Providing palatable nutritional food of appropriate size to marine larvae at different development stages is one of the most challenging tasks in marine hatcheries. For some larvae with small mouth gapes, the traditional live prey of rotifers (mean length 135 μm of SS-type rotifer Brachionus rotundiformis) and Artemia nauplii (typically between 400–500 μm) could be too large to be ingested by them (Fielder et al., 2000; Assavaaree et al., 2003; Gopakumar and Santhosi, 2009; Wullur et al., 2009; Conceição et al., 2010; Lindley et al., 2011; Hagiwara et al., 2014), and lacking of appropriate live prey to feed such larvae has been a crucial technical bottleneck. Copepods are the natural food for most marine larvae and nauplii of many copepods are less than 100 μm in size, which can meet the first feeding requirement of even small mouth- gaped fish larvae (McKinnon et al., 2003; Camus and Zeng, 2010; Bradley et al., 2013; Hill et al., 2020). Moreover, copepods typically develop through six naupliar and six copepodite stages with each stage of different size, making them suitable prey for fish larvae with different sizes of mouth gapes, as well as at different developmental stages (Schipp et al., 1999).

Several paracalanid species, including Parvocalanus crassirostris (Alajmi and Zeng, 2015) and Bestiolina similis (McKinnon et al., 2003; Camus et al., 2009; VanderLugt et al., 2009; Camus and Zeng, 2010), have been recommended as good candidates as larvae prey for fish larvae with small mouth gape. B. amoyensis, a species belonging to the same genus of B. similis, has been reported to co-exists with B. similis in the tropical coastal and estuary waters (McKinnon et al., 2003). The species distributes widely in Pacific Ocean, especially in western Pacific Ocean, and it is a dominant species during autumn (August – November) in subtropical Xiamen Harbor and Dongshan Bay of China (Yang, 2007; Zheng, 2009). However, no attempts have been made to culture the species as the live prey for feeding fish larvae previously.

Comparing to the traditional live prey, the main problem of copepods as live feed for larval culture is their difficulty for intensive culture and/or low culture productivity. Therefore, optimizing the culture conditions has been the focus of previous research. Many factors could affect copepod culture reproductivity and past research has been focused on temperature, salinity, and both quality and quantity of their food (e.g., Holste and Peck, 2006; Camus et al., 2009; Camus and Zeng, 2010; Nogueira et al., 2017; Nguyen et al., 2020; Choi et al., 2021; Dayras et al., 2021), while the effects of photoperiod is less studied despite photoperiod has been shown to significantly affect culture productivity of two Acartia species, i.e., Acartia tonsa (Peck and Holste, 2006) and Acartia sinjiensis (Camus and Zeng, 2008). Photoperiod is well known as one of the most important environmental factors regulating reproductive activity of aquatic animals, including copepods, and it has been reported to influence number of eggs produced by females, as well as the type (i.e., subitaneous vs. diapause eggs) of eggs produced (Marcus, 1982; Peck and Holste, 2006; Camus, 2012; Fereidouni et al., 2015). Furthermore, the physiological “clock” of aquatic animals allows them to response to changes in light condition, which could affect their feeding and reproductive activity (Miliou, 1992). For example, marine zooplankton, especially copepods, are known to undertake diurnal vertical migration (Haney, 1988) with a typical feeding pattern showing higher ingestion rate during night (Fuller, 1937; Petipa, 1958; Dagg and Grill, 1980; Stearns, 1986; Saito and Taguchi, 1996; Calbet et al., 1999), and some calaniod copepods, such as P. crassirostris (Sun, 2008) and Calanus (Harding et al., 1951; Zhang, 2003), spawn occurs more frequently at night. Hence, the goal of the current study was to evaluate the effects of photoperiod and microalgae feeding concentration and their interaction effects on culture productivity of B. amoyensis, seeking to answer the questions such as is there an optimum photoperiod regime for the cultivation of B. amoyensis? Whether there exist interactions between algae feeding concentration and photoperiod? For instance, whether longer darkness hours leading to enhanced egg production may require more abundant food supply to sustain higher productivity?

Reproductive ability of copepods is affected by the nutritional condition of mature females (Castro-Longoria, 2003), and food quantity is an important factor that can significantly affect female condition. For example, the egg production has been reported to typically increase with the food concentration, reach a maximum level at a certain concentration (Runge, 1984; Hirche et al., 1997; Camus, 2012), but tend to decrease when the food concentration exceeds a threshold level (Zhao et al., 2014). Therefore, determining the optimal microalgae feeding quantity is crucial for optimizing the culture protocol of B. amoyensis.

