Effects of antennal segments defects on blood-sucking behavior in Aedes albopictus.

After mating, female mosquitoes need a blood meal to promote the reproductive process. When mosquitoes bite infected people and animals, they become infected with germs such as viruses and parasites. Mosquitoes rely on many cues for host selection and localization, among which the trace chemical cues emitted by the host into the environment are considered to be the most important, and the sense of smell is the main way to perceive these trace chemical cues. However, the current understanding of the olfactory mechanism is not enough to meet the needs of mosquito control. Unlike previous studies that focused on the olfactory receptor recognition spectrum to reveal the olfactory mechanism of mosquito host localization. In this paper, based on the observation that mosquitoes with incomplete antennae still can locate the host and complete blood feeding in the laboratory, we proposed that there may be some protection or compensation mechanism in the 13 segments of antennae flagella, and only when the antennae are missing to a certain threshold will it affect the mosquito's ability to locate the host. Through rational-designed behavioral experiments, we found that the 6th and 7th flagellomeres on the Aedes albopictus antenna are important in the olfactory detection of host searching. This study preliminarily screened antennal segments important for host localization of Ae. albopictus, and provided a reference for subsequent cell biology and molecular biology studies on these segments. Meanwhile, the morphology and distribution of sensilla on each antenna flagellomere were also analyzed and discussed in this paper.

Aedes yunnanensis (Diptera: Culicidae): transferred from the subgenus Hulecoeteomyia to Orohylomyia subg. nov., with descriptions of the species and the new subgenus.

Aedes yunnanensis (Gaschen), currently classified in the subgenus Hulecoeteomyia Theobald, is transferred to a new monobasic subgenus, Orohylomyia Somboon & Harbach, subg. nov., based on morphological assessment of adults, male and female genitalia, larvae, and pupae, and phylogenetic analysis. The new subgenus and its type species are described in detail.

Aedes pertinax, Anopheles perplexens, Culex declarator, and Cx. interrogator: An Update of Mosquito Species Records for Charlotte County, Florida.

Understanding the geographic occurrence of mosquito species is an important element to addressing public health and nuisance mosquito-related issues, particularly as changing climates and increased global connectivity is likely to facilitate changes in the distribution of mosquitoes and other species. In Charlotte County, FL, routine surveillance of mosquito species for public health in 2019-21 identified 4 mosquito species not previously documented in the county. Aedes pertinax, Anopheles perplexens, Culex declarator, and Cx. interrogator adults were collected and verified to species level. Aedes pertinax and Cx. declarator and were collected in 2019, whereas An. perplexens and Cx. interrogator were documented from collections in 2021. All 4 species were initially visually identified by external morphology and confirmed by sequencing the DNA barcoding region of the cytochrome c oxidase subunit I gene. Apart from native An. perplexens, in which only 1 specimen has been confirmed to date, the 3 newly documented nonnative species are now recognized throughout the county.

The Acquisition and Retention of Lumpy Skin Disease Virus by Blood-Feeding Insects Is Influenced by the Source of Virus, the Insect Body Part, and the Time since Feeding.

