UV-irradiated Eggs Research Articles

<p class="Body"><strong>Comparison of different pollen grains and a factitious prey as food sources for <em>Amblyseius swirskii</em> (Acari: Phytoseiidae)</strong></p>

Pollen used as an alternative or supplementary food source for a predatory mite not only provides important nutrients but also allows the predator to establish a population before prey becomes available. In this study, we compared the pollen of apricot, pear, hollyhock, opium poppy, and date palm, and a factitious prey, UV-irradiated eggs of Ephestia kuehniella, as food sources for Amblyseius swirskii. Different food items affected all development stages differently, except for hollyhock pollen, where eggs failed to develop to maturity. Food type affected the reproductive ability of females, which did not reproduce when fed on opium poppy during immature stages. Most eggs per female were produced when feeding on the factitious prey, and second most with apricot pollen. Feeding on factitious prey compared with pollen, resulted in greater longevity of both female and male adults. Feeding on pear pollen or factitious prey caused, respectively, the lowest and highest population growth rates (r and λ). Both net (R0) and gross (GRR) reproductive rates were highest when the factitious prey was the food source. Among the pollen diets, apricot was the most beneficial food item. Because of their effects on rates of population increase, either apricot pollen or the factitious prey could be further utilized as cost-effective alternative food diets for both experimental and commercial mass rearing of A. swirskii.

Morphology and performance specifications of Blaptosthethus pallescens Poppius (Heteroptera: Anthocoridae) when reared on two alternate laboratory hosts

The present research paper provides detailed morphology and morphometrics of an anthocorid predator, Blaptostethus pallescens Poppius. To identify the alternate laboratory host to be utilized for mass production, biological parameters of the anthocorid were evaluated by rearing on UV irradiated eggs of two alternate laboratory hosts, Corcyra cephalonica (Stainton) and Sitotroga cerealella (Motsch.). Five nymphal instars were recorded on both hosts. No significant differences were observed in incubation period, nymphal duration, total developmental period and sex ratio when reared on the two hosts. However, significant differences were observed in two biological parameters i.e., adult longevity and fecundity. Longer life span and higher fecundity of B. pallescens was recorded when reared on C. cephalonica eggs in comparison to S. cerealella eggs, indicating the suitability of C. cephalonica eggs for mass rearing B. pallescens in the laboratory. B. pallescens could be reared continuously for 7 generations on C. cephalonica eggs.

The occurrence of hypertetraploid and other unusual polyploid loaches Misgurnus anguillicaudatus among market specimens in Japan

Exotic animals can cause genetic contamination of indigenous species if they escape and reproduce in wild populations. The loach Misgurnus anguillicaudatus and species related to it have been imported into Japan for commercial use. We collected live loach specimens from the central wholesale market in Tokyo. For the 451 specimens collected, ploidy status was examined by DNA content flow cytometry, and polyploid loaches with triploid, tetraploid, and other higher ploidy ranges were detected. Hypertriploid and hypertetraploid individuals were easily detected by flow cytometry using standard eudiploid, eutriploid, and eutetraploid controls, and the reproductive capacities of these hyperpolyploid males were then examined. Sperm of hypertriploid males did not exhibit active progressive motility, and major populations of spermatozoa or spermatozoon-like cells were detected in the triploid and hexaploid ranges. Motile haploid spermatozoa were very rare in sperm from hypertriploid males. Therefore, hypertriploid males were sterile, whereas hypertetraploid males produced fertile hyperdiploid spermatozoa with active progressive motility after contact with ambient water. Viable progeny occurred in crosses between normal wild-type diploid females and hypertetraploid males, but androgenotes induced by the fertilization of UV-irradiated eggs with the sperm of hypertetraploid males were inviable hyperdiploids. Cytogenetic analyses of those androgenotes indicated that hypertetraploid males should produce hyperdiploid spermatozoa with 2n = 54 (i.e., four supernumerary microchromosomes).

