Apomixis beyond trees in the Brazilian savanna: new insights from the orchid Zygopetalum mackayi.

n the Neotropics, the focus of apomictic studies predominantly centres on trees within the Brazilian savanna, characterized, mostly as sporophytic and facultative, associated with polyploidy and polyembryony. To enhance our understanding of the mechanisms governing apomixis and sexual reproduction in tropical herbaceous plants, we clarify the relationship between apomixis, chromosome counts, and polyembryony in the epiphytic orchid Zygopetalum mackayi, which forms a polyploid complex within rocky outcrops in both the Brazilian savanna and the Atlantic forest. To define embryo origins and describe megasporogenesis and megagametogenesis, we performed manual self-pollinations in first-day flowers of cultivated plants, considering all three cytotypes (2x, 3x, 4x) of this species. Flowers and fruits at different stages were collected to describe the development and morphology of ovules and seeds considering sexual and apomictic processes. As self-pollination treatments resulted in high fruit abortion in diploids, we also examined pollen tube development in aborted flowers and fruits to search for putative anomalies. Megasporogenesis and megagametogenesis occur regularly in all cytotypes. Apomixis is facultative and sporophytic, and associated with polyploid cytotypes, while diploid individuals exclusively engage in sexual reproduction. Polyembryony is caused mainly by the production of adventitious embryos from nucellar cells of triploids and tetraploids, but also by the development of multiple archesporia in all cytotypes. Like other apomictic angiosperms within the Brazilian savanna, our findings demonstrate that apomixis in Z. mackayi relies on pollinators for seed production. We also consider the ecological implications of these apomictic patterns in Z. mackayi within the context of habitat loss and its dependence on pollinators.

Reproductive biology of harvestmen (Arachnida: Opiliones): a review of a rapidly evolving research field.

Harvestmen are a major arachnid order that has experienced a dramatic increase in biological knowledge in the 21st century. The publication of the book Harvestmen: The Biology of Opiliones in 2007 stimulated the development of many behavioral studies. Although the book is relatively recent, our understanding of the reproductive biology of harvestmen is already outdated due to the fast accumulation of new data. Our goal is to provide an updated review of the subject to serve as a benchmark for the following years. In the pre-copulatory phase, we explore the evolution of facultative parthenogenesis, the factors that may affect the types of mating system, and the role of nuptial gifts in courtship. Regarding the copulatory phase, harvestmen are unique arachnids because they have aflagellate spermatozoa and a penis with complex morphology. We discuss the implications of these two features for sperm competition and cryptic female choice. In the post-copulatory phase, we connect oviposition site selection and climate conditions to the widespread occurrence of resource defense polygyny, alternative reproductive tactics, and sexual dimorphism in several clades of tropical harvestmen. Finally, we present the different forms of parental care in the order, and discuss the benefits and costs of this behavior, which can be performed either by females or males. Throughout the review, we indicate gaps in our knowledge and subjects that deserve further studies. Hopefully, the information synthesized here will stimulate researchers worldwide to embrace harvestmen as a study system and to improve our effort to unravel the mysteries of their reproductive biology.

A comprehensive review on the documented characteristics of four Reticulitermes termites (Rhinotermitidae, Blattodea) of China / Uma revisão abrangente sobre as características documentadas de quatro cupins Reticulitermes da China

Termites are known as social insects worldwide. Presently in China 473 species, 44 genera and 4 families of termites have been reported. Of them, 111 Reticulitermes species are widely spread in different zones of China. The dispersion flight season of these Chinese Reticulitermes species are usually started from February to June, but in some regions different species are distributed, sharing their boundaries and having overlapping flight seasons. These reasons become important sources of hybridization between two different heterospecific populations of termites. It was confirmed that the fertilized eggs and unfertilized eggs of some Reticulitermes termites have the capacity of cleavage. While the unfertilized eggs of R. aculabialis, R. chinensis and R. labralis cleaved normally and the only R. aculabialis unfertilized eggs develop in embryos. While, the R. flaviceps and R. chinensis were observed with their abnormal embryonic development, and not hatching of eggs parthenogenetically. They were reported more threatening to Chinese resources as they propagate with parthenogenesis, hybridization and sexual reproduction. Eggshell and macrophiles of eggs play important roles in species identification and control. Although, they are severe pests and cause a wide range of damages to wooden structures and products in homes, buildings, building materials, trees, crops, and forests in China's Mainland.

