Phylogeny And Experimental Ecology Of Gambierdiscus Spp

Ciguatera is a serious seafood poisoning syndrome caused by the consumption of ciguatoxin-contaminated finfish from the tropical and subtropical Caribbean Sea, Pacific, Atlantic and Indian Ocean. Benthic dinoflagellates in the genus Gambierdiscus spp. produce the ciguatoxin precursors responsible for ciguatera toxicity. Other genera in the benthic community, including Prorocentrum, Ostreopsis, Amphidinium and Coolia, also produce toxins and co-occur with Gambierdiscus spp.; however, their involvement in ciguatera is not well known. This dissertation examined the phylogeny and experimental ecology of Gambierdiscus populations from ciguatera-endemic regions of the central Pacific and Caribbean Sea.The Republic of Kiribati is an atoll nation in the central Pacific, and has one of the highest rates of ciguatera in the world; however, few ecological studies have been conducted in this country and the ecology of Gambierdiscus populations has yet to be determined. To fill this research gap, this study examined the community structure of ciguatera-associated and potentially associated dinoflagellates and the distribution, taxonomy and toxicity of Gambierdiscus spp. from a high-risk area of western Marakei, Republic of Kiribati.Results of field surveys in May 7-11,2011 showed that the dinoflagellate genera Gambierdiscus, Prorocentrum, Ostreopsis, Amphidinium and Coolia were present in Marakei Island, and generally the former three dominated the dinoflagellate assemblage. Among these three, Gambierdiscus was the most abundant dinoflagellate genus, ranging from 0-175 cells g"1 Halimeda sp., at three of the four sites sampled. The following patterns of abundance were observed among sites:(1) Average Gambierdiscus spp. abundance at the Sites 1 and 2 exceeded Sites 3 and 4 by a factor of 19-54; and (2) Gambierdiscus spp. abundance at shallow sites (2-3 m) exceeded deeper sites (10-15 m). The distribution of Gambierdiscus spp. at Marakei corresponded with previously observed patterns of fish toxicity, with fish from locations near Site 3 and 4 being much less toxic than fish sampled north of the central channel. Differences in levels of irradiance may help explain the difference in abundance observed between the shallow and deep sties.DNA sequencing identified three Gambierdiscus species (G. belizeanus, G. carpenteri, G. pacificus) and three previously unreported ribotypes (Gambierdiscus sp. type 4, Gambierdiscus sp. type 5, Gambierdiscus sp. type 6) in the samples; Gambierdiscus sp. type 4 may represent a Pacific clade of Gambierdiscus sp. ribotype 1. To our knowledge, this is the first report of G. belizeanus in Micronesia. Morphological examination by scanning electron microscopy showed that Gambierdiscus clones from Marakei featured typical lenticular, antero-posteriorly compressed shape with the plate tabulation formula of Po,3’,7",5’", 1p,2"", similar to previous studies. Differences in the cell depth and width, apical pore plate Po, apical plate 2’, posterior intercalary plate 1p, and postcingular plate 4’" were observed among species. Mouse neuroblastoma assay determined that Gambierdiscus sp. type 4 isolates were more toxic than the Gambierdiscus sp. type 5 and G. pacificus isolates, with toxin contents of 2.6-6.0 (mean: 4.3±1.4),0.010 and 0.011 fg P-CTX-1 eq cell’1, respectively. In a general, toxicity of Gambierdiscus spp. tested in Marakei was low and moderate level in this genus.Due to uncertainty regarding Gambierdiscus taxonomy, growth response on this organism to environmental conditions prior to 2009 neither provides information on the species involved nor addresses intra-versus inter-specific diversity. Physiological studies of Gambierdiscus spp. carried out after 2009 describe inter-specific diversity; however, variability within species has not been studied. Here under unialgal laboratory conditions, single factor experiments on the growth of eight Gambierdiscus species (17 strains) examined the influence of temperature 16-38℃, salinity 10-60 and irradiance 55-400 μmol photons·m-2·s-1. Gambierdiscus isolates used in these experiments were established from Marakei Island, Republic of Kiribati, the Florida Keys, and St. Thomas, United States Virgin Islands (USVI). The growth characteristics under changing environmental regimes were further used to predict its distribution and explain the previous ecological observations in these regions.The 17 strains of Gambierdiscus used in these experiments included the following species and ribotypes:G. belizeanus, G. caribaeus, G. carolinianus, G. carpenteri, G. pacificus, Gambierdiscus sp. ribotype 1, Gambierdiscus sp. type 4 and Gambierdiscus sp. type 5. All strains examined exhibited the typical growth phases (exponential phase, stationary phase and decline phase). Growth rates measured in this study fell within the range of 0-0.48 divisions day-1, which are comparable to previous findings. In the temperature and salinity studies, Gambierdiscus spp. responded in a Gaussian non-linear way, with optimum growth occurred in the middle range of temperature and salinity, whereas growth were generally inhibited at temperatures<17.5℃ and> 32.5℃, and at salinities≤20 and ≥50. In the irradiance experiment, no mortality was observed; growth from 110-400 μmol photons·m-2 ·s-1 was relatively stable but faster than growth at 55 μmol photons·m-2· s-1.High coefficient of variability of Gambierdiscus growth rates was primarily found at the end points of temperatures, salinities and irradiances tested, indicating differences among strains in their range of tolerance to these environmental conditions. In contrast, statistical differences in intraspecific growth rates were mostly encountered at optimum growth conditions, indicating that environmental conditions may help determine Gambierdiscus population structure. Maximum growth occurred at temperature and salinity ranges of 23.8-29.2℃ and 30.1-38.5 psu, respectively. Mortality was observed at temperatures of 15.9-20.1℃ and 29.8-35.4℃, and at salinities of 10.4-27.2 and 47.6-57.0. The range tolerance of Gambierdiscus to temperature and salinity was broader than previous laboratory studies and provide ecological support to field observations of Gambierdiscus, and is useful in estimating potential range extensions and understanding periodicity patterns.Gambierdiscus growth was generally strain-dependent instead of species-dependent; however, species of G. belizeanus, G. caribaeus, G. carpenteri and G. pacificus were comparatively better adapted to a range of environmental conditions, especially to extreme temperature and salinity levels, which helps interpret their broad geographic distribution. In contrast, the three Gambierdiscus ribotypes all displayed a narrow range of tolerance. Given the results of these experiments, the water temperature, salinity and irradiance levels measured at the isolation sites promote optimal growth of the Gambierdiscus populations from these locations.