Caribbean Isolates Research Articles

Characterisation of South African field Ehrlichia ruminantium using multilocus sequence typing.

Heartwater, one of the major tick-borne diseases of some domestic and wild ruminants in Africa, is caused byEhrlichia ruminantium. The genetic diversity ofE. ruminantiumisolates renders the available vaccine ineffective against certain virulent isolates. To better understand theE. ruminantiumgenotypes in South Africa, a total of 1004Amblyomma hebraeumtick deoxyribonucleic acid (DNA) samples from cattle in three South African provinces were tested by pCS20 Sol1 real-time polymerase chain reaction (qPCR) and characterised by multilocus sequence typing (MLST) using five housekeeping genes. Out of 1004 samples tested, 222 (22%) were positive forE. ruminantium. The occurrence ofE. ruminantiumin Mpumalanga, KwaZulu-Natal and Limpopo provinces was 19%, 22% and 27%, respectively. TheE. ruminantiumpositive samples were screened for housekeeping genes and sequenced. Phylogenetic analysis revealed three main lineages: clade 1 made up of worldwide isolates (eastern, southern Africa, and Caribbean isolates), clade 2 comprised only West African isolates and clade 3 consisted of Omatjenne, Kümm2 and Riverside. Some study sample sequences were not identical to any of the reference isolates. However, they could all be grouped into the worldwide clade. Genetic variation in the sequenced regions was observed in the form of single nucleotide polymorphisms (SNPs). Using MLST to characteriseE. ruminantiumfield isolates allowed the South African genotypes to be clearly distinguished from the distinct West African isolates.Contribution:Characterisation ofE. ruminantiumfield isolates is important for the control of heartwater and contributes to preliminary knowledge required for the development of a more practical vaccine against heartwater.

Ubiquitous Selfish Toxin-Antidote Elements in Caenorhabditis Species

Toxin-antidote elements (TAs) are selfish genetic dyads that spread in populations by selectively killing non-carriers. TAs are common in prokaryotes, but very few examples are known in animals. Here, we report the discovery of maternal-effect TAs in both C.tropicalis and C.briggsae, two distant relatives of C.elegans. In C.tropicalis, multiple TAs combine to cause a striking degree of intraspecific incompatibility: five elements reduce the fitness of >70% of the F2 hybrid progeny of two Caribbean isolates. Weidentified the genes underlying one of the novel TAs, slow-1/grow-1, and found that its toxin, slow-1, is homologous to nuclear hormone receptors. Remarkably, although previously known TAs act during embryonic development, maternal loading of slow-1 in oocytes specifically slows down larval development, delaying the onset of reproduction by several days. Finally, we found that balancing selection acting on linked, conflicting TAs hampers their ability to spread in populations, leading to more stable genetic incompatibilities. Our findings indicate that TAs are widespread in Caenorhabditis species and target a wide range of developmental processes and that antagonism between them may cause lastingincompatibilities in natural populations. We expect that similar phenomena exist in other animal species.

Burkholderia pseudomallei, the causative agent of melioidosis, is rare but ecologically established and widely dispersed in the environment in Puerto Rico.

BackgroundBurkholderia pseudomallei is a soil-dwelling bacterium and the causative agent of melioidosis. The global burden and distribution of melioidosis is poorly understood, including in the Caribbean. B. pseudomallei was previously isolated from humans and soil in eastern Puerto Rico but the abundance and distribution of B. pseudomallei in Puerto Rico as a whole has not been thoroughly investigated.Methodology/Principal findingsWe collected 600 environmental samples (500 soil and 100 water) from 60 sites around Puerto Rico. We identified B. pseudomallei by isolating it via culturing and/or using PCR to detect its DNA within complex DNA extracts. Only three adjacent soil samples from one site were positive for B. pseudomallei with PCR; we obtained 55 isolates from two of these samples. The 55 B. pseudomallei isolates exhibited fine-scale variation in the core genome and contained four novel genomic islands. Phylogenetic analyses grouped Puerto Rico B. pseudomallei isolates into a monophyletic clade containing other Caribbean isolates, which was nested inside a larger clade containing all isolates from Central/South America. Other Burkholderia species were commonly observed in Puerto Rico; we cultured 129 isolates from multiple soil and water samples collected at numerous sites around Puerto Rico, including representatives of B. anthina, B. cenocepacia, B. cepacia, B. contaminans, B. glumae, B. seminalis, B. stagnalis, B. ubonensis, and several unidentified novel Burkholderia spp.Conclusions/SignificanceB. pseudomallei was only detected in three soil samples collected at one site in north central Puerto Rico with only two of those samples yielding isolates. All previous human and environmental B. pseudomallei isolates were obtained from eastern Puerto Rico. These findings suggest B. pseudomallei is ecologically established and widely dispersed in the environment in Puerto Rico but rare. Phylogeographic patterns suggest the source of B. pseudomallei populations in Puerto Rico and elsewhere in the Caribbean may have been Central or South America.

