Experiments inducing prospective polar body nuclei to participate in embryogenesis of the sawfly Athalia rosae (Hymenoptera)

Summary Mature eggs dissected from ovaries of unmated females of Athalia rosae (Hymenoptera: Tenthredinidae), if placed on a filter-paper soaked with distilled water, are activated and develop to haploid males. Occasionally, however, diploid females develop from these artificially activated eggs. Treatment of mature unfertilized eggs dissected from diploid females with ice-cold temperatures immediately before activation and with a high temperature (36° C) upon and immediately after activation resulted in the production of diploid males, diploid females, triploid females and gynandromorphs at high frequency. The same treatment of mature unfertilized eggs dissected from triploid females resulted in the production of only triploid survivors. These results, together with the results on the segregation of a marker mutation, yellow fatbody (yfb), appear to indicate that meiotic divisions were complete in the treated eggs, and that all four nuclei became potentially capable of participating in development with or without automictic fusion.Studies on the sawfly, Athalia rosae (Insecta, Hymenoptera, Tenthredinidae), part V     

Production of haploid-haploid chimeras by sperm injection in the sawfly,Athalia rosae (Hymenoptera)

Mature unfertilized eggs (oocytes) dissected from the ovary of the sawfly Athalia rosae (Hymenoptera) begin parthenogenetic development if exposed to distilled water and produce haploid males. Injection of sperm into mature oocytes through the anterior pole resulted in karyogamy in a fraction of cases which developed as diploid females. No haploid-haploid chimeras due to independent participation of the injected sperm in development were produced. When sperm were injected through the posterior pole, however, fertilization never occurred but haploid-haploid chimeras were produced in a smaller fraction of cases. Both egg nucleus-derived and injected sperm-derived nuclei contributed in forming the germ cells of the chimeric males.     

The production of cloned fish in the medaka (Oryzias latipes)

The measurement of cellular DNA content by DNA microfluorometry revealed that medaka embryos that were fertilized with normal sperm and exposed to heat shock (41 degrees C for 3 min) or hydrostatic pressure (700 kg/cm2 for 10 min) at 85-95 min after insemination were tetraploid. Embryos fertilized with normal sperm and exposed to heat shock (41 degrees C for 2 min at 2-3 min after insemination) were triploid. These results suggest that heat shock or hydrostatic pressure at 85-95 min after insemination arrests the first cleavage, while heat shock at 2-3 min after insemination arrests the second meiotic division. Medaka clones have been produced by the following method: Eggs from orange-red or variegated variety were activated by UV-irradiated, genetically impotent sperm of wild-type fish (UV sperm). The haploid eggs obtained were diploidized by preventing the first cleavage with heat shock or hydrostatic pressure to produce homozygous females. Each of the two homozygous females was mated with vasectomized male in isotonic balanced salt solution to collect unfertilized eggs. The collected eggs were activated with UV sperm and converted from haploid to diploid by arrest of the second meiotic division with heat shock. Hatched fry of each homozygous diploid (all females) were fed with a methyltestosterone-containing diet (40 micrograms/gm diet) to produce sex-reversed males, which were mated with brood females, and thus two cloned lines were obtained.     

Reproductive capacity of triploid loaches obtained from Hokkaido Island, Japan

Reproductive capacity was investigated in naturally occurring triploid individuals of the loach Misgurnus anguillicaudatus collected from Memanbetsu Town, Abashiri County, Hokkaido Island, Japan. These triploids have been considered to appear by accidental incorporation of the haploid sperm genome from normal diploid into unreduced diploid eggs from the clonal lineage that usually reproduces unisexually. By fertilization with sperm from the normal male, one triploid female gave many inviable aneuploid (2.1–2.7n) and very few tetraploid progeny, whereas the other produced both diploid and triploid progeny. The results suggest that at least four different types of eggs can be formed in triploid females in this locality. In contrast, no progeny hatched when eggs of the normal female were fertilized with sperm or sperm-like cells obtained from triploid males. These gametes exhibited inactive or no motility after adding ambient water. They had larger head sizes than those of normal haploid sperm and had a short or no tail. Although their ploidy was triploid or hexaploid, a small number of haploid cells were detected in the semen by flow cytometry. Thus, triploid males were generally sterile, but they have a little potential for producing very few haploid sperm.     

