2 nauplii/female) compared to other concentrations. For microalgal experiments, I. galbana ­104 cells/ml seemed to be optimal that produced NPR of 65.6 ± 1.7 nauplii/female. Compared to the B. subtilis, the I. galabana concentrations such as 1­ 03, ­105, ­106, and 1­ 07 have exerted low NPR yield of 58.3 ± 2.6, 53 ± 2, 33 ± 5.7, and 17 ± 3.7 nauplii/female, respectively. SR of P. annandalei was experimented with sole diets of, I. galbana ­(104 cells/ml) and probiotic bacteria B. subtilis ­(106 CFU/mL). The results (Fig. 8) of the probiotic-treated copepod P. annandalei had a high SR of 77.14% compared to the I. galbana 60.15%. From day 1 to day 3, no mortality was recorded in both treatments. I. galbana fed copepods showed slow decrease in SR to 91.66% on 4th day, followed by 72.18% around day 9, and after day 10 no mortality was recorded. In probiotic-treated copepod, P. annandalei SR no mortality was observed till day 6. On day 9, the SR decreased to 77.14%, no mortality was recorded thereafter. In comparison to the control, this data demonstrates that the probiotic bacterium B. subtilis has been better diet in terms of survival of copepod P. annandalei. Biomass Conversion and Biorefinery Fig. 3  Regression between the 18°C CSB and 26°C NSB selected mean nauplii production (days 3 to 12 pooled) across five generations and cumulative selection differential 4 Discussion The copepods, particularly P. annandalei have been proven to be high-value live feed to tropical finfish larvae [19, 31]. Copepods are the superior nutrition exchanger in aquatic ecosystems [38]. If they are cultivated in high density, that would lead to an excellent pathway for the live feed/aquaculture industry. But the main challenge lies in continuous maintenance of the copepod stock culture [39, 40] as opposed to other live feeds like rotifers and artemia. However, as the aquaculture business grows, there will be greater demand for high-density cultures of small prey items that are ideal for high-value finfish [11, 41, 42]. The P. annandalei is a promising copepod for mass culture and continuous production through selective and induced breeding technology. The copepod, P. annandalei, is a model organism for these technologies; in the future, as these technologies would support high density and continuous production of other copepods also. The present selective breeding findings are in line with the reports of earlier researchers [29], who have demonstrated that a simple selective breeding method (applied over a short period) could significantly increase the reproductive capacity of Parvocalanus crassirostris. During the present experiment, the temperatures of 26°C NSB and 18°C CSB were found to be conducive for high copepod production through selective breeding. Furthermore, the study by Pan [26] provided evidence that cold selective breeding enhances the productivity of the copepod, Apocyclops royi. In the selective breeding, the top 30% of the cohort was selected to improve the NPR of P. annandalei. This selection criterion was based on the previous study [29], and the selecting a smaller percentage (e.g. 10% or 20%) may not yield enough genetic gain as it would give very minimal number of progeny for next generation. While selecting a larger percentage(>40) may reduce genetic variability and increase the risk of inbreeding depression and this 30% would give much more animals for next-generation development. Maybe, the smaller percentage also gives better result in the future that needs to be studied. This study reveals that the selective breeding of the copepod P. annandalei provides a significant result at 26°C NSB as compared to the 18°C CSB and nonselective breeding (control). Both the 18°C CSB and the 26°C NSB had higher chosen mean nauplii production during five generations. While the genetic gain (∆G) for G5 at 26°C was raised to 29.6%, the ∆G at 18°C was enhanced to 31.9%. But the mean nauplii production of the select line in G5 at 18°C was 53.6 ± 2.1 nauplii/female, and at 26°C, it was 78.6 ± 1.8. So, generation-wise, the 26°C temperature has produced the best result. The performance of 18°C CSB was lower than the 26°C NSB, but this is not surprising given that 26°C is the optimal temperature for P. annandalei. However, it would be interesting to investigate the production rate of cold-selected strain (18°C) back at 26°C. Nonetheless, the study provides valuable insights into the potential of selective breeding to enhance copepod production under different temperature regimes. Across five generations of selective breeding were only studied here, maybe studies covering more than 5 generations of selective breeding (18°C), in future, would provide positive results because the heritability ­(h2) at 26°C was 0.30, but it was 0.64 at 18°C. It was twofold higher than 26°C. The result of 26°C ­h2 for P. annandalei was slightly matched with the earlier findings of Aljami [29] with P. crassirostris. It is important to undertake more research to better understand the impact of employing small volumes of water in selective breeding trials and to see if the findings can be extrapolated on a larger scale. Future study may also concentrate on enhancing copepod mass culture conditions to increase their potential as a live feed supply for larval stages. 