Transgene-induced cell death following dengue-2 virus infection in Aedes aegypti.

Dengue viruses (DENVs) are mosquito-borne flaviviruses causing millions of human infections each year and pose a challenge for public health systems worldwide. Aedes aegypti is the principal vector species transmitting DENVs to humans. Controlling Ae. aegypti is difficult due to the abundance of breeding sites and increasing insecticide resistance in the vector populations. Developing new vector control strategies is critical for decreasing the disease burden. One potential approach is genetically replacing Ae. aegypti populations with vector populations highly resistant to DENV transmission. Here, we focus on an alternative strategy for generating dengue 2 virus (DENV-2) resistance in genetically-modified Ae. aegypti in which the mosquitoes express an inactive form of Michelob_x (Mx), an antagonist of the Inhibitor of Apoptosis (IAP), to induce apoptosis in those cells in which actively replicating DENV-2 is present. The inactive form of Mx was flanked by the RRRRSAG cleavage motif, which was recognized by the NS2B/NS3 protease of the infecting DENV-2 thereby releasing and activating Mx which then induced apoptosis. Our transgenic strain exhibited a significantly higher mortality rate than the non-transgenic control when infected with DENV-2. We also transfected a DNA construct containing inactive Mx fused to eGFP into C6/36 mosquito cells and indirectly observed Mx activation on days 3 and 6 post-DENV-2 infections. There were clear signs that the viral NS2B/NS3 protease cleaved the transgene, thereby releasing Mx protein into the cytoplasm, as was confirmed by the detection of eGFP expression in infected cells. The present study represents proof of the concept that virus infection can be used to induce apoptosis in infected mosquito cells.

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.

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.

A Metagenomic Approach Identified a Novel Phasi Charoen-Like Virus Coinfecting a Chikungunya Virus-Infected Aedes aegypti Mosquito in Brazil.

Insect-specific viruses do not replicate in vertebrates. Here, we report the genome sequence of a novel strain of a Phasi Charoen-like virus (PCLV) that was isolated from a wild Aedes aegypti mosquito collected in Aracajú, Sergipe State, Brazil. The coding-complete genome of the PCLV is described in this report.

Polymorphism in male genitalia of Aedes (Ochlerotatus) scapularis Rondani, 1848

Morphology of male genitalia of culicids is generally species-specific and often used as a taxonomic marker. However, some characters of the male genitalia vary intraspecifically and are not taxonomically diagnostic. This might be the case of Aedes scapularis, a Neotropical culicid with vector competence for arboviruses and filarial worms. Males of this species may or not present a retrorse process (RP) in the genitalic claspette filaments, which led authors to suspect that this variance might be indicative of population divergence or incipient speciation process. This suspicion has not been investigated hitherto and it is not known if there are variable patterns of RPs. We hypothesized that the presence of the RP varies intraspecifically in Ae. scapularis and then we statistically evaluated the variability of this character in a single population. To this study the genitalia of 73 males of Ae. scapularis were prepared, and their RPs were meristically quantified and categorized according to the phenotypes observed. We noted that the presence or RPs is a polymorphic character because it varied inter and intra-individually. The presence of a single RP on each claspette filament was the predominant pattern (77%), but absent or multiple RPs in each filament were also found either in bilateral symmetry or asymmetry. Thus, we conclude that the presence of RPs owing to its high variability is not indicative of populational divergence or diagnostic of species complex within Ae. scapularis.

Polymorphism in male genitalia of Aedes (Ochlerotatus) scapularis Rondani, 1848

Morphology of male genitalia of culicids is generally species-specific and often used as a taxonomic marker. However, some characters of the male genitalia vary intraspecifically and are not taxonomically diagnostic. This might be the case of Aedes scapularis, a Neotropical culicid with vector competence for arboviruses and filarial worms. Males of this species may or not present a retrorse process (RP) in the genitalic claspette filaments, which led authors to suspect that this variance might be indicative of population divergence or incipient speciation process. This suspicion has not been investigated hitherto and it is not known if there are variable patterns of RPs. We hypothesized that the presence of the RP varies intraspecifically in Ae. scapularis and then we statistically evaluated the variability of this character in a single population. To this study the genitalia of 73 males of Ae. scapularis were prepared, and their RPs were meristically quantified and categorized according to the phenotypes observed. We noted that the presence or RPs is a polymorphic character because it varied inter and intra-individually. The presence of a single RP on each claspette filament was the predominant pattern (77%), but absent or multiple RPs in each filament were also found either in bilateral symmetry or asymmetry. Thus, we conclude that the presence of RPs owing to its high variability is not indicative of populational divergence or diagnostic of species complex within Ae. scapularis.

