Evidence of Melanoma in Wild Marine Fish Populations
2012

M Sweet
N Kirkham
M Bendall
L Currey
J Bythell
Evidence of Melanoma in Wild Marine Fish Populations

PLoS ONE
78419892012Received May 11, 2012; Accepted June 27, 2012;Editor: Jean-Pierre Vartanian, Institut Pasteur, France Funding: The work was funded by a Natural Environment Research Council grant (NE/E006949). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. *
The increase in reports of novel diseases in a wide range of ecosystems, both terrestrial and marine, has been linked to many factors including exposure to novel pathogens and changes in the global climate. Prevalence of skin cancer in particular has been found to be increasing in humans, but has not been reported in wild fish before. Here we report extensive melanosis and melanoma (skin cancer) in wild populations of an iconic, commercially-important marine fish, the coral trout Plectropomus leopardus. The syndrome reported here has strong similarities to previous studies associated with UV induced melanomas in the well-established laboratory fish model Xiphophorus. Relatively high prevalence rates of this syndrome (15%) were recorded at two offshore sites in the Great Barrier Reef Marine Park (GBRMP). In the absence of microbial pathogens and given the strong similarities to the UV-induced melanomas, we conclude that the likely cause was environmental exposure to UV radiation. Further studies are needed to establish the large scale distribution of the syndrome and confirm that the lesions reported here are the same as the melanoma in Xiphophorus, by assessing mutation of the EGFR gene, Xmrk. Furthermore, research on the potential links of this syndrome to increases in UV radiation from stratospheric ozone depletion needs to be completed.

Introduction

Prevalence and occurrence of novel diseases are reported to be increasing in many organisms worldwide. Understanding the etiology of these diseases, the host organisms they affect and potential causes and consequences are a vital first step in the development of control and management strategies. Many diseases are caused by microbial pathogens, and fish diseases in particular have been shown to be caused by a diversity of such pathogens including bacteria, parasitic copepods, viruses and fungi [1,2,3]. Historically, diseases in fish have been recorded more commonly in species of commercial value, usually farmed fish. This may be due to the higher than normal stocking densities which in turn can lead to higher levels of infections and/or the ease of sampling large numbers and continuous monitoring capabilities. Furthermore, there is also significant economic benefit to identifying pathogens of these commercially reared fish with the aim of ultimately curing them. In aquaculture systems, diseases cause a significant economic loss, with bacteria, viruses and fungi being the dominant pathogens involved [1,3]. In contrast, diseases of wild fish have received considerably less attention and their economic impact on commercial and recreational fisheries is unknown. In addition to microbial diseases common in fish, other diseases such as carcinomas have been extensively studied in the laboratory using fish model systems, including the Xiphophorus (swordtail) [4,5] and, more recently, the Danio (zebrafish) models [5,6]. To date, however, there are no reports of cancers occurring in wild fish populations. This study aimed to describe a previously unknown disease lesion, which was observed affecting large numbers of a commercially important reef fish, the coral trout Plectropomus leopardus.

Methods

Sampling

Individual coral trout, Plectropomus leopardus, were line caught with barbless 8/0 hooks using pilchard bait, following methods employed by commercial fishers. Four fishing trips were completed between Aug 2010 and Feb 2012 off the east coast of Australia, at Heron Island (23.4uS, 151.9uE) and One Tree Island (23.5uS, 152.0uE). In total 136 fish were sampled and photographed, 20 of which showed signs of skin abnormalities. From healthy individuals and those with the syndrome, two sets of samples were taken; one for microbial analysis and the other for histological examination. Additional affected individuals were observed during snorkel and dive activities, but only those individuals captured via fishing were included in this analysis.

To test for differences in bacterial, fungal and ciliate molecular diversity between healthy and lesion samples, we analyzed tissue sections collected from individuals captured in August 2011. Three replicate tissue sections (,10l63 w63 d cm), separated by ,5 cm were cut using a sterile scalpel blade from n = 5 non-diseased (ND) fish, n = 5 diseased (D) fish (Fig. 1a) at the lesion interface and n = 5 apparently healthy (AH) tissues adjacent to the lesion on a disease fish. Samples were placed directly into 100% EtOH and stored at 220uC until extraction and further analysis. A further set of samples, aimed at sampling the surface associated microbes, utilised sterile swabs. The surface of the fish scales were swabbed and these were placed directly into sterile micro-centrifuge tubes with 100% EtOH, stored at 220uC until extraction and further analysis. Further samples, aimed at sampling the surface associated microbes used sterile swabs. These were placed in sterile microcentrifuge tubes and stored in 100% EtOH at 220uC until extraction. Samples for histology were collected as for microbial analysis (see above), with the same sample number of samples however, they were preserved in 5% paraformaldehyde made up with Phosphate Buffer Saline (PBS). Samples were fixed for 24 hr, dehydrated in a dilution series of EtOH from 50 to 100% and stored at 4uC until embedding and sectioning. DNA was extracted using the QIAGEN DNeasy Blood and tissue extraction kit.

Fungal PCR amplification and denaturing gradient gel electrophoresis (DGGE) of tissue samples/swabs For DGGE analysis a portion of the fungal ITS rRNA gene was amplified using universal fungal primers; a nested PCR approach was utilised to yield the most complete diversity [7]. 1 st round; fungal primers ITS1F (59CTTGGTCATTTAGAGGAAGTAA-39) and ITS4 (59-TCCTCCGCTTATTGATATGC-39) [8] were used following the protocol described by [9] (94uC for 5 min; 35 cycles at; 94uC for 30 sec, 55uC for 30 sec, 72uC for 30 sec then elongation at 72uC for 5 min). 20 ml PCR reactions were routinely used (final PCR buffer contained: 1 mM MgCl 2 , and 1 U Taq DNA polymerase (QBiogene); 100 mM dNTPs; 0.2 mM of each of the forward and reverse primers; and 0.4% BSA, with 20 ng of template DNA). A 1:100 dilution of the PCR product was then used in a further PCR with the primers ITS3 (59-GCATCGAT-GAAGAACGCAGC-39) and ITS4-GC (59-CGCCCGCCGCG-CCCCGCGCCCGGCCCGC CGCCCCCGCCCC-TCCTCC-GCTTATTGATATGC -39) [10]. All reactions were performed using a Hybaid PCR Express thermal cycler. PCR products were verified by agarose gel electrophoresis [1.6% (w/v) agarose] with ethidium bromide staining and visualized using a UV transilluminator. DGGE was performed using the D-Code universal mutation detection system (Bio-Rad). PCR products were resolved on 10% (w/v) polyacrylamide gels that contained a 30-60% formamide (denaturant) gradient for 13 h at 60uC and a constant voltage of 50 V. Gels were stained with SYBER gold as described by [11]. Bands of interest (those which explained the greatest differences/similarities between samples) were excised from DGGE gels, left overnight in Sigma molecular grade water, vacuum centrifuged, re-amplified with the specific primers, labelled using Big Dye (Applied biosystems) transformation sequence kit and sent to Genevision (Newcastle University, UK) for sequencing. Fungal operational taxonomic units (OTUs) were defined from DGGE band-matching analysis using BioNumerics 3.5 (Applied Maths BVBA).

