Stable Transmission of Borrelia burgdorferi Sensu Stricto on the Outer Banks of North Carolina.

The spirochaete (Borrelia burgdorferi) associated with Lyme disease was detected in questing ticks and rodents during a period of 18 years, 1991-2009, at five locations on the Outer Banks of North Carolina. The black-legged tick (Ixodes scapularis) was collected at varied intervals between 1991 and 2009 and examined for B. burgdorferi. The white-footed mouse (Peromyscus leucopus), house mouse (Mus musculus) marsh rice rat (Oryzomys palustris), marsh rabbit (Sylvilagus palustris), eastern cottontail (Sylvilagus floridanus) and six-lined racerunner (Cnemidophorus sexlineatus) were live-trapped, and their tissues cultured to isolate spirochaetes. Borrelia burgdorferi isolates were obtained from questing adult I. scapularis and engorged I. scapularis removed from P. leucopus, O. palustris and S. floridanus. The prevalence of B. burgdorferi infection was variable at different times and sites ranging from 7 to 14% of examined questing I. scapularis. Mitochondrial (16S) rRNA gene phylogenetic analysis from 65 adult I. scapularis identified 12 haplotypes in two major clades. Nine haplotypes were associated with northern/Midwestern I. scapularis populations and three with southern I. scapularis populations. Sixteen isolates obtained from tick hosts in 2005 were confirmed to be B. burgdorferi by amplifying and sequencing of 16S rRNA and 5S-23S intergenic spacer fragments. The sequences had 98-99% identity to B. burgdorferi sensu stricto strains B31, JD1 and M11p. Taken together, these studies indicate that B. burgdorferi sensu stricto is endemic in questing I. scapularis and mammalian tick hosts on the Outer Banks of North Carolina.

Innovative sugar-insecticide feeding bioassay for adult female Anopheles gambiae (Diptera: Culicidae).

The primary malaria vector in sub-Saharan Africa, Anopheles gambiae Giles (Diptera: Culicidae), is an anthropophagic and endophilic mosquito targeted for control with insecticides applied to interior resting surfaces and impregnated onto bed net materials. Effective malaria vector management involves monitoring the insecticide susceptibility of mosquito populations. Contemporary bioassays are based on mosquito contact with insecticide residues. We developed an innovative insecticide bioassay system that involves mosquito ingestion of a sugar-insecticide solution. The sucrose-permethrin solution in our bioassay system contained Trypan blue dye, creating a visual marker of insecticide ingestion in the mosquito's abdomen. Blue fecal spots deposited in the bioassay container provided further evidence of mosquito feeding. We used our bioassay to characterize the permethrin susceptibility of adult females of two strains of A. gambiae, one of which was susceptible and the other exhibited reduced susceptibility to permethrin. We compared the dose-response of both strains to permethrin in a forced-contact filter paper bioassay. Both assay approaches produced similar dose-dependent mortality, indicating that the feeding bioassay had appropriately characterized permethrin susceptibility for both mosquito strains.

Responses of Amblyomma americanum and Dermacentor variabilis to odorants that attract haematophagous insects.

Carbon dioxide (CO(2) ), 1-octen-3-ol, acetone, ammonium hydroxide, L-lactic-acid, dimethyl trisulphide and isobutyric acid were tested as attractants for two tick species, Amblyomma americanum and Dermacentor variabilis (Acari: Ixodidae), in dose-response bioassays using Y-tube olfactometers. Only CO(2) , acetone, 1-octen-3-ol and ammonium hydroxide elicited significant preferences from adult A. americanum, and only CO(2) was attractive to adult D. variabilis. Acetone, 1-octen-3-ol and ammonium hydroxide were separately evaluated at three doses against CO(2) (from dry ice) at a field site supporting a natural population of A. americanum nymphs and adults. Carbon dioxide consistently attracted the highest number of host-seeking ticks. However, for the first time, acetone, 1-octen-3-ol and ammonium hydroxide were shown to attract high numbers of A. americanum. Further research is needed to determine the utility of these semiochemicals as attractants in tick surveillance and area-wide management programmes.

