Larval competition alters susceptibility of adult Aedes mosquitoes to dengue infection

Dengue, the most important human arboviral disease, is transmitted primarily by Aedes aegypti and, to a lesser extent, by Aedes albopictus. The current distributions of these invasive species overlap and are affected by interspecific larval competition in their container habitats. Here we report that competition also enhances dengue infection and dissemination rates in one of these two vector species. We determined the effects of competition on adult A. aegypti and A. albopictus, comparing their susceptibility to infection with a Southeast Asian strain of dengue-2 virus. High levels of intra- or interspecific competition among larvae enhanced the susceptibility of A. albopictus to dengue virus infection and potential for transmission, as indicated by disseminated infections. Doubling the number of competing larvae (A. albopictus or A. aegypti), led to a significant (more than 60%) increase in the proportion of A. albopictus with disseminated dengue-2 infection. Competition-enhanced vector competence appears to result from a reduction in 'barriers' (morphological or physiological) to virus infection and dissemination and may contribute to the importance of A. albopictus in dengue transmission. Similar results for other unrelated arboviruses suggest that larval competition, common in mosquitoes, should be considered in estimates of vector competence for pathogens that infect humans.     

Additional morphological and physiological heterogeneity within the midgut of larval Aedes aegypti (Diptera: Culicidae) revealed by histology, electrophysiology, and effects of Bacillus thuringiensis endotoxin

Analysis of larval Aedes aegypti midgut using scanning electron microscopy, nuclear and mitochondrial dyes, response to Bacillus thuringiensis israelensis CryIVB toxin, and electrophysiology is described. The anterior ventriculus ("stomach") region is found to have much lower mitochondrial densities than other midgut regions. The transitional region is distinguished by apical surface architecture, and by region-specific effects of CryIVB endotoxin. In this region CryIVB causes holes ranging from 1.0 to 7.0 microm in diameter (mean 3.3+/-0.53 microm, N=12), blisters 16.9+/-1.54 microm in diameter (N=10), and separation of adjacent cells. The holes are not consistent with damage due to the colloid osmotic lysis model of delta-endotoxin activity. The posterior ventriculus possesses a distinctive cellular architecture consisting of hemispherical, domed apical membranes surrounded by deep clefts. Functional and morphological heterogeneity is revealed within the posterior ventriculus, with the anterior end dominating the electrical profile of isolated, perfused preparations and showing the greatest response to serotonin. Hyperpolarization of the transepithelial potential by serotonin occurred in conjunction with a decrease in the space constant lambda, ruling out closure of ion channels as the mechanism of action of serotonin.     

Isolation of spore-forming bacilli from mosquitoes in natural breeding habitats in Iraq

New isolates of spore-forming bacilli from larvae and pupae of 3 species of mosquitoes are recorded in central Iraq.Bacillus sphaericus Meyer and Neide was isolated fromCulex pipiens (L.) larva.Bacillus carotarum Koch andBacillus cereus Frankland & Frankland were isolated fromTheobaldia longiareolata (Macquart) pupae.Bacillus laterosporus Laubach andBacillus thuringiensis (H 18) were isolated fromAedes caspius (Pallas) larvae. In addition, unidentifiedBacillus spp. were isolated fromCx. pipiens, T. longiareolata andAe. caspius larvae. Examination of soil samples collected from mosquito natural breeding habitats revealed isolates ofB. cereus, Bacillus thuringiensis H4a 4b; H 12 and H 16 and an unidentifiedBacillus sp.

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Résumé Des souches bactériennes sporogènes sont isolées de moustiques qui se trouvent dans la région centrale de l'Irak. Les résultats obtenus sont les suivants:Bacillus sphaericus Meyer & Neide [Culex pipiens (L.), larve],Bacillus carotarum Koch etBacillus cereus Frankland & Frankland [Theobaldia longiareolata (Macquart), nymphe],Bacillus laterosporus Laubach etBacillus thuringiensis (H 18) [Aedes caspius (Pallas), larvel]. L'examen des larves deCulex pipiens. T. longiareolata etAe. caspius, ainsi que l'analyse des échantillons du sol prélevés dans la région montrent la présence deBacillus cereus, Bacillus thuringiensis H4a 4b; H12 plus H16 et d'autresBacillus non identifiés.

