Draft-genome sequences of Leptospira santarosai strains isolated from urogenital tract of cows.

Leptospira santarosai serovar Guaricura is adapted to bovines and is associated with a chronic disease that causes reproductive disorders, leading to important economic losses. Here, we present the draft genomes of three L. santarosai strains isolated from vaginal fluid and one from the urine of cows with reproductive disorders from Brazil.

In silico prospection of Lactobacillus acidophilus strains with potential probiotic activity.

Lactic acid bacteria (LAB) are fermentative microorganisms and perform different roles in biotechnological processes, mainly in the food and pharmaceutical industries. Among the LAB, Lactobacillus acidophilus is a species that deserves to be highlighted for being used both in prophylaxis and in the treatment of pathologies. Most of the metabolites produced by this species are linked to the inhibition of pathogens. In this study, we utilized a pangenomic and metabolic annotation analysis using Roary and BlastKOALA, ML-based probiotic activity prediction with iProbiotic and whole-genome similarity using ANI to identify strains of L. acidophilus with potential probiotic activity. According to the results in BlastKOALA and iProbiotics, L. acidophilus NCTC 13721 had the greatest potential among the 64 strains tested, both in terms of its ability to be a Lactobacillus spp. probiotic, when in the amount of genes involved in the metabolism of organic acids and quorum sensing. In addition, DSM 20079 proved to be promising for prospecting new probiotic Lactobacillus from BlastKOALA analyses, as they presented similar results in the number of genes involved in the production of lactic acid, acetic acid, hydrogen peroxide, except for quorum sensing where the NCTC 13721 strain had 14 more genes. L. acidophilus NCTC 13721 and L. acidophilus La-5 strains showed greater ability to be Lactobacillus spp. probiotic capacity, showing 84.8% and 51.9% capacity in the iProbiotics tool, respectively. When analyzed in ANI, none of the evaluated strains showed genomic similarity with NCTC 13721. In contrast, the DSM 20079 strain showed genomic similarity with all evaluated strains except NCTC 13721. Furthermore, eight strains with characteristics with approximately 100% genomic similarity to La-5 were listed: S20_1, LA-5, FSI4, APC2845, LA-G80-111, DS1_1A, LA1, and BCRC 14065. Therefore, according to the findings in iProbiotics and BlastKoala, among the 64 strains evaluated, NCTC 13721 is the most promising strain to be used for future in vitro studies.

Genetic Analysis of LigA, LipL32, Loa22, and OmpL1 Antigens from Leptospira spp. Sejroe Serogroup: How Close We Are To a Vaccine Against Bovine Leptospirosis?

Bovine leptospirosis has as main causative agents Leptospira spp. from Sejroe serogroup. Vaccination is a crucial step to control this infection. The use of conserved proteins among Leptospira spp. is of great importance for a protective immune response. The aim of the present study is to genetically analyze antigens of Leptospira spp. from Sejroe serogroup strains isolated from cattle for a preliminary evaluation of vaccine candidates. Genes associated with antigenicity-LigA, LipL32, Loa22, and OmpL1-were analyzed through bioinformatic and immunoinformatic tools. Despite high diversity observed in strains, on an amino acid level, highly conserved regions were observed (> 90%), particularly in LipL32 gene. Moreover, highly conserved amino acid regions (> 30 aa) were observed in all genes, regardless of species, geographical origin or biological source of isolation. Superposed structures of protein fragments including all the predicted MHC-II and B-Cell epitopes were demonstrated. Results presented herein are preliminary, but a fundamental step towards the development of an efficient vaccine against bovine leptospirosis, a silent but enormously concerning disease.

Bambu and its applications in the discovery of active molecules against melanoma.