Comparative virulence of Caribbean, Brazilian and European isolates of Toxoplasma gondii

BackgroundToxoplasma gondii is a zoonotic parasite of global importance. The outcome of infection in humans can depend on a number of factors including the infecting stage of the parasite, inoculating dose and virulence of the infecting strain. Molecular epidemiological studies have demonstrated an abundance of atypical strains of T. gondii in South America, many of which have been associated with more severe sequelae of infection. The aim of this study was to compare the virulence of T. gondii strains isolated in the Caribbean to a virulent Brazilian strain and an avirulent European strain.MethodsOne hundred and twenty Swiss CD-1 mice were split into 8 groups of 15 mice and each group was inoculated with 200 tachyzoites of one of 8 isolates, comprising ToxoDB genotypes #1, #141, #265, #13, #3 and #6. Five mice per group were euthanized at day 8 post-inoculation (p.i.) and parasite burden was determined in heart, lungs and eyes using quantitative PCR. Lungs and brain were also examined by histopathology and immunohistochemistry. The remaining 10 mice per group were part of a survival experiment to assess virulence. DNA was extracted from tachyzoites of each of the 8 T. gondii isolates and genotyped at four ROP gene loci, including ROP5, ROP16, ROP17 and ROP18 to look for association with markers of virulence.ResultsInfection with ToxoDB genotype #13 from the Caribbean resulted in 100% of mice being euthanized which was comparative to infection with the virulent Brazilian strain (ToxoDB genotype #6). Significantly higher parasite burdens were recorded in the lungs and eyes of mice infected with ToxoDB genotypes #13 and #6. Genotyping of ROP loci revealed that the virulent Caribbean isolates had a different ROP18/ROP5 allelic profile (3/1) to the virulent Brazilian isolate (1/3); however, the avirulent Caribbean isolate (ToxoDB genotype #1) had the same ROP18/ROP5 profile as the avirulent European isolate (ToxoDB #3) (both 2/2). Caribbean isolates of intermediate virulence (ToxoDB #141 and #265) all had the same ROP18/ROP5 allelic profile (2/2).ConclusionsIsolates from the Caribbean with ToxoDB genotype #13 were acutely virulent for mice and comparable to a known virulent Brazilian isolate. The ROP protein allelic profile of the virulent Caribbean and Brazilian isolates differed indicating that perhaps other factors are involved in predicting virulence. Understanding virulence is important for predicting disease outcome in humans and may also aid vaccine design as well as drug discovery.

Distribution and phylodynamics of papaya ringspot virus on Carica papaya in Cuba

Orchard and garden papaya crops grown in 47 Cuban municipalities were surveyed from 2008 to 2013, revealing the widespread distribution of papaya ringspot virus (PRSV) in Cuba. Phylodynamic analyses performed with the partial coat protein gene of all Cuban PRSV‐P isolates (34 sequences) and 107 sequences of isolates from the American continent and the Caribbean islands showed a most recent common ancestor in 1942 (95% highest posterior density, HPD 95% = 1911–1967). The substitution rate was estimated to be 7.7 × 10−4 substitutions per site per year (HPD 95% = 4.6 × 10−4 to 1.1 × 10−3), which is equivalent to those detected in other RNA viruses. Demographic reconstruction of PRSV showed that viral diversity increased in the 1985–1990 period, which coincides with the implementation of extensive production practices. Moreover in Cuba, viral dispersion occurred from Mexico and other unknown ancestral locations. The spatiotemporal diffusion analysis proposed Mexico as an ancestral area for the origin of diversification in the American continent and suggests new dispersion events between American and Caribbean isolates. The observed widespread distribution, clear geographic grouping of Cuban isolates, virus growth and genetic diversity provide strong evidence of the PRSV dispersion patterns, which has implications for the control strategies of PRSV.

