Distinct roles of the Hox genes Ultrabithorax and abdominal-A in scorpionfly embryonic proleg development.

The abdominal appendages of larval insects have a complex evolutionary history of gain and loss, but the regulatory mechanisms underlying the abdominal appendage development remain largely unclear. Here, we investigated the embryogenesis of abdominal prolegs in the scorpionfly Panorpa liui Hua (Mecoptera: Panorpidae) using in situ hybridization and parental RNA interference. The results show that RNAi-mediated knockdown of Ultrabithorax (Ubx) led to a homeotic transformation of the first abdominal segment (A1) into the third thoracic segment (T3) and changed the distributions of the downstream target Distal-less (Dll) expression but did not affect the expression levels of Dll. Knockdown of abdominal-A (abd-A) resulted in malformed segments, abnormal prolegs and disrupted Dll expression. The results demonstrate that the gene Ubx maintains an ancestral role of modulating A1 appendage fate without preventing Dll initiation, and a secondary adaptation of abd-A evolves the ability to specify abdominal segments and proleg identity. We conclude that changes in abdominal Hox gene expression and their target genes regulate abdominal appendage morphology during the evolutionary course of holometabolous larvae.

Flagellar sensilla of the hangingflies Bittacus planus Cheng and Bittacus sinicus Issiki (Mecoptera: Bittacidae).

Adult hangingflies are very sensitive to humidity, thereby frequently being used as ecological indicators to assess the degradation of the environment, especially forest ecosystems. Studies on the sense organs associated with hygro- and thermo-sensitivity, however, have been scant. Here, the ultramorphology and distribution of the flagellar sensilla were investigated in the adult hangingflies Bittacus planus Cheng, 1949 and Bittacus sinicus Issiki, 1931 using scanning electron microscopy. Four types of sensilla are identified in B. planus, including sensilla campaniformia, sensilla chaetica (SC1, SC2), sensilla basiconica (SB1, SB2), and sensilla coeloconica. In B. sinicus, sensilla chaetica III are present additionally from 7th to 14th flagellomeres. Abundant sensilla coeloconica are present on the flagella of Bittacus. Sensilla basiconica I are situated at the joints of flagellomeres. Sensilla campaniformia are densely distributed on the basal half of the first flagellomere, but scarcely on other flagellomeres. Sensilla chaetica II are present on the distal three flagellomeres. The size, abundance, and distribution of flagellar sensilla differ considerably between B. planus and B. sinicus. We infer that the abundance of sensilla coeloconica is likely associated with the hygro- and thermo-sensitivity in Bittacidae. Studies of flagellar sensilla can provide valuable information for subsequent electrophysiological, behavioral, biogeographical, and phylogenetic analyses of Bittacidae.

Two new species of the genus Terrobittacus Tan & Hua, 2009 (Mecoptera, Bittacidae).

Two new species of Terrobittacus Tan & Hua, 2009 are described and illustrated from Sichuan and Guangxi provinces of China, increasing the species number of Terrobittacus to eight. Terrobittacusemeishanicussp. nov. is differentiated from its congeners by wings with distinct markings and a female subgenital plate with a V-shaped carina. Terrobittacuslaoshanicussp. nov. can be recognized by the black terga VI-IX. A key to species of Terrobittacus is updated. The species distribution and the relationship between adult morphology and mating behavior were briefly discussed.

Ultrastructure of the dorsal ocellus of Bittacus planus larvae (Mecoptera: Bittacidae) with evolutionary significance.

