Screening of entomopathogenic Metarhizium anisopliae isolates and proteomic analysis of secretion synthesized in response to cowpea weevil (Callosobruchus maculatus) exoskeleton

Cowpea crops are severely attacked by Callosobruchus maculatus, a Coleopteran that at the larval stage penetrates into stored seeds and feeds on cotyledons. Cowpea weevil control could be based in utilization of bacteria and fungi to reduce pest development. Entomopathogenic fungi, such as Metarhizium anisopliae, are able to control insect-pests and are widely applied in biological control. This report evaluated ten M. anisopliae isolates according to their virulence, correlating chitinolytic, proteolytic and alpha-amylolytic activities, as well proteomic analysis by two dimensional gels of fungal secretions in response to an induced medium containing C. maculatus shells, indicating novel biotechnological tools capable of improving cowpea crop resistance.     

Resistance to the cowpea weevil (Callosobruchus maculatus) larva in pods of cowpea (Vigna unguiculata)

Thirty cowpea, Vigna unguiculata (L.) Walpers, cultivars were evaluated as intact pods to determine if any possessed resistance to the cowpea weevil, Callosobruchus maculatus (Fabr.). Pod resistance was measured as pre-establishment larval mortality (PreM); those larvae dying after egg hatch but before penetrating into the seeds, and as post-establishment within-seed mortality (PostM); those larvae dying after penetrating into the seeds. Among the 30 varieties examined, PreM ranged from 57.9% to 99.4% and PostM ranged from 6.7% to 82.6%. Ten varieties exhibited total intact pod mortality (mortality from egg hatch to adult emergence from the seed) greater than 95%.Physical measurements were made of several pod and seed characteristics to ascertain whether the observed pod resistance was due to seed factors, pod-wall factors, or to interactions between the pod and seeds. Although resistance to breakage (from handling plus pod dehiscence) is a necessary characteristic for overall pod protection against C. maculatus; other factors are also important. Among the other pod and seed characteristics measured to identify major resistance factors, seed coat thickness was the one most highly correlated with pod resistance. Our results suggest that interactions between pod-wall and seed coat characteristics play a large role in pod resistance of cowpeas to C. maculatus.     

Vicilin variants and the resistance of cowpea (Vigna unguiculata) seeds to the cowpea weevil (Callosobruchus maculatus)

1. A globulin fraction prepared from the meal of Callosobruchus maculatus-resistant cowpea (Vigna wiguiculata) seeds was shown to be detrimental to this bruchid when incorporated in artificial seeds.2. The performance of C. maculatus was also shown to be strongly hindered by vicilins from resistant seeds when these storage proteins were incorporated in artificial seeds at the level of 2%.3. The purified vicilins from seeds of both resistant and susceptible cowpea varieties were shown to have the same SDS-PAGE pattern but different mobilities in non-denaturing polyacrylamide gel electrophoresis.4. These results and previous ones obtained by us (Silva and Xavier-Filho, 1991; Sales et al., 1992) strongly suggest that the resistance of cowpea seeds from the cultivar TVu 2027 and from others bred from it is associated with the presence of vicilin molecules which are refractory to digestion by bruchid midgut proteinases.     

Adaptive plasticity of egg size in response to competition in the cowpea weevil,Callosobruchus maculatus (Coleoptera: Bruchidae)

