Ultrastructure of the male reproductive system of Cotesia vestalis (Hymenoptera: Braconidae) with preliminary characterization of the secretions

The morphology and ultrastructure of testes and accessory glands along with the characterization of their secretions were investigated for a braconid species Cotesia vestalis (Hymenoptera: Braconidae) using light and electron microscopes. The male internal reproductive system of this species is distinguished by a pair of testes, one vas deferens, and a pair of male accessory glands. The testes are separate, and the accessory glands are oval and not fused. It was observed that the secretory cells of testes have characteristic smooth and rough endoplasmic reticulum, and that the cytoplasm is filled with an array of granule droplets usually of two to three types. The secretory cells in the case of accessory glands are typified by the presence of microvilli on their apical cell surfaces and numerous mitochondria in their cytoplasm. SDS-PAGE profile when performed depicted a similarity in most bands of the secretions from both testes and accessory glands, except for four proteins of which two were present only in testes, while the other two only appeared in accessory glands. Their molecular weights were 117 and 55 kDa for testes and 196 and 30 kDa for accessory glands, respectively. This study gives new insights into the intriguing features of male internal reproductive system and it especially constitutes the first report of its kind about the secretion properties of these organs in C. vestalis.     

Structural and ultrastructural features of the Malpighian tubules of Dolycoris baccarum (Linnaeus 1758), (Heteroptera: Pentatomidae)

The morphology and ultrastructure of the Malpighian tubules of Dolycoris baccarum were analyzed by scanning (SEM) and transmission (TEM) electron microscopy in order to determine their functional organization. The Malpighian tubules are compared with similar structures of other insects based on cell structure and functional organization. The Malpighian tubules of D. baccarum extend from the midgut–hindgut region of the digestive tract. The Malpighian tubules are divided into two regions: the proximal segment is short and flattened and the distal segment is long, stringy in shape and free in hemolymph. The tubules are generally long and narrow. There is a large number of trachea around the tubules. They consist of a single layer of epithelial cells. It is observed in the TEM observation that the epithelial cells have numerous microvilli at the apical side of the cells. At the basal side of the cells, there is a great number of membrane foldings and mitochondria among them. Besides some spherites, mitochondria, lysosome‐like bodies, and large or small granules can be distinguished in the cells. With this study, we aimed to demonstrate the ultrastructure of the Malpighian tubules of D. baccarum and differences or similarities with other species.     

Ontogeny of the testicular excurrent duct system of male Japanese quail (Coturnix japonica): A histological,ultrastructural, and histometric study

The testicular excurrent duct system undergoes several physiological and morphological changes during the reproductive stage or breeding season in mammals, birds, and reptiles. Studies on normal age-related histomorphological changes in the excurrent duct system of Japanese quails (Coturnix japonica) remain unreported, despite the extensive use of this bird as an avian model in research studies. The current study investigated the histological, ultrastructural, and histometric changes in the testicular excurrent duct system of the Japanese quail during three reproductive stages, namely prepubertal, pubertal, and adult. Simple squamous to low cuboidal cells formed the epithelia of the rete testis in prepubertal and pubertal birds, while in adult birds the lining was low cuboidal to cuboidal. In pubertal and adult birds, the nonciliated Type I epithelial cells of the proximal efferent duct displayed a subapical endocytotic apparatus comprising coated pits, coated apical tubules, and endosomes. There was a significant increase (p ≤ .001) in epithelial heights of all ducts of the excurrent duct system in the mature, sexually active, adult birds when compared to the other age groups. The luminal and tubular diameters, and the cross-sectional areas of efferent ducts and the epididymal duct unit increased significantly (p ≤ .001) with age. It is concluded that the morphology and morphometry of the excurrent ducts of the testis of the Japanese quail change as birds mature.     

Morphological and histological structure of the male reproductive system of the water strider Gerris lacustris (Linnaeus 1758) (Gerridae,Heteroptera)

This work presents the male reproductive system morphology and histology of the water strider Gerris lacustris (Linnaeus 1758) (Gerridae, Heteroptera) using light microscopy and scanning electron microscopy. The male reproductive system of G. lacustris comprise of a pair of testes, two vasa deferentia, two seminal vesicles, an ejaculatory duct. There is no bulbus ejaculatorius and the long vas deferantia uniting to form a simple ductus ejaculatorius which is connected to the aedeagus. The testes are white colored and this cylindiric‐shaped structure lies along genital abdominal segment. The testicular follicles have three different development zones (growth zone, maturation zone, differentiation zone). Each testis has two follicles, which are not lined by a common peritoneal sheath and involving many cysts arranged in a progressive order of maturation from the distal to the proximal region; spermiogenesis occurs in mature males, finishing with the organization of sperm bundles. The testes are connected to the seminal vesicles, specialized sperm storage places, by the vas deferentia.     

