Species and populations of the Anopheles gambiae complex in Cameroon with special emphasis on chromosomal and molecular forms of Anopheles gambiae s.s

We studied the geographical distribution of species, chromosomal, and molecular forms of the Anopheles gambiae Giles (Diptera: Culicidae) complex in 23 sites in Cameroon, Central Africa. Almost all the specimens collected in the four northern-most arid sites were Anopheles arabiensis. Anopheles melas was found in a rural locality surrounded by mangrove swamps, on the Atlantic Coast. In total, 1,525 An. gambiae s.s. females were identified down to their molecular form, and inversion polymorphisms on polytene chromosomes were scored from 186 half-gravid females. The Forest chromosomal form, with standard arrangements almost fixed on both arms of chromosome-2, was the only one observed in the southern, more humid localities. Karyotypes typical of Savanna and Mopti were recorded northwards, in the humid savannas of the Adamawa Province. The molecular forms M and S were widespread throughout Cameroon, and assort independently from the chromosomal forms. S-form populations were characterized by karyotypes typical of Forest and Savanna chromosomal forms, and M-form populations were characterized by karyotypes typical of Forest, Savanna, and Mopti. No M/S hybrid patterns were detected, although M and S mosquitoes were sympatric in 15 sites, providing further evidence for positive assortative mating within molecular forms. The observed ecogeographical distribution of M and S was peculiar: the ecological parameters involved in this distribution still need to be clarified as well as the possible role of competitive exclusion between chromosomally homosequential molecular forms. No difference was observed in host preference or in Plasmodium falciparum infection rates between sympatric M and S populations.     

Relationship between kdr mutation and resistance to pyrethroid and DDT insecticides in natural populations of Anopheles gambiae

The spread of insecticide resistance genes in Anopheles gambiae Giles sensu stricto threatens to compromise vector-based malaria control programs. Two mutations at the same locus in the voltage-gated sodium channel gene are known to confer knockdown resistance (kdr) to pyrethroids and DDT. Kdr-e involves a leucine-serine substitution, and it was until recently thought to be restricted to East Africa, whereas kdr-w, which involves a leucine-phenylalanine substitution, is associated with resistance in West Africa. In this study, we analyze the frequency and relationship between the kdr genotypes and resistance to type I and type II pyrethroids and DDT by using WHO test kits in both the Forest-M and S molecular forms of An. gambiae in Cameroon. Both kdr-w and kdr-e polymorphisms were found in sympatric An. gambiae, and in many cases in the same mosquito. Kdr-e and kdr-w were detected in both forms, but they were predominant in the S form. Both kdr-e and kdr-w were closely associated with resistance to DDT and weakly associated with resistance to type II pyrethroids. Kdr-w conferred greater resistance to permethrin than kdr-e. We also describe a modified diagnostic designed to detect both resistant alleles simultaneously.     

Distribution of Anopheles daciae and other Anopheles maculipennis complex species in Serbia

Parasitology Research - Malaria is one of the most severe health problems facing the world today. Until the mid-twentieth century, Europe was an endemic area of malaria, with the Balkan countries...     

Occurrence of an unusual polymerase chain reaction product during identification of Anopheles gambiae sibling species (Diptera: Culicidae)

 Using the polymerase chain reaction (PCR) for species differentiation within the Anopheles gambiae complex, we examined several hundred Cameroonian mosquito specimens. Applying an approved routine protocol for DNA extraction and PCR conditions, apart from the indubitable identification of most of the specimens, we came across ten PCR products that did not correspond in length to any of the hitherto known amplification products of the sibling species. Sequencing experiments showed the ten products to be of identical length (117 bp) and nucleotide sequence. The total sequence of the novel product is included in the PCR product specific for A. melas, which is known to occur in the same collection area as the ten unidentifiable mosquito specimens. On alignment of the novel PCR product sequence and the A. melas one starting at the 3"-end primer annealing site, the last 20 nucleotides of the novel product, reflecting the sequence of the 2^nd PCR primer, showed only 60% homology with the then-corresponding A. melas DNA site. Explanations for the occurrence of the unusual PCR products are given and discussed. Received: 10 July 1995 / Accepted: 27 October 1995     

Species composition of the Anopheles gambiae complex (diptera: Culicidae) at two sites in western Kenya

