First neurotranscriptome of adults Tambaquis (Colossoma macropomum) with characterization and differential expression between males and females.

The Tambaqui is one of the most representative Amazon fish species, being highly exploited in fisheries, aquaculture and as a research model. Nonetheless, data about functional genome are still required to evaluate reproductive and nutrition parameters as well as resistance to pathogens. The of next-generation sequencing has allows assessing the transcriptional processes in non-model species by providing comprehensive gene collections to be used as a database in further genomic applications and increased performance of captive populations. In this study, we relied on RNAseq approach to generate the first transcriptome of the telencephalon from adult males and females of Colossoma macropomum, resulting in a reference dataset for future functional studies. We retrieved 896,238 transcripts, including the identification of 267,785 contigs and 203,790 genes. From this total, 91 transcripts were differentially expressed, being 63 and 28 of them positively regulated for females and males, respectively. The functional annotation resulted in a library of 40 candidate genes for females and 20 for males. The functional enrichment classes comprised reproductive processes (GO:0,048,609; GO:0,003,006; GO:0,044,703; GO:0,032,504; GO:0,019,953) being related to sex differentiation (e.g., SAFB) and immune response (e.g., SLC2A6, AHNAK, NLRC3, NLRP3 and IgC MHC I alpha3), thus indicating that the genes in the neurotranscriptome of Tambaqui participate in sex differentiation and homeostasis of captive specimens. These data are useful to design the selection of genes related to sex determination and animal welfare in raising systems of Tambaqui.

Genes, inflammatory response, tolerance, and resistance to virus infections in migratory birds, bats, and rodents.

Normally, the host immunological response to viral infection is coordinated to restore homeostasis and protect the individual from possible tissue damage. The two major approaches are adopted by the host to deal with the pathogen: resistance or tolerance. The nature of the responses often differs between species and between individuals of the same species. Resistance includes innate and adaptive immune responses to control virus replication. Disease tolerance relies on the immune response allowing the coexistence of infections in the host with minimal or no clinical signs, while maintaining sufficient viral replication for transmission. Here, we compared the virome of bats, rodents and migratory birds and the molecular mechanisms underlying symptomatic and asymptomatic disease progression. We also explore the influence of the host physiology and environmental influences on RNA virus expression and how it impacts on the whole brain transcriptome of seemingly healthy semipalmated sandpiper (Calidris pusilla) and spotted sandpiper (Actitis macularius). Three time points throughout the year were selected to understand the importance of longitudinal surveys in the characterization of the virome. We finally revisited evidence that upstream and downstream regulation of the inflammatory response is, respectively, associated with resistance and tolerance to viral infections.

Molecular Changes in the Brain of the Wintering Calidris pusilla in the Mangroves of the Amazon River Estuary.

Migrant birds prepare differently to fly north for breeding in the spring and for the flight to lower latitudes during autumn, avoiding the cold and food shortages of the Northern Hemisphere's harsh winter. The molecular events associated with these fundamental stages in the life history of migrants include the differential gene expression in different tissues. Semipalmated sandpipers (Calidris pusilla) are Arctic-breeding shorebirds that migrate to the coast of South America during the non-breeding season. In a previous study, we demonstrated that between the beginning and the end of the wintering period, substantial glial changes and neurogenesis occur in the brain of C. pusilla. These changes follow the epic journey of the autumn migration when a 5-day non-stop transatlantic flight towards the coast of South America and the subsequent preparation for the long-distance flight of the spring migration takes place. Here, we tested the hypothesis that the differential gene expressions observed in the brains of individuals captured in the autumn and spring windows are consistent with the previously described cellular changes. We searched for differential gene expressions in the brain of the semipalmated sandpiper, of recently arrived birds (RA) from the autumnal migration, and that of individuals in the premigratory period (PM) in the spring. All individuals were collected in the tropical coastal of northern Brazil in the mangrove region of the Amazon River estuary. We generated a de novo neurotranscriptome for C. pusilla individuals and compared the gene expressions across libraries. To that end, we mapped an RNA-Seq that reads to the C. pusilla neurotranscriptome in four brain samples of each group and found that the differential gene expressions in newly arrived and premigratory birds were related with neurogenesis, metabolic pathways (ketone body biosynthetic and the catabolic and lipid biosynthetic processes), and glial changes (astrocyte-dopaminergic neuron signaling, astrocyte differentiation, astrocyte cell migration, and astrocyte activation involved in immune response), as well as genes related to the immune response to virus infections (Type I Interferons), inflammatory cytokines (IL-6, IL-1ß, TNF, and NF-κB), NLRP3 inflammasome, anti-inflammatory cytokines (IL-10), and cell death pathways (pyroptosis- and caspase-related changes).

