Evaluation of an experimental product based on Bacillus thuringiensis sorovar. israelensis against Aedes aegypti larvae (Diptera:Culicidae)

The larvicidal activity of an experimental formulation of Bacillus thuringiensis israelensis (Bti) against Aedes aegypti larvae was evaluated under laboratory and simulated field conditions (SFC). Samples of technical powder (TP) were assayed to establish the LC₅₀ and the potency of the product. The larvicidal activity of the TP and the tablet (T) were evaluated under SFC to assess the efficacy and the residual activity, measured against Ae. aegypti larvae. Either a T or 250 mg of TP were added to 50 L of water in plastic containers. Containers were exposed to sunlight or kept in the shade. Results showed a LC₅₀ of 0.26 mg/L and a potency of 750 ITU/mg. In spite of differences in the toxicity amongst TP and T samples, all of them killed 98–100% of the larvae and the mortality remained high for six months, in the shade. The replacement of 20% or 60% of the water volume did not affect the activity of the product. Seasonal differences influenced the persistence of the product in containers exposed to sunlight. Both formulations showed an excellent performance, especially when kept in the shade. The Bti tablet evaluated in this study is potentially very useful in programs to control dengue vectors.     

Some effects of overcrowding on the respiration of larval Aedes aegypti

Changes in the respiratory rates of standard, overcrowded, and starved fourth-instar Aedes aegypti are compared. The respiratory effects of varying larval densities during and prior to measurement are investigated. In addition, the results of exposure of standard and over-crowded larvae to growth-retarding factors (GRF) at various concentrations are also evaluated.

---

Résumé Le but de cette étude est d'étudier les modifications de l'activité respiratoire chez des larves du 4ème stade mises respectivement en condition d'élevage standard, en condition de surpeuplement, et en condition de restriction alimentaire.Les conditions de surpeuplement et de restriction alimentaire entrainent un abaissement significatif du niveau du métabolisme respiratoire. L'activité respiratoire des larves élevées en condition standard, prend des valeurs plus élevées quand la mesure est faite sur un plus grand nombre de larves rassemblées dans le tube-test; par contre les conditions de mesure (15, 25 ou 35 larves dans le tube-test) ne modifient pas la valeur de l'activité respiratoire des larves élevées en condition de surpeuplement. Quand des larves élevées en milieu surpeuplé et en milieu standard sont soumises à de fortes concentrations des facteurs retardant la croissance (Growth-retardant factors; GRF de Moore & Fisher 1969) l'activité respiratoire est stimulée. Dans les conditions de surpeuplement et de restriction alimentaire, deux types facteurs pourraient agir sur le métabolisme respiratoire, des facteurs chimiques et des facteurs mécaniques tels que l'agitation des individus dans un espace étroit (par exemple le tube-test).

     

Development of a cost-effective medium for the large scale production of Bacillus thuringiensis var israelensis

Bacillus thuringiensis var israelensis (B.t.i.) has been widely used in mosquito control programs, but the large scale production of this bacillus is expensive because of the high cost of the medium. In this study, we attempted to develop a cost-effective medium, based on inexpensive, locally available raw materials including soybean flour (Glycine max), groundnut cake powder (Arachis hypogea), and wheat bran extract (Triticum aestivum) by using 100-L fermentor. Sporulation, toxicity, and biomass were satisfactory after B.t.i. was produced on all the three media. Use of the soybean culture medium resulted in maximum toxicity (LC₅₀ 8.89 ng/ml against Culex quinquefasciatus IIIrd instar larvae), highest spore count (0.48 × 10¹¹ c.f.u./ml), and maximum biomass (7.8 g/L) within a short fermentation time of 24 h. Hence, this soybean-based culture medium was considered most economical for the large scale industrial production of B.t.i.     

Sensitivity to plating of Escherichia coli cells expressing the cryA gene from Bacillus thuringiensis var. israelensis

Summary The gene (cytA) coding for the 27 kDa polypeptide of the Bacillus thuringiensis var. israelensis mosquito larvicidal -endotoxin, was cloned into a plasmid containing the T7 bacteriophage promoter. The plasmid was used to transform an Escherichia coli strain containing the T7 RNA polymerase gene 1, under the control of lacP. Loss of colony-forming ability without substantial lysis, associated with immediate inhibition of DNA synthesis, was observed after induction of transformed cells. The cytA gene product may kill E. colicells by disrupting their chromosome replicating apparatus.     

