Susceptibility of preimaginal western cherry fruit fly, Rhagoletis indifferens (Diptera: Tephritidae) to Beauveria bassiana (Balsamo) Vuillemin Clavicipitaceae (Hypocreales)

Last-instar larvae of the western cherry fruit fly, Rhagoletis indifferens, were subjected to Beauveria bassiana GHA incorporated into sterile sand and non-sterile orchard soil. Mycosis in the pupal stage was observed in >20% of buried R. indifferens pupae and >80% of larvae entering sand treated with either of two B. bassiana isolates. When pre-pupal larvae burrowed into conidium-treated non-sterile cherry orchard soil, the incidence of mycosis, on both the puparia and internally developing pupae, increased with dose. Internal pupal tissues were found to contain B. bassiana. Increasing the soil moisture level from 20% to 35% water holding capacity did not have an effect on the percentage of mycosed pupae. This is the first evidence that the preimaginal stages of R. indifferens are susceptible to infection by B. bassiana.     

Can larval epidermis omit the secretion of a pupal cuticle in a saturniid moth, Samia cynthia ricini?

When the larval tissue is exposed to the hormonal milieu lacking juvenile hormone, adult characters appear directly omitting the pupal stage in some insects but not in others. In Samia cynthia ricini, a species belonging to the latter group, a possible omission of pupal characters was tested by previously untried experiments. Firstly, the possibility that the larval epidermis of only some stages is capable of responding so as to omit to secrete the pupal cuticle was tested. Pieces of larval integument taken from various developmental stages were implanted into developing (pharate) adults. None of these failed to secrete the pupal cuticle. Secondly, pieces of larval integument were first implanted into brainless pupae and left there for a month to eliminate the effect of a trace of juvenile hormone which might have been carried over by the implants. They were then caused to develop, and they again secreted pupal cuticle. It is concluded that the larval epidermis cannot omit secreting pupal cuticle in this species.     

The vitellogenin of the bumblebee, Bombus hypocrita: studies on structural analysis of the cDNA and expression of the mRNA

In this present study, the cDNA of Bombus hypocrita vitellogenin (Vg) was cloned and sequenced. It is composed of 5,478 bp and contains an ORF of 1,772 amino acids within a putative signal peptide of 16 residues. The deduced amino acid sequence shows significant similarity with Bombus ignitus (95%) and Apis mellifera (52%) and a high number of conserved motifs. Close to the C terminus there is a GL/ICG motif followed by nine cysteines, and a DGXR motif is located 18 residues upstream from the GL/ICG motif. Moreover, we predicted the 3D structure of B. hypocrita Vg. Furthermore, the Vg mRNA of B. hypocrita was spatio-temporally analyzed in different castes (such as queen, worker and drone) from pupae to adult. The Vg mRNA was found in the white-eyed pupal (Pw) stage in queens, and the expression increased during the entire pupal development and attained its peak in the dark brown pupal stage. It also had a high expression in the adult fat body. In workers, the Vg expression was detected in the Pw stage, and its levels increased with age with the highest in 15 days. Afterward, it decreased progressively. Vg mRNA was also observed in drones, with a higher level of expression shown in only freshly molted adult drones.     

Factors influencing the developmental capacity of Galleria larval epidermis

The nature of the cuticle secreted by integument from a day-1 penultimate instar larval Galleria when cultured in vivo in the abdomen of a last instar larva varied with the age of the host. When placed in a day-5 last instar larva, the implanted integument secreted a pupal cuticle at the time the host metamorphosed and became a pupa. However, when placed in a day-7 last instar larva the implant, from the same stage donor, secreted a larval cuticle at the time the host pupated. Experimental studies involving implantation of the integument for a 24 hr period, into various developmental stages of normal and ligated last instar larvae, pupae, and pharate adults, prior to placing it in a day-7 last instar larva suggest that a non-hormonal factor present in day-4 and -5 last instar larvae is important to initiate pupal syntheses.     

