Morphology and phylogenetic relationships of a remarkable new genus and two new species of Neotropical freshwater stingrays from the Amazon basin (Chondrichthyes: Potamotrygonidae)

The morphology and phylogenetic relationships of a new genus and two new species of Neotropical freshwater stingrays, family Potamotrygonidae, are investigated and described in detail. The new genus, Heliotrygon, n. gen., and its two new species, Heliotrygon gomesi, n. sp. (type-species) and Heliotrygon rosai, n. sp., are compared to all genera and species of potamotrygonids, based on revisions in progress. Some of the derived features of Heliotrygon include its unique disc proportions (disc highly circular, convex anteriorly at snout region, its width and length very similar), extreme subdivision of suborbital canal (forming a complex honeycomb-like pattern anterolaterally on disc), stout and triangular pelvic girdle, extremely reduced caudal sting, basibranchial copula with very slender and acute anterior extension, and precerebral and frontoparietal fontanellae of about equal width, tapering very little posteriorly. Both new species can be distinguished by their unique color patterns: Heliotrygon gomesi is uniform gray to light tan or brownish dorsally, without distinct patterns, whereas Heliotrygon rosai is characterized by numerous white to creamy-white vermiculate markings over a light brown, tan or gray background color. Additional proportional characters that may further distinguish both species are also discussed. Morphological descriptions are provided for dermal denticles, ventral lateral-line canals, skeleton, and cranial, hyoid and mandibular muscles of Heliotrygon, which clearly corroborate it as the sister group of Paratrygon. Both genera share numerous derived features of the ventral lateral-line canals, neurocranium, scapulocoracoid, pectoral basals, clasper morphology, and specific patterns of the adductor mandibulae and spiracularis medialis muscles. Potamotrygon and Plesiotrygon are demonstrated to share derived characters of their ventral lateral-line canals, in addition to the presence of angular cartilages. Our morphological phylogeny is further corroborated by a molecular phylogenetic analysis of cytochrome b based on four sequences (637 base pairs in length), representing two distinct haplotypes for Heliotrygon gomesi. Parsimony analysis produced a single most parsimonious tree revealing Heliotrygon and Paratrygon as sister taxa (boot-strap proportion of 70%), which together are the sister group to a clade including Plesiotrygon and species of Potamotrygon. These unusual stingrays highlight that potamotrygonid diversity, both in terms of species composition and undetected morphological and molecular patterns, is still poorly known.

Tenacity and silk investment of two orb weavers: considerations about diversification of the Araneoidea

Orbiculariae consists of two major clades: the cribellate Deinopidea and the much more diverse ecribellate Araneoidea. It has been hypothesized that the higher diversity of Araneoidea is a consequence of the superiority of the viscid orb web. However, this explanation seems incomplete: for example, cribellate silk may perform better than viscid silk in some contexts. Here, we consider the hypothesis that the diversification of Araneoidea was facilitated by changes in microhabitat occupation behavior due to the cheaper viscid orb web. In the present work we investigate the idea that the reduction in site tenacity caused by the emergence of the viscid orb web has led to an increase in the exploration of different resources and to a greater diversification of the Arancoidea through the evolutionary time. To test this idea, we evaluated the response of one cribellate orb web spider (Zosis geniculata Olivier 1789, Uloboridae) and one ecribellate orb web spider (Metazygia rogenhoferi Keyserling 1878, Arancidae) to abrupt prey absence. The changes in site tenacity and the day-to-day investment in web silk were evaluated. Spiders with three-dimensional webs tend to exhibit greater site tenacity than spiders making orb webs. Zosis geniculata and M. rogenhoferi show similar site tenacity when prey is ample. When prey is unavailable, the tenacity of the cribellate species increases while the tenacity of the ecribellate remains unchanged, and the silk investment of both species decreases. However, this decrease in silk investment is more extensive in Z. geniculata. These results coincide with the idea that a less costly ecribellate orb web leads to a lower tenacity and suggest that more frequent microhabitat abandonment in a context of insect radiation (Neiptera) leads to more diverse and opportunistic exploration of microhabitats that, in the long term, may be one explanation for the greater Araneoidea diversification.

