Neonatal GABAergic transmission primes vestibular gating of output for adult spatial navigation.

GABAergic interneurons are poised with the capacity to shape circuit output via inhibitory gating. How early in the development of medial vestibular nucleus (MVN) are GABAergic neurons recruited for feedforward shaping of outputs to higher centers for spatial navigation? The role of early GABAergic transmission in assembling vestibular circuits for spatial navigation was explored by neonatal perturbation. Immunohistochemistry and confocal imaging were utilized to reveal the expression of parvalbumin (PV)-expressing MVN neurons and their perineuronal nets. Whole-cell patch-clamp recording, coupled with optogenetics, was conducted in vitro to examine the synaptic function of MVN circuitry. Chemogenetic targeting strategy was also employed in vivo to manipulate neuronal activity during navigational tests. We found in rats a neonatal critical period before postnatal day (P) 8 in which competitive antagonization of GABAergic transmission in the MVN retarded maturation of inhibitory neurotransmission, as evidenced by deranged developmental trajectory for excitation/inhibition ratio and an extended period of critical period-like plasticity in GABAergic transmission. Despite increased number of PV-expressing GABAergic interneurons in the MVN, optogenetic-coupled patch-clamp recording indicated null-recruitment of these neurons in tuning outputs along the ascending vestibular pathway. Such perturbation not only offset output dynamics of ascending MVN output neurons, but was further accompanied by impaired vestibular-dependent navigation in adulthood. The same perturbations were however non-consequential when applied after P8. Results highlight neonatal GABAergic transmission as key to establishing feedforward output dynamics to higher brain centers for spatial cognition and navigation.

The secondary somatosensory cortex gates mechanical and heat sensitivity.

The cerebral cortex is vital for the processing and perception of sensory stimuli. In the somatosensory axis, information is received primarily by two distinct regions, the primary (S1) and secondary (S2) somatosensory cortices. Top-down circuits stemming from S1 can modulate mechanical and cooling but not heat stimuli such that circuit inhibition causes blunted perception. This suggests that responsiveness to particular somatosensory stimuli occurs in a modality specific fashion and we sought to determine additional cortical substrates. In this work, we identify in a mouse model that inhibition of S2 output increases mechanical and heat, but not cooling sensitivity, in contrast to S1. Combining 2-photon anatomical reconstruction with chemogenetic inhibition of specific S2 circuits, we discover that S2 projections to the secondary motor cortex (M2) govern mechanical and heat sensitivity without affecting motor performance or anxiety. Taken together, we show that S2 is an essential cortical structure that governs mechanical and heat sensitivity.

The Secondary Somatosensory Cortex Gates Mechanical and Thermal Sensitivity.

The cerebral cortex is vital for the perception and processing of sensory stimuli. In the somatosensory axis, information is received by two distinct regions, the primary (S1) and secondary (S2) somatosensory cortices. Top-down circuits stemming from S1 can modulate mechanical and cooling but not heat stimuli such that circuit inhibition causes blunted mechanical and cooling perception. Using optogenetics and chemogenetics, we find that in contrast to S1, an inhibition of S2 output increases mechanical and heat, but not cooling sensitivity. Combining 2-photon anatomical reconstruction with chemogenetic inhibition of specific S2 circuits, we discover that S2 projections to the secondary motor cortex (M2) govern mechanical and thermal sensitivity without affecting motor or cognitive function. This suggests that while S2, like S1, encodes specific sensory information, that S2 operates through quite distinct neural substrates to modulate responsiveness to particular somatosensory stimuli and that somatosensory cortical encoding occurs in a largely parallel fashion.

The Secondary Somatosensory Cortex Gates Mechanical and Thermal Sensitivity.