Rapid Extraction and Identification of Maitotoxin and Ciguatoxin-Like Toxins from Caribbean and Pacific Gambierdiscus Using a New Functional Bioassay

BackgroundCiguatera is a circumtropical disease produced by polyether sodium channel toxins (ciguatoxins) that enter the marine food chain and accumulate in otherwise edible fish. Ciguatoxins, as well as potent water-soluble polyethers known as maitotoxins, are produced by certain dinoflagellate species in the genus Gambierdiscus and Fukuyoa spp. in the Pacific but little is known of the potential of related Caribbean species to produce these toxins.MethodsWe established a simplified procedure for extracting polyether toxins from Gambierdiscus and Fukuyoa spp. based on the ciguatoxin rapid extraction method (CREM). Fractionated extracts from identified Pacific and Caribbean isolates were analysed using a functional bioassay that recorded intracellular calcium changes (Ca2+) in response to sample addition in SH-SY5Y cells. Maitotoxin directly elevated Ca2+i, while low levels of ciguatoxin-like toxins were detected using veratridine to enhance responses.ResultsWe identified significant maitotoxin production in 11 of 12 isolates analysed, with 6 of 12 producing at least two forms of maitotoxin. In contrast, only 2 Caribbean isolates produced detectable levels of ciguatoxin-like activity despite a detection limit of >30 pM. Significant strain-dependent differences in the levels and types of ciguatoxins and maitotoxins produced by the same Gambierdiscus spp. were also identified.ConclusionsThe ability to rapidly identify polyether toxins produced by Gambierdiscus spp. in culture has the potential to distinguish ciguatoxin-producing species prior to large-scale culture and in naturally occurring blooms of Gambierdiscus and Fukuyoa spp. Our results have implications for the evaluation of ciguatera risk associated with Gambierdiscus and related species.

Diversity of Papaya ringspot virus Isolates in Puerto Rico

Papaya ringspot virus (PRSV) devastates papaya production worldwide. In Puerto Rico, papaya fields can be completely infected with PRSV within a year of planting. Information about the diversity of the Puerto Rican PRSV (PR-PRSV) population is relevant to establish a control strategy in the island. The coat protein gene (cp) of PRSV was sequenced from 62 isolates from different regions in Puerto Rico. The viral population of PRSV in Puerto Rico has 4% nucleotide and 5% amino acid diversity. Analysis of the coat protein (CP) amino acid sequence showed a variable amino terminal (N-terminal) region with a conserved aphid transmission motif and a variable EK repeat region. The core and carboxyl terminal (C-terminal) region were conserved. In the phylogenetic analysis, Puerto Rican isolates grouped independently of their geographical origin, with the exception of southern isolates that formed two separate subgroups and were the most divergent. Sequences of the cp from the Puerto Rican isolates, when compared with sequences from other countries, showed least genetic distance with isolates from the United States and Australia, followed by other American and Caribbean isolates. The U.S. and Australian isolates are sister taxa to the Puerto Rican isolates in the phylogenetic tree. This suggests that PRSV from Puerto Rico and the isolates from the United States and Australia have a common origin thought to be from a Mexican population.

Chikungunya Virus as Cause of Febrile Illness Outbreak, Chiapas, Mexico, 2014.

Since chikungunya virus (CHIKV) was introduced into the Americas in 2013, its geographic distribution has rapidly expanded. Of 119 serum samples collected in 2014 from febrile patients in southern Mexico, 79% were positive for CHIKV or IgM against CHIKV. Sequencing results confirmed CHIKV strains closely related to Caribbean isolates.

