Ovariole Sheath Structure and Its Relationship With Developing Embryos in a Parthenogenetic Viviparous Aphid

Couchman, J. R., King, P. E. 1980. Ovariole sheath structure and its relationship with developing embryos in a parthenogenetic viviparous aphid. (Zoology Department, University College, Swansea, U.K.) — Acta zool. (Stockh.) 61(3): 147–155. The ovariole sheath of the viviparous parthenogenetic aphid Brevicoryne brassicae has been the subject of an ultrastructural examination, particularly with regard to its relationship with embryos at various stages of ontogeny. The sheath is a unilaminar, squamous epithelial layer enclosing germarium and embryos, but does not have an intrinsic trophic role until the connection between the embryo and germarial trophocytes, in the form of the trophic cord, is lost. At this time there is a degree of thickening and an increase in the number of cell organelles with ultrastructural indications of metabolic activity, a situation paralleled in the embryonic serosa. There is also evidence that haemolymph components may have direct access to the embryos through intercellular spaces in the sheath. Intercellular junctions, microtubules and cell overlap in regions of the sheath adjacent to embryos at a more advanced state of development are all implicated in a mechanism which resists deformation, maintains sheath integrity and allows embryo passage. A tunica propria and microfilament bundles are absent in the ovariole sheath of this species.     

Moderate expression of Wnt signaling genes is essential for porcine parthenogenetic embryo development

Parthenogenetic embryos are invariably lost in mid-gestation, possibly due to the lack of the paternal genome and the consequent induction of aberrant gene expression. Wnt signaling is essential for embryonic development; however, the studies of this pathway in porcine parthenogenetic embryos have been limited. Here, the role of Wnt signaling in porcine parthenogenetic embryos was studied. In vivo embryos were used as controls. Single cell quantitative real-time PCR showed that Wnt signaling was down-regulated in porcine parthenogenetic embryos. Furthermore, immunofluorescence staining and real-time PCR demonstrated that porcine parthenogenetic embryo development was largely unaffected by the inhibition of Wnt signaling with IWP-2, but blastocyst hatching and trophectoderm development was blocked. In addition, parthenogenetic blastocyst hatching was improved by the activation of Wnt signaling by BIO. However, the developmental competency of porcine embryos, including blastocyst hatching, was impaired and apoptosis was induced upon the excessive activation of Wnt signaling. These findings constitute novel evidence that Wnt signaling is important for porcine pre-implantation development and that its down-regulation may lead to the low hatching rate of porcine parthenogenetic blastocysts.     

Tripsacum dactyloides (Poaceae): a natural model system to study parthenogenesis

Diplosporous apomeiosis, formation of unreduced embryo sacs primarily of the Antennaria type, followed by parthenogenetic embryo development and pseudogamy (fertilization of the central cell) describe gametophytic apomixis within the Tripsacum agamic complex. Tripsacum dactyloides (Eastern gamagrass) is a close relative of domesticated maize and was chosen as a natural model system to investigate gene expression patterns associated with parthenogenesis. The genome size of diploid sexual and polyploid apomictic T. dactyloides was estimated by flow cytometry to be 7.37 pg (2C), 14.74 pg (4C) and 22.39 pg (6C), respectively. The diploid genome size is thus approximately 1.352 larger than that of maize. The apomeiotic-pseudogamous pathway of seed formation was demonstrated at a rate of 92% by the flow cytometric seed screen (FCSS) with single mature seeds in tetraploid accessions. This number includes twin embryos which were detected in 13% of the seeds analyzed. Fertilization of unreduced egg cells (B_III hybrids) was measured in 10% of apomictic seeds. Autonomous (fertilization-independent) embryo development and fertilization-dependent endosperm formation were confirmed by pollination of tetraploid T. dactyloides with a diploid transgenic maize line carrying an actin::#-glucuronidase (GUS) reporter construct. GUS expression was detected after pollination in the developing endosperm, but not in the embryo. In similar intraspecific crossing experiments with maize, GUS expression was detected in both the embryo and endosperm. A protocol was established for microdissection of embryo sacs and early parthenogenetic embryos of T. dactyloides. Together, these techniques provide new tools for future studies aimed at comparing gene expression patterns between sexual maize and sexual or apomictic T. dactyloides.     

