Hemoparasites Do Not Affect Life-History Traits and Cellular Immune Response in Treefrog Hosts Boana cordobae.

We provide the first evidence for hemoparasites in the endemic Cordoba treefrog Boana cordobae. We collected 37 adult frogs at 1200 m a.s.l. in the Comechingones Mountains in the Córdoba province (Argentina). Each individual was sexed, then snout-vent length and body mass were recorded, a toe was collected for skeletochronological age determination, and a slide with a blood smear was prepared for hemoparasite screening, before releasing the frogs in situ. A total of 81% (n = 30) of the frogs were infected by hemogregarines and trypanosomes with a high intensity of infections. Dactylosoma was found for the first time in Argentina. Hemoparasites had no significant effect on the leukocyte profile, which we assessed from the May-Grünwald-Giemsa-stained blood smears. The neutrophils/lymphocytes ratio, indicative of stress, was insignificantly higher (0.06) in parasitized frogs than in parasite-free individuals (0.04). Infected frogs were larger than the controls, but this effect vanished when correcting size data for age. Young frogs (first-breeders) dominated the age distribution of parasite-free individuals, suggesting that infection of frogs takes usually place after sexual maturation. Vectors transmitting hemoparasites to B. cordobae remain to be identified. We demonstrate that moderate to high intensities of hemoparasites do not significantly affect the cellular immune response of B. cordobae, or any of the life-history traits studied, nor did they show any external sign of disease.

Metabolic changes associated with chilling injury tolerance in tomato fruit with hot water pretreatment.

Hot water treatment (HWT) of tomato (Solanum lycopersicum L.) fruit reduces the symptoms of chilling injury (CI). The aim of this study was to identify metabolites associated with HWT-induced CI tolerance in tomato fruit cv. Imperial. Mature green tomatoes with HWT (42°C/5 min) and control were stored under chilling conditions (5°C/20 days) and then ripened (21°C/7 days). Methanol extracts from pericarp were analyzed for total phenolics (TP), antioxidant activity (AoxA), and metabolic profiling by UPLC-DAD-MS and GC-MS. After cold storage and ripening, HWT fruit showed less CI, higher TP, and AoxA than control. It also showed an increased accumulation of phenolics, sugars, and some alkaloids that may be mediated by azelaic acid, glutamine, and tryptophan. The levels of N-feruloyl putrescine, esculeoside AII, and hydroxy-α-tomatine II were reduced. The better metabolic performance of HWT fruit under cold storage was associated with a higher accumulation of several metabolites (e.g., antioxidants and osmolytes) in ripening fruit. PRACTICAL APPLICATION: The identification of metabolites associated with the reduction of chilling injury (CI) symptoms in HWT tomato fruit extends the understanding of the mechanisms involved in CI tolerance. This information provides targets that could be used to develop strategies for preventing CI (e.g., genetic improvement of tomato, direct application of key metabolites). The application of such strategies will increase the economic value and decrease postharvest losses.

Body size, age and growth in males populations of Boana pulchella (Anura, Hylidae).

Age determination in amphibians is crucial to the investigation of life-history traits. In this context, we studied, for the first time, the life-history traits of Boana pulchella from a sample (63 adult males) of three populations (Las Acequias, Río Cuarto, Alejandro Roca) in central Argentina using the skeletochronological method. All adults of B. pulchella studied showed recognizable bone structures that allowed age determination. The average snout-vent length of sexually mature males was 43.39 mm. The maximum observed longevity was 5 years (5 Las Acequias, 4 Río Cuarto, 3 Alejandro Roca) and minimum age at sexual maturity was 2 years (same in the three populations), with mean of 2.96 years. Body size and age were positively correlated (except in Alejandro Roca populations). The growth patterns, estimated by the von Bertalanffy growth equation, do not show differences between populations, and the growth rate decrease after the attainment of sexual maturity. In conclusion, the determination of the individual age of the different populations of B. pulchella allowed us for the first time to establish the characteristics of the life history of the species, important for future comparisons whit others populations and future conservation biology studies.

Characterization of peptides with antioxidant activity and antidiabetic potential obtained from chickpea (Cicer arietinum L.) protein hydrolyzates.