General morphology and ultrastructure of the female reproductive apparatus of Trichomalopsis shirakii crawford (Hymenoptera,Pteromalidae)

The morphology and ultrastructure of the female reproductive system were examined for a larval–pupal parasitoid Trichomalopsis shirakii Crawford of Oulema oryzae Kuwayama using light and electron microscopes. The reproductive system includes two ovaries, two pairs of accessory glands, an unbranched venom gland, a large venom reservoir and a Dufour gland. Each ovariole contains follicles and oocytes at different stages of maturation. A fibrous layer covers the surface of mature egg. The accessory glands are made up of a layer of secretory cells surrounded by muscle fibers. In these secretory cells, numerous mitochondria, electron‐dense secretory granules and vesicles filled with dense granular particles are present. These granular particles appear as virus‐like particles (VLPs). The venom gland consists of a single layer of secretory cells which are organelle rich with abundant rough endoplasmic reticulum, mitochondria and vesicular organelles, a layer of duct cells and an inner intima. The reservoir consists of a muscular sheath, epidermal cells with few organelles and an intima layer. The Dufour gland has a relatively large lumen surrounded by a single layer of columnar epithelial cells which are characterized by clusters of smooth endoplasmic reticulum and lipid droplets. Aside from the venom, the fibrous layer coating the egg and the granular particles which may be VLPs have been discovered in our study. They may serve as one of the parasitoid‐associated factors in their host–parasitoid relationship and play a role in host immune suppression. Microsc. Res. Tech. 79:625–636, 2016. © 2016 Wiley Periodicals, Inc.     

Ultrastructure of sensory equipments on the heads of Kallitaxila granulata (Stål) (Hemiptera: Fulgoromorpha: Tropiduchidae)

The polyphagous planthopper, Kallitaxila granulata (Stål) (Hemiptera: Fulgoromorpha: Tropiduchidae), has been recently introduced into southeastern China, the Philippine islands, and Hawaii, where it has done significant damage to agricultural and forest ecosystems. The external morphology of the heads of adult male and female K. granulata was examined using scanning electron microscopy. Seven types of sensilla were reported: trichoid sensilla and campaniform sensilla on the antennal pedicel, antennal scape and maxilla; plate organs on the antennal pedicel; coeloconic sensilla in Bourgoin's organ on the expanded flagellar base; ampullaceal sensilla on the antennal pedicel; wavy‐pit sensilla on the antennal pedicel and antennal scape; and coin‐shaped sensilla on each lateral side of the labium. Evans' organ was described as placoid sensilla sunk into shallow cuticular cavities below the antennae. The external morphology, distribution, and abundance of sensilla located on antennae, maxillae, and labium in K. granulata were illustrated, with a brief discussion of their taxonomic, phylogenetic, and putative functional significance. Microsc. Res. Tech., 2012. © 2012 Wiley Periodicals, Inc.     

Morphology and morphometry of the reproductive system of female Saguinus midas (Linnaeus, 1758)

In this article, the reproductive system's morphology of three young animals of the species Saguinus midas, from the bauxite mine in Paragominas, is described. The specimens were fixed and preserved in a solution of 10% aqueous formaldehyde, followed by dissection, measurement of the genital organs (uterus, vagina, ovaries, and uterine tubes), and histological processing. The vulva is delimited by the labia, with a clitoris. It is lined by keratinized stratified squamous epithelium with sebaceous glands of holocrine secretion. The vagina is an elongated tube with an average length of 26 mm and diameter of 1 mm, presenting a non-keratinized squamous epithelium, disposed between the vestibule of the vagina and cervix, the latter being relatively short. The uterus is simple, has globular shape and is located in the caudal portion of the abdominal cavity, with an average length of 14 mm and average width of 7 mm. It is formed by vascular and serous layers of muscles, and undergoes a bifurcation to form two structures on the bottom of blind sac. The uterine tubes are long and convoluted with an average length of 35 mm (right) and 36 mm (left), consisting of loose connective tissue and muscle layer lined by simple ciliated columnar epithelium. The ovaries are large and ellipsoid with smooth surface. Histologically, one animal showed ovulation fosse.     

