Wolbachia infection in West Nile Virus vectors of northwest Iran

Mosquito-borne viral diseases are serious health problems in many countries. Various methods have been used for controlling the vectors of these diseases. Among symbiotic bacteria, the members of the genus Wolbachia are the most ubiquitous symbionts of arthropods and play key roles in their host biological characteristics with various effects on their hosts. The identification of these bacteria in Iranian mosquitoes is limited to a few studies. The current study was carried out to determine (1) the Wolbachia infection of probable arbovirus vectors (Aedes caspius, Culex pipiens, Culex theileri and Culiseta longiareolata), (2) the Wolbachia strain(s) infecting the mosquitoes, and (3) the geographical distribution of the Wolbachia strain(s) in the northwest of Iran. Eight species including Ae. caspius, Anopheles hyrcanus, An. maculipennis, Cx. hortensis, Cx. modestus, Cx. pipiens, Cx. theileri, and Cs. longiareolata were identified, amongst which Ae. caspius with 63.1% and An. hyrcanus with 0.3% were the most and the least abundant species, respectively. The results of semi-nested PCR using Wolbachia surface protein (wsp) fragment assays showed that Wolbachia infection was present in three out of the four above mentioned arboviral vector species (Aedes caspius, Culex pipiens, Culex theileri and Culiseta longiareolata), where the highest infection rate was seen in Cx. pipiens. The infection rates of mosquitoes with Wolbachia in the species of Cx. pipiens, Cs. longiareolata, Cx. theileri, and Ae. caspius were 96.9%, 11.5%, 5.2% and 0%, respectively.

     

Activité comparée de 22 variétés deBacillus thuringiensis sur 3 espèces deCulicidae

Résumé Le pouvoir larvicide des cultures totales de 22 variétés deBacillus thuringiensis Berliner représentant 15 sérotypes H a été testé sur larves L4 d'Aedes aegypti (L.)Culex pipiens pipiens (L.) etAnopheles stephensi (Liston). Seul le sérotype H 14, variétéisraelensis, est réellement actif, provoquant 100% de mortalité à la dilution 10⁻⁵. Avec des doses beaucoup plus fortes, 10⁻², une certaine toxicité peut être manifestée par les variétésentomocidus, galleriae etkyushuensis en ce qui concerneAe. aegypti etC. pipiens pipiens, ou par les variétésentomocidus, tolworthi, kyushuensis etaizawai, pourAn. stephensi. Cependant cette activité n'a rien de comparable avec celle de la variétéisraelensis.

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Summary We have studied the 15 H serotypes ofBacillus thuringiensis Berliner including 22 varieties. The larvicidal potency of the whole cultures of these varieties is evaluated on 4th instar larvae ofAedes aegypti (L.),Culex pipiens pipiens (L.) andAnopheles stephensi (Liston). The H-14 serotype, varietyisraelensis is the only one to show a true toxicity at 10⁻⁵ dilution on larvae of the 3 mosquito species. A low mortality at 10⁻² dilution is observed onAe. aegypti andCx. pipiens pipiens larvae withentomocidus, galleriae andkyushuensis varieties; onAn. stephensi withentomocidus, tolworthi, kyushuensis andaizawa? varieties. Nevertheless, this activity cannot be compared to the extremely high toxicity of theisraelensis variety.

     

Cloning and characterization of an insecticidal crystal protein gene from Bacillus thuringiensis subspecies kenyae

A sporulating culture ofBacillus thuringiensis subsp.kenyae strain HD549 is toxic to larvae of lepidopteran insect species such asSpodoptera litura, Helicoverpa armigera andPhthorimaea operculella, and a dipteran insect,Culex fatigans. A 1.9-kb DNA fragment, PCR-amplified from HD549 using cryII-gene-specific primers, was cloned and expressed inE. coli. The recombinant protein produced 92% mortality in first-instar larvae ofSpodoptera litura and 86% inhibition of adult emergence inPhthorimaea operculella, but showed very low toxicity againstHelicoverpa armigera, and lower mortality against third-instar larvae of dipteran insectsCulex fatigans, Anopheles stephensi andAedes aegypti. The sequence of the cloned crystal protein gene showed almost complete homology with a mosquitocidal toxin gene fromBacillus thuringiensis var.kurstaki, with only five mutations scattered in different regions. Amino acid alignment with different insecticidal crystal proteins using the MUTALIN program suggested presence of the conserved block 3 region in the sequence of this protein. A mutation in codon 409 of this gene that changes a highly conserved phenylalanine residue to serine lies in this block.     