Restoration of the loach, Misgurnus anguillicaudatus, from cryopreserved diploid sperm and induced androgenesis

In the present study, we evaluated the feasibility of using cryopreserved diploid sperm as a repository genebank for the loach, Misgurnus anguillicaudatus, along with a rederivation strategy utilizing induced-androgenesis. Firstly, we evaluated three types of media for egg inactivation: Hank's saline solution + 0.5% bovine serum albumin (BSA), Ringer's solution + 0.5% BSA, and masu salmon seminal plasma. Haploid and diploid sperm were taken from diploid and tetraploid loaches, respectively. Fresh and cryopreserved haploid or diploid sperm were then used to fertilize intact or UV-irradiated eggs from wild diploid females. The irradiation media evaluated here successfully maintained the egg quality over 2 h. Fertilization and hatching rates of eggs fertilized with cryopreserved diploid sperm were 11.68 ± 6.74% and 7.14 ± 6.29% respectively, compared to 63.51 ± 10.68% and 45.19 ± 16.2% for intact eggs fertilized by fresh haploid sperm. All-male inheritance was confirmed by determination of larval morphology, ploidy status and microsatellite genotypes of putative androgenetic progeny.

Reproductive capacity of neo-tetraploid loaches produced using diploid spermatozoa from a natural tetraploid male

Tetraploid loaches have been discovered amongst specimens recovered from fish markets in Japan. These tetraploids can be used as a source of diploid gametes to assist the further expansion of ploidy manipulation. Here, we produced a first generation neo-tetraploid strain by fertilizing eggs from a normal diploid female with diploid spermatozoa from a natural tetraploid male, followed 5 min later by heat shock (42 °C, 2 min duration) to inhibit release of the second polar body. Diploid spermatozoa from the neo-tetraploid males produced were then used to create androgenetic diploid progeny by fertilizing UV-irradiated eggs from a normal diploid female. Triploid progeny were produced by crossing a normal diploid female with a neo-tetraploid male. Tetraploid progeny were produced by cold shock (1 °C, 40 min duration), beginning 5 min after fertilizing normal eggs with diploid spermatozoa from first generation neo-tetraploid males. Reproductive performance of second generation progeny was also examined. Androgenetic diploid males generated fertile haploid spermatozoa. Triploid males were sterile, but a triploid female laid fertile haploid eggs. Second generation neo-tetraploid males were considered sterile.

Evaluation of different doses of UV irradiation to loach eggs for Genetic inactivation of the maternal genome

Genetic inactivation of the egg nucleus is an indispensable step in the production of androgenetic embryos in teleosts. However, few experimental studies have focused on determining the most effective means of achieving complete inactivation of the maternal genome. Here, we sought to identify the optimum conditions of ultraviolet (UV) irradiation for complete inactivation of the loach egg nucleus. Unfertilized eggs were UV irradiated from above with a dose in the range 0-200 mJ/cm2. Successful inactivation of the maternal genome was evaluated by the exclusive expression of a paternally inherited color phenotype. The presence or absence of putative maternal chromosome fragments was screened by flow cytometry of DNA content and by cytogenetic analysis. The majority of the larvae derived from irradiated eggs had an abnormal appearance. Haploid individuals were detected by measurement of DNA content flow cytometry and by chromosome counting in the groups that received more than 75 mJ/cm2 groups. Although the coefficient of variation of DNA content was apparently reduced in the 125-200 mJ/cm2 groups, chromosome fragments were still detected in all the groups from irradiated eggs. Inactivation of the egg nucleus was also histologically elucidated by the presence or absence of residual nuclear material in anuclear embryos that developed from UV-irradiated eggs fertilized with UV-irradiated sperm. Embryos that were completely or near-completely anuclear were found in the 150 and 200 mJ/cm2 groups. We conclude that the optimum UV dose for complete genetic inactivation of the egg nucleus is more than 150 mJ/cm2.