     

Distributions of larval mosquitoes among bamboo-stump pools which vary in persistence and resource input

We studied the seasonal occurrence and the distribution patterns of larval mosquitoes in a bamboo grove in northern Kyushu, Japan. The number of pools was large from June to August and was small in winter. Deep stumps and semi-closed-type stumps held water more persistently than shallow and open-type stumps, respectively. Open-type stumps trapped more leaves than semi-closed-type stumps and the number of leaves trapped in the open-type stump was positively correlated with the area of the pool. The incidences ofTripteroides bambusa andAedes albopictus were low early in their breeding season and gradually increased thereafter. In August, the density ofT. bambusa larvae per pool was higher in the old (>2 months) pools than in the new pools and was positively correlated with the depth of the stumps. In contrast, the density ofA. albopictus did not differ significantly between the new-and old pools and was not significantly correlated with the depth of the stump. The density ofA. albopictus was positively correlated with the number of leaves that had been trapped in the stump, whereas the density ofT. bambusa was not. In addition to the seasonal fluctuation in habitat-patch availability, the variations in habitat persistence and resource input among bamboo stumps may facilitate the coexistence of the two mosquitoes that were differentially limited by these factors.     

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).     

Invasive mosquitoes, larval competition, and indirect effects on the vector competence of native mosquito species (Diptera: Culicidae)

Invasive arthropods that vector pathogens have the potential to influence pathogen transmission both directly, by becoming a novel pathogen vector, or indirectly, by interacting with native vectors. Adult mosquito size is influenced by food availability in the larval stage, and smaller, nutrient-deprived mosquitoes are, in some studies, more efficient viral vectors in the laboratory. This is the first study to examine the indirect impacts that larval competition between Aedes albopictus, an introduced mosquito species, and Ochlerotatus triseriatus, a native mosquito species and the primary vector for La Crosse virus (LACV) in the US, has on native mosquito larval survival, adult size, and vector competence. A. albopictus presence decreased Oc. triseriatus larval survival, but surviving Oc. triseriatus females were larger, potentially owing to a release from intraspecific competition. These larger, native females were more likely to develop both midgut and disseminated LACV infections than females emerging from monospecific treatments. Collectively, these results suggest a need to better understand the ecology of both native and invasive vector species, their interactions, and the potential for those interactions to alter vector-borne disease transmission.     

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.     

Predation potential of three flatworm species (Platyhelminthes: Turbellaria) on mosquitoes (Diptera: Culicidae)

We conducted a field survey for flatworms to select species as potential biological control agents against Aedes aegypti and Culex pipiens (Diptera, Culicidae) breeding in artificial containers. Laboratory experiments were performed to determine the daily predation rate, differential predation on each mosquito larval instar, selective predation on either A. aegypti or C. pipiens, and predator tolerance to water from artificial containers. Girardia anceps (Tricladida, Paludicola, Dugesiidae), Mesostoma ehrenbergii and Bothromesostoma cf. evelinae (Rhabdocoela, Typhloplanoida, Typhloplanidae) were found in temporary puddles and permanent pools. In the laboratory, they killed between 52% and 100% of immature mosquitoes coexisting in the same habitat. No preference of flatworms for mosquito preys was detected. Predation rate was related to predator size and instar of preys. Girardia anceps and B. evelinae survived after a dry period and when re-flooding occurred, they laid eggs. Tolerance to water from artificial containers was highest in G. anceps and this species could be a suitable predator to reduce mosquito populations from artificial containers using an inoculative approach.     

Electrophysiological and behavioural responses of mosquitoes to volatiles of Silene otites (Caryophyllaceae)

In order to understand the biological significance of flower odour for attraction of mosquitoes, electrophysiological responses to headspace flower odour samples of Silene otites (L.) Wibel were investigated on Culex pipiens pipiens biotype molestus Forskal 1775 and Aedes aegypti L. using coupled gas chromatographic-electroantennographic detection (GC-EAD). No remarkable differences in antennal responses to the odour compounds have been found between these two mosquito species. Further, the behavioural attractiveness of the electrophysiologically active compounds, singly or as multiple odour mixtures, was evaluated with bioassay experiments with C. pipiens molestus. In bioassays, C. pipiens responded to 14 electrophysiologically active compounds in different magnitudes (65–20%) and acetophenone, linalool oxide (pyranoid), phenyl acetaldehyde and phenylethyl alcohol were found as more attractive in comparison to the least attractive compound, hexanol. In two-stimulus choice test, mosquitoes were significantly more attracted to the mixture of the four most attractive compounds compared to the mixture of all 14 compounds. The results of present study confirm that floral odours are attractive cues for mosquitoes.     

