Brian D. Byrd

Field assessments in western Kenya link malaria vectors to environmentally disturbed habitats during the dry season

BackgroundNumerous malaria epidemics have occurred in western Kenya, with increasing frequency over the past 20 years. A variety of hypotheses on the etiology of these epidemics have been put forth, with different implications for surveillance and control. We investigated the ecological and socioeconomic factors promoting highland malaria vectors in the dry season after the 2002 epidemic.MethodsInvestigations were conducted in Kisii District during the dry season. Aquatic habitats in were surveyed for presence of malaria vectors. Brick-making pits were further investigated for co-associations of larval densities with emergent vegetation, habitat age, and predator diversity. Indoor spray catches were completed in houses near aquatic habitats. Participatory rural appraisals (PRAs) were conducted with 147 community members.ResultsThe most abundant habitat type containing Anopheles larvae was brick-making pits. Vegetation and habitat age were positively associated with predator diversity, and negatively associated with mosquito density. Indoor spray catches found that houses close to brick-making sites had malaria vectors, whereas those next to swamps did not. PRAs revealed that brick-making has grown rapidly in highland swamps due to a variety of socioeconomic pressures in the region.ConclusionBrick-making, an important economic activity, also generates dry season habitats for malaria vectors in western Kenya. Specifically, functional brick making pits contain less that 50% as many predator taxa and greater than 50% more mosquito larvae when compared with nearby abandoned brick making pits. Further evaluations of these disturbed, man-made habitats in the wet season may provide information important for malaria surveillance and control.

IMPACT OF WEST NILE VIRUS OUTBREAK UPON ST. TAMMANY PARISH MOSQUITO ABATEMENT DISTRICT

ABSTRACT St. Tammany Parish, Louisiana, experienced an outbreak of West Nile virus (WNV) in 2002, with 40 human cases and 4 deaths, most occurring from June to August. Culex pipiens quinquefasciatus was believed to be the primary vector of WNV during the outbreak, although circumstantial evidence suggests that Aedes albopictus also may have been involved in transmission. Dead bird reports were the 1st indication of the outbreak and were an excellent indicator of WNV activity; however, sentinel chickens were the most effective in tracking viral activity. Although sentinel chickens tested positive 2–3 wk after the 1st dead bird, they peaked at about the same time as human cases, and continued testing positive when viral activity was no longer detected in birds and mosquito pools. Lag time between the 1st positive sentinel chicken and the peak in human cases was 6 wk. If this trend continues in the future, sentinel chickens could be used to predict the peak in number of human cases. The 2002 WNV outbreak had a strong impact on operational budget of the St. Tammany Parish Mosquito Abatement District (88% increase above the 3-year average). Vector control activities accounted for most of the operational increase and consisted of targeted population reduction of known WNV-competent mosquito species. The goal of these activities was to prevent new human WNV cases. The 3- to 10-fold reduction in vector mosquito populations from May to August, together with a dramatic drop in number of new human cases by the end of August, indicated that our strategy was effective.

PCR detection of malaria parasites in desiccated Anopheles mosquitoes is uninhibited by storage time and temperature

BackgroundReliable methods to preserve mosquito vectors for malaria studies are necessary for detecting Plasmodium parasites. In field settings, however, maintaining a cold chain of storage from the time of collection until laboratory processing, or accessing other reliable means of sample preservation is often logistically impractical or cost prohibitive. As the Plasmodium infection rate of Anopheles mosquitoes is a central component of the entomological inoculation rate and other indicators of transmission intensity, storage conditions that affect pathogen detection may bias malaria surveillance indicators. This study investigated the effect of storage time and temperature on the ability to detect Plasmodium parasites in desiccated Anopheles mosquitoes by real-time polymerase chain reaction (PCR).MethodsLaboratory-infected Anopheles stephensi mosquitoes were chloroform-killed and stored over desiccant for 0, 1, 3, and 6 months while being held at four different temperatures: 28, 37, -20 and -80°C. The detection of Plasmodium DNA was evaluated by real-time PCR amplification of a 111 base pair region of block 4 of the merozoite surface protein.ResultsVarying the storage time and temperature of desiccated mosquitoes did not impact the sensitivity of parasite detection. A two-way factorial analysis of variance suggested that storage time and temperature were not associated with a loss in the ability to detect parasites. Storage of samples at 28°C resulted in a significant increase in the ability to detect parasite DNA, though no other positive associations were observed between the experimental storage treatments and PCR amplification.ConclusionsCold chain maintenance of desiccated mosquito samples is not necessary for real-time PCR detection of parasite DNA. Though field-collected mosquitoes may be subjected to variable conditions prior to molecular processing, the storage of samples over an inexpensive and logistically accessible desiccant will likely ensure accurate assessment of malaria parasite presence without diminishing PCR-detection of parasites in mosquitoes stored for at least six months.

