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Research Article
Chromosomes of four species of dobsonflies, in the genus Protohermes (Megaloptera, Corydalidae, Corydalinae) from East Asia
expand article infoYoshinori Takeuchi‡, Koji Iizuka§, Hiroyuki Koishi§, Hidehiro Hoshiba|
‡ Bohkai Junior High School, Hyogo, Japan
§ Matsue 4th Junior High School, Tokyo, Japan
| Tamagawa Univesity, Tokyo, Japan

Corresponding author: Yoshinori Takeuchi ( takeyoshiaki3022@outlook.jp )

Academic editor: Vladimir Lukhtanov
© 2025 Yoshinori Takeuchi, Koji Iizuka, Hiroyuki Koishi, Hidehiro Hoshiba.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation: Takeuchi Y, Iizuka K, Koishi H, Hoshiba H (2025) Chromosomes of four species of dobsonflies, in the genus Protohermes (Megaloptera, Corydalidae, Corydalinae) from East Asia. Comparative Cytogenetics 19: 75-82. https://doi.org/10.3897/compcytogen.19.146501
ZooBank: urn:lsid:zoobank.org:pub:7EAB9C15-4ED3-4E0C-8C4C-3CE881B676ED
Open Access

Abstract

We analyzed chromosomes of four species of East Asian dobsonflies (Megaloptera: Corydalidae): Protohermes grandis (Thunberg, 1781), P. immaculatus Kuwayama, 1964, P. disjunctus Liu, Hayashi et Yang, 2007 and P. costalis (Walker, 1853). The chromosome number in all species was 2n = 24, consisting of 11 pairs of autosomes plus the XX chromosomes in females and the Xyp in males. The karyotype of P. immaculatus which occurs in near the central part of the Ryukyu Islands, and is a vicarious species of P. grandis, was similar to the karyotype of P. grandis. On the other hand, the karyotype of P. disjunctus, which is from the Sakishima Islands, and is a vicarious species of P. costalis, resembled that of P. costalis.

The X chromosomes are submetacentric, while the Y is the smallest, dot-like chromosome of the set. The sex chromosomes of the first meiotic metaphase (MI) spermatocytes in all species invariably appear as a bivalent-like structure known as parachut bivalents Xyp, suggesting that the species in this genus share a common sex-bivalent mechanism.

Keywords

Chromosomes, dobsonflies, Protohermes, sex chromosomes, XX/Xyp

Introduction

Megaloptera is composed of two families, Corydalidae and Sialidae (= alderflies). Corydalidae is further divided into two subfamilies, Corydalinae (= dobsonflies) and Chauliodinae (= fishflies). Corydalinae larvae are aquatic, inhabiting deeper waters in streams and rivers and using tracheal gills on the ventral surface of their abdomens to absorb dissolved oxygen. On the other hand, Chauliodinae larvae live near the banks of rivers and are primarily air breathers, using a pair of respiratory tubes on the dorsal surface of the abdomen. Studies on Megaloptera chromosomes have been reported by Itoh (1933a, b), Hughes-Schrader (1980), and Takeuchi et al. (2002, 2012a, b). Two species of Corydalinae, one each from Japan and North America, both had chromosome number 2n = 24 (22+XY) (Hughes-Schrader 1980; Takeuchi et al. 2002). By contrast, four Japanese species of Chauliodinae had chromosome number 2n = 20 (18+XY) (Takeuchi et al. 2012a, b) and one species from North America had chromosome number 2n = 22 (20+XY) (Hughes-Schrader 1980). There are no reports on the chromosomes of Sialidae.

There are about 90 species of Protohermes Weele, 1907 recognized worldwide, with all occurring in Asia (Lacewing Digital Library 2025). Four of these species have been studied in this report: Protohermes grandis (Thunberg, 1781), P. immaculatus Kuwayama, 1964, P. disjunctus Liu, Hayashi et Yang, 2007, and P. costalis (Walker, 1853). P. costalis is native to continental China, Taiwan, and India (Lacewing Digital Library 2025). The remaining three species are only known from Japan, with P. disjunctus known from the Yaeyama Islands, P. grandis from throughout Japan, and P. immaculatus from the Ryukyu Islands.

Specimens of P. immaculatus are small in size and considered a vicarious species of P. grandis (Hayashi 1989a). Vicarious species are considered to be closely related, having a common ancestor, but are geographically separated. Similarly, specimens of P. disjunctus are small in size and considered a vicarious species of P. costalis (Hayashi 1989b, c). Hayashi (1989a, b, c) reported that dwarfism resulted in dobsonflies from warm water temperatures and shortage of food on some islands. We studied chromosomes of these four Protohermes dobsonflies, using larval gonads. To date, of these four species the chromosome number has only been studied in P. grandis — 2n = 24 (Takeuchi et al. 2002). Our objective was to study and compare the karyotypes of these four Protohermes dobsonflies species and to discuss the finding in relationship to phylogenetic evolution caused by geographical isolation.

