Population Density Affects Drosophila Male Pheromones in Laboratory-Acclimated and Natural Lines.

In large groups of vertebrates and invertebrates, aggregation can affect biological characters such as gene expression, physiological, immunological and behavioral responses. The insect cuticle is covered with hydrocarbons (cuticular hydrocarbons; CHCs) which reduce dehydration and increase protection against xenobiotics. Drosophila melanogaster and D. simulans flies also use some of their CHCs as contact pheromones. In these two sibling species, males also produce the volatile pheromone 11-cis-Vaccenyl acetate (cVa). To investigate the effect of insect density on the production of CHCs and cVa we compared the level of these male pheromones in groups of different sizes. These compounds were measured in six lines acclimated for many generations in our laboratory - four wild-type and one CHC mutant D. melanogaster lines plus one D. simulans line. Increasing the group size substantially changed pheromone amounts only in the four D. melanogaster wild-type lines. To evaluate the role of laboratory acclimation in this effect, we measured density-dependent pheromonal production in 21 lines caught in nature after 1, 12 and 25 generations in the laboratory. These lines showed varied effects which rarely persisted across generations. Although increasing group size often affected pheromone production in laboratory-established and freshly-caught D. melanogaster lines, this effect was not linear, suggesting complex determinants.

Excess Dally-like Induces Malformation of Drosophila Legs.

Glypicans are closely associated with organ development and tumorigenesis in animals. Dally-like (Dlp), a membrane-bound glypican, plays pivotal roles in various biological processes in Drosophila. In this study, we observed that an excess of Dlp led to the malformation of legs, particularly affecting the distal part. Accordingly, the leg disc was shrunken and frequently exhibited aberrant morphology. In addition, elevated Dlp levels induced ectopic cell death with no apparent cell proliferation changes. Furthermore, Dlp overexpression in the posterior compartment significantly altered Wingless (Wg) distribution. We observed a marked expansion of Wg distribution within the posterior compartment, accompanied by a corresponding decrease in the anterior compartment. It appears that excess Dlp guides Wg to diffuse to cells with higher Dlp levels. In addition, the distal-less (dll) gene, which is crucial for leg patterning, was up-regulated significantly. Notably, dachshund (dac) and homothorax (hth) expression, also essential for leg patterning and development, only appeared to be negligibly affected. Based on these findings, we speculate that excess Dlp may contribute to malformations of the distal leg region of Drosophila, possibly through its influence on Wg distribution, dll expression and induced cell death. Our research advances the understanding of Dlp function in Drosophila leg development.

Syntaxin5 is essential for survival by ensuring midgut epithelial homeostsis and regulating feeding in Locusta migratoria.

Syntaxin5 (Syx5) belongs to SNAREs family, which play important roles in fusion of vesicles to target membranes. Most of what we know about functions of Syx5 originates from studies in fungal or vertebrate cells, how Syx5 operates during the development of insects is poorly understood. In this study, we investigated the role of LmSyx5 in the gut development of the hemimetabolous insect Locusta migratoria. LmSyx5 was expressed in many tissues, with higher levels in the gut. Knockdown of LmSyx5 by RNA interference (RNAi) considerably suppressed feeding in both nymphs and adults. The dsLmSyx5-injected locusts lost body weight and finally died at a mortality of 100%. Furthermore, hematoxylin-eosin staining indicated that the midgut is deformed in dsLmSyx5-treated nymphs and the brush border in midgut epithelial cells is severely damaged, suggesting that LmSyx5 is involved in morphogenesis of the midgut. TEM further showed that the endoplasmic reticulum of midgut cells have a bloated appearance. Taken together, these results suggest that LmSyx5 is essential for midgut epithelial homeostsis that affects growth and development of L. migratoria. Thus, Syx5 is a promising RNAi target for controlling L. migratoria, and even other pests.

Extra-corporeal detoxification in insects.

