Biomass Conversion and Biorefinery

https://doi.org/10.1007/s13399-023-04348-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 concen-
tration 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.
1 Introduction
Highlights
1. The P. annandalei copepod at 26°C NSB yield was significant In recent years, there has been an increase in global
through RASC, with a total population of 17181.6 ± 371.2 ind/l in 2 protein demand [1, 2]. The average edible fish required
months. Whereas, 18°C CSB, and the control yielded a population of
10237.6 ± 225.8 and 12030.6 ± 364.4 ind/l.
per person has increased from 9.0 kg to 20.5 kg in
2. The genetic gain (∆G) for G5 increased by 31.9% and 29.6% at 2018. Between 1986 and 1995, aquaculture production
18°C and 26°C, respectively. But the mean nauplii production of the increased to 82.1 million tonnes, up from 14.9 mil-
select line in G5 at 18°C was 53.6 ± 2.1 nauplii/female only, whereas lion tonnes [3]. Aquaculture is considered to be a most
the 26°C produced 78.6 ± 1.8 nauplii/female.
3. In hormone induction, high NPR was achieved in bisphenol A at a
excellent option to meet the growing demand for fish
concentration of 20 μg/L (83 ± 2.8 nauplii/female) and 17β-estradiol protein [4–6]. But the major bottleneck in marine fish
at 40 μg/L (75.3 ± 1.4 nauplii/female). But long-term hormone larviculture is mass larval mortality owing to insuffi-
effect needs to be studied because it severely affects the survival rate cient nutrition during the first feeding stage [7]. Copep-
of the copepod.
4. The current work demonstrates B. subtilis probiotic induction can
ods act as a promising crustacean as live feed; they have
improve copepod productivity and which provides excellent results at small naupliar stages, showing a high concentration of
a concentration of ­106 CFU/mL (74.3 ± 1.8 nauplii/female) and also essential fatty acids and nutrients, and they possess a
increased the survival rate of copepod when compared to control I. unique swimming motion that encourages the fish fry
galbana.
to eat [8–10].
Extended author information available on the last page of the article

13
Vol.:(0123456789)
 Biomass Conversion and Biorefinery

While calanoid copepods are considered to be an effective stock was maintained in our laboratory without interruption
live feed for marine fish larviculture [8, 11, 12], the calanoid since 2018.
copepod, Pseudodiaptomus annandalei, is an excellent model The copepods were transferred to the laboratory and
organism that is used as a live feed for aquatic animals in the given gentle aeration with aerators. The copepod samples
aquafeed business, and it is one of the most frequent copepods were then coarsely filtered to exclude the nauplii and other
seen in the wild [13–16]. P. annandalei is numerous and can larval forms using the superimposed sieves. The males and
adjust to changes in salinity, temperature, nutritional concen- females of P. annandalei were separated from the diluted
tration, oxygen levels, and suspended particles and, thus, a samples and the mother stock cultured in 1-L glass beakers
potential one for outdoor culture [14, 17–19]. To date, high- with 700 mL of saline water. The copepods were then mass
density copepod culture has been difficult when compared to cultured in 100-L fibre-reinforced plastic (FRP) tanks filled
other live feeds such as artemia and rotifers [20–23]. The con- with saltwater filtered through a 1-μm filter bag to remove
tinuous production of copepods is also complicated, and main- the additional impurities from the water. The salinity and
taining stock culture is also a significant problem here [24, 25]. temperature of seawater were adjusted to 26 PSU and 26
Given the above constraint, we need to develop new tech- ± 2°C, respectively 8 pH, 1000 lux light intensity, 12:12
niques and methods for the high-density and continuous light-and-dark photoperiod, and 4 mg/L dissolved oxygen.
production of copepods. Therefore, presently the following The I. galbana microalga was fed to P. annandalei every 2
methods have been tried for the tropical calanoid copepod days at 3×104 cells/ml concentration. The faecal pellets and
P. annandalei in high-density and continuous production by detritus were siphoned out once in 4 days, and total water
selective breeding [21, 26] and induced breeding through was exchanged at 15-day intervals.
hormone induction [27], microbial induction [28], and envi-
ronmental induction by changing and optimizing the differ- 2.3 Research design
ent parameters, viz., salinity, temperature, pH, light intensity,
photo-period or light regime, and dissolved oxygen. The research was conducted to develop high density and con-
tinuous production of marine copepod P. annandalei using
various methods like (a) selective breeding normal 26°C and
2 Material and methods cold selective breeding 18°C, (b) environmental induction, (c)
hormone induction, and (d) microbial induction. Each experi-
2.1 Microalgal culture ment consisted of triplicates (n=3). All the experiments were
conducted under indoor conditions and maintained the same
In this study, the experimental copepod P. annanda- parameters as for stock culture (except different experiments
lei was fed with the microalga Isochrysis galbana. The were under different parameters depending on the study). For
microalga strain, I. galbana was collected, isolated, and each experiment with copepod, we took care to acclimate all
maintained at the Marine Planktonology and Aquacul- copepods to the experimental conditions for at least 24 h prior to
ture Laboratory (MPAL), Department of Marine Science, conducting any experiments to minimize any potential effects of
Bharathidasan University, Tiruchirappalli, Tamil Nadu, acclimation on our results. Copepods and developmental stages
India. Algal stock and mass cultures were kept in 1-L coni- were enumerated by an inverted microscope (Micros Austria
cal flasks and 20-L carboys indoors at a temperature of 23 SUNDEW MCX1600, India, with Tuscan Discovery CH30 3.0
± 2°C with a cycle of 12:12 light: dark hours. Fluorescent MP Camera).
tubes were used as a light source, with a light intensity The experiment was performed in the Marine Plank-
of 2000 lux. For algal cultivation, filtered and sterilized tonology and Aquaculture Laboratory (MPAL), Depart-
seawater 25 PSU enriched with Conway’s medium was ment of Marine Science, Bharathidasan University, located
utilized (Walne, 1970). For mass cultivation, I. galbana in Tiruchirappalli, Tamil Nadu, India.
was cultivated in a 1000-L FRP tank.
2.3.1 Recirculation aquaculture system for copepods
(RASC)
2.2 Copepod culture
The working principle of Recirculation Aquaculture Sys-
The copepod species, P. annandalei (KY982586), was cul- tem for Copepods (RASC) to get high-density production of
tured and maintained at the MPAL laboratory; The strain copepods is shown in Fig. 1. In brief, freshwater or saltwa-
was initially collected using 158-μm mesh size plank- ter (according to need) was pumped to 5000-L and 3000-L
ton nets (0.35-m diameter opening) from Nagore (Lat. capacity storage tanks (ST). The raw water was pumped
10°49′37.804″ N; Long. 79°51′49.060″ E), Southeast Coast from the ST using the pump (P) and filtered through a
of India, and has been cultured, isolated, identified and the series of filters such as sand filter (S), biological filter (B),

