A new orthopteran-parasitizing horsehair worm, Acutogordius taiwanensis sp. n., with a redescription of Chordodes formosanus and novel host records from Taiwan (Nematomorpha, Gordiida)

Abstract A description of a new species of horsehair worm, Acutogordius taiwanensis sp. n., a redescription of Chordodes formosanus, and novel host records for the latter are provided. Acutogordius taiwanensis sp. n. is morphologically similar to A. protectus with moderately flat areoles on its tail tips, but is distinguishable by small mid-body ornamentations. Despite the distinct differences in the post-cloacal crescents between 14 male samples, their conspecific status, along with that of nine female samples, was upheld by a phylogenetic comparison of partial cytochrome oxidase subunit I (COI) sequences. Chordodes formosanus is another common horsehair worm species in Taiwan, which was previously believed to specifically parasitize Hierodula mantids. However, in this study, five C. formosanus were observed emerging from an Acromantis mantid, and two long-horned grasshopper hosts (Leptoteratura sp. and Holochlora japonica). These five worms showed high degrees of similarity in COI sequences and morphology, but one of these individuals bore abnormal crowned areoles, which has never been observed in C. formosanus, and may be attributed to the incomplete development of this particular individual.


Introduction
Horsehair worms (phylum Nematomorpha) are aquatic parasites whose life cycle typically contains a free-living, aquatic phase, including mating and early larval development, and two parasitic stages, including an aquatic paratenic host stage and a terrestrial definitive host stage (Schmidt-Rhaesa 2014, Bolek et al. 2015). Over 350 freshwater species have been described worldwide , but only one, Chordodes formosanus Chiu, 2011, is known in Taiwan (Chiu et al. 2011). Acutogordius taiwanensis sp. n. is another commonly encountered species, which is usually sympatric with C. formosanus at low altitudes in Taiwan. In the aquatic paratenic host stage, cysts of Acutogordius and Chordodes have been found sympatrically in aquatic chironomids (Chiu et al. 2016), whereas the adults generally parasitize various terrestrial hosts.
The definitive hosts of C. formosanus are Hierodula mantids (Chiu et al. 2011), whereas adults of Acutogordius taiwanensis sp. n. in Taiwan generally emerge from orthopteran insects. Acutogordius is a small genus that consists of ten described species (de Miralles and de Villalobos 1998, Schmidt-Rhaesa and Geraci 2006, Schmidt-Rhaesa and Schwarz 2016 in the family Gordiidae (Poinar 2008). Only two genera in the Gordiidae, Acutogordius, and Gordius, are characterized by a post-cloacal crescent located at the base of the two tail lobes, but are distinguishable by the distinctly pointed tips on the Acutogordius male's tail lobes (Schmidt-Rhaesa 2002). Classification of Acutogordius species is primarily based on the characters of the males' tails. However, interspecific variation has not been well defined, and distinction between the species is still not clear (Schmidt-Rhaesa and Geraci 2006).
In the present study, the conspecific status of 23 Acutogordius samples is established according to minor differences observed in the sequences of their partial mitochondrial DNA cytochrome oxidase subunit I (mtDNA-COI) genes, collected from eleven species of orthopteran hosts, and the morphological differences among these samples is determined as intraspecific variation. Furthermore, the morphology of the immature stages of this species was also described. Five horsehair worm samples that had emerged from Acromantis mantids and two long-horned grasshopper species, i.e., Leptoteratura sp. and Holochlora japonica, were identified as C. formosanus based on morphological and molecular evidence, and thus added these insect species as novel definitive hosts of C. formosanus.