Bacterial PCR amplification and denaturing gradient gel electrophoresis (DGGE) of tissue samples/swabs Extraction was the same as above. For DGGE analysis a portion of the bacterial 16S rRNA gene was amplified using universal eubacterial primers [12]; (357F-GC) (59-CCTACGGGAGGCAG-CAG-39) and (518R) (59-CGCCCGCCGCGCGCGGCGG-GCGGGGCGGGGGCAGCACGGGGGG-ATTACCGCGGC-TGCTGG-39). PCR reaction mixtures and program were as described by [11]. PCR products were resolved on 10% (w/v) polyacrylamide gels that contained a 30-60% formamide (denaturant) gradient for 13 h at 60uC and a constant voltage of 50 V. Gels were stained as above and bands of interest were excised from DGGE gels, labelled and sent to Genevision (Newcastle University, UK) for sequencing. Bacterial OTUs were defined from DGGE band-matching analysis using Bionumerics 3.5 (Applied Maths BVBA).

Ciliate PCR amplification and denaturing gradient gel electrophoresis (DGGE) of tissue samples/swabs Ciliates 18S rRNA genes were amplified with an un-nested PCR approach. PCR mixture was as above with the forward primer CilF (59-TGGTAGTGTATTGGACWACCA-39) with a 36-bp GC clamp [13] attached to the 59 end and CilDGGE-r (59TGAAAACATCCTTGGCAACTG-39). Initial denaturation was at 94uC for 5 min, followed by 26 cycles of 94uC for 1 min, 52uC for 1 min, and 72uC for 1 min and a final elongation step of 10 min at 72uC to reduce double bands in the DGGE patterns. The DGGE was carried out using a D-code system (Bio-Rad) with 0.75 mm thick 6% polyacrylamide gels in 16 TAE buffer. Electrophoresis was carried out for 16 h at 60uC and 50 V in a linear 32 to 42% denaturant (formamide) gradient. Gels were stained as above.

Statistic analysis of microbial communities

In order to assess variation in microbial assemblages (bacterial, fungal and ciliate), matrices consisting of OTU's and samples were generated using both presence/absence and band intensity data, using marker lanes for between-gel comparisons. Changes in microbial assemblages were evaluated with a one-way analysis of similarity (Primer, ANOSIM) and multi dimensional scaling (MDS), based on Bray-Curtis similarities, which was performed on all samples sets, healthy, apparently healthy and diseased.

Histology

Samples were collected as for microbial analysis; however tissue samples were preserved with 5% paraformaldehyde for 24 hrs then stored in 100% EtOH until resin embedding in LR white (r) . For each tissue type, the location of bacteria was recorded using fluorescent in situ hybridisation (FISH) and the general fluorescent stain, 496-diamidino-2-phenylindole (DAPI). For FISH, samples were stained and sectioned following the protocols in [14], with the addition of an equimolar mix (EUBMIX). Oligonucleotide probes were purchased from Interactiva (http://www.interactiva. de) with an aminolink C6/MMT at the 59 end. Four probes were used: the 'universal' eubacterial probes EUB338 (59-GCT GCC TCC CGT AGG AGT-39), EUB338-II (59-GCA GCC ACC CGT AGG TGT-39), EUB338-III (59-GCT GCC ACC CGT AGG TGT-39) and the 'non-sense probe' NONEUB (59-ACT CCT ACG GGA GGC AGC-39), which has the complementary sequence to EUB338, used to determine non-specific binding of EUB338. The three eubacterial probes were used in an equimolar mix (EUBMIX) and the NONEUB probe was used singly. DAPI staining followed the protocol by [11], whereby each section was stained with 100 ml of 4% PBS buffered paraformaldehyde solution containing 496-diamidino-2-phenylindole (final concentration 5 mg ml 21 ) for 10 minutes, rinsed with filtered 16 PBS pH 7.4. All sections were viewed under epiflourescence microscopy with an FITC-specific filter block (Nikon UK Ltd, Surrey, UK) and images recorded using an integrating camera (Model JVC KY-SSSB: Foster Findlay and Associates, Newcastle upon Tyne, UK). Samples of pure cultured E.coli were run alongside each section and for each staining protocol as a positive stain.

Further histological samples were stained with the melanin specific stain, Fontana-Mason, melanin granules reduce silver nitrate to metallic silver, which results in a histochemical reaction that participates black material wherever melanin is located [15].

Samples for Scanning Electron Microscopy (SEM) were dehydrated using EtOH and PBS; 25% EtOH, 50% EtOH, 75% EtOH (30 mins each), then a further (261 hr) in 100% EtOH, with final dehydration using carbon dioxide in a Baltec Critical Point Dryer. Specimens were then mounted on an aluminium stub with Achesons Silver Dag (dried overnight) and coated with gold (standard 15 nm) using a Polaron SEM Coating Unit. Specimens were examined using a Stereoscan 240 Scanning Electron Microscope, and digital images collected by Orion6.60.6 software.

