Mung bean (<Emphasis Type="Italic">Vigna radiata</Emphasis>) cultivars mediated oviposition preference and development of <Emphasis Type="Italic">Callosobruchus chinensis</Emphasis> (Coleoptera: Chrysomelidae: Bruchinae)

The Azuki bean weevil, Callosobruchus chinensis (L.), is a destructive pest of stored mung bean [Vigna radiata (L.) Wilczek] as well as other leguminous seeds. The development of resistant seeds to manage this pest is of current great interest to plant breeders. In this study, we investigated the oviposition preference and development of C. chinensis on two susceptible mung bean cultivars (Seonhwa and Gyeongseon) and one previously reported resistant cultivar (Jangan), compared to the susceptible cowpea (Vigna unguiculata L.), cultivar (Yeonbun) using both multiple-choice and no-choice tests. In addition, the development of C. chinensis was also examined at four constant temperatures (20, 25, 30, and 35 °C). Both tests found cowpea to be the most suitable seed for oviposition. Total developmental time from oviposition to adult emergence ranged from 27.01 to 38.2 days, being shortest on cowpea and longest on the mung bean, cv. Jangan. However, no successful development of C. chinensis larvae on mung bean, cv. Jangan, occurred at any temperature. The highest rate of adult emergence and the longest adult longevity both occurred on cowpea and certain mung bean cultivars (Seonhwa and Gyeongseon), with the dramatic exception of cv. Jangan. These results suggest that the higher preference and performance of C. chinensis on cowpea (3.3 egg/seed) and least on mung bean, cv. Jangan (0.4 egg/seed). This information may facilitate the exploration of resistant genetic materials and chemicals associated with seeds for successful breeding. Further studies should examine the chemicals associated with mung bean cultivars and its resistant mechanism to develop a control method against bruchines.     

Overwintering stages of Callosobruchus chinensis (L.) in the field

Adzuki bean beetle, Callosobruchus chinensis, is a worldwide oligophagous and a field‐to‐storage pest of legumes including adzuki beans (Vigna angularis). Its over‐wintering stage is yet to be confirmed. A series of experiments was carried out at Jinju, in the southern part of the Republic of Korea, to elucidate the overwintering stages. Top‐ and sub‐soil samplings followed by pod and seed samplings from left over plants from adzuki bean fields in the winter of 2015, and an outdoor overwintering test (during Nov 2018 to Jun 2019) by exposing bean seeds harboring different stages (eggs, larvae, pupae, pharates and adults) of laboratory reared C. chinensis to the winter environment were carried out. None of the C. chinensis stages was detected from the top‐ and sub‐soil samples. When the pods and seeds from left over plants were sampled, only a few live pupae were obtained. In the outdoor test with laboratory reared C. chinensis, all the stages (larvae, pupae, and adults), except eggs, were detected on every other weekly samples of infested adzuki bean seeds throughout the winter. These results showed that the overwintering stages of C. chinensis in Jinju, southern Korea, are larvae, pupae, pharates and adults.     

Studies on interspecies competition between the azuki bean weevil,Callosobruchus chinensis,and the southern cowpea weevil,C. maculatus II. Competition under different environmental conditions

The experimental studies was done on the interspecies competition between the azuki bean weevil, Callosobruchus chinensis, and the southern cowpea weevil, C. maculatus, under the different environmental conditions.

1. At 30°C, 70% R. H., the change of food replenishment interval had no influence upon the competition results, and the competition always ended in the extinction of C. maculatus. But there was a tendency for the period of co-existence to be elongated as the interval of replenishment lengthened.
2. At 30°C, 70% R. H., there were no differences in the competition processes and results between the competition using the dry bean (water content about 11%) or the normal one (about 15%).
3. At 32°C, 64% R. H., the competition result was reversed from that at 30°C, 70% R. H. This is thought to be due to the differences of the developmental rates and the numbers of eggs oviposited between the two species at these conditions.
4. In some cases the competition result could be understood from the point of the phenomena of single species populations of both species, but in other cases, the results were in conflict with the expectation from those phenomena. To analyze the competition mechanism, it is necessary to investigate the interaction between species.

