The medico-legal section of forensic entomology focuses on the analysis of insects associated with a corpse. Such insects are identified, and their life history characteristics are evaluated to provide information related to the corpse, such as postmortem interval and time of colonization. Forensically important insects are commonly identified using dichotomous keys, which rely on morphological characteristics. Morphological identifications can pose a challenge as local keys are not always available and can be difficult to use, especially when identifying juvenile stages. If a specimen is damaged, certain keys cannot be used for identification. In contrast, molecular identification can be a better instrument to identify forensically important insects, regardless of life stage or specimen completeness. Despite more than 20 yr since the first use of molecular data for the identification of forensic insects, there is little overlap in gene selection or phylogenetic methodology among studies, and this inconsistency reduces efficiency. Several methods such as genetic distance, reciprocal monophyly, or character-based methods have been implemented in forensic identification studies. It can be difficult to compare the results of studies that employ these different methods. Here we present a comprehensive review of the published results for the molecular identification of Diptera of forensic interest, with an emphasis on evaluating variation among studies in gene selection and phylogenetic methodology.

The common bed bug, Cimex lectularius (Linnaeus 1758), is a nocturnal blood-sucking ectoparasite of humans that is highly prevalent in the northeast of Iran. In recent years, the efficacy of those insecticides that have been frequently used to control bed bugs in Iran has not been studied. Due to frequent complaints about bed bug treatment failures in Mashhad city (northeastern Iran), this study assessed the susceptibility of C. lectularius collected from a student residence hall to Diazinon, Malathion, and λ-cyhalothrin. The desired concentrations of each insecticide were prepared in acetone, and bioassays were performed using insecticide-impregnated filter paper method. The concentration–response data were subjected to POLO-PC software and data were analyzed by the log-probit procedure. The LC50 values of Diazinon and λ-cyhalothrin for examined bed bugs were 1,337.40 and 2,022.36 ppm, respectively. Malathion at the highest concentration (10,000 ppm) did not exhibit any toxicity to examined C. lectularius. Comparing these results to the same previous studies showed that susceptibility of examined bed bugs to these insecticides has been highly decreased.This study revealed an occurrence of insecticide resistance in bed bug populations in northeastern Iran. It also suggests that Malathion, Diazinon, and λ-cyhalothrin are ineffective against bed bugs in this region.

Variation in the structure of phlebotomine (sand fly) communities in forest fragments with different degrees of preservation and human occupation (peridomicile) in eastern Amazonia was studied. We identified 43 species of sand flies in our study, of which 38 occurred in both preserved forest areas and in the peridomiciles of short-term settlements, while another 28 species occurred in altered forest fragments and long-term settlements. The composition of the community at each site changed with the type of environment (forest or peridomicile), with the species Lutzomyia evandroi, L. whitmani, L. choti, L. serrana, L. triacantha, L. migonei, L. hirsuta, L. shannoni, and L. brachyphylla accounting for more than 54% of the differences among environments. The quality of the environment exerted a significant influence on the structure of phlebotomine communities, and affected their species composition, richness, and abundance.

Genetic profiling has been used to link mosquito bloodmeals to the individual humans, but this analysis has not been done for other mammalian bloodmeals. In this study, we describe a microsatellite-based method for identifying individual pigs in mosquito bloodmeals based on their unique multilocus genotypes. Eleven tetranucleotide microsatellites and a sex-specific marker were selected based on Smith-Waterman DNA sequence alignment scores from the reference genome and primers were designed with features that reduce primer dimers, promote complete adenylation, and enable fluorescent labeling of amplicons. A multiplex polymerase chain reaction (PCR) assay was optimized and validated by analyzing DNA of individual pigs from several nuclear families and breeds before it was used to analyze genomic DNA of pig-derived mosquito bloodmeals from villages of Papua New Guinea. Population analysis of the nuclear families showed high expected and observed heterozygosity. The probability of observing two unrelated or sibling individuals sharing the same genotype at a single microsatellite locus or a combination of loci was vanishingly low. Samples had unique genotypes and gender was accurately predicted. Analysis of 129 pig bloodmeals identified 19 unique genotypes, which varied greatly in frequency in the mosquito bloodmeal samples. The high allelic diversity of the microsatellite loci and low probability of false attribution of identity show that this genotyping method reliably distinguishes distantly and closely related pigs and can be used to identify individual pigs from genotyped mosquito bloodmeals.

Determining the age of an insect collected in a corpse is a key element to estimate the minimum postmortem interval in forensic entomology. Along with models of accumulated degree-hours, the estimation of the age based on larval weight and length is among the most common methods for this purpose. Sarconesia chlorogaster (Wiedemann, 1830), along with other Calliphoridae, is an important species in forensics in South America. However, studies analyzing the weight and length of S. chlorogaster larvae during development have not yet been done. In this work, we develop growth models based on the weight and length of larvae in different temperatures and provide formulas that can be used to estimate the age of the larvae based on the weight and length for each temperature. Larvae were reared at six temperatures (10, 15, 20, 25, 30, 35°C), and the measurements of larval length and weight were taken during all development until they stopped feeding. For the measurements, we attempted to use methodologies that can be easily replicated and do not require equipment that could be of difficult access. The results of this work come to complement and improve the use of S. chlorogaster in forensic applications.

The decomposition rate and insects attraction to carcasses are affected by a number of factors such as temperature, body mass, the presence of poisons, humidity, and insect activity. The present study was planned to investigate the influence of clothing on both the decomposition process and the presence of insects on rabbit carcasses. The study was performed in May 2016 and involved the use of 18 live mature rabbits (Oryctolagus cuniculus L.), which were euthanized by chloroform. Rabbit carcasses were divided into three replicates with six rabbits in each replicate. Rabbits in each replicate were further subdivided, so that three rabbits were wrapped with cloth, whereas the remaining three were left unclothed. Both clothed and unclothed carcasses reached the dry stage within 6 d. The results of this study showed that clothes neither affect the decomposition process nor the presence of insects (flies and beetles). The pattern of flies and beetles, however, did vary significantly in the different decomposition stages. In total, 2,849 insects, including 2,459 flies and 390 beetles, were collected and sorted during the decomposition process. Among the dominant species, flies were assigned to the families Calliphoridae and Muscidae, whereas beetles were assigned to the families Dermestidae and Histeridae. Lucilia sericata Meigen, Musca domestica L., Dermestes maculatus DeGeer, and Saprinus moyses Marseul were the most abundant species occurring on both the clothed and unclothed carcasses. In conclusion, this study emphasized that, the clothes have no effect when dealing with decay process and insect attraction to the carrions.

