MELANOMACRÓFAGOS HEPÁTICOS Y ACTIVIDAD DE LA ENZIMA GLUTATION S-TRANSFERASA EN Leptodactylus chaquensis (ANURA, LEPTODACTYLIDAE) COMO BIOMARCADORES DE ESTRÉS OXIDATIVO POR LA EXPOSICIÓN A CLORPIRIFOS / Liver Melanomacrophages and Gluthation S-Transferase Activity in Leptodactylus chaquensis (Anura, Leptodactylidae) as Biomarkers of Oxidative Stress Due to Chlorpyrifos Exposition

RESUMEN Se cuantificaron y compararon los melanomacrófagos (MM) y la actividad de la enzima Glutatión S-Transferasa (GST) en hígados de ejemplares adultos de Leptodactylus chaquensis (Anura, Leptodactylidae) colectados en un cultivo de arroz (CA) (siete días posteriores a la aplicación de clorpirifos, CPF) y en un sitio de referencia (SR) en el departamento San Javier, provincia de Santa Fe (Argentina). El análisis histológico reveló una mayor cantidad (p = 0.028) y área ocupada por MM (p = 0.017) y se registró una inhibición en la actividad de la GST (p = 0.030) en individuos del CA respecto a los del SR. Los efectos descriptos evidencian el riesgo ecotoxicológico para los anuros en los cultivos de arroz con aplicaciones de CPF.

ABSTRACT We quantified and compared the hepatic melanomacrophage (MM) and glutathione S-transferase (GST) enzyme activity (two oxidative stress biomarkers) in the liver of Leptodatylus chaquensis adults (Anura, Leptodactylidae) collected in a rice field (CA) in San Javier department, Santa Fe (Argentina), seven days after the application of chlorpyrifos and in a reference site (SR). The histological analysis revealed a significant amount (p = 0.028) and area occupied by MM (p = 0.017) in livers of CA compared to SR. Furthermore, a significant inhibition of GST activity was recorded in the CA frogs compared to the SR (p = 0.030). The histopathological and enzymatic effects provide evidences of ecotoxicological risk for anurans in rice field with CPF application.

Toxicity of Bacillus thuringiensis var. israelensis in aqueous suspension on the South American common frog Leptodactylus latrans (Anura: Leptodactylidae) tadpoles.

The effects of commercial formulations of Bacillus thuringiensisvar.israelensis (Bti) on non-target organisms are still a matter of debate; in amphibians, the risks of Bti are little known. To evaluate the toxicity of a commercial liquid (aqueous suspension, AS) formulation of Bti (Introban(®)) on Leptodactylus latrans tadpoles, including median lethal concentration (LC50) and no-and lowest-observed-effect concentrations (NOEC and LOEC, respectively), as well as the possible effects of Bti on oxidative responses, erythrocytes genotoxicity, and histology of the intestines. In the laboratory, tadpoles were exposed to nominal concentrations of 0 (control), 2.5, 5, 10, 20 and 40 mg/L of formulated Bti-AS. Glutathione S-transferase (GST) and catalase (CAT) activities, as well as formation of erythrocyte nuclear abnormalities (ENAs), and histological effect were measured in tadpoles displaying survival rates >85%. L. latrans tadpoles were sensitive to exposure to Bti-AS, reaching 100% mortality after 48 h of exposure at the highest concentration. Bti-AS induced GST and CAT enzymes and genotoxicity (erythrocyte's nuclear abnormalities), and caused intestine's histopathology. Our results demonstrate that toxicity of Bti-AS is dose-dependent for L. latrans tadpoles and that sublethal exposure alters enzymes of oxidative stress, induces genotoxicity, and causes intestine damage. Further research is needed to evaluate the ecotoxicological risk of the massive use of Bti formulations on amphibian populations that commonly used suburban wastewater or urban waterbodies to reproduce and where this biopesticide is frequently applied.

Biochemical changes in certain enzymes of Lysapsus limellium (Anura: Hylidae) exposed to chlorpyrifos.