Phylogenetic position of Dendropsophus gaucheri (Lescure and Marty 2000) highlights the need for an in-depth investigation of the phylogenetic relationships of Dendropsophus (Anura: Hylidae)

Dendropsophus gaucheri is a recently described species which inhabits open areas of the eastern part of the Guiana Shield and is currently assigned to the D. parviceps species group based on the presence of a subocular cream spot. Herein we investigate its phylogenetic position including material from the type locality and newly documented populations from Suriname and Brazil based on mtDNA sequences. The species, as well as D. riveroi which is assigned to the D. minimus species group, were recovered nested within the D. microcephalus species group which implies the paraphyly of the three Dendropsophus species groups. Such result, along with other evidences, highlights the need for a thorough revision of the genus. The genetic distances among D. gaucheri samples studied are low confirming their conspecificity and suggesting recent connections among populations from open areas currently isolated by rainforest in the lowlands of the Guiana Shield.

Phylogenetic relationships of the Neotropical spider genus Itatiaya (Araneae)

A cladistic analysis using parsimony under equal weights is applied to test the phylogenetic relationships of Itatiaya Mello-Leitao, previously described in Ctenidae. The data matrix comprised 25 taxa scored for a total of 47 characters. The cladistic analysis yielded two equally parsimonious trees of 124 steps. The consensus of the two most parsimonious trees is used to discuss the phylogenetic relationships and justify taxonomic modifications. The results indicate that this genus is a representative of Zoropsidae, which is newly recorded from the Neotropical region. The monophyly of Itatiaya is supported by three non-ambiguous synapomorphies and three homoplastic synapomorphies. A new diagnosis is provided for Itatiaya. Itatiaya pucupucu is placed as sister species to the remaining species of the genus. A polytomic clade composed of Itatiaya modesta, Itatiaya iuba, Itatiaya apipema and the clade formed by Itatiaya tacamby + Itatiaya pykyyra is supported by the presence of modified cylindrical gland spigots. Additionally, the male of I. pykyyra Polotow & Brescovit is described for the first time.

Molecular phylogenetics and character evolution of the ""sacaca"" clade: Novel relationships of Croton section Cleodora (Euphorbiaceae)

Phylogenetic relationships of Croton section Cleodora (Klotzsch) Baill. were evaluated using the nuclear ribosomal ITS and the chloroplast trnl-F and trnH-psbA regions. Our results show a strongly supported clade containing most previously recognized section Cleodora species, plus some other species morphologically similar to them. Two morphological synapomorphies that support section Cleodora as a clade include pistillate flowers in which the sepals overlap to some degree, and styles that are connate at the base to varying degrees. The evolution of vegetative and floral characters that have previously been relied on for taxonomic decisions within this group are evaluated in light of the phylogenetic hypotheses. Within section Cleodora there are two well-supported clades, which are proposed here as subsections (subsection Sphaerogyni and subsection Spruceani). The resulting phylogenetic hypothesis identifies the closest relatives of the medicinally important and essential oil-rich Croton cajucara Benth. as candidates for future screening in phytochemical and pharmacological studies. (C) 2011 Elsevier Inc. All rights reserved.

Acquisition of Pavlovian Fear Conditioning Using beta-Adrenoceptor Activation of the Dorsal Premammillary Nucleus as an Unconditioned Stimulus to Mimic Live Predator-Threat Exposure

In the present work, we sought to mimic the internal state changes in response to a predator threat by pharmacologically stimulating the brain circuit involved in mediating predator fear responses, and explored whether this stimulation would be a valuable unconditioned stimulus (US) in an olfactory fear conditioning paradigm (OFC). The dorsal premammillary nucleus (PMd) is a key brain structure in the neural processing of anti-predatory defensive behavior and has also been shown to mediate the acquisition and expression of anti-predatory contextual conditioning fear responses. Rats were conditioned by pairing the US, which was an intra-PMd microinjection of isoproterenol (ISO; beta-adrenoceptor agonist), with amyl acetate odor-the conditioned stimulus (CS). ISO (10 and 40 nmol) induced the acquisition of the OFC and the second-order association by activation of beta-1 receptors in the PMd. Furthermore, similar to what had been found for contextual conditioning to a predator threat, atenolol (beta-1 receptor antagonist) in the PMd also impaired the acquisition and expression of OFC promoted by ISO. Considering the strong glutamatergic projections from the PMd to the dorsal periaqueductal gray (dPAG), we tested how the glutamatergic blockade of the dPAG would interfere with the OFC induced by ISO. Accordingly, microinjections of NMDA receptor antagonist (AP5, 6 nmol) into the dPAG were able to block both the acquisition, and partially, the expression of the OFC. In conclusion, we have found that PMd beta-1 adrenergic stimulation is a good model to mimic predatory threat-induced internal state changes, and works as a US able to mobilize the same systems involved in the acquisition and expression of predator-related contextual conditioning. Neuropsychopharmacology (2011) 36, 926-939; doi:10.1038/npp.2010.231; published online 5 January 2011

