# The immune system of sturgeons and paddlefish (Acipenseriformes): a review with new data from a chromosome-scale sturgeon genome

Christin Höhne, Dmitry Prokopov, Heiner Kuhl, Kang Du, Christophe Klopp, Sven Wuertz, Vladimir Trifonov, Matthias Stöck

<figure><img src="image_1.png" /></figure>

## To cite this version:

Christin Höhne, Dmitry Prokopov, Heiner Kuhl, Kang Du, Christophe Klopp, et al.. The immune system of sturgeons and paddlefish (Acipenseriformes): a review with new data from a chromosome-scale sturgeon genome. Reviews in Aquaculture, 2021, 13 (3), pp.1709-1729. 10.1111/raq.12542. hal-04126879

HAL Id: hal-04126879

https://hal.inrae.fr/hal-04126879

Submitted on 13 Jun 2023

HAL is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

<figure><img src="image_2.png" /></figure>
<figure><img src="image_3.png" /></figure>

# The immune system of sturgeons and paddlefish (Acipenseriformes): a review with new data from a chromosome-scale sturgeon genome

Christin Höhne¹,* ID, Dmitry Prokopov² ID, Heiner Kuhl¹ ID, Kang Du³,⁴ ID, Christophe Klopp⁵ ID, Sven Wuertz¹ ID, Vladimir Trifonov² ID and Matthias Stöck¹ ID

1. Leibniz-Institute of Freshwater Ecology and Inland Fisheries (IGB), Berlin, Germany

2. Institute of Molecular and Cellular Biology, Siberian Branch of the Russian Academy of Sciences, Novosibirsk, Russia

3. Developmental Biochemistry, Biocenter, University of Wuerzburg, Wuerzburg, Germany

4. Xiphophorus Genetic Stock Center, Texas State University, San Marcos, TX, USA

5. SIGENAE, Plate-forme Bio-informatique Genotoul, Mathématiques et Informatique Appliquées de Toulouse, INRAE, Castanet-Tolosan, France

*Present address: State Research Center of Agriculture and Fisheries Mecklenburg-Western Pomerania, Institute of Fisheries, Rostock, Germany

**Correspondence**

Matthias Stöck, Leibniz-Institute of Freshwater
Ecology and Inland Fisheries (IGB),
Müggelseedamm 301, D-12587 Berlin,
Germany.

Email: matthias.stoeck@igb-berlin.de

Received 9 November 2020; accepted 20 January 2021.

**Abstract**

Sturgeon immunity is relevant for basic evolutionary and applied research, including caviar- and meat-producing aquaculture, protection of wild sturgeons and their re-introduction through conservation aquaculture. Starting from a comprehensive overview of immune organs, we discuss pathways of innate and adaptive immune systems in a vertebrate phylogenetic and genomic context. The thymus as a key organ of adaptive immunity in sturgeons requires future molecular studies. Likewise, data on immune functions of sturgeon-specific pericardial and meningeal tissues are largely missing. Integrating immunological and endocrine functions, the sturgeon head kidney resembles that of teleosts. Recently identified pattern recognition receptors in sturgeon require research on downstream regulation. We review first acipenseriform data on Toll-like receptors (TLRs), type I transmembrane glycoproteins expressed in membranes and endosomes, initiating inflammation and host defence by molecular pattern-induced activation. Retinoic acid-inducible gene-I-like (RIG-like) receptors of sturgeons present RNA and key sensors of virus infections in most cell types. Sturgeons and teleosts share major components of the adaptive immune system, including B cells, immunoglobulins, major histocompatibility complex and the adaptive cellular response by T cells. The ontogeny of the sturgeon innate and onset of adaptive immune genes in different organs remain understudied. In a genomics perspective, our new data on 100 key immune genes exemplify a multitude of evolutionary trajectories after the sturgeon-specific genome duplication, where some single-copy genes contrast with many duplications, allowing tissue specialization, sub-functionalization or both. Our preliminary conclusion should be tested by future evolutionary bioinformatics, involving all >1000 immunity genes. This knowledge update about the acipenseriform immune system identifies several important research gaps and presents a basis for future applications.

**Key words:** evolution, genomics, immune genes, immune organs, immune system, sturgeon.

**Introduction**

Research on sturgeon immunity is highly relevant for basic evolutionary and applied research, including caviar- and meat-producing aquaculture, protection of wild sturgeons

and their re-introduction through conservation aquaculture. While many single aspects of sturgeon immunology have been addressed, these data are scattered and a comprehensive overview is missing. In this review, starting from a synopsis of sturgeon immune organs, we focus on pathways
of the innate and adaptive immune systems, and discuss them in a vertebrate phylogenetic and genomic context.

In the evolutionary arms race between hosts and pathogens, vertebrates evolved a complex immunity, a decentralized system, composed of a variety of humoral and cellular components (Murphy & Weaver 2016). While humoral immunity is mediated by macromolecules in extracellular fluids, such as secreted antibodies, complement proteins and antimicrobial peptides, cell-mediated immunity comprises, for example the activation of phagocytes, antigen-specific cytotoxic T-lymphocytes and the release of various cytokines in response to antigen components. The immune response is conceptually categorized into and realized via the interrelated mechanisms of the innate and the adaptive immune system (Yuan et al. 2014; Iwasaki & Medzhitov 2015). Both immune systems are thought to co-evolve in vertebrates, a paradigm recently challenged by Swann et al. (2020) in anglerfish, where co-evolution of innate and adaptive immunity has been disentangled.

Molecules of the innate immune system are encoded by genes in the germline DNA and do not require somatic gene rearrangement. Thus, the innate immune system represents the first line of defence by recognition of a broad range of pathogen-associated structures and, within minutes to hours, protection against most pathogens (Netea et al. 2019). Physical, chemical and biological barriers, and cellular and humoral components, such as phagocytes and complement system, are part of this innate immunity (Beutler 2004).

The adaptive immune system is the second line of defence that acts in the immune response, days to weeks after the first antigenic contact, activated via innate immunity signalling pathways, amplifying several innate immune mechanisms (Netea et al. 2019). It has the ability to generate a long-lasting immunological memory, allowing fast intervention upon re-stimulation with the same antigen, thus exhibiting high specificity against pathogens (Cooper & Alder 2006). Somatic diversification and clonal expression of antigen receptors form the basis for the adaptive immune system, but during vertebrate evolution convergently emerged twice, in agnathans (VLRs, variable lymphocyte receptors) and gnathostomes (BCRs, B-cell receptors; TCRs, T-cell receptors; Cooper & Alder 2006; Płytycz 2008; Boehm et al. 2012b; Yuan et al. 2014). Adaptive immunity appears to have arisen abruptly in evolution (Murphy & Weaver 2016), correlating with the two whole-genome duplication events (WGDs) in early vertebrate evolution (1R and 2R; Cooper & Alder 2006; Rolff 2007; Boehm 2011; Yuan et al. 2014).

Cheng et al. (2019) published a premature sturgeon draft genome. Recently, Du et al. (2020) published a chromosome-scale, well-annotated and well-analysed sterlet sturgeon genome and uncovered an ancient, 180 My old

whole-genome duplication (WGD, Ars3R) in the sturgeon lineage (Acipenseriformes). After submission of the first version of our paper, a genome of the American paddlefish (*Polyodon spathula*) has also been assembled (Cheng et al. 2020) but this was too late to be considered in our analyses. Sturgeon and paddlefish branched off early from the actinopterygian lineage (Fig. 1), which gives them a special position in the phylogenetic tree after the ancient 2R duplication and before the 3R event, specific to the teleost lineage (Du et al. 2020). Rolff (2007) stated that selection for gene repair mechanisms and immune genes is crucial for genome duplication events, as they are often associated with an increased risk of mutations and genomic imbalance, which can lead to cancer. Partial tetraploidy in salmonids, which independently underwent an additional WGD (4R) after the ancient teleost WGD (3R), indicates beneficial adaptation to new disease pressures (Kjærner-Semb et al. 2016). Therefore, Du et al. (2020) hypothesized that the host defence system of *Acipenser ruthenus*, in which they proved segmental tetraploidy, may also have benefited from the sturgeon WGD event.

The first aim of our study was to provide an update on the current knowledge about the sturgeon immune system. We secondly supplement this summary with new genomic and transcriptomic data to understand whether and how the functional tetraploidy, resulting from the sturgeon-specific genome duplication (Ars3R), is still mirrored and conserved in the sturgeon immune genes and whether this could have fundamental effects on their immune system. We intend to identify knowledge and research gaps and to open up new fields of research on sturgeon immunity in both basic and applied contexts.

## Materials and methods

### Identification of a subset of key immune genes

To get a first overview of genes involved in immunity, we started with schemes of immune pathways from the KEGG (Kyoto Encyclopedia of Genes and Genomes) database. These list 1136 genes for all 20 vertebrate immune pathways (Table S1). In addition, we included the highly relevant FoxO signalling pathway, containing *Rag1* and *Rag2*, which is essential for V(D)J recombination in developing lymphocytes and leads to the diversity of both immunoglobulins and T-cell receptors. To identify key immune genes, which occupy indispensable interface positions in the immune network, we quantified the role of each gene in all immune pathways (Table S1). Those top 10 key genes per pathway (*n* = 49) were complemented by another 51 genes playing key roles in the vertebrate immune system (Uribe et al. 2011; Zhu et al. 2013; Murphy & Weaver 2016; Smith et al. 2019). For these 100 key immune genes, we counted the paralogous copies that can be traced back to the sturgeon WGD (Ars3R), to learn, whether they are present and
<figure><img src="image_4.png" /><figcaption>**Figure 1** Main immune organs of sturgeon (Acipenseridae) and paddlefish (Polyodontidae) and other vertebrate clades in phylogenetic and genomic context, as discussed in this paper. Note that whole-genome duplications may or may not be identical in the sturgeon and paddlefish lineages. (●) Thymoids (in Agnatha), Thymus (in Gnathostomata); (●) Spleen; (●) Gut-associated lymphoid tissue; (●) Leydig organ; (●) Epigonal organ; (●) Meningeal lymphoid tissue; (●) Head kidney (rudimental in Actinistia); (●) Pericardial lymphoid tissue; (■) Whole genome duplication.</figcaption></figure>

expressed in various tissues, and what this implies for the evolution of the sturgeon immune system.

## Bioinformatics pipeline to examine the evolutionary fate of key immune genes after the sturgeon-specific genome duplication

*Identification and validation of the immune system gene-coding sequences (CDS)*

Sterlet-annotated CDS were obtained earlier from genome annotation in combination with *de novo* annotation,

homology annotation and transcriptional analysis (Du *et al.* 2020). We cross-checked our results on the paternal assembly of *A. ruthenus* (GCF_902713435.1) and found four paralogs that were potentially missed, when using the reference genome (Du *et al.* 2020). On the other hand, the GCF_902713435.1 assembly missed 13 paralogs (Table S1). To avoid incorrect automatic annotation, we applied a phylogenetic approach to find orthologs with OrthoFinder 2.3.11 (Emms & Kelly 2019). For phylogenetic analysis, we used sequences of all sterlet proteins (proteomes) from Du *et al.* (2020), as well as the proteomes of *Callorhinchus milii*,
Danio rerio, Erpetoichthys calabaricus, Gallus gallus, Homo sapiens, Latimeria chalumnae, Lepisosteus oculatus, Petromyzon marinus, Salmo salar and Xenopus tropicalis from the Ensembl genome database (Hubbard et al. 2002). As most genes encode different transcripts, a script from the OrthoFinder package *primary_transcript.py* was run, which extracted only the longest transcript for each gene. A list of orthogroups (File S1) was obtained with the following parameters: -t 16 -a 16 -M msa -S blast, and phylogenetic trees were reconstructed. In accordance with the configuration of the phylogenetic tree for each protein of interest, sterlet orthologous proteins were obtained from the genomic annotation data.

### Copy number estimation of genes in the sterlet genome

Some paralogous genes may be missing from the genomic annotation results due to the fact that not all expressed gene sequences were revealed and some may not be expressed at all. To calculate the exact number of genes in the genome, for each gene of interest, coding sequences (CDS) were extracted from the genomic annotation data. If the obtained paralogous CDS for a gene is greater than 1, the longest CDS was left. The CDS were then aligned to the sterlet genome assembly using blastn 2.9.0 with options -ungapped -outfmt 6 qseqid sseqid pident length qstart qend sstart send sseq -num_threads 20 (Altschul et al. 1990). Alignments covering less than 30% of the query CDS, exhibiting low identity values (<50%), and processed pseudogenes were discarded. The number of such alignments was calculated for each gene.