Germline transformation of the sawfly, Athalia rosae (Hymenoptera: Symphyta), mediated by a piggyBac-derived vector

A piggyBac construct carrying two green fluorescent protein (GFP)-coding sequences one driven by Bombyx mori actin gene promoter and the other by Drosophila melanogaster heat-shock protein 70 (hsp70) promoter were injected together with a nonautonomous helper plasmid containing an active piggyBac transposase gene into the posterior end of mature unfertilized eggs dissected from the ovaries of Athalia rosae (Hymenoptera: Symphyta). These injected eggs, which developed as haploid male embryos upon artificial activation, were cultured to adulthood. Of 278 injected eggs, 61 grew to G(0) haploid adult males. These G(0) haploid males were individually mated to diploid females. The progeny embryos (G(1) generation) were examined for GFP expression. Four GFP-positive embryos (from three independent G(0) matings) were obtained. Two eclosed as diploid adult G(1) females. Mature unfertilized eggs dissected from the GFP-positive G(1) diploid females were activated artificially, and the resultant embryos were examined for GFP expression, separated and cultured to adulthood (G(2) generation). The G(2) haploid embryos segregated to GFP-positive and -negative individuals. By mating the G(2) adult haploid males individually to diploid females, stocks were established in which the piggyBac construct was stably integrated into the genome, as evidenced by GFP expression and Southern blot hybridization. The piggyBac transposition occurred at its canonical target TTAA sequence. These results, which demonstrate the first successful stable transposon-mediated germline transformation in Hymenoptera, will expand the usefulness of the piggyBac vector.     

Phenotypic fitness effects of the selfish B chromosome,paternal sex ratio (PSR) in the parasitic waspNasonia vitripennis

Summary B chromosomes are often considered genomic parasites. Paternal sex ratio (PSR) is an extreme example of a parasitic B chromosome in the parasitoid waspNasonia vitripennis. PSR is transmitted through the sperm of carrier males and destroys the other paternal chromosomes in early fertilized eggs. PSR disrupts the normal haplodiploid sex determination in this wasp by converting diploid (female) eggs into haploid (male) eggs that bear PSR. In this study I compare a number of phenotypic fitness aspects of PSR and standard (non-PSR) males. In general, PSR males were as fit as standard males. No significant differences were found in longevity (with one exception), ability to compete for mates and sperm depletion rates. PSR males produced 11–22% larger family sizes and developed slightly faster than standard males. Under conditions of sperm competition, females who mated with both types of males fertilized a constant proportion of eggs with each sperm type over their lifetime. PSR males produced fewer offspring among progenies from double-inseminated females. Phenotypic fitness effects are believed to play a minor role in determining PSR frequencies in natural populations.     

Diploid males sire triploid daughters and sons in the parasitoid wasp Cotesia vestalis

In the Hymenoptera, males develop as haploids from unfertilized eggs and females develop as diploids from fertilized eggs. In species with complementary sex determination (CSD), however, diploid males develop from zygotes that are homozygous at a highly polymorphic sex locus or loci. We investigated mating behavior and reproduction of diploid males of the parasitoid wasp Cotesia vestalis (C. plutellae), for which we recently demonstrated CSD. We show that the behavior of diploid males of C. vestalis is similar to that of haploid males, when measured as the proportion of males that display wing fanning, and the proportion of males that mount a female. Approximately 29% of diploid males sired daughters, showing their ability to produce viable sperm that can fertilize eggs. Females mated to diploid males produced all-male offspring more frequently (71%) than females mated to haploid males (27%). Daughter-producing females that had mated to diploid males produced more male-biased sex ratios than females mated to haploid males. All daughters of diploid males were triploid and sterile. Three triploid sons were also found among the offspring of diploid males. It has been suggested that this scenario, that is, diploid males mating with females and constraining them to the production of haploid sons, has a large negative impact on population growth rate and secondary sex ratio. Selection for adaptations to reduce diploid male production in natural populations is therefore likely to be strong. We discuss different scenarios that may reduce the sex determination load in C. vestalis.     

