Plankton Benthos Res 12(3): 145–150, 2017 Plankton & Benthos Research © The Plankton Society of Japan

Life cycle and precopulatory mate guarding of Goidelia japonica (Copepoda: Poecilostomatoida: Echiurophilidae) associated with the echiuran Urechis unicinctus

1,†, 1,‡ 1,$ Minoru Ijichi *, Gyo Itani & Hiroshi Ueda

1 Center for Marine Environmental Studies, Ehime University, 2–5 Bunkyo-cho, Matsuyama 790–8577, Japan † Present address: Atmosphere and Ocean Research Institute, the University of Tokyo, 5–1–5 Kashiwanoha, Kashiwa 277–8564, Japan ‡ Present address: Faculty of Education, Kochi University, 2–5–1 Akebono, Kochi 780–8520, Japan $ Present address: 1–13–22–703 Tanaka-cho, Higashinada-ku, Kobe 658–0081, Japan Received 7 February 2017; Accepted 24 March 2017 Responsible Editor: Koichi Ara

Abstract: The life cycle and precopulatory mate guarding behavior of the poecilostomatoid copepod Goidelia ja- ponica associated with the spoon worm Urechis unicinctus (Echiura) are described based on specimens from 19 worms collected from western Japan. A total of 676 copepods were collected from the host’s rectum (494 copepods) and body surface (182 copepods). Copepod numbers were significantly correlated with host weight. Copepods in the rectum consisted almost completely of copepodids (35%) and adult males (65%), with only one non-ovigerous adult female recorded. In contrast, those on the body surface were all adult males (24%) or females (76%), of which 58% were ovig- erous. No nauplii or CI occurred. In the rectum, 36% of the adult males guarded 67% of the copepodids, consisting of CII–CV. From these results, their life cycle is considered as follows: nauplii and CI live as plankton; just before or after molting to CII, they enter the host’s rectum and females are probably soon guarded by males; they grow up to CV there; CV females guarded by males move to the body surface just before the final molt; on the body surface females molt to adults and spawn eggs. Guarding the first symbiotic stage (CII) is a unique precopulatory behavior for symbiotic poe- cilostomatoid copepods. This can be explained from the viewpoint of evolutionary resolution of the intersexual conflict and advantage for early copepodids in precopula.

Key words: Goidelia japonica, life cycle, precopulatory mate guarding, symbiosis, Urechis unicinctus

postnaupliar stages and mating behavior of the poecilosto- Introduction matoid copepod Goidelia japonica Embleton, 1901 (Echiu- The crustacean subclass Copepoda contains about rophilidae) associated with the spoon (or innkeeper) worm 13,000 species (Boxshall & Defaye 2008). According to Urechis unicinctus (von Drasche, 1881) (Annelida, Echi- Ho (2001), more than one-third of known copepods are ura). This echiuran lives in a U-shaped burrow in muddy symbiotic with other organisms, and their hosts range from and/or sandy sediments in lower inter- and subtidal zones sponges to marine mammals, including all major aquatic of the Chinese continent and Japan, and is used as food in taxonomic groups. He noted that all aquatic animals are Japan, China and especially Korea (Abe et al. 2014). Echi- potential hosts for symbiotic copepods. Furthermore, sym- uran burrows are known to serve as habitats for various biotic copepods can be very abundant on or in their hosts. symbionts, such as shrimps, crabs, bivalves and demersal Despite such an ubiquitous distribution, studies on the bi- fishes (Fisher & MacGinitie 1928, Anker et al. 2005, Itani ology of symbiotic copepods are still very limited, com- et al. 2005, Goto 2017). In addition to the commercial and pared to studies on their taxonomy. ecological importance of U. unicinctus, it has the potential The present study describes the life cycle during the to improve water quality by filtering suspended materials (Abe et al. 2014) and the quality of the organically contam- * Corresponding author: Minoru Ijichi; E-mail, nitrifi[email protected] inated coastal sediment by reducing acid volatile sulfide, tokyo.ac.jp COD, and total ignition loss (Kang et al. 2010). In Japan, 146 M. Ijichi et al.