Proof of concept for a novel insecticide bioassay based on sugar feeding by adult Aedes aegypti (Stegomyia aegypti).

Aedes aegypti L. (Stegomyia aegypti) (Diptera: Culicidae) is the principal vector of dengue and yellow fever viruses in tropical and subtropical regions of the world. Disease management is largely based on mosquito control achieved by insecticides applied to interior resting surfaces and through space sprays. Population monitoring to detect insecticide resistance is a significant component of integrated disease management programmes. We developed a bioassay method for assessing insecticide susceptibility based on the feeding activity of mosquitoes on plant sugars. Our prototype sugar-insecticide feeding bioassay system was composed of inexpensive, disposable components, contained minimal volumes of insecticide, and was compact and highly transportable. Individual mosquitoes were assayed in a plastic cup that contained a sucrose-permethrin solution. Trypan blue dye was added to create a visual marker in the mosquito's abdomen for ingested sucrose-permethrin solution. Blue faecal spots provided further evidence of solution ingestion. With the sugar-insecticide feeding bioassay, the permethrin susceptibility of Ae. aegypti females from two field-collected strains was characterized by probit analysis of dosage-response data. The field strains were also tested by forced contact of females with permethrin residues on filter paper. Dosage-response patterns were similar, indicating that the sugar-insecticide feeding bioassay had appropriately characterized the permethrin susceptibility of the two strains.

First report of the repellency of 2-tridecanone against ticks.

2-Tridecanone and 2-undecanone are both found naturally in the trichomes of wild tomato plants and are important in plant resistance to herbivory. 2-Undecanone is the U.S. Environmental Protection Agency (EPA)-registered active ingredient in the commercially available arthropod repellent, BioUD(®) . The goal of this study was to examine the tick repellency of 2-tridecanone. Two-choice bioassays were conducted using 8% 2-tridecanone vs. the repellent carrier (absolute ethanol) and compared with two-choice studies using 8% 2-undecanone vs. absolute ethanol. Unfed, host-seeking adult (mixed sex) Amblyomma americanum (L.) (Acari: Ixodidae) and Dermacentor variabilis Say (Acari: Ixodidae) were used to evaluate repellency and time to repellent failure at room temperature. The present study shows in filter paper assays (0.63 mg test compound/cm(2) ) that 2-tridecanone was 87% repellent to A. americanum at 12 h after application, but had no statistically significant repellency at 15 h and 24 h, and was 72% repellent to D. variabilis at 15 h, but had no statistically significant repellency at 24 h. By contrast, 2-undecanone was 74% and 75% repellent to A. americanum and D. variabilis, respectively, at 2 h after application, but no statistically significant repellency was noted at 2.5 h and 3 h. In two-choice assays on cheesecloth, 2-tridecanone at 0.25 mg/cm(2) was 85% repellent to A. americanum 6 h after application, demonstrating its potential use as an arthropod repellent that can be used on clothing without the need for formulation. No statistically significant repellency was found at 9 h or 12 h. The potential use of 2-tridecanone as a tick repellent is discussed.

Novel field assays and the comparative repellency of BioUD(®) , DEET and permethrin against Amblyomma americanum.

