4378 (4): 581 588 Article

ZOOTAXA

ISSN 1175-5326 (print edition)

ISSN 1175-5334 (online edition)Copyright © 2018 Magnolia Press

Zootaxa 4378 (4): 581–588

http://www.mapress.com/j/zt/Article

https://doi.org/10.11646/zootaxa.4378.4.9

http://zoobank.org/urn:lsid:zoobank.org:pub:C83D6D14-D1D5-474B-91FC-A776D58771E2

Description of the final-instar larva and pupa of Neoneuromus ignobilis Navás,

1932 (Megaloptera: Corydalidae)

CHENGQUAN CAO1,2,3, FANGQING LIU2, CHAO TONG1, YAQIAN FANG1,

HUIMIN XU1 & XIN LI1 & XINGYUE LIU4,5

1College of Life Science, Leshan Normal University, Leshan, Sichuan 614004, China2Key Laboratory of Sichuan Institute for Protecting Endangered Birds in the Southwest Mountains, Leshan Normal University,

Leshan, Sichuan 614004, China3College of Biology and Environmental Sciences, China hellgrammites Research Center, Jishou University, Jishou, Hunan 416000,

China4Department of Entomology, China Agricultural University, Beijing 100193, China4Corresponding author. E-mail: [email protected]

Abstract

The immature stages of only one species of the dobsonfly genus Neoneuromus van der Weele are known. The final-instar

larva and pupa of N. ignobilis Navás, 1932, the most common and widespread species of the genus, is herein described

for the first time. Several morphological features of the larvae were found to distinguish N. ignobilis from other dobsonfly

species with similar large-sized mature larvae.

Key words: Corydalinae, dobsonfly, immature, taxonomy, morphology

Introduction

The genus Neoneuromus van der Weele, 1909 (Megaloptera: Corydalidae: Corydalinae) is one of the Asian

endemic dobsonfly genera. The genus is primarily distributed in the Oriental Region (Yang & Liu 2010). Nine

valid species are currently known (Liu & Yang 2004; Yang & Liu 2010). Despite the well-studied adult taxonomy

of this genus, the immature stages are poorly known. Currently, the final-instar larva and pupa of only one species,

N. sikkimmensis (van der Weele, 1907) has been described (Cao et al. 2016). It is noteworthy to mention that

different Neoneuromus species can be often sympatric (unpublished data). Additionally, the mature larvae of

Neoneuromus are often difficult to distinguish from the larvae of Acanthacorydalis van der Weele, 1907 (Cao &

Liu 2013; Cao 2016). Larvae of these two genera are often used as food and medicine in southwestern China,

especially in Sichuan Province (Cao 2014, 2016). In this paper, we describe the final-instar larva and pupa of

Neoneuromus ignobilis Navás, 1932, which is the most common species of Neoneuromus and widely distributed in

China, Vietnam, and Laos.

Material and methods

Morphological study. All larvae for the present study were collected from streams in the vicinity of Kaili City,

Guizhou Province of southwestern China in 2017 and are deposited in Leshan Normal University, Leshan, Sichuan

Province, China. Following the rearing method in Cao and Liu (2013) and Cao et al. (2016), all examined

specimens were placed in several plastic containers measuring 60×40×20 cm (water 3-5 cm deep) and covered by

plastic net with 30 mm mesh. Cobblestones were put on the bottom, and a mixture of sand and soil (ratio of 1:1)

Accepted by B. Kondratieff: 18 Dec. 2017; published: 12 Feb. 2018 581

mailto:[email protected]

was placed at one side of containers over water surface for the pupation site. Larvae were fed chironomid larvae

and the small crustacean Macrobranchium nipponense (de Haan, 1849). Each emerged adult was identified to

species by Xingyue Liu based on external morphology including genitalic characters (Liu & Yang 2004; Yang &

Liu 2010). Sexes of all the final-instar larvae could not be determined in the present study, whereas the sex of

pupae were determined on the presence or absence of the ovipositor, a pair of valvate structures at posteroventral

portion of the abdominal apex. The present description of the pupa is based only on female pupae, the only sex

available for this study.

