The Role of Complement in Cnidarian-Dinoflagellate Symbiosis and Immune Challenge in the Sea Anemone Aiptasia pallida

Abstract

The complement system is an innate immune pathway that in vertebrates, is responsible for initial recognition and ultimately phagocytosis and destruction of microbes. Several complement molecules including C3, Factor B, and mannose binding lectin associated serine proteases (MASP) have been characterized in invertebrates and while most studies have focused on their conserved role in defense against pathogens, little is known about their role in managing beneficial microbes. The purpose of this study was to (1) characterize complement pathway genes in the symbiotic sea anemone Aiptasia pallida, (2) investigate the evolution of complement genes in invertebrates, and (3) examine the potential dual role of complement genes Factor B and MASP in the onset and maintenance of cnidarian-dinoflagellate symbiosis and immune challenge using qPCR based studies. The results demonstrate that A. pallida has multiple Factor B genes (Ap_Bf-1, Ap_Bf-2a, and Ap_Bf-2b) and one MASP gene (Ap_MASP). Phylogenetic analysis indicates that the evolutionary history of complement genes is complex, and there have been many gene duplications or gene loss events, even within members of the same phylum. Gene expression analyses revealed a potential role for complement in both onset and maintenance of cnidarian-dinoflagellate symbiosis and immune challenge. Specifically, Ap_Bf-1 and Ap_MASP are significantly upregulated in the light at the onset of symbiosis and in response to challenge with the pathogen Serratia marcescens suggesting that they play a role in the initial recognition of both beneficial and harmful microbes. Ap_Bf-2b in contrast, was generally downregulated during the onset and maintenance of symbiosis and in response to challenge with S. marcescens. Therefore, the exact role of Ap_Bf-2b in response to microbes remains unclear, but the results suggest that the presence of microbes leads to repressed expression. Together, these results indicate functional divergence between Ap_Bf-1 and Ap_Bf-2b, and that Ap_Bf-1 and Ap_MASP may be functioning together in an ancestral hybrid of the lectin and alternative complement pathways. Overall, this study provides information on the role of the complement system in a basal metazoan and its role in host-microbe interactions.

Keywords: Aiptasia; Serratia marcescens; Symbiodinium; cnidarians; complement; innate immunity; symbiosis.

Figure 1

Diagrammatic representation of the complement system. In vertebrates there are three activation mechanisms including the classical, lectin, and alternative pathways, which all converge at cleavage of the central protein C3. Invertebrates only possess components of the lectin and alternative pathways. Red boxes surround sequences that have been characterized in invertebrate genomes and transcriptomes. The question mark by C1q indicates that proteins with the C1q domain have been characterized in invertebrates, but a direct link to their role in the complement system has not been established.

Figure 2

Summary of Distribution of complement in the invertebrate phyla. Presence or absence of complement proteins genes C3, Factor B, and MASP is shown for each invertebrate phylum for which sequence databases were searched. A checkmark indicates presence while an empty box indicates absence based on available data.

Figure 3

Maximum likelihood phylogenetic analysis of Factor B proteins in invertebrates. Sequences are color coded by phylum (subphylum for chordates) and circles at each node represent bootstrap support. Blue, Porifera; Red, Cnidaria; Yellow, Mollusca; Purple, Arthropoda; Orange, Echinodermata; Green, Hemichordata; Magenta, Cephalochordata (subphylum); Aqua, Urochordata (subphylum). Filled circles = 85–100% bootstrap support, half-filled = 70–84% bootstrap support, and open circles = 50–69% bootstrap support.

Figure 4

Domain structures of A. pallida Factor B and MASP sequences. A. pallida has three Factor B and one MASP gene. EGF, epidermal growth factor; CCP, complement control protein motif; VWA, Von Willabrand Factor type A domain; Trypsin, serine protease domain; CUB, complement C1r/C1s, Uegf, Bmp1.

Figure 5

Expression of Ap_Bf-1, Ap_Bf-2b, and Ap_MASP as a function of symbiotic state. The relative quantities from qPCR on the log₂ scale are shown for aposymbiotic organisms (solid bars) and symbiotic organisms (bars with lines). Bars represent means ±SE (n = 3).

Figure 6

Expression of Ap_Bf-1, Ap_Bf-2b, and Ap_MASP during the recolonization experiment for symbiont+light (SL) and no symbiont+light (NSL) treatments. (A) Ap_Bf-1, (B) Ap_Bf-2b, and (C) Ap_MASP). Bars represent means ± SE (n = 3) and stars (^*) represent expression differences that are significantly different between the SL and NSL treatments at a particular time point (ANOVA, Tukey's post-hoc test). ^*p < 0.05, ^**p < 0.01, ^***p < 0.001.

Figure 7

Expression of Ap_Bf-1, Ap_Bf-2b, and Ap_MASP during the recolonization experiment for symbiont+dark treamtment (SD). Bars represent means ± SE (n = 3) and letters represent expression differences that are significantly different between time points within each gene (ANOVA, Tukey's post-hoc test).

Figure 8

Comparison of symbiont 28S rDNA in symbiotic, aposymbiotic and recolonized A. pallida. For each treatment group, replicate relative quantities are presented with the mean relative log₂ quantity indicated by the bar. Points are slightly offset for clarity. Groups that have unique letters (a, b, or c) are significantly different (p ≤ 0.05) as indicated by a 1-tailed Wilcoxon rank-sum test. In addition those samples within group B indicated by a star (^*) are significantly different from each other, but not from the other group B samples.

Figure 9

Expression Ap_Bf-1, Ap_Bf-2b, and Ap_MASP in and aposymbiotic (A,C,E) and symbiotic A. pallida (B,D,F) in response to challenge with S. marcescens for low bacterial concentrations (10⁴ cells/mL) or high bacterial concentrations (10⁷ cells/mL). The relative quantities on the log2 scale are shown and bars represent ±SE (n = 3). Letters indicate significant differences in expression within a time point (ANOVA, Tukey's post-hoc test).