Persistent organic pollutants in sediments and fish from Lake Victoria, Uganda

This thesis describes the occurrence and sources of selected persistent organic pollutants (POPs) such as polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), polychlorinated biphenyls (PCBs), polybrominated diphenyl ethers (PBDEs) and hexachlorocyclohexanes (HCHs) in the northern watershed of Lake Victoria. Sediments and fish were collected from three highly polluted embayments (i.e. Murchison Bay, Napoleon Gulf and Thurston Bay) of the lake. The analysis for PCDD/Fs, PCBs and PBDEs was done using a high resolution mass spectrometer coupled to a gas chromatograph (GC), and a GC equipped with an electron capture detector was used for HCHs. Total (Σ) PCDD/Fs, PCBs and PBDEs in sediments ranged from 3.19 to 478, 313 to 4325 and 60.8 to 179 pg g-1 dry weight (dw), respectively. The highest concentrations of pollutants were found at sites close to industrial areas and wastewater discharge points. The maximum concentrations of PCDD/Fs, PCBs, PBDEs and HCHs in fish muscle homogenates were 49, 779, 495 and 45,900 pg g-1 wet weight (ww), respectively. The concentrations of the pollutants in Nile perch (Lates niloticus) were significantly greater than those in Nile tilapia (Oreochromis niloticus), possibly due to differences in trophic level and dietary feeding habits among fish species. World Health Organization-toxic equivalency quotient (WHO2005-TEQ) values in the sediments were up to 4.24 pg g-1 dw for PCDD/Fs and 0.55 pg TEQ g-1 dw for the 12 dioxin-like PCBs (dl-PCBs). 23.1% of the samples from the Napoleon Gulf were above the interim sediment quality guideline value of 0.85 pg WHO-TEQ g-1 dw set by the Canadian Council for Ministers of the Environment. The WHO2005-TEQs in fish were 0.001-0.16 pg g-1 for PCDD/Fs and 0.001-0.31 pg g-1 ww for dl- PCBs. The TEQ values were within a permissible level of 3.5 pg g−1 ww recommended by the European Commission. Based on the Commission set TEQs and minimum risk level criteria formulated by the Agency for Toxic Substances and Disease Registry, the consumption of fish from Lake Victoria gives no indication of health risks associated to PCDD/Fs and PCBs. Principal component analysis (PCA) indicated that anthropogenic activities such as agricultural straw open burning, medical waste incinerators and municipal solid waste combustors were the major sources of PCDD/Fs in the watershed of Lake Victoria. The ratios of α-/γ-HCH varied from 0.89 to 1.68 suggesting that the highest HCH residues mainly came from earlier usage and fresh γ-HCH (lindane). In the present study, the concentration of POPs in fish were not significantly related to those in sediments, and the biota sediment accumulation factor (BSAF) concept was found to be a poor predictor of the bioavailability and bioaccumulation of environmental pollutants.

Effect of chronic fish oil supplementation on renal function of normal and cachectic rats

In the present study we determined the effect of chronic diet supplementation with n-3 PUFA on renal function of healthy and cachectic subjects by providing fish oil (1 g/kg body weight) to female rats throughout pregnancy and lactation and then to their offspring post-weaning and examined its effect on renal function parameters during their adulthood. The animals were divided into four groups of 5-10 rats in each group: control, control supplemented with fish oil (P), cachectic Walker 256 tumor-bearing (W), and W supplemented with fish oil (WP). Food intake was significantly lower in the W group compared to control (12.66 ± 4.24 vs 25.30 ± 1.07 g/day). Treatment with fish oil significantly reversed this reduction (22.70 ± 2.94 g/day). Tumor growth rate was markedly reduced in the P group (16.41 ± 2.09 for WP vs 24.06 ± 2.64 g for W). WP group showed a significant increase in mean glomerular filtration rate compared to P and control (1.520 ± 0.214 ml min-1 kg body weight-1; P < 0.05). Tumor-bearing groups had low urine osmolality compared to control rats. The fractional sodium excretion decreased in the W group compared to control (0.43 ± 0.16 vs 2.99 ± 0.87%; P < 0.05), and partially recovered in the WP group (0.90 ± 0.20%). In summary, the chronic supplementation with fish oil used in this study increased the amount of fat in the diet by only 0.1%, but caused remarkable changes in tumor growth rate and cachexia, also showing a renoprotective function.

