Functional morphology and movements of the proboscis of Lepidoptera (Insecta)

Summary The mouthparts of Lepidoptera were investigated in a number of species by morphological and cinematographical methods. Both the galeae (which compose the proboscis) and the basal maxillary components (stipites) were studied in the resting position, in motion, and during feeding. In the resting position the proboscis is coiled so tightly that the surfaces of the consecutive coils are in close contact and the outermost coil touches the ventral side of the head. Cuticular processes of the galeal wall interlock between the coils in this position. In the investigated species they occur on the galeal wall and on the ventral side of the head in varying number and distribution. By the extension of the basal galeal joint, the coiled proboscis is released from its resting position and is elevated continuously. It uncoils in 3–5 steps which effect the entire length simultaneously. Each uncoiling step occurs synchronously with a compression of the stipital tubes on either side of the body. These compression movements pump hemolymph into the galeae. In all investigated Lepidoptera the uncoiled proboscis shows a distinct downward bend at a certain point which is also detectable in anaesthetized or freshly killed animals in some species. This feeding position and the movements of the uncoiled proboscis are similar in all species despite the intrinsic galeal muscles being variously arranged in the galeal lumen in different Lepidoptera. When comparing cross-sections through corresponding regions of coiled and uncoiled proboscises, the curvatures of the dorsal galeal walls remain unchanged. Coiling of the proboscis starts at the tip and progresses to the base. After coiling the proboscis tightly beneath the head, the diameter of the spiral widens due to its elastic properties until the proboscis props itself against the ventral side of the head. This elastic effect combined with the interlocking cuticular processes seems to be responsible for the resting position of the proboscis.Abbreviations an antenna - bre bend region - ca cardo - ci cibarium - cl clypeus - co complex eye - cp cuticular process - dre distal region - esm external tentoriostipital muscle - fc food canal - fst flat part of the stipes - ga galea - hs horizontal septum - igm intrinsic galeal muscles - ism internal tentoriostipital muscle - la labium - lap labial palpus - lr labrum - mxp maxillary palpus - ne nerve - pi pilifer - pom primary oblique galeal muscles - pr proboscis - pre proximal region - sa salivarium - se sensillum - som secondary oblique galeal muscles - st stipes - stl stipital lamella - te tentorium - tr trachea - tst tubular part of the stipes - vm ventral membrane - vs vertical septum     

Gross morphology of multiporous plate sensilla in the Hymenoptera (Insecta)

Both external and internal gross morphology of multiporous plate sensilla were investigated throughout the Hymenoptera, with particular reference to nonaculeate families, using SEM. Several scenarios concerning the origin of multiporous plate sensilla are discussed. Shape, attachment, position, and reinforcing structures, were found to vary between the families, and the possible phylogenetic implications of these are discussed.     

Zur morphometrie der antenne des seidenspinners,Bombyx mori L.: Zahl und Verteilung der Riechsensillen (Insecta,Lepidoptera)

A light and scanning electron microscope study of the silk moth antenna investigated the general morphology and the distribution pattern of sensilla which are spezialized for the detection of the female sex pheromone. Receptor physiology and morphogenetic aspects are discussed.     

Morphology of the Pylorus of Bombyx mori (Linnaeus) (Lepidoptera: Bombycidae)

The pylorus is a segment of the hindgut of great morphological diversity, which is subdivided into the posterior interstitial ring, pyloric cone, and pyloric valve. There is no morphological information on Bombyx mori (L.) hybrids, which is an insect of economic interest to Brazil. Consequently, this study aimed to analyze the morphology of the pylorus of hybrid larvae of fifth instars of B. mori from crossbreeding of Chinese and Japanese races. These were anesthetized, and the pylorus was fixed and processed by light microscopy and scanning electron microscopy. The pylorus presented as a separate compartment, consisting of a simple epithelium, with large variation in cell morphology, covered on the luminal side by an intima with specializations in some areas, the spicules. The musculature was formed by bundles of striated muscle fibers in longitudinal and circular arrangement. These fibers were multinucleated with central and peripheral nuclei, operating in peristalsis. The morphology of the pylorus is a reflection of functional dynamism, and this study provided a better understanding of its physiology, which promotes the regulated movement of the bolus, preventing reflux and directing it towards the ileum in the formation of fecal pellets.     

