Identification of candidate genes responsible for innate fear behavior in the chicken.

Identifying the genes responsible for quantitative traits remains a major challenge. We previously found a major QTL on chromosome 4 affecting several innate fear behavioral traits obtained by an open-field test in an F2 population between White Leghorn and Nagoya breeds of chickens (Gallus gallus). Here, an integrated approach of transcriptome, haplotype frequency, and association analyses was used to identify candidate genes for the QTL in phenotypically extreme individuals selected from the same segregating F2 population as that used in the initial QTL analysis. QTL mapping for the first principal component, which summarizes the variances of all affected behavioral traits in the F2 population, revealed the behavioral QTL located at 14-35 Mb on chromosome 4 with 333 genes. After RNA-seq analysis using two pooled RNAs from extreme F2 individuals, real-time qPCR analysis in the two parental breeds and their F1 individuals greatly reduced the number of candidate genes in the QTL interval from 333 to 16 genes. Haplotype frequency analysis in the two extreme F2 groups further reduced the number of candidate genes from 16 to 11. After comparing gene expression in the two extreme groups, a conditional correlation analysis of diplotypes between gene expression and phenotype of extreme individuals revealed that NPY5R and LOC101749214 genes were strong candidate genes for innate fear behavior. This study illustrates how the integrated approach can identify candidate genes more rapidly than fine mapping of the initial QTL interval and provides new information for studying the genetic basis of innate fear behavior in chickens.

Effects of Testicular and Non-Testicular Testosterone on Territorial and Isolation-induced Aggressive Behavior of Male Layer Chicks.

Testosterone (T) is known to induce aggressive behavior, particularly in male animals. However, our recent results showed that a certain kind of aggressive behavior is T-independent; moreover, the role of T in chicken territorial and isolation-induced aggressive behavior has not yet been investigated. In addition, castration alone is insufficient to evaluate the role of T in aggressive behavior because we found that non-testicular T concentration, probably derived from the adrenal gland, in the blood of castrated chicks was low, but not zero. In the present study, therefore, the role of testicular T in chicken aggressive behavior was evaluated through castration, and the role of nontesticular T was assessed using the subcutaneous implantation of flutamide, a non-steroidal antiandrogen, in the castrated male layer chicks. Resident-intruder (R-I) and social interaction (SI) tests were used to quantitatively monitor territorial and isolation-induced aggressive behavior, respectively. Castration and drug implantation of the chicks were performed at 14 days of age. The R-I test was performed at 29 and 30 days of age, and the SI test was performed at 31 and 32 days of age. The total aggression frequencies (TAFs) and aggression establishment rate (AER) were used as indices of chick aggressive behavior. In the R-I test, castration significantly decreased the TAFs but the AER was not affected by castration or flutamide implantation. In the SI test, on the other hand, there were no significant differences in the TAFs, but the AER tended to increase in the intact chicks and decrease in the flutamide-implanted, castrated male chicks. These results suggest that the role of T in chicken aggression depends on the differences in social context of the behavior, and that both testicular and non-testicular T play an important role in the occurrence of isolation-induced aggression in male layer chicks.

Blood Testosterone Concentration and Testosterone-induced Aggressive Behavior in Male Layer Chicks: Comparison between Isolated- and Grouped-Raising.

Testosterone (T) is known to induce aggressive behavior, mainly in male animals. Subcutaneous implantation of T-filled silastic tubes, rather than intramuscular injection of T, is generally recommended for long-term treatment using exogenous T. However, the effect of T implantation on chicken aggressive behavior has not been investigated. In addition, the concentration of T required to induce aggressive behavior or whether rearing conditions such as isolated- or grouped-raising affect T-induced aggressive behavior in chickens is not known. The present study aimed to examine the relationship between the lengths of T-filled tubes, blood T concentration, and aggressive behavior in group- and isolation-raised male layer chicks. The testes were bilaterally removed and silactic tubes of various lengths filled with crystalline T were subcutaneously implanted at 14 days of age. A social interaction test was performed to quantitatively assess chick aggressive behavior at 32 days of age. Comb weight and size were used to assess the activation of endogenous androgen receptors. Total aggression frequencies (TAF) and aggression establishment rate (AER) were used to evaluate aggressiveness. Significant positive correlations (P<0.001) were observed between the comb parameters and plasma T concentration. In the isolation-raised chicks, the TAF and AER were high irrespective of the lengths of the implanted T tubes or the corresponding plasma T concentrations. However, in the group-raised chicks, the AER tended to differ between the T-implanted aggressors (P=0.0902), and the AER significantly increased with implantation of 1.0-cm-long T-filled tubes (P<0.05), which corresponded to approximately 47 pg/mL plasma T concentration. These results suggest that both grouped raising and approximately 47 pg/mL plasma T concentration are required for the induction of T-dependent aggressive behavior, and that isolation-induced aggressive behavior is T-independent in male layer chicks.

A New Classification of the Dictyostelids.