     

Population-level analysis on reduction in equilibrium population size of the azuki bean beetle

Responses of laboratory population of the azuki bean beetle Callosobruchus chinensis to temperature conditions of 30°C and 32°C are compared. Equilibrium population size was found to be lower at 32°C. A one-generation process examined by reproduction-curve experiments is divided into two consecutive life stages specified by different habitats, i.e., outside and inside beans. Modified logistic difference equations are used to describe these population-level responses at each life stages. Sensitivity analysis is used to determine parameters of importance, thus determining the main cause of the reduced equilibrium population size at 32°C. The reduction resulted from the decrease in maximum population size of emerged adults which have experienced inside-bean process. The application and limitation of this population-level analysis to different levels are discussed.     

Experimental evolution of competing bean beetle species reveals long‐term reversals of short‐term evolution,but no consistent character displacement

Interspecific competition for shared resources should select for evolutionary divergence in resource use between competing species, termed character displacement. Many purported examples of character displacement exist, but few completely rule out alternative explanations. We reared genetically diverse populations of two species of bean beetles, Callosobruchus maculatus and Callosobruchus chinensis, in allopatry and sympatry on a mixture of adzuki beans and lentils, and assayed oviposition preference and other phenotypic traits after four, eight, and twelve generations of (co)evolution. C. maculatus specializes on adzuki beans; the generalist C. chinensis uses both beans. C. chinensis growing in allopatry emerged equally from both bean species. In sympatry, the two species competing strongly and coexisted via strong realized resource partitioning, with C. chinensis emerging almost exclusively from lentils and C. maculatus emerging almost exclusively from adzuki beans. However, oviposition preferences, larval survival traits, and larval development rates in both beetle species did not vary consistently between allopatric versus sympatric treatments. Rather, traits evolved in treatment‐independent fashion, with several traits exhibiting reversals in their evolutionary trajectories. For example, C. chinensis initially evolved a slower egg‐to‐adult development rate on adzuki beans in both allopatry and sympatry, then subsequently evolved back toward the faster ancestral development rate. Lack of character displacement is consistent with a previous similar experiment in bean beetles and may reflect lack of evolutionary trade‐offs in resource use. However, evolutionary reversals were unexpected and remain unexplained. Together with other empirical and theoretical work, our results illustrate the stringency of the conditions for character displacement.     

不同生态环境条件沙生植物沙鞭的结实研究

为了阐明不同生态环境条件下沙生植物沙鞭的结实规律,该文对沙鞭137个种群结实情况进行了实地观察,发现沙鞭种子的结实情况可被划分为无种子、种子饱满和种子不饱满3种类型;在此基础上,该文采用聚类分析、Kruskal-Wallis检验和典范对应分析(CCA)等方法探究沙鞭种群结实情况与22个地理气候因子的相关性。结果表明:(1)沙鞭137个种群按照地理气候因子不同聚为3个组;(2) Kruskal-Wallis检验显示沙鞭3个组间种子结实情况差异不显著(P=0.269),即沙鞭种群间种子结实与其所处的地理气候因子无直接相关性;(3)典范对应分析(CCA)表明沙鞭种群间种子结实情况差异也不显著(P0.05),但地理气候因子与种子饱满度以及无种子特征具有显著相关性,其中海拔和降雨因子(bio12-bio19)与种子饱满度呈正相关,而经纬度和温度因子(bio4,bio7)与种子饱满度呈负相关,无种子特征仅与最湿季平均温度(bio8)呈正相关。地理气候因子对沙鞭天然种群有性繁殖(有种子)重要性高于无性繁殖(无种子),表明制约沙生植物沙鞭有性繁殖的环境因子复杂,其无性繁殖可能是种群数量稳定的适应性表现。     

Studies on the interspecies competition between the azuki bean weevil and the southern cowpea weevil IV. Competition between strains

This study analyzes results of interspecies competition between the azuki bean weevil, Callosobruchus chinensis and the southern cowpea weevil, C. maculatus using the four geographical strains of each species.