Effects of Diflubenzuron on the Development of Bombyx mori (Lepidoptera: Bombycidae)

ABSTRACT This study was conducted to determine the effects of diflubenzuron (DFB) on the growth and development of the silkworm, Bombyx mori (L.). The DFB treatment extended abnormally the larval duration and affected negatively on larval spinning of the 5th instar. All the larvae treated with high DFB concentration (2.5 × 10^-1 and 2.5x 10^-1μg/μl) lost their spinning capability and finally died, whereas 62% of larvae treated with low DFB concentration (2.5 × 10^-1μg/μl)) spinned cocoon. The larval weights depended sensitively on the DFB treatment period rather than on the DFB concentration. The DFB treatment decreased the larval maturity less than 6% without regard to the concentration and treatment period. All the larva, when treated with DFB before the 5th days of the 5th instar, were not matured.     

Pachytene mapping in the female silkworm,Bombyx mori L. (Lepidoptera)

Pachytene preparations of the chromosome complement of female larvae of Bombyx mori were improved to give a distinct chromomere pattern of the bivalents suitable for chromosome mapping. Six of the 28 bivalents are described and can be identified regularly in the bivalent complements, among them the bivalent containing the nucleolus organizer. The relative lengths of these bivalents compared with one another change during development of pachytene. In contrast to other members of the Lepidoptera there is no conspicuous heterochromatic W-chromosome, which corresponds to the female-specific heterochromatin body present in the nuclei of somatic tissues. This tissue-specific heteropycnosis indicates a different functional state of the responsible chromosome or chromosomal segment in germ line and somatic cells.     

Male meiosis in polyploid silkworms,Bombyx mori L. (Lepidoptera : Bombycidae)

Tetraploid and hexaploid silkworms, Bombyx mori L. (Lepidoptera Bombycidae) were induced by applying a cold shock to diploid and triploid eggs at the first cleavage stage. Male meiosis in the primary spermatocytes of these silkworms having a different ploidy was observed. In the polyploid cells, chromosome bridges, 2 for the tetraploid and 3 for the hexaploid, occurred between the newly formed daughter nuclei at telophase. Observation in the living spermatocytes showed that tetraploid cells needed a longer time than the diploid ones to complete the first meiotic division. The delay in the cell division may be responsible for the high sterility of the tetraploid males.     

Behavior of centrioles during meiosis in the male silkworm, Bombyx mori (Lepidoptera)

The behavior of centrioles during eupyrene and apyrene meiosis was examined in the silkworm, Bombyx mori , by transmission electron microscopy and indirect immunofluorescence for tubulin. In eupyrene spermatocytes the centrioles, accompanied by axonemes, attached temporarily to the nucleus at diplotene, then detached from the nucleus in diakinesis. After the separation, a beret-shaped structure consisting of a double membrane covered the proximal region of the pair of centrioles. The structure disappeared after breakdown of the nuclear membrane. The centriole, with the axoneme, reattached to the nucleus at telophase I. The process was repeated during meiosis II until the centrioles maintained their nuclear attachment in newly developed spermatids. In stark contrast to their eupyrene counterparts, apyrene spermatocytes were conspicuously devoid of any attachment of the centrioles to the nucleus. These eupyrene-specific and apyrene-specific relationships were consistently and repeatedly found between the nuclear membrane and centrioles, giving rise to suspicion that the behavioral phenomena may be related to differentiation of the dimorphic sperm types.     

Rab14 from Bombyx mori (Lepidoptera: Bombycidae) shows ATPase activity

Rab GTPases are essential for vesicular transport, whereas adenosine triphosphate (ATP) is the most important and versatile of the activated carriers in the cell. But there are little reports to clarify the connection between ATP and Rab GTPases. A cDNA clone (Rab14) from Bombyx mori was expressed in Escherichia coli as a glutathione S-transferase fusion protein and purified. The protein bound to [³H]-GDP and [³⁵S]-GTPγS. Binding of [³⁵S]-GTPγS was inhibited by guanosine diphosphate (GDP), guanosine triphosphate (GTP) and ATP. Rab14 showed GTP- and ATP-hydrolysis activity. The Km value of Rab14 for ATP was lower than that for GTP. Human Rab14 also showed an ATPase activity. Furthermore, bound [³H]-GDP was exchanged efficiently with GTP and ATP. These results suggest that Rab14 is an ATPase as well as GTPase and gives Rab14 an exciting integrative function between cell metabolic status and membrane trafficking.     

