Comprehensive analysis of the isomiRome in the vegetative organs of the conifer Pinus pinaster under contrasting water availability.

An increasing number of microRNAs (miRNAs) and miRNA-related sequences produced during miRNA biogenesis, comprising the isomiRome, have been recently highlighted in different species as critical mediators of environmental stress responses. Conifers have some of the largest known genomes but an extensive characterization of the isomiRome from any conifer species has been lacking. We provide here a comprehensive overview of the Pinus pinaster isomiRome expressed in roots, stem and needles under well-watered and drought conditions. From the 13,441 unique small RNA sequences identified, 2,980 were annotated as canonical miRNAs or miRNA* and the remaining were classified as isomiRNA or miRNA-like sequences. A survey of their expression patterns highlighted roots as the most responsive organ under drought, where specific sequences of which a 24-nt novel miRNA stood out, were strongly down-regulated. Given the putative roles of the miRNA-targeted transcripts validated specifically in root tissues, some of the miRNAs, conserved and novel, are shortlisted as potential regulators of drought response. These results provide a valuable resource for comparative studies between gymnosperms and angiosperms. Furthermore, it evidences high transferability of the isomiRome between pine species being a useful basis for further molecular regulation and physiological studies, and especially those focused on adaptation to drought conditions.

Stem metabolism under drought stress - a paradox of increasing respiratory substrates and decreasing respiratory rates.

Metabolic changes underpinning drought-induced variations in stem respiration (Rs ) are unknown. We measured Rs rates and metabolite and gene expression profiles in Ulmus minor Mill. and Quercus ilex L. seedlings subjected to increasing levels of drought stress to better understand how carbon, nitrogen and energy metabolism interact during drought. In both species, only plants showing extreme stress symptoms - i.e. negligible rates of leaf stomatal conductance and photosynthesis, and high stem dehydration (30-50% of maximum water storage) and contraction (50-150 µm week-1 ) - exhibited lower Rs rates than well-watered plants. Abundance of low-molecular weight sugars (e.g. glucose and fructose) and sugar alcohols (e.g. mannitol) increased with drought, at more moderate stress and to a higher extent in Q. ilex than U. minor. Abundance of amino acids increased at more severe stress, more abruptly, and to a higher extent in U. minor, coinciding with leaf senescence, which did not occur in Q. ilex. Organic acids changed less in response to drought: threonate and glycerate increased, and citrate decreased although slightly in both species. Transcripts of genes coding for enzymes of the Krebs cycle decreased in Q. ilex and increased in U. minor in conditions of extreme drought stress. The maintenance of Rs under severe growth and photosynthetic restrictions reveals the importance of stem mitochondrial activity in drought acclimation. The eventual decline in Rs diverts carbon substrates from entering the Krebs cycle that may help to cope with osmotic and oxidative stress during severe drought and to recover hydraulic functionality afterwards.

Gene expression trade-offs between defence and growth in English elm induced by Ophiostoma novo-ulmi.

Wilt diseases caused by vascular pathogens include some of the most damaging stresses affecting trees. Dutch elm disease (DED), caused by the fungus Ophiostoma novo-ulmi, destroyed most of North American and European elm populations in the 20th century. The highly susceptible English elm, also known as Atinian clone, suffered the highest mortality rates during the last pandemic event, probably due to its lack of genetic diversity. To study the DED pathosystem, we inoculated English elm ramets with O. novo-ulmi and evaluated xylem anatomy, molecular response, and disease symptoms. The high DED susceptibility of the clone was linked to xylem structure. The transcript levels changed significantly for 1,696 genes during O. novo-ulmi invasion. Genes covering different steps of the plant immune system were identified, many of which showed homology with Arabidopsis thaliana genes involved in systemic acquired resistance. Induction of several pathogenesis-related proteins and repression of fasciclin-like arabinogalactan proteins and other cell wall biosynthesis pathways evidence unbalanced costs between growth and defence mechanisms far from the inoculation point. This study sheds light on elm molecular defence mechanisms against DED.

