Parthenogenetic Research Articles

Genetic mapping of the LOSS OF PARTHENOGENESIS locus in Pilosella piloselloides and the evolution of apomixis in the Lactuceae.

Pilosella piloselloides var. praealta (syn. P. praealta; Hieracium praealtum) is a versatile model used to study gametophytic apomixis. In this system apomixis is controlled by three loci: one that controls the avoidance of meiosis (LOA), one that controls the avoidance of fertilization (LOP) and a third that controls autonomous endosperm formation (AutE). Using a unique polyhaploid mapping approach the LOP locus was mapped to a 654 kb genomic interval syntenic to linkage group 8 of Lactuca sativa. Polyhaploids form through the gametophytic action of a dominant determinant at LOP, so the mapped region represents both a functional and a physical domain for LOP in P. piloselloides. Allele sequence divergence (ASD) analysis of the PARTHENOGENESIS (PAR) gene within the LOP locus revealed that dominant PAR alleles in Pilosella remain highly similar across the genus, whilst the recessive alleles are more divergent. A previous report noted that dominant PAR alleles in both Pilosella and Taraxacum are modified by the presence of a class II transposable element (TE) in the promoter of the gene. This observation was confirmed and further extended to the related genus Hieracium. Sufficient differences were noted in the structure and location of the TE elements to conclude that TE insertional events had occurred independently in the three genera. Measures of allele crossover amongst the polyhaploids revealed that P. piloselloides is an autopolyploid species with tetrasomic inheritance. It was also noted that the dominant determinant of LOP in P. piloselloides could transmit via a diploid gamete (pollen or egg) but not via a haploid gamete. Using this information, a model is presented of how gametophytic apomixis may have evolved in several members of the Lactuceae, a tribe of the Asteraceae.

A new hypothesis may explain human parthenogenesis and ovarian teratoma: A review study.

Parthenogenesis (PG) is a rare phenomenon occurring in humans, and understanding this may help us develop an explanation for such occurrences. Moreover, it may help reveal the cause of idiopathic ovarian teratoma (OT). We aim to explain the occurrence of PG and OT in humans based on a new hypothesis. Previous literature has been searched through relevant scientific websites and international journals on the causes and mechanisms of PG and OT in humans. The previous literature on human PG was sparse and mostly contained case reports. It appears that human PG is not as rare as previously reported but may occur spontaneously, resulting in OT formation. The difference between PG and sexual reproduction is that PG has no embryonic diversity. The biopsied embryonic samples in the PG correspond exclusively to those of the maternal side. Spontaneous PG in humans often degrades or leads to formation of OT. The cause and mechanism of spontaneous PG remain unclear in the available literature. Here, we hypothesized that in some cases the secondary oocyte and first polar body enclosed in the zona pellucida may fuse together to form a single cell that restores the diploid number of chromosomes and initiates cell division to form PG. It may go unnoticed or be represented by the OT. Future studies are recommended to investigate this hypothesis.

Effect of Growth Factors and Hormones during In Vitro Growth Culture of Cumulus-Oocyte-Complexes Derived from Small Antral Follicles in Pigs

This study evaluated the effect of various growth factors and hormones in an in vitro growth (IVG) medium on the in vitro maturation (IVM) and developmental competence of oocytes derived from small antral follicles (SAFs) in pigs. Cumulus-oocyte complexes (COCs) derived from SAFs were either untreated or treated with epidermal growth factor (EGF), insulin-like factor-1 (IGF-1), insulin, or growth hormone (GH) for 2 days of IVG. Following IVG, COCs were cultured for maturation, and IVM oocytes were induced for parthenogenesis (PA). During IVG, the nuclear maturation of oocytes was significantly increased by the insulin treatment compared to other treatments. Moreover, the insulin treatment significantly increased blastocyst formation after PA relative to the No-IVG, control, EGF, and GH treatments. The cumulus expansion score after IVG-IVM was significantly higher in the insulin group than in the other groups. The glutathione (GSH) contents in IVM oocytes were increased through treatment with IGF, insulin, and GH compared to those of No-IVG oocytes. The level of reactive oxygen species (ROS) in IVM oocytes in all treatment groups was significantly lower after IVG culture than in the No-IVG group. The maturation-promoting factor (MPF) activity after IVM in the insulin-treated oocytes was significantly higher than that of the oocytes treated with EGF, IGF-1, and GH. In conclusion, this study demonstrates that insulin treatment during IVG culture improves the maturational and developmental competence of oocytes derived from SAFs in pigs through its effect on cumulus cell expansion and cytoplasmic microenvironments, such as GSH, ROS, and MPF activity.

