Molecular genetic evidence for alternative reproductive strategies in North American pitvipers (Serpentes: Viperidae): long‐term sperm storage and facultative parthenogenesis

The first documentation of facultative parthenogenesis (FP) in non‐avian reptiles (snakes) occurred in 1997, following the application of molecular genetic methods for parentage analysis, and since has been described in only four additional snake species. Here, in two species of live‐bearing (viviparous) North American pitviper snakes, using microsatellite DNA fingerprinting, we describe the first record of a virgin birth by FP in the copperhead (Agkistrodon contortrix), and the first genetically confirmed case of long‐term sperm storage (LTSS) of exceptional duration (5 years) in the eastern diamond‐backed rattlesnake (Crotalus adamanteus). The capacity of female squamate reptiles (snakes, lizards, and amphisbaenians) to store viable sperm for extended periods of time is a competing hypothesis to FP, and it is often inferred when interpreting certain births where adult females have been isolated from adult male mates for prolonged periods. On reviewing the literature of certain cases of LTSS in snakes, we provide compelling support for the prevalence of FP, casting doubt over the widespread acceptance of LTSS. Accordingly, with the discovery of FP in squamates and the results of the present study, we advocate that in order to differentiate between LTSS and FP, especially under natural conditions, rigorous molecular testing will be required. © 2011 The Linnean Society of London, Biological Journal of the Linnean Society, 2011, 104 , 934–942.     

Annually recurring parthenogenesis in a zebra shark Stegostoma fasciatum

A zebra shark, Stegostoma fasciatum, held in captivity at the Burj Al Arab aquarium, produced embryos and pups in the absence of a male. A total of 15 pups were produced from eggs laid within the aquarium over a period of four consecutive years commencing 2007. Parthenogenesis was confirmed through DNA analysis for three pups sampled during the first two consecutive egg cycles and is presumed to be the method of reproduction responsible thereafter.     

No evidence for Wolbachia‐induced parthenogenesis in the social Hymenoptera

In some parasitoid wasps, infection by the micro‐organism Wolbachia leads to asexual reproduction. Within the Hymenoptera, the limits of distribution of parthenogenesis inducing Wolbachia have not yet been established. To address this issue, we screened all known thelytokous social hymenopteran species using a PCR assay. None was infected, and therefore we conclude that worker thelytoky evolves independently of Wolbachia in ants and bees. This supports the previously proposed hypothesis that a sex determining system based on heterozygosity may form a proximate limitation to Wolbachia‐induced parthenogenesis.     

New insights on facultative parthenogenesis in pythons

In vertebrates, facultative parthenogenesis (i.e. asexual reproduction by a sexually reproducing species) has been documented in four diverse taxonomic groups, namely sharks, birds, lizards, and snakes. With a single exception, the mode is terminal fusion automixis, where the second polar body fuses with the nucleus of the gamete, restoring diploidy and triggering cell division. The deviating case involves a report of a captive Burmese python (Python bivittatus), a giant Asiatic species common in zoological gardens and the pet trade. Although terminal fusion automixis produces half‐clones of the mother, under this unique case in P. bivittatus, the foetuses were reported as full clones. This conclusion is an apparent anomaly with respect to the mechanism of facultative parthenogenesis reported in all other snakes. In the present study, using genotyping methods, we analyze facultative parthenogenesis in two other species of pythonids and report results that challenge the abovementioned conclusions regarding clonality. Specifically, we report new findings comparable to those reported in other primitive snakes (namely boids), which support the hypothesis of terminal fusion automixis as the mode of facultative parthenogenesis. Furthermore, in light of our new data, we re‐examine the previous report of facultative parthenogenesis in the Burmese python and suggest an intriguing alternative explanation for the earlier findings. © 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 112 , 461–468.     

