Multistage mechanism of reproductive sympatric isolation of the closely-related species, Ixodes persulcatus and I. Pavloskyi (Ixodidae)

The closely related species Ixodes persulcatus and I. pavlovskyi are vectors of agents tick-born encephalitis and Lyme disease. These species have two great disjunctive sympatric distribution areas (fig. 1), where both species inhabit the same biotopes, and during the same season. In these conditions there are prerequisites of contacts of opposite sexes of the different species during ambushing the hosts on vegetation. Sequence of "switching on" of the morphological, physiological, and hostparasite factors of reproductive isolation has been established. The features and the range of manifestation of these factors at different combinations of sexes and species of the partners have been revealed. The studied factors, such as precopulative and postcopulative components of the mechanism of reproductive isolation of I. persulcatus and I. pavlovskyi were considered. A hypothesis for sympatric origin of the species as a result of specialization the adults of I. persulcatus to mammals, and I. pavlovskyi to birds is proposed.     

Long-term dynamics of density and diversity of ticks (Ixodidae) on the natural and disturbed territories

As result of long-years monitoring of density and diversity of Ixodidae living on Tomsk territory and in suburbs it has been shown that at least two species of ticks (I. persulcatus and I. pavlovskyi) inhabit there simultaneously. It has been studied that percent of I. pavlovskyi increased nearby town buildings. In the natural biotopes I. persulcatus is dominated, while I. pavlovskyi is founded rare. It has been shown, that during last 15 years (since 1996 till 2010) density of ticks increase on the town suburbs. The combination of ecological and chemical methods of tick suppression on the limited territory may leads to significant decreasing of ticks density. There is one biotope only, where non-typical for this region species (Dermacentor reticulatus and Haemaphysalis concinna).     

Detection of the West Nile Virus and its genetic typing in ixodid ticks (Parasitiformes: Ixodidae) in Tomsk City and its suburbs

Four tick species, Ixodes persulcatus, I. pavlovskyi, I. trianguliceps, and Dermacentor reticulatus, were found in Tomsk and its suburbs in 2006. The species I. pavlovskyi was found to be dominant in the localities situated in Tomsk City, and I. persulcatus was dominant in its suburbs. Viral RNA and viral antigen of the West Nile virus (WNV) were detected in the ticks I. pavlovskyi and I. persulcatus collected in the city and its suburbs by the RT PCR method and enzyme immunoassay with monoclonal antibodies against protein E of the WNV. Average rate of the WNV infected ticks varied from 5.2 up to 11.7% in different localities. Identification of the nucleotide sequence of the protein E gene fragment allowed classifying the cDNA obtained as genotype Ia of the WNV. The sequences are proved similar to the strain LEIV-Vlg99-27889-human of the WNV isolated in Volgograd. The obtained data showed that natural foci of the WNV virus can appear in the city and its suburbs probably involving two dominant tick species. The WNV infected imagoes, larvae, and nymphs of I. persulcatus and I. pavlovskyi were collected from small mammals, lizards, and birds. Therefore we presume that these hosts can be involved in the circulation and distribution of WNV on the territory of Tomsk Region.     

The infection of ixodid ticks collected from humans with the tick-borne encephalitis virus in Tomsk city and its suburbs

The ticks Ixodes persulcatus and I. pavlovskyi collected from people visited gardens and suburban forests have been examined by the IFA methods on the presence of tick-borne encephalitis virus (TEV). It was established, that the most part of ticks collected from humans belongs to I. persulcatus, despite the fact that I. pavlovskyi dominates on the territory of the city and its suburbs. TEV infection was registered more often in fed ticks, in comparison with those without signs of preceding feeding. Infected specimens constituted 9.4% of 53 I. persulcatus individuals examined. In I. pavlovskyi 3.7% of 27 examined specimens were infected. In fed ticks (41 specimens of I. persulcatus and 20 I. pavlovskvi) the rates of infected specimens were higher and constituted 48.8 and 35%, respectively. In suburban forests, as well as in the city, I. persulcatus was found to attack humans more often than I. pavlovskyi. TEV infection was recorded in 12.7% of 220 unfed I. persulcatus specimens examined and in 41.6% of 142 fed individuals. In I. pavlovskyi, 6.6% of unfed and 25% of fed specimens was infected (33 unfed and 24 fed specimens were examined). Thus, in all territories examined, percent of infected specimens was significantly lower in I. pavlovskyi, and in fed ticks of both species, TEV was recorded more often.     