1. Fifteen combinations of one strain from each species were set up to compete. The strains of C. chinensis, rather than of C. maculatus, characteristically determine the eventual outcomes of competition. Some strains of C. chinensis (jC and kC) invariably won every encounter with C. maculatus. Another strain (nC) always lost its encounters, while one other strain (iC) won some encounters (:cQ and :cQ) and lost others (:tQ and :bQ). For any combination the competition results were deterministic and no reversal in outcome was observed among the replicates.
2. The weakness of strain nC in competition is explained by its characteristics in single species populations, different greatly from other strains of C. chinensis, although it had the similar primary (individual) characteristics as other strains of C. chinensis. Strain iC had the shortest adult longevity in the four strains of C. chinensis although it behaved similar to strain jC at the single species population level. Especially under the food supply schedule of the present experiment the shortness of adult longevity might be the important factor determining the outcome.
3. The relationship of the eight strains based on the individual and single species population characteristics, and that based on the competition results were very similar. Even when the competition results could not be explained by any one or two of these characteristics alone, the outcome would be largely decided by the overall biotic profile made by several characteristics.

     

Transgenic cowpea (<Emphasis Type="Italic">Vigna unguiculata</Emphasis>) seeds expressing a bean α-amylase inhibitor 1 confer resistance to storage pests,bruchid beetles

Cowpea is one of the important grain legumes. Storage pests, Callosobruchus maculatus and C. chinensis cause severe damage to the cowpea seeds during storage. We employ a highly efficient Agrobacterium-mediated cowpea transformation method for introduction of the bean (Phaseolus vulgaris) α-amylase inhibitor-1 (αAI-1) gene into a commercially important Indian cowpea cultivar, Pusa Komal and generated fertile transgenic plants. The use of constitutive expression of additional vir genes in resident pSB1 vector in Agrobacterium strain LBA4404, thiol compounds during cocultivation and a geneticin based selection system resulted in twofold increase in stable transformation frequency. Expression of αAI-1 gene under bean phytohemagglutinin promoter results in accumulation of αAI-1 in transgenic seeds. The transgenic protein was active as an inhibitor of porcine α-amylase in vitro. Transgenic cowpeas expressing αAI-1 strongly inhibited the development of C. maculatus and C. chinensis in insect bioassays.     

Rapid Development of Microsatellite Markers for Callosobruchus chinensis Using Illumina Paired-End Sequencing

Background

The adzuki bean weevil, Callosobruchus chinensis L., is one of the most destructive pests of stored legume seeds such as mungbean, cowpea, and adzuki bean, which usually cause considerable loss in the quantity and quality of stored seeds during transportation and storage. However, a lack of genetic information of this pest results in a series of genetic questions remain largely unknown, including population genetic structure, kinship, biotype abundance, and so on. Co-dominant microsatellite markers offer a great resolving power to determine these events. Here, we report rapid microsatellite isolation from C. chinensis via high-throughput sequencing.

Principal Findings

In this study, 94,560,852 quality-filtered and trimmed reads were obtained for the assembly of genome using Illumina paired-end sequencing technology. In total, the genome with total length of 497,124,785 bp, comprising 403,113 high quality contigs was generated with de novo assembly. More than 6800 SSR loci were detected and a suit of 6303 primer pair sequences were designed and 500 of them were randomly selected for validation. Of these, 196 pair of primers, i.e. 39.2%, produced reproducible amplicons that were polymorphic among 8 C. chinensis genotypes collected from different geographical regions. Twenty out of 196 polymorphic SSR markers were used to analyze the genetic diversity of 18 C. chinensis populations. The results showed the twenty SSR loci were highly polymorphic among these populations.

Conclusions

This study presents a first report of genome sequencing and de novo assembly for C. chinensis and demonstrates the feasibility of generating a large scale of sequence information and SSR loci isolation by Illumina paired-end sequencing. Our results provide a valuable resource for C. chinensis research. These novel markers are valuable for future genetic mapping, trait association, genetic structure and kinship among C. chinensis.     

