Larval Cuticle Research Articles

Alpha-amylase as the culprit in an occupational mealworm allergy case

BackgroundOccupational allergy has been described in employees working in contact with mealworms in pet stores, live fish bait or infested stored grains and recently, in mealworm farming for animal feed and human consumption. Mealworm allergens linked to occupational allergy are troponin C, cockroach-like allergen, tropomyosin, arginine kinase, early-staged encapsulation inducing- and larval cuticle proteins.ObjectiveWe report a case of occupational mealworm allergy and studied the culprit component.MethodsDiagnosis was done by skin prick, specific IgE, basophil activation and lung function testing. Allergen purification was performed by anion-exchange chromatography and immunoblotting with patient IgE. Allergens were identified by in-gel trypsin digest and tandem mass spectrometry. Allergenicity and specificity further confirmed by IgE inhibition and passive basophil activation experiments.ResultsWe describe a new case of occupational mealworm allergy in a laboratory worker, with sensitization to different developmental stages and derivates of the mealworm. In basophil activation tests, the majority of patient's basophils (69%–91%) degranulated upon stimulation with the lowest concentration of mealworm extracts (0.16 µg/ml). Despite strong sensitization to mites, the patient did not show cross-reactivity to other insects. We were able to identify alpha-amylase as the main allergen and through inhibition experiments, we demonstrated that low amounts (0.1 µg/ml) of this allergen could strongly inhibit mealworm specific IgE by 79.1%. Moreover, passive BAT experiments demonstrated the IgE-alpha-amylase interaction to be functional, inducing up to 25.5% degranulation in healthy donor basophils.ConclusionAlpha-amylase can be identified as the responsible allergen in this specific case of occupational mealworm allergy.

Parasitoid venom alters the lipid composition and development of microorganisms on the wax moth cuticle

AbstractEctoparasitoids may change host metabolism, making the environment more favorable for the development of their larvae. These alterations may also be suitable for growth and proliferation of commensal microorganisms and pathogens. This is especially important in relation to cuticular microbiota, with which ectoparasitoids interact directly. However, biochemical and microbiological changes occurring on insect cuticles in response to parasitoid venoms are insufficiently understood. We assayed the biochemical and microbiological changes in cuticles of larvae of the wax moth,Galleria mellonella(L.) (Lepidoptera: Pyralidae), after envenomation byHabrobracon brevicornis(Wesmael) (Hymenoptera: Braconidae).In particular, lipid composition in the epicuticle, total nitrogen (N) and carbon (C) amounts in the cuticle, bacterial colony‐forming unit (CFU) counts, and bacterial community structure (16S rRNA gene‐based metagenomics) on the insect surface were analyzed. The susceptibility of wax moth larvae to the entomopathogenic fungus (EPF)Metarhizium robertsiiJ.F. Bisch., S.A. Rehner & Humber (Hypocreales: Clavicipitaceae) was also assessed.Envenomation led to an increase in the hydrocarbon amount, as well as alterations in the fatty acid composition of the epicuticle; in particular, a large decrease in the amount of ω‐1 hydroxy fatty acids. The total N and C amounts in the cuticle also slightly increased after envenomation. These changes were correlated with a decrease in bacterial diversity and an increase inEnterococcusabundance on the surface of envenomated larvae. Envenomation also led to a substantial increase in larval susceptibility toM. robertsiiinfection; differences between the LC50values of envenomated and control larvae were 505 000‐fold. We suggest that the hyper‐lipidation of the cuticle is related to processes occurring in the host’s hemocoel after envenomation by the parasitoid. The effects of the changes in the lipid composition on the proliferation and development of microorganisms are discussed.

Insecticidal efficacy of nanomaterials used to control mosquito, Culex quinquefasciatus Say, 1823 with special reference to their hepatotoxicity in rats.

