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Background Anopheles darlingi is a primary malaria vector in the Peruvian Amazon, yet characterization of behavioural traits contributing to human-vector contact is limited. Additionally, studies comparing key behaviours of wild-type to colonized An. darlingi populations are minimal. This study compared resting and entry behaviour between these two types of populations. Specific objectives were to use experimental huts to (1) evaluate and compare indoor resting behaviours of wild-type and colonized An. darlingi populations; (2) quantify An. darlingi house entry rates into interception traps in relation to protected Human Landing collection (HLC). Methods The study was conducted in Zungarococha village, Loreto, Peru. Prior to hut evaluations, An. darlingi biting activity and population dynamics were evaluated using HLC outside local homes from June 2014 to May 2015. Indoor resting location (window, door, wall, roof) of wild caught and colonized An. darlingi was evaluated in three experimental huts. Controlled indoor releases were performed for 6 days each month from March to August 2015. Wild An. darlingi hourly house-entry rates were quantified using interception traps affixed to an experimental hut and indoor HLC from May to August 2015. Two collectors were positioned inside huts to generate host-seeking cues during evaluations. Results Anopheles darlingi had a bimodal outdoor biting pattern with two peaks at 1800 h and 2200 h. HLC densities were associated with Amazon River levels measured nearby Iquitos city. Colonized An. darlingi preferred to rest in lower parts of the door (29.8%), roof (12.7%), and window (11.8%) which was similar to wild caught An. darlingi which preferred to rest in low door (32.7%), window (14.0%), and roof (13.0%). Wild An. darlingi entry behaviour peaked from 2200 to 2300 h, this was clearly observed when collection densities increased, varying between 2300 and 2400 h at low collection densities. Capture rates from interception traps were lower compared to indoor HLC rates from adjacent experimental huts and local houses. Conclusions Results from this study provide useful insights into An. darlingi resting and entry behaviour in a malaria endemic area in the Peruvian Amazon and inform on the use of colonized mosquitos in vector behavioural studies. This information is relevant to malaria epidemiology and will be useful to evaluate new tools for malaria control programmes.

Background In Peru, the information regarding sand fly vectors of leishmaniasis and bartonellosis in the Amazon region is limited. In this study, we carried out sand fly collections in Peruvian lowland and highland jungle areas using different trap type configurations and screened them for Leishmania and Bartonella DNA. Methodology/Principal findings Phlebotomine sand flies were collected in Peruvian Amazon jungle and inter Andean regions using CDC light trap, UV and color LED traps, Mosquito Magnet trap, BG Sentinel trap, and a Shannon trap placed outside the houses. Leishmania spp. screening was performed by kDNA PCR and confirmed by a nested cytochrome B gene (cytB) PCR. Bartonella spp. screening was performed by ITS PCR and confirmed by citrate synthase gene (gltA). The PCR amplicons were sequenced to identify Leishmania and Bartonella species. UV and Blue LED traps collected the highest average number of sand flies per hour in low jungle; UV, Mosquito Magnet and Shannon traps in high jungle; and Mosquito Magnet in inter Andean region. Leishmania guyanensis in Lutzomyia carrerai carrerai and L. naiffi in Lu. hirsuta hirsuta were identified based on cytB sequencing. Bartonella spp. related to Bartonella bacilliformis in Lu. whitmani, Lu. nevesi, Lu. hirsuta hirsuta and Lu. sherlocki, and a Bartonella sp. related to Candidatus B. rondoniensis in Lu. nevesi and Lu. maranonensis were identified based on gltA gene sequencing. Conclusions/Significance UV, Blue LED, Mosquito Magnet and Shannon traps were more efficient than the BG-Sentinel, Green, and Red LED traps. This is the first report of L. naiffi and of two genotypes of Bartonella spp. related to B. bacilliformis and Candidatus B. rondoniensis infecting sand fly species from the Amazon region in Peru.

