Comparative histology of the testes of the spined loach Cobitis taenia L. and natural allotetraploids of Cobitis (Pisces, Cobitidae)

The spined loach Cobitis taenia L. creates exclusively diploid and mixed diploid–polyploid populations. Allotriploid females, which co-exist with C. taenia or C. elongatoides and a few tetraploid males and females dominate in most Cobitis mixed populations. They reproduce gynogenetically and produce triploid eggs that are stimulated to development by sperm from Cobitis males. Some of these eggs are fertilized, which leads to the production of bisexual tetraploids. Males of C. taenia (2n = 48) from a diploid population in Lake Klawój, Northern Poland (46 individuals) and from a mixed Cobitis population in the Bug River, Eastern Poland (7 individuals), and three tetraploid males (4n = 98) from the same mixed population were examined. All the fish were analyzed karyologically and histologically. Tubules with cysts of the testes of C. taenia from both populations were filled with germ cells at various developmental stages. Among fishes from Lake Klawój sperm maturation in batches simultaneous with the batch spawning of C. taenia females was found. The testes of the loach C. taenia, from a mixed population in the Bug River, were filled with spermatozoa over the entire reproductive season. Sperm maturation in batches was not observed. Sperm maturation in batches seems to be only connected with a few diploid males in this population. So, a continuous process of spermatogenesis in their testes is required. Only in the testes of all tetraploid Cobitis males were cells characteristic of the early stages of spermatogenesis observed, i.e. without spermatids and spermatozoa. Furthermore, the histological sections of the testis of a male captured in August, revealed fragments with connective tissue between the germ cells. However the participation of tetraploid, infertile Cobitis males in the process of reproduction in the investigated mixed population remains controversial. The results obtained so far, reveal that even the infertile sperm of tetraploid males may induce gynogenesis in Cobitis triploid females.     

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.     

Viable diploid progeny induced from sperm of Chinese tetraploid pond loach by cold‐shock androgenesis

Natural tetraploid loach (Misgurnus anguillicaudatus) living in the Changjiang River basin and adjacent area in China has been considered a true genetic tetraploidy (4n = 100) with four sets of homologous chromosomes. Here, we reported its further supporting evidence provided by the cold‐shock androgenesis. Viable androgenetic progeny appeared when eggs were cold‐shocked at 3°C for 60 min, starting 5 min after fertilization with sperm of natural tetraploid males, although the survival rate was significantly lower than control group. The majority (87%) of androgenetic embryos were diploid (2n = 50) and all‐paternal inheritance was verified in larval stage by microsatellite genotypes. Microscopic observations confirmed the elimination of both egg nucleus and second polar body from a fertilized egg, followed by the cleavage exclusively with sperm nucleus. Thus, the appearance of normal diploid androgenetic progeny from sperm of natural tetraploid loach revealed the presence of four chromosome sets in tetraploid males.     

Ploidy-dependent survival of progeny arising from crosses between natural allotriploid Cobitis females and diploid C. taenia males (Pisces,Cobitidae)

Crosses between 21 triploid hybrid Cobitis females and 19 C. taenia (2n = 48) males led to viable progeny; whereas no embryonic development was observed in crosses with tetraploid males (4n = 98). The ploidy status of 491 progenies randomly selected with flow cytometry (316) or chromosome analysis (175) revealed an average of 55.2 % triploids and 44.8 % tetraploids, but the ratio of 3n versus 4n fish did change during development. In the first 2 days after hatching, approximately 65.1 % of tetraploid larvae were observed. Their number decreased significantly to 30.8 and 6.2 % on average during 2–5 and 10–15 months of life, respectively. The karyotype of tetraploid progeny (4n = 98) included 3n = 74 chromosomes of the parental female and n = 24 of C. taenia male. The number of tetraploid progeny indicated indirectly that about 66 % of eggs from 3n females were fertilized with C. taenia. The rest of the eggs developed clonally via gynogenesis or hemiclonally via hybridogenesis into triploids of the same karyotype structure as parental females. We have documented for the first time that (at least under experimental conditions) tetraploids are commonly formed, but are less viable than triploids, and a ratio similar to what is found under natural conditions is finally attained. The current explanation concerning the ploidy and karyotype structure of the progeny confirms that the eggs of 3n Cobitis females are not only capable of maintaining all chromosomes but are also capable of incorporating the sperm genome, thus creating the potential to produce tetraploids.     