Life history theory predicts that larger propagules should be produced when the offspring are expected to experience intense competition. This study tested whether female cowpea weevils responded to high larval or adult density by producing larger eggs. In a splitbrood design I measured the effect of density experienced by females at their larval stage (1 vs. 4–6 larvae/cowpea) on the size of eggs produced just after emergence. The females were then kept either at low adult density (1 female+1 male per vial), or at high adult density (10 females+10 males) for 2 days, and tested for the effect of this adult density treatment on the size of eggs laid subsequently. I measured egg length and width, as well as the diameter of the entrance tunnel made by the larva, which can be regarded as a crude measure of larval size. Females that experienced high adult density subsequently laid slightly wider eggs than those kept at low density. This difference, albeit small (about 1–4% after correction for female weight and the effect of family, depending on the statistical model used), was statistically significant and robust to alterations of the statistical model. It may be a remnant of a larger plastic response of egg size to competition that has become eroded during many generations in high-density laboratory cultures. There was no difference in egg length or the diameter of the entrance tunnel. Eggs laid just after emergence by females reared at high larval density also tended to be wider than those produced by females that had no competitors. This effect was only marginally significant, however, and sensitive to the statistical model. Both egg length and width and the diameter of the entrance tunnel increased with female weight and decreased with female age. The tunnel diameter was positively correlated with both egg length and width, but the effect of width was larger.     

Deterrent activity of plant lectins on cowpea weevil Callosobruchus maculatus (F.) oviposition

A set of 14 plant lectins was screened in a binary choice bioassay for inhibitory activity on cowpea weevil Callosobruchus maculatus (F.) oviposition. Coating of chickpea seeds (Cicer arietinum L.) with a 0.05% (w/v) solution of plant lectins caused a significant reduction in egg laying. Control experiments with heat inactivated lectin and BSA indicated that the observed deterrent effects are specific and require carbohydrate-binding activity. However, no clear correlation could be established between deterrent activity and sugar-binding specificity/molecular structure of the lectins. Increasing the insect density reduced the inhibitory effect of the lectins confirming that female insects are capable of adjusting their oviposition rates as a function of host availability.     

Genetic architecture of differences between populations of cowpea weevil (Callosobruchus maculatus) evolved in the same environment

We investigated the genetic architecture underlying differentiation in fitness-related traits between two pairs of populations of the seed beetle Callosobruchus maculatus (Coleoptera: Bruchidae). These populations had geographically distant (> 2000 km) origins but evolved in a uniform laboratory environment for 120 generations. For each pair of populations (Nigeria x Yemen and Cameroon x Uganda) we estimated the means of five fitness-related characters and a measure of fitness (net reproductive rate R0) in each of the parental populations and 12 types of hybrids (two F1 and two F2 lines and eight backcrosses). Models containing up to nine composite genetic parameters were fitted to the means of the 14 lines. The patterns of line means for all traits in the Nigeria x Yemen cross and for four traits (larval survival, developmental rate, female body weight, and fecundity) in the Cameroon x Uganda cross were best explained by models including additive, dominance, and maternal effects, but excluding epistasis. We did not find any evidence for outbreeding depression for any trait. An epistatic component of divergence was detected for egg hatching success and R0 in the Cameroon x Uganda cross, but its sign was opposite to that expected under outbreeding depression, that is, additive x additive epistasis had a positive effect on the performance of F2 hybrids. All traits except fecundity showed a pattern of heterosis. A large difference of egg-hatching success between the two reciprocal F1 lines in that cross was best explained as fertilization incompatibility between Cameroon females and sperm carrying Uganda genes. The results suggest that these populations have not converged to the same life-history phenotype and genetic architecture, despite 120 generations of uniform natural selection. However, the absence of outbreeding depression implies that they did not evolve toward different adaptive peaks.     

The toxicity of Jack bean (Canavalia ensiformis) cotyledon and seed coat proteins to the cowpea weevil (Callosobruchus maculatus)

The seeds of the Jack bean, Canavalia ensiformis (L) DC are known to contain several toxic substances that prevent their utilisation as food for humans and animals. The lectin concanavalin A and the enzyme urease are the best known of these proteins. We have found that many proteins present in the seeds of the Jack bean, like trypsin inhibitors and canatoxin, are detrimental to the development of the bruchid insect Callosobruchus maculatus (F) (Coleoptera: Bruchidae). Among these proteins, canavalin (vicilin, 7S globulin) was found to be expressed in the seed coat. We suggest that seed coat canavalin, in addition to other detrimental proteins expressed in this tissue, may have been of importance in the evolutionary discrimination of the seeds of this legume by non-pest bruchids.     