Alcalase hydrolyzates were prepared from the albumin (AH) and globulin (GH) fractions of eight chickpea (Cicer arietinum L.) genotypes from Mexico and 10 from other countries. Protein content, antioxidant activity (AA) (ABTS, DPPH), and degree of hydrolysis were evaluated and the best genotype was selected by principal component analysis. The hydrolyzates of the chosen genotype were analyzed for its antidiabetic potential measured as inhibition of α-amylase, α-glucosidase, and dipeptidyl peptidase-4 (DPP4). Peptide profiles were obtained by liquid chromatography-mass spectrometry (UPLC-DAD-MS), and the most active peptides were analyzed by molecular docking. The average antioxidant activity of albumin hydrolyzates was higher than that of globulin hydrolyzates. ICC3761 was the selected genotype and peptides purified from the albumin hydrolyzate showed the best antioxidant activity and antidiabetic potential (FEI, FEL, FIE, FKN, FGKG, and MEE). FEI, FEL, and FIE were in the same chromatographic peak and this mixture showed the best ABTS scavenging (78.25%) and DPP4 inhibition (IC50  = 4.20 µg/ml). MEE showed the best DPPH scavenging (47%). FGKG showed the best inhibition of α-amylase (54%) and α-glucosidase (56%) and may be a competitive inhibitor based on in silico-predicted interactions with catalytic amino acids in the active site of both enzymes. These peptides could be used as nutraceutical supplements against diseases related to oxidative stress and diabetes. PRACTICAL APPLICATION: This study showed that chickpea protein hydrolyzates are good sources of peptides with antidiabetic potential, showing high antioxidant activity and inhibition of enzymes related to carbohydrate metabolism and type 2 diabetes. These hydrolyzates could be formulated in functional foods for diabetes.

Copepods and ostracods associated with bromeliads in the Yucatán Peninsula, Mexico.

A substantial fraction of the freshwater available in the Neotropical forests is enclosed within the rosettes of bromeliads that form small aquatic islands within a terrestrial landscape. These aquatic oases provide shelter, water, nutrients and resting of aggregation sites for several aquatic organisms, among them crustaceans. However, in comparison with the multitude of studies on open aquatic systems, our knowledge on crustaceans inhabiting semi-terrestrial habitats and phytotelmata is limited and their presence in such environments is poorly understood. The present study was carried out in two natural protected areas of the Yucatán Peninsula aiming to understand the diversity and dispersal strategies of crustaceans living in bromeliads. Sediment and water contained in four species of bromeliads have been collected in order to understand the diversity and dispersal strategies of crustaceans living in such habitats. From a total of 238 bromeliads surveyed, 55% were colonized by crustaceans. Sixteen copepod, three ostracod and one branchiopod species were recorded during this study, however only seven species are considered as true bromeliad inhabitants. Different degrees of association between crustaceans and bromeliad species were assessed with an indicator species analysis, where significant associations were found for all crustaceans. We found significant differences between bromeliad species and reserves and their associated fauna. In order to analyze the genetic diversity of this fauna, we sequenced several individuals of each species with two genetic markers (18S rRNA and COI mtDNA). Bayesian analyses and the Generalized Mixed Yule Coalescent method (GMYC), delimited 7 well supported species. A comparison of the dispersal strategies used by different species, including passive dispersal, phoretic behavior and active dispersal, is included. This study stresses the need of studying meiofauna of phytotelms, which could be used as an indicator of local diversity in Neotropical forests.

Bioguided isolation of N-malonyl-(+)-tryptophan from the fruit of Pithecellobium dulce (Roxb.) Benth. that showed high activity against Hymenolepis nana.

Pithecellobium dulce is distributed in America and Asia where is widely used in traditional medicine. This study describes the bioguided fractionation of the methanol extract (ME) obtained from the P. dulce fruit that showed in vitro activity against Hymenolepis nana; Artemia salina assay was used to determine toxicity; and the purified compound was computationally analysed to obtain its absorption-distribution-metabolism-excretion-and-toxicity properties (ADMET). The ME and its fractions were more active than praziquantel (PZQ), and the purified compound was characterized as N-malonyl-(+)-tryptophan (NMT). Parasites treated with NMT showed shorter paralysis and death times (5 and 7 min) than those treated with PZQ (15 and 30 min), both used at 20 mg/mL. Toxicity and ADMET prediction results supported the slight-hazardousness and efficacy of the assayed fractions/compound. This is the first report of the antiparasitary activity of both the P. dulce ME and NMT, showing their potential to treat human H. nana infections.

