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Talisin is a storage protein from Talisia esculenta seeds that presents lectin-like and peptidase inhibitor properties. These characteristics suggest that talisin plays a role in the plant defense process, making it a multifunctional protein. This work aimed to investigate the effects of chronic intake of talisin on fifth instar larvae of Spodoptera frugiperda, considered the main insect pest of maize and the cause of substantial economic losses in several other crops. The chronic intake of talisin presented antinutritional effects on the larvae, reducing their weight and prolonging the total development time of the insects. In addition, talisin-fed larvae also showed a significant reduction in the activity of trypsin-like enzymes. Midgut histology analysis of talisin-fed larvae showed alterations in the intestinal epithelium and rupture of the peritrophic membrane, possibly causing an increase of aminopeptidase activity in the midgut lumen. Talisin also proved to be resistant to degradation by the digestive enzymes of S. frugiperda. The transcription profile of trypsin, chymotrypsin and aminopeptidase genes was also analyzed through qPCR technique. Talisin intake resulted in differential expression of at least two genes from each of these classes of enzymes. Molecular docking studies indicated a higher affinity of talisin for the less expressed enzymes.

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OBJECTIVE: To analyze the transcription pattern of neuropeptides in the ontogeny of a malaria vector, the mosquito Anopheles albimanus. MATERIALS AND METHODS: The transcription pattern of Crustacean CardioActive peptide (CCAP), corazonin, Ecdysis Triggering Hormone (ETH), allatostatin-A, orcokinin, Insulin Like Peptide 2 (ILP2), Insulin Like Peptide 5 (ILP5) and bursicon was evaluated using qPCR on larvae (1st - 4th instar), pupae and adult mosquitoes. RESULTS: Unlike in other insects, transcripts of CCAP (70.8%), ETH (60.2%) and corazonin (76.5%) were expressed in 4th instar larvae, probably because these three neuropeptides are associated with the beginning of ecdysis. The neuropeptide ILP2 showed higher transcription levels in other stages and orcokinin decreased during the development of the mosquito. CONCLUSIONS: The CCAP, corazonin and ETH neuropeptidesare potential targets for the design of control strategies aimed at disrupting An. albiamnus larval development.

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Abstract: Objective: To analyze the transcription pattern of neuropeptides in the ontogeny of a malaria vector, the mosquito Anopheles albimanus. Materials and methods: The transcription pattern of Crustacean CardioActive peptide (CCAP), corazonin, Ecdysis Triggering Hormone (ETH), allatostatin-A, orcokinin, Insulin Like Peptide 2 (ILP2), Insulin Like Peptide 5 (ILP5) and bursicon was evaluated using qPCR on larvae (1st - 4th instar), pupae and adult mosquitoes. Results: Unlike in other insects, transcripts of CCAP (70.8%), ETH (60.2%) and corazonin (76.5%) were expressed in 4th instar larvae, probably because these three neuropeptides are associated with the beginning of ecdysis. The neuropeptide ILP2 showed higher transcription levels in other stages and orcokinin decreased during the development of the mosquito. Conclusion: The CCAP, corazonin and ETH neuropeptides are potential targets for the design of control strategies aimed at disrupting An. albiamnus larval development.

Resumen: Objetivo: Describir la expresión de neuropéptidos durante la ontogenia del mosquito vector de la malaria Anopheles albimanus. Material y métodos: Se midió la expresión de CCAP, corazonina, ETH, allatostatina, orcokinina, ILP2, ILP5 y bursicon en larvas de primer (2mm), segundo (4mm), tercer (5mm) y cuarto (6mm) estadio, pupas y mosquitos adultos, mediante qPCR. Resultados. A diferencia de otros insectos en donde, CCAP, corazonina y ETH se expresan principalmente en estadios pupales, en An. albimanus se expresaron mayoritariamente en larvas de cuarto estadio, CCAP tuvo 70.8% de expresión relativa, corazonina 76.5% y ETH 60.2%. ILP2 fue el neuropéptido que más se expresó en el primer, segundo y tercer estadio y orcokinina disminuyó durante el desarrollo del mosquito. Conclusión. Los péptidos estudiados se expresaron en todos los estadios de desarrollo del mosquito. Sin embargo, su expresión varió en cada uno de ellos. Los neuropéptidos CCAP, corazonina y ETH, que son esenciales para la transformación de lavas a pupas, pueden ser blancos potenciales para el diseño de estrategias de control dirigidas a interrumpir el desarrollo larvario de An. albimanus.