No patrigenes required for femaleness in the haplodiploid wasp Nasonia vitripennis

The parasitoid wasp Nasonia vitripennis is an emerging model organism for developmental and behavioral genetics. It reproduces by haplodiploidy; males typically develop parthenogenetically from haploid eggs and females from fertilized diploid eggs. A polyploid mutant strain is available in which females are triploid and lay haploid and diploid eggs that normally develop into males when unfertilized. In contrast to previous reports, approximately 2% of triploid females were found to occasionally produce daughters as well as gynandromorphs from diploid unfertilized eggs. Daughter production increased with age and differed among familial lineages. This is the first report of parthenogenetic female development in Nasonia. The results show that a paternally provided genome is not required for femaleness and call for modifications of existing models of sex determination in Nasonia.     

Effects of ploidy and sex-locus genotype on gene expression patterns in the fire ant Solenopsis invicta

Males in many animal species differ greatly from females in morphology, physiology and behaviour. Ants, bees and wasps have a haplodiploid mechanism of sex determination whereby unfertilized eggs become males while fertilized eggs become females. However, many species also have a low frequency of diploid males, which are thought to develop from diploid eggs when individuals are homozygous at one or more sex determination loci. Diploid males are morphologically similar to haploids, though often larger and typically sterile. To determine how ploidy level and sex-locus genotype affect gene expression during development, we compared expression patterns between diploid males, haploid males and females (queens) at three developmental timepoints in Solenopsis invicta. In pupae, gene expression profiles of diploid males were very different from those of haploid males but nearly identical to those of queens. An unexpected shift in expression patterns emerged soon after adult eclosion, with diploid male patterns diverging from those of queens to resemble those of haploid males, a pattern retained in older adults. The finding that ploidy level effects on early gene expression override sex effects (including genes implicated in sperm production and pheromone production/perception) may explain diploid male sterility and lack of worker discrimination against them during development.     

Egg activation and timing of sperm acceptance by an egg in honeybees (Apis mellifera L.)

Summary. Mechanical stresses by a narrow glass capillary were applied to unfertilized eggs of honeybees to determine whether the removal of meiotic blocks of the eggs could be caused by simple mechanical stimuli. The treated eggs developed into the anaphase of the first meiotic division at 15 min after treatment, whereas the untreated eggs remained arrested at the metaphase of the first meiotic division. The results of histological examination of the common oviduct showed that its inner widths were sufficiently narrow to cause the distortion of eggs passing through it. The distorted eggs could be fertilized and develop into diploid embryos if they were exposed to the semen immediately (within 30 sec) after egg distortion. However, this would not happen if the distorted eggs were exposed to semen later (30 min). The eggs exposed to the semen but not given mechanical stimuli could initiate the embryonic development with diploid chromosomes. The interval between mechanical distortion and sperm acceptance by eggs in vitro is compatible with that of natural oviposition of fertilized eggs by honeybee queens. These results suggest that egg activation by mechanical stresses in the common oviduct is valid for the natural oviposition in honeybees.     

Participation of testicular spermatids in development upon intracytoplasmic injection into eggs of the sawfly, Athalia rosae (Insecta, hymenoptera)

Fertilization by intracytoplasmic injection of mature sperm into mature eggs has previously been achieved in the sawfly, Athalia rosae (Insecta, Hymenoptera). In the present study, we examined the potential of spermatids, premature male gametes, for participating in development. When round spermatids and elongating spermatids from pupal testes were injected into the anterior end of mature eggs, about 5% of the total injected eggs developed into chimeric embryos (independent participation in development of the egg and spermatid nuclei). Some of them developed further, hatched, and pupated, with 1-2% of the total injected eggs becoming haploid chimeric male adults in which both the egg-derived and injected spermatid-derived nuclei contributed to the germline. No fertilized embryos were obtained by these injections. Elongated spermatids (immature sperm) from newly eclosed adult male testes upon injection did produce fertilized embryos that developed into normal diploid females (about 7% of the total injected). These results indicate that insect spermatids (round and elongating) have the potential to participate in development, but only independently of the egg nucleus. J. Exp. Zool. 286:181-192, 2000.     