四纹豆象不同地理种群的遗传分化

【目的】通过对四纹豆象Callosobruchus maculatus Fabricius不同地理种群mtDNA-Cytb和COⅠ基因部分序列进行比较, 分析其不同地理种群间的遗传分化情况, 为揭示其与生物入侵的关系及入侵过程中种群系统发育地理格局与演变机制提供依据。【方法】用PCR产物直接测序法对分别来自中国海南、喀麦隆、韩国和泰国的四纹豆象4个地理种群的mtDNA-Cytb和COⅠ序列进行测序, 运用软件MEGA3.1对四纹豆象不同地理种群mtDNA-Cytb和COⅠ序列进行序列分析, 以绿豆象C. chinensis为外群构建了不同单倍型的分子系统树。【结果】34条420 bp Cytb序列中共检测到14个多态位点和5种单倍型, 33条822 bp COⅠ序列中检测到28个多态位点和9种单倍型, 其中4种单倍型为独享单倍型, 其余为全部或部分种群的共享单倍型。AMOVA分析结果显示, 四纹豆象4个地理种群间的遗传结构差异并不明显, 遗传差异主要发生在地理种群内。对4个地理种群进行了Fst值和基因流动统计, 结果表明4个地理种群间既存在着一定数量的基因交流, 也存在一定程度的遗传分化。【结论】根据单倍型分布格局初步推测, 中国不可能是四纹豆象的原产地, 而喀麦隆有可能是原产地之一, 并且喀麦隆种群与泰国种群之间的基因交流比较充分, 而中国种群与其他种群之间的遗传分化相对较大。     

Insecticidal activity of Poppy (Papaver somniferum L.) seed oil against cowpea weevil (Callosobruchus maculatus F.) in stored cowpea

In this study, the insecticidal effect of Poppy (Papaver somniferum) seed oil was investigated against Callosobruchus maculatus (Bruchidae) in cowpea, at different concentrations and exposure time. The Poppy seed oil at 2, 4, 6, 8 and 10?ml/kg was tested against adults of C. maculatus, and the mortality was counted after 24, 48 and 72?h of exposure. All tests were conducted at 27–30?°C and 65?±?5% r.h. The experiments were carried out based on the factorial experiment by randomized complete design with four replications. Twenty-five insects with 0–24?h old were used for each replication. The effect of Poppy seed oil on the reduction of emergence of insects in next generation (F₁) was also assayed. The results of experiments indicate the significant differences between concentrations and exposure time (p?<?0.0001), and the increase of concentrations and exposure time increased mortality. The amount of mortality at high concentrations was remarkable, and highest mortality rate (96.91%) was recorded at 10?ml/kg, after 72?h of exposure. The application of oil significantly reduced F₁ progeny production and even at lowest concentration (2?ml/kg), the amount of F₁ production was reduced more than 70%, compared with control treatment. Complete (100%) reduction in progeny production was recorded at the rates of 8 and 10?ml/kg. The results of seed germination assay showed no significant differences between control and treated seed, and no harmful effect was observed on the seed germination. These results proved that Poppy seed oil can be used as a controlling agent of storage pests, especially C. maculatus, although more detailed studies are necessary.     

Use of commercial freezers to control cowpea weevil, Callosobruchus maculatus (Coleoptera: Bruchidae), in organic garbanzo beans

One California processor of organic garbanzo beans (Cicer arietinum L.), unable to use chemical fumigants, relies on 30-d storage at -18 degrees C to disinfest product of the cowpea weevil, Callosobruchus maculatus (F). To determine whether the storage period may be shortened, the most cold-tolerant life stage of the cowpea weevil was identified. Laboratory studies showed that the egg stage was most tolerant to -18 degrees C and that adults were most susceptible. To examine the efficacy of cold storage disinfestation, bags of black-eyed peas, Vigna unguiculata (L.) Walp., infested with cowpea weevil eggs were buried within garbanzo bean bins placed in a commercial cold storage facility kept at approximately -18 degrees C and removed after 7, 14, and 21 d. Survival was highest in eggs located at the center of the bins and coincided with the slowest cooling rate. Although temperatures within the bins did not reach -18 degrees C until after 14-19 d, egg mortality was estimated to be >98% after just 7 d of exposure. Complete mortality of eggs occurred after 14 d of cold storage. A 2-wk treatment regimen may be sufficient for control of cowpea weevil in organic legumes.     

