Physiological changes during torpor favor association with Endozoicomonas endosymbionts in the urochordate Botrylloides leachii

Abstract

Environmental perturbations evoke down-regulation of metabolism in some multicellular organisms, leading to dormancy, or torpor. Colonies of the urochordate Botrylloides leachii enter torpor in response to changes in seawater temperature and may survive for months as small vasculature remnants that lack feeding and reproductive organs but possess torpor-specific microbiota. Upon returning to milder conditions, the colonies rapidly restore their original morphology, cytology and functionality while harboring re-occurring microbiota, a phenomenon that has not been described in detail to date. Here we investigated the stability of B. leachii microbiome and its functionality in active and dormant colonies, using microscopy, qPCR, in situ hybridization, genomics and transcriptomics. A novel lineage of Endozoicomonas, proposed here as Candidatus Endozoicomonas endoleachii, was dominant in torpor animals (53-79% read abundance), and potentially occupied specific hemocytes found only in torpid animals. Functional analysis of the metagenome-assembled genome and genome-targeted transcriptomics revealed that Endozoicomonas can use various cellular substrates, like amino acids and sugars, potentially producing biotin and thiamine, but also expressing various features involved in autocatalytic symbiosis. Our study suggests that the microbiome can be linked to the metabolic and physiological states of the host, B. leachii, introducing a model organism for the study of symbioses during drastic physiological changes, such as torpor.

Keywords: Endozoicomonas; aestivation; ascidians; hibernation; metabolism; symbiosis; torpor.

Figure 1

Botrylloides leachii torpor. (A) An active colony was grown on a glass slide in the laboratory. The zooids are peripherally surrounded by extended ampullae, the blind termini of vasculature that are loaded with hemocyte. Each zooid is approximately 1–1.5 mm in length and contains an oral siphon (a black arrowhead), whereas a system of 8 zooids (in this figure) shares a common atrial siphon (a blue arrowhead). The whole colony is embedded in a gelatinous matrix – the tunic. (B) A colony in a full hibernation state, following exposure to 15°C water temperature for 15 days (ambient seawater = 20°C). The resorbed zooids are replaced by a ‘carpet’ of opaque lacunae and dilated vasculature (a white arrowhead) loaded with pigment cells that give this colonial remnant its deep color. (C–E) From active (C), mid torpor (D) to fully torpor (E) states in the same B. leachii colony. Functional units (zooids, red dash line) are located only in active and mid-torpor states. Bars = 2 mm, am = ampulla, zo = zooid.

Figure 2

TEM sections of B. aff. leachii vasculature and hemocytes in active, mid-torpor and fully torpid states. (A) An active phenotype, no cell-containing bacteria. Bar-10 μm. (B) A torpid colony, bacteria cells within the cytoplasm of some circulated hemocytes (a blue arrowhead). Bar-10 μm. (C) Bacteria-containing cells, phenotype 1-a 12 μm animal cell containing 2 vacuoles, the inner vacuole holds 3 intact bacteria cells. Bar-2 μm. (D) A bacteria-containing cell, phenotype 2- a 22 μm cell holding 22 intact bacteria cells. (D’) The bacteria cell wall is double layered, as some of the naked DNA that is at the state of binary fission (yellow asterisks). Bar-0.5 μm.

Figure 3

Histological observations of vasculature and hemocytes in active, mid-torpor and fully torpid colonies. (A) Active colony- histological section through a peripheral ampulla stained with hematoxylin & eosin. (B) In vitro Hoechst stain of hemocytes isolated from the active colony. (C) Mid-torpor colony- histological section through a peripheral ampulla stained with hematoxylin & eosin. Several hemocytes containing multiple DNA spots are marked (red arrowheads). (D) Torpid colony- Histological section through an ampulla (hematoxylin & eosin). A wide range of hemocytes with multiple DNA spots (red arrowheads). (E) In vitro Hoechst-stained hemocytes isolated from a torpid colony (E’), many of them contain multiple DNA spots (marked with red arrowheads). Bars- 10 μm.

Figure 4

The relative read abundance of 54 bacterial taxa in the vasculature of torpid colonies, mid-torpor state (5 days) and active colonies (3 different biological replicates for either physiological state, each replicate marked by its ID at the x-axis base). Empty slots: relative abundance <0.1%. Taxonomy is based on the annotation of the full, SPAdes-assembled rRNA sequences, classified using the best BLAST hit in the Silva 138 database.

