Live Mosquitoes Research Articles

Efficacy of dinotefuran, permethrin and pyriproxyfen combination spot-on against Aedes aegypti mosquitoes on dogs

A spot-on formulation combining permethrin, dinotefuran and pyriproxyfen (Vectra 3D™ spot-on solution for dogs – one 10–25kg pipette contains 196mg dinotefuran, 1429mg permethrin and 17mg pyriproxyfen) was evaluated in adult Beagle dogs in a study designed to measure its efficacy to control Aedes aegypti (anti-feeding effect and mortality effect).The trial was performed according to Animal Welfare and Good Clinical Practice.Twelve dogs (five males and seven female, >3 years old, weighing 8.8–13.0kg) were randomly allocated to treatment groups on pre-treatment mosquito counts: six dogs served as untreated controls, and six dogs were treated with the test formulation. Treatment consisted of applying a combination formulation to deliver at least 46.6mgkg−1 permethrin, 6.40mgkg−1 dinotefuran and 0.57mgkg−1 pyriproxyfen. The combination is designed to control fleas, ticks, sand flies and mosquitoes. Each dog was infested with approximately 100 adult unfed A. aegypti once before treatment (day 6) then at 1, 7, 14, 21 and 28 days post-treatment. Counts and engorgement determination of dead and live mosquitoes were performed after 1h exposure period. In the treated group (group A), the repellency effect of the product based on engorgement status (anti-feeding effect), was 91.5%, 94%, 94.7%, 94% and 87% at 1, 7, 14, 21 and 28 days post-treatment. Mortality effect or insecticidal efficacy calculated at the end of the 1-h exposure was almost identical when calculated 24h after the 1-h exposure and remained above 93% until the end of the in-life phase. No adverse events were observed following treatment, including observations conducted 2, 4 and 24h after the last dog was treated.

A modified experimental hut design for studying responses of disease-transmitting mosquitoes to indoor interventions: the Ifakara experimental huts.

Differences between individual human houses can confound results of studies aimed at evaluating indoor vector control interventions such as insecticide treated nets (ITNs) and indoor residual insecticide spraying (IRS). Specially designed and standardised experimental huts have historically provided a solution to this challenge, with an added advantage that they can be fitted with special interception traps to sample entering or exiting mosquitoes. However, many of these experimental hut designs have a number of limitations, for example: 1) inability to sample mosquitoes on all sides of huts, 2) increased likelihood of live mosquitoes flying out of the huts, leaving mainly dead ones, 3) difficulties of cleaning the huts when a new insecticide is to be tested, and 4) the generally small size of the experimental huts, which can misrepresent actual local house sizes or airflow dynamics in the local houses. Here, we describe a modified experimental hut design - The Ifakara Experimental Huts- and explain how these huts can be used to more realistically monitor behavioural and physiological responses of wild, free-flying disease-transmitting mosquitoes, including the African malaria vectors of the species complexes Anopheles gambiae and Anopheles funestus, to indoor vector control-technologies including ITNs and IRS. Important characteristics of the Ifakara experimental huts include: 1) interception traps fitted onto eave spaces and windows, 2) use of eave baffles (panels that direct mosquito movement) to control exit of live mosquitoes through the eave spaces, 3) use of replaceable wall panels and ceilings, which allow safe insecticide disposal and reuse of the huts to test different insecticides in successive periods, 4) the kit format of the huts allowing portability and 5) an improved suite of entomological procedures to maximise data quality.

