Molecular biological approaches to the study of vectors in relation to malaria control

To a large extent, control of malaria vectors relies on the elimination of breeding sites and the application of chemical agents. There are increasing problems associated with the use of synthetic insecticides for vector control, including the evolution of resistance, the high cost of developing and registering new insecticides and an awareness of pollution from insecticide residues. These factors have stimulated interest in the application of molecular biology to the study of mosquito vectors of malaria; focussing primarily on two aspects. First, the improvement of existing control measures through the development of simplified DNA probe systems suitable for identification of vectors of malaria. The development of synthetic, non-radioactive DNA probes suitable for identification of species in the Anopheles gambiae complex is described with the aim of defining a simplified methodology wich is suitable for entomologist in the field. The second aspect to be considered is the development of completely novel strategies through the development of completely novel strategies through the genetic manipulation of insect vectors of malaria in order to alter their ability to transmit the disease. The major requirements for producing transgenic mosquitoes are outlined together with the progress wich has been made to date and discussed in relation to the prospects which this type of approach has for the future control of malaria.

Household-based malaria control in a highly endemic area of Africa (Tanzania): determinants of transmission and disease and indicators for monitoring - Kilombero Malaria Project

The Kilombero Malaria Project (KMP) attemps to define opperationally useful indicators of levels of transmission and disease and health system relevant monitoring indicators to evaluate the impact of disease control at the community or health facility level. The KMP is longitudinal community based study (N = 1024) in rural Southern Tanzania, investigating risk factors for malarial morbidity and developing household based malaria control strategies. Biweekly morbidity and bimonthly serological, parasitological and drug consumption surveys are carried out in all study households. Mosquito densities are measured biweekly in 50 sentinel houses by timed light traps. Determinants of transmission and indicators of exposure were not strongly aggregated within households. Subjective morbidity (recalled fever), objective morbidity (elevated body temperature and high parasitaemia) and chloroquine consumption were strongly aggregated within a few households. Nested analysis of anti-NANP40 antibody suggest that only approximately 30% of the titer variance can explained by household clustering and that the largest proportion of antibody titer variability must be explained by non-measured behavioral determinants relating to an individual's level of exposure within a household. Indicators for evaluation and monitoring and outcome measures are described within the context of health service management to describe control measure output in terms of community effectiveness.

Transmission immunity in malaria: reflections on the underlying immune mechanisms during natural infections and following artificial immunization

Malaria transmission-blocking immunity has been studied in natural malaria infections in man, during infections in animals and following artificial immunization of animals with sexual stage malaria parasites. Effective immunity, which prevents infectivity of a malarial infection to mosquitoes, has been observed under all of these circumstances. Two general types of effector mechanism have been identified. One is an antibody mediated mechanism which acts against the extracellular sexual stages of the parasite within the midgut of a blood feeding mosquito. The other is a cytokine mediated mechanism which inactivates the gametocytes of the parasites while in the circulation of the vertebrate host. Both effects have been observed during natural infections and following artificial immunization. The basis of induction of transmission-blocking immunity, including the nature of the memory for such immunity, however, may be very different in different host/parasite systems and during natural infection of following artificial immunization. Following artificial immunization a strong immune memory for transmission blocking immunity has been observed in animal systems. By contrast, following natural infections in man immune memory for transmission blocking immunity has been found to be weak and short lived if it occurs at all. It is suggested that the immunogens which induce natural transmission blocking immunity may be CD4+ independent.

