Human granulocytic ehrlichiosis (HGE) is an emerging tick-borne disease recently recognized in the United States. The blacklegged tick, Ixodes scapularis Say, is the principle vector in the eastern United States. The disease has been commonly reported in the northeastern and upper midwestern states; however, suitable vectors and reservoir hosts exist in the southeast. To assay the prevalence of the HGE agent in vector ticks, we screened 818 individual I. scapularis from 15 locations in South Carolina, Georgia, and Florida using nested polymerase chain reaction, which targets the HGE agent 16S rRNA gene. Prevalence among locations ranged from 0 to 20%. The overall average prevalence of 15 sites was 1.6% (n = 818). Verification by sequencing the 16S rDNA from the positive samples showed 99.8–100% nucleotide identities with the sequences of the HGE agent in GenBank. These results were supported by the phylogenetic analysis using 16S rDNA sequences.

A chemical, electrophysiological and behavioral study was carried out to analyze the volatile compounds of the Brindley’s gland of Rhodnius prolixus Stål. Six compounds were found in the Brindley’s gland of males and females: acetic, isobutyric, caproic acids and three compounds not identified. The mass spectra of the unidentified compounds have the appearance of a propionate ester, a butyrate ester and a valerate ester. There was no difference in the concentration of these compounds in the glands of males and females. The major component was isobutyric acid. The electroantennographic (EAG) evaluation of isobutyric, acetic, and caproic acids elicited weak responses. With both sexes, the EAG responses for the acids were no different from the control response at any of the doses evaluated (1, 10, 100, and 500 μg). The behavioral response of males and females to acetic, isobutyric, and caproic acids at 1, 5, and 10 μg and binary and tertiary mixtures of the three compounds was evaluated in a Y-olfactometer. Males showed preference for acetic acid at a dose of 1 μg, but not at 5 or 10 μg. Females showed preference for this compound at the dose of 5 μg. Males showed preference for isobutyric acid at 1 and 5 μg, but not at 10 μg. Females did not show any preference for isobutyric acid. Males were attracted to caproic acid at the dose of 1 and 10 μg. Females were attracted to the caproic acid. Males, but not females, preferred the mixture of acetic and isobutyric acids over control. Neither males nor females showed preference for the mixture of acetic and caproic acids or hexane control. Females, but not males, were attracted to the mixture of the isobutyric and caproic acids. Only males showed a positive response for the tertiary mixture of the acids.

Hemolymph coagulation began almost immediately after wounding in mosquito, Armigeres subalbatus, (Coquillett) larvae. Immunocytochemical localization showed that prophenoloxidase (pro-PO) was distributed in the wound site. In the initial wounding, coagulation and wound plug formation occurred with granulocyte migration. The hemocytes lysed and released granular materials around the wound site, prophenoloxidase being mostly localized in granules and cuticle. In the second phase of wound healing, melanin accumulation occurred at the wound site along the margin of the cuticle and rapidly increased in thickness. Immunogold-labeled pro-PO was localized in vacuoles, melanins, and cuticle, with the gold particles labeled intensely on the undarkened cuticle and weakly on the darkened cuticle. It is believed that pro-PO is activated upon wound initiation to produce melanin product and deposited on the cuticle. In the final phase of healing, scab melanization and pro-PO immunogold localization were reduced and accompanied by epithelial cell regeneration. This proenzyme was localized in vesicles and flocculent materials, but was absent in the melanized scab. Our results further indicate that pro-PO was present in granules, cuticles, epithelial cells, vacuoles, and flocculent materials but not in melanized scab and coagulated clot. The pro-PO immunogold particles labeled intensely in the initial wounding but weakly in the final phase. Our observations also suggest that pro-PO is released from granulocytes by cell rupture, synthesized or stored in granulocytes, and then is released into the wound site via the cytoplasmic granules. This study indicates that the pro-PO is involved in numerous physiological roles in the process of wound healing in this mosquito.

peruensis is described as new from specimens collected from Oryzomys yunganus, Proechimys quadruplicatus, and Oryzomys megacephalus taken in Peru. A new character of a nude tactile seta on femur III is noted for the genus and a key to the species of Polylopadium is presented.

