Frequency Of Immature Erythrocytes Research Articles

Addressing the impact of mercury estuarine contamination in the European eel (Anguilla anguilla L., 1758) – An early diagnosis in glass eel stage based on erythrocytic nuclear morphology

The decline of the European eel (Anguilla anguilla L., 1758) population throughout Europe has been partially attributed to pollution. As glass eel estuarine migration may represent a considerable threat, the impact of mercury (Hg) contamination at this stage was evaluated through an in situ experiment (7days). Total Hg (tHg) bioaccumulation was evaluated concomitantly with erythrocytic nuclear morphology alterations: erythrocytic nuclear abnormalities assay (ENA), frequency of immature erythrocytes (IE) and the erythrocytic maturity index (EMI). The ENA results suggested a genotoxic pressure at the most contaminated sites, in line with the tHg increase. The EMI data, together with IE frequency, showed that fish exposed to high levels of Hg exhibited alterations of haematological dynamics, translated into an erythropoiesis increment. Despite the presence of these compensatory mechanisms, the present findings suggest a harmful impact of Hg on genome integrity at this early development stage, potentially affecting eels' condition and ultimately the population sustainability.

Erythrocyte morphology of the Critically Endangered Geometric Tortoise Psammobates geometricus: effects of season, sex and age

Land transformation reduced the habitat of Psammobates geometricus to small isolated fragments, rendering the species vulnerable to extinction. Field biologists often use erythrocyte metrics to assess wildlife health but need baseline values to distinguish normal variations from disease or chronic stress. Here we characterise erythrocyte types and features of P. geometricus and assess if age, sex and environmental fluctuations influence their frequencies and metrics. We collected blood from males, females and juveniles over four seasons to prepare smears for cytological evaluation. We treated the smears with May–Grünwald–Giemsa stains and used imaging software to measure size, shape and colour intensity of erythrocyte types. The blood of all groups contained immature, mature and senescent erythrocytes, and the appearance of each cell type corresponded to descriptions for other chelonians. Season had a strong effect on the occurrence of erythrocyte types and features with few group differences. The frequency of immature erythrocytes was high in winter and spring, the wet season, reflecting increased erythropoiesis. Mature erythrocytes dominated in summer and autumn, but frequencies of senescent cells, poikilocytosis, cytoplasmic inclusions and cytoplasmic vacuoles increased in autumn, indicating that water and food shortages caused physiological stress. The small size and elongated shape of summer and autumn erythrocytes would facilitate oxygen exchange when high temperatures increase metabolic needs of ectotherms. Lower temperatures and energy demands in the wet season counter the lower oxygen capacity of round, immature cells. Our results provide baseline information on erythrocyte physiology and metrics for health assessments of geometric tortoises.

Assessment of chromosomal damage induced by a deltamethrin-based insecticide in fish (Anguilla anguilla L.) – A follow-up study upon exposure and post-exposure periods

The pyrethroid insecticide Decis®, containing deltamethrin as active ingredient, is among the most popular broad-spectrum biocides, with wide application in agriculture and home pest control. The occurrence of deltamethrin in the aquatic environment is well-established, but the possible genotoxic effects of Decis® in non-target organisms, namely fish, remain unknown. Hence, this work aimed to evaluate the cytogenetic damaging potential of Decis® in European eel (Anguilla anguilla L.), adopting the erythrocytic nuclear abnormalities (ENAs) assay. In addition, it was intended to investigate the damage progression in the post-exposure period. The frequency of immature erythrocytes (IE) was also determined to provide indirect information on the erythrocyte catabolism and erythropoiesis rate. Fish were exposed to 17.5 and 35μgL−1 of Decis® (equivalent to 0.05 and 0.1μgL−1 of deltamethrin, respectively) during 1 and 3days. Thereafter, fish were transferred to clean water and kept for 1, 7 and 14days. The results demonstrated a clear potential to induce chromosomal damage following 3days exposure, depicted in an ENA frequency increase for both Decis® concentrations. The transient nature of this cytogenetic damage was also demonstrated, as ENA frequency returned to the control level 1 and 7days after cessation of the exposure, respectively for the higher and the lower Decis® concentration. Moreover, this response pattern suggested a rapid metabolization and elimination of the formulation constituents by A. anguilla, combined with an increased erythrocyte turnover in fish exposed to the higher Decis® concentration, as pointed out by the IE frequency rise. Overall, the demonstrated genotoxic properties of Decis® pointed out increased risk factors to fish exposed to this insecticide.