A molecular approach to drought-induced reduction in leaf CO2 exchange in drought-resistant Quercus ilex.

Drought-induced reduction of leaf gas exchange entails a complex regulation of the plant leaf metabolism. We used a combined molecular and physiological approach to understand leaf photosynthetic and respiratory responses of 2-year-old Quercus ilex seedlings to drought. Mild drought stress resulted in glucose accumulation while net photosynthetic CO2 uptake (Pn ) remained unchanged, suggesting a role of glucose in stress signaling and/or osmoregulation. Simple sugars and sugar alcohols increased throughout moderate-to-very severe drought stress conditions, in parallel to a progressive decline in Pn and the quantum efficiency of photosystem II; by contrast, minor changes occurred in respiration rates until drought stress was very severe. At very severe drought stress, 2-oxoglutarate dehydrogenase complex gene expression significantly decreased, and the abundance of most amino acids dramatically increased, especially that of proline and γ-aminobutyric acid (GABA) suggesting enhanced protection against oxidative damage and a reorganization of the tricarboxylic cycle acid cycle via the GABA shunt. Altogether, our results point to Q. ilex drought tolerance being linked to signaling and osmoregulation by hexoses during early stages of drought stress, and enhanced protection against oxidative damage by polyols and amino acids under severe drought stress.

Single-cell atlas of rainbow trout peripheral blood leukocytes and profiling of their early response to infectious pancreatic necrosis virus.

The recent development of single cell sequencing technologies has revolutionized the state-of-art of cell biology, allowing the simultaneous measurement of thousands of genes in single cells. This technology has been applied to study the transcriptome of single cells in homeostasis and also in response to pathogenic exposure, greatly increasing our knowledge of the immune response to infectious agents. Yet the number of these studies performed in aquacultured fish species is still very limited. Thus, in the current study, we have used the 10x Genomics single cell RNA sequencing technology to study the response of rainbow trout (Oncorhynchus mykiss) peripheral blood leukocytes (PBLs) to infectious pancreatic necrosis virus (IPNV), an important trout pathogen. The study allowed us to obtain a transcriptomic profile of 12 transcriptionally distinct leukocyte cell subpopulations that included four different subsets of B cells, T cells, monocytes, two populations of dendritic-like cells (DCs), hematopoietic progenitor cells, non-specific cytotoxic cells (NCC), neutrophils and thrombocytes. The transcriptional pattern of these leukocyte subpopulations was compared in PBL cultures that had been exposed in vitro to IPNV for 24 h and mock-infected cultures. Our results revealed that monocytes and neutrophils showed the highest number of upregulated protein-coding genes in response to IPNV. Interestingly, IgM+IgD+ and IgT+ B cells also upregulated an important number of genes to the virus, but a much fainter response was observed in ccl4 + or plasma-like cells (irf4 + cells). A substantial number of protein-coding genes and genes coding for ribosomal proteins were also transcriptionally upregulated in response to IPNV in T cells and thrombocytes. Interestingly, although genes coding for ribosomal proteins were regulated in all affected PBL subpopulations, the number of such genes transcriptionally regulated was higher in IgM+IgD+ and IgT+ B cells. A further analysis dissected which of the regulated genes were common and which were specific to the different cell clusters, identifying eight genes that were transcriptionally upregulated in all the affected groups. The data provided constitutes a comprehensive transcriptional perspective of how the different leukocyte populations present in blood respond to an early viral encounter in fish.

Pleiotropic Role of Rainbow Trout CXCRs in Response to Disease and Environment: Insights from Transcriptional Signatures and Structure Analysis.

