Prevailing triple infection with Wolbachia in Callosobruchus chinensis (Coleoptera: Bruchidae)

Prevailing triple infection with three distinct Wolbachia strains was identified in Japanese populations of the adzuki bean beetle, Callosobruchus chinensis. When a polymerase chain reaction (PCR) assay was conducted using universal primers for ftsZ and wsp, Wolbachia was detected in all the individuals examined, 288 males and 334 females from nine Japanese populations. PCR-restriction fragment length polymorphism (RFLP) analysis of cloned wsp gene fragments from single insects revealed that three types of wsp sequences coexist in the insects. Molecular phylogenetic analysis of the wsp sequences unequivocally demonstrated that C. chinensis harbours three phylogenetically distinct Wolbachia, tentatively designated as wBruCon, wBruOri and wBruAus, respectively. Diagnostic PCR analysis using specific primers demonstrated that, of 175 males and 235 females from nine local populations, infection frequencies with wBruCon, wBruOri and wBruAus were 100%, 96.3% and 97.0%, respectively. As for the infection status of individuals, triple infection (93.7%) dominated over double infection (6.1%) and single infection (0.2%). The amounts of wBruCon, wBruOri and wBruAus in field-collected adult insects were analysed by using a quantitative PCR technique in terms of wsp gene copies per individual insect. Irrespective of original populations, wBruCon and wBruOri (107 -108 wsp copies/insect) were consistently greater in amount than wBruAus (106 -107 wsp copies/insect), suggesting that the population sizes of the three Wolbachia strains are controlled, although the mechanism is unknown. Mating experiments suggested that the three Wolbachia cause cytoplasmic incompatibility at different levels of intensity.     

Contact and fumigant activities of Citrullus colocynthis formulations against Callosobruchus chinensis (Coleoptera: Bruchidae)

The efficacy of two different formulations of Citrullus colocynthis extracts (emulsifiable and powder) were tested as contact and fumigant toxicants against cowpea weevil adults, Callosobruchus chinensis. The emulsifiable concentrate showed repellent activity against the adults. No eggs were laid by females in the choice test at 1.0% concentration compared with 150.0 eggs in control. Using the same concentration, the females deposited 1.4 eggs in the non-choice test compared with 78.2 eggs in control. The concentration of 2.5 μl/38.5 ml air of citrullus emulsifiable caused 100% mortality to adults during one day in the fumigation test. Also, the vapor of citrullus emulsifiable was highly effective against eggs of cowpea weevil, where, at 2.5 μl/38.5 ml air, no eggs hatched. Both formulations affect the different biological aspects of cowpea weevil, however, citrullus emulsifiable concentrate was more potent than the citrullus powder.     

Sources of semiochemicals mediating host finding in Callosobruchus chinensis (Coleoptera: Bruchidae)

Bruchid pests such as Callosobruchus chinensis (Linnaeus) endanger stored legume seeds throughout the tropical belt. The chemical composition of the headspace volatiles from healthy and fourth instar larvae-infested cowpea seeds were identified, characterized, and compared using gas chromatography-mass spectrometry. Y-tube olfactometer bioassays were performed to evaluate the effect of these chemicals on the orientation of conspecific adult females. Analysis of volatiles released from healthy and infested seeds revealed qualitative differences for three out of the 17 compounds identified. Dimethyl disulphide, isobutenyl methyl ketone and methyl trisulphide were found only in the blend emitted from infested but not from healthy seeds. Quantitative differences were apparent for tridecane which was released in larger amounts from infested seeds. While volatiles collected from healthy seeds were attractive to female bruchids, volatiles collected from infested seeds were repellent. To test the hypothesis that the qualitative differences in the chemical composition found may be due to insect-derived components, the volatiles from frass and fourth instar larvae combined were analysed. These volatiles contained both of the sulphides emitted from infested seed but not from healthy seeds. Although a limited induction of volatiles from cowpea seeds cannot be excluded, it is postulated that behavioural differences of the female weevils are largely due to insect-derived semiochemicals. The potential use of such semiochemicals as part of an integrated pest management strategy is discussed.     

