The discovery of Aedes albopictus Skuse infestation in Texas in 1985 and subsequent global spread of this mosquito have spawned a large number of publications worldwide. The unique discovery in this episode was used tire trade as mechanism by which this exotic mosquito was introduced to the United States. While most researchers have focused their attention primarily on recent events and scientific progress, far fewer examined in depth early contributions made in the United States still very much relevant for contemporary discussion and other important subjects associated with its infestation. The first part of this review covers early background histories of research on Ae. albopictus in the institutions in the United States and of introduction of Ae. albopictus and Ae. aegypti to the U.S. territories in the eastern Pacific as well as dengue outbreaks therein. The second part covers evolution of recognition of the importance of tire as breeding site of domesticated or peridomestic dengue vectors and of its transport in the dispersal of these mosquitoes. In the third part, the significance of vector-borne viral disease outbreak potential of Ae. albopictus and Ae. aegypti in temperate regions and a variety of problems concerning vector spread and commercial practices learned in the past are reassessed in the context of contemporary research. I further identified as byproducts of this review two potentially important epidemiological mechanisms in the transmission of dengue in temperate regions, increasing tendency of indoor activity of Ae. albopictus and massive human movement associated with unpredictable political development.

We demonstrate the utility of models as aids in the design and development of experiments aimed at measuring the effects of proposed vector population control strategies. We describe the exploration of a stochastic, age-structured model that simulates field cage experiments that test the ability of a female-killing strain of the mosquito Aedes aegypti (L.) to suppress a wild-type population. Model output predicts that choices of release ratio and population size can impact mean extinction time and variability in extinction time among experiments. We find that unless fitness costs are >80% they will not be detectable in experiments with high release ratios. At lower release ratios, the predicted length of the experiment increases significantly for fitness costs >20%. Experiments with small populations may more accurately reflect field conditions, but extinction can occur even in the absence of a functional female-killing construct because of stochastic effects. We illustrate how the model can be used to explore experimental designs that aim to study the impact of density dependence and immigration; predictions indicate that cage population eradication may not always be obtainable in an operationally realistic time frame. We propose a method to predict the extinction time of a cage population based on the rate of population reduction with the goal of shortening the duration of the experiment. We discuss the model as a tool for exploring and assessing the utility of a wider range of scenarios than would be feasible to test experimentally because of financial and temporal restraints.

Mosquitoes of the genus Orthopodomyia (Diptera: Culicidae) are little known and of uncertain epidemiological importance. In the United States, there are three Orthopodomyia species (i.e., Or. signifiera (Coquillett), Or. alba Baker, and Or. kummi Edwards); they are all members of the Signifera Group based on the current morphological taxonomy. In the course of identifying recently collected specimens, a problem was found with the current key morphological characters for separating the fourth instar larvae of Or. signifera and Or. kummi. Internal transcribed spacer two sequences of the rDNA were obtained to resolve the identities. The Orthopodomyia internal transcribed spacer two ranged in size from 193 (Or. kummi) to 244 bp (Or. signifera) (mean = 218 bp) and were slightly Adenine/Thymine enriched (44.7% Guanine/Cytosine on average). Putative secondary structures reveal structural homologies (four domains) consistent between species that also feature conserved sequences specific to mosquitoes (e.g., a conserved motif on the 3′ aspect of the longest helix: GARTACATCC). Sequence analyses suggest that in certain areas of southwestern North America, hybridization may occur between Or. kummi and Or. signifera. Furthermore, our analyses confirm that Or. califomica (a junior synonym of Or. signifera) is indeed Or. signifera. To our knowledge, this is the first sequence-based phylogenetic and molecular analysis of the Orthopodomyia.

Simulium (Simulium) kisapense sp. nov. is described on the basis of reared adult, pupal, and larval specimens collected from Langkawi Island, Peninsular Malaysia, and placed in the multistriatum species-group in the subgenus Simulium. This new species is characterized by the bare basal section of the female radial vein, the male ventral plate with setae, the eight pupal gill filaments divergent at an angle of >90 degrees, and the shoe-shaped cocoon with small lateral window(s). Taxonomic notes are given to separate this new species from S. (S.) hirtinervis Edwards and S. (S.) malayense Takaoka and Davies, both from Peninsular Malaysia, and several other known species from Bhutan, India, Nepal, and Thailand.

Horse flies (Diptera: Tabanidae) are ecologically important pollinators and vectors of many disease-causing organisms, as adult females are known to mechanically transfer multiple disease agents during feeding affecting humans, livestock, and many native mammals. Scaptia (Pseudoscione) Lutz in Lutz, Araujo, & Fonseca 1918 has the widest distribution of all genera in the tribe Scionini, occurring in Australia, New Guinea, New Zealand, and South America. Seven new species of Australian S. (Pseudoscione) are described and included in an updated key to the subgenus. The new species are: S. (Pseudoscione) baylessi sp. nov. Lessard, S. (Pseudoscione) casseli sp. nov. Lessard, S. (Pseudoscione) mackerrasi sp. nov. Lessard, S. (Pseudoscione) moritae sp. nov. Lessard, S. (Pseudoscione) turcatelae sp. nov. Lessard, S. (Pseudoscione) turneri sp. nov. Lessard, and S. (Pseudoscione) wiegmanni sp. nov. Lessard. In addition, S. (Pseudoscione) occidentalis Mackerras, 1960, previously described as a subspecies, has been raised to species level. One new species significantly extends the known distribution of Scaptia into central Australia, >1,200 km NW from the nearest recorded species within the subgenus.

Adults of an exotic mosquito, Aedes (Finlaya) koreicus (Edwards) (Diptera: Culicidae) were identified by morphology and genotyping from one site in Belgium in 2008. In late summer of that year, the occurrence of adults and immature stages reconfirmed its presence. This is the first record of this species outside its native range and in particular in Europe. Two subsites of the original location were prospected from April until October 2009 with different traps to evaluate the extent of its presence and establishment in the area and to understand the dynamics of the species' population. Next to Ae. koreicus, 15 other mosquito species were collected. Adult individuals of Ae. koreicus were found from May to September and larvae were still found early October. Larvae were mainly retrieved from artificial containers both in 2008 as in 2009. Containers with eggs and/or larvae were found up to 4 km away from the initial location, indicating the species is spreading locally. Though the introduction route is unknown, it may have occurred via international trade as a large industrial center was located nearby. A comparison of different climatic variables between locations in Belgium with Ae. koreicus and putative source locations in South Korea, revealed similarities between winter temperatures and the number of freezing days and nights in four consecutive years (2004–2008), while humidity and precipitation values differed strongly. The introduction of a new potential disease vector into Europe seems to be a result of proper entrance points, created by intense worldwide trade and suitable environmental conditions.