Sequence, Secondary Structure, and Phylogenetic Analyses of the Ribosomal Internal Transcribed Spacer 2 (ITS2) in Members of the North American Signifera Group of Orthopodomyia (Diptera: Culicidae)

ABSTRACT 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.

La Crosse Encephalitis A Persistent Arboviral Threat in North Carolina

Mosquito-borne diseases remain a significant cause of economic, social, and health burdens in North Carolina. Although recently overshadowed by emerging threats such as chikungunya virus and Zika virus, La Crosse virus and other endemic arboviruses remain persistent environmental health hazards. Indeed, La Crosse virus, West Nile virus, and Eastern equine encephalitis virus accounted for more than 98% of the reported human arboviral diseases acquired in North Carolina in the past decade. Arbovirus infection is increasingly prevalent in Western North Carolina, with La Crosse encephalitis being endemic in this area. While infections are often asymptomatic and seldom fatal, the long-term neurologic sequelae of La Crosse encephalitis represent a significant burden.

Aedes pertinax, a Newly Recognized Mosquito Species in the United States

Abstract Specimens of a mosquito new to the continental USA, Aedes pertinax, were retrospectively identified from 2 collections made in 2011 in Indian River County, FL. Routine mosquito surveillance in subsequent years yielded more than 700 specimens appearing in 100 collections. The distribution of this mosquito in Florida and the United States is currently unknown, and recognition of the adult female is likely hampered by morphological similarities to Ae. atlanticus and Ae. tormentor.

Molecular Identification of Aedes triseriatus and Aedes hendersoni by a Novel Duplex Polymerase Chain Reaction Assay

Abstract Aedes triseriatus is the principal vector of La Crosse virus (LACv), which is the most common cause of pediatric arboviral encephalitis in North America. Here we report a novel species-specific polymerase chain reaction (PCR) assay that differentially identifies Ae. triseriatus and Ae. hendersoni. Because these 2 sibling species differ in their abilities to transmit LACv, accurate identification is critical for surveillance, research, and control programs. This duplex assay can detect the presence of both species in a single PCR reaction and is therefore simpler and faster than previously reported methods.

Molecular Identification of Aedes bahamensis (Diptera: Culicidae)