Cutaneous leishmaniasis (CL) is an important health problem in the New World affecting civilian and military populations that are frequently exposed in endemic settings. The Peruvian region of Madre de Dios located near the border with Brazil is one of the most endemic CL regions in South America with more than 4,451 reported cases between 2010 and 2015 according to the Peruvian epidemiology directorate. However, little is known regarding the diversity and distribution of sand fly vectors in this region. In this study, we aimed to characterize the sand fly fauna in this endemic setting and identify sand fly species naturally infected with Leishmania possibly involved in pathogen transmission. Sand fly collections were carried out during 2014 and 2015 in the communities of Flor de Acre, Villa Primavera, Mavila and Arca Pacahuara using CDC light traps and Shannon traps. Collected specimens were identified and non-blood-fed females were selected for Leishmania infection screening using kinetoplastid DNA-PCR (kDNA-PCR) and nested Real time PCR for species identification. A total of 10,897 phlebotomines belonging to the genus Lutzomyia (58 species) and Brumptomyia (2 species) were collected. Our study confirmed the widespread distribution and abundance of Lutzomyia (Trichophoromyia) spp. (24%), Lu. whitmani (19.4%) and Lu. yucumensis (15.8%) in the region. Analysis of Shannon diversity index indicates variability in sand fly composition across sites with Villa Primavera presenting the highest sand fly diversity and abundance. Leishmania screening by kDNA-PCR resulted in 45 positive pools collected from Flor de Acre (34 pools), Mavila (10 pools) and Arca Pacahuara (1 pool) and included 14 species: Lu. yucumensis, Lu. aragoi, Lu. sallesi, Lu. sherlocki, Lu. shawi, Lu. walkeri, Lu nevesi, Lu. migonei, Lu. davisi, Lu. carrerai, Lu. hirsuta, Lu. (Trichophoromyia) spp., Lu. llanosmartinsi and Lu. whitmani. Lutzomyia sherlocki, Lu. walkeri and Lu. llanosmartinsi had the highest infection rates (8%, 7% and 6%, respectively). We identified Leishmania guyanensis in two Lu. whitmani pools, and L. braziliensis in two Lu. llanosmartinsi pools and one Lu. davisi pool. Based on our collections there is high sand fly diversity in Madre de Dios, with differences in sand fly abundance and species composition across sites. We identified 14 sand fly species naturally infected with Leishmania spp., having detected natural infection with L. (V.) guyanensis and L. (V.) braziliensis in three sand fly species. These results suggest the presence of several potential vectors that vary in their spatial and geographical distribution, which could explain the high prevalence of CL cases in this region.

Background Outdoor malaria transmission hinders malaria elimination efforts in the Amazon region and novel vector control tools are needed. Ivermectin mass drug administration (MDA) to humans kills wild Anopheles , targets outdoor-feeding vectors, and can suppress malaria parasite transmission. Laboratory investigations were performed to determine ivermectin susceptibility, sporontocidal effect and inhibition of time to re-feed for the primary Amazonian malaria vector, Anopheles darlingi . Methods To assess ivermectin susceptibility, various concentrations of ivermectin were mixed in human blood and fed to An. darlingi . Mosquito survival was monitored daily for 7 days and a non-linear mixed effects model with Probit analysis was used to calculate lethal concentrations of ivermectin that killed 50% (LC 50 ), 25% (LC 25 ) and 5% (LC 5 ) of mosquitoes. To examine ivermectin sporonticidal effect, Plasmodium vivax blood samples were collected from malaria patients and offered to mosquitoes without or with ivermectin at the LC 50 , LC 25 or LC 5 . To assess ivermectin inhibition of mosquito time to re-feed, concentrations of ivermectin predicted to occur after a single oral dose of 200 μg/kg ivermectin were fed to An. darlingi . Every day for 12 days thereafter, individual mosquitoes were given the opportunity to re-feed on a volunteer. Any mosquitoes that re-blood fed or died were removed from the study. Results Ivermectin significantly reduced An. darlingi survivorship: 7-day-LC 50  = 43.2 ng/ml [37.5, 48.6], -LC 25  = 27.8 ng/ml [20.4, 32.9] and -LC 5  = 14.8 ng/ml [7.9, 20.2]. Ivermectin compound was sporontocidal to P. vivax in An. darlingi at the LC 50 and LC 25 concentrations reducing prevalence by 22.6 and 17.1%, respectively, but not at the LC 5 . Oocyst intensity was not altered at any concentration. Ivermectin significantly delayed time to re-feed at the 4-h (48.7 ng/ml) and 12-h (26.9 ng/ml) concentrations but not 36-h (10.6 ng/ml) or 60-h (6.3 ng/ml). Conclusions Ivermectin is lethal to An. darlingi , modestly inhibits sporogony of P. vivax , and delays time to re-feed at concentrations found in humans up to 12 h post drug ingestion. The LC 50 value suggests that a higher than standard dose (400-μg/kg) is necessary to target An. darlingi . These results suggest that ivermectin MDA has potential in the Amazon region to aid malaria elimination efforts.