Upon uptake of toxins, insects launch a detoxification program. This program is deployed in multiple organs and cells to raise their tolerance against the toxin. The molecular mechanisms of this program inside the insect body have been studied and understood in detail. Here, we report on a yet unexplored extra-corporeal detoxification of insecticides in Drosophila melanogaster. Wild-type D. melanogaster incubated with DDT, a contact insecticide, in a closed environment died as expected. However, incubation of a second cohort in the same environment after removal of the dead flies was not lethal. The effect was significantly lower if the flies of the two cohorts were unrelated. Incubation assays with Chlorpyrifos, another contact insecticide, yielded identical results, while incubation assays with Chlorantraniliprole, again a contact insecticide, was toxic for the second cohort of flies. A cohort of flies incubated in a DDT environment after an initial incubation of a honeybee survived treatment. Together, our data suggest that insects including Apis mellifera and D. melanogaster have the capacity to modify their proximate environment. Consequently, in their ecological niche, following individuals might be saved from intoxication thereby facilitating colonisation of an attractive site.

Role of CYP311A1 in wing development of Drosophila melanogaster.

Lipid homeostasis is crucial for growth and development of organisms. Several cytochrome P450 monooxygenases (CYPs) are involved in lipid metabolism. The function of Cyp311a1 in the anterior midgut as a regulator of phosphatidylethanolamine (PE) metabolism in Drosophila melanogaster has been demonstrated, as depletion of Cyp311a1 caused larval growth arrest that was partially rescued by supplying PE. In this study, we investigated the role of CYP311A1 in wing morphogenesis in Drosophila. Using the GAL4-UAS system, Cyp311a1 was selectively knocked down in the wing disc. A deformed wing phenotype was observed in flies with reduced Cyp311a1 transcripts. BODIPY and oil red O staining revealed a reduction of neutral lipids in the wing disc after the depletion of Cyp311a1. In addition, we observed an enhanced sensitivity to Eosin Y penetration in the wings of Cyp311a1 knocked-down flies. Moreover, the reduction of CYP311A1 function in developing wings does not affect cell proliferation and apoptosis, but entails disordered Phalloidin or Cadherin distribution, suggesting an abnormal cell morphology and cell cortex structure in wing epithelial cells. Taken together, our results suggest that Cyp311a1 is needed for wing morphogenesis by participating in lipid assembly and cell homeostasis.

RNA interference-mediated silencing of coat protein II (COPII) genes affects the gut homeostasis and cuticle development in Locusta migratoria.

The coat protein II (COPII) complex consists of five primary soluble proteins, namely the small GTP-binding protein Sar1, the inner coat Sec23/Sec24 heterodimers, and the outer coat Sec13/Sec31 heterotetramers. COPII is essential for cellular protein and lipid trafficking through cargo sorting and vesicle formation at the endoplasmic reticulum. However, the roles of COPII assembly genes remain unknown in insects. In present study, we identified five COPII assembly genes (LmSar1, LmSec23, LmSec24, LmSec13 and LmSec31) in Locusta migratoria. RT-qPCR results revealed that these genes showed different expression patterns in multiple tissues and developmental days of fifth-instar nymphs. Injection of double-stranded RNA against each LmCOPII gene induced a high RNAi efficiency, and considerably suppressed feeding, and increased mortality to 100 %. Results from the micro-sectioning and hematoxylin-eosin staining of midguts showed that the brush border was severely damaged and the number of columnar cells was significantly reduced in dsLmCOPII-injected nymphs, as compared with the control. The dilated endoplasmic reticulum phenotype of columnar cells was observed by transmission electron microscopy. RT-qPCR results further indicated that silencing any of the five genes responsible for COPII complex assembly repressed the expression of genes involved in insulin/mTOR-associated nutritional pathway. Therefore, COPII assembly genes could be promising RNAi targets for insect pest management by disrupting gut and cuticle development.

Determination of the larval precursor configuration of the Drosophila adult hindgut by G-TRACE analysis.

