﻿Description of the larva of Cybisterlewisianus Sharp, 1873 (Coleoptera, Dytiscidae, Cybistrinae)

﻿Abstract We describe for the first time, the larvae of Cybister (Cybister) lewisianus Sharp, 1873, an endangered species of diving beetle in Japan, emphasizing the chaetotaxy of the cephalic capsule, head appendages, legs, last abdominal segment, and urogomphi. Cybisterlewisianus larvae are characterized by a longer third article of antenna 3 than the sum of the first and second articles; rounded apex of parietal setae 1–3; labium seta 8 absent; elongated trochanter seta 4, not multi-branched; rounded apex of abdominal setae 1, 12, and 13 (instar I); narrow lateral projections of the frontoclypeus; pronotum without two dark-brown longitudinal stripes dorsally (instar III); and the base of the thick row of small setae on the inner edge of the mandible angulate and projecting medially (all instars).


Introduction
Compared to adults, coleopteran larvae lack morphological information (Hammond et al. 2019).This trend is also present in Dytiscidae, in which information on larval morphology is generally limited (Alarie and Michat 2023).A nomenclature system of Dytiscidae larval chaetotaxy was developed to provide a detailed description and comparison of their larval morphology and to allow for phylogenetic analysis (Alarie and Michat 2023).Particularly, primary sensilla (setae and pores) have been important for the diagnosis and study of phylogenetic relationships among the species (Michat et al. 2017).
The genus Cybister Curtis, 1827 includes large diving beetles (adult length: 13-43 mm) and belongs to the subfamily Cybistrinae (Miller and Bergsten 2016).Cybister includes 97 species and is distributed in all major biogeographic regions (Miller and Bergsten 2016;Nilsson and Hájek 2023).Seven species are distributed in Japan (Nakajima et al. 2020;Watanabe and Yoshitomi 2022), and six of these species (approximately 86%) are on the national Red List and are threatened with extinction (Ministry of the Environment of Japan 2020).
Cybister lewisianus Sharp, 1873 (adult length 21-26 mm) is listed as "Critically Endangered" on the Japanese Red List (Ministry of the Environment of Japan 2015) and is designated a "nationally endangered species of wild fauna and flora" by the Japanese "Conservation of Endangered Species of Wild Fauna and Flora" Act (Ministry of the Environment of Japan 2022).Identifying the larvae of Japanese Cybister to species level is challenging due to their similar body shape and length.They have not been described in detail, and morphological information is limited to third-instar larvae (Ichikawa 1984;Kamite 2008;Mitamura et al. 2017).Recently, Inoda et al. (2022) stated that the shape of the larval clypeus could be useful to identify these species.Similarly, Watanabe (2024) suggested color of the antennal segments, ratio of each antennal segment and article, and shape of the mandible are helpful for identification in the field.Herein, we describe in detail the morphometric and chaetotaxic characteristics of C. lewisianus larvae for the first time, according to the now generalized system used to describe aquatic Adephaga larvae (Alarie and Michat 2023).

Material and methods
All larvae used for description were obtained through rearing following the methods used by Watanabe et al. (2021Watanabe et al. ( , 2023)).A wild individual (Fig. 4) was captured with official permission of the Wildlife Division, Chubu Regional Environmental Office, Ministry of the Environment of Japan (2303313) and the Ishikawa Prefecture, Japan (4-45), and was released at the site after it was photographed.
The larvae were fixed in boiling water, transferred to 70% ethanol in glass vials with caps and subsequently mounted on slides with 70% ethanol or euparal.The specimens were observed using an optical microscope (Nikon ECLIPSE E400) up to 1000-fold magnification and were sketched using a microscope equipped with a Nikon Y-IDT drawing tube.Figures of the line drawing were prepared using an iPad Pro 11-inch (4 th generation) after scanning the sketch.Photographs of the living larvae were captured using a Nikon D500 digital camera equipped with a Nikon AF-S Micro NIKKOR 60 mm f/2.8GED lens.The examined larvae were deposited in the larval collections of the Ishikawa Insect Museum (Ishikawa, Japan) and the Hoshizaki Green Foundation (Shimane, Japan).

Description of larvae of Cybister (Cybister) lewisianus Sharp, 1873
Figs 1-30, Table 1 Material source.The description of larvae of Cybister lewisianus was based on four instar I, three instar II, and three instar III specimens, reared ex ovo in the laboratory at the Ishikawa Insect Museum from adults collected in Suzu-shi, Ishikawa Prefecture, with official permission for exhibition and research at the Ishikawa Insect Museum.
Diagnosis.Ratio HL/HW = 1.26-1.32(instar I), 1.30-1.33(instar II), and 1.32-1.34(instar III) (Table 1 Description, instar I (Figs 1, 5-30).Color (Fig. 1).Head capsule pale-yellow; anterolateral part of frontclypeus, coronal line, and lateral margin light-yellowbrown; several brown small maculae; stemmata dark-brown; antennae pale-yellow except for third article of A3 and A4 dark-brown; mandible pale-yellow except for distal portion light-red-brown and thick row of small setae on the inner margin dark-brown; maxilla pale-yellow except for third article of palpomere 3;   labium pale-yellow; thoracic tergites light-yellow-brown except for pale-yellow membranous area, several small brown maculae, slightly larger pair of brown maculae on anterior quarter side of protergite; one pair of brown maculate on each side of membranous region of meso-and metathorax; abdominal tergites I-VI pale-yellow with several small brown maculae; one pair of slightly larger brown maculae on middle of dorsolateral margin of membranous region, five pairs of ventral brown maculae; abdominal tergites VII-VIII light-yellow-brown with several small brown maculae; four pairs of brown maculae on sterna of abdominal tergite VII; legs pale-yellow; urogomphus light-yellow-brown.Color as shown in Fig. 1 and in Watanabe (2024: figs 1B, 2C).Body (Fig. 1).Elongate, subcylindrical; measurements and body shape ratios are shown in Table 1.
Abdomen.Spiracles present on segments I-VII.
Remarks.A study on Laccophilus yoshitomii Watanabe & Kamite, 2018 reported that larvae raised in captivity differed in color from those collected in the field (Watanabe and Okada 2023); however, no pronounced differences in color were observed between captive and wild individuals of Cybister lewisianus in the present study (Figs 3,4).

Table 1 .
Measurements and ratios for the larvae of Cybister lewisianus Sharp, 1873.N = number of specimens examined.