Integration of entomopathogenic fungi and eco-friendly insecticides for management of red palm weevil,Rhynchophorus ferrugineus (Olivier)

Red palm weevil (Rhynchophorus ferrugineus) is a voracious pest of date palm worldwide. Pakistan ranks sixth in date palm production globally. Losses to date palm plantations in Pakistan sometimes surpass 10%-20%. Most of the traditional management strategies used by farmers have been found insignificant to combat this voracious pest. The entomopathogenic fungi, Beauveria bassiana [QA-3(L) and QA-3(H)] and insecticides, Nitenpyram (Active 10% SL) [NIT (L) and NIT (H)] were applied to larval (2nd, 4th, and 6th), pupal and adult stages of R. ferrugienus. Integration or alone application of fungi with insecticides at different concentration under laboratory conditions. Combined application was depicted additive and synergistic interactions. Contrarily, highest cumulative mortality (100%) was recorded in 2nd instar larvae as compared to later instar larvae at combined application. The maximum pupal and adult mortality remained 89% and 66% respectively after treatment with [QA-3 (H) + NIT (L)]. The combination of B. bassiana at higher concentration whereas Nitenpyram at lower dose was found more lethal to larvae, pupae and adults of R. ferrugineus. This signifies the need of combining B. bassiana and bio-rational insecticides that can reduce the cost of management with least harm to environment and natural enemies.     

Armigeres subalbatus prophenoloxidase III: Cloning,characterization and potential role in morphogenesis

It has long been suggested that phenoloxidases (POs) play key roles in various physiological functions in insects, e.g., cuticular sclerotization, wound healing, egg tanning and melanotic encapsulation of pathogens. Here we report that a mosquito PO, designated Armigeres subalbatus prophenoloxidase III (As-pro-PO III), is likely involved in the morphogenesis in mosquito. Expression profile analysis found that As-pro-PO III mRNA is persistently expressed in adult mosquitoes and is not significantly affected by blood feeding, microfilariae inoculation, or Escherichia coli inoculation, but expression levels of As-pro-PO III fluctuated in larval and pupal stages. Knockdown of As-pro-PO III expression in pupae using double-stranded RNA resulted in high pupal mortality and deformed adults that subsequently died following emergence. Promoter activity analyses by electrophoretic mobility-shift assays and transfection assays suggest that the As-pro-PO III gene is positively regulated by a putative Zeste motif, a developmental regulatory element. These results suggest that As-pro-PO III is associated with morphogenesis of mosquitoes.     

Convergent evolution of neuroendocrine control of phenotypic plasticity in pupal colour in butterflies

Phenotypic plasticity in pupal colour occurs in three families of butterflies (the Nymphalidae, Papilionidae and Pieridae), typically in species whose pupation sites vary unpredictably in colour. In all species studied to date, larvae ready for pupation respond to environmental cues associated with the colour of their pupation sites and moult into cryptic light (yellow–green) or dark (brown–black) pupae. In nymphalids and pierids, pupal colour is controlled by a neuroendocrine factor, pupal melanization-reducing factor (PMRF), the release of which inhibits the melanization of the pupal cuticle resulting in light pupae. In contrast, the neuroendocrine factor controlling pupal colour in papilionid butterflies results in the production of brown pupae. PMRF was extracted from the ventral nerve chains of the peacock butterfly Inachis io (Nymphalidae) and black swallowtail butterfly Papilio polyxenes (Papilionidae). When injected into pre-pupae, the extracts resulted in yellow pupae in I. io but brown pupae in P. polyxenes. These results suggest that the same neuroendocrine factor controls the plasticity in pupal colour, but that plasticity in pupal colour in these species has evolved independently (convergently).     