Materials and methods
The morphologies and DNA sequences of 29 adult horsehair worms (24 Acutogordius and 5 Chordodes) were examined. Two pairs of Acutogordius were reared in the laboratory for two weeks to breed and lay eggs, and the morphology of the resulting larvae was examined using a light microscope (Olympus BH-2, PM-10AD, Tokyo, Japan). Specimens (partial bodies with their hosts) were preserved at the Department of En-tomology, National Taiwan University, Taipei; National Museum of Natural Science, Taichung, Taiwan; and Lake Biwa Museum, Shiga, Japan. Specimen examination followed our previously published methods described in Chiu et al. (2011).

Collection and preservation of horsehair worms
Insect hosts infected with horsehair worms were hand-collected from a riparian environment. To determine if a host was infected with worms, its posterior was examined, and then the worms were collected by immersing the host in water or by host dissection. Except for the two pairs of Acutogordius that were kept for breeding, all other horsehair worms were killed with hot water (>80°C), fixed in a 75% alcohol solution with their hosts for few days, and preserved in a 95% alcohol solution. The collection and host data are given in Tables 1 and 2, respectively. Breeding Acutogordius pairs were reared in 800 mL aerated tap water and maintained at room temperature (~28°C). After mating, the males were removed and fixed and preserved in 75% followed by 95% alcohol solutions. After the mated females laid eggs for one month, they were also removed and fixed and preserved in 75% followed by 95% alcohol solutions. Egg strings were detected at approximately three days, and hatched larvae were detected at approximately 2-3 weeks after egg laying. Live larvae were observed under a light microscope.
Snails (Physa sp.) infected by horsehair worms were collected with nine other non-infected snails from a small pond in Wufengqi Waterfalls, Jiaushi Township, Yilan County, Taiwan, where free-living adult horsehair worms have been seen. Live snails were maintained together in 2000 mL aerated tap water and were dissected after five days.
Immature stages. Eggs and newly hatched larvae were examined and photographed alive on microslides using a light microscope (Olympus BH-2, PM-10AD, Olympus, Tokyo, Japan) at magnifications of 200× and 400×. Larvae, for examination under SEM, were first killed, fixed by 75% alcohol solution, and collected in a paper envelope soaked in 75% alcohol solution. The protocol of dehydration, critical point drying, and gold sputter coating followed that applied in the examination of the adult fragments and they were examined at a magnification of 500-9,000×. To examine the cysts inside the snail hosts, the snail shells were removed, the soft tissue flattened by two glass slides, and the slides were examined under a light microscope at 200× magnification.
The measurements of each characteristic were performed using the segmented line function in ImageJ 1.47 (Abràmoff et al. 2004), and calibrated spatially to the scale included in each picture. The terminology for larval stages used in this study primarily followed that of Hanelt and Janovy (2002) and Szmygiel et al. (2014).

Phylogenetic analysis
Genomic DNA from adult horsehair worms was extracted using an ALS Tissue Genomic DNA Extraction Kit (Pharmigene, Kaohsiung, Taiwan). A set of universal primers (LCO1490 and HC02198) (Folmer et al. 1994) were applied to amplify and sequence the partial COI sequence. Fourteen Acutogordius samples with COI sequences that could not be well amplified with the universal primers were prepared for use with a newly designed primer set (AcCOiF: TGAGCTGCCTTTTTAG, AcCOiR: TGTATTAATGTTTCGGTC). The PCR for both primer sets was initiated at 95°C for 5 min, and amplification was conducted for 40 cycles of 95°C for 1 min, 50°C for 1 min, and 72°C for 1 min, with a final extension at 72°C for 10 min.
Type material. Partial bodies of the holotype and allotype were deposited with their hosts at the National Museum of Natural Science. Paratypes were deposited at  Table 2 for detailed information.
Etymology. The specific name refers to the type locality, Taiwan. Diagnosis. Acutogordius taiwanensis sp. n. is morphologically similar to A. protectus Schmidt-Rhaesa and Geraci, 2006 with regards to the (1) distribution pattern of tiny bristles on the ventral posterior end, (2) moderately flat areoles (rounded in elevation) covering the tail tips, and (3) cone-shaped spines scattered on the base of the tail lobes of the male samples. However, it is distinct because of the small ornamentations on the mid-body.
