Phylogenetic implications based on an ultrastructural study with emphasis on the tracheal system of the compound eyes of three species of nymphalid butterflies.

Compound eyes are the prominent visual organs of insects and can provide valuable information for the reconstruction of insect phylogeny. Although the largest butterfly family (Nymphalidae) has been well defined, the infrafamilial phylogenetic relationships remain controversial hitherto. In the present study the ultrastructure of the compound eyes of three nymphalids Neptis beroe, Childrena zenobia, and Palaeonympha opalina was investigated using light and transmission electron microscopy in an attempt to seek potentially important phylogenetic characters. The compound eyes of the nymphalids share a tracheal system in a "1-4-8" branching pattern. The eight tracheal subbranches exhibit distinct distribution patterns along the basal retinula cell as follows: the tracheal subbranches of Palaeonympha opaline are close to the rhabdom in the distance from the distalmost part of the basal retinula cell to the rhabdom end, while those of N. beroe and C. zenobia are on the periphery of the retinula along almost the whole basal retinula cell and become close to the rhabdom just at the proximal end of the basal retinula cell. The tracheal structure of the three nymphalids is discussed for their potential phylogenetic implications.

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.

Spermiogenic chromatin condensation patterning in several hexapods may involve phase separation dynamics by spinodal decomposition or microemulsion inversion (nucleation).

We have examined published transmission electron microscopy (TEM). photomicrographs of chromatin condensation patterning in developing sperm nuclei from five species of entognathous hexapods within the Classes Protura, Collembola, Diplura and five species of ancestral wingless insects in the Orders Archaeognatha and Zygentoma as well as in fifteen species of the winged insects. Each species reproduces by internal fertilization. Spatially quantitative analysis indicates that spermiogenic chromatin condensation patterning in several of these species may be due to spinodal decomposition (SD) or to microemulsion inversion (chromatin-in-nucleoplasm → nucleoplasm-in-chromatin), also known as nucleation (Nc). These are two different dynamic mechanisms of liquid-liquid phase separation (LLPS). They might either occur independently or co-exist during the chromatin condensation associated with insect spermiogenesis. For example, the chromatin condensation pattern such as that observed in transverse sections of developing sperm nuclei from the wingless insect Anurida maritima (Collembola) is: granules → fibers → lamellae (SD) → nucleation (Nc) → condensed nuclei. Similar transitions are also observed in other more recently evolved species within the Class Insecta. From the limited but comprehensive sample of entognathus and ectognathus hexapods analyzed here, it appears that LLPS of sperm chromatin during spermiogenesis has occurred quite pervasively within the subphylum Hexapoda, including insects.

Protocol for preparation of heterogeneous biological samples for 3D electron microscopy: a case study for insects.

Modern morphological and structural studies are coming to a new level by incorporating the latest methods of three-dimensional electron microscopy (3D-EM). One of the key problems for the wide usage of these methods is posed by difficulties with sample preparation, since the methods work poorly with heterogeneous (consisting of tissues different in structure and in chemical composition) samples and require expensive equipment and usually much time. We have developed a simple protocol allows preparing heterogeneous biological samples suitable for 3D-EM in a laboratory that has a standard supply of equipment and reagents for electron microscopy. This protocol, combined with focused ion-beam scanning electron microscopy, makes it possible to study 3D ultrastructure of complex biological samples, e.g., whole insect heads, over their entire volume at the cellular and subcellular levels. The protocol provides new opportunities for many areas of study, including connectomics.

The histology and ultrastructure of the salivary glands of Neopanorpa longiprocessa (Mecoptera: Panorpidae).

