Toll-Like Receptor 5 Promotes the Neurogenesis From Embryonic Stem Cells and Adult Hippocampal Neural Stem Cells in Mice.

Toll-like receptors (TLRs) make a crucial contribution to the innate immune response. TLR5 was expressed in embryoid body derived from mouse embryonic stem cells (mESCs) and ßIII-tubulin-positive cells under all-trans retinoic acid-treated condition. TLR5 was upregulated during neural differentiation from mESCs and augmented the neural differentiation of mESCs via nuclear factor-κB and interleukin 6/CREB pathways. Besides, TLR5 was expressed in SOX2- or doublecortin-positive cells in the subgranular zone of the hippocampal dentate gyrus where adult neurogenesis occurs. TLR5 inhibited the proliferation of adult hippocampal neural stem cells (NSCs) by regulating the cell cycle and facilitated the neural differentiation from the adult hippocampal NSCs via JNK pathway. Also, TLR5 deficiency impaired fear memory performance in mice. Our data suggest that TLR5 is a crucial modulator of neurogenesis from mESCs and adult hippocampal NSCs in mice and represents a new therapeutic target in neurological disorders related to cognitive function.

Identification and sequencing of the gene encoding DNA methyltransferase 3 (DNMT3) from sea cucumber, Apostichopus japonicus.

The sea cucumber Apostichopus japonicus is well known as a traditional tonic food and as a commercially important cultured aquatic species. This species produces saponins, and has a high potential to cope with environmental stress, such as aestivation, organ regeneration, and wound healing. Recently, several studies have shown that cellular reprogramming and the physiological responses of the sea cucumber to environmental changes, including aestivation, are potentially mediated by epigenetic DNA methylation. The DNA methyltransferase (DNMT)1 and DNMT3 genes are independent participants in the maintenance and de novo methylation of specific sequences. Sea urchin (Strongylocentrotus purpuratus) and starfish (Asterina pectinifera), which belong to the same phylum as A. japonicus, have both DNMT1 and DNMT3 genes. However, it was previously reported that DNMT1 is present, but DNMT3 is absent, in A. japonicus. In the present study, we sequenced the full-length cDNA of the A. japonicus DNMT3 gene. The newly sequenced DNMT3 gene comprises three major conserved domains (Pro-Trp-Trp-Pro (PWWP), plant homeodomain (PHD), and S-adenosylmethionine-dependent methyltransferase (AdoMet-MTase)), indicating that the DNMT3 possibly has de novo DNA methylation catalytic activity. Gene structure and phylogenetic analysis showed that sea cucumber DNMT3 is evolutionarily conserved in the Echinodermata. Next, we demonstrated the conservation of DNMT3 gene expression in sea cucumber and starfish belong to same phylum, echinoderm. Using reverse transcription-polymerase chain reaction, sea cucumber DNMT3 mRNA was detected in testis tissue, but not in other tissues tested, including the respiratory tree, muscle, tentacle, intestine, and ovary. This is inconsistent with previous reports, which showed the expression of DNMT3 in ovary, but not in testis of the starfish A. pectinifera, indicating the tissue- and species-specific expression of DNMT3 gene. Although further studies are needed to clarify the epigenetic regulatory mechanisms of DNMT3 and its application to the aquaculture industry, our findings may provide insights into the sea cucumber biology.

State-of-the-art housekeeping proteins for quantitative western blotting: Revisiting the first draft of the human proteome.

Western blotting (WB) analysis is the most popular and widely used methodology for protein detection and characterization over recent decades. In accordance with the advancement of the technologies for the acquisition of WB signals, a quantitative value is used to present the abundance of target proteins in a complex sample, thereby requiring the use of specific proteins as internal references that represent total proteins. Heretofore, proteins encoded by housekeeping genes such as GAPDH, ß-tubulin and ß-actin have been commonly used as loading controls without any hesitation because their mRNA expression levels tend to be high and constant in many different cells and tissues. Experimentally, however, some of the housekeeping reference proteins are often displayed with inconsistent expression levels in both homogeneous and heterogeneous tissues, and, in terms of mRNA levels, they have a weak correlation to the abundance of proteins. To estimate accurate, reliable, and reproducible protein quantifications, it is crucial to define appropriate reference controls. For this paper, we explored the recently released large-scale, human proteomic database ProteomicsDB including 16 857 liquid chromatography tandem-mass-spectrometry data from 27 human tissues, and suggest 20 ubiquitously- and constitutively-expressed, putative internal-reference controls for the quantification of differential protein expressions. Intriguingly, the most commonly used, known housekeeping genes were entirely excluded in our newly defined candidates. Although the applications of the candidates under many different biological conditions and in other organisms are yet to be empirically verified, we propose reliable, potential loading controls for a WB analysis in this paper.