Toxicity of trifluralin herbicide in a representative of the edaphic fauna: Histopathology of the midgut of Rhinocricus padbergi (Diplopoda)

The aim of this study was to use the diplopod Rhinocricus padbergi as test organism to verify the possible toxic effects of the herbicide trifluralin in a representative of the edaphic fauna. Histological and histochemical techniques were applied in the midgut of the animals after exposure to different concentrations of trifluralin for 7 and 90 days (acute and subchronic exposure, respectively). The individuals from the control group did not present alterations to any of the exposures. In the acute exposure, the group exposed to a quarter and half of the field dose presented an accumulation of cytoplasmic granules in the hepatic cells; the field dose group presented an increase in the rate of epithelial renewal and the group exposed to double the field concentration presented an increase in the release of secretory vesicles. The subchronic response was obtained with the field dose and double field dose because the animals from a quarter and half groups did not survive until the end of the experiment. In both groups, the animals presented disruption of the epithelium and higher occurrence of hemocytes among the hepatic cells. The group exposed to double the field dose presented sites of cytoplasmic vacuolization, dilatation of the intercellular space, increase in the epithelial renewal, and release of secretory vesicles. The results alert for a careful use of this herbicide, since it promoted alterations in the midgut cells of the test organism used. Microsc. Res. Tech. 2012. © 2012 Wiley Periodicals, Inc.     

Ultrastructure and distribution of antennal sensilla of the chilli thrips Scirtothrips dorsalis hood (Thysanoptera: Thripidae)

The chilli thrips, Scirtothrips dorsalis Hood, is a serious pest of numerous important vegetable and ornamental crops. Various signals, especially phytochemical cues, determine the behavior of the phytophagous thrips at host selection. The sensory abilities of S. dorsalis are poorly understood although the antennae of adult are known to possess important sensory structures in orther insects. In this study, the morphology, distribution, and ultrastructure of the antennal sensilla of the S. dorsalis were examined by using scanning and transmission electron microscopy. Microscopy observations revealed that adult male and female S. dorsalis possess filiform antennae. Each antenna comprises a scape, a pedicel, and a flagellum composed of six segments without clear sexual dimorphism in the number and distribution of antennal sensilla. The scape and pedicel exhibit Böhm's bristles, sensilla chaetica, and sensilla campaniform. The external structures of these organs reveal their mechanosensory function. In the flagellum, the most represented sensilla are the multiporous sensilla basiconica, which can be divided into three types of single‐walled olfactory sensilla; three types of sensilla chaetica with mechanosensory and gustatory functions; sensilla coeloconica, which possess hollow cuticular spoke channels and represent double‐walled olfactory sensilla; sensilla capitula and sensilla cavity with thermo‐hygrosensory functions; and aporous sensilla trichodea with smooth cuticula and mechanosensory function. The putative function of described sensilla is discussed in ralation to host plant selection behavior of S. dorsalis.     

Ultrastructural observations on antennal sensilla of Cnaphalocrocis medinalis (Lepidoptera: Pyralidae)

The morphology and ultrastructure of the antennal sensilla of Cnaphalocrocis medinalis (Lepidoptera: Pyralidae) was studied by scanning and transmission electron microscopic techniques. Eight morphological types of sensillum were recorded in both sexes: sensilla trichodea (S. trichodea), sensilla basiconc (S. basiconc), sensilla coeloconica (S. coeloconica), sensilla styloconica (S. styloconica), sensilla squamous (S. squamous), sensilla auricillica (S. auricillica), B?hm bristles (B. bristles), and sensilla cavity (S. cavity). S. trichodea were the most abundant sensilla and were distributed over the entire antennal surface. Four different types of S. trichodea and S. basiconc were observed. The number of S. basiconc and S. coeloconica of males were greater than those of females of C. medinalis. S. squamous formed on the dorsal part of the antenna, as a cluster in females and as in a line in males. Higher magnification revealed that S. basiconc had an olfactory function, while the character of longer length of these sensilla suggested that they also played a rolein sensing mechanical or other chemical stimuli. Transmission electron microscopy (TEM) of S. squamous revealed nonporous walls suggesting a non-olfactory function. S. coeloconica, S. styloconica, and S. cavity may be involved in the perception of humidity, temperature, heat, and CO?. Because of their particular location, we infer that B. bristles may function in sensing the position and movements of the antennae, while the function of S. auricillica is as yet unknown. The results obtained provide direct morphological evidence that antennae possess structures that can play a role in finding mates and locating host plants.     

