African Vector Research Articles

Plasmodium gallinaceum: A Refractory Mechanism of Ookinete Killing in the Mosquito, Anopheles gambiae

We have identified a mechanism for refractoriness to a bird malaria, Plasmodium gallinaceum, in the African vector of human malaria, Anopheles gambiae. Oocysts fail to develop in the refractory mosquitoes as a result of ookinete death which occurs within 27 hr of midgut invasion. Ultrastructural studies showed that parasite death occurs while the ookinete lies free in the midgut epithelial cell cytosol, usually surrounded by an organelle-free zone that consists of finely fibrillar material. The mechanism of parasite killing does not involve a previously described refractory mechanism of parasite encapsulation. We selected genetic lines which are refractory and susceptible to midgut infection. Genetic crossing of the lines suggests that the refractory trait is inherited as a single dominant genetic locus. Other loci probably influence oocyst number in susceptible mosquitoes. Intracellular ookinete killing appears to involve a previously unrecognized host defense mechanism against malaria parasites that involves direct destruction of the invading organism.

Salivary Apyrase in African and New World Vectors of Plasmodium Species and its Relationship to Malaria Transmission

The salivary gland activities of apyrase, an enzyme that prevents platelet aggregation by eliminating ADP, were compared among five members of the Anopheles gambiae species complex and An. albimanus. Within the An. gambiae group, An. quadriannulatus exhibited the lowest amount of enzyme activity at all pH levels measured. Apyrase activity could be separated into three groups at pH 7.5 and 8.0. The two most anthropophilic species (An. gambiae and An. arabiensis) exhibited higher activity at pH 9.0. Anopheles merus and An. melas, both saltwater taxa, and An. albimanus, a New World species, exhibited peak apyrase activity at pH 8.0. When the effects of divalent cations (Ca++, Mg++) on enzyme activity were compared at pH 8.5, apyrase activity in the presence of Mg++ could be separated into three levels. Anopheles gambiae and An. quadriannulatus exhibited reduced activity in the presence of Mg++. Anopheles arabiensis, An. merus, and An. melas displayed the highest relative levels of activity. Anopheles albimanus, with a Mg:Ca ratio of 0.80, was most similar to An. arabiensis. These biochemical differences suggest that different isoenzymes of apyrase have developed within the genus Anopheles.

Detection ofCowdria ruminantiumby means of a DNA probe, pCS20 in infected bont ticks,Amblyomma hebraeum, the major vector of heartwater in Southern Africa

A DNA probe, pCS20, previously described for use in detection of Cowdria ruminantium infections in Amblyomma variegatum (the principal vector of heartwater) hybridized with C. ruminantium DNA in organs of laboratory-infected A. hebraeum adult ticks (the major southern African vector of heartwater). The probe hybridized with C. ruminantium DNA in 46/49 midguts from male ticks and 26/29 from females, thus indicating infection. Corresponding salivary glands were less heavily infected, but infections were more numerous in glands from males. Infection in ticks was confirmed by transmission of the disease to susceptible goats. The probe did not hybridize with DNA from uninfected ticks or with DNA from a spotted fever group rickettsia commonly associated with A. hebraeum in Zimbabwe. The C. ruminantium specific pCS20 DNA probe can be applied to determine accurately the infection rates in the two major vectors of heartwater and the risk of exposure of ruminants in endemic areas.

Comparative host usage by Amblyomma hebraeum and Amblyomma marmoreum, (Acari, Ixodidae) the South African vectors of the disease heartwater

AbstractA comparative, quantitative study is made of the overlap in host range of and relative infestation levels of the bont tick, Amblyomma hebraeum and the South African tortoise tick, Amblyomma marmoreum.Although there is very little overlap in the adult host ranges, immatures occur on a similar range of species. Niche overlap of between 30% and 90% is common for both wild and domestic hosts.This overlap suggests that A. marmoreum may be of more significance in the epidemiology of the disease heartwater than has previously been considered.ZusammenfassungVergleichende Untersuchungen zum Wirtsspektrum von Amblyomma hebraeum und Amblyomma marmoreum (Acari, Ixodidae), zwei südafrikanische Vektoren der HerzwassersuchtEs wurde eine vergleichende, quantitative Untersuchung über die Kongruenz des Wirtsspektrums und den Grad des Wirtsbefalls bei der “bont” Zecke Amblyomma hebraeum und der südafrikanischen Schildkrötenzecke Amblyomma marmoreum erstellt. Obwohl es bei ausgewachsenen Zecken nur eine geringe Kongruenz des Wirtsbefalls gibt, treten Larven und Nymphen bei einem ähnlichen Spektrum von Arten auf. Eine Nischenkongruenz von 30% bis 90% ist sowohl bei Wirten unter Wildtieren als auch unter Haustieren verbreitet. Diese Kongruenz läßt vermuten, daß Amblyomma marmoreum eine größere Bedeutung bei der Epidemiologie der Herzwassersucht haben könnte, als man bisher angenommen hat.

