Wufengqi Waterfalls (24°49'55.62"N, 121°44'50.10"E), Jiaushi Township, Yilan County, Taiwan (Holotype). Dachijieu (24°56'59.21"N, 121°34'2.12"E), Sindian (New Taipei City) (allotypes). Paratypes collected from Taiwan and Japan: Taipei Zoo (Taipei City), Sindian (New Taipei City), Taroko National Park (Hualien County), Wufengqi Waterfalls (Yilan County), Taiwan and Miyazaki Prefecture and Sakado (Saitama Prefecture), Japan. For detailed data, see Table 1.

Partial bodies of holotype (male, 167 mm), and allotype (female, 282 mm) deposited at the Department of Entomology, National Taiwan University with the hosts. Paratypes deposited at the Department of Entomology, National Taiwan University, Taipei, and National Museum of Natural Science, Taichung, Taiwan and Lake Biwa Museum, Shiga, Japan. For detailed information, see Table 1.

Hierodula formosana Giglio-Tos (Mantodea: Mantidae). Hierodula formosana endemic to Taiwan, and the adult always emerging from late June to early August. Hosts of some samples belonging to Hierodula patellifera which distributed in both Taiwan and Japan. Their adults usually emerging in late autumn, about 2 months later than Hierodula formosana.

The specific name refers to Taiwan, the collection locality of the type specimens.

(Figs 1–5)

Male adult (n = 17) (Figs 1, 2). Body length 74–277 mm, width (widest) 0.7–1 mm (after dehydration). In alcohol-preserved specimens, body rough and flat with dorsal and ventral grooves; dark-brown with bright lengthwise regions on both dorsal and ventral sides and darkly pigmented line on ventral side in most specimens (Fig. 1D).

Posterior end (Fig. 1C) not lobed, with short spines (ca. 5–12 μm) among areoles on margin. Cloacal opening subterminal, oval, 27–78 μm long and 17–63 μm wide. A pair of oval regions without areoles posterior to cloacal opening, each with scattered bristles extending as two rows of ventral strips (155–160 μm wide), structured by cord-like folds or flat areoles; flat areoles ornamented with short filaments in a cluster on top or scattered on cord-like folds, or absent. Paired oval bristlefields (70–77 μm wide and 145–243 μm long) bearing bristles on borders between flat areoles and normal areoles on lateral side of cloacal opening; bristles in bristlefields varying among individuals; some bearing only shorter or thinner unbranched bristles and some with both branched and unbranched bristles (Figs 2D–F). Anterior end tapered, same color as body, with white tip (white cap) but no dark collar under a stereomicroscope. Under SEM, anterior end round with moderately flat areoles and short bristles on surface; about 10 of them elevated and cone-like near anterior terminal; long thick bristles scattered among areoles, some between areoles and some penetrating areoles (Fig. 2C). Anterior end on one individual with residual larval cuticle tapered but flat terminally (Fig. 2A); also flat surrounding ornamentations and bristles (Fig. 2B). Mouth opens terminally in some individuals.

Entire body covered by areoles with cord-like folds in between. Areoles characterized into five types (simple, tubercle, thorn, circumcluster, and crowned areoles). Simple areoles (Fig. 1A), most abundant, covering most of body surface except anterior end and ventral side of posterior end; each 5–8 μm in diameter, more or less circular or oval, generally with a smooth surface but some with dots, grooves, or short bristles on surface. Simple areoles varying in height and some significantly elevated areoles in clusters of two to ten, looking like bulging areoles as mentioned by Schmidt-Rhaesa et al. (2008); but darker under light microscopy (Fig. 1D). Tubercle areoles (Fig. 1A) scattered among simple areoles, each shaped similarly to simple areole but with a tubercle (6–9 μm long) on apically concave center. Thorn areoles (Fig. 1A) distributed slightly along dorsal and ventral middle lines, similar to tubercle areoles but with a long solid thorn (22–57 μm long) instead of a tubercle. Thorn areoles small or absent in two samples. Crowned areoles clustered in pair with a central tubercle in between and surrounded by 12–20 circumcluster areoles with short filaments on apical surface (short-crowned areoles) (Figs 1A, B); scattered over trunk except anterior and posterior ends; each with medium filaments (10–15 μm) originating from apical center and sidelong to edges; only one male with a few crowned areoles containing a few filaments of around 100 μm.

Female adult (n = 14) (Fig. 3). Length 263.7 (78–440) mm; body width (widest) 1–1.5 mm (after dehydration); body rough, flattened, dorsal and ventral grooves present; light to dark-brown with lengthwise regions on both dorsal and ventral sides, and darkly pigmented line on ventral side in most specimens. Some individuals with dark patches on bodies.

