Effects of temperature on mating duration, sperm transfer and remating frequency in Callosobruchus chinensis

Insect body temperature is usually determined by ambient temperature. Therefore, most biochemical and physiological processes underlying behavioural patterns are temperature dependent. Mating duration is also dependent on temperature, and therefore temperature should influence on sperm transfer and female remating frequency. In the adzuki bean beetle, Callosobruchus chinensis, we found negative relationships between ambient temperature and mating duration, sperm transfer and sperm transfer duration. Female remating frequency at lower temperature (17 °C) was lower than at other temperatures (25 °C and 33 °C). The physiological and behavioural significance of these results is discussed. The number of ejaculated sperm was significantly lower at 33 °C than at 17 °C; the effect of temperature on sperm transfer is discussed in relation to the intensity of female refusal behaviour directed against males.     

Intra-specific variation in strategic ejaculation according to level of polyandry in Callosobruchus chinensis

Optimal sperm allocation should differ according to the level of polyandry within a population, because the risk of sperm competition depends on the re-mating frequency of females. We compared the number of sperm ejaculated by males into the female reproductive organ between strains with different levels of polyandry in the adzuki bean beetle, Callosobruchus chinensis (Coleoptera: Bruchidae) when males were reared in different larval densities in a bean. The results showed that males derived from a population with a higher level of polyandry increased ejaculatory expenditure when they were reared under higher larval densities. We discuss the evolutionary correlation of ejaculatory expenditure to the level of polyandry.     

Female mating receptivity inhibited by injection of male-derived extracts in Callosobruchus chinensis

The effects of male-derived extracts on female receptivity to remating were investigated in Callosobruchus chinensis (Coleoptera: Bruchidae). Injection of aqueous extracts of male reproductive tracts into the abdomen of females reduced receptivity. When aqueous extracts of male reproductive tracts were divided to three molecular weight (MW) fractions by ultrafiltration: <3, 3-14, and >14 kDa, the filtrate containing MW substances <3 kDa reduced female receptivity 3h and 1 day after injection, whereas the fraction containing MW substances >14 kDa inhibited receptivity 2 and 4 days after injection. Finally, male reproductive tract organs were divided into accessory gland, seminal vesicle, and testis. Aqueous extracts of testis reduced receptivity of females on the second day and at 3h, and aqueous extracts of accessory gland reduced receptivity of females on the second day after injection. On the other hand, aqueous extracts of seminal vesicle did not reduce female receptivity. The results indicate that more than one mechanism may be involved in producing the effects of male-derived substances on female receptivity; low MW male-derived substances, which possibly exist in testis, cause short-term inhibition, while high MW substances, which possibly exist in the accessory gland, inhibit female mating later than low MW substances in C. chinensis.     

Costs of mating and egg production in female Callosobruchus chinensis

Costs of reproduction include the costs of mating and egg production. Specific techniques such as irradiation or genetic mutation have been used to divide the expense into costs of mating and egg production in previous studies. We tried to divide the costs in the adzuki bean beetle, Callosobruchus chinensis (Coleoptera: Bruchidae), which needs some kinds of bean as an oviposition substrate. Mated females that were not allowed to lay eggs had a shorter life span than virgin females, but they had a longer life span than mated females that were allowed to lay eggs. The results showed two independent significant costs, mating and egg production, on the life span in C. chinensis. Costs of mating, however, include the costs of sexual harassment by males and copulation itself, and we need further studies to divide the costs. The present method for dividing the cost of reproduction into costs of mating and egg production can be applied to a broad taxonomic range of insect species, and thus it will be a useful model system for inter-specific comparisons of costs of mating and egg production.     

Female mating receptivity after injection of male-derived extracts in Callosobruchus maculatus

The effects of male-derived extracts on female receptivity were investigated in Callosobruchus maculatus (Coleoptera: Bruchidae). Injection of aqueous extracts of the male reproductive tract into the abdomen of females reduced receptivity. Aqueous extracts of male reproductive tracts were divided to three molecular weight (MW) fractions by ultrafiltration: Fractions: (I) MW<3 kDa, (II) 3-14 kDa, and (III)>14 kDa. Fraction II reduced female receptivity from 3h after injection, and Fraction III reduced female receptivity from 2 days after injection. On the other hand, no effect on receptivity was found for Fraction I. Furthermore, male reproductive tract organs were divided into accessory gland, testis, and seminal vesicle including the ejaculatory duct. Aqueous extracts of the seminal vesicle reduced receptivity of females immediately following injection, while aqueous extracts of the accessory gland reduced receptivity at the second day. The results suggest that the components of Fraction II existed in the seminal vesicle, and those of Fraction III in the accessory gland. The results of the present and the previous studies in Callosobruchus chinensis, a species closely related to C. maculatus, were compared and are discussed from the viewpoint of the significance of ejaculation in the two species.     

