Species‐specific PCR assays for Gambierdiscus excentricus and Gambierdiscus silvae (Gonyaulacales,Dinophyceae)

The two most toxic Gambierdiscus species identified from the Caribbean are G. excentricus and G. silvae. These species are the primary causes of ciguatera fish poisoning and likely contribute disproportionately to the toxicity of marine food webs. While Gambierdiscus species are difficult to distinguish using light or scanning electron microscopy, reliable species‐specific molecular identification methods have been developed and used successfully to identify a number of other Gambierdiscus species. Corresponding species‐specific assays are not yet available for G. excentricus and G. silvae, which imposes limitations on species identification and related ecological studies. The following note describes species‐specific polymerase chain reaction assays for G. excentricus and G. silvae that can be used for these purposes.     

Gambierdiscus (Dinophyceae) species diversity in the Flower Garden Banks National Marine Sanctuary,Northern Gulf of Mexico,USA

Globally, ciguatera fish poisoning (CFP) is the principal cause of non-bacterial illness associated with seafood consumption. The toxins (ciguatoxins) responsible for CFP are produced by dinoflagellates in the genus Gambierdiscus, which are endemic to tropical and sub-tropical areas. Ciguatoxins are lipophilic and bioaccumulate in marine food webs, typically reaching their highest concentrations in fish. Following a CFP event in 2008, the U.S. Food and Drug Administration (USFDA) issued a ciguatera toxin alert that included fish harvested in the northern Gulf of Mexico in and near the Flower Garden Banks National Marine Sanctuary (FGBNMS). The East Flower Garden Bank (EFGB) and West Flower Garden Bank (WFGB) are characterized by thriving coral communities that support Gambierdiscus growth. This study was undertaken specifically to document the diversity of Gambierdiscus species present in the sanctuary that may be sources of ciguatoxins entering the food web. Samples collected from the FGBNMS over a three year period were screened using species-specific polymerase chain reaction assays. A diverse assemblage of Gambierdiscus species was distributed to depths of >45 m, a new depth record for Gambierdiscus. Gambierdiscus belizeanus, Gambierdiscus caribaeus, Gambierdiscus carolinianus, Gambierdiscus carpenteri and Gambierdiscus ribotype 2 were all found on both East and West FGB with Gambierdiscus ruetzleri also recorded from the WFGB. The most common species was G. carolinianus, originally identified from samples collected between 35 and 40 m off the coast of NC, USA. Our findings are consistent with recent physiological studies showing that some Gambierdiscus species can grow year round at the temperatures and salinities at the FGBNMS and at light levels as low as 10 μmol photons m⁻² s⁻¹. Such irradiances are estimated to occur in the FGBNMS at depths of ∼70–80 m. The consistent recovery of Gambierdiscus species from deep sampling sites in areas known to produce ciguatoxic fish signals a substantial change in our concept of suitable habitats for Gambierdiscus to include depths greater than 50 m.     

Evaluation of Gambierdiscus survival after exposure to ballast water

The dinoflagellate Gambierdiscus was exposed to ballast water from a trans-oceanic vessel, and maintained at a variety of temperatures in the dark to determine if viability would be maintained. Logarithmically growing Gambierdiscus inocula were admixed (1:6, vol:vol) with ballast water, maintained in the dark at 22.6 °C, 24.6 °C, 26.8 °C and 29.0 °C and assessed for numerical abundance over six days. Calculated growth rates from the biomass time series showed no indication that ballast water negatively impacted Gambierdiscus viability; accompanying microscopic inspections supported this conclusion. Filtration of large volumes of collected ballast water failed to show the presence of any Gambierdiscus cells contained therein. Recovery and microscopic examination of the experimental inocula after 10 weeks in the dark, failed to show cyst development at any temperature regime. This examination of ballast water showed no evidence of cytotoxicity to Gambierdiscus spp.     

