Reproductive output of female Anopheles gambiae (Diptera: Culicidae): comparison of molecular forms.

Knowledge of ecological differences between the molecular forms of Anopheles gambiae Giles (Diptera: Culicidae) might lead to understanding of their unique contribution to disease transmission, to better vector control, and to identification of the forces that have separated them. We compared female fecundity measured as egg batch size in relation to body size between the molecular forms in Mali and contrasted them with their sibling species, Anopheles arabiensis Patton. To determine whether eggs of different egg batches are of similar "quality," we compared the total protein content of first-stage larvae (L1s), collected < 2 h after hatching in deionized water. Egg batch size significantly varied between An. gambiae and An. arabiensis and between the molecular forms of An. gambiae (mean batch size was 186.3, 182.5, and 162.0 eggs in An. arabiensis and the M and the S molecular form of An. gambiae, respectively). After accommodating female body size, however, the difference in batch size was not significant. In the S molecular form, egg protein content was not correlated with egg batch size (r = -0.08, P > 0.7) nor with female body size (r = -0.18, P > 0.4), suggesting that females with more resources invest in more eggs rather than in higher quality eggs. The mean total protein in eggs of the M form (0.407 microg per L1) was 6% higher than that of the S form (0.384 microg per L1), indicating that the M form invests a greater portion of her resources into current (rather than future) reproduction. A greater investment per offspring coupled with larger egg batch size may reflect an adaptation of the M form to low productivity larval sites as independent evidence suggests.

Multistage multiantigen heterologous prime boost vaccine for Plasmodium knowlesi malaria provides partial protection in rhesus macaques.

A nonhuman primate model for malaria vaccine development allowing reliable, stringent sporozoite challenge and evaluation of both cellular and antibody responses is needed. We therefore constructed a multicomponent, multistage DNA vaccine for the simian malaria species Plasmodium knowlesi including two preerythrocytic-stage antigens, the circumsporozoite protein (PkCSP) and sporozoite surface protein 2 (PkSSP2), and two blood stage antigens, apical merozoite antigen 1 (PkAMA1) and merozoite surface protein 1 (PkMSP1p42), as well as recombinant canarypox viruses encoding the four antigens (ALVAC-4). The DNA vaccine plasmids expressed the corresponding antigens in vitro and induced antiparasite antibodies in mice. Groups of four rhesus monkeys received three doses of a mixture of the four DNA vaccine plasmids and a plasmid encoding rhesus granulocyte-monocyte colony-stimulating factor, followed by boosting with a single dose of ALVAC-4. Three groups received the priming DNA doses by different routes, either by intramuscular needle injection, by intramuscular injection with a needleless injection device, the Biojector, or by a combination of intramuscular and intradermal routes by Biojector. Animals immunized by any route developed antibody responses against sporozoites and infected erythrocytes and against a recombinant PkCSP protein, as well as gamma interferon-secreting T-cell responses against peptides from PkCSP. Following challenge with 100 P. knowlesi sporozoites, 1 of 12 experimental monkeys was completely protected and the mean parasitemia in the remaining monkeys was significantly lower than that in 4 control monkeys. This model will be important in preclinical vaccine development.

Linkage of a gene causing malaria refractoriness to Diphenol oxidase-A2 on chromosome 3 of Anopheles gambiae.

An inbred line of the African malaria vector Anopheles gambiae is refractory to development of malaria parasites. It is homozygous for a 4.3-kb Sal I restriction fragment at the Dox-A2 locus, whereas the parent population is polymorphic at this locus, and a susceptible line is homozygous for an alternate 3.85-kb fragment. The Dox-A2 locus is located in the middle of chromosome 3R, in division 33B, and is tightly linked to a cluster of genes including Dopa decarboxylase that are involved in the production of melanin. Because the refractoriness phenotype, melanotic encapsulation of ookinete/oocysts, might involve activation of or alteration in one or more of these genes, we performed genetic crosses to determine whether a previously identified Plasmodium cynomolgi Ceylon refractoriness gene, Pif-C, is linked to Dox-A2. Backcross mosquitoes fed on one infected monkey developed infections of < or = 100 oocysts. About 50% of these mosquitoes appeared phenotypically refractory, as expected for the backcross performed, but gave slight evidence of linkage between a refractoriness gene and Dox-A2. In contrast, females fed on a monkey that yielded higher infection levels, up to > 300 oocysts, showed clear evidence of linkage between a refractoriness gene and Dox-A2. We conclude that this Dox-A2-linked refractoriness gene is expressed under conditions particular to the higher infection levels, or that environmental factors obscured the genetic effect of this gene at lower infection levels.

Genetic approaches to malaria control: how long the road?