Impact of male condition on his spermatophore and consequences for female reproductive performance in the Glanville fritillary butterfly

In butterflies, male reproductive success is highly related to the quality and the size of the spermatophore transferred to the female. The spermatophore is a capsule produced by the male during copulation, which in many species contains sperm in addition to a nuptial gift, and which is digested by the female after copulation. The nuptial gift may contribute to egg production and offspring quality, and in some cases also to female body maintenance. The production of the spermatophore, however, represents a cost for the male and, in polyandrous species, ejaculates are sometimes allocated adaptively across matings. Nonetheless, although the ecological factors affecting the reproductive success of female butterflies have been the topic of numerous studies, little information exists on the factors affecting males’ contribution to reproduction, and the indirect impacts on female fecundity and fitness. We used the Glanville fritillary butterfly, Melitaea cinxia (Linnaeus, 1758) (Nymphalidae), in order to assess variation in male allocation to matings. In this species, smaller males produce smaller spermatophores, but variation in spermatophore size is not correlated with female reproductive success. We show that spermatophore size increases with male age at first mating, decreases with mating frequency and adult food‐deprivation, and is not influenced by developmental food‐limitation. The length of copulation period does not influence the spermatophore size nor influences the polyandrous mating behavior in this species. Male contribution to his spermatophore size is clearly influenced by his condition and adult‐resource at the time of mating. Despite this variation, spermatophore size does not seem to have a direct impact on female reproductive output or mating behavior.     

Peritrophic matrix in the midgut of tick females of the genus Ixodes (Acari: Ixodidae)

The formation of the peritrophic matrix in the midgut of females of 5 ixodid tick species (Ixodes pacificus, I. pavlovskyi, I. persulcatus, I. ricinus and I. scapularis) was studied by means of light and electron microscopy in different periods of the feeding and after detachment. The formation of the peritrophic matrix started when the first food portions came into the gut lumen, 9-12 hours after the attachment. Renovation of the peritrophic matrix took place during the whole feeding period; every new generation of midgut cells synthesized their own matrix. It was deposited on the apical surface of every midgut cell in the beginning of differentiation, and was functioning during the life of the cell. The peritrophic matrix separates spaces of the cavitary and cytozoic digestions.     

Spermatophore transfer in the hermit crab Clibanarius vittatus (Crustacea,Anomura, Diogenidae)

Although mating has been described in several hermit crab species, the mechanics of spermatophore transfer have not previously been demonstrated. Evidence from pleopod and gonopore morphology, video observations, and inseminated females indicates that in Clibanarius vittatus the male applies a spermatophoric mass directly onto the female via the gonopores rather than with modified pleopods 1-2 (gonopods) and/or genital papillae as in many other decapods. The single second pleopod of males of C. vittatus has a simple endopod with no apparent modifications for sperm transfer. There are no genital papillae extending from the male gonopores. The globular spermatophores are aligned in rows surrounded by a seminal secretion in the male ducts (vasa deferentia that terminate in ejaculatory ducts opening to the exterior via the gonopores). During copulation, described from time-lapse video recordings, the ventral surface of the last thoracic segment of the male, bearing the gonopores, was apposed to the ventral cephalothorax of the female. A massive amount of seminal secretion containing spermatophore ribbons, termed here the spermatophoric mass and described for the first time in a hermit crab species, was observed covering the sternites and coxae of pereopods 1-5 of a recently copulated female. It is suggested that during copulation the male emits the contents of the ejaculatory ducts directly onto the female without the aid of gonopods or genital papillae. Although spermatophore transfer is simple in C. vittatus, the presence of modified anterior pleopods or elongate genital papillae (sexual tubes) in other paguroidean species suggests the possibility of a more complex insemination process in these other hermit crabs.     

