IDENTIFICATION AND TOXICITY OF ALEXANDRIUM TAMARENSE (DINOPHYCEAE) IN SCOTTISH WATERS1

Contamination of shellfish with paralytic shellfish poisoning (PSP) toxins produced by Alexandrium species poses a potential threat to the sustainability of the Scottish aquaculture industry. Routine LM analysis of water samples from around the Scottish coast has previously identified Alexandrium (Dinophyceae) as a regular part of the spring and summer phytoplankton communities in Scottish coastal waters. In this study, Alexandrium tamarense (M. Lebour) Balech isolated from sediment and water samples was established in laboratory culture. Species identification of these isolates was confirmed using thecal plate dissections and by molecular characterization based on their LSU and, in some cases, ITS rDNA sequence. Molecular characterization and phylogenetic analysis showed the presence of two ribotypes of A. tamarense: Group I (North American ribotype) and Group III (Western European ribotype). Assessment of PSP toxin production using hydrophilic interaction liquid chromatography–tandem mass spectrometry (HILIC–MS/MS) showed that A. tamarense Group I produced a complex array of toxins (～2,000 fg STX equivalents · cell^?1) with the major toxins being C2, neosaxitoxin (NEO), saxitoxin (STX), gonyautoxin‐4 (GTX‐4), and GTX‐3, while A. tamarense Group III did not produce toxins. Historically, it was considered that all Alexandrium species occurring in Scottish waters produce potent PSP toxins. This study has highlighted the presence of both PSP toxin‐producing and benign species of A. tamarense and questions the ecological significance of this finding.     

Isolation of 18 polymorphic microsatellite loci from the North American red squirrel,Tamiasciurus hudsonicus (Sciuridae,Rodentia), and their cross‐utility in other species

We isolated 18 polymorphic microsatellite loci to be used for pedigree analysis in a wild population of North American red squirrels, Tamiasciurus hudsonicus. Allelic diversity and observed heterozygosity ranged from six to 13 and 0.39 to 0.89, respectively, in a sample of 93 individuals. Up to 13 sets of primers also amplify in other rodent species.     

Bioactive compounds of marine dinoflagellate isolates from western Greenland and their phylogenetic association within the genus Alexandrium

The diversity and biogeography of populations of the toxigenic marine dinoflagellate genus Alexandrium, a major global cause of paralytic shellfish poisoning (PSP), are represented by only a few studies based upon a low number of cultured isolates and remain poorly described in Arctic and sub-Arctic waters. Multiple clonal isolates (n = 22) of the Alexandrium tamarense species complex, and a single isolate of A. tamutum, were collected from the water column while on board an oceanographic expedition to the west coast of Greenland. After culturing of these isolates under controlled conditions, their phylogenetic affinities within the genus Alexandrium were characterized by sequence analysis of nuclear large sub-unit (LSU) rDNA. Based upon morphological and molecular genetic criteria, all isolates of the A. tamarense species complex were consistent with membership in the Group I ribotype (previously known as the North American ribotype). Phenotypic signatures were also analyzed based upon their respective profiles of paralytic shellfish toxins (PST) and allelochemical interactions against a target cryptophyte Rhodomonas, as determined by lytic potency. All isolates conforming to the A. tamarense Group I produced PST, but no toxins were detected in A. tamutum P2E2. Unusually, only carbamoyl toxins were produced among the A. tamarense Group I isolates from Greenland; sulfocarbamoyl derivatives, generally present in A. tamarense population from other locations, including the Arctic, North Pacific and North Atlantic, were absent from all isolates. Allelochemical activity, causing cell lysis of Rhodomonas, but generally being unrelated to cellular PST, was expressed by all A. tamarense isolates and also by A. tamutum, but varied widely in potency. Comparison of the genotypic (rDNA) and phenotypic (PST profile, allelochemical activity) characteristics of Greenland isolates with those of other Arctic populations reveals a complex pattern of intra-specific diversity. Estimation of diversity relationships is problematic because of the distinct patterns of divergence and lack of evidence of linkage among the alternative biomarkers and morphology. Nevertheless, such studies are necessary as the basis for constructing hindcasting scenarios and predicting changes in Alexandrium species distribution in the Arctic from the regional to the global scale.     

Development and optimization of microsatellite DNA primers for boreal owls (Aegolius funereus)

We developed 22 microsatellite loci for boreal owls (Aegolius funereus). We genotyped 275 unrelated boreal owls (Aegolius f. richardsoni) and 36 unrelated Tengmalm's owls (Aegolius f. funereus) using seven loci that were polymorphic and did not have detectable null alleles. Among North American and Scandinavian boreal owls, respectively, allelic diversity ranged from three to 11 alleles and from one to 11 alleles, observed heterozygosity ranged from 0.31 to 0.80 and from 0.00 to 0.81, and expected heterozygosity ranged from 0.28 to 0.81 and from 0.00 to 0.81. These markers appeared to amplify DNA in six other Strigidae species.     

