Luciana R. Souza-Bastos

A multibiomarker evaluation of urban, industrial, and agricultural exposure of small characins in a large freshwater basin in southern Brazil

Iguaçu River is the second most polluted river of Brazil. It receives agrochemicals and contaminants of urban and industrial sources along its course. A multibiomarker approach was employed here to evaluate the health of a small characin (Astyanax spp.) at two sites along the river, sampled during a dry (autumn) and a rainy (spring) season. Biomarkers were condition factor and somatic indices (gonads and liver); genetic damage (comet assay and micronucleus test); enzyme activities such as hepatic catalase (CAT) and glutathione S-transferase (GST), lipoperoxidation (LPO), branchial and renal carbonic anhydrase (CA), acetylcholinesterase (AChE) in the muscle and the brain, histopathology of the liver and gills, and concentrations of polycyclic aromatic hydrocarbons (PAHs) in bile. There were no consistent differences in biomarker responses between the two study sites. Some biomarkers revealed greater potential impact in the rainy season, when increased amounts of contaminants are washed into the river (combined CAT inhibition and LPO increase, CA upregulation). Other biomarkers, however, revealed potential greater impact in the dry season, when contaminants potentially concentrate (GST induction, AChE inhibition, and liver histopathological alterations). Although of a complex nature, field experiments such as this provide rich data for monitoring protocols and assessment of general risk of exposure to pollutants of river systems.

Regulation of muscle hydration upon hypo- or hyper-osmotic shocks: differences related to invasion of the freshwater habitat by decapod crustaceans.

Decapod crustaceans have independently invaded freshwater habitats from the sea/estuaries. Tissue hydration mechanisms are necessary for the initial stages of habitat transitions but can be expected to diminish, as the capacity for extracellular homeostasis increases in hololimnetic species. Six decapod species have been compared concerning the maintenance of muscle hydration in vitro: Hepatus pudibundus (marine); Palaemon pandaliformis (estuarine resident), Macrobrachium acanthurus (freshwater diadromous), and the three hololimnetic Macrobrachium potiuna, Dilocarcinus pagei, and Aegla parana. The effects of inhibitors of potassium channels (barium chloride) and NKCC (furosemide) were evaluated under isosmotic, and respectively hypo- (50% below iso) or hyper- (50% above iso) conditions. There was high muscle hydration control in H. pudibundus with a possible role of NKCC in isosmotic conditions. Shrimps consistently showed small deviations in muscle hydration under anisosmotic conditions; P. pandaliformis has shown evidence of the presence of NKCC; M. potiuna was the species less affected by both inhibitors, under iso- or anisosmotic conditions. In the two hololimnetic crab species, both independent long-time inhabitants of freshwater, while the capacity to deal with hyper-osmotic shock is decreased, the capacity to deal with hyposmotic shock is retained, possibly because of hemolymph dilution during molting in fresh water. D. pagei apparently depends on potassium channels for volume recovery after swelling, whereas A. parana shows some dependence on NKCC to minimize volume loss in hyper-osmotic conditions. Although no molecular screening techniques have been tried here, data point to distinct cell/tissue transport mechanisms acting upon hydration/volume challenges in decapods of different habitats and lineages.

Osmoregulation of the resident estuarine fish Atherinella brasiliensis was still affected by an oil spill (Vicuña tanker, Paranaguá Bay, Brazil), 7 months after the accident.

An oil tanker loaded with methanol and bunker oil has exploded in November 2004 in Paranaguá Bay, in front of Paranaguá Harbor, southern Brazil. In order to investigate the chronic effects of an oil spill on a resident estuarine fish, the Brazilian silverside Atherinella brasiliensis was sampled 1, 4, and 7 months after the spill, from 2 sites inside Paranaguá Bay, and also from a reference site inside nearby Guaratuba Bay, non-affected by the spill. Increases in plasma osmolality (reaching ~525 mOsm/kg H₂O, or ~70% above values in reference fish) and chloride (reaching 214 mM in site C, or ~51% above values in reference fish) were detected 4 months after the spill, in parallel with branchial carbonic anhydrase inhibition (to 56% of the activity measured in reference fish) in silversides obtained from the contaminated sites. Plasma cortisol concentration increased progressively in samples from fish obtained 4 (462 ng/mL) and 7 (564-650 ng/mL) months after the spill, when compared to values in reference fish (192 ng/mL). Osmoregulation of a resident estuarine fish is still affected by an oil spill, months after the accident. It is, thus, a sensitive tool for the evaluation of the chronic effects of oil spills inside tropical estuarine systems, and A. brasiliensis is proposed as an adequate sentinel species for monitoring protocols.