Two new field bioassay methods were developed to compare the repellent activity of BioUD(®) (containing 7.75% 2-undecanone), 98.1% DEET and 0.5% permethrin against natural populations of nymphal Amblyomma americanum (L.) (Acari: Ixodidae). In a cloth sheet assay, pieces of material measuring 41 × 58 cm, separately treated with one of the test materials or the appropriate solvent carrier, were placed at random on the ground and baited with dry ice for 1 h. Mean numbers of ticks on repellent-treated sheets were significantly lower than on control sheets. There was no significant difference in the number of ticks collected between sheets treated with BioUD(®) and those treated with DEET. However, significantly fewer ticks were found on sheets treated with BioUD(®) or DEET than on permethrin-treated sheets. In a sock test, over-the-calf tube socks were treated with one of the test materials or the appropriate solvent carrier. Human volunteers wore a repellent-treated and a corresponding carrier-treated sock on either leg and walked randomly over an area of approximately 4000 m(2) for 15 min. Significantly fewer ticks were collected from socks treated with BioUD(®) or DEET than from socks treated with the carrier and there was no significant difference in repellency between these two agents. No difference in the mean number of ticks collected was found between permethrin-treated and corresponding carrier-treated socks. To examine the mechanism of repellency of BioUD(®) , a four-choice olfactometer was used to assess spatial repellency against adult A. americanum. As expected in the absence of a repellent, when all choices were represented by water-treated filter paper, ticks were equally distributed among the choices. When one choice consisted of BioUD(®) -treated filter paper and the remaining choices of water-treated paper, the distribution of ticks on the repellent-treated paper was significantly lower than might be expected to occur by chance, suggesting that repellency is at least partly achieved by an olfactory mechanism.

Efficacy of the new repellent BioUD against three species of ixodid ticks.

BioUD with the active ingredient 2-undecanone originally derived from wild tomato plants is a new repellent recently registered by the US EPA. Repellent efficacy of BioUD (7.75% 2-undecanone) and DEET (98.11%) was examined in the laboratory using a choice test between repellent-treated and control filter paper surfaces for Amblyomma americanum, Dermacentor variabilis, and Ixodes scapularis. BioUD provided greater repellency against A. americanum and I. scapularis than DEET. No difference was found between BioUD and DEET against D. variabilis. In head-to-head assays between BioUD and DEET, undiluted and 50% dilutions of BioUD were more repellent than undiluted DEET against all three species tested. Similarly, a 25% dilution of BioUD was more repellent than DEET against A. americanum while no difference in mean percentage repellency was found between a 25% dilution of BioUD and DEET against I. scapularis. Based on regression analysis, the concentration of BioUD required for equivalent repellency to 98.11% DEET was 39.5% for D. variabilis and 29.7% for I. scapularis. A log-probit model could not be constructed for A. americanum from the dosages tested. Based on filter paper head-to-head assays, BioUD is at least 2-4 times more active as a repellent than DEET against three species of ixodid ticks under the conditions of our laboratory bioassays.

Novel arthropod repellent, BioUD, is an efficacious alternative to deet.

For >50 yr, N,N-diethyl-3-methylbenzamide (deet) has been the standard for arthropod repellents and has been an important tool to protect people from disease agents carried by ticks, mosquitoes, and other arthropods. However, some people avoid using deet because of concerns about adverse health effects. In 2007, a new repellent, BioUD, with the active ingredient 7.75% 2-undecanone, originally derived from wild tomato (Lycopersicon hirsutum Dunal f. glabratum C. H. Müll) plants, was registered by the U.S. Environmental Protection Agency. In the current study, repellent efficacy of BioUD was compared using arm-in-cage studies with 7 and 15% deet against the mosquitoes Aedes aegypti (L.) and Aedes albopictus Skuse. No differences were found in mean repellency over 6 h after application between BioUD versus 7 and 15% deet for Ae albopictus. For Ae. aegypti, no differences were found over the same time period for 7% deet. Compared with 15% deet, BioUD mean repellency was lower over the 6-h test period. Human subject field trials were conducted in North Carolina, United States, and Ontario, Canada, comparing the repellency of BioUD to products containing 25 and 30% deet. BioUD provided the same repellency or was more efficacious than 25 and 30% deet, respectively, in these studies. Laboratory trials were conducted to determine the repellent activity of BioUD against the American dog tick, Dermacentor variabilis (Say), on human skin and cloth. BioUD repelled ticks at least 2.5 h after application to human skin. On cloth, no differences in mean repellency were found through 8 d after application between BioUD and 7% deet. In a two-choice test for BioUD versus 15% deet on filter paper, ticks spent significantly more time on the deet-treated surface than the BioUD-treated surface. Based on these studies in toto, BioUD is an efficacious alternative to deet in its repellent activity.