Blow fly (Diptera: Calliphoridae) larvae are commonly used in forensic cases to determine postmortem intervals using development rates and successional changes in community composition. Studies are conducted from different regions to provide these data. We wanted to know how widely applicable these data are. We examined whether urbanized landscapes have distinct urban blow fly communities or whether the community composition in urbanized areas is simply a variation of that found in the surrounding habitat or ecozone. Using liver baited traps, we sampled 7,272 flies from 32 sites across Canada and used mapping analysis to assess urban and rural landcover classifications, and compared urban and rural species abundance and composition. Blow fly species communities from urban areas across Canada were made up of similar species and differed from the communities found in nearby rural sites. Trapping at rural sites caught more blow flies compared with urban sites (mean flies/site 59.5 and 12.4). Of the 14 species caught, 8 were caught at urban sites, 61% of these being Cynomya cadaverina Robineau-Desvoidy, 14% Phormia regina Meigen, and 11% Lucilia sericata (Meigen). In rural sites, all 14 species were caught, 41% of specimens caught were P. regina, 21% C. cadaverina, 10% Calliphora vomitoria (Linnaeus), with only 4% L. sericata. These data suggest that regional studies are appropriate for forensic entomology applications in urban landscapes, given the similar trends across Canada, less so for wilderness or rural landscapes.

Transgenic Anopheles gambiae Giles (Diptera: Culicidae) mosquitoes have been developed that confer sexual sterility on males that carry a transgene encoding a protein which cuts ribosomal DNA. A relevant risk concern with transgenic mosquitoes is that their capacity to transmit known pathogens could be greater than the unmodified form. In this study, the ability to develop two human pathogens in these transgenic mosquitoes carrying a homing endonuclease which is expressed in the testes was compared with its nontransgenic siblings. Infections were performed with Plasmodium falciparum (Welch) and o'nyong-nyong virus (ONNV) and the results between the transgenic and nontransgenic sibling females were compared. There was no difference observed with ONNV isolate SG650 in intrathoracic infections or the 50% oral infectious dose measured at 14 d postinfection or in mean body titers. Some significant differences were observed for leg titers at the medium and highest doses for those individuals in which virus titer could be detected. No consistent difference was observed between the transgenic and nontransgenic comparator females in their ability to develop P. falciparum NF54 strain parasites. This particular transgene caused no significant effect in the ability of mosquitoes to become infected by these two pathogens in this genetic background. These results are discussed in the context of risk to human health if these transgenic individuals were present in the environment.

The surge in tropical bed bug Cimex hemipterus (Fabricius) (Hemiptera: Cimicidae) infestations has led to an increase in genomic studies. In this study, the population genetics and breeding patterns of 22 Malaysian populations were analyzed, including genetic differentiation and genetic distance. For seven microsatellite loci, the number of alleles varied from 6 to 14.The allelels per loci contrasted sharply between the overall population and within the populations. The average observed and expected heterozygosity was 0.280 and 0.828 for the overall population and 0.281 and 0.657 among the populations, respectively. Based on polymorphic information criteria, the markers with a value >0.5 were highly polymorphic. In the Hardy–Weinberg equilibrium, the loci of Ch 09ttn, Ch 01dn, and Ch 13dn of the overall population showed signs of a null allele. The stutter peaks caused no scoring errors; large allele dropouts were not detected for any loci; and a correlation imbalance was not indicated. The genetic differentiation among populations was moderate, with a coefficient of genetic differentiation (F_ST) of 0.144.The bed bug populations showed strong inbreeding, with highly positive coefficients of inbreeding (F_IS). The molecular variation attributed to inbreeding was 83% within the populations, compared with 17% among the populations.The admixture individuals in STRUCTURE and neighbor-joining phylogenetic trees also indicated weak genetic structure in the geographical populations, suggesting moderate gene flows between populations. Thus, moderately active dispersion and human-mediated transport shaped the genetic structure of C. hemipterus populations in Malaysia.

Resistance to pyrethroid insecticides is widespread in Indonesian Aedes aegypti (Linnaeus), the primary vector of dengue viruses. This study aims to investigate the mutations in the voltage-gated sodium channel (Vgsc) conferring pyrethroid resistance against Ae. aegypti populations from Indonesia. Molecular genotyping of mutations using polymerase chain reaction assay and direct DNA sequencing were performed at positions 989 and 1,016 in IIS6 region, and 1,534 in IIIS6 region of the voltage-gated sodium channel (Vgsc) in nine populations of Indonesian Ae. aegypti. The V1016G and S989P genotyping identified the RR genotype to be predominant in six out of nine populations of Ae. aegypti, whereas the SS genotype occurred only in minority. Interestingly, co-occurrence of the V1016G and S989P mutations was detected in the aforementioned six populations with high frequency. Genotyping of F1534C showed all nine populations exhibited the SS genotype, with merely two individuals from a population were heterozygous (RS). Significant correlations were demonstrated between the allele frequencies of the V1016G mutation and the survivability rates as well as resistance ratios in pyrethroid adult bioassays. This signifies the V1016G can contribute more to the insensitivity of Vgsc than the F1534C. Homozygous 1016G mosquitoes were likelier to survive pyrethroid exposure. Identification of underlying mechanisms resulting in insecticide resistance is advantageous in developing effective mosquito control programs in Indonesia.