Development of polymorphic microsatellite loci for North American river otters (Lontra canadensis) and amplification in related Mustelids

In order to complement ecological research on native and reintroduced populations of the North American river otter (Lontra canadensis), we developed 10 polymorphic microsatellite loci that would permit us to evaluate population genetic structure and conduct fecal DNA analyses. The number of alleles per locus ranged from 3 to 14 and observed heterozygosity ranged from 0.23 to 0.68. Preliminary screening revealed that primers were polymorphic in a subset of other Mustelids.     

Isolation and characterization of microsatellite markers in Phytophthora ramorum,the causal agent of sudden oak death

We describe specific primers and conditions to amplify two dinucleotide and five trinucleotide microsatellite DNA loci isolated from the oomycete Phytophthora ramorum, the causal agent of sudden oak death. The primer sets were tested on 14–30 isolates from North America and Europe. Seven of 14 loci differentiated between A1 and A2 mating types. All seven loci successfully amplified DNA isolated from infected plant tissue. Four loci may be useful for the diagnosis of P. ramorum because they do not amplify closely related Phytophthora species.     

Isolation and characterization of microsatellites in Aphidius ervi (Hymenoptera: Braconidae) and their applicability to related species

Aphidius ervi (Hymenoptera: Braconidae) is an important biological control agent of aphids, and a model organism for the study of host–parasitoid ecology and evolution. We isolated microsatellite loci from A. ervi to examine the genetic effects of its introduction from Europe to North America. We present primers for 11 microsatellites from A. ervi. We have assayed six of these loci for variability and found from two to 37 alleles. These six primer pairs were tested on 17 related species and appear broadly applicable. These microsatellite loci provide a new tool for research on A. ervi and its relatives.     

ANALYSIS OF ALEXANDRIUM (DINOPHYCEAE) SPECIES USING SEQUENCES OF THE 5.8S RIBOSOMAL DNA AND INTERNAL TRANSCRIBED SPACER REGIONS1

The 5.8S ribosomal RNA gene (rDNA) and flanking internal transcribed spacers 1 and 2 (ITS1 and ITS2) from 7 isolates of Alexandrium catenella (Wedon et Kofoid) Taylor, 13 isolates of A. tamarense (Lebour) Balech, 2 isolates of A. affine (Fukuyo et Inoue) Balech, and single isolates of A. fundyense Balech, A. insuetum Balech, and A. pseudogonyaulax (Biecheler) Horiguchi ex Yuki et Fukuyo comb. nov. from Japan, Thailand, and the United States were amplified using the polymerase chain reaction (PCR), sequenced, and subjected to phylogenetic analysis. The sequences ranged from 518 to 535 base pairs (bp) exclusive of the 18S and 28S rDNA coding regions. Sequence comparisons revealed seven divergent “ITS types” designated as follows: 1) catenella type, 2) tamarense type, 3) WKS-1 type, 4) Thai type, 5) affine type, 6) insuetum type, and 7) pseudogonyaulax type. Isolates of the tamarense type from various locations in Japan and the United States and of A. fundyense from the United States were closely related to each other and were clearly divergent from isolates of A. tamarense WKS-1 (WKS-I type) or A. tamarense CU-15 (Thai type). These latter two strains carried unique ITS types, although they were not distinguishable from isolates of the tamarense type by morphological criteria. Distance values between isolates of the tamarense type and the WKS-1 or Thai type were quite high (about 0.21 and 0.39, respectively). Seven isolates of A. catenella from Japan (catenella type) clearly diverged from the other ITS types already mentioned. Distance values between isolates of the catenella type were extremely low (<0.01), whereas distance values of ITS between the catenella type and the tamarense, WKS-1, or Thai type were 0.17, 0.18, and 0.40, respectively. Isolates of A. affine, A. insuetum, and A. pseudogonyaulax all carried unique ITS types. The ITSs of the tamarense type exhibited two distinct ITS sets, the “A gene” and the “B gene.” The two sequences occurred in a 1:1 ratio in PCR products. In contrast, the ITSs of all other isolates appeared homogeneous. Sequence comparisons also showed that the variations in the 3′ end of ITS1 (150-177 bp) were low within each ITS type but extremely high between ITS types. The number of different nucleotides among the seven Alexandrium types in this 28-bp region is more than 10. High diversity of this region may facilitate the design of DNA probes specific for each ITS type/species of Alexandrium.     