13
Biomass Conversion and Biorefinery

membrane filter (M), and UV filter (UV), and filtered water by using pump (P) and passed through the sand filter (S),
and was stored in reservoir tank (RT). From the RT, the fil- biological filter (B), and degasser cum skimmer (D) where
tered water was supplied to all 6 copepod culture tanks (CC) the vigorous aeration was supplied by using the blower (BL)
and microalga culture tanks (MC). A known volume (5 L) to remove the foam and ammonia. Finally, the purified water
of needful species of microalga was obtained from indoor from degasser cum skimmer (D) was recirculated to all the
stock culture facility and was inoculated in 1000-L capacity culture tanks. The different experiment copepods, such as
FRP tanks filled with filtered water and fertilized with com- 26°C non-selective breeding (control), 18°C cold selective
mercial fertilizers such as ammonium sulphate, urea, and breeding (18°C CSB), and 26°C normal selective breeding
super phosphate in the ratio of 10:1:1 gm/L of water. The (26°C NSB) adults, were maintained in the separate tanks
microalgal culture was developed prior to copepod inocula- such as CC1, CC2, and CC3, respectively. The cultured
tion. A known numbers of copepods (>1000 ind/l); both copepods were harvested by draining the water through one
including male and female or egg carrying female (based on end of the outlet fitted with mechanical filters made up of
experiment) was inoculated in CC tanks. For the selective plankton sieve holders. The wastewater seeped from the cul-
breeding, selection of male and female was done according ture tanks during harvest was drained through another end
to Alajmi & Zeng [21]. The aeration for both copepods and of the outlet after one crop is over, and the wastewater was
microalgal tanks was supplied through the PVC pipeline and discharged through the drainage canal (Fig. 1).
silicon tube attached with the air stones by using industrial Overall, the RASC was designed to provide a stable
air compressor (AC). The cultured microalga was supplied and controlled environment for the copepods, allowing
as feed for copepods using an algal dosing pump (ADP). The precise manipulation of environmental variables such as
water quality parameters such as temperature, salinity, pH, temperature, salinity, pH, photoperiod, photo regimes, and
and dissolved oxygen in the culture tanks were maintained water quality. While RASC for copepod culture is uncom-
using a probe connected with automatic controller (ATC). mon, it has several advantages over other culture methods,
The available wastewater in the CC tanks was drained including the ability to maintain consistent water qual-
through the wastewater pipeline and collected at recircula- ity and temperature and to reduce water usage and waste
tion tank (RCT). From the RCT, the wastewater was pumped 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 days for nauplii counting under the specific experimen-
tal conditions of each study.
A consistent selective breeding approach, slightly modi- To track total nauplii production, nauplii generated
fied from Alajmi and Zeng [29] was adopted, for high by each G0 female were collected on a 25-μm mesh and
density RASC culture system, for raising the copepod recorded every day until the nauplii production stopped.
P. annandalei at the different temperatures of 26°C [29] These nauplii were cultivated until they matured. The
and 18°C [26]. In this study, the selection was based on nauplii were taken from each G0 pair to guarantee that a
the rate of nauplii production of P. annandalei (Fig. 2). sufficient number of offspring were collected to start the
For selective breeding, 50 active pairs of adult P. next generation. The G1 was developed by selecting the
annandalei were randomly selected from the laboratory progeny of the top 30% of G0 females with the highest
stock cultures to produce the base population (G0). From nauplii production on the selected days as parents and an
the base population, 150 pre-matured female P. annan- equivalent number of male individuals from these top 30%
dalei at copepodite stage V (CV) were individually iso- of females. The control copepod was formed by randomly
lated in Petri dishes and observed for sexual matura- pairing male and female individuals from G0’s offspring.
tion. The animals from 16th day of the culture or 2 days By mating newly matured adults, fifty pairs were produced
after reaching the CV stage were considered as sexu- for each control and experiment. To eliminate inbreeding,
ally mature and further confirmed by the moulting of according to Nomura and Yonezawa study [32], a circular
the animal according to the standard protocol [30]. An mating design was used, which allows males from each
adult male was partnered with mature adult female. Each group to be transferred to the neighbouring group in a rota-
pair was maintained in a 50-ml glass beaker submerged tional fashion for mating. The same selection criteria were
in filtered saltwater at a temperature of 26°C normal used in all five generations of selection. A control line was
selective breeding (26°C NSB) in one group and another kept alongside each succeeding generation with random
18°C cold selective breeding (18°C CSB), 25 ± 2 PSU mating without bias for a particular trait.
salinity, 8–8.5 pH, 4 mg/l dissolved oxygen, 1000 lux After the final G5, the top 30% was inoculated into a
light intensity, and 12:12 h of light: dark photoperiod, 1-L beaker for further culture. After obtaining an ade-
and the copepods were fed with microalga I. galbana, 2 quate number (copepods >1000 ind/L) of adult copepods,
days once 3×10 4 cells/ml concentration. A 90-μm mesh their progenies were transported into a 100-L FRP tank
was inserted at the bottom so that only the nauplii were for mass production of P. annandalei. Then the cultures
permitted through the bottom mesh. A Bogorov count- were transferred to a 2000-L FRP tank controlled by
ing chamber and a stereomicroscope were used to count RASC (Recirculation Aquaculture System for Copepod).
the nauplii that accumulated at the bottom of the beak- After reaching the adequate density of copepod P. annan-
ers. Throughout the experiment, copepods were given dalei (days 20 to 28 from inoculation to the RASC), the
I. galbana. The family selection was used to develop total population consisting of nauplii, copepodite, and
a copepod strain with a higher nauplii production. A adults were counted by the Bogorov counting chamber
pilot-scale experiment was conducted to determine the under a stereomicroscope by random sampling of 1 L
nauplii production pattern of P. annandalei. The study from each 2000-L RASC tank. The salinity, pH, oxygen,
identified the highest nauplii output period based on the photoperiod, and light intensity were kept at the same
high nauplii production rate (NPR) over the lifespan of optimal levels as that of the stock culture.
the copepod. As per the study findings, the days between
day 3 and day 12 were determined as the optimal days Calculation of response to selection (R) genetic gain (∆G)
for taking nauplii counts. Previous studies have also and realized heritability (­ h 2 ) The difference in mean
reported higher nauplii output during the early stages phenotype (nauplii production) between the progeny
of copepod culture [31]. However, it is important to keep generation and the previous generation determined the
in mind that the optimal days for nauplii counting may response (R). The selection differential (S) was deter-
vary depending on the specific experimental conditions mined using the mean phenotypic difference between
and research objectives. Therefore, it is always recom- the chosen parents and the overall parent population.
mended to conduct pilot studies to determine the optimal The slope of the generation representing the regression