The Bittacidae are unique in holometabolous insects in that their larvae bear a dorsal ocellus on the frons. The fine structure of the dorsal ocellus, however, has not been investigated to date. Here, the ultrastructure of the larval dorsal ocellus was studied in the hangingfly Bittacus planus Cheng, 1949 using light, scanning, and transmission electron microscopy. The dorsal ocellus of the larvae comprises a cornea, corneagenous cells, and retinula cells. The cornea is a laminated structure. A layer of corneagenous cells is located below the cornea. Numerous retinula cells are arranged tightly beneath the corneagenous cells. The retinula cells modify their adjacent membranes into numerous linear microvilli, which form an analogue of the rhabdom among adjacent retinula cells. The results show that the dorsal ocellus of larval Bittacidae is a highly vestigial organ and appears to be degenerating during the postembryonic development. The presence of the vestigial dorsal ocellus is likely to represent an ancestral plesiomorphy of holometabolous insects, providing new evidence for exploring the evolutionary origin of holometabolous larvae.

Mouthpart structure of the adult Bicaubittacus appendiculatus () (Mecoptera: Bittacidae).

The structure and functional morphology of the mouthparts were investigated in adult hangingfly Bicaubittacus appendiculatus (Esben-Petersen, 1927) by scanning electron microscopy and histological serial sections. The mandibulate mouthparts consist of a labrum-epipharynx, paired mandibles and maxillae, and unpaired labium and hypopharynx. The labrum is elongated and tapered toward the apex. The epipharynx is furnished with numerous sensilla. The mandibles are sword-shaped, with an outer sharp tooth curved mesad and an inner blunt corner. The basal region of each mandible processes a conical projection. The maxillae are well-developed, each consisting of a sclerotized cardo, an elongated stipes, which bears an inner lacinia, an outer galea, and laterally a five-segmented maxillary palp. The labium is formed by a postmentum, a prementum and a pair of two-segmented labial palps. The hypopharynx is concave inward on the anterior side, with numerous microtrichia on the posterior surface. Seven types of sensilla were found on the mouthparts: sensilla basiconica on the epipharynx, and maxillary and labial palps; sensilla chaetica on the epipharynx; sensilla palmata, sensilla placoidea and sensilla trichodea on the epipharynx and maxillary palp; sensilla campaniformia and hair plates on the basal joints of palps. The sensillar function and the feeding mechanism of mouthparts in bittacids are briefly discussed.

The Hox gene Abdominal-B regulates the appendage development during the embryogenesis of scorpionflies.

The Homeotic Complex (Hox) genes encode conserved homeodomain transcription factors that specify segment identity and appendage morphology along the antero-posterior axis in bilaterian animals. The Hox gene Abdominal-B (Abd-B) is mainly expressed in the posterior segments of the abdomen and plays an important role in insect organogenesis. In Mecoptera, the potential function of this gene remains unclear yet. Here, we performed a de novo transcriptome assembly and identified an Abd-B ortholog in the scorpionfly Panorpa liui. Quantitative real-time reverse transcription PCR showed that Abd-B expression increased gradually in embryos 76 h post oviposition, and was mainly present in the more posterior abdominal segments. Embryonic RNA interference of Abd-B resulted in a set of abnormalities, including developmental arrest, malformed suckers and misspecification of posterior segment identity. These results suggest that Abd-B is required for the proper development of the posterior abdomen. Furthermore, in Abd-B RNAi embryos, the expression of the appendage marker Distal-less (Dll) was up-regulated and was additionally present on abdominal segments IX and X compared with wild embryos, suggesting that scorpionfly Abd-B may act to suppress proleg development and has gained the ability to repress Dll expression on the more posterior abdominal segments. This study provides additional information on both the functional and evolutionary roles of Abd-B across different insects.

Ultrastructure of the larval eyes of the hangingfly Terrobittacus implicatus (Mecoptera: Bittacidae).