Occurrence and relative abundance of mosquitoes in stormwater retention facilities in North Carolina, U.S.A.

Throughout the 2004 mosquito season, 52 stormwater retention facilities were sampled to characterize the seasonal occurrence and relative abundance of mosquito species in relation to the structural complexity and biological diversity of the facilities. The three different types of facilities included standard wet ponds (n=20), innovative ponds (n=14), and wetland ponds (n=18). All retention structures were sampled at the beginning, middle and end of the mosquito season so that seasonal changes in mosquito production could be characterized. Overall samplings, mosquitoes were collected from 34% of the retention structures. Fourteen species representing 7 genera were collected, but only 5 species were commonly collected in all three types of stormwater management facilities. In general, the seasonal prevalence and relative abundance of mosquito species did not vary among three types of retention structures. A significant association (P < 0.01) between the presence of mosquito larvae or pupae and the absence of mosquitofish was found for innovative and wetland stormwater retention facilities but not for standard retention facilities (P > 0.05).

Horizontal transfer of the insect growth regulator pyriproxyfen to larval microcosms by gravid Aedes albopictus and Ochlerotatus triseriatus mosquitoes in the laboratory.

The insect growth regulator (IGR) pyriproxyfen is highly active against mosquitoes (Diptera: Culicidae). Through continuous emersion of large larvae (instars 3-4) the concentration causing 50% inhibition of adult emergence (EI50) was determined as 0.200 p.p.b. for Aedes albopictus (Skuse) and 3.5 to 7 times less for Ochlerotatus triseriatus (Say): IE50 0.0288 p.p.b. As a possible method of application to larval microcosms of these species that oviposit in water containers and phytotelmata, the horizontal transfer of pyriproxyfen to larval microcosms by adult mosquitoes was evaluated under laboratory conditions. Gravid females were forced to walk on surfaces treated with pyriproxyfen (tarsal contact exposure) and then allowed to oviposit in larval microcosms. Using replicate bioassay cages, each with an oviposition container, and a factorial experimental design, we assessed Ae. albopictus for the effects of (i) pyriproxyfen concentration (0.2, 0.3 and 0.4 mg/cm2) contacted by gravid females, and (ii) the number of treated gravid females added to bioassay cages (one, three or five females/cage), on the mortality of larvae in oviposition containers. Only 0.2 mg/cm2 treatment rate was tested on Oc. triseriatus. A significant (P < 0.05) curvilinear response in inhibition of emergence (IE) was achieved on both species. Densities of one or three treated Oc. triseriatus females/cage yielded IE rates of only 21-27%, whereas five treated females/cage resulted in 70% inhibition. With Ae. albopictus, densities of three or five treated females/cage yielded 48-67% and 59-73% IE, respectively, whereas one treated female/cage gave only 4-30% inhibition. Use of IGR-treated oviposition containers to achieve horizontal transfer of pyriproxyfen to mosquito oviposition sites could be a field management technique based on mosquito biology and behaviour. In binary choice tests with Ae. albopictus, horizontal transfer of pyriproxyfen from a container with a treated ovistrip (0.3 or 0.4 mg/cm2) to an untreated microcosm resulted in 14-38% inhibition. In larval bioassays, pyriproxyfen activity declined markedly within 10 days. Forcibly exposing gravid female mosquitoes to pyriproxyfen-treated paper surface did not affect their fecundity. However, from the 1st to 2nd gonotrophic cycles the egg hatch rate declined by 30% (P < 0.05). Some variation of results could be due to interactions between females at the oviposition site, possibly causing disproportionate transfer of pyriproxyfen to larval microcosms. Comparative studies of the oviposition behaviour of each mosquito are warranted and would potentially provide information needed to improve the technique.

Hemolymph proteins in ticks.