Improving Polyphenolic Compounds: Antioxidant Activity in Chickpea Sprouts through Elicitation with Hydrogen Peroxide.

Elicitation appears to be a promising alternative to enhance the bioactive compound content and biological activities of legume sprouts. Multi-response optimization by response surface methodology (RSM) with desirability function (DF) was used to optimize the elicitor concentration (hydrogen peroxide (H2O2)) and germination time in order to maximize total phenolic content (TPC), total flavonoids content (TFC), and antioxidant activity (AOX) of chickpea sprouts. Chemical, antinutritional, and nutraceutical properties of optimized chickpea sprouts (OCS) were also determined. The predicted regression models developed were efficiently fitted to the experimental data. The results of the desirability function revealed that optimum attributes in chickpea sprouts can be achieved by the application of 30 mM H2O2 and 72 h of germination time, with global desirability value D = 0.893. These OCS had higher (p < 0.05) TPC (7.4%), total iso-flavonoids (16.5%), AOX (14.8%), and lower phytic acid (16.1%) and saponins (21.8%) compared to H2O2 non-treated chickpea sprouts. Optimized germination conditions slightly modified the flavonoid profile in chickpea; eight iso-flavonoids were identified in OCS, including formononetin and biochanin A, which were identified as the major compounds. Results from this study support elicitation with H2O2 as an effective approach to improve phytochemical content and antioxidant activity in chickpea sprouts.

Genus level molecular phylogeny of Aegisthidae Gisbrecht, 1893 (Copepoda: Harpacticoida) reveals morphological adaptations to deep-sea and plagic habitats.

BACKGROUND: The family Aegisthidae is known as typical component of deep-sea hyperbenthic waters that gradually colonized other marine environments. The phylogenetic relationships within this family have been examined here including hyperbenthic, planktonic, benthic forms and two associated Aegisthidae species. RESULTS: Ninety four specimens belong to 14 genera were studied using 18S and 28S rRNA and COI mtDNA. Bayesian analysis supports the monophyly of 10 genera whereas Andromastax, Jamstecia, Nudivorax and Aegisthus revealed to be paraphyletic. The first offshoot of the phylogenetic tree is a clade consists of the undescribed genus Aegisthidae gen.1 sister to the two monophyletic genera Cerviniella and Hase, whereas the other Cerviniinae members (represented by Cervinia and Expansicervinia) assemble a monophylum, sister to the hyperbenthic and planktonic aegisthid genera, resulting in the paraphyly of the subfamily Cerviniinae. Hence, we defined the new subfamily Cerviniellinae subfam. nov. encompassing the three benthic genera Cerviniella, Hase and Eucanuella. The subfamily Cerviniinae has been re-defined to include Cervinia, Expansicervinia and Paracerviniella. Members of the subfamily Pontostratiotinae were clustered into two clades, one consists of the genus Stratiopontotes sister to an undescribed genus + Cerviniopsis and Siphonis. The second contains Pontostratiotes sister to the members of the planktonic subfamily Aegisthinae, resulting in the paraphyly of the Pontostratiotinae. Therefore, the Pontostratiotinae has been re-defined to include only members of the genus Pontostratiotes; whereas the subfamily Cerviniopseinae has been re-erected and re-defined containing Stratiopontotes, Cerviniopsis, Siphonis, Aegisthidae gen. 2, Herdmaniopsis, Hemicervinia and Tonpostratiotes. Within this subfamily, the associated Siphonis clusters as sister to the Cerviniopsis represents an example of convergent evolution in which the possession of a stylet-like mandible and an oral cone reminiscent of the Siphonostomatoida. The planktonic Aegisthus, Andromastax, Jamstecia, Nudivorax and Scabrantenna confirm the monophylom Aegisthinae, sister to the Pontostratiotinae. CONCLUSIONS: Our DNA based phylogeny reveals the deep-sea origin of Aegisthidae by placing benthic Aegisthidae gen.1 and Cerviniellinae subfam. nov. as the most basal lineages. Secondary adaptations to hyperbenthic and planktonic realms, as well as associated lifestyle were discovered here by the derived positions of Pontostratiotinae, Aegisthinae and Siphonis respectively.