Body light brown, smooth, and slightly mucous covered (liquid on the body surface slightly viscous, light on live worms usually refracted) before fixed in alcohol, alcoholpreserved specimens significantly flat and hard.
Anterior end columnar and slightly narrowed at tip; anterior tip white (white cap) with a dark-brown collar ( Cuticle in mid-body smooth, slightly wrinkled, or cracked; short or cone-like bristles (Fig. 3D, E) scattered on some samples (6/14).
Posterior end divided into two tail lobes, each 360.25 ± 53.30 (303.70-489.58) µm in length; lobe tips generally tapered, wrinkled, or covered by moderately flat areoles with short spines amongst areoles; inner side of tail lobes smooth; tiny spines scattered around tip; cone-shaped spines or flat areoles scattered on base behind postcloacal crescent.
Under SEM (worm-form larvae), larvae superficially annulated with 13 segments on pre-septum and 35 on post-septum, ectodermal septum not distinguishable (Fig.  6E). Hooks arranged in three rings on anterior pre-septum: outer ring containing seven hooks, including ventral double hooks close to each other; middle and inner rings containing six hooks, and six inner spines, located between each outer hook (Fig.  6F). Proboscis inside the pre-septum covered by sheath, ornamented with two sets of spines: seven larger spines arranged laterally in two lines, except the largest terminal spine; seven smaller spines on dorsal side, no spines on ventral proboscis (Fig. 6H). One single posterior spine located on end of post-septum (Fig. 6E); exterior openings of pseudo-intestine may be present, but not clear (Fig. 6C).
Phylogeny. Except for one female with insufficient DNA for sequencing, the 23 Acutogordius COI sequences (GenBank numbers KX591922, KX591926-KX591935, KX591937-KX591948) contained eight haplotypes with 442 invariable sites, six singletons, and two parsimoniously informative sites. The genetic distance among them was 0.0025 with a range of 0.0000-0.0112. The phylogenetic tree had a polytomic topology in which some clades were not highly supported because of low bootstrap values and short genetic distances (Fig. 7). The genetic distance between the COI sequences of these 23 Acutogordius individuals and that of G. balticus was 0.27948 compared to 0.25455 and 0.27439 for G. attoni and G. cf. robustus, respectively. Comments. The 23 Acutogordius samples from orthopteran hosts were determined to be from a single species based on their low genetic distances, which was similar to the intraspecific pairwise distances found within G. cf. robustus (0.64-2.63%)  and C. formosanus (0-1.92%) (Chiu et al. 2011) and lower than the interspecific pairwise distances among species of the genera Gordius (8.0-24.3%)  and Chordodes (16.84%) (Chiu et al. 2011).  All three morphological types of post-cloacal crescents identified in A. protectus were apparent in the Acutogordius taiwanensis sp. n. samples. Nevertheless, post-cloacal crescents significantly extending onto the tail lobes were only described in Acutogordius taiwanensis sp. n. and previously in A. acuminatus de Miralles and de Villalobos 1998, A. feae (Camerano, 1897), A. obesus (Camerano, 1895), and A. sulawensis Schmidt-Rhaesa and Geraci, 2006. High intraspecific variation in the post-cloacal crescent makes this structure unsuitable as a diagnostic characteristic at the species level, despite that it is the most obvious structure that can be examined by stereomicroscope.