The salivary glands of Panorpidae usually exhibit distinct sexual dimorphism and are closely related to the nuptial feeding behavior. In this study, the salivary glands of Neopanorpa longiprocessa were investigated using light microscopy and transmission electron microscopy. The salivary glands are tubular labial glands and consist of a scoop-shaped salivary pump, a common salivary duct, and a pair of salivary tubes. The male and female salivary glands are remarkably different in the bifurcation position of the common salivary duct and the length and shape of the secretory tubes. Compared with the simple female salivary glands, the male's are more developed as their paired elongated salivary tubes can be divided into two parts, the glabrate anterior tube and the posterior tube with many secretory tubules. The ultrastructural study shows that the male salivary tubes have strong secretory function. The existence of different secretion granules indicates that there are some chemical reactions or mixing occurring in the lumen. Based on the ultrastructural characteristics, the functions of the different regions of the salivary tube have been speculated. The relationship between the salivary glands and nuptial feeding behavior of N. longiprocessa has been briefly discussed based on the structure of the salivary glands.

Molecular mechanisms of olfactory detection in insects: beyond receptors.

Insects thrive in diverse ecological niches in large part because of their highly sophisticated olfactory systems. Over the last two decades, a major focus in the study of insect olfaction has been on the role of olfactory receptors in mediating neuronal responses to environmental chemicals. In vivo, these receptors operate in specialized structures, called sensilla, which comprise neurons and non-neuronal support cells, extracellular lymph fluid and a precisely shaped cuticle. While sensilla are inherent to odour sensing in insects, we are only just beginning to understand their construction and function. Here, we review recent work that illuminates how odour-evoked neuronal activity is impacted by sensillar morphology, lymph fluid biochemistry, accessory signalling molecules in neurons and the physiological crosstalk between sensillar cells. These advances reveal multi-layered molecular and cellular mechanisms that determine the selectivity, sensitivity and dynamic modulation of odour-evoked responses in insects.

Comparative study of sensilla and other tegumentary structures of Myrmeleontidae larvae (Insecta, Neuroptera).

Antlion larvae have a complex tegumentary sensorial equipment. The sensilla and other kinds of larval tegumentary structures have been studied in 29 species of 18 genera within family Myrmeleontidae, all of them with certain degree of psammophilous lifestyle. The adaptations for such lifestyle are probably related to the evolutionary success of this lineage within Neuroptera. We identified eight types of sensory structures, six types of sensilla (excluding typical long bristles) and two other specialized tegumentary structures. Both sensilla and other types of structures that have been observed using scanning electron microscopy show similar patterns in terms of occurrence and density in all the studied species (with few exceptions). The sensilla identified are: coeloconica, placoidea, basiconica, trichodea type I, trichodea type II, and campaniformia. All these sensilla have mechano- or chemosensorial functions. Some regions of the larval body have been studied using SEM for the first time, such as the surface of the food canal, which bears sensilla coeloconica, and the abdominal segment X, that bears three types of sensilla: coeloconica, basiconica, and campaniformia. Sensilla placodea are newly reported on antlion larvae, being present on the mandibular base, pronotum, mentum, and cardum. Also, new locations of sensilla coeloconica (e.g., on rastra) and sensilla campaniformia (e.g., on odontoid processes) are noted. A novel porous texture with chemoreceptor function has been identified in the base of mandibles. A mechanism of dentate-notched surfaces that anchor maxillae and mandible, reinforcing the food canal, is detailed. All these sensorial structures, in addition to ocular tubercles for light caption and their great muscular system, confer to these larvae an extraordinary predation capacity to success hunting and living in such harsh environments.

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.

Centrioles and Ciliary Structures during Male Gametogenesis in Hexapoda: Discovery of New Models.

Centrioles are-widely conserved barrel-shaped organelles present in most organisms. They are indirectly involved in the organization of the cytoplasmic microtubules both in interphase and during the cell division by recruiting the molecules needed for microtubule nucleation. Moreover, the centrioles are required to assemble cilia and flagella by the direct elongation of their microtubule wall. Due to the importance of the cytoplasmic microtubules in several aspects of the cell life, any defect in centriole structure can lead to cell abnormalities that in humans may result in significant diseases. Many aspects of the centriole dynamics and function have been clarified in the last years, but little attention has been paid to the exceptions in centriole structure that occasionally appeared within the animal kingdom. Here, we focused our attention on non-canonical aspects of centriole architecture within the Hexapoda. The Hexapoda is one of the major animal groups and represents a good laboratory in which to examine the evolution and the organization of the centrioles. Although these findings represent obvious exceptions to the established rules of centriole organization, they may contribute to advance our understanding of the formation and the function of these organelles.

