Bacillus sphaericus for the control of mosquitoes

A survey was conducted to evaluate the number of tree-hole breeding mosquito species and their distribution in the six principal woodland types in Israel. Out of approximately 3,000 mature trees examined, only 38 contained holes that retained water for extended periods of time, and breeding mosquitoes were observed in 27 of them. Two specialized tree-hole breeders, Aedes pulchritarsis Rondani and Aedes geniculatus Oliver, were found breeding at several sites in northern Israel, always at locations 500 m above sea level (a.s.l) and with high annual precipitation. Aedes albopictus Skuse which, in Israel, is known as an opportunistic container breeder, was found in this study to have adapted remarkably well to breeding in tree holes and was found in most forest types investigated and in most tree species which had adequate tree holes. Two other species, Culiseta annulata Schrank and Culex pipiens Linnaeus instars, were found in one of the tree holes, but did not survive to reach maturity.

Samples collected from the mangroves of Vellar estuary yielded a mosquitocidal bacterium, whose secondary metabolites exhibited mosquito larvicidal and pupicidal activity. The bacterium was isolated using standard microbiological methods and identified using classical biochemical tests. The mosquitocidal bacterium was identified as Bacillus subtilis, Bacillus thuringiensis, Bacillus sphaericus and Bacillus cereus. Mosquitocidal metabolite(s) was separated from the culture supernatant of the bacterium and its efficacy was against the larval and pupal stages of two different species of mosquitoes and determined in terms of LC50 and LC90. Mosquito larvicidal activity in terms of LC50 against Anopheleus stephensi and Aedes aegypti was 4.374 and 7.406μl/ml and its pupicidal activity was 4.928 and 9.865μl/ml, respectively. The present study proved that the mosquitocidal properties of the Bacillus species isolated from mangroves of Vellar estuary was evaluated as target species of mosquito vectors. This is an ideal eco-friendly approach for the vector control programs.

Background and Objectives The genus Piper (family Piperaceae) includes aromatic plants widely used as spices and in traditional medicine. Essential oils from Piper species are known for their antimicrobial and pesticidal properties. This study aims to characterize the chemical profiles of the stem bark and leaf essential oils from two Vietnamese Piper species, Piper hainanense and P. thomsonii, and evaluate their biological activities. Methods Essential oil components were identified using gas chromatography-mass spectrometry (GC-MS). Antimicrobial activity was assessed using the broth microdilution method, while mosquito larvicidal activity was evaluated against fourth instar larvae of Aedes aegypti. Results The major constituents of P. hainanense essential oils were sabinene (14.4-15.9%), δ-selinene (6.7-14.6%), β-pinene (13.5-13.9%), β-selinene (5.9-12.4%), α-pinene (7.0-9.0%), and β-elemene (6.1-6.6%). In P. thomsonii stem bark essential oil, elemicin (23.8%), spathulenol (14.5%), and caryophyllene oxide (7.4%) predominated, while its leaf essential oil contained elemicin (23.0%), β-pinene (15.6%), and γ-elemene (13.6%). Antimicrobial assays revealed that P. thomsonii essential oils exhibited strong antifungal activity against Aspergillus niger (MIC = 32 µg/mL). P. hainanense essential oils demonstrated strong mosquito larvicidal activity, with 24-h LC50 values of 26.72–32.57 µg/mL and LC90 values of 34.15–43.48 µg/mL. Conclusion P. hainanense and P. thomsonii essential oils exhibit potential as natural agents for creating antimicrobial medications and mosquito-control strategies. These results serve as a foundation for additional investigation into the medicinal and insecticidal uses of essential oils from Piper.

The peritrophic matrix (PM) of Plutella xylostella larvae was found to contain twelve integral and eighteen loosely associated proteins. An antiserum against Mamestra configurata integral PM proteins cross-reacted with several P. xylostella PM proteins and was used to isolate a partial cDNA encoding an insect intestinal mucin (PxIIM). PxIIM was expressed primarily in the larval midgut. The deduced protein sequence of the partial cDNA contained three potentially glycosylated, mucin-like domains and six cysteine-rich chitin-binding domains (CBDs). An additional chitin-binding domain was proposed to reside at the amino terminus of the protein based on comparison with other IIM. The organization of mucin domains and CBDs exhibited features, including an internal triplet of regularly spaced CBDs and a carboxyl terminal CBD with two additional conserved cysteine residues, that were found to be common to other lepidopteran IIMs.

