De novo assembly of the freshwater prawn Macrobrachium carcinus brain transcriptome for identification of potential targets for antibody development.

Crustaceans are major constituents of aquatic ecosystems and, as such, changes in their behavior and the structure and function of their bodies can serve as indicators of alterations in their immediate environment, such as those associated with climate change and anthropogenic contamination. We have used bioinformatics and a de novo transcriptome assembly approach to identify potential targets for developing specific antibodies to serve as nervous system function markers for freshwater prawns of the Macrobrachium spp. Total RNA was extracted from brain ganglia of Macrobrachium carcinus freshwater prawns and Illumina Next Generation Sequencing was performed using an Eel Pond mRNA Seq Protocol to construct a de novo transcriptome. Sequencing yielded 97,202,662 sequences: 47,630,546 paired and 1,941,570 singletons. Assembly with Trinity resulted in 197,898 assembled contigs from which 30,576 were annotated: 9,600 by orthology, 17,197 by homology, and 3,779 by transcript families. We looked for glutamate receptors contigs, due to their main role in crustacean excitatory neurotransmission, and found 138 contigs related to ionotropic receptors, 32 related to metabotropic receptors, and 18 to unidentified receptors. After performing multiple sequence alignments within different biological organisms and antigenicity analysis, we were able to develop antibodies for prawn AMPA ionotropic glutamate receptor 1, metabotropic glutamate receptor 1 and 4, and ionotropic NMDA glutamate receptor subunit 2B, with the expectation that the availability of these antibodies will help broaden knowledge regarding the underlying structural and functional mechanisms involved in prawn behavioral responses to environmental impacts. The Macrobrachium carcinus brain transcriptome can be an important tool for examining changes in many other nervous system molecules as a function of developmental stages, or in response to particular conditions or treatments.

Alterations of larval photo-dependent swimming responses (PDR): New endpoints for rapid and diagnostic screening of aquatic contamination.

Detection and toxicity assessment of waterborne contaminants are crucial for protecting human health and the environment. Development of easy-to-implement, rapid and cost-effective tools to measure anthropogenic effects on watersheds are critical for responsible management, particularly in times of increasing development and urbanization. Traditionally, environmental toxicology has focused on limited endpoints, such as lethality and fertility, which are directly affecting population levels. However, more sensitive readings are needed to assess sub-lethal effects. Monitoring of contaminant-induced behavior alterations was proposed before, but is difficult to implement in the wild and performing it in aquatic laboratory models seem more suited. For this purpose, we adapted a photo-dependent swimming response (PDR) that was previously described in zebrafish larva. We first asked if PDR was present in other aquatic animals. We measured PDR in larvae from two freshwater prawn species (Macrobrachium rosenbergii, MR, and Macrobrachium carcinus, MC) and from another fish the fathead minnow (FHM, Pimephales promelas). In all, we found a strong and reproducible species-specific PDR, which is arguing that this behavior is important, therefore an environmental relevant endpoint. Next, we measured PDR in fish larvae after acute exposure to copper, a common waterborne contaminant. FHM larvae were hyperactive at all tested concentrations in contrast to ZF larvae, which exhibited a concentration-dependent hyperactivity. In addition to this well-accepted anxiety-like behavior, we examined two more: photo-stimulated startle response (PSSR) and center avoidance (CA). Both were significantly increased. Therefore, PDR measures after acute exposure to this waterborne contaminant provided as sensitive readout for its detection and toxicity assessment. This approach represents an opportunity to diagnostically examine any substance, even when present in complex mixtures like ambient surface waters. Mechanistic studies of toxicity using the extensive molecular tool kit of ZF could be a direct extension of such approaches.

Assessment of Concentrations of Heavy Metals and Phthalates in Two Urban Rivers of the Northeast of Puerto Rico.