Losses of immunoreactive parvalbumin amacrine and immunoreactive alphaprotein kinase C bipolar cells caused by methylmercury chloride intoxication in the retina of the tropical fish Hoplias malabaricus

To quantify the effects of methylmercury (MeHg) on amacrine and on ON-bipolar cells in the retina, experiments were performed in MeHg-exposed groups of adult trahiras (Hoplias malabaricus) at two dose levels (2 and 6 µg/g, ip). The retinas of test and control groups were processed by mouse anti-parvalbumin and rabbit anti-alphaprotein kinase C (alphaPKC) immunocytochemistry. Morphology and soma location in the inner nuclear layer were used to identify immunoreactive parvalbumin (PV-IR) and alphaPKC (alphaPKC-IR) in wholemount preparations. Cell density, topography and isodensity maps were estimated using confocal images. PV-IR was detected in amacrine cells in the inner nuclear layer and in displaced amacrine cells from the ganglion cell layer, and alphaPKC-IR was detected in ON-bipolar cells. The MeHg-treated group (6 µg/g) showed significant reduction of the ON-bipolar alphaPKC-IR cell density (mean density = 1306 ± 393 cells/mm²) compared to control (1886 ± 892 cells/mm²; P < 0.001). The mean densities found for amacrine PV-IR cells in MeHg-treated retinas were 1040 ± 56 cells/mm² (2 µg/g) and 845 ± 82 cells/mm² (6 µg/g), also lower than control (1312 ± 31 cells/mm²; P < 0.05), differently from the data observed in displaced PV-IR amacrine cells. These results show that MeHg changed the PV-IR amacrine cell density in a dose-dependent way, and reduced the density of alphaKC-IR bipolar cells at the dose of 6 µg/g. Further studies are needed to identify the physiological impact of these findings on visual function.

Agonistic-like responses from the torus semicircularis dorsalis elicited by GABA A blockade in the weakly electric fish Gymnotus carapo

Findings by our group have shown that the dorsolateral telencephalon of Gymnotus carapo sends efferents to the mesencephalic torus semicircularis dorsalis (TSd) and that presumably this connection is involved in the changes in electric organ discharge (EOD) and in skeletomotor responses observed following microinjections of GABA A antagonist bicuculline into this telencephalic region. Other studies have implicated the TSd or its mammalian homologue, the inferior colliculus, in defensive responses. In the present study, we explore the possible involvement of the TSd and of the GABA-ergic system in the modulation of the electric and skeletomotor displays. For this purpose, different doses of bicuculline (0.98, 0.49, 0.245, and 0.015 mM) and muscimol (15.35 mM) were microinjected (0.1 µL) in the TSd of the awake G. carapo. Microinjection of bicuculline induced dose-dependent interruptions of EOD and increased skeletomotor activity resembling defense displays. The effects of the two highest doses showed maximum values at 5 min (4.3 ± 2.7 and 3.8 ± 2.0 Hz, P < 0.05) and persisted until 10 min (11 ± 5.7 and 8.7 ± 5.2 Hz, P < 0.05). Microinjections of muscimol were ineffective. During the interruptions of EOD, the novelty response (increased frequency in response to sensory novelties) induced by an electric stimulus delivered by a pair of electrodes placed in the water of the experimental cuvette was reduced or abolished. These data suggest that the GABA-ergic mechanisms of the TSd inhibit the neural substrate of the defense reaction at this midbrain level.

Effects of mercury intoxication on the response of horizontal cells of the retina of thraira fish (Hoplias malabaricus)

Methyl mercury (MeHg) is highly neurotoxic, affecting visual function in addition to other central nervous system functions. The effect of mercury intoxication on the amplitude of horizontal cell responses to light was studied in the retina of the fish Hoplias malabaricus. Intracellular responses were recorded from horizontal cells of fish previously intoxicated with MeHg by intraperitoneal injection (IP group) or by trophic exposure (T group). Only one retina per fish was used. The doses of MeHg chloride administered to the IP group were 0.01, 0.05, 0.1, 1.0, 2.0, and 6.0 mg/kg. The amplitudes of the horizontal cell responses were lower than control in individuals exposed to 0.01 (N = 4 retinas), 0.05 (N = 2 retinas) and 0.1 mg/kg (N = 1 retina), whereas no responses were recorded in the 1.0, 2.0, and 6.0 mg/kg groups. T group individuals were fed young specimens of Astyanax sp previously injected with MeHg corresponding to 0.75 (N = 1 retina), 0.075 (N = 8 retinas) or 0.0075 (N = 4 retinas) mg/kg fish body weight. After 14 doses, one every 5 days, the amplitude of the horizontal cell response was higher than control in individuals exposed to 0.075 and 0.0075 mg/kg, and lower in individuals exposed to 0.75 mg/kg. We conclude that intoxication with MeHg affects the electrophysiological response of the horizontal cells in the retina, either reducing or increasing its amplitude compared to control, and that these effects are related to the dose and/or to the mode of administration.

