Within-population variation in ejaculate characteristics in a prolonged breeder,Peron’s tree frog, <Emphasis Type="Italic">Litoria peronii</Emphasis>

Sperm number is often a good predictor of success in sperm competition; however, it has become increasingly clear that, for some species, variation in probability of paternity cannot be explained by sperm number alone. Intraspecific variation in ejaculate characteristics, such as the number of viable sperm and sperm longevity, may play an equally important role in determining fertilization success. Here, we assess variation among ejaculates in three factors that may contribute to fertilization success (number of sperm per ejaculate, viability, and longevity), in a population of Peron’s tree frog (Litoria peronii). We detected large variation among males in the number of sperm per ejaculate and the proportion of viable sperm within ejaculates, which could not be explained by variation in either male size or body condition. However, the proportion of viable sperm released by males increased over the season. Finally, we assessed sperm longevity (proportion viable sperm determined using a dual-fluorochrome vital dye) at two different temperatures. At 23°C, on average, 75% of sperm remained viable after 2 h, but there were significant differences amongst males with the percentage of viable sperm ranging from 43% to 95%. For sperm incubated at 4°C, ejaculates varied fivefold in sperm longevity with some males having 50% viable sperm after 5 days. Our data suggest that ejaculate characteristics (sperm number, viability, and longevity) vary widely in Peron’s tree frog and may therefore play an important role in determining siring success both in the presence and absence of sperm competition. We discuss the results in relation to selection on ejaculate traits via natural and sexual selection in this and other amphibians.     

Extraordinarily long sperm in the socially monogamous cichlid fish <Emphasis Type="Italic">Pelvicachromis taeniatus</Emphasis>

The main function of the spermatozoon is the transfer of the male haploid genome during fertilisation. In animals in general and in fishes in particular, there is huge variation in sperm size. In fishes, sperm size ranges from 13 μm in Mugil cephlus to nearly 100 μm in the channel catfish Ictalurus punctatus. We examined intra-specific variation in sperm morphometry in the socially monogamous cichlid Pelvicachromis taeniatus using scanning electron microscopy. The mean total sperm length of nearly 70 μm was extraordinarily large for cichlids. Furthermore, within-male variation was remarkably high. To our knowledge, P. taeniatus produces the longest cichlid sperm ever documented. Several hypotheses concerning the adaptive significance of these results are presented.     

Dynamics of sperm transfer in the ant Leptothorax gredleri

Mating tactics differ remarkably between and within species of social Hymenoptera (bees, wasps, ants) concerning, e.g., mating frequencies, sperm competition, and the degree of male sperm limitation. Although social Hymenoptera might, therefore, potentially be ideal model systems for testing sexual selection theory, the dynamics of mating and sperm transfer have rarely been studied in species other than social bees, and basic information needed to draw conclusions about possible sperm competition and female choice is lacking. We investigated sperm transfer in the ant Leptothorax gredleri, a species in which female sexuals attract males by “female calling.” The analysis of 38 female sexuals fixed immediately or up to 7 days after copulation with a single male each revealed that the sperm is transferred into the female bursa copulatrix embedded in a gelatinous mass, presumably a spermatophore. Sperm cells rapidly start to migrate from the tip of the spermatophore towards the spermatheca, but transfer is drastically slowed down by an extreme constriction of the spermathecal duct, through which sperm cells have to pass virtually one by one. This results in the spermatheca being filled only between one and several hours after mating. During this time, the posterior part of the spermatophore seals the junction between bursa copulatrix and spermathecal duct and prevents sperm loss. The prolonged duration of sperm transfer might allow female sexuals to chose between ejaculates and explain previously reported patterns of single paternity of the offspring of multiply mated queens. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

No evidence for sperm priming responses under varying sperm competition risk or intensity in guppies

Sperm competition theory predicts that males should tailor their investment in ejaculates according to the number of rival males competing to fertilize a female’s eggs. Research spanning several taxa supports this prediction by showing that males are often sensitive to the level of sperm competition and adjust their investment in sperm numbers accordingly. More recent work has revealed that males may also tailor the quality of sperm according to the number of males competing for fertilization. Here I test for both effects in guppies (Poecilia reticulata) in an experiment that simultaneously evaluates the risk and intensity models of sperm competition. The experiment determined whether male guppies adjust the number (stripped ejaculate size) and quality (sperm velocity and viability) of sperm that are primed over a 3-day period according to experimental changes in the perceived level of sperm competition. A total of 136 focal males were initially stripped of all retrievable sperm and assayed for these sperm traits before being allocated at random to one of four treatments simulating different levels of sperm competition risk and intensity. During the 3-day treatment phase, focal males had visual and olfactory access to a sexually receptive (initially virgin) female maintained with different numbers of stimulus males to simulate variation in the risk and intensity of sperm competition. Following this, males were assayed again for the sperm traits. Contrary to predictions, there was no significant change in any of the measured variables among treatments, although qualitatively the patterns for sperm velocity and viability did conform to expectation. The lack of any trend for the number of sperm primed was unequivocal and future work examining the effects of sperm competition on sperm production should focus on whether males differentially allocate sperm numbers among matings that differ in the level of sperm competition.     

