The utility of reptile blood transcriptomes in molecular ecology

Abstract

Reptiles and other non-mammalian vertebrates have transcriptionally active nucleated red blood cells. If blood transcriptomes can provide quantitative data to address questions relevant to molecular ecology, this could circumvent the need to euthanize animals to assay tissues. This would allow longitudinal sampling of animals' responses to treatments, as well as sampling of protected taxa. We developed and annotated blood transcriptomes from six reptile species. We found on average 25,000 proteins are being transcribed in the blood, and there is a CORE group of 9,282 orthogroups that are found in at least four of six species. In comparison to liver transcriptomes from the same taxa, approximately two-thirds of the orthogroups were found in both blood and liver; and a similar percentage of ecologically relevant gene groups (insulin and insulin-like signaling, electron transport chain, oxidative stress, glucocorticoid receptors) were found transcribed in both blood and liver. As a resource, we provide a user-friendly database of gene ids identified in each blood transcriptome. Although, on average 37% of reads mapped to hemoglobin, importantly, the majority of non-hemoglobin transcripts had sufficient depth (e.g., 97% at ≥10 reads) to be included in differential gene expression analysis. Thus, we demonstrate that RNAseq blood transcriptomes from a very small blood sample (<10 >μl) is a minimally invasive option in non-mammalian vertebrates for quantifying expression of a large number of ecologically relevant genes that would allow longitudinal sampling and sampling of protected populations.

A chromosome-level genome assembly for the Eastern Fence Lizard (Sceloporus undulatus), a reptile model for physiological and evolutionary ecology

“…The mitochondrial genome was not captured by the genome sequencing approaches, likely due to how these types of libraries are prepared. Mitochondrial sequence data obtained via RNAseq can be effectively assembled into whole mtDNA genomes [70-73]. We used RNAseq reads from 18 S. undulatus individuals from the RNAseq Dataset 4 (Table 2), which are from the same population as the individuals used for the genome sequencing.…”

Testing the Stability of Plasma Protein and Whole Blood RNA in Archived Blood of Loggerhead Sea Turtles, Caretta caretta

“…RNAlater is a standard RNA preservative for tissues, intended to be used immediately at sample collection, and has been used for transcriptomic studies of reptilian nucleated red blood cells. 11 Heparinized blood from Turtle 12 was divided into three treatments (no replicates per treatment) immediately after collection. Six milliliters were removed and split equally between two vials each containing 7.5 mL of RNAlater.…”

“…Likewise, RNA is increasingly used for the evaluation of gene expression from blood transcriptomes. [9][10][11] Storage temperature can affect concentrations of plasma proteins, 12,13 and RNA degrades rapidly if not properly stored. 14 However, few studies have assessed stability of whole blood RNA and plasma proteins of reptiles.…”

Immune and sex-biased gene expression in the threatened Mojave desert tortoise, Gopherus agassizii

“…allow for the molecular characterization of the host's systemic response to mycoplasma infection. Second, in reptiles erythrocytes are nucleated and transcriptionally active [40,41] and thrombocytes remain as intact cells instead of producing anuclear platelet cytoplasmic fragments [42]. Genes involved in insulin signaling, electron transport chain, stress, and oxidative response are shared between whole blood and liver [41], making whole blood a non-invasive sample to assess immunological and physiological response to infection.…”

“…Second, in reptiles erythrocytes are nucleated and transcriptionally active [40,41] and thrombocytes remain as intact cells instead of producing anuclear platelet cytoplasmic fragments [42]. Genes involved in insulin signaling, electron transport chain, stress, and oxidative response are shared between whole blood and liver [41], making whole blood a non-invasive sample to assess immunological and physiological response to infection. We extracted~2.5 mL whole blood from all tortoises via jugular venipuncture [43] using either a 1.91-cm, 25-gauge needle-IV infusion set and 3 mL syringe, or subcarapacial venipuncture [37] using a 3.81-cm, 23-gauge needle and 3 mL syringe.…”