Comparative histopathology of some Diptera and Crustacea of aquatic alpine ecosystems, after treatment with Bacillus thuringiensis var. israelensis

Species of Diptera (larval Culicidae, Chironomidae and Simuliidae) and Crustacea (cladoceran adults) that are representative of the fauna of aquatic alpine ecosystems are routinely treated with Bacillus thuringiensis var. israelensis (Bti) for mosquito control. The toxicity of Bti on these organisms was investigated. Bioassays indicated that Bti used at the concentration for operational field application is deleterious to all dipteran species, but not to Cladocera. Histopathological observations reveal that, in all cases, the midgut epithelium is affected by Bti treatment. However, the vulnerability of epithelial cells to the microbial insecticide is different from one cell-type to another, clear cells being more vulnerable than dark cells. Because of the concentration of clear cells within the anterior midgut of dipteran larvae, this region appears to be the first to show symptoms of intoxication through enhancing a natural process of cell turnover. The relative importance of such a vulnerable region in the midgut of dipteran larvae may account for the general efficacy of Bti to these species. Its harmlessness to Cladocera may be correlated to the relative scarcity of clear cells and their patchy distribution along the whole midgut together with important cellular renewal capacity of the epithelium.     

Identification and characterization of Aedes aegypti aminopeptidase N as a putative receptor of Bacillus thuringiensis Cry11A toxin

Bacillus thuringiensis subsp. israelensis, which is used worldwide to control Aedes aegypti larvae, produces Cry11Aa and other toxins during sporulation. In this study, pull-down assays were performed using biotinylated Cry11Aa toxin and solubilized brush border membrane vesicles prepared from midguts of Aedes larvae. Three of the eluted proteins were identified as aminopeptidease N (APN), one of which was a 140 kDa protein, named AaeAPN1 (AAEL012778 in VectorBase). This protein localizes to the apical side of posterior midgut epithelial cells of larva. The full-length AaeAPN1 was cloned and expressed in Eschericia coli and in Sf21 cells. AaeAPN1 protein expressed in Sf21 cells was enzymatically active, had a GPI-anchor but did not bind Cry11Aa. A truncated AaeAPN1, however, binds Cry11Aa with high affinity, and also Cry11Ba but with lower affinity. BBMV but not Sf21 expressed AaeAPN1 can be detected by wheat germ agglutinin suggesting the native but Sf21 cell-expressed APN1 contains N-acetylglucosamine moieties.     

Structural features of crystal-forming proteins produced by Bacillus thuringiensis subspecies israelensis

Entomocidal crystals produced by Bacillus thuringiensis ssp. israelensis are formed by two proteins with molecular masses of 130 and 28 kDa, whereas the protein with a molecular mass of 70 kDa appears as a result of 130 kDa protein limited proteolysis by admixtures of bacterial proteinases in the course of its dissolution. The comparison of the N-terminal sequences of the protein with molecular mass of 70 kDa (Met-Glu-Asn-Xaa-Pro-Leu-Asp-Thr-Leu-Ser-Ile-Val-Asn-Glu-Thr-Asp) and that of 28 kDa (Met-Glu-Asn-Leu-Asn-[Phe]-[Trp]-Pro-Leu-Gln-Asp-Ile-Lys-Val-Asn-Pro) reveals only marginal similarity between them (only 4 identical residues among 16 aligned). Both B. thuringiensis israelensis crystal-forming proteins appear hardly related to those contained in the crystal produced by other B. thuringiensis subspecies, e.g. kurstaki. It might be concluded that at least some of the entomocidal proteins found in the crystalline inclusion bodies of various B. thuringiensis subspecies revealed rather strong variations in their primary structures that facilitate their adaptation to different hosts.Bacillus thuringiensis ssp. israelensis δ-EndotoxinEntomocidal crystalInsecticideMosquito     

The effects of various oxygen tensions on embryogeny and larval responses of Aedes aegypti

Eggs of Aedes aegypti (L.) were submerged in water containing dissolved oxygen at levels ranging from less than 1 to 14 parts per million, at 1, 24, 48, 72 and 96 hours after being laid. After a 4.5 day exposure period, which encompasses the normal period of embryogeny, the eggs were subjected to the hatching stimulus as a measure of maturity.The whole of embryogeny occurred at a normal rate under levels of 3.8 to 14 ppm dissolved oxygen. An oxygen level of 0.95 ppm was lethal to all eggs except those exposed only in the advanced stages of development. A level of 1.9 ppm dissolved oxygen caused a retardation of developmental rate, with 6.5 days being required to achieve maturation.Immature, but advanced, embryos could be hatched artificially, with completion of development to normal adults.

