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[This corrects the article DOI: 10.1371/journal.pone.0313227.].

[This corrects the article DOI: 10.1371/journal.pone.0309199.].

Anopheles baileyi species D of the Baileyi Complex, subgenus Anopheles (Diptera: Culicidae) in Thailand is diagnosed and formally named An. inthanonensis Somboon & Harbach, n. sp. Morphological characters of the adults, and the pupal and larval stages with their chaetotaxy, are provided and compared with other species of the complex. Phylogenetic analysis of COI sequences revealed that An. inthanonensis appears to be more closely related to An. monticola in Bhutan and China than it is to other members of the Baileyi Complex.

In temperate regions of the United States, female Anopheles mosquitoes respond to low temperatures and short photoperiods by entering an overwintering dormancy or diapause. Diapause in Anopheles results in reduced frequency of blood-feeding and reproductive arrest, indicating a period when pathogen transmission by these mosquitoes is unlikely. However, it is unclear precisely how late into the fall and how early in the spring these mosquitoes are biting, reproducing, and potentially transmitting pathogens. This is further complicated by the lack of clear markers of diapause in Anopheles (e.g., changes in egg follicle length). Our goal was to characterize the seasonal reproductive activity of female Anopheles in central Ohio, United States and evaluate egg follicle length as an indicator of Anopheles diapause. We used traditional mosquito traps and aspirators to collect Anopheles from urban woodlots and culverts, respectively, from late September 2021 through mid-May 2022 in central Ohio. By measuring their egg follicle length, reproductive status, and bloodfeeding status, we found that egg follicle length is not a reliable indicator of Anopheles diapause. We also found that a small proportion of An. punctipennis (Say), An. perplexens (Ludlow), and An. quadrimaculatus (Say) continued to bite and reproduce into early November 2021 and that females of these species terminated reproductive dormancy and began biting by mid-March 2022. This period of reproductive activity extends beyond current mosquito surveillance and control in Ohio. Our findings suggest that within temperate regions of North America, Anopheles have the capacity to transmit pathogens throughout the spring, summer, and fall.

This article deals with the morphological and molecular assessments of 3 species of mosquitoes collected in Yunnan Province, China, i.e Anopheles monticola Somboon, Namgay & Harbach, the only species of the Baileyi Complex identified, Culex sasai Kano, Nitahara &Awaya, and Lutzia vorax Edwards. Interestingly, some adult specimens of An. monticola exhibited a variant form in having pale fringe spots, as found in An. simlensis James, another species of the Baileyi Complex. Culex sasai was recorded for the first time in Yunnan and mainland China. The prevalence of Lt. vorax was confirmed, and previous records of Lt. halifaxii in China are regarded as misidentifications of Lt. vorax.

Five sibling species of the Lindesayi Complex of the genus Anopheles have been discovered in Bhutan: An. druki Somboon, Namgay & Harbach, An. himalayensis Somboon, Namgay & Harbach, An. lindesayi Giles, An. lindesayi species B, and An. thimphuensis Somboon, Namgay & Harbach. The species are morphologically similar in the adult and/or immature stages. This study aimed to develop a multiplex PCR assay to identify the 5 species. Allele-specific primers were designed for specific nucleotide segments of ITS2 sequences previously reported for each species. The assay provided products of 183 bp for An. druki, 338 bp for An. himalayensis, 126 bp for An. lindesayi, 290 bp for An. lindesayi species B, and 370 bp for An. thimphuensis. The use of the assay produced consistent results. The assay is relatively inexpensive, enables the rapid identification of a large number of specimens, and will foster further studies of the Lindesayi Complex.

Constant and extensive use of chemical insecticides has created a selection pressure and favored resistance development in many insect species worldwide. One of the most important pyrethroid resistance mechanisms is classified as target site insensitivity, due to conformational changes in the target site that impair a proper binding of the insecticide molecule. The voltage-gated sodium channel (NaV) is the target of pyrethroids and DDT insecticides, used to control insects of medical, agricultural and veterinary importance, such as anophelines. It has been reported that the presence of a few non-silent point mutations in the Na V gene are associated with pyrethroid resistance, termed as ‘kdr’ (knockdown resistance) for preventing the knockdown effect of these insecticides. The presence of these mutations, as well as their effects, has been thoroughly studied in Anopheles mosquitoes. So far, kdr mutations have already been detected in at least 13 species (Anopheles gambiae, Anopheles arabiensis, Anopheles sinensis, Anopheles stephensi, Anopheles subpictus, Anopheles sacharovi, Anopheles culicifacies, Anopheles sundaicus, Anopheles aconitus, Anopheles vagus, Anopheles paraliae, Anopheles peditaeniatus and Anopheles albimanus) from populations of African, Asian and, more recently, American continents. Seven mutational variants (L1014F, L1014S, L1014C, L1014W, N1013S, N1575Y and V1010L) were described, with the highest prevalence of L1014F, which occurs at the 1014 site in NaVIIS6 domain. The increase of frequency and distribution of kdr mutations clearly shows the importance of this mechanism in the process of pyrethroid resistance. In this sense, several species-specific and highly sensitive methods have been designed in order to genotype individual mosquitoes for kdr in large scale, which may serve as important tolls for monitoring the dynamics of pyrethroid resistance in natural populations. We also briefly discuss investigations concerning the course of Plasmodium infection in kdr individuals. Considering the limitation of insecticides available for employment in public health campaigns and the absence of a vaccine able to brake the life cycle of the malaria parasites, the use of pyrethroids is likely to remain as the main strategy against mosquitoes by either indoor residual spraying (IR) and insecticide treated nets (ITN). Therefore, monitoring insecticide resistance programs is a crucial need in malaria endemic countries.

