Detecting and locating a carrion resource is critical for the reproduction of necrophagous insects and initiating forensically important timelines. Blow flies (Diptera: Calliphoridae) primarily use olfactory cues in the form of volatile organic compounds to locate a suitable resource. Factors governing detecting and locating a resource have been studied using various behavior assays with modifications to suit the experiment design, such as the dual-choice cube olfactometer, which was examined in the current study. Systems optimization ensures biologically relevant and consistent results across replicates. In this study, two responses were measured: 1) leaving cube for either control or treatment and 2) choice between control and treatment. Phenotype (e.g., male, non-gravid, gravid) and total blow fly, Cochliomyia macellaria (Fabricius) (Diptera: Calliphoridae), number to respond were measured. Four parameters were assessed for their impact on response: 1) adjustment time in cube before trial, 2) trial length, 3) sugar/water presence, and 4) screening type in arms. Approximately, 70% of all phenotypes responded to liver with the 30-min adjustment period where only 50% responded with other adjustment periods.Trial length had a significant impact on response (35% increase in the 8 h trial compared to shorter durations); however, significant response to treatment was lost by increasing trial length.The presence of sugar/water decreased gravid and non-gravid response by 35% but did not impact males. Screening had no influence on overall or treatment response. Data indicate experiment design impacts fly response. Future studies should optimize parameters for their given fly population prior to initiating experiments.

The current study aimed to evaluate the effects of methoxyfenozide (RH-2485), an insect growth disrupter (IGD) belonging to molting hormone agonist class, against female adults of Culex pipiens L. under laboratory conditions. Lethal concentrations (LC₅₀ = 24.54 µg/liter and LC₉₀ = 70.79 µg/liter), previously determined against fourth instar larvae, were tested for adult female fertility, fecundity and oviposition after tarsal contact before mating and any bloodmeal. Methoxyfenozide was found to alter negatively their autogeny capacity and oviposition. A strong reduction of 56% and 72% (P < 0.001) in females' autogeny capacity was observed in both treated series, respectively. Alteration in oviposition were found to be higher with LC₉₀ (OAI-LC₉₀ = –0.62) than with the LC₅₀ (OAI-LC50 = –0.42). Also fecundity and hatching rate (fertility) were significantly reduced in treated series as compared to controls. A significant reduction of 37.65 and 28.23% in fecundity and decrease of 56.85 and 71.87% in fertility were found, respectively in LC₅₀ and LC₉₀ treated series. Obtained data clearly demonstrated that methoxyfenozide have significant depressive effect on reproductive potential against medically important vector with minimizing ecotoxicological risks in mosquitoes management.

Because of its importance as a malaria vector, Anopheles coluzzii's Coetzee & Wilkerson olfactory system has been studied extensively. Among this work is a series of studies comparing the expression of chemosensory genes in olfactory organs in females and/or males of these species. These have identified species- and female-biased chemosensory gene expression patterns. However, many questions remain about the role of chemosensation in male anopheline biology. To pave the way for future work we used RNAseq to compare chemosensory gene expression in the male maxillary palps of An. coluzzii and its sibling species An. quadriannulatus Theobald. As expected, the chemosensory gene repertoire is small in the male maxillary palps. Both species express the tuning receptors Or8 and Or28 at relatively high levels. The CO₂ receptor genes Gr22-Gr24 are present in both species as well, although at much lower level than in females. Additionally, several chemoreceptors are species-specific. Gr37 and Gr52 are exclusive to An. coluzzii, whereas Or9 and Gr60 were detected only in An. quadriannulatus. Furthermore, several chemosensory genes show differential expression between the two species. Finally, several Irs, Grs, and Obps that show strong differential expression in the female palps, are absent or lowly expressed in the male palps. While many questions remain about the role of chemosensation in anopheline male biology, these results suggest that the male maxillary palps could have both a sex- and species-specific role in the perception of chemical stimuli. This work may guide future studies on the role of the male maxillary palp in these species.

The mosquito Aedes aegypti (L.) is the primary vector of several arboviruses. Mosquito control and surveillance are essential to restrict disease transmission, the effectiveness of which depends on our understanding of the mosquito's behaviors, including oviposition. Previous studies have identified a variety of oviposition cues. However, most of these studies involved only Ae. aegypti outside of the species' native range, Africa. Populations outside Africa differ in their genetics and some behaviors from their African counterparts, suggesting possibly different oviposition preferences. Within Africa, Ae. aegypti can be found in both ancestral forest habitats and domestic habitats. The African domestic populations may represent an intermediate state between the forest and the truly domesticated non-African populations. Comparing mosquitoes from these three habitats (African forest, African domestic, and non-African domestic) might provide insight into the evolution of oviposition behavior. In this study, I examined the oviposition choices of multiple Ae. aegypti colonies from all three habitats in laboratory settings. I applied a two-choice assay to test four oviposition cues: the preexistence of conspecific larvae, salinity, shading, and microbiome. A subset of African colonies showed similar oviposition choices as their non-African counterparts, whereas the rest show little response to the factors tested. Within the African colonies, oviposition choices of the domestic colonies were significantly different from the forest colonies in most experiments.Yet, their preferences were not always intermediate between that of mosquitoes from the other two habitats. Collectively, this study adds to our understanding of Ae. aegypti oviposition, especially in previously understudied African populations.

The ability to escape predation modulates predator–prey interactions and represents a crucial aspect of organismal life history, influencing feeding, mating success, and survival. Thanatosis, also known as death feigning or tonic immobility (TI), is taxonomically widespread, but understudied in blood-feeding vectors. Hematophagous arthropods, such as ticks, are unique among animals as their predators (birds, mice, lizards, frogs, and other invertebrates) may also be their source of food. Therefore, the trade-off between predator avoidance and host-seeking may shift as the time since the last bloodmeal increases. Because ticks are slow-moving and unable to fly, or otherwise escape, we predicted that they may use TI to avoid predation, but that TI would be influenced by time since the last bloodmeal (starvation). We therefore aimed to quantify this relationship, examining the effect of starvation, body mass, and ontogeny on TI for two tick species: Dermacentor variabilis (Say) (Acari: Ixodidae) and Rhipicephalus sanguineus (Latreille) (Acari: Ixodidae). As we predicted, the duration and use of TI decreased with time since feeding and emergence across species and life stages. Therefore, ticks may become more aggressive in their search for a bloodmeal as they continue to starve, opting to treat potential predators as hosts, rather than avoiding predation by feigning death. Antipredator behaviors such as TI may influence the intensity and amount of time ticks spend searching for hosts, driving patterns of tick-borne pathogen transmission. This identification and quantification of a novel antipredation strategy add a new component to our understanding of tick life history.

Carrion beetles of genus Thanatophilus (Leach, 1815) are an important group of necrophagous insects, with great potential for forensic entomology in temperate zones of Africa, America, Asia, and Europe. Developmental models for majority of Thanatophilus species remain unknown. In this study, we will provide new thermal summation models for all the developmental stages of Thanatophilus sinuatus (Fabricius, 1775), one of the most abundant and widespread species of the genus. The beetles were bred at seven different constant temperatures, and developmental time was measured for each developmental stage (egg, three larval instars, postfeeding stage, and pupa). Temperature–sex influence was tested, and thermal summation constants were calculated to be used for postmortem interval estimation during criminal investigations.

Chrysomya albiceps (Wiedemann 1819) is one of the most important insects in forensic entomology. Its larval developmental and survival rates are influenced by nutritional resources, temperature, humidity, and geographical regions. The present study investigated the possibility of relying on body size and cuticular hydrocarbon composition as indicators for age estimation of the different larval instars of C. albiceps. Larvae were maintained in standardized laboratory conditions at different experimental temperatures. All larval instars (first, second, and third) were randomly collected for measuring their body sizes and for estimating their cuticular hydrocarbons at different rearing temperatures (30, 35, 40, and 45°C) using gas chromatography–mass spectrometry (GC–MS). Results indicated that the duration of larval stage was temperature dependent as it gradually decreased on increasing the rearing temperature (30, 35, and 40°C) except 45°C at which larval development was ceased. In contrary, larval body size, in terms of length, width, and weight, was temperature dependent as it gradually increased with larval development on increasing rearing temperature except at 45°C at which larval development was ceased. The GC–MS showed a significant difference in the extracted components of cuticular hydrocarbons between different larval instars reared in the same temperature and between the same larval instar that reared at different temperatures. Furthermore, the highest and lowest amounts of cuticular hydrocarbons were detected at 35 and 40°C, respectively. Overall, larval body size and cuticular hydrocarbon components were temperature dependent within the range 30–40°C, which may suggest them as possible reliable age indicators for estimating the postmortem interval in the field of medicolegal entomology.

