Paul K. Hayes

Sensitive detection of trypanosomes in tsetse flies by DNA amplification

African trypanosome species were identified using the Polymerase Chain Reaction (PCR) by targeting repetitive DNA for amplification. Using oligonucleotide primers designed to anneal specifically to the satellite DNA monomer of each species/subgroup, we were able to accurately identify Trypanosoma simiae, three subgroups of T. congolense, T. brucei and T. vivax. The assay was sensitive and specific, detecting one trypanosome unequivocally and showing no reaction with non-target trypanosome DNA or a huge excess of host DNA. The assay was used to identify developmental stage trypanosomes in the tsetse fly. The use of radioisotopes was not necessary and mixed infections could be detected easily by incorporating more than one set of primers in a single reaction. The use of crude preparations of template made the process very rapid. The methodology should be suitable for large-scale epidemiological studies.

Representing key phytoplankton functional groups in ocean carbon cycle models: Coccolithophorids

Carbonates are the largest reservoirs of carbon on Earth. From mid-Mesozoic time, the biologically catalyzed precipitation of calcium carbonates by pelagic phytoplankton has been primarily due to the production of calcite by coccolithophorids. In this paper we address the physical and chemical processes that select for coccolithophorid blooms detected in Sea-viewing Wide Field-of-view Sensor (SeaWiFS) ocean color imagery. Our primary goal is to develop both diagnostic and prognostic models that represent the spatial and temporal dynamics of coccolithophorid blooms in order to improve our knowledge of the role of these organisms in mediating fluxes of carbon between the ocean, the atmosphere, and the lithosphere. On the basis of monthly composite images of classified coccolithophorid blooms and global climatological maps of physical variables and nutrient fields, we developed a probability density function that accounts for the physical chemical variables that predict the spatiotemporal distribution of coccolithophorids in the world oceans. Our analysis revealed that areas with sea surface temperatures (SST) between 3° and 15°C, a critical irradiance between 25 and 150 µmol quanta m-2 s-1, and decreasing nitrate concentrations (N/t < 0) are selective for upper ocean large-scale coccolithophorid blooms. While these conditions favor both Northern and Southern Hemisphere blooms of the most abundant coccolithophorid in the modern oceans, Emiliania huxleyi, the Northern and Southern Hemisphere populations of this organism are genetically distinct. Applying amplified fragment length polymorphism as a marker of genetic diversity, we identified two major taxonomic clades of E. huxleyi; one is associated with the Northern Hemisphere blooms, while the other is found in the Southern Hemisphere. We suggest a rule of “universal distribution and local selection”: that is, coccolithophorids can be considered cosmopolitan taxa, but their genetic plasticity provides physiological accommodation to local environmental selection pressure. Sea surface temperature, critical irradiance, and N/t were predicted for the years 2060–2070 using the NCAR Community Climate System Model to generate future monthly probability distributions of coccolithophorids based upon the relationships observed between the environmental variables and coccolithophorid blooms in modern oceans. Our projected probability distribution analysis suggests that in the North Atlantic, the largest habitat for coccolithophorids on Earth, the areal extent of blooms will decrease by up to 50% by the middle of this century. We discuss how the magnitude of carbon fluxes may be affected by the evolutionary success of coccolithophorids in future climate scenarios.

Intraspecific genetic diversity in the marine coccolithophore Emiliania huxleyi (Prymnesiophyceae): the use of microsatellite analysis in marine phytoplankton population studies

Using primer pairs for seven previously described microsatellite loci and three newly characterized microsatellite loci from the coccolithophore Emiliania huxleyi (Lohm.) Hay and Mohler, we assessed genetic variation within this species. Analysis of microsatellite length variants (alleles) was conducted for 85 E. huxleyi isolates representative of different ocean basins. These results revealed high intraspecific genetic variability within the E. huxleyi species concept. Pairwise comparison of a 1992 Coastal Fjord group (FJ92) (n=41) and a North East Atlantic (NEA) group (n=21), using FST as an indicator of genetic differentiation, revealed moderate genetic differentiation (FST=0.09894; P=0; significance level=0.05). Gene flow between the FJ92 and NEA groups was estimated to be low, which is in agreement with the moderate levels of genetic differentiation revealed by the microsatellite data. A genetic assignment method that uses genotype likelihoods to draw inference about the groups to which individuals belong was tested. Using FJ92 and NEA as reference groups, we observed that all the E. huxleyi groups tested against the two reference groups were unrelated to them. On a global biogeographical scale, E. huxleyi populations appear to be highly genetically diverse. Our findings raise the question of whether such a high degree of intraspecific genetic diversity in coccolithophores translates into variability in ecological function.