Adult abundance, biting behavior and parity of Anopheles aquasalis, Curry 1932 in two malarious areas of Sucre State, Venezuela

The principal vector of malaria in eastern Venezuela, Anopheles aquasalis, is exophagic and exophilic. Control using indoor insecticide house sprays has failed to lower the number of malaria cases. Therefore, studies were initiated in two villages of the eastern coastal state of Sucre to better understand this vector's biology and develop a more integrated control program. An. aquasalis was found to have a crepuscular biting behavior with a major peak at dusk and a minor peak at dawn. Mosquitos were collected more outdoors than indoors. Forty-seven percent of the biting took place before people went to bed (22:30 hr) and 69% of the mosquitos biting during this time period bite outdoors. Outdoor biting could be the reason why indoor spraying alone did not lower malaria cases. Seasonal abundance was greater in the rainy season compared to the dry season. Seasonal parous rates were high (78.3%-100%) and similar indoors and outdoors and between dry and wet season in Santa F e. In Guayana, the seasonal parity was lower (34.6%-42.2%) than Santa F e with indoor parity slightly higher than outdoors. Malaria cases were higher in Santa F e, but adult mosquito density was much lower than in Guayana. This difference could have been due to higher parity in Santa F e compared to Guayana. The greater distance to the nearest breeding site and presence of alternative hosts in Guayana can not be discounted as factors which contributed to the difference in malaria transmission between locations. We concluded that knowledge on seasonal occurrence, biting activity, resting behavior and breeding site location can be used to design a new control strategy for this vector.

Ultrastructural aspects of virus replication in one fatal case and several other isolates from a dengue type 2 outbreak in Rio de Janeiro

Dengue virus replication in mosquito cell cultures was observed by electron microscopy in one fatal and 40 classical isolates from a dengue type 2 outbreak in Rio de Janeiro and compared with the prototype New Guinea C strain. All the Brazilian isolates presented, beside the classical structured dengue virus particles, fuzzy coated virus-like particles, never observed in thereferencial New Guinea C virus strain. more numerous DEN-2 virus particles, fuzzy coated virus-like particles, defective virus particles and smooth membrane structures inside the rough endoplasmic reticulum characterized the unique fatal isolate examined.

Biochemistry and mode of action of the Bacillus sphaericus toxins

Bacillus sphaericus produces at least two toxins which are highly toxic to mosquito larvae. The binary toxin, which is comprised of proteins of 51.4 and 41.9 kDa, is present in all highly insecticidal strains. The 100 kDa SSII-1 toxin is present in most highly insecticidal as well as the weakly insecticidal strains. The current status of studies on biochemistry and mode of action of these toxins is reviewed.

Integrated control measures against Culex quinquefasciatus, the vector of filariasis in Recife

Integrated control measures against Culex quinquefasciastus have been implemented in a pilot urban area in Recife, Brazil. About 3,000 breeding sites found within the operational area were responsible for very high mosquito densities recorded during the pretrial period. Physical control measures have been applied to cess pits before starting a series of 37 treatments of the other sites with Bacillus sphaericus strain 2362, over 27 months. In spite of the difficulties due to environmental conditions, very significant reductions in preimaginal population of C. quinquefasciatus were achieved and, as a consequence, low adult mosquito densities were maintained for a relatively long period of time. Entomological and environmental data gathered in this pilot project can contribute to design an integrated mosquito control program in Recife city.

Life history correlates of adult size in the malaria vector Anopheles darlingi

Adult dry weights of laboratory-reared Anopheles darlingi were highly correlated with wing lengths, which were used to estimate size variation in natural populations of this species. Significant differences in mean wing lengths of females trapped at baits were detected among collections in the same week at one site, but not between three sites in Brazil and Boliva. Relatively higher variability of wing lengths, compared to collections of other Anopheles (Nyssorhynchus), and platykurtic size distributions in large, single-night collections suggested that An. darlingi females caught at baits emerged from heterogenous larval habitats. No relationship was detected between parous state and the body size of wild-caught females. Adult males and females of laboratory-reared An. darlingi did not differ in body size. This absence of sexual size dimorphism is rare among mosquitoes and has not been noted previously in the genus Anopheles.