Samples for Transmission Electron Microscopy (TEM) were dehydrated using 25% acetone, 50% acetone, 75% acetone, (30 min each) and 100% acetone (261 h). Then impregnated with 25% LR White resin in acetone, 50% resin/acetone, 75% resin/ acetone (1 h each), then 100% resin for minimum of 3 changes over 24 h, with final embedding in 100% resin at 60uC for 24 hrs. Survey sections of 1 m were cut and stained with 1% Toluidine Blue in 1% Borax. Ultrathin sections (80 nm approx) were then cut using a diamond knife on a RMC MT-XL ultramicrotome. These were then stretched with chloroform to eliminate compression and mounted on Pioloform filmed copper grids. Staining was with 2% aqueous Uranyl Acetate and Lead Citrate (Leica). The grids were then examined using a Philips CM 100 Compustage (FEI) Transmission Electron Microscope and digital images were collected using an AMT CCD camera (Deben) at the Electron Microscopy Research Services Laboratory, Newcastle University.

Results and Discussion

Approximately 15% of samples from a population of Plectropomus leopardus line caught at two locations in the southern Great Barrier Reef Marine Park -Heron Island and One Tree Island -showed evidence of a dark growth lesion (Fig. 1a,b), similar in appearance to those reported from laboratory induced melanomas seen in the fish Xiphophorus [16]. Prevalence of skin lesions was not significantly different (Chi Square = 0.063, df = 1, p = 0.803) between reef platforms with 14.1% of individuals at Heron Island and 15.7% of individuals at One Tree Island affected. In this study, the fish displaying these skin lesions struck fishing hooks as strongly as healthy individuals, appeared to have good muscle tone and were assessed by external examination as healthy aside from the skin discolouration. Coverage of the lesion on individual fish varied from ,10% of body surface (Fig. 1a) to almost complete coverage (Fig. 1b). Although the size range of individuals sampled was limited (344-639 mm fork length), there was no relationship between percent cover and fish size (r 2 = 0.02). Small individuals (468 mm) could show up to 98% lesion cover and larger individuals (639 mm) showed as little as 30% cover, indicating that prevalence can occur at varying sizes and ages. Lesions affected the surface of the fish caught, with a change from the characteristic blue-spotted patterning (Fig. 1c) in healthy individuals to raised lesions which were darker black/brown in coloration (Fig. 1d). Location of the lesions on the body varied between individuals.

Associated microbial (rRNA gene) communities

Analysis of microbial communities associated with healthy (nondiseased) and diseased fish, which would highlight potential pathogenic agents (those present in lesions and absent in healthy samples [17,18]), was conducted using culture-independent (rRNA gene) molecular screening techniques. Swabs of the mucus and tissue samples from healthy fish, apparently healthy tissues on affected fish, and the lesion itself were sampled. Microbial (rRNA gene) diversity assessed using bacterial-, fungal-and ciliate-specific PCR primers showed no significant difference (p.0.45) between the sample types. No known microbial pathogen sequences were found in lesion samples that were absent or in lower numbers within healthy and/or apparently healthy samples (Fig. 2a). The technique utilised in this study has routinely been used successfully in other studies to highlight potential microbial pathogens [19,20,21]. Furthermore, no significant differences (p = 0.12) was found between tissue sections and non-invasive surface mucus swabs, suggesting that the microbial communities detected were mainly present on the surface of the fish and not in the dermis or muscle tissues, where the lesion recorded. Histological sections visualised with either Fluorescence In Situ Hybridisation (FISH) using eubacterial probes [14] or the general nucleic acid stain DAPI (Fig. 2b,c), showed no microbial populations within the dermis, which supports the conclusion that the microbes detected using culture-independent screening were associated with the surface mucus layer of the fish. No evidence of these or other microbes such as virus like particles (VLPs) were detected using either Scanning Electron Microscopy (SEM) for surface microbes (Fig. 3a,b), or Transmission Electron Microscopy (TEM) (Fig. 4e,  f, g, h) for those within the tissues. Processing for SEM and TEM would have removed the surface mucus layer; again supporting the conclusion that few, if any, microbes were present within the dermis at the sites of pathogenesis.

Histopathological analyses

Melanin-containing cells (melanosomes) were found to be in higher density, more widespread and with a deeper distribution within the lesions than compared to healthy tissue sections (Fig. 1c,  d and Fig. 3c, d). In normal fish skin these cells are restricted to the immediate subepithelial dermis and are responsible for the pigment patterns in the integument [22]. Melanosomes are normally found to be well organised and clustered in tight groups throughout the dermis beneath the epidermal basement mem-brane (Fig. 4a, c, e, g). Sections from apparently healthy areas of skin from affected fish showed this normal pattern, whilst samples from lesional plaques, which often occurred in areas that are not normally pigmented, showed a tumourous appearance of disorganised pleomorphic cells containing melanosomes (Fig. 4b, d, f, h). Melanosomes in the lesions contained more pigment and were thought to be mature, older cells [5]. The number of melanosomes, and hence pigmentation, in the cells varied from completely absent (Cell B) to cells with plentiful melanosomes (Cell A). Melanin-specific Masson-Fontana-stained sections (Fig. 4c, d) were used to visualise these melanin-producing pigmented cells. A thickening of the integument (Fig. 4a,b double headed arrows) and extensive melanosis (development of melanotic overgrowths, which in turn is a consequence of extreme pigment cell proliferation), can clearly be seen in the cases of all lesion samples in this study and are characteristic of laboratory induced-melanomas in the Xiphophorus model [16]. Usually there is little distinction between premalignant melanosis and melanomas, whereby in the former the number of pigmented cells (melanophores) is increased but restricted to the dermis (as was the case for most of the lesions in this study), and in the latter the melanophores invade the underlying tissues. However, 5 prominent types of melanomas have previously been distinguished [16], one of which Melanophorous-Macromelanophorous Polymorphic Melanoma (MMPM) is known to be heterogeneous, with heavily and lightly pigmented areas, as observed here. Lesions contained different cell types, including melanocytes, epitheliod-like cells, melanophores and macromelanophore cells (Fig. 4f,h and Fig. 5b), consistent with MMPM. Interestingly, the majority of P. leopardus examined, exhibited lower density and coverage of skin lesions (Fig. 1a). However, this may be due to the sampling regime utilised in this paper. This was further reflective histopathologically, with stage I or stage II melanomas as described by [23], where the macromelanophores were restricted to the dermis, the meninges, the peritoneum, and the perivascular connective tissue of the blood vessels (Fig. 5b). No fish analysed in this study showed a more advanced stage of melanoma development, stage III, IV and V, where the macromelanophores penetrate the stratum compactum of the dermis and invade the underlying muscles. Fishes exhibiting this more advanced stage may show behavioural differences in the wild and may therefore have not been caught using the techniques utilised in this study. Further work to follow disease progression on captive held individuals would highlight the spread of the lesion, show the different stages of cancer, and show whether this type of melanoma is benign or malignant.

