Quantification of permethrin resistance and kdr alleles in Florida strains of Aedes aegypti (L.) and Aedes albopictus (Skuse).

Recent outbreaks of locally transmitted dengue and Zika viruses in Florida have placed more emphasis on integrated vector management plans for Aedes aegypti (L.) and Aedes albopictus Skuse. Adulticiding, primarily with pyrethroids, is often employed for the immediate control of potentially arbovirus-infected mosquitoes during outbreak situations. While pyrethroid resistance is common in Ae. aegypti worldwide and testing is recommended by CDC and WHO, resistance to this class of products has not been widely examined or quantified in Florida. To address this information gap, we performed the first study to quantify both pyrethroid resistance and genetic markers of pyrethroid resistance in Ae. aegypti and Ae. albopictus strains in Florida. Using direct topical application to measure intrinsic toxicity, we examined 21 Ae. aegypti strains from 9 counties and found permethrin resistance (resistance ratio (RR) = 6-61-fold) in all strains when compared to the susceptible ORL1952 control strain. Permethrin resistance in five strains of Ae. albopictus was very low (RR<1.6) even when collected from the same containers producing resistant Ae. aegypti. Characterization of two sodium channel kdr alleles associated with pyrethroid-resistance showed widespread distribution in 62 strains of Ae. aegypti. The 1534 phenylalanine to cysteine (F1534C) single nucleotide polymorphism SNP was fixed or nearly fixed in all strains regardless of RR. We observed much more variation in the 1016 valine to isoleucine (V1016I) allele and observed that an increasing frequency of the homozygous V1016I allele correlates strongly with increased RR (Pearson corr = 0.905). In agreement with previous studies, we observed a very low frequency of three kdr genotypes, IIFF, VIFF, and IIFC. In this study, we provide a statewide examination of pyrethroid resistance, and demonstrate that permethrin resistance and the genetic markers for resistance are widely present in FL Ae. aegypti. Resistance testing should be included in an effective management program.

Nighttime Applications of Two Formulations of Pyrethroids are Effective Against Diurnal Aedes albopictus.

The successful control of Aedes albopictus requires a multifaceted approach using a variety of integrated pest management techniques. Because this species is diurnal, nighttime ultra-low volume adulticide applications seem likely to miss resting mosquitoes and, therefore, are often met with skepticism. The goal of this study was to compare the efficacy of nighttime applications of pyrethroids with and without prallethrin to control caged and field populations of Ae. albopictus. During August and September of 2015, 2 adulticide applications were performed, treating 4 urban sites in the city of Trenton. We compared Anvil®, which contains sumithrin and piperonyl butoxide (PBO), to Duet™, which contains sumithrin, prallethrin, and PBO. Because prallethrin excites resting mosquitoes to flight, we hypothesized that Duet would kill more mosquitoes, especially those resting in cryptic harborages. Comparing pretreatment and posttreatment adult mosquito numbers, Biogents Sentinel trap collections revealed twice as many mosquitoes were killed by Duet than by Anvil. For caged Ae. albopictus, both products performed comparably, with Duet achieving a slightly higher mortality in front yards and Anvil achieving a slightly higher mortality in backyards. It is clear that nighttime adulticide applications are effective against Ae. albopictus, and the need to continue efficacy data collection is important because adulticiding is a key component of disease control response.

New Introductions, Spread of Existing Matrilines, and High Rates of Pyrethroid Resistance Result in Chronic Infestations of Bed Bugs (Cimex lectularius L.) in Lower-Income Housing.

Infestations of the common bed bug (Cimex lectularius L.) have increased substantially in the United States in the past 10-15 years. The housing authority in Harrisonburg, Virginia, conducts heat-treatments after bed bugs are detected in a lower-income housing complex, by treating each infested unit at 60°C for 4-6 hours. However, a high frequency of recurrent infestations called into question the efficacy of this strategy. Genetic analysis using Bayesian clustering of polymorphic microsatellite loci from 123 bed bugs collected from 23 units from May 2012 to April 2013 in one building indicated that (a) 16/21 (73%) infestations were genetically similar, suggesting ineffective heat-treatments or reintroductions from within the building or from a common external source, followed by local spread of existing populations; and (b) up to 5 of the infestations represented new genotypes, indicating that 5 new populations were introduced into this building in one year, assuming they were not missed in earlier screens. There was little to no gene flow among the 8 genetic clusters identified in the building. Bed bugs in the U.S. often possess one or both point mutations in the voltage-gated sodium channel, termed knockdown resistance (kdr), from valine to leucine (V419L) and leucine to isoleucine (L925I) that confer target-site resistance against pyrethroid insecticides. We found that 48/121 (40%) bed bugs were homozygous for both kdr mutations (L419/I925), and a further 59% possessed at least one of the kdr mutations. We conclude that ineffective heat treatments, new introductions, reintroductions and local spread, and an exceptionally high frequency of pyrethroid resistance are responsible for chronic infestations in lower-income housing. Because heat treatments fail to protect from reintroductions, and pesticide use has not decreased the frequency of infestations, preventing new introductions and early detection are the most effective strategies to avoid bed bug infestations in multistory apartment buildings.