Regulation of Progeny Sex by Anisopteromalus calandrae (Hymenoptera: Pteromalidae) in Relation to Host Preference,Host Vulnerability and Host Size

Regulation of progeny sex by Anisopteromalus calandrae (Howard) in relation to host preference, host vulnerability and host size was studied with two host species, Sitophilus oryzae (L.) and Callosobruchus chinensis L. A. calandrae preferred S. oryzae to C. chinensis (preference index = 0.87). The progeny sex ratio (females/total) was significantly higher on C. chinensis, the less preferred host, than on S. oryzae regardless of the instar age of the two host species. A. calandrae could locate only a few young larvae of C. chinensis (≤third instar), which could be apparently due to the low vulnerability of the young larvae locating deeper in the adzuki bean kernels. The larval instar age of Sitophilus oryzae affect the progeny sex ratio of A. calandrae, but that of Callosobruchus chinensis did not except invulnerable ones. The progeny sex ratio of A. calandrae produced from C. chinensis, ranged from 0.66±0.05 to 0.82±0.02. The invulnerability of the young larval instar of C. chinensis and regulation of the progeny sex ratio by A. calandrae based on host size in a manner of absolute rule could explain the higher sex ratio on the young larval instar of C. chinensis, less preferred and less vulnerable host, than the old larval instar of S. oryzae, highly preferred host. The differences in host vulnerability could be another factor for A. calandrae assigning progeny sex ratio.     

Systems analysis of density-dependent population processes in the azuki bean weevil,Callosobruchus chinensis

Systems analysis of density-dependent population processes was conducted in an experimental population of the azuki bean weevil,Callosobruchus chinensis. Density-dependent population change was formulated for two separate processes; adult production of eggs and hatching of the eggs outside the beans, and survival of the first instar larvae inside the beans and their emergence as new adults. The formulation of each process was based on a common basic equation modified from the logistic-difference equation. Parametric values were estimated from experimental data onC. chinensis in a laboratory population. Simulations of the model showed good fits not only to observed population changes both outside and inside the beans, but also to the observed reproduction curve reflecting the population change as a whole within one generation in an experimental population. Furthermore, population dynamics from generation to generation were simulated from the model reproduction curve. The simulation showed a slight dumped oscillation and converged to a carrying capacity, which fitted well with the experimental population dynamics.     

Response of Callosobruchus maculatus (F.) to varying temperature and relative humidity under laboratory conditions

The effect of temperature and relative humidity were determined on the development of Callosobruchus maculatus (F.) on stored bean (Vigna unguiculata) seeds exposed to five temperatures (20, 25, 30, 35 and 40 °C) and six relative humidity levels (40, 50, 60, 70, 80 and 90%). Oviposition and total adult progeny responded in a curvilinear pattern to temperature and relative humidity while developmental period presented a linear response. Egg laying (117.33 ± 3.21) and adult emergence (35.00 ± 1.70) were least at 20 °C and 90% R.H. but those showed the optimal values at 30 °C and 70% R.H. The implication of these findings is that the growth attributes of C. maculatus relate with the weather variables studied in a curvilinear manner and thus quadratic equations generated could be used for the prediction of optimum temperature and relative humidity in a given area for the management of C. maculatus in stored beans.     

Age specificity of inbreeding depression during a life cycle ofCallosobruchus chinensis (Coleoptera: Bruchidae)

Age-specific effects of inbreeding on fecundity were assayed for adzuki bean weevil Callosobruchus chinensis by comparing inbred lines and their cross. Four consecutive full-sib matings reduced only 10.3 percent in total fecundity, and did not decrease early fecundity at all until third day from the onset of reproduction. It is suggested that recessive detrimental genes have been eliminated from the early period of adult life span when reproductive value is high. There was a slight tendency that inbreeding depression increased as age proceeded though not statistically significant.     

Synergistic effect of fumigation with ethyl formate and methyl salicylate on mortality of life stages of adzuki bean beetle,Callosobruchus chinensis (L.)

