Knockdown of Vacuolar ATPase Subunit G Gene Affects Larval Survival and Impaired Pupation and Adult Emergence in Henosepilachna vigintioctopunctata

The vATPase holoenzyme consists of two functional subcomplexes, the cytoplasmic (peripheral) V1 and the membrane-embedded V0. Both V1 and V0 sectors contain eight subunits, with stoichiometry of A3B3CDE3FG3H in V1 and ac8c’c”def(Voa1p) in V0 respectively. However, the function of G subunit has not been characterized in any non-Drosophilid insect species. In the present paper, we uncovered that HvvATPaseG was actively transcribed from embryo to adult in a Coleopteran pest Henosepilachna vigintioctopunctata. Its mRNA levels peaked in larval hindgut and Malpighian tubules. RNA interference (RNAi)-mediated knockdown of HvvATPaseG significantly reduced larval feeding, affected chitin biosynthesis, destroyed midgut integrity, damaged midgut peritrophic membrane, and retarded larval growth. The function of Malpighian tubules was damaged, the contents of glucose, trehalose, lipid, total soluble amino acids and protein were lowered and the fat bodies were lessened in the HvvATPaseG RNAi larvae, compared with those in the PBS- and dsegfp-fed beetles. In contrast, the amount of glycogen was dramatically increased in the HvvATPaseG depletion ladybirds. As a result, the development was arrested, pupation was inhibited and adult emergence was impaired in the HvvATPaseG hypomorphs. Our results demonstrated that G subunit plays a critical role during larval development in H. vigintioctopunctata.

Synthesis, molecular docking and mosquitocidal efficacy of lawsone and its derivatives against the dengue vector Aedes aegypti L. (Diptera: Culicidae)

Background: Aedes aegypti is the primary dengue vector, a significant public health problem in many countries. Controlling the growth of Ae. aegypti is the biggest challenge in the mosquito control program, and there is a need for finding bioactive molecules to control Ae. aegypti in order to prevent dengue virus transmission. Objective: To assess the mosquitocidal property of lawsone and its 3-methyl-4H-chromen-3-yl-1-phenylbenzo[6,7]chromeno[2,3,c]pyrazole-dione derivatives (6a-6h) against various life stages of Ae. aegypti. Besides, to study the mode of action of the active compound by molecular docking and histopathological analysis. Methods: All derivatives were synthesized from the reaction between 2-hydroxy-1,4-naphthoquinone, chromene-3-carbaldehyde, and 1-phenyl-3-methyl-pyrazol-5-one by using one pot sequential multicomponent reaction. The mosquito life stages were subjected to diverse concentrations ranging from 1.25, 2.5, 5.0, and 10 ppm for lawsone and its derivatives. The structure of all synthesized compounds was characterized by spectroscopic analysis. Docking analysis was performed using autodock tools. Midgut sections of Ae. aegypti larvae were analyzed for histopathological effects. Results: Among the nine compounds screened, derivative 6e showed the highest mortality on Ae. aegypti life stages. The analyzed LC50 and LC90 results of derivative 6e were 3.01, 5.87 ppm, and 3.41, 6.28 ppm on larvae and pupae of Ae. aegypti, respectively. In the ovicidal assay, the derivative 6e recorded 47.2% egg mortality after 96-hour post-exposure to 10 ppm concentration. In molecular docking analysis, the derivative 6e confirmed strong binding interaction (-9.09 kcal/mol and -10.17 kcal/mol) with VAL 60 and HIS 62 of acetylcholinesterase 1 (AChE1) model and LYS 255, LYS 263 of kynurenine aminotransferase of Ae. aegypti, respectively. The histopathological results showed that the derivative 6e affected the columnar epithelial cells (CC) and peritrophic membrane (pM). Conclusion: The derivative 6e is highly effective in the life stages of Ae. aegypti mosquito and it could be used in the integrated mosquito management programme.

