A genotyping array for the globally invasive vector mosquito, Aedes albopictus.

BACKGROUND: Although whole-genome sequencing (WGS) is the preferred genotyping method for most genomic analyses, limitations are often experienced when studying genomes characterized by a high percentage of repetitive elements, high linkage, and recombination deserts. The Asian tiger mosquito (Aedes albopictus), for example, has a genome comprising up to 72% repetitive elements, and therefore we set out to develop a single-nucleotide polymorphism (SNP) chip to be more cost-effective. Aedes albopictus is an invasive species originating from Southeast Asia that has recently spread around the world and is a vector for many human diseases. Developing an accessible genotyping platform is essential in advancing biological control methods and understanding the population dynamics of this pest species, with significant implications for public health. METHODS: We designed a SNP chip for Ae. albopictus (Aealbo chip) based on approximately 2.7 million SNPs identified using WGS data from 819 worldwide samples. We validated the chip using laboratory single-pair crosses, comparing technical replicates, and comparing genotypes of samples genotyped by WGS and the SNP chip. We then used the chip for a population genomic analysis of 237 samples from 28 sites in the native range to evaluate its usefulness in describing patterns of genomic variation and tracing the origins of invasions. RESULTS: Probes on the Aealbo chip targeted 175,396 SNPs in coding and non-coding regions across all three chromosomes, with a density of 102 SNPs per 1 Mb window, and at least one SNP in each of the 17,461 protein-coding genes. Overall, 70% of the probes captured the genetic variation. Segregation analysis found that 98% of the SNPs followed expectations of single-copy Mendelian genes. Comparisons with WGS indicated that sites with genotype disagreements were mostly heterozygotes at loci with WGS read depth < 20, while there was near complete agreement with WGS read depths > 20, indicating that the chip more accurately detects heterozygotes than low-coverage WGS. Sample sizes did not affect the accuracy of the SNP chip genotype calls. Ancestry analyses identified four to five genetic clusters in the native range with various levels of admixture. CONCLUSIONS: The Aealbo chip is highly accurate, is concordant with genotypes from WGS with high sequence coverage, and may be more accurate than low-coverage WGS.

Origins of high latitude introductions of Aedes aegypti to Nebraska and Utah during 2019.

Aedes aegypti (L.), the yellow fever mosquito, is also an important vector of dengue and Zika viruses, and an invasive species in North America. Aedes aegypti inhabits tropical and sub-tropical areas of the world and in North America is primarily distributed throughout the southern US states and Mexico. The northern range of Ae. aegypti is limited by cold winter months and establishment in these areas has been mostly unsuccessful. However, frequent introductions of Ae. aegypti to temperate, non-endemic areas during the warmer months can lead to seasonal activity and disease outbreaks. Two Ae. aegypti incursions were reported in the late summer of 2019 into York, Nebraska and Moab, Utah. These states had no history of established populations of this mosquito and no evidence of previous seasonal activity. We genotyped a subset of individuals from each location at 12 microsatellite loci and ~ 14,000 single nucleotide polymorphic markers to determine their genetic affinities to other populations worldwide and investigate their potential source of introduction. Our results support a single origin for each of the introductions from different sources. Aedes aegypti from Utah likely derived from Tucson, Arizona, or a nearby location. Nebraska specimen results were not as conclusive, but point to an origin from southcentral or southeastern US. In addition to an effective, efficient, and sustainable control of invasive mosquitoes, such as Ae. aegypti, identifying the potential routes of introduction will be key to prevent future incursions and assess their potential health threat based on the ability of the source population to transmit a particular virus and its insecticide resistance profile, which may complicate vector control.

Insecticide resistance and genetic structure of Aedes aegypti populations from Rio de Janeiro State, Brazil.