The health signalling of ornamental traits in the Grey Partridge (Perdix perdix)

Birds express various secondary ornaments that can indicate individual condition and health. Amongst these, red-coloured carotenoid-based ornaments are supposed to be particularly valuable predictors of individual quality, due to their sensitivity to oxidative stress. Nevertheless, melanin-pigmented traits might also signal health and immune functions. Both types of ornaments may be either skin-based or feather-based, each differing in their dynamics. In the present study, we compared the health- and stress-indicating capacity of melanin-based feather ornamentation and putatively carotenoid-based skin ornamentation in a single species—the Grey Partridge (Perdix perdix), a vulnerable avian species of the European agricultural landscape. In captive males, we firstly verified the carotenoid content of the red-coloured skin tissue behind the eye by chromatography (HPLC). Secondly, we assessed the individual health status of all males by examining differential leukocyte count, the frequency of immature erythrocytes, malaria prevalence and proinflammatory immune responsiveness to phytohaemagglutinin (PHA). Both the size of the melanin-based ornament and red chroma of the carotenoid-based ornament were related to the heterophil:lymphocyte (H/L) ratio. Hence, in the Grey Partridge, both redness of the skin ornament and area of the feather ornament may serve as honest indicators of individual health and long-term stress. However, the two ornamental components were unrelated to each other, and the directions of their association to the H/L ratio were opposite. We therefore propose that, in this species, larger melanin-based feather ornamentation size is linked to higher levels of stress (possibly caused by more intensive social interactions with other males), while the level of expression of the carotenoid-based skin ornamentation more reliably signals actual individual health status. Our results are potentially valuable from the perspective of Grey Partridge conservation efforts, as they indicate a simple method for assessing individual quality in this species.

B-esterase activities and blood cell morphology in the frog Leptodactylus chaquensis (Amphibia: Leptodactylidae) on rice agroecosystems from Santa Fe Province (Argentina)

Activity of B-esterases (BChE: butyrylcholinesterase and CbE: carboxylesterase using two model substrates: α-naphthyl acetate and 4-nitrophenyl valerate) in a native frog, Leptodactylus chaquensis from rice fields (RF1: methamidophos and RF2: cypermethrin and endosulfan sprayed by aircraft) and non-contaminated area (pristine forest) was measured. The ability of pyridine-2-aldoxime methochloride (2-PAM) to reactivate BChE levels was also explored. In addition, changes in blood cell morphology and parasite infection were determined. Mean values of plasma BChE activities were lower in samples from the two rice fields than in those from the reference site. CbE (4-nitrophenyl valerate) levels varied in the three sites studied, being highest in RF1. Frog plasma from RF1 showed positive reactivation of BChE activity after incubation with 2-PAM. Blood parameters of frogs from RF2 revealed morphological alterations (anisochromasia and immature erythrocytes frequency). Moreover, a major infection of protozoan Trypanosoma sp. in individuals from the two rice fields was detected. We suggest that integrated use of several biomarkers (BChE and CBEs, chemical reactivation of plasma with 2-PAM, and blood cell parameters) may be a promising procedure for use in biomonitoring programmes to diagnose pesticide exposure of wild populations of this frog and other native anuran species in Argentina.

