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.     

Non-larvicidal effects of Bacillus thuringiensis israelensis and Bacillus sphaericus on oviposition and adult mortality of Culex quinquefasciatus Say (Diptera: Culicidae).

Two microbial mosquito larvicides, Bacillus thuringiensis ssp. israelensis (Bti) and Bacillus sphaericus (Bsph), have been shown to be highly effective in controlling mosquito larvae and have been used in larvicidal programs for many years. In exploring other modes of action of these agents, we studied the ovipositional response of Bsph susceptible and resistant Culex quinquefasciatus to aqueous suspensions of Bti and Bsph water dispersible granules (WDG). We quantified the level of mortality of adult mosquitoes caused by exposure to Bti and Bsph suspensions during oviposition. Significantly lower numbers of egg rafts were laid and collected from the treatments than the control regimen. There was an inverse relationship between Bacillus product concentrations and oviposition. As the concentration of Bti or Bsph increased from 0.0 to 2.0 mg/L, treated waters received progressively fewer egg rafts. In addition to the negative effects of Bacillus on oviposition, both male and female adult mosquitoes suffered high mortality on landing and imbibing on Bti and Bsph suspensions, the extent of mortality directly proportional to concentration. These two microbial agents used solely as mosquito larvicides thus have the additional benefits of reducing mosquito oviposition and killing adult mosquitoes, especially gravid females that come in contact with the treated water either for oviposition or drinking. Reducing the number of gravid females may also result in reduced transmission rates of pathogens. The combined effects of reduced oviposition and adult mortality could result in higher control potential of these microbial agents.     

Biological control of mosquitoes by the larvicidal activity of Bacillus thuringiensis var. israelensis delta endotoxin

Bacillus thuringiensis var. israelensis (B.t.i.) is a promising, safe toxic agent for control of mosquitoes, but the rapid disappearance of its toxicity makes its use in practice economically unattractive. The lack of evidence for B.t.i. multiplication in water also makes the natural ecology of B.t.i. puzzling. The observation that mosquito larvae readily cannibalize carcasses of B.t.i.-killed larvae, and that the carcasses become toxic to scavenging larvae provides a possible solution to this puzzle. Several experimental techniques have been developed to study these phenomena (e.g., quantitative determination of spore numbers despite aggregation, protocols for following toxicity development, etc.). Results suggest a cycle involving larval poisoning and death due to delta-endotoxin of ca. 1,000 spores, germination, vegetative growth and sporulation (with toxin production) of B.t.i. after multiplication to several million in the carcass. Implications of these results for the ecology of B.t.i., and for practical applications in mosquito control, are discussed.     

BIOLOGICAL CONTROL OF MOSQUITO LARVAE WITH MESOCYCLOPS LEUKARTI1)

Abstract  The control efficacy of a predatory copepod, Mesocyclops leukarti , collected in Jining, and its compatibility with Bacillus sphaericus (B.s. ) and B. thuringiensis var. csraefensis H-14 ( B. t. i. ) against mosquito larvae were evaluated in the laboratory and in the field. In the laboratory, M. leukarti showed higher predatory activity toward first instar larvae of Aedes albopictus than toward that of Anopheles sinensis and Culex pipiens pallens. Cage simulation test showed that larvae of Cx. pipiens pallens, An. sinensis and Ae. albopictus could be eliminated within 9, 10 and 7 weeks after interaction with the copepods respectively. M. leukarti population can grow quickly, and its mass production is easy and economic. However, pilot field trials indicated that M. leukarti alone could not eradicate mosquito larvae of the three species rapidly and completely as by chemical insecticides, although they could persist in mosquito breeding habitats longer than the mosquito season. When Mesocyclops was used in combination with B. s. and B. t. i. , higher and sustainable control effects were achieved. So this combination, which achieved the rapid larvicidal and sustainable control effect, was presumed to be practical and could be integrated into mosquito control program.     

