Comparison of arginine vasotocin immunoreactivity differences in dominant and subordinate green anole lizards

The neuropeptide arginine vasotocin (AVT) and its mammalian homologue arginine vasopressin (AVP) are believed to be involved in many social behaviors including territorial aggression. Testosterone (T) is also important for controlling territorial aggression, and it is believed to be involved in modulating AVT/AVP levels in the brain. In this study, male Anolis carolinensis were paired (n = 11 pairs) in a neutral cage and were allowed to establish a dominant-subordinate relationship for 10 days (experimental groups) or housed in a neutral cage with or without a female (control groups; each n = 4). On 10th day animals were sacrificed and their brain sections were processed for AVT immunohistochemistry and their serum was analyzed for testosterone levels. AVT immunoreactive (AVT-ir) cell numbers were counted in the anterior hypothalamus (AH), paraventricular nucleus (PN), posterior hypothalamus (PH), preoptic area (POA), and supra optic nuclei (SON). 2-way randomized block design was conducted to assess AVT-ir cell number differences between dominant and subordinate animals and Pearson's correlations were used to determine if a relationship existed between T levels and AVT-ir cell numbers. Dominant animals had more AVT-ir cells in the POA compared to subordinate animals, and subordinate animals had fewer AVT-ir cells in the POA compared to males housed either singly or with a female. There were no differences in AVT-ir cell numbers between dominant and subordinate animals in other areas. T levels were not correlated with the AVT-ir cell numbers in any area. Thus dominant animals have increased AVT-ir cell numbers compared to subordinate animals in a brain region known to be important in male sexual behavior. However, this difference is not related to differences in T.     

Arborization of dewlap motoneurons in the green anole lizard (Anolis carolinensis) is not sexually dimorphic

Male anoles extend a bright red throat fan, called a dewlap, during both courtship and aggressive encounters. Female anoles perform this behavior less often than males and only in aggression towards both sexes. The cartilage, muscle fibers, and motoneuron somata controlling the display are larger in males than females. In the present study, we used the Golgi technique in an effort to characterize more completely the morphology of these dewlap motoneurons, and to investigate whether the dendritic arborization is different between the sexes. In addition to describing the morphology, we report that the length of processes, and numbers of primary processes and branch points are comparable in males and females. This similarity in arborization represents an intriguing contrast to other sexually dimorphic neuromuscular systems.     

Social status differentiates rapid neuroendocrine responses to restraint stress

Male Anolis carolinensis that win aggressive interactions mobilize neuroendocrine responses to social stress more rapidly than defeated lizards. We initially examined temporal patterns of neuroendocrine response to restraint stress in lizards of unknown status, and then investigated whether winning males respond more rapidly to this non-social stressor. Size-matched male pairs interacted to establish social status, and then were returned to individual home cages for 3 days. Plasma and brains were collected from non-restrained dominants and subordinates, and from a non-interacting control group. Additional groups of dominants and subordinates underwent 90 s restraint stress, with plasma and brains collected either immediately or 300 s after restraint. In lizards of unknown social status restraint stimulated rapid monoaminergic responses in nucleus accumbens, hippocampus, amygdala, and locus ceruleus, with delayed responses seen in VTA and raphé. Non-restrained dominants and subordinates had lower levels of raphé serotonergic activity and lower hippocampal dopaminergic activity 3 days after interacting, compared to controls. Dominants had higher corticosterone levels, both immediately and 300 s after restraint, than either non-restrained dominants or restrained subordinates. Restraint induced higher raphé serotonergic activity in dominants. However, subordinates also showed rapid responses to restraint; exhibiting lower hippocampal dopamine (DA) levels than non-restrained subordinates. At 300 s after the stress, amygdalar serotonin levels increased in dominants, while subordinates showed higher amygdalar DA levels. These results suggest that stressful aggressive interactions will not only alter basal neurochemical activity, but also influence neuroendocrine responses to non-social stressors according to individual social status.     

