Bacterial diversity in Haemagogus leucocelaenus (Diptera: Culicidae) from Vale do Ribeira, São Paulo, Brazil.

INTRODUCTION: Mosquitoes (Diptera: Culicidae) are vectors that transmit numerous pathogens to humans and other vertebrates. Haemagogus leucocelaenus is a mosquito associated with transmission of yellow fever virus. The insect gut harbors a variety of microorganisms that can live and multiply within it, thus contributing to digestion, nutrition, and development of its host. The composition of bacterial communities in mosquitoes can be influenced by both biotic and abiotic factors. The goal of this study was to investigate the bacterial diversity of Hg. leucocelaenus and verify the differences between the bacterial communities in Hg. leucocelaenus from three different locations in the Atlantic tropical rain forest and southeastern state of São Paulo State, Brazil. RESULTS: The phylum Proteobacteria was found in mosquitoes collected from the three selected study sites. More than 50% of the contigs belong to Wolbachia, followed by 5% Swaminathania, and 3% Acinetobacter. The genus Serratia was found in samples from two locations. CONCLUSIONS: Wolbachia was reported for the first time in this species and may indicates that the vector competence of the populations of the species can vary along its geographical distribution area. The presence of Serratia might facilitate viral invasion caused by the disruption of the midgut barrier via action of the SmEnhancin protein, which digests the mucins present in the intestinal epithelium.

The potential of genome-wide RAD sequences for resolving rapid radiations: a case study in Cactaceae.

The reconstruction of relationships within recently radiated groups is challenging even when massive amounts of sequencing data are available. The use of restriction site-associated DNA sequencing (RAD-Seq) to this end is promising. Here, we assessed the performance of RAD-Seq to infer the species-level phylogeny of the rapidly radiating genus Cereus (Cactaceae). To examine how the amount of genomic data affects resolution in this group, we used datasets and implemented different analyses. We sampled 52 individuals of Cereus, representing 18 of the 25 species currently recognized, plus members of the closely allied genera Cipocereus and Praecereus, and other 11 Cactaceae genera as outgroups. Three scenarios of permissiveness to missing data were carried out in iPyRAD, assembling datasets with 30% (333 loci), 45% (1440 loci), and 70% (6141 loci) of missing data. For each dataset, Maximum Likelihood (ML) trees were generated using two supermatrices, i.e., only SNPs and SNPs plus invariant sites. Accuracy and resolution were improved when the dataset with the highest number of loci was used (6141 loci), despite the high percentage of missing data included (70%). Coalescent trees estimated using SVDQuartets and ASTRAL are similar to those obtained by the ML reconstructions. Overall, we reconstruct a well-supported phylogeny of Cereus, which is resolved as monophyletic and composed of four main clades with high support in their internal relationships. Our findings also provide insights into the impact of missing data for phylogeny reconstruction using RAD loci.

Occurrence of Cyanobacteria and microcystins in hydroelectric reservoirs used for fish farming.

Fish farming can have a negative impact on water quality and aquatic organisms due to emerging blooms of Cyanobacteria and the production of cyanotoxins. In this study, the effect of aquaculture in hydroelectric reservoirs in Brazil was evaluated in six fish farms and in upstream and downstream water through analysis of the microbiome, Cyanobacteria and microcystin concentrations. Synechococcus and Microcystis were observed at all six locations, while Limnothrix was also observed abundantly at two locations. An increase in the relative abundance of Cyanobacteria inside the fish farms was observed at two locations, while an increase of Cyanobacteria was observed in downstream at five of the six locations. Microcystins were detected in significant and high values in all locations, with concentrations up to 1.59 µg/L. The trend in microcystin concentrations was mirrored in copy numbers of the mcyE gene (encodes microcystin synthetase) and presence of Microcystis, but not in any of the other observed cyanobacterial groups. In summary, the study shows that aquaculture production influenced the water microbiome inside and downstream the fish farms, and a direct correlation was found between mcyE gene copies, microcystin production and abundance of Microcystis, but not for the total abundance of Cyanobacteria.