Worldwide invasion of Aedes albopictus (Skuse) has become a serious concern in vector control. In temperate regions, an important goal has been to predict northern range limits after new introductions or global climate change. We propose a simple thermal suitability index (TSI) that can be calculated from monthly mean temperatures and may predict the northern distributional limits of Ae. albopictus. Analyses of known distributions revealed that TSI yields a common criterion (TSI = 1) below which establishment is thermally prohibited throughout the northern hemisphere. However, temperature conditions around northern limits differ among regions, depending on thermal continentality (the degree of seasonal temperature variation). Thermal contfnentality is high on the Asian Continent, intermediate in North America and Japan, and low (i.e., oceanic) in Europe. On the Asian Continent, there may be maximal winter coldness above which establishment is prohibited irrespective of summer reproduction. In Europe, establishment is possible under summer warmth lower than in other regions but there may be minimal summer warmth below which establishment is difficult despite high winter survival during mild winters. Among the thermal indices that have been commonly used as establishment criteria, the annual mean temperature of 10°C proved to be a common criterion irrespective of thermal continentality, whereas coldest month means and warmth during the reproductive season (often expressed as accumulated degree-days above 11°C) are strongly influenced by thermal continentality. Although annual mean temperatures can be a practical criterion, TSI was more informative in evaluating thermal suitability for this species.

We report on the collection of immatures of Aedes (Ochlerotatus) epactius Dyar & Knab from artificial containers during July through September 2011 in 12 communities located along an elevation and climate gradient extending from sea level in Veracruz State to high elevations (>2,000 m) in Veracruz and Puebla States, México. Ae. epactius was collected from ff of the 12 study communities; the lone exception was the highest elevation community along the transect (>2,400 m). This mosquito species was thus encountered at elevations ranging from near sea level in Veracruz City on the Gulf of México to above 2,100 m in Puebla City in the central highlands. Collection sites included the city of Cordoba, located at ≈850 m, from which some of the first described specimens of Ae. epactius were collected in 1908. Estimates for percentage of premises in each community with Ae. epactius pupae present, and abundance of Ae. epactius pupae on the study premises, suggest that along the transect in central México, the mosquito is present but rare at sea level, most abundant at mid-range elevations from 1,250–1,750 m and then decreases in abundance above 1,800 m. Statistically significant parabolic relationships were found between percentage of premises with Ae. epactius pupae present and average minimum daily temperature, cumulative growing degree-days, and rainfall. We recorded Ae. epactius immatures from a wide range of container types including cement water tanks, barrels/ drums, tires, large earthen jars, small discarded containers, buckets, cement water troughs, flower pots, cement water cisterns, and larger discarded containers. There were 45 documented instances of co-occurrence of Ae. epactius and Aedes aegypti (L.) immatures in individual containers.

Triatomine insects are a problem for human health in southwestern United States because of the moderate-to-severe allergic reactions their bites can cause and because they are potential vectors of Chagas Disease. Although both infected insects and wild mammalian reservoirs are plentiful in southern U.S., only seven cases of autochthonous transmission (plus 16 new presumed cases) of this disease have been reported to date. Therefore, the purpose of this study was to investigate triatomine distribution and abundance in a metropolitan area in southern Arizona. Species, life-stage, locality, and date of collection were recorded for 1,878 triatomine insects collected during 4 yr inside and around houses. For both sexes of the most abundant species, Triatoma rubida (Uhler) (>95% of triatomines collected), dispersal followed a typical year-to-year pattern: dispersal started at the beginning of May and peaked during the first-second week of June. T. rubida was found widely distributed in suburban areas. Triatomines of the two less abundant species, T. recurva (Stal) and T. protracta (Uhler), were collected in all suburban areas throughout the 4-yr survey. All of these population characteristics were observed both at a large (i.e., all collection sites pooled) and a small (i.e., single collection sites) scale. In total, ≈55–60% of the triatomines were collected inside houses, and 30–35% of those were found in or near beds; thus, it is likely that they fed on humans. To our knowledge, this study is the first comprehensive multi-year analysis of triatomine distribution and abundance in the U.S., providing data that allow inferences about risks to human health.

We sampled stable fly, Stomoxys calcitrans (L.), populations using a CO₂ baited cloth trap (Nzi trap) each day throughout the summer and autumn at a beef farm near Peterborough, Ontario, Canada, from 1997 to 2001. Females (2,512) were dissected for ovarian age-grading, to produce a demographic profile of farm populations. The number of follicles were counted to produce fecundity estimates. The developmental periods of adult female stages, measured as accumulated degree-days above 10°C, were determined for a lab colony of stable flies. These measurements were used to calculate survival in terms of degree-days of the farm populations each year. Of the 2,512 females caught, 42.4% were nulliparous on average each year. The median follicle size at insemination was 305 µm in the field populations, and 495 µm in the colony. Farm caught females had an average of 49.15 follicles per ovary overall, with the body size (leg length) and fecundity increasing slightly with age. On average, 44.5% (SE 3.2%) of nulliparous females survived to become parous, and of these, 45.7% (SE 2.1%) survived the uniparous state to become multiparous. Years of higher rainfall had increased fecundity; rainfall did not appear to affect survival.

Leptotrombidium chiangraiensis Tanskul & Linthicum, and Leptotrombidium imphalum Vercammen—Grandjean are important vectors of scrub typhus in rice field habitats in northern Thailand. The developmental biology of all stages of the life cycle of two generations of these species of mites infected with Orientia tsutsugamushi (Hayashi) and uninfected mites is reported. The development of the infected lines of both F₁ and F₂ L. chiangraiensis were significantly longer than their respective uninfected lines (P < 0.05). The developmental times of uninfected and infected F₁ lines of L. imphalum were not significantly different; however, F₂ infected lines took significantly longer to develop (P < 0.05). Both F₁ and F₂ generations of infected L. imphalum and L. chiangraiensis oviposited on average >150 fewer eggs than uninfected mites.