Epigallocatechin-3-gallate rescues LPS-impaired adult hippocampal neurogenesis through suppressing the TLR4-NF-κB signaling pathway in mice.

Adult hippocampal dentate granule neurons are generated from neural stem cells (NSCs) in the mammalian brain, and the fate specification of adult NSCs is precisely controlled by the local niches and environment, such as the subventricular zone (SVZ), dentate gyrus (DG), and Toll-like receptors (TLRs). Epigallocatechin-3-gallate (EGCG) is the main polyphenolic flavonoid in green tea that has neuroprotective activities, but there is no clear understanding of the role of EGCG in adult neurogenesis in the DG after neuroinflammation. Here, we investigate the effect and the mechanism of EGCG on adult neurogenesis impaired by lipopolysaccharides (LPS). LPS-induced neuroinflammation inhibited adult neurogenesis by suppressing the proliferation and differentiation of neural stem cells in the DG, which was indicated by the decreased number of Bromodeoxyuridine (BrdU)-, Doublecortin (DCX)- and Neuronal Nuclei (NeuN)-positive cells. In addition, microglia were recruited with activatingTLR4-NF-κB signaling in the adult hippocampus by LPS injection. Treating LPS-injured mice with EGCG restored the proliferation and differentiation of NSCs in the DG, which were decreased by LPS, and EGCG treatment also ameliorated the apoptosis of NSCs. Moreover, pro-inflammatory cytokine production induced by LPS was attenuated by EGCG treatment through modulating the TLR4-NF-κB pathway. These results illustrate that EGCG has a beneficial effect on impaired adult neurogenesis caused by LPSinduced neuroinflammation, and it may be applicable as a therapeutic agent against neurodegenerative disorders caused by inflammation.

Appetitive learning requires the alpha1-like octopamine receptor OAMB in the Drosophila mushroom body neurons.

Associative learning is a fundamental form of behavioral plasticity. Octopamine plays central roles in various learning types in invertebrates; however, the target receptors and underlying mechanisms are poorly understood. Drosophila provides a powerful system to uncover the mechanisms for learning and memory. Here, we report that OAMB in the mushroom body neurons mediates the octopamine's signal for appetitive olfactory learning. The octopamine receptor OAMB has two isoforms (OAMB-K3 and OAMB-AS), differing in the third cytoplasmic loop and downstream sequence. The activation of each OAMB isoform increases intracellular Ca(2+) similar to the alpha1 adrenergic receptor, while OAMB-K3 additionally stimulates cAMP production. The oamb-null mutants showed severely impaired learning in appetitive olfactory conditioning that tests flies' capacity to learn and remember the odor associated with sugar reward. This deficit was also seen in the hypomorphic mutant with reduced OAMB expression in the mushroom bodies, the brain structure crucial for olfactory conditioning. Consistently, the oamb mutant's learning phenotype was fully rescued by conditional expression of either OAMB isoform in the mushroom body αß and γ neurons. These results indicate that the OAMB receptor is a key molecule mediating the octopamine's signal for appetitive olfactory learning and its functional site is the mushroom body αß and γ neurons. This study represents a critical step forward in understanding the cellular mechanism and neural circuit mediating reward learning and memory.

Research Note: Welfare and stress responses of broiler chickens raised in conventional and animal welfare-certified broiler farms.