用刘氏中剑水蚤防治蚊幼虫(英文)

本研究在室内评价了在济宁采集的捕食性刘氏中剑水蚤单用及与球形芽孢杆菌(B.s.)或苏云金杆菌以色列变种(B.t.i.)合用对蚊幼虫的控制效果。在室内,刘氏中剑水蚤对白蚊伊蚊Ⅰ龄幼虫的捕食效果好于对中华按蚊和淡色库蚊幼虫的效果。蚊笼模拟试验表明,刘氏中剑水蚤可分别于9、10和7周内消灭淡色库蚊、中华按蚊和白蚊伊蚊的幼虫种群。该剑水蚤增长速率快,大量培养经济易行。现场试验证明,刘氏中剑水蚤虽可在蚊虫孳生地存活时间长于蚊虫季节,但单用该剑水蚤不能象化学杀虫剂一样快速完全地控制三种蚊虫。刘氏中剑水蚤与B.s.或B.t.i.合用,应用刘氏中剑水蚤的长持效和生物杀虫剂的速效,取得了高效、持久的防治效果。因此二者合用可用于蚊虫防治并可作为蚊虫综合防治的一个组成部分。     

Monitoring temporal fluctuations of Culex quinquefasciatus using oviposition traps containing attractant and larvicide in an urban environment in Recife, Brazil

The use of attractants and larvicides in oviposition traps is of practical interest for the surveillance and control of urban mosquitoes. In addition to increasing the safety of the traps, this combination is essential for an attract-and-kill control strategy based on trapping mosquito eggs. The combination of Bacillus thuringiensis var. israelensis (Bti) and grass infusion (GI) vs. GI alone were tested for their ability to attract in paired BR-OVT traps in the backyards of 10 houses in Recife, Brazil, for a period of 45 days. Results show that females prefer to oviposit in traps containing Bti (363 compared with 251 egg rafts over 45 days). Results from a one-year trial on the efficacy of BR-OVT traps loaded with GI and Bti as a sampling tool to monitor temporal fluctuations in the population densities of Culex quinquefasciatus in an urban environment are also reported. From December 2006-January 2007, one trap per home was installed and maintained for 348 consecutive days in 134-151 houses located in three urban blocks. Throughout the one-year field trial a total of 43,151 Culex egg rafts were collected in the traps. The data show that BR-OVT loaded with GI and Bti is sensitive enough to demonstrate continuous reproductive activity of Cux. quinquefasciatus in the study area throughout the year and to monitor temporal fluctuations in population density.     

Clostridium bifermentans serovar malaysia, a new anaerobic bacterium pathogen to mosquito and blackfly larvae

A strain of Clostridium bifermentans individualized as serovar malaysia (C.b.m.) according to its specific H antigen is toxic to mosquito and blackfly larvae when given orally. The toxicity occurs in sporulated cells which contain, in addition to spores, proteinic parasporal inclusion bodies and feather-like appendages; the amino acid content of the inclusion bodies is similar to that of Bacillus thuringiensis serovar israelensis (B.t.i.) and B. sphaericus crystals. The toxicity to Anopheles stephensi is as high as that of B.t.i. and the best strains of B. sphaericus. Culex pipiens is somewhat less susceptible, and Aedes aegypti much less. Pure parasporal inclusion bodies, isolated by ultracentrifugation on sucrose gradients, are highly toxic to mosquito larvae. The larvicidal power is destroyed by heating at 80 degrees C or by treatment with 50 mM NaOH. It is preserved by freeze-drying. The innocuity to mice of the sporulated cells is shown by different routes of administration: force-feeding, percutaneous, subcutaneous, intraperitoneal or intravenous injections. The potential for the biological control of mosquito and blackfly larvae is suggested.     