In comparison to insects and Crustacea, our knowledge of the predominant hemolymph proteins in ticks is minimal. The hemolymph protein most studied in ticks has been vitellogenin (Vg). Vg is synthesized by the tick fat body after female adults obtain a blood meal, is released into the hemolymph and is absorbed by developing oocytes as vitellin (Vn). Much of what we know about Vg is from studies of Vn. In general, the carbohydrate, lipid and amino acid composition is similar to insects except that in the tick, Vg contains heme, most likely from the digestion of host hemoglobin. In the American dog tick, Dermacentor variabilis, Vg is comprised of two native proteins and seven subunits on SDS-PAGE. Vg has been characterized in five tick species but the amino acid sequence is not yet available. Another predominant hemolymph protein, apparently a carrier protein (CP), has recently been studied in two tick species. This protein is found in the hemolymph of both male and females adults, in adult tissues outside of the hemolymph in some tick species, in coxal fluid of soft ticks and in whole body homogenates from eggs, larvae and nymphs. CP from the hard tick, D. variabilis, contains cholesterol, phospholipids, monoacylglycerides, triacylglycerides, free fatty acids, carbohydrate and heme. Under identical assay conditions, the analogous protein in the soft tick, Ornithodoros parkeri, did not contain heme. CP in the American dog tick consists of two subunits, one of which has 61% identity to the biliprotein, artemocyanin, from the fairy shrimp. CP is identical to a heme-lipoprotein (HeLp) from Boophilus microplus. The exact roles of CP and HeLp have not yet been fully determined, but they apparently are important in heme sequestration and as a storage depot for protein and lipid. Macroglobulin, lectin, antimicrobial, JH binding, JH esterase, and other tick hemolymph proteins are also discussed.

Tissue distribution and characterization of predominant hemolymph carrier proteins from Dermacentor variabilis and Ornithodoros parkeri.

The tissue distribution of the predominant hemolymph protein found throughout tick development was examined in the hard tick, Dermacentor variabilis, and in the soft tick, Ornithodoros parkeri. In D. variabilis, the predominant (purified) hemolymph protein was a lipoglycoheme-carrier protein (DvCP) with a molecular weight of 200K. A protein with a similar mobility on native-PAGE was found in fat body, salivary gland, muscle and ovary from partially fed females which was most abundant in the plasma and salivary gland. DvCP from plasma, salivary gland and fat body of partially fed females consisted of two subunits on SDS-PAGE (98 and 92K). In replete females, only salivary gland exhibited protein subunits equivalent to hemolymph CP. CP in salivary gland and fat body stained positive for lipids. The concentration of CP in tissues varied between partially fed and replete females, indicating a difference in the expression and/or sequestration of CP during adult development. The predominant hemolymph carrier protein from O. parkeri (OpCP) was purified to homogeneity for the first time and is presumed to have similar functions to CP from D. variabilis. Purified OpCP exhibited a molecular weight of 668K by native-PAGE. Unlike CP from D. variabilis, OpCP was not detected in fat body or salivary gland tissues but occurred abundantly in coxal fluid. By SDS-PAGE, purified hemolymph OpCP consisted of two major subunits (114 and 93K) and a less abundant protein with an apparent molecular weight of 48K. Purified native OpCP was a lipoprotein like DvCP. A spectral analysis of purified OpCP failed to demonstrate the presence of heme like that found for CP from D. variabilis, purified by the same methods. However, plasma from O. parkeri contained heme with a lambda(max) of 410nm.

First isolation of La Crosse virus from naturally infected Aedes albopictus.

La Crosse (LAC) virus, a California serogroup bunyavirus, is the leading cause of pediatric arboviral encephalitis in the United States and an emerging disease in Tennessee, West Virginia, and North Carolina. Human cases of LAC encephalitis in Tennessee and North Carolina have increased above endemic levels during 1997 to 1999 and may represent an expansion of a new southeastern endemic focus. This report describes the isolation of LAC virus from the exotic mosquito Aedes albopictus. The discovery of LAC virus in wild populations of Ae. albopictus coupled with its expanding distribution in the southeastern United States, suggests that this mosquito may become an important accessory vector, potentially increasing the number of human cases in endemic foci or expanding the range of the disease.