PsA inhibits the development of bovine embryos through epigenetic and oxidative stress

Histone deacetylases (HDACs) are the key regulators involved in the process of embryo development and tumor progression and are often dysregulated in numerous disordered cells, including tumor cells and somatic cell nuclear transfer (SCNT) embryos. Psammaplin A (PsA), a natural small-molecular therapeutic agent, is a potent histone deacetylase inhibitor (HDACi) that alters the regulation of histone. Approximately 2,400 bovine parthenogenetic (PA) embryos. To investigate the effect of PsA on bovine preimplanted embryos, we analyzed the preimplantation development of PA embryos treated with PsA in this study. The blastocyst formation rate of bovine PA embryos decreased sharply with an increase in concentration and duration. Furthermore, the expression of the pluripotency-related gene Nanog was decreased, and the inhibitory effects on histone deacetylases 1 (HDAC1) and DNA methylation transferase 1 (DNMT1) were observed in bovine PA embryos. The acetylation level of histone H3 lysine 9 (H3K9) was enhanced by a PsA treatment of 10 μM for 6 h, while the DNA methylation appeared unchanged. Interestingly, we also found that PsA treatment enhanced the intracellular reactive oxygen species (ROS) generation and decreased the intracellular mitochondrial membrane potential (MMP)- and superoxide dismutase 1 (SOD1)-induced oxidative stress. Our findings improve the understanding of HDAC in embryo development and provide a theoretical basis and reproduction toxicity evaluation for the application of PsA. These results indicate that PsA inhibits the development of bovine preimplantation PA embryos, supplying data for the PsA clinical application concentration to avoid reproductive toxicity. In addition, the reproduction toxic effect of PsA may be modulated through increased oxidative stress on the bovine PA embryo, suggesting that PsA in combination with antioxidants, for example, melatonin, might be an effective clinical application strategy.

Human zygotic genome activation is initiated from paternal genome

Although parental genomes undergo extensive epigenetic reprogramming to be equalized after fertilization, whether they play different roles in human zygotic genome activation (ZGA) remains unknown. Here, we mapped parental transcriptomes by using human parthenogenetic (PG) and androgenetic (AG) embryos during ZGA. Our data show that human ZGA is launched at the 8-cell stage in AG and bi-parental embryos, but at the morula stage in PG embryos. In contrast, mouse ZGA occurs at the same stage in PG and AG embryos. Mechanistically, primate-specific ZNF675 with AG-specific expression plays a role in human ZGA initiated from paternal genome at the 8-cell stage. AG-specifically expressed LSM1 is also critical for human maternal RNA degradation (MRD) and ZGA. The allelic expressions of ZNF675 and LSM1 are associated with their allelically epigenetic states. Notably, the paternally specific expressions of ZNF675 and LSM1 are also observed in diploid embryos. Collectively, human ZGA is initiated from paternal genome.

Paratanytarsus grimmii (Chironomidae) Larvae in Drinking Water Distribution Systems: Impairment or Disaster?

The occurrence and mass development of macroinvertebrates in drinking water networks is a challenge for drinking water pipe management. A current problem is the development of larvae of the chironomid Paratanytarsus grimmii (mosquito), a so-called pest organism that can have severe impacts on water quality due to mass accumulation from parthenogenic reproduction, biofouling and other aesthetic reasons. The aim of this study was to develop a new practical method for determining larvae size classes and analyzing the growth of the larvae. Knowledge of the dimensions, life cycle and fertility of these larvae within drinking water networks is essential for any risk analysis and the development of P. grimmii pest-control strategies. A two-year study of P. grimmii in a drinking water distribution system in Northern Germany was conducted, and P. grimmii population dynamics are presented. The parthenogenetic reproduction of P. grimmii without any pharate females (facultative flying stage) within the drinking water distribution system was proofed. In 2020 and 2021, five generations of P. grimmii were observed per year, with a maximum abundance of 6350 ind. m−3. Mass accumulation occurred in the late-summer/autumn period.

