Possible Aplanochytrid (Labyrinthulea) Prey Detected Using

Plankton Benthos Res 13(2): 75–82, 2018 Plankton & Benthos Research © The Plankton Society of Japan

Possible aplanochytrid (Labyrinthulea) prey detected using 18S metagenetic diet analysis in the key copepod species Calanus sinicus in the coastal waters of the subtropical western North Paciﬁc

1,†, 2,3 3,4 1 Junya Hirai *, Yoko Hamamoto , Daiske Honda & Kiyotaka Hidaka

1 National Research Institute of Fisheries Science, Japan Fisheries Research and Education Agency, 2–12–4 Fukuura, Kanazawa, Yokohama, Kanagawa 236–8648, Japan, 2 Graduate School of Natural Science, Konan University, 8–9–1 Okamoto, Higashinada, Kobe, Hyogo 658–8501, Japan 3 Institute for Integrative Neurobiology Konan University, 8–9–1 Okamoto, Higashinada, Kobe, Hyogo 658–8501, Japan 4 Faculty of Science and Engineering, Konan University, 8–9–1 Okamoto, Higashinada, Kobe, Hyogo 658–8501, Japan † Present address: Atmosphere and Ocean Research Institute, The University of Tokyo, 5–1–5 Kashiwanoha, Kashiwa, Chiba, 277–8564, Japan Received 19 September 2017; Accepted 20 February 2018 Responsible Editor: Ryuji Machida

Abstract: Metagenetic diet analyses of the 18S V9 region were conducted in 40 adult female Calanus sinicus during winter in Tosa Bay (Japan). The majority of prey items were small crustaceans (of Copepoda and Cirripedia) and diatoms, taxa that are dominant in the environment and have been previously reported as important prey items of Cala- nus. The abundance of sequences attributable to Dinophyta and Chlorophyta was significantly lower in C. sinicus gut contents than in environmental plankton communities, suggesting that C. sinicus avoids prey from these groups. Hy- drozoans were also observed, and aplanochytrids (Labyrinthulea) were detected for the first time as a major prey of C. sinicus. Additionally, high proportions of unclassified eukaryote material were observed, suggesting undetected predator–prey relationships in key copepod species in marine ecosystems. The dietary importance of aplanochytrids, heterotrophic protists that accumulate unsaturated fatty acids such as docosahexaenoic acid, has been overlooked in previous research. Calanus sinicus is a key copepod species in the subtropical coastal regions of the western North Pacific, and a major food source for the larvae of commercially important fish; therefore, further investigation into novel prey items such as aplanochytrids is recommended to understand the complex food web structures in marine ecosystems.

