Adult female mosquitoes within the subgenus Culex (Melanoconion) Theobald (Diptera: Culicidae) are difficult to identify to species using external morphological features. We present two multiplexed polymerase chain reaction assays that quickly and accurately identify specimens from the southeastern United States based on sequence differences in the internal transcribed spacers of the ribosomal DNA gene array. One assay identifies all species that occur only in Florida, whereas the second assay identifies species that may occur in other southeastern states. These assays require small amounts of DNA, such as DNA from two sonicated legs, or an individual specimen. These assays also may be run as multiple singleplex reactions to determine the mosquito species composition of virus-positive mosquito pools. Reaction volumes may be as low as 10 µl, which reduces assay cost.

At temperate latitudes, vectors and pathogens must possess biological mechanisms for coping with cold temperatures and surviving from one transmission season to the next. Mosquitoes that overwinter in the adult stage have been proposed as winter maintenance hosts for certain arboviruses. In the cases of West Nile virus (family Flaviviridae, genus Flavivirus) and St. Louis encephalitis virus (family Flaviviridae, genus Flavivirus), discovery of infected overwintering females lends support to this hypothesis, but for other arboviruses, in particular Eastern equine encephalomyelitis virus (family Togaviridae, genus Alphavirus, EEEV), overwintering of the virus in mosquito hosts as not been demonstrated. In the current study, we collected overwintering mosquitoes from a focus of EEEV transmission in the southeastern United States to determine whether mosquitoes serve as winter maintenance hosts for EEEV and to document overwintering biologies of suspected vectors. No virus was detected via reverse transcription-polymerase chain reaction of >500 female mosquitoes collected during three winters. Investigation into the winter biologies indicated that Anopheles punctipennis (Say), Culex erraticus (Dyar & Knab), Culex peccator Dyar & Knab, and Uranotaenia sapphirina (Osten Sacken) overwinter as females. Females of these species were collected from hollow trees and emergence traps placed over ground holes. Southern magnolia, Magnolia grandiflora L., trees were preferred overwintering sites of culicine mosquitoes. Emergence from underground overwintering sites peaked in mid-March, when air temperatures reached 18–22°C, and the first bloodengorged females of Cx. erraticus and Cx. peccator were collected during this same period. Blood-fed Culex territans Walker females were collected as early as mid-February. This work provides insight into the overwintering biologies of suspected virus vectors at a site of active EEEV transmission and provides limited evidence against the hypothesis that EEEV persists through intertransmission periods in overwintering mosquitoes.

Variation in temperature and food availability in larval habitats can influence the abundance, body size, and vector competence of the mosquito Aedes aegypti. Although increased temperature has energetic costs for growing larvae, how food resources influence the developmental response of this mosquito species to thermal conditions is unknown. We explored how rearing temperature and food affect allometric scaling between wing size and epidermal cell size in Ae. aegypti. Mosquitoes were reared at 22 and 28°C across a gradient of field-collected detritus designed to simulate commonly observed natural larval food resources. Overall, reduced temperature and increased food level increased wing size, but only temperature affected cell size. Females fed the least food had the longest time to maturation, and their increases in wing size induced by cold temperature were associated with larger, rather than more, cells. By contrast, males fed the most food had the shortest time to maturation, and their increases in wing size induced by cold temperature were associated with more, rather than larger, cells. Therefore, food levels can alter the underlying physiological mechanisms generating temperature-size patterns in mosquitoes, suggesting that the control of development is sensitive to the combination of nutrient and thermal conditions, rather than each independently. Conditions prolonging development time may favor increased cell division over growth. We suggest that understanding the effects of climate change on Ae. aegypti vectorial capacity requires an improved knowledge of how water temperature interacts with limited food resources and competition in aquatic container habitats.