The cerebral cortex is vital for the perception and processing of sensory stimuli. In the somatosensory axis, information is received by two distinct regions, the primary (S1) and secondary (S2) somatosensory cortices. Top-down circuits stemming from S1 can modulate mechanical and cooling but not heat stimuli such that circuit inhibition causes blunted mechanical and cooling perception. Using optogenetics and chemogenetics, we find that in contrast to S1, an inhibition of S2 output increases mechanical and heat, but not cooling sensitivity. Combining 2-photon anatomical reconstruction with chemogenetic inhibition of specific S2 circuits, we discover that S2 projections to the secondary motor cortex (M2) govern mechanical and thermal sensitivity without affecting motor or cognitive function. This suggests that while S2, like S1, encodes specific sensory information, that S2 operates through quite distinct neural substrates to modulate responsiveness to particular somatosensory stimuli and that somatosensory cortical encoding occurs in a largely parallel fashion.

Brainstem serotonin neurons selectively gate retinal information flow to thalamus.

Retinal ganglion cell (RGC) types relay parallel streams of visual feature information. We hypothesized that neuromodulators might efficiently control which visual information streams reach the cortex by selectively gating transmission from specific RGC axons in the thalamus. Using fiber photometry recordings, we found that optogenetic stimulation of serotonergic axons in primary visual thalamus of awake mice suppressed ongoing and visually evoked calcium activity and glutamate release from RGC boutons. Two-photon calcium imaging revealed that serotonin axon stimulation suppressed RGC boutons that responded strongly to global changes in luminance more than those responding only to local visual stimuli, while the converse was true for suppression induced by increases in arousal. Converging evidence suggests that differential expression of the 5-HT1B receptor on RGC presynaptic terminals, but not differential density of nearby serotonin axons, may contribute to the selective serotonergic gating of specific visual information streams before they can activate thalamocortical neurons.

Functional convergence of on-off direction-selective ganglion cells in the visual thalamus.

In the mouse visual system, multiple types of retinal ganglion cells (RGCs) each encode distinct features of the visual space. A clear understanding of how this information is parsed in their downstream target, the dorsal lateral geniculate nucleus (dLGN), remains elusive. Here, we characterized retinogeniculate connectivity in Cart-IRES2-Cre-D and BD-CreER2 mice, which labels subsets of on-off direction-selective ganglion cells (ooDSGCs) tuned to the vertical directions and to only ventral motion, respectively. Our immunohistochemical, electrophysiological, and optogenetic experiments reveal that only a small fraction (<15%) of thalamocortical (TC) neurons in the dLGN receives primary retinal drive from these subtypes of ooDSGCs. The majority of the functionally identifiable ooDSGC inputs in the dLGN are weak and converge together with inputs from other RGC types. Yet our modeling indicates that this mixing is not random: BD-CreER+ ooDSGC inputs converge less frequently with ooDSGCs tuned to the opposite direction than with non-CART-Cre+ RGC types. Taken together, these results indicate that convergence of distinct information lines in dLGN follows specific rules of organization.

The Enhanced Immune Protection in Chinese Mitten Crab Eriocheir sinensis Against the Second Exposure to Bacteria Aeromonas hydrophila.

Accumulating evidences suggest that the enhanced immune responses and increased protection against bacteria-induced mortality can be initiated after the primary exposure to various microbial communities and their components in various organisms including commercially valuable crustaceans. In the present study, the survival rate and immune responses of Chinese mitten crab Eriocheir sinensis were determined after an immune priming (IP) with formalin-killed Aeromonas hydrophila and an immune challenge (ICH) with the same but live pathogen (Ah group). A group in which the animals received a salt injection prior to challenge was maintained as control (Ns group). In the present study, it was shown that an IP with killed A. hydrophila can significantly protect the crabs against the ICH with a lethal dose of the live pathogen. The increased survival was associated with elevated rate and duration of phagocytosis. The antibacterial activity of the serum was significantly increased in Ah group compared to that in Ns group. Significant changes of phenoloxidase (PO) activities were also found between Ah and Ns group but not in Ah group between IP and ICH. No significant changes of lysozyme were found in Ah and NS group during the whole experiment except 3 h after IP. In addition, the levels of transcripts and protein of Dscam were increased in hemocytes of the crabs from Ah group. All the results suggested that a primary immune priming with a particular killed pathogen could induce an enhanced immunity in crabs when they were encountered secondly with the same live pathogen. The evidences of elevated immune protections in crabs would contribute to better understand the mechanism of immune priming in invertebrates.

