Frequency of micronuclei and of other nuclear abnormalities in erythrocytes of the grey mullet from the Mondego, Douro and Ave estuaries—Portugal

Fish are bioindicators of water pollution, and an increased rate of their erythrocyte nuclear morphological abnormalities (ENMAs)—and particularly of erythrocyte micronuclei (EMN)—is used as a genotoxicity biomarker. Despite the potential value of ENMAs andMN, there is scarce information about fish captured in Iberian estuaries. This is the case of the Portuguese estuaries of the Mondego, Douro and Ave, suffering from different levels of environmental stress and where chemical surveys have been disclosing significant amounts of certain pollutants. So, the aim of this study was to evaluate genotoxicants impacts and infer about the exposure at those ecosystems, using the greymullet (Mugil cephalus) as bioindicator and considering the type and frequency of nuclear abnormalities of erythrocytes as proxies of genotoxicity. Sampling of mullets was done throughout the year in the important Mondego, Douro and Ave River estuaries (centre and north-western Portugal). The fish (total n=242) were caught in campaigns made in spring–summer and autumn– winter, using nets or fishing rods. The sampled mullets were comparable between locations in terms of the basic biometric parameters. Blood smears were stained with Diff-Quik to assess the frequencies of six types of ENMAs and MN (given per 1,000 erythrocytes). Some basic water physicochemical parameters were recorded to search for fluctuations matching the ENMAs. Overall, the most frequent nucleus abnormality was the polymorphic type, sequentially followed by the blebbed/ lobed/notched, segmented, kidney shaped, vacuolated,MNand binucleated. The total average frequency of the ENMAs ranged from 73‰in the Mondego to 108‰in the Ave. The polymorphic type was typically ≥50 % of the total ENMAs, averaging about 51‰, when considering all three estuaries. The most serious lesion—the MN—in fish from Mondego and Douro had a similar frequency (≈0.38‰), which was significantly lower than that in the Ave (0.75‰). No significant seasonal differences existed as to the MN rates and seasonal differences existed almost only in the Douro, with the higher values in AW. In general, the pattern of ENMAs frequencies was unrelated with the water physicochemical parameters. Considering the data for both the total ENMAs and for each specific abnormality, and bearing in mind that values of MN in fish erythrocytes >0.3‰ usually reflect pollution by genotoxicants, it is suggested that mullets were likely being chronically exposed to such compounds, even in the allegedly less polluted ecosystem (Mondego). Moreover, data supported the following pollution exposure gradient: Mondego<Douro<Ave. The scenario and inferences nicely agree with the published data from chemical monitoring.This work was partially supported by the European Regional Development Fund (ERDF) funds through the Competitiveness and Trade Expansion Program (COMPETE), and by National Funds provided by Fundação para a Ciência e a Tecnologia (FCT), via the research projects PTDC/MAR/70436/2006, PEst-C/MAR/LA0015/ 2011, PEst-C/MAR/LA0015/2013, and also by the PhD grant SFRH/BD/25746/2005

Alterações histológicas em brânquias de tilápia nilotica Oreochromis niloticus causadas pelo cádmio Histological alterations in gills of Nile tilapia Oreochromis niloticus caused by cadmium

Os efeitos histopatológicos do cádmio nas brânquias de tilápia Oreochromis niloticus foram estudados por microscopia óptica, usando 25mgl-1 de CdCl2 durante quatro dias, com o objetivo de identificar seus efeitos agudos na estrutura das brânquias. A morfologia geral das brânquias de O. niloticus é idêntica à de outros teleósteos, apresentando quatro pares de arcos branquiais com filamentos bem desenvolvidos. Situadas lateralmente, encontram-se as lamelas provenientes do eixo central dos filamentos. No epitélio filamentar foi possível identificar células de cloro, pavimentosas e mucosas. Os peixes expostos ao cádmio mostraram sinais de lesões epiteliais; edema intersticial, vasodilatação das lamelas, destacamento do epitélio lamelar e proliferação do epitélio filamentar. As alterações observadas também incluíram fusão nas lamelas como resultado de hiperplasia e hipertrofia epitelial, ruptura do sistema de células pilar, aneurismas e necroses.<br>The histopathogical effects of cadmium on the gills of tilapia Oreochromis niloticus were studied by light microscopy, using 25mgl-1 of CdCl2 during four days to identified the effects of short-term exposure on gills structure. The general morphology of O. niloticus gills is similar to the other teleostean fishes, showing four pairs of gills arches with well developed filaments. Bilaterally situated, secondary lamellae branches are found from the central axis of the filaments. The filamentar epithelium showed the chloride cells, the pavement cells and mucous cells. Fish exposed to cadmium showed signs of epithelial lesion, namely the interstitial edema, swollen of the lamellae, lifting and cellular proliferation of the filamentar epithelium. The changes of the gills also included lamellar fusion as a result of epithelial hyperplasia and hypertrophy, the breakdown of pillar cell system, and aneurisms with some ruptures and necrosis, especially in the filamentar epithelium