Stress responses of the fish Nile tilapia subjected to electroshock and social stressors

Plasma cortisol and glucose levels were measured in 36 adult Nile tilapia males, Oreochromis niloticus (standard length, mean ± SD, 14.38 ± 1.31 cm), subjected to electroshock and social stressors. Pre-stressor levels were determined 5 days after the adjustment of the fish to the experimental aquaria (1 fish/aquarium). Five days later, the effects of stressors on both cortisol and glucose levels were assessed. The following stressors were imposed for 60 min: pairing with a larger resident animal (social stressor), or a gentle electroshock (AC, 20 V, 15 mA, 100 Hz for 1 min every 4 min). Each stressor was tested in two independent groups, one in which stress was quantified immediately after the end of the 60-min stressor imposition (T60) and the other in which stress was quantified 30 min later (T90). Pre-stressor values for cortisol and glucose were not statistically different between groups. Plasma cortisol levels increased significantly and were of similar magnitude for both electroshock and the social stressor (mean ± SD for basal and final samples were: electroshock T60 = 65.47 ± 15.3, 177.0 ± 30.3; T90 = 54.8 ± 16.0, 196.2 ± 57.8; social stress T60 = 47.1 ± 9.0, 187.6 ± 61.7; T90 = 41.6 ± 8.1, 112.3 ± 26.8, respectively). Plasma glucose levels increased significantly for electroshock at both time points (T60 and T90), but only at T90 for the social stressor. Initial and final mean (± SD) values are: electroshock T60 = 52.5 ± 9.2, 115.0 ± 15.7; T90 = 35.5 ± 1.1, 146.3 ± 13.3; social stress T60 = 54.8 ± 8.8, 84.4 ± 15.0; T90 = 34.5 ± 5.6, 116.3 ± 13.6, respectively. Therefore, electroshock induced an increase in glucose more rapidly than did the social stressor. Furthermore, a significant positive correlation between cortisol and glucose was detected only at T90 for the social stressor. These results indicate that a fish species responds differently to different stressors, thus suggesting specificity of fish stress response to a stressor.

Automatic recognition of fish from video sequences

The problem of automatic recognition of the fish from the video sequences is discussed in this Master’s Thesis. This is a very urgent issue for many organizations engaged in fish farming in Finland and Russia because the process of automation control and counting of individual species is turning point in the industry. The difficulties and the specific features of the problem have been identified in order to find a solution and propose some recommendations for the components of the automated fish recognition system. Methods such as background subtraction, Kalman filtering and Viola-Jones method were implemented during this work for detection, tracking and estimation of fish parameters. Both the results of the experiments and the choice of the appropriate methods strongly depend on the quality and the type of a video which is used as an input data. Practical experiments have demonstrated that not all methods can produce good results for real data, whereas on synthetic data they operate satisfactorily.

Spatial and temporal variation in fish populations and assemblages in coastal waters of the northern Baltic Proper

The distribution and traits of fish are of interest both ecologically and socio-economically. In this thesis, phenotypic and structural variation in fish populations and assemblages was studied on multiple spatial and temporal scales in shallow coastal areas in the archipelago of the northern Baltic Proper. In Lumparn basin in Åland Islands, the fish assemblage displayed significant seasonal variation in depth zone distribution. The results indicate that investigating both spatial and temporal variation in small scale is crucial for understanding patterns in fish distribution and community structure in large scale. The local population of Eurasian perch Perca fluviatilis L displayed habitat-specific morphological and dietary variation. Perch in the pelagic zone were on average deeper in their body shape than the littoral ones and fed on fish and benthic invertebrates. The results differ from previous studies conducted in freshwater habitats, where the pelagic perch typically are streamlined in body shape and zooplanktivorous. Stable isotopes of carbon and nitrogen differed between perch with different stomach contents, suggesting differentiation of individual diet preferences. In the study areas Lumparn and Ivarskärsfjärden in Åland Islands and Galtfjärden in Swedish east coast, the development in fish assemblages during the 2000’s indicated a general shift towards higher abundances of small-bodied lower-order consumers, especially cyprinids. For European pikeperch Sander lucioperca L., recent declines in adult fish abundances and high mortalities (Z = 1.06–1.16) were observed, which suggests unsustainably high fishing pressure on pikeperch. Based on the results it can be hypothesized that fishing has reduced the abundances of large predatory fish, which together with bottom-up forcing by eutrophication has allowed the lower-order consumer species to increase in abundances. This thesis contributes to the scientific understanding of aquatic ecosystems with new descriptions on morphological and dietary adaptations in perch in brackish water, and on the seasonal variation in small-scale spatial fish distribution. The results also demonstrate anthropogenic effects on coastal fish communities and underline the urgency of further reducing nutrient inputs and regulating fisheries in the Baltic Sea region.