The Drosophila hindgut is a classical model to study organogenesis. The adult hindgut originates from the precursor cells in the larval hindgut. However, the territory of these cells has still not been well determined. A ring of wingless (wg)-expressing cells lies at the anterior zone of both the larval and adult hindgut. The larval Wg ring was thought as a portion of precursor of the adult hindgut. By applying a cell lineage tracing tool (G-TRACE), we demonstrate that larval wg-expressing cells have no cell lineage contribution to the adult hindgut. Additionally, adult Wg ring cells do not divide and move posteriorly to replenish the hindgut tissue. Instead, we determine that the precursors of the adult pylorus and ileum are situated in the cubitus interruptus (ci)-expressing cells in the anterior zone, and deduce that the precursor stem cells of the adult rectum locate in the trunk region of the larval pylorus including hedgehog (hh)-expressing cells. Together, this research advances our understanding of cell lineage origins and the development of the Drosophila hindgut.

Spatio-temporal distribution and genetic background of elastic proteins inside the chitin/chitosan matrix of insects including their functional significance for locomotion.

In insects, cuticle proteins interact with chitin and chitosan of the exoskeleton forming crystalline, amorphic or composite material structures. The biochemical and mechanical composition of the structure defines the cuticle's physical properties and thus how the insect cuticle behaves under mechanical stress. The tissue-specific ratio between chitin and chitosan and its pattern of deacetylation are recognized and interpreted by cuticle proteins depending on their local position in the body. Despite previous research, the assembly of the cuticle composites in time and space including its functional impact is widely unexplored. This review is devoted to the genetics underlying the temporal and spatial distribution of elastic proteins and the potential function of elastic proteins in insects with a focus on Resilin in the fruit fly Drosophila. The potential impact and function of localized patches of elastic proteins is discussed for movements in leg joints, locomotion and damage resistance of the cuticle. We conclude that an interdisciplinary research approach serves as an integral example for the molecular mechanisms of generation and interpretation of the chitin/chitosan matrix, not only in Drosophila but also in other arthropod species, and might help to synthesize artificial material composites.

Dynamics of cuticle-associated transcript profiles during moulting of the bed bug Cimexlectularius.

The bed bug Cimex lectularius is a worldwide human pest. The sequenced genome allows molecular analyses of all aspects of bed bug biology. The present work was conducted to contribute to bed bug cuticle biology. As in other insect species, the C. lectularius cuticle consists of the three horizontal layers procuticle, epicuticle and envelope. To analyse the genes needed for the establishment of the stratified cuticle, we studied the expression pattern of 42 key cuticle-related genes at the transition of the penultimate nymphal stage to adult animals when a new cuticle is formed. Based on gene expression dynamics, in simplified model, we distinguish two key events during cuticle renewal in C. lectularius. First, upon blood feeding, modulation of ecdysone signalling culminates in the transcriptional activation of the transcription factor Clec-Ftz-F1 that possibly controls the expression of 32 of the 42 genes tested. Second, timed expression of Clec-Ftz-F1 seems to depend also on the insulin signalling pathway as RNA interference against transcripts of the insulin receptor delays Clec-Ftz-F1 expression and stage transition. An important observation of our transcript survey is that genes needed for the construction of the three cuticle layers are largely expressed simultaneously. Based on these data, we hypothesise a considerable synchronous mechanism of layer formation rather than a strictly sequential one. Together, this work provides a basis for functional analyses of cuticle formation in C. lectularius.

Osiris17 is essential for stable integrin localization and function during insect wing epithelia remodeling.

The dynamic adhesion between cells and their extracellular matrix is essential for the development and function of organs. During insect wing development, two epithelial sheets contact each other at their basal sites through the interaction of ßPS integrins with the extracellular matrix. We report that Osiris17 contributes to the maintenance of ßPS integrins localization and function in developing wing of Drosophila and locust. In flies with reduced Osiris17 expression the epithelia sheets fail to maintain the integrity of basal cytoplasmic junctional bridges and basal adhesion. In contrast to the continuous basal integrin localization in control wings, this localization is disrupted during late stages of wing development in Osiris17 depleted flies. In addition, the subcellular localization revealed that Osiris17 co-localizes with the endosomal markers Rab5 and Rab11. This observation suggests an involvement of Osiris17 in endosomal recycling of integrins. Indeed, Osiris17 depletion reduced the numbers of Rab5 and Rab11 positive endosomes. Moreover, overexpression of Osiris17 increased co-localization of Rab5 and ßPS integrins and partially rescued the detachment phenotype in flies with reduced ßPS integrins. Taken together, our data suggest that Osiris17 is an endosome related protein that contributes to epithelial remodeling and morphogenesis by assisting basal integrins localization in insects.

