Chin-Gi Huang

Parallel infection of Japanese encephalitis virus and Wolbachia within cells of mosquito salivary glands.

Abstract The endosymbiont Wolbachia usually causes cytoplasmic incompatibility in dipteran hosts, including mosquitoes. However, some important arbovirus-transmitting mosquitoes such as Aedes aegypti (L.) are not heritably infected by Wolbachia. In Wolbachia-harboring mosquito Armigeres subalbatus Coquillett, colocalization of Wolbachia and inoculated Japanese encephalitis virus (family Flaviviridae, genus Flavivirus, JEV) in salivary gland (SG) cells was shown by electron microscopy. The infection rate of JEV in SGs, detected with either immunofluorescent antibody test or reverse transcription-polymerase chain reaction, did not show significant differences between Wolbachia-infected and -free colonies. It is suggested that Wolbachia did not mediate resistance of SG cells to superinfection by JEV, although both microorgamisms coexist in the same niche, i.e., the same SG cell. Therefore, a SG escape barrier may not be elevated due to Wolbachia infection, which presumably has no deleterious effects on vector competence in Wolbachia-harboring mosquitoes.

Molecular (sub) grouping of endosymbiont Wolbachia infection among mosquitoes of Taiwan.

Abstract Wolbachia are maternally inherited bacteria that infect a wide range of arthropods as well as filarial worms. The infection usually results in reproductive distortions of the host, primarily cytoplasmic incompatibility, parthenogenesis, and feminization. This study showed that Wolbachia infection (15/29; 51.72%) was prevalent among field-caught mosquitoes in Taiwan. Three mosquito species were identified as having Wolbachia A infection, eight species as having Wolbachia B, and four other species were dually infected by both groups. Each Wolbachia isolate from different mosquitoes was further divided into a specific subgroup. However, there were still some isolates that did not belong to any known subgroup, suggesting that more subgroups remain to be identified. Investigation of tissue tropism in either Aedes albopictus (Skuse) or Armigeres subalbatus (Coquillett) revealed that Wolbachia were extensively distributed within the host, although the ovary was most susceptible to infection. This report provides preliminary features of molecular relationships among Wolbachia groups of mosquitoes from Taiwan.

Rickettsia felis in cat fleas in Taiwan.

We describe the first detection of Rickettsia felis in cat fleas (Ctenocephalides felis) in Taiwan. Natural infections of R. felis in cat fleas were isolated and confirmed using polymerase chain reaction (PCR) and an immunofluorescence assay. The infection rate in individual fleas and the minimum infection rate in pooled fleas detected by the PCR method were found to be 18.8% (13/69) and 8.2% (8/97), respectively. Partial sequences of the plasmid pRF, 17-kDa antigen, and outer membrane protein A genes obtained from the samples are identical to those of R. felis URRWXCal2. Serological studies confirmed R. felis infection in two stray cats, as demonstrated by the presence of serum IgG antibodies against R. felis with an immunofluorescence assay titer of 1:320.

Intestinal Expression of H+ V‐ATPase in the Mosquito Aedes albopictus is Tightly Associated with Gregarine Infection

ABSTRACT. Vacuolar ATPase (V‐ATPase) is a family of ATP‐dependent proton pumps expressed on the plasma membrane and endomembranes of eukaryotic cells. Acidification of intracellular compartments, such as lysosomes, endosomes, and parasitophorous vacuoles, mediated by V‐ATPase is essential for the entry by many enveloped viruses and invasion into or escape from host cells by intracellular parasites. In mosquito larvae, V‐ATPase plays a role in regulating alkalization of the anterior midgut. We extracted RNA from larval tissues of Aedes albopictus, cloned the full‐length sequence of mRNA of V‐ATPase subunit A, which contains a poly‐A tail and 2,971 nucleotides, and expressed the protein. The fusion protein was then used to produce rabbit polyclonal antibodies, which were used as a tool to detect V‐ATPase in the midgut and Malpighian tubules of mosquito larvae. A parasitophorous vacuole was formed in the midgut in response to invasion by Ascogregarina taiwanensis, confining the trophozoite(s). Acidification was demonstrated within the vacuole using acridine orange staining. It is concluded that gregarine sporozoites are released by ingested oocysts in the V‐ATPase‐energized high‐pH environment. The released sporozoites then invade and develop in epithelial cells of the posterior midgut. Acidification of the parasitophorous vacuoles may be mediated by V‐ATPase and may facilitate exocytosis of the vacuole confining the trophozoites from the infected epithelial cells for further extracellular development.

Prevalence of Rickettsia felis and the first identification of Bartonella henselae Fizz/CAL-1 in cat fleas (Siphonaptera: Pulicidae) from Taiwan.