Comparison of the potential activities of viral and bacterial chitinases

Background Chitin, a long-chain polymer of N-acetylglucosamine, is a major structural component of the insect exoskeleton and the peritrophic membrane (PM). Chitinases are able to effectively break down glycosidic bonds of chitin polymer thus can be used in agriculture to control plant pathogen insects. These enzymes can be synthesized by higher plants, animals, protista, bacteria, and viruses. Results In this study, viral and bacterial chitinases were compared for their potential activity on a laboratory test insect. The genes encoding chitinases of Autographa californica nucleopolyhedrovirus (AcNPV) and Cydia pomonella granulovirus (CpGV) were amplified from genomic DNAs by PCR and cloned into the pET-28a (+) expression vector. The chitinase proteins of these 2 viruses (AcNPV-Chi, CpGV-Chi) and Serratia marcescens chitinase C (ChiC) protein which was previously cloned were overexpressed in Escherichia coli. Expressed proteins were purified and confirmed by western blot analysis as 50, 63, and 68 kDa for AcNPV, CpGV, and S. marcescens chitinases, respectively. Enzyme activities of the chitinases were confirmed. Chitinases were also compared to each other in silico. The insecticidal effects of these proteins were evaluated on Galleria mellonella L. larvae. Bioassays were performed on the 3rd instar larvae for each chitinase protein in triplicate. The results showed that although there were differences in enzymatic activities and domain organizations, all 3 microbial chitinases produced almost the same level of insecticidal activity on the test insect. LC50 and LT50 values were compatible with the mortality results. These results were a preanalysis for comparing the effects of microbial chitinases. Conclusion Potential activity experiments should be carried out on more insects to provide detailed information on the insecticidal effects of bacterial and viral chitinases.

Growth inhibition of Spodoptera frugiperda larvae by camptothecin correlates with alteration of the structures and gene expression profiles of the midgut

Background Spodoptera frugiperda is a serious pest that causes devastating losses to many major crops, including corn, rice, sugarcane, and peanut. Camptothecin (CPT) is a bioactive secondary metabolite of the woody plant Camptotheca acuminata, which has shown high toxicity to various pests. However, the effect of CPT against S. frugiperda remains unknown. Results In this study, bioassays have been conducted on the growth inhibition of CPT on S. frugiperda larvae. Histological and cytological changes were examined in the midgut of larvae fed on an artificial diet supplemented with 1.0 and 5.0 µg/g CPT. The potential molecular mechanism was explored by comparative transcriptomic analyses among midgut samples obtained from larvae under different treatments. A total of 915 and 3560 differentially expressed genes (DEGs) were identified from samples treated with 1.0 and 5.0 µg/g CPT, respectively. Among the identified genes were those encoding detoxification-related proteins and components of peritrophic membrane such as mucins and cuticle proteins. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses indicated that part of DEGs were involved in DNA replication, digestion, immunity, endocrine system, and metabolism. Conclusions Our results provide useful information on the molecular basis for the impact of CPT on S. frugiperda and for future studies on potential practical application.

Characterization and Functional Analysis of trehalase Related to Chitin Metabolism in Glyphodes pyloalis Walker (Lepidoptera: Pyralidae)

