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Muscle melanisation of southern sand flathead (Platycephalus bassensis) in the Tamar Estuary, Tasmania, Australia
Environmental Pollution xxx (xxxx) xxx
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Environmental Pollution journal homepage: www.elsevier.com/locate/envpol
Muscle melanisation of southern sand flathead (Platycephalus bassensis) in the Tamar Estuary, Tasmania, Australia* Clayton W. Stocker a, James Haddy a, Jeremy Lyle b, Barbara F. Nowak a, * a
Fisheries and Aquaculture Centre, Institute for Marine and Antarctic Studies, University of Tasmania, Locked Bag 1370, Launceston, 7250, Tasmania, Australia b Fisheries and Aquaculture Centre, Institute for Marine and Antarctic Studies, 15-21 Nubeena Crescent, Taroona, 7053, Tasmania, Australia
a r t i c l e i n f o
a b s t r a c t
Article history: Received 11 August 2019 Received in revised form 6 October 2019 Accepted 20 October 2019 Available online xxx
Tasmanian recreational fishers have reported the presence of dark pigmentations in the usually white fillets of southern sand flathead (Platycephalus bassensis), a phenomenon known as muscle melanisation. Based on histology, it is suggested that eumelanin and pheomelanin are involved in the occurrence of the phenomenon. A gross melanisation scoring system was validated through a comparison with an image analysis technique, that quantified the percentage surface area of the fillets affected by muscle melanisation. The occurrence of muscle melanisation was most severe in fish inhabiting Deceitful Cove, Tamar Estuary. This indicated that muscle melanisation in P. bassensis may be caused by yet to be identified site specific factors. No significant relationships were evident between the percentage surface area of melanised muscle with condition index, age, sex, maturation stage, fish weight, fish length and size of melano-macrophage centres in the liver or spleen. Overall, this study has provided critical information that will frame the direction and focus of future P. bassensis muscle melanisation research. © 2019 Elsevier Ltd. All rights reserved.
Keywords: Muscle melanisation Melano-macrophage centres Melanin Tamar estuary Southern sand flathead
1. Introduction Dark pigmentation of the usually white fillets in fish is a phenomenon known as muscle melanisation. There are two reported forms of muscle melanisation; the diffuse deposition of melanin (fillet greying) and the localised deposition of melanin (black pigmentation) in fillets. The former was reported in farmed barramundi (Lates calcarifer) where melanin was the cause of ‘fillet greying’ (Howieson et al., 2013). Examples of the latter have been reported in a range of species, including; farmed Atlantic salmon (Salmo salar) (Bjorgen et al., 2015), wild and farmed Atlantic cod (Gadus morhua) (Cooper et al., 2011), wild Portuguese pouting (Trisopterus luscus) (Esteves et al., 2009; Ramos et al., 2019) and wild southern sand flathead (Platycephalus bassensis) (Ooi et al., 2019). The causes of the phenomenon are varied, including: a response to vaccination (Bjorgen et al., 2015; Larsen et al., 2014) or increased immune response to bacterial or viral infections (Bjorgen et al., 2015; Krasnov et al., 2016) in S. salar, excessive levels of dietary copper (Cu) in G. morhua (Cooper et al., 2011), and a parasitic
* This paper has been recommended for acceptance by Dr. Sarah Harmon. * Corresponding author. E-mail address: [email protected] (B.F. Nowak).
infection in T. luscus (Esteves et al., 2009; Ramos et al., 2019). In the case of P. bassensis, the cause of muscle melanisation remains unknown (Ooi et al., 2019). Melanin pigments are complex polymers that are found in three forms within vertebrates, including eumelanin, pheomelanin and neuromelanin (Adachi et al., 2005; Slominski et al., 2004). Eumelanin is commonly recorded in fish species (Cal et al., 2017), whereas there are only a few reports of pheomelanin in teleosts (Adachi et al., 2005; Dang et al., 2019; Kottler et al., 2015; Kumar et al., 2016). There are many factors that can influence the level of melanin production (melanogenesis) within fish, including; injury or infection (Fagerland et al., 2013; Larsen et al., 2012), heavy metals (Bowness and Morton, 1953; McGraw, 2003), ultraviolet radiation (Adachi et al., 2005) and dietary imbalances (Adachi et al., 2005). Melano-macrophages (pigment-producing leukocytes) capable of melanogenesis, often cluster together forming melanomacrophage centres (MMCs) (Agius and Roberts, 2003; Evans and Nowak, 2016; Kumar et al., 2016; Thorsen et al., 2006). Located in fish haemopoietic organs, MMCs are commonly found in the spleen, head kidney and to a lesser extent, the liver (Agius and Roberts, 2003; Evans and Nowak, 2016; Kumar et al., 2016). Factors that alter the size, prevalence and morphology of MMCs vary
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Please cite this article as: Stocker, C.W et al., Muscle melanisation of southern sand flathead (Platycephalus bassensis) in the Tamar Estuary, Tasmania, Australia, Environmental Pollution, https://doi.org/10.1016/j.envpol.2019.113452
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between different species and internal organs, including; seasonal variation (Kumar et al., 2016), age (Agius, 1981; Passantino et al., 2014), sex (Coffey et al., 2009; Ferreira, 2011), parasite loading and infectious diseases (Evans and Nowak, 2016; Steinel and Bolnick, 2017). There have been previous reports of MMCs in the liver and spleen of P. bassensis showing links between an increase in the size and abundance of MMCs with suboptimal health of P. bassensis (Fu et al., 2017; Langdon, 1986). Currently, it is unknown if there is any association between melanised muscle and the presence of MMCs in the internal organs. Commonly living over sand or mud, P. bassensis is a benthic species, endemic to the southern regions of Australia and abundant in both coastal and estuarine waters (Bani et al., 2009; Ewing et al., 2014; Imamura, 2015; Jordan, 2001). In both Tasmania’s recreational and commercial fishing, P. bassensis is one of the key target species (Ewing et al., 2014; Frijlink and Lyle, 2013). Recently, there has been an increase number of inquiries from the recreational fishers regarding the presence of dark pigmentation in P. bassensis fillets (Ooi et al., 2019). The occurrence of the phenomenon in Tasmanian waters could affect recreational fishing activities, tourism industry and commercial fishing sector (Attard et al., 2012; Stocker et al., 2019). Preliminary histological studies into P. bassensis muscle melanisation have classified the phenomenon as the localised deposition of melanin (Ooi et al., 2019). The previously reported causes of the localised deposition of melanin in wild G. morhua and T. luscus have been investigated in the case of P. bassensis (Cooper et al., 2011: Esteves et al., 2009; Ramos et al., 2019). A parasitological investigation and heavy metal analysis found no association of parasites or copper with the presence of P. bassensis muscle melanisation (Ooi et al., 2019). Studies based in the Tamar Estuary have suggested site specificity at Deceitful Cove, and a higher concentration of zinc in melanised muscle compared to non-melanised muscle (Ooi et al., 2019). There is a widespread distribution of P. bassensis with muscle melanisation around Tasmania, with notable prevalence within the Tamar Estuary and south-eastern Tasmania (including; Derwent Estuary, D’Entrecasteaux Channel,
Norfolk-Frederick Henry Bays and the Tasman Peninsula) (Ooi et al., 2019). This was further emphasised through a state-wide questionnaire-based survey of recreational fishers (Stocker et al., 2019). This study aimed to develop a method to quantify the severity of observed muscle melanisation in P. bassensis (as a percentage surface area of the fillet) and implement this technique to; validate a gross melanisation scoring system, quantify the site specific occurrence and severity of muscle melanisation in the Tamar Estuary, and explore relationships between muscle melanisation severity and variables that directly or indirectly influence the production of melanin in fish. A histological examination was implemented to characterise P. bassensis melanised muscle. 2. Materials and methods 2.1. Study area The sampling locations were all in the Tamar Estuary, Tasmania and included; Deceitful Cove, Inspection Head, West Arm and Middle Island (Fig. 1). The P. bassensis sampling was approved by the University of Tasmania Animal Ethics Committee (Permit no. A0017197). Sampling locations and procedures were selected based on previous studies (Ooi et al., 2019). 2.2. Sample collection A total of 60 P. bassensis were caught with a baited hook and line from the Tamar Estuary (n ¼ 15 from each sampling location) during three sampling trips in April and May of 2018 (Autumn). Once an individual was landed it was euthanised by immersion in an AQUI-S bath (American Veterinary Medical Association, 2013; AQUI-S, 2013). Following euthanasia, fish were processed to collect data on total fish length (cm ± 1 mm) and total fish weight (g ± 0.1g). Both otoliths were extracted from the ventral brain case (Ewing et al., 2007; Jordan, 1998), and the sex (male, female, indeterminate) and maturation stage (1 ¼ immature, 2 ¼ early developing, 3 ¼ developing, 4 ¼ late developing, 5 ¼ ripe,
Fig. 1. Tamar Estuary sampling locations: A map showing the sampling locations (Deceitful Cove, Inspection Head, Middle Island and West Arm) within the Tamar Estuary, Tasmania. Black square ¼ Tamar Estuary.
