Defective gut function in drop-dead mutant Drosophila

Mutation of the gene drop-dead (drd) causes adult Drosophila to die within 2 weeks of eclosion and is associated with reduced rates of defecation and increased volumes of crop contents. In the current study, we demonstrate that flies carrying the strong allele drdlwf display a reduction in the transfer of ingested food from the crop to the midgut, as measured both as a change in the steady-state distribution of food within the gut and also in the rates of crop emptying and midgut filling following a single meal. Mutant flies have abnormal triglyceride (TG) and glycogen stores over the first 4 days post-eclosion, consistent with their inability to move food into the midgut for digestion and nutrient absorption. However, the lifespan of mutants was dependent upon food presence and quality, suggesting that at least some individual flies were able to digest some food. Finally, spontaneous motility of the crop was abnormal in drdlwf flies, with the crops of mutant flies contracting significantly more rapidly than those of heterozygous controls. We therefore hypothesize that mutation of drd causes a structural or regulatory defect that inhibits the entry of food into the midgut.     

Factors controlling in vitro hatching of Vairimorpha plodiae (Microspora) spores and their infectivity to Plodia interpunctella,Heliothis virescens, and Pieris brassicae

Factors which influence the hatching of spores and proliferation of stages of the microsporidium Vairimorpha plodiae in two susceptible insects, Plodia interpunctella and Heliothis virescens, and one nonsusceptible insect, Pieris brassicae, were investigated. Spores hatched in 0.1 and 1 m KCl solutions when subjected to a change in pH, from pH 11 to pH 8. K⁺ was essential for hatching; NaCl solutions were not effective. Ca²⁺ and Mg²⁺ inhibited hatching, and calcium and magnesium chelating agents enhanced it. All three insect species had alkaline midgut contents and smooth, fragile peritrophic membranes. Spores hatched inside the midguts of all three insect species (P. interpunctella: maximum rate, 92.5%; H. virescens, 91.5%; P. brassicae, 82%). Sporoplasms were observed in the midgut epithelial and associated tracheole cells of P. brassicae. Both H. virescens and P. brassicae became infected when injected intrahemocoelically with spores.     

Mode of infection of the corn earworm (Heliothis zea) by Beauveria bassiana as revealed by scanning electron microscopy

Conidia from highly pathogenic mutants of Beauveria bassiana germinate quickly (within 18 hr) on the surface of corn earworm larvae (Heliothis zea) and immediately begin penetration of the cuticle. Enzymes produced by the penetrating hyphae degrade the cuticle since holes are formed at the point of entry. Clustering of conidia around nodules of larvae is often seen, but penetration is not restricted to such areas. Direct evidence is presented to show that conidia can also germinate inside of spiracle openings and could invade larvae by this route. Once inside the hemocoel, the fungus multiplies extensively; however, larval death occurs with only minimal breakdown of internal tissues. During mummification, outgrowths of fungal hyphae occur first and most extensively in the intersegmental regions of larvae. Conidia from mutants exhibiting low levels of pathogenicity are either significantly delayed or do not germinate on larval surfaces. When germination does occur, hyphae from such mutants do not penetrate immediately; instead, extensive surface hyphal growth, with or without eventual penetration of the integument, is evident.     

Inhibition of Metarhizium anisopliae in the alimentary tract of the eastern subterranean termite Reticulitermes flavipes

Reticulitermes flavipes workers were individually inoculated with 10,000 conidia of the entomopathogenic fungus Metarhizium anisopliae. After being kept in groups of 20 individuals for 1-6 d, histopathological approach showed that most of the inoculated conidia were groomed from the surface of the cuticle by nestmates within 24 h, and that a large number of conidia was subsequently found in different parts of the gut of the groomers. Our observations showed that, among thousands of conidia found in the termite’s gut, conidial germination never occurred in all inspected specimens, even when the conidia had the chance to bind to the surface of the cuticular lining of the gut. In addition, when termites were left for decomposition several days after death caused by an external infection of M. anisopliae, the hyphal growth was generalized in the body cavity of the cadaver, but was never observed in the lumen of the gut even 2 d post-mortem. Our observation suggests that the putative biochemicals involved in the termite’s gut defense against fungal pathogens are from multiple origins.     