The development of eight microsatellite loci in the wild daffodil Narcissus triandrus (Amaryllidaceae)

We report microsatellite primer pairs for the wild tristylous daffodil, Narcissus triandrus (Amaryllidaceae). From enriched libraries, we identified 58 unique microsatellite loci. We designed primer pairs for 27 of these loci and screened genomic DNA from 38 to 40 adults from a single population. For eight polymorphic loci, the number of alleles per locus ranged from five to 17. As six primers also amplified loci in three other Narcissus species, including two horticultural varieties, we expect that some of these markers will be transferable to other Narcissus species.     

Probable origin and toxin profile of Alexandrium tamarense (Lebour) Balech from southern Brazil

The distribution of the toxic dinoflagellate Alexandrium tamarense Lebour has apparently expanded within the southern hemisphere during the last 2 decades. Toxic blooms of A. tamarense were recorded in Argentinean coastal waters since 1980; however, the first documented bloom in southern Brazil was in 1996. In this study, 13 strains of A. tamarense from southern Brazil were isolated and kept in culture. Phylogenetic analysis using RFLP and DNA sequences of the D1–D2 region of large subunit ribosomal DNA (rDNA) clearly indicates that Brazilian strains are most closely related to other South American strains. The strains from South America are placed firmly within a phylogenetic clade which contains strains from North America, northern Europe and northern Asia, previously called the North American clade. Possible dispersal hypotheses are discussed. The cultures were also analyzed for saxitoxin and its derivatives by high performance liquid chromatography (HPLC). The main saxitoxin groups found were the low toxicity N-sulfocarbamoyl group, C1, 2 (30–84%), followed by the high potency carbamate toxins, gonyautoxins 1, 4 (6.6–55%), gonyautoxins 2, 3 (0.3–29%), neosaxitoxin (1.4–24%) and saxitoxin (0–4.4%). The toxin composition is similar to that of other strains from South America, supporting a close relationship between A. tamarense from southern Brazil and other areas of South America. Toxicity values were variable (7.07–65.92 pg STX cell⁻¹), with the higher range falling among the most toxic values recorded for cultures of A. tamarense, indicating the significant risk for shellfish contamination and human intoxication during blooms of this species along the southern Brazilian coast.     

Microsatellite markers for two species of dampwood termites in the genus Zootermopsis (Isoptera: Termopsidae)

Dampwood termites in the genus Zootermopsis inhabit forested areas in western North America. To better understand the colony composition and breeding structure of Zootermopsis, we identified polymorphic microsatellite loci to use in population analysis. Microsatellite loci were isolated from Zootermopsis nevadensis nevadensis (Hagen); however, all primers amplified homologous loci in Zootermopsis angusticollis (Hagen) and Zootermopsis nevadensis nuttingi (Hagen). Twelve loci were polymorphic in one or more of the above subspecies and species. The number of alleles per locus ranged from one to six, with some allelic differences among subspecies and species. We are currently utilizing the microsatellite markers to investigate the population genetics of Zootermopsis.     

Isolation and characterization of microsatellite loci in the sugar beet cyst nematode Heterodera schachtii

We report the isolation of five microsatellites loci from the sugar beet cyst nematode (Heterodera schachtii). Multilocus genotypes were obtained on individual larvae freshly emerged from cysts. Allelic diversity ranged from four to 27 among the five loci. The primers were tested for cross‐species amplification in six other species of phytoparasitic nematodes of the Heterodera genus. Those molecular markers will be used to study the genetic structure of this obligatory parasite and how it is affected by the use of resistant plants.     

Isolation and characterization of microsatellite loci in the black rat snake (Elaphe obsoleta)

We obtained molecular markers useful for population level studies of the black rat snake (Elaphe obsoleta) by screening genomic DNA libraries enriched for dinucleotide, tetranucleotide, and pentanucleotide microsatellite repeats. Following sequencing of the positive clones, 11 pairs of primers were designed for polymorphic loci and their variability assessed in > 350 individuals from four populations in North America. The loci had between 9 and 40 alleles and observed heterozygosities ranged from 0.071 to 0.87. Some of these pairs of primers also successfully amplified DNA from two other snake species.     

Development of 22 new microsatellite loci for fishers (Martes pennanti) with variability results from across their range

We developed 22 new microsatellite loci for the fisher (Martes pennanti), a North American mesocarnivore. The loci were developed with samples from the southern Sierra Nevada Mountains in California, and were screened with samples from this population and four other populations. We observed a range of six to 21 polymorphic loci per population, with the Sierra Nevada population exhibiting markedly lower levels of variation compared to the other four.     

