Voltage-gated ion channels are expressed in the Malpighian tubules and anal papillae of the yellow fever mosquito (Aedes aegypti), and may regulate ion transport during salt and water imbalance.

Vectors of infectious disease include several species of Aedes mosquitoes. The life cycle of Aedes aegypti, the yellow fever mosquito, consists of a terrestrial adult and an aquatic larval life stage. Developing in coastal waters can expose larvae to fluctuating salinity, causing salt and water imbalance, which is addressed by two prime osmoregulatory organs - the Malpighian tubules (MTs) and anal papillae (AP). Voltage-gated ion channels (VGICs) have recently been implicated in the regulation of ion transport in the osmoregulatory epithelia of insects. In the current study, we: (i) generated MT transcriptomes of freshwater-acclimated and brackish water-exposed larvae of Ae. aegypti, (ii) detected expression of several voltage-gated Ca2+, K+, Na+ and non-ion-selective ion channels in the MTs and AP using transcriptomics, PCR and gel electrophoresis, (iii) demonstrated that mRNA abundance of many altered significantly following brackish water exposure, and (iv) immunolocalized CaV1, NALCN, TRP/Painless and KCNH8 in the MTs and AP of larvae using custom-made antibodies. We found CaV1 to be expressed in the apical membrane of MTs of both larvae and adults, and its inhibition to alter membrane potentials of this osmoregulatory epithelium. Our data demonstrate that multiple VGICs are expressed in osmoregulatory epithelia of Ae. aegypti and may play an important role in the autonomous regulation of ion transport.

Laboratory scale evaluation of the feasibility of locally found bladderworts as biological agents to control dengue vector, Aedes aegypti in Sri Lanka.

BACKGROUND: The carnivorous genus Utricularia also includes aquatic species that have the potential to trap a wide range of prey, leading its death due to anoxia. However, the effectiveness of such an approach with carnivorous plants for vector control has not been evaluated in Sri Lanka. METHODS: Early instar (i & ii) and late instar (iii & iv) larvae of Aedes aegypti were exposed to locally found bladderwort (U. aurea Lour and Utricularia sp.). The experimental design was set with 10 larvae (both early and late instars separately) in 250 mL of water with bladderworts containing approximately 100 bladders in plant segments of both species, separately. Each treatment and control were repeated 50 times. The survival status of larvae was recorded daily until death or adult emergence. The larvae found whole or partially inside the bladders were attributed to direct predation. The Cox-regression model and Mantel-Cox log rank test were carried out to assess the survival probabilities of larvae in the presence of two bladderworts separately. RESULTS: The highest predation was observed when using early instar larvae in both U. aurea (97.8%) and Utricularia sp. (83.8%). The mortality caused due to predation by U. aurea was observed to be significantly higher according to the Mantel-Cox log-rank test (HR = 60.71, CI; 5.69-999.25, P = 0.004). The mortality rates of late instar stages of Ae. aegypti were observed to be lower in both U. aurea (82.6%) and Utricularia sp. (74.8%). Overall, the highest predation efficacy was detected from U. aurea (HR = 45.02; CI: 5.96-850.51, P = 0.017) even in late instar stages. The results suggested the cumulative predation in both plants on Ae. aegypti larvae was > 72%. CONCLUSIONS: Utricularia aurea is a competent predator of Ae. aegypti larvae. Further, it is recommended to evaluate the feasibility of this plant to be used in the field as a control intervention in integrated vector management programmes.

Infection of the malaria vector Anopheles coluzzii with the entomopathogenic bacteria Chromobacterium anophelis sp. nov. IRSSSOUMB001 reduces larval survival and adult reproductive potential.