The goblet cavity matrix in the larval midgut of Hyalophora cecropia

The larval midgut of the silkmoth Hyalophora cecropia was examined using scanning electron microscopy. Goblet cells were observed to contain within their cavities a matrix plug. This matrix material was extruded onto the lumen side of the epithelium when the tissue was stretched. The rôle of this matrix material in maintenance of the capacity of the midgut to transport ions in vivo and in vitro is discussed.     

Degradation of haemoglobin in Chironomus during metamorphosis

The degradation of haemoglobin (Hb) during the metamorphosis of Chironomus pallidivitatus was studied by means of spectrophotometry, disc electrophoresis, and isotopic tracer methods. Hb concentration was maximal during the late fourth instar larva and exhibited a continual decline throughout the pharate adult periods; in the mature adult Hb is almost entirely absent. Coupled with this reduction in Hb is an increase in the quantity of bile pigment (tentatively identified as bililatrenes) most probably representing products of the Hb degradation. These pigments are found in considerable quantity within the meconium at the time of adult emergence.Larval, ⁵⁹Fe-labelled, Hb injected into late fourth instar larvae was found to be most concentrated in the midgut portion of the early pharate adult. The quantity of ⁵⁹Fe-haemoglobin in the midgut of early pharate adult was at least five times the concentration found in late pharate pupae. Spectrophotometric and electrophoretic analysis of the midgut contents of untreated larvae showed that Hb was one of the major soluble proteins in the midgut at the time of pupation. This rapid intestinal uptake of Hb suggests that the intestine is involved in the mechanism for the degradation and removal of the Hb, no longer required in the adult stage, a seemingly important step in the metamorphosis of this insect.     

Phenotypic changes and the fate of digestive enzymes during induction of amber disease in larvae of the New Zealand grass grub (Costelytra zealandica)

Amber disease of the New Zealand grass grub Costelytra zealandica (Coleoptera: Scarabaeidae) is caused by ingestion of pADAP plasmid carrying isolates of Serratia entomophila or Serratia proteamaculans (Enterobacteriaceae) and causes infected larvae to cease feeding and clear their midgut to a pale amber colour where midgut serine protease activities are virtually eliminated. Using bacterial strains and mutants expressing combinations of the anti-feeding (afp) and gut clearance (sep) gene clusters from pADAP, we manipulated the disease phenotype and demonstrated directly the relationship between gene clusters, phenotype and loss of enzyme activity. Treatment with afp-expressing strains caused cessation of feeding without gut clearance where midgut protease activity was maintained at levels similar to that of healthy larvae. Treatment with strains expressing sep-genes caused gut clearance followed by a virtual elimination of trypsin and chymotrypsin titre in the midgut indicating both the loss of pre-existing enzyme from the lumen and a failure to replenish enzyme levels in this region by secretion from the epithelium. Monitoring of enzymatic activity through the alimentary tract during expression of disease showed that loss of serine protease activity in the midgut was matched by a surge of protease activity in the hindgut and frass pellets, indicating a flushing and elimination of the midgut contents. The blocking of enzyme secretion through amber disease appears to be selective as leucine aminopeptidase and α-amylase were still detected in the midgut of diseased larvae.     

Collection of highly germinative pseudochain conidia of Oidium neolycopersici from conidiophores by electrostatic attraction

A population of simultaneously germinating conidia is an ideal inoculum of the powdery mildew pathogen, Oidium neolycopersici. In conditions of no or low wind velocity, O. neolycopersici successively stacks mature conidia on conidiophores in a chain formation (pseudochain), without releasing the precedent mature conidia. These pseudochain conidia represent a perfect inoculum, in which all conidia used for inoculation germinate simultaneously. However, we found that conidia must be collected before they fall to the leaf surface, because the germination rate was lower among conidia deposited on the leaf surface. We used an electrostatic spore collector to collect the pseudochain conidia, and their high germination rate was not affected by this treatment. The spore collector consisted of an electrified insulator probe, which created an electrostatic field around its pointed tip, and attracted conidia within its electric field. The attractive force created by the probe tip was directly proportional to voltage, and was inversely proportional to the distance between the tip and a target colony on a leaf. Pseudochain conidia were successfully collected by bringing the electrified probe tip close to target colonies on leaves. In this way, conidia were collected from colonies at 3-d intervals. This effectively collected all conidia from conidiophores before they dropped to the leaf surface. A high germination rate was observed among conidia attracted to the probe tip (95.5 ± 0.6 %). Conidia were easily suspended in water with added surfactant, and retained their germination ability. These conidia were infective and produced conidia in pseudochains on conidiophores after inoculation. The electrostatic spore collection method can be used to collect conidia as they form on conidiophores, thus obtaining an inoculum population in which all of the conidia germinate simultaneously.     