Resistance development in mosquito larvae Culex pipiens to the bacterial agent Bacillus thuringiensis var. israelensis

Abstract:  Mosquito larvae of Culex pipiens were subjected repeatedly to selection pressure with the bacterial agent Bacillus thuringiensis var. israelensis ( B.t.i .) in the laboratory. Only 2.78-fold increase in tolerance to B.t.i . was induced in C. pipiens as a result of 20 generations of selection. The tolerance of C. pipiens to B.t.i . decreased by about 58% after stopping the selection for three generations. Larval selection with B.t.i. caused a reduction in the reproductive potential of mosquito adult survivors but did not affect the adult longevity and the time of blood meal digestion ingested by female mosquitoes.     

Toxicity of insecticides for control of freshwater Culex annulirostris (Diptera: Culicidae) to the nontarget shrimp, Caradina indistincta (Decapoda: Atyidae)

Laboratory evaluations were conducted in southeastern Queensland, Australia, to determine the toxicities of two organophosphate compounds (temephos and pirimiphos-methyl), an insect growth regulator (s-methoprene), and an entomopathogenic bacterium (Bacillus thuringiensis variety israelensis de Barjac [B.t.i.]) to Culex annulirostris (Skuse), an Australian freshwater mosquito vector of arboviruses, and to Caradina indistincta Calman, a co-habiting nontarget shrimp species. S-methoprene and B.t.i. were safest for Cx. annulirostris control with lethal dose ratios (LC95 nontarget/LC95 target) of 3,300 and 846,000, respectively. In contrast, lethal dose ratios for temephos and pirimiphos-methyl were 0.05 and 0.00005, respectively, suggesting that they are environmentally unsuitable. Based on their high lethal dose ratios, s-methoprene and B.t.i. are recommended for control of larval Cx. annulirostris in Australian freshwater habitats.     

Selection of cyanobacteria isolated from mosquito breeding sites as a potential food source for mosquito larvae.

One way to increase the persistence of larvicidal toxins in mosquito breeding sites is to clone the corresponding genes in microorganisms, such as cyanobacteria, which could serve as a source of food for the larvae. We isolated and cultured 10 strains of cyanobacteria from three mosquito breeding sites along the French Mediterranean coast. Most of the strains were tolerant to a relatively wide range of salt concentrations, and all of them were totally or partially resistant to at least four of the five biological or chemical larvicides used in the local mosquito control program. Six unicellular strains from these habitats and Synechococcus strain PCC 7942, a strain maintained for more than 10 years under laboratory conditions, were assessed for ingestion and digestion by larvae Culex pipiens and Anopheles gambiae mosquitoes. The numbers of cells ingested and digested were dependent on the cyanobacterial strain and varied with the mosquito species. Three of the new isolates, Synechococcus strain PCC 8905 and Synechocystis strains PCC 8906 and PCC 8912, were ingested and digested rapidly by larvae of both mosquito species. Since these strains are also tolerant to larvicides and relatively resistant to elevated salt concentrations, they meet the basic requirements for potential recipients of bacterial genes that encode endotoxins.     

Selection of cyanobacteria isolated from mosquito breeding sites as a potential food source for mosquito larvae.

One way to increase the persistence of larvicidal toxins in mosquito breeding sites is to clone the corresponding genes in microorganisms, such as cyanobacteria, which could serve as a source of food for the larvae. We isolated and cultured 10 strains of cyanobacteria from three mosquito breeding sites along the French Mediterranean coast. Most of the strains were tolerant to a relatively wide range of salt concentrations, and all of them were totally or partially resistant to at least four of the five biological or chemical larvicides used in the local mosquito control program. Six unicellular strains from these habitats and Synechococcus strain PCC 7942, a strain maintained for more than 10 years under laboratory conditions, were assessed for ingestion and digestion by larvae Culex pipiens and Anopheles gambiae mosquitoes. The numbers of cells ingested and digested were dependent on the cyanobacterial strain and varied with the mosquito species. Three of the new isolates, Synechococcus strain PCC 8905 and Synechocystis strains PCC 8906 and PCC 8912, were ingested and digested rapidly by larvae of both mosquito species. Since these strains are also tolerant to larvicides and relatively resistant to elevated salt concentrations, they meet the basic requirements for potential recipients of bacterial genes that encode endotoxins.     