Novel microsatellite markers suggest the mechanism of parthenogenesis in Extatosoma tiaratum is automixis with terminal fusion

Parthenogenetic reproduction is taxonomically widespread and occurs through various cytological mechanisms, which have different impact on the genetic variation of the offspring. Extatosoma tiaratum is a facultatively parthenogenetic Australian insect (Phasmatodea), in which females oviposit continuously throughout their adult lifespan irrespective of mating. Fertilized eggs produce sons and daughters through sexual reproduction and unfertilized eggs produce female offspring via parthenogenesis. Here, we developed novel microsatellite markers for E. tiaratum and characterized them by genotyping individuals from a natural population. We then used the microsatellite markers to infer the cytological mechanism of parthenogenesis in this species. We found evidence suggesting parthenogenesis in E. tiaratum occurs through automixis with terminal fusion, resulting in substantial loss of microsatellite heterozygosity in the offspring. Loss of microsatellite heterozygosity may be associated with loss of heterozygosity in fitness related loci. The mechanism of parthenogenetic reproduction can therefore affect fitness outcomes and needs to be considered when comparing costs and benefits of sex versus parthenogenesis.     

Allelic inheritance in naturally occurring parthenogenetic offspring of the gonochoric sea star Patiria miniata

Abstract. In laboratory studies of fertilization using the gonochoric broadcast-spawning asterinid sea star Patiria miniata , we found many cases in which some mature eggs spawned by females formed cleavage-stage embryos and feeding bipinnaria larvae without fertilization by sperm. Segregation of maternal microsatellite alleles among the parthenogenetic offspring of known heterozygous females was consistent with several specific modes of asexual reproduction, including polar body suppression. Cryptic outcrossing by sperm contamination was ruled out by the failure to observe non-maternal alleles. The potential for asexual reproduction by the normally outcrossing members of P. miniata may suggest a shared propensity for asexuality among asterinid species from several clades in which isolated adults can produce offspring without outcrossing.     

Negative evidence of parthenogenesis induction by Wolbachia in a gallwasp species, Dryocosmus kuriphilus

The alpha‐proteobacteria of the genus Wolbachia is a widespread group of maternally inherited endosymbionts of arthropod and nematode hosts. Wolbachia infection induces a range of host phenotypes, including cytoplasmic incompatibility, male killing, feminization, and induction of thelytokous parthenogenesis. Heterogony (cyclical parthenogenesis) is a remarkable characteristic of oak gallwasps, Cynipini, the largest tribe of the Cynipidae. A few species of Cynipini are exceptional in that they are univoltine and exhibit thelytokous parthenogenesis, probably because they lost the arrhenotokous generation of their heterogonic ancestor species due to Wolbachia infection. In this study, the presence of Wolbachia was detected using polymerase chain reaction primers for the wsp genes in a thelytokous parthenogenetic species [Dryocosmus kuriphilus (Yasumatsu)] (Hymenoptera: Cynipidae: Cynipini). Approximately 29.8 and 87.1% of adults of the Zhuzhou and Fuzhou strains, respectively, were infected with Wolbachia while all females of the remaining four strains collected from other localities in China were Wolbachia free. The length of the wsp fragment of Zhuzhou and Fuzhou strains was found to be 573 and 561 bp, respectively. The nucleotide sequence of the bacterial wsp fragment indicated that the endosymbiotic bacteria of the Zhuzhou and Fuzhou strains are members of supergroup A, but belong to different clades; they probably originated from two independent infection events. In conclusion, thelytokous parthenogenesis of D. kuriphilus is not caused by Wolbachia infection and the deletion of the arrhenotokous generation is thus not associated with such an infection.     

Molecular evidence for two vitellogenin genes and processing of vitellogenins in the American cockroach, Periplaneta americana