Posterior end (Fig. 3B) rounded, slightly swollen, covered by moderately flat areoles with cord-like folds surrounding cloacal opening; short bristles (10–27 μm) scattered between borders of moderately flat areoles and cord-like folds. Cloacal opening on terminal end, circular, 18–33 μm in diameter, no circumcloacal spine.

Anterior end with similar structure and color to males except lower cone-like areoles; terminally flat anterior end also appearing in one individual. Pattern and distribution of areoles (Fig. 3A) also similar to those of males but much more crowded in most individuals. Thorns of areoles shorter than those of most males (11–30 μm) but small or absent in three females. Cord-like folds present between areoles. Crowned areoles scattered over trunk as in males while roughly arranged in two lines on ventral and dorsal midlines, bearing significantly longer filaments (longest apical filaments ranging 65.57–392.25 μm (237.47 ± 66.22 μm, for details see “Diagnosis”)) (Figs 3A, C).

Eggs (Fig. 4).In laboratory, egg strings stuck onto substrate or drifting on bottom. Eggs (6 days after being laid) (Fig. 4B) nearly circular, 30.39 ± 1.15 μm (n = 10) in diameter. Egg strings white when laid and becoming light-brown within 1 day, turning dark-gray just before hatching. Eggs collected in field (Fig. 4C) all stuck onto rocks; mostly brown to gray, but some light-brown as those just laid in laboratory.

Larvae (Figs 4, 5). Larvae remaining near egg strings after hatching, not active. Under light microscopy, larval preseptum (Fig. 4A) averaging 20.55 (16.32–24.78) μm long and 13.21 (10.93–16.34) μm wide; postseptum averaging 24.91 (22.52–27.44) μm long and 10.06 (9.25–11.49) μm wide, stylet averaging 11.04 (9.59–13.25) μm long and 3.36 (2.76–3.91) μm wide. Pseudointestines V-shaped (Fig. 4A) with one small and one large branch, both with a swelling on posterior ends. Large branch averaging 8.27 (7.28–9.82) μm, small branch averaging 6.70 (5.43–7.59) μm long. Under SEM, larvae superficially annulated with 13 segments on preseptum and 10 on postseptum, ectodermal septum as a single segment between them. Three sets of hooks arranged in three rings on anterior preseptum (Fig. 5A): outer ring containing seven hooks (outer hooks), two ventrally positioned and closely together on base (ventral double hook); six hooks on second ring located between each outer hook (middle hook); inner ring containing at least three inner spines, but real number unknown. A stylet (Figs 5A, C) appearing inside preseptum, ornamented with two sets of spines: nine spines on dorsal and ventral sides of stylet, five small lateral papillae on left side. A pair of anterior and posterior terminal spines (Fig. 5B) on posterior of postseptum. Pseudointestine exterior opening (Fig. 5B) centrally located between anterior terminal spines on ventral body. Several larvae covered by residual skin: one observed in broken egg suggesting that molting had occurred before emergence (Fig. 4D).

Horsehair worms from the mantids Hierodula formosana and Hierodula patellifera were characterized by all six types of areoles, including simple, tubercle, thorn, circumcluster, short-crowned, and long-crowned areoles in the female. The same six areole types are similar to those of Chordodes japonensis described by Inoue (1952) and Baek (1993). Nevertheless, the significantly longer filaments on female crowned areoles suggest they belong to a new species, Chordodes formosanus sp. n. By the way, the absence of long-crowned areoles in our male sample of Chordodes formosanus sp. n. probably implies their potential for distinguishing these two different species. However, since the dimorphism of male crowned areoles has not been mentioned in Chordodes japonensis, more studies are needed to uncover this phenomenon.

The crowned areole is an autapomorphy of the genus Chordodes. In Chordodes formosanus sp. n. and Chordodes japonensis, it is composed of two major areoles ornamented with apical filaments and several surrounding circumcluster areoles. The dimorphic length of the apical filaments divides the crowned areoles into two types, short-crowned areoles with short ornamental filaments and long-crowned areoles with long ones. All samples we checked (both sexes of Chordodes formosanus sp. n. and one male Chordodes japonensis) had short-crowned areoles scattered all over the body trunk, with the long-crowned areoles only appearing on the ventral and dorsal midlines of the female Chordodes formosanus sp. n. andmale Chordodes japonensis, but not themale Chordodes formosanus sp. n. We did not personally observe the female Chordodes japonensis, but these dimorphic crowned areoles must be present according to the descriptions of Inoue (1952) and Baek (1993). Additionally, the apical filament lengths of long-crowned areoles were significantly longer on Chordodes formosanus sp. n. We randomly chose two to five sets of long-crowned areoles from our female samples and measured each of their longest apical filaments. In the 68 sets of long crowned areoles, the longest apical filaments ranged 65.57–392.25 (237.47 ± 66.22) μm. Fifty-one of these 68 (75%) sets of crowned areoles had apical filaments of > 200 μm. The longest apical filaments in our male Chordodes japonensis were 92.03–139.70 μm. Significantly shorter filaments in Chordodes japonensis were also described by Inoue (1952) and Baek (1993). Chordodes japonensis has long-crowned areoles with filaments of around 50 μm (Inoue 1952; fig. 1a) and filaments of < 200 μm in the description by Baek (1993). For other differences and detailed comparisons, see Table 2 and the “Discussion”.