Contrasting patterns in the spread of two seed-borne viruses in pea embryos

The temporal and spatial patterns of pea seed-borne mosaic potyvirus (PSbMV) and pea early browning tobra-virus (PEBV) accumulation in pea embryos were analyzed using in-situ hybridization and immunohistochemistry. For PSbMV, which infects embryos after fertilization, the distribution changed as the embryo developed and some tissues remained free of virus infection. In contrast, embryos were uniformly infected with PEBV from the earliest stages of embryo development, and PEBV was detected in the egg cell and pollen grains, indicative of gametic transmission into the embryo. These observations suggest that gametically transmitted viruses may be appropriate as potential vectors for the ectopic and uniform expression of novel genes in embryonic tissues. Functional complementarity in the two processes of embryo invasion was tested following co-inoculation with PSbMV and PEBV. Instead of complementation, interference in PSbMV seed transmission by PEBV was observed; PEBV seed transmission remained unaffected by PSbMV.     

Early patterning of the mouse embryo--contributions of sperm and egg

The first cleavage of the fertilised mouse egg divides the zygote into two cells that have a tendency to follow distinguishable fates. One divides first and contributes its progeny predominantly to the embryonic part of the blastocyst, while the other, later dividing cell, contributes mainly to the abembryonic part. We have previously observed that both the plane of this first cleavage and the subsequent order of blastomere division tend to correlate with the position of the fertilisation cone that forms after sperm entry. But does sperm entry contribute to assigning the distinguishable fates to the first two blastomeres or is their fate an intrinsic property of the egg itself? To answer this question we examined the distribution of the progeny of early blastomeres in embryos never penetrated by sperm - parthenogenetic embryos. In contrast to fertilised eggs, we found there is no tendency for the first two parthenogenetic blastomeres to follow different fates. This outcome is independent of whether parthenogenetic eggs are haploid or diploid. Also unlike fertilised eggs, the first 2-cell blastomere to divide in parthenogenetic embryo does not necessarily contribute more cells to the blastocyst. However, even when descendants of the first dividing blastomere do predominate, they show no strong predisposition to occupy the embryonic part. Thus blastomere fate does not appear to be decided by differential cell division alone. Finally, when the cortical cytoplasm at the site of sperm entry is removed, the first cleavage plane no longer tends to divide the embryo into embryonic and abembryonic parts. Together these results indicate that in normal development fertilisation contributes to setting up embryonic patterning, alongside the role of the egg.     

Centrosome dynamics and inheritance in related sexual and parthenogenetic Bacillus (Insecta Phasmatodea)

In animals, some general features of centrosome dynamics and inheritance have been widely recognized. The most acknowledged model assigns to sperm the contribution of a centriole to the fertilized egg, which in turn provides the pericentriolar materials, including gamma-tubulin, recruiting them from the cytoplasm: the main zygote microtubule organizing center (MTOC) is thus reconstituted to organize first the spermaster and then the full first embryonic spindle. Obviously the model cannot apply to parthenogenetic systems, which actually rely on egg components alone. In stick insects of the Bacillus genus, the spindle of both somatic and germ cells is clearly anastral, therefore we have been investigating their centrosome in sexual and parthenogenetic taxa by analyzing its component dynamics and transmission through the use of monoclonal beta- and gamma-tubulin antibodies and transmission electron microscopy (TEM). It has been shown that in sexually reproducing species the spermatozoon does not contribute the centriole, so that the egg wholly provides the MTOC and the ensuing anastral spindle of the embryo: MTs appear to derive from pronuclear chromatin surroundings and no asters are observed. The parthenogenetic embryo development is the same as the sexual one if syngamy is excepted. The parthenogenetic mechanism realized by these panoistic insects appears to differ from that observed in the meroistic hymenopteran and drosophilid species, where the embryo spindle derives from asters formed in the egg cortex. In stick insects, the lack of sperm contribution to embryonic centrosome appears to be a major trait accounting for the widespread occurrence of facultative and obligate parthenogenesis within the order.     

Inducing the sexual forms and hatching the eggs of pea aphids

In temperate climates, pea aphids (Acyrthosiphon pisum) produce a single sexual generation each year in response to declining photoperiod and temperature. Mating occurs in the fall and the eggs have an obligatory winter diapause. Genetic recombination during the sexual phase is thought to be an important source of genetic variability within cyclically parthenogenetic aphid populations. Methods for reliably producing sexual forms and hatching the eggs of aphids are therefore central not only to the study of evolutionary change in aphid populations, but also for a general understanding of the origin of agriculturally important variation in destructiveness within pest species.Here, sexual forms of six pea aphid clones were induced in the laboratory and eggs were successfully hatched by creating conditions that closely mimicked those found in field situations. A declining photoperiod was produced by controlling artificial lighting using a timer with variable cycle length. Using these conditions, sexual forms were successfully produced for all six clones tested, which were then mated in all combinations. Eggs were exposed to a daily cycle of freezing and thawing in an incubator under a short-day photoperiod. Egg hatch averaged 60%, but was as high as 89% for some crosses. These methods will permit testing of evolutionary hypotheses and execution of detailed genetic studies of sources of variability within pea aphid populations. They are thus important tools for both evolutionary and agricultural studies.     

