Malaria infection of the mosquito Anopheles gambiae activates immune-responsive genes during critical transition stages of the parasite life cycle

Six gene markers have been used to map the progress of the innate immune response of the mosquito vector, Anopheles gambiae, upon infection by the malaria parasite, Plasmodium berghei. In addition to four previously reported genes, the set of markers included NOS (a nitric oxide synthase gene fragment) and ICHIT (a gene encoding two putative chitin-binding domains separated by a polythreonine-rich mucin region). In the midgut, a robust response occurs at 24 h post-infection, at a time when malaria ookinetes traverse the midgut epithelium, but subsides at later phases of malaria development. In contrast, the salivary glands show no significant response at 24 h, but are activated in a prolonged late phase when sporozoites are released from the midgut into the haemolymph and invade the glands, between 10 and 25 days after blood feeding. Furthermore, the abdomen of the mosquito minus the midgut shows significant activation of immune markers, with complex kinetics that are distinct from those of both midgut and salivary glands. The parasite evidently elicits immune responses in multiple tissues of the mosquito, two of which are epithelia that the parasite must traverse to complete its development. The mechanisms of these responses and their significance for malaria transmission are discussed.     

Distribution of genetic diversity in relation to chromosomal inversions in the malaria mosquito Anopheles gambiae

The epidemiology of malaria in Africa is complicated by the fact that its principal vector, the mosquito Anopheles gambiae, constitutes a complex of six sibling species. Each species is characterized by a unique array of paracentric inversions, as deduced by karyotypic analysis. In addition, most of the species carry a number of polymorphic inversions. In order to develop an understanding of the evolutionary histories of different parts of the genome, we compared the genetic variation of areas inside and outside inversions in two distinct inversion karyotypes of A. gambiae. Thirty-five cDNA clones were mapped on the five arms of the A. gambiae chromosomes with divisional probes. Sixteen of these clones, localized both inside and outside inversions of chromosome 2, were used as probes in order to determine the nucleotide diversity of different parts of the genome in the two inversion karyotypes. We observed that the sequence diversity inside the inversion is more than three-fold lower than in areas outside the inversion and that the degree of divergence increases gradually at loci at increasing distance from the inversion. To interpret the data we present a selectionist and a stochastic model, both of which point to a relatively recent origin of the studied inversion and may suggest differences between the evolutionary history of inversions in Anopheles and Drosophila species.     

The malaria parasite, Plasmodium falciparum, increases the frequency of multiple feeding of its mosquito vector, Anopheles gambiae

It has often been suggested that vector-borne parasites alter their vector's feeding behaviour to increase their transmission, but these claims are often based on laboratory studies and lack rigorous testing in a natural situation. We show in this field study that the malaria parasite, Plasmodium falciparum, alters the blood-feeding behaviour of its mosquito vector, Anopheles gambiae s.l., in two ways. First, mosquitoes infected with sporozoited, the parasite stage that is transmitted from the mosquito to a human, took up larger blood meals than uninfected mosquitoes. Whereas 72% of the uninfected mosquitoes had obtained a full blood meal, 82% of the infected ones had engorged fully. Second, mosquitoes harbouring sporozoites were more likely to bite several people per night. Twenty-two per cent of the infected mosquitoes, but only 10% of the uninfected mosquitoes, contained blood from at least two people. We conclude that the observed changes in blood-feeding behaviour allow the parasite to spread more rapidly among human hosts, and thus confirm that the parasite manipulates the mosquito to increase its own transmission.     