Phenotypic and genotypic heterogeneity among Streptococcus iniae isolates recovered from cultured and wild fish in North America, Central America and the Caribbean islands.

Streptococcus iniae, the etiological agent of streptococcosis in fish, is an important pathogen of cultured and wild fish worldwide. During the last decade outbreaks of streptococcosis have occurred in a wide range of cultured and wild fish in the Americas and Caribbean islands. To gain a better understanding of the epizootiology of S. iniae in the western hemisphere, over 30 S. iniae isolates recovered from different fish species and geographic locations were characterized phenotypically and genetically. Species identities were determined biochemically and confirmed by amplification and sequencing of the 16S rRNA gene. Repetitive-element palindromic PCR fingerprinting as well as biochemical and antimicrobial susceptibility profiles suggest that a single strain of S. iniae was responsible for two different disease outbreaks among reef fishes in the Caribbean, one in 1999 and another in 2008. Interestingly, a majority of the isolates recovered from cultured fish in the Americas were genetically distinct from the Caribbean isolates and exhibited a trend toward higher minimal inhibitory concentration with respect to several antibiotics as well as greater genetic variability. The biological significance of this genetic variability is unclear, but it could have implications for future vaccine development and treatment.

Mosquito-associated Dengue Virus, Key West, Florida, USA, 2010

To the Editor: Except for sporadic cases along the Texas–Mexico border, local transmission of dengue virus (DENV) has not occurred in the contiguous United States since 1946. In 2009, DENV was diagnosed in a vacationer to Key West, Florida (1). Subsequently, 25 other cases were reported that year, transmission was confirmed by detection of DENV serotype 1 (DENV-1) in local mosquitoes, and a random serosurvey showed evidence of recent DENV infection in 5.4% of Key West residents (1). Transmission continued in 2010, and an additional 63 cases were confirmed (2). We used PCR amplification and sequence analysis of virus identified from mosquito collections during 2010 to identify the closest relatives, probable geographic origin, and divergence time of the Key West DENV. A total of 1,178 pools of Aedes aegypti mosquitoes were collected in Monroe County, Florida, during January 27–December 17, 2010 (Figure A1, panel A), by using BG-Sentinel (Biogents, Regensburg, Germany) or CDC (Clarke, Roselle, IL, USA) light traps, and stored at −80°C. Reverse transcription PCR was conducted on each pool by using primers designed to amplify all 4 DENV serotypes, followed by seminested PCR with serotype-specific primers (3). Results from 2 Key West mosquito pools collected on June 25 and 30 showed a positive first-round reverse transcription PCR and a positive second-round PCR specific for DENV-1. A third Key West pool collected on August 27 showed only a positive second-round PCR specific for DENV-1. No other DENV serotypes were detected. DENV-1–specific primers (5′-GGGCCTTGAGACACCCAGG-3′ and 5′-CCTCCCATGCCTTCCCAATGGC-3′) were used to amplify products encompassing the envelope (E) gene region and parts of the premembrane and nonstructural 1 genes from the pools collected on June 25 and 30. PCR products were sequenced by using amplification and internal primers to provide double or triple coverage (Functional Biosciences, Madison, WI, USA). The sequences from these 2 pools were identical (GenBank accession no. {type:entrez-nucleotide,attrs:{text:JF519855,term_id:355344079}}JF519855). We used ClustalX (4) to align the Key West sequences with 175 nonredundant American DENV-1 sequences from the National Center for Biotechnology Information Virus Variation database (5) and 9 additional DENV-1 subgenomic E sequences from GenBank, which provided a comprehensive set of American DENV-1 sequences, including several isolates from Hawaii (USA) and Easter Island (Chile) that grouped with Asian DENV-1 clades as an outgroup. Maximum-likelihood phylogenetic analysis of the 1,484-nt E gene region was conducted by using SeaView software (6). The analysis showed that American DENV-1 strains clustered by geography and by year of collection (Figure A1, panel B). This clustering might be the result of lineage replacement that has been described in DENV-1 (7). Additionally, clustering might be influenced by serotype prevalence or sampling bias. For example, the sequence database contains few Caribbean isolates from after 2000 and few Central or South American isolates from before 2000. The Key West sequence grouped as a member of a large clade of recent viruses from Central America that was separated from Caribbean and South American viruses with a well-supported bootstrap value (86%) and relatively long branch length. The closest relatives were 2 strains isolated in Nicaragua in 2006 and 2008. The bootstrap support for this grouping was 100%. Phylogenetic analyses with neighbor-joining, maximum-parsimony, and Bayesian methods gave trees with similar topologies, including clear separation of most recent Central American isolates into 1 clade, as well as grouping of the Key West sequence with the same 2 isolates from Nicaragua (data not shown). No protein coding changes between these strains were identified, which suggests purifying selection for an optimum phenotype. There were 8 synonymous differences over the 1,708-nt amplified region between the Key West and Nicaragua 2008 sequences. Previous molecular clock determinations for DENV-1 provided a range of 2.5–7.0 × 10–4 substitutions per nucleotide per year (8). This calculation produced an estimate of a 6.7–18.7-year divergence time between the Key West virus and the most closely related Nicaragua strain. When during this time the ancestor of Key West DENV was introduced to Florida is unknown. Analysis of the entire Key West DENV-1 genome may help pinpoint the origin and address the possibility of selective pressure on other genes or recombination events (9). Given the recent reports of DENV in residents of other Florida counties who had no travel histories (2), monitoring of Key West and other nearby urban areas for evidence of local DENV transmission should continue.