A study on the ecological patterns characterizing the community of arthropods parasitic of sigmodontine rodents was carried out in an area that represents the environmental conditions of the Argentinean Chaco. Four species of mites (Laelapidae and Macronyssidae), one of tick (Ixodidae), and five of fleas (Rhopalopsyllidae and Stephanocircidae) were collected on six species of sigmodontine rodents (Cricetidae: Sigmodontinae). As a generality, the parasite taxa studied in this work presented similar patterns of host specificity and aggregation. With the exception of laelapid mites, the remainder taxa exhibited low levels of host specificity. All parasites show a tendency toward aggregation, and the pattern of host usage appears to be constant on geographical scales. Ticks (immature stages of A. tigrinum) were found on sigmodontines during the whole year with peaks of abundance in different seasons, while mites and fleas were detected from late summer to late spring, with the peak of abundance in winter. A significant positive correlation between monthly abundance of rodents and monthly abundance of mites and fleas was found, suggesting that the pattern of seasonality of these two taxa could be determined by the seasonal abundance of hosts. Finally, the potential epidemiological implications related to the lack of strict host specificity showed by most of the parasites studied in this work were discussed.

The biting activity and host attractancy of vector mosquitoes are important in assessing the risk of arbovirus transmission, especially where migratory and nonmigrating bird species congregate, such as in Hulun Lake, Manzhouli. In 2009, the population distribution, species diversity, biting activity, and host attractancy of mosquitoes were investigated in Hulan Lake and its associated prairie area. The adult mosquitoes were captured either by human volunteers using aspirators in mosquito nets, by CO₂-baited light traps, or by animal-baited traps. In total, 27,004 mosquitoes, representing three genera and 10 species, were collected from Manzhouli, China, in July 2009, of which Aedes dorsalis (Meigen) were most predominant species, followed by Ae. vexans (Meigen). Biting activity peaks by Ae. flavescens (Muller), Ae. dorsalis, and Culex modestus (Facalbi) on human subjects were investigated. Four mosquito species were captured from different animal sheds (sheep, cattle, and goose). Ae. flavescens was more abundant in the cattle shed than in the other two sheds. The Ae. dorsalis in the sheep shed was much higher than in the other animal sheds. The Ae. flavescens collected via chicken-baited traps were significantly higher than those collected via rabbit-baited and pigeon-baited traps. There were no significant differences in the number of Ae. dorsalis and Ae. vexans collected using the three different animal traps.

The mosquito fauna in many areas of western Uganda has never been studied and is currently unknown. One area, Bwamba County, has been previously studied and documented but the species lists have not been updated for >40 yr. This paucity of data makes it difficult to determine which arthropod-borne viruses pose a risk to human or animal populations. Using CO₂ baited-light traps, from 2008 through 2010, 67,731 mosquitoes were captured at five locations in western Uganda including Mweya, Sempaya, Maramagambo, Bwindi (BINP), and Kibale (KNP). Overall, 88 mosquito species, 7 subspecies, and 7 species groups in 10 genera were collected. The largest number of species was collected at Sempaya (65 species), followed by Maramagambo (45), Mweya (34), BINP (33), and KNP (22). However, species diversity was highest in BINP (Simpson's Diversity Index 1-D = 0.85), followed by KNP (0.80), Maramagambo (0.79), Sempaya (0.67), and Mweya (0.56). Only six species Aedes (Aedimorphus) cumminsii (Theobald), Aedes (Neomelaniconion) circumluteolus (Theobald), Culex (Culex) antennatus (Becker), Culex (Culex) decens group, Culex (Lutzia) tigripes De Grandpre and De Charmoy, and Culex (Oculeomyia) annulions (Theobald), were collected from all five sites suggesting large differences in species composition among sites. Four species (Aedes (Stegomyia) metallicus (Edwards), Anopheles (Cellia) rivulorum Leeson, Uranotaenia (Uranotaenia) chorleyi (Edwards), and Uranotaenia (Uranotaenia) pallidocephala (Theobald) and one subspecies (Aedes (Stegomyia) aegypti formosus (Walker)) were collected in Bwamba County for the first time. This study represents the first description of the mosquito species composition of Mweya, Maramagambo, BINP, and KNP. A number of morphological variations were noted regarding the postspiracular scales, hind tibia, and sternites that make Culex (Culex) neavei (Theobald) challenging to identify. At least 50 species collected in this study have previously been implicated in the transmission of arboviruses of public health importance suggesting a high potential for maintenance and transmission of a wide variety of arboviruses in western Uganda.

Oviposition dynamics and colonization of container habitats by the invasive species, Aedes (Finlaya) japonicus japonicus (Theobald) were examined through the use of ovistrips placed in buckets, and larval surveys of tree holes and tires at sites in central Michigan. In general, oviposition and colonization increased during the study periods, with several sites showing large increases from <10% Ae. j. japonicus initially to over 60% in the following years. Seasonally, higher proportions of Ae. j. japonicus were found in spring and fall collection periods. Ae. j. japonicus larvae co-occurred in the artificial containers with Ae. triseriatus, Ae. hendersoni, several Culex spp., and Anopheles spp. Recent surveys of tire and tree hole habitats at our study areas in mid-Michigan revealed that Ae. j. japonicus had colonized most of these habitats, but maintained relatively low populations in tree holes occupied by Ae. triseriatus. Trends seen in tires from 2008 to 2011, and from gravid trap and New Jersey light traps in 2005–2011, suggest that Ae. j. japonicus populations are stabilizing as they integrate into native Michigan mosquito communities.

The success of an invasive species in a new region depends on its interactions with ecologically similar resident species. Invasions by disease vector mosquitoes are important as they may have ecological and epidemiological consequences. Potential interactions of a recent invasive mosquito, Aedes japonicus Theobald, with resident species in Virginia were evaluated by sampling larvae from containers and trapping adults. Distinct species compositions were observed for artificial containers and rock pools, with Ae. albopictus most abundant in the former and Ae. japonicus in the latter. However, these two species were found to co-occur in 21.2% of containers sampled. Among the six mosquito species most common in containers from May through September, 2006, only interspecific associations of Ae. japonicus with Aedes albopictus (Skuse) and Aedes triseriatus (Say) were significant, and both were negative. In addition to differences in habitat preference, mean crowding estimates suggest that interspecific repulsion may contribute to the significant negative associations observed between these species. High relative abundances of late instars and pupae of Ae. japonicus seem to provide this species with a mechanism of evading competition with Ae. albopictus, facilitating their coexistence in artificial containers. Although annual fluctuations were observed, trends in adult populations over a 6-yr period provide no evidence of declines. In summary, this survey of diverse container types and all life stages provided only limited evidence for competitive displacements or reductions of resident container species by Ae. japonicus, as observed elsewhere in its invasive range.

