Journal of Marine Science and Engineering Article Small Jellyfish as a Supplementary Autumnal Food Source for Juvenile Chaetognaths in Sanya Bay, China Lingli Wang 1,2,3, Minglan Guo 1,3, Tao Li 1,3,4, Hui Huang 1,3,4,5, Sheng Liu 1,3,* and Simin Hu 1,3,* 1 CAS Key Laboratory of Tropical Marine Bio-resources and Ecology, Guangdong Provincial Key Laboratory of Applied Marine Biology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510301, China; [email protected] (L.W.); [email protected] (M.G.); [email protected] (T.L.); [email protected] (H.H.) 2 College of Earth and Planetary Sciences, University of Chinese Academy of Sciences, Beijing 100049, China 3 Innovation Academy of South China Sea Ecology and Environmental Engineering, Chinese Academy of Sciences, Guangzhou 510301, China 4 Tropical Marine Biological Research Station in Hainan, Chinese Academy of Sciences, Sanya 572000, China 5 Key Laboratory of Tropical Marine Biotechnology of Hainan Province, Sanya 572000, China * Correspondence: [email protected] (S.L.); [email protected] (S.H.) Received: 10 October 2020; Accepted: 19 November 2020; Published: 24 November 2020 Abstract: Information on the in situ diet of juvenile chaetognaths is critical for understanding the population recruitment of chaetognaths and their functional roles in marine food web. In this study, a molecular method based on PCR amplification targeted on 18S rDNA was applied to investigate the diet composition of juvenile Flaccisagitta enflata collected in summer and autumn in Sanya Bay, China. Diverse diet species were detected in the gut contents of juvenile F. enflata, including copepods, small jellyfish, anthozoa, polychaetes, echinoderms, diatoms and dinoflagellates. The diet composition showed obvious differences between summer and autumn. Copepod, such as Temora turbinata, Canthocalanus pauper and Subeucalanus crassus, dominated the diet in summer, representing up to 61% of the total prey items. However, small jellyfish, mainly consisting of Bougainvillia fulva, Solmissus marshalli and Pleurobrachia globosa, was the main food group (72.9%) in autumn. Environmental parameters showed no significant difference between summer and autumn. The mean abundance of juvenile chaetognaths in autumn was about eight times higher than that in summer, while the abundance of potential food prey was similar in both seasons. Our results suggested that juveniles chaetognaths might consume small jellyfish as a supplementary food source under enhanced feeding competition in autumn. Keywords: juvenile chaetognaths; diet; small jellyfish; supplementary food; tropical bay 1. Introduction Chaetognaths are among the most abundant macro-zooplankton in coastal ecosystems worldwide. They contribute 5% to 15% of the total zooplankton biomass [1]. As main predator of mesozooplankton, such as copepods and cladocerans, chaetognaths play a central role in the planktonic food web [2,3]. They exert considerable influence on the population dynamics of their prey groups by predation [4,5]. Besides which, chaetognaths are also a food source for fish [6–8]. Except for the link role between small zooplankton and top predators, chaetognaths also play an essential role in the biogeochemical cycling by making a substantial contribution to vertical carbon flux through producing large, fast-sinking fecal pellets [9,10]. Simultaneously, chaetognaths can also be competitors of fish larvae because they feed on similar preys [11,12]. Thus, it is important to study population dynamics of chaetognaths and analyze the related influencing factors. J. Mar. Sci. Eng. 2020, 8, 956; doi:10.3390/jmse8120956 www.mdpi.com/journal/jmse J. Mar. Sci. Eng. 2020, 8, 956 2 of 20 Chaetognaths are usually present year-round and reach their highest densities in some seasons. For example, in the Ionian Sea and the Cretan Passage, chaetognaths reach the highest densities in autumn after the dominant species F. enflata breeds in late summer [2,13]. The highest abundance could 3 reach 310 ind m− in October in the Gullmar fjord [14]. Among the most abundant period, juvenile individuals (mostly stage I) contribute to almost 64.9%–97.7% of total chaetognath abundance [15]. Several studies showed that the high abundance of juvenile chaetognath usually coincides with the occurrence of large numbers of their food (mostly copepods) [16–20]. As food was believed to be one of the most critical parameters impacting the dominance of chaetognath juveniles, it is essential to study their feeding strategy to better understand the mechanisms sustaining a thriving chaetognath population in some seasons. Small copepods/copepod nauplii were considered to be the main prey for juveniles chaetognath [2,11, 17,21,22]. Also, non-copepod prey such as tintinnids and rotifers were reported to be important in the diets of juvenile chaetognaths in the South Atlantic Bight when they reach a high abundance to obtain sufficient energy [11,23,24]. Moreover, several studies suggest that chaetognaths can feed on detritus (or