Chordodes japonensis Inoue, 1952

Chiu, Ming-Chung, Huang, Chin-Gi, Wu, Wen-Jer & Shiao, Shiuh-Feng, 2011, A new horsehair worm, Chordodes formosanus sp. n. (Nematomorpha, Gordiida) from Hierodula mantids of Taiwan and Japan with redescription of a closely related species, Chordodes japonensis, ZooKeys 160, pp. 1-22 : 9-14

Chordodes japonensis Inoue, 1952

Material examined.

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.

Host.

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

Redescription.

(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).

Phylogeny.

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.

Discussion.

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 .

Morphological comparison of Chordodes formosanus sp. n. and Chordodes japonensis

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.

Molting and environmental effects on morphology

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.

Host specificity

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.

Molecular approach and perspectives

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.