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.     

Reduction of resistance of Culex pipiens larvae to the binary toxin from Bacillus sphaericus by coexpression of cry4Ba from Bacillus thuringiensis subsp. israelensis with the binary toxin gene

The cry4Ba gene from Bacillus thuringiensis subsp. israelensis and the binary toxin gene from B. sphaericus C3-41 were cloned together into a shuttle vector and expressed in an acrystalliferous strain of B. thuringiensis subsp. israelensis 4Q7. Transformed strain Bt-BW611, expressing both Cry4Ba protein and binary toxin protein, was more than 40-fold more toxic to Culex pipiens larvae resistant to B. sphaericus than the transformed strains expressing Cry4Ba protein or binary toxin protein independently. This result showed that the coexpression of cry4Ba of B. thuringiensis subsp. israelensis with B. sphaericus binary toxin gene partly suppressed more than 10,000-fold resistance of C. pipiens larvae to the binary toxin. It was suggested that production of Cry4Ba protein and binary toxin protein interacted synergistically, thereby increasing their mosquito-larvicidal toxicity.     

Functional characterizations of residues Arg-158 and Tyr-170 of the mosquito-larvicidal Bacillus thuringiensis Cry4Ba

The insecticidal activity of Bacillus thuringiensis (Bt) Cry toxins involves toxin stabilization, oligomerization, passage across the peritrophic membrane (PM), binding to midgut receptors and pore-formation. The residues Arg-158 and Tyr-170 have been shown to be crucial for the toxicity of Bt Cry4Ba. We characterized the biological function of these residues. In mosquito larvae, the mutants R158A/E/Q (R158) could hardly penetrate the PM due to a significantly reduced ability to alter PM permeability; the mutant Y170A, however, could pass through the PM, but degraded in the space between the PM and the midgut epithelium. Further characterization by oligomerization demonstrated that Arg-158 mutants failed to form correctly sized high-molecular weight oligomers. This is the first report that Arg-158 plays a role in the formation of Cry4Ba oligomers, which are essential for toxin passage across the PM. Tyr-170, meanwhile, is involved in toxin stabilization in the toxic mechanism of Cry4Ba in mosquito larvae. [BMB Reports 2014; 47(10): 546-551]     

Lethality of chlorine, chlorine dioxide, and a commercial fruit and vegetable sanitizer to vegetative cells and spores of Bacillus cereus and spores of Bacillus thuringiensis

Chlorine, chlorine dioxide (ClO₂), and a commercial raw fruit and vegetable sanitizer (Fit powder) were evaluated for their effectiveness in killing vegetative cells and spores of Bacillus cereus and spores of Bacillus thuringiensis. The ultimate goal was to use one or both species as a potential surrogate(s) for Bacillus anthracis in studies that focus on determining the efficacy of sanitizers in killing the pathogen on food contact surfaces and foods. Treatment with alkaline (pH 10.5–11.0) ClO₂ (200 mg/mL) produced by electrochemical technologies reduced populations of a five-strain mixture of vegetative cells and a five-strain mixture of spores of B. cereus by more than 5.4 and more than 6.4 log cfu/mL, respectively, within 5 min. This finding compares with respective reductions of 4.5 and 1.8 log cfu/mL resulting from treatment with 200 mg/mL chlorine. Treatment with a 1.5% acidified (pH 3.0) solution of Fit powder product was less effective, causing 2.5-log and 0.4-log cfu/mL reductions in the number of B. cereus cells and spores, respectively. Treatment with alkaline ClO₂ (85 mg/mL), acidified (pH 3.4) ClO₂ (85 mg/mL), and a mixture of ClO₂ (85 mg/mL) and Fit powder product (0.5%) (pH 3.5) caused reductions in vegetative cell/spore populations of more than 5.3/5.6, 5.3/5.7, and 5.3/6.0 log cfu/mL, respectively. Treatment of B. cereus and B. thuringiensis spores in a medium (3.4 mg/mL organic and inorganic solids) in which cells had grown and produced spores with an equal volume of alkaline (pH 12.1) ClO₂ (400 mg/mL) for 30 min reduced populations by 4.6 and 5.2 log cfu/mL, respectively, indicating high lethality in the presence of materials other than spores that would potentially react with and neutralize the sporicidal activity of ClO₂.Published by permission of the International Association for Food Protection: Journal of Food Protection (2004) 60:1702–1708This revised version was published online in April 2005 with corrections to the text and the section heading.In section Preparation of treatment solutions the phrase 22-28°C was replaced by 22±2°C.     