Combined sewage overflows have created favorable conditions for the establishment of the southern house mosquito, Culex quinquefasciatus Say (Diptera: Culicidae), larvae in natural creeks that would otherwise be unsuitable for the development of this mosquito species. Here, we show the results from a seminatural experiment carried over the three seasons of mosquito activity (spring, summer, and fall) in Tanyard Creek, Atlanta, GA. In this study we manipulated the amount of nutrients by further enriching combined sewage overflow water, and tracked weather variables, organic nutrient concentration, exposure time to conspecifics, and the number of egg rafts collected in experimental containers. We found season and nutrient enrichment to be the most important variables explaining the differences in egg rafts counts. Further analyses suggest that temperature may also play a role in seasonal oviposition patterns. The results from this study suggest that nutrient enrichment and adequate temperatures are important factors shaping Cx. quinquefasciatus oviposition seasonality in combined sewage overflows.

Quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) is a widely used laboratory tool to quantify mRNA levels of target genes involved in various biological processes. The most commonly used method for analyzing qRT-PCR data are the normalizing technique where a housekeeping gene is used to determine the transcriptional regulation of the target gene. The choice of a reliable internal standard is pivotal for relative gene expression analysis to obtain reproducible results, especially when measuring small differences in transcriptional expression. In this study, we used geNorm, NormFinder, and BestKeeper programs to analyze the gene expression results using qRT-PCR. Five candidate reference genes, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), β-actin, α-tubulin, elongation factor 1-α, and glutathione s-transjerase, were used to evaluate the expression stability during prolonged blood-feeding on the vertebrate host. These five genes were evaluated in all life stages of Amblyomma maculatum (Koch) as well as in the salivary gland and midgut tissues of adult females to determine which are the most stably expressed gene for use in qRT-PCR studies. β-Actin is the most stably expressed gene in salivary glands and midguts of A. maculatum, and throughout all developmental stages both Actin and GAPDH were found to have the most stable expression with the lowest degree of variance. We recommend the use of β-actin and/or GAPDH as reference genes for qRT-PCR analysis of gene expression in A. maculatum.

The continuous culture of mosquitoes is a costly endeavor for vector biology laboratories. In addition to the resources that must be committed to colony maintenance, biological costs, including genetic drift and accidental colony loss, also can occur. Although alternatives do exist, their application to mosquitoes is limited. Mosquito cryopreservation remains elusive, and many important species lack a well-defined diapause. Previously, we demonstrated that cold storing nondiapausing mated adult females of the northern house mosquito, Culex pipiens L. resulted in a nearly four-fold increase in longevity when measured at the LT₅₀, allowing for cold storage for up to 10 wk. In the current study, we used sugar feeding during cold storage to significantly improve cold storage longevity. At 6°C, the LT₅₀ of cold stored females was 23 wk, and 100% mortality was not realized until 43 wk. Cold-stored females did exhibit reduced fecundity, but egg production returned to normal levels within two generations. These results suggest that cold storage without diapause induction is a viable option for Cx. pipiens, and with the addition of sugar feeding, a colony could be maintained with less than two generations per year.

Triatoma infestans (Klug, 1834) (Hemiptera, Reduviidae) is the main vector of Chagas disease in the southern cone South America. Chemical control to the vectors appears to be the best option to reduce the incidence of the disease. However, since 2002, high resistance to insecticides that correlated with field control failures was detected in T. infestans from Argentina and Bolivia. In this paper, we analyzed three T. infestans populations whose pyrethroid-resistance had been recently detected, and we defined at least three resistant profiles according to the toxicological and biochemical characteristics of the studied resistant populations. The resistance profiles were identified as Ti-R1, Ti-R2, and Ti-R3, corresponding to the Argentinean Acambuco, and the Bolivians Entre Ríos and Mataral. Ti-R1 exhibited nymphs and eggs with medium resistance level to deltamethrin (RR = 32.5 and 28.6; respectively). Pyrethroid-esterases played a relevant role in deltamethrin resistance. Ti-R2 exhibited nymphs with high resistance to deltamethrin (RR = 173.8) and low resistance to fipronil (RR = 12.4). Pyrethroid-esterases were involved in resistance. Moreover, eggs showed medium resistance level to deltamethrin (RR = 39.1). Ti-R3 had nymphs with low resistance to deltamethrin (RR = 17.4), and medium resistance to fipronil (RR = 66.8). Pyrethroid-esterases showed increased activity, and eggs possessed low resistance to deltamethrin (RR = 8.4). The characterization of the resistance to pyrethroid in these T. infestans populations from Argentina and Bolivia do not permit the generalization of three forms of resistance profile. So far as we appear to know, the forms of mechanisms and their frequencies reported here are selected independently, so additional sites might well show additional combinations of resistance mechanisms and their frequencies.

Larval management of the malaria vector, Anopheles gambiae Giles s.s., has been successful in reducing disease transmission. However, pesticides are not affordable to farmers in remote villages in Mali, and in other material resource poor countries. Insect resistance to insecticides and nontarget toxicity pose additional problems. Neem (Azadirachta indica A. Juss) is a tree with many beneficial, insect bioactive compounds, such as azadirachtin. We tested the hypothesis that neem leaf slurry is a sustainable, natural product, anopheline larvicide. A field study conducted in Sanambele (Mali) in 2010 demonstrated neem leaf slurry can work with only the available tools and resources in the village. Laboratory bioassays were conducted with third instar An. gambiae and village methods were used to prepare the leaf slurry. Experimental concentration ranges were 1,061–21,224 mg/L pulverized neem leaves in distilled water. The 50 and 90% lethal concentrations at 72 h were 8,825 mg/L and 15,212 mg/L, respectively. LC concentrations were higher than for other parts of the neem tree when compared with previous published studies because leaf slurry preparation was simplified by omitting removal of fibrous plant tissue. Using storytelling as a medium of knowledge transfer, villagers combined available resources to manage anopheline larvae. Preparation of neem leaf slurries is a sustainable approach which allows villagers to proactively reduce mosquito larval density within their community as part of an integrated management system.