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

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

Growth,sporulation and toxin production byBacillus thuringiensis subsp.israelensis andB. sphaericus in media based on mustard-seed meal

Bacillus thuringiensis subsp.israelensis andB. sphaericus strains 2362 and 1593 were grown in media based on defatted mustard-seed meal (MSM). The meal contains 40% (w/w) protein, with glutamic acid and arginine as the major amino acids. The toxic potencies of the final bacterial powders towardsCulex pipens quinquefasciatus Say, compared with those of the respective international reference standards, were 46% forB. thuringiensis subsp.israelensis, 62% forB. sphaericus 2362 and 88% forB. sphaericus 1593 when 2% (w/v) MSM was used for growth. With 4% (w/v) MSM,B. thuringiensis subsp.israelensis grew better but had undetectable larvicidal activity, whereas theB. sphaericus strains not only grew better but gave a higher degree of sporulation and toxicity. The potencies ofB. sphaericus in medium with 4% MSM were comparable with those of international reference standards.The authors are with the Department of Life Sciences, University of Bombay, Bombay 400 098, India.     

Screening of the insecticidal activity ofBacillus thuringiensis strains against the egyptian cotton leaf wormSpodoptera littoralis

A screening of the larvicidal activity of the more than 900 strains ofBacillus thuringiensis strains, combining the Institut Pasteur collection was realized. A quick bioassay using 1st instar larvae and semi-synthetic medium was developed. Many strains were toxic toSpodoptera littoralis, but only a few belonging mainly to serovarsaizawai, kenyae andentomocidus showed high level of toxicity. The profiles of strain activities differed from serovar to serovar, but within the same serovar toxicity can vary with different strains. Oneaizawai strain tested in the field gave satisfactory results, better than a commercially used strain, tested in the same experiment.

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Résumé Le criblage de notre collection deB. thuringiensis (plus de 900 souches) a été effectué contreSpodoptera littoralis. Une technique rapide de bioessai utilisant des chenilles néonates et un milieu semisynthétique a été mise au point. Beaucoup de souches se sont montrées actives à forte dose, mais seulement quelques-unes appartenant principalement aux sérovaraizawai etkenyae à doses plus faibles. Les différents sérotypes ont un profil d'activité différent mais un même sérotype peut comprendre des souches de toxicité variée. Une souche du sérovaraizawai a été testée en champs et a donné de bons résultats, son activité ayant été évaluée à environ 5 fois celle d'une souche couramment commercialisée.

     

The identification ofBacillus thuringiensis

Résumé Deux nouvelles méthodes, analyses d'antigènes H et d'estérases, ont été récemment ajoutées aux tests biochimiques pour l'identification deBacillus thuringiensis. Certains des tests biochimiques se sont montrés valables, d'autres non. Deux études utilisant les antigènes H concordent dans la reconnaissance de 9 groupes qui correspondent étroitement à la classification basée sur les estérases. En utilisant les 3 méthodes, douze types bactériens peuvent être distingués, certains à distribution géographique restreinte. Des antisérums standards pour diagnostics devraient être rendus disponibles à tous les chercheurs intéressés. Des problèmes liés à l'identification sont discutés. La parenté des groupes de bactériesB. cereus etB. thuringiensis est étroite mais une connaissance plus approfondie est nécessaire pour résoudre la question de la nomenclature.

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This paper was presented at the ?International Symposium on the identification and assay of viruses and Bacillus thuringiensisused for insect control?, London 13 th July 1964.     

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.     

Sensibilité desCulicidae a la β-exotoxine deBacillus thuringiensis

Résumé Cette étude vise à élargir les connaissances du potentiel toxique de la β-exotoxine deB. thuringiensis Berliner sur lesCulicidae et particulièrement surAedes aegypti (L.),Anopheles stephensi (Liston) etCulex pipiens (L.). La toxicité par ingestion de la β-exotoxine deB. thuringiensis H 1 à 1 mg/ml est totale, sur les adultes males et femelles. La sensibilité des stades larvaires est proportionnelle à la concentration. La descendance des adultes, issus de larves traitées à une dose sublétale, présente une sensibilité accrue à l'exotoxine. La β-exotoxine induit un effet retard sur les mues larvaires ainsi que des effets tératogènes à la nymphose.