Urbanization adjacent to rivers has increased in recent years and is considered a source of environmental contamination. The resulting increase in number of urban rivers in highly populated areas, such as the Caribbean island of Puerto Rico, has led to the appearance of synthetic as well as naturally occurring chemicals not previously observed nor regularly monitored in freshwater habitats. Some of these chemicals, such as heavy metals and plasticizers, have been shown to affect endocrine, respiratory, and nervous system function in animals and humans, even at relatively low concentrations. The purpose of this study was to measure concentrations of such emergent contaminants on rivers of urbanized areas on the northeast of Puerto Rico, as one element in the assessment of the impact of urbanism on water quality in these communities. To accomplish this, we used Inductively Coupled Plasma and Gas Chromatography Mass Spectrometry to measure amounts of heavy metals and phthalates, respectively, in superficial water of three rivers of Puerto Rico: Mameyes (non-urban), Río Piedras (urban river without a dam), and La Plata (urban river with a dam). The urban rivers had significantly higher concentrations of heavy metals arsenic, barium, cadmium, manganese, and antimony, when compared with the reference non-urban river. Manganese was the only metal found in concentrations higher than limits established by the EPA for drinking water. Of eight phthalates amenable to measurement with the chosen protocol and instrumentation, only dibutyl phthalate was detected, only in the La Plata river, and at concentrations ranging from 3 to 8 parts-per-billion. These findings suggest that urbanism close to rivers of Puerto Rico is likely having an impact on water quality and thus further study to identify the potential sources, as well as the inclusion of these emergent contaminants on the list of chemicals regularly monitored by government agencies is justified.

Characterization of a prawn OA/TA receptor in Xenopus oocytes suggests functional selectivity between octopamine and tyramine.

Here we report the characterization of an octopamine/tyramine (OA/TA or TyrR1) receptor (OA/TAMac) cloned from the freshwater prawn, Macrobrachium rosenbergii, an animal used in the study of agonistic social behavior. The invertebrate OA/TA receptors are seven trans-membrane domain G-protein coupled receptors that are related to vertebrate adrenergic receptors. Behavioral studies in arthropods indicate that octopaminergic signaling systems modulate fight or flight behaviors with octopamine and/or tyramine functioning in a similar way to the adrenalins in vertebrate systems. Despite the importance of octopamine signaling in behavioral studies of decapod crustaceans there are no functional data available for any of their octopamine or tyramine receptors. We expressed OA/TAMac in Xenopus oocytes where agonist-evoked trans-membrane currents were used as readouts of receptor activity. The currents were most effectively evoked by tyramine but were also evoked by octopamine and dopamine. They were effectively blocked by yohimbine. The electrophysiological approach we used enabled the continuous observation of complex dynamics over time. Using voltage steps, we were able to simultaneously resolve two types of endogenous currents that are affected over different time scales. At higher concentrations we observe that octopamine and tyramine can produce different and opposing effects on both of these currents, presumably through the activity of the single expressed receptor type. The pharmacological profile and apparent functional-selectivity are consistent with properties first observed in the OA/TA receptor from the insect Drosophila melanogaster. As the first functional data reported for any crustacean OA/TA receptor, these results suggest that functional-selectivity between tyramine and octopamine is a feature of this receptor type that may be conserved among arthropods.

[Genotyping of Cryptococcus neoformans/Cryptococcus gattii complex clinical isolates from Hospital "Dr. Julio C. Perrando", Resistencia city (Chaco, Argentina)]. / Genotipificación de aislamientos clínicos del complejo Cryptococcus neoformans/Cryptococcus gattii obtenidos en el Hospital «Dr. Julio C. Perrando¼, de la ciudad de Resistencia (Chaco, Argentina).

Cryptococcosis is a fungal infection caused by yeast species of Cryptococcus genus, particularly Cryptococcus neoformans/Cryptococcus gattii species complex. The knowledge of the cryptococcosis casuistic in northeastern Argentina is scarce and there is no information about the molecular types circulating in this area. The aim of this study was to genotyping C. neoformans/C. gattii complex clinical isolates obtained at Hospital "Dr. Julio C. Perrando", Resistencia city (Chaco, Argentina), in order to determine species, variety and molecular type. During two years and one month 26 clinical isolates were studied. Using conventional and molecular methods one isolate was identified as C. gattii VGI type, and 25 isolates as C. neoformans var. grubii; 23 of these belonged to VNI type and two belonged to VNII type. This data is a contribution to the knowledge of cryptococcosis epidemiology in Argentina and the first report about C. neoformans/ C. gattii complex molecular types from clinical isolates in northeastern Argentina.