Habitat-related mtDNA polymorphism in the stored-bean pest Callosobruchus chinensis (Coleoptera: Bruchidae)

The genetic diversity of populations of the azuki bean beetle, Callosobruchus chinensis (Linnaeus) from natural, pre-harvest and post-harvest sites, was investigated to understand population structure and gene flow. A 522-bp fragment of the mitochondrial gene COI was sequenced for eight populations of C. chinensisfrom Japan, Korea and Taiwan collected from different habitats. Six haplotypes were detected, one of which, U1, occurred most frequently and widely. The following hypotheses were tested as a cause of the wide distribution of haplotype U1; (i) topographical separation (by national boundaries), (ii) host plant species, and (iii) habitat type (natural, pre-harvest crop, or post-harvest storage). Categorization of collection sites by country or by host species did not yield differences in the occurrence of haplotype U1, but habitat type did. Populations utilizing cultivated post-harvest hosts that were mass stored were highly likely to be the common haplotype, whereas host plants in natural habitats away from agriculture were utilized by populations with locally characteristic haplotypes. Sampling of commercial beans for quarantine and export purposes indicated that gene flow in C. chinensis was largely unidirectional into Japan at the present time.     

昆虫病原真菌与硅藻土混用对四纹豆象的毒力效果(英文)

在室内条件下检测了白僵菌Beauveria bassiana (Balsamo) Vuillemin 菌株IRAN-187C 和金龟子绿僵菌Metarhizium anisopliae (Metschnikow) Sorokin菌株A-396分别与硅藻土（DE）制剂InsectoSec混用对四纹豆象Callosobruchus maculatus成虫的毒力效果。结果表明：硅藻土与致死剂量和亚致死剂量的真菌分离物混用导致了四纹豆象成虫的高死亡率,且这些处理均缩短了真菌的致死中时(LT₅₀)。LC₅₀ 的白僵菌B. bassiana菌株IRAN-187C和 LC₅₀ 的DE混用对四纹豆象的LT₅₀ (122.2 h)最低,在95%置信限上与LC50 的金龟子绿僵菌M. anisopliae菌株A-396 与LC₅₀的DE混用得到的LT₅₀ (128.2 h) 相当,说明两种真菌分离物与DE混用对四纹豆象成虫都具有较好的毒力效果。用四纹豆象成虫检测3个温度（22, 27和30℃）和2个相对湿度（40%和55%）组合对DE活性的影响,结果显示30℃和40%相对湿度条件下得到的LT₅₀ (110.11 h)最低,说明高温低湿条件下DE活性较高。     

Toxic and deterrent effects of two volatile oils against cowpea weevil,Callosobruchus chinensis (Coleoptera: Bruchidae)

The fumigant effect of Mentha piperita and Ocimum basilicum oils and their mixture against adults and eggs of Callosobruchus chinensis (L.) was evaluated. Bioassay experiments showed that Ocimum basilicum oil was significantly effective against adult and egg stage which was the least effective. However, in the fumigant toxicity experiments against adult stage, Ocimum basilicum oil at a dose of 1.0 μl/38.5 ml air caused 100% mortality (LC₅₀ = 1.88), the mixture of both oils at a dose of 6.0 μl/38.5 ml air caused 100% mortality (LC₅₀ = 10.3) and Mentha piperita oil at a dose of 80.0 μl/38.5 ml air caused 80.0% mortality (LC₅₀ = 41.224) during a one day exposure period. Regarding the oviposition deterrent activity, Ocimum basilicum oil achieved 100% oviposition deterrent (at a dose of 0.5 μl/38.5 ml air) followed by the mixture of Mentha piperita and Ocimum basilicum oils, which achieved 71.22% oviposition deterrent (at a dose of 1.0 μl/38.5 ml air) and Mentha piperita oil, which achieved 39.6% oviposition deterrent (at a dose of 5.0 μl/38.5 ml air). The essential oils and their mixtures studied here determined a significant decrease in the number of eggs hatched and in the emergence of adults. The eggs failed to hatch on using Ocimum basilicum oil at a dose of 0.6 μl/38.5 ml air. However, the number of eggs hatched decreased to 7.4 on using a mixture of oils at a dose of 2.0 μl/38.5 ml air and 14.0 with Mentha piperita oil at a dose of 80.0 μl/38.5 ml air compared with 25.0 eggs hatched in the control experiments. On the other hand, the percentage reduction in emerging adults were 100% for Ocimum basilicum oil, 90.9% for a mixture of the two oils and 72.7% for Mentha piperita oil. Results showed that Ocimum basilicum oil and Ocimum basilicum oils plus Mentha piperita oils in blend are potential alternatives to synthetic fumigants in the treatment of durable agricultural products. Successful adoption of plant oils in the protection of food commodities promises an eco-friendly option compatible with international biosafety regulations.     

