Comparative Phylogeography and Integrative Taxonomy of Ochlerotatus caspius (Dipera: Culicidae) and Ochlerotatus dorsalis.

Given that accurately identifying pathogen vectors is vital for designing efficient mosquito control programs based on the proper surveillance of the epidemiologically important species, it has been suggested the complementary use of independently evolving genes and morphometric traits as a reliable approach for the characterization and delimitation of related species. Hence, we examined the spatial distribution of COI mtDNA and ITS2 rDNA variation from the historical perspective of Ochlerotatus caspius (Pallas, 1771) and O. dorsalis (Meigen, 1830), while simultaneously testing the utility of the two markers in integrative species delimitation when combined with phenotypic character analyses of larvae and adults. Despite the striking difference in haplotype diversity (high in COI mtDNA, low in ITS2 rDNA), no evident phylogeographic structure was apparent in the Palearctic O. caspius. The Holarctic O. dorsalis species was subdivided into two highly distinctive COI mtDNA phylogroups which corresponded to the Nearctic and Palearctic regions. Strong support for the independence of the two allopatric evolutionary lineages suggested that geographical barrier and climatic changes during Pleistocene caused vicariance of the ancestral range. COI mtDNA reliably distinguished O. caspius and O. dorsalis, while ITS2 rDNA yet again lacked the proper resolution for solving this problem. An integrative approach based on the larval and adult morphological traits have varying taxonomic applications due to their differential diagnostic values. Thus, by the implementation of an integrative taxonomic approach, we successfully detected species borders between the two epidemiologically relevant species and uncovered the presence of cryptic diversity within O. dorsalis.

Establishment of Aedes (Ochlerotatus) scapularis (Diptera: Culicidae) in Mainland Florida, With Notes on the Ochlerotatus Group in the United States.

Aedes scapularis (Rondani), a widespread neotropical vector mosquito species, has been included in the mosquito fauna of Florida on the basis of just three larval specimens that were collected in the middle Florida Keys in 1945. Here, we report numerous recent collections of immature and adult Ae. scapularis from multiple locations in two counties of southern Florida. These specimens represent the first records of Ae. scapularis from mainland Florida and the first records of the species in the state since the initial detection of the species 75 yr ago. Collections of both larvae and adults across several years indicate that Ae. scapularis is now established in Broward and Miami-Dade Counties. These contemporary records of this species in Florida may represent novel dispersal and subsequent establishment events from populations outside the United States or a recent reemergence of undetected endemic populations. To confirm morphological identification of Ae. scapularis specimens from Florida, the DNA barcoding region of the cytochrome c oxidase subunit I gene (COI) was sequenced and compared to all other Ochlerotatus Group species from the United States, specifically Aedes condolescens Dyar and Knab (Diptera: Culicidae), Aedes infirmatus Dyar and Knab (Diptera: Culicidae), Aedes thelcter Dyar (Diptera: Culicidae), Aedes tortilis (Theobald) (Diptera: Culicidae), and Aedes trivittatus (Coquillett) (Diptera: Culicidae). Molecular assays and sequencing confirm morphological identification of Ae. scapularis specimens. Maximum likelihood phylogenetic analysis of COI and ITS2 sequences place Florida Ae. scapularis in a distinct clade, but was unable to produce distinct clades for Florida specimens of Ae. condolescens and Ae. tortilis.

Nine years of mosquito monitoring in Germany, 2011-2019, with an updated inventory of German culicid species.

