Morphology of the immature stages of Dasyhelea silvatica Wang, Zhang & Yu with redescriptions of adults (Diptera, Ceratopogonidae)

Abstract The immatures of the biting midge Dasyhelea silvatica are described and illustrated for the first time and a complete description of the adult male and female are provided using scanning electron and compound microscopes. The specimens were collected from flooded soil near a pond in Guizhou Province, China, and reared in the laboratory.


Introduction
Biting midges of the genus Dasyhelea Kieffer, 1911 (Diptera, Ceratopogonidae) are a large and complex group of Ceratopogonidae with diverse morphology and biology, and are cosmopolitan in distribution except in Antarctica (Grogan and Wieners 2006). At present there are 192 extant species of Dasyhelea in China (Duan et al. 2019;Nie et al. 2019), but only eight of these species are known by their immature stages (Yu et al. 2005(Yu et al. , 2013Duan et al. 2019). This may be an indication that China has been under-sampled historically compared to other countries. Therefore, efforts were made in order to study the immature stages of biting midges in China. During a recent entomological survey carried out in the vicinity of Xiaojiawan, a village in the Guizhou Province, immature specimens of Dasyhelea silvatica Wang, Zhang & Yu, 2014 were collected. The purpose of this paper is to provide a complete description, with illustrations, of the fourth instar larva and pupa of D. silvatica and a redescription of the adult male and female using a compound and a scanning electron microscope (LM and SEM).

Materials and methods
Larvae and pupae of D. silvatica were collected from flooded soil in Xiaojiawan, Guizhou Province in 2018, using a small shovel, and transferred to the laboratory. The larvae were individually placed in 24-well plates and fed with a sterile nutrient solution of Chlorella. Once they pupated, they were isolated in ampoule bottles on filter paper with sugar water. They were reared in an environmental chamber maintained at a temperature of 28 ± 2 °C, a relative humidity of 75 ± 2%, and a photoperiod of 12 h light and 12 h dark, and observed daily until adult emergence. The emergent adults, whole larvae, and pupae were preserved in ethanol at each stage. The specimens were mounted in Canada balsam following the technique described by Yu et al. (2005).
For the SEM study, one larva of D. silvatica was prepared following the technique of Ronderos et al. (2000Ronderos et al. ( , 2008. Ink illustrations were made using an attached camera lucida. Photographs were taken with a digital system adapted to an Olympus BX43 with a digital camera DP26. The studied material was deposited in the Insect Collection, Zunyi Medical University, Guizhou Province, China (ICZU). The morphological terms and identification methods of larvae, pupae and adults used follow Díaz et al. (2013), Borkent (2014) and Yu et al. (2005). The abbreviations used in this paper follow Duan et al. (2019). cylindrical, with seven or eight apical papillae. Hypostoma (Fig. 1F) with three large mesal teeth, flanked with four strong, lanceolate lateral teeth each side. Lacinial sclerite 1 with one seta; lacinial sclerite 2 without seta (Fig. 1F). Epipharynx (Fig. 1H-J) strongly sclerotized, dorsal comb moderately wide, round, subequally elongate, the middle of trailing edge with a large number of spines on the semicircular transparent membranous structure; lateral arms stout, elongate, with two auxiliary sclerites; LAW 0.10-0.13 (0.12, N = 2) mm, DCW 0.03-0.04 (0.04, N = 3). Hypopharynx (Fig. 1H, I) stout, heavily sclerotized, posterior comb straight with fringe, labium sclerotized. Thoracic pigmentation diffused, pale brown. Abdominal segments whitish, with diffused pale brown pigmentation. Caudal segment (Fig. 1G) with long stout hooks with pointed tips.

Discussion
Dasyhelea silvatica belongs to the subgenus D. (Dasyhelea) (Wang et al. 2014). The fourth instar larva of D. silvatica is very similar to its congeners D. azteca Huerta & Grogan by virtue of the antenna being short, the lateral arms of the epipharynx stout and the anterior portion of palatum with four pairs of campaniformia sensilla, but it can be distinguished from the former by the posterior portion of palatum bearing two pairs of coeloconica sensilla, the MD with three teeth, and the MP with only three small papillae. In addition, the fourth instar larva of D. silvatica is also similar to that of D. flavifrons (Guérin-Méneville), recently described by Díaz et al. (2019) with the brown head capsule, the posterior portion of palatum with four pairs of campaniformia sensilla and three pairs of coeloconica sensilla, the posterior comb of the hypopharynx has a fringe; however, D. flavifrons differs by the galeolacinia with 5-6 papillae and the MP with only three or four papillae. The common characteristics of the pupae of D. silvatica and D. azteca are as follows: the small rounded tubercles on cephalothorax; the surface of dorsal apotome also has rounded tubercles; the abdominal segments covered with spinules, and the sensilla of the fourth abdominal segment are all located on flattened tubercles. But the pupa of D. azteca differs from D. silvatica by having a single ocular sensillum, the exuviae is brown in general coloration, and the RO has 22-24 apical and three or four lateral pores, and without a pedicel. The pupa of D. silvatica is similar to D. flavifrons by virtue of the three dorsolateral cephalic sclerite sensilla and the tergite of the first abdominal segment with the L-1-IV represented by a long and stout seta, but the one ocular sensilla, the RO bearing 14-16 apical and four or five lateral pores, anterolateral sensilla with AL-1-T, AL-2-T long, thin seta and AL-3-T short, stout seta distinguish from D. silvatica. Furthermore, the pupa of D. silvatica is similar to that of Dasyhelea eloyi Díaz & Ronderos, 2013 with small rounded tubercles on cephalothorax surface and the RO with scale-like spines, but the latter has 6-8 lateral pores compared to D. silvatica with 5 lateral pores. Duan et al. (2019) described the larvae and pupae of D. alula collected in the same small wetland as D. silvatica. The fourth instar larva of D. silvatica shows similarities with that of D. alula in the rear comb of the hypopharynx with a fringe and two auxiliary sclerites on the lateral arms of the epipharynx. However, the fourth instar larva of D. alula is distinctly distinguished by its head capsule being yellowish, short, and wide; three teeth of the mandible are the same size; and hypostoma has the mesal portion smooth. The pupa of D. alula differs in the smaller total length (1.97 mm); RO having 7-8 apical and three lateral pores; anterolateral sensilla with AL-1-T medium-sized, thin seta and AL-3-T short, stout seta; the tergite I without L-2-I and L-3-I setae.
Finally, we found a semicircular, transparent membrane, strongly varying in shape, present at the trailing edge of the epipharynx of D. silvatica.