Malaria control schemes based on the yet untested concept of replacing vector populations with mosquitoes of the same species unable to transmit the parasite offer one more means of attacking this important public health problem. Research is underway in several laboratories aimed at defining factors that can act to interrupt the development of the malaria parasite in the mosquito host, the genetic basis for such refractory mechanisms, methods for introducing genes coding for refractory mechanisms into the mosquito genome, and methods for replacing vector populations in the field. The complexities of such an undertaking are many and varied, but the potential impact of a successful replacement strategy on the epidemiology of malaria in the target area could be significant.

Evidence through crossmating experiments of a species complex in Anopheles pseudopunctipennis sensu lato: a primary malaria vector of the American continent.

Crossmating experiments were conducted to determine if postmating reproductive barriers are involved in the maintenance of genetic divergence among populations of Anopheles pseudopunctipennis sensu lato, a primary malaria vector of the American continent. Reciprocal crosses were conducted between colony and wild strains from Mexico, Bolivia, and Peru. Hybridization experiments revealed unidirectional male/female hybrid sterility in crosses between Mexican females and South American males. The data presented provide the first evidence that genetic differences exist among geographic strains of An. pseudopunctipennis in neotropical America. There is a consistent pattern suggesting the presence of at least two allopatric sibling species. One species occurs in central Mexico, the other in the South American Andean Cordillera.

Genetic evidence of a species complex in Anopheles pseudopunctipennis sensu lato.

This preliminary report provides information concerning genetic variation in the vector mosquito Anopheles pseudopunctipennis sensu lato that could have malaria control implications. Because of inconsistencies in malaria transmission patterns within geographic zones inhabited by this vector, the possibility that it represents a species complex was investigated. Hybrid crossing studies, electrophoretic analysis of enzyme variation, and DNA restriction studies were carried out in mosquitoes captured in nine areas of Mexico, Bolivia, and Peru. The findings demonstrated the existence of a species complex believed to have resulted from allopatric speciation. This research points to a need for establishing the geographic distribution of the newly recognized species because of their potential influence on malaria control.

Exoerythrocytic development of Plasmodium gallinaceum sporozoites in a chicken fibroblast cell line and inhibition of the cell invasion by specific anti-sporozoite monoclonal antibodies.

Cultivation of the Plasmodium gallinaceum exoerythrocytic forms from sporozoites was attempted in three different cell lines: HEPG2-A16 (from a human hepatoma), VERO (monkey kidney epithelial cells) and SL-29 (chicken embryo fibroblast cells). The sporozoites invaded all three cells types but their development into exoerythrocytic forms occurred only in the SL-29 cells. In the presence of specific monoclonal antibodies against the major circumsporozoite protein, there were varying degrees of inhibition of parasite invasion of the SL-29 cells. Of seven monoclonal antibodies tested, two completely inhibited cell invasion at high concentrations and caused intense inhibition at concentrations as low as 2.5 micrograms/ml, four caused intense inhibition at these various concentrations, and one had no effect on sporozoite invasion.

[Genetic evidence of a species complex in Anopheles pseudopunctipennis pseudopunctipennis]. / Evidencia genética de un complejo de especie en Anopheles pseudopunctipennis pseudopunctipennis.

In light of inconsistencies in the pattern of malaria transmission within geographical areas inhabited by Anopheles pseudopunctipennis pseudopunctipennis, a study was carried out to investigate the possibility that this vector constitutes a species complex. Hybrid crossing studies, electrophoretic analysis of enzyme loci, and DNA restriction analysis were conducted on mosquitoes captured at nine sites in Mexico, Bolivia, and Peru. The sterility of generations resulting from cross-mating of Mexican female mosquitoes and South American male mosquitoes; the results of electrophoretic analysis, which showed differences at two loci; and a genetic distance value of 0.13 confirmed the existence of a species complex, probably produced by allopatric speciation. It is concluded that the geographic distribution of this newly discovered species complex should be defined, in view of its potential effect on malaria control.

An RFLP map of the Plasmodium falciparum genome, recombination rates and favored linkage groups in a genetic cross.

We report a genetic linkage map of the Plasmodium falciparum genome, using the inheritance patterns of nearly 90 RFLP markers in a genetic cross. Markers were assigned to polymorphic loci on all 14 nuclear chromosomes. Genetic recombination between parental markers was detected in each of the progeny, indicating that progeny from cross-fertilization events were favored over progeny from self-fertilization of either parent alone. Inheritance patterns among the markers suggested that certain parental linkage groups on chromosomes 2, 3, 12 and 13 were favored in the cross. Recombination frequencies on five chromosomes indicated an approximate map unit size of 15-30 kb per centiMorgan for P. falciparum.

Induction of Plasmodium falciparum transmission-blocking antibodies by recombinant vaccinia virus.