marine snow) when high population abundance occurred [25–28]. Therefore, exact dietary analysis of chaetognaths, especially juveniles, is essential to understand their food source sustaining such high abundance. However, methodological limitations result in biased inferences on the composition of juvenile chaetognaths diets, because of low fractions of foods [2,29], and large amounts of “unidentified food items” (their sizes (mostly < 6 mm) in their guts) [30–32]. Besides this, the unidentified food in diet of chaetognath juveniles (stage I of F. enflata and Parasagitta setosa) could reach as high as 35.1% [31,33]. Molecular methods based on DNA markers provide a powerful tool to resolve this problem by retrieving DNA fragments and accurately identifying their origin from partially digested or broken pieces of food items [34]. Further, this method is particularly suitable for diet identification of small-sized zooplankton, such as copepods and larval fish, which are difficult to process for gut dissection [35,36]. Bonnet et al. (2010) successfully detected copepod Calanus helgolandicus in the gut content of chaetognath Sagitta setosa using a Calanus specific primers [37], indicating that a molecular method can be effective when unraveling the diets of chaetognaths. Sanya Bay is a typical tropical bay in the north of South China Sea, characterized by abundant marine resources and high biodiversity [38]. Chaetognaths are distributed widely in Sanya Bay, and the highest abundance usually occurs in summer and autumn. In autumn, chaetognaths could reach 3 an abundance of 79.68 ind m− , with F. enflata being the most dominant species accounting for as high as 90% of the total chaetognath abundance, especially juvenile individuals [39,40]. Meanwhile, the dominant food for chaetognaths, especially copepods, was relatively low during autumn [40]. As food was believed to be one of the most critical parameters impacting the survival of chaetognath juveniles, we hypothesized that there might be other food sources other than already known preys to sustain a high abundance of juvenile-dominated chaetognath community in this tropical bay. Therefore, we analyze the diet composition of juvenile F. enflata collected from summer and autumn in Sanya Bay by molecular method, with the purpose of revealing the potential resource supporting the high abundance of chaetognaths based on the precise food detection of the molecular method. 2. Materials and Methods 2.1. Sample Collection Sampling was conducted at the intermediate zone (W3 station) and off-shore areas (W4 station; W9 station) of Sanya Bay in July 29 and October 26 of 2014 (Figure1)[ 41]. The water depth was ~19 m in W3, 25 m in W4 and 28 m in W9. J. Mar. Sci. Eng. 2020, 8, 956 3 of 20 Figure 1. Sampling stations in Sanya Bay separately visited on July and October, 2014. Physical and chemical parameters in the environment (temperature, salinity, dissolved oxygen concentration, pH and dissolved organic carbon concentration) were measured using the YSI6600 Water Quality System. Chaetognath juveniles were collected using a cylindro-conical net (50 cm aperture, 145 cm height, and 505 µm mesh size) towed vertically from the bottom to the surface without replicate. The towing 1 speed was 1 m s− . To prevent any possible changes in chaetognath juvenile gut content as an artifact of sampling, collected samples were fixed after transfer to the bottle by adding neutral Lugol’s solution at 2% final concentration. All sampling and preservation processes were completed within two minutes. Neutral Lugol’s (no acetic acid added) had been shown to effectively preserve samples for DNA analysis in zooplankton [42,43]. Other sets of zooplankton samples were collected and preserved in a 5% formalin-seawater solution for species identification using stereomicroscope. The sample IDs were as follows: W3-Jul-J, W4-Jul-J, W9-Jul-J, W3-Oct-J, W4-Oct-J and W9-Oct-J (Wx means the sample station, Jul means sample was collected in July, Oct means sample was collected in October, J means F. enflata juveniles). 2.2. Zooplankton Identification and Statistical Analysis To obtain the information of potential food organisms, zooplankton samples were split using a Folsom splitter as they were abundant, and then were identified and counted under the 3 stereomicroscope [2]. Zooplankton abundance was expressed as individuals per cubic meter (ind m− ). One-way ANOVA analysis in SPSS22.0 data analysis software was used to test for significant difference of environmental parameters. 2.3. DNA Extraction of F. enflata Juveniles F. enflata juveniles (in stage I which had no visible ova) were identified and sorted using a wide-bore plastic pipette under stereomicroscope Leica S8APO in the laboratory [44–46]. The length of juvenile specimens used in this study were in the range of 3–6 mm. To avoid artifacts of prey J. Mar. Sci. Eng. 2020, 8, 956 4 of 20 items from cod-end feeding, 1/3 of the forward gut of all chaetognaths under examination was cut and thrown away.