A shared genetic mechanism for melanotic encapsulation of CM-Sephadex beads and a malaria parasite, Plasmodium cynomolgi B, in the mosquito, Anopheles gambiae

Dialysis dose and malnutrition have a great impact on the clinical out come of chronic hemodialysis patients. The interrelationships between them, however, remain undefined. Thus, we performed a study to determine the effects of increasing the dialysis dose on serum albumin concentrations and mortality in hemodialysis patients. We examined urea kinetic modeling, biochemical nutritional indices, comorbid conditions, patient survival time, and annual mortality rate. Dialysis dose, measured by Kt/V, significantly increased from 1.3 +/- 0.3 in 1987 to 1.5 +/- 0.4 in 1990 and to 1.7 +/- 0.4 in 1993. Serum albumin level also increased from 3.8 +/- 0.4 g/dL in 1987 to 4.0 +/- 0.4 in 1990 and to 1.7 +/- 0.4 in 1993. In 1993, 76% of patients had Kt/V > or = 1.50 compared with 45% in 1990 and 28% in 1987, whereas 82% of patients had a serum albumin level > or 4.0 g/dL in 1993 compared with 58% in 1990 and 29% in 1987. Protein catabolic rate and hematocrit also increased from 1987 to 1993, but not serum cholesterol or triglyceride. The annual mortality rate declined from 16.1% in 1987 to 13.2% in 1990 and to 8.0% in 1993. The decrease in mortality appeared to be unrelated to differences in patient selection or comorbid conditions. Serum albumin levels, hematocrit, Kt/V, and protein catabolic rate were significantly related to patient survival after age, sex, and diabetic status had been adjusted. Furthermore, there was a positive correlation between Kt/Vs and serum albumin concentration (r = 0.216, P < 0.001). Thus it appears that increasing the dose of dialysis improves serum albumin levels and perhaps survival rate in hemodialysis patients as well.     

A cuticular protein from the moulting stages of an insect

A 22 kDa peptide was purified from prepupal cuticles of 5th instar Calpodes ethlius caterpillars. It was absent earlier in the stadium and from the egg and adult, i.e. it is related to cuticle turnover rather than cuticle structure. It was present at larval and metamorphic moults, showing that it is related to moulting not just metamorphosis. The cDNA corresponding to the 22 kDa peptide was isolated by antibody screening of an epidermal cDNA expression library. Hybridization to Calpodes genomic DNA showed that the gene was present as a single copy. The deduced amino acid sequence is not like any of the sequences of cuticular structural proteins that have been published, but has a 47 amino acid sequence similar to bacteriophage T7 N-acetylmuramoyl-L-alanine amidase (34% identical, 51% similar). The amino acid sequence, the timing of expression in development, and the similarity between the substrate of the bacteriophage amidase and components of insect cuticle, all suggest that the 22 kDa protein may have a role in cleaving chitin-peptide bonds as a prerequisite for digestion of the cuticle by chitinases and proteases.     

Activation of Anopheles gambiae mosquitoes by carbon dioxide and human breath

Female Anopheles gambiae Giles mosquitoes were observed individually in a cage within a wind tunnel and their responses to pulses of carbon dioxide recorded on video tape. The range of concentrations tested revealed an 'activation' threshold concentration of carbon dioxide in the region of 0.01% above background. At this concentration, approximately 60% of the mosquitoes took off and flew upwind. Pulses of human breath, diluted with wind tunnel air to reproduce equivalent concentrations of carbon dioxide, elicited similar levels of response and the same 'activation' threshold concentration. These findings are discussed in relation to the activation of host-seeking mosquitoes.     

Detection of chromosome races among the malarial mosquito Anopheles sacharovi]

Depression in the elderly is a public health problem that will increase in magnitude as a progressively larger proportion of our population consists of those over the age of 65. Epidemiologic studies indicate that the elderly are in need of physical and mental health care. New data from 1,645 respondents in a Southeastern county show the high frequency of depressive symptomatology in the elderly and a significant relationship between poverty and depressive symptomatology. Predominant symptoms were lowered spirits, feeling helpless, a gloomy outlook on the future, a sense of powerlessness, and difficulties with sleep and appetite. A social psychiatric orientation is needed to supplement the more traditional psychodynamic formulations of depression in the aged. Adverse social conditions and cultural biases deprive the elderly person of a requisite level of self-esteem and place him at risk for depression. Treatment included humanistically based psychotherapy which can be carried out by doctors, the use of antidepressants, medications, and the development of social support systems.     