Given the strong histopathological similarities between the lesions described here in P. leopardus and the UV radiation-induced melanomas in the laboratory model Xiphophorus [5,22], along with a lack of any evidence for a pathogenic cause, we conclude that this represents the first case of melanoma in a wild fish population. As the sampled fish were collected offshore in a marine protected area with no reports of pollution, the likelihood of potential carcinogenic pollutants being the causal factor is low, at least in this reported case. UV radiation, in comparison, is known to be a causal factor in skin damage in many animals and therefore is a likely driving factor of prevalence of melanoma in P. leopardus. There is a significant correlation between average solar radiation (i.e. latitude) and melanoma mortality in humans [5,24]. UV-B (l = 280-320 nm) appears to be the most damaging radiation [25] and has previously been shown to increase in intensity as stratospheric ozone levels have decreased [26]. UV radiation in aquatic systems has previously been reported to have detrimental effects on marine and freshwater organisms, with UV penetrating as deep as 60 m in the sea [27,28,29]. Therefore P. leopardus inhabiting the clear waters of the Great Barrier Reef would be  exposed to UV radiation over a wide depth range. Individuals in this study were all captured in less than 20 m depth, well within the UV-B exposure range of 30 m [27]. Interestingly, juvenile hammerhead sharks have been shown to have the ability to 'sun tan' [30], whereby integumental pigments such as melanin increased in direct response to increases in solar radiation. The juvenile shark's skin responded similarly to that observed in humans and other vertebrates in response to direct sunlight, turning from brown to black. Although a similar melanin response was seen in this study (i.e. increased melanin concentration), the sharks in this previous study showed no visible lesions or growths and were therefore not shown to contract melanomas or dermal carcinomas.

With regard to the Xiphophorus induced melanoma model, it had long been assumed that only hybrid crosses of Xiphophorus (those bred in captivity), could be induced to contract melanomas or experience extensive melanosis. The wild (parental) types of these species in comparison, were non-susceptible to neoplasia, even after exposure to high doses of physical and chemical carcinogens [4,31]. However, in addition to this study illustrating melanosis/ melanoma induction in wild type Plectropomus, one further study on Xiphophorus also showed non-hybrid melanoma formation in a wild caught fish, however this was accredited to a build up of androgen metabolites within the holding tank [22]. Hybrid strains of Xiphophorus have been noted to have differing susceptibility to carcinogens suggesting a genetic basis for susceptibility to melanoma formation [32]. Furthermore, it has been shown that melanoma in Xiphophorus is caused by a mutated EGFR gene, Xmrk, with constitutive expression of growth factors. When Xmrk, is transplanted into another fish Oryzias latipes, they subsequently contract melanomas themselves [33,34]. Therefore, this suggests an underlying genetic predisposition to the disease that is expressed with the loss of tumour suppressor genes caused by the onset of hybridisation. The occurrence of melanoma in a wild population, particularly, at the levels observed in this study is unusual. The relatively high (15%) prevalence of this syndrome within the sampled P. leopardus population may be indicative of a similar genetic defect as that experienced during hybridisation in the laboratory, or alternatively it may be due to potential inbreeding in this portion of the P. leopardus population resulting in recessive susceptibility genes becoming homozygous. In the latter instance, inbreeding may be potentially proliferated in the local area due to recruitment of genetically related individuals to the same reef system [35]. However, hybridisation has frequently been shown to occur in wild populations of many fish species [36,37], including populations of P. leopardus which have been shown to hybridise with other Plectropomus species, such as the Barcheeked coral trout, P. maculatus [38]. Frisch and van Herwerden (2006) concluded that despite behavioural barriers to reproduction (such as assortative mating), there was considerable opportunity for hybridisation between different species of coral trout. Indeed, the same macroscopic signs of this disease have been noticed on P. maculates and one further species, the blue spotted coral trout, P. laevis, suggesting hybridisation between these species may be the most likely cause of predisposition of Plectropomus to melanomas. Current information suggests this syndrome is present throughout the Great Barrier Reef (MRH unpublished data), but prevalence appears to be highest in the southern Great Barrier Reef. This high prevalence recorded in this study further supports the presence of a genetic component to this syndrome, yet detailed, broader sampling is required to confirm the extent of prevalence in other Great Barrier Reef regions.

Coral trout, P. leopardus, is an iconic and highly valued species and the Great Barrier Reef is one of the world's most pristine and carefully managed reef habitats. Successful management of these resources is a crucial and challenging task [39]. The implications of extensive melanosis/melanoma in wild coral trout will depend on the prevalence of the syndrome outside the study region, the causal factors and the proportion which develop into fatal melanomas. However, this syndrome will no doubt have implications for the management of fish populations and the GBR marine park. Beyond health implications for individual fish, this syndrome may have implications for the population as a whole and the commercial and recreational fisheries that exploit this species. In Xiphophorus, fish with tumours usually survive around 6 months, compared to an average of 4 years in healthy fish, but any change in their environment, such as a drop in temperature can rapidly lead to death [5]. It is unclear whether future changes in the ocean environment or climate will similarly exacerbate the effect of melanomas in wild P. leopardus populations, but clearly further research is urgently needed to understand the distribution, prevalence, ecological and fisheries significance of this syndrome.