For photographing, the larvae and pupae were placed in a covered petri dish and kept for a few minutes in a

refrigerator at ca. 5˚C to reduce movements. Habitus photographs and measurements were taken using a

microscopic system of LY-WN. The naturally extended larvae were measured with a caliper calibrated in

millimeters. Body length was measured from clypeus to posterior end of the abdomen. All measurements are

reported as mean ± standard deviation (SD).

Molecular identification. We selected three specimens of N. ignobilis, including a final-instar larva, an early

phase pupa, and a pre-emergence pupa, for molecular identification of species. Specimens were stored in 100%

ethanol after collection. For comparison, three other species of Neoneuromus [N. tonkinensis (van der Weele,

1907), N. sikkimmensis (van der Weele, 1907), and N. orientalis Liu & Yang, 2004] as well as two outgroups

[Acanthacorydalis orientalis (McLachlan, 1899) and Protohermes costalis (Walker, 1853)] were included for the

analysis.

The total genomic DNA of this species was extracted from the muscle tissues of abdomen using the TIANamp

Genomic DNA kit (TIANGEN BIOTECH, Beijing, China). Partial fragments of the cytochrome C oxidase subunit

1 (COI) gene of the mitochondrial genome were amplified using the universal primers C1-J-2183 (5’-

CAACATTTATTTTGATTTTTTGG-3’) and TL2-N-3014 (5’-TCCATTGCACTAATCTGC CATATTA-3’) (Simon

et al. 1994). The thermal cycling program consisted of an initial denaturation at 94°C for 5 min, followed by 35

cycles of 94°C denaturation for 30 s, 50°C annealing for 30 s, 72°C extension for 1 min, and a final extension at

72°C for 10 min. PCR products were sequenced bidirectionally by PCR-based method in Sangon Biotech

(Shanghai) company. GenBank accession numbers of the presently sequenced samples of N. ignobilis are:

MG583673, MG583674, and MG583675. DNA sequences of outgroups and other ingroup taxa were downloaded

from GenBank (N. tonkinensis (van der Weele, 1907): KF944566; N. sikkimmensis: KF944565; N. orientalis:

KF944564; N. ignobilis: KF944563; A. orientalis: KF944552; P. costalis: KF944577).

Consensus sequences were created with the program DNAman (Woffelman 2004) using sequence data from

both DNA strands. Sequences were double-checked by eye and aligned with BioEdit (Alzohairy 2011). Genetic

distances among ingroup species based on COI sequences (769 bp) were calculated using MEGA 6.0 (Tamura et

al. 2013) with the Kimura 2 Parameter (K2P) model (Kimura 1980). We used the neighbour-joining (NJ) approach,

which was performed in MEGA 6.0 to investigate the grouping of the present samples. Branch supporting values

were assessed using 1000 bootstraps replication.

Results

Molecular identification

The COI interspecific genetic divergence among the four species of Neoneuromus ranged from 0.068 to 0.102

(Table 1). The genetic divergence between N. ignobilis and each of the other three species of Neoneuromus ranged

from 0.068 to 0.094. The maximum interspecific genetic divergence (0.102) was found between N. orientalis and

N. tonkinensis. The minimum interspecific genetic divergence (0.068) was found between N. ignobilis and N.

orientalis. The intraspecific genetic divergence among the four samples of N. ignobilis ranged from 0.007 to 0.015.

In the NJ tree, the four samples of N. ignobilis are clustered as a monophyletic group that is the sister to N.

orientalis. Neoneuromus tonkinensis and N. sikkimmensis form another clade, being the sister to the clade of N.

ignobilis + N. orientalis.

CAO ET AL.582 · Zootaxa 4378 (4) © 2018 Magnolia Press

TABLE 1. Genetic divergence among samples based on COI sequence data.

FIGURE 1. Neighbour-joining tree of Neoneuromus species based on COI sequence data. Bootstrap values are shown at

nodes.