Traditional morphology-based taxonomy of dictyostelids is rejected by molecular phylogeny. A new classification is presented based on monophyletic entities with consistent and strong molecular phylogenetic support and that are, as far as possible, morphologically recognizable. All newly named clades are diagnosed with small subunit ribosomal RNA (18S rRNA) sequence signatures plus morphological synapomorphies where possible. The two major molecular clades are given the rank of order, as Acytosteliales ord. nov. and Dictyosteliales. The two major clades within each of these orders are recognized and given the rank of family as, respectively, Acytosteliaceae and Cavenderiaceae fam. nov. in Acytosteliales, and Dictyosteliaceae and Raperosteliaceae fam. nov. in Dictyosteliales. Twelve genera are recognized: Cavenderia gen. nov. in Cavenderiaceae, Acytostelium, Rostrostelium gen. nov. and Heterostelium gen. nov. in Acytosteliaceae, Tieghemostelium gen. nov., Hagiwaraea gen. nov., Raperostelium gen. nov. and Speleostelium gen. nov. in Raperosteliaceae, and Dictyostelium and Polysphondylium in Dictyosteliaceae. The "polycephalum" complex is treated as Coremiostelium gen. nov. (not assigned to family) and the "polycarpum" complex as Synstelium gen. nov. (not assigned to order and family). Coenonia, which may not be a dictyostelid, is treated as a genus incertae sedis. Eighty-eight new combinations are made at species and variety level, and Dictyostelium ammophilum is validated.

Screening of the Behavioral Tests for Monitoring Agonistic Behavior of Layer Chicks.

Chicken agonistic behavior, a type of social behavior related to threatening and fighting, is among the most serious problems in the poultry industry. However, due to luck of effective models for investigating the brain mechanisms of the behavior, no effective measures have been taken. This study, therefore, aimed to select the behavioral tests available for monitoring chicken agonistic behavior. Two behavioral tests, resident-intruder (R-I) test and social interaction (SI) test, were performed for 10 minutes in 10 pairs of male layer chicks at 8, 12, 16, 20, and 24 days of age, and total agonistic frequencies (TAF: Sum of the frequencies of agonistic displays like pecking, biting, kicking, threatening, and leaping) and latency (the period of time from the beginning of the behavioral test to the occurrence of the first agonistic behavior) were measured as indices of agonistic behavior. Two-way repeated measures ANOVA revealed significant differences in TAF and latency between aggressors and opponents in both the behavioral tests. In the R-I test, the TAF of aggressors significantly increased from 8 to 20 days of age, and the latency significantly decreased from 8 to 24 days of age. In the SI test, however, the TAF of aggressors significantly increased and the latency significantly decreased only from 16 to 20 days of age. When the criterion of high agonistic behavior was defined as the TAF, where aggressors showed more than 30 times of TAF and the opponents did less than one-third TAF of aggressors, the aggression establishment rate (AER), which is equal to the number of aggressors showing high agonistic behavior per total behavioral trials, was significantly higher in the R-I test than in the SI test. These results suggest that the R-I test, rather than the SI test, is an effective tool for monitoring agonistic behavior of layer chicks.

Comparisons of insulin related parameters in commercial-type chicks: Evidence for insulin resistance in broiler chicks.

The aim of this study is to elucidate whether insulin acts differentially within the central nervous system (CNS) of two types of commercial chicks to control ingestive behavior. Male layer and broiler chicks (4-day-old) were intracerebroventricularly (ICV) injected with saline or insulin under satiated and starved conditions. Feed intake was measured at 30, 60 and 120 min after treatment. Secondly, blood and hypothalamus were collected from both chick types under ad libitum feeding and fasting for 24 h. Plasma insulin concentration was measured by time-resolved fluoro-immunoassay. Hypothalamic insulin receptor mRNA expression levels were measured by quantitative RT-PCR. The ICV injection of insulin significantly inhibited feed consumption in layer chicks when compared with saline (P<0.05), but not broiler chicks (P>0.1). Plasma insulin concentration of both chick types significantly decreased following 24 h of fasting, while insulin concentrations in the broiler chicks were significantly higher compared to the layers fed under ad libitum conditions. Hypothalamic insulin receptor mRNA expression levels were significantly lower (P<0.05) in broiler chicks than in layer ones under ad libitum feeding. Feed deprivation significantly decreased insulin receptor mRNA levels in layer chicks (P<0.01), but not in broiler chicks (P>0.1). Moreover, plasma insulin concentrations correlated negatively with hypothalamic insulin receptor protein expression in the two types of chicks fed ad libitum (P<0.05). These results suggest that insulin resistance exists in the CNS of broiler chicks, possibly due to persistent hyperinsulinemia, which results in a down-regulation of CNS insulin receptor expression compared to that in layer chicks.

Localization of hypothalamic insulin receptor in neonatal chicks: evidence for insulinergic system control of feeding behavior.