Influence of Environmental Variables on Gambierdiscus spp. (Dinophyceae) Growth and Distribution

Benthic dinoflagellates in the genus Gambierdiscus produce the ciguatoxin precursors responsible for the occurrence of ciguatera toxicity. The prevalence of ciguatera toxins in fish has been linked to the presence and distribution of toxin-producing species in coral reef ecosystems, which is largely determined by the presence of suitable benthic habitat and environmental conditions favorable for growth. Here using single factor experiments, we examined the effects of salinity, irradiance, and temperature on growth of 17 strains of Gambierdiscus representing eight species/phylotypes (G. belizeanus, G. caribaeus, G. carolinianus, G. carpenteri, G. pacificus, G. silvae, Gambierdiscus sp. type 4–5), most of which were established from either Marakei Island, Republic of Kiribati, or St. Thomas, United States Virgin Island (USVI). Comparable to prior studies, growth rates fell within the range of 0–0.48 divisions day^-1. In the salinity and temperature studies, Gambierdiscus responded in a near Gaussian, non-linear manner typical for such studies, with optimal and suboptimal growth occurring in the range of salinities of 25 and 45 and 21.0 and 32.5°C. In the irradiance experiment, no mortality was observed; however, growth rates at 55μmol photons · m^-2 · s^-1 were lower than those at 110–400μmol photons · m^-2 · s^-1. At the extremes of the environmental conditions tested, growth rates were highly variable, evidenced by large coefficients of variability. However, significant differences in intraspecific growth rates were typically found only at optimal or near-optimal growth conditions. Polynomial regression analyses showed that maximum growth occurred at salinity and temperature levels of 30.1–38.5 and 23.8–29.2°C, respectively. Gambierdiscus growth patterns varied among species, and within individual species: G. belizeanus, G. caribaeus, G. carpenteri, and G. pacificus generally exhibited a wider range of tolerance to environmental conditions, which may explain their broad geographic distribution. In contrast, G. silvae and Gambierdiscus sp. types 4–5 all displayed a comparatively narrow range of tolerance to temperature, salinity, and irradiance.     

The prevalence of benthic dinoflagellates associated with ciguatera fish poisoning in the central Red Sea

This study confirms the presence of the toxigenic benthic dinoflagellates Gambierdiscus belizeanus and Ostreopsis spp. in the central Red Sea. To our knowledge, this is also the first report of these taxa in coastal waters of Saudi Arabia, indicating the potential occurrence of ciguatera fish poisoning (CFP) in that region. During field investigations carried out in 2012 and 2013, a total of 100 Turbinaria and Halimeda macroalgae samples were collected from coral reefs off the Saudi Arabian coast and examined for the presence of Gambierdiscus and Ostreopsis, two toxigenic dinoflagellate genera commonly observed in coral reef communities around the world. Both Gambierdiscus and Ostreopsis spp. were observed at low densities (<200 cells g⁻¹ wet weight algae). Cell densities of Ostreopsis spp. were significantly higher than Gambierdiscus spp. at most of the sampling sites, and abundances of both genera were negatively correlated with seawater salinity. To assess the potential for ciguatoxicity in this region, several Gambierdiscus isolates were established in culture and examined for species identity and toxicity. All isolates were morphologically and molecularly identified as Gambierdiscus belizeanus. Toxicity analysis of two isolates using the mouse neuroblastoma cell-based assay for ciguatoxins (CTX) confirmed G. belizeanus as a CTX producer, with a maximum toxin content of 6.50 ± 1.14 × 10⁻⁵ pg P-CTX-1 eq. cell⁻¹. Compared to Gambierdiscus isolates from other locations, these were low toxicity strains. The low Gambierdiscus densities observed along with their comparatively low toxin contents may explain why CFP is unidentified and unreported in this region. Nevertheless, the presence of these potentially toxigenic dinoflagellate species at multiple sites in the central Red Sea warrants future study on their possible effects on marine food webs and human health in this region.     