Microbial larvicides for mosquito control: Impact of long lasting formulations of Bacillus thuringiensis var. israelensis and Bacillus sphaericus on non‐target organisms in western Kenya highlands

The microbial larvicides Bacillus thuringiensis var. israelensis and Bacillus sphaericus have been used extensively for mosquito control and have been found to be effective and safe to non‐target organisms cohabiting with mosquito larvae. Recently developed long lasting microbial larvicides (LLML), although evading the previous challenge of short duration of activity, increase the risk of persistence of toxins in the treated larval habitats. This study monitored the impact of LLML FourStar^® and LL3 on non‐target organisms cohabiting with mosquito larvae in an operational study to control malaria vectors in western Kenya highlands. A total of 300 larval habitats were selected in three highland villages. The habitats were first monitored for 5 weeks to collect baseline data on non‐target organisms cohabiting with mosquito larvae and then randomized into two treatment arms (respective FourStar^® and LL3) and one control arm. Non‐target organisms were sampled weekly for 5 months after treatment to assess the impact of LLML intervention. Before treatment, the mean density of all non‐target organisms combined in the control, LL3 and FourStar^® treated habitats was 1.42, 1.39 and 1.49 individuals per habitat per sampling occasion, respectively. Following treatment, this density remained fairly unchanged for 21 weeks at which time it was 1.82, 2.11, and 2.05 for the respective control, LL3 and FourStar^® treated habitats. Statistical analysis revealed that LL3 and FourStar^® did not significantly alter abundance, richness or diversity of the 11 taxa studied, when comparing the intervention and control larval habitats. However, both FourStar^® and LL3 significantly reduced the density of malaria vectors. In conclusion, one round of label rate application of FourStar^® or LL3 in natural larval habitats did not alter richness, abundance or diversity of the monitored aquatic non‐target organisms cohabiting with mosquito larvae to an ecologically significant level.     

应用氯氰菊酯和B.t.i H-14混配杀虫剂现场防治城市蚊幼虫的效果评价

采用常量喷雾或注射器滴注方法观察氯氰菊酯、B .t.iH 1 4混配杀虫剂对城市中小型蚊幼虫孳生地现场防治蚊幼虫的效果。结果表明用 2 5mg L、5 0mg L混配杀虫剂分别处理蚊幼虫孳生地 ,1天后蚊幼虫下降率均为 1 0 0 %。容器指数由处理前的 5 3 5和 5 1 6下降至零的持效期分别为 1 4和 2 1天。2 5mg L混配杀虫剂在 2 1天内的毒杀持效与单独使用 5 0mg LB t iH 1 4、1 0mg L氯氰菊酯相当 ,而5 0mg L混配杀虫剂持效比单独使用 5 0mg LB .t.iH 1 4,1 0mg L氯氰菊酯延长 7天 ,表明混配杀虫剂在现场能有效防治蚊幼虫 ,减少化学杀虫剂的用量。     

Effects of methoprene and Bti (Bacillus thuringiensis var. israelenis) on non-target insects

The effects of mosquito larvicides Bacillus thuringiensis israelensis (Bti) and methoprene, on non-target benthic invertebrates were studied in a divided pond experiment in south-central Minnesota, USA, during the spring and summer of 1989. Density and biomass of insects and other benthic macroinvertebrates were quite low in all sections of the three ponds studied, which may have been due to three consecutive years of drought, but richness appeared to be high and we observed no evidence of dominance by a particular group. We also saw no evidence of negative effects of larvicide treatment on density or biomass of any invertebrate group, nor was there a treatment-related decrease in richness of benthic invertebrate taxa under these drought conditions.     

Influence of Bacillus thuringiensis var. israelensis on oviposition of Aedes albopictus (Skuse).

