Corresponding author: Shiuh-Feng Shiao (
Academic editor: A. Schmidt-Rhaesa
A new species of horsehair worm,
The host range of an organism is an important ecological character since it reflects the survival and reproduction of parasites (
However, in April 2007 to February 2008, we conducted a field survey of horsehair worms in Taiwan and found that the mantids,
Specimen information examined in the present study
Host | Horsehair worms | |||||||
---|---|---|---|---|---|---|---|---|
Species | Collecting date | Locality | Longitude and latitude | Collector | Species | Sex | GenBank no. | Deposition |
10-VII-2008 | Xindian, New Taipei City, Taiwan |
|
Ming-Chung Chiu |
|
Male | HM044112 | NTU | |
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|
|
|
|
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Female | HM044113 | NTU |
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|
|
|
|
|
Female | HM044114 | NTU |
|
|
|
|
|
|
Female | HM044115 | NTU |
|
12-VII-2008 | Xindian, New Taipei City, Taiwan |
|
Ming-Chung Chiu |
|
Female | HM044119 | LBM |
|
20-VII-2008 | Xindian, New Taipei City, Taiwan |
|
Ming-Chung Chiu |
|
Male | HM044116 | NMNS |
|
|
|
|
|
|
Male | HM044104 | NMNS |
|
|
|
|
|
|
Female2/ |
|
NMNS |
|
10-VII-2009 | Shimen, New Taipei City, Taiwan | NA | Chun-Kai Wang |
|
Male | HM044123 | LBM |
|
2-VIII-2007 | Taipei Zoo, Taipei City, Taiwan |
|
Ming-Chung Chiu |
|
Female | HQ322115 | NTU |
|
|
|
|
|
|
Female | HQ322116 | NTU |
|
29-I-2008 | Taipei Zoo, Taipei City, Taiwan |
|
Ming-Chung Chiu |
|
Male | HM044122 | NTU |
|
|
|
|
|
|
Female | HM044121 | NTU |
|
23-VII-2008 | Jiaushi, Yilian, Taiwan |
|
Ming-Chung Chiu |
|
Male | HM044111 | NTU |
|
23-VII-2008 | Jiaushi, Yilian, Taiwan |
|
Ming-Chung Chiu |
|
Male | HM044118 | LBM |
|
|
|
|
|
|
Female | HM044108 | LBM |
|
24-VI-2009 | Jiaushi, Yilian, Taiwan |
|
Ming-Chung Chiu |
|
Male3/ |
|
NMNS |
|
|
|
|
|
|
Male | HM044125 | NMNS |
|
16-VII-2009 | Jiaushi, Yilian, Taiwan |
|
Ming-Chung Chiu |
|
Male | HM044126 | NTU |
|
3-VIII-2009 | Jiaushi, Yilian, Taiwan |
|
Ming-Chung Chiu |
|
Male | HM044127 | NMNS |
|
16-VII-2009 | Jiaushi, Yilian, Taiwan |
|
Ming-Chung Chiu |
|
Male | HM044128 | NMNS |
|
23-VII-2008 | Jiaushi, Yilian, Taiwan |
|
Ming-Chung Chiu |
|
Female | HM044117 | NMNS |
|
10-VII-2008 | Jiaushi, Yilian, Taiwan |
|
Ming-Chung Chiu |
|
Female | HM044120 | NTU |
|
23-VII-2008 | Jiaushi, Yilian, Taiwan |
|
Ming-Chung Chiu |
|
Female | HM044106 | NTU |
|
23-VII-2008 | Jiaushi, Yilian, Taiwan |
|
Ming-Chung Chiu |
|
Female | HM044109 | NTU |
|
23-VII-2008 | Jiaushi, Yilian, Taiwan |
|
Ming-Chung Chiu |
|
Female | HM044110 | NTU |
|
5-VII-2008 | Taroko National Park, Hualien, Taiwan | NA | Tsung-Hung Yang |
|
Male | HM044107 | NTU |
|
30-IX-2006 | Taipei Zoo, Taipei City, Taiwan |
|
Ming-Chung Chiu |
|
Male | JF808204 | NTU |
|
X-2008 | Hsinchu City, Taiwan | NA | Ju-Chun Hsu |
|
Female | JF808197 | NTU |
18-VII-2003 | Lyudao, Taitung, Taiwan | NA | Hsing-Yu Chou |
|
Male | JF808203 | NTU | |
|
|
|
|
|
|
Male | JF808205 | NTU |
|
16-X-2010 | Sakado, Saitama, Japan |
|
Etsuko Suzuki |
|
Female | JF808194 | NTU |
|
1-XI-2010 | Kijo, Miyazaki, Japan |
|
Yasukuni Ono |
|
Female | JF808198 | LBM |
|
1-XI-2010 | Kijo, Miyazaki, Japan |
|
Yasukuni Ono |
|
Female | JF808199 | NTU |
|
10-XI-2010 | Kijo, Miyazaki, Japan |
|
Yasukuni Ono |
|
Female | JF808202 | NTU |
|
11-XI-2010 | Miyazaki, Miyazaki, Japan |
|
Yasukuni Ono |
|
Female | JF808200 | NTU |
|
26-XI-2010 | Kijo, Miyazaki, Japan |
|
Yasukuni Ono |
|
Male | JF808196 | NTU |
|
26-XI-2010 | Kijo, Miyazaki, Japan |
|
Yasukuni Ono |
|
Female | JF808201 | NTU |
|
16-X-2010 | Sakado, Saitama, Japan |
|
Wataru Toki |
|
Female | JF808195 | NTU |
|
5-XI-2010 | Kijo, Miyazaki, Japan |
|
Yasukuni Ono |
|
Male | JF808206 | NTU |
LBM: Lake Biwa Museum; NMNS: National Museum of Natural Science; NTU: National Taiwan University.