Material and methods

Insects

Final-instar larvae of Protohermes grandis, P. immaculatus, P. disjunctus, and P. costalis, were collected from June 1994 to February 1995 in rivers in Japan and Taiwan by the first author (Fig. 1). Collection sites, sampling dates, and the numbers of studied larvae are given in Table 1. In the field, larvae were placed individually in cups, filled with water and brought back to the laboratory alive. The larvae were fed aquatic insects until used in the experiment.

Table 1.
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Material used. Collection sites, sampling dates, and number of studied final-instar larvae of four Protohermes dobsonflies species.

Protohermes taxon Sampling locality and date of collection No. of studied larvae
P. grandis Japan, Honshu, Hyogo Prefecture, Sugihara River 35°05'N, 134°53'E; VI.1994-VI.1995 13
P. immaculatus Japan, Kagoshima Prefecture, Amami-Oshima Island, Kawauchi River; 28°17'N, 129°28'E; XI.1994 5
P. disjunctus Japan, Okinawa Prefecture, Ishigaki Island, Nagura River; 24°24'N, 124°09'E; Iriomote Island, Takana River; 24°22'N, 123°54'E; VIII-XI.1994 6
P. costalis Taiwan, Wulai, Tunhou Valley; 24°51'N, 121°29'E; II-1995 6
Figure 1.

Locations where the larvae of four Protohermes were collected A Protohermes grandis B P. immaculatus C P. disjunctus D P. costalis. (See Table 1 for location details.)

Chromosome preparation

For all four species, the gonads of the final-instar larvae were used in the experiments. The dates of the experiment were from June 1994 to June 1995. Sex of each larva was determined by the width of the head (Takeuchi and Hoshiba 2012a). The method used in the present study is a modification of the Hoshiba et al. (1989) method. The tissues (testes and ovaries) were dissected from the larvae in 1% sodium citrate, and then placed in a colchicine solution (0.005%) for 30 minutes. The tissues were then placed on individual clean glass slides, after which three drops of a fixative I (F1: 3 parts glacial acetic acid, 3 parts of 99% ethanol, 4 parts of water) were added using a pipette. Next, a tiny drop of dissociation solution (1 part of 30% lactic acid diluted in glacial acetic acid and 2 or 3 parts of F1) was put on the tissue. The tissue samples were dissociated using a needle for 10–15 seconds. Then, two drops of another second fixative (F2: 1 part of glacial acetic acid: 1 part of 99% ethanol) were added to the cells by pipette, after which excess fixative and dissociation solution were removed with filter paper. Each slide was then dried, but just before the slide was completely dry, a drop of glacial acetic acid was added to the cells. To complete drying, the slides were then placed in an incubator at 40–50 °C overnight.

Chromosome staining

The chromosomes were stained with 3% Gimsa’s solution in Sorensen’s phosphate buffer at pH 6.8 for 20 min (Hoshiba et al. 1989).

Microscopy and imaging

Microscopic photography of chromosomal preparations was performed using an optical microscope (OL-IM) connected to a Microflex Afx-dx (both manufactured by Japan Optical Industry Co., Ltd.). Photographs of selected chromosome spreads were made using a 100× oil immersion lens. Photographs were taken using Mini-copy film ISO25 and Sencia ISO100 (both manufactured by Fujifilm Co., Ltd.) and printed on Fuji WP FM2~3 photographic paper.

Results and discussion

The karyotypes were described following the nomenclature of Levan et al. (1964). The number of chromosomes for all four species was 2n = 24 (11 autosomal pairs +XX in females and 11 autosomal pairs + XY in males, Fig. 2). Takeuchi et al. (2002) reported that the autosomes of P. grandis consisted of one pair of large submetacentric chromosomes (no. 1), two pairs of metacentric chromosomes (no. 2 and 5), seven pairs of telocentric chromosomes (no. 3, 4, 6, 7, 8, 9, and 10), one pair of small chromosomes (no. 11) and the sex chromosomes consisting of a small submetacentric X chromosome and a small dot-like Y chromosome (Fig. 2A). In the present study, the autosomes of P. immaculatus consisted of one pair of large submetacentric chromosomes (no. 1), two pairs of metacentric chromosomes (no. 2 and 5), two pairs of subtelocentric chromosomes (no. 3 and 7), five pairs of telocentric chromosomes (no. 4, 6, 8, 9, and 10), one pair of small chromosomes and the sex chromosomes consisting of submetacentric X chromosome and a small dot-like Y chromosome (Fig. 2B). The X chromosome of P. immaculatus was about 5 µm long, which was considerably longer than the X chromosomes in the other three species (Fig. 2). A secondary constriction was observed in long-arm of chromosome number 3 of P. immaculatus. Overall, the karyotype of P. immaculatus was similar to that of P. grandis based on chromosome number and the position of the centromere on the chromosomes (Table 2). The autosomes of P. disjunctus consisted of one pair of large submetacentric chromosomes (no. 1), nine pairs of telocentric chromosomes (no. 2, 3, 4, 5, 6, 7, 8, 9, and 10), one pair of small chromosomes and the sex chromosomes consisting of a small submetacentric X chromosome and a small dot-like Y chromosome (Fig. 2C). The autosomes of P. costalis consisted of one pair of large submetacentric chromosomes (no. 1), nine pairs of telocentric chromosomes (no. 2, 3, 4, 5, 6, 7, 8, 9, and 10), one pair of small chromosomes and the sex chromosomes consisting of a small submetacentric X chromosome and a small dot-like Y chromosome (Fig. 2D). Overall, the karyotype of P. disjunctus was similar to that of P. costalis based on chromosome number and the position of the centromere on the chromosomes (Table 2).