Developmental profile, isolation, and biochemical characterization of a novel lipoglycoheme-carrier protein from the American dog tick, Dermacentor variabilis (Acari: Ixodidae) and observations on a similar protein in the soft tick, Ornithodoros parkeri (Acari: Argasidae).

A novel lipoglycoheme-carrier protein (CP) in the American dog tick, Dermacentor variabilis (Say) has been purified and characterized. CP was purified by native-PAGE from partially fed virgin females. CP has a density of 1.25 g/ml with a molecular weight of 200 K by native-PAGE and 340 K by gel filtration chromatography. CP is comprised of two majour subunits, 98 K and 92 K in molecular weight by SDS-PAGE. Separate amino acid composition of the two subunits indicated high contents of As(x), Gl(x) and leucine. However, the N-terminal amino acid sequence of the two subunits was only 13% identical. The lower molecular weight subunit showed 61% identity to artemocyanin (biliprotein) in fairy shrimps, 46% identity to minor vitellogenin in chickens and 13% identity to vitellin of the black-legged tick. No similarity match was found for the other subunit. CP is a lipoglycoheme-protein as indicated by selective staining of native-PAGE gel for lipids, carbohydrates and heme. Lipid analysis by thin layer chromatography revealed the presence of cholesterol, phospholipids, monoacylglycerides, triacylglycerides and free fatty acids. Heme associated with purified CP demonstrated a lambda(max) of 397.5 nm while the lambda(max) of crude hemolymph plasma was 402.5 nm. The presence of CP in whole body homogenates of eggs, unfed and fed larvae and fed nymphs as well as in the plasma of unfed and fed adults including vitellogenic females was demonstrated by native-PAGE. Although a protein of analogous size was not found in the soft tick, Ornithodoros parkeri Cooley, a high molecular weight protein (500 K) is the predominant plasma protein in both unfed and fed male and female adults of that species as determined by native-PAGE. Also, CP appears to function as a biliprotein which sequesters heme.

Bunyavirus infections in North Carolina white-tailed deer (Odocoileus virginianus).

We analyzed serum from white-tailed deer (Odocoileus virginianus) collected in southeastern North Carolina in 1991 for neutralizing antibodies to six mosquito-borne bunyaviruses (Lacrosse, Jamestown Canyon, Keystone,Cache Valley, Potosi, and Tensaw), including several of public health importance. Evidence was found for all six to be locally transmitted, although greatest seroprevalence was found for Potosi, Jamestown Canyon, and Cache Valley viruses.

Development and survival of immature Aedes albopictus and Aedes triseriatus (Diptera: Culicidae) in the laboratory: effects of density, food, and competition on response to temperature.

