Orientation and ingestion rates of larval Anopheles albimanus in response to floating particles

Response of fourth-instar larvae of Anopheles albimanus Wiedemann (Diptera: Culicidae) to food and inert particles floating at the water surface was studied. In a choice test, larvae aggregated at powdered organic materials (blood meal, liver powder alfalfa flour and wheat flour) but not at inert materials (kaolin, chalk or charcoal). Larvae responded positively to proteins as well as some carbohydrates, but not to cellulose. Retention of larvae at food sources found by random locomotion was found to be responsible for larval aggregation. Larvae ingested food particles 6–9 times faster than insert particles. The significance of Anopheline feeding behavior in the development of formulations of stomach toxins (bacterial agents) used in larval control is discussed.

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Zusammenfassung Die vorliegende Studie befasst sich mit Verhaltensreaktionen von Anopheles albimanus Viertlarven auf an der Wasseroberfläche schwimmende Partikel. Verteilung und Orientierung der Larven wurde in einer Wahlapparatur quantifiziert. Nach Auftrag von Alfalfamehl, Weizenmehl, Stärke, Blutmehl, Leberpulver und Fischmehl wurde Aggregation der Larven in den beköderten Fächern der Apparatur beobachtet. Sowohl Proteine (Casein) als auch einige Kohlehydrate (Amylose, Amylopectin) lösten Aggregationen der Larven aus. Im Unterschied dazu führte Auftrag von Kreide, Kaolin, Polyaethylenpulver, Talcum oder Cellulose nicht zu Aggregationen. Zur Beschreibung der Entstehung larvaler Aggregationen bei Futterstoffen wurden die Schwimmbewegungen der Larven in Anwesenheit von Weizenmehl als Ködersubstanz quantifiziert. Da keine Attraktion der Larven im Sinne einer gerichteten Schwimmbewegung beobachtet werden konnte, wird geschlussfolgert, dass sofortige Beendung der Suchaktivität der Larven bei zufällig gefundenen Futterquellen für die beobachteten Aggregationen bei organischen Substanzen verantwortlich ist.Die Fressraten der Larven bei Angebot verschiedener Substanzen im Überschuss wurde bestimmt. Larven fülten drei von insgesamt sechs Darmabschnitten innerhalb von 15–30 min bei Angebot von Futtersubstanzen, während die Füllung von nur zwei Darmabschnitten mit inerten Materialien erst nach 90–120 min zu beobachten war. Die Resultate werden in Bezug auf wasseroberflächengebundene Formulierungen von Frassgiften diskutiert. Inerte Trägersubstanzen werden wahrscheinlich wesentlich langsamer aufgenommen als Futtersubstanzen. Da An. albimanus Larven nicht von Futterquellen angezogen werden, ist eine rasche und wirksame Giftaufnahme besonders dann zu erwarten, wenn die gesamte Oberfläche der Brutgewässer mit toxinhaltigen Trägerpartikeln bedeckt werden kann.

     

Distribution and habitats of mosquito larvae in the Kingdom of Tonga

Abstract  Mosquitoes are a significant pest and human health issue in the Kingdom of Tonga. The occurrence of species and habitats used by mosquito larvae were investigated to determine the potential for control through larval habitat management. Forty-two sites, including 22 villages and 20 farm plantations on the six islands of Tongatapu, Pangaimotu, Vava'u, Pangaimotu (Vava'u group), 'Utungake and Nuku, were surveyed in April 2006. A total of eight mosquito species were collected: Aedes aegypti (Linnaeus), Ae. horrescens (Edwards), Ae. nocturnus (Theobold), Ae. tongae (Edwards), Culex albinervis (Edwards), Cx. annulirostris (Skuse), Cx. quinquefasciatus (Say) and Cx. sitiens (Wiedemann). Several species were widespread, particularly Ae. aegypti and Ae. nocturnus on the main island of Tongatapu, whereas Ae. aegypti dominated sites on islands of the Vava'u group. Comparative sampling of 17 village and 17 rural sites showed that larval habitat was more abundant in towns than in rural areas. Larvae were found in a wide range of habitats but were particularly abundant in artificial water bodies (e.g. disused concrete water tanks, 44-gallon drums and used car tyres). In rural sites, habitats were generally sparse except in rain-filled branch stems of giant taro plants. Mosquito populations in artificial habitats could be markedly reduced by seeding disused water tanks with aquatic predators already present in Tonga, using mesh-net covers over 44-gallon drums, and drilling holes in used car tyres.     