Comparative single-cell transcriptomic profiles of human androgenotes and parthenogenotes during early development

To unravel the differential transcriptomic behavior of human androgenotes (AGs) and parthenogenotes (PGs) throughout the first cell cycles, analyze the differential expression of genes related to key biologic processes, and determine the time frame for embryonic genome activation (EGA) in AGs and PGs. Laboratory study. Private fertility clinic. Mature oocytes were retrieved from healthy donors and subjected to artificial oocyte activation using calcium ionophore and puromycin to generate PGs (n = 6) or enucleated and subjected to intracytoplasmic sperm injection to generate AGs (n = 10). Uniparental constructs at different early stages of development were disaggregated into constituent single cells (we suggest the terms parthenocytes and androcytes) to characterize the single-cell transcriptional landscape using next-generation sequencing. Transcriptomic profiles comparison between different stages of early development in AGs and PGs. The uniparental transcriptomic profiles at the first cell cycle showed 68 down-regulated and 26 up-regulated differentially expressed genes (DEGs) in PGs compared with AGs. During the third cell cycle, we found 60 up-regulated and 504 down-regulated DEGs in PGs compared with AGs. In the fourth cell cycle, 1,771 up-regulated and 1,171 down-regulated DEGs were found in PGs compared with AGs. The AGs and PGs had reduced EGA profiles during the first 3 cell cycles, and a spike of EGA at the fourth cell cycle was observed in PGs. Transcriptomic analysis of AGs and PGs revealed their complementary behavior until the fourth cell cycle. Androgenotes undergo a low wave of transcription during the first cell cycle, which reflects the paternal contribution to cell cycle coordination, mechanics of cell division, and novel transcription regulation. Maternal transcripts are most prominent in the third and fourth cell cycles, with amplification of transcription related to morphogenic progression and embryonic developmental competence acquisition. Regarding EGA, in PGs, a primitive EGA begins at the 1-cell stage and gradually progresses until the 4-cell stage, when crucial epigenetic reprogramming (through methylation) is up-regulated. In addition, our longitudinal single-cell transcriptomic analysis challenges that the zygote and early cleavage stages are the only totipotent entities, by revealing potential totipotency in cleavage-stage AGs and implications of paternal transcripts.

Evolutionary Potential of Parthenogenesis-Bisexual Lineages within Triploid Apomictic Thelytoky in Cacopsylla ledi (Flor, 1861) (Hemiptera, Psylloidea) in Fennoscandia.

A widely accepted hypothesis is that parthenogenesis is an evolutionary dead end since it is selectively advantageous in the short term only but results in lowered diversification rates. Triploid apomictic parthenogenesis might represent an exception, as in favorable environments, triploid females are able to produce rare males and diploid females. The aim of the present study was to analyze the modes of reproduction and their evolutionary implications in the parthenogenetic psyllid Cacopsylla ledi (Flor, 1861) from Fennoscandia. The cytogenetic assessment of ploidy levels and the analysis of the COI haplotype revealed two geographically separated bisexual lineages implying genuine bisexual populations. The southern lineage occurring south of latitude 65° N in Finland showed a COI haplotype different from that of parthenogenetic triploids in the same population but identical to the haplotype of specimens in a genuine bisexual population in the Czech Republic. This allows us to suggest that bisexuals in southern Fennoscandia represent the original bisexual C. ledi. By contrast, in the northern bisexual lineage north of latitude 65° N, rare males and diploid females carried the same haplotype as triploids in the same population, having been produced by the triploids. In the Kola Peninsula, a genuine bisexual population of presumably rare male/diploid female origin was discovered. As this population is geographically isolated from populations of the ancestral bisexual C. ledi, it can develop into a new bisexual species through peripatric speciation during evolution. Our findings demonstrate that apomictic triploid parthenogenesis is not necessarily an evolutionary dead end but is able to lead to the emergence of a new bisexual species of parthenogenetic origin.