Factors Predicting the Response to a Vitamin D-Fortified Milk in Healthy Postmenopausal Women.

BACKGROUND: Milk products fortified with vitamin D may constitute an alternative to pharmacological supplements for reaching the optimal levels of serum 25-hydroxyvitamin D [25(OH)D]. Our aim was to analyze the response of serum 25(OH)D and its predictive factors in postmenopausal healthy women after a dietary intervention with a milk fortified with vitamin D and calcium. METHODS: We designed a prospective study including 305 healthy postmenopausal women who consumed a fortified milk with calcium (900 mg/500 mL) and vitamin D3 (600 IU/500 mL) daily for 24 months. RESULTS: The 25(OH)D concentrations at 24 months were correlated to weight, to body mass index, to the percentage of fat, triglycerides and to baseline 25(OH)D levels. We found significant differences in the levels of 25(OH)D at 24 months according to baseline 25(OH)D levels (p < 0.001) and body mass index (p = 0.019) expressed at quartiles. Multivariate analysis showed an association between levels of 25(OH)D after the intervention and at baseline 25(OH)D (Beta = 0.47, p < 0.001) and percentage of body fat (Beta = -0.227, p = 0.049), regardless of the body mass index. CONCLUSIONS: In healthy postmenopausal women, the improvement in 25(OH)D after an intervention with a fortified milk for 24 months depends mainly on the baseline levels of serum 25(OH)D and on the percentage of body fat.

Anuran tadpoles inhabiting a fluoride-rich stream: diets and morphological indicators.

We in situ assessed the influence of natural fluoride concentrations in lotic freshwater ecosystems on diet and morphology of Boana cordobae tadpoles. Two streams were sampled in Argentina: Los Vallecitos stream (LF-LV) and Los Cerros Negros stream (HF-CN) with low and high fluoride levels, respectively. We captured tadpoles of B. cordobae in each stream using nets. Body weight (BW), total length (TL) and body condition (BC) of tadpoles was registered. Food items were identified to genus level and assigned to functional traits. Tadpoles showed significant differences in TL between streams, with smaller individuals in HF-CN, while did not show differences in BW and BC. The diet of tadpoles consisted mostly of microalgae. In tadpoles from HF-CN stream the proportion of cyanobacteria was lower than tadpoles from LF-LV. In relation to functional traits, small algae, high profile and colonial algae were more abundant in HF-CN. Algae attached by pads showed a higher proportion in HF-CN diets and stalked algae were more abundant in LF-LV. The differences in TL and diet of tadpoles can be attributed to differences in algal community composition, with genera that are affected by high concentrations of natural fluoride; for example cyanobacteria. The low algal richness registered in HF-CN stream does not affect the physiological state of the tadpoles, possibly because of a higher algal density in HF-CN diets. However, in further studies it would be important to evaluate the population status of B. cordobae from the HF-CN, because a small body length of tadpoles could have consequences at the population level.

Molecular evidence for the retention of the Thaumatopsyllidae in the order Cyclopoida (Copepoda) and establishment of four suborders and two families within the Cyclopoida.

