The ghost sex-life of the paedogenetic beetle Micromalthus debilis.

Genetic and sexual systems can be evolutionarily dynamic within and among clades. However, identifying the processes responsible for switches between, for instance, sexual and asexual reproduction, or cyclic and non-cyclic life histories remains challenging. When animals evolve parthenogenetic reproduction, information about the sexual mating system becomes lost. Here we report an extraordinary case where we have been able to resurrect sexual adults in a species of beetle that reproduces by parthenogenetic paedogenesis, without the production of adults. Via heat treatment, we were able to artificially induce adult beetles of Micromalthus debilis in order to describe its pre-paedogenetic mating system. Adults showed a highly female biased sex ratio, out-breeding behaviour, and sex-role reversal. Paedogenetic larvae of Micromalthus are infected with the endosymbiotic bacteria Rickettsia and Wolbachia. Clear signs of vestigialization in adults are concurrent with the loss of adults. Our data suggest an ancient female sex ratio bias that predates the loss of adults, perhaps associated with endosymbionts. We propose a model for the transition from a haplodiploid cyclical parthenogenetic life history to parthenogenetic paedogenesis. Paedogenetic development induces a new mechanism of sex ratio bias in midges, wasps and beetles.

Toxic effects of Hoechst staining and UV irradiation on preimplantation development of parthenogenetically activated mouse oocytes.

Parthenogenetic activation of oocytes is a helpful tool to obtain blastocysts, of which the inner cell mass may be used for derivation of embryonic stem cells. In order to improve activation and embryonic development after parthenogenesis, we tried to use sperm injection and subsequent removal of the sperm head to mimic the natural Ca2+ increases by release of the oocyte activating factor. Visualization of the sperm could be accomplished by Hoechst staining and ultraviolet (UV) light irradiation. To exclude negative effects of this treatment, we examined toxicity on activated mouse oocytes. After activation, oocytes were incubated in Hoechst 33342 or 33258 stain and exposed to UV irradiation. The effects on embryonic development were evaluated. Our results showed that both types of Hoechst combined with UV irradiation have toxic effects on parthenogenetically activated mouse oocytes. Although activation and cleavage rate were not affected, blastocyst formation was significantly reduced. Secondly, we used MitoTracker staining for removal of the sperm. Sperm heads were stained before injection and removed again after 1 h. However, staining was not visible anymore in all oocytes after intracytoplasmic sperm injection. In case the sperm could be removed, most oocytes died after 1 day. As MitoTracker was also not successful, alternative methods for sperm identification should be investigated.

Effects of salinity and ultraviolet radiation on the bioaccumulation of mycosporine-like amino acids in Artemia from Lake Urmia (Iran).

We investigated the effects of salinity and artificial UV radiation on the accumulation of mycosporine-like amino acids (MAAs) in sexual and parthenogenetic Artemia from Lake Urmia. The nauplii hatched from the cysts were cultured until adulthood under two salinities (150 and 250 g L(-1) ) and two light treatments (PAR and PAR+UVR) in the laboratory. Finally, the Artemia were analyzed for their concentration of MAAs. In most of the cases, the higher salinity level applied was found to increase the MAA concentrations in both Artemia populations significantly. The acquisition efficiency of MAAs in both Artemia populations increased under exposure to UVR-supplemented photosynthetically active radiation (PAR) compared to those raised under PAR, except for Porphyra-334. It was observed that combination of UV radiation and elevated salinity significantly increased the bioaccumulation of MAAs. Thus, the presence of these compounds in these populations of Artemia may increase their adaptability for living in high-UV and high-salinity conditions prevailing in Lake Urmia. Higher concentrations of MAAs in the parthenogenetic population of Artemia could be probably attributed to its mono sex nature and higher adaptation capacities to extreme environmental conditions.

Femtosecond laser-induced fusion of nonadherent cells and two-cell porcine embryos.

