Microsatellite DNA markers for the ground beetle Carabus insulicola

Twelve microsatellite DNA loci were newly isolated from the ground beetle Carabus insulicola, endemic to Japan, for studying mating systems. Phage vector was used for establishing genomic DNA library, and positive clones were screened with ³²P‐labelled probes. Primer sequences and annealing temperature for PCR amplification were determined for each locus. Allelic polymorphism of each locus, number of alleles and observed and expected heterozygosities, were investigated based on 24 individuals. Similarity between observed and expected heterozygosities suggests random mating, and relatively low observed heterozygosity found in two loci may be due to the presence of null alleles.     

Spermatophore displacement and male fertilization success in the ground beetle Carabus insulicola

Sperm displacement is an effective male mating tactic of spermcompetition to ensure paternity. However, the effectivenessof sperm displacement may depend on defensive mating tacticsemployed by rival males and also may be affected by females.To test this hypothesis, I conducted double-mating experimentswith microsatellite paternity analyses using the ground beetleCarabus insulicola. First, I examined how often spermatophoredisplacement by the second male occurs. Spermatophore depositionsuccess of the second male was high (85%) as was that of thefirst male (100%), in which 80% of the second males displacedthe first male's spermatophore, suggesting that spermatophoredisplacement is a highly effective offensive tactic of spermcompetition in C. insulicola. Second, I examined the effectsof postcopulatory guarding by the first male and remating intervaland sperm utilization pattern of the female on fertilizationsuccess of the second male. P₂ values were moderate on average(0.30–0.57) and negatively correlated with postcopulatoryguarding duration of the first male. Sperm use by the femalewas temporally nonrandom, such that sperm of the second maletended to be used first, although there was no significant effectof female-remating interval. These results suggest that theeffectiveness of spermatophore displacement depends on postcopulatoryguarding by the first male and the sperm utilization patternof the female. These findings provide empirical evidence forthe conflicting nature of selection for male offensive and defensivetactics in sperm competition and significant contribution offemales therein.     

Mating behavior and the function of the male genital spine in the ground beetle Carabus clathratus

The morphologies of male genitalia often appear harmful or aggressive, as if they may inflict physical damage upon females during copulation. Such male genitalia are often thought to function in intra- and intersexual interactions during mating. In the carabid genus Carabus, division Spinulati, males possess a spine (spinula) on the intromittent organ, of which function is unknown. To reveal the function of the spinula, we studied the mating behavior and genital coupling of a Spinulati species, Carabus (Limnocarabus) clathratus. The males positioned the spinula along the inner wall of the vaginal opening throughout copulation. This placement created a small dent and subsequently a melanized patch (wound) on the vaginal wall, but the spinula rarely penetrated the vaginal wall. The spinula did not reach the innermost part of the vagina where the spermatophore is deposited. These results suggest that the spinula is not used for inflicting damage on female genitalia or manipulating spermatophores of rival males. During spermatophore formation, the male partially withdrew the aedeagus, and only the aedeagal tip and endophallus remained within the female. By placing the spinula against the vaginal wall, the male could hold the endophallus within the vaginal chamber in the unstable copulatory posture. Thus, our observations suggest that the spinula primarily functions as an "anchor" to maintain the coupling of the male and female genitalia and thereby ensure insemination.     

Isolation and characterization of microsatellite loci in the ground beetle Carabus problematicus (Coleoptera,Carabidae)

Six microsatellite loci were isolated from the ground beetle Carabus problematicus. The polymorphism ranged from two to 17 alleles and observed and expected heterozygosities ranged from 0.137 to 0.676 and 0.147 to 0.710, respectively.     

Isolation and characterization of microsatellite loci in the ground beetle Carabus nemoralis (Coleoptera,Carabidae)

We isolated and characterized 10 microsatellite loci in the ground beetle Carabus nemoralis (Coleoptera, Carabidae), an ubiquist species largely distributed in Europe. Polymorphism ranged from 3 to 12 alleles on the 30 individuals from the same locality examined.     

Age-related sperm transfer and sperm competitive ability in the male hide beetle

The influence of male age on reproductive success after a singlemating has been explored widely; however, few studies have investigatedwhether quantitative or qualitative differences in male spermare responsible for the observed patterns. Moreover, the roleof male age on sperm competitive ability has been largely ignored.We examined the importance of male age on the probability andamount of sperm transferred during a single mating and exploredwhether sperm competitive ability varies with male age in thehide beetle Dermestes maculatus, a species where sperm viabilitydoes not vary with male age. We also investigated whether spermtransfer rates varied with female age. We found that the probabilityof sperm transfer and the amount of sperm transferred variedwith male, but not female, age. All males performed behaviorallysuccessful copulations, but intermediate-age and old males weremore likely to transfer sperm successfully and also transferreda greater quantity of sperm than young males. Old males wereless likely to transfer sperm than intermediate-age males, butif they did transfer sperm successfully, they transferred comparableamounts. Sperm competitive ability varied with male age andreflected the quantity of sperm transferred. On average, intermediate-agemales achieved greater fertilization success when competingagainst young or old males than when competing against otherintermediate-age males. Old males were poor competitors againstintermediate-age males, but they achieved significantly higherrates of fertilization when competing against young males. Ourfindings suggest that quantitative differences in the amountof sperm transferred determine male success in sperm competitionin the hide beetle.     