Complexities in the genetic structure of Anopheles gambiae populations in west Africa as revealed by microsatellite DNA analysis

Chromosomal forms of Anopheles gambiae, given the informal designations Bamako, Mopti, and Savannah, have been recognized by the presence or absence of four paracentric inversions on chromosome 2. Studies of karyotype frequencies at sites where the forms occur in sympatry have led to the suggestion that these forms represent species. We conducted a study of the genetic structure of populations of An. gambiae from two villages in Mali, west Africa. Populations at each site were composed of the Bamako and Mopti forms and the sibling species, Anopheles arabiensis. Karyotypes were determined for each individual mosquito and genotypes at 21 microsatellite loci determined. A number of the microsatellites have been physically mapped to polytene chromosomes, making it possible to select loci based on their position relative to the inversions used to define forms. We found that the chromosomal forms differ at all loci on chromosome 2, but there were few differences for loci on other chromosomes. Geographic variation was small. Gene flow appears to vary among different regions within the genome, being lowest on chromosome 2, probably due to hitchhiking with the inversions. We conclude that the majority of observed genetic divergence between chromosomal forms can be explained by forces that need not involve reproductive isolation, although reproductive isolation is not ruled out. We found low levels of gene flow between the sibling species Anopheles gambiae and Anopheles arabiensis, similar to estimates based on observed frequencies of hybrid karyotypes in natural populations.     

Enzyme variation at the aspartate aminotransferase locus in members of the Anopheles gambiae complex (Diptera: Culicidae)

The enzyme aspartate aminotransferase (AAT) currently is used to identify Anopheles quadriannulatus Theobald, the animal-biting, nonmalaria vector species of the Anopheles gambiae complex. Samples of An. quadriannulatus from South Africa and An. gambiae Giles s.str. from the island of Grand Comoros and the People's Republic of Congo have shown variation in electromorph frequencies that indicate that AAT has five alleles. The three slowest alleles are found in An. quadriannulatus and the three fastest in An. gambiae, An. arabiensis Patton and An. merus D?nitz. One of these is common to both An. quadriannulatus and An. gambiae. This overlap indicates a potential misidentification of 0.3% of unknown females with a further 2.1% being unidentifiable. However, all of the specimens in the overlap area were classified correctly using octanol dehydrogenase (ODH). Variation at the ODH locus in An. quadriannulatus is recorded for the first time, with four of 157 specimens being heterozygous for the fast allele. The probability of both AAT and ODH giving an incorrect or indecisive identification is 0.0005. The slowest AAT alleles were present in samples from a single locality, indicating the lack of gene flow between subpopulations of An. quadriannulatus in close geographic proximity in the Shingwedzi area, South Africa. A modified method for multiple gel casting is given.     

Induced immunity against the mosquito Anopheles stephensi Liston (Diptera: Culicidae): effects on mosquito survival and fecundity

Mice were immunised three to five times with extracts of Anopheles stephensi heads, midguts, ovaries or fat bodies. At each immunisation the effects of feeding An. stephensi on the mice was determined, and changes in mosquito longevity and fecundity examined as the immune response developed. Although variability was common between control cages, significant and consistent reductions in mosquito longevity were observed when midguts were used as immunogens. Other extracts caused transient reductions in mortality. Fecundity was reduced significantly in mosquitoes fed upon mice immunised with each extract in at least one experiment. Mosquitoes fed upon fat-body-immunised mice showed delayed egg-laying as well as overall reduction in fecundity. The results confirm the feasibility of targeting mosquito antigens for novel vaccine development, but the "shotgun" approach used probably fails to successfully hit a suitable target antigen with any consistency. The natural variation in mosquito mortality can be countered by rigorous statistical analysis which can identify subtle effects in a very "noisy" experimental system. The midgut is the obvious target organ for anti-mosquito vaccine development and future work will focus on targeting components of this tissue for further immunisations.     