Effect of rapamycin treatment on oocyte in vitro maturation and embryonic development after parthenogenesis in yaks

Autophagy plays an important role in mammalian oocyte maturation and early embryonic development and rapamycin is well known for inducing autophagy. Although previous studies have reported the effects of rapamycin on oocytes in vitro maturation (IVM) in different species, few studies have been reported on the role of rapamycin in yak oocytes IVM and embryonic development. Therefore, the objective of this study was to examine the effect of rapamycin treatment on yak oocytes IVM and early embryonic development. Specifically, immature yak oocytes during IVM or parthenogenetic (PA) embryos were treated with different rapamycin concentrations to select an optimal dose. Then evaluated its effect on maturation rates, cleavage, and blastocyst formation rates, mitochondrial membrane potential, ROS levels. Related genes and proteins expression in matured oocytes and blastocysts were also evaluated. The results show that 10 nM rapamycin treatment during IVM significantly improved oocyte maturation rates of oocytes and blastocyst formation rates. Treatment with 10 nM rapamycin reduced ROS level but increased mitochondrial membrane potential. Correspondingly, mRNA and protein expressions of LC3, Beclin-1, and Bcl-2 up-regulated while Bax down-regulated in matured yak COCs. When parthenogenetic embryos were treated with different rapamycin concentrations, 10 nM rapamycin treatment showed higher 8-cell and blastocyst formation rates. Also, CDX2, POU5F1, SOX2, and Nanog levels in blastocysts were upregulated. In summary, our findings demonstrate that rapamycin treatment improves oocytes maturation probably by increasing mitochondrial membrane potential, reducing ROS levels, and regulating the apoptosis in mature yak oocytes. Rapamycin treatment also improves embryonic developmental competence in the yak.

Recent speciation and phenotypic plasticity within a parthenogenetic lineage of levantine whip spiders (Chelicerata: Amblypygi: Charinidae)

Caves constitute ideal study systems for investigating adaptation and speciation, as the abiotic conditions shared by aphotic habitats exert a set of environmental filters on their communities. Arachnids constitute an important component of many cave ecosystems worldwide. We investigated the population genomics of two whip spider species: Sarax ioanniticus, a widely distributed parthenogenetic species found across the eastern Mediterranean; and S. israelensis, a recently described troglomorphic species that is endemic to caves in Israel. Here, we show that S. israelensis is completely genetically distinct from S. ioanniticus and most likely also a parthenogen. Counterintuitively, despite the lack of genetic variability within S. ioanniticus and S. israelensis, we discovered considerable variation in the degree of median eye reduction, particularly in the latter species. Natural history data from captive-bred specimens of S. israelensis validated the interpretation of parthenogenesis. Our results are most consistent with a scenario of a sexual ancestral species that underwent speciation, followed by independent transitions to apomictic parthenogenesis in each of the two daughter species. Moreover, the lack of genetic variability suggests that variation in eye morphology in S. israelensis is driven exclusively by epigenetic mechanisms.

A new genus and species of ?parthenogenic anostracan (Pancrustacea, Branchiopoda, ?Thamnocephalidae) from the Lower Cretaceous Koonwarra Fossil Bed in Australia

The fossil record of fairy shrimp, or anostracan crustaceans, is poor with only six species formally described to date. The group stratigraphically ranges from the Devonian to recent. The Lower Cretaceous (middle–upper Aptian) Koonwarra Fossil Bed in Australia contains a new species of anostracan, which we describe here. The Koonwarra Fossil Bed is famous for its rich fossil assemblage of avian-line dinosaur feathers, bony fish, insects and aquatic invertebrates that were preserved in the fine bottom sediments of a restricted montane waterbody. Previously, the Koonwarra Fossil Bed anostracans were classified as taxonomically indeterminate; however, we show that they are potentially attributable to Thamnocephalidae based on the egg shape found in gravid female individuals. Notably, all of the recovered Koonwarra Fossil Bed anostracans (∼40 individuals) are either females or juveniles. This suggests that the species might have undergone parthenogenic reproduction. Furthermore, the preservation of the fossils resembles experimentally decayed modern anostracans, evidencing decay-induced fabrics that retained remnants of the cuticle, digestive tract and internal anatomy to varying degrees. Emma Van Houte [vanh1629@fredonia.edu], Department of Geology and Environmental Sciences, SUNY Fredonia, Fredonia, NY 14063, USA; Thomas A. Hegna [thomas.hegna@fredonia.edu], Department of Geology and Environmental Sciences, SUNY Fredonia, Fredonia, NY 14063, USA; Aodhán D. Butler [aodhan.b@gmail.com], Palaeobiology Programme, Department of Earth Sciences: Uppsala University, Villavägen 16, Uppsala 75236, Sweden.