Functional morphology of the reproductive system of Galathea intermedia (Decapoda: Anomura)

Spermatophore formation in Galathea intermedia begins in the proximal part of the vas deferens. The contents subsequently form a spermatophoric ribbon, the so-called "secondary spermatophore," in its distal part. A strongly muscular ductus ejaculatorius is present in the coxa of the fifth pereiopod which builds up pressure for the extrusion of the spermatophoric ribbon. After extrusion, the ribbon is caught by the first gonopod, while the second gonopod dissolves the matrix of the ribbon. During copulation the spermatophores are randomly placed on the sternum of the female, near the genital opening, by the fifth pereiopods of the male. Subsequent ovulation of the female via the genital opening, an active process accomplished through muscular activity, results in fertilization of the eggs by the exploding spermatophores. External intersexes are characterized by both male and female external sexual characters, but in all individuals only male gonads are present. No trace of a female reproductive system could be detected. Thus, these external intersexes are exclusively functional males.     

Sexual transmission of Borrelia garinii by male Ixodes persulcatus ticks (Acari, Ixodidae)

We investigated the transmission of Borrelia garinii and Borrelia afzelii between male and female Ixodes persulcatus ticks. For this purpose the infection rate of partners from tick couples was determined by polymerase chain reaction and reverse line blot. In couples, where the male tick was infected with B. garinii, four out of nine female partners carried B. garinii. In eight couples, male ticks had a dual infection of B. afzelii and B. garinii and three female partners were infected by Borrelia spirochetes. Two female ticks carried B. garinii, and one female tick had a dual infection. No evidence for transmission of B. afzelii from male to female ticks was found among seven couples. In 45 couples where the female tick was infected, not one male tick carried spirochetes. The difference in the B. garinii infection rate between male and female ticks among these couples is highly significant. Our data suggest that transmission of B. garinii from male ticks to female ticks does occur. Sexual transmission of this pathogen may play an important role in the maintenance of B. garinii in I. persulcatus. © Rapid Science Ltd. 1998     

Increased copulation duration before ejaculate transfer is associated with larger spermatophores, and male genital titillators, across bushcricket taxa

Copulation duration varies considerably across species, but few comparative studies have examined factors that might underlie such variation. We examined the relationship between copulation duration (prior to spermatophore transfer), the complexity of titillators (sclerotized male genital contact structures), spermatophore mass and male body mass across 54 species of bushcricket. Using phylogenetic comparative analyses, we found that copulation duration was much longer in species with titillators than those without, but it was not longer in species with complex compared with simple titillators. A positive relationship was found between spermatophore size and copulation duration prior to ejaculate transfer, which supports the hypothesis that this represents a period of mate assessment. The slope of this relationship was steeper in species with simple rather than complex titillators. Although the data suggest that the presence of titillators is necessary to maintain long copulation prior to ejaculate transfer, mechanisms underlying this association remain unclear.     

How the length of genital parts affects copulation performance in a carabid beetle: implications for correlated genital evolution between the sexes

To identify factors leading to the correlated evolution of exaggerated male and female genitalia, we studied the effects of the variable dimensions of corresponding functional genital parts (male copulatory piece and female vaginal appendix) on copulatory performance in the polygamous carabid beetle Carabus (Ohomopterus) maiyasanus. We used mating pairs of individuals from two populations to increase the variances in genital dimensions and determined the copulation performance (insemination and spermatophore replacement, and copulation time) in single‐ and double‐mating situations. In single mating, insemination success was not affected by genital dimensions, although the copulation time was significantly shorter when the male aedeagus was longer. In the double‐mating experiment, insemination and replacement of spermatophores by the second male succeeded more frequently when the copulatory piece was shorter and the vaginal appendix was longer, and when the difference between the length of the copulatory piece and the vaginal appendix was smaller. Thus, a matching of the corresponding genital parts between the sexes increases the male's reproductive success in sperm competition, but elongation of the copulatory piece cannot be explained simply by the improvement in male reproductive success. We discuss possible factors for the elongation of genital parts in terms of sexual conflict and reproductive interference through interspecific copulation.     

A new observation on spermateleosis in ticks.