Chemokines are cytokines with chemoattractant capacities that exert their physiological functions through the binding of chemokine receptors. Thus, chemokine and receptor complexes exert important roles in regulating development and homeostasis during routine immune surveillance and inflammation. Compared to mammals, the physiology and structure of chemokine receptors in fish have not been systematically studied. Furthermore, the salmonid-specific whole genome duplication has significantly increased the number of functional paralogs of chemokine receptors. In this context, in the current study, trout exhibited 17 cxcr genes, including 12 newly identified and 5 previously identified receptors. Interestingly, gene expression of brain cxcr1 and cxcr4, kidney cxcr3 and cxcr4, and spleen cxcr3, cxcr4, and cxcr5 subtypes were altered by bacterial infection, whereas brain cxcr1, kidney cxcr1 and cxcr7, and liver cxcr2, cxcr3, and cxcr4 subtypes were changed in response to environmental changes. Based on protein structures predicted by ColabFold, the conserved amino acids in binding pockets between trout CXCR4.1 subtypes and human CXCR4 were also analyzed. Our study is valuable from a comparative point of view, providing new insights into the identification and physiology of salmonid chemokine receptors.

Endoplasmic reticulum expansion throughout the differentiation of teleost B cells to plasmablasts.

The differentiation of B cells into antibody-secreting cells is fundamental for the generation of humoral immunity. In mammals, this process involves a series of metabolic and intracellular changes, not studied to date in teleost fish, where a clear distinction between naive B cells and plasmablasts/plasma cells (PCs) is still missing. Thus, in the current study, we have established that upon activation, teleost B cells undergo an expansion of the endoplasmic reticulum (ER) but experience no significant changes in mitochondria content. In parallel, the transcription of genes implicated in B cell differentiation increases, while that of mitochondrial genes decreases. In this context, ER monitoring has allowed us to distinguish between small cells with low amounts of ER (FSCloERlo B cells), that correspond to undifferentiated cells, and large cells with expanded ER (FSChiERhi B cells), characterized as plasmablasts. The results shed new light on the B cell differentiation process in teleosts and provide us with novel tools to study B cell function in these species.

Novel conserved segments are associated with differential expression patterns for Pinaceae dehydrins.

Dehydrins are thought to play an essential role in the response, acclimation and tolerance to different abiotic stresses, such as cold and drought. These proteins have been classified into five groups according to the presence of conserved and repeated motifs in their amino acid sequence. Due to their putative functions in the response to stress, dehydrins have been often used as candidate genes in studies on population variability and local adaptation to environmental conditions. However, little is still known regarding the differential role played by such groups or the mechanism underlying their function. Based on the sequences corresponding to dehydrins available in public databases we have isolated eight different dehydrins from cDNA of Pinus pinaster. We have obtained also their genomic sequences and identified their intron/exon structure. Quantitative RT-PCR analysis of their expression pattern in needles, stems and roots during a severe and prolonged drought stress, similar to the ones trees must face in nature, is also reported. Additionally, we have identified two amino acid motifs highly conserved and repeated in Pinaceae dehydrins and absent in angiosperms, presumably related to the divergent expression profiles observed.

miR160 Interacts in vivo With Pinus pinaster AUXIN RESPONSE FACTOR 18 Target Site and Negatively Regulates Its Expression During Conifer Somatic Embryo Development.