The neuroendocrine immunomodulatory axis-like pathway mediated by circulating haemocytes in pacific oyster Crassostrea gigas.

The neuroendocrine-immune (NEI) regulatory network is a complex system, which plays an indispensable role in the immunity of host. In this study, a neuroendocrine immunomodulatory axis (NIA)-like pathway mediated by the nervous system and haemocytes was characterized in the oyster Crassostrea gigas Once invaded pathogen was recognized by the host, the nervous system would temporally release neurotransmitters to modulate the immune response. Instead of acting passively, oyster haemocytes were able to mediate neuronal immunomodulation promptly by controlling the expression of specific neurotransmitter receptors on cell surface and modulating their binding sensitivities, thus regulating intracellular concentration of Ca2+ This neural immunomodulation mediated by the nervous system and haemocytes could influence cellular immunity in oyster by affecting mRNA expression level of TNF genes, and humoral immunity by affecting the activities of key immune-related enzymes. In summary, though simple in structure, the 'nervous-haemocyte' NIA-like pathway regulates both cellular and humoral immunity in oyster, meaning a world to the effective immune regulation of the NEI network.

Transcriptional activation and translocation of ancient NOS during immune response.

NOS is the key component of the NO system, which plays an indispensable role in many physiologic and immunologic processes; however, the process that underlies the activation of ancient NOSs and their functions remains unclear. Expression of Crassostrea gigas NOS (CgNOS) mRNA in hemocytes was examined after stimulating oysters with LPS and TNF-α. Expression level of CgNOS mRNA was increased significantly, by 2.61-fold (P < 0.05), at 24 h poststimulation. A positive CgNOS signal was detected via immunoprecipitation, and only one protein was detected in oyster hemocytes. Shifting and supershifting bands were observed in EMSAs between the CgNOS promoter and the transcription factors CgNF-κB1 and Cg-signal transducer and activator of transcription (STAT). CgNF-κB1 was detected in the nucleus only at 12 h, whereas CgSTAT was observed in the cytoplasm and nucleus at 12 and 24 h. Expression levels of tyrosine-protein kinase receptor Tie-1, phosphatidylinositide phosphatase SAC2, phosphatidylinositol-4-phosphate 5-kinase type-1α, diacylglycerol kinase θ, LPS-induced TNF-α factor-like protein, cAMP-dependent transcription factor-2, NF-κB1, and STAT6 were significantly elevated in a transcriptome analysis after 12 h of LPS and TNF-α stimulation. An immunoreactive CgNOS signal was observed in both the cell membrane and cytoplasm at 12 h, whereas it was mainly localized to the cytoplasm at 24 h post-LPS and -TNF-α stimulation. These findings revealed that CgNOS could be transcriptionally activated by CgNF-κB1 and CgSTAT via the PI3K-Akt pathway, similar to what occurs for iNOS, but CgNOS translocated to the cytoplasm, similar to neuronal NOS, to modulate downstream signals during an immune defense. These results collectively provide crucial knowledge about the evolution of NOS structure and function.-Jiang, Q., Liu, Z., Zhou, Z., Wang, L., Wang, L., Yue, F., Wang, J., Wang, H., Song, L. Transcriptional activation and translocation of ancient NOS during immune response.

A novel multi-domain C1qDC protein from Zhikong scallop Chlamys farreri provides new insights into the function of invertebrate C1qDC proteins.

