Bloom of the cyanobacterium Moorea bouillonii on the gorgonian coral Annella reticulata in Japan

Coral populations are in decline due to environmental changes and biological attacks by predators and infectious diseases. Here, we report a localized bloom of the benthic filamentous cyanobacterium Moorea bouillonii (formerly Lyngbya bouillonii) observed exclusively on the gorgonian (sea fan) coral Annella reticulata at around 20 m depth in Japan. The degree of infection has reached 26% among different sizes of Annella colonies. Thick and continuous growth of Moorea may be sustained partly by symbiotic alpheid shrimp, which affix Moorea filaments to gorgonian corals for use as food and shelter. Most filaments get entangled on the coral colony, some penetrate into the stem of the coral with a swollen end like a root hair, which appears to function as an anchor in Annella. In addition to the cyanobacterium–shrimp interaction, the new trait of anchoring by the cyanobacterium into gorgonian coral may contribute to persistence of this bloom.

Percentage of infected colonies of M. bouillonii on A. reticulata reached 26%, and some algal cover was found on every size class of coral colony (Fig. 3). A small amount of other algal species (filamentous green or attached diatoms) was observed, but M.bouillonii was the most abundant and entangled on branches of Annella. The sewing (tube-forming) shrimp, Alpheus frontalis H. Milne Edwards 1958, was identified by the presence of tubular cyanobacteria, but quantitative measurement was not performed because most shrimp escaped from the cyanobacterial tubes during collection. Other small organisms found in the cyanobacterial mat, which were considered to be secondarily attached, included foraminiferas, nematodes, copepods, gastropods, and tunicates etc. Nutrient concentrations measured from the sea surface to 25 m in depth were 0.17 mmol for NH 4 , 0.04 mmol for NO 2 , 0.99 mmol for NO 3 , and 0.09 mmol for PO 4 .
Coral branches overgrown by Moorea mats ultimately die, which results in collapse of the branch (Fig. 2E) followed by gradual detachment of outer sclerites and then the loss of successively longer sclerites that form the inner axis. Most Moorea bouillonii filaments were entangled on coral colonies, although some were loosely attached and lying on the branch surface. Some filaments penetrated directly into the coral branch and reached the outermost region of the central axis (Fig. 4A). The terminal end of the boring filament was swollen like a hair root, and consisted of a multilayered sheath (Fig. 4B) which functions as an anchor.