PURPOSE OR OBJECTIVE: Melanoma is one of the most dangerous forms of skin cancer and the discovery of novel drugs is an ongoing effort. Quantitative Structure Activity Relationship (QSAR) is a computational method that allows the estimation of the properties of a molecule, including its biological activity. QSAR models have been widely employed in the search for potential drug candidates, but also for agrochemicals and other molecules with applications in different branches of the industry. Here we present Bambu, a simple command line tool to generate QSAR models from high-throughput screening bioassays datasets. METHODS: The tool was developed using the Python programming language and relies mainly on RDKit for molecule data manipulation, FLAML for automated machine learning and the PubChem REST API for data retrieval. As a proof-of-concept we have employed the tool to generate QSAR models for melanoma cell growth inhibition based on HTS data and used them to screen libraries of FDA-approved drugs and natural compounds. Additionally, Bambu was compared to QSAR-Co, another automated tool for QSAR model generation. RESULTS: based on the developed tool we were able to produce QSAR models and identify a wide variety of molecules with potential melanoma cell growth inhibitors, many of which with anti-tumoral activity already described. The QSAR models are available through the URL http://caramel.ufpel.edu.br, and all data and code used to generate its models are available at Zenodo (https://doi.org/10.5281/zenodo.7495214). Bambu source code is available at GitHub (https://github.com/omixlab/bambu-v2). In the benchmark, Bambu was able to produce models with higher accuracy, recall, F1 and ROC AUC when compared to QSAR-Co for the selected datasets. CONCLUSIONS: Bambu is an free and open source tool which facilitates the creation of QSAR models and can be futurely applied in a wide variety of drug discovery projects.

A paraventricular thalamus to insular cortex glutamatergic projection gates "emotional" stress-induced binge eating in females.

It is well-established that stress and negative affect trigger eating disorder symptoms and that the brains of men and women respond to stress in different ways. Indeed, women suffer disproportionately from emotional or stress-related eating, as well as associated eating disorders such as binge eating disorder. Nevertheless, our understanding of the precise neural circuits driving this maladaptive eating behavior, particularly in women, remains limited. We recently established a clinically relevant model of 'emotional' stress-induced binge eating whereby only female mice display binge eating in response to an acute "emotional" stressor. Here, we combined neuroanatomic, transgenic, immunohistochemical and pathway-specific chemogenetic approaches to investigate whole brain functional architecture associated with stress-induced binge eating in females, focusing on the role of Vglut2 projections from the paraventricular thalamus (PVTVglut2+) to the medial insular cortex in this behavior. Whole brain activation mapping and hierarchical clustering of Euclidean distances revealed distinct patterns of coactivation unique to stress-induced binge eating. At a pathway-specific level, PVTVglut2+ cells projecting to the medial insular cortex were specifically activated in response to stress-induced binge eating. Subsequent chemogenetic inhibition of this pathway suppressed stress-induced binge eating. We have identified a distinct PVTVglut2+ to insular cortex projection as a key driver of "emotional" stress-induced binge eating in female mice, highlighting a novel circuit underpinning this sex-specific behavior.

Bioprospection of the trichomonacidal activity of lipid extracts derived from marine macroalgae Gigartina skottsbergii.

Algal extracts are sources of bioactive substances with applications in the development of novel alternative drugs against several diseases, including trichomoniasis sexually transmitted infection caused by Trichomonas vaginalis. Factors such as clinical failures and resistant strains limit the success of the existing drugs available for treating this disease. Therefore, searching for viable alternatives to these drugs is essential for the treatment of this disease. The present study was conducted for, in vitro and in silico characterization of extracts obtained from marine macroalgae Gigartina skottsbergii at stages gametophidic, cystocarpic, and tetrasporophidic. In addition, antiparasitic activity of these extracts against the ATCC 30236 isolate of T. vaginalis, their cytotoxicity, and gene expression of trophozoites after treatment were evaluated. The minimum inhibitory concentration and 50% inhibition concentration were determined for each extract. Results: In vitro analysis of the extracts' anti-T. vaginalis activity revealed an inhibitory effect of 100%, 89.61%, and 86.95% for Gigartina skottsbergii at stages gametophidic, cystocarpic, and tetrasporophidic, respectively, at 100 µg/mL. In silico analysis revealed the interactions between constituents of the extracts and enzymes from T. vaginalis, with significant free energy values obtained for the binding. None of the extract concentrations exhibited cytotoxic effects on VERO cell line compared to control, while cytotoxicity on HMVII vaginal epithelial cells line was observed at 100 µg/mL (30% inhibition). Gene expression analysis revealed differences in the expression profile of T. vaginalis enzymes between the extract-treated and control groups. According to these results, Gigartina skottsbergii extracts exhibited satisfactory antiparasitic activity.

An exploratory data analysis on genetic architecture in Bos taurus through miRNAs within QTLs and their target genes.