Short bristles on the mid-body were a newly described character, which were first found in A. finni (Schmidt-Rhaesa and Schwarz 2016). This character is not likely to be examined by stereomicroscope, but in Acutogordius taiwanensis sp. n., the short bristles were still not consistently present in all individuals examined under SEM. One of the possible reasons is that the bristles were covered by mucus on the cuticle surface. The surface of Acutogordius has been generally described as totally smooth (de Miralles andde Villalobos 1998, Schmidt-Rhaesa et al. 2006). However, various structures were found on the surface of Acutogordius taiwanensis sp. n., including wrinkled, cracked, or indented structures. A similar structure (fine grooves as described in Schmidt-Rhaesa and Schwarz (2016) have been found in A. finni (Figs. 4D, E in Schmidt-Rhaesa and Schwarz (2016)), and some of the bristles look like "sticks" on the cuticle surface. In addition, the areole-like structure on the anterior end of one female, also suggested the possibility that the moderately flat areoles covering male tail tips were caused by mucus. Thus, although the moderately flat areoles and short bristles were applied as the main diagnostic characters for Acutogordius taiwanensis sp. n. and A. protectus, more information may be necessary to confidently distinguish between the two species.
Comments. The five male horsehair worms were all determined to be C. formosanus because of the low genetic distances between their COI sequences and those of C. formosanus individuals as described in Chiu et al. (2011). Their morphology was also similar to that of the species described in Chiu et al. (2011), which consisted of five areole types in male adults with slight differences found in the bristlefield and crowned areoles.
The size of the bristlefields was smaller in individuals in the present study than in those described in Chiu et al. (2011) (70-77 µm wide and 145-243 µm long, respectively) and were not found at all in one extremely small individual. Although the difference in the bristlefields in the male Chordodes species has not been used to distinguish the species, this character has been used to distinguish Gordionus kii from G. chinensis (Schmidt-Rhaesa and Sato 2009).
Another abnormal morphological feature was the similar-sized paired crowned areoles and their surrounding circumcluster areoles. These "abnormal crowned areoles" were only found in one extremely small individual, but not in the other horsehair worms, including the two large ones that emerged from the same host individual. Because the molecular data suggested that this individual was conspecific with C. formosanus, we believe the abnormal crowned areoles may have been caused by incomplete development during synchronized maturation (see Discussion for details).

Discussion
In this article, a new species, Acutogordius taiwanensis sp. n. and its immature stages were described, and 11 species of orthopteran insect hosts of this new species were identified. In addition, three novel hosts of C. formosanus, Acromantis japonica, Leptoteratura sp., and Holochlora japonica, were identified.

Acutogordius taiwanensis sp. n.
Intraspecific variation. Finding stable diagnostic characters is a crucial step in distinguishing horsehair worm species Geraci 2006, Hanelt et al. 2015). This process includes two main challenges: 1) finding a stable diagnostic character and 2) setting the boundary between intra-and interspecific variation. The postcloacal crescent was the main diagnostic character distinguishing the Acutogordius and Gordius species (Schmidt-Rhaesa 2001). This structure, despite its function being unclear, is easily examined under both an SEM and a stereomicroscope. However, its morphology may be more unstable than previously understood. The potential polymorphism has been documented in A. protectus, which has primarily been characterized by tail areoles, instead of the postcloacal crescent (Schmidt-Rhaesa and Geraci 2006). In Acutogordius taiwanensis sp. n., distinct intraspecific variation in the postcloacal crescent was further confirmed. Likewise, the small ornamentations on the body cuticle currently described as a diagnostic character in Acutogordius taiwanensis sp. n. and A. finni (Schmidt-Rhaesa and Schwarz 2016) may also appear in other Acutogordius species, especially those that were described without SEM examination before the descriptions of de Miralles and de Villalobos (1998) and .
Using SEM to describe nematormorph species has become standard practice (Schmidt-Rhaesa 2001). However, mucus, which may be present in Acutogordius (as well as Gordius), is likely to obstruct various morphological features under SEM. This is the first report suggesting that horsehair worms may secrete mucus on its body surface, and the function of this mucus is unknown. As an aquatic animal that parasitizes terrestrial hosts, the horsehair worm is known to avoid emerging on land where it could become dehydrated by manipulating its hosts (Thomas et al. 2002). The mucus secretion could be an additional strategy for retaining moisture caused by the high risk of being out of the water.