The American cockroach, Periplaneta americana has two vitellins (Vn1 and Vn2) and corresponding vitellogenins (Vg1 and Vg2). Vns/Vgs were separated on the SDS-PAGE as three major polypeptide bands [170, 100 (multisubunits), and 50 kD] and a minor polypeptide band (150 kD) both in the egg (mature terminal oocyte) extract and in the female hemolymph. We previously cloned one Vg (Vg1) cDNA and showed that the 170-kD polypeptide originated from the C-terminus of the Vg1. In the present study, we cloned the other Vg (Vg2) cDNA. It is 5,826 bp long encoding 1,876 amino acid residues (including 16 residues for putative signal peptide) in a single ORF. The deduced amino acid sequences of both Vgs (Vg1 and Vg2) of P. americana showed 30% identity. The GL/ICG motif is followed by eight cysteine residues at conserved locations near the C-terminal and the DGXR motif starts 18 residues upstream of the GL/ICG motif. The chemically determined N-terminal amino acid sequences of the 150-kD and of the 50-kD polypeptides matched exactly with each other and with the deduced N-terminal amino acid sequence of the Vg2 cDNA. The pattern of processing in P. americana Vns/Vgs is discussed.     

On the function of sex pheromone components in Periplaneta americana: improved odour source localization with periplanone-A

ABSTRACT. The female American cockroach, Periplaneta americana L., produces two sex pheromone components: periplanone-A (PA) and periplanone-B (PB). PB induces male locomotion and upwind orientation. PA, when presented alone, has the same effect but with a higher threshold concentration. When added to PB in the natural ratio, however, PA reduces rather than increases locomotory activity. To examine whether PA affects male orientation in the natural blend, a pheromone source was suspended above the centre of a circular arena. Efforts were made to exclude air movements so that pheromone concentration declined centrifugally. Males walking in a PB gradient spent more time near the source than in the peripheral areas when low doses were presented. At emission rates approximately equivalent to those of a female, however, they were not able to locate the pheromone source as precisely as at lower rates. In contrast, males walking in a PA gradient spent more time in the centre of the arena when the pheromone dosage was increased. When PA was added to natural levels of PB, males walked more slowly and stopped more frequently near the source than with PB alone. Thus PA improves chemo-orientation of males when presented in a natural ratio with PB. A reduction of linear velocity near the pheromone source (an orthokinetic effect) is involved in this process.     

Parthenogenesis in Hexapoda: Entognatha and non‐holometabolous insects

In hexapods, unlike the majority of animals, development without fertilization is a common phenomenon. They evolved a striking diversity of unisexual reproductive types that include a variety of modes starting from spontaneous parthenogenesis in females to the production of impaternate males with different variants in between. Many reports about parthenogenetic species have accumulated over time. Here, we present a review of various parthenogenetic hexapod groups with a particular focus on their chromosome systems and ploidy level. We show that conclusions about the reproductive mode often lack solid evidence and sometimes inefficiently demonstrate how parthenogenesis is maintained in corresponding groups. In this review, basal hexapods (Protura, Collembola, Diplura), primarily wingless insect groups (‘Apterygota’) and non‐holometabolous insects are listed with references to a variety of their unisexual reproductive modes.     

First record of second‐generation facultative parthenogenesis in a vertebrate species,the whitespotted bambooshark Chiloscyllium plagiosum

In this study, two parthenogenetic events within a family of the whitespotted bambooshark Chiloscyllium plagiosum are reported. A captive female produced multiple parthenogens. Unexpectedly, a single specimen of a total of nine parthenogens displayed external claspers characterizing the male sex in chondrichthyans. Upon dissection, internal sexual organs of this specimen were malformed or absent; however, the presence of claspers in this study challenges the as yet assumed sex determination system in this shark species. Even more remarkable was that one of the female parthenogens reproduced asexually again producing viable offspring. As far as is known, this is the first genetically confirmed evidence for second‐generation facultative parthenogenesis in vertebrates. These results support the evolutionary significance of parthenogenesis as an alternative to sexual reproduction.     

Observable and efficient rearing system for wood-feeding cockroaches

Plant decomposition is an essential component of forest ecosystems. Wood-feeding cockroaches are responsible for the decomposition of rotten logs. However, the ecology of wood-feeding cockroaches is not well understood. To understand how they decompose rotten logs in the gallery inside the logs, it is necessary to observe them in laboratories. Here, I present an observable and cost-effective for rearing these log decomposers using a culture dish and an artificial fungal meal. The culture dish is traditionally used to cultivate fungi or bacteria with the lid on. The artificial fungal meal is a commercial product for stage beetle larvae, which are a kind of log decomposers. These let researchers easy to deal with wood-feeding cockroaches. In the course of keeping over 1,000 individuals of the wood-feeding cockroaches, I have developed effective techniques to rear them under the observable system for several years. I present these techniques along with several tips to keep the wood-feeding cockroaches healthy and observe them using video cameras. These techniques remove many of the limitations for study on the plant decomposers. When researchers use this observable rearing system, we can conduct rearing experiments easily and well understand the ecology of wood-feeding cockroaches and insects associating with them.     

