Differential regulation of degradation and immune pathways underlies adaptation of the ectosymbiotic nematode Laxus oneistus to oxic-anoxic interfaces

Abstract

Eukaryotes may experience oxygen deprivation under both physiological and pathological conditions. Because oxygen shortage leads to a reduction in cellular energy production, all eukaryotes studied so far conserve energy by suppressing their metabolism. However, the molecular physiology of animals that naturally and repeatedly experience anoxia is underexplored. One such animal is the marine nematode Laxus oneistus. It thrives, invariably coated by its sulfur-oxidizing symbiont Candidatus Thiosymbion oneisti, in anoxic sulfidic or hypoxic sand. Here, transcriptomics and proteomics showed that, whether in anoxia or not, L. oneistus mostly expressed genes involved in ubiquitination, energy generation, oxidative stress response, immune response, development, and translation. Importantly, ubiquitination genes were also highly expressed when the nematode was subjected to anoxic sulfidic conditions, together with genes involved in autophagy, detoxification and ribosome biogenesis. We hypothesize that these degradation pathways were induced to recycle damaged cellular components (mitochondria) and misfolded proteins into nutrients. Remarkably, when L. oneistus was subjected to anoxic sulfidic conditions, lectin and mucin genes were also upregulated, potentially to promote the attachment of its thiotrophic symbiont. Furthermore, the nematode appeared to survive oxygen deprivation by using an alternative electron carrier (rhodoquinone) and acceptor (fumarate), to rewire the electron transfer chain. On the other hand, under hypoxia, genes involved in costly processes (e.g., amino acid biosynthesis, development, feeding, mating) were upregulated, together with the worm's Toll-like innate immunity pathway and several immune effectors (e.g., bactericidal/permeability-increasing proteins, fungicides). In conclusion, we hypothesize that, in anoxic sulfidic sand, L. oneistus upregulates degradation processes, rewires the oxidative phosphorylation and reinforces its coat of bacterial sulfur-oxidizers. In upper sand layers, instead, it appears to produce broad-range antimicrobials and to exploit oxygen for biosynthesis and development.

Figure 1

Relative transcript abundance and expression levels of the top 100 expressed genes of L. oneistus across all conditions. (A) Relative transcript abundance (%) of the top 100 expressed genes with a manually curated functional category. The top 100 expressed genes were collected by averaging the expression values (log₂TPM) across all replicates of all incubations (Fig. S1A, Data S1, and S2). Functional classifications were extracted from UniProt and from comprehensive literature search focused mainly on C. elegans, and confirmed with the automatically annotated eggNOG classification (Data S1). (B) Median gene expression levels of selected L. oneistus manually annotated functional categories of the top 100 expressed genes. Metabolic processes include both differentially and constitutively expressed genes. Each dot represents the average log₂TPM value per gene across all replicates of all incubations. All gene names (or locus tags for unidentified gene names) are listed in Data S2.

Figure 2

Median gene expression levels of selected L. oneistus metabolic processes among the differentially expressed genes between the hypoxic (H) and anoxic sulfidic (AS) conditions after 24 h. Individual processes among the differentially expressed genes are ordered according to their difference in median expression between the AS and H incubations. Namely, detoxification (far left) had the largest difference in median expression in the AS condition, whereas immune response (far right) had the largest median expression difference in the H condition. The absolute number of genes are indicated at the top of each process. Metabolic processes were manually assigned and confirmed with the automatic annotated eggNOG classification. For specific gene assignments see Data S1. Some genes are present in more than one functional category and processes comprising only one gene are not displayed in the figure but listed in Data S1.

Figure 3

Genes involved in detoxification, ubiquitin–proteasome, autophagy, apoptosis, and amino acids degradation were predominantly expressed in AS worms. Heatmap displaying genes upregulated in AS (anoxic sulfidic) relative to H (hypoxic) worms after 24 h-long incubations under one of the two conditions (1.5-fold change, FDR ≤ 0.05). Expression levels are displayed as mean-centered log₂TPM value (transcripts per kilobase million). Genes are ordered by function in their respective metabolic pathways. For each process, the minority of genes that were upregulated in H worms is shown in Data S1. Red denotes upregulation, and blue downregulation. Prot. protein, COP9: Constitutive photomorphogenesis 9. dcp: domain-containing proteins; Put. glut. peroxid.: putative glutamate peroxidase; Put. sarc. oxid.: putative sarcosine oxidase.

Figure 4

Genes involved in translation and energy generation and genes encoding for C-type lectins and mucins were predominantly expressed in AS worms. Heatmap displaying genes upregulated in AS (anoxic sulfidic) relative to H (hypoxic) worms, upon 24 h-long incubations under one of the two conditions (1.5-fold change, FDR ≤ 0.05). Expression levels are displayed as mean-centered log₂TPM values (transcripts per kilobase million). Genes are ordered by function in their respective metabolic pathways. For each process, the minority of genes that were upregulated in H worms is shown in Data S1. Red denotes upregulation, and blue downregulation. Fp, family-containing protein; Cytoch. C ox. su. II: cytochrome c oxidase subunit II; Ubiq./rhodoq biosynth.: Ubiquinone or rhodoquinone biosynthesis.

Figure 5

Genes involved in immune response, development and nervous system were predominantly expressed in hypoxic (H) worms. Heatmap displaying genes upregulated in H relative to AS worms, upon 24 h-long incubations under one of the two conditions (1.5-fold change, FDR ≤ 0.05). Expression levels are displayed as mean-centered log₂TPM value (transcripts per kilobase million). Genes are ordered by function in their respective metabolic pathways. For each process, the minority of genes that were upregulated in AS worms is shown in Data S1. Red denotes upregulation and blue downregulation. MN, mechanosensory neurons; Embr. body wall muscle posit.: Embryonic body wall muscle positioning; Put.; putative.

Figure 6

Genes involved in carbohydrate, lipid- and sulfur-metabolism, amino acids biosynthesis, and transport were predominantly expressed in hypoxic (H) worms. Heatmap displaying genes upregulated in H relative to AS worms, upon 24 h-long incubations under one of the two conditions (1.5-fold change, FDR ≤ 0.05). Expression levels are displayed as mean-centered log₂TPM values (transcripts per kilobase million). Genes are ordered by function in their respective metabolic pathways. For each process, the minority of genes that were upregulated in AS worms is shown in Data S1. Red denotes upregulation, and blue downregulation. FA, fatty acids; PC phosphatidylcholine; PL, phospholipids; metab: metabolism; synth: synthesis; assim: assimilation, oxid: oxidation; Transp: transporters.

Figure 7

Schematic representation of Laxus oneistus physiology in anoxic or hypoxic  conditions. In anoxic sulfidic  conditions (left), L. oneistus does not enter suspended animation. Instead, it upregulates the expression of genes mediating inhibitory neurotransmission, involved in symbiosis establishment (e.g., lectins, mucins) and in ribosome biogenesis. Metabolism may be supported by the degradation of starch and by rewiring the electron transfer chain: rhodoquinone (RQ) is used as electron carrier and fumarate as electron acceptor. Moreover, the worm activates degradation pathways (e.g., ubiquitin–proteasome system (UPS), autophagy, and apoptosis) and may anticipate reoxygenation by upregulating superoxide dismutase (SOD) and glutathione peroxidase (GP). In hypoxic  conditions (right), instead, L. oneistus appears to use trehalose and cellulose for energy generation, while engaging in costly processes such as development, molting, feeding, and mating. Genes involved in excitatory neurotransmission are also upregulated, together with Toll-like receptors and immune effectors (e.g., fungicides, BPIs).