Lumpy skin disease virus (LSDV) is a poxvirus that causes severe systemic disease in cattle and is spread by mechanical arthropod-borne transmission. This study quantified the acquisition and retention of LSDV by four species of Diptera (Stomoxys calcitrans, Aedes aegypti, Culex quinquefasciatus, and Culicoides nubeculosus) from cutaneous lesions, normal skin, and blood from a clinically affected animal. The acquisition and retention of LSDV by Ae. aegypti from an artificial membrane feeding system was also examined. Mathematical models of the data were generated to identify the parameters which influence insect acquisition and retention of LSDV. For all four insect species, the probability of acquiring LSDV was substantially greater when feeding on a lesion compared with feeding on normal skin or blood from a clinically affected animal. After feeding on a skin lesion LSDV was retained on the proboscis for a similar length of time (around 9 days) for all four species and for a shorter time in the rest of the body, ranging from 2.2 to 6.4 days. Acquisition and retention of LSDV by Ae. aegypti after feeding on an artificial membrane feeding system that contained a high titer of LSDV was comparable to feeding on a skin lesion on a clinically affected animal, supporting the use of this laboratory model as a replacement for some animal studies. This work reveals that the cutaneous lesions of LSD provide the high-titer source required for acquisition of the virus by insects, thereby enabling the mechanical vector-borne transmission. IMPORTANCE Lumpy skin disease virus (LSDV) is a high consequence pathogen of cattle that is rapidly expanding its geographical boundaries into new regions such as Europe and Asia. This expansion is promoted by the mechanical transmission of the virus via hematogenous arthropods. This study quantifies the acquisition and retention of LSDV by four species of blood-feeding insects and reveals that the cutaneous lesions of LSD provide the high titer virus source necessary for virus acquisition by the insects. An artificial membrane feeding system containing a high titer of LSDV was shown to be comparable to a skin lesion on a clinically affected animal when used as a virus source. This promotes the use of these laboratory-based systems as replacements for some animal studies. Overall, this work advances our understanding of the mechanical vector-borne transmission of LSDV and provides evidence to support the design of more effective disease control programmes.

First description of the mitogenome and phylogeny:Aedes vexansand Ochlerotatus caspius of the Tribe Aedini (Diptera: Culicidae).

Culicidae, the mosquito family, includes more than 3600 species subdivided into the subfamilies Anophelinae and Culicinae. One-third of mosquitoes belong to the Aedini tribe, which is subordinate to the subfamily Culicinae, which comprises common vectors of viral zoonoses. The tribe of Aedini is extremely diverse in morphology and geographical distribution and has high ecological and medical significance. However, knowledge about the systematics of the Aedini tribe is still limited owing to its large population and the similar morphological characteristics of its species. This study provides the first description of the complete mitochondrial (mt) genome sequence of Aedes vexans and Ochlerotatus caspius belonging to the Aedini tribe. The mt genomes of A. vexans and O. caspius are circular molecules that are 15,861 bp and 15,954 bp in size, with AT contents of 78.54% and 79.36%, respectively. Both the circular mt genomes comprise 37 functional subunits, including 13 protein-coding genes (PCGs), two ribosomal RNA genes, 22 transfer RNA genes (tRNAs), and a control region (also known as the AT-rich region). The most common start codons are ATT/ATG, apart from cox1 (TCG) and nad5 (GTG), while TAA is the termination codon for all PCGs. All tRNAs have a typical clover leaf structure, except tRNA Ser1. Phylogenetic analysis of the concatenated, aligned amino acid sequences of the 13 PCGs showed that A. vexans gathered with Aedes sp. in a sister taxon, and O. caspius gathered with Ochlerotatus sp. in a sister taxon. The findings from the present study support the concept of monophyly of all groups, ratify the current taxonomic classification, and provide vital molecular marker resources for further studies of the taxonomy, population genetics, and systematics of the Aedini tribe.

Using a pupal exuvia to designate the undamaged neotype of a species belonging to a complex of sibling species - the case of Aedes coluzzii (Diptera, Culicidae).

The mosquito species Aedes (Ochlerotatus) coluzzii Rioux, Guilvard & Pasteur, 1998 was distinguished from its sibling species Aedes detritus (Haliday, 1833) using an isoenzymatic method that required the destruction of the entire specimen, therefore no holotype was designated by the species authors. We aimed to designate a neotype for Ae. coluzzii from specimens collected from the type-locality and individually reared up to adult stage. Genomic DNA was extracted from pupal exuvia and ITS2 was sequenced, enabling verification of the identity of each specimen as Ae. coluzzii or Ae. detritus. Among the series of Ae. coluzzii, a male was designated as neotype and deposited in a collection. To our knowledge, this is the first time the type of a mosquito species is deposited thanks to its molecular identification from its pupal exuvia. The set of identified specimens allowed additional phylogenetic and morphologic studies.