In this study we evaluated the biting deterrent effects of a series of saturated and unsaturated fatty acids against Aedes aegypti (L), yellow fever mosquito (Diptera: Culicidae) using the K &D bioassay module system. Saturated (C_6:0 to C_16:0 and C_18:0) and unsaturated fatty acids (C_11:1 to C_14:1, C_16:1, C_18:1, and C_18:2) showed biting deterrence index (BDI) values significantly greater than ethanol, the negative control. Among the saturated fatty acids, mid chain length acids (C_10:0 to C_13:0) showed higher biting deterrence than short (C_6:0 to C_9:0) and long chain length acids (C_14:0 to C_18:0), except for C_8:0 and C_16:0 that were more active than the other short and long chain acids. The BDI values of mid chain length acids (C_10:0 to C_13:0) were not significantly less than N, N-diethyl-meta-toluamide (DEET), the positive control. Among the unsaturated fatty acids, C_11:1 showed the highest activity (BDI = 1.05) and C_18:2 had the lowest activity (BDI = 0.7). In C_11:1, C_12:1, and C_14:1 BDI values were not significantly less than DEET. After the preliminary observations, residual activity bioassays were performed on C_11:0, C_12:0, C_11:1, and C_12:1 over a 24-h period. All the fatty acids (C_11:0, C_12:0, C_11:1, and C_12:1) and DEET showed significantly higher activity at all test intervals than the solvent control. At treatment and 1-h posttreatment, all fatty acids showed proportion not biting (PNB) values not significantly less than DEET. At 3-, 6-, and 12-h posttreatment, all fatty acids showed PNB values significantly greater than DEET. At 24-h posttreatment, only the PNB value for C_12:0 was significantly higher than DEET. The dose-responses of C_12:0 and DEET were determined at concentrations of 5–25 nmol/cm². As in the residual activity bioassays, the PNB values for C_12:0 and DEET at 25 nmol/cm² were not significantly different. However, at lower concentrations, the PNB values for C_12:0 were significantly greater than DEET. These results clearly indicate that mid chain length fatty acids not only have levels of biting deterrence similar to DEET at 25 nmol/cm² in our test system, but also appeared to be more persistent than DEET. In contrast, in vivo cloth patch assay system showed that the mid-chain length fatty acids, C_11:0, C_11:1 C_12:0, and C_12:1had minimum effective dose (MED) values greater than DEET against Ae. aegypti and their relative repellency varied according to species tested. The MED values of 120 (C_11:0), 145 (C_12:0) and 116 (C_11:1) nmol/cm² against Anopheles quadrimaculatus Say, indicated that these acids were not as potent as DEET with a MED of 54 nmol/cm². The MED ratio of the C_11:0 and C_11:1 for all three mosquito species indicated the C₁₁ saturated and unsaturated acids as more repellent than their corresponding C_12:0 and C_12:1homologues.

Effectiveness of the elimination efforts against Triatoma infestans (Klug) in South America through residual application of pyrethroid insecticides has been highly variable in the Gran Chaco region. We investigated apparent vector control failures after a standard community-wide spraying with deltamethrin SC in a rural area of northeastern Argentina encompassing 353 houses. Insecticide spraying reduced house infestation less than expected: from 49.5% at baseline to 12.3 and 6.7% at 4 and 8 mo postspraying, respectively. Persistent infestations were detected in 28.4% of houses, and numerous colonies with late-stage bugs were recorded after the interventions. Laboratory bioassays showed reduced susceptibility to pyrethroids in the local bug populations. Eleven of 14 bug populations showed reduced mortality in diagnostic dose assays (range, 35 ± 5% to 97 ± 8%) whereas the remainder had 100% mortality. A fully enclosed residual bug population in a large chicken coop survived four pyrethroid sprays, including two double-dose applications, and was finally suppressed with malathion. The estimated resistance ratio of this bug population was 7.17 (range, 4.47–11.50). Our field data combined with laboratory bioassays and a residual foci experiment demonstrate that the initial failure to suppress T. infestans was mainly because of the unexpected occurrence of reduced susceptibility to deltamethrin in an area last treated with pyrethroid insecticides 12 yr earlier. Our results underline the need for close monitoring of the impact of insecticide spraying to provide early warning of possible problems because of enhanced resistance or tolerance and determine appropriate responses.

A protocol has been developed for the indoor evaluation of candidate spatial repellents intended for use in push and pull systems. Single treatments (catnip oil, 1-methylpiperazine, and homopiperazine) and a mixture of catnip oil and homopiperazine were tested with yellow-fever mosquitoes (Aedes aegypti) in Y-tube olfactometers to determine 1) if these compounds inhibited mosquito host-seeking at short distances and 2) if results obtained in olfactometer tests can be correlated with a larger scale set-up, that is, a room test. All test materials significantly decreased the ability of mosquitoes to find host odors (from a human finger) by up to 96.7% (2.5% catnip and homopiperazine mix). Similar effects could be observed within a new room test set-up, which involved a repellent dispensing system and an attractive trap (BG-Sentinel). Mosquitoes captured by the BGS trap had to fly through a treatment-containing air curtain created by the dispensing system. Compared with the use of a control (ethanol solvent without candidate repellent), trap catch rates were significantly reduced when 5% catnip, 5% 1-methylpiperazine, and 5% homopiperazine were dispensed. Homopiperazine produced the greatest level of host-seeking inhibition with a 95% reduction in the trap catches. The experimental set-up was modified to test the viability of those technologies in a simple push & pull situation.. The combination of BGS trap and a 10% mix of catnip and homopiperazine helped to reduce human landing rates by up to 44.2% with a volunteer sitting behind the air curtain and the trap running in front of the curtain.

Essential oils of Hyptis suaveolens (Lamiaceae), Croton roxburghii (Euphorbiaceae), and Litsea cubeba (Lauraceae) were tested in the field near Vientiane city, Lao PDR, on humans for repellent activity against mosquitoes. Landing mosquitoes were collected and later identified. The most abundant mosquitoes captured belonged to the genera Armigeres, Culex, and Aedes. All the plant oils tested at concentrations of 1.7 µg/cm², 3.3 µg/cm², and 6.3 µg/cm² were significantly more mosquito repellent than the negative control. Croton oil was significantly repellent against mosquitoes of the three genera at the highest (6.3 µg/cm²) concentration tested. Litsea oil was significantly repellent against Annigeres at all (1.7 µg/cm², 3.3 µg/cm², and 6.3 µg/cm²) concentrations tested. Hyptis oil was significantly repellent against Armigeres at 3.3 µg/cm² and 6.3 µg/cm² and against Culex at 1.7 µg/cm²and 6.3 µg/cm². The oils were analyzed for chemical content of volatiles, mainly terpenes. Main constituents were β-pinene, sabinene, and 1,8-cineol from oils of the green parts of H. suaveolens; α-pinene, β-pinene, and α-phellandrene from fresh bark of C. roxburghii; and α-pinene, β-phellandrene, sabinene, and 1,8-cineol from fresh fruits of L. cubeba.