Research on the drag reduction mechanism of antlion (Myrmeleon sagax) larvae nonsmooth structural surface.

Antlion (Myrmeleon sagax) larvae live in sandy soil and possess the ability to enter soil quickly. In this article, the hierarchical structure of the nonsmooth surface of antlion larvae was obtained using a scanning electron microscope (SEM). Based on the results, a bionic nonsmooth structure model was established to investigate the friction and movement of soil particles above it. Then the relationship between drag reduction characteristics and the antlion larvae's nonsmooth structural surface was discussed, which would be helpful to design soil-engaging components. When the height of each nonsmooth structure is proportional to the square of their interval distance, and is proportional to the velocity of movement, it is shown that a nonsmooth structural surface contributes to improving the bulk coefficient of granular materials, which leads to substantial drag reduction.

Morphogenesis of the ovarian follicular epithelium during initial stages of embryogenesis of the viviparous earwig, Hemimerus talpoides.

Representatives of the highly specialized earwig family Hemimeridae are epizoic and viviparous. Their embryos develop inside terminal ovarian follicles (termed also embryonic follicles) and rely solely on nutrients transferred from mother tissues. In this report, we present results of ultrastructural and histochemical studies of the initial stage of Hemimerus talpoides development. Our results show that the follicular cells surrounding fully grown oocyte of Hemimerus do not degenerate after initiation of embryogenesis, but transform and gradually form the wall of the incubation chamber in which the embryo develops. We also show that amniotic cells of the early embryo remain in direct contact with transformed follicular cells. In the region of contact, short outgrowths of the amniotic cells associate with irregular surface specializations of the transformed follicular cells. We suggest that extended "postfertilization" activity of hemimerid follicular cells represents an adaptation to viviparity and matrotrophy in this insect lineage.

Adaptive morphology of the host-seeking first-instar larva of Stylops advarians Pierce (Strepsiptera, Stylopidae), a parasite of Andrena milwaukeensis Graenicher (Hymenoptera, Andrenidae).

The morphology of the prognathous, host-seeking first-instar larvae of Stylops advarians was examined to advance our understanding of their adaptations to reach immature bee hosts, a process requiring temporal phoresy on an adult bee. Sensory structures on the larval head, including eye spots and two pairs of olfactory pits, evidently assist recognition of an adult bee and eventual detection of a permanent host within a nest cell. First-instar larvae utilize various features of their appendages to travel securely on their phoretic host. Flexible adhesive tarsi of the pro- and mesothoracic legs allow them to embark and be retained on a flying bee. The tips of the pair of caudal filaments appear modified for a similar purpose. Spinulae of two lengths, and arranged in distinct patterns, cover the posterior edges of the thoracic and abdominal segments both dorsally and ventrally. These projections can cause lodging of larvae in the plumose hairs of the phoretic host, and may lock into the exine of pollen collected by the foraging bee. Discovery of a first-instar larva partially packed into a pollen load and in the crop of Andrena milwaukeensis demonstrates that Stylops is adapted to travel with a phoretic host both externally and internally.

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.

Molecular characterization, ultrastructure, and transovarial transmission of Tremblaya phenacola in six mealybugs of the Phenacoccinae subfamily (Insecta, Hemiptera, Coccomorpha).