Queue up, please! Spermathecal filling in the rove beetle Drusilla canaliculata (Coleoptera, Staphylinidae)

Most studies on insect sperm motility have been conducted in vitro using artificial environments outside the animal’s body. Only little is known about the function of motile insect sperm at different sites within the male or female genital tracts. We dissected genital tracts of female rove beetles (Drusilla canaliculata) to show that spermatozoa use their own motility to migrate from the spermatophore into the spermatheca. Our dissection method allowed direct observation and filming of the spermathecal filling process inside the female’s genital tract. Spermatozoa were found to enter the spermatheca individually, sometimes in groups of two or three. Although exhibiting only weak motility and no progressive motion in buffer solution, the spermatozoa inside the female show vigorous lashing and reach an average velocity of 47.5 μm s⁻¹. To gain mobility and speed, the spermatozoa likely utilize the relatively small diameter of the spermathecal duct to push themselves off the duct walls, rather than swimming freely in seminal fluid. The spermatozoa (approximately 1,250 μm) are considerably longer than the distance they have to travel along the spermathecal duct (approximately 800 μm). Our study provides the first direct observation of active sperm migration within the female of an insect stressing the importance of the genital tract as a prerequisite for functional sperm motility.     

汽车尾气对人精子运动能力影响探讨

采用［日］岛津AA－670型原子吸收分光光度计,四唑氮蓝（Nitro－BT）染色法,及图象分析仪（MIAS－300）分析了交通警察和男性师生两人群精液的精子运动能力参数和血铅水平。结果显示：与对照组比较,交警组精子活动能力降低（P＜0.05）,精子琥珀酸脱氢酶（SDH）活性明显抑制（P＜0.001）。而且SDH活性与血铅水平呈负相关。认为交警精子活动能力下降可能和铅干扰精子线粒体能量代谢有关。     

Millennium-old farm breeding of Chinese softshell turtles (<Emphasis Type="Italic">Pelodiscus</Emphasis> spp.) results in massive erosion of biodiversity

Chinese softshell turtles (Pelodiscus spp.) are widely distributed, ranging from the Amur and Ussuri Rivers in the Russian Far East through the Korean Peninsula, Japan, and eastern, central, and southern China to southern Vietnam. In East and Southeast Asia, Chinese softshell turtles are traditionally exploited for food and have been farm-bred in China since the Spring and Autumn Period, more than 2400 years ago. Currently, the annual production of Pelodiscus amounts to 340,000 t in China alone. Using mitochondrial DNA (2428 bp) and five nuclear loci (3704 bp), we examined broad sampling of wild and farm-bred Pelodiscus to infer genetic and taxonomic differentiation. We discovered four previously unknown mitochondrial lineages, all from China. One lineage from Jiangxi is deeply divergent and sister to the mitochondrial lineage of Pelodiscus axenaria. The nuclear loci supported species status for P. axenaria and the new lineage from Jiangxi. Pelodiscus maackii and P. parviformis, both harboring distinct mitochondrial lineages, were not differentiated from P. sinensis in the studied nuclear markers. The same is true for two new mitochondrial lineages from Zhejiang, China, represented by only one individual each, and another new lineage from Anhui, Guangdong, Jiangxi and Zhejiang, China. However, Vietnamese turtles yielding a mitochondrial lineage clustering within P. sinensis were distinct in nuclear markers, suggesting that these populations could represent another unknown species with introgressed mitochondria. Its species status is also supported by the syntopic occurrence with P. sinensis in northern Vietnam and by morphology. In addition, we confirmed sympatry of P. axenaria and P. parviformis in Guangxi, China, and found evidence for sympatry of P. sinensis and the new putative species from Jiangxi, China. We also discovered evidence for hybridization in turtle farms and for the occurrence of alien lineages in the wild (Zhejiang, China), highlighting the risk of genetic pollution of native stock. In the face of the large-scale breeding of Pelodiscus, we claim that the long-term survival of distinct genetic lineages and species can only be assured when an upscale market segment for pure-bred softshell turtles is established, making the breeding of pure lineages lucrative for turtle farms. Our findings underline that the diversity of Pelodiscus is currently underestimated and threatened by anthropogenic admixture. We recommend mass screening of genetic and morphological variation of Chinese softshell turtles as a first step to understand and preserve their diversity.     

