Larval Cuticle Research Articles

Dynein directs prophase centrosome migration to control the stem cell division axis in the developing Caenorhabditis elegans epidermis.

The microtubule motor dynein is critical for the assembly and positioning of mitotic spindles. In Caenorhabditis elegans, these dynein functions have been extensively studied in the early embryo but remain poorly explored in other developmental contexts. Here, we use a hypomorphic dynein mutant to investigate the motor's contribution to asymmetric stem cell-like divisions in the larval epidermis. Live imaging of seam cell divisions that precede formation of the seam syncytium shows that mutant cells properly assemble but frequently misorient their spindle. Misoriented divisions misplace daughter cells from the seam cell row, generate anucleate compartments due to aberrant cytokinesis, and disrupt asymmetric cell fate inheritance. Consequently, the seam becomes disorganized and populated with extra cells that have lost seam identity, leading to fatal epidermal rupture. We show that dynein orients the spindle through the cortical GOA-1Gα-LIN-5NuMA pathway by directing the migration of prophase centrosomes along the anterior-posterior axis. Spindle misorientation in the dynein mutant can be partially rescued by elongating cells, implying that dynein-dependent force generation and cell shape jointly promote correct asymmetric division of epithelial stem cells.

Knockdown of Thitarodes host genes influences dimorphic transition of Ophiocordyceps sinensis in the host hemolymph.

The Chinese cordyceps, a unique parasitic complex of Thitarodes/Hepialus ghost moths and Ophiocordyceps sinensis fungus in the Tibetan Plateau, is a highly valuable biological resource for medicine and health foods in Asian countries. Efficient system for artificial cultivation of Chinese cordyceps relies on understanding the gene functions involved in the induction of growing blastospores into hyphae in the larval hemolymph of insect host, during O. sinensis infection. Transcriptome analysis and ribonucleic acid interference (RNA interference) method were employed to identify the key differentially expressed genes and to demonstrate their functions in Thitarodes xiaojinensis. Key larval genes critical for O. sinensis blastospore development or filamentation were identified. Nine of the 20 top upregulated genes encoded cuticles proteins, indicating that these proteins highly activated when the larval hemolymph was full of blastospores. Small interfering RNA (siRNA) knockdown of five larval genes such as Flightin, larval cuticle protein LCP-30, 26-hydroxylase (CYP18A1), cuticle protein 18.6, isoform B, and probable chitinase 3 significantly stimulated the dimorphic transition from blastospores to prehyphae in O. sinensis in the larval hemolymph after 120 h after injection. The expressions of these genes determined by quantitative real-time PCR were suppressed in various levels from 38.64% to 91.54%, compared to the controls. These results demonstrated that injection of the siRNAs of key upregulated genes into the larval hemolymph containing high load of blastospores caused the gene silence in T. xiaojinensis larvae and induced the fungal transition from blastospores to prehyphae, providing novel knowledge on the regulation of O. sinensis fungal dimorphism by Thitarodes host and cues for further study of Thitarodes biology and commercial cultivation of Chinese cordyceps.

The armoured cuticle of the black soldier fly Hermetia illucens

We characterise in detail the larval and pupal cuticle of the black soldier fly Hermetia illucens L. (Diptera: Stratiomyidae), a key insect species in circular economy. In particular, we focus on ultrastructure using scanning and transmission electron microscopy, material characterization and composition (elements and minerals) with confocal laser scanning microscope, energy dispersive X-ray microanalysis, powder X-ray diffraction and mechanical properties with nanoindentation measurements. Calcium carbonate crystallizes on the epicuticle as blocks of calcite in the pupal cuticle. Calcium carbonate granules are stored in two specialised Malpighian tubules. CaCO3 is already present in the cuticle of young larval instars, but it is mainly in the form of amorphous calcium carbonate while the amount of calcite increases during larval development. The presence of calcite leads to cuticle hardening. Larval and pupal cuticles contain large amounts of resilin which guarantee cuticle flexibility.