### Paralog-specific expression

RNA-seq data for undifferentiated gonads (SRR13376048), brain (SRR11013458, SRR11013452), liver (SRR11013453, SRR11013455), ovary (SRR13378073), muscles (SRR11013457), testes (SRR13378074), spleen (SRR11013456) and skin (SRR11013454) were taken from Du et al. (2020), and data for the head kidney were taken from a *de novo* transcriptome (SRR13009649). Raw reads were pre-processed with fastp 0.20.0 with options detect_adapters_for_pe -g -c -l 50 -5 -3, and low-quality sequences and adapters were removed (Chen et al. 2018). The trimmed sequencing data were aligned on the sterlet genome sequence using hisat2 2.2.0 with default settings (Kim et al. 2019). Using the samtools 1.9 (Li et al. 2009), the resulting .sam files were converted to .bam with options view -b, sorted with option sort and filtered by alignment quality, leaving only alignments with MAPQ > 30 with view -h -q 30 options. Potential transcripts for each tissue were assembled using stringtie 2.1.4 with standard settings and FPKM (fragments per kilobase of exon per million fragments mapped) calculated for each transcript (Pertea et al. 2015). To calculate the differences in expression, genes with two paralogs were selected. The CDS

coordinates of these genes in the genome were obtained in the previous step using the blastn alignment and written to the .bed file. The resulting stringtie .gtf file, containing the expression data for each gene, was intersected by coordinates with the bed file with CDS coordinates using bedtools 2.27.1 with Intersect option (Quinlan & Hall 2010). For each gene, FPKM values for their transcripts were extracted. FPKM values for each gene were transformed as follows for plotting: lg (1 + FPKM), and a heat map was built in MATLAB 9.8.0.

## Results and discussion

### The sturgeon immune system—a knowledge update

#### Overview of sturgeon immune organs

**Hematopoietic and lymphoid organs.** Despite some variation, haematopoiesis seems widely conserved in vertebrates (Liu et al. 2017b). All immune effector cells differentiate in haematopoietic tissues from haematopoietic stem cells into myeloid and lymphoid lineages, except T cells that only develop in the thymus (Boehm et al. 2012a; Boehm & Swann 2014). In adult teleosts, haematopoietic organs comprise head kidney and trunk kidney, as well as spleen, thymus, liver and intestinal mucosa (Zapata et al. 2006). Sturgeon, however, possess additional haematopoietic pericardial tissue and haematopoietic meningeal tissue in cartilaginous skull capsules, located above the *medulla oblongata* and the anterior notochord (Fänge 1986; Lange et al. 2000; Icardo et al. 2002; Gradil et al. 2014b; Liu et al. 2017b).

Lymphoid haematopoietic organs aggregate lymphoid immune cell lines, which play a pivotal role in the adaptive immune responses (Murphy & Weaver 2016) and occur in all vertebrates (Boehm & Swann 2014). While primary lymphoid organs provide a microenvironment that promotes the lymphopoiesis in the absence of antigens, such contact and clonal proliferation occur in secondary lymphoid organs, which coordinate immune responses by spatial organization of immune effector cell interactions (e.g. antigen-presenting cells and lymphocytes; Ruddle & Akirav 2009; Hofmann et al. 2010; Boehm & Swann 2014). The main lymphoid organs of sturgeon present thymus, head kidney, spleen, pericardial and meningeal tissues (Fänge 1986; Lange et al. 2000; Gradil et al. 2014b). As in teleosts, in the gut of sturgeon, mucosa-associated lymphoid tissue (MALT) has been observed (Fänge 1986; Lange et al. 2000), but studies on its presence in other organs are scarce in Acipenseriformes.

**Thymus.** In this primary lymphoid and key organ of the adaptive immune system, located in the pharynx of gnathostomes (Boehm & Swann 2014), T cells—named after their origin—are produced and differentiated, thereby
playing major roles in cell-mediated immunity (Boehm *et al.* 2012a; Meštanová & Varga 2016). There is a true homology between the thymoids of lampreys (agnathans) and the thymus of gnathostomes (Meštanová & Varga 2016). The paired structure and localization of the thymus gland of sharks, sturgeons and teleosts are the same, and because of the homology, their morphology is even similar to that of mammals (Luer *et al.* 1995; Boehm *et al.* 2012a; Smith *et al.* 2014; Gradil *et al.* 2014a,b). The sturgeon thymus comprises a paired organ within cartilaginous skull capsules behind the eyes, adjacent to the gill openings (Fänge 1986; Lange *et al.* 2000; Gradil *et al.* 2014a,b; Salkova & Flajshans 2016; Salkova *et al.* 2020). In ontogenetic studies in *Acipenser oxyrinchus*, the thymus was visible at 768 GDD (i.e. growing degree days (GDD), °C-day, the time integral of the daily temperature measured above some temperature threshold, according to Neuheimer & Taggart, 2007) in most specimens and found in all from 950 GDD onwards (Gradil *et al.* 2014b). Since the ontogeny of sturgeons and paddlefish is almost identical (Dettlaff *et al.* 1993), this study of Gradil *et al.* (2014b) regarding the timing of the immune organ development can likely be extrapolated to other Acipenseriformes. Hassall's corpuscles, also known as thymic corpuscles, are multicellular components of the non-lymphocytic microenvironment of the *medulla* of mammalian thymi (Bodey *et al.* 2000). Hassall's corpuscle-like structures have been observed in a wide range of vertebrate taxa such as elasmobranchs, teleosts and lungfish (Bowden *et al.* 2005; Mohammad *et al.* 2007; Smith *et al.* 2014). Fänge (1986) could not find Hassall's corpuscles in *A. transmontanus*, whereas Salkova and Flajshans (2016) claimed their first description in sturgeon (*A. brevirostrum*, *A. ruthenus*). Still, Lange *et al.* (2000) had already mentioned them from the thymic *medulla* of *A. baerii* as did Petrie-Hanson and Peterman (2005) in *Polyodon spathula*, suggesting that Hassall's corpuscles present a distinct feature of the acipenseriform thymus. Age-associated thymic involution or regression, shrinkage of thymus mass, loss of tissue structure and a reduction in thymocyte numbers contribute to immunosenescence and occur in all thymus-possessing vertebrates (Shanley *et al.* 2009). In elasmobranchs, this is well documented (Luer *et al.* 1995). Despite reports of age-related changes in cell composition and relative organ size of the thymus (Fänge 1986; Gradil *et al.* 2014b), no study has analysed thymic regression in sturgeon in-depth. Furthermore, to date, neither classical thymus gene expression studies nor transcriptionics are known in Acipenseriformes.

**Head kidney.** In teleosts, this blood-filtering, immune-responsive, secondary lymphoid organ is responsible for antigen trapping like the spleen (Pastoret *et al.* 1998), while also presenting an important endocrine organ (Wendelaar

Bonga 1997; Geven & Klaren 2017). Harbouring two main endocrine cell types, the corticosteroid-secreting and the catecholamine-secreting (chromaffin) ones, the head kidney is homologous to the mammalian adrenal glands and unique in teleosts (Gallo & Civinini 2003; Tort *et al.* 2003; Geven & Klaren 2017). In elasmobranchs, the head kidney is not considered a major immune organ, as lymphoid tissue is absent in most species (Pastoret *et al.* 1998; Smith *et al.* 2014). In an ontogenetic study, Gradil *et al.* (2014b) detected the head kidney at least in 165 GDD old *A. oxyrinchus* juveniles. Sturgeon head kidneys morphologically share adrenocortical features with teleosts, even though cellular arrangement slightly differs between Chondrostei and Teleostei (Gallo & Civinini 2003). Together with trunk kidney and meningeal tissue, the head kidney is the most important haematopoietic organ of sturgeon (Fänge 1986; Lange *et al.* 2000; Liu *et al.* 2017b). Not surprisingly, in *A. dabryanus*, all types and developmental stages of blood cells have been found therein (Liu *et al.* 2017b). Compared with other organs, expression of immune-related genes is often found the highest in kidney and spleen. Transcriptome analysis and qPCR of selected immune genes revealed this in different tissues of healthy *A. sinensis* and in bester hybrids (*Huso huso* x *A. ruthenus*), with spleen showing the highest response after stimulation with synthetic double-stranded RNA (Zhu *et al.* 2016; Mugue *et al.* 2019). However, since not all immune organs were studied, additional organs (e.g. lymphoid pericardial or meningeal tissue) may relativize these expression analyses. Transcriptome profiles of head kidney from *A. dabryanus*, infected with *Aeromonas hydrophila*, revealed expression patterns of the immune response and defence against bacterial infections (Luo *et al.* 2018a). The KEGG pathway assignment confirmed the dual endocrine and immunological functions of the sturgeon head kidney. Immune unigenes (i.e. clustered transcripts per gene) could be assigned to 16 immune-related pathways. After *A. hydrophila* stimulation compared with control animals, the five immune-signalling pathways with the most genes differentially expressed are the chemokine, the Toll-like receptor (TLR), the RIG-I-like receptor and the NOD-like receptor signalling pathway, as well as antigen processing and presentation. All of this proves the important role of the head kidney for innate and adaptive defence in sturgeon, which integrates important immunological and endocrine functions in one organ, and thus occupies the same position as the teleost head kidney.

**Spleen.** In vertebrates, this is the main secondary lymphoid organ, which serves as blood reservoir, is involved in lymphopoiesis and has several immune functions including trapping circulating antigens (Boehm & Swann 2014; Liu *et al.* 2017b; Chesneau 2018). As thymus, the spleen as a
distinct organ phylogenetically first appears in elasmobranchs (Smith *et al.* 2014). In lampreys, small foci of lymphoid cells in intestinal haematopoietic areas are considered as splenic equivalents (Boehm & Swann 2014). The basic structure of the sturgeon spleen is remarkably similar to that in other gnathostomes and consists of easily detectable white and red pulp, the white containing lymphocytes, granulocytes and macrophages (Fänge 1986; Smith *et al.* 2014). It slightly differs histologically from the splenic tissue of modern bony fish (Teleostei) by the presence of large lymphoid follicles with mainly basophilic cells (white pulp) and large central arteries, surrounded by ellipsoidal blood vessels, containing erythrocytes and forming the red pulp (Lange *et al.* 2000; Gradil *et al.* 2014b). In *A. oxyrinchus*, the spleen was first visible in one 541 GDD juvenile, but is consistently seen from 768 GDD onwards (Gradil *et al.* 2014b).

Transcriptome profiles from *Acipenser schrenckii*, infected with Gram-negative *Yersinia ruckeri*, uncovered the spleen immune response (Li *et al.* 2017a). Unigenes were assigned to 20 KEGG immune pathways. For 125 genes from 16 KEGG immune pathways, differential expression after *Y. ruckeri* infection was shown. Transcriptome analysis of the spleen of *A. dabryanus* also revealed upregulation of immune-related signalling pathways after feeding different fat sources for seven weeks (Chen *et al.* 2019b). Differentially expressed unigenes revealed enriched pathways mainly related to the immune system, with NOD-like receptor signalling, platelet activation, Fc gamma R-mediated phagocytosis, Th17 cell differentiation, and Th1 and Th2 cell differentiation among the most important ones. The comparative study by Tang *et al.* (2019) about immune-related genes after *Aeromonas hydrophila* infection of *A. sinensis* and *A. dabryanus* also focused on spleen. Of the 96 immune genes identified from transcriptomes, 25 showed low similarity between both sturgeon species. Both belong to the Pacific phylogenetic clade (Peng *et al.* 2007), characterized by octoploidy, except for the presumably tetraploid (Yin *et al.* 2009) *A. dabryanus* that other authors considered octoploid (Rajkov *et al.* 2014; see also Liu *et al.* 2017a). Thus, ploidy levels and the immune-related gene copy numbers should be in the focus of future analysis. It should also be checked, if these results are not an interpretation of potential artefacts, as analysis of the octoploid *A. sinensis* gene products might have been challenging.

*Pericardial tissue.* The subepicardium of the sturgeon heart contains nodular structures, separated by connective or adipose tissue. In young specimens, it contains thymus-like lympho-haematopoietic cells (Fänge 1986; Lange *et al.* 2000; Icardo *et al.* 2002). Icardo *et al.* (2002) suggest the subepicardium to establish and maintain the immune

responses in sturgeon. This structure is changing with age, losing distinct organization with sexual maturity. The lympho-haematopoietic tissue and the nodular structure disappear and the remaining tissue is infiltrated by lymphocytes (Icardo *et al.* 2002). Gene expression analysis of selected immune genes of the heart, liver and intestine of healthy *A. sinensis* reveals an upregulation, compared with muscle (Zhu *et al.* 2016), suggesting that these organs should be considered for future studies on sturgeon immunity.