Standardization of DNA extraction from sand flies: Application to genotyping by next generation sequencing.

Standardization of the methods for extraction of DNA from sand flies is essential for obtaining high efficiency during subsequent molecular analyses, such as the new sequencing methods. Information obtained using these methods may contribute substantially to taxonomic, evolutionary, and eco-epidemiological studies. The aim of the present study was to standardize and compare two methods for the extraction of genomic DNA from sand flies for obtaining DNA in sufficient quantities for next-generation sequencing. Sand flies were collected from the municipalities of Campo Grande, Camapuã, Corumbá and Miranda, state of Mato Grosso do Sul, Brazil. Three protocols using a silica column-based commercial kit (ReliaPrep™ Blood gDNA Miniprep System kit, Promega®), and three protocols based on the classical phenol-chloroform extraction method (Uliana et al., 1991), were compared with respect to the yield and quality of the extracted DNA. DNA was quantified using a Qubit 2.0 fluorometer. The presence of sand fly DNA was confirmed by PCR amplification of the IVS6 region (constitutive gene), followed by electrophoresis on a 1.5% agarose gel. A total of 144 male specimens were analyzed, 72 per method. Significant differences were observed between the two methods tested. Protocols 2 and 3 of phenol-chloroform extraction presented significantly better performance than all commercial kit extraction protocols tested. For phenol-chloroform extraction, protocol 3 presented significantly better performance than protocols 1 and 2. The IVS6 region was detected in 70 of 72 (97.22%) samples extracted with phenol, including all samples for protocols 2 and 3. This is the first study on the standardization of methods for the extraction of DNA from sand flies for application to next-generation sequencing, which is a promising tool for entomological and molecular studies of sand flies.

Lipoprotein lipase and PPAR alpha gene polymorphisms, increased very-low-density lipoprotein levels, and decreased high-density lipoprotein levels as risk markers for the development of visceral leishmaniasis by Leishmania infantum.

In visceral leishmaniasis (VL) endemic areas, a minority of infected individuals progress to disease since most of them develop protective immunity. Therefore, we investigated the risk markers of VL within nonimmune sector. Analyzing infected symptomatic and, asymptomatic, and noninfected individuals, VL patients presented with reduced high-density lipoprotein cholesterol (HDL-C), elevated triacylglycerol (TAG), and elevated very-low-density lipoprotein cholesterol (VLDL-C) levels. A polymorphism analysis of the lipoprotein lipase (LPL) gene using HindIII restriction digestion (N = 156 samples) (H+ = the presence and H- = the absence of mutation) revealed an increased adjusted odds ratio (OR) of VL versus noninfected individuals when the H+/H+ was compared with the H-/H- genotype (OR = 21.3; 95% CI = 2.32-3335.3; P = 0.003). The H+/H+ genotype and the H+ allele were associated with elevated VLDL-C and TAG levels (P < 0.05) and reduced HDL-C levels (P < 0.05). An analysis of the L162V polymorphism in the peroxisome proliferator-activated receptor alpha (PPARα) gene (n = 248) revealed an increased adjusted OR when the Leu/Val was compared with the Leu/Leu genotype (OR = 8.77; 95% CI = 1.41-78.70; P = 0.014). High TAG (P = 0.021) and VLDL-C (P = 0.023) levels were associated with susceptibility to VL, whereas low HDL (P = 0.006) levels with resistance to infection. The mutated LPL and the PPARα Leu/Val genotypes may be considered risk markers for the development of VL.

Identification of blood meal sources of Lutzomyia longipalpis using polymerase chain reaction-restriction fragment length polymorphism analysis of the cytochrome B gene.

