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The work of collecting mosquitoes in the field is essential for improving knowledge about species of known and unknown vector status, their invasion dynamics, and further understanding their involvement in circulating pathogens of medical and veterinary importance. Over the years, different techniques have been developed to capture mosquitoes for entomological, pathogen transmission, and surveillance studies. For entomological and transmission dynamic research to be reliable, it is essential for mosquito specimens to be correctly identified so that their role in pathogen transmission can be appropriately assessed. Currently, modern molecular techniques support traditional morphological taxonomic identification and provide faster and more assertive identification. It is also important to test the susceptibility of potentially relevant mosquitoes or mosquitoes with unknown vector status in the laboratory to identify which species could participate in pathogen transmission. This last step toward fully performing vector incrimination of mosquito species and understanding their interactions with relevant pathogens requires proper handling of live specimens and laboratory colonization under artificial conditions to perform artificial infection studies. In this work, we aim to underscore the significance of the available tools for entomological studies and pathogen transmission research while also offering insights into the principles behind recent technological advancements that enhance the effectiveness and reliability of these studies. Graphical Abstract

Background Anopheles squamosus is a widespread mosquito species in sub-Saharan Africa. It is a potential vector for human malaria parasites and has been found naturally infected with Plasmodium falciparum and Plasmodium vivax . Morphological identification is challenging even with pristine specimens and current molecular methods such as the use of the internal transcribed spacer 2 (ITS2) polymerase chain reaction (PCR) cannot distinguish An. squamosus from morphologically similar Anopheles species . Described in the following methods is the development and validation of a new PCR assay that will reliably identify An. squamosus . Methods Multiple alignments of previously published ITS2 contig sequences in NCBI from An. squamosus and An. species 11 and 15, were used to identify candidate ITS2 regions for primer design. Six sets of primers were evaluated overall for specificity of species identification. The one set with An. squamosus species-specific amplification was tested using 78 specimens morphologically identified from Zambia and South Africa. Results A new assay consisting of a forward (ITS2-ASQ-R10, 5’-CCC TCG AAG GGT GCT GTG-3’) and reverse (ITS2-ASQ-R10 5’-AAT CCA CGG TGT GAT GGC-3’) primer reliably (> 94.9%) amplified an ITS2 fragment of 301 bp length for An. squamosus . The An. squamosus- specific primer set can be multiplexed with existing ITS2 assays frequently used for anopheline species identification. Conclusions The development of this robust PCR assay for An. squamosus is vital to accurate identification of this species in malaria vector surveillance efforts. Improved understanding of the anopheline community composition will lead to better targeted methods of vector eradication and malaria prevention. To further the validation of this ITS2 PCR assay, more species of Anopheles should be compared in addition to An. squamosus collected in different regions. To refine and optimize the PCR process with these primers, touchdown PCR can be used to increase specificity. Applying genomic tools to correctly identify An. squamosus will allow for a better understanding of their role in malaria transmission and may lead to genomic insights into what influences their behaviour, thus leading to new innovations in malaria elimination.

More than 3550 species of mosquitoes are known worldwide, and only a fraction is involved in the transmission of arboviruses. Mosquitoes in sylvatic and semi-sylvatic habitats may rapidly adapt to urban parks and metropolitan environments, increasing human contact. Many of these mosquitoes have been found naturally infected with arboviruses from the Alphaviridae, Flaviviridae, and Bunyaviridae families, with many being the cause of medically important diseases. However, there is a gap in knowledge about the vector status of newly invasive species and their potential threat to human and domestic animal populations. Due to their rapid distribution, adaptation to urban environments, and anthropophilic habits, some neglected mosquito species may deserve more attention regarding their role as secondary vectors. Taking these factors into account, we focus here on Aedes (Ochlerotatus) scapularis (Rondani), Aedes japonicus japonicus (Theobald), and Aedes (Fredwardsius) vittatus (Bigot) as species that have the potential to become important disease vectors. We further discuss the importance of these neglected mosquitoes and how factors such as urbanization, climate change, and globalization profoundly alter the dynamics of disease transmission and may increase the participation of neglected species in propagating diseases.

The main mosquito species capable of transmitting arboviruses belong to the genera Aedes spp., Psorophora spp., Anopheles spp., Culex spp., Mansonia spp., Coquillettidia spp., Haemagogus spp., Sabethes spp., Culiseta spp., and Wyeomyia spp. Some neglected mosquito species have the potential to become significant disease vectors due to parameters such as global distribution, rapid adaptation to urban areas, and anthropophilic habits. This review discusses the epidemiological importance and biology of three neglected mosquitoes, Aedes scapularis , Aedes vittatus , and Aedes japonicus japonicus , in the context of vectorial capacity and how urbanization, climate change, and globalization alter disease transmission dynamics and may increase the participation of neglected species in propagating diseases. More than 3550 species of mosquitoes are known worldwide, and only a fraction is involved in the transmission of arboviruses. Mosquitoes in sylvatic and semi-sylvatic habitats may rapidly adapt to urban parks and metropolitan environments, increasing human contact. Many of these mosquitoes have been found naturally infected with arboviruses from the Alphaviridae , Flaviviridae , and Bunyaviridae families, with many being the cause of medically important diseases. However, there is a gap in knowledge about the vector status of newly invasive species and their potential threat to human and domestic animal populations. Due to their rapid distribution, adaptation to urban environments, and anthropophilic habits, some neglected mosquito species may deserve more attention regarding their role as secondary vectors. Taking these factors into account, we focus here on Aedes (Ochlerotatus ) scapularis (Rondani), Aedes japonicus japonicus (Theobald), and Aedes (Fredwardsius ) vittatus (Bigot) as species that have the potential to become important disease vectors. We further discuss the importance of these neglected mosquitoes and how factors such as urbanization, climate change, and globalization profoundly alter the dynamics of disease transmission and may increase the participation of neglected species in propagating diseases.

Ades aegypti is the most important arbovirus vector in the world, and new strategies are under evaluation. Biological studies mentioning the occurrence of a second mate in Aedes aegypti can interfere with vector control program planning, which involves male mosquito release technique. This study presents different experiments to show the occurrence of mixed progeny. Mixed male crosses (using a combination of different type of males in confinement with virgin females) showed no polyandric female. Individual crosses with male substitution in every gonotrophic cycle also did not show any polyandric female. Individual crosses with a 20 minutes interval, with subsequent male change, showed that only a few females presented mixed offspring. The copulation breach in three different moments, group A with full coitus length, group B the coitus was interrupted in 5-7 seconds after the start; and group C, which the copulation was interrupted 3 seconds after started. In summary, group A showed a majority of unique progeny from the first male; group B showed the higher frequency of mixed offspring and group C with the majority of the crosses belonging to the second male. To conclude, the occurrence of a viable second mate and mixed offspring is only possible when the copulation is interrupted; otherwise, the first mate is responsible for mixed progeny.

[This corrects the article DOI: 10.1371/journal.pone.0193164.].