Background The World Health Organization (WHO) recommends mass distribution of insecticide-treated nets (ITNs) to prevent malaria transmission. Unfortunately, resistance to pyrethroids affects the efficacy of standard ITNs. To overcome this resistance and continue to protect the population, the WHO has recommended new types of ITNs that combine a pyrethroid insecticide with either a synergist (PBO) or a second insecticide, such as chlorfenapyr. This study examines the baseline characteristics of malaria vectors prior to the distribution of three types of insecticide-treated nets as part of a three-arm randomised controlled trial: Interceptor G2 (pyrethroid–chlorfenapyr), VEERALIN (pyrethroid–PBO), and MAGNet (pyrethroid only). Methods The study was carried out in 40 villages (grouped into 33 clusters) of Tiébissou district in central Côte d’Ivoire. To assess biting rate and biting behaviour, human landing catches were conducted hourly indoors and outdoors in six randomly selected houses in each cluster, starting at 18:00 and continuing until 08:00 the next morning. Adult mosquitoes collected were morphologically identified, and a subset of Anopheles gambiae sensu lato (s.l.) and An. funestus s.l. were speciated by quantitative PCR (qPCR). Plasmodium sporozoite infections were detected by qPCR to estimate infection rates. The entomological inoculation rate was calculated as the product of the mosquito biting rate and the sporozoite infection rate. Results Among the 10,698 mosquitoes collected, An. gambiae s.l. was the predominant species, accounting for 62.5% ( n  = 6683) of the catch, followed by An. funestus s.s., which accounted for 19.8% ( n  = 2120). Of the sub-sample of An. gambiae s.l. processed by PCR, 79.0% ( n  = 1291/1635) were An. coluzzii and the remaining were Anopheles gambiae s.s. Malaria vectors were highly aggressive, with an average of 14.8 bites/person/night for An. coluzzii , 2.0 b/p/n for An. gambiae s.s. and 5.4 b/p/n for An. funestus , representing an overall average of 22.2 b/p/n (95% CI 17.2–27.2 b/p/n). No significant difference was found in biting activity between indoor and outdoor environments ( Z  = −0.25, P  = 0.803). Plasmodium sporozoite infection rate was 2.4% (95% CI 1.3–3.6%) for An. coluzzii , 1.5% (95% CI 0.3–2.6%) for An. gambiae s.s. and 2.7% (95% CI 1.2–4.3%) for An. funestus . The estimated overall entomological inoculation rate was 0.4 infected b/p/n (95% CI 0.3–0.6) and varied between 0.0 and 0.2 infective bites/person/night according to species. There was no difference observed in entomological infection rate (EIR) between capture locations (indoors versus outdoors; Z  = 1.521, P  = 0.128). Conclusions This study shows that An. coluzzii and An. funestus were the main malaria vectors and showed similar biting patterns both indoors and outdoors. Anopheles funestus was found in high density in a limited number of villages. Malaria transmission was high despite universal distribution of pyrethroid-ITN in the district. Graphical Abstract

This paper deals with species A, C, D, and E of the Lindesayi Complex of Anopheles subgenus Anopheles (Diptera: Culicidae) that were recently recorded from Bhutan. Species B is not included due to insufficient data. Species A is Anopheles lindesayi sensu stricto, and species C, D, and E are new species that are formally described and named herein as Anopheles druki Somboon, Namgay & Harbach, Anopheles himalayensis Somboon, Namgay & Harbach and Anopheles thimphuensis Somboon, Namgay & Harbach, respectively. Morphological characteristics of the adults, larval and pupal stages of each species are provided and compared with An. lindesayi from Bhutan.