Please cite this article as: Stocker, C.W et al., Muscle melanisation of southern sand flathead (Platycephalus bassensis) in the Tamar Estuary, Tasmania, Australia, Environmental Pollution, https://doi.org/10.1016/j.envpol.2019.113452
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6 ¼ spent) were determined according to adopted criteria detailed in Bani et al. (2009) and Haddy (2000). Liver and spleen samples were collected and fixed in 10% neutral buffered formalin (NBF). After filleting, the observed muscle melanisation in each individual was recorded based on a visual interpretation (gross melanisation scoring system) of the extent of dark pigmentation in the fillets (gross scores: 0 ¼ 0%; 1 ¼ < 5%; 2 ¼ 5e20%; 3 ¼ > 20%). Melanised individuals with highly concentrated localised areas of dark pigmentation, had small samples (5 5 mm) of melanised and non-melanised muscle tissues collected and fixed in NBF (n ¼ 11). Fillets were transported on ice in individual plastic bags, prior to being frozen and stored at 20 C. Histological samples were not collected for Middle Island due to logistics. 2.3. Histological processing Following the initial transfer of samples from NBF to 70% ethanol, samples were processed for histology. Briefly, samples were embedded in paraffin blocks (Shandon Histocentre 3) and a section of each sample was cut at 5 mm (Microm HM340) and stained with haematoxylin and eosin (H&E). H&E was considered the standard method for MMCs quantification. Melanised and non-melanised muscle samples that were collected from the 11 P. bassensis had two sections prepared, one stained with H&E and the second one with Fontana Masson. The combination of the two stains has been previously implemented to indicate an initial detection of two forms of melanin (eumelanin and pheomelanin) (Dang et al., 2019; Okazaki et al., 2015). These samples were observed under a light microscope at 400x magnification (Leica DM 500) and were examined for pigmentations. The association of pigments with muscle fibres or blood vessels in Fontana Masson stained sections were recorded. 2.4. Image capture 2.4.1. Histology image capture From each H&E slide, 5 images were captured of each liver and spleen with a light microscope at 100x magnification (Leica DM 500 with Leica ICC50W Camera) and LAS EZ software (brightness ¼ 50%, Gamma ¼ 3.00, Saturation ¼ 64.00) (Leica Microsystems, 2008). A systematic approach was implemented, with an image captured in each corner and centre of every section. Images were required to have the section covering a minimum of 50% of the field of view and no overlap between images captured from the same organ.
2.5.1. Histology images The protocols implemented for the image analysis of liver and spleen histology samples were adaptations of those previously described (Dang et al., 2019; Evans and Nowak, 2016). Initially, the image was calibrated and a colour deconvolution with H&E vectors was completed. Total surface area of the liver and spleen in the field of view was measured (mm2), through adjustments to the image grey value threshold on the second deconvolution window, until all pixels within the organ were selected (grey value threshold ranges: Liver ¼ 143e165, Spleen ¼ 140-160). The surface area of MMCs in the liver and spleen were subsequently measured (mm2), through adjustments to the image grey value threshold on the third colour deconvolution window, until all MMCs (no size or shape restrictions) were selected (threshold ranges: Liver ¼ 140e180, Spleen ¼ 140-170). Voids and artefacts (over 1000 mm2) were individually selected and removed from all measurements. 2.5.2. Fillet images Initially, the original RGB colour image was calibrated and transformed into an 8-bit image. Total surface area of the entire fillet was measured (mm2) through adjustments to the image grey value threshold until all pixels within the fillet area were selected (grey value threshold range ¼ 85e107). The surface area of melanised muscle was measured (mm2) by reducing the image grey value threshold until the remaining selected pixels mirrored the extent of dark pigments observed in the original image (grey value threshold range ¼ 186e218). 2.6. Otolith processing and interpretation Each otolith had three transverse sections prepared through standard otolith processing (Ewing et al., 2014; Jordan, 1998). To maintain consistency in the ageing process, the otoliths selected for processing were from the right side of the fish where available. Otolith sections were examined under a light microscope (Leica M28) at 16x magnification and a validated ageing protocol for P. bassensis was implemented (Ewing et al., 2014; Jordan, 1998). 2.7. Data analysis 2.7.1. Data preparation The condition index for P. bassensis was calculated by applying the Fulton’s Condition Index (Nash et al., 2006):
K ¼ 100
2.4.2. Fillet image capture All fillets were thawed, and excess skin was removed from the edges. A light box with a white background was used for scaling and lighting consistency (Grunenwald et al., 2018) and an image of the fillets captured with a FujiFilm X20 camera (WB ¼ 3200, F ¼ 2.8, SS ¼ 640, ISO ¼ 800).
Surface Area Melanised Muscle ð%Þ ¼
3
W L3
where K ¼ Condition Index, W ¼ Fish Weight (g), L ¼ Total Fish Length (cm). Ratios for sex and maturation stage were established and means or percentages were calculated for all the variables at each sampling location (Table 1). The percentage surface area of melanised muscle on P. bassensis fillets was quantified:
Surface Area Melanised Muscle * 100: Total Fillet Area
2.5. Image analysis Image J (Fiji) software was used for image analysis (Schindelin et al., 2012).
The percentage surface area of MMCs in the liver and spleen of P. bassensis was calculated:
Please cite this article as: Stocker, C.W et al., Muscle melanisation of southern sand flathead (Platycephalus bassensis) in the Tamar Estuary, Tasmania, Australia, Environmental Pollution, https://doi.org/10.1016/j.envpol.2019.113452
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Table 1 Characteristics of P. bassensis sampled from the sampling locations in the Tamar Estuary. Different letters indicate significant differences between means across sampling locations (p 0.05). (mean ± SE). Variables
Deceitful Cove
Inspection Head
West Arm
Middle Island
n Condition Index Age Sex (M:F:I) Maturation Stage (1:2) Fish Weight (g) Fish Length (cm)
15 0.55 ± 0.01ab 1.93 ± 0.07a (4:11:0) (0:15) 148.40 ± 10.96ab 29.65 ± 0.64a
15 0.53 ± 0.01a 2.33 ± 0.23a (5:10:0) (0:15) 124.27 ± 11.92a 28.18 ± 0.72a
15 0.60 ± 0.01c 2.00 ± 0.00a (4:10:1) (1:14) 164.27 ± 10.58b 29.87 ± 0.68a
15 0.59 ± 0.01bc 2.13 ± 0.09a (8:7:0) (0:15) 161.40 ± 7.51ab 30.02 ± 0.38a
Surface Area MMC ð%Þ ¼
Surface Area MMC * 100: Total Tissue Area
Masson and H&E stained consecutive sections. Furthermore, the presence of pigments associated with muscle fibres or blood vessels was recorded and compared between the sampling locations and the gross scores (gross melanisation scoring system). 3. Results
2.7.2. Statistical analysis All statistical analyses were completed using the statistical program SPSS, with a set p-value of 0.05 (IBM Corp, 2016). Nonparametric tests were applied when assumptions were violated (Shapiro-Wilk test; Levene statistic; scatter plot), including; Kruskal-Wallis tests, median tests and Spearman’s correlations. To identify any site specificity within the Tamar Estuary, the difference in the percentage surface area of melanised muscle between sampling locations was statistically analysed with a KruskalWallis test and pairwise comparison post hoc test (adjusted by Bonferroni correction). The statistical analyses of P. bassensis variables with the percentage surface area of melanised muscle was completed for Deceitful Cove and Inspection Head separately, and then also for all the Tamar Estuary sampling locations pooled together. Statistical analysis for individual locations for West Arm and Middle Island were excluded due to the limited presence of muscle melanisation. There was an indeterminate individual sampled in West Arm that was excluded from statistical analyses relating to sex. A multiple regression analysis was employed to determine the relationships of P. bassensis condition index, sex, age and maturation stage, with the percentage surface area of melanised muscle. The fish weight and fish length were excluded from this statistical analysis due to multicollinearity with condition index and age. Therefore, Spearman’s correlations were implemented to statistically analyse these associations. Spearman’s correlations were applied to test the associations between the percentage surface area of MMCs in the liver and spleen, with the percentage surface area of melanised muscle. The differences in the percentage surface area of MMCs in the liver and spleen between Tamar Estuary sampling locations were statistically analysed using both a one-way analysis of variances (ANOVAs) and a Kruskal-Wallis test with a pairwise comparison post hoc test (adjusted by Bonferroni correction). Gross melanisation scoring system was statistically compared with the percentage surface area of melanised muscle with a Kruskal-Wallis test and pairwise comparison post hoc test (adjusted by Bonferroni correction). Furthermore, ANOVAs, Kruskal-Wallis tests, median tests and Pearson’s chi-squared tests were implemented to assess the relationship of sampling location, condition index, sex, age, maturation stage, fish weight, fish length and MMCs in the liver and spleen with the gross scores. These statistical results were compared to those recorded for the percentage surface area of melanised muscle. Based on histology, the presence or absence of pigments within melanised and non-melanised muscle samples was recorded. The extent of pigments present was then compared between Fontana
3.1. Gross muscle melanisation The highest prevalence of P. bassensis with muscle melanisation was in Deceitful Cove (80% of individuals), followed by Inspection Head (40% of individuals). The occurrence of the phenomenon was relatively low in fish from West Arm (13.3% of individuals) and Middle Island (6.7% of individuals). There was a significant difference in the percentage surface area of melanised muscle between the sampling locations in the Tamar Estuary (Kruskal-Wallis: x2 ¼ 26.938, df ¼ 3, p < 0.001). Pairwise comparisons revealed the significant differences were between Deceitful Cove and Inspection Head (p ¼ 0.030), West Arm (p < 0.001) and Middle Island (p < 0.001) and there were no significant differences between any of the three other sampling locations. 3.2. Associations between P. bassensis variables and melanised muscle There were no significant relationships between the percentage surface area of melanised muscle and condition index, age, sex and maturation stage of P. bassensis in Deceitful Cove, Inspection Head and the Tamar Estuary (Table 2). The overall fit of each multiple regression model had no statistical significance (Table 2). Fish weight, fish length and percentage surface area of MMCs in the liver and spleen were not significantly associated with the percentage surface area of melanised muscle in Deceitful Cove, Inspection Head and the Tamar Estuary (Table 3). 3.3. MMCs and sampling location There was a significant difference in the percentage surface area of MMCs in the liver of P. bassensis, between the Tamar Estuary sampling locations (Kruskal-Wallis: x2 ¼ 7.282, df ¼ 2, p ¼ 0.026). Pairwise comparisons revealed the significant difference was between Inspection Head and West Arm (p ¼ 0.037). In contrast, there was no significant difference in the percentage surface area of MMCs in the spleen between the Tamar Estuary sampling locations (one-way ANOVA: F ¼ 2.546, df ¼ 44, p ¼ 0.090). 3.4. Validation of gross melanisation scoring system 3.4.1. Melanised muscle and gross scores There was a significant difference in the percentage surface area of melanised muscle between the gross scores (Kruskal-Wallis Test: x2 ¼ 25.118, df ¼ 3, p < 0.001) (Fig. 2). A pairwise comparison revealed that the significant difference was between gross score
Please cite this article as: Stocker, C.W et al., Muscle melanisation of southern sand flathead (Platycephalus bassensis) in the Tamar Estuary, Tasmania, Australia, Environmental Pollution, https://doi.org/10.1016/j.envpol.2019.113452
0.012 0.079 0.023
p F
0.043 0.313 0.439
r2
Model Fit
0.987 0.816 0.726
C.W. Stocker et al. / Environmental Pollution xxx (xxxx) xxx
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0 and scores 1 (p ¼ 0.010), 2 (p < 0.001), and 3 (p ¼ 0.002) and there was no significant differences between any of the three other gross scores. 3.4.2. Comparing statistical analyses results of muscle melanisation assessment methods Both muscle melanisation assessment methods gave consistent results. A significant relationship was identified between the Tamar Estuary sampling locations and the gross scores (Table 4). Furthermore, there were no significant relationships between any P. bassensis variables (condition index, age, sex, maturation stage, fish weight, fish length and MMCs in the liver and spleen) and the gross scores within Deceitful Cove, Inspection Head and the Tamar Estuary (Table 4). These results directly correspond with the results obtained using the percentage surface area of melanised muscle.
0.047 0.144 0.086 0.898 0.592 0.660 0.794 0.632 0.396 Deceitful Cove Inspection Head Tamar Estuary
0.081 0.171 0.114
0.045 0.193 0.059
0.891 0.658 0.523
Constant Constant Constant
b b p
b p
b
p
Maturation Stage Sex Age Condition Index
Multiple Regression Analysis
Table 2 Multiple regression analysis results for the relationships between P. bassensis variables and the percentage surface area of melanised muscle.
p
3.5. Histology of melanised muscle No pigmentation was observed within the non-melanised muscle samples. In contrast, all melanised muscle samples had pigments visible in the two consecutive histological sections, stained with Fontana Masson or H&E (Figs. 3 and 4). The absence of pigments in the non-melanised muscle samples acts as a control, confirming the pigments observed are melanin and not formalin. There were occasionally melanin positive areas in the Fontana Masson stained sections, that were absent in H&E (Figs. 3 and 4). This suggests both eumelanin (Fontana Masson positive, H&E present) and pheomelanin (Fontana Masson positive, H&E absent) contribute to P. bassensis muscle melanisation. Melanin association with muscle fibres was present in 10 of the 11 P. bassensis sampled (Fig. 3). Blood vessels were not present in the histology sections of four P. bassensis. Melanin association with blood vessels was observed in all the remaining P. bassensis sampled (n ¼ 7) (Fig. 4). The results observed were consistent across all sampling locations and gross scores (gross melanisation scoring system). 4. Discussion Melanin was only observed in histological sections of melanised muscle samples and not at all in those non-melanised. This confirmed that the localised deposition of melanin is responsible for the dark pigmentations reported in P. bassensis fillets (Ooi et al., 2019). The presence of melanin in both Fontana Masson and H&E stained samples, suggests that the pigment present is eumelanin (Dang et al., 2019). Melanin that was observed in Fontana Masson staining but not in H&E stained samples, is most likely pheomelanin (Dang et al., 2019). The results of this suggest that both eumelanin and pheomelanin are involved in the occurrence of P. bassensis muscle melanisation. This discovery conforms with recent studies suggesting the presence of pheomelanin in teleosts (Adachi et al., 2005; Dang et al., 2019; Kottler et al., 2015; Kumar et al., 2016). It is still uncertain whether pheomelanin is present in teleosts species and a more qualitative method (High-performance liquid chromatography) is needed in future studies to confirm the present of pheomelanin in P. bassensis muscle melanisation (Dang et al., 2019; Ito and Wakamatsu, 2003). The association of melanin with muscle fibres or blood vessels was observed in almost all melanised muscle samples. These findings agree with a previous histological investigation into P. bassensis muscle melanisation, where melanin association was reported with either muscle fibres or blood vessels (Ooi et al., 2019). Melanin association with muscle fibres or blood vessels were consistent, and not influenced by sampling location or gross score (gross melanisation scoring system). However, a small sample
Please cite this article as: Stocker, C.W et al., Muscle melanisation of southern sand flathead (Platycephalus bassensis) in the Tamar Estuary, Tasmania, Australia, Environmental Pollution, https://doi.org/10.1016/j.envpol.2019.113452
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Table 3 Spearman’s correlation results for the relationship between P. bassensis variables and the percentage surface area of melanised muscle. Spearman’s Correlations
Deceitful Cove Inspection Head Tamar Estuary
Fish Weight
Fish Length
MMCs (Liver)
MMCs (Spleen)
r
p
r
p
r
p
r
p
0.004 0.181 0.119
0.990 0.519 0.363
0.014 0.166 0.042
0.960 0.554 0.749
0.209 0.147 0.122
0.454 0.602 0.424
0.129 0.380 0.063
0.648 0.162 0.682
Fig. 2. Comparison of the two methods of muscle melanisation quantification: Percentage surface area of melanised muscle at the different gross scores. Different letters indicate significant differences between means (p 0.05). (mean ± SE).
size and absence of blood vessels from some melanised muscle samples, means that the results should be interpreted with caution.