Microsatellite-Based Parentage Analysis ofAedes aegypti(Diptera: Culicidae) Using Nonlethal DNA Sampling

To track Aedes aegypti (L.) egg-laying behavior in the field in Iquitos, Peru, we developed methods for 1) sampling DNA from live mosquitoes and 2) high through-put parentage analysis using microsatellite markers. We were able to amplify DNA extracted from a single hind leg, but not from the pupal exuvia. Removal of a leg from teneral females caused no significant changes in female behavioral or life history traits (e.g., longevity, blood feeding frequency, fecundity, egg hatch rate, gonotrophic cycle length, or oviposition behavior). Using a panel of nine microsatellite markers and an exclusion-based software program, we matched offspring to parental pairs in 10 Ae. aegypti test families in which parents originated from natural development sites in Iquitos. By mating known individuals in the laboratory, retaining the male, sampling the female's DNA before release, and collecting offspring in the field, the technique we developed can be used to genotype large numbers of Ae. aegypti, reconstruct family relationships, and track the egg-laying behavior of individual Ae. aegypti in nature.

Chitosan microparticles incorporating gold as an enhanced contrast flow tracer in dynamic X-ray imaging

In situ monitoring of a biofluid can provide important information on circulatory disorders and a basic understanding on the metabolic mechanisms of living organisms. X-ray imaging has significant advantages as one of the most popular diagnostic tools to seethrough various biological systems. Particle traced velocity field measurement is one of the most popular methods for quantitative analysis of dynamic flow motion. In this study we have developed chitosan microparticles incorporating gold nanoparticles (AuNP) as a new enhanced contrast flow tracer for dynamic X-ray imaging. Gold is a useful material possessing high X-ray absorption ability and also biocompatibility. We chose chitosan as an AuNP delivery system because it can effectively trap AuNPs at high yield. In particular, the unique gold ion reduction ability of and compatibility with surface-modified chitosans are effectively utilized. The physical properties of the Au–chitosan microparticles can be controlled by varying the molecular weight of the chitosan employed and the AuNP incorporation methodology. The environment of the particles and the type of applied X-ray essentially determine the imaging efficiency. The designed chitosan microparticles incorporating Au have been successfully applied to track the digestive mechanisms occurring in delicate insects such as live mosquitoes.

Anopheles mortality is both age- and Plasmodium-density dependent: implications for malaria transmission

BackgroundDaily mortality is an important determinant of a vector's ability to transmit pathogens. Original simplifying assumptions in malaria transmission models presume vector mortality is independent of age, infection status and parasite load. Previous studies illustrate conflicting evidence as to the importance of Plasmodium-induced vector mortality, but very few studies to date have considered the effect of infection density on mosquito survival.MethodsA series of three experiments were conducted, each consisting of four cages of 400-1,000 Anopheles stephensi mosquitoes fed on blood infected with different Plasmodium berghei ookinete densities per microlitre of blood. Twice daily the numbers of dead mosquitoes in each group were recorded, and on alternate days a sample of live mosquitoes from each group were dissected to determine parasite density in both midgut and salivary glands.ResultsSurvival analyses indicate that mosquito mortality is both age- and infection intensity-dependent. Mosquitoes experienced an initially high, partly feeding-associated, mortality rate, which declined to a minimum before increasing with mosquito age and parasite intake. As a result, the life expectancy of a mosquito is shown to be dependent on both insect age and the density of Plasmodium infection.ConclusionThese results contribute to understanding in greater detail the processes that influence sporogony in the mosquito, indicate the impact that parasite density could have on malaria transmission dynamics, and have implications for the design, development, and evaluation of transmission-blocking strategies.

Using green fluorescent malaria parasites to screen for permissive vector mosquitoes

