Influence of Microalgae Diets on the Biological and Growth Parameters of Oithona nana (Copepoda: Cyclopoida)

Simple Summary The success of marine fish farming is primarily determined by diet in early life. While both artemia and rotifers are commonly used as live feed in aquaculture laboratories in Peru, there has been little work studying the use of native food species. Here, we report our research on the use of a native copepod (Oithona nana) for its potential use in Peruvian marine aquaculture. We collected specimens of the native copepod O. nana and performed culturing experiments with two microalgae that have been widely used in aquaculture. Results show that this species adapted positively to our culture conditions, achieving high densities. We also report on the copepod’s reproduction and growth characteristics. Because of these promising results, we recommend that O. nana be studied further and propose it as a species with potential use as a live feed. Abstract Several species of the planktonic free-living genus Oithona have been successfully used in the larviculture of marine fish and shrimp. However, few studies have been published that allow us to estimate the potential of Oithona nana culture under controlled conditions. This work evaluated the effect of the microalgae Isochrysis galbana and Chaetoceros calcitrans as single (200,000 cells/mL) and mixed diets (100,000 + 100,000 cells/mL) on population and individual growth, ingestion rate, number of spawnings, fertility, development time by stage, and sex ratio of O. nana. We cultured this copepod at 28 ± 0.5 °C, 35 PSU salinity, 125 lux, and 12:12 photoperiod. Results showed that diet had no effect on the final population level (6273–7966 ind/L) or on individual growth, nor on sex ratio, with less males than females. With C. calcitrans, O. nana had a higher filtration rate (57 ng C/ind/day). On the other hand, a mixed diet induced a higher number of spawns (0.4 events/day) and nauplii per spawn (23 ind). Similarly, a single or mixed diet, containing I. galbana, accelerated the development rate by 6.33–7.00 days. We concluded that O. nana can be cultured with both microalgae, indicating its potential use in an intensive system for production. However, more research is required to improve the productivity of O. nana rearing.


Introduction
Zooplanktonic copepods form part of the diet of many aquatic organisms [1], and because of this they have been employed as live prey in fish larviculture [2]. Copepods The transformation suggested by Menden-Deuer and Lessard [56] for converting cell volume to carbon concentration was employed, which consists of using the equation 1: Log (C) = -0.541 + 0.811 (Log V), where, C is the cellular carbon concentration (pg C cell -1 ), and V is the cell volume (µm 3 ). ARA: arachidonic acid, EPA: eicosapentaenoic acid, DHA: docosahexanoic acid. 1 [57]. 2 [58].
The copepod O. nana was captured in the summer of 2019 from the rocky beaches of CAMOSA, using a 55 µm plankton collector, and they were transported to the live feed stock of the Universidad Nacional Jorge Basadre Grohmann (UNJBG), which is also located in CAMOSA. The copepods were identified by the Ornithology and Biodiversity Center (CORBIDI) in Lima, in accordance with Murphy [48] and Ferrari and Bowman [59]. The copepods were kept in 1 L flasks at 28 ± 0.5 • C, 35 PSU salinity, 125 lux, and a 12:12 photoperiod, at a density of less than 5 ind/mL, with a low level of constant aeration. We fed the copepod cultures a mixed diet of I. galbana and C. calcitrans at a density of 10 6 cells/mL of each species, with 100% water exchange every 15 days. Seawater was filtered through a 0.45 µm nitrocellulose membrane using a vacuum pump (Vacuubrand MD-1, Wertheim, Germany) and then treated with UV (American Ultraviolet AL-PVC-160W, Lebanon, IN 46052, USA).

Experimental Design
For each experiment, the copepods were fed with only one of the three experimental diets at a density of 200,000 cells/mL. The experimental diets were: Iso, I. galbana (2400 µg C/L); Ch, C. calcitrans (3100 µg C/L); and Iso + Ch, I. galbana plus C. calcitrans (1200 + 1550 µg C/L). Assays were used to determine the required concentration of microalgae before conducting the experiments. All cultures were obtained at 28 ± 0.5 • C, 35 PSU salinity, 125 lux, and a 12:12 photoperiod, with a slight and constant aeration in UV-treated filtered (0.45 µm) seawater. The algal concentration was measured daily in all cultures using a Neubauer chamber (mean of six-cell counts), and corresponding volumes of micro-algae were administered to maintain stability in the experimental concentration.