Characterization of HSP70 expression in drones of Apis cerana (Hymenoptera: Apidae)

Heat shock proteins (HSPs) constitute a superfamily of molecular chaperones that are rapidly biosynthesized in response to various biotic and abiotic factors. In this study, we first cloned the full-length HSP70 gene of the Eastern honeybee Apis cerana. Then, using real-time quantitative PCR, we explored HSP70 expression profiles in drones at different developmental stages, ages, and reproductive statuses (with and without semen). The full-length HSP70 cDNA is 2421 bp, including a 1953-bp open reading frame (ORF) that encodes a polypeptide of 650 amino acids. The HSP70 gene consists of one intron and two exons. The phylogenetic analysis revealed that the HSP70 genes of A. cerana and Apis mellifera are the most closely related. We observed HSP70 expression at all selected developmental stages and detected the highest expression in pupae with an unpigmented body cuticle and brown eyes (Pb) and much lower expression in larvae hatched within 72 h. In adult drones of different ages, the highest expression level of HSP70 was observed in 16-day-old drones; significantly lower accumulation of HSP70 mRNA was detected in 4-day-old drones. There was no significant difference in HSP70 expression between drones with and without semen captured at the entrance, while the HSP70 gene expression level strikingly differed between drones captured at the entrance and the drones collected within the hive. Our study suggests that HSP70 might play a critical role in drone development and during reproductive mating events.     

Immune function differences between two color morphs of the red palm weevil Rhynchophorus ferrugineus (Coleoptera: Curculionidae) at different life stages

Several studies demonstrated that in insects cuticle melanism is interrelated with pathogen resistance, as melanin‐based coloration and innate immunity possess similar physiological pathways. For some insects, higher pathogen resistance was observed in darker individuals than in individuals with lighter cuticular coloration. Here, we investigated the difference in immune response between two color morphs (black and red) and between the life stages (pupa and adult) of the red palm weevil Rhynchophorus ferrugineus (Coleoptera: Curculionidae). Here in this study, cuticle thickness, microbial test (antimicrobial activity, phenoloxidase activity, and hemocyte density), and immune‐related gene expression were evaluated at different stages of RPW. Study results revealed that cuticle thickness of black phenotype was thicker than red phenotype at old‐pupa stage, while no significant difference found at adult stage. These results may relate to the development processes of epidermis in different stages of RPW. The results of antimicrobial activity, phenoloxidase (PO) activity, and hemocyte density analyses showed that adults with a red phenotype had stronger pathogen resistance than those with a black phenotype. In addition to antimicrobial activity and PO activity, we tested relative gene expression in the fat body of old pupae. The results of hemolymph antimicrobial analysis showed that old pupae with a red phenotype were significantly different from those with a black phenotype at 12 hr after Staphylococcus aureus injection, suggesting that red phenotype pupae were more sensitive to S. aureus. Examination of gene expression in the fat body also revealed that the red phenotype had a higher immune response than the black phenotype. Our results were inconsistent with the previous conclusion that dark insects had increased immune function, suggesting that the relationship between cuticle pigmentation and immune function in insects was not a direct link. Additional possible factors that are associated with the immune response, such as life‐history, developmental, physiological factors also need to be considered.     

Cuticular cycle and molting hormone levels during the metamorphosis of Tenebrio molitor (Insecta Coleoptera)

The cuticular cycle of Tenebrio molitor (apolysis, synthesis of outer and inner epicuticle, fibrous cuticle deposition) was studied during the last larval and pupal stages by electron microscopy. Concurrently, molting hormone (MH) titers in the hemolymph were determined by a radioimmunoassay method. It appears, both in larvae and in pupae, that the MH peak coincides with the initiation of pre-ecdysial cuticle deposition (i.e., outer epicuticle synthesis). Thus MH is involved in the induction of cuticular synthesis; however, its role in inducing larval-pupal apolysis is questionable. We note that this peculiar apolysis occurs long before MH release.     