Identification of Critical Conditions for Immunostaining in the Pea Aphid Embryos: Increasing Tissue Permeability and Decreasing Background Staining

The pea aphid Acyrthosiphon pisum, with a sequenced genome and abundant phenotypic plasticity, has become an emerging model for genomic and developmental studies. Like other aphids, A. pisum propagate rapidly via parthenogenetic viviparous reproduction, where the embryos develop within egg chambers in an assembly-line fashion in the ovariole. Previously we have established a robust platform of whole-mount in situ hybridization allowing detection of mRNA expression in the aphid embryos. For analyzing the expression of protein, though, established protocols for immunostaining the ovarioles of asexual viviparous aphids did not produce satisfactory results. Here we report conditions optimized for increasing tissue permeability and decreasing background staining, both of which were problems when applying established approaches. Optimizations include: (1) incubation of proteinase K (1 µg/ml, 10 min), which was found essential for antibody penetration in mid- and late-stage aphid embryos; (2) replacement of normal goat serum/bovine serum albumin with a blocking reagent supplied by a Digoxigenin (DIG)-based buffer set and (3) application of methanol rather hydrogen peroxide (H₂O₂) for bleaching endogenous peroxidase; which significantly reduced the background staining in the aphid tissues. These critical conditions optimized for immunostaining will allow effective detection of gene products in the embryos of A. pisum and other aphids.     

Partitioning of Symbiotic Bacteria between Generations of an Insect: a Quantitative Study of a Buchnera sp. in the Pea Aphid (Acyrthosiphon pisum) Reared at Different Temperatures

The population of symbiotic Buchnera bacteria in parthenogenetic females of the pea aphid Acyrthosiphon pisum was determined by quantitative hybridization of a DNA probe (groESL) to aphid homogenates. The aphids bore 1 x 10(sup7) to 2 x 10(sup7) bacterial cells per mg (fresh weight). In teneral aphids (i.e., aphids that had moulted to adulthood but that had not initiated reproduction), >75% of the bacteria were in the embryos, and the density of bacteria in the embryos was consistently greater than that in the maternal tissues. The bacterial density in teneral aphids increased from 1.3 x 10(sup7) to 2.0 x 10(sup7) cells mg (fresh weight) of aphids(sup-1) with temperature between 15 and 25(deg)C. This variation could be attributed to a temperature-dependent increase in both the density of bacteria in the embryos and embryo content of the aphids.     

Facultative symbiont infections affect aphid reproduction

Some bacterial symbionts alter their hosts reproduction through various mechanisms that enhance their transmission in the host population. In addition to its obligatory symbiont Buchnera aphidicola, the pea aphid Acyrthosiphon pisum harbors several facultative symbionts influencing several aspects of host ecology. Aphids reproduce by cyclical parthenogenesis whereby clonal and sexual reproduction alternate within the annual life cycle. Many species, including the pea aphid, also show variation in their reproductive mode at the population level, with some lineages reproducing by cyclical parthenogenesis and others by permanent parthenogenesis. While the role of facultative symbionts has been well studied during the parthenogenetic phase of their aphid hosts, very little is known on their possible influence during the sexual phase. Here we investigated whether facultative symbionts modulate the capacity to produce sexual forms in various genetic backgrounds of the pea aphid with controlled symbiont composition and also in different aphid genotypes from natural populations with previously characterized infection status and reproductive mode. We found that most facultative symbionts exhibited detrimental effects on their hosts fitness under sex-inducing conditions in comparison with the reference lines. We also showed that the loss of sexual phase in permanently parthenogenetic lineages of A. pisum was not explained by facultative symbionts. Finally, we demonstrated that Spiroplasma infection annihilated the production of males in the host progeny by inducing a male-killing phenotype, an unexpected result for organisms such as aphids that reproduce primarily through clonal reproduction.     