Genetic variation in natural honeybee populations,<Emphasis Type="Italic"> Apis mellifera capensis</Emphasis>

Genetic variation in honeybee, Apis mellifera, populations can be considerably influenced by breeding and commercial introductions, especially in areas with abundant beekeeping. However, in southern Africa apiculture is based on the capture of wild swarms, and queen rearing is virtually absent. Moreover, the introduction of European subspecies constantly failed in the Cape region. We therefore hypothesize a low human impact on genetic variation in populations of Cape honeybees, Apis mellifera capensis. A novel solution to studying genetic variation in honeybee populations based on thelytokous worker reproduction is applied to test this hypothesis. Environmental effects on metrical morphological characters of the phenotype are separated to obtain a genetic residual component. The genetic residuals are then re-calculated as coefficients of genetic variation. Characters measured included hair length on the abdomen, width and length of wax plate, and three wing angles. The data show for the first time that genetic variation in Cape honeybee populations is independent of beekeeping density and probably reflects naturally occurring processes such as gene flow due to topographic and climatic variation on a microscale.     

Cytogenetic analysis of human oocytes after subzonal insemination

Subzonal insemination has been proposed to achieve fertilization in cases where standard in vitro fertilization has failed. We present the results of chromosome analysis of oocytes after subzonal insemination. Our data suggest that the main cause (76 per cent) of the absence of cleavage after subzonal insemination is the total absence of sperm nucleus evolution of the injected spermatozoa. Our results also suggest that spermatozoa chromatin development is normal after subzonal insemination. Aneuploidy does not seem to be increased in zygotes after subzonal insemination. However, polyploidy was often more important than predicted by the observation of pronuclei (PN). Pronucleus development might be asynchronous and can appear earlier or later than after standard IVF. The cytogenetic risk after subzonal insemination might therefore be triploidy (if a triploid egg is transferred, because only 2 PN were seen) rather than aneuploidy or structural abnormalities.     

5,000 year-old spermatozoa in Quaternary Ostracoda (Crustacea)

Fossil spermatozoa are recorded for the first time in freshwater ostracods (small bivalved crustaceans) from Holocene sediments at sites in the United Kingdom and Germany. Exceptional conditions at these sites have resulted in the preservation of chitinous "soft parts", including limbs, the remains of Zenker's Organs (sperm pumps that are part of the male reproductive apparatus in cypridoidean ostracods) and spermatozoa from eight different species. Comparisons are made with spermatozoa from living ostracods and the implications of these discoveries for evolutionary studies of reproductive modes are discussed.     

兽药阿苯达唑的蚯蚓繁殖毒性研究

阿苯达唑是目前应用广泛的高效抗寄生虫兽药,其残留对蚯蚓等土壤动物的繁殖毒性鲜见报道.本研究选择了阿苯达唑的5个染毒剂量(1、3、6、9和12 mg·kg-1),检测染毒56 d阿苯达唑对蚯蚓产茧、孵化及其幼蚓产生的影响,以及染毒14 d蚯蚓精子畸形率和精子细胞超微结构的变化.结果表明,染毒28 d时,卵茧数量比其他繁殖参数(卵茧重和孵化率)对低剂量阿苯达唑敏感,随着染毒剂量(3~12 mg·kg-1)增加,卵茧数量显著降低(p0.01),呈现明显的剂量-效应关系;染毒56 d时,幼蚓重量比其他繁殖参数(幼蚓数量和孵化率)敏感,产生有毒作用的最低染毒剂量为3 mg·kg-1(p0.01).短期染毒14 d时,精子细胞的超微结构在染毒剂量为3~12 mg·kg-1时发生明显变化,尤其是线粒体,而精子畸形率发生明显变化的最低剂量为6 mg·kg-1,可见,短期染毒时精子细胞的超微结构比光镜下的精子畸形率敏感.该研究结果可为阿苯达唑残留的土壤生态毒理及早期监测提供依据.     