Most of paracalanid copepods are herbivorous (Paffenhöfer, 1984) although a few of them have been reported as could be carnivorous (Suzuki et al., 1999). Similar to its congener B. similis (Vineetha et al., 2018; Siddique et al., 2021), we found B. amoyensis as herbivorous, feeding mainly on phytoplankton. In the present study, the microalgae Isochrysis spp., one of the most commonly used marine unicellular algae for feeding calanoid copepods (Payne and Rippingale, 2000; Vengadeshperumal et al., 2010; Santhanam et al., 2013; El-Tohamy et al., 2021), was confirmed could fulfill the growth and reproduction needs of B. amoyensis. Isochrysis spp. is known for their high nutritional value, such as rich in polyunsaturated fatty acids (PUFAs), e.g., DHA and EPA (Sukenik and Wahnon, 1991; Gouveia et al., 2008) and grows well in mass cultures, either indoors or outdoors (Kaplan et al., 1986; Fidalgo et al., 1998). When at exponential phase of growth, the flagellated microalgae are not easily accumulated at the bottom like some other microalgae species, such a feature benefited the present experiments: the concentrations of Isochrysis spp. largely remained at designated levels during the 24 h period between daily water changes as confirmed by intermittent sampling of culture waters from replicates to estimate the algal concentrations from different treatments (see Supplementary Table 1).

Copepods productivity has commonly been indicated by daily egg production of females (Klepper et al., 1998). However, Camus and Zeng (2010) have adopted total egg production over female lifespan to estimate female productivity, and noted that this method is more accurate since it takes female life expectancy and reproductivity changes during lifespan into account. In this study, we have measured both daily egg production and egg production over female lifespan to provide a comprehensive information on egg production of B. amoyensis under different culture conditions. Our results showed that under all 3 photoperiods used, average daily egg production of B. amoyensis was significantly higher when they were fed Isochrysis spp. at a concentration ≥3 × 10⁵ cells ml^–1 when compared to that at ≤2 × 10⁵ cells ml^–1. In the case of total egg production over female lifespan, under all photoperiods used, the lowest total egg production was found in the lowest algal concentration of 1 × 10⁵ cells mL^–1, and total egg production increased by the algal concentration, reaching a maximum level at 4 × 10⁵ cells mL^–1 before decreased with further increase of food concentration. Similar results were also reported for other copepod species, such as Calanus pacificus (Runge, 1984), Calanus finmarchicus (Hirche et al., 1997), and Tisbe furcate (Zhao et al., 2014). Such a result likely can be explained by that when food concentration is low, copepods need to move actively to catch more algae and with increase movement frequency of feeding appendage to enhance feeding (Paffenhöfer and Lewis, 1990), which increases energy expenditure. As the result, less energy can be channeled for egg production. Indeed, it has been observed that in some calanoids, clearance rate, i.e., the water volume sweeps clear by a copepod per unit time, increased with decreasing food concentration (Corner et al., 1972). Clearly, clearance rate increase is associated with higher energy expenditure. With higher food concentrations, copepods could obtain enough food with a lower clearance rate, which reduces energy consumption while still meeting the energy demands for both maintenance and reproduction (Zhao et al., 2014). However, when the food concentration is too high, it was found that microalgae could adhere to the feeding appendages and body surface of copepods, which could impair their feeding and motility, leading to lower food ingestion, and subsequent lower egg output and shorter female life expectancy. While Garrido et al. (2013) found that ingestion rate of calanoid copepod Centropages chierchiae increased linearly with the range of food concentrations they used (6.4–393.8 μC L^–1), but out results suggest that the ingestion rate of B. amoyensis does not increase infinitely with food concentration and a threshold exists, beyond which the clearance rate would not further increase, or even decrease.

Female life expectancy is one of the factors that could affect how much a female contributes to the total pool of eggs produced in the culture, and hence culture population growth. When food concentrations were ≥3 × 10⁵ cells mL^–1, adult females cultured under 16 h L:8 h D photoperiod had a shorter life expectancy than other two photoperiods. The prolonged light phase likely kept B. amoyensis active for longer, and the higher metabolic rate may result in shorter female lifespan. Similarly, A. sinjiensis cultured under constant light had a relative shorter life expectancy than other photoperiods (Camus and Zeng, 2008).