BackgroundThe Plasmodium species that infect rodents, particularly Plasmodium berghei and Plasmodium yoelii, are useful to investigate host-parasite interactions. The mosquito species that act as vectors of human plasmodia in South East Asia, Africa and South America show different susceptibilities to infection by rodent Plasmodium species. P. berghei and P. yoelii infect both Anopheles gambiae and Anopheles stephensi, which are found mainly in Africa and Asia, respectively. However, it was reported that P. yoelii can infect the South American mosquito, Anopheles albimanus, while P. berghei cannot.MethodsP. berghei lines that express the green fluorescent protein were used to screen for mosquitoes that are susceptible to infection by P. berghei. Live mosquitoes were examined and screened for the presence of a fluorescent signal in the abdomen. Infected mosquitoes were then examined by time-lapse microscopy to reveal the dynamic behaviour of sporozoites in haemolymph and extracted salivary glands.ResultsA single fluorescent oocyst can be detected in live mosquitoes and P. berghei can infect A. albimanus. As in other mosquitoes, P. berghei sporozoites can float through the haemolymph and invade A. albimanus salivary glands and they are infectious in mice after subcutaneous injection.ConclusionFluorescent Plasmodium parasites can be used to rapidly screen susceptible mosquitoes. These results open the way to develop a laboratory model in countries where importation of A. gambiae and A. stephensi is not allowed.

Imaging movement of malaria parasites during transmission by Anopheles mosquitoes.

Malaria is contracted when Plasmodium sporozoites are inoculated into the vertebrate host during the blood meal of a mosquito. In infected mosquitoes, sporozoites are present in large numbers in the secretory cavities of the salivary glands at the most distal site of the salivary system. However, how sporozoites move through the salivary system of the mosquito, both in resting and feeding mosquitoes, is unknown. Here, we observed fluorescent Plasmodium berghei sporozoites within live Anopheles stephensi mosquitoes and their salivary glands and ducts. We show that sporozoites move in the mosquito by gliding, a type of motility associated with their capacity to invade host cells. Unlike in vitro, sporozoite gliding inside salivary cavities and ducts is modulated in speed and motion pattern. Imaging of sporozoite discharge through the proboscis of salivating mosquitoes indicates that sporozoites need to locomote from cavities into ducts to be ejected and that their progression inside ducts favours their early ejection. These observations suggest that sporozoite gliding allows not only for cell invasion but also for parasite locomotion in host tissues, and that it may control parasite transmission.

Rationale and plans for developing a non-replicating, metabolically active, radiation-attenuated Plasmodium falciparum sporozoite vaccine.

Annually, malaria causes >300 million clinical cases and 1 million deaths, is responsible for the loss of >1% of gross domestic product (GDP) in Africa and is a serious concern for travelers. An effective vaccine could have a dramatic impact on the disease. For 20 years, scientists have tried to develop modern, recombinant 'subunit' malaria vaccines. This has been difficult. In fact, there is only one recombinant protein vaccine on the market for any disease, and no vaccines based on synthetic peptides, recombinant viruses, recombinant bacteria or DNA plasmids. Most vaccines are based on attenuated or inactivated whole pathogens or material derived directly from the infectious agent. It is in that context that our recent report summarizing the protection of humans with attenuated Plasmodium falciparum (Pf) sporozoites produced at four different sites over 25 years is important. In studies utilizing live mosquitoes as the vaccine delivery mechanism, there was complete protection against malaria in 93% of volunteers (13/14) and 94% of challenges (33/35). Sanaria's goal is to develop and commercialize a non-replicating, metabolically active Pf sporozoite vaccine. Three practical questions must be addressed before manufacturing for clinical trials: (1) can one administer the vaccine by a route that is clinically practical; (2) can one produce adequate quantities of sporozoites; and (3) can sporozoites be produced with the physical characteristics that meet the regulatory, potency and safety requirements of regulatory authorities? Once these questions have been answered, Sanaria will demonstrate that the vaccine protects >90% of human recipients against experimental challenge with Pf sporozoites, can be produced with an efficiency that makes it economically feasible, and protects >90% of African infants and children from infection, and has severe morbidity and mortality. By producing a vaccine for travelers, Sanaria will provide the infrastructure, regulatory foundation and funds necessary to speed licensure, manufacturing and deployment of the vaccine for the infants and children who need it most.