Discussion
Seaweeds negatively impact corals via multiple mechanisms such as shading, abrasion, vectoring of coral diseases, and release of metabolites 8,24 . Cyanobacterial blooms in coral reefs are due to excessive anthropogenic nutrient loading, and have been reported from Florida, USA 4,13 , Guam 25 , and Queensland, Australia 11,26 . The ambient nutrient concentrations measured at this study site (0.99 mmol for NO 3 and 0.09 mmol for PO 4 ) were slightly below the levels prev-    iously reported for sustaining macroalgal blooms (1.0 mmol for NO 3 and 0.1 mmol for PO 4 27 or cyanobacterial growth in enrichment experiments 26,[28][29] . On the other hand, it is widely accepted that reefs are not limited to low-nutrient areas 1,27,30 . Thus, to accurately address algal growth, we must consider nutrients, producers (algae), and consumers (herbivores, predators).
Herbivores consume algae, affecting algae-coral interactions. In a marine protected area in Fiji, the amount of macroalgae is better controlled than in an adjacent fished reef 7 . In general, however, filamentous cyanobacteria and gorgonian corals are generally not preferred food for predators such as benthivorous fish [31][32][33] . Furthermore, L. majuscula produces feeding deterrents, such as ypaoamide 34 and lyngbyatoxin 35 . Cytotoxic macrolides and peptides have been identified from samples of Moorea bouillonii associated with Alpheus frontalis shrimp in Guam 36 . Similarly, gorgonian corals are not suitable food because they possess chemical metabolites and mechanical sclerites as defenses against fish 37,38 , as well as antifungal secondary compounds 39 . Furthermore, among cnidarian animals, sea fan corals develop cell-based immune defenses (amoebocytes) 40 . Nevertheless, gorgonian corals, as well as hard corals, are facing a crisis of fungal infestations (e.g., aspergillosis disease in the Caribbean Sea 17,41 ) and algal blooms due to eutrophication.
At our study site, how the cyanobacteria initially settled on the coral is unknown, but the important question is how the bloom is maintained in oligotrophic water. Engene et al. 18 showed that Moorea bouillonii lacks heterocysts and genes for nitrogen fixation. Nutrient concentrations at the study site were not high enough from the water surface to 25 m deep to cause algal blooms. Furthermore, cyanobacterial coverage was observed exclusively on Annella. The tubeforming or sewing shrimp Alpheus frontalis H. Milne Edwards have been found in cyanobacterial tubes which they made to live and to eat [20][21] . Thus, the sewing shrimp Alpheus may play an important role in perpetuating continuous blooms by attaching cyanobacterial filaments to coral branches to form tube-like mats that it uses for food and shelter. NH 4 and PO 4 excreted from the shrimp were absorbed by Moorea determined in a laboratory experiment (not shown, Yamashiro unpublished data). In addition, this shrimp, like other alpheid shrimp, uses its large claws to snap at other animals and protect its nest made of Moorea 42,43 . This symbiosis also seems to have a synergistic relationship in respect to nutrition (between photosynthetic cyanobacteria and nitrogen/phosphorus-emitting shrimp).
Concerning the mortality of Annella due to Moorea cover, there are several possible mechanisms. Coral death can be caused by metabolic decline including oxygen depletion or a reduction of food supply to the corals by algal (including cyanobacteria) coverage [44][45][46][47] . In addition to physical stress, biochemical effects such as allelopathic terpenes secreted by algae have been reported as causing coral death 48 . Titlyanov 49 performed direct contact experiment using cyanobacteria Lyngbya (Moorea) bouillonii on live coral Porites, and demonstrated that M. bouillonii acted as a one-sided inhibitor for scleractinian corals inducing bleaching and severe damage of live coral tissue. Similar interaction is often observed in the field of Okinawa Island between M. bouillonii and branching corals such as Montipora nested by sewing shrimp. Coral tissue where filamentous M. bouillonii was tied by the shrimp showed bleached and partial death (not shown). The main cause of octocoral death was not identified in this study, physical effects such as abrasion or oxygen depletion, or biochemical (toxic or allelopathic) effects must be involved.
Some cyanobacteria associated with the coral are able to penetrate into the soft tissue and skeleton 50 . Our study highlights that Moorea bouillonii is capable of penetrating tissues of gorgonian coral branches by changing its shape at the terminal end. A swollen structure of multiple layers of sheath appears to function as an anchor (Fig. 4B), firmly attaching the cyanobacterium to Annella. Strong persistence of M. bouillonii to the host coral should exist, but this trait has previously been unrecognized. The origin and transmission of the cyanobacterium is still unknown, but the synergy between filamentous Moorea and sewing shrimp, and the special trait of penetration found in this M. bouillonii, must allow the persistence of year-round blooms on the sea fan Annella reticulata.

Methods
We first collected filamentous algae entangled on the gorgonian coral colony on 3 March 2009. We measured the concentrations of nutrients (NH 4 , NO 3 , NO 2 , and PO 4 ) using a nutrient autoanalyzer (BL Tech Co.) of triplicate seawater samples collected every 5 m down to 25 m depth on 10 April 2009. We identified the filamentous algae by morphology and molecular information (16S rRNA sequence, see below), and the gorgonian coral by morphological observation of colony structure and sclerites on live or formalin-fixed samples using dissecting or digital microscopes (VHX-1000, Keyence Co.). We also made histological sections to determine the method of attachment of the filamentous algae.
On 17 September 2009, we recorded the height of all gorgonian corals within a 2m-wide 3 20-m-long transect at 18 m depth on a nearly vertical reef with the highest local density of cyanobacteria infection. We recorded the overgrowth (infection) by filamentous cyanobacteria on the coral on all colonies (n 5 91) within the transect and classified them into size classes.
Cyanobacteria-specific PCR primers CYA106F (CGGACGGGTGAGTAACGC-GTGA) and CYA781R (an equimolar mixture of CYA 781R(a) (GACTACTGG-GGTATCTAATCCCATT) and CYA781R(b) (GACTACAGGGGTATCTAATC-CCTTT) 52 were used to amplify an about 680-bp region of the 16S rRNA gene. Reaction mixture of 25 ml contained 0.6 mM of each primer, 0.2 mM of each dNTP, 1X PCR Reaction Buffer (TaKaRa), 1.5 mM of MgCl 2 solution, 0.08% (w/v) bovine serum albumin, 0.2 U of ExTaq DNA Polymerase (TaKaRa) and 20 ng of template DNA. Amplification was performed with initial melting at 94uC for 3 min, followed by 30 cycles of 94uC for 1.5 min, 59uC for 1 min and 72uC for 2 min, and a final extension at 72uC for 5 min. After electrophoresis, PCR products were purified with DNA Cleaner (Wako). The purified PCR products were cloned using TOPO cloning kit (Invitrogen). The totals of twenty clones of sequences were carried out on an automated sequencer CEQ8800 (Beckman Coulter).