Feeding behavior is managed by various neuropeptides and/or neurotransmitters within the central nervous system in vertebrates. It is proposed that central insulin acts as the negative-feedback regulator of appetite via the central melanocortin system in neonatal chicks. The present study investigated the localization of insulin receptors in the chick hypothalamus using immunohistochemistry. Immunostaining revealed hypothalamic neuron expressing insulin receptors in the paraventricular nucleus, ventromedial hypothalamus, lateral hypothalamus and infundibular nucleus, the avian equivalent of the mammalian arcuate nucleus. Additionally, double-staining immunohistochemistry in the infundibular nucleus revealed the presence of insulin receptors in both α-melanocyto stimulating hormone and neuropeptide Y neurons. Immunohistological analysis indicates that the insulinergic system in the chick hypothalamus contributes to feeding behavior and this system regulates both anorexigenic and orexigenic neuropeptides. Furthermore, the mechanisms of central insulin induced-feeding behavior contributes to the regulation of the melanocortin system in the chick infundibular nucleus.

Fat depot-specific differences in angiogenic growth factor gene expression and its relation to adipocyte size in cattle.

Adipocytes derived from different anatomical sites vary in the expression of adipocytokines and growth factor genes. Adipogenesis is tightly associated with angiogenesis, although the regional variation of angiogenic growth factor gene expression in adipose tissues remains unclear. In this experiment, we studied the fat depot-specific differences (subcutaneous, intramuscular, intermuscular, renal, and mesenteric) in the expression of angiogenic growth factor mRNA [vascular endothelial growth factor (VEGF), fibroblast growth factor-2 (FGF-2), fibroblast growth factor-10 (FGF-10), hepatocyte growth factor (HGF), and leptin], as well as the relationship between angiogenic growth factor mRNA level and adipocyte size in bovine adipose tissues. Intermuscular, renal, and mesenteric adipose tissues expressed significantly higher VEGF, FGF-2, and leptin mRNA levels than did subcutaneous and intramuscular adipose tissues. Mesenteric adipose tissue also expressed higher FGF-10 mRNA levels than did subcutaneous and intramuscular adipose tissues. There was no significant difference in the expression of HGF mRNA among adipose tissue depots. A significant correlation existed between adipocyte size and VEGF, FGF-2, FGF-10, and leptin mRNA levels. These results indicate that fat depot-specific difference in angiogenic growth factor gene expression results from the difference in adipocyte size.

Polysphondylium multicystogenum sp. nov., a new dictyostelid species from Sierra Leone, West Africa.

Polysphondylium multicystogenum, a new heterothallic species of dictyostelids, is described based on three isolates collected from soils in Sierra Leone, West Africa. This species is characterized by sorophores with a combination of clavate base and ovoid to oblong tip cell, smaller spores and abundant microcyst production under the usual culture conditions for sorocarp formation at 20 C. This is the first report of Polysphondylium producing such abundant microcysts.

A taxonomic revision of two dictyostelid species, Polysphondylium pallidum and P. album.

To reevaluate two dictyostelid species, namely, Polysphondylium pallidum and P. album, 92 isolates of the P. pallidum complex from their type localities were examined based on mating relationships and morphological characteristics. In the mating tests three heterothallic mating groups were found among the isolates. They also were different morphologically from each other. These results suggested that they belonged to distinct taxa. By comparison of the three mating groups with the type specimens of P. pallidum and P. album, two of them were identified as P. pallidum and P. album. Based on the examined isolates P. pallidum and P. album were redescribed in detail.

Genetic variation in the chloroplast genome suggests multiple domestication of cultivated Asian rice (Oryza sativa L.).

Two hundred and seventy-five accessions of cultivated Asian rice and 44 accessions of AA genome Oryza species were classified into 8 chloroplast (cp) genome types (A-H) based on insertion-deletion events at 3 regions (8K, 57K, and 76K) of the cp genome. The ancestral cp genome type was determined according to the frequency of occurrence in Oryza species and the likely evolution of the variable 57K region of the cp genome. When 2 nucleotide substitutions (AA or TT) were taken into account, these 8 cp types were subdivided into 11 cp types. Most indica cultivars had 1 of 3 cp genome types that were also identified in the wild relatives of rice, O. nivara and O. rufipogon, suggesting that the 3 indica cp types had evolved from distinct gene pools of the O. rufipogon - O. nivara complex. The majority of japonica cultivars had 1 of 3 different cp genome types. One of these 3 was identified in O. rufipogon, suggesting that at least 1 japonica type is derived from O. rufipogon with the same cp genome type. These results provide evidence to support a polyphyletic origin of cultivated Asian rice from at least 4 principal lineages in the O. rufipogon - O. nivara complex.

Localization of microtubule-associated protein (MAP) 1B in the postsynaptic densities of the rat cerebral cortex.

1. Although microtubule-associated protein (MAP) 1B and its phosphorylation have been suggested to be important for synapse formation among cortical neurons, the localization of MAP1B in synapses has not yet been confirmed. In this report, we examine the localization of MAP1B in synaptic regions. 2. The localization of MAP1B was observed by immunohistochemical and electron microscopic techniques using specific antibodies against MAP1B. 3. MAP1B immunoreactivities were widely distributed in the cerebral cortex and were observed in the postsynaptic area but not in presynaptic terminals. 4. These synapses were classified as the asymmetrical type. 5. Only some synapses exhibited MAP1B immunoreactivities. MAP1B-immunopositive synapses accounted for about half of the total synapses. 6. Such a localization suggests MAP1B's important roles in synaptic functions.