Figure 3 in this Journal of Medical Entomology article is central to the authors' warning about an exploding white-tailed deer population but conflicts in important aspects with the relevant deer research. Among other problems, it shows a 60% increase in the white-tailed deer density from 1500 to 2020 when the research consensus is that the population is about the same. It shows an exploding population from 2000 to 2020 without supporting data when the population peaked around the year 2000 according to evidence-based research.

This review describes an innovative and efficient modification of a type of Malaise trap meme overlooked in a recent review of such insect traps. It further identifies the large variety of dipteran parasites of vertebrates caught in the traps when CO₂ was added as an attractant that otherwise rarely were caught in unbaited traps. Baited trap catches of parasitic flies mimicked those caught attacking cervid hosts. This review particularly focuses on comprehensive studies of: 1) several hematophagous species of largely unknown snipe flies (Diptera: Rhagionidae: Symphoromyia [Artiodactyla: Cervidae]) found host specific for Columbian black-tailed deer (Odocoileus hemionus columbianus Richardson), and 2) the biology of oestrid fly (Diptera: Oestridae) parasites of black-tailed deer and caribou/reindeer [Rangifer tarandus (L.) (Artiodactyla: Cervidae)], after it was discovered that the non-hematophagous females are guided to their vertebrate hosts by tracking a source of CO₂, as done by many hematophagous flies.

Mosquitoes and other blood feeding arthropods are vectors of pathogens causing serious human diseases, such as Plasmodium spp. (malaria), Wuchereria bancrofti (lymphatic filariasis), Borrelia burgdorferi (Lyme disease), and viruses causing dengue, Zika, West Nile, chikungunya, and yellow fever. Among the most effective strategies for the prevention of vector-borne diseases are those aimed at reducing human–vector interactions, such as insecticide applications and insecticide-treated bed nets (ITNs). In some areas where ITNs are widely used, behavioral adaptations have resulted in mosquitoes shifting their time of blood feeding to earlier or later in the night when the bed nets are not being employed. Little is known about the genetic basis of these behavioral shifts. We conducted quantitative trait locus (QTL) analysis using two strains of Culex pipiens sensu lato with contrasting blood feeding behaviors, wherein the lab adapted Shasta strain blood feeds at any time of the day or night, while the newly established Trinidad strain feeds only at night. We identified a single locus on chromosome 2 associated with the observed variation in feeding times. None of the core clock genes period, timeless, clock, cycle, PAR-domain protein 1, vrille, discs overgrown, cryptochrome 1, or cryptochrome 2 were located within the QTL region. We then monitored locomotor behavior to determine if they differed in their flight activity. The highly nocturnal Trinidad strain showed little daytime activity while the day-feeding Shasta strain was active during the day, suggesting blood feeding behavior and flight activity are physiologically linked.

Species belonging to the Muscidae family are important in the medical-sanitary and forensic fields due to their biology. The intrapuparial stage of Hydrotaea aenescens (Wiedemann, 1830), which represents about 50% of the immature development cycle in muscoid Diptera, has not yet been studied in detail. This study identifies and describes the main temporal morphological changes that occur in the pupal body of H. aenescens during its development at 22, 27, and 32 ± 1°C, RH 60 ± 10% with a 12-hr photoperiod. Ten pupae were collected, sacrificed, and fixed every 3 hr after pupariation up to 24 hr, and every 6 hr thereafter until the first emergence. Emergence took place at 282, 174, and 126 hr at 22, 27, and 32°C, with 520, 340, and 260 pupae, respectively. External morphology was described and recorded at eight development stages: pupariation, pre-pupae, early and late cryptocephalic pupae, phanerocephalic pupae, pupae-adult apolysis, pharate adults, and imago, employing 18 key morphological characters for pupae age. These morphological pupae characters, comprise, therefore an alternative and/or additional method to aid in determining the minimum Postmortem Interval (_minPMI).

Graphical Abstract

Espécies da família Muscidae são importantes no campo médico-sanitário e forense devido a sua biologia. O estágio intrapuparial do Hydrotaea aenescens (Wiedemann, 1830), que representa cerca de 50% do ciclo de desenvolvimento imaturo de Diptera muscóide, ainda não foi estudado em detalhes. Este estudo identifica e descreve as principais alterações morfológicas temporais que ocorrem no corpo da pupa de H. aenescens durante seu desenvolvimento sob as temperaturas de 22, 27 e 32 ± 1°C, 60 ± 10% de umidade relativa e fotoperíodo de 12 horas. Dez pupas foram coletadas, mortas e fixadas a cada 3 horas a partir da pupariação até 24 horas e depois a cada 6 horas até a primeira emergência. A emergência ocorreu a 282, 174 e 126 horas sob 22, 27 e 32°C, com 520, 340 e 260 pupas, respectivamente. A morfologia externa foi descrita e registrada em oito fases de desenvolvimento: pupariação; pré-pupa; pupa criptocefálica anterior e posterior; pupa fanerocefálica; apólise pupa-adulto; adulto farato e imago, usando 18 caracteres morfológicos chave para a idade das pupas. Assim, estes caracteres morfológicos das pupas são uma alternativa e/ou método adicional para ajudar a determinar o Intervalo Pós-Morte mínimo (IPM_mín).

Dipetalogaster maxima (Uhler) is a triatomine species that has been found to be infected by Trypanosoma cruzi Chagas in the habitats of the most important tourist areas of Mexico. Its behavior and vectorial capacity have been scarcely studied, although such information is necessary to reliably estimate the importance of this species as a vector of T. cruzi in its distribution area. This study reports biological parameters related to the vectorial capacity of D. maxima. In particular, the egg-to-adult development time, number of blood meals required to molt, accumulative mortality, time to beginning of feeding, feeding and defecation times, fecundity, and fertility were examined. D. maxima took a median of 211 d to develop from egg to adult, requiring 11 meals in total. Almost two-thirds (63%) of specimens died during the cycle. The time to beginning of feeding was 1 min in all instars. Feeding times varied from 14 to 27 min. Most nymphs (except first-instar) defecated when feeding or immediately thereafter. A mean of 0.7 eggs/♀/day was recorded, with an eclosion rate of 27.3%. Five of the eight studied parameters (mainly defecation delay) suggest the remarkable potential vectorial capacity of D. maxima, so it is necessary to maintain permanent surveillance of domiciliary populations of D. maxima, because they may be infected with T. cruzi.