New Perspectives on beta-Adrenergic Mediation of Innate and Learned Fear Responses to Predator Odor

In the present study, we investigated the role of noradrenergic transmission in unconditioned and conditioned responses to predatory threats. First, we examined the effects of systemically injected beta-blockers on unconditioned and contextual conditioned response to cat odor. The centrally acting beta-blocker (propranolol) was able to impair unconditioned responses, as well as the acquisition of the contextual fear to cat odor; however, the peripherally acting (nadolol) was not effective. Next, we examined the neural substrate underlying the noradrenergic modulation of the defensive response to cat odor and focused on the dorsal premammillary nucleus (PMd), because it represents the hypothalamic site most responsive to predatory threats and, at the same time, presents a dense plexus of noradrenergic fibers. We were able to see that propranolol significantly reduced PMd-Fos expression in response to cat odor and that beta-adrenoceptor blockade in the PMd, before cat odor exposure, reduced defensive responses to the cat odor and to the cat odor-related environment. We have also shown that beta-adrenoceptor blockade in the PMd, before the exposure to cat odor-related context, impaired the contextual conditioned responses. Overall, the present results provide convincing evidence suggesting that central noradrenergic mediation is critical for the expression of unconditioned and contextual conditioned antipredatory responses. We have further shown that the PMd appears to be an important locus to mediate these beta-adrenoceptor effects.

Investigation of the hypothalamic defensive system in the mouse

The hypothalamus plays especially important roles in various endocrine, autonomic, and behavioral responses that guarantee the survival of both the individual and the species. In the rat, a distinct hypothalamic defensive circuit has been defined as critical for integrating predatory threats, raising an important question as to whether this concept could be applied to other prey species. To start addressing this matter, in the present study, we investigated, in another prey species (the mouse), the pattern of hypothalamic Fos immunoreactivity in response to exposure to a predator (a rat, using the Rat Exposure Test). During rat exposure, mice remained concealed in the home chamber for a longer period of time and increased freezing and risk assessment activity. We were able to show that the mouse and the rat present a similar pattern of hypothalamic activation in response to a predator. Of particular note, similar to what has been described for the rat, we observed in the mouse that predator exposure induces a striking activation in the elements of the medial hypothalamic defensive system, namely, the anterior hypothalamic nucleus, the dorsomedial part of the ventromedial hypothalamic nucleus and the dorsal premammillary nucleus. Moreover, as described for the rat, predator-exposed mice also presented increased Fos levels in the autonomic and parvicellular parts of the paraventricular hypothalamic nucleus, lateral preoptic area and subfornical region of the lateral hypothalamic area. In conclusion, the present data give further support to the concept that a specific hypothalamic defensive circuit should be preserved across different prey species. (C) 2008 Elsevier B.V. All rights reserved.

Hypothalamic sites responding to predator threats - the role of the dorsal premammillary nucleus in unconditioned and conditioned antipredatory defensive behavior

In this study we provide a comprehensive analysis of the hypothalamic activation pattern during exposure to a live predator or an environment previously associated with a predator. Our results support the view that hypothalamic processing of the actual and the contextual predatory threats share the same circuit, in which the dorsal premammillary nucleus (PMd) plays a pivotal role in amplifying this processing. To further understand the role of the PMd in the circuit organizing antipredatory defensive behaviors, we studied rats with cytotoxic PMd lesions during cat exposure and examined the pattern of behavioral responses as well as how PMd lesions affect the neuronal activation of the systems engaged in predator detection, in contextual memory formation and in defensive behavioral responses. Next, we investigated how pharmacological blockade of the PMd interferes with the conditioned behavioral responses to a context previously associated with a predator, and how this blockade affects the activation pattern of periaqueductal gray (PAG) sites likely to organize the conditioned behavioral responses to the predatory context. Behavioral observations indicate that the PMd interferes with both unconditioned and conditioned antipredatory defensive behavior. Moreover, we have shown that the PMd influences the activation of its major projecting targets, i.e. the ventral part of the anteromedial thalamic nucleus which is likely to influence mnemonic processing, and PAG sites involved in the expression of antipredatory unconditioned and conditioned behavioral responses. Of particular relevance, this work provides evidence to elucidate the basic organization of the neural circuits integrating unconditioned and contextual conditioned responses to predatory threats.