A PARTHENOGENESIS allele from apomictic dandelion can induce egg cell division without fertilization in lettuce.

Apomixis, the clonal formation of seeds, is a rare yet widely distributed trait in flowering plants. We have isolated the PARTHENOGENESIS (PAR) gene from apomictic dandelion that triggers embryo development in unfertilized egg cells. PAR encodes a K2-2 zinc finger, EAR-domain protein. Unlike the recessive sexual alleles, the dominant PAR allele is expressed in egg cells and has a miniature inverted-repeat transposable element (MITE) transposon insertion in the promoter. The MITE-containing promoter can invoke a homologous gene from sexual lettuce to complement dandelion LOSS OF PARTHENOGENESIS mutants. A similar MITE is also present in the promoter of the PAR gene in apomictic forms of hawkweed, suggesting a case of parallel evolution. Heterologous expression of dandelion PAR in lettuce egg cells induced haploid embryo-like structures in the absence of fertilization. Sexual PAR alleles are expressed in pollen, suggesting that the gene product releases a block on embryogenesis after fertilization in sexual species while in apomictic species PAR expression triggers embryogenesis in the absence of fertilization.

In vitro maturation using αMEM with reduced NaCl enhances maturation and developmental competence of pig oocytes after somatic cell nuclear transfer

BackgroundCompared to medium containing 108 mM sodium chloride (NaCl), in vitro maturation (IVM) using a simple medium with reduced (61.6 mM) NaCl increases the cytoplasmic maturation and embryonic development of pig oocytes.ObjectivesThis study determines the effect of a complex medium containing reduced NaCl on the IVM and embryonic development of pig oocytes.MethodsPig oocytes were matured in Minimum Essential Medium Eagle-alpha modification (αMEM) supplemented with 61.6 (61αMEM) or 108 (108αMEM) mM NaCl, and containing polyvinyl alcohol (PVA) (αMEMP) or pig follicular fluid (PFF) (αMEMF). Medium-199 (M199) served as the control for conventional IVM. Cumulus cell expansion, nuclear maturation, intra-oocyte glutathione (GSH) contents, size of perivitelline space (PVS), and embryonic development after parthenogenesis (PA) and somatic cell nuclear transfer (SCNT) were evaluated after IVM.ResultsRegardless of PVA or PFF supplementation, oocytes matured in 61αMEM showed increased intra-oocyte GSH contents and width of PVS (p < 0.05), as well as increased blastocyst formation (p < 0.05) after PA and SCNT, as compared to oocytes matured in 108αMEMP and M199. Under conditions of PFF-enriched αMEM, SCNT oocytes matured in 61αMEMF showed higher blastocyst formation (p < 0.05), compared to maturation in 108αMEMF and M199, whereas PA cultured oocytes showed no significant difference.ConclusionsIVM in αMEM supplemented with reduced NaCl (61.6 mM) enhances the embryonic developmental competence subsequent to PA and SCNT, which attributes toward improved oocyte maturation.

265 Awardee Talk: Maternal and Paternal Contributions to Fertility

Abstract Using over 56,000 pregnancy diagnostic records in beef cattle, a meta-analysis was conducted to objectively determine that 28.4 % of embryos will not develop past day 7 of gestation with most embryonic losses occurring before day 4. Furthermore, by day 30 of gestation, 47.9 % of cows submitted to a single insemination at day 0 will not be pregnant and pregnancy loss between days 32 and 100 was 5.8%. Reproductive success, however, is highly variable and influenced by maternal and paternal factors. Maternal characteristics, including subspecies, parity and reproductive tract size, intersect with breeding management decisions regarding estrus expression and detection to influence conception and pregnancy rates. The maternal endocrine environment, from estradiol associated with estrus around fertilization to elevated prostaglandins during the embryonic to fetal transition period, play critical roles in pregnancy success. Despite the numerous maternal factors involved with pregnancy establishment, sire contribution to reproductive failure should not be overlooked. Sires are reported to influence pregnancy loss during the second month of gestation in both beef and dairy herds. These differences in sire field fertility could not be explained by differences in semen characteristics nor differences in sire conception rate (SCR) score. To understand parental contribution to pregnancy development during the second month of gestation we developed parthenogenetic (PA) embryos (embryos lacking paternal genome). We observed that PA conceptus had well developed trophectoderm tissue at day 31 of gestation but no site of embryo attachment to the endometrium. Moreover, conceptus secretion such as pregnancy-associated glycoproteins and interferon-stimulated genes were not found in maternal circulation. These results suggest that paternal genetics is required for post-elongated embryo attachment to endometrium. Further exploring the maternal vs paternal contribution to pregnancy development can help elucidate the mechanism that drives reproductive failure in cattle.

