Isolation and development of microsatellite markers for the Japanese dormouse, Glirulus japonicus

Eight microsatellite markers were developed for the Japanese dormouse (Glirulus japonicus), a natural monument and near‐threatened species in Japan. The markers amplify in individuals from all of the mitochondrial lineages detected in a previous study. Numerous polymorphisms were detected in specimens from a local population in central Honshu (11–21 alleles per locus; n = 31) and from the entire distribution range of the species (19–41 alleles per locus; n = 152). These microsatellites will be useful in conservation genetic studies of G. japonicus.     

The hitchhiker's guide to becoming invasive: exotic mosquitoes spread across a US state by human transport not autonomous flight

Not all exotic species establish and expand aggressively (i.e. become invasive). As potential vectors of disease agents, invasive mosquitoes can have considerable impact on public health, livestock and wildlife; therefore, understanding the species characteristics and ecological circumstances promoting their invasiveness is important. The mosquito Aedes japonicus japonicus, originally from north‐east Asia, was introduced at least two separate times to the north‐eastern USA, as surmised from the initial existence of two populations with distinct nuclear and mitochondrial genetic signatures that later intermixed. Since these original introductions in the late 1990s, Ae. j. japonicus has expanded across 31 US states, two Canadian provinces and five European countries. Although some of the expanded range was due to other independent introductions, to understand what drove the postintroduction expansion of Ae. j. japonicus within the north‐eastern USA, we performed a high‐resolution landscape genetic analysis of 461 specimens collected across Virginia, a state south of the original introductions. All specimens were genotyped at seven pre‐optimized microsatellite loci, and a subsample was sequenced at one mitochondrial locus. We concluded that throughout Virginia this species has primarily expanded in association with humans: genetic distance and distance along roads remained correlated after controlling for geographic distance, and proximity to I‐95, a major interstate highway, strongly predicted nuclear ancestry. In contrast, there was very limited evidence of diffusion even at distances potentially suitable for autonomous mosquito flight. This implies that its association with humans (rather than innate species characteristics) is the single most important determinant of invasiveness in this mosquito.     

Development of microsatellite markers for an extremely limited distributed rare diving beetle species,Acilius kishii,and a widely distributed species,Acilius japonicus (Coleoptera: Dytiscidae)

Acilius kishii Nakane, 1963 (Coleoptera: Dytiscidae) is an endangered diving beetle species distributed in only one location, Lake Yashaga‐Ike, Honshu Island, Japan. Acilius japonicus, which is related to A. kishii, is distributed widely in northern Honshu Island and Hokkaido Island in Japan. In this study, we identified 14 microsatellite loci for A. kishii and A. japonicus, including both polymorphic and monomorphic loci, using the next‐generation sequencing method. We observed that 5 and 10 loci showed polymorphisms in 31 and 32 individuals of A. kishii and A. japonicus, respectively. The observed and expected heterozygosities were 0.00–1.00 and 0.00–0.74, respectively. These microsatellite loci could be useful for future conservation genetic studies, including monitoring of genetic diversity and extinction risk of A. kishii.     

Identification and characterization of microsatellite markers derived from expressed sequence tags of the sea cucumber Stichopus japonicus

Eleven polymorphic microsatellite markers were identified in expressed sequence tags generated from Stichopus japonicus cDNA libraries. The numbers of alleles ranged from three to 10, and the expected and observed heterozygosities ranged from 0.378 to 0.870 and from 0.077 to 0.690, respectively. Significant deviations from Hardy–Weinberg expectations were observed at eight loci due to homozygote excess, suggesting the widespread occurrence of null alleles. The microsatellite markers will be useful for examining genetic population structure, parentage analysis and mapping studies of S. japonicus.     

Characterization of expressed sequence tag‐derived microsatellites from the kuruma prawn,Marsupenaeus japonicus

The kuruma prawn, Marsupenaeus japonicus, is a commercially important benthic marine crustacean in East Asia. Understanding the species’ population structures will be very important for its proper stock assessment and management strategy. Herein, 13 polymorphic microsatellite loci originating from expressed sequence tag libraries of M. japonicus were isolated and characterized. Number of alleles per locus ranged from two to 15, observed and expected heterozygosities ranged from 0.250 to 0.906 and from 0.310 to 0.932, respectively. The adequate level of variability within the population renders these microsatellite loci ideal markers for population genetic analysis of M. japonicus.     