The classification of the Thaumatopsyllidae within the Copepoda has been an issue of ongoing discussion since the discovery of Thaumatopsyllus paradoxus G.O. Sars, 1913 from the Norwegian coast. The family has been formally placed in the Monstrilloida, the Cyclopoida and even in its own order, the Thaumatopsylloida, based on different morphological criteria. We examined for the first time, the phylogenetic position of the Thaumatopsyllidae using gene sequences of 28S and 18S rRNA, as well as COI mtDNA, obtained from two thaumatopsyllid species occurring off the coast of southern California. We also fortuitously explored the phylogenetic relationships of the Cyclopoida in more detail than Khodami et al. (2017) by including a wider sample of key families such as the Erebonasteridae and Gisellinidae. Both Maximum Likelihood and Bayesian analyses revealed the Thaumatopsyllidae is nested in the Cyclopoida and is related to the marine Speleoithonidae. In addition, 16 families of the Cyclopoida are supported to be monophyletic, but surprisingly, the Cyclopidae is paraphyletic. The Cyclopicinidae is the first monophyletic offshoot of the cyclopoid tree, followed by two derived clades. The first clade contains a monophylum consisting of the Schminkepinellidae + Giselinidae which is sister to a clade including the monophyletic Erebonasteridae and all other poecilostome families. The second clade is divided into two main, well-supported family clusters. One includes the Cyclopidae encompassing two subfamilies (Eucylopinae and Cyclopinae), but unexpectedly the parasitic Lernaeidae cluster as a sister-group to the brackish water Halicyclops (subfamily Halicyclopinae) and the Euryteinae is the sister to all the rest of Cyclopidae s. l., making the Cyclopidae paraphyletic. To resolve this conundrum, we erected two families, Euryteidae and Halicyclopidae. The Cyclopidae s. str. retains the subfamilies Eucyclopinae and Cyclopinae, although our phylogeny does not support the reciprocal monophyly of these subfamilies. Our results support the gradual invasion of fresh water by the four families in this cluster. The highly supported monophyletic marine Euryteidae is the first offshoot followed by the brackish-water, free-living Halicyclopidae and the freshwater, parasitic Lernaeidae. The Cyclopidae fulfilled the colonization of freshwater bodies. The other clade of families comprises 12 monophyletic families recovered by our analysis, including the Pterinopsyllidae (at first offshoot), the Smirnovipinidae sister to the Hemicyclopinidae + Psammocyclopinidae, the Thaumatopsyllidae + Speleoithonidae, an undescribed family sister to the Archinotodelphyidae + Notodelphyidae and the Cyclopinidae sister to the Oithonidae + Cyclopettidae. We propose suborder ranks for each of the four main phylogenetic subdivisions of the Cyclopoida. These are named Cyclopicinida, Ergasilida, Cyclopida and Oithonida after the type genus of the oldest described family in the respective group.

Morphological abnormalities in natural populations of the common South American toad Rhinella arenarum inhabiting fluoride-rich environments.

Morphological abnormalities in amphibians may be attributed to contaminants, ultraviolet radiation and trematode parasites, or a synergistic effect between them. In the present study, morphological abnormalities in Rhinella arenarum adults from natural and artificial fluoride-rich environments were identified and evaluated. Three sites were sampled in central Argentina: Los Vallecitos stream (LF-LV), Los Cerros Negros stream (MF-CN), and Decantation ponds (HF-DP), with low (0.33 mg/L), middle (2.03 mg/L) and high (14.0 mg/L) fluoride levels respectively; the latter site is associated with a fluoride mine. Abnormal individuals were photographed and then standard radiographs were taken. Abnormality frequencies and relative percentage of abnormal individuals were calculated for each site. In addition, skeletochronology was used to estimate toad's age. Five abnormality types were identified: syndactyly, ectrodactyly, polydactyly, microphthalmia and ectromelia. Percentages of abnormal individuals per site were: LF-LV = 4%, MF-CN = 21.2% and HF-DP = 6.4%. The MF-CN and HF-DP populations had morphological abnormality frequencies that exceeded the reference value (5%) reported in the literature. The average age did not differ between sites. The results of this study indicate that there is an association between frequency of morphological abnormalities and high fluoride levels.

Evaluation in situ of genotoxic and cytotoxic response in the diploid/polyploid complex Odontophrynus (Anura: Odontophrynidae) inhabiting agroecosystems.

Polyploidization has been documented across a wide range of vertebrates. Gene duplication could promote better adaptation to environmental changes and to chronic injury or stress. We investigated if genotoxic and cytotoxic responses to agricultural impact are affected by ploidy. We evaluate syntopic populations of the cryptic diploid/polyploid complex Odontophrynus cordobae/O. americanus breeding in an agroecosystem from Central Argentina. The blood of 72 adult anurans was analysed. We used erythrometry to distinguish Odontophrynus individuals with different ploidy levels. We calculated micronucleus frequencies (Mn) and erythrocytic nuclear abnormalities (ENAs) as genotoxic effects and enucleated, mitotic, pyknotic and immature erythrocytes as cytotoxic endpoints (CYT). Mn, ENAs and CYT frequencies were significantly different between diploid and polyploid organisms. The higher frequencies of Mn and CYT were recorded in polyploid organisms, and the higher frequency of ENAs was recorded in diploids. These results indicate that stress response, as indicated by most genotoxic and cytotoxic endpoints, was higher in polyploids respect to diploids. Polyploidy could provide greater genetic flexibility increasing buffering against exogenous DNA-damaging agents and thus confer an advantage over diploids under certain environmental conditions.