Title: Utilisation d'une exuvie nymphale pour désigner le néotype intact d'une espèce appartenant à un complexe d'espèces jumelles - le cas d'Aedes coluzzii (Diptera, Culicidae). Abstract: L'espèce de moustique Aedes (Ochlerotatus) coluzzii Rioux, Guilvard & Pasteur, 1998 a été distinguée de son espèce jumelle Aedes detritus (Haliday, 1833) par une méthode isoenzymatique qui a nécessité la destruction de l'ensemble du spécimen, et donc aucun holotype n'a été désigné par les auteurs de l'espèce. Notre objectif était de désigner un néotype pour Ae. coluzzii à partir de spécimens collectés dans la localité-type et élevés individuellement jusqu'au stade adulte. L'ADN génomique a été extrait de l'exuvie nymphale et l'ITS2 a été séquencé, permettant la vérification de l'identité de chaque spécimen comme Ae. coluzzii ou Ae. détritus. Parmi la série d'Ae. coluzzii, un mâle a été désigné comme néotype et déposé dans une collection. À notre connaissance, c'est la première fois que le type d'une espèce de moustique est déposé grâce à l'identification moléculaire à partir de son exuvie nymphale. L'ensemble des spécimens identifiés a permis des études phylogénétiques et morphologiques complémentaires.

Genetic and morphological polymorphisms of Aedes scapularis (Diptera: Culicidae), vector of filariae and arboviruses.

BACKGROUND: Aedes scapularis is a neotropical mosquito that is competent to vector viruses and filariae. It is reputed to be highly morphologically and genetically polymorphic, facts that have raised questions about whether it is a single taxonomic entity. In the last five decades, authors have posed the hypothesis that it could actually be a species complex under incipient speciation. Due to its epidemiological importance, its taxonomic status should be determined with confidence. AIM AND METHOD: Our objective was to investigate more deeply the polymorphism of Ae. scapularis to detect any evidence of incipient speciation of cryptic species. We then compared populational samples from the Southeastern, Northern and Northeastern regions of Brazil. The biological markers used in the comparison were: the complete mitochondrial DNA, the isolated mitochondrial gene cytochrome oxidase subunit I (COI) and wing geometry. RESULTS AND DISCUSSION: As expected, high morphological/genetic polymorphism was observed in all Ae. scapularis populations, however it was not indicative of segregation or incipient speciation. There was no correlation between wing shape and the geographical origin of the populations analysed. A congruent observation resulted from the analysis of the COI gene, which revealed a high number of haplotypes (51) and no clusterization of populational samples according to the original biomes. In the phylogenetic analysis of the 13 mitochondrial protein-coding genes, the Ae. scapularis clade clustered with maximum support (100% bootstrap support and posterior probability of 1). No significant internal structure was observed in the Ae. scapularis clade, which was nearly a polytomy. Taken together, our results indicate that this species is not a species complex. CONCLUSION: We conclude that there was no indication, in the analysed regions, of the occurrence of more than one taxon in the species Ae. scapularis, despite it being highly polymorphic. By ruling out the former species complex hypothesis, our phylogenetic results reinforce that Ae. scapularis is a single taxonomic unit and should be monitored with standardized surveillance and control methods.

First Record of Natural Transovarial Transmission of Dengue Virus in Aedes albopictus from Cuba.

Transovarial transmission (TOT) of dengue virus (DENV) in Aedes spp. is an important mechanism for DENV maintenance in nature and may be important in initiating outbreaks. The objective of this study was to explore the occurrence of TOT in wild Aedes albopictus populations in Cuba. Mosquito larvae were collected in Cotorro municipality, Havana, Cuba, and identified to species. Fifteen pools of Ae. albopictus each containing 30 larvae were processed for DENV detection by using conventional reverse transcription polymerase chain reaction (RT-PCR) and nested PCR. Four out of 15 pools processed were positive for DENV-3, but no other DENV serotype was detected. This is the first time TOT of DENV detected in Cuban field populations of Ae. albopictus, and this suggests that this species may be an important vector of DENV in Cuba.

Modeling the effects of Aedes aegypti's larval environment on adult body mass at emergence.