Melanin pigmentation variations in the larval cuticle of almond moth, Ephestia cautella caused by gamma radiation

To determine the effects of gamma radiation on the melanization process and phenoloxidase activity, Ephestia cautella larvae were exposed to dosages of 200, 400, 600, 800, and 1000 Gy. After irradiation, the number of non melanized larvae and the number exhibiting a slight melanization usually increased. The degree of melanization in treated larvae differed significantly from untreated larvae. The amount of melanin usually decreases as the dosage increases and as time passes after the treatment. The results of the phenoloxidase assay indicate that the enzyme activity responds differently to radiation. For instance, at doses of 200, 400, and 800 Gy, the enzyme activity remained consistent in both control and irradiated larvae. However, at doses of 600 and 1000 Gy, the enzyme activity increased to 14.92 and 13.37 O.D. units, respectively, compared to 8.81 O.D. units in the control. In order to determine if irradiated larvae have been previously exposed to ionizing radiation, a quick and easy test based on phenoloxidase activity or the melanization response is presented for use in quarantine treatment. Histological changes, specifically in the pigment granules of melanin, were studied using a light microscope. Upon inspection of the unirradiated larvae, it was observed that brown melanin pigment granules were deposited in the epicuticle and exocuticle layers of the cuticle. When gamma radiation dosages were administered to larvae, it was observed that the melanin pigment gradually diminished until it vanished at the highest dose (1000 Gy).

Growth performance and dynamic copper accumulation in tissues of black soldier fly (Hermetia illucens) larvae under copper exposure

Abstract To figure out the copper metabolism features in vivo and evaluate the potential risk of copper residue in tissues of black soldier fly larvae (BSFL) and their by-products, the effects of oral copper exposure of 0, 50, 500 and 1000 mg/kg (Control, Cu50, Cu500 and Cu1000) on growth performance and dynamic copper accumulation in the fat body, cuticle of BSFL and the rearing residue were investigated in a 12-days rearing trial. The results showed that, the body weight and length, and body protein content of BSFL were improved by exposure to 50 mg/kg copper, but reduced by exposure to 500 and 1000 mg/kg copper, as well as that of the pre-pupa rate. Under the copper exposure of 500 and 1000 mg/kg, the copper concentrations in the larval fat body were high at the early developmental stage, but sharply reduced to be 8.1 ± 0.3 mg/kg within 12 days with no significant differences among groups. Similarly, the copper concentrations in the cuticle also decreased rapidly to be 46.3 ± 2.9 and 91.3 ± 9.4 mg/kg in the Cu500 and Cu1000 groups within 12 days, but they were significantly higher than those of the control (8.1 ± 0.3 mg/kg) and Cu50 (10.1 ± 1.1 mg/kg) groups. The linear regression analysis between copper exposure doses and copper concentrations in the rearing residues indicated that the latter are dose- and time-dependent. Overall, the growth performance and body protein content of BSFL were improved by low copper exposure, but reduced by high copper exposure. The oral copper could be excluded from larval fat body and cuticle rapidly, and be largely accumulated in the rearing residues. Therefore, little risk of copper residue in the main tissues of post larvae or pre-pupae of black soldier fly (BSF), but higher risk of it in the rearing residues could be anticipated. Our results provide valuable information for safety assessment of copper residues in BSF products and by-products.

Functional characterization of Bombyx mori (Lepidoptera: Bombycidae) C-type lectin 5.

C-type lectins (CTLs) are an important family of pattern recognition receptors (PRRs) that regulate immune responses. The CTL5 gene of the silkworm Bombyx mori L. (Lepidoptera: Bombycidae) encodes a protein comprised of 223 amino acids, containing a signal peptide and a carbohydrate recognition domain (CRD). Our previous study showed that CTL5 can facilitate the clearance of bacteria from larval hemocoel but the underlying mechanisms are unclear. In this study, we found that CTL5 was mainly expressed in fourth-instar larvae, adult moths, and the larval epidermis. CTL5 expression showed differential responses to both pathogenic stimuli and the molting hormone 20-hydroxyecdysone. The full-length (FL) and truncated (ΔN/ΔC/ΔNC) CTL5 recombinant proteins can bind to hemocytes, polysaccharides, bacteria, and spores of the entomopathogenic fungus Beauveria bassiana. Yeast 2-hybrid assays showed that the recombinant proteins can interact with integrin β2-β5 subunits. Recombinant proteins increased the phagocytic rate of hemocytes. Injection of recombinant CTL5 stimulated the expression of many immune genes in hemocytes, mainly antimicrobial peptides and immune signaling molecules. Additionally, transcriptomic sequencing of CTL5-stimulated hemocytes revealed 265 upregulated and 580 downregulated genes. Functional enrichment and the gene set enrichment analyses showed that differentially expressed genes were mainly enriched in innate immune responses and signaling. Our study suggests that CTL5 may act as an opsonin to enhance the clearance of pathogens by regulating both humoral and cellular responses.