BACKGROUND: Vector control tools are urgently needed to control malaria transmission in Africa. A native strain of Chromobacterium sp. from Burkina Faso was recently isolated and preliminarily named Chromobacterium anophelis sp. nov. IRSSSOUMB001. In bioassays, this bacterium showed a promising virulence against adult mosquitoes and reduces their blood feeding propensity and fecundity. The current study assessed the entomopathogenic effects of C. anophelis IRSSSOUMB001 on larval stages of mosquitoes, as well as its impacts on infected mosquitoes reproductive capacity and trans-generational effects. METHODS: Virulence on larvae and interference with insemination were assayed by co-incubation with C. anophelis IRSSSOUMB001 at a range of 104 to 108 cfu/ml. Trans-generational effects were determined by measuring body size differences of progeny from infected vs. uninfected parent mosquitoes using wing size as a proxy. RESULTS: Chromobacterium anophelis IRSSSOUMB001 killed larvae of the pyrethroid-resistant Anopheles coluzzii with LT80 of ~ 1.75 ± 0.14 days at 108 cfu/ml in larval breeding trays. Reproductive success was reduced as a measure of insemination rate from 95 ± 1.99% to 21 ± 3.76% for the infected females. There was a difference in wing sizes between control and infected mosquito offsprings from 2.55 ± 0.17 mm to 2.1 ± 0.21 mm in infected females, and from 2.43 ± 0.13 mm to 1.99 ± 0.15 mm in infected males. CONCLUSIONS: This study showed that C. anophelis IRSSSOUMB001 was highly virulent against larvae of insecticide-resistant Anopheles coluzzii, and reduced both mosquito reproduction capacity and offspring fitness. Additional laboratory, field, safety and social acceptance studies are needed to draw firm conclusions about the practical utility of this bacterial strain for malaria vector control.

Breeding site selection and co-existing patterns of tropical mosquitoes.

We investigated the physicochemical properties and the biotic interactions of breeding sites of tropical mosquito species. Field sampling was done in 12 study areas in Sri Lanka covering areas with secondary natural forests and human settlements. A total of 226 breeding sites were investigated to determine the biotic interactions and physiochemical properties of breeding water (pH, Conductivity, Dissolved Oxygen, Total Dissolved Solids and Temperature). A total of 80.5% of breeding sites from both habitats were positive for mosquito larvae of seven genera and 24 species. Orthopodomyia flavithorax (297) and Aedes albopictus (295) were dominated in tree holes of Alstonia macrophylla, Vateria copallifera and Artocarpus nobilis. Diversity indices showed that the diversity of mosquitoes is high in wet zone habitats of Sri Lanka compared to dry and intermediate zone habitats. Aedes albopictus coexisted with 11 different mosquito species while it avoided larvae of Culex fuscanus, Cx. uniformis and Tripteroides affinis. Strong positive associations were reported between Ae. albopictus and Ar. subalbatus while larvae of Or. flavithorax mosquitoes were not co-occurred with the larvae of Ae. vittatus, Ae. aegypti, Cx. sitiens, Ar. subalbatus, Anopheles spp and Tr. affinis. The findings identified the breeding adaptability and tolerance to a wide range of physiochemical properties of tropical mosquito communities.

Comparative efficacy of natural aquatic predators for biological control of mosquito larvae: A neglected tool for vector control.

Mosquitoes are a dominant fraction of dipteran fauna, occupying a variety of niches. The most common method deployed for their control is the use of insecticides. Throughout their life cycle they are exposed to a wide range of predators in different habitats, thus biological control of mosquitoes by using aquatic predators has been suggested. Therefore, the present study was carried out to explore the type of natural predators coexisting with the mosquito larvae in still water bodies and to determine their efficacy as predators for mosquito larvae. A coexistence of different predators with mosquito larvae was observed in 27 standing water bodies of Chandigarh, India. The predation efficiency of tadpoles of frog was comparable to Gambusia fish, as 97% of the mosquito larvae of all instars of the medically important mosquito genera Anopheles, Aedes, Culex and Armigeres were preyed. The toad tadpoles were found to be least effective and their predation rate was found to be negligible. Further studies on larval source management by frog tadpoles in combination with insecticides or stand-alone would be worthwhile.

A delay suppression model with sterile mosquitoes release period equal to wild larvae maturation period.