The determination of necrophagous fly specie's development time is considered an accurate method for estimating postmortem interval (PMI). However, pesticides and other chemicals can alter the flies' life cycle, inducing errors in PMI estimation. Thus, this work aimed to evaluate the effect of different doses of Terbufos (Organophosphates) on the temporal dispersion pattern and development of immature dipterans associated with decaying rat carcasses. For this, 150 g female Wistar rats received, via gavage, 200 µl of Terbufos (5 or 10 mg/kg) or distilled water (control) and, after 30 min of administration, the animals were euthanized and distributed in suspended traps to decompose under environmental conditions. The dispersing immatures were collected daily, and their development time was monitored until the emergence of adult flies. After data analysis, it was observed that Terbufos altered 1) the temporal pattern of larval dispersion; 2) the composition and structure of the colonizing assemblage (emerged adults); 3) species' development time, accelerating or delaying their cycle, depending on the dose used; and 4) the califorids and sarcophagids emergence rate, increasing the mortality of pupae from intoxicated carcasses. Thus, this work demonstrates experimentally that Terbufos directly influences the development of flies with forensic potential and discusses the implications for PMI estimation, which can assist in future investigative processes with suspected poisoning by this organophosphate.

Obtaining the specific development time of each species of forensic interest is crucial for the estimation of an accurate and reliable Minimum Postmortem Interval (PMI_min). In Argentina, Lucilia ochricornis (Wiedemann) and Lucilia purpurascens (Walker) (Diptera: Calliphoridae) were masked under the name Lucilia cluvia (Walker) for a long time still in forensic expertise. For this reason, the objective of this work is to deepen the study of the development time of these species and utilize this relevant information in the generation of different associated methods that can be used in forensics to estimate the PMI. Immature stages of L. ochricornis and L. purpurascens were reared in a brood chamber according to the following temperature treatments: 13.4, 15.1, 22.3, and 23.6°C. The development time of each stage/state of these flies was recorded as well as the resulting accumulated degree-hours (ADH), to build isomorphen diagrams and thermal summation models for each species. The development time and ADH were different between both species and their development stages. On the other hand, the methods provided for estimating PMI_min provide the forensic entomologist more tools to reach accurate and reliable estimates.

Fire ants (Solenopsis spp.) have increasingly been reported from carrion in the southeastern United States and are now a part of the normal succession community. There have been previous observations of these ants altering carrion and preying on other carrion-attendant fauna; however, the overall effects of these activities on carrion decomposition rates, community composition, and blow fly larval development are poorly understood. Alteration of these ecological processes by fire ants could affect the forensic interpretation of entomological data. We conducted a study in Mississippi and Florida whereby portions of the succession fauna were excluded from access to pig carrion to study the relative effects of fire ants and blow flies on carrion decomposition and succession: a control with all fauna having access, a second treatment where fire ants and other geophilic taxa were excluded, and a third treatment in which blow flies and other large organisms were excluded. Fire ants inflicted lesions in the carrion, buried portions that touched the ground, and preyed on some members of the succession fauna. Their exclusion did not affect carrion decomposition rates that were measured but slightly affected the overall carrion community, and strongly affected the oviposition and development of blow flies. Despite the presence of fire ants early in the control, blow flies were eventually able to overcome predation of eggs and larvae, continue colonization, and complete development; however, the delay in the colonization of blow flies on carrion could affect the determination of postmortem intervals when development rates of blow flies are considered in the calculation.

The American dog tick, Dermacentor variabilis (Say) (Acari: Ixodidae), is a vector for several human disease-causing pathogens such as tularemia, Rocky Mountain spotted fever, and the understudied spotted fever group rickettsiae (SFGR) infection caused by Rickettsia montanensis. It is important for public health planning and intervention to understand the distribution of this tick and pathogen encounter risk. Risk is often described in terms of vector distribution, but greatest risk may be concentrated where more vectors are positive for a given pathogen. When assessing species distributions, the choice of modeling framework and spatial layers used to make predictions are important. We first updated the modeled distribution of D. variabilis and R. montanensis using maximum entropy (MaxEnt), refining bioclimatic data inputs, and including soil variables. We then compared geospatial predictions from five species distribution modeling frameworks. In contrast to previous work, we additionally assessed whether the R. montanensis positive D. variabilis distribution is nested within a larger overall D. variabilis distribution, representing a fitness cost hypothesis. We found that 1) adding soil layers improved the accuracy of the MaxEnt model; 2) the predicted ‘infected niche’ was smaller than the overall predicted niche across all models; and 3) each model predicted different sizes of suitable niche, at different levels of probability. Importantly, the models were not directly comparable in output style, which could create confusion in interpretation when developing planning tools. The random forest (RF) model had the best measured validity and fit, suggesting it may be most appropriate to these data.

Pedrowygomyia is a Neotropical genus of Simuliidae composed of four species; all were described in 1989 from high-elevation (above 3,000 m) areas in the Andean region. In this article, a new species for this genus, Pedrowygomyia hanaq n. sp., is described based on all stages of development. The new species was collected in the south-central Andes of Peru at an altitude above 4,000 m, and its known distribution is currently restricted to the type-locality. Based on the pupal stage, the new species appears to be more closely related to Pedrowygomyia punapi (Wygodzinsky & Coscarón) (Diptera: Simuliidae), a species known from Argentina, Bolivia, and Chile.

Graphical Abstract

The taxonomic identity of two species of sand flies, Psathyromyia pradobarrientosi (Le Pont, Matias, Martinez & Dujardin, 2004) and Psathyromyia runoides (Fairchild & Hertig, 1953) (Diptera, Psychodidae), was evaluated morphologically and molecularly based upon specimens collected in Brazilian states. The morphological component compared collected specimens with paratypes of Pa. runoides and Pa. pradobarrientosi and their descriptions. Phylogenetic analysis of coI sequences of Pa. pradobarrientosi showed a well-supported group distinct from Pa. runoides. Morphologically, Psathyromyia runoides and Pa. pradobarrientosi males are distinguished by characteristics of the aedeagal ducts and parameral sheath in the genitalia; females are distinguished by the number and shape of the teeth in the cibarium and by the shape of the spermathecae. Given the morphological similarity between the males and the absence of the description of the female of Pa. pradobarrientosi, it is possible that specimens previously identified as Pa. runoides in Brazil are in fact Pa. pradobarrientosi.

Graphical Abstract

Lice pose major public and veterinary health problems with economic consequences. Their identification is essential and requires the development of an innovative strategy. MALDI-TOF MS has recently been proposed as a quick, inexpensive, and accurate tool for the identification of arthropods. Alcohol is one of the most frequently used storage methods and makes it possible to store samples for long periods at room temperature. Several recent studies have reported that alcohol alters protein profiles resulting from MS analysis. After preliminary studies on frozen lice, the purpose of this research was to evaluate the influence of alcohol preservation on the accuracy of lice identification by MALDI-TOF MS. To this end, lice stored in alcohol for variable periods were submitted for MS analysis and sample preparation protocols were optimized. The reproducibility and specificity of the MS spectra obtained on both these arthropod families allowed us to implement the reference MS spectra database (DB) with protein profiles of seven lice species stored in alcohol. Blind tests revealed a correct identification of 93.9% of Pediculus humanus corporis (Linnaeus, 1758) and 98.4% of the other lice species collected in the field. This study demonstrated that MALDI-TOF MS could be successfully used for the identification of lice stored in alcohol for different lengths of time.

The aim of the present study was to describe the morphology of the eggs of Culex (Culex) saltanensis Dyar that occurs in the Neotropical region. Eggs of the Cx. (Cux.) saltanensis were collected at the Mata Atlântica FIOCRUZ campus, fixed in 1% osmium tetroxide, prepared for mounting on metal supports, observed under a scanning electron microscope, and described morphologically. The eggs had a coniform shape with a length of approximately 0.5 mm (505–510 µm) and a width in the median portion of 117 µm (113–123 µm). Upper portion is lined with tubers of irregular shape and varying sizes (0.64–1.31 µm), located on a cross-linked matrix forming bands observed under optical microscopy. The micropyle is encased in a necklace of approximately 6.6-µm plates arranged in a flower-like shape. Comparing Cx. (Cux.) saltanensis eggs with several species of different genera, important divergent characteristics can be observed. However, this study points to the need for new descriptions of eggs of species belonging to the same subgenus in order to analyze if there will be differences between them. Culex (Cux.) saltanensis eggs have particular characteristics not observed in eggs of other Culicidae genera.