Anguilla anguilla L. liver ethoxyresorufin O-deethylation, glutathione S-tranferase, erythrocytic nuclear abnormalities, and endocrine responses to naphthalene and β-naphthoflavone

The effects of naphthalene (NAP) and β-naphthoflavone (BNF) on phase I biotransformation and genotoxicity in Anguilla anguilla L. were evaluated. Phase II biotransformation and cortisol levels were also assessed in NAP-treated fish. Two groups of eels were exposed to either a NAP or a BNF concentration range (0.1–2.7 μM) for different exposure periods (2–72 h). An early significant ethoxyresorufin O-deethylation (EROD) activity inhibition was observed, especially for the highest NAP concentrations at 2–6 h exposure and for BNF at 2 h exposure. However, a significant EROD activity increase was detected from 16 to 72 h exposure for NAP and from 4 to 72 h exposure for BNF. The cytochrome P450 (P450) content was not dose related. However, with regard to BNF exposure, P450 was the first biomarker to respond. Liver alanine transaminase (ALT) activity was measured as an indicator of hepatic health condition. ALT results demonstrated that the EROD activity decrease, previously described for NAP, was not related to tissue damage. Nevertheless, the highest BNF concentrations were demonstrated to induce liver damage and to impair the EROD activity response. An increased genotoxic response, measured as erythrocytic nuclear abnormalities (ENA), was observed during the first 8 h NAP exposure. However, for exposures longer than 8 h, ENA frequency returned to the control levels. This response profile may reflect a considerable DNA repair capacity and/or a metabolic adaptation providing an efficient NAP biotransformation and consequent detoxification. BNF revealed no ENA alterations for all concentrations and exposure lengths. In the NAP experiment a causal relationship between immature erythrocytes (IE) and ENA frequency disappearance was not found. BNF results with regard to IE frequency revealed an ability to alter the balance between erythropoiesis and removal of erythrocytes. Liver glutathione S-transferase activity was significantly induced after 2 and 48 h NAP exposure. A cortisol-impaired response seems to occur from 4 to 24 h NAP exposure, demonstrating an endocrine disruption. However, an adaptation process seems to occur after 48 h, since the plasma cortisol had a tendency to increase. The present findings confirm the usefulness of the adopted biomarkers. The ecological risk associated with aquatic contamination by NAP was also confirmed by the present data.

Biotransformation, genotoxic, and histopathological effects of environmental contaminants in European eel ( Anguilla anguilla L.)

A prolonged toxicity study was carried out in young European eel ( Anguilla anguilla L.) to evaluate the effects of environmental contaminants, namely, two individual standard compounds, benzo[ a]pyrene (BaP) and dehydroabietic acid (DHAA), and a complex mixture, bleached kraft pulp mill effluent (BKPME). Fish were exposed to BaP (0.22, 0.45, and 0.9 μM) and BKPME (3.12%, 6.25%, and 12.5% (v/v)) for 3, 7, and 30 days and to DHAA (0.07, 0.15, and 0.30 μM) for 3, 7, 30, 90, and 180 days. The biomarkers include biotransformation and genotoxicity indicators, such as total ethoxyresorufin O-deethylase (EROD) activity and frequency of erythrocytic nuclear abnormalities (ENAs), respectively. Hematological dynamics was assessed as frequency of immature erythrocytes (IEs). Histopathological examinations were carried out for the highest concentrations and for 30 days and longer exposures. Total EROD increases significantly only after 180 days of DHAA exposure. However, significant ENA induction was generally observed during exposure to all contaminants tested. Nevertheless, some of the ENA results suggest an altered genotoxic response, which may arise either from short-term exposures to the highest contaminant levels or long-term exposures to the lowest contaminant levels. IE frequency decreased significantly after 30 days of exposure to 0.45 μM BaP and 180 days of exposure to the entire DHAA concentration range. Increased density of pigmented macrophage aggregates in 30-day BaP- and BKPME-exposed fish as well as in 90- and 180-day DHAA-exposed fish confirmed histopathological liver alterations. Bile accumulation in hepatocytes after BaP treatment, cytoplasmic vacuolization and cell atrophy following DHAA exposure, as well as liver loss of parenchymal cells in BKPME-exposed fish, were also detected. Dispersed necrosis and focal inflammation were observed in the livers of all treated groups. Fish exposed to DHAA and BKPME showed skin and gill disruption as well as kidney Malpighian corpuscle alterations. All 30-day-treated groups revealed intense spleen hemosiderosis, indicating increased erythrophagia. This splenic effect may be strongly correlated with the observed disappearance of ENAs. Neoplastic lesions were not found. A multibiomarker strategy, which includes EROD, ENA, and IE assays as well as histopathological studies, contributed to a better understanding of the global toxic process.