The velvetbean caterpillar Anticarsia gemmatalis is one of the main soybean defoliators in Brazil. Currently, the main biopesticide used to control insect pests worldwide is the bacteria Bacillus thuringiensis (Bt), which produces entomopathogenic Crystal toxins (Cry) that act in the midgut of susceptible insects, leading them to death. The mode of action of Cry toxins in the midgut involves binding to specific receptors present on the brush border of epithelial cells such as aminopeptidase N (APN), alkaline phosphatase (ALP), cadherin, and others. Mutations in these receptors, among other factors, may be involved in the development of resistance; identification of functional Cry receptors in the midgut of A. gemmatalis is crucial to develop effective strategies to overcome this possible scenario. This study’s goal is to characterize APNs of A. gemmatalis and identify a receptor for Cry1Ac in the midgut. The interaction of Bt spores with the midgut epithelium was observed in situ by immunohistochemistry and total aminopeptidase activity was estimated in brush border membrane vesicle (BBMV) samples, presenting higher activity in challenged individuals than in control ones. Ten APN sequences were found in a A. gemmatalis’ transcriptome and subjected to different in silico analysis, such as phylogenetic tree, multiple sequence alignment and identification of signal peptide, activity domains and GPI-anchor signal. BBMV proteins from 5th instar larvae were submitted to a ligand blotting using activated Cry1Ac toxin and a commercial anti-Cry polyclonal antibody; corresponding bands of proteins that showed binding to Cry toxin were excised from the SDS-PAGE gel and subjected to mass spectrometry analysis, which resulted in the identification of seven of those APNs. Quantitative PCR was realized to compare expression levels between individuals subjected to sublethal infection with Bt spores and control ones, presenting up- and downregulations upon Bt infection. From these results, we can infer that aminopeptidases N in A. gemmatalis could be involved in the mode of action of Cry toxins in its larval stage.

Background: Insects can be found in numerous diverse environments, being exposed to pathogenic organisms like fungi and bacteria. Once these pathogens cross insect physical barriers, the innate immune system operates through cellular and humoral responses. Antimicrobial peptides are small molecules produced by immune signaling cascades that develop an important and generalist role in insect defenses against a variety of microorganisms. In the present work, a cecropin B-like peptide (AgCecropB) sequence was identified in the velvetbean caterpillar Anticarsia gemmatalis and cloned in a bacterial plasmid vector for further heterologous expression and antimicrobial tests. Methods: AgCecropB sequence (without the signal peptide) was cloned in the plasmid vector pET-M30-MBP and expressed in the Escherichia coli BL21(DE3) expression host. Expression was induced with IPTG and a recombinant peptide was purified using two affinity chromatography steps with Histrap column. The purified peptide was submitted to high-resolution mass spectrometry (HRMS) and structural analyses. Antimicrobial tests were performed using gram-positive (Bacillus thuringiensis) and gram-negative (Burkholderia kururiensis and E. coli) bacteria. Results: AgCecropB was expressed in E. coli BL21 (DE3) at 28 degrees C with IPTG 0.5 mM. The recombinant peptide was purified and enriched after purification steps. HRMS confirmed AgCrecropB molecular mass (4.6 kDa) and circular dichroism assay showed alpha-helix structure in the presence of SDS. AgCrecropB inhibited almost 50% of grampositive B. thuringiensis bacteria growth. Conclusions: The first cecropin B-like peptide was described in A. gemmatalis and a recombinant peptide was expressed using a bacterial platform. Data confirmed tertiary structure as predicted for the cecropin peptide family. AgCecropB was capable to inhibit B. thuringiensis growth in vitro.

Zika is a vector-borne disease caused by an arbovirus (ZIKV) and overwhelmingly transmitted by Ae. aegypti . This disease is linked to adverse fetal outcomes, mostly microcephaly in newborns, and other clinical aspects such as acute febrile illness and neurologic complications, for example, Guillain-Barré syndrome. One of the actual most promising strategies to mitigate arbovirus transmission involves releasing Ae. aegypti mosquitoes carrying the maternally inherited endosymbiont bacteria Wolbachia pipientis . The presence of Wolbachia is associated with a reduced susceptibility to arboviruses and a fitness cost in mosquito life-history traits as fecundity and fertility. However, the mechanisms by which Wolbachia influences metabolic pathways leading to differences in egg production remains poorly known. To investigate the impact of co-infections on the reproductive tract of the mosquito, we applied an isobaric labeling-based quantitative proteomic strategy to investigate the influence of Wolbachia w Mel and ZIKV infection in Ae. aegypti ovaries. To the best of our knowledge, this is the most complete proteome of Ae. aegypti ovaries reported so far, with a total of 3,913 proteins identified, also,were able to quantify a total of 1,044 Wolbachia proteins in complex sample tissue of Ae. aegypti ovary.. Furthermore, we discuss proteins and pathways altered in Ae. aegypti during ZIKV infections, Wolbachia infections, co-infection Wolbachia/ZIKV, and compared with no infection, focusing on immune and reproductive aspects of Ae. aegypti . The modified aspects were mostly related to the immune priming enhancement by Wolbachia presence and the modulation of the Juvenile Hormone pathway caused by both microorganisms infection. Highlights: Proteome changes in Ae. aegypti , Wolbachi a, and ZIKV interactions A great diversity of Wolbachia proteins were quantified in Ae. aegypti ovary Juvenile Hormone pathway is modulated by both infections Wolbachia enhances Ae. aegypti immune priming mechanism ZIKV unsettles host immune response by reducing antimicrobial peptides production Coinfection triggers oxidative stress and a lack of vitellogenin precursors

Zika virus is a global public health emergency due to its association with microcephaly, Guillain-Barré syndrome, neuropathy, and myelitis in children and adults. A total of 87 countries have had evidence of autochthonous mosquito-borne transmission of Zika virus, distributed across four continents, and no antivirus therapy or vaccines are available. Therefore, several strategies have been developed to target the main mosquito vector, Aedes aegypti , to reduce the burden of different arboviruses. Among such strategies, the use of the maternally-inherited endosymbiont Wolbachia pipientis has been applied successfully to reduce virus susceptibility and decrease transmission. However, the mechanisms by which Wolbachia orchestrate resistance to ZIKV infection remain to be elucidated. In this study, we apply isobaric labeling quantitative mass spectrometry-based proteomics to quantify proteins and identify pathways altered during ZIKV infection; Wolbachia infection; co-infection with Wolbachia/ ZIKV in the Ae. aegypti heads and salivary glands. We show that Wolbachia regulates proteins involved in ROS production, regulates humoral immune response, and antioxidant production. The reduction of ZIKV polyprotein in the presence of Wolbachia in mosquitoes was determined by mass spectrometry and corroborates the idea that Wolbachia helps to block ZIKV infections in Ae. aegypti . The present study offers a rich resource of data that may help to elucidate mechanisms by which Wolbachia orchestrate resistance to ZIKV infection in Ae. aegypti , and represents a step further on the development of new targeted methods to detect and quantify ZIKV and Wolbachia directly in complex tissues. Highlights: The abundance of ZIKV polyprotein is reduced in the presence of Wolbachia Shotgun proteomics quantifies ZIKV and Wolbachia proteins directly in tissues Wolbachia regulates proteins involved in ROS production Wolbachia regulates humoral immune response and antioxidant production Metabolism and detoxification processes were associated with mono infections