13
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 differen- successive generations of selection were calculated
tial was used to estimate the realized heritability ­( h 2 ) according to the following equation:
[33]. Genetic gain (∆G) achieved throughout every two

13
 Biomass Conversion and Biorefinery

ΔGn = (Sn − Cn) using the Bogorov counting chamber under the stereo micro-
scope on selected days (days 3 to 12).
where S is the select line’s mean phenotypic value, C is the
control line’s mean phenotypic value, and n is the genera- 2.3.5 Microbial induction
tion number.
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
2.3.3 Environmental induction and optimization centrifuged at 5000g for 10 min at 4°C. The supernatants
were removed, and the cell pellets were washed three times
Responses of the P. annandalei to various environmental before being resuspended in a sterile saline solution (0.9 %
factors such as temperature (15, 18, 21, 23, 26, 29, 32, and NaCl). The spread plate method was used to determine the
35°C), salinity (15, 20, 25, 30, 35, and 40 PSU), pH (6.5, concentration of bacterium by colony-forming unit (CFU/
7, 7.5, 8, 8.5, and 9 pH), light intensity (500, 1000, 1500, mL). The bacterium was quantified and stored in suspen-
2000, 2500, and 3000 lux), photoperiod (12:12, 14:10, sion form at 4°C for use in feed preparation. The prepared
10:14, 08:16, and 16:08 light-and-dark hours), and dis- probiotic bacteria cells were inoculated in a 50-ml beaker
solved oxygen (1, 2, 3, 4, 5 mg/L) were observed (in order containing two pairs of P. annandalei Copepodite V (CV)
to increase NPR and to optimize culture parameters. For and were maintained in the same environmental condition as
each experiment, a pair of P. annandalei male and female stock. One treatment contained I. galbana alga at a concen-
CV stages were isolated from a stock culture with the help tration of 30,000 cells/mL; the actual concentration added
of a stereomicroscope and transferred into a 50-ml glass was adjusted daily by determining cell density with the par-
beaker. The nauplii were counted on selected days (days ticle counter (control). The probiotic bacterium B. subtilis
3 to 12). Conditions similar to stock maintenance were was adjusted to the concentration of 1­ 05, ­106, ­107, and 1­ 08
employed except for parameter-specific experiments. CFU/mL for (treatment). The copepod nauplii were counted
for NPR on selected days (days 3 to 12).
2.3.4 Hormonal induction
2.3.6 Survival rate (SR)
The induced breeding method was followed by adopting the
method of [27], with slight alterations. In brief, the estro- The survival rate of copepod P. annandalei was inves-
genic chemicals such as bisphenol A (CAS 80-05-7; >97) tigated for hormone induction and microbial induction.
and 17β-estradiol (CAS 50-28-2; ≥98) were purchased Experiments i, were done in triplicates using 10 active
from Sigma-Aldrich. Stock solutions were prepared using adults of P. annandalei for each experiment. For hormone
dimethyl sulfoxide (DMSO; CAS 67-68-5; ≥99.9%) (Sigma- induction, the copepods were treated with bisphenol A and
Aldrich) as carrier solvent. The solutions were stirred and 17β-estradiol with which concentration produces high NPR,
stored at 4°C in the dark until used. A ten-fold dilution series vis., bisphenol-A 20 μg/L, and 17β-estradiol was 40 μg/L.
in 100% DMSO was made from each stock solution. Ten For microbial induction, the probiotic bacterium B. subtilis
microliters of the dilution series was mixed with 100 mL was taken at ­106 CFU/mL, and I. galabana ­104 cells/ml was
seawater to make a nominal concentration. In the solvent taken and induced to the copepod. The Kaplan-Meier sur-
control, bisphenol A, and 17β-estradiol treatments, the final vival curve method was used for analysis SR [37].
concentration of DMSO was 0.0001% (v/v). Two pairs of
CV stage P. annandalei were inoculated into the beaker 2.4 Statistical analysis
filled with filtered seawater. The induced nauplii production
experiment was started with the test chemicals bisphenol A SPSS statistic software version 26 was used for statisti-
and 17β-estradiol with concentrations much less than acute cal studies. To assess the significant differences between
toxic test values, viz., 10, 20, 30, 40, and 50 μg/L. Prior stud- treatments and controls, the data were analysed using
ies [27, 34–36] on various copepod species, have reported ANOVA followed by the Tukey post hoc test. For the sur-
higher effective concentration (EC) and lethal concentration vival study, the Kaplan–Meier survival analysis (Log-rank
(LC) values (>0.5 mg/L) for bisphenol-A and 17β-estradiol. and Gehan-Breslow-Wilcoxon tests) method was used to
So, in comparison, this study used a very less concentration compare and estimate adult copepod’s survival duration
of hormone to avoid toxicity to the animal. The number of (days). All graphs were plotted with the help of GraphPad
nauplii released by the copepod P. annandalei was counted prism version 8.