Based on the idea that only sexually active sterile mosquitoes are included in the modeling process, we study the dynamics of the interactive wild and sterile mosquito model with time delay, which consists of three sub-equations. Due to the fact that the maturation period of sterile mosquitoes bred in the lab or mosquito factories is almost the same time period of wild adult mosquitoes matured from larvae, we particularly assume that the waiting period for two consecutive releases of sterile mosquitoes equals the maturation period of wild mosquitoes, as a new practical sterile mosquito release strategy. We first ingeniously solve the delay model with the initial functions that are solutions of the corresponding equation without delay and we call them "good" solutions. Using these "good" solutions, we then surprisedly obtain sufficient and necessary conditions for the trivial solution and a unique periodic solution of the delay model to be globally asymptotically stable, respectively. We provide a numerical example to demonstrate the model dynamics and brief discussions of our findings as well.

Docking studies and thiourea-mediated reduced graphene oxide nanosheets' larvicidal efficacy against Culexquinquefasciatus.

The larvicidalproperty of graphene oxide (GO) and thiourea-reduced graphene oxide (T-rGO)was assessed against Culexquinquefasciatuslarvae. A simple water-soluble material synthesis method was used. The transformation of graphene into graphene oxide was accomplished in a single step. Under mild conditions, grapheneoxidewasdissolved in water to form a solution. Structure, optical, and microstructural features of the synthesized samples wereevaluatedusing a variety of analytical tools to compare the samples. Both GO and RGO, as well as GO, showed strong larvicidal potential when used against the third instar larvae of the Culexquinquefasciatus mosquito, with LC50and LC90values of 1.71 and 5.17 ppm and 1.89 and 5.00 ppm, respectively. As a result, our study showed that all of the GO and T-rGO under investigation create larvicidal compounds that could be employed to support efforts to control mosquito populations. It also offers an alternative method for producing GO and rGO on a big scale, which may be used in the future for a variety of biomedical applications.The binding efficacy of the active compounds against AChE1 was studied using Auto dock and the results were observed to be highly promising.

Purification, characterization and toxicity assessment of PirAB toxins from Photorhabdus akhurstii subsp. akhurstii K-1.

Photorhabdus insect related proteins A & B (PirA, PirB) from Photorhabdus and Xenorhabdus bacteria exhibit both oral and injectable toxicity against lepidopteran and dipteran insect pest. The pirA, pirAt (encoding 6 N-terminal truncated PirA), pirB genes, pirA-pirB (with ERIC sequences), pirA-pirB-mERIC (modified pirA-pirB with mutated ERIC sequences) and polycistronic-pirAB were cloned and expressed in Escherichia coli. However, PirA protein was expressed in insoluble form and therefore the pirA gene was modified to produce PirAt. Moreover, pirA-pirB-mERIC, polycistronic-pirAB and co-transformed pirA/pirB genes were not expressed in the studied prokaryotic expression systems. None of the single purified proteins or mixtures of the individually expressed and purified proteins were toxic to mosquito larvae of Aedes aegypti and Culex quinquefasciatus. However, PirA-PirB protein mixtures purified from pirA-pirB operon plasmid were toxic to A. aegypti and C. quinquefasciatus larvae with LC50 values of 991 and 614 ng/ml, respectively. The presence of ERIC sequences between the two orfs of the pirA-pirB operon could help to obtain the proteins in biologically active form. Further, results confirm that PirA-PirB proteins of P. akhurstii subsp. akhurstii K-1 are binary insecticidal toxins and ERIC sequences could play an important role in expression of Pir proteins. Reports of biophysical characterization of individually purified PirAt, PirB and expressed PirA-PirB toxin mixture could provide the structural insight into these proteins.

Behavioural changes and flight response of a mosquito (Culicidae) and an earthworm (Lumbricidae), respectively, after exposure to imidacloprid.