Stable integration and expression of a bacterial gene in the mosquito Anopheles gambiae.

Foreign DNA was successfully introduced into the germline of the African mosquito vector of malaria Anopheles gambiae. Stable integration of genes into the germlines of insects had been achieved previously only in Drosophila melanogaster and related species and required the use of the P element transposon. In these experiments with Anopheles gambiae, the plasmid pUChsneo was used, which contains the selectable marker neo gene flanked by P element inverted repeats. Mosquitoes injected with this plasmid were screened for resistance to the neomycin analog G-418. A single event of plasmid insertion was recovered. Integration appears to be stable and, thus far, resistance to G-418 has been expressed for eight generations. The transformation event appears to be independent of P.

Genetic selection of a Plasmodium-refractory strain of the malaria vector Anopheles gambiae.

The anopheline mosquito is the target in most malaria control programs, primarily through the use of residual insecticides. A mosquito was studied that is refractory to most species of malaria through a genetically controlled mechanism. A strain of Anopheles gambiae, which was selected for complete refractoriness to the simian malaria parasite Plasmodium cynomolgi, also has varying degrees of refractoriness to most other malaria species examined, including the human parasites P. falciparum, P. ovale, and P. vivax for which this mosquito is the principal African vector. Furthermore, the refractoriness extends to other subhuman primate malarias, to rodent malaria, and to avian malaria. Refractoriness is manifested by encapsulation of the malaria ookinete after it completes its passage through the mosquito midgut, approximately 16 to 24 hours after ingestion of an infective blood meal. Fully encapsulated ookinetes show no abnormalities in parasite organelles, suggesting that refractoriness is due to an enhanced ability of the host to recognize the living parasite rather than to a passive encapsulation of a dead or dying parasite. Production of fully refractory and fully susceptible mosquito strains was achieved through a short series of selective breeding steps. This result indicates a relatively simple genetic basis for refractoriness. In addition to the value these strains may serve in general studies of insect immune mechanisms, this finding encourages consideration of genetic manipulation of natural vector populations as a malaria control strategy.

Heartwater in the Caribbean

Cowdria ruminantium , causal rickettsia of heartwater in ruminants, was until recently only known to occur in Africa. it is transmitted by Amblyomma ticks. Amblyomma variegatum , one of its African vectors, was introduced into the Caribbean area with cattle from Senegal about 1830. Before 1900 only Guadeloupe and Antigua were known to be infested. Martinique was invaded as late as 1948. The spread of the tick accelerated afterwards, especially during the last decade. It has now been found on Guadeloupe (Grande Terre and Basse Terre), Antigua, Martinique, St. Croix, Puerto Rico, St. Lucia, Nevis, St. Kitts, St. Maarten/St. Martin, Vieques, Anguilla, Marie-Galante and La Désirade. All of these islands are probably still infested today, except possibly St. Croix and La Désirade. The existence of heartwater on Guadeloupe has been suspected since 1966, and was at last confirmed in 1980, having almost certainly been present for a century and a half. A strain of C. ruminantium was isolated in 1982. Retrospectively, a bovine disease with symptoms like those of heartwater has been known on the island since 1932. Two other African infections of which A. variegatum is a vector, Theileria mutans and T. velifera , also occur in cattle on Guadeloupe. Heartwater has so far been diagnosed only in Guadeloupe and in near-by Marie-Galante. An investigation for its presence has been started on other islands where A.variegatum occurs. However, it has been shown that the American tick Amblyomma maculatum is a good experimental vector, and that A.cajennense can also transmit C.ruminantium , but probably not very efficiently. If the agent can adapt to these and other American Amblyomma species, it could spread to large areas in the western hemisphere, well outside those where the strictly tropical A.variegatum would find a suitable habitat. An assessment of the extent of this serious threat to the livestock industry of the Americas should be followed by urgent action while there is still hope of removing the danger as long as the disease remains limited to a few of the smaller islands. Apart from a survey of the distribution of the disease and Amblyomma ticks, information is needed on animal movements between islands, on migration routes of birds (hosts of immature stages), animal husbandry methods, and species and numbers of domestic animals, as well as wild hosts which may interfere with tick eradication. Severe dermatophilosis (cutaneous streptothricosis) of cattle appears to be closely linked with the presence of A.variegatum and causes high losses on the infested islands. This will make it easier to obtain the collaboration of livestock owners and authorities for eradication campaigns, even on islands where heartwater does not (yet) occur.

