Limits...
RNA-Seq analysis of seasonal and individual variation in blood transcriptomes of healthy managed bottlenose dolphins

View Article: PubMed Central - PubMed

ABSTRACT

Background: The blood transcriptome can reflect both systemic exposures and pathological changes in other organs of the body because immune cells recirculate through the blood, lymphoid tissues, and affected sites. In human and veterinary medicine, blood transcriptome analysis has been used successfully to identify markers of disease or pathological conditions, but can be confounded by large seasonal changes in expression. In comparison, the use of transcriptomic based analyses in wildlife has been limited. Here we report a longitudinal study of four managed bottlenose dolphins located in Waikoloa, Hawaii, serially sampled (approximately monthly) over the course of 1 year to establish baseline information on the content and variation of the dolphin blood transcriptome.

Results: Illumina based RNA-seq analyses were carried out using both the Ensembl dolphin genome and a de novo blood transcriptome as guides. Overall, the blood transcriptome encompassed a wide array of cellular functions and processes and was relatively stable within and between animals over the course of 1 year. Principal components analysis revealed moderate clustering by sex associated with the variation among global gene expression profiles (PC1, 22 % of variance). Limited seasonal change was observed, with < 2.5 % of genes differentially expressed between winter and summer months (FDR < 0.05). Among the differentially expressed genes, cosinor analysis identified seasonal rhythmicity for the observed changes in blood gene expression, consistent with studies in humans. While the proportion of seasonally variant genes in these dolphins is much smaller than that reported in humans, the majority of those identified in dolphins were also shown to vary with season in humans. Gene co-expression network analysis identified several gene modules with significant correlation to age, sex, or hematological parameters.

Conclusions: This longitudinal analysis of healthy managed dolphins establishes a preliminary baseline for blood transcriptome analysis in this species. Correlations with hematological parameters, distinct from muted seasonal effects, suggest that the otherwise relatively stable blood transcriptome may be a useful indicator of health and exposure. A robust database of gene expression in free-ranging and managed dolphins across seasons with known adverse health conditions or contaminant exposures will be needed to establish predictive gene expression profiles suitable for biomonitoring.

Electronic supplementary material: The online version of this article (doi:10.1186/s12864-016-3020-8) contains supplementary material, which is available to authorized users.

No MeSH data available.


Weighted gene expression co-variance network analysis (WGCNA) identified 15 co-expressed gene modules. a Average link hierarchical clustering dendrogram of the network with color bands identifying module membership. b Hierarchical cluster of module eigengenes identifies closely related modules. c Correlation matrix of modules with sample traits and hematological parameters: red is positively correlated, green is negatively correlated. The correlation coefficient between the module eigengene and the measured trait is listed for each pairwise correlation, with significance in parentheses (p-value). Number of genes in each module is listed at left
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC5016863&req=5

Fig5: Weighted gene expression co-variance network analysis (WGCNA) identified 15 co-expressed gene modules. a Average link hierarchical clustering dendrogram of the network with color bands identifying module membership. b Hierarchical cluster of module eigengenes identifies closely related modules. c Correlation matrix of modules with sample traits and hematological parameters: red is positively correlated, green is negatively correlated. The correlation coefficient between the module eigengene and the measured trait is listed for each pairwise correlation, with significance in parentheses (p-value). Number of genes in each module is listed at left

Mentions: The utility of the blood transcriptome to identify physiological perturbations, such as those resulting from disease or toxic exposure, requires insight into the stability of the healthy transcriptome over time, as well as differences between individuals that may relate to differences in age, sex, or hematological parameters. We therefore constructed a gene co-expression network in WGCNA using all samples (n = 31) as independent measures. Fifteen co-expressed gene modules were identified (Fig. 5a and b), while the majority of genes, represented by the grey module, were not significantly co-regulated. Gene membership in these modules is listed in Additional file 4: Table S4. Pairwise correlations between each module eigengene and each of the physical or hematological parameters measured revealed several modules with significant associations (Fig. 5c). There were no co-expressed gene modules associated with temperature or day length, and only modest correlation to season (blue module, r = 0.38, p = 0.04). Only two modules showed strong correlation to individual animal, the blue module (352 transcripts, r = 0.54, p = 2e−03) and the greenyellow module (115 transcripts, p = 0.61, p = 3e−04). These modules also correlated significantly with the sex and age, and were negatively correlated with hematocrit and alkaline phosphatase. Hematocrit values were significantly different between sexes (mean ± SEM = 38.7 ± 0.49 in females; 43.75 ± 0.53 in males). The correlation with alkaline phosphatase likely reflects the high alkaline phosphatase levels observed in the young male (Hua, 5 years; range 570–848 U/L) and the low alkaline phosphatase levels found in the older female (Pele, 28 years; range 107–190 U/L). Alkaline phosphatase levels have previously been shown to be high in juvenile dolphins [44]. The KEGG pathway for map kinase signaling (p = 0.015) was enriched in the blue module while KEGG pathways for hematopoeitic cell lineage (p = 7.2e-03) and regulation of the actin cytoskeleton (p = 7.2e-03) were enriched in the yellowgreen module. The salmon module, although more weakly associated with sex and age, was also strongly associated with alkaline phosphatase (r = −0.68, p = 2e−05), however the small number of annotated transcripts in this module prevented the identification of any enrichment within the module.Fig. 5