Spermatozoa are among the most diverse cell types, and their morphologies often provide data that can be used to reliably evaluate phylogenetic relationships. They can also help to clarify the nature of ‘specific complexes’, which are common among triatomines. In the present study, we evaluated the copulation behavior of Triatoma rubrovaria Blanchard 1843 (Hemiptera: Reduviidae: Triatominae) and the structural morphology of sperm from T. carcavalloi Jurberg Rocha & Lent, 1998, T. infestans Klug, 1834, T. pintodiasiJurberg Cunha & Rocha, 2013, and T. rubrovaria. Copulatory behavior was described from the moment males and females genitalia joined until they separated. Insemination was confirmed by the presence of a spermatophore in the female's bursa copulatrix. To measure their sperm, males were dissected and their seminal vesicles were removed, squashed on glass slides, and then spread, fixed, and observed under a photomicroscope. The images obtained were analyzed to measure the sperm. Seminal vesicles were also prepared for transmission electron microscopy. We performed K-means clustering separately for each species to group their sperm based on morphology. The differences in spermatozoa length among species of Triatominae, sperm types, and the interaction between species and sperm type were assessed with a two-way analysis of variance (ANOVA). The copulation time in T. rubrovaria was 3 to 5 min, which was sufficiently long for spermatophore transfer. All taxa showed polymorphic (short and long) sperm, with significant differences in the lengths of sperm among taxa. Using electron microscopy, the sperm cells of the four taxa examined were found to have similar ultrastructural morphology, confirming the hypothesized synapomorphies of sperm within the suborder Heteroptera (Hemiptera).

Many morphologically similar species of the simuliid (Diptera: Simuliidae) subgenus Wilhelmia, Enderlein are difficult to distinguish. Thus, the revision of the subgenus using various morphological, cytogenetic, and genetic analyses has been attempted. Neglected until now, the Balkan Peninsula, a crossroad between Europe and Anatolia, provides insight which could resolve problematic interrelationships of the taxa within this subgenus. To uncover the status and relations within the subgenus Wilhelmia, mtDNA was extracted from 47 individuals of six morphospecies: Simulium balcanicum (Enderlein, 1924), Simulium turgaicum Rubtsov, 1940, Simulium lineatum (Meigen, 1804), Simulium pseudequinum Séguy, 1921, Simulium equinum (Linnaeus, 1758), and Simulium paraequinum Puri, 1933 from 21 sites throughout the Balkan Peninsula. Phylogenetic analysis of the Wilhelmia species using mitochondrial DNA barcoding (COI) gene showed two major branches, the lineatum branch, which includes the lineages sergenti, paraequinum, and lineatum, and the equinum branch. In the equinum branch, the mtDNA sequences formed six clades, with high genetic distances, suggesting the existence of different species. Historically, the clades of the equinum branch appeared at numerous islands, perhaps as a result of allopatric speciation. The paraequinum lineage (lineatum branch) is composed of two species. However, six clades of the lineatum lineage overlapped with intra- and interspecific genetic distances. Our results revealed that the species S. balcanicum, S. pseudequinum B, and S. equinum were omnipresent in the Balkans. The results point to not only the fair diversity of Wilhelmia species in the Balkans, but also indicate that most Wilhelmia species live in sympatry.

Dermacentor parumapertus Neumann (Acari: Ixodidae) is a rather obscure tick found on jackrabbits in the western United States and parts of Canada and Mexico. Specimens from the northern part of their range are consistently different morphologically from ones found in southern and eastern parts of their range (particularly west Texas), leading some researchers to declare the southern form a variety or subspecies. This study examined field-collected adult D. parumapertus from two main locations—Utah and Texas—within its geographic distribution to ascertain the degree of genetic divergence in the two populations based upon both nuclear and mitochondrial DNA sequences. In total, 30 D. parumapertus were analyzed by PCR using both mtDNA and nDNA genes, and one D. nitens was included for comparison. Trees were constructed for all mtDNA genes individually, as well as after concatenating mtDNA (COI, COII, 12S) and nDNA (2 ITS2 primers), respectively. All constructed trees were exported to FigTree v1.4.3 and TreeGraph v2.14.1–771 beta for visualization. The majority of the Utah and Texas populations of D. parumapertus separated molecularly in both mtDNA and nDNA trees; however, analysis with mtDNA genes showed that 3/13 (23%) of Utah tick specimens were removed molecularly from other specimens collected at the same location. Thus, there was not enough evidence to declare these two disparate and morphologically different populations as distinct and separate species.

The heart is a pivotal organ in insects because it performs a number of different tasks, such as circulating nutrients, hormones, and excreta. In this study, the morphologies of the heart and associated tissues, including pericardial cells (PCs) and alary muscles (AMs), in the hematophagous mosquitoes Anopheles aquasalis Curry (Diptera: Culicidae), Aedes aegypti L. (Diptera: Culicidae), and Culex quinquefasciatus Say (Diptera: Culicidae), and the phytophagous Toxorhynchites theobaldi Dyar & Knab (Diptera: Culicidae) were compared using different microscopy techniques. Mosquito hearts are located across the median dorsal region of the whole abdomen. Paired incurrent openings in the heart wall (ostia) are found in the intersegmental regions (segments 2–7) of the abdomen, while an excurrent opening is located in the terminal cone of Ae. aegypti. The sides of the heart contain PC that are more numerous in An. aquasalis and Th. theobaldi. In these two species, PC form a cord of as closely aggregated cells, but in Ae. aegypti and Cx. quinquefasciatus, PC occur in pairs with two or four PC pairs per intersegmental region. In Th. theobaldi, AM binds to all regions of the heart, whereas in other mosquitoes they only bind in the intersegmental regions. The basic plan of the adult heart was conserved across all the adult mosquitoes investigated in this study. This conserved organization was expected because this organ plays an important role in the maintenance of individual homeostasis. However, the species had different PC and of AM morphologies. These morphological differences seem to be related to distinct physiological requirements of mosquito circulatory system.