Ethyl formate (EF) is becoming an insecticide of choice for protecting stored commodities. Along with EF, methyl salicylate (MS) is also naturally occurring, and both are generally regarded as safe fumigants for their higher threshold limit values (TLV) and residual status below minimum residue limit (MRL). Sub-lethal doses of EF and MS were evaluated to determine if there was a synergistic effect on fumigation against life stages of the adzuki bean beetle, Callosobruchus chinensis (L.) infesting adzuki bean grains. Based on lethal dose (LD) values, the C. chinensis eggs and adults were equally susceptible to EF followed by larvae and pupae. Adults were the most MS-sensitive followed by eggs and larvae, and pupae were the most tolerant stage. The mortalities of all the life stages of C. chinensis by combined fumigation with sub-lethal EF and MS doses were significantly higher compared to the sum of their separate fumigations revealing a notable synergistic effect. Moreover, the maximum doses of EF and MS tested for synergistic fumigation test did not hamper the germination potential of adzuki been seeds. The synergistic effect between EF and MS can significantly reduce the dose rate of flammable EF against C. chinensis thereby offering a good prospect for EF + MS formulation as an alternative to safeguard stored adzuki bean.     

The dynamics of laboratory populations ofCallosobruchus chinensis andC. Maculatus (Coleoptera: Bruchidae) in patchy environments

Experiments are described showing the long-term dynamics of two species of bruchid beetles (Callosobruchus chinensis and C. maculatus) in arenas in which the resource of 50 black-eyed beans is divided between 5, 10 or 50 ‘patches’. Both species of adult beetles exhibit clumped distributions between patches. Within a patch there is a tendency for a density dependent reduction in (1) eggs laid per female, (2) the proportion of eggs hatching per bean (C. chinensis only) and (3) larval survival which is strongly overcompensating (particularly in C. maculatus). A discrete generation model is used as a framework to draw these results together and show how the different factors affecting natality and mortality can influence the population dynamics. Finally, the importance of the resource renewal interval in influencing the period of the population cycles is discussed.     

Sperm precedence in <Emphasis Type="Italic">Callosobruchus chinensis</Emphasis> estimated using the sterile male technique

P₂, the proportion of offspring sired by the second male to mate, is an indicator of the outcome of postcopulatory sexual selection, which occurs through sperm competition and/or cryptic female choice. We determined the appropriate dose of gamma radiation for sterilization of adult males and, using the sterile male technique, measured P₂ in the adzuki bean beetle, Callosobruchus chinensis. Adult males of C. chinensis were almost completely sterilized when irradiated at 80 Gy. Thus, we obtained sterile males through irradiation at this dose. Neither the probability of female first mating nor the probability of female remating was affected by whether females were paired with normal or sterile males. The P₂ calculated from the hatching success of eggs laid by females that mated both with normal and sterile males did not differ between reciprocal mating sequences, indicating that the sterilization has no effect on sperm fertilizing ability. The P₂ was estimated at 0.25. This study shows that female remating in C. chinensis means the coexistence of sperm from two males and thus the occurrence of postcopulatory sexual selection within the female reproductive tract, resulting in first-male sperm precedence.     

Competitive exclusion between contest and scramble strategists in <Emphasis Type="Italic">Callosobruchus</Emphasis> seed–beetle modeling

Interspecific competition between an intermediate contest strain of Callosobruchus maculatus and a scramble strain of C. chinensis was investigated on two types of resource beans: the azuki (Vigna anguralis, small seed) and the black-eye bean (Vigna unguiculata, large seed). We conducted both single-generation competition experiments and multiple-generation experiments and then analyzed the system with a discrete version of the Lotka–Volterra competition model. The estimated competition coefficients showed the competitive dominance of the C. maculatus strain over the C. chinensis strain in both types of bean. Zero-growth isocline analysis based on the predicted model showed that an overall dominance of the contest species, C. maculatus, in azuki beans, whereas the outcome of competition was dependent on the initial population size of each species in the black-eye beans. In the multiple-generation experiments, C. maculatus was the successful competitor irrespective of the ratio of the two beans, whereas C. chinensis overcame C. maculatus in one of five replicates with the black-eye beans system. When we incorporated the cost of scramble competition, or density-dependent reduction in body mass into the predicted model, the model alteration decreased the survival region of C. chinensis in phase space on the black-eye bean. Thus, the competitive equations, which do not consider the density-dependent body size reduction, tend to overestimate the advantages of scramble strategists. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Bioefficacy of plant essential oils against pulse beetles Callosobruchus spp. (Coleoptera: Bruchidae) in pigeon pea seeds with particular reference to Clausena pentaphylla (Roxb.) DC.

