Different approaches to the response of benthic foraminifera to heavy metal pollution

Resumo

This PhD thesis consists of three different experiments based on the same premise, that recent benthic foraminifera experience changes when facing heavy metal pollution, and therefore they are excellent organisms to be used as bioindicators of marine ecosystems. Nevertheless, in natural conditions is difficult to attribute their alterations to one single parameter; due to that, the works set for this thesis were based on laboratory conditions. Foraminifera are an extremely successful taxon, which can be defined as singlecellular organisms that develop a shell to isolate them from the environment. Mostly marine, they had colonized all latitudes in the world, and there are species that inhabit even soil and fresh water environments. Both individuals and populations show changes and alterations linked to changes in their environment, due to that they have been used as bioindicators since decades ago. These alterations can be changes in population parameters, density, species diversity or dominance relationships for instance. Apart from that, benthic foraminifera at an individual level show ultrastructural and geochemical changes due to alterations in their environment. However, in natural locations sometimes is not possible to address changes in foraminifera to one single parameter or pollutant, as there are many factors influencing. Different methodologies were employed to achieve results from different points of view. In the first case, mesocosms containing populations of benthic foraminifera underwent different concentrations of mercury (Hg) pollution, in order to understand their response in population terms. Then, traditional methods based on morphology and eDNA metabarcoding were compared one to each other and used to identify the individuals and analyze the detrimental effects of the pollutant on them. The second experiment was based on electronic microscopy. Individuals of two different species, Ammonia parkinsoniana and Pseudotriloculina rotunda, were exposed to lead (Pb) and mercury (Hg) and zinc (Zn) respectively, with the aim to assess the detrimental effects of these elements in the ultrastructure of the cell. Polluted samples and control specimens were photographed using Transmission Electronic Microscopy (TEM) in order to document and compare both healthy and damaged organelles within the foraminiferal cell. With the aim of having a new methodology that allows qualitative and quantitative studies of the foraminiferal cell, the third experiment consisted in the test of a battery of fluorescence-based probes that targeted different structures within the cell. The tested probes were CellHunt Blue, as a viability test of the individuals; CellRox in order to be able to notice changes in the production of reactive oxygen species (ROS); LipidTox and Nile Red to label lipid droplets and neutral and polar lipids respectively; LysoSensor and Acridine Orange were employed to observe the acidic vesicles within the cytoplasm; ActinGreen was used to make visible the actin microfilaments of the cytoskeleton; and MitoTracker and Nonyl Acridine orange were tested to make visible the mitochondria within the foraminifera.