The present study was conducted to assess the animal welfare status of broiler chickens raised in conventional and welfare-certified farms. One conventional farm (30,000 birds/house, 1,488 m2/house, 2 houses) and one animal welfare-certified farm (32,000 birds/house, 1,920 m2/house, 2 houses) were selected to measure productivity, stress responses, and animal welfare indicators in 3 broiler flocks (2 farms/season, n = 6 flocks/farm type) during summer, autumn, and spring. Upon farm visits, body weight, uniformity, and animal welfare indicators (i.e., fecal and feather corticosterone, footpad dermatitis, hock burn, feather dirtiness, and gait score) were measured at 26 d posthatch. Also, moisture, nitrogen, and pH of litter, light intensity, ammonia concentration, and body surface temperature of head, chest, and legs were measured. There was no difference in body weight and uniformity between farm types. Fecal corticosterone concentrations were higher (P = 0.021) in welfare-certified vs. conventional farm, but no significant difference was found in feather corticosterone. Welfare-certified vs. conventional farm had lower percentages of hock burn (P = 0.018), feather dirtiness scores (P = 0.009), and gait score (P = 0.040), and there was no difference in footpad dermatitis. Nitrogen content in litter samples tended to be higher in conventional vs. welfare-certified farms (P = 0.094), and there was no difference in moisture and pH between farm types. Ammonia concentration within the broiler houses was not different between 2 farms. However, animal welfare farm was found to be brighter than conventional farm (P < 0.001). The body surface temperature of head, chest, and legs was not different between farm types. In conclusion, the welfare-certified farm had higher welfare measures, including lower hock burn, feather dirtiness, and gait score, confirming an overall improvement in welfare indicators. However, the observation on the elevated feather corticosterone noted in welfare vs. conventionally raised chickens warrants further studies.

Effects of grain-based pecking blocks on productivity and welfare indicators in commercial broiler chickens.

OBJECTIVE: This experiment was conducted to investigate the effect of grain-based pecking blocks on productivity and welfare status at two commercial broiler welfare-certified farms. METHODS: Production and welfare indicators were assessed at two farms (designated Farm A and B). Both farms had two windowless houses with forced tunnel-type ventilation and housed broilers at stocking densities of approximately 16.7 birds/m2 (Farm A) and 16.8 birds/m2 (Farm B). Each house was divided into two or three equal sections and was provided with or without pecking blocks. Grain-based pecking blocks, measuring 25 × 25 × 25 cm, were given to broilers in both farms at 1 block per 1,000 birds. Various parameters including productivity (body weight and flock uniformity), corticosterone levels (in fecal droppings and feathers), footpad dermatitis, hock burn, feather dirtiness, gait score, litter quality, body surface temperature, and volatile fatty acids in fecal samples were assessed at 26 days of age, whereas litter quality was analyzed at 13 and 26 days of age. RESULTS: There were no significant effects of providing pecking blocks on productivity (body weight and uniformity), fecal and feather corticosterone, welfare indicators (i.e., footpad dermatitis, hock burn, feather cleanliness, and gait score), and litter quality (i.e., moisture, nitrogen, and pH). No differences in body surface temperature between the control and enrichment treatments were noted in Farm B, but body surface temperatures of the head (p = 0.029) and legs (p = 0.011) in the enrichment vs. control group were elevated in Farm A. Butyrate concentration in the enrichment vs control group was higher in Farm B (p = 0.023), but this effect was not detected in Farm A. CONCLUSION: It is concluded that grain-based pecking blocks did not affect performance and welfare indicators. Further studies are warranted to elucidate the potential impact of grain-based pecking blocks on gut health indicators.

Dietary probiotic Lacticaseibacillus paracasei NSMJ56 modulates gut immunity and microbiota in laying hens.

This study was performed to investigate supplementary effects of probiotic Lacticaseibacillus paracasei NSMJ56 strain on laying performance, egg quality, intestinal histology, antioxidant status, gut immunity and microbiota in laying hens. A total of ninety-six 21-wk-old Hy-Line Brown laying hens were randomly subjected to one of 2 dietary treatments: a control group fed a non-supplemented diet, or a probiotic group fed with a diet supplemented with 1 g of Lacticaseibacillus paracasei NSMJ56 (5 × 108 CFU/kg of diet). The trial lasted for 4 wk. Egg weight was increased (P < 0.05) in laying hens fed probiotic-fed diet compared with the control group. Dietary probiotics did not affect egg quality except for Haugh unit, which was improved (P < 0.05) in the probiotic-fed group. Neither jejunal histology nor cecal short-chain fatty acids were affected by dietary treatments. Dietary probiotics increased the activity of catalase compared with the control group. Flow cytometry analysis revealed that dietary probiotics elevated the CD4+ T cells, but not CD8+ T cells, in jejunal lamina propria. Based on the LEfSe analysis at the phylum and genus levels, Erysipelotrichales, Erysipelotrichia, Flintibater, Dielma, Hespellia, Coprobacter, Roseburia, Anaerotignum, and Coprococcus were enriched in the probiotic group compared with the control group. Taken together, our study showed that dietary probiotics could be used to improve some parameters associated with egg freshness and antioxidant capacity, and to partially alter T cell population and microbial community in laying hens.

