Food contamination with fipronil alters gene expression associated with foraging in Africanized honey bees.

Taking into consideration that bees can be contaminated by pesticides through the ingestion of contaminated floral resources, we can utilize genetic techniques to assess effects that are scarcely observed in behavioral studies. This study aimed to investigate the genetic effects of ingesting lethal and sublethal doses of the insecticide fipronil in foraging honey bees during two periods of acute exposure. Bees were exposed to fipronil through contaminated honey syrup at two dosages (LD50 = 0.19 µg/bee; LD50/100 = 0.0019 µg/bee) and for two durations (1 and 4 h). Following exposure, we measured syrup consumption per bee, analyzed the transcriptome of bee brain tissue, and identified differentially expressed genes (DEGs), categorizing them functionally based on gene ontology (GO). The results revealed a significant genetic response in honey bees after exposure to fipronil, regardless of the dosage used. Fipronil affected various metabolic, transport, and cellular regulation pathways, as well as detoxification processes and xenobiotic substance detection. Additionally, the downregulation of several DEGs belonging to the olfactory-binding protein (OBP) family was observed, suggesting potential physiological alterations in bees that may lead to disoriented behaviors and reduced foraging efficiency.

Impact of Long-Term Pyriproxyfen Exposure on the Genetic Structure and Diversity of Aedes aegypti and Aedes albopictus in Manaus, Amazonas, Brazil.

Aedes aegypti and Aedes albopictus are responsible for transmitting major human arboviruses such as Dengue, Zika, and Chikungunya, posing a global threat to public health. The lack of etiological treatments and efficient vaccines makes vector control strategies essential for reducing vector population density and interrupting the pathogen transmission cycle. This study evaluated the impact of long-term pyriproxyfen exposure on the genetic structure and diversity of Ae. aegypti and Ae. albopictus mosquito populations. The study was conducted in Manaus, Amazonas, Brazil, where pyriproxyfen dissemination stations have been monitored since 2014 up to the present day. Double digest restriction-site associated DNA sequencing was performed, revealing that despite significant local population reductions by dissemination stations with pyriproxyfen in various locations in Brazil, focal intervention has no significant impact on the population stratification of these vectors in urban scenarios. The genetic structuring level of Ae. aegypti suggests it is more stratified and directly affected by pyriproxyfen intervention, while for Ae. albopictus exhibits a more homogeneous and less structured population. The results suggest that although slight differences are observed among mosquito subpopulations, intervention focused on neighborhoods in a capital city is not efficient in terms of genetic structuring, indicating that larger-scale pyriproxyfen interventions should be considered for more effective urban mosquito control.

Intake of imidacloprid in lethal and sublethal doses alters gene expression in Apis mellifera bees.

Bees are important pollinators for ecosystems and agriculture; however, populations have suffered a decline that may be associated with several factors, including habitat loss, climate change, increased vulnerability to diseases and parasites and use of pesticides. The extensive use of neonicotinoids, including imidacloprid, as agricultural pesticides, leads to their persistence in the environment and accumulation in bees, pollen, nectar, and honey, thereby inducing deleterious effects. Forager honey bees face significant exposure to pesticide residues while searching for resources outside the hive, particularly systemic pesticides like imidacloprid. In this study, 360 Apis mellifera bees, twenty-one days old (supposed to be in the forager phase) previously marked were fed syrup (honey and water, 1:1 m/v) containing a lethal dose (0.081 µg/bee) or sublethal dose (0.00081 µg/bee) of imidacloprid. The syrup was provided in plastic troughs, with 250 µL added per trough onto each plastic Petri dish containing 5 bees (50 µL per bee). The bees were kept in the plastic Petri dishes inside an incubator, and after 1 and 4 h of ingestion, the bees were euthanised and stored in an ultra-freezer (-80 °C) for transcriptome analysis. Following the 1-h ingestion of imidacloprid, 1516 genes (73 from lethal dose; 1509 from sublethal dose) showed differential expression compared to the control, while after 4 h, 758 genes (733 from lethal dose; 25 from sublethal) exhibited differential expression compared to the control. All differentially expressed genes found in the brain tissue transcripts of forager bees were categorised based on gene ontology into functional groups encompassing biological processes, molecular functions, and cellular components. These analyses revealed that sublethal doses might be capable of altering more genes than lethal doses, potentially associated with a phenomenon known as insecticide-induced hormesis. Alterations in genes related to areas such as the immune system, nutritional metabolism, detoxification system, circadian rhythm, odour detection, foraging activity, and memory in bees were present after exposure to the pesticide. These findings underscore the detrimental effects of both lethal and sublethal doses of imidacloprid, thereby providing valuable insights for establishing public policies regarding the use of neonicotinoids, which are directly implicated in the compromised health of Apis mellifera bees.

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.

High Parasitic Loads Quantified in Sylvatic Triatoma melanica, a Chagas Disease Vector.