Os cupins são conhecidos como insetos sociais em todo o mundo. Atualmente na China foram relatadas 473 espécies, 44 gêneros e 4 famílias de cupins. Destas, 111 espécies de Reticulitermes estão amplamente distribuídas em diferentes zonas da China. A temporada de voo de dispersão dessas espécies chinesas de Reticulitermes geralmente começa de fevereiro a junho, mas em algumas regiões diferentes espécies são distribuídas, compartilhando seus limites e tendo temporadas de voo sobrepostas. Essas razões tornam-se importantes fontes de hibridização entre duas populações heteroespecíficas de cupins. Foi confirmado que os ovos fertilizados e não fertilizados de alguns cupins Reticulitermes possuem capacidade de clivagem. Já os ovos não fertilizados de R. aculabialis, R. chinensis e R. labralis clivaram normalmente, e os únicos ovos não fertilizados de R. aculabialis se desenvolvem em embriões. R. flaviceps e R. chinensis foram observados com desenvolvimento embrionário anormal, e não eclosão de ovos por partenogênese. Eles foram relatados como mais ameaçadores para os recursos chineses à medida que se propagam com partenogênese, hibridização e reprodução sexual. Casca de ovo e macrófilos de ovos desempenham papéis importantes na identificação e controle de espécies, embora sejam pragas graves e causem uma ampla gama de danos a estruturas e produtos de madeira em residências, edifícios, materiais de construção, árvores, plantações e florestas na China continental.

Combined Exogenous Activation of Bovine Oocytes: Effects on Maturation-Promoting Factor, Mitogen-Activated Protein Kinases, and Embryonic Competence.

Oocyte activation via dual inhibition of protein synthesis and phosphorylation has improved in vitro embryo production in different mammalian species. In this study, we evaluated the effects of the combination of cycloheximide (CHX), dimethyl amino purine (DMAP), and anisomycin (ANY) on the activation of bovine oocytes, particularly on dynamics of MPF and MAPKs, embryonic developmental potential, and quality. The results showed that the cleavage and blastocyst rates, as well as levels of CCNB1, CDK1, p-CDK1Thr161, and p-CDK1Thr14-Tyr15, were similar among groups; ANY and ANY + CHX reduced the expression of ERK1/2 compared to DMAP-combinations (p < 0.05), whereas ANY + DMAP, CHX + DMAP, and ANY + CHX + DMAP reduced p-ERK1/2 compared to ANY and ANY + CHX treatments (p < 0.05). The quality of blastocysts in terms of cell counts, their allocation, and the numbers of TUNEL-positive cells did not differ among groups. However, transcript levels of POU5F1 were higher in embryos derived from ANY + CHX + DMAP treatment compared to other groups, while expression levels of CDX2 did not show differences. In addition, the BCL2A1/BAX ratio of the ANY + CHX + DMAP treatment was significantly low compared to the ANY treatment (p < 0.05) and did not differ significantly from the other treatments. In conclusion, oocyte activation by dual inhibition of protein synthesis and phosphorylation induces MPF inactivation without degradation of CCNB1, while MAPK inactivation occurs differentially between these inhibitors. Thus, although the combined use of these inhibitors does not affect early developmental competence in vitro, it positively impacts the expression of transcripts associated with embryonic quality.

Phylogeography of Eriotheca species complex: insights into the origin and range expansion of apomictic and polyploid trees in Neotropical Savannas.

Polyploidy and whole genome duplication are major evolutionary drivers in plants. Climate variations during the Pleistocene have influenced distribution and range expansion worldwide. Similar trends have been reported for Cerrado plants, but no attempt has been made to link phylogeography with ploidy and breeding changes. Thus, we aimed to (i) assess ploidy and genome size of Eriotheca estevesiae Carv.-Sobr., and compare it with E. pubescens (Mart.) Schott & Endl. (Both included into the Eriotheca Stellate Trichome Species Complex - ESTSC). (ii) Subsequently, we investigated their phylogeography to see whether genetic structure and range expansion trends were similar to those previously described for the Cerrado biome. Finally (iii), we discuss whether ESTSC phylogeographic patterns could be associated with geographic parthenogenesis processes. Common cytogenetic techniques and flow cytometry were used to confirm chromosome number and genome size of E. estevesiae. We used three cpDNA regions to analyse 14 ESTSC Cerrado populations, for which we also obtained ploidy level and breeding information. We investigated haplotype diversity, population structure and tested neutrality, aiming to reconstruct phylogeographic scenarios. We found three ploidy levels and eight cpDNA haplotypes in ESTSC, one shared by most populations. Haplotype and ploidy distribution corroborated that E. pubescens, the widely distributed polyploid and apomictic species, may have originated from northern diploid and probably sexual E. estevesiae. Matrilinear cpDNA links support the idea that apomixis and polyploidy in ESTSC may have allowed range expansion during the Pleistocene, in a process analogous to the geographic parthenogenesis described elsewhere.

Taxonomic study of Scytosiphon (Phaeophyceae) from temperate coasts of Argentina.