Figure 1.

Male adult of Chordodes formosanus sp. n. A Cuticular surface with five types of areole B paired crowned areoles with a central tubercle C posterior end of the male D bright lengthwise regions with a darkly pigmented line on the ventral side of male body. Cir, circumcluster areole; Clo, cloacal opening; P, pigmented line; Sc, short-crowned areole; Sim, simple areoles; T, central tubercle; Th, thorn areole; Tu, tubercle areole.

Figure 2.

Details of ornamentations on anterior and posterior ends of male Chordodes formosanus sp. n. A Anterior end with larval cuticle B flat ornamentations and bristles on top of anterior end C cone-like areoles with bristles on top of anterior end D–F bristlefields with branched and unbranched bristles (D), short and unbranched bristles (E), or thin and unbranched bristles (F). Bri, bristle; Coa, cone-like areole; Lc, residual of larval cuticle; Fla, flat ornamentations.

Figure 3.

Female adult of Chordodes formosanus sp. n. A Cuticular surface with six types of areole B posterior end of female C ventral side of female body. Bri, bristle; Cir, circumcluster areole; Clo, cloacal opening; Lc, long crowned areole; Sc, short-crowned areole; Sim, simple areoles; Th, thorn areole; Tu, tubercle areole.

Figure 4.

Eggs and larvae of Chordodes formosanus sp. n. A Live larva in water B eggs (6 days after being laid) C egg strings stuck onto a rock D larva in an egg with residual cuticle. EctoS, ectodermal septum; Ho, hooklet; PostS, postseptum; PreS, preseptum; PsI, pseudointestine gland; Rc, residual cuticle; Sty, stylet.

Figure 5.

Detail of larvae of Chordodes formosanus sp. n. A Anterior view of a larva showing stylet and hook arrangement B posterior view of a larva C style with spines and lateral papillae. Asp, anterior terminal spine; Lp, lateral papillae; Is, inner spines; Mh, middle hook; Oh, outer hook; Peo, Pseudointestine exterior opening; Psp, posterior terminal spine; S, stylet; Ssp, stylet spines; Vdh, ventral double hook.

Examined male collected with its host from Miyazaki Prefecture, Japan (32°10'21.50"N, 131°27'36.53"E) by Yasukuni Ono on 5-XI-2010. Partial body of horsehair worm deposited with its host at Department of Entomology, National Taiwan University, Taipei, Taiwan. Accession number of partial COI sequence in GenBank: JF808206.

Chinese mantids, Tenodera sinensis (Mantodea: Mantidae), which are sometimes classified as Tenodera aridifolia.

(Fig. 6)

Male adult (n = 1). Body length 220 mm, width (widest) 0.94 mm (after dehydration). In alcohol-preserved specimens, body rough and flat with dorsal and ventral grooves, dark-brown with a darkly pigmented line on ventral side.

Posterior end (Fig. 6A) not lobed, with short spines (ca. 5–13 μm) among areoles on margin. Cloacal opening subterminal, oval 44 μm long and 25 μm wide, with circumcloacal spines. A pair of oval regions without areoles posterior to cloacal opening, each with scattered bristles extending as two rows of ventral strips (115–120 μm wide) structured by cord-like folds or flat areoles. Paired oval bristlefields (82 μm wide and 231 μm long) bearing numerous branched and unbranched bristles on borders between flat areoles and normal areoles on lateral side of cloacal opening. Anterior end tapered, same color as body, with white tip (white cap) but no dark collar under stereomicroscopy. Under SEM, anterior end (Fig. 6F) smooth with short, thick bristles and small spines; mouth open on cone at anterior extremity.