Understanding the genetic architecture is important because it allows us to understand the relationship between molecular markers, like miRNAs, QTLs and SNPs, and traits of interest such as milk yield, lipidic content in milk, heat tolerance etc. In the present study we performed an exploratory analysis that found an association of 480 miRNAs within QTLs related to 155 traits. We also discovered that those 480 miRNAs might modulate the expression of 12.374 genes. The most predominant traits available in the data were milk related, such as milk yield and fatty acids content in milk. Besides, the functional enrichment analysis revealed that some of those genes were linked to sensory perception, olfactory perception, perception of stimulus and chemical stimulus, localization, establishment of localization, transport etc. Furthermore, the discovery of miRNAs within QTLs and their target analysis suggests that they might be associated with characteristics of interest. By analyzing these relationships, we strongly encourage that future QTL studies should include miRNAs analysis. Altogether, our analysis enabled us to explore the association between multiple complex traits, miRNAs within QTLs and their target genes in Bos taurus that might have implications in breeding programs.

Pathogenesis and Genomic Analysis of a Virulent Leptospira Interrogans Serovar Copenhageni Isolated from a Dog with Lethal Infection.

Dogs are highly susceptible to leptospirosis and are a public health concern due to their important role as a source of spreading disease, particularly in urban settings. In this study, we present the pathogenesis, serological characterization, and complete genome sequencing of a virulent Brazilian strain (NEG7) of L. interrogans serovar Copenhageni isolated from the urine of a dog that died due to acute leptospirosis. Clinical investigation showed that the dog was presented with icteric mucous membranes, weakness, dehydration, anorexia, and kidney and liver failures. Necropsy followed by histopathological evaluation revealed lesions compatible with liver and kidney leptospirosis. The leptospires recovered from the urine were further characterized by genome analysis, which confirmed that the isolate belonged to L. interrogans serogroup icterohaemorrhagiae serovar Copenhageni. Multiple bioinformatics tools were used to characterize the genomic features, and comparisons with other available Copenhageni strains were performed. Characterization based on absence of an INDEL in the gene lic12008, associated with phylogenetic and ANI (99.99% identity) analyses, confirmed the genetic relatedness of the isolate with L. interrogans serovar Copenhageni. A better understanding of the diversity of the pathogenic Leptospira isolates could help in identifying genotypes responsible for severe infections. Moreover, it can be used to develop control and prevention strategies for Leptospira serovars associated with particular animal reservoirs.

Polymerase chain reaction and loop-mediated isothermal amplification targeting lic13162, lic20239, and lipL32 genes for leptospirosis diagnosis.

Leptospirosis is a zoonotic disease caused by pathogenic species of Leptospira. Due to the similarity with clinical signs of other febrile diseases, early diagnosis remains challenging. Real-time PCR has been used for direct detection of Leptospira, but it requires thermocyclers and highly trained personnel. Loop-mediated isothermal amplification (LAMP) is a simple and rapid DNA-based assay. Therefore, here we have developed PCR and LAMP assays targeting two novel genes, lic13162 and lic20239, and also lipL32 gene to detect pathogenic Leptospira. Analytical and diagnostic performances were compared with bacterial isolates (including different Leptospira species and serovars) and clinical samples. The results demonstrated that PCR assays targeting lic13162 and lic20239 were successful to amplify Leptospira, but LAMP not. However, both PCR and LAMP targeting lipL32 could detect pathogenic Leptospira. LAMP lipL32 could be performed in 30 min with a detection limit of 156 cells/mL. Diagnostic performance of lipL32-LAMP presented 84.2% sensitivity and 93.2% specificity. In conclusion, lipL32 PCR and LAMP are effective methods to detect pathogenic Leptospira directly from clinical samples.

Reconstruction of regulatory network predicts transcription factors driving the dynamics of zebrafish heart regeneration.

The limited regenerative capacity in mammals has serious implications for cardiac tissue damage. Meanwhile, zebrafish has a high regenerative capacity, but the regulation of the heart healing process has yet to be elucidated. The dynamic nature of cardiac regeneration requires consideration of the inherent temporal dimension of this process. Here, we conducted a systematic review to find genes that define the regenerative cell state of the zebrafish heart. We then performed an in silico temporal gene regulatory network analysis using transcriptomic data from the zebrafish heart regenerative process obtained from databases. In this analysis, the genes found in the systematic review were used to represent the final cell state of the transition process from a non-regenerative cell state to a regenerative state. We found 135 transcription factors driving the cellular state transition process during zebrafish cardiac regeneration, including Hand2, Nkx2.5, Tbx20, Fosl1, Fosb, Junb, Vdr, Wt1, and Tcf21 previously reported for playing a key role in tissue regeneration. Furthermore, we demonstrate that most regulators are activated in the first days post-injury, indicating that the transition from a non-regenerative to a regenerative state occurs promptly.