Isolation and characterization of 22 polymorphic microsatellite DNA markers of Japanese sea bass (Laterolabrax japonicus)

Japanese sea bass (Laterolabrax japonicus) inhabits the coast of East Asia and is cage‐cultured currently in China as well. Twenty‐two microsatellite DNA markers were developed and used to type 35 individuals collected along the Chinese coast. The number of alleles per locus ranged from three to 23. The expected heterozygosity ranged from 0.111 to 0.938, while the observed heterozygosity ranged from 0.114 to 0.914. All 22 loci are neutral and independent of each other; two deviated significantly from Hardy–Weinberg equilibrium. These microsatellite DNA markers are moderately informative, which will certainly facilitate the management and exploitation of the genetic resource of L. japonicus.     

First record and spreading of the invasive mosquito Aedes japonicus japonicus (Theobald, 1901) in Croatia

Aedes (Hulecoeteomyia) japonicus japonicus (Theobald, 1901) has recently established across North America and Central Europe. A 3‐year survey was conducted in northwestern Croatian regions from 2013 to 2015 using mosquito ovitraps at possible points of entry and house yards, occasionally complemented by larval collections from cemetery vases. In the first year, the survey investigated the county bordering Slovenia, where the first detection of Ae. j. japonicus had taken place on 28 August 2013. During the next 2 years, Ae. j. japonicus was detected in this area from early May until late October. In 2015, several counties further to the east were included in the survey, leading to the detection of Ae. j. japonicus approximately 100 km eastward from the initially surveyed region. Given a moderate continental climate and homogeneous climatic conditions in this part of Europe, the eastward spread of Ae. j. japonicus can be expected to continue.     

Development and characterization of 45 novel microsatellite markers for sea cucumber (Apostichopus japonicus)

Simple sequence repeat‐enriched library screening and expressed sequence tag database mining were adopted to develop microsatellite markers for sea cucumber (Apostichopus japonicus). Eighty‐three microsatellite loci were selected for polymorphism assessment using 48 individuals. The results showed that 45 novel loci were polymorphic. The number of alleles ranged from two to 16, and the values of observed and expected heterozygosities varied from 0 to 0.9375 and from 0.1135 to 0.9674, respectively. No significant linkage disequilibrium between pairs of loci was found and 26 loci conformed to the Hardy–Weinberg equilibrium. These markers are therefore a potential tool for studies in the population structure and linkage map construction for A. japonicus.     

Large Scale Structural Analysis of cDNAs in the Model Legume, Lotus japonicus

Lotus japonicus possesses certain characteristics suited to molecular genetic and genomic analyses and has been adopted as a model species in the study of legume plants. To make a catalogue of genes expressed in L. japonicus and understand biological processes specific to legume plants, large scale EST analyses have been performed. To date, more than 26,000 EST sequences of L. japonicus have been deposited in the public databases. These sequences were developed by five laboratories using different organs. In this review, information obtained from two EST projects carried out in Japan is presented. Some 7137 non-redundant EST groups from young plants and 718 groups from flower buds were generated. A similarity search revealed that homologues of nodulin genes in other legume plants, as well as genes related to secondary metabolism, seed development and the reproductive process, were included in the EST collection, indicating the usefulness of the EST clones in the study of biological phenomena distinctive to legume plant species. Received 23 August 2000/ Accepted in revised form 22 September 2000     

Life history,distribution and molecular phylogenetics of the Upward-Mouth Spikefish Atrophacanthus japonicus (Teleostei: Tetraodontiformes: Triacanthodidae)

Ninety-six juvenile specimens (37–54 mm standard length; L_S) of the rarely collected Upward-Mouth Spikefish Atrophacanthus japonicus (Triacanthodidae) were obtained from the stomachs of three Yellowfin Tuna Thunnus albacares collected off Guam in the Mariana Islands in the central Pacific Ocean. These specimens extend the range of A. japonicus eastward into Oceania. We review the systematic characters of the monotypic genus Atrophacanthus and present colour photographs of freshly collected specimens. The diet of the juvenile specimens of A. japonicus consisted of thecosome pteropods and foraminiferans. We present a range map of A. japonicus based on all known specimens and show that specimen size is related to whether specimens were collected in the pelagic zone or on the bottom. Our results support that, compared to all other Triacanthodidae, A. japonicus has an unusually extended pelagic larval and juvenile period, up to 54 mm L_S, before settling to the bottom as adults. Lastly, we provide a multilocus phylogeny addressing the phylogenetic placement of Atrophacanthus based on eight of 11 triacanthodid genera and six genetic markers. Our results reveal that Atrophacanthus is the sister group of Macrorhamphosodes and they provide new insights about the evolutionary history of the family.     