Post-injury hydraulic fracturing drives fissure formation in the zebrafish basal epidermal cell layer

The skin epithelium acts as the barrier between an organism's internal and external environments. In zebrafish and other freshwater organisms, this barrier function requires withstanding a large osmotic gradient across the epidermis. Wounds breach this epithelium, causing a large disruption to the tissue microenvironment due to the mixing of isotonic interstitial fluid with the external hypotonic fresh water. Here, we show that, following acute injury, the larval zebrafish epidermis undergoes a dramatic fissuring process that resembles hydraulic fracturing, driven by the influx of external fluid. After the wound has sealed-preventing efflux of this external fluid-fissuring starts in the basal epidermal layer at the location nearest to the wound and then propagates at a constant rate through the tissue, spanning over 100μm. During this process, the outermost superficial epidermal layer remains intact. Fissuring is completely inhibited when larvae are wounded in isotonic external media, suggesting that osmotic gradients are required for fissure formation. Additionally, fissuring partially depends on myosin II activity, as myosin II inhibition reduces the distance of fissure propagation away from the wound. During and after fissuring, the basal layer forms large macropinosomes (with cross-sectional areas ranging from 1 to 10μm2). We conclude that excess external fluid entry through the wound and subsequent closure of the wound through actomyosin purse-string contraction in the superficial cell layer causes fluid pressure buildup in the extracellular space of the zebrafish epidermis. This excess fluid pressure causes tissue to fissure, and eventually the fluid is cleared through macropinocytosis.

Resistance to the larvicide temephos and altered egg and larval surfaces characterize salinity-tolerant Aedes aegypti

Aedes aegypti, the principal global vector of arboviral diseases and previously considered to oviposit and undergo preimaginal development only in fresh water, has recently been shown to be capable of developing in coastal brackish water containing up to 15 g/L salt. We investigated surface changes in eggs and larval cuticles by atomic force and scanning electron microscopy, and larval susceptibility to two widely-used larvicides, temephos and Bacillus thuringiensis, in brackish water-adapted Ae. aegypti. Compared to freshwater forms, salinity-tolerant Ae. aegypti had rougher and less elastic egg surfaces, eggs that hatched better in brackish water, rougher larval cuticle surfaces, and larvae more resistant to the organophosphate insecticide temephos. Larval cuticle and egg surface changes in salinity-tolerant Ae. aegypti are proposed to respectively contribute to the increased temephos resistance and egg hatchability in brackish water. The findings highlight the importance of extending Aedes vector larval source reduction efforts to brackish water habitats and monitoring the efficacy of larvicides in coastal areas worldwide.

The histone H4K20 methyltransferase SUV4-20H1/KMT5B is required for multiciliated cell differentiation in Xenopus.

H4 lysine 20 dimethylation (H4K20me2) is the most abundant histone modification in vertebrate chromatin. It arises from sequential methylation of unmodified histone H4 proteins by the mono-methylating enzyme PR-SET7/KMT5A, followed by conversion to the dimethylated state by SUV4-20H (KMT5B/C) enzymes. We have blocked the deposition of this mark by depleting Xenopus embryos of SUV4-20H1/H2 methyltransferases. In the larval epidermis, this results in a severe loss of cilia in multiciliated cells (MCC), a key component of mucociliary epithelia. MCC precursor cells are correctly specified, amplify centrioles, but ultimately fail in ciliogenesis because of the perturbation of cytoplasmic processes. Genome-wide transcriptome profiling reveals that SUV4-20H1/H2-depleted ectodermal explants preferentially down-regulate the expression of several hundred ciliogenic genes. Further analysis demonstrated that knockdown of SUV4-20H1 alone is sufficient to generate the MCC phenotype and that its catalytic activity is needed for axoneme formation. Overexpression of the H4K20me1-specific histone demethylase PHF8/KDM7B also rescues the ciliogenic defect in a significant manner. Taken together, this indicates that the conversion of H4K20me1 to H4K20me2 by SUV4-20H1 is critical for the formation of cilia tufts.