Historias de vida y demografía de Physalaemus biligonigerus (Anura: Leptodactylidae) en una charca periurbana del centro de Argentina / Demography and life histories of Physalaemus biligonigerus (Anura: Leptodactylidae) in a peri-urban pond in central Argentina

Resumen El conocimiento de la dinámica de poblaciones provee información sobre la historia reciente, el estado actual y la tendencia futura de una población. Physalaemus biligonigerus es un anuro con una amplia distribución en el Neotrópico, presenta una actividad reproductiva elevada y explosiva, pero sus poblaciones parecen estar compuestas por individuos jóvenes. El objetivo de este estudio fue analizar la demografía poblacional de P. biligonigerus en una charca semipermanente en Río Cuarto, Córdoba, Argentina y realizar una proyección poblacional a 30 generaciones. Se determinó la estructura de edad utilizando esqueletocronología, y posteriormente se construyó una tabla de vida estática. Se calculo la fecundidad y las tasas de supervivencia para cada estadío con los que se construyó una Matriz de Leslie y se realizó la proyección poblacional. La proyección muestra oscilaciones en el número de individuos pero a pesar de esto, la tendencia final de la población es a un aumento en su tamaño. La mayor proporción de supervivientes se da en las fases de huevos a larvas, y la fuerza de mortalidad actúa con intensidad sobre la fase de larvas. La tasa neta reproductiva indica que la población de P. biligonigerus de la charca en estudio está en incremento.

Abstract Knowledge of population dynamics provides information on the recent history, current status, and future trends of a population. Physalaemus biligonigerus is widely distributed in the Neotropics, this anuran has a high and explosive reproductive activity, but its populations seem to be composed of young individuals. The objective of this study was to analyze the population demography of P. biligonigerus in a semipermanent pond in Río Cuarto, Córdoba, Argentina and to carry out a population projection through 30 generations. The age structure was determined from the use of skeletochronology, and a static life table was later made. Fertility and survival rates were calculated for each stage, by which a Leslie Matrix was constructed and the population projection was performed. The projection shows oscillations in the number of individuals but in spite of this, the final tendency of the population is an increase in its size. The highest proportion of survivors occurs in the egg-to-larval stages and the mortality force acts intensively on the larval phase. The net reproductive rate indicates that the population of P. biligonigerus of the study pond is increasing. Rev. Biol. Trop. 66(2): 765-775. Epub 2018 June 01.

Effects of Daily Intake of Calcium and Vitamin D-Enriched Milk in Healthy Postmenopausal Women: A Randomized, Controlled, Double-Blind Nutritional Study.

OBJECTIVE: To determine the effect of the daily intake of calcium and vitamin D-enriched milk (with or without fructooligosaccharides [FOS]) on vitamin D, bone metabolism, and cardiovascular risk factors. MATERIALS AND METHODS: Two-year randomized controlled study, including 500 healthy postmenopausal women, assigned to 500 mL/day of skimmed milk to one of three groups: Low-dose (L): (120 mg/100 mL calcium, vitamin D3 30 UI/100 mL), group A: calcium and vitamin D (180 mg/100 mL and 120 UI/100 mL), and group B: calcium and vitamin D (180 mg/100 mL and 120 UI/100 mL) and FOS (5 g/L). We evaluated serum 25(OH)D, bone mineral density (BMD) by Dual Energy X-ray Absorptiometry, and biochemical data of glucose and lipid metabolism. RESULTS: After 24 months, vitamin D concentrations did not change in the control group, but increased in group A and group B, p < 0.001. We observed an increase in femoral neck BMD and an improvement in fasting plasma glucose, HbA1c, total cholesterol, low-density lipoprotein cholesterol, and apolipoprotein B 100. CONCLUSIONS: Daily intake of milk enriched with calcium and vitamin D in postmenopausal healthy women induces a significant improvement in vitamin D status, a significant increase in BMD at femoral neck, and also favorable effects on glucose and lipid profile.

