Ultrastructural and morphological observations of the external and internal structures of the adult midge Culicoides grisescens (Diptera: Ceratopogonidae)

Abstract

The external and internal structures of adult males and females of Culicoides grisescens Edwards were examined using scanning electron microscopy (SEM) and tissue sectioning techniques. Specimens were collected from Qilian Mountain National Park in Qinghai Province, China. The ultrastructure and morphology of the compound eyes, antennae, maxillary palpi, spermathecae, genitalia, and other structural features both male and female adults of C. grisescens are described. Significant morphological differences between the two sexes were observed. The female has a pair of large mandibles with 16 teeth arranged in two rows. The first row contains 16 teeth, while the second row is located near the terminal teeth at the lower part. In contrast, the male mandibles have 2 to 3 teeth, totaling over 20, with a slender structure and small teeth distributed near the center at the terminal end of the mandibles. The tip of the male tongue is narrower and triangular than in shape compared to the female. Additionally, there are significant differences in the shape of the antennae and the types of antennal sensilla between males and females. This study also observed that the middle lobe of the male masculine stem is V-shaped, and the terminal end of the masculine lateral process is bifurcated, featuring 5 to 7 slender, finger-like branches. These unique morphological structures are useful for distinguishing male Culicoides from each other. For the first time, this paper describes the internal structures of this species, including histological sections of the digestive, nervous, respiratory, and reproductive systems. The findings presented herein provide both visual and descriptive details for the systematic taxonomic study of Ceratopogonidae, highlighting their significant economic and medical importance.

Introduction

Culicoides are small, blood-sucking biting midges belonging to the family Ceratopogonidae (Diptera: Culicomorpha). Currently, 1,347 species have been identified worldwide, with 480 species recorded in China (Yang et al. 2020, Borkent et al. 2022). Female adults of many Culicoides species are pests to humans and livestock, causing painful lesions and triggering acute allergic reactions, such as eczema in horses (van der Rijt R et al. 2008). In addition, they can transmit both viral and non-viral pathogens, such as Bluetongue virus, Schmallenberg virus, African horse sickness virus, and the malaria-like parasites Haemoproteus (Borkent 2004, Wang et al. 2010, Veiga et al. 2018, Mignotte et al. 2021). Therefore, Culicoides represent a group of medically significant insects.

Culicoides grisescens Edwards is a common species distributed in Europe and Asia (Yu et al. 2005). In China, its primary distribution includes regions such as Xinjiang, Inner Mongolia, and other high-altitude areas (Yu et al. 2005, Paslaru et al. 2021), which are characterized by numerous farms and well-developed animal husbandry. Thus, these areas face an increased risk of midge-borne disease transmission. Previously, the identification of C. grisescens has largely relied on hand-drawn figures of taxonomic features, which often overlook or inadequately describe subtle and distinguishing characteristics. The cryptic species C. grisescens G1 and G2 are morphologically indistinguishable by light microscopy (Wenk et al. 2012). Thus, it is essential to investigate more detailed and subtle morphological features to enhance and refine their descriptions. In this study, the morphology and ultrastructure of both adult sexes of C. grisescens were observed and described using by scanning electron microscopy (SEM). Additionally, the internal anatomical structures of C. grisescens were systematically analyzed for the first time through hamatoxylin-esoin (HE) staining and continuous paraffin sectioning. This approach provides a robust basis for the accurate classification and identification of Culicoides species.

Materials and Methods

Entomological Sampling

Adults of C. grisescens were collected using light trapping by the research team in Huzhu, Menyuan, and Qilian counties within the Qilian Mountain National Park, Qinghai Province, from July to August 2020. Two UV-light trap lamps were employed for specimen collection. The lamps were positioned near livestock and a water source, in an area devoid of other light sources and wind, approximately 1.5 m above the ground. The lights were set up at sunset and retrieved early the following morning. The specimens were preserved in 70% ethanol and transported to the laboratory for species identification, based on Ceratopogonidae of China (Yu et al. 2005).