Graphical Abstract

The mosquito Aedes aegypti is the primary vector of the dengue, yellow fever, and chikungunya viruses. Evidence shows that Ae. aegypti males are polyandrous whereas Ae. aegypti females are monandrous in mating. However, the degree to which Ae. aegypti males and females can mate with different partners has not been rigorously tested. Therefore, this study examined the rates of polyandry via parentage assignment in three sets of competitive mating experiments using wild-type male and female Ae. aegypti. Parentage assignment was monitored using nine microsatellite DNA markers. All Ae. aegypti offspring were successfully assigned to parents with 80% or 95% confidence using CERVUS software. The results showed that both male and female Ae. aegypti mated with up to 3–4 different partners. Adults contributed differentially to the emergent offspring, with reproductive outputs ranging from 1 to 25 viable progeny. This study demonstrates a new perspective on the capabilities of male and female Ae. aegypti in mating. These findings are significant because successful deployment of reproductive control methods using genetic modification or sterile Ae. aegypti must consider the following criteria regarding their mating fitness: 1) choosing Ae. aegypti males that can mate with many different females; 2) testing how transformed Ae. aegypti male perform with polyandrous females; and 3) prioritizing the selection of polyandrous males and/or females Ae. aegypti that have the most offspring.

Bed bugs, Cimex lectularius (L.) (Hemiptera: Cimicidae) and Cimex hemipterus (F.), have become established worldwide in recent years largely due to the development of insecticide resistance. However, limited attention has been given to ongoing morphological and macroevolutionary changes within the species and their populations, which could have implications for their control. Here, we evaluated whether bed bugs of the species C. hemipterus inhabiting different communities in Cape Coast, Ghana are undergoing segregation, which could lead to possible speciation. We also aimed to provide a morphometric description of all nymphal stages. Nine-bed bug populations of C. hemipterus were field-collected in Cape Coast and were subjected to geometric morphometric analysis. The multivariate parameters applied distinguished various populations from each of the locations, indicating the presence of morphologically distinct subpopulations of C. hemipterus. Shape-based segregation and shape changes associated with the insect pronotum (which is an important taxonomic character in the Cimicidae) were evident across the populations. Through this comparative study of C. hemipterus, we showed that possible subpopulations of this bed bug are being spread from Ghana. The nymphal stages (first–fifth) of C. hemipterus were distinguished by the length of the last three antennal segment and pronota width; such information contributes to the taxonomic knowledge of the species.

The dipteran fauna from the Brazilian Amazon remains poorly known, including that of forensic interest, such as the Sepsidae. The sepsid fauna of two secondary forests in the eastern Amazon was surveyed utilizing rotting bovine lung and feces (humans, bovines, and monkeys).We obtained 17 sespid species in six genera, most of them on bovine dung and rotting bovine lung. Two new species are described herein: Archisepsis bosque sp. nov. and Archisepsis verae sp. nov. The genus Palaeosepsis Duda, 1926 and the species Archisepsis polychaeta (Ozerov, 1993), Archisepsis diversiformis (Ozerov, 1993), Microsepsis mitis (Curran, 1927), Meropliosepsis sexsetosa Duda, 1926, and Palaeosepsis cf. golovastik Ozerov, 2004 are newly recorded from the Brazilian Amazon.

Chalcid Brachymeria podagrica (Fabricius), formerly known as Brachymeria fonscolombei (Dufour), is a parasitoid wasp that infests dipteran larvae of fly, Sarcophaga dux (Thomson), (Diptera: Sarcophagidae) recovered from the dead body of sheep in Bisha City, Asir region–Saudi Arabia. S. dux larvae were infested by B. podagrica. The first instar larvae and pupae of B. podagrica were found inside the larvae and pupae of S. dux. The adults of the parasitic wasp emerged from the pupae of the host. Adults and larvae of the parasitoid wasp were observed under a dissecting microscope. Identification was made by following the identification keys. It was confirmed that it was B. podagrica, which is herewith reported for the first time in Saudi Arabia.

In this paper, we review the quill mite fauna of the family Syringophilidae Lavoipierre, 1953 (Acariformes: Prostigmata) associated with New World and African parrots (Aves: Psittaciformes: Psittacidae), and describe eight new species including: Neoaulobia unsoeldi Marciniak-Musial & Sikora sp. nov. from the Burrowing Parakeet Cyanoliseus patagonus in Argentina; Lawrencipicobia arini Marciniak-Musial & Sikora sp. nov. from the Black-headed Parrot Pionites melanocephalus in Surinam; L. ararauna Marciniak-Musial & Sikora sp. nov. from the Black-headed Parrot Ara ararauna in Brazil; L. touiti Marciniak-Musial & Sikora sp. nov. from the Golden-tailed Parrotlet Touit surdus in Brazil; Rafapicobia valdiviana Marciniak-Musial & Sikora sp. nov. from the Burrowing Parrot Cyanoliseus patagonus in Brazil; R. pyrrhura Marciniak-Musial & Sikora sp. nov. from the Green-cheeked Parakeet Pyrrhura molinae in Bolivia; R. xanthopterygius Marciniak-Musial & Sikora sp. nov. from the Blue-winged Parrotlet Forpus xanthopterygius in Brazil; and R. trainidadi Marciniak-Musial & Sikora sp. nov. from the Lilac-tailed Parrotlet Touit batavicus in Trinidad and Tobago. Additionally, we note fifteen new host species and many new locality records for the previously described taxa, and provide the keys for all species associated with psittaciform birds. Finally, we discuss the host–parasite relationships between syringophilid mites and parrots.

Culicoides Latreille (Diptera: Ceratopogonidae) biting midges are vectors of important animal pathogens including bluetongue virus (BTV) and epizootic hemorrhagic disease virus (EHDV). While some Culicoides species present in the southern California desert are implicated in the transmission of these viruses to ruminant animals, these species have not been extensively studied due in part to the challenge of identifying Culicoides to species and to the lack of published gene sequences for these species to support their molecular identification. In this study, Culicoides were captured using suction traps baited with either carbon dioxide or UV light from transitional habitat between the southern California peninsular mountain ranges and the Colorado desert of southeastern California. Captured midges were initially identified using traditional morphological methods, with species identification subsequently confirmed by sequence analysis of COI and 28S rDNA genes. Phylogenetic analyses support that some Culicoides subgenera are not monophyletic. Two recognized species (C. sitiens Wirth and Hubert and C. bakeri Vargas) shared the same COI and 28S sequences. An additional cryptic species may be present within C. sitiens. Two additional recognized species (C. cacticola Wirth and Hubert and C. torridus Wirth and Hubert) may be conspecific or cryptic to each other. In total, 19 Culicoides species (or species aggregate) were collected in this study, with genetic sequences published for the first time for 16 of them. Published genetic sequences will support future research on these species, including studies on the ecology and habits of their immature stages which are often tedious to identify using morphology.