13 Biomass Conversion and Biorefinery Fig. 4  Values are expressed as Mean ± SD, n=3. NPR (nauplii/female) by P. annandalei with different environmental shock. The values are significantly different determined by one-way ANOVA; A-temperature (F(7,16)=48.055, P<0.001), B-salinity (F(5,12)=31.498, P<0.001), C-oxygen (F(4,10)=47.842, P<0.001), D-pH (F(5,12)=15.884, P<0.001), E-light intensity (F(5,12)=11.868, P<0.001, F-photo period (F(4,10)=5,728, P=0.012). The different letters above each bar represent the significant variations within the treatment identified by Tukey’s post hoc analysis At 26°C NSB through RASC the yield was massive, with a total population of 17181.6 ± 371.2 ind/l in two months. The same duration 18°C CSB and control yielded a total population of 10237.6 ± 225.8, and 12030.6 ± 364.4 ind/l, respectively. The RASC system, which was shown to be an effective method in an aquaculture system, has been adopted in live feed industries [43], and it is necessary to improve the RASC system-automation. The demographic structure of copepod populations can have a significant impact on population growth and naupliar Fig. 5  Values are expressed as mean ± SE, n=3. Bisphenol A and 17-estradiol were used to induce hormones in the copepod P. annandalei at concentrations of 10, 20, 30, 40, and 50 μg/L. The different letters above each bar represent the significant variations between treatment groups identified by Tukey’s post hoc analysis Fig. 6  Kaplan-Meier survival curve shows the survival rate of P. annandalei by survival percentage on hormone induction by bisphenol A and 17β-estradiol 13 Biomass Conversion and Biorefinery NPR(Nauplii/Female) 100 I. galbana B. subtilis 80 60 A AB a a C BC a D b 40 c 20 0 10 3 10 4 10 5 10 6 10 7 I. galbana (cells/ml) B. subtilis (cfu/ml) Fig. 7  Mean ± SE, n=3. The copepod P. annandalei was subjected to microbial induction by probiotic B. subtilis bacterium at various doses (CFU/mL), with I. galbana serving as the control. The different letters (a, b, c I. galbana; A, B, C B. subtilis) above each bar indicate the significant differences among treatment groups identified by Tukey’s post hoc test production. In our study, we have observed differences in the demographic structure of copepod populations between the two different temperature treatments with selective and non-selective breeding copepods. Specifically, at 26°C in the selective breeding treatment, we observed a significantly higher number of nauplii, copepodites, and adults, resulting in a significantly higher total population size as compared to the other treatments such as 26°C non-selective breeding, and 18°C selective breeding. These results suggest that temperature, and selective breeding may play a role in shaping the demographic structure of copepod populations and that may have implications for optimizing copepod culture protocols. Further research is needed to fully understand the mechanisms underlying these observed differences on demographic structure and their implications for copepod culture. Fig. 8  Kaplan-Meier survival curve shows the SR of P. annandalei survival percentage by probiotic bacteria B. subtilis induction During our environmental induction and optimization, the copepod P. annandalei showed an enhanced NPR. The maximum NPR was attained under the following conditions: 26°C temperature, 20 PSU salinity, 4 mg/L oxygen, 8 pH, 1000 lux light intensity, and a 10:14 light: dark hours photoperiod. The P. annandalei culture was similarly optimized using the aforementioned conditions. The recorded decreased naupliar production at high oxygen level (5 mg/L) could be caused to the copepods by high ­O2, leading to a decrease in their reproductive performance [45] and also high oxygen levels can increase metabolic rates. Anderson and others have stated that estrogens like 17β-estradiol could stimulate female sexual maturation and egg production [27] and they have reported the bisphenol A and 17β-estradiol accelerated the maturation of ovaries and increased egg production of copepod Acartia tonsa. Presently, the hormone induction produced high NPR at 20 μg/L concentration 83 ± 2.8 nauplii/female and 17β-estradiol 40 μg/L concentration 75.3 ± 1.4 nauplii/female. However, the survival rate under bisphenol A and 17β-estradiol was low compared to the control. Bisphenol A has 21.8% and 17β-estradiol 32.08% on the ­15th day. As a result, additional hormone study is required. Earlier more studies were conducted on toxicity of endocrine-disrupting hormones, including bisphenol A and 17β-estradiol, but the toxicity occurs at concentration greater than 0.5 mg/L [34, 36, 46]. However, modest concentrations like 10, 20, 30, 40, and 50 μg/L also have an impact on the copepod P. annandalei survival rate; this may require further research. Even with the possible harmful effects of these chemicals on the environment and copepods, we believe that this section still holds value in the context of aquaculture. Hormonal induction is a widely used technique in aquaculture practices, and our study offers significant information on the possible effects of using endocrine disruptors in such practices. Furthermore, our research also explored the impact of different concentrations of these chemicals and the findings will be useful for developing more effective and sustainable hormonal induction protocols. Although we recognize the concerns about the potential ecological impact of endocrine disruptors, we believe that our study provides crucial insights that can help advance aquaculture practices in a more sustainable manner. However, in the context of