Reproductive endpoints of Rhinella arenarum (Anura, Bufonidae): Populations that persist in agroecosystems and their use for the environmental health assessment.

Degradation of the environment by agriculture affects the persistence and health of the amphibian populations. Characteristics related to reproduction of anuran can be used to evaluate the status of populations and as endpoints in environmental health assessment. In this in situ study the aspects related to the amplexus and ovipositions of the bioindicator species Rhinella arenarum that inhabits agroecosystems were analyzed. The hypothesis of this study is that perturbations of agroecosystems have a negative impact on the size of reproductive adults, on the size of ovipositions and eggs, and on the survival of eggs and embryos. Study area is located in the rural landscape of central Argentina. Four sampling sites were selected: C1, C2 and C3 are ponds on agroecosystems; and SM is a reference site that is not affected by agriculture or livestock. Abundance of amplexus pairs, oviposition and tadpoles per site was recorded. Individuals´ snout-vent length (SVL) in amplexus was measured. The fecundity was calculated like number of eggs per oviposition. The eggs' Gosner stage, the diameter eggs and the frequency of dead and abnormal eggs were recorded by oviposition. Killing-power between egg-embryo and egg-tadpole was calculated. The higher phosphate concentration was detected in all agroecosystems and nitrate was detected in C1 and C2. Conductivity, salinity and SDT were higher in C1 site Male SVL from the SM site was lower than the other sites while the largest SVL was of female from the C3 site. The higher frequencies of sprouted eggs and of dead eggs were recorded in the C2 site. Egg diameter was associated with SM and correlated negatively to SVL of the male and female. No correlation between female SVL and oviposition size was recorded. Killing-power in the passage from egg to tadpole classes was higher in the three agroecosystems. The hypothesis of this study was corroborated in part. Reproductive adults in agroecosystems did not have smaller body size. However, in the agroecosystem ponds, the eggs with smaller diameter were registered, the oviposition had higher frequency of abnormal eggs and the higher mortality was registered. This confirms the high sensitivity of the early stages to environmental disturbances and sustains their use as endpoints for the environmental health assessment.

Morphology and size of blood cells of Rhinella arenarum (Hensel, 1867) as environmental health assessment in disturbed aquatic ecosystem from central Argentina.

Four populations of Rhinella arenarum from aquatic environments with different degrees of disturbance in central Argentina were compared to assess the ability of cytomorphology and cytomorphometry of blood cells as a hematological biomarker. A total of 93 specimens of R. arenarum (adults sexually mature) were captured during the spring. From the analysis of cell, no variations were found in terms of morphology, whereas in nuclear and cell areas and Price-Jones curves, we observed a smaller size in erythrocytes of individuals inhabiting the site most altered, "Villa Dalcar," as well as for leukocytes, lymphocytes, neutrophils, and eosinophils for the same site. This could be caused by presence of different pollutants in the lake. Furthermore, this was confirmed by the high levels of environmental variables (conductivity, total dissolved solids, and salinity) show that Villa Dalcar is the site most affected by human activities.

Evaluation in situ of genotoxicity and stress in South American common toad Rhinella arenarum in environments related to fluorite mine.

Little attention has been paid to the impact of wastewater generated by mining activities on fluoride. In this study, we evaluated the hematology responses of common South American toad Rhinella arenarum inhabiting natural and artificial environments associated with a fluorite mine from central Argentina. We analyzed three sampling stations associated with the fluorite mine: (I) Los Cerros Negros stream (CN), which runs on granitic rock with a high fluorite content; (II) Los Vallecitos stream (LV), which runs on metamorphic rock with low fluorite content; and (III) artificial decantation ponds (DP) containing sediments produced by fluorite flotation process. We calculated frequencies of micronuclei, erythrocyte nuclear abnormalities, mitosis, and immature erythrocytes. In addition, we performed a differential leukocyte count and determined neutrophils/lymphocyte ratio as a stress response estimator. We found high micronucleus (MN) and erythrocyte nuclear abnormality (ENA) frequencies in DP and CN but low frequencies in LV. The neutrophil/lymphocyte ratio was different among sites, with a significant increase in individuals from DP. Values registered in DP could be caused by exposure to mixture of compounds registered in dams that hold wastewater, while high values registered in CN stream might be due to natural concentrations of fluoride. Our results suggest that blood is an effective and non-destructive sensitive indicator for monitoring genotoxic agents in freshwater ecosystems.