Genotipificación de aislamientos clínicos del complejo Cryptococcus neoformans/Cryptococcus gattii obtenidos en el Hospital ½Dr. Julio C. Perrando», de la ciudad de Resistencia (Chaco, Argentina) / [Genotyping of Cryptococcus neoformans/Cryptococcus gattii complex clinical isolates from Hospital \"Dr. Julio C. Perrando\", Resistencia city (Chaco, Argentina)].

Cryptococcosis is a fungal infection caused by yeast species of Cryptococcus genus, particularly Cryptococcus neoformans/Cryptococcus gattii species complex. The knowledge of the cryptococcosis casuistic in northeastern Argentina is scarce and there is no information about the molecular types circulating in this area. The aim of this study was to genotyping C. neoformans/C. gattii complex clinical isolates obtained at Hospital "Dr. Julio C. Perrando", Resistencia city (Chaco, Argentina), in order to determine species, variety and molecular type. During two years and one month 26 clinical isolates were studied. Using conventional and molecular methods one isolate was identified as C. gattii VGI type, and 25 isolates as C. neoformans var. grubii; 23 of these belonged to VNI type and two belonged to VNII type. This data is a contribution to the knowledge of cryptococcosis epidemiology in Argentina and the first report about C. neoformans/ C. gattii complex molecular types from clinical isolates in northeastern Argentina.

Genotipificación de aislamientos clínicos del complejo Cryptococcus neoformans/Cryptococcus gattii obtenidos en el Hospital ½Dr. Julio C. Perrando», de la ciudad de Resistencia (Chaco, Argentina). / [Genotyping of Cryptococcus neoformans/Cryptococcus gattii complex clinical isolates from Hospital "Dr. Julio C. Perrando", Resistencia city (Chaco, Argentina)].

Cryptococcosis is a fungal infection caused by yeast species of Cryptococcus genus, particularly Cryptococcus neoformans/Cryptococcus gattii species complex. The knowledge of the cryptococcosis casuistic in northeastern Argentina is scarce and there is no information about the molecular types circulating in this area. The aim of this study was to genotyping C. neoformans/C. gattii complex clinical isolates obtained at Hospital "Dr. Julio C. Perrando", Resistencia city (Chaco, Argentina), in order to determine species, variety and molecular type. During two years and one month 26 clinical isolates were studied. Using conventional and molecular methods one isolate was identified as C. gattii VGI type, and 25 isolates as C. neoformans var. grubii; 23 of these belonged to VNI type and two belonged to VNII type. This data is a contribution to the knowledge of cryptococcosis epidemiology in Argentina and the first report about C. neoformans/ C. gattii complex molecular types from clinical isolates in northeastern Argentina.

Cloning and distribution of a putative octopamine/tyramine receptor in the central nervous system of the freshwater prawn Macrobrachium rosenbergii.

There is ample evidence linking octopamine (OA) and tyramine (TA) to several neurophysiological functions in arthropods. In our laboratory we use the freshwater prawn Macrobrachium rosenbergii to study the neural basis of aggressive behavior. As a first step towards understanding the possible role of these amines and their receptors in the modulation of interactive behaviors, we have cloned a putative octopamine/tyramine receptor. The predicted sequence of the cloned OA/TA(Mac) receptor consists of 1,579 base pairs (bp), with an open reading frame of 1,350bp that encodes a 450 amino acid protein. This putative protein displays sequence identities of 70% to an Aedes aegypti mosquito TA receptor, followed by 60% to a Stegomyia aegypti mosquito OA receptor, 59% and 58% to the migratory locust TA-1 and -2 receptors respectively, and 57% with the silkworm OA receptor. We also mapped the OA/TA(Mac) receptor distribution by in-situ hybridization to the receptor's mRNA, and by immunohistochemistry to its protein. We observed stained cell bodies for the receptor's mRNA, mainly in the midline region of the thoracic and in the abdominal ganglia, as well as diffuse staining in the brain ganglia. For the receptor's protein, we observed extensive punctate staining within the neuropil and on the membrane of specific groups of neurons in all ganglia throughout the CNS, including the brain, the midline region and neuropiles of the thoracic ganglia, and ventral part and neuropiles of the abdominal ganglia. The same pattern of stained cells was observed on the thoracic and abdominal ganglia in both in-situ hybridization and immunohistochemistry experiments. Diffuse staining observed with in-situ hybridization also coincides with punctate staining observed in brain, SEG, thoracic, and abdominal ganglia in immunohistochemical preparations. This work provides the first step towards characterizing the neural networks that mediate octopaminergic signaling in prawn.