High efficiency of meiotic gynogenesis in sea lamprey Petromyzon marinus

Induction of androgenesis and gynogenesis by applying a pressure (PS) or heat shock (HS) to double the haploid chromosomal set results in progenies possessing only chromosomes from a single parent. This has never been accomplished in representatives of Agnatha. The objective of this study was to induce gynogenesis and androgenesis in sea lamprey Petromyzon marinus. For gynogenesis experiments, ultraviolet (UV)-irradiated sperm was used to activate sea lamprey eggs and HS or PS were applied to inhibit the second meiotic division and consequently induce diploidy in the embryos. The UV irradiation of immobilized sperm was performed for 1 min at 1,719 J m(-2). HS of 35+/-1 degrees C for 2 min and PS of 9,000 psi for 4 min were applied at different times after egg activation (8, 12, 20, and 24 min or 8, 16, and 24 min for HS or PS, respectively). Regardless of the induction time of the HS, survivals at pre-hatching stage were similar. In contrast, PS applied 8 min after activation appears to increase survival rate of pre-hatched embryos in comparison to 16 and 24 min after activation. In control groups, without shock treatment (no diploidization), there were no survivors. All deformed, gynogenetic embryos were confirmed to be haploids and died prior to burying themselves in the sand. We confirmed by flow cytometry that progenies produced using both shock methods surviving to the next stage, burying in the substrate, were diploid gynogenetic. For the androgenesis experiments, UV-irradiated eggs (1,719 J m(-2) for 1 min) were fertilized with non-treated sperm and HS was applied to restore diploidy of the eggs. Several attempts have been made to optimize the parameters used. HS of 35+/-1 degrees C was applied 110, 140, 170, 200, and 230 min after activation for 2 min. Low yields of androgens were obtained and all animals died within a week after hatching. These techniques will allow to establish meiotic gynogenetic lines of sea lamprey for determining sex differentiation in this species and to analyze its hormonal and environmental regulation.

Production of Androgenetic Diploid Loach <i>Misgurnus anguillicaudatus</i> from UV Irradiated Eggs by Suppression of the First Cleavage

Production of Androgenetic Diploid Loach <i>Misgurnus anguillicaudatus</i> from UV Irradiated Eggs by Suppression of the First Cleavage

Presumptive Primordial Germ Cells (pPGCs) and PGCs in Tadpoles from UV-irradiated embryos of Xenopus: (UV-irradiation/primordial germ cell/monoclonal antibody/complete sterility/Xenopus laevis).

In order to determine whether or not tadpoles that once lacked primordial germ cells (PGCs) in the genital ridges and dorsal mesentery as a result of ultraviolet (UV) irradiation subsequently contained germ cells at more advanced stages of larval development, the numbers of presumptive PGCs or PGCs were carefully examined in Xenopus tadpoles at Nieuwkoop and Faber's stage 35/36-52 that developed normally from UV-irradiated eggs. No late-appearing germ cells were observed in almost all the UV-irradiated tadpoles examined at stages 49-52. This same population had completely lacked PGCs at about stage 46. Moreover, presumptive PGCs (pPGCs) or cells with granular cytoplasm that reacted with a monoclonal antibody specific for the germ plasm of cleaving Xenopus eggs stayed in the central part of the endoderm cell mass in the irradiated tadpoles at stage 35/36, when the majority of those cells were located in the dorsal part of the endoderm in unirradiated controls. Furthermore, in the irradiated embryos pPGCs were demonstrated to decrease in number with development and eventually to disappear in tadpoles at about stage 40. The results strongly suggest that UV irradiation under the conditions used here totally eliminated germline cells from the irradiated animals.

The effect of immunization of pigs with Ascaris suum cuticle components on the development of resistance to parenteral migration during a challenge infection

The development of immunity to Ascaris suum was studied in pigs immunized with isolated cuticle fragments from A. suum second and third stage larvae (L2/L3) and adult worms, and compared with other methods that stimulate a strong protective response in pigs. A significant protective response was seen in animals immunized with isolated cuticle fragments from A. suum L2/L3 and adults, but it was less than that seen in animals inoculated with UV-irradiated eggs or naturally exposed to eggs on a dirt lot. Significant IgG responses to 2-mercaptoethanol (2ME)-soluble cuticle components were seen in all groups, but the level of the antibody response did not relate to protection. Group differences in antibody and lymphocyte blastogenic responses to cuticle proteins indicated quantitative and qualitative stage specific differences in 2ME-soluble and insoluble cuticular proteins. Intestinal immunity was notably absent from cuticle immunized pigs because a marked liver white spot response was observed following the challenge inoculation. Thus, cuticle fragments from larval and adult A. suum are capable of inducing a protective response to larval migration; however, the development of intestinal immunity is not a direct function of exposure to these antigens.