Vector control largely relies on neurotoxic chemicals, and insecticide resistance (IR) directly threatens their effectiveness. In some cases, specific alleles cause IR, and knowledge of the genetic diversity and gene flow among mosquito populations is crucial to track their arrival, rise, and spread. Here we evaluated Aedes aegypti populations' susceptibility status, collected in 2016 from six different municipalities of Rio de Janeiro state (RJ), to temephos, pyriproxyfen, malathion, and deltamethrin. We collected eggs of Ae. aegypti in Campos dos Goytacazes (Cgy), Itaperuna (Ipn), Iguaba Grande (Igg), Itaboraí (Ibr), Mangaratiba (Mgr), and Vassouras (Vsr). We followed the World Health Organization (WHO) guidelines and investigated the degree of susceptibility/resistance of mosquitoes to these insecticides. We used the Rockefeller strain as a susceptible positive control. We genotyped the V1016I and F1534C knockdown resistance (kdr) alleles using qPCR TaqMan SNP genotyping assay. Besides, with the use of Ae. aegypti SNP-chip, we performed genomic population analyses by genotyping more than 15,000 biallelic SNPs in mosquitoes from each population. We added previous data from populations from other countries to evaluate the ancestry of RJ populations. All RJ Ae. aegypti populations were susceptible to pyriproxyfen and malathion and highly resistant to deltamethrin. The resistance ratios for temephos was below 3,0 in Cgy, Ibr, and Igg populations, representing the lowest rates since IR monitoring started in this Brazilian region. We found the kdr alleles in high frequencies in all populations, partially justifying the observed resistance to pyrethroid. Population genetics analysis showed that Ae. aegypti revealed potential higher migration among some RJ localities and low genetic structure for most of them. Future population genetic studies, together with IR data in Ae aegypti on a broader scale, can help us predict the gene flow within and among the Brazilian States, allowing us to track the dynamics of arrival and changes in the frequency of IR alleles, and providing critical information to improving vector control program.

Evolution of kdr haplotypes in worldwide populations of Aedes aegypti: Independent origins of the F1534C kdr mutation.

Aedes aegypti is the primary vector of dengue, chikungunya, Zika, and urban yellow fever. Insecticides are often the most effective tools to rapidly decrease the density of vector populations, especially during arbovirus disease outbreaks. However, the intense use of insecticides, particularly pyrethroids, has selected for resistant mosquito populations worldwide. Mutations in the voltage gated sodium channel (NaV) are among the principal mechanisms of resistance to pyrethroids and DDT, also known as "knockdown resistance," kdr. Here we report studies on the origin and dispersion of kdr haplotypes in samples of Ae. aegypti from its worldwide distribution. We amplified the IIS6 and IIIS6 NaV segments from pools of Ae. aegypti populations from 15 countries, in South and North America, Africa, Asia, Pacific, and Australia. The amplicons were barcoded and sequenced using NGS Ion Torrent. Output data were filtered and analyzed using the bioinformatic pipeline Seekdeep to determine frequencies of the IIS6 and IIIS6 haplotypes per population. Phylogenetic relationships among the haplotypes were used to infer whether the kdr mutations have a single or multiple origin. We found 26 and 18 haplotypes, respectively for the IIS6 and IIIS6 segments, among which were the known kdr mutations 989P, 1011M, 1016I and 1016G (IIS6), 1520I, and 1534C (IIIS6). The highest diversity of haplotypes was found in African samples. Kdr mutations 1011M and 1016I were found only in American and African populations, 989P + 1016G and 1520I + 1534C in Asia, while 1534C was present in samples from all continents, except Australia. Based primarily on the intron sequence, IIS6 haplotypes were subdivided into two well-defined clades (A and B). Subsequent phasing of the IIS6 + IIIS6 haplotypes indicates two distinct origins for the 1534C kdr mutation. These results provide evidence of kdr mutations arising de novo at specific locations within the Ae. aegypti geographic distribution. In addition, our results suggest that the 1534C kdr mutation had at least two independent origins. We can thus conclude that insecticide selection pressure with DDT and more recently with pyrethroids is selecting for independent convergent mutations in NaV.

Species and sex-specific chemosensory gene expression in Anopheles coluzzii and An. quadriannulatus antennae.

