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.     

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.     

Leukocytes in a Plasmodium falciparum-infected blood meal reduce transmission of malaria to Anopheles mosquitoes

Mosquitoes are infected with Plasmodium falciparum by taking a blood meal from a gametocyte carrier. Since a mosquito takes a volume of 1 to 2 microl, a blood meal may contain 1 x 10(4) to 3 x 10(4) leukocytes (WBC). The majority of WBC are composed of neutrophils which may phagocytose and kill developing gametes inside the mosquito midgut. Phagocytosis was measured in vitro by a luminol-dependent chemiluminescence (CL) assay. In the presence of P. falciparum gametes, sera from areas of endemicity had an increased CL response compared to controls. In mosquito membrane feeding experiments some such sera showed a transmission reduction which was related to the presence of viable WBC. The results of this study suggest that phagocytosis of opsonized gametes inside the mosquito midgut occurs and can contribute to a reduction in the transmission of P. falciparum parasites.     

The Anopheles gambiae tryptophan oxygenase gene expressed from a baculovirus promoter complements Drosophila melanogaster vermilion

An Anopheles gambiae cDNA encoding tryptophan oxygenase was placed under the control of the constitutive baculovirus promoter, ie-1. The chimeric construct, expressed transiently in vermilion (tryptophan oxygenase) mutants of Drosophila melanogaster, partially rescued adult eye color. The successful genetic complementation by this construct demonstrated both the proper function of the tryptophan oxygenase product and the effectiveness of the ie-1 promoter in directing expression of foreign genes in live insects. The functionality of An. gambiae tryptophan oxygenase in a higher fly fulfils predictions based on its structural conservation throughout millions of years of independent evolution.     

Effect of body size on host seeking and blood meal utilization in Anopheles gambiae sensu stricto (Diptera: Culicidae): the disadvantage of being small

1. Ranolazine shifts ATP production away from fatty acid oxidation toward glucose oxidation. 2. Because more oxygen is required to phosphorylate a given amount of ATP during fatty acid oxidation than during carbohydrate oxidation, the ranolazine-induced shift in substrate selection reduces the cell's demand for oxygen without decreasing its ability to do work. The shift also maintains coupling of glycolysis to glucose oxidation during ischemia, thus reducing tissue acidosis. 3. This unique, non-hemodynamic mechanism offers the potential to treat angina without reducing blood pressure, heart rate or myocardial contractility. 4. At least three double-blind, randomized, placebo-controlled clinical trials have yielded data consistent with this hypothesis.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

The Anopheles albimanus white gene: molecular characterization of the gene and a spontaneous white gene mutation

We have cloned and characterized the white gene of Anopheles albimanus. Comparison of the deduced amino acid sequence of this white gene with its homologs from six species of Diptera show that the An. albimanus gene is most similar to the white gene of An. gambiae (92% identity). A spontaneous white-eyed mutant An. albimanus was caused by an approximately 10 kb insertion into a CT dinucleotide repeat region of intron 2 of the white locus. The flanks of this insertion are long (at least 400 bp), nearly perfect inverted terminal repeat sequences. This cloned white gene should be useful as a marker for germ line transformation of An. albimanus.     

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.     

Effects of a dietary mixture of meat and bone meal, feather meal, blood meal, and fish meal on nitrogen utilization in finishing Holstein steers

Our objective was to determine to what extent rate and efficiency of protein gain in finishing cattle can be enhanced by feeding an amino acid-balanced mixture of undegraded intake proteins. The Cornell Net Carbohydrate and Protein System (CNCPS) model was used to formulate a corn-based diet that would meet the rumen requirements for 410-kg large-framed steers with an estrogen implant and fed an ionophore. The CNCPS model was also used to formulate a highly undegradable intake protein (UIP) mixture from meat and bone meal, blood meal, fish meal, and hydrolyzed feather meal to provide the amino acids needed to supplement those derived from microbial protein to better meet amino acid requirements for growth. Four Holstein steers weighing 407 kg were offered a 90:10 concentrate-forage diet at hourly intervals at 95% of ad libitum intake. The steers were injected with 500 microg of estradiol-17beta at 12-h intervals to mimic the effects of an estrogenic implant. Treatments planned consisted of inclusion of the UIP mixture at 0, 2.5, 5, and 7.5% of the diet DM. Dry matter intake was fixed at 6.4 kg/d, and DM digestibility was not significantly affected by varying the amount of UIP addition. Apparent digestibility of N increased (P = .011) from 63.8 to 65.8, 70.7, and 71.5%, the amount of N absorbed increased (P = .001) from 73 to 84, 100, and 106 g/d, and N balance increased (P = .003) from 20 to 30, 33, and 39 g/d when UIP was fed at 0, 2.6, 5.2, and 7.8% of diet DM, respectively. The efficiency of N use increased 39.7%, and biological value increased 31.6% when the UIP mixture was added to the diet. Circulating concentrations of plasma urea N (PUN) were increased (P = .017) from 4.5 for the control diet to 5.7, 6.2, and 6.1 mg/dL when the UIP mixture was added at 2.6, 5.2, and 7.8%, respectively. Corresponding IGF-I concentrations were also increased from 491 to 558 and 624 ng/mL with 2.6 and 5.2% levels of UIP addition. Plasma glucose, NEFA, and insulin concentrations were not affected by feeding the UIP mix. The rate and efficiency of N use for growth improved with addition of an amino acid-balanced UIP mixture to the diet.     

Permethrin-treated bed nets do not have a ''mass-killing effect'' on village populations of Anopheles gambiae s.l. in The Gambia

Juvenile hyaline fibromatosis is a multisystemic disorder characterized by a triad of cephalic fibrous outgrowths, gingival hyperplasia, and flexion contractures. The aim of this study was to find new ultrastructural features that could be useful for differentiating this entity from other types of fibromatosis. Mucosal lesions processed for light and electron microscopy by routine techniques showed hyperactive-appearing spindle-shaped fibroblasts and dysplastic mesenchymal cells. Dilated rough endoplasmic reticulum, prominent Golgi complexes, and multivesicular bodies as well as single membrane vesicles filled with fibrillogranular material were seen within the cytoplasm of dysplastic mesenchymal cells. Many fibrillogranular vesicles contained smaller vesicles. There were also invaginations of the cell membrane that contained fibrillogranular material similar to that seen in the single membrane vesicles, suggesting engulfment of an extracellular substance. The stroma contained both normal and serrated collagen fibrils, microfibrils, and two types of fibrillogranular material, one of them with a characteristic banding pattern. Our clinical and histopathologic findings resemble those previously described in cases of infantile systemic hyalinosis and juvenile hyaline fibromatosis. So many features of these two conditions overlap that it is difficult not to consider them as parts of a spectrum of the same disorder.     

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.