Before the background of increasingly frequent outbreaks and cases of mosquito-borne diseases in various European countries, Germany recently realised the necessity of updating decade-old data on the occurrence and spatiotemporal distribution of culicid species. Starting in 2011, a mosquito monitoring programme was therefore launched with adult and immature mosquito stages being collected at numerous sites all over Germany both actively by trapping, netting, aspirating and dipping, and passively by the citizen science project 'Mueckenatlas'. Until the end of 2019, about 516,000 mosquito specimens were analysed, with 52 (probably 53) species belonging to seven genera found, including several species not reported for decades due to being extremely rare (Aedes refiki, Anopheles algeriensis, Culex martinii) or local (Culiseta alaskaensis, Cs. glaphyroptera, Cs. ochroptera). In addition to 43 (probably 44 including Cs. subochrea) out of 46 species previously described for Germany, nine species were collected that had never been documented before. These consisted of five species recently established (Ae. albopictus, Ae. japonicus, Ae. koreicus, An. petragnani, Cs. longiareolata), three species probably introduced on one single occasion only and not established (Ae. aegypti, Ae. berlandi, Ae. pulcritarsis), and a newly described cryptic species of the Anopheles maculipennis complex (An. daciae) that had probably always been present but not been differentiated from its siblings. Two species formerly listed for Germany could not be documented (Ae. cyprius, Ae. nigrinus), while presence is likely for another species (Cs. subochrea), which could not be demonstrated in the monitoring programme as it can neither morphologically nor genetically be reliably distinguished from a closely related species (Cs. annulata) in the female sex. While Cs. annulata males were collected in the present programme, this was not the case with Cs. subochrea. In summary, although some species regarded endemic could not be found during the last 9 years, the number of culicid species that must be considered firmly established in Germany has increased to 51 (assuming Cs. subochrea and Ae. nigrinus are still present) due to several newly emerged ones but also to one species (Ae. cyprius) that must be considered extinct after almost a century without documentation. Most likely, introduction and establishment of the new species are a consequence of globalisation and climate warming, as three of them are native to Asia (Ae. albopictus, Ae. japonicus, Ae. koreicus) and three (Ae. albopictus, An. petragnani, Cs. longiareolata) are relatively thermophilic. Another thermophilic species, Uranotaenia unguiculata, which had been described for southwestern Germany in 1994 and had since been found only at the very site of its first detection, was recently documented at additional localities in the northeastern part of the country. As several mosquito species found in Germany are serious pests or potential vectors of disease agents and should be kept under permanent observation or even be controlled immediately on emergence, the German mosquito monitoring programme has recently been institutionalised and perpetuated.

MOSQUITOES (DIPTERA: CULICIDAE) OF THE WESTERN COASTAL AREA, KINGDOM OF SAUDI ARABIA: SPECIES COMPOSITION, ABUNDANCE, DIVERSITY AND MEDICAL IMPORTANCE.

An entomological survey was undertaken for two years to update the mosquito fauna of the 3 regions (Mecca, Al Madinah and Tabouk), of the western coast, Kingdom of Saudi Arabia. Six localies representing such Regions (lhif, Mecca, Jeddah, Yanbu, Daba and Haql) were sampled for larvae and adults. Nineteen soecies of six genera were reported of which genus Culex (57.89% sp.) were the most commo6, followed by Anopheles (21.05%) and Stegomyia, Ochlerotatus, Aedes and Culesita (5.26% each). Three species (Cx. sitiens, Cx. torrenlium and Am. v. arabiensis) were new reports in study area. A total of 10930 larvae and 33108 adults were collected of which those representing genus Culex were predominating (92.74% larva, 93.46% adult). Of the reported species, Cx. quinquefasciatus (47.11% larva, 57.52% adult) and Cx. pipiens (22.24% larva, 27.42% adult) Were the common ones. Both Taif and Tabouk reported the highest number of species (13 sp., 68.42%), followed by Jeddah (12 sp., 63.16%), Mecca (10 sp., 52.63%) and Yanbu (8 sp., 42.11%). Tabouk represented the maximum diversity with the highest Simpson index (1-D=0.65) and Shannon index (H=1.25) followed by Taif (1- D=0.54, H=1.01). Per sampling site, Jeddah reported the highest number of larvae (313.70), and Mecca had the highest adult number (670.18). A complete list of mosquito fauna of the western coast including 35. spp. was presented based on the present and previous surveys. The role of the reported mosquito species as disease vectors was discussed. The study concluded that the diversity and abundance of the different mosquito species, among which several vectors of diseases mainly malaria, Rift Valley fever, lymphatic filariasis and dengue fever may pause a risk of transmission and more spread of such disease in this part that contains the holy places which are visited all year round by millions of people from several countries.

Morphological Differentiation of the Eggs of North American Container-Inhabiting Aedes Mosquitoes.

Aedes aegypti , Ae. albopictus, Ae. japonicus japonicus, and Ae. triseriatus eggs are described with the aid of color images and images from scanning electron microscopy. All eggs were broadly cigar shaped with Ae. triseriatus and Ae. j japonicus eggs being dull or matte black, while Ae. albopictus and Ae. aegypti eggs were shiny jet black. Aedes triseriatus eggs were lighter in color and had a rougher appearance when compared to those of Ae. j. japonicus. Aedes albopictus and Ae. aegypti eggs can be differentiated by the distinct presence of micropylar collars in Ae. aegypti. Our results reduce the need to rear these mosquitoes, reducing labor and time while providing an accurate identification of the vector mosquitoes.