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BACKGROUND: Antimicrobial peptides (AMPs) are the first line of host immune defense against pathogens. Among AMPs from the honeybee Apis mellifera, abaecin is a major broad-spectrum antibacterial proline-enriched cationic peptide. RESULTS: For heterologous expression of abaecin in Pichia pastoris, we designed an ORF with HisTag, and the codon usage was optimized. The gene was chemically synthetized and cloned in the pUC57 vector. The new ORF was sub-cloned in the pPIC9 expression vector and transformed into P. pastoris. After selection of positive clones, the expression was induced by methanol. The supernatant was analyzed at different times to determine the optimal time for the recombinant peptide expression. As a proof-of-concept, Escherichia coli was co-incubated with the recombinant peptide to verify its antimicrobial potential. DISCUSSION: Briefly, the recombinant Abaecin (rAbaecin) has efficiently decreased E. coli growth (P < 0.05) through an in vitro assay, and may be considered as a novel therapeutic agent that may complement other conventional antibiotic therapies.

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Chagas disease is one of the main parasitic diseases found in Latin America and it is estimated that between six and seven million people are infected worldwide. Its etiologic agent, the protozoan Trypanosoma cruzi, is transmitted by triatomines, some of which from the genus Rhodnius. Twenty species are currently recognized in this genus, including some closely related species with low levels of morphological differentiation, such as Rhodnius montenegrensis and Rhodnius robustus. In order to investigate genetic differences between these two species, we generated large-scale RNA-sequencing data (consisting of four RNA-seq libraries) from the heads and salivary glands of males of R. montenegrensis and R. robustus. Transcriptome assemblies produced for each species resulted in 64,952 contigs for R. montenegrensis and 70,894 contigs for R. robustus, with N50 of approximately 2,100 for both species. SNP calling based on the more complete R. robustus assembly revealed 3,055 fixed interspecific differences and 216 transcripts with high levels of divergence which contained only fixed differences between the two species. A gene ontology enrichment analysis revealed that these highly differentiated transcripts were enriched for eight GO terms related to AP-2 adaptor complex, as well as other interesting genes that could be involved in their differentiation. The results show that R. montenegrensis and R. robustus have a substantial quantity of fixed interspecific polymorphisms, which suggests a high degree of genetic divergence between the two species and likely corroborates the species status of R. montenegrensis.

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A multi-generational approach was used to investigate the persistent effects of a sub-lethal dose of spinosad in Plutella xylostella. The susceptibility of various sub-populations of P. xylostella to spinosad and the effects of the insecticide on the gene expression of γ-aminobutyric acid receptor (GABAR) were determined. The results of a leaf dip bioassay showed that the sensitivity of P. xylostella to spinosad decreased across generations. The sub-strains had been previously selected based on a determined LC25 of spinosad. Considering that GABA-gated chloride channels are the primary targets of spinosad, the cDNA of P. xylostella was used to clone GABARα by using reverse transcription-polymerase chain reaction (RT-PCR). The mature peptide cDNA was 1477-bp long and contained a 1449-bp open reading frame encoding a protein of 483 amino acids. The resulting amino acid sequence was used to generate a neighbor-joining dendrogram, and homology search was conducted using NCBI BLAST. The protein had high similarity with the known GABAR sequence from P. xylostella. Subsequent semi-quantitative RT-PCR and real-time PCR analyses indicated that the GABAR transcript levels in the spinosad-resistant strain (RR, 145.82-fold) and in Sub1 strain (selected with LC25 spinosad for one generation) were the highest, followed by those in the spinosad-susceptible strain, the Sub10 strain (selected for ten generations), and the Sub5 strain (selected for five generations). This multi-generational study found significant correlations between spinosad susceptibility and GABAR gene expression, providing insights into the long-term effects of sub-lethal insecticide exposure and its potential to lead to the development of insecticide-resistant insect populations.

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The Receptor for Activated C Kinase 1 (RACK1), a scaffold protein member of the tryptophan-aspartate (WD) repeat family, folds in a seven-bladed ß-propeller structure that permits the association of proteins to form active complexes. Mosquitoes of the genus Aedes sp., are vectors of virus producing important diseases such as: dengue, chikungunya and yellow fever. Based on the highly conserved gene sequence of AeaeRACK1 of the mosquito Aedes aegypti we characterized the mRNA and protein of the homologous AealRACK1 from the Ae. albopictus-derived cell line C6/36 HT. Two protein species differing in MW/pI values were observed at 35kDa/8.0 and 36kDa/6.5. The behavior of AealRACK1 was studied inducing stress with serum deprivation and the glucocorticoid dexamethasone. Both stressors induced increase of the expression of AealRACK1 mRNA and proteins. In serum-deprived cells AealRACK1 protein was located cortically near the plasma membrane in contrast to dexamethasone-treated cells where the protein formed a dotted pattern in the cytoplasm. In addition, 33 protein partners were identified by immunoprecipitation and mass spectrometry. Most of the identified proteins were ribosomal, involved in signaling pathways and stress responses. Our results suggest that AealRACK1 in C6/36 HT cells respond to stress increasing its synthesis and producing phosphorylated activated form. BIOLOGICAL SIGNIFICANCE: Insect cells adapt to numerous environmental stressors, including chemicals and invasion of pathogenic microorganisms among others, coordinating cellular and organismal responses. Individual cells sense the environment using receptors that trigger signaling pathways that regulate expression of specific effector proteins and/or cellular responses as movement or secretion. In the coordination of responses to stress, scaffold proteins are pivotal molecules that recruit other proteins forming active complexes. The Receptor for Activated C Kinase 1 (RACK1) is the best studied member of the conserved tryptophan-aspartate (WD) repeat family. RACK1 folds in a seven-bladed ß-propeller structure and it could be activated during stress, participating in different signaling pathways. The presence and activities of RACK1 in mosquitoes had not been documented before, in this work the molecule is demonstrated in an Aedes albopictus-derived cell line and its reaction to stress is observed under the effect of serum deprivation and the presence of glucocorticoid analog dexamethasone, a chemical used to cause stress in vitro.