Sperm competition promotes diversity of sperm bundles in <Emphasis Type="Italic">Ohomopterus</Emphasis> ground beetles

Diversification of sperm morphology has been investigated in the context of sperm competition, but the adaptive significance of sperm bundles is still unclear. In analyzing 10 taxa of the genus Carabus subgenus Ohomopterus and one related Carabus ground beetles, we found that dimorphic sperm bundles occurred in most species with varied degrees of bimodality, whereas sperm were generally monomorphic. Comparative analyses with phylogenetically independent contrasts revealed that the sizes of large and small sperm bundles evolved more rapidly than, and were not correlated with, the length of sperm, suggesting more intense selection on sperm bundle sizes and their independent responses to different evolutionary forces. The size of large sperm bundles was positively correlated with male genital morphology (pertinent to displacement of rival spermatophores) and postcopulatory guarding duration as well as male body length, suggesting that larger sperm bundles have been favored when the risk of spermatophore displacement is high. Larger sperm bundles may be advantageous because of their ability to migrate more rapidly into the spermatheca. In contrast, no clear association was detected between the small sperm bundle size and mating traits despite its rapid diversification. The present study provides the first record of heteromorphic sperm bundles, the diversity of which may be promoted by sperm competition.     

Chromosome 18 analysis by fluorescence in situ hybridization (fish) in human blastomeres of abnormal embryos after in vitro fertilization (ivf) attempt

We performed fluorescence in situ hybridization (FISH) with a chromosome 18-specific probe on human abnormal cleaved embryos, fertilized either by two spermatozoa and exhibiting three pronuclei (3 PN) or normally fertilized and exhibiting two pronuclei (2 PN) with subsequent severe fragmentation and/or blocking. The aim of the study was to evaluate the incidence of chromosome 18 anomalies among these embryos, in order to evaluate the FISH efficiency on such material and to obtain more precise and complete data than those obtained with classical cytogenetic analysis. For the 3 PN cleaved embryos, FISH confirmed the frequent regulation towards diploidy (25 per cent) and the high frequency of mosaics (53 per cent). For the 2 PN blocked or damaged embryos, FISH permitted chromosome evaluation, which was otherwise impossible with classical cytogenetic techniques: we also found a high mosaic frequency (45 per cent) with these embryos. If this frequency were the same for normally developing embryos, it would be a major obstacle to the reliability of either chromosomal or genetic preimplantation diagnosis.     

How is a giant sperm ejaculated? Anatomy and function of the sperm pump, or “Zenker organ,” in Pseudocandona marchica (Crustacea, Ostracoda, Candonidae)

‘Giant sperm’, in terms of exceptionally long spermatozoa, occur in a variety of taxa in the animal kingdom, predominantly in arthropod groups, but also in flatworms, mollusks, and others. In some freshwater ostracods (Cypridoidea), filamentous sperm cells reach up to ten times the animal’s body length; nonetheless, during a single copulation several dozen sperm cells can be transferred to the female’s seminal receptacle. This highly effective ejaculation has traditionally been credited to a chitinous-muscular structure within the seminal duct, which has been interpreted as a sperm pump. We investigated this organ, also known as the Zenker organ, of a cypridoid ostracod, Pseudocandona marchica, utilizing light and electron microscope techniques and produced a three-dimensional reconstruction based on serial semi-thin histological sections. This paper shows that numerous muscle fibers surround the central tube of the Zenker organ, running in parallel with the central tube and that a thin cellular layer underlies the muscular layer. A cellular inner tube exists inside the central tube. A chitinous-cellular structure at the entrance of the organ has been recognized as an ejaculatory valve. In male specimens during copulation, we confirmed a small hole derived from the passage of a single spermatozoon through the valve. The new data allowed for proposing a detailed course of operation of the Zenker organ during giant sperm ejaculation.     

Multiple paternity in<Emphasis Type="Italic"> Rana dalmatina</Emphasis>, a monogamous territorial breeding anuran

Polyandry and sperm competition in anurans have rarely been documented. We investigated the genetic paternity inferred from allozyme variations in 650 tadpoles from four natural ponds in a territorial breeding anuran, Rana dalmatina. Multiple paternity was demonstrated, although R. dalmatina is regarded as a monogamous species. Polyandrous mating was not a common event, occurring only in 17.9% of clutches, with no significant differences among clutches. The proportions of tadpoles fathered by a second male did not significantly differ among ponds, showing that multipaternity was not restricted to a single site. Such a polyandry may result from synchronous multiple amplexus and should reduce the heterozygote deficit related to the breeding-pond fidelity usually exhibited by most anurans.     

