Mosquito Passage Research Articles

FIELD ASSESSMENT OF AUTODISSEMINATION OF PYRIPROXYFEN BY CONTAINER-INHABITING AEDES MOSQUITOES IN FLORIDA

Domestic mosquito control for container-inhabiting Aedes vectors of Zika, chikungunya, yellow fever, and dengue viruses is challenging, and novel methods are needed. Autodissemination strategies are one such method. In this control method, females are attracted to stations treated with an insect growth regulator (IGR), become treated, and subsequently deposit the IGR in natural oviposition sites, preventing pupal emergence. We developed and tested treatment stations in semi-field conditions based on number of captured mosquitoes in the treatment stations. The modified treatment station attracted gravid females with oak leaf infusion and mosquito passage through exit chutes granted access to sucrose and topical contamination of IGR (pyriproxyfen) for 25% of released mosquitoes. Although a majority of released mosquitoes were uncaptured (75%), sufficient amounts of pyriproxyfen contaminated female mosquitoes to result in 75% inhibition of adult emergence in larval bioassays. These stations were then used in a field experiment to test the efficacy using sentinel cups with mosquito larvae. Three treatments which included varied numbers of autodissemination stations (control, low density, and high density) were compared. Both low and high density of stations provided high inhibition of adult emergence from sentinel cups relative to controls. We did not observe differences in mosquito emergence inhibition whether sites contained low or high densities of stations (i.e., similar rates of mosquito inhibition). Two additional field trials showed that topically contaminated mosquitoes were traveling further than expected and causing mortality in sentinel cups at least as far as 80 meters from the autodissemination stations. The development and implementation of autodissemination of IGRs is an additional tool for use in integrated mosquito management.

Mosquito Passage Dramatically Changes var Gene Expression in Controlled Human Plasmodium falciparum Infections.

Virulence of the most deadly malaria parasite Plasmodium falciparum is linked to the variant surface antigen PfEMP1, which is encoded by about 60 var genes per parasite genome. Although the expression of particular variants has been associated with different clinical outcomes, little is known about var gene expression at the onset of infection. By analyzing controlled human malaria infections via quantitative real-time PCR, we show that parasite populations from 18 volunteers expressed virtually identical transcript patterns that were dominated by the subtelomeric var gene group B and, to a lesser extent, group A. Furthermore, major changes in composition and frequency of var gene transcripts were detected between the parental parasite culture that was used to infect mosquitoes and Plasmodia recovered from infected volunteers, suggesting that P. falciparum resets its var gene expression during mosquito passage and starts with the broad expression of a specific subset of var genes when entering the human blood phase.

Tricks in Plasmodium’s molecular repertoire – Escaping 3′UTR excision-based conditional silencing of the chloroquine resistance transporter gene

In the human malaria parasite Plasmodium falciparum, the major determinant of chloroquine resistance, P. falciparum chloroquine resistance transporter (pfcrt), likely plays an essential role in asexual blood stages, thus precluding conventional gene targeting approaches. We attempted to conditionally silence the expression of its ortholog in Plasmodium berghei (pbcrt) through Flp recombinase-mediated excision of the 3′untranslated region (UTR) during mosquito passage. However, parasites maintained pbcrt expression despite 3′UTR excision. Characterisation of these pbcrt mRNAs, by 3′rapid amplification of cDNA ends, identified several replacement 3′UTR sequences. Our observations demonstrate the astounding genetic plasticity of this parasite when faced with the loss of an essential gene.

Experimental Passage of St. Louis Encephalitis Virus In Vivo in Mosquitoes and Chickens Reveals Evolutionarily Significant Virus Characteristics

St. Louis encephalitis virus (SLEV; Flaviviridae, flavivirus) was the major cause of epidemic flaviviral encephalitis in the U.S. prior to the introduction of West Nile virus (WNV) in 1999. However, outbreaks of SLEV have been significantly more limited then WNV in terms of levels of activity and geographic dispersal. One possible explanation for these variable levels of activity is that differences in the potential for each virus to adapt to its host cycle exist. The need for arboviruses to replicate in disparate hosts is thought to result in constraints on both evolution and host-specific adaptation. If cycling is the cause of genetic stability observed in nature and arboviruses lack host specialization, then sequential passage should result in both the accumulation of mutations and specialized viruses better suited for replication in that host. Previous studies suggest that WNV and SLEV differ in capacity for both genetic change and host specialization, and in the costs each accrues from specializing. In an attempt to clarify how selective pressures contribute to epidemiological patterns of WNV and SLEV, we evaluated mutant spectra size, consensus genetic change, and phenotypic changes for SLEV in vivo following 20 sequential passages via inoculation in either Culex pipiens mosquitoes or chickens. Results demonstrate that the capacity for genetic change is large for SLEV and that the size of the mutant spectrum is host-dependent using our passage methodology. Despite this, a general lack of consensus change resulted from passage in either host, a result that contrasts with the idea that constraints on evolution in nature result from host cycling alone. Results also suggest that a high level of adaptation to both hosts already exists, despite host cycling. A strain significantly more infectious in chickens did emerge from one lineage of chicken passage, yet other lineages and all mosquito passage strains did not display measurable host-specific fitness gains. In addition, increased infectivity in chickens did not decrease infectivity in mosquitoes, which further contrasts the concept of fitness trade-offs for arboviruses.

