Histone H1-like, lysine-rich low complexity amino acid extensions in mosquito ribosomal proteins RpL23a and RpS6 have evolved independently

Histone H1-like amino acid extensions have been described at the amino terminus of Drosophila RpL22 and RpL23a, and at the carboxyl terminus of mosquito ribosomal protein RpS6. An in silico search suggested that RpL23a, but not RpL22, in Anopheles gambiae has an amino-terminal extension. Because low complexity amino acid extensions are not common on eukaryotic ribosomal proteins, and their functions are unknown, we cloned cDNAs encoding RpL23a from Aedes albopictus and Anopheles stephensi mosquito cell lines. RpL23a proteins in Aedes and Anopheles mosquitoes are rich in lysine (approximately 25%), alanine (approximately 21%), and proline (approximately 8%), have a mass of approximately 40 kDa, a pI of 11.4 to 11.5, and contain an N-terminal extension of approximately 260 amino acid residues. The N-terminal extension in mosquito RpL23a is about 100 amino acids longer than that in the Drosophila RpL23a homolog, and contains several repeated amino acid motifs. Analysis of exon-intron organization in the An. gambiae and in D. melanogaster genes suggests that a short first exon encodes a series of 11 amino acid residues conserved in RpL23a proteins from Drosophila, mosquitoes, and the moth, Bombyx mori. The histone H1-like sequence in RpL23a is encoded entirely within the second exon. The C-terminal 126 amino acid residues of the RpL23a protein, encoded by exon 3 in Drosophila, and by exons 3 and 4 in Anopheles gambiae, are well conserved, and correspond to Escherichia coli RpL23 with the addition of the eukaryotic N-terminal nuclear localization sequence. Sequence comparisons indicate that the histone H1-like extensions on mosquito RpS6 and RpL23a have evolved independently of each other, and of histone H1 proteins.     

The dynamics of interactions between Plasmodium and the mosquito: a study of the infectivity of Plasmodium berghei and Plasmodium gallinaceum,and their transmission by Anopheles stephensi,Anopheles gambiae and Aedes aegypti

Knowledge of parasite-mosquito interactions is essential to develop strategies that will reduce malaria transmission through the mosquito vector. In this study we investigated the development of two model malaria parasites, Plasmodium berghei and Plasmodium gallinaceum, in three mosquito species Anopheles stephensi, Anopheles gambiae and Aedes aegypti. New methods to study gamete production in vivo in combination with GFP-expressing ookinetes were employed to measure the large losses incurred by the parasites during infection of mosquitoes. All three mosquito species transmitted P. gallinaceum; P. berghei was only transmitted by Anopheles spp. Plasmodium gallinaceum initiates gamete production with high efficiency equally in the three mosquito species. By contrast P. berghei is less efficiently activated to produce gametes, and in Ae. aegypti microgamete formation is almost totally suppressed. In all parasite/vector combinations ookinete development is inefficient, 500-100,000-fold losses were encountered. Losses during ookinete-to-oocyst transformation range from fivefold in compatible vector parasite combinations (P. berghei/An. stephensi), through >100-fold in poor vector/parasite combinations (P. gallinaceum/An. stephensi), to complete blockade (>1,500 fold) in others (P. berghei/Ae. aegypti). Plasmodium berghei ookinetes survive poorly in the bloodmeal of Ae. aegypti and are unable to invade the midgut epithelium. Cultured mature ookinetes of P. berghei injected directly into the mosquito haemocoele produced salivary gland sporozoites in An. stephensi, but not in Ae. aegypti, suggesting that further species-specific incompatibilities occur downstream of the midgut epithelium in Ae. aegypti. These results show that in these parasite-mosquito combinations the susceptibility to malarial infection is regulated at multiple steps during the development of the parasites. Understanding these at the molecular level may contribute to the development of rational strategies to reduce the vector competence of malarial vectors.     

Switchover to the mated state by spermathecal activation in female Anopheles gambiae mosquitoes

Both Aedes aegypti and Anopheles gambiae mosquitoes undergo physiological and behavioral changes after the females mate, but unlike Ae. aegypti, the mated state in An. gambiae is not attained through the action of male accessory gland substances. Experiments in which the spermathecae of mated females were manipulated suggest that a spermatheca filled with sperm is responsible for triggering oviposition behavior. An estimated 48% of the previously mated An. gambiae females mated again when the interval between encounters with males was less than 24h. Unlike male Ae. aegypti that can inseminate up to seven females after their testes are removed, An. gambiae do not store sperm in the vas deferens and without testes would be unable to inseminate any females.     