Geometric morphometrics in mosquitoes: What has been measured?

The field of morphometrics is developing quickly. Recent advances have enabled geometric techniques to be applied to many zoological problems, particularly those involving epidemiologically-relevant mosquitoes. Herein, we briefly introduce geometric morphometric (GM) techniques and then review selected groups of mosquitoes (Culicidae) to which those techniques have been applied. In most of the reviewed cases, GM was capable of satisfactorily discriminating among the tested groups primarily when the studies considered differences within and among species, sexual dimorphism, treatments and the separation of laboratory strains. Although GM approaches have developed quite rapidly, some caution must be taken during data processing for a reliable biometrical approach, such as allometry and asymmetry analyses, scale removal and wing clarification staining for landmark digitization. We also critically forecast directions in this field and discuss how the creation of image databases should enhance species identification in culicids.

First report of naturally infected Aedes aegypti with chikungunya virus genotype ECSA in the Americas.

BACKGROUND: The worldwide expansion of new emergent arboviruses such as Chikungunya and Zika reinforces the importance in understanding the role of mosquito species in spreading these pathogens in affected regions. This knowledge is essential for developing effective programs based on species specificity to avoid the establishment of endemic transmission cycles sustained by the identified local vectors. Although the first autochthonous transmission of Chikungunya virus was described in 2014 in the north of Brazil, the main outbreaks were reported in 2015 and 2016 in the northeast of Brazil. METHODOLOGY/PRINCIPAL FINDINGS: During 5 days of February 2016, we collected mosquitoes in homes of 6 neighborhoods of Aracaju city, the capital of Sergipe state. Four mosquito species were identified but Culex quinquefasciatus and Aedes aegypti were the most abundant. Field-caught mosquitoes were tested for Chikungunya (CHIKV), Zika (ZIKV) and Dengue viruses (DENV) by qRT-PCR and one CHIKV-infected Ae. aegypti female was detected. The complete sequence of CHIKV genome was obtained from this sample and phylogenetic analysis revealed that this isolate belongs to the East-Central-South-African (ECSA) genotype. CONCLUSIONS: Our study describes the first identification of a naturally CHIKV-infected Ae. aegypti in Brazil and the first report of a CHIKV from ECSA genotype identified in this species in the Americas. These findings support the notion of Ae. aegypti being a vector involved in CHIKV outbreaks in northeast of Brazil.

First report of naturally infected Aedes aegypti with chikungunya virus genotype ECSA in the Americas

Background The worldwide expansion of new emergent arboviruses such as Chikungunya and Zika reinforces the importance in understanding the role of mosquito species in spreading these pathogens in affected regions. This knowledge is essential for developing effective programs based on species specificity to avoid the establishment of endemic transmission cycles sustained by the identified local vectors. Although the first autochthonous transmission of Chikungunya virus was described in 2014 in the north of Brazil, the main outbreaks were reported in 2015 and 2016 in the northeast of Brazil. Methodology/Principal findings During 5 days of February 2016, we collected mosquitoes in homes of 6 neighborhoods of Aracaju city, the capital of Sergipe state. Four mosquito species were identified but Culex quinquefasciatus and Aedes aegypti were the most abundant. Field-caught mosquitoes were tested for Chikungunya (CHIKV), Zika (ZIKV) and Dengue viruses (DENV) by qRT-PCR and one CHIKV-infected Ae. aegypti female was detected. The complete sequence of CHIKV genome was obtained from this sample and phylogenetic analysis revealed that this isolate belongs to the East-Central-South-African (ECSA) genotype. Conclusions Our study describes the first identification of a naturally CHIKV-infected Ae. aegypti in Brazil and the first report of a CHIKV from ECSA genotype identified in this species in the Americas. These findings support the notion of Ae. aegypti being a vector involved in CHIKV outbreaks in northeast of Brazil.

