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Ultrastructural Analysis in the Elucidation of Disease in Corals

Authors:
D. Abigail Renegar at Nova Southeastern University
D. Abigail Renegar
Patricia Lurie Blackwelder at University of Miami
Patricia Lurie Blackwelder
Deborah Gochfeld at University of Mississippi
Deborah Gochfeld
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Abstract

of a paper presented at Microscopy and Microanalysis 2007 in Ft. Lauderdale, Florida, USA, August 5 – August 9, 2007
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Ultrastructural Analysis in the Elucidation of Disease in Corals
D. A. Renegar*, P. L. Blackwelder*
,
**, D. J. Gochfeld***
*Nova Southeastern University Oceanographic Center, 8000 N. Ocean Dr., Dania, FL 33004
**RSMAS, University of Miami, 600 Rickenbacker Causeway, Miami, FL 33149
***Natl. Ctr. for Natural Products Research, University of Mississippi, University, MS 38677
An increase in the incidence of coral disease has been observed worldwide, and may play a
significant role in the demise of critical reef-building species. Dark Spot Syndrome (DSS) typically
affects the reef-building coral Siderastrea siderea and manifests as lesions of varying color, size,
shape and location that can result in tissue death and skeletal changes. A cause has not yet been
identified. The greater resolution possible with TEM (transmission electron microscopy) and SEM
(scanning electron microscopy) can reveal microbial activity and initial tissue changes not resolvable
utilizing histology. The objective of this study is ultrastructural investigation of the cellular and
skeletal characteristics, and possible pathogenic microbes in affected and healthy S. siderea.
Coral tissue samples underwent primary fixation in glutaraldehyde, postfixation in osmium tetroxide,
and dehydration in a graded series of ethanols. A sample subset was decalcified before dehydration.
SEM samples were further dehydrated with HMDS, mounted on carbon adhesive covered aluminum
stubs, and sputter coated (Pd). TEM samples were embedded in Spurr
TM
resin and sectioned using
an ultramicrotome fitted with a diamond knife. The sections were retrieved on formvar carbon
coated 200 mesh copper grids and stained with lead citrate.
Compared to healthy tissue (Fig. 1), DSS affected tissue has less integrity, with increasing cellular
degradation and vacuolization. The zooxanthellae population was in decline, characterized by many
abnormal or necrotic cells with internal organelle disruption and debris. In calcified tissue, aragonite
crystals appear diminished and abnormal, with little to no organic matrix present. A high
concentration of electron dense inclusions, believed to be zymogen granules based on morphology
and staining properties, was concentrated in the calicodermis and adjacent gastrodermal layer (Fig.
2). Zymogen is an acidophilic digestive enzyme [1] not previously observed concentrated in the
tissue types examined here, and may be used as a defensive mechanism against the numerous fungal
cells observed directly beneath the tissue in close proximity to the calicodermis in all of the DSS
affected samples (Fig. 3). These cells are dimensionally and morphologically consistent with the
genus Aspergillus. Other etiologic organisms were not observed. Skeletal changes observed
included darkened areas of skeleton both directly beneath dark spots in the tissue and some distance
beneath the coral surface, possibly caused by the increased concentration of zymogen.
These observations support the hypothesis that DSS is likely a stress response instead of a true
disease [2]. If DSS can be positively linked with skeletal discoloration, it may be possible to identify
and correlate past stress events with elemental predictors of climate and ocean chemistry changes
associated with coral skeletal composition, thus providing a unique insight into coral reef resiliency.
References
[1] W. M. Goldberg, Tissue and Cell 34 (2002) 246.
[2] J. L. Borger, Coral Reefs 24 (2005) 139.
272 CD
Copyright 2007 Microscopy Society of America
Microsc Microanal 13(Suppl 2), 2007
DOI: 10.1017/S1431927607074892
Fig. 1. Siderastrea siderea. A) Colony with DSS, B) TEM micrograph, normal tissue, and C) TEM
micrograph, normal gastrodermal tissue and zooxanthellae. Scale bars: A = 10 cm, B and C = 10 µm.
Fig. 2. Siderastrea siderea. A) TEM micrograph, normal coral calicodermal tissue, with normal
organic matrix (OM) and zymogen (ZY), B) TEM micrograph, DSS affected calicodermal tissue,
with higher concentration of zymogen and lack of organic matrix, and C) TEM micrograph, DSS
affected tissue with abnormal zooxanthellae (ZO). Scale bars: A = 5 µm, B = 3 µm, C = 10 µm.
Fig. 3. Siderastrea siderea. A) TEM micrograph, fungal cell (FC) in close proximity to DSS
affected tissue with abnormal zooxanthellae (ZO), and B) TEM micrograph, fungal cell. Scale bars:
A and B = 5 µm.