An analysis of the dietary content of haematophagous insects can provide important information about the transmission networks of certain zoonoses. The present study evaluated the potential of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the mitochondrial cytochrome B (cytb) gene to differentiate between vertebrate species that were identified as possible sources of sandfly meals. The complete cytb gene sequences of 11 vertebrate species available in the National Center for Biotechnology Information database were digested with Aci I, Alu I, Hae III and Rsa I restriction enzymes in silico using Restriction Mapper software. The cytb gene fragment (358 bp) was amplified from tissue samples of vertebrate species and the dietary contents of sandflies and digested with restriction enzymes. Vertebrate species presented a restriction fragment profile that differed from that of other species, with the exception of Canis familiaris and Cerdocyon thous. The 358 bp fragment was identified in 76 sandflies. Of these, 10 were evaluated using the restriction enzymes and the food sources were predicted for four: Homo sapiens (1), Bos taurus (1) and Equus caballus (2). Thus, the PCR-RFLP technique could be a potential method for identifying the food sources of arthropods. However, some points must be clarified regarding the applicability of the method, such as the extent of DNA degradation through intestinal digestion, the potential for multiple sources of blood meals and the need for greater knowledge regarding intraspecific variations in mtDNA.

Bacterial Microbiota from Lab-Reared and Field-Captured Anopheles darlingi Midgut and Salivary Gland.

Anopheles darlingi is a major malaria vector in the Amazon region and, like other vectors, harbors a community of microorganisms with which it shares a network of interactions. Here, we describe the diversity and bacterial composition from the midguts and salivary glands of lab-reared and field-captured An. darlingi using metagenome sequencing of the 16S rRNA gene. The libraries were built using the amplification of the region V3-V4 16S rRNA gene. The bacterial community from the salivary glands was more diverse and richer than the community from the midguts. However, the salivary glands and midguts only showed dissimilarities in beta diversity between lab-reared mosquitoes. Despite that, intra-variability was observed in the samples. Acinetobacter and Pseudomonas were dominant in the tissues of lab-reared mosquitoes. Sequences of Wolbachia and Asaia were both found in the tissue of lab-reared mosquitoes; however, only Asaia was found in field-captured An. darlingi, but in low abundance. This is the first report on the characterization of microbiota composition from the salivary glands of An. darlingi from lab-reared and field-captured individuals. This study can provide invaluable insights for future investigations regarding mosquito development and interaction between mosquito microbiota and Plasmodium sp.

Host Feeding Patterns of Mansonia (Diptera, Culicidae) in Rural Settlements near Porto Velho, State of Rondonia, Brazil.

Mosquito females of the genus Mansonia (Blanchard) can be a nuisance to humans and animals since they are voraciously hematophagous and feed on the blood of a variety of vertebrates. Despite their relevance, there is a lack of investigation into the blood-feeding patterns of the Mansonia species. Knowledge of the host preference is crucial in establishing the public health importance of a mosquito species and its potential to be involved in the transmission dynamics of pathogens. Species that are primarily anthropophilic can be more effective in spreading vector-borne pathogens to humans. In this study, we used an Illumina Nextera sequencing protocol and the QIIME2 workflow to assess the diversity of DNA sequences extracted in the ingested blood of mosquito species to evaluate the overall and local host choices for three species: Ma. titillans, Ma. Amazonensis, and Ma. humeralis, in rural areas alongside the Madeira River in the vicinities of the Santo Antonio Energia (SAE) reservoir in the municipality of Porto Velho, Rondônia, Western Brazil. By performing our analysis pipeline, we have found that host diversity per collection site showed a significant heterogeneity across the sample sites. In addition, in rural areas, Ma. amazonensis present a high affinity for B. taurus, Ma. humeralis shows an overall preference for C. familiaris and B. taurus, but also H. sapiens and E. caballus in urban areas, and Ma. titillans showed more opportunistic behavior in rural areas, feeding on wild animals and G. gallus, though with an overall preference for H. sapiens.

Genetic Structuring of One of the Main Vectors of Sylvatic Yellow Fever: Haemagogus (Conopostegus) leucocelaenus (Diptera: Culicidae).