A major point of concern in ecotoxicology is the effects of pesticides on nontarget organisms. This can impact the ecological role played by certain beneficial species in nature. Regarding neonicotinoid insecticides such as imidacloprid (IMI), several measures, including limited trade, restrictive use, and ban have been implemented in Europe and the USA but not globally. The goal of our study was to evaluate the potential risk of this still widely used agrochemical on the behaviour of mosquito larvae (Culicidae) and the escape behaviour of earthworms (Lumbricidae). Changes in breathing, swimming and resting were recorded in mosquitoes postexposure to 0, 1 and 2 mg IMI/L for 10 min. Earthworms were topically exposed in water for 2 minutes to 0, 5, 10 and 20 mg IMI/L. The escape behaviour (initial escape distance and speed) of the earthworms were recorded. In culicids, resting particularly was significantly increased by the exposure to imidacloprid (p < 0.05). In earthworms, the initial escape distance was statistically longer (p < 0.05) when fleeing from the 5 mg IMI/L solution than the solutions with the two highest concentrations. The worms exposed to the 5 mg IMI/L reacted faster than those exposed to the higher concentrations, which explained the long distance covered in the same amount of time. These results point to the relatively quick onset of the neurotoxic effects of imidacloprid, crippling earthworms and altering the buoyancy of mosquito larvae. The ecological consequences of these findings on the completion of life cycles and the survival of these species in nature are yet to be established.

Diversity of native larvivorous fish fauna during pre- and post-tsunami in Car Nicobar, Andaman & Nicobar Islands, India.

BACKGROUND & OBJECTIVES: Malaria is a serious public health problem in Car Nicobar Island, Andaman & Nicobar Islands, India. Using larvivorous fishes has proved to be the cheapest method for vector management approach, with long suppression of mosquito population. This study aims to scale-up the diversity of larvivorous fishes and their potential larvivoracity to evolve an appropriate biological intervention strategy against the immature stages of malaria vectors. METHODS: During 2003 (pre-tsunami) and 2014-2016 (post-tsunami), an ecological descriptive survey was carried out in the water bodies around Car Nicobar Island. Fishes were captured using fishing nets and cast nets; and placed in plastic jars and preserved in formalin solution. Fishes were identified and classified according to the available keys. Their abundance and data on the current conservation status was recorded and analyzed. Their potential larvivoracity was graded according to their feeding intensity. RESULTS: Pre-tsunami, a total of 27 larvivorous fish species belonging to 11 orders, 23 families and 23 genera were identified. Order Perciformes and the family Cyprinidae were the most ascendant group constituting 51.9 and 11.1%, respectively. While, 80.8% of species were preferred to inhabit the freshwater region, only 37% of species were surface feeders (SF). According to the conservation, assessment and management plan (CAMP, 1998), 44.4% species were at lower risk least concern (LRlc), while 55.6% species were at least concerned (LC) as per the IUCN, 2017 categorization. However, after the tsunami, 17 fish species had gone extinct and 10 fish species were found to remain alive in the local water bodies. INTERPRETATION & CONCLUSION: Post-tsunami, the larvivoracity analysis indicated that Ophiocara aporos, Ophiocara procephala, Valamugil seheli, Channa punctata, Kuhlia rupestris, Khulia mugil, and Terapon jarbua possess high-level larvivorous potentiality in nature and are recommended for vector control in the study area. These fish species were facing several anthropogenic threats, such as human interference, loss of habitat, trade, overexploitation, and fishing. Therefore, it is important to protect the water bodies from external impact and implement the conservation strategies. Further, periodic fish fauna surveys, identification of breeding sites, scale-up of the larvivorous potentiality at the field level, creating public awareness through health education on establishment of larvivorous fish ponds and planning for mass rearing of the native fish species should be adopted as part of vector management approach in the endemic malarious region of Car Nicobar Island.

Channel-pore cation selectivity is a major determinant of Bacillus thuringiensis Cry46Ab mosquitocidal activity.

