Isolation and characterization of 49 polymorphic microsatellite loci in the ayu,Plecoglossus altivelis

A total of 49 novel polymorphic microsatellite markers in the ayu fish, Plecoglossus altivelis, were successfully isolated and characterized. The number of alleles per locus, observed heterozygosity, and expected heterozygosity ranged from two to 25 (11.3 on average), from 0.125 to 1.000 (0.648 on average), and from 0.123 to 0.957 (0.751 on average), respectively. These microsatellite markers will be useful for both studying population structure and identifying quantitative trait loci that control evolutionarily and economically important traits of ayu.     

Development and multiplex PCR amplification of new microsatellite markers for the freshwater fish,ayu (Plecoglossus altivelis)

Multiplex polymerase chain reaction (PCR) technique has recently been applied to gain many advantages in molecular genetics. The present study focused on the development of 15 new microsatellite markers with multiplex PCR systems in ayu, Plecoglossus altivelis, an important freshwater fish in Japan. All loci were followed Mendelian inheritance in 27 F1 progeny except for the one locus. The number of alleles per locus ranged from nine to 44 and the observed heterozygosities ranged from 0.680 to 0.980 in 50 unrelated individuals. The results indicate that these new microsatellite markers are useful for studies of linkage mapping and population genetics for the species.     

Population genetic structure and effective population size of ayu (Plecoglossus altivelis), an amphidromous fish

The temporal and spatial population genetic structure of ayu Plecoglossus altivelis (Salmoniformes: Plecoglossidae), an amphidromous fish, was examined using analysis of variation at six microsatellite DNA loci. Intracohort genetic diversities, as measured by the number of alleles and heterozygosity, were similar among six cohorts (2001–2006) within a population (Nezugaseki River), with the mean number of alleles per cohort ranging from 11·0 to 12·5 and the expected heterozygosity ranging from 0·74 to 0·77. Intrapopulational genetic diversities were also similar across the three studied populations along the 50 km coast, with the mean number of alleles and the expected heterozygosity ranging from 11·33 to 11·67 and from 0·75 to 0·76, respectively. The authors observed only one significant difference in pair-wise population differentiation ( F _ST-value) between the cohorts within a population and among three populations. Estimates of the effective population size ( N _e) based on maximum-likelihood method yielded small values (ranging from 94·8 to 135·5), whereas census population size ranged from c. 4800 to 24 000. As a result, the ratio of annual effective population sizes to census population size ( N _e/ N ) ranged from 0·004 to 0·023. These estimates of N _e/ N agree more closely with estimates for marine fishes than that of the larger estimates for freshwater fishes. The present study suggests that ayu which is highly fecund and shows low survival during the early life stages is also characterized by having low value of N _e/ N , similar to marine species with a pelagic life cycle.     

Inheritance of amplified fragment length polymorphism markers and their utility in population genetic analysis of Plecoglossus altivelis

The reproducibility, mode of inheritance and polymorphism of amplified fragment length polymorphism (AFLP) markers were examined in ayu Plecoglossus altivelis (Salmoniformes: Plecoglossidae). The AFLP markers were highly reproducible, their inheritance following Mendelian expectations. The number of fragments amplified (34–134), polymorphic ratio (0·15–0·78) and average heterozygosity (0·02–0·25) of the AFLP markers showed significant variation among six primer pairs and among ayu populations, including a landlocked Lake‐Biwa population, two amphidromous populations ( P. a. altivelis ) and two Ryukyu‐ayu populations ( P. a. ryukyuensis ). Although AFLP analysis provided similar results in intra‐population diversity and relationships among populations to those found by analyses of allozymes, microsatellites and mitochondrial DNA sequences, AFLPs showed higher polymorphisms and hence greater distinction between genetically close populations.     