Scytosiphon is a common intertidal genus widely distributed on temperate coasts worldwide. Recently, eight species have been delimited with molecular tools. Although S. lomentaria is the only species that predominates in the macroalgal literature of the Southwestern Atlantic Ocean (SwAO), unpublished molecular data obtained for a population study of S. lomentaria revealed hidden species diversity of Scytosiphon among the individuals collected from four localities at the SwAO. The aim of this study was to revise the identity and phylogenetic relationships of Scytosiphon from temperate coasts of the SwAO using DNA data. Thalli were collected from the Argentinean coast between 39° S and 43° S, from which cox1 and rbcL gene sequences were obtained. Phylogenies and haplotype networks were inferred and morphology of gametophytes was studied. Four species were recognized, S. lomentaria, S. promiscuus, S. shibazakiorum, and one species that belongs to a complex of species known as "Scytosiphon Atlantic complex." This complex was known to occur only in the North Atlantic, however, the results found in this study revealed that it has an extended distribution range that includes the southern hemisphere, where its populations have high genetic diversity and unique haplotypes. The morphological differences among the four species were subtle; denoting that previous Scytosiphon records from the SwAO attributed to the renowned S. lomentaria could represent different species. In addition, sex ratio and genome-wide single nucleotide polymorphisms (SNPs) analyses were done for populations of S. promiscuus presumably introduced to the SwAO, and the results indicated that they included female-dominant parthenogenetic populations, which were probably introduced from Japan.

Hunger for sex: Abundant, heterogeneous resources select for sexual reproduction in the field.

Major hypotheses on sex evolution predict that resource abundance and heterogeneity should either select for or against sexual reproduction. However, seldom have these predictions been explicitly tested in the field. Here, we investigated this question using soil oribatid mites, a diverse and abundant group of soil arthropods whose local communities can be dominated by either sexual or asexual species. First, we refined theoretical predictions by addressing how the effects of resource abundance, heterogeneity and abiotic conditions could modify each other. Then, we estimated the strength of selection for sexual species in local communities while controlling for phylogeny and neutral processes (ecological drift and dispersal), and tested its relation to resource and abiotic gradients. We show that sexual species tended to be favoured with increasing litter amount, a measure of basal resource abundance. Further, there was some evidence that this response occurred mainly under higher tree species richness, a measure of basal resource heterogeneity. This response to resources is unlikely to reflect niche partitioning between reproductive modes, as sexual and asexual species overlapped in trophic niche according to a comparative analysis using literature data on stable isotope ratios. Rather, these findings are consistent with the hypothesis that sex facilitates adaptation by breaking unfavourable genetic associations, an advantage that should increase with effective population size when many loci are under selection and, thus, with resource abundance.

Mates Matter: Gametophyte Kinship Recognition and Inbreeding in the Giant Kelp, Macrocystis pyrifera (Laminariales, Phaeophyceae).

Inbreeding, the mating between genetically related individuals, often results in reduced survival and fecundity of offspring, relative to outcrossing. Yet, high inbreeding rates are commonly observed in seaweeds, suggesting compensatory reproductive traits may affect the costs and benefits of the mating system. We experimentally manipulated inbreeding levels in controlled crossing experiments, using gametophytes from 19 populations of Macrocystis pyrifera along its Eastern Pacific coastal distribution (EPC). The objective was to investigate the effects of male-female kinship on female fecundity and fertility, to estimate inbreeding depression in the F1 progeny, and to assess the variability of these effects among different regions and habitats of the EPC. Results revealed that the presence and kinship of males had a significant effect on fecundity and fertility of female gametophytes. Females left alone or in the presence of sibling males express the highest gametophyte size, number, and size of oogonia, suggesting they were able to sense the presence and the identity of their mates before gamete contact. The opposite trend was observed for the production of embryos per female gametes, indicating higher costs of selfing and parthenogenesis than outcrossing on fertility. However, the increased fecundity compensated for the reduced fertility, leading to a stable overall reproductive output. Inbreeding also affected morphological traits of juvenile sporophytes, but not their heatwave tolerance. The male-female kinship effect was stronger in high-latitude populations, suggesting that females from low-latitude marginal populations might have evolved to mate with any male gamete to guarantee reproductive success.

Life cycle of Ixodes schulzei (Acari: Ixodidae) in the laboratory, and demonstration of reproduction by parthenogenesis.