Consumption of food and spatial organization of digestion in the cassava hornworm, Erinnyis ello

Fifth-instar Erinnyis ello larvae eat 2.1 times their own weight per day of Euphorbia pulcherrima leaves, with a coefficient of digestibility of 45% and an efficiency of food conversion into tissue of 25%. The food takes about 150 min to go through the gut. Midgut contents have a pH of 9.3–9.8, depending on the region. Cellulase is absent from the gut in E. ello. Significant gut hydrolase activities are found only in midgut. Amylase and trypsin occur in the midgut tissue and contents and in regurgitated material, whereas aminopeptidase, α-glucosidase, β-glucosidase and trehalase are found in major amounts in the midgut tissue, in minor amounts in the midgut contents and are absent from regurgitated material. The results support the hypothesis that digestion starts in the endoperitrophic space under the action of amylase and trypsin and is largely completed in the ectoperitrophic space through the catalytic action of several oligomer and dimer hydrolases. Involvement of a membrane-bound aminopeptidase in the terminal digestion of oligopeptides cannot, at present, be excluded. The finding that less than 7% of the total amylase and trypsin are excreted, after a time identical to the passage time of the food bolus, leads to the proposal for the existence of some mechanism by which those enzymes are recovered from the undigested food before it is excreted.     

Evolutionary relationships between aquatic anamorphs and teleomorphs: Tricladium and Varicosporium

Tricladium, with 21 accepted species, is the largest genus of aquatic hyphomycetes. It encompasses species with dematiaceous as well as mucedinaceous colonies. Conidiogenesis is thalloblastic; conidiogenous cells proliferate percurrently or sympodially. Conidia have typically two alternate primary lateral branches. Fontanospora and Variocladium are segregates of Tricladium, differing by conidial branching. Varicosporium comprises nine species, one not well known. Conidiogenesis is blastic or thalloblastic, conidiogenous cells proliferate sympodially or are determinate; conidia regularly produce primary and secondary branches and often fragment into part conidia. Molecular analyses on the 28S rDNA of 86 isolates, including 16 species of Tricladium, five species of Varicosporium, two species of Fontanospora and one species of Variocladium, place these hyphomycetes within Helotiales. Tricladium is polyphyletic and placed in six clades; Varicosporium is polyphyletic and placed in three clades; Fontanospora is polyphyletic within a single clade. Variocladium is placed with poor support as a sister taxon to Varicosporium giganteum, Hymenoscyphus scutula and Torrendiella eucalypti.     

Exploring the midgut proteome of partially fed female cattle tick (Rhipicephalus (Boophilus) microplus)

The continued development of effective anti-tick vaccines remains the most promising prospect for the control of the cattle tick, Rhipicephalus (Boophilus) microplus. A vaccine based on midgut proteins could interfere with successful tick feeding and additionally interfere with midgut developmental stages of Babesia parasites, providing opportunities for the control of both the tick and the pathogens it transmits. Midgut proteins from partially fed adult female cattle ticks were analysed using a combination of 2-DE and gel-free LC-MS/MS. Analysis of the urea-soluble protein fraction resulted in the confident identification of 105 gut proteins, while the PBS-soluble fraction yielded an additional 37 R. microplus proteins. The results show an abundance of proteins involved in mitochondrial ATP synthesis, electron transport chain, protein synthesis, chaperone, antioxidant and protein folding and transport activities in midgut tissues of adult female ticks. Among the novel products identified were clathrin-adaptor protein, which is involved in the assembly of clathrin-coated vesicles, and membrane-associated trafficking proteins such as syntaxin 6 and surfeit 4. The observations allow the formulation of hypotheses regarding midgut physiology and will serve as a basis for future vaccine development and tick-host interaction research.     