Opponent recognition and social status differentiate rapid neuroendocrine responses to social challenge

Individual social status discriminates rapid neuroendocrine responses to non-social stress in male Anolis carolinensis, but whether such status-influenced reactions are retained in response to subsequent social stress is unknown. Dominant and subordinate males modify their behavioral responses to social challenge according to familiarity of the opponent, suggesting that accompanying neuroendocrine responses may differ according to opponent recognition despite social rank. We examined endocrine and neurochemical correlates of prior social status and opponent recognition during the opening stages of social challenge. Male pairs interacted and established dominant/subordinate status, followed by 3 days separation. Subsequently, subjects were paired with either the same opponent or an unfamiliar male according to rank (dominant with subordinate). After 90 s of social exposure, subjects were caught and brains and plasma collected for measurement of circulating corticosterone and limbic monoamines. Controls included pairs experiencing just one 90 s encounter plus a group of non-interacting subjects. Opponent recognition differentiated status-influenced responses, such that dominant lizards paired with familiar subordinate opponents had increased hippocampal dopamine and epinephrine, but showed increased plasma corticosterone and ventral tegmental area (VTA) norepinephrine when challenged with an unfamiliar opponent. Subordinate lizards encountering familiar opponents also had increased corticosterone, along with decreased hippocampal dopamine and increased VTA epinephrine, but showed no changes in response to an unfamiliar opponent. Such plasticity in status-influenced rapid neuroendocrine responses according to opponent recognition may be necessary for facilitating production of behavioral responses adaptive for particular social contexts, such as encountering a novel versus familiar opponent.     

Androgen dependent seasonal changes in muscle fiber type in the dewlap neuromuscular system of green anoles

Green anoles (Anolis carolinensis) possess two sexually dimorphic neuromuscular systems involved in reproductive behaviors. One controls extension of a red throat fan (dewlap), which males employ during courtship, and the other controls intromission of copulatory organs (hemipenes). Although seasonal changes in circulating androgens mediate both courtship and copulatory behaviors, testosterone has differential effects on the underlying neuromuscular morphology. The present experiments were designed to test whether changes in muscle fiber type correspond to seasonal and androgenic regulation of reproductive behaviors in gonadally intact males (Experiment 1) or castrated males treated with either testosterone propionate or vehicle (Experiment 2). Gonadally intact males housed in breeding environmental conditions had a higher percentage of fast oxidative glycolytic fibers in the dewlap muscle than non-breeding males, but no effect of season on copulatory fibers was detected. Interestingly, testosterone treatment increased the percentage of fast oxidative glycolytic dewlap fibers independent of season, suggesting that routine changes in this hormone may mediate fiber type in gonadally intact males. In contrast, testosterone manipulation had little to no effect on copulatory muscle fiber type, demonstrating that a change in this feature is not the primary mediator for seasonal changes in male copulatory behaviors.     

Sociality,stress, and the corpus striatum of the green anolis lizard

The green anolis lizard, Anolis carolinensis, is a uniquely convenient species with great potential for providing insights about the causes and consequences of social behavior from an evolutionary perspective. In this species, social interactions are mediated by visual displays in which specific units of behavior are combined in various ways to communicate several more-or-less specific messages. Two related research programs that utilize this species converge in provocative ways to provide insight into this phenomenon. The first program is centered on the basal ganglia, now known to be crucial to the expression of aggressive territoriality in this species, and the second research program examines the way the physiological stress response is involved in aggression and its subsequent adaptive outcomes. Both the neural and the neuroendocrine systems affect the progress of social interactions as well as the subsequent social dominance relationships when combatants subsequently live together. Further, because body color depends almost exclusively on the stress response, skin color provides a unique in situ bioassay of otherwise inaccessible information about the animal's internal state. The fullest understanding of the physiological ethology of this model species will depend on an interdisciplinary approach that considers both proximate (physiological) and ultimate (evolutionary) causes of displays. Questions thus arising include how the nervous system controls and assembles the specific units of behavior-motor patterns and autonomic reflexes-into displays that are adaptive in specific contexts.     

Social isolation stress down-regulates cortical early growth response 1 (Egr-1) expression in mice

The G protein-coupled receptor 39-b (GPR39-1b) is a splice variant of which is expressed in the central nervous and gastrointestinal systems. Previously, GPR39-1b was proposed to be the receptor for obestatin, but current evidence does not support this hypothesis. The purpose of the present work was to identify the role of GPR39-1b in anxiety and eating behaviors. Antisense oligonucleotides were infused at a constant rate into the cerebral lateral ventricles of rats and their effect on anxiety-like behavior and food intake was monitored. GPR39-1b antisense oligonucleotides produced anxiolytic-like effects in the elevated-plus maze test and in the black and white box test. Antisense oligonucleotides also decreased food intake. These results indicate that inhibition of GPR39-1b induces a decrease in anxiety-related behaviors and disturbs appetite.     