Population and Individual Growth and Ingestion Rate
To begin with, 10 sexually mature pairs of O. nana were placed into five 1-L flasks, each containing 800 mL of filtered seawater. The experimental conditions were kept constant for 16 days, with manual shaking every 12 h from the third day onwards. Finally, the population of each experimental unit was harvested using a 50 µm sieve and preserved in a 3.7% formalin solution of 10 mL to be counted later by stage (nauplii, copepodites, adults: male and female) in a Sedgwick-Rafter chamber. The total length (prosome + urosome) of 600 individuals, collected from each flask (150 per stage), was measured with a microscope (Optika B-500Ti, OPTIKA S.r.l., Ponteranica, BG, Italy) equipped with a previously graduated micrometer eyepiece. Microalgae consumption was measured in 240 adults of O. nana between 24 to 48 h in the 1 L flasks, under the same conditions and at the same time as the growth experiment. This data was compared to the growth of single algae cultures, to assess the actual ingestion of microalgae. The ingestion rates (I, cells/mL h) were determined according to equation, indicated by Frost [60]: where, C1 and C2 are the concentrations of microalgae (cells/mL), N is the number of copepods, for a duration of 24 h.

Number of Spawning and Fertility
Females who had reached their first sexual maturation (i.e., possessing ovigerous sacs) were isolated from a stock culture and placed in a 100 × 20 mm Petri dish filled with 40 mL of media (one individual per dish), with single or mixed algal food at the experimental density, and sealed with parafilm. The experiment was carried out in triplicate. When the females spawned, the nauplii that were released were quickly collected and placed on a new plate with a new medium. The number of spawning events for each female was recorded over the course of 20 days. We counted the nauplii for each spawn in each treatment to determine female fertility (number of nauplii per spawning).

Development Time by Stage and Sex Ratio
One egg-bearing female was removed from a stock culture and placed in a Petri dish with 40 mL of culture media, with single or mixed algal food at the experimental density. The experiment was carried out in triplicate. The female was examined twice a day to determine when the sac ruptured and the nauplii were released, and at this point, the female was then transferred to another Petri dish with the culture medium, leaving the nauplii alone. The development time by stage was determined by examining the Petri dishes containing nauplii every 12 h for 20 days under a stereo microscope, differentiating the molts until they reached adulthood, when sexual dimorphism was recognizable. The sex ratio (male/female) was calculated based on the number of adult copepods.

Analysis of Data
The normal distribution of the data was determined using the Shapiro-Wilk test, and the variance homogeneity was determined using the Levene test. The (t-test for independent samples was used for the single algal ingestion rate and a one-way ANOVA was used to obtain the other data. A Tukey HSD test was used for results that were found to be significant (p < 0.05). When the data did not meet the statistical assumptions of ANOVA, the Kruskal-Wallis test was applied, and non-parametric multiple comparisons of the p-values were performed for significant results (p < 0.05) [61]. Statistical analyses were performed with STATISTICA 10.0 (StatSoft, Tulsa, OK, USA).

Population and Individual Growth and Ingestion Rate
The population density on day 16 did not differ significantly between treatments (Figure 1), nor did the densities in the percentage of nauplii, copepodites, adult female, and male stages ( Table 2). Tables 3 and 4 show the length values of O. nana in the minimummaximum intervals for each stage fed with different diets. Furthermore, there was no difference in the average length between the experimental groups. The ingestion rates, measured over 24 h, revealed no differences between single algae diets ( Table 3).

Number of Spawning and Fertility
The number of spawnings of O. nana during a 20-day period differed significantly depending on the diet supplied (p < 0.05). The single I. galbana diet resulted in the lowest number of spawnings. Similarly, the number of nauplii per spawning showed the lowest values for the I. galbana diet (p < 0.05) ( Figure 2).      N.E. Adriatic sea 520 520 [69] Fresh and decomposed algae + Navicula

Development Time by Stage and Sex Ratio
The development time per stage of O. nana during the 20-day period showed a significant difference in all stages (p < 0.05). In the nauplii phase, the Iso diet provided the shortest time (p < 0.05), while in the copepod phase (p < 0.05), the Iso and mixed diets provided a shorter development time, and thus a shorter time to obtain adult copepods. At the end of the experiment, the proportion of sex did not differ between treatments (p < 0.05) ( Table 5).

Discussion
Our study provided information on the growth parameters of O. nana under specific, constant conditions as recommended by Kaviyarasan et al. [52] and Cornwell et al. [70]. As a result, we have a better understanding of how this species may be successfully cultured.