Sample Preparation

For the SEM study, adults of C. grisescens were prepared according to the techniques described by Jiang et al. (2018). The adult C. grisescens samples were washed in distilled water for 10 min with gentle agitation. After cleaning, the specimens were dehydrated in ethanol solutions of 75%, 85%, and 100% concentrations for 15 min at each concentration and subsequently preserved in 100% ethanol. Drying was performed using a Leica EM CPD300. Following the drying process, the specimens were dissected under a stereoscope, and the dissected parts were then affixed with double-sided conductive adhesive.Their positions were recorded and the samples were coated with gold using a Leica EM ACE600. Finally, the ultrastructure was examined with an SEM (SU8010), and images were captured and preserved. The abbreviations employed in this paper adhere to the conventions established Jiang et al. (2019).

For the histological study, adults of C. grisescens were prepared following the techniques described by Ning et al. (2022) and Jiang et al. (2019). Wings and legs were carefully removed using fine needles, and the remaining body was immersed in fixative for 24 h at room temperature. Specimens were then dehydrated using a graded ethanol series with concentrations of 80%, 85%, 90%, 90%, 95%, 95%, 100%, and 100%, with each concentration for 30 min. After dehydration, the specimens were placed in benzene and then embedded in paraffin at 55 °C. Both longitudinal and transverse sections (3 µm thick) were prepared, stained with HE, and subsequently soaked in xylene for 10 min. The specimens were mounted on slides with neutral glue, a coverslip was added, and the samples were dried. The tissues were observed and photographed using a digital system attached to an Olympus BX43 microscope with a DP26 digital camera.

For the descriptions, 3 specimens were measured for each character and ratio. Measurements of the antennae (AR), palpi (PR _(III)), wings, and costal vein (CR) were obtained following the methods described by Dominiak (2012).

Results

Culicoides grisescens Edwards, 1939

Figs 1–6

Material examined

Following classification and identification (Yu et al. 2005), over 360 C. grisescens specimens were obtained. This species was the dominant one in all 3 counties, accounting for 33.60%, 85.85%, and 97.95% of the total Culicoides midge population in Huzhu, Menyuan, and Qilian counties, respectively, within the Qilian Mountain National Park, Qinghai Province, China. The coordinates are 35°27'13.77"N, 101°93'25.62"E, with an elevation of 2941 m, recorded on August 2020. Nine females and 9 males with intact limbs were selected for the experiment.

Redescription of Adults (Figs. 1–3)

Female (Figs. 1A–O, 2A–I)

Head (

Fig. 1A–O). The general view of the head is dark brown (Fig. 1A). The eyes are contiguous, abutting medially for a length equivalent to 1.0 ommatidia, without interommatidial spicules, and the surface is smooth (Fig. 1A, B). The fontal sclerite is nearly round, with a long, slender ventral projection (Fig. 1B). The antennae (Fig. 1C) are brown, with 13 vasiform-shaped segments, the distal 5 flagellomeres are elongate, with an AR 1.14–1.21 (1.17, n = 3). Antennal sensilla are distributed on flagellomeres 1-13 increasing in number gradually from the base to apex. Numerous sensillum trichodea are present, arranged irregularly. Sensilla coeloconica are primarily present on flagellomeres 1 and 9–13 (Fig. 1C–F): two on segments 1 and 12, one on each of segments 9–11, and 3 on segment 13. Sensilla basiconica are present on the distal 5 segments, straight, or slightly curved at the basal third (Fig. 1C–E). The mouthparts are well developed and consist of the labrum, mandible, maxilla, hypopharynx, and labium (Fig. 1G). The labrum is sunken in the middle, tapering apically, with the distal portion the teeth being flame-shaped (Fig. 1H–I). The mandible features a narrow and short protuberance in the middle, while the apical portion is triangular and possesses 16 teeth (Fig. 1J). The lacinia contains two rows of teeth: 16 in the first row and 1 in the second row, with the apical 3 teeth is triangular in shape (Fig. 1K). The hypopharynx is sharp, long and sword-shaped (Fig. 1L). The labium consists of 4 segments, with the fourth segment bearing sensilla trichodea, sensilla chaetica, and sensilla basiconica (Fig. 1M). The maxillary palpus consists of 5 segments, with a PR value ranging from 3.34 to 3.59. The third segment is slender, with the apical third slightly swollen, featuring multiple irregular pits that contain11-13 hollow capitate sensilla scattered (Fig. 1N–O).