Delayed sperm injection and fertilization in parthenogenetically activated insect egg (Athalia rosae,Hymenoptera)

Summary Mature eggs dissected from the ovary of unmated females of Athalia rosae ruficornis Jakovlev (Hymenoptera, Tenthredinidae) can be activated to develop (into haploid parthenogenetic males) simply by exposing them to distilled water. These eggs, which are primary oocytes arrested at the first meiotic metaphase, resume meiosis upon activation and reach the first meiotic telophase in 20 min. Mature eggs immediately upon dissection have previously been shown to complete karyogamy and develop as fertilized diploid females if injected with sperm. We show here that the eggs activated in water for 20 min have a much higher rate of successful fertilization if injected with sperm, and that the eggs activated for 40 min, upon sperm injection, though at a reduced frequency still develop as diploid fertilized females. Eggs left in water for 60 min, however, are no longer fertilized upon sperm injection and develop as haploid males.     

Induction of diapausing amictic eggs in Synchaeta pectinata

Amictic females of a clone of S. pectinata from Star Lake (Norwich, Vermont) may produce diapausing as well as non-diapausing (subitaneous) eggs. The proportion of diapausing eggs produced in cultures was unaffected by temperature (12 vs 19 °C) or rotifer population density (minima of 0.33 vs 3 ind. ml^–1) at 19 °C. However, at 19 °C this proportion was higher in cultures maintained at a low food level suppressing reproduction (5 × 10³ cells ml^–1 Cryptomonas erosa) than in those maintained at a high food level (2 × 10⁴ cells ml^–1); the treatment effect was marginally significant (p=0.067). Consistent with the effect of low food availability, a period of starvation was very effective in inducing the development of diapausing eggs. None of 19 females cultured individually from hatching at 19 °C on C. erosa (2 × 10⁴ cells ml^–1) in 1-ml volumes produced any diapausing eggs in 4 days (0 out of 349 eggs), while 13 out of 16 females subjected to a 15-hour starvation period 6 hours after birth produced one or more diapausing eggs during that time (34% of the 158 eggs produced by the 16 females were diapausing). Diapausing eggs produced and left at 19 °C hatched after 4 to 13 days. Those produced in cultures with a low food level took significantly longer to hatch (9.7 days) than those produced in cultures with a high food level (8.1 days) (p=0.022). In natural communities, S. pectinata should be able to respond directly and rapidly to poor food conditions by producing eggs that undergo an obligatory dormant period before resuming development.     

Effect of female aging on the morphology and hatchability of resting eggs in the rotifer <Emphasis Type="Italic">Brachionus plicatilis</Emphasis> Müller

This study examined the morphology and hatchability of Brachionus plicatilis resting eggs as a function of the aging of maternal fertilized mictic females. One-hundred twenty fertilized B. plicatilis (Australian strain) were individually cultured and monitored daily until death. All cultures were maintained at 25°C, 11 ppt, and fed the micro-algae Tetraselmis tetrathele. Resting eggs produced by the females were investigated using two parameters: egg morphology, and hatching rate. Under these culture conditions, females normally produce 1–6 (mean ± SD = 2.7 ± 1.2) resting eggs during their lifetime. However, the number of resting eggs with abnormal morphology increased as a function of maternal age. Among resting eggs with normal morphology (n = 225), 82.2% were produced during the first and second spawning, and had hatching rates of more than 60%, while the hatching rates were below 30% in resting eggs with a spawning order of >2. Thus, the quality of B. plicatilis resting eggs was negatively correlated with maternal age.     

The ms1 mutation in soybean: involvement of gametes in crosses with tetraploid soybean

Summary Previous studies indicated that ms1ms1 malesterile female-fertile soybean (Glycine max [L.] Merr.) plants can produce seeds with different ploidy levels. The codominant chlorophyll-deficient mutant y11 was used in attempts to understand the embryo-endosperm relationship in seed production in ms1ms1 plants and to determine the mechanism of gamete formation in the ms1 mutation. Crosses were conducted between yellow-green male-sterile plants (ms1ms1Y11y11) and green fertile tetraploid cultivars (Ms1Ms1Ms1Ms1Y11Y11Y11Y11) in the greenhouse in the summers of 1987 and 1988. A total of 2,007 cross-pollinations were made. Thirty hybrid seeds were obtained, and plants were analyzed for chromosome number, fertility, and color. All the hybrid seedlings were tetraploid and fertile. No triploids were found. Among the 30 F₁ plants, 7 were green (Y11Y11Y11Y11), 17 were green-yellow (Y11Y11Y11y11), and 6 were yellow-green (Y11Y11y11y11). The segregation ratio was close to the expected 1 green: 2 green-yellow: 1 yellow-green (X² = 0.38; 0.90>p>0.75). From the results of this experiment, we conclude that: (1) triploids were not produced by crossing diploid ms1ms1 soybean plants with tetraploid plants; (2) tetraploid progeny can be produced from these crosses by the fusion of 2n ms1 eggs, or fusion of other 2n gametophyte cells in the embryo sac with a 2x sperm from tetraploid plants; (3) the megaspore mother cell of male-sterile plants undergoes meiotic division without cytokinesis after telophase II and forms more than the normal number of gametes, which can fuse with each other to generate tetraploid gametophyte cells.Joint contribution: U.S. Department of Agriculture, Agricultural Research Service, Cereal and Soybean Research Improvement Unit, Midwest Area, and Journal Paper No.-13838 of the Iowa Agricultural and Home Economics Experiment Station, Ames, Iowa     