In particular, further studies should focus on UV exposure as a risk factor and confirm whether there is a genetic effect to susceptibility of the syndrome. Utilising molecular markers used to study melanomas in humans and laboratory fish models e.g. those that target the B-Raf protein [40], the EGFR gene, Xmrk, or other mitochondrial DNA status markers [41] would highlight this genetic aspect.

Figure 1 .
1Lesions were present on approximately 15% of the sampled population of Plectropomus leopardus; a) affected individual showing ,10% coverage of body surface; b) P. leopardus with almost complete coverage .90%; c) healthy tissue under light microscope and d) the lesion. Scale bars = 20 mm. doi:10.1371/journal.pone.0041989.g001

Figure 2 .
2Microbial analysis of Plectropomus leopardus samples; a) Bacterial 16S rRNA gene fingerprints (represented on Denaturing Gradient Gel Electrophoresis) of fish mucus (SWB) and tissue samples (TSU), standardised for gel-to-gel comparison using BioNumerics; b) resin embed histological section of a healthy fish, stained with the general DNA stain 496-diamidino-2-phenylindole (DAPI); c) histological section of the lesion on a diseased fish stained with DAPI, both showing no bacteria within the dermis suggesting the bacteria present in (a) are localised within the mucus layer not within the tissues. Scale bars = 10 mm. doi:10.1371/journal.pone.0041989.g002

Figure 3 .
3Microscopic images of Plectropomus leopardus tissues; a) Scanning Electron Micrograph (SEM) of the healthy tissue; b) SEM of the lesion. MGC = mucus goblet cells, M = mucus. c) Light microscope image of a healthy scale and d) light microscope image of a diseased scale, showing disorganisation of natural melanin patterns seen in (c). Scales bars = 10 mm. doi:10.1371/journal.pone.0041989.g003

Figure 4 .
4Histological section of LR white resin embedded samples of healthy and diseased Plectropomus leopardus; a) Healthy section stained with toluidine blue; b) lesion stained with toluidine blue; c) healthy section stained with melanin specific stain Masson-Fontana; d) lesion stained with Masson-Fontana; e) Transmission Electron Micorgraph (TEM) of healthy section; f) TEM of lesion; g) higher magnification of TEM in (e); h) higher magnification of TEM in (f). Scale bars for (a-f) = 10 mm; scale bars for (g) and (h) = 2 mm. D = dermis (cologne of stroma), E = epithelium, M = melanosome, N = cell nucleus, CBM = caliginous basal membrane. Double headed arrows shows thickening of the integument, characteristic of laboratory induced-melanomas in the Xiphophorus model. doi:10.1371/journal.pone.0041989.g004

Figure 5 .
5Transmission Electron Micrographs of different samples of P. leopardus exhibiting; (a) healthy tissue showing the two cell types (A & B) associated in the dermis along the collagenous basal membrane (CBM). Cell A shows localisation of melanosomes and Cell B shows absence of melanosomes in the same area. (b) Lesion showing disorganisation of pleomorphic cells (A & B) with an increase in number and spread of melanosomes. This lesion is an example of a P. leopardus suffering from stage II melanoma, where the melanosomes are restricted to the dermis. Scale bars = 10 mm. doi:10.1371/journal.pone.0041989.g005
PLoS ONE | www.plosone.org
August 2012 | Volume 7 | Issue 8 | e41989
AcknowledgmentsThe work was conducted under a permit registered to AIMS via GBRMPA with permit No: G10/33440.1. Thanks to Lyndon Llewellyn and Colin Simpfendorfer for reviewing this manuscript before submission and helpful comments and guidance.Author Contributions
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The effects of 8 weeks of whey or rice protein supplementation on body composition and exercise performance

Jordan M Joy
Ryan P Lowery
Jacob M Wilson
Martin Purpura
Eduardo O De Souza
Stephanie Mc Wilson
Douglas S Kalman
Joshua E Dudeck
Ralf Jäger
The effects of 8 weeks of whey or rice protein supplementation on body composition and exercise performance
R E S E A R C HProtein QualityLeucineWheyRice
Background: Consumption of moderate amounts of animal-derived protein has been shown to differently influence skeletal muscle hypertrophy during resistance training when compared with nitrogenous and isoenergetic amounts of plant-based protein administered in small to moderate doses. Therefore, the purpose of the study was to determine if the post-exercise consumption of rice protein isolate could increase recovery and elicit adequate changes in body composition compared to equally dosed whey protein isolate if given in large, isocaloric doses.Methods: 24 college-aged, resistance trained males were recruited for this study. Subjects were randomly and equally divided into two groups, either consuming 48 g of rice or whey protein isolate (isocaloric and isonitrogenous) on training days. Subjects trained 3 days per week for 8 weeks as a part of a daily undulating periodized resistance-training program. The rice and whey protein supplements were consumed immediately following exercise. Ratings of perceived recovery, soreness, and readiness to train were recorded prior to and following the first training session. Ultrasonography determined muscle thickness, dual emission x-ray absorptiometry determined body composition, and bench press and leg press for upper and lower body strength were recorded during weeks 0, 4, and 8. An ANOVA model was used to measure group, time, and group by time interactions. If any main effects were observed, a Tukey post-hoc was employed to locate where differences occurred.Results: No detectable differences were present in psychometric scores of perceived recovery, soreness, or readiness to train (p > 0.05). Significant time effects were observed in which lean body mass, muscle mass, strength and power all increased and fat mass decreased; however, no condition by time interactions were observed (p > 0.05).Conclusion: Both whey and rice protein isolate administration post resistance exercise improved indices of body composition and exercise performance; however, there were no differences between the two groups.

Background

Recommended levels for an adequate dietary protein intake for an adult is 0.8 grams per kilogram of body weight, the average daily intake level that is sufficient to meet the nutrient requirement of nearly all healthy individuals. The protein requirements are based on nitrogen balance, trying to achieve a balance between nitrogen intake and excretion. Protein recommendations for endurance and strength trained athletes range from 1.2 to 2.0 g/kg bw/d, reflecting the athlete's nutritional goal to increase lean body mass [1,2]. The athlete has a choice of different animal (e.g. whey, casein, egg, beef, fish) or plant protein (e.g. soy, rice, pea, hemp) sources, differing in numerous ways such as the presence of allergens (lactose, soy), cholesterol, saturated fats, digestion rate (fast, intermittent, slow absorption of amino acids), or the relative amount of individual amino acids. In contrast to dairy protein, plant protein sources are more often lower in one or more essential amino acids failing to match the requirements of a complete protein (Table 1).