Evaluation of sticky light traps for sampling mosquito larvae

Very simply constructed aquatic sticky light traps employing a chemical solution emitting a bright light attracted large numbers of all larval instars, but few pupae of Aedes cantans in trials in England. During April numbers of larvae per trap ranged from 73±14.2 to 163±19.9, whereas dipping with a ladle yielded only 6.9±5.5 to 11.8±7.7 larvae/dip. The traps were also effective in sampling larvae of Culiseta morsitans and in Sierra Leone larvae of Aedes aegypti. It was concluded that sticky light traps could be valuable in sampling relative numbers of mosquito larvae and merited further evaluation.

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Résumé Des pièges adhésifs lumineux faciles à réaliser sont construits pour les captures des larves de moustiques. Ils sont formés de paires de boîtes de Pétri en plastique contenant une solution émettrice de lumière provenant de bâtons lumineux chimiques Cyalume^®. Chaque piège est recouvert d'une feuille de plastique de 16×16 cm enduite d'une substance qui reste adhésive sous l'eau. Les larves de moustiques attirées vers la source lumineuse viennent se coller à la feuille adhésive.Ces pièges ont été utilisés avec succès en Angleterre pour l'échantillonnage de tous les stades larvaires d'Aedes cantans, mais pas avec le même efficacité pour les nymphes. Des tests plus restreints ont montré que la technique était également utilisable pour les larves de Culiseta morsitans. Les nombres moyens d'A. cantans capturés pendant une piode de 12 h (73±14,2–163±19,9) étaient supérieux à ceux obtenus en plongeant une louche (6,9±5,5–11,8±7,7). En utilisant les pièges adhésifs lumineux, les captures moyennes de C. morsitans étaient également supérieures (12,6±4,7–35,8±8,9) contre (5,7±6,1–8,9±7,8) avec la méthode de la louche.En Sierra Leone, des stades larvaires immatures d'A. aegypti se développant dans des containers de stockage d'eau ont également pu être capturés à l'aide de ces pièges.Bien qu'une évaluation plus poussée soit encore nécessaire, il ne fait aucun doute que ces pièges adhésifs lumineux aient un intérêt considérable pour l'échantillonnage larvaire dans les sites inaccessibles, tels que les trous d'arbres ou les trous de crabes, où les méthodes d'échantillonage classiques sont souvent d'un emploi difficile.

     

Microplate assay of elevated esterase activity in individual pyrethroid-resistant mosquitoes

Involvement of esterase-mediated hydrolysis as a mechanism of pyrethroidresistance in three species of mosquitoes,viz., Aedes aegypti, Culex quinquefasciatus andAnopheles Stephensi was investigated by microplate assay of Β-esterases in individual larva and adult female and male mosquitoes. Assuming an absorbance value of 0.4 and above at 555 nm as the threshold level of elevated esterase activity which confers resistance, frequency distributions of such individual test mosquitoes were constructed in resistant and susceptible populations. The results indicate the involvement of ester hydrolysis of Pyrethroids as a predominant mechanism of pyrethroid-resistance in the larvae ofCulex quinquefasciatus but not inAedes aegypti. However, a marginal role of esterases is indicated in the larvae ofAnopheles stephensi.     

