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A new copepod with transformed body plan and unique phylogenetic position parasitic in the acorn worm Ptychodera flava.


Copepods are one of the most diverse and abundant animal groups in marine ecosystems and also the most common and widespread crustaceans living in symbiotic relationship with other organisms (Humes, 1985a, 1994; Ho, 2001). They comprise 10 orders (Calanoida, Cyclopoida, Gelyelloida, Harpacticoida, Thaumatopsylloida, Mormonilloida, Platycopioida, Poecilostomatoida, and Siphonostomatoida) based on the morphological characters (Huys and Boxshall, 1991; Ho et al, 2003; Huys et al., 2007), with a total of approximately 14,617 valid species (Walter and Boxshall, 2013). Among them, symbiotic or parasitic copepods constitute about one-third of the known copepods (Ho, 2001), and the hosts include all major groups of marine animals, including Porifera (Bandera et al., 2005), Cnidaria (Humes, 1985b), Mollusca (Huys, 2001), Crustacea (Humes and Ho, 1969), Echinodermata (Boxshall and Ohtsuka, 2001), Urochordata (Ooishi, 2001), Hemichordata (Mayer, 1879; Kesteven, 1913), and Chordata (Abaunza et al., 2001). Although more than 4000 symbiotic copepod species have been named, it is apparent that many species are yet to be found (Ho, 2001). Recently, molecular studies based on DNA sequences have provided important insights into the phylogenetic relationships within copepods (Huys et al., 2006, 2007, 2009, 2012). Parasitic copepods usually exhibit transformed or reduced body plans with fewer morphological characters for taxonomic identification. Therefore, when new parasitic copepods are discovered from previously unexplored habitats or hosts, molecular data may provide useful information to determine the phylogenetic affiliation of the new species.

Acorn worms (Enteropneusta), a solitary class of hemichordates, are marine vermiform invertebrates that generally live concealed in burrows (Hyman, 1959). There are about 70 species of acorn worms, and most of them belong to the families Harrimaniidae and Ptychoderidae (Cannon et al., 2009). Until now, only two species of copepods, Ive balanoglossi Mayer, 1879 and Ubius hilli Kesteven, 1913, have been described as parasites in acorn worms. Ive balanoglossi was collected from Glossobalanus minutus Kowalevsky, 1866 (= Balanoglossus minutus) in the Mediterranean Sea (Mayer, 1879); Ubius hilli was described as a parasite of Balanoglossus australiensis Hill, 1894 (= Ptychodera australis or Ptychodera australiensis) in Australian waters (Kesteven, 1913). Both parasitic copepods lack typical morphological features and cannot be allocated to any family of the Copepoda (Boxshall and Halsey, 2004). They were temporarily grouped together into the Ive-group due to the similarity in body transformation and number of appendages. Willey (1897) and Nishikawa (1977) also observed parasitic copepods from a widespread acorn worm, Ptychodera flava Eschscholtz, 1825, in the Marshall Islands and Kushimoto, Japan, respectively. These parasitic copepods were assumed to be I. balanoglossi even though no clear description was provided. The effects of the copepods on the acorn worm host are also not known.

Parasites can play important roles in all ecosystems and exert strong selection pressure on their hosts (Anderson, 1980; Price, 1980; Anderson and May, 1981; Michalakis and Hochberg, 1994). It has been inferred that parasitic copepods may feed on mucus, tissues, blood, and symbiotic algae as food sources from their hosts or associated animals (Humes, 1985a; Huys and Boxshall, 1991; Johnson et al., 2004; Cheng and Dai, 2010). They may also cause variable levels of localized lesions to their hosts and elicit host tissue responses through the process of attachment and feeding activities (Bron et al., 1991; Johnson and Albright, 1992; Roubal, 1994). Although many copepod species have been well documented for their potential influences upon their hosts, most studies have focused on the species causing significant mortality on economically important hosts, such as salmon and trout (Huys and Boxshall, 1991; Boxshall and Bravo, 2000; Johnson et al, 2004).

In this study, we present a full description of a new copepod species, Ive ptychoderae, collected from the acorn worm Ptychodera flava in Taiwan. Our study combines morphological features and molecular approaches to determine the taxonomic identification and phylogenetic position of this copepod. We also report infestation parameters (prevalence and intensity) of I. ptychoderae, which are considered to be the primary information for understanding the possible roles of parasites on their host populations (Bush et al, 1997; Rozsa et al, 2000; Smallridge and Bull, 2000; Mihalca et al., 2008). Moreover, we examined histological sections of the infected tissues of the acorn worms and the gut contents of their parasites to better understand the parasite-host relationships.

Materials and Methods

Sample collection and preparation

Acorn worms Ptychodera flava were collected monthly from September to December 2009, as well as in June 2010 and June 2011, with a shovel in the sandy beach at Chito, Penghu Islands, Taiwan (23[degrees]38'54.17"N, 119[degrees]36'14.40"E). To isolate the parasitic copepods, acorn worms were anesthetized with 0.2 mol [l.sup.-1] magnesium chloride in seawater for 15 min, and the copepods were dissected from cysts of hosts by using insect needles. Location of cysts, number and sex of copepods, and sex of hosts were determined under a dissecting microscope (Olympus SZ61). Length and width of copepods were measured in 120 (60 [female][female] and 60 [male][male]) individuals. All copepod specimens were preserved in 70% ethanol except for those used for scanning electron microscopic (SEM) observations and DNA extraction.

Morphological studies

Morphological observations of parasitic copepods were performed as described in Humes and Gooding (1964). Three copepods of each sex were cleared in 85% lactic acid for 1 h and dissected on a wooden slide under a dissecting microscope. The removed body parts and appendages were examined under a compound microscope (Zeiss AXIOS-KOP-40) with a series of magnifications up to 1000X. All drawings were made with the aid of a drawing tube.