Effects of temperature,salinity and their interaction on growth of Japanese Gambierdiscus spp. (Dinophyceae)

Marine toxic dinoflagellates of the genus Gambierdiscus are the causative agents of ciguatera fish poisoning (CFP), a seafood poisoning that is widespread in tropical, subtropical and temperate regions of the world. In the main island of Japan, distributions of Gambierdiscus australes, Gambierdiscus scabrosus and two phylotypes of Gambierdiscus spp. type 2 and type 3, have been reported. To discuss the bloom dynamics of these Japanese species/phylotypes of Gambierdiscus, first we tested six culture media to optimize growth conditions and then clarified the effects of temperature and salinity and temperature–salinity interactions on growth. All strains of the species/phylotypes tested showed the highest cell yields when they were cultivated in IMK/2 medium. G. australes, G. scabrosus and Gambierdiscus sp. type 2 grew in the range 17.5–30 °C, whereas Gambierdiscus sp. type 3 grew in 15–25 °C. The semi-optimal temperature ranges (≥80% of the maximal growth rate) of the former three species/phylotypes were 19–28 °C, 24–31 °C and 21–28 °C, respectively, whereas that of the latter phylotype was 22–25 °C. Hence, Gambierdiscus sp. type 3 may be adapted to relatively lower water temperatures of ≤25 °C. In contrast, G. australes, G. scabrosus and Gambierdiscus sp. type 2 presumably possess adaptability to relatively high water temperatures. The optimal temperature for G. scabrosus was 30 °C, whereas the optimal temperature for the others was 25 °C. G. australes and Gambierdiscus sp. type 3 grew in a salinity range of 25–40 whereas G. scabrosus and Gambierdiscus sp. type 2 grew in salinity 20–40. Furthermore, the semi-optimal salinity range of G. australes, G. scabrosus, Gambierdiscus spp. type 2 and type 3 were salinity 27–38, 24–36, 22–36 and 29–37, respectively. Among the species/phylotypes, G. scabrosus and Gambierdiscus sp. type 2 grew even at salinity 20 where the others did not grow, thus possessing adaptability to low salinity waters. Our results clearly demonstrate that the optimal and tolerable temperature–salinity conditions differ among Japanese Gambierdiscus species/phylotypes. Considering these results, temperature–salinity interactions may play an important role in bloom dynamics and the distribution of the Gambierdiscus species/phylotypes in Japanese coastal waters.     

Characterization of Gambierdiscus lapillus sp. nov. (Gonyaulacales,Dinophyceae): a new toxic dinoflagellate from the Great Barrier Reef (Australia)

Gambierdiscus is a genus of benthic dinoflagellates found worldwide. Some species produce neurotoxins (maitotoxins and ciguatoxins) that bioaccumulate and cause ciguatera fish poisoning (CFP), a potentially fatal food‐borne illness that is common worldwide in tropical regions. The investigation of toxigenic species of Gambierdiscus in CFP endemic regions in Australia is necessary as a first step to determine which species of Gambierdiscus are related to CFP cases occurring in this region. In this study, we characterized five strains of Gambierdiscus collected from Heron Island, Australia, a region in which ciguatera is endemic. Clonal cultures were assessed using (i) light microscopy; (ii) scanning electron microscopy; (iii) DNA sequencing based on the nuclear encoded ribosomal 18S and D8‐D10 28S regions; (iv) toxicity via mouse bioassay; and (v) toxin profile as determined by Liquid Chromatography‐Mass Spectrometry. Both the morphological and phylogenetic data indicated that these strains represent a new species of Gambierdiscus, G. lapillus sp. nov. (plate formula Po, 3′, 0a, 7″, 6c, 7‐8s, 5?, 0p, 2″″ and distinctive by size and hatchet‐shaped 2′ plate). Culture extracts were found to be toxic using the mouse bioassay. Using chemical analysis, it was determined that they did not contain maitotoxin (MTX1) or known algal‐derived ciguatoxin analogs (CTX3B, 3C, CTX4A, 4B), but that they contained putative MTX3, and likely other unknown compounds.     