A new subgenus, Reinertia Somboon, Namgay & Harbach, of the genus Aedes Meigen and its type species, Ae. suffusus Edwards, are described from specimens reared from larvae and pupae found in a tree hole in Bhutan. The scutum of the adults is mostly covered with narrow pale falcate scales. The proboscis, maxillary palpus, tibiae, and tarsi are dark-scaled. The gonocoxite of the male genitalia bears a unique setose basomesal sclerite. The larva closely resembles larvae of the subgenus Downsiomyia Vargus in having setae 4–6-C with numerous branches and inserted more or less on level with seta 7-C, abdominal seta 12-I is present and the comb is composed of 6–10 spine-like scales arranged in an irregular row. Surprisingly, Reinertia shares features of the adult habitus, male genitalia, and larva with the Palearctic subgenus Dahliana Reinert, Harbach & Kitching. However, in phylogenetic analyses of the mitochondrial COI gene of species representing 38 subgenera of Aedes and six other genera of the tribe Aedini Neveu-Lemaire, Reinertia was not associated with Dahliana or Downsiomyia. In both maximum likelihood and Bayesian analyses of the data, Ae. suffusus was recovered as the weakly supported sister of a clade composed of five species of the subgenus Protomacleaya Theobald. In the absence of strong support, and because Protomacleaya is an unnatural group of species that resemble each other phenetically by virtue of what they lack, Ae. suffusus cannot be placed in the subgenus Protomacleaya. Thus, the morphological and molecular data attest the uniqueness of Ae. suffusus and its recognition as a monobasic subgeneric lineage.

Sand flies are vector of several diseases, mostly cutaneous and visceral leishmaniasis (CL and VL). Also, 29 sand fly species have been identified in previous fauna studies carried out in 40 provinces of Turkey. Totally, 24 sand flies species belonging to Phlebotomus (Ph.) (Diptera: Psychodidae) genus have been proven or reported as possible vector species. This study aimed to develop a new software which could contribute to researchers' decision making about the identification of sand flies with obtained data from entomological surveys conducted before in Turkey. Developed software called TRsandflies included 35 textbox created with parameters obtained from caught sand flies specimens by the above-mentioned surveys. It also contained 130 photos and distribution maps related to 24 sand flies species. In addition, C# language and MYSQL database were used in the program. TRsandflies had three different forms (pages) allowing the user to compare the specimens and known species. In the species identification trials with three repetitions carried out in the program, except for the specimens belonging to the Transphlebotomus Artemiev & Neronov, 1984 subgenus, morphometric data of all previously collected sand fly species specimens were included. The process of running the morphometric measurement results of predetermined specimens in the program provided us with an accurate prediction rate of 86.66% in male specimens and 71.66% in female specimens. We concluded that the web-based software developed could play an important role in reducing the rate of possible errors that might be encountered by conventional identification methods.

We describe two new species of sucking lice in the genus Hoplopleura Enderlein, 1904 (Psocodea: Phthiraptera: Hoplopleuridae) from Australia: Hoplopleura gracilicaudatusa n. sp. from the eastern chestnut mouse Pseudomys gracilicaudatus (Gould) (Rodentia: Muridae), and Hoplopleura nanusa n. sp. from the western chestnut mouse Pseudomys nanus (Gould) (Rodentia: Muridae). Pseudomys Gray is the most speciose genus of rodents endemic to Australia with 24 species; however, only two Pseudomys species have been reported previously to be hosts of sucking lice. The description of the new species in the present study doubles the number of sucking louse species known to parasitize Pseudomys mice and increases the total number of sucking louse species known from endemic Australian rodents from 21 to 23. Pseudomys gracilicaudatus and P. nanus are closely related murines that diverged ∼1 MYA with distinct and widely separated extant geographic distributions. The two new Hoplopleura species described in the present study share some morphological characters and likely co-evolved and co-speciated with their chestnut mouse hosts.

South American chiggers have historically been poorly studied, and this has continued into present times. Of the 33 genera in the family Leeuwenhoekiidae Womersley, only Odontacarus Ewing and Sasacarus Brennan & Jones have been reported in Peru. Here, we describe a new genus, Peruacarus n. gen., and a new species, Peruacarus anthurium n. sp., parasitizing Koford's grass mouse, Akodon kofordi Myers & Patton, in Peru.

Timber harvest may impact tick-borne disease by affecting small mammal and tick community structures. We assessed tick and small mammal populations in older second-growth redwood (Sequoia sempervirens (D. Don) Endl) habitat at two harvested sites in Santa Cruz County, California, where local risk of tick-borne disease is high and determined the prevalence of tick-borne pathogens in ticks. After single-tree removal harvest in 2014, there was a modest reduction in canopy, primarily toward the end of the study. Harvested sites showed strong reductions in California mouse (Peromyscus californicus, (Gambel)) captures 2-yr after harvest, resolving such that treatments and controls were comparable by the end of the study. Following harvest, treated sites experienced a transient decreased tick infestation while control plots experienced an increase. Ixodes angustus (Neumann) infestation probability on harvested plots decreased immediately after harvest, increasing with time but remaining lower than control plots, whereas I. pacificus (Cooley and Kohls) prevalence was higher shortly after the harvest on harvested plots, and continued to increase. Mean abundance of ticks on vegetation increased on control plots. We detected Borrelia burgdorferi ((Johnson et al.) Baranton) and Anaplasma phagocytophilum ((Foggie 1949) Dumler) in 3.8 and 3.1% of ticks on rodents, but no differences were associated with harvest. Impacts of forest harvest on tick-borne disease depend on removal practice and intensity, whether or not hosts are habitat specialists, and whether or not ticks are host specialists.

Although parasites are by definition costly to their host, demonstrating that a parasite is regulating its host abundance in the field can be difficult. Here we present an example of a gregarine parasite, Ascogregarina taiwanensis Lien and Levine (Apicomplexa: Lecudinidae), regulating its mosquito host, Aedes albopictus Skuse (Diptera: Culicidae), in Bermuda. We sampled larvae from container habitats over 2 yr, assessed parasite prevalence, and estimated host abundance from egg counts obtained in neighboring ovitraps. We regressed change in average egg count from 1 yr to the next on parasite prevalence and found a significant negative effect of parasite prevalence. We found no evidence of host density affecting parasite prevalence. Our results demonstrate that even for a parasite with moderate virulence, host regulation can occur in the field.

Non-lethal predator-prey interactions during the immature stages can cause significant changes to mosquito life history traits and their ability to transmit pathogens as adults.Treatment manipulations using mosquitoes Aedes aegypti (L.) and Toxoryhnchites rutilus (Coquillett) were performed during the immature stages to explore the potential impacts of non-lethal interactions on adult susceptibility to infection, disseminated infection and saliva infection of Ae. aegyptifollowing ingestion of Zika virus-infected blood.Treatments inducing density reduction resulted in reduced development time and survivorship to adulthood. However, effects of treatment did not alter infection, dissemination, or saliva infection.These observations indicate that, while non-lethal predation may impact some traits that influence population dynamics and transmission of pathogens, there were no direct effects on mosquito-arbovirus interactions.

The wide distribution of Culex (Cx.) pipiens complex mosquitoes makes it difficult to prevent the transmission of mosquito-borne diseases in humans. Gene editing using CRISPR/Cas9 is an effective technique with the potential to solve the growing problem of mosquito-borne diseases. This study uses the ReMOT Control technique in Culex pipiens pallens (L.) to produce genetically modified mosquitoes. A microinjection system was established by injecting 60 adult female mosquitoes—14 µl injection mixture was required, and no precipitation occurred with ≤1 µl of endosomal release reagents (chloroquine or saponin). The efficiency of delivery of the P2C-enhanced green fluorescent protein-Cas9 (P2C-EGFP-Cas9) ribonucleoprotein complex into the ovary was 100% when injected at 24 h post-bloodmeal (the peak of vitellogenesis). Using this method for KMO knockout, we found that gene editing in the ovary could also occur when P2C-Cas9 RNP complex was injected into the hemolymph of adult Cx. pipiens pallens by ReMOT Control. In the chloroquine group, of the 2,251 G₀ progeny screened, 9 individuals showed with white and mosaic eye phenotypes. In the saponin group, of the 2,462 G₀ progeny screened, 8 mutant individuals were observed. Sequencing results showed 13 bp deletions, further confirming the fact that gene editing occurred. In conclusion, the successful application of ReMOT Control in Cx. pipiens pallens not only provides the basic parameters (injection parameters and injection time) for this method but also facilitates the study of mosquito biology and control.

The rapid and economical monitoring of mosquitos is imperative to understanding the dynamics of both disease vectors and nuisance species. In light of technological advances in mosquito sampling and DNA sequencing, health agencies can now utilize the full potential of metabarcoding pipelines for rapid and standardizable surveillance. Here, we describe mosquito spatial and temporal variation, with particular focus on Mansonia Blanchard species, in the Madeira (Rondônia State) and the Ribeira (São Paulo) watersheds, Brazil using metabarcoding of the D2 rDNA marker. Sampling and molecular pipelines were used to evaluate the taxonomic contribution of mosquitos in pools of culicids collected en masse from macrophyte-roots (immatures) and from Mosquito Magnet traps and protected human landings (adults). Results for adult captures are comparable to morphological diagnoses and clarify previously unknown temporal and spatial species turnover. Metabarcoding of immature stages also confirmed the extent of the geographical distribution of some species and each taxon's association with macrophyte species. Given the benefits of metabarcoding, such as taxonomic acuity, high throughput processing, and objectivity, we suggest such techniques should be more fully incorporated into culicid monitoring schemes. The metabarcoding protocol described herein paired with standardized field sampling schemes, when used by mosquito monitoring professionals, offers substantial improvements in terms of practicality, speed and cost.

