The first five variable microsatellite DNA loci for mangrove crab, Scylla paramanosain, were developed. Allelic variation and other characteristics at these loci were examined in this species captured at the Urado Bay, Japan. The number of alleles per locus ranged from 24 to 44. The expected heterozygosity across loci ranged from 0.900 to 0.999 and probability of identity (PI) ranged from 2.8 × 10−3 to 1.7 × 10−2. Therefore, these microsatellite markers could be useful for estimating effect of stock enhancement release and population genetic structure of mangrove crab.

The first five microsatellite markers for the ide, Leuciscus idus , and four microsatellite markers for the Siberian roach, Rutilus rutilus , were designed. Cross-amplification of ide markers was examined in Siberian roach and vice versa. The number of alleles per locus ranged from three to 13 in ide and from two to eight in roach. The expected heterozygosity ranged from 0.313 to 0.909 in ide and from 0.119 to 0.775 in roach. These markers could be used to evaluate the genetic population structure of these species and other fish from the Cyprinidae family.

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The first microsatellite markers were isolated from the golden cuttlefish, Sepia esculenta Hoyle. Eleven primer sets were designed to amplify the marker sequences via polymerase chain reaction. The 45 -50 individuals from one wild population in the coastal waters of Ehime Prefecture, Japan were used to screen polymorphism in the 11 microsatellite loci. All the microsatellite loci were polymorphic, with the range of alleles from seven to 27 per locus. The observed and expected heterozygosities ranged from 0.380 to 0.980 and from 0.654 to 0.940, respectively. These marker loci except for one locus showing significant deviation from Hardy-Weinberg equilibrium will be useful for the assessment of genetic variation and population structure of this species.

Threeline grunt (Parapristipoma trilineatum) distributes around the southwestern coast of Japan and the east coast of China. The Chinese P. trilineatum was imported by Japan as an aquacultural seed because of its rapid growth compared with that of the Japanese P. trilineatum. The Japanese P. trilineatum differs from the Chinese P. trilineatum in some quantitative traits, and it has been suggested that these two P. trilineatum populations are genetically different. In order to identify the population structures around Japan and China, 5 local populations of the Japanese P. trilineatum and 2 local populations of the Chinese P. trilineatum were analyzed using 4 microsatellite DNA markers. Significant differences were detected between Japanese and Chinese P. trilineatum and among samples of Chinese P. trilineatum; however, among the samples of Japanese P. trilineatum, no significant differences were detected. These results suggest that care must be taken to prevent the escape of the Chinese P. trilineatum from culture cages around the Japanese coast, in order to preserve the genetically different population structures of Japanese and Chinese P. trilineatum.

Spondyloarthritis (SpA) comprises a number of inflammatory rheumatic diseases with overlapping clinical manifestations. Strong association with several HLA-I alleles and T cell infiltration into an inflamed joint suggest involvement of T cells in SpA pathogenesis. In this study, we performed high-throughput T cell repertoire profiling of synovial fluid (SF) and peripheral blood (PB) samples collected from a large cohort of SpA patients. We showed that synovial fluid is enriched with expanded T cell clones that are shared between patients with similar HLA genotypes and persist during recurrent synovitis. Using an algorithm for identification of TCRs involved in immune response we discovered several antigen-driven CD8+ clonal groups associated with risk HLA-B*27 or HLA-B*38 alleles. We further show that these clonal groups were enriched in SF and had higher frequency in PB of SpA patients vs healthy donors, implying their relevance to SpA pathogenesis. Several of the groups were shared among patients with different SpAs that suggests a common immunopathological mechanism of the diseases. In summary, our results provide evidence for the role of specific CD8+ T cell clones in pathogenesis of SpA.

The change of gene frequencies at the AAT-1 * and PGM-1 * loci was examined in the two guppy strains, S and S3, which had been maintained as a closed colony for a period of 16 years. These strains had been separated from an original strain in 1981. In the S strain, the *a allele frequency at AA T-1 * locus changed from 0.282 in 1981 to 0.602 in 1997, and the *a allele at the PGM-1 * locus changed from 0.772 to 0.430. On the other hand, in the S3 strain, the *a allele frequency at the AA T-1 * locus changed to 0.395 in 1997, and the *a allele at the PGM-1 * locus changed to 0.528.The effective population size (Ne) during the maintenance period of the guppy strain was estimated based on the magnitude of the random drift in the gene frequency. The estimated Ne was initially 623 in the S strain and 413 in the S3 strain. The decrease of Ne over a period of 16 years was demonstrated to be 18 in the S strain and 24 in the S3 strain, and these phenomenon may be led by the sampling of mature fish.