*Meningeal tissue.* Sturgeon possess haematopoietic tissue in cartilaginous skull capsules above the *medulla oblongata* and the anterior notochord. A similar structure is described in gar (*Lepisosteus osseus*) and bowfin (*Amia calva*; Lange *et al.* 2000), two Holostei that present the sister taxon of modern Teleostei, and, together with Acipenseriformes and Polypteriformes, belong to the early branches of ray-finned fish (Hughes *et al.* 2018). In elasmobranchs (sharks, rays), brain meninges are also known to be lymphoid (Smith *et al.* 2014). The meningeal tissue of sturgeon contains cells of erythroid, lymphoid and granulocyte haematopoietic lineage, and is histologically similar to the organization of mammalian haematopoietic bone marrow (Lange *et al.* 2000). In *A. oxyrinchus*, the meningeal tissue was first visible in 768 GDD old juveniles as undifferentiated and reticular cells; most cell types were found from 950 GDD onwards (Gradil *et al.* 2014b). To our knowledge, gene expression and transcriptome studies have never been performed in sturgeon meningeal tissue; only its interaction with spleen and thymus was examined (Gradil *et al.* 2014a).

*Mucosa-associated lymphoid tissue.* Mucosa-associated lymphoid tissues (MALTs) are physical barriers and active immunological sites (Rombout *et al.* 2014). Unlike the organized structures of mammals (e.g. Peyer's patches) and to the exception of interbranchial lymphoid tissue in salmons, in most other teleosts, MALTs comprise the gut (GALT)-, skin (SALT)-, gill (GIALT)- and nasopharynx (NALT)-associated lymphoid tissues. They are characterized by bacterial microbiota and a network of diffusely distributed leucocytes on mucosal surfaces (Foey & Pichietti 2014; Salinas 2015). In sturgeons, accumulations of lymphoid follicles were found in the serosa of the midgut and the spiral valve of the hindgut (Fänge 1986; Lange *et al.* 2000). Under bacterial infection with *Edwardsiella tarda*, mucosal sites (skin, gills, hindgut) acted earlier and stronger than systemic sites, as revealed by gene expression of antimicrobial peptides (AMPs) in *Acipenser dabryanus* (Chen *et al.* 2019a). However, comprehensive studies on sturgeon MALTs are still required, as the currently available studies mainly refer to the gastrointestinal tract.
## The sturgeon innate immune system: genes and signalling pathways

### Monocytes, macrophages and neutrophils

Vertebrates' innate immune cells are evolutionarily relatively conserved (Gourbal et al. 2018). Accordingly, morphogenesis and morphology of sturgeon blood cells are similar to that of other fish and higher vertebrates (Zexia et al. 2007; Gradil et al. 2014b; Liu et al. 2017b). Most cells in the innate immune response present the myeloid lineage, but lymphoid natural killer cells (NKs) also have important functions, as do some myeloid immune cells, such as macrophages and dendritic cells in adaptive immunity by antigen presentation (Murphy & Weaver 2016). In vertebrates, monocytes and NKs can exert a nonspecific innate immune memory, called trained immunity, protecting against re-infection with identical pathogens and cross-protection against different ones (Gourbal et al. 2018). Therefore, strict division into innate and adaptive cells is difficult. Neutrophils fulfil key tasks in the innate immune response, such as phagocytosis, chemotaxis, cytokine and chemokine production, reactive oxygen species (ROS) production and enzyme secretion like myeloperoxidase (MPO; Palić et al. 2011; Malech et al. 2020). Sturgeon neutrophils lack MPO but contain alkaline phosphatase instead and are classified as heterophils (Palić et al. 2011). In comparison with teleost or mammalian neutrophils, heterophils of shovelnose sturgeon (*Scaphirhynchus platorynchus*) exhibit decreased oxidative burst upon phorbol myristate acetate stimulation, similar to avian heterophils (Palić et al. 2011). Heterophiles are the predominant type in meningeal tissue (see above), observed in developing *A. oxyrinchus* from 950 GDD onwards, and from 768 GDD in spleen, increasing in numbers with size and age in both organs (Gradil et al. 2014b). Monocytes migrate from blood into most tissues to form macrophages. With neutrophils, they encounter intruding pathogens as initial defence of innate immunity (Gradil et al. 2014b; Hodgkinson et al. 2015; Murphy & Weaver 2016; Malech et al. 2020). They share functions with neutrophils but can also activate an adaptive immune response by presenting antigen to CD4+ T cells via MHC (major histocompatibility complex) class II antigens on macrophages (Beutler 2004). The differentiation of macrophages in mammals is controlled by binding of the cytokine CSF to the corresponding CSF 1R and has been identified in Teleostei and Chondrichthyes (Hodgkinson et al. 2015; Smith et al. 2019) but not in Acipenseriformes.

### Pattern recognition receptors (PRRs)

At pathogen invasion, these crucial innate immune components recognize many pathogen- or damage-associated molecular patterns (Hari et al. 2019). After ligand binding, PRRs activate downstream cascades of signal transduction

pathways, leading to innate and adaptive immune responses. The main receptor classes include Toll-like (TLRs), RIG-I-like (retinoic acid-inducible gene-I-like receptors, RLRs), NOD-like (nucleotide-binding oligomerization domain-like receptors, NLRs) and C-type lectin receptors (He et al. 2019; Hari et al. 2019). A recent comparative study on the evolution of these receptor classes in non-teleost actinopterygians includes Acipenseriformes (He et al. 2019). Beyond the framework of our study, additional classes comprise cytoplasmic DNA sensors and scavenger receptors (Zhu et al. 2016).

Toll-like receptors (TLRs). Toll-like receptors present type I transmembrane glycoproteins, expressed in cell membranes and endosomes. Pathogen-associated molecular pattern-induced activation of TLRs initiate inflammation and host defence via two signalling cascades: the myeloid differentiation primary response gene 88 (MyD88)-dependent pathway and TIR-domain-containing adapter-inducing interferon-β (TRIF) pathway, comprehensively reviewed by Fitzgerald and Kagan (2020). TLR signalling is linked to the adaptive immune response. Each TLR gene is structured into three parts: varying numbers of N-terminal leucine-rich repeats (LRRs) for extracellular pathogen-associated molecular pattern recognition, and a highly conserved intracellular C-terminal Toll-interleukin 1 receptor (TIR) domain, required for signal transduction, spaced by a short conserved transmembrane domain (Leulier & Lemaitre 2008; Kawai & Akira 2010; Khan et al. 2019; Fitzgerald & Kagan 2020). Liu et al. (2020) evaluated the phylogenetic relationships of 1726 TLRs from 200 species from most vertebrate clades (for overview Table 1), and defined eight TLR subfamilies, two more than classically distinguished (Roach et al. 2005; Khan et al. 2019; He et al. 2019). Depending on the ligand binding, TLRs can be classified as viral or non-viral TLRs, recognizing either bacterial components or other microbial nucleic acids, or rarely both (Liu et al. 2020).

To date, 13 TLRs are known from mammals (*TLR1-13*), numerous more in other vertebrates (Table 1). Teleosts possess at least 22 TLRs, among these *TLR5S* appears teleost-specific (Zhu et al. 2013; Smith et al. 2019; Li et al. 2020). During evolution, TLRs seemed gained and lost (He et al. 2019), for example *TLR6* and *TLR10* were considered mammalian, since missing in birds, reptiles and amphibians (He et al. 2019; Liu et al. 2020). However, a *TLR6* homolog was discovered in the transcriptome of a shark (*Chiloscyllium griseum*; Krishnaswamy Gopalan et al. 2014), while *TLR6* seems missing in elephant shark (Holocephali, *Callorhinchus milii*; He et al. 2019). A teleost *TLR6* homolog also occurred in a *Liza haematocheila* spleen transcriptome (Qi et al. 2016). In Acipenseriformes, a *TLR6* homolog was characterized in healthy *A. sinensis*, in which
<table><caption>**Table 1** Comparative overview of pattern recognition receptors (PRRs) in Acipenseriformes and various clades of vertebrates</caption><thead><tr><th>Clade/gene</th><th>Agnatha (lampreys, hagfish)</th><th>Chondrichthyes (sharks, rays)</th><th>Polypteriformes (bichirs, reedfishes)</th><th>Acipenseriformes (sturgeon, paddlefish)</th><th>Holostei (gars, bowfins)</th><th>Teleostei (teleosts)</th><th>Actinista (coelacanths)</th><th>Comments</th></tr></thead><tbody><tr><td rowspan="10">TLRs</td><td>TLR1</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td rowspan="4">* Not in gars (Atractosteus spatula, pseudogene; Lepisosteus oculatus, loss of function)</td></tr><tr><td>TLR2</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td></tr><tr><td>TLR3</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td></tr><tr><td>TLR4</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td></tr><tr><td>TLR5</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td></tr><tr><td>TLR6</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td></tr><tr><td>TLR7</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td rowspan="6">* Found in grey bamboo shark (Chiloscyllium griseum), Chinese sturgeon (Acipenser sinensis), Dabry's sturgeon (A. dabryanus), redlip mullet (Liza haematocheila) but not in elephant shark (Callorhinchus mili), American paddlefish (Polyodon spatula), zebrafish (Danio rerio)</td></tr><tr><td>TLR8</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td></tr><tr><td>TLR9</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td></tr><tr><td>TLR10</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td></tr><tr><td>TLR13/22</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td></tr><tr><td>TLR14/18</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td></tr><tr><td>TLR19</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>* Not in gars (Atractosteus spatula, Lepisosteus oculatus)</td></tr><tr><td>TLR20</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>* Not in gars (Atractosteus spatula, Lepisosteus oculatus)</td></tr><tr><td>TLR21</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>* Not in bowfin (Amia calva)</td></tr><tr><td>TLR25</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>* Not in bowfin (Amia calva)</td></tr><tr><td>TLR27</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>* Not in bowfin (Amia calva)</td></tr></tbody></table>
<table><caption>Table 1 (continued)</caption><thead><tr><th>Clade/gene</th><th>Agnatha (lampreys, hagfish)</th><th>Chondrichthyes (sharks, rays)</th><th>Polypteriformes (bichirs, reedfishes)</th><th>Acipenseriformes (sturgeon, paddlefish)</th><th>Holostei (gars, bowfins)</th><th>Teleostei (teleosts)</th><th>Actinistia (coelacanths)</th><th>Comments</th></tr></thead><tbody><tr><td>TLR downstream</td><td>CD14<br>CHUK<br>IKBKB<br>IKBKE</td><td>—<br>—<br>—<br>—</td><td>+</td><td>—<br>+</td><td>—<br>+</td><td>—<br>+</td><td>—<br>+</td><td>* Not in American paddlefish (Polyodon spatulatus) but in sterlet sturgeon (Acipenser ruthenus)<br>* Not in American paddlefish (Polyodon spatulatus) but in sterlet sturgeon (Acipenser ruthenus)<br>* Not in American paddlefish (Polyodon spatulatus) but in sterlet sturgeon (Acipenser ruthenus)</td></tr><tr><td>IKBKG</td><td>—</td><td>—</td><td>+</td><td>—<br>+</td><td>+</td><td>+</td><td>+</td><td></td></tr><tr><td>IRAK1<br>IRAK2<br>IRAK4<br>IRF3<br>IRF7<br>LBP<br>LY96<br>MAI<br>MYD88<br>NFKB1<br>NFKB2<br>NFKBIA<br>NFKBIAA</td><td>—<br>—<br>—<br>—<br>—<br>—<br>—<br>—<br>—<br>—<br>—<br>—<br>—<br>—</td><td>—<br>—<br>—<br>—<br>—<br>—<br>—<br>—<br>—<br>—<br>—<br>—<br>—</td><td>—<br>—<br>—<br>—<br>—<br>—<br>—<br>—<br>—<br>—<br>—<br>—<br>—</td><td>+</td><td>+</td><td>+</td><td>+</td><td></td></tr><tr><td>NFKBIAB</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—<br>+</td><td>+</td><td>—<br>+</td><td>* Not in spotted gar (Lepisosteus ocularatus) and bowfin (Amia calva), but in alligator gar (Atractosteus spatulatus)<br>* Not in alligator gar (Atractosteus spatulatus), but in spotted gar (Lepisosteus ocularatus) and bowfin (Amia calva)</td></tr><tr><td>NFKBIB<br>NFKBID<br>NFKBIE<br>NFKBIZ</td><td>—<br>—<br>—<br>—</td><td>—<br>—<br>—<br>—</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td></td></tr><tr><td>RIPK1<br>RIPK1L<br>TAB1</td><td>—<br>—<br>—</td><td>—<br>—<br>—</td><td>—<br>+</td><td>—<br>+</td><td>—<br>+</td><td>—<br>+</td><td>—<br>+</td><td></td></tr><tr><td>TAB2<br>TAB3<br>TBK1<br>TICAM1</td><td>—<br>—<br>—<br>+</td><td>+</td><td>+</td><td>—<br>+</td><td>+</td><td>+</td><td>+</td><td></td></tr><tr><td>TICAM2<br>TIRAP<br>TMEM173</td><td>—<br>—<br>—</td><td>+</td><td>—<br>+</td><td>—<br>+</td><td>+</td><td>+</td><td>—<br>+</td><td>* Gene fragments in lamprey (Petromyzon marinus)<br>* Not in spotted gar (Lepisosteus ocularatus)</td></tr><tr><td>TRAF3<br>TRAF6</td><td>—<br>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td></td></tr></tbody></table>
<table><caption>Table 1 (continued)</caption><thead><tr><th></th><th>Clade/gene</th><th>Agnatha (lampreys, hagfish)</th><th>Chondrichthyes (sharks, rays)</th><th>Polypteriformes (bichirs, reedfishes)</th><th>Acipenseriformes (sturgeon, paddlefish)</th><th>Holostei (gars, bowfins)</th><th>Teleostei (teleosts)</th><th>Actinistia (coelacanths)</th><th>Comments</th></tr></thead><tbody><tr><td rowspan="3">RLRs</td><td>RIG - I</td><td>+</td><td>+</td><td>-</td><td>+</td><td>+</td><td>+</td><td>+</td><td>* Not in Acanthopterygii</td></tr><tr><td>LGP2</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>* Gene fragments in lamprey (Fetromyzon marinus)</td></tr><tr><td>MDA5</td><td>-*</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td></td></tr><tr><td rowspan="6">RLR downstream</td><td>DDX41</td><td>+</td><td>+</td><td>-</td><td>+</td><td>+</td><td>+</td><td>+</td><td></td></tr><tr><td>MAVS</td><td>-</td><td>-</td><td>+</td><td>+</td><td>+</td><td>+</td><td>-</td><td></td></tr><tr><td>MB21D1</td><td>-</td><td>-</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td></td></tr><tr><td>NAP1</td><td>-</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td></td></tr><tr><td>NLRC3</td><td>-</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td></td></tr><tr><td>TANK</td><td>-</td><td>-</td><td>-</td><td>+</td><td>+</td><td>+</td><td>+</td><td></td></tr><tr><td rowspan="8">NLRs</td><td>TRM25</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td></td></tr><tr><td>CIITA</td><td>-</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td></td></tr><tr><td>NAIP</td><td>-</td><td>-</td><td>+</td><td>+</td><td>-</td><td>-</td><td>+</td><td></td></tr><tr><td>NLRB1</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>+</td><td>-</td><td></td></tr><tr><td>NLRB5</td><td>+</td><td>-</td><td>-</td><td>-</td><td>+</td><td>+</td><td>-</td><td></td></tr><tr><td>NOD1</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>* Not in spotted gar (Lepisosteus oculatus)</td></tr><tr><td>NOD2</td><td>-</td><td>-</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td></td></tr><tr><td>NLRC3</td><td>-</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td></td></tr><tr><td rowspan="3">NLRC4</td><td>NLRC3L</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>+</td><td>-</td><td></td></tr><tr><td>NLRP1</td><td>-</td><td>-</td><td>+</td><td>+</td><td>+</td><td>-</td><td>+</td><td></td></tr><tr><td>NLRP4</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td></td></tr><tr><td rowspan="8">NLRP3</td><td>NLRP2</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td></td></tr><tr><td>NLRP2P</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td></td></tr><tr><td>NLRP5</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td></td></tr><tr><td>NLRP6</td><td>-</td><td>-</td><td>-</td><td>-</td><td>+</td><td>+</td><td>-</td><td>* Not in American paddlefish (Polyodon spathula) but in Chinese sturgeon (Acipenser sinensis)</td></tr><tr><td>NLRP7</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td></td></tr><tr><td>NLRP8</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td></td></tr><tr><td>NLRP9</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td></td></tr><tr><td>NLRP10</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td></td></tr><tr><td>NLRP11</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td></td></tr><tr><td>NLRP12</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td></td></tr><tr><td>NLRP13</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td></td></tr><tr><td>NLRP14</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td>-</td><td></td></tr></tbody></table>
<table><caption>Table 1 (continued)</caption><thead><tr><th>Clade/gene</th><th>Agnatha (lampreys, hagfish)</th><th>Chondrichthyes (sharks, rays)</th><th>Polypteriformes (bichirs, reedfishes)</th><th>Acipenseriformes (sturgeon, paddlefish)</th><th>Holostei (gars, bowfins)</th><th>Teleostei (teleosts)</th><th>Actinistia (coelacanths)</th><th>Comments</th></tr></thead><tbody><tr><th>References</th><td>He et al. (2019)</td><td>He et al. (2019), Krishnaswamy Gopalan et al. (2014), Kagan (2017)</td><td>He et al. (2019)</td><td>He et al. (2019), Zhu et al. (2016), Luo et al. (2018), Qi et al. (2018), Tang et al. (2020), This study</td><td>He et al. (2019)</td><td>He et al. (2019), Qi et al. (2016), Chen et al. (2017)</td><td>He et al. (2019)</td><td></td></tr></tbody></table>