Thorax

Wing length 1.86–2.15 mm (2.03 mm, n = 3), width 0.74–0.96 mm (0.85 mm, n = 3), CR 0.63–0.65 (0.65, n = 3). The wing surface is densely covered with both microtrichia and macrotrichia, with the basal cell lacking macrochaetae (Fig. 2A–B). The scutellum is trapezoid, bearing 4 stout and 8 fine setae (Fig. 2C). The postscutellum is pubescent on the surface (Fig. 2C). The hind tibia comb consists of 6 terminal bristles, with the first and second spines being the longest (Fig. 2D). The tarsal claws are horn-shaped (Fig. 2E-F).

Abdomen

The abdomen is brown. The ventral surface is densely covered with a large number of bristles and spicules (Fig. 2G). The cercus is semicircular in shape (Fig. 2I). The spermatheca consists of two ovules of different sizes (71.02 μm × 54.82 μm, 68. 62 μm × 57.28 μm).

Male (Fig. 3A–L)

Head

Similar to the female, but with the following sexual dimorphism. Antennae are brown. AR 1.02-1.17 (1.07, n = 3) (Fig. 3A). PR 3.18-3.52 (3.41, n = 3). The dorsal surface of the basal 1/3 of the labrum is covered with microtrichia arranged in a triangle (Fig. 3B). The maxilla is slender and bifurcate, with smaller teeth in the middle, and has 20 teeth in total (Fig. 3C). The mandible has two teeth of different sizes (Fig. 3D). The hypopharynx is long and sword-shaped, with a slender groove in the middle and a slender tooth at the apex edge (Fig. 3E).

Thorax

Wing length 1.90–2.04 mm (1.99 mm, n = 3), width 0.65–0.68 mm (0.67 mm, n = 3), CR 0.60–0.63 (0.62, n = 3). The wing surface is densely covered with microtrichia and macrotrichia, and the basal cell lacks macrochaeta.

Abdomen

Genitalia. The ninth tergite is trapezoidal, gradually narrowing from the base (Fig. 3E, I). The ninth sternite has spicules medially (Fig. 3G). The gonocoxite is stout, with both coarse bristles and fine bristles on its surface (Fig. 3G–H). The two cerci are separate and located on the hypoproct (Fig. 3I). The parameres (Fig. 3G, J) are separate, with a short and thin median lobe, as well as a curved and slender lateral lobe that gradually narrows to the apex. The apex features 5 to 7 branches and slender, finger-like processes (Fig. 3J–K). The aedeagus is a tower-like, with lateral separation and a shallow V-shaped concavity near the apex (Fig. 3L).

Description of Internal Structures of Adults (Figs. 4–6)

Digestive System (Fig. 4A–L)

Foregut

(Fig. 4A–F). The mouthparts (Fig. 4A), including the labellum, labrum, hypopharynx, maxilla, and mandible, are located anterior to the digestive tract. The pharynx (Fig. 4B) exhibits thickening of the pharyngeal tube wall, with a cibarium that is protuberance-shaped and connected to the upper and lower tube walls by muscles. The esophagus (Fig. 4C–D) is slender, tubular structure, mainly composed of a monolayer of epithelial cells, and connects to the posterior of the pharynx, at the junction between the head and thorax. The cardia (Fig. 4C, E–F) is located at the end of the foregut, where part of the tube wall is sunken inward to form the cardia valve. Salivary glands are located ventrally and anteriorly in the thorax, and each gland was positioned laterally to the foregut, between the layers of thoracic muscles (Fig. 4G). These glands are gathered by the salivary gland duct, which leads to the saliva pump near the mouthparts. Each gland is divided into 2 parts: the main gland, which is grape-like in shape with an acinar structure surrounded by large secretory cells, and the accessory gland, which is located at the base of the ducts.

Midgut

(Fig. 4H–I). The midgut is well-developed and extends from the thorax to the abdomen, located posterior to the cardiac valve and anterior to the pyloric node. The intestinal parietal cells are composed of a monolayer of columnar epithelial cells, which are neatly arranged with large, round nuclei, typically located in the middle or base of the cells. The midgut consists of two regions: the first is long and narrow, while the second is the stomach, which is dilated and located within the enlarged abdominal lumen (Fig. 4H).