The protein L-isoaspartyl-O-methyltransferase functions in the Caenorhabditis elegans stress response

The efficient use of nutrients is important in development and aging. In this study, we asked if the protein repair methyltransferase has a related or additional role in energy metabolism and stress response in the nematode Caenorhabditis elegans. Worms lacking the pcm-1 gene encoding this enzyme exhibit reduced longevity as SDS-isolated dauer larvae and as arrested L1 larvae under starvation stress, while overexpression leads to increased adult longevity. These findings led us to question whether pcm-1 deficient C. elegans may have inappropriate metabolic responses to stress. We assayed dauer and dauer-like larvae for starvation survival and observed a two-fold reduction of median survival time for pcm-1 mutants compared to N2 wild-type worms. Under these conditions, pcm-1 deficient dauer larvae had reduced fat stores, suggesting that PCM-1 may have a role in the initiation of the correct metabolic responses to stress starvation. We show expression of the pcm-1 gene in neurons, body wall and reproductive tissues. Upon heat shock and dauer formation-inducing conditions, we observe additional pcm-1 expression in body wall muscle nuclei and actomyosin filaments and in hypodermal cells. These results suggest that this enzyme may be important in stress response pathways, including proper decision making for energy storage.     

Behavioral sensitivity to corticosterone in juveniles of the wall lizard, Podarcis muralis

Reptiles exposed to stressful conditions respond with increases in plasma levels of corticosterone (CORT). The outcome of such hormonal fluctuations can affect the organism's physiology and behavior; however, relatively few studies in reptiles have progressed past the effects of stress on the plasma levels of CORT. The purpose of this study was to investigate the effects of CORT on lizard behavior. We focused on activity and thermoregulation of juveniles of the wall lizard, Podarcis muralis. Juveniles with experimentally elevated plasma CORT concentrations showed a higher moving rate and spent a greater amount of time moving. They also spent more time being involved in attempts to escape from a terrarium. This enhanced locomotor activity was associated with significant changes in thermoregulatory behavior, as CORT-increased individuals had reduced thermoregulatory activity than the controls. The same pattern of results was obtained in nature and under laboratory conditions. Thus, CORT levels appear to mediate an important trade-off between locomotor activity and thermoregulation. To our knowledge, this is the first time that the effect of CORT on lizard thermoregulation has been examined.     

Histopathological alterations in the brain (optic tectum) of the fresh water teleost Channa punctatus in response to acute and subchronic exposure to the pesticide Chlorpyrifos

In the present investigation an attempt was made to assess the toxicity of the organophosphate pesticide Chlorpyrifos (0,0-diethyl-0-3,5,6-trichloro-2-pyridyl phosphorothioate; CPF) on the brain (optic tectum) of the teleost Channa punctatus (Bloch). Fish were exposed to acute (1.5, 3.0, 4.5, 6.0 and 7.5 μl/l) for 24 h and sublethal concentrations of CPF 1.79 μl/l (1/3 of LC₅₀) and 0.538 μl/l (1/10 of LC₅₀) for 3 and 7 days respectively. Several endpoints related to the histoarchitectural profile in the optic tectum were evaluated. Histological examination showed detachment in the superficial zone of the Stratum opticum, Str. marginale due to degeneration of neuronal cells. Spongiosis, congestion, necrosis and appearance of clear areas around the nucleus of mononuclear cells in the lining of the Str. fibrosum grisium superficiale, Str. griseum centrale, Str. album centrale were seen. Granular cells found in the innermost layer of optic tectum, i.e. the Str. periventriculare, were severely degenerated and vacuolized and they migrated toward the Torus semicircularis. The histopathological changes were more pronounced with higher concentrations of CPF. The degree of neurodegeneration found in the deep layers of the optic tectum in higher concentration treatment (6.0 and 7.5 μl/l) was more pronounced. These alterations of the optic tectum affected the functioning of motor coordination of the fish body, because CPF inhibited acetylcholine in neuronal synapses. Due to apoptosis in the superficial zone of the optic tectum, normal visual response was affected. Fish showed microphthalmia (reduced size and eye shrinkage in the eye orbit) because of detachment, necrosis, degeneration and vacuolization in different regions after CPF treatment. This study shows that CPF is highly toxic to fish and affect their population survival in environment.     