Field Evaluation of Bacillus thuringiensis var. israelensis as a Black Fly Biocontrol Agent and its Effect on Nontarget Stream Insects12

J. Econ. Entomol. 74: 314--3 18 ( 1981} When a small stream was treated at 0.5 ppm/15 min (l3°C) with an aqueous suspension of unformulated Bellon primary powder of Bacillus thuringiensis Berliner var. israelensis, 96, 86, 53, and 11% reductions occurred , respectively , in larval populations of Simulium spp . black flies at 20, 180, 350, and 705 m below the treatment point. In contrast, the number of black fly larvae declined by only 5% in the upstream control. Mortality occurred within 2-3 days of treatment , with few dead larvae remaining attached by the 5th day posttreatment. The mortality rates of larvae transported to and reared in the laboratory for 48 h posttreatment closely corresponded to the rates recorded in the stream. Significantly higher mortality rates occurred among smaller larvae . In contrast to the sharp reduction (89%) in black fly larvae in the 20-350 m area below the treatment point, Surber samples indicated increases in mayfly (35%), caddisfly (47%), stonefly (75%) , chironomid (19%), and elmid (242%) populations. Thus , no adverse effect on any of these nontarget populations was evident following stream treatment. Varieties of Bacillus thuringiensis Berliner which are active against lepidopterans were shown to be relatively ineffective against mosquitoes (Hall et al. 1977) and black flies (Lacey and Mull a 1977). Consequently, this bacterium had been considered to have little potential as a biological control agent of these medically impor­ tant insects. The discovery of a new strain of B. thurin­ giensis, however, and the demonstration of its high pathogenicity to mosquitoes renewed interest in this bac­ terium (Goldberg and Margalit 1977) . The strain was determined to be a new serotype (H-14) and was de­ scribed as B. t. var. israelensis (de Barjac 1978). The laboratory effectiveness of this new variety as a black fly larvicide was subsequently demonstrated against Cnephia, Prosimulium, and Simulium spp., including the West African vector of onchocerciasis S. damnosum Theobald s. I. (Undeen and Berl 1979, Undeen and Nagel 1978). Our laboratory studies have shown that the effectiveness of the bacterium as a larvicide can be influenced by several factors, such as the species and in star of black fly, temperature, formulation, and prod­ uct manufacturer (Molloy et al. 1981). Reported here are results of a field trial with B. t. var . israelensis against Simulium spp. in the Adirondack Mountains of New York. Our primary objectives were: to determine the effective carry of the treatment (i.e., the distance downstream in which the bacteria continued to produce high mortality); to examine instar suscepti­ bility; and to monitor the effect of the treatment on non­ targt insect populations.

Tests of susceptibility of Liberian Culex quinquefasciatus to Tanzanian Wuchereria bancrofti

Culex quinquefasciatus strains of Liberian and Tanzanian origin were fed on Tanzanian donors with Wuchereria bancrofti microfilaraemias. 11 to 17 days later the mosquitoes were dissected and scored for the presence and numbers of immature and mature filarial larvae. In one experiment a significantly lower susceptibility was found in a Liberian compared with a Tanzanian strain, but in the other cases there was no significant difference. The susceptibility of the Liberian strains was higher than in published data on the same mosquito strains tested with Liberian W. bancrofti. It is doubtful whether the Liberian mosquito strains could be a useful source of genes for the construction of a refractory strain with which to replace an East African vector population.

Loa loa: development to the infective stage in an American deerfly, Chrysops atlanticus.

Chrysops atlanticus, an American species of deerfly collected along the Mississippi Gulf Coast, will support the development of the microfilaria of human Loa loa to the infective stage. Development takes place in the fat body of the fly and requires 9 to 10 days of development and pattern of morphogenesis of the parasite in C. atlanticus is virtually identical to that described in natural African vectors. Chrysops atlanticus will support the development of large numbers of L. loa to the infective stage without apparent ill effects. Two uninfected patas nonkeys each were given 75 third-stage larvae obtained from experimentally infected C. atlanticus. Both animals developed patent infections in approximately 5 months, clearly demonstrating that the entire life cycle of Loa loa can be maintained in the laboratory outside endemic areas.