For SEM observation, six copepod individuals (3 [female][female] and 3 [male][male]) were preserved in 2.5% glutaraldehyde in 0.1 mol [l.sup.-1] MOPS buffer (0.1 mol [l.sup.-1] MOPS, 2 mmol [1.sup.-1] MgS[0.sub.4], 1 mmol [l.sup.-1] EGTA, 0.5 mol [l.sup.-1] NaCl, pH 7.5) at 4[degrees]C overnight and then post-fixed with 2% osmid (WAKO) in phosphate buffered saline (pH 7.4) overnight. Fixed copepods were dehydrated through a graded series of ethanol concentrations followed by critical-point-drying in a critical point dryer (Peleo CPD2). Dry individuals were coated with gold using a Cressington sputter coater (TED Pella, USA) and observed in an FEI Quanta 200 scanning electron microscope.

For histological observations, 10 infected acorn worms were anesthetized with 0.2 mol [l.sup.-1] magnesium chloride in seawater for 15 min, fixed with 4% paraformaldehyde in 0.1 mol [l.sup.-1] MOPS buffer at 4[degrees]C overnight, and then dehydrated through a graded series of ethanol concentrations. Samples were embedded in paraffin and sectioned at 5 [micro]m, or were embedded in low-viscosity Spurr's resin (EMS Cat. 14300) and sectioned at 1 [micro]m using a Leica Ultracut UCT microtome. Hematoxylin and eosin and toluidine blue (1% in 2% sodium borate solution) stains were applied to the sections, and the images were documented using a Zeiss Axio Imager.A1 microscope.

Phylogenetic analysis

For molecular phylogenetic analysis, a DNeasy Blood and Tissue kit (Qiagen) was used to extract genomic DNA from a single adult female parasite. For amplifying the 18S rDNA gene, PCR was carried out using primers 18-e (5'CTGGTTGATCCTGCCAGT-3') (Hillis and Dixon, 1991) and 18-p-c (5'-TAATGATCCTTCCGCAGGTTCACCT3') (Winchell et al, 2002) with the following conditions: 94 [degrees]C for 30 s, 55[degrees]C for 30 s, 72[degrees]C for 1.5 min. The PCR products were cloned and sequenced. The sequence was aligned with published copepod 18S sequences (Huys et al, 2006, 2007, 2009, 2012) using MUSCLE ver. 3.8.31 (Edgar, 2004). Alignments were used to construct phylogenetic trees with two phylogenetic methods, maximum likelihood (ML) and Bayesian inference (BI). ML was performed using PHYML ver. 20120412 with default settings (Guindon et al., 2010) and BI analysis with MrBayes ver. 3.1.2 (Ronquist and Huelsenbeck, 2003). The level of bootstrap support for ML was calculated by 10,000 resamplings using SEQBOOT in the PHYLIP ver. 3.69 package. For BI analysis, the nucleotide substitution model was set to mixed with gamma-distributed rate variation across sites and a proportion of invariable sites. The Markov chain Monte Carlo analysis was set to run for 1,000,000 generations and sampled every 100 generations. The first 25% of the samples were discarded as the burn-in. Parameters not specified were set to the default. The phylogenetic trees were illustrated with the FigTree ver. 1.4.0 program. GenBank accession number for the 18S rDNA sequence of I. ptychoderae is JF417992 (under the name "Ive sp. YHS-2012").

Statistical procedures

Total numbers of parasites were determined directly by numerical count. Prevalence, mean intensity, and median intensity were recorded as described in Bush et al. (1997) and Rozsa et al. (2000). Monthly differences in parasite prevalence were determined using Fisher's exact test in the Quantitative Parasitology 1.0 software (Rozsa et al., 2000). One-way ANOVA and Duncan's multiple comparison test in the SPSS 12 package programs were used for analyzing the differences of the mean intensities. Differences in median intensity were analyzed using Mood's median test in the Quantitative Parasitology 1.0 software.



Genus Ive Mayer, 1879

Ive ptychoderae, n. sp.

Type-host: Ptychodera flava Eschscholtz, 1825

Site: Parasitic copepods mostly appear in the cysts at the edges of the genital wings or the branchial region of the host (Fig. 1A, B). Male holds the female with clawlike maxilliped in the same cyst. Females were frequently accompanied by one or several males, which are smaller than the female (Fig. 1C).

Type-locality: Chito, Penghu Islands, Taiwan.

Etymology: The specific name, ptychoderae, is derived from the generic name of its host.

Type-material: Twenty-six [female][female] and 31 [male][male] collected on 24 September 2009; 60 [female][female] and 12 [male][male] collected on 26 October 2009, 20 [female][female] and 40 [male][male] collected on 17 November 2009, 24 [female][female] and 40 [male][male] collected on 2 December 2009, 31 [female][female] and 50 [male][male] collected on 30 June 2010, and 23 [female][female] and 21 [male][male] collected on June 2011 (Table 1). Holotype (SINICA-COPEPOD 001), allotype (SINICA-COPEPOD 002), and paratypes (SINICA-COPEPOD sOOl) were deposited at the Institute of Cellular and Organismic Biology, Academia Sinica, Taipei, Taiwan. Remaining specimens are retained in the first author's private collection.