U. unicinctus occurred previously as a common species in sals with a tendency to kleptoparasitism. Copepoda is one mud and sandy tidal flats, but its population has decreased of the few taxa of host-specific associates living on or in dramatically due to reduction of their habitats during re- echiurans, and it appears that they are dominant endo- and cent decades (Nishikawa 2012, Abe et al. 2014). ectosymbionts. However, there have, as yet, been no stud- Goidelia japonica was first described from the rec- ies on the life cycle and behaviors of copepod symbionts tum of Urechis unicinctus by Embleton (1901) based on associated with echiurans. Japanese specimens, and subsequently by Gooding (1963) based on specimens collected in Ehime, northwestern Shi- Materials and Methods koku Island, Japan, and Tokyo Bay, and by Kim (1998) on Korean specimens. The following other five poecilostoma- Nineteen individuals of Urechis unicinctus were col- toid species are known to be associates with echiurans: lected from a tidal flat of the Kamogawa River (33.935°N, Echiurophilus fizei Delamare-Deboutteville & Nunes- 133.155°E) on the northern coast of Ehime Prefecture, Shi- Ruivo, 1955 with Anelassorhynchus inansensis (Ikeda, koku Island, Japan, during a spring ebb tide on 4 June 1904) from Vietnam (Delamare-Deboutteville & Nunes- 2004. The sediment at the collection site was sandy mud. Ruivo 1955), Hemicyclops mortoni Boxshall & Humes, The worms were dug up from burrows using a shovel, and 1987 with Ochetostoma erythrogrammon Leuckart & Rüp- individually fixed and preserved in 5% formalin-seawater pell, 1828 from Hong Kong (Boxshall & Humes 1987), solution immediately after collection. In the laboratory, Goidelia pelliviva Kim I.H., 2000 with Thalassema sp. sample bottles were shaken to separate copepods off the from Korea (Kim 2000), and the recently described Echiu- body surface of the worm and all copepods in the sample ricopus aprilis Kim I.H., 2016 and Echiuricopus tenuipes waters were collected and examined under a stereoscopic Kim I.H., 2016 with unidentified echiurans in Korea (Kim microscope; these copepods are regarded as those associ- 2016). The number of known copepod species associated ated with the host body surface. Then the rectums of the with Echiura is very few compared to those with other worms were dissected to collect copepods; prior to this op- aquatic invertebrate groups such as Mollusca, Cnidaria, eration, we dissected the whole digestive tracts of several Echinodermata, Urochordata, Crustacea, and Polychaeta, worms and confirmed that copepods were found restricted- with which more than a hundred copepod species have ly in the rectums. We examined a dissected rectum twice been reported as symbionts (Humes 1994). for each worm under a stereoscopic microscope to collect Anker et al. (2005) reviewed symbiotic animals associ- copepods completely. No other copepods were found in ated with echiurans. Most of these symbionts are consid- the samples from the rectum and/or the body surface. Wet ered as inhabitants in echiurans’ burrows and commen- weights of the 19 worms, which were measured using an

Table 1. The numbers of Goidelia japonica collected from Urechis unicinctus. Values in brackets and parentheses indicate the numbers of individuals in precopula and of ovigerous females, respectively. Life cycle and precopula of Goidelia japonica 147

Fig. 1. The relationships between the number of Goidelia japonica and the wet weight of the host Urechis unicinctus. The Fig. 3. Precopulatory adult male of Goidelia japonica guarding solid, dotted, and broken regression lines represent regressions of a CII individual. the numbers of total copepods from the host, from the host rectum, and from the host body surface, respectively.