Elevation of Pseudoskusea, Rusticoidus and Protomacleaya to valid subgenera in the mosquito genus Aedes based on taxon naming criteria recently applied to other members of the Tribe Aedini (Diptera: Culicidae).

BACKGROUND: Pseudoskusea, Rusticoidus and Protomacleaya were well-recognized, morphologically distinct subgenera within the genus Aedes prior to a series of taxonomic changes over the past 15 years by Reinert, Harbach and Kitching, when they were recognized as subgenera of the genus Ochlerotatus. In our recent effort to stabilize the Tribe Aedini, we synonymized these subgenera and associated species back into the genus Aedes, but incorrectly assigned them as putative informal groups, instead of reinstating them to subgenera. CONCLUSION: Here we formally elevate three traditionally recognized subgenera (Pseudoskusea, Rusticoidus and Protomacleaya) within the genus Aedes.

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.

Effectiveness of Mosquito Magnet® trap in rural areas in the southeastern tropical Atlantic Forest

Traps are widely employed for sampling and monitoring mosquito populations for surveillance, ecological and fauna studies. Considering the importance of assessing other technologies for sampling mosquitoes, we addressed the effectiveness of Mosquito Magnet® Independence (MMI) in comparison with those of the CDC trap with CO2 and Lurex3® (CDC-A) and the CDC light trap (CDC-LT). Field collections were performed in a rural area within the Atlantic Forest biome, southeastern state of São Paulo, Brazil. The MMI sampled 53.84% of the total number of mosquitoes, the CDC-A (26.43%) and CDC-LT (19.73%). Results of the Pearson chi-squared test (χ2) showed a positive association between CDC-LT and species of Culicini and Uranotaeniini tribes. Additionally, our results suggested a positive association between CDC-A and representatives of the Culicini and Aedini tribes, whereas the MMI was positively associated with the Mansoniini and Sabethini as well as with Anophelinae species. The MMI sampled a greater proportion (78.27%) of individuals of Anopheles than either the CDC-LT (0.82%) or the CDC-A traps (20.91%). Results of the present study showed that MMI performed better than CDC-LT or CDC-A in sampling mosquitoes in large numbers, medically important species and assessing diversity parameters in rural southeastern Atlantic Forest.

Effectiveness of Mosquito Magnet® trap in rural areas in the southeastern tropical Atlantic Forest.

Traps are widely employed for sampling and monitoring mosquito populations for surveillance, ecological and fauna studies. Considering the importance of assessing other technologies for sampling mosquitoes, we addressed the effectiveness of Mosquito Magnet® Independence (MMI) in comparison with those of the CDC trap with CO2 and Lurex3® (CDC-A) and the CDC light trap (CDC-LT). Field collections were performed in a rural area within the Atlantic Forest biome, southeastern state of São Paulo, Brazil. The MMI sampled 53.84% of the total number of mosquitoes, the CDC-A (26.43%) and CDC-LT (19.73%). Results of the Pearson chi-squared test (χ2) showed a positive association between CDC-LT and species of Culicini and Uranotaeniini tribes. Additionally, our results suggested a positive association between CDC-A and representatives of the Culicini and Aedini tribes, whereas the MMI was positively associated with the Mansoniini and Sabethini as well as with Anophelinae species. The MMI sampled a greater proportion (78.27%) of individuals of Anopheles than either the CDC-LT (0.82%) or the CDC-A traps (20.91%). Results of the present study showed that MMI performed better than CDC-LT or CDC-A in sampling mosquitoes in large numbers, medically important species and assessing diversity parameters in rural southeastern Atlantic Forest.

Molecular identification of Aedes triseriatus and Aedes hendersoni by a novel duplex polymerase chain reaction assay.

Aedes triseriatus is the principal vector of La Crosse virus (LACv), which is the most common cause of pediatric arboviral encephalitis in North America. Here we report a novel species-specific polymerase chain reaction (PCR) assay that differentially identifies Ae. triseriatus and Ae. hendersoni. Because these 2 sibling species differ in their abilities to transmit LACv, accurate identification is critical for surveillance, research, and control programs. This duplex assay can detect the presence of both species in a single PCR reaction and is therefore simpler and faster than previously reported methods.

A multiplex PCR to differentiate the two sibling species of mosquitoes Ochlerotatus detritus and Oc. coluzzii and evidence for further genetic heterogeneity within the Detritus complex.