Different enzyme biomarkers (AChE: acetylcholinesterase, CbE: carboxylesterase, GST: glutathione-S-transferase, CAT: catalase) were measured in digestive tissues of Lysapsus limellum frogs collected from a rice field (RF: chlorpyriphos sprayed by aircraft) and a non-contaminated area (RS: reference site), immediately (24h) and 168 h after aerial spraying with chlorpyrifos (CPF). CPF degradation was also searched in water samples collected from RF and RS, and found that insecticide concentration was reduced to≈6.78% of the original concentration in RF at 168 h. A significant reduction of AChE and CbE activities was detected in L. limellum from RF in stomach and liver at 24 and 168 h, and in intestine only at 24h, with respect to RS individuals. CAT activity decreased in intestine of L. limellum from RF 24h and 168 h after exposure to CPF, whereas GST decreased in that tissue only at 24h. In stomach and liver, a decrease was observed only at 168 h in both CAT and GST. The use of biomarkers (AChE, CbE, GST, and CAT) provides different lines of evidences for ecotoxicological risk assessment of wild frog populations at sites contaminated with pesticides.

Induction of micronuclei and nuclear abnormalities in tadpoles of the common toad (Rhinella arenarum) treated with the herbicides Liberty® and glufosinate-ammonium.

The assessment of micronucleated erythrocytes (ME) in blood represents a widely used method for the detection of chromosomal damage by chemical agents, such as herbicides that may occur as water contaminants. We investigated the changes in some circulating blood-cell parameters of tadpoles of the common toad (Rhinella arenarum) that were exposed during 48 or 96 h to three sub-lethal concentrations (3.75, 7.5, and 15 mg/L) of a commercial formulation of a glufosinate-ammonium (GLA)-based herbicide (Liberty(®), LY(®)) as well as to the corresponding active ingredient GLA. The frequency of ME and other erythrocyte nuclear abnormalities (ENA, i.e., lobed nuclei, binucleates or segmented nuclei, kidney-shaped nuclei, notched nuclei, and picnotic nuclei) were evaluated and compared with positive (cyclophosphamide, CP, 40 mg/L) and negative (de-chlorinated tap water) controls. The results indicate that the exposure of R. arenarum tadpoles to LY(®) induces a concentration-dependent increase in ME frequency. The ENA frequency at 48 h was also significantly higher than that in the negative control group for all the chemicals assayed (CP, LY(®) and GLA) whereas at 96 h, increases in ENA over the negative control group were found only for CP and GLA (7.5 mg/L). Our study demonstrates that the commercial formulation of a GLA-based herbicide induces micronucleus formation in R. arenarum tadpoles, in contrast to the active ingredient. According to these results, the inert ingredients of the commercial formulation played an important role in the production of genotoxic damage in erythrocytes of amphibian tadpoles.

Biological endpoints, enzyme activities, and blood cell parameters in two anuran tadpole species in rice agroecosystems of mid-eastern Argentina.

Different biological variables of tadpoles, including survival, development and growth rates, and biomarkers [cholinesterases, glutathione-S-transferases (GST), and blood cell morphology] were evaluated in two anuran species, Scinax squalirostris (Hylidae) and Leptodactylus mystacinus (Leptodactylidae), using in situ experimental chambers in a rice field (RF) sprayed with insecticide Lambda-cyhalothrin (LTC) by aircraft in Santa Fe Province, Argentina. We found a significant decrease in body weight (0.62 ± 0.04 g) of L. mystacinus and an increased development rate of S. squalirostris in individuals from RF (41 ± 1; Gosner) with respect to individuals from the reference site (RS: 0.93 ± 0.04 g and 37 ± 0; respectively). In S. squalirostris, individuals from RF mean values of butyrylcholinesterase activities decreased at 48 (4.09 ± 0.32 nmol min-1 mg-1 of TP) and 96 h (3.74 ± 0.20 nmol min-1 mg-1 of TP), whereas inhibition of acetylcholinesterase was observed at 96 h (47.44 ± 2.78 nmol min-1 mg-1 of TP). In L. mystacinus from RF, an induction of acetylcholinesterase activity was observed at 96 h (36.01 ± 1.09 nmol min-1 mg-1 of TP). Glutathione-S-transferase levels varied between species, being higher in L. mystacinus individuals but lower in S. squalirostris from RF at 48 (272.29 ±11.78 and 71.87 ± 1.70 nmol min-1 mg-1 of TP; respectively) and 96 h (279.25 ± 13.06 and 57.62 ± 4.58 nmol min-1 mg-1 of TP, respectively). Blood cell parameters revealed a lower number of mitotic cells (MC: 0.36 ± 0.31%o for S. squalirostris and 0.08 ± 0.05 %o for L. mystacinus) and higher number of eosinophils (E: 3.45 ± 1.75 %o for S. squalirostris and 7.64 ± 0.98 %o for L. mystacinus) in individuals from the RF than in individuals from the RS (MC: 2.55 ± 0.74 %o for S. squalirostris and 1.87 ± 0.72%o for L. mystacinus; and E: 0.13 ± 0.09 for S. squalirostris and 3.20 ± 0.80 for L. mystacinus). Overall, our results demonstrate the existence of apparent differences in sensitivity between species in a series of sublethal responses to short-term exposure in RF after the application of Lambda-cyhalothrin. We suggest that the integral use of biological endpoints (development and growth) together with biomarkers (cholinesterase, GST, and blood cell parameters) may be a promising integral procedure for investigating pesticide exposure in wild frog populations.