Detection of Gambierdiscus and Fukuyoa single cells using recombinase polymerase amplification combined with a sandwich hybridization assay

Dinoflagellates of the genera Gambierdiscus and Fukuyoa are known to produce several bioactive compounds including the potent neurotoxic ciguatoxins (CTXs) which are able to accumulate in fish and through the food web. When humans ingest fish contaminated with CTXs, it can result in an intoxication named ciguatera. Although not all the currently recognized species are able to produce toxins, G. australes and G. excentricus have been highlighted to be the most abundant and toxic among the species present in the Atlantic. Even though the genera Gambierdiscus and Fukuyoa are endemic to tropical areas, recently their presence was recorded in subtropical and temperate regions. In this work, the development of three molecular assays for the detection of the Gambierdiscus and Fukuyoa genera and for G. australes and G. excentricus species, based on the combination of recombinase polymerase amplification with detection via hybridization, is successfully described. Furthermore, a remarkable limit of detection of a single cell was achieved. Additionally, six different species have been used to check the ability of each primer set to give an amplified product, even in presence of potentially interfering non-target DNAs. Therefore, these developments provide a rapid and cost-effective strategy for detection of both genera and two of the most toxic species, which will undoubtedly contribute to reliable screening of samples and ciguatera risk assessment, guaranteeing seafood safety and protection of human health.

     

Associations between fish health and Pfiesteria spp. in Chesapeake Bay and mid-Atlantic estuaries

In response to concerns that there may be an association between harmful algal bloom (HAB) species and fish health, including the widespread use of fish health as one indicator of a possible HAB warranting further investigation, evidence for such an association was evaluated in Chesapeake Bay and other mid-Atlantic estuaries (1999–2001). A statistical approach was used, without invoking causality, to test whether there is an association between the prevalence of externally-visible lesions in fish populations above background levels and the presence of Pfiesteria spp. in co-located water and fish samples. Externally visible anomalies (e.g. ulcers, necrosis, parasites, etc.) were recorded for Atlantic menhaden (Brevoortia tyrannus) and all other fish collected. Polymerase chain reaction (PCR) techniques were used to test for the presence of Pfiesteria spp. in water samples collected at routine and rapid response sampling events. No actively toxic Pfiesteria was found during this study. Fine-scale (within a given sample site) and broad-scale (estuary-wide sampling) comparisons showed positive associations between externally-visible fish lesions in menhaden populations and the presence of Pfiesteria spp. in co-located samples. Logistic regression modeling of Pfiesteria detection probabilities as a function of prevalence of menhaden with lesions was significant (P = 0.0096). Reductions in the false positive (tests indicating Pfiesteria presence when its absent) and false negative (tests indicating Pfiesteria is absent when it is actually present) rates occurred when the minimum sample size threshold increased from 1 to 30 fish (P = 0.003–0.001). This association served as a useful field indicator of potential HAB activity that could warrant further field investigation and testing.     

Effects of temperature,salinity and composition of the dinoflagellate assemblage on the growth of Gambierdiscus carpenteri isolated from the Great Barrier Reef