Many candidate antigens of malaria vaccines have limited immunological recognition. One exception is Pfs25, a cysteine-rich, 25-kilodalton sexual stage surface protein of Plasmodium falciparum. Pfs25 is a target of monoclonal antibodies that block transmission of malaria from vertebrate host to mosquito vector. The surface of mammalian cells infected with a recombinant vaccinia virus that expressed Pfs25 specifically bound transmission-blocking monoclonal antibodies. Furthermore, major histocompatibility complex-disparate congenic mouse strains immunized with recombinant Pfs25 elicited transmission-blocking antibodies, demonstrating that the capacity to develop transmission-blocking antibodies is not genetically restricted in mice. Live recombinant viruses may provide an inexpensive, easily administered alternative to subunit vaccines prepared from purified recombinant proteins to block transmission of malaria in developing countries.

Use of a restriction fragment length polymorphism (RFLP) as a genetic marker in crosses of Anopheles gambiae (Diptera: Culicidae): independent assortment of a diphenol oxidase RFLP and an esterase locus.

Analysis of restriction fragment length polymorphism (RFLP) is a powerful tool for analyzing linkage relationships in species where few genetic markers have been described and where conduct of crosses is difficult. It also permits integration of genetic and physical (cytogenetic) data when the probes have been mapped by in situ hybridization. To illustrate the utility of the method, and because some mutations of a diphenol oxidase gene might conceivably produce the malaria refractoriness phenotype of ookinete-oocyst encapsulation, backcrosses between two inbred lines of Anopheles gambiae Giles were carried out to determine the linkage relationship between the diphenol oxidase A2 (Dox) gene and the esterase locus associated with refractoriness to Plasmodium cynomolgi NIH. The Dox alleles were a Sal I restriction fragment length polymorphism visualized by probing Southern blotted DNA from portions of individual mosquitoes with a cloned Dox gene probe. The two genes were shown to segregate independently.

Differentiation of Anopheles gambiae and An. arabiensis (Diptera: Culicidae) by ELISA using immunoaffinity-purified antibodies to vitellogenin.

Yolk proteins (vitellogenin and vitellin) proved to be excellent marker molecules for separating Anopheles gambiae Giles and An. arabiensis Patton, two morphologically indistinguishable members of the An. gambiae species complex. A rabbit polyclonal antibody directed against An. gambiae yolk proteins was made species-specific by removing immunoglobulins that crossreacted with An. arabiensis by immunoaffinity chromatography. The resultant antibody was 400 times more sensitive to An. gambiae and was employed as the secondary antibody in a modified double antibody "sandwich" ELISA, which also used monoclonal antibodies to anopheline vitellogenin as the primary or coating antibody. This ELISA easily differentiated soluble yolk protein samples from An. gambiae and An. arabiensis. A field study with 628 females of An. gambiae complex collected in western Kenya demonstrated that the ELISA results were 98.4% in agreement with the standard cytotaxonomic method.

Chloroquine resistance not linked to mdr-like genes in a Plasmodium falciparum cross.

Chloroquine is thought to act against falciparum malaria by accumulating in the acid vesicles of the parasite and interfering with their function. Parasites resistant to chloroquine expel the drug rapidly in an unaltered form, thereby reducing levels of accumulation in the vesicles. The discovery that verapamil partially reverses chloroquine resistance in vitro led to the proposal that efflux may involve an ATP-driven P-glycoprotein pump similar to that in mammalian multidrug-resistant (mdr) tumor cell lines. Indeed, Plasmodium falciparum contains at least two mdr-like genes, one of which has been suggested to confer the chloroquine resistant (CQR) phenotype. To determine if either of these genes is linked to chloroquine resistance, we performed a genetic cross between CQR and chloroquine-susceptible (CQS) clones of P. falciparum. Examination of 16 independent recombinant progeny indicated that the rapid efflux phenotype is controlled by a single gene or a closely linked group of genes. But, there was no linkage between the rapid efflux, CQR phenotype and either of the mdr-like P. falciparum genes or amplification of those genes. These data indicate that the genetic locus governing chloroquine efflux and resistance is independent of the known mdr-like genes.

An ultrastructural study of the exoerythrocytic schizonts of Plasmodium cynomolgi and P. knowlesi in Macaca mulatta.

Exoerythrocytic schizonts of Plasmodium cynomolgi and P. knowlesi were examined by electron microscopy in biopsy samples of primate livers. With maturity the parasitophorous vacuole membrane becomes highly sculptured by the addition of a discontinuous dense thickening, the distribution of which can be a distinguishing character between these two species. The parasitophorous vacuole membrane follows the contours of the parasite faithfully with a minimal surrounding vacuole. The marked destruction of the cytoplasm of the host hepatocyte by most of the parasites studied however gave the distinct, but erroneous, appearance of a large parasitophorous vacuole at the light microscope level. The mature parasite often exhibited a highly invaginated surface contour with the result that the cytoplasm of the host cell and parasite became intimately interdigitated, this interweaving is unlikely to be recognized in light microscopic studies.

