Cellular Responses in Sea Fan Corals: Granular Amoebocytes React to Pathogen and Climate Stressors

BACKGROUND: Climate warming is causing environmental change making both marine and terrestrial organisms, and even humans, more susceptible to emerging diseases. Coral reefs are among the most impacted ecosystems by climate stress, and immunity of corals, the most ancient of metazoans, is poorly known. Although coral mortality due to infectious diseases and temperature-related stress is on the rise, the immune effector mechanisms that contribute to the resistance of corals to such events remain elusive. In the Caribbean sea fan corals (Anthozoa, Alcyonacea: Gorgoniidae), the cell-based immune defenses are granular acidophilic amoebocytes, which are known to be involved in wound repair and histocompatibility. METHODOLOGY/PRINCIPAL FINDINGS: We demonstrate for the first time in corals that these cells are involved in the organismal response to pathogenic and temperature stress. In sea fans with both naturally occurring infections and experimental inoculations with the fungal pathogen Aspergillus sydowii, an inflammatory response, characterized by a massive increase of amoebocytes, was evident near infections. Melanosomes were detected in amoebocytes adjacent to protective melanin bands in infected sea fans; neither was present in uninfected fans. In naturally infected sea fans a concurrent increase in prophenoloxidase activity was detected in infected tissues with dense amoebocytes. Sea fans sampled in the field during the 2005 Caribbean Bleaching Event (a once-in-hundred-year climate event) responded to heat stress with a systemic increase in amoebocytes and amoebocyte densities were also increased by elevated temperature stress in lab experiments. CONCLUSIONS/SIGNIFICANCE: The observed amoebocyte responses indicate that sea fan corals use cellular defenses to combat fungal infection and temperature stress. The ability to mount an inflammatory response may be a contributing factor that allowed the survival of even infected sea fan corals during a stressful climate event

Histological images of sea fans infected with <i>Aspergillus sydowii</i> and presence of melanin both as a band and in amoebocytes as melanosomes.

<p>A) Histological preparation of an infected coral stained with H&E showing individual fungal hyphae (F) in skeleton and surrounded by melanin (M). Note granular amoebocytes (ga) aggregated near melanin and fungal hyphae. Scale bar = 25 µm. B) Histological preparation of an infected coral showing multiple amoebocytes containing melanin granules in contact with the thick layer of melanin preventing <i>A. sydowii</i> hyphae within the axial skeleton (at left) from contacting the sea fan tissue (on right). Fontana-Masson staining procedure, scale bar = 25 µm. C) Close up of an amoebocyte containing melanin granules (Mg) and black stained melanin (M) layer surrounding fungus (F). Fontana-Masson staining procedure, scale bar = 10 µm D) Uninfected coral stained with Fontana-Masson's procedure showing lack of melanin and melanin granules. Scale bar = 10 µm.</p

Amoebocytes in mesoglea (connective tissue) of naturally diseased sea fan corals.

<p>A) Healthy coral with granular amoebocytes dispersed in mesoglea as indicated by arrows. B) Diseased coral with an increase in granular amoebocytes in the mesoglea. Scale bar = 25 µm.</p

Prophenoloxidase activity in healthy and diseased sea fans as measured by the oxidation of L-dopa to dopachrome.

<p>Data presented are mean±s.e.m n = 12, F = 4.7, p = 0.040.</p

Picture of a sea fan coral (<i>Gorgonia ventalina</i>) infected with <i>Aspergillus sydowii</i>, multifocal purple annular lesions are indicative of infection (photo by Ernesto Weil).

<p>Picture of a sea fan coral (<i>Gorgonia ventalina</i>) infected with <i>Aspergillus sydowii</i>, multifocal purple annular lesions are indicative of infection (photo by Ernesto Weil).</p

Increase in amoebocyte surface area in sea fans exposed to elevated temperature (31.5°C) for 8 days.

<p>Data presented are mean±s.e.m, n = 6, <i>X</i>² = 305.11, p<0.0001.</p

Quantitative analysis of amoebocytes in mesoglea of sea fans experimentally exposed to <i>Aspergillus sydowii</i>.

<p>A) Increase in amoebocytes in sea fans exposed to fungal hyphae, n = 14, <i>X</i>² = 41.85, p<0.0001. B) Fine spatial analysis of amoebocyte surface area at point of fungus exposure and 1, 4, and 8 mm away. Dashed line indicated control or basal % amoebocyte surface area. Data presented are mean±s.e.m, n = 8, F = 18.95, p = 0.015. Asterisk denotes significant differences at p<0.05.</p

Amoebocytes are heterogeneously distributed within individual naturally infected sea fan colonies.

<p>Data presented are mean±s.e.m, n = 8, <i>X</i>² = 12.43, p = 0.0004.</p

Amoebocytes in mesoglea (connective tissue) of sea fan corals exposed to experimental heat stress.

<p>Images are of the same coral colony, with A) one fragment kept at 29°C and B) fragment kept at 31.5°C. Scale bar = 25 µm.</p