Increases in reported incidence of ciguatera fish poisoning (hereafter ciguatera) have been linked to warmer sea temperatures that are known to trigger coral bleaching events. The drivers that trigger blooms of ciguatera-causing dinoflagellates on the Great Barrier Reef (GBR) are poorly understood. This study investigated the effects of increased temperatures and lowered salinities, often associated with environmental disturbance events, on the population growth of two strains of the potentially ciguatera-causing dinoflagellate, Gambierdiscus carpenteri (NQAIF116 and NQAIF380). Both strains were isolated from the central GBR with NQAIF116 being an inshore strain and NQAIF380 an isolate from a stable environment of a large coral reef aquarium exhibit in ReefHQ, Townsville, Australia. Species of Gambierdiscus are often found as part of a mixed assemblage of benthic toxic dinoflagellates on macroalgal substrates. The effect of assemblage structure of dinoflagellates on the growth of Gambierdiscus populations has, however, not been explored. The study, therefore investigated the growth of G. carpenteri within mixed assemblages of benthic dinoflagellates. Population growth was monitored over a period of 28 days under three salinities (16, 26 and 36) and three temperature (24, 28 and 34 °C) conditions in a fully crossed experimental design. Temperature and salinity had a significant effect on population growth. Strain NQAIF380 exhibited significantly higher growth at 28 °C compared to strain NQAIF116, which had highest growth at 24 °C. When strain NQAIF116 was co-cultured with the benthic dinoflagellates, Prorocentrum lima and Ostreopsis sp., inhibitory effects on population growth were observed at a salinity of 36. In contrast, growth stimulation of G. carpenteri (strain NQAIF116) was observed at a salinity of 26 and particularly at 16 when co-cultured with Ostreopsis-dominated assemblages. Range expansion of ciguatera-causing dinoflagellates could lead to higher frequency of reported ciguatera illness in populated temperate Australian regions, outside the tropical range of the GBR. Therefore, the findings on salinity and temperature tolerance of two strains of G. carpenteri indicates potential adaptability to different local environmental conditions. These are baseline data for future investigations into the potential southward range expansion of ciguatera-causing dinoflagellates originating from the GBR.     

An examination of the epiphytic nature of Gambierdiscus toxicus, a dinoflagellate involved in ciguatera fish poisoning

Twenty-four specimen of macroalgae were collected in nearshore waters of the island of Hawaii, identified, and maintained to examine how the epiphytic relationship between Gambierdiscus toxicus (isolate BIG12) varied among the macroalgal species. Gambierdiscus cells were introduced to Petri dishes containing 100 g samples of each macroalgal host, which were examined at two, 16, 24, and every 24–72 h thereafter, over a 29-day period. Gambierdiscus proliferated in the presence of some host species (e.g., Galaxaura marginata and Jania sp.), but grew little in the presence of other species (e.g., Portieria hornemannii). Gambierdiscus exhibited high survival rates (>99%) in the presence of Chaetomorpha sp., but died before the end of the experiment (after 21 days) with other host species (e.g., Dictyota and Microdictyon spp.). Gambierdiscus avoided contact with P. hornemannii, but averaged up to 30% attachment with other host species. The numbers of Gambierdiscus cells belonging to one of three classes (alive and attached; alive and unattached; and dead) were determined for each time point. The 24 algal hosts were grouped according to their commonalities relative to these three classes using a Bray-Curtis similarity index, similarity profile (SIMPROF) permutation tests, and Multi-Dimensional Scaling (MDS) analysis (PRIMER 6). The resultant six groupings were used to construct different Gambierdiscus growth profiles for the different algal hosts. Group A is characterized by a preponderance of unattached cells and high mortality rates. Groups B, C, E, and F also displayed high proportions of unattached cells, but mortality either occurred later (Groups B and C) or rates were lower (Groups E and F). Group D had the highest proportion of attached cells. Group E contained three out of the four chlorophyte species, while Group F contained the majority of the rhodophytes. Over 50% of the species in Group F are considered to be palatable, whereas Groups A, B, and C are composed of species that exhibit chemical defenses against herbivory. The results of this study coupled with previous findings indicate that Gambierdiscus is not an obligate epiphyte; it can be free-swimming and found in the plankton. The conditions that lead to changes between epiphytic and planktonic stages need to be better studied in order to determine how they affect Gambierdiscus growth and physiology, connectivity and dispersion mechanisms, and toxin movement up into the foodweb.     

First report of the benthic dinoflagellate,Gambierdiscus belizeanus (Gonyaulacales: Dinophyceae) for the east coast of Sabah,Malaysian Borneo

Species of the genus Gambierdiscus Adachi & Fukuyo, in particular G. toxicus Adachi & Fukuyo are known producers of neurotoxins associated with ciguatera fish poisoning (CFP). In this study live samples were collected from seaweed beds of the east coast of Sabah, Malaysian Borneo and a strain of Gambierdiscus was isolated and cultured. Examination of the thecal fine morphology was undertaken using light, epifluorescence, and scanning electron microscopy. Observed morphological features and their associated morphometric information enabled identification to Gambierdiscus belizeanus Faust. This represents the first report for the occurrence of G. belizeanus in the Asia Pacific region.     