Grouper (Epinephelus coioides) CXCR4 is expressed in response to pathogens infection and early stage of development

Chemokine (C-X-C motif) receptor 4 (CXCR4) from orange-spotted grouper (Epinephelus coioides) was identified and characterized in this study. gCXCR4 shared common features in protein sequence and predicted structure of CXCR4 family. This suggested that gCXCR4 is a member of G protein-coupled receptors with seven transmembrane domains. The expression patterns revealed that gCXCR4 may play a key role in early development of grouper. Furthermore, overexpression of gCXCR4-GFP for 48 h had significant effects on the GF-1 cell viability. gCXCR4 protein was mainly expressed in the marginal zone of head kidney and on the surface of intestinal villi. gCXCR4 expression can be detected in all the examined tissues and significantly up-regulated in eye and brain, which are the main targets for nervous necrosis virus (NNV) infection and replication. gCXCR4 gene expression can be induced in the spleen and eye by lipopolysaccharide and NNV, respectively. Our data suggested that gCXCR4 may not only play a role in the early immune response to microbial infection but also restrain to the immune system and central nervous system.     

Compartmentalized expression of light-induced clock genes in the suprachiasmatic nucleus of the diurnal grass rat (Arvicanthis niloticus)

Photic responses of the circadian system are mediated through light-induced clock gene expression in the suprachiasmatic nucleus (SCN). In nocturnal rodents, depending on the timing of light exposure, Per1 and Per2 gene expression shows distinct compartmentalized patterns that correspond to the behavioral responses. Whether the gene- and region-specific induction patterns are unique to nocturnal animals, or are also present in diurnal species is unknown. We explored this question by examining the light-induced Per1 and Per2 gene expression in functionally distinct SCN subregions, using diurnal grass rats Arvicanthis niloticus. Light exposure during nighttime induced Per1 and Per2 expression in the SCN, showing unique spatiotemporal profiles depending on the phase of the light exposure. After a phase delaying light pulse (LP) in the early night, strong Per1 induction was observed in the retinorecipient core region of the SCN, while strong Per2 induction was observed throughout the entire SCN. After a phase advancing LP in the late night, Per1 was first induced in the core and then extended into the whole SCN, accompanied by a weak Per2 induction. This compartmentalized expression pattern is very similar to that observed in nocturnal rodents, suggesting that the same molecular and intercellular pathways underlying acute photic responses are present in both diurnal and nocturnal species. However, after an LP in early subjective day, which induces phase advances in diurnal grass rats, but not in nocturnal rodents, we did not observe any Per1 or Per2 induction in the SCN. This result suggests that in spite of remarkable similarities in the SCN of diurnal and nocturnal rodents, unique mechanisms are involved in mediating the phase shifts of diurnal animals during the subjective day.     

Investigation of Van Gogh-like 2 mRNA regulation and localisation in response to nociception in the brain of adult common carp (Cyprinus carpio)

The Van Gogh-like 2 (vangl2) gene is typically associated with planar cell polarity pathways, which is essential for correct orientation of epithelial cells during development. The encoded protein of this gene is a transmembrane protein and is highly conserved through evolution. Van Gogh-like 2 was selected for further study on the basis of consistent regulation after a nociceptive stimulus in adult common carp and rainbow trout in a microarray study. An in situ hybridisation was conducted in the brain of mature common carp (Cyprinus carpio), 1.5 and 3 h after a nociceptive stimulus comprising of an acetic acid injection to the lips of the fish and compared with a saline-injected control. The vangl2 gene was expressed in all brain regions, and particularly intensely in neurons of the telencephalon and in ependymal cells. In the cerebellum, a greater number (P = 0.018) of Purkinje cells expressed vangl2 after nociception (n = 7) compared with controls (n = 5). This regulation opens the possibility that vangl2 is involved in nociceptive processing in the adult fish brain and may be a novel target for central nociception in vertebrates.     

Shrimp Pm-fortilin inhibits the expression of early and late genes of white spot syndrome virus (WSSV) in an insect cell model

Fortilin plays an important role in anti-apoptotic mechanisms and cell proliferation in many eukaryotic organisms. This work confirmed previous reports that Sf9 can support the replication of white spot syndrome virus (WSSV) genomic material by using immunohistochemistry with a specific antibody to detect the immediate early gene 1 (ie1) and by amplification of WSSV DNA and mRNA products. Using this insect-cell model system, we show that overexpression of Pm-fortilin in Sf9 cells inhibited the expression of WSSV early genes and late genes (WSSV-DNA polymerase, VP15 and VP28) but not an immediate early gene ie1. This is the first time that an insect cell line has been used to demonstrate interaction between a shrimp gene and genes of a shrimp virus.     