Diploid arrhenotoky and automictic thelytoky in soft scale insects (Lecaniidae: Coccoidea: Homoptera)

Parthenogenesis is reported in three soft scales with 2n=16. In the unfertilized eggs of all three, oogenesis is normal and diploidy is restored by the fusion of the division products of the haploid female pronucleus. In Lecanium putmani Phillips 12 of 13 uninseminated females collected in the wild produced only males. The 21 inseminated females produced 15% males. The males were diploid but contained one euchromatic (E) and one heterochromatic (H) chromosome set. Most of the eggs produced by the inseminated females contained sperm but a few did not. It was concluded, therefore, that females develop from fertilized eggs and males from unfertilized eggs and that the species was diploid arrhenotokous. In L. cerasifex Fitch only 18 of 56 females collected in the wild had been inseminated. The frequency of males among their embryos was 22%. The males were again diploid with one E and one H set of chromosomes. Among the 38 uninseminated females, 27 produced only males, and 10 produced only females. All the female producers contained needle-like bacterial symbionts. Most of the male producers, and most of the inseminated females contained no symbionts; the rest contained rod-like symbionts. It was concluded, therefore, that the females of L. cerasifex studied belonged to two races, a diploid arrhenotokous race and an obligate automictic thelytokous race. Eucalymantus tessellatus (Signoret) is obligate automictic thelytokous. All the females examined were uninseminated and produced only females.Supported by Grant GB 23665 from the National Science Foundation, Washington, D.C.     

Cytological mechanisms of gynogenesis and sperm incorporation in unreduced diploid eggs of the clonal loach, Misgurnus anguillicaudatus (Teleostei: Cobitidae)

The loach Misgurnus anguillicaudatus comprises diploid clonal, triploid and diploid-triploid mosaic individuals in a wild population on Hokkaido island, Japan. When diploid eggs of clonal loaches are fertilized by haploid sperm of normal bisexual loaches, both diploid clonal and non-diploid aclonal individuals occur in the progeny. Flow cytometry and microsatellite analyses revealed that the occurrence of triploid, diploid-triploid and other progeny was essentially due to the genetic incorporation of sperm to diploid clonal genomes of unreduced eggs. In this study, we examined the influence of water temperature from fertilization to early embryogenesis on frequencies of diploid clonal and other progeny and observed that progeny of three out of four clonal females examined exhibited approximately constant rates of diploid clonal individuals (54.2-68.9%) at hatching stage. Thus, no drastic increase of non-diploid progeny was detected. However, the 28 degrees C group of the fourth clonal female gave significantly lower rate (28.1%) of diploid clonal progeny, suggesting that this temperature might be a critical or a borderline temperature inducing sperm incorporation. We also examined the cytological process by which diploid clonal and other aclonal progeny develop after fertilization. In some fertilized eggs, the sperm nucleus remained condensed throughout fertilization and early embryogenesis and never fused with the female pronucleus. This cytological observation concludes that clonal eggs develop by the mechanism of gynogenesis. However, some other eggs showed the cytological process of syngamy between the female pronucleus and an accidentally formed male nucleus, suggesting the formation of triploid progeny. The syngamy between an accidentally activated sperm nucleus with a male pronucleus-like structure and nucleus of a blastomere of gynogenetically developing clonal diploid embryo might produce a diploid-triploid mosaic individual.     

Intracytoplasmic Sperm Injection Using Cryopreserved, Fixed, and Freeze-dried Sperm in Eggs of Nile Tilapia

Gamete preservation techniques are essential in animal husbandry as well as in assisted reproduction for humans. In this research we attempted to use 3 different sperm preservation techniques in combination with newly developed techniques for intracytoplasmic sperm injection (ICSI) to fertilize eggs of a teleost fish, the Nile tilapia (Oreochromis niloticus). Of 47 eggs injected with fresh sperm, 11 (23%) were fertilized, 5 developed abnormally, and 4 developed normally and hatched; from these, one grew to adulthood. Nuclear DNA content of 4 of the abnormal embryos indicated that they were diploid. Flow cytometric analysis of a blood sample from the surviving ICSI fish collected 2 months after fertilization indicated that the fish was diploid. Of 45 eggs injected with cryopreserved sperm, 9 (20%) developed to the blastula stage. Of 40 eggs injected with sperm preserved in 70% methanol, none were fertilized. No injections were possible with freeze-dried Nile tilapia sperm owing to technical difficulties during manipulation. Although the findings described here are limited, they provide the first steps toward using sperm preservation methods in addition to cryopreservation for fertilization in fishes.     