Application of one step RT-PCR assay for detection of flavivirus RNA in mosquitoes

The conditions of one step RT-PCR method for detection of virus RNA in field-collected mosquitoes, and preservation period of infected mosquitoes for one step RT-PCR were examined. We compared several virus RNA extraction methods with artificially contaminated mosquito pools with dengue virus (DV), Japanese encephalitis virus (JEV), and yellow fever virus (YFV) with a known amount of plaque forming unit (PFU) to establish the condition of one step RT-PCR. In this study, most effective RNA extraction method was ISOGEN-LS extraction combined with supernatant of centrifuged mosquito homogenates. Detection limit of one step RT-PCR using flavivirus universal primer in ten mosquitoes/tube (pool) was 10 PFU of DV, JEV and YFV, 1 PFU of each viruses using species-specific primer respectively, in one hundred mosquitoes/tube, 100 PFU/tube using universal primer pairs, 10 PFU/tube using species-specific primer pairs respectively. Dengue virus infected single mosquito was mixed with 99 un-infected mosquitoes, and tested by one step RT-PCR. We could detect single infected mosquito in pools containing 99 un-infected mosquitoes. Aedes aegypti and Aedes albopictus were inoculated intrathoracically with a mouse-adapted strain of dengue-1 virus and were kept up to 30 days at different temperature. Then examined by one step RT-PCR to determine the appropriate mosquito handling method and the condition of transportation. Positive result was obtained up to 30 days after the mosquito died naturally. These results suggested that we could detect flavivirus RNA tested not only from live mosquitoes but also dead mosquitoes as well, and could apply one step RT-PCR as a rapid, specific, and highly sensitive tool for flavivirus surveillance.

Application of a polymerase chain reaction-ELISA to detect Wuchereria bancrofti in pools of wild-caught Anopheles punctulatus in a filariasis control area in Papua New Guinea.

Chemotherapy-based eradication programs are aimed at stopping transmission of Wuchereria bancrofti by its obligatory mosquito vector. This study compares one year post-treatment W. bancrofti infection rates of Anopheles punctulatus, the main vector of lymphatic filariasis in Papua New Guinea, using traditional dissection techniques and a polymerase chain reaction (PCR)-based ELISA of a parasite-specific Ssp I repeat. A total of 633 mosquitoes in 35 batches were dissected. Six batches contained W. bancrofti-infected mosquitoes, giving a minimum infection rate of 0.9%. This value was not different than the actual infection rate, which was 9 (1.4%) of 633 mosquitoes (P = 0.48). The DNA was extracted from 47 pools containing a mean of 13.2 mosquitoes per pool. A total of 621 mosquitoes were processed for the PCR-ELISA, including 486 caught by human bait and 135 by light trap, which included both dead and live mosquitoes. Of 23 pools of alcohol-preserved human-bait mosquitoes, seven were positive by the PCR-ELISA, giving an infection rate identical to that obtained by dissection of individual mosquitoes (1.4%). The minimum infection rates for pools of light-trap mosquitoes found dead and alive were 2.7% (2 of 74) and 4.9% (3 of 61), respectively. These values did not differ from each other (P = 0.84), but the overall infection rate of light-trap mosquitoes was greater than that of mosquitoes captured by human bait (3.7% versus 1.4%; P = 0.09). These data indicate that the PCR-ELISA of a W. bancrofti Ssp I repeat using pools of mosquitoes is comparable to traditional dissection techniques for monitoring transmission intensity following introduction of mass chemotherapy. This approach may also be useful for rapid and cost-effective assessment of transmission in endemic areas where the frequency of overt lymphatic pathology is low.

Why aircraft disinsection?