IkB genes encoded in Cotesia plutellae bracovirus suppress an antiviral response and enhance baculovirus pathogenicity against the diamondback moth, Plutella xylostella

An endoparasitoid wasp, Cotesia plutellae, parasitizes larvae of the diamondback moth, Plutella xylostella, with its symbiotic polydnavirus, C. plutellae bracovirus (CpBV). This study analyzed the role of Inhibitor-kB (IkB)-like genes encoded in CpBV in suppressing host antiviral response. Identified eight CpBV-IkBs are scattered on different viral genome segments and showed high homologies with other bracoviral IkBs in their amino acid sequences. Compared to an insect ortholog (e.g., Cactus of Drosophila melanogaster), they possessed a shorter ankyrin repeat domain without any regulatory domains. The eight CpBV-IkBs are, however, different in their promoter components and expression patterns in the parasitized host. To test their inhibitory activity on host antiviral response, a midgut response of P. xylostella against baculovirus infection was used as a model reaction. When the larvae were orally fed the virus, they exhibited melanotic responses of midgut epithelium, which increased with baculovirus dose and incubation time. Parasitized larvae exhibited a significant reduction in the midgut melanotic response, compared to nonparasitized larvae. Micro-injection of each of the four CpBV genome segments containing CpBV-IkBs into the hemocoel of nonparasitized larvae showed the gene expressions of the encoded IkBs and suppressed the midgut melanotic response in response to the baculovirus treatment. When nonparasitized larvae were orally administered with a recombinant baculovirus containing CpBV-IkB, they showed a significant reduction in midgut melanotic response and an enhanced susceptibility to the baculovirus infectivity.     

A new microsporidian species, Vairimorpha ocinarae n. sp., isolated from Ocinara lida Moore (Lepidoptera: Bombycidae) in Taiwan

A new microsporidium was isolated from Ocinara lida Moore (Lepidoptera: Bombycidae), a pest of Ficus microcarpa L. f. in Taiwan. The microsporidium produces systemic infections in O. lida larvae; the midgut epithelium, Malpighian tubules, and midgut muscle tissues were the target tissues for this isolate, and atrophied fat body tissues were found in heavily infected larvae. Two types of spores were observed, diplokaroytic spores with 11-13 coils of polar tube, and monokaryotic spores with 12 coils of the polar tube that developed within a sporophorous vesicle to form octospores. Electron-dense granules were abundant in the episporontal space of the sporophorous vesicles, and were similar to those of Vairimorpha invictae isolated from Solenopsis invicta, but different from granules or inclusions of other Vairimorpha species. Based on the phylogenetic analysis of the small subunit ribosomal DNA sequence, this isolate is unique within the Vairimorpha complex. Morphological and genetic characters showed this isolate to be a new species. It is placed in the genus Vairimorpha and is described as Vairimorpha ocinarae n. sp.     

The proteases and the protease inhibitor in the midgut of Leucophaea maderae

The caseinolytic enzymes of the midgut lumina and epithelia of Leucophaea were purified through precipitation by 60% saturated (NH₄)₂SO₄, followed by gel permeation on Sephadex G-200 and subsequent DEAE anionexchange chromatography. At least four peaks with enzyme activity were eluted from anionexchange chromatography columns. Gregarines of the midgut lumen apparently do not contribute to the caseinolytic activity within the midgut. Elution profiles of lumen and epithelial enzymes were nearly identical. The same enzymes were identified in the lumina of epithelial microsomal vesicles. This allows the conclusion that these enzymes are produced by the midgut epithelia.Practically all protease activity of the midgut was found in the posterior half, both in the lumen and epithelium. Feeding stimulated protease production primarily in the posterior midgut. The pH optimum of the proteases lay between 9.0 and 9.5 which was closely matched by the observed pH of the posterior midgut where most of the activity is seen. The anterior midgut pH was determined to be around 8.0.The anterior midgut of Leucophaea contained a heatstable protease inhibitor with characteristics of a competitive inhibitor. This inhibitor was precipitable by 60% saturated (NH₄)₂SO₄ and eluted from a Sephadex G-200 column more or less together with the proteases. From a DEAE anionexchange column it was eluted by 0.8 M NaCl, i.e. after the main portion of the proteases. The biological significance of the protease inhibitor in the anterior portion of the midgut is obscure.     