GYRKPPFNGSIFamide (Gly-SIFamide) modulates aggression in the freshwater prawn Macrobrachium rosenbergii.

The freshwater prawn Macrobrachium rosenbergii is a tropical crustacean with characteristics similar to those of lobsters and crayfish. Adult males develop through three morphological types-small (SC), yellow (YC), and blue claws (BC)-with each representing a level in the dominance hierarchy of a group, BC males being the most dominant. We are interested in understanding the role played by neuropeptides in the mechanisms underlying aggressive behavior and the establishment of dominance hierarchies in this type of prawn. SIFamides are a family of arthropod peptides recently identified in the central nervous system of insects and crustaceans, where it has been linked to olfaction, sexual behavior, and gut endocrine functions. One of the six SIFamide isoforms, GYRKPPFNGSIFamide (Gly-SIFamide), is highly conserved among decapod crustaceans such as crabs and crayfish. We wanted to determine whether Gly-SIFamide plays a role in modulating aggression and dominant behavior in the prawn. To do this, we performed behavioral experiments in which interactions between BC/YC pairs were recorded and quantified before and after injecting Gly-SIFamide directly into the circulating hemolymph of the living animal. Behavioral data showed that aggression among interacting BC/YC prawns was enhanced by injection of Gly-SIFamide, suggesting that this neuropeptide does have a modulatory role for this type of behavior in the prawn.

Cloning and immunoreactivity of the 5-HT 1Mac and 5-HT 2Mac receptors in the central nervous system of the freshwater prawn Macrobrachium rosenbergii.

Biogenic amines are implicated in several mental disorders, many of which involve social interactions. Simple model systems, such as crustaceans, are often more amenable than vertebrates for studying mechanisms underlying behaviors. Although various cellular responses of biogenic amines have been characterized in crustaceans, the mechanisms linking these molecules to behavior remain largely unknown. Observed effects of serotonin receptor agonists and antagonists in abdomen posture, escape responses, and fighting have led to the suggestion that biogenic amine receptors may play a role in modulating interactive behaviors. As a first step in understanding this potential role of such receptors, we have cloned and fully sequenced two serotonin receptors, 5-HT(1Mac) and 5-HT(2Mac), from the CNS of the freshwater prawn Macrobrachium rosenbergii and have mapped their CNS immunohistochemical distribution. 5-HT(1Mac) was found primarily on the membranes of subsets of cells in all CNS ganglia, in fibers that traverse all CNS regions, and in the cytoplasm of a small number of cells in the brain and circum- and subesophageal ganglia (SEG), most of which also appear to contain dopamine. The pattern of 5-HT(2Mac) immunoreactivity was found to differ significantly; it was found mostly in the central neuropil area of all ganglia, in glomeruli of the brain's olfactory lobes, and in the cytoplasm of a small number of neurons in the SEG, thoracic, and some abdominal ganglia. The observed differences in terms of localization, distribution within cells, and intensity of immunoreactive staining throughout the prawn's CNS suggest that these receptors are likely to play different roles.

Arthropod 5-HT2 receptors: a neurohormonal receptor in decapod crustaceans that displays agonist independent activity resulting from an evolutionary alteration to the DRY motif.