The fine structure of the larval eyes of the hangingfly Terrobittacus implicatus (Huang & Hua) was investigated using scanning and transmission electron microscopy. The results show that the larval eyes of T. implicatus each consist of seven spaced ommatidia. Each ommatidium is composed of a corneal lens with about 45 lamellae, a tetrapartite eucone type of crystalline cone, eight retinula cells, two primary pigment cells, and an undetermined number of secondary pigment cells. The rhabdomeres of eight retinula cells effectively fuse into a centrally-fused, tiered funnel-shaped rhabdom extending from the base of the crystalline cone deeply into the ommatidium. In light of different positions in the ommatidium, the retinula cells can be divided into four distal and four proximal retinula cells. Pigment cells envelop the entire ommatidium. Electron-lucent vesicles are abundant throughout the cytoplasm of the eight retinula cells. The larval ommatidia of T. implicatus are similar to those of the Panorpidae, except for the distal retinula cells that also participate in the formation of the proximal rhabdom. In this case, the larval eyes of T. implicatus may lie in the transitional stage during the larval eye evolution of insects from ommatidia to stemmata.

A new species of the genus Cerapanorpa (Mecoptera, Panorpidae) from the eastern Bashan Mountains.

BACKGROUND: Cerapanorpa Gao, Ma & Hua, 2016, an endemic genus of Panorpidae in central China's mountain regions, currently comprises 21 described species. Recently, the short-horned scorpionfly C.brevicornis (Hua & Li, 2007) was confirmed to contain two valid species by phylogeographic and morphological data. Individuals from the highlands of the eastern Bashan Mountains were suggested as a good species, separated from the original short-horned C.brevicornis. NEW INFORMATION: Cerapanorpaalpina sp. nov. was described from the alpine zone of the eastern Bashan Mountains in central China. The new species differs from its congeners by the following combination of characters: male bearing an extra-short anal horn on posterior margin of tergum VI; paramere elongate, extending beyond the median tooth of gonostylus and curved laterally at basal half; female medigynium slightly constricted medially without dorsal basal plate. The species number of Cerapanorpa is raised to 22.

Traumatic mating increases anchorage of mating male and reduces female remating duration and fecundity in a scorpionfly species.

Traumatic mating is the male wounding his mate during mating using specialized anatomy. However, why males have evolved to injure their mates during mating remains poorly understood. We studied traumatic mating in Dicerapanorpa magna to determine its effects on male and female fitness. The sharp teeth on male gonostyli penetrate the female genitalia and cause copulatory wounds, and the number of scars on the female genitals is positively related to the number of times females mated. When the injurious teeth were encased with low-temperature wax, preventing their penetration of the female's genitalia during mating, male mating success and copulation duration were reduced significantly, indicating the importance of the teeth in allowing the male to secure copulation, remain in copula and effectively inseminate his mate. The remating experiments showed that traumatic mating had little effect on the female mating refractory period, but significantly reduced female remating duration with subsequent males, probably benefiting the first-mating male with longer copulation duration and transferring more sperm into the female's spermatheca. The copulatory wounds reduced female fecundity, but did not accelerate the timing of egg deposition. This is probably the first report that traumatic mating reduces female remating duration through successive remating experiments in animals. Overall, our results provide evidence that traumatic mating in the scorpionfly helps increase the male's anchoring control during mating and provides him advantage in sperm competition, but at the expense of lowering female fecundity.

Spermatheca of the scorpionfly Sinopanorpa tincta (Navás, 1931) (Mecoptera: Panorpidae).

The spermathecal structure of the scorpionfly Sinopanorpa tincta (Navás, 1931) was investigated using light microscopy, scanning, and transmission electron microscopy. The spermatheca consists of a bean-shaped spermathecal reservoir and an elongated spermathecal duct. The spermathecal reservoir can be subdivided into a distal portion with well-developed muscles and a proximal transitional portion connected to the spermathecal duct. The spermathecal duct is slender for its basal three-fourths and is greatly thicker for its distal one-fourth, which is mainly responsible for secretory function. A spermathecal pump formed from longitudinal muscle fibers was attached to the reservoir to control the transport of sperm. The lumen of the spermathecal reservoir is lined with a cuticle and filled with spermatozoa after copulation. The epithelium of the distal reservoir comprises a single type of epithelial cells, while the epithelium of the proximal reservoir and the spermathecal duct has three types of cells: secretory cells, duct-forming cells, and common epithelial cells. The secretory cells are rich in mitochondria, rough endoplasmic reticulum, and electron-dense secretory vesicles. The duct-forming cells form cuticular ducts, which connect the secretory cells to the lumen of the spermatheca. The spermathecal reservoir mainly serves as the storage of sperm, but the proximal reservoir as well as the spermathecal duct serves as secretory functions that maintain sperm viability.