Key words: aplanochytrid, Calanus sinicus, diet, Labyrinthulea, metagenetics

sinicus is omnivorous, and also feeds on various micro- Introduction zooplankton (Uye & Murase 1997, Yang et al. 2009). It has Calanus sinicus (Brodsky, 1962) is a large calanoid co- also been reported that feeding habits, such as the propor- pepod (approximately 2.1–3.3 mm in total length) common tion of phytoplankton contributing to its diet, vary tem- to the coastal waters of the subtropical western North Pa- porally and spatially, according to food availabilities in cific (Hulsemann 1994, Chihara & Murano 1997). Calanus ambient waters (Zhang et al. 2006). In addition to water sinicus is a key species in the food webs adjacent to the temperature, food availability is one of the primary factors Kuroshio Current, off Japan, where it makes up most of controlling development and egg production in C. sinicus the zooplankton biomass, and where its immature stages (Uye & Murase 1997), suggesting that detailed investiga- are the primary prey for larvae of commercially important tion of predator–prey relationships involving C. sinicus is fish (Nakata & Hidaka 2003, Hirai et al. 2017). Although important to understand planktonic food webs in the west- phytoplankton is considered its primary food source, C. ern North Pacific. Various approaches have been used to analyse the diets of copepods, including morphological observations, stable * Corresponding author: Junya Hirai; E-mail, [email protected] isotope analyses, and pigment measurements; however, it 76 J. Hirai et al. is difficult to obtain detailed taxonomic information from damaged gut contents using such methods. For example, observations using scanning electron microscopy (SEM) frequently detect fragments of diatoms in the gut contents of C. sinicus; however, the majority of gut contents remain unidentifiable (Chen et al. 2010). In contrast, a molecular- based method can detect detailed taxonomic information in damaged gut contents. The metagenetic method, which uses high-throughput sequencing, is a particularly power- ful approach to investigating predator–prey relationships at high taxonomic resolutions in marine taxa such as copepods (Pompanon et al. 2012, Cleary et al. 2016). Diets of C. sinicus have been investigated in the South Yellow Sea and the Bohai Sea using the cloning technique (Yi et al. 2017), and in the coastal waters around Taiwan and Hong Kong using metagenetic analysis (Ho et al. 2017). These studies confirm that C. sinicus is omnivorous and that feed- Fig. 1. Sampling locations in the present study. Black dots show ing habits vary spatially and temporally, and furthermore sampling stations. Isobaths are indicated by grey lines. identified novel prey items in the gut contents belonging to gelatinous taxa classified as Hydrozoa and Ctenophora. High-throughput sequencing In the present study, we collected C. sinicus during win- Frozen bulk zooplankton samples were thawed in the ter in Tosa Bay (Japan). 18S metagenetic analyses of gut laboratory, and we identified 10 adult female C. sinicus contents were carried out in adult female C. sinicus to individuals, morphologically, from each sample. In total, characterise the diet and to detect novel prey items previ- 40 individuals (10 at each station, day and night) were dis- ously undetectable using conventional means. Consider- sected, and the whole gut of each specimen was collected. ing its strong ecological role in linking higher and lower Genomic DNA was extracted from the guts of each C. trophic levels, investigating in detail the prey items taken sinicus individual using 30 µL 5% Chelex buffer (Bio-Rad by C. sinicus is an important step in understanding marine Laboratories) according to the methods proposed by Nagai ecosystems in the coastal waters of the subtropical western et al. (2012). Protocols of library preparations for high- North Pacific. throughput sequencing were performed for gut contents of C. sinicus, according to the method of Hirai et al. (2017). The 18S V9 region (approximately 130 bp) was amplified Materials and Methods by the polymerase chain reaction (PCR) using eukaryotic universal primers 1389F (5′-TTG TAC ACA CCG CCC- Samplings 3′) and 1510R (5′-CCT TCYGCA GGT TCA CCT AC-3′; Samples were collected during the cruise SY-15-02 Amaral-Zettler et al. 2009). Adaptors for Illumina MiSeq aboard the research vessel (R/V) Soyo-maru (National Re- and index sequences for discriminating samples were search Institute of Fisheries Science, Japan Fisheries Re- attached by second and third PCRs (Hirai et al. 2017). search and Education Agency), at two stations, L3250 (32° DNA concentrations of PCR products were measured us- 49.8′N; 133° 10.2′E) and N3320 (33° 20.1′N; 133° 29.9′E), ing a Qubit 3.0 Fluorometer (Life Technologies). An equal in the coastal area of Tosa Bay, off the southern coast of amount of DNA was combined from each of the PCR prod- Japan (Fig. 1). Samples were collected on 15 and 17 Febru- ucts of gut contents, before being purified using the QIA- ary 2015, respectively, to correspond with the spawning quick PCR Purification Kit (QIAGEN). Finally, the PCR seasons of the Japanese sardine Sardinops melanostictus products of gut contents were transferred to Bioengineer- and the Pacific round herring Etrumeus teres (Hirai et al. ing Lab. Co. Ltd. (Atsugi, Japan), where a single sequenc- 2017). Bulk zooplankton samples were collected using ORI ing procedure was carried out using MiSeq Reagent Kit or NORPAC nets