The present study aimed to develop a novel methodology for controlling the mosquito larvae using different nanoparticles, with special reference to their effect on rats (a non-target mammalian model). The mosquito species of Culex quinquefasciatus was reared in the laboratory. Chitosan, silver nanoparticles and their combination as well as lavender (Lavandula officinalis) nanoemulsion with different concentrations were tested as biological insecticides against the mosquito larvae. Mammalian toxicity of the used nanoparticles were evaluated using 27 adult male rats, experimental rats were divided into 9 equal groups (n=3). The nanoparticles were added to the drinking water for 30 days. At the end of the study, blood and tissue samples were collected to assess the levels of the serum alanine aminotransferase and aspartate aminotransferase, different genes expression as interleukin 6 (IL-6) and IL-1β activity. Histopathological and immunohistochemical studies using two markers (TNF-α and BAX expression) were also applied. The LC50 and LC90 were recorded for each tested nanoparticles, and also the changes of the treated mosquito larvae cuticle were assessed using the scanning electron microscopy. Green nanoemulsion (Lavandula officinalis) was more effective than metal (silver) or even biodegradable (chitosan) nanoparticles in controlling of Culex quiquefasciatus mosquito larvae, and also it proved its safety by evaluation of the mammalian hepatotoxicity of the tested nanoparticles.

Taxonomic significance of microlepides on subimaginal tarsi of Ephemeroptera.

In each mayfly individual, the surface of the subimaginal cuticle significantly differs both from imaginal and larval cuticles, being different on different parts of the body. Most of the subimaginal body, including femora and tibiae, is covered with microtrichia. Tarsal segments are either also covered with microtrichia, or their microtrichia are transformed into microlepides of various shapes. The most usual forms of microlepides are the pointed and the blunt ones. Arrangements of microtrichia, pointed microlepides, blunt microlepides and other forms of microlepides on certain tarsomeres represent good taxonomic characters, which in some cases allow characterization of high level taxa, and in some cases distinguish closely related species. Arrangement of microlepides and microtrichia on subimaginal tarsi of examined mayfly species is given. The following new synonyms are proposed: Afroptilum boettgeri (Kopelke 1980) = Xyrodromeus africanus Lugo-Ortiz McCafferty 1997, syn. n.; Labiobaetis vinosus (Barnard 1932) = L. tenuicrinitus (Kopelke 1980), syn. n.; Baetis parvulus Crass 1947 = Baetis permultus Kopelke 1980, syn. n.; Epeorus gilliesi Braasch 1981 = Epeorus petersi Sivaruban et al. 2013, syn. n.; Dicercomyzon femorale Demoulin 1954 = Dicercomyzon costale Kimmins 1957, syn. n.

Ultrasound-assisted nanoemulsion of Trachyspermum ammi essential oil and its constituent thymol on toxicity and biochemical aspect of Aedes aegypti.

Aedes aegypti is the main vector of yellow fever, chikungunya, Zika, and dengue worldwide and is managed by using chemical insecticides. Though effective, their indiscriminate use brings in associated problems on safety to non-target and the environment. This supports the use of plant-based essential oil (EO) formulations as they are safe to use with limited effect on non-target organisms. Quick volatility and degradation of EO are a hurdle in its use; the present study attempts to develop nanoemulsions (NE) of Trachyspermum ammi EO and its constituent thymol using Tween 80 as surfactant by ultrasonication method. The NE of EO had droplet size ranging from 65 ± 0.7 to 83 ± 0.09nm and a poly dispersity index (PDI) value of 0.18 ± 0.003 to 0.20 ± 0.07 from 1 to 60days of storage. The NE of thymol showed a droplet size ranging from 167 ± 1 to 230 ± 1nm and PDI value of 0.30 ± 0.03 to 0.40 ± 0.008 from 1 to 60days of storage. The droplet shape of both NEs appeared spherical under a transmission electron microscope (TEM). The larvicidal effect of NEs of EO and thymol was better than BEs (Bulk emulsion) of EO and thymol against Ae. aegypti. Among the NEs, thymol (LC50 34.89ppm) had better larvicidal action than EO (LC50 46.73ppm). Exposure to NEs of EO and thymol causes the shrinkage of the larval cuticle and inhibited the acetylcholinesterase (AChE) activity in Ae. aegypti. Our findings show the enhanced effect of NEs over BEs which facilitate its use as an alternative control measure for Ae. aegypti.

Ultrastructural analysis of beetle larva cuticles during infection with the entomopathogenic fungus, Beauveria bassiana.