Phlebotomine sand flies (Diptera, Psychodidae) are important vectors of human pathogens. Moreover, they possess monoxenous parasites, including gregarines of the genus Psychodiella Votypka, Lantova, and Volf, which can negatively affect laboratory-reared colonies, and have been considered as potential candidates in biological control. In this study, effects of the gregarine Psychodiella sergenti Lantova, Volf, and Votypka on its natural host Phlebotomus sergenti Parrot were evaluated. The gregarines increased the mortality of immature sand fly stages, and this effect was even more apparent when the infected larvae were reared in more dense conditions. Similarly, the gregarines negatively affected the survival of adult males and females. However, no impact was observed on the mortality of blood-fed females, the proportion of females that laid eggs, and the number of eggs oviposited. The 10-times higher infection dose (50 versus five gregarine oocysts per one sand fly egg) led to ∼10 times more gamonts in fourth-instar larvae and two or three times more gamonts in females and males, respectively. Our study clearly shows that Ps. sergenti is harmful to its natural host under laboratory conditions. However, its potential for use in biological control is questionable as a result of several factors, including this parasite's strict host specificity.

Characterizing mosquito larval habitats is essential for understanding the complex interactions between immatures and the biotic and abiotic components of their environment. Using generalized linear mixed models, we studied the environmental predictors of the presence of three ubiquitous mosquito species breeding in ground water habitats in the Paraná Lower Delta, Argentina. During a year-round survey, 34.1% of the 419 ground water habitats inspected were positive for either Culex dolosus s.l. (Lynch Arribálzaga 1891), Aedes crinifer (Theobald 1903), or Culex intrincatus Brèthes 1916. Univariate analysis showed that the former two occurred throughout the year, whereas the latter during the summer and fall. Ae. crinifer and Cx. intrincatus were more frequently collected in secondary forests, whereas Cx. dolosus s.l. was homogeneously distributed among land uses. Best generalized linear mixed models included the sampling period and landscape variables in different combinations for each species. Spatial dependence of the data was evident for Cx. dolosus s.l. and Ae. crinifer. Our results showed that the most widespread species presented different spatio-temporal distribution patterns, related with land use, anthropic intervention, and seasonality, highlighting the complexity of the wetland under study. This methodological approach could aid in the selection of priority areas for vector control and disease risk management.

Diseases transmitted by mosquitoes could be controlled if vector populations were replaced with strains that have reduced vector competency. Such a strategy is being developed for control of dengue virus which is transmitted by Aedes aegypti (L.) (Diptera: Culicidae). Mosquitoes artificially infected with the bacterium, Wolbachia pipientis Hertig, are being assessed as candidates for release at the adult stage with the aim of replacement of the wild population. Wolbachia can reduce the capacity of Ae. aegypti to transmit dengue virus and has potential to be driven through the natural population via a system of cytoplasmic incompatibility. Deployment of benign mosquito strains will be influenced by population size and structure of wild-type Ae. aegypti in proposed release areas, as well as rates of gene flow among populations in the wet and dry tropical seasons. Mosquitoes from northern Queensland were screened with genetic markers to find an optimal locality for release of a benign strain of Ae. aegypti. The inland towns of Chillagoe and Charters Towers and the coastal town of Ingham had mosquito populations that were partly genetically isolated from mosquitoes in other areas across both seasons. These locations may be suitable release sites if it is important for the released strain to be restricted during initial phases of implementation. Smaller genetic differences were also evident among other regions and were consistent over two seasons (wet and dry).

The endosymbiotic bacteria Wolbachia manipulate host reproduction by inducing a form of sterility known as cytoplasmic incompatibility (CI), promoting the invasion of infection into natural host populations. CI has received attention for use in applied strategies to control insect vectors of disease. Thus, to understand both naturally occurring Wolbachia invasions and evaluate potential applied strategies, it is important to understand Wolbachia interactions with its host, including impacts on fitness and the CI level. In this study, we examined for an effect of Wolbachia on survivorship, developmental time, sex ratio, longevity, fecundity, and egg hatch of Aedes polynesiensis Marks, which is the primary vector of Wuchereria bancrofti in the South Pacific. In this study, we have compared strains of A. polynesiensis that are naturally and artificially infected with Wolbachia and additional strains that are aposymbiotic (Wolbachia removed to generate an uninfected strain). Artificially infected strains were observed to have increased larval mortality and decreased adult longevity when compared with aposymbiotic strains. Naturally infected strains were observed to have decreased larval mortality, pupal mortality, increased adult longevity, and a larger adult size when compared with aposymbiotic strains. Artificially infected males that were 4 wk old were able to induce high rates of CI, similar to young males. We discuss the results in relation to the natural spread of Wolbachia and Wolbachia-based applied strategies to modify A. polynesiensis populations.