Taxonomic description

Figures 2, 3 and 4

Female: Body (Figs. 2A and 4A) vermiform, lacking external segmentation. Average length 8.84 [+ or -] 0.51 mm (mean [+ or -] SD), ranging from 2.61 to 23.27 mm; average width 1.26 [+ or -] 0.04 mm, ranging from 0.45 to 1.94 mm, based on 60 specimens. Prosome (Figs. 2A and 4A) with 4 pairs of annular swellings. Urosome (Figs. 2B and 4A) much shorter than prosome, bearing only 1 pair of swellings. Caudal ramus (Fig. 2C) 1 and 4 lateral tubercles.

Rostral area (Fig. 2D) unarmed. Cephalic region swollen laterally. Antennule (Fig. 2E) short, unsegmented, with scattered spinules, bearing 3 (2 subterminal and 1 terminal) spines and 5 tubercles in distal region. Chelate antenna (Fig. 2F) robust, indistinctly 3-segmented; basal segment largest, bearing 4 patches of spinules; second segment with pointed process and basal spiniform seta; distal segment tipped with short claw in addition to carrying 2 spiniform setae. Oral aperture (Figs. 2D and 4C) distinct, with slightly raised rim (lip), which is divided into 2 halves (anterior and posterior) by a pair of tiny, bifid, lateral knobs (Fig. 4C, indicated with p).

Mandible, maxillule, and maxilla absent. Maxilliped (Fig. 2D) sexually dimorphic, reduced to tiny lobe located not far posterior to oral aperture.

Leg 1 (Fig. 2G) biramous; 2-segmented large protopod with strongly arched intercoxal sclerite, bearing bifid, unsegmented exopod and endopod. Leg 2 similar to leg 1. Legs 3-5 absent.

Male: Body (Figs. 3A and 4B) highly transformed, average length 3.40 [+ or -] 1.50 mm (mean [+ or -] SD) ranging from 1.79 to 7.54 mm; average width 0.53 [+ or -] 0.19 ranging from 0.32 to 1.13 mm, based on 60 specimens. Segmentation of body indistinct. Body without annular swellings. Caudal ramus (Fig. 3B) generally as in female but much smaller.

Antennule and antenna generally like those in female. Rim of oral aperture with more prominent lateral bifid knobs (Fig. 4D). Mandible, maxillule, and maxilla absent. Maxilliped (Fig. 3C) 2-segmented, first segment larger than the second, with scattered spinules; distal segment small, carrying 3 spines. Legs 1-2 as in female. Legs 3-5 absent.

Remarks: Inasmuch as the original species description of I. balanoglossi, the only existing congener of the present new species, lacked the morphological details of the appendages, it is impossible to make a close comparison with 1. ptychoderae. Although I. balanoglossi has been sighted twice since Mayer's (1879) report, once by Willey (1897) found in P. flava in the Marshall Islands and another time by Nishikawa (1977) also found in P. flava but from Kushimoto, Japan, the parasite was only mentioned without addition of any morphological information. Nevertheless, the difference in the number of annular swellings, four in I. balanoglossi and five in I. ptychoderae, clearly indicates that the species of Ive from Taiwan is different from the species reported by Mayer (1879) from the Mediterranean.

Molecular phylogenetic analysis

In an attempt to determine the phylogenetic position of I. ptychoderae within the Copepoda, we performed molecular phylogenetic analyses using the 18S rDNA sequences (Fig 5). Both the Bayesian and ML analyses showed that I. ptychoderae groups with Poecilostomatoida, although the supporting values are low (Bayesian posterior probability 0.84 and bootstrap value 32). In addition, I. ptychoderae is not closely related to any copepod family with known 18S sequences. We noted that the Clausidiiform complex of Poecilostomatoida does not form a monophyletic group with other Poecilostomatoida species and I. ptychoderae. This result is similar to a previous published study (Huys et al., 2012). Therefore, we concluded that I. ptychoderae is possibly a member of Poecilostomatoida and may belong to a distinct clade within the order.

Biology of Ive ptychoderae n. sp.

According to our sampling records in Penghu Islands from September 2009 to June 2011 (Table 1), 444 individuals of I. ptychoderae were obtained from 290 acom worms.

The average prevalence of I. ptychoderae in the P. flava population during the collecting period was 42.41% (123/ 290), ranging from 32.89% (June 2010) to 69.23% (November 2009). The prevalence values were not significantly different between months during the collecting period (Fisher's exact test, P = 0.0575). The mean intensity of I. ptychoderae varied significantly between the months (P < 0.05), and a maximum mean intensity (6.67 [+ or -] 2.45) was recorded in November 2009. The median intensity, ranging from 2.0 to 7.0 parasites per infected acorn worm, also showed a similar trend with the mean intensities observed between each month (Table 1).

Individuals of I. ptychoderae were commonly found near the edges of the genital wings (86%, n = 106, Fig. 1 A), in the branchial region (5%, n = 6, Fig. 1B), or in both regions (9%, n = 11) of the acorn worms. The number of cysts varied among acorn worm individuals. In most cases, the infected acorn worms contained one (60%, n = 74) or two (33%, n = 41) cysts. A few acorn worms were found to have three (4.88%, n = 6) or four (1.63%, n = 2) cysts. In general, a single cyst contains one female of I. ptychoderae accompanied by one or more (up to 7) males. The most common cases were 1 9/1 [male] (37%, n = 46) followed by 1 [female]/2 [male][male] (21.86%, n = 27). We also found that 15.85% (n = 19) of the cysts contained only a single female without any males, while cysts containing only male I. ptychoderae were rare.