---

Die wirkungen unterschiedlicher sauerstoffspannungen auf die embryogenese und larvalreaktionen von Aedes aegypti

Zusammenfassung Eier von Aedes aegypti wurden 1, 24, 48, 72 und 96 Stunden nach der Ablage in Wasser getaucht, das gelösten Sauerstoff in Mengen von weniger als 1 bis 14 Teilen pro Million enthielt. Nach einer Behandlungszeit von 4,5 Tagen, die dem normalen Zeitraum der Embryonalentwicklung entspricht, wurden die Eier als Maß ihrer Reife dem Schlüpfreiz unterworfen.Die gesamte Embryonalentwicklung verlief bei Sauerstoffspannungen von 3,8 bis 14 ppm in normalem Ausmaß. Eine Sauerstoffspannung von 0,95 ppm war für alle Eier lethal mit Ausnahme derjenigen, die ihr erst in fortgeschrittenen Entwicklungsstadien ausgesetzt wurden. Eine Menge von 1,9 ppm gelösten Sauerstoffs verursachte eine Verzögerung der Entwicklungsgeschwindigkeit, bei der 6,5 Tage zur Erreichung der Schlüpfreife benötigt wurden.Unreife, aber fortgeschrittene Embryonen konnten künstlich zum Schlüpfen gebracht werden, bei vollständiger Weiterentwicklung zu normalen Imagines.

     

Lecithin in synthetic larval diet for Aedes aegypti improves larval and adult performance

A chemically defined synthetic rearing medium was used to compare larval growth of Aedes aegypti with or without crude animal lecithin or synthetic dipalmitoyl lecithin. Pupal weights, adult female life spans and oviposition histories of pupae and adults derived from synthetic diet rearings and from crude culture (liver powder) rearings were also compared. Both lecithins improved larval growth rate; optimal concentrations reduced time to pupation by 2 and 1 days for animal and synthetic lecithins respectively, and animal lecithin was better tolerated by the mosquito larvae at the higher concentrations tested. Addition of crude animal lecithin to the basal synthetic rearing medium had little effect on the weights of male and female pupae, but it increased adult female life spans and improved weekly egg production. In no case did mosquitoes from axenic rearings in synthetic media deviate greatly from limits specified by earlier workers for field-derived mosquitoes.

---

Résumé Les développements larvaires d'Aedes aegypti ont été comparés sur des substrats alimentaires avec ou sans lecithine animale brute ou DL-a-dipalmitoyle lecithine synthétique.Les comparaisons ont porté aussi sur les poids nymphaux, la longé-vité et la fécondité de femelles élevées sur régime synthétique ou à partir de poudre de foie.Les deux types de lecithines améliorent le développement larvaire; les concentrations optimales des deux régimes (lecithine animale et lecithine synthétique) avancent respectivement la pupaison de 2 et 1 jour; les lecithines animale et synthétique sont tolérées par les larves respectivement jusqu'aux concentrations de 0,008 et 0,006%.L'addition de lecithine animale brute au régime contenant de la lecithine synthétique a eu peu d'effets sur les poids des nymphes mâles et femelles mais a augmenté la longévité et la fécondité hebdomadaire des femelles.Les performances des moustiques élevés sur substrat aseptique synthétique n'ont jamais été très éloignées des limites indiquées antérieurement par les travaux sur moustiques provenant de la nature.

     

Refined, Circular Restriction Map of the Bacillus thuringiensis subsp. israelensis Plasmid Carrying the Mosquito Larvicidal Genes

All the genetic elements responsible for the mosquito larval toxicity of Bacillus thuringiensis subsp. israelensis are located on one of its largest plasmids, nicknamed pBtoxis. Two linkage groups (with sizes of about 75 and 55 kb) have previously been mapped partially with respect to SacI and BamHI restriction sites (Ben-Dov et al., 1996), but linking them to a single circular plasmid unambiguously was impossible with the available data. To finalize the plasmid map, another rare cutting restriction endonuclease, AlwNI, was used in addition. The two linkage groups and the fragments generated by AlwNI were aligned on the circular plasmid, and known insertion sequences were localized on the refined map. Pulsed-field electrophoresis revealed that the total size of pBtoxis (137 kb) was larger than thought before.     