Effect of Dietary Chlorella vulgaris or Tetradesmus obliquus on Laying Performance and Intestinal Immune Cell Parameters.

A feeding trial was conducted to investigate the effect of dietary supplementation of Chlorella vulgaris (CV) or Tetradesmus obliquus (TO) on laying performance, egg quality, and gut health indicators of laying hens. A total of 144 Hy-Line Brown laying hens aged 21 weeks were randomly assigned to one of three dietary treatments with eight replicates of six hens. Dietary treatments were as follows: CON, basal diet; CV, basal diet + 5 g C. vulgaris/kg of diet; TO, basal diet + 5 g T. obliquus/kg of diet. The results showed that diets supplemented with CV or TO had insignificant effects on laying performance, egg quality (i.e., Haugh unit and eggshell strength and thickness), jejunal histology, cecal short-chain fatty acids, and antioxidant/immune markers in ileal mucosa samples of laying hens. Compared with the control group, the egg yolk color score was higher (p < 0.05) in laying hens fed on diets containing CV and TO, although the former was a more intense yellow than the latter. Small intestinal lamina propria cells were isolated using flow cytometry to examine the percentages of immune cell subpopulations. Dietary microalgae did not affect B cells or monocytes/macrophages but altered the percentage of CD4+ T cells and CD8- TCR γδ T cells. Collectively, diets supplemented with C. vulgaris or T. obliquus can improve egg yolk color and would modulate host immune development and competence in laying hens.

The cell adhesion molecule Roughest depends on beta(Heavy)-spectrin during eye morphogenesis in Drosophila.

Cell junctions have both structural and morphogenetic roles, and contain complex mixtures of proteins whose interdependencies are still largely unknown. Junctions are also major signaling centers that signify correct integration into a tissue, and modulate cell survival. During Drosophila eye development, the activity of the immunoglobulin cell adhesion molecule Roughest (also known as Irregular chiasm C-roughest protein) mediates interommatidial cell (IOC) reorganization, leading to an apoptotic event that refines the retinal lattice. Roughest and the cadherin-based zonula adherens (ZA) are interdependent and both are modulated by the apical polarity determinant, Crumbs. Here we describe a novel relationship between the Crumbs partner beta(Heavy)-spectrin (beta(H)), the ZA and Roughest. Ectopic expression of the C-terminal segment 33 of beta(H) (betaH33) induces defects in retinal morphogenesis, resulting the preferential loss of IOC. This effect is associated with ZA disruption and Roughest displacement. In addition, loss-of-function karst and roughest mutations interact to cause a synergistic and catastrophic effect on retinal development. Finally, we show that beta(H) coimmunoprecipitates with Roughest and that the distribution of Roughest protein is disrupted in karst mutant tissue. These results suggest that the apical spectrin membrane skeleton helps to coordinate the Cadherin-based ZA with Roughest-based morphogenesis.

Effect of Dietary Organic and Inorganic Sulfur on the Performance of Coccidiosis Vaccine Challenged Broiler Chickens.

