Eduardo Costas

Detection of Infectious Cryptosporidium parvum Oocysts in Mussels (Mytilus galloprovincialis) and Cockles (Cerastoderma edule)

ABSTRACT Infective Cryptosporidium parvum oocysts were detected in mussels (Mytilus galloprovincialis) and cockles (Cerastoderma edule) from a shellfish-producing region (Gallaecia, northwest Spain, bounded by the Atlantic Ocean) that accounts for the majority of European shellfish production. Shellfish were collected from bay sites with different degrees of organic pollution. Shellfish harboring C. parvum oocysts were recovered only from areas located near the mouths of rivers with a high density of grazing ruminants on their banks. An approximation of the parasite load of shellfish collected in positive sites indicated that each shellfish transported more than 103 oocysts. Recovered oocysts were infectious for neonatal mice, and PCR-restriction fragment length polymorphism analysis demonstrated a profile similar to that described for genotype C or 2 of the parasite. These results demonstrate that mussels and cockles could act as a reservoir ofC. parvum infection for humans. Moreover, estuarine shellfish could be used as an indicator of river water contamination.

Revised dinoflagellate phylogeny inferred from molecular analysis of large-subunit ribosomal RNA gene sequences

The nucleotide sequence analysis of the PCR products corresponding to the variable large-subunit rRNA domains D1, D2, D9, and D10 from ten representative dinoflagellate species is reported. Species were selected among the main laboratory-grown dinoflagellate groups: Prorocentrales, Gymnodiniales, and Peridiniales which comprise a variety of morphological and ecological characteristics. The sequence alignments comprising up to 1,000 nucleotides from all ten species were employed to analyze the phylogenetic relationships among these dinoflagellates. Maximum parsimony and neighbor joining trees were inferred from the data generated and subsequently tested by bootstrapping. Both the D1/D2 and the D9/D10 regions led to coherent trees in which the main class of dinoflagellates, Dinophyceae, is divided in three groups: prorocentroid, gymnodinioid, and peridinioid. An interesting outcome from the molecular phylogeny obtained was the uncertain emergence of Prorocentrum lima. The molecular results reported agreed with morphological classifications within Peridiniales but not with those of Prorocentrales and Gymnodiniales. Additionally, the sequence comparison analysis provided strong evidence to suggest that Alexandrium minutum and Alexandrium lusitanicum were synonymous species given the identical sequence they shared. Moreover, clone Gg1V, which was determined Gymnodinium catenatum based on morphological criteria, would correspond to a new species of the genus Gymnodinium as its sequence clearly differed from that obtained in G. catenatum. The sequence of the amplified fragments was demonstrated to be a valuable tool for phylogenetic and taxonomical analysis among these highly diversified species.

Did algal toxins cause monk seal mortality

The population of Mediterranean monk seals off the coast of the western Sahara has recently suffered a sudden mortality. A morbillivirus was isolated post-mortem from the tissues of three seals, and it has been proposed that the virus was the agent responsible. This conclusion is called into question by epidemiological, clinical, pathological and toxicological considerations. We suggest here that intoxication by algal toxins is a more likely cause of the deaths.

Resistance of microalgae to modern water contaminants as the result of rare spontaneous mutations

Modern chemical residues from water pollution (such as herbicides, antibiotics and even chemical substances of military use) are a challenge to survival of microalgal populations. Adaptation of three microalgal species – Pseudanabaena planctonica (Cyanophyceae), Dunaliella tertiolecta (Polyblepharidaceae, Chlorophyceae) and Scenedesmus sp. (Chlorophyceae) – to growth and survival in the presence of erythromycin, 3-(3,4-dichlorophenyl)-1,1-dimethylurea, and 2,4,6-trinitrotoluene was analysed as an experimental model. Growth of these species was restricted even by micromolar concentrations of such xenobiotics. When such contaminants are added to a dense microalgal culture, the cell density will be reduced after a few days due to the death of sensitive cells. However, after further incubation for several days, the culture will sometimes increase in density again due to the growth of a cell variant which is resistant to the contaminants. A Luria–Delbrück fluctuation analysis was carried out to distinguish between resistant cells arising from rare spontaneous mutation and resistant cells arising from other mechanisms of adaptation. In all cases, the contaminant-resistant cells arise randomly by rare spontaneous mutation during replication of cells prior to the addition of the contaminant (pre-selective mutations). Since wild-type microalgal genotypes are unable to survive in the presence of such contaminants, spontaneous pre-selective mutation (i.e. mutation from herbicide sensitivity to herbicide resistance) offers insights into the evolutionary capabilities of microalgal populations in contaminated environments. The rate of spontaneous mutation from sensitivity to resistance ranged from 2.0 × 10−6 mutants per cell division for erythromycin sensitive→ erythromycinresistant P. planctonica to 8.2 × 10−6 mutants per cell division for TNTsensitive→ TNTresistant Scenedesmus sp. Since contaminant-resistant mutants have a diminished fitness, the resistant variants are maintained in the absence of the contaminants as the result of balance between new resistant cells arising from spontaneous mutation and resistant cells eliminated by natural selection. Thus recurrence of rare spontaneous pre-selective mutations ensures the survival of microalgal population in suddenly polluted environments.