Ixodes schulzei is an ixodid tick that parasitizes Cricetidae rodents, chiefly the South American water rat, Nectomys squamipes, in Brazil and Argentina. In the present study, we evaluated the life cycle of I. schulzei by exposing larvae and nymphs to feed on two rodent species, N. squamipes and Calomys callosus (large vesper mouse),while adult ticks were exposed to feed on N. squamipes. Off-host developmental periods were observed in an incubator at 27 °C, 95% relative humidity, and 0:24 (light:dark) regimen. Larvae and nymphs successfully fed on either C. callosus or N. squamipes. Mean larval and nymphal feeding periods were 8.8 and 8.7 days on N. squamipes and 8.5 and 9.7 days on C. callosus. The majority of engorged larvae (79.0-80.8%) and nymphs (67.0-86.0%) successfully molted to nymphs and adults, respectively. Mean premolt periods were 11.5-11.7 days for engorged larvae and 22.5-23.7 days for engorged nymphs. Only adult females emerged from engorged nymphs, regardless of host species, i.e., none of 120 engorged nymphs molted to male. Around 18% of the unfed females presented teratologies compatible with the metagynander type of gynandromorphism. Ixodes schulzei adult females successfully fed (mean feeding period, 9.4 days), oviposited, and presented high reproductive performance (high engorged weight, egg mass weight, and % egg mass hatching), in the absence of male ticks. Our results showed that I. schulzei successfully reproduces by parthenogenesis, and corroborate field data that indicate N. squamipes as the most important host for this tick species. The male of I. schulzei remains unknown.

Effect of single and combined treatments with MPF or MAPK inhibitors on parthenogenetic haploid activation of bovine oocytes.

In bovine, correct oocyte artificial activation is a key step in ICSI and other reproductive biotechnologies, and still needs to be improved. The current study was designed to compare the activating efficiency of ionomycin (Io) followed by: a 4 h time window and ethanol (4h-Et), roscovitine (Rosc), dehydroleucodine (DhL), cycloheximide (CHX) or PD0325901 (PD), each as a single treatment, and then combine them in novel protocols. Parthenogenetic haploid activation was evaluated in terms of pronuclear (PN) formation, second polar body (2PB) extrusion, ploidy of day 2 embryos and in vitro development. Combined treatments with Io-4h-Et-Rosc and Io-Rosc/CHX increased PN formation (92.2% and 96%, respectively) compared with Io-Rosc, Io-CHX or Io-4h-Et, which were equally efficient at inducing PN formation (82-84%) and 2PB extrusion (62.1-70.5%). Oocyte activation with Io-DhL and Io-Rosc/DhL resulted in higher 2PB extrusion rates (90% and 95.9%, respectively) but lower PN formation (49.4-58.8%) and cleavage rates (36-57.9%), as occurred with Io-CHX/DhL (76.4% and 70.4%, respectively). For the first time, results show that Io followed by the MAPK inhibitor PD induces PN formation and 2PB extrusion, but PD combined with Rosc or CHX resulted in low rates of haploid day 2 embryos. In conclusion, DhL strongly induces 2PB extrusion but leads to poor PN formation and embryo development. PD induces bovine oocyte activation but results in low rates of haploid embryos. In contrast, the improved PN formation rates after treatment with combined Io-4h-Et-Rosc and Io-Rosc/CHX suggest they should be further evaluated in ART, aiming to increase success rates in bovine.

Vitrification of in vitro produced porcine blastocysts: influence of cryoprotectants toxicity and embryo age

Background: Porcine embryos are sensible to all assisted reproduction manipulations, especially the ones that involvecryopreservation. Despite the high cryoprotectant concentrations routinely applied, vitrification is the most effective technique to date. These substances toxicity can also play a negative role in embryo viability. During in vitro porcine embryoproduction, the speed of development is often unevenly distributed. It is possible that their development speed, affectsembryo tolerance to cryoprotectants. This study aimed to evaluate the toxicity of porcine embryos of days 5 or 6 of cultureto cryoprotectant agents; as well as to assess embryo survival to vitrification.Materials, Methods & Results: Parthenogenetic porcine blastocysts and expanded blastocysts of days 5 and 6 of culturewere exposed to toxicity tests (experiments 1 and 2) and vitrification (experiment 3) using different protocols. In the firstexperiment, three different cryoprotectants were used (Dimethyl sulfoxide - DMSO, Ethylene glycol - EG, and Sucrose- SUC), combined in three different associations (G1: 15% EG + 15% DMSO with 0.5 M SUC; G2: 16% EG + 16%DMSO with 0.4 M SUC; G3: 18% EG + 18% DMSO with 0.5 M SUC). In the fresh Control, embryos of day 6 are moresensible than the ones of day 5, whom showed a lower hatching rate (39.7 vs. 60.8%). After the toxicity (Experiment 1)test, the G1 showed better expansion rates in day 6 (50.0 vs 31.0 and 3.6% for G2 and G3) and higher hatching of day 6compared to G2 and G3 (23.2, vs. 8.6 and 0.0% for G2 and G3). The fresh non hatched embryos at day 8, derived at day6, had a lower percentage of cells with cleaved caspase-3 (20.2%) compared with the G1 (30.5%), G2 (31.4%) and G3(30.5%). The hatched embryos of day 5 from G2 had lower total cell number (TCN) compared with the day 6 hatchedembryos, whereas in G1 the TCN was not affected. The second experiment compared EG combined to one of these threeextracellular...(AU)

Translocation of a parthenogenesis gene candidate to an alternate carrier chromosome in apomictic Brachiaria humidicola.