Disruption of normal meiosis in artificial inter-populational hybrid females of <Emphasis Type="Italic">Misgurnus</Emphasis> loach

Artificial cross between two genetically different populations of Japanese Misgurnus loach was made to examine the reproductive capacity of the artificial inter-populational hybrid females. Ploidy status and microsatellite genotypes of the eggs laid by these hybrids were inferred from those determined in progenies developed by normal fertilization with haploid loach sperm, induced gynogenesis with UV-irradiated goldfish sperm and/or hybridization with intact goldfish sperm. Some hybrid females laid unreduced diploid eggs genetically identical to the mother. However, these diploid eggs could not develop by spontaneous gynogenesis, but grow to triploid by incorporation of a sperm nucleus. Other hybrid females laid haploid eggs together with diploid eggs and/or various aneuploid and polyploid eggs. Thus, a disruption of normal meiosis occurred in inter-populational hybrid females. The results suggested that the two populations should be so distant as to give rise to atypical formation of unreduced and other unusual eggs in their hybrids.     

Single-locus complementary sex determination in the inbreeding wasp Euodynerus foraminatus Saussure (Hymenoptera: Vespidae)

The Hymenoptera have arrhenotokous haplodiploidy in which males normally develop from unfertilized eggs and are haploid, while females develop from fertilized eggs and are diploid. Multiple sex determination systems are known to underlie haplodiploidy, and the best understood is single-locus complementary sex determination (sl-CSD) in which sex is determined at a single polymorphic locus. Individuals heterozygous at the sex locus develop as females; individuals that are hemizygous (haploid) or homozygous (diploid) at the sex locus develop as males. sl-CSD can be detected with inbreeding experiments that produce diploid males in predictable proportions as well as sex ratio shifts due to diploid male production. This sex determination system is considered incompatible with inbreeding because the ensuing increase in homozygosity increases the production of diploid males that are inviable or infertile, imposing a high cost on matings between close relatives. However, in the solitary hunting wasp Euodynerus foraminatus, a species suspected of having sl-CSD, inbreeding may be common due to a high incidence of sibling matings at natal nests. In laboratory crosses with E. foraminatus, we find that sex ratios and diploid male production (detected as microsatellite heterozygosity) are consistent with sl-CSD, but not with other sex determination systems. This is the first documented example of sl-CSD in a hymenopteran with an apparent natural history of inbreeding, and thus presents a paradox for our understanding of hymenopteran genetics.     

Meiotic hybridogenesis in triploid Misgurnus loach derived from a clonal lineage

Triploid loaches Misgurnus anguillicaudatus are derived from unreduced diploid gametes produced by an asexual clonal lineage that normally undergoes gynogenetic reproduction. Here, we have investigated the reproductive system of two types of triploids: the first type carried maternally inherited clonal diploid genomes and a paternally inherited haploid genome from the same population; the second type had the same clonal diploid genomes but a haploid genome from another, genetically divergent population. The germinal vesicles of oocytes from triploid females (3n=75) contained only 25 bivalents, that is, 50 chromosomes. Flow cytometry revealed that the majority of the progeny resulting from fertilization of eggs from triploid females with normal haploid sperm were diploid. This indicates that triploid females mainly produced haploid eggs. Microsatellite analyses of the diploid progeny of triploid females showed that one allele of the clonal genotype was not transmitted to haploid eggs. Moreover, the identity of the eliminated allele differed between the two types of triploids. Our results demonstrate that there is preferential pairing of homologous chromosomes as well as the elimination of unmatched chromosomes in the course of haploid egg formation, that is, meiotic hybridogenesis. Two distinct genomes in the clone suggest its hybrid origin.     

Life‐history traits of the Whiting polyploid line of the parasitoid Nasonia vitripennis