Atlantic sturgeons (<Emphasis Type="Italic">Acipenser sturio</Emphasis>, <Emphasis Type="Italic">Acipenser oxyrinchus</Emphasis>): American females successful in Europe

Recent molecular data on the maternally inherited mitochondrial (mt) DNA have challenged the traditional view that the now extinct Baltic sturgeon population belonged to the European sturgeon Acipenser sturio. Instead, there is evidence that American sea sturgeon Acipenser oxyrinchus historically immigrated into the Baltic Sea. In this study, we test the hypothesis that A. oxyrinchus introgressed into, rather than replaced, the A. sturio population in the Baltic. We established four single nucleotide polymorphisms (SNPs) in the nuclear MHC II antigen gene with a species-specific SNP pattern. Using an ancient DNA approach and two independent lines of molecular evidence (sequencing of allele-specific clones, SNaPshot), we detected both A. sturio and A. oxyrinchus alleles in the available museum material of the now extinct Baltic sturgeon population. The hybrid nature of the Baltic population was further confirmed by very high levels of heterozygosity. It had been previously postulated that the immigration of the cold-adapted A. oxyrinchus into the Baltic occurred during the Medieval Little Ice Age, when temperature likely dropped below the degree inducing spawning in A. sturio. Under this scenario, our new findings suggest that the genetic mosaic pattern in the Baltic sturgeon population (oxyrinchus mtDNA, sturio and oxyrinchus MHC alleles) is possibly caused by sex-biased introgression where spawning was largely restricted to immigrating American females, while fertilization was predominantly achieved by abundant local European males. The hybrid nature of the former Baltic sturgeon population should be taken into account in the current reintroduction measures. Electronic supplementary material  Supplementary material is available in the online version of this article at and is accessible for authorized users.     

湘江流域传统村落景观基因变异及其分异规律

传统村落在历史、文化、经济、旅游和科学等多个方面价值颇高,保护传统村落的空间格局和文化内涵极为重要。以中国湘江流域上中下游六个典型传统村落为例,以景观基因理论为基础,运用景观基因分析法识别了湘江流域传统村落景观基因,分析了上中下游传统村落显性物质景观基因与隐性非物质文化基因的变异特征,总结了湘江流域传统村落景观基因变异特征及分异规律,研究结果显示：（1）湘江流域上游传统村落平面基因变异相悖于本土的情况侧重发生在平面结构变异上;立面基因的变异相悖于本土、突变后直接消亡的情况较多;文化基因上受外来冲击的不适感较强,有加剧景观基因变异的风险和趋势。（2）湘江流域中游传统村落对立面基因的改动需求较大,且改动时间较早,造成立面基因变异以及平面基因中街巷格局、平面布局变异较大,变异后相悖于本土的特征显著;而在文化基因中,对文化的变异融合接受能力强,变异相融于本土的情况显著。（3）湘江流域下游传统村落在形态、布局特征明显的平面基因中产生的变异不大显著;在立面基因中遭遇突变后直接消亡的特征明显;在文化基因中,对家风信仰的基因变异以相融于本土为主,习俗礼仪的基因变异呈现分化趋势,单民族聚居式的家族以相悖于本土的特征为主,多民族聚居式的家族以相融于本土的特征为主。     

导数同步荧光法测定硒

对硒与２,３－二氨基萘（ＤＡＮ）络合物在有机溶剂中的同步荧光光增进行了研究,当同步荧光法谱的△λ选择１４０ｎｍ时,可获得一锐形单隆光谱,在此基础上获得了一阶导数同步荧光和二阶导数同步荧光的最佳实验条件。用一阶导数同步荧光定量测定硒可提高的灵敏度和改善精密度,在仪器的中挡灵敏范围,硒的检测限为１．５ｎｇ,硒浓度在０－２５０ｎｇ时,硒浓度与荧光强度的直线关系是ｙ＝０．４４ｘ－０．１８,相关系数（ｒ）０     

Prenatal diagnosis of respiratory chain deficiency by direct mutation screening

Respiratory chain deficiency (RCD) is responsible for a clinically heterogeneous group of early-onset untreatable disorders. Enzymological prenatal diagnosis (PD) can only be offered to a fraction of families. Moreover, due to the two-fold genetic origin of the respiratory chain (nuclear and mitochondrial DNA) and owing to the large number of nuclear genes involved in the respiratory chain assembly, maintenance and functioning, the identification of the disease causing gene in a given family remains challenging. Here, we report on PD of RCD by direct screening of NDUFV1, SDH-Fp, SCO1 and SURF1 mutations in five unrelated families with complex I, II and IV deficiency, respectively. The identification of the disease-causing gene in a given family with RCD is a major issue to provide both adequate genetic counselling and early, reliable PD. Copyright © 2001 John Wiley & Sons, Ltd.