AMYGDALAR ROLES DURING EXPOSURE TO A LIVE PREDATOR AND TO A PREDATOR-ASSOCIATED CONTEXT

The amygdala plays a critical role in determining the emotional significance of sensory stimuli and the production of fear-related responses. Large amygdalar lesions have been shown to practically abolish innate defensiveness to a predator; however, it is not clear how the different amygdalar systems participate in the defensive response to a live predator. Our first aim was to provide a comprehensive analysis of the amygdalar activation pattern during exposure to a live cat and to a predator-associated context. Accordingly, exposure to a live predator up-regulated Fos expression in the medial amygdalar nucleus (MEA) and in the lateral and posterior basomedial nuclei, the former responding to predator-related pheromonal information and the latter two nuclei likely to integrate a wider array of predatory sensory information, ranging from olfactory to non-olfactory ones, such as visual and auditory sensory inputs. Next, we tested how the amygdalar nuclei most responsive to predator exposure (i.e. the medial, posterior basomedial and lateral amygdalar nuclei) and the central amygdalar nucleus (CEA) influence both unconditioned and contextual conditioned anti-predatory defensive behavior. Medial amygdalar nucleus lesions practically abolished defensive responses during cat exposure, whereas lesions of the posterior basomedial or lateral amygdalar nuclei reduced freezing and increased risk assessment displays (i.e. crouch sniff and stretch postures), a pattern of responses compatible with decreased defensiveness to predator stimuli. Moreover, the present findings suggest a role for the posterior basomedial and lateral amygdalar nuclei in the conditioning responses to a predator-related context. We have further shown that the CEA does not seem to be involved in either unconditioned or contextual conditioned anti-predatory responses. Overall, the present results help to clarify the amygdalar systems involved in processing predator-related sensory stimuli and how they influence the expression of unconditioned and contextual conditioned anti-predatory responses. (C) 2011 IBRO. Published by Elsevier Ltd. All rights reserved.

Evidence for the thalamic targets of the medial hypothalamic defensive system mediating emotional memory to predatory threats

Previous studies from our laboratory have documented that the medial hypothalamic defensive system is critically involved in processing actual and contextual predatory threats, and that the dorsal premammillary nucleus (PMd) represents the hypothalamic site most responsive to predatory threats. Anatomical findings suggest that the PMd is in a position to modulate memory processing through a projecting branch to specific thalamic nuclei, i.e., the nucleus reuniens (RE) and the ventral part of the anteromedial nucleus (AMv). In the present study, we investigated the role of these thalamic targets in both unconditioned (i.e., fear responses to predatory threat) and conditioned (i.e., contextual responses to predator-related cues) defensive behaviors. During cat exposure, all experimental groups exhibited intense defensive responses with the animals spending most of the time in the home cage displaying freezing behavior. However, during exposure to the environment previously associated with a cat, the animals with combined RE + AMv lesions, and to a lesser degree, animals with single AMv unilateral lesions, but not animals with single RE lesions, presented a reduction of contextual conditioned defensive responses. Overall, the present results provide clear evidence suggesting that the PMd`s main thalamic targets (i.e., the nucleus reuniens and the AMv) seem to be critically involved in the emotional memory processing related to predator cues. (C) 2010 Elsevier Inc. All rights reserved.

THE ROLE OF THE SUPERIOR COLLICULUS IN PREDATORY HUNTING

Combining the results of behavioral, neuronal immediate early gene activation, lesion and neuroanatomical experiments, we have presently investigated the role of the superior colliculus (SC) in predatory hunting. First, we have shown that insect hunting is associated with a characteristic large increase in Fos expression in the lateral part of the intermediate gray layer of the SC (Wig). Next, we have shown that animals with bilateral NMDA lesions of the lateral parts of the SC presented a significant delay in starting to chase the prey and longer periods engaged in other activities than predatory hunting. They also showed a clear deficit to orient themselves toward the moving prey and lost the stereotyped sequence of actions seen for capturing, holding and killing the prey. Our Phaseolus vulgaris-leucoagglutinin analysis revealed that the lateral SCig, besides providing the well-documented descending crossed pathway to premotor sites in brainstem and spinal cord, projects to a number of midbrain and diencephalic sites likely to influence key functions in the context of the predatory behavior, such as general levels of arousal, motivational level to hunt or forage, behavioral planning, appropriate selection of the basal ganglia motor plan to hunt, and motor output of the primary motor cortex. In contrast to the lateral SC lesions, medial SC lesions produced a small deficit in predatory hunting, and compared to what we have seen for the lateral SCig, the medial SCig has a very limited set of projections to thalamic sites related to the control of motor planning or motor output, and provides conspicuous inputs to brainstem sites involved in organizing a wide range of anti-predatory defensive responses. Overall, the present results served to clarify how the different functional domains in the SC may mediate the decision to pursue and hunt a prey or escape from a predator. (C) 2010 IBRO. Published by Elsevier Ltd. All rights reserved.