Bacillus sphaericus produces a mosquito-larvicidal binary toxin composed of BinB and BinA subunits. BinA is important for toxicity, whereas BinB acts as a specific receptor-binding component. To study the functional significance of two regions that are only present in BinB, four block mutations and two single mutations were initially introduced: (111)YLD(113)-->(111)AAA(113), (115)NNH(117)-->(115)AAA(117), (143)GEQ(145)-->(143)AAA(145), (147)FQFY(150)-->(147)AAAA(150), N114A and F146A. Only the replacements at (147)FQFY(150) resulted in a total loss of toxicity to Culex quinquefasciatus larvae. Further single alanine substitutions in this region, F147A, Q148A, F149A and Y150A, were introduced to identify residues playing a critical role in mosquito-larvicidal activity. Larvicidal activity assays revealed that only F149A and Y150A mutants exhibited a total loss of toxicity. The in vitro interaction assays demonstrated that all BinB mutants are able to interact with BinA. Immunohistochemistry analysis revealed that only the Y150A mutant was unable to bind to the larval midgut, suggesting an important role of this residue in receptor binding of the BinB subunit. Conservative aromatic substitutions at F149 and Y150 resulted in full recovery of larvicidal activity, indicating that the aromaticity of F149 and Y150 is a key determinant of larvicidal activity, possibly playing a key role in the membrane interaction and receptor binding.

Assessment of the impact of the biological larvicide VectoMax G: Combination of Bacillus thuringiensis and Lysinibacillus sphaericus on non-target aquatic organisms in Yaoundé-Cameroon

Mercury (Hg) is a toxic metal frequently used in illegal and artisanal extraction of gold and silver which makes it a cause of environmental poisoning. Since biosorption of other heavy metals has been reported for several Lysinibacillus sphaericus strains, this study investigates Hg removal. Three L. sphaericus strains previously reported as metal tolerant (CBAM5, Ot4b31, and III(3)7) were assessed with mercury chloride (HgCl2). Bacteria were characterized by scanning electron microscopy coupled with energy dispersive spectroscopy (EDS-SEM). Sorption was evaluated in live and dead bacterial biomass by free and immobilized cells assays. Hg quantification was achieved through spectrophotometry at 508 nm by reaction of Hg supernatants with dithizone prepared in Triton X-114 and by graphite furnace atomic absorption spectroscopy (GF-AAS). Bacteria grew up to 60 ppm of HgCl2. Non-immobilized dead cell mixture of strains III(3)7 and Ot4b31 showed a maximum sorption efficiency of 28.4 µg Hg/mg bacteria during the first 5 min of contact with HgCl2, removing over 95% of Hg. This process was escalated in a semi-batch bubbling fluidized bed reactor (BFB) using rice husk as the immobilization matrix leading to a similar level of efficiency. EDS-SEM analysis showed that all strains can adsorb Hg as particles of nanometric scale that can be related to the presence of S-layer metal binding proteins as shown in previous studies. These results suggest that L. sphaericus could be used as a novel biological method of mercury removal from polluted wastewater.

Unlike Bacillus thuringiensis, Bacillus sphaericus was first recognized as a saprophytic microorganism, the spores of which currently contaminate soil and aquatic environments. Since the first reports of strains moderately toxic to mosquito larvae (strain K of Kellen and Meyers 1964; strain SSII-1 [= 1321] of Singer 1973), researchers have isolated a series of very potent B. sphaericus strains, beginning in 1977 with strain 1593 (Singer 1977). Strain 1593 was followed by strain 2297 ( = MR-4) (Wickremesinghe and Mendis 1980), strain 2362 (= 290-8) (Weiser 1984), and strain Iab 59 (de Barjac et al. 1988). In addition to the numerous atoxic strains, in 1989 more than 150 mosquito pathogenic isolates with various toxicity levels have become available. This has created problems in their identification and differentiation—first from the atoxic B. sphaericus, and second from each other within the toxic group.