Efficient and stereo-selective syntheses of three stereoisomers of the sex pheromone of the cowpea weevil, Callosobruchus maculatus

An efficient and stereoselective synthesis of three stereoisomers of the sex pheromone of the cowpea weevil, Callosobruchus maculatus, is reported.     

RAPD-PCR characterization of Beauveria bassiana isolates from the rice water weevil Lissorhoptrus oryzophilus

B.E. Urtz and W.C. Rice. 1997. Random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) was used to examine the genetic variability among Beauveria bassiana isolates from infected rice water weevil (RWW), Lissorhoptrus oryzophilus , collected in south-western Louisiana. Most of the B. bassiana isolates tightly clustered into one or the other of two groups that diverged at the 65% similarity level. Three soil isolates also clustered within the two groups while one soil isolate did not tightly cluster (mean similarity = 65%) with any of the RWW isolates. The results suggest that certain genotypes of B. bassiana commonly infect RWW while others do not.     

Effects of Phaseolus vulgaris (Fabaceae) seed coat on the embryonic and larval development of the cowpea weevil Callosobruchus maculatus (Coleoptera: Bruchidae)

Bruchid beetles infest various seeds. The seed coat is the first protective barrier against bruchid infestation. Although non-host seed coats often impair the oviposition, eclosion and survival of the bruchid Callosobruchus maculatus larvae, morphological and biochemical aspects of this phenomenon remain unclear. Here we show that Phaseolus vulgaris (non-host) seed coat reduced C. maculatus female oviposition about 48%, increased 83% the seed penetration time, reduced larval mass and survival about 62 % and 40 % respectively. Interestingly, we found no visible effect on the major events of insect embryogenesis, namely the formation of the cellular blastoderm, germ band extension/retraction, embryo segmentation, appendage formation and dorsal closure. Larvae fed on P. vulgaris seed coat have greater FITC fluorescence signal in the midgut than in the feces, as opposed to what is observed in control larvae fed on Vigna unguiculata. Cysteine protease, α-amylase and α-glucosidase activities were reduced in larvae fed on P. vulgaris natural seed coat. Taken together, our results suggest that although P. vulgaris seed coat does not interfere with C. maculatus embryonic development, food digestion was clearly compromised, impacting larval fitness (e.g. body mass and survivability).     

Use of micro-CAT scans to understand cowpea seed resistance to Callosobruchus maculatus

The cowpea bruchid, Callosobruchus maculatus (F.) (Coleoptera: Chrysomelidae: Bruchini), is a worldwide pest of stored cowpea grain [ Vigna unguiculata (L.) Walp. (Leguminosae)], causing tens of millions of dollars' worth of damage annually. One method of managing this pest involves planting cultivars whose seeds are resistant to bruchids. Despite extensive research, we still do not know the mechanism by which TVu 2027, the original resistant cowpea line, resists C. maculatus . Using micro-CAT (computerized axial tomography) scan imaging, we visualized the feeding patterns of bruchids living and growing within cowpea seeds. We present evidence that an interior zone in TVu 2027 seeds is responsible for the high mortality and developmental delays experienced by avirulent larval bruchids. We observed that both virulent as well as avirulent bruchid strains have different feeding patterns in TVu 2027 seeds compared to susceptible seeds. It appears that the resistance factor is most concentrated in a zone in each cotyledon adjacent to the air space separating the two seed halves.     