Long term, periodized resistance training (RT) results in increases in skeletal muscle size and, ultimately, force generating capacity [3,4]. Sports nutrition scientists have attempted to increase training induced gains through a number of protocols, which generally attempt to augment and/or speed skeletal muscle regeneration. One such intervention has been to increase the provision of the branched chain amino acids (BCAAs), leucine, isoleucine, and valine, which make up more than one third of muscle protein [5]. The BCAAs are unique among the essential amino acids (EAAs) for their roles in protein metabolism [6], neural function [7][8][9], and blood glucose and insulin regulation [10]. Moreover, Garlick and colleagues [11] have found that BCAAs were able to stimulate skeletal muscle protein synthesis (MPS) to the same degree as all 9 EAAs. Of the BCAAs, only leucine was able to independently stimulate MPS [11]. It is well known that vigorous exercise can induce a net negative protein balance in response to both endurance and resistance training [12]. Norton and Layman proposed that consumption of BCAAs, namely leucine, could turn individuals from a negative to a positive whole-body protein balance after intense exercise [6]. In support, the consumption of a protein or EAA complex that contains sufficient leucine has been shown to shift protein balance to a net positive state after intense exercise training [6,13]. These findings led Norton and Wilson [14] to suggest that optimal protein intake per meal should be based on the leucine content of the protein consumed.

Early research indicates that 2-3 g, or up to 0.05 g/kg bodyweight, of leucine are required to maximize MPS [14][15][16]. However once this threshold has been reached, a protein's beneficial effects on MPS effectively plateaus. For example, consuming 40 grams of egg protein (4 grams of leucine) did not enhance MPS over 20 grams of egg protein (2 grams of leucine) [17].

Plant-based proteins contain approximately 6-8% leucine, and in low doses, they do not increase MPS compared to animal-based proteins, which contain approximately 8-11% leucine [18,19]. However, if leucine is added to a plant-based protein, MPS rates are not significantly different from animal-based proteins [20]. Moreover at lower doses of protein (10% of energy), animal sources stimulate MPS to a greater degree than plant sources. However, at higher doses (30% of energy), both plant and animal-based proteins have reached the amount of leucine needed to optimize MPS, resulting in no differences between the sources [21].

To date however, no research has compared higher doses of plant to animal based protein following a resistance training intervention. Large doses of rice protein isolate, an allergen-free plant protein, containing 8% leucine may be a suitable form of protein to support muscle hypertrophy in combination with RT. Based on the available data, we hypothesize that higher doses of rice protein (48 g) will be comparable to an equally high dose of whey protein in its effects on lean mass and strength when given following RT. Therefore, the purpose of this study was to investigate the effects of higher doses of rice protein compared to equally high doses of whey protein on skeletal muscle hypertrophy, lean body mass, strength and power when given following 8 weeks of periodized RT in those individuals with previous RT experience.

Methods

Experimental design

Our study consisted of a randomized, double blind protocol consisting of individuals given either 48 grams of rice or 48 grams of whey protein isolate following an acute resistance exercise bout (phase 1) and following each session during an 8 week periodized training protocol (Phase 2). Phase 1 of the study investigated the effects of protein sources on recovery 48 hours following a high volume, hypertrophy oriented resistance-training session. Phase two occurred for the remaining eight-week RT protocol, which consisted of training each muscle group twice per week using a non-linear periodized RT model. Direct ultrasound determined muscle mass, dual emissions x-ray absorptiometery (DXA) determined body composition, maximal strength, and power were assessed collectively at the end of weeks 0, 4, and 8.

Subjects

Twenty-four healthy males (21.3 ± 1.9 years, 76.08 ± 5.6 kg, 177.8 ± 12.3 cm) participated in the study. As inclusion criteria, it was required that all subjects cease taking nutritional supplements for three months prior to the study, had participated in RT at least 3 times per week for the past six months, and had a minimum of 1 year of RT experience. Subjects were carefully matched by age, body mass, strength, and resistance training experience, then randomly placed into either the rice (n = 12) or the whey (n = 12) group. All procedures were approved by the University of Tampa's Institutional Review Board.

Phase 1 resistance training protocol

All subjects participated in a high volume resistance training session consisting of 3 sets of leg press, bench press, and military press, pull-ups, bent over rows, barbell curls and extensions. Immediately following the workout, subjects consumed 48 grams of RPI or WPI respectively. Immediately prior to the exercise session and 48 hours post exercise, soreness, perceived readiness to train, and perceived recovery scale (PRS) measurements were taken. Soreness was measured on a visual analogue scale ranging from 0-10. With zero representing no soreness in the muscles at all, and 10 representing the worst muscle soreness ever experienced. PRS consists of values between 0-10, with 0-2 being very poorly recovered with anticipated declines in performance, 4-6 being low to moderately recovered with expected similar performance, and 8-10 representing high perceived recovery with expected increases in performance. Perceived readiness indicates how ready the subject felt they were to train. In this scale a 10 is the most ready an individual could be to train, while a 0 indicates the subject feels they are not ready at all to train.

Resistance training protocol

Our resistance training protocol was a modified combination from Kraemer et al. [22] and Monteiro et al. [3]. These researchers found that a non-linear resistancetraining program yielded greater results than a traditional or non periodized program in athletes. The program was designed to train all major muscle groups using mostly compound movements for the upper and lower body. The programmed, non-linear training split was divided into hypertrophy days consisting of 8-12 RM loads for 3 sets, with 60-120 seconds rest and strength days consisting of 2 to 5 RM loads for 3 sets for all exercises except the leg press and bench press which received 5 total sets. Weights were progressively increased by 2-5% when the prescribed repetitions could be completed. All training sessions were closely monitored by the researchers to ensure effort and intensity were maximal each training session.