Permethrin-impregnated bednets: behavioural and killing effects on mosquitoes

Permethrin-treated pieces of netting and simulated bednets were evaluated against Anopheles gambiae Giles and Aedes aegypti (L.) in the laboratory. When female mosquitoes were allowed to feed on a human arm through pieces of impregnated netting fastened at the end of tubes, doses above 2 g/m2 were required to stop blood-feeding of both An. gambiae and Ae. aegypti. A much lower dose prevented Ae. aegypti from feeding on mice through impregnated netting. When mosquitoes were released in a room and a human subject sat under a permethrin-impregnated (0.2 g/m2) bednet with an arm pressed against the net (mesh 1.5 mm), mosquitoes failed to bite through the net. All the mosquitoes trying to bite through or entering the net through holes cut in it were knocked down within 30 min of release and ultimately died. Permethrin-impregnated wide-mesh (4 or 8 mm) bednets similarly prevented entry and caused high mortality-rates of An. gambiae.     

Ovicidal and larvicidal activity against Aedes aegypti and Anopheles gambiae complex mosquitoes of essential oils extracted from three spontaneous plants of Burkina Faso

Essential oils extracted from dried leaves of three spontaneous plants naturally growing in Burkina Faso, i.e. Cymbopogon proximus, Lippia multiflora and Ocimum canum, exhibited larvicidal activity by the WHO standard protocol against 3rd and 4th instar F1-larvae of field-collected mosquitoes vectors of human disease, namely Aedes aegypti and members of the Anopheles gambiae complex, An. arabiensis and An. gambiae. The median lethal concentration (LC50) for Ae. aegypti and An. gambiae s.l. larvae ranged between 53.5-258.5 ppm and 61.9-301.6 ppm, respectively. The LC90 estimates ranged 74.8-334.8 ppm for Ae. aegypti, and 121.6-582.9 ppm for An. gambiae s.l. Ovicidal activity against eggs of An. gambiae s.l. was also demonstrated. The LC50 values for An. gambiae s.l. eggs ranged between 17.1-188.7 ppm, while LC90 values ranged between 33.5-488 ppm. Lippia multiflora showed the highest activity against An. gambiae s.l. eggs and Ae. aegypti larvae, whereas no difference was found among C. proximus and L. multiflora in their activity against An. gambiae s.l. larvae. Of the three plants, essential oils from O. canum had the lowest activity against both eggs and larvae. Eggs were more susceptible than larvae. Ae. aegypti larvae were more susceptible than larvae of An. gambiae s.l.     

An in vivo transfection approach elucidates a role for Aedes aegypti thioester-containing proteins in flaviviral infection

Mosquitoes transmit pathogens that cause infectious diseases of global importance. Techniques to easily introduce genes into mosquitoes, however, limit investigations of the interaction between microbes and their arthropod vectors. We now show that a cationic liposome significantly enhances delivery and expression of plasmid DNA in Aedes aegypti and Anopheles gambiae mosquitoes. We then introduced the genes for Ae. aegypti thioester-containing proteins (AeTEPs), which are involved in the control of flaviviral infection, into mosquitoes using this technique. In vivo transfection of AeTEP-1 into Ae. aegypti significantly reduced dengue virus infection, suggesting that the approach can further our understanding of pathogen-mosquito interactions.     

Effects of variations in a formulated protein meal on the fecundity and fertility of female mosquitoes

Abstract. A formulated protein meal developed by Kogan (1990) for adult female Aedes aegypti mosquitoes was evaluated and modified to increase egg and pupal yield. A vigorous laboratory colony was maintained with the females fed exclusively on this dietary formula for about twenty-five generations over more than 2 years. Extra modifications were made to produce a diet suitable for Anopheles arabiensis and Anopheles stephensi females to produce eggs. Both formulations contain bovine albumin, haemoglobin and globulin in a ringer based solution, plus ATP as a phagostimulant for Ae.aegypti. Compared to Kogan's original, our Aedes formula doubled the production of pupae per female after a single meal, although the yield was still significantly lower than from mosquitoes fed on animal hosts or defibrinated pig blood. In varying the proportions of different constituents during attempts to optimize the formula, no relationship was found between total protein content (within the range 80–220 mg/ml) and fecundity, percentage hatch or pupal yield of Ae.aegypti. Equivocal results were found when an isoleucine supplement was added to the formula.     