Fish hemoglobins

Vertebrate hemoglobin, contained in erythrocytes, is a globular protein with a quaternary structure composed of 4 globin chains (2 alpha and 2 beta) and a prosthetic group named heme bound to each one. Having myoglobin as an ancestor, hemoglobin acquired the capacity to respond to chemical stimuli that modulate its function according to tissue requirements for oxygen. Fish are generally submitted to spatial and temporal O2 variations and have developed anatomical, physiological and biochemical strategies to adapt to the changing environmental gas availability. Structurally, most fish hemoglobins are tetrameric; however, those from some species such as lamprey and hagfish dissociate, being monomeric when oxygenated and oligomeric when deoxygenated. Fish blood frequently possesses several hemoglobins; the primary origin of this finding lies in the polymorphism that occurs in the globin loci, an aspect that may occasionally confer advantages to its carriers or even be a harmless evolutionary remnant. On the other hand, the functional properties exhibit different behaviors, ranging from a total absence of responses to allosteric regulation to drastic ones, such as the Root effect.

Vitamin B1 and B6 in the malaria parasite: requisite or dispensable?

Vitamins are essential compounds mainly involved in acting as enzyme co-factors or in response to oxidative stress. In the last two years it became apparent that apicomplexan parasites are able to generate B vitamers such as vitamin B1 and B6 de novo. The biosynthesis pathways responsible for vitamin generation are considered as drug targets, since both provide a high degree of selectivity due to their absence in the human host. This report updates the current knowledge about vitamin B1 and B6 biosynthesis in malaria and other apicomplexan parasites. Owing to the urgent need for novel antimalarials, the significance of the biosynthesis and salvage of these vitamins is critically discussed in terms of parasite survival and their exploitation for drug development.

Immunodominance: a new hypothesis to explain parasite escape and host/parasite equilibrium leading to the chronic phase of Chagas' disease?

Intense immune responses are observed during human or experimental infection with the digenetic protozoan parasite Trypanosoma cruzi. The reasons why such immune responses are unable to completely eliminate the parasites are unknown. The survival of the parasite leads to a parasite-host equilibrium found during the chronic phase of chagasic infection in most individuals. Parasite persistence is recognized as the most likely cause of the chagasic chronic pathologies. Therefore, a key question in Chagas' disease is to understand how this equilibrium is established and maintained for a long period. Understanding the basis for this equilibrium may lead to new approaches to interventions that could help millions of individuals at risk for infection or who are already infected with T. cruzi. Here, we propose that the phenomenon of immunodominance may be significant in terms of regulating the host-parasite equilibrium observed in Chagas' disease. T. cruzi infection restricts the repertoire of specific T cells generating, in some cases, an intense immunodominant phenotype and in others causing a dramatic interference in the response to distinct epitopes. This immune response is sufficiently strong to maintain the host alive during the acute phase carrying them to the chronic phase where transmission usually occurs. At the same time, immunodominance interferes with the development of a higher and broader immune response that could be able to completely eliminate the parasite. Based on this, we discuss how we can interfere with or take advantage of immunodominance in order to provide an immunotherapeutic alternative for chagasic individuals.

Comparison of I-FISH and G-banding for the detection of chromosomal abnormalities during the evolution of myelodysplastic syndrome

Myelodysplastic syndrome (MDS) patients with a normal karyotype constitute a heterogeneous group from a biological standpoint and their outcome is often unpredictable. Interphase fluorescence in situ hybridization (I-FISH) studies could increase the rate of detection of abnormalities, but previous reports in the literature have been contradictory. We performed I-FISH and conventional karyotyping (G-banding) on 50 MDS patients at diagnosis, after 6 and 12 months or at any time if a transformation to acute myeloid leukemia (AML) was detected. Applying a probe-panel targeting the centromere of chromosomes 7 and 8, 5q31, 5p15.2 and 7q31, we observed one case with 5q deletion not identified by G-banding. I-FISH at 6 and 12 months confirmed the karyotype results. Eight cases transformed to AML during follow-up, but no hidden clone was detected by I-FISH in any of them. The inclusion of I-FISH during follow-up of MDS resulted in a small improvement in abnormality detection when compared with conventional G-banding.