Recombinant Vaccine Design Against Clostridium spp. Toxins Using Immunoinformatics Tools.

The emergence of recombinant DNA technology has led to the exploration of the use of the technology to develop novel vaccines. With a fundamental role in vaccines design, several immunoinformatics tools have been created to identify isolated epitopes that stimulate a specific immune response, contributing to effective vaccines development. In the past, vaccine development projects relied entirely on animal experimentation, a relatively expensive and time-consuming process. Currently, use of immunoinformatics tools play a vital role in the antigen analysis and refinement, allowing the identification of possible protective epitopes capable of stimulating convenient humoral or cellular immune responses, in addition to facilitating time and cost reduction of vaccine production. The vaccination aimed at bacterial species of Clostridium spp. has been considered a promising example of use of these approaches in recent years. Based on the literature search, it is possible to understand the best immunoinformatics software used by researchers that facilitate recombinant vaccine antigens design and development. This chapter presents an overview of how these tools are supporting the antigen engineering, aiming at increasing the efficiency of inducing protective immune response in animals.

In silico analyses and design of a chimeric protein containing epitopes of SpaC, PknG, NanH, and SodC proteins for the control of caseous lymphadenitis.

Caseous lymphadenitis (CLA) is an infectious disease that affects goats and sheep causing drastic impacts on milk and meat production and is caused by Corynebacterium pseudotuberculosis. The disease can be prevented through vaccination but currently, vaccines demonstrate limited efficacy consequently leading to a need for the development of new ones. Here, we described the in silico development of a new chimeric protein constructed with epitopes identified from the sequences of the genes nanH, pknG, spaC, and sodC, previously described as potential vaccinal targets against C. pseudotuberculosis. The chimera was expressed, purified, and its immunogenicity was evaluated using sera of immunized mice. Results indicate the chimeric protein was able to stimulate antibody production. Additionally, analysis using serum from naturally infected goats showed that the protein is recognized by sera from these animals, indicating the possibility for using this chimera in new diagnostic methods. KEY POINTS: • The chimera was expressed with 52 kDa and a yield of 7 mg/L after purification. • The chimera was recognized by the sera of animals immunized with this formulation. • Chimera reacted with the serum of goats naturally infected with C. pseudotuberculosis.

Three challenging cases of infections by multidrug-resistant Serratia marcescens in patients admitted to intensive care units.

The occurrence of multidrug-resistant Serratia marcescens strains represents a serious public health threat. The purpose here is to report three cases of carbapenem-resistant S. marcescens infections with unfavorable clinical outcomes and provide a molecular description of the antibiotic resistance determinants at a genomic level. We performed bacterial identification by VITEK 2 and MALDI-TOF. The minimal inhibitory concentrations of antimicrobials were determined according to the Clinical and Laboratory Standards Institute guidelines, except for tigecycline, for which they were determined using Etest strips. Preliminary screening for the presence of carbapenemases was performed by ertapenem hydrolysis using MALDI-TOF MS. Whole-genome sequencing was provided to identify genes responsible for virulence and antimicrobial resistance. Here we report three challenging cases of S. marcescens that were resistant to the most commonly used antibiotics. Otherwise, we performed a genome description, which includes several genes involved in the resistance and virulence. These cases illustrate serious infection due to multidrug-resistant organisms and the complexity of treatment. Our results highlight the need to evaluate isolates regularly during long-term hospital stay to achieve optimal quality of clinical care and thus improve patient outcomes.

Seroepidemiological investigation of animal leptospirosis and molecular characterization of the first Leptospira strain isolated from Fernando de Noronha archipelago, Brazil.

Leptospirosis has been widely reported in insular environments worldwide, characterizing a major public health threat. Although low-genetic biodiversity is expected in these regions, the introduction of domestic and synanthropic mammals may contribute to the wider diversity of leptospiral strains in insular settings. This study proposes a large-scale seroepidemiological investigation of Leptospira infection in animals from Fernando de Noronha archipelago and describes the characterization of the first leptospiral strain ever isolated from an insular setting in Brazil. A total of 1,265 blood samples from domestic (n = 682), synanthropic (n = 133) and wild (n = 450) animals were collected between 2007 and 2014, totalling 12 species. The presence of anti-Leptospira spp. antibodies was investigated by the microscopic agglutination test (MAT), and kidney samples from 20 synanthropic rodents were collected for the isolation of Leptospira spp. The leptospires recovered were further characterized by serogrouping with polyclonal antibodies, whole-genome sequencing and multilocus sequence typing (MLST). The MAT results revealed the presence of agglutinins in 90 samples (7.1%) and the most frequently found serogroup was Icterohaemorrhagiae (n = 57) in practically all species included. Viable leptospires were recovered from one brown rat, and characterization revealed that the isolate belongs to L. interrogans serogroup Pyrogenes. The results suggest that synanthropic rodents might play an important role in leptospiral infection among wildlife and domestic species in the archipelago.