Taxonomy

Final-instar larva of Neoneuromus ignobilis Navás

(Fig. 2)

Description. Body length 72.45±6.05 mm (n = 8).

Head subquadrate in dorsal view, with distinct epicranial suture, black with intricate yellow markings. Clypeus

nearly ⅓Χ head width, faint yellow with a pair of blackish dot markings posterolaterally. Antennae with five

segments; distal two segments slightly shorter than 3rd segment; distal three segments much thinner, brown,

second segment black. Mouthparts generally blackish-brown; labrum with anterior margin yellowish brown;

mandibles slightly shorter than head, acutely tapering at tip, with three shorter inner teeth. Stemmata present. Frons

medially with three subtriangular yellow markings, and laterally with two pairs of smaller yellow markings. Vertex

with numerous yellow dots that are arranged into several reticulate patches at middle and on lateral portions.

Ventral portion of head slightly paler, posterolaterally with two broad, reticulate, yellow patches.

N.

tonkinensis

N.

sikkimmensis

N.

orientalis

N.

ignobilis

N.

ignobilis-1

N.

ignobilis-2

N.

ignobilis-3

P.

costalis

A.

orientalis

N. tonkinensis

N. sikkimmensis 0.079

N. orientalis 0.102 0.078

N. ignobilis 0.090 0.077 0.070

N. ignobilis-1 0.094 0.078 0.075 0.013

N. ignobilis-2 0.090 0.072 0.075 0.013 0.007

N. ignobilis-3 0.092 0.074 0.068 0.015 0.009 0.007

P. costalis 0.168 0.163 0.156 0.155 0.155 0.156 0.152

A. orientalis 0.128 0.126 0.131 0.129 0.131 0.129 0.125 0.150

Zootaxa 4378 (4) © 2018 Magnolia Press · 583LARVAE AND PUPAE OF NEONEUROMUS IGNOBILIS

FIGURE 2. The final-instar larva of Neoneuromus ignobilis Navás. A. habitus, dorsal view; B. habitus, ventral view. Scale bar

= 10 mm.


Pronotum blackish brown, glossy, with some short setae and a few longer conical setae laterally; complex

reticulate yellow patches present, laterally ones generally arranged into a pair of oblique bands, and medial ones

present as a pair of narrow vittae along posteromedian line; prosternum yellowish brown, but blackish laterally,

laterally with several yellow markings. Meso- and metathorx dorsally blackish brown, each notum with a pair of

comma-shaped yellow markings and a few yellow markings around them; ventrally yellow, with black setae. Legs

dark brown except for coxae, trochanters, ventral part of femora slightly paler; meso- and metafemora with a few

yellow stripes.

Abdomen blackish brown dorsally, with a few yellow, narrow, transverse or longitudinal lines; venter yellow,

with numerous blackish brown setae; within setose region of sterna 1–7 a glabrous sinuate transverse line present at

middle, and two pairs of glabrous longitudinal lines present laterally. Lateral filaments yellow with blackish long

setae. Segments 9 and 10 ventrally reddish brown. Claws of prolegs reddish brown, but black at tip. Ventral tufts

slightly pinkish.

Female pupa of Neoneuromus ignobilis Navás

(Figs. 3–4)

Description. Body length 48.25±5.65 mm (n = 6). Exarate, curved inward in natural condition (Fig. 3A).

Head subquadrate with some long setae, postocular spines slightly prominent; clypeus with anterior margin

truncate with a triangular labrum. Antennae filiform, long, curving to ventral part of head. Compound eyes ovoid.

Mandibles slightly shorter than head, with similar teeth to that in final-instar larva. Pronotum slightly longer than

width. Meso- and metathorax robust. Wing pads with feebly developed veins. Genitalia with weakly developed

gonocoxites 9 and ectoprocts; gonocoxites 9 paired, small and subtriangular; ectoprocts much larger than

gonocoxites 9, with posterodorsal portion distinctly produced.