Thirty essential oils from higher plants of Gorakhpur Division (India) were evaluated at 0.36?μl/ml against two pulse beetles, Callosobruchus chinensis L. and C. maculatus F., causing infestation of pigeon pea seeds during storage. Clausena pentaphylla oil was more effective and exhibited absolute repellency against both the insects followed by Ocimum canum, Salvia plebeia and Zingiber zerumbet oils. Among these four oils, C. pentaphylla oil was most toxic and showed 100% mortality of both the insects at 10-μl dosage and 24-h exposure (LD₅₀?=?2.7?μl for C. chinensis & 2.4?μl for C. maculatus). Physical factors, viz. temperature, storage and autoclaving, did not cause any adverse effect on the toxicity of Clausena oil. During in vivo investigation, the oil protected 1?kg of pigeon pea seeds completely without reducing weight loss and seed damage up to 6?months when stored in gunny bags and glass containers. The oil was standardised by determining its various physicochemical properties. Thus, C. pentaphylla oil can be judiciously exploited as herbal insecticide against pulse beetles of pigeon pea seeds during storage.     

Seed germination temperatures of eight Mexican Agave species with economic importance

The genetic diversity of Agave plants is threatened by clonal commercial reproduction and climatic change. Sexual reproduction is uncommon and research on seed germination is scarce. The present study evaluated the seed germination of Agave lechuguilla, Agave striata, Agave americana var. marginata, Agave asperrima, Agave cupreata, Agave duranguesis, Agave angustifolia ssp. tequilana and Agave salmiana at constant temperatures (10, 15, 20, 25, 30, 35 and 40°C). Initial imbibition (after the first 12 h) was significantly variable among species, positively correlated with seed weight (r = 0.6560, P < 0.001) and increased with temperature (from 35% at 10°C to 66% at 40°C). Temperature affected maximum imbibition (83–150%) for A. asperrima, A. lechuguilla, A. salmiana and A. striata; other species averaged 110%. Most germination kinetics best fitted a logistic model, whereas only a few treatments fit a Weibull model. The time to germination onset diminished (P < 0.05) from 125–173 h at 15°C to 68–84 h at 25°C, and then ascended to 84–196 h at 35°C. The mean germination rate and seed germination percentage after 312 h peaked at 25°C (0.50–0.95% seeds/h and 85–99%, respectively) and fell (P < 0.05) to near zero at 10 and 40°C. Temperatures of 10, 35 and 40°C were partially lethal to A. asperrima, A. duranguensis and A. salmiana seeds. The time to germination onset, seed germination percentage after 312 h and mean germination rate are best described by a Gaussian distribution, with its optimum at approximately 25°C. Thus, optimum temperatures are related to the ecological characteristics of each species area.     

Avoidance of reproductive interference causes resource partitioning in bean beetle females

Reproductive interference is any interspecific sexual interaction that adversely affects female fitness through indiscriminate reproductive activities. It can be a driving force of resource partitioning in conjunction with resource competition. We previously showed that the bean beetle Callosobruchus maculatus is superior in larval resource competition, but vulnerable in reproductive interference, compared with its congener C. chinensis. We hypothesized that these two species might use two resources differently if one of the resources modified the intensity of reproductive interference or resource competition. We observed that C. maculatus females often enter gaps between beans to avoid mating attempts of heterospecific males, and hypothesized that removing bean gaps would strengthen reproductive interference. Therefore, we provided normal beans with gaps and split beans without gaps to females of the two species housed with conspecific or heterospecific males or no males and compared the number of eggs on each bean type among treatments. Callosobruchus maculatus females housed with heterospecific males were more likely to oviposit on normal beans than C. chinensis females. As a result, more C. chinensis adults hatched from split beans and more C. maculatus hatched from normal beans when females and males of both species were housed together. Thus, oviposition resource partitioning resulted from the combination of female avoidance of reproductive interference and resource competition.