香鱼肌肉与免疫器官6种同工酶的表达

采用聚丙烯酰胺电泳方法,对香鱼(Plecoglossus altivelis)肌肉和肝脏、脾脏及肾脏3种免疫器官的6种与防御机能相关的同工酶的表达进行研究,共分析了SOD、POD、ACP、ALP、CAT、EST等6种同工酶的表达模式。结果表明,香鱼已经具有了完善的机体防御系统,肝脏和肾脏在香鱼的防御系统中发挥着十分重要的作用。本研究能为筛选具有较强抗病力和适应性的香鱼良种提供有价值的理论参考。     

Photoreceptor development in the pineal organ and the eye of Plecoglossus altivelis and Paralichthys olivaceus (Teleostei)

Summary The ontogenetic apperance of pineal photo-receptors was compared with that of retinal photoreceptors in the ayu Plecoglossus altivelis and the lefteye flounder Paralichthys olivaceus, which hatched 10 days and 3 days after fertilization, respectively. Despite the disparity in incubation time, the outer segments (containing membranous lamellae) of the pineal photoreceptors first appeared from 3 to 4 days after fertilization in both species. In contrast, the outer segments of the retinal photoreceptors first became visible 5 to 6 days after fertilization, although a characteristic retinal stratification and the optic tract leaving the ganglion cell layer were already found 4 days after fertilization in both species. The functional significance of these temporal disparities and/or similarities in photoreceptor development are discussed with special reference to the timing of daily rhythmic activities during the early developmental period of the teleosts.The results were presented at the Annual Meeting of the Japanese Society of Scientific Fisheries on April 2, 1990 (Tokyo)     

Distribution of two sympatric amphidromous grazing fish Plecoglossus altivelis Temminck & Schlegel and Sicyopterus japonicus (Tanaka) along the course of a temperate river

Densities of two sympatric amphidromous grazing fishes, Plecoglossus altivelis and Sicyopterus japonicus, and gross primary production of benthic algae were investigated at the second, third and fourth-order sites located, respectively, 7.0, 6.7 and 3.6 km from the mouth of the Choshi River, Japan. Gross primary production estimated by multiplying gross photosynthetic rates by hours of insolation on the streambed increased downstream with decreasing canopy cover by valley walls and trees standing along the river. Density of P. altivelis was greatest at the fourth-order site with the higher primary production, while the distribution pattern of S. japonicus differed between adult (> ca. 7 cm in total length) and young individuals. Density of adult of S. japonicus was greater at the third-order site, although the young were more abundant at the fourth-order site. Aggressive acts against conspecifics and different species were observed only by adults of S. japonicus and most frequently observed at the third-order site. The upstream shift in the distribution of the adults of S. japonicus would mitigate interference competition with P. altivelis and contribute to their coexistence in the river. Handling editor: K. Martens     

香鱼主要组织相容性复合体MHCIIB的分子克隆及其在鳗弧菌感染下的功能鉴定

主要组织相容性复合体(major histocompatibility complex classⅡ, MHC Ⅱ)在脊椎动物免疫反应中发挥重要作用。本研究从香鱼(Plecoglossus altivelis) 单核/巨噬细胞(monocytes/ macrophages,MO/MФ)转录组中获得了MHC II基因β链(PaMHCIIB) cDNA序列。PaMHCIIB由920个核苷酸组成,包含一个大的开放阅读框,编码251个氨基酸,预测分子质量为28.23 kD。氨基酸序列分析表明,PaMHCIIB具有MHC IIB的典型特征,主要包括信号肽、2个胞外区和1个保守的connecting peptide/transmembrane/cytoplasmic (CP/TM/CYT)结构域,与北极红点鲑(Salvelinus alpinus) MHC IIB同源性最高,为65.04%;系统发育分析表明,PaMHCIIB与北极红点鲑MHC IIB进化相关性最高。实时荧光定量PCR(quantitative real-time PCR, qPCR)结果显示,PaMHCIIB mRNA主要在香鱼鳃、肠和脾中表达;鳗弧菌感染(Vibrio anguillarum)后香鱼肝中PaMHCIIB mRNA在感染后12 h(hours post infection, hpi)时上调显著,在24 hpi达到峰值,为对照组的3.18倍,脾、头肾、肠和鳃中PaMHCIIB mRNA均在4 hpi时上调显著,分别在4、8、24和12 hpi时达到峰值,分别为对照组的85.18、2.06、4.21和6.81倍(P<0.05)。香鱼MO/MФ经鳗弧菌感染后,PaMHCIIB mRNA的表达水平在4 hpi时上调显著,在12 hpi时达到峰值,为对照组的3.35倍(P<0.05)。原核表达了PaMHCIIB胞外区并制备其抗体。Western 印迹分析结果表明,香鱼MO/MФ中PaMHCIIB具有N糖基化修饰,且鳗弧菌感染后其蛋白表达水平在12 hpi时显著增加,在24 hpi时达到峰值,为对照组的3.19倍(P<0.05)。抗体封闭PaMHCIIB后,香鱼MO/MФ吞噬活性被抑制,为对照组的0.28倍(P<0.05),而且鳗弧菌诱导的4个细胞因子TNF-α、IL-1β、IL-10和TGF-β表达均受到抑制,均在8 hpi时被抑制最明显,表达量分别为对照组的0.23、0.41、0.51和0.20倍(P<0.05)。本研究结果揭示PaMHCIIB可能参与香鱼MO/MФ抵抗病原菌感染的免疫防御。     