Hindgut

(Fig. 4J–L). The hindgut is positioned between the midgut and the anus, consists of the ileum, colon, and rectum (Fig. 4J). The Malpighian tubules are located separately from the connection between the hindgut and the midgut (Fig. 4K). The differentiation between the ileum and colon is not easily to distinguish. The anterior part of the rectum swells to form a rectal capsule, which contains oval glands on the wall known as rectal pads (Fig. 4L). The top of the rectal pad extends deep into the rectal cavity, and the surface of the intima is thin. The rectal sac narrows gradually and ends at the anus.

Central Nervous System (Fig. 5A–H)

The central nervous system consists of the brain and the ventral nerve cord. The internal structure of the brain (Fig. 5A) is complex, located in the head above the pharynx, covered by the cuticle of the head and surrounded by muscle tissue. It can be divided into 3 functional regions: the protocerebrum, deutocerebrum and tritocerebrum. The protocerebrum, which constitutes the majority of the brain, along with the protocerebral and optic lobes (Fig. 5B) sends nerves that connect to the compound eye (Fig. 5C). The protocerebral lobe is extremely complex, containing a pair of mushroom bodies, a central complex, and a pair of accessory protocerebral lobes (Fig. 5A). Its medullary layer is formed by bundles of nerve cells and fibers. The optic lobes are located on both sides of the protocerebrum (Fig. 5C), consist of the anterior and posterior lobe of the medulla interna, the medulla externa, and the lamina. An inner chiasma is formed between the medulla externa and the medulla interna, while an outer chiasma is formed between the medulla externa and the lamina (Fig. 5C). The lamina sends out nerve fibers that connected to the compound eye (Fig. 5C–D). The deutocerebrum (Fig. 5A) lies below the protocerebrum and consists of a pair of dorsal lobes and a pair of antennal lobes (Fig. 5E). It is connects to the protocerebrum, anteriorly and to the antennal nerve bundle posteriorly. The tritocerebrum (Fig. 5F) sends out the circumoesophageal connectives that link to the subesophageal ganglion.

The ventral nerve cord (Fig. 5F–H) has a complex structure, formed by the fusion of the left and right nerve trunks. It includes one subesophageal ganglion, 3 thoracic ganglia, and 5 abdominal ganglia. The subesophageal ganglion (Fig. 5F) contains numerous nerve fiber poles. The thoracic ganglia (Fig. 5G), located in the anterior, middle, and posterior chest respectively, correspond to the anterior, middle and posterior legs, respectively. They send out nerve fibers to the ventral side of legs and toward the back into the base of the wings. The thoracic ganglion (Fig. 5H) is highly fused within the thoracic cavity, well-developed and easily distinguishable. The abdominal ganglion are underdeveloped, and the nerve fibers primarily innervate the abdominal internal organs.

Respiratory System (Fig. 6A–B)

The tracheal system is distributed throughout the body, consisting of the trachea, bronchi and microtrachea, which gradually decrease in diameter and branch continuously. The system communicates with the external environment through the spiracles. Two pairs of spiracles are located in the mesothorax (Fig. 6A) and the metathorax (Fig. 6B). The trachea is associated with expanding air sacs, including both bubble-like sacs and regular air sacs.

Reproductive System (Fig. 6C–F)

The female reproductive system (Fig. 6C–D) consists of the ovaries, oviducts, spermatheca, and accessory gland. A pair of the symmetrical ovaries are each connected to the two oviducts. The basal portions of the oviducts converge and lead to the genital opening (Fig. 6C). The spermatheca are spherical in shape and connected to the spermatheca duct and the vesicle duct, which in turn connected to the genital opening (Fig. 6D).

The male reproductive system (Fig. 6E–F) includes the testes, spermaducts, ejaculatory duct, and accessory gland. The testes are symmetrical and connected to the ejaculatory duct through the vas deferens, containing spermatogonia at various stages of spermatogenesis, which exhibit light blue coloration on HE staining (Fig. 6E). A pair of reproductive accessory glands are located on either side of the ejaculatory duct and open into the ejaculatory duct (Fig. 6F).