Production of females by parthenogenesis in the ant,Cerapachys biroi

Summary Groups of virginCerapachys biroi workers produced females (workers) in the laboratories. This is the first report of thelytoky in Cerapachyinae.     

Asexual reproduction in introduced and native populations of the ant Cerapachys biroi

Asexual reproduction is particularly common among introduced species, probably because it helps to overcome the negative effects associated with low population densities during colonization. The ant Cerapachys biroi has been introduced to tropical and subtropical islands around the world since the beginning of the last century. In this species, workers can reproduce via thelytokous parthenogenesis. Here, we use genetic markers to reconstruct the history of anthropogenic introductions of C. biroi, and to address the prevalence of female parthenogenesis in introduced and native populations. We show that at least four genetically distinct lineages have been introduced from continental Asia and have led to the species' circumtropical establishment. Our analyses demonstrate that asexual reproduction dominates in the introduced range and is also common in the native range. Given that C. biroi is the only dorylomorph ant that has successfully become established outside of its native range, this unusual mode of reproduction probably facilitated the species' worldwide spread. On the other hand, the rare occurrence of haploid males and at least one clear case of sexual recombination in the introduced range show that C. biroi has not lost the potential for sex. Finally, we show that thelytoky in C. biroi probably has a genetic rather than an infectious origin, and that automixis with central fusion is the most likely underlying cytological mechanism. This is in accordance with what is known for other thelytokous eusocial Hymenoptera.     

Thelytokous parthenogenesis in the exotic dacetine ant Strumigenys rogeri (Hymenoptera: Formicidae) in Taiwan

Only recently has it become clear that several species of eusocial hymenopterans regularly reproduce by thelytokous parthenogenesis, that is, the production of diploid female offspring by unmated females. This phenomenon suggests that parthenogenetic reproduction might be advantageous to organisms under certain environmental conditions. Here the occurrence of asexual reproduction is reported for the first time in the dacetine ant, Strumigenys rogeri, at least for the focal populations in Taiwan. Virgin queens of S. rogeri maintained with several workers produced both workers and young queens from unfertilized eggs under laboratory conditions in as short as 39 days, whereas workers were strictly sterile as no spermatheca was discovered after dissection. Combined with additional evidence (i.e. absence of males in field colonies), queen thelytoky is confirmed. Such a reproductive mode and short development time may jointly help explain the success of this tramp ant species in Taiwan and elsewhere.     

The molecular and metabolic essentials for long-distance flight in insects

Summary The mechanism of long-distance flight in insects was investigated by comparing lipid mobilization and transport in gregarious- and solitary-phase locusts and in the American cockroach. Unlike the gregarious-phase locust, both the American cockroach and the solitary locust were unable to form low-density lipophorin (loaded with increased amount of diacylglycerol) even when injected with adipokinetic hormone (AKH). The cockroach fat body responded to AKH. However, not only does the American cockroach lack apolipophorin-III (apoLp-III) in the haemolymph, but the fat body contains only an extremely small amount of diacylglycerol and a relatively large triacylglycerol pool. By contrast, the solitary-phase locust had apoLp-III in the haemolymph, but the fat body was only one-seventh or less in weight of the fat body of the gregarious locust. Furthermore, the fat body of the solitary locust contains a very small amount of triacylglycerol (1/20 or less of that of the gregarious locust) with only a trace of diacylglycerol. It was concluded that in the American cockroach and the solitary locust, the stores of fuel in the fat body are insufficient to maintain prolonged flight.Abbreviations AKII adipokinetic hormone - apoLp-III apolipophorin III - HDLp high-density lipophorin - LDLp low-density lipophorin - LTP lipid transfer particle - MW molecular weight - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis     