with a mesh size of 335 µm at depths of v2 on an Illumina MiSeq platform to obtain 2×250 bp 0–50 m, once during the day and once at night at each sta- paired-end sequence reads (DDBJ Sequence Read Archive tion, and were preserved at −20°C. Water samples were accession number DRA005779). The sequence data for en- used to characterise the environmental plankton commu- vironmental seawater samples were already available from nities at the sampling sites, according to the methods de- a previous study (Hirai et al. 2017); therefore, sequencing tailed by Hirai et al. (2017). We collected a water sample at data were not newly obtained for environmental plankton three depths (0, 10, and 30 m) at each station. Immediately communities in the present study. after collection, 1 L of each sample was filtered using ster- ile polycarbonate filters with pores 1 µm wide (Nuclepore Data analyses membrane; GE Healthcare). The raw sequence data for C. sinicus gut contents were Aplanochytrid prey for copepods 77 combined with those of the environmental plankton com- individual to a category based on the proportion of se- munities reported by Hirai et al. (2017). According to the quence reads in the gut contents: low (0–1%), medium (1– methods described by Hirai et al. (2017), sequences with 10%), or high (>10%). The number of individuals assigned low quality and short fragment length were removed using to each category for each taxonomic group was counted Trimmomatic (Bolger et al. 2014). The remaining paired- both for Eukaryota as a whole and for Eukaryota after ex- end sequences were merged and further quality-filtered in cluding Opisthokonta. All eukaryotic sequence reads were MOTHUR (Schloss et al. 2009). These high-quality se- clustered into MOTUs at a similarity threshold of 99%. For quences were aligned using Silva 119 databases (Quast et obtaining detailed taxonomic information of major prey, al. 2013) in MOTHUR. After single-linkage pre-clustering the Basic Local Alignment Search Tool (BLAST) in the (Huse et al. 2010), we removed singleton reads accord- National Center for Biotechnology Information (NCBI) da- ing to Unno (2015), in order to remove redundant and er- tabase was used for the 10 most abundant MOTUs in the roneous molecular operational taxonomic units (MOTUs). gut contents. The putative taxon of each MOTU was inves- Chimeric sequences were removed using UCHIME (Ed- tigated based on the results of the BLAST analysis. gar et al. 2011). We added four sequences from the Gen- Bank database (Paracalanus parvus, GenBank accession Results numbers KX364939.1 and JF326205.1; Labidocera japon- ica, AB200178.1; and Calanus sinicus, KR048707.1) to the Massive sequence data V9_ PR2 reference database of the Tara Oceans project (de Vargas et al. 2015) to carry out taxonomic classification of After quality filtering all sequence data, a total of sequence reads using a naϊve Bayesian classifier (Wang 2,919,386 reads (average 72,985 reads per C. sinicus indi- et al. 2007). Following selections of Eukaryota, sequence vidual) were obtained in the gut content analysis. Among reads belonging to the family Calanidae, unclassified Co- the gut content data, large proportions (>94%) of sequence pepoda, or unclassified calanoid copepods were removed reads were attributed to the host (Table 1). Sequence reads accordingly from the subsequent analysis, because those classified as parasites or mammals were comparatively rare sequences might be derived from the hosts of the present (<1%; Table 1). The remainder were attributed to prey spe- study. The sequences were also removed if they were clas- cies. The number of sequence reads in each of six environ- sifiable into parasitic or mammalian sources. The taxo- mental plankton community samples ranged from 19,778 nomic groups of parasites were based on those detected in to 42,720. In total, 287,165 sequence reads from 40 gut 18S metagenetic analysis of environmental seawaters in content samples and six environmental community sam- Cleary & Durbin (2016), who reviewed the literature to de- ples were used for the subsequent analysis. termine parasitic taxa. The proportions of sequence reads in the gut contents were calculated for all eukaryotic su- Opisthokont taxa pergroups as well as for major taxonomic groups after ex- High proportions of Opisthokonta were detected among cluding the supergroup Opisthokonta. The proportions of the sequence reads of Eukaryota (Fig. 2A). In Opistho- sequence reads attributable to each taxonomic group in the konta, sequences belonging to Copepoda were particu- gut contents were averaged across the day and night sam- larly prominent, accounting on average for 28.9–57.1% of ples, respectively, at each station. Moreover differences in sequences in the gut content and 28.0–45.4% in the envi- the proportional contribution of taxonomic groups between ronmental samples (Fig. 2A). Cirripedia were abundant in all gut contents and environmental samples were tested us- the gut contents during the day at station L3250, accounting Mann-Whitney U tests. In addition to averaged propor- ing for 24.2% of the sequences; however, the abundance of tions of sequence reads, detection frequencies for each ma- Cirripedia sequences in the gut contents was lower in other jor taxonomic group were investigated in all 40 C. sinicus samples, accounting for up to 4.8% of the sequence reads. individuals. In this frequency analysis, we assigned each Hydrozoa were relatively abundant at station L3250 (day:

Table 1. Numbers and percentages of sequence reads detected in the gut contents of Calanus sinicus taken in Tosa Bay, off Japan, at two stations (L3250 and N3320) during the day and at night. Sequence read numbers and percentages represent averages calculated from 10 individuals collected during each sampling. Sequence reads were classiﬁed into host, parasite or mammal, or prey materials. Average sequence reads (%) N All Host Parasite or Mammal Prey L3250 Day 10 74,351 70,405 (94.7%) 449 (0.63%) 3,497 (4.65%) L3250 Night 10 74,094 72,234 (97.6%) 39 (0.05%) 1,821 (2.33%) N3320 Day 10 68,016 64,883 (95.3%) 126 (0.19%) 3,007 (4.49%) N3320 Night 10 75,478 73,051 (96.9%) 39 (0.05%) 2,388 (3.09%) All 40 72,985 70,143 (96.1%) 163 (0.23%) 2,678 (3.64%) 78 J. Hirai et al.

6.2%, night: 5.6%); however, smaller proportions were ob- of C. sinicus individuals had high abundances of sequence served at station N3320 (day: 1.3%; night: 1.5%). Other reads (>10%) in the gut contents attributable to Copepoda Opisthokonta material was relatively common, account- (Table 2). Low proportions (<1%) were observed in one in- ing for 10.3–24.3% of sequence reads in the gut contents. dividual. Cirripedia and Hydrozoa frequently contributed When all sequence data were combined, Cirripedia were moderate proportions (1–10%); however, 25% and 42.5% proportionally more abundant in gut contents than in envi- of C. sinicus individuals had low proportions of Cirripedia ronmental plankton communities, although the difference and Hydrozoa, respectively. was not signiﬁcant (Fig. 3). The abundances of copepods and hydrozoans were approximately equal in gut contents Stramenopile taxa and environmental plankton communities. Ninety percent Sequences classiﬁed as Stramenopiles were also abundant among all Eukaryota, comprising 8.6–38.1% and 7.7–17.1% in gut contents and in environmental plankton, respectively (Fig. 2A). After removing Opisthokonta, the proportion of Bacillariophyta (Stramenopiles) material was particularly high both in gut contents (33.1–51.0%) and in environmental plankton (13.6–23.8%; Fig. 2B). Laby- rinthulea (Stramenopiles) was also abundant. After removing Opisthokonta, the highest observed proportion, 32.7%, was measured in the night sample from station L3250; a

Fig. 2. Average sequence compositions of major taxonomic groups detected in the gut contents of female Calanus sinicus sampled at two stations (L3250 and N3320) in Tosa Bay, off Japan, Fig. 3. Comparisons of relative abundances of major taxonomic sorted by sampling time (day: N=10 per station; night: N=10 per groups between all Calanus sinicus gut contents and environmental station). Average environmental plankton compositions (Environ- seawater. The abundance of sequence reads are presented as logx+1- ment) at both sites are also included (seawater sample N=3 for transformed averages of all available samples both during day and each station). (A) Supergroups within Eukaryota. Opisthokont night at two sampling sites (gut contents: N=40; seawater samples: materials are further classiﬁed into the major taxonomic groups N=6). Scale bars indicate standard deviation. Asterisks (*) indicate Cirripedia, Copepoda, and Hydrozoa. (B) Major taxa after remov- a signiﬁcant difference (p<0.01) detected by the Mann–Whitney ing Opisthokonta. U test.