Comparative morphology of the spiracles of the Papilionidae,Sphingidae, and Saturniidae (Insecta: Lepidoptera)

The morphology, ultrastructure, and innervation of the spiracles of the instars and adults of representatives of three lepidopteran families were examined: Ornithoptera priamus poseidon and Pachliopta aristolochiae (Papilionidae), Attacus atlas (Saturniidae), and Acherontia atropos (Sphingidae).Peritreme and atrium show stage- and family-specific structures for protecting the internal valve apparatus. The gross morphology of the cuticular valve mechanism is uniform within the three families, consisting of a rigid bow and a movable bar with a lever. In adult Papilionidae, all cuticular parts (bow, bar and lever) of the valve are innervated by multipolar dendrites. Internal or external cuticular chemo- or hygroreceptors, which could participate in the regulation of respiration, could not be detected in any stage. The closing muscle inserts between the tip of the lever and the base of the bar, and is innervated only by motor neurons. The elasticity of the cuticular system and an opener are the antagonists to the closing muscle. The spiracular opener of the adult Papilionidae and of all instars of the moths is an elastic ligament. The opener of the larval and pupal spiracles of the Papilionidae, however, is a single thickened muscle fiber surrounded by an elastic sheath of connective tissue. As it contains motor and multipolar sensory neurons, we assume that it may function as a stretch receptor for controlling the spiracular opening state.     

BAC selection of the large white butterfly,Pieris brassicae (Lepidoptera: Pieridae) containing orthologs of the silkworm,Bombyx mori (Lepidoptera: Bombycidae)

Applied Entomology and Zoology - We constructed a bacterial artificial chromosome (BAC) library from the large white butterfly, Pieris brassicae (L.) (Lepidoptera: Pieridae), which...     

Arginine kinase is highly expressed in a resistant strain of silkworm (Bombyx mori, Lepidoptera): Implication of its role in resistance to Bombyx mori nucleopolyhedrovirus

A gene encoding Bombyx mori arginine kinase (BmAK) has been indentified differentially expressed in the midguts of Bombyx mori strain NB which is resistant to nucleopolyhedrovirus (BmNPV), strain 306 which is susceptible to NPV and a near isogenic line BC(8) with similar genetic background to 306 but resistant to NPV by two-dimensional gel electrophoresis (2-DE). In this study, we characterized the expression profiles of BmAK using RT-PCR and real-time quantitative PCR. The expression level of BmAK fluctuated in various developing stage and various tissue. Remarkably, the expression level of BmAK increased more than 10-fold 24 hours post inoculation (h p.i.) of NPV in strain NB and BC(8), while such increment was abraded in strain 306 although the basal expression level of BmAK in strain 306 was higher than that of strain NB and BC(8). Western blotting analysis using polyclonal antibody against BmAK verified such observation, and immunofluoresence analysis indicated for the first time that BmAK was mainly located to the cytoplasm or some structures in cytoplasm. These findings suggest that arginine kinase is involved in the antiviral process of Bombyx mori larvae against NPV infection.     

phiC31-integrase-mediated, site-specific integration of transgenes in the silkworm, Bombyx mori (Lepidoptera: Bombycidae)

Transgenic silkworms can be useful for investigating the functions of genes in the post-genomic era. However, the common method of using a transposon as an insertion tool may result in the random integration of a foreign gene into the genome and suffer from a strong position effect. To overcome these problems, it is necessary to develop a site-specific integration system. It is known that phiC31 integrase has the capacity to mediate recombination between the target sequences attP and attB. To test the availability of site-specific integration in the silkworm, we first examined the efficiency of recombination between the target sites of the two plasmids in silkworm embryos and found that the frequency of recombination was very high. Then we constructed a host strain that possessed the target sequence attP using the common method. We injected the donor plasmid together with the phiC31 integrase mRNA into the embryos of the host strain and obtained positive lines. Structural analysis of the lines showed that site-specific integration occurred by recombination between the genomic attP site and the attB site of the donor plasmid. We can conclude from the results that phiC31 integrase has the ability to mediate the site-specific integration of transgenes into the silkworm chromosome.     