The salivary purine nucleosidase of the mosquito,Aedes aegypti

A cDNA clone originating from adult female Aedes aegypti mosquitoes was found with substantial similarity to nucleosidases of the EC 3.2.2.1 enzyme class. Although this type of enzyme is unusual in animals, abundant enzyme activity was found in salivary homogenates of this mosquito, but not in salivary homogenates of the mosquitoes Anopheles gambiae and Culex quinquefasciatus, or the sand fly Lutzomyia longipalpis. Aedes salivary homogenate hydrolyses inosine and guanosine to hypoxanthine and xanthine plus the ribose moiety, but does not hydrolyse the pyrimidines uridine and cytidine, thus characterizing the presence of a purine nucleosidase activity. The enzyme is present in oil-induced saliva, indicating that it is secreted. Male Ae. aegypti salivary gland homogenates (SGH) have very low purine nucleosidase activity, suggesting that the enzyme plays a role in mosquito blood feeding. A novel isocratic HPLC method to separate nucleosides and their bases is described.     

Alternative splicing generates multiple transcripts of the inhibitor of apoptosis protein 1 in Aedes and Culex spp. mosquitoes

We determined the sequences of cDNA encoding Inhibitor of Apoptosis Protein 1 (IAP1) homologues from Aedes triseriatus, Aedes albopictus, Aedes aegypti, Culex pipiens and Culex tarsalis. The cDNAs encode translation products that share > or = 84% sequence similarity. The IAP1 mRNA of each mosquito species exists as 3-5 distinct variants due to the presence of heterogeneous sequences at the distal end of their 5'UTRs. Partial genomic sequencing upstream of the 5' end of the Ae. triseriatus IAP1 gene, and analysis of the Ae. aegypti genomic sequence, suggest that these mRNA variants are generated by alternative splicing. Each IAP1 mRNA variant from Ae. triseriatus and Cx. pipiens was detected by RT-PCR in all mosquito life-stages and adult tissues examined, and the relative concentration of each Ae. triseriatus IAP mRNA variant in various tissues was determined.     

Effect of entomopathogenic fungus, Beauveria bassiana on larvae of three species of mosquitoes

Beauveria bassiana, an entomopathogenic fungus, was evaluated for its potential against second and third instar larvae of Culex quinquefasciatus, Anopheles stephensi and Aedes aegypti. Conidiospores of this fungus were effective in causing infection leading to mortality of different larval instars. Larvae of Cx. quinquefasciatus were more susceptible to infection than An. stephensi and the second instar larvae of these two species were more susceptible than third instar larvae. Larvae of Ae. aegypti were resistant to infection by B. bassiana.     

Susceptibility of ten species of mosquito larvae to the parasitic nematode Romanomermis iyengari and its development

Ten species of mosquitoes (Diptera: Culicidae) from five genera were exposed to preparasites of the tropical mermithid nematode species Romanomermis iyengari (Welch) (Nematoda: Mermithidae), a strain isolated in 1978 from Pondicherry. By exposing mosquito larvae during the second instar, nematode infection was invariably lethal, the rate being highest in Culex sitiens Wiedemann (95%) followed by Cx. quinquefasciatus Say (90%), Aedes aegypti (L.) (79%), Anopheles subpictus Grassi (64%), Ae. albopictus (Skuse) and Armigeres subalbatus Coquillett (62%), Cx. tritaeniorhynchus Giles (57%), Mansonia annulifera (Theobald) (46%), An. stephensi Liston (40%) and An. culicifacies Giles (36%). When fourth-instar larvae were exposed, the infection was highest in Ar. subalbatus (66%), followed by An. stephensi (52%), Cx. quinquefasciatus (47%), Ae. aegypti and An. subpictus (42%), Ae. albopictus (30%), An. culicifacies (29%), Cx. sitiens (24%), Cx. tritaeniorhynchus (19%) and Ma. annulifera (8%), with 2-45% of infected culicines surviving to adulthood. The parasitic phase of the nematode lasted 5-7 days in all the host species, yielding 1.1-3.2 parasites per II instar and 1.1-2.5 parasites per IV instar. The overall output of parasites per 100 mosquito larvae (infected + uninfected) was highest for Ae. aegypti when mosquitoes were exposed during II instar (2.53 parasites/larva) and for Ar. subalbatus when mosquitoes were exposed during IV instar (1.65/larva), and lowest for Ma. annulifera exposed during IV instar (0.09/larva). For routine laboratory culture of R. iyengari it is convenient to employ Cx. quinquefasciatus as the host yielding 90-190 parasites/100 larva.     

