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Diversity within diversity: molecular approaches to studying microbial interactions with insects.

DNA sequence information has greatly augmented the number of characters available for analysis in phylogenetic research. Nowhere is this more evident than in studies of microbial evolution. Ribosomal DNA sequence data has simultaneously permitted the distinction between individual species and the inference of their phylogenetic relationships in many cases where both were formerly impossible. These have contributed to our understanding of the ecology of particular microbe-host interactions and the history of these relationships over evolutionary time. We describe examples from two ends of the ecological spectrum in insect/bacterial associations: one in which bacteria mediate host cytoplasmic incompatibility and parthenogenesis, and the other in which mycetocyte bacteria augment host nutrition. In the former, the pattern of bacterial interaction is general, with the same or closely related strains of the genus Wolbachia associating with a wide range of insect taxa. In the latter, concordance between host and microbe phylogenies suggests cospeciation between bacteria and host, although it is as yet unclear whether this process has involved step-wise, reciprocal coevolution. We conclude with a discussion of how developments in molecular techniques may aid in analyzing more complex interactions between insects and microbes.

Animals↗

Haplo-diploid locust embryos arising by accidental thelytoky in Chortoicetes terminifera investigated by G-banding.

Two mosaic sibling embryos of the Australian plague locust, Chortoicetes terminifera are reported with haploid and diploid cell lines in widely differing proportions. One small chromosome pair involved in the two cases has alternative morphology and a B-chromosome is present in one. In addition, G-banding identifies two medium-sized chromosome pairs and alternative states of a second small pair. Using these markers it is clear that both diploid cell lines are homozygous for the chromosomes of the corresponding haploid line. These embryos have thus developed by accidental parthenogenesis from haploid cells, some of which were duplicated by endomitosis after development began.

Animals↗

Effects of superoxide dismutase on mouse in vitro fertilization and embryo culture system.

PURPOSE: We recently found that, for mouse embryos fertilized in vivo, the two-cell block could be attenuated by adding superoxide dismutase (SOD), a scavenger of superoxide radicals, to the culture medium. In this study, we evaluated the effects of SOD on the process of fertilization and on the further development of the embryos fertilized in vitro. METHODS: We performed incubation of mouse epididymal spermatozoa, in vitro fertilization, and further cultivation in Biggers-Whitten-Whittingham's medium supplemented with various concentrations of Cu.Zn-SOD. RESULTS: High concentrations (2000 micrograms/ml or more) of SOD prevented loss of motility in mouse sperm over time. The addition of SOD (less than 2000 micrograms/ml) to the basic medium showed no significant difference in the fertilization rate. Also, no significant difference was observed in the rate of polyspermy or parthenogenesis between the basic and the SOD-supplemented media. However, 18% of the two-cell-stage embryos developed to the expanded blastocyst stage in the 500 micrograms/ml SOD-supplemented medium, while no blastocysts were found in the basic medium. Furthermore, the addition of SOD 7 hr after insemination increased the expanded blastocyst rate (28%). CONCLUSIONS: These results indicate that the addition of SOD exerts a protecting effect from oxidative stress both on sperm viability and on the development of embryos fertilized in vitro as well as in vivo, while its addition showed no effect on the process of fertilization.

Animals↗

Parthenogenetic activation of human oocytes by puromycin.

OBJECTIVE: Our objective was to obtain parthenogenetic activation of unfertilized human oocytes by puromycin and to try to use this procedure for cytogenetic purposes. SETTING: The setting was our IVF laboratory. METHODS: Eighty-two spare oocytes from 34 IVF patients were used. In the first series of experiments 39 unfertilized oocytes were cultured in medium containing 100, 50, or 10 micrograms/ml puromycin for 6 to 24 hr. After the appearance of pronuclei they were transferred to plain medium, further cultured, and cytogenetically analyzed. In the second series of experiments 43 oocytes were cultured for 5 to 10 hr in 10 micrograms/ml puromycin, transferred to plain medium, and fixed for cytogenetic analysis 2 hr after nuclear envelope breakdown. RESULTS: Ninety-one percent of the oocytes in the first experiment showed the presence of one or more nuclei. From the pronucleate oocytes additionally cultured in puromycin-free medium, 46% developed further to the metaphase of the first mitotic division or the two-cell stage and 54% remained arrested at the pronuclear stage. In the second experiment 88% of the treated oocytes showed pronuclei or had cleaved, and after withdrawal from puromycin 96% of the pronucleate oocytes entered mitosis. CONCLUSION: Puromycin induces haploid as well as diploid parthenogenesis in aged human oocytes. A 5- to 10-hr treatment of oocytes with 10 micrograms/ml puromycin yields the highest percentage of activation, and almost all parthenogenetically activated oocytes enter or develop beyond the first cleavage mitosis. Analysis of mitotic metaphase chromosomes from parthenogenetically activated human oocytes may be a promising new approach to preimplantation cytogenetics.