We observed that 21.74% to 55.00% of I. ptychoderae females carried egg strings containing eggs or embryos (ovigerous females; Table 1), suggesting that they can produce offspring during our sampling period. Each egg string contained approximately 637.97 [+ or -] 307.90 eggs (n = 30) (Fig. 6A). When mature egg strings were dissected from hosts in seawater, nauplius lavae hatched rapidly (Fig. 6B). Swimming nauplius larvae maintained in seawater with mixed algae at room temperature moulted into copepodid larvae (Fig. 6C) in 3 to 4 days. In situ observations showed that I. ptychoderae went through embryonic development until the nauplius stage inside the egg strings within hosts (Fig. 6D), and the nauplius larvae were later released into seawater.

To observe the relationship between the parasitic copepod and acorn worm host in situ, we examined histological sections of the infected tissues of acorn worms and found that cysts in the genital wing of the infected acorn worm were enclosed by the hypertrophied epithelium and connective tissues of the host (Fig. 6E, F). We also observed significant degeneration of host muscular tissue in the cystcontaining regions (Fig. 6F). Inside the digestive tracts of I. ptychoderae, we observed chyme containing disintegrated host tissues (Fig. 6G, H) and host eggs covered with multilayered peritrophic membranes (Fig. 61). These results suggest that I. ptychoderae feeds on somatic and gonadal tissues of its acorn worm host.


Phylogenetic affiliation of Ive ptychoderae n. sp.

Since 1879, only two parasitic copepods, Ive balanoglossi and Ubius hilli, have been described from acorn worms. Boxshall and Halsey (2004) suggested that these two genera (Ive and Ubius) are clearly not related to Siphonostomatoida since they lacked an oral cone. With the absence of the labrum and concomitant modification of thoracopods into exopods and endopods, these two genera are also excluded from any of the existing poecilostome families. However, the presence of sexually dimorphic maxilliped in I. ptychoderae suggests that this new species (and the two genera) may belong to the Poecilostomatoida (Huys and Boxshall, 1991; Kim, 2004). Our molecular analyses placed I. ptychoderae with non-Clausidiiform Poecilostomatoida, although the supporting values are not confident enough (Fig. 5). Nevertheless, both morphological and molecular features suggest that I. ptychoderae may represent a member of a distinct copepod clade. Further molecular phylogenetic analysis using 18S rDNA sequences or other molecular markers from related species, such as I. balanoglossi and U. hilli, would be necessary to understand the exact phylogenetic position of these parasitic copepods. In addition, uncovering and comparing morphological features of postembryonic stages of I. ptychoderae may provide more insights into its phylogenetic position.

Because of the highly distinct morphological and molecular character of I. ptychoderae, we propose herein to establish a new family, Iveidae, to accommodate both Ive and Ubius. The diagnosis of this new family is as follows.

Female: Body highly transformed, vermiform, large (may be up to 23 mm), and lacking external segmentation; trunk cylindrical in both, but with annular swellings along body in Ive. Body tapering posteriorly in Ubius, but with paired caudal rami in Ive. First leg-bearing somite separates from cephalosome. Mouth aperture distinct, without labrum or labium.

Antennule stubby, unsegmented, and tipped with elements. Antenna uniramous, 3-segmented; forming chelate apparatus with pointed process of middle segment lying against claw of terminal segment. Mandible, maxillule, and maxilla absent. Maxilliped sexually dimorphic, reduced to tiny lobe in Ive, but retained as 2-segmented functional appendage in Ubius.

Protopod of first and second thoracopods large, carrying biramous rami, with each ramus transformed into movable chela. Legs 3 to 5 absent.

Male: Similar to female but with relatively much shorter body (up to 7.5 mm). Maxilliped 2-segmented, with large proximal segment and small distal segment tipped with 2 or 3 processes.

Prevalence and intensity of Ive ptychoderae n. sp. within the hemichordate Ptychodera flava

During our sampling period we found more than one-third of the acorn worms collected in Penghu Islands to be infected by I. ptychoderae. It suggests that I. ptychoderae is a common parasite within the acorn worm population. Our results also show that the highest infestation of I. ptychoderae occurred in November. Since sexual reproduction of P. flava is restricted primarily from October to December in Taiwan (unpubl. data), the highest infestation is possibly related to the weakening of the defense mechanisms of acorn worms during their reproductive season. These results support the hypothesis of a trade-off between reproductive effort and the ability to defend against parasitic infection (Festa-Bianchet, 1989; Norris et al., 1994). The investment of acorn worms in reproduction may result in higher susceptibility to parasite infection. However, the ecological conditions may also play critical roles in altering the parasite community (Mouritsen and Poulin, 2002; Poulin and Mouritsen, 2005; Mpller, 2010). Further studies with monthly sampling and long-term prevalence and intensity analyses in the wild are required to address these issues.

The same acorn worm species P. flava is commonly found in Hawaii and has been used for biological research, especially developmental studies, during the past 15 years (Lowe et al., 2004; Rottinger and Lowe, 2012). However, the occurrence of parasitic copepods has as yet not been recorded from this population. If I. ptychoderae is indeed absent in Hawaiian P. flava, it would suggest that there may be some barriers preventing the long-distance dispersal of this copepod parasite. Alternatively, parasitic copepods are present within the P. flava population in Hawaii Islands but were not noticed by the researchers. It would require careful reexamination of the P. flava from Hawaii Islands to confirm this issue. This issue is particularly important since P. flava has recently become one of the major hemichordate model species for evolutionary developmental biology research (review in Rottinger and Lowe, 2012), and studies of its genome have provided new insights into the evolution of deuterostomes (Freeman et al, 2012; Ikuta et al, 2013). Therefore, to avoid the potential problem of contamination, it would be necessary to make sure that there is no parasitic copepod present in the biological materials of P. flava for future genomic studies. Similar care should be taken for other acom worm species, such as Saccoglossus kowa levskii, which has also been used frequently for developmental and genomic studies in recent years (Freeman et al., 2008; Cameron and Bishop, 2012), although no parasites have been reported yet from this species.