Bed bug detection: current technologies and future directions.

Technologies to detect bed bugs have not kept pace with their global resurgence. Early detection is critical to prevent infestations from spreading. Detection based exclusively on bites is inadequate, because reactions to insect bites are non-specific and often misdiagnosed. Visual inspections are commonly used and depend on identifying live bugs, exuviae, or fecal droplets. Visual inspections are inexpensive, but they are time-consuming and unreliable when only a few bugs are present. Use of a dog to detect bed bugs is gaining in popularity, but it can be expensive, may unintentionally advertise a bed bug problem, and is not foolproof. Passive monitors mimic natural harborages; they are discreet and typically use an adhesive to trap bugs. Active monitors generate carbon dioxide, heat, a pheromone, or a combination to attract bed bugs to a trap. New technologies using DNA analysis, mass spectrometry, and electronic noses are innovative but impractical and expensive for widespread use.

Vector competence of Aedes albopictus from Virginia and Georgia for chikungunya virus isolated in the Comoros Islands, 2005.

We evaluated recently colonized samples from Virginia and Georgia (USA) of Aedes albopictus, an important vector of chikungunya virus (CHIKV), to determine whether they became infected with and transmitted COM125, a CHIKV isolate from the Comoros Islands. Seven days after imbibing an infective blood meal containing a minimum of 8.5×10(4) plaque-forming units (pfu)/mL, 31-86% of A. albopictus from the counties of Fairfax, Loudon, Rockingham, and Suffolk, Virginia, and Fulton, Georgia were infected. The average viral titer per mosquito was 1.1×10(4) pfu/mL (2×10(2)-3.3×10(4)). We detected CHIKV in salivary expectorate of infected mosquitoes from Rockingham (8%), Fulton (22%), and Loudon (48%) counties 7 days after blood feeding. Because CHIKV has no vaccines or specific antiviral treatments, vector control and education are critical to prevent its transmission. We discuss how local populations of A. albopictus could transmit CHIKV introduced to the southeastern USA from the Indian Ocean or Indian Subcontinent.

Nutritional stress affects mosquito survival and vector competence for West Nile virus.

Most anautogenous female mosquitoes ingest plant carbohydrates for flight energy and survival, and they imbibe vertebrate blood for egg development. We evaluated the effect of different sucrose meals following a blood meal containing West Nile virus (WNV) on Culex pipiens pipiens survival, nutritional status, and susceptibility to viral infection and transmission. Ten days after blood feeding, no mosquitoes survived on distilled water, 55% survived on 2% sucrose, 61% on 10 and 20% sucrose meals, and over 70% survived on 40% sucrose. There was a positive correlation between sucrose meal concentration and detectable sugars, glycogen, and lipid in whole-body homogenates. Average sugar values increased from 0 microg per starved mosquito (range 0-1.0 microg) to an average of 392 microg per mosquito fed on 40% sucrose (85-1088 microg). Average glycogen values increased from 0 microg (0-5.7 microg) to an average of 620 microg (118-1421 microg). Average lipid values were identical for mosquitoes in the starved and 2% sucrose series (38 microg) and increased to 172 microg per mosquito fed on 40% sucrose (92-266 microg). Mosquitoes in all sucrose series were equally susceptible to WNV infection (p > 0.5), but mosquitoes with lower nutrient reserves as a result of lower sucrose meals were more likely to orally transmit virus (p < 0.05). We discuss how mosquito nutritional status influences probability of daily survival, susceptibility to infection, and vectorial capacity. We conclude that maintaining C. p. pipiens on standard 10% sucrose is justified in light of these results.

Geographic variation in vector competence for West Nile virus in the Culex pipiens (Diptera: Culicidae) complex in California.

We evaluated the susceptibility to infection and transmission of West Nile virus (WNV) in seven populations of Culex pipiens pipiens (L.), Cx. p. quinquefasciatus Say, and from populations containing Cx. pipiens/quinquefasciatus hybrids in a north-south transect of California. Samples were identified to species or as hybrid forms based on morphology of male terminalia. After 7 and 14 days of extrinsic incubation, few females were infected and none transmitted WNV from samples of Cx. p. pipiens from northern Shasta County and of Cx. p. quinquefasciatus from southern Los Angeles County. Seven days after infective feeding, 13%-36% of mosquitoes from the counties of Merced, Fresno, Kern, and San Bernardino were infected, and 12%-40% of infected mosquitoes expressed WNV in salivary expectorate. Fourteen days after infective feeding, 18%-43% of mosquitoes from these counties were infected, and 50%-69% of infected mosquitoes transmitted WNV in salivary expectorate. A sample of Cx. p. quinquefasciatus from Riverside County did not transmit WNV after 7 days, but 71% transmitted 14 days after infective feeding. These results reveal extensive geographic variation in vector competence for WNV in the Culex pipiens complex in California.