Because of high intraspecific variation, the conspecific status of the 28 examined samples of Acutogordius taiwanensis sp. n. was primarily based on the comparison of DNA barcodes and secondarily supported by the similarity of hosts and habitat. DNA sequencing, with the application of SEM since 1980 (reviewed in Chandler and Wells 1989), provides a new and standard tool in horsehair worm taxonomy. Although the database of horsehair worm sequences is not yet complete enough to enable determination of a new species, it is useful in judging the conspecific status of a set of samples. The combination of molecular and morphological data has also improved our understanding of intraspecific variation and cryptic species (Chiu et al. 2011. Thus, while here we suggested that Acutogordius taiwanensis sp. n. is a newly described species to science, its phylogenetic relationship with nine other Acutogordius species (or seven species, see Schmidt-Rhaesa 2002) is still unclear and worth further investigation.
The immature stages of Acutogordius. Studies of the immature stages of horsehair worms have received more attention in recent years. Although morphological identification of the immature stage to the genus level is only now roughly possible (Szmygiel et al. 2014), cysts from a wide range of aquatic hosts have recently been used to estimate geographic distributions (Hanelt et al. 2001, Harkins et al. 2016, species composition (Bolek et al. 2013a), and annual reproductive seasons (Chiu et al. 2016). The morphology of the immature stages of Acutogordius in the present study was similar to that of the cysts we found in a field survey (Chiu et al. 2016, Fig. 1, type 2 cyst), which suggests the distribution of Acutogordius, despite the fact that no adult worms were found during that survey.
Newly hatched larvae that fold their bodies outside of hosts have rarely been described (Dorier 1930. This might not be typical, because larvae of numerous gordiid species have never been observed to encyst in air or water, and species folding their larvae have only occasionally been observed in some individuals . In the case of Acutogordius taiwanensis sp. n., the folded larvae (cyst form) were found among the crushed egg strings. As there were some dead larvae near the egg strings, it seems the eggs had already hatched for a few days before being examined. It is uncertain if the larvae folded outside of the eggs or inside the eggs and then were pushed out. Nevertheless, because all the larvae inside the eggs were found to be unfolded, we believe they folded themselves at the egg strings after hatching. To date, we do not yet know which factors triggered newly hatched larvae to become worm-shaped or cyst-shaped because both were found under similar living conditions. Larval worms are thought to stay on the river bottom and be passively ingested by paratenic hosts (Hanelt et al. 2005). Folding their bodies may provide a benefit by reducing the water flow or by making them easy to ingest because the postseptum in Acutogordius, as well as in Gordius, is significantly longer than in other genera (Hanelt andJanovy 2002, Szmygiel et al. 2014).
Acutogordius taiwanensis sp. n. hosts. Prior to this study, the only reported host of Acutogordius was Acanthodis sp. (reviewed in Schmidt-Rhaesa 2013). In the case of Acutogordius taiwanensis sp. n., it was found to emerge from several families of Orthoptera with different foraging behaviors. Most of the hosts are obligate or facultative predators and are frequently found to prey on small insects in the field (e.g., Eugryllacris sp., Neanias magnus, Hexacentrus japonicus, and H. unicolor), but some of them are primarily herbivorous in Taiwan (e.g., Deflorita apicalis, Elimaea sp., Isopsera sp., and Phaulula sp.). Adult horsehair worms have emerged from non-carnivorous hosts, herbivores (Barbitistes serricauda, Leptophyes punctatissima), or detritivores (Cambala annulata) , 2009). These horsehair worms challenge our current knowledge because these worms typically invade their definitive hosts through paratenic hosts, which require that a carnivorous host ingests a paratenic host carrying the horsehair worm's cyst (Hanelt et al. 2005). A possible pathway for parasitizing these non-carnivorous hosts is via consumption of larvae/cysts in the water or on vegetation , 2009). Horsehair worm cysts have known to maintain partial infectiousness after the paratenic host dies and after they are dry for 30 days (Bolek et al. 2013b). This makes them capable of being accidentally ingested by herbivorous or detritivorous, if the dead paratenic host is in the water or on the vegetation. In addition, it is also possible that herbivorous hosts facultatively prey on weak or newly emerged paratenic hosts. Regardless of the pathway the horsehair worm cysts follow, the infection rates of non-carnivorous hosts are theoretically lower than that of predators, and this supports our observations in the field in Taiwan.