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Procecidochares utilis is a tephritid gall fly, which is known to be an effective biological agent that can be used to control the notoriously widespread crofton weed Eupatorium adenophorum. Despite its importance, genetic resources for P. utilis remain scarce. In this study, 1.2 Gb sequences were generated using Illumina paired-end sequencing technology. De novo assemblies yielded 491,760 contigs, 90,474 scaffolds, and 58,562 unigenes. Among the unigenes, 34,809 (59.44%) had a homologous match against the National Center for Biotechnology Information non-redundant protein database by translated Basic Local Alignment Search Tool (BlastX) with a cut-off E-value of 10(-5). Among the unigenes, 57,627 were classified in the Gene Ontology database, 15,910 were assigned to Clusters of Orthologous Groups, and 38,565 were found in Kyoto Encyclopedia of Genes and Genomes. In addition, 5723 simple sequence repeats (SSRs) were discovered based on the unigene sequences. The transcriptome sequences and SSRs obtained represent a major molecular resource for P. utilis, which will extend our knowledge of the comparative and functional genomics of this organism and enable population genomic and gene-based association studies of the gall fly.

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Pectin methylesterase removes the methyl groups from the main chain of pectin, the major component of the middle lamella of the plant cell wall. The enzyme is involved in plant cell-wall development, is part of the enzymatic arsenal used by microorganisms to attack plants and also has a wide range of applications in the industrial sector. Therefore, there is a considerable interest in studies of the structure and function of this enzyme. In this work, the pectin methylesterase from Sphenophorus levis was produced in Pichia pastoris and purified. Crystals belonging to the monoclinic space group C2, with unit-cell parameters a = 122.181, b = 82.213, c = 41.176 Å, ß = 97.48°, were obtained by the sitting-drop vapour-diffusion method and an X-ray diffraction data set was collected to 2.1 Šresolution. Structure refinement and model building are in progress.

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BACKGROUND: Dinoponera quadriceps is a predatory giant ant that inhabits the Neotropical region and subdues its prey (insects) with stings that deliver a toxic cocktail of molecules. Human accidents occasionally occur and cause local pain and systemic symptoms. A comprehensive study of the D. quadriceps venom gland transcriptome is required to advance our knowledge about the toxin repertoire of the giant ant venom and to understand the physiopathological basis of Hymenoptera envenomation. RESULTS: We conducted a transcriptome analysis of a cDNA library from the D. quadriceps venom gland with Sanger sequencing in combination with whole-transcriptome shotgun deep sequencing. From the cDNA library, a total of 420 independent clones were analyzed. Although the proportion of dinoponeratoxin isoform precursors was high, the first giant ant venom inhibitor cysteine-knot (ICK) toxin was found. The deep next generation sequencing yielded a total of 2,514,767 raw reads that were assembled into 18,546 contigs. A BLAST search of the assembled contigs against non-redundant and Swiss-Prot databases showed that 6,463 contigs corresponded to BLASTx hits and indicated an interesting diversity of transcripts related to venom gene expression. The majority of these venom-related sequences code for a major polypeptide core, which comprises venom allergens, lethal-like proteins and esterases, and a minor peptide framework composed of inter-specific structurally conserved cysteine-rich toxins. Both the cDNA library and deep sequencing yielded large proportions of contigs that showed no similarities with known sequences. CONCLUSIONS: To our knowledge, this is the first report of the venom gland transcriptome of the New World giant ant D. quadriceps. The glandular venom system was dissected, and the toxin arsenal was revealed; this process brought to light novel sequences that included an ICK-folded toxins, allergen proteins, esterases (phospholipases and carboxylesterases), and lethal-like toxins. These findings contribute to the understanding of the ecology, behavior and venomics of hymenopterans.

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