TLRs – toll-like receptors; RLRs – retinoic acid-inducible gene-I-like receptors, or RIG-I-like receptors; NLRs – NOD-like (nucleotide-binding oligomerization domain-like) receptors. (–) not detected so far in representatives of the clade, (+) detected in at least one representative of the clade; (*) refers to comment in right column. Note that recent information from a bowfin (*Amia calva*, Thompson et al. 2020), a paddlefish (*Polyodon spathula*, Cheng et al. 2020) and a lungfish genome (*Meyer et al. 2021*) could not be considered yet as all three genomes were unavailable upon submission of this paper.

Zhu *et al.* (2016) identified 11 *TLRs* in liver, spleen and kidney transcriptomes (*TLR1-9*, *TLR13*, *TLR22*). Luo *et al.* (2018a) also found *TLR6* in the head kidney of *A. dabryanus*, additionally to *TLR1-2*, *TLR4-5*, *TLR8*, *TLR13*, *TLR21-22* and *TLR25*, of which *TLR1-2* and *TLR4-5* were upregulated by bacterial infection. In *P. spathula*, He *et al.* (2019) identified 26 *TLR* homologs, including duplicated genes and pseudogenes: *TLR1-5*, *TLR7-9*, *TLR13/22*, *TLR14/18*, *TLR21*, *TLR25*, *TR27* and one unknown *TLR* from the *TLR1* subfamily. Seven *TLRs* were in-depth characterized in *A. dabryanus*, that is *TLR1* and *TLR4* (Han *et al.* 2018), *TLR21-22* and *TLR25* (Qi *et al.* 2018), and *TLR2* and *TLR13* (Tang *et al.* 2020). *TLR4* of amniotes recognizes bacterial lipopolysaccharides (LPSs). Dysregulation of the *TLR4/MD-2* complex may lead to LPS-induced septic shock (Sepulcre *et al.* 2009; Loes *et al.* 2019). Teleosts were long suggested insensitive to LPS, turning them insensitive to this toxicity (Sepulcre *et al.* 2009). In fact, many teleost species lack *TLR4* and also the genes encoding its critical cofactors have not been identified in any actinopterygians (Sepulcre *et al.* 2009; Khan *et al.* 2019; Smith *et al.* 2019; Liu *et al.* 2020; Li *et al.* 2020). However, a pre-print by Loes *et al.* (2019) claims the identification of a single copy of an orthologous *MD-2* gene in *Danio rerio* macrophage-like cells, whereas *CD14* could not be detected. If confirmed, zebrafish exhibit an ancestral, low-sensitivity *TLR4/MD-2* complex acting in parallel to other LPS sensing pathways. In Cladistia, He *et al.* (2019) identified *CD14* in bichir (*Polypterus senegalus*) as well as the downstream adapter *TRIF* used by *TLR3* and *TLR4* but did not find any *MD-2* and *TLR4* genes. The elephant shark (*C. milii*) genome lacks *CD14* and *MD-2* homologs (Table 1); *TLR4* is a pseudogene with many stop codons (Kagan 2017; He *et al.* 2019), similar as in Holostei (gars, *Atractosteus spatula*, *Lepisosteus oculatus* and bowfin, *Amia calva*). In Acipenseriformes (*P. spathula*), two *TLR4* genes and one pseudogene (stop codons) were detected. Further homologs of the LPS sensing pathway via *TLR4* could not be found in *P. spathula* (He *et al.* 2019). However, the opinion that Chondrichthyes and Actinopterygii are LPS-insensitive should be reconsidered. *In vivo* stimulation of sharks (*Scyliorhinus canicula*) led to an upregulation of *IL-1β* (interleukin-1β; Smith *et al.* 2014). In teleosts, *TLR5* might be a potent LPS recognition receptor, as shown by *A. hydrophila* and *Escherichia coli* stimulations (Li *et al.* 2020). In *A. dabryanus*, qPCR revealed upregulation of *TLR1-2*, *TLR4*, *TLR13*, *TRL21-22* and *TLR25* in head kidney leucocytes upon LPS stimulation, suggesting also other TLRs may be involved in LPS recognition (Han *et al.* 2018; Qi *et al.* 2018; Tang *et al.* 2020), although, except for *TLR4*, recognition of other TLRs has not been shown in Teleostei or Acipenseriformes. Vertebrate *TLR5* recognizes bacterial flagellin, triggering the MyD88-dependent signalling
pathway, which activates nuclear factor κB (NF-κB) to stimulate transcription of pro-inflammatory genes, such as interleukin 6 (IL-6) and tumour necrosis factor-α (TNF-α; Pietretti & Wiegertjes 2014; Hwang et al. 2010). Some teleosts (Ictalurus punctatus, Oncorhynchus mykiss, Paralichthys olivaceus) have a flagellin-sensitive, membrane-located TLR5 and a soluble TLR5S in functional interaction, where TLR5S is an amplifier for TLR5-mediated cellular responses in positive feedback (Pietretti & Wiegertjes 2014). He et al. (2019) identified TLR5 in Holostei (gars, A. spatula, L. oculatus and bowfin, A. calva), Cladistia (bichir, P. senegalus) and Chondrostei (P. spathula), but not in Chondrichthyes (elephant shark, C. milii). Representatives of Polypteryiformes (Cladistia) and Acipenseriformes (Chondrostei) showed an additional TLR5, suggesting TLR5S, but whether both forms interact, as demonstrated in teleosts, requires functional studies.

Retinoic acid-inducible gene-I-like receptors or RIG-I-like Receptors (RLRs). These present RNA and key sensors of virus infections in most cell types. Activation leads to the transcription of type I interferons (IFNs) and additional genes, provoking an antiviral response (Rehwinkel & Gack 2020). In mammals, the RLR family contains three members (Chen et al. 2017): RIG-I, MDA5 and LGP2, sharing the structure to detect immunostimulatory RNAs. Additionally, two caspase activation and recruitment domains (CARDs) are found in RIG-I and MDA5, mediating downstream signal transduction. LGP2 lacks these CARD domains and regulates RIG-I and MDA5 rather than detecting viruses (Zhu et al. 2013; Rehwinkel & Gack 2020). After activation, CARD domains of RIG-I or MDA5 interact with that of MAVS, transmitting the signal to TBK1 and IκB kinase-ε (IKKε), mediating the activation of IRF3, IRF7 and NF-κB, which in turn induce the expression of type I IFNs, other cytokines and IFN-stimulated genes (reviewed in Rehwinkel & Gack 2020, and Eisenächer & Krug 2012). Orthologous genes of mammalian RIG-I, MDA5 and LGP2 have been found in several teleosts (Table 1): MDA5 and LGP2 in Cypriniformes, Siluriformes, Salmoniformes and some Acanthopterygii, in which RIG-I seems absent (Chen et al. 2017). In Holostei, two RIG-I homologs were identified in gars (Atractosteus spatula, L. oculatus), while only one gene copy occurs in bowfin (Amia calva; He et al. 2019). Single copies of LGP2, MDA5 and MAVS homologs were found in all investigated Holostei. Bichirs (Cladistia, Polypterus senegalus) lack RIG-I and MAVS but possess one copy of MDA5 and LGP2. The acipenseriform P. spathula, like gars, has two gene copies of RIG-I and one copy each of LGP2, MDA5 and MAVS. A transcriptome from octoploid Acipenser sinensis revealed homologs of all three RLR members with two potential RIG-I genes (RIG-I-1/2) and several genes of downstream

components such as MAVS, TBK1, IKKε, IRF3/7 and TRAF2/3/6 (Zhu et al. 2016). Structural similarity to double-stranded RNA of some viruses makes poly I:C (polyinosinic:polycytidylic acid) an immunostimulant to study antiviral responses. Injection with poly I:C upregulated RIG-I-2 in spleen, head kidney and gill transcriptomes of hybrid sturgeons (bester: Huso huso x Acipenser ruthenus; Mugue et al. 2019). Likewise, bacterial stimulation with Aeromonas hydrophila activated the RLR pathway in A. dabryanus head kidney (Luo et al. 2018a). The teleost (Danio rerio) RNF135 gene enhances poly I:C-induced expression of RIG-I, suggesting that zbRNF135 might be a modulator (Lai et al. 2019). Expression patterns and function of RNF114 in innate immune response through regulation of RIG-I was also reported in A. sinensis (Liao et al. 2014), in accordance with studies of human RNF114, which modulates RIG-I/MDA5 signalling through a positive feedback loop, enhancing dsRNA-induced production of type I IFNs. Even though teleosts and sturgeon data suggest conserved RLR pathways (Chen et al. 2017; He et al. 2019), detailed regulatory mechanisms require further investigation.