Lyme disease is the most common vector-borne disease in the United States. While Lyme disease vectors are widespread, high incidence states are concentrated in the Northeast, North Central and Mid-Atlantic regions. Mapping the distribution of Lyme disease spirochetes in ticks may aid in providing data-driven explanations of epidemiological trends and recommendations for targeting prevention strategies to communities at risk. We compiled data from the literature, publicly available tickborne pathogen surveillance databases, and internal CDC pathogen testing databases to map the county-level distribution of Lyme disease spirochetes reported in host-seeking Ixodes pacificus and Ixodes scapularis across the contiguous United States. We report B. burgdorferi s.s.-infected I. scapularis from 384 counties spanning 26 eastern states located primarily in the North Central, Northeastern, and Mid-Atlantic regions, and in I. pacificus from 20 counties spanning 2 western states, with most records reported from northern and north-coastal California. Borrelia mayonii was reported in I. scapularis in 10 counties in Minnesota and Wisconsin in the North Central United States, where records of B. burgdorferi s.s. were also reported. In comparison to a broad distribution of vector ticks, the resulting map shows a more limited distribution of Lyme disease spirochetes.

The increase in malaria transmission in the Amazon region motivated vector control units of the Ministry of Health of Ecuador and Peru to investigate Anopheles (Diptera: Culicidae) species present in transmission hotspots. Mosquitoes were collected using prokopack aspirators and CDC light traps (Ecuador) and human landing catch in Peru. In Ecuador, 84 Anopheles were captured from Pastaza, Morona Santiago, and Orellana provinces and identified morphologically [An. (An.) apicimacula Dyar and Knab, An. (Nys.) near benarrochi, An. (Nys.) near oswaldoi, An. (Nys.) near strodei, An. (An.) nimbus (Theobald, 1902), and An. (Nyssorhynchus) sp.]. In Peru, 1,150 Anopheles were collected in Andoas District. A subsample of 166 specimens was stored under silica and identified as An. near oswaldoi, An. darlingi, and An. (An.) mattogrossensis Lutz and Neiva. COI barcode region sequences were obtained for 137 adults (107 from Peru, 30 from Ecuador) identified by ITS2 PCR-RFLP as An. benarrochi Gabaldon, Cova Garcia, and Lopez and retained in the final analysis. Haplotypes from the present study plus An. benarrochi B GenBank sequences grouped separately from Brazilian An. benarrochi GenBank sequences by 44 mutation steps, indicating that the present study specimens were An. benarrochi B. Our findings confirm the presence of An. benarrochi B in Ecuador and reported here for the first time from the Amazonian provinces of Orellana and Morona Santiago. Furthermore, we confirm that the species collected in Andoas District in the Datem del Maranon Province, Peru, is An. benarrochi B, and we observed that it is highly anthropophilic. Overall, the known distribution of An. benarrochi B has been extended and includes southern Colombia, much of Peru and eastern Ecuador.

Throughout North America, Dermacentor spp. ticks are often found feeding on animals and humans, and are known to transmit pathogens, including the Rocky Mountain spotted fever agent. To better define the identity and distribution of Dermacentor spp. removed from dogs and cats in the United States, ticks submitted from 1,457 dogs (n = 2,924 ticks) and 137 cats (n = 209 ticks) from veterinary practices in 44/50 states from February 2018-January 2020 were identified morphologically (n = 3,133); the identity of ticks from regions where Dermacentor andersoni (Stiles) have been reported, and a subset of ticks from other regions, were confirmed molecularly through amplification and sequencing of the ITS2 region and a 16S rRNA gene fragment. Of the ticks submitted, 99.3% (3,112/3,133) were Dermacentor variabilis (Say), 0.4% (12/3,133) were D. andersoni, and 0.3% (9/3,133) were Dermacentor albipictus (Packard). While translocation of pets prior to tick removal cannot be discounted, the majority (106/122; 87%) of Dermacentor spp. ticks removed from dogs and cats in six Rocky Mountain states (Montana, Idaho, Wyoming, Nevada, Utah, and Colorado) were D. variabilis, suggesting this species may be more widespread in the western United States than is currently recognized, or that D. andersoni, if still common in the region, preferentially feeds on hosts other than dogs and cats. Together, these data support the interpretation that D. variabilis is the predominant Dermacentor species found on pets throughout the United States, a finding that may reflect recent shifts in tick distribution.

Graphical Abstract

The blacklegged tick (Ixodes scapularis Say) vectors several bacterial, protozoan, and viral human pathogens. The known distribution, abundance, and phenology of I. scapularis within its estimated range are incomplete. This gap in knowledge is problematic because these factors are important for determining acarological risk of exposure to infected ticks. Consequently, enhanced surveillance of I. scapularis is being promoted and supported in the United States. Although the most common method for collecting I. scapularis is by dragging a sturdy cloth along the ground, there are no published empirical data showing which drag fabric is most effective. We used a randomized block design to directly compare the relative efficiencies of canvas, corduroy, and flannel drags for the collection of larval, nymphal, and adult I. scapularis. Overall, flannel was the most effective fabric and canvas was the least effective. Significantly more adults were collected on flannel than on canvas or corduroy, and the same number of nymphs was collected on flannel and corduroy. Significantly more larvae were collected on flannel than on canvas, but one-third of larvae could not be removed from the former fabric by lint-rolling, and handpicking was difficult. Our findings support the use of flannel drags to maximize sampling effort for collection of I. scapularis, especially adults to determine the presence of ticks and tick-borne pathogens when density and infection prevalence are low, with the caveat that detection and removal of larvae are time-consuming.

Metabolomics can indicate the physiological and biochemical responses of mosquitoes to different stimulants, including insecticides, which allow them to adapt to different inhospitable environments. Though metabolic differences between insecticide-resistant and -susceptible strains have been established for other mosquito species, such as Anopheles and Culex, it is yet to be done for Aedes albopictus (Skuse). In this study, nuclear magnetic resonance (NMR) spectroscopy–based metabolomic analysis performed on Ae. albopictus deltamethrin-resistant and -susceptible strains showed significant differences in amino acid, organic acid, and sugar metabolism. Concentrations of neutral amino acids and sugars tended to be lower in the deltamethrin-resistant strain than in the deltamethrin-suceptible strain, but the concentration of basic and acidic amino acids and organic acids increased. All these changes might accommodate biochemical and physiological needs in deltamethrin-resistant mosquitoes, such as enzyme synthesis and detoxification. This was further confirmed by the predictable draft metabolic map. This is the first report using NMR spectroscopy to investigate the metabolic differences between deltamethrin-resistant and -susceptible strains of Ae. albopictus. To a certain degree, this demonstrates how Ae. albopictus develop insecticide resistance by metabolic reprograming to survive under the insecticide pressure.

In the search for new strategies to control Aedes aegypti Linnaeus (Diptera: Culicidae), several studies have successfully related pyriproxyfen (PPF) tarsal transference to breeding sites (autodissemination), as well as the sterilization potential of females exposed to PPF. Potential PPF autodissemination by mosquito feces after the ingestion of sugar baits has also been proposed. Therefore, the present work evaluated several parameters, e.g., fecal production, residuality under dry and aqueous conditions, PPF excretion affecting emergence inhibition (EI) by fecal deposits of Ae. aegypti fed with attractive toxic sugar baits (ATSBs) containing PPF as well as their reproductive potential. Females were fed with ATSBs offered as droplets and the feces were collected using filter paper and transferred to plastic cups with L3 larvae to evaluate EI. The residual effect of feces in aqueous and dry conditions and PPF excretion on EI was obtained by keeping the feces in water or dried for different time intervals and using feces collected at 24-h intervals, respectively. Females received a bloodmeal after feeding on ATSBs, eggs and larval counting expressed the reproductive potential. The fecal mass was not affected by PPF concentration, but EI increased from 33 to 54% as the PPF concentration increased. The PPF excretion in the feces exceeded 96 h. The residual effect in the EI for feces kept in water was reduced by more than 60% after 30 d but was not affected under dry conditions. The fecundity and fertility of the females were reduced up to 51% and 97%, respectively.