Discussion

In this study, the morphological ultrastructure of the male and female adults of C. grisescens was observed using SEM to obtain detailed morphological information and to provide a more accurate redescription of its characteristics. Additionally, the internal structure of the digestive, nervous, respiratory and reproductive systems of C. grisescens were analyzed using fixation, sectioning, and staining techniques.

Adult Ceratopogonidae have a small body size (1–4 mm) and include a wide variety of species, including complex groups or species complexes consisting of closely related species with similar morphology (Gopurenko et al. 2015). For taxonomists, it is crucial to comprehensively and accurately describe and interpret their morphological traits. In this study, the aedeagus was found to be V-shaped, with the distal portion of the paramere bifurcated and possessing 5 to 7 branches, supplementing some of the morphological characteristics reported in previous studies.

This study observed significant morphological differences between the female and male mouthparts of C. grisescens, as well as differences in antennal morphology, antennal sensilla types, and other morphological characteristics. Due to its pronounced sexual dimorphism, the species may exhibit synonymy. Although this study provides more comprehensive, detailed, and vivid structural information on C. grisescens, it lacks comparable morphological characteristic for its cryptic species. Therefore, future work will involve comparing the species morphology from different regional populations of C. grisescens and related species to obtain more stable and reliable morphological data.

Although numerous studies have been conducted on the morphology of other insects (Xie et al. 2000, Wang et al. 2006, Xun et al. 2011, 2013, Hu et al. 2013), however, there are fewer reports are available on the tissue structure of biting midges. This study is the first to describe the internal structure of this species using the slicing technique, focusing on 4 systems: the digestive system, nervous system, respiratory system, and reproductive system. C. grisescens lacks a gastric cecum in its digestive tract, and the presence and number of this structure vary significantly among Diptera (Bo et al. 2000, Ning et al. 2021). Since the main function of the gastric cecum is to increase the surface area of the midgut to aid in digestion and absorption, insects that feed on liquid food do not possess this structure. The brain of C. grisescens is similar to that of most insects (Ning et al. 2021), typically consisting of 3 distinguishable functional regions: the forebrain, midbrain, and hindbrain. The thoracic ganglion is highly fused within the thorax, indicating its significant role in controlling movement, with variations in the fusion of thoracic ganglia among different groups. Significant differences in the structure of internal reproductive organs are observed between sexes and species. Two round spermathecae are visible in female adults, and the color inside the testes of male adults differs, suggesting varying degrees of sperm maturity.

Although the respiratory system of C. grisescens is distributed throughout the body, a complete representation of the entire organ system was not provided due to the small size of the species and limitations in the staining process. This study only focused on the structure of the spiracles, and the further research is needed. In conclusion, the tissue structure of insect plays a critical role in basic insect physiology, vector-borne disease transmission, and even human diseases, and should not be ignored. However, a major limitation is that morphology-based methods for Culicoides species identification are time-consuming and require taxonomic expertise. Due to the limitations of morphological identification, the integration of molecular data is clearly warranted in further study (Nielsen and Kristensen 2015, Bakhoum et al. 2018).

Acknowledgments

We cordially thank Dr. Art Borkent for invaluable advice and comments on the manuscript, and to Dr. Phillip Shults for linguistic and professional assistance during the preparation of this manuscript.

Funding

This research was financially supported by the grant from the National Natural Science Foundation of China (31960102).

Author contributions

Fengyue Wang (Conceptualization [equal], Data curation [equal], Investigation [equal], Validation [equal], Writing - original draft [equal], Writing - review & editing [lead]), Xue Lu (Conceptualization [equal], Investigation [equal], Validation [equal], Writing - original draft [lead], Writing - review & editing [supporting]), Zhuang Fei Wang (Conceptualization [equal], Resources [lead], Validation [equal], Writing - original draft [equal], Writing - review & editing [supporting]), Jing Ma (Conceptualization [supporting], Resources [supporting], Writing - original draft [supporting]), Nanling Zhou (Conceptualization [supporting], Resources [supporting], Writing - original draft [supporting]), and Xiaohui Hou (Conceptualization [lead], Funding acquisition [lead], Methodology [lead], Project administration [lead], Supervision [lead], Writing - review & editing [supporting])

Conflicts of interest. The authors declare no conflicts of interest.

Data Availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

References

Author notes