13
Biomass Conversion and Biorefinery

3 Results a 31.9% genetic gain over the 18°C control. At 26°C NSB,
nauplii production was significantly increased (P<0.001)
3.1 Selective breeding when compared to the 18°C CSB and both controls.
Overall, after five generations of selection at 26°C and
During selective breeding, the copepod P. annandalei was cul- 18°C, the percentage of pooled nauplii production for days
tured two different methods viz, non-selective breeding (con- 3 and 12 was increased from 16.1% (G1) to 29.6% (G5) in
trol) and selective breeding. The selective breeding was done 26°C NSB and 18.4% (G1) to 31.9% (G5) in 18°C CSB.
in two different temperatures viz., 26°C and 18°C. The cope- Based on the observed selection responses, the heritability
pods were eventually mass cultured using the Recirculation (h2) for nauplii production in P. annandalei at 18°C was
Aquaculture System for Copepods (RASC) to validate the 0.64 ± 0.05 and at 26°C was 0.30 ± 0.05 (Fig. 3).
increase in population. As a result (Table 1), copepods reared
through 26°C NSB produced a high-density population of 3.2 Environmental induction
17181.6 ± 371.2 ind./l, consisting of 10430 ± 351.5 nauplii,
3985.3 ± 77.4 copepodites, and 2766.3 ± 103.4 adults, fol- Environmental induction was used to gauge the NPR
lowed by non-selective breeding (control) with a total popula- response of copepod P. annandalei. This will help to
tion of 12030.6 ± 364.4 ind./l, consisting of 7260.3 ± 189.2 release the nauplii immediately to the water column. The
nauplii, 3014 ± 136.5 copepodites, and 1756.3 ± 68.4 adults, highest rate of NPR was achieved at 26°C yielded 67
and at 18°C CSB, the yield was low when compared to the ± 4.5 nauplii/female, salinity at 20 PSU gave 69 ± 3
control and at 26°C NSB a total population of 10237.6 ± nauplii/female, oxygen shock at 4 mg/L produced 70 ±
225.8 ind/l, comprising 6638 ± 201.3 nauplii, 2414 ± 259.4 7.5 nauplii/female, pH shock at 8 pH yielded 68.6 ± 3.5
copepodites, and 1185.6 ± 155.5 adults. A total population nauplii/female, light intensity at 1000 lux produced 68
between the non-selective breeding (control), 26°C NSB, and ± 3 nauplii/female, and photoperiod at 10:14 dark: light
18°C CSB are extremely significant (P < 0.001). Also, 26°C hours produced 71.3 ± 2 nauplii/female. All these param-
NSB was extremely significant when compared to 18°C CSB eters were taken into consideration for the optimization
(P < 0.001). But there was no statistically significant differ- of P. annandalei. So, the parameters optimized were;
ence in nauplii, copepodite, and adult production between 26°C temperature, 20 PSU salinity, 4 mg/L oxygen, 8
control and 18°C CSB (P > 0.05). pH, 1000 lux light intensity, and 10:14 light: dark hours
photoperiod, for better survival and high NPR. Lower
3.1.1 Genetic gain NPR of P. annandalei was recorded at temperature 15°C
cold and 35°C hot, 15 PSU salinity, 1 mg/L oxygen, 6.5
The results show (Table 2) that at 26°C, the mean nauplii pH, 3000 lux light intensity, and 16:08 light: dark hours
production before selection (G0) was 59.6 ± 2 nauplii/ photoperiod (Fig. 4).
female (days 3 to 12 pooled). The 26°C select line copepod
nauplii output has been significantly enhanced as a result of 3.3 Hormone induction
selective breeding. In 26°C NSB G5 P. annandalei, nauplii
production was increased by 78.6 ±1.8 nauplii/female which The copepod P. annandalei was tested with the hor-
was a 29.6% genetic gain as compared to the control. In mones, bisphenol A and 17β-estradiol for continu-
contrast, the initial nauplii production (G0) of 18°C CSB ous production of copepod. Bisphenol-A significantly
was 31.6 ± 2 nauplii/female (days 3–12 pooled), which was (P<0.001; Fig. 5) increased NPR of the copepod P.
then increased to 53.6 ± 2.1 nauplii/female, by representing annandalei, with the maximum NPR 83 ± 2.8 nauplii/

Table 1  The total population, Stages Control (non-selective 18°C CSB 26°C NSB
nauplii, copepodites, and adults breeding) 26°C
(ind/L) of P. annandalei through
non-selective breeding (control Nauplii (ind/l) 7260.3 ± 189.2b 6638 ± 201.3b(ns) 10430 ± 351.5a(***)
26°C), selective breeding 26°C
Copepodite (ind/l) 3014 ± 136.5b 2414 ± 259.4b(ns) 3985.3 ± 77.4a(*)
NSB and 18°C CSB
Adult (ind/l) 1756.3 ± 68.4b 1185.6 ± 155.5b(ns) 2766.3 ± 103.4a(*)
Total population (ind/l) 12030.6 ± 364.4b 10237.6 ± 225.8b(***) 17181.6 ± 371.2a(***)

Values are expressed as mean ± SE (n=3). Different letters in the same row are indicated significant dif-
ferences 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 Control line (C) R (Sn-Cn) ∆G (%) *Selection
differential

26°C G0 59.6 ± 2 61.6 ± 1.2 2

G1 62.3 ± 0.8a 65.6 ± 3.1 53.6 ± 1.2 b
8.6 16.1 3.3
G2 62 ± 1.1a 72.6 ± 2 54 ± 2.5b 8 14.8 10.6
G3 66.6 ± 1.8a 75.3 ± 5.7 57.6 ± 2.9b 9 15.6 8.6
G4 70.6 ± 2.8a 81 ± 1.5 56 ± 1.1b 14.6 26.1 10.3
G5 78.6 ± 1.8a 90.3 ± 2.7 60.6 ± 1.2b 18 29.6
18°C G0 31.6 ± 2 38.3 ± 5 6.6
G1 36.3 ± 1.4a 40.6 ± 1.4 30.6 ± 1.4 b 5.6 18.4 4.3
G2 38.3 ± 1.4a 45.3 ± 1.7 32 ± 1.7 b 6.3 19.7 7
G3 44.6 ± 0.6a 51.3 ± 1.4 36.3 ± 0.8 b 8.3 22.9 6.6
G4 49.3 ± 2.4a 58 ± 1.5 38.6 ± 1.7 b 10.6 27.5 8.6
G5 53.6 ± 2.1a 61.3 ± 1.3 40.6 ± 1.4 b 13 31.9