Entire body covered by areoles with slightly cord-like folds in between. Areoles characterized into six types (simple, tubercle, thorn, circumcluster, and two types of crowned areoles). Simple areoles (Fig. 6C), most abundant, covering entire body surface except anterior end and ventral side of posterior end; each 5–11 μm in diameter, more or less circular or oval, surface uneven, some areas with short bristles. Simple areoles varying in height and some significantly elevated areoles in clusters of two to five, appearing as bulging areoles but darker under light microscopy. Tubercle areoles (Fig. 6C) scattered among simple areoles, each similarly shaped to simple areole but with a tubercle (about 7 μm long) on apically concave center. Thorn areoles (Fig. 6E) similar to tubercle areoles but with a long solid thorn (15 μm long) instead of tubercle; number of thorn areoles much fewer than tubercle areoles. Crowned areoles (Figs 6B, D) clustered in pairs with a central tubercle in between and surrounded by 7–14 circumcluster areoles with short filaments on apical surface; scattered over trunk; each with medium filaments (12–20 μm) originating from apical center and sidelong to edges. Crowned areoles roughly arranged in two lines on ventral and dorsal midlines, bearing significantly longer filaments (most around 100 μm, none > 150 μm) (Figs 6B, C).

Figure 6.

Male adult of Chordodes japonensis. A Posterior end of male B ventral side of male body C cuticular surface with four types of areole D short-crowned areoles E thorn areole F anterior end. Bri, bristle; Cir, circumcluster areole; Clo, cloacal opening; Lc, long crowned areole; M, mouth; Sc, short-crowned areole; Sim, simple areoles; Tu, tubercle areole.

A phylogenetic tree of the 40 samples of horsehair worms collected from three species of mantids Hierodula formosana, Hierodula patellifera, and Tenodera sinensis from Taiwan and Japan with one outgroup (Paragordius sp.) is shown in Fig. 7.

Comparison of the 40 horsehair worm samples (39 Chordodes formosanus and one Chordodes japonensis) revealed there were 31 haplotypes with 432 invariable sites, 64 singletons, and 32 parsimoniously informative sites. Newly sequenced COI data were deposited in the GenBank database (see Table 1 for accession nos.). Samples from the host mantids of Hierodula and Tenodera (which are considered two species, Chordodes formosanus sp. n. and Chordodes japonensis, respectively)were significantly separated into two groups based on the phylogenetic tree restructured by the NJ method (Fig. 7). The genetic distance between these two groups was 0.16840. This result supports that theybelong to different species as we suggested based on morphology. The phylogenetic tree also revealed a polytomic topology among the 39 horsehair worms parasitizing Hierodula. Although some clades were observed, they were not highly supported due to low bootstrap values and short genetic distances; the mean genetic distances among them was 0.00979 with a range of 0.000-0.01922. Since the genetic distance between the larvae collected in the field and Chordodes formosanus was only 0.00759, we suggest that those larvae belong to the same species.

Figure 7.

Neighbor-joining tree of Chordodes formosanus sp. n. and Chordodes japonensis with genetic distances between each group. Abbreviations in the table indicate the horsehair worms’ mantid hosts (Hf, Hierodula formosana; Hp, Hierodula patellifera; Ts, Tenodera sinensis) and collecting localities (Tw, Taiwan; Ja, Japan). Numbers at the nodes represent the percentage of 1000 bootstrap values of > 70%. The outgroup (out) was a Paragordius sp.

In this article, a new species, Chordodes formosanus sp. n., which parasitizes Hierodula formosana and Hierodula patellifera, was proposed and described including the morphology of the egg and larval stages. Because of a similar morphological description by Schmidt-Rhaesa (2004), we categorized the species in that study into Chordodes formosanus sp. n. and also limited the mantid host range of Chordodes japonensis to the genus Tenodera.

The two species, Chordodes formosanus sp. n. and Chordodes japonensis, can be distinguished by the presence of dimorphism in male crowned areoles and the length of long filaments on female crowned areoles. A comparison of areolar types in Chordodes formosanus sp. n. and Chordodes japonensis is given in Table 2. Both Chordodes formosanus sp. n. and Chordodes japonensis have two forms of crowned areoles on their cuticles, one with moderate attachments on the top (short-crowned areoles) and the other with significantly longer attachments (long-crowned areoles). Both types of crowned areoles were found on these two species except for the male Chordodes formosanus sp. n. (the short-crowned areoles were not mentioned in the description of Chordodes japonensis by Inoue (1952)), which indicates sexual dimorphism in Chordodes formosanus sp. n. We believe the female worm which was previously considered to be Chordodes japonensis by Schmidt-Rhaesa (2004) actually belongs to the species, Chordodes formosanus n. sp, described here. The reason why the sexual dimorphism was not mentioned by Schmidt-Rhaesa (2004) is probably that the free-living male is not the same species as the female. In addition, the length of the filaments on the long-crowned areoles is also a significant character differentiating these two species; they are always > 200 μm in Chordodes formosanus sp. n. (or > 6-fold that of paired crowned areoles) but < 200 μm in Chordodes japonensis (or < 5-fold that of paired crowned areoles, and most of them are around 100 μm).