The Dynamic Changes of Transcription Factors During the Development Processes of Human Biparental and Uniparental Embryos.

Previous studies have revealed that transcription factors (TFs) play important roles in biparental (BI) early human embryogenesis. However, the contribution of TFs during early uniparental embryo development is still largely unknown. Here we systematically studied the expression profiles of transcription factors in early embryonic development and revealed the dynamic changes of TFs in human biparental and uniparental embryogenesis by single-cell RNA sequencing (scRNA-seq). In general, the TF expression model of uniparental embryos showed a high degree of conformity with biparental embryos. The detailed network analysis of three different types of embryos identified that 10 out of 17 hub TFs were shared or specifically owned, such as ZNF480, ZNF581, PHB, and POU5F1, were four shared TFs, ZFN534, GTF3A, ZNF771, TEAD4, and LIN28A, were androgenic (AG) specific TFs, and ZFP42 was the only one parthenogenetic (PG) specific TF. All the four shared TFs were validated using human embryonic stem cell (hESC) differentiation experiments; most of their target genes are responsible for stem cell maintenance and differentiation. We also found that Zf-C2H2, HMG, and MYB were three dominant transcription factor families that appeared in early embryogenesis. Altogether, our work provides a comprehensive regulatory framework and better understanding of TF function in human biparental and uniparental embryogenesis.

Evaluating Invasive Marbled Crayfish as a Potential Livestock for Sustainable Aquaculture

The marbled crayfish (Procambarus virginalis) is a recently discovered freshwater crayfish species, which reproduces by apomictic parthenogenesis, resulting in a monoclonal, and all-female population. The animals were widely distributed through the aquarium trade and have established numerous stable wild populations through anthropogenic releases. They are highly prevalent in Madagascar, where they have become a popular source of nutritional protein. As freshwater crayfish aquaculture in open systems is a thriving, but ecologically damaging global industry, alternatives are urgently needed. Although marbled crayfish are often branded by their invasive mode of reproduction, their overall invasiveness is not higher than for other cultured crayfish species. Furthermore, their resiliency and high adaptability provide a strong rationale for evaluating them for closed, and environmentally safe aquaculture approaches. Here we describe a novel population of marbled crayfish in a former German coal mining area that is characterized by acid and polluted water. Even under these adverse conditions, animals grew to sizes, and weights that are comparable to commercially farmed freshwater crayfish. Tailored feed development and laboratory testing demonstrated highly efficient feed conversion, suggesting a considerable capacity for sustainable production in closed systems. We further show that marbled crayfish meat can be readily introduced into European meals. Finally, chemical analysis of marbled crayfish exoskeletons revealed comparably high amounts of chitin, which is a valuable source for the synthesis of chitosan and bioplastics. Our results thus suggest that production of marbled crayfish in closed systems may represent a sustainable alternative for crayfish aquaculture.

Optimal Treatment of 6-Dimethylaminopurine Enhances the In Vivo Development of Canine Embryos by Rapid Initiation of DNA Synthesis.

Artificial activation of oocytes is an important step for successful parthenogenesis and somatic cell nuclear transfer (SCNT). Here, we investigated the initiation of DNA synthesis and in vivo development of canine PA embryos and cloned embryos produced by treatment with 1.9 mM 6-dimethylaminopurine (6-DMAP) for different lengths of time. For experiments, oocytes for parthenogenesis and SCNT oocytes were cultured for 4 min in 10 μM calcium ionophore, and then divided into 2 groups: (1) culture for 2 h in 6-DMAP (DMAP-2h group); (2) culture for 4 h in DMAP (DMAP-4h group). DNA synthesis was clearly detected in all parthenogenetic (PA) embryos and cloned embryos incorporated BrdU 4 h after activation in DMAP-2h and DMAP-4h groups. In vivo development of canine parthenogenetic fetuses was observed after embryo transfer and the implantation rates of PA embryos in DMAP-2h were 34%, which was significantly higher than those in DMAP-4h (6.5%, p < 0.05). However, in SCNT, there was no significant difference in pregnancy rate (DMAP-2h: 41.6% vs. DMAP-4h: 33.3%) and implantation rates (DMAP-2h: 4.94% vs. DMAP-4h: 3.19%) between DMAP-2h and DMAP-4h. In conclusion, the use of DMAP-2h for canine oocyte activation may be ideal for the in vivo development of PA zygotes, but it was not more effective in in vivo development of canine reconstructed SCNT oocytes. The present study demonstrated that DMAP-2h treatment on activation of canine parthenogenesis and SCNT could effectively induce the onset of DNA synthesis during the first cell cycle.