Effects of embryonic responses to clutch mates on egg hatching patterns of Pentatomidae (Heteroptera)

Stink bugs and shield bugs of the family Pentatomidae (Heteroptera) generally produce a clutch of densely deposited eggs. In a few species of this family, embryos hatch in response to some form of cues associated with the preceding hatching to synchronize egg hatching with clutch mates. The aim of the present study is to obtain a family‐wide understanding of the extent to which the hatching response to clutch mates accelerates hatching within egg clutches. Accordingly, the hatching patterns in intact egg clutches and eggs individually detached from egg clutches are compared in eight species among different genera. In Halyomorpha halys, hatching is significantly and highly synchronized by the effect of the hatching response: when eggs are not attached to each other, the hatching rate is only 3.8% at 15 min and exceeds 95% at 200 min. By contrast, when eggs are attached to each other, the hatching rate reaches more than 95% at 15 min. Hatching is also significantly synchronized by the hatching response in Nezara viridula (which shows relatively high hatching synchronization) and in Piezodorus hybneri and Plautia stali (both of which show milder hatching synchronization). Synchronization of hatching is not found to be promoted by a hatching response in Aelia fieberi, Dolycoris baccarum, Eurydema rugosum or Palomena angulosa. These findings reveal that the hatching response varies depending on the species in Pentatomidae, with a wide spectrum of effects on the hatching patterns of the egg clutches.     

Antibiotic curing of parthenogenesis in Eretmocerus mundus (Australian parthenogenic form)

The thelytokous parasitoid, Eretmocerus mundus (Australian parthenogenetic form) was infected with a species of Group B Wolbachia. Antibiotic treatment of the parasitoid cured parthenogenesis and lead to male production and reduced egg hatching by virgin females. For each generation infected females produced more progeny than cured females. The descendants of individuals treated with antibiotic were in most cases free of a detectable infection. Both treated females and descendant females mated and received sperm, but these were not used as mated and unmated cured females produced offspring with a similar sex ratio. Males produced as a result of the antibiotic treatment were at least partially functional as they produced sperm and copulated with and inseminated females. However, no sexual line could be established.     

Suppression of first egg mitosis induced by heat shocks in the rainbow trout

Chromosome doubling by mitotic interference was achieved by heat-shocking rainbow trout ( Oncorhynchus mykiss ) eggs fertilized with either intact or genetically inactivated sperm. Tetraploid and mitotic gynogenetic individuals resulted from these treatments respectively. The temperature (27–33° C), duration (2–30min) and application time (2–4 h 40min after egg activation, at 10° C) of the thermal shock were investigated. The best yields of gynogenetics usually resulted from shocks of medium intensity (30° C for 9 min, 31° C for 5 min, 32° C for 4 min). A range of optimal application times was determined between 3 and 4 h after egg activation. A strong maternal effect on gynogenetic yield, irrespective of the application time, was observed. The treatments found to produce the highest yields of gynogenetics (up to 23% survival at hatching, relative to that of the diploid control) should not be used for tetraploid induction because of their rather low efficiency in chromosome doubling (around 70%). Tetraploid populations where viable residual diploids are undesirable should be produced by more intense shocks.     

Resting eggs of the marine rotifer Brachionus plicatilis Müller: development,and effect of irradiation on hatching

Diapause and hatching of Brachionus plicatilis Müller resting eggs were examined through histological and optical approaches. Compound microscope observations on 1% toluidine blue-stained embryo sections suggests that the total number of nuclei in an embryo during the internal diapause period increased from 22 on Day 2 to 39 (each n = 1) on Day 6. The outer layer of embryo membrane gradually thickens from 1.2 (Day 0) to 4.0 µm (Day 8) (each n = 10).Resting eggs that have completed maturation and are in the external diapause period require light for hatching. The threshold of light (halogen lamp) intensity for hatching was estimated to be 4400 lux for 30 min. Hatching rate decreased with longer wavelength irradiation (mercury lamp). Irradiation at more than 350 nm caused 1–25% hatching, but it reached 50–60% at 250–310 nm light. The addition of hydrogen peroxide or prostaglandins (E ₁, E ₂ or F ₂) caused resting egg hatching even in darkness. The production of peroxide in seawater caused by light as well as the oxidation of fatty acid to prostaglandins inside the embryo is a possible mechanism of resting egg hatching.     