Plasticity in the hippocampal formation of shorebirds during the wintering period: Stereological analysis of parvalbumin neurons in Actitis macularius.

The number of parvalbumin neurons can be modified by social, multisensory, and cognitive stimuli in both mammals and birds, but nothing is known about their plasticity in long-distance migratory shorebirds. Here, in the spotted sandpiper (Actitis macularius), we investigated the plasticity of parvalbumin neurons of two brain areas during this species' wintering period at a lower latitude. We compared individuals in a nonmigratory rest period (November-January) and premigration (May-July) period. We used parvalbumin as a marker for counting a subpopulation of inhibitory neurons in the hippocampal formation (HF), with the magnocellular nucleus of the tectal isthmus (IMC) as a control area. Because the HF is involved in learning and memory and social interaction and the IMC is essential for control of head, neck, and eye movements, we hypothesized that parvalbumin neurons would increase in the HF and remain unchanged in the IMC. We used an optical fractionator to estimate cell numbers. Compared with the nonmigratory rest birds, parvalbumin neuron count estimates in the premigration birds increased significantly in the HF but remained unchanged in IMC. We suggest that the greater number of parvalbuminergic neurons in the HF of A. macularius in the premigration period represents adaptive circuitry changes involved in the migration back to reproductive niches in the northern hemisphere.

Microglial Morphology Across Distantly Related Species: Phylogenetic, Environmental and Age Influences on Microglia Reactivity and Surveillance States.

Microglial immunosurveillance of the brain parenchyma to detect local perturbations in homeostasis, in all species, results in the adoption of a spectrum of morphological changes that reflect functional adaptations. Here, we review the contribution of these changes in microglia morphology in distantly related species, in homeostatic and non-homeostatic conditions, with three principal goals (1): to review the phylogenetic influences on the morphological diversity of microglia during homeostasis (2); to explore the impact of homeostatic perturbations (Dengue virus challenge) in distantly related species (Mus musculus and Callithrix penicillata) as a proxy for the differential immune response in small and large brains; and (3) to examine the influences of environmental enrichment and aging on the plasticity of the microglial morphological response following an immunological challenge (neurotropic arbovirus infection). Our findings reveal that the differences in microglia morphology across distantly related species under homeostatic condition cannot be attributed to the phylogenetic origin of the species. However, large and small brains, under similar non-homeostatic conditions, display differential microglial morphological responses, and we argue that age and environment interact to affect the microglia morphology after an immunological challenge; in particular, mice living in an enriched environment exhibit a more efficient immune response to the virus resulting in earlier removal of the virus and earlier return to the homeostatic morphological phenotype of microglia than it is observed in sedentary mice.

Shorebirds' Longer Migratory Distances Are Associated With Larger ADCYAP1 Microsatellites and Greater Morphological Complexity of Hippocampal Astrocytes.

For the epic journey of autumn migration, long-distance migratory birds use innate and learned information and follow strict schedules imposed by genetic and epigenetic mechanisms, the details of which remain largely unknown. In addition, bird migration requires integrated action of different multisensory systems for learning and memory, and the hippocampus appears to be the integration center for this task. In previous studies we found that contrasting long-distance migratory flights differentially affected the morphological complexity of two types of hippocampus astrocytes. Recently, a significant association was found between the latitude of the reproductive site and the size of the ADCYAP1 allele in long distance migratory birds. We tested for correlations between astrocyte morphological complexity, migratory distances, and size of the ADCYAP1 allele in three long-distance migrant species of shorebird and one non-migrant. Significant differences among species were found in the number and morphological complexity of the astrocytes, as well as in the size of the microsatellites of the ADCYAP1 gene. We found significant associations between the size of the ADCYAP1 microsatellites, the migratory distances, and the degree of morphological complexity of the astrocytes. We suggest that associations between astrocyte number and morphological complexity, ADCYAP1 microsatellite size, and migratory behavior may be part of the adaptive response to the migratory process of shorebirds.

Environmental Enrichment Improved Learning and Memory, Increased Telencephalic Cell Proliferation, and Induced Differential Gene Expression in Colossoma macropomum.

