Using Bacterial Extract along with Differential Gene Expression in Acropora millepora Larvae to Decouple the Processes of Attachment and Metamorphosis

Abstract

Biofilms of the bacterium Pseudoalteromonas induce metamorphosis of acroporid coral larvae. The bacterial metabolite tetrabromopyrrole (TBP), isolated from an extract of Pseudoalteromonas sp. associated with the crustose coralline alga (CCA) Neogoniolithon fosliei, induced coral larval metamorphosis (100%) with little or no attachment (0–2%). To better understand the molecular events and mechanisms underpinning the induction of Acropora millepora larval metamorphosis, including cell proliferation, apoptosis, differentiation, migration, adhesion and biomineralisation, two novel coral gene expression assays were implemented. These involved the use of reverse-transcriptase quantitative PCR (RT-qPCR) and employed 47 genes of interest (GOI), selected based on putative roles in the processes of settlement and metamorphosis. Substantial differences in transcriptomic responses of GOI were detected following incubation of A. millepora larvae with a threshold concentration and 10-fold elevated concentration of TBP-containing extracts of Pseudoalteromonas sp. The notable and relatively abrupt changes of the larval body structure during metamorphosis correlated, at the molecular level, with significant differences (p<0.05) in gene expression profiles of 24 GOI, 12 hours post exposure. Fourteen of those GOI also presented differences in expression (p<0.05) following exposure to the threshold concentration of bacterial TBP-containing extract. The specificity of the bacterial TBP-containing extract to induce the metamorphic stage in A. millepora larvae without attachment, using a robust, low cost, accurate, ecologically relevant and highly reproducible RT-qPCR assay, allowed partially decoupling of the transcriptomic processes of attachment and metamorphosis. The bacterial TBP-containing extract provided a unique opportunity to monitor the regulation of genes exclusively involved in the process of metamorphosis, contrasting previous gene expression studies that utilized cues, such as crustose coralline algae, biofilms or with GLW-amide neuropeptides that stimulate the entire onset of larval metamorphosis and attachment.

Gene Expression Patterns during the Early Stages of Chemically Induced Larval Metamorphosis and Settlement of the Coral Acropora millepora

“…All three cues showed a similar trend in the regulation of MEKK-1 , ERK-2 , MAPK-p38 , AP-1/cFos , and cAMP/ATF2 (Figure 1), indicating common activation of the immunity response in the early developmental (1–3 hpi) phase [5], [6], [8], [10]–[12], [21], [22]. However, AP-1/cJun exhibited an opposite response to J010-E over time but showed the same response at 12 hpi and with the two CCA-derived cues.…”

“…The suggested roles of the GOI in relation to marine invertebrate larval settlement and metamorphosis have been summarised and reported previously (Table 1 in [12]). One negative template (T−) and one negative Reverse Transcriptase (RT−; Kan r only) control was used in each assay to test for cDNA and RNA contaminants, respectively, according to the GenomeLab™ GeXP manual (a detailed description for this procedure is reported in [17]).…”

Stepwise metamorphosis of the tubeworm Hydroides elegans is mediated by a bacterial inducer and MAPK signaling

“…Hydroides up-regulated SPI-encoding genes, which have potent bactericidal activity implicated in host innate immunity in Hydra (49) and black tiger shrimp (50). Similar differential expression of immunity genes during metamorphosis was observed in corals (25,28) and ascidians (14,27,61). VWF domain-encoding genes were up-regulated and might be important for tube building.…”

“…These systems include hormones (16)(17)(18), neurotransmitters (19)(20)(21), and nitric oxide (22)(23)(24). Additionally, diverse animals regulate metamorphosis at the transcriptional and posttranslational levels, such as differential expression of metamorphosis-associated genes (25)(26)(27) and MAPK signaling (28)(29)(30)(31), respectively. Although a number of signaling systems and regulatory networks orchestrate metamorphosis in animals from diverse taxa, comparatively little is known about how bacteria stimulate these systems and networks.…”

The c-Jun N-terminal kinase prevents oxidative stress induced by UV and thermal stresses in corals and human cells

“…To prevent or to fight oxidative stress, corals have implemented defence strategies such as the synthesis of fluorescent pigments, heat-shock proteins, mycosporin-like amino acids2728 and the production of anti-oxidant enzymes26. An increasing number of studies indicate that corals express genes encoding MAPKs, including those encoding JNK293031323334. This suggests the presence of a conserved stress response between coral and human cells at the molecular level, although the phylum cnidaria diverged from bilateria 550 million years ago35.…”