FIGURE 3. Female pupa of Neoneuromus ignobilis Navás. A. habitus, lateral view in natural condition; B. habitus, dorsal

view, with abdomen stretched; C. habitus, ventral view, with abdomen stretched; D. apex of abdomen, dorsal view; E. apex of

abdomen, ventral view; F. apex of abdomen, lateral view. Scale bar = 10 mm (A–C) and 4 mm (D–F).


FIGURE 4. Female pupa of Neoneuromus ignobilis Navás at the pre-emergence stage. A. habitus, dorsal view, with abdomen

stretched; B. habitus, ventral view, with abdomen stretched. Scale bar = 10 mm.


Coloration and markings. Body coloration and some markings varying very seriously depending on the

developmental stages. At early stage (Fig. 3B, C), body golden yellow generally; compound eyes grayish black;

head yellow and immaculate; antennae yellow and slightly brown; pronotum almost immaculate with some

glabrous areas that are generally similar in shape to pronotal marking pattern in final-instar larva; wing pads yellow

to brown; legs pale yellow; abdomen largely brown dorsally but yellow ventrally. At pre-emergence stage (Fig. 4),

body coloration in general yellowish brown, pronotum laterally with a pair of blackish vittae; wing pads blackish;

legs with apex of femora, base and apex of tibia, and tarsomeres 2–4 blackish.

Materials examined. CHINA: Guizhou Province: Kaili City, 26°35′N, 107°58′E, 10.II.2017, Chengquan Cao

& Yu Deng, 8 final-instar larvae and 6 pupae reared from larvae collected in the field.

Discussion

The present description of the final-instar larva and pupa of N. ignobilis provides supplementary information on the

knowledge of Neoneuromus immature stages. A morphological comparison of the final-instar larvae of N. ignobilis

with the final-instar larva of N. sikkimmensis, we found several characters useful for species identification, at least

for distinguishing N. ignobilis from N. sikkimmensis. First, the N. ignobilis larvae have three median yellow

markings and two pairs of rather small yellow markings around median markings, while in N. sikkimmensis the

median frontal markings are slightly larger and another pair of broad yellow markings is present laterally. Second,

the larvae of N. ignobilis lack the median yellow vittae on anteromedian portion of pronotum, while in N.

sikkimmensis these vittae are present. Third, the abdomen is blackish brown dorsally in N. ignobilis but yellowish

brown in N. sikkimmensis. Cao et al. (2016) noted that the Neoneuromus larvae can be distinguished from the co-

occurring Acanthacorydalis larvae by the presence of a pair of distinct pale longitudinal markings on anteromedian

portion of pronotum and by the absence of a V-shaped elevated bare area on sterna of abdominal segments.

However, this comparison was made based only on one species of each genus. As the median yellow vittae on

anteromedian portion of pronotum are absent in the larvae of N. ignobilis, this character is not useful for

distinguishing larvae of Neoneuromus and Acanthacorydalis. Nevertheless, the second character mentioned in Cao

et al. (2016), the presence/absence of V-shaped elevated bare area on sterna of abdominal segments is still valid for

distinguishing the two genera.

Adults of N. ignobilis and N. sikkimmensis differ also in cephalic and pronotal markings. In N. ignobilis, at

least for the studied adults of this species from Guizhou Province of southwestern China (distributional area of the

presently studied larvae), the adult head of N. ignobilis has broad black markings on lateral portion of vertex, and

the pronotum has a pair of wide black vittae. In contrast, the adult head of N. sikkimmensis has only narrow black

lateral margins of vertex, and the pronotum has a pair of narrower black vittae, which are usually interrupted

anteriorly. Interestingly, the reduced condition of yellow markings on head and pronotum in N. ignobilis,

alternatively shown with expanded black areas of the larvae, has similar tendency of development of dark markings

to the conspecific adults. This may indicate possible corresponding development of coloration and markings

between larva and adults in Neoneuromus. Many species of Neoneuromus, including N. ignobilis, are well-known

to be polymorphic intraspecifically in body and wing coloration/marking patterns (unpublished data). It is

suggested that in future studies, a comparison of larval morphologies among intraspecific color forms of

Neoneuromus is warranted.