Mosquitoes vector harmful pathogens that infect millions of people every year, and developing approaches to effectively control mosquitoes is a topic of great interest. However, the success of many control measures is highly dependent upon ecological, physiological, and life history traits of mosquito species. The behavior of mosquitoes and their potential to vector pathogens can also be impacted by these traits. One trait of interest is mosquito body mass, which depends upon many factors associated with the environment in which juvenile mosquitoes develop. Our experiments examined the impact of larval density on the body mass of Aedes aegypti mosquitoes, which are important vectors of dengue, Zika, yellow fever, and other pathogens. To investigate the interactions between the larval environment and mosquito body mass, we built a discrete time mathematical model that incorporates body mass, larval density, and food availability and fit the model to our experimental data. We considered three categories of model complexity informed by data, and selected the best model within each category using Akaike's Information Criterion. We found that the larval environment is an important determinant of the body mass of mosquitoes upon emergence. Furthermore, we found that larval density has greater impact on body mass of adults at emergence than on development time, and that inclusion of density dependence in the survival of female aquatic stages in models is important. We discuss the implications of our results for the control of Aedes mosquitoes and on their potential to spread disease.

Evidence for the spread of the alien species Aedes koreicus in the Lombardy region, Italy.

BACKGROUND: Aedes koreicus is a mosquito species characterized by marked anthropophilic behavior, and a potential vector of nematodes and viruses. It is native to East Asia, but its presence has recently been reported in many regions of Europe. In Italy, these mosquitoes had been detected in the northeast since 2011 and are now spreading towards the southwest of the country. METHODS: In 2020, during a surveillance program for invasive mosquito species in the district of Bergamo (Lombardy Region, Italy), about 6000 mosquito larvae were collected. Emerged adults were assigned to mosquito species according to morphological analyses, followed by amplification and sequencing of genetic markers (COI, ND4, ITS2 and D2). RESULTS: According to the morphological and genetic data, about 50 individuals belonged to the species Ae. koreicus. CONCLUSION: We report the presence of Ae. koreicus in the district of Bergamo, which confirms the spread of this species in the north of Italy and raises concerns about its possible role as a vector of diseases in the Alpine area.

Distinct Roles of Hemocytes at Different Stages of Infection by Dengue and Zika Viruses in Aedes aegypti Mosquitoes.

Aedes aegypti mosquitoes are vectors for arboviruses of medical importance such as dengue (DENV) and Zika (ZIKV) viruses. Different innate immune pathways contribute to the control of arboviruses in the mosquito vector including RNA interference, Toll and Jak-STAT pathways. However, the role of cellular responses mediated by circulating macrophage-like cells known as hemocytes remains unclear. Here we show that hemocytes are recruited to the midgut of Ae. aegypti mosquitoes in response to DENV or ZIKV. Blockade of the phagocytic function of hemocytes using latex beads induced increased accumulation of hemocytes in the midgut and a reduction in virus infection levels in this organ. In contrast, inhibition of phagocytosis by hemocytes led to increased systemic dissemination and replication of DENV and ZIKV. Hence, our work reveals a dual role for hemocytes in Ae. aegypti mosquitoes, whereby phagocytosis is not required to control viral infection in the midgut but is essential to restrict systemic dissemination. Further understanding of the mechanism behind this duality could help the design of vector-based strategies to prevent transmission of arboviruses.

Aedes albopictus diversity and relationships in south-western Europe and Brazil by rDNA/mtDNA and phenotypic analyses: ITS-2, a useful marker for spread studies.