Genetic diversity in the US ex situ populations of the endangered Puerto Rican Boa,Chilabothrus inornatus

The Puerto Rican Boa (Chilabothrus inornatus) was placed on the US Endangered Species List in 1970. Progress has been made since to clarify the recovery status of this species, though the design of a new recovery plan must include information regarding genetic variation within and among populations of this species. While measures of genetic diversity in wild populations of this species are finally becoming available, relative genetic diversity represented in ex situ populations is unknown, which hampers efforts to develop an ex situ species management plan. Here, we provide an analysis of genetic diversity in US public and private collections (zoos and breeders) using mitochondrial sequence data and five highly polymorphic nuclear microsatellite loci. We analyzed 50 boas from the US ex situ population and determined overall genetic diversity and relatedness among these individuals. We then compared these data to mitochondrial and microsatellite data obtained from 176 individuals from wild populations across the native range of the species. We found little inbreeding and a large amount of retained genetic diversity in the US ex situ population of C. inornatus relative to wild populations. Genetic diversity in the ex situ population is similar to that found in wild populations. Ours is only the second explicit attempt to characterize genetic diversity at the molecular level in ex situ populations of boid snakes. We anticipate that these results will inform current breeding strategies as well as offer additional information that will facilitate the continuation of ex situ conservation breeding or management in boas.     

Novel Jefferson salamander,Ambystoma jeffersonianum,microsatellite DNA markers detect population structure and hybrid complexes

Twenty polymorphic microsatellite DNA markers were isolated and characterized in Ambystoma jeffersonianum collected from three vernal pools in the mid‐Atlantic region of the U.S. These markers revealed a high degree of genetic diversity (7–23 alleles per locus), heterozygosity (46.7% to 100%), and allelic heterogeneity (96% of comparisons were statistically significant). Genetic distances were greatest in comparisons between collections, intermediate within collections, and least among sibling pairs. Six markers were trisomic in A. jeffersonianum‐A. laterale hybrids. These microsatellite DNA loci should allow delineation of genetic structure within and among populations of the diploid A. jeffersonianum and provide an effective method for identification of triploid hybrid individuals.     

Unexpected Patterns of Admixture in German Populations of Aedes japonicus japonicus (Diptera: Culicidae) Underscore the Importance of Human Intervention

The mosquito Aedes japonicus japonicus, originally restricted to temperate East Asia, is now widespread in North America and more recently has become established in Europe. To ascertain the putative number of separate introductions to Europe and examine patterns of expansion we analyzed the genetic makeup of Ae. j. japonicus populations from five cemeteries in North Rhine-Westphalia and Rhineland-Palatinate, two western German federal states, as well as of specimens from populations in Belgium, Switzerland, and Austria/Slovenia. To do so, we genotyped individual specimens at seven pre-existing polymorphic microsatellite loci and sequenced part of the nad4 mitochondrial locus. We found evidence of two different genotypic signatures associated with different nad4 mitochondrial haplotypes, indicating at least two genetically differentiated populations of Ae. j. japonicus in Europe (i.e. two distinct genotypes). Belgian, Swiss, and Austrian/Slovenian populations all share the same genotypic signature although they have become differentiated since isolation. Contrary to expectations, the German Ae. j. japonicus are not closely related to those in Belgium which are geographically nearest but are also highly inbred. German populations have a unique genotype but also evidence of mixing between the two genotypes. Also unexpectedly, the populations closest to the center of the German infestation had the highest levels of admixture indicating that separate introductions did not expand and merge but instead their expansion was driven by punctuated human-mediated transport. Critically, the resulting admixed populations have higher genetic diversity and appear invasive as indicated by their increased abundance and recent spread across western Germany.     

Aquatic thermal conditions predict the presence of native and invasive rock pool Aedes (Diptera: Culicidae) in the southern Appalachians,U.S.A.

The native rock pool mosquito, Aedes atropalpus (Coquillett), and the invasive Aedes japonicus (Theobald) have been found in many types of artificial and natural containers throughout North America. Little is known about the ecology of these two species in habitats where they co‐occur, although multiple investigators have reported the decline of the native species concurrent with the introduction and spread of the invasive species. Here we report the results of riverine rock pool collections (n=503) in the southern Appalachian Mountains between 2009‐2015. Surface water temperatures strongly predicted the presence of each species across a broad range of observed temperatures (11‐39.8° C). For every unit of increase in temperature (°C) the odds of collecting Ae. atropalpus larvae increased by 0.34 while the odds of collecting Ae. japonicus larvae decreased by 0.28. No Ae. japonicus larvae or pupae were collected at temperatures greater than 36° C; however, immature Ae. atropalpus were found in rock pools with temperatures up to 39.8° C. In contrast, Ae. japonicus were highly abundant in cooler rock pools (<17° C) where Ae. atropalpus were infrequent or absent. Our findings suggest that in spite of the successful invasion by Ae. japonicus, Ae. atropalpus remains well established in the southern Appalachian Mountains. Given the strong correlation of temperature with the presence of the two species and the contrasting absence of each species at observed temperature extremes, the role of thermal conditions should be carefully explored in the context of other ecological factors likely influencing the range and abundance of these mosquitoes.     