High-frequency twinning of Xenopus laevis embryos from eggs centrifuged before first cleavage

In embryos of the frog Xenopus laevis, the dorsal structures normally develop from regions of the egg opposite the side of sperm entry. Gravity and centrifugal force, applied at an angle of 90° to the animal-vegetal axis of the egg, can override this topographic relationship and can cause the dorsal structures to be positioned according to the force vector ( S. Black and J. Gerhart, 1985, Dev. Biol. 108, 310–324) . We report here that at time 0.40 (40% of the first cleavage interval), an average of 60% of eggs centrifuged at 30 g for 4 min in this orientation form conjoined twins with one body axis arising from the centripetal side of the egg and one arising from the centrifugal side of the egg. This positioning is observed regardless of the orientation of the side of sperm entry in the centrifugal field. If, after the 0.40 centrifugation, the eggs are inclined with the centripetal side up, they do not make twins; instead, they make only a single axis at the centripetal side. This indicates that the second axis in twins is caused to form by postcentrifugation gravity-driven internal rearrangements of materials that were displaced by the centrifugation. Twins also form at high frequency in eggs centrifuged twice, first at an inclination of 90°, and then at an inclination of 0°. The second centrifugation yields secondary axes even when it is begun midway in the second cell cycle, well after the time of grey crescent formation. Double centrifugation also causes twinning (“double rescue”) of uv-irradiated eggs which otherwise would not develop axial structures. This suggests that the internal displacements caused by the centrifugations can substitute for a step in the normal axis specification process that is impaired in irradiated eggs.

Rearing Trichogramma nubilale (Hymenoptera: Trichogrammatidae) on Ultraviolet-Irradiated Eggs of the European Corn Borer (Lepidoptera: Pyralidae)1

Trichogramma nubilale Ertle and Davis may be reared on eggs of European corn borer (ECB), ostrinia nubilalis (Hubner), which have been killed by ultraviolet (UV) radiation. A dosage of 30 min is sufficient to cause 100% mortality in ECB eggs 0 to 4 hold. T. nubilale reared for five consecutive generations on dead, UV-irradiated eggs exhibit a slightly shortened life span, but there were no significant changes in the number of progeny per female, adult emergence, sex ratio, and progeny produced per ECB egg.

Replication and recovery of nuclear DNA in UV-irradiated eggs of Lymnaea stagnalis (Mollusca, Gastropoda)

Synchronized and oriented egg cells of L. stagnalis have been irradiated with UV light during the G2 phase of the 2nd cleavage cycle; next the egg cells were treated with caffeïne (3 × 10 −3M) for varying periods during the 2nd and the 3rd cleavage cycle. In a second experiment the egg cells were irradiated at the same stage in the G2-phase with different UV doses, and then treated with caffeïne until the beginning of the 3rd cleavage. In the caffeïne-treated egg cells the two cleavage cycles following irradiation appeared to be lengthened, whereas in non-caffeïne controls only the first cleavage cycle after irradiation is longer. It is concluded that the extension of the 3rd cleavage cycle is to be attributed to a prolongation of the S phase, which is not caused by an inhibition by caffeïne of the initiation of the DNA synthesis. The results point to an effect of caffeïne on the post-replication mode of repair; lesions in the DNA are not rendered innocuous in the presence of caffeïne. At the beginning of the next cleavage cycle these lesions still have an inhibiting effect on the progression of DNA synthesis. Morphogenesis was reduced by increasing UV doses; with caffeïne less than 7% of the embryos developed normally. These results may indicate an error-prone repair mechanism.

Gonadal differentiation in frogs, Rana japonica and R. brevipoda, raised from UV irradiated eggs.

The gonadal differentiation of anurans, Rana japonica and R. brevipoda, was examined in animals raised from eggs which had been irradiated at the vegetal hemisphere with UV (9300 erg/mm2) at the 2-cell stage. In R. japonica about 70% of the larvae at stage I from the pressed and UV-irradiated eggs were germ cell free, but at a stage immediately after metamorphosis all animals had at least some germ cells, although their gonads often were extremely small and poorly differentiated. When male animals matured sexually, many of them had abnormal gonads. However, all of them were shown by artificial means to be capable of fertilization. In the nonpressed and irradiated group, no larvae were germ cell free and the animals immediately after metamorphosis showed nearly normal gonadal differentiation except for the presence of a few degenerate oocytes in the ovaries. The results in R. brevipoda were basically similar to those in R. japonica. In both species, sex ratios were determined at two stages, the first immediately after metamorphosis and the other when the animals matured, as based on gonad morphology and histology and on external sexually dimorphic characters as well. Sex ratios at these two stages in frogs from the pressed and irradiated eggs differed markedly in R. brevipoda. The ratio was normal at metamorphosis but high M/F ratios occurred when animals became mature. That sex reversal took place in this species as well as in R. japonica (in which sex-ratio deviation was not statistically significant) was supported by the sex ratios of the progenies of these supernumerary males.

