Full length articleTranscriptome sequencing of hybrid bester sturgeon: Responses to poly (I:C) in the context of comparative immunogenomics
Introduction
Order Acipenseriformes contains 27 extant species distributed across the northern hemisphere, including so-called “living fossil” species of paddlefish and sturgeons. The order has a basal position within Actinopteri and is characterized by many specific morphological and genomic features, including high diploid chromosome numbers, various levels of ploidy between species, conserved morphology, and slow molecular evolution. Sturgeons are well known for the delicacy of their eggs, the caviar, one of the most valuable products on the food market, and now considered as one of the most imperilled group of animals worldwide (IUCN press release of 18, Mar 2010, https://www.iucn.org/press/news-releases). Overfishing of wild stocks, encouraged by high profits from illegal markets, is the major cause that has led all sturgeon species to the brink of extinction, inducing the International Union for Conservation of Nature (IUCN) to list them. Depletion of wild stocks and high demand on caviar led to development of industrial scale sturgeon aquaculture. The first FAO-recorded harvest from aquaculture was in 1984 with 150 tonnes. Since then, it gradually increased to the beginning of the 2000's when it started increasing rapidly year by year. In both 2015 and 2016 the world aquaculture production of sturgeon was about 105.000 tonnes. Subsequently, nearly all caviars on the market today are harvested from farmed sturgeon.(The caviar market. Production, trade and consumption in and outside the EU. https://www.eumofa.eu/documents/20178/84590/The+caviar+market_EU.pdf).
High fish density in intensive aquaculture requires constant monitoring of immune status of stocks to prevent massive losses by pathogens, however, immune system of these species is heavily understudied. Therefore, immunogenomics of sturgeons is interesting and important both from practical side and in the context of comparative genomics and evolution. Rapid progress in knowledge of the immune system of sturgeons was achieved recently through application of parallel RNA sequencing [[1], [2], [3], [4]]. A large number of immune genes was identified and annotated and responses to bacterial pathogens Yersinia ruckeri [1] and Aeromonas hydrophila [2]. We performed sequencing of bester sturgeon, a fertile hybrid between sterlet, Acipenser ruthenus and beluga, Huso huso. We assume that the results are relevant for both genera, because all Acipenseriformes are known for high conservation of genomic sequences [5] including non-coding regions such as microsatellites [6] and, as a consequence, for extraordinary viability of hybrids from distantly related species [7].To enhance transcription of immune genes and explore innate responses, fish was injected with synthetic double stranded RNA – poly(I:C), which is commonly used for stimulation of innate antiviral immunity in fish [8,9]. The immune genes and responses to surrogate infection were explored in the context of comparative genomics, Atlantic salmon (Salmo salar) was used as a reference species. Owing to long-term research experience in functional genomics of fish, we know that many immune genes are not satisfactorily covered with public resources, which are commonly used for de novo annotation of sequence data: multiple genes are either absent from mammals or have changed their functional roles. Therefore information on functional categories and pathways from public databases was supplemented with own annotations, which are to large extent based on experimental results. This assisted interpretation of sturgeon immune responses and added to understanding of the origin and evolution of the piscine immune system.
Section snippets
Fish, treatment
Six specimens of 3 month old bester of aquaculture breed “Burtzevskaya” (Huso huso x Acipenser ruthenus, 5th generation) with average length 21 cm and weight 70g were randomly assigned into control and treatment group and marked with individual external tag. Fish from the treatment group were injected intraperitoneally with 2 ml of poly(I:C) (Sigma Aldrige Cat# P1530) diluted to 2,5 mg/ml in Ringer solution. The control group were injected in 2 ml of Ringer solution. Fish were individually
Composition of transcriptome
Blastx comparison of bester sturgeon contigs with vertebrates from three classes produced similar numbers of matches: 20.7 k for Latimeria and human and 21.5 k for teleosts, being lower for jawless species (18 k). Using annotations of Atlantic salmon genes as a reference, we evaluated presentation of the functional groups and pathways in sturgeon sequences. For immune genes, the ratio (number of identified bester sturgeon genes to number of salmon genes per group) was slightly higher than for
Discussion
The number of contigs with length greater than 700 bp was 57 k, and similar to previous studies with sturgeons [[1], [2], [3]], a large fraction of bester sturgeon contigs remained unidentified. However, only 20 unidentified contigs (less one per mille) responded to treatment with poly(I:C), while a large fraction of protein identified genes showed differential expression in at least one of analysed tissues. Expression rates of identified and unidentified contigs were markedly different: median
Acknowledgements
The study was supported by Russian Science Foundation grant # 16-14-00221.
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