Alterações do epitélio branquial e das lamelas de tilápias (Oreochromis niloticus) causadas por mudanças do ambiente aquático em tanques de cultivo intensivo The influence of the aquatic environment in tanks sequetially interconnected with PVC pipes on the gill epithelium and lamellas of tilapia (Oreochromis niloticus)

Objetivou-se analisar o comportamento do epitélio branquial de tilápias, cultivadas em tanques posicionados em diferentes altitudes e interconectados por tubos de PVC. Filamentos branquiais de quatro espécimes de quatro tanques (T1, T2, T3 e T4) interconectados seqüencialmente foram submetidos à rotina histológica, para obtenção de cortes de 5µm de espessura, os quais foram corados com Hematoxilina-Eosina, ou submetidos à técnica histoquímica para glicoconjugados: PAS + solução de diástase ou Alcian Blue pH 2,5 ou Alcian Blue pH 1,0. Considerando as regiões basal, intermediária e apical dos filamentos, mensurou-se a área lamelar e contou-se o número de células mucosas em cada uma dessas regiões, o que correspondia a 0,56 mm². Verificou-se que a concentração de oxigênio, pH e a temperatura se reduziam progressivamente com a passagem da água de um tanque para outro. Em função disso, constatou-se um aumento abrupto do número de células mucosas e da área lamelar no T2, e uma redução progressiva destas medidas nos tanques que recebiam água do T2. Além disso, observou-se nos animais do T2, T3 e T4, descolamento do epitélio branquial, hiperplasia celular no espaço interlamelar e telangectasias. Conclui-se que o ambiente aquático de tanques interconectados seqüencialmente por tubos de PVC se altera ao passar de um tanque para o outro, e que estas flutuações físico-químicas se refletem no comportamento do epitélio branquial através de variações da área lamelar e do número de células mucosas.<br>The behavior of the gill epithelium of tilapias cultured in tanks at different altitudes and interconnected with PVC pipes was analyzed. Gill filaments of four specimens from four tanks (T1, T2, T3 e T4) sequentially interconnected were submitted to histological routine to obtain 5-mm-thick cuts that were stained with HE or submitted to histochemistry reactions PAS + diastase solution or Alcian Blue pH 2.5 or Alcian Blue pH 1.0. Considering the intermediary, apical and basal regions of the filaments, the lamellar area was measured and the amount of mucous cells was counted. It was verified that oxygen, pH, and temperature decreased progressively as the water flew from one tank to another. Thus, an increase was realized of the amount of mucous cells and the lamellar area in T2, as well as a progressive decrease of these measures on the tanks which received water from T2. Moreover, detachment of the gill epithelium, cellular hyperplasia in the interlamellar space, and telangectasias were observed in the fishes from T2, T3 e T4. It was concluded that the aquatic environment in tanks sequentially interconnected with PVC pipes suffers alterations from one tank to another, as that physico-chemical fluctuations reflect on the behavior of the gill epithelium through variations of the lamellar area and the amount of mucous cells

Effects of partial or total fish meal replacement by agricultural by-product diets on gonad maturation, sex steroids and vitellogenin dynamics of African catfish (Clarias gariepinus)

The establishment of the first sexual maturation was characterized in African catfish (Clarias gariepinus) in order to study the efficiency of replacement of fish meal (FM) by diets composed of local vegetable ingredients. Four diets were formulated containing decreasing levels of FM (50-0% for diet 1 to diet 4) and increasing proportions of vegetable ingredients (50-100%). Gonadosomatic index (GSI), diameter and percentages of developmental stages of oocytes, plasma sex steroids and vitellogenin dynamics were investigated from February to June using one-year-old fish. Fish were individually tagged, and 12 individuals from each diet were investigated monthly. Replacement of FM with plant ingredients did not affect the GSI neither in males, nor in females. All males were spermiating, and no abnormal gonads were found. In females, GSI and percentages of advanced stages of oocytes decreased during the dry season, indicating seasonal changes in gonad development. Moreover, oocytes were in late exogenous vitellogenesis, but no final maturation stages were observed, whatever the diet. Higher plasma levels of E2 in females and of androgens (T and 11-KT) in both sexes were observed in fish fed diet 4 than in those receiving diet 1 depending on the season. Levels of plasma E2 and ALP (indicator for vitellogenin) in males did not differ among treatments and seasons suggesting no phytoestrogenic activity. The results showed that total replacement of FM by vegetable diets composed of groundnut oilcakes, bean and sunflower meals has no deleterious effect on the onset of sexual maturation in African catfish but, may stimulate the sex steroid production and in turns may potentially exert some positive actions on reproductive success