Infection sites of the entomopathogenic fungus Beauveria bassiana in the larvae of the mosquito Aedes aegypti

A series of experiments were conducted to determine the infection sites of the entomopathogenic fungus Beauveria bassiana after ingestion by the larvae of Aedes aegypti. The timing of the host death in relation to larval molting, the principle sites of fungal infection and development in host tissues were studied. Fungal conidiospores (CS) and blastospores (BS) were used separately for treatment of mosquito larvae. Although in most instances CS germinated and developed within the host, in others there was a premature abortion of the fungal development cycle. On the other hand, BS ingested by the larvae showed differences in the fungal development stages in the larval tissues. While the two primary infection sites were the head and the anal region, the most preferred site for fungal development was the larval gut. No more than two cycles of fungal development can occur in the host. Although both CS and BS are effective as larvicides, BS is far more pathogenic.

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Résumé Une série d'expériences a été menée afin de déterminer les emplacements d'infection du fongus entomopathogénique Beauveria bassiana (souche GK 2016) lequel avait été ingesté des larves d'Aedes aegypti. On a étudié le moment de la mort des hôtes en relation des mues des larves, les emplacements principaux où se conduit l'infection fongale et le développement du fongus dans des tissus divers d'hôte. On a utilisé les conidiospores (CS) et les blastospores (BS) séparément pour traitement des larves des moustiques. Bien que dans la plupart des cas CS ait germiné et se soit développé chez l'hôte, en d'autres cas, CS a germiné mais n'avait pas produit du BS évidemment grâce à l'avortement prématuré du cycle de développement des fongus chez l'hôte. Le BS qui avait été ingesté dans les larves présentait des stages différents de développement du fongus dans les tissus des larves. Quoique les deux emplacements placements principaux étaient la tête et la région anale, l'emplacement préféré pour le développement du fongus était l'intestin des larves. Nous montrons qu'il n'y a plus de deux cycles de développement du fongus chez l'hôte. Bien que CS et BS soient efficaces comme larvicides, BS est de loin plus pathogène.

     

Growth of Anopheles mosquito larvae on dietary microbiota in aquatic surface microlayers

Abstract. Hydrophobic organic matter accumulates under the surface film of water bodies to form the surface microlayers. Heterotrophic microorganisms use this organic matter for growth, and they, in turn, are fed upon by Anopheles mosquito larvae and other animals. From laboratory experiments we show that two species of mosquito larvae, Anopheles gambiae and An.quadrimaculatus , grew most rapidly where surface micro-layers were present and, especially, where labile dissolved organic matter was added to promote growth of microorganisms. The importance of microorganisms was confirmed by the addition of gentamicin antibiotic, which suppressed the microbiota and reduced the growth of larvae feeding on surface microlayers. Anopheles larvae grew well on a suspension of finely ground fish food to which the antibiotic had been added, showing that reduced growth was not due to gentamicin itself. Because sub-surface microorganisms are the components of the larval diet that most affect growth, we discuss their relevance to strategies for larval control of Anopheles mosquitoes.     

Resistance to theBacillus sphaericus toxin in cultured mosquito cells

Summary Increasing doses ofBacillus sphaericus toxin were used to select a toxin-resistant cell line from theCulex quinquefasciatus line. This resistant cell line was proven to beC. quinquefasciatus in origin by isozyme analysis and karyotype. The resistant line bound fluorescent-labeled toxin as did the unselected susceptible line. A high level of resistance was quickly achieved, and this level was maintained after 4 mo. culture in the absence of toxin. This work was supported by grant number RO1 AI 22702 from the National Institutes of Health, Bethesda, MD.     

Development of low-cost media for the culture of mosquito larvicides, Bacillus sphaericus and Bacillus thuringiensis serovar. israelensis

Mosquito larvicides like Bacillus sphaericus and Bacillus thuringiensis serovar. israelensis have been widely and effectively used in mosquito control programs, but the industrial production of these bacilli is expensive. Here we have attempted to develop three cost-effective media, based on cheap sources, potato, common sugar and bengalgram. Growth and production of the insecticidal proteins from these bacteria were satisfactory. Bioassay studies with different mosquito larvae showed considerable toxicity. Therefore the investigation suggests that potato-based culture media are more economical for the industrial production of B. sphaericus and B. thuringiensis serovar. israelensis.     