Clone worth? Genetic diversity in obligate apomictic Miconia albicans (Melastomataceae).

Apomixis is the asexual production of seeds by plants and, in theory, would render low genetic diversity and even clonal lineages. However, recent studies have shown otherwise, although is not always clear where the genetic diversity of obligate apomicts comes from. We evaluated the genetic diversity among sister seedlings of M. albicans, an obligate apomictic species in Cerrado, Neotropical Savannas in Central Brazil. A total of 50 seedlings from five individuals were analysed using ISSR primers. We obtained 107 fragments, all with good resolution, consistently observed and replicable. The percentage of polymorphic loci ranged from 28.04% to 33.64% and Shannon's information index (I) averaged 0.173. The expected heterozygosity (He) averaged 0.117, similar to the observed for populations of M. albicans and other selfed species. Only two seedlings showed the same genotype (possible clones), but most differed at least for five loci. Most of variance was among progenies (62%), but we found that 38% was within progenies. Genetic distances separated the progenies in two groups, and analogous analyses between individuals reconstructed the original progenies clustering. The results confirmed a relatively high genetic diversity among sister seedling of this obligatory apomictic plant and clones were rare. This diversity can be generated during development, probably by restitutional meiosis or other recombination processes. These differences may accumulate into lineages and populations well adapted to heterogenous Cerrado environment.

DNA barcoding of selected alpine beetles with focus on Curculionoidea (Coleoptera)

Selected beetles, mainly weevils, from the Alpine Arc were barcoded. From 187 samples of 106 assigned species of the families Curculionidae (152 samples, mainly Entiminae, Cyclominae and Hyperinae), Carabidae (18), Apionidae (6), Chrysomelidae and Staphylinidae (each 1 sample), sequences from the COI (subunit 1 of the cytochrome oxydase gene) were obtained, with a success of more than 86% (162 samples). In the cases of Otiorhynchus pupillatus Gyllenhal, 1834, O. nodosus (O. F. Muller, 1764), O. meridionalis Gyllenhal, 1834, Dichotrachelus koziorowicziDesbrochers des Loges, 1873, D. augusti F. Solari, 1946 and D. maculosus Fairmaire, 1869 more diversity was hidden than foreseen in the beginning, suggesting partly cryptic (not yet described) species. One name is thus resurrected from junior synonymy (O. civisStierlin, 1861stat. rev. from synonymy with O. meridionalis). In another case with strictly parthenogenetically reproducing populations of O. pupillatus and O. nodosus in the Swiss Alps, several lineages from hypothetical postglacial immigration events, or alternatively complexes of species in statu nascendi might explain the results observed. Moreover, some morphologically debated species-pairs/triples confirmed to be problematic too, even with our COI sequence data [Hypera nigrirostris (Fabricius, 1775) – ononidis (Chevrolat, 1863) – melarynchus (Olivier, 1807)]. On the other hand, in some cases the lspecies' identity, based on the monophyly of the investigated populations, could be confirmed [Anthonomus rubi (Herbst, 1795), Polydrusus chaerodrysius Gredler, 1866, P. paradoxus Stierlin, 1859]. In the hyperdiverse genus Otiorhynchus Germar, 1822, some preliminary insights into the systematics at the subgenus-level could be made, suggesting that many changes of the present morphologically based systematic structure will be necessary.

Non-dualism: Vedāntic and Āgamic (Advaita as Expounded by Śaṅkara and Abhinavagupta)

Non-dualism: Vedāntic and Āgamic (Advaita as Expounded by Śaṅkara and Abhinavagupta)