Nucleotide-binding oligomerization domain-like or NOD-like receptors (NLRs). NLRs are cytosolic PRRs that mediate the initial innate immune response to cellular injury and stress through detection of a wide range of pathogen- and damage-associated molecular patterns (Platnich & Muruve 2019). Mammalian NLRs comprise five subfamilies (NLRA, NLRB, NLRC, NLRP and NLRx) all sharing two structures: a central nucleotide-binding domain (NBD) within the larger NACHT domain and C-terminal LRR domains (Ting et al. 2008; Platnich & Muruve 2019). Some NLRs form multi-protein complexes (inflammasomes), responsible for maturation and secretion of pro-inflammatory cytokines, others possess inflammasome-independent functions, but their specific functions remain poorly known (Laing et al. 2008; Platnich & Muruve 2019). The Danio rerio-NLR orthologs were categorized into three subfamilies: NLRA (like mammalian NOD1-5, mostly NLRC-subfamily, five members), NLRB (like mammalian NLRPs, six members) and NLRC (a large subfamily containing several hundred NLRC genes), supposedly unique to teleosts (Laing et al. 2008). He et al. (2019) identified NLRA, NLRB, NLRC1-4 and NLRx1 in Holostei (gars, A. spatula, L. oculatus and bowfin, A. calva) and Cladistia (bichir, P. senegalus), with the exception of NLRC2 that is unknown in the spotted gar (L. oculatus). In the NLRP family, NLRP3 was only found in bichir (Holostei, P. senegalus) with high copy numbers, and NLRP6 only in gars (Holostei, A. spatula, L. oculatus), but no NLRP gene is known from bowfin (Holostei, A. calva). One NLRP12 copy was found in bichir (Holostei, P. senegalus). In Acipenseriformes (P. spathula), NLRA,
NLRB, NLRC1-4, NLRx1 and TLRP12 homologs were detected. In an A. sinensis transcriptome, 12 NLR homologs were identified (NLRA, NLRB, NLRC1-5, NLRx1, NLRP1/3/6) and several genes of downstream signalling molecules (Zhu et al. 2016). Transcriptomics of A. schrenckii spleen revealed an upregulation of NLRP3 and caspase-1, while other NLR genes were down-regulated (Li et al. 2017a).

### Cytokines

Cytokines, such as interferons (IFNs), interleukins (ILs) and tumour necrosis factors (TNFs), mediate immune cell homeostasis and coordinate innate and adaptive immune responses. They are produced and secreted by immune-related cells after pathogen recognition via PRRs (Murphy & Weaver 2016). TLRs and RLRs are directly associated with the activation of the IFN system (Langevin et al. 2013). In mammals, three IFN family types (I-III) are known, only types I and II are known in sharks, sturgeon and teleosts (Langevin et al. 2013; Secombes & Zou 2017; Xu et al. 2019). Types I and III may be specialized antiviral IFNs, while type II acts as a regulatory and antibacterial cytokine (Langevin et al. 2013), as also shown in sturgeon (Xu et al. 2019). This study revealed that LPS-induced mRNA expression type II IFNs in A. dabryanus was higher than for type I after poly I:C stimulation. Although in sharks, sturgeon and teleosts a more complex receptor system is suggested than in mammals (Secombes & Zou 2017; Luo et al. 2018b), all signalling pathways, downstream of IFNRs (interferon receptors) appear to be evolutionarily conserved between all these groups (Langevin et al. 2013). Regulators, such as IRFs (interferon regulating factors) and TRIM (tripartite motif-containing) proteins are also described for these taxa (Ozato et al. 2008; Secombes & Zou 2017; Li et al. 2017c; Li et al. 2019).

### Complement system

The complement system provides protection early in infection through a tightly regulated network of more than 30 proteins in blood or other body fluids. Its activation leads to the opsonization of pathogens, coating them with antibodies and complement proteins, and removal by phagocytes and cell lysis (Sarma & Ward 2011; Zhu et al. 2013; Murphy & Weaver 2016). Three pathways of complement activation comprise alternative (pathogen-triggered), classical (antibody-triggered) and lectin pathways (lectin-type protein-triggered). All of them generate a C3 convertase that cleaves C3, leaving C3b to the pathogen surface and releasing C3a (Murphy & Weaver 2016). The complement system plays essential roles in adaptive immunity and immunologic memory (Sarma & Ward 2011; Zhu et al. 2013). Teleosts have fully developed classical and alternative pathways, plus some evidence of the lectin pathway. C3 levels, as an important marker for innate immunity, are

often measured in experimental and developmental studies. In octoploid A. baerii, C3 was most concentrated in unfertilized eggs and two-month juveniles, decreasing towards four months and older (Valipour et al. 2018); similarly, C3 may be produced by juveniles, as shown in Salmo salar (Løvoll et al. 2007). In the tetraploid A. dabryanus head kidney, 163 unigenes of the KEGG complement and coagulation pathway were identified upon bacterial Aeromonas hydrophila infection (Luo et al. 2018a). Compared to that, in the octoploid A. schrenckii spleen, 490 unigenes of this KEGG pathway were identified after Y. ruckeri stimulation (Li et al. 2017b).

### Antimicrobial peptides (AMPs) and lysozymes

AMPs and lysozymes are bactericidal agents that are produced or released by phagocytes after microorganism intake. They act as PRRs and effector molecules at the same time (Murphy & Weaver 2016). AMPs comprise agents that directly lyse bacterial cell membranes and are secreted by epithelial cells into fluids of mucosal surfaces, and by phagocytes in tissues. Important mammalian AMPs are the defensins, cathelicidins and histatins (Murphy & Weaver 2016). Shamova et al. (2014) isolated and characterized six novel AMPs, called acipensins (Ac1-Ac6), from leucocytes of the octoploid Acipenser gueldenstaedtii, histone derivatives with a broad spectrum of antibacterial activity. In A. dabryanus, three novel AMPs (cathelicidin, hepcidin and defensin) were identified by transcriptome analysis and tested by infection with the gram-negative Edwardsiella tarda (Chen et al. 2019a).

Lysozymes are glycosidases that cleave the β-linkage between N-acetylglucosamine and its acid in the bacterial peptidoglycan cell wall. Of the three main types of lysozymes, only the c- and g-types occur in chordates, the i-type is restricted to invertebrates (Callewaert & Michiels 2010). Zhang et al. (2018) first identified and characterized c- and g-type lysozymes, previously only known from teleosts (Callewaert & Michiels 2010), in Acipenseriformes, specifically in A. dabryanus head kidney transcriptomes. After stimulation of A. dabryanus with Aeromonas hydrophila, both enzymes suggested different functions by different expression levels and time courses in identical tissues (Zhang et al. 2018).

### The adaptive immune system of sturgeons

#### B cells and immunoglobulins (Igs)

Igs are crucial molecules of the adaptive immune system and explicitly expressed by B cells in two forms, that is membrane-bound Igs that are also known as B-cell receptors (BCRs), as well as antibodies that are secreted by differentiated B cells (plasma cells) into the blood plasma (Zhu et al. 2013; Murphy & Weaver 2016). Ig molecules
consist of heavy (IgH) and light (IgL) chains, each of which
possesses an antigen-specific variable (V) and a constant
(C) region. The exons of the variable regions are assembled
by RAG-mediated somatic gene rearrangement from a clus-
ter of numerous V, (D) and J segments during B-cell devel-
opment (Bengtén & Wilson 2015), a process that first
appeared in Chondrichthyes (Smith *et al.* 2014). Different
isotypes of IgH and IgL chains have evolved during phy-
logeny. Mammals possess five IgH isotypes, that is IgM,
IgD, IgG, IgA and IgE. Teleosts express IgM, IgD and IgT/Z
(Zhu *et al.* 2013; Zhu *et al.* 2014). Sharks possess three
types, IgM, IgW and IgNAR (Smith *et al.* 2014), whereas in
sturgeon IgM and IgD (Zhu *et al.* 2014) as well as a putative
IgW homolog were described (Zhu *et al.* 2016). Phyloge-
netic analysis of IgL isotypes by Guselnikov *et al.* (2018)
showed the subdivision into five major groups (κ, λ, λ-2,
σ, σ-2), with Acipenseriformes having three, Holostei four
and Cladistia at least four isotypes. In *A. ruthenus*, the sple-
nic κ transcripts comprised 99 % of IgL transcripts, sug-
gesting λ and σ playing minor roles in sturgeon immunity.
In contrast to Teleostei, natural antibodies of Chon-
drichthyes and Acipenseriformes are found to bind to a
broad range of antigens (Gonzalez *et al.* 1988). The anti-
gen-binding capacity of bester (*A. ruthenus x H. huso*) anti-
bodies was higher compared with that of teleosts and
increased with age (Yasumoto *et al.* 2020). Sturgeons also
produced specific antibodies without immunization (Yasu-
moto *et al.* 2020).

The MHC and antigen presentation

Major histocompatibility complex in jawed vertebrate
(Gnathostoma) genomes is a well-known linked set of poly-
morphic genes, which encode cell-surface glycoproteins,
essential for the adaptive immune system. MHC molecules
present peptides to T-lymphocytes and stimulate several
immune responses, such as the activation of macrophages
to kill bacteria or B cells to produce antibodies. They are
divided into two functionally and structurally different sub-
families, MHC class I (MHC I) and MHC class II (MHC II;
Murphy & Weaver 2016). MHC I molecules are expressed
on all nucleated somatic cells and present endogenous anti-
gens to CD8+ T cells (cytotoxic T-lymphocytes). They are
heterodimers with a heavy chain (α-chain), encoded by
class I genes, and β2-microtubulin, encoded outside the
MHC complex. MHC II are expressed only on certain cells,
such as B-lymphocytes or macrophages, and present extra-
cellular antigens to CD4+ T cells (helper T-lymphocytes).
They are also heterodimers that consist of an α-chain and
β-chain, which are encoded by class II A and B genes
(Wang et al. 2010; Li et al. 2017b). Wang et al. (2010) char-
acterized six and 11 MHC I gene sequences in P. spathula
and A. sinensis, which show high polymorphism and posi-
tive selection. Li et al. (2017b) characterized MHC II α-, β-,

and γ-chain from molecular features in *A. sinensis*. Expression analysis upon stimulation with poly I:C and with the inactivated Gram-negative bacterium *Vibrio anguillarum* revealed a wide tissue distribution, as *MHC II* α, β and γ genes were expressed in brain, heart, intestine, head kidney, liver, muscle and spleen, with highest expression levels in spleen followed by intestine and head kidney.

T cells and the adaptive cellular response

Upon antigen presentation by MHC I or MHC II, T cells
recognize antigens via membrane-bound T-cell receptors
(TCRs), which are related to the Igs both in their protein
structure and in the genetic mechanism of V(D)J recombi-
nation (Murphy & Weaver 2016). Aforementioned CD8+
cytotoxic T cells (Tc) interact with MHC I, whereas CD4+
helper T cells (Th) interact with MHC II. There are two
types of TCRs, that is a heterodimer of α- and β-polypep-
tide chains (αβ-TCR) or of γ- and δ-chains (γδ-TCR), each
having only one antigen-binding site compared with B-cell
receptors (Smith et al. 2019). All four TCR loci (α-δ) are
present in Chondrichthyes, Teleostei and also in Acipenser-
iformes (Castro et al. 2011; Smith et al. 2014; Zhu et al.
2016; Smith et al. 2019). When activated, CD4+ T cells
secrete various cytokines, like the B-cell growth factors IL-
4, IL-5, IL-9 and IL-13, while CD8+ T cells release mainly
cytotoxins to induce apoptosis of the target cell (Murphy &
Weaver 2016). Wang et al. (2019) characterized sturgeon
(A. baerii) IL-8, which is a small cytokine produced by
many cell types, such as monocytes, macrophages and
epithelial cells, and is involved in local inflammatory
response by attraction of neutrophils, basophils and naive
T cells to the site of infection (Murphy & Weaver 2016).
Sturgeon *IL-8* was ubiquitously expressed in all examined
organs with the highest level in the liver, where it was sig-
nificantly upregulated 12 h post-*A. hydrophila* injection.
Intraperitoneal injection of IL-8 led to an upregulation of
*IL-6*, *IgM* and *MCHIIβ* in the spleen of *A. baerii*.

