1 Vol. 1, no. 3-4, Molecular phylogenetic analysis in ecogenotoxicological studies D. Davolos*,V. Iannilli**, E. De Matthaeis**, B. Pietrangeli* * ISPESL, Department of Production Plants and Environmental Interaction, Rome ** University of Rome La Sapienza, Department of Animal and Human Biology ABSTRACT The usefulness of taxa belonging to the Amphipoda (Crustacea) in environmental toxicology is welldocumented in literature and a growing number of studies are addressing this line of research. However, as with most ecotoxicology studies, this research is rarely supported by analyses of the genetic structure and evolutionary history of the taxa under examination.the availability of such information becomes crucial when research is being carried out on the effects, such as genotoxic ones, of exposure to particular substances. In this work, two crustaceans (Orchestia garbinii and Gammarus aequicauda;amphipoda) from aquatic habitats and with different ecological characteristics were chosen as potential candidates for ecogenotoxicological assessment for water, as well as for sediments in toto. Data are presented on the genetic structure and on the relationships among populations of these Crustaceans, inferred by using sequences of mitochondrial genes. Furthermore, various methods are discussed, which are helpful in assessing genotoxicity in tissues of organisms exposed in the laboratory and in populations taken from contaminated sites. 69 (Key words: ecogenotoxicology, molecular phylogeny,amphipoda) BOW PO/base indexing: EUOSHA - OSH: Genetic toxicology (26601D), Environmental pollution (05481D), Measurement and assessment (12561D) CIS: Ecogenetics (Wopg), Ecotoxicology (Sepe), Crustacea (Fidu),Water pollution (Bubue), Sampling and analysis (Qe) Reviewed and accepted: 20/02/2006 by Giovanni Alfredo Zapponi; 17/03/2006 by Laura Mancini - National Institute of Health (ISS)
2 INTRODUCTION 70 The development of Community and national norms on the subject of water protection, geared initially towards the protection of drinking water, bathing water and of the consumption of edible aquatic organisms, is currently geared towards an approach of integrated protection, taking into account the necessity to safeguard the entire aquatic ecosystem. In fact, defence of the whole hydric environment from pollution caused by the introduction of dangerous substances from specific and widespread sources is necessary in order to ensure the protection of human health (e.g. from risks deriving from the transfer of contaminants through processes of bioaccumulation and biomagnification). Criteria for toxicity and ecotoxicity are therefore extremely useful for defining environmental quality standards that are necessary to achieve a good chemical state in bodies of water 1-2. The usefulness of determined taxa in environmental toxicology is well-documented in scientific literature 3-5. However, ecotoxicology studies are rarely supported by analyses of the genetic structure or the evolutionary history of the taxa under examination.the availability of this information 6-14 turns out to be indispensable when research is being carried out on the effects (e.g. the genotoxic effects) from exposure to particular substances Two Crustaceans belonging to Amphipoda and linked to aquatic environments were chosen for this study, as potential candidates for ecogenotoxicological assessment (direct, indirect and long-term effects) both for water and for sediments in toto.analyses were carried out on the talitrid, Orchestia garbinii (Fig. 1), a semiterrestrial species found along the banks of lakes and rivers in Europe, and the gammarid, Gammarus aequicauda (Fig. 2), found in salt water and the lagoon systems of the Mediterranean. Figure 1 - Orchestia garbinii (Crustacea,Amphipoda,Talitridae): adult male, 20 mm Figure 2 - Gammarus aequicauda (Crustacea,Amphipoda, Gammaridae): adult male, 15 mm In this paper, we present phylogenetic results on those crustaceans with the focus on gene sequences of mitochondrial DNA (mtdna). We will also discuss various methods that are helpful for evaluating genotoxicity and mutagenesis in tissues of organisms exposed in the laboratory and in populations taken from contaminated sites.
3 1. MATERIALS AND METHODS The sampling sites of O. garbinii were: Lake Garda (Verona ) and Lake Bracciano (Rome) (Fig. 3a,b).The samples of G. aequicauda were taken from the following sites: Lake Patria (Caserta); the mouth of the River Ombrone (Grosseto); the mouth of the River Mignone (Viterbo); the mouth of the River Candeloro, Manfredonia (Foggia). Figure 3 - Sampling sites of Orchestia garbinii: (a) Lake Garda and (b) Lake Bracciano Lake Garda (a) 71 Lake Bolsena Lake Vico Lake Bracciano (b)
