Mauro de Freitas Rebelo

New Insights from the Oyster Crassostrea rhizophorae on Bivalve Circulating Hemocytes

Hemocytes are the first line of defense of the immune system in invertebrates, but despite their important role and enormous potential for the study of gene-environment relationships, research has been impeded by a lack of consensus on their classification. Here we used flow cytometry combined with histological procedures, histochemical reactions and transmission electron microscopy to characterize the hemocytes from the oyster Crassostrea rhizophorae. Transmission electron microscopy revealed remarkable morphological characteristics, such as the presence of membranous cisternae in all mature cells, regardless of size and granulation. Some granular cells contained many cytoplasmic granules that communicated with each other through a network of channels, a feature never previously described for hemocytes. The positive reactions for esterase and acid phosphatase also indicated the presence of mature cells of all sizes and granule contents. Flow cytometry revealed a clear separation in complexity between agranular and granular populations, which could not be differentiated by size, with cells ranging from 2.5 to 25 µm. Based on this evidence we suggest that, at least in C. rhizophorae, the different subpopulations of hemocytes may in reality be different stages of one type of cell, which accumulates granules and loses complexity (with no reduction in size) as it degranulates in the event of an environmental challenge.

Antioxidant responses in different body regions of the polychaeta Laeonereis acuta (Nereididae) exposed to copper

Antioxidant enzymes, total antioxidant capacity (TOSC) and concentration of reactive oxygen species (ROS) were measured in anterior (A), middle (M) and posterior (P) body regions of Laeonereis acuta after copper (Cu; 62.5 microg/l) exposure. A catalase (CAT) activity gradient observed in control group (lowest in A, highest in P) was not observed in Cu exposed group. Glutathione-S-transferase (GST) activity in A region of Cu group was higher than in A region of the control group. DNA damage (comet assay) was augmented in the A region of Cu group. Since copper accumulation was similar in the different body regions, sensitivity to copper in A regions seems to be related to lowest CAT activity. In sum, copper exposure lowered TOSC, a result that at least in part can be related to lowering of antioxidant enzymes like CAT. DNA damage was induced in the anterior region, where a lower CAT activity was observed.

Heavy metals in benthic organisms from Todos os Santos Bay, Brazil

The marine ecosystems of Todos os Santos Bay (TSB, The State of Bahia, Brazil) have been impacted by the presence on its coast of a large metropolitan area as well as of chemical and petrochemical activities. Despite its ecological importance, there is a lack of scientific information concerning metal contamination in TSB marine biota. Thus, we analyzed concentrations of metals in four species of marine benthic organisms (two seaweeds, Padina gymnospora and Sargassum sp. one seagrass, Halodule wrightii and one oyster, Crassostrea rhizophorae) in three sites from the TSB region that have been most affected by industrial activities. The concentrations of Al, Cd, Cr, Cu, Fe, Mn, Ni, Pb and Zn were determined by Atomic Absorption Spectrophometry. The obtained data indicates that cadmium and copper in seaweeds, oysters and seagrass, as well as Ni concentrations in oysters, were in range of contaminated coastal areas. Cadmium and copper are available to organisms through suspended particles, dissolved fraction of water column and bottom sediment interstitial water. As oysters and other mollusks are used as food sources by the local population, the metal levels found in oysters in TSB may constitute a health risk for this population. Our results suggest implanting a heavy metals biomonitoring program in the TSB marine ecosystems.

Expression analysis of the molluscan p53 protein family mRNA in mussels (Mytilus spp.) exposed to organic contaminants

In this study, we report the tissue expression analysis of the p53 protein family mRNA in mussels (Mytilus spp.) by means of quantitative RT-PCR. The tissue specific response was evaluated after 24 h exposure to a sublethal benzo[a]pyrene (B[a]P) concentration (75 nM), showing a 2.6 fold induction in digestive gland cells and a dramatic gene down regulation in circulating hemocytes. The comet assay and DNA gel diffusion tests showed significant effects in hemocytes and negligible differences in the digestive gland nuclei, implicating p53 in DNA damage of molluscan hemocytes. Finally, the kinetics of p53 protein family mRNA expression in the digestive gland of animals exposed to B[a]P and crude oil (0.5 ppm) showed partially overlapping trends, characterised by a common down regulation after 1 week exposure. These data should be carefully considered in view of the biological effects of organic pollutants and particularly following spills.

Cloning and detection of metallothionein mRNA by RT-PCR in mangrove oysters (Crassostrea rhizophorae)

A semi-quantitative RT-PCR protocol was developed to directly evaluate metallothionein (MT) mRNA expression in different tissues of mangrove oysters (Crassostrea rhizophorae), using beta-Actin (ACT) as a normalizing gene. Clones with high degree of identity from partial coding sequences were obtained for both MT and ACT. Although not statistically significant, high relative accumulation of MT mRNA was observed in the digestive gland (DGG), but not in the gills, from samples collected from both control and contaminated sites. Nevertheless, MT expression was not comparable to the high levels of metal in the contaminated oysters. Results indicate that the variation in relative MT mRNA levels from different samples of the same site could be due to multiple gene copies or different MT isoform induction.

Gene discovery through transcriptome sequencing for the invasive mussel Limnoperna fortunei.