Table 4 Statistical analyses results relating to the gross scores. p-value (p) in bald indicates a result of interest. Characteristics Pearson’s Chi-square Tests Location (Tamar Estuary Sites) Sex (Inspection Head) Sex (Deceitful Cove) Sex (Tamar Estuary Sites) Maturation Stage (Tamar Estuary Sites) One-way ANOVAs Fish Weight (Deceitful Cove) Fish Weight (Tamar Estuary Sites) Fish Length (Deceitful Cove) Fish Length (Tamar Estuary Sites) Condition Index (Inspection Head) Condition Index (Deceitful Cove) Condition Index (Tamar Estuary Sites) Age (Tamar Estuary Sites) MMC Liver (Deceitful Cove) MMC Spleen (Inspection Head) MMC Spleen (Deceitful Cove) MMC Spleen (Tamar Estuary Sites) Kruskal-Wallis Tests Fish Weight (Inspection Head) Fish Length (Inspection Head) Age (Deceitful Cove) MMC Liver (Inspection Head) Median Tests Age (Inspection Head) MMC Liver (Tamar Estuary Sites)
X2/F
df
p
26.051 0.600 0.938 2.710 0.548
9 2 3 3 3
0.002 0.741 0.816 0.439 0.908
0.742 0.147 0.725 0.074 0.328 0.180 0.830 1.054 0.263 1.303 0.429 0.347
14 59 14 59 14 14 59 59 14 14 14 44
0.549 0.931 0.558 0.974 0.726 0.908 0.483 0.376 0.851 0.307 0.737 0.791
0.951 0.920 2.750 1.054
2 2 3 2
0.622 0.631 0.432 0.590
1.538 3.802
2 3
0.463 0.284
Overall, the characterisation of P. bassensis melanised muscle, confirmed the presence of localised deposition of melanin, and melanin association with either muscle fibres or blood vessels, sometimes in the same individual. These findings therefore justify future research into specific factors that are reported to influence melanogenesis (melanin synthesis), including; heavy metals (Bowness and Morton, 1953; McGraw, 2003), ultraviolet radiation (Adachi et al., 2005) and dietary imbalances (Adachi et al., 2005). Increased sampling of P. bassensis from the Tamar estuary could provide further insights on these findings. This study has confirmed site specificity within the Tamar Estuary. Deceitful Cove had both a higher proportion of affected P. bassensis and a significant difference in the percentage surface area of melanised muscle, when compared to Inspection Head, West Arm and Middle Island. This is consistent with previous P. bassensis muscle melanisation research (Ooi et al., 2019; Stocker et al., 2019) and indicates that the phenomenon may be caused by site-specific factors. A state-wide questionnaire-based survey of recreational fishers proposed that in addition to the Tamar Estuary, there are potentially other melanisation ‘hotspots’ around Tasmania (Stocker et al., 2019). The verification of site specificity within the Tamar Estuary emphasises the need for further sampling across Tasmania, to compare and confirm site specificity in other locations and identify similar factors that are responsible for the increased occurrence and severity of P. bassensis muscle melanisation. The presence of muscle melanisation in P. bassensis was unrelated to condition index, age, sex, maturation stage, fish weight, fish length and MMCs in the liver and spleen. The P. bassensis sampled in this study did not vary substantially in age, maturation stage, fish length or splenic MMCs (Table 1). Sampling of P. bassensis with
Please cite this article as: Stocker, C.W et al., Muscle melanisation of southern sand flathead (Platycephalus bassensis) in the Tamar Estuary, Tasmania, Australia, Environmental Pollution, https://doi.org/10.1016/j.envpol.2019.113452
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Fig. 3. Melanin association with muscle fibres (A, B): Black dashed arrows demonstrate the location where melanin is positive in Fontana Masson (A) and absent in H&E (B).
broader ranges for those variables would allow for a clearer evaluation of these relationships. Melanogenesis has been reported to be influenced by factors that adversely affect the health of the fish, including; injury or infection (Fagerland et al., 2013; Larsen et al., 2012), heavy metals (Bowness and Morton, 1953; McGraw, 2003), ultraviolet radiation (Adachi et al., 2005) and dietary imbalances (Adachi et al., 2005). The condition index (a function of fish weight and fish length) is commonly implemented as a biomarker for fish health (Nash et al., 2006). In P. bassensis specifically, sub-optimal health has been reported to increase the size and abundance of MMCs in the liver and spleen (Fu et al., 2017; Langdon, 1986). MMCs are capable of melanogenesis in fish (Agius and Roberts, 2003;
Kumar et al., 2016). Age (Agius, 1981; Passantino et al., 2014) and sex (Coffey et al., 2009; Ferreira, 2011) are reported factors that alter the size and prevalence of MMCs. Variables that are reported to either directly or indirectly influence melanogenesis were found unrelated to P. bassensis muscle melanisation. Therefore, the origin of the phenomenon remains unknown and further studies on causation are required. The gross melanisation scoring system was successful in identifying the presence or absence of P. bassensis muscle melanisation (significant difference between score 0 and scores 1, 2 and 3). However, the system does not accurately allocate P. bassensis into different gross scores (no significant difference between scores 1, 2
Please cite this article as: Stocker, C.W et al., Muscle melanisation of southern sand flathead (Platycephalus bassensis) in the Tamar Estuary, Tasmania, Australia, Environmental Pollution, https://doi.org/10.1016/j.envpol.2019.113452
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Fig. 4. Melanin association with blood vessels (A, B): Black dashed arrows demonstrate the location where melanin is positive in Fontana Masson (A) and absent in H&E (B).
and 3). A comparison of the statistical analysis results of both methods of quantifying muscle melanisations (gross melanisation scoring system and the percentage surface area of melanised muscle), suggests they are equally accurate in testing associations with the phenomenon. This signifies that although a more complex method is needed to precisely quantify the severity of muscle melanisation (percentage surface area of melanised muscle), the gross melanisation scoring system is a more efficient and equally statistically accurate method. This study has indicated the limitations and strengths of the gross melanisation scoring system, enabling the application of the appropriate methodology in future muscle melanisation research.
5. Conclusion Eumelanin and pheomelanin were both most likely involved in the occurrence of muscle melanisation in P. bassensis. Site specificity within the Tamar Estuary was confirmed and variables that were reported to directly or indirectly influence levels of melanin in fish, were unrelated to the occurrence of P. bassensis muscle melanisation. The successful development of an image analysis method for quantifying the severity of muscle melanisation in P. bassensis, and the resulting validation of a gross melanisation scoring system will allow for accurate quantification in future muscle melanisation studies.