The objective of this study was to evaluate the effects of dietary sulfur from either organic (methyl sulfonyl methane, MSM) or inorganic (sodium sulfate, SS) sources on the growth performance of broiler chickens challenged against a high-dose coccidiosis vaccine. A total of 320 day-old Ross 308 broiler chicks were randomly placed into 32 pens of 10 birds each (keeping 16 pens/control group and 8 pens/treatment group until 21 days post-hatch) and reared for 28 days. The experimental diets were formulated by mixing a corn and soybean meal-based control diet with MSM or SS. At 21 days post-hatch, half (n = 8) of the control and all of the sulfur-added diet-fed (i.e., MSM and SS) groups were challenged with a 30-fold dose of a commercially available Eimeria vaccine (Livacox® T coccidiosis vaccine). Unchallenged control chicks (n = 8) were considered as the negative control group. At 21 days (before coccidiosis vaccine challenge), the production parameters and cecal short-chain fatty acids were not affected by dietary treatments. The concentrations of total antioxidant capacity in liver samples were elevated in both the MSM and SS groups compared with the control group (p = 0.001). During 21 to 28 days (i.e., one week post coccidiosis vaccine challenge), challenge tended to lower body weight and feed intake by an average of 5.3% (p = 0.262) and 2.8% (p = 0.504), respectively, but to increase the feed conversion ratio by an average of 2.7% (p = 0.087) compared with the non-challenged control groups. None of dietary sulfur groups affected the body weight gain, feed intake, or feed conversion ratio of vaccine-challenged chickens. Mild Eimeria-specific lesions were noted in duodenum (p = 0.006), jejunum (p = 0.017), and ceca (p = 0.047), but dietary sulfur treatments did not affect the Eimeria-induced gut lesion scores. At 28 days, Eimeria challenge significantly impaired (p = 0.001) the apparent ileal digestibility of crude protein and crude ash compared with the naïve control group. Dietary MSM increased the apparent ileal digestibility of crude ash by 15.5% on average compared with the coccidiosis vaccine control group. We conclude that dietary antioxidant sulfur of organic or inorganic origins at the inclusion level (i.e., 0.7 g sulfur/kg of diet) has a limited effect on the growth performance of chickens challenged with coccidiosis vaccine.

Incorporation of Dietary Methyl Sulfonyl Methane into the Egg Albumens of Laying Hens.

This study evaluated the effects of graded levels of dietary methyl sulfonyl methane (MSM) on the laying performance, egg quality, antioxidant capacity, and the incorporation of MSM into the egg albumen of laying hens. A total of 240 73-week-old laying hens (Lohmann Brown Lite) were randomly allotted to 1 of 5 dietary treatments, with 8 replicates of 6 birds per replicate. The experimental diets were formulated by mixing corn and soybean meal-based diets with MSM to reach 0.0, 1.0, 2.0, 3.0, and 4.0 g per kg of diet, and were fed to the birds for 12 weeks. Increasing dietary MSM led to a significant quadratic effect on the feed intake and feed conversion ratio at 4 weeks (p < 0.05). However, none of the egg qualities and egg components were altered by dietary MSM. The deposition of MSM in egg albumens increased in a linear manner (p < 0.05) in response to the increasing dietary MSM levels. The concentration of malondialdehyde in the egg yolk decreased at 12 weeks (linear and quadratic effect; p < 0.05), as the dietary MSM levels increased. Increasing dietary MSM affected the indicators of antioxidant/oxidative stress in the serum samples, such as superoxide dismutase at 12 weeks (linear and quadratic effect; p < 0.05), total antioxidant capacity at 8 and 12 weeks (linear effect; p < 0.05), and malondialdehyde at 8 weeks (linear effect; p < 0.05). Taken together, our study shows that dietary MSM has potential to be used as an antioxidant feed additive for laying hens, and can be used to produce functional eggs with health benefits for humans.

In Vivo Recovery of Bacteriophages and Their Effects on Clostridium perfringens-Infected Broiler Chickens.

The objectives of this study were to recover bacteriophages (BPs) from the intestinal digesta of BP-fed broilers and to evaluate the antibacterial effects of encapsulated or powdered BPs in broiler chickens challenged with Clostridium perfringens. Day-old broiler chicks (n = 320/experiment) were randomly assigned to 32 pens (n = 10 broilers/pen) and allocated to one of four dietary groups: (1) unchallenged group (NEG); (2) C. perfringens-challenged group (POS); (3) POS group fed a diet supplemented with powdered BPs; and (4) POS group fed a diet supplemented with encapsulated BPs. On days 21, 22, and 23 post-hatch, all chickens except NEG were orally inoculated twice a day with 2 mL C. perfringens (1.0 × 108 cfu/mL). Varying BP levels were detected in gut digesta at all ages and were numerically or significantly higher in the encapsulated BP group than in the powdered BP group. Dietary powder or encapsulated BPs reversed the C. perfringens-mediated increase in crypt depth. In addition, villus height to crypt depth ratio was elevated in the NEG and BP-treated/challenged groups compared with that in the POS group. C. perfringens counts in the cecum were significantly lower in the BP-fed chickens than in the POS group. The encapsulated BP-supplemented diet-fed chickens had the highest serum IgA levels. Collectively, our results suggest that dietary BP remains viable in intestinal digesta upon ingestion and can inhibit cecal C. perfringens counts.