Mealybugs (Hemiptera, Coccomorpha: Pseudococcidae) are plant sap-sucking insects which require close association with nutritional microorganisms for their proper development and reproduction. Here, we present the results of histological, ultrastructural, and molecular analyses of symbiotic systems of six mealybugs belonging to the Phenacoccinae subfamily: Phenacoccus aceris, Rhodania porifera, Coccura comari, Mirococcus clarus, Peliococcus calluneti, and Ceroputo pilosellae. Molecular analyses based on bacterial 16S rRNA genes have revealed that all the investigated species of Phenacoccinae are host to only one type of symbiotic bacteria-a large pleomorphic betaproteobacteria-Tremblaya phenacola. In all the species examined, bacteria are localized in the specialized cells of the host-insect termed bacteriocytes and are transovarially transmitted between generations. The mode of transovarial transmission is similar in all of the species investigated. Infection takes place in the neck region of the ovariole, between the tropharium and vitellarium. The co-phylogeny between mealybugs and bacteria Tremblaya has been also analyzed.

Bacterial associates of Orthezia urticae, Matsucoccus pini, and Steingelia gorodetskia - scale insects of archaeoccoid families Ortheziidae, Matsucoccidae, and Steingeliidae (Hemiptera, Coccomorpha).

The biological nature, ultrastructure, distribution, and mode of transmission between generations of the microorganisms associated with three species (Orthezia urticae, Matsucoccus pini, Steingelia gorodetskia) of primitive families (archaeococcoids = Orthezioidea) of scale insects were investigated by means of microscopic and molecular methods. In all the specimens of Orthezia urticae and Matsucoccus pini examined, bacteria Wolbachia were identified. In some examined specimens of O. urticae, apart from Wolbachia, bacteria Sodalis were detected. In Steingelia gorodetskia, the bacteria of the genus Sphingomonas were found. In contrast to most plant sap-sucking hemipterans, the bacterial associates of O. urticae, M. pini, and S. gorodetskia are not harbored in specialized bacteriocytes, but are dispersed in the cells of different organs. Ultrastructural observations have shown that bacteria Wolbachia in O. urticae and M. pini, Sodalis in O. urticae, and Sphingomonas in S. gorodetskia are transovarially transmitted from mother to progeny.

The sperm pump and genital coupling of Panorpodes kuandianensis (Mecoptera: Panorpodidae).

Males of Panorpodidae possess a special sperm pump, through which they directly transfer seminal fluid to the female spermatheca. However, the sperm pump has not been studied in Panorpodes to date. Here, the structure of the sperm pump and the internal coupling of genitalia were investigated in the short-faced scorpionfly Panorpodes kuandianensis Zhong, Zhang, and Hua, 2011 using light and scanning electron microscopy. The sperm pump mainly consists of a piston, a pumping chamber, the anterior region of the aedeagal complex, the posterior region of the ejaculatory sac, and associated muscles. The piston as a propulsion apparatus is controlled by levator and depressor muscles. Its posterior region connects dorsally to the aedeagus via a joint. The pumping chamber is located between the piston and the aedeagus. The dorsal and ventral parameres were attached by retractor muscles. During copulation, the male phallotreme connects to the female copulatory pore to transfer sperm. Male gonostyli and parameres grasp the female to restrict the genitalia movement and impede her medigynium from retreating. The sperm ejaculatory mechanism of Panorpodes and the evolution of sperm transfer mode in insects are briefly discussed based on the structure of the sperm pump and the internal coupling of genitalia.

Small, but oh my! Head morphology of adult Aleuropteryx spp. and effects of miniaturization (Insecta: Neuroptera: Coniopterygidae).

We present the first morphological study of the internal head structures of adults of the coniopterygid genus Aleuropteryx, which belong to the smallest known lacewings. The head is ventrally closed with a gula, which is unique in adult Neuroptera and otherwise developed in Megaloptera, the sister group of Neuroptera. The dorsal tentorial arms are directed posteriorly and fused, forming an arch that fulfills functions otherwise taken by the tentorial bridge. A newly found maxillary gland is present in both sexes. Several structural modifications correlated with miniaturization are recognized: a relative increase in the size of the brain, a reduction in the number of ommatidia and diameter of the facets, a countersunken cone-shaped ocular ridge, and a simplification of the tracheal system. The structure of the head differs strikingly from that of the previously studied species Coniopteryx pygmaea, indicating a greater variability in the family Coniopterygidae, which might be another effect of miniaturization.