Host suitability, respiration rate and the outcome of larval competition in strains of the cowpea weevil, Callosobruchus maculatus

Abstract.  Variation between strains of Callosobruchus maculatus in several life-history traits is well known. Differences in functional anatomy of egg pores and larval respiration rates have also been reported in strains from Brazil and Yemen. The response of five strains of C. maculatus to seeds of two host species, cowpea ( Vigna unguiculata ) and mung bean ( Vigna radiata ), was measured along with the larval respiration rates of the same strains on both hosts. There was significant variability of response to the two hosts. Strains with higher larval respiration rate (µL O₂/insect/day) showed higher seed consumption, which significantly affected adult emergence per seed on both hosts. This finding provides support for the hypothesis that differential feeding rate is an important mechanistic component of the larval competition outcome observed in strains of C. maculatus .     

Farm-scale trials to compare the entomopathogenic fungus Beauveria bassiana with pirimiphos methyl + deltamethrin and essential oil of lemon grass for protection of stored cowpea against Callosobruchus maculatus (Coleoptera: Bruchidae)

In trials conducted in Benin, conidia of Beauveria bassiana were evaluated as a control method against the cowpea weevil, Callosobruchus maculatus , in stored cowpea. In the first trial using a high artificial infestation of C.   maculatus in 8-kg batches of cowpea in jerry cans under typical conditions, concentrations of 1 × 10⁹ and 1 × 10¹¹ conidia kg⁻¹ were compared with lemon grass oil at 2 mL kg⁻¹ and the synthetic pesticide mixture of 1.5% pirimiphos methyl + 0.05% deltamethrin at 0.5 g kg⁻¹. After 2 months of storage, seed losses (SD) were 20.63 (5.3)% in the untreated control, 8.04 (3.2)% in the low-dose B.   bassiana group, 3.12 (1.3)% in the high-dose B.   bassiana group, 2.52 (0.4)% in the lemon grass oil group and 0% in the pirimiphos methyl + deltamethrin group. In a second trial with natural infestations in 50-kg batches of cowpea in 200-L drums, treatment with B.   bassiana 1 × 10¹¹ conidia kg⁻¹ was compared with pirimiphos methyl + deltamethrin at 0.5 g kg⁻¹. After 6 months of storage on six farms, losses reached 30.76 (1.5)% in the control, 1.28 (0.2)% in the pirimiphos methyl + deltamethrin group and 3.69 (0.6)% in the B.   bassiana group.     

Behavioral processes and egg-laying decisions of the bean weevil, Callosobruchus maculatus

Empirical studies of behavioral processes were designed in a seed beetle, Callosobruchus maculatus, to distinguish between two alternative tactical models, namely, comparison tactics and threshold tactics of seed choice, and also to deduce the behavioral rule of its oviposition decisions. Search sequences and the oviposition process of the female bruchid beetle were observed and recorded. Analyzing data of the search sequence showed that the female bruchid beetle repeatedly encountered seeds randomly and tended to oviposit on a newly encountered seed in an ovposition bout. These results contradict the usual comparison models, which predict that the female will return to choose any one of the previously encountered seeds after sampling. In addition, the oviposition decision (rejection or acceptance of an encountered seed to oviposit) was analyzed with a generalized linear modeling (GLIM) technique. Modeling showed that the probability of accepting a seed with different numbers of eggs on it changed during her egg-laying process. This evidence supports the idea that the female is using a threshold tactic and that her acceptance threshold is being adjusted by experience gained during the egg-laying process. The analysis of statistical modeling also showed that both the time since the last oviposition and the number of eggs which had been laid by the female had a significant effect on the probability of accepting seeds with different numbers of eggs. Therefore, a time measuring system and the physiological state variable, e.g., eggload of the female, should be included in the behavioral rule to explore how the female makes her decision in the egg-laying process and to examine the importance of choice behavior as a component of selection and adaptation. Received: May 8, 1998 / Accepted: September 20, 1999     

Evaluation of different botanical for the management of cowpea bruchid (Callosobruchus maculatus) in north-eastern Ethiopia

