Formalin-fixed paraffin-embedded (FFPE) samples help to investigate transcriptomic responses in wildlife disease

Abstract

Infectious diseases impact numerous organisms. Knowledge of host-pathogen interactions and host responses to infection is crucial for conservation and management. Obtaining this knowledge quickly is made increasingly possible by a variety of genomic approaches, yet, for many species the bottleneck to understanding this, remains access to appropriate samples and data. Lack of sample availability has also limited our understanding of how pathogens and the immune responses of hosts change over time. Archival materials may provide a way to explore pathogen emergence and host responses over multiple-possibly hundreds-of years. Here, we tested whether formalin-fixed paraffin-embedded (FFPE) tissue samples could be used to understand an unknown pathology, lamprey reddening syndrome (LRS), affecting pouched lampreys (Geotria australis). Our differential expression analyses of dermal tissues from four unaffected lampreys and eight affected lampreys collected in 2012 alluded to several potential agents associated with LRS. Interestingly, the pathways associated with viral infections were overrepresented in affected versus unaffected lamprey. Gene ontology analyses of the affected and non-affected lampreys also provided new insights into the largely understudied immune responses of pouched lampreys. Our work confirms that FFPE samples can be used to infer information about the transcriptional responses of a wildlife species affected by unknown historical pathologies/syndromes. In addition, the use of FFPE samples for transcriptomics offers many opportunities to investigate the genomic responses of a species to a variety of environmental changes. We conclude with a discussion about how to best sample and utilize these unique archival resources for future wildlife transcriptomic studies.

Keywords: conservation; fish; historical pathologies; immune response; lamprey reddening syndrome; pouched lamprey.

FIGURE 1

Pouched lamprey (Geotria australis) upstream‐migrating adult displaying symptoms of lamprey reddening syndrome (LRS): (a) Upstream‐migrating adult displaying LRS anterior haemorrhaging. (b) FFPE blocks of tissue collected from 2012 pouched lamprey submitted to the MPI facility. (c) Upstream‐migrating adults with LRS displaying cloudy eyes (top) and posterior haemorrhaging (bottom). (d) FFPE blocks displaying various embedded tissue types. Photo credit: A. C Brosnahan, B & D. A Miller, C. A Pande.

FIGURE 2

Rivers affected by lamprey reddening syndrome (LRS) and sampling locations for the current study. Coloured circles represent Southland, Aotearoa New Zealand rivers affected by LRS (Brosnahan et al.,  and JK, personal communication, July 2022). The circle colours denote the years that the LRS incidences were reported. Additional informal reports of LRS have occurred since 2013 but, except for one incidence in 2022, they were not well documented and are not shown here. White borders denote Aotearoa New Zealand regional boundaries. Coloured stars represent sampling locations of the LRS treatment (yellow stars) and control samples (blue stars) used in this study. The red box denotes the location of the enlarged map.

FIGURE 3

Gene expression patterns of pouched lamprey affected by LRS (LRSall) and not affected by LRS (Control). (a) Heatmap showing differentially expressed transcripts (DETs) in rows for LRSall and Control replicate samples (shown in columns) and hierarchical clustering of samples (top) and transcripts (rows). Yellow denotes genes that were upregulated and purple denotes genes that were downregulated. (b) Multi‐dimensional scaling (MDS) plot of the top 500 DETs of the LRSall versus Control replicate comparison.

FIGURE 4

Expression matrices (transcript cluster plots) of signifcantly differentially expressed (FDR < 0.05) transcripts from LRSall versus Control exact test comparisons. (a) Subcluster 1 = 4491 transcripts. (b) Subcluster 2 = 3160 transcripts. 2. (c) Subcluster 3 = 74 transcripts. (d) Subcluster 4 = 16 transcripts. A 60% cut‐off of the transcript dendrogram was used with a <2 and >−2 log2 expression setting. Transcripts are plotted in grey with the mean expression profile in dark blue. LRSall = all lamprey reddening syndrome (LRS) affected samples (denoted in red). Control = lampreys not affected with LRS (denoted in light blue). Panels (a) and (b) are moderately (<2 and >−2 log2 expressed) upregulated/downregulated significant (FDR < 0.05) transcripts. Panels (c) and (d) are strongly (>2 and <−2 log2 expressed) upregulated/downregulated significant LRSall transcripts.

FIGURE 5

Heatmap displaying the number of transcript counts (normalized and filtered by expression level) of 37 transcripts that received BLAST hits (UniProt TrEMBL) to viruses. One asterisk (“*”) denotes a transcript that was upregulated in the LRSall samples and not upregulated, or upregulated only in very low amounts, in the control samples. Two asterisks (“**”) denotes a transcript that was greatly upregulated in the LRSall samples. LRSall = all lamprey reddening syndrome (LRS) affected samples. Control = lampreys not affected with LRS.

FIGURE 6

Heatmap displaying the number of transcript counts (normalized and filtered by expression level) of 36 transcripts that received BLAST hits (UniProt TrEMBL) to bacteria. An asterisk (“*”) denotes a transcript that was upregulated in the LRSall samples and not upregulated, or upregulated in very low amounts, in the control samples. LRSall = all lamprey reddening syndrome (LRS) affected samples. Control = lampreys not affected with LRS.

FIGURE 7

Results from the PANTHER v17.0 Reactome Pathways statistical overrepresentation test of the LRSall versus Control differentially expressed transcripts. LRSall = all lamprey reddening syndrome (LRS) affected samples. Control = lampreys not affected with LRS. Dot sizes correspond to fold enrichment values and colours correspond to raw p‐values.