At two sites in the Kisumu area of western Kenya, the species composition of the Anopheles gambiae complex was determined by analysis of ovarian polytene chromosomes. Of 1,915 females, 26.1% were An. arabiensis Patton and 73.9% were An. gambiae Giles; one arabiensis x gambiae hybrid was identified. No major differences in the proportions of An. arabiensis and An. gambiae were observed between sites or between years. The ratio of An. arabiensis/An. gambiae was 6.7:1 (n = 231) in cow-baited traps, 0.2:1 (n = 1,525) in indoor resting samples, and 0.5:1 (n = 145) in all-night human bait catches. The proportion of An. arabiensis decreased progressively from 50.0% to 8.3% (n = 1,129) during 11 wk from September to November 1987; this change was correlated negatively with night temperature and positively with temperature range. In cow-baited traps, 97.4% (n = 194) of An. arabiensis were cow-fed and 95.8% (n = 1,054) of An. gambiae from indoor resting collections were human-fed. In indoor collections, 37.2% (n = 215) of An. arabiensis were cow-fed and 23.1% (n = 26) of An. gambiae from cow traps were human-fed. This demonstrates post-blood-feeding endophily by An. arabiensis and suggests post-blood-feeding exophily by An. gambiae. Malaria infection rates were higher for An. gambiae than for An. arabiensis by a ratio of 3:1 in 1986 (by Plasmodium falciparum ELISA) and 2.3:1 in 1987 (by dissection). Despite the higher proportion of infective An. gambiae, both species in this area serve as efficient vectors through their remarkably stable contact with the human population as demonstrated by their blood feeding and resting behavior.     

The immunogenic properties of protozoan glycosylphosphatidylinositols in the mosquito Anopheles gambiae

In contrast to humans, mosquitoes do not have an adaptive immune response to deal with pathogens, and therefore must rely on their innate immune system to deal with invaders. This facilitates the recognition of different microbes on the basis of surface components or antigens. Such antigens have been identified in various types of microbe such as bacteria and fungi, yet none has been identified in the genus protozoa, which includes pathogens such as the malaria parasite, Plasmodium falciparum and Toxoplasma gondii. This study allowed us to test the antigenic properties of protozoan glycosylphosphatidylinositol (GPI) on the mosquito immune system. We found that both P. falciparum GPI and T. gondii GPI induce the strong expression of several antimicrobial peptides following ingestion, and that as a result of the immune response against the GPIs, the number of eggs produced by the mosquito is reduced dramatically. Such effects have been associated with malaria infected mosquitoes, but never associated with a Plasmodium specific antigen. This study demonstrates that protozoan GPIs can be considered as protozoan specific immune elicitors in mosquitoes, and that P. falciparum GPI plays a critical role in the malaria parasite manipulation of the mosquito vector to facilitate its transmission.     

Thyrotropin-releasing hormone analogue and serotonin interact within the dorsal vagal complex to augment gastric acid secretion.

The effects of serotonin (5HT) and a thyrotropin-releasing hormone (TRH) analogue, RX77368, on vagal control of gastric acid secretion were studied. Microinjection of RX77368 (0.66 pmol in 10 nl), but not 5HT (8 pmol in 10 nl), into the dorsal vagal complex (DVC) evoked a significant increase in acid output. When the same doses of RX77368 and 5HT were co-injected, the amount of acid secreted was significantly greater than that due to RX77368 alone. Thus, 5HT and the TRH analogue interact within the DVC to enhance vagal stimulation of acid secretion. The study suggests a possible functional significance of raphe TRH/serotonergic tracts projecting to the DVC.     

Combining piperonyl butoxide and dinotefuran restores the efficacy of deltamethrin mosquito nets against resistant Anopheles gambiae (Diptera: Culicidae)