Japanese encephalitis is a disease caused by the Japanese encephalitis virus (JEV) and is a concern for U.S. military personnel stationed in the Republic of Korea (ROK). The recent literature reports a potential shift from GI to GV as the dominant genotype circulating in east Asia. In the ROK, GV has been reported in a few Culex spp., but not in the main JEV vector, Cx. tritaeniorhynchus. The goal of this surveillance was to shed light on the current knowledge of the epidemiology of JEV in the ROK by analyzing mosquito collection data from three consecutive years, 2021–2023, and molecularly detecting and genotyping JEV in all Culex spp. collected in several military locations across the ROK. In this study, we detected only JEV GI in Cx. tritaeniorhynchus in 2021 samples. In contrast, all 2022 and 2023 positive samples were GV and detected in Cx. bitaeniorhynchus, Cx. orientalis, and Cx. pipiens. Results support a shift in JEV genotype in the ROK and suggest that for GV, Culex spp. other than Cx. tritaeniorhynchus may be playing an important role.

U.S. military troops are exposed to mosquito-borne pathogens when deployed to endemic regions. Personal protective measures such as permethrin-treated uniforms and dermal repellents are the cornerstones of mosquito-borne disease prevention for the U.S. military. These measures have limitations and additional personal protection tools, such as spatial repellent devices to decrease the risk of vector-borne pathogen transmission, are required. Novel spatial repellent controlled-release devices formulated with metofluthrin were evaluated in an outdoor setting in the northern Amazon of Peru to evaluate performance under field conditions. The metofluthrin emitting devices lowered the number of mosquitoes captured in protected human landing collections (HLC) compared to blank devices, although there were effect differences between Anopheles spp. and species in other mosquito genera. A computational-experimental model was developed to correlate HLC and active ingredient (AI) concentrations as a function of time and space. Results show a strong correlation between the released AI and the decrease in HLC. This model represents the first effort to obtain a predictive analytical tool on device performance using HLC as the entomological endpoint.

Barrier insecticide treatments have a long history in mosquito control programs but have been used more frequently in the United States in recent years for control of invasive “backyard” species (eg, Aedes albopictus) and increases in incidence of vector-borne diseases (eg, Zika). We reviewed the published literature for studies investigating barrier treatments for mosquito control during the last 74 years (1944-2018). We searched databases such as PubMed, Web of Science, and Google Scholar to retrieve worldwide literature on barrier treatments. Forty-four studies that evaluated 20 active ingredients (AIs) and 21 formulated products against multiple mosquito species are included. Insecticides investigated for efficacy included organochlorines (dichlorodiphenyltrichloroethane [DDT], β-hexachlorocyclohexane [BHC]), organophosphates (malathion), and pyrethroids (bifenthrin, deltamethrin, permethrin, lambda-cyhalothrin) as AIs. Study design varied with multiple methods used to evaluate effectiveness of barrier treatments. Barrier treatments were effective at lowering mosquito populations although there was variation between studies and for different mosquito species. Factors other than AI, such as exposure to rainfall and application equipment used, also influenced control efficacy. Many of the basic questions on the effectiveness of barrier insecticide applications have been answered, but several important details still must be investigated to improve precision and impact on vector-borne pathogen transmission. Recommendations are made to assist future evaluations of barrier treatments for mosquito control and to limit the potential development of insecticide resistance.