Temperature is recognized as the most influential abiotic factor on the distribution and dispersion of most insect species including Rhodnius prolixus (Stål, 1859) and Triatoma infestans (Klug, 1834), the two most important Chagas disease vectors. Although, these species thermotolerance range is well known their plasticity has never been addressed in these or any other triatomines. Herein, we investigate the effects of acclimation on thermotolerance range and resistance to stressful low temperatures by assessing thermal critical limits and ‘chill-coma recovery time’ (CCRT), respectively. We found positive effects of acclimation on thermotolerance range, especially on the thermal critical minimum of both species. In contrast, CCRT did not respond to acclimation in either. Our results reveal the plasticity of these Triatomines thermal tolerance in response to a wide range of acclimation temperatures. This presumably represents a physiological adaptation to daily or seasonal temperature variation with concomitant improvement in dispersion potential.

The aim of this study was to investigate whether temperature, rainfall, and humidity influence the annual distribution of Culicoides Latreille 1809, species abundance and richness in rural areas on São Luís Island in the state of Maranhão, Brazil. Biting midges were collected in housing for domestic animals in the peridomestic areas of 10 homes. A CDC light trap was installed in each peridomestic area from 6:00 p.m. to 6:00 a.m. once a month for 12 mo. In total, 23,633 individuals belonging to 26 Culicoides species were captured. The most abundant species were Culicoides diabolicus Hoffman, 1925 (27.43%), C. ignacioi Forattini, 1957 (25.9%), C. flavivenulus Costa Lima, 1937 (15.53%), C. insignis Lutz, 1913 (10.66%), C. filariferus Hoffman, 1939 (6.21%), C. boliviensis Spinelli & Wirth, 1984 (6.07%), C. foxi Ortíz, 1950 (2.83%), and C. leopoldoi Ortíz, 1951 (2.54%). Species richness and abundance were greater during the rainy season (24 species; 88% of the individuals) than during the dry season (18 species; 12% of individuals). However, persistent rain on the days the midges were collected or in the preceding 24 h adversely affected abundance. Mean monthly maximum temperature and rainfall on the day of the collection adversely affected Culicoides abundance and richness.

Rhodnius prolixus Stål, a major Chagas disease vector, often colonizes in houses, whereas its sister species, Rhodnius robustus Larrousse genotype I, does not colonize in houses and has little medical relevance. Factors potentially underlying this crucial difference remain largely uncharted. The ‘microclimate-adaptation hypothesis' notes that R. prolixus is adapted to the dry microclimate of small-crowned Copernicia palms, whereas R. robustus I exploits the high-moisture microclimate of large-crowned Attalea and Acrocomia. Hence, R. prolixus, but not R. robustus I, would be (pre)adapted to the relatively dry microclimate typical of man-made habitats. This hypothesis predicts that, while severe dehydration should harm both species similarly, R. prolixus should withstand moderate-to-mild dehydration stress better than R. robustus I. To test this prediction, we compared fitness metrics of genotyped R. prolixus and R. robustus I kept at 28°C and under severe (20% relative humidity, RH), moderate (40% RH), or mild dehydration stress (75% RH). Egg-hatching success increased with decreasing dehydration stress in R. robustus I (0% → 19% → 100%), but was high across treatments in R. prolixus (78% → 100% → 100%). Both species underwent high, early mortality under severe dehydration; under moderate and mild stress, R. prolixus experienced less mortality and survived longer than R. robustus I. Our results suggest that adaptation to distinct palm-crown microclimates may partly underlie the so far unexplained differences in house-colonization ability among Rhodnius Stål species. Experimental replication across additional species/populations will be required to further probe this adaptive hypothesis—which, if supported, may also provide insight into the likely responses of Chagas disease vectors to climate change.

Hard ticks are among the most important blood sucking arthropods that transmit pathogens to humans and animals. This study was designed to determine prevalence, mapping, geographical distribution, and seasonal activity of hard tick species infesting the most common domestic and wild mammals in various districts of Riyadh Province, Saudi Arabia, during the period January to December 2017. In total, 10,832 adult hard ticks were collected from the bodies of 8,435 animals belonging to 18 different mammalian species. The ticks were preserved in 70% alcohol and microscopy was used to identify species. Two genera, Hyalomma and Rhipicephalus, were identified, comprising 10 species of hard ticks, with Hyalomma comprising 68.3% and Rhipicephalus comprising 31.7% of species. The most common species on domestic mammalian hosts was Hyalomma dromedarii (Koch 1844) (39.9%) followed by Rhipicephalus turanicus (Pomerantsev, Matikashvili & Lotosky 1936) (34.9%), whereas on wild mammalian hosts Rhipicephalus sanguineus (Latreille 1806) was by far the most prevalent species (83.0%). However, ticks were most abundant during May through July (36.0%) in the studied areas, and tick intensity and abundance differed among seasons. Our results provide information for human and animal health service managers, as well as governmental authorities, to gain a better understanding of hard ticks infesting mammalian hosts in Riyadh Province, Saudi Arabia, which can help improve prevention and control of tick-borne diseases, especially during outbreaks.

This investigation was initiated to control Aedes aegypti and Zika virus transmission in Caguas City, Puerto Rico, during the 2016 epidemic using Integrated Vector Management (IVM), which included community awareness and education, source reduction, larviciding, and mass-trapping with autocidal gravid ovitraps (AGO). The epidemic peaked in August to October 2016 and waned after April 2017. There was a preintervention period in October/November 2016 and IVM lasted until August 2017. The area under treatment (23.1 km²) had 61,511 inhabitants and 25,363 buildings. The city was divided into eight even clusters and treated following a cluster randomized stepped-wedge design. We analyzed pools of female Ae. aegypti adults for RNA detection of dengue (DENV), chikungunya (CHIKV), and Zika (ZIKV) viruses using 360 surveillance AGO traps every week. Rainfall, temperature, and relative humidity were monitored in each cluster. Mosquito density significantly changed (generalized linear mixed model; F_{8, 14,588} = 296; P < 0.001) from 8.0 ± 0.1 females per trap per week before the intervention to 2.1 ± 0.04 after the percentage of buildings treated with traps was 60% and to 1.4 ± 0.04 when coverage was above 80%. Out of a total 12,081 mosquito pools, there were 1 DENV-, 7 CHIKV-, and 49 ZIKV-positive pools from October 2016 to March 2017. Afterward, we found only one positive pool of DENV in July 2017. This investigation demonstrated that it was possible to scale up effective Ae. aegypti control to a medium-size city through IVM that included mass trapping of gravid Ae. aegypti females.