BACKGROUND: Olfactory cues drive mosquito behaviors such as host-seeking, locating sugar sources and oviposition. These behaviors can vary between sexes and closely related species. For example, the malaria vector Anopheles coluzzii is highly anthropophilic, whereas An. quadriannulatus is not. These behavioral differences may be reflected in chemosensory gene expression. METHODS: The expression of chemosensory genes in the antennae of both sexes of An. coluzzii and An. quadriannulatus was compared using RNA-seq. The sex-biased expression of several genes in An. coluzzii was also compared using qPCR. RESULTS: The chemosensory expression is mostly similar in the male antennae of An. coluzzii and An. quadriannulatus, with only a few modest differences in expression. A handful of chemosensory genes are male-biased in both species; the highly expressed gustatory receptor AgGr33, odorant binding proteins AgObp25, AgObp26 and possibly AgObp10. Although the chemosensory gene repertoire is mostly shared between the sexes, several highly female-biased AgOrs, AgIrs, and one AgObp were identified, including several whose expression is biased towards the anthropophilic An. coluzzii. Additionally, the expression of several chemosensory genes is biased towards An. coluzzii in both sexes. CONCLUSIONS: Chemosensory gene expression is broadly similar between species and sexes, but several sex- biased/specific genes were identified. These may modulate sex- and species-specific behaviors. Although the male behavior of these species remains poorly studied, the identification of sex- and species-specific chemosensory genes may provide fertile ground for future work.

Toxicological and ultrastructural analysis of the impact of pesticides used in temperate fruit crops on two populations of Chrysoperla externa (Neuroptera, Chrysopidae) / Análises toxicológica e ultra-estrutural do impacto de agrotóxicos usados no cultivo de frutíferas de clima temperado sobre duas populações de Chrysoperla externa (Neuroptera, Chrysopidae)

This study aimed to evaluate the effects of (g a.i. L-1) abamectin (0.02), carbaryl (1.73), sulphur (4.8), fenitrothion (0.75), methidathion (0.4), and trichlorfon (1.5) on the survival of larvae and pupae, on the oviposition of adults and hatching of eggs from treated Chrysoperla externa third-instar larvae from two different populations (Bento Gonçalves and Vacaria, Rio Grande do Sul State, Brazil). Morphological changes caused by abamectin to eggs laid by C. externa from Vacaria population were evaluated by mean of ultrastructural analysis. The pesticides were applied on glass plates. Distilled water was used as control. For the evaluation of larvae mortality, a fully randomized experimental design in a 2 x 7 (two populations x seven treatments) factorial scheme was used, whereas for the effects of the compounds on oviposition capacity and egg viability, a 2 x 4 factorial scheme was used. Carbaryl, fenitrothion, and methidathion caused 100 percent mortality of larvae. Abamectin reduced the hatching of eggs from treated third-instar larvae of both populations; however, this pesticide presented highest toxicity on insects from Vacaria. The ultrastructural analysis showed that abamectin caused malformations in micropyle and in chorion external surface of C. externa eggs. Based in the total effect (E), carbaryl, fenitrothion, and methidathion are harmful to C. externa; trichlorfon is harmless to third-instar larvae, while abamectin and sulphur are harmless and slightly harmful to third-instar larvae from Bento Gonçalves and Vacaria, respectively.

Avaliaram-se os efeitos de (g i.a. L-1) abamectina (0,02), carbaril (1,73), enxofre (4,8) fenitrotiona (0,75), metidationa (0,4) e triclorfom (1,5) sobre a sobrevivência de larvas e pupas, na oviposição de adultos e viabilidade de ovos de Chrysoperla externa de duas populações (Bento Gonçalves e Vacaria, Rio Grande do Sul). Alterações morfológicas causadas por abamectina em ovos depositados por C. externa da população de Vacaria foram avaliadas por meio de análises ultra-estruturais. Os agrotóxicos foram aplicados sobre placas de vidro. Água destilada foi utilizada como testemunha. Para avaliação da mortalidade de larvas utilizou-se delineamento experimental inteiramente ao acaso, em esquema fatorial 2 x 7 (duas populações x sete tratamentos) e para avaliação dos efeitos dos compostos sobre a capacidade de oviposição e viabilidade de ovos utilizou-se esquema fatorial 2 x 4. Carbaril, fenitrotiona e metidationa causaram 100 por cento de mortalidade das larvas. Abamectina reduziu a viabilidade de ovos de C. externa, provenientes de larvas de terceiro ínstar de ambas as populações; entretanto, apresentou toxicidade mais elevada sobre insetos de Vacaria. Análises ultra-estruturais evidenciaram que abamectina causou deformações na micrópila e na superfície externa do córion de ovos de C. externa. Baseando-se no efeito total (E), carbaril, fenitrotiona e metidationa são prejudiciais a C. externa; triclorfom é inócuo, enquanto abamectina e enxofre são inócuos e levemente prejudiciais a larvas de terceiro ínstar oriundas de Bento Gonçalves e Vacaria, respectivamente.