Ribosomal protein S6 cDNA from two Aedes mosquitoes encodes a carboxyl-terminal extension that resembles histone H1 proteins

Ribosomal protein S6 (rpS6) is the major phosphorylated protein on the eukaryotic ribosome. Because electrophoretic evidence suggested that the homolog of rpS6 from the mosquitoes Aedes albopictus and Aedes aegypti was measurably larger than Drosophila rpS6, we have now isolated full-length cDNAs encoding Aedes albopictus and Aedes aegypti rpS6. The mosquito rpS6 cDNAs encoded a 100 amino acid extension at the carboxyl-terminus, relative to rpS6 from humans and Drosophila. This region had homology to cDNAs encoding histone H1 from various species and accounted for the larger size of the mosquito protein on polyacrylamide gels. On Northern blots, the mosquito cDNA hybridized to a single band measuring approximately 1.2 kb. This revised version was published online in July 2006 with corrections to the Cover Date.     

Structural organization of posterior midgut muscles in mosquitoes, Aedes aegypti and Anopheles gambiae

In order to adapt to diverse feeding behavior, animal intestines have evolved with distinct differences. Such adaptation may include the structure of the longitudinal and circular muscles that maintain the integrity and the tensile strength of the gut tissue in higher metazoans. Here we examined the structural organization of the posterior midgut muscles of two insects, Aedes aegypti and Anopheles gambiae. We found the estimated number of longitudinal muscles in a cross-section to be 168 in Ae. aegypti and 37 in An. gambiae. Within the region, the estimated number of circular muscles is 77 in Ae. aegypti and 57 in An. gambiae. In An. gambiae, longitudinal muscles appear as sets of parallel bundles. Each set overlaps its neighbor to form a continuous tube. We found that this novel mode of muscle fiber sharing makes all circular muscles interconnected. Both types of muscle lie orthogonally to form a grid that holds the epithelium of the posterior midgut. In Ae. aegypti, the muscle fibers between the bundles are shared extensively, making the organization more intricate. This study implies that, because of its simple structure, the insect midgut may provide a powerful tool with which to study the structural evolution and function of animal intestines.     

Wolbachia induces density-dependent inhibition to dengue virus in mosquito cells

Wolbachia is a maternal transmitted endosymbiotic bacterium that is estimated to infect up to 65% of insect species. The ability of Wolbachia to both induce viral interference and spread into mosquito vector population makes it possible to develop Wolbachia as a biological control agent for dengue control. While Wolbachia induces resistance to dengue virus in the transinfected Aedes aegypti mosquitoes, a similar effect was not observed in Aedes albopictus, which naturally carries Wolbachia infection but still serves as a dengue vector. In order to understand the mechanism of this lack of Wolbachia-mediated viral interference, we used both Ae. albopictus cell line (Aa23) and mosquitoes to characterize the impact of Wolbachia on dengue infection. A serial of sub-lethal doses of antibiotic treatment was used to partially remove Wolbachia in Aa23 cells and generate cell cultures with Wolbachia at different densities. We show that there is a strong negative linear correlation between the genome copy of Wolbachia and dengue virus with a dengue infection completely removed when Wolbacha density reaches a certain level. We then compared Wolbachia density between transinfected Ae. aegypti and naturally infected Ae. albopictus. The results show that Wolbachia density in midgut, fatbody and salivary gland of Ae. albopictus is 80-, 18-, and 24-fold less than that of Ae. aegypti, respectively. We provide evidence that Wolbachia density in somatic tissues of Ae. albopictus is too low to induce resistance to dengue virus. Our results will aid in understanding the mechanism of Wolbachia-mediated pathogen interference and developing novel methods to block disease transmission by mosquitoes carrying native Wolbachia infections.