Despite previous research demonstrating the benefits of including growth factors and antioxidants to maturation medium to support embryo production, to date the effect of epidermal growth factor (EGF) and melatonin (Mel) on oocyte competency has not been studied. This study supplemented in vitro maturation (IVM) medium with EGF (10 ng/ml) and Mel (50 ng/ml) alone, or in combination, and evaluated cumulus cell (CC) gene expression and the development and quality of parthenogenetic blastocysts. No differences in CC gene expression levels indicative of developmental potential were found among the treatment groups. Antioxidant gene CuZnSOD was significantly (P < 0.05) decreased in CCs from the Mel group. Moreover, blastocyst rates on day 7 were significantly increased in EGF or Mel (P < 0.05), but not EGF+Mel. Significant decrease (P < 0.05) in GPX1, CuZnSOD, SLC2A1 and HSPA1A (P = 0.07) mRNA levels was observed in blastocysts from the Mel group. OCT4 gene expression was significantly increased (P < 0.05) in EGF+Mel and confirmed using immunofluorescence. Our results indicate that, despite the lack of changes of competence-related genes in CCs, IVM medium supplemented with Mel improved the culture environment sufficiently, resulting in improved blastocysts. Moreover, EGF and Mel combined during maturation increased OCT4 gene and protein expression in blastocysts, indicating its potential for stem cells.

Dibothriocephalus latus is the most frequent causative agent of fish-borne zoonosis (diphyllobothriosis) in Europe, where it is currently circulating mainly in the Alpine lakes region (ALR) and Russia. Three mitochondrial genes (cox1, cob and nad3) and 6 microsatellite loci were analysed to determine how is the recently detected triploidy/parthenogenesis in tapeworms from ALR displayed at the DNA level. A geographically distant population from the Krasnoyarsk Reservoir in Russia (RU-KR) was analysed as a comparative population. One or 2 alleles of each microsatellite locus was detected in plerocercoids from RU-KR, corresponding to the microsatellite pattern of a diploid organism. In contrast, 1–3 alleles were observed in tapeworms from ALR, in accordance with their triploidy. The high diversity of mitochondrial haplotypes in D. latus from RU-KR implied an original and relatively stable population, but the identical structure of mitochondrial genes of tapeworms from ALR was probably a consequence of a bottleneck typical of introduced populations. These results indicated that the diploid/sexually reproducing population from RU-KR was ancestral, located within the centre of the distribution of the species, and the triploid/parthenogenetically reproducing subalpine population was at the margin of the distribution. The current study revealed the allelic structure of the microsatellite loci in the triploid tapeworm for the first time.

Across Far East Asia, aspermic Fasciola forms are found endemically. They have abnormal spermatogenesis and oogenesis, and it is presumed that their progeny are produced parthenogenetically and clonally. Because of this, they are also termed parthenogenic Fasciola forms. Currently, there is no evidence that they do indeed reproduce parthenogenetically and clonally. In this study, the multilocus genetic type (MLG) in 12 microsatellite markers of adult flukes and their subsequent progeny larvae were analysed using two laboratory aspermic Fasciola triploid strains. The MLGs of adults and their larvae were identical for all markers evaluated, suggesting that these strains reproduce their progeny clonally. The deviation between theoretical and actual frequency within the larvae genotype of the Fh_6 locus resulted in the inability for self-fertilization within individual adult flukes. These findings strongly suggested that aspermic Fasciola forms reproduce their progeny by means of parthenogenesis, possibly gynogenesis.

The large-sized tapeworm Dibothriocephalus latus is known as the broad or fish-borne cestode of mammals that is capable to infect humans and cause diphyllobothriosis. Recently, molecular data on D. latus has been accumulating in the literature and a complete genome sequence has been published; however, little is known about the karyotype and chromosome architecture. In this study, an in-depth karyological analysis of 2 D. latus specimens was carried out. The plerocercoids originated from a perch caught in subalpine Lake Iseo (Italy) and the tapeworms were reared in hamsters. Both specimens contained cells with a highly variable number of chromosomes ranging from18 to 27. Nevertheless, the largest portion of mitotic figures (47%) showed a number corresponding to the triploid set, 3n = 27. Accordingly, the karyotype of the analyzed specimens consisted of 9 triplets of metacentric chromosomes. Fluorescence in situ hybridization (FISH) with the 18S rDNA probe clearly demonstrated the presence of 3 clusters of hybridization signals on the triplet of chromosome 7, thus confirming the triploid status of the specimens. FISH with a telomeric (TTAGGG)n probe confined hybridization signals exclusively to the terminal chromosomal regions, supporting the earlier findings that this repetitive motif is a conserved feature of tapeworm telomeres.