Internal transcribed spacer regions of ribosomal DNA were sequenced, and species-specific primers were designed to simplify the identification of two morphologically similar species of the Detritus complex, Ochlerotatus detritus and Ochlerotatuscoluzzii. Each newly designed primer was able to amplify a species-specific fragment with a different size. Samples from France and Germany were successfully tested. This new tool prompts for bio-ecological studies to refine basic knowledge on the bionomics of this species complex, towards a better control and prevention of ensuing nuisances. Moreover, ITS2 sequencing revealed the existence of (1) two distinct haplotypes of Oc. detritus that are sympatric and widely distributed along the French Atlantic and Mediterranean littorals and (2) a specific haplotype in mosquitoes sampled from Tunisia, raising the question of the taxonomic status of this North-African population.

A pictorial key to the species of Aedes (Ochlerotatus and Coetzeemyia) in the Afrotropical Region (Diptera: Culicidae).

Six species of the subgenus Ochlerotatus and one species of the subgenus Coetzeemyia of the genus Aedes in the Afrotropical Region are treated in a pictorial key based on diagnostic morphological features. Brief remarks on the new reclassification of the genus Aedes are also included.

Description of Himalayan mosquito pupae III, Ochlerotatus albolateralis and Ochlerotatus deccanus.

The pupae of Ochlerotatus albolateralis and Oc. deccanus are described and illustrated for the first time from specimens collected in the Himalayan Mountains in Nepal, breeding in tree holes and bamboo stumps. Chaetotaxal tables and illustrations show the morphology of their setae. Characters to recognize each species are explained.

Descriptions of the pupae of Ochlerotatus aloponotum and Ochlerotatus campestris.

Pupae of 2 western species, Ochlerotatus aloponotum and Oc. campestris, are described and illustrated. Sources of the original descriptions and known stages are given. The relation of Oc. aloponotum to Oc. excrucians and characters to separate them are discussed.

Description of the pupae of Ochlerotatus flavescens and Ochlerotatus melanimon.

The previously unknown pupae of Ochlerotatus flavescens and Oc. melanimon are described and illustrated. They will both be included in a forthcoming key to known pupae of genera Aedes and Ochlerotatus in the Nearctic Region. Characters to recognize each are explained.

Redescription of the pupae of Ochlerotatus dorsalis and Ochlerotatus squamiger.

The pupae of Ochlerotatus dorsalis and Oc. squamiger are completely described and illustrated for the first time. Considering the entire pupal morphology, characters to separate the 2 pupae are discussed.

On the evolution of saline tolerance in the larvae of mosquitoes in the genus Ochlerotatus.

We examined physiological and morphological parameters associated with saline tolerance in the larvae of 11 species of mosquito in the genus Ochlerotatus that were collected from the wild in a variety of sites around North America. Saline tolerance was assayed, and all of the species were osmoregulators. Six of the species examined were found to be physiologically restricted to freshwater habitats, while the other five could successfully osmoregulate in both freshwater and saline water, including seawater. All larvae that were obligate freshwater forms had only one rectal segment, while all of the euryhaline osmoregulators had two. We were interested in the evolutionary pathway by which saline tolerance arose in this lineage. DNA sequence data were obtained by polymerase chain reaction amplification and sequencing of the D2 region of the 28s rDNA gene in all of the freshwater and saline-tolerant Ochlerotatus species we studied. When the morphological and physiological characters were mapped on the resultant cladogram, they revealed a complex pattern, with freshwater and saline-water forms being adjacent and interspersed through the tree. The data also demonstrate that saline tolerance has been gained and then lost at least once in this lineage. Two possible evolutionary scenarios are presented, but the one we favor is that saline tolerance arose one time in this lineage and repeated reversions to the freshwater condition have occurred.

Description of Himalayan mosquito pupae III. Ochlerotatus gubernatoris and Ochlerotatus unicinctus.

The pupae of Ochlerotatus gubernatoris and Oc. unicinctus are described and illustrated for the first time. They were collected in the Himalaya Mountains of Nepal during field trips to the region. Both breed in natural and artificial containers.

Description of Himalayan mosquito pupae II. Ochlerotatus shortti and Oc. elsiae.

The pupae of Ochlerotatus shortti and Oc. elsiae are described and illustrated. These species occur in the Himalaya Mountains at middle range altitudes. Characters to recognize each are explained.

Description of the pupa of the Himalayan mosquito, Ochlerotatus pulchriventer.

The pupa of Ochlerotatus pulchriventer (=Aedes pulchriventer) is described and illustrated for the first time. It was collected at midrange altitudes in the Himalaya Mountains of Nepal. A subspecies from Taiwan is discussed.