Hybridization, developmental stability, and functionality of morphological traits in the ground beetle Carabus solieri (Coleoptera, Carabidae)

The assessment of developmental stability in hybrids can provide valuable information in the study of species formation because it allows an evaluation of the degree of incompatibility of genetic systems that control developmental processes. The present study assessed the impact of two hybridization events, assumed to have occurred at different times, on developmental instability in the ground beetle Carabus solieri . Developmental instability was estimated in 678 individuals from 27 populations from the fluctuating asymmetry (FA) levels of four morphological traits: the tibia length of middle and hind legs, which are functional structures, and the length and the proximal width of the hind wings, which are vestigial and thus nonfunctional structures. Significant variations of FA levels between populations were shown only for the wing width. For this trait, FA levels in hybrids were higher than in their parental entities for both hybridization events, indicating a significant divergence of the gene systems controlling development between the parental entities in the two hybridization cases. As expected, wing traits exhibited FA levels at least three times higher than leg trait. Finally, the potential interest of vestigial traits in the particular context of hybridization is discussed.  © 2006 The Linnean Society of London, Biological Journal of the Linnean Society , 2006, 89 , 151–158.     

Sex differences in morphology across an expanding range edge in the flightless ground beetle,Carabus hortensis

1. Species’ ranges are dynamic, changing through range shifts, contractions, and expansions. Individuals at the edge of a species’ shifting range often possess morphological traits that increase movement capacity, that are not observed in individuals farther back within the species’ range. Although morphological traits that increase in proportion toward the range edge may differ between the sexes, such sex differences are rarely studied.
2. Here, we test the hypotheses that body size and condition increase with proximity to an expanding range edge in the flightless ground beetle, Carabus hortensis, and that these trait changes differ between the sexes.
3. Male, but not female, body size increased with proximity to the range edge. Body size was positively correlated with male front and mid tibia length and to female hind tibia length, indicating that body size is indicative of movement capacity in both sexes. Body condition (relative to body size) decreased with increasing population density in males but not females. Population density was lowest at the range edge.
4. Our results indicate that sex is an important factor influencing patterns in trait distribution across species’ ranges, and future studies should investigate changes in morphological traits across expanding range margins separately for males and females. We discuss the implications for sex differences in resource allocation and reproductive rates for trait differentiation across species’ shifting ranges.

     

Phylogeography and introgressive hybridization of the ground beetle Carabus yamato in Japan based on mitochondrial gene sequences

To study the phylogeography of the ground beetle Carabus yamato in Japan, we compared 1,020-bp sequences of the mitochondrial NADH dehydrogenase subunit 5 (ND5) gene from 373 specimens from 37 localities with those of three parapatric species (C. albrechti, C. kimurai, and C. japonicus) that might share mitochondrial lineages with C. yamato through introgressive hybridization. We found 81 haplotypes from C. yamato. Of these, 17 haplotypes were considered to be of an introgressed lineage from C. albrechti, based on the phylogeny and geographic distribution. In addition, one haplotype of C. kimurai was likely an introgressant from C. yamato. Putative introgression events among the four species were restricted to these two directional cases. We analyzed the phylogeography of C. yamato using nested clade phylogeographical analysis and population genetic parameters. The mitochondrial lineages of C. yamato were estimated to have diverged no more than approximately 1.12 million years ago, implying that the estimated historical events occurred after the Early Pleistocene. Carabus yamato was inferred to have experienced a contraction of its distribution range, followed by recent range expansion. Populations in the western and eastern regions, segregated by Ise Bay and the Nobi and Okazaki Plains, diverged in the mitochondrial clades. The northern and most western populations possessed one clade only (except an introgressed lineage), whereas eastern and some southwestern populations possessed several diverged clades, which were considered to be ancestral; these populations may have been associated with refugia during glacial periods.     