The stomatogastric nervous system (STNS) is a premiere model for studying modulation of motor pattern generation. Whereas the cellular and network responses to monoamines have been particularly well characterized electrophysiologically, the transduction mechanisms that link the different monoaminergic signals to specific intracellular responses are presently unknown in this system. To begin to elucidate monoaminergic signal transduction in pyloric neurons, we used a bioinformatics approach to predict the existence of 18 monoamine receptors in arthropods, 9 of which have been previously cloned in Drosophila and other insects. We then went on to use the two existing insect databases to clone and characterize the 10th putative arthropod receptor from the spiny lobster, Panulirus interruptus. This receptor is most homologous to the 5-HT2 subtype and shows a dose-dependent response to 5-HT but not to any of the other monoamines present in the STNS. Through a series of pharmacological experiments, we demonstrate that this newly described receptor, 5-HT2betaPan, couples with the traditional G(q) pathway when expressed in HEK293 cells, but not to G(s) or G(i/o). Moreover, it is constitutively active, because the highly conserved DRY motif in transmembrane region 3 has evolved into DRF. Site-directed mutagenesis that reverts the motif back to DRY abolishes this agonist-independent activity. We further demonstrate that this receptor most likely participates in the modulation of stomatogastric motor output, because it is found in neurites in the synaptic neuropil of the stomatogastric ganglion as well as in the axon terminals at identified pyloric neuromuscular junctions.

A crustacean serotonin receptor: cloning and distribution in the thoracic ganglia of crayfish and freshwater prawn.

Serotonin (5-HT) is involved in regulating important aspects of behavior and a variety of systemic physiological functions in both vertebrates and invertebrates. These functions are mediated through binding to 5-HT receptors, of which approximately 13 have been characterized in mammals. In crustaceans, important model systems for the study of the neural basis of behaviors, 5-HT is also linked with higher-order behaviors, associated with different 5-HT receptors that have been identified at the physiological and pharmacological levels. However, no crustacean 5-HT receptors have been identified at the molecular level. We have cloned a putative 5-HT(1) receptor (5-HT(1crust)) from crayfish, prawn, and spiny lobster and have raised antibodies that recognize this protein in all three organisms. 5-HT(1crust) immunoreactivity (5-HT(1crust)ir) was observed surrounding the somata of specific groups of neurons and as punctate staining within the neuropil in all thoracic ganglia of crayfish and prawn. In the crayfish, 5-HT(1crust)ir was also found in boutons surrounding the first and second nerves of each ganglion and on the 5-HT cells of T1-4. In the prawn, 5-HT(1crust)ir was also found in axons that project across the ganglia and along the connectives. We found examples of colocalization of 5-HT(1crust) with 5-HT, consistent with the short-term modulatory role of 5-HT, as well as cases of serotonergic staining in the absence of a 5-HT(1crust) signal, which might imply that other 5-HT receptors are found at these locations. We also observed receptors that did not possess counterpart 5-HT staining, suggesting that these may also mediate long-term neurohormonal functions of serotonin.

Microhongos anemofilos en ambientes abiertos de dos ciudades del nordeste argentino / Anemophilous microfungi in outdoor environments of two cities of northern Argentina

Con el objetivo de estudiar la aeromicota de las ciudades de Resistencia y Corrientes, ubicadas en el nordeste argentino, se muestrearon por la técnica de deposición gravitacional, dos zonas en cada ciudad. El muestreo se realizó en primavera y otoño, en dos horarios diferentes. Existen diferencias significativas entre los aislamientos de ambas ciudades. En Resistencia se aisló un mayor número de taxa (323) que en Corrientes, (248) y también se obtuvo mayor diversidad de géneros (34/28). Los géneros mas frecuentes fueron Cladosporium, Aspergillus, Penicillium y Phoma. Los hongos aislados son de interés médico por que pueden causar infecciones, alergias o micotoxocosis. La información obtenida puede, por lo tanto, ser útil para alergistas, clínicos y epidemiólogos

Microhongos anemofilos en ambientes abiertos de dos ciudades del nordeste argentino / Anemophilous microfungi in outdoor environments of two cities of northern Argentina