ABSTRACT Cat fleas (Ctenocephalides felis [Bouché]) are the primary ectoparasites of dog and cat populations. In this study, we report the monthly population dynamics of Rickettsia felis and Bartonella spp. (two zoonotic pathogens that can cause human disease) in cat fleas collected from dogs and cats in Taipei, Taiwan, from December 2006 to December 2007. Natural R. felis infection in individual cat fleas was assessed by polymerase chain reaction (PCR) using pRF-, ompB-, and gltA-specific primer pairs. Samples positive by PCR were confirmed with DNA sequencing. R. felis was detected in cat fleas year round, and the average infection rate was 21.4% (90 of 420) in 2007. Cat fleas also play an important role in the transmission of Bartonella between reservoirs and other mammalian hosts. In this study, we used primer pairs specific for the Bartonella gltA and rpoB genes to detect Bartonella infections. Of the 420 cat fleas tested, 38 were positive by PCR for Bartonella. Sequence similarities to Bartonella henselae, Bartonella clarridgeiae, and Bartonella koehlerae were observed in 6.2% (26 of 420), 2.1% (9 of 420), and 0.7% (3 of 420) of the fleas, respectively. Based on the pap31 gene sequence, several amplicons of the B. henselae detected in the cat fleas could be subgrouped into three strains: Fizz/CAL-1 (n = 18), Marseille (n = 5), and Houston-1 (n = 3). These results demonstrate that cat fleas infected with R. felis are endemic to Taiwan, and highlight the role of C. felis in Bartonella transmission between reservoirs and other mammal hosts and demonstrate the genetic variability of B. henselae in Taiwan.

Using In Situ Hybridization to Detect Endosymbiont Wolbachia in Dissected Tissues of Mosquito Host

Abstract The endosymbiont Wolbachia, extensively occurring in arthropods, usually causes reproductive distortions of the host, such as mosquitoes. In past years, detection of Wolbachia in host tissues has highly relied on transmission electron microscopy (TEM) that is tedious and usually unable to gain satisfactory results without experienced techniques and expensive instruments. Polymerase chain reaction (PCR) recently has become popular in Wolbachia identification. However, necessity of DNA extraction from host individuals or dissected tissues has limited its application in extensiveness and versatility. At present, in situ hybridization has increased its role in examination of various microbes. This report provides a technique for rapid detection and localization of Wolbachia in tissues dissected from mosquitoes and possibly other infected organisms. To detect Wolbachia and to localize them in host tissues more precisely, in situ hybridization by using digoxigenin (DIG)-labeled probes was invented and applied to Wolbachia detection in this study. The results showed that Wolbachia preferentially aggregate in ovarioles, which is consistent with previous observations by TEM. The endobacteria also were detected in salivary glands, mostly in lateral lobes. Ultrastructurally, Wolbachia has been shown to occur in the cytoplasma of salivary gland cells.

Rapid Identification of the Mediterranean Fruit Fly (Diptera: Tephritidae) by Loop-Mediated Isothermal Amplification

ABSTRACT The Mediterranean fruit fly, Ceratitis capitata (Wiedemann) (Diptera: Tephritidae), ranks as one of the worlds most destructive agricultural pests. This pest is also widespread and highly invasive; thus, it is a high priority for pest detection and quarantine programs. Although Mediterranean fruit fly adult and third-instar larvae can usually be identified and distinguished from other species by morphological keys, it is often difficult or impossible to identify or distinguish this species from other tephritids by using material from other stages of development. In such situations, use of a molecular technique known as loop-mediated isothermal amplification (LAMP) would be valuable as a rapid and robust alternative species diagnostic tool. This method uses isothermal conditions and requires only relatively inexpensive equipment. In this study we have developed a simple and rapid procedure that combines a Chelex-based DNA extraction procedure with LAMP to rapidly detect the presence of Mediterranean fruit fly DNA and discriminate it from other species, by using material from different stages of development. Amounts of DNA as little as that recovered from a single egg were shown to be adequate for the analysis, and LAMP itself required only 45 min to complete.

Development of a Larval Bioassay Method Using 96-Well Microtiter Plates for Evaluation of Susceptibility of the Cat Fleas (Siphonaptera: Pulicidae) to Insecticides.

Abstract Cat fleas (Ctenocephalides felis (Bouché)) are a common flea species mostly found on cats and dogs. They not only cause discomfort to pets and their owners but also act as important vectors for human and pet zoonoses. Over the past 15 yr, the control of cat fleas on pets has been revolutionized through the use of various treatments. At present, fipronil and imidacloprid are used for flea control in Taiwan and are available in spot-on and spray formulations. Outside Taiwan, spinosad in tablet form is also available. In this study, we examined the effects of the aforementioned three insecticides on laboratory-reared and field-collected cat flea larvae. We developed a new technique for detecting flea susceptibility using a single larval bioassay with 96-well microtiter plates via contact and oral applications. Compared to the lab strain, the field strain exhibited lower susceptibility to fipronil, with the latter showing resistance levels two to four times higher than that of the former. By contrast, no difference in susceptibility was found between the two strains when tested with spinosad and imidacloprid. Our new technique was found to be stable, standardized, more efficient, convenient, and reproducible when compared to present techniques.