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 3.4 Microbial induction

bisphenol-A. The minimal nauplii production was 37.6
± 2 nauplii/female at a dosage of 50 μg/L, and 65.3 The microbial induction was done by the using probiotic
± 3.1 and 50 ± 1.7 nauplii/female NPR were achieved bacterium B. subtilis as sole diet to the copepod P. annan-
at 10 and 40 μg/L of bisphenol A, respectively. Mean- dalei. The microbial induction had significant (P<0.001;
while, NPR was found to be higher at 75.3 ± 1.4 nau- Fig. 7) effect on nauplii production of the copepod. The
plii/female in the 40 μg/L dose and 65.6 ± 1.4, 60.3 highest NPR was attained at 1­ 06 CFU/mL NPR 74.3 ± 1.8
± 2.7 nauplii/female in the 30 μg/L, and 50 μg/L nauplii/female. The remaining B. subtilis concentrations of
doses of 17β-estradiol, respectively. In 10 and 20 μg/L ­105, ­107, and ­104 CFU/mL produced 68.6 ±1.4, 65 ± 2,
17β-estradiol was not significantly produced NPR 51 and 57.6 ±1.8 nauplii/female. ­103 CFU/mL showed least
± 1.5, 56 ±1.5 nauplii/female. Overall bisphenol A 20 NPR (47 ± 2 nauplii/female) compared to other concentra-
μg/L and 17β-estradiol 40 μg/L concentration induced tions. For microalgal experiments, I. galbana ­104 cells/ml
better nauplii production of copepod P. annandalei, for seemed to be optimal that produced NPR of 65.6 ± 1.7 nau-
continuous production. plii/female. Compared to the B. subtilis, the I. galabana con-
The SR of P. annandalei (Fig. 6), in hormones bis- centrations such as 1­ 03, ­105, ­106, and 1­ 07 have exerted low
phenol A (20 μg/L) and 17β-estradiol (40 μg/L) was NPR yield of 58.3 ± 2.6, 53 ± 2, 33 ± 5.7, and 17 ± 3.7
investigated. The survival percentage of the control was nauplii/female, respectively.
high compared to the hormones (test), throughout the SR of P. annandalei was experimented with sole diets of,
experiment. Both, control and treatment had a 100% sur- I. galbana ­(104 cells/ml) and probiotic bacteria B. subtilis
vival till day 3. From day 4, a steady decrease in SR was ­(106 CFU/mL). The results (Fig. 8) of the probiotic-treated
observed in both the hormone treatments. The control SR copepod P. annandalei had a high SR of 77.14% compared
was stable from day 6 to day 12(82%) but, dropped to to the I. galbana 60.15%. From day 1 to day 3, no mortality
55% on days 13 to 15. Despite the drop, SR of control was recorded in both treatments. I. galbana fed copepods
was always higher than the hormone treatments. The showed slow decrease in SR to 91.66% on 4th day, followed
treatment of bisphenol A dropped the survival rate rap- by 72.18% around day 9, and after day 10 no mortality
idly, day 5 to 6 83.3%, day 7 to 8 74.07%, day 9 to 11 was recorded. In probiotic-treated copepod, P. annandalei
63.49%, day 12 and 13 47.61%, and finally, day 14 to SR no mortality was observed till day 6. On day 9, the SR
day 15 have 21.8%. In 17β-estradiol from day 6 to 7 has decreased to 77.14%, no mortality was recorded thereafter.
82.5%, on day 8 to 9 72.18%, on day 10 60.15%, on 11 to In comparison to the control, this data demonstrates that the
12 48.12%, and on the last day 13 to 15 survival percent- probiotic bacterium B. subtilis has been better diet in terms
age declined to 32.08%. of survival of copepod P. annandalei.

13
Biomass Conversion and Biorefinery

technologies; in the future, as these technologies would sup-

port high density and continuous production of other cope-
pods also.
The present selective breeding findings are in line with
the reports of earlier researchers [29], who have demon-
strated that a simple selective breeding method (applied
over a short period) could significantly increase the repro-
ductive 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 produc-
tion 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 selec-
tion 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 num-
ber 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 non-
selective 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
Fig. 3  Regression between the 18°C CSB and 26°C NSB selected best result. The performance of 18°C CSB was lower than the
mean nauplii production (days 3 to 12 pooled) across five generations 26°C NSB, but this is not surprising given that 26°C is the
and cumulative selection differential optimal temperature for P. annandalei. However, it would be
interesting to investigate the production rate of cold-selected
4 Discussion strain (18°C) back at 26°C. Nonetheless, the study provides
valuable insights into the potential of selective breeding to
The copepods, particularly P. annandalei have been proven enhance copepod production under different temperature
to be high-value live feed to tropical finfish larvae [19, 31]. regimes. Across five generations of selective breeding were
Copepods are the superior nutrition exchanger in aquatic only studied here, maybe studies covering more than 5 genera-
ecosystems [38]. If they are cultivated in high density, that tions of selective breeding (18°C), in future, would provide
would lead to an excellent pathway for the live feed/aqua- positive results because the heritability ­(h2) at 26°C was 0.30,
culture industry. But the main challenge lies in continu- but it was 0.64 at 18°C. It was twofold higher than 26°C. The
ous maintenance of the copepod stock culture [39, 40] as result of 26°C ­h2 for P. annandalei was slightly matched with
opposed to other live feeds like rotifers and artemia. How- the earlier findings of Aljami [29] with P. crassirostris. It is
ever, as the aquaculture business grows, there will be greater important to undertake more research to better understand
demand for high-density cultures of small prey items that are the impact of employing small volumes of water in selective
ideal for high-value finfish [11, 41, 42]. The P. annandalei is breeding trials and to see if the findings can be extrapolated on
a promising copepod for mass culture and continuous pro- a larger scale. Future study may also concentrate on enhancing
duction through selective and induced breeding technology. copepod mass culture conditions to increase their potential as
The copepod, P. annandalei, is a model organism for these 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. annan-
dalei with different environ-
mental 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 an effective method in an aquaculture system, has been
a total population of 17181.6 ± 371.2 ind/l in two months. adopted in live feed industries [43], and it is necessary to
The same duration 18°C CSB and control yielded a total improve the RASC system-automation.
population of 10237.6 ± 225.8, and 12030.6 ± 364.4 ind/l, The demographic structure of copepod populations can
respectively. The RASC system, which was shown to be 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. annan-
dalei at concentrations of 10, 20, 30, 40, and 50 μg/L. The different Fig. 6  Kaplan-Meier survival curve shows the survival rate of P.
letters above each bar represent the significant variations between annandalei by survival percentage on hormone induction by bisphe-
treatment groups identified by Tukey’s post hoc analysis nol A and 17β-estradiol

13
Biomass Conversion and Biorefinery

100 I. galbana
During our environmental induction and optimization,
B. subtilis the copepod P. annandalei showed an enhanced NPR. The
80 A maximum NPR was attained under the following condi-
tions: 26°C temperature, 20 PSU salinity, 4 mg/L oxygen,
NPR(Nauplii/Female)