闽浙地区香鱼线粒体Cyt b基因和D-loop区序列多态性分析

对浙江瑞安、福建宁德、福建东张水库3个地理群体共31例香鱼(Plecoglossus altivelis)的线粒体细胞色素b(Cyt b)基因和线粒体D-loop区序列进行了PCR扩增、序列测定、核苷酸组成和多态性分析。Cyt b基因中, A、T、C和G 4种核苷酸的比例分别为19.72%、29.71%、32.25%和18.32%, A + T含量为49.43%, G + C含量为50.57%。D-loop区序列中, A、T、C和G 4种核苷酸的比例分别为29.99%、29.29%、23.80%和16.92%, A + T含量为59.28%, G + C含量为40.72%。在长度为1 141 bp的Cyt b基因序列中, 仅存在1个变异位点, 核苷酸多样性指数(π值)为0.00028, 31个样本中仅出现两种单倍型; 857 bp长的D-loop区序列中, 仅存在5个变异位点, 核苷酸多样性指数(π值)为0.00199, 仅出现5种单倍型。这表明闽浙地区香鱼的遗传多样性水平很低, 应当加大对香鱼的保护力度。     

Molecular cloning and characterization of rhodopsin in a teleost (Plecoglossus altivelis,Osmeridae)

Amplified fragments encoding exon-4 of opsin cDNAs were cloned from the retina of landlocked ayu (Plecoglossus altivelis), and sequenced. On the basis of the sequence homology to previously characterized fish visual pigments, one clone was identified as rod opsin (AYU-Rh), and two clones as green (AYU-G1, -G2), one as red (AYU-R) and two as ultraviolet (AYU-UV1, -UV2) cone opsins. The 335-amino acid sequence deduced from the full-length cDNA of AYU-Rh included residues highly conserved in vertebrate rhodopsins and showed the greatest degree (88%) of similarity with salmon rhodopsin. Southern blotting analysis indicated that ayu possess two rhodopsin genes, one encoding visual rhodopsin (AYU-Rh) and the other non-visual extra-ocular rhodopsin (AYU-ExoRh). RT-PCR experiments revealed that AYU-Rh was expressed in the retina and AYU-ExoRh in the pineal gland. In situ hybridization experiments showed that the mRNA of AYU-Rh was localized only in rod cells not in cone cells. Lake and river type landlocked ayu having different amounts of retinal and 3-hydroxyretinal in their retinas expressed a rhodopsin (AYU-Rh) of identical amino acid sequence.     

Existence of an extra-retinal and extra-pineal photoreceptive organ that regulates photoperiodism in gonadal development of an Osmerid teleost, ayu (Plecoglossus altivelis)

Ayu (Plecoglossus altivelis) is an Osmerid teleost whose gonadal development exhibits clear photoperiodism: it is stimulated and prevented under short and long photoperiod, respectively. However, the photoreceptor organ involved in this process remains to be identified. In the present study, we examined whether gonads of ophthalmectomized (Ex) and pinealectomized (Px) ayu respond to short photoperiod to test whether photoreceptors other than lateral eyes and pineal complex are involved in the photoperiodic response of gonadal development. Gonadosomatic index (GSI) and plasma levels of sex steroids (testosterone and 11-ketotestosterone for males and testosterone and estradiol 17-beta for females) were significantly increased in the Ex+Px ayu kept under short photoperiod in both males and females as compared with the initial control. On the other hand, there were no significant increases in GSI and sex steroids in the Ex+Px ayu kept under long photoperiod. Histological observation of gonads in the Ex+Px ayu revealed that oocytes undergoing final maturation in females and proliferation of germ cells in males were observed under short photoperiod but not under long photoperiod. These results indicate that extra-retinal and extra-pineal photoreceptive organ regulates photoperiodic gonadal development in this species.     