Beet soil-borne mosaic virus RNA-3 is replicated and encapsidated in the presence of BNYVV RNA-1 and -2 and allows long distance movement in Beta macrocarpa

Beet soil-borne mosaic virus (BSBMV) and Beet necrotic yellow vein virus (BNYVV) belong to the Benyvirus genus. BSBMV has been reported only in the United States, while BNYVV has a worldwide distribution. Both viruses are vectored by Polymyxa betae and possess similar host ranges, particle number and morphology. BNYVV and BSBMV are not serologically related but they have similar genomic organizations. Field isolates usually consist of four RNA species but some BNYVV isolates contain a fifth RNA. RNAs 1 and 2 are essential for infection and replication while RNAs 3 and 4 play important roles in plant and vector interactions, respectively. Nucleotide and amino acid analyses revealed that BSBMV and BNYVV are sufficiently different to be classified as two species. Complementary base changes found within the BSBMV RNA-3 5′ UTR made it resemble to BNYVV 5′ RNA-3 structure whereas the 3′ UTRs of both species were more conserved. cDNA clones were obtained, and allowed complete copies of BSBMV RNA-3 to be trans-replicated, trans-encapsidated by the BNYVV viral machinery. Long-distance movement was observed indicating that BSBMV RNA-3 could substitute BNYVV RNA-3 for systemic spread, even though the p29 encoded by BSBMV RNA-3 is much closer to the RNA-5-encoded p26 than to BNYVV RNA-3-encoded p25. Competition occurred when BSBMV RNA-3-derived replicons were used together with BNYVV-derived RNA-3 but not when the RNA-5-derived component was used. Exploitation of the similarities and divergences between BSBMV and BNYVV should lead to a better understanding of molecular interactions between Benyviruses and their hosts.     

PIK3CA mutations and amplification in endometrioid endometrial carcinomas: relation to other genetic defects and clinicopathologic status of the tumors

Alterations of the PIK3CA gene occur in endometrial carcinomas, but their role in the carcinogenesis of those malignancies is still poorly understood. In this study, PIK3CA mutations and amplification in 196 endometrioid endometrial carcinomas and 20 endometrial hyperplasias were assessed by single-strand conformation polymorphism (PCR-SSCP), sequencing, and quantitative polymerase chain reaction. Results were correlated with mutations in the PTEN, KRAS, and CTNNB1 genes and with the clinicopathologic parameters of the tumors. PIK3CA mutations were found in 39 (20%) carcinomas. Six new mutations were identified. No PIK3CA mutations were found in endometrial hyperplasias. PIK3CA amplification was observed in 24 (12.2%) carcinomas and in 2 (10%) hyperplasias. The PIK3CA mutations and amplifications (with the exception of 6 cases) occurred independently. PIK3CA mutations were significantly associated with PTEN mutations (P = .0414) and tended to be associated with CTNNB1 (P = .0833), but not with KRAS mutations. Conversely, the PIK3CA amplifications significantly negatively correlated with PTEN mutations (P = .0038) and did not coexist with CTNNB1 and KRAS mutations. The PIK3CA mutations were significantly associated with poorly differentiated tumors (P = .0423). Interestingly, PIK3CA amplifications, but not mutations, were strongly associated with older age (≥63 years, P = .0018). Our data show that mutations and amplification of PIK3CA are significant genetic alterations in endometrioid endometrial carcinomas associated with adverse clinicopathologic parameters (grade and stage). These data also demonstrate that PIK3CA mutations cooperate with PTEN mutations, suggesting an additive effect to PTEN, whereas PIK3CA amplification can, as an isolated event, enable the development of those tumors. Moreover, for the first time, a possible role of PIK3CA amplification in initiation and progression of endometrial carcinomas in older women is suggested, but this preliminary suggestion requires further research.     

Characterization of Culex Flavivirus (Flaviviridae) strains isolated from mosquitoes in the United States and Trinidad

Recent reports indicate that flaviviruses similar to the cell fusing agent virus (CFAV) naturally infect a wide variety of mosquito species. These newly recognized insect-specific viruses comprise a distinct CFAV complex within the genus Flavivirus. Here, we describe the isolation and characterization of nine strains of Culex flavivirus (Cx FV), a member of the CFAV complex, from mosquitoes collected in the United States (East Texas) and Trinidad. Phylogenetic analyses of the envelope protein gene sequences of these nine mosquito isolates with those of other CFAV complex flaviviruses in GenBank indicate that the U.S. isolates group with CxFV isolates from Asia (Japan and Indonesia), while the Trinidad isolates are more similar to CxFV isolates from Central America. A discussion follows on the possible biological significance of the CFAV complex flaviviruses.