Hormonal control of pupal coloration in the painted lady butterfly Vanessa cardui

Pupae of the painted lady butterfly Vanessa cardui exhibit pupal color polyphenism consisting of white, dark and intermediate types. We investigated environmental factors affecting pupal coloration and the physiological mechanisms underlying the control of pupal color polyphenism in this species. Over 80% of larvae reared at 16 °C developed into pupae of dark types, whereas over 82% of larvae at 32 °C developed into pupae of white types irrespective of long/short-day photoperiod conditions. When mature larvae reared at 32 °C were ligatured between thoracic and abdominal parts at three different pharate pupal stages, all of the head-thoracic parts developed into white pupae regardless of pupal stage, but all abdominal parts ligatured at the early pharate pupal stage only developed into dark pupae. These results indicate that temperature during larval stages is an important element affecting pupal coloration as an environmental cue in V. cardui, and that a factor(s) inducing white pupae is released from head-thoracic parts under conditions of high temperature. Additionally, when ligatured abdomens destined to develop into dark pupae were treated with crude extracts prepared from the central nervous system, all of the ligatured abdomens developed into white pupae at a level dependent on dose and pupal stage. These results suggest that the factor inducing white pupae is a key molecule controlling pupal color polyphenism in V. cardui.     

The initial analysis of a serine proteinase gene (<Emphasis Type="Italic">AccSp10</Emphasis>) from <Emphasis Type="Italic">Apis cerana cerana</Emphasis>: possible involvement in pupal development,innate immunity and abiotic stress responses

Serine proteinases play important roles in innate immunity and insect development. We isolated a serine proteinase gene, designated AccSp10, from the Chinese honeybees (Apis cerana cerana). RT-qPCR and a Western blot analysis at different pupal development stages indicated that AccSp10 might be involved in melanin formation in pupae and promote pupal development. In adult workers, the expression of AccSp10 was upregulated by treatments mimicking harmful environments such as the presence of Bacillus bombysepticus, different temperatures (4, 24 and 42 °C), HgCl₂, H₂O₂ and paraquat; the exception was treatment with VC (vitamin C), which did not upregulate AccSp10 expression. Western blot confirmed the results. A disc diffusion assay indicated that recombinant AccSp10 accelerated E. coli cell death during stimulation with harmful substances (HgCl₂, paraquat and cumene hydroperoxide). These findings suggest that AccSp10 may be involved in the pupal development of Chinese honeybees and protection against microorganisms and abiotic harms.     

Conserved roles of Osiris genes in insect development,polymorphism and protection

Much of the variation among insects is derived from the different ways that chitin has been moulded to form rigid structures, both internal and external. In this study, we identify a highly conserved expression pattern in an insect‐only gene family, the Osiris genes, that is essential for development, but also plays a significant role in phenotypic plasticity and in immunity/toxicity responses. The majority of Osiris genes exist in a highly syntenic cluster, and the cluster itself appears to have arisen very early in the evolution of insects. We used developmental gene expression in the fruit fly, Drosophila melanogaster, the bumble bee, Bombus terrestris, the harvester ant, Pogonomyrmex barbatus, and the wood ant, Formica exsecta, to compare patterns of Osiris gene expression both during development and between alternate caste phenotypes in the polymorphic social insects. Developmental gene expression of Osiris genes is highly conserved across species and correlated with gene location and evolutionary history. The social insect castes are highly divergent in pupal Osiris gene expression. Sets of co‐expressed genes that include Osiris genes are enriched in gene ontology terms related to chitin/cuticle and peptidase activity. Osiris genes are essential for cuticle formation in both embryos and pupae, and genes co‐expressed with Osiris genes affect wing development. Additionally, Osiris genes and those co‐expressed seem to play a conserved role in insect toxicology defences and digestion. Given their role in development, plasticity, and protection, we propose that the Osiris genes play a central role in insect adaptive evolution.     