Formamidines are unique insecticides and acaricides that elicit multiple effects in controlling insects. Here, we tested two formamidines, amitraz, and chlordimeform, for their synergistic actions on type II pyrethroids and neonicotinoids to increase their larvicidal actions on the fourth instars of Aedes aegypti L. An organophosphate insecticide was used as a negative control. After 24 h, the synergism of formamidines was highest on imidacloprid, followed by two type II pyrethroids, deltamethrin and fenvalerate. After 48 h, the synergism of formamidines on imidacloprid decreased, remained unchanged on type II pyrethroids, and increased noticeably on two of the newer type neonicotinoids, dinotefuran and thiamethoxam. By 72 h, synergism of formamidines on dinotefuran reached the maximum, while that on imidacloprid was at a minimum. Both formamidines did not show synergistic effects on permethrin or fenitrothion. In all cases, the synergistic effects of amitraz on the two major classes of larvicides were greater than for chlordimeform. These results indicate that amitraz is a promising synergist that shows the potential to increase the efficacy of certain members of type II pyrethroids as well as neonicotinoids to control Ae. aegypti larvae.

Stored product mites commonly occur in agricultural work environments and sometimes in homes in significant numbers. They are a source of allergens that sensitize and induce allergic reactions. This may include atopic dermatitis. The purpose of this investigation was to determine if the common species of storage mites are the sources of molecules that influence the function of human dermal microvascular endothelial cells that regulate the trafficking of inflammatory and immune cells into the dermis during allergic reactions and other skin diseases. Human dermal microvascular endothelial cells were challenged with varying doses of extracts of the storage mites Acarus siro L., Chortoglyphus arcuatus (Troupeau), Lepidoglyphus destructor (Schrank), or Tyrophagus putrescentiae (Schrank) and the secretion of cytokines and expression of adhesion molecules were measured. The role of endotoxin and protein in inducing these responses was evaluated. These stored product mite extracts induced secretion of interleukin-6, interleukin-8, monocyte chemotactic protein-1, and granulocyte/monocyte colony stimulating factor. Some of these effects were induced by protein present in the extracts, some were induced by endotoxin, and some were induced by other substances. C. arcuatus and T. putrescentiae extracts also down-regulated tumor necrosis factor α-induced vascular cell adhesion molecule-1 expression. Stored product mite extracts contain an assortment of molecules, including endotoxins and proteins, which modulate the expression of cell adhesion molecules and the secretion of cytokines by microvascular endothelial cells. These modulating properties varied among mite species indicating that each mite species has a unique set of molecules that is responsible for its activity.

To identify the host range of potential vectors of Dirofilaria immitis Leidy, the causal agent of canine dirofilariasis, we studied the bloodmeal origin of mosquitoes trapped on two of the Canary Islands, Gran Canaria and Tenerife, where this disease is considered hyperendemic. On Gran Canaria, mosquitoes were captured using Centers for Disease Control and Prevention (CDC) traps (outdoors) and resting in a bathroom (indoors). Only CDC traps were used to capture mosquitoes in Tenerife. The species captured in decreasing order of abundance were Culex theileri Theobald, Culex pipiens L., Culiseta longiareolata Macquart, Anopheles atroparvus van Thiel, and Anopheles cinereus Theobald. The origins of bloodmeals were identified for 121 Cx. theileri and 4 Cx. pipiens after amplification and sequencing of a fragment of the vertebrate cytochrome oxidase I (COI) gene. Cx. theileri fed on goats, sheep, dogs, cattle, cats, humans, and chickens, and Cx. pipiens fed on goats and chickens. A lower success of bloodmeal identification was obtained in mosquitoes captured resting indoors than outdoors in CDC traps, probably because of a longer time period between feeding and capture. Although most Cx. theileri fed on ruminants, this species also fed on different mammal species susceptible to dirofiliarasis, including humans, suggesting it could play a role on parasite transmission.

The effects of La Crosse virus (LACV) infection on blood feeding behavior in Aedes triseriatus (Say) and Aedes albopictus (Skuse) were investigated in the laboratory by measuring the size of the bloodmeal imbibed and the extent of refeeding by virus-infected and uninfected mosquitoes. LACV-infected Ae. triseriatus and Ae. albopictus took significantly less blood compared with uninfected mosquitoes. Twice as many virus-infected Ae. triseriatus mosquitoes refed compared with uninfected individuals (18 vs. 9%; P < 0.05); however, virus infection had no significant effect on the refeeding rate of Ae. albopictus. Reduction in bloodmeal size followed by an increased avidity for refeeding may lead to enhanced horizontal transmission of the LACV by its principal vector, Ae. triseriatus.

Vertebrate reservoirs of arboviruses are often infected with microfilariae (MF). Laboratory studies have shown that MF can enhance the infectivity of arboviruses to mosquitoes. Soon after being ingested, MF penetrate the mosquito midgut. If the host blood also contains virus (i.e., vertebrate is dually infected), penetrating MF may introduce virus into the hemocoel. This can transform otherwise virus-incompetent mosquito species into virus-competent species and simultaneously accelerate viral development, allowing mosquitoes to transmit virus sooner than normal. This phenomenon is termed microfilarial enhancement of arboviral transmission. The prevalence of MF is very high in many passerine populations in North America. Therefore, we investigated if microfilarial enhancement could have facilitated the establishment and rapid spread of West Nile virus (WNV) across the mid-western United States. Our investigations revealed that mosquitoes, WNV, and passerine MF do interact in nature because; 1) 17% of 54 common grackles (Quiscalus quiscula L.), 8% of 26 American robins (Turdus migratorius L.), and 33% of three eastern kingbirds (Tyrannus tyrannus L.) were concurrently microfilaremic and seropositive to WNV; 2) feeding activities of mosquitoes overlapped temporally with the appearance of MF in the blood of common grackles; 3) mosquitoes fed on common grackles and American robins in nature; and 4) mosquito ingestion of two taxonomically distant species of passerine MF (i.e., Chandlerella quiscali and Eufilaria spp.) resulted in penetration of mosquito midguts. To estimate the theoretical effect that MF enhancement could have on WNV transmission in areas of high MF prevalence, vectorial capacity values were calculated for Culex mosquitoes feeding on common grackles, whereby MF enhancement was either invoked or ignored. For Cx. pipiens, vectorial capacity increased over three-fold when potential effects of MF were included in the calculations. For Cx. tarsalis, the effect was less (i.e., 1.4-fold increase). Closer attention should be paid to the potential of MF to enhance mosquito transmission of arboviruses.