Figure 5

qPCR quantification of the two key bacteria associated with B. leachii hibernation– the ratio between the copy numbers of the rRNAs: bacterial 16S to the host 18S. Endozoicomonas was significantly enriched in the hibernation state (n = 5), compared to freshly collected (wild n = 5) and laboratory-grown samples (active n = 5). Fodinicurvata was highly abundant in some freshly collected samples. One-way ANOVA: Endozoicomonas along different stages F (2,12) = 9.915, p = 0.0038 Fodinicurvata F(2,12) = 1.913, p = 0.19.

Figure 6

Microbiota varies between different physiological states (active, mid-torpor, and full-torpor colonies) and tissue types (vasculature and zooids) in Botrylloides leachi. Principal Coordinates Analysis (PCoA) based on the Bray–Curtis dissimilarity matrix of transcriptomic rRNA full-length read abundances.

Figure 7

Phylogeny of Endozoicomonas species based on amino acid sequences of 90 single-copy gene markers (maximum likelihood, JTT + CAT model). The scale bar represents the number of substitutions per site. Branch bootstrap support values are shown. NCBI BioSample accession numbers are indicated for the respective genomes.

Figure 8

Key metabolic pathways of Ca. Endozoicomonas endoleachii, based on genome-centered transcriptomics. The enzymes that catalyze each reaction are marked with the name of the encoding gene. Colors represent gene expression only in torpor colonies, as log transcripts per million reads (TPM). The following enzymes and respective genes are shown: PTS system, N-acetylgalactosamine-specific IIC component agaW; PTS system, N-acetylgalactosamine-specific IID component agaE; PTS N-acetylgalactosamine transporter subunit IIB agaV; N-acetylgalactosamine transporter subunit IIA agaF; N-acetylgalactosamine-6-phosphate deacetylase agaA; D-galactosamine-6-phosphate deaminase AgaS; 6-phosphofructokinase pfkA; tagatose-bisphosphate aldolase agaY; triosephosphate isomerase tpi; glyceraldehyde-3-phosphate dehydrogenase gapdH; phosphoglycerate kinase pgk; phosphoglucomutase pgm; enolase eno; pyruvate kinase pyk; pyruvate dehydrogenase aceEF-lpd; citrate synthase gltA; aconitase acnA; isocitrate dehydrogenase idh; 2-oxoglutarate dehydrogenase sucAB-lpd; succinyl-CoA ligase sucCD; succinate dehydrogenase sdhABCD; fumarate hydratase fumA; malate dehydrogenase mdh; F₀F₁-type ATP synthase atpABCDEFGH; Na(+)-translocating NADH-quinone reductase nqrSBCDEF; Na(+)/H(+) antiporters nhaA and nhaP; cytochrome bc1 complex petABC; cytochrome bd-I ubiquinol oxidase cydABX; cytochrome bo₃ ubiquinol oxidase cyoABC; ba₃-type cytochrome c oxidase caaABC; nranched-chain amino acid permeases brnQ; branched-chain-amino-acid aminotransferase ilvE; branched-chain alpha-keto acid dehydrogenase complex bkdA1A2B; isovaleryl-CoA dehydrogenase liuA; methylcrotonyl-CoA carboxylase liuBD; methylglutaconyl-CoA hydratase liuC; hydroxymethylglutaryl-CoA lyase liuE; acyl-CoA dehydrogenase short/branched chain acadsb; enoyl-CoA hydratase ech; 3-hydroxyisobutyryl-CoA hydrolase bch; 3-hydroxyisobutyrate dehydrogenase mmsB; methylmalonate-semialdehyde dehydrogenase mmsA; phosphate transport system pstABC-phoU-pltA; oligopeptide ABC transporter oppABCF; putrescine transporter potFGHI; ABC transporters dctMPQ; vitamin B12 transporter btuBF.

Figure 9

Volcano plot showing the key features upregulated in the mid-torpor and torpid states. Each dot represents a gene, blue if the adjusted p value < 0.01, red if log2 fold change >1 and adjusted p value < 0.05. Selected genes were annotated using the protein product nomenclature. GPDH is glycerol-3-phosphate dehydrogenase [NAD(P)⁺] (EC 1.1.1.94). CSP is a cold shock protein. PAP2 is a putative membrane-associated phospholipid phosphatase, PAP2 superfamily.