Histopathological changes of Ceroplastes japonicus infected by Lecanicillium lecanii

The infection process and pathological changes of Japanese wax scale, Ceroplastes japonicus Green, by the hyphomycete Lecanicillium lecanii (Zimmermann) Gams & Zare were investigated by light, scanning and transmission electron microscopy. The results showed that L. lecanii generally infected the wax scale by penetrating the integument. The anal area, the body margin, around the base of mouthparts and legs, over the stigmatic furrow and the area around the vulva were susceptible places, while the wax test had an inhibitory effect on L. lecanii. Within 24 h after inoculation, conidia became attached to the cuticle, and within 48 h, hyphae adhered to the integument of the scale and their tips differentiated into specialized infection pegs. Penetration of the cuticle occurred within 72 h of inoculation; the fungus caused the insect cuticle to rupture and hyphae entered the insect body through these openings. Within 72 h after inoculation, L. lecanii entered the hemocoele of the scale and formed blastospores. After 96 h, blastospores were dispersed throughout the hemolymph and completely disrupted the hemocytes, resulting in damage of the cell nucleus and agglutination of chromatin. Concomitant to colonization of the hemolymph, the internal organs and tissues, e.g., tracheae, malpighian tubules and muscle fibers, were also infected. As the infection progressed, the wax test and body changed color from white and red, respectively, to yellowish. After 144 h, the internal tissue structure was totally compromised and the insects died. After this time, new conidiophores bearing conidia were produced on the surface of the cadavers.     

A putative kinase-related protein (PKRP) from Plasmodium berghei mediates infection in the midgut and salivary glands of the mosquito

The completion of the Plasmodium (malaria) life cycle in the mosquito requires the parasite to traverse first the midgut and later the salivary gland epithelium. We have identified a putative kinase-related protein (PKRP) that is predicted to be an atypical protein kinase, which is conserved across many species of Plasmodium. The pkrp gene encodes a RNA of about 5300 nucleotides that is expressed as a 90 kDa protein in sporozoites. Targeted disruption of the pkrp gene in Plasmodium berghei, a rodent model of malaria, compromises the ability of parasites to infect different tissues within the mosquito host. Early infection of mosquito midgut is reduced by 58-71%, midgut oocyst production is reduced by 50-90% and those sporozoites that are produced are defective in their ability to invade mosquito salivary glands. Midgut sporozoites are not morphologically different from wild-type parasites by electron microscopy. Some sporozoites that emerged from oocysts were attached to the salivary glands but most were found circulating in the mosquito hemocoel. Our findings indicate that a signalling pathway involving PbPKRP regulates the level of Plasmodium infection in the mosquito midgut and salivary glands.     

Histopathological effects of Bacillus thuringiensis on the midgut of tobacco hornworm larvae (Manduca sexta): Low doses compared with fasting

The cytology and ultrastructure of the midgut cells of Manduca sexta larvae are described for untreated controls, larvae which fed on a spore preparation of Bacillus thuringiensis, and larvae which were fasted for either 24 or 48 hr. New observations on the ultrastructure of midgut cells in Manduca larvae included the finding of specialized Golgi vesicles in anteriormost columnar cells and of regular arrays of expanded rough endoplasmic reticulum in goblet cells of the posterior midgut region. The present observations reveal that the columnar cells of the midgut responded cytologically in the same way to fasting as they did to exposure to the toxic spores of B. thuringiensis. The goblet cells, however, appeared unaffected by fasting but became swollen in response to feeding of B. thuringiensis spore preparation.     