Mouse and lamb oocytes were vitrified with, or exposed to, different cryoprotectants and evaluated for their effects on their survival and developmental competence after in vitro fertilization (IVF) and activation treatments. Control oocytes remained untreated, whilst the remainder were exposed to three different combinations of vitrification solutions [dimethyl sulfoxide (DMSO) + ethylene glycol (EG), EG only, or propanediol (PROH) + EG] and either vitrified or left unfrozen (exposed groups). Oocytes in the control and vitrified groups underwent IVF and developmental competence was assessed to the blastocyst stage. In lambs, survival rate in vitrified oocytes was significantly lower than for oocytes in the exposed groups (P <0.05). Blastocyst development was low in vitrified oocytes compared with controls (<6% vs 38.9%, P <0.01). Parthenogenetic activation was more prevalent in vitrified lamb oocytes compared with controls (P <0.05). No evidence of zona pellucida hardening or cortical granule exocytosis could account for reduced fertilization rates in vitrified lamb oocytes. Mouse oocytes demonstrated a completely different response to lamb oocytes, with survival and parthenogenetic activation rates unaffected by the vitrification process. Treatment of mouse oocytes with DMSO + EG yielded significantly higher survival and cleavage rates than treatment with PROH + EG (87.8% and 51.7% vs 32.7% and 16.7% respectively, P <0.01), however cleavage rate for vitrified oocytes remained lower than for the controls (51.7% vs 91.7%, P <0.01) as did mean blastocyst cell number (33 ± 3.1 vs 42 ± 1.5, P <0.05). From this study, it is clear that lamb and mouse show different tolerances to cryoprotectants commonly used in vitrification procedures, and careful selection and testing of species-compatible cryoprotectants is required when vitrifying oocytes to optimize survival and embryo development.

Populations of the recently described black oak gall wasp, Zapatella davisae Buffington (Hymenoptera: Cynipidae), have been identified as the cause of extensive tree damage and mortality to black oaks, Quercus velutina Lamarck (Fagales: Fagaceae), in the northeastern United States. Relatively little is known, however, about the distribution, phylogenetic placement, and lifecycle of this important tree pest. Therefore, we conducted next-generation sequencing using the Ion Torrent™ PGM (ThermoFisher Scientific, Inc.) platform to develop genomic resources for the study of Z. davisae and for other closely related species of oak gall wasps. Individual sequence reads were aligned, assembled into unique contigs, and the contigs were then utilized for the in silico isolation and development of microsatellite markers. In total, we screened 36 candidate microsatellite loci, of which 23 amplified consistently (five polymorphic and 18 monomorphic). We then examined whether the polymorphic loci could be used to infer whether populations of Z. davisae from Cape Cod and Nantucket are sexual or asexual by calculating several metrics of genetic diversity that might indicate the mode of reproduction. These included testing for statistical deviations from Hardy–Weinberg equilibrium (HWE) and for linkage disequilibrium (LD), observations for the presence of the Meselson effect, and by calculating the probability that clonal individuals are more prevalent than would be expected in a randomly mating population. While we found significant deviations from HWE and more clonal individuals than expected, our estimates of the Meselson effect were inconclusive due to limited sampling, and we found no evidence of LD. Therefore, the sexual/asexual status of Z. davisae populations remains uncertain.