Lutzomyia longipalpis (Diptera: Psychodidae) is the principal vector of American visceral leishmaniasis. Several studies have indicated that the Lu. longipalpis population structure is complex. It has been suggested that genetic divergence caused by genetic drift, selection, or both may affect the vectorial capacity of Lu. longipalpis. However, it remains unclear whether genetic differences among Lu. longipalpis populations are directly implicated in the transmission features of visceral leishmaniasis. We evaluated the genetic composition and the patterns of genetic differentiation among Lu. longipalpis populations collected from regions with different patterns of transmission of visceral leishmaniasis by analyzing the sequence variation in the mitochondrial cytochrome b gene. Furthermore, we investigated the temporal distribution of haplotypes and compared our results with those obtained in a previous study. Our data indicate that there are differences in the haplotype composition and that there has been significant differentiation between the analyzed populations. Our results reveal that measures used to control visceral leishmaniasis might have influenced the genetic composition of the vector population. This finding raises important questions concerning the epidemiology of visceral leishmaniasis, because these differences in the genetic structures among populations of Lu. longipalpis may have implications with respect to their efficiency as vectors for visceral leishmaniasis.

Bedbugs are found in many countries around the world, and in some regions they are resistant to numerous insecticides. This study surveyed bedbugs in Thailand and determined their resistance to insecticides. The surveys were carried out in six provinces that attract large numbers of foreign tourists: Bangkok, Chonburi, Chiang Mai, Ubon Ratchathani, Phuket, and Krabi. Bedbugs were collected from hotels and colonized in the laboratory to evaluate their resistance to insecticides. Cimex hemipterus (F.) was found in some hotels in Bangkok, Chonburi, Phuket, and Krabi, whereas Cimex lectularius L. was found only in hotels in Chiang Mai. No bedbugs were found in Ubon Ratchathani. The colonized bedbugs showed resistance to groups of insecticides, including organochlorines (dichlorodiphenyl trichloroethane, dieldrin), carbamates (bendiocarb, propoxur), organophosphates (malathion, fenitrothion), and pyrethroids (cyfluthrin, deltamethrin, permethrin, λ-cyhalothrin, etofenprox) in tests using World Health Organization insecticide-impregnated papers. The new insecticides imidacloprid (neonicotinoid group), chlorfenapyr (pyrrole group), and fipronil (phenylpyrazole group) were effective against the bedbugs; however, organophosphate (diazinon), carbamates (fenobucarb, propoxur), and pyrethroids (bifenthrin, cypermethrin, esfenvalerate, etofenprox) were ineffective. Aerosols containing various pyrethroid insecticides with two to four different active ingredients were effective against the bedbugs. The results obtained from this study suggested that both species of bedbugs in Thailand have developed marked resistance to various groups of insecticides, especially those in the pyrethroid group, which are the most common insecticides used for pest control. Therefore, an integrated pest management should be implemented for managing bedbugs in Thailand.

This study determined the transovarial transmission (TOT) potential and tissue tropisms of Culex flavivirus (CxFV), an insect-specific flavivirus, in Culex pipiens (L.). Several hundred mosquito egg rafts were collected in the field, transferred to the insectaries, reared to the fourth larval instar, and identified using morphological characteristics. Cx. pipiens were reared to adults, allowed to oviposit in individual containers, and tested for CxFV RNA by reverse transcription-polymerase chain reaction (RT-PCR) and nucleotide sequencing. Eighteen CxFV RNA-positive females were identified from 26 females that oviposited viable egg rafts. Thirty F₁ adults from each positive female were individually tested by RT-PCR for CxFV RNA. Viral RNA was detected in 526 of 540 progeny, and thus, the filial infection rate was 97.4%. Because all 18 positive females produced infected offspring, the TOT prevalence was 100%. These data indicated that efficient TOT of CxFV occurs in nature. To define the tissue tropisms of CxFV, different tissues (salivary glands, ovaries, testes, head, fat bodies, and midguts) were removed from the remainder of the F₁ and tested by RT-PCR for CxFV RNA. Viral RNA was detected in all tissues. Additionally, uninfected laboratory-colonized Cx. pipiens were infected with CxFV by needle inoculation, and ovaries were collected at 4, 6, 8, and 12 d postinoculation and tested for CxFV RNA by RT-PCR. Viral RNA was detected at all time points, demonstrating that CxFV infects the ovaries as early as 4 d postinoculation. Surprisingly, however, we were unable to demonstrate transovarial transmission despite the presence of viral RNA in the ovaries. Nevertheless, the experiments performed with field-infected Cx. pipiens demonstrate that TOT is an efficient mechanism by which CxFV is maintained in mosquitoes in nature.