Los mosquitos mordedores del género Culicoides Latreille (Diptera: Ceratopogonidae) son vectores de patógenos animales importantes que incluyen los virus de enfermedad hemorrágica. Mientras algunas especies de Culicoides presentes en el desierto del sur de California están implicadas en la transmisión de estos virus a animales rumiantes, estas especies no han sido ampliamente estudiadas debido en parte al desafío de identificar Culicoides a las especies y a la falta de secuencias genéticas publicadas para estas especies que podrían apoyar su identificación molecular. En este estudio, los Culicoides fueron capturados del hábitat de transición entre las cadenas montañosas peninsulares del sur de California y el desierto de Colorado en el sureste de California usando trampas de succión cebadas con dióxido de carbono o luz ultravioleta. Los mosquitos capturados se identificaron inicialmente utilizando métodos morfológicos tradicionales, con la identificación de especies confirmada posteriormente mediante el análisis de secuencia de los genes COI y 28S ADN Ribosómico. Los análisis filogenéticos apoyan que algunos subgéneros de Culicoides no son monofiléticos. Dos especies reconocidas (C. sitiens Wirth y Hubert y C. bakeri Vargas), aunque morfológicamente distinguibles, compartían las mismas secuencias COI y 28S. Una especie críptica adicional puede estar presente dentro de C. sitiens. Dos especies adicionales reconocidas (C. cacticola Wirth y Hubert y C. torridus Wirth y Hubert) son morfológicamente inseparables y pueden ser conespecíficas o crípticas entre sí. Un total de 19 especies de Culicoides (o agregado de especies) fueron recolectadas en este estudio, con secuencias genéticas publicadas por primera vez a 16 de estas especies. Las secuencias genéticas publicadas apoyarán futuras investigaciones sobre estas especies, y pueden incluir estudios sobre la ecología y los hábitos de sus etapas inmaduras que a menudo son tediosos de identificar utilizando la morfología.

A new species of Sarcophagidae (Diptera) in the genus Helicobia Coquillett, Helicobia neuzalmeidae sp. nov., is described based on two male specimens obtained from pig carcasses in savanna-like vegetation in northeastern Brazil (state of Maranhão) during a forensic study. The holotype was obtained from a larva that abandoned the pig carcass to pupate, whereas the paratype was collected with a trap placed above the cage containing the carcass. This is the first record of a Helicobia species reared from a vertebrate carcass.

Taxonomic uncertainties and morphologic and molecular similarities may lead to incorrect or incomplete species identifications of immature ticks. This problem can be compounded when the diagnostic morphological features used are unreliable, unstable, or difficult to view. Our study attempts to sort out the similar morphologies of three Amblyomma spp. nymphs with partially overlapping geographic distributions in South Texas. Amblyomma americanum (L.), Amblyomma mixtum Koch, and Amblyomma tenellum Koch are three closely related species with similar nymphs and larvae. Previous attempts to morphologically distinguish among nymphs of these species have relied on difficult-to-observe and unreliable characters. Over the course of decades of routine diagnostic work with these ticks, we have observed and discovered consistent and useful morphologic features sufficient to practically distinguish among these nymphs. We confirmed our morphological studies with molecular genetic evidence (i.e., 12S mitochondrial genes), and we propose that practical, rapid morphological species-level identification of nymphs of the three named species is now possible with confidence.

To glean more information on mosquito diversity and distribution in Auroville, a cross-sectional study was carried out by mapping the distribution of water bodies and habitats supporting immature stages on the one hand and the distribution of water bodies/habitats supporting mosquito immature stages on the other. A satellite image covering an area of 8.08 km² was overlaid with a grid of 500 × 500 m. Fifteen modules were selected and the area of each module served as the sampling site for the entomological survey. Adult and larval stages were sampled. Diversity indices were analyzed to compare mosquito diversity. Rarefaction estimations were used to compare abundance and richness of the mosquito species between different zones. In total, 750 mosquito larvae and 84 resting adults were sampled. Eighteen species of mosquitoes belonging to 11 subgenera and 7 genera were documented. Genera included Aedes (Johann Wilhelm Meigen 1818, Diptera, Culicidae), Anopheles (Johann Wilhelm Meigen 1818, Diptera, Culicidae), Armigeres (Theobald 1901, Diptera, Culicidae), Culex (Carl Linnaeus 1758, Diptera, Culicidae), Lutzia (Theobald 1903, Diptera, Culicidae), and Mimomyia (Theobald 1903, Diptera, Culicidae). Of the 18 mosquito species identified, 8 species are new records for Auroville. The Alpha (α) biodiversity indices show that the mosquito fauna is diverse (S = 18; D_Mg = 2.732 [95% CI: 2.732–2.732]). The Shannon-Weiner (H′ = 2.199 [95% CI: 2.133–2.276]) and Simpson indices (λ = 0.8619 [95% CI: 0.8496–0.8723]) measured species richness, evenness, and dominance. The values of these indices suggest high species richness, evenness, and dominance. Prevailing conditions can provide suitable environment for establishment of different mosquito species in this ecosystem. Given the sociodemographic characteristics of this area, research on mosquito diversity and risk of vector-borne diseases will be of great use.

In the United States, the Gulf Coast tick (Amblyomma maculatum Koch) is a species of growing medical and veterinary significance, serving as the primary vector of the pathogenic bacterium, Rickettsia parkeri (Rickettsiales: Rickettsiaceae), in humans and the apicomplexan parasite, Hepatozoon americanum, in canines. Ongoing reports of A. maculatum from locations outside its historically reported distribution in the southeastern United States suggest the possibility of current and continuing range expansion. Using an ecological niche modeling approach, we combined new occurrence records with high-resolution climate and land cover data to investigate environmental drivers of the current distribution of A. maculatum in the United States. We found that environmental suitability for A. maculatum varied regionally and was primarily driven by climatic factors such as annual temperature variation and seasonality of precipitation. We also found that presence of A. maculatum was associated with open habitat with minimal canopy cover. Our model predicts large areas beyond the current distribution of A. maculatum to be environmentally suitable, suggesting the possibility of future northward and westward range expansion. These predictions of environmental suitability may be used to identify areas at potential risk for establishment and to guide future surveillance of A. maculatum in the United States.