Two new species of Cerapanorpa (Mecoptera, Panorpidae) from the Qinling and Minshan mountains.

Two new species of Cerapanorpa Gao, Ma & Hua, 2016 are described from the Qinling and Minshan mountains, respectively. Cerapanorpa qinlingensis sp. nov. can be readily distinguished from its congeners by the elongate hypovalves and the extremely developed basal process of gonostylus in male genitalia. Cerapanorpa minshana sp. nov. is characterized by its bifurcated parameres and a cluster of long black bristles on the inner apex of the gonocoxite. The number of species of Cerapanorpa is raised to 21. An updated key to species of Cerapanorpa is presented.

The highly rearranged karyotype of the hangingfly Bittacus sinicus (Mecoptera, Bittacidae): the lowest chromosome number in the order.

Cytogenetic features of the hangingfly Bittacus sinicus Issiki, 1931 were investigated for the first time using C-banding and DAPI (4',6-diamidino-2-phenylindole) staining. The karyotype analyses show that the male B. sinicus possesses the lowest chromosome number (2n = 15) ever observed in Mecoptera, and an almost symmetric karyotype with MCA (Mean Centromeric Asymmetry) of 12.55 and CVCL (Coefficient of Variation of Chromosome Length) of 19.78. The chromosomes are either metacentric or submetacentric with their sizes decreasing gradually. Both the C-banding and DAPI+ patterns detect intermediate heterochromatin on the pachytene bivalents of B. sinicus, definitely different from the heterochromatic segment at one bivalent terminal of other bittacids studied previously. The male meiosis of B. sinicus is chiasmate with two chiasmata in metacentric bivalents and one in the submetacentric bivalent. The sex determination mechanism is X0(♂), which is likely plesiomorphic in Bittacidae. Two alternative scenarios of karyotype origin and evolution in Bittacus Latreille, 1805 are discussed.

Three new species of the genus Bittacus Latreille, 1805 (Mecoptera: Bittacidae), with a key to the species of Bittacidae in South China.

The Bittacidae of South China consist of two species of Bicaubittacus and eight species of Bittacus, among which three species are described here as new to science. Bittacus acutus sp. n. from Guangxi is recognized mainly by the hat-shaped epandrial lobe with an acute ventro-distal process in the male genitalia. Bittacus shaoguanensis sp. n. from Guangdong is distinguished by the triangular epandrial lobe in the male genitalia, and a V-shaped streak in the central part of wings. Bittacus longilobus sp. n. from Guangdong is recognizable by the main body of the epandrial lobe trapezoid, with an elongate ventro-distal lobe twice as long. The number of described species of the Chinese Bittacus is now raised to 41. A key to the species of Bittacidae in South China is provided.

Vasa deferentia and associated structures of the male Panorpodes kuandianensis (Mecoptera: Panorpodidae).

