David A. Florin

Discovery, Distribution, and Abundance of the Newly Introduced Mosquito Ochlerotatus japonicus (Diptera: Culicidae) in Connecticut, USA

Abstract The earliest documented specimen of an exotic east Asian mosquito Ochlerotatus (Finlaya) japonicus japonicus (Theobald) in the Western Hemisphere is reported along with the results of a state wide survey to determine the distribution and abundance of this mosquito in Connecticut. Ochlerotatus japonicus was collected from 87 locations in eight counties. It is established throughout the state and occurs in a variety of natural and artificial container habitats including discarded tire casings, bird baths, wooden barrels, porcelain bath tubs (used for watering animals), plastic milk cartons, toys, vinyl tarpaulins (covering wood piles and swimming pools), exposed rock holes in stream beds, tree holes, subterranean catch basins, surface water rain pools, and spring-fed depressions. Larvae were particularly common in containers with water, decaying leaves, and algae, in shaded and sunlit areas and, in rock-pool habitats along streambeds, in association with Ochlerotatus atropalpus (Coquillett). Adult females were collected in sod grass-infused gravid and CO2-baited light traps, from early June through October, with peak collections in September. Biting females were collected by human bait method augmented with CO2, verifying its capacity to feed on humans. The ovitraps used in this study were not effective for recovering this species. Our results suggest that Oc. japonicus was introduced into Connecticut between 1992 and 1998. Because of the ability of Oc. japonicus to transmit West Nile virus, and because of the recent detection of this virus in field-collected specimens, the introduction of Oc. japonicus is considered a significant public health development.

New State Records for Lutzomyia shannoni and Lutzomyia vexator

ABSTRACT Two species of phlebotomine sand flies, Lutzomyia shannoni (Dyar) and Lutzomyia vexator (Coquillett), are reported for the first time from Kentucky and Ohio. L. vexator also is reported for the first time from Tennessee. These insects were found in a northeasterly band extending from southwestern Kentucky to southwestern Ohio. Both species were consistently captured from mid-July through September in 2006 and 2007 by using CO2-baited Center for Disease Control light traps. Weekly sampling revealed that these flies are more abundant in the southern part of this band than in the northern part, but increasing densities throughout this new range indicate that the flies are currently expanding their range. Although both species have been reported further north along the Atlantic coast, and L. vexator along the Pacific coast, neither of them had been reported this far north along the Mississippi Valley. Previous reports established L. shannoni as far north as west central Tennessee and L. vexator in a similar spatial pattern in the eastern part of its range, extending as far north as northern Alabama. Whether the new records reported herein represent a northerly expansion of the geographic range of these species or are reflective of sampling changes is inconclusive. However, the former scenario could presage an increased prevalence of the diseases associated with this group of insects.

Detection of Rickettsia parkeri from within Piura, Peru, and the First Reported Presence of Candidatus Rickettsia andeanae in the Tick Rhipicephalus sanguineus

Domestic farm animals (n=145) were sampled for the presence of ectoparasites in northwestern Peru during March, 2008. Ninety domestic animals (62%) were positive for the presence of an ectoparasite(s) and produced a total collection of the following: 728 ticks [Amblyomma maculatum, Anocentor nitens, Rhipicephalus (Boophilus) microplus, Rhipicephalus sanguineus, and Otobius megnini], 12 lice (Haematopinus suis), and 3 fleas (Ctenocephalides felis). A Rickettsia genus-specific qPCR assay was performed on nucleic acid preparations of the collected ectoparasites that resulted in 5% (37/743, 35 ticks and 2 fleas) of the ectoparasites positive for the presence of Rickettsia. DNA from the positive individual ticks was tested with 2 other qPCR assays for the presence of the ompB gene in Candidatus Rickettsia andeanae or Rickettsia parkeri. Candidatus R. andeanae was found in 25 A. maculatum ticks and in two Rh. sanguineus ticks, whereas R. parkeri was detected in 6 A. maculatum ticks. Two A. maculatum were co-infected with both Candidatus R. andeanae and R. parkeri. Rickettsia felis was detected in 2 fleas, Ctenocephalides felis, by multilocus sequence typing of the 17-kD antigen and ompA genes. These findings expand the geographic range of R. parkeri to include Peru as well as expand the natural arthropod vector of Candidatus R. andeanae to include Rhipicephalus sanguineus.