BACKGROUND: The apomictic reproductive mode of Brachiaria (syn. Urochloa) forage species allows breeders to faithfully propagate heterozygous genotypes through seed over multiple generations. In Brachiaria, reproductive mode segregates as single dominant locus, the apospory-specific genomic region (ASGR). The AGSR has been mapped to an area of reduced recombination on Brachiaria decumbens chromosome 5. A primer pair designed within ASGR-BABY BOOM-like (BBML), the candidate gene for the parthenogenesis component of apomixis in Pennisetum squamulatum, was diagnostic for reproductive mode in the closely related species B. ruziziensis, B. brizantha, and B. decumbens. In this study, we used a mapping population of the distantly related commercial species B. humidicola to map the ASGR and test for conservation of ASGR-BBML sequences across Brachiaria species. RESULTS: Dense genetic maps were constructed for the maternal and paternal genomes of a hexaploid (2n = 6x = 36) B. humidicola F1 mapping population (n = 102) using genotyping-by-sequencing, simple sequence repeat, amplified fragment length polymorphism, and transcriptome derived single nucleotide polymorphism markers. Comparative genomics with Setaria italica provided confirmation for x = 6 as the base chromosome number of B. humidicola. High resolution molecular karyotyping indicated that the six homologous chromosomes of the sexual female parent paired at random, whereas preferential pairing of subgenomes was observed in the apomictic male parent. Furthermore, evidence for compensated aneuploidy was found in the apomictic parent, with only five homologous linkage groups identified for chromosome 5 and seven homologous linkage groups of chromosome 6. The ASGR mapped to B. humidicola chromosome 1, a region syntenic with chromosomes 1 and 7 of S. italica. The ASGR-BBML specific PCR product cosegregated with the ASGR in the F1 mapping population, despite its location on a different carrier chromosome than B. decumbens. CONCLUSIONS: The first dense molecular maps of B. humidicola provide strong support for cytogenetic evidence indicating a base chromosome number of six in this species. Furthermore, these results show conservation of the ASGR across the Paniceae in different chromosomal backgrounds and support postulation of the ASGR-BBML as candidate genes for the parthenogenesis component of apomixis.

Apomixis in flowering plants: Developmental and evolutionary considerations.

Apomixis refers to a set of reproductive mechanisms that invariably rely on avoiding meiotic reduction and fertilization of the egg cell to generate clonal seeds. After having long been considered a strictly asexual oddity leading to extinction, the integration of more than 100 years of embryological, genetic, molecular, and ecological research has revealed apomixis as a widely spread component of the dynamic processes that shape flowering plant evolution. Apomixis involves several flexible and versatile developmental pathways that can be combined within the ovule to produce offspring. Here we review the large body of classic and contemporaneous contributions that have addressed unreduced gamete formation, haploid induction, and parthenogenesis in flowering plants. We emphasize similarities and differences between sexual and apomictic reproduction, and highlight their implications for the evolutionary emergence of asexual reproduction through seeds. On the basis of these comparisons, we propose a model that associates the developmental origin of apomixis to a dynamic epigenetic landscape, in which environmental fluctuations reversibly influence female reproductive development through mechanisms of hybridization and polyploidization.

Game of clones: Is Wolbachia inducing speciation in a weevil with a mixed reproductive mode?

Parthenogenesis is widely distributed in Metazoa but it is especially frequent in weevils (Coleoptera, Curculionidae) with one fifth of all known cases. Previous studies have shown that in the tribe Naupactini parthenogenetic reproduction most likely originated with an infection of the endoparasitic bacterium Wolbachia pipientis. In particular, Pantomorus postfasciatus possess a mixed reproductive mode: some populations have males while in others they are absent, and females produce clones by thelytoky. To better understand this scenario, we studied the population structure and infection status in 64 individuals of P. postfasciatus from Argentina and Brazil. We sequenced two mitochondrial (COI and COII) and one nuclear (ITS-1) fragments and obtained two very divergent haplogroups, one corresponding to the sexual populations uninfected with Wolbachia, and another conforming a monophyletic parthenogenetic (or presumptively parthenogenetic) and infected clade. Each of these haplogroups was identified as an independently evolutionary unit by all species delimitation analyses accomplished: multilocus *BEAST and BP&P, and single locus GMYC and K/θ rule. Additionally, present evidence suggests that Wolbachia infection occurred at least twice in all-female populations of P. postfasciatus with two different bacterial strains. Speciation mediated by Wolbachia is a recently described phenomenon and the case of P. postfasciatus is the first known case in a diplo-diploid insect. A model that describes how thelytoky-inducing phenotypes of Wolbachia could generate new lineages is discussed.