Cell Division↗

Parthenogenetic reptiles: new subjects for laboratory research.

Problems preventing establishment of laboratory colonies of parthenogenetic lizards have been solved. Now, productive colonies of these lizards, which have remarkably little genetic variation, can be readily established and used not only for research on parthenogenesis but also for many kinds of experiments for which reptile systems are desirable. Research colonies can provide valuable specimens while reducing the exploitation of natural populations.

Animals↗

A non-policing honey bee colony (Apis mellifera capensis).

In the Cape honey bee Apis mellifera capensis, workers lay female eggs without mating by thelytokous parthenogenesis. As a result, workers are as related to worker-laid eggs as they are to queen-laid eggs and therefore worker policing is expected to be lower, or even absent. This was tested by transferring worker- and queen-laid eggs into three queenright A. m. capensis discriminator colonies and monitoring their removal. Our results show that worker policing is variable in A. m. capensis and that in one colony worker-laid eggs were not removed. This is the first report of a non-policing queenright honey bee colony. DNA microsatellite and morphometric analysis suggests that the racial composition of the three discriminator colonies was different. The variation in policing rates could be explained by differences in degrees of hybridisation between A. m. capensis and A. m. scutellata, although a larger survey is needed to confirm this.

Animals↗

Centrosome inheritance in the parthenogenetic egg of the collembolan Folsomia candida.

Unfertilized eggs commonly lack centrioles, which are usually provided by the male gamete at fertilization, and are unable to assemble functional reproducing centrosomes. However, some insect species lay eggs that develop to adulthood without a contribution from sperm. We report that the oocyte of the parthenogenetic collembolan Folsomia candida is able to self-assemble microtubule-based asters in the absence of pre-existing maternal centrosomes. The asters, which develop near the innermost pole of the meiotic apparatus, interact with the female chromatin to form the first mitotic spindle. The appearance of microtubule-based asters in the cytoplasm of the activated Folsomia oocyte might represent a conserved mechanism for centrosome formation during insect parthenogenesis. We also report that the architecture of the female meiotic apparatus and the structure of the mitotic spindles during the early embryonic divisions are unusual in comparison with that of insects.

Animals↗

Human embryo cloning prohibited in Hong Kong.

Since the birth of Dolly (the cloned sheep) in 1997, debates have arisen on the ethical and legal questions of cloning-for-biomedical-research (more commonly termed "therapeutic cloning") and of reproductive cloning using human gametes. Hong Kong enacted the Human Reproductive Technology Ordinance (Cap 561) in 2000. Section 15(1)(e) of this Ordinance prohibits the "replacing of the nucleus of a cell of an embryo with a nucleus taken from any other cell," i.e., nucleus substitution. Section 15(1)(f) prohibits the cloning of any embryo. The scope of the latter, therefore, is arguably the widest, prohibiting all cloning techniques such as cell nucleus replacement, embryo splitting, parthenogenesis, and cloning using stem cell lines. Although the Human Reproductive Technology Ordinance is not yet fully operative, this article examines how these prohibitions may adversely impact on basic research and the vision of the Hong Kong scientific community. It concludes that in light of recent scientific developments, it is time to review if the law offers a coherent set of policies in this area.

Cloning, Organism↗

Isolation of candidate genes for apomixis in Poa pratensis L.