Beauveria bassiana is one of the commercially available entomopathogenic fungi (EPF), and a number of isolates with high virulence and broad host spectrum have been used to control agricultural and forest pests. Although the functional importance of genes in EPFs' pathogenesis have been extensively studied, the precise ultrastructural mechanism of the fungal infection, particularly penetration of the host insect cuticles, is not well understood. In this study, we investigated the morphology and ultrastructure of the larval cuticle of the red flour beetle, Tribolium castaneum, after treatment with B. bassiana ERL1170 expressing an enhanced green fluorescent protein (Bb-eGFP). The Bb-eGFP showed high virulence against the larvae, with approximately 90% mortality at 48 h after treatment (HAT) and 100% at 72 HAT under our infection conditions. In these larvae, the regions of the body wall with flexible cuticles, such as the ventral and ventrolateral thorax and abdomen, became darkly melanized, but there was little to no melanization in the rigid dorsal cuticular structures. Confocal microscopy and transmission electron microscopy (TEM) indicated that germinated conidia on the surface of the larval cuticle were evident at 6 HAT, which formed penetration pegs and began to penetrate the several cuticle layers/laminae by 12 HAT. The penetration pegs then developed invading hyphae, some of which passed through the cuticle and reached the epidermal cells by 24 HAT. The larval cuticle was aggressively and extensively disrupted by 48 HAT, and a number of outgrowing hyphae were observed at 72 HAT. Our results indicate that Bb-eGFP is capable of infection and penetrating T. castaneum larvae shortly after inoculation (~24 HAT) at the body regions with apparently flexible and membranous cuticles, such as the ventral intersegmental regions and the ventrolateral pleura. This study provides details on the histopathogenesis of the host cuticle by infection and penetration of EPFs, which can facilitate the management of insect pests. © 2022 Society of Chemical Industry.

Silencing tyrosine hydroxylase or dopa decarboxylase gene disrupts cuticle tanning during larva-pupa-adult transformation in Henosepilachna vigintioctopunctata.

The 28-spotted potato ladybird, Henosepilachna vigintioctopunctata, is a notorious defoliator of many solanaceous and cucurbitaceous plants. Tyrosine hydroxylase (TH) and dopa decarboxylase (DDC) are responsible for cuticle tanning pathway in insects. We identified HvTH and HvDDC in H. vigintioctopunctata, and found that high levels of them were accumulated just before or right after molting. Injection of dsHvTH or feeding 3-iodo-tyrosine (3-IT) at the third instar larval stage repressed tanning of the larval cuticle, reduced larval feeding, inhibited larval growth, and consequently caused 100% of larval mortality. Knockdown of HvDDC at the third instar larval stage hardly affected the coloration of larval head, and partially inhibited pigmentation of larval bodies and around 80% of the HvDDC RNAi larvae developed into albino pupae and adults. Moreover, depletion of HvTH or HvDDC at the fourth instar larval stage resulted in albino pupae and adults. The HvTH or HvDDC hypomorph adults fully or partially failed to remove the larval/pupal exuviae, possessed pale and abnormal wings, and poorly tanned heads and bodies, and eventually, struggled for several days without feeding on leaves before death. These results show that TH and DDC play key roles in larval and adult cuticle tanning and development in H. vigintioctopunctata. Also, these findings suggest that dopa- and dopamine-originated pigments are essential for larval and adult feeding behavior and the molting process during emergence. © 2022 Society of Chemical Industry.

Octanoic Acid-An Insecticidal Metabolite of Conidiobolus coronatus (Entomopthorales) That Affects Two Majors Antifungal Protection Systems in Galleria mellonella (Lepidoptera): Cuticular Lipids and Hemocytes.

The food flavour additive octanoic acid (C8:0) is also a metabolite of the entomopathogenic fungus Conidiobolus coronatus, which efficiently infects and rapidly kills Galleria mellonella. GC-MS analysis confirmed the presence of C8:0 in insecticidal fraction FR3 extracted from C. coronatus filtrate. Topical administration of C8:0 had a dose-dependent effect on survival rates of larvae but not on pupation or adult eclosion times of the survivors. Topically applied C8:0 was more toxic to adults than larvae (LD100 for adults 18.33 ± 2.49 vs. 33.56 ± 2.57 µg/mg of body mass for larvae). The administration of C8:0 on the cuticle of larvae and adults, in amounts corresponding to their LD50 and LD100 doses, had a considerable impact on the two main defense systems engaged in protecting against pathogens, causing serious changes in the developmental-stage-specific profiles of free fatty acids (FFAs) covering the cuticle of larvae and adults and damaging larval hemocytes. In vitro cultures of G. mellonella hemocytes, either directly treated with C8:0 or taken from C8:0 treated larvae, revealed deformation of hemocytes, disordered networking, late apoptosis, and necrosis, as well as caspase 1–9 activation and elevation of 8-OHdG level. C8:0 was also confirmed to have a cytotoxic effect on the SF-9 insect cell line, as determined by WST-1 and LDH tests.