MicroRNAs (miRNAs) are key regulators of several plant developmental processes including embryogenesis. Most miRNA families are conserved across major groups of plant species, but their regulatory roles have been studied mainly in model species like Arabidopsis and other angiosperms. In gymnosperms, miRNA-dependent regulation has been less studied since functional approaches in these species are often difficult to establish. Given the fundamental roles of auxin signaling in somatic embryogenesis (SE) induction and embryo development, we investigated a previously predicted interaction between miR160 and a putative target encoding AUXIN RESPONSE FACTOR 18 in Pinus pinaster (PpARF18) embryonic tissues. Phylogenetic analysis of AUXIN RESPONSE FACTOR 18 (ARF18) from Pinus pinaster and Picea abies, used here as a model system of conifer embryogenesis, showed their close relatedness to AUXIN RESPONSE FACTOR (ARF) genes known to be targeted by miR160 in other species, including Arabidopsis ARF10 and ARF16. By using a luciferase (LUC) reporter system for miRNA activity in Arabidopsis protoplasts, we have confirmed that P. pinaster miR160 (ppi-miR160) interacts in vivo with PpARF18 target site. When the primary miR160 from P. pinaster was overexpressed in protoplasts under non-limiting levels of ARGONAUTE1, a significant increase of miR160 target cleavage activity was observed. In contrast, co-expression of the primary miRNA and the target mimic MIM160 led to a decrease of miR160 activity. Our results further support that this interaction is functional during consecutive stages of SE in the conifer model P. abies. Expression analyses conducted in five stages of development, from proembryogenic masses (PEMs) to the mature embryo, show that conifer ARF18 is negatively regulated by miR160 toward the fully developed mature embryo when miR160 reached its highest expression level. This study reports the first in vivo validation of a predicted target site of a conifer miRNA supporting the conservation of miR160 interaction with ARF targets in gymnosperms. The approach used here should be useful for future characterization of miRNA functions in conifer embryogenesis.

EuroPineDB: a high-coverage web database for maritime pine transcriptome.

BACKGROUND: Pinus pinaster is an economically and ecologically important species that is becoming a woody gymnosperm model. Its enormous genome size makes whole-genome sequencing approaches are hard to apply. Therefore, the expressed portion of the genome has to be characterised and the results and annotations have to be stored in dedicated databases. DESCRIPTION: EuroPineDB is the largest sequence collection available for a single pine species, Pinus pinaster (maritime pine), since it comprises 951 641 raw sequence reads obtained from non-normalised cDNA libraries and high-throughput sequencing from adult (xylem, phloem, roots, stem, needles, cones, strobili) and embryonic (germinated embryos, buds, callus) maritime pine tissues. Using open-source tools, sequences were optimally pre-processed, assembled, and extensively annotated (GO, EC and KEGG terms, descriptions, SNPs, SSRs, ORFs and InterPro codes). As a result, a 10.5× P. pinaster genome was covered and assembled in 55 322 UniGenes. A total of 32 919 (59.5%) of P. pinaster UniGenes were annotated with at least one description, revealing at least 18 466 different genes. The complete database, which is designed to be scalable, maintainable, and expandable, is freely available at: http://www.scbi.uma.es/pindb/. It can be retrieved by gene libraries, pine species, annotations, UniGenes and microarrays (i.e., the sequences are distributed in two-colour microarrays; this is the only conifer database that provides this information) and will be periodically updated. Small assemblies can be viewed using a dedicated visualisation tool that connects them with SNPs. Any sequence or annotation set shown on-screen can be downloaded. Retrieval mechanisms for sequences and gene annotations are provided. CONCLUSIONS: The EuroPineDB with its integrated information can be used to reveal new knowledge, offers an easy-to-use collection of information to directly support experimental work (including microarray hybridisation), and provides deeper knowledge on the maritime pine transcriptome.

Diversity of Rainbow Trout Blood B Cells Revealed by Single Cell RNA Sequencing.

Single-cell sequencing technologies capable of providing us with immune information from dozens to thousands of individual cells simultaneously have revolutionized the field of immunology these past years. However, to date, most of these novel technologies have not been broadly applied to non-model organisms such as teleost fish. In this study, we used the 10× Genomics single cell RNA sequencing technology and used it to analyze for the first time in teleost fish the transcriptional pattern of single B cells from peripheral blood. The analysis of the data obtained in rainbow trout revealed ten distinct cell clusters that seem to be associated with different subsets and/or maturation/differentiation stages of circulating B cells. The potential characteristics and functions of these different B cell subpopulations are discussed on the basis of their transcriptomic profile. The results obtained provide us with valuable information to understand the biology of teleost B cells and offer us a repertoire of potential markers that could be used in the future to differentiate trout B cell subsets.

Pro-Inflammatory and B Cell Regulating Capacities of TWEAK in Rainbow Trout (Oncorhynchus mykiss).