ecotoxicology, the adverse effect of endocrine disrupting hormones is high. So, we must find out the alternative methods for inducing reproduction in copepods future. The probiotic strain B. subtilis can enhance feed digestion and assimilation, water bioremediation and reduce disease development in aquaculture [47]. In addition, they have unique properties, like antibacterial activity against aquatic pathogens [48], which would aid copepods in better survival. In microbial induction, the probiotic bacterium B. subtilis at ­106 CFU/mL concentration, significantly increased 13 Biomass Conversion and Biorefinery NPR (74.3 ± 1.8 nauplii/female), and with I. galbana NPR was 65.6 ± 1.7 nauplii/female. Sun and others [49] have suggested that the copepod P. annandalei is an appropriate probiotic vector for marine fish larvae. Earlier researchers have also reported the B. subtilis induced larval production in some copepods including P. annandalei [44]. However, the potential causes of the drop-in survival rate observed on day 4 in I. galbana treatment, could be due to multiple causative factors. One possibility could be related to the ecophysiology of copepods and the development. Previous studies have shown that copepods may require specific types of food or feeding regimes [50, 51]. In our study, we used I. galbana as the sole food source for copepod, which may not have provided all the necessary nutrients for copepod. Another possibility is that some unknown factors or random fluctuations in environmental conditions could have influenced the results. Overall, we believe that further investigation is needed to determine the specific causes of the observed drop-in survival rate and to explore alternative feeding regimes that may be more suitable for P. annandalei. These findings would be valuable for improving the efficiency and sustainability of copepod aquaculture practices. curation, formal analysis, writing—original draft, writing—review and editing. Shanmugam Gunabal: Investigation, formal analysis. Palani Sridhar: Investigation, formal analysis. Piliyan Raju: Formal analysis, writing—review and editing. Selvaraj Ananth: Formal analysis. Ravichandran Nandakumar: Writing—review and editing. Moorthy Kaviyarasan: Formal analysis. Ayyanar Shenbaga Devi: Formal analysis. Selvakumaran Jeyanthi: Formal analysis. Meril Divya: Formal analysis. Nagarajan Krishnaveni: Formal analysis. Ayyasamy Gowthami: Formal analysis. Pachiappan Perumal: Writing—review and editing Funding The Department of Biotechnology (DBT), Govt. of India, New Delhi, is gratefully acknowledged for its financial support to this work through an extramural research project (BT/PR28606/ AAQ/3/921/2018; dated-). Authors (MSM, SG, and PS) thank the DBT for the fellowship provided. Data availability The corresponding author will provide the datasets created during the current study upon reasonable request. Declarations Ethical approval Not applicable. Competing interests The authors declare no competing interests. References 5 Conclusion According to our findings, the experimental copepod P. annandalei appears to be a promising species for the live feed sector and an excellent choice for further investigation. The high-density copepod production was significantly achieved at 26°C NSB through RASC. Nauplii production was increased in P. annandalei at 26°C NSB G5 by 29.6% ∆G. Only five generations of selected breeding were examined in this study; however, given that heritability ­(h2) at 18°C was 0.64 and at 26°C was 0.30, and there foremore generations of selective-breeding study may provide the positive outcome of 18°C. Continuous breeding of P. annandalei was achieved by high NPR environmental induction with 26°C temperature, 20 PSU salinity, 4 mg/L oxygen, 8 pH, 1000 lux light intensity, and 10:14 light: dark hours. The current work demonstrates that the B. subtilis probiotic induction can improve copepod productivity. In future, the combination of microalgae and probiotics as well as the long-term effects of the hormones have to be studied. Acknowledgements The authors are extremely thankful to the Head, Dept. of Marine Science, and authorities of the Bharathidasan University for providing the necessary facilities. Authors’ contributions Perumal Santhanam: Conceptualization, methodology, writing—review and editing, supervision. Mohammed Syed Marjuk: Investigation, methodology, validation, software, data 13 1. Bairagi S, Zereyesus Y, Baruah S, Mohanty S (2022) Structural shifts in food basket composition of rural and urban Philippines: implications for the food supply system. 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Authors and Affiliations Perumal Santhanam1 · Mohammed Syed Marjuk1 · Shanmugam Gunabal1 · Palani Sridhar1 · Piliyan Raju1 · Selvaraj Ananth1,2 · Ravichandran Nandakumar1 · Moorthy Kaviyarasan1 · Ayyanar Shenbaga Devi1 · Selvakumaran Jeyanthi1,3 · Meril Divya1,4 · Nagarajan Krishnaveni1 · Ayyasamy Gowthami1 · Pachiappan Perumal1 * Perumal Santhanam-1 Department of Marine Science, School of Marine Sciences, Bharathidasan University, Tiruchirappalli 620 024, Tamil Nadu, India 2 Department of Fisheries, Ministry of Fisheries, Animal Husbandry and Dairying, New Delhi 110 001, India 13 3 Department of Zoology, Ethiraj College for Women, Chennai 600 008, Tamil Nadu, India 4 TNJFU‑Fisheries Business School, Tamil Nadu Dr. J. Jayalalithaa Fisheries University, Vaniyanchavadi, Chennai 603 103, Tamil Nadu, India