A Blueprint of Microstructures and Stage-Specific Transcriptome Dynamics of Cuticle Formation in Bombyx mori.

Insect cuticle is critical for the environmental adaptability and insecticide resistance of insects. However, there is no clear understanding of the structure and protein components of the cuticle during each developmental stage of holometabolous insects, and knowledge about the protein components within each layer is vague. We conducted serial sectioning, cuticular structure analysis, and transcriptome sequencing of the larval, pupal, and adult cuticles of Bombyx mori. The deposition processes of epicuticle, exocuticle, and endocuticle during larval, pupal, and adult cuticle formation were similar. Transcriptome analysis showed that these cuticle formations share 74% of the expressed cuticular protein (CP) genes and 20 other structural protein genes, such as larval serum protein and prisilkin. There are seven, six, and eleven stage-specific expressed CP genes in larval, pupal, and adult cuticles, respectively. The types and levels of CP genes may be the key determinants of the properties of each cuticular layer. For example, the CPs of the RR-2 protein family with high contents of histidine (His) are more essential for the exocuticle. Functional analysis suggested that BmorCPAP1-H is involved in cuticle formation. This study not only offers an in-depth understanding of cuticle morphology and protein components but also facilitates the elucidation of molecular mechanisms underlying cuticle formation in future studies.

Autophagy–mediated plasma membrane removal promotes the formation of epithelial syncytia

Epithelial wound healing in Drosophila involves the formation of multinucleate cells surrounding the wound. We show that autophagy, a cellular degradation process often deployed in stress responses, is required for the formation of a multinucleated syncytium during wound healing, and that autophagosomes that appear near the wound edge acquire plasma membrane markers. In addition, uncontrolled autophagy in the unwounded epidermis leads to the degradation of endo‐membranes and the lateral plasma membrane, while apical and basal membranes and epithelial barrier function remain intact. Proper functioning of TORC1 is needed to prevent destruction of the larval epidermis by autophagy, in a process that depends on phagophore initiation and expansion but does not require autophagosomes fusion with lysosomes. Autophagy induction can also affect other sub‐cellular membranes, as shown by its suppression of experimentally induced laminopathy‐like nuclear defects. Our findings reveal a function for TORC1‐mediated regulation of autophagy in maintaining membrane integrity and homeostasis in the epidermis and during wound healing.

ITRAQ Proteomic Analysis of Interactions Between 20E and Phospholipase C in Apolygus lucorum (Meyer-Dür).

Polyphagous Apolygus lucorum has become the dominant insect in Bacillus thuringiensis (Bt) cotton fields. Hormone 20-hydroxyecdysone (20E) regulates multiple insect development and physiology events. 20E responses are controlled by pathways triggered by phospholipase C (PLC)-associated proteins. However, 20E-modulated genes and related proteins that can be affected by PLC still remain unknown. Here, isobaric tag for relative and absolute quantitation (iTRAQ) and immunoblotting techniques were used to compare differentially expressed proteins (DEPs) in A. lucorum in response to the treatment of 20E and the PLC inhibitor U73122 as well as their combination. A total of 1,624 non-redundant proteins and 97, 248, 266 DEPs were identified in the 20E/control, U73122/control, and 20E + U73122/control groups, respectively. Only 8 DEPs, including pathogenesis-related protein 5-like, cuticle protein 19.8, trans-sialidase, larval cuticle protein A2B-like, cathepsin L1, hemolymph juvenile hormone-binding protein, ATP-dependent RNA helicase p62-like, and myosin-9 isoform X1, were detected in all three groups. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that the DEPs were involved in diverse signaling pathways. The results were validated by immunoblotting, which highlighted the reliability of proteomics analysis. These findings provided novel insights into the function of PLC in 20E signaling pathway in A. lucorum.