Please cite this article as: Stocker, C.W et al., Muscle melanisation of southern sand flathead (Platycephalus bassensis) in the Tamar Estuary, Tasmania, Australia, Environmental Pollution, https://doi.org/10.1016/j.envpol.2019.113452
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Acknowledgements The authors of this study wish to acknowledge Dr Melanie Leef for her expertise and assistance in sampling and laboratory procedures. Thanks also go to PhD students Mai Dang and Chun Kit Ooi, for providing critical background information on laboratory techniques. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.envpol.2019.113452. References Adachi, k., Kato, K., Wakamatsu, K., Ito, S., Ishimaru, K., Hirata, T., Murata, O., Kumai, H., 2005. The histological analysis, colorimetric evaluation, and chemical quantification of melanin content in ’suntanned’ fish. Pigment Cell Melanoma Res. 18, 465e468. https://doi.org/10.1111/j.1600-0749.2005.00272.x. Agius, C., 1981. Preliminary studies on the ontogeny of the melano-macrophages of teleost haemopoietic tissues and age-related changes. Dev. Comp. Immunol. 5, 597e606. https://doi.org/10.1016/S0145-305X(81)80034-1. Agius, C., Roberts, R.J., 2003. Melano-macrophage centres and their role in fish pathology. J. Fish Dis. 26, 499e509. https://doi.org/10.1046/j.13652761.2003.00485.x. American Veterinary Medical Association, 2013. AVMA Guidelines for the Euthanasia of Animals, 2013 Edition. AQUI-S, 2013. Preparation of Anaesthetic Baths. AQUI-S New Zealand LTD. Attard, M., Thompson, M., Kelly, R., Locatelli, A., 2012. Tamar Estuary Ecosystem Health Assessment Program, Monitoring Report 2012 Monitoring period October 2010 e September 2011. Bani, A., Moltschaniwskyj, N., Pankhurst, N., 2009. Reproductive strategy and spawning activity of sand flathead, Platycephalus bassensis (Platycephalidae). Cybium: Int. J. Ichthyol. 33, 151e162. Bjorgen, H., Wessel, O., Fjelldal, P.G., Hansen, T., Sveier, A., Saebo, H.R., Enger, K.B., Monsen, E., Kvellestad, A., Rimstad, E., Koppang, E.O., 2015. Piscine orthoreovirus (PRV) in red and melanised foci in white muscle of Atlantic salmon (Salmo salar). Vet. Res. 46 https://doi.org/10.1186/s13567-015-0244-6. Bowness, J.M., Morton, R.A., 1953. The association of zinc and other metals with melanin and a melanin-protein complex. Biochem. J. 53, 620e626. Cal, L., Suarez-Bregua, P., Cerda-Reverter, J., Braasch, I., Rotllant, J., 2017. Fish pigmentation and the melanocortin system. Comp. Biochem. Physiol. Part A Mol. Integr. Physiol. 211, 26e33. https://doi.org/10.1016/j.cbpa.2017.06.001. Coffey, M.J., Tillitt, D., Papoulias, D., Nicks, D., Candrl, J., Annis, M., 2009. Organochlorine Chemical Hazards for Sturgeon Larvae in the Middle Mississippi River National Wildlife Refuge On-Refuge Contaminants Investigation Report. Cooper, M., Olsen, R.L., Seliussen, J., Gannefors, C., 2011. Dietary trace metal supplements promote blood vessels melanosis in fillets of juvenile farmed atlantic cod, Gadus morhua L. J. World Aquac. Soc. 42, 222e229. https://doi.org/10.1111/ j.1749-7345.2011.00458.x. Dang, M., Nowell, C., Nguyen, T., Bach, L., Sonne, C., Norregaard, R., Stride, N., Nowak, B., 2019. Characterisation and 3D structure of melanomacrophage centers in shorthorn sculpins (Myoxocephalus scorpius). Tissue Cell 57, 34e41. https://doi.org/10.1016/j.tice.2019.02.003. Esteves, A., Seixas, F., Carvalho, S., Nazario, N., Mendes, M., Martins, C., 2009. Huffmanela sp. (Nematoda: trichosomoididae) muscular parasite from Trisopterus luscus captured off the Portuguese coast. Dis. Aquat. Org. 84, 251e255. https://doi.org/10.3354/dao02048. Evans, D., Nowak, B., 2016. Effect of ranching time on melanomacrophage centres in anterior kidney and spleen of Southern bluefin tuna, Thunnus maccoyii. Fish Shellfish Immunol. 59, 358e364. https://doi.org/10.1016/j.fsi.2016.11.014. Ewing, G.P., Lyle, J.M., Mapleston, A., 2014. Developing a Low-Cost Monitoring Regime to Assess Relative Abundance and Population Characteristics of Sand Flathead. Institute for Marine and Antarctic Studies, Hobart TAS. Ewing, G.P., Lyle, J.M., Murphy, R.J., Kalish, J.M., Ziegler, P.E., 2007. Validation of age and growth in a long-lived temperate reef fish using otolith structure, oxytetracycline and bomb radiocarbon methods. Mar. Freshw. Res. 58, 944e955. https://doi.org/10.1071/MF07032. Fagerland, H.A.S., Austbo, L., Fritsvold, C., Alarcon, M., Rimstad, E., Falk, K., Taksdal, T., Koppang, E.O., 2013. Pathological pigmentation in cardiac tissue of Atlantic salmon (Salmo salar L.) with cardiomyopathy syndrome. Vet. Res. 44 https://doi.org/10.1186/1297-9716-44-107. Ferreira, C.M.H., 2011. Can Fish Liver Melanomacrophages Be Modulated by Xenoestrogenic and Xenoandrogenic Pollutants? Experimental Studies on the Influences of Temperature, Sex, and Ethynylestradiol, Using the Platyfish as the Model Organism. Instituto De Ciencias Biomedicas Abel Salazar. Frijlink, S., Lyle, J.M., 2013. Establishing Historical Baselines for Key Recreational and Commercial Fish Stocks in Tasmania. Institute for Marine and Antarctic Studies, Hobart TAS. Fu, D., Bridle, A., Leef, M., Gagnon, M.M., Hassell, K.L., Nowak, B.F., 2017. Using a
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Fish Shellfish Immunol. 33, 277e285. https://doi.org/10.1016/j.fsi.2012.05.012. Larsen, H.A.S., Austbo, L., Nodtvedt, A., Fraser, T.W.K., Rimstad, E., Fjelldal, P.G., Hansen, T., Koppang, E.O., 2014. The effect of vaccination, ploidy and smolt production regime on pathological melanin depositions in muscle tissue of Atlantic salmon, (Salmo salar L.). J. Fish Dis. 37, 327e340. https://doi.org/10.1111/ jfd.12106. Leica Microsystems, 2008. Leica Application Suite, Version 3.3.0. McGraw, K.J., 2003. Melanins, metals, mate quality. Oikos 102, 402e406. https:// doi.org/10.1034/j.1600-0579.2003.12513.x. Nash, R.D.M., Valencia, A.H., Geffen, A.J., 2006. The Origin of Fulton’s Condition Factor - Setting the Record Straight. Fisheries History. American Fisheries Society, pp. 236e238. Okazaki, s., Funasaka, Y., Wakamatsu, K., Kawana, S., Saeki, H., 2015. Effect of infrared radiation A on pgotoaged hairless mice harbouring eumelanin and pheomelanin in the epidermis. J. Dermatol. 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Please cite this article as: Stocker, C.W et al., Muscle melanisation of southern sand flathead (Platycephalus bassensis) in the Tamar Estuary, Tasmania, Australia, Environmental Pollution, https://doi.org/10.1016/j.envpol.2019.113452
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Muscle melanisation of southern sand flathead (Platycephalus bassensis) in the Tamar Estuary, Tasmania, Australia* Clayton W. Stocker a, James Haddy a, Jeremy Lyle b, Barbara F. Nowak a, * a
Fisheries and Aquaculture Centre, Institute for Marine and Antarctic Studies, University of Tasmania, Locked Bag 1370, Launceston, 7250, Tasmania, Australia b Fisheries and Aquaculture Centre, Institute for Marine and Antarctic Studies, 15-21 Nubeena Crescent, Taroona, 7053, Tasmania, Australia
a r t i c l e i n f o
a b s t r a c t
Article history: Received 11 August 2019 Received in revised form 6 October 2019 Accepted 20 October 2019 Available online xxx
Tasmanian recreational fishers have reported the presence of dark pigmentations in the usually white fillets of southern sand flathead (Platycephalus bassensis), a phenomenon known as muscle melanisation. Based on histology, it is suggested that eumelanin and pheomelanin are involved in the occurrence of the phenomenon. A gross melanisation scoring system was validated through a comparison with an image analysis technique, that quantified the percentage surface area of the fillets affected by muscle melanisation. The occurrence of muscle melanisation was most severe in fish inhabiting Deceitful Cove, Tamar Estuary. This indicated that muscle melanisation in P. bassensis may be caused by yet to be identified site specific factors. No significant relationships were evident between the percentage surface area of melanised muscle with condition index, age, sex, maturation stage, fish weight, fish length and size of melano-macrophage centres in the liver or spleen. Overall, this study has provided critical information that will frame the direction and focus of future P. bassensis muscle melanisation research. © 2019 Elsevier Ltd. All rights reserved.