Glyphodes pyloalis Walker (G. pyloalis) is a serious pest on mulberry. Due to the increasing pesticide resistance, the development of new and effective environmental methods to control G. pyloalis is needed. Trehalase is an essential enzyme in trehalose hydrolysis and energy supply, and it has been considered a promising target for insect pest control. However, the specific function of trehalase in G. pyloalis has not been reported. In this study, two trehalase genes (GpTre1 and GpTre2) were identified from our previous transcriptome database. The functions of the trehalase in chitin metabolism were studied by injecting larvae with dsRNAs and trehalase inhibitor, Validamycin A. The open reading frames (ORFs) of GpTre1 and GpTre2 were 1,704 bp and 1,869 bp, which encoded 567 and 622 amino acid residues, respectively. Both of GpTre1 and GpTre2 were mainly expressed in the head and midgut. The highest expression levels of them were in 5th instar during different development stages. Moreover, knockdown both of GpTre1 and GpTre2 by the dsRNAs led to significantly decreased expression of chitin metabolism pathway-related genes, including GpCHSA, GpCDA1, GpCDA2, GpCHT3a, GpCHT7, GpCHSB, GpCHT-h, GpCHT3b, GpPAGM, and GpUAP, and abnormal phenotypes. Furthermore, the trehalase inhibitor, Validamycin A, treatment increased the expressions of GpTre1 and GpTre2, increased content of trehalose, and decreased the levels of glycogen and glucose. Additionally, the inhibitor caused a significantly increased cumulative mortality of G. pyloalis larvae on the 2nd (16%) to 6th (41.3%) day, and decreased the rate of cumulative pupation (72.3%) compared with the control group (95.6%). After the activities of trehalase were suppressed, the expressions of 6 integument chitin metabolism-related genes decreased significantly at 24 h and increased at 48 h. The expressions of GpCHSB and GpCHT-h, involved in chitin metabolism pathway of peritrophic membrane in the midgut, increased at 24 h and 48 h, and there were no changes to GpCHT3b and GpPAGM. These results reveal that GpTre1 and GpTre2 play an essential role in the growth of G. pyloalis by affecting chitin metabolism, and this provides useful information for insect pest control in the future.

Alterations in the expression of certain midgut genes of Spodoptera littoralis (Boisd.) (Lepidoptera: Noctuidae) larvae and midgut histopathology in response to Bacillus thuringiensis Cry1C toxin

Background Bacillus thuringiensis (Bt) utilization as a biological control agent is highly recommended due to its safety, specificity, and efficiency. Importance of the entomocidal Cry proteins secreted by Bt is dramatically increased subsequent Cry genes transformation into a number of economic crops, rendering them protection against insect attack. In the last decade, insect resistance against transgenic Bt crops is gradually raised in several lepidopteran pests. A better understanding of the processing of Bt Cry1C toxin in the larval midgut of the lepidopteran pest species, the cotton leaf worm, Spodoptera littoralis (Boisd.), is very important to characterize the main regulatory elements of Bt tolerance. Results The present study aimed to define factors that are involved in insect tolerance toward Bt Cry1C through evaluating the mRNA level of trypsin (Try), aminopeptidase N (APN), alkaline phosphatase (ALP), cadherin (Cad), and cytochrome P450 (CYP) in both susceptible and cry1C tolerant strains of S. littoralis. Total RNAs were extracted from susceptible and tolerant strains to construct cDNAs. Quantitative real-time polymerase chain reaction (qPCR) showed a significant upregulation of CYP gene in tolerant strain. In contrast, the levels of expression of Try, ALP, and Cad were significantly downregulated in tolerant strain. APN relative mRNA expression did not show significant differences between susceptible and tolerant strains. Histologically, the midgut of late third-instar larvae of tolerant population S. littoralis showed vacuolization of the epithelium and disruption of both the peritrophic membrane and the striated boarder compared to the susceptible strain. Conclusions Obtained data indicated a relationship between exposing to Bt Cry1C toxin and alteration of CYP, Try, ALP, and Cad expression in midgut of S. littoralis. These results may be an evidence for the important roles of CYP, Try, ALP, and Cad in the resistance development and toxicity to Bt Cry1C. The results are useful for further illustrating of Bt Cry1C processing and S. littoralis tolerance.

Vickermania gen. nov., trypanosomatids that use two joined flagella to resist midgut peristaltic flow within the fly host

Background The family Trypanosomatidae encompasses parasitic flagellates, some of which cause serious vector-transmitted diseases of humans and domestic animals. However, insect-restricted parasites represent the ancestral and most diverse group within the family. They display a range of unusual features and their study can provide insights into the biology of human pathogens. Here we describe Vickermania, a new genus of fly midgut-dwelling parasites that bear two flagella in contrast to other trypanosomatids, which are unambiguously uniflagellate. Results Vickermania has an odd cell cycle, in which shortly after the division the uniflagellate cell starts growing a new flagellum attached to the old one and preserves their contact until the late cytokinesis. The flagella connect to each other throughout their whole length and carry a peculiar seizing structure with a paddle-like apex and two lateral extensions at their tip. In contrast to typical trypanosomatids, which attach to the insect host’s intestinal wall, Vickermania is separated from it by a continuous peritrophic membrane and resides freely in the fly midgut lumen. Conclusions We propose that Vickermania developed a survival strategy that relies on constant movement preventing discharge from the host gut due to intestinal peristalsis. Since these parasites cannot attach to the midgut wall, they were forced to shorten the period of impaired motility when two separate flagella in dividing cells interfere with each other. The connection between the flagella ensures their coordinate movement until the separation of the daughter cells. We propose that Trypanosoma brucei, a severe human pathogen, during its development in the tsetse fly midgut faces the same conditions and follows the same strategy as Vickermania by employing an analogous adaptation, the flagellar connector.