One strategy suggested for the management of mosquito insecticide resistance consists of combining a pyrethroid with an insecticide that has a different mode of action. To restore the efficacy of deltamethrin (pyrethroid) against pyrethroid-resistant strain of Anopheles gambiae Giles (VKPR: homozygous Kdr), deltamethrin was combined with the neonicotinoid insecticide dinotefuran and piperonyl butoxide (PBO). Bednets impregnated with deltamethrin, dinotefuran, and PBO alone and in combination were tested in the laboratory. Knockdown (KD) and mortality were measured using WHO cone tests on susceptible and pyrethroid-resistant adult mosquitoes. The combination of deltamethrin and PBO was synergistic against resistant female An. gambiae (58.2% mortality). Both mortality and knockdown time (KDt(50/95) values) of the tricomponent mixture on the VKPR strain were similar to the insecticidal activity of deltamethrin on the pyrethroid-susceptible KIS strain (98.8 and 100% mortality, respectively). The three-compound mixture of deltamethrin + PBO + dinotefuran showed an insecticidal efficacy greater than the deltamethrin + PBO mixture to the extent of completely restoring the efficacy of deltamethrin on pyrethroid-resistant An. gambiae.     

Larvicidal activity of metabolites from the endophytic Podospora sp. against the malaria vector Anopheles gambiae

In a screening for natural products with mosquito larvicidal activities, the endophytic fungus Podospora sp. isolated from the plant Laggera alata (Asteraceae) was conspicuous. Two xanthones, sterigmatocystin (1) and secosterigmatocystin (2), and an anthraquinone derivative (3) 13-hydroxyversicolorin B were isolated after fermentation on M₂ medium. These compounds were characterised using spectroscopic and X-ray analysis and examined against third instar larvae of Anopheles gambiae. The results demonstrated that compound 1 was the most potent one with LC₅₀ and LC₉₀ values of 13.3 and 73.5 ppm, respectively. Over 95% mortality was observed at a concentration 100 ppm after 24 h. These results compared farvourably with the commercial larvicide pylarvex? that showed 100% mortality at the same concentration. Compound 3 was less potent and had an LC₅₀ of 294.5 ppm and over 95% mortality was achieved at a concentration of 1,000 ppm. Secosterigmatocystin (2) revealed relatively weak activity and therefore LC values were not determined.     

Insecticide susceptibility status of Anopheles gambiae s.l. (Diptera: Culicidae) in the Republic of Cameroon

A large-scale survey of Anopheles gambiae Giles, 1902 susceptibility to DDT, dieldrin, permethrin, and deltamethrin was conducted in the Republic of Cameroon. 15 field populations from various geographical areas were tested using World Health Organization test kits for adult mosquitoes. The laboratory Kisumu susceptible reference strain was tested as a control. Results showed that dieldrin and DDT resistance was still present in some populations, and indicated permethrin or deltamethrin resistance. Within the Anopheles gambiae complex, resistant individuals belonged to An. gambiae s.s. and An. arabiensis species. Both M and S molecular forms of An. gambiae s.s. were found resistant. In most of resistant populations, the knockdown times were 2-5-folds increased. However, none of the surviving mosquitoes was positive to the kdr "Leu-Phe" mutation using polymerase chain reaction (PCR) diagnostic test. These results likely suggested involvement of other resistance mechanism(s), such as enzyme detoxification or kdr "Leu-Ser" mutation. Researches on An. gambiae s.l. resistance should be promoted in Cameroon, to improve malaria vector control programs and to implement resistance management strategies.     

Evidence for a new malaria vector species, species E, within the Anopheles culicifacies complex (Diptera: Culicidae).

Female Anopheles culicifacies Giles from Ramanathapuram district, Tamil Nadu state, India, were examined for oocysts and sporozoites and their larval progeny for mitotic karyotype. Collections were made from Mandapam and Uchipuli on the mainland, and Thangachimadam and Pamban on Rameshwaram Island. Of the 451 An. culicifacies females that were collected and dissected, 24 were found positive for Plasmodia (21 for sporozoites and 3 for oocysts). Both acrocentric and submetacentric Y-chromosome karyotypes were observed among the progeny of females from all villages. All 11 iso-female lines whose parental females were positive for sporozoites or oocysts had progeny with submetacentric Y-chromosomes. Total absence of sporozoite-positives among mothers of acrocentric males was evidence of assortative mating between these 2 sympatric populations (i.e., 2 species). We propose that the nonvector population with acrocentric Y-chromosome sons retain the original designation of species B and that the vector population with the submetacentric Y-chromosome sons be designated as species E, a new species.     