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Summary The β-exotoxine ofB. thuringiensis H 1 acts as a larvicide and as an adulticide when ingested at high concentrations byAedes aegypti L.,Anopheles stephensi Liston orCulex pipiens L. Sublethal concentrations of β-exotoxin induce a delay of larval moulting and teratological effects on larvae and pupae. After sublethal application of β-exotoxine to larval stage the next larval generation presents an enhanced sensibility to this toxin.

     

Isolation of novel Bacillus species showing high mosquitocidal activity against several mosquito species

Two novel mosquitocidal bacteria, VB17 and VB24, identified as new Bacillus species were isolated from dead mosquito larvae obtained in Florida aquatic habitats. Gas chromatographic analysis of fatty acid methyl esters (GC-FAME) and 16S rRNA sequencing indicated that VB24 is closely related to Bacillus sphaericus whereas VB17 does not have a close relationship with either Bacillus thuringiensis or B. sphaericus. Both isolates were significantly more active than B. sphaericus 2362 against Aedes taeniorhynchus, Anopheles quadrimaculatus, Culex quinquefasciatus larvae, and as active as B. sphaericus 2362 against Anopheles gambiae. Interestingly, however, both were not active against Aedes aegypti larvae, indicating some level of insecticidal specificity.     

Response of the Carrot Weevil,Listronotus oregonensis(Coleoptera: Curculionidae), to Strains ofBacillus thuringiensis

Mortality and frass production bioassays were used to investigate the toxicity of seven strains ofBacillus thuringiensisagainst the adult carrot weevil,Listronotus oregonensis(Le Conte). A semi-artificial diet of carrot foliage with 4% agar was selected to maximize feeding by the insects.Bacillus thuringiensissubsp.tenebrionis(Krieg, Huger, Langenbruch, and Schnetter) (BTT) and two unidentifiedB. thuringiensisstrains, A30 and A429, gave the lowest LC₅₀values. The frass bioassay supported the conclusions of the mortality assay. Mortality of adults continued after their removal from the insecticidal medium, with the highest mortality being caused by strains A429 and BTT. Survivors from the frass bioassay, initially exposed to strains A30, A429, and BTT, did not resume normal levels of feeding after their removal from the insecticidal medium.     

Studies on the effect ofbacillus thuringiensis Berl. on the spiny bollworm,Earias insulana Boisd. and other lepidopterous insects

Résumé Bacillus thuringiensis Berl. tue 50 % des chenilles d'Earias insulana après 72 heures. Le traitement est appliqué avec une suspension de 4 g par litre d'eau (à raison de 30 milliards de spores par gramme). Dans les mêmes conditions, la mortalité atteint 100 % pour le ver à soie. Une mortalité plus élevée est constatée lors des applications sur chenilles jeunes. La poudre de larves mortes après infection parB. thuringiensis détermine une mortalité faible, tandis que les chenilles nourries de feuilles de muriers préalablement traitées par une suspension d'excrétats de vers à soie infectés deBacillus thuringiensis, ne sont pas tuées. Dans les champs de coton, leBacillus détermine une réduction considérable de l'infestation parEarias mais cette réduction n'atteint pas celle obtenue par traitement chimique. LeBacillus est sans effet sur la population d'Aleurodes et sur celle des Acariens (Tetranychus atlanticus Mcgregor) contrairement à l'endrine qui favorise la pullulation de ces derniers.      

The pathogenic fungi of the spittlebug in Mexico. III. Innocuity ofErynia neoaphidis andConidiobolus major in experimental animals

Previous research concerning theEntomophthorales has demonstrated their capabilities as biological control agents. To use them widely and at industrial levels, studies to demonstrate their innocuity in animals must be made. The present study concerns the intraperitoneal and subcutaneous inoculation of 274 animals (mice and guinea pigs) withErynia neoaphidis Remaudière & Hennebert andConidiobolus major (Thaxter) Remaudière & Keller, fungi pathogenic for the spittlebug [Aeneolamia postica (Walk) andProsapia simulans (Walk)] in Mexico. The alterations found were macroscopic tissue lesions consisting of nodules, abscesses and adhesions in the liver, spleen, kidneys and intraperitoneal cavity. No fungi could be recovered from organs at any time and only lysed filaments surrounded by polymorphonuclear cells could be seen at day 8 post-inoculation. The histopathological findings showed no important pathological alterations. These results suggest the innocuity ofErynia neoaphidis Remaudière & Hennebert andConidiobolus mamor (Thaxter) Remaudière & Keller in mice and guinea pigs.