It should be noted that the differences in levels of parasite infection between sexes is a common phenomenon in host species (Morand et al., 2004; Christe et al., 2007). In our studies, when the sex of the infested acorn worms can be identified during the gametogenesis period, we observed that the sex ratio (male/female) of infested acorn worms was similar to the sex ratio of acorn worms in the wild population at Chito, Penghu Islands (3:2, unpubl. data). Previous studies pointed out that the sex-specific parasitism may be mediated by gender differences in susceptibility to parasitism and that male hosts seem to be more susceptible than females (Zuk, 1990; Schalk and Forbes, 1997; Klein, 2000; Moore and Wilson, 2002). Our observations, to a certain extent, suggest that the infection of P. flava by I. ptychoderae in Penghu is not sex-biased and is a common phenomenon within this population.

Infection by Ive ptychoderae causes damage in host tissues

Ive ptychoderae may elicit defense reactions by its acorn worm host and trigger the formation of cystic dilatation in host tissues. We suspect that the process of cyst formation in acorn worms by I. ptychoderae may be similar to that of gall-forming copepods, whose appendages attach to soft tissues of their coral hosts and elicit defense reactions by depositing a calcareous barrier (gall) (Dojiri, 1988; BuhlMortensen and Mortensen, 2004). In addition, we observed that mature eggs hatched and directly developed into larval stages inside the cyst (Fig. 6D), and the larvae were subsequently released from their hosts, possibly through "birth" pores. The movements of the parasites may cause localized lesions in the tissues of their host. In our study a variety of such lesions were observed, including degeneration of muscular tissue, thickening of vascularized basement membranes, mesenchymal cell aggregation, and expansion of epidermis.

Sex ratio and sex determination in Ive ptychoderae

Although the sex ratio (male/female) of I. ptychoderae in the acorn worm population ranged from 1.17 to 2.00 (Table 1), suggesting a male-biased population, we rarely found cysts containing only male I. ptychoderae in our collection. In most cases, we observed one female, either with or without males. This observation raises the possibility that the sex determination in I. ptychoderae may be based on the environmental conditions experienced by different individuals after they infect the host. The environmental sex determination mechanism has been reported in the parasitic copepod Pachypygus gibber of the tunicate Ciona intestinalis (Becheikh et al., 1998; Michaud et al., 2004). In P. gibber, it has been demonstrated that in a rich environment (abundant food resource) the larvae tend to develop into females; in addition, it was reported that an existing sexual partner in a particular environment exerts a strong influence on sex determination of the newcomer (Becheikh et al., 1998). It is possible that I. ptychoderae also employs a similar mechanism for sex determination. We hypothesize that P. flava represents a rich host environment, and the initial individuals of I. ptychoderae entering this environment tend to differentiate into females. On the other hand, if a female I. ptychoderae is already present inside an acorn worm, when the newcomers encounter this female they may differentiate into males and attach to the female with a maxilliped.

Parasite-host coevolution

Ptychodera flava is considered a widespread species across the Indo-Pacific Ocean (Lowe et al., 2004). Previously, Willey (1897) and Nishikawa (1977) reported the occurrence of parasitic copepods from P. flava in the Marshall Islands and Japan, respectively; however, detailed descriptions of the parasites were not provided, and the name "Ive balanoglossi" was given in the literature. In the present study, we show that the parasitic copepod found within P. flava in Penghu Islands is a new species, I. ptychoderae. Further re-sampling would be required to confirm whether the previously reported parasitic copepods from P. flava in the Marshall Islands and Japan are different species or indeed belong to I. ptychoderae. In addition, I. ptychoderae found in P. flava in the Pacific Ocean represents an additional parasite-host pair to the two previously described cases, I. balanoglossi of Glossobalanus minutus in the Mediterranean Sea and U. hilli of Balanoglossus australiensis in Australian waters. Although the morphology of I. ptychoderae is distinguishable from I. balanoglossi and U. hilli, they are similar in their highly transformed body plans. Further studies on the phylogenetic relationships and interactions between parasitic copepods and acorn worms among the three pairs will be needed to investigate whether the parasites coevolve with their hosts in different geographic areas.


We thank Mr. Tai-Lang Lin and the personnel in the ICOB core facility for excellent technical assistance with SEM. We are grateful to Hao-Hsiang Lee for his help in collecting acorn worm samples. This work was supported by intramural funding from the Institute of Cellular and Organismic Biology, Academia Sinica, Taiwan (to YHS and JKY), and a Career Development Award from Academia Sinica, Taiwan (to JKY). YHS and JKY were also supported by the National Science Council, Taiwan (1012627-B-001-001 to YHS and 101-2923-B-001-004-MY2 to YHS and JKY). Completion of this manuscript was aided by a grant from the Paramitas Foundation (to JSH).

Received 3 July 2013; accepted 3 February 2014.

Literature Cited

Abaunza, P., N. L. Arroyo, and I. Preciado. 2001. A contribution to the knowledge on the morphometry and the anatomical characters of Pennella balaenopterae (Copepoda, Siphonostomatoida, Pennellidae), with special reference to the buccal complex. Crustaceana 74: 193-210.

Anderson, R. M. 1980. Depression of host population abundance by direct life cycle macroparasites. J. Theor. Biol. 82: 293-311.

Anderson, R. M., and R. M. May. 1981. The population dynamics of microparasites and their invertebrate hosts. Philos. Trans. R. Soc. Lond. B 291: 451-524.