Genes of cathepsin L-like proteases in Trypanosoma rangeli isolates: Markers for diagnosis, genotyping and phylogenetic relationships

We have sequenced genes encoding cathepsin L-like (CatL-like) cysteine proteases from isolates of Trypanosoma rangeli from humans, wild mammals and Rhodnius species of Central and South America. Phylogenetic trees of sequences encoding mature CatL-like enzymes of T rangeli and homologous genes from other trypanosomes, Leishmania spp. and bodonids positioned sequences of T rangeli (rangelipain) closest to T cruzi (cruzipain). Phylogenetic tree of kinetoplastids based on sequences of CatL-like was totally congruent with those derived from SSU rRNA and gGAPDH genes. Analysis of sequences from the CatL-like catalytic domains of 17 isolates representative of the overall phylogenetic diversity and geographical range of T rangeli supported all the lineages (A-D) previously defined using ribosomal and spliced leader genes. Comparison of the proteolytic activities of T rangeli isolates revealed heterogeneous banding profiles of cysteine proteases in gelatin gels, with differences even among isolates of the same lineage. CatL-like sequences proved to be excellent targets for diagnosis and genotyping of T rangeli by PCR. Data from CatL-like encoding genes agreed with results from previous studies of kDNA markers, and ribosomal and spliced leader genes, thereby corroborating clonal evolution, independent transmission cycles and the divergence of T rangeli lineages associated with sympatric species of Rhodnius. (c) 2009 Elsevier B.V. All rights reserved.

Cathepsin L-like genes of Trypanosoma vivax from Africa and South America - characterization, relationships and diagnostic implications

We characterized sequences from genes encoding cathepsin L-like (CatL-like) cysteine proteases from African and South American isolates of Trypanosoma vivax and T. vivax-like organisms, and evaluated their suitability as genetic markers for population structure analysis and diagnosis. Phylogenetic analysis of sequences corresponding to CatL-like catalytic domains revealed substantial polymorphism, and clades of sequences (TviCatL1-9) were separated by large genetic distances. TviCatL1-4 sequences were from cattle isolates from West Africa (Nigeria and Burkina Faso) and South America (Brazil and Venezuela), which belonged to the same T. vivax genotype. T. vivax-like genotypes from East Africa showed divergent sequences, including TviCatL5-7 for isolates from Mozambique and TviCatL8-9 for an isolate from Kenya. Phylogenetic analysis of CatL-like gene data supported the relationships among trypanosome species reflected in the phylogenies based on the analysis of small subunit (SSU) of ribosomal RNA gene sequence data. The discovery of different CatL-like sequences for each genotype, defined previously by ribosomal DNA data, indicate that these sequences provide useful targets for epidemiological and population genetic studies. Regions in CatL-like sequences shared by all T. vivax genotypes but not by other trypanosomes allowed the establishment of a specific and sensitive diagnostic PCR for epidemiological studies in South America and Africa. (C) 2008 Elsevier Ltd. All rights reserved.

Role of glutamate NMDA receptors and nitric oxide located within the periaqueductal gray on defensive behaviors in mice confronted by predator

The midbrain periaqueductal gray (PAG) is part of the brain system involved in active defense reactions to threatening stimuli. Glutamate N-methyl-d-aspartate (NMDA) receptor activation within the dorsal column of the PAG (dPAG) leads to autonomic and behavioral responses characterized as the fear reaction. Nitric oxide (NO) has been proposed to be a mediator of the aversive action of glutamate, since the activation of NMDA receptors in the brain increases NO synthesis. We investigated the effects of intra-dPAG infusions of NMDA on defensive behaviors in mice pretreated with a neuronal nitric oxide synthase (nNOS) inhibitor [N omega-propyl-l-arginine (NPLA)], in the same midbrain site, during a confrontation with a predator in the rat exposure test (RET). Male Swiss mice received intra-dPAG injections of NPLA (0.1 or 0.4 nmol/0.1 mu l), and 10 min later, they were infused with NMDA (0.04 nmol/0.1 mu l) into the dPAG. After 10 min, each mouse was placed in the RET. NMDA treatment enhanced avoidance behavior from the predator and markedly increased freezing behavior. These proaversive effects of NMDA were prevented by prior injection of NPLA. Furthermore, defensive behaviors (e.g., avoidance, risk assessment, freezing) were consistently reduced by the highest dose of NPLA alone, suggesting an intrinsic effect of nitric oxide on defensive behavior in mice exposed to the RET. These findings suggest a potential role of glutamate NMDA receptors and NO in the dPAG in the regulation of defensive behaviors in mice during a confrontation with a predator in the RET.