Effects of crowding on populations of Aedes albifasciatus larvae under laboratory conditions

Aedes (Ochlerotatus) albifasciatus(Macquart 1838) (Diptera: Culicidae) is a neotropical flood water mosquito, incriminated as the main vector of the western equine encephalitis virus, and which affects beef and milk production in central Argentina. The short time required to hatch and develop from egg to adult, usually in temporary pools, suggests a strategy which allows for exploitation of transient pools, thus evading predation and interspecific competition. Under these conditions intra specific competition could represent a major density-dependent source of larval mortality, but the relative importance of density-dependent regulation of mosquito populations has generated controversy. Therefore we examined the effects of larval density on basic population characteristics of Ae. albifasciatusin the laboratory. Larvae were obtained by synchronous hatching of eggs laid by field-trapped females. Emerging larvae (L1) were used to build cohorts of different initial densities, kept in plastic trays with 400 ml of distilled water, and food supplied daily during the first 10 days (0.1 g per larvae day^–1). Age-specific development time and specific and relative mortality were estimated, and their relation to initial larval density was assessed through linear and non-linear regressions and correlation analysis. First hatching was registered 3 h after flooding the eggs. Higher levels of pre-adult mortality were detected in groups with higher densities. Specific mortality and average time to enter a stage of L1 to L3 could directly be related to initial larval density, but no significant relations were found for L4 and pupae. Results suggest that crowding could be a factor capable of regulating the density of natural populations of Ae. albifasciatus.     

Immunostaining for allatotropin and allatostatin-A and -C in the mosquitoes <Emphasis Type="Italic">Aedes aegypti</Emphasis> and <Emphasis Type="Italic">Anopheles albimanus</Emphasis>

Confocal laser-scanning microscopy was used to carry out a comparative study of the immunostaining for three families of neuropeptides, viz., allatostatin-A (AS-A), allatostatin-C (AS-C) and allatotropin (AT), in adult female mosquitoes of Aedes aegypti and Anopheles albimanus. The specific patterns of immunostaining for each of the three peptides were similar in both species. The antisera raised against AT, AS-A, and AS-C revealed intense immunoreactivity in the cells of each protocerebral lobe of the brain and stained cells in each of the ventral ganglia and neuronal projections innervating various thoracic and abdominal tissues. Only the AS-A antiserum labeled immunoreactive endocrine cells in the midgut. The distribution of the peptides supports the concept that they play multiple regulatory roles in both species.This work was supported by NIH grant AI 45545 to F.G.N. and CONACyT G-37186-M to M.H.R.     

The ecology of mosquito larvae (Diptera: Culicidae) in the subalpine zone of the eastern Sierra Nevada Mountains,California, U.S.A.

Mosquito larvae were collected from the subalpine region of the eastern Sierra Nevada Mountains from 2011 to 2014. Two watersheds were sampled and sites selected were mainly vernal snow‐melt pools and wet meadows. Seven Aedes species, Culiseta incidens (Thomson), and Culex tarsalis Coquillett were collected. The most abundant and widely distributed species were Ae. hexodontus Dyar and Ae. tahoensis Dyar. Aedes tahoensis was the predominate species in woodland snow‐melt habitats. Some species were found at most elevations while others were found more often at specific elevations. The most restrictive species was Ae. ventrovittis Dyar which occurred almost exclusively between 3,219 m a.s.l. and 3,390 m a.s.l. Shannon and Simpson species diversity indices demonstrated that species diversity was greater in meadow habitats compared to woodland habitats. Mixed woodland/meadows, rock pools, and shallow grass pools were intermediate in species diversity. Abiotic factors such as snowpack and water temperature impacted species development times and when habitats dried. It was concluded that spatial and temporal patterns of habitats, along with elevation, influenced species presence and larval development. The results of the present study and previous work in the eastern Sierras will help guide future research that focuses on the potential change in the distribution and seasonality of subalpine mosquitoes and disease potential in the eastern Sierras as climatic conditions change.     