Genetic variability among isolates of Coconut lethal yellowing phytoplasmas determined by Heteroduplex Mobility Assay (HMA)

Heteroduplex mobility assay (HMA) was used to determine genomic diversity among African isolates of coconut lethal yellowing phytoplasmas causing Cape St. Paul wilt disease (CSPD, Ghana), lethal disease (LD, Tanzania), and lethal yellowing (LYM, Mozambique). They were also compared to the Caribbean phytoplasma associated with coconut lethal yellowing (LY). A DNA fragment of 1850 bp covering the 16S rRNA gene and 16/23S intergenic spacer region of each isolate was amplified with primers P1 and P7 and subsequently submitted to HMA analysis for sequence variation. A PCR product amplified from GH5D (CSPD isolate) as a reference was combined with each PCR product and electrophoresed on polyacrylamide gels. Three groups of phytoplasmas associated with various coconut lethal yellowing diseases were identified by HMA. The samples from Mozambique (LYM) and Ghana (CSPD) formed one group, which was different from the second group, LD from Tanzania. These two groups were different from the third group of Caribbean isolates. This grouping was consistent with the genetic diversity described in the coconut yellowing-associated phytoplasmas detected after cloning, sequencing, and phylogenetic analyses. The HMA technique described here has the potential to provide a simple and rapid means to identify and to establish the diversity of isolates within the coconut lethal yellowing disease group.

Phylogenetic relationships among Ehrlichia ruminantium isolates.

Ehrlichia ruminantium, the causative agent of heartwater, is a tick-borne pathogen infecting ruminants throughout sub-Saharan Africa and on some Caribbean islands. The most reliable test for E. ruminantium is PCR-based, but this gives positive results in some areas free of clinical heartwater and of the known Amblyomma spp. tick vectors. To investigate the molecular basis for this finding we have sequenced and carried out phylogenetic analysis of a range of genes from a number of E. ruminantium isolates. The genes include ribonuclease III and cytochrome c oxidase assembly protein genes (the pCS20 region), groESL, citrate synthase (gltA), and 16S ribosomal RNA. Relationships among major antigenic protein (map1) genes have been exhaustively investigated in a previous study that showed that the genes are variable in length, have non-synonymous mutations, and show no geographical specificity among isolates. The 16S sequences are highly conserved, except in the V1 loop region. The pCS20, groESL, and gltA genes show only single nucleotide polymorphisms (SNPs) dispersed throughout the sequenced regions. Phylogenetic analysis using pCS20 data differentiates the western African isolates into a single clade, which also includes a southern African isolate. All other southern African isolates and a Caribbean isolate fall into a further clade, which is subdivided into two groups. Sequence variation within this clade is greater than that within the western African clade, suggesting that E. ruminantium originated in southern Africa.