a AB BC
a C 8 pH, 1000 lux light intensity, and a 10:14 light: dark hours
60 a
D photoperiod. The P. annandalei culture was similarly opti-
b mized using the aforementioned conditions. The recorded
40 decreased naupliar production at high oxygen level (5 mg/L)
c could be caused to the copepods by high ­O2, leading to a
20 decrease in their reproductive performance [45] and also
high oxygen levels can increase metabolic rates.
0 Anderson and others have stated that estrogens like
10
3
10
4
10
5
10
6
10
7
17β-estradiol could stimulate female sexual maturation and
egg production [27] and they have reported the bisphenol A
I. galbana (cells/ml) B. subtilis (cfu/ml)
and 17β-estradiol accelerated the maturation of ovaries and
increased egg production of copepod Acartia tonsa. Pres-
Fig. 7  Mean ± SE, n=3. The copepod P. annandalei was subjected ently, the hormone induction produced high NPR at 20 μg/L
to microbial induction by probiotic B. subtilis bacterium at various
doses (CFU/mL), with I. galbana serving as the control. The different
concentration 83 ± 2.8 nauplii/female and 17β-estradiol 40
letters (a, b, c I. galbana; A, B, C B. subtilis) above each bar indi- μg/L concentration 75.3 ± 1.4 nauplii/female. However,
cate the significant differences among treatment groups identified by the survival rate under bisphenol A and 17β-estradiol was
Tukey’s post hoc test low compared to the control. Bisphenol A has 21.8% and
17β-estradiol 32.08% on the ­15th day. As a result, addi-
production. In our study, we have observed differences in tional hormone study is required. Earlier more studies were
the demographic structure of copepod populations between conducted on toxicity of endocrine-disrupting hormones,
the two different temperature treatments with selective and including bisphenol A and 17β-estradiol, but the toxicity
non-selective breeding copepods. Specifically, at 26°C in occurs at concentration greater than 0.5 mg/L [34, 36, 46].
the selective breeding treatment, we observed a significantly However, modest concentrations like 10, 20, 30, 40, and 50
higher number of nauplii, copepodites, and adults, resulting μg/L also have an impact on the copepod P. annandalei sur-
in a significantly higher total population size as compared vival rate; this may require further research. Even with the
to the other treatments such as 26°C non-selective breed- possible harmful effects of these chemicals on the environ-
ing, and 18°C selective breeding. These results suggest that ment and copepods, we believe that this section still holds
temperature, and selective breeding may play a role in shap- value in the context of aquaculture. Hormonal induction is
ing the demographic structure of copepod populations and a widely used technique in aquaculture practices, and our
that may have implications for optimizing copepod culture study offers significant information on the possible effects of
protocols. Further research is needed to fully understand the using endocrine disruptors in such practices. Furthermore,
mechanisms underlying these observed differences on demo- our research also explored the impact of different concentra-
graphic structure and their implications for copepod culture. tions of these chemicals and the findings will be useful for
developing more effective and sustainable hormonal induc-
tion 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 man-
ner. 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 diges-
tion and assimilation, water bioremediation and reduce dis-
ease development in aquaculture [47]. In addition, they have
unique properties, like antibacterial activity against aquatic
pathogens [48], which would aid copepods in better sur-
Fig. 8  Kaplan-Meier survival curve shows the SR of P. annandalei vival. In microbial induction, the probiotic bacterium B. sub-
survival percentage by probiotic bacteria B. subtilis induction tilis at ­106 CFU/mL concentration, significantly increased

13
 Biomass Conversion and Biorefinery

NPR (74.3 ± 1.8 nauplii/female), and with I. galbana NPR curation, formal analysis, writing—original draft, writing—review and
was 65.6 ± 1.7 nauplii/female. Sun and others [49] have editing. Shanmugam Gunabal: Investigation, formal analysis. Palani
Sridhar: Investigation, formal analysis. Piliyan Raju: Formal analysis,
suggested that the copepod P. annandalei is an appropriate writing—review and editing. Selvaraj Ananth: Formal analysis. Ravi-
probiotic vector for marine fish larvae. Earlier researchers chandran Nandakumar: Writing—review and editing. Moorthy Kavi-
have also reported the B. subtilis induced larval production yarasan: Formal analysis. Ayyanar Shenbaga Devi: Formal analysis.
in some copepods including P. annandalei [44]. However, Selvakumaran Jeyanthi: Formal analysis. Meril Divya: Formal analysis.
Nagarajan Krishnaveni: Formal analysis. Ayyasamy Gowthami: Formal
the potential causes of the drop-in survival rate observed analysis. Pachiappan Perumal: Writing—review and editing
on day 4 in I. galbana treatment, could be due to multiple
causative factors. One possibility could be related to the Funding The Department of Biotechnology (DBT), Govt. of India,
ecophysiology of copepods and the development. Previ- New Delhi, is gratefully acknowledged for its financial support to
this work through an extramural research project (BT/PR28606/
ous studies have shown that copepods may require specific AAQ/3/921/2018; dated 16.05.2019). Authors (MSM, SG, and PS)
types of food or feeding regimes [50, 51]. In our study, thank the DBT for the fellowship provided.
we used I. galbana as the sole food source for copepod,
which may not have provided all the necessary nutrients for Data availability The corresponding author will provide the datasets
created during the current study upon reasonable request.
copepod. Another possibility is that some unknown factors
or random fluctuations in environmental conditions could Declarations
have influenced the results. Overall, we believe that further
investigation is needed to determine the specific causes of Ethical approval Not applicable.
the observed drop-in survival rate and to explore alternative
Competing interests The authors declare no competing interests.
feeding regimes that may be more suitable for P. annandalei.
These findings would be valuable for improving the effi-
ciency and sustainability of copepod aquaculture practices.

References

5 Conclusion 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. PLoS One 17:e0264079.
According to our findings, the experimental copepod P. https://​doi.​org/​10.​1371/​journ​al.​pone.​02640​79
annandalei appears to be a promising species for the live 2. Goedde L, Horii M, Sanghvi S (2015) Pursuing the global oppor-
feed sector and an excellent choice for further investiga- tunity in food and agribusiness. McKinsey Global Institute
3. FAO (2020) The State of World Fisheries and Aquaculture 2020.
tion. The high-density copepod production was signifi- FAO
cantly achieved at 26°C NSB through RASC. Nauplii pro- 4. Hua K, Cobcroft JM, Cole A et al (2019) The future of aquatic
duction was increased in P. annandalei at 26°C NSB G5 protein: implications for protein sources in aquaculture diets. One
by 29.6% ∆G. Only five generations of selected breeding Earth 1:316–329. https://​doi.​org/​10.​1016/j.​oneear.​2019.​10.​018
5. Turchini GM, Trushenski JT, Glencross BD (2019) Thoughts
were examined in this study; however, given that herit- for the future of aquaculture nutrition: realigning perspectives
ability ­(h2) at 18°C was 0.64 and at 26°C was 0.30, and to reflect contemporary issues related to judicious use of marine
there foremore generations of selective-breeding study resources in aquafeeds. N Am J Aquac 81:13–39. https://​doi.​org/​
may provide the positive outcome of 18°C. 10.​1002/​naaq.​10067
6. Jiang Q, Bhattarai N, Pahlow M, Xu Z (2022) Environmental sus-
Continuous breeding of P. annandalei was achieved by tainability and footprints of global aquaculture. Resour Conserv
high NPR environmental induction with 26°C temperature, Recycl 180:106183. https://​doi.​org/​10.​1016/j.​resco​nrec.​2022.​
20 PSU salinity, 4 mg/L oxygen, 8 pH, 1000 lux light 106183
intensity, and 10:14 light: dark hours. The current work 7. Hamre K, Yúfera M, Rønnestad I et al (2013) Fish larval nutri-
tion and feed formulation: knowledge gaps and bottlenecks for
demonstrates that the B. subtilis probiotic induction can advances in larval rearing. Rev Aquac 5:S26–S58. https://d​ oi.o​ rg/​
improve copepod productivity. In future, the combina- 10.​1111/j.​1753-​5131.​2012.​01086.x
tion of microalgae and probiotics as well as the long-term 8. Wilson JM, Ignatius B, Santhosh B et al (2022) Effect of adult
effects of the hormones have to be studied. density on egg production, egg hatching success, adult mortality,
nauplii cannibalism and population growth of the tropical calanoid
Acknowledgements The authors are extremely thankful to the Head, copepod Acartia tropica. Aquaculture 547:737508. https://d​ oi.o​ rg/​
Dept. of Marine Science, and authorities of the Bharathidasan Univer- 10.​1016/j.​aquac​ulture.​2021.​737508
sity for providing the necessary facilities. 9. Gopakumar G (2009) Use of copepods as live feed for larviculture
of damselfishes. Asian Fish Sci 22. https://​doi.​org/​10.​33997/j.​afs.​
Authors’ contributions Perumal Santhanam: Conceptualization, 2009.​22.1.​001
methodology, writing—review and editing, supervision. Mohammed 10. Kleppel G (1993) On the diets of calanoid copepods. Mar Ecol
Syed Marjuk: Investigation, methodology, validation, software, data Prog Ser 99:183–195. https://​doi.​org/​10.​3354/​meps0​99183