Aedes aegypti (Linnaeus) and Aedes albopictus (Skuse) are worldwide vectors of dengue and yellow fever viruses. These species coexist in many countries and the biotic interactions between them can influence their abundances and distributions. In Argentina, Ae. aegypti is widely distributed in the north and center regions of the country, with temperate and subtropical climate, while both are sympatric only in the northeastern area of the subtropical region. Interspecific and intraspecific larval competition for food was evaluated to assess if their interaction influences on patterns of abundance and distribution. Finite rates of increase and survivorship for each species were estimated and the effects of mosquito density ratio and detritus availability were determined. The Lambda (λ′) index of population performance of both showed there is no competitive exclusion pattern. However, survival of Ae. albopictus was negatively affected by the presence of Ae. aegypti. These results suggest one possible explanation for the codominance pattern of both species display in rural regions of the southernmost distribution of Ae. albopictus in South America. They also show Ae. aegypti as a potential biotic barrier for the expansion of Ae. albopictus as was reported in regions of the United States.

Amblyomma americanum (Linnaeus) (Acari: Ixodidae) (lone star tick) is an aggressive, generalist parasite that vectors numerous important human and animal pathogens. In recent decades its geographic range has expanded northwards from endemic regions in the southeastern and southcentral United States. In 2019 five questing A. americanum ticks, comprising two life stages were detected at one site in southwestern Michigan, satisfying one CDC criterium for an established population for the first time in recent history in the state. To better characterize the extent of emerging A. americanum, we conducted active surveillance (i.e., drag sampling) in summer 2020 throughout Michigan's southern counties and detected one adult A. americanum from each of six widespread sites, including where they had been detected in 2019. A larger established population was identified at another site in Berrien County, which yielded 691 A. americanum comprising three life stages, and questing phenologies here were similar to that reported for other endemic regions. Statewide surveillance in 2021 revealed no A. americanum outside of Berrien County, but establishment criteria were met again at the two sites where established populations were first detected respectively in 2019 and 2020. These observations may represent the successful invasion of A. americanum into Michigan. Data from passive (1999–2020) and active surveillance (2004–2021) efforts, including a domestic animal sentinel program (2015–2018), are reported to provide context for this nascent invasion. Continued active surveillance is needed to help inform the public, medical professionals, and public health officials of the health risks associated with this vector.

The larval ecology of Culicoides (Diptera: Ceratopogonidae) influences their spatial distributions and the pathogens they transmit. These features are of special concern for deer farmers in Florida where epizootic hemorrhagic disease virus (EHDV) is a major source of mortality in captive herds. Rarity of larval morphological expertise leads many researchers to study larval ecology by quantifying emergence, either with field emergence traps or removing substrate from the field for observation under laboratory conditions. We investigated the comparability of these methods in Florida seepages where two recently implicated EHDV vectors, Culicoides stellifer Coquillett and Culicoides venustus Hoffman, are common. We compared the abundance and composition of emerging Culicoides collected from emergence traps with removed substrate samples (soil plugs) at three seepages. Soil plugs were sampled adjacent to the emergence trap and from underneath the trap footprint, and then monitored under laboratory conditions for 11–13 wk to compare the methods and to assess the role of incubation period for removed substrate samples. Emergence traps and removed substrate sampling largely agreed on community compositions and trends within different seepages. However, comparatively large numbers of C. stellifer emerged later than expected and well into the incubation period with emergence still occurring after 13 wk (90 d). Removed substrate samples were more similar to emergence traps at shorter incubation times. The importance of time for the capture of Culicoides in removed substrate sampling was more pronounced than we anticipated and is important from both a methodological and biological perspective.

Aedes albopictus (Skuse) is a vector of several arboviruses, such as dengue, chikungunya, West Nile, and Zika viruses. At present, the use of synthetic insecticides is the main vector control strategy. However, the widespread and long-term use of insecticides has aroused several problems, including insecticide resistance, environmental pollution, and non-target species effects, thereby encouraging researchers to search for new alternatives derived from natural products. In recent decades, essential oils (EOs) as natural alternatives to control mosquitoes have received increasing attention. In the initial larvicidal activity screen, two Rutaceae plants (Citrus aurantium and Citrus paradisi) EOs were selected and evaluated for killing Ae. albopictus larvae. The LC₅₀ values of C. aurantium and C. paradisi EOs against Ae. albopictus were 91.7 and 100.9 ppm, respectively. The main components of C. aurantium EO include diethyl o-phthalate (37.32%), limonene (10.04%), and methyl dihydrojasmonate (6.48%). The main components of C. paradisi EO include limonene (60.51%), diethyl o-phthalate (11.75%), linalool (7.90%), and styralyl acetate (6.28%). Among these main components of the two EOs, limonene showed potent larvicidal activity, with the LC₅₀ value of 39.7 ppm. The nanoemulsions of limonene were prepared and characterized. The duration of larvicidal activity was greater in the limonene nanoemulsions than when limonene was applied in solvent. This study demonstrates that EOs of plants in family Rutaceae are a potential resource to develop new larvicides, and nanoemulsification is an effective method for improving the physicochemical properties and efficacy of natural products as larvicides.

The insecticide sensitivity and resistance status of both adults and larvae from six Aedes albopictus samples collected in Tuskegee, Tuscaloosa, Birmingham, Dothan, Mobile, and Montgomery, Alabama, were evaluated for the levels of sensitivity and resistance to eight insecticides: β-cyfluthrin, chlorpyrifos, deltamethrin, etofenprox, fenitrothion, permethrin, resmethrin, and malathion. Adult Ae. albopictus from all locations showed similar results for the difference between the time to 100% mortality and the diagnostic time in the CDC bottle bioassay, although Ae. albopictus survive longer than the diagnostic time to permethrin, fenitrothion, and resmethrin treatments. The larval bioassay indicated that malathion was the least toxic to Ae. albopictus from all locations (LC₅₀: ranging from 0.1 ppm to 1.2 ppm), followed by resmethrin and etofenprox (LC₅₀: 0.05 ppm–0.4 ppm), and deltamethrin and fenitrothion (LC₅₀: 0.01 ppm–0.06 ppm). Chlorpyrifos exhibited the highest larval toxicity (LC₅₀: 0.003 ppm–0.05 ppm). The resistance status of Ae. albopictus from all six locations was similar to the resistance levels found in a previous survey in 2004, indicating that in Alabama the development of resistance is slow in this strain, although comparing the resistance of Ae. albopictus from Tuskegee to that of a susceptible strain showed that it is resistant to chlorpyrifos. The slopes of the dose–response curves to most of the insecticides tested for these field populations of Ae. albopictus were generally similar to or slightly higher than those measured eighteen years previously, indicating that these populations are relatively homozygous in response to all the insecticides tested.