Effect of exposure to contaminated pond sediments on survival, development, and enzyme and blood biomarkers in veined treefrog (Trachycephalus typhonius) tadpoles.

Sediments are important elements of aquatic ecosystems and in general sediments accumulate diverse toxic substances. Amphibians potentially have a greater risk of exposure to contaminants in sediments, and the test of sediments provides first lines of evidences. Sediment outdoor microcosm experiments were conducted to analyze biological endpoints (survival, development, growth, and morphological and organ malformation), enzyme activity (butyrylcholinesterase, BChE; glutathione-S-transferase, GST; and catalase, CAT) and blood biomarkers in veined treefrog Trachycephalus typhonius tadpoles, a widespread neotropical species. Hatching (stage 23) of T. thyphonius was exposed until they reached metamorphosis (stage 46). Sediment tests were performed and four different treatments were used: three ponds (LTPA, ISP, and SSP) influenced by industrial and agricultural activities and a reference treatment from a forest (RFS). Physical and chemical variables and concentration of nutrients, pesticide residues, and metals were determined. One treatment was metal-rich (LPTA) and two were nutrient-rich (ISP and SSP). Sediment treatments had no significant effect on survival; in contrast they had significant sublethal effects on T. typhonius larval development and growth rates, and affected overall size and shape at stage 38. Principally, in LPTA animals were significantly larger than in RFS, exhibiting swollen bodies, tail muscles and tail fin. In addition, metamorphs from LPTA, ISP, and SSP were smaller and showed signs of emaciation by the end of the experiment. Statistical comparisons showed that the proportions of each type of morphological abnormalities (swollen bodies and diamond shape, gut uncoiling, diverted gut, stiff tails, polydactyly, and visceral and hindlimb hemorrhaging) were significantly greater in metal- and nutrient-rich sediment treatments. Moreover, activities of BChE, GST and CAT, as well as and presence of micronuclei, immature, mitotic, anucleated erythrocytes varied significantly among treatments. Our biological effects-based sediment study highlights the use of different biological endpoints and biomarkers on anuran larvae at sites where pond sediment is risky and sediment management should be considered. Finally, the information of those biological endpoints and biomarkers would be useful as a management tool to decide if there are sufficient exposures of tadpoles to suspected pollutants on sediment.

Trombiculid mites (Hannemania sp.) in Leptodactylus chaquensis (Amphibia: Anura) inhabiting selected soybean and rice agroecosystems of Argentina.

Trombiculid mites are known to parasitize a variety of amphibian species; however, few comparisons of mite parasitism among sites have been made. Here, Hannemania sp. parasitism in frogs (Leptodactylus chaquensis) inhabiting agroecosystems from mideastern Argentina was described. A total of 40 adult frogs (22 females and 18 males) were analyzed to detect ectoparasite Hannemania spp. larvae. Prevalence and mean abundance of Hannemania sp. were consistently higher in frogs from the agroecosystems (rice and soybean fields) than from two reference sites. Leptodactylus chaquensis might be considered an important host species of Hannemania sp., particularly in agricultural areas.