Complex patterns of hybridization and introgression across evolutionary timescales in Mexican whiptail lizards (Aspidoscelis).

Identifying patterns of introgression across the tree of life is foundational to understanding general mechanisms that govern the impacts of gene flow on the speciation process. There are few vertebrate groups in which hybridization is associated with as large a diversity of outcomes as in North American whiptail lizards (Aspidoscelis). Of particular interest is that hybridization among divergent whiptail species has repeatedly led to the formation of unisexual (parthenogenetic) lineages. Understanding the hybrid origin of these unisexual lineages requires an accurate understanding of species boundaries among gonochoristic whiptails. Doing so has historically been an extremely challenging problem which, in part, may be a consequence of widespread hybridization and incomplete reproductive isolation among lineages. The lack of a robust phylogenetic framework and uncertainty in species boundaries precludes studies of general patterns and mechanisms of introgression among whiptail species. Here, we use genomic data to reconstruct a robust estimate of evolutionary history in the largest clade of whiptail lizards (A. sexlineatus species group) and use it to identify patterns of introgression. Our results indicate substantial introgressive hybridization and admixture has occurred among multiple lineages of whiptails across diverse evolutionary time scales, and illustrate their impact on phylogenetic inference. Thus, hybridization among whiptail species appears to have been a prominent feature throughout their evolutionary history, which could, in part, explain why parthenogenesis has evolved so many times in whiptails in comparison to other vertebrate groups.

Vitrification of in vitro produced porcine blastocysts: influence of cryoprotectants toxicity and embryo age

Background: Porcine embryos are sensible to all assisted reproduction manipulations, especially the ones that involvecryopreservation. Despite the high cryoprotectant concentrations routinely applied, vitrification is the most effective technique to date. These substances toxicity can also play a negative role in embryo viability. During in vitro porcine embryoproduction, the speed of development is often unevenly distributed. It is possible that their development speed, affectsembryo tolerance to cryoprotectants. This study aimed to evaluate the toxicity of porcine embryos of days 5 or 6 of cultureto cryoprotectant agents; as well as to assess embryo survival to vitrification.Materials, Methods & Results: Parthenogenetic porcine blastocysts and expanded blastocysts of days 5 and 6 of culturewere exposed to toxicity tests (experiments 1 and 2) and vitrification (experiment 3) using different protocols. In the firstexperiment, three different cryoprotectants were used (Dimethyl sulfoxide - DMSO, Ethylene glycol - EG, and Sucrose- SUC), combined in three different associations (G1: 15% EG + 15% DMSO with 0.5 M SUC; G2: 16% EG + 16%DMSO with 0.4 M SUC; G3: 18% EG + 18% DMSO with 0.5 M SUC). In the fresh Control, embryos of day 6 are moresensible than the ones of day 5, whom showed a lower hatching rate (39.7 vs. 60.8%). After the toxicity (Experiment 1)test, the G1 showed better expansion rates in day 6 (50.0 vs 31.0 and 3.6% for G2 and G3) and higher hatching of day 6compared to G2 and G3 (23.2, vs. 8.6 and 0.0% for G2 and G3). The fresh non hatched embryos at day 8, derived at day6, had a lower percentage of cells with cleaved caspase-3 (20.2%) compared with the G1 (30.5%), G2 (31.4%) and G3(30.5%). The hatched embryos of day 5 from G2 had lower total cell number (TCN) compared with the day 6 hatchedembryos, whereas in G1 the TCN was not affected. The second experiment compared EG combined to one of these threeextracellular...

Aquatic long-distance dispersal and vicariance shape the evolution of an ostracod species complex (Crustacea) in four major Brazilian floodplains.

Cladogenesis is often driven by the interplay of dispersal and vicariance. The importance of long-distance dispersal in biogeography and speciation is increasingly recognised, but still ill-understood. Here, we study faunal interconnectivity between four large Brazilian floodplains, namely the Amazon, Araguaia, Pantanal (on Paraguay River) and Upper Paraná River floodplains, investigating a species complex of the non-marine ostracod genus Strandesia. We use DNA sequence data from the mitochondrial COI and the nuclear Elongation Factor 1 alpha genes to construct molecular phylogenies and minimum spanning networks, to identify genetic species, analyse biogeographic histories and provide preliminary age estimates of this species complex. The Strandesia species complex includes five morphological and eleven genetic species, which doubles the known diversity in this lineage. The evolutionary history of this species complex appears to comprise sequences of dispersal and vicariance events. Faunal and genetic patterns of connectivity between floodplains in some genetic species are mirrored in modern hydrological connections. This could explain why we find evidence for (aquatic) long-distance dispersal between floodplains, thousands of kilometres apart. Our phylogenetic reconstructions seem to mostly indicate recent dispersal and vicariance events, but the evolution of the present Strandesia species complex could span up to 25 Myr, which by far exceeds the age of the floodplains and the rivers in their current forms.