Effects of Dietary Organic and Inorganic Sulfur on Laying Performance, Egg Quality, Ileal Morphology, and Antioxidant Capacity in Laying Hens.

The present study was conducted to investigate the comparative effects of organic and inorganic forms of sulfur, methyl sulfonyl methane (MSM) and sodium sulfate (SS), on laying performance, egg quality, ileal morphology, ileal volatile fatty acids, and antioxidant and stress markers in various biological samples in aged laying hens. A total of 144, 73-week-old Lohman Brown-Lite laying hens were randomly assigned to one of three experimental diets: basal diet (CONT), CONT + 0.2% MSM (MSM), and CONT + 0.3% SS (SS). The trial lasted for 12 weeks. MSM and SS diets contained 0.07% of sulfur, either organic or inorganic. Dietary MSM did not affect egg production or feed conversion ratio at 12 weeks compared with the CONT group. Dietary sulfur did not affect egg quality except for the Haugh unit at 4 weeks, which was lowered (p < 0.05) in the SS group. Compared with the CONT group, a higher (p < 0.05) villus height to crypt depth ratio was observed in the SS group. Dietary sulfur did not affect the percentages of short-chain fatty acids in the ileum. Total antioxidant capacity of the liver increased (p < 0.05) in laying hens fed MSM- and SS-added diets compared with the CONT group. The MSM and SS groups were found to have lowered (p < 0.05) malondialdehyde (MDA) concentration in serum samples compared with CONT. Finally, dietary MSM had the lowest (p < 0.05) MDA concentrations in yolk samples. Taken together, our study showed that dietary organic and inorganic sulfur have positive effects on ileal morphology and antioxidant capacity in laying hens. However, SS-mediated inhibition in laying performance needs to be clarified.

Quantification of the paralytic shellfish poisoning dinoflagellate Alexandrium species using a digital PCR.

A ubiquitous dinoflagellate, Alexandrium, produces paralytic shellfish toxin (PST), and its outbreaks have negative impacts on aquaculture, fisheries, human health, and the marine ecosystem. To minimize such damages, a routine monitoring program of toxic species must be implemented with a suitable analytical technique for their identification and quantification. However, the taxonomic identification and cell quantification of Alexandrium species based on their external morphology under a light microscope, or by using conventional molecular approaches have limited sensitivity and reproducibility. To address these challenges, we have developed an advanced protocol using droplet-digital PCR (ddPCR) for the discrimination and enumeration of three co-occurring Alexandrium species (A. affine, A. catenella, and A. pacificum) in environmental samples. Copies of species-specific internal transcribed spacer (ITS) per cell, which were calculated from environmental samples spiked with various numbers of culture cells, were used to estimate the abundance of species in the field samples. There were no significant differences in ITS copies estimated by the digital PCR assay between environmental samples from different localities, spiked artificially with a consistent number of cells from Alexandrium cultures. This sensitive assay was applied to determine the abundance and vertical distribution of those populations in the southern coastal waters of Korea. In spring, A. catenella was the dominant species, followed by the non-toxic A. affine in summers. A novel digital PCR assay can also be used to monitor other harmful marine protists that require high sample throughput and low detection limit with high accuracy and precision.

D1 dopamine receptor dDA1 is required in the mushroom body neurons for aversive and appetitive learning in Drosophila.

Drosophila has robust behavioral plasticity to avoid or prefer the odor that predicts punishment or food reward, respectively. Both types of plasticity are mediated by the mushroom body (MB) neurons in the brain, in which various signaling molecules play crucial roles. However, important yet unresolved molecules are the receptors that initiate aversive or appetitive learning cascades in the MB. We have shown previously that D1 dopamine receptor dDA1 is highly enriched in the MB neuropil. Here, we demonstrate that dDA1 is a key receptor that mediates both aversive and appetitive learning in pavlovian olfactory conditioning. We identified two mutants, dumb1 and dumb2, with abnormal dDA1 expression. When trained with the same conditioned stimuli, both dumb alleles showed negligible learning in electric shock-mediated conditioning while they exhibited moderately impaired learning in sugar-mediated conditioning. These phenotypes were not attributable to anomalous sensory modalities of dumb mutants because their olfactory acuity, shock reactivity, and sugar preference were comparable to those of control lines. Remarkably, the dumb mutant's impaired performance in both paradigms was fully rescued by reinstating dDA1 expression in the same subset of MB neurons, indicating the critical roles of the MB dDA1 in aversive as well as appetitive learning. Previous studies using dopamine receptor antagonists implicate the involvement of D1/D5 receptors in various pavlovian conditioning tasks in mammals; however, these have not been supported by the studies of D1- or D5-deficient animals. The findings described here unambiguously clarify the critical roles of D1 dopamine receptor in aversive and appetitive pavlovian conditioning.