Keywords: Muscle melanisation Melano-macrophage centres Melanin Tamar estuary Southern sand flathead
1. Introduction Dark pigmentation of the usually white fillets in fish is a phenomenon known as muscle melanisation. There are two reported forms of muscle melanisation; the diffuse deposition of melanin (fillet greying) and the localised deposition of melanin (black pigmentation) in fillets. The former was reported in farmed barramundi (Lates calcarifer) where melanin was the cause of ‘fillet greying’ (Howieson et al., 2013). Examples of the latter have been reported in a range of species, including; farmed Atlantic salmon (Salmo salar) (Bjorgen et al., 2015), wild and farmed Atlantic cod (Gadus morhua) (Cooper et al., 2011), wild Portuguese pouting (Trisopterus luscus) (Esteves et al., 2009; Ramos et al., 2019) and wild southern sand flathead (Platycephalus bassensis) (Ooi et al., 2019). The causes of the phenomenon are varied, including: a response to vaccination (Bjorgen et al., 2015; Larsen et al., 2014) or increased immune response to bacterial or viral infections (Bjorgen et al., 2015; Krasnov et al., 2016) in S. salar, excessive levels of dietary copper (Cu) in G. morhua (Cooper et al., 2011), and a parasitic
* This paper has been recommended for acceptance by Dr. Sarah Harmon. * Corresponding author. E-mail address: [email protected] (B.F. Nowak).
infection in T. luscus (Esteves et al., 2009; Ramos et al., 2019). In the case of P. bassensis, the cause of muscle melanisation remains unknown (Ooi et al., 2019). Melanin pigments are complex polymers that are found in three forms within vertebrates, including eumelanin, pheomelanin and neuromelanin (Adachi et al., 2005; Slominski et al., 2004). Eumelanin is commonly recorded in fish species (Cal et al., 2017), whereas there are only a few reports of pheomelanin in teleosts (Adachi et al., 2005; Dang et al., 2019; Kottler et al., 2015; Kumar et al., 2016). There are many factors that can influence the level of melanin production (melanogenesis) within fish, including; injury or infection (Fagerland et al., 2013; Larsen et al., 2012), heavy metals (Bowness and Morton, 1953; McGraw, 2003), ultraviolet radiation (Adachi et al., 2005) and dietary imbalances (Adachi et al., 2005). Melano-macrophages (pigment-producing leukocytes) capable of melanogenesis, often cluster together forming melanomacrophage centres (MMCs) (Agius and Roberts, 2003; Evans and Nowak, 2016; Kumar et al., 2016; Thorsen et al., 2006). Located in fish haemopoietic organs, MMCs are commonly found in the spleen, head kidney and to a lesser extent, the liver (Agius and Roberts, 2003; Evans and Nowak, 2016; Kumar et al., 2016). Factors that alter the size, prevalence and morphology of MMCs vary
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Please cite this article as: Stocker, C.W et al., Muscle melanisation of southern sand flathead (Platycephalus bassensis) in the Tamar Estuary, Tasmania, Australia, Environmental Pollution, https://doi.org/10.1016/j.envpol.2019.113452
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between different species and internal organs, including; seasonal variation (Kumar et al., 2016), age (Agius, 1981; Passantino et al., 2014), sex (Coffey et al., 2009; Ferreira, 2011), parasite loading and infectious diseases (Evans and Nowak, 2016; Steinel and Bolnick, 2017). There have been previous reports of MMCs in the liver and spleen of P. bassensis showing links between an increase in the size and abundance of MMCs with suboptimal health of P. bassensis (Fu et al., 2017; Langdon, 1986). Currently, it is unknown if there is any association between melanised muscle and the presence of MMCs in the internal organs. Commonly living over sand or mud, P. bassensis is a benthic species, endemic to the southern regions of Australia and abundant in both coastal and estuarine waters (Bani et al., 2009; Ewing et al., 2014; Imamura, 2015; Jordan, 2001). In both Tasmania’s recreational and commercial fishing, P. bassensis is one of the key target species (Ewing et al., 2014; Frijlink and Lyle, 2013). Recently, there has been an increase number of inquiries from the recreational fishers regarding the presence of dark pigmentation in P. bassensis fillets (Ooi et al., 2019). The occurrence of the phenomenon in Tasmanian waters could affect recreational fishing activities, tourism industry and commercial fishing sector (Attard et al., 2012; Stocker et al., 2019). Preliminary histological studies into P. bassensis muscle melanisation have classified the phenomenon as the localised deposition of melanin (Ooi et al., 2019). The previously reported causes of the localised deposition of melanin in wild G. morhua and T. luscus have been investigated in the case of P. bassensis (Cooper et al., 2011: Esteves et al., 2009; Ramos et al., 2019). A parasitological investigation and heavy metal analysis found no association of parasites or copper with the presence of P. bassensis muscle melanisation (Ooi et al., 2019). Studies based in the Tamar Estuary have suggested site specificity at Deceitful Cove, and a higher concentration of zinc in melanised muscle compared to non-melanised muscle (Ooi et al., 2019). There is a widespread distribution of P. bassensis with muscle melanisation around Tasmania, with notable prevalence within the Tamar Estuary and south-eastern Tasmania (including; Derwent Estuary, D’Entrecasteaux Channel,
Norfolk-Frederick Henry Bays and the Tasman Peninsula) (Ooi et al., 2019). This was further emphasised through a state-wide questionnaire-based survey of recreational fishers (Stocker et al., 2019). This study aimed to develop a method to quantify the severity of observed muscle melanisation in P. bassensis (as a percentage surface area of the fillet) and implement this technique to; validate a gross melanisation scoring system, quantify the site specific occurrence and severity of muscle melanisation in the Tamar Estuary, and explore relationships between muscle melanisation severity and variables that directly or indirectly influence the production of melanin in fish. A histological examination was implemented to characterise P. bassensis melanised muscle. 2. Materials and methods 2.1. Study area The sampling locations were all in the Tamar Estuary, Tasmania and included; Deceitful Cove, Inspection Head, West Arm and Middle Island (Fig. 1). The P. bassensis sampling was approved by the University of Tasmania Animal Ethics Committee (Permit no. A0017197). Sampling locations and procedures were selected based on previous studies (Ooi et al., 2019). 2.2. Sample collection A total of 60 P. bassensis were caught with a baited hook and line from the Tamar Estuary (n ¼ 15 from each sampling location) during three sampling trips in April and May of 2018 (Autumn). Once an individual was landed it was euthanised by immersion in an AQUI-S bath (American Veterinary Medical Association, 2013; AQUI-S, 2013). Following euthanasia, fish were processed to collect data on total fish length (cm ± 1 mm) and total fish weight (g ± 0.1g). Both otoliths were extracted from the ventral brain case (Ewing et al., 2007; Jordan, 1998), and the sex (male, female, indeterminate) and maturation stage (1 ¼ immature, 2 ¼ early developing, 3 ¼ developing, 4 ¼ late developing, 5 ¼ ripe,
Fig. 1. Tamar Estuary sampling locations: A map showing the sampling locations (Deceitful Cove, Inspection Head, Middle Island and West Arm) within the Tamar Estuary, Tasmania. Black square ¼ Tamar Estuary.