Differential gene flow of mitochondrial and nuclear DNA markers among chromosomal races of Australian morabine grasshoppers (Vandiemenella, viatica species group)

Recent theoretical developments have led to a renewed interest in the potential role of chromosomal rearrangements in speciation. Australian morabine grasshoppers (genus Vandiemenella, viatica species group) provide an excellent study system to test this potential role of chromosomal rearrangements because they show extensive chromosomal variation and formed the basis of a classic chromosomal speciation model. There are three chromosomal races, viatica19, viatica17, and P24(XY), on Kangaroo Island, South Australia, forming five parapatric populations with four putative contact zones among them. We investigate the extent to which chromosomal variation among these populations may be associated with barriers to gene flow. Population genetic and phylogeographical analyses using 15 variable allozyme loci and the elongation factor-1alpha (EF-1alpha) gene indicate that the three races represent genetically distinct taxa. In contrast, analyses of the mitochondrial cytochrome c oxidase subunit I (COI) gene show the presence of three distinctive and geographically localized groups that do not correspond with the distribution of the chromosomal races. These discordant population genetic patterns are likely to result from introgressive hybridization between the chromosomal races and range expansions/contractions. Overall, these results suggest that reduction of nuclear gene flow may be associated with chromosomal variation, or underlying genetic variation linked with chromosomal variation, whereas mitochondrial gene flow appears to be independent of this variation in these morabine grasshoppers. The identification of an intact contact zone between P24(XY) and viatica17 offers considerable potential for further investigation of molecular mechanisms that maintain distinct nuclear genomes among the chromosomal races.     

Hierarchical analysis of population genetic structure in the monogynous ant Cataglyphis cursor using microsatellite and mitochondrial DNA markers

Despite having winged queens, female dispersal in the monogynous ant Cataglyphis cursor is likely to be restricted because colonies reproduce by fission. We investigated the pattern of population genetic structure of this species using eight microsatellite markers and a mitochondrial DNA (mtDNA) sequence, in order to examine the extent of female and nuclear gene flow in two types of habitat. Sampling was carried out at a large spatial scale (16 sites from 2.5 to 120 km apart) as well as at a fine spatial scale (two 4.5-km transects, one in each habitat type). The strong spatial clustering of mtDNA observed at the fine spatial scale strongly supported a restricted effective female dispersal. In agreement, patterns of the mtDNA haplotypes observed at large and fine spatial scales suggested that new sites are colonized by nearby sites. Isolation by distance and significant nuclear genetic structure have been detected at all the spatial scales investigated. The level of local genetic differentiation for mitochondrial marker was 15 times higher than for the nuclear markers, suggesting differences in dispersal pattern between the two sexes. However, male gene flow was not sufficient to prevent significant nuclear genetic differentiation even at short distances (500 m). Isolation-by-distance patterns differed between the two habitat types, with a linear decrease of genetic similarities with distance observed only in the more continuous of the two habitats. Finally, despite these low dispersal capacities and the potential use of parthenogenesis to produce new queens, no signs of reduction of nuclear genetic diversity was detected in C. cursor populations.     

Distribution of mitochondrial DNA variation in lake sturgeon (Adpenser fulvescens) from the Moose River basin, Ontario, Canada

We analysed mitochondrial DNA (mtDNA) variation of lake sturgeon ( Adpenser fulvescens ) from the Moose River basin. Our objective was to address various proximate and ultimate factors which may influence the distribution of lake sturgeon mtDNA haplotype lineages in this watershed. The lake sturgeon sampled were characterized by only two mtDNA hapiotypes based on a restriction fragment length polymorphism analysis with 40 restriction endonucleases and direct sequencing of 275 nucleotides in the mtDNA control region. We detected no heterogeneity in the mtDNA haplotype frequencies of lake sturgeon captured from different sites within rivers including those separated by major hydroelectric installations. However, lake sturgeon from one tributary had significantly different haplotype frequencies than those from other tributaries suggesting that they composed a discrete genetic stock. These results suggest that gene flow among most sites is significant and is an important factor affecting the distribution of mtDNA variation in this species. The genetic structuring and diversity are discussed in relation to lake sturgeon management and conservation.     