Genetic diversity and population structuring for the species Haemogogus leucocelaenus, a sylvatic vector of yellow fever virus, were found to vary with the degree of agricultural land use and isolation of fragments of Atlantic Forest in municipalities in the state of São Paulo where specimens were collected. Genotyping of 115 mitochondrial SNPs showed that the populations with the highest indices of genetic diversity (polymorphic loci and mean pairwise differences between the sequences) are found in areas with high levels of agricultural land use (northeast of the State). Most populations exhibited statistically significant negative values for the Tajima D and Fu FS neutrality tests, suggesting recent expansion. The results show an association between genetic diversity in this species and the degree of agricultural land use in the sampled sites, as well as signs of population expansion of this species in most areas, particularly those with the highest forest edge densities. A clear association between population structuring and the distance between the sampled fragments (isolation by distance) was observed: samples from a large fragment of Atlantic Forest extending along the coast of the state of São Paulo exhibited greater similarity with each other than with populations in the northwest of the state.

Next-Generation High-Throughput Sequencing to Evaluate Bacterial Communities in Freshwater Ecosystem in Hydroelectric Reservoirs.

The quality of aquatic ecosystems is a major public health concern. The assessment and management of a freshwater system and the ecological monitoring of microorganisms that are present in it can provide indicators of the environment and water quality to protect human and animal health. with bacteria is. It is a major challenge to monitor the microbiological bacterial contamination status of surface water associated with anthropogenic activities within rivers and freshwater reservoirs. Understanding the composition of aquatic microbial communities can be beneficial for the early detection of pathogens, improving our knowledge of their ecological niches, and characterizing the assemblages of microbiota responsible for the degradation of contaminants and microbial substrates. The present study aimed to characterize the bacterial microbiota of water samples collected alongside the Madeira River and its small tributaries in rural areas near the Santo Antonio Energia hydroelectric power plant (SAE) reservoir in the municipality of Porto Velho, Rondonia state, Western Brazil. An Illumina 16s rRNA metagenomic approach was employed and the physicochemical characteristics of the water sample were assessed. We hypothesized that both water metagenomics and physicochemical parameters would vary across sampling sites. The most abundant genera found in the study were Acinetobacter, Deinococcus, and Pseudomonas. PERMANOVA and ANCOM analysis revealed that collection points sampled at the G4 location presented a significantly different microbiome compared to any other group, with the Chlamidomonadaceae family and Enhydrobacter genus being significantly more abundant. Our findings support the use of metagenomics to assess water quality standards for the protection of human and animal health in this microgeographic region.

Mansonia spp. population genetics based on mitochondrion whole-genome sequencing alongside the Madeira River near Porto Velho, Rondonia, Brazil.

In high abundance, females of the genus Mansonia (Blanchard) can be a nuisance to humans and animals because they are voraciously hematophagous and feed on the blood of a myriad of vertebrates. The spatial-temporal distribution pattern of Mansonia species is associated with the presence of their host plants, usually Eichhornia crassipes, E. azurea, Ceratopteris pteridoides, Limnobium laevigatum, Pistia stratiotes, and Salvinia sp. Despite their importance, there is a lack of investigation on the dispersion and population genetics of Mansonia species. Such studies are pivotal to evaluating the genetic structuring, which ultimately reflects populational expansion-retraction patterns and dispersal dynamics of the mosquito, particularly in areas with a history of recent introduction and establishment. The knowledge obtained could lead to better understanding of how anthropogenic changes to the environment can modulate the population structure of Mansonia species, which in turn impacts mosquito population density, disturbance to humans and domestic animals, and putative vector-borne disease transmission patterns. In this study, we present an Illumina NGS sequencing protocol to obtain whole-mitogenome sequences of Mansonia spp. to assess the microgeographic genetic diversity and dispersion of field-collected adults. The specimens were collected in rural environments in the vicinities of the Santo Antônio Energia (SAE) hydroelectric reservoir on the Madeira River.

Nyssorhynchus darlingi genome-wide studies related to microgeographic dispersion and blood-seeking behavior.