Cry46Ab from Bacillus thuringiensis TK-E6 is a new mosquitocidal toxin with an aerolysin-type architecture, and it is expected to be used as a novel bioinsecticide. Cry46Ab acts as a functional pore-forming toxin, and characteristics of the resulting channel pores, including ion selectivity, have been analyzed. However, the relationship between channel-pore ion selectivity and insecticidal activity remains to be elucidated. To clarify the effects of charged amino acid residues on the ion permeability of channel-pores and the resulting insecticidal activity, in the present study, we constructed Cry46Ab mutants in which a charged amino acid residue within a putative transmembrane ß-hairpin region was replaced with an oppositely charged residue. Bioassays using Culex pipiens mosquito larvae revealed that the mosquitocidal activity was altered by the mutation. A K155E Cry46Ab mutant exhibited toxicity apparently higher than that of wild-type Cry46Ab, but the E159K and E163K mutants exhibited decreased toxicity. Ions selectivity measurements demonstrated that the channel pores formed by both wild-type and mutant Cry46Abs were cation selective, and their cation preference was also similar. However, the degree of cation selectivity was apparently higher in channel pores formed by the K155E mutant, and reduced selectivity was observed with the E159K and E163K mutants. Our data suggest that channel-pore cation selectivity is a major determinant of Cry46Ab mosquitocidal activity and that cation selectivity can be controlled via mutagenesis targeting the transmembrane ß-hairpin region. KEY POINTS: • Cry46Ab mutants were constructed by targeting the putative transmembrane ß-hairpin region. • Charged residues within the ß-hairpin control the flux of ions through channel pores. • Channel-pore cation selectivity is correlated with insecticidal activity.

Artisanal fishing supports breeding of malaria mosquitoes in Western Kenya.

BACKGROUND: Everyday hundreds of people, mainly men, set out to take part in a vibrant artisanal capture fishing (ACF) industry on Lake Victoria. It is not known whether actions of artisanal fishers, in their unrelenting quest for existence, surpass ecosystems' sustainability thresholds with potentially negative repercussions on human health with respect to malaria transmission potential. This article sought to fill this information gap. METHODS: This study used an ecosystem approach to find out how ACF processes facilitate the breeding of mosquitoes. The observational study adopted a cross-sectional design and was carried out on Mageta Island situated inside Lake Victoria in western Kenya. RESULTS: Of the 87 mosquito larval habitats identified 27 (31%) were created through ACF activities. The ACF-related habitats, hereafter collectively referred to as 'fishing habitats', included fishing boats (24), trenches (1) and fish bait mines (2). About half (48%) of Anopheles larvae were recovered from fishing habitats. The mean larval density in the fishing habitats (35.7 ± 1.15) was double that in non-fishing habitats (17.4 ± 0.539). Despite being the most common 'non-fishing habitat' type (N = 32), the mean number of Anopheles larvae present in rock pools (30.81 ± 10.54) was significantly less than those found inside fishing boats (N = 24; 40.08 ± 10.16). Overall, man-made habitats and those used to support livelihoods contained significantly more Anopheles larvae. CONCLUSIONS: These data show that artisanal capture fishing is a key driver of malaria epidemiology on Mageta Island. This suggests that larval source management strategies in the global south should pay attention to the heterogeneity in Anopheles breeding habitats created through livelihood activities.

PEGylated cyanine dye nanoparticles as photothermal agents for mosquito and cancer cell control.

Conversion of light energy to heat via photothermal conversion agents (PTCAs) is of great interest and has potential applications. Here, we described a heptamethine cyanine (Cy7) dye nanoparticles (Cy7-PEG NPs) prepared from heptamethine cyanine and poly(ethylene glycol) (PEG400) via a simple solvothermal process as novel PTCA. Cy7-PEG NPs have absorption maximum at about 808 nm and good photothermal conversion ability. Upon irradiation, Cy7-PEG NPs can effectively kill living mosquito larva (Aedes albopictus) through heat generation. Furthermore, Cy7-PEG NPs have excellent phototoxic activity to Sf9, HeLa and MCF-7 cells. Our results indicated that Cy7-PEG NPs can be used as controlling agent for mosquito larvae and cancer cells.