The essential feature of apomixis is that an embryo is formed autonomously by parthenogenesis from an unreduced egg of an embryo sac generated through apomeiosis. The genetic constitution of the offspring is, therefore, usually identical to the maternal parent, a trait of great interest to plant breeders. If apomixis were well understood and harnessed, it could be exploited to indefinitely propagate superior hybrids or specific genotypes bearing complex gene sets. A fundamental contribution to the understanding of the genetic control of the apomictic pathway could be provided by a deep knowledge of molecular mechanisms that regulate the reproductive events. In Poa pratensis the cDNA-AFLP method of mRNA profiling allowed us to visualize a total of 2248 transcript-derived fragments and to isolate 179 sequences that differed qualitatively or quantitatively between apomictic and sexual genotypes at the time of flowering when the primary stages of apomixis occur. Three ESTs were chosen for further molecular characterization because of their cDNA-AFLP expression pattern and BLAST information retrieval. The full-lengths of the newly isolated genes were recovered by RACE and their temporal expression patterns were assessed by RT-PCR. Their putative role in cell signaling transduction cascades and trafficking events required during sporogenesis, gametogenesis and embryogenesis in plants is reported and discussed.

Cell Cycle Proteins↗

High conservation of the differentially amplified MPA2 satellite DNA family in parthenogenetic root-knot nematodes.

Sequence variability and distribution of a newly characterized MPA2 satellite DNA family are described in five root-knot nematode species of the genus Meloidogyne, the mitotic parthenogens M. paranaensis, M. incognita, M. arenaria and M. javanica, and the meiotic/mitotic M. hapla (isolates A and B, respectively). The lack of distinctive mutations and the considerable contribution (40.8%) of ancestral changes disclose an ancient satellite DNA which existed in the common ancestor of extant parthenogenetic species in the same or similar form and remained preserved for a period of at least 43 My. Nonuniformly distributed polymorphic sites along the satellite monomer suggest differences in constraints acting on particular sequence segments. Sequence diversity is clearly unaffected by significant differences in genomic abundance of the MPA2 satellite DNA in the examined species. Observed results suggest that the dynamics of this satellite DNA family might be in the first instance a consequence of characteristics of its nucleotide sequence and possible constraints imposed on it. Under conditions of mitotic and meiotic parthenogenesis, slow accumulation of mutations and slow replacement of old MPA2 sequence variants with new ones may be equivalent to the dynamics of some satellite DNA sequences conserved for extremely long evolutionary periods in sexual species.

Animals↗

Coexistence of a sexual and an unisexual form stabilized by parasites.

An unisexual species (either parthenogenetic or gynogenetic form) often coexists sympatrically with a closely related anisogamous sexual species, forming a unisexual complex. This is puzzling because, all else being equal, a sexual population with the two-fold cost of sex (or the cost of producing males) cannot coexist with a unisexual population. This suggests that some ecological processes are at work to make possible the coexistence of the unisexual complex. Field and laboratory studies suggest that parasitism in a gynogenetic complex of the Japanese crucian carp (Carassius auratus) may play an important role in realizing the coexistence by giving frequency-dependent benefit to sexual population. Here, we study the simple dynamics of host-parasite interactions in which non-specific immune reaction of a sexual host is more effective than that of an unisexual host. We simply assume that the infective individuals are sterile. Stable coexistence of a unisexual species with their sexual relative is possible if pathogens are virulent and if the susceptibility of the unisexual form is more than twice as high as that of the sexual form. The coexistence is more difficult, when the fertility of males is relatively low in gynogenetic complexes. This implies that the coexistence of gynogenetic complexes is more difficult than that of parthenogenetic complexes in which parthenogenesis has no constraint on males. We conclude that parasitism is a promising candidate mechanism for the coexistence of unisexual complexes.

Animals↗

Infection dynamics of different Wolbachia-types within one host population.

Wolbachia are widespread intracellular symbionts of arthropods which are known to cause several reproductive manipulations in their hosts, the commonest of which being cytoplasmic incompatibility (CI), male killing (MK), and the induction of parthenogenesis (PI). Strains of endosymbionts inducing one of these effects can be referred to as 'Wolbachia-types'. Here, we try to ascertain whether two of these Wolbachia-types can stably coexist within one population. We investigate this question by means of two discrete-time mathematical models which describe the dynamics of an infection of a host population with either CI- and MK- or CI- and PI-Wolbachia. We derive analytical solutions for two special cases of each model showing that stable coexistence of the respective Wolbachia-types is not possible if no doubly infected individuals occur within the population and that stable coexistence is possible when doubly infected hosts do exist and transmission of the endosymbionts is perfect. Moreover, we show that a population infected with either CI- or MK-Wolbachia at equilibrium can resist invasion of the respective other Wolbachia-type as a single infection. In contrast, a population infected with CI-Wolbachia can be invaded by PI-Wolbachia as a single infection with the CI-Wolbachia going extinct. Computer simulations confirmed these findings for the general models. We discuss our results with respect to the prevalence of the Wolbachia-types considered here and the emergence of PI- from CI-Wolbachia.