Application of different downstream processing methods and their comparison for the large-scale preparation of Bacillus thuringiensis var. israelensis after fermentation for mosquito control

Bacillus thuringiensis var. israelensis, a gram positive, spore-forming bacillus, produces parasporal crystal protein during sporulation, which is toxic in the mosquito larvae gut. An efficient downstream processing method for separating the spore crystal complex (SCC) from the fermented broth of B. thuringiensis var. israelensis is required to achieve maximum mosquitocidal activity. The different downstream processing methods, viz., tangential flow ultra-filtration, continuous centrifugation and acid precipitation were compared for their efficiency in separating SCC from broth obtained from a pilot-scale fermentor (100 l capacity). Among the three downstream processing methods, tangential flow ultra-filtration yielded the maximum amount of biomass (53.3 g/l), maximum number of spores (2.30 × 10¹⁸ CFU/ml) and highest level of larvicidal activity (LC₅₀ 28 nl/ml) against Aedes aegypti Bora-Bora strain followed by continuous centrifugation and acid precipitation methods.     

苏云金杆菌以色列亚种杀蚊蛋白Cyt1Aa在离中不粘柄菌中的表达

在蚊幼虫生活水域里的离中不粘柄菌(Asticcacaulis excentricus,Ae)中已成功表达苏云金芽孢杆菌以色列亚种(Bacillus thuringiensis subsp.israelensis,Bti)杀蚊蛋白基因cry11Aa的基础上,将另一Bti杀蚊蛋白基因cyt1Aa转化入Ae中表达。构建并转化了分别单独含有cyt1Aa基因、及同时含有cry11Aa基因的表达质粒pSODCyt20和pSODCryCyt20,蛋白免疫杂交检测相应的Ae重组子分别表达产生了Cyt1Aa和Cry11Aa蛋白。为了探究Ae(pSODCryCyt20)重组子不能表达cyt1Aa的原因,提取了重组子总RNA、并与同是革兰氏染色阴性的大肠杆菌的总RNA比较,结果显示两者RNA系统显著不同,推测Ae中多个外源基因的表达,可能要求每个基因必需一个启动子。     

Synergism in mosquitocidal activity of 26 and 65 kDa proteins from Bacillus thuringiensis subsp. israelensis crystal

Three major protein components, 130, 65 and 26 kDa of solubilized Bacillus thuringiensis subsp. israelensis crystal were separated by gel filtration. The isolated 26 kDa protein is inactive against mosquito larvae at 6.4 μg/ml protein. The 65 kDa protein is also inactive towards mosquito larvae at low concentrations and shows but weak activity at higher concentrations. However, 26 and 65 kDa proteins simultaneously present at low concentrations exhibit high mosquitocidal activity. Similar synergistic effects are observed between 26 and 130 kDa proteins. These results differ from the findings of other investigators who have reported that a single protein component is sufficient for mosquitocidal activity [(1984) FEBS Lett. 175, 377-382; (1985) Biochem. Biophys. Res. Commun. 126, 961-965]. Our data suggest that the simultaneous presence of at least two proteins is required for activity.

Synergism Mosquitocidal activity Crystal protein     

Iron loaded ferritin secretion and inhibition by CI-976 in Aedes aegypti larval cells

Ferritin is a multimer of 24 subunits of heavy and light chains. In mammals, iron taken into cells is stored in ferritin or incorporated into iron-containing proteins. Very little ferritin is found circulating in mammalian serum; most is retained in the cytoplasm. Female mosquitoes, such as Aedes aegypti (yellow fever mosquito, Diptera), require a blood meal for oogenesis. Mosquitoes receive a potentially toxic level of iron in the blood meal which must be processed and stored. We demonstrate by ⁵⁹Fe pulse-chase experiments that cultured A. aegypti larval CCL-125 cells take up iron from culture media and store it in ferritin found mainly in the membrane fraction and secrete iron-loaded ferritin. We observe that in these larval cells ferritin co-localizes with ceramide-containing membranes in the absence of iron. With iron treatment, ferritin is found associated with ceramide-containing membranes as well as in cytoplasmic non-ceramide vesicles. Treatment of CCL-125 cells with iron and CI-976, an inhibitor of lysophospholipid acyl transferases, disrupts ferritin secretion with a concomitant decrease in cell viability. Interfering with ferritin secretion may limit the ability of mosquitoes to adjust to the high iron load of the blood meal and decrease iron delivery to the ovaries reducing egg numbers.     

ERIC-PCR鉴别苏云金芽胞杆菌与蜡状芽胞杆菌的研究

利用ERIC-PCR技术对苏云金芽孢杆菌(Bt)、蜡状芽孢杆菌(Bc)和对照菌基因组DNA进行扩增,回收、标记BtPCR扩增片段,分别与各菌株的基因组DNA进行斑点杂交和Southern杂交,筛选Bt标识序列。结果显示:Bt各菌株均可扩增得到250bp的特异片段;Bt和Bc均可得到600bp的共有扩增片段;以筛选得到的569bp片段为探针,可特异性地与Bt基因组DNA杂交;ERIC-PCR技术可以在DNA指纹图谱水平区分鉴别Bt与Bc菌,正确反映出两者的亲缘关系。结果表明ERIC-PCR技术在Bt的检测及在Bt与Bc的鉴定中具有较强的实用性。     

Chemical composition and larvicidal effects of essential oil of Dendropanax morbifera against Aedes aegypti L.