The C1q domain containing (C1qDC) proteins are a family of proteins possessing globular C1q (gC1q) domains, and they rely on this domain to recognize various ligands such as PAMPs, immunoglobulins, ligands on apoptotic cell. In the present study, a novel multi-domain C1qDC protein (CfC1qDC-2) was identified from scallop Chlamys farreri, and its full length cDNA was composed of 1648 bp, encoding a signal peptide and three typical gC1q domains. BLAST analysis revealed significant sequence similarity between CfC1qDC-2 and C1qDC proteins from mollusks. Three gC1q domains were predicted in its tertiary structure to form a tightly packed bell-shaped trimer, and each one adopted a typical 10-stranded sandwich fold with a jelly-roll topology and contained six aromatic amino acids forming the hydrophobic core. The mRNA transcripts of CfC1qDC-2 were mainly detected in the tissues of hepatopancreas and gonad of adult scallops, and the expression level was up-regulated in hemocytes after stimulated by LPS, PGN and ß-glucan. During the embryonic development of scallop, the mRNA transcripts of CfC1qDC-2 were presented in all the detected stages, and the expression level was up-regulated from D-hinged larvae and reached the highest at eye-spot larvae. The recombinant protein of MBP-CfC1qDC-2 (rCfC1qDC-2) could bind various PAMPs including LPS, PGN, LTA, ß-glucan, mannan as well as polyI:C, and different microorganisms including three Gram-negative bacteria, three Gram-positive bacteria and two yeasts, as well as scallop apoptotic cells. Meanwhile, rCfC1qDC-2 could interact with human heat-aggregated IgG and IgM, and inhibit the C1q-dependent hemolysis of rabbit serum. All these results indicated that CfC1qDC-2 could recognize not only PAMPs as a PRR, but also the apoptotic cells. Moreover, the similar structures and functions shared by CfC1qDC-2 and complement C1q provided a new insight into the evolution of C1qDC proteins in complement system.

Mutual modulation between norepinephrine and nitric oxide in haemocytes during the mollusc immune response.

Nitric oxide (NO) is one of the most important immune molecules in innate immunity of invertebrates, and it can be regulated by norepinephrine in ascidian haemocytes. In the present study, the mutual modulation and underlying mechanism between norepinephrine and NO were explored in haemocytes of the scallop Chlamys farreri. After lipopolysaccharide stimulation, NO production increased to a significant level at 24 h, and norepinephrine concentration rose to remarkable levels at 3 h and 12~48 h. A significant decrease of NO production was observed in the haemocytes concomitantly stimulated with lipopolysaccharide and α-adrenoceptor agonist, while a dramatic increase of NO production was observed in the haemocytes incubated with lipopolysaccharide and ß-adrenoceptor agonist. Meanwhile, the concentration of cyclic adenosine monophosphate (cAMP) decreased significantly in the haemocytes treated by lipopolysaccharide and α/ß-adrenoceptor agonist, while the content of Ca(2+) was elevated in those triggered by lipopolysaccharide and ß-adrenoceptor agonist. When the haemocytes was incubated with NO donor, norepinephrine concentration was significantly enhanced during 1~24 h. Collectively, these results suggested that norepinephrine exerted varied effects on NO production at different immune stages via a novel α/ß-adrenoceptor-cAMP/Ca(2+) regulatory pattern, and NO might have a feedback effect on the synthesis of norepinephrine in the scallop haemocytes.

The comparative proteomics analysis revealed the modulation of inducible nitric oxide on the immune response of scallop Chlamys farreri.

Nitric oxide (NO) is an important gasotransmitter which plays a key role on the modulation of immune response in all vertebrates and invertebrates. In the present study, the modulation of inducible NO on immune response of scallop Chlamys farreri was investigated via proteomic analysis. Total proteins from hepatopancreas of scallops treated with lipopolysaccharide (LPS) and/or the inhibitor of vertebrate inducible NO synthase (S-methylisothiourea sulfate, SMT) for 12 h were analyzed via 2-D PAGE and ImageMaster 2D Platinum. There were 890, 1189 and 1046 protein spots detected in the groups treated by phosphate buffered saline (PBS), LPS and LPS+SMT, respectively, and 26 differentially expressed protein spots were identified among them. These proteins were annotated with binding or catalytic activity, and most of them were involved in metabolic or cellular processes. Some immune-related or antioxidant-related molecules such as single Ig IL-1-related receptor, guanine nucleotide-binding protein subunit beta-like protein and peroxiredoxin were identified, and the changes of their expression levels in LPS group were intensified significantly after adding SMT. The decreased expression level of tyrosinase and increased level of glutathione S-transferase 4 in LPS group were diametrically reversed by appending SMT. Moreover, interferon stimulated exonuclease gene 20-like protein and copper chaperone for superoxide dismutase were only induced by LPS+SMT stimulation but not by LPS stimulation. These data indicated that NO could modulate many immunity processes in scallop, such as NF-κB transactivation, cytoskeleton reorganization and other pivotal processes, and it was also involved in the energy metabolism, posttranslational modification, detoxification and redox balance during the immune response.