The peridomestic anthropophilic Aedes aegypti L. (Diptera: Culicidae) is originated from the wild zoophilic subspecies Aedes aegypti formosus in sub-Saharan Africa, and currently has a broad distribution in human-modified environments of the tropics and subtropics worldwide. In California, breeding populations were initially detected in 2013 in the cities of Fresno, Madera, and San Mateo, and now can be found in 188 cities of 12 counties in the state. Recent genetic studies suggest that this species invaded California on multiple occasions from several regions of the United States and northern Mexico prior to initial detection. As an invasive species and vector for numerous arboviruses, Ae. aegypti is a primary target of surveillance and control in California. In southern California city of Montclair, a population was identified in September 2015, from which a short-term colony was established in an insectary. The susceptibility of this field population to commonly used pesticides with various modes of action, including 15 formulations against larvae and four against adults, was determined, in reference to a susceptible laboratory colony of the same species. No resistance was shown to most pesticides tested. However, tolerance or reduced susceptibility to spinosad, spinetoram, diflubezuron, and fipronil was detected, and modest levels of resistance to pyriproxyfen (resistance ratio = 38.7-fold at IE₅₀ and 81.5-fold at IE₉₀) was observed. Results are discussed based on the field usage and modes of action of the pesticides tested. Strategic selection and application of pesticides against this population of Ae. aegypti in the urban environments should be taken into consideration.

The yellow fever mosquito, Aedes aegypti (Diptera: Culicidae) transmits several devastating arboviruses, including dengue, chikungunya, and Zika virus, making development of inexpensive and eco-friendly strategies for its control an urgent priority. We evaluated the lethality of 13 commonly used plant-derived edible oils against late-third instar Ae. aegypti and then tested the three most lethal oils for stage-specific differences in lethality. We also examined the effects of the most lethal (hempseed), moderately lethal (sunflower and peanut), and least lethal (olive) oils on survival to adulthood and oviposition behavior of gravid females. We hypothesized that the insecticidal activity of edible oils is a function of the content of their linoleic acid, a key fatty acid component with film-forming properties. Among the 13 oils tested, hempseed oil was the most lethal, with an LC₅₀ of 348.25 ppm, followed by sesame (670.44 ppm) and pumpkinseed (826.91 ppm) oils. Oils with higher linoleic acid content were more lethal to larvae than those with low linoleic acid content. Furthermore, pure concentrated linoleic acid was more lethal to larvae compared to any edible oil. In comparison to early instars, late instars were more susceptible to hempseed, sunflower, peanut, and olive oils; these oils also acted as oviposition deterrents, with effective repellency ≥63%.The proportion of larvae surviving to adulthood was significantly reduced in hempseed, sunflower, peanut, and olive oil treatments relative to controls. Our results suggest that some edible plant oils have potential as effective, eco-friendly larvicides, and oviposition deterrents for controlling container-dwelling mosquitoes, especially in resource-limited settings.

The horn fly, Haematobia irritans irritans (L.) (Diptera: Muscidae), is an important and cosmopolitan blood feeding ectoparasite of cattle. Resistance to conventional insecticides is increasingly problematic and alternative pesticides, including natural products, are being investigated. Limonene is a cyclic monoterpene repellent to some insects that occurs in citrus fruit rinds and in other plants. We assessed laboratory grade limonene and a commercial product, Orange Guard (5.8% AI limonene), against H. irritans irritans in terms of their contact effects upon contact on egg mortality, adults, and larval and pupal development; adult repellency as well as sublethal and fumigation effects. Egg viability declined when they were exposed to Orange Guard at concentrations of 1.45%, 2.9%, and 5.8% whereas laboratory grade limonene at 5.8% and 11.6% was ovicidal. Contact exposure of adult H. irritans irritans to 5.8% laboratory grade limonene and 2.9% Orange Guard caused up to 100 and 88% knockdown (immobilization), respectively. At higher concentrations, laboratory grade limonene and Orange Guard resulted in less, and often shorter periods of knockdown. Although direct contact of 2.9 and 5.8% laboratory grade limonene caused mortality it was negligible when flies were sprayed directly with undiluted Orange Guard. Female H. irritans irritans exposed to sublethal concentrations of Orange Guard did not reduce the numbers of eggs produced, but the undiluted product reduced egg hatchability. Interestingly, limonene and Orange Guard attracted adult H. irritans irritans at concentrations <0.1%. We suggest that the attractancy of unformulated pure limonene might be useful for trapping H. irritans irritans adults.

Sustaining high levels of indoor residual spraying (IRS) coverage (≥85%) for community protection against malaria remains a challenge for IRS campaigns. We examined biting rates and insecticide resistance in Culex species and Anopheles gambiae s.l., and their potential effect on community adherence to IRS. The average IRS coverage in urban Malabo between 2015 and 2017 remained at 80%. Culex biting rate increased 6.0-fold (P < 0.001) between 2014 and 2017, reaching 8.08 bites per person per night, whereas that of An. gambiae s.l. remained steady at around 0.68. Although An. gambiae s.l. was susceptible to carbamates and organophosphates insecticides, Culex spp. were phenotypically resistant to all four main classes of WHO-recommended IRS insecticides. Similarly, the residual activity of the organophosphate insecticide used since 2017, ACTELLIC 300CS, was 8 mo for An. gambiae s.l., but was almost absent against Culex for 2 mo post-spray. A survey conducted in 2018 within urban Malabo indicated that 77.0% of respondents related IRS as means of protection against mosquito bites, but only 3.2% knew that only Anopheles mosquitoes transmit malaria. Therefore, the increasing biting rates of culicines in urban Malabo, and their resistance to all IRS insecticides, is raising concern that a growing number of people may refuse to participate in IRS as result of its perceived failure in controlling mosquitoes. Although this is not yet the case on Bioko Island, communication strategies need refining to sensitize communities about the effectiveness of IRS in controlling malaria vectors in the midst of insecticide resistance in nonmalaria vector mosquitoes.