High levels of genetic diversity and low levels of genetic differentiation in North Sea Pseudo-nitzschia pungens (Bacillariophyceae) populations

Six microsatellite markers were used to investigate the genetic structure of North Sea Pseudo‐nitzschia pungens (Grunow ex P. T. Cleve) Hasle populations. Isolates were collected on 42 separate occasions from waters surrounding the German islands of Helgoland and Sylt over the course of three sampling periods: spring 2002, spring 2003, and autumn 2003. In total, 464 isolates were genotyped, of which 453 were different (i.e. clonal diversity was 98%). The numbers of alleles per locus ranged from 6 to 24 and the observed heterozygosities from 0.59 to 0.87 (mean Ho and He were 0.73); there were no significant departures from Hardy‐Weinberg equilibrium at any of the six loci. Sexual reproduction therefore appears to be important in the production of genetic variation. Over the temporal and spatial scales sampled (18 months and 100 km), weak genetic differentiation was detected both within and between sampling periods (significant FST values ranged from 0.0018 to 0.0389), suggesting that the German North Sea supports a single largely unstructured population of P. pungens.

Genetic characterisation of Emiliania huxleyi (Haptophyta)

Amongst the coccolithophorids, Emiliania huxleyi is the most successful and can form large scale blooms under a variety of environmental conditions. This implies extensive genetic variation within this taxon. Physiological, morphological and antigenic differences between clonal isolates support this suggestion. Our investigations into the level of genetic variation within the morphological species concept of E. huxleyi indicate that it is such a young taxon that sequence comparisons of both coding and non-coding regions cannot resolve the issue of how many separate taxonomic entities are involved. However, PCR-based genetic fingerprinting techniques do reveal extensive genetic diversity, both on a global scale and within major bloom populations in both space and time. Cell DNA content can also separate cells with morphotype A coccoliths from those with morphotype B coccoliths. Taken together with physiological and morphological evidence, these data suggest that the morphotypes of E. huxleyi should be separated at the variety level. We have used both nuclear and plastid rRNA sequence comparisons to confirm the place of E. huxleyi within the Haptophyta.

The gas vesicles, buoyancy and vertical distribution of cyanobacteria in the Baltic Sea

The mean pressures required to collapse gas vesicles in turgid cells of cyanobacteria from the Baltic Sea were 0·91 MPa (9·1 bar) in Aphanizomenon flos-aquae, 0·83 MPa in Nodularia sp. collected from the main deep basins and 0·34 MPa in Nodularia from shallower coastal regions. The gas vesicles were strong enough to withstand the depth of winter mixing, down to the permanent halocline (60 m in the Bornholm Sea, 90 m in the Eastern Gotland Sea) or to the sea bottom (30 m or less in the shallow Arkona Sea and Mecklenburg Bight). The cyanobacteria had low cell turgor pressures, within the range 0·08–0·18 MPa. The colonies were highly buoyant: the Aphanizomenon colonies floated up at a mean velocity of 22 m per day and the Nodularia colonies at 36 m per day. The colonies remained floating when up to half of the gas vesicles had been collapsed. In summer the cyanobacteria were mostly restricted to the water above the thermocline and in calm conditions their concentration increased towards the top of the water ...

Permanent Genetic Resources added to Molecular Ecology Resources Database 1 August 2009-30 September 2009

This article documents the addition of 229 microsatellite marker loci to the Molecular Ecology Resources Database. Loci were developed for the following species: Acacia auriculiformis × Acacia mangium hybrid, Alabama argillacea, Anoplopoma fimbria, Aplochiton zebra, Brevicoryne brassicae, Bruguiera gymnorhiza, Bucorvus leadbeateri, Delphacodes detecta, Tumidagena minuta, Dictyostelium giganteum, Echinogammarus berilloni, Epimedium sagittatum, Fraxinus excelsior, Labeo chrysophekadion, Oncorhynchus clarki lewisi, Paratrechina longicornis, Phaeocystis antarctica, Pinus roxburghii and Potamilus capax. These loci were cross‐tested on the following species: Acacia peregrinalis, Acacia crassicarpa, Bruguiera cylindrica, Delphacodes detecta, Tumidagena minuta, Dictyostelium macrocephalum, Dictyostelium discoideum, Dictyostelium purpureum, Dictyostelium mucoroides, Dictyostelium rosarium, Polysphondylium pallidum, Epimedium brevicornum, Epimedium koreanum, Epimedium pubescens, Epimedium wushanese and Fraxinus angustifolia.