Acknowledgments

We thank Dr. Yu Wang from Panzhihua University, Yu Deng and Suting Li for collecting, rearing and photograph.

This research was funded by the National Natural Science Foundation of China (Nos. 31322051, 31301902,

31672322) and the project of the Science and Technology Department of Sichuan Province (2015NZ0096).

References

Alzohairy, A.M. (2011) BioEdit: An important software for molecular biology. GERF: Bulletin of Biosciences, 2, 60–61.


Cao, C.Q. (2014) The current development and utilization and species definition of sand crawling worm in China. Hubei

Agricultural Science, 53, 5061–5064.

Cao, C.Q. (2016) Rearing hellgrammites for food and medicine in China. Journal of Insects as Food and Feed, 2, 263–267.

https://doi.org/10.3920/JIFF2016.0009

Cao, C.Q., Huang, M.Y., Qing, Y.T., Chen, S.Z., Huang, C.J. & Ge, Y.Y. (2011) Respiratory system and respiratory behaviors of

the larva of Neoneuromus ignobilis. Chinese Journal of Applied Entomology, 2, 392–396.

Cao, C.Q. & Liu, X.Y. (2013) Description of the final-instar larva and pupa of Acanthacorydalis orientalis (McLachlan, 1899)

(Megaloptera: Corydalidae) with some life history notes. Zootaxa, 3691 (1), 145–152.


Cao, C.Q., Tong, C., Chen, S.Z., Liu, Z.W. & Liu, X.Y. (2016) First description and bionomic notes for the final-instar larva

and pupa of an Oriental dobsonfly species, Neoneuromus sikkimmensis (van der Weele, 1907) (Megaloptera: Corydalidae).

Zootaxa, 4179 (2), 288–294.


Kimura, M. (1980) A simple method for estimating evolutionary rates of base substitutions through comparative studies of

nucleotide sequences. Journal of Molecular Evolution, 16, 111–120.

https://doi.org/10.1007/BF01731581

Liu, X.Y. & Yang, D. (2004) A revision of the genus Neoneuromus in China (Megaloptera: Corydalidae). Hydrobiologia, 517,

147–159.

https://doi.org/10.1023/B:HYDR.0000027343.41538.a1

McLachlan R. (1899) A second Asiatic species of Corydalis. Transactions of the Entomological Society of London, 1899, 281–

283.

https://doi.org/10.1111/j.1365-2311.1899.tb00984.x

Navás, L. (1932) Decadas de insectos nuevos. Brotéria (Zoológica), 1, 145–155.

Simon, C., Frati, F., Beckenbach, A. Crespi, B., Liu, H. & Flook, P. (1994) Evolution, weighting, and phylogenetic utility of

mitochondrial gene sequences and a compilation of conserved polymerase chain reaction primers. Annals of the

Entomological Society of America, 87, 651–701.

https://doi.org/10.1093/aesa/87.6.651

Tamura, K., Stecher, G., Peterson, D., Filipski, A. & Kumar, S. (2013) MEGA6: molecular evolutionary genetics analysis.

Version 6.0. Molecular Biology and Evolution, 30, 272–2729.

https://doi.org/10.1093/molbev/mst197

van der Weele, H.W. (1907) Notizen uber Sialiden und Beschreibung einiger neuen Arten. Notes from the Leyden Museum, 28,

227–264.

Woffelman, C. (2004) DNAMAN for Windows, Version 5.2.10. Lynon Biosoft, Institute of Molecular Plant Sciences. Leiden

University, Leiden. [software]

Walker, F. (1853) Catalogue of the specimens of neuropterous insects in the collection of the British Museum. Part II (Sialides–

Nemopterides). Newman, London, 284 pp. [pp. 193–476]

Yang, D. & Liu, X.Y. (2010) Fauna Sinica Insecta. Vol. 51. Megaloptera. Science Press, Beijing, 457 pp., XIV pls.


4378 (4): 581 588 Article

Documents

Transcript of 4378 (4): 581 588 Article