In hymenopterans, males are normally haploid (1n) and females diploid (2n), but individuals with divergent ploidy levels are frequently found. In species with ‘complementary sex determination’ (CSD), increasing numbers of diploid males that are often infertile or unviable arise from inbreeding, presenting a major impediment to biocontrol breeding. Non‐CSD species, which are common in some parasitoid wasp taxa, do not produce polyploids through inbreeding. Nevertheless, polyploidy also occurs in non‐CSD Hymenoptera. As a first survey on the impacts of inbreeding and polyploidy of non‐CSD species, we investigate life‐history traits of a long‐term laboratory line of the parasitoid Nasonia vitripennis (Walker) (Hymenoptera: Pteromalidae) (‘Whiting polyploid line’) in which polyploids of both sexes (diploid males, triploid females) are viable and fertile. Diploid males produce diploid sperm and virgin triploid females produce haploid and diploid eggs. We found that diploid males did not differ from haploid males with respect to body size, progeny size, mate competition, or lifespan. When diploid males were mated to many females (without accounting for mating order), the females produced a relatively high proportion of male offspring, possibly indicating that these males produce less sperm and/or have reduced sperm functionality. In triploid females, parasitization rate and fecundity were reduced and body size was slightly increased, but there was no effect on lifespan. After one generation of outbreeding, lifespan as well as parasitization rate were increased, and a body size difference was no longer apparent. This suggests that outbreeding has an effect on traits observed in an inbred polyploidy background. Overall, these results indicate some phenotypic detriments of non‐CSD polyploids that must be taken into account in breeding.     

What haplodiploids can teach us about hybridization and speciation

Most evolutionary theory focuses on species that reproduce through sexual reproduction where both sexes have a diploid chromosome count. Yet a substantial proportion of multicellular species display complex life cycles, with both haploid and diploid life stages. A classic example is haplodiploidy, where females develop from fertilized eggs and are diploid, while males develop from unfertilized eggs and are haploid. Although haplodiploids make up about 15% of all animals (de la Filia et al. 2015 ), this type of reproduction is rarely considered in evolutionary theory. In this issue of Molecular Ecology, Patten et al. ( 2015 ) develop a theoretical model to compare the rate of nuclear and mitochondrial introgression in haplodiploid and diploid species. They show that when two haplodiploid species hybridize, nuclear genes are much less likely to cross the species barrier than if both species were to be diploids. The reason for this is that only half of the offspring resulting from matings between haplodiploid species are true hybrids: sons from such mating only inherit their mother genes and therefore only contain genes of the maternal species. Truly, hybrid males can only occur through backcrossing of a hybrid female to a male of one of the parental species. While this twist of haplodiploid transmission genetics limits nuclear introgression, mitochondrial genes, which are maternally inherited, are unaffected by the scarcity of hybrid males. In other words, the rate of mitochondrial introgression is the same for haplodiploid and diploid species. As a result, haplodiploid species on average show a bias of mitochondrial compared to nuclear introgression.     

POPULATION GENETICS OF A PARASITIC CHROMOSOME: EXPERIMENTAL ANALYSIS OF PSR IN SUBDIVIDED POPULATIONS

Nasonia vitripennis is a parasitoid wasp that harbors several non-Mendelian sex-ratio distorters. These include MSR (Maternal Sex Ratio), a cytoplasmic element that causes nearly all-female families, and PSR (Paternal Sex Ratio), a supernumerary chromosome that causes all-male families. As in other hymenoptera, N. vitripennis has haplodiploid sex determination. Normally, unfertilized (haploid) eggs develop into males and fertilized (diploid) eggs develop into females. The PSR chromosome violates this normal pattern; it is inherited through sperm, but then causes destruction of the paternal chromosomes (except itself), thus converting diploid fertilized eggs (normally females) into haploid eggs that develop into PSR-bearing males. PSR is an extreme example of “parasitic” or “selfish” DNA. Because N. vitripennis has a highly subdivided population structure in nature, population-level selection may be important in determining the dynamics of PSR in natural populations. A theoretical analysis shows that subdivided population structure reduces PSR frequency, whereas high fertilization proportion (such as produced by the MSR element) increases PSR frequency. Population experiments using two deme sizes (3- and 12-foundress groups) and strains producing two fertilization proportions [wild-type (LabII)–57–67% female, and MSR (MI)–90–93% female] confirm these predictions. PSR achieved frequencies over 0.90 in 12–foundress group MSR populations in contrast to 0.20–0.40 in wild-type 12–foundress populations. PSR was selected against in wild-type populations composed of three-foundress groups. In MSR populations with three-foundress groups, presence of PSR selected against the MSR cytoplasmic element, eventually leading to low frequencies of both PSR and MSR. Complicated dynamics may occur when these two sex-ratio distorters are both present in highly subdivided populations. The existence of PSR in natural populations may depend on the presence of MSR. Results indicate that population subdivision could be important in determining the frequency of sex ratio distorters in N. vitripennis.