Identifying and Characterizing a Novel Peritrophic Matrix Protein (MdPM-17) Associated With Antibacterial Response From the Housefly, Musca domestica (Diptera: Muscidae)

Peritrophic matrix/membrane (PM) critically prevents the midgut of insects from external invasion by microbes. The proteins in the peritrophic membrane are its major structural components. Additionally, they determine the formation and function of this membrane. However, the role of PM proteins in immune regulation is unclear. Herein, we isolated a novel PM protein (MdPM-17) from Musca domestica larvae. Further, the function of MdPM-17 in regulating host innate immunity was identified. Results showed that the cDNA of MdPM-17 full is 635 bp in length. Moreover, it consists of a 477-bp open reading frame encoding 158 amino acid residues. These amino acid residues are composed of two Chitin-binding type-2 domain (ChtBD2) and 19 amino acids as a signal peptide. Moreover, tissue distribution analysis indicates that MdPM-17 was enriched expressed in midgut, and moderate levels in the fat body, foregut, and malpighian tubule. Notably, MdPM-17 recombinant protein showed high chitin-binding capacity, thus belongs to the Class III PM protein group. MdPM-17 protein silencing via RNA interference resulted in the expression of antimicrobial peptide (defensin, cecropins, and diptericin) genes, and this occurred after oral inoculation with exogenous microbes Escherichia coli (Enterobacteriales:Enterobacteriaceae), Staphylococcus aureus (Bacillales:Staphylococcaceae), and Candida albicans (Endomycetales:Saccharomycetaceae)). Therefore, all the antimicrobial peptide (AMP) gene expression levels are high in MdPM-17-depleted larvae during microbial infection compared to controls. Consequently, these findings indicate that MdPM-17 protein is associated with the antibacterial response from the housefly.

Identification of Peritrophins and Antiviral Effect of Bm01504 against BmNPV in the Silkworm, Bombyx mori

The insect midgut secretes a semi-permeable, acellular peritrophic membrane (PM) that maintains intestinal structure, promotes digestion, and protects the midgut from food particles and pathogenic microorganisms. Peritrophin is an important PM protein (PMP) in the PM. Here, we identified 11 peritrophins with 1–16 chitin binding domains (CBDs) comprising 50–56 amino acid residues. Multiple CBDs in the same peritrophin clustered together, rather than by species. The CBD contained six highly conserved cysteine residues, with the key feature of amino acids between them being CX11-15CX5CX9-14CX11-12CX6-7C. Peritrophins with 2 and 4 CBDs (Bm09641 and Bm01504, respectively), and with 1, 8, and 16 CBDs (Bm11851, Bm00185, and Bm01491, respectively) were mainly expressed in the anterior midgut, and throughout the midgut, respectively. Survival rates of transgenic silkworms with Bm01504 overexpression (Bm01504-OE) and knockout (Bm01504-KO) infected with B. morinucleopolyhedrovirus (BmNPV) were significantly higher and lower, whereas expression of the key viral gene, p10, were lower and higher, respectively, compared with wild type (WT). Therefore, Bm01504-OE and Bm01504-KO transgenic silkworms were more and less resistant, respectively, to BmNPV. Bm01504 plays important roles in resisting BmNPV invasion. We provide a new perspective for studying PM function, and reveal how the silkworm midgut resists invasive exogenous pathogenic microorganisms.