Associations between household environmental factors and immature mosquito abundance in Quetzaltenango, Guatemala.

BACKGROUND: Aedes aegypti-borne diseases are becoming major public health problems in tropical and sub-tropical regions. While socioeconomic status has been associated with larval mosquito abundance, the drivers or possible factors mediating this association, such as environmental factors, are yet to be identified. We examined possible associations between proximity to houses and roads and immature mosquito abundance, and assessed whether these factors and mosquito prevention measures mediated any association between household environmental factors and immature mosquito abundance. METHODS: We conducted two cross-sectional household container surveys in February-March and November-December, 2017, in urban and rural areas of Quetzaltenango, Guatemala. We used principal components analysis to identify factors from 12 variables to represent the household environment. One factor which included number of rooms in house, electricity, running water, garbage service, cable, television, telephone, latrine, well, and sewer system, was termed "environmental capital." Environmental capital scores ranged from 0 to 5.5. Risk factors analyzed included environmental capital, and distance from nearest house/structure, paved road, and highway. We used Poisson regression to determine associations between distance to nearest house/structure, roads, and highways, and measures of immature mosquito abundance (total larvae, total pupae, and positive containers). Using cubic spline generalized additive models, we assessed non-linear associations between environmental capital and immature mosquito abundance. We then examined whether fumigation, cleaning containers, and distance from the nearest house, road, and highway mediated the relationship between environmental capital and larvae and pupae abundance. RESULTS: We completed 508 household surveys in February-March, and we revisited 469 households in November-December. Proximity to paved roads and other houses/structures was positively associated with larvae and pupae abundance and mediated the associations between environmental capital and total numbers of larvae/pupae (p ≤ 0.01). Distance to highways was not associated with larval/pupal abundance (p ≥ 0.48). Households with the lowest and highest environmental capital had fewer larvae/pupae than households in the middle range (p < 0.01). CONCLUSIONS: We found evidence that proximity to other houses and paved roads was associated with greater abundance of larvae and pupae. Understanding risk factors such as these can allow for improved targeting of surveillance and vector control measures in areas considered at higher risk for arbovirus transmission.

Field application of MALDI-TOF MS on mosquito larvae identification.

In recent years, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has emerged as an efficient tool for arthropod identification. Its application for field monitoring of adult mosquitoes was demonstrated, but identification of larvae has been limited to laboratory-reared specimens. Study aim was to test the success of MALDI-TOF MS in correctly identifying mosquito larvae collected in the field. Collections were performed at 13 breeding sites in urban areas of Marseille, a city in the South of France. A total of 559 larvae were collected. Of these, 73 were accurately morphologically identified, with confirmation either by molecular identification (n = 31) or analysis with MALDI-TOF MS (n = 31) and 11 were tested using both methods. The larvae identified belonged to six species including Culiseta longiareolata, Culex pipiens pipiens, Culex hortensis, Aedes albopictus, Ochlerotatus caspius and Anopheles maculipennis. A high intra-species reproducibility and inter-species specificity of whole larva MS spectra was obtained and was independent of breeding site. More than 92% of the remaining 486 larvae were identified in blind tests against the MS spectra database. Identification rates were lower for early and pupal stages, which is attributed to lower protein abundance and metamorphosis, respectively. The suitability of MALDI-TOF MS for mosquito larvae identification from the field has been confirmed.

Native larvivorous fish diversity as a biological control agent against mosquito larvae in an endemic malarious region of Ranchi district in Jharkhand, India.