Graphical Abstract

Entomopathogenic fungi allow chemical-free and environmentally safe vector management. Beauveria bassiana (Balsamo-Crivelli) Vuillemin is a promising biological control agent and an important component of integrated vector management. We investigated the mortality of Aedes albopictus (Skuse) larvae exposed to five concentrations of B. bassiana using Mycotrol ESO and adult oviposition behavior to analyze the egg-laying preferences of wild Ae. albopictus in response to different fungal concentrations. We examined the mortality of mid-instars exposed to B. bassiana concentrations of 1 × 10⁴, 1 × 10⁵, 1 × 10⁶, 1 × 10⁷, and 1 × 10⁸ conidia/ml every 24 h for 12 d. In the oviposition behavior study, the fungus was applied to wooden paddles at 1 × 10⁵, 1 × 10⁷, and 1 × 10⁹ conidia/ml, and the paddles were individually placed into quad-ovitraps. Both experiments contained control groups without B. bassiana. Kaplan–Meier survival analysis revealed that larval mortality was concentration dependent. The median lethal concentration was 2.43 × 10⁵ conidia/ml on d 12. The median lethal time was 3.68 d at 1 × 10⁶ conidia/ml. Oviposition monitoring revealed no significant difference in egg count between the control and treatment paddles. We observed an inverse relationship between the concentration of B. bassiana and the percentage of paddles with eggs. We concluded that concentrations above 1 × 10⁶ conidia/ml are larvicidal, and Ae. albopictus laid similar numbers of eggs on fungus-impregnated and control wooden substrates; however, they were more likely to oviposit on substrates without B. bassiana. With these findings, we suggest that B. bassiana-infused ovitraps can be used for mosquito population monitoring while also delivering mycopesticides to adult mosquitoes.

Three compounds synthetically-derived from botanicals sources, ethyl perillyl carbonate, geranyl isovalerate, and citronellyl cyclobutane carboxylate, were tested for repellent activity against Ixodes scapularis Say in a field trial. Tick drags were treated with the compounds or with N, N-diethyl-m-toluamide (DEET) at high (0.25 mg/cm²) or low (0.16 mg/cm²) concentrations. Negative controls included untreated drags and drags treated with acetone, the carrier for all repellents. Freshly treated drags (within 20 min) were used to collect I. scapularis ticks at a county park in Wisconsin. To assess effectiveness, we measured tick encounter rates, detachment rate, and time to detachment. None of the repellent treatments, including DEET, resulted in significantly fewer encounters compared to both control treatments. However, the percentage of ticks that detached within 3 min was significantly higher on drags treated with repellents compared to controls. DEET was the most effective, repelling 69.7–87% of ticks by 3 min, but the effectiveness of the three test compounds was still high, with 42–87% of ticks detaching by 3 min. For time to detachment, there were no significant differences between DEET and the three test compounds. We conclude that these synthetically-derived repellents were effective against I. scapularis in a field trial and could be viable alternatives to DEET.

Systemic parasiticides in livestock can control zoophilic malaria vectors that contribute to residual malaria transmission. Membrane feeding techniques were used to screen seven systemic parasiticidic drugs currently in veterinary use for livestock and dogs. Drugs were tested in two laboratory strains of zoophilic Anopheles –A. stephensi (South Asian vector) and A. albimanus (Central American vector). To assess the relative potentials of these drugs, the resultant LC-50 for each drug was compared with what is known about the pharmacokinetic of the drug. Drugs with LC-50 values below the reported maximum plasma concentration of treated animals were considered as showing the most promise for use in the field. Ivermectin and fipronil showed the greatest promise for use in cattle against A. stephensi. Fipronil showed the greatest promise for use in cattle against A. albimanus. Both fluralaner and afoxolaner were highly effective against both mosquito species but pharmacokinetic data for these drugs in cattle are lacking. Eprinomectin, moxidectin and abamectin showed marginal to no promise for either mosquito species. At sublethal doses, ivermectin, fipronil, and afoxolaner (but not fluralaner) significantly reduced the larval production of surviving A. stephensi and A. albimanus. Further testing of candidate systemic parasiticides, including their product formulations, in livestock against field-collected populations of Anopheles is the next logical step toward full implementation of this strategy to manage zoophilic vectors.

Attractive toxic sugar baits (ATSBs) require target insects to locate, orient toward, and feed on an insecticidal sugar solution to control populations. Formulating these baits with different attractants and phagostimulants can increase their efficacy by causing insects to choose the ATSB over competing natural sugar sources, and to ingest more of the bait solution. We tested formulations of a 20% sodium ascorbate (SA) ATSB solution using different sugars, adenosine triphosphate (ATP), gallic acid, and six plant volatile compounds to determine their effect on adult Aedes aegypti (L.) and Anopheles stephensi Liston mortality. Baits formulated with fructose or sucrose had no effect on either species, neither did the addition of ATP. Gallic acid increased the survival of Ae. aegypti. Four of the six volatile compounds increased mortality in at least one species. We also examined An. stephensi response to baits formulated with each of the six volatile compounds. Anisaldehyde significantly increased the number of mosquitoes responding toward the SA-ATSB, but increasing the amount had no effect. Addition of anisaldehyde also significantly increased An. stephensi feeding rates on the SA-ATSB, though mosquitoes will avoid the toxic bait if a nontoxic sugar source is available. Formulation of SA-ATSBs with synthetic blends of attractive compounds can increase bait efficacy and consistency, though further research is needed to assess their performance in the field in the presence of natural sugar sources.

Populations of Blattella germanica (L.) (German cockroach) have been documented worldwide to be resistant to a wide variety of insecticides with multiple modes of action. The phenylpyrazole insecticide fipronil has been used extensively to control German cockroach populations, exclusively in baits, yet the highest reported fipronil resistance is 38-fold in a single population. We evaluated five populations of German cockroaches, collected in 2018–2019 in apartments in North Carolina and assayed in 2019, to determine the status of fipronil resistance in the state. Resistance ratios in field-collected strains ranged from 22.4 to 37.2, indicating little change in fipronil resistance over the past 20 yr. In contrast, resistance to pyrethroids continues to escalate. We also assessed the roles of detoxification enzymes in fipronil resistance with four synergists previously shown to diminish metabolic resistance to various insecticides in German cockroaches—piperonyl butoxide, S,S,S-tributyl phosphorotrithioate, diethyl maleate, and triphenyl phosphate. These enzymes appear to play a variable role in fipronil resistance. We also sequenced a fragment of the Rdl (resistant to dieldrin) gene that encodes a subunit of the GABA receptor. Our findings showed that all field-collected strains are homozygous for a mutation that substitutes serine for an alanine (A302S) in Rdl, and confers low resistance to fipronil. Understanding why cockroaches rapidly evolve high levels of resistance to some insecticides and not others, despite intensive selection pressure, will contribute to more efficacious pest management.