High rates of dengue morbidity occur in southern Thailand. The intensive application of insecticides in orchards could affect not only agricultural insect pests, but also nontarget mosquitoes or beneficial insects. In this study, the type and quantity of insecticides commonly used across durian plantations in southern Thailand were characterized, along with the population density of Aedes albopictus (Skuse) (Diptera: Culicidae). Our primary aim was to determine the susceptibility status of field-derived Ae. albopictus to typical application concentrations of four agrochemical insecticides; cypermethrin, chlorpyrifos, carbaryl, and imidacloprid. Mosquito eggs were collected from durian cultivation sites in five provinces in southern Thailand and used to generate adults for susceptibility tests. The cultivation sites were categorized into three groups based on insecticide application: intensive application of insecticides, low application of insecticides, and no application of insecticides. Twenty ovitraps were deployed for at least three consecutive days at each study site to collect mosquito eggs and to determine Ae. albopictus population density. WHO tube assays were used to determine the susceptibility of adult mosquitoes derived from field-collected eggs to selected insecticides. This represents the first report of the susceptibility status of Ae. albopictus from durian orchards in southern Thailand to agrochemical insecticides. Results showed complete susceptibility of these Ae. albopictus to chlorpyrifos, but reduced mortality following exposure to λ-cyhalothrin, carbaryl, and imidacloprid, which is suggestive of the development of resistance. These findings provide new insights into the status of insecticide susceptibility in Ae. albopictus populations, with important implications for mosquito and mosquito-borne disease control in Thailand.

Malaria is a leading public health challenge causing mortality and morbidity in sub-Saharan Africa. Prominent malaria vector control methods employed in sub-Saharan Africa include Long Lasting Insecticide Nets (LLINs) and Indoor Residual spraying (IRS). This study investigated knowledge, attitude and practices (KAP) of malaria vector control methods in Lagos, South-West Nigeria. Structured questionnaires were employed for the cross-sectional survey which was carried out between May and August 2018. Multi-stage sampling technique was used to select Lagos Mainland, Kosofe, and Ojo local government areas (LGAs). Five hundred and twenty questionnaires were used for the study. Data were analyzed for descriptive statistics, whereas χ² was used to determine influence of respondents' LGA, level of education and type of dwelling on respondents' attitude and practice. Respondents' LGAs have no significant impact on attitude and practice to malaria vector control methods. However, ‘level of education' as well as ‘type of dwelling structure' impacted significantly on some practices and attitude. Basically, IRS is the major tool employed in malaria vector control, but sometimes it is used in combination with other methods. A good number of residents also use LLINs. ‘Choice of method’ employed is mainly based on the effectiveness of method. General perception about LLINs and IRS is that they are effective, cheap and safer. Considering the widespread use of IRS and LLINs for malaria vector control in Lagos, implementation of malaria control programs should consider KAP to these two strategies.

Vector-borne diseases are a worldwide threat to human health. Often, no vaccines or treatments exist. Thus, personal protection products play an essential role in limiting transmission. The World Health Organization (WHO) arm-in-cage (AIC) test is the most common method for evaluating the efficacy of topical repellents, but it remains unclear whether AIC testing conditions recreate the mosquito landing rates in the field. This study aimed to estimate the landing rate outdoors, in an area of Europe highly infested with the Asian tiger mosquito (Aedes albopictus (Skuse, 1894, Diptera: Culididae)), and to determine how to replicate this rate in the laboratory. To assess the landing rate in the field, 16 individuals were exposed to mosquitoes in a highly infested region of Italy. These field results were then compared to results obtained in the laboratory: 1) in a 30 m³ room where nine volunteers were exposed to different mosquito abundances (ranges: 15–20, 25–30, and 45–50) and 2) in a 0.064 m³ AIC test cage where 10 individuals exposed their arms to 200 mosquitoes (as per WHO requirements). The highest mosquito landing rate in the field was 26.8 landings/min. In the room test, a similar landing rate was achieved using 15–20 mosquitoes (density: 0.50–0.66 mosquitoes/m³) and an exposure time of 3 min. In the AIC test using 200 mosquitoes (density: 3,125 mosquitoes/m³), the landing rate was 229 ± 48 landings/ min. This study provides useful reference values that can be employed to design new evaluation standards for topical repellents that better simulate field conditions.

Graphical Abstract

Previous work presented the profound antimosquito potential of Petroselinum crispum essential oil (PEO) against either the pyrethroid-susceptible or resistant strains of Aedes aegypti. This plant oil also inhibited the activity of acetylcholinesterase and mixed-function oxidases significantly, thus suggesting its potential as a synergist for improving mosquitocidal efficacy of insecticidal formulations. This study investigated the chemical composition, larvicidal activity, and potential synergism with synthetic insecticides of PEO and its main compounds for the purpose of interacting with insecticide resistance in mosquito vectors. The chemical profile of PEO, obtained by GC-MS analysis, showed a total of 17 bioactive compounds, accounting for 99.09% of the whole oil, with the most dominant constituents being thymol (74.57%), p-cymene (10.73%), and γ-terpinene (8.34%). All PEO constituents exhibited promising larvicidal effects, with LC₅₀ values ranging from 19.47 to 59.75 ppm against Ae. aegypti, in both the pyrethroid-susceptible and resistant strains. Furthermore, combination-based bioassays revealed that PEO, thymol, p-cymene, and γ-terpinene enhanced the efficacy of temephos and deltamethrin significantly. The most effective synergist with temephos was PEO, which reduced LC₅₀ values to 2.73, 4.94, and 3.28 ppb against MCM-S, PMD-R, and UPK-R, respectively, with synergism ratio (SR) values of 1.33, 1.38, and 2.12, respectively. The best synergist with deltamethrin also was PEO, which reduced LC₅₀ values against MCM-S, PMD-R, and UPK-R to 0.008, 0.18, and 2.49 ppb, respectively, with SR values of 21.25, 9.00, and 4.06, respectively. This research promoted the potential for using essential oil and its principal constituents as not only alternative larvicides, but also attractive synergists for enhancing efficacy of existing conventional insecticides.

In total, 341 fleas belonging to 16 species were collected from 78 host mammals belonging to 10 species in Panamá from 2010 to 2016. The cat flea, Ctenocephalides felis (Bouché) predominated on domestic dogs and was also recorded from domestic cats, the raccoon, Procyon lotor (Linnaeus) and the common opossum, Didelphis marsupialis Linnaeus. The largest number of flea species (7) was recorded from D. marsupialis and the most common flea on that host was the ctenophthalmid, Adoratopsylla intermedia copha Jordan. One Oriental rat flea, Xenopsylla cheopis (Rothschild), was collected from D. marsupialis. Native rodents were parasitized by indigenous ceratophyllid, rhopalopsyllid, and stephanocircid fleas. The Mexican deermouse, Peromyscus mexicanus (Saussure), was parasitized by six species of ceratophyllids belonging to the mostly Central American genera, Baculomeris, Jellisonia, Kohlsia and Plusaetis. The long-tailed singing mouse, Scotinomys xerampelinus (Bangs), was parasitized by Plocopsylla scotinomi Tipton and Méndez, the only species of stephanocircid flea known from Central America. Twenty-six pools of extracted flea DNA representing 5 flea species (C. felis, Pulex echidnophagoides (Wagner), Pulex simulans Baker, A. intermedia copha, and P. scotinomi) and 79 individual fleas were all real-time polymerase chain reaction negative for Rickettsia felis, Rickettsia typhi, and Bartonella henselae.

West Nile virus (WNV) is the most prevalent arbovirus found throughout the United States. Surveillance of surface breeding Culex vectors involved in WNV transmission is primarily conducted using CDC Gravid traps. However, anecdotal claims from mosquito abatement districts in Louisiana assert that other trap types may be more suited to WNV surveillance. To test the validity of these assertions, we conducted a series of trapping trials and WNV surveillance over 3 yr to compare the efficacy of multiple trap types. First, we compared the CDC Gravid trap, CO₂-baited New Standard Miniature Blacklight traps, and CO₂-baited CDC light traps with either an incandescent light, a red light, or no light. We found that the CDC Gravid trap and CO₂-baited no-light CDC Light trap collected the most mosquitoes. Second, we conducted additional, long-term trapping and WNV surveillance to compare these two trap types. We found that CO₂-baited no-light CDC traps collected more of the local WNV vector, Culex quinquefasciatus (Say, Diptera, Culicidae), and detected WNV with greater sensitivity. Finally, we conducted trapping to compare the physiological states of Cx. quinquefasciatus and diversity of collected mosquitoes. CO₂-baited no-light CDC light traps collected more unfed Cx. quinquefasciatus while Gravid traps collected more blooded Cx. quinquefasciatus; both traps collected the same number of gravid Cx. quinquefasciatus. Additionally, we found that CO₂-baited no-light CDC light traps collected a larger diversity of mosquito species than Gravid traps.

Borrelia is a genus of spirochetal bacteria with several species known to cause disease in humans. The distribution of Borrelia has rarely been studied in Thailand. In this study, a retrospective survey of Borrelia was conducted in ticks and wild rodents to better characterize the prevalence, diversity, and distribution of Borrelia across Thailand. Several pools of DNA from tick samples were positive for Borrelia spp. (36/258, 13.9%). Borrelia theileri/B. lonestari was found in 17 tick samples (16 pools of Haemaphysalis bandicota and 1 pool of Rhipicephalus sp.), and Borrelia yangtzensis was found in 8 tick samples (2 pools of H. bandicota and 6 pools of Ixodes granulatus). Borrelia spp. were detected at low prevalence levels in rodent tissue samples (24/2001, 1.2%), with 19 identified as B. theileri or B. lonestari and 5 identified as B. miyamotoi. Several geographic and species-specific infection trends were apparent, with Ixodes ticks infected with B. yangtzensis and Haemaphysalis and Rhipicephalus ticks infected with both B. yangtzensis and B. theileri/B. lonestari. Notably, B. yangtzensis showed a similar geographic distribution to B. miyamotoi, which was identified in new areas of Thailand in this study. The flagellin gene sequence from B. miyamotoi was more similar to European (99.3–99.9%) than Japanese (96.9–97.6%) genotypes. This study greatly expands the knowledge of Borrelia in Thailand and identified several Borrelia species for the first time. It also found several ticks and rodents infected with the pathogen that were not previously known to carry Borrelia.