Aedes (Howardina) bahamensis Berlin (or Howardina bahamensis of Reinert et al. 2004) is an exotic species first detected in the United States in 1986 from eggs and adult females collected in Miami-Dade and Broward counties, Florida (Berlin 1969; Pafume et al. 1988; O’Meara et al. 1989). In the U.S., immature Ae. bahamensis are chiefly found in artificial containers (e.g., abandoned tires, cemetery vases, etc.) and exotic tank bromeliads (O’Meara et al. 1995a). Although Ae. bahamensis readily feeds on humans, it is not a major pest or disease vector. However, the immature stages are frequently found in habitats that are also known to harbor 2 medically important mosquito species: Ae. albopictus (Skuse) and Ae. aegypti (L.) (O’Meara et al. 1995b; Lounibos et al. 2010). Morphological differences between these 3 species provide useful characters that readily distinguish the egg, larval, and adult life stages (Linley 1989; Darsie & Ward 2005). Nevertheless, a molecular method to identify Ae. bahamensis would be useful to confirm the identity of damaged adult specimens collected in fan-based traps and would allow rapid identification of the species at any life stage. Here we report the rDNA second internal transcribed spacer (ITS2) sequence and a species-specific PCR primer for the identification of Ae. bahamensis. Specimens of Aedes bahamensis were obtained from colony material maintained by GFO at the Florida Medical Entomology Laboratory (Vero Beach, Florida) and field samples collected by BDB. Field collected specimens were identified using key characters described by Darsie (1992). Total DNA was obtained from adult specimens (8 colony and 14 field) using the Qiagen DNeasy kit (Qiagen, Valencia, California) or the DNAzol reagent (Molecular Research Center, Inc., Cincinnati, Ohio) per the manufacturers’ instructions. The resulting extractions were PCR amplified in 50 μL reactions using the Invitrogen PCR Supermix (Invitrogen, Carsbad, California). Each reaction mixture contained 3 μLs of DNA template (435 ng/μL), 1 μL of each forward and reverse primer (200 nM final concentration), and 45 μL of the PCR supermix. Amplification cycling conditions were 94 °C for 5 min followed by 35 cycles of 94 °C for 1 min, 54 °C for 30 s and 72 °C for 1 min. The CP-P1A/P1B primer pair was used to amplify the complete ITS2 (Fig. 1) (Wesson et al. 1992). A negative control (H2O en lieu of DNA template) was included in each run. PCR amplicons were visualized on a 1.5% agarose gel. There were no obvious intraspecific amplicon size polymorphisms. The PCR products were gel purified with the Qiaquick Gel Extraction kit (Qiagen) and subsequently cloned into the pCR 2.1 TOPO vector (Invitrogen). Purified plasmids were obtained using the Promega Wizard Plus SV miniprep kit (Promega, Madison, WI) and then sequenced (n = 10) using the Applied Biosystems (Carlsbad, CA) Big Dye Terminator V3.0 chemistry by the Davis Sequencing Facility, University of California (Davis, California). The sequences were verified as ITS2 after evaluating the results of an NCBI BLAST query, secondary structure analysis, and the identification of specific sequence motifs known to exist on the ITS2 of mosquitoes (Coleman 2007). Novel ITS2 sequences, partial 5.8S, and partial 28S sequences for Aedes bahamensis were annotated and representative samples were submitted to the NCBI GenBank (Accession numbers: JN020552, JN020553) (Keller et al. 2009). The CP-P1A/P1B primer pair produces a 380 base pair (bp) amplicon for Ae. bahamensis that differs from the amplicons of Ae. albopictus (600 bp), Ae. aegypti (360 bp), and Ae. triseriatus (Say)

Insecticide Susceptibility Screening Against Culex and Aedes (Diptera: Culicidae) Mosquitoes From the United States

Abstract Mosquitoes exposed to sublethal doses of insecticides may be selected for resistance to insecticide active ingredients (AIs). Mosquitoes are exposed to AIs through agricultural, public/private mosquito control programs, homeowners, and other sources. Hence, mosquito control programs should routinely measure the resistance/susceptibility status of mosquito populations of public health concern.The objectives here were to determine resistance status for six AIs used in adult mosquito control in the United States to assess how resistance/susceptibility differs between AI, mosquito species (states where > 1 species collected), and between years (some populations sampled for 2 yr). Field-collected eggs from 21 mosquito populations of six different species or hybrid species (Aedes albopictus Skuse [Diptera: Culicidae], Aedes aegypti L. [Diptera: Culicidae], Culex nigripalpus Theobald, Culex pipiens L. [Diptera: Culicidae], Culex quinquefasciatus Say [Diptera: Culicidae], Cx. pipiens/quinquefasciatus) were obtained. Centers for Disease Control and Prevention bottle bioassays were used to assess the resistance/susceptibility status for six AIs (bifenthrin, deltamethrin, etofenprox, malathion, permethrin, and phenothrin). World Health Organization guidelines were used to classify mosquitoes as susceptible (98–100% mortality at diagnostic time [DT]), possibly resistant (80–97% mortality at DT), or resistant (<80% mortality at DT). Significant differences were observed in mosquito susceptibility/resistance between species and AIs. In states where both Aedes and Culex were collected, the odds of exhibiting resistance in Culex were 68–69 times higher than Aedes (Texas odds ratio: 69.30; 95% confidence interval: 5.86, 819.44; P = 0.001; North Carolina odds ratio: 67.99; 95% confidence interval: 15.21, 303.94; P < 0.0001). Some level of resistance was detected against all tested AIs in several mosquito populations and some varied between 2015 and 2016.

Differentiation of Aedes atlanticus and Aedes tormentor by restriction fragment length polymorphisms of the second internal transcribed spacer.

Abstract Using novel DNA sequence data, we designed a restriction enzyme assay that distinguishes Aedes atlanticus and Ae. tormentor, based on size polymorphisms. The restriction endonuclease Hpy188I digests polymerase chain reaction–amplified 2nd internal transcribed spacer products once for Ae. atlanticus and twice for Ae. tormentor, thus providing a useful method for identifying adult female collections that are generally considered morphologically indistinguishable.