Polymorphic population genetic markers for the Australian wood cockroach Panesthia australis

Primers for five polymorphic nuclear DNA (nDNA) markers and one mitochondrial DNA (mtDNA) gene (COI) were developed for the Australian wood cockroach Panesthia australis using a range of techniques. Eight mitochondrial haplotypes and four to 36 alleles per nuclear locus were detected in 744 cockroaches. Observed heterozygosity ranged from 0.117 to 0.816 in a sample of 30 animals from one population. The markers will be useful for population biology and for the measurement of the effects of habitat fragmentation on this ecologically important forest‐dependent species.     

Molecular genetic evidence for parthenogenesis in the Burmese python,Python molurus bivittatus

Parthenogenesis among reptiles is rare. Only a few species have the ability to reproduce asexually. Most of these are obligate parthenogenetic species that consist (almost) entirely of females, which can reproduce solely through parthenogenesis. Rarer are sexual species that only sporadically reproduce through parthenogenesis. A female Python molurus bivittatus (Reptilia, Boidae) from the Artis Zoo, Amsterdam, produced eggs in five consecutive years that contained embryos while she was isolated from males. These eggs might be fertilized with stored sperm, or might be the product of parthenogenesis. Parthenogenesis has not been shown for the Boidae family before. We performed parentship analyses on the snake and seven of her embryos using microsatellites and AFLP. Four microsatellite loci developed for this species combined with three loci developed previously for different snake species revealed too little variation to discriminate between sperm retention and parthenogenesis. With AFLP we were able to confirm that the Artis Zoo female reproduced parthenogenetically. Because the offspring are genetically identical to their mother, whereas in previous studies on sporadic parthenogenesis in snakes a loss of genetic information was reported, we conclude that the meiotic pathways that produce the diploid egg cells are different.     

Repetitive DNA in the automictic fungus <Emphasis Type="Italic">Microbotryum violaceum</Emphasis>

The small genomes of fungi are expected to have little repetitive content other than rDNA genes. Moreover, among asexual or highly selfing lineages, the diversity of repetitive elements is also expected to be very low. However, in the automictic fungus Microbotryum violaceum, a very large proportion of random DNA fragments from the autosomes and the fungal sex chromosomes are repetitive in nature, either as retrotransposon or helicase sequences. Among the retrotransposon sequences, examples were found from each major kind of elements, including copia, gypsy, and non-LTR sequences. The most numerous were copia-like elements, which are believed to be rare in fungi, particularly among basidiomycetes. The many helicase sequences appear to belong to the recently discovered Helitron type of transposable elements. Also, sequences that could not be identified as a known type of gene were also very repetitive within the database of random fragments from M. violaceum. The differentiated pair of fungal sex chromosomes and suppression of recombination may be the major forces determining the highly repetitive content in the small genome of M. violaceum.     

Dynamic linkage relationships to the mating‐type locus in automictic fungi of the genus Microbotryum

Regions of the chromosomes determining mating compatibility in some fungi, including Microbotryum lychnidis‐dioicae and Neurospora tetrasperma, exhibit suppressed recombination similar to sex chromosomes in plants and animals, and recent studies have sought to apply basic theories of sex chromosome evolution to fungi. A phylogeny of the MTL1 locus in Microbotryum indicates that it has become part of the nonrecombining regions of the mating‐type chromosomes in multiple independent events, and that recombination may have been subsequently restored in some cases. This illustrates that fungal mating‐type chromosomes can exhibit linkage relationship that are quite dynamic, adding to the list of similarities to animal or plant sex chromosomes. However, fungi such as M. lychnidis‐dioicae and N. tetrasperma exhibit an automictic mating system, for which an alternate theoretical framework exists to explain the evolution of linkage with the mating‐type locus. This study encourages further comparative studies among fungi to evaluate the role of mating systems in determining the evolution of fungal mating‐type chromosomes.