Con el objetivo de estudiar la aeromicota de las ciudades de Resistencia y Corrientes, ubicadas en el nordeste argentino, se muestrearon por la técnica de deposición gravitacional, dos zonas en cada ciudad. El muestreo se realizó en primavera y otoño, en dos horarios diferentes. Existen diferencias significativas entre los aislamientos de ambas ciudades. En Resistencia se aisló un mayor número de taxa (323) que en Corrientes, (248) y también se obtuvo mayor diversidad de géneros (34/28). Los géneros mas frecuentes fueron Cladosporium, Aspergillus, Penicillium y Phoma. Los hongos aislados son de interés médico por que pueden causar infecciones, alergias o micotoxocosis. La información obtenida puede, por lo tanto, ser útil para alergistas, clínicos y epidemiólogos (AU)

Microhongos anemofilos en ambientes abiertos de dos ciudades del nordeste argentino / Anemophilous microfungi in outdoor environments of two cities

Con el objetico de estudiar los mirohongos anÚmofilos de las ciudades de Resistencia y Corrientes, ubicadas en el nordeste argentino, se muestrearon por la tÚcnica de depòsitación gravitacional, dos zonas en cada ciudad. El muestreo se realizó en primavera y otoño, en dos horarios diferentes. Existe diferencvia entre los aislamientos de ambas ciudades. En Resistencia se observó un mayor número de taxa (323) que enn Corrientes, (248) siendo mßs notoria esta diferencia en otoño junto a una mayor diversidad de gÚneros (34/28). Los gÚneros mßs frecuentes en ambas ciudades fueron: Cladosporium, Aspergillus, Penicillium, Phona, Fusarium , Alternaria, Curvularia y Trichoderma. Un buen número de los hongos aislados son de interÚs en salud publica por sus capacidades oportunistas en diversos cuadros clinicos, asi comno en alergias o micotoxocosis.

Distribución altitudinal de hongos queratinofilos, epífitos y endófitos en suelos semiáridos del noreste argentino (Prov. de Jujuy, 23°L. S y 66°L. W) / Altitudinal distribution of keratinophilic, epiphytic and endophytic fungi from northwestern argentinian semiarid soils (Prov. of Jujuy, 23°L. S and 66°L. W)

En zonas semidesérticas del noreste argentino, entre los 200 y 3700m de altura y en época veraniega, se colectaron mediante anzuelo queratínico y vegetal (epífitos y endófitos), 26 muestras de suelos y 41 de flora autóctona, obteniéndose un total de 555 aislamientos fúngicos repartidos en 60 géneros y 100 especies. Los géneros con mayor número de especies correspondieron a: Acremonium, Aspergillus, Chaetomium, Fusarium, Phoma y Ulocladium. En las 3 metodologías y entre los 2000 y 3000m, se obtuvo el mayor número de taxa y los mayores aislamientos en todas las altitudes, correspondieron a los epífitos y a los endófitos, donde los Hyphomycetes constituyeron el grupo más representativo en géneros y especies. Los Onygenales fueron poco representados en el sustrato queratínico, y la mayor presencia correspondió a Chrysosporium keratinophilum, Gymnascella aurantiaca y Aphanoascus fulvescens, mientras en otros grupos taxonómicos, la especie más frecuente fue Paecilomyces lilacinus. Debe destacarse la capacidad de este substrato para detectar la presencia de Fonsecaea pedrosoi un importante agente de cromoblastomicosis en Argentina. En los epífitos, el 41,8 porciento de los aislamientos fueron representados por: Alternaria alternata, Cladosporium cladosporioides, Epicoccum nigrum, Penicillium spp. y Ulocladium atrum complex. En los endófitos, los integrantes de los géneros Chaetomium, Aspergillus, Phoma y Fusarium, presentaron la mayor diversidad de especies bajo los 3000m, a mayor altitud, sólo Phoma mantuvo una alta diversidad. A.alternata complex, fue la especie con mayor capacidad adaptativa en las 3 metodologías y en las 2 altitudes. En el área estudiada, la altitud parece no afectar la diversidad de especies sobre los substratos analizados, donde la flora autóctona de la región, más que el suelo, parece representar el reservorio y hábitat más propicio.