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Résumé L'utilisation des Entomophthorales dans la lutte biologique contre les insectes n'est possible qu'après avoir vérifié leur innocuité vis-à-vis des vertébrés. Dans ce but, des injections sous-cutanées et intrapéritonéales ont été réalisées chez 274 animaux (cobayes et souris) à l'aide de mycélium et spores deErynia neoaphidis etConidiobolus major pathogènes de la “mosca pinta”. Les seules lésions tissulaires observées sont des nodules, des abcès et des adhérences au niveau de la rate, du foie, des reins et du péritoine. Les champignons n'ont jamais pu être réisolés à partir des divers organes. Les observations histopathologiques ne montrent aucune altération pathologique importante; seuls des filaments lysés entourés de polymorphonucléaires ont pu être retrouvés. Ces résultats indiquent l'innocuité deE. neoaphidis etC. major vis-à-vis des souris et des cobayes.

     

Host specificity of the two lace bugs,Dictyla echii andDictyla nassata [Hem.: Tingidae] potential biological control agents for paterson's curse (Echium plantagineum)

Two species of lace bugs,Dictyla echii (Schrank) andDictyla nassata (Puton), were tested for host specificity as potential biological control agents for Paterson's curse,Echium plantagineum L. A range of test plants from the family Boraginacea, from related families in the order Laminales and from species of economic importance in Australia which have had inadequate exposure in their countries of origin, were tested under both choice and non-choice conditions. These bugs proved to be oligophagous within the Boraginaceae and attacked a number of Australian native species. Because of this,D. echii andD. nassata are not considered sufficiently specific to release from quarantine and tests were abandoned before completion of the test plant list.

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Résumé La spécificité parasitaire deDictyla echii (Schrank) etD. nassata (Puton) a été étudiée en tant qu'agents de lutte biologique potentiels contreEchium plantagineum L. Dans les conditions de choix et d'absence de choix, une série de plantes ont été testées provenant de la famille des Boraginacées, des familles voisines de l'ordre des Laminales et d'espèces d'importance économique en Australie qui n'avaient pas fait l'objet de tests suffisants dans leur pays d'origine. Ces punaises se révèlent oligophages en ce qui concerne les Boraginacées et s'attaquent à de nombreuses espèces indigènes d'Australie. Pour cette raison,D. echii etD. nassata ne sont pas considérées comme suffisamment spécifiques pour les relacher à partir de la quarantaine et les tests ont été abandonnés avant d'épuiser la totalité des plantes prévues sur la liste.

     

Interpreting the SDS-PAGE protein patterns with self-organizing maps: application for the characterization of mosquito-pathogenic Bacillus strains

Aims: To present the pairwise comparison of potential mosquito‐pathogenic Bacillus strains based on their SDS‐PAGE protein patterns and to evaluate their characteristic toxicity patterns. Methods and Results: In this work, 20 Bacillus strains were subjected to qualitative toxicity tests against Aedes aegypti and Culex quinquefasciatus larvae. The selected strains were then characterized by SDS‐PAGE protein profiles. The highly heterogeneous multiple protein components of protein patterns were analysed using self‐organizing map (SOM), a ‘visualization and clustering’ tool. Members of mosquitocidal Bacillus species were classified in four distinct clusters, and then toxicity patterns were examined. Cluster (1, 1) comprised of three highly toxic strains of Bacillus sphaericus: SPH88, 1593 and KSD‐4; cluster (1, 2) consisted of two B. sphaericus strains: SSII‐1 and Bsp‐R that showed weak larvicidal activity; cluster (2, 1) constituted two B. sphaericus strains: WHO2297 and ISPC‐5 that possessed moderate toxicity; and cluster (2, 2) contained four B. thuringiensis ssp. israelensis strains: ONR‐60A, HD500, IPS70 and IPS82 belonging to serotype H14 but exhibited moderate to high mosquito larvicidal toxicity. Conclusions: SOM served as a colour‐coded alternate for easy visualization of similarities or dissimilarities between the strains even at the infra subspecies level. Furthermore, characteristic toxicity patterns of Bacillus strains of different clusters were determined. Significance and Impact of the Study: Analysis of electrophoretic protein patterns using SOM provides a better insight into the inter‐relationships of bacterial strains through similarity‐based clustering and pairwise comparison of two strains.     

The functional response and preference ofMacrolophus caliginosus [Heteroptera: Miridae] for two of its prey:Myzus persicae andTetranychus urticae

Functional response studies of females ofMacrolophus caliginosus Wagner for 2 prey speciesMyzus persicae Sulzer andTetranychus urticae Koch showedHolling's type II response (1959). Estimations of handling time (Th) and attack rate (a) obtained for the aphid (Th=.055 day; a=1.035) and for the mite (Th=.025 day; a=.947) enabled the development of a mathematical model which describes the predation rate on the 2 prey simultaneously. This mirid shows a sharp preference for aphids over mites.