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.     

Interpopulation variation in female remating is attributable to female and male effects in <Emphasis Type="Italic">Callosobruchus chinensis</Emphasis>

The evolution of female multiple mating is best understood by consideration of male and female reproductive perspectives. Females should usually be selected to remate at their optimum frequencies whereas males should be selected to manipulate female remating to their advantage. Female remating behavior may therefore be changed by variation of male and female traits. In this study, our objective was to separate the effects of female and male strains on female remating for the adzuki bean beetle, Callosobruchus chinensis, for which there is interstrain variation in female remating frequency. We found that interstrain variation in female remating is primarily attributable to female traits, suggesting genetic variation in female receptivity to remating in C. chinensis. Some interstrain variation in female remating propensity was attributable to an interaction between female and male strains, however, with the males of some strains being good at inducing nonreceptivity in females from one high-remating strain whereas others were good at inducing copulation in nonvirgin females from the high-remating strain. There is, therefore, interstrain variation in male ability to deter females from remating and in male ability to mate successfully with nonvirgin females. These results suggest that mating traits have evolved along different trajectories in different strains of C. chinensis.     

Presence and partial characterisation of a major proteolytic enzyme in the larval gut of Callosobruchus maculatus

Protease activity in the gut of larvae of the bruchid beetle, Callosobruchus maculatus (a storage pest of cowpea seeds), has been investigated to help clarify nutritional mechanisms in view of reports that these insects carry out little or no proteolysis (Applebaum, 1964). Larval gut homogenates showed protease activity against a variety of different protein substrates, but did not hydrolyse a synthetic trypsin substrate. The proteolytic activity had a pH optimum of 5.4. It was not inhibited by serine protease inhibitors, but was inhibited by reagents reactive against-SH groups. Protein trypsin inhibitors from legume seeds which are not hosts to C. maculatus (soybean, limabean) were not effective inhibitors of the larval proteolytic activity but a cowpea protease inhibitor preparation and aprotinin partially inhibited proteolysis. The latter two inhibitors also inhibited the plant thiol protease papain. It is suggested that C. maculatus has replaced the normal insect proteases with an enzyme similar to plant proteases to evade the antimetabolic effects of trypsin/chymotrypsin inhibitors in seeds. Besides trypsin/chymotrypsin inhibitors, cowpea seeds also contain proteins which inhibit papain; these inhibitors were purified and were shown to be effective inhibitors of C. maculatus larval protease.

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Mise en évidence et caractérisation partielle d'une enzyme protéolytique importante du tube digestif des larves de Callosobruchus maculatus

Résumé Callosobruchus maculatus attaque les graines stockées particulièrement de Vigna unguiculata. L'activité protéolytique du tube digestif des larves a été examinée. Aucune hydrolyse n'a été observée contre la N-benzoyl arginine p-nitroanilide (substrat de la trypsine synthétique), mais la protéolyse a été mise en évidence en utilisant la viciline de V. unguiculata comme substrat naturel ou la myoglobine comme substrat artificiel, le p.H optimum est de 5,4. Les inhibiteurs chimiques des protéases de la sérine n'ont pas altéré l'activité enzymatique, mais les réactifs du groupe SH l'ont inhibée. Nous suggérons que cette protéase est une thiol protéase, c'est à dire avec un groupe actif semblable à celui de la papaïne, protéase végétale. En accord avec cette hypothèse, les inhibiteurs de protéine efficaces seulement contre la trypsine, c'est à dire les inhibiteurs de la trypsine de Glycine max et de la trypsine de Phaseolus lunatus n'affectent pas cette protéase larvaire, tandis que les protéines avec une faible action inhibitrice contre la papaïne (préparation inhibitrice de la protéase de V. unguiculata, aprotinine) inhibent partiellement la protéolyse par des extraits de tube digestif larvaire.Des inhibiteurs protéiques de papaïne extraits de graines de V. unguiculata sont des inhibiteurs très efficaces de la protéase larvaire. On peut penser que C. maculatus contient une protéase semblable aux protéases végétales, plutôt qu'aux protéases classiques, d'insectes, ce qui permet d'éviter les effets antimétaboliques directs des inhibiteurs de protéase (inhibiteurs de trypsine et de chymotrypsine) trouvés en quantité relativement importantes dans les graines de légumineuses.

