Abstract
While copepods are important prey for many wild marine fish larvae and are preferred as a live feed for culturing larval fish, their low productivity and the costs associated with culturing them typically limit their use as a live food source in all but experimental situations. In a series of experiments with four readily and commercially available microalgae (Thalassiosira weissflogii Grunow, Rhodomonas salina Wislouch, Tetraselmis suecica Kylin Butch, and Isochrysis galbana Parke), we determine which of them, when exclusively fed to a commonly occurring copepod, Oithona oculata, over a 15-day period, results in the greatest copepod survival and egg production rates. We posit that species of Oithona might be an ideal candidate for copepod taxa for commercial-scale copepod culture and that the microalga Rhodomonas salina is an ideal food source to culture them.
1. Introduction
Copepods are natural prey for most marine fish larvae and comprise nearly 80% of their stomach contents [1, 2]. In aquaculture and ornamental fish industries, copepods are preferred over other commonly used species (e.g., Artemia, rotifers) as a live feed for marine fish larvae [3]. Marine fish 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 fish larvae [8], and are preyed upon by commercially significant 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 first food item for many small-mouthed larval marine fish species, including ornamentals [10].
Food quality is a major bottleneck in the mass cultivation of planktonic copepods. Diet affects copepod egg production, survival and growth rates, hatching success, and population growth. One difficulty 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 fishes, we fed adult females four different microalgal diets to determine which of them was the most favourable for the survival and reproduction of this copepod in culture.
2. Materials and Methods
Cultures of four often-used and readily available microalgae (Thalassiosira weissflogii Grunow, Rhodomonas salina Wislouch, Tetraselmis suecica Kylin Butch, and Isochrysis galbana Parke) were established in f/2 medium [14] in 50 mL conical flasks maintained at 20°C in an incubator (FLI-301N, EYELA), with a 12 : 12 h light: dark cycle and light intensity 120 μmol·m−2·s−1. Microalgae fed to copepods were harvested during the mid-to-late logarithmic growth phase.
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, identified 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 filtered (65 μm) ambient seawater, then acclimatised at 25°C (near-ambient seawater temperature), fed sufficient (1000 μg C·L−1) microalgal food comprising a 1 : 1 : 1 : 1 carbon ratio of T. weissflogii, R. salina, T. suecica, and I. galbana, and incubated (CN-25C, Mitsubishi) in darkness for 24 h to negate the effects of the prior natural food environment [16]. After 24 h, 10 females carrying egg sacs were fixed in a 5% buffered 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 filtered (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. The 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 filtered seawater (FSW) to remove hatched nauplii. The numbers of dead females and egg sacs were recorded daily. The survival rate (%) was calculated from the total number of individuals in each food treatment and the number of individuals that died. Survival analysis was conducted by the Kaplan–Meier method. The ovigerous rate (O, %) was calculated as where Fovi is the abundance of ovigerous females and Fall 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 significant differences at .
3. Results and Discussion
Some planktonic copepods cease producing eggs when fed mono-microalgal diets [19–21], 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. weissflogii and 50% when fed R. salina (Figure 2). The ovigerous rate varied for each diet (Figure 3). There was no significant difference 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 significant differences 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 specific 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]. Therefore, 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. weissflogii) sacs female−1·d−1 (Figures 6(a)–6(d)). Production rates are greatest for females fed R. salina and T. suecica. To identify differences in egg production in females fed different diets, females were both reared and monitored separately (Figures 7(a)–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. weissflogii 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.
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Data Availability
The data supporting the findings 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.
Conflicts of Interest
The authors declare that they have no conflicts of interest.
Authors’ Contributions
Yoshiki Takayama, Taisei Yamasaki, and Tatsuki Toda designed the study; Yoshiki Takayama and Taisei Yamasaki conducted field sampling, the experiment, and sample measurements; and Yoshiki Takayama drafted the manuscript. All authors have read and accepted the final manuscript before submission.
Acknowledgments
The authors thank the Manazuru Marine Center for Environmental Research and Education, Yokohama National University, for the assistance in sample collection. This work was partly supported by the Japan Society for the Promotion of Science KAKENHI (JP21K14902) to the first author, and KAKENHI (JP19H03035) to the last author.