Transfection of exogenous DNA complexed to cationic dendrimer induces alterations of bovine sperm microRNAome.

MicroRNAs have been hypothesized to be involved in the regulation of male fertility potential. The primary aim of our study was to demonstrate the effects of transfection with dendrimer nanostructure on the parameters of bovine sperm quality and to investigate whether the microRNA profile could be disturbed after cationic dendrimer-mediated exogenous DNA transfection of bovine spermatozoa. The binding of exogenous DNA was significantly increased when dendrimer-based transfection was implemented. However, cationic dendrimer transfection induced detrimental changes in the kinetics and sperm quality parameters, such as membrane integrity, acrosome reaction, and mitochondrial membrane potential, when compared to the control group. Sperm microRNA sequencing revealed 218 known and 106 novel microRNAs in the sperm samples, among which nine were dysregulated after transfection (one was upregulated and eight were downregulated), in comparison to the non-transfected sperm. All the dysregulated microRNAs were related to sperm quality and embryonic development. These results suggest that the transfection process using the dendrimer nanostructure has an impact on the quality and microRNA profile of bovine sperm.

Transcriptome Analysis Identifies Candidate Target Genes Involved in Glyphosate-Resistance Mechanism in Lolium multiflorum.

Italian ryegrass (Lolium multiflorum; LOLMU) is one of the most troublesome weeds in temperate regions in the world. This weed species interfere with wheat, corn, rye, and oat, causing significant crop yield losses. This species has evolved glyphosate resistance, making it difficult to control. The mechanisms of glyphosate resistance are still unknown, and an understanding thereof will favor the development of new strategies of management. The present study is the first transcriptome study in LOLMU using glyphosate-resistant and -sensitive biotypes, aiming to identify and to provide a list of the candidate target genes related to glyphosate resistance mechanism. The transcriptome was assembled de novo, producing 87,433 contigs with an N50 of 740 bp and an average length of 575 bp. There were 92 and 54 up- and down-regulated genes, respectively, in the resistant biotype, while a total of 1683 were differentially expressed in the sensitive biotype in response to glyphosate treatment. We selected 14 highly induced genes and seven with repressed expression in the resistant biotype in response to glyphosate. Of these genes, a significant proportion were related to the plasma membrane, indicating that there is a barrier making it difficult for glyphosate to enter the cell.

First transcriptome of the Neotropical pest Euschistus heros (Hemiptera: Pentatomidae) with dissection of its siRNA machinery.

Over the past few years, the use of RNA interference (RNAi) for insect pest management has attracted considerable interest in academia and industry as a pest-specific and environment-friendly strategy for pest control. For the success of this technique, the presence of core RNAi genes and a functional silencing machinery is essential. Therefore, the aim of this study was to test whether the Neotropical brown stinkbug Euschistus heros has the main RNAi core genes and whether the supply of dsRNA could generate an efficient gene silencing response. To do this, total mRNA of all developmental stages was sequenced on an Illumina platform, followed by a de novo assembly, gene annotation and RNAi-related gene identification. Once RNAi-related genes were identified, nuclease activities in hemolymph were investigated through an ex vivo assay. To test the functionality of the siRNA machinery, E. heros adults were microinjected with ~28 ng per mg of insect of a dsRNA targeting the V-ATPase-A gene. Mortality, relative transcript levels of V-ATPase-A, and the expression of the genes involved in the siRNA machinery, Dicer-2 (DCR-2) and Argonaute 2 (AGO-2), were analyzed. Transcriptome sequencing generated more than 126 million sequenced reads, and these were annotated in approximately 80,000 contigs. The search of RNAi-related genes resulted in 47 genes involved in the three major RNAi pathways, with the absence of sid-like homologous. Although ex vivo incubation of dsRNA in E. heros hemolymph showed rapid degradation, there was 35% mortality at 4 days after treatment and a significant reduction in V-ATPase-A gene expression. These results indicated that although sid-like genes are lacking, the dsRNA uptake mechanism was very efficient. Also, 2-fold and 4-fold overexpression of DCR-2 and AGO-2, respectively, after dsRNA supply indicated the activation of the siRNA machinery. Consequently, E. heros has proven to be sensitive to RNAi upon injection of dsRNA into its hemocoel. We believe that this finding together with a publically available transcriptome and the validation of a responsive RNAi machinery provide a starting point for future field applications against one of the most important soybean pests in South America.