BACKGROUND: Aedes albopictus is a very invasive mosquito, which has recently colonized tropical and temperate regions worldwide. Of concern is its role in the spread of emerging or re-emerging mosquito-borne diseases. Ae. albopictus from south-western Europe and Brazil were studied to infer genetic and phenetic diversity at intra-individual, intra-population and inter-population levels, and to analyse its spread. METHODS: Genotyping was made by rDNA 5.8S-ITS-2 and mtDNA cox1 sequencing to assess haplotype and nucleotide diversity, genetic distances and phylogenetic networks. Male and female phenotyping included combined landmark-and outlined-based geometric morphometrics of wing size and shape. RESULTS: Specimens from seven populations from Spain, France and Brazil provided 12 cox1 and 162 5.8S-ITS-2 haplotypes, with great genetic variability difference between both markers (0.9% vs 31.2%). Five cox1 haplotypes were shared with other countries, mainly Italy, USA and China, but none was shared between Europe and Brazil. The 5.8S-ITS-2 showed 2-7 intra-individual (mean 4.7) and 16-34 intra-/inter-population haplotypes (24.7), including haplotypes shared between Spain, France and Brazil. A 4.3% of ITS-2 haplotypes were shared, mainly with Italy, USA and Thailand, evidencing worldwide spread and introductions from areas where recent outbreaks of Ae. albopictus-transmitted pathogens occurred. Wing size showed sex differences. Wing shape distinguished between Brazilian and European specimens. Both genetic and morphometric markers showed differences between insular Spain and continental Spain, France and Brazil. CONCLUSIONS: ITS-2 proves to be a useful marker to assess Ae. albopictus spread, providing pronouncedly more information than cox1, including intra-individual, intra-population and inter-population levels, furnishing a complete overview of the evolutionary exchanges followed by this mosquito. Wing morphometry proves to be a useful phenotyping marker, allowing to distinguish different populations at the level of both male and female specimens. Results indicate the need for periodic surveillance monitorings to verify that no Ae. albopictus with high virus transmission capacity is introduced into Europe.

Implementation of a deep learning model for automated classification of Aedes aegypti (Linnaeus) and Aedes albopictus (Skuse) in real time.

Classification of Aedes aegypti (Linnaeus) and Aedes albopictus (Skuse) by humans remains challenging. We proposed a highly accessible method to develop a deep learning (DL) model and implement the model for mosquito image classification by using hardware that could regulate the development process. In particular, we constructed a dataset with 4120 images of Aedes mosquitoes that were older than 12 days old and had common morphological features that disappeared, and we illustrated how to set up supervised deep convolutional neural networks (DCNNs) with hyperparameter adjustment. The model application was first conducted by deploying the model externally in real time on three different generations of mosquitoes, and the accuracy was compared with human expert performance. Our results showed that both the learning rate and epochs significantly affected the accuracy, and the best-performing hyperparameters achieved an accuracy of more than 98% at classifying mosquitoes, which showed no significant difference from human-level performance. We demonstrated the feasibility of the method to construct a model with the DCNN when deployed externally on mosquitoes in real time.

Oviposition behavior of wild yellow fever vector mosquitoes (Diptera: Culicidae) in an Atlantic Forest fragment, Rio de Janeiro state, Brazil.

Although there are many studies on the control of mosquito vectors of the yellow fever virus (YFV) in tropical forests, there are still few ecological studies regarding abiotic factors effect on these mosquitoes. Here we characterize these effects on oviposition behavior, abundance, and diversity of mosquito vectors of YFV. The study was conducted in Córrego da Luz Municipal Park, in Casimiro de Abreu, Rio de Janeiro state, Brazil, from July 2018 to December 2019. Ovitraps were placed at ground level and 3 m high. The data were tested for normality using the Shapiro-Wilk test, followed by an independent sample analysis, the Mann-Whitney test. The Shannon Diversity Index was used to evaluate the abundance of mosquitos' eggs collected at both ground level and 3 m high. We highlight the presence of Haemagogus janthinomys and Hg. leucocelaenus, primary YFV vectors in forest areas. The abundance of Hg. leucocelaenus (63%), Hg. janthinomys (75%), and Aedes terrens (58%) was higher at the height of 3 m, while Ae. albopictus (52%) was higher at ground level. Aedes albopictus was positively correlated with temperature. Culicidae monitoring is essential for assessing the YFV transmission cycle in Atlantic forest fragments.

From Aedes to Zeugodacus: a review of dipteran body coloration studies regarding evolutionary developmental biology, pest control, and species discovery.