Programs that aim to control vector-borne zoonotic diseases require information on zoonotic hosts and on the feeding behavior of bridging vectors that are capable of transmitting pathogens from those hosts to humans. Here we describe an assay developed to identify bloodmeals in field-collected cat fleas (Ctenocephalides felis Bouché) to assess this species' potential role as a Yersinia pestis bridging vector in a plague-endemic region of Uganda. Our assay uses a single primer set and SYBR Green I-based real-time polymerase chain reaction to amplify a segment of the 12S mitochondrial ribosomal RNA gene for identification by sequencing. The assay capitalizes on the sensitivity of real-time polymerase chain reaction and the specificity of sequencing and can be used to differentiate vertebrate bloodmeals to the genus or species level without a priori knowledge of the host community. Because real-time assays that detect vertebrate DNA are highly sensitive to human DNA contamination, we analyzed detection in artificially fed and unfed fleas to establish a Ct cutoff that optimized specificity without completely sacrificing sensitivity. Using the established cutoff, our assay detected human, rat, and goat DNA in artificially fed C. felis up to 72 h postfeeding.

The seasonal abundance of Dinopsyllus lypusus Jordan and Rothschild and Ctenophthalmus calceatus Waterson (potential vectors of plague in southern Africa) were studied on rodent hosts captured in selected habitat types of two periurban suburbs of Harare, Zimbabwe. Removal trapping was used to capture the rodents, from which fleas were collected and identified. Prevalence (proportion of animals infested) and specific flea index (SFI = number of fleas per animal) were calculated for each species of rodent host. Cohabitation of the two flea species on the host and its implications were also assessed. In total, 1,083 rodents belonging to nine species were trapped and over 97% of the total captures comprised of four species; Mastomys nataknsis Smith, Rattus rattus L., Tatera leucogaster Peters, and Rhabdomys pumilio Sparrman. In total, 735 D. lypusus and 335 C. calceatus were recorded on these four common rodent species. Population density of D. lypusus as measured by prevalence and SFI varied from 13.4 to 53.3% and 0.2–1.5, respectively, while that of C. calceatus varied from 8.2 to 26.7% and 0.2–0.6, respectively. For all rodent species captured, both prevalence and SFI of D. lypusus and C. calceatus were highest during the cold-dry season, followed by the hot dry season, with the hot-wet season recording the lowest indices. Overall cohabitation was highest during the cold-dry season and nonexistent during the hot-wet season. Our findings on the abundance and ecology of D. lypusus and C. calceatus suggest that their roles in the transmission of plague in Zimbabwe need further investigation.

Sand flies Lutzomyia (Psathyromyia) shannoni (Dyar) and Lu. (Hekocyrtomyia) vexator (Coquillet) were collected for the first time in southwest Missouri and southeast Kansas, expanding the known range of these species in North America. Altogether, 680 sand flies (356 males and 324 females) were collected during trapping from May through October 2011 and identified using morphological characters. Of the total sand flies collected, 315 were identified as Lu. shannoni, with 181 individuals (or 26.6% of all sand flies) trapped in Missouri and 134 individuals (or 19.7%) trapped in Kansas. Whereas 358 Lu. vexator were identified from southwest Missouri, only a single specimen was trapped in southeast Kansas. One male Lu. vexator with asymmetric gonostyli was trapped in Missouri. We also developed a polymerase chain reaction protocol to consistently and accurately distinguish Lu. shannoni from Lu. vexator based on presence or absence of a 416 bp fragment from the cytochrome oxidase c subunit 1 gene.

Laboratory and field research was conducted to determine if Culex tarsalis Coquillett expectorated West Nile virus (WNV) during sugar feeding and if a lure or bait station could be developed to exploit this behavior for WNV surveillance. Experimentally infected Cx. tarsalis repeatedly expectorated WNV onto filter paper strips and into vials with wicks containing sucrose that was readily detectable by a quantitative reverse transcriptase-polymerase chain reaction assay. Few females (33%, n = 27) became infected by imbibing sugar solutions spiked with high concentrations (10⁷ plaque forming units/ml) of WNV, indicating sugar feeding stations probably would not be a source of WNV infection. In nature, sugar bait stations scented with the floral attractant phenyl acetaldehyde tracked WNV transmission activity in desert but not urban or agricultural landscapes in California. When deployed in areas of the Coachella Valley with WNV activity during the summer of 2011, 27 of 400 weekly sugar samples (6.8%) tested positive for WNV RN A by reverse transcriptase-polymerase chain reaction. Prevalence of positives varied spatially, but positive sugar stations were detected before concurrent surveillance measures of infection (mosquito pools) or transmission (sentinel chicken seroconversions). In contrast, sugar bait stations deployed in urban settings in Los Angeles or agricultural habits near Bakersfield in Kern County supporting WNV activity produced 1 of 90 and 0 of 60 positive weekly sugar samples, respectively. These results with sugar bait stations will require additional research to enhance bait attractancy and to understand the relationship between positive sugar stations and standard metrics of arbovirus surveillance.

Lyme disease (LD), caused by the bacterium Borrelia burgdorferi and transmitted in the eastern United States by blacklegged ticks, Ixodes scapularis Say, is classified as nonendemic in Tennessee and surrounding states in the Southeast. Low incidence of LD in these states has been attributed, in part, to vector ticks being scarce or absent; however, tick survey data for many counties are incomplete or out of date. To improve our knowledge of the distribution, abundance, and Borrelia spp. prevalence of I. scapularis, we collected ticks from 1,018 hunter-harvested white-tailed deer (Odocoileus virginianus (Zimmerman)) from 71 of 95 Tennessee counties in fall 2007 and 2008. In total, 160 deer (15.7%) from 35 counties were infested with adult I. scapularis; 30 of these counties were new distributional records for this tick. The mean number of I. scapularis collected per infested deer was 5.4 ±0.6 SE. Of the 883 I. scapularis we removed from deer, none were positive for B. burgdorferi and one tested positive for B. miyamotoi. Deer are not reservoir hosts for B. burgdorferi; nevertheless, past surveys in northern LD-endemic states have readily detected B. burgdoreferi in ticks collected from deer. We conclude that I. scapularis is far more widespread in Tennessee than previously reported. The absence of detectable B. burgdorferi infection among these ticks suggests that the LD risk posed by I. scapularis in the surveyed areas of Tennessee is much lower than in LD-endemic areas of the Northeast and upper Midwest.