Pri peptides temporally coordinate transcriptional programs during epidermal differentiation.

To achieve a highly differentiated state, cells undergo multiple transcriptional processes whose coordination and timing are not well understood. In Drosophila embryonic epidermal cells, polished-rice (Pri) smORF peptides act as temporal mediators of ecdysone to activate a transcriptional program leading to cell shape remodeling. Here, we show that the ecdysone/Pri axis concomitantly represses the transcription of a large subset of cuticle genes to ensure proper differentiation of the insect exoskeleton. The repression relies on the transcription factor Ken and persists for several days throughout early larval stages, during which a soft cuticle allows larval crawling. The onset of these cuticle genes normally awaits the end of larval stages when the rigid pupal case assembles, and their premature expression triggers abnormal sclerotization of the larval cuticle. These results uncovered a temporal switch to set up distinct structures of cuticles adapted to the animal lifestyle and which might be involved in the evolutionary history of insects.

Colorimetric surface lipid quantification in Drosophila.

Insects are covered with free neutral cuticular hydrocarbons (CHC) that may be linear, branched, and unsaturated and vary in their chain length. The CHC composition is species-specific and contributes to the adaptation of the animal to its ecological niche. Commonly, CHCs contribute substantially to the inward and outward barrier function of the cuticle and serve pheromonal communication. They are generally determined by gas-chromatography, a time-consuming method requiring detailed expertize, but it is not available in many laboratories. Here, we report on the establishment of a colorimetric method allowing semi-quantitative determination of unsaturated CHCs in Drosophila flies. This method is based on the in vitro reaction of vanillin with double bounds in lipid molecules in an acidic solution to generate a reddish color. We found a robust correlation between gas chromatographic and vanillin-colorimetric data on unsaturated CHCs amounts in single flies. As the role of unsaturated CHCs in the performance of insects in their environment is only partly understood, we think that this novel method would allow fast and broad analyses of this type of CHCs in insects both in the field and in laboratories and thereby contribute to a substantial improvement in the investigation of this matter.

Replenishment of Drosophila Male Pheromone After Mating.

Insect exocrine gland products can be involved in sexual communication, defense, territory labelling, aggregation and alarm. In the vinegar fly Drosophila melanogaster the ejaculatory bulb synthesizes and releases 11-cis-Vaccenyl acetate (cVa). This pheromone, transferred to the female during copulation, affects aggregation, courtship and male-male aggressive behaviors. To determine the ability of male flies to replenish their cVa levels, males of a control laboratory strain and from the desat1 pheromone-defective mutant strain were allowed to mate successively with several females. We measured mating frequency, duration and latency, the amount of cVa transferred to mated females and the residual cVa in tested males. Mating duration remained constant with multiple matings, but we found that the amount of cVa transferred to females declined with multiple matings, indicating that, over short, biologically-relevant periods, replenishment of the pheromone does not keep up with mating frequency, resulting in the transfer of varying quantities of cVa. Adult responses to cVa are affected by early developmental exposure to this pheromone; our revelation of quantitative variation in the amount of cVa transferred to females in the event of multiple matings by a male suggests variable responses to cVa shown by adults produced by such matings. This implies that the natural role of this compound may be richer than suggested by laboratory experiments that study only one mating event and its immediate behavioral or neurobiological consequences.

Spirotetramat reduces fitness of the spotted-wing Drosophila, Drosophila suzukii.