An optimized microsatellite marker set for detection of Metarhizium anisopliae genotype diversity on field and regional scales

Thirty-three Metarhizium anisopliae isolates sampled across Switzerland as well as 35 and 36 M. anisopliae isolates sampled from two field sites were assembled in three isolate collections. All isolates were analyzed using 27 newly developed and 14 previously published microsatellite markers. The 41 markers allowed for detection of 25 genotypes in the Swiss collection while 30 and 11 genotypes were detected in the two field collections. This indicated high genetic diversity on a regional as well as on a field scale. In order to improve genotyping efficiency, an optimized marker set, which allows discrimination of a large number of genotypes with as few markers as possible was developed. The optimized marker set consisted of 16 common markers, which provided resolution close to maximal resolution in all three collections (91–93 %). The results demonstrated that optimized marker sets have to be validated before large scale application to previously unassessed collections in order to avoid suboptimal resolution. This genetic tool will be valuable for analyses of genetic population structure of M. anisopliae in different habitats on a regional as well as on a field scale.     

Development and characterization of 33 polymorphic microsatellite markers in Trachurus japonicus by SLAF-seq technology

Trachurus japonicus is an economically important fish in the northwestern Pacific Ocean. However, its resources have declined seriously and there is an urgent need for a wide-range of investigations of the existing genetic resources. This requires a large number of diverse molecular markers with high discriminating power. In this study, we identified 43,264 perfect SSRs in T. japonicus genome using SLAF-seq technology. Of these, we randomly selected 106 SSRs (tri-nucleotide to hexa-nucleotide) to test for polymorphism. Eventually, we successfully developed a total of 33 loci including 8 tri-nucleotide and 25 long repeat motifs (tetra-nucleotide to hexa-nucleotide). The number of alleles (Na) of these loci ranged from 4 to 24 (mean 12.6). The observed heterozygosity (Ho) and expected heterozygosity (He) varied from 0.258 to 0.969 (mean 0.723) and from 0.452 to 0.962 (mean 0.827), respectively. All loci except TJ6-7 were highly informative (PIC > 0.5). These results showed that the shortlisted 33 loci exhibited moderate to relatively high genetic diversity, of which 18 were regarded as highly polymorphic and well-resolved. In summary, these diverse and potential microsatellites detected in our study provide substantial genetic basis for the screening of polymorphic SSR markers of T. japonicus and also provide a powerful tool to perform further studies on the genetic resource assessment and conservation of T. japonicus.     

Comparative studies on the seasonal occurrence,nymphal development and food menu in two giant water bugs,Diplonychus japonicus vuillefroy andDiplonychus major esaki (Hemiptera: Belostomatidae)

We compared the seasonal changes in population density and nymphal development at different water temperatures and under different food conditions between two giant water bugs, Diplonychus japonicus and D. major, in Okayama, Japan. D. japonicus produced 1–2 generations a year, while D. major was strictly inivoltine. The developmental velocity was higher in D. japonicus than in D. major. The thermal constant of D. japonicus was less than that of D. major. These results suggest that D. japonicus is adapted, to higher water temperature than D. major. In the field, D. japonicus preyed predominately on Lymnaeidae and Physidae snails, while D. major preyed on aquatic insects such as dragonfly nymphs.     

Invasion of the Hawaiian Islands by a parasite infecting imperiled stream fishes

Points of origin and pathways of spread are often poorly understood for introduced parasites that drive disease emergence in imperiled native species. Co‐introduction of parasites with non‐native hosts is of particular concern in remote areas like the Hawaiian Islands, where the introduced nematode Camallanus cotti has become the most prevalent parasite of at‐risk native stream fishes. In this study, we evaluated the prevailing hypothesis that C. cotti entered the Hawaiian Islands with poeciliid fishes from the Americas, and spread by translocation of poeciliid hosts across the archipelago for mosquito control. We also considered the alternative hypothesis of multiple independent co‐introductions with host fishes originating from Asia. We inferred conduits of introduction and spread of C. cotti across the archipelago from geographic patterns of mtDNA sequence variation and allelic variation across 11 newly developed microsatellite markers. The distribution of haplotypes suggests that C. cotti spread across the archipelago following an initial introduction on O'ahu. Approximate Bayesian Computation modeling and allelic variation also indicate that O'ahu is the most likely location of introduction, from which C. cotti dispersed to Maui followed by spread to the other islands in the archipelago. Evidence of significant genetic structure across islands indicates that contemporary dispersal is limited. Our findings parallel historical records of non‐native poeciliid introductions and suggest that remediating invasion hotspots could reduce the risk of infection in native stream fishes, which illustrates how inferences on parasite co‐introductions can improve conservation efforts by guiding responses to emerging infectious disease in species of concern.