Evaluation of Microalgal Diet to Culture Adult Oithona oculata Farran (Copepoda, Cyclopoida)

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Introduction
Copepods are natural prey for most marine fsh larvae and comprise nearly 80% of their stomach contents [1,2].In aquaculture and ornamental fsh industries, copepods are preferred over other commonly used species (e.g., Artemia, rotifers) as a live feed for marine fsh larvae [3].Marine fsh larvae fed copepods survive better [4] and have both better pigmentation [5] and growth [6].However, despite the obvious advantages of using copepods as a food source, their low productivity and cost of culture limit their use for this purpose.
Species of Oithona, marine planktonic copepods that occur widely, can dominate coastal waters [7], provide important links between primary producers and fsh larvae [8], and are preyed upon by commercially signifcant marine species [9].Consequently, some Oithona species are used as experimental live feed in marine aquaculture [10].Oithona oculata Farran, an annually dominant species that occurs in Sagami Bay at high densities [11], has small nauplii that are suitable as a frst food item for many small-mouthed larval marine fsh species, including ornamentals [10].Food quality is a major bottleneck in the mass cultivation of planktonic copepods.Diet afects copepod egg production, survival and growth rates, hatching success, and population growth.One difculty with the mass cultivation of copepods is, however, the varied dietary requirements of individual species [12,13].Because of the potential suitability of O. oculata as a prey source for larval fshes, we fed adult females four diferent microalgal diets to determine which of them was the most favourable for the survival and reproduction of this copepod in culture.
Zooplankton was collected on 14 August 2020, by a plankton net (180 μm mesh) towed gently and obliquely from the seabed to the surface at Manazuru Port (35 °09′49″N, 139 °10′33″E), northwestern Sagami Bay, Japan.Surface seawater was also collected by the bucket for copepod culture.Within 20 min of sampling, zooplankton samples were transferred to the laboratory, where adult female O. oculata with egg sacs, identifed following Nishida [15], were sorted under a dissecting microscope (WILD M10, Leica Co., Ltd.).Sixty female O. oculata were placed into a bottle containing 2 L of fltered (65 μm) ambient seawater, then acclimatised at 25 °C (near-ambient seawater temperature), fed sufcient (1000 μg C•L −1 ) microalgal food comprising a 1 : 1 : 1 : 1 carbon ratio of T. weissfogii, R. salina, T. suecica, and I. galbana, and incubated (CN-25C, Mitsubishi) in darkness for 24 h to negate the efects of the prior natural food environment [16].After 24 h, 10 females carrying egg sacs were fxed in a 5% bufered formalin-seawater solution, and the number of eggs within each egg sac was counted under the microscope to estimate the initial egg sac size (eggs sac −1 ).
After acclimatisation, 10 healthy female O. oculata were placed into separate culture chambers, within beakers with 10 mL fltered (0.22 μm membrane (Merck Millipore)) seawater (Figure 1).Each culture chamber had a 180 μm nylon mesh placed 2 mm above its base to allow nauplii to pass through and minimise their possible cannibalism [17].
Copepods were fed a monomicroalgal diet at 1000 μg C•L −1 (10 μg C ind.• −1 ) daily and kept in darkness at 25 °C in an incubator for 15 d.Te culture period was based on the average survival time of O. oculata at the adult stage collected from the sampling site.Because females can prey on nauplii [18], 10 mL seawater in each chamber was replaced daily with fresh fltered seawater (FSW) to remove hatched nauplii.Te numbers of dead females and egg sacs were recorded daily.Te survival rate (%) was calculated from the total number of individuals in each food treatment and the number of individuals that died.Survival analysis was where F ovi is the abundance of ovigerous females and F all is the abundance of all females (ovigerous and not ovigerous).
After day 15 of culture, the egg sac size in each diet condition was measured.
All the data met the parametric test assumptions.Differences in egg production between dietary treatments were analysed using one-way analysis of variance (ANOVA).A Tukey-Kramer post hoc test was performed when ANOVA revealed signifcant diferences at p < 0.05.

Results and Discussion
Some planktonic copepods cease producing eggs when fed mono-microalgal diets [19][20][21], possibly because of nutrient limitations in (for example) elemental ratios [22], highly unsaturated fatty acids [23], sterols [24], and/or amino acids [25].While we do not use the number of nauplii produced as a metric of copepod production (because they could be underestimated due to cannibalism), we removed nauplii from cultures because they could contribute to the diet of cannibalistic parents.After 15 d of culture on each diet, female survival rates were 70% when fed T. suecica, I. galbana, and T. weissfogii and 50% when fed R. salina (Figure 2).Te ovigerous rate varied for each diet (Figure 3).Tere was no signifcant diference in the numbers of egg sacs produced per female over 15 d among diets, except for starvation (FSW) conditions (Figure 4).Similarly, there were no signifcant diferences in egg sac size among diets (Figure 5).Oithona oculata continued to produce eggs in all diet treatments.Because the culture of this species does not require the preparation of any specifc diet, it may be a candidate species for mass culture [26].Populations of the congeneric O. nana can also be maintained on a wide range of phytoplankton, microzooplankton, and alternative diets such as soybeans, yeast, rice bran, and corn starch [26,27].
Terefore, Oithona copepods may be highly adaptable to a variety of diets and therefore suitable for mass culture.
To estimate egg sac production rates in females fed each diet, linear regressions were performed between incubation duration and cumulative egg sac number.Egg sac production rates (slopes) are 1.36 (R. salina), 1.39 (T.suecica), 0.94 (I.galbana), and 1.21 (T.weissfogii) sacs female −1 •d −1 (Figures 6(a)-6(d)).Production rates are greatest for females fed R. salina and T. suecica.To identify diferences in egg production in females fed diferent diets, females were both reared and monitored separately (Figures 7(a

Data Availability
Te data supporting the fndings of this study are available from the corresponding author upon reasonable request.

Ethical Approval
Copepods were collected in accordance with national legislation in Japan, with all necessary permits obtained prior to conducting research.Aquaculture Research

Figure 1 :
Figure 1: Culture chamber used in egg production experiments.
)-7(d)).Almost all females fed R. salina produced egg sacs throughout the 15 d culture period (Figure 7(a)), whereas half of the females fed T. suecica and (especially) T. weissfogii stopped producing egg sacs late in the culture period (Figures 7(b) and 7(d)).Because R. salina was accepted by many individual copepods and resulted in continuous egg production, we conclude that it represents the most appropriate of the four trialled microalgae to feed O. oculata.