Ammonia excretion and expression of transport proteins in the gills and skin of the intertidal fish Lipophrys pholis.

Intertidal pools are intensely challenging environments, due to rapid and extreme fluctuations in water conditions during the tidal cycle. Emersion is another challenge intertidal fishes may face. Mechanisms of ammonia excretion and ion regulation were studied in the resident amphibious blennid Lipophrys pholis. The ammonia transporters Rhcg1 and Rhcg2 were cloned and characterized. Fish were challenged for 24h to 1) emersion, 2) fresh water (FW), and 3) high environmental ammonia (HEA; 1mM NH4Cl), or 4) ammonia loading (1.5μmol/g NH4HCO3). When air exposed, L. pholis maintained aquatic ammonia excretion rates (JAmm) while branchial Na(+)/K(+)-ATPase (NKA) activity increased, but no changes at the protein or mRNA levels of transporters were noted. In FW, JAmm decreased and osmotic problems were encountered. Skin NKA activity decreased, branchial Rhcg2, and skin Rhcg1 and Rhcg2 increased. Exposure to HEA only increased branchial Rhcg2 levels. Although internal ammonia loading only led to a modest non-significant increase in JAmm, skin NKA (activity and α-subunit), carbonic anhydrase protein levels, and branchial Rhcg1 levels increased. In summary, variable responses were observed involving both gill and skin but given the instability of its habitat, the constitutive expression of transporters is likely also of importance.

Skin extract from Rhamdia quelen(Siluriformes: Heptapteridae) does not promote stress in conspecifics

Chemical communication is widely used in aquatic environments, where visual or auditory signals may not be always effective. Fish of the superorder Ostariophysi are known to display epidermal cells (club cells) that produce and store alarm substances, which are released to the water when the skin is damaged. Responses to alarm substances range widely, between active searches for refuge to a complete stop in any locomotor activity. In this study a large number of binucleated club cells (average density of 11 cells /5m2) were histologically observed in the skin of the catfish Rhamdia quelen (known as jundia). Skin extract (2, 5, and 10% w/v) applied for 15 minutes to conspecifics elicited increase in swimming activity and in the area visited by the fish inside the tank. However, exposure to the epithelial alarm cue did not evoke any stress response: plasma osmolality, ions (sodium, chloride, magnesium, and potassium), glucose and cortisol remained unchanged. In conclusion, the conspecific alarm cue of the jundia induces behavioral responses but not an acute stress response upon short-term exposure, compatible with its role in fostering physical integrity without representing major stress activation. Considering that in the natural environment such stimuli must quickly disappear due to dilution and that rapid protection responses may be necessary upon the possibility of an approaching predator, a faster mechanism to assure survival may come into play, such as sympathetic nervous system activation.

Different abilities to regulate tissue hydration upon osmotic challenge in vitro, in the cephalopods Octopus vulgaris and O. insularis

The spatial distribution of cephalopods is influenced by salinity and temperature. In marine osmoconformers, one reason for different spatial distributions maybe the putative distinct capacity to regulate tissue hydration. Adult Octopus vulgaris (n = 6) and O. insularis (n = 6) were obtained by divers from sites along the southern and northeastern Brazilian coasts, respectively. In the laboratory, octopuses were acclimated to full-strength seawater (salinity 34), for 5–10 days. They were then anesthetized (10 min in 4 °C seawater), a portion of the second right arm was removed, and cut into three portions. Tissues were placed in control isosmotic saline (osmolality 1072 mOsm/kg H2O). Fragments were weighed and transferred to either isosmotic, hyposmotic (50%), or hyperosmotic (150%) salines, and their weight was observed for 120 min. Tissue from both species maintained their hydration/weight following the hyposmotic shock, but differed in their response to the hyperosmotic challenge, where hydration remained unchanged in O. insularis, but was significantly reduced in O. vulgaris. This result could partially explain why O. insularis is more abundant than O. vulgaris throughout the shallow reefs and tide pools in the warmer and salty tropical waters of the northeastern Brazilian coast.