BACKGROUND: In Brazil, malaria is concentrated in the Amazon Basin, where more than 99% of the annual cases are reported. The main goal of this study was to investigate the population structure and genetic association of the biting behavior of Nyssorhynchus (also known as Anopheles) darlingi, the major malaria vector in the Amazon region of Brazil, using low-coverage genomic sequencing data. METHODS: Samples were collected in the municipality of Mâncio Lima, Acre state, Brazil between 2016 and 2017. Different approaches using genotype imputation and no gene imputation for data treatment and low-coverage sequencing genotyping were performed. After the samples were genotyped, population stratification analysis was performed. RESULTS: Weak but statistically significant stratification signatures were identified between subpopulations separated by distances of approximately 2-3 km. Genome-wide association studies (GWAS) were performed to compare indoor/outdoor biting behavior and blood-seeking at dusk/dawn. A statistically significant association was observed between biting behavior and single nucleotide polymorphism (SNP) markers adjacent to the gene associated with cytochrome P450 (CYP) 4H14, which is associated with insecticide resistance. A statistically significant association between blood-seeking periodicity and SNP markers adjacent to genes associated with the circadian cycle was also observed. CONCLUSION: The data presented here suggest that low-coverage whole-genome sequencing with adequate processing is a powerful tool to genetically characterize vector populations at a microgeographic scale in malaria transmission areas, as well as for use in GWAS. Female mosquitoes entering houses to take a blood meal may be related to a specific CYP4H14 allele, and female timing of blood-seeking is related to circadian rhythm genes.

Susceptibility of Field-Collected Nyssorhynchus darlingi to Plasmodium spp. in Western Amazonian Brazil.

Mosquito susceptibility to Plasmodium spp. infection is of paramount importance for malaria occurrence and sustainable transmission. Therefore, understanding the genetic features underlying the mechanisms of susceptibility traits is pivotal to assessing malaria transmission dynamics in endemic areas. The aim of this study was to investigate the susceptibility of Nyssorhynchus darlingi-the dominant malaria vector in Brazil-to Plasmodium spp. using a reduced representation genome-sequencing protocol. The investigation was performed using a genome-wide association study (GWAS) to identify mosquito genes that are predicted to modulate the susceptibility of natural populations of the mosquito to Plasmodium infection. After applying the sequence alignment protocol, we generated the variant panel and filtered variants; leading to the detection of 202,837 SNPs in all specimens analyzed. The resulting panel was used to perform GWAS by comparing the pool of SNP variants present in Ny. darlingi infected with Plasmodium spp. with the pool obtained in field-collected mosquitoes with no evidence of infection by the parasite (all mosquitoes were tested separately using RT-PCR). The GWAS results for infection status showed two statistically significant variants adjacent to important genes that can be associated with susceptibility to Plasmodium infection: Cytochrome P450 (cyp450) and chitinase. This study provides relevant knowledge on malaria transmission dynamics by using a genomic approach to identify mosquito genes associated with susceptibility to Plasmodium infection in Ny. darlingi in western Amazonian Brazil.

Selenization of S. cerevisiae increases its protective potential in experimental autoimmune encephalomyelitis by triggering an intestinal immunomodulatory loop.

Multiple sclerosis is an autoimmune disease that affects the myelinated central nervous system (CNS) neurons and triggers physical and cognitive disabilities. Conventional therapy is based on disease-modifying drugs that control disease severity but can also be deleterious. Complementary medicines have been adopted and evidence indicates that yeast supplements can improve symptoms mainly by modulating the immune response. In this investigation, we evaluated the therapeutic potential of Saccharomyces cerevisiae and its selenized derivative (Selemax) in experimental autoimmune encephalomyelitis (EAE). Female C57BL/6 mice submitted to EAE induction were orally supplemented with these yeasts by gavage from day 0 to day 14 after EAE induction. Both supplements determined significant reduction in clinical signs concomitantly with diminished Th1 immune response in CNS, increased proportion of Foxp3+ lymphocytes in inguinal and mesenteric lymph nodes and increased microbiota diversity. However, Selemax was more effective clinically and immunologically; it reduced disease prevalence more sharply, increased the proportion of CD103+ dendritic cells expressing high levels of PD-L1 in mesenteric lymph nodes and reduced the intestinal inflammatory process more strongly than S. cerevisiae. These results suggest a clear gut-brain axis modulation by selenized S. cerevisiae and suggest their inclusion in clinical trials.