BmSUC1 is essential for glycometabolism modulation in the silkworm, Bombyx mori

Sucrose is the most commonly transported sugar in plants and is easily assimilated by insects to fulfill the requirement of physiological metabolism. BmSuc1 is a novel animal β-fructofuranosidase (β-FFase, EC 3.2.1.26)-encoding gene that was firstly cloned and identified in silkworm, Bombyx mori. BmSUC1 was presumed to play an important role in the silkworm-mulberry enzymatic adaptation system by effectively hydrolyzing sucrose absorbed from mulberry leaves. However, this has not been proved with direct evidence thus far. In this study, we investigated sucrose hydrolysis activity in the larval midgut of B. mori by inhibition tests and found that sucrase activity mainly stemmed from β-FFase, not α-glucosidase. Next, we performed shRNA-mediated transgenic RNAi to analyze the growth characteristics of silkworm larvae and variations in glycometabolism in vivo in transgenic silkworms. The results showed that in the RNAi-BmSuc1 transgenic line, larval development was delayed, and their body size was markedly reduced. Finally, the activity of several disaccharidases alone in the midgut and the sugar distribution, total sugar and glycogen in the midgut, hemolymph and fat body were then determined and compared. Our results demonstrated that silencing BmSuc1 significantly reduced glucose and apparently activated maltase and trehalase in the midgut. Together with a clear decrease in both glycogen and trehalose in the fat body, we conclude that BmSUC1 acts as an essential sucrase by directly modulating the degree of sucrose hydrolysis in the silkworm larval midgut, and insufficient sugar storage in the fat body may be responsible for larval malnutrition and abnormal petite phenotypes.     

Effects of culture media on hydrophobicity and thermotolerance of Bb and Ma conidia, with description of a novel surfactant based hydrophobicity assay

Hypocrealean entomopathogenic fungal conidia are made up of multi-aged groups given their chronological conidiogenesis. Most thermotolerance assays have been conducted using mixed-age conidia. The present work exploited a polysiloxane polyether copolymer (siloxane) (Silwet L-77®) mediated conidial collection method, validated by a hydrophobicity assay. This was done to divide mixed-age conidia into two groups based on hydrophobicity and test their thermotolerance, relying on the relationship of conidial age with hydrophobicity. Beauveria bassiana GHA and ERL1170 and Metarhizium anisopliae ERL1171 and ERL1540 conidia, produced on millet agar, whey permeate agar, and ¼SDAY were subjected to hydrophobicity assays that included data on yield of conidia/unit of surface area. Conidia were also collected using 0.01% siloxane, and those remaining with 0.08% siloxane. Hydrophobicity was correlated with percent conidia collected in the two siloxane solutions and yield, suggesting a relationship between percent conidia collected and conidial age (maturation). The conidial suspensions were exposed to 45 °C for 45 min, and conidial germination was examined. Overall, conidia which were collected in 0.08% siloxane had lower germination after heat exposure than those collected in the 0.01% solution. Conidia of both fungi produced by incubation on millet or whey permeate for 14 d were more hydrophobic and exhibited greater thermotolerance than those produced on ¼SDAY. These results suggest that conidia can be divided into two groups with different thermotolerance by using a siloxane-mediated conidial collection method based on hydrophobicity. This depends on the types of substrates used that could influence conidial maturation.     

iTRAQ-based quantitative proteomic analysis of conidia and mycelium in the filamentous fungus Metarhizium robertsii

Metarhizium robertsii is widely applied in biological control via conidia application. To clarify the proteomic differences between conidia and mycelia and explore the underlying mechanisms of conidia as a unit responsible for dispersal and environmental stress, we carried out an iTRAQ (isobaric tags for relative and absolute quantitation)-based quantitative proteomic analysis for two developmental stages from M. robertsii. A total of 2052 proteins were detected, and 90 showed differential protein abundance between the conidia and mycelia. These 90 proteins were primarily associated with stress resistance, amino acid and protein metabolism, and energy metabolism. Further bioinformatics analysis showed that these proteins could be mapped to 52 pathways, five of which were significantly enriched after mapping to KEGG pathways. Interestingly, many proteins involved in the significantly enriched pathway of peroxisome, biosynthesis of secondary metabolites and glyoxylate and dicarboxylate metabolism, including catalase, peroxisomal membrane anchor protein, formate dehydrogenase and isocitrate lyase, were identified with higher abundance in conidia. The results deepened our understanding of the conidia proteome in M. robertsii and provide a basis for further exploration for improving the efficiency of the fungi as biocontrol agents.