Fish oil ingestion reduces the number of aberrant crypt foci and adenoma in 1,2-dimethylhydrazine-induced colon cancer in rats

We determined the effect of fish oil (FO) ingestion on colonic carcinogenesis in rats. Male Wistar rats received 4 subcutaneous injections (40 mg/kg body weight each) of 1,2-dimethylhydrazine (DMH) at 3-day intervals and were fed a diet containing 18% by weight FO (N = 10) or soybean oil (SO, N = 10) for 36 weeks. At sacrifice, the colon was removed, aberrant crypt foci were counted and the fatty acid profile was determined. Intestinal tumors were removed and classified as adenoma or carcinoma. Liver and feces were collected and analyzed for fatty acid profile. FO reduced the mean (± SEM) number of aberrant crypt foci compared to SO (113.55 ± 6.97 vs 214.60 ± 18.61; P < 0.05) and the incidence of adenoma (FO: 20% vs SO: 100%), but carcinoma occurred equally in FO and SO rats (2 animals per group). The polyunsaturated fatty acid (PUFA) profile of the colon was affected by diet (P < 0.05): total ω-3 (FO: 8.18 ± 0.97 vs SO: 1.71 ± 0.54%) and total ω-6 (FO: 3.83 ± 0.59 vs SO: 10.43 ± 1.28%). The same occurred in the liver (P < 0.05): total ω-3 (FO: 34.41 ± 2.6 vs SO: 6.46 ± 0.59%) and total ω-6 (FO: 8.73 ± 1.37 vs SO: 42.12 ± 2.33%). The PUFA profile of the feces and liver polyamine levels did not differ between groups (P > 0.05). In conclusion, our findings indicate that chronic FO ingestion protected against the DMH-induced preneoplastic colon lesions and adenoma development, but not against carcinoma in rats.

Control of respiration in fish, amphibians and reptiles

Fish and amphibians utilise a suction/force pump to ventilate gills or lungs, with the respiratory muscles innervated by cranial nerves, while reptiles have a thoracic, aspiratory pump innervated by spinal nerves. However, fish can recruit a hypobranchial pump for active jaw occlusion during hypoxia, using feeding muscles innervated by anterior spinal nerves. This same pump is used to ventilate the air-breathing organ in air-breathing fishes. Some reptiles retain a buccal force pump for use during hypoxia or exercise. All vertebrates have respiratory rhythm generators (RRG) located in the brainstem. In cyclostomes and possibly jawed fishes, this may comprise elements of the trigeminal nucleus, though in the latter group RRG neurons have been located in the reticular formation. In air-breathing fishes and amphibians, there may be separate RRG for gill and lung ventilation. There is some evidence for multiple RRG in reptiles. Both amphibians and reptiles show episodic breathing patterns that may be centrally generated, though they do respond to changes in oxygen supply. Fish and larval amphibians have chemoreceptors sensitive to oxygen partial pressure located on the gills. Hypoxia induces increased ventilation and a reflex bradycardia and may trigger aquatic surface respiration or air-breathing, though these latter activities also respond to behavioural cues. Adult amphibians and reptiles have peripheral chemoreceptors located on the carotid arteries and central chemoreceptors sensitive to blood carbon dioxide levels. Lung perfusion may be regulated by cardiac shunting and lung ventilation stimulates lung stretch receptors.

Autonomic control of cardiorespiratory interactions in fish, amphibians and reptiles

Control of the heart rate and cardiorespiratory interactions (CRI) is predominantly parasympathetic in all jawed vertebrates, with the sympathetic nervous system having some influence in tetrapods. Respiratory sinus arrhythmia (RSA) has been described as a solely mammalian phenomenon but respiration-related beat-to-beat control of the heart has been described in fish and reptiles. Though they are both important, the relative roles of feed-forward central control and peripheral reflexes in generating CRI vary between groups of fishes and probably between other vertebrates. CRI may relate to two locations for the vagal preganglionic neurons (VPN) and in particular cardiac VPN in the brainstem. This has been described in representatives from all vertebrate groups, though the proportion in each location is variable. Air-breathing fishes, amphibians and reptiles breathe discontinuously and the onset of a bout of breathing is characteristically accompanied by an immediate increase in heart rate plus, in the latter two groups, a left-right shunting of blood through the pulmonary circuit. Both the increase in heart rate and opening of a sphincter on the pulmonary artery are due to withdrawal of vagal tone. An increase in heart rate following a meal in snakes is related to withdrawal of vagal tone plus a non-adrenergic-non-cholinergic effect that may be due to humoral factors released by the gut. Histamine is one candidate for this role.