Gene expression profile of Aedes aegypti females in courtship and mating.

Aedes aegypti is the most synanthropic and anthropophilic mosquito of Culicidae. This species always cohabits with humans and is extremely opportunistic. Vector dispersal is directly related to the ability of the females on successfully finding a mate in a generally patchy urban scenario. In the present work, we investigate transcriptional changes in Ae. aegypti females during the courtship process and after mating. We observe a substantial alteration in gene expression triggered just upon contact with Ae. aegypti males, which in turn was not fully correlated to the changes triggered by the contact. After analysing shared significant differentially regulated genes between conspecific contact and insemination, the major part of the observed transcriptomic change triggered by contact is reversed after mating, indicating an intermediary situation between naive and mating conditions that we hypothesize to be crucial for mating success. Upon contact, several chemosensory related genes are repressed, especially odorant binding proteins. Most of these genes return to higher expression rates after mating. None of these genes are significantly regulated by the encounter of a different species, Aedes albopictus. The results presented here might be applied to an innovative control approach focusing on the semiochemical systems of mosquitoes in an effort to disrupt undesirable host-insect interaction to reduce the risk of pathogen transmission to humans.

Genetic diversity of Nyssorhynchus (Anopheles) darlingi related to biting behavior in western Amazon.

BACKGROUND: In the Amazon Basin, Nyssorhynchus (Anopheles) darlingi is the most aggressive and effective malaria vector. In endemic areas, behavioral aspects of anopheline vectors such as host preference, biting time and resting location post blood meal have a key impact on malaria transmission dynamics and vector control interventions. Nyssorhynchus darlingi presents a range of feeding and resting behaviors throughout its broad distribution. METHODS: To investigate the genetic diversity related to biting behavior, we collected host-seeking Ny. darlingi in two settlement types in Acre, Brazil: Granada (~ 20-year-old, more established, better access by road, few malaria cases) and Remansinho (~ 8-year-old, active logging, poor road access, high numbers malaria cases). Mosquitoes were classified by the location of collection (indoors or outdoors) and time (dusk or dawn). RESULTS: Genome-wide SNPs, used to assess the degree of genetic divergence and population structure, identified non-random distributions of individuals in the PCA for both location and time analyses. Although genetic diversity related to behavior was confirmed by non-model-based analyses and FST values, model-based STRUCTURE detected considerable admixture of these populations. CONCLUSIONS: To our knowledge, this is the first study to detect genetic markers associated with biting behavior in Ny. darlingi. Additional ecological and genomic studies may help to understand the genetic basis of mosquito behavior and address appropriate surveillance and vector control.

Genome Reduction in the Mosquito Symbiont Asaia.

Symbiosis is now recognized as a driving force in evolution, a role that finds its ultimate expression in the variety of associations bonding insects with microbial symbionts. These associations have contributed to the evolutionary success of insects, with the hosts acquiring the capacity to exploit novel ecological niches, and the symbionts passing from facultative associations to obligate, mutualistic symbioses. In bacterial symbiont of insects, the transition from the free-living life style to mutualistic symbiosis often resulted in a reduction in the genome size, with the generation of the smallest bacterial genomes thus far described. Here, we show that the process of genome reduction is still occurring in Asaia, a group of bacterial symbionts associated with a variety of insects. Indeed, comparative genomics of Asaia isolated from different mosquito species revealed a substantial genome size and gene content reduction in Asaia from Anopheles darlingi, a South-American malaria vector. We thus propose Asaia as a novel model to study genome reduction dynamics, within a single bacterial taxon, evolving in a common biological niche.

Macrogeographic genetic structure of Lutzomyia longipalpis complex populations using Next Generation Sequencing.