Oviposition deterrents of Callosobruchus maculatus (Coleoptera: Bruchidae)

Abstract Many bruchids distribute their eggs uniformly among the host seeds available to them and tend to avoid adding eggs to seeds bearing any eggs if pristine seeds remain. Several authors have postulated that the adult cowpea seed beetles, Callosobruchus maculatus (F.) (Coleoptera: Bruchidae), produce pheromones inhibiting oviposition by females exposed to them. It is shown that both sexes produce compounds which can be washed from glass surfaces in a variety of organic solvents which do not deter oviposition as such, but seeds coated with such washings are avoided by ovipositing females when they are selecting seeds for egg-laying and uncoated seeds are available. Some components, at least, of the products persist for at least 30 days, longer than the normal development time of insects in the seeds. The compounds affect the distribution of eggs among the available seeds. At least three different biotypes of the species produce, recognize, and respond in the same way to each other's products. It is emphasized that the eggs themselves may not be the site of the product produced by females but that the physical presence of eggs cannot be overlooked in any discussion of the possible role of pheromones in oviposition and egg distribution by bruchids.     

绿豆象气味结合蛋白基因的克隆及表达分析

[目的]对绿豆象Callosobruchus chinensis气味结合蛋白(odorant binding proteins,OBPs)基因进行克隆、鉴定和组织表达分析,为研究OBPs在绿豆象嗅觉感受过程中的功能奠定基础.[方法]基于绿豆象触角转录组数据,通过RT-PCR克隆绿豆象6个OBP基因并进行生物信息学分析;...     

Bionomics and polymorphism in Callosobruchus subinnotatus (Coleoptera: Bruchidae)

Callosobruchus subinnotatus (Pic) is the major insect pest of stored bambara groundnuts, Vigna subterranea (L.) Verdcourt, in sub-Saharan West Africa, but little is currently known about its biology or how it may be controlled. A series of laboratory studies was performed to investigate the bionomics of and differences between two apparently different morphs of adult of each sex of this species, here termed 'active' and 'normal'. Major differences in their morphology, physiology and behaviour were identified and are described in detail for the first time. They provide clear evidence of the existence of an adult polymorphism among populations of this species, which is comparable in certain respects to that previously described for C. maculatus (Fabricius) and C. chinensis Linnaeus. Adults can be separated into the correct morph based on characteristic differences in elytral and pygidial colour and pattern. 'Normal' adults are characterized by having high fecundity, short adult life and are relatively sedentary while 'active' adults exhibit reproductive diapause (suspension of reproductive activity), are long lived, and show (at least in females) increased dispersal tendencies. These characteristics suggest adaptation of the 'active' and 'normal' morphs respectively to the different environments of field and seed stores, and the significance of the polymorphism in the life history of C. subinnotatus is discussed in this context. The design of any effective control regime for this bruchid needs to take account of and could potentially be based upon the existence of polymorphism in C. subinnotatus.     