Cell fusion is a fundamental biological process that can be artificially induced by different methods. Although femtosecond (fs) lasers have been successfully employed for cell fusion over the past few years, the underlying mechanisms are still unknown. In our experimental study, we investigated the correlation between fs laser-induced cell fusion and membrane perforation, and the influence of laser parameters on the fusion efficiency of nonadherent HL-60 cells. We found that shorter exposure times resulted in higher fusion efficiencies with a maximum of 21% at 10 ms and 100 mJ/cm(2) (190 mW). Successful cell fusion was indicated by the formation of a long-lasting vapor bubble in the irradiated area with an average diameter much larger than in cell perforation experiments. With this knowledge, we demonstrated, for the first time, the fusion of very large parthenogenetic two-cell porcine embryos with high efficiencies of 55% at 20 ms and 360 mJ/cm(2) (670 mW). Long-term viability of fused embryos was proven by successful development up to the blastocyst stage in 70% of cases with no significant difference to controls. In contrast to previous studies, our results indicate that fs laser-induced cell fusion occurs when the membrane pore size exceeds a critical value, preventing immediate membrane resealing.

Effect of fluorescent mercury light irradiation on in vitro and in vivo development of mouse oocytes after parthenogenetic activation or sperm microinjection.

The detection of specific cellular components using fluorescent agents such as green fluorescent protein (GFP), red fluorescent protein or Hoechst dyes provides a powerful tool for studying cell biology. However, specimens must be exposed to high-intensity light, which might cause cellular damage. Here, we exposed mouse metaphase stage (M) II oocytes to fluorescent mercury vapor light at three wavelengths (539 nm, 488 nm and 341 nm) to determine the maximum exposure time that would avoid damage. When oocytes were activated parthenogenetically after exposure to these wavelengths for more than 20 min, 5 min or 4 sec, respectively, the percentages of dead oocytes after activation increased, and none of the surviving embryos developed to blastocysts. However, embryos fertilized by intracytoplasmic sperm injection (ICSI) were more tolerant to light damage, even though the quality of blastocysts, judged by cell number and cell allocation to the inner cell mass and trophectoderm measured by immunostaining for Oct4 and Cdx2, was reduced as exposure times increased. Live, healthy offspring were obtained when these exposed embryos were transferred into recipient pseudopregnant females at the 2-cell stage. In addition, MII oocytes collected from GFP-expressing transgenic mice after 5 min of irradiation with 488-nm light were also able to develop to full term following ICSI. Thus, we determined the safe period of exposure to several wavelengths for oocyte manipulation or observation that would permit subsequent development.

Development of an optimized zona-free method of somatic cell nuclear transfer in the goat.

The purpose of this study was to develop an improved zona-free method of goat somatic cell nuclear transfer (SCNT) that has both ease of operation and efficiency. The main steps involved were: (1) optimization of in vitro oocyte maturation, (2) parthenogenetic activation of zona-free oocytes, (3) SCNT of zona-free anaphase II-telophase II (AII-TII) oocytes that subverted the need for long term UV-exposure of the oocytes, and (4) in vitro culture of groups of cloned embryos in wells in a highly efficient continuous serum-free embryo medium to the blastocyst stage before transfer to the recipients. Percentages of transgenic blastocyst production were 22.3 and 33.1% for adult and fetal cell lines, respectively. After transfer of cloned and transgenic blastocysts, 28.6 and 36.4% of the recipients were confirmed pregnant and 75 and 33.3% of the pregnancies resulted in the delivery of viable offspring, respectively. To our knowledge, this is the first report of successful live and survived birth of cloned and transgenic offspring through a whole procedure of in vitro oocyte maturation and embryo development to the blastocyst stage, and in this study the in vitro efficiencies of cloned and transgenic embryo production were higher than the available reports.

Effects of irradiation on Planococcus minor (Hemiptera: Pseudococcidae).