Effects of food, density, and heterospecific interactions on temperature-dependent development of Aedes albopictus (Skuse) and Aedes triseriatus (Say) larvae and pupae were described using a degree-day model. Under all conditions, the predicted number of degree-days (DD(T0)) to complete larval development was less, and the threshold temperature (T0) for initiation of larval development was higher for Ae. albopictus than for Ae. triseriatus. The DD(T0) for both species was food and density dependent. However, the per capita food ration appeared to exert a greater influence on the developmental times of Ae. triseriatus immatures, whereas for Ae. albopictus effects of density were not completely eliminated by an increase in the per capita food ration. The presence of heterospecific larvae did not prolong the DD(T0) of either species. At the low food ration, DD(T0) for both species were significantly greater under conspecific conditions. The DD(T0) for Ae. triseriatus increased directly as the proportion of conspecific larvae increased. However, when a higher per capita food ration was provided, conspecific effects on DD(T0) were mitigated. For Ae. albopictus, estimated DD(T0) values for larvae reared in pure culture were significantly higher than when heterospecific larvae were present, regardless of the per capita amount of food provided. Survivorship of immatures was density and food-dependent for both species. Ae. albopictus exhibited higher immature survivorship under all conditions relative to Ae. triseriatus. For both species, survival probabilities were lowest under conditions of high density and low food. Addition of food improved survival for both species. The presence of heterospecific larvae exerted a differential effect on the survivorship response of Ae. albopictus immatures to temperature. Lowest probability of survival for Ae. albopictus was occurred at low temperature when the proportion of heterospecific larvae in containers was the highest. In contrast, at high temperatures, survivorship of immatures improved, but was lowest for pure species cultures. For Ae. triseriatus, survivorship of immatures was consistently lower for pure species cultures regardless of the temperature. Standing crop production of adults of both species was primarily food rather that density-dependent. Under field conditions, Ae. triseriatus were predicted to initiate development sooner and exhibit faster population growth early in the season than Ae. albopictus. However, because of the shorter DD(T0), Ae. albopictus population growth was predicted to surpass that of Ae. triseriatus populations later in the season. Based on optimal DD(T0) values from laboratory experiments, spring emergence dates of Ae. triseriatus females in western North Carolina for 1989 and 1990 were predicted from accumulated degree-days calculated from local air temperature records. Predicted emergence dates were congruent with results of a previous survey for the same locality, indicating that accumulated degree-days can be used to accurately predict the seasonal occurrence of Ae. triseriatus. The utility of the degree-day approach in predicting the phenology of Ae. albopictus remains to be established.

Absence of insect juvenile hormones in the American dog tick, Dermacentor variabilis (Say) (Acari:Ixodidae), and in Ornithodoros parkeri Cooley (Acari:Argasidae).

Synganglia, salivary gland, midgut, ovary, fat body and muscle alone and in combination from the ixodid tick, Dermacentor variabilis (Say), or the argasid tick, Ornithodoros parkeri Cooley, were incubated in vitro in separate experiments with L-[methyl-(3)H]methionine and farnesoic acid or with [1-(14)C]acetate. Life stages examined in D. variabilis were 3 and 72 h old (after ecdysis) unfed nymphs, partially fed nymphs (18 and 72 h after attachment to the host), fully engorged nymphs (2 d after detachment from host), 3 and 72 h old (after eclosion) unfed females, partially fed unmated females (12-168 h after attachment to host) and mated replete females (2 d after detachment from the host). Those from O. parkeri were third and fourth stadium nymphs and female O. parkeri, 1-2 d after detachment. Corpora allata from Diploptera punctata, Periplaneta americana and Gromphadorina portentosa were used as positive controls in these experiments. No farnesol, methyl farnesoate, JH I, JH II, JH III, or JHIII bisepoxide was detected by radio HPLC from any tick analysis while JH III, methyl farnesoate, and farnesol were detected in the positive controls. To examine further for the presence of a tick, insect-juvenilizing agent, Galleria pupal-cuticle bioassays were conducted on lipid extracts from 10 and 15 d old eggs, unfed larvae (1-5 d after ecdysis), unfed nymphs (1-7 d after ecdysis), and partially fed, unmated female adults (completed slow feeding phase) of D. variabilis. Whole body extracts of fourth stadium D. punctata and JH III standard were used as positive controls. No juvenilizing activity in any of the tick extracts could be detected. Electron impact, gas chromatography-mass spectrometry of hemolymph extracts from fed, virgin (forcibly detached 7 d after attachment) and mated, replete (allowed to drop naturally) D. variabilis and fully engorged (1-2 d after detachment) O. parkeri females also failed to identify the common insect juvenile hormones. The same procedures were successful in the identification of JH III in hemolymph of fourth stadium D. punctata. Last stadium nymphal (female) O. parkeri implanted with synganglia from second nymphal instars underwent normal eclosion to the adult. The above studies in toto suggest that D. variabilis and O. parkeri do not have the ability to make the common insect juvenile hormones, and these juvenile hormones do not regulate tick metamorphosis or reproduction as hypothesized in the literature.