Primary fibroblast cell cycle synchronization and effects on handmade cloned (hmc) bovine embryos / Sincronização do ciclo celular de fibroblastos primários e efeitos na produção de embriões bovinos clonados por handmade (hmc)

Spatial and temporal synchrony and compatibility between the receptor oocyte and the donor cell nucleus are necessary for the process of embryo cloning to allow nuclear reprogramming and early embryonic development. The objective of the present study was to evaluate three cell cycle synchronization methods on a primary bovine fibroblast culture for 24, 48, or 72 h. These fibroblasts were used as nuclear donors to evaluate their in vitro developmental potential and the quality of the embryos produced through handmade cloning (HMC). No differences were found between the methods used for fibroblast synchronization in G0/G1 (p > 0.05). Production of clones from fibroblasts in four groups- no treatment at 0 h and using serum restriction SR, high culture confluence HCC, and SR+HCC at 24 h- resulted in high cleavage rates that were not different. Embryo production rates were 37.9%, 29.5%, and 30.9% in the 0h, SR24h, and SR+HHC24h groups, respectively, and 19.3% in the HCC group, which was significantly different from the other three (p < 0.05). There were no differences in the quality parameter among the clones produced with fibroblasts subjected to the different synchronization. Finally, when overall clone production was compared versus parthenotes and IVF embryos, the only difference was between clones and parthenogenetic embryos with zona pellucida (30.2% vs 38.6%). The number of blastomeres from the blastocytes produced through IVF was significantly greater than those from embryos activated parthenogenetically and from clones (117, 80, 75.9, and 67.1, respectively). The evaluation of three synchronization methods at different time points did not demonstrate an increase in the percentage of fibroblasts in the G0/G1 phases of the cell cycle; however, good quality and high cloning rates were obtained, suggesting that it is not always necessary to subject the cells to any synchronization treatments, as they would yield equally good cloning results.(AU)

A sincronia espacial e temporal e a compatibilidade entre o oócito receptor e o núcleo celular doador são necessárias para o processo de clonagem de embriões a fim de permitir a reprogramação nuclear e o desenvolvimento embrionário precoce. O objetivo do presente estudo foi avaliar três métodos de sincronização do ciclo celular em uma cultura primária de fibroblastos bovinos durante 24, 48 ou 72 h. Estes fibroblastos foram utilizados como doadores nucleares para avaliar o seu potencial de desenvolvimento in vitro e a qualidade dos embriões produzidos por meio da técnica de Handmade cloning (HMC). Não foram encontradas diferenças entre os métodos utilizados para a sincronização de fibroblastos em G0 / G1 (p> 0,05). Produção de clones de fibroblastos nos quatro grupos - sem tratamento a 0 h e com restrição de soro RS, alta confluência celular ACC e RS + ACC às 24 h - resultou em altas taxas de clivagem que não foram diferentes. As taxas de produção de embriões foram de 37,9%, 29,5% e 30,9% nos grupos 0h, RS24h e RS + ACC24h, respectivamente, e 19,3% no grupo ACC, que foi significativamente diferente dos outros três (p <0,05). Não houve diferenças no parâmetro de qualidade entre os clones produzidos com fibroblastos submetidos à sincronização diferente. Finalmente, quando a produção geral de clones foi comparada versus partenotos e embriões de FIV, a única diferença foi entre clones e embriões partenogênicos com zona pelúcida (30,2% vs 38,6%). O número de blastômeros dos blastocitos produzidos através da FIV foi significativamente maior do que os de embriões ativados partenogeneticamente e de clones (117, 80, 75,9 e 67,1, respectivamente).(AU)

Primary fibroblast cell cycle synchronization and effects on handmade cloned (hmc) bovine embryos / Sincronização do ciclo celular de fibroblastos primários e efeitos na produção de embriões bovinos clonados por handmade (hmc)