Envelope sequence variation and phylogenetic relations of human T cell lymphotropic virus type 1 from endemic areas of Colombia.

The HTLV-1 envelope gene of 12 TSP/HAM patients from two endemic areas of southwest Colombia (Tumaco and Buenaventura) was amplified by nested PCR, sequenced, and compared with previously reported HTLV-1 envelope sequences from isolates worldwide. In general, the sequence divergences among all Colombian samples ranged from 0.1 to 1.6%. Some amino acid substitutions, referring to the ATK-1 prototype strain in the surface domain gp46 and in p21, were highly prevalent in southwest Colombia, suggesting a geographical clustering of mutations in the envelope gene. The phylogenetic analysis showed that the Colombian isolates belong to the HTLV-1a lineage with minor subgroups. The genetic distance between Colombian and Japanese isolates ranged from 0.1 to 1.8%; in comparison, the genetic distance between Colombian and Caribbean isolates ranged from 0.4 to 2.2%. Our results strongly suggest that the actual quasispecies populations in southwest Colombia have been generated by separate, differently timed introductions of virus.

PHYLOGEOGRAPHIC STUDIES IN THE TROPICAL SEAWEED CLADOPHOROPSIS MEMBRANACEA (CHLOROPHYTA, ULVOPHYCEAE) REVEAL A CRYPTIC SPECIES COMPLEX1

The seaweed Cladophoropsis membranacea (Hofman Bang ex. C. Agardh) Børgesen is a widely distributed species on coral reefs and along rocky coastlines throughout the tropics and subtropics. In a recent population‐level survey openface>1600 individuals with eight microsatellite loci, a number of isolates from biogeographically disjunct locations could not be amplified for any of the loci. Nonamplifiable and amplifiable isolates co‐occurred within the Canary Islands, Cape Verde Islands, and in the Caribbean. These unexpected results led to question whether or not C. membranacea is a single species. Phylogenetic relationships were evaluated using rDNA ITS1 and ITS2 sequence comparisons from 42 isolates sampled from a subset of 30 of the 66 locations. Four well‐supported clades were identified. Sequence divergence within clades was <1%, whereas between‐clade divergence was 2%–3%. Intraindividual variation was extremely low with no effects on the analysis. A strong, but imperfect, correspondence was found between ITS clades and amplifiable microsatellite loci. It is concluded that C. membranacea consists of three cryptic species. Using Pacific isolates as an outgroup, the most basal clade included the Central Canary Islands, Cape Verde, and Bonaire (Caribbean) isolates and thus spanned the widest latitude. Two derived sister clades consisted of a southern transtropical group stretching across the SE Caribbean to the Cape Verde Islands and African coast (but not the Canary Islands) and a NE‐Canary Island‐Mediterranean clade that also included the Red Sea. The detection of overlapping biogeographic distributions highlights the importance of ecotypic differentiation with respect to temperature and the importance of shifting sea surface isotherms that have driven periodic extinctions and recolonizations of the Canary Islands—a crossroads of marine floral exchange—since the last glacial maximum.

PHYLOGEOGRAPHIC STUDIES IN THE TROPICAL SEAWEED CLADOPHOROPSIS MEMBRANACEA (CHLOROPHYTA, ULVOPHYCEAE) REVEAL A CRYPTIC SPECIES COMPLEX1