Table 2. Numbers of Calanus sinicus individuals with low (0–1%), medium (1–10%), or high (>10%) proportions of sequence reads attributable to major taxonomic groups in the gut contents. For each major taxonomic group, all specimens of female Calanus sinicus (n=40) were categorised into one of the three frequency groups based on the proportions of sequence reads of a given taxonomic group in the gut contents. This frequency analysis was carried out for taxonomic groups identiﬁed in all taxonomic groups of Eukaryota (left), and for Eu- karyota excluding Opisthokonta (right). All-Eukaryota Non-Opisthokonta <1% 1–10% >10% <1% 1–10% >10% Bacillariophyta 8 22 10 3 7 30 Chlorophyta 34 5 1 28 7 5 Cirripedia 10 23 7 / / / Copepoda 1 3 36 / / / Dinophyta 27 10 3 19 12 9 Hydrozoa 17 18 5 / / / Labyrinthulea 22 13 5 19 5 16 Rhizaria 36 3 1 35 4 1 Aplanochytrid prey for copepods 79

Table 3. BLAST results for the 10 most abundant MOTUs identified in the gut contents of Calanus sinicus. The average percentage of sequence reads was calculated from data obtained from 40 female C. sinicus collected from Tosa Bay, off Japan. Query coverage and sequence identity are provided as percentages. Asterisks (*) denote cases where multiple best-hit species were detected with the same score; representa- tive species are listed in such cases. Rank Average Best hit species Coverage Identity Accession Putative taxon 1 10.09% Pseudocalanus sp. 95% 98% GU594644.1 Copepoda (Clausocalanidae) 2 8.39% Oithona similis 100% 99% KF153700.1 Copepoda (Oithonidae) 3 7.67% Thalassiosira mala 100% 100% HM991693.1 Bacillariophyta (Thalassiosirales) 4 7.64% Galkinius sp.* 100% 100% KM217491.1 Cirripedia 5 5.62% Paracalanus sp. 100% 99% KX364940.1 Copepoda (Paracalanidae) 6 3.96% Aegyria foissneri* 30% 100% KX364493.1 Unclassifiable (Eukaryota) 7 3.49% Aplanochytrium kerguelense 100% 98% AB022103.1 Labyrinthulea (Aplanochytrium) 8 3.27% Acartia hongi 27% 100% GU969195.1 Unclassifiable (Eukaryota) 9 2.85% Muggiaea sp.* 100% 100% AF358073.1 Hydrozoa 10 2.80% Sinocalanus tenellus* 100% 95% GU969144.1 Calanoid copepod proportion of 6.3% was measured in the day sample from <1% Chlorophyta material in the gut (70% after removing station L3250, and proportions of 9.3% and 6.9% were Opisthokonta), 67.5% had <1% Dinophyta material (47.5% measured in the day and night samples, respectively, from after removing Opisthokonta), and 90% had <1% Rhiz- station N3320. In a comparison of all samples, the propor- aria in the gut (87.5% after removing Opisthokonta; Table tion of Bacillariophyta was lower in gut contents than in 2). Unclassifiable Eukaryota material comprised 0.3–9.9% environmental plankton communities, whereas the propor- of all gut contents, or 9.3–25.3% after removing Opistho- tion of Labyrinthulea was higher; however, these differ- konta. Other taxa, including Apusozoa, Excavata, Hacro- ences were not significant (Fig. 3). Other Stramenopiles, bia, and Picozoa, were comparatively uncommon across all including unclassified taxa, comprised 3.2–24.9% of se- sequence reads. quences in gut contents, after removing Opisthokonta. Fifty-five percent of C. sinicus individuals had medium Taxonomy of abundant MOTUs proportions of Bacillariophyta sequence reads; however, BLAST results indicated that several of the 10 most high proportions were observed in 75.0% of individuals af- abundant MOTUs in the gut contents were copepods (Table ter removing Opisthokonta from the analysis (Table 2). Al- 3). The most abundant MOTU belonged to the copepod most half of C. sinicus individuals had low proportions of family Clausocalanidae. The families Oithonidae (rank 2) Labyrinthulea both in the all-Eukaryota (22/40 individuals) and Paracalanidae (rank 5), as well as unclassified Calanoid and non-Opisthokonta analyses (19/40 individuals). Large copepods (rank 10) and another crustacean, attributable proportions of Labyrinthulea sequence reads were found to Cirripedia (rank 4), also appeared among the 10 most in 12.5% of C. sinicus in the all-Eukaryota analysis, and in abundant MOTUs. The third most abundant Bacillariophy- 40.0% after removing Opisthokonta. ta MOTU was attributed to Thalassiosirales. The remaining MOTUs comprised Labyrinthulea material with a high Other taxa similarity to Aplanochytrium (rank 7), two unclassifiable Alveolata and Archaeplastida were relatively abundant eukaryotes (ranks 6 and 8), and one hydrozoan (rank 9). in environmental samples, comprising 15.5–24.5% and 12.2–13.1% sequences, respectively, of all sequences (Fig. Discussion 2A). These supergroups were primarily represented by Dinophyta and Chlorophyta, comprising 25.1–47.0% and The major finding in the present study was the detection 20.8–22.4%, respectively, of environmental plankton com- of aplanochytrids (Labyrinthulea) previously unrecognised munities, after removing Opisthokonta (Fig. 2B). Howev- as playing a role in C. sinicus diets; however, the results of er, Dinophyta and Chlorophyta comprised 3.5–13.9% and the present study also supports those of previous reports 1.0–8.0% sequences in the gut contents, respectively, even that diatoms (Bacillariophyta) and small crustaceans are after removing Opisthokonta. Rhizaria comprised 0.1– major prey groups in C. sinicus. Although Eukaryota-wide 1.7% of all gut contents, or 0.4–6.0% after removing Opis- use of the metagenetic method is possibly prone to bias thokonta. The relative abundances of Rhizaria, Dinophyta, (e.g. copy numbers of rRNA genes, primer mismatches), and Chlorophyta were significantly lower in gut contents metagenetic analysis concerning 18S rRNA has been sug- than in environmental samples (p<0.01; Fig. 3). These gested as a viable, semi-quantitative method for providing taxonomic groups were also rare in the frequency analy- snapshots of eukaryotic community compositions useful sis, which showed that 85% of C. sinicus individuals had for accurately characterising predator–prey relationships 80 J. Hirai et al.