Gene expression and immunolocalization of chitin deacetylase BmCDA2 in silkworm

Deacetylation of chitin is closely related to insect development and metamorphosis. Chitin deacetylase (CDA) is a key enzyme in the process. However, to date, the CDAs of Bombyx mori (BmCDAs), which is a model Lepidopteran insect, were not well studied. In order to better understand the role of BmCDAs in the metamorphosis and development of silkworm, the BmCDA2 which is highly expressed in epidermis was selected to study by bioinformatics methods, protein expression purification and immunofluorescence localization. The results showed that the two mRNA splicing forms of BmCDA2, namely BmCDA2a and BmCDA2b, were highly expressed in the larval and pupal epidermis, respectively. Both genes had chitin deacetylase catalytic domain, chitin binding domain and low density lipoprotein receptor domain. Western blot showed that the BmCDA2 protein was mainly expressed in the epidermis. Moreover, fluorescence immunolocalization showed that BmCDA2 protein gradually increased and accumulated with the formation of larval new epidermis, suggesting that BmCDA2 may be involved in the formation or assembly of larval new epidermis. The results increased our understandings to the biological functions of BmCDAs, and may facilitate the CDA study of other insects.

Genome-Wide Identification and Transcriptome-Based Expression Profile of Cuticular Protein Genes in Antheraea pernyi.

Antheraea pernyi is one of the most famous edible and silk-producing wild silkworms of Saturniidae. Structural cuticular proteins (CPs) are the primary component of insect cuticle. In this paper, the CPs in the genome of A. pernyi were identified and compared with those of the lepidopteran model species Bombyx mori, and expression patterns were analyzed based on the transcriptomic data from the larval epidermis/integument (epidermis in the following) and some non-epidermis tissues/organs of two silkworm species. A total of 217 CPs was identified in the A. pernyi genome, a comparable number to B. mori (236 CPs), with CPLCP and CPG families being the main contribution to the number difference between two silkworm species. We found more RR-2 genes expressed in the larval epidermis of fifth instar of A. pernyi than B. mori, but less RR-2 genes expressed in the prothoracic gland of A. pernyi than B. mori, which suggests that the hardness difference in the larval epidermis and prothoracic gland between the two species may be caused by the number of RR-2 genes expressed. We also revealed that, in B. mori, the number of CP genes expressed in the corpus allatum and prothoracic gland of fifth instar was higher than that in the larval epidermis. Our work provided an overall framework for functional research into the CP genes of Saturniidae.

Antixenosis of Thodan (AAB), a resistant Musa cultivar to Odoiporus longicollis (Olivier) and characterization of larvicide molecules

Banana pseudostem borer (BPB) Odoiporus longicollis (Olivier) (Coleoptera, Curculionidae) is a serious pest of Musa cultivars. Experimental maintenance of larvae in the live pseudostem of cultivar Thodan, a resistant AAB Musa cultivar has resulted antixenosis followed by death of larvae within a week. Antixenosis was characterized by significant decrease of total hemocytes and sharp changes on the proportionate distribution of different types of hemocytes in larvae. As the number of plasmatocytes, prohemocytes, splenocytes and adipohemocytes decreased the number of granulocytes and oenocytes increased. Antixenosis also caused accumulation of 20-hydroxyecdysone (20E) and significant inhibition on the activities of trypsin like serine protease (TlSP) and phenoloxidase (PO). Phytochemical analysis of Thodan resulted characterization of three larvicides such as Betulinic acid (BA), Stigmasterol-3-O-glucoside (SOG) and Sulfoquinovosyl diacyl glycerol (SQDG), and the content in the pseudostem ranged 0.0027 to 0.007 per cent. All the three larvicides were highly toxic to the larvae with LD50 of 0.38 ppm for SOG, 0.41 ppm for SQDG and 0.83 ppm for BA. Simultaneous action of three larvicides in the live pseudostem resulted resistance in Thodan against infestation by BPB. Susceptible Nendran showed negligibly low content of SOG (0.0011%) and SQDG (0.0013%) but no detectable quantity of BA. Intoxication by all the three larvicides caused significant changes on the proportionate distribution of hemocytes, accumulation of 20-hydroxy ecdysone (20E) and inhibition of TlSP and PO, the enzymes involved in larval metamorphosis and cuticle sclerotisation. This study demonstrated that resistance of Thodan against O. longicollis is due to adverse effect of these larvicides on endocrine system, cuticle development and cytotoxicity of hemocytes. As these larvicide molecules are stable compounds, there is scope for them to be used as substitutes in place of deleterious insecticides for the management BPB.