The seaweed Cladophoropsis membranacea (Hofman Bang ex. C. Agardh) Børgesen is a widely distributed species on coral reefs and along rocky coastlines throughout the tropics and subtropics. In a recent population-level survey openface>1600 individuals with eight microsatellite loci, a number of isolates from biogeographically disjunct locations could not be amplified for any of the loci. Nonamplifiable and amplifiable isolates co-occurred within the Canary Islands, Cape Verde Islands, and in the Caribbean. These unexpected results led to question whether or not C. membranacea is a single species. Phylogenetic relationships were evaluated using rDNA ITS1 and ITS2 sequence comparisons from 42 isolates sampled from a subset of 30 of the 66 locations. Four well-supported clades were identified. Sequence divergence within clades was <1%, whereas between-clade divergence was 2%–3%. Intraindividual variation was extremely low with no effects on the analysis. A strong, but imperfect, correspondence was found between ITS clades and amplifiable microsatellite loci. It is concluded that C. membranacea consists of three cryptic species. Using Pacific isolates as an outgroup, the most basal clade included the Central Canary Islands, Cape Verde, and Bonaire (Caribbean) isolates and thus spanned the widest latitude. Two derived sister clades consisted of a southern transtropical group stretching across the SE Caribbean to the Cape Verde Islands and African coast (but not the Canary Islands) and a NE-Canary Island-Mediterranean clade that also included the Red Sea. The detection of overlapping biogeographic distributions highlights the importance of ecotypic differentiation with respect to temperature and the importance of shifting sea surface isotherms that have driven periodic extinctions and recolonizations of the Canary Islands—a crossroads of marine floral exchange—since the last glacial maximum.

THE PHYLOGENY OF CAULERPA BASED ON RDNA INTERNAL TRANSCRIBED SPACER SEQUENCES

Phylogenetic hypotheses for the pantropical marine green algal genus, Caulerpa, were inferred based on analyses of nuclear‐encoded rDNA internal transcribed spacer (ITS) sequences. Results of these analyses were used to assess the correspondence between rDNA phylogeny and traditional sectional taxonomy, to identify synapomorphic morphological characters (including assimilator morphology and chloroplast ultrastructure), and to examine marine biogeographic hypotheses for the genus. Ribosomal DNA ITS sequences were aligned for thirty‐three species and intraspecific taxa of Caulerpa. Results indicate limited correspondence between phylogeny and sectional taxonomy for the genus, (e.g., the sections Filicoideae and Sedoideae were not monophyletic). In contrast, chloroplast morphology could be mapped to the tree topology with limited homoplasy. Pantropical isolates of the filicoidean species, Caulerpa sertularioides and Caulerpa mexicana each formed monophyletic groups. Caulerpa reyesii was included as a derived taxon within the Caulerpa taxifolia clade, suggesting that these species were conspecific and affirmed the lack of correspondence between phylogeny and assimilator morphology. Isolates and various intraspecific taxa of Caulerpa racemosa did not form a monophyletic group. Instead, these taxa formed a heterogeneous assemblage with other sedoidean and filicoidean taxa. Within the C. sertularioides clade, Caribbean and Atlantic isolates formed a basal paraphyletic group, whereas eastern and western Pacific isolates formed a more derived monophyletic group. Therefore, these results are not consistent with an Indo‐West Pacific origin of this species.

Description of a novel Mycobacterium simiae allelic variant isolated from Caribbean AIDS patients by PCR-restriction enzyme analysis and sequencing of hsp 65 gene

A sudden upsurge in the isolation of Mycobacterium simiae from terminally ill AIDS patients was recently reported on the Caribbean island of Guadeloupe. Identification of these M. simiae isolates was achieved using biochemical tests and further confirmed by PCR-restriction analysis (PRA) of a 439-bp fragment of hsp 65. A novel PRA profile III (three Bst EII fragments of 240/125/80 bp and four Hae III fragments of 145/125/40/25 bp) was observed in four blood isolates from two patients. The 16 S rRNA gene sequencing of the hypervariable A region confirmed that all the pattern III isolates were indeed M. simiae species, and the hsp 65 sequencing confirmed the existence of a new hsp 65 allele in these caribbean isolates. A hsp 65 sequence-based phylogenetic tree was also created for 39 species including M. simiae and related mycobacterial species as well as other rapid and slow growing mycobacteria, and may serve as an useful tool for identification of mycobacteria to species level.

Characterization of novel extrachromosomal DNA from giant-celled marine green algae.