The male reproductive system may provide significant evidence for the taxonomic and phylogenetic analyses of insects. However, current knowledge of the male reproductive system in Mecoptera is mainly concentrated on the external genitalia, and is rarely involved in the internal reproductive system. Here, we investigated the morphology and the fine structure of the vasa deferentia and associated structures of the male reproductive system of Panorpodes kuandianensis Zhong et al., 2011 (Panorpodidae) using light, scanning, and transmission electron microscopy. The male reproductive system of P. kuandianensis consists of a pair of symmetrical testes with three tubular testicular follicles, two epididymides, two distinctly partitioned vasa deferentia, a pair of mesadenia, one ejaculatory sac, and the external genitalia. A pair of expanded seminal vesicles are modified from the median part of the vasa deferentia, and evolve into secretory organs. The seminal vesicles have elongated cylindrical epithelial cells, which contain abundant secretory materials in the cytoplasm and form a small central lumen, likely serving a secretory function rather than provisionally storing sperm as in most other insects. Alternatively, the sperm are stored temporarily in the epididymis, the greatly coiled portion of the vasa deferentia. The morphology of the male reproductive system supports the close relationships of Panorpidae and Panorpodidae.

How Does the Male Penisfilum Enter the Female Copulatory Pore in Hangingflies?

Hangingflies are characterized by the interesting nuptial feeding behavior and unusual belly-to-belly hanging mating position. However, the mating behavior and the copulatory mechanism remain poorly known for Bittacidae, especially how the elongated male penisfilum enters the copulatory pore of the female. In this study, the mating behavior and copulatory mechanism of Terrobittacus implicatus (Huang and Hua, 2006) were investigated to reveal the functional morphology of hangingfly genitalia. The results show that the male provides a prey as a nuptial gift to the female and twists his abdomen about 180° to form a belly-to-belly hanging mating position. During the penisfilum-entering process, the male epandrial lobes clamp the female subgenital plate with the aid of the female abdomen swelling. Then the male locates the female copulatory pore through his upper branch of the proctiger and inserts his penisfilum into the female spermathecal duct in cooperation with the short setae on the groove of the proctiger. The female subgenital plate where the epandrial lobes clamp is strongly sclerotized and melanized. The copulatory mechanism of Terrobittacus is briefly discussed.

Two new species of the genus Panorpa (Mecoptera, Panorpidae) from eastern China and a new synonym.

Panorpa Linnaeus, 1758 is the largest genus in the scorpionfly family Panorpidae. Herein we describe two new species from eastern China, Panorpa jinhuaensis sp. nov. from Jinhua, Zhejiang Province and Panorpa menqiuleii sp. nov. from Yuexi and Huoshan, Anhui Province. Panorpa wrightae Cheng, 1957 from Mount Mogan, Zhejiang Province is considered to be a junior subjective synonym of Panorpa mokansana Cheng, 1957 from the same locality. Panorpa mokansana Cheng, 1957 is redescribed and illustrated in detail. A key to species of Panorpa from eastern China is also provided.

Habitat divergence shapes the morphological diversity of larval insects: insights from scorpionflies.

Insects are the most diverse group of organisms in the world, but how this diversity was achieved is still a disputable and unsatisfactorily resolved issue. In this paper, we investigated the correlations of habitat preferences and morphological traits in larval Panorpidae in the phylogenetic context to unravel the driving forces underlying the evolution of morphological traits. The results show that most anatomical features are shared by monophyletic groups and are synapomorphies. However, the phenotypes of body colorations are shared by paraphyletic assemblages, implying that they are adaptive characters. The larvae of Dicerapanorpa and Cerapanorpa are epedaphic and are darkish dorsally as camouflage, and possess well-developed locomotory appendages as adaptations likely to avoid potential predators. On the contrary, the larvae of Neopanorpa are euedaphic and are pale on their trunks, with shallow furrows, reduced antennae, shortened setae, flattened compound eyes on the head capsules, and short dorsal processes on the trunk. All these characters appear to be adaptations for the larvae to inhabit the soil. We suggest that habitat divergence has driven the morphological diversity between the epedaphic and euedaphic larvae, and may be partly responsible for the divergence of major clades within the Panorpidae.

Complete mitochondrial genomes of Bittacus strigosus and Panorpa debilis and genomic comparisons of Mecoptera.