Physical mapping of rDNA genes in the ground beetle Carabus and related genera (Carabidae: Coleoptera)

The major ribosomal DNA (rDNA) loci were localized on meiotic and mitotic chromosomes and in interphase nuclei of 18 ground-beetle species belonging to three tribes of the supertribe Carabitae by fluorescence in situ hybridization (FISH), using a PCR-amplified 18S rDNA as a probe. Meiotic observations indicate that the 18S rDNA sequences are located on the largest autosomal bivalent in 12 species of Carabus , two species of Calosoma (both genera belonging to the tribe Carabini), and three sibling species of Ceroglossus chilensis (tribe Ceroglossini). The data suggest the occurrence of a conservative pattern in these three genera despite the chromosomal rearrangements that have led to karyotypes with higher chromosome numbers in Ceroglossus . A different result is found in Cychrus caraboides (tribe Cychrini), where ribosomal cistrons are located in two medium-sized autosomal pairs. Further species of Cychrini should be studied for corroborating the occurrence of molecular and karyotypical apomorphies in Cychrus with regard to the genera Carabus, Calosoma and Ceroglossus .     

Genetic differences and phenotypic plasticity in body size between high- and low-altitude populations of the ground beetle Carabus tosanus

The body size of a univoltine carabid beetle Carabus tosanus on Shikoku Island, Japan, was clearly smaller in higher‐altitude populations (subspecies), which possibly represents incipient speciation. To explore the determinants of altitudinal differences in body size in this species, we studied the degree of phenotypic plasticity by conducting rearing experiments at two constant temperatures and examined genetic differences through interpopulation crosses. At 15 °C, C. tosanus had a longer developmental period and a shorter adult body than at 20 °C. Nevertheless, variation in body size due to temperature effects (phenotypic plasticity) was small compared to the interpopulation differences, which suggests substantial genetic differences between populations (subspecies) at different altitudes. In F₁ offspring from crosses between a low‐altitude (subspecies tosanus) and a high‐altitude population (subspecies ishizuchianus), adult body length was affected by the genotypes of both parents, with an interaction effect of parental genotype and offspring sex. Further analyses revealed that adult body length was affected by sex‐linked factors in addition to autosomal factors. These genetic differences in body size may have resulted from adaptations to different altitudes and may be important for the process of incipient speciation because body size differences could contribute to premating reproductive isolation.     

Historical divergence of mechanical isolation agents in the ground beetle Carabus arrowianus as revealed by phylogeographical analyses

In the carabid genus Carabus subgenus Ohomopterus , diverged body size and genital morphology serve as mechanical reproductive barriers. To elucidate the diverging process of body and genital sizes in Carabus arrowianus , which exhibits marked morphological diversity among geographical populations and may represent an early stage of speciation, we analysed a mitochondrial gene sequence for 1051 individuals from 63 populations and male morphology for 359 individuals from 47 populations. Two discrete morphological groups segregated by geographical barriers were distinguished, one of which possessed smaller bodies and shorter genitalia ( S group) than the other ( L group), which exhibited larger bodies and exaggerated genitalia. Genetic divergence between the two groups was significant but not large. Phylogeographical and population genetic analyses indicated that the L group was derived from the S group, and a coalescent simulation revealed that the two groups diverged during the latest middle Pleistocene (0.13 million years ago), with a much larger effective population size in the L group than the S group. Because the body size divergence could not be explained by adaptation to climatic conditions and genital morphology is considered to be subject to sexual selection, we postulated that a population division and colonization in favourable habitats caused by the Pleistocene climatic and geographical change might facilitate natural and sexual selection for enlarged body and genital sizes in the L group.     

Mating behavior of the cabbage beetle, Colaphellus bowringi (Coleoptera: Chrysomelidae)

Abstract  Circadian mating rhythms, mating frequency, mating duration, and the effect of mating duration on fecundity and fertility in the cabbage beetle, Colaphellus bowringi were investigated in the laboratory. Mating occurred throughout the 24-h cycle but the majority of copulations occurred in the photophase with two apparent peaks, one at 8:00 and another at 16:00. Mating frequency observations for 10 consecutive days indicated that pre-mating period of C. bowringi was about 4 days, and pairs mated an average of 5 times per day and an average of 40 times during the first 10 days. There was a negative correlation between mating frequency and mating duration during the consecutive mating. The mean duration of the first copulation (136.24 ± 4.62 min) was significantly longer than those of the second (57.87 ± 2.03 min), third (53.05 ± 2.05 min) and fourth copulation (30.86 ± 2.98 min). Fecundity showed a slight increase with increasing mating duration but no significant difference among treatments. However, fertility was significantly influenced by the mating duration in this species. Mating of 20-min duration did not produce viable eggs. The mean percentage of fertile eggs with completed mating duration (204.43 ± 18.96 min, 56.75% fertile eggs) was significantly higher than those with 60 min (39.55%) and 30 min (17.91%) mating duration, suggesting that the longer mating duration might be associated with transfer of more sperm that are used to increase the fertility of eggs.     