Fish use spatial cognition based on allocentric cues to navigate, but little is known about how environmental enrichment (EE) affects learning and memory in correlation with hematological changes or gene expression in the fish brain. Here we investigated these questions in Colossoma macropomum (Teleostei). Fish were housed for 192 days in either EE or in an impoverished environment (IE) aquarium. EE contained toys, natural plants, and a 12-h/day water stream for voluntary exercise, whereas IE had no toys, plants, or water stream. A third plus maze aquarium was used for spatial and object recognition tests. Compared with IE, the EE fish showed greater learning rates, body length, and body weight. After behavioral tests, whole brain tissue was taken, stored in RNA-later, and then homogenized for DNA sequencing after conversion of isolated RNA. To compare read mapping and gene expression profiles across libraries for neurotranscriptome differential expression, we mapped back RNA-seq reads to the C. macropomum de novo assembled transcriptome. The results showed significant differential behavior, cell counts and gene expression in EE and IE individuals. As compared with IE, we found a greater number of cells in the telencephalon of individuals maintained in EE but no significant difference in the tectum opticum, suggesting differential plasticity in these areas. A total of 107,669 transcripts were found that ultimately yielded 64 differentially expressed transcripts between IE and EE brains. Another group of adult fish growing in aquaculture conditions were either subjected to exercise using running water flow or maintained sedentary. Flow cytometry analysis of peripheral blood showed a significantly higher density of lymphocytes, and platelets but no significant differences in erythrocytes and granulocytes. Thus, under the influence of contrasting environments, our findings showed differential changes at the behavioral, cellular, and molecular levels. We propose that the differential expression of selected transcripts, number of telencephalic cell counts, learning and memory performance, and selective hematological cell changes may be part of Teleostei adaptive physiological responses triggered by EE visuospatial and somatomotor stimulation. Our findings suggest abundant differential gene expression changes depending on environment and provide a basis for exploring gene regulation mechanisms under EE in C. macropomum.

Differential Change in Hippocampal Radial Astrocytes and Neurogenesis in Shorebirds With Contrasting Migratory Routes.

Little is known about environmental influences on radial glia-like (RGL) α cells (radial astrocytes) and their relation to neurogenesis. Because radial glia is involved in adult neurogenesis and astrogenesis, we investigated this association in two migratory shorebird species that complete their autumnal migration using contrasting strategies. Before their flights to South America, the birds stop over at the Bay of Fundy in Canada. From there, the semipalmated sandpiper (Calidris pusilla) crosses the Atlantic Ocean in a non-stop 5-day flight, whereas the semipalmated plover (Charadrius semipalmatus) flies primarily overland with stopovers for rest and feeding. From the hierarchical cluster analysis of multimodal morphometric features, followed by the discriminant analysis, the radial astrocytes were classified into two main morphotypes, Type I and Type II. After migration, we detected differential changes in the morphology of these cells that were more intense in Type I than in Type II in both species. We also compared the number of doublecortin (DCX)-immunolabeled neurons with morphometric features of radial glial-like α cells in the hippocampal V region between C. pusilla and C. semipalmatus before and after autumn migration. Compared to migrating birds, the convex hull surface area of radial astrocytes increased significantly in wintering individuals in both C. semipalmatus and C. pusilla. Although to a different extent we found a strong correlation between the increase in the convex hull surface area and the increase in the total number of DCX immunostained neurons in both species. Despite phylogenetic differences, it is of interest to note that the increased morphological complexity of radial astrocytes in C. semipalmatus coincides with the fact that during the migratory process over the continent, the visuospatial environment changes more intensely than that associated with migration over Atlantic. The migratory flight of the semipalmated plover, with stopovers for feeding and rest, vs. the non-stop flight of the semipalmated sandpiper may differentially affect radial astrocyte morphology and neurogenesis.

Hippocampal neurogenesis and volume in migrating and wintering semipalmated sandpipers (Calidris pusilla).

Long distance migratory birds find their way by sensing and integrating information from a large number of cues in their environment. These cues are essential to navigate over thousands of kilometers and reach the same breeding, stopover, and wintering sites every year. The semipalmated sandpiper (Calidris pusilla) is a long-distance migrant that breeds in the arctic tundra of Canada and Alaska and winters on the northeast coast of South America. Its fall migration includes a 5,300-kilometer nonstop flight over the Atlantic Ocean. The avian hippocampus has been proposed to play a central role in the integration of multisensory spatial information for navigation. Hippocampal neurogenesis may contribute to hippocampal function and a variety of factors including cognitive activity, exercise, enrichment, diet and stress influence neurogenesis in the hippocampus. We quantified hippocampal neurogenesis and volume in adult migrating and wintering semipalmated sandpipers using stereological counts of doublecortin (DCX) immunolabeled immature neurons. We found that birds captured in the coastal region of Bragança, Brazil during the wintering period had more DCX positive neurons and larger volume in the hippocampus than individuals captured in the Bay of Fundy, Canada during fall migration. We also estimate the number of NeuN immunolabeled cells in migrating and wintering birds and found no significant differences between them. These findings suggest that, at this time window, neurogenesis just replaced neurons that might be lost during the transatlantic flight. Our findings also show that in active fall migrating birds, a lower level of adult hippocampal neurogenesis is associated with a smaller hippocampal formation. High levels of adult hippocampal neurogenesis and a larger hippocampal formation found in wintering birds may be late occurring effects of long distance migratory flight or the result of conditions the birds experienced while wintering.