The polymorphism in the promoter of HSP70 gene is associated with heat tolerance of two congener endemic bay scallops (Argopecten irradians irradians and A. i. concentricus).

BACKGROUND: The heat shock protein 70 (HSP70) is one kind of molecular chaperones, which plays a key role in protein metabolism under normal and stress conditions. METHODOLOGY: In the present study, the mRNA expressions of HSP70 under normal physiological condition and after acute heat stress were investigated in gills of two bay scallop populations (Argopecten irradians irradians and A. i. concentricus). The heat resistant scallops A. i. concentricus showed significantly lower basal level and higher induction of HSP70 compared with that of the heat sensitive scallops A. i. irradians. The promoter sequence of HSP70 gene from bay scallop (AiHSP70) was cloned and the polymorphisms within this region were investigated to analyze their association with heat tolerance. Totally 11 single nucleotide polymorphisms (SNPs) were identified, and four of them (-967, -480, -408 and -83) were associated with heat tolerance after HWE analysis and association analysis. Based on the result of linkage disequilibrium analysis, the in vitro transcriptional activities of AiHSP70 promoters with different genotype were further determined, and the results showed that promoter from A. i. concentricus exhibited higher transcriptional activity than that from A. i. irradians (P<0.05). CONCLUSIONS: The results provided insights into the molecular mechanisms underlying the thermal adaptation of different congener endemic bay scallops, which suggested that the increased heat tolerance of A. i. concentricus (compared with A. i. irradians) was associated with the higher expression of AiHSP70. Meanwhile, the -967 GG, -480 AA, -408 TT and -83 AG genotypes could be potential markers for scallop selection breeding with higher heat tolerance.

The immunomodulation of inducible hydrogen sulfide in Pacific oyster Crassostrea gigas.

Hydrogen sulfide (H2S) is an important gasotransmitter, which plays indispensable roles in cardiovascular, nervous and immune systems of vertebrates. However, the information about the immunomodulation of H2S in invertebrates is still very limited. In the present study, the temporal expression profile of cystathionine γ lyase in oyster Crassostrea gigas (CgCSE) was investigated after the oysters were stimulated by lipopolysaccharide. The expression levels of CgCSE mRNA transcripts in hemocytes increased significantly at 12h (1.31-fold of the PBS group, P<0.05) after LPS stimulation. The immunomodulation of inducible H2S in oyster was examined by monitoring the alterations of both cellular and humoral immune parameters in response to the stimulations of LPS, LPS+Na2S and LPS+propargylglycine (PAG). The total hemocyte counts (THC) and hemolymph PO activity increased significantly after LPS stimulation, and the increase could be further enhanced by adding PAG, while inhibited by appending Na2S. The phagocytosis activity of hemocytes was also increased firstly after LPS treatment, and the increase was enhanced by adding Na2S but inhibited after appending PAG. The anti-bacterial activity in hemolymph increased at 3h post LPS treatment, and then decreased after adding PAG. The total SOD activity of hemolymph was also elevated at 6h post LPS treatment, and the elevated activity was depressed by adding Na2S. These results collectively indicated that H2S might play crucial roles in the immune response of oyster via modulating the turnover and phagocytosis of hemocytes, and regulating the anti-bacterial activity and proPO activation in the hemolymph.