Tick-borne pathogens are of growing concern. The U.S. Centers for Disease Control and Prevention (CDC) developed guidelines standardizing surveys of tick vectors to better monitor the changes in their occurrences. Unbiased surveillance data, from standardized surveys, are presumed critical to generate valid species distribution models (SDMs). We tested previously generated SDMs from standardized protocols for three medically important ticks [Amblyomma americanum (Linnaeus, Ixodida, Ixodidae), Ixodes scapularis (Say, Ixodida, Ixodidae), and Dermacentor variabilis (Say, Ixodida, Ixodidae)]. These previous models ruled out a quarter to half of the state as having these species, with consensus occurrence in about a quarter of the state. New surveys performed throughout 2019 on 250 transects at 43 sites indicated the rule-out functions were 100% accurate for I. scapularis and D. variabilis and 91.9% for A. americanum. As SDM concordance increased, the proportion of transects yielding ticks increased. Independent surveys of SDMs provide external validation—an aspect missing from many SDM studies.

Lyme and other tick-borne diseases are increasing in the eastern United States and there is a lack of research on integrated strategies to control tick vectors. Here we present results of a study on tick-borne pathogens detected from tick vectors and rodent reservoirs from an ongoing 5-yr tick suppression study in the Lyme disease-endemic state of Maryland, where human-biting tick species, including Ixodes scapularis Say (Acari: Ixodidae) (the primary vector of Lyme disease spirochetes), are abundant. During the 2017 tick season, we collected 207 questing ticks and 602 ticks recovered from 327 mice (Peromyscus spp. (Rodentia: Cricetidae)), together with blood and ear tissue from the mice, at seven suburban parks in Howard County. Ticks were selectively tested for the presence of the causative agents of Lyme disease (Borrelia burgdorferi sensu lato [s.l.]), anaplasmosis (Anaplasma phagocytophilum), babesiosis (Babesia microti), ehrlichiosis (Ehrlichia ewingii, Ehrlichia chaffeensis, and ‘Panola Mountain’ Ehrlichia) and spotted fever group rickettsiosis (Rickettsia spp.). Peromyscus ear tissue and blood samples were tested for Bo. burgdorferi sensu stricto (s.s), A. phagocytophilum, Ba. microti, and Borrelia miyamotoi. We found 13.6% (15/110) of questing I. scapularis nymphs to be Bo. burgdorferi s.l. positive and 1.8% (2/110) were A. phagocytophilum positive among all sites. Borrelia burgdorferi s.s. was found in 71.1% (54/76) of I. scapularis nymphs removed from mice and 58.8% (194/330) of captured mice. Results from study on tick abundance and pathogen infection status in questing ticks, rodent reservoirs, and ticks feeding on Peromyscus spp. will aid efficacy evaluation of the integrated tick management measures being implemented.

Spotted fever group rickettsiae, mainly maintained and transmitted by ticks, are important etiological agents of (re)emerging zoonotic diseases worldwide. It is of great significance to investigate spotted fever group rickettsiae in ticks in different areas for the prevention and control of rickettsioses. In this study, a total of 305 ticks were collected from wild and domestic animals in Chongqing, Guizhou, Yunnan, and Guangxi provinces of southwestern China during 2017–2019 and examined for the presence of spotted fever group rickettsiae by PCR with primers targeting the partial gltA, ompA, rrs, and htrA genes. Results showed that two spotted fever group rickettsiae species, including the pathogenic Candidatus Rickettsia jingxinensis (Rickettsiales: Rickettsiaceae) and a potential novel species Rickettsia sp. sw (Rickettsiales: Rickettsiaceae), were identified. The Ca. R. jingxinensis sequences were recovered from Rhipicephalus microplus (Ixodida: Ixodidae) and Haemaphysalis longicornis (Ixodida: Ixodidae) ticks and phylogenetically clustered with previous Ca. R. jingxinensis, Ca. R. longicornii (Rickettsiales: Rickettsiaceae), and Rickettsia sp. XY118 (Rickettsiales: Rickettsiaceae) strains. Rickettsia sp. sw was detected in Amblyomma geoemydae (Ixodida: Ixodidae) and Rh. microplus. Interestingly, as far as we know, this was the first report of Rickettsia (Rickettsiales: Rickettsiaceae) in A. geoemydae. Phylogenetic analyses indicated that this potential novel species was closely related to R. aeschlimannii (Rickettsiales: Rickettsiaceae) with gltA and ompA genes and grouped in a cluster composed of R. montanensis (Rickettsiales: Rickettsiaceae), R. raoultii (Rickettsiales: Rickettsiaceae), R. aeschlimannii, R. massiliae (Rickettsiales: Rickettsiaceae), and R. rhipicephali (Rickettsiales: Rickettsiaceae) with htrA, while formed a separate clade with rrs. The pathogenicity of Rickettsia sp. sw should be further confirmed. These results expand the knowledge of the geographical distribution and vector distribution of spotted fever group rickettsiae in China and are useful for assessing the potential public health risk.

Rickettsia and Anaplasma are bacteria that can be transmitted by hematophagous arthropods such as ticks infesting animals in close proximity to humans. The main objective of the present study was to investigate abundance of common tick species infesting domestic animals and presence of Rickettsia and Anaplasma in tick populations. Adult ticks were collected from domestic animals in rural areas and screened by molecular detection of bacterial DNA for these two genera of bacteria. A total of 1,778 adult ixodid tick specimens were collected from 200 cattle, 200 buffaloes, 200 goats, and 40 dogs. The collection consisted of four species of ixodid ticks, Rhipicephalus (Boophilus) microplus (Canestrini) (83.8%), Hyalomma kumari (Sharif) (7.1%), Rhipicephalus sanguineus (Latreille) (6.4%), and Dermacentor auratus (Supino) (2.7%) infesting the domestic animals. The prevalence of all the collected tick species was highest in the month of October. Anaplasma spp. was the most frequently identified bacteria (3.3%) in tested ticks. Of 17 positive tick pools for Anaplasma spp., 14 pools were from ticks infesting cattle, 2 pools of ticks collected from buffalo, and the remaining pool were ticks infesting a goat at the time of collection. Although 1.6% tick pools of R. microplus collected from cattle tested positive for Rickettsia spp., present investigation provides evidence of the most prevalent ixodid ticks infesting domestic animals and the presence of obligate intracellular bacteria, Rickettsia and Anaplasma, in these ticks collected in the Gorakhpur division of Northern India.

In a follow-up to the investigations of soft ticks identified from seabird nest soil and litter collected from coastal islands of the Republic of Korea (ROK), Ornithodoros sawaii and Ornithodoros capensis were assessed for the presence and identification of rickettsiae. Ticks collected from samples of 50–100 g of nest litter and soil from seabird nests were identified individually by morphological techniques, and species confirmed by sequencing of the mt-rrs gene. Subsequently, tick DNA preparations were screened for the presence of rickettsiae using a genus-specific nested PCR (nPCR) assay targeting the 17 kDa antigen gene. The amplicons from the 17 kDa assay and two additional nPCR assays targeting the gltA and ompB gene fragments were sequenced and used to identify the rickettsiae. A total of 134 soft ticks belonging to two species, O. sawaiiKitaoka & Suzuki 1973 (n = 125) and O. capensis Neumann 1901 (n = 9), were collected. Rickettsia lusitaniae DNA was detected and identified among O. sawaii ticks (n = 11, 8.8%) collected from nest litter and soil of the Japanese murrelet (Synthliboramphus wumizusume Temminck 1836) at Gugul Island along the western coastal area of the ROK. This study confirmed for the first time the presence of R. lusitaniae associated with O. sawaii collected from migratory seabird nests in the ROK.