Nuclear receptor FTZ-F1 is required for larval-pupal molting by regulating ecdysteroidogenesis and chitin metabolism in Lasioderma serricorne

Fushi tarazu factor 1 (FTZ-F1) is a typical nuclear receptor transcription factor that plays crucial roles during the molting process in insects. However, the role of FTZ-F1 in Lasioderma serricorne, an important stored insect pest, remains unclear. In this study, the FTZ-F1 gene from L. serricorne was identified and cloned. The role of FTZ-F1 in regulating ecdysteroidogenesis and chitin metabolism was analyzed by RNA interference (RNAi) during larval–pupal transition of L. serricorne. The expression of LsFTZ-F1 varied during development and peaked in the final stage of fourth-instar larvae. The LsFTZ-F1 was mainly expressed in larval epidermis. The expression of LsFTZ-F1 was induced by 20-hydroxyecdysone (20E), but it was suppressed by silencing of ecdysone receptor genes LsEcR and LsUSP. RNAi-mediated knockdown of LsFTZ-F1 in the fourth-instar larvae significantly decreased 20E titers and the expression of 20E synthesis and signaling pathway genes. Suppression of LsFTZ-F1 disturbed the larval–pupal transition and resulted in 100% death, which could be rescued by supplementary 20E. The chitin contents of L. serricorne larvae treated with dsLsFTZ-F1 were decreased, as was the expression of chitin synthesis and degrading genes. Hematoxylin and eosin staining analysis indicated that LsFTZ-F1 depletion prevented the larval–pupal molt by inhibiting the synthesis of new cuticle. These results indicate that LsFTZ-F1 is critical for chitin metabolism and ecdysone synthesis during the larva to pupa molt, and that it is a promising RNAi target for L. serricorne control.

Poly(ADP-ribosyl)ating enzymes cooperate to coordinate development

The transcriptome is subject to rapid and massive changes during the transition between developmental stages. These changes require tight control to avoid the undesired reactivation of gene expression that is only important for previous developmental stages and, if unchecked during transition between developmental stages, could lead to anarchic proliferation and formation of malignant tumors. In this context, the involvement of chromatin factors is important since they can directly regulate the expression of multiple genes at the same time. Poly(ADP-ribose) enzymes, involved in several processes from DNA repair to transcription regulation, might play a role in this regulation. Here, we report that PARP-1 and PARG cooperate to temporally regulate the gene expression profile during the larval/pupa transition. PARP-1 and PARG are both essential in repressing the expression of genes coding for digestive enzymes and larval cuticle proteins, while PARG positively regulate the expression of defense response genes. These results suggest a cooperative coordination between PARP-1 and PARG that specifically maintains the integrity of expression profile between developmental stages.

A sigma class glutathione S-transferase gene regulated by the CncC pathway is required for phytochemical tolerance in the red flour beetle, Tribolium castaneum

Insect glutathione S-transferases (GSTs) play a crucial role in the detoxification of exogenous compounds, especially insecticides and plant allelochemicals. A sigma class GST gene, TcGSTS7, mediates the response to eugenol in Tribolium castaneum. However, the mechanism underlying this effect remains largely unknown. In this study, TcGSTS7, which exhibits a structural motif and domain organization characteristic of GSTs, was cloned from the T. castaneum genome. Spatiotemporal expression analysis revealed that TcGSTS7 was most highly expressed at the late larva stage and was mainly expressed in the fat body and epidermis of larvae and adults, suggesting that TcGSTS7 may play a potential role in the protection against toxic xenobiotics in T. castaneum. Furthermore, the expression of TcGSTS7 was significantly induced after exposure to eugenol, while RNA interference (RNAi) targeting TcGSTS7 enhanced the sensitivity of the beetle to eugenol, indicating that TcGSTS7 is involved in the tolerance of T. castaneum to this insecticide. Interestingly, the depletion of TcCncC, which encodes a transcription factor of the CncC pathway that has been associated with the regulation of detoxification-related genes in insects, led to a reduction in the TcGSTS7 transcript level following exposure to eugenol, which suggests that TcGSTS7 acts downstream of the CncC pathway. Combined, these results indicated that TcGSTS7 participates in the tolerance of T. castaneum to phytochemicals in a CncC pathway-dependent manner. These findings have implications for the development of novel drugs for use in pest control.