Aldehyde oxidases mediate plant toxicant susceptibility and fecundity in the red flour beetle, Tribolium castaneum

Aldehyde oxidases (AOXs) are a group of metabolic enzymes that play critical roles in the degradation of xenobiotics and chemicals. However, the physiological function of this enzyme in insects remains poorly understood. In this study, three TcAOX genes (TcAOX1, TcAOX2, TcAOX3) were identified and characterized from Tribolium castaneum genome. Spatiotemporal expression profiling showed that TcAOX1 expression was most highly expressed at the early pupal stage and was predominantly expressed in the antennae of adults, indicating that TcAOX1 was involved in the degradation of chemical signals; TcAOX2 expression was most highly expressed at the late pupal stage and was mainly expressed in the fat body, epidermis of larvae and adults, respectively; and TcAOX3 expression was in all stages and was primarily expressed in the head of adults. Moreover, the transcripts of TcAOX2 and TcAOX3 were significantly induced after exposure to plant oil, and RNA interference (RNAi) targeting of each of them enhanced the susceptibility of beetles to this plant toxicant, suggesting that these two genes are associated with plant toxicant detoxification. Intriguingly, knockdown of the TcAOX1 led to reductions in female egg-laying but unchanged the hatchability and the development of genital organs, suggesting that this gene may mediate fecundity by effecting the inactivation of chemical signals in T. castaneum. Overall, these results shed new light on the function of AOX genes in insects, and could facilitate the development of research on pest control management.

Annona (Annonaceae) by-products derivatives: Toxicity to the European pepper moth and histological assessment

Obtaining derivatives with insecticidal properties from abundant plant biomass (waste) represents a good opportunity to transform environmental problems into innovative solutions for pest management programs. In the present study, we evaluated the toxicity of the aqueous emulsions of the ethanolic extract of Annona mucosa and Annona sylvatica (Annonaceae) seeds on the European pepper moth Duponchelia fovealis (Lepidoptera: Crambidae), a major pest of strawberry plants. In addition, we evaluated anti-feeding and developmental inhibitory effects, as well as macro and microscopic damage to the excretory and nervous systems of European pepper moth larvae. In the contact bioassay, the aqueous emulsion of A. mucosa extract provided the fastest action on larvae with LT50 of 6.10 h, followed by chlorfenapyr (Pirate®) (LT50 = 8.2 h) and the aqueous emulsion of the A. sylvatica extract (LT50 = 10.3 h). In contrast, the commercial bioinsecticide based on acetogenins (Anosom® 1 EC) had a higher LT50 (32.8 h). In addition to the lethal effects, the aqueous emulsions tested caused significant phagodeterrence (>90%) and inhibited the development of D. fovealis. Macroscopic observation revealed the presence of edemas as well as the darkening on the surface of the larvae's cuticle. Histological analyses detected alterations. Our results suggest that aqueous emulsions produced from ethanolic extract of A. mucosa and A. sylvatica seeds, a residue in fruit processing industrial complexes (pulp factories), are alternatives for the management of D. fovealis, especially in organic and/or ecological strawberry production systems.

Jumu is required for the activation of JAK/STAT in Drosophila lymph gland development and epidermal wounds

The regulatory mechanism of hematopoiesis and innate immunity in Drosophila is highly similar to that in mammals, and Drosophila has become a suitable model to understand vertebrate hematopoiesis and the immune response. JAK-STAT signaling pathway components are widely conserved during evolution, and contribute to hematopoiesis and multiple tissue damage and immune responses. Here, we demonstrate that Stat92E is widely expressed in the lymph gland, and the loss of jumu inhibits the maintenance of the JAK/STAT pathway in the CZ and MZ but not in the PSC of the lymph gland. Furthermore, we found that clean puncture wounding of the larval epidermis can lead to the activation of JAK/STAT signaling and the generation of lamellocytes, and Jumu is required for the activation of JAK/STAT in response to epidermal wounds.

Yellow-b, -c, -d, and -h are required for normal body coloration of Henosepilachna vigintioctopunctata.