Morphometric and molecular analyses of the sand fly species Lutzomyia shannoni (Diptera: Psychodidae: Phlebotominae) collected from seven different geographical areas in the southeastern United States.

ABSTRACT A morphometric and molecular study of adult male and female Lutzomyia shannoni (Dyar 1929) collected at seven different locations within the southeastern United States was conducted to assess the degree of divergence between the grouped specimens from each location. The collection locations were as follows: Fort Bragg, NC; Fort Campbell, KY; Fort Rucker, AL; Ossabaw Island, GA; Patuxent National Wildlife Research Refuge, MD; Suwannee National Wildlife Refuge, FL; and Baton Rouge, LA. Forty males and forty females from each location were analyzed morphometrically from 54 and 49 character measurements, respectively. In addition, the molecular markers consisting of the partial cytochrome c oxidase subunit I (from 105 sand flies: 15 specimens/collection site) and the partial internal transcribed spacer 2 (from 42 sand flies: six specimens/collection site) were compared. Multivariate analyses indicate that the low degree of variation between the grouped specimens from each collection site prevents the separation of any collection site into an entity that could be interpreted as a distinct population. The molecular analyses were in concordance with the morphometric study as no collection location grouped into a separate population based on the two partial markers. The grouped specimens from each collection site appear to be within the normal variance of the species, indicating a single population in the southeast United States. It is recommended that additional character analyses of L. shannoni based on more molecular markers, behavioral, ecological, and physiological characteristics, be conducted before ruling out the possibility of populations or a cryptic species complex within the southeastern United States.

Molecular Identification of Sand Flies (Diptera: Psychodidae) in Eastern North America by Using PCR-RFLP

ABSTRACT Sand flies (Diptera: Psychodidae) are small blood-feeding dipterans that are primary vectors of numerous human and livestock pathogens. Effective surveillance programs with accurate identification tools are critical in development and implementation of modern integrated pest management programs. Although morphological keys are available for North American species, identification can still be challenging owing to the nature of sample preparation and incompatibility with molecular or biochemical-based pathology assays. Further, the potential for introduction of Old World or other exotic species is not accounted for by current keys. Herein, we present the development and validation of a restriction fragment-length polymorphism-based molecular identification method. Specifically, cytochrome c oxidase subunit I, a mitochondrial DNA marker, was used to distinguish two species of adult sand flies indigenous to eastern North America with two exotic species not yet known to occur in the United States.

Morphological Anomalies in Two Lutzomyia (Psathyromyia) shannoni (Diptera: Psychodidae: Phlebotominae) Specimens Collected From Fort Rucker, Alabama, and Fort Campbell, Kentucky

ABSTRACT This report describes two male specimens of the sand fly species Lutzomyia shannoni (Dyar) (Diptera: Psychodidae: Phlebotominae) collected at Fort Rucker, AL, and Fort Campbell, KY, in dry ice-baited light traps during September 2005. The specimens were observed to have anomalies to the number of spines on the gonostyli. The taxonomic keys of Young and Perkins (Mosq. News 44: 263–285; 1984) use the number of spines on the gonostylus in the first couplet to differentiate two major groupings of North American sand flies. The two anomalous specimens were identified as L. shannoni based on the following criteria: 1) both specimens possess antennal ascoids with long, distinct proximal spurs (a near diagnostic character of L. shannoni in North America), 2) the sequences of the partial cytochrome c oxidase subunit 1 gene from both specimens indicated L. shannoni, and 3) the sequences of the internal transcribed spacer 2 molecular marker from both specimens indicated L. shannoni. The anomalous features are fundamentally different from each other as the Fort Rucker specimen possesses a fifth spine (basally located) on just one gonostylus, whereas the Fort Campbell specimen possesses five spines (extra spines subterminally located) on both gonostyli. Because the gonostyli are part of the external male genitalia, anomalies in the number of spines on the gonostyli may have serious biological consequences, such as reduced reproductive success, for the possessors. These anomalies are of taxonomic interest as the specimens could easily have been misidentified using available morphological keys.