The first phase of spermiogenesis in argasid and ixodid ticks is described. The inner core of the spermatid passes through the outer sheath so that speriogenesis is permitted to follow its course. The outer sheath is not ruptured in the process, but a well-defined operculum opens to let the inner core through. The opening of the operculum is a clear-cut indication of the beginning of spermiogenesis. In the examined species speriogenesis begins in Argasidae when the male transfers the spermatophore to the female genital opening, whereas in Ixodidae it starts only after the endospermatophore has reached the female genital tract.     

Female Reproductive Fitness Declines with Increasing Male Density in the Polyandrous Weevil Sitophilus oryzae

The effect of male density on female reproductive fitness is still poorly understood in many insect species. Here we examined how male density influenced female mating behaviour, fecundity, longevity and damage to the genital tract in Sitophilus oryzae (L.) (Coleoptera: Curculionidae), an important pest of whole grains worldwide. We show that with increasing male density, (1) both male mate competition and female copulation duration significantly increased and (2) female fecundity, longevity and feeding time significantly decreased. Dissection indicates that the backward pointing spines on the reversed internal sac of male genitalia may grip and damage the female genital tracts. These results suggest that longer copulation and more frequent matings under higher male densities reduce the time for feeding and oviposition and increase the damage to female genital tracts. We conclude that the decreased female reproductive fitness under high male densities is most likely caused by a combination of the decreased feeding and oviposition time and increased damage to female genital tracts.     

The taxonomic aspects of the transmission of the causative agent of Lyme disease

According to world literature data 17 species of ixodid ticks have been studied for natural infection with the Lyme disease agent. Analysis of the data on the level of the infection, transovarial and transphase transmission has shown that main biological vectors of Borrelia burgdorferi are the species of the subgenus Ixodes s. str. - I. ricinus, I. persulcatus (Eurasia), I. dammini, I. pacificus (North America). Potential vectors are I. scapularis, I. dentatus, Amblyomma americanum, Dermacentor variabilis. Single isolations were registered for I. neotomae, Haemaphysalis leporispalustris, D. occidentalis. Nonidentified spirocheta was isolated from A. americanum, D. variabilis, D. parumapertum, Rhipicephalus sanguineus. No agent was isolated from I. cookey, D. albipictus, R. reticulatus, H. concinna. On the basis of comparative and ontogenetic data the species from a group of main vectors: I. ricinus, I. persulcatus, I. pacificus had been attributed by me to the phyletic group persulcatus before Lyme disease was discovered and its causative agent isolated. The question whether I. scapularis belongs to the group persulcatus was also discussed at that time but left open due to somewhat aberrant structure of gnathosoma at preimaginal phases (Filippova, 1969, 1971, 1973). 6 Palaearctic, 2 Indomalayan and 3 Nearctic species were referred to the group persulcatus at the time. I. dammini was described later, in 1979. Gnathosoma of its preimaginal phases has an intermediate structure between I. scapularis and other species of the group persulcatus. Sexually mature phase and nymph of I. dentatus have much in common with Palaearctic members of the group, I. pavlovskyi, I. kazakstani, I. kashmicus. Preimaginal phases of I. scapularis and nymph of I. dentatus were studied by me on the collection material. Thus, it is possible to speak of the belonging of main vectors of B. burgdorferi to a common phyletic group within the subgenus Ixodes s. str. and, therefore, of common origin of ecological medium for the agent. At the same time each species of the vector is an evolutionally developed difference of ecological medium for B. burgdorferi. Roots of the group persulcatus could originate as far as in Paleocene before the land connection between North America and Europe disappeared. Conditions for the existence of recent species, however, appeared considerably later and their flourishing is dated by Pliocene. The main epidemiological role belongs now to I. ricinus, I. persulcatus, I. dammini, I. pacificus.(ABSTRACT TRUNCATED AT 400 WORDS)     

Characteristics of the distribution of ticks Ixodes persulcatus and Ixodes pavlovskyi at the border between the forest and forest-steppe zones in the territory near Ob River

Field investigations performed in 2009 and 2010 in the Novosibirskiy, Toguchinskiy, and Sovetskiy districts of Novosibirsk Province showed, that at present, Ixodes persulcatus, I. pavlovskyi (subspecies I. pavlovskyi occidentals Filippova et Panova, 1998), and Dermacentor reticulatus (Fabricius, 1794) permanently cohabitate in the woodland park of the Novosibirsk Akademgorodok, and I. pavlovskyi is the dominating species there. The highest abundance of I. pavlovskyi was recorded in pine forests subjected to intensive recreational load. At the same time, I. pavlovskyi was not found in the Novosibirskiy District and in the plane part of Toguchinskiy District, while the abundance of I. persulcatus is almost three times higher in the above mentioned territories, than in the Akademgorodok.     