The involvement of yellow genes y-b, y-c, y-e, and y-h in cuticle tanning has poorly been clarified. In the present paper, six putative yellow (y-y, y-b, y-c, y-e y-f, and y-h) genes were identified in Henosepilachna vigintioctopunctata. Hvy-b, Hvy-c, Hvy-e, and Hvy-h were abundantly transcribed at early larval and late pupal stages, especially in the epidermis. Accordingly, RNA interference (RNAi) experiments were performed by an injection of dsy-b, dsy-c, dsy-e, or dsy-h into the second instar larvae and 1-day-old pupae. The head capsule, scoli and strumae, and legs in the fourth-instar larvae became blacker; the blackish spots in the pupae were darkened and widened after RNAi of Hvy-b, compared with those of dsegfp-treated controls. Depletion of Hvy-b at the 1-day-old pupal stage expanded two pair of black markings on the sternum of the metathorax, and darkened the black patched on the sterna of the abdomen segments I-VI in the resultant adults. Depletion of Hvy-e caused darker pigmented adult body and elytral cuticles than those of dsegfp-introduced controls. However, there was no obvious difference in pigmentation of the black markings. Hvy-h-deficient larvae displayed dark yellow body color, whereas the body color of the dsegfp-injected control was pale yellow. There was no obvious difference in coloration of larval specific-black markings or pupal cuticle between dsHvy-h- and dsegfp-treated animals. Moreover, silence of Hvy-c at the second instar larval stage lightened black markings in the resulting larvae and pupae, but had no influence on pale yellow body color. Our results demonstrated their different roles of the four yellow genes during body pigmentation: HvY-b and HvY-c, respectively, inhibit and facilitate the coloration within dark markings, whereas HvY-e and HvY-h, respectively, repress the pigmentation in adult and larval body cuticles outside the black patches in H. vigintioctopunctata.

Pvr and distinct downstream signaling factors are required for hemocyte spreading and epidermal wound closure at Drosophila larval wound sites.

Tissue injury is typically accompanied by inflammation. In Drosophila melanogaster larvae, wound-induced inflammation involves adhesive capture of hemocytes at the wound surface followed by hemocyte spreading to assume a flat, lamellar morphology. The factors that mediate this cell spreading at the wound site are not known. Here, we discover a role for the platelet-derived growth factor/vascular endothelial growth factor-related receptor (Pvr) and its ligand, Pvf1, in blood cell spreading at the wound site. Pvr and Pvf1 are required for spreading in vivo and in an in vitro spreading assay where spreading can be directly induced by Pvf1 application or by constitutive Pvr activation. In an effort to identify factors that act downstream of Pvr, we performed a genetic screen in which select candidates were tested to determine if they could suppress the lethality of Pvr overexpression in the larval epidermis. Some of the suppressors identified are required for epidermal wound closure (WC), another Pvr-mediated wound response, some are required for hemocyte spreading in vitro, and some are required for both. One of the downstream factors, Mask, is also required for efficient wound-induced hemocyte spreading in vivo. Our data reveal that Pvr signaling is required for wound responses in hemocytes (cell spreading) and defines distinct downstream signaling factors that are required for either epidermal WC or hemocyte spreading.

Comprehensive characterization of internal and cuticle surface microbiota of laboratory-reared F1Anopheles albimanus originating from different sites

BackgroundResearch on mosquito-microbe interactions may lead to new tools for mosquito and mosquito-borne disease control. To date, such research has largely utilized laboratory-reared mosquitoes that typically lack the microbial diversity of wild populations. A logical progression in this area involves working under controlled settings using field-collected mosquitoes or, in most cases, their progeny. Thus, an understanding of how laboratory colonization affects the assemblage of mosquito microbiota would aid in advancing mosquito microbiome studies and their applications beyond laboratory settings.MethodsUsing high throughput 16S rRNA amplicon sequencing, the internal and cuticle surface microbiota of F1 progeny of wild-caught adult Anopheles albimanus from four locations in Guatemala were characterized. A total of 132 late instar larvae and 135 2–5 day-old, non-blood-fed virgin adult females that were reared under identical laboratory conditions, were pooled (3 individuals/pool) and analysed.ResultsResults showed location-associated heterogeneity in both F1 larval internal (p = 0.001; pseudo-F = 9.53) and cuticle surface (p = 0.001; pseudo-F = 8.51) microbiota, and only F1 adult cuticle surface (p = 0.001; pseudo-F = 4.5) microbiota, with a more homogenous adult internal microbiota (p = 0.12; pseudo-F = 1.6) across collection sites. Overall, ASVs assigned to Leucobacter, Thorsellia, Chryseobacterium and uncharacterized Enterobacteriaceae, dominated F1 larval internal microbiota, while Acidovorax, Paucibacter, and uncharacterized Comamonadaceae, dominated the larval cuticle surface. F1 adults comprised a less diverse microbiota compared to larvae, with ASVs assigned to the genus Asaia dominating both internal and cuticle surface microbiota, and constituting at least 70% of taxa in each microbial niche.ConclusionsThese results suggest that location-specific heterogeneity in filed mosquito microbiota can be transferred to F1 progeny under normal laboratory conditions, but this may not last beyond the F1 larval stage without adjustments to maintain field-derived microbiota. These findings provide the first comprehensive characterization of laboratory-colonized F1An. albimanus progeny from field-derived mothers. This provides a background for studying how parentage and environmental conditions differentially or concomitantly affect mosquito microbiome composition, and how this can be exploited in advancing mosquito microbiome studies and their applications beyond laboratory settings.