In addition to the crowned areoles, other minor differences in Table 2 (thorn areoles and bulging areoles) are much more difficult to use in discriminating these two species. Although the presence of thorn areoles is always questionable due to their small number, it is still considered a key character for distinguishing different species (Schmidt-Rhaesa et al. 2008). Thorn areoles were not reported by Schmidt-Rhaesa (2004) or Baek (1993) but appeared in the description by Inoue (1952). In our study, thorn areoles were not found in nine Chordodes formosanus sp. n. but appeared in the other 31 Chordodes formosanus sp. n. and one Chordodes japonensis. Contrary to the thorn areoles which can be easily distinguished from other types of areoles, bulging areoles are much easier to be confused with simple areoles (Schmidt-Rhaesa et al. 2008). Although bulging areoles were consistently ignored in descriptions of Chordodes japonensis, we observed various heights of simple areoles, and some of them clustered in groups similar to bulging areoles as described by Schmidt-Rhaesa et al. (2008). This renders the presence of bulging areoles in Chordodes japonensis as well as Chordodes formosanus sp. n. questionable.

About 90 species belong to the genus Chordodes which were proposed using only five characteristic types of areoles (Schmidt-Rhaesa et al. 2008). Unfortunately, most characters other than these areole types have seldom been mentioned. According to our observations, the complex ornamentations of the structure of the anterior end were previously considered to be smooth. A horsehair worm explores unknown environments with its head, and the head is also the first part which contacts outer environments when they emerge from a host. Schmidt-Rhaesa and Gerke (2006) suggested that the apical filaments of the crowned areoles may have sensory functions. It is rational that the anterior and posterior ends of horsehair worms with the complex ornamentations play important roles in exploration and mating. Therefore, the smooth anterior ends in some descriptions (e.g., de Villalobos and Zanca 2001) and some of our samples may have been caused by damage from the environment. In addition to the damage, some samples were found to have been covered by residual juvenile skins, indicating that the worms had just molted. Shapes of the ornamentations on the ends under the larval cuticle were flat. The molting of hairworms was observed (Schmidt-Rhaesa et al. 2003), and its potential effects on the morphology should be carefully considered in the future studies. Residual skin was also found on newly hatched larvae, and on a larva still inside an egg, indicating that molting occurred before it emerged from the egg. As a group of Ecdysozoa (Aguinaldo et al. 1997), horsehair worms molt between each instar. According to our observations of metamorphosis from cysts to wormlike juveniles and from juveniles to adults, we suspect those horsehair worms may proceed through at least three molts before maturing.

Compared to the wide range of paratenic hosts, horsehair worms’ definitive host range is limited to one or a few species. Because nematomorphs are sometimes found after they have emerged from their hosts, definitive information on hosts is unknown in some species. Chordodes japonensis was reported to be parasites of the mantids, Tenodera sinensis, Tenodera angustipennis (Inoue 1952), Hierodula patellifera (Schmidt-Rhaesa 2004), and longhorn grasshoppers, Hexacentrus japonicus japonicus (Inoue 1955) in Japan. Since the hairworm described by Schmidt-Rhaesa (2004) was here considered to be Chordodes formosanus sp. n., the mantid host of Chordodes japonensis is now limited to two species of Tenodera. Until now, we have no evidence to exclude Hexacentrus japonicus japonicus from being a potential host of Chordodes japonensis. However, in our investigation, the host range of these two species seems to be limited to the generic level. Therefore, we still question the ability of Chordodes japonensis to develop inside hosts other than mantids.

In the 40 samples from Taiwan and Japan we examined, the taxonomic status was supported not only by their morphologies, but also by the partial COI sequences. COI sequences were used to study inter- and intraspecific relationships due to the high mutation rate (Hebert et al. 2003). The low genetic divergence among our hairworm samples suggests their conspecific relationship, and our samples can also be separated from those hairworms that emerged from Tenodera by theirsignificantly divergent sequences. Since species of immature stages can only be conjectured by adults in the same area (Winterbourn 2005), molecular data were herein proven to be a useful tool for identifying both adults and immatures. As molecular information for the phylum Nematomorpha is still limited, we believe more molecular data would be helpful and can be used to uncover uncertain relationships among horsehair worms in the near future.