We evaluated the potential for mosquitoes collected in the Amazon Basin, near Iquitos, Peru, to become infected with and transmit Murutucu (MURV) and Itaqui viruses (ITQV) (Order Bunyavirales, Family: Peribunyaviridae, Genus: Orthobunyavirus). Viremia levels in Syrian hamsters peaked 2 d after infection with either virus, and both viruses were highly lethal in hamsters with virtually all hamsters dying prior to 3-d postinfection. For almost all of the mosquito species tested some individuals were susceptible to infection and some developed a disseminated infection after oral exposure to either MURV or ITQV. However, only the Culex species (Culex (Culex) coronator Dyar and Knab [Diptera, Culicidae], Culex (Melanoconian) gnomatos Sallum, Huchings, and Ferreira [Diptera, Culicidae], Culex (Mel.) pedroi Sirivanakarn and Belkin [Diptera, Culicidae], and Culex (Mel.) vomerifer Komp [Diptera, Culicidae]) successfully transmitted virus by bite. However, even among these species, only about 37% of the individuals with a disseminated infection successfully transmitted these viruses, indicating a significant salivary gland barrier. Although little is known about the medical or veterinary importance of many members of the genus Orthobunyavirus, we have demonstrated that Culex spp. (Diptera, Culicidae) could be potential vectors.

Microfilariae (MF) are the immature stages of filarial nematode parasites and inhabit the blood and dermis of all classes of vertebrates, except fish. Concurrent ingestion of MF and arboviruses by mosquitoes can enhance mosquito transmission of virus compared to when virus is ingested alone. Shortly after being ingested, MF penetrate the mosquito's midgut and may introduce virus into the mosquito's hemocoel, creating a disseminated viral infection much sooner than normal. This phenomenon is known as microfilarial enhancement. Both American Robins and Common Grackles harbor MF—that is, Eufilaria sp. and Chandlerella quiscali von Linstow (Spirurida: Onchocercidae), respectively. We compared infection and dissemination rates in Culex pipiens L. mosquitoes that fed on birds with and without MF infections that had been infected with West Nile virus (WNV). At moderate viremias, about 10⁷ plaque-forming units (pfu)/ml of blood, there were no differences in infection or dissemination rates among mosquitoes that ingested viremic blood from a bird with or without microfilaremia. At high viremias, >10^8.5 pfu/ml, mosquitoes feeding on a microfilaremic Grackle with concurrent viremia had significantly higher infection and dissemination rates than mosquitoes fed on viremic Grackles without microfilaremia. Microfilarial enhancement depends on the specific virus, MF, and mosquito species examined. How virus is introduced into the hemocoel by MF differs between the avian/WNV systems described here (i.e., leakage) and various arboviruses with MF of the human filarid, Brugia malayi (Brug) (Spirurida: Onchocercidae) (i.e., cotransport). Additional studies are needed to determine if other avian species and their MF are involved in the microfilarial enhancement of WNV in nature.

The Musca domestica salivary gland hypertrophy virus (MdSGHV) substantially enlarges the house fly's salivary glands and prevents or delays ovarian development in its adult host, but the effect that MdSGHV has on the house fly's food consumption is currently unknown. Using house flies from a laboratory-reared colony, we evaluated the effect of MdSGHV infection on food consumption over a 7-d period. Both treatment (virus-infected) and control (saline-injected) flies were provided with a choice of 8% sucrose solution and 4% powdered milk solution to determine food preferences. Quantities of each solution consumed were measured every 24 h for each fly to measure food consumptions. Infected house flies were shown to consume less overall of both solutions than house flies injected with saline. The largest consumption discrepancy was seen between female house flies. Healthy female flies with developing ovaries continued to consume a sugar and protein diet, whereas infected female flies fed predominantly on a sugar diet. Additionally, infected male and female flies consumed significantly lower quantities of protein and sucrose than control flies. This suggests that MdSGHV has a negative consumption effect (e.g., hunger, starvation) on its host. Thus, differences in food consumption of infected and control flies probably represent differences in the nutritional requirements of flies resulting from viral infection.

Recurrence of local transmission of Zika virus in Puerto Rico is a major public health risk to the United States, where mosquitoes Aedes aegypti (Linnaeus) and Aedes mediovittatus (Coquillett) are abundant. To determine the extent to which Ae. mediovittatus are capable of transmitting Zika virus and the influence of viremia, we evaluated infection and transmission in Ae. mediovittatus and Ae. aegypti from Puerto Rico using serial dilutions of infectious blood. Higher doses of infectious blood resulted in greater infection rates in both mosquitoes. Aedes aegypti females were up to twice as susceptible to infection than Ae. mediovittatus, indicating a more effective midgut infection barrier in the latter mosquito species. Aedes aegypti exhibited higher disseminated infection (40–95%) than Ae. mediovittatus (<5%), suggesting a substantial midgut escape barrier in Ae. mediovittatus. For Ae. aegypti, transmission rates were low over a range of doses of Zika virus ingested, suggesting substantial salivary gland barriers.

The dengue viruses (DENVs) occur throughout tropical and subtropical regions of the world where they infect 100s of millions of people annually. In Australia, the dengue receptive zone is confined to the northern state of Queensland where the principal vector Aedes aegypti (L.) is present. In the current study, two populations of Ae. aegypti from north Queensland were exposed to two urban outbreak strains and one sylvatic strain of dengue virus type 2 (DENV-2). The titer of virus required to infect 50% of mosquitoes was between 10⁵ and 10⁶ 50% tissue culture infectious dose (TCID)₅₀/ml and was influenced by the combination of the origin of Ae. aegypti population and virus strain. When exposed to infectious bloodmeal titers > 10⁶TCID₅₀/ml, infection and dissemination rates were all > 50% and were significantly affected by the origin of the mosquito population but not by the strain of DENV-2. Replication of DENV-2 was also significantly affected by the mosquito population and the titer of the infectious bloodmeal that mosquitoes were exposed to. The results of this study are discussed in the context of DENV transmission dynamics in northern Australia and the relative fitness of the sylvatic virus strain in urban Ae. aegypti populations.

The Asian longhorned tick, Haemaphysalis longicornis Neumann (Acari: Ixodidae), was recently introduced into the United States and is now established in at least 15 states. Considering its ability for parthenogenetic propagation and propensity for creating high-density populations, there is concern that this tick may become involved in transmission cycles of endemic tick-borne human pathogens. Human granulocytic anaplasmosis (HGA) caused by Anaplasma phagocytophilum is one of the more common tick-borne diseases in the United States, especially in the northeastern and midwestern states. There is considerable geographical overlap between HGA cases and the currently known distribution of H. longicornis, which creates a potential for this tick to encounter A. phagocytophilum while feeding on naturally infected vertebrate hosts.Therefore, we evaluated the ability of H. longicornis to acquire and transmit the agent of HGA under laboratory conditions and compared it to the vector competence of I. scapularis. Haemaphysalis longicornis nymphs acquired the pathogen with the bloodmeal while feeding on infected domestic goats, but transstadial transmission was inefficient and PCR-positive adult ticks were unable to transmit the pathogen to naïve goats. Results of this study indicate that the Asian longhorned tick is not likely to play a significant role in the epidemiology of HGA in the United States.

The temperate United States has experienced increasing incidence of mosquito-borne diseases. Recent studies conducted in Baltimore, MD have demonstrated a negative relationship between abundances of Aedes albopictus (Skuse) and Culex mosquitoes and mean neighborhood income level, but have not looked at the presence of pathogens. Mosquitoes collected from five socioeconomically variable neighborhoods were tested for infection by West Nile, chikungunya, and Zika viruses in 2015 and 2016, and again from four of the neighborhoods in 2017. Minimum infection rates of pooled samples were compared among neighborhoods for each year, as well as among individual blocks in 2017. West Nile virus was detected in both Ae. albopictus and Culex pools from all neighborhoods sampled in 2015 and 2017. No infected pools were detected in any year for chikungunya or Zika viruses, and none of the target viruses were detected in 2016. Infection rates were consistently higher for Culex than for Ae. albopictus. Minimum infection rate was negatively associated with mean neighborhood income for both species in 2015. Although earlier work has shown a positive association between block-level abandonment and mosquito abundance, no association was detected in this study. Still, we demonstrate that viral infection in mosquito pools can differ substantially across adjacent urban neighborhoods that vary by income. Though trap security and accessibility often inform city sampling locations, detecting and managing arboviral risk requires surveillance across neighborhoods that vary in socioeconomics, including lower income areas that may be less accessible and secure but have higher infection rates.

Dirofilaria immitis, the causative agent of dog heartworm disease, is an important cause of canine morbidity and mortality, expensive to treat, and severe infections are often fatal. Much is known about the pathogen in the canine host, yet little is known on the basic ecology of the nematode in the mosquito vector.Thus, to evaluate the effectiveness of collection techniques on ability to capture dog heartworm-infected mosquitoes (Diptera Culicidae), we conducted a field study spanning 111 wk. Four methods were used: two aspirators types, sweep netting, and a CDC trap. All sites had canines present in either residential yards (n = 4) or dog kennel facilities (n = 3). Collected mosquitoes were sorted by site, trap, species, and date, then pooled into groups of up to 25 individuals. Mosquito head and thorax pools were extracted for DNA, that was screened using currently available protocols. These protocols were found unreliable; thus, we developed a novel qPCR primer and probe set. Using this method, the original samples were re-assayed and provided 494 positive pools. Approximately 10% of positive samples were confirmed by Sanger sequencing.Twenty-two mosquito species tested positive for dog heartworm DNA, including a new association with Wyeomyia mitchellii (Theobald). Although Aedes atlanticus (Dyar and Knab), Anopheles crucians Wiedemann, and Culiseta melanura (Coquillett) composed nearly 36% of the total collection, these species represented 42% of the qPCR positive pools. Infection rates within commonly collected mosquitoes ranged up to 2.5%, with more rarely collected species ranging up to 14%.The CDC trap was the most effective collection method at trapping infected mosquitoes.