Over the past two decades, evo-devo (evolution of development) studies have elucidated genetic mechanisms underlying novel dipteran body color patterns. Here we review the most recent developments, which show some departure from the model organism Drosophila melanogaster, leading the field into the investigation of more complex color patterns. We also discuss how the robust application of transgenic techniques has facilitated the study of many non-model pest species. Furthermore, we see that subtle pigmentation differences guide the discovery and description of new dipterans. Therefore, we argue that the existence of new field guides and the prevalence of pigmentation studies in non-model flies will enable scientists to adopt uninvestigated species into the lab, allowing them to study novel morphologies.

Reinertia, a New Subgenus of the Genus Aedes Meigen and Its Type Species Aedes (Reinertia) suffusus (Diptera: Culicidae), Newly Recorded From Bhutan.

A new subgenus, Reinertia Somboon, Namgay & Harbach, of the genus Aedes Meigen and its type species, Ae. suffusus Edwards, are described from specimens reared from larvae and pupae found in a tree hole in Bhutan. The scutum of the adults is mostly covered with narrow pale falcate scales. The proboscis, maxillary palpus, tibiae, and tarsi are dark-scaled. The gonocoxite of the male genitalia bears a unique setose basomesal sclerite. The larva closely resembles larvae of the subgenus Downsiomyia Vargus in having setae 4-6-C with numerous branches and inserted more or less on level with seta 7-C, abdominal seta 12-I is present and the comb is composed of 6-10 spine-like scales arranged in an irregular row. Surprisingly, Reinertia shares features of the adult habitus, male genitalia, and larva with the Palearctic subgenus Dahliana Reinert, Harbach & Kitching. However, in phylogenetic analyses of the mitochondrial COI gene of species representing 38 subgenera of Aedes and six other genera of the tribe Aedini Neveu-Lemaire, Reinertia was not associated with Dahliana or Downsiomyia. In both maximum likelihood and Bayesian analyses of the data, Ae. suffusus was recovered as the weakly supported sister of a clade composed of five species of the subgenus Protomacleaya Theobald. In the absence of strong support, and because Protomacleaya is an unnatural group of species that resemble each other phenetically by virtue of what they lack, Ae. suffusus cannot be placed in the subgenus Protomacleaya. Thus, the morphological and molecular data attest the uniqueness of Ae. suffusus and its recognition as a monobasic subgeneric lineage.

Identification of mosquitoes (Diptera: Culicidae) from Mexico State, Mexico using morphology and COI DNA barcoding.

Mosquitoes are commonly identified to species level using morphological traits, but complementary methods for identification are often necessary when specimens are collected as immature stages, stored inadequately, or when delineation of species complexes is problematic. DNA-barcoding using the mitochondrial cytochrome c oxidase subunit 1 (COI) gene is one such tool used for the morphological identification of species. A comprehensive entomological survey of mosquito species in Mexico State identified by COI DNA barcoding and morphology is documented in this paper. Specimens were collected from all the physiographic provinces in Mexico State between 2017 and 2019. Overall, 2,218 specimens were collected from 157 localities representing both subfamilies Anophelinae and Culicinae. A species checklist that consists of 6 tribes, 10 genera, 20 subgenera, and 51 species, 35 of which are new records for Mexico State, is provided. Three hundred and forty-two COI sequences of 46 species were analysed. Mean intraspecific and interspecific distances ranged between 0% to 3.9% and from 1.2% to 25.3%, respectively. All species groups were supported by high bootstraps values in a Neighbour-Joining analysis, and new COI sequences were generated for eight species: Aedes chionotum Zavortink, Ae. vargasi Schick, Ae. gabriel Schick, Ae. guerrero Berlin, Ae. ramirezi Vargas and Downs, Haemagogus mesodentatus Komp and Kumm, Culex restrictor Dyar and Knab, and Uranotaenia geometrica Theobald. This study provides a detailed inventory of the Culicidae from Mexico State and discusses the utility of DNA barcoding as a complementary tool for accurate mosquito species identification in Mexico.

Wing geometry and genetic analyses reveal contrasting spatial structures between male and female Aedes aegypti (L.) (Diptera: Culicidae) populations in metropolitan Manila, Philippines.