The results of population growth experiment showed that feeding on Isochrysis spp. alone, B. amoyensis could significantly expanded its population at all concentrations tested, ranging from 1 × 10⁵ to 5 × 10⁵ cells ml^–1 and under different photoperiods. Interestingly, at all 5 algal feeding concentrations, B. amoyensis cultured under 12 h L:12 h D photoperiod consistently had the maximum and significantly bigger population sizes at the end of the experiment than other two photoperiods, indicating the important of photoperiod in regulating population growth. A possible explanation of such a result is that 12 h L:12 h D mimics typical photoperiod of Xiamen region during autumn when B. amoyensis reportedly is a dominant copepod species in Xiamen Harbor of China (Yang, 2007) where B. amoyensis used in this study were collected from. Indeed, the photoperiod of Xiamen region in spring and autumn ranges from 12 h L:12 h D, hence B. amoyensis may have adapted to such a photoperiod condition for maximum production. Indeed, under 12 h L:12 h D photoperiod, female B. amoyensis typically had the longest life expectancy among 3 photoperiods except at 1 × 10⁵ cells ml^–1 algal concentration, which was also not significant different from the longest life expectancy under 16 h L:8 h D. This should also lead to higher overall egg production while suggesting that 12 h L:12 h D is in fact optimal photoperiod for B. amoyensis.

The relative numbers of nauplii and copepodites in a culture population typically indicate the status of the culture (VanderLugt et al., 2009). VanderLugt and Lenz (2009) pointed out that populations dominated by nauplii typically indicate high reproductive rates of females. The maximum numbers and proportion of nauplii in the final populations of the population growth experiment were also found from the 12 h L:12 h D photoperiod, and at all 5 algae concentrations exceeding 50% of the total population. In particular, the number of nauplii of the 4 × 10⁵ cells mL^–1 treatment achieved 75% (Figure 3), indicating that B. amoyensis had a high reproductive rate under 12 h L:12 h D photoperiod and when fed Isochrysis spp. at 4 × 10⁵ cells mL^–1.

Sex-determining in copepods are related to the population size and sex ratio (Mauchline, 1998; Voordouw and Anholt, 2002). In planktonic calanoid copepods, sex may also be controlled by environmental factors (Mauchline, 1998). The female-biased adult sex ratios had been reported in many species, including Acrocalanus sp., Parvocalanus sp., Paracalanus indicus, B. similis (McKinnon and Duggan, 2001; McKinnon et al., 2005; Duggan et al., 2008; Gusmão and McKinnon, 2009), Paracalanus nanus (McKinnon and Duggan, 2001; Gusmão and McKinnon, 2009), and Paracalanus aculeatus (McKinnon and Duggan, 2001; McKinnon et al., 2005; Gusmão and McKinnon, 2009). The sex ratios of Calanus spp. vary seasonally, and the food quality and quantity could also significantly affect sex ratio. For example, more males could be observed during the phytoplankton boom season comparing to the pre-phytoplankton boom season (Irigoien et al., 2000). Similarly, there were more male B. amoyensis found in the populations of 4 × 10⁵ cells mL^–1 treatments under 12 h L:12 h D photoperiod with the sex ratio approaching 1:1, indicating the culture population was at a stable status (Fisher, 1930) under the culture condition.

The results of this study showed that Isochrysis spp. is a suitable diet for B. amoyensis culture as the population expanded significantly when it is fed to the copepod at concentrations ranging from 1 × 10⁵ to 5 × 10⁵ cells ml^–1, and under photoperiods of 8 h L:16 h D, 12 h L:12 h D and 16 h L:8 h D. However, summarizing the outcomes of a range productivity related parameters measured in B. amoyensis, including egg production, female life expectancy, and population growth and composition, the optimum Isochrysis spp. feeding concentration and photoperiod for the culture productivity of the species was determined to be 4 × 10⁵ cells mL^–1 and 12 h L:12 h D, respectively.

The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author.

SW and LW conceived the original idea of this study and designed the experiments. LW conducted the experiments and collected the data. LW, SW, and CsZ performed the data analyses. LW wrote the draft of the article and revised it with SW and CsZ. YW and CxZ attended the field works and performed part of data collection. SW supervised the research. All authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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