The vector competence of Culex quinquefasciatus from five localities in Brazil to Dirofilaria immitis was evaluated experimentally. Females from each locality were fed on an infected dog (~ 6 microfilariae/µl blood). A sample of blood fed mosquitoes were dissected approximately 1 h after blood meal. These results demonstrated that all had ingested microfilariae (mean, 4.8 to 24.6 microfilariae/mosquito). Fifteen days after the infected blood meal, the infection and infective rates were low in all populations of Cx. quinquefasciatus. The mean number of infective larvae detected in the head and proboscis of these mosquitoes was 1-1.5. The vector efficiency, the number of microfilariae ingested/number of infective larvae, was low for all populations of Cx. quinquefasciatus. However, the survival rate for all populations was high (range 50-75%). The survival rate of Aedes aegypti assayed simultaneously for comparison was low (24.7%), while the vector efficiency was much higher than for Cx. quinquefasciatus. These data suggest that the vector competence of all assayed populations of Cx. quinquefasciatus to D. immitis in Brazil is similar and that this species is a secondary vector due to its low susceptibility. Nevertheless, vector capacity may vary between populations due to differences in biting frequency on dogs that has been reported in Brazil.

/pdf/vector-competence-of-culex-quinquefasciatus-say-from-4965nuyqe3.pdf

Vector competence of Culex quinquefasciatus say from different regions of Brazil to Dirofilaria immitis.

Xenomonitoring (detection of filarial larvae or their DNA in mosquitoes) is a sensitive marker for assessing the endemicity of filariasis and a useful tool for evaluating elimination programs. To examine the fate of microfilariae (mf) and filarial DNA in vector competent and non-competent mosquito strains, we compared the detection of Brugia malayi parasites by dissection and by quantitative real-time polymerase chain reaction (PCR) in three different mosquito strains. We conclude that PCR is much more sensitive than dissection for detecting filarial larvae, especially their remnants in mosquitoes. However, parasite DNA can be detected in both vector and non-vector mosquitoes for two weeks or longer after they ingest mf-positive blood. Thus, although xenomonitoring with vector and non-vector mosquito species may be a sensitive method for indirectly detecting filarial parasites in human populations, positive test results for parasite DNA in mosquitoes do not necessarily prove that transmission is ongoing in the study area.

/pdf/persistence-of-brugia-malayi-dna-in-vector-and-non-vector-3s24t2zmvt.pdf

Persistence of Brugia malayi DNA in vector and non-vector mosquitoes: implications for xenomonitoring and transmission monitoring of lymphatic filariasis.

Culex pipiens pipiens: characterization of immune peptides and the influence of immune activation on development of Wuchereria bancrofti.

Background 
Detection of filarial DNA in mosquitoes by PCR cannot differentiate infective mosquitoes from infected mosquitoes. In order to evaluate transmission risk an assay is needed that can specifically detect infective L3 stage parasites. We now report the development of an assay that specifically detects the infective stage of Wuchereria bancrofti in mosquitoes. The assay detects an L3-activated mRNA transcript by reverse-transcriptase PCR (RT-PCR).


Methodology/Principal Findings 
W. bancrofti cuticle-related genes were selected using bioinformatics and screened as potential diagnostic target genes for L3 detection in mosquitoes. Expression profiles were determined using RT-PCR on RNA isolated from mosquitoes collected daily across a two-week period after feeding on infected blood. Conventional multiplex RT-PCR and real-time multiplex RT-PCR assays were developed using an L3-activated cuticlin transcript for L3 detection and a constitutively expressed transcript, tph-1, for ‘any-stage’ detection.


Conclusions/Significance 
This assay can be used to simultaneously detect W. bancrofti infective stage larvae and ‘any-stage’ larvae in pooled vector mosquitoes. This test may be useful as a tool for assessing changes in transmission potential in the context of filariasis elimination programs.

/pdf/detection-of-wuchereria-bancrofti-l3-larvae-in-mosquitoes-a-4evoz44e9w.pdf

Detection of Wuchereria bancrofti L3 larvae in mosquitoes: A reverse transcriptase PCR assay evaluating infection and infectivity

Mosquitoes in the Culex pipiens complex thrive in temperate and tropical regions worldwide, and serve as efficient vectors of Bancroftian lymphatic filariasis (LF) caused by Wuchereria bancrofti in Asia, Africa, the West Indies, South America, and Micronesia. However, members of this mosquito complex do not act as natural vectors for Brugian LF caused by Brugia malayi, or for the cat parasite B. pahangi, despite their presence in South Asia where these parasites are endemic. Previous work with the Iowa strain of Culex pipiens pipiens demonstrates that it is equally susceptible to W. bancrofti as is the natural Cx. p. pipiens vector in the Nile Delta, however it is refractory to infection with Brugia spp. Here we report that the infectivity barrier for Brugia spp. in Cx. p. pipiens is the mosquito midgut, which inflicts internal and lethal damage to ingested microfilariae. Following per os Brugia exposures, the prevalence of infection is significantly lower in Cx. p. pipiens compared to susceptible mosquito controls, and differs between parasite species with <50% and <5% of Cx. p. pipiens becoming infected with B. pahangi and B. malayi, respectively. When Brugia spp. mf were inoculated intrathoracically to bypass the midgut, larvae developed equally well as in controls, indicating that, beyond the midgut, Cx. p. pipiens is physiologically compatible with Brugia spp. Mf isolated from Cx. p. pipiens midguts exhibited compromised motility, and unlike mf derived from blood or isolated from the midguts of Ae. aegypti, failed to develop when inoculated intrathoracically into susceptible mosquitoes. Together these data strongly support the role of the midgut as the primary infection barrier for Brugia spp. in Cx. p. pipiens. Examination of parasites recovered from the Cx. p. pipiens midgut by vital staining, and those exsheathed with papain, suggest that the damage inflicted by the midgut is subcuticular and disrupts internal tissues. Microscopic studies of these worms reveal compromised motility and sharp bends in the body; and ultrastructurally the presence of many fluid or carbohydrate-filled vacuoles in the hypodermis, body wall, and nuclear column. Incubation of Brugia mf with Cx. p. pipiens midgut extracts produces similar internal damage phenotypes; indicating that the Cx. p. pipiens midgut factor(s) that damage mf in vivo are soluble and stable in physiological buffer, and inflict damage on mf in vitro.