Aedes aegypti mosquitoes are capable of vectoring a wide range of diseases including dengue, yellow fever, and Zika viruses, with approximately half of the worlds' population at risk from such diseases. Development of combined predator–parasite treatments for the control of larvae consistently demonstrates increased efficacy over single-agent treatments, however, the mechanism behind the interaction remains unknown. Treatments using the natural predator Toxorhynchites brevipalpis and the entomopathogenic fungus Metarhizium brunneum were applied in the laboratory against Ae. aegypti larvae as both individual and combined treatments to determine the levels of interaction between control strategies. Parallel experiments involved the removal of larvae from test arenas at set intervals during the course of the trial to record whole body caspase and phenoloxidase activities. This was measured via luminometric assay to measure larval stress factors underlying the interactions. Combined Metarhizium and Toxorhynchites treatments were seen to drastically reduce lethal times as compared to individual treatments. This was accompanied by increased phenoloxidase and caspase activities in combination treatments after 18 h (p < 0.001). The sharp increases in caspase and phenoloxidase activities suggest that combined treatments act to increase stress factor responses in the larvae that result in rapid mortality above that of either control agent individually. This work concludes that the underlying mechanism for increased lethality in combined parasite–predator treatments may be related to additive stress factors induced within the target host larvae.

The outbreak of yellow fever virus transmitted by Aedes aegypti has been of major concern in Nigeria, this mosquito also transmits several other arboviruses globally. The control of many vectors of mosquito-borne diseases relies heavily on the use of insecticides. Therefore, constant monitoring of insecticide resistance status and associated mechanisms is crucial within the local mosquito population. Here, we determined the resistance profile of adult Ae. aegypti from Majidun and Oke Ota communities, Ikorodu Local Government Area of Lagos State, Nigeria to different classes of insecticides using WHO procedures. The resistant phenotypes of Ae. aegypti were screened for the presence of kdr mutations F1534C, S989P, and V1016G, which have been implicated in insecticide resistance in yellow fever vectors. A high level of resistance to DDT and pyrethroids was recorded in Ae. aegypti in this study, although possible resistance to deltamethrin, one of the pyrethroids was reported in one of the locations. Resistance to bendiocarb was recorded in the Majidun community while Ae. aegypti in both locations were susceptible to malathion. The presence of F1534C mutation associated with DDT and deltamethrin resistance in Ae. aegypti population, and the presence of S989P mutation were detected singly and in co-occurrence with F1534C for the first time in Africa, while V1016G mutation was not detected in this study. The role of these mutations in resistance phenotype expressed in Ae. aegypti in larger populations needs to be established.

Data on the prevalence and distribution of ticks and tick-borne diseases in Belize are lacking. Ticks (n = 564) collected from dogs, horses, and vegetation in two villages in Stann Creek District in southeastern Belize in 2018, were molecularly identified and screened for tick-borne nonviral human pathogens. The identity of 417 ticks was molecularly confirmed by DNA barcoding as Rhipicephalus sanguineus (Latreille) (66.43%), Amblyomma ovale Koch (15.59%), Dermacentor nitens Neumann (11.51%), Amblyomma sp. ADB0528 (3.6%), and the remainder being small records (2.87%) of Amblyomma coelebs Neumann, Amblyomma imitator Kohls, Amblyomma tapirellum Dunn, Amblyomma auricularium Conil, and Amblyomma maculatum Koch. Individual tick extracts were screened for the presence of Rickettsia spp., Babesia spp., Babesia microti, Borrelia spp., Ehrlichia spp., and Anaplasma spp. using available conventional polymerase chain reaction (PCR) assays. Rickettsia parkeri strain Atlantic Rainforest was identified in five specimens of A. ovale, and one other unidentified tick, all collected from dogs. Another unidentified tick—also collected from a dog—tested positive for an undefined but previously detected Ehrlichia sp. With the exception of D. nitens, all eight other tick species identified in this study were collected on dogs, suggesting that dogs could be usefully employed as sentinel animals for tick surveillance in Belize.

West Nile virus (WNV) is a zoonotic disease that is endemic in North America and is known to cause a range of symptoms from mild to life threatening in humans. Culex quinquefasciatus is one of the most prominent vectors of WNV in Southern California. The goal of this study was to identify which animal species are most fed upon by these mosquitoes in various habitats in the West Valley area of San Bernardino County, California, and determine the relationship between blood-feeding patterns and WNV activity in the region. Culex quinquefasciatus specimens were collected by West Valley Mosquito and Vector Control District during 2011 from 32 different sites. The bloodmeals of 683 individuals (92.4% of those tested) were identified using the mitochondrial gene cytochrome c oxidase 1 (COI). These bloodmeals comprised 29 vertebrate species across four different habitats. Species richness (ranging from 10 to 17) was not significantly different between habitats when rarified to account for sample size. Across habitats, the highest percentage of avian bloodmeals were taken from house sparrows (18.8–39.1%) and house finches (2.6–31.5%). Bloodmeals were identified from five mammalian species, accounting for 5.1–59.2% of bloodmeals by habitat, including humans (0–4.1%). A seasonal shift towards increased mammalian bloodmeal prevalence, specifically for domestic dog and human bloodmeals, was observed in urban habitats. The WNV activity during 2011 in San Bernardino County occurred mostly in urban and suburban areas as indicated by minimum infection rate (MIR) in Culex quinquefasciatus, notable as all human bloodmeals were identified from these two habitats.

The mosquito-borne yellow fever virus (YFV) is the cause of a zoonotic disease, with both sylvatic and urban cycles. Some mosquito species have been associated directly with transmission of the virus in South America, although the importance given to each species varies depending on the bibliography, geographical location, or year of publication. For Argentina, few publications have assessed the involvement of mosquito species, especially those included in the sylvatic cycle. Therefore, the goal of our paper was to gather all the information available in South America and categorize all mosquito species potentially involved in the YFV transmission cycle in Argentina according to incriminating vector criteria. Based on three main characteristics (‘Hosts’, ‘Mosquito vector’, and ‘YF outbreak’), we generated scales of importance by species, one exclusively for Argentina and another for all of South America, Sabethes albiprivus Theobald, 1903 was the most important species in Argentina; whereas the most important species in South America were Haemagogus janthinomys Dyar 1921, Hg. leucocelaenus Dyar and Shannon, 1924, and Sa. chloropterus Von Humboldt, 1819. Our review highlights the lack of research that evaluates the importance of these species for YFV transmission in Argentina, while serving as a starting point to establish priorities for research on the bionomics and vector status of these species.