Campylobacter jejuni isolated from poultry meat in Brazil: in silico analysis and genomic features of two strains with different phenotypes of antimicrobial susceptibility.

Campylobacter jejuni is the most common bacterial cause of foodborne diarrheal disease worldwide and is among the antimicrobial resistant "priority pathogens" that pose greatest threat to public health. The genomes of two C. jejuni isolated from poultry meat sold on the retail market in Southern Brazil phenotypically characterized as multidrug-resistant (CJ100) and susceptible (CJ104) were sequenced and analyzed by bioinformatic tools. The isolates CJ100 and CJ104 showed distinct multilocus sequence types (MLST). Comparative genomic analysis revealed a large number of single nucleotide polymorphisms, rearrangements, and inversions in both genomes, in addition to virulence factors, genomic islands, prophage sequences, and insertion sequences. A circular 103-kilobase megaplasmid carrying virulence factors was identified in the genome of CJ100, in addition to resistance mechanisms to aminoglycosides, beta-lactams, macrolides, quinolones, and tetracyclines. The molecular characterization of distinct phenotypes of foodborne C. jejuni and the discovery of a novel virulence megaplasmid provide useful data for pan-genome and large-scale studies to monitor the virulent C. jejuni in poultry meat is warranted.

TargeTALE: A Web Resource to Identify TALEs in Xanthomonas Genomes and Their Respective Targets.

The Xanthomonas genus, comprises more than 30 species of gram-negative bacteria, most of which are pathogens of plants with high economic value, such as rice, common bean, and maize. Transcription activator-like effectors (TALEs), which act by regulating the host gene expression, are some of the major virulence factors of these bacteria. We present a novel tool to identify TALE genes in the genome of Xanthomonas strains and their respective targets. The analysis of the results obtained by TargeTALE in a proof-of-concept validation demonstrate that, at optimum setting, approximately 93% of the predicted target genes with available expression data were confirmed as upregulated during the infection, indicating that the tool might be useful for researchers in the field.

The South American Fruit Fly: An Important Pest Insect With RNAi-Sensitive Larval Stages.

RNA interference (RNAi) technology has been used in the development of approaches for pest control. The presence of some essential genes, the so-called "core genes," in the RNAi machinery is crucial for its efficiency and robust response in gene silencing. Thus, our study was designed to examine whether the RNAi machinery is functional in the South American (SA) fruit fly Anastrepha fraterculus (Diptera: Tephritidae) and whether the sensitivity to the uptake of double-stranded RNA (dsRNA) could generate an RNAi response in this fruit fly species. To prepare a transcriptome database of the SA fruit fly, total RNA was extracted from all the life stages for later cDNA synthesis and Illumina sequencing. After the de novo transcriptome assembly and gene annotation, the transcriptome was screened for RNAi pathway genes, as well as the duplication or loss of genes and novel target genes to dsRNA delivery bioassays. The dsRNA delivery assay by soaking was performed in larvae to evaluate the gene-silencing of V-ATPase, and the upregulation of Dicer-2 and Argonaute-2 after dsRNA delivery was analyzed to verify the activation of siRNAi machinery. We tested the stability of dsRNA using dsGFP with an in vitro incubation of larvae body fluid (hemolymph). We identified 55 genes related to the RNAi machinery with duplication and loss for some genes and selected 143 different target genes related to biological processes involved in post-embryonic growth/development and reproduction of A. fraterculus. Larvae soaked in dsRNA (dsV-ATPase) solution showed a strong knockdown of V-ATPase after 48 h, and the expression of Dicer-2 and Argonaute-2 responded with an increase upon the exposure to dsRNA. Our data demonstrated the existence of a functional RNAi machinery in the SA fruit fly, and we present an easy and robust physiological bioassay with the larval stages that can further be used for screening of target genes at in vivo organisms' level for RNAi-based control of fruit fly pests. This is the first study that provides evidence of a functional siRNA machinery in the SA fruit fly.