/pdf/midgut-barrier-imparts-selective-resistance-to-filarial-worm-1twwv58wmx.pdf

Midgut Barrier Imparts Selective Resistance to Filarial Worm Infection in Culex pipiens pipiens

Studies were conducted to compare early phenomena associated with W. bancrofti infection and further development in the filaria vector Culex pipiens and the refractory mosquito Aedes caspius. Ingestion rates evaluated immediately after simultaneous feeding on an infected human were 69.1% for 43 Cx. pipiens and 35.7% for 28 Ae. caspius. The observed number of mf ingested by either mosquito did not vary significantly (2.7 +/- 1.4, and 2.3 +/- 0.9, respectively) and, based on the size of the blood meal ingested (2.8 and 2.3 microliters respectively), a two-fold mf concentration factor was recorded for both species. Blood ingested by Cx. pipiens (N = 16) and Ae. caspius (N = 10) clotted within 120 and 90 min post-feeding respectively. The time difference observed, however, did not affect significantly the rates of migration into the hemocele (56 and 67% respectively). Comparison of initial infection rates with those obtained after the extrinsic incubation period of the parasite was completed, indicated that the proportion of infected Cx. pipiens was reduced by 3.9% and that of Ae. caspius by 30.0%. Furthermore, the observed infectivity ratio of 265 Cx. pipiens that had an infective blood meal was 0.74 and only 0.009 for 70 Ae. caspius. It is concluded that refractoriness of Ae. caspius to W. bancrofti is expressed through the feeding mechanism itself, by severely limiting the mf ingestion rate, and through physiological processes that inhibited the development of ingested worms.

Uptake and development of Wuchereria bancrofti in Culex pipiens L. and Aedes caspius pallas.

We compared the defense mechanisms directed against Wuchereria bancrofti by a filaria susceptible mosquito, Culex pipiens, and a refractory one, Aedes caspius. The reciprocal deleterious effects of both the mosquito host and its parasite are reported. Anatomical and histological examinations of mosquitoes revealed minor differences in the general structure of the foregut of either species. The cibarial pump of Ae. caspius, however, lacked a cibarial armature and damage inflicted to some of the microfilariae (mf) ingested by this mosquito was not mechanical. In contrast, the cibarial armature of Cx. pipiens consisted of 24 delicate teeth which did not seem to affect ingested mf. The peritrophic membrane (pm) did not constitute a significant barrier to mf migrating through the midgut wall of either mosquito. Indeed, mf forced their way out into the hemocoel, immediately after they have been ingested, and up to 60 min post-feeding, when the pm was still thin and soft. It hardened 16 h post-feeding. Traversing mf perforated the pm and displaced the basement membrane. Within the thorax of Cx. pipiens, the parasite completed its development synchronously. In contrast, mf ingested by Ae. caspius did not develop beyond the sausage stage, and many of these larvae were vacuolated. In both mosquitoes, parasitized thoracic muscle fibers were tunnelled and, only in Ae. caspius, their nuclei were pyknotic. No worm encapsulation or melanization was observed in either mosquito. It is concluded that defense lines to invading parasites in Cx. pipiens are insignificant, and that in Ae. caspius, the main barrier to parasite development resides within the thoracic muscles. In the absence of cellular evidence, it is suggested that such a barrier is physiological in nature.

Host-parasite relationships of Wuchereria bancrofti and mosquito hosts, Culex pipiens L. and Aedes caspius pallas.

Mosquitoes transmit a variety of pathogens that have devastating consequences for global public and veterinary health. Despite their capacity to serve as vectors, these insects have a robust capacity to respond to invading organisms with strong cellular and humoral immune responses. In Egypt, Aedes caspius (Pallas, 1771) has been suspected to act as a bridge vector of Rift Valley Fever virus between animals and humans. Microscopic analysis of Ae. caspius hemolymph revealed the presence of phagocytic cells called granulocytes. We further evaluated cellular immune responses produced by Ae. caspius as a result of exposure to a Gram-negative, and Gram-positive bacterium, and to latex beads. After challenge, a rapid and strong phagocytic response against either a natural or synthetic invader was evident. Hemocyte integrity in bacteria-inoculated mosquitoes was not morphologically affected. The number of circulating granulocytes decreased with age, reducing the overall phagocytic capacity of mosquitoes over time. The magnitude and speed of the phagocytic response suggested that granulocytes act as an important force in the battle against foreign invaders, as has been characterized in other important mosquito vector species.

R. E. Hammad

Papers

Uptake and development of Wuchereria bancrofti in Culex pipiens L. and Aedes caspius pallas.

Host-parasite relationships of Wuchereria bancrofti and mosquito hosts, Culex pipiens L. and Aedes caspius pallas.

Innate Cellular Immune Responses in Aedes caspius (Diptera: Culicidae) Mosquitoes.