Table 2.
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Chromosome numbers of Corydalinae (Megaloptera) species so far studied with karyotype descriptions. LM: large metacentric; LSM: large submetacentric; M: metacentric; SM: submetacentric; ST: subtelocentric; T: telocentric; dot: a very small chromosome.

Species Chromosome number (2n) Morphology Method Authors
Autosomes X Y
CORYDALINAE (Dobsonflies)
Protohermes grandis 24 1LSM+2M+7T+1dot SM dot drying-1*1 Takeuchi et al. 2002
drying-2*2 Present study
Protohermes immaculatus 24 1LSM+2M+2ST+5T+1dot SM dot drying-2*2 Present study
Protohermes disjunctus 24 1LSM+9T+1dot SM dot drying-2*2 Present study
Protohermes costalis 24 1LSM+9T+1dot SM dot drying-2*2 Present study
Corydalus cornutus 24 1LM+1M+8T+1dot SM dot squash Hughes-Schrader 1980

*1 = method by Kezer and Sessions (1979); *2 = method by Hoshiba et al. (1989).
Figure 2.

Karyotypes from spermatogonia of four species of dobsonflies in the genus Protohermes A P. grandis (male: 2n = 22 + XY) B P. immaculatus (male: 2n = 22+XY) C P. disjunctus (male: 2n = 22 + XY) D P. costalis (male: 2n = 22 + XY). Scale bar: 5 μm.

According to the paleogeographic map in and around the Ryukyu Arc since the late Pliocene, non-marine water was represented in the inland region during 2–1.7 Mya. In addition, the Ryukyu Arc ran from Kyushu to Taiwan several times during 0.4–0.02 Mya (Kimura 1996). Since 0.02 Mya, the Ryukyu Islands have been formed by sea immersion and some gaps (Fig. 1). Although speciation occurred due to geographic isolation, karyotypes remained similar between P. grandis and P. immaculatus, as well as between P. costalis and P. disjunctus. The cytological data in the present study supports the current concepts on taxonomy and phylogeny that P. immaculatus is a vicariant in the Ryukyu islands of P. grandis and P. disjunctus is a vicariant in the Sakishima islands of P. costalis. In the dobsonflies there appears to be a link between phylogenetic evolution and karyotype similarity.

Takeuchi et al. (2002) reported the parachute-type bivalent “Xyp” sex chromosomes of P. grandis. We found similar Xyp sex chromosomes in three additional Protohermes species in the present study (Fig. 3). This bivalent type of chromosomes has also been reported in two species of Parachauliodes fishflies (Takeuchi et al. 2012a), two species of Neochauliodes fishflies (Takeuchi et al. 2012b), and in the American fishfly species Neohermes fillicornis (Banks, 1903) (Hughes-Schrader 1980). This type of sex chromosome bivalent is also well-known and common in Coleoptera (Smith and Virkki 1978).

Figure 3.

Male spermatocyte of three species of dobsonflies in the genus Protohermes. Arrows indicate X and Y chromosomes that form parachute-type bivalents (“Xyp”) A P. immaculatus B P. disjunctus C P. costalis. Scale bar: 5 μm.

As in the present study, Hughes-Schrader (1980) reported that the North American dobsonfly Corydalus cornutus Linnaeus (1758) had 2n = 24 (11 autosomal pairs +XX in the female and 11 autosomal pairs + Xyp in the male). This similarity between East Asian and North American Corydalinae demonstrates that chromosome number is highly conserved among dobsonflies on different continents.

Acknowledgments

We are grateful to Dr. Stanley K. Sessions (Hartwick College, New York) and Dr. Robert A. Haack (USDA Forest Service, East Lansing, Michigan, emeritus) for reviewing this manuscript and contributing helpful comments. We also thank Dr. Fumio Hayashi (Tokyo Metropolitan University) and Dr. John Applegarth (retired, US Bureau of Land Management) for providing valuable information.

References

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