Three organophosphate resistant Boophilus microplus Canestrini cell lines were generated by exposing B. microplus VIII-SCC cell line to incrementally increased toxic concentrations of the acaricide coumaphos. The development of resistance was evidenced by LC50 values elevated over those of control cells. The resistant cell lines selected in higher concentrations of organophosphate, designated C44 and C54, also had significantly slower duplication rates than a resistant cell line selected in lower concentrations of coumaphos (C34) and the nonresistant control cells. Resistant cell lines C44 and C54 also had significantly higher levels of esterase after exposure to coumaphos than resistant cell line C34 and the nonresistant controls. These in vitro results agree with reports of increased esterase activity associated with organophosphate resistance in B. microplus ticks in vivo.

We evaluated an artificial capillary feeding method to infect nymphal Ixodes scapularis (Say) ticks with Borrelia burgdorferi, the causative agent of Lyme disease. Thirty to 70% of the nymphs were infected after feeding for 2.5 h from glass capillary tubes filled with a solution of spirochetes. Capillary infection was stable and persisted in the nymphs for at least 10 d after feeding. Capillary feeding also maintained natural vector competence patterns because I. scapularis ticks acquired infection unlike Dermacentor variablis (Say), which did not become infected. Capillary infected I. scapularis nymphs were capable of transmitting the infection to naive mice although not as efficiently as naturally infected nymphs. The capillary infection method is convenient and is a better alternative to syringe inoculation as a means of infecting animals with B. burgdorferi.

Laboratory colonies of the black fly Simulium vittatum Zetterstedt cytospecies IS-7 were analyzed cytogenetically and compared with the original New York population from which source material was collected 18 yr earlier. All sex chromosomes and major autosomal polymorphisms that were present in the source population were still represented in the laboratory colonies. However, the extent of sex linkage and the frequencies of four of the five major autosomal inversions changed significantly in at least one colony, perhaps because of bottlenecks experienced by the colony. A complete absence of males homozygous for the IS-7 inversion in both field and colony material is explained by postulating that the Y₂ chromosome, representing the inverted condition for the IS-7 sequence, is absent from the population or acts as a rare reproductive lethal. This example possibly represents pseudo-partial sex linkage involving the X-linked sequences.

Two new species of spinturnicid mites of the genus Periglischrus are described and illustrated from phyllostomid bats from southeastern Mexico: The female, male, and protonymph of Periglischrus steresotrichus, new species, from Tonatia evotis Davis & Carter, and the female and male of Periglischrus eurysternus, new species, from Tonatia saurophila Koopman & Williams. A supplementary description of the male deutonymph of P. eurysternus from T. saurophila from Panama is given. The morphological features of the two new species of Periglischrus are used as a basis for discussing their phylogeny and its potential relationship to that of their hosts.