Heat-shock proteins 90 (hsp90s) are a class of molecules able to stabilize a network of ‘client’ proteins that are involved in several processes. Furthermore, recent studies indicated that mutations in the hsp90-encoding gene induce a wide range of phenotypic abnormalities, which have been interpreted as an increased sensitivity of different developmental pathways to hidden/cryptic mutations. In order to verify the role of hsp90 in aphids, we amplified and sequenced the hsp90 gene in 17 lineages of the peach potato aphid Myzus persicae (Sulzer, 1776) looking for the presence of mutations. In particular, we compared lineages with different reproductive modes (obligate vs. cyclical parthenogenesis), propensity to develop winged females and karyotype stability. Differently from the cyclical parthenogenetic lineages that possessed functional hsp90 genes, the seven analysed asexual lineages showed severe mutations (including frameshift and non-sense mutations). In vivo functional assays with the hsp90-inhibitor geldanamycin showed that some lineages with cyclical parthenogenesis may lose their ability to induce sexuales in the absence of active hsp90 revealing the presence of cryptic mutations in their genomes. As a whole, our data suggest that hsp90 could play in aphids a role in buffering hidden/cryptic mutations that disrupt cyclical parthenogenesis.

The aim of this study was to compare the effects of various activating factors on feline oocytes. The study included activation within the ovary (natural), activation during in vitro maturation (spontaneous activation), chemical activation (ionomycin + 6-DMAP), activation by spermatozoa and injection (ICSI) and mechanical activation (sham ICSI). According to our results, parthenogenetic embryos could emerge at every step of in vitro embryo production (IVP) procedures. After oocyte collection, 6% of parthenogenetic embryos were observed, mainly at the 2–4-blastomere stages. After 24 h of in vitro maturation, parthenogenetic activation was observed in 7% of oocytes. Using ionomycin and 6-DMAP to artificially activate oocytes, 53% of cleaved embryos were obtained. The results after ICSI (54% cleaved embryos) were not significantly different from the results in Group III using chemical activation (53% cleaved embryos). But only after ICSI were blastocysts obtained (5/73.7%) as a result of in vitro culture. Moreover, embryos after ICSI were of the best morphological quality with minor levels of fragmentation evident in the embryos. After sham mechanical activation, ‘sham ICSI’, 8% of cleaved embryos were noted. Therefore, it is advised to maintain a negative control in parallel with each step of IVP techniques, to avoid misleading results. Chemical methods for artificial activation of feline oocytes are the most promising for application to the cloning and production of parthenogenetic embryos for experimental studies.

In a pioneer application of acarology to Quaternary fossil-bearing sediments in southern Africa, the oribatid composition in the Florisbad Quaternary sediments was determined and compared to the currently known distribution of those species. Nine species of oribatid mites were recorded in the Holocene aeolian deposits of the third test pit, three species from the Middle Stone Age (MSA) horizon sediments of the third test pit, and thirteen species from the Holocene spring sediments. The Florisbad results indicate a better agreement between the oribatid fauna of the last interglacial MSA horizon of the third test pit and the organic-rich mid-Holocene deposits near the spring than between either of these and early- and late-Holocene aeolian sediments of the third test pit, suggesting some similarity in microsedimentary environments. The majority of the species recorded in the sediments are parthenogenetic and can be regarded as pioneer species.

Frankliniella schultzei Trybom is a polyphagous pest and vector of tospoviruses worldwide. It occurs in dark and pale colour forms that are morphologically similar but differ in vector competency and geographic spread. In Kenya and other tropical regions, mixed populations of both colour forms are observed in similar habitats, so are considered as one species. To ascertain the taxonomic status of the two colour forms, they were characterized using morphological, molecular, biological and ecological approaches. Morphological characterization revealed differences between the colour forms on eight features and they separated into distinct clusters through principal component analysis. Restriction fragment length polymorphism of the internal transcribed spacer region (ITS-RFLP) analysis revealed differences between the two colour forms and was confirmed by differences in ITS2 sequences. Virgin pale females had female offspring (thelytoky), while virgin dark females had male offspring (arrhenotoky). Interbreeding of dark males with pale females resulted in pale females, indicating absence of interbreeding between the two colour forms. Laboratory colonies of pale forms lacked males and further analysis of F. schultzei males from Ipomoea setosa flowers in the field indicated the presence of dark males and the absence of pale males. Field surveys in Kenya indicated differences in distribution and host plant preferences among the colour forms. Lack of interbreeding, distinct host preferences and distribution, and morphological and molecular differences indicate that the two colour forms of F. schultzei could be different species. The results highlight the need for combining morphological, biological, molecular and ecological characteristics for resolving taxonomic status of closely related insects.