Mutation of algae from sensitivity to resistance against environmental selective agents: the ecological genetics of Dictyosphaerium chlorelloides (Chlorophyceae) under lethal doses of 3-(3,4-dichlorophenyl)-1, 1-dimethylurea herbicide

Abstract Residues contribute to water pollution and pose a challenge to microalgal populations because numerous contaminants are toxic to microalgae, even in the micromolar range. Adaptation of microalgae from herbicide sensitivity to herbicide resistance was analysed by an ecological genetic approach, using the unicellular alga Dictyosphaerium chlorelloides (Chlorophyceae) as an experimental model. A dose–effect study showed that the Malthusian parameter under conditions of r selection in an uncrowded environment and the carrying capacity under conditions of K selection in an crowded environment were both restricted even by low concentrations (< 1 μM) of 3-(3,4-dichlorophenyl)-1, 1-dimethylurea (DCMU) herbicide. When a culture was treated with 50 μM DCMU, it cleared after a few days, as a result of destruction of sensitive cells by the herbicide. However, after further incubation for several days, the culture sometimes regained colour, owing to the growth of cell variants resistant to the herbicide. A fluctuation analysis was carried out to distinguish between (1) herbicide-resistant cells arising by direct and specific acquired adaptation in response to the herbicide and (2) herbicide-resistant cells arising by rare spontaneous mutations occurring randomly during replication of organisms prior to the incorporation of the herbicide. The fluctuation analysis unequivocally demonstrated that DCMU is not facilitating the development of DCMU-resistant cells; rather, we found that DCMU-resistant cells occur spontaneously by mutation in nonselective conditions prior to the incorporation of the herbicide (preselective mutations). The rate of spontaneous mutation from DCMU sensitivity to DCMU resistance was ∼ 2.2 × 10−6 mutants per cell division. Mutation was recurrent from a normal wild-type DCMU-sensitive allele to an DCMU-resistant allele, but such herbicide-resistant alleles were detrimental in terms of fitness in the absence of the herbicide. A competition experiment between wild-type DCMU-sensitive cells and DCMU-resistant mutants showed that, in small populations, the DCMU-resistant mutants are driven to extinction. The resistant variants are maintained in the absence of the herbicide as the result of a balance between new resistant cells arising by rare spontaneous mutation and resistant cells eliminated by natural selection. In our case, the average frequency of DCMU-resistant mutants in the absence of the DCMU is about five DCMU-resistant mutants per million cells. The results of our experimental model suggest that spontaneous mutation from herbicide sensitivity to herbicide resistance is sufficient in itself to assure the survival of microalgae populations in herbicide-contaminated environments when the population size is large.

ADAPTATION OF NEOSPORA CANINUM ISOLATES TO CELL-CULTURE CHANGES: AN ARGUMENT IN FAVOR OF ITS CLONAL POPULATION STRUCTURE

Neospora caninum, a recently recognized protozoan parasite of animals, is considered to be a major cause of bovine abortion worldwide. Although its life cycle is not completely known, recent studies suggest that the sexual stage occurs in dogs. The prevalence of sexual reproduction in N. caninum, however, is unknown. We investigated the ability of 3 N. caninum isolates (NC-1, NC-SweB1, and NC-Liverpool) to propagate asexually for approximately 250 parasite generations in a cell line in which they had not been cultured previously. The malthusian parameter of fitness was estimated for each isolate from 10 independent replicates of tachyzoites at the beginning as well as at the end of the experimental period. Derived and ancestral values for mean fitness were compared both within and among NC-1, NC-SweB1, and NC-Liverpool isolates. Results showed a significant increase in mean fitness for the 3 N. caninum isolates at the end of the experimental period. These findings indicate that N. caninum can adapt to new environmental conditions without the help of sexual recombination, supporting the idea that this parasite has, at least potentially, the capacity for maintaining clonal propagation in nature.

