Bacterial symbionts in animal development: arginine biosynthesis complementation enables larval settlement in a marine sponge

Abstract

Larval settlement and metamorphosis are regulated by nitric oxide (NO) signalling in a wide diversity of marine invertebrates (1-10). It is surprising, then, that in most invertebrates, the substrate for NO synthesis - arginine - cannot be biosynthesized but instead must be exogenously sourced (11). In the sponge Amphimedon queenslandica, vertically-inherited proteobacterial symbionts in the larva are able to biosynthesize arginine (12,13). Here we test the hypothesis that symbionts might provide arginine to the sponge host so that nitric oxide synthase expressed in the larva can produce NO, which induces metamorphosis (8), and the byproduct citrulline (Fig. 1). First, we find support for an arginine-citrulline biosynthetic loop in this sponge larval holobiont using stable isotope tracing. In symbionts, incorporated 13C-citrulline decreases as 13C-arginine increases, consistent with the use of exogenous citrulline for arginine synthesis. In contrast, 13C-citrulline accumulates in larvae as 13C-arginine decreases, demonstrating the uptake of exogenous arginine and its conversion to NO and citrulline. Second, we show that while Amphimedon larvae can derive arginine directly from seawater, normal settlement and metamorphosis can occur in artificial sea water lacking arginine. Together, these results support holobiont complementation of the arginine-citrulline loop and NO biosynthesis in Amphimedon larvae, suggesting a critical role for bacterial symbionts in the development of this marine sponge. Given that NO regulates settlement and metamorphosis in diverse animal phyla (1-10) and arginine is procured externally in most animals (11), we propose that symbionts may play a equally critical regulatory role in this essential life cycle transition in other metazoans. O_FIG O_LINKSMALLFIG WIDTH=95 HEIGHT=200 SRC="FIGDIR/small/240770v1_fig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@1625afcorg.highwire.dtl.DTLVardef@438543org.highwire.dtl.DTLVardef@e1fd20org.highwire.dtl.DTLVardef@b155de_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 1.C_FLOATNO Amphimedon queenslandica cannot synthesize arginine, which is required for nitric oxide-induced settlement and metamorphosis. A. In A. queenslandica, nitric oxide (NO) is necessary for completion of larval settlement (Ueda et al.). B. Arginine, which is a necessary precursor for NO synthesis, cannot be synthesized the sponge. Its genome does not code for argininosuccinate synthase and argininosuccinate lyase, but both genes are present in its primary bacterial symbionts. Colored dots next to the enzymes comprising the arginine-citrulline synthesis loop correspond to presence in sponge and symbiont genomes. C. Fluorescence in situ hybridization (FISH) with an AqS1-specific 16S probe showing the distribution and abundance of this symbiont in the larval anterior. The outer epithelial layer (OEL), middle subepithelial layer (SEL) and inner cell mass (ICM) are shown. This symbiont is enriched in the inner cell mass (red; arrows pointing to examples); blue, DAPI-stained sponge cell nuclei. Scale bar 10 m. C_FIG