Fig. 2. Goidelia japonica. A: living individuals (indicated by arrows) on the body surface of Urechis unicinctus; B: enlarged photo of an ovigerous female (indicated with a big arrow in A); C: ovigerous female on glass slide (scale bar 0.50 mm). electronic balance, ranged from 4.9–14.4 g (average 7.4 g). Developmental stages of copepods were distinguished Fig. 4. Goidelia japonica. A: a male clasping a CV female; B: under a stereoscopic microscope. Adult copepods were enlarged photo of A, showing a male clasping with maxillipeds. identified by the genital compound somite or presence of egg clutches for females and six urosomites for males. Co- worm and the total number of copepods from the 19 pepodid stages were distinguished by the number of leg worms was 676, of which 494 (73%) were from the rec- pairs on the prosome (two for CI, three for CII, and four tum and 182 (27%) were from the body surface (Table for the later stages) and the number of urosomites (three 1). Copepod nauplii and CI were not found at all. The for CI–CIII, four for CIV, and five for CV) following the stage-specific individual numbers of CII–CV and adults, description of copepodid stages of the poecilostomatoid excluding two copepodids for which the stages could not copepod Hemicyclops japonicus Itoh & Nishida, 1993 be distinguished, were 37, 74, 18, 40, and 505, respectively, (Itoh & Nishida 1995). of which the ratios to the total numbers of copepods were 5.5, 11.0, 2.7, 6.0, and 75.1%, respectively. Adult males (365 individuals) were 2.6 times more numerous than adult fe- Results males (140 individuals). The number of total copepods from a worm showed a Abundance of copepods significantly positive correlation with the wet weight of Goidelia japonica was collected from every spoon the worm (r=0.77, p<0.001) (Fig. 1). Significant positive 148 M. Ijichi et al. relationships with the wet weight of the worm were also that more than a half of the females on the body surface observed for copepods from the rectum (r=0.76, p<0.001) were ovigerous and that most copepodids, including CII and from the body surface (r=0.66, p<0.01). stage, in the rectum were guarded by males in precopula. In the rectum, copepods consisted of 171 copepodids These results lead us to propose the following scenario for (35%) and 323 adult males (65%), along with only one the life cycle of Goidelia japonica. They hatch out from non-ovigerous adult female. In contrast to the number of females on the body surface of Urechis unicinctus and live copepodids in the rectum, no copepodids were found on apart from the host as plankton until CI. Just before or af- the body surface. About one-fourth (24%) of the adult co- ter molting to CII, they enter the rectum of a host through pepods on the body surface were male and three-fourths the anus, and female copepodids are probably soon guard- (76%) were female, of which more than a half (58%) were ed by adult males. They grow up to CV in the rectum. CV ovigerous (Fig. 2). females in precopula with adult males move to the body surface before the final molt. On the body surface, they Precopulatory mate guarding quickly molt to adult females, copulate with males, and A total of 115 pairs of copepods in precopula were ob- spawn eggs. served in 14 rectums among the 15 worms, which had both In her original description of Goidelia japonica from the adult males and copepodids in their rectums (Table 1). No rectum of Urechis unicinctus, Embleton (1901) described precopula pairs were found in the surface copepods. Co- nothing about copepodids or precopulatory behavior, but pepods guarding other copepods were all adult males and stated that no adult females have egg-sacs. Gooding (1963) guarded copepods were various copepodid stages from CII observed only two females of G. japonica, in contrast to to CV. The precopulatory guarding of CII (Fig. 3) was not many males (107) and copepodids (159), collected from the rare; the stages of guarded copepodids were not completely rectum of a single specimen of U. unicinctus. Since their recorded in this study, but five of the six CII individuals re- collections were from the rectums, their observations are corded were guarded. Precopula of one copepodid guarded consistent with the present results. by two males were observed in worms #7 and #13. In the The characteristic life cycle of Goidelia japonica, i.e. rectum, 36% of adult males guarded copepodids, and these planktonic from nauplius to CI and symbiotic from CII comprised 67% of total copepodids. The number of precop- to the adult stage, is common to the poecilostomatoid ula pairs per host showed a significantly positive correlation Hemicyclops spp., of which planktonic CI has a charac- with the wet weight of the worm (r=0.803, p<0.001). teristic body shape different from the later stages and had Precopulatory guarding was performed by clasping the therefore previously been identified as a different genus dorsal joint between the first and second prosomal somites “Saphirella” (Itoh & Nishida 1995, 2007). Do et al. (1984) using maxillipeds (Fig. 4). described the complete life cycle of Pseudomyicola spino- sus (Raffaele & Monticelli, 1885), one of the most com- mon poecilostomatoid copepods associated with bivalve Discussion mollusks. This copepod is planktonic during all naupliar The present study revealed that Goidelia japonica lives stages and symbiotic during the copepodid and adult stag- not only in the rectum but also on the body surface of es. The body shape of CI of this species is not character- Urechis unicinctus. In previous studies, poecilostomatoid istic but is similar to those of the later stages. Compared copepods associated with echiurans were collected either with this, the characteristic shape of CI in Hemicyclops from hosts’ digestive tubes (Embleton 1901, Delamare-De- is considered to be due to a planktonic life different from boutteville & Nunes-Ruivo 1955, Gooding 1963) or body the symbiotic life led by the later stages. This suggests that surfaces, the latter specimens being collected from burrow CI of G. japonica also has a different body shape from the waters (Boxshall & Humes 1987), washings of echiurans later stages, and thereby that it is impossible to find CI in (Kim 2016), or directly from the hosts’ skin (Kim 2000). field samples and, instead, culture from eggs in the labo- The present result that they inhabit both the internal and ratory or molecular biological analyses are necessary for external surfaces of echiurans is the first finding for poeci- identification. lostomatoid copepod symbionts with echiurans. In the above scenario, we consider that most copepodids No nauplii or CI stages were collected from the worms, in the rectum are guarded by adult males, sometimes from indicating that only the stages from CII to adult are symbi- CII. The basis for this is the high ratio of copepodids in otic. However, the developmental stage and adult sex com- precopula to all copepodids. Given that the sex ratio of position were quite different between those in the rectum copepodids is not so different from that of the adult stages and on the body surface. Copepods in the rectum consisted (male/female=2.6) and that most copepodids in precopula completely of adult males and copepodids with the excep- are female, at least from CIV and later stages, which are tion of a single non-ovigerous female, whereas on the body sexually dimorphic stages (Do et al. 1984, Itoh & Nishida surface they were mostly adult females and much smaller 1995), the high ratio (67%) of copepodids in precopula im- numbers of adult males. No copepodids were found on the plies that almost all female copepodids, including CII, are body surface. Noteworthy findings in the present study are guarded by males. Life cycle and precopula of Goidelia japonica 149