Hippocampal Astrocytes in Migrating and Wintering Semipalmated Sandpiper Calidris pusilla.

Seasonal migratory birds return to the same breeding and wintering grounds year after year, and migratory long-distance shorebirds are good examples of this. These tasks require learning and long-term spatial memory abilities that are integrated into a navigational system for repeatedly locating breeding, wintering, and stopover sites. Previous investigations focused on the neurobiological basis of hippocampal plasticity and numerical estimates of hippocampal neurogenesis in birds but only a few studies investigated potential contributions of glial cells to hippocampal-dependent tasks related to migration. Here we hypothesized that the astrocytes of migrating and wintering birds may exhibit significant morphological and numerical differences connected to the long-distance flight. We used as a model the semipalmated sandpiper Calidris pusilla, that migrates from northern Canada and Alaska to South America. Before the transatlantic non-stop long-distance component of their flight, the birds make a stopover at the Bay of Fundy in Canada. To test our hypothesis, we estimated total numbers and compared the three-dimensional (3-D) morphological features of adult C. pusilla astrocytes captured in the Bay of Fundy (n = 249 cells) with those from birds captured in the coastal region of Bragança, Brazil, during the wintering period (n = 250 cells). Optical fractionator was used to estimate the number of astrocytes and for 3-D reconstructions we used hierarchical cluster analysis. Both morphological phenotypes showed reduced morphological complexity after the long-distance non-stop flight, but the reduction in complexity was much greater in Type I than in Type II astrocytes. Coherently, we also found a significant reduction in the total number of astrocytes after the transatlantic flight. Taken together these findings suggest that the long-distance non-stop flight altered significantly the astrocytes population and that morphologically distinct astrocytes may play different physiological roles during migration.

Visuospatial learning and memory in the Cebus apella and microglial morphology in the molecular layer of the dentate gyrus and CA1 lacunosum molecular layer.

We investigated whether the morphology of microglia in the molecular layer of the dentate gyrus (DG-Mol) or in the lacunosum molecular layer of CA1 (CA1-LMol) was correlated with spatial learning and memory in the capuchin monkey (Cebus apella). Learning and memory was tested in 4 monkeys with visuo-spatial, paired associated learning (PAL) tasks from the Cambridge battery of neuropsychological tests. After testing, monkeys were sacrificed, and hippocampi were sectioned. We specifically immunolabeled microglia with an antibody against the adapter binding, ionized calcium protein. Microglia were selected from the middle and outer thirds of the DG-Mol (n=268) and the CA1-LMol (n=185) for three-dimensional reconstructions created with Neurolucida and Neuroexplorer software. Cluster and discriminant analyses, based on microglial morphometric parameters, identified two major morphological microglia phenotypes (types I and II) found in both the CA1-LMol and DG-Mol of all individuals. Compared to type II, type I microglia were significantly smaller, thinner, more tortuous and ramified, and less complex (lower fractal dimensions). PAL performance was both linearly and non-linearly correlated with type I microglial morphological features from the rostral and caudal DG-Mol, but not with microglia from the CA1-LMol. These differences in microglial morphology and correlations with PAL performance were consistent with previous proposals of hippocampal regional contributions for spatial learning and memory. Our results suggested that at least two morphological microglial phenotypes provided distinct physiological roles to learning-associated activity in the rostral and caudal DG-Mol of the monkey brain.

Hippocampus and dentate gyrus of the Cebus monkey: architectonic and stereological study.

Behavioral, electrophysiological, and anatomical assays of non-human primates have provided substantial evidence that the hippocampus and dentate gyrus are essential for memory consolidation. However, a single anatomical and stereological investigation of these regions has been done in New World primates to complement those assays. The aim of the present study was to describe the cyto-, myelo-, and histochemical architecture of the hippocampus and dentate gyrus, and to use the optical fractionator method to estimate the number of neurons in the hippocampal pyramidal and granular neurons in the dentate gyrus of the Cebus monkey. NeuN immunolabeling, lectin histochemical staining with Wisteria floribunda agglutinin (WFA), enzyme-histochemical detection of NADPH-diaphorase activity and Gallyas silver staining were used to define the layers and limits of the hippocampal fields and dentate gyrus. A comparative analysis of capuchin (Cebus apella) and Rhesus (Macaca mulatta) monkeys revealed similar structural organization of these regions but significant differences in the regional distribution of neurons. C. apella were found to have 1.3 times fewer pyramidal and 3.5 times fewer granular neurons than M. mulatta. Taken together the architectonic and stereological data of the present study suggest that hippocampal and dentate gyrus neural networks in the C. apella and M. mulatta may contribute to hippocampal-dentate gyrus-dependent tasks in different proportions.