     

Negative relationship between ambient temperature and death-feigning intensity in adult Callosobruchus maculatus and Callosobruchus chinensis

Abstract.  Although the effects of temperature on insect behaviours are studied frequently, few studies report on the relationship between temperature and anti-predator behaviours. A negative relationship between ambient temperature and the intensity of death-feigning is found in adults of two seed beetle species, Callosobruchus maculatus (F.) and C. chinensis (L.) (Coleoptera: Bruchidae). Two traits representing the intensity of immobility, the frequency and the duration of death-feigning, are measured at different temperatures. Almost all adults feign death at 15 °C, but the frequency of death-feigning decreases at higher temperatures in C. maculatus , whereas all C. chinensis adults show this behaviour at 15 and 20 °C and almost all show it at 25 °C, but the frequency of death-feigning decreases at 30 and 35 °C. The difference between the two species might be due to the specific strain of each species used in the experiment. The duration of death-feigning is correlated negatively with the increase in ambient temperature in both species. The frequency at which adults feigned death is higher in females than in males in both species, but the duration of death-feigning is higher in females than in males only in C. maculatus . The relationships between temperature and death-feigning behaviours are discussed from physiological and ecological viewpoints.     

Offspring production with cryopreserved sperm from a live-bearing fish Xiphophorus maculatus and implications for female fecundity

Xiphophorus fishes are well-established models for biomedical research of spontaneous or induced tumors, and their use in research dates back to the 1930s. Currently, 58 well-pedigreed lines exist among 24 Xiphophorus species housed as live animals at the Xiphophorus Genetic Stock Center. The technique of sperm cryopreservation has been applied to preserve these valuable genetic resources, and production of offspring has been reported with cryopreserved sperm in two species (X. helleri and X. couchianus). The goal of this research was to establish protocols for sperm cryopreservation and artificial insemination that yield live young in X. maculatus, a widely used research species. The objectives were to: 1) collect basic biological characteristics of males, and quantify the sperm production yield after crushing of dissected testis; 2) cryopreserve sperm from X. maculatus by adapting as necessary the protocols for sperm cryopreservation of X. helleri and X. couchianus; 3) use cryopreserved sperm to inseminate virgin females of X maculatus and other species (X. helleri and X. couchianus), and 4) compare experimental trials over a 3-year period to identify opportunities for improving female fecundity. In total, 117 males were used in this study with a standard length of 2.5 ± 0.3 cm (mean ± SD), body weight of 0.474 ± 0.149 g, and dissected testis weight of 7.1 ± 3.7 mg. Calculation of sperm availability showed 5.9 ± 2.8 × 10(6) sperm cells per mg of testis weight. Offspring were produced from cryopreserved sperm. Male-to-male variation (1-70%) was observed in post-thaw motility despite little variation in motility before freezing (60-90%) or genetic variation (~100 generations of sib-mating). Comparisons of biological factors of males did not have significant correlations with the production of live young, and the influence of females on production of young was identified from the comparison of artificial insemination over 3 years. Overall, this study describes offspring production from cryopreserved sperm in a third species of Xiphophorus fishes, and identifies the opportunities for improving female fecundity which is essential for establishment of germplasm repositories for Xiphophorus fishes.     

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.     

Intraspecific variation in the egg-spacing behavior of the seed beetleCallosobruchus maculatus

Females of Callosobruchus maculatustend to distribute their eggs uniformly among host seeds and, thereby, reduce competition among larvae within seeds. We investigated variability in this behavior by assaying beetles from seven geographic strains on each of three host species. To quantify egg-spacing behavior,we devised an index (U)that estimates the uniformity of the egg distribution but is independent of the number of eggs laid. Egg-spacing behavior was highly variable among beetle strains and legume hosts. Females from an Indian strain frequently achieved the most uniform distribution possible (maximum hyperdispersion), whereas females from two Brazilian strains frequently deposited eggs randomly. Differences among strains were evident throughout the period of oviposition;some strains were sloppy when even egg densities were low. Variation in egg-spacing behavior appears to be influenced by prior host associations and by host size. The most well-developed spacing behavior was observed in a strain associated with the smallest host, which supports only one or two larvae per seed. Realized fecundity also varied significantly among beetle strains. We found no evidence, however, of a predicted tradeoff between the number of eggs laid and the uniformity of the egg dispersion.     