Diagnostic assays for the detection of St. Louis encephalitis (SLE) and western equine encephalomyelitis (WEE) viruses in mosquito pools and avian tissues were compared for sensitivity, accuracy and specificity. The in situ enzyme immunoassay (EIA), plaque assay on Vero cells, passage in Aedes albopictus Skuse C6/36 and C7/10 cells, antigen capture enzyme immunoassay (AC-EIA), and single and multiplex reverse transcription-polymerase chain reactions (RT-PCR) were evaluated using pools of 50 mosquitoes containing 1–2 experimentally infected individuals. RT-PCR was the most sensitive assay, with a detection limit of <0.1 plaque forming unit. AC-EIA was the fastest and most economical procedure, but was the least sensitive, detecting only 38% of positive pools. The in situ EIA included initial virus amplification on Vero cells, thereby improving assay sensitivity to detect 68% of positive pools. Passage in C6/36 and/or C7/10 cell culture revealed the presence of infectious virus in samples positive by RT-PCR, but initially negative by plaque assay on Vero cell culture, indicating that detection was related to assay sensitivity and not to the absence of intact infectious virus. Combining WEE and SLE RT-PCR assays into a multiplex assay reduced sensitivity, but still detected viral RNA at titers below plaque assay sensitivity. Plaque assay on Vero cells, mosquito cell passage, and several RT-PCR procedures were evaluated for their ability to detect WEE and SLE in white-crowned sparrow tissues during acute and chronic stages of infection. All assays detected virus during acute infection at times of high viremia; however, only RT-PCR assays were positive by day 7 when virus was not detected in sera. RT-PCR detected SLE RNA in spleen tissue from one bird 51 d after infection. Assay sensitivity also was compared using extracts of homogenized bird organs spiked with known titers of WEE and SLE. Trizol RNA extraction followed by Qiagen one-step RT-PCR was the most sensitive method, but occasionally resulted in the presence of secondary bands confounding interpretation and requiring confirmatory assays. A balanced surveillance program should combine systems that allow the detection of new agents and the sensitive monitoring of endemic agents to provide an early warning of pending health risks.

The determination of the presence or absence of malaria sporozoites in wild-caught Anopheles mosquitoes remains an integral component to the understanding of the transmission dynamics in endemic areas. To improve that capability, there has been on-going development of a new device using dipstick immunochromatographic technology for simplifying the testing procedure and reducing the time required to obtain results. As part of a larger multi-center effort, we evaluated the sensitivity and specificity of a prototype malaria sporozoite antigen panel assay (Medical Analysis Systems, Camarillo, CA) against three human Plasmodium species/polymorphs. The wicking (dipstick) assay was compared against a standard parasite antigen capture enzyme-linked immunosorbent assay (ELISA) for the detection of human circumsporozoite protein (CSP) in wild-caught mosquitoes. Over 6,800 Anopheles mosquitoes, representing 20 species collected from malaria endemic areas of Indonesia were tested either individually or in pools of up to 10 mosquitoes each. From 1,442 pooled test strip assays and ELISA formats, nine mosquito pools were found reactive for P. falciparum, P. vivax 210, or P. vivax 247 CSP. There was complete concordance between test strip results and ELISA results. Sensitivity was 100% and given some minor problems with false positives or negatives, specificity (n = 488) was 97%. Most strips judged as false positive produced very weak signals compared with negative control blank strips and paired ELISA-negative samples. The dipstick test proved technically simpler to perform and interpret than the ELISA and results were obtained within 15 min of exposure to mosquito suspension. This qualitative assay appears an attractive alternative to the CSP ELISA for detection of sporozoites in fresh or dried mosquitoes.

The sequence and tissue expression of the gene encoding a peptide hormone Aea-HP-I, known to inhibit host-seeking behavior, has been characterized for the yellowfever mosquito, Aedes aegypti (L.). The open reading frame reveals a prepropeptide that would be processed into three identical peptides. The gene contains four short introns and exists as a single genomic copy. Transcripts of the gene were present in the brain, terminal ganglion, and midgut of adults, and in females, its expression profile differed for each tissue before and during a reproductive cycle. Peptides resulting from this expression were identified in the female tissues by immunoassays. Numerous neurosecretory cells and neurons in the nervous system were immunostained by an Aea-HP-I antiserum. Hundreds of endocrine cells were stained similarly in the midgut, thus contributing to the 10 times greater amount of immunoreactive peptide in an abdomen than in a head, as determined with an Aea-HP-I radioimmunoassay. Based on these results, neurosecretory cells and midgut endocrine cells are likely sources of Aea-HPs shown to reach highest hemolymph titer at the same time as host seeking is inhibited in female Ae. aegypti during a reproductive cycle.