Ultrastructure of the vasa deferentia of Terrobittacus implicatus and Cerapanorpa nanwutaina (Insecta: Mecoptera).

The fine structures of vasa deferentia and postvesicular vasa deferentia were investigated in the hangingfly Terrobittacus implicatus (Cai et al. 2006) and the scorpionfly Cerapanorpa nanwutaina (Chou 1981) using light and transmission electron microscopy, and schematic diagrams were drawn accordingly. The vasa deferentia of both species comprise muscular layers, a basal lamina, and a mono-layered epithelium, but the postvesicular vasa deferentia contain muscular layers, a basal lamina, a single-layered epithelium, a subcuticular cavity, and an inner cuticle respectively. The vas deferens releases secretions into the lumen directly, probably by means of merocrine production. On the contrary, the cells of the postvesicular vas deferens correspond to class I glandular cells, discharging secretions into the subcuticular cavity first, and then into the lumen through an inner cuticle. The epithelium in both structures of Bittacidae is well developed and contains more microvilli, organelles, and more types of secretions than in Panorpidae. In Panorpidae, the spine of the postvesicular vas deferens may serve as a barricade for the reflow of the sperm and to protect the extraordinarily long structure from being collapsed or injured.

Can exposure to neem oil affect the spermatogenesis of predator Ceraeochrysa claveri?

Novel biological control methods and integrated pest management strategies are basic requirements for the development of sustainable agriculture. As a result, there is a growing demand for research on the use of plant extracts and natural enemies such as the green lacewing, Ceraeochrysa claveri, as natural pest control methods. Studies have shown that although natural compounds such as neem oil (Azadirachta indica) are effective as pest control strategies, they also cause sublethal effects on nontarget insects, such as C. claveri. The aim of this study was to examine the effects of neem oil on C. claveri testes. C. claveri larvae were fed Diatraea saccharalis eggs, which were pretreated with 0.5%, 1%, and 2% neem oil. Testes were collected from larvae, pupae, and adults and analyzed using light and electron (transmission and scanning) microscopy. Changes in cellular stress and possible cell death were also determined by TUNEL assay and the marker HSP-70. The results showed that neem oil affects the organization and distribution of cysts in the testes and the normal sequence of cyst development, causing a delay in spermatogenesis in the testes of treated insects. Tests for cellular stress and DNA fragmentation indicated there was no cellular alteration in the treated groups. Although neem oil does not induce cell death or changes in HSP-70 expression, this biopesticide negatively impacts the process of spermatogenesis and could decrease the perpetuation of this species in the agroecosystem, indicating that the use of neem oil in association with green lacewings as a biological control should be carefully evaluated.

Copulatory mechanism and functional morphology of genitalia and anal horn of the scorpionfly Cerapanorpa dubia (Mecoptera: Panorpidae).

The scorpionfly genus Cerapanorpa is characterized by the male possessing a single finger-like anal horn on the posterior portion of tergite VI. However, the functional morphology of this anal horn and the genitalia have not been studied to date. Herein, we investigated the functional morphology of the genitalia and the nongenital structures of the scorpionfly Cerapanorpa dubia by observing the mating process and dissecting the freeze-fixated pairs in copula to reveal the copulatory mechanism. The male C. dubia provides a solid salivary mass to the female as a nuptial gift prior to copulation. When the female starts to feed on the gift, the male uses his notal organ and complex genital structures to control the closest wing and genitalia of the female to establish a V-shaped mating position. In the maintenance phase of copulation, the male uses his anal horn in cooperation with the basally-constricted abdominal segment VII to clamp female abdominal segment VIII. The male hypovalves grasp female cerci, and move up and down rhythmically. The paired parameres clasp both sides of female tergite IX. The basal processes on male gonostyli grip the pleural membranes of the female genital chamber. In the sperm transfer phase, the male aedeagus directly couples with the female medigynium to transmit sperm by connecting his phallotreme to the female's copulatory pore. The evolution of the male complex grasping structures in Panorpidae is also briefly discussed.