[Hematology and blood cell cytochemistry of Rhinella fernandezae (Amphibia: Anura) from Espinal and Delta-Islands of Paraná River, Argentina]. / Hematología y citoquímica de las células sanguíneas de Rhinella fernandezae (Anura: Bufonidae) en Espinal y Delta-Islas del río Paraná, Argentina.

The description of amphibian hematology is scarce and most of these studies have been done in species from North America, Asia and Europe. With the purpose to obtain basic hematological information of Rhinella fernandezae, 23 blood samples from Santa Fe and Entre Rios natural reserves were studied. Blood of each individual was extracted by cardiac puncture and hemograms were carried out. Morphological and cytochemical description of blood cells were analyzed in slides and were inspected for extra and intra cellular parasites. Five leucocytes types were observed, being lymphocytes the predominant ones followed by basophiles. Heterophils and eosinophils were positive to PAS, Sudan B and peroxidase. The erythrocytes and its precursors were negative for cytochemical reactions. Micronuclei and nuclear alterations frequencies were scarce. No significant differences (p > 0.05) were observed between sexes neither in hemograms nor in blood cells morphology. Microfilarias were the only hemoparasites found with a relative low prevalence and infection intensity. The hematological characteristics studied were similar to those reported for other amphibians, suggesting that R. fernandezae individuals present optimal nutritional and immunological status.

Hematología y citoquímica de las células sanguíneas de Rhinella fernandezae (Anura: Bufonidae) en Espinal y Delta-Islas del río Paraná, Argentina

Hematology and blood cell cytochemistry of Rhinella fernandezae (Amphibia: Anura) from Espinal and Delta-Islands of Paraná River, Argentina. The description of amphibian hematology is scarce and most of these studies have been done in species from North America, Asia and Europe. With the purpose to obtain basic hematological information of Rhinella fernandezae, 23 blood samples from Santa Fe and Entre Ríos natural reserves were studied. Blood of each individual was extracted by cardiac puncture and hemograms were carried out. Morphological and cytochemical description of blood cells were analyzed in slides and were inspected for extra and intra cellular parasites. Five leucocytes types were observed, being lymphocytes the predominant ones followed by basophiles. Heterophils and eosinophils were positive to PAS, Sudan B and peroxidase. The erythrocytes and its precursors were negative for cytochemical reactions. Micronuclei and nuclear alterations frequencies were scarce. No significant differences (p>0.05) were observed between sexes neither in hemograms nor in blood cells morphology. Microfilarias were the only hemoparasites found with a relative low prevalence and infection intensity. The hematological characteristics studied were similar to those reported for other amphibians, suggesting that R. fernandezae individuals present optimal nutritional and immunological status. Rev. Biol. Trop. 59 (1): 17-28. Epub 2011 March 01.

La descripción de la hematología de los anfibios anuros es escasa, habiéndose realizado la mayoría de los trabajos en especies de Norteamérica, Asia y Europa. Con el propósito de obtener datos hematológicos para Rhinella fernandezae, fueron estudiados 23 especímenes provenientes de zonas protegidas de las provincias de Santa Fe y Entre Ríos. Se les extrajo sangre por punción cardíaca y se realizaron hemogramas. En los extendidos sanguíneos, se efectuaron la descripción morfológica y citoquímica de las células sanguíneas y búsqueda de parásitos. Se observaron cinco tipos de leucocitos, donde predominaron los linfocitos pequeños. Heterófilos y eosinófilos resultaron positivos para PAS, Sudan B y peroxidasa; contrariamente, los eritrocitos y sus precursores fueron negativos. Los puntajes de las reacciones citoquímicas fueron variables para basófilos, linfocitos, monocitos y trombocitos. Las frecuencias de micronúcleos y de alteraciones nucleares fueron escasas. No se observaron diferencias significativas (p>0.05) entre sexos en el hemograma ni en la morfología sanguínea. Los únicos hemoparásitos encontrados fueron microfilarias (Nematoda: Filaroidea), cuya prevalencia e intensidad de infección fueron bajas. Las características hematológicas estudiadas fueron semejantes a los valores reportados para otros anfibios, pudiendo inferir que los individuos de R. fernandezae estudiados se encuentran en un buen estado nutricional e inmunológico.

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.