Eudusbabekia provirilia new species was found on the bat Leptonycteris nivalis (Saussure) in the central part of Mexico. The female, male, protonymph, and larva are described and illustrated.

A 2-yr longitudinal malaria study was undertaken in a suburb of Yaounde, the capital city of Cameroon, in the village of Simbock, ≈2 km from the city limits. This study allowed assessment of malaria transmission intensity and dynamics in this region before implementation of pyrethroid impregnated bed nets through the national vector control program. Anophelines were captured on human volunteers by pyrethrum spray collections and in resting sites outdoors. Malaria vectors were Anopheles funestus Giles, Anopheles gambiae s.s. Giles (M and S forms), Anopheles moucheti Evans, and Anopheles nili Theobald. An. moucheti was the most abundant mosquito captured during the study, accounting for >54% of total anophelines caught. The annual Plasmodium falciparum Welch entomological inoculation rates measured by enzyme-linked immunosorbent assay were 277 infected bites per human for the first year and 368 for the second year. An. gambiae s.s., An. funestus, An. moucheti, and An. nili were responsible for 23.8%, 26.8%, 39.2%, and 10.2% of malaria transmission, respectively. Malaria transmission is perennial throughout the year. All these vectors were highly anthropophagous because only two out of 566 mosquitoes blood-meal tested were not taken on humans.

At semiarid Charters Towers, north Queensland, Australia, the importance of Aedes aegypti (L.) in wells was assessed in relation to the colonization of surface habitats during the wet season. From April to July 1999, 10 wells (five positive for Ae. aegypti) were monitored to assess their status and larvae population numbers therein. All surface containers located within a 100 m radius of each well were removed, treated with s-methoprene or sealed to prevent the utilization of these containers by mosquitoes. These inner cores were surrounded by outer zones for a further 100 m in which surface containers were left untreated but all subterranean habitats were treated. Ovitraps were monitored monthly in the inner cores for 36 wk from August 1999 to April 2000 and differences in the proportions of ovitraps positive for Ae. aegypti and Ochlerotatus notoscriptus (Skuse) were analyzed by logistic regression. Analysis of the proportions of ovitraps positive for Ae. aegypti near positive wells indicated significantly greater colonization from November to March (the wet season), compared with those situated near Ae. aegypti negative wells. As Oc. notoscriptus were not produced from subterranean sites, comparisons of the proportions of ovitraps positive for Oc. notoscriptus in positive and negative inner cores provided an indication of the relative productivity of the uncontrolled surface containers in the outer zones. Differences in the utililization of ovitraps by Oc. notoscriptus among positive and negative cores were observed during only one month (March), when oviposition was greater in ovitraps in the negative cores, compared with the positive cores. Best subsets linear regression analysis of the proportion of ovitraps positive for Ae. aegypti against meteorological variables (rainfall, mean wind speed, mean relative humidity, mean minimum, and maximum temperature) during the week of ovitrapping indicated that minimum temperature and wind speed accounted for 63.4% of the variability. This study confirms that for semiarid towns such as Charters Towers, the practice of treating a relatively small number of key subterranean habitats during winter will significantly affect Ae. aegypti recolonization of surface container habitats during summer, the period of greatest risk for dengue.

Members of the Culex sitiens subgroup are important vectors of arboviruses, including Japanese encephalitis virus, Murray Valley encephalitis virus and Ross River virus. Of the eight described species, Cx. annulirostris Skuse, Cx. sitiens Wiedemann, and Cx. palpalis Taylor appear to be the most abundant and widespread throughout northern Australia and Papua New Guinea (PNG). Recent investigations using allozymes have shown this subgroup to contain cryptic species that possess overlapping adult morphology. We report the development of a polymerase chain reaction–restriction fragment-length polymorphism (PCR-RFLP) procedure that reliably separates these three species. This procedure utilizes the sequence variation in the ribosomal DNA ITS1 and demonstrates species-specific PCR-RFLP profiles from both colony and field collected material. Assessment of the consistency of this procedure was undertaken on mosquitoes sampled from a wide geographic area including Australia, PNG, and the Solomon Islands. Overlapping adult morphology was observed for Cx. annulirostris and Cx. palpalis in both northern Queensland and PNG and for all three species at one site in northwest Queensland.