Strength, power, body composition and skeletal muscle hypertrophy testing

Strength was assessed via 1-RM testing of the leg press and bench press. Each lift was deemed successful as described by International Powerlifting Federation rules [23]. Body composition (lean body mass, fat mass, and total mass) was determined on a Lunar Prodigy DXA apparatus (software version, enCORE 2008, Madison, Wisconsin, U.S.A.). Skeletal muscle hypertrophy was determined via changes in ultrasonography determined combined muscle thickness of the biceps brachii and vastus lateralis (VL) and vastus intermedius (VI) muscles (General Electric Medical Systems, Milwaukee, WI, USA).

Power was assessed during a maximal cycling ergometry test. During the cycling test, the volunteer was instructed to cycle against a predetermined resistance (7.5% of body weight) as fast as possible for 10 seconds [24]. The saddle height was adjusted to the individual's height to produce a 5-10°knee flexion while the foot was in the low position of the central void. A standardized verbal stimulus was provided to the subjects. Power output was recorded in real time by a computer connected to the Monark standard cycle ergometer (Monark model 894e, Vansbro, Sweden) during the 10-second sprint test. Peak power (PP) was recorded using Monark Anaerobic test software (Monark Anaerobic Wingate Software, Version 1.0, Monark, Vansbro, Sweden). From completion of wingate tests performed over several days, interclass correlation coefficient for peak power was 0.96.

Supplementation and diet control

Two weeks prior to and throughout the study, subjects were placed on a diet consisting of 25% protein, 50% carbohydrates, and 25% fat by a registered dietician who specialized in sport nutrition. Subjects met as a group with the dietitian, and they were given individual meal plans at the beginning of the study. Daily total of calories were determined by the Harris-Benedict equation and tracked by weekly logs to ensure compliance. The protein supplement was administered under supervision of a laboratory assistant following resistance training, and it consisted of either The amino acid profile of the study material was analyzed by an independent analytical laboratory (Eurofins Analytical Laboratories, Metairie, LA) and is displayed in Table 2. Both the whey protein supplement and rice protein supplement were isonitrogenous, isocaloric, and macronutrient ratio matched. All supplements were tested by HFL Sports Science prior to use to ensure no contamination with steroids or stimulants according to ISO 17025 accredited tests.

Statistics

An ANOVA model was used to measure group, time, and group by time interactions for both phase 1 and 2. If any main effects were observed, a Tukey post-hoc was employed to locate where differences occurred. All statistics were run using Statistica software (Statsoft, 2011).

Results

Phase 1

No differences existed between groups at baseline for any measure. There were no differences in the total amounts of weight lifted by the RPI (12296.3 ± 2412.6 kg) or WPI (11831.6 ± 2611.3 kg) group during the resistance training session. There was a significant time effect (p <0.05) for soreness, which increased in both the RPI (0.3 ± 0.6 to 5.6 ± 2.2) and WPI (0.3 ± 0.5 to 6.0 ± 1.9) groups, with no differences between groups (no condition X time effect). There was a significant time effect (p <0.05) for PRS, which decreased in both the RPI (9.1 ± 1.5 to 5.45 ± 1.5) and WPI (8.7 ± 2.6 to 5.6 ± 1.4) groups, with no differences between groups (no condition X time effect). There were no significant time or condition x time effects for perceived readiness to train, indicating that the subject's perceived readiness had recovered within 48 hours.

Phase 2

There was a significant time effect (p <0.01) for lean body mass, which increased in both the rice (58.5 ± 5.5 (baseline) to 59.5 ± 4.5 (week 4) to 61.0 ± 5.6 kg (week 8) and whey protein (59.6 ± 5.2 to 61.9 ± 4.5 to 62.8 ± 5.2 kg) conditions, with no differences between conditions (no condition X time effect). There was a significant time effect for body fat (p < 0.05), which decreased in both conditions, 17.8 ± 6.0 to 16.6 ± 4.8 to 15.6 ± 4.9 kg in the rice protein condition and 16.3 ± 5.1 to 15.7 ± 4.8 to 15.6 ± 4.9 kg in the whey protein condition, from pre to post training, with no differences between conditions (no condition X time effect). There was a significant time effect for quadriceps and biceps thickness (p < 0.05), which increased from pre to post training in the rice protein (5.0 ± 0.4 to 5.1 ± 0.4 to 5.2 ± 0.5 cm and 3.6 ± 0.3 to 3.9 ± 0.3 to 4.1 ± 0.4 cm, respectively) and whey protein (4.8 ± 0.7 to 5.0 ± 0.5 to 5.1 ± 0.5 cm and 3.6 ± 0.2 to 4.0 ± 0.3 to 4.1 ± 0.3 cm, respectively) conditions, with no differences between conditions (no condition X time effect). Body composition data is displayed in Figure 1.

There was a significant time effect (p < 0.01) for 1-RM bench press strength, which increased from baseline to week 8 in both the rice protein (85.9 ± 20.5 to 95.5 ± 21.4 kg) and whey protein (89.5 ± 18.5 to 98.5 ± 16.4 kg ) conditions, with no differences between groups (no condition X time effect). There was a significant time effect (p < 0.01) for 1-RM leg press strength, which increased from baseline to week 8 in both the rice (220.0 ± 38.5 to 286.8 ± 37.2 kg) and whey (209.5 ± 35.0 to 289.7 ± 40.1 kg) conditions, with no differences between conditions (no condition X time effect). There was a significant time effect for wingate peak power (p < 0.01), which increased from baseline to week 8 in both the rice protein (638.4 ± 117.2 to 753.9 ± 115.6 watts) and whey protein (687.1 ± 125.3 to 785.0 ± 101.1 watts) conditions, with no differences between conditions. Performance data is displayed in Table 3.