Spatial and temporal synchrony and compatibility between the receptor oocyte and the donor cell nucleus are necessary for the process of embryo cloning to allow nuclear reprogramming and early embryonic development. The objective of the present study was to evaluate three cell cycle synchronization methods on a primary bovine fibroblast culture for 24, 48, or 72 h. These fibroblasts were used as nuclear donors to evaluate their in vitro developmental potential and the quality of the embryos produced through handmade cloning (HMC). No differences were found between the methods used for fibroblast synchronization in G0/G1 (p > 0.05). Production of clones from fibroblasts in four groups- no treatment at 0 h and using serum restriction SR, high culture confluence HCC, and SR+HCC at 24 h- resulted in high cleavage rates that were not different. Embryo production rates were 37.9%, 29.5%, and 30.9% in the 0h, SR24h, and SR+HHC24h groups, respectively, and 19.3% in the HCC group, which was significantly different from the other three (p < 0.05). There were no differences in the quality parameter among the clones produced with fibroblasts subjected to the different synchronization. Finally, when overall clone production was compared versus parthenotes and IVF embryos, the only difference was between clones and parthenogenetic embryos with zona pellucida (30.2% vs 38.6%). The number of blastomeres from the blastocytes produced through IVF was significantly greater than those from embryos activated parthenogenetically and from clones (117, 80, 75.9, and 67.1, respectively). The evaluation of three synchronization methods at different time points did not demonstrate an increase in the percentage of fibroblasts in the G0/G1 phases of the cell cycle; however, good quality and high cloning rates were obtained, suggesting that it is not always necessary to subject the cells to any synchronization treatments, as they would yield equally good cloning results.

A sincronia espacial e temporal e a compatibilidade entre o oócito receptor e o núcleo celular doador são necessárias para o processo de clonagem de embriões a fim de permitir a reprogramação nuclear e o desenvolvimento embrionário precoce. O objetivo do presente estudo foi avaliar três métodos de sincronização do ciclo celular em uma cultura primária de fibroblastos bovinos durante 24, 48 ou 72 h. Estes fibroblastos foram utilizados como doadores nucleares para avaliar o seu potencial de desenvolvimento in vitro e a qualidade dos embriões produzidos por meio da técnica de Handmade cloning (HMC). Não foram encontradas diferenças entre os métodos utilizados para a sincronização de fibroblastos em G0 / G1 (p> 0,05). Produção de clones de fibroblastos nos quatro grupos - sem tratamento a 0 h e com restrição de soro RS, alta confluência celular ACC e RS + ACC às 24 h - resultou em altas taxas de clivagem que não foram diferentes. As taxas de produção de embriões foram de 37,9%, 29,5% e 30,9% nos grupos 0h, RS24h e RS + ACC24h, respectivamente, e 19,3% no grupo ACC, que foi significativamente diferente dos outros três (p <0,05). Não houve diferenças no parâmetro de qualidade entre os clones produzidos com fibroblastos submetidos à sincronização diferente. Finalmente, quando a produção geral de clones foi comparada versus partenotos e embriões de FIV, a única diferença foi entre clones e embriões partenogênicos com zona pelúcida (30,2% vs 38,6%). O número de blastômeros dos blastocitos produzidos através da FIV foi significativamente maior do que os de embriões ativados partenogeneticamente e de clones (117, 80, 75,9 e 67,1, respectivamente).

Influence of host preference, mating, and release density on the parasitism of Telenomus remus (Nixon) (Hymenoptera, Platygastridae)

ABSTRACT We evaluated the influence of host preference, mating, and release density on Telenomus remus (Nixon, 1937) (Hymenoptera: Platygastridae) parasitizing eggs of Spodoptera frugiperda (Smith, 1797) (Lepidoptera: Noctuidae). First, we tested host preference of T. remus (free choice test) offered a choice between eggs of Corcyra cephalonica (Stainton, 1865) (Lepidoptera: Pyralidae) and S. frugiperda. Parasitism capacity and host preference (S. frugiperda) of T. remus reared on either of the two hosts did not differ. Secondly, we evaluated the influence of mating behavior of T. remus females on its parasitism. Only the offspring sex ratio differed between treatments, indicating that the species reproduces by parthenogenesis of the arrhenotoky type. Finally, we evaluated the influence of release density on T. remus parasitism. This was tested by releasing different numbers of the parasitoid per S. frugiperda egg using T. remus reared for different numbers of generations on C. cephalonica eggs. The regression analysis between percentage of parasitism and density of released T. remus females showed a quadratic effect for all tested parasitoid generations (F35, F40, and F45) with maximum parasitism from 65.07% to 71.69%. Our results allow the conclusion that (a) T. remus prefers S. frugiperda eggs, regardless of the host on which this parasitoid was reared, showing no preimaginal conditioning; (b) Mating does not affect the number of eggs parasitized by T. remus or the development of its offspring; and (c) The optimal T. remus release density when reared on C. cephalonica is between 0.133 and 0.150 females/S. frugiperda.