Mitochondrial genomes play a significant role in reconstructing phylogenetic relationships and revealing molecular evolution of insects. However, only four mitochondrial genomes were reported in Mecoptera to date. Here, we obtained two new complete mitochondrial genomes of the hangingfly Bittacus strigosus Hagen, 1861 and the scorpionfly Panorpa debilis Westwood, 1846. The results show that the complete mitogenome sequences of B. strigosus and P. debilis are 15,825 and 17,018 bp, respectively, both containing 37 genes and one control region. The mecopteran mitogenomes are highly similar in A + T bias, AT-skew, and GC-skew. Tandem repeats of the control region were discovered in Mecoptera for the first time. The sliding window, genetic distance, and Ka/Ks ratio analyses indicate the purifying selection of 13 protein-coding genes, the lowest evolutionary rate of cox1, and the highest sequence variability of atp8. Considering the sufficiently large size, fast evolution, and high ratio of Ka/Ks, nad4L and nad6 are regarded as potential markers for future phylogenetic analyses, population genetics, and species delimitations in Mecoptera. The phylogenetic relationships were reconstructed for four families of Mecoptera based on all six available mitogenomes using Bayesian inference and maximum likelihood methods. The phylogeny is presented as Boreidae + (Nannochoristidae + (Bittacidae + Panorpidae)).

Spermiogenesis of the hangingfly Terrobittacus implicatus (Huang and Hua) (Mecoptera: Bittacidae).

The structure of spermatozoa is able to provide valuable characters in resolving phylogenic relationships in Metazoa, especially in insects. Such data, however, are greatly deficient in Mecoptera. Here, we studied the spermiogenesis and ultrastructure of sperm in the hangingfly Terrobittacus implicatus (Huang and Hua) using transmission electron microscopy. The results show that the spermatogenesis of T. implicatus occurs within sperm cysts, following a pattern commonly found in insects. The microtubular doublets of spermatid axoneme exhibit a hooklike projection from the B-subtubule in the early period, but the projection disappears in the mature stage. The mature spermatozoon of T. implicatus is a filiform cell that is pronouncedly elongated and has a bi-layered acrosome, a nucleus with two lateral longitudinal grooves, a neck region with the centriole adjunct, a flagellum with a simple 9 + 2 axoneme, two extra-axonemal accessory structures, two accessory bodies, and two mitochondrial derivatives of unequal size, and a prominent glycocalyx. The basic structure of spermatozoa of T. implicatus is similar to that of other Mecoptera studied. However, this species shows characteristics unique in Bittacidae, such as the reniform appearance of the centriole adjunct, two triangular accessory bodies with granular materials, and two asymmetric mitochondrial derivatives with a circular profile in cross-section. The potential utilization of the sperm ultrastructure for understanding the phylogeny of Bittacidae is briefly discussed.

Ultrastructure of the rectum of the soil-spraying larva in Bittacus cirratus (Mecoptera: Bittacidae).

The larvae of Bittacidae have an interesting behavior of spraying soil particles on their body surface through the anus. However, the hindgut specialization associated with this behavior has rarely been studied hitherto. Here, we investigated the fine structure of the larval rectum in the hangingfly Bittacus cirratus Tjeder using light and transmission electron microscopy. The results show that the larvae of B. cirratus have a tubular rectum without rectal pads or papillae. The rectum consists of well-developed visceral muscle layers, a non-cellular basal lamina, a single-layer epithelium with a cuticular intima, and a central lumen. The folded rectal epithelium consists of two types of flattened epithelial cells: electron-dense type I cells and electron-lucent type II cells. The apical and basal plasma membranes are infolded and are associated with mitochondria in the epithelial cells. The epithelial cells are held by septate and scalariform junctions. The lateral cell membranes are combined with mitochondria among type I cells and generate mitochondria-scalariform junction complexes. These features suggest that the epithelial cells are active in water and ion reabsorption. We conclude that the absence of rectal pads or papillae and the presence of developed circular muscles are likely morphological adaptations of these larvae to the soil-spraying behavior.