Discussion

The novel finding in the present study is that no significant condition by time interactions were observed between the rice protein and whey protein supplements on short term recovery or training-induced adaptations. Our findings support the proposed hypothesis that higher doses of rice protein (48 g) will be comparable to an equally high dose of whey protein in its effects on body composition and exercise performance after periodized RT. In other words, RPI supports changes in strength and body composition similarly to WPI. Subjects were given either 48 g of protein in the form of a rice or whey protein supplement. At these doses, the rice protein supplement contained approximately 3.8 g of leucine whereas the whey protein supplement contained 5.5 g of leucine. At these doses, both supplements are predicted to reach levels necessary to optimize muscle protein accretion [14] they are also greater than the amounts observed in prior research [25,26]. Moore et al. [25] conducted a dose response study of an egg protein supplement comparing 0 g, 5 g, 10 g, 20 g, and 40 g of egg protein delivered after a bout of exercise. After consumption of the supplement, MPS rates were monitored for four hours. Their results suggested that MPS was maximally stimulated with 20 g of egg protein, which contains 1.7 g of leucine. It was also observed that at double that dose (40 g, 3.4 g of leucine), no significant differences in MPS occurred.

Chronic free leucine supplementation alone did not improve lean body or muscle mass during resistance training in the elderly, whereas it was able to limit the weight loss induced by malnutrition. Leucine-rich amino acid mixtures or proteins appeared more efficient than leucine alone to improve muscle mass and performance, thereby suggesting the efficacy of leucine depends on the presence of other amino acids. Small differences in protein digestion rates, differences in branched-chain amino acid content can impact the ability of the protein to maximize post exercise MPS. Available data on soy protein suggests that plant proteins might differ in their ability to support muscle protein  accretion after resistance exercise [17,26]. For example, post exercise consumption of fat-free milk promotes greater hypertrophy during the early stages of resistance training in novice weightlifters when compared with isonitrogenous and isoenergetic fat-free soy protein [26].

Hartman et al. [26] conducted research comparing milk protein, to soy protein, to a maltodextrin control in untrained individuals. In Hartman's study, 17.5 g of protein in the form of milk or soymilk was given immediately and one hour following exercise, while the control group received an isocaloric maltodextrin beverage. 17.5 g of protein from milk contains approximately 1.7 g of leucine, and 17.5 g of protein from soymilk would contain 1.4 g of leucine. Following a twelve week RT program, the milk protein group experienced greater increases in type II muscle fiber area. This study suggests that a moderate dose of milk protein increases lean mass to a greater extent than soy or a maltodextrin control when given following exercise. Soy proteins appear to support greater splanchnic rather than peripheral (i.e., muscle) protein synthesis and are converted to urea to a greater extent than are milk proteins. Alternatively, observed differences might be explained by differences in leucine content or absorption kinetics.

In the present study, the combined muscle thickness of the VI and VL increased in both the rice protein (0.2 cm) and whey protein (0.5 cm) conditions. Lean body mass increased in the rice protein condition by 2.5 kg, and it also increased in the whey protein condition by 3.2 kg. Combined bench press and leg press 1-RM strength increased in the rice protein condition by 76.4 kg and in the whey protein condition by 89.5 kg. However, no significant differences were observed between the two conditions for any measure. The collective findings of our study and others suggests that as the amount of protein consumed increases, the importance of the relative leucine content of the protein diminishes (see Figure 2) [21,27].

Study limitations

Limitations of this study include the duration of the research and the lack of a non-supplemented control group. While no significant effects were observed between groups, potential differences in effects on body composition and exercise performance between groups may be more evident if examined over a longer duration. Without a nonsupplemented control group, we cannot conclude how beneficial protein supplementation was to resistance training in this study.

Conclusion

The present results suggest that differences in protein composition are of less relevance when protein is consumed in high doses throughout periodized RT. Rice protein isolate consumption post resistance exercise decreases fat-mass and increases lean body mass, skeletal muscle hypertrophy, power and strength comparable to whey protein isolate.

Figure 1
1Changes in (A) Lean Body mass, (B) Body fat, (C) Biceps muscle thickness and (D) Quadriceps muscle thickness. * Indicates significantly different from baseline. # Indicates significantly different from week 4.

Table 1
1Essential amino acids profile of a complete protein in comparison to whey protein isolate and rice protein isolate used in this study (Eurofins Analytical Laboratories, Metairie, LA)Essential amino acid
[mg/g of protein]

Complete
protein

Whey protein
isolate

Rice protein
isolate

Tryptophan
7
22
14

Threonine
27
76
35

Isoleucine
25
70
41

Leucine
55
115
80

Lysine
51
101
31

Methionine + Cystine
25
48
49

Phenylalanine + Tyrosine
47
64
101

Valine
32
64
58

Histidine
18
18
22

Table 2
2Amino acid profile of the study materialsAmino acid [mg/g of protein]
Whey protein
isolate

Rice protein
isolate

Alanine
54
54

Arginine
23
77

Aspartic Acid
118
87

Cystine
25
21

Glutamic Acid
191
174

Glycine
19
43

Histidine
18
22

Isoleucine
70
41

Leucine
115
80

Lysine
101
31

Methionine
23
28

Phenylalanine
33
53

Proline
64
45

Serine
52
49

Threonine
76
35

Tryptophan
22
14

Tyrosine
31
47

Valine
64
58

Table 3
3Changes in strength and powerData is displayed as mean ± standard deviation, * denotes significant change from baseline (p ≤ 0.05).Baseline
Week 4
Week 8

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AcknowledgementThe authors would like to thank a dedicated group of subjects. The authors would like to thank Increnovo LLC, Milwaukee, WI, for funding this research.Competing interestsThe authors declare that they have no competing interests.Authors' contribution JMJ, RPL and JMW developed the study design, supervised and trained subjects, performed the statistical analysis, participated in data acquisition and drafting the manuscript. RJ developed the study design and participated in drafting the manuscript. MP assisted with the design of the study and drafting the manuscript. EODS assisted in drafting of the manuscript and performing the statistical analysis. SMCW was responsible for diet control and also assisted in drafting the manuscript. JED supervised subjects and assisted in drafting the manuscript. DSK assisted in study conception and writing of the manuscript. All authors read and approved the final manuscript.Submit your next manuscript to BioMed Central and take full advantage of:
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The effects of 8 weeks of whey or rice protein supplementation on body composition and exercise performance. Joy, Nutrition Journal. 1286Cite this article asCite this article as: Joy et al.: The effects of 8 weeks of whey or rice protein supplementation on body composition and exercise performance. Nutrition Journal 2013 12:86.