Mating behavior of an aphidophagous ladybird beetle,Propylea dissecta （Mulsant）

Abstract The mating behavior of Propylea dissecta (Mulsant) was investigated in detail. Protandry was evident, as males mature earlier than females. Predicted age of sexual maturation of male and female ladybirds was 7.12 and 9.33 days, respectively. Males performed courtship in six steps, viz. approach, watch, examine, embrace, mount and attempt. Embrace is possibly an appeasement act, while examine serves to recognize mate. Chemical signals initiate male attraction toward females. Visual and tactile cues appear to secondarily help in mate recognition. Mating lasted longest (275.40±12.23 min) when it occurred between unmated individuals. It was much shorter (176.60±5.60 min) when prior mated individuals copulated. There was a significant decrease in mating durations when adults were subjected to five successive matings. Fecundity and percent egg viability increased significantly with increase in the number of matings.     

Marked sperm dimorphism in the ground beetle Scarites terricola: a novel type of insect sperm polymorphism

Sperm polymorphism describes the phenomenon of male ejaculates containing two or more distinct types of sperm. In insects, four types of sperm polymorphism are recognized in species from the orders Diptera, Hemiptera, Hymenoptera, Lepidoptera and Coleoptera. The present study describes dimorphic sperm of the ground beetle Scarites terricola (Coleoptera: Carabidae) as a novel type of sperm polymorphism in insects. Sperm from the spermatophore and male seminal vesicles are examined at the light‐microscopic level, and both display marked dimorphism. One type has sperm formed into bundles, in which the head of numerous spermatozoa are ‘glued’ together, with tails free‐moving. The other type are free as single spermatozoa and have a disproportionately large‐sized head and an elongated tail. Both types are motile in Ringer's solution. The adaptive and phylogenetic importance of these findings is discussed.     

Intraperitoneal insemination, sperm transport and capacitation in the pig

Intraperitoneal insemination was studied in a total of 58 pigs, both to ascertain the success of this route of sperm deposition with the eventual use of frozen-thawed boar semen in mind and to estimate the timing of capacitation in the absence of uterine exposure of spermatozoa. Ovulation was controlled in mature gilts, and 5–20 ml freshly collected semen containing approximately 10⁸ spermatozoa per ml introduced through the peritoneum either by means of mid-ventral laparotomy or using a 3.5-in (ca. 9 cm) × 18-gauge hypodermic needle.Embryo development to the morula and blastocyst stage appeared chronologically and cytologically normal after intraperitoneal insemination, but the timing of semen deposition was critical: optimal levels of fertilization (60%) arose from insemination in the 12 h preceding ovulation. Fertility was never comparable to that found after natural mating due to the inefficiency of sperm transport into the oviducts and the absence of significant sperm reservoirs. The timing of sperm capacitation after intraperitoneal insemination was not reduced when compared with that found after insemination directly into the oviducts, indicating a negligible contribution of peritoneal exposure to this process. Spermatozoa were not phagocytosed in the oviducts, but rather descended to the uterus at the same time as the developing embryos or degenerating eggs, the sperm flagellum usually being separated from the head by this stage.     

Spernathecal morphology,sperm transfer and a novel mechanism of sperm displacement in the rove beetle,Aleochara curtula (Coleoptera,Staphylinidae)

Summary The mechanism by which sperm are transferred from the male's spermatophore to the female's storing cage is described for the rove beetle Aleochara curtula, emphasizing a novel mechanism of sperm displacement by competing males. The cuticular, U-shaped spermatheca is equipped with a valve structure and two sclerotized teeth. The tube of the spermatophore extends into the spermathecal duct through the guidance of the flagellum of the male endophallus. Further elongation of the spermatophore tube, however, occurs only after separation of the pair. A primary tube bursts at its tip after passing through the valve. Within the lumen of the primary tube, a second tube passes through the valve and continues to extend up to the apical bulb of the spermatheca, doubles back on itself and swells to form a balloon filling most of the spermatheca. The balloon of the spermatophore is pierced within the spermatheca by tooth-like structures pressed against the spermatophore through contraction of the spermathecal muscle. The same process of spermatophore growing and swelling is also observed in mated females. Sperm from previous copulations are backflushed through the valve and the spermathecal duct, indicative of last-male sperm predominance.Abbreviations ad adhesive secretion covering the sperm - sac am amorphous secretion of the spermatophore - as ascending portion of the spermatophore - ds descending portion of the spermatophore - end parts of the male endophallus - ext extended tube - f flagellum - gs genital segment - lt large tooth - m muscle of the spermatheca - nsc non sclerotized cuticle - op opening of the spermathecal gland - pt primary tube - sc sclerotized cuticle - sd spermathecal duct - se secretion of the spermathecal gland - sf secretion flowing out of the primary tube - sg spermathecal gland - sm sperm - smt small tooth - sp spermatheca - ss sperm sac - st secondary tube - vm vaginal muscle