With climate change, the spotted-wing Drosophila (SWD, Drosophila suzukii) invades a great number of fruit production regions worldwide. A plethora of insecticides are being applied for management of this pest. As expected, SWD develops resistance against some potent insecticides or is rather insensitive to some others. Therefore, there is an urgent need to identify and characterize alternative insecticides to control SWD populations. Here, we have studied the effects of the orally applied inhibitor of the fatty acid synthesis pathway spirotetramat on SWD fitness with respect to fecundity and surface barrier function at different stages. In our experiments, we applied spirotetramat mixed with baker's yeast and showed that females and males were not repelled by effective concentrations of this insecticide. We found that spirotetramat, by trend, lowers egg numbers laid by fed females. These eggs rapidly desiccate, and only a few larvae hatch. Spirotetramat is lethal to larvae and reduces survival of adult flies under low-humidity conditions. Taken together, based on our data, we propose to use yeast supplemented with spirotetramat and additional SWD-specific attractants in traps in non-crop areas in order to eradicate SWD populations before they infest crop production sites.

Pb exposure causes non-linear accumulation of Pb in D. melanogaster controlled by metallothionein B and exerts ecological effects.

Pb pollution can harm human health and the ecosystem. Therefore, it is worthwhile to study the metabolic processes of heavy metals in individual bodies and their influence on ecological systems. In this work, we analyzed the genetic responses and physiological changes of D. melanogaster which took diets exposed to different doses of Pb using transcriptomic analysis, ICP-MS, and various other physiological methods. We found that the Pb accumulated in D. melanogaster in a nonlinear pattern with the increase of Pb content in food. Metallothioneins (Mtns), especially the MtnB directly affects the accumulation and excretion of metal Pb in D. melanogaster, and causes the nonlinear accumulation. Metal regulatory transcription factor-1 (MTF-1) is involved in the regulation of Pb-induced high expressions of Mtns. Furthermore, an interaction between the metal metabolism pathway and xenobiotic response pathway leads to the cross-tolerances of Pb-exposed D. melanogaster to insecticides and other toxins. The oxidative stress induced by Pb toxicity may be the bridge between them. Our findings provide a physiological and molecular genetic basis for further study of the accumulation and metabolism of Pb in D. melanogaster.

Insights into spermatogenesis in the migratory locust, Locusta migratoria (Linnaeus, 1758) (Orthoptera: Acrididae), following histological and ultrastructural features of the testis.

The migratory locust, Locusta migratoria (Linnaeus, 1758), is one of the most destructive agricultural pests globally, and this species is particularly localized in several regions of Egypt. However, so far, very little attention has been paid to the characteristics of the testes. Furthermore, spermatogenesis requires careful analysis to characterize and track developmental episodes. We thus investigated, for the first time, the histological and ultrastructural properties of the testis in L. migratoria employing a light microscope, a scanning electron microscope (SEM), and a transmission electron microscope (TEM). Our results revealed that the testis comprises several follicles, emerging with distinct outer surface wrinkle patterns for each follicle throughout the length of the follicular wall. Furthermore, histological examination of the follicles showed that each has three developmental zones. Each zone has cysts with characteristic spermatogenic elements, beginning with the spermatogonia at the distal end of each follicle and ending with the spermatozoa at the proximal end. Moreover, spermatozoa are arranged in spermatozoa bundles called spermatodesms. Overall, this research provides novel insights into the structure of the testes of L. migratoria, which will significantly contribute to formulating effective pesticides against locusts.

Serpentine and Vermiform Are Produced Autonomously to Fulfill Their Function in Drosophila Wings.

Group I chitin deacetylases (CDAs), CDA1 and CDA2, play an essential role in cuticle formation and molting in the process of insect wing development. A recent report showed that trachea are able to take up a secreted CDA1 (serpentine, serp) produced in the fat body to support normal tracheal development in the fruit fly Drosophila melanogaster. However, whether CDAs in wing tissue were produced locally or derived from the fat body remains an open question. To address this question, we applied tissue-specific RNAi against DmCDA1 (serpentine, serp) and DmCDA2 (vermiform, verm) in the fat body or the wing and analyzed the resulting phenotypes. We found that repression of serp and verm in the fat body had no effect on wing morphogenesis. RT-qPCR showed that RNAi against serp or verm in the fat body autonomously reduced their expression levels of serp or verm in the fat body but had no non-autonomous effect on the expression in wings. Furthermore, we showed that inhibition of serp or verm in the developing wing caused wing morphology and permeability deficiency. Taken together, the production of Serp and Verm in the wing was autonomous and independent of the fat body.