Genetic Control of Contagious Asexuality in the Pea Aphid

Although evolutionary transitions from sexual to asexual reproduction are frequent in eukaryotes, the genetic bases of such shifts toward asexuality remain largely unknown. We addressed this issue in an aphid species where both sexual and obligate asexual lineages coexist in natural populations. These sexual and asexual lineages may occasionally interbreed because some asexual lineages maintain a residual production of males potentially able to mate with the females produced by sexual lineages. Hence, this species is an ideal model to study the genetic basis of the loss of sexual reproduction with quantitative genetic and population genomic approaches. Our analysis of the co-segregation of ∼300 molecular markers and reproductive phenotype in experimental crosses pinpointed an X-linked region controlling obligate asexuality, this state of character being recessive. A population genetic analysis (>400-marker genome scan) on wild sexual and asexual genotypes from geographically distant populations under divergent selection for reproductive strategies detected a strong signature of divergent selection in the genomic region identified by the experimental crosses. These population genetic data confirm the implication of the candidate region in the control of reproductive mode in wild populations originating from 700 km apart. Patterns of genetic differentiation along chromosomes suggest bidirectional gene flow between populations with distinct reproductive modes, supporting contagious asexuality as a prevailing route to permanent parthenogenesis in pea aphids. This genetic system provides new insights into the mechanisms of coexistence of sexual and asexual aphid lineages.     

Leukemia inhibitory factor (LIF) improves and prolongs the development of parthenogenetic mouse embryos

We studied the effect of the growth factor LIF on the development of parthenogenetic mouse embryos (CBA x C57BL/6)F1. LIF was added to the culture medium at 10, 50, 100, and 250 ng/ml at the morula stage and parthenogenetic embryos were cultivated in vitro until the late blastocysts stage and then transplanted in the uterus of pseudopregnant females, which were then sacrificed on day 12 of pregnancy. All the LIF doses used improved the development of parthenogenetic mouse embryos at the preimplantation stages and increased the amount of blastocysts by 16%, on average, as compared to the control. LIF at 50 and 100 ng/ml increased approximately twice the number of embryos that reached the somatic stages. Some of them reached the stage of 32-45 somites and had fore and hind limb buds. No such embryos were found in the control. Well formed placenta was observed in 6% of the embryos treated with LIF and the most pronounced effect was recorded at 100 ng/ml. The data we obtained suggest that exogenous LIF can improve pre- and postimplantation development of parthenogenetic mouse embryos due, possibly, to increased survival rate of embryonic stem cells derived from the inner cell mass of blastocysts. LIF improves not only the development of the parthenogenetic embryo per se, but also the formation of its extraembryonic envelopes, which leads to the development of a larger placenta in LIF-treated parthenogenetic embryos, as compared to the control.     

Development of sheep androgenetic embryos is boosted following transfer of male pronuclei into androgenetic hemizygotes

Androgenetic embryos are useful model for investigating the contribution of the paternal genome to embryonic development. Little work has been done with androgenetic embryo production in domestic animals. The aim of this study was the production of diploid androgenetic sheep embryos. In vitro matured sheep oocytes were enucleated and fertilized in vitro; parthenogenetic and normally fertilized embryos were also produced as a control. Fifteen hours after in vitro fertilization (IVF), presumptive zygotes were centrifuged and scored for the number of pronucleus. IVF, parthenogenetic, and androgenetic embryos (haploid, diploid, and triploid) were cultured in SOFaa medium with bovine serum albumin (BSA). The proportion of oocytes with polyspermic fertilization increased linearly with increasing sperm concentration. After IVF, there was no significant difference in early cleavage and morula formation rates between the groups, while there was a significant difference on blastocyst development between IVF, parthenogenetic, and androgenetic embryos, the last ones displaying poor developmental potential (IVF, parthenogenetic, and haploid, diploid, and triploid androgenetic embryos: 43%, 38%, 0%, 2%, and 2%, respectively). In order to boost androgenetic embryonic development, we produced diploid androgenetic embryos through pronuclear transfer. Single pronuclei were aspirated with a bevelled pipette from haploid or diploid embryos and transferred into the perivitelline space of other haploid embryos, and the zygotes were reconstructed by electrofusion. Fusion rates approached 100%. Pronuclear transfer significantly increased blastocyst development (IVF, parthenogenetic, androgenetic: Diploid into Haploid, and Haploid into Haploid: 42%, 42%, 19%, and 3%, respectively); intriguingly, the Haploid + Diploid group showed the highest development to blastocyst stage. The main findings of our study are: (1) sheep androgenetic embryos display poor developmental ability compared with IVF and parthenogenetic embryos; (2) diploid androgenetic embryos produced by pronuclear exchange developed in higher proportion to blastocyst stage, particularly in the Diploid-Haploid group. In conclusion, pronuclear transfer is an effective method to produce sheep androgenetic blastocysts.     