A metalloproteinase Tsdpy31 from Trichinella spiralis participates in larval molting and development

Trichinellosis is a serious food-borne zoonotic parasitic disease with global distribution, causing serious harm to public health and food safety. Molting is prerequisite for intestinal larval development in the life cycle of T. spiralis. Metalloproteinases play an important role in the molting process of T. spiralis intestinal infective larvae (IIL). In this study, the metalloproteinase Tsdpy31 was cloned, expressed and characterized. The results revealed that the Tsdpy31 was expressed at various T. spiralis stages and it was principally located in cuticle, hypodermis and embryos of the nematode. Recombinant Tsdpy31 (rTsdpy31) had the catalytic activity of natural metalloproteinase. Silencing of Tsdpy31 increased the permeability of larval new cuticle. When the mice were orally challenged with dsRNA treated- muscle larvae, the burden of intestinal adult and muscle larvae in Tsdpy31 dsRNA treatment group was significantly reduced, compared with the control green fluorescent protein (GFP) dsRNA and PBS groups (P < 0.05). Tsdpy31 may play a major role in the new cuticle synthesis and old cuticle shedding. Tsdpy31 also participates in T. spiralis embryonic development. We conclude that Tsdpy31 could be a candidate vaccine target molecule against intestinal T. spiralis ecdysis and development.

Effect of Tagetes minuta oil on larval morphology of Plutella xylostella through scanning electron microscopy and mechanism of action by enzyme assay

The larvae of Plutella xylostella treated with Tagetes minuta oil were processed for SEM and detoxification enzyme activities. In treated larvae, reduction in the length of the setae (15.93 ± 3.46 to 30.59 ± 3.35 µm) was observed as compared to untreated setae. The cuticular setae were clubbed, swollen, and shaft of the hair was broken after 72 h of treatment and then hair socket was fragmented the bloated setae. Similarly, the larval cuticle, thoracic and abdominal legs after treatment were also presented. Higher concentration of T. minuta oil significantly inhibited the GST and AChE activity in P. xylostella.

Neonicotinoids can cause arrested pupal ecdysis in Lepidoptera

Recently, we reported a novel mode of action in monarch butterfly (Danaus plexippus) larvae exposed to neonicotinoid insecticides: arrest in pupal ecdysis following successful larval ecdysis. In this paper, we explore arrested pupal ecdysis in greater detail and propose adverse outcome pathways to explain how neonicotinoids cause this effect. Using imidacloprid as a model compound, we determined that final-instar monarchs, corn earworms (Helicoverpa zea), and wax moths (Galleria mellonella) showed high susceptibility to arrested pupal ecdysis while painted ladies (Vanessa cardui) and red admirals (Vanessa atalanta) showed low susceptibility. Fall armyworms (Spodoptera frugiperda) and European corn borers (Ostrinia nubilalis) were recalcitrant. All larvae with arrested ecdysis developed pupal cuticle, but with incomplete shedding of larval cuticle and unexpanded pupal appendages; corn earworm larvae successfully developed into adults with unexpanded appendages. Delayed initiation of pupal ecdysis was also observed with treated larvae. Imidacloprid exposure was required at least 26 h prior to pupal ecdysis to disrupt the molt. These observations suggest neonicotinoids may disrupt the function of crustacean cardioactive peptide (CCAP) neurons, either by directly acting on their nicotinic acetylcholine receptors or by acting on receptors of inhibitory neurons that regulate CCAP activity.