Phenotypic and genotypic features of new autoagglutinating Bacillus thuringiensis strains

A total of 28 autoagglutinating strains of Bacillus thuringiensis were isolated from different ecologic niches and distinct sites. Twenty-six strains demonstrated toxicity to mosquito larvae of Aedes aegypti and Culex quinquefasciatus. The electrophoretic protein profiles of the crystal components were studied. Twenty-three out of the 28 strains showed the same larvicidal activity and the same protein profiles as B. thuringiensis serovar israelensis. Using isoenzyme analysis (MLEE), it was observed the presence of three electrophoretic types (ETs). The mosquitocidal strains grouped into one ET. The random amplified polymorphic DNA analysis (RAPD) was evaluated using six primers, which demonstrated three different patterns for the 28 autoagglutinating strains, allowing correlation of the profiles obtained with the toxicity observed in the bioassays. The RAPD patterns for mosquitocidal strains were identical to the one of serovar israelensis. However, to strains of low toxicity, each primer generated distinctive RAPD patterns, which demonstrated that these strains belong to different serovars. Although the antigenic classification the 26 autoagglutinating strains of B. thuringiensis could not be determined by classical flagellar serotyping, MLEE and RAPD profiles proved these strains to be compatible with B. thuringiensis serovar israelensis.     

Differential susceptibility of Cotesia orobenae, a parasitoid of the cross-striped cabbageworm, to commonly used insecticides in Cruciferae

The larval endoparasitoid Cotesia orobenae Forbes (Hymenoptera: Braconidae) is an effective natural enemy of the cross-striped cabbageworm, Evergestis rimosalis (Guenée) (Lepidoptera: Pyralidae), in southwest Virginia. Routine use of insecticides to control Plutella xyostella (L.) and other lepidopterous larvae in commercial plantings of cabbage, cauliflower and broccoli can disrupt populations of C. orobenae, causing localized outbreaks of the cross-striped cabbageworm to occur. Since the use of insecticides is the choice insect pest management tactic of growers in southwest Virginia, we examined the differential susceptibility of this hymenopteran parasitoid to four frequently used pesticides, methomyl (carbamate), permethrin and esfenvalerate (pyrethroids), and Bacillus thuringiensis Berliner subsp. Kurstaki (bacterium). Filter paper dipped into four different concentrations of methomyl, permethrin, and esfenvalerate were left to dry and then exposed to C. orobenae. Two concentrations of B. thuringiensis were also tested by contact bioassay, and an additional test was conducted by mixing B. thuringiensis with honey and water as a food source for the insects. Ten adult parasitoids were exposed to each treatment, replicated 10 times. Significant differences in mortality among insecticides tested (p < 0.05) were found. Most toxic to C. orobenae was permethrin, followed by esfenvalerate and methomyl. The parasitoid was not affected by B. thuringiensis when placed in contact with B. thuringiensis-treated broccoli leaves, or by ingestion of honey mixed with it (p < 0.05).     

Novel toxicity of native and HD Bacillus thuringiensis strains against to the sugarcane borer Diatraea saccharalis

Diatraea saccharalis (F.) (Lepidoptera: Pyralidae) is a pest that causes great economic losses to sugarcane producers in Mexico. In order to obtain alternatives for control of this pest, several Bacillus thuringiensis strains (native and from the Howard Dulmage collection) were tested. In bioassays, strains HD-133, HD-551, GM-7, GM-10, and GM-34 caused more than 50% mortality with a 50 g/ml spore-crystal complex concentration, and were selected as toxic strains. The lowest LC₅₀ value corresponded to GM-34 (33.21 g/ml). Cry1B and cry1C genes were detected by PCR analysis in the toxic strains. HD-133 and GM-10 habored cry1C gene, HD-551 and GM-7 strains harbored cry1B gene, while GM34 strain did not contain cry1B nor cry1C. An additional PCR analysis was performed to detect cry1A-type genes. All the toxic strains habor at least one cry1A-type gene. Immunoblotting revealed that all strains cross-reacted with an antiCry1A, and only the HD-551 gave a positive signal with antiCry1B polyclonal antisera. GM-7 crystal protein showed no cross-reaction with polyclonal Cry1B antiserum. The toxicity of these strains may be related to some member of the Cry1A toxin class.     