Spatio-temporal dynamics and biotic substrate preferences of benthic dinoflagellates in the Lesser Antilles,Caribbean sea

Epibenthic dinoflagellates were monitored monthly over an 18 month period in Guadeloupe and Martinique (Lesser Antilles, Caribbean Sea). These islands are located in the second most affected ciguatera fish poisoning (CFP) region of the world. Guadeloupe presented five times more total epibenthic dinoflagellates and two times less abundant Gambierdiscus spp. compared to Martinique, although the area of frequent CFP outbreaks covers Guadeloupe and not Martinique. Results did not show any clear seasonal variations of benthic dinoflagellates abundances. Temperature and salinity were not driving parameters in the evolution of total benthic dinoflagellate abundances. Preferential associations were found between macrophyte species and epibenthic dinoflagellates. The Phaeophyceae Dictyota spp. hosted the highest abundances of total epibenthic dinoflagellates, composed mainly of Ostrepsis and Prorocentrum genera. The seagrass Halophila stipulacea hosted the highest abundances of Gambierdiscus spp. and Sinophysis spp. whilst the highest abundance of Coolia was determined on Galaxaura spp. The pelagic Sargassum spp. hosted the lowest abundances of benthic dinoflagellates including the genus Gambierdiscus.     

Note on the occurrence of Pseudo-nitzschia australis and domoic acid in squid from Monterey Bay, CA (USA)

Over the past decade diatom blooms of domoic acid (DA)-producing Pseudo-nitzschia spp. have been responsible for numerous marine mammal and bird mortalities in Monterey Bay, CA. One possible toxin vector is the market squid, Loligo opalescens, a small pelagic mollusk that plays an important role in the near-shore food web of the California Current ecosystem as a favored vertebrate prey species. This study examined the trophic link between toxic Pseudo-nitzschia and L. opalescens using toxin and stomach content analyses of animals collected from Monterey Bay, CA in 2000. Receptor binding assay data (confirmed by tandem mass spectrometry), demonstrated the presence of DA in squid during a toxic Pseudo-nitzschia event, with P. australis frustules observed in stomach samples. Though DA levels were low (<0.5 μg DA g⁻¹ tissue) in L. opalescens during the study period, it is now clear that this potent neurotoxin can occur in squid and is likely delivered through its krill prey species, which are known to retain DA after feeding on toxic Pseudo-nitzschia. Our findings suggest that further study of the relationship between Pseudo-nitzschia blooms and DA contamination of squid is warranted to better evaluate the potential health risk to humans and wildlife associated with this major commercial seafood species and important prey item.     

Role of symbiotic and non-symbiotic bacteria in carbon dioxide production from hosts infected with Steinernema riobrave

Entomopathogenic nematodes of the family Steinernematidae and their mutualistic bacteria (Xenorhabdus spp.) are lethal endoparasites of insects. We hypothesized that growth of the nematode’s mutualistic bacteria in the insect host may contribute to the production of cues used by the infective juveniles (IJs) in responding to potential hosts for infection. Specifically, we tested if patterns of bacterial growth could explain differences in CO₂ production over the course of host infection. Growth of Xenorhabdus cabanillasii isolated from Steinernema riobrave exhibited the characteristic exponential and stationary growth phases. Other non-nematode symbiotic bacteria were also found in infected hosts and exhibited similar growth patterns to X. cabanillasii. Galleria mellonella larvae infected with S. riobrave produced two distinct peaks of CO₂ occurring at 25.6–36 h and 105–161 h post-infection, whereas larvae injected with X. cabanillasii alone showed only one peak of CO₂, occurring at 22.8–36.2 h post-injection. Tenebrio molitor larvae infected with S. riobrave or injected with bacteria alone exhibited only one peak of CO₂ production, which occurred later during S. riobrave infection (41.4–64.4 h post-infection compared to 20.4–35.9 h post-injection). These results indicate a relationship between bacterial growth and the first peak of CO₂ in both host species, but not for the second peak exhibited in G. mellonella.     