BACKGROUND & OBJECTIVES: Mosquitoes are responsible for transmitting several diseases, including malaria, dengue, chikungunya, filariasis, and yellow fever, etc. Release of larvivorous fishes is one of the cheapest method of vector management approach, with long suppression of mosquito population. The present study identifies the native larvivorous fishes and evaluates their potential larvivoracity for biological control of mosquito larvae in an endemic malarious region. METHODS: During the year 2012-13, an ecological descriptive study was carried out in diverse aquatic habitats of fish species found in different areas of Ranchi district, in Jharkhand state of India. Fishes were captured using fishing nets, and identified and classified according to the available keys. Their larvivorous potential was graded according to their feeding potential. Data on current conservation status as well as their abundance were also recorded and analysed. RESULTS: In total, 30 larvivorous fish species belonging to seven orders, 10 families and 21 genera were identified. Order Cypriniformes and the family Cyprinidae were the most ascendant group constituting 66.7 and 60%, respectively. The grading assessment of larvivorous potential for different fish species revealed that, Colisa fasciatus possess maximum larvivoracity (+ + + + +). According to the conservation, assessment and management plan (CAMP, 1998), 60% species were at lower risk near threat (LRnt), while 86.7% species were at least concerned (LC) as per the IUCN, 2017 categorisation. All fish species preferred to inhabit in freshwater. Maximum species occurrence was found in the river (63.3%). Only 30% species were bottom feeders (BF). INTERPRETATION & CONCLUSION: The larvivoracity and habitat distribution analysis indicated that C. fasciatus, Oreochromis mossambica, Esomus danricus, Oryzias melastigma, Puntius sophore, P. ticto, Rasbora daniconius, R. elegans, Aplocheilus panchax, and Danio (B) rerio possess high-level larvivorous potentiality in nature and are recommended for malaria control in the study area. There is an increasing pressure on the fish fauna, of facing several threats, such as fishing, human interference, loss of habitat, overexploitation, pollution, siltation, trade, and diseases. Therefore, periodic survey and monitoring of fish biodiversity, demarcation of breeding sites, field level research study on the efficacy of these fishes, and public awareness on establishment of larvivorous fish ponds should be adopted as a part of the vector management approach in the endemic malarious region of Ranchi district in Jharkhand, India.

Do Multi-year Applications of Bacillus thuringiensis subsp. israelensis for Control of Mosquito Larvae Affect the Abundance of B. cereus Group Populations in Riparian Wetland Soils?

Bacillus thuringiensis subsp. israelensis (Bti) is a soil-borne bacterium affiliated to the Bacillus cereus group (Bcg) and has been used in biocontrol products against nematoceran larvae for several decades. However, knowledge is limited on whether long-term Bti application can affect the structure of indigenous communities of Bcg and the overall abundance of Bti. Using species- and group-specific quantitative PCR assays, we measured the Bcg- and Bti-abundances in riparian wetlands in the River Dalälven floodplains of central Sweden. On five occasions during one vegetative season, soil samples were collected in alder swamps and wet meadows which had been treated with Bti for mosquito larvae control during the preceding 11 years, as well as in untreated control sites and well-drained forests in the same area. The average abundance of Bcg in alder swamps was around three times higher than in wet meadows. Across all sites and habitats, the Bti treatments had no effect on the Bcg-abundance, whereas the Bti-abundance was significantly higher in the treated than in the control sites. However, for individual sampling sites, abundances of Bti and Bcg were not correlated with the number of Bti applications, indicating that added Bti possibly influenced the total population of Bti in the short term but had only a limited effect in the longer term. The findings of this study increase the understanding of the ecology of Bti applications for mosquito control, which can facilitate environmental risk assessment in connection with approval of microbiological control agents.

New insights into the in vitro development and virulence of Culicinomyces spp. as fungal pathogens of Aedes aegypti.