Indirect effects of the fairy shrimp,Branchipus schaefferi and two ostracod species on Bacillus thuringiensis var Israelensis-induced mortality in mosquito larvae

Bacillus thuringiensis var israelensis (BTI), when ingested by mosquito larvae, is highly toxic to them. Many other aquatic invertebrates feed on bacteria but, in general, BTI is not toxic to them. We tested in the laboratory the hypothesis that certain crustaceans indirectly benefit mosquito larvae by reducing mortality caused by BTI. We presumed the mechanism to be ingestion of the bacteria by the crustaceans resulting in a lower concentration available to the mosquito larvae.Mortality of Aedes aegypti larvae exposed to BTI was reduced in the presence of the fairy shrimp, Branchipus schaefferi and the ostracod, Cypridopsis vidua (only during summer trials and not autumn trials for the latter species) but was not reduced in the presence of the ostracod, Heterocypris incongruens. By contrast, H. incongruens preyed upon infected, though still-moving, larvae. Feeding on the bacteria by the crustaceans may not be an important mechanism; our data indicates that the crustaceans did not reduce BTI in the water. Moreover, B. schaefferi, introduced into water and then removed prior to the introduction of BTI and mosquitoes, also reduced mosquito mortality.The mechanism for the protective effect of B. schaefferi and C. vidua is unknown. However, these results suggest that the abundance of certain organisms co-occurring with mosquito larvae may partially explain why the effective concentration of BTI varies among habitats.     

The effect of oxygen tension on the production of Bacillus thuringiensis subsp. israelensis toxin active against Aedes aegypti larvae

An optimized batch production of Bacillus thuringiensis subsp. israelensis was made in a stirred Bioflo III reactor using a previously selected medium, and operating conditions in the range of 100–500 rev/min stirrer speeds and 0.2–1 air flow/culture medium volume/minute (v/v/m) aeration rates, including five combinations; at the end of fermentation, dry powders were recovered and evaluated against Aedes aegypti larvae at 0.05 mg/l. Later, the lethal concentration inducing 50% mortality (LC₅₀) was determined for the two most toxic powders. The bioinsecticide yields varied from 9.1 to 15.7 g/l and the total fermentation times ranged between 18 and 30.3 h. The toxicity varied for two powders much more than for the others. These were for combinations with 300 rev/min:1 v/v/m and 500 rev/min:0.6 v/v/m, giving mortality percentages of 47.2 and 59.7, with LC₅₀ values of 0.2675 and 0.0685 mg/l, respectively. A t test showed no significant difference. However, the larvicidal powder produced with 300 rev/min:1 v/v/m gave more variable toxicity than the powder obtained with 500 rev/min:0.6 v/v/m.     

Reconstruction of Bacillus thuringiensis ssp. israelensis Cry11A Endotoxin from Fragments Corresponding to Its N- and C-Moieties Restores Its Original Biological Activity

Subtilisin hydrolyzes Cry11A endotoxin (of 70 kD) produced by Bacillus thuringiensis ssp. israelensis to fragments of 33- and 36-kD, which correspond to N- and C-terminal halves of the endotoxin molecule. Thermitase (a serine protease from Thermoactinomyces vulgaris) and insect gut proteases from Diptera and Lepidoptera exhibit the same hydrolytic effect on Cry11A. Hydrolyzates maintain high toxicity with respect to larvae of Aedes aegypti, Anopheles stephensi, and Culex pipiens. The 33- and 36-kD Cry11A endotoxin components purified by ion-exchange chromatography from the subtilisin hydrolyzate were inactive; however, equimolar mixture of these proteins exhibited almost the same activity as the initial hydrolyzate.