Population Growth
An evaluation of how diet effects the growth of copepod populations provides information for their potential use in aquaculture [71,72]. This was confirmed for O. nana using the oceanic algae Nannochloropsis in experiments at 2 × 10 6 cells/mL and 5 × 10 6 cells/mL [46]. Similar results were reported for O. rigida [73]. In our study, single and mixed diets with I. galbana and C. calcitrans did not affect the final population of O. nana. This could indicate the easy adaptation of this copepod to these algae. Chilmawati and Suminto [3] reported results similar to ours; they reported maximum population densities of 6.96 ± 0.38 ind/mL with C. calcitrans (2 × 10 6 cells/mL) and 6.28 ± 0.25 ind/mL with I. galbana (2 × 10 6 cells/mL) in Oithona sp.
In our study, we did not find significant differences among experimental algae diets, meaning that O. nana can be cultivated with single and mixed diets. In addition, the use of a single diet means less algae rearing hatchery work and diminishes possibility of cross-contamination in copepod cultures vessels. According to Santhanam et al. [24], I. galbana (30,000 cels/mL) alone is sufficient in the diet for O. rigida due to the high content of DHA, and the high DHA:EPA ratio required for its proper growth. The positive effect of Iso and Ch as single diets with a high content of DHA and EPA, respectively, could be attributed to the synthesis and bioaccumulation capacity of highly unsaturated fatty acids (HUFA) in copepods [8,[74][75][76]. This biotransformation of essential fatty acids has been demonstrated for cyclopoid copepods [77,78], calanoid copepods [75,79], and harpacticoid copepods [74,80].
Because organisms' behavior and growth can vary even within the same taxonomic group, a mixed algal diet may be preferable for rearing copepods such as O. nana, which has six naupliar phases and five copepodite stages [81]. However, because the aim of our study was to measure copepod production and the growth time between stages as a function of food, we did not characterize each stage by each growth stage.
In terms of the overall population, the final density of copepods obtained in our experiments (5.25-6.61 ind/mL) is lower than that indicated by Magouz et al. [46], who reported concentrations of more than 9 ind/mL, grown on an alternative cornstarch diet. This difference in densities of the final population could be attributed to the initial inoculum density, which included 10 adult female O. nana at their first sexual maturity per 800 mL in this study, as opposed to Horne et al. [81] and Magouz et al. [46], who started with 1875 nauplii/L and 1000 ind/L, respectively. Oftentimes, live feed laboratories start their mass cultures with high concentrations because it is sufficient for harvesting higher densities. However, to avoid overcrowding and negative outcomes, the maximum rearing densities must be determined for this species. High rearing densities are known to have a negative impact on growth, behavior [82], fertilization [83], adult cannibalism of nauplii [2,[84][85][86][87], female reproductive capacity, egg hatching rate, and to induce the collapse of the reared stocks [84], causing the nauplii population to decline. Holm et al. [88] reported that maintaining a high density of O. nana males minimizes mate-seeking behavior after a period of competition for food and starvation, which also may limit the growth of the species [89]. To avoid this behavior, it would be necessary to first conduct high stoking density experiments with O. nana to determine whether a problem exists in the mass culture.

Individual Growth
Diet plays an important role in determining copepod size [90]. Our data showed no difference in body size between O. nana that were fed single and mixed algae diets. This is not the case in other cyclopoids, such as Paracyclopina nana, which were found to have larger body sizes when fed mixed diets [91]. The body size range for each stage of O. nana recorded in this study corresponds to the sizes reported in natural and cultured conditions by other authors, indicated in Table 4.