Mutation of a lepidopteran-specific PMP-like protein, BmLSPMP-like, induces a stick body shape in silkworm, Bombyx mori.

Lepidoptera is one of the largest orders of insects, some of which are major pests of crops and forests. The cuticles of lepidopteran pests play important roles in defense against insecticides and pathogens, and are indispensable for constructing and maintaining extracellular structures and locomotion during their life cycle. Lepidopteran-specific cuticular proteins could be potential targets for lepidopteran pest control. But information on this is limited. Our research aimed to screen the lepidopteran-specific cuticular proteins using the lepidopteran model, the silkworm, to explore the molecular mechanism underlying the involvement of cuticular proteins in body shape construction. Positional cloning showed that BmLSPMP-like, a gene encoding a lepidopteran-specific peritrophic matrix protein (PMP) like protein which includes a peritrophin A-type chitin-binding domain (CBM_14), is responsible for the stick (sk) mutation. BmLSPMP-like is an evolutionarily conserved gene that exhibits synteny in Lepidoptera and underwent purifying selection during evolution. Expression profiles demonstrated that BmLSPMP-like is expressed in chitin-forming tissues, testis and ovary, and accumulates in the cuticle. BmLSPMP-like knockout, generated with CRISPR/Cas9, resulted in a stick-like larval body shape phenotype. Over-expression of BmLSPMP-like in the sk mutant rescued its abnormal body shape. The results showed that BmLSPMP-like may be involved in assemblage in the larval cuticle. Our results suggested that the dysfunction of BmLSPMP-like may result in a stick body shape phenotype in silkworm, through the regulation of the arrangement of the chitinous laminae and cuticle thickness. Our study provides new evidence of the effects of LSPMP-likes on lepidopteran body shape formation, metamorphosis and mortality, which could be an eco-friendly target for lepidopteran pest management. © 2022 Society of Chemical Industry.

Editor's Choice: October 2022

Parasitoid venom alters the lipid composition and development of microorganisms on the wax moth cuticle – V. Y. Kryukov, E. I. Chernyak, N. Kryukova, M. Tyurin, A. Krivopalov, O. Yaroslavtseva, I. Senderskiy, O. Polenogova, E. Zhirakovskaia, V. V. Glupov & S. V. Morozov (https://doi.org/10.1111/eea.13219). Parasitoid venom alters the lipid composition and development of microorganisms on the wax moth cuticle – V. Y. Kryukov, E. I. Chernyak, N. Kryukova, M. Tyurin, A. Krivopalov, O. Yaroslavtseva, I. Senderskiy, O. Polenogova, E. Zhirakovskaia, V. V. Glupov & S. V. Morozov (https://doi.org/10.1111/eea.13219) Parasitoids are in a continuous arms race with their host for survival. This battle has led to numerous evolutionary innovations, including the recruitment of additional parties, such as the host microbiome. Kryukov and colleagues demonstrate that parasitization by Habrobracon brevicornis (Hymenoptera: Braconidae) leads to differences in the microbial composition of larvae of the wax moth, Galleria mellonella (Lepidoptera: Pyralidae). They envision that the parasitoid's venom alters the lipid composition of the host larval cuticle, which increases its susceptibility to infection, thereby putting an extra challenge on the host immune system, which in turn may increase the survival chance of the parasitoid's offspring. This study is an intriguing example of the intricate interactions between parasitoids and their hosts.