Wolbachia-infected Aedes aegypti (L.) mosquitoes for control of dengue transmission are being released experimentally in tropical regions of Australia, south-east Asia, and South America. To become established, the Wolbachia Hertig (Rickettsiales: Rickettsiaceae) strains used must induce expression of cytoplasmic incompatibility (CI) in matings between infected males and uninfected females so that infected females have a reproductive advantage, which will drive the infection through field populations. Wolbachia is a Rickettsia-like alphaproteobacterium which can be affected by tetracycline antibiotics. We investigated whether exposure of Wolbachia-infected mosquitoes to chlortetracycline at environmentally relevant levels during their aquatic development resulted in loss or reduction of infection in three strains, wAlbB, wMel, and wMelPop. Wolbachia density was reduced for all three strains at the tested chlortetracycline concentrations of 5 and 50 μg/liter. Two of the strains, wMel and wMelPop, showed a breakdown in CI. The wAlbB strain maintained CI and may be useful at breeding sites where tetracycline contamination has occurred. This may include drier regions where Ae. aegypti can utilize subterranean water sources and septic tanks as breeding sites.

Spatial repellents can reduce fecundity and interrupt oviposition behavior in Aedes aegypti. Yet, it is unclear if short exposure times, resistant phenotypes, and other aspects of spatial repellents can impact these effects on mosquito reproduction. To address these issues, pyrethroid susceptible, pyrethroid resistant, and field strains of Ae. aegypti were used to evaluate the extent to which fecundity and oviposition behavior are affected following metofluthrin exposure. Mosquitoes were exposed for 60 s to a sub-lethal dose (LC₃₀) of metofluthrin before blood feeding and allowed 72 h to become gravid before evaluation in an oviposition bioassay for an additional 72 h. Metofluthrin-exposed susceptible, field, and to a lesser extent resistant strain Ae. aegypti showed oviposition across fewer containers, less egg yield, less egg viability, and reduced larval survivorship in hatched eggs compared to unexposed cohorts. Susceptible mosquitoes retained some eggs at dissection following bioassays, and in one case, melanized eggs retained in the female. Treated resistant and field strain F₁ larvae hatched significantly earlier than unexposed cohorts and resulted in increased larval mortality in the first 3 d after oviposition. Upon laying, the treated field strain had incompletely melanized eggs mixed in with viable eggs. The treated field strain also had the lowest survivorship of larvae reared from bioassay eggs. These results indicate that metofluthrin could succeed in reducing mosquito populations via multiple mechanisms besides acute lethality. With the available safety data, pre-existing spatial repellent registration, and possibilities for other outdoor delivery methods, metofluthrin is a strong candidate for transition into broader mosquito abatement operations.

Host-targeted technologies provide an alternative to the use of conventional pesticide applications to reduce the abundance of Ixodes scapularis Say, the vector for an array of tick-associated human diseases. We compared the ability of Damminx Tick Tubes (Damminix) and SELECT Tick Control System (Select TCS) bait boxes to control host-seeking I. scapularis nymphs in a wooded residential environment. Small mammals accepted and used Select TCS bait boxes with greater frequency compared to Damminix tubes over the course of the 2-yr trial. Nymphal tick infestation prevalence and intensity on captured mice and chipmunks provided no conclusive evidence of a treatment effect during May–June of both years. However, both treatments had a measurable effect on larval tick burdens in July–August and the magnitude of the effect was greater at the Select TCS-treated area and reflected the fact that Select TCS effectively treated chipmunks, while Damminix did not. Deployment of Damminix resulted in 27.6 and 20.3% control of questing nymphs in treated areas at 1 yr and 2 yr postintervention, while Select TCS bait boxes provided 84.0 and 79.1% control, respectively. The economics of residential tick control using these products in wooded residential landscapes is discussed.

As the incidence of arboviral diseases such as dengue, Zika, chikungunya, and yellow fever increases globally, controlling their primary vector, Aedes aegypti (L.) (Diptera: Culicidae), is of greater importance than ever before. Mosquito control programs rely heavily on effective adult surveillance to ensure methodological efficacy. The Biogents Sentinel (BGS) trap is the gold standard for surveilling adult Aedes mosquitoes and is commonly deployed worldwide, including during modern ‘rear and release’ programs. Despite its extensive use, few studies have directly assessed environmental characteristics that affect BGS trap catches, let alone how these influences change during ‘rear and release’ programs. We assessed male and female Ae. aegypti spatial stability, as well as premises condition and trap location influences on BGS trap catches, as part of Debug Innisfail ‘rear and release’ program in northern Australia. We found similar trends in spatial stability of male and female mosquitoes at both weekly and monthly resolutions. From surveillance in locations where no males were released, reduced catches were found at premises that contained somewhat damaged houses and unscreened properties. In addition, when traps were located in areas that were unsheltered, more than 10 m from commonly used sitting areas or more visually complex catches were also negatively affected. In locations where males were released, we found that traps in treatment sites, relative to control sites, displayed increased catches in heavily shaded premises and were inconsistently influenced by differences in house sets and building materials. Such findings have valuable implications for a range of Ae. aegypti surveillance programs.

Aedes albopictus (Skuse) is a widespread invasive mosquito vector species with a distribution including tropical and temperate climates; its range is still expanding. Aedes albopictus populations were recently detected in Morocco and Algeria, the countries neighboring Tunisia, but never in Tunisia. In 2018, we initiated an intensive field study using BG-Sentinel Traps, ovitraps, larval surveys, and citizens' reports to determine whether Ae. albopictus populations exist in Tunisia. In October 2018, we collected adults and larval stages of Ae. albopictus in Carthage, Amilcar, and La Marsa, less than 20 km, northeast of Tunis, the Tunisian capital. These Ae. albopictus larvae were primarily collected from Phoenician funeral urns at the archeological site of Carthage. This is, to our knowledge, the first detection of Ae. albopictus in Tunisia.