A serious problem is posed by the inadvertent transport of live mosquitoes aboard aircraft arriving from tropical countries where vector-borne diseases are endemic. Surveys at international airports have found many instances of live insects, particularly mosquitoes, aboard aircraft arriving from countries where malaria and arboviruses are endemic. In some instances mosquito species have been established in countries in which they have not previously been reported. A serious consequence of the transport of infected mosquitoes aboard aircraft has been the numerous cases of "airport malaria" reported from Europe, North America and elsewhere. There is an important on-going need for the disinsection of aircraft coming from airports in tropical disease endemic areas into nonendemic areas. The methods and materials available for use in aircraft disinsection and the WHO recommendations for their use are described.

Pyrethroid‐treated bednet effects on mosquitoes of the Anopheles gambiae complex in The Gambia

The response of Anopheles gambiae complex mosquitoes to men sleeping under insecticide-impregnated or untreated bednets in six verandah trap huts was studied during the dry season in The Gambia. With this type of hut it was possible to collect live and dead indoor-resting mosquitoes and estimate the number of wild mosquitoes which entered, bloodfed on man, and exited each night. Bednets were treated with emulsions targetted to leave deposits of 25 mg/m2 lambda-cyhalothrin, or 5, 50 or 500 mg/m2 permethrin, diluted from emulsifiable concentrates (EC), or a blank formulation similar to the EC except that the permethrin was omitted; the sixth net was left untreated. Nets and sleepers were rotated between huts on different nights, the design being based on a series of Latin squares and conducted double-blind. Permethrin-impregnated bednets deterred mosquitoes from entering the huts. The degree of deterrency was proportional to the dosage of permethrin. This effect was also caused by the blank formulation and therefore attributed to other components of the formulation, rather than to the permethrin itself. The net impregnated with 500 mg permethrin per square metre gave the best individual protection, reducing mosquito bloodfeeding by 91% compared with untreated nets. However, lambda-cyhalothrin was proportionately more insecticidal than permethrin at doses of equivalent deterrency. At this stage of research, it remains conjectural whether chemical deterrency or killing of malaria vectors is better for community protection.

Detection of mature malaria infections in live mosquitoes

A method has been developed which detects malaria parasites in the salivary glands of live Anopheles stephensi. The method exploits the sugar feeding behaviour of the mosquito and requires only routine Western blotting techniques on nitrocellulose membrane (NCM). Infectivity can be determined without any direct manipulation of individual mosquitoes. Female A. stephensi were infected with the rodent malaria parasite, Plasmodium berghei, and after 14–16 d were starved of fructose overnight (12–18 h), then resupplied with fructose presented through a small piece of NCM. Mosquitoes were allowed to probe the membrane for several hours; the NCM was then removed and subjected to a standard immunoblotting protocol using an anti- P. berghei circumsporozoite protein (CSP) monoclonal antibody as the primary reagent, and a horseradish peroxidase-coupled secondary antibody. NCMs taken from cages containing infected mosquitoes showed a variable number of small black dots where individual females had probed and deposited either CSP or sporozoites. Infectivity could be detected easily from 13–14 d after feeding, and in as few as 10 mosquitoes at 19 d after infection; in one instance, infection in a single mosquito was clearly determined. After blocking with goat serum, the NCMs could be stored for 3–4 months and still provided positive reactions, offering some potential for applicability to field research studies.

Technique for Isozyme Analysis of Live Mosquitoes (Diptera: Culicidae)

For isozyme characterization of live mosquitoes, one hind leg of an etherized mosquito is removed and triturated, and then the tissue homogenate is used for cellulose acetate electrophoresis. This allows genotype determination of live, virgin mosquitoes so that it is possible to sel planned genetic crosses, thus accelerating selection for homozygous enzyme markers and reducing negative effects of repeated inbreeding.