The effectiveness of CO₂-baited and human-baited mosquito traps for the sampling of Anopheles darlingi Root was evaluated and compared with human landing collections in Suriname. Biting preferences of this mosquito on a human host were studied and related to trapping data. Traps used were the Centers for Disease Control and Prevention Miniature Light trap, the BG Sentinel mosquito trap, the Mosquito Magnet Liberty Plus mosquito trap (MM-Plus), and a custom-designed trap. Carbon dioxide and humans protected by a bed net were used as bait in the studies. The number of An. darlingi collected was greater with human landing collections than with all other collection methods. An. darlingi did not show a preference for protected humans over CO₂ bait. The BG Sentinel mosquito trap with CO₂ or human odor as bait and the MM-Plus proved the best alternative sampling tools for An. darlingi. The BG Sentinel mosquito trap with CO₂ or human odor as bait was also very efficient at collecting Culex spp. In a field study on biting preferences of wild An. darlingi, the females showed directional biting behavior (P < 0.001), with a majority of females (93.3%) biting the lower legs and feet when approaching a seated human host. Higher efficiency of the closer-to-the-ground collecting MM-Plus and BG Sentinel mosquito trap when compared with the other trapping methods may be a result of a possible preference of this mosquito species for low-level biting. It is concluded that odor-baited sampling systems can reliably collect An. darlingi, but the odor bait needs to be improved, for instance, by including host-specific volatiles, to match live human baits.

The dynamics of West Nile virus (family Flaviviridae, genus Flavivirus, WNV) infection in mosquitoes, sentinel pheasants, and wild dead birds were evaluated during 2003–2006 in Saginaw Co., MI. Mosquitoes were collected by New Jersey Light Traps at 22 sites during May–September, pooled by species and sample location, and tested for presence of WNV RNA by using a real-time reverse transcription-polymerase chain reaction assay. Oral swabs from wild dead birds submitted by the public were tested by Vec-Test assay. Sentinel pheasants were bled weekly, and serum was tested for antibodies with an inhibition enzyme immunoassay. In total, 37,225 mosquitoes [Aedes vexans (Meigen), Culex pipiens L., and Culex restuans Theobald] were tested in 5,429 pools, of which 59 (1.1%) were positive. Ae. vexans was most abundant but had a comparatively low infection rate (0.06–2.11) compared with Cx. pipiens (1.75–4.59) and Cx. restuans (1.22–15.67). Mosquito abundances were temporally related to variations in 2-wk average weather variables. Infected dead crows appeared earlier each transmission season than blue jays, but infection prevalence for both peaked approximately mid-August. Space-time clusters were found in different locations each year. Sentinel pheasant seroprevalence was 19.3% (16/83), 12.7% (10/79), and 7.7% (5/65) during 2003–2005, respectively. We demonstrated temporal patterns of WNV activity in corvid birds and Culex spp. mosquitoes during the study period, suggesting virus transmission within an enzootic cycle. Despite the absence of human case reports nearby, this surveillance system demonstrated WNV transmission and possible human risk. Maintained surveillance using more appropriate gravid traps and CDC CO₂ light traps could improve sensitivity of vector collection and virus detection.