A Complementary Method for Production of Tetraploid Crassostrea gigas Using Crosses Between Diploids and Tetraploids with Cytochalasin B Treatments

We present a new method to produce tetraploid Crassostrea gigas by cytochalasin B inhibition of polar body 2 expulsion in diploid females crossed with tetraploid males. This offers a means of direct introgression of genetic characters from selected diploid to tetraploid lines, avoiding a triploid step. Offspring larval ploidy shifted over time and depended on size, with tetraploids more frequent among the smaller larvae and triploids among the large. Viable tetraploids were found at 4 and 6 months, indicating the technique was successful. The possibility that gynogenesis occurred was tested by microsatellite analysis to confirm the presence of paternally inherited alleles. These were present in all animals of the 2n × 4n + CB (female first) cross. However, a 4n × 2n + CB cross produced triploids, including some gynogens. Our method illustrates for the first time that diploid C. gigas eggs, if selected for large size, can give viable tetraploid offspring.     

The evolution of polyandry: an examination of the genetic incompatibility and good‐sperm hypotheses

I have examined the adaptive significance of polyandry using the Australian field cricket Teleogryllus oceanicus. Previous studies of polyandry have examined differences in offspring production by females mated multiply to a single male or females mated multiply to different males. Here I combine this approach with a study of parentage of offspring produced in the later group. Females mated to two different males had a higher proportion of their eggs hatching than did females mating twice with a single male. Offspring fitness parameters were not effected. There was little evidence to suggest that females elevate their hatching success via fertilizing their eggs with sperm from genetically compatible males. Although the average paternity points towards random sperm mixing, there was considerable individual variation in sperm competition success. Patterns of parentage were consistent across females mating twice or four times. Sperm competition success was not related to offspring viability or performance. Thus, the notion that competitively superior sperm produce competitively superior offspring is not supported either. The mechanism underlying increased hatching success with polyandry requires further study.     

休眠卵孵化对策与食物浓度对萼花臂尾轮虫(Brachionus calyciflorus)两品系干雌体种群增长的影响

周期性孤雌繁殖的轮虫靠休眠卵度过不良环境,等环境适宜时休眠卵孵化出干雌体,再次通过孤雌繁殖建立种群。通常休眠卵要经历一段休眠期再孵化,但也有些休眠卵生成后很快孵化,称为早孵化现象。有关休眠卵不同孵化对策如何影响其干雌体克隆种群的增长,目前尚不清楚。分别观测了萼花臂尾轮虫（Brachionus calyciflorus）的两个品系H1（窄温度生态位）和D1（宽温度生态位）的休眠卵在不同孵化对策和不同食物浓度下所生产的干雌体克隆群的种群增长差异。孵化对策包括早孵化（early hatching,EH）和晚孵化（late hatching,LH）;食物浓度包括高食物浓度（high food concentration,HF：2×10⁶个细胞/mL）和低食物浓度（low food concentration,LF：5×10⁵个细胞/mL）,每个品系下各设置4个实验组（LH-HF、LH-LF、EH-HF、EH-LF）,每组10个重复。结果发现,对D1品系来说：孵化对策和食物浓度对最大种群数量具有显著的综合影响（P=0.002）,但两因素间不存在交互作用（P=0.911）;早孵化的干雌体种群在最大种群数量上显著高于晚孵化的干雌体种群（P=0.001）。对H1品系：孵化对策和食物浓度对最大种群数量综合影响显著（P<0.001）,且两个因素之间存在交互作用（P<0.001）;高食物浓度下,EH干雌体克隆群的最大种群数量显著低于LH干雌体克隆群（P<0.001）。高食物浓度下干雌体克隆群开始有性生殖的密度阈值,D1品系EH组显著高于LH组（P=0.041）;而H1品系EH组却显著低于LH组（P=0.022）。最高种群密度下,H1品系的有性生殖率在两种孵化对策之间存在显著差异（P=0.044）,EH种群的有性生殖率低于LH种群,而D品系却未见有性生殖率在不同孵化对策间存在明显差异。本研究结果显示休眠卵的孵化对策会影响其后代干雌体种群的增长特性,其影响的结果可能与种群的生境适应相关。     

The contribution of multiple mating and spermatophore consumption to the lifetime reproductive success of female field crickets (Gryllus bimaculatus)

ABSTRACT.