Mosquito-borne diseases are a major public health concern globally and early detection of pathogens is critical to implement vector management and control strategies. Existing methods for pathogen detection include screening sentinel animals for antibodies and analyzing mosquitoes for pathogen presence. While these methods are effective, they are also expensive, labor-intense, and logistically challenging. To address these limitations, a new method was developed whereby mosquito saliva is collected on honey-coated nucleic acid preservation cards which are analyzed by molecular assays for detection of pathogens. However, mosquitoes only expel small amounts of saliva when feeding on these cards, potentially leading to false negatives. Another bodily fluid that is expelled by mosquitoes in larger volumes than saliva is excreta, and recent laboratory experiments have demonstrated that a range of mosquito-borne pathogens can be detected in mosquito excreta. In the current study, we have modified light and passive mosquito traps to collect their excreta and assessed their efficacy in field evaluations. From these field-collections, we detected West Nile, Ross River, and Murray Valley encephalitis viruses. Our findings suggest that mosquito traps are easily modified to collect excreta and, that this system has the potential to enhance detection of pathogens.

Questing ticks were surveyed by dragging in forested habitats within the Lehigh Valley region of eastern Pennsylvania for four consecutive summers (2015–2018). A high level of inter-annual variation was found in the density of blacklegged tick nymphs, Ixodes scapularis Say, with a high density of host-seeking nymphs (DON) in summer 2015 and 2017 and a relatively low DON in summer 2016 and 2018. Very few American dog ticks (Dermacentor variabilis Say) and Ixodes cookei Packard were collected. Lone star ticks (Amblyomma americanum L.) and longhorned ticks (Haemaphysalis longicornis Neumann) were not represented among the 6,398 ticks collected. For tick-borne pathogen surveillance, DNA samples from 1,721 I. scapularis nymphs were prepared from specimens collected in summers 2015–2017 and screened using qPCR, high resolution melting analysis, and DNA sequencing when necessary. The overall 3-yr nymphal infection prevalence of Borrelia burgdorferi was 24.8%, Borrelia miyamotoi was 0.3%, Anaplasma phagocytophilum variant-ha was 0.8%, and Babesia microti was 2.8%. Prevalence of coinfection with B. burgdorferi and B. microti as well as B. burgdorferi and A. phagocytophilum variant-ha were significantly higher than would be expected by independent infection. B. burgdorferi nymphal infection prevalence is similar to what other studies have found in the Hudson Valley region of New York, but levels of B. microti and A. phagocytophilum variant-ha nymphal infection prevalence are relatively lower. This study reinforces the urgent need for continued tick and pathogen surveillance in the Lehigh Valley region.

Arbovirus surveillance is crucial for the implementation of vector-borne disease control measures. Recently, it has been demonstrated that mosquitoes with a disseminated arbovirus infection excrete viral RNA, which can be detected by molecular methods. Thereby, mosquito excreta has been proposed as a sample type that could be utilized for arbovirus surveillance. In this study, we evaluated if West Nile virus (Kunjin strain, WNV_KUN) RNA in Culex annulirostris Skuse (Diptera: Culicidae) excreta deposited on different substrates could be detected after storage for up to 2 wk at tropical conditions of high heat and humidity. No significant drop in relative quantity of WNV_KUN RNA (determined by comparison of Ct values) in excreta deposited on Flinders Associate Technologies (FTA) cards was observed over 14 d, suggesting that RNA was stable for that time. There was no significant difference in relative quantity of WNV_KUN RNA in excreta deposited on FTA cards or polycarbonate substrates after 24 h. However, after 7 and 14 d, there was a significant decline in the relative quantity of viral RNA in the excreta stored on polycarbonate substrates. For incorporation in arbovirus surveillance programs, we recommend the use of polycarbonate substrates for excreta collection in mosquito traps deployed overnight, and the integration of FTA cards in traps serviced weekly or fortnightly. Polycarbonate substrates facilitate the collection of the majority of excreta from a trap, and while FTA cards offer limited area coverage, they enable preservation of viral RNA in tropical conditions for extended periods of time.

The seasonal abundance of Haemaphysalis longicornis Neumann and H. flava Neumann (Acari: Ixodidae) was monitored from 2015 through 2017 in Gyeonggi-do, South Korea. The hard ticks were surveyed monthly using CO₂ bait traps from April to November and flagging from July to September in a coniferous forest, a deciduous forest, shrubs, and a grassland vegetation types. Haemaphysalis longicornis and H. flava were most abundant in the grassland and shrubs, which yielded over 50% of the total number of ticks in all 3 yr. Furthermore, H. longicornis comprised up to 90% of the ticks collected. Generally, peaks of nymph, adult, and larva numbers were observed from April to June, from June to July, and from August to September, respectively. Half of the ticks were pooled and tested for the presence of the SFTS virus with negative results.

Anecdotal evidence of pyrethroid insecticide product failure for the control of stable fly [Stomoxys calcitrans (L.)] populations in the United States and worldwide prompted us to evaluate the frequency of knockdown resistance (kdr)-type polymorphisms within the voltage-sensitive sodium channel (Vssc) gene of field collected specimens from the United States, France, Costa Rica, and Thailand. The kdr-his allele (L1014H), associated with permethrin resistance, was detected in stable flies from the 10 states sampled in the United States, as well as from Costa Rica and France (Toulouse). Field collections of stable flies from California (Modesto) and New York (Cliffton Springs) exhibited reduced susceptibility upon exposure to a diagnostic permethrin concentration of 10× LC₉₉, but survival did not appear to strictly associate with frequency of the kdr-his allele. This suggests that there are additional resistance mechanisms contributing to the phenotype in these states. The kdr allele (L1014F) was detected for the first time in stable flies originating in France and Thailand, and an improved, DNA-based diagnostic assay was developed and validated for use in future screens for kdr and kdr-his allele frequencies from field collections. The absence of kdr in United States and Costa Rica populations suggests that the allele is currently restricted to Europe and Asia.