Genetic diversity and purifying selection in West Nile virus populations are maintained during host switching

To investigate differential evolutionary rates and selective forces of WNV in hosts and vectors, we measured the genetic diversity that arose during alternating passage in mosquitoes and birds. Within-host genetic diversity was monitored in each of three experimentally passed replicates, and the complete genome sequence of each WNV strain was determined after passage. The intrahost genetic diversity that arose during alternating passage was significantly greater than the diversity generated during chicken-only passage and similar to mosquito-only passage. d N / d S ratios suggested purifying selection similar to chick-passed virus, but not to mosquito-passed virus. Thus, the abundant genetic variation contributed to WNV populations through infection of mosquitoes and the strong purifying selection contributed by infection of birds may be maintained despite frequent host switching.

The West Nile virus mutant spectrum is host-dependant and a determinant of mortality in mice

To define the impact of mosquitoes and birds on intrahost WNV population dynamics, the mutant spectra that arose as a result of 20 serial in vivo passages in Culex pipiens and young chickens were examined. Genetically homogeneous WNV was serially passaged 20 times in each host. Genetic diversity was greater in mosquito-passaged WNV compared to chicken-passaged WNV. Changes in the viral consensus sequence occurred in WNV passaged in mosquitoes earlier and more frequently than in chicken-passaged WNV. Analysis of synonymous and nonsynonymous variation suggested that purifying selection was relaxed during passage in mosquitoes. Mortality in mice was significantly negatively correlated with the size of the WNV mutant spectrum. These studies suggest that mosquitoes serve as sources for WNV genetic diversity, that birds are selective sieves, and that both the consensus sequence and the mutant spectrum contribute to WNV phenotype.

Dynamics of susceptibility and transmissibility of the live, attenuated, candidate vaccines dengue-1 PDK13, dengue-3 PGMK30F3, and dengue-4 PDK48 after oral infection in Aedes aegypti.

Dengue-1 virus PDK13 and isolates from vaccinees (dengue-1 Ib1 and dengue-1 Ib 10), dengue-3 PGMK30F3, and dengue-4 PDK48 were studied for their abilities to infect, disseminate, and replicate in Aedes aegypti mosquitoes by the oral route. In general, infection and dissemination rates were poorer for the vaccine compared with the parent viruses. The transmissibility was also lower for dengue-1 PDK13 than the parent virus, whereas it was not detected for isolates from vaccinees and dengue-3 PGMK30F3. Transmissibility of dengue-4 PDK48 was not determined because no dissemination occurred. Replication rates of vaccine strains were also found to be less efficient than the parent viruses. These imply that vaccination with the candidate vaccine is safe. Moreover, vector attenuation of vaccine viruses was consistent with its phenotypic markers of attenuation, which remained stable after a mosquito passage or after human and mosquito passage.

Dengue 3 Virus Infection of Aedes Albopictus and Aedes Aegypti: Comparison of Parent and Progeny Candidate Vaccine Viruses

DEN-3 parent (strain CH53489) and progeny candidate vaccine (clone 24/28) viruses were compared in their abilities to interact with Aedes aegypti and Ae. albopictus. The parent and progeny virus were equivalent in their ability to infect, to replicate in, and to be transmitted by both species of mosquitoes. The candidate vaccine DEN-3 clone was temperature sensitive and had small plaque morphology. These phenotypic markers remained stable during mosquito passage. Thus, temperature sensitivity and small plaque morphology are not reliable biological markers for attenuation.

Plasmodium berghei NK65 in the inbred A-J mouse: variations in virulence of P. berghei demes.

SYNOPSIS. Plasmodium berghei NK65 was passed thru Anopheles stephensi to golden hamsters and mice. The percent cumulative mortality was compared after each mosquito passage in early (7–13) blood passages. The strain was found to have divided into demes (populations) retaining original virulence and demes which were less virulent. The separation of virulent and less virulent demes was traced to its origin. Both virulent and less virulent demes can be passed thru mosquitoes and hamsters. No concomitant organism has been identified.