Please cite this article as: Stocker, C.W et al., Muscle melanisation of southern sand flathead (Platycephalus bassensis) in the Tamar Estuary, Tasmania, Australia, Environmental Pollution, https://doi.org/10.1016/j.envpol.2019.113452
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6 ¼ spent) were determined according to adopted criteria detailed in Bani et al. (2009) and Haddy (2000). Liver and spleen samples were collected and fixed in 10% neutral buffered formalin (NBF). After filleting, the observed muscle melanisation in each individual was recorded based on a visual interpretation (gross melanisation scoring system) of the extent of dark pigmentation in the fillets (gross scores: 0 ¼ 0%; 1 ¼ < 5%; 2 ¼ 5e20%; 3 ¼ > 20%). Melanised individuals with highly concentrated localised areas of dark pigmentation, had small samples (5 5 mm) of melanised and non-melanised muscle tissues collected and fixed in NBF (n ¼ 11). Fillets were transported on ice in individual plastic bags, prior to being frozen and stored at 20 C. Histological samples were not collected for Middle Island due to logistics. 2.3. Histological processing Following the initial transfer of samples from NBF to 70% ethanol, samples were processed for histology. Briefly, samples were embedded in paraffin blocks (Shandon Histocentre 3) and a section of each sample was cut at 5 mm (Microm HM340) and stained with haematoxylin and eosin (H&E). H&E was considered the standard method for MMCs quantification. Melanised and non-melanised muscle samples that were collected from the 11 P. bassensis had two sections prepared, one stained with H&E and the second one with Fontana Masson. The combination of the two stains has been previously implemented to indicate an initial detection of two forms of melanin (eumelanin and pheomelanin) (Dang et al., 2019; Okazaki et al., 2015). These samples were observed under a light microscope at 400x magnification (Leica DM 500) and were examined for pigmentations. The association of pigments with muscle fibres or blood vessels in Fontana Masson stained sections were recorded. 2.4. Image capture 2.4.1. Histology image capture From each H&E slide, 5 images were captured of each liver and spleen with a light microscope at 100x magnification (Leica DM 500 with Leica ICC50W Camera) and LAS EZ software (brightness ¼ 50%, Gamma ¼ 3.00, Saturation ¼ 64.00) (Leica Microsystems, 2008). A systematic approach was implemented, with an image captured in each corner and centre of every section. Images were required to have the section covering a minimum of 50% of the field of view and no overlap between images captured from the same organ.
2.5.1. Histology images The protocols implemented for the image analysis of liver and spleen histology samples were adaptations of those previously described (Dang et al., 2019; Evans and Nowak, 2016). Initially, the image was calibrated and a colour deconvolution with H&E vectors was completed. Total surface area of the liver and spleen in the field of view was measured (mm2), through adjustments to the image grey value threshold on the second deconvolution window, until all pixels within the organ were selected (grey value threshold ranges: Liver ¼ 143e165, Spleen ¼ 140-160). The surface area of MMCs in the liver and spleen were subsequently measured (mm2), through adjustments to the image grey value threshold on the third colour deconvolution window, until all MMCs (no size or shape restrictions) were selected (threshold ranges: Liver ¼ 140e180, Spleen ¼ 140-170). Voids and artefacts (over 1000 mm2) were individually selected and removed from all measurements. 2.5.2. Fillet images Initially, the original RGB colour image was calibrated and transformed into an 8-bit image. Total surface area of the entire fillet was measured (mm2) through adjustments to the image grey value threshold until all pixels within the fillet area were selected (grey value threshold range ¼ 85e107). The surface area of melanised muscle was measured (mm2) by reducing the image grey value threshold until the remaining selected pixels mirrored the extent of dark pigments observed in the original image (grey value threshold range ¼ 186e218). 2.6. Otolith processing and interpretation Each otolith had three transverse sections prepared through standard otolith processing (Ewing et al., 2014; Jordan, 1998). To maintain consistency in the ageing process, the otoliths selected for processing were from the right side of the fish where available. Otolith sections were examined under a light microscope (Leica M28) at 16x magnification and a validated ageing protocol for P. bassensis was implemented (Ewing et al., 2014; Jordan, 1998). 2.7. Data analysis 2.7.1. Data preparation The condition index for P. bassensis was calculated by applying the Fulton’s Condition Index (Nash et al., 2006):
K ¼ 100
2.4.2. Fillet image capture All fillets were thawed, and excess skin was removed from the edges. A light box with a white background was used for scaling and lighting consistency (Grunenwald et al., 2018) and an image of the fillets captured with a FujiFilm X20 camera (WB ¼ 3200, F ¼ 2.8, SS ¼ 640, ISO ¼ 800).
Surface Area Melanised Muscle ð%Þ ¼
3
W L3
where K ¼ Condition Index, W ¼ Fish Weight (g), L ¼ Total Fish Length (cm). Ratios for sex and maturation stage were established and means or percentages were calculated for all the variables at each sampling location (Table 1). The percentage surface area of melanised muscle on P. bassensis fillets was quantified:
Surface Area Melanised Muscle * 100: Total Fillet Area
2.5. Image analysis Image J (Fiji) software was used for image analysis (Schindelin et al., 2012).
The percentage surface area of MMCs in the liver and spleen of P. bassensis was calculated:
Please cite this article as: Stocker, C.W et al., Muscle melanisation of southern sand flathead (Platycephalus bassensis) in the Tamar Estuary, Tasmania, Australia, Environmental Pollution, https://doi.org/10.1016/j.envpol.2019.113452
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Table 1 Characteristics of P. bassensis sampled from the sampling locations in the Tamar Estuary. Different letters indicate significant differences between means across sampling locations (p 0.05). (mean ± SE). Variables
Deceitful Cove
Inspection Head
West Arm
Middle Island
n Condition Index Age Sex (M:F:I) Maturation Stage (1:2) Fish Weight (g) Fish Length (cm)
15 0.55 ± 0.01ab 1.93 ± 0.07a (4:11:0) (0:15) 148.40 ± 10.96ab 29.65 ± 0.64a
15 0.53 ± 0.01a 2.33 ± 0.23a (5:10:0) (0:15) 124.27 ± 11.92a 28.18 ± 0.72a
15 0.60 ± 0.01c 2.00 ± 0.00a (4:10:1) (1:14) 164.27 ± 10.58b 29.87 ± 0.68a
15 0.59 ± 0.01bc 2.13 ± 0.09a (8:7:0) (0:15) 161.40 ± 7.51ab 30.02 ± 0.38a
Surface Area MMC ð%Þ ¼
Surface Area MMC * 100: Total Tissue Area
Masson and H&E stained consecutive sections. Furthermore, the presence of pigments associated with muscle fibres or blood vessels was recorded and compared between the sampling locations and the gross scores (gross melanisation scoring system). 3. Results
2.7.2. Statistical analysis All statistical analyses were completed using the statistical program SPSS, with a set p-value of 0.05 (IBM Corp, 2016). Nonparametric tests were applied when assumptions were violated (Shapiro-Wilk test; Levene statistic; scatter plot), including; Kruskal-Wallis tests, median tests and Spearman’s correlations. To identify any site specificity within the Tamar Estuary, the difference in the percentage surface area of melanised muscle between sampling locations was statistically analysed with a KruskalWallis test and pairwise comparison post hoc test (adjusted by Bonferroni correction). The statistical analyses of P. bassensis variables with the percentage surface area of melanised muscle was completed for Deceitful Cove and Inspection Head separately, and then also for all the Tamar Estuary sampling locations pooled together. Statistical analysis for individual locations for West Arm and Middle Island were excluded due to the limited presence of muscle melanisation. There was an indeterminate individual sampled in West Arm that was excluded from statistical analyses relating to sex. A multiple regression analysis was employed to determine the relationships of P. bassensis condition index, sex, age and maturation stage, with the percentage surface area of melanised muscle. The fish weight and fish length were excluded from this statistical analysis due to multicollinearity with condition index and age. Therefore, Spearman’s correlations were implemented to statistically analyse these associations. Spearman’s correlations were applied to test the associations between the percentage surface area of MMCs in the liver and spleen, with the percentage surface area of melanised muscle. The differences in the percentage surface area of MMCs in the liver and spleen between Tamar Estuary sampling locations were statistically analysed using both a one-way analysis of variances (ANOVAs) and a Kruskal-Wallis test with a pairwise comparison post hoc test (adjusted by Bonferroni correction). Gross melanisation scoring system was statistically compared with the percentage surface area of melanised muscle with a Kruskal-Wallis test and pairwise comparison post hoc test (adjusted by Bonferroni correction). Furthermore, ANOVAs, Kruskal-Wallis tests, median tests and Pearson’s chi-squared tests were implemented to assess the relationship of sampling location, condition index, sex, age, maturation stage, fish weight, fish length and MMCs in the liver and spleen with the gross scores. These statistical results were compared to those recorded for the percentage surface area of melanised muscle. Based on histology, the presence or absence of pigments within melanised and non-melanised muscle samples was recorded. The extent of pigments present was then compared between Fontana