Animals↗

Rabbit embryonic stem cell lines derived from fertilized, parthenogenetic or somatic cell nuclear transfer embryos.

Embryonic stem cells were isolated from rabbit blastocysts derived from fertilization (conventional rbES cells), parthenogenesis (pES cells) and nuclear transfer (ntES cells), and propagated in a serum-free culture system. Rabbit ES (rbES) cells proliferated for a prolonged time in an undifferentiated state and maintained a normal karyotype. These cells grew in a monolayer with a high nuclear/cytoplasm ratio and contained a high level of alkaline phosphate activity. In addition, rbES cells expressed the pluripotent marker Oct-4, as well as EBAF2, FGF4, TDGF1, but not antigens recognized by antibodies against SSEA-1, SSEA-3, SSEA-4, TRA-1-10 and TRA-1-81. All 3 types of ES cells formed embryoid bodies and generated teratoma that contained tissue types of all three germ layers. rbES cells exhibited a high cloning efficiency, were genetically modified readily and were used as nuclear donors to generate a viable rabbit through somatic cell nuclear transfer. In combination with genetic engineering, the ES cell technology should facilitate the creation of new rabbit lines.

Animals↗

Gyrodactylid developmental biology: historical review, current status and future trends.

In the viviparous gyrodactylids, embryos develop one inside another within the parental uterus, a phenomenon with major implications for the biology of this species-rich group. Development occurs via two routes: first-born daughters develop at the centre of an embryo cluster in utero, whereas all other daughters develop from oocytes. The resulting offspring are, however, morphologically indistinguishable. We review here the history of gyrodactylid embryology in the context of current knowledge and, present additional cytogenetic and ultrastructural observations of embryonic development. These progenetic parasites are highly modified for viviparity; oocyte maturation and sperm storage occur in a single chamber, the Egg Cell Forming Region, and a mature oocyte passes into the uterus after the birth of the preceding, fully developed offspring. The uterus has a syncytial lining derived from anterior and posterior cap cells. These cells are the first to differentiate in the female reproductive system and may be involved in controlling development. Embryos receive nutrients via the uterus rather than from vitelline cells. Traditionally, development of the first-born daughter has been considered a form of polyembryony, although paedogenesis has also been suggested. In contrast to previous studies, we could not trace lineage of the first-born daughter to a single quiescent macromere. However, only mitotic divisions have been conclusively observed in the intraembryonic generation, indicating an asexual origin. All other daughters are formed from meiotically derived oocytes by sexual reproduction or automictic parthenogenesis. The latter may involve pre-meiotic doubling of chromosomes, but the precise mechanism and the relative proportion of sexual and parthenogenetic offspring are unknown. Exceptionally, cleavage in Gyrodactylus spp. occurs by duets rather than quartets (a pattern previously only recorded in acoels) and is characterised by extensive cell rearrangements. Blastomeres may be connected by fine cytoplasmic processes or completely disassociated and are readily redistributed by the muscular actions of the parental uterus. This process resembles 'Blastomeren-Anarchie' of rhabdocoels but without the structural support of vitelline cells. It prevents generation of early cell fate maps and indicates regulative, rather than mosaic, development. Structures such as the gut and excretory system differentiate late, and are highlighted, together with the attachment apparatus, as examples of post-embryonic differentiation. Molecular and cellular techniques are now essential to further elucidate mechanisms of gyrodactylid reproduction, which will in turn contribute to current debates with animal embryology.

Animals↗

The genetic outcomes of sex and recombination in long-term functionally parthenogenetic lineages of Australian Sitobion aphids.