Effects of temperature, pH and salinity on the infection of Leptolegnia chapmanii Seymour (Peronosporomycetes) in mosquito larvae

The effects of temperature, pH, and NaCl concentrations on the infectivity of zoospores of Leptolegnia chapmanii (Argentine isolate) were determined for Aedes aegypti and Culex pipiens under laboratory conditions. Zoospores of L. chapmanii were infectious at temperatures between 10 and 35 degrees C but not at 5 or 40 degrees C. At the permissive temperatures, mortality rates in young instars were much higher than in older instars and larvae of Ae. aegypti were more susceptible to L. chapmanii than larvae of Cx. pipiens. At 25 degrees C, Ae. aegypti larvae challenged with L. chapmanii zoospores resulted in 100% infection at pH levels ranging from 4 to 10. Larvae of Cx. pipiens exposed to similar pH and zoospore concentrations resulted in increasing mortality rates from 62% to 99% at pH 4 to 7, respectively, and then decreased to 71% at pH 10. Aedes aegypti larvae exposed to L. chapmanii zoospores in NaCl concentrations ranging from 0 to 7 parts per thousand (ppt) at 25 degrees C resulted in 100% mortality while mortality rates for Cx. pipiens decreases from 96% in distilled water to 31.5% in water with 6 ppt NaCl. Control Cx. pipiens larvae died when exposed at a NaCl concentration of 7 ppt. Vegetative growth of L. chapmanii was negatively affected by NaCl concentrations. These results have demonstrated that the Argentinean isolate of L. chapmanii tolerated a wide range of temperatures, pH, and salinity, suggesting that it has the potential to adapt to a wide variety of mosquito habitats.     

Laboratory evaluation of deet and permethrin impregnated wide-mesh netting against mosquitoes

Behaviour of two species of mosquitoes was studied by releasing them into a tunnel of cages in which either deet or permethrin impregnated wide-mesh cotton or nylon netting was stretched across the tunnel between the mosquito entry point and an animal bait.Di-ethyl toluamide (deet) was found to be more effective in repelling Culex quinquefasciatus say when pieces of impregnated thick cotton netting were placed at a distance from the bait rather than close to the bait. Anopheles gambiae Giles was repelled at lower deet dosages than was Cx. quinquefasciatus. One impregnation remained effective for 7 weeks when the netting was impregnated with 25 ml per m² held in still air in a room. When it was held in a plastic bag the shelf life was at least one year.Permethrin impregnated nylon netting of 4 mm or 6 mm mesh-size had a strong toxic and excitorepellent effect on An. gambiae. However, the effects were much weaker when the mesh size was 13 mm. Permethrin impregnated 8 mm-mesh thick, cotton netting offered very good protection against mosquitoes for at least 30 weeks. There was no marked difference in effectiveness between doses of 0.2 or 0.5 g/m².

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Résumé Le comportement de deux espèces de moustiques a été examiné en les lâchant dans un tunnel formé par 6 cages successives dans lesquelles étaient tendus, entre l'entrée et un animal proie, des filets de coton ou de nylon imprégnés de deet ou de permethrine. La répulsion par le deet (di-éthyl toluamide) s'est révélé plus efficace contre Culex quinquefasciatus avec les filets de coton tendus à une certaine distance de la proie plutôt qu'à proximité. Anopheles gambiae a été repoussé par des doses plus faibles de deet que C. quinquefasciatus. Une imprégnation du filet avec 25 ml/m² est resté efficace pendant 7 semaines dans une pièce où l'air était calme. Conservé dans un emballage plastique la durée de conservation a été au moins d'un an.La permethrine imprégnant un filet de nylon de maille 4 à 6 mm a présenté un effet toxique et repulsif-excitant très fort contre A. gambiae. L'effet a été beaucoup plus faible lorsque les mailles étaient de 18 mm. La permethrine, imprégnant un filet de coton de maille 8 mm, a fourni une très bonne protection contre les moustiques pendant au moins 30 semaines. Il n'y a pas eu de différences significatives entre des doses de 0,2 et 0,5 g/m².