(de Vargas et al. 2015, Ray et al. 2016). There is a possibil- the Yellow Sea (Huo et al. 2008). ity of feeding in cod-ends of the plankton net in the analy- The metagenetic method is particularly effective in dis- ses of the gut contents; however, the mesh size used in the covering novel prey items. The present study identified a present study was larger than the major size of prey in C. gelatinous zooplankton taxa (Hydrozoa) as a major source sinicus (Yang et al. 2009). In addition, data on plankton of prey for C. sinicus, and this taxa occurred among the communities in the environment are valuable for inves- individuals sampled at a similar frequency as observed tigating prey preference. Therefore, quantitative data ob- in previous molecular-based studies (Yi et al. 2017, Ho et tained from sequence reads detected in both gut contents al. 2017). Although the metagenetic analysis used in this and environmental waters in the present study provide a study was unable to provide information concerning the representation of the relative contributions of different developmental stages of hydrozoans selected by C. sinicus, prey taxa present in C. sinicus diets. the morphological analysis is almost entirely unable to de- Diatoms were identified as a common prey item in most tect gelatinous organisms in the gut contents of C. sinicus individuals and contributed relatively large proportions of (Chen et al. 2010). Therefore, this study indicated that ge- sequence reads, a result that supports those of previous latinous organisms, including Hydrozoa, may be an impor- studies. Although overall proportions were lower in the gut tant food source during winter in Tosa Bay. Furthermore, contents than in the environmental samples, possibly be- aplanochytrids (Labyrinthulea), previously unrecognised cause of their toxic effects on copepods (Ianora & Miralto as playing a role in C. sinicus diets, were also identified as 2010), diatoms, particularly Thalassiosirales, nevertheless a possible major food source in the present study. Aplano- contributed substantially to gut contents in this study. Al- chytrids are unicellular heterotrophic protists belonging to though cannibalism could not be evaluated using the meta- Labyrinthulea in Stramenopiles (Honda et al. 1999), and, genetic methods deployed in the present study, crustacean interestingly, are characterised by the ability to accumu- material attributable to Copepoda and Cirripedia com- late unsaturated fatty acids such as docosahexaenoic acid prised substantial proportions of the diet in almost all in- (DHA; Ellenbogen et al. 1969, Gomathi et al. 2013). Al- dividuals of C. sinicus analysed. Predation on crustaceans though the ecological role of Labyrinthulea is poorly un- by C. sinicus has been reported only rarely (Uye & Murase derstood in natural marine environments, the community 1997); however, such behaviour is frequently observed in structures of such taxa change seasonally, occasionally other Calanus species (Landry 1981). The BLAST results reaching high abundances in coastal waters (Ueda et al. reported in the present study indicate that small copepods 2015), suggesting that they may play an important eco- functioned as important prey items for adult female C. logical role. The biomass of Labyrinthulea sometimes ex- sinicus, suggesting that the role played by small copepods ceeded that of bacterioplankton, and Aplanochytrids have in marine ecosystems has been underestimated (Turner commonly been observed in the oceans, including coastal 2004). Previous molecular-based diet studies used methods waters of southern China (Liu et al. 2017) and oceanic Ha- to prevent amplifying host sequences during PCR (Yi et al. waiian waters (Li et al. 