Phylogeographic structure in mitochondrial DNA of a South-east Asian freshwater fish, Hemibagrus nemurus (Siluroidei; Bagridae) and Pleistocene sea-level changes on the Sunda shelf

We postulated that the biogeographical history of South-east Asia contributed to extensive admixture during Pleistocene low sea levels of genetic groups of an obligate freshwater fish (the river catfish, Hemibagrus nemurus) isolated during periods of high sea levels. During Pleistocene glacial maxima, the sea level was lower than at present and the islands of the Sunda shelf (Sumatra, Borneo and Java) and the Asian mainland were connected by lowlands traversed by rivers. Restriction fragment length polymorphisms in mitochondrial DNA were documented for 140 putative H. nemurus analysed from 13 sampling sites resulting in the definition of 35 haplotypes. The high level of haplotype differentiation (mean P × 100 = 2.22, SD = 1.33) indicates that the subdivision of the ancestral H. netnurus group was extensive and probably occurred early in the Pleistocene. The occurrence of some genetically divergent groups of the H. netnurus complex occurring in sympatry in widely separated locations supports the proposition that low sea levels aided the dispersion and mingling of genetic groups. Based on both genetic and morphological evidence, the main H. nemurus line gave rise to three regional groups: (1) a morphologically distinct ‘Indochinese’ group composed of two mtDNA clades overlapping in east peninsular Malaysia; (2) a ‘Sundaic’ group composed of various lineages of differing morphology and genetic identity; (3) a genetically distinct ‘Sarawak’ group in west Borneo, similar in morphology to the ‘Sundaic’ and ‘Indochinese’ groups, but including a small, golden colour morph as a distinct dade. The morphologically similar Sundaic forms from west Java, Sumatra and west Borneo show some degree of genetic divergence, but their phylogenetic relationships are poorly resolved. The most genetically and morphologically distinct Sundaic dade, assigned to H. hoevenii, colonized the Kapuas river (west Borneo), east Sumatra and south peninsular Malaysia. Contrary to our original hypothesis and present biogeographical theory, little exchange of genetic groups has apparently occurred between the mainland and the Sunda Islands during recent glaciations.     

Differentiation and levels of genetic variation in northern European lynx (Lynx lynx) populations revealed by microsatellites and mitochondrial DNA analysis

The European lynx (Lynx lynx) hasexperienced significant decline in populationnumbers over large parts of its formerdistribution area in central and northernEurope. In Scandinavia (Sweden and Norway), thespecies has been subject to intense hunting and inthe early 20th century the population size mayhave been as low as about 100 animals. Duringthe rest of the century there have beenalternating periods of restricted hunting andtotal protection. Future management of theScandinavian lynx population will requireinsight into what effects demographicbottlenecks may have had on genetic variabilityand structure. For this purpose, 276 lynxesfrom Sweden, Norway, Finland, Estonia andLatvia were analysed for polymorphism at 11feline microsatellite loci and at themitochondrial DNA (mtDNA) control region.Scandinavian lynxes were found to be fixed fora single mtDNA haplotype, while this and threeadditional haplotypes were seen in Finland andthe Baltic States (Estonia and Latvia); thehaplotypes were all very similar, onlydiffering at 1–4 sites within a 700 bp regionsequenced. Microsatellite variability wasmoderate (H_e = 0.51–0.62) with lowerheterozygosity and fewer alleles in Scandinaviathan in Finland and the Baltic States together,though significant so only for the latter.Heterozygosity data in Scandinavia wereconsistent with a recent population bottleneck.Various analyses (e.g. F_st, individual-basedtree, assignment test) revealed distinctgenetic differentiation between Scandinavianlynxes and animals from Finland and the BalticStates. Some structure was evident withinScandinavia as well, suggesting an isolation bydistance. The observed partition of geneticvariability between Scandinavia and the easterncountries thereof indicates that lynxpopulations from the two regions may need to beseparately managed. We discuss what factors canhave contributed to the population geneticstructure seen in northern European lynxpopulations of today.     