Geometric morphometrics in mosquitoes: what has been measured?

The field of morphometrics is developing quickly. Recent advances have enabled geometric techniques to be applied to many zoological problems, particularly those involving epidemiologically-relevant mosquitoes. Herein, we briefly introduce geometric morphometric (GM) techniques and then review selected groups of mosquitoes (Culicidae) to which those techniques have been applied. In most of the reviewed cases, GM was capable of satisfactorily discriminating among the tested groups primarily when the studies considered differences within and among species, sexual dimorphism, treatments and the separation of laboratory strains. Although GM approaches have developed quite rapidly, some caution must be taken during data processing for a reliable biometrical approach, such as allometry and asymmetry analyses, scale removal and wing clarification staining for landmark digitization. We also critically forecast directions in this field and discuss how the creation of image databases should enhance species identification in culicids.

Assessment of the correlation between wing size and body weight in captive Culex quinquefasciatus.

INTRODUCTION: Mass production of mosquitoes under laboratory conditions allows implementing methods to control vector mosquitoes. Colony development depends on mosquito size and weight. Body size can be estimated from its correlation with wing size, whereas weight is more difficult to determine. Our goal was to test whether wing size can predict the weight. METHODS: We compared dry weight and wing centroid size of Culex quinquefasciatus reared at different temperatures and four diets. RESULTS: Weight and wing size were strongly correlated. The diets did not influence wing size. CONCLUSIONS: Wing centroid size is a good predictor of Cx. quinquefasciatus body weight.

Assessment of the correlation between wing size and body weight in captive Culex quinquefasciatus

Introduction: Mass production of mosquitoes under laboratory conditions allows implementing methods to control vector mosquitoes. Colony development depends on mosquito size and weight. Body size can be estimated from its correlation with wing size, whereas weight is more difficult to determine. Our goal was to test whether wing size can predict the weight. Methods: We compared dry weight and wing centroid size of Culex quinquefasciatus reared at different temperatures and four diets. Results: Weight and wing size were strongly correlated. The diets did not influence wing size. Conclusions: Wing centroid size is a good predictor of Cx. quinquefasciatus body weight

High morphological and genetic variabilities of Ochlerotatus scapularis, a potential vector of filarias and arboviruses.

BACKGROUND: Ochlerotatus scapularis is a potential vector of filarias and arboviruses in the Neotropics. This species was once typically associated with sylvatic environments; however, cases of synanthropy and urbanization of this species have been increasingly reported in southeast Brazil. Despite the medical relevance of Oc. scapularis, its populational variability is not yet known. To our knowledge, this is the first report describing the morphological and genetic variabilities of this species. METHODS: Population samples were characterized using the cytochrome oxidase subunit I (COI) mitochondrial gene and wing geometrics. Adult mosquitoes were collected from five sampling sites from remnants of the Atlantic forest embedded in the urban or rural areas of southeast Brazil. RESULTS: In the 130 individuals analyzed, 46 COI haplotypes were detected. Haplotype diversity was high and ranged from 0.66 to 0.97. Six haplotypes were present in 61% of the individuals, whereas the remaining haplotypes were less frequent (39%). Wing shape was also highly polymorphic. Differentiation of populations across sampling sites according to genetic distances (Fst = -0.009 to 0.060) and morphological distances (Qst = 0.47) indicated that populations were not identical. No correlations were noted for phenetic and genetic diversities (p = 0.19) or for genetic or phenetic distances with geographical distances (p = 0.2 and p = 0.18, respectively). CONCLUSIONS: Our study results suggest that Oc. scapularis has a rich genetic patrimony, even though its habitat is fragmented. Implications of such genetic richness with respect to vectorial competence, plasticity, and ability to exploit urbanized areas need to be further investigated.