The insecticidal potential of Bacillus velezensis CE 100 against Dasineura jujubifolia Jiao & Bu (Diptera: Cecidomyiidae) larvae infestation and its role in the enhancement of yield and fruit quality of jujube (Zizyphus jujuba Miller var. inermis Rehder)

Jujube (Zizyphus jujuba Miller var. inermis Rehder) is highly susceptible to jujube gall midge (Dasineura jujubifolia Jiao & Bu) larvae infestation, which causes rolled leaf damage, leaf galls, discoloration, and a decline in fruit yield. In this study, we demonstrate the larvicidal potential of cuticle-degrading chitinase and protease produced by Bacillus velezensis CE 100 against jujube gall midge larvae and the plant growth-promoting effect on jujube fruit yield. The laboratory insecticidal assay involving bacterial broth culture and crude enzymes of B. velezensis CE 100 revealed that both treatments caused gall midge larvae mortality rates of up to 85.5% and 96.7%, respectively. Both treatments caused severe degradation of the larvae cuticle, such as deformation of the exoskeleton and loss of sensilla. Foliar application of the bacterial broth culture in the jujube field reduced the rate of rolled leaf damage by 3.8-fold more than that in the control group. In addition, B. velezensis CE 100 produced indole-3-acetic acid (IAA) up to 3.5 mg/mL, which improved root development, and exhibited the potential for nitrogen-fixation and phosphate solubilization to improve nutrient availability and uptake. Compared to the control, applying the bacterial treatment in the jujube field increased the leaf chlorophyll content by 1.3-fold and improved the fruit yield by 2.4-fold. For the first time, we demonstrate the dual larvicidal and bio-stimulation potential of entomopathogenic B. velezensis CE 100 in managing gall midge larvae and the improvement of jujube fruit production, respectively.

Abnormal overexpression of SoxD enhances melanin synthesis in the Ursa mutant of Bombyx mori

The pigment and structural color of insects play crucial roles in body protection, ecological adaptation, and signal communication. Epidermal melanization is a common and main coloring pattern, which results in broad phenotypic diversity. Melanin is one of the compounds contributing to dark brown-black pigmentation, which is synthesized from dopamine and tyrosine by the melanin metabolism pathway. The Ursa mutant of the silkworm Bombyx mori is a body-color mutant characterized by excessive melanin pigmentation in the larval epidermis. However, the exact gene responsible for this phenotype remains unclear. Here, we performed positional cloning of the gene responsible for Ursa, which was mapped to an 83-kb region on chromosome 14. The genomic region contains a protein-coding gene encoding a transcription factor, which was designated BmSoxD. The mutation site was determined by analysis of nucleotide sequences of the genomic region corresponding to BmSoxD, which identified a 449-bp transposable sequence similar to that of the B. mori transposon Helitron inserted into the sixth intron. BmSoxD was dramatically overexpressed in the epidermis of Ursa at the end of the molting stage compared with that of wild-type B. mori. Overexpression of BmSoxD led to upregulation of genes involved in the melanin metabolism pathway, whereas knocking down BmSoxD via small interfering RNAs blocked melanin pigment production in the larval epidermis. These data indicate that the mutation in BmSoxD is responsible for the Ursa mutant phenotype. We propose that the transposable sequence insertion causes abnormal overexpression of BmSoxD at the molting stage in the Ursa mutant, resulting in excessive melanin synthesis and its accumulation in epidermal cells.

The invasion process of the entomopathogenic fungus Ophiocordyceps sinensis into the larvae of ghost moths (Thitarodes xiaojinensis) using a GFP-labeled strain.

Chinese cordyceps is a well-known and valuable traditional Chinese medicine that forms after Ophiocordyceps sinensis parasitizes ghost moth larvae. The low natural infection rate of O. sinensis limits large-scale artificial cultivation of Chinese cordyceps, and the invasion process is unclear. To investigate the temporal and spatial regulation when O. sinensis enters ghost moths, we constructed an O. sinensis transformant that stably expresses green fluorescent protein (GFP). Inoculating Thitarodes xiaojinensis larvae with a high concentration of GFP-labeled O. sinensis, we observed that O. sinensis conidia could adhere to the host cuticle within 2 days, germinate penetration pegs within 4 days, and produce blastospores in the host hemocoel within 6 days. The reconstructed three-dimensional (3D) structures of the invasion sites showed that penetration pegs germinated directly from O. sinensis conidia at the joining site with the larval cuticle. Differentiated appressoria or hyphae along the host epicuticle are not required for O. sinensis to invade ghost moths. Overall, the specific invasion process of O. sinensis into its host is clarified, and we provided a new perspective on the invasion process of entomopathogenic fungi.