Effects of magainins and cecropins on the sporogonic development of malaria parasites in mosquitoes.

Magainins and cecropins are families of peptides with broad antimicrobial and antiparasitic activities derived respectively from the skin of frogs or from giant silk moths. In insects, cecropins function as part of an inducible immune system against a number of bacterial infections. When injected into anopheline mosquitoes previously infected with a variety of Plasmodium species, both magainins and cecropins disrupt sporogonic development by aborting the normal development of oocysts; sporozoites are not formed and the vector cannot transmit the parasite to another host. It may be possible to induce effective transmission-blocking immunity in the mosquito vector by the introduction and expression of genes coding for magainins, cecropins, or similarly acting parasiticidal peptides into the mosquito genome.

A general system of resistance to malaria infection in Anopheles gambiae controlled by two main genetic loci.

Genetic analysis of a system of Plasmodium refractoriness in Anopheles gambia suggests that the joint action of 2 unlinked genetic loci substantially controls expression of the susceptible and refractory phenotypes. One genetic component, here named Pif-B (for Plasmodium infectivity factor), is closely linked or identical to a polymorphic autosomal esterase locus which can be visualized by gel electrophoresis. This locus exerts the major controlling effect on susceptibility to Plasmodium cynomolgi B. The other genetic component is independent of esterase and exerts major control over refractoriness to the P. cynomolgi Ceylon strain parasite. Genetic assortment of the esterase-independent component suggests that it is controlled by 1 principal locus, here named Pif-C. The 2 genetic components of Plasmodium refractoriness appear to contribute to the same phenotype through physiologically independent means.

Anopheles sergenti (Theobald) a potential malaria vector in Egypt.

Two immunoassays for malaria sporozoite detection and identification, the immunoradiometric assay (IRMA) and the enzyme-linked immunosorbent assay (ELISA) using the species-specific monoclonal antibodies are routinely performed in our laboratory. We analyzed (573) anopheline mosquitoes of A. sergenti (463), A. pharoensis (81) and A. multicolor (29) collected from Siwa-oases and Faiyum Governorate (two known active malaria foci in Egypt), for detection of P. falciparum and P. vivax sporozoites. P. falciparum sporozoites were detected by both IRMA and ELISA tests in two A. sergenti mosquitoes (one from Siwa 1/389 = (0.26%) and one from Faiyum Governorate 1/74 = (1.35%)). No P. vivax sporozoites were detected. This finding is important in explaining the malaria transmission and provide first incrimination of An. sergenti as the responsible vector of malaria in Siwa-oasis, Egypt.

The genetics and expression of an esterase locus in Anopheles gambiae.

The main polymorphic system of esterase isoenzymes in adults of the G3 laboratory strain of Anopheles gambiae consists of two to five major bands of activity per individual. The bands are designated 5S, 5F, 13, 14, and 15. In genetic crosses, the genes which coded for the bands assorted as three codominant alleles, Est A, Est B, and Est C, at a single autosomal locus. Homozygotes for the Est C allele were significantly underrepresented among backcross progeny. The developmental pattern of esterase expression was examined. Esterase gene expression in embryos was first detectable between 2 and 12 hr after oviposition. The initiation or termination of expression of some of the bands corresponded to boundaries between developmental stages. Most of the esterase fractions were not specifically localized within the tissues tested, with the exception of a series of bands which were restricted largely to adult male testes.

The genome of Plasmodium cynomolgi is partitioned into separable domains which appear to differ in sequence stability.

The genome of Plasmodium cynomolgi is partitioned into at least 7 distinct genetic domains. Each domain is apparently uniform in DNA density and is separable from the others by CsCl density centrifugation in the presence of Hoechst dye. The protein-encoding genes that were tested are localized in the two heaviest density domains (isochores). The ribosomal genes are in two lighter isochores as well as in one of the isochores that contains protein encoding genes. Telomeric sequences are mainly, if not exclusively, in the lightest isochores, indicating that position with regard to chromosome ends may correlate with density. Blocks of a tandemly-repeating sequence which mark genetically hypervariable chromosome regions in malaria parasites are located in all isochores. However, the rate of change associated with the blocks of sequence is much slower in some isochores than in others. This indicates that the rate of genetic change in these parasites may differ with isochore and chromosomal position. These results may also have more general biological implications since they suggest that the genetic instability often noted for tandem repeat sequences in the eukaryotic genome may be limited to only a distinct subset of the genomic complement of such sequence blocks.