The pesticide efficacy of locally available seven botanicals “Gime” (Chenopodium ambrosioides), “Ayderke” (Jatropha curcas), Neem (Azadirachta indica), Melia (Melia azadirach), “Chobe” (Cisus rotonifolia), “Kelewa” (Maesa lanceolata) and “Antharfa” at a rate of 4% weight by weight was evaluated for the control of cowpea bruchid using 200?g cowpea seeds in 500?cm³ volume jar in 2010 and 2011 under laboratory condition at Sirinka Agricultural Research Centre. The combined analysis showed that “Gime” (both leaf and seed) had high speed of pesticide effect as it showed significantly higher per cent of parent adult mortality (84 and 71%, respectively) as to the standard check Malathion 5% dust (100%) in the first day of adult mortality count. “Antharfa” and “Ayderke” also have shown significantly higher per cent of parent adult mortality as to Malathion in the third and fifth day of adult mortality count, respectively. In the seventh day, all botanicals showed significantly higher per cent of adult mortality as compared to untreated check. “Gime” leaf and seed powder, “Ayderke” seed, Neem seed and “Antharfa” leaf powders significantly reduced the number of progeny emergence per day as compared to all other treatments. Moreover, these botanicals were effective in reducing per cent seed damage (0.00, 0.00, 0.57, 5.86 and 10.86%, respectively) and storage loss (0.00, 0.00, 0.073, 1.02 and 2.27%, respectively). Gime (both leaf and seed powder), “Ayderke” seed and Neem seed are locally available, simple for preparation and environmentally friendly. Therefore, these three botanicals can be recommended to satisfy the demand for organic food and save the seed damage and yield loss of cowpea caused by Callosobruchus maculates and bean bruchid (Zabrotes subfasciatus) which has similar biology with Callosobruchus maculatus.     

Selection of Beauveria bassiana isolates to control Alphitobius diaperinus

This paper presents results on isolates of the entomopathogenic fungus Beauveria bassiana for Alphitobius diaperinus (Coleoptera: Tenebrionidae) control. Of the 30 isolates tested, four (CG 71, CG 152, UNIOESTE 4, and UNIOESTE 40) resulted in mortality rates >or= 40% within 10 days. These mortality rates could be considered high because of the resistance of these species to B. bassiana. Tests were conducted using these isolates to estimate LC(50), mortality rate over time, vegetative growth, and conidial production on artificial medium and on insects. Isolates CG 71, CG 152, and UNIOESTE 4 showed the best performance and great potential to be used in an integrated management program in poultry farms to control A. diaperinus. Also, the molecular profiles of 12 isolates were analyzed using the RAPD technique. The high-virulence isolates presented a more homogeneous RAPD pattern than the others. Genetic sequencing of the ITS region was performed for one of the virulent isolates (UNIOESTE 4) and compared with sequences deposited at the NCBI database, confirming its taxonomical position as belonging to B. bassiana Clade A.     

基于分生孢子热胁迫反应的球孢白僵菌耐热菌株筛选

将寄主和原产地不同的16株球孢白僵菌Beauveriabassiana的分生孢子分别悬浮于1mL萌发液中,接受48℃恒温水浴45min的热胁迫后,再在25℃下振荡培养24h,以残存活孢率作为各菌株对热胁迫反应的耐热力指标。结果显示,供试菌株的残存活孢率在4.6%~87.1%之间变化,相互间差异极显著。热胁迫后有三株菌的残活孢率均在40%以上,耐热力强;四株菌的残存活孢率低于10%,耐热力弱;其余菌株的残存活孢率为11.0%~23.6%,耐热力居中。从上述菌株中挑选耐热力强、居中和弱的菌株各一株(残存活孢率分别为40.8%、15.8%和4.6%),分别置于25℃、30℃及35℃下平板培养,其菌落生长表现明显与耐热力的强弱相关。虽然三菌株在25℃下的菌落生长无显著差异,但只有耐热力强的菌株能在35℃下正常生长并产孢,是罕见的球孢白僵菌耐热菌株。