1 No host specimen preserved.
2 Allotype.
3 Holotype.
In total, the morphologies of 40 adult horsehair worms (including two females which laid eggs in the laboratory) were examined, and these worms were used for a DNA analysis. The morphologies of larvae laid in the laboratory were examined by light microscopy. Eggs and larvae collected in 2010 were examined by an SEM, and their COI sequences were analyzed to determine their taxonomic status. After studying the specimens, the partial bodies of these 40 samples were preserved in the Department of Entomology, National Taiwan University, Taipei; National Museum of Natural Science, Taichung, Taiwan; and Lake Biwa Museum, Shiga, Japan.
Mantids (
Two pairs of adult horsehair worms (two males from Sindian, New Taipei City, and two females from Taipei Zoo) were collected on August 2, 2007. They were reared together in a plastic container (20 cm in diameter and 10.5 cm high) filled with 800 ml of aerated tap water, and maintained at 27 ± 1°C. The females were kept in water to lay eggs for 1 month, then fixed in a 75% alcohol solution and preserved in a 95% alcohol solution. Egg strings were found after 5 days and had hatched to larvae by 1 month later. Larvae were kept alive until being observed under light microscopy. Egg strings stuck on rocks were collected from Wufengqi Waterfalls, Yilan County, on July 21, 2010. They were brought back to the laboratory and kept in a tank with 20 L of tap water under the same conditions as described above. Eggs hatched about 8 weeks later and were then fixed and preserved as described above.
For adult specimens, the body surface was examined under light microscopy (Olympus BH-2, PM-10AD, Tokyo, Japan). For each specimen, a fragment of about 1 cm long of the mid-body was removed and cut longitudinally. Instead of using a scalpel to directly remove the internal tissues, we dipped the fragment into a 1% KOH solution for 2 h. The internal tissues became transparent and removable. The cuticles were placed on a microslide and observed under a microscope at a magnification of 40–200×. Eggs and newly hatched larvae were placed on the microslides, each with a drop of water and a cover glass. They were observed alive under the light microscope (at a magnification of 400×).
SEM was also used to examine adult and larval specimens. Its preparation protocol followed that of
The terminology of larvae follows that of
Genomic DNA was extracted from fragments of horsehair worms and whole larvae using an ALS Tissue Genomic DNA Extraction Kit (Kaohsiung, Taiwan). A partial COI sequence was amplified by a polymerase chain reaction (PCR) with a set of universal primers (LCO1490 and HC02198) (
For the phylogenetic analysis, the COI sequence of
Wufengqi Waterfalls (
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
The specific name refers to Taiwan, the collection locality of the type specimens.
(
Posterior end (
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 (
Posterior end (
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 (
Horsehair worms from the mantids
The crowned areole is an autapomorphy of the genus
Comparison of areolar types between
Areolar type |
|
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This study |
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This study |
|
|
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Sample size | 17 ♂♂, 22 ♀♀ | 1 ♂**, 1 ♀ | 1 ♂ | 49 ♂♂, 37 ♀♀ | 17 ♂♂, 7 ♀♀ |
Collecting locality | Taiwan, Japan | Japan | Japan | Japan | Korea |
Host |
|
Not mentioned | Not mentioned | ||
Crowned areoles with short projections | + | + | + | - | + |
Crowned areoles with long projections | + (most areoles with filaments > 200 μm) | + (> 200 μm) | + (mostly around 100 μm, maximum 140 μm) | + (around 50 μm in |
+ (< 200 μm) |
Sexual dimorphism in crowned areoles | + | - | ? (Only males were investigated) | - | - |
Circumcluster areoles | + (12-20) | + (at least 12 in |
+ (7-14) | + (10 in |
+ (7-17) |
Tubercle areoles | + | + | + | + | + |
Thorn areoles (spine areoles) | + (small or absent in some samples; 9/40) | - | + | + | - |
Bulging areoles | * (some elevated simple areoles in clusters of 2-10) | - (simple areoles elevated differently in |
* (some elevated simple areoles in clusters of 2-5) | - (paired elevated areoles) | - (simple areoles elevated differently in Fig1) |
Simple areoles | + | + | + | + | + |
Terminology based on
+, present; -, absent; *, difficult to determine; ?, unknown.
**, According to the description, we consider the specimen not to be
Male adult of
Details of ornamentations on anterior and posterior ends of male
Female adult of
Eggs and larvae of
Detail of larvae of
Examined male collected with its host from Miyazaki Prefecture, Japan (
Chinese mantids,
(
Posterior end (
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 (
Male adult of
A phylogenetic tree of the 40 samples of horsehair worms collected from three species of mantids
Comparison of the 40 horsehair worm samples (39
Neighbor-joining tree of
In this article, a new species,
The two species,
In addition to the crowned areoles, other minor differences in
About 90 species belong to the genus
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.
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 (
We would like to thank Chu-fa Tsai and Huai-Sheng Fang for revising the manuscript. Advice from George O. Poinar of the Department of Zoology, Oregon State University, Corvallis, OR, resolved many questions in this study. The molecular analysis was supported by Hurng-Yi Wang and Chia-Hung Hsieh. Samples from Japan were collected by Yasukuni Ono, Etsuko Suzuki, and Wataru Toki. Much help received from Ju-Chun Hsu, Hsing-Yu Chou, Tsung-Hung Yang, and Chun-Kai Wang who offered their collections from Taiwan is also highly appreciated. This work was supported by the National Science Council, Taiwan (NSC96-2815-C-002-104-B, NSC99-2321-B-002-040, and NSC 100-2631-H-002-018).