Tumor necrosis factor (TNF)-like weak inducer of apoptosis or TWEAK is a member of the TNF superfamily involved in the regulation of many biological processes. In mammals, TWEAK has been shown to play a role in some autoimmune or inflammatory conditions, but its immune role is not yet clearly defined. In teleost fish, although a few studies have identified homologues to mammalian TWEAK, their biological effects have never been investigated. In the current study, we have studied the transcriptional regulation of two TWEAK homologues (TWEAK 1 and 2) identified in rainbow trout (Oncorhynchus mykiss) throughout different tissues, in response to parasitic or viral infections, or in head kidney (HK) leukocytes stimulated with different stimuli. Although the transcription of both homologues was modulated when HK leukocytes were exposed to several immune stimuli, only TWEAK 1 was significantly modulated upon pathogenic exposure. Thus, we performed a characterization of the functions exerted by this cytokine in HK leukocytes. Recombinant TWEAK 1 strongly up-regulated the transcription of pro-inflammatory genes and antimicrobial peptides in HK leukocytes, with differential transcriptional effects in IgM+ B cells, IgM- lymphocytes and myeloid cells. TWEAK 1 also increased the survival and promoted the differentiation of B cells in HK leukocyte cultures. Our results demonstrate that in teleost fish, TWEAK 1 is involved in the response to different types of pathogens, through the modulation of antimicrobial and pro-inflammatory genes in different leukocytes subsets. Furthermore, a role for TWEAK as a B cell differentiation factor has also been established in rainbow trout.

Individual B cells transcribe multiple rearranged immunoglobulin light chains in teleost fish.

B cells express a unique antibody protein which comprises two pairs of immunoglobulin (Ig) heavy (H) and light (L) chains. In addition to an invariable constant (C) region, IgH and IgL chains encompass a variable (V) region mediating antigen binding. This unique region stems from Ig V(D)J gene recombination, which generates diversity by assembling these gene segments into VHDJH and VLJL genes. To ensure that one B cell only expresses one antibody, VHDJH rearrangement occurs only in one IgH locus (allelic exclusion), whereas VLJL rearrangement only in either the κ or λ locus (isotype exclusion). However, teleosts express multiple IgLs encoded by distinct CL genes. Using single-cell transcriptomics, we have demonstrated the transcription of distinct rearranged VLJLCL genes in single rainbow trout B cells. Our results highlight the laxity of isotype exclusion in teleosts and strongly suggest that fish B cells can produce antibodies of different specificities.

CD38 Defines a Subset of B Cells in Rainbow Trout Kidney With High IgM Secreting Capacities.

CD38 is a multifunctional molecule that functions both as a transmembrane signaling receptor and as an ectoenzyme with important roles in cell adhesion, calcium regulation and signal transduction. Within the B cell linage, CD38 is expressed in diverse murine B cell subsets, with highest levels in innate B cell subpopulations such as marginal zone (MZ) B cells or B1 cells. In humans, however, CD38 is transiently expressed on early lymphocyte precursors, is lost on mature B cells and is consistently expressed on terminally differentiated plasma cells. In the present work, we have identified two homologues of mammalian CD38 in rainbow trout (Oncorhynchus mykiss), designating them as CD38A and CD38B. Although constitutively transcribed throughout different tissues in homeostasis, both CD38A and CD38B mRNA levels were significantly up-regulated in head kidney (HK) in response to a viral infection. In this organ, after the generation of a specific monoclonal antibody (mAb) against CD38A, the presence of CD38A+ populations among IgM+ B cells and IgM- leukocytes was investigated by flow cytometry. Interestingly, the percentage of IgM+CD38A+ B cells increased in response to an in vitro stimulation with inactivated Aeromonas salmonicida. Finally, we demonstrated that HK IgM+CD38A+ B cells had an increased IgM secreting capacity than that of cells lacking CD38A on the cell surface, also showing increased transcription levels of genes associated with B cell differentiation. This study strongly suggests a role for CD38 on the B cell differentiation process in teleosts, and provides us with novel tools to discern between B cell subsets in these species.