13
Biomass Conversion and Biorefinery

11. Ajiboye O, Yakubu AF, Adams TE et al (2011) A review of the Copepoda). J Plankton Res 39:994–1003. https://d​ oi.o​ rg/1​ 0.1​ 093/​
use of copepods in marine fish larviculture. Rev Fish Biol Fish plankt/​fbx041
21:225–246. https://​doi.​org/​10.​1007/​s11160-​010-​9169-3 27. Andersen HR, Halling-Sørensen B, Kusk KO (1999) A parameter
12. Chen JY, Zeng C, Jerry DR, Cobcroft JM (2019) Recent for detecting estrogenic exposure in the copepod Acartia tonsa.
advances of marine ornamental fish larviculture: broodstock Ecotoxicol Environ Saf 44:56–61. https://​doi.​org/​10.​1006/​eesa.​
reproduction, live prey and feeding regimes, and comparison 1999.​1800
between demersal and pelagic spawners. Rev Aquac raq.12394. 28. Alajmi F, Zeng C (2015) Evaluation of microalgal diets for the
https://​doi.​org/​10.​1111/​raq.​12394 intensive cultivation of the tropical calanoid copepod, Parvoca-
13. Rayner TA, Jørgensen NOG, Blanda E et al (2015) Biochemi- lanus crassirostris. Aquac Res 46:1025–1038. https://​doi.​org/​10.​
cal composition of the promising live feed tropical calanoid 1111/​are.​12254
copepod Pseudodiaptomus annandalei (Sewell 1919) cultured in 29. Alajmi F, Zeng C, Jerry DR (2014) Improvement in the reproduc-
Taiwanese outdoor aquaculture ponds. Aquaculture 441:25–34. tive productivity of the tropical calanoid copepod Parvocalanus
https://​doi.​org/​10.​1016/j.​aquac​ulture.​2015.​01.​034 crassirostris through selective breeding. Aquaculture 420–421:18–
14. Kaviyarasan M, Santhanam P, Ananth S et al (2020) Population 23. https://​doi.​org/​10.​1016/j.​aquac​ulture.​2013.​10.​031
growth, nauplii production and post-embryonic development 30. Golez MN, Takahashi T, Ishimarul T, Ohno A (2004) Post-
of Pseudodiaptomus annandalei (Sewell, 1919) in response embryonic development and reproduction of Pseudodiaptomus
to temperature, light intensity, pH, salinity and diets. IJMS annandalei (Copepoda: Calanoida). Plankton Biol Ecol 51:15–25
49(6):1000–1009 31. Chen Q, Sheng J, Lin Q et al (2006) Effect of salinity on repro-
15. Kadiene EU, Ouddane B, Gong H-Y et al (2022) Multigenera- duction and survival of the copepod Pseudodiaptomus annanda-
tional study of life history traits, bioaccumulation, and molec- lei Sewell, 1919. Aquaculture 258:575–582. https://​doi.​org/​10.​
ular responses of Pseudodiaptomus annandalei to cadmium. 1016/j.​aquac​ulture.​2006.​04.​032
Ecotoxicol Environ Saf 230:113171. https://​doi.​org/​10.​1016/j.​ 32. Nomura T, Yonezawa K (1996) A comparison of four systems of
ecoenv.​2022.​113171 group mating for avoiding inbreeding. Genet Sel Evol 28:141–
16. Pan Y-J, Dahms H-U, Hwang J-S, Souissi S (2022) Recent 159. https://​doi.​org/​10.​1051/​gse:​19960​202
trends in live feeds for marine larviculture: a mini review. Front 33. Falconer DS (1996) Introduction to quantitative genetics. Pearson
Mar Sci 9. https://​doi.​org/​10.​3389/​fmars.​2022.​864165 Education India
17. GrØnning J, Doan NX, Dinh NT et al (2019) Ecology of 34. Andersen HR, Wollenberger L, Halling-Sørensen B, Kusk KO
Pseudodiaptomus annandalei in tropical aquaculture ponds (2001) Development of copepod nauplii to copepodites-a param-
with emphasis on the limitation of production. J Plankton Res eter for chronic toxicity including endocrine disruption. Environ
41:741–758. https://​doi.​org/​10.​1093/​plankt/​fbz053 Toxicol Chem 20:2821–2829. https://​doi.​org/​10.​1002/​etc.​56202​
18. Beyrend-Dur D, Kumar R, Rao TR et al (2011) Demographic 01222
parameters of adults of Pseudodiaptomus annandalei (Copepoda: 35. Marcial HS, Hagiwara A, Snell TW (2003) Estrogenic compounds
Calanoida): temperature–salinity and generation effects. J Exp Mar affect development of harpacticoid copepod Tigriopus japonicus.
Biol Ecol 404:1–14. https://​doi.​org/​10.​1016/j.​jembe.​2011.​04.​012 Environ Toxicol Chem 22:3025. https://​doi.​org/​10.​1897/​02-​622
19. Blanda E, Drillet G, Huang C-C et al (2015) Trophic interac- 36. Djebbi E, Yahia MND, Farcy E et al (2022) Acute and chronic tox-
tions and productivity of copepods as live feed from tropical icity assessments of 17β-estradiol (E2) and 17α-ethinylestradiol
Taiwanese outdoor aquaculture ponds. Aquaculture 445:11–21. (EE2) on the calanoid copepod Acartia clausi: effects on sur-
https://​doi.​org/​10.​1016/j.​aquac​ulture.​2015.​04.​003 vival, development, sex-ratio and reproduction. Sci Total Environ
20. Camus T, Zeng C (2009) The effects of stocking density on egg 807:150845. https://​doi.​org/​10.​1016/j.​scito​tenv.​2021.​150845
production and hatching success, cannibalism rate, sex ratio 37. Kaplan EL, Meier P (1958) Nonparametric estimation from
and population growth of the tropical calanoid copepod Acartia incomplete observations. J Am Stat Assoc 53:457–481. https://​
sinjiensis. Aquaculture 287:145–151. https://​doi.​org/​10.​1016/j.​ doi.​org/​10.​1080/​01621​459.​1958.​10501​452
aquac​ulture.​2008.​10.​005 38. Runge JA (1988) Should we expect a relationship between pri-
21. Alajmi F, Zeng C (2014) The effects of stocking density on mary production and fisheries? The role of copepod dynamics
key biological parameters influencing culture productivity of as a filter of trophic variability. Hydrobiologia 167–168:61–71.
the calanoid copepod, Parvocalanus crassirostris. Aquaculture https://​doi.​org/​10.​1007/​BF000​26294
434:201–207. https://​doi.​org/​10.​1016/j.​aquac​ulture.​2014.​08.​029 39. Cutts CJ (2003) Culture of harpacticoid copepods: potential as
22. Vu MTT, Hansen BW, Kiørboe T (2017) The constraints of high live feed for rearing marine fish. Elsevier, pp 295–316
density production of the calanoid copepod Acartia tonsa Dana. 40. Stottrup JG (2000) The elusive copepods: their production and
J Plankton Res 39:1028–1039. https://​doi.​org/​10.​1093/​plankt/​ suitability in marine aquaculture. Aquac Res 31:703–711. https://​
fbx056 doi.​org/​10.​1046/j.​1365-​2109.​2000.​00488.x
23. Torres Valencia GA, Merino GE, Prieto-Guevara MJ et al 41. McKinnon AD, Duggan S, Nichols PD et al (2003) The potential of
(2022) Spawning Parvocalanus crassirostris at a high adult tropical paracalanid copepods as live feeds in aquaculture. Aquacul-
density: explaining low adult population numbers and means ture 223:89–106. https://​doi.​org/​10.​1016/​S0044-​8486(03)​00161-3
for improving their intensive culture. Aquaculture 546:737347. 42. Hagiwara A, Marcial HS (2019) The use of non-Brachionus
https://​doi.​org/​10.​1016/j.​aquac​ulture.​2021.​737347 plicatilis species complex rotifer in larviculture. Hydrobiologia
24. Kline MD, Laidley CW (2015) Development of intensive cope- 844:163–172. https://​doi.​org/​10.​1007/​s10750-​018-​3837-z
pod culture technology for Parvocalanus crassirostris: optimiz- 43. Rurangwa E, Verdegem MCJ (2015) Microorganisms in recir-
ing adult density. Aquaculture 435:128–136. https://​doi.​org/​10.​ culating aquaculture systems and their management. Rev Aquac
1016/j.​aquac​ulture.​2014.​09.​022 7:117–130. https://​doi.​org/​10.​1111/​raq.​12057
25. Das P, Mandal SC, Bhagabati SK, Akhtar MS, Singh SK (2012) 44. Drillet G, Frouël S, Sichlau MH et al (2011) Status and recom-
Important live food organisms and their role in aquaculture. mendations on marine copepod cultivation for use as live feed.
Frontiers in Aquaculture 5(4):69–86 Aquaculture 315:155–166. https://​doi.​org/​10.​1016/j.​aquac​ulture.​
26. Pan Y-J, Souissi A, Sadovskaya I et al (2017) Effects of cold 2011.​02.​027
selective breeding on the body length, fatty acid content, and pro- 45. Barreto FS, Burton RS (2013) Elevated oxidative damage is cor-
ductivity of the tropical copepod Apocyclops royi (Cyclopoida, related with reduced fitness in interpopulation hybrids of a marine