Vibrations mediate prudent resource exploitation by competing larvae of the bruchid bean weevil Callosobruchus maculatus

Larvae of the bean weevil C. maculatus feeding in a single bean apparently respond to vibrations from each other's chewing: one larva feeds normally, the other is inhibited. If the burrows of the larvae intersect, the inhibited larva dies. If the dominant larva pupates or dies without the burrows intersecting, the inhibited larvae then feeds and matures if enough food remains. Since females add second eggs to the largest beans after most beans carry a single egg, competition is most common in the largest available beans, precisely where inhibited larvae can benefit from avoiding a contest.

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Résumé Lors de la compétition, les larves réagissent l'une par rapport à l'autre, même lorsque leurs galeries sont séparées. L'une est inhibée, tandis que l'autre se comporte comme si elle était seule. Le signal provoquant ces réactions, qui peut traverser du papier métallique, est dû vraisemblablement aux vibrations de mastication des larves; ces dernières perçoivent ces signaux tout au long de leur vie active. La larve la plus âgée ne l'emporte pas obligatoirement, ce qui signifie que le signal indique plus que l'âge.Les conditions expérimentales peuvent inverser les réponses des unes et des autres, bien que la larve la plus âgée puisse souvent mourir de ces conditions anormales de compétition. L'avantage potentiel associé à l'inhibition du futur perdant est mis en évidence en collant ensemble des graines. Quand la larve dominante se nymphose, la larve inhibée recommence à s'alimenter et achève son développement. En ayant eu son alimentation interrompue et en restant dans une galerie superficielle, la larve inhibée évite les affrontements et peut se développer lorsque la larve dominante meurt ou se nymphose, puisque la galerie de la larve inhibée n'avait pas été sectionnée.La stratégie de ponte réduit la fréquence de la compétition en uniformisant la distribution des oeufs; elle augmente les chances de survie des larves vaincues avec l'addition des oeufs sur les graines les plus grosses quand les disponibilités sont limitées. L'étude de l'évolution de ces adaptations a été possible puisque, comme on le sait, C. maculatus est génétiquement polymorphe pour les caractères déterminant le comportement de ponte des femelles et la compétition larvaire.

     

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.     

Independence of genetic variation between circadian rhythm and development time in the seed beetle, Callosobruchus chinensis

A positive genetic correlation between periods of circadian rhythm and developmental time supports the hypothesis that circadian clocks are implicated in the timing of development. Empirical evidence for this genetic correlation in insects has been documented in two fly species. In contrast, here we show that there is no evidence of genetic correlation between circadian rhythm and development time in the adzuki bean beetle, Callosobruchus chinensis. This species has variation that is explained by a major gene in the expression and period length of circadian rhythm between strains. In this study, we found genetic variation in development time between the strains. The development time was not covaried with either the incidence or the period length of circadian rhythm among the strains. Crosses between strains suggest that development time is controlled by a polygene. In the F₂ individuals from the crosses, the circadian rhythm is attributable to allelic variation in the major gene. Across the F₂ individuals, development time was not correlated with either the expression or the period length of circadian rhythm. Thus, we found no effects of major genes responsible for variation in the circadian rhythm on development time in C. chinensis. Our findings collectively give no support to the hypothesis that the circadian clock is involved in the regulation of development time in this species.     

Performance of bean bruchids Callosobruchus maculatus and Zabrotes subfasciatus (Coleoptera: Bruchidae) reared on resistant (IT81D-1045) and susceptible (Epace 10) Vigna unguiculata seeds: relationship with trypsin inhibitor and vicilin excretion

Callosobruchus maculatus (Cm) and Zabrotes subfasciatus (Zs) were reared on resistant (IT81D-1045) and on susceptible (Epace 10) cowpea seeds. The emergence of adult insects, total developmental period (TDP) and excretion of trypsin inhibitor and vicilin were determined for both bruchid populations. Parameter evaluation showed that the Zs populations emerged from both seeds had no significant differences in emergence and TDP. The Cm population raised from resistant seeds had lower emergence (5.6+/-1.3%) and delayed TDP (46+/-1.25 days) than those emerged from susceptible seeds. The excretion of defense proteins showed that Zs reared in resistant seeds excreted 1.7 times more trypsin inhibitor, but this did not affect emergence or TDP. Furthermore, Cm population emerged from resistant seeds excreted 7 times higher vicilin and 0.4 times less trypsin inhibitor than that emerged from susceptible seeds. These results indicate that vicilins from resistant seeds are involved to significantly longer TDP (46 days) and also drastic reduction of insect emergence ( approximately 5%) of C. maculatus.