Bandera, M. E., M. Conradi, and P. J. Lopez-Gonzalez. 2005. Asterocheres hirsulus, a new species of parasitic copepod (Siphonostomatoida: Asterocheridae) associated with an Antarctic hexactinellid sponge. Helgol. Mar. Res. 59: 315-322.

Becheikh, S., M. Michaud, F. Thomas, A. Raihaut, and F. Renaud. 1998. Roles of resource and partner availability in sex determination in a parasitic copepod. Proc. R. Soc. Lond. B 265: 1153-1156.

Boxshall, G. A., and S. Bravo. 2000. The identity of the common Caligus (Copepoda: Siphonostomatoida: Caligidae) from salmonids net pen systems in southern Chile. Contrib. Zool. 69: 137-146.

Boxshall, G. A., and S. H. Halsey. 2004. An Introduction to Copepod Diversity. Ray Society, London.

Boxshall, G. A., and S. Ohtsuka. 2001. Two new families of copepods (Copepoda: Siphonostomatoida) parasitic on echinoderms. J. Crustac. Biol. 21: 96-105.

Bron, J. E., C. Sommervilie, M. Jones, and G. H. Rae. 1991. The settlement and attachment of early stages of the salmon louse Lepeophtheirus salmonis (Copepoda: Caligidae) on the salmon host, Salmo salar. J. Zool. 224: 201-212.

Buhl-Mortensen, L., and P. B. Mortcnsen. 2004. Gorgonophilus canadensis n. gen., n. sp. (Copepoda: Lamippidae), a gall forming endoparasite in the octocoral Paragorgia arborea (L., 1758) from the Northwest Atlantic. Symbiosis 37: 155-168.

Bush, A. O., K. D. Lafferty, J. M. Lotz, and A. W. Shostak. 1997. Parasitology meets ecology on its own terms: Margolis et al. revisited. J. Parasitol. 83: 575-583.

Cameron, C. B., and C. D. Bishop. 2012. Biomineral ultrastructure, elemental constitution and genomic analysis of biomineralization-related proteins in hemichordates. Proc. R. Soc. Lond. B 279: 3041-3048.

Cannon, J. T., A. L. Rvchel, H. Eccleston, K. M. Halanych, and B. J. Swalla. 2009. Molecular phylogeny of hemichordata, with updated status of deep-sea enteropneusts. Mol. Phylogenet. Evol. 52: 17-24.

Cheng, Y. R., and C. F. Dai. 2010. Endosymbiotic copepods may feed on zooxanthellae from their coral host, Pocillopora damicornis. Coral Reefs 29: 13-18.

Christe, P., O. Glaizot, G. Evanno, N. Bruyndonckx, G. Devevey, G. Yannic, P. Patthey, A. Maeder, P. Vogel, and R. Arlettaz. 2007. Host sex and ectoparasites choice: preference for, and higher survival on female hosts. J. Anim. Ecol. 76: 703-710.

Dojiri, M. 1988. Isomolgus desmotes, new genus, new species (Lichomolgidae), a gallicolous poecilostome copepod from the scleractinian coral Seriatopora hystrix Dana in Indonesia, with a review of gall-inhabiting crustaceans of anthozoans. J. Crustac. Biol. 8: 99-109.

Edgar, R. C. 2004. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 32: 1792-1797.

Festa-Bianchet, M. 1989. Individual differences, parasites, and the cost of reproduction in bighorn ewes (Ovis Canadensis). J. Anim. Ecol. 58: 785-795.

Freeman, R., T. Ikuta, M. Wu, R. Koyanagi, T. Kawashima, K. Tagawa, T. Humphreys, G.-C. Fang, A. Fujiyama, H. Saiga et al. 2012. Identical genomic organization of two hemichordate Hox clusters. Curr. Biol. 22: 2053-2058.

Freeman, R. M., Jr., M. Wu, M.-M. Cordonnier-Pratt, L. H. Pratt, C. E. Gruber, M. Smith, E. S. Lander, N. Stange-Thomann, C. J. Lowe, J. Gerhart, and M. Kirschner. 2008. cDNA sequences for transcription factors and signaling proteins of the hemichordate Saccoglossus kowalevskii: efficacy of the expressed sequence tag (EST) approach for evolutionary and developmental studies of a new organism. Biol. Bull. 214: 284-302.

Guindon, S., J. S. Dufayard, V. Lefort, M. Anisimova, W. Hordijk, and O. Gascuel. 2010. New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0. Syst. Biol. 59: 307-321.

Hillis, D. M., and M. T. Dixon. 1991. Ribosomal DNA: molecular evolution and phylogenetic inference. Q. Rev. Biol. 66: 411-453.

Ho, J. S. 2001. Why do symbiotic copepods matter? Hydrobiologia 453/454: 1-7.

Ho, J. S., M. Dojiri, G. Hendler, and G. B. Deets. 2003. A new species of copepoda (Thaumatopsyllidae) symbiotic with a brittle star from California, U.S.A., and designation of a new order Thaumatopsylloida. J. Crustac. Biol. 23: 582-593.

Humes, A. G. 1985a. Cnidarians and copepods: a success story. Trans. Am. Microsc. Soc. 104: 313-320.

Humes, A. G. 1985b. A review of the Xarifiidae (Copepoda, Poecilostomatoida), parasites of scleractinian corals in the Indo-Pacific. Bull. Mar. Sci. 36: 467-632.

Humes, A. G. 1994. How many copepods? Hydrobiologia 292/293: 1-7.

Humes, A. G., and R. U. Gooding. 1964. A method for studying the external anatomy of copepods. Crustaceana 6: 238-240.

Humes, A. G., and J. S. Ho. 1969. The genus Sunaristes (Copepoda, Harpacticoida) associated with hermit crabs in the western Indian Ocean. Crustaceana 17: 1-18.