New data on the evolutionary fate of 100 key immune
genes following the sturgeon-specific genome duplication

Number of orthologous genes

Out of the 100 chosen key immune genes, 84 orthologous
genes were identified in the sterlet genome (Table S1). For
16 genes, no orthologs were found (C1S, CSF2, IL2, IL3,
IL4, IL5, MAPK3, MAPK11, HLA-A, HLA-B, HLA-C, HLA-
E, HLA-F, HLA-G, HLA-DMA and HLA-DMB). For most
genes, this indicates a specific loss in the sturgeon lineage,
while in some other cases, these genes are specific to the
tetrapod/mammalian clade.

Despite the sturgeon WGD (Ars3R), 12 genes were
reverted to single copy per genome, and we did not find
their traces on the paralogous genome region, suggesting
they were degenerated or deleted after the Ars3R. Another 45 genes retained two paralogs. In most cases, these genes are located in the respective regions of paralogous chromosomes, resulting from the Ars3R, although in three cases (IKBKG, MAPK12 and STING1), two copies are tandemly arranged at one of the paralogous chromosomes and absent on the other. Furthermore, 20 genes were found to have more than two copies due to additional gene amplification.

### Gene expression patterns

Thirteen genes with identified paralogs showed considerable differential expression between both copies in all studied tissues, with one dominating paralog demonstrating higher expression in one or several tissues (Fig. 2). In some cases (e.g. CHUK, conserved helix-loop-helix ubiquitous kinase), this is accompanied by the fact that the lower expressed paralog is truncated. Interestingly, this group of

genes is mostly expressed in one to several tissues only (e.g. IL6, IL11 and RAG1 were detected in a single tissue only). All of this suggests expression diversification and tissue-specific specialisation.

Both paralogs of three genes (NFKBIA, PIK3R2 and JAK1) showed expression in all tissues studied, with almost no difference between expression levels of paralogs. Both paralogs of TNF were expressed at similar levels only in the spleen.

For 23 genes, one paralog was slightly higher expressed in some tissues, while the second paralog in the other tissues, suggesting sub-functionalization. Both paralogs of some genes retained high expression profiles (e.g. C6). However, in tissues with reduced transcription, the difference was noticeable, where the C6_1 paralog was higher transcribed in liver and skin, and C6_2 in muscle and ovary.

<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /><figcaption>**Figure 2** (a, b) Heat map of FPKM (fragments per kilobase of exon per million fragments mapped) distributions for paralogous genes in sterlet, Acipenser ruthenus in nine different organs. FPKM values for each gene were transformed by plotting lg (1 + FPKM) and building the heat map using MATLAB 9.8.0. Each column in the subfigures a and b represents an organ's tissue, and each row a gene. The colours in the graph show the levels of gene expression, ranging from dark red, indicating that a gene is highly expressed in an organ, to dark blue, showing low expression.</figcaption></figure>
For nine genes (FGA, MBL, F2, MASP, CD8A, IL7, PIK3R1, IKBKE and PIK3R3), the signal of sub-functional-
ization is much more prominent. For example, one paralog
of FGA (fibrinogen alpha chain) revealed an almost 50x
higher expression in liver, while the second paralog had 8x
higher expression in kidney and also 3-5x higher expression
in all other studied tissues (brain, muscle, skin, spleen,
gonads). One paralog of MBL2 (mannose-binding lectin 2)
was highly expressed in undifferentiated gonads, while the
second paralog was active in the kidney.

Paralogs of four genes (IL10, MASP2, MAP2K1 and JAK3) were co-expressed in some tissues, with a trend of one paralog to dominate. For example, one paralog of JAK3 was differentially expressed in undifferentiated gonads, testis, liver and brain, while in kidney and spleen both paralogs were active. This latter group may indicate combined sub-functionalization accompanied with tissue specificity. We detected only very low expression of RAG2 in the kidney and no expression in all other tissues.

Taken together, our initial genomic examination of the
100 key immune genes shows a multitude of evolutionary
fates after the sturgeon-specific genome duplication. These
reach from single-copy maintenance, potentially due to ini-
tial functional conflicts between paralogs in key player posi-
tions, and many true duplications, followed by either tissue
specialization, sub-functionalization or both. All of this
might have fundamental effects on the acipenseriform
immune system, possibly contributing to evolutionary age,
slow molecular evolution and longevity of sturgeon.

Conclusions: A basis for future applications and
research perspectives

In this paper, we aimed at providing a review of basic
research data on sturgeon immunology. Applications to
sturgeon aquaculture, including aspects of probiotic treat-
ments (e.g. Song *et al.* 2014) and immunostimulation (e.g.
Dawood *et al.* 2018) have been reviewed previously. While
this also applies to vaccination (e.g. Khoshbavar-Rostami
*et al.* 2007; Chesneau 2018), we try to exemplify how our
review might be relevant for this field. In sturgeon breed-
ing, disease outbreaks are well documented (e.g. Kayiş
*et al.* 2017; Bigarré *et al.* 2017; Ciulli *et al.* 2020). While fre-
quent bacterial infections may require antibiotics if other
countermeasures fail, increasingly, viral diseases become an
issue (e.g. Waltzek *et al.* 2014; Mugetti *et al.* 2020). Vacci-
nation contributes to sustainable aquaculture by cutting
down on antibiotics (Pridgeon & Klesius 2012; Gudding
2014) and seems the only protection against severe viral
diseases. Biering and Salonius (2014) recommend stimula-
tion of cellular immunity by DNA vaccines, which mimic
viral infections better than common inactivated vaccines
and may improve protective immune responses.

Determination of DNA-vaccine efficiency requires knowl-
edge about construct design, transfection or cell transport
of the target organisms. Therefore, comprehensive research
on immune-signalling pathways and their induction is
highly relevant for vaccine development. Namely, as the
first components in contact with pathogen-associated
molecular patterns, PRRs and their ligands are of greatest
interest. Many established and experimental vaccines eval-
uated in humans and mice also contain ligands for TLRs
(Van Duin *et al.* 2006).

As discussed here for vaccination as an example, our
comprehensive synopsis with new genomic data will further
contribute to a better understanding of sturgeon and pad-
dlefish immunity and may lead to other future applications
in aquaculture practice, which are, however, beyond the
framework of this paper. Several published transcriptomic
data stem from sturgeon species that are so far without
high-quality, well-annotated genomes. Especially in the
highly polyploid genomes of some species of Acipenseris-
formes (Du *et al.* 2020), this remains an important future
research and application challenge.

Indeed, our comprehensive research update on innate and
adaptive immunology in sturgeon and paddlefish highlights
new findings, for example on PRRs and antibodies. Basic struc-
tures of the acipenseriform immunity, including morphology
and ontogeny of immune organs, haematopoiesis and immune
responses to various stimuli and stressors, are relatively well-
studied. However, many mechanistic and molecular details of
sturgeon immunity remain unknown. Future research should
focus on functional studies of key players of the innate and
adaptive immune system. Here, the interlinkage of the repro-
ductive and the immune systems during maturation will be
important to analyse as well as the ontogeny and timing of
innate and onset of adaptive immune system, which translate
into vulnerability of larvae and juveniles. Immune genes in dif-
ferent organs, especially the role of the thymus, have not at all
been studied in Acipenseriformes. Ontogenetic fates of innate
and adaptive immune markers should be in focus of future
research. One hundred key immune genes exemplify a multi-
tude of evolutionary trajectories after the sturgeon genome
duplication, where some single-copy genes contrast with many
duplications, allowing tissue specialization, sub-functionaliza-
tion or both. This preliminary conclusion should be tested by
future evolutionary bioinformatics involving all the >1000
KEGG immunity genes.

Acknowledgements

This study has been funded through COFASP/ERANET (STURGEONOMICS) by the German Federal Ministry of Food and Agriculture through the Federal Office for Agriculture and Food (Grant No. 2816ERA04G) to M.S. and S.W.
V.T. and D.P. were funded by the Russian Science Foun-
dation, Project No. 18-44-04007.

We thank the Leibniz-Institute of Freshwater Ecology and Inland Fisheries (IGB) for supporting open access publication of this paper. Open Access funding enabled and organized by ProjektDEAL.

Data availability statement

Transcriptomic data for all tissues analysed are accessible at
GenBank (NCBI): SRR13376048, SRR11013452 to
SRR11013458 and SRR13378073 to SRR13378074 from Du
et al. (2020); SRR13009649: de novo RNA-seq.

References

Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990)
Basic local alignment search tool. *Journal of Molecular Biology*
**215**: 403–410.

Bengtén E, Wilson M (2015) Antibody repertoires in fish. *Results and Problems in Cell Differentiation* **57**: 193–234.

Beutler B (2004) Innate immunity: an overview. *Molecular Immunology* **40**: 845–859.

Biering E, Salonius K (2014) DNA vaccines. In: Gudding R, Lillehaug A, Evensen Ø (eds) *Fish Vaccination*, pp. 47–55. John Wiley & Sons, West Sussex.

Bigarré L, Lesne M, Lautraite A, Chesneau V, Leroux A, Jamin M *et al.* (2017) Molecular identification of iridoviruses infecting various sturgeon species in Europe. *Journal of Fish Diseases* **40**: 105–118.

Bodey B, Bodey B Jr, Siegel SE, Kaiser HE (2000) Novel insights into the function of the thymic Hassall's bodies. *In vivo* (*Athens, Greece*) **14**: 407–418.

Boehm T (2011) Design principles of adaptive immune systems. *Nature Reviews Immunology* **11**: 307–317.

Boehm T, Hess I, Swann JB (2012a) Evolution of lymphoid tissues. *Trends in Immunology* **33**: 315–321.

Boehm T, Iwanami N, Hess I (2012b) Evolution of the immune system in the lower vertebrates. *Annual Review of Genomics and Human Genetics* **13**: 127–149.

Boehm T, Swann JB (2014) Origin and evolution of adaptive immunity. *Annual Review of Animal Biosciences* **2**: 259–283.

Bowden TJ, Cook P, Rombout JH (2005) Development and function of the thymus in teleosts. *Fish and Shellfish Immunology* **19**: 413–427.

Callewaert L, Michiels CW (2010) Lysozymes in the animal kingdom. *Journal of Biosciences* **35**: 127–160.

Castro R, Bernard D, Lefranc MP, Six A, Benmansour A, Boudi-
not P (2011) T cell diversity and TcR repertoires in teleost
fish. *Fish and Shellfish Immunology* **31**: 644–654.

Chen H, Wang B, Yu N, Qi J, Tang N, Wang S *et al.* (2019b) Transcriptome analysis and the effects of polyunsaturated fatty acids on the immune responses of the critically endan- gered Yangtze sturgeon (*Acipenser dabryanus*). *Fish and Shell- fish Immunology* **94**: 199–210.

Chen S, Zhou Y, Chen Y, Gu J (2018) fastp: an ultra-fast all-in-one FASTQ preprocessor. *Bioinformatics* **34**: 884–890.

Chen SN, Zou PF, Nie P (2017) Retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) in fish: current knowledge and future perspectives. *Immunology* **151**: 16–25.

Chen Y, Gong Q, Song M, Lai J, Sun J, Liu Y (2019a) Identifica-
tion and characterization of three novel antimicrobial pep-
tides from *Acipenser dabryanus*. *Fish and Shellfish Immunology*
**88**: 207–216.

Cheng P, Huang Y, Du H, Li C, Lv Y, Ruan R *et al.* (2019) Draft genome and complete Hox-cluster characterization of the sterlet (*Acipenser ruthenus*). *Frontiers in Genetics* **10**: 776.

Cheng P, Huang Y, Lv Y, Du H, Ruan Z, Li C *et al.* (2020) The American paddlefish genome provides novel insights into chromosomal evolution and bone mineralization in early vertebrates. *Molecular Biology and Evolution*. https://doi.org/10.1093/molbev/msaa326.

Chesneau V (2018) Immunology in sturgeons with a focus on the siberian sturgeon mechanisms, responses to stress and stimulation. In: Williot P, Nonnotte G, Chebanov M (eds) *The Siberian sturgeon (Acipenser baerii, Brandt, 1869) Vol. 2, Farming*, pp. 373–402. Springer, Cham.

Ciulli S, Volpe E, Sirri R, Tura G, Errani F, Zamperin G *et al.* (2020) Multifactorial causes of chronic mortality in juvenile sturgeon (*Huso huso*). *Animals* **10**: 1866.

Cooper MD, Alder MN (2006) The evolution of adaptive immune systems. *Cell* **124**: 815–822.

Dawood MAO, Koshio S, Esteban MA (2018) Beneficial roles of feed additives as immunostimulants in aquaculture: a review. *Reviews in Aquaculture* **10**: 950–974.

Dettlaff TA, Ginsburg AS, Schmalhausen OI (1993) *Sturgeon Fishes: Developmental Biology and Aquaculture*, 1st edn. Springer Science & Business Media, Berlin.