An Altitude Biting Study of Culex tritaeniorhynchus (Giles) and Other Associated Mosquitoes in Japan

At Sagiyama, Japan, >Culex tritaeniorhynchus (Giles), the vector of Japanese encephalitis virus in the Fart East, was found to bite black crowned night herons (BCNH) (a species known to host the virus of .Japanese encephalitis) at all levels up to 50 feet. The biting rate was overwhelmingly higher at ground level with a decreasing biting rate up to 50 feet. Culex pipiens (L.) and C. bitaeniorhynchus (Giles) biting rates suggested the same trend as that of C. tritaeniorhynchus. Armigeres subalbatus was collected from the ground level only. Other associated mosquito species attracted to BCNH-baited traps were too few to he considered in this study. The 1956 Professional Report of the 406th Medical General Laboratory, APO 343, San Francisco, California, contains information pertaining to the relationship of the high biting rate of C. tritaeniorhynchus at ground level to susceptible nestliing birds that fell to thc ground before the age of flight. For trapping live mosquitoes at different altitude levels, aerial traps modified after that described by Magoon (1935)and Bates (1944) Were used.

The Development of Public Health Service Quarantine Entomology and Its Program in South Florida

Guarding against the introduction of diseases from other countries is a responsibility of the Division of Foreign Quarantine of the U. S. Public Health Service. The Service has been charged with this duty since 1798. Control measures against Aedes aegypti mosquitoes became an accepted Public Health procedure on ships with the discovery in 1900 of the relationship of this mosquito with the yellow fever virus. However, it was not until the early days of international air travel that Public Health quarantine entomology as such, had its inception. Even in these early days epidemiologists of the Service foresaw an era in which there would be increased dangers to our country through the possible importation of insects from foreign countries by airplane. Their realization of this potential threat to our country led to the first reported studies of insects on aircraft. Griffitts (1931) inspected 102 aircraft arriving at Miami, Florida, from Central and South America and the Caribbean area during 1931 and reported the occurrence of 29 mosquitoes on 21 planes. This observation posed a logical question. Can insects survive at the high altitudes and cold temperatures in which planes fly? Subsequent tests by Griffitts (1933) demonstrated the ability of mosquitoes to survive for approxiinately 80 hours of flying time and at 14,000 feet altitude in these prototypes of the modern plane. Carnahan (1938) reported on insects recovered from aircraft at Miami, Florida, while Welch (1939) was the only worker up to that time to pay any attention to insects other than those of medical importance. Denning et al. (1947) and Hughes (1949) have admirably summarized the findings made by Public Health Service inspectors. Since those days much has been said of the accidental transport of insects in aircraft. Observations made today indicate that live insects, including mosquitoes, are frequently found on the modern planes which fly at higher altitudes and often for greater lengths of time than the early planes. The knowledge that mosquitoes were able to survive at the altitudes these early planes sometimes reached in their international flights was cause for deep concern. It could have been pigeon-holed as just another observation in the gathering of scientific information, but, remembering the not too far removed outbreak of yellow fever in New Orleans, our officials were not anxious to allow foreign mosquitoes access to our shores. The development of heated and pressurized cabins further increased the possibilities of transporting live mosquitoes. Consequently, studies were commenced to find the best way to prevent these insects from establishing themselves in this country. The earliest control measures made use of hydrocyanic acid gas fumigations, rotenone dusts and formaldehyde sprays, but none of these were

A Technic for Shipping Live Mosquitoes with Particular Reference to Culex tarsalis

A Technic for Shipping Live Mosquitoes with Particular Reference to <i>Culex tarsalis</i>

Mosquitoes and Encephalitis in the Yakima Valley, Washington: IV. A Trap for Collecting Live Mosquitoes

In May of 1941 an extensive field survey of the mosquitoes of the Yakima Valley, Washingron was begun as part of a study of epidemic encephalitis. An essential part of this investigation was the collection of large representative samples of live specimens, by hand after inoculation into mice, the viruses of St. Louis and Western equine encephalomyelitis. This has been subsequently confirmed.2 The trap was constructed so the insects did not have to pass through the blades of a fan, as in the New Jersey

Live Mosquitoes Replaced by Glands in Paresis Treatment

Live Mosquitoes Replaced by Glands in Paresis Treatment