Association of vectors and environmental conditions during the emergence of Peruvian horse sickness orbivirus and Yunnan orbivirus in northern Peru

ABSTRACT: Since 1983, cases of diseased donkeys and horses with symptoms similar to those produced by alphaviruses were identified in two departments in northern Peru; however serological testing ruled out the presence of those viruses and attempts to isolate an agent were also unproductive. In 1997, also in northern Peru, two new orbiviruses were discovered, each recognized as a causative agent of neurological diseases in livestock and domestic animals and, at the same time, mosquitoes were found to be infected with these viruses. Peruvian horse sickness virus (PHSV) was isolated from pools of culicid mosquitoes, Aedes serratus and Psorophora ferox, and Yunnan virus (YUOV) was isolated from Aedes scapularis in the subtropical jungle (upper jungle) located on the slope between the east side of the Andes and the Amazonian basin in the Department of San Martín. Both viruses later were recovered from mosquitoes collected above the slope between the west side of the Andes and the coast (Department of Piura) in humid subtropical areas associated with the Piura River basin. In this region, PHSV was isolated from Anopheles albimanus and YUOV was isolated from Ae. scapularis. We discuss the ecology of vector mosquitoes during the outbreaks in the areas where these mosquitoes were found.

Divergence of Lutzomyia (Psathyromyia) shannoni (Diptera: Psychodidae: Phlebotominae) is Indicated by Morphometric and Molecular Analyses when Examined between Taxa from the Southeastern United States and Southern Mexico

ABSTRACT The medically important sand fly Lutzomyia shannoni (Dyar 1929) was collected at eight different sites: seven within the southeastern United States and one in the state of Quintana Roo, Mexico. A canonical discriminant analysis was conducted on 40 female L. shannoni specimens from each of the eight collection sites (n = 320) using 49 morphological characters. Four L. shannoni specimens from each of the eight collection sites (n = 32) were sent to the Barcode of Life Data systems where a 654-base pair segment of the cytochrome c oxidase subunit 1 (CO1) genetic marker was sequenced from each sand fly. Phylogeny estimation based on the COI segments, in addition to genetic distance, divergence, and differentiation values were calculated. Results of both the morphometric and molecular analyses indicate that the species has undergone divergence when examined between the taxa of the United States and Quintana Roo, Mexico. Although purely speculative, the arid or semiarid expanse from southern Texas to Mexico City could be an allopatric barrier that has impeded migration and hence gene flow, resulting in different morphology and genetic makeup between the two purported populations. A high degree of intragroup variability was noted in the Quintana Roo sand flies.

Population Dynamics of Lutzomyia shannoni (Diptera: Psychodidae) at the Patuxent National Wildlife Research Refuge, Maryland

Abstract The seasonal abundance and temporal patterns of the adult sand fly (Lutzomyia shannoni Dyar) were examined at the Patuxent National Wildlife Research Refuge, MD, from August 3, 2005, to July 29, 2006. A total of 138 (53 males, 85 females) L. shannoni was collected from 4 dry ice–baited traps set at the same 4 locations throughout the study. The male∶female ratio was 1∶2.4. All 4 traps, separated by a maximum distance of approximately 1.6 km, operated simultaneously on the collection dates. The collection dates were spaced apart by near weekly intervals during the months of expected sand fly activity. No collections occurred in December–February. August was clearly the period of peak adult abundance as the numbers collected were significantly greater during this month than any other month of collection. Results indicate the existence of a unimodal pattern of abundance with adult emergence beginning in June and ending by September. The temporal pattern and abundance differ from what has been observed for the species on Ossabaw Island, a barrier island located along coastal Georgia, and at San Felasco Hammock Preserve State Park, Gainesville, FL. Continued research is needed to compile multiyear data to confirm the temporal abundance patterns of this species in Maryland.