B C A
B
A
OM
ZY
B
ZY
C
ZO
A
ZO
FC
ZO
273 CD
Microsc Microanal 13(Suppl 2), 2007
... Few studies have examined coral ultrastructure as a means to understand disease and most have not been extensive (Renegar et al., 2007;Ainsworth et al., 2007a). The use of transmission electron microscopy (TEM) to describe the ultrastructure of healthy coral has only covered a limited number of reef-building or scleractinian coral species (Chapman, 1974;Goldberg 2001aGoldberg , 2001b. ...
Characterizations of the Major Coral Diseases of the Philippines: Ulcerative White Spot Disease and Novel Growth Anomalies of Porites
Article
Full-text available
Coral reefs are in decline worldwide and coral disease is a significant contributing factor. However, etiologies of coral diseases are still not well understood. In contrast with the Caribbean, extremely little is known about coral diseases in the Philippines. In 2005, off Southeast Negros Island, Philippines, I investigated relationships between environmental parameters and prevalence of the two most common coral diseases, ulcerative white spot (UWS) and massive Porites growth anomalies (MPGAs). Samples were collected along a disease prevalence gradient 40.5 km long. Principal component analyses showed prevalence of MPGAs was positively correlated with water column nitrogen, organic carbon of surface sediments, and colony density. UWS was positively correlated with water column phosphorus. This is the first quantitative evidence linking anthropogenically-impacted water and sediment to a higher prevalence of these diseases.Histological and cytological alterations were investigated by comparing tissues from two distinct types of MPGA lesions (types 1 and 2) and healthy coral using light and electron microscopy. Skeletal abnormalities and sloughing, swelling, thinning, and loss of tissues in MPGAs resembled tissues exposed to bacterial or fungal toxins. Both lesion types had decreases in symbiotic zooxanthellae, which supply nutrients to corals. Notable alterations included migrations of chromophore cells (amoebocytes) (1) nocturnally to outer epithelia to perform wound-healing, including plugging gaps and secreting melanin in degraded tissues, and (2) diurnally to the interior of the tissue possibly to prevent shading zooxanthellae in order to maximize photosynthate production. Depletion of melanin (active in wound healing) in type 2 lesions suggested type 2 tissues were overtaxed and less stable. MPGAs contained an abundance of endolithic fungi and virus-like particles, which may result from higher nutrient levels and play roles in disease development. Swollen cells and mucus frequently blocked gastrovascular canals (GVCs) in MPGAs. Type 1 lesions appeared to compensate for impeded flow of wastes and nutrients through these canals with proliferation of new GVCs, which were responsible for the observed thickened tissues. In contrast, type 2 tissues were thin and more degraded. Dysplasia and putative neoplasia were also observed in MPGAs which may result from the tissue regeneration capacity being overwhelmed.
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Gastrodermal structure and feeding responses in the scleractinian Mycetophyllia reesi, a coral with novel digestive filaments
Article
  • Sep 2002
  • TISSUE CELL
Mycetophyllia reesi Wells is a colonial scleractinian coral whose outer surface consists of a series of oral-pharyngeal openings that lack tentacles. The polyps also lack a column and cannot protrude from the colonial surface. Correspondingly, there is no central digestive cavity. Instead, the pharynx is directly connected to a series of radially arranged mesenterial ducts lying parallel to the skeleton. The ducts, composed primarily of ciliated cells with small mucus inclusions and large, compartmentalized mucocytes, house filaments that protrude through the oral apertures during feeding. The filaments may or may not be directly connected with or originate from the mesenterial ducts and are histologically distinct from them. They are therefore referred to as digestive, rather than mesenterial filaments. In contrast with other scleractinians, the digestive filaments are thin, unequally bilobed stalks with spatulate ends. The cnidoglandular (CG) lobe, the larger of the two, exhibits a distinct cellular zonation. Large mastigophore cnidae and elongated zymogen-like cells are clustered at its distal end. Neither of these cells appear to respond to particulate food material, suggesting that they may be employed in alternative modes of nutrition and/or competition. Behind the distal region, the CG lobe exhibits typical zymogen, mucus, and collar cells as well as numerous atrichous nematocysts. The atrichs and zymogen cells discharge during particulate feeding. Tracts of collar cells with particularly well-defined cilia, elongated rootlets, and mucus inclusions are found at the outer edge of the CG lobe. These cells disgorge their contents during feeding and appear to function in food transport. The smaller lobe of the filament is a muscular sheet containing well-defined fields of circular and longitudinal myofibrils along with associated neurons. Collar cells with lysosome-like inclusions and large, compartmentalized mucocytes are also characteristic of this region. There are no zooxanthellae in the filaments, but these endosymbionts are present as a thin layer in the oral-most portion of the gastrodermis. The cellular zonation and multi-functionality of these digestive filaments suggest another example of a cnidarian structure at the organ level of complexity.
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139. 272 CD Copyright
  • Jan 2005
  • J L Borger
J. L. Borger, Coral Reefs 24 (2005) 139. 272 CD Copyright 2007 Microscopy Society of America Microsc Microanal 13(Suppl 2), 2007 DOI: 10.1017/S1431927607074892