Weight gain by adult cat fleas, Ctenocephalides felis (Bouché), was influenced primarily by the concentrations of protein and sodium chloride in the feeding solution. After 48 h of feeding, fleas fed whole blood weighed almost twice as much as fleas fed plasma or hemolyzed blood and 1.25 times as much as fleas fed 0.15 M sodium chloride. When fleas were fed sodium chloride solutions ranging from 0 to 0.5 M, weight gain was greatest on the 0.15- or 0.2-M solutions. Weight gain decreased significantly when fleas were fed plasma, hemolyzed blood or 0.3 or 0.5 M sodium chloride in place of whole blood, but improved when plasma was diluted 100% and when hemolyzed blood was diluted 10% with distilled water. Adenosine-5′-triphosphate did not appear to stimulate weight gain in cat fleas; weight gain was unchanged in fleas fed hemolyzed blood or 0.15 M sodium chloride to which 0.005 M ATP was added. Insemination did not occur in starved fleas or those fed protein-free diets. When fleas were starved or fed distilled water, sodium chloride, or other salt solutions, sperm was transferred from the testes to the vas deferens in 91–94% of males, but no females were inseminated. In contrast, when fleas were fed whole blood, hemolyzed blood, plasma, or bovine serum albumin (3.5 or 7.0 g/deciliter) dissolved in 0.15 M saline, 80, 80, 10, and 10% of the females were inseminated, respectively.

In addition to a soluble response, many invertebrates control bacterial infections by means of phagocytosis or melanotic encapsulation. In some insects, Escherichia coli growth is reported to be inhibited by aggregation/encapsulation. Soluble and phagocytic responses to bacterial challenge have been reported in ticks, but evidence of an aggregation/encapsulation response was reported only for inanimate (araldite) implants. Ticks were challenged by direct inoculation of bacteria into the hemocoel cavity. By plating, no viable E. coli were detected 6 h postinoculation. A direct fluorescence assay (DFA) revealed aggregated bacteria 1 h postinoculation. Furthermore, DFA showed aggregated bacteria at 6, 24, and 48 h postinoculation associated with masses of tissue, presumably of cellular origin, suggesting events similar to those described as nodulation. These findings suggest that encapsulation/nodulation may be an important component of the immune response in ticks.

Psoroptes ovis (Hering), the sheep scab mite, is responsible for psoroptic scabies of cattle and sheep. Reverse translation of 30 N-terminal amino acids of the major P. ovis allergen, previously chosen as a candidate immunogen and identified as a 16 kDa protein yielded a degenerate sequence used to design oligodeoxynucleotide polymerase chain reaction (PCR) primers. Use of the PCR primers with a P. ovis cDNA library succeeded in amplification of a 90 bp cDNA gene fragment that was cloned, sequenced, and used to select unique sequencing/PCR primers. Primer walking generated overlapping subclones which yielded the 588 nucleotide consensus sequence of the cDNA encoding the 143 amino acid P. ovis allergen precursor. Nucleotide and translated sequences of the cDNA were compared with sequences in GenBank and found to be homologous to mite group II allergens Lep d II (formerly Lep d I) of Lepidoglyphus destructor Schrank, Der f II of Dermatophagoides farinae Hughes, Der p II of Dermatophagoides pteronyssinus (Trouessart), Tyr p II of Tyrophagus putrescentiae (Schrank), Eur m II of Euroglyphus maynei (Cooreman) and Gly d II of Glycophagus domesticus (De Geer). The mature P. ovis allergen is composed of 126 amino acids with a calculated molecular mass of 13,468 Da, three disulfide bonds, and pI of 6.06 with one potential o-glycosylation site at Thr116. We designate the P. ovis 16 kDa protein as Pso o II in conformity with nomenclature for mite group II allergens.

