Understanding and Integrating Resolution, Accuracy and Sampling Rates of Temperature Data Loggers Used in Biological and Ecological Studies

International audienceDuring the 5 th Workshop about Temperature-Dependent Sex Determination held in the 38 th International Sea Turtles Symposium (16-22 February 2018) in Kobe, Japan, we discussed the uncertainty of temperatures recorded by data logger and their calibration. We report here an extension of this discussion. First, we propose a way to estimate the uncertainty of the average temperature recorded using data loggers considering the accuracy of the data logger (repeatability of measurements), resolution of the data logger (resolution of its indicating device) and period of sampling temperature. Second, a general procedure of calibration is described. Functions to perform the estimates are provided in R package embryo growth freely available

Establishment and characterization of turtle liver organoids provides a potential model to decode their unique adaptations

Painted turtles are remarkable for their freeze tolerance and supercooling ability along with their associated resilience to hypoxia/anoxia and oxidative stress, rendering them an ideal biomedical model for hypoxia-induced injuries (including strokes), tissue cooling during surgeries, and organ cryopreservation. Yet, such research is hindered by their seasonal reproduction and slow maturation. Here we developed and characterized adult stem cell-derived turtle liver organoids (3D self-assembled in vitro structures) from painted, snapping, and spiny softshell turtles spanning ~175My of evolution, with a subset cryopreserved. This development is, to the best of our knowledge, a first for this vertebrate Order, and complements the only other non-avian reptile organoids from snake venom glands. Preliminary characterization, including morphological, transcriptomic, and proteomic analyses, revealed organoids enriched in cholangiocytes. Deriving organoids from distant turtles and life stages demonstrates that our techniques are broadly applicable to chelonians, permitting the development of functional genomic tools currently lacking in herpetological research. Such platform could potentially support studies including genome-to-phenome mapping, gene function, genome architecture, and adaptive responses to climate change, with implications for ecological, evolutionary, and biomedical research

RNAi-Mediated Gene Silencing in a Gonad Organ Culture to Study Sex Determination Mechanisms in Sea Turtle

The autosomal Sry-related gene, Sox9, encodes a transcription factor, which performs an important role in testis differentiation in mammals. In several reptiles, Sox9 is differentially expressed in gonads, showing a significant upregulation during the thermo-sensitive period (TSP) at the male-promoting temperature, consistent with the idea that SOX9 plays a central role in the male pathway. However, in spite of numerous studies, it remains unclear how SOX9 functions during this event. In the present work, we developed an RNAi-based method for silencing Sox9 in an in vitro gonad culture system for the sea turtle, Lepidochelys olivacea. Gonads were dissected as soon as the embryos entered the TSP and were maintained in organ culture. Transfection of siRNA resulted in the decrease of both Sox9 mRNA and protein. Furthermore, we found coordinated expression patterns for Sox9 and the anti-Müllerian hormone gene, Amh, suggesting that SOX9 could directly or indirectly regulate Amh expression, as it occurs in mammals. These results demonstrate an in vitro method to knockdown endogenous genes in gonads from a sea turtle, which represents a novel approach to investigate the roles of important genes involved in sex determination or differentiation pathways in species with temperature-dependent sex determination

Molecular and Genomic Characterization of Vibrio mimicus Isolated from a Frozen Shrimp Processing Facility in Mexico.

Vibrio mimicus is a gram-negative bacterium responsible for diseases in humans. Three strains of V. mimicus identified as V. mimicus 87, V. mimicus 92 and V. mimicus 93 were isolated from a shrimp processing facility in Guaymas, Sonora, Mexico. The strains were analyzed using several molecular techniques and according to the cluster analysis they were different, their similarities ranged between 51.3% and 71.6%. ERIC-PCR and RAPD (vmh390R) were the most discriminatory molecular techniques for the differentiation of these strains. The complete genomes of two strains (V. mimicus 87, renamed as CAIM 1882, and V. mimicus 92, renamed as CAIM 1883) were sequenced. The sizes of the genomes were 3.9 Mb in both strains, with 2.8 Mb in ChI and 1.1 Mb in ChII. A 12.7% difference was found in the proteome content (BLAST matrix). Several virulence genes were detected (e.g. capsular polysaccharide, an accessory colonization factor and genes involved in quorum-sensing) which were classified in 16 categories. Variations in the gene content between these genomes were observed, mainly in proteins and virulence genes (e.g., hemagglutinin, mobile elements and membrane proteins). According to these results, both strains were different, even when they came from the same source, giving an insight of the diversity of V. mimicus. The identification of various virulence genes, including a not previously reported V. mimicus gene (acfD) in ChI in all sequenced strains, supports the pathogenic potential of this species. Further analysis will help to fully understand their potential virulence, environmental impact and evolution

RNAi-Mediated Gene Silencing in a Gonad Organ Culture to Study Sex Determination Mechanisms in Sea Turtle

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Maximum likelihood (ML) and Neighbor joining (NJ) phylogenetic trees of 28 core genome virulence genes.

<p>The ML and NL phylogenetic trees of both chromosomes of <i>V</i>. <i>mimicus</i> CAIM 1882, 1883, 602 and <i>V</i>. <i>mimicus</i> 451 were obtained using MEGA, where <i>V</i>. <i>cholerae</i> O1 biovar El Tor N16961 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144885#pone.0144885.ref034" target="_blank">34</a>] was used as outgroup.</p

Genome Atlas obtained of <i>V</i>. <i>mimicus</i> CAIM 602 (green), CAIM 1882 (red), CAIM 1883 (blue) and <i>V</i>. <i>mimicus</i> 451 (as control strain).

<p>The atlas was constructed with GeneWiz Browser 0.94. From the inner ring to the outer ring: Percent AT, GC Skew, Global Inverted Repeats, Global Direct Repeats, Position Preference, Stacking Energy and Intrinsic Curvature.</p

BLAST matrix (proteome comparison) of <i>V</i>. <i>mimicus</i> 451, <i>V</i>. <i>mimicus</i> CAIM 602, <i>V</i>. <i>mimicus</i> CAIM 1882 and <i>V</i>. <i>mimicus</i> CAIM 1883.

<p>The BLAST matrix was obtained using CMG-biotools [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144885#pone.0144885.ref035" target="_blank">35</a>].</p