Passive Immunization Delays Disease Outcome in Gilthead Sea Bream Infected With Enteromyxum leei (Myxozoa), Despite the Moderate Changes in IgM and IgT Repertoire.

Passive immunization constitutes an emerging field of interest in aquaculture, particularly with the restrictions for antibiotic use. Enteromyxum leei is a myxozoan intestinal parasite that invades the paracellular space of the intestinal epithelium, producing a slow-progressing disease, leading to anorexia, cachexia and mortalities. We have previously demonstrated that gilthead sea bream (GSB, Sparus aurata) that survive E. leei infection become resistant upon re-exposure, and this resistance is directly related to the presence of high levels of specific IgM in serum. Thus, the current work was aimed to determine if passive immunization could help to prevent enteromyxosis in GSB and to study in detail the nature of these protective antibodies. Serum from a pool of resistant (SUR) or naïve (NAI) animals was intracoelomically injected 24 h prior to the E. leei-effluent challenge and at 9 days post-challenge (dpc). Effluent challenge lasted for 23 days, and then the injected groups were allocated in separate tanks with clean water. A non-lethal parasite diagnosis was performed at 56 dpc. At the final sampling (100 dpc), blood, serum and tissues were collected for histology, molecular diagnosis and the detection of circulating antibodies. In parallel, we performed an immunoglobulin repertoire analysis of the fish generating SUR and NAI sera. The results showed that, fish injected with parasite-specific antibodies (spAbs) became infected with the parasite, but showed lower disease signs and intensity of infection than the other groups, indicating a later establishment of the parasite. Repertoire analysis revealed that E. leei induced a polyclonal expansion of diverse IgM and IgT subsets that could be in part an evasion strategy of the parasite. Nonetheless, GSB was able to produce sufficient levels of parasite-spAbs to avoid re-infection of surviving animals and confer certain degree of protection upon passive transfer of antibodies. These results highlight the crucial role of spAb responses against E. leei and set the basis for the development of effective treatment or prophylactic methods for aquaculture.

Insights Into the Evolution of the prdm1/Blimp1 Gene Family in Teleost Fish.

In mammals, Blimp1 (B lymphocyte-induced maturation protein 1) encoded by the prdm1 gene and its homolog Hobit (homolog of Blimp1 in T cells) encoded by znf683, represent key transcriptional factors that control the development and differentiation of both B and T cells. Despite their essential role in the regulation of acquired immunity, this gene family has been largely unexplored in teleosts to date. Until now, one prdm1 gene has been identified in most teleost species, whereas a znf683 homolog has not yet been reported in any of these species. Focusing our analysis on rainbow trout (Oncorhynchus mykiss), an in silico identification and characterization of prdm1-like genes has been undertaken, confirming that prdm1 and znf683 evolved from a common ancestor gene, acquiring three gene copies after the teleost-specific whole genome duplication event (WGD) and six genes after the salmonid-specific WGD. Additional transcriptional studies to study how each of these genes are regulated in homeostasis, in response to a viral infection or in B cells in different differentiation stages, provide novel insights as to how this gene family evolved and how their encoded products might be implicated in the lymphocyte differentiation process in teleosts.

Phellem versus xylem: genome-wide transcriptomic analysis reveals novel regulators of cork formation in cork oak.

Cork cambium (or phellogen) is a secondary meristem responsible for the formation of phelloderm and phellem/cork, which together compose the periderm. In Quercus suber L., the phellogen is active throughout the entire life of the tree, producing a continuous and renewable outer bark of cork. To identify specific candidate genes associated with cork cambium activity and phellem differentiation, we performed a comparative transcriptomic study of Q. suber secondary growth tissues (xylem and phellogen/phellem) using RNA-seq. The present work provides a high-resolution map of all the transcripts identified in the phellogen/phellem tissues. A total of 6013 differentially expressed genes were identified, with 2875 of the transcripts being specifically enriched during the cork formation process versus secondary xylem formation. Furthermore, cork samples originating from the original phellogen (`virgin' cork) and from a traumatic phellogen (`amadia' cork) were also compared. Our results point to a shortlist of potentially relevant candidate genes regulating phellogen activity and phellem differentiation, including novel genes involved in the suberization process, as well as genes associated to ethylene and jasmonate signaling and to meristem function. The future functional characterization of some of the identified candidate genes will help to elucidate the molecular mechanisms underlying cork cambium activity and phellem differentiation.