4 The methods used for amplification by Polymerase Chain Reaction (PCR) and the sequencing of mitochondrial genes of the subunits I and II of the cytochrome oxidase (COI and COII) 23-5 and of 16S ribosomal RNA (16S rrna) are shown in 14, Homologous sequences of the mtdna of talitrids 14 (one sequence is deposited in GenBank, NCBI, with access number AY555730), of gammarids 28,29 and of other Crustacea (extracted from GenBank) were used to carry out phylogenetic analyses by the Neighbour- Joining and Maximum Parsimony methods 14. Molecular phylogenetic inferences were carried out by using nucleotidic sequences (examining both transitions and transversions) and the deduced amino acid sequences (with Poisson correction) 14. From 500 to 0 bootstrap replications were calculated for each phylogenetic reconstruction RESULTS 72 Mitochondrial regions encoding protein and ribosomal RNA of O. garbinii and of G. aequicauda were amplified through the PCR method and then sequenced.the alignments with homologous sequences of Crustacea generally resulted as unambiguous.the gene trna LeuUUR was found between the genes for the subunits I and II of the cytochrome oxidase (COI and COII) in G. aequicauda, while COI-NC-COII 14 rearrangement was found in O. garbinii. Inferences on the evolutionary relationships of O. garbinii and G. aequicauda (potential candidates for ecogenotoxicological assessment for water as well as for sediments) were obtained through phylogenetic reconstruction using mitochondrial sequences. Figs. 4 and 5 show some results of a phylogenetic analysis of the two Amphipods examined and of other Crustacea, based on sequences of nucleotides and amino acids encoded by the COI and COII genes. Fig. 6 shows a phylogenetic study of O. garbinii and G. aequicauda based on nucleotide sequences of ribosomal RNA of the large subunit (16S rrna). Figure 4 - Molecular phylogeny of O. garbinii, talitrids and other Crustacea (sequences extracted from GenBank) obtained through (a) Neighbour-Joining on 121 amino acids (Poisson correction) encoded by a region of the COI gene and (b) Maximum Parsimony (consensus tree) on 367 nucleotides of the COI gene Panulirus japonicus Pagurus longicarpus Penaeus monodon Orchestia gammarellus (I. Cumbrae) Orchestia gammarellus (I.Wight) Orchestia mediterranea (I.Wight) Talorchestia deshayesii (I.Wight) Talitrus saltator (I.Wight) Talitrus ssltator (I. Cumbrae) Orchestia stephenseni Orchestia garbinii (L. Garda) Orchestia garbinii (L. Bracciano) Parhjale hawaiiensis Triops cancriformis Daphnia pulex Artemia francescana Tigriopus japonicus Hyalidae Talitridae Talitroidea 0,05 (a)
5 66 Triops cancriformis Daphnia pulex Artemia francescana Panulirus japonicus Penaeus monodon Pagurus longicarpus Parhyale hawaiiensis Hyalidae Orchestia garbinii (L. Garda) Orchestia garbinii (L. Bracciano) Orchestia gammarellus (I. Cumbrae) Orchestia gammarellus (I.Wight) Orchestia mediterranea (I.Wight) Orchestia stephenseni (AY555730) Talitrus saltator (I.Wight) Talitrus saltator (I. Cumbrae) Talorchestia deshayesii (I.Wight) Talitridae Talitroidea (b) Figure 5 - Molecular phylogeny of O. garbinii, of G. aequicauda and other Crustacea (sequences extracted from Genbank) obtained through Maximum Parsimony (consensus tree) on 114 amino acids encoded by regions of the COI and COII genes Daphnia pulex Triops cancriformis Pagurus longicarpus Penaeus monodon Gammarus aequicauda (Mignone) Gammarus aequicauda (L. Patria) Gammarus aequicauda (Ombrone) Gammarus aequicauda (Candeloro) Parhyale hawaiiensis Hyalidae Orchestia garbinii (L. Bracciano) Orchestia garbinii (L. Garda) Talitrus saltator (I.Wight) Talitrus saltator (I. Cumbrae) Talorchestia deshayesii (I.Wight) Orchestia gammarellus (I.Wight) Orchestia gammarellus (I. Cumbrae) Orchestia mediterranea (I.Wight) Gammaridae Talitridae Talitroidea 73 Figure 6 - Molecular phylogeny of O. garbinii, of G. aequicauda and of other Crustacea (sequences extracted from GenBank) obtained through Maximum Parsimony (consensus tree) on 246 nucleotides of the 16S rrna gene (500 bootstrap replications) Pagurus longicarpus Penaeus monodon Gammarus locusta Gammarus aequicauda (Black Sea) Gammarus aequicauda (L. Patria) Gammarus aequicauda (Mignone) Gammarus balcanicus Gammarus fasciatus Gammarus mucronatus Gammarus annulatus Gammarus oceanicus Gammarus elvirae Gammarus lacustris (L. Hovsgol) Chaetogammarus marinus Orchestia garbinii (L. Garda) Orchestia garbinii (L. Bracciano) Orchestia cavimana (AY744911) Parhyale hawaiiensis Hyalidae Gammaridae Talitridae Talitroidea
6 3. DISCUSSION 74 Many human activities (in the industrial, urban, agricultural sectors, etc.) have caused an increase in the environmental concentration of particular pollutants including heavy metals, mutagenic substances, etc.the negative effects (direct and indirect) of xenobiotic agents can be significant in natural populations 12-30, with a potential risk of exposure and repercussions on human health 31. In recent years, many research projects have focussed attention on the identification of species that are useful in ecogenotoxicological assessments on different types of matrices. However, the lack of phylogenetic analyses of the taxa under examination may entail a margin of error in the interpretation of the research results, e.g. environmental-genotoxicological 16,17,21,22. Low levels of genetic divergence found among the populations of O. garbinii validate this organism as a suitable subject for ecotoxicological investigation on various geographical scales It should be noted that our research group is involved in further molecular studies to examine populations of O. garbinii and G. aequicauda sampled in other geographical areas, analysing especially the control region of mtdna which generally presents hypervariable portions 36 and non-coding mitochondrial segments 14,37,38. Furthermore, studies into genotoxicology 39 and mutagenesis (Fig. 7) are underway on organisms exposed in the laboratory. Figure 7 - Nucleotidic mutation in the COI gene highlighted by sequencing of mtdna. (Davolos, study pending).the chromatograms are visualized with Chromas software version
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