Antennal morphology and sensilla ultrastructure of three tomicus species (coleoptera: Curculionidae,scolytinae)

The antennal morphology and sensilla ultrastructure of Tomicus yunnanensis, T. minor, and T. brevipilosus were studied by scanning electron microscopy and transmission electron microscopy. Eight common sensilla types were recorded: (1) sensilla trichodea (S.tr.) types 1 and 2 were located on the club and were innervated by five and eight dendrites, respectively; (2) sensilla chaetica (S.ch.) types 1 and 2 had no dendrites in the sensilla lymph lumen; (3) sensilla basiconica (S.b.), top‐protuberated S.b. and fluted cones (Fl.c.) occurred on the club; and Böhm bristles (B.b.) occurred on the funicle. S.b. were the most abundant and were innervated by 10–14 dendritic branches. Top‐protuberated S.b., a new sensilla type, were innervated by one dendrite. Fl.c. were innervated by five dendrites. Only three sensilla furcatea were visible on the antennae of female T. yunnanensis. The possible functions of these sensilla are discussed in relation to their morphology and ultrastructure. No statistical differences between sexes were found in the size and numbers of each sensilla type. Although the three species had similar antennal morphology and sensilla type, sensilla on sub‐segments 3 and 4 of the antennal club of T. minor were much sparser than those of T. yunnanensis or T. brevipilosus. Concerning the antennae of T. yunnanensis, there were more S.tr. type 2, S.ch. type 2 and S.b. and the size of S.tr. type 1 and S.b. were significantly greater than those of T. minor. Microsc. Res. Tech., 2012. © 2012 Wiley Periodicals, Inc.     

Morphology and ultrastructure of antennal sensilla of the parasitic wasp Baryscapus dioryctriae (Hymenoptera: Eulophidae)

Baryscapus dioryctriae is an endoparasitic wasp in the pupae of many Pyralidae pests, such as Dioryctria mendacella, Ostrinia furnacalis, and Chilo suppressalis. To provide requisite background for our ongoing research on the mechanisms of host location in B. dioryctriae, the morphology, abundance, distribution, and ultrastructure of the antennal sensilla were investigated using scanning and transmission electron microscopy. The geniculate antennae of B. dioryctriae are composed of scape, pedicel, and flagellum. Eight types of sensilla including Böhm sensilla, chaetica, trichodea, basiconic capitate peg, campaniformia, placodea, coeloconica, and sensilla styloconicum with a long hair were identified on both sexes. Sexual dimorphism exists in the antennae of B. dioryctriae. The number of flagellomere in males is over females, and the subtypes and abundance of sensilla are also different between the sexes. Additionally, the possible functions of distinct sensilla were discussed, which varies from olfaction, contact chemoreceptive, mechanoreception to hygro-/thermoreception, especially, the sensilla trichodea and placodea might be involved in olfactory perception in B. dioryctriae. These results provide an essential basis for further study on chemical communication between B. dioryctriae and their hosts, and contribute to the development of B. dioryctriae becoming an effective biocontrol agent against the pests of agriculture and forestry.     

Morphological characterization of the nymphs Rhipicephalus sanguineus ticks (Latreille, 1806) (Acari: Ixodidae). Description of the testes, integument, Malpighian tubules, and midgut on the detachment day

This study presents the morpho-histological and histochemical characterization of the testes, integument, Malpighian tubules, and midgut of engorged Rhipicephalus sanguineus nymphs on the detachment day, showing the morphological and physiological characteristics to this phase in the life cycle of these individuals. The testis is constituted by germinative cells (only spermatogonia) with large, round-shaped and strongly stained nuclei which are organized into cysts by a thin layer of somatic cells. The integument consists of a cuticle subdivided into epicuticle (lipoprotein) and procuticle (glycoproteic), and a layer of epithelial cells which present glycolipoprotein elements. The procuticle presents two distinct regions: the exocuticle (next to the epicuticle) and the endocuticle (next to the epithelial layer). The Malpighian tubules present a simple epithelium with small flat and/or cubic cells, which form its wall and delimitates a lumen full of lipoprotein material. The midgut consists of an epithelial wall formed by two types of digestive cells, spent cells and empty digest cells, and by generative cells supported by a basal lamina and a thin layer of muscular tissue. This study described the main organs of engorged nymphs of R. sanguineus, to generate information that can help researchers to better understand the biology of these ectoparasites; which is fundamental for the development of compounds that are less aggressive to the environment. In addition, if the immature stages of the ticks are controlled, the number of adult ticks able to cause damages to the animals--and to the man as well--is also under control.     