The essential roles of core binding factors CfRunt and CfCBFß in hemocyte production of scallop Chlamys farreri.

Core binding factor (CBF) is a family of heterodimeric transcription factors composed of a DNA-binding CBFα subunit and a non-DNA-binding CBFß subunit, which plays critical roles in regulating hematopoiesis, osteogenesis and neurogenesis. In the present study, two genes encoding Runt (designed as CfRunt) and CBFß (designed as CfCBFß) were cloned and characterized from scallop Chlamys farreri. The full-length cDNA of CfRunt and CfCBFß consists of 2128 bp and 1729 bp encoding a predicted polypeptide of 530 and 183 amino acids with a conserved Runt domain and CBFß domain, respectively. Electrophoretic mobility shift assay demonstrated that the recombinant CfRunt protein (rCfRunt) exhibited solid ability to bind specific DNA, whereas rCfCBFß could remarkably increase the DNA-binding affinity of rCfRunt. The mRNA transcripts of CfRunt and CfCBFß could be detected in all tested tissues, especially in hemocytes, heart, hepatopancreas or muscle. After bacterial challenge, the circulating total hemocyte count (THC) of scallop reduced to the lowest level at 6h (P<0.05), and then it recovered gradually to the control level at 48-96 h, while the mRNA expressions of CfRunt and CfCBFß were significant up-regulated between 6 and 48 h (P<0.05). After CfRunt gene was silenced by RNA interference, the hemocyte renewal rate and circulating THC both decreased significantly (P<0.05). However, following the RNA interference of CfRunt, the mRNA expression of CfRunt was significantly induced (P<0.05) and the attenuated hemocyte renewal rate and circulating THC could be repaired partially by LPS stimulation in the CfRunt-silenced scallops. The results collectively indicated that CfRunt and CfCBFß, as conserved transcription factors, played essential roles in regulating hemocyte production of scallop.

A scallop nitric oxide synthase (NOS) with structure similar to neuronal NOS and its involvement in the immune defense.

BACKGROUND: Nitric oxide synthase (NOS) is responsible for synthesizing nitric oxide (NO) from L-arginine, and involved in multiple physiological functions. However, its immunological role in mollusc was seldom reported. METHODOLOGY: In the present study, an NOS (CfNOS) gene was identified from the scallop Chlamys farreri encoding a polypeptide of 1486 amino acids. Its amino acid sequence shared 50.0~54.7, 40.7~47.0 and 42.5~44.5% similarities with vertebrate neuronal (n), endothelial (e) and inducible (i) NOSs, respectively. CfNOS contained PDZ, oxygenase and reductase domains, which resembled those in nNOS. The CfNOS mRNA transcripts expressed in all embryos and larvae after the 2-cell embryo stage, and were detectable in all tested tissues with the highest level in the gonad, and with the immune tissues hepatopancreas and haemocytes included. Moreover, the immunoreactive area of CfNOS distributed over the haemocyte cytoplasm and cell membrane. After LPS, ß-glucan and PGN stimulation, the expression level of CfNOS mRNA in haemocytes increased significantly at 3 h (4.0-, 4.8- and 2.7-fold, respectively, P < 0.01), and reached the peak at 12 h (15.3- and 27.6-fold for LPS and ß-glucan respectively, P < 0.01) and 24 h (17.3-fold for PGN, P < 0.01). In addition, TNF-α also induced the expression of CfNOS, which started to increase at 1 h (5.2-fold, P < 0.05) and peaked at 6 h (19.9-fold, P < 0.01). The catalytic activity of the native CfNOS protein was 30.3 ± 0.3 U mgprot(-1), and it decreased significantly after the addition of the selective inhibitors of nNOS and iNOS (26.9 ± 0.4 and 29.3 ± 0.1 U mgprot(-1), respectively, P < 0.01). CONCLUSIONS: These results suggested that CfNOS, with identical structure with nNOS and similar enzymatic characteristics to nNOS and iNOS, played the immunological role of iNOS to be involved in the scallop immune defense against PAMPs and TNF-α.