Complex copulatory behavior and the proximate effect of genital and body size differences on mechanical reproductive isolation in the millipede genus Parafontaria

The role of species-specific genitalia in reproductive isolation is unclear. Males of the millipede genus Parafontaria use gonopods (modified eighth legs) charged with sperm from the genital openings of the second legs as intromittent organs. Males perform both preliminary and true intromission during mating. During preliminary intromission, a male attempts to insert his gonopods into the female genitalia before charging the gonopods with sperm. If this intromission is completed, it is followed by the ejaculation of sperm to the gonopods and true intromission for insemination. In two sympatric species of Parafontaria that lack effective precopulatory isolation, copulation was terminated without insemination because of preliminary intromission failure caused by mismatched genital and body sizes. Thus, mechanical isolation between these sympatric species resulted from morphological differentiation mediated by the obligatory preliminary intromission. These findings demonstrate the proximate importance of genital and body size differences for reproductive isolation within this genus of millipede.     

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.     

The evolution of male and female mating preferences in Drosophila speciation*

The relative importance of male and female mating preferences in causing sexual isolation between species remains a major unresolved question in speciation. Despite previous work showing that male courtship bias and/or female copulation bias for conspecifics occur in many taxa, the present study is one of the first large‐scale works to study their relative divergence. To achieve this, we used data from the literature and present experiments across 66 Drosophila species pairs. Our results revealed that male and female mate preferences are both ubiquitous in Drosophila but evolved largely independently, suggesting different underlying evolutionary and genetic mechanisms. Moreover, their relative divergence strongly depends on the geographical relationship of species. Between allopatric species, male courtship and female copulation preferences diverged at very similar rates, evolving approximately linearly with time of divergence. In sharp contrast, between sympatric species pairs, female preferences diverged much more rapidly than male preferences and were the only drivers of enhanced sexual isolation in sympatry and Reproductive Character Displacement (RCD). Not only does this result suggest that females are primarily responsible for such processes as reinforcement, but it also implies that evolved female preferences may reduce selection for further divergence of male courtship preferences in sympatry.     

No evidence for external genital morphology affecting cryptic female choice and reproductive isolation in Drosophila

Genitalia are one of the most rapidly diverging morphological features in animals. The evolution of genital morphology is proposed to be driven by sexual selection via cryptic female choice, whereby a female selectively uptakes and uses a particular male's sperm on the basis of male genital morphology. The resulting shifts in genital morphology within a species can lead to divergence in genitalia between species, and consequently to reproductive isolation and speciation. Although this conceptual framework is supported by correlative data, there is little direct empirical evidence. Here, we used a microdissection laser to alter the morphology of the external male genitalia in Drosophila, a widely used genetic model for both genital shape and cryptic female choice. We evaluate the effect of precision alterations to lobe morphology on both interspecific and intraspecific mating, and demonstrate experimentally that the male genital lobes do not affect copulation duration or cryptic female choice, contrary to long‐standing assumptions regarding the role of the lobes in this model system. Rather, we demonstrate that the lobes are essential for copulation to occur. Moreover, slight alterations to the lobes significantly reduced copulatory success only in competitive environments, identifying precopulatory sexual selection as a potential contributing force behind genital diversification.     

Longevity of forest and taiga ticks (Ixodidae) infected and non-infected with Borrelia burgdorferi groups

A life longevity of Ixodes ricinus and I. persulcatus have been examined in laboratory conditions. It was found that specimens infected with borreliae live slightly longer than non-infected ones (difference is not reliable); females live 1.5 times longer than males; I. ricinus lives 2-4 times longer than I. persulcatus. It is suggested that I. persulcatus has a higher rate of metabolism than I. ricinus and that the latter species obtains an ability to survive after the last moult two winters and keep an activity in two summer seasons not counting the year of moulting.