Mapping a quantitative trait locus involved in melanotic encapsulation of foreign bodies in the malaria vector, Anopheles gambiae

A Plasmodium-refractory strain of Anopheles gambiae melanotically encapsulates many species of Plasmodium, whereas wild-type mosquitoes are usually susceptible. This encapsulation trait can also be observed by studying the response of refractory and susceptible strains to intrathoracically injected CM-Sephadex beads. We report the results of broad-scale quantitative trait locus (QTL) mapping of the encapsulation trait using the bead model system. Interval mapping using the method of maximum likelihood identified one major QTL, Pen1. The 13.7-cM interval containing Pen1 was defined by marker AGH157 at 8E and AGH46 at 7A on 2R. Pen1 was associated with a maximum LOD score of 9.0 and accounted for 44% of the phenotypic variance in the distribution of phenotypes in the backcross. To test if this QTL is important for encapsulation of Plasmodium berghei, F2 progeny were infected with P. berghei and evaluated for degree of parasite encapsulation. For each of the two markers that define the interval containing Pen1, a significant difference of encapsulation was seen in progeny with at least one refractory allele in contrast with homozygous susceptible progeny. These results suggest that Pen1 is important for melanotic encapsulation of Plasmodium as well as beads.     

Escape reactions and variation in some genetic parameters in larva from the malaria mosquito Anopheles messeae

S-Methyl N,N-diethylthiolcarbamate sulfoxide (DETC-MeSO) and sulfone (DETC-MeSO2) both inhibit rat liver low Km aldehyde dehydrogenase (ALDH2) in vitro and in vivo (Nagendra et al., Biochem Pharmacol 47: 1465-1467, 1994). DETC-MeSO has been shown to be a metabolite of disulfiram, but DETC-MeSO2 has not. Studies were carried out to further investigate the inhibition of ALDH2 by DETC-MeSO and DETC-MeSO2. In an in vitro system containing hydrogen peroxide and horseradish peroxidase, the rate of DETC-MeSO oxidation corresponded to the rate of DETC-MeSO2 formation. Carbamoylation of GSH by both DETC-MeSO and DETC-MeSO2 was observed in a rat liver S9 fraction. Carbamoylation of GSH was not observed in the presence of N-methylmaleimide. In in vitro studies, DETC-MeSO and DETC-MeSO2 were equipotent ALDH2 inhibitors when solubilized mitochondria were used, but DETC-MeSO was approximately four times more potent than DETC-MeSO2 in intact mitochondria. In studies with rats, the dose (i.p. or oral) required to inhibit 50% ALDH2 (ED50) was 3.5 mg/kg for DETC-MeSO and approximately 35 mg/kg for DETC-MeSO2, approximately a 10-fold difference. Furthermore, maximum ALDH2 inhibition occurred 1 hr after DET(-MeSO administration, whereas maximal ALDH2 inhibition occurred 8 hr after DETC-MeSO2 dosing. DETC-MeSO is, therefore, not only a more potent ALDH2 inhibitor than DETC-MeSO2 in vivo, but also in vitro when intact mitochondria are utilized. The in vitro results thus support the in vivo findings. Since oxidation of DETC-MeSO can occur both enzymatically and non-enzymatically, it is possible that DETC-MeSO2 is formed in vivo. DETC-MeSO2, however, is not as effective as DETC-MeSO in inhibiting ALDH2, probably because it has difficulty penetrating the mitochondrial membrane. Thus, even if DETC-MeSO2 is formed in vivo from DETC-MeSO, it is the metabolite DETC-MeSO that is most likely responsible for the inhibition of ALDH2 after disulfiram administration.     

Molecular identification of sympatric chromosomal forms of Anopheles gambiae and further evidence of their reproductive isolation

In presented paper a review of recent articles regarding applicability of computerized rhinomanometry in modern rhinological diagnostics was done. A conclusion of discussed opinions is that at the time it is no agreement about clinical value of rhinomanometry. There are however some applications, in which rhinomanometry seems to have proved its usefulness.     