Revision of the Remaneicaris argentina-group (Copepoda, Harpacticoida, Parastenocarididae): supplementary description of species, and description of the first semi-terrestrial Remaneicaris from the tropical forest of Southeast Mexico.

Remaneicaris is a species-rich Neotropical monophyletic group, easily recognized by the synapomorphic position of the outer seta of the third exopodite of leg 4, localized at 2/3 of the outer margin. The genus, comprising 35 species in five monophyletic groups, plus R. ignotus and R. meyerabichi, retains an unusual set of plesiomorphic characters. Herein we supplement the descriptions of the species belonging to the Remaneicaris argentina-group, and describe a new species from the tropical forest of Southeast Mexico. The present study extends the geographic distribution of the genus, with the northernmost record until now being from El Salvador. The genus having hitherto been known from interstitial groundwater habitats, this is its first record in epigean semi-terrestrial habitats. Remaneicaris siankaan sp. nov. was found in phytotelmata (bromeliads), leaf litter, moist soil, permanent ponds (known locally as "aguadas"), and temporal and permanent wetlands (savannahs). The new species can be easily characterized by its finely pitted cuticle, the ε (epsilon)-shaped thumb of the male P3 and the bifid accessory spine with distal hyaline inner tip, which precedes the thumb. A new method for the 3D reconstruction of microcrustaceans is described.

Chemical composition and biological activities of Helicteres vegae and Heliopsis sinaloensis.

CONTEXT: Helicteres vegae Cristóbal (Sterculiaceae) (Hv) and Heliopsis sinaloensis B.L. Turner (Asteraceae) (Hs) are endangered and poorly studied plant species; related plants have been used against chronic-degenerative and infectious diseases. Therefore, Hv and Hs could be sources of bioactive compounds against these illnesses. OBJECTIVE: To determine the chemical composition and biological activities (antioxidant, antimutagenic and antimicrobial) of Hv and Hs leaves (L) and stems (S). MATERIALS AND METHODS: Methanol extracts (ME) of each plant/tissue were evaluated for their phytochemicals; phenolics (HPLC-DAD-ESI-MS); antioxidant activity (AA) (0.125-4 mg/mL) (DPPH, ABTS, ORAC and ß-carotene discoloration); antimutagenicity (0.5 and 1 mg/plate) (Ames assay, tester strain Salmonella enterica serovar Typhimurium YG1024, 1-nitropyrene as mutagen); activity against human pathogens (1 mg/mL); and toxicity (0.01-2 mg/mL) (Artemia salina assay). RESULTS: All ME showed flavonoids and triterpenes/steroids. The ME-SHv had the highest content of total phenolics (TP) (2245.82 ± 21.45 mg GAE/100 g d.w.) and condensed tannins (603.71 ± 1.115 mg CE/100 g d.w.). The compounds identified were flavonoids (kaempferol 7-O-coumaroylhexoside, and two kaempferol 7-O-rhamnosylhexosides) and phenolics [rosmarinic acid, and 3'-O-(8″-Z-caffeoyl) rosmarinic acid]. The ME-LHs showed the highest content of flavonoids (357.88 mg RE/g d.w.) and phenolic acids (238.58 mg CAE/g d.w.) by HPLC. The ME-SHv showed the highest AA. All ME were strong antimutagens (63.3-85.7%). Only the Hs extracts were toxic (ME-LHs, LC50 = 94.9 ± 1.7 µg/mL; ME-SHs, LC50 = 89.03 ± 4.42 µg/mL). DISCUSSION AND CONCLUSIONS: Both Hv and Hs are potential sources of preventive and therapeutic agents against chronic-degenerative diseases.