The population genetic structure of Aedes aegypti (Linnaeus, 1762) has been studied in order to understand its role as an efficient vector. Several studies utilized an integrative approach; to combine genetic and phenotypic data to determine its population structure but these studies have only focused on female populations. To address this particular gap, our study compared the population variability and structuring between its male and female populations using phenotypic and genetic data from a highly-urbanized and dengue-endemic region of the Philippines, Metropolitan Manila. Five mosquito populations comprised of female (n = 137) and male (n = 49) adult mosquitoes were used in this study. All mosquito individuals underwent geometric morphometric (26 landmarks), and genetic (11 microsatellite loci) analyses. Results revealed that FST estimates (genetic) were 0.055 and 0.009 while QST estimates (phenotypic) were 0.318 and 0.309 in in male and female populations, respectively. Wing shape variation plots showed that male populations were distinctly separated from each other while female populations overlapped. Similarly, discriminant analysis of principal components using genetic data revealed that male populations were also distinctly separated from each other while female populations showed near-overlapping populations. Genetic and phenetic dendrograms showed the formation of two groups in male populations but no groups in female populations. Further analysis indicated a significant correlation (r = 0.68, p = 0.02) between the genetic and phenetic distances of male populations. Bayesian analysis using genetic data also detected multiple clusters in male (K = 3) and female (K = 2) populations, while no clusters were detected using the phenotypic data from both sexes. Our results revealed contrasting phenotypic and genetic patterns between male and female Ae. aegypti, indicating that male populations were more spatially structured than female populations.

Incursion and establishment of the Old World arbovirus vector Aedes (Fredwardsius) vittatus (Bigot, 1861) in the Americas.

Routine biosurveillance efforts at the Naval Station Guantanamo Bay, Cuba, on 18 June 2019, detected two unusual mosquitos in a CO2-baited CDC light trap. Morphological and molecular analysis confirmed the presence of Aedes (Fredwardsius) vittatus (Bigot, 1861) - the first record of the Old World dengue, chikungunya, Zika and yellow fever virus vector into the Americas - and provides evidence for its establishment in Cuba. Newly submitted GenBank sequences from Dominican Republic further evidence its establishment in the Caribbean, and a median-joining network analysis using mitochondrial COI gene sequences clearly supports multiple introductions of Ae. vittatus into the Caribbean from the Indian subcontinent. It was determined that many Ae. vittatus COI barcode sequences in GenBank are currently misidentified as Aedes (Fredwardsius) cogilli Edwards, 1922.

Crude extract of the tropical tree Gallesia integrifolia (Phytolaccaceae) for the control of Aedes aegypti (Diptera: Culicidae) larvae / Extracto crudo del árbol tropical Gallesia integrifolia (Phytolaccaceae) para el control de larvas de Aedes aegypti (Diptera: Culicidae)

Introduction: Phytoinsecticides are alternatives to control insects in different stages, Gallesia integrifolia (Spreng.) Harms, Phytolacaceae family, popularly known as pau d'alho, garlic tree, and guararema in Brazil, is known due to its strong alliaceous odor because of the presence of sulfur molecules in the plant. This species presents biological activity and potential insecticide effect that is still unexploited. Objective: This study aimed to evaluate the biological activity of the ethanolic crude extract from G. integrifolia leaves, flowers, and fruits on the control of Aedes aegypti third-stage larvae and pupae. Methods: The botanical material was collected in city Umuarama, Paraná, Brazil at the coordinates (23º46'16" S & 53º19'38" WO), and altitude of 442 m, the fruits of G. integrifolia were collected in May and the leaves and flowers in December 2017. The crude extracts of G. integrifolia leaves, flowers, and fruits were prepared by dynamic maceration technique. The chemical composition of the extracts was determined by gas chromatography coupled to a mass spectrometry. The insecticidal activity of the crude extracts of G. integrifolia were carried out on larvae and pupae of A. aegypti in concentrations between 0.001 to 25 000 mg/mL, and afterwards the lethal concentrations that kill 50 % (LC50) and 99.9 % (LC99.9) were determined by probit analysis. Anticholinesterase activity was determined by bioautographic method at concentrations from 0.000095 to 50 mg/mL. Results: The yield of G. integrifolia crude extracts were 8.2, 9.1, and 17.3 % for flowers, fruits, and leaves, respectively. The chemical composition of G. integrifolia extracts was characterized by presence of fatty acid esters, phytosterols, vitamins, oxygenated diterpenes and organosulfur compounds. The flower extract presented the high amount of sulfur compounds (20.2 %) such as disulfide, bis (2-sulfhydryl ethyl) (11.9 %), 2,3,5-trithiahexane (6.2 %), 1,2,4-trithiolane (1.1 %), and 2,4-dithiapentane (1.1 %). Regarding the insecticidal activity, flower extract showed highly active with LC99.9 of 0.032 mg/mL and LC99.9 of 0.969 mg/mL on A. aegypti larvae and pupae, respectively, and the highest inhibition of acetylcholinesterase enzyme (0.00019 mg/mL) ex situ. The flower extract presented anticholinesterase and larvicide activity, respectively, 12.8 % and 35.6 % greater than the control temephos. Conclusions: This study opens new perspectives on the use of extracts from G. integrifolia as a bioinsecticide alternative for the control of A. aegypti larvae and pupae.