Catastrophic mortality of flamingos in a Spanish national park caused by cyanobacteria

However, no other such case has been recorded either on the affected farm or with specimens tested retrospectively at the Onderstepoort Veterinary Institute. Further investigation into such an occurrence is underway. In countries where both forms of the disease occur, and where it is not unusual to find wildebeest, cattle and sheep in close proximity to each other, the distinction between the two forms has an epidemiological value and may help in instituting control measures. PCR assays that can be used for reliable routine and differential diagnoses of these two infections are therefore important. The nested PCR described by Baxter and others (1993) has the advantage of being sensitive and specific for SA-MCF. Nested PCRS, however, are notoriously prone to DNA cross-contamination during the transfer of amplicons from the first to the second round (nested PCR mix). This problem is exacerbated when large numbers of samples are processed. The STN-PCR alleviates this problem since all primers needed are included in the initial mix, and, thus, this protocol is shorter and cheaper. It is suggested that the STN-PCR protocol for SA-MCF could be used as a screening tool for large numbers of specimens in 96-tube matrix plates designed for use in a thermocycler.

Evolution of microalgae in highly stressing environments: An experimental model analyzing the rapid adaptation of Dictyosphaerium chlorelloides (chlorophyceae) from sensitivity to resistance against 2,4,6-trinitrotoluene by rare preselective mutations

The increasing rates of global extinction due to human activities necessitate studies of the ability of organisms to adapt to the new environmental conditions resulting from human disturbances. We investigated the evolutionary adaptation of a microalga to sudden environmental change resulting from exposure to novel toxic chemical residues. A laboratory strain of Dictyosphaerium chlorelloides (Naum.) Kom. and Perm. (Chlorophyceae) was exposed to increasing concentrations of the modern contaminant 2,4,6‐trinitrotoluene (TNT). When algal cultures were exposed to 30 mg·L− 1 TNT, massive lysis of microalgal cells was observed. The key to understanding the evolution of microalgae in such a contaminated environment is to characterize the TNT‐resistant variants that appear after the massive lysis of the TNT‐sensitive cells. A fluctuation analysis demonstrated unequivocally that TNT did not facilitate the appearance of TNT‐resistant cells; rather it was found that TNT‐resistant cells appeared spontaneously by rare mutations under nonselective conditions, before exposure to TNT. The estimated mutation rate was 1.4 × 10−5 mutants per cell division. Isolated resistant mutants exhibited a diminished fitness in the absence of TNT. Moreover, the gross photosynthetic rate of TNT‐resistant mutants was significantly lower than that of wild‐type cells. Competition experiments between resistant mutants and wild‐type cells showed that in small populations, the resistant mutants were driven to extinction. The balance between mutation rate and the rate of selective elimination determines the occurrence of about 36 TNT‐resistant mutants per million cells in each generation. These scarce resistant mutants are the guarantee of potential for adaptation.

IDENTIFICATION OF MARINE DINOFLAGELLATES USING FLUORESCENT LECTINS1

Toxic and nontoxic species of marine dinoflagellates were characterized using fluorescent lectins. Lectin binding was detected by epifluorescence as well as spectrofluorometry. The binding assay of fluorescent lectins readily differentiated between morphologically similar species (i.e the toxic dinoflagellate Gymnodinium catenatum and the nontoxic Gymnodinium sp.). Lectins appear to be a useful tool to distinguish among different clones of the same species and, thus, possibly as a tool in dinoflagellate identification. Moreover, the lectins used show that thecate species have more binding sites and diversity in glycan moieties than athecate species.

Short-term adaptation of microalgae in highly stressful environments : an experimental model analysing the resistance of Scenedesmus intermedius (Chlorophyceae) to the heavy metals mixture from the Aznalcollar mine spill

The toxic spill of acid wastes rich in heavy metals/metalloids (AWHM) from the Aznalcóllar mine in April 1998, threatening the Doñana National Park, is considered to be the worst environmental disaster related to acute pollution in Spanish history. The aim of this work was to study the adaptation of microalgae (which play an important role as primary producers) from AWHM sensitivity to AWHM resistance by using the alga Scenedesmus intermedius as an experimental model. The Malthusian parameter (m) and the carrying capacity (K) were reduced by mud and soil samples collected from the affected area. A dose–effect analysis showed that fitness progressively diminished with increasing sample concentration. A fluctuation analysis demonstrated that AWHM-resistant cells arose by rare spontaneous mutations that occurred randomly prior to the incorporation of the AWHM. The rate of spontaneous mutation from AWHM sensitivity to AWHM resistance was 2·12×10−5 mutants per cell division. A competition experiment between wild-type AWHM-sensitive cells and AWHM-resistant mutants showed that in small populations the AWHM-resistant mutants are driven to extinction in the absence of selection for AWHM resistance. The resistant phenotypes are maintained in the absence of AWHM as the result of a balance between spontaneous mutation and natural selection, so that about 43 AWHM-resistant mutants per million cells are present in the absence of AWHM. Our experimental model suggests that mutation is essential for adaptation of microalgal populations to environmental changes. Rare spontaneous pre-adaptive mutation is enough to ensure the survival of microalgal populations in contaminated environments when the population size is large enough.