During the course of screening Bacillus species from food and water in Norway, we isolated a strain of Bacillus sphaericus of DNA homology group V, not previously recognized to contain entomopathogenic strains, that was cytotoxic to Vero cell epithelia. Peptide mass fingerprinting of a protein purified from the culture supernatant of B. sphaericus B354 identified a cholesterol-dependent cytolysin (CDC) with high amino acid sequence identity with sphaericolysin, a CDC identified recently in B. sphaericus DNA homology group IIA. The toxin was haemolytic against erythrocytes from a range of species. Haemolysis was potentiated by dithiothreitol and inhibited by preincubation with cholesterol. The toxin induced lactate dehydrogenase release from Vero cells and formed pores in planar lipid bilayers. The distribution of CDC genes in B. sphaericus was examined, with CDC gene products obtained in 13 out of 17 strains representing four of the six DNA homology groups. Thus, we demonstrate the presence of a CDC in a nonentomopathogenic DNA homology group of B. sphaericus (group V) with typical CDC characteristics. CDCs appear to be present in a high proportion of B. sphaericus strains and are not restricted to group IIA insecticidal strains.

The chitin synthase B gene is a key enzyme in the chitin synthesis of insect peritrophic matrix (PM), which affects insects' feeding behavior. The chitin synthase B gene was cloned from the transcription library of Heortia vitessoides Moore. RT-qPCR showed that HvChsb was highly expressed in the larval stage of H. vitessoides, especially on the first day of the pre-pupal stage, as well as in the midgut of larvae and the abdomen of adults. After starvation treatment, HvChsb was found to be significantly inhibited over time. After 48 h of starvation, the feeding experiment showed that HvChsb increased with the prolongation of the re-feeding time. The experimental data showed that feeding affected the expression of HvChsb. HvChsb was effectively silenced via RNA interference; thus, its function was lost, significantly decreasing the survival rate of H. vitessoides. The survival rate from larval-to-pupal stages was only 43.33%, and this rate was accompanied by abnormal phenotypes. It can be seen that HvChsb plays a key role in the average growth and development of H. vitessoides.

The fate of Bacillus sphaericus strain SSII-1 cells ingested by Culex pipiens quinquefasciatus (= C. pipiens fatigans, C. fatigans, C. quinquefasciatus of authors; Diptera: Culicidae) larvae and the cytological events preceding death of the host were observed using electron microscopy. Bacillus sphaericus cells were digested rapidly in the anterior and central midgut. The outer cell wall layer and cytoplasmic ground substance disappeared soon after ingestion. Cytolysosomes became prominent in midgut cells as these cells gradually separated from one another. All bacteria, including B. sphaericus, were confined within the peritrophic membrane until after death of the host. Digestion by the larval host is confirmed as a possible mechanism for release of B. sphaericus toxin from the bacterial cells.

Background and Objective: Most strains of B. thuringiensis produce delta-endotoxin crystals toxic to lepidopteran insects such moth. But some strains of B. thuringiensis produce delta-endotoxin crystals toxic to dipteran insects such as mosquitoes and blackflies. To compare B. thuringiensis isolate from soil and mulberry leaves. Material and Methods:One gram of soil samples was suspended in 9 ml of sterile distilled water and shaken for 5 min. the upper layer of the soil suspension was transferred to a test tube and heated at 80°C for 5 min in water bath to kill non-spore-forming organism and vegetative cells to prepare the sporulated culture, bacteria were grown on nutrient agar pH 7.2, at 30°C for 4 days. The isolates were obtained from mulberry leaves collected in West Java, Indonesia, using the leaf-lift technique. Leaves were trimmed to fit inside a 100 mm petri dish. Abaxial leaf surfaces were placed in contact with nutrient agar, and a sterile, per forated stainless steel disk was placed on the leaf sections to ensure maximum contact with the agar. After the sample was coated with carbon and gold, it was observed and photographed with (SEM). However they had differences form crystal protein, but they had unique as insecticidal to control the same orders (mosquitocidal). Results:The study found that B. thuringiensis serovar entomocidus INA288 produced large cuboidal-form crystals and B. thuringiensis serovar aizawai BUN 1-14 a little homology with serovar entomocidus INA288 but, B. thuringiensis israelensis ONR60A has irregular shaped crystal protein. Toxicity results that serovar entomocidus INA288, serovar aizawai BUN 1-14 and serovar israelensis ONR60A showed toxic to mosquito insects. which was composed of major protein of 130 kDa peptides. B. thuringiensis serovar entomocidus INA288 has 70 kDa and aizawai BUN 1-14 had 69 kDa. Conclusion: B. thuringiensis strain had different crystal protein form but had same toxicity.