The polymorphism in the promoter region of metallothionein 1 is associated with heat tolerance of scallop Argopecten irradians.

Metallothioneins (MTs), a superfamily of cysteine-rich proteins, perform multiple functions, such as maintaining homeostasis of essential metals, detoxification of toxic metals and scavenging of oxyradicals. In this study, the promoter region of a metallothionein (MT) gene from Bay scallop Argopecten irradians (designed as AiMT1) was cloned by the technique of genomic DNA walking, and the polymorphisms in this region were screened to find their association with susceptibility or tolerance to high temperature stress. One insert-deletion (ins-del) polymorphism and sixteen single nucleotide polymorphisms (SNPs) were identified in the amplified promoter region. Two SNPs, -375 T-C and -337 A-C, were selected to analyze their distribution in the two Bay scallop populations collected from southern and northern China coast, which were identified as heat resistant and heat susceptible stocks, respectively. There were three genotypes, T/T, T/C and C/C, at locus -375, and their frequencies were 25%, 61.1% and 13.9% in the heat susceptible stock, while 34.2%, 42.1% and 23.7% in the resistant stock, respectively. There was no significant difference in the frequency distribution of different genotypes between the two stocks (P>0.05). In contrast, at locus -337, three genotypes A/A, A/C and C/C were revealed with the frequencies of 11.6%, 34.9% and 53.5% in the heat susceptible stock, while 45.7%, 32.6% and 21.7% in the heat resistant stock, respectively. The frequency of C/C genotype in the heat susceptible stock was significantly higher (P<0.01) than that in the heat resistant stock, while the frequency of A/A in the heat resistant stock was significantly higher (P<0.01) than that in the heat susceptible stock. Furthermore, the expression of AiMT1 mRNA in scallops with C/C genotype was significantly higher than that with A/A genotype (P<0.05) after an acute heat treatment at 28°C for 120min. These results implied that the polymorphism at locus -337 of AiMT1 was associated with the susceptibility/tolerance of scallops to heat stress, and the -337 A/A genotype could be a potential marker available in future selection of Bay scallop with heat tolerance.

A tailless Dscam from Eriocheir sinensis diversified by alternative splicing.

Dscam (Down syndrome cell adhesion molecule), a member of the immunoglobulin superfamily (IgSF), plays an essential role in pathogen recognition and further involves in the innate defense of invertebrates. In the present study, the cDNA of a Dscam from Chinese mitten crab Eriocheir sinensis (designated EsDscam) was cloned and characterized. It contained a 5-terminal untranslated region (UTR) of 60 bp, a 3-UTR of 216 bp with a poly (A) tail, and an open reading frame (ORF) of 4848 bp encoding a polypeptide of 1615 amino acids with the putative molecular mass of 178.4 kDa and theoretical isoelectric point of 6.31. The EsDscam protein shared higher sequence identities and similar domain architecture with Dscams from other invertebrate, including typical 10 immunoglobulin (Ig) domains, 6 fibronectin type 3 domains (FNIII) and one cell attachment sequence (RGD) in extracellular region, while it lacked the expected transmembrane domain and cytoplasmic tail compared with other members of Dscam family. After sequencing 80 separate clones of Ig2, 3 and Ig7 regions from pooled cDNA libraries constructed from normal and bacterial-infected crabs, 44 alternative sequences were detected in the N-terminal of Ig2, 39 ones in Ig3, and 31 ones in Ig7 domain, suggesting that EsDscam could potentially encode at least 53196 unique isoforms. Furthermore, two 3'UTR isoforms and two 5'UTR isoforms of EsDscam were also identified by RACE strategy. EsDscam mRNA was most abundantly expressed in the tissues of nerve, muscle, hepatopancreas and gill, and weakly expressed in heart, gonad and hemocytes. Western blotting and immunofluorescence analysis revealed that EsDscam protein was mainly distributed in serum, and few on the membrane of crab hemocytes. These results suggested that this tailless EsDscam was one member of crustacean Dscam family, and the generation of diverse isoforms through alternative splicing allowed it to recognize various pathogens and play an active role in immune defense of crabs.