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Résumé L'étude en laboratoire des réponses fonctionnelles des femelles adultes deMacrolophus caliginosus Wagner vis-à-vis des deux proiesMyzus persicae Sulzer etTetranychus urticae Koch montre dans les 2 cas une réponse de type II deHolling (1959). L'estimation du temps de manipulation (Th) et du taux d'attaque instantané (a) pour l'aphide (Th=.055 jour; a=1.035) et pour l'acarien (Th=.025 jour; a=.947) permet d'établir un modèle mathématique décrivant la prédation sur les 2 proies présentes simultanément et de préciser ainsi, au regard des résultats expérimentaux, l'existence d'une préférence du miride pour le puceronM. persicae.

     

Preparation of a flowable liquid bacterial insecticide based on Bacillus sphaericus

Fermenter-produced Bacillus sphaericus 2362 was formulated into a thick, dark flowable liquid concentrate containing 4.8×10⁹ c.f.u./ml and charcoal as protector against ultraviolet light. The potencies of the formulation against L₄ Culex pipiens quinquefasciatus before and after storage for 2 years were 5714 and 5862 International Toxic Units (ITU), respectively, when compared with a standardized B. sphaericus from the WHO at 1000 ITU. In field trials, treatment at 1.01/ha gave 96 to 100% control of mosquito larvae. B. sphaericus could be re-isolated in 5% of the samples 9 months after application.The authors are at the Department of Applied Microbiology & Brewing, Anambra State University of Technology, P.M.B. 5025, Awka, Nigeria.     

Investigation of the cyt gene in Bacillus thuringiensis and the biological activities of Bt isolates from the soil of China

The presence of cyt genes was investigated in 80 type strains of Bacillus thuringiensis and 143 isolates obtained from soil samples of China by PCR amplification using two pairs of primers for the cyt1 and cyt2 genes. Three type strains of serotypes H11ac, H14 and H36, eight isolates belonging to H3, H14, H18 and H21, and one isolate of unknown serotype harbored cyt genes. We also tested the cytolytic activity for mammal cells, the hemolytic activity for sheep erythrocytes and insecticidal activity against mosquitoes of five isolates that contained cyt genes but did not belong to B. thuringiensis serovar israelensis. The protein profiles of the five isolates were different from those of the type strains of B. thuringiensis serovar israelensis, and among the five isolates, only Y-5 showed mosquitocidal activity against larvae of Culex quinquefasciatus. All five of the isolates exhibited hemolytic activity, but only three could cause the cell death of A549 cells. The cytopathological changes induced by NX-4 in some A549 cells were characterized with cell-ballooning.     

Étude du phytoseiideCydnodromus chilenensis en vue de son utilisation contreTetranychus urticae en culture protégée de fraisier

Résumé La comparaison de deux espèces de PhytoseiidesP. persimilis etC. chilenensis sur le terrain et en laboratoire, montre qu'elles sont très semblables mais différent principalement pour leur résistance au je?ne et leur fécondité. Des lachers des deux espèces conjointement, ou deP. persimilis seul lorsqueC. chilenensis est présent, peuvent permettre d'utiliser le premier plut?t pour son effet de choc et le deuxième plut?t pour son effet de régulation.

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Summary The comparison between two species of predatory mites,Phytoseilus persimilis andC. chilenensis in the fields as well as in the laboratory shows that they differ from each other primarily in voracity. ReleasesP. persimilis, C. chilenensis being present, afford a good combination between the drastic effect of the former and the regulating one of the latter.

     

Naturally occurring antibiotic resistance inBacillus sphaericus andBacillus licheniformis

Bacillus sphaericus is an aerobic, spore-forming, gram-variable bacillus. Certain strains of this organism are extremely pathogenic for mosquito larvae. Strains from four different serotypes ofB. sphaericus were found to be naturally resistant to streptomycin and to chloramphenicol. Both entomocidal and nonentomocidal strains of this organism exhibited similar patterns of resistance to these antimicrobial agents. In contrast, other members of the genusBacillus, includingB. subtilis, B. thuringiensis, B. laterosporus, andB. amyloliquefaciens, proved to be quite sensitive to these antibiotics. Four strains ofB. licheniformis were found to be resistant to chloramphenicol but sensitive to streptomycin.