Biochemical, immunological and toxicological characteristics of the crystal proteins of Bacillus thuringiensis subsp. medellin

Characterization of the insecticidal and hemolytic activity of solubilized crystal proteins of Bacillus thuringiensis (Bt) subsp. medellin (Btmed) was performed and compared to solubilized crystal proteins of isolates 1884 of B. thuringiensis subsp. israelensis (Bti) and isolate PG-14 of B. thuringiensis subsp. morrisoni (Btm). In general, at acid pH values solubilization of the Bt crystalline parasporal inclusions (CPI) was lower than at alkaline pH. The larvicidal activity demonstrated by the CPI of Btmed indicated that optimal solubilization of CPI takes place at a pH value of 11.3, in Bti at pH values from 5.03 to 11.3 and in Btm at pH values from 9.05 to 11.3. Hemolytic activity against sheep red blood cells was mainly found following extraction at pH 11.3 in all Bt strains tested. Polyacrylamide gel electrophoresis under denaturing conditions revealed that optimal solubilization of the CPI in all Bt strains takes place at the alkaline pH values from 9.05 to 11.3. An enriched preparation of Btmed crystals was obtained, solubilized and crystal proteins were separated on a size exclusion column (Sephacryl S-200). Three main protein peaks were observed on the chromatogram. The first peak had two main proteins that migrate between 90 to 100 kDa. These proteins are apparently not common to other Bt strains isolated to date. The second and third peaks obtained from the size exclusion column yielded polypeptides of 68 and 28-30 kDa, respectively. Each peak independently, showed toxicity against 1st instar Culex quinquefasciatus larvae. Interestingly, combinations of the fractions corresponding to the 68 and 30 kDa protein showed an increased toxicity. These results suggest that the 94 kDa protein is an important component of the Btmed toxins with the highest potency to kill mosquito larvae. When crystal proteins of Bti were probed with antisera raised independently against the three main protein fractions of Btmed, the only crystal protein that showed cross reaction was the 28 kDa protein. These data suggest that Btmed could be an alternative bacterium for mosquito control programs in case mosquito larval resistance emerges to Bti toxic proteins.

Simian malaria at two sites in the Brazilian Amazon - II: Vertical distribution and frequency of anopheline species inside and outside the forest

An anopheline survey was carried out in two simian malaria areas in the Brazilian Amazon, Balbina and Samuel, to determine the potential vectors of Plasmodium brasilianum. The most abundant and/or acrodendrophilic anophelines in the forest and the most likely vector were Anopheles mediopunctatus, An. nuneztovari, An. oswaldoi, An. triannulatus and An. shannoni. An. darlingi and An. marajoara were captured essentially in anthropic habitats outside the forest and are unlikely to be involved in the transmission of P. brasilianum among monkeys within the forests and from monkeys to man in their surroundings in the Amazon.

Cloning, Expression and Toxicity of a Mosquitocidal Toxin Gene of Bacillus thuringiensis subsp. medellin

Bacillus thuringiensis (Bt) subsp. medellin (Btmed) produces parasporal crystalline inclusions which are toxic to mosquito larvae. It has been shown that the inclusions of this bacterium contain mainly proteins of 94, 68 and 28-30 kDa. EcoRI partially digested total DNA of Btmed was cloned by using the Lambda Zap II cloning kit. Recombinant plaques were screened with a mouse policlonal antibody raised against the 94 kDa crystal protein of Btmed. One of the positive plaques was selected, and by in vivo excision, a recombinant pBluescript SK(-) was obtained. The gene encoding the 94 kDa toxin of Btmed DNA was cloned in a 4.4 kb DNA fragment. Btmed DNA was then subcloned as a EcoRI/EcoRI fragment into the shuttle vector pBU4 producing the recombinant plasmid pBTM3 and used to transform by electroporation Bt subsp. israelensis (Bti) crystal negative strain 4Q2-81. Toxicity to mosquito larvae was estimated by using first instar laboratory reared Aedes aegypti, and Culex quinquefasciatus larvae challenged with whole crystals. Toxicity results indicate that the purified inclusions from the recombinant Bti strain were toxic to all mosquito species tested, although the toxicity was not as high as the one produced by the crystal of the Btmed wild type strain. Poliacrylamide gel electrophoresis indicate that the inclusions produced by the recombinant strain Bti (pBTM3) were mainly composed of the 94 kDa protein of Btmed, as it was determined by Western blot