Expression and purification of snustorr snarlik protein from Plutella xylostella.

Snustorr snarlik (Snsl) is a type of extracellular protein essential for insect cuticle formation and insect survival, but is absent in mammals, making it a potential selective target for pest control. Here, we successfully expressed and purified the Snsl protein of Plutella xylostella in Escherichia coli. Two truncated forms of Snsl protein, Snsl 16-119 and Snsl 16-159, were expressed as a maltose-binding protein (MBP) fusion protein and purified to a purity above 90% after a five-step purification protocol. Snsl 16-119, forming stable monomer in solution, was crystallized, and the crystal was diffracted to a resolution of ∼10 Å. Snsl 16-159, forming an equilibrium between monomer and octamer in solution, was shown to form rod-shaped particles on negative staining electron-microscopy images. Our results lay a foundation for the determination of the structure of Snsl, which would improve our understanding of the molecular mechanism of cuticle formation and related pesticide resistance and provide a template for structure-based insecticide design.

Entomotherapeutic Role of Periplaneta americana Extract in Alleviating Aluminum Oxide Nanoparticles-Induced Testicular Oxidative Impairment in Migratory Locusts (Locusta migratoria) as an Ecotoxicological Model.

In this study, we shed light for the first time on the usage of migratory locusts (Locusta migratoria) as an insect model to investigate the nanotoxicological influence of aluminum oxide (Al2O3) nanoparticles at low doses on testes, and evaluate the capacity of a whole-body extract of American cockroaches (Periplaneta americana) (PAE) to attenuate Al2O3 NPs-induced toxicity. Energy dispersive X-ray microanalyzer (EDX) analysis verified the bioaccumulation of Al in testicular tissues due to its liberation from Al2O3 NPs, implying their penetration into the blood-testis barrier. Remarkably, toxicity with Al engendered disorders of antioxidant and stress biomarkers associated with substantial DNA damage and cell apoptosis. Furthermore, histopathological and ultrastructural analyses manifested significant aberrations in the testicular tissues from the group exposed to Al2O3 NPs, indicating the overproduction of reactive oxygen species (ROS). Molecular docking analysis emphasized the antioxidant capacity of some compounds derived from PAE. Thus, pretreatment with PAE counteracted the detrimental effects of Al in the testes, revealing antioxidant properties and thwarting DNA impairment and cell apoptosis. Moreover, histological and ultrastructural examinations revealed no anomalies in the testes. Overall, these findings substantiate the potential applications of PAE in preventing the testicular impairment of L. migratoria and the conceivable utilization of locusts for nanotoxicology studies.

Lipophorin receptor is required for the accumulations of cuticular hydrocarbons and ovarian neutral lipids in Locusta migratoria.

Lipophorin is the most abundant lipoprotein particle in insect hemolymph. Lipophorin receptor (LPR) is a glycoprotein that binds to the lipophorin and mediates cellular uptake and metabolism of lipids by endocytosis. However, the roles of LPR in uptake of lipids in the integument and ovary remain unknown in the migratory locust (Locusta migratoria). In present study, we characterized the molecular properties and biological roles of LmLPR in L. migratoria. The LmLPR transcript level was high in the first 2 days of the adults after eclosion, then gradually declined. LmLPR was predominately expressed in fat body, ovary and integument. Using immuno-detection methods, we revealed that LmLPR was mainly localized in the membrane of oenocytes, epidermal cells, fat body cells and follicular cells. RNAi-mediated silencing of LmLPR led to a slight decrease of the cuticle hydrocarbon contents but with little effect on the cuticular permeability. However, the neutral lipid content was significantly decreased in the ovary after RNAi against LmLPR, which led to a retarded ovarian development. Taken together, our results indicated that LmLPR is involved in the uptake and accumulation of lipids in the ovary and plays a crucial role in ovarian development in L. migratoria. Therefore, LmLPR could be a promising RNAi target for insect pest management by disrupting insect ovarian development.