• 1 The adaptive significance of multiple mating by female Gryllus bimaculatus (De Geer) was investigated.
• 2 Multiple mating prevented the depletion of sperm stores and, therefore, maintained high hatching success. This may not, however, explain the high frequency of remating in this species.
• 3 Male-derived egg stimulants known to be passed with sperm at mating increased the number of eggs produced only when females mated throughout their lifespans.
• 4 Spermatophore consumption appeared to provide nutrients which, while they did not increase the quantity of eggs, increased egg quality as indicated by weight. Females who consumed spermatophores had a greater hatching success.
• 5 While females may derive non-genetic benefits from mating, these are apparently long-term benefits; females must mate throughout their lives in order to accrue them.
• 6 Since the benefits of mating may not be derived from individual males, the spermatophores and their contents in this species are best considered as mating effort.

     

Receptivity of female remating and sperm number in the sperm storage organ in the bean bug,Riptortus clavatus (Heteroptera: Alydidae)

This study examines the relationship between the number of sperm in the seminal receptacle (spermatheca) and the receptivity of female remating in the bean bugRiptortus clavatus Thunberg. On the 21 st day after the first mating when receptivity to remating was > 70%, females receptive to remating had significantly fewer sperm ( < 40 on average) in the spermathecae than females reluctant to do (about 150 on average). However, averages of the number of eggs laid by receptive and reluctant females within 21 days were almost same. The proportion of fertilized eggs for receptive females at 15–21 days after copulation was significantly lower than that for reluctant females. Spermatozoa transferred from a male to a female’s spermatheca were detected 5 min after copulation and then increased continuously to about 500 with the first hour. When copulation durations were manipulated artificially, the shorter the copulation period (=females had less sperm in their spermathecae), the higher the remating rate became. Females may perceive the number of sperm in their seminal receptacles and then determine whether they copulate or not. These results support the hypothesis that females mate multiply in order to replenish inadequate sperm supplies to fertilize all eggs produced.     

Field and laboratory studies on the timing of oviposition and hatching of the western black-legged tick, Ixodes pacificus (Acari: Ixodidae)

The timing of oviposition and hatching of Ixodes pacificus was investigated in the field and at constant temperatures in the laboratory. Replete females held at temperatures between 9 and 29°C began depositing eggs a mean of 9–70 days after drop off. Egg masses held between 12 and 25°C commenced hatching 25–178 days after the onset of oviposition. Eggs held at 9 or 29°C did not hatch. The lower temperature thresholds for development (LTD) for oviposition and hatching were 6.5 and 9°C, respectively. The number of degree days required for oviposition and hatching was 173 and 588, respectively. Replete females placed in the field on 2 December through to 8 March deposited eggs from 2 February through to 24 April; the eggs commenced hatching between 2 July and 21 August. Unfed larvae from two of 20 egg masses survived through the winter and fed readily when exposed to deer mice (Peromyscus maniculatus) on 22 April. Replete larvae were returned to the field and moulted between 9 and 21 August. Larvae exposed to deer mice in August, 4 weeks after hatching, also fed readily. Although further studies are needed to clarify the timing of nymphal development, the present study suggests that I. pacificus requires more than 1 year to complete its life cycle.     

Phenotypic Variations in the Life History of Two Clones of Macrobiotus richtersi (Eutardigrada, Macrobiotidae)

A comparative study of life history traits of two clones (CDMr01 and CDMr02) of a triploid thelytokous apomictic population of the eutardigrade Macrobiotus richtersi has been carried out. Both clones were reared under the same lab conditions: temperature of 14 °C, photoperiod of 12 h/12 h (L/D), and nematodes ad libitum as food. Statistical analysis of the life history traits considered has indicated interclonal variability. The two clones were significantly different in the number of eggs per clutch (fertility), number of eggs laid per female per life span (fecundity), hatching percentage of eggs and hatching time. Similarities between clones have been observed with regard to life span, total number of ovipositions per life span, and age at first oviposition. In addition, significant differences in hatching time, with eggs hatched over a long period, were found within each clone. Interclonal variability in life history traits indicated the effects of genetic factors, whereas intraclonal variability reflected the effects of environmental factors. The evolutionary and adaptive significance of the life history phenotypic variations is discussed.     

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.