Gelechioidea (Insecta: Lepidoptera) systematics: a reexamination using combined morphology and mitochondrial DNA data

Phylogenies of Gelechioidea (Insecta: Lepidoptera) historically have been in disagreement and definitions vary at the family and subfamily levels. Addition of new taxa or new characters drastically changes relationships indicating that current phylogenetic schemes require more investigation. This study is the first phylogenetic analysis of Gelechioidea to include molecular data. Here we present a combined analysis using Maximum Parsimony to investigate sister-group relationships within Gelechioidea. The addition of Cytochrome oxidase I and II to revised published morphological matrices gives 453 parsimony informative characters for the 42 taxa for which we have sequence data. The combined analysis resulted in two trees with mostly novel sister-group relationships. These results challenge current concepts of Gelechioidea, suggesting that traditional morphological characters that have united taxa may not be homologous structures and are in need of further investigation. A combination of morphological data with new molecular data will be the most robust method of study for Gelechioidea phylogenetics.     

Involvement of calcineurin in the signal transduction of PBAN in the silkworm, Bombyx mori (Lepidoptera)

In several moth species sex pheromone production in the pheromone gland is regulated by a neurohormone, pheromone biosynthesis activating neuropeptide (PBAN). In Bombyx mori it is suggested that PBAN, after binding to the cell-surface receptor, primarily activates a plasma membrane receptor-activated Ca2+ channel to increase cytosolic levels of Ca2+, and Ca2+/calmodulin complex directly or indirectly activates a phosphoprotein phosphatase, which in turn elicits activation of acyl CoA reductase (the key enzyme under PBAN control) through dephosphorylation, resulting in pheromone (bombykol) production. The effect of cyclosporin A (CsA) and FK 506, specific inhibitors of calcineurin (phosphoprotein phosphatase 2B) was studied on the sex pheromone production, in B. mori. The in vitro experiments showed that both chemicals exerted a dose-dependent inhibitory action when they were co-incubated with TKYFSPRL amide (Hez-PBAN fragment peptide). Practically, no difference was detected between the two chemicals in the tested doses (0.025-1250 microM). When effects of CsA or FK 506 were studied on cell-free production of bombykol by using microsomal fraction no inhibition was detected. Since microsomal fraction contains the acyl CoA synthetase, the rate-limiting acyl CoA reductase and the precursor, bombykol is produced if supplied with CoA, ATP and NADPH. Thus, the inhibitory action of CsA and FK506 under in vitro conditions should occur before the step of acyl group reduction and the effect is likely to be attributable to the inhibition of calcineurin in the signal transduction cascade mechanism of PBAN, in B. mori. The existence of calcineurin in the pheromone gland by using Western blot analysis is also demonstrated.     

Functional morphology of the glandula prostatica,ejaculatory valve,and ductus ejaculatorius of the silkworm Bombyx mori

The penis of the silkmoth, Bombyx mori, consists of two parts covered with cuticle, the corpus penis and crus penis, and a third part, the radix penis, without a cuticle but surrounded by a thick sphincter. The radix penis is divisible into anterior and posterior parts. The ductus (d.) ejaculatorius passing through the penis has no secretory cells. In the anterior radix penis, the wall of the d. ejaculatorius is thin and without folds; in the posterior section, it is thick, with folds in its lumen. The glandula (g.) prostatica is divisible into anterior and posterior parts according to differences in the histological and morphological characteristics of the cells and their secretions, which contain many heterogeneous substances. In the anterior g. prostatica, secretions accumulate separately in the anterior and posterior sections before ejaculation. Unlike the posterior region, the anterior region displays a large mass(es) at the periphery of the lumen along the secretory cell layer. Judging from staining properties, the pearly body and the first layer of the spermatophore wall, which, after copulation, form in the female bursa copulatrix, seem to be derived from the secretions of the anterior and posterior regions of the g. prostatica, respectively. The secretion of the posterior g. prostatica contains initiatorin, which acts as a sperm-activating factor in the inner and outer matrices of the spermatophore. An ejaculatory valve is found between the radix penis and the g. prostatica. The opening of this valve is regulated by the surrounding sphincter, thus impeding the back-flow of secretions and seminal fluid in the radix penis and resulting in their transport outwards during ejaculation. The musculature of the d. ejaculatorius and the corpus penis promotes further transport of these secretions into the female bursa copulatrix.     