Seasonal testicular changes in <i>Dendropsophus minutus</i> Peters, 1872 (Anura,Hylidae)

The reproductive cycle in anurans may be either continuous or discontinuous. These differences may be connected to seasonal climate changes and/or to anthropic activity. Forty adult male individuals of the Dendropsophus minutus species were collected during one year, in the municipality of Chapada dos Guimarães (Mato Grosso, Brazil). The testicles were studied under light and transmission electron microscopy. No variations were observed when the diameter of the seminiferous tubules and the thickness of the interstitial tissue were studied. However, changes in spermatogenesis were conspicuous and indicated that the reproductive cycle of D. minutus in Chapada dos Guimarães is discontinuous and seems related to variations in air temperature and rainfall.     

Ultrastructural characteristics of the spermatogenesis during the four phases of the annual reproductive cycle of the black myotis bat,Myotis nigricans (Chiroptera: Vespertilionidae)

Myotis nigricans is an endemic species of vespertilionid bat, from the Neotropical region, that resembles temperate zone bats in their reproductive cycle; presenting an annual reproductive cycle with two periods of testicular regression, which are not linked to the apoptotic process and seems to be not directly linked to any seasonal abiotic variation. Thus, this study aimed to ultrastructurally evaluate their reproductive cycle. The process of testicular regression could be divided into four periods: active; regressing; regressed and recrudescence; with all presenting distinct characteristics. The active period was similar to that of other bats, presenting the complete occurrence of spermatogenesis, with three main types of spermatogonia (A_d, A_p, and B) and 12 steps in spermatid differentiation; however, it differed in having the outer dense fibers 1, 5, 6, and 9 larger than the others. These three types of spermatogonia undergo considerable morphologic changes from regressing to the regressed period, and in the recrudescence, they return to the basic morphology, which reactivates spermatogenesis. In conclusion, our study described the process of spermatogenesis, the ultrastructure of the spermatozoa and the distinct morphologic variations in the ultrastructure of the testicular cells of M. nigricans during the four different periods of its annual reproductive cycle. Microsc. Res. Tech., 76:1035–1049, 2013. © 2013 Wiley Periodicals, Inc.     

Ultrastructure of differentiating oocytes and vitellogenesis in the giant freshwater prawn, Macrobrachium rosenbergii (de man)

The ultrastructure of oogenesis in Macrobrachium rosenbergii, with reference to vitellogenesis, has not been reported. We used light and electron microscopy, as well as vitellin (Vn) purification and antibody production, to study the temporal and spatial production of Vn in the ovary by immunofluorescence. Histologically, the ovary is subdivided into cone‐shaped ovarian pouches with a central core containing layers of oogonia. These divide to produce oocytes that migrate outwardly and differentiate into mature oocytes. During the course of differentiation, oocytes undergo modifications, including the rearrangement of nuclear chromatin, the accumulation of ribosomes, rough endoplasmic reticulum (RER), and lipid, and the formation of secretory and yolk granules, resulting in four stages. Ultrastructurally, early previtellogenic oocytes (Oc₁) are characterized by the accumulation of new ribosomal aggregates, translocated from the nucleus. Late previtellogenic oocytes (Oc₂) show nuclear heterochromatin with a “clock face” pattern, the presence of RER, and three types of secretory granules. Follicular cells occupy the intercellular spaces and surround the Oc₂. Early vitellogenic oocytes (Oc₃) are larger, with nuclei containing predominantly decondensed euchromatin, and cytoplasm with yolk and secretory granules, and few lipid droplets. Late vitellogenic oocytes (Oc₄) are characterized by completely euchromatic nuclei, an indistinct plasma membrane, yolk platelets and secretory granules, and abundant lipid. Vitellogenin (Vg) in ovaries of M. rosenbergii consist of two main bands at MW 90 and 102 kDa. Our data indicates that Vn is present, and probably synthesized in Oc₃ and Oc₄, but there may be some undetected exogenous Vg production. Microsc. Res. Tech. 2012. © 2012 Wiley Periodicals, Inc.     