Background:Recently a group of T-cell clones with characteristic T-cell receptor (TCR) motif was identified in peripheral blood and synovial fluid of HLA-B*27+ patients with ankylosing spondylitis (AS) [1-2] - a prototypic disease from a wider group of spondyloarthropathies (SpAs). Extraarticular manifestations of AS could involve skin, intestine or eye. Emerging data indicate linkage between intestinal and joint inflammation, including expression of gut-associated integrins on synovial T-cells [3-4]. However, clonal T-cell composition and presence of identical clones in different inflamed sites in SpAs remains poorly studied.Objectives:To investigate clonal T-cell repertoire and presence of AS-related TCR motif in different sites of inflammation of patients with SpA.Methods:Samples of synovial fluid (SF) were obtained from HLA-B*27+ and HLA-B*27- patients with ankylosing spondylitis (AS) and psoriatic arthritis (PsA), as well as gut biopsy samples from patients with AS and Crohn’s disease (AS/CD) or ulcerative colitis (AS/UC), and conjunctival swabs from patients with uveitis (Uv) and with or without articular manifestations (Table 1). Also SF and gut biopsy samples were obtained from HLA-B*27+ patients with juvenile idiopathic arthritis (JIA). For one patient PsA patient paired samples of SF and gut biopsy were obtained.Table 1.Detection of the AS-related motif TRBV9_CASS[V/A/L/P][G/A] [L/T/V][F/Y]STDTQYF_TRBJ2-3 in bTCR repertoires of samples from different inflamed sites of patients with SpATissueDiagnosisB27+B27-AS-related TCR motif+ among all samples from B27+ donorsSynovial fluidAS2012PsAJIAIntestinal biopsyAS/CD433 / 4AS/UCJLAConjunctival swabUv804 / 8SF and gut samples were processed to isolate mononuclear cells, while conjunctival swabs were directly lysed in the lysis buffer. CD3+ β7-intergin+ cells were isolated from SF by fluorescence-activated cell sorting. Deep TCR repertoire profiling was carried out using UMI-based cDNA library preparation technology [1].Results:Identical T-cell clonotypes were detected between paired SF and gut samples of the same patient with psoriatic arthritis and intestinal inflammation. The subpopulation of β7-intergin+ SF T-cells shared significantly more identical clonotypes with gut biopsy repertoire compared to the bulk SF T-cell repertoire.Clonotypes belonging to the AS-related TCR beta motif TRBV9_CASS[V/A/L/P][G/A][L/T/V][F/Y]STDTQYF_TRBJ2-3 were detected in all inflamed tissues tested: synovial fluid, intestinal biopsies and conjunctival swabs of SpA patients (Table 1). Importantly, we observed these clonotypes exclusively in samples from HLA-B*27+ donors (n=26), but not in HLA-B27- context (n=15) with comparable analysis depth, thus confirming strong HLA-B*27-restriction of the clonotypes. The AS-related clonotypes were detected in the subpopulation of β7-intergin+ SF T-cells from HLA-B*27+ patients with PsA.Conclusion:For the first time we directly report the T-cell clonal sharing between synovial fluid and inflamed gut tissue of SpA patients. In sum our data suggests involvement of identical T-cell clones in inflammation in different anatomical sites in SpA.References:[1]Komech et al. Rheumatology (Oxford). 2018;57(6):1097-1104.[2]Faham et al. Arthritis Rheumatol. 2016;11(10):300-308.[3]Guggino et al.Ann Rheum Dis. Published Online First: 18 October 2019.doi:10.1136/annrheumdis-2019-216456.[4]Qaiyum et al Ann Rheum Dis. 2019;78(11):1566-1575.Acknowledgements:We thanks all the patients and medical personnel involved in the studyDisclosure of Interests:None declared