In this scenario, precopula males and female CV stages (Jormalainen 1998). Based on this theory, the reason why move to the body surface just before the final molt, where G. japonica males guard mates from the youngest symbi- she copulates with the precopula males on the body sur- otic stage could be explained as follows. Adult males are face. This supposition is based on the result that one-fourth much more numerous than copepodids in the rectum and of the copepods on the body surface were male. If females therefore competition between adult males for precopula- molt and copulate in the rectum, males would not need to tory mates is severe. On the other hand, the costs of guard- go out from the rectum and as a result males on the body ing females are low because there is no predatory risk in surface would be much less numerous. If this were the case the echiuran rectum; no predatory animals were found in then the question arises as to the reason for the lower num- our observation. Guarding by adult males may give advan- ber of males than females on the body surface, because tages rather than risks, especially to early copepodid stag- if females move from the rectum to the body surface to- es, because guarded early copepodids could retain them- gether with males then the sex ratio should be even on the selves more easily against excretory action in the rectum body surface. Two possible reasons for this situation pres- than non-guarded copepodids. Embleton (1901) noted that ent themselves. One is that some males could return to the G. japonica specimens adhered very firmly to the epithe- rectum after copulation. The much higher number of adult lial lining of the host’s rectum. This suggests that retention males than copepodids in the rectum seems to corroborate in the rectum is not easy for early copepodids. this answer. The other is that females could live on the body surface longer than males. This answer is also highly Acknowledgements plausible, especially if females could spawn repeatedly us- ing sperm in the seminal receptacle. We thank Dr. Susumu Ohtsuka of Hiroshima Univer- Precopula is widespread in podoplean copepods, es- sity for providing literature related to the present study, pecially Harpacticoida and Siphonostomatoida (Boxshall and Mika Kawaguchi and Aiko Toda Mitsuzawa of Ehime 1990). For example, Kern et al. (1984) listed reported University for help in field sampling. claspings by males for 17 harpacticoid species, of which 13 included precopulatory mate guarding. As for Sipho- References nostomatoida, precopulatory mate guarding is a common behavior, and their life cycles with precopulatory guard- Abe H, Sato-Okoshi W, Tanaka M, Okoshi K, Teramoto W, Kon- ing have been studied for species, such as Cardiodectes doh T, Nishitani G, Endo Y (2014) Swimming behavior of the sp., Lernaeocera branchialis (Linnaeus, 1767), and Lep- spoon worm Urechis unicinctus (Annelida, Echiura). Zool 117: eophtheirus pectoralis (Müller O.F., 1776) (Boxshall 1990). 216–223. In Poecilostomatoida, only four species have hitherto Anker A, Murina GV, Lira C, Vera Caripe JA, Palmer AR, Jeng been known to have precopulatory guarding behavior, i.e. MS (2005) Macrofauna associated with echiuran burrows: a Pseudomyicola spinosus, Hemicyclops spinulosus Itoh & review with new observations of the innkeeper worm, Ocheto- Nishida, 1998, Pseudanthessius tortuosus Stock, Humes stoma erythrogrammon Leuckart and Rüppel, in Venezuela. & Gooding, 1964 and Pennatulicola pterophilus (Stock, Zool Stud 44: 157–190. 1962) (Itoh & Nishida 2008). These species are all symbi- Boxshall GA (1990) Precopulatory mate guarding in copepods. onts with benthic invertebrates. A remarkable difference of Bijdr Dierk 60: 209–213. G. japonica from the four other species is that G. japonica Boxshall GA, Defaye D (2008) Global diversity of copepods (Crustacea: Copepoda) in freshwater. Hydrobiologia 595: 195– males guard copepodids from the first symbiotic stage II 207. onwards. The youngest stages of the four other species Boxshall GA, Humes AG (1987) A new species of Hemicyclops guarded by adult males are CIII (Stock et al. 1962, Gotto (Copepoda: Poecilostomatoida) associated with an echiuran 1979, Do et al. 1984), except for H. spinulosus, of which worm in Hong Kong. Asian Mar Biol 4: 61–66. the youngest guarded stage was unknown because obser- Delamare Deboutteville C, Nunes-Ruivo LP (1955) Echiu- vation of precopulatory guarding behavior was made only rophilus fizei n. g. n. sp. Copépode parasite d’un Echiuride on a CV female and two adult males collected from the d’Indochine. Vie Milieu 6: 101–112. field (Itoh & Nishida 2008). Pseudomyicola spinosus and Do TT, Kajihara T, Ho J-S (1984) The life history of Pseudomy- H. spinulosus start symbiotic life at CI (Do et al. 1984, Itoh icola spinosus (Raffaele & Monticelli, 1885) from the blue & Nishida 2008), indicating that adult males do not guard mussel, Mytilus edulis galloprovincialis in Tokyo Bay, Japan, CI or CII, even if they encounter them. Precopulatory mate with notes on the production of a typical male. Bull Ocean Res guarding from mid- or late copepodid stages seems com- Inst Univ Tokyo 17: 1–65. mon also in harpacticoid copepods (Kern et al. 1984). Embleton AL (1901) Goidelia japonica̶a new entozoic cope- The timing to start precopulatory guarding is interpret- pod from Japan, associated with an Infusorian (Trichodina). J ed as an evolutionary resolution of the intersexual conflict Linn Soc Zool 28: 211–229, pls. 21–22. that males favor a longer duration to assure paternity in Fisher WK, MacGinitie GE (1928) The natural history of an case of potential sperm competition while females favor a echiuroid worm. Ann Mag Nat Hist 1: 204–213. shorter duration because of the potential costs of guarding Gooding RU (1963) External morphology and classification of 150 M. Ijichi et al.