RNA-Seq analysis of seasonal and individual variation in blood transcriptomes of healthy managed bottlenose dolphins
Weighted gene expression co-variance network analysis (WGCNA) identified 15 co-expressed gene modules. a Average link hierarchical clustering dendrogram of the network with color bands identifying module membership. b Hierarchical cluster of module eigengenes identifies closely related modules. c Correlation matrix of modules with sample traits and hematological parameters: red is positively correlated, green is negatively correlated. The correlation coefficient between the module eigengene and the measured trait is listed for each pairwise correlation, with significance in parentheses (p-value). Number of genes in each module is listed at left
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC5016863&req=5

Fig5: Weighted gene expression co-variance network analysis (WGCNA) identified 15 co-expressed gene modules. a Average link hierarchical clustering dendrogram of the network with color bands identifying module membership. b Hierarchical cluster of module eigengenes identifies closely related modules. c Correlation matrix of modules with sample traits and hematological parameters: red is positively correlated, green is negatively correlated. The correlation coefficient between the module eigengene and the measured trait is listed for each pairwise correlation, with significance in parentheses (p-value). Number of genes in each module is listed at left
Mentions: The utility of the blood transcriptome to identify physiological perturbations, such as those resulting from disease or toxic exposure, requires insight into the stability of the healthy transcriptome over time, as well as differences between individuals that may relate to differences in age, sex, or hematological parameters. We therefore constructed a gene co-expression network in WGCNA using all samples (n = 31) as independent measures. Fifteen co-expressed gene modules were identified (Fig. 5a and b), while the majority of genes, represented by the grey module, were not significantly co-regulated. Gene membership in these modules is listed in Additional file 4: Table S4. Pairwise correlations between each module eigengene and each of the physical or hematological parameters measured revealed several modules with significant associations (Fig. 5c). There were no co-expressed gene modules associated with temperature or day length, and only modest correlation to season (blue module, r = 0.38, p = 0.04). Only two modules showed strong correlation to individual animal, the blue module (352 transcripts, r = 0.54, p = 2e−03) and the greenyellow module (115 transcripts, p = 0.61, p = 3e−04). These modules also correlated significantly with the sex and age, and were negatively correlated with hematocrit and alkaline phosphatase. Hematocrit values were significantly different between sexes (mean ± SEM = 38.7 ± 0.49 in females; 43.75 ± 0.53 in males). The correlation with alkaline phosphatase likely reflects the high alkaline phosphatase levels observed in the young male (Hua, 5 years; range 570–848 U/L) and the low alkaline phosphatase levels found in the older female (Pele, 28 years; range 107–190 U/L). Alkaline phosphatase levels have previously been shown to be high in juvenile dolphins [44]. The KEGG pathway for map kinase signaling (p = 0.015) was enriched in the blue module while KEGG pathways for hematopoeitic cell lineage (p = 7.2e-03) and regulation of the actin cytoskeleton (p = 7.2e-03) were enriched in the yellowgreen module. The salmon module, although more weakly associated with sex and age, was also strongly associated with alkaline phosphatase (r = −0.68, p = 2e−05), however the small number of annotated transcripts in this module prevented the identification of any enrichment within the module.Fig. 5

View Article: PubMed Central - PubMed

ABSTRACT

Background: The blood transcriptome can reflect both systemic exposures and pathological changes in other organs of the body because immune cells recirculate through the blood, lymphoid tissues, and affected sites. In human and veterinary medicine, blood transcriptome analysis has been used successfully to identify markers of disease or pathological conditions, but can be confounded by large seasonal changes in expression. In comparison, the use of transcriptomic based analyses in wildlife has been limited. Here we report a longitudinal study of four managed bottlenose dolphins located in Waikoloa, Hawaii, serially sampled (approximately monthly) over the course of 1 year to establish baseline information on the content and variation of the dolphin blood transcriptome.

Results: Illumina based RNA-seq analyses were carried out using both the Ensembl dolphin genome and a de novo blood transcriptome as guides. Overall, the blood transcriptome encompassed a wide array of cellular functions and processes and was relatively stable within and between animals over the course of 1 year. Principal components analysis revealed moderate clustering by sex associated with the variation among global gene expression profiles (PC1, 22 % of variance). Limited seasonal change was observed, with < 2.5 % of genes differentially expressed between winter and summer months (FDR < 0.05). Among the differentially expressed genes, cosinor analysis identified seasonal rhythmicity for the observed changes in blood gene expression, consistent with studies in humans. While the proportion of seasonally variant genes in these dolphins is much smaller than that reported in humans, the majority of those identified in dolphins were also shown to vary with season in humans. Gene co-expression network analysis identified several gene modules with significant correlation to age, sex, or hematological parameters.

Conclusions: This longitudinal analysis of healthy managed dolphins establishes a preliminary baseline for blood transcriptome analysis in this species. Correlations with hematological parameters, distinct from muted seasonal effects, suggest that the otherwise relatively stable blood transcriptome may be a useful indicator of health and exposure. A robust database of gene expression in free-ranging and managed dolphins across seasons with known adverse health conditions or contaminant exposures will be needed to establish predictive gene expression profiles suitable for biomonitoring.

Electronic supplementary material: The online version of this article (doi:10.1186/s12864-016-3020-8) contains supplementary material, which is available to authorized users.

No MeSH data available.