Essential oils obtained from the flowers of Dendropanax morbifera were extracted and the chemical composition and larvicidal effects were studied. The analyses were conducted by gas chromatography and mass spectroscopy (GC–MS) revealed that the essential oil of D. morbifera contained 27 compounds. The major chemical components identified were γ-elemene (18.59%), tetramethyltricyclohydrocarbon (10.82%), β-selinene (10.41%), α-zingibirene (10.52%), 2-isopropyl-5-methylbicylodecen (4.2%), β-cubebene (4.19), and 2,6-bis(1,1-Dimethylethyl)-4-phenol (4.01%). The essential oil had a significant toxic effect against early fourth-stage larvae of Aedes aegypti L. with an LC₅₀ value of 62.32 ppm and an LC₉₀ value of 131.21 ppm. The results could be useful in search for newer, safer, and more effective natural larvicidal agents against A. aegypti.     

Suppression of RNA interference increases alphavirus replication and virus-associated mortality in Aedes aegypti mosquitoes

Background  

Arthropod-borne viruses (arboviruses) can persistently infect and cause limited damage to mosquito vectors. RNA interference (RNAi) is a mosquito antiviral response important in restricting RNA virus replication and has been shown to be active against some arboviruses. The goal of this study was to use a recombinant Sindbis virus (SINV; family Togaviridae; genus Alphavirus) that expresses B2 protein of Flock House virus (FHV; family Nodaviridae; genus Alphanodavirus), a protein that inhibits RNAi, to determine the effects of linking arbovirus infection with RNAi inhibition.     

Effects of AeDNV infection on Aedes aegypti lifespan and reproduction

The effects of Aedes Densovirus (AeDNV) infections on survival, fertility, fecundity and vertical transmission in Aedes aegypti (Diptera: Culicidae) were measured in laboratories in Kiev, Ukraine and Colorado, USA and incorporated into a predictive model of the effects of AeDNV on vector capacity. Adult lifespan and daily survival were reduced in AeDNV infected mosquitoes. This effect was dependent on the dose of the virus. Infected females had decreased fecundity. The oviposition rate was less in infected females and the hatch rate declined in eggs laid by infected females. The amounts of AeDNV in infected females and the infection rate of their offspring were measured with real-time PCR. The average filial transmission rate was 70% and larval infection rates from infected females varied between 42 and 62%. Vertically infected larvae, and individual eggs contained 1 × 10⁵ AeDNV genome equivalents (geq). Modeling the effects of AeDNV infection on Ae. aegypti populations suggested a large decrease in the numbers of eggs, larvae, pupae, and adults arising from infected mothers and suggested that AeDNV treatment of larvae could cause up to a 76% reduction of infectious mosquito days.     

Vitelline envelope genes of the yellow fever mosquito, Aedes aegypti

Vitelline envelope genes from the mosquito Aedes aegypti were analyzed with respect to their DNA sequences, genomic representation, temporal and spatial expression profiles and response to 20-hydroxyecdysone. Genomic clones of three vitelline envelope genes, 15a-1, 15a-2 and 15a-3 were isolated. Southern analysis indicates that all three genes are represented by a single copy in the genome. The deduced amino acid sequences of all three vitelline envelope genes contain a conserved region of 46 residues that overlaps with a region that is conserved in four Drosophila melanogaster vitelline envelope genes. DNA was sequenced flanking the 15a-1, 15a-2 and 15a-3 coding regions. A 360 bp sequence 5′ of the 15a-2 coding region was identified with 72% identity to a sequence upstream of the Ae. aegypti VgA1 vitellogenin gene. The temporal patterns of 15a-1, 15a-2 and 15a-3 expression, as determined by Northern analysis, were similar. The spatial patterns of expression, as determined by whole-mount in situ hybridization, differed between the three genes. 15a-1 and 15a-3 were only expressed in the middle and posterior regions of the follicle, while 15a-2 was also expressed at the anterior region. Vitelline envelope gene expression was higher in ovaries that were dissected at 0, 2 and 10 h following a blood meal and then incubated in vitro for 10 h in medium containing 10⁻⁵ M 20-hydroxyecdysone, compared to ovaries that were incubated without hormone.