Further Studies on the Plasmodium berghei-Anopheles stephensi: Rodent System of Mammalian Malaria

Techniques for laboratory maintenance of the Plasmodium berghei-Anopheles stephensi system for rodent malaria are described. The system permits the regular production of sporozoite-induced blood infections in susceptible rodents, as well as the regular attainment of preerythrocytic schizonts in the livers of young white rats. The most sensitive measures of the infectivity of a pool of sporozoites are the time to patency of infected animals, and the percentage of sporozoites which become preerythrocytic schizonts. Three kinds of variability in this infectivity were noted: 1) a variability between different pools of sporozoites, 2) an interspecific variability with tree rats, young white rats, and mice being susceptible in that order, 3) an intraspecific variability, with some animals of a given species being less susceptible than others. Repetitive sporozoite induction of rodent malaria in the laboratory by means of the Plasmodium berghei-Anopheles stephensi system was reported by us in a previous paper (Nussenzweig et al., 1966). Hamsters, tree rats (Thamnomys surdaster), and young white rats were shown to be suitable laboratory hosts, whereas white mice (CF#1 strain) were found to be less readily infected. This difficulty in infecting mice was partially circumvented with the use of an inbred strain of mouse (A/J from Jackson Memorial Laboratories) that was more susceptible to sporozoite-induced infection (Most et al., 1966). Since then, improvements in our technique have enabled us to produce and harvest a greater number of more infective sporozoites. Our experiences with this new system have demonstrated its value in both basic and applied investigations. The sporozoites have been used in chemotherapeutic studies (Most, Herman, and Schoenfeld, 1967), as well as in studies on specific immunity (Nussenzweig et al., 1967) and nonspecific resistance (Nussenzweig, 1967) to sporozoiteinduced infection. The relative ease with which this model may be set up leads us to report our techniques and the characteristics of the sporozoite-induced infections. This paper forms a background for other studies: modification of sporozoites by X-radiation Received for publication 14 May 1968. * This work, contribution no. 384 from the Army Research Program on Malaria, was sponsored by the Commission on Malaria, Armed Forces Epidemiological Board, and was supported by the United States Army Medical Research and Development Command. (Vanderberg et al., 1968), and studies on the use of these irradiated sporozoites as agents for immunization (Nussenzweig et al., 1967). MATERIALS AND METHODS Rearing of mosquitoes Anopheles stephensi were reared in an insectary maintained at 28 C and about 85% relative humidity. Rearing pans containing third and fourth instar larvae were moved to an insectary maintained at 21 C and about 85% relative humidity. The slowing down of development resulting from this drop in temperature allowed us to harvest pupae every other day, instead of daily. Pupae were harvested by means of the cold water technique of Weathersby (1963). Emerged adults were fed 10% glucose solution prior to their infective feeding on hamsters. All mosquitoes were kept on a daily cycle of 16 hr of light and 8 hr of darkness. Infection of hamsters to be used as sources of gametocytes Hamsters used for mosquito feeding were 3 to 5 weeks old. They were infected with an intraperitoneal inoculation of heart blood from a hamster infected with the NK strain of P. berghei berghei. Blood from a donor animal was found to be most useful when the animal was in the rapidly rising phase of an infection in which 20 to 35% of its erythrocytes were parasitized. To guard against senescence of the P. berghei strain only a few hamster to hamster passages of the parasite were used before interposition of a sexual cycle by mosquito passage. More recently we have been fee ng mosquitoes only on hamsters which had been infected with blood from a sporozoite-induced infection. Infection of mosquitoes P ior to their infective blood meal,. mosquitoes were kept at 21 C. The hamsters were anesthetized with an intraperitoneal injection of thialbarbitone sodium (Kemithal?), and placed on top of a cage containing about 250 mosquitoes in the 21 C insectary. The mosquitoes were allowed to feed through

Amodiaquine and hydroxychloroquine resistance in Plasmodium falciparum.

Summary and ConclusionsA strain of Plasmodium falciparum from the Magdalena Valley, Colombia, South America, which is resistant to chloroquine, has been shown to be resistant also to amodiaquine (Camoquin®) and to hydroxychloroquine (Plaquenil®), which are other members of the 4-aminoquinoline group. The resistant quality persisted after mosquito passage. The infections responded to quinine and to mepacrine, but relapses occurred. In areas where there is resistance to one of the above 4-aminoquinolines, it would appear unwise to depend upon another of the group for treatment, especially in acute cases; until evidence to the contrary is found, it seems desirable to use some other drug, perhaps quinine or mepacrine.