Mechanisms of the insecticidal action of TEL (Talisia esculenta lectin) against Callosobruchus maculatus (Coleoptera: Bruchidae)

Plant lectins have insecticidal activity that is probably mediated through their ability to bind carbohydrates. To examine the influence of sugars on the insecticidal activity of a lectin from Talisia esculenta seeds (TEL), the lectin was mixed with mannose, glucose, or mannose plus glucose. Mannose abolished the insecticidal activity. Affinity chromatography showed that TEL bound to midgut proteins of the insect Callosobruchus maculatus. Immunoblotting showed that TEL recognized some proteins, probably glycoproteins, present in the midgut membrane of this insect. The principal proteases responsible for digestive proteolysis in fourth instar larvae of C. maculatus were purified by chromatography on activated thiol-Sepharose. These purified proteases were unable to digest TEL after a 15-h incubation. These results suggest that the insecticidal activity of TEL involves a specific carbohydrate-lectin interaction with glycoconjugates on the surface of digestive tract epithelial cells, as well as binding to assimilatory glycoproteins present in midgut extracts and resistance to enzymatic digestion by cysteine proteinases.     

Varietal susceptibility of certain broad bean seeds to infestation with Callosobruchus maculatus (F.) and Callosobruchus chinensis (L.) (Coleoptera: Bruchidae)

Seeds susceptibility of eight broad bean varieties to Callosobruchus maculatus (F.) and Callosobruchus chinensis (L.) infestation were studied for the first time in free- and no-choice methods in the laboratory of Plant Protection Department, Faculty of Agriculture, Zagazig University. The relation between certain seed physical characteristics and some biological and and infestation parameters of both insects in the two studied methods were evaluated. None of these varieties were resistant to both insects, showing various levels of susceptibility. Biological and infestation parameters were significantly different among varieties except the developmental period. In free- choice method, Giza 3 was the most susceptible variety to both insects, since produced the highest progeny of 246.67 and 75.67 adults and susceptibility index (SI) of 10.25 and 7.42, respectively, while the least susceptible variety was Giza 716. In no– choice method, Nubaria 5 and Sakha 1 were the most susceptible varieties to C. chinensis, while Nubaria 3 and Giza 3 to C. maculatus. Differences between physical characters of varieties were significant. Seed hardness were correlated negatively and seed coat thickness positively with laid eggs, progeny and (SI) of both insects in free-choice method. Also seed coat thickness correlated positively with weight loss and seed damage (%) of C. chinensis and negatively of C. maculatus. To reduce seed losses the cultivation of the least susceptible variety (Giza 716) is encouraged and considered for breeding purposes to avoid insecticide usage.     

微波处理对绿豆象的杀虫效果及对红小豆发芽率的影响

通过设置不同时间和功率的微波对裸露绿豆象Callosobruchus chinensis (L.)成虫、与红小豆混合的成虫、绿豆内幼虫及卵进行处理,以研究微波对绿豆象的杀虫作用及对绿豆象成虫产卵量、幼虫羽化率及卵孵化率等生物学特性的影响。试验结果表明：微波处理裸成虫的死亡率随处理时间及功率增加而升高。成虫在中高火和高火分别处理60 s和50 s以上时间时死亡率过半,为51.21%～99.92%。处理与红小豆混合的成虫死亡率随功率和时间变化趋势同相同处理的裸虫,但相同条件下与红小豆混合的成虫死亡率明显高于相同处理的裸虫。低火至中低火处理60 s、解冻50 s及中火至高火40 s以上时间时成虫死亡都过半,为55.75%～100%,而中火处理70 s、中高火至高火60～70 s可使成虫全部死亡。微波处理具一定后续效应,处理的成虫虽没立刻死亡但随后死亡率仍比对照高。成虫高火处理50 s后的第3天校正死亡率可达87.74%。微波处理还可降低绿豆象成虫产卵量、幼虫羽化率、卵孵化率及红小豆发芽率。成虫、卵和豆内幼虫对微波敏感性依次增高。红小豆在中低火处理60 s、解冻50 s、中火40 s、中高火至高火30 s以上时间时发芽率都低于一半,为0.21%～48.33%。微波对绿豆象杀虫效果显著,但使用时需考虑微波仪器、处理功率和时间、产品用途及含水量等诸多因素的影响并进行优化试验以取得最佳效果。     

Conspecific sperm precedence in Callosobruchus subinnotatus (Coleoptera: Bruchidae): mechanisms and consequences