A molecular and phenotypic analysis of Nodularia (Cyanobacteria) from the Baltic Sea

The filamentous diazotrophic cyanobacterium Nodularia forms water blooms each year in the Baltic Sea. Filaments isolated from such water blooms vary in their trichome width, degree of coiling, and properties of their gas vesicles; previously, these characters have been used to classify individuals to species level. To test the validity of such a phenotypic classification, we determined the nucleotide sequences for a region of the phycocyanin locus that includes a noncoding intergenic spacer (PC‐IGS), the IGS between two adjacent copies of the gvpA gene (which encodes the main structural gas vesicle protein) and the rDNA internal transcribed spacer (rDNA‐ITS), for 13 clonal Nodularia isolates from the Baltic Sea during August 1994. The complete 16S‐rDNA sequence was determined for three isolates and was found to be identical in each of them. Molecular sequences for noncoding regions of the genome were used to assign isolates to three groups on the basis of PC‐IGS, two groups on the basis of gvpA‐IGS, and three groups on the basis of rDNA‐ITS. No consistent correlation was found between genotype and any of the phenotypic features examined, and no link was found between any of these features themselves, indicating that these characters are not useful for placing Nodularia isolates into meaningful taxonomic groups. The PC‐IGS, gvpA‐IGS, and rDNA‐ITS genotypic groupings were not congruent. This might indicate that gene flow occurs between individuals in Nodularia populations.

A REAPPRAISAL OF PORPHYRA AND BANGIA (BANGIOPHYCIDAE, RHODOPHYTA) IN THE NORTHEAST ATLANTIC BASED ON THE rbcL–rbcS INTERGENIC SPACER

Sequence data of the rbcL–rbcS noncoding intergenic spacer of the plastid genome for 47 specimens of Porphyra and Bangia from the northeast Atlantic reveal that they fall into 11 distinct sequences: P. purpurea, P. dioica (includes a sample of P. “ochotensis” from Helgoland), P. amplissima (includes P. thulaea and British records of P. “miniata”), P. linearis, P. umbilicalis, P. “miniata”, B. atropurpurea s.l. from Denmark and B. atropurpurea s.l. from Wales, P. drachii, P. leucosticta (includes a British record of P. “miniata var. abyssicola”), and P. “insolita” (includes P. “yezoensis” from Helgoland). Of these, data obtained for P. purpurea, P. dioica, P. amplissima, P. linearis, P. umbilicalis, P. drachii, and P. leucosticta were based on type specimens or material compared with types. Comparison of sequence data for Porphyra spp. and Bangia atropurpurea s.l. (including B. fuscopurpurea, the type species of Bangia) confirms that the species are congeneric. The data also confirm that the number of layers that make up the Porphyra thallus are not taxonomically significant. Comparison of sequence data for species from the northeast Atlantic with those for material of two species from the Pacific reveals that the species fall into two distinct groupings: an Atlantic group, containing P. purpurea, P. dioica, P. amplissima, P. linearis, P. umbilicalis, P. “miniata”, and B. atropurpurea, and a Pacific group, containing P. “pseudolinearis”, P. drachii, P. leucosticta, P. “yezoensis” (including a sample of P. “tenera”), and P. “insolita” (including P. “yezoensis” from Helgoland). The possibility of alien species in the northeast Atlantic is discussed.

Microsatellite marker development and genetic variation in the toxic marine diatom Pseudo-nitzschia multiseries (Bacillariophyceae)

The genetic structure of phytoplankton populations is largely unknown. In this study we developed nine polymorphic microsatellite markers for the domoic acid–producing marine diatom Pseudo‐nitzschia multiseries (Hasle) Hasle. We then used them in the genotyping of 25 physiologically diverse field isolates and six of their descendants: 22 field isolates originated from eastern Canadian waters, two from European waters, and one from Russian waters. The numbers of alleles per locus ranged from three to seven and the observed heterozygosities from 0.39 to 0.70. A substantial degree of genetic variation was observed within the field isolates, with 23 different genotypes detected. The Russian isolate was the most genetically distinct, although there was also evidence of genetic differentiation at a more local scale. Mating experiments demonstrated that alleles were inherited in a Mendelian manner. Pseudo‐nitzschia multiseries primer pairs were tested on DNA from four congeners: P. calliantha Lundholm, Moestrup et Hasle; P. fraudulenta (P. T. Cleve) Hasle; P. pungens (Grunow ex P. T. Cleve) Hasle; and P. seriata (P. T. Cleve) H. Peragallo. Cross‐reactivity was only observed in P. pungens. Our results are a first step in understanding the genetic variation present at the Pseudo‐nitzschia“species” level and in determining the true biogeographic extent of Pseudo‐nitzschia species.