Du K, Stöck M, Kneitz S, Klopp C, Woltering JM, Adolfi MC *et al.* (2020) The sterlet sturgeon genome sequence and the mechanisms of segmental rediploidization. *Nature Ecology & Evolution* **4**: 841–852.

Eisenächer K, Krug A (2012) Regulation of RLR-mediated innate immune signaling – It is all about keeping the balance. *European Journal of Cell Biology* **91**: 36–47.

Emms DM, Kelly S (2019) OrthoFinder: phylogenetic orthol-
ogy inference for comparative genomics. *Genome Biology*
**20**: 238.

Fänge R (1986) Lymphoid organs in sturgeons (Acipenseridae). *Veterinary Immunology and Immunopathology* **12**: 153–161.

Fitzgerald KA, Kagan JC (2020) Toll-like Receptors and the Control of Immunity. *Cell* **180**: 1044–1066.

Foey A, Picchietti S (2014) Immune defences of teleost fish. In: Merrifield D, Ringø E (eds) *Aquaculture Nutrition: Gut Health, Probiotics and Prebiotics*, pp. 14–52. John Wiley & Sons, West Sussex.

Gallo VP, Civinini A (2003) Survey of the adrenal homolog in teleosts. *International Review of Cytology* **230**: 89–187.
Geven E, Klaren P (2017) The teleost head kidney: Integrating thyroid and immune signalling. *Developmental and Comparative Immunology* **66**: 73–83.

Gonzalez R, Charlemagne J, Mahana W, Avrameas S (1988) Specificity of natural serum antibodies present in phylogenetically distinct fish species. *Immunology* **63**: 31–36.

Gourbal B, Pinaud S, Beckers G, Van Der Meer J, Conrath U, Netea MG (2018) Innate immune memory: an evolutionary perspective. *Immunological Reviews* **283**: 21–40.

Gradil AM, Wright GM, Speare DJ, Wadowska DW, Purcell S, Fast MD (2014a) The effects of temperature and body size on immunological development and responsiveness in juvenile shortnose sturgeon (*Acipenser brevirostrum*). *Fish and Shellfish Immunology* **40**: 545–555.

Gradil AM, Wright GM, Wadowska DW, Fast MD (2014b) Ontogeny of the immune system in Acipenserid juveniles. *Developmental and Comparative Immunology* **44**: 303–314.

Gudding R (2014) Vaccination as a preventive measure. In: Gudding R, Lillehaug A, Evensen Ø (eds) *Fish Vaccination*, pp. 12–21. John Wiley & Sons, West Sussex.

Guselnikov SV, Baranov KO, Najakshin AM, Mechetina LV, Chikaev NA, Makunin AI *et al.* (2018) Diversity of immunoglobulin light chain genes in non-teleost ray-finned fish Uncovers IgL subdivision into five ancient isotypes. *Frontiers in Immunology* **9**: 1079.

Han P, Wang S, Zhang Q, Zhang S, Shao R, Xu W *et al.* (2018) Molecular characterization and expression analysis of TLR1 and TLR4 from the endangered fish Dabry's sturgeon (*Acipenser dabryanus*). *Developmental and comparative Immunology* **86**: 180–188.

Hari P, Millar FR, Tarrats N, Birch J, Quintanilla A, Rink CJ *et al.* (2019) The innate immune sensor Toll-like receptor 2 controls the senescence-associated secretory phenotype. *Science Advances* **5**: eaaw0254.

He Y, Pan H, Zhang G, He S (2019) Comparative study on pattern recognition receptors in non-teleost ray-finned fishes and their evolutionary significance in primitive vertebrates. *Science China. Life Sciences* **62**: 566–578.

Hodgkinson JW, Grayfer L, Belosevic M (2015) Biology of bony fish macrophages. *Biology* **4**: 881–906.

Hofmann J, Greter M, Du Pasquier L, Becher B (2010) B-cells need a proper house, whereas T-cells are happy in a cave: the dependence of lymphocytes on secondary lymphoid tissues during evolution. *Trends in Immunology* **31**: 144–153.

Hubbard T, Barker D, Birney E, Cameron G, Chen Y, Clark L *et al.* (2002) The Ensembl genome database project. *Nucleic Acids Research* **30**: 38–41.

Hughes LC, Ortí G, Huang Y, Sun Y, Baldwin CC, Thompson AW *et al.* (2018) Comprehensive phylogeny of ray-finned fishes (Actinopterygii) based on transcriptomic and genomic data. *Proceedings of the National Academy of Sciences of the United States of America* **115**: 6249–6254.

Hwang SD, Asahi T, Kondo H, Hirono I, Aoki T (2010) Molecular cloning and expression study on Toll-like receptor 5

paralogs in Japanese flounder, *Paralichthys olivaceus*. *Fish and Shellfish Immunology* **29**: 630–638.

Icardo JM, Colvee E, Cerra MC, Tota B (2002) The structure of the conus arteriosus of the sturgeon (*Acipenser naccarii*) heart: II. The myocardium, the subepicardium, and the conus-aorta transition. *The Anatomical Record* **268**: 388–398.

Iwasaki A, Medzhitov R (2015) Control of adaptive immunity by the innate immune system. *Nature Immunology* **16**: 343–353.

Kagan JC (2017) Lipopolysaccharide Detection across the Kingdoms of Life. *Trends in Immunology* **38**: 696–704.

Kawai T, Akira S (2010) The role of pattern-recognition receptors in innate immunity: update on Toll-like receptors. *Nature Immunology* **11**: 373–384.

Kayiş Ş, Er A, Kangel P, Kurtoğlu İZ (2017) Bacterial pathogens and health problems of *Acipenser gueldenstaedtii* and *Acipenser baeri* sturgeons reared in the eastern Black Sea region of Turkey. *Iranian Journal of Veterinary Research* **18**: 18.

Khan I, Maldonado E, Silva L, Almeida D, Johnson WE, O'Brien SJ *et al.* (2019) The vertebrate TLR supergene family evolved dynamically by gene gain/loss and positive selection revealing a host-pathogen arms race in birds. *Diversity* **11**: 131.

Khoshbavar-Rostami HA, Soltani M, Hassan HMD (2007) Immune responses of great sturgeon *Huso huso* to *Aeromonas hydrophila* bacterin. *Journal of Fish Biology* **70**: 1931–1938.

Kim D, Paggi JM, Park C, Bennett C, Salzberg SL (2019) Graph-based genome alignment and genotyping with HISAT2 and HISAT-genotype. *Nature Biotechnology* **37**: 907–915.

Kjærner-Semb E, Ayllon F, Furmanek T, Wennevik V, Dahle G, Niemelä E *et al.* (2016) Atlantic salmon populations reveal adaptive divergence of immune related genes - a duplicated genome under selection. *BMC Genomics* **17**: 610.

Krishnaswamy Gopalan T, Gururaj P, Gupta R, Gopal DR, Rajesh P, Chidambaram B *et al.* (2014) Transcriptome profiling reveals higher vertebrate orthologous of intra-cytoplasmic pattern recognition receptors in grey bamboo shark. *PLoS One* **9**: e100018.

Lai Y, Liang M, Hu L, Zeng Z, Lin H, Yi G *et al.* (2019) RNF135 is a positive regulator of IFN expression and involved in RIG-I signaling pathway by targeting RIG-I. *Fish and Shellfish Immunology* **86**: 474–479.

Laing KJ, Purcell MK, Winton JR, Hansen JD (2008) A genomic view of the NOD-like receptor family in teleost fish: identification of a novel NLR subfamily in zebrafish. *BMC Evolutionary Biology* **8**: 42.

Lange MA, Goviadinova AA, Khrushchov NG (2000) Issledovanie lokalizatsii krovetvornoï tkani u osetrovykh ryb [Localization of hematopoietic tissue in sturgeon]. *Ontogenez* **31**: 440–444.

Langevin C, Aleksejeva E, Passoni G, Palha N, Levraud JP, Bouzinot P (2013) The antiviral innate immune response in fish: evolution and conservation of the IFN system. *Journal of Molecular Biology* **425**: 4904–4920.
Leulier F, Lemaitre B (2008) Toll-like receptors - taking an evolutionary approach. *Nature Reviews. Genetics* **9**: 165-178.

Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N *et al.* (2009) The sequence alignment/map format and SAM-tools. *Bioinformatics* **25**: 2078-2079.

Li S, Zhang Y, Cao Y, Wang D, Liu H, Lu T (2017a) Transcriptome profiles of Amur sturgeon spleen in response to *Yersinia ruckeri* infection. *Fish and Shellfish Immunology* **70**: 451-460.

Li X, Du H, Liu L, You X, Wu M, Liao Z (2017b) MHC class II alpha, beta and MHC class II-associated invariant chains from Chinese sturgeon (*Acipenser sinensis*) and their response to immune stimulation. *Fish and Shellfish Immunology* **70**: 1-12.

Li Y, Xia P, Wu J, Huang A, Bu G, Meng F *et al.* (2020) The potential sensing molecules and signal cascades for protecting teleost fishes against lipopolysaccharide. *Fish and Shellfish Immunology* **97**: 235-247.

Li Y, Zhang S, Luo K, Xia L, Hu W, Tian G *et al.* (2017c) First study on interferon regulatory factor in sturgeon: expression pattern of interferon regulatory factor in Dabry's sturgeon *Acipenser dabryanus*. *Journal of Interferon and Cytokine Research* **37**: 503-512.

Li Y, Zhang S, Han P, Luo K, Hu W, Zhang W *et al.* (2019) Bioinformatic analysis of antivirus-related TRIM genes in Dabry's sturgeon *Acipenser dabryanus*. *Journal of Applied Ichthyology* **35**: 226-234.

Liao Z, Chen X, Nie D, Wang J, Wu M (2014) A RING finger protein 114 (RNF114) homolog from Chinese sturgeon (*Acipenser sinensis*) possesses immune-regulation properties via modulating RIG-I signaling pathway-mediated interferon expression. *Fish and Shellfish Immunology* **41**: 507-516.

Liu G, Zhang H, Zhao C, Zhang H (2020) Evolutionary history of the toll-like receptor gene family across vertebrates. *Genome Biology and Evolution* **12**: 3615-3634.

Liu Y, Chen Y, Gong Q, Lai J, Du J, Deng X (2017a) Paternity assignment in the polyploid *Acipenser dabryanus* based on a novel microsatellite marker system. *PLoS One* **12**: e0185280.

Liu Y, Xiao Q, Yang S, Zhao L, Fu H, Du J (2017b) Characterization of hematopoiesis in Dabry's sturgeon (*Acipenser dabryanus*). *Aquaculture and Fisheries* **2**: 262-268.

Loes AN, Hinman MN, Farnsworth DR, Miller AC, Guillememin K, Harms MJ (2019) Identification and characterization of zebrafish Tlr4 co-receptor Md-2. *BioRxiv*. https://doi.org/10.1101/817528.

Lovoll M, Johnsen H, Boshra H, Bogwald J, Sunyer JO, Dalmo RA (2007) The ontogeny and extrahepatic expression of complement factor C3 in Atlantic salmon (*Salmo salar*). *Fish and Shellfish Immunology* **23**: 542-552.

Luer CA, Walsh CJ, Bodine AB, Wyffels JT, Scott TR (1995) The elasmobranch thymus: anatomical, histological, and preliminary functional characterization. *Journal of Experimental Zoology* **273**: 342-354.

Luo K, Di J, Han P, Zhang S, Xia L, Tian G *et al.* (2018a) Transcriptome analysis of the critically endangered Dabry's sturgeon (*Acipenser dabryanus*) head kidney response to

*Aeromonas hydrophila*. *Fish and Shellfish Immunology* **83**: 249-261.

Luo K, Zhang S, Tang D, Xia L, Gao W, Tian G *et al.* (2018b) Analysis of the expression patterns of the cytokine receptor family B (CRFB) and interferon gamma receptor (IFNGR) in Dabry's sturgeon (*Acipenser dabryanus*). *Developmental and Comparative Immunology* **84**: 420-426.

Malech HL, DeLeo FR, Quinn MT (2020) The role of neutrophils in the immune system: an overview. In: Quinn M., DeLeo F. (eds) *Neutrophil. Methods in Molecular Biology*, vol. 2087, pp. 3-10, Humana, New York, NY.

Meštanová V, Varga I (2016) Morphological view on the evolution of the immunity and lymphoid organs of vertebrates, focused on thymus. *Biologia* **71**: 1080-1097.

Meyer A, Schloissnig S, Franchini P, Du K, Irisarri I *et al.* (2021) Giant lungfish genome elucidates the conquest of land by vertebrates. *Nature*. https://doi.org/10.1038/s41586-021-03198-8.

Mohammad M, Chilmonczyk S, Birch D, Aladaileh S, Raftos D, Joss J (2007) Anatomy and cytology of the thymus in juvenile Australian lungfish, *Neocertodus forsteri*. *Journal of Anatomy* **211**: 784-797.