Apoptosis in mosquito midgut epithelia associated with West Nile virus infection.

The mosquito Culex pipiens pipiens is a documented vector of West Nile virus (WNV, Flaviviridae, Flavivirus). Our laboratory colony of C. p. pipiens, however, was repeatedly refractory to experimental transmission of WNV. Our goal was to identify if a cellular process was inhibiting virus infection of the midgut. We examined midguts of mosquitoes fed control and WNV-infected blood meals. Three days after feeding, epithelial cells from abdominal midguts of mosquitoes fed on WNV fluoresced under an FITC filter following Acridine Orange staining, indicating apoptosis in this region. Epithelial cells from experimental samples examined by TEM exhibited ultrastructural changes consistent with apoptosis, including shrinkage and detachment from neighbors, heterochromatin condensation, nuclear degranulation, and engulfment of apoptotic bodies by adjacent cells. Virions were present in cytoplasm and within cytoplasmic vacuoles of apoptotic cells. No apoptosis was detected by TEM in control samples. In parallel, we used Vero cell plaque assays to quantify infection after 7 and 10 day extrinsic incubation periods and found that none of the mosquitoes (0/55 and 0/10) which imbibed infective blood were infected. We propose that programmed cell death limits the number of WNV-infected epithelial cells and inhibits disseminated viral infections from the mosquito midgut.

Isolation of a myoinhibitory peptide from Leishmania major (Kinetoplastida: Trypanosomatidae) and its function in the vector sand fly Phlebotomus papatasi (Diptera: Psychodidae).

Protozoan parasites in the genus Leishmania are ingested by sand flies with blood and multiply in the gut until they are transmitted to a vertebrate host when the sand fly blood feeds again. Infections of the enzootic vector Phlebotomus papatasi Scopoli result in distended midguts with no spontaneous gut contractions. Using a P. papatasi hindgut contraction bioassay, a paralytic factor sensitive to trypsin, chymotrypsin, proteinase-K, and heating at 56 degrees C was detected in crude lysates of Leishmania major promastigotes. Application of parasite lysate to isolated hindguts resulted in reversible, dose-dependent inhibition of spontaneous contractions. Mean volume of isolated midguts and hindguts increased by 50-60% after application of L. major lysate. L. major paralytic factor was purified 10(4)-fold over the total protein preparation and yielded a hydrophobic 12-kDa peptide. Myoinhibitory activity eluted as a single peak in reverse phase-high-pressure liquid chromatography. Tandem mass spectrometry resulted in 15 amino acid sequences, three of them sharing 45-73% homology with short hypothetical gene products of undefined function from Pseudomonas, Halobacterium, and Drosophila. This unique protozoan peptide mimics the function of endogenous insect neuropeptides that control visceral muscle contractions. By this novel mechanism, parasites persist in the expanded, relaxed midgut after blood meal and peritrophic matrix digestion. This allows time for development and migration of infective forms, facilitating sand fly vector competence and parasite transmission.

Leishmania parasites (Kinetoplastida: Trypanosomatidae) reversibly inhibit visceral muscle contractions in hemimetabolous and holometabolous insects.

Female sand flies can acquire protozoan parasites in the genus Leishmania when feeding on an infected vertebrate host. The parasites complete a complex growth cycle in the sand fly gut until they are transmitted by bite to another host. Recently, a myoinhibitory peptide was isolated from Leishmania major promastigotes. This peptide caused significant gut distension and reversible, dose-dependent inhibition of spontaneous hindgut contractions in the enzootic sand fly vector, Phlebotomus papatasi. The current study further characterizes myoinhibitory activity in L. major and other kinetoplastid parasites, using the P. papatasi hindgut and other insect organ preparations. Myoinhibitory activity was greatest in cultured promastigotes and in culture medium in late log-phase and early stationary-phase, coinciding with development of infective Leishmania morphotypes in the sand fly midgut. L. major promastigote lysates inhibited spontaneous contractions of visceral muscle preparations from hemimetabolous (Blattaria and Hemiptera) and holometabolous (Diptera) insects. Inhibition of visceral muscle contractions in three insect orders indicates a conserved mode of action. Myoinhibitory activity was detected also in Leishmania braziliensis braziliensis, a Sudanese strain of Leishmania donovani, and the kinetoplastid parasite Leptomonas seymouri. Protozoan-induced myoinhibition mimics the effect of insect myotropins. Inhibiting host gut contractions protects Leishmania parasites from being excreted after blood meal and peritrophic matrix digestion, allowing development and transmission of infective forms.