Conspecific sperm precedence (CSP) has been identified as an important post-copulatory, pre-zygotic mechanism that can act to reduce gene flow between populations. The evolution of CSP is thought to have arisen as a by-product of male and female coevolution in response to intraspecific post-copulatory sexual selection. However, little is known about the mechanisms that generate CSP. When Callosobruchus subinnotatus females copulate with both C. subinnotatus and Callosobruchus maculatus males, regardless of mating order, the majority of eggs are fertilized by conspecific sperm. The low number of heterospecific fertilizations does not result from general differences in the viability of sperm in the female reproductive tract, as heterospecific sperm fertilized equivalent numbers of eggs as conspecific sperm in the absence of sperm competition. Instead, CSP results from disadvantages to heterospecific sperm that are manifest only when in competition with conspecific sperm. CSP in C. subinnotatus appears to result from two, not mutually exclusive, mechanisms. First, conspecific sperm are better able to displace heterospecific sperm from female storage. Second, conspecific sperm achieve disproportionately higher numbers of fertilizations relative to their proportional representation in the fertilization set. Thus, we provide evidence of differential sperm use from the female spermatheca.     

Polymorphism of competition type and its genetics in Callosobruchus maculatus (Coleoptera: Bruchidae)

Callosobruchus maculatus has both contest and scramble competition strategies. The currently existing theoretical models using game theory suggest that the contest strategy should be selected for. However, most geographic strains of C. maculatus show scramble competition. We experimentally crossed the representative strains of contest and scramble. We expressed the degree of contest competition by a continuous value named the C-value, which ranges from zero (pure scramble) to unity (pure contest). The competition types expressed in the C-value were genetically additive. Their larval developmental rates were negatively correlated with C-values. Multiple-generation experiments of the mixed strains confirmed that there were no overwhelming advantages of contest over scramble type. Most of the mixed strains remained in the intermediate states. We discuss the results in terms of the resource size necessary for developmental success and developmental speed. Received: April 19, 2001 / Accepted: November 2, 2001     

Insecticidal and biological efficacy of two vegetable oils against Callosobruchus maculatus (Fabricius) (Coleoptera: Bruchidae) under laboratory conditions

Bioefficacy of two vegetable oils, namely ethyl oleate and Acorus calamus, were conducted against the pulse beetle Callosobruchus maculatus (Fabr.) under a constant temperature of 29 ± 1 °C and a relative humidity of 65 ± 5% R.H. Four concentrations (0.125, 0.25, 0.5 and 1.0%) of each oil were used against the adult and tested their latent effect on the immature stages of the beetle. The results obtained show insecticidal effect of both ethyl oleate and A. calamus oil; however, A. calamus oil proved to be more toxic to the adults at all the concentrations tested in comparison to the ethyl oleate oil. As the concentration of the oils decreased, the percentage mortality decreased. The obtained results concentrated on biological efficacy of the oils tested against the adults. Both oils affected the longevities of both male and female C. maculatus adults in comparison to the control. The oviposition periods of the adult female were affected significantly in comparison to the control. A. calamus oil prohibited oviposition completely at high concentrations of 0.5 and 1.0% and caused complete sterility to the females. Lower concentrations decreased the oviposition period greatly and reduced the number of eggs laid in comparison to the control. Ethyl oleate had an inconsiderable latent effect on the incubation period as well as the percentage of hatching of the resulting eggs. A. calamus oil decreased the percentage of egg hatchability with serious increase in the percent sterility. The two tested oils had slight extension in either larval or pupal stage of the resulting progeny of C. maculatus. Both oils show oviposition deterrency towards C. maculatus adults. The oviposition deterrent index was 100% when mung bean seeds were treated with A. calamus oil. The bruchid females laid all eggs on control seeds and no eggs were laid on treated seeds.     