Introducción: Los fitoinsecticidas son alternativas para el control de insectos en diferentes etapas, Gallesia integrifolia (Spreng.) Harms, familia Phytolacaceae, conocida popularmente como pau d'alho, árbol de ajo y guararema en Brasil, es conocida por su fuerte olor aliáceo debido a la presencia de moléculas de azufre en la planta. Esta especie presenta actividad biológica y potencial efecto insecticida que aún no está explotado. Objetivo: El objetivo de este estudio fue evaluar la actividad biológica del extracto crudo etanólico de las hojas, flores y frutos de G. integrifolia en el control de las larvas y pupas de la tercera etapa de Aedes aegypti. Métodos: El material botánico fue recolectado en la ciudad de Umuarama, Paraná, Brasil (23º46'16" S & 53º19'38" W), a una altitud de 442 m y los frutos de G. integrifolia fueron recolectados en mayo de 2017 y las hojas y flores en diciembre del mismo año. Los extractos crudos de hojas, flores y frutos de G. integrifolia se prepararon por técnica de maceración dinámica. La composición química de los extractos se determinó por cromatografía de gases acoplada a espectrometría de masas. La actividad insecticida de los extractos crudos de G. integrifolia fue evaluada en larvas y pupas de A. aegypti en concentraciones entre 0.001 a 25 000 mg/mL, y las concentraciones letales que matan 50 % (CL50) y 99.9 % (CL99.9) de larvas fueron determinados por análisis de probit. La actividad anticolinesterasa fue evaluada por método bioautográfico en concentraciones de 0.000095 a 50 mg/mL. Resultados: El rendimiento de los extractos crudos de G. integrifolia fue de 8.2, 9.1 y 17.3 % para flores, frutos y hojas, respectivamente. La composición química de los extractos de G. integrifolia se caracterizó por la presencia de ésteres de ácidos grasos, fitosteroles, vitaminas, diterpenos oxigenados y compuestos organosulfurados. El extracto de las flores presentó alta cantidad de compuestos de azufre (20.2 %) como disulfuro, bis(2-sulfhidril etilo) (11.9 %), 2,3,5-tritiahexano (6.2 %), 1,2,4-tritiolano (1.1 %) y 2,4-ditiapentano (1.1 %). En relación con la actividad insecticida, el extracto de las flores mostró una gran actividad con CL99.9 de 0.032 mg/mL y CL99.9 de 0.969 mg/mL en larvas y pupas de A. aegypti, respectivamente, y la inhibición más alta de la enzima acetilcolinesterasa (0.00019 mg/mL) ex situ. El extracto de las flores presentó actividad anticolinesterasa y larvicida, 12.8 y 35.6 %, respectivamente, mayor que el control temephos. Conclusiones: Este estudio abre nuevas perspectivas sobre el uso de extractos de G. integrifolia como alternativa bioinsecticida para el control de larvas y pupas de A. aegypti.