Huys, R. 2001. Splanchnotrophid systematics: a case of polyphyly and taxonomic myopia. J. Crustac. Biol. 21: 106-156.

Huys, R., and G. A. Boxshall. 1991. Copepod Evolution. Ray Society, London.

Huys, R., J. Llewellyn-Hughes, P. D. Olson, and K. Nagasawa. 2006. Small subunit rDNA and Bayesian inference reveal Pectenophilus ornatus (Copepoda incertae sedis) as highly transformed Mytilicolidae, and support assignment of Chondracanthidae and Xarifiidae to Lichomolgoidea (Cyclopoida). Biol. J. Linn. Soc. 87: 403-425.

Huys, R., J. Llewellyn-Hughes, S. Conroy-Dalton, P. D. Olson, J. N. Spinks, and D. A. Johnston. 2007. Extraordinary host switching in siphonostomatoid copepods and the demise of the Monstrilloida: integrating molecular data, ontogeny and antennulary morphology. Mol. Phylogenet. Evol. 43: 368-378.

Huys, R., J. Mackenzie-Dodds, and J. Llewellyn-Hughes. 2009. Cancrincolidae (Copepoda, Harpacticoida) associated with land crabs: a semiterrestrial leaf of the ameirid tree. Mol. Phylogenet. Evol. 51: 143-156

Huys, R., F. Fatih, S. Ohtsuka, and J. Llewellyn-Hughes. 2012. Evolution of the bomolochiform superfamily complex (Copepoda: Cyclopoida): New insights from ssrDNA and morphology, and origin of umazuracolids from polychaete-infesting ancestors rejected. Int. J. Parasitol. 42: 71-92.

Hyman, L. H. 1959. The Invertebrates: Smaller Coelomate Groups: Chaetognatha, Hemichordata, Pogonophora, Phoronida, Ectoprocta, Brachipoda, Sipunculida, the Coelomate Bilateria. McGraw-Hill, New York.

Ikuta, T., Y. C. Chen, R. Annunziata, H. C. Ting, C. H. Tung, R. Koyanagi, K. Tagawa, T. Humphreys, A. Fujiyama, H. Saiga et al. 2013. Identification of an intact ParaHox cluster with temporal colinearity but altered spatial colinearity in the hemichordate Ptychodera flava. BMC Evol. Biol. 13: 129.

Johnson, S. C., and L. J. Albright. 1992. Comparative susceptibility and histopathology of the host response of native Atlantic, chinook, and coho salmon to experimental infection with Lepeophtheirus salmonis (Copepoda: Caligidae). Dis. Aquat. Org. 14: 179-193.

Johnson, S. C., J. W. Treasurer, S. Bravo, K. Nagasawa, and Z. Kabata. 2004. A review of the impact of parasitic copepods on marine aquaculture. Zool. Stud. 43: 229-243.

Kesteven, H. L. 1913. A new endoparasitic copepod: morphology and development. Proc. Linn. Soc. NSW 37: 673-688.

Kim, I. H. 2004. Copepodid stages of Ergasilus hypomesi Yamaguti (Copepoda, Poecilostomatoida, Ergasilidae) from a brackish lake in Korea. Korean J. Biol. Sci. 8: 1-12.

Klein, S. L. 2000. The effects of hormones on sex differences in infection: from genes to behavior. Neurosci. Biobehav. Rev. 24: 627-638.

Lowe, C. J., K. Tagawa, T. Humpreys, M. Kirschner, and J. Gerhart. 2004. Hemichordate embryos: procurement, culture, and basic methods. Methods Cell Biol. 74: 171-194.

Mayer, P. 1879. Ein neuer parasitischer Copepode. Mitt. Zool. Stn. Neapel 1: 515-521.

Michalakis, Y., and M. E. Hochberg. 1994. Parasitic effects on host life-history traits: a review of recent studies. Parasite 1: 291-294.

Michaud, M., T. de Meeus, and F. Renaud. 2004. Environmental sex determination in a parasitic copepod: checking heterogeneity and unpredictability of the environment. Mar. Ecol. Prog. Ser. 269: 163-171.

Mihalca, A. D., K. Racka, C. Gherman, and D. T. Ionescu. 2008. Prevalence and intensity of blood apicomplexan infections in reptiles from Romanis. Parasitol. Res. 102: 1081-1083.

Moller, A. P. 2010. Host-parasite interactions and vectors in the barn swallow in relation to climate change. Glob. Change Biol. 16: 1158-1170.

Moore, S. L., and K. Wilson. 2002. Parasites as a viability cost of sexual selection in natural populations of mammals. Science 297: 2015-2018.

Morand, S., J. G. De Beliocq, M. Stanko, and D. Miklisova. 2004. Is sex-biased ectoparasitism related to sexual size dimorphism in small mammals of Central Europe? Parasitology 129: 505-510.

Mouritsen, K. N., and R. Poulin. 2002. Parasitism, climate oscillations and the structure of natural communities. Oikos 97: 462-468.

Nishikawa, T. 1977. Preliminary report on the biology of the enteropneust, Ptychodera flava Eschscholtz, in the vicinity of Kushimoto, Japan. Publ. Seto Mar. Biol. Lab. 23: 393-419.

Norris, K., M. Anwar, and A. F. Read. 1994. Reproductive effort influences the prevalence of haematozoan parasites in great tits. J. Anim. Ecol. 63: 601-610.

Ooishi, S. 2001. Two ascidicolous copepods, Haplostomides otagoensis n. sp. and Botryllophilus cf. banyulensis Brement, living in compound ascidians from Otago Harbor, New Zealand. Hydrobiologia 453/454: 417-426.