Since the recent introduction of the Asian longhorned tick (Haemaphysalis longicornis Neumann) in the United States, quantitative surveillance information remains lacking, which hinders accurate estimates of population structure and entomological risk. We conducted statewide, active tick surveillance from May to August 2019 and report data on H. longicornis geographical distribution and population density in Pennsylvania. In total, 615 H. longicornis were collected from four counties. Across samples recovering H. longicornis, mean density of H. longicornis was 9.2/100 m², comparably greater than Ixodes scapularis Say (8.5/100 m²). Density of H. longicornis was also significantly greater in August, largely driven by larvae, and greater in recreational habitat types (12.6/100 m²) and in Bucks County (11.7/100 m²), situated adjacent to the location of the first U.S. discovery of intense infestations. These data are among the first to document H. longicornis from statewide tick surveillance and provide initial measures of population density enabling more quantitative characterizations of distributional patterns.

There are many records of the family Sarcoptidae in bats. The species Notoedres (Notoedres) yunkeri has been reported only once, parasitizing a molossid bat in Panama. In the present study, we expand the occurrence of the species to Brazil.

Dengue virus infection, transmitted via mosquito bites, poses a substantial risk to global public health. Studies suggest that the mosquito's microbial community can profoundly influence vector-borne pathogen transmissions, including dengue virus. Ascogregarina culicis (Ross) of the phylum Apicomplexa is among the most common parasites of Aedes aegypti (Linnaeus), the principal vector of dengue. Despite a high prevalence worldwide, including in the areas where dengue is endemic, the impact of A. culicis on Ae. aegypti vector competence for dengue virus is unknown.This study aimed to investigate the effects of A. culicis infection on mosquito size and fitness, as measured by wing length, and the susceptibility to dengue virus infection in Ae. aegypti. Our results showed that there was no statistically significant difference in wing lengths between Ae. aegypti infected and not infected with A. culicis. Furthermore, A. culicis infection did not significantly affect dengue virus infection or disseminated infection rate. However, there was a significant association between shorter wings and higher dengue virus infection rate, whereby a 0.1-mm increase in wing length decreased the odds of the mosquito being infected by 32%.Thus, based on our result, A. culicis infection does not influence the body size and dengue virus infection in Ae. aegypti.This study helps to shed light on a common but neglected eukaryotic mosquito parasite.

The blacklegged tick, Ixodes scapularis Say, is the primary vector of several tick-borne pathogens, including those causing Lyme disease and babesiosis, in the eastern United States and active collection methods for this species include dragging or wild animal sampling. Nest boxes targeting mice may be an alternative strategy for the surveillance and collection of immature I. scapularis feeding on these hosts and would be much safer for animals compared to small mammal trapping. We constructed double-walled insulated nest boxes (DWINs) with collection tubes mounted below the nesting chamber and deployed eleven in southern Wisconsin from June until September of 2020. The DWINs were occupied by Peromyscus spp. and birds (wren species, Troglodytidae family). We collected 192 ticks from collection tubes, all of which were identified as either I. scapularis (95%) or Dermacentor variabilis Say (Acari: Ixodidae) (5%). Only 12% (21/182) and 20% (2/10) of I. scapularis and D. variabilis were blood-fed, respectively. The high proportion of unfed ticks found in collection tubes may be due to grooming by hosts inside the nest boxes. Alternatively, immature ticks may have climbed trees and entered the DWIN seeking a host. Results suggest that nest boxes could be a tool for finding ticks in areas of low density or at the leading edge of invasion, when small mammal trapping or drag sampling is not feasible.

The subgenus Culex L. includes species involved in summer–autumn arbovirus transmission but studies during winter are scarce in temperate Argentina. Female specimens were collected host-seeking at dry-ice-baited traps during autumn–winter–spring at two sites in Córdoba City during 2016 and 2017. The specimens were morphologically identified and dissected to determine the follicular developmental stage (gonotrophic activity). Females with advanced follicular stages (≥III) were subjected to molecular procedures to confirm or re-identify previous morphological identification. Five species (Culex apicinus Philippi (Diptera: Culicidae), Culex dolosus (Lynch-Arribálzaga) (Diptera: Culicidae), Culex maxi Dyar (Diptera: Culicidae), Culex pipiens pipiens L. (Diptera: Culicidae), and Culex quinquefasciatus Say (Diptera: Culicidae)) were collected and found gonoactive during winter; showing that a high proportion of Culex (Culex) females remain reproductively active during the unfavorable season for mosquito populations. Among them, it is worth noting the collection of Cx. quinquefasciatus, vector of the St. Louis encephalitis virus (endemic in the city), a specimen of Cx. p. pipiens, and a hybrid of Cx. p. pipiens/Cx. quinquefasciatus (during autumn).The study of this community during winter should continue because a high gonoactive female proportion with advanced follicular stages was found: 29.12 and 13.07% in 2016 and 2017, respectively. Local studies such as this one provide evidence about ornithophilic Culex species with active year-round life cycles, species that could favor arbovirus overwintering.

We identified an established population of the Gulf Coast tick (Amblyomma maculatum Koch) infected with Rickettsia parkeri in Connecticut, representing the northernmost range limit of this medically relevant tick species. Our finding highlights the importance of tick surveillance and public health challenges posed by geographic expansion of tick vectors and their pathogens.

Specific identification of oestrid larvae is usually problematic not only when using morphobiometric features, but also when applying molecular criteria, since very few molecular markers have been described for this group of flies. New molecular markers for oestrid are needed for more reliable species identification, diagnostic purposes, and epidemiological surveys; moreover, they can help in phylogenetic reconstruction. Here, we report the characterization of COI, 28S rDNA, ITS1, and ITS2 in Cephenemyia stimulator from roe deer and in Cephenemyia auribarbis and Pharyngomyia picta from red deer. The COI and 28S rDNA are very uniform in length, while the ITSs sequences are highly variable at both intraspecific and interspecific levels. The described ITSs sequences were longer than those described for other dipteran species by the presence of simple repeats and tandem repeat sequences. In C. auribarbis both ITS1 and ITS2 appeared as two variants, one short and the other long. In general, the analyzed markers present low intraspecific genetic variation and high interspecific variation. ITSs showed the greatest amount of intraspecific and interspecific variation. Phylogenetic analysis demonstrated that the characterized sequences differentiate the species and genera of Oestridae.

Investigations that analyze interspecific associations of vectors on their hosts are important for understanding community structure and implementing ways to comprehend mechanisms of pathogen transmission. We assessed the interspecific association of two tick species (Ixodes scapularis Say (Ixodida: Ixodidae) and Dermacentor variabilis Say (Ixodida: Ixodidae)) on the rodent host Peromyscus leucopus Rafinesque (Rodentia: Cricetidae) at the Hobart Ames Research and Education Center in southwesternTennessee. Of the rodents captured, 95 (63%) had neither species of tick, 6 (4%) had both tick species, 25 (16%) had I. scapularis only, and 26 (17%) had D. variabilis only. A coefficient of association (C₇ = –0.08) was calculated, which suggested there was competition between the two species of ectoparasites, but this value was not significant, indicating that there was a neutral relationship between the tick species on P. leucopus. The co-occurrence of both tick species on their host at the same time suggested that the two tick species can occupy the same host and use the same resources without competing.

Aminopeptidase N1 (APN) is one of the important enzymes involved in blood digestion and is up-regulated along with several other enzymes in response to bloodmeal ingestion. APN is a zinc metalloprotease that cleaves one amino acid residue at a time from the amino terminus of the protein. The APN1 gene of the Indian malaria vector Anopheles culicifacies Giles was cloned and characterized. The An. culicifacies APN1 (AcAPN1) gene has an Open Reading Frame of 3084 basepairs which encodes a putative protein of 1027 amino acids. The coding region of the gene shares 81% and 78% similarity to the APN1 genes found in An. stephensi (Diptera: Culicidae) and An. gambiae (Diptera: Culicidae), respectively. The organization of the APN1 gene was studied in available mosquito genomes and a three-dimensional structure of AcAPN1 modeled using homology structure modeling. The enzymatic active site was predicted to consist of HEYAH and GAMEN amino acid residues, and a comparison of the protein sequences among different genera revealed the conservation of zinc-binding residues. The expression pattern of AcAPN1 showed that the gene was expressed rapidly in response to the ingestion of the bloodmeal and therefore this gene may be used to exploit its promoter region as an antiparasite candidate molecule.