Triatoma melanica is a sylvatic vector species in Brazil. In We aimed to characterize the Trypanosoma cruzi discrete typing units (DTUs), the parasitic loads, and the blood meal sources of insects collected in rocky outcrops in rural areas in the state of Minas Gerais. An optical microscope (OM) and kDNA-PCR were used to examine natural infection by T. cruzi, and positive samples were genotyped by conventional multilocus PCR. Quantification of the T. cruzi load was performed using qPCR, and the blood meal sources were identified by Sanger sequencing the 12S rRNA gene. A total of 141 T. melanica were captured. Of these, ~55% (61/111) and ~91% (63/69) were positive by OM and KDNA-PCR, respectively. We genotyped ~89% (56/63) of the T. cruzi-positive triatomines, with TcI (~55%, 31/56) being the most prevalent DTU, followed by TcIII (~20%, 11/56) and TcII (~7%, 4/56). Only TcI+TcIII mixed infections were detected in 10 (~18%) specimens. A wide range of variation in the parasitic loads of T. melanica was observed, with an overall median value of 104 parasites/intestine, with females having higher T. cruzi loads than N2, N4, and N5. TcII showed lower parasitic loads compared to TcI and TcIII. The OM positive diagnosis odds ratio between T. cruzi infection when the parasite load is 107 compared to 103 was approximately 29.1. The most frequent blood meal source was Kerodon rupestris (~58%), followed by Thrichomys apereoides (~18%), Wiedomys cerradensis (~8%), Galactis cuja (~8%) and Gallus gallus (~8%). Our findings characterize biological and epidemiological aspects of the sylvatic population of T. melanica in the study area, highlighting the need to extend surveillance and control to this vector.

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.

Zinc supplementation modifies brain tissue transcriptome of Apis mellifera honeybees.

BACKGROUND: Bees are the most important group of pollinators worldwide and their populations are declining. In natural conditions, Apis mellifera depends exclusively on food from the field to meet its physiological demands. In the period of scarcity, available resources are insufficient and artificial supplementation becomes essential for maintaining the levels of vitamins, proteins, carbohydrates, and minerals of colonies. Among these minerals, zinc is essential in all living systems, particularly for the regulation of cell division and protein synthesis, and is a component of more than 200 metalloenzymes. RESULTS: The total RNA extracted from the brain tissue of nurse bees exposed to different sources and concentrations of zinc was sequenced. A total of 1,172 genes in the treatment that received an inorganic source of zinc and 502 genes that received an organic source of zinc were found to be differentially expressed among the control group. Gene ontology enrichment showed that zinc can modulate important biological processes such as nutrient metabolism and the molting process. CONCLUSIONS: Our results indicate that zinc supplementation modulates the expression of many differentially expressed genes and plays an important role in the development of Apis mellifera bees. All the information obtained in this study can contribute to future research in the field of bee nutrigenomics.

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.

Genotypic Trypanosoma cruzi distribution and parasite load differ ecotypically and according to parasite genotypes in Triatoma brasiliensis from endemic and outbreak areas in Northeastern Brazil.

This study aimed to identify the Trypanosoma cruzi genotypes and their relationship with parasitic load in distinct geographic and ecotypic populations of Triatoma brasiliensis in two sites, including one where a Chagas disease (ChD) outbreak occurred in Rio Grande do Norte state, Brazil. Triatomine captures were performed in peridomestic and sylvatic ecotopes in two municipalities: Marcelino Vieira - affected by the outbreak; and Currais Novos - where high pressure of peridomestic triatomine infestation after insecticide spraying have been reported. The kDNA-PCR was used to select 124 T. cruzi positive triatomine samples, of which 117 were successfully genotyped by fluorescent fragment length barcoding (FFLB). Moreover, the T. cruzi load quantification was performed using a multiplex TaqMan qPCR. Our findings showed a clear ecotypic segregation between TcI and TcII harboured by T. brasiliensis (p<0.001). Although no genotypes were ecotypically exclusive, TcI was predominant in peridomestic ecotopes (86%). In general, T. brasiliensis from Rio Grande do Norte had a higher T. cruzi load varying from 3.94 to 7.66 x 106T. cruzi per insect. Additionally, TcII (median value=299,504 T. cruzi/intestine unit equivalents) had more than twice (p=0.1) the parasite load of TcI (median value=149,077 T. cruzi/intestine unit equivalents), which can be attributed to a more ancient co-evolution with T. brasiliensis. The higher prevalence of TcII in the sylvatic T. brasiliensis (70%) could be associated with a more diversified source of bloodmeals for wild insect populations. Either TcI or TcII may have been responsible for the ChD outbreak that occurred in the city of Marcelino Vieira. On the other hand, a smaller portion of T. brasiliensis was infected by TcIII (3%) in the peridomicile, in addition to T. rangeli genotype A (1%), often found in mixed infections. Our results highlight the need of understanding the patterns of T. cruzi genotype´s development and circulation in insect vectors and reservoirs as a mode of tracking situations of epidemiologic importance, as the ChD outbreak recently recorded for Northeastern Brazil.

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.

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.