     

The roles of history: age and prior exploitation in aquatic container habitats have immediate and carry‐over effects on mosquito life history

1. Per‐capita resource availability in aquatic habitats is influenced directly by consumer density via resource competition and indirectly via delayed resource competition when temporally non‐overlapping cohorts of larvae exploit the same resources. In detritus‐based systems, resources are likely to be influenced by the age of the aquatic habitat, as detritus changes in quality over time and may be replenished by new inputs. 2. For aquatic insects that exploit detritus‐based habitats, feeding conditions experienced during immature stages can influence fitness directly via effects on development and survivorship, but also indirectly by influencing adult traits such as fecundity and longevity. 3. Larval habitat age and prior resource exploitation were manipulated in a field experiment using the container mosquito Aedes triseriatus. 4. It was found that A. triseriatus from older habitats had greater larval survival, faster development and greater adult longevity. Exploitation of larval habitats by a prior cohort of larvae had a significant negative effect on subsequent cohorts of larvae by delaying development. 5. It is suggested that extended conditioning of detritus probably resulted in conversion of recalcitrant resources to more available forms which improved the quality of the habitat. 6. In a parallel study, evidence was found of carry‐over effects of habitat age and prior exploitation on adult longevity for A. triseriatus and Aedes japonicus collected from unmanipulated aquatic habitats. 7. These results indicate the importance of detritus dynamics and the discontinuous nature of resource competition in these mosquito‐dominated aquatic systems.     

Biological differences in reproductive strategy between the mosquito sibling species Anopheles gambiae sensu stricto and An. quadriannulatus

Females of the afrotropical mosquito species Anopheles gambiae Giles sensu stricto and An. quadriannulatus (Theobald) (Diptera: Culicidae) were studied for the effect of blood meal size and the frequency of blood feeding on reproductive development during the first gonotrophic cycle. To standardize the blood meals, meals were administered by enema in some experiments. The effects of insemination, mosquito size, and metabolic reserves at emergence on egg development were also investigated. Maximum insemination was reached after seven days, varying from 62% in An. quadriannulatus to 95% in An. gambiae and was significantly different (P<0.05) between the two species. Insemination had no effect on feeding success. Females of An. quadriannulatus were significantly larger than An. gambiae females (mean wing size 2.90 ±0.01 mm versus 2.82 ±0.01 mm), but the protein, glycogen, and lipid content of newly emerged females of the two species were not significantly different. Without a blood meal, larger females of both species were significantly more likely to develop oocytes up to Christopher's stage II. With one blood meal, 27% of An. gambiae became pre-gravid and 73% matured eggs. In contrast, all An. quadriannulatus females remained in the pre-gravid stage following ingestion of one blood meal. Vitellogenesis was significantly reduced in smaller-sized pre-gravid An. quadriannulatus compared to larger individuals. When given the opportunity to feed up to three times on three successive days, all females of An. gambiae matured eggs but only 85% of An. quadriannulatus did so. When 1 l of human blood was administered by enema, none of the females of either species developed eggs. With a single enema of 1.5 l of human blood, only An. gambiae developed eggs. A similar result was observed with 1 l and 1.5 l enemas of bovine blood although some An. gambiae also developed eggs with 1 l of blood. Anopheles quadriannulatus developed eggs only when given two 1 l enemas on successive days. However, the percentage of females developing eggs was significantly lower than that of An. gambiae. The implications of these differences in reproductive strategy are discussed in the light of behavioural traits in the field.     

Cytogenetic analysis of metaphase chromosomes from pupal testes of four mosquito species using fluorescence <Emphasis Type="Italic">in situ</Emphasis> hybridization technique (FISH)