Restoration of fertility in sterilized Drosophila eggs by transplantation of polar cytoplasm

A technique for transplantation of cytoplasm between Drosophila eggs is described. Polar cytoplasm of newly laid eggs was first made ineffective for the determination of germ cells by UV irradiation. The sterility which results from this UV irradiation could be prevented by the injection of polar cytoplasm, but not by the injection of anterior cytoplasm from unirradiated donor eggs. The results provide the first demonstration of transplantation of agents causing determination in an insect and also provide a bioassay for these agents. Microscopical observations of UV irradiated eggs with and without transplanted polar cytoplasm revealed that UV irradiation delays the migration of cleavage nuclei into the posterior periplasm and prevents cytoplasmic protrusions at the posterior pole from becoming isolated from the periplasm to form pole cells. Transplantation of polar cytoplasm repairs these defects.

Ultrastructural changes associated with UV irradiation in the “germinal plasm” of Xenopus laevis

To detect structural changes following UV irradiation in the “germinal plasm,” ultrastructure of the “germinal plasm” was studied in normal and UV-irradiated eggs of Xenopus laevis at the following stages: prior to fertilization, early 2-cell, 32-cell, and late blastula. It was revealed that ultrastructural features of the “germinal plasm” were essentially common between Xenopus laevis and Rana pipiens. That is, the “germinal plasm” is composed primarily of a large aggregation of mitochondria and distinctive electron dense bodies (germinal granules). Irregularly shaped cylinderlike granules (giant germinal granules), having the same internal characteristics as the germinal granules, were found in the “germinal plasm” of all eggs examined. Comparison between normal and UV-irradiated eggs has demonstrated that UV irradiation causes swelling and vacuolation of mitochondria and fragmentation of germinal granules. The suggestion is that the integrity of certain UV-sensitive factor(s), which is involved in maintaining normal structure of germinal granules, is indispensable for the determination of the primordial germ cells.

Temperature effects on embryogenesis in Smittia spec. (Diptera, chironomidae): Q10-values of normal development and frequency of "double abdomens" after UV irradiation.

1. The effect of different temperatures (8° to 24° C) upon the rate of normal development and upon the frequency of the malformation "double abdomen" in partially UV irradiated eggs ofSmittia was tested. 2. The Q10-value of the rate of normal development increased with decreasing temperature. A value of 1.8 was found between 24° and 20° C, a value of 5.4 was reached between 12° and 8° C. 3. The frequency of double abdomens in UV irradiated eggs decreased with increasing temperature on dark cultivation after UV irradiation. This effect is named "thermoreversion" 4. Dose-response curves obtained at cultivation temperatures of 8° and 20° C, respectively, show a constant dose reduction factor of 0.58. 5. Thermoreversion occurred after UV irradiation at all stages from egg deposition to formation of preblastoderm. 6. The kinetics of thermoreversion show significant, stage dependent deviations from a simple exponential rise. This finding indicates complicated interrelations between thermoreversion and developmental changes within the egg. 7. Incubation of irradiated eggs in a 50 μM NaCN solution caused a significant increase of the frequency of double abdomens. 8. Some speculations about mechanisms possibly underlying thermoreversion are presented.

Behaviour of sulfhydryl groups of sea urchin eggs under the blockage of cell division by UV and heat shock

1. 1. In the sea urchins Hemicentrotus pulcherrimus, Anthocidaris crassispina, and Clypeaster japonicus, cleavage of the eggs can be suppressed by short treatment with higher temperature (34 °C) or by UV irradiation. 2. 2. While the division of the cell body is blocked ( a) in the heat treatment, nuclear division is not affected ( b) by UV irradiation, but it is suppressed. 3. 3. During heat treatment ( a), usual cyclic change in SH amounts of 0.6 M KCl-soluble protein stops while in UV treatment ( b), it persists. 4. 4. There are two cycles present in the cell which are independent from each other: one is the nuclear cycle and the other is the cytoplasmic SH cycle. 5. 5. Since cleavage does not take place if one of the two cycles is blocked or they are pushed out of pace, synchronization of the two cycles is a necessary condition for a cleavage to occur. 6. 6. From the fact that the time of micromere formation in UV irradiated eggs always occurs simultaneously with the fourth cleavage of the untreated controls and at the fourth peak of SH, a possibility is considered that the cyclic change of SH may be a pace-maker for differentiation.