The success of the Asian bivalve Limnoperna fortunei as an invader in South America is related to its high acclimation capability. It can inhabit waters with a wide range of temperatures and salinity and handle long-term periods of air exposure. We describe the transcriptome of L. fortunei aiming to give a first insight into the phenotypic plasticity that allows non-native taxa to become established and widespread. We sequenced 95,219 reads from five main tissues of the mussel L. fortunei using Roche’s 454 and assembled them to form a set of 84,063 unigenes (contigs and singletons) representing partial or complete gene sequences. We annotated 24,816 unigenes using a BLAST sequence similarity search against a NCBI nr database. Unigenes were divided into 20 eggNOG functional categories and 292 KEGG metabolic pathways. From the total unigenes, 1,351 represented putative full-length genes of which 73.2% were functionally annotated. We described the first partial and complete gene sequences in order to start understanding bivalve invasiveness. An expansion of the hsp70 gene family, seen also in other bivalves, is present in L. fortunei and could be involved in its adaptation to extreme environments, e.g. during intertidal periods. The presence of toll-like receptors gives a first insight into an immune system that could be more complex than previously assumed and may be involved in the prevention of disease and extinction when population densities are high. Finally, the apparent lack of special adaptations to extremely low O2 levels is a target worth pursuing for the development of a molecular control approach.

A description of mercury in fishes from the Madeira River Basin, Amazon, Brazil

Over the last 20 years several projects carried on the Madeira River basin in the Amazon produced a great amount data on total Hg concentration in different fish species. In this paper we discuss temporal trends in Hg contamination and its relation to body weight in some of those fishes, showing that even within similar groups, such as carnivorous and non-migratory fish, the interspecies variability in Hg accumulation is considerable.

Oyster condition index in Crassostrea rhizophorae (Guilding, 1828) from a heavy-metal polluted coastal lagoon

The condition index (CI) of oysters represents an ecophysiological approach to estimate meat quality and yield in cultured bivalve mollusks. In the present study, the CI of oysters from a heavy-metal polluted bay was analyzed with respect to Zn and Cd contamination in soft tissues, spawning, and polychaete infestation. The CI was calculated through a new technique based on molds made to measure the volume of oyster-shell internal cavities. The higher CI values (over 9 in the dry season) were probably related availability of suspended particles rich in organic matter in the bay, while the rapid reduction in the CI from one season to the next at some stations suggests the effect of spawning. Polychaete infestation was considered low (18.7%) and produced no clear CI effects. The Cd in the oyster tissue collected during the rainy season was weak, although still significantly correlated with the CI (r = -0.36; p < 0.05). All other comparisons of CI and metal concentrations demonstrated a non-significant correlation. The CI variations observed on the temporal and spatial scale were likely to have been caused by availability of organic matter and spawning, rather than spionid infestation or metal body burdens.

Organochlorine pesticides residues in feed and muscle of farmed Nile tilapia from Brazilian fish farms

Organochlorine pesticide (OCP) concentrations were determined in fish muscle and feed collected from four different fish farms in Brazil. Nile tilapia from two growth stages, juveniles and adults, collected at two intensive tanks farms (IT1 and IT2) and two net cage farms (NC1 and NC2), were analyzed by High Resolution Gas Chromatography/High Resolution Mass Spectrometry. Pesticides were detected in almost all samples, but no samples exceeded international maximum limits for safe fish consumption. ΣDDT was the predominant pesticide in fish muscle, found in all fish samples, and endosulfan was the most predominant pesticide in feed, found in all feed samples. No significant correlation (p>0.05) was observed between the different growth stages and OCP concentrations, although slightly higher OCP concentrations were observed in adults. Among the rearing systems, NC farmed fish presented higher lipid levels and, consequently, higher OCP concentrations than fish from IT farms. Some OCPs (ΣHCH, aldrin, dieldrin and endrin) presented strong positive correlations (p<0.05) between feed and fish muscle concentrations, while others (ΣDDT, mirex, chlordane, ΣHCB and endosulfan) presented no correlation. However, the low levels of the sum of contaminants found in most of the feed samples may explain the low contaminant levels in fish tissue.

Mercury accumulation and metallothionein expression from aquafeeds by Litopenaeus vannamei Boone, 1931 under intensive aquaculture conditions

This study describes the accumulation of Hg and metallothionein gene expression in Litopenaeus vannamei Boone, 1931 with aquafeeds as the major source of Hg. Trials were conducted under controlled conditions in experimental tank facilities with high (indoor tanks) and low (outdoor tanks) Hg aquafeeds concentrations. Aquafeeds were the sole source of Hg for the shrimps and concentrations varied from 5.4 to 124 ng.g-1 d.w.. In the three animal fractions analysed; muscle (6,3 - 15,9 ng.g-1); hepatopancreas (5,1 - 22,0 ng.g-1) and exoskeleton (3,0 - 16,2 ng.g-1), Hg concentrations were significantly lower in the outdoor trials submitted to Hg-poor aquafeeds. Maximum shrimp muscle Hg concentrations were low (36.4 ng.g-1 w.w.) relative to maximum permissible concentrations for human consumption and Hg content in muscle and hepatopancreas were significantly correlated with Hg content in aquafeeds. Highest Hg concentrations in the exoskeleton of animals exposed to Hg-richer aquafeed, suggested that a detoxification mechanism is taking place. On the other hand the metallothionein suffered no variation in its relative expression in any of the experiments, meaning that the contact with feed containing the observed Hg concentrations were not sufficient to activate gene transcription. It was not possible, under the experimental design used, to infer Hg effects on the biological performance of the animals.