Nuclear and mitochondrial DNA reveal contrasting evolutionary processes in populations of deer mice (Peromyscus maniculatus)

We investigated a major geographic break in mitochondrial DNA (mtDNA) haplotypes in deer mice, Peromyscus maniculatus, by analysing spatial variation in a 491‐bp fragment of the mtDNA control region from 455 samples distributed across a north–south transect of 2000 km in Western North America. To determine whether the mtDNA break was reflected in the nuclear genome, we then compared spatial variation in 13 nuclear microsatellites of 95 individuals surrounding the mtDNA break. Using a canonical correlation analysis we found that nuclear genomic variation was not correlated with mtDNA differentiation. The contrasting patterns of variation in mtDNA and nuclear DNA are consistent with a hypothesis of historic genetic drift that occurred in isolated refugia combined with recent gene flow between the formerly isolated refugial populations. A Mantel test of genetic vs. geographic distance revealed that recent gene flow between deer mouse populations has been high. We conclude that past vicariant events associated with Pleistocene climate changes together with recent gene flow have created the observed intra‐specific cytonuclear discordance in Western North America.     

Population genetics of Scirtothrips perseae: tracing the origin of a recently introduced exotic pest of Californian avocado orchards, using mitochondrial and microsatellite DNA markers

The Californian avocado industry has recently been impacted by the establishment of three exotic arthropod pests that are native to Mexico and Central America. Establishment of all three pests is thought to have resulted from illegal movement of host plants (avocado) into California. To identify likely sources and routes of entry of such pest invasions, we examined the population genetics of the most recent invader, avocado thrips [Scirtothrips perseae Nakahara (Thysanoptera: Thripidae)], using mitochondrial DNA (mtDNA) and microsatellite markers. The mtDNA sequences revealed three geographically distinct and divergent lineages, of which the mtDNA haplotypes of Californian individuals were most closely related to populations in the center of the pest's native range. Analysis of allele frequencies at four microsatellite loci added resolution, indicating Coatepec Harinas, Mexico, as the most likely source of the Californian population. Statistically, we did not detect any bottleneck in population size associated with the invasion of California. However, estimates of the effective population size of the invading population suggest that a severe bottleneck occurred, indicating that the quantity of host‐plant material entering California was small. Our findings implicate Coatepec Harinas, a large avocado germplasm and breeding center, as the most likely source of the introduced Californian population of S. perseae and as a likely source of previous and future avocado pest introductions. Efforts to identify natural enemies of S. perseae for biological control should focus on Coatepec Harinas and immediate surrounds. Moreover, identification of the source of invasive pests enables the establishment and enforcement of plant quarantine and free‐trade protocols.     

Analysis of mitochondrial DNA: Taxonomic and phylogenetic relationships in two fish taxa (Pisces: Mugilidae and Cyprinidae)

To solve some systematic questions as well as to study genetic variability and evolutionary relationships in two groups of fish belonging to the Mugilid (Mugilidae) and Cyprinid (Cyprinidae) families, we have used restriction fragment length polymorphism analysis of mitochondrial DNA (mtDNA) fragments amplified in polymerase chain reaction. The analysis of three mtDNA fragments of 7220 bp total length of six Mugilid species has shown that Mediterranean Liza aurata, L. ramada, L. saliens, and Chelon labrosus form a common cluster, L. aurata and C. labrosus being the closest relatives, whereas L. haematocheilus (syn. C. haematocheilus) of the Sea of Japan forms a sister group to the Mediterranean cluster. It was found that Chelon and Liza genera are paraphyletic, and therefore their division into two genera is unnatural and they should be synonymized. According to priority, Liza species should be ascribed to Chelon genus. Mugil cephalus is the most distant compared to the rest of the species studied. The level of genetic divergence between allopatric samples of M. cephalus from the Sea of Japan and the Mediterranean Sea has proved to be very high—4.5% of nucleotide substitutions. The analysis of four mtDNA fragments of 9340 bp total length of six Cyprinid species has shown that L. waleckii is the most genetically distant. Pseudaspius leptocephalus is a sister group to Tribolodon species. All Tribolodon species form a common cluster with T. sachalinensis as a root. The remaining species form two branches, one of which includes T. nakamurai and T. brandtii, another one combines T. hakonensis and a new form of Tribolodon revealed that is close to T. hakonensis by its mtDNA (2.4% of nucleotide substitutions). This new form might be an independent species. Published in Russian in Biokhimiya, 2007, Vol. 72, No. 12, pp. 1651–1658.