Interaction between three types of variation and fluctuating asymmetry in the population of malaria mosquito Anopheles messeae Fall

In the population of the malaria mosquito Anopheles messeae, interrelations of three types of variation (polymorphism for inversions, dorsal color of abdomen and thorax, and number of dents in the crests of the stigmal plate) and fluctuating asymmetry were studied. The presence of white pigmentation on the larval dorsum did not correlate with inversion polymorphism and fluctuating asymmetry, but correlated with the number of small and large dents in the crests of stigmal plate. Asymmetry was shown to have a weak positive correlation with the number of dents. In larvae having a small number of dents, a decrease in the frequency of autosomal homozygotes 3R00 and an increase in the frequency of autosomal heterozygotes 3R01 were found. The dents in the crests of the stigmal plate had two morphological forms: a large one, which is associated with arms 2R and XL, and a small one, which is associated with arms 3R, XL, and 3L. Correlation between inversion polymorphism and bilateral (fluctuating) asymmetry was more complex. The group of individuals with a difference by one dense differed from that with a difference by three or more dents in the frequency of chromosome sequences XL0, XL1, and 2R0. Chromosome sequences XL1, 2R0, 3R0, and 3L1 were classified as those increasing the developmental stability of individuals; XL2, XL0, 2R1, 3R1, and 3L0, as those decreasing it.     

The mosquito Anopheles stephensi limits malaria parasite development with inducible synthesis of nitric oxide

We have discovered that the mosquito Anopheles stephensi, a natural vector of human malaria, limits parasite development with inducible synthesis of nitric oxide (NO). Elevated expression of A. stephensi NO synthase (NOS), which is highly homologous to characterized NOS genes, was detected in the midgut and carcass soon after invasion of the midgut by Plasmodium. Early induction is likely primed by bacterial growth in the blood meal. Later increases in A. stephensi NOS expression and enzyme activity occurred at the beginning of sporozoite release. Circulating levels of nitrite/nitrate, end-products of NO synthesis, were significantly higher in Plasmodium-infected mosquitoes. Dietary provision of the NOS substrate L-arginine reduced Plasmodium infections in A. stephensi. In contrast, dietary provision of a NOS inhibitor significantly increased parasite numbers in infected mosquitoes, confirming that A. stephensi limits Plasmodium development with NO.     

Polymorphism of pericentromere heterochromatin of polytene chromosomes of ovarian trophocytes in natural populations of the malaria mosquito Anopheles messeae Fall

The HBsAg status and demographic data of 2480 pregnant women who attended antenatal clinics at Maternal and Child Health Centres in Hong Kong were collected by means of a self-administered questionnaire over a 1-week period in July 1996, to explore the underlying reason of a higher than expected HBsAg prevalence. Local women constituted 49.2% of the sample, mainland Chinese 39.7% and others 11.1%. The overall HBsAg prevalence was 10.0%. When related to place of birth, those born in Hong Kong had a prevalence of 8.4% whereas the prevalence of those born in mainland China was 13.1% (P < 0.001). The overall HBsAg carriage rate is high because of a higher rate in immigrants in the community. It is apparent that the HBsAg prevalence of local people in Hong Kong has been decreasing in the past decade. Overall, the current HBsAg carriage rate in the local adult population is estimated to have declined to about 8%.     

The genetics of black larva, an autosomal recessive lethal mutation located on chromosome 3 in the mosquito Anopheles albimanus

Lung tissue from 14 normal residents of high altitude regions, 10 patients with chronic bronchitis and emphysema, and 1 patient with Pickwickian syndrome was studied with regard to the occurrence of pulmonary vascular changes. In addition to the well-known pulmonary arterial alterations, lesions in small pulmonary veins were found in the great majority of the cases. These changes, consisting of medial hypertrophy and arterialization and of bundles of smooth muscle cells within the venous intima, have not been described before in man. These findings suggest that alveolar hypoxia acts not only on small pulmonary arteries and arterioles but also on veins of small caliber, probably by inducing venoconstriction.