Hematophagous arthropod bloodmeal identification has remained a challenge in the field of vector biology, but these studies are important to understand blood feeding patterns of arthropods, spatial, and temporal patterns in arbovirus transmission cycles, and risk of human and veterinary disease. We investigated the use of an existing vertebrate primer set for use on the droplet digital polymerase chain reaction (ddPCR) platform, to explore the use of this technology in the identification and quantification of vertebrate DNA in mosquito blood meals. Host DNA was detectable 48-h post-engorgement in some mosquitoes by ddPCR, compared with 24-h post-engorgement using traditional PCR. The capability of ddPCR for absolute quantification of template DNA offers unique potential applications of this new technology to field studies on the ecology of vector-borne diseases, but currently with limited scope.

The Togolese Republic has a tropical and humid climate which constitutes an ideal environment for mosquitoes to breed and transmit diseases. The Aedes mosquito is known to transmit yellow fever (YF), dengue, chikungunya, and Zika viruses in West Africa. Togo has been suffering from YF virus transmission, despite vaccination efforts. Unfortunately, there is scarcity in the data that reflect mosquito spatial distribution in Togo, specifically possible YF vectors. In the current study, mosquito surveillance efforts targeted areas with confirmed YF cases between July and August 2012. Indoor mosquitoes were collected using knockdown insecticide spraying, whereas Biogents (BG) traps were used to collect outdoor mosquito adults. Mosquito larval surveillance was conducted as well. In total, 17 species were identified. This investigation revealed the presence of medically important vectors in Togo, especially the Aedes aegypti (Linnaeus) (Diptera: Culicidae) which was collected in the four regions. Screening of all pools of female Aedes mosquitoes for YF, by real-time PCR, showed negative results. This is the first record for Coquillettidia flavocincta (Edwards) (Diptera: Culicidae) species in West Africa. This preliminary work serves as a baseline for further mosquito distribution studies in Togo.

A survey of susceptibility to DDT, deltamethrin, bendiocarb, and chlorpyrifos-methyl was conducted in five localities in 2011 in Togo, West Africa, to assess the insecticide resistance status of Anopheles gambiae s.l. (Diptera: Culicidae). Female populations of An. gambaie s.l. emerged from collected larvae (F0) were exposed to insecticide-impregnated papers using World Health Organization test kits for adult mosquitoes; the susceptible reference strain Kisumu was used as a control. Resistance to DDT and deltamethrin was observed within the mosquito populations tested. Anopheles gambiae s.s. and Anopheles coluzzii represented the only species recorded in the study sites. The frequency of knockdown resistance (kdr L1014F) mutation determined using polymerase chain reaction diagnostic tests was lower in An. gambiae than in An. coluzzii in all of the localities except Kolokopé. Further investigations of An. gambiae s.l. resistance are needed in Togo to help the National Malaria Control Programme in vector control decision making and implementation of resistance management strategy.

Constructed wetlands are popular tools for managing threatened flora and fauna in urban settings, but there are concerns that these habitats may increase mosquito populations and mosquito-related public health risks. Understanding the interactions occurring between mosquitoes of public health concern and co-occurring organisms is critical to informing management of these habitats to mitigate potential health risks and balance the multiple values of urban wetlands. This study examined how oviposition behavior of Culex annulirostris Skuse, the most important pest mosquito species associated with freshwater wetland habitats in Australia, is influenced by the presence of Gambusia holbrooki Girard, a widespread invasive fish. Water was collected from urban wetlands that are intensively managed to reduce G. holbrooki populations to assist conservation of locally threatened frogs, and adjacent unmanaged wetlands where G. holbrooki was abundant. Laboratory experiments were conducted to examine the oviposition response by Cx. annulirostris to water samples from these two habitats. Experiments were conducted on two occasions, once in February following draining and refilling of the urban wetlands, and repeated following a substantial rainfall event in March. The results clearly demonstrate that ovipositing mosquitoes were able to detect and avoid water derived from habitats containing fish, even in the absence of the fish themselves. Understanding how invasive species affect the behavior and spatial distribution of pest species such as Cx. annulirostris will enable future wetland design and management to maximize benefits of urban wetlands and minimize potential public health risks.

Blood-feeding patterns of mosquitoes affect the transmission and maintenance of arboviral diseases. In the Caribbean, Aedes aegypti (L.) and Culex quinquefasciatus Say mosquitoes are the dominant mosquito species in developed areas. However, no information is available on the bloodmeal hosts of these invasive vectors in Grenada, where arboviral pathogens such as dengue, chikungunya, and Zika viruses cause significant human suffering. To this end, Ae. aegypti and Cx. quinquefasciatus mosquitoes were investigated from five semirural locations near houses in St. George's Parish, from 2017 to 2018. Polymerase chain reaction was conducted on DNA extracted from individual blood-fed mosquitoes using vertebrate-specific cytochrome b primers. The 32 Ae. aegypti bloodmeals included humans (70%), mongooses (18%), domestic dogs (6%), a domestic cat (3%), and an unidentified bird (3%).Thirty-seven Cx. quinquefasciatus mosquitoes took bloodmeals from seven species of birds (51%), humans (27%), domestic cats (8%), iguanas (5%), a domestic dog (3%), a rat (3%), and a common opossum (3%).The high percentage of human bloodmeal hosts in our study, especially by the normally anthropophilic Ae. aegypti, is expected. The bloodmeal sources and the percentage of nonhuman bloodmeals (30%) taken by Ae. aegypti are comparable to other studies. The large range of hosts may be explained in part by the semirural nature of most local housing. Accordingly, this may contribute to an exchange of pathogens between domestic, peridomestic, and sylvatic transmission cycles.