Chordodes formosanus
Novel hosts. The Acromantis mantid and Leptoteratura sp. are general predators that can easily ingest the cysts of C. formosanus in paratenic hosts, but C. formosanus was previously thought to develop specifically inside Hierodula mantids (Chiu et al. 2011). The novel hosts suggest flexibility in C. formosanus host use. Such a phenomenon may also occur in C. japonensis, which primarily parasitizes Tenodera mantids (Inoue 1952(Inoue , 1955Chiu et al. 2011), but has also been found in long-horn grasshoppers, Hexacentrus japonicus japonicus (Inoue 1955).
In the present study, we recorded long-horn grasshoppers and an Acromantis mantid as hosts of C. formosanus. However, we do not think these novel hosts are the general ones used by C. formosanus. Horsehair worm hosts have been recorded for most of horsehair worm genera (Schmidt-Rhaesa 2013), but knowledge of host preference, the extent to which a particular host taxon is used by a parasite (Lymbery 1989), is generally lacking. We believe that Acromantis mantids and tettigoniids, compared to Hierodula mantids, are rarely parasitized by C. formosanus because of the seasonal infection rates of aquatic paratenic hosts in Taiwan. Our previous survey of horsehair worm infections in aquatic paratenic hosts suggested a single infection peak after the adult C. formosanus emerge from Hierodula formosana individuals. Such an infection peak did not appear, at least not significantly, after the spring when adult worms emerged from Acromantis mantids and tettigoniids (Chiu et al. 2016, Fig. 1). Thus, the contribution of Acromantis mantids and tettigoniids to the population of C. formosanus may be less than that of Hierodula mantids. It is not clear if development in novel hosts is ecologically significant or accidental, but flexibility in host use may improve our understanding of the physiological mechanisms triggering cyst metamorphosis.
Abnormal morphology of the smallest individual. Horsehair worm length is strongly correlated to the host's size and to the number of individuals in a single host (Hanelt 2009). However, the relationship between horsehair worm size and its morphology has not been evaluated. The only individual bearing "abnormal crowned areoles" and lacking bristlefields was half the length (58 mm) of the other two (125 and 115 mm) individuals from the same host individual. One hypothesis explaining the abnormal morphology, even though we have no direct evidence, may be related to incomplete development. Thus, the morphological similarity of the abnormal crowned areole and the simple areole might suggest the crowned areole is differentiated from the simple areole. The similarity of the (well-developed) crowned areole and the simple areole were also suggested by the ultrastructure of Chordodes nobilii examined by transmission electron microscopy (Schmidt-Rhaesa and Gerke 2006).
The possible reason causing incomplete development may be resource competition and synchronized maturation. Horsehair worms inside a host individual may compete for resources to increase their fecundity, or they may ensure their survival by synchronizing maturation before the host performs the suicide behavior. These actions would subsequently cause the horsehair worm, which may enter the host later than its neighbors, to mature without completing its development. Thus, we suggest that the abnormal crowned areoles may be a result of incomplete development rather than small size. This hypothesis could also be supported by another extremely small C. formosanus (43 mm in length) with crowned areoles that were more likely to be normal in this study. This small C. formosanus singly developed in a host individual. Without the influence from neighbor horsehair worms, its small size might have been the result of the small host (9.63 mm in length), instead of incomplete development.