Culicinomyces spp. (Hypocreales: Cordycipitaceae) are facultative fungal pathogens affecting the larval stages from a range of mosquito species and are especially notable in their ability to infect hosts through the digestive tract after conidial ingestion. While Culicinomyces spp. were studied mainly in the 1980s, little is yet known about inter- and intraspecific variability of the in vitro development of these fungi at different temperatures, and nothing is known about the impact of serial host-passage on the development or virulence against Aedes aegypti larvae. The development of ten isolates of C. clavisporus (ARSEF 372, 582, 644, 706, 964, 1260, 2471, 2478, 2479 and 2480) and one of C. bisporalis (ARSEF 1948) was assessed on solid SDAY/4 and liquid SDY/4 at 15, 20, 25, 30 and 35°C. Based on the results of these assays, three isolates were selected (ARSEF 644, 964 and 2479) for three serial host-passage/reisolation cycles, and comparison of the reisolates with the original stock isolates for their virulence, vegetative growth and conidiogenesis. The highest germination rates (≥95%) after 48h incubation were obtained at 25 and 20°C, and the lowest germination (≤12%) at 35°C after the same time. The optimal temperature for radial growth was 25°C (≥11.8mm), followed by 20°C for all isolates. ARSEF 706, 582 and 372 showed the greatest vegetative growth (≥20mm). In general, there was little radial growth of colonies at 30°C (≤2.5mm), and none at 35°C. Isolates, especially ARSEF 964, 2479, and 644, generally produced the highest numbers of conidia at 25°C (≥1.42×105 conidia/plate) after 15days. After two host-passages, conidiogenesis increased significantly on SDAY/4 for ARSEF 2479 but not for ARSEF 644 or 964. All larvae exposed to these three isolates of C. clavisporus died within 7days regardless of the concentration or host-passage; C. bisporalis was not tested in these experiments. The virulence of ARSEF 964 increased at lower concentrations (106-3×105conidia/ml) after the first host-passage.

Interaction of Lysinibacillus sphaericus binary toxin with mosquito larval gut cells: Binding and internalization.

The binary toxin produced by Lysinibacillus sphaericus is composed of BinA and BinB subunits. Together, but not separately, the two subunits are highly toxic to Culex quinquefasciatus larvae, but show no toxicity to Aedes aegypti. The molecular mechanism underlying intoxication has not been clearly elucidated. The present study compares the binding and the internalization of binary toxin into the midgut epithelial cells of susceptible C. quinquefasciatus mosquito larvae with those of Bin-refractory A. aegypti. The guts from larvae fed with fluorescently labeled toxin were dissected and analyzed using a confocal laser scanning microscope. When fed with a mixture of both components, co-localization of BinA and BinB was detected both on the cell surface and in the cytoplasm of Culex larval gut cells. However, administration of BinA alone resulted in localization only on the cell membrane, whereas BinB alone was detected both on the cell membrane and inside the cytoplasm. In contrast, when a mixture of both components, or each individual component, was fed to Aedes larvae, BinA and BinB were unable to reach the cytoplasm and were localized only on the cell membrane. These results are consistent with the suggestion that the internalization of BinA is essential for toxicity, and that BinB is required for this internalization into susceptible larval gut cells.

Single-reversal charge in the ß10-ß11 receptor-binding loop of Bacillus thuringiensis Cry4Aa and Cry4Ba toxins reflects their different toxicity against Culex spp. larvae.

Bacillus thuringiensis Cry4Aa toxin was previously shown to be much more toxic to Culex mosquito-larvae than its closely related toxin - Cry4Ba, conceivably due to their sequence differences within the ß10-ß11 receptor-binding loop. Here, single-Ala substitutions of five residues (Pro(510), Thr(512), Tyr(513), Lys(514) and Thr(515)) within the Cry4Aa ß10-ß11 loop revealed that only Lys(514) corresponding to the relative position of Cry4Ba-Asp(454) is crucial for toxicity against Culex quinquefasciatus larvae. Interestingly, charge-reversal mutations at Cry4Ba-Asp(454) (D454R and D454K) revealed a marked increase in toxicity against such less-susceptible larvae. In situ binding analyses revealed that both Cry4Ba-D454R and D454K mutants exhibited a significant increase in binding to apical microvilli of Culex larval midguts, albeit at lower-binding activity when compared with Cry4Aa. Altogether, our present data suggest that a positively charged side-chain near the tip of the ß10-ß11 loop plays a critical role in determining target specificity of Cry4Aa against Culex spp., and hence a great increase in the Culex larval toxicity of Cry4Ba was obtained toward an opposite-charge conversion of the corresponding Asp(454).