Lutzomyia longipalpis is the main vector of Leishmania infantum, the causative agent of visceral leishmaniasis in the Neotropical realm. Its taxonomic status has been widely discussed once it encompasses a complex of species. The knowledge about the genetic structure of insect vector populations helps the elucidation of components and interactions of the disease ecoepidemiology. Thus, the objective of this study was to genotypically analyze populations of the Lu. longipalpis complex from a macrogeographic perspective using Next Generation Sequencing. Polymorphism analysis of three molecular markers was used to access the levels of population genetic structure among nine different populations of sand flies. Illumina Amplicon Sequencing Protocol® was used to identify possible polymorphic sites. The library was sequenced on paired-end Illumina MiSeq platform. Significant macrogeographical population differentiation was observed among Lu. longipalpis populations via PCA and DAPC analyses. Our results revealed that populations of Lu. longipalpis from the nine municipalities were grouped into three clusters. In addition, it was observed that the levels of Lu. longipalpis population structure could be associated with distance isolation. This new sequencing method allowed us to study different molecular markers after a single sequencing run, and to evaluate population and inter-species differences on a macrogeographic scale.

Partial characterization of proteolytic activity in Giardia duodenalis purified proteins.

This report describes a preliminary characterization of proteolytic activity of proteins isolated from lysate of Giardia trophozoites of an axenic Brazilian strain. Fractions obtained by high-performance liquid chromatography (FPLC) were tested in SDS-polyacrylamide gel for the protein profiles, and the proteases activity was analyzed using gelatin impregnated SDS-PAGE. The proteases characterization was based on inhibition assays employing synthetic inhibitors for cysteine (E-64, IAA), serine (PMSF, TPCK, TLCK, and elastatinal), metalo (EDTA) and aspartic (pepstatin) proteases. Among thirty eluted fractions, polypeptide bands were observed in eight of them, however, proteolytic activity was detected in four ones (F23, F24, F25 and F26). Protein profiles of these fractions showed a banding pattern composed by few bands distributed in the migration region of 45 to < 18 kDa. The zymograms revealed proteolytic activity in all the four fractions assayed, mainly distributed in the migration region of 62 to 35 kDa. Among the profiles, the main pronounced zones of proteolysis were distinguished at 62, 55, 53, 50, 46 and 40 kDa. In inhibition assays, the protease activities were significantly inhibited by cysteine (E-64) and serine proteases (TPCK, TLCK and elastatinal) inhibitors. Gels incubated with other cysteine and serine protease inhibitors, IAA and PMSF, respectively, showed a decrease in the intensity of hydrolysis zones. Indeed, in the assays with the inhibitors EDTA for metalloproteases and pepstatin for aspartic proteases, none inhibition was detected against the substrate. These observations are relevants, especially if we consider that to define the real role of the proteases in host-parasite interaction, the purification of these enzymes for detailed studies may be warranted.

Microgeographical structure in the major Neotropical malaria vector Anopheles darlingi using microsatellites and SNP markers.

BACKGROUND: In recent decades, throughout the Amazon Basin, landscape modification contributing to profound ecological change has proceeded at an unprecedented rate. Deforestation that accompanies human activities can significantly change aspects of anopheline biology, though this may be site-specific. Such local changes in anopheline biology could have a great impact on malaria transmission. The aim of this study was to investigate population genetics of the main malaria vector in Brazil, Anopheles darlingi, from a microgeographical perspective. METHODS: Microsatellites and ddRADseq-derived single nucleotide polymorphisms (SNPs) were used to assess levels of population genetic structuring among mosquito populations from two ecologically distinctive agricultural settlements (~60 km apart) and a population from a distant (~700 km) urban setting in the western Amazon region of Brazil. RESULTS: Significant microgeographical population differentiation was observed among Anopheles darlingi populations via both model- and non-model-based analysis only with the SNP dataset. Microsatellites detected moderate differentiation at the greatest distances, but were unable to differentiate populations from the two agricultural settlements. Both markers showed low polymorphism levels in the most human impacted sites. CONCLUSIONS: At a microgeographical scale, signatures of genetic heterogeneity and population divergence were evident in Anopheles darlingi, possibly related to local environmental anthropic modification. This divergence was observed only when using high coverage SNP markers.