13
 Biomass Conversion and Biorefinery

copepod. Proc Royal Soc B: Biol Sci 280. https://d​ oi.o​ rg/1​ 0.1​ 098/​ 50. Kandathil Radhakrishnan D, AkbarAli I, Schmidt BV et al (2020)
RSPB.​2013.​1521 Improvement of nutritional quality of live feed for aquaculture:
46. Dahms H-U, Lee SH, Huang D-J et al (2017) The challenging an overview. Aquac Res 51:1–17. https://​doi.​org/​10.​1111/​ARE.​
role of life cycle monitoring: evidence from bisphenol A on the 14357
copepod Tigriopus japonicus. Hydrobiologia 784:81–91. https://​ 51. Meunier CL, Boersma M, Wiltshire KH et al (2016) Zooplankton
doi.​org/​10.​1007/​s10750-​016-​2859-7 eat what they need: copepod selective feeding and potential con-
47. Olmos J, Acosta M, Mendoza G, Pitones V (2020) Bacillus sub- sequences for marine systems. Oikos 125:50–58. https://​doi.​org/​
tilis, an ideal probiotic bacterium to shrimp and fish aquaculture 10.​1111/​OIK.​02072
that increase feed digestibility, prevent microbial diseases, and
avoid water pollution. Arch Microbiol 202:427–435. https://​doi.​ Publisher’s note Springer Nature remains neutral with regard to
org/​10.​1007/​s00203-​019-​01757-2 jurisdictional claims in published maps and institutional affiliations.
48. Nair AV, Leo Antony M, Praveen NK et al (2021) Evaluation of
in vitro and in vivo potential of Bacillus subtilis MBTDCMFRI Springer Nature or its licensor (e.g. a society or other partner) holds
Ba37 as a candidate probiont in fish health management. Microb exclusive rights to this article under a publishing agreement with the
Pathog 152:104610. https://​doi.​org/​10.​1016/j.​micpa​th.​2020.​104610 author(s) or other rightsholder(s); author self-archiving of the accepted
49. Sun Y-Z, Yang H-L, Huang K-P et al (2013) Application of manuscript version of this article is solely governed by the terms of
autochthonous Bacillus bioencapsulated in copepod to grouper such publishing agreement and applicable law.
Epinephelus coioides larvae. Aquaculture 392–395:44–50. https://​
doi.​org/​10.​1016/j.​aquac​ulture.​2013.​01.​037

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

3
* Perumal Santhanam Department of Zoology, Ethiraj College for Women,
[email protected] Chennai 600 008, Tamil Nadu, India
4
1 TNJFU‑Fisheries Business School, Tamil Nadu
Department of Marine Science, School of Marine Sciences,
Dr. J. Jayalalithaa Fisheries University, Vaniyanchavadi,
Bharathidasan University, Tiruchirappalli 620 024,
Chennai 603 103, Tamil Nadu, India
Tamil Nadu, India
2
Department of Fisheries, Ministry of Fisheries, Animal
Husbandry and Dairying, New Delhi 110 001, India

13