marine poecilostome copepods belonging to the families Clau- copulatory mate guarding. Plankton Benthos Res 3: 189–201. sidiidae, Clausiidae, Nereicolidae, Eunicicolidae, Synaptiphi- Jormalainen V (1998) Precopulatory mate guarding in crusta- lidae, Catiniidae, Anomopsyllidae, and Echiurophilidae. PhD ceans: Male competitive strategy and intersexual conflict. thesis, University of Washington, USA, 275 pp. Quart Rev Biol 73: 275–304. Goto R (2017) The Echiura of Japan: In: Diversity, Classification, Kang KH, Zhang Z, Kim JM, Seon SC, Shao M (2010) Effects of Phylogeny, and Their Associated Fauna. Species Diversity of Urechis unicinctus juveniles on chemical characteristics of or- Animals in Japan (eds Motokawa M, Kajihara H), Springer ganically contaminated coastal sediment. J Ocean Univ China Japan, Tokyo, pp. 513–542. 9: 48–52. Gotto RV (1979) The association of copepods with marine inver- Kern JC, Edwards NA, Bell SS (1984) Precocious clasping of tebrates. Adv Mar Biol 16: 1–109. early copepodite stages: a common occurrence in Zausodes Ho J-S (2001) Why do symbiotic copepods matter? Hydrobiolo- arenicolus Wilson (Copepoda: Harpacticoida). J Crustac Biol gia 453/454: 1–7. 4: 261–265. Humes AG (1994) How many copepods? Hydrobiologia 292/293: Kim I-H (1998) Illustrated Encyclopedia of Fauna and Flora of 1–7. Korea. Vol. 38. Cirripedia, Symbiotic Copepoda and Pycnogo- Itani G, Izichi M, Ueda H (2005) Crab species collected from the nida. Ministry of Education, Korea 1038 pp. (In Hangul) burrows of Urechis unicinctus in Hiuchi-nada, the central Seto Kim I-H (2000) Poecilostomatoid copepods from an intertidal Inland Sea, Japan. Cancer 14: 1–4. (in Japanese with English mud flat in the Yellow Sea. J Nat Hist 34: 367–432. abstract) Kim I-H (2016) Two new species of Echiuricopus n. gen. (Co- Itoh H, Nishida S (1995) Copepodid stages of Hemicyclops ja- pepoda, Poecilostomatoida) associated with echiurans from ponicus Itoh and Nishida (Poecilostomatoida: Clausidiidae) the Yellow Sea. Anim Syst Evol Divers 32: 9–20. reared in the laboratory. J Crustac Biol 15: 134–155. Nishikawa T (2012) Urechis unicinctus (Drasche,1881). In: Itoh H, Nishida S (2007) Life history of the copepod Hemicy- Threatened Animals of Japanese Tidal Flats: Red Data Book clops gomsoensis (Poecilostomatoida, Clausidiidae) associated of Seashore Benthos (ed Japanese Association of Benthology), with decapod burrows in the Tama-River estuary, central Ja- Tokai University Press, Kanagawa, pp. 238. (In Japanese) pan. Plankton Benthos Res 3: 134–146. Stock JH, Humes AG, Gooding RU (1962) Copepoda associated Itoh H, Nishida S (2008) Life history of the copepod Hemicy- with West Indian invertebrates - I. The genus Nanaspis (Sipho- clops spinulosus (Poecilostomatoida, Clausidiidae) associated nostoma, Nanaspidae). Stud Fauna Curaçao Carib Isl 13: 1–20. with crab burrows with notes on male polymorphism and pre-