Germ-plasm specification and germline development in the parthenogenetic pea aphid Acyrthosiphon pisum: Vasa and Nanos as markers

The germarium, oocytes and embryos of the parthenogenetic viviparous pea aphid Acyrthosiphon pisum are contained within a single ovariole. This species provides an excellent model for studying how maternally-inherited germ plasm is specified and how it is transferred to primordial germ cells. Previous studies have shown that germ cells are first segregated at the embryonic posterior after formation of the blastoderm. We used two cross-reacting antibodies against the conserved germline markers Vasa and Nanos, which specifically identified these presumptive germ cells, to investigate whether germ cells were determined during early development. We observed randomly-distributed weak expression of Vasa signals in the developing oocyte but no localization in the oocyte segregated from the germarium. Localized Vasa was not apparent until it was detected at the posterior in the embryo undergoing the second nuclear division. Nanos, on the other hand, was localized to a nuage-like structure surrounding the nucleus in the developing and segregated oocytes. At the beginning of the oocyte maturation division, Nanos localization shifted to the posterior and could be identified in successive stages until it was incorporated into the germ cells. Taken together, our results suggest that germ plasm is specified in the developing oocyte and that Nanos is an earlier germline marker than Vasa. Germ cells stained for Vasa remained at a dorsal location in the egg during mid-development and then were guided into abdominal segments A1 to A6 during germ-band retraction. We infer that germ cells coalesce with segmented gonadal mesoderm during this period.     

Differential regulations of wing and ovarian development and heterochronic changes of embryogenesis between morphs in wing polyphenism of the vetch aphid

SUMMARY In wing polyphenisms that produced alternative wing morphs depending on environmental conditions, the developmental regulations to balance between flight and reproductive abilities should be important. Many species of aphids exhibit wing polyphenisms, and the development of wing and flight muscles is thought to incur costs of reproductive ability. To evaluate the relationship between flight and reproduction, the fecundity and the wing- and ovarian development in the parthenogenetic generations were compared between winged and wingless aphids in the vetch aphid Megoura crassicauda . Although no differences in offspring number and size were detected, the onset of larviposition after imaginal molt was delayed in winged adults. The comparison of growth in flight apparatus revealed that, after the second-instar nymphs, the flight-apparatus primordia of presumptive wingless aphids were degenerated while those of winged nymphs rapidly developed. In the ovaries of winged line, the embryo size was smaller and the embryonic stages were delayed from third to fifth instars, although these differences had disappeared by the time of larviposition. It is therefore likely that the delay in larviposition in winged aphids is due to the slower embryonic development. The correlation between embryo size and developmental stage suggests that the embryos of winged aphids are better developed than similarly sized embryos in wingless aphids. These heterochronic shifts would facilitate the rapid onset of larviposition after the dispersal flight. This developmental regulation of embryogenesis in the aphid wing polyphenism is suggested to be an adaptation that compensates the delay of reproduction caused by the wing development.     

Comparing fecundity in parthenogenetic versus sexual populations of the freshwater snail Campeloma limum: is there a two‐fold cost of sex?

Abstract. The predominance of sexuality in eukaryotes remains an evolutionary paradox, given the "two-fold cost of sex" also known as the "cost of males." [Correction added after online publication 29 January 2009: in the preceding sentence, extraneous words were deleted.] As it requires two sexual parents to reproduce and only one parthenogenetic parent, parthenogens should have twice the reproductive rate compared with their sexual counterparts and their genes should spread twice as fast, if all else is equal. Yet, parthenogenesis is relatively rare and considered an evolutionary dead-end, while sexuality is the dominant form of reproduction in multicellular eukaryotes. Many studies have explored short-term benefits of sex that could outweigh its two-fold cost, but few have compared fecundity between closely related sexuals and parthenogens to first verify that "all else is equal" reproductively. We compared six fecundity measures between sexual and parthenogenetic populations of the freshwater snail, Campeloma limum , during a brooding cycle (1 year) across two drainages. Drainages were analyzed separately because of a significant drainage effect. In the Savannah drainage, fecundity was not significantly different between sexuals and parthenogens, even though parthenogens had significantly more empty egg capsules per brood. In the Ogeechee drainage, parthenogens had significantly more egg capsules with multiple embryos and more hatched embryos than sexuals. Taken over 1 year, embryo size was not significantly different between parthenogens and sexuals in either drainage. Given these results and the close proximity of sexual and parthenogenetic populations, it is perplexing why parthenogenetic populations have not completely replaced sexual populations in C. limum.