The DNA probes, P1887, P2405, P2056 (being specific tags for Aedes aegypti genes coding for ribosomal RNA) and a centric heterochromatin probe, K20-1A5, were chosen to hybridize the metaphase chromosomes from the testes of four mosquito species, Culex pipiens, Aedes aegypti, Aedes albopictus and Aedes triseriatus. In addition, a single plasmid, P2392, which contained the three probes, P1887, P2405 and P2056, was also used as chromosome landmark in aedine species. Only the Aedes aegypti metaphase chromosome 1-specific tag, P1887, was conserved in Aedes albopictus, Aedes triseriatus, and Culex pipiens metaphase chromosomes. Aedes triseriatus exhibited two gene loci, on chromosomes 1 and 3, coding for ribosomal RNA per haploid genome. When the specific probes for chromosomes 2 and 3, 2405 and 2056, were used in the fluorescence in situ hybridization technique against the metaphase chromosomes the fluorescent signals were not seen in Aedes albopictus, Aedes triseriatus or Culex pipiens. Also the centric heterochromatin probe, K20-1A5, exhibited strong fluorescent signals on chromosomes 1, 2 and 3 of Aedes aegypti. These fluorescent signals were not observed in metaphase chromosomes derived from the other aedine species, indicating that the centromere sequence can vary within the species.This paper was presented at the Second Arab Conference on Biotechnology and Genetic Engineering, held in the Kingdom of Bahrain, 15–17 April, 2002 and is published here with the endorsement of the Co-ordinator of the Scientific Committee, Professor Essam H. Ghanem, University of Bahrain. Its publication has been delayed because of the ill health of the senior author. Other papers from this conference were published in the July 2003 issue (vol. 19, no. 5).     

Sodium channel genes and their differential genotypes at the L-to-F kdr locus in the mosquito Culex quinquefasciatus

The para-type sodium channel in insects is the primary target of pyrethroid and DDT insecticides. However, modifications in the target protein structure such as point mutations or substitutions, resulting from single nucleotide polymorphisms (SNP), cause insensitivity of the insect’s nervous system to pyrethroids and DDT and, in turn, result in insecticide resistance. Among these mutations, substitution of leucine to phenylalanine (L to F) in the 6th segment of domain II (IIS6) has been clearly associated with pyrethroid and DDT resistance in many insect species, including mosquitoes. Here, multiple copies of the sodium channel gene were identified in the mosquito Culex quinquefasciatus by Southern blot analysis and polymerase chain reaction (PCR) analysis. Two genomic DNA fragments of the mosquito sodium channel gene (509 and 181 bp) were detected by a single PCR primer pair. Sequence analysis indicated the lack of an intron sequence in the 181 bp sodium channel fragment. Single nucleotide polymorphism (SNP) analysis revealed a strong correlation among the frequencies of L-to-F allelic (T) expression at the RNA level, the frequencies and resistance allele (T) at the L-to-F site of the 509 bp genomic DNA fragment, which did include an intron sequence, and the levels of insecticide resistance. Taking together, this study, for the first time, not only revealed multiple copies of the sodium channel gene presented in the Culex mosquito genome but also suggested that the one with the intro sequence may be a functional copy of the sodium channel gene in the Culex mosquitoes.     

Time and site of assembly of the peritrophic membrane of the mosquito Aedes aegypti

Summary We determined the time and site of secretion of the precursors of the peritrophic membrane (PM) in Aedes aegypti and when the structure is assembled. The fine structure of the developing membrane of blood-feed females was described, and the pattern of secretion of injected tritiated glucosamine analyzed autoradiographically. Immediately following blood feeding, ingested red cells rapidly become compressed, such that the surrounding plasma is extruded to the margin of the midgut contents. Thereby, ingested fluids form a narrow margin separating the blood mass from the midgut epithelium. By electron microscopy, the PM first becomes evident at about 4 to 8 h after blood is ingested, and the membrane attains mature texture by 12 h. The compacted mass of ingested erythrocytes seems to serve as a template for the forming structure. In contrast, tritiated glucosamine, injected into freshly engorged mosquitoes, begins to concentrate on the midgut microvilli by 2 h after feeding. By 8 h the label assumes the layered appearance that characterizes the fine structure of the mature membrane. In contrast to the prevailing concept that the PM of mosquitoes first assumes texture anteriorly immediately after blood is ingested, we find that this potential barrier to pathogen development forms no earlier than 4 h after feeding and that it is formed from precursors secreted along the entire length of the epithelium overlying the food mass.