Mugetti D, Pastorino P, Menconi V, Pedron C, Prearo M (2020) The old and the new on viral diseases in sturgeon. *Pathogens* **9**: 146.

Mugue N, Terekhanova N, Afanasyev S, Krasnov A (2019) Transcriptome sequencing of hybrid bester sturgeon: responses to poly (I:C) in the context of comparative immunogenomics. *Fish and Shellfish Immunology* **93**: 888-894.

Murphy K, Weaver C (2016) *Janeway's Immunobiology*, 9th edn. Garland Science, Taylor & Francis Group, New York, NY.

Netea MG, Schlitzer A, Placek K, Joosten L, Schultze JL (2019) Innate and adaptive immune memory: an evolutionary continuum in the host's response to pathogens. *Cell host and Microbe* **25**: 13-26.

Neuheimer AB, Taggart CT (2007) The growing degree-day and fish size-at-age: the overlooked metric. *Canadian Journal of Fisheries and Aquatic Sciences* **64**: 375-385.

Ozato K, Shin DM, Chang TH, Morse HC 3rd (2008) TRIM family proteins and their emerging roles in innate immunity. *Nature reviews. Immunology* **8**: 849-860.

Palić D, Beck LS, Palić J, Andreasen CB (2011) Use of rapid cytochemical staining to characterize fish blood granulocytes in species of special concern and determine potential for function testing. *Fish and Shellfish Immunology* **30**: 646-652.

Pastoret PP, Griebel P, Bazin H, Govaerts A (1998) *Handbook of vertebrate immunology*, 1st edn. Academic Press, London.

Peng Z, Ludwig A, Wang D, Diogo R, Wei Q, He S (2007) Age and biogeography of major clades in sturgeons and paddlefishes (Pisces: Acipenseriformes). *Molecular Phylogenetics and Evolution* **42**: 854-862.

Pertea M, Pertea GM, Antonescu CM, Chang TC, Mendell JT, Salzberg SL (2015) StringTie enables improved reconstruction of a transcriptome from RNA-seq reads. *Nature Biotechnology* **33**: 290-295.
Petrie-Hanson L, Peterman AE (2005) American paddlefish leukocytes demonstrate mammalian-like cytochemical staining characteristics in lymphoid tissues. *Journal of Fish Biology* **66**: 1101–1115.

Pietretti D, Wiegertjes GF (2014) Ligand specificities of Toll-like receptors in fish: indications from infection studies. *Developmental and Comparative Immunology* **43**: 205–222.

Platnich JM, Muruve DA (2019) NOD-like receptors and inflammasomes: a review of their canonical and non-canonical signaling pathways. *Archives Biochemistry Biophysics* **670**: 4–14.

Płtycz B (2008) Convergent evolution of acquired immunity. *Central European Journal of Immunology* **33**: 83–86.

Pridgeon JW, Klesius PH (2012) Major bacterial diseases in aquaculture and their vaccine development. *CAB Reviews* **7**: 1.

Qi Z, Wang S, Zhu X, Yang Y, Han P, Zhang Q *et al.* (2018) Molecular characterization of three toll-like receptors (TLR21, TLR22, and TLR25) from a primitive ray-finned fish Dabry's sturgeon (*Acipenser dabryanus*). *Fish and Shellfish Immunology* **82**: 200–211.

Qi Z, Wu P, Zhang Q, Wei Y, Wang Z, Qiu M *et al.* (2016) Transcriptome analysis of soiny mullet (*Liza haematocheila*) spleen in response to *Streptococcus dysgalactiae*. *Fish and Shellfish Immunology* **49**: 194–204.

Quinlan AR, Hall IM (2010) BEDTools: a flexible suite of utilities for comparing genomic features. *Bioinformatics* **26**: 841–842.

Rajkov J, Shao Z, Berrebi P (2014) Evolution of polyploidy and functional diploidization in sturgeons: microsatellite analysis in 10 sturgeon species. *Journal of Heredity* **105**: 521–531.

Rehwinkel J, Gack MU (2020) RIG-I-like receptors: their regulation and roles in RNA sensing. *Nature Reviews Immunology* **20**: 537–551.

Roach JC, Glusman G, Rowen L, Kaur A, Purcell MK, Smith KD *et al.* (2005) The evolution of vertebrate Toll-like receptors. *Proceedings of the National Academy of Sciences* **102**: 9577–9582.

Rolff J (2007) Why did the acquired immune system of vertebrates evolve? *Developmental & Comparative Immunology* **31**: 476–482.

Rombout JH, Yang G, Kiron V (2014) Adaptive immune responses at mucosal surfaces of teleost fish. *Fish and Shellfish Immunology* **40**: 634–643.

Ruddle NH, Akirav EM (2009) Secondary lymphoid organs: responding to genetic and environmental cues in ontogeny and the immune response. *The Journal of Immunology* **183**: 2205–2212.

Salinas I (2015) The mucosal immune system of teleost fish. *Biology* **4**: 525–539.

Salkova E, Flajshans M (2016) The first finding of Hassall's corpuscles in the thymi of cultured sturgeons. *Veterinarní Medicina* **61**: 464–466.

Salkova E, Flajshans M, Steinbach C (2020) Immunohistochemical mapping of thymic microenvironment in sterlet (*Acipenser ruthenus*). *Veterinární Medicína* **65**: 301–308.

Sarma JV, Ward PA (2011) The complement system. *Cell and Tissue Research* **343**: 227–235.

Secombes CJ, Zou J (2017) Evolution of interferons and interferon receptors. *Frontiers in Immunology* **8**: 209.

Sepulcre MP, Alcaraz-Pérez F, López-Muñoz A, Roca FJ, Meseguer J, Cayuela ML *et al.* (2009) Evolution of lipopolysaccharide (LPS) recognition and signaling: fish TLR4 does not recognize LPS and negatively regulates NF-kappaB activation. *Journal of Immunology* **182**: 1836–1845.

Shamova OV, Orlov DS, Balandin SV, Shramova EI, Tsvetkova EV, Panteleev PV *et al.* (2014) Acipensins novel antimicrobial peptides from leukocytes of the Russian sturgeon *Acipenser gueldenstaedtii*. *Acta Naturae* **6**: 23.

Shanley DP, Aw D, Manley NR, Palmer DB (2009) An evolutionary perspective on the mechanisms of immunosenescence. *Trends in Immunology* **30**: 374–381.

Smith NC, Rise ML, Christian SL (2019) A comparison of the innate and adaptive immune systems in cartilaginous fish ray-finned fish and lobe-finned fish. *Frontiers in Immunology* **10**: 2292.

Smith SL, Sim RB, Flajnik MF (eds) (2014) *Immunobiology of the Shark*, 1st edn. CRC Press Taylor and Francis, Boca Raton.

Song SK, Beck BR, Kim D, Park J, Kim J, Kim HD *et al.* (2014) Prebiotics as immunostimulants in aquaculture: a review. *Fish and Shellfish Immunology* **40**: 40–48.

Swann JB, Holland SJ, Petersen M, Pietsch TW, Boehm T (2020) The immunogenetics of sexual parasitism. *Science* **369**: 1608–1615.

Tang D, Deng D, Zhang S, Yuan H, Wenbing Z, Xu Q *et al.* (2019) Similarity analysis of the immune-related genes from Chinese Sturgeon (*Acipenser sinensis*) and Dabrys sturgeon (*Acipenser dabryanus*). *Turkish Journal of Fisheries and Aquatic Sciences* **19**: 865–871.

Tang R, Wang S, Han P, Zhang Q, Zhang S (2020) Toll-like receptor (TLR) 2 and TLR13 from the endangered primitive-ray finned fish Dabry's sturgeon (*Acipenser dabryanus*) and their expression profiling upon immune stimulation. *Aquaculture Reports* **16**: 100247.

Thompson A, Hawkins M, Parey E, Wcisel D, Ota T, Kawasaki K *et al.* (2020) The genome of the bowfin (*Amia calva*) illuminates the developmental evolution of ray-finned fish. https://doi.org/10.21203/rs.3.rs-92055/v1

Ting JP, Lovering RC, Alnemri ES, Bertin J, Boss JM, Davis BK *et al.* (2008) The NLR gene family: a standard nomenclature. *Immunity* **28**: 285–287.

Tort L, Balasch JC, Mackenzie S (2003) Fish immune system A crossroads between innate and adaptive responses. *Immunología* **22**: 277–286.

Uribe C, Folch H, Enríquez R, Moran G (2011) Innate and adaptive immunity in teleost fish: a review. *Veterinarní Medicina* **56**: 486–503.

Valipour A, Heidari B, Hadavi M, Yousefi A (2018) Changes in immune parameters (lysozyme IgM C3) in early life stages and broodstock of Siberian sturgeon *Acipenser baerii*. *Fisheries and Aquatic Life* **26**: 21–30.
Van Duin D, Medzhitov R, Shaw AC (2006) Triggering TLR signaling in vaccination. *Trends in Immunology* **27**: 49–55.

Waltzek TB, Miller DL, Gray MJ, Drecktrah B, Briggsler JT, MacConnell B *et al*. (2014) New disease records for hatchery-reared sturgeon *I* Expansion of frog virus 3 host range into *S* *caphirhynchus albus*. *Diseases of Aquatic Organisms* **111**: 219–227.

Wang D, Zhong L, Wei Q, Gan X, He S (2010) Evolution of MHC class I genes in two ancient fish paddlefish (*Polyodon spathula*) and Chinese sturgeon (*Acipenser sinensis*). *FEBS Letters* **584**: 3331–3339.

Wang X, Ma G, Zhang R, Liu L, Zhu J, Zhu H (2019) Molecular characterization and biological functioning of interleukin-8 in Siberian sturgeon (*Acipenser baeri*). *Fish and Shellfish Immunology* **90**: 91–101.

Wendelaar Bonga SE (1997) The stress response in fish. *Physiological Reviews* **77**: 591–625.

Xu Q, Luo K, Zhang S, Gao W, Zhang W, Wei Q (2019) Sequence analysis and characterization of type I interferon and type II interferon from the critically endangered sturgeon species *A* *dabryanus* and *A*. *sinensis*. *Fish and Shellfish Immunology* **84**: 390–403.

Yasumoto K, Koiwai K, Hiraoka K, Hirono I, Kondo H (2020) Characterization of natural antigen-specific antibodies from naive sturgeon serum. *Developmental & Comparative Immunology*. https://doi.org/10.1016/j.dci.2020.103770.

Yin H, Sun Z, Sun D, Ge Y, Ma A (2009) Cytogenetical study on karyotype and ploidy of *Acipenser dabryanus* Dumeril. *Chinese Journal of Fisheries* **3**: 11.

Yuan S, Tao X, Huang S, Chen S, Xu A (2014) Comparative immune systems in animals. *Annual Review of Animal Biosciences* **2**: 235–258.

Zapata A, Diez B, Gutiérrez-de Frías CT, Cortés A (2006) Ontogeny of the immune system of fish. *Fish and Shellfish Immunology* **20**: 26–136.

Zexia G, Weimin W, Yi Y, Abbas K, Dapeng L, Guiwei Zet *et al*. (2007) Morphological studies of peripheral blood cells of the Chinese sturgeon *Acipenser sinensis*. *Fish Physiology and Biochemistry* **33**: 213–222.

Zhang S, Xu Q, Boscari E, Du H, Qi Z, Li Y *et al*. (2018) Characterization and expression analysis of g-and c-type lysozymes in Dabry's sturgeon (*Acipenser dabryanus*). *Fish and Shellfish Immunology* **76**: 260–265.

Zhu LY, Nie L, Zhu G, Xiang LX, Shao J (2013) Advances in research of fish immune-relevant genes: a comparative overview of innate and adaptive immunity in teleosts. *Developmental and Comparative Immunology* **39**: 39–62.

Zhu L, Yan Z, Feng M, Peng D, Guo Y, Hu X *et al*. (2014) Identification of sturgeon IgD bridges the evolutionary gap between elasmobranchs and teleosts. *Developmental & Comparative Immunology* **42**: 138–147.

Zhu R, Du HJ, Li SY, Li YD, Ni H, Yu XJ *et al*. (2016) *De novo* annotation of the immune-enriched transcriptome provides insights into immune system genes of Chinese sturgeon (*Acipenser sinensis*). *Fish and Shellfish Immunology* **55**: 699–716.

## Supporting Information

Additional supporting information may be found online in the Supporting Information section at the end of the article.

**File S1.** Orthogroups (phylogenetic trees) of selected immune genes inferred using the OrthoFinder2pipeline; for details: Material and Methods.

**Table S1.** (a) Number of orthologous key immune genes in the sterlet genome based on two annotations. (b) The top 10 genes per pathway (rated via numbers of involved pathways per gene). (c) Immune system pathways and genes (KEGG classification).