RESUMEN Psoroptes ovis (Hering), el ácaro de la sarna de los ovinos, es responsable de la sarna psoróptica del ganado y las ovejas. Traducción reversa de 30 amino ácidos de la región N-terminal del alérgeno mayor de P. ovis, fue previamente escogido como un candidato a immunógeno e identificado como una proteína de 16 kDa, de la cual se diseñaron oligonucleótidos iniciadores degenerados para PCR. El uso de los iniciadores con una geneteca de cDNA de P. ovis, fueron exitosos para amplificar un fragmentode cDNA de 90 pares de bases, los cuales fueron clonados, secuenciados y usados para seleccionar iniciadores específicos para PCR. Iniciadores detraslape de secuencias generaron subclonas, las cuales resultaron en una secuencia consenso de cDNA de 588 nucleótidos, que codifica el alergeno precursor de 143 amino ácidos de P. ovis. Las secuencias traducidas de nucleótidos de cDNA fueron comparadas con secuencias en el GenBank y se encontraron homólogas al grupo II de alergenos de ácaros Lep d II (antes Lep d I) de Lepidoglyphus destructor (Schrank), Der f II de Dermatophagoides farinae (Hughes), Der p II de Dermatophagoides pteronyssinus (Trouessart), Tyr p II de Tyrophagus putrescentiae (Schrank), Eur m II de Euroglyphus maynei (Cooreman) y Gly d II de Glycophagus domesticus (De Geer). El alergeno maduro de P. ovis esta compuesto de 126 amino ácidos con una masamolecular calculada de 13,468 Da, three puentes disulfuro y un pI de 6.06, con un sitio potencial de o-glicosilación en Thr116. Nosotros designamos la proteínade 16 kDa de P. ovis como Pso o II para concordar con la nomenclatura para el grupo II de alergenos de ácaros.

Larvae of Phormia regina (Meigen), Phaenicia sp., and Sarcophaga sp. were identified from raccoon carcasses placed in sunlit and shaded areas at a southwestern West Virginia site in May of 2000. Samples of larvae were taken from each carcass at 3-h intervals over a 153-h experimental period. Phormia regina was clearly the dominant species with large numbers of third instars observed at every 3-h collection period from 81 to 153 h on both carcasses. Mean lengths of third-instar P. regina larvae collected from the sunlit carcass were significantly greater than mean lengths of larvae collected from the shaded carcass. Third-instar Phaenicia sp. also appeared at 81 h on both carcasses, but relatively few (≤4) individuals were present in each 3-h collection sample from 81 through 126 h. Larvae of this species were not present in samples from either carcass in those 3-h intervals from 129 to 147 h. Sarcophaga sp. larvae were also collected, but only in samples taken from the sunlit carcass at 81 and 93 h. Ambient temperatures were recorded throughout the experimental period, whereas maggot mass temperatures were not recorded until the appearance of large numbers of second instars at 48 h. From 48 to 69 h, maggot mass temperatures were equivalent to ambient temperatures; but after 69 h, maggot mass temperatures were considerably elevated over ambient temperatures.

The male and female of Amblyomma geochelone n. sp. are described and illustrated by both scanning electron micrographs and line drawings. Specimens of this new tick species were recovered from the endangered ploughshare tortoise, Geochelone yniphora (Vaillant), in northwestern Madagascar. This relatively large tick is morphologically most similar to Amblyomma nuttalli Dönitz, which occurs in mainland sub-Saharan Africa where it mainly parasitizes other species of tortoises. However, several characters distinguish the new species from A. nuttalli including the scutal ornamentation in both sexes and the characteristic patterns of shallow grooves on the alloscutum of the female of A. geochelone. Because the adult stages of A. geochelone are almost certainly host specific ectoparasites of the ploughshare tortoise, this new tick species is also probably endangered.