Ingestion Rate
The ingestion rate is an important metric through which to determining copepod food preferences in both their environment and under laboratory conditions [92,93]. Even though C. calcitrans had larger dimensions and a larger biovolume than I. galbana ( Table 1), adults of O. nana did not show a difference in ingestion rate in our study. This could indicate a preference for specific nutrients found in both microalgae. However, because developing O. nana nauplii may have difficulty consuming and digesting the diatom C. calcitrans [3]-perhaps due to its long setae-they were not considered in this evaluation.
Species of Oithona are classified as diet-mixed consumers; adults can be carnivorous [94]. Carnivory was confirmed for adults of O. nana when supplied with Acartia clausi nauplii [95]. According to Lampitt and Gamble [47], O. nana can consume phytoplankton or nauplii in monospecific diets in the range of 1-300 um, as well as non-traditional foods such as soybeans, yeast, rice bran, and corn starch [46]. Thus, O. nana possess a high adaptability to consume a wide variety of foods.
According to our findings ( Table 3), adults of O. nana consumed high amounts of microalgae, as evidenced by the carbon consumption of 34.07 ng C/ind/day for I. galbana and of 56.99 ng C/ind/day for C. calcitrans. These values are higher than those reported by Lampitt and Gamble [47], who evaluated the diet of the following microalgae, ranging in size from 2.7-10 µm to 10-67 µm: I. galbana and Dunaliella euchlora, Cricosphaera elongata, Thalassiosira fluviatilis, and Prorocentrurn micans reported a consumption of 9.2 ng C/ind/day with I. galbana and the highest consumption of 51.5 ng C/ind/day with Cricosphaera elongata, at an ambient temperature of 10 • C. In our case, we used a higher temperature (28 ± 1 • C), which could have resulted in a greater rate of ingestion of smaller microalgae. Another factor that may lead to the reduced ingestion of tiny cells is hydrodynamic disruption, which has been found in the culture of other species to alter swimming and food perception [87].

Number of Spawning
The development rate, longevity, and fertility of copepod are all influenced by the type and availability of food [96,97]. In our experiment, single and mixed diets containing Chaetoceros sp. produced greater spawning, perhaps due to the high concentration of polyunsaturated fatty acids [98]. According to Lora-Vilchis et al. [99], unlike I. galbana, Chaetoceros sp. has a high concentration of HUFAs and approximately five times the content of EPA (Table 1). However, to achieve optimal fertility, the 2:1 DHA/EPA ratio must be considered in copepod diets [96].
In our cultures, O. nana spawned at a high frequency during periods of 1 to 4 days when cultivated at 28 • C, with longer periods as the females' ages increased. Haq [68] reported that embryonic phase of O. nana in cultures with Phaeodactylum tricornutum was 4 to 5 days at 18 • C and 2.5 to 3.5 days at 20 • C, and Temperoni [100] reported times of 3.22, 2.37, and 2.05 days for this species in oceanographic sampling at 12, 17 and 21 • C, respectively. Hence, we found that temperature influences embryonic and reproductive development in Oithona [36]. Given these data, it would be appropriate to cultivate O. nana at temperatures that allow it to adapt to local hatchery conditions, ideally at 28 • C, which corresponds to the temperature detected during O. nana collection on Morro Sama's rocky beaches.
The high temperature of 28 • C may have contributed to the fact that O. nana was the only copepod species found at the sampling site, which allowed us to obtain a monospecific sample. In addition to being an organism found worldwide, O. nana can also be categorized as a eurytherm, as it has been found in South America at temperatures ranging from 18.5 to 37 • C [33].

Fecundity
According to Støttrup and Jensen [101], the size, quantity and quality of food, have a direct influence on the number of copepod eggs produced per female. Our results show that the mixed diet produced the most nauplii of O. nana per egg laying event. On the contrary, Chilmawati and Suminto [3] found no differences in egg production in Oithona sp. utilizing four single algae diets: 1-2 × 10 6 cells/mL for C. calcitrans and I. galbana, and 2-4 × 10 6 cells/mL for Chlorella vulgaris and Nannochloropsis oculata. This could be because different algal diets produce different reproductive responses in copepods [102]. Suminto et al. [103] successfully tested the inclusion of organic ferments in single diets with C. calcitrans (2 × 10 6 cells/mL) in Oithona sp. to enhance egg production, and thus the number of nauplii produced. Similarly, Chilmawati et al. [104] obtained a higher level of egg production in O. similis fed with an organic feed containing 30% protein.
The number of nauplii per liter produced at 28 • C in our cultures was greater than that reported by Haq [68], who used the diatom Phaeodactylum tricornutum. Haq found 21, 22, 15, and 6 nauplii/L of cultured O. nana at 11, 18, 18 and 20 • C, respectively. This demonstrates that nutrition and rearing temperature affect both the number of nauplii per liter and the number of spawning events. However, as shown for the herbivorous copepod, P. crassirostris, eggs can be damaged or eaten by O. nana [47], which might cause issues in nauplii production when the density of adults increases [105].