Immunochemical evidence for the transport of haemolymph protein into the cuticle of Manduca sexta

Larvae and pupae of Manduca sexta were utilized to determine whether haemolymph proteins can traverse the epidermal cell and enter the newly deposited pupal cuticle in an unaltered state. The proteins examined were those that function in carrying dopamine (or a dopamine metabolite) from the haemolymph into the cuticle. Radiotracer studies and electrophoretic analysis suggested that the haemolymph carrier proteins were indeed transported into the cuticle. Antibodies against the haemolymph carrier proteins reacted with proteins extracted from the cuticle. Further work demonstrated that proteins extracted from the cuticle are immunologically similar to the haemolymph carrier proteins.     

Identification of a cuticle protein with unique repeated motifs in the silkworm,Bombyx mori

The insect cuticle is non-cellular matrix secreted from a monolayer of epidermal cells. After abrasion of the larval cuticle of the silkworm, Bombyx mori, a protein with molecular mass of 135 kDa is newly detected in the cuticle. Mass spectrometric analysis of the tryptic fragments from this protein revealed that the 135-kDa protein is encoded by the Cb10 gene. In the predicted amino acid sequence of Cb10, three repeated motifs with [YxGGFGGppG(L/V)L] sequence are found in the C-terminal region. In addition to the repeated motifs, Cb10 has seventeen CxxxxC motifs randomly distributed throughout the polypeptide chain and serine rich region at the N-terminal region. The Cb10 gene is strongly expressed in epidermal cells after pupal ecdysis, and its expression in the larval epidermal cells is induced not only by cuticular abrasion, but also by bacterial infection. These expression patterns suggest some specific roles of this protein in pupal cuticle formation and defense reactions.     

Expression and characteristic of the Cu/Zn superoxide dismutase gene from the insect parasitizing fungus Cordyceps militaris

A Cu/Zn-superoxide dismutase (SOD) gene was characterized from Cordycepes militaris by gene cloning, heterogeneous expression and function analysis. This 154-aa SOD (CmSOD) was deduced from a 465-bp gene cloned, showing 72–95?% sequence identity to Cu/Zn-SODs from other fungi. The deduced amino acid sequence of the cDNA is highly similar to Beauveria bassiana (95?%), Isaria tenuipes (94?%) and Claviceps purpurea (88?%), respectively. The SOD gene of C. militaris spin 589?bp and consisted of two introns and three exons. The CmSOD coding region sequence was inserted into plasmid pQE-30 in order to construct prokaryotic expression vector, then transformed into Escherichia coli M15 cells for expression, and a mass of rCmSOD was obtained by IPTG induction. The enzyme activity of the purified rCmSOD was approximately 714.48 U/mg after the assay. The study provided a way for in-depth research on the expression and regulation of the CmSOD, and the molecular mechanism of anti-oxidative effect in C. militaris.     

Cloning and expression analysis of the chitinase gene Ifu-chit2 from Isaria fumosorosea

Entomopathogenic fungi can produce a series of chitinases, some of which function synergistically with proteases and other hydrolytic enzymes to degrade the insect cuticle. In the present study, the chitinase gene Ifu-chit2 from Isaria fumosorosea was investigated. The Ifu-chit2 gene is 1,435-bp long, interrupted by three short introns, and encodes a predicted protein of 423 amino acids with a 22 residue signal peptide. The predicted Ifu-Chit2 protein is highly homologous to Beauveria bassiana chitinase Bbchit2 and belongs to the glycohydrolase family 18. Ifu-Chit2 was expressed in Escherichia coli to verify chitinase activity, and the recombinant enzyme exhibited activity with a colloidal chitin substrate. Furthermore, the expression profiles of Ifu-chit2 were analyzed at different induction times under in vivo conditions. Quantitative real-time PCR analysis revealed that Ifu-chit2 expression peaked at two days post-induction. The expression of chitinase Ifu-chit2 in vivo suggests that the chitinase may play a role in the early stage of pathogenesis.