There are currently >300 malaria cases reported annually in the Republic of Korea (ROK), with most cases attributed to exposure in northern Gangwon and Gyeonggi provinces near the demilitarized zone (DMZ). The species diversity and malaria infection rate were determined for a sample of Anopheles mosquitoes collected from May to early November 2020 for six sites in a malaria high-risk area in/near the DMZ and two malaria low-risk areas in southern Gyeonggi province using Mosquito Magnet traps in the ROK. A total of 1864 Anopheles spp. were identified to species by PCR. Overall, An. kleini (31.4%, 510/1622) was the most frequently species assayed, followed by An. pullus (25.5%, 413/1622), An. sineroides (23.9%, 387/1622), and An. sinensis (10.2%, 165/1622), while the other four species only accunted for 9.1% (147/1622) collected in/near the DMZ. Only three species, An. pullus, An. sinensis, and An. sineroides were collected at Humphreys US Army Garrison (USAG) (235 individuals), while only An. sinensis was collected atYongsan USAG (7 individuals). A total of 36 Anopheles specimens belonging to five species collected in/near the DMZ were positive for Plasmodium vivax by PCR. Anopheles kleini (9) was the most frequent species positive for P. vivax, followed by An. belenrae (8), An. pullus (8), An. sinensis (5), An. sineroides (5), and a member of the Anopheles Lindesayi Complex in the ROK (1). This is the first report of P. vivax in a member of the An. Lindesayi Complex in the ROK. These findings can assist in guiding future malaria vector management in the ROK.

Tularemia is a highly infectious, potentially fatal disease of humans and animals caused by the gram negative, intracellular bacterium Francisella tularensis. The San Mateo County Mosquito and Vector Control District conducted surveillance for F. tularensis from 2017 to 2021 in Dermacentor occidentalis (Marx) (Ixodida: Ixodidae), D. variabilis (Say) (Ixodida: Ixodidae), and Haemaphysalis leporispalustris (Packard) (Ixodida: Ixodidae) ticks in coastal southwestern San Mateo County, California. A total of 3,021 D. occidentalis and 1,019 D. variabilis were collected. Of those, 25 positive pools of F. tularensis were detected (five ticks per pool, overall minimum infection prevalence: 0.62%). Twenty-two of the 25 positive pools (88%) contained D. occidentalis. Eighty-eight percent (88%) of all positive pools were collected from the western half of the site, nearest to the ocean. We did not detect a seasonal effect on the probability of detecting a positive tick pool. There were two human cases of tularemia during the summers of 2019 and 2021. We conducted rodent surveillance in June of 2019, before the human case report. Twenty-four small mammals were collected, but none of their sera tested positive for F. tularensis. It is clear that tularemia is endemic to this region of San Mateo County, but the extent of its range and its ecology is not currently well understood.

Graphical Abstract

Active surveillance was conducted by collecting questing ticks from vegetation through a 2-yr survey in Pike County, Pennsylvania. Over a thousand blacklegged ticks (Ixodes scapularis Say) and American dog ticks (Dermacentor variabilis Say) were collected. A single specimen of the following species was collected: lone star tick (Amblyomma americanum L.), rabbit tick (Haemaphysalis leporispalustris Packard), and an Asian longhorned tick (Haemaphysalis longicornis Neumann). This study represents the largest county-wide study in Pennsylvania, surveying 988 questing I. scapularis adult and nymphs. Molecular detection of five distinct tick-borne pathogens was screened through real-time PCR at a single tick resolution. Respectively, the overall 2-yr adult and nymph prevalence were highest with Borrelia burgdorferi (Spirochaetales: Spirochaetacceae) (45.99%, 18.94%), Anaplasma phagocytophilum (Rickettsiales: Anaplasmataceae) (12.29%, 7.95%) where the variant-ha (8.29%, 3.03%) was overall more prevalent than the variant-v1 (2.49%, 4.17%), Babesia microti (Piroplasmida: Babesiidae) (4.97%, 5.30%), Borrelia miyamotoi (Spirochaetales: Spirochaetaceae) (1.38%, 1.89%), and Powassan virus lineage II [POWV]/deer tick virus (DTV) (2.07%, 0.76%). Adult and nymph coinfection prevalence of B. burgdorferi and B. microti (3.03%, 4.97%) and adult coinfection of B. burgdorferi and A. phagocytophilum or A. phagocytophilum and B. microti were significantly higher than the independent infection rate expected naturally. This study highlights the urgency to conduct diverse surveillance studies with large sample sizes to better understand the human risk for tick-borne diseases within small geographical areas.

Eastern equine encephalitis virus (EEEV) is a mosquito-borne virus that affects humans and horses, with a high case fatality rate in both species. The virus can be transmitted by several mosquito species and maintained in multiple reservoir hosts, making EEEV dynamics difficult to anticipate. In this study, we identified spatial and temporal factors that precede EEEV detection using sentinel chicken surveillance data from Orange County, Florida, from 2003 to 2017. We first examined the land cover and mosquito species composition associated with sentinel chicken sites. We then fit distributed lag nonlinear models of EEEV detection at the county scale, using monthly temperature, precipitation, and Southern Oscillation Index values, and at the sentinel flock-scale, using remotely sensed temperature and wetness indicators. We found positive associations between the percent wooded wetlands and the count of EEEV detections. We found Culiseta melanura (Diptera: Culicidae) were more abundant at positive sites in winter and summer, but Coquillettidia perturbans (Walker) were more abundant at positive sites in spring. In the county-wide model, precipitation, temperature, and Southern Oscillation Index values at lags of two, nine, and twelve months were significant, respectively, while temperature and wetness were significant at lags of eight and six months in the flock-specific models.