Molecular study of drought response in the Mediterranean conifer Pinus pinaster Ait.: Differential transcriptomic profiling reveals constitutive water deficit-independent drought tolerance mechanisms.

Adaptation of long-living forest trees to respond to environmental changes is essential to secure their performance under adverse conditions. Water deficit is one of the most significant stress factors determining tree growth and survival. Maritime pine (Pinus pinaster Ait.), the main source of softwood in southwestern Europe, is subjected to recurrent drought periods which, according to climate change predictions for the years to come, will progressively increase in the Mediterranean region. The mechanisms regulating pine adaptive responses to environment are still largely unknown. The aim of this work was to go a step further in understanding the molecular mechanisms underlying maritime pine response to water stress and drought tolerance at the whole plant level. A global transcriptomic profiling of roots, stems, and needles was conducted to analyze the performance of siblings showing contrasted responses to water deficit from an ad hoc designed full-sib family. Although P. pinaster is considered a recalcitrant species for vegetative propagation in adult phase, the analysis was conducted using vegetatively propagated trees exposed to two treatments: well-watered and moderate water stress. The comparative analyses led us to identify organ-specific genes, constitutively expressed as well as differentially expressed when comparing control versus water stress conditions, in drought-sensitive and drought-tolerant genotypes. Different response strategies can point out, with tolerant individuals being pre-adapted for coping with drought by constitutively expressing stress-related genes that are detected only in latter stages on sensitive individuals subjected to drought.

Rainbow Trout IgM+ B Cells Preferentially Respond to Thymus-Independent Antigens but Are Activated by CD40L.

In the absence of class switch recombination and germinal centers, the mechanisms through which B cells from teleost fish mount extrafollicular immunoglobulin M (IgM) memory responses remains mostly unexplored. In this report, we demonstrate that teleost IgM+ B cells respond to CD40L, a thymus-dependent activation signal, similarly to mammalian B2 cells. However, when stimulated with different types of antigens, fish IgM+ B cells only reach a general activation state in response to antigens cataloged as thymus-independent 1 (TI-1) in mammals, as established through both functional assays and RNA sequencing. Interestingly, fish IgM+ B cells remained completely unresponsive to TI-2 antigens, suggesting that the engagement of innate receptors provided by TI-1 antigens is required for the activation of teleost B cells. Finally, a synergy between CD40L and TI-1 antigens was also demonstrated, further supporting that there is no clear dichotomy between thymus-dependent and TI responses in teleost fish.

Teleost IgD+IgM- B Cells Mount Clonally Expanded and Mildly Mutated Intestinal IgD Responses in the Absence of Lymphoid Follicles.

Immunoglobulin D (IgD) is an ancient antibody with dual membrane-bound and fluid-phase antigen receptor functions. The biology of secreted IgD remains elusive. Here, we demonstrate that teleost IgD+IgM- plasmablasts constitute a major lymphocyte population in some mucosal surfaces, including the gut mucosa. Remarkably, secreted IgD binds to gut commensal bacteria, which in turn stimulate IgD gene transcription in gut B cells. Accordingly, secreted IgD from gut as well as gill mucosae, but not the spleen, show a V(D)J gene configuration consistent with microbiota-driven clonal expansion and diversification, including mild somatic hypermutation. By showing that secreted IgD establishes a mutualistic relationship with commensals, our findings suggest that secreted IgD may play an evolutionary conserved role in mucosal homeostasis.