The aim of this study was to determine the incidence of bacteria of public health interest transmitted by cockroaches in different food-related environments. From April to November, cockroaches were trapped in 11 buildings in different urban areas of Western Andalusia (Spain): three hotels, four grocery stores, a catering establishment, a food-industry plant, a health center, and a care home. The presence of a number of bacterial species, including Salmonella, in these food-related environments was confirmed; these species included microorganisms listed in European Union regulations, such as Salmonella spp., Enterobacter sakazakii (Cronobacter spp.), and Escherichia coli. A wide variety of species were isolated, some belonging to different genera that have a significant impact on public health and hygiene, such as Enterobacter and Klebsiella. To ensure adequate elimination of these microorganisms in food-related environments, the control of vectors such as Blattella germanica, Periplaneta americana, and Blatta orientalis, together with a thorough review of hygiene strategies, appears to be fundamental. It is clearly essential to compare the results of hygiene regulations implemented in food-related environments.

Results of an environmental assessment conducted in a newly emergent focus of murine typhus in southern California are described. Opossums, Didelphis virginiana Kerr, infested with cat fleas, Ctenocephalides felis Buché, in the suburban area were abundant. Animal and flea specimens were tested for the DNA of two flea-borne rickettsiae, Rickettsia typhi and Rickettsia felis. R. felis was commonly detected in fleas collected throughout this area while R. typhi was found at a much lower prevalence in the vicinity of just 7 of 14 case-patient homes identified. DNA of R. felis, but not R. typhi, was detected in renal, hepatic, and pulmonary tissues of opossums. In contrast, there were no hematologic polymerase chain reaction findings of R. felis or R. typhi in opossums, rats, and cats within the endemic area studied. Our data suggest a significant probability of human exposure to R. felis in the area studied; however, disease caused by this agent is not recognized by the medical community and may be misdiagnosed as murine typhus using nondiscriminatory serologic methods.

The screwworm, Cochliomyia hominivorax (Coquerel), remains one of the most damaging parasites of livestock in South America, causing millions of dollars in annual losses to producers. Recently, South American countries demonstrated interest in controlling this pest using the Sterile Insect Technique, and a pilot-project was conducted near the Brazil-Uruguay border in 2009. Since molecular studies have suggested the existence of C. hominivorax regional groups, crossing tests were conducted to evaluate mating competitiveness, mating preference and reproductive compatibility between a C. hominivorax strain from the Caribbean (Jamaica-06) and one from Brazil. Mating rates between Jamaican males and Brazilian females ranged between 82 and 100%, and each male inseminated from 3.3 to 3.95 females. Sterile males, regardless of the strain, competed equally against the fertile males for Brazilian females. Jamaican sterile males and Brazilian fertile males mated randomly with fertile or sterile females. No evidence of genetic incompatibility or hybrid dysgenesis was found in the hybridization crosses. Mating barriers should not compromise the use of Jamaican sterile males for Sterile Insect Technique campaigns in Brazil.

Culex nigripalpus Theobald is a primary vector of St. Louis encephalitis virus in the southeastern United States. Cx. nigripalpus females were fed blood containing a low (4.0 ± 0.01 log₁₀ plaque-forming unit equivalents (PFUeq) /ml) or high (4.7 ± 0.1 log10 PFUeq/ml) St. Louis encephalitis virus dose and maintained at extrinsic incubation temperatures (EIT) of 25 or 28°C for 12 d. Vector competence was measured via quantitative real-time reverse transcriptase polymerase chain reaction to estimate PFUeq using rates of infection, dissemination, and transmission. There were no differences in infection rates between the two EITs at either dose. The low dose had higher infection rates at both EITs. Dissemination rates were significantly higher at 28°C compared with 25°C at both doses. Virus transmission was observed (<7%) only at 28°C for both doses. The virus titer in body tissues was greater at 28°C compared with 25°C at both doses. The difference between the EITs was greater at the low dose, resulting in a higher titer for the low dose than the high dose at 28°C. Virus titers in leg tissues were greater in mosquitoes fed the high versus low dose, but were not influenced by EIT. Further investigations using a variety of environmental and biological factors would be useful in exploring the complexity of vector competence.

In total, 656 sand flies were collected in June 2008 from Louata, a locality of Sefrou province, Morocco. Testing was conducted for the presence of phlebovirus by nested reverse transcriptase polymerase chain reaction and sequencing. We detected Toscana virus in four pools of male Phlebotomus perniciosus. This virus belongs to the genotype B previously recognized in France and Spain. This is the first time that Toscana virus has been detected in Morocco.

During 2008–2010, ticks were collected from road-killed wild animals within the Serra dos Órgãos National Park area in the state of Rio de Janeiro, Brazil. In total, 193 tick specimens were collected, including Amblyomma dubitatum Neumann and Amblyomma cajennense (F.) from four Hydrochoerus hydrochaeris (L.), Amblyomma calcaratum Neumann and A. cajennense from four Tamandua tetradactyla (L.), Amblyomma aureolatum (Pallas) and A. cajennense from five Cerdocyon thous L., Amblyomma longirostre (Koch) from one Sphiggurus villosus (Cuvier), Amblyomma varium Koch from three Bradypus variegatus Schinz, and A. cajennense from one Buteogallus meridionalis (Latham). Molecular analyses based on polymerase chain reaction targeting two rickettsial genes (gltA and ompA) on tick DNA extracts showed that 70.6% (12/17) of the A. dubitatum adult ticks, and all Amblyomma sp. nymphal pools collected from capybaras were shown to contain rickettsial DNA, which after DNA sequencing, revealed to be 100% identical to the recently identified Rickettsia sp. strain Pampulha from A. dubitatum ticks collected in the state of Minas Gerais, Brazil. Phylogenetic analysis with concatenated sequences (gltA-ompA) showed that our sequence from A. dubitatum ticks, referred to Rickettsia sp. strain Serra dos Órgãos, segregated under 99% bootstrap support in a same cluster with Old World rickettsiae, namely R. tamurae, R. monacensis, and Rickettsia sp. strain 774e. Because A. dubitatum is known to bite humans, the potential role of Rickettsia sp. strain Serra dos Órgãos as human pathogen must be taken into account, because both R. tamurae and R. monacencis have been reported infecting human beings.

PCR-Restriction fragment length polymorphism is a time saving and accurate technique to differentiate closely related organisms. In the regions endemic for visceral leishmaniasis in India, various species of morphological similar sand fly exist but only female Phlebotomus argentipes is the vector for visceral leishmaniasis. In the current study primers were designed targeting the 18S rRNA encoding gene that showed amplification in all the major sand fly species found in India. The amplified fragments were further digested using the HinfI or HpaII restriction enzymes. Each of the restriction enzyme produced a species specific restriction patterns, which can easily be used to identify specific sand fly species. This technique can be used in the identification of sand fly species.