The presence of mosquitoes in an area is dependent on the availability of suitable breeding sites that are influenced by several environmental factors. Identification of breeding habitats for vector surveillance and larval source management is key to disease control programs. We investigated water quality parameters and microbial composition in selected mosquito breeding sites in urban Accra, Ghana and associated these with abundance of Anopheles (Diptera: Culicidae) and Culex (Diptera: Culicidae) larvae. Physicochemical parameters and microbial composition explained up to 72% variance among the breeding sites and separated Anopheles and Culex habitats (P < 0.05). Anopheles and Culex abundances were commonly influenced by water temperature, pH, nitrate, and total hardness with contrasting impacts on the two mosquito species. In addition, total dissolved solids, biochemical oxygen demand, and alkalinity uniquely influenced Anopheles abundance, while total suspended solids, phosphate, sulphate, ammonium, and salinity were significant determinants for Culex. The correlation of these multiple parameters with the occurrence of each mosquito species was high (R² = 0.99, P < 0.0001). Bacterial content assessment of the breeding ponds revealed that the most abundant bacterial phyla were Patescibacteria, Cyanobacteria, and Proteobacteria, constituting >70% of the total bacterial richness. The oligotrophic Patescibacteria was strongly associated with Anopheles suggestive of the mosquito's adaptation to environments with less nutrients, while predominance of Cyanobacteria, indicative of rich nutritional source was associated with Culex larval ponds. We propose further evaluation of these significant abiotic and biotic parameters in field identification of larval sources and how knowledge of these can be harnessed effectively to reduce conducive breeding sites for mosquitoes.

Mosquito control programs have increasingly used Centers for Disease Control and Prevention bottle bioassays (BB) to evaluate insecticide resistance (IR). The reported utility, benefits, and limitations of BB and other methods were assessed via electronic survey of U.S. mosquito control professionals to identify potential areas for improvement, future study, and professional training. Opportunities were identified to improve BB operational value and IR detection.

Recently, the endobacteria Wolbachia has emerged as a biological tool for the control of arboviruses. Thus, we investigated the rate of natural infection by Wolbachia in Culicidae species from Maranhão, Brazil. For this, we amplified the Wolbachia surface protein gene (wsp) from mosquitoes collected in six localities of Maranhão, and positive samples were subjected to new analysis using group-specific primers. In total, 448 specimens comprising 6 genera and 18 species of mosquitoes were analyzed. Wolbachia DNA was PCR-detected in 7 species, three of which are new records: Aedes scapularis (Rondani, 1848), Coquillettidia juxtamansonia (Chagas, 1907) and Cq. venezuelensis (Theobald, 1912), in addition to Ae. albopictus (Skuse, 1894) and Culex quinquefasciatus Say, 1823, which are commonly described as permissive to maintain this bacterium in natural environments, and two species of the subgenera Anopheles (Nyssorhynchus) Blanchard, 1902 and Culex (Melanoconion) Theobald, 1903 which could not be identified at species level. The infection rate of all species ranged from 0 to 80%, and the average value was 16.5%. This study increases the knowledge about the prevalence of Wolbachia in the culicid fauna and may help in selecting strains for biological control purposes.

Open-environment poultry farms that allow chickens to forage outdoors are becoming increasingly common throughout the United States and Europe; however, there is little information regarding the diversity and prevalence of ectoparasites in these farming systems. Eight to 25 birds were captured and surveyed for ectoparasites on each of 17 farms across the states of Washington, Idaho, Oregon, and California. Among the farms sampled, six louse species (Phthiraptera: Ischnocera & Amblycera) and two parasitic mite species (Acari: Mesostigmata) were collected and identified: Goniodes gigas (Taschenberg, 1879; Phthiraptera: Menoponidae) on one farm, Menacathus cornutus (Schömmer, 1913; Phthiraptera: Menoponidae) on one farm, Menopon gallinae (Linnaeus, 1758; Phthiraptera: Menoponidae) on six farms, Lipeurus caponis (Linnaeus, 1758; Phthiraptera: Philopteridae) on five farms, Menacanthus stramineus (Nitzsch, 1818; Phthiraptera: Menoponidae) on nine farms, Goniocotes gallinae De Geer (Phthiraptera: Philopteridae) on 11 farms, Dermanyssus gallinae (De Geer, 1778; Mesostigmata: Dermanyssidae) on two farms, and Ornithonyssus sylviarum (Canestrini & Fanzago, 1877; Mesostigmata: Macronyssidae) on one farm. The diversity of ectoparasites on these open environment poultry farms highlights a need for additional research on ectoparasite prevalence and intensity in these poultry farming systems.

The invasive Asian longhorned tick, Haemaphysalis longicornis, has rapidly spread across the northeastern United States and is associated with pathogens of public health and veterinary concern. Despite its importance in pathogen dynamics, H. longicornis blood-feeding behavior in nature, specifically the likelihood of interrupted feeding, remains poorly documented. Here, we report the recovery of partially engorged, questing H. longicornis from active tick surveillance in Pennsylvania. Significantly more engorged H. longicornis nymphs (1.54%) and adults (3.07%) were recovered compared to Ixodes scapularis nymphs (0.22%) and adults (zero). Mean Scutal Index difference between unengorged and engorged nymph specimens was 0.65 and 0.42 for I. scapularis and H. longicornis, respectively, suggesting the questing, engorged H. longicornis also engorged to a comparatively lesser extent. These data are among the first to document recovery of engorged, host-seeking H. longicornis ticks and provide initial evidence for interrupted feeding and repeated successful questing events bearing implications for pathogen transmission and warranting consideration in vector dynamics models.

Present work aimed to identify blood feeding sources and attempt to detect Leishmania DNA in Nyssomyia antunesi, suspected vector of Leishmania sp., from a park in the urban center of Belém, the capital of Pará State, in the Brazilian Amazon. Entire bodies and gut contents of Ny. antunesi engorged females, previously captured in the urban park with Centers for Disease Control (CDC) light traps and aspiration on tree bases, were subjected to Leishmania and vertebrate DNA detection through amplification of the Leishmania mini-exon and vertebrate cytochrome b (cyt b) gene regions, respectively. The quality of DNA extraction from entire bodies was ensured through amplification of the dipteran cyt b region. The vertebrate cyt b amplicons were sequenced and compared with those available on GenBank. A maximum likelihood phylogenetic tree was constructed to assess the clustering patterns of these sequences. Leishmania DNA was not detected. The sequences of 13 vertebrate cyt b amplicons were considered informative, exhibiting similarity and clustering with the following six vertebrate species: Dasyprocta leporina (1), Cuniculus paca (1), Tamandua tetradactyla (4), Choloepus didactylus (4), Pteroglossus aracari aracari (2), Homo sapiens (1). The samples of D. leporina and C. paca were obtained from the CDC canopy, whereas the others were by aspiration from tree bases. The present results revealed the eclectic and opportunist blood-feeding behavior of Ny. antunesi, with birds and mammals, these last ones acting as potential reservoirs for Leishmania species, distributed throughout the vertical forest strata.