Nucleopolyhedrovirus infected central nervous system cells of Bombyx mori (L.) (Lepidoptera: Bombycidae)

BmMNPV, a Nucleopolyhedrovirus isolated from infected Bombyx mori (L.) larvae in Paraná State--Brazil, was used to inoculate healthy 5th-instar B. mori larvae and examine the infection on central nervous system (CNS) cells. Samples of nervous tissue were removed from the infected insects, at different sampling times, and processed for cytopathology studies by light and transmission electron microscopy using routine techniques. The experiment included both inoculated and non-inoculated larvae (control). BmMNPV infection was detected on the 5th day after inoculation in CNS cells. Initially, infection was characterized by nuclear hypertrophy and the presence of virogenic stroma, in which the progeny virions were produced. Virions are enveloped and occluded into protein crystal, the polyhedra. Lyses of infected CNS cells were undetected; however, free mature polyhedra were seen in spaces inside the CNS. These polyhedra possibly came from trachea that penetrate the CNS and its cells, which are susceptible to BmMNPV and lyses after infection. We conclude that the tracheal system is responsible for disseminating BmMNPV infection in B. mori CNS and that the tracheal branches allow non-occluded virions to pass through the blood-brain barrier.     

Functional analysis of a survivin-like gene in Bombyx mori

The survivin (svv) gene is a newly discovered member of the inhibitors of apoptosis gene family. In recent years, svv has been confirmed to have an anti-apoptosis function and to play a critical role in cell division. We identified a survivin-like gene in the silkworm, Bombyx mori (Bm-svv). In this study, to gain insight into its function, a baculovirus expression system was used to express the Bm-svv gene in insect cell lines. The recombinant viruses were then used as a vector to transform insect cells, and cell activity was determined using the Cell Counting Kit-8 (CCK-8), which is usually employed for detecting mammalian cell number. The results indicated that the Bm-svv gene plays a role in the cell growth arrest or apoptosis induced by viruses. Furthermore, the CCK-8 kit is effective in determining the activity of insect cells.     

Deletion of the Bombyx mori odorant receptor co-receptor (BmOrco) impairs olfactory sensitivity in silkworms

Olfaction plays an essential role in many important insect behaviors such as feeding and reproduction. To detect olfactory stimuli, an odorant receptor co-receptor (Orco) is required. In this study, we deleted the Orco gene in the Lepidopteran model insect, Bombyx mori, using a binary transgene-based clustered regulatory interspaced short palindromic repeats (CRISPR)/Cas9 system. We initially generated somatic mutations in two targeted sites, from which we obtained homozygous mutants with deletion of a 866 base pair sequence. Because of the flight inability of B. mori, we developed a novel method to examine the adult mating behavior. Considering the specialization in larval feeding, we examined food selection behavior in Orco somatic mutants by the walking trail analysis of silkworm position over time. Single sensillum recordings indicated that the antenna of the homozygous mutant was unable to respond to either of the two sex pheromones, bombykol or bombykal. An adult mating behavior assay revealed that the Orco mutant displayed a significantly impaired mating selection behavior in response to natural pheromone released by a wild-type female moth as well as an 11:1 mixture of bombykol/bombykal. The mutants also exhibited a decreased response to bombykol and, similar to wild-type moths, they displayed no response to bombykal. A larval feeding behavior assay revealed that the Orco mutant displayed defective selection for mulberry leaves and different concentrations of the volatile compound cis-jasmone found in mulberry leaves. Deletion of BmOrco severely disrupts the olfactory system, suggesting that BmOrco is indispensable in the olfactory pathway. The approach used for generating somatic and homozygous mutations also highlights a novel method for mutagenesis. This study on BmOrco function provides insights into the insect olfactory system and also provides a paradigm for agroforestry pest control.