A survey of epiphytic dinoflagellates from the coastal waters of the island of Hawai‘i

Three hundred and sixty-nine macroalgal and non-algal samples were collected from six coastal sites around the island of Hawai‘i on a biweekly basis over a 1-year period to ascertain (1) the presence of potentially toxigenic benthic dinoflagellates, and (2) substrate and environmental preferences of the dinoflagellates. Twenty-six genera/species of dinoflagellates were encountered including the (potentially) toxigenic species Amphidinium sp., Coolia monotis, Gambierdiscus sp., Ostreopsis ovata, Prorocentrum concavum, Prorocentrum hoffmannianum, Prorocentrum lima, and Prorocentrum mexicanum. Twenty of the species are being reported for the first time as present in Hawaiian coastal waters. There was some evidence of macroalgal host preference (e.g., C. monotis on Tolypiocladia glomerulata), although host morphology preferences was greater (e.g., Gambierdiscus sp., and P. lima on filamentous turfs; O. sp. 1 on sheet-like macroblades). While some dinoflagellate groups were significantly correlated with nutrient concentrations (e.g., total dinoflagellates with nitrate + nitrite and phosphate concentrations), others were not (e.g., Gambierdiscus sp., Prorocentrum emarginatum, P. lima, and Sinophysis microcephalus). The presence of several potentially toxigenic dinoflagellate species in Hawai‘i merits future study on possible impacts of these dinoflagellates on coastal food webs and human health.     

Occurrence and toxicity of Amphidinium carterae Hulburt in the North Arabian Sea

The occurrence and toxicity of Amphidinium carterae Hulburt is hereby reported for the first time from the North Arabian Sea on the coast of Pakistan. The concentrations of 1.2 × 10⁴ cells ml⁻¹ were found in intertidal pools that were also inhabited by the brown macroalga Sargassum wightii. Both wild and cultured A. carterae cells were tested for ciguatera toxicity through exposure to brine shrimp nauplii (Artemia salina) and albino mice. Although the brine shrimp did not appear to be affected mortalities in mice ranged between 13 and 16% at doses of 7.2 × 10⁴ and 2.5 × 10⁵ cells ml⁻¹, respectively. When mice were affected pharmacological effects such as muscle contraction in lower back area, increased respiration, immobility and paralysis in hind limbs were observed for 2 h. These effects appeared to be reversible and gradually disappeared within 24 h.     

Biological and Integrated Control of Botrytis Bunch Rot of Grape UsingTrichodermaspp.

Field trials were carried out in upstate New York in 1990, 1992, 1993, and 1994 and in Chile in 1992–1993 and 1993–1994 in order to evaluate the ability of various strains ofTrichodermaspp. to control bunch rot of grape, to assess the compatibility and possible additive effects of selected biocontrol fungi and dicarboximide fungicides, and to determine factors affecting biocontrol efficacy. In 1990, three strains ofTrichodermaspp. were evaluated for their biocontrol ability, and all provided significant control ofBotrytis cinerea.As few as two late applications of the biocontrol fungi were nearly as effective as up to five applications throughout bloom and fruit development. Trials in New York in 1992 and in Chile in 1992–1993 indicated thatTrichoderma harzianumcould replace some applications of iprodione or vinclozolin with little reduction in efficacy. In New York in 1993, we found that applications ofT. harzianumat bloom and early fruit development followed by a tank-mix application ofT. harzianumand half rates of iprodione gave extremely effective control of bunch rot. In 1994, less effective control was obtained than in earlier years. Addition of a nutritive adhesive (Pelgel, a mixture of carboxymethyl cellulose and gum arabic) applied with the biocontrol agent tended to improve results. Thus, biological control of bunch rot of grape withT. harzianumcan be an effective method of management of this disease.