Potential Vectors of Dirofilaria immitis (Leidy, 1856) in Itacoatiara, Oceanic Region of Niterói Municipality, State of Rio de Janeiro, Brazil

Dirofilaria immitis is a widespread mosquito-borne parasite that causes dirofilariasis, a commonly diagnosed disease of dogs that is rarely reported in cats and humans. A mosquito survey was conducted in Itacoatiara in the State of Rio de Janeiro, from March 1995 to February 1996, using canine, feline and human baits. A total of 3,667 mosquitoes were dissected for D. immitis larvae, representing 19 species and 10 genera. From those, Ae. scapularis, Ae taeniorhynchus, Cx. quinquefasciatus, Cx. declarator, Cx. saltanensis and Wy. bourrouli were found infected with D. immitis parasites, and among those, only the first three harbored infective larvae. The majority of larvae were found in the Malpighian tubules (889/936), and larval melanization was observed in the two Aedes species. In descending order, the best vectors were Ae. scapularis, Ae. taeniorhynchus, and Cx. quinquefasciatus which alternate seasonally in importance. Cx. quinquefasciatus is suggested to be a vector to cats. The potential transmission of D. immitis parasites by these three vectors to man is discussed.

Studies on the Bacillus sphaericus Larvicidal Activity against Malarial Vector Species in Amazonia

In this work, bioassays were carried out in laboratory conditions (average temperature 26 ± 2ºC) to test ten strains of Bacillus sphaericus, isolated from Brazilian soils against third instar larvae from anopheline species recorded as malaria vectors in Amazonian - Anopheles nuneztovari and An. darlingi. With the former mosquito, three strains - S2, S20 and S46 showed relative activity, in 24 and 48 hr exposure to the B. spahericus strains. With the latter only the S2 and S20 were effective in the 48 hr reading. The studied strains that showed the most adequate response in the Amazonian region were S2 and S20 showing broader and more efficient results. Therefore, S2 was the most effective when the 24 and 48 hr readings were considered, because it showed the greatest relative activity values.

Molecular Epidemiology and Emergence of Rift Valley Fever

Rift Valley fever (RVF) is a mosquito-borne viral disease which manifested itself during recent epidemics and revealed its significant potential of emergence. Studies on molecular epidemiology undertaken to better understand the factors leading to RVF emergence, have confirmed the mode of circulation of the virus and highlithted probable risks and obstacles for prevention and control. As for several other viral agents, molecular epidemiology is becoming a useful tool in the study of the emergence of RVF as a serious infectious disease.

Bloodmeal microfilariae density and the uptake and establishment of Wuchereria bancrofti infections in Culex quinquefasciatus and Aedes aegypti

The relationship between ingestion of microfilariae (mf), production of infective larvae (L3) and mf density in human blood has been suggested as an important determinant in the transmission dynamics of lymphatic filariasis. Here we assess the role of these factors in determining the competence of a natural vector Culex quinquefasciatus and a non vector Aedes aegypti to transmit Wuchereria bancrofti. Mosquitoes were infected via a membrane feeding procedure. Both mosquito species ingested more than the expected number of microfilariae (concentrating factor was 1.28 and 1.81 for Cx. quinquefasciatus and Ae. aegypti, respectively) but Cx. quinquefasciatus ingested around twice as many mf as Ae. aegypti because its larger blood meal size. Ae. aegypti showed a faster mf migration capacity compared to Cx. quinquefasciatus but did not allow parasite maturation under our experimental conditions. Similar proportions of melanized parasites were observed in Ae. aegypti (2.4%) and Cx. quinquefasciatus (2.1%). However, no relationship between rate of infection and melanization was observed. We conclude that in these conditions physiological factors governing parasite development in the thorax may be more important in limiting vectorial competence than the density of mf ingested.