Genome Sequences of Two Shewanella spp. Isolated from the Gut of the Sea Cucumber Apostichopus japonicus (Selenka, 1867).

In this study, we sequenced the genomes of two Shewanella spp., newly isolated from the gut of the sea cucumber Apostichopus japonicus (Selenka, 1867). The whole-genome sequences reported here will expand the repertoire of genomic information for the members of the genus Shewanella and will provide important insights into their roles within microbial communities.

Draft Genome Sequences of Pseudoalteromonas tetraodonis CSB01KR and Pseudoalteromonas lipolytica CSB02KR, Isolated from the Gut of the Sea Cucumber Apostichopus japonicus.

We present here the complete genome sequences of two newly isolated Pseudoalteromonas tetraodonis and Pseudoalteromonas lipolytica strains, isolated from the gut of the sea cucumber Apostichopus japonicus, to provide a useful means for facilitating the study of antibacterial, bacteriolytic, agarolytic, and algicidal activities of marine Pseudoalteromonas species.

Draft genome of the sea cucumber Apostichopus japonicus and genetic polymorphism among color variants.

The Japanese sea cucumber (Apostichopus japonicus Selenka 1867) is an economically important species as a source of seafood and ingredient in traditional medicine. It is mainly found off the coasts of northeast Asia. Recently, substantial exploitation and widespread biotic diseases in A. japonicus have generated increasing conservation concern. However, the genomic knowledge base and resources available for researchers to use in managing this natural resource and to establish genetically based breeding systems for sea cucumber aquaculture are still in a nascent stage. A total of 312 Gb of raw sequences were generated using the Illumina HiSeq 2000 platform and assembled to a final size of 0.66 Gb, which is about 80.5% of the estimated genome size (0.82 Gb). We observed nucleotide-level heterozygosity within the assembled genome to be 0.986%. The resulting draft genome assembly comprising 132 607 scaffolds with an N50 value of 10.5 kb contains a total of 21 771 predicted protein-coding genes. We identified 6.6-14.5 million heterozygous single nucleotide polymorphisms in the assembled genome of the three natural color variants (green, red, and black), resulting in an estimated nucleotide diversity of 0.00146. We report the first draft genome of A. japonicus and provide a general overview of the genetic variation in the three major color variants of A. japonicus. These data will help provide a comprehensive view of the genetic, physiological, and evolutionary relationships among color variants in A. japonicus, and will be invaluable resources for sea cucumber genomic research.

Comparative transcriptome analysis of three color variants of the sea cucumber Apostichopus japonicus.

The sea cucumber Apostichopus japonicus Selenka 1867 represents an important resource in biomedical research, traditional medicine, and the seafood industry. Much of the commercial value of A. japonicus is determined by dorsal/ventral color variation (red, green, and black), yet the taxonomic relationships between these color variants are not clearly understood. We performed the first comparative analysis of de novo assembled transcriptome data from three color variants of A. japonicus. Using the Illumina platform, we sequenced nearly 177,596,774 clean reads representing a total of 18.2Gbp of sea cucumber transcriptome. A comparison of over 0.3 million transcript scaffolds against the Uniprot/Swiss-Prot database yielded 8513, 8602, and 8588 positive matches for green, red, and black body color transcriptomes, respectively. Using the Panther gene classification system, we assessed an extensive and diverse set of expressed genes in three color variants and found that (1) among the three color variants of A. japonicus, genes associated with RNA binding protein, oxidoreductase, nucleic acid binding, transferase, and KRAB box transcription factor were most commonly expressed; and (2) the main protein functional classes are differently regulated in all three color variants (extracellular matrix protein and phosphatase for green color, transporter and potassium channel for red color, and G-protein modulator and enzyme modulator for black color). This work will assist in the discovery and annotation of novel genes that play significant morphological and physiological roles in color variants of A. japonicus, and these sequence data will provide a useful set of resources for the rapidly growing sea cucumber aquaculture industry.