Poulin, R., and K. N. Mouritsen. 2005. Climate change, parasitism and structure of intertidal ecosystems. J. Helminthol. 80: 183-191.

Price, P. W. 1980. Evolutionary Biology of Parasites. Princeton University Press, Princeton, NJ.

Ronquist, F.. and J. P. Huelsenbeck. 2003. MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19: 1572-1574.

Rottinger, E., and C. J. Lowe. 2012. Evolutionary crossroads in developmental biology: hemichordates. Development 139: 2463-2475.

Roubal, F. R. 1994. Histopathology caused by Caligus epidemicus Hewitt (Copepoda: Caligidae) on captive Acanthopagrus australis (Giinther) (Pisces: Sparidae). J. Fish Dis. 17: 631-640.

Rozsa, L., J. Reiczigel, and G. Majoros. 2000. Quantifying parasites in samples of hosts. J. Parasitol. 86: 228-232.

Schalk, G., and M. R. Forbes. 1997. Male biases in parasitism of mammals: effects of study type, host age, and parasite taxon. Oikos 78: 67-74.

Smallridge, C. J., and C. M. Bull. 2000. Prevalence and intensity of the blood parasite Hemolivia mariae in a field population of the skink Tiquila rugosa. Parasitol. Res. 86: 655-660.

Walter, T. C., and G. Boxshall. 2013. World Copepods database. [Online], Available: [2013, July 1].

Willey, A. 1897. Memoirs: On Ptychodera flava, Eschscholtz. Q. J. Microsc. Sci. s2-40: 165-184.

Winchell, C. J., J. Sullivan, C. B. Cameron, B. J. Swalla, and J. Mallatt. 2002. Evaluating hypotheses of deuterostome phylogeny and chordate evolution with new LSU and SSU ribosomal DNA data. Mol. Biol. Evol. 19: 762-776.

Zuk, M. 1990. Reproductive strategies and disease susceptibility: an evolutionary viewpoint. Parasitol. Today 6: 231-233.


(1) Institute of Cellular and Organismic Biology, Academia Sinica, Taipei, Taiwan; (2) Institute of Oceanography, National Taiwan University, Taipei, Taiwan; (3) Department of Biological Sciences, California State University, Long Beach, California; and (4) Institute of Plant and Microbial Biology, Academia Sinica, Taipei, Taiwan

* To whom correspondence should be addressed. E-mail: jkyu@gate.;

(#) Authors contributed equally to this work.

Table 1
Summary of statistics for the parasite Ive ptychoderae found in the
corn worm Ptychodera flave

                                           Sep. 2009

No. of collected acorn worms                  47
No. of infected acorn worms                   16
Parasite abundance (male/female)          57 (31/26)
Sex ratio of parasites                       1.19
No. of ovigerous females                       7
Percentages of ovigerous females             26.92
Prevalence (%)                               34.04
Mean ([+ or -] SD) intensity        3.56 [+ or -] 1.50 (ab)
Median intensity                              4.0

                                          Oct. 2009

No. of collected acorn worms                 103
No. of infected acorn worms                   46
Parasite abundance (male/female)         132 (72/60)
Sex ratio of parasites                       1.20
No. of ovigerous females                      33
Percentages of ovigerous females            55.00
Prevalence (%)                              44.66
Mean ([+ or -] SD) intensity        2.81 [+ or -] 1.80 (b)
Median intensity                             2.0

                                          Nov. 2009

No. of collected acorn worms                  13
No. of infected acorn worms                   9
Parasite abundance (male/female)          60 (40/20)
Sex ratio of parasites                       2.00
No. of ovigerous females                      6
Percentages of ovigerous females            30.00
Prevalence (%)                              69.23
Mean ([+ or -] SD) intensity        6.67 [+ or -] 2.45 (a)
Median intensity                             7.0

                                           Dec. 2009

No. of collected acorn worms                  30
No. of infected acorn worms                   16
Parasite abundance (male/female)          64 (40/24)
Sex ratio of parasites                       1.67
No. of ovigerous females                       9
Percentages of ovigerous females             37.50
Prevalence (%)                               53.33
Mean ([+ or -] SD) intensity        4.00 [+ or -] 1.83 (ab)
Median intensity                              4.0

                                          Jun. 2010

No. of collected acorn worms                  76
No. of infected acorn worms                   25
Parasite abundance (male/female)          81 (50/31)
Sex ratio of parasites                       1.61
No. of ovigerous females                      11
Percentages of ovigerous females            35.48
Prevalence (%)                              32.89
Mean ([+ or -] SD) intensity        3.24 [+ or -] 1.42 (b)
Median intensity                             3.0

                                           Jun. 2011

No. of collected acorn worms                  21
No. of infected acorn worms                   11
Parasite abundance (male/female)          50 (27/23)
Sex ratio of parasites                       1.17
No. of ovigerous females                       5
Percentages of ovigerous females             21.74
Prevalence (%)                               52.38
Mean ([+ or -] SD) intensity        4.55 [+ or -] 3.42 (ab)
Median intensity                              4.0

During the sampling periods, 290 acorn worms were examined and 444
parasitic copepods (184 [female][female] and 260 [male][male]) were
found. Various superscripts denote significant differences (P < 0.05)
between groups.
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Author:Tung, Che-Huang; Cheng, Yu-Rong; Lin, Ching-Yi; Ho, Ju-Shey; Kuo, Chih-Horng; Yu, Jr.-Kai; Su, Yi-Hs
Publication:The Biological Bulletin
Article Type:Report
Geographic Code:1USA
Date:Feb 1, 2014
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