Selection of suitable reference genes for normalization of quantitative RT-PCR in peripheral blood samples of bottlenose dolphins (Tursiops truncatus)

Quantitative RT-PCR is often used as a research tool directed at gene transcription. Selection of optimal housekeeping genes (HKGs) as reference genes is critical to establishing sensitive and reproducible qRT-PCR-based assays. The current study was designed to identify the appropriate reference genes in blood leukocytes of bottlenose dolphins (Tursiops truncatus) for gene transcription research. Seventy-five blood samples collected from 7 bottlenose dolphins were used to analyze 15 candidate HKGs (ACTB, B2M, GAPDH, HPRT1, LDHB, PGK1, RPL4, RPL8, RPL18, RPS9, RPS18, TFRC, YWHAZ, LDHA, SDHA). HKG stability in qRT-PCR was determined using geNorm, NormFinder, BestKeeper and comparative delta Ct algorithms. Utilization of RefFinder, which combined all 4 algorithms, suggested that PGK1, HPRT1 and RPL4 were the most stable HKGs in bottlenose dolphin blood. Gene transcription perturbations in blood can serve as an indication of health status in cetaceans as it occurs prior to alterations in hematology and chemistry. This study identified HKGs that could be used in gene transcript studies, which may contribute to further mRNA relative quantification research in the peripheral blood leukocytes in captive cetaceans

Skin pigmentation provides evidence of convergent melanism in extinct marine reptiles

Throughout the animal kingdom, adaptive colouration serves critical functions ranging from inconspicuous camouflage to ostentatious sexual display, and can provide important information about the environment and biology of a particular organism1, 2. The most ubiquitous and abundant pigment, melanin, also has a diverse range of non-visual roles, including thermoregulation in ectotherms3, 4. However, little is known about the functional evolution of this important biochrome through deep time, owing to our limited ability to unambiguously identify traces of it in the fossil record2. Here we present direct chemical evidence of pigmentation in fossilized skin, from three distantly related marine reptiles: a leatherback turtle5, a mosasaur6 and an ichthyosaur7. We demonstrate that dark traces of soft tissue in these fossils are dominated by molecularly preserved eumelanin, in intimate association with fossilized melanosomes. In addition, we suggest that contrary to the countershading of many pelagic animals8, 9, at least some ichthyosaurs were uniformly dark-coloured in life. Our analyses expand current knowledge of pigmentation in fossil integument beyond that of feathers2, 10, allowing for the reconstruction of colour over much greater ranges of extinct taxa and anatomy. In turn, our results provide evidence of convergent melanism in three disparate lineages of secondarily aquatic tetrapods. Based on extant marine analogues, we propose that the benefits of thermoregulation and/or crypsis are likely to have contributed to this melanisation, with the former having implications for the ability of each group to exploit cold environments