Isolation,characterization and cross‐species amplification of microsatellite DNA loci in the tropical American yam Dioscorea trifida

We developed microsatellite markers in American yam (Dioscorea trifida). A microsatellite sequence‐enriched genomic library was screened, and after sequencing, primers were designed for 20 microsatellites. Among these, eight primer pairs yielded amplification products that were both interpretable and polymorphic in 24 yam cultivars. The number of alleles per locus ranged from two to 13 and the overall expected heterozygosity was around 0.5. Six of the eight Dioscorea trifida microsatellite loci gave amplification products in other Dioscorea species.     

Development of a novel molecular marker on the mitochondrial genome of a toxic dinoflagellate, <Emphasis Type="Italic">Alexandrium</Emphasis> spp., and its application in single-cell PCR

Although the molecular data currently used for identifying dinoflagellates are generally limited to nuclear ribosomal RNA genes, some dinoflagellates cannot be identified by their gene sequence or morphotype, suggesting that additional effective molecular makers are required. We report here a novel species-specific marker on the mitochondrial (mt) genome of dinoflagellates belonging to six Alexandrium spp., namely, A. tamarense, A. catenella, A. tamiyavanichii, A. affine, A. hiranoi, and A. pseudogonyaulax. This new mt marker was able to clearly differentiate these six species. PCR analysis using a primer set for the A. tamarense-specific sequence confirmed that this sequence is conserved in A. tamarense strains but not in other dinoflagellate species. We also sequenced the mt genome containing the developed molecular marker using a single cell from a field sample, which suggests that this marker is a powerful tool for identifying unculturable dinoflagellates. The sequenced molecular region was also used to identify Alexandrium-like cells isolated from environmental seawater as A. tamarense and A. affine.     

Development of microsatellite markers in the toxic dinoflagellate Alexandrium tamarense (Dinophyceae)

Outbreaks of paralytic shellfish poisoning caused by the toxic dinoflagellate Alexandrium tamarense (Dinophyceae) are currently a serious problem from an economic and food hygiene point of view throughout the world. We isolated 13 polymorphic microsatellite loci from this species. These loci provided microsatellite markers with high polymorphism ranging from four to 15 alleles per locus and gene diversity between 0.632 and 0.974. The markers are available for more detailed investigations of genetic structure and gene flow of A. tamarense populations.     

Identification and characterization of nine polymorphic microsatellite loci in the North American pika,Ochotona princeps

Nine polymorphic microsatellite loci were developed for the North American pika (Ochotona princeps) from di‐ and tetranucleotide repeat‐enriched genomic libraries. Polymorphism was assessed for 165 individuals from eight geographical locations in the western United States. All loci were polymorphic. The number of alleles per locus ranged from three to 14, with observed heterozygosity between 0.189 and 0.822. All loci were in Hardy–Weinberg equilibrium (< 0.05). Regional differences were evident with unique alleles at multiple loci in six of eight populations.     

Isolation and characterization of eight microsatellite loci from the house finch (Carpodacus mexicanus)

I isolated the first set of polymorphic microsatellite markers from the house finch, Carpodacus mexicanus, a well‐studied North American bird species, as part of an effort to compare levels of genetic diversity in introduced and native populations. Here, I describe eight independently assorting microsatellite loci screened for polymorphism using 40 house finches. Polymorphism levels ranged from six to 14 alleles (mean = 10.6), making these markers a powerful tool for paternity and population level analyses of this widely distributed North American species.     

Genetic diversity of Valsa malicola isolates assessed by microsatellite-primed PCR (MP-PCR)

Genetic diversity of 40 isolates of Valsa malicola in Iran was investigated by MP-PCR. Isolates were obtained from different host plants in diverse regions during 2004–2010. Of the six microsatellite primers tested, only four primers; (ACTG)₄, (GACA)₄, (AC)₈ and (CGA)_5, were able to amplify DNA fragments. With these four primers, 120 loci were identified; of which eight were monomorphic (6.7%) and 112 were polymorphic (93.3%). Approximate size of amplified DNA fragments ranged from 0.2 to 3?kb. Observed high genetic diversity in isolates of V. malicola indicates the eligibility of the marker to investigate ranks below the species level. The results of cluster analysis indicated that isolates are related to each other with 45.63% similarity and four groups (1, 2, 3 and 4) were identified in the dendrogram. Group 3 included 37 isolates that were obtained from different hosts and geographical regions and each of groups 1, 2 and 4 only had one isolate. Some correlations between identified groups and host origins or geographic distributions of the isolates were found.