An opioid growth factor receptor (OGFR) for [Met5]-enkephalin in Chlamys farreri.

Opioid growth factor receptor (OGFR) is a receptor for [Met(5)]-enkephalin and plays important roles in the regulation of cell growth and embryonic development. In the present study, a cDNA of 2381 bp for the scallop Chlamys farreri OGFR (designated as CfOGFR) was identified by rapid amplification of cDNA ends (RACE) approach and expression sequence tag (EST) analysis. The complete cDNA sequence of CfOGFR contained an open reading frame (ORF) of 1200 bp, which encoded a protein of 399 amino acids. The amino acid sequence of CfOGFR shared 33-64% similarity with other OGFRs. There was a low complexity domain and a conserved OGFR_N domain at the N-terminal of CfOGFR. The mRNA transcripts of CfOGFR were constitutively expressed in the tested tissues with the highest expression level in hepatopancreas. During the early embryonic development, the mRNA transcripts of CfOGFR could be detected in different development stages, where the expression level presented a downward trend as a whole. The stimulations of LPS, Glu and poly (I:C) significantly induced the expression of CfOGFR mRNA in hemocytes (P < 0.05), while PGN stimulation exerted no influence. Co-IP and western blot results revealed that the CfOGFR in hemocytes displayed high affinity and specificity to [Met(5)]-enkephalin. Exogenous [Met(5)]-enkephalin was observed to inhibit the proliferation of HEK293T cells transfected with pcDNA3.1(+)-CfOGFR in a time and dosage dependent manner. These results collectively indicated that CfOGFR, as a homolog of OGFRs in C. farreri, played an important role in cells proliferation, and might be involved in the immune response of scallops.

The response of mRNA expression upon secondary challenge with Vibrio anguillarum suggests the involvement of C-lectins in the immune priming of scallop Chlamys farreri.

The enhanced immunity against a second encounter with the particular pathogen has suggested the presence of "immune priming" in scallop. In the present study, the survival rate and expression patterns of five C-lectin isoforms from scallop Chlamys farreri were explored after "vaccination" of heat-killed Vibrio anguillarum or successively challenge with V. anguillarum and Micrococcus luteus. When scallops were challenged with live bacteria, the survival rate increased significantly only in the group firstly "vaccinated" with inactivated V. anguillarum and then challenged with live V. anguillarum compared with naive scallops (from 41% to 63.6%, P<0.05), showing enhanced protective effects of inactivated bacteria with "specificity". When scallops received the challenge with V. anguillarum, the mRNA expression level of five C-lectins in scallops which were immuned previously with heat-killed V. anguillarum peaked significantly higher (26.7-, 121.7-, 60.1-, 27.4-, 16.3-fold to 0h, respectively, P<0.01) than that in non-immuned scallops (7.6-fold, P<0.05; 6.4-, 3.9-fold, P>0.05; 5.7-fold, P<0.05; 11.7-fold, P<0.01, to 0h, respectively). A significantly higher peak and 3-9h earlier response of all C-lectins mRNA expression were observed after challenge with live V. anguillarum (26.7-, 121.7-, 60.1-, 26.4- and 16.3-fold to 0h, respectively, P<0.01), compared with those only received first injection with heat-killed V. anguillarum (1.6-fold, P>0.05; 8.3-fold, P<0.05; 5.2-fold, P>0.05; 14.5-fold, P<0.01; 4.3-fold, P>0.05, to 0h, respectively). The response of mRNA expression to the secondary encounter with the same bacteria was stronger than that of successively encounter with the different class of bacteria. It was obvious that the mRNA expression of C-lectins in scallops was significantly enhanced by the successive challenge of same species of bacteria with a certain degree of specificity. All the results suggested that C-lectins might be involved in some form of immune priming, and it might provide new insights into mechanism of invertebrate immune priming.