The effect of Bacillus thuringiensis var israelensis (B.t.i.) on the oviposition behavior of Aedes albopictus was evaluated in the field and laboratory in Clemson, SC, U.S.A. In the field, water taken from containers in which mosquito larvae were reared (conditioned water) was placed in 16 containers. Eight containers received 50 jld of B.t.i., and eight with water only were kept as controls. In the laboratory, field-collected females of Ae. albopictus were placed in rearing chambers and provided two containers for oviposition, one with 50 microl of B.t.i and one a control with water only. Eight cage experiments were conducted, five using filtered tap water and three with conditioned water. In the field over the 13 trials, more eggs were laid in the containers with B.t.i. although no significant difference was found in the number of eggs between the treatment and controls over 72 h. In the laboratory, more eggs were laid in the containers with B.t.i. versus the controls. The containers had filtered tap water and B.t.i. had significantly more eggs laid in them compared to the controls.     

Evolution of resistance toward Bacillus sphaericus or a mixture of B. sphaericus+Cyt1A from Bacillus thuringiensis, in the mosquito, Culex quinquefasciatus (Diptera: Culicidae)

The 2362 strain of Bacillus sphaericus (Bs) Neide is a highly mosquitocidal bacterium used in commercial bacterial larvicides primarily to control mosquitoes of the genus Culex. Unfortunately, Bs is at high risk for selecting resistance in mosquito populations, because its binary toxin apparently only binds to a single receptor type on midgut microvilli. A potential key strategy for delaying resistance to insecticidal proteins is to use mixtures of toxins that act at different targets within the insect, especially mixtures that interact synergistically. We tested this hypothesis for delaying the phenotypic expression of resistance by exposing Culex quinquefasciatus Say larvae to Bs alone or in combination with Cyt1A from Bacillus thuringiensis subsp. israelensis. Two laboratory lines of Cx. quinquefasciatus, one sensitive to Bs and the other containing Bs resistance alleles, were subjected to intensive selection pressure for 20 generations with either Bs 2362 or a 3:1 mixture of Bs 2362+Cyt1A. At the end of the study, the sensitive line had evolved >1000-fold resistance when selected with Bs alone, whereas the parallel line selected with Bs+Cyt1A exhibited only low resistance toward this mixture (RR95, 1.4). Similar results were observed in the lines containing Bs resistance alleles. Both lines selected with Bs+Cyt1A exhibited substantial resistance to Bs in the absence of Cyt1A. Although selection with Bs+Cyt1A did not prevent the underlying evolution of resistance to Bs, these results suggest that a mixture of Bs with other endotoxins, particularly one like Bs+Cyt1A in which the components interact synergistically, will provide longer lasting and more effective mosquito control than Bs alone.     

Biocidal activity of Bacillus species for Anopheles larvae

It is generally accepted that Bacillus thuringiensis (B.t.) is specifically toxic to some insects but does not pose any threat to the environment, operators, or consumers. There are several other Bacillus species which can be used as effective bioinsecticides. In this study four different species of Bacillus, i.e., B. coagulans, B. megaterium, B. brevis, and B. sphaericus were isolated from soil samples collected from Kala Shah Kakoo and Kasur areas, in the suburbs of Lahore. Isolated Bacillus species were administered to mosquito larvae to evaluate their biocidal activity. B. coagulans I from Kala Shah Kakoo showed 93% mortality, while B. coagulans III from Kasur showed 70% mortality. Bacterial isolates most toxic to Anopheles larvae showed optimum growth at 37 degrees C and pH 7. These isolates have a great potency to controlling anopheline population.     