Cloned HinfI fragments of the plasmid-like 2.2-kb DNA from the green alga Ernodesmis verticillata (Kützing) Borgesen hybridized solely to single or double bands within the 2.2-kb DNA in genomic Southern blots. Heterologous probes for nuclear and chloroplast genes hybridized only to high-molecular-weight (HMW) DNA. Thus, the low-molecular-weight (LMW) DNA is extrachromosomal and lacks extensive homology to nuclear or chloroplast genes. There was cross-hybridization to LMW DNA from other Caribbean isolates of E. verticillata, but not to that from a Pacific isolate. Under reduced stringency, cross-hybridization to LMW DNA from the related green alga Boergesenia forbesii (Harvey) Feldmann was also observed, suggesting that the LMW DNA may have a common origin and/or function in these algae. Six out of sixteen unique clones hybridized to discrete bands in northern blots, indicating that the LMW DNA may be actively transcribed in vivo. Four of the putatively transcribed clones have regions with significant deduced amino-acid sequence identity to psa and psb gene products, implying that the plasmid-like molecules might have originated from chloroplast DNA. Sequencing data also indicated a high G/C content, as well as the presence of frequent tandem and direct repeats in many of the cloned fragments. Sequencing and restriction analyses suggest that most of the cloned fragments are portions of different DNA molecules, providing evidence that the 2.2-kb extrachromosomal DNA in Ernodesmis is novel in that it represents a fairly heterogeneous population of molecules.

Molecular characterization of Mycobacterium avium complex isolates from Caribbean patients by DT1/DT6-PCR, nonradioactive Southern hybridization, and the Accuprobe system.

A genetic fingerprinting analysis of Caribbean isolates of M. avium complex (MAC) from AIDS patients by a Southern blotting technique after Pstl digestion with nonradioactive DNA probes coding for single-copy sequences DT1 and DT6 was performed. In parallel, a selective amplification of a 187-bp fragment within the DT6 sequence with AV6/AV7 primers for Mycobacterium avium and of a 666-bp fragment within the DT1 sequence of M. intracellulare with the IN38/IN41 primers was also performed, and the molecular speciation with these two methods was compared with results obtained with DNA probes of the Accuprobe system. 66 strains investigated comprised 31 international reference isolates of MAC belonging to serovars 1-28 and 42-44, and 35 clinical isolates including 24 strains from Caribbean AIDS patients. 91.43% of the clinical isolates tested gave concordant data with the DT1/DT6 Southern hybridization and PCR as compared with 74.28% for PCR and Accuprobe, and 71.43% for Accuprobe and Southern hybridization. Our results corroborated previous findings showing that the DT1 probe was specific for M. intracellulare, whereas the DT6 probe was specific for M. avium (reference serovars 2 and 3 probed positive both with DT1 and DT6 probes). Contrary to DT1 probe, which did not reveal sufficient polymorphism to discriminate between MAC isolates, DT6 probe showed an interesting polymorphism giving four distinct clusters. Three clusters corresponded to profiles previously reported for reference and/or clinical isolates; however, a fourth cluster was discovered in five Caribbean isolates from four AIDS patients that did not correspond to previously published genetic patterns. When probed with the insertion sequence IS1245, this cluster retained its homogeneity.

AMPHIDINIUM OPERCULATUM VAR. NOV. GIBBOSUM (DINOPHYCEAE), A FREE‐SWIMMING MARINE SPECIES PRODUCING CYTOTOXIC METABOLITES1

ABSTRACTA single‐cell isolate of Amphidinium from the northern Caribbean Sea was identified as A. operculatum Clap. et Lack. var. nov. gibbosum Maranda et Shimizu based on a morphological and ultrastructural study. This free‐swimming dinoflagellate, which was found to produce potent antitumor metabolites, is elongate and asymmetrical. It is compared with two close relatives A. klebsii Kofoid et Swezy and A. carterae Hulburt. Amphidinium operculatum var. gibbosum can be distinguished from A. klebsii on the basis of shape while it differs from A. carterae on the basis of size, shape, cell surface, and chloroplast arrangement. The shape and size of the nucleus and the presence of mucocysts also differentiate the gibbosum variety from its two relatives. The architecture of the pusule may ultimately provide the best ultrastructural discriminating character. Otherwise, the general ultra‐structure of the Caribbean isolate is typical of many unarmored photo synthetic dinoflagellates with amphiesma, condensed chromosomes, trichocysts, mitochondria with tubular cristae, and trilamellar chloroplasts.