Laboratory and field evaluations of oviposition responses of Aedes albopictus and Aedes triseriatus (Diptera: Culicidae) to oak leaf infusions.

Organic infusions created by fermenting white oak (Quercus alba L.) leaves in water were evaluated as sources of attractant odorants and contact oviposition stimulants for gravid Aedes albopictus (Skuse) and Aedes triseriatus (Say). Infusions were bioassayed in the laboratory by giving single females a choice of ovipositing in 1 container with infusion and 7 containers with water. Ae. albopictus laid significantly more eggs in containers with infusion, regardless of concentration (dilutions ranging from 10 to 100%) or age (fermentation periods of 7, 28, 60 d), than in containers holding water. The largest proportion of eggs (76.8%) was deposited in response to a 60% concentration of 7-d-old infusion. In contrast, Ae. triseriatus exhibited variable oviposition responses but generally deposited the largest number of eggs in only a few concentrations of older age infusions. In binary "sticky screen" bioassays, there was no difference between the numbers of females attracted to infusion or water, indicating that oviposition responses to infusion were mediated by contact chemostimulants rather than by attraction to odorants. Oviposition responses to infusions by field populations of Ae. albopictus and Ae. triseriatus in Raleigh, NC, were evaluated with pairs of oviposition traps, one containing infusion and the other containing water. Generally, Ae. albopictus laid significantly more eggs in ovitraps containing infusion regardless of its age (7, 28, and 60 d old) or the mass of leaves fermented (126 g = 1x or 504 g = 4x) than in water. In contrast, Ae. triseriatus deposited an equivalent number of eggs in traps containing water or 1x, 80% infusion regardless of its age; however, the oviposition response to ovitraps containing 4x, 7-d-old, 50% infusion was significant. Placement of an automobile tire behind an ovitrap did not increase the number of Ae. albopictus eggs laid in ovitraps containing 4x, 7-d-old, 50% infusion or water relative to ovitraps without a tire. Our research indicates that baiting ovitraps with oak leaf infusion would increase the sensitivity of surveillance efforts for Ae. albopictus and Ae. triseriatus.

An experimental chain of infection reveals that distinct Borrelia burgdorferi populations are selected in arthropod and mammalian hosts.

The prokaryotic, spirochaetal microorganism Borrelia burgdorferi is the causative agent of Lyme disease, an arthropod-borne disease of a variety of vertebrates and the most prevalent arthropod-borne disease of humans in the United States. In order to understand better the normal life cycle of B. burgdorferi, an experimental chain of infection was devised that involved multiple sequential arthropod and mammalian passages. By examining populations of B. burgdorferi emerging from different points in this infectious chain, we demonstrate that selection of B. burgdorferi populations peculiar to arthropod or vertebrate hosts is a property of at least one of the two ecologically distinct strains we examined. Distinct B. burgdorferi populations were identified using an antigenic profile, defined by a set of monoclonal antibodies to eight B. burgdorferi antigens, and a plasmid profile, defined by the naturally occurring plasmids in the starting clonal populations. These two profiles constituted the phenotypical signature of the population. In the strain exhibiting selection in the different hosts, transition from one host to another produced a striking series of alternating phenotypical signatures down the chain of infection. At the molecular level, the alternating signatures were manifested as a reciprocal relationship between the expression of certain antigenic forms of outer surface protein (Osp) B and OspC. In the case of OspC, the antigenic changes could be correlated to the presence of one of two distinctly different alleles of the ospC gene in a full-length and presumably transcriptionally active state. In the case of OspB, two alleles were again identified. However, their differences were minor and their relationship to OspB antigenic variation more complicated. In addition to the reciprocating changes in the antigenic profile, a reciprocating change in the size (probably the multimeric state) of a 9.0 kbp supercoiled plasmid was also noted. Selection of distinct populations in the tick may be responsible for the microorganism's ability to infect a wide range of vertebrate hosts efficiently, in that the tick might provide selective pressure for the elimination of the population selected in the previous host.