The typical life cycle of an aphid is cyclical parthenogenesis which involves the alternation of sexual and asexual reproduction. However, aphid life cycles, even within a species, can encompass everything on a continuum from obligate sexuality, through facultative sexuality to obligate asexuality. Loss of the sexual cycle in aphids is frequently associated with the introduction of a new pest and can occur for a number of environmental and genetic reasons. Here we investigate loss of sexual function in Sitobion aphids in Australia. Specifically, we aimed to determine whether an absence of sexual reproduction in Australian Sitobion results from genetic loss of sexual function or environmental constraints in the introduced range. We addressed our aims by performing a series of breeding experiments. We found that some lineages have genetically lost sexual function while others retain sexual function and appear environmentally constrained to asexuality. Further, in our crosses, using autosomal and X-linked microsatellite markers, we identified processes deviating from normal Mendelian segregation. We observed strong deviations in X chromosome transmission through the sexual cycle. Additionally, when progeny genotypes were examined across multiple loci simultaneously we found that some multilocus genotypes are significantly over-represented in the sample and that levels of heterozygosity were much higher than expected at almost all loci. This study demonstrates that strong biases in the transmission of X chromosomes through the sexual cycle are likely to be widespread in aphids. The mechanisms underlying these patterns are not clear. We discuss several possible alternatives, including mutation accumulation during periods of functional asexuality and genetic imprinting.

Animals↗

Schistosoma mansoni: vitelline gland development in females from single sex infections.

Vitelline gland development and incomplete parthenogenesis in 65-week-old female Schistosoma mansoni from single sex infections is described. Vitelline gland development was restricted to the extreme posterior end of each worm and the developed vitelline lobules showed the normal sequence of development and maturation. The ovary, however, was always in an immature state. Egg production occurred although the eggs were non-viable. The results suggest that, whilst male stimulation is not necessary to initiate vitelline gland development and egg production, it appears to be essential for the complete maturation of the ovary. The possible importance of ovarian maturation to the production of viable eggs is discussed.

Animals↗

A genetic analysis of reproduction in Strongyloides ratti.

Strongyloides ratti has a complex life-cycle with two adult generations, one free-living and dioecious and one parasitic and female only. The parasitic females reproduce by parthenogenesis, but it is unclear whether this is mitotic or meiotic in nature. This question has been addressed genetically by analysing the progeny of parasitic females that were heterozygous at an actin locus for evidence of allelic segregation. Such progeny were similarly heterozygous showing that segregation had not occurred. It was therefore concluded that reproduction in the parasitic female of S. ratti is functionally mitotic.

Actins↗

Morphological features of lipid droplet transition during porcine oocyte fertilisation and early embryonic development to blastocyst in vivo and in vitro.

Lipid content in mammalian oocytes or embryos differs among species, with bovine and porcine oocytes and embryos showing large cytoplasmic droplets. These droplets are considered to play important roles in energy metabolism during oocyte maturation, fertilisation and early embryonic development, and also in the freezing ability of oocytes or embryos; however, their detailed distribution or function is not well understood. In the present study, changes in the distribution and morphology of porcine lipid droplets during in vivo and in vitro fertilisation, in contrast to parthenogenetic oocyte activation, as well as during their development to blastocyst stage, were evaluated by transmission electron microscopy (TEM). The analysis of semi-thin and ultra-thin sections by TEM showed conspicuous, large, electron-dense lipid droplets, sometimes associated with mitochondrial aggregates in the oocytes, irrespective of whether the oocytes had been matured in vivo or in vitro. Immediately after sperm penetration, the electron density of the lipid droplets was lost in both the in vivo and in vitro oocytes, the reduction being most evident in the oocytes developed in vitro. Density was restored in the pronculear oocytes, fully in the in vivo specimens but only partially in the in vitro ones. The number and size of the droplets seemed, however, to have decreased. At 2- to 4-cell and blastocyst stages, the features of the lipid droplets were almost the same as those of pronuclear oocytes, showing a homogeneous or saturated density in the in vivo embryos but a marbled or partially saturated appearance in the in vitro embryos. In vitro matured oocytes undergoing parthenogenesis had lipid droplets that resembled those of fertilised oocytes until the pronuclear stage. Overall, results indicate variations in both the morphology and amount of cytoplasmic lipid droplets during porcine oocyte maturation, fertilisation and early embryo development as well as differences between in vivo and in vitro development, suggesting both different energy status during preimplantation development in pigs and substantial differences between in vitro and in vivo development.

Animals↗