Internal spatiotemporal population dynamics of infection with three Wolbachia strains in the adzuki bean beetle,Callosobruchus chinensis (Coleoptera: Bruchidae)

The adzuki bean beetle, Callosobruchus chinensis, is infected with three distinct lineages of endosymbiotic bacteria belonging to the genus Wolbachia, which were designated wBruCon, wBruOri, and wBruAus. In an attempt to understand the mechanisms underlying the infection with these three organisms, the spatiotemporal infection dynamics of the three Wolbachia strains was investigated in detail by using a quantitative PCR technique. During the development of C. chinensis, the wBruCon, wBruOri, and wBruAus infection levels consistently increased but the growth patterns were different. The levels of infection plateaued at the pupal stage at approximately 3 x 10(8), 2 x 10(8), and 5 x 10(7) wsp copy equivalents per insect for wBruCon, wBruOri, and wBruAus, respectively. At the whole-insect level, the population densities of the three Wolbachia types did not show remarkable differences between adult males and females. At the tissue level, however, the total densities and relative levels of the three Wolbachia types varied significantly when different tissues and organs were compared and when the same tissues derived from males and females were compared. The histological data obtained by in situ hybridization and electron microscopy were concordant with the results of quantitative PCR analyses. Based on the histological data and the peculiar Wolbachia composition commonly found in nurse tissues and oocytes, we suggest that the Wolbachia strains are vertically transmitted to oocytes not directly, but by way of nurse tissue. On the basis of our results, we discuss interactions among the three coinfecting Wolbachia types, reproductive strategies of Wolbachia, and factors involved in the different cytoplasmic incompatibility phenotypes.     

Internal Spatiotemporal Population Dynamics of Infection with Three Wolbachia Strains in the Adzuki Bean Beetle, Callosobruchus chinensis (Coleoptera: Bruchidae)

The adzuki bean beetle, Callosobruchus chinensis, is infected with three distinct lineages of endosymbiotic bacteria belonging to the genus Wolbachia, which were designated wBruCon, wBruOri, and wBruAus. In an attempt to understand the mechanisms underlying the infection with these three organisms, the spatiotemporal infection dynamics of the three Wolbachia strains was investigated in detail by using a quantitative PCR technique. During the development of C. chinensis, the wBruCon, wBruOri, and wBruAus infection levels consistently increased but the growth patterns were different. The levels of infection plateaued at the pupal stage at approximately 3 × 10⁸, 2 × 10⁸, and 5 × 10⁷ wsp copy equivalents per insect for wBruCon, wBruOri, and wBruAus, respectively. At the whole-insect level, the population densities of the three Wolbachia types did not show remarkable differences between adult males and females. At the tissue level, however, the total densities and relative levels of the three Wolbachia types varied significantly when different tissues and organs were compared and when the same tissues derived from males and females were compared. The histological data obtained by in situ hybridization and electron microscopy were concordant with the results of quantitative PCR analyses. Based on the histological data and the peculiar Wolbachia composition commonly found in nurse tissues and oocytes, we suggest that the Wolbachia strains are vertically transmitted to oocytes not directly, but by way of nurse tissue. On the basis of our results, we discuss interactions among the three coinfecting Wolbachia types, reproductive strategies of Wolbachia, and factors involved in the different cytoplasmic incompatibility phenotypes.     

Location and ultrastructure of sex pheromone glands in female Callosobruchus maculatus (Fabricius) (Coleoptera : Bruchidae)

The ultrastructure of the female sex pheromone glands in Callosobruchus maculatus (Coleoptera : Bruchidae) were localized using a masking technique, combined with eiectro-antennography and by a comparison of the glandular cells of sexually active (flightless) females and non-sexually active (flight-form) females. Each unicellular gland is an invagination of the integumental membrane capped by a single secretory cell. These glands are situated on the fine intersegmental membrane, which joins the pygidium to the ovipositor. The secretory cells of the glands of active females are characterized by well-developed microvilli, with many elongated mitochondria among the latter. The high metabolic activity of these cells is revealed by the presence of heterogeneous secretion vesicles, some of which contain abundant crystallized material. Deep basal invaginations indicate the uptake of substances from the haemolymph. The receptor canal is a network of fine cuticular filaments which have the same structure regardless of the female's sexual status. Cells from the glands of non-sexually active females are underdeveloped and show no invaginations of the basal membrane and very few microvilli. The localization of these glands was made possible by the use of SEM, TEM and EAG as well as by masking the suspected zones and by comparing females in different physiological states: flightless females, which were sexually active and producing pheromones; and flight-form females, non-sexually active and producing no sex pheromones. Only by adopting such a stringent method was it possible to confirm the function of the glands whose ultrastructure was studied.