Development Time Per Stage
Food quality clearly affects growth of copepods [92]. Our results indicate a longer copepod development period when they are fed with a monospecific diet of C. calcitrans. The nauplii and early stages of copepodites were unable to digest this diatom due to their diameter and cell wall composition. On the other hand, I. galbana microalgae does not have rigid cell wall, which, coupled with its smaller size may be important for food ingestion and digestion by nauplii. Of course, as the organisms grow, they may consume larger cells, thereby providing them with more energy. We found that nauplii required 4 to 7 days to grow, whereas copepodite required 2.33 to 4.67 days. Conversely, Murphy [48] demonstrated the requirement of 26 days for growth in the naupliar stage and 28 days for the copepod stage of O. nana fed with fresh and decomposed algae diets with the addition of Navicula. This would mean that diets that do not satisfy the nutritional requirements of the copepod could lead to a prolonged development time in copepods. Due to the identification of non-optimum temperatures, the results must be corroborated by additional studies, i.e., by determining the temperature coefficient (Q10) and thermal dependence. For example, Almeda et al. [106] report a Q10 of 2.4 for O. davisae nauplii cultured between 12 and 28 • C. Species in the genus Oithona have a short development time, for instance, Oithona rigida cultured at 28 • C presented development times from 3.8 to 5.2 days for nauplii and 7.1 to 8.4 days for copepodites, both fed with I. galbana at 40 × 10 3 cells/mL [24].

Sex Ratio
Various studies report that the sex ratios obtained in zooplankton are not of a ratio of 1:1 [107]. Although there is a propensity for a higher percentage of females than males in O. nana, little variation was observed in the sex ratio in our experimental treatments ( Figure 2 and Table 5). Temperoni [100] found that females outnumbered males in more than 90% of his oceanographic samplings, with females outnumbering males by 60 to 100% of the total population of O. nana. This also coincides with the results reported by Holm et al. [88], who claim that the male to female ratio is even lower than 20% under growing conditions. Temperature [81] and salinity [44,108,109] are two potential environmental factors that may influence the development of O. nana, and manifest in the predominance of one of the sexes. Another possibility that has been reported is the change of sex in planktonic copepods during their development under adverse conditions [107], which is primarily related to quantity [110] and food quality [111]. Although there have been no reports of a change in sex for O. nana, we recommend additional research aimed at increasing fecundity and the proportion of females. In this way, the cultivation of O. nana can increase in output and productivity.
Although Murphy [48] and Haq [68] were the first to study O. nana in culture, their objectives were to develop an understanding of the morphological description of the life cycle and the nauplii stage, respectively. Following their study, only a few studies used biological parameters to assess the viability of their cultures. Our findings are significant because they demonstrate the possibility of culturing O. nana with two microalgae species that are often used in aquaculture production facilities. They are also proof of the metabolic adaptability of O. nana in obtaining and assimilating essential nutrients from different diets to perform its complete biological cycle. This could indicate a bioaccumulation possible bioconversion of essential nutrients at various stages of development, which is important in the transfer of these compounds to higher levels of the food web. These factors have been reported on for other groups of copepods, but have yet to be investigated for O. nana.

Conclusions
Our study provides valuable information about culturing the copepod O. nana in small-scale settings. We found that I. galbana and C. calcitrans are suitable food sources across all development stages of O. nana. This constitutes an advantage for producing live feed for copepods that cultivate both species of microalgae as food for zooplankton. The next logical steps are to (1) assess growth while varying conditions of temperature, food quality and quantity, and water chemistry and (2) scale the culture. Institutional Review Board Statement: Our work does not apply the evaluation of the methodology by an ethics committee, because we, from a water sample, select a zooplanktonic organism (copepod) and cultivate it under controlled conditions, similar to those reported. in its natural environment. The intention of our work was to know its biological processes, to propose it as a potential live food for the larval phase of fish, for possible future work. (In this work we did not work with fish). No morphological or genetic modification was made, and no attempt was made to feed with food that is not part of their natural diet.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author. The data are not publicly available for privacy reasons.
Acknowledgments: This work constitutes the thesis research to qualify for the degree of Magíster en Acuicultura mención Cultivo de Recursos Hidrobiológicos, from the la Universidad Arturo Prat, Arica, Chile. We thank to the Fondo Nacional de Desarrollo Pesquero by the rearing facilities provided in the Centro de Acuicultura de Morro Sama, to O. Morales-Olín for supplying the microalgae. We thank the SEA-SOS network and anonymous reviewers for their suggestions and recommendations that have improved the quality of the manuscript.

Conflicts of Interest:
The authors declare no conflict of interest.