2013). Aplanochytrid material was 2017, Ho et al. 2017), which may prevent amplification of also detected in the guts and faecal pellets of copepods in crustacean DNA other than the host species; therefore, the the tropical Indian Ocean (Damare & Raghukumar 2006, contributions of crustaceans might not be appropriately re- 2010), supporting our findings that aplanochytrids are an flected in these previous studies. The metagenetic analysis important food source for C. sinicus. Given the higher pro- we used, which did not prevent amplifying host sequences, portions of aplanochytrids in gut contents than in the envi- was capable of detecting small non-host crustaceans; thus, ronmental samples in the present study, we suggest that C. we suggest that the high contributions of small crustaceans sinicus positively selects aplanochytrids as prey. Because to the proportions reported reflect the predation patterns of metagenetic data provide no information on the distribu- adult female C. sinicus. The small-bodied crustaceans we tion of different aplanochytrids forms in the environment, detected in the C. sinicus samples were generally abundant it is unclear precisely how C. sinicus consumed aplano- in the environmental plankton community (Hirai et al. chytrids. Labyrinthulea have been frequently observed to 2017), indicating that prey availability plays an important attach to aggregations in the environment (Li et al. 2013; role in determining gut content composition. As clearly ob- Bochdansky et al. 2017); therefore, these aggregations may served in Cirripedia, spatial and temporal differences in C. be consumed by zooplankton. Calanus sinicus uses DHA sinicus diets were also suggested in this study. Taxa attrib- in lipid metabolism (Wang et al. 2017), suggesting that utable to Dinophyta and Chlorophyta were relatively abun- aplanochytrids may act as important sources of DHA for dant in the environment; however, they were significantly C. sinicus. Finally, we recommend investigating the role less abundant in C. sinicus gut contents. The same pattern of aplanochytrid prey in larvae of commercially important was observed in Rhizaria, indicating that adult female C. fish, including Japanese sardine and Pacific round herring. sinicus avoid feeding on these taxa. Thus, contributions of The results we obtained using the 18S metagenetic ap- these taxa as prey were small for adult female C. sinicus proach in this study support those of previous studies in- during the sampling period. The scarcity of Dinophyta ma- dicating that diatoms, small crustaceans, and hydrozoans terial in the gut contents of C. sinicus was also observed in play important roles in the diet of C. sinicus, and further Aplanochytrid prey for copepods 81 suggest a new predator–prey interaction involving aplano- ages and a potential mechanism of niche partitioning. Deep- chytrids. However, the scope of the present study was con- Sea Res II 134: 181–189. fined to a single copepod species during the winter in Tosa Damare V, Raghukumar S (2006) Morphology and physiology Bay, off Japan; therefore, further studies are recommended of the marine straminipilan fungi, the aplanochytrids isolated to investigate the ecological roles of the prey organisms from the equatorial Indian Ocean. Indian J Mar Sci 35: 326– newly detected in this study. Additionally, we frequently 340. encountered large proportions of eukaryote, opisthokont, Damare V, Raghukumar S (2010) Association of the strameno- and stramenopile material that could not be identified to pilan protists, the aplanochytrids, with zooplankton of the major taxa, indicating that further DNA barcoding efforts equatorial Indian Ocean. 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