Functional redundancy of structural proteins of the peritrophic membrane in Trichoplusia ni

This paper focuses on some factors that affect the gut bacterial communities of Plodia interpunctella and their role in the removal of plastic polymer concentrations to make a clean environment. Counting of midgut bacteria from P. interpunctella reared on different diets exhibited that wheat bran gave a significantly higher number of colonies than other diets (84 × 102 CFU/gut). Histological midgut studies on control fully grown larvae showed different shapes of epithelial cells, elongated or pyramidal, and few numbers of oval regenerative cells. Various secretions are released from the apical margin of the cells. The peritrophic membrane cannot be detected and large numbers of bacteria occupied the midgut lumen. While, irradiated larvae with 250 Gy revealed sever damaged midgut cells, some cells appeared swollen and the others fused as heterogeneous mass. Besides, ruptured of peritrophic membrane and very reduction in the bacterial population were appeared in the lumen. In this respect, two bacterial isolates were isolated from the midgut larvae and identified by 16S rRNA gene sequencing with accession number NR 146667.2 and NR 112686.1, as Enterobacter tabaci strain YIM Hb-3(B1) and Bacillus subtilis subsp. Spizizenii strain NBRC 101239(B2), respectively. Scanning electron microscopy analysis revealed alterations in the polyethylene during 60 days of incubation. Two bacterial strains showed damaging effects on plastic including cavities and pits on the surface of plastic pieces increased with the time of treatment. Moreover, the formation of carbonyl groups was verified using a fourier transform infrared imaging microscope.

BackgroundFailure in detecting naturally occurring breeding sites of Aedes mosquitoes can bias the conclusions drawn from field studies, and hence, negatively affect intervention outcomes. We characterized the habitats of immature Aedes mosquitoes and explored species dynamics along a rural-to-urban gradient in a West Africa setting where yellow fever and dengue co-exist.MethodologyBetween January 2013 and October 2014, we collected immature Aedes mosquitoes in water containers in rural, suburban, and urban areas of south-eastern Côte d’Ivoire, using standardized sampling procedures. Immature mosquitoes were reared in the laboratory and adult specimens identified at species level.Principal findingsWe collected 6,159, 14,347, and 22,974 Aedes mosquitoes belonging to 17, 8, and 3 different species in rural, suburban, and urban environments, respectively. Ae. aegypti was the predominant species throughout, with a particularly high abundance in urban areas (99.374%). Eleven Aedes larval species not previously sampled in similar settings of Côte d’Ivoire were identified: Ae. albopictus, Ae. angustus, Ae. apicoargenteus, Ae. argenteopunctatus, Ae. haworthi, Ae. lilii, Ae. longipalpis, Ae. opok, Ae. palpalis, Ae. stokesi, and Ae. unilineatus. Aedes breeding site positivity was associated with study area, container type, shade, detritus, water turbidity, geographic location, season, and the presence of predators. We found proportionally more positive breeding sites in urban (2,136/3,374, 63.3%), compared to suburban (1,428/3,069, 46.5%) and rural areas (738/2,423, 30.5%). In the urban setting, the predominant breeding sites were industrial containers (e.g., tires and discarded containers). In suburban areas, containers made of traditional materials (e.g., clay pots) were most frequently encountered. In rural areas, natural containers (e.g., tree holes and bamboos) were common and represented 22.1% (163/738) of all Aedes-positive containers, hosting 18.7% of the Aedes fauna. The predatory mosquito species Culex tigripes was commonly sampled, while Toxorhynchites and Eretmapodites were mostly collected in rural areas.Conclusions/significanceIn Côte d’Ivoire, urbanization is associated with high abundance of Aedes larvae and a predominance of artificial containers as breeding sites, mostly colonized by Ae. aegypti in urban areas. Natural containers are still common in rural areas harboring several Aedes species and, therefore, limiting the impact of systematic removal of discarded containers on the control of arbovirus diseases.