Mosquito-borne pathogens are a threat to US troops stationed in the Republic of Korea. From April 2023 until December 2024, larvae and pupae of Aedes albopictus , Ae. koreicus , and Culex pipiens collected from U.S. Army garrisons were screened for resistance to chlorpyrifos, deltamethrin, etofenprox and permethrin using the Centers for Disease Control and Prevention bottle bioassay. Colonies of Cx. pipens var. molestus from Camp Humphreys, Camp George, and the Busan Storage Facility were established and tested against permethrin and permethrin + piperonyl butoxide (PBO). No resistance to deltamethrin or chlorpyrifos was detected in Ae. albopictus populations, but 1 population from Camp George showed possible resistance to permethrin . Aedes koreicus indicated possible resistance to etofenprox from Camp Casey in 2023 and resistance to etofenprox from Camp Casey in 2024. All populations of Ae. koreicus tested were susceptible to chlorpyifos, permethrin, and deltamethrin. Culex pipiens field-collected populations were susceptible to chlorpyrifos and resistant to permethrin, etofenprox, and deltamethrin. Exposing laboratory Cx. pipiens var. molestus to PBO and then permethrin restored susceptibility to all 3 colonies. Screening will be ongoing to track changes in resistance status for these species.

Mosquito-borne pathogens are a threat to US troops stationed in the Republic of Korea (ROK). Insecticide resistance has been reported in mosquito vectors in the ROK, highlighting the need for a sustained ROK-wide resistance surveillance program. To address this need from April 2022 until October 2022, larvae and pupae of Aedes albopictus , Ae. koreicus , and Culex pipiens were collected from US Army Garrison (USAG) Daegu (Camps Carroll and Henry), USAG Yongsan-Casey (Camp Casey), and USAG Humphreys (Camp Humphreys) and screened for resistance to insecticides using the Centers for Disease Control and Prevention (CDC) bottle bioassay. No resistance to deltamethrin or chlorpyrifos was detected in Ae. albopictus populations, but one population showed possible resistance to permethrin. Aedes koreicus populations were found to be resistant to etofenprox and permethrin with possible resistance to deltamethrin but were susceptible to chlorpyrifos. Culex pipiens populations were found to be resistant to chlorpyrifos, permethrin, and deltamethrin. Screening using CDC bottle bioassays will continue, and efforts will be made to determine the operational impact of the assay results on military installation mosquito control programs.

The Asian longhorned tick (ALT), Haemaphysalis longicornis, is a three‐host hard tick native to East Asia. Its opportunistic feeding habits make it an acute agricultural and medical threat, capable of spreading various zoonotic pathogens. An affinity for livestock and companion animals has allowed parthenogenetic populations of ALT to travel to and establish in overseas locations including the United States. To better understand the population dynamics of this rapidly expanding species, we sequenced the complete mitogenome of specimens collected from native and invasive ranges and performed phylogeographic analyses. As well as illustrating the diversity of Australasian and US ALT haplotypes, these methods have allowed us to estimate the source and frequency of successful introductions to the US. We highlight four potential introductions of parthenogenetic ALT, with likely origin populations identified in the Republic of Korea and Japan. These findings provide insight into potential routes of entry for ALT and other invasive tick species. The Asian longhorned tick, Haemaphysalis longicornis, is a significant global vector and threat to both agriculture and public health. Formerly known only in the Eastern Hemisphere, parthenogenetic ALT have been reported from 20 states across the eastern and central US, with a pattern of expansion suggestive of multiple distinct populations. Our mitogenomic analysis of specimens from both native and invasive populations provides evidence for the first time of at least four separate US introductions of parthenogenetic ALT and directly links each lineage to respective and putative source populations identified in East Asia.