Irradiation has been recognized and endorsed as a potential phytosanitary measure that could be an alternative to current quarantine treatments. Dosages of 50, 100, 150, 200, and 250 Gy were used to irradiate three different life stages (eggs, immatures, and adults) of Planococcus minor (Maskell) (Hemiptera: Pseudococcidae), focusing on females due to its parthenogenesis ability, with an aim to find the most tolerant stage and the most optimal dose to control P. minor. Cobalt 60 was the source of irradiation used. Irradiation of 150-250 Gy has a significant effect on all life stages of P. minor, decreasing its survival rate, percentage of adult reproduction, oviposition, and fertility rate. The adult was the most tolerant life stage in both mortality and fertility rate. All the different irradiated target life stage groups oviposited eggs, but none of the F2 eggs hatched at the most optimal dosage of 150-250 Gy.

Effects of cycloheximide on parthenogenetic development of pig oocytes activated by ultrasound treatment.

The present study was carried out to examine the parthenogenetic development of pig oocytes treated with different concentrations of cycloheximide for different durations following activation by ultrasound stimulation. When oocytes were treated with 10 microg/ml cycloheximide for different durations, the blastocyst formation rate of oocytes treated for 5 h was significantly (P<0.05) higher than those of oocytes treated for 0-2 h. The blastocyst formation rate of oocytes treated with 10 microg/ml cycloheximide for 5 h was significantly (P<0.05) higher than those of oocytes treated with 0-5 or 15-20 microg/ml cycloheximide for the same duration. When oocytes were treated with different concentrations of cycloheximide for 2 h, however, the blastocyst formation rate of oocytes treated with 40 microg/ml cycloheximide was significantly (P<0.05) higher than those of oocytes treated with 0-10 or 50 microg/ml cycloheximide. The blastocyst formation rate of oocytes treated with 10 microg/ml cycloheximide for 5 h was not significantly different from that of oocytes treated with 40 microg/ml cycloheximide for 2 h. These treatments did not affect the activation status of oocytes compared with controls that were not treated with cycloheximide. The results of the present study showed that cycloheximide improves the parthenogenetic development of pig oocytes activated by ultrasound stimulation.

[The effect of heterosis on inheritance of quantitative traits in silkworm exposed to electromagnetic irradiation]. / Effekt geterozisa i nasledovanie kolichestvennykh priznakov u tutovogo shelkopriada v usloviiakh élektromagnitnogo oblucheniia.

The effect of heterosis was studied in several quantitative traits of clone breed and interbreed silkworm hybrids exposed to electromagnetic irradiation (lambda = 1.6 cm, power density 700 microW/cm2) during postdiapause embryonic development. The influence of the type of reproduction on the manifestation of irradiation effects in the next generation was also examined. In hybrids, the resistance to low-intensity high-frequency irradiation was higher than in the parental forms. Unlike the latter, the hybrids showed no significant modification of the traits after the exposure to electromagnetic irradiation. In the second generation, the modifying effect of irradiation is retained in the case of parthenogenetic silkworm development but not after mating.

Ultrastructural morphometry of precompacted bovine embryos produced in vivo and in vitro after activation by electric pulse AC/DC.

Early bovine precompacted embryos (at 1- to 8-blastomere stage) were analyzed by electron microscopy. The volume density of cellular components was determined by morphometric analysis to quantify the ultrastructure of early bovine embryos produced either in vivo or parthenogenetically after stimulation of oocytes by electric pulse AC/DC. In embryos obtained in vivo, most of cellular volume was occupied by cytoplasm (82.93%). The relative volume of lipids, vacuoles, mitochondria was relatively low (5.46; 5.07; 3.78%, respectively), and the relative volume of Golgi apparatus and cell inclusions was the lowest (1.51%). AC/DC-derived parthenogenotes had a relative high area occupied by vacuoles and lipids (18.68 vs. 14.33%) and a significantly lower relative volume was occupied by cytoplasm (60.63%) when compared with the control in vivo embryos. These observations demonstrated that parthenogenetic embryos had significantly altered ultrastructure, indicating extensive subcellular damages. These findings are discussed from the physiological and functional point of view.