Comparative and functional genomics of the innate immune system in the malaria vector Anopheles gambiae

Summary: In much of Africa, the mosquito Anopheles gambiae is the major vector of human malaria, a devastating infectious disease caused by Plasmodium parasites. Vector and parasite interact at multiple stages and locations, and the nature and effectiveness of this reciprocal interaction determines the success of transmission. Many of the interactions engage the mosquito's innate immunity, a primitive but very effective defense system. In some cases, the mosquito kills the parasite, thus blocking the transmission cycle. However, not all interactions are antagonistic; some represent immune evasion. The sequence of the A. gambiae genome revealed numerous potential components of the innate immune system, and it established that they evolve rapidly, as summarized in the present review. Their rapid evolution by gene family expansion diversification as well as the prevalence of haplotype alleles in the best‐studied families may reflect selective adaptation of the immune system to the exigencies of multiple immune challenges in a variety of ecologic niches. As a follow‐up to the comparative genomic analysis, the development of functional genomic methodologies has provided novel opportunities for understanding the immune system and the nature of its interactions with the parasite. In this context, identification of both Plasmodium antagonists and protectors in the mosquito represents a significant conceptual advance. In addition to providing fundamental understanding of primitive immune systems, studies of mosquito interactions with the parasite open unprecedented opportunities for novel interventions against malaria transmission. The generation of transgenic mosquitoes that resist malaria infection in the wild and the development of antimalarial ‘smart sprays’ capable of disrupting interactions that are protective of the parasite, or reinforcing others that are antagonistic, represent technical challenges but also immense opportunities for improvement of global health.     

Evidence that prostate gonadotropin-releasing hormone receptors mediate an anti-tumourigenic response to analogue therapy in hormone refractory prostate cancer

Gonadotropin-releasing hormone analogue (GnRHa) therapy is an established method of androgen withdrawal in the treatment of prostate cancer. The present study investigated if the expression of prostate GnRH receptors (GnRHRs) might influence the response to GnRHa. GnRHR protein expression was first studied in a panel of prostate cancer cell lines. In androgen-dependent cells, GnRHR expression was unchanged following acute or chronic androgen withdrawal. In these cells, GnRHa significantly inhibited androgen-induced cell proliferation (p = 0.01). In contrast, GnRHa was unable to further suppress basal levels of cell proliferation induced by androgen withdrawal. In androgen-independent prostate cancer cells, variable levels of GnRHR expression were observed. In these cells, GnRHa treatment blocked cell proliferation (p = 0.001) and invasion (up to 70%) induced by fibroblast growth factor stimulation. Crucially, this effect was only evident in cells that expressed high levels of the GnRHR. GnRHa treatment also significantly inhibited the ability of these cells to recover from a cytotoxic insult (50% inhibition). The clinical significance of prostate GnRHR was tested by immunohistochemistry in a preliminary cohort of patients treated with GnRHa or surgical castration. There was no association between GnRHR expression and pathological grade, clinical stage, time to PSA nadir (p = 0.82) (n = 35) or progression to hormone refractory disease (p = 0.22) (n = 21), irrespective of the treatment method. GnRHa therapy in the presence of high GnRHR expression however, was found to be associated with longer disease-specific survival (mean survival 85 months, p = 0.002). In contrast, high GnRHR expression was not associated with survival among surgically castrated patients (mean survival 50 months, p = 0.7). Taken together, these data support the notion of a functional interaction between GnRHa and the GnRHR, which results in an anti-tumourigenic effect on prostate cancer cells. Findings from this report have direct implications for the use of GnRHR as a novel therapeutic target in hormone refractory prostate cancer.     

Flavobacterium odoratum: a species resistant to a wide range of antimicrobial agents.

During the period 1966-77, 24 strains of Flavobacterium odoratum were identified from among strains of Gram-negative, non-fermentative bacteria submitted to the National Collection of Type Cultures for computer-assisted identification. The F. odoratum strains showed resistance to therapeutic levels of gentamicin, tobramycin, amikacin, and carbenicillin as well as to several other antimicrobial agents generally useful in the treatment of infections caused by Gram-negative, non-fermentative bacteria. Two strains isolated from amputation stumps and another three strains isolated in significant numbers from urine specimens were possibly opportunist pathogens. The biochemical characteristics of the 24 strains, the proposed neotype strain of F. odoratum, and three strains representative of a group, referred to at the Center for Disease Control, Atlanta as group M-4f, were compared with those of biochemically similar species which may be isolated from clinical material.