Lighted Centers for Disease Control and Prevention (CDC) light traps were baited with carbon dioxide (CO₂) produced from three different sources to compare the efficacy of each in collecting phlebotomine sand flies in Bahrif village, Aswan Governorate, Egypt. Treatments consisted of compressed CO₂ gas released at a rate of 250 ml/min, 1.5 kg of dry ice (replaced daily) sublimating from an insulated plastic container, CO₂ gas produced from a prototype FASTGAS (FG) CO₂ generator system (APTIV Inc., Portland, OR), and a CDC light trap without a CO₂ source. Carbon dioxide was released above each treatment trap's catch opening. Traps were placed in a 4 × 4 Latin square designed study with three replications completed after four consecutive nights in August 2007. During the study, 1,842 phlebotomine sand flies were collected from two genera and five species. Traps collected 1,739 (94.4%) Phlebotomus papatasi (Scopoli), 19 (1.0%) Phlebotomus sergenti, 64 (3.5%) Sergentomyia schwetzi, 16 (0.9%) Sergentomyia palestinensis, and four (0.2%) Sergentomyia tiberiadis. Overall treatment results were dry ice (541) > FG (504) > compressed gas (454) > no CO₂ (343). Total catches of P. papatasi were not significantly different between treatments, although CO₂-baited traps collected 23–34% more sand flies than the unbaited (control) trap. Results indicate that the traps baited with a prototype CO₂ generator were as attractive as traps supplied with CO₂ sources traditionally used in sand fly surveillance efforts. Field-deployable CO₂ generators are particularly advantageous in remote areas where dry ice or compressed gas is difficult to obtain.

Understanding how ecological conditions influence physiological responses is fundamental to forensic entomology. When determining the minimum postmortem interval with blow fly evidence in forensic investigations, using a reliable and accurate model of development is integral. Many published studies vary in results, source populations, and experimental designs. Accordingly, disentangling genetic causes of developmental variation from environmental causes is difficult. This study determined the minimum time of development and pupal sizes of three populations of Lucilia sericata Meigen (Diptera: Calliphoridae; from California, Michigan, and West Virginia) at two temperatures (20°C and 33.5°C). Development times differed significantly between strain and temperature. In addition, California pupae were the largest and fastest developing at 20°C, but at 33.5°C, though they still maintained their rank in size among the three populations, they were the slowest to develop. These results indicate a need to account for genetic differences in development, and genetic variation in environmental responses, when estimating a postmortem interval with entomological data.

In addition to feeding on carrion tissues and fluids, social wasps can also prey on immature and adult carrion flies, thereby reducing their populations and retarding the decomposition process of carcasses. In this study, we report on the occurrence and behavior of social wasps attracted to vertebrate carrion. The collections were made monthly from September 2006 to October 2007 in three environments (rural, urban, and forest) in six municipalities of southeast Brazil, using baited bottle traps. We collected Agelaia pallipes (Olivier, 1791) (n = 143), Agelaia vicina (Saussure, 1854) (n = 106), Agelaia multipicta (Haliday, 1836) (n = 18), and Polybia paulista Ihering, 1896 (n = 3). The wasps were observed feeding directly on the baits and preying on adult insects collected in the traps. Bait and habitat associations, temporal variability of social wasps, and possible forensic implications of their actions are discussed.

Host-feeding patterns play a key role in the transmission of vector-borne diseases such as West Nile fever, which involves two kinds of vertebrates, birds and mammals. In this study, we propose a theoretical formulation for mosquito host-feeding patterns using three quantities, as follows: the apparent attractiveness/contact probabilities, the conditional host (-feeding) preferences, and the enzootic versus bridge probabilities. Using results from host-baited trap collections, the quantities defined above were assessed for the most abundant mosquito species in the main West Nile virus focus of southern France. We found that host availability is important in determining the efficiency of bridge vectors, and that even ornithophilic mosquitoes like Culex species, classically classified as enzootic vectors, may turn out to be efficient bridge vectors in certain contexts of host abundance. Our developed theoretical framework can easily be adapted and applied to other experimental data and other vector-borne diseases.

From May through November 2007, intensive weekly surveys at the site of a previously reported autochthonous human case of Chagas parasite infection resulted in the collection of 298 Triatoma sanguisuga (Leconte) specimens, of which 60.4% (180) were polymerase chain reaction positive for Trypanosoma cruzi Chagas. All were adults, in a ratio of ≈1:1 female to male, indicating that the domicile was not colonized, but was a destination for these host-seeking adults. We report on seasonal activity pattern, T. cruzi prevalence in T. sanguisuga, and attempts at insect exclusion and control at the case residence.