Mosquito Population Regulation and Larval Source Management in Heterogeneous Environments

An important question for mosquito population dynamics, mosquito-borne pathogen transmission and vector control is how mosquito populations are regulated. Here we develop simple models with heterogeneity in egg laying patterns and in the responses of larval populations to crowding in aquatic habitats. We use the models to evaluate how such heterogeneity affects mosquito population regulation and the effects of larval source management (LSM). We revisit the notion of a carrying capacity and show how heterogeneity changes our understanding of density dependence and the outcome of LSM. Crowding in and productivity of aquatic habitats is highly uneven unless egg-laying distributions are fine-tuned to match the distribution of habitats’ carrying capacities. LSM reduces mosquito population density linearly with coverage if adult mosquitoes avoid laying eggs in treated habitats, but quadratically if eggs are laid in treated habitats and the effort is therefore wasted (i.e., treating 50% of habitat reduces mosquito density by approximately 75%). Unsurprisingly, targeting (i.e. treating a subset of the most productive pools) gives much larger reductions for similar coverage, but with poor targeting, increasing coverage could increase adult mosquito population densities if eggs are laid in higher capacity habitats. Our analysis suggests that, in some contexts, LSM models that accounts for heterogeneity in production of adult mosquitoes provide theoretical support for pursuing mosquito-borne disease prevention through strategic and repeated application of modern larvicides.     

Mosquitocidal bacterial toxins: diversity, mode of action and resistance phenomena

Bacteria active against dipteran larvae (mosquitoes and black flies) include a wide variety of Bacillus thuringiensis and B. sphaericus strains, as well as isolates of Brevibacillus laterosporus and Clostridium bifermentans. All display different spectra and levels of activity correlated with the nature of the toxins, mainly produced during the sporulation process. This paper describes the structure and mode of action of the main mosquitocidal toxins, in relationship with their potential use in mosquito and/or black fly larvae control. Investigations with laboratory and field colonies of mosquitoes that have become highly resistant to the B. sphaericus Bin toxin have shown that several mechanisms of resistance are involved, some affecting the toxin/receptor binding step, others unknown.     

Disturbance to a stream food web by a bacterial larvicide specific to black flies: feeding responses of predatory macroinvertebrates

1. A field study was conducted to: (i) assess feeding habit changes of two predatory stoneflies following the loss of larval black fly (Diptera: Simuliidae) prey from two streams; and (ii) determine the relative importance of black fly larvae as prey for these and other selected predatory benthic macroinvertebrates. 2. Acroneuria lycorias and Paragnetina media (Plecoptera: Perlidae) diets were monitored in response to local reductions in larval black fly populations caused by Bacillus thuringiensis var. israelensis (B.t.i.) in two Michigan streams. These predators were collected from B.t.i.-treated and control sections of the streams, and their foreguts inspected for prey. 3. Black flies were the major dietary component of both predators collected from the control sections, but the number of black flies ingested was significantly less for predators collected from B.t.i.-treated habitats. Total number of prey ingested significantly decreased for A. lycorias, but not for P. media, and non-black fly prey consumption significantly increased for P. media, but not for A, lycorias, following B.t.i. applications. 4. In prey choice trials conducted in experimental channels, A. lycorias and P. media showed no preference between prey types (black flies and mayflies). Body mass gain of individual A. lycorias nymphs was measured, and was similar for nymphs in black fly-rich and black fly-poor environments. Conversely, Isoperla signata and I. dicala (Plecoptera: Perlodidae) ingested significantly more Simulium vittatum (Diptera: Simuliidae) than Baetis flavistriga (Ephemeroptera: Baetidae) or Epeorus vitrea (Heptageniidae) prey. Boyeria vinosa (Odonata: Aeshnidae) ingested significantly more B. flavistriga than S. vittatum prey. 5. Reducing black fly densities in these streams, using B.t.i., indirectly and differentially affected predators. In black fly-poor environments, feeding habits of specialist predators were most affected, and generalist predators least affected because the latter consumed alternative prey. Predator—predator and predator-prey interactions, and prey community structure may be affected indirectly by disturbances such as B.t.i. applications by reducing food resources and forcing predation onto less preferred prey.