Ultrastructure of multicellular dwarf males with external gametangium in Oedogonium macrandrium (Oedogoniales,Chlorophyta)

This is the first comprehensive ultrastructural study on dwarf males with external gametangia in the ge in the genus Oedogonium, from androspore germination to the liberation of mature male gametes. The ultrastructure of the process in O. macrandrium Wittrok is similar to that of Bulbochaete hiloensis (Nordstedt) Tiffany, butwith two remarkable differences. In O. macrandrium : 1) instead of a true transverse wall, only condensed mucilage appears between the gametes of each antheridial cell, and 2) the cell wall between the basal cell and the basal most antheridial cell has simple plasmodesmata similar to those present in the transverse walls of vegetative cells, which are absent in B. hiloensis.     

The histomorphological structure of the male reproductive system of maize leaf weevil Tanymecus dilaticollis Gyllenhal, 1834 (Coleoptera: Curculionidae)

The histomorphology of the reproductive system and the germ cells has been useful to establish phylogenetic relationships in many insects. However, these elements remain little known in the Curculionidae. In this study, histomorphological structure of the male reproductive system of Tanymecus dilaticollis, which is economically important, is described, illustrated using stereomicroscopy, light microscopy, and scanning electron microscopy techniques, and discussed in relation to other Coleoptera species. Results showed that distinctive features of the male reproductive system of T. dilaticollis consist of a pair of yellowish testes, a pair of seminal vesicles, a pair of vasa deferentia, an ejaculatory duct, accessory glands, prostate glands, and aedeagus. Each testis is subdivided into two testicular follicles, enclosed by a peritoneal sheath. Each follicle of the mature testes is full sperm cysts with germ cells at various stages development of spermatogenesis. The testes have four types of germ cells (spermatogonia, spermatocytes, spermatids, and spermatozoa). They are occupied by the growth zone containing spermatogonia and spermatocytes, the maturation zone containing spermatids, while differentiation zone containing spermatozoa. There is a seminal vesicle at the center of each testis. Most mature sperms are stored in the seminal vesicle. Each testis is attached to the vas deferens by a stalk‐like seminal vesicle. In the distal part, vasa deferentia fuse with the ejaculatory duct. It is linked to the aedeagus. The provided results will contribute to the understanding of the reproductive cell biology of Curculionidae.     

Phylogenetic relationship of seven Dendrolimus (lepidoptera: Lasiocampidae) species based on the ultrastructure of male moths' antennae and antennal sensilla

The morphology and ultrastructure of the antennae and antennal sensilla of seven male Dendrolimus species and a male Trabala vishnou gigantina (Yang) (Lepidoptera: Lasiocampidae) were examined by light microscope and scanning electron microscope. Six morphological types of antennal sensilla were identified: Sensilla trichodea, Sensilla chaetica, Sensilla styloconica, Sensilla coeloconica, Böhm bristles, and foot‐like sensilla. Six of the Dendrolimus moths and Trabala vishnou gigantina Yang share the same antennal sensilla type, as do various geographic populations of the same species. The exception, Dendrolimus spectabilis Butler, has foot‐like sensilla. However, the antennal sensilla subtypes were significantly different among species and/or populations. There were no remarkable differences in the width of the scape, pedicel, subflagellum, and the side‐branches between the eight male species studied. However, we observed significant differences in the number of flagellomere and the length of scape, pedicel, subflagellums, and side‐branches. The length and basal diameter of various types of antennal sensilla did not vary significantly among Dendrolimus moths. Beyond that, there were no differences among populations of the same kind of species. Hierarchical cluster analysis found two clusters: the first contained D. punctata punctata (Walker), D. punctata wenshanensis (Tsai et Liu), D. tabulaeformis (Tsai et Liu), and D. spectabilis and D. superans (Butler), and the second contained D. grisea (Moore) and D. kikuchii kikuchii (Matsumura). Trabala vishnou gigantina was placed separately from the two clusters. We conclude that D. punctata wenshanensis,D. tabulaeformis, and D. spectabilis are geographic subspecies of D. punctata punctata. Microsc. Res. Tech. 2012. © 2012 Wiley Periodicals, Inc.