3.1. Gross muscle melanisation The highest prevalence of P. bassensis with muscle melanisation was in Deceitful Cove (80% of individuals), followed by Inspection Head (40% of individuals). The occurrence of the phenomenon was relatively low in fish from West Arm (13.3% of individuals) and Middle Island (6.7% of individuals). There was a significant difference in the percentage surface area of melanised muscle between the sampling locations in the Tamar Estuary (Kruskal-Wallis: x2 ¼ 26.938, df ¼ 3, p < 0.001). Pairwise comparisons revealed the significant differences were between Deceitful Cove and Inspection Head (p ¼ 0.030), West Arm (p < 0.001) and Middle Island (p < 0.001) and there were no significant differences between any of the three other sampling locations. 3.2. Associations between P. bassensis variables and melanised muscle There were no significant relationships between the percentage surface area of melanised muscle and condition index, age, sex and maturation stage of P. bassensis in Deceitful Cove, Inspection Head and the Tamar Estuary (Table 2). The overall fit of each multiple regression model had no statistical significance (Table 2). Fish weight, fish length and percentage surface area of MMCs in the liver and spleen were not significantly associated with the percentage surface area of melanised muscle in Deceitful Cove, Inspection Head and the Tamar Estuary (Table 3). 3.3. MMCs and sampling location There was a significant difference in the percentage surface area of MMCs in the liver of P. bassensis, between the Tamar Estuary sampling locations (Kruskal-Wallis: x2 ¼ 7.282, df ¼ 2, p ¼ 0.026). Pairwise comparisons revealed the significant difference was between Inspection Head and West Arm (p ¼ 0.037). In contrast, there was no significant difference in the percentage surface area of MMCs in the spleen between the Tamar Estuary sampling locations (one-way ANOVA: F ¼ 2.546, df ¼ 44, p ¼ 0.090). 3.4. Validation of gross melanisation scoring system 3.4.1. Melanised muscle and gross scores There was a significant difference in the percentage surface area of melanised muscle between the gross scores (Kruskal-Wallis Test: x2 ¼ 25.118, df ¼ 3, p < 0.001) (Fig. 2). A pairwise comparison revealed that the significant difference was between gross score
Please cite this article as: Stocker, C.W et al., Muscle melanisation of southern sand flathead (Platycephalus bassensis) in the Tamar Estuary, Tasmania, Australia, Environmental Pollution, https://doi.org/10.1016/j.envpol.2019.113452
0.012 0.079 0.023
p F
0.043 0.313 0.439
r2
Model Fit
0.987 0.816 0.726
C.W. Stocker et al. / Environmental Pollution xxx (xxxx) xxx
5
0 and scores 1 (p ¼ 0.010), 2 (p < 0.001), and 3 (p ¼ 0.002) and there was no significant differences between any of the three other gross scores. 3.4.2. Comparing statistical analyses results of muscle melanisation assessment methods Both muscle melanisation assessment methods gave consistent results. A significant relationship was identified between the Tamar Estuary sampling locations and the gross scores (Table 4). Furthermore, there were no significant relationships between any P. bassensis variables (condition index, age, sex, maturation stage, fish weight, fish length and MMCs in the liver and spleen) and the gross scores within Deceitful Cove, Inspection Head and the Tamar Estuary (Table 4). These results directly correspond with the results obtained using the percentage surface area of melanised muscle.
0.047 0.144 0.086 0.898 0.592 0.660 0.794 0.632 0.396 Deceitful Cove Inspection Head Tamar Estuary
0.081 0.171 0.114
0.045 0.193 0.059
0.891 0.658 0.523
Constant Constant Constant
b b p
b p
b
p
Maturation Stage Sex Age Condition Index
Multiple Regression Analysis
Table 2 Multiple regression analysis results for the relationships between P. bassensis variables and the percentage surface area of melanised muscle.
p
3.5. Histology of melanised muscle No pigmentation was observed within the non-melanised muscle samples. In contrast, all melanised muscle samples had pigments visible in the two consecutive histological sections, stained with Fontana Masson or H&E (Figs. 3 and 4). The absence of pigments in the non-melanised muscle samples acts as a control, confirming the pigments observed are melanin and not formalin. There were occasionally melanin positive areas in the Fontana Masson stained sections, that were absent in H&E (Figs. 3 and 4). This suggests both eumelanin (Fontana Masson positive, H&E present) and pheomelanin (Fontana Masson positive, H&E absent) contribute to P. bassensis muscle melanisation. Melanin association with muscle fibres was present in 10 of the 11 P. bassensis sampled (Fig. 3). Blood vessels were not present in the histology sections of four P. bassensis. Melanin association with blood vessels was observed in all the remaining P. bassensis sampled (n ¼ 7) (Fig. 4). The results observed were consistent across all sampling locations and gross scores (gross melanisation scoring system). 4. Discussion Melanin was only observed in histological sections of melanised muscle samples and not at all in those non-melanised. This confirmed that the localised deposition of melanin is responsible for the dark pigmentations reported in P. bassensis fillets (Ooi et al., 2019). The presence of melanin in both Fontana Masson and H&E stained samples, suggests that the pigment present is eumelanin (Dang et al., 2019). Melanin that was observed in Fontana Masson staining but not in H&E stained samples, is most likely pheomelanin (Dang et al., 2019). The results of this suggest that both eumelanin and pheomelanin are involved in the occurrence of P. bassensis muscle melanisation. This discovery conforms with recent studies suggesting the presence of pheomelanin in teleosts (Adachi et al., 2005; Dang et al., 2019; Kottler et al., 2015; Kumar et al., 2016). It is still uncertain whether pheomelanin is present in teleosts species and a more qualitative method (High-performance liquid chromatography) is needed in future studies to confirm the present of pheomelanin in P. bassensis muscle melanisation (Dang et al., 2019; Ito and Wakamatsu, 2003). The association of melanin with muscle fibres or blood vessels was observed in almost all melanised muscle samples. These findings agree with a previous histological investigation into P. bassensis muscle melanisation, where melanin association was reported with either muscle fibres or blood vessels (Ooi et al., 2019). Melanin association with muscle fibres or blood vessels were consistent, and not influenced by sampling location or gross score (gross melanisation scoring system). However, a small sample
Please cite this article as: Stocker, C.W et al., Muscle melanisation of southern sand flathead (Platycephalus bassensis) in the Tamar Estuary, Tasmania, Australia, Environmental Pollution, https://doi.org/10.1016/j.envpol.2019.113452
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Table 3 Spearman’s correlation results for the relationship between P. bassensis variables and the percentage surface area of melanised muscle. Spearman’s Correlations
Deceitful Cove Inspection Head Tamar Estuary
Fish Weight
Fish Length
MMCs (Liver)
MMCs (Spleen)
r
p
r
p
r
p
r
p
0.004 0.181 0.119
0.990 0.519 0.363
0.014 0.166 0.042
0.960 0.554 0.749
0.209 0.147 0.122
0.454 0.602 0.424
0.129 0.380 0.063
0.648 0.162 0.682
Fig. 2. Comparison of the two methods of muscle melanisation quantification: Percentage surface area of melanised muscle at the different gross scores. Different letters indicate significant differences between means (p 0.05). (mean ± SE).
size and absence of blood vessels from some melanised muscle samples, means that the results should be interpreted with caution.
Table 4 Statistical analyses results relating to the gross scores. p-value (p) in bald indicates a result of interest. Characteristics Pearson’s Chi-square Tests Location (Tamar Estuary Sites) Sex (Inspection Head) Sex (Deceitful Cove) Sex (Tamar Estuary Sites) Maturation Stage (Tamar Estuary Sites) One-way ANOVAs Fish Weight (Deceitful Cove) Fish Weight (Tamar Estuary Sites) Fish Length (Deceitful Cove) Fish Length (Tamar Estuary Sites) Condition Index (Inspection Head) Condition Index (Deceitful Cove) Condition Index (Tamar Estuary Sites) Age (Tamar Estuary Sites) MMC Liver (Deceitful Cove) MMC Spleen (Inspection Head) MMC Spleen (Deceitful Cove) MMC Spleen (Tamar Estuary Sites) Kruskal-Wallis Tests Fish Weight (Inspection Head) Fish Length (Inspection Head) Age (Deceitful Cove) MMC Liver (Inspection Head) Median Tests Age (Inspection Head) MMC Liver (Tamar Estuary Sites)
X2/F
df
p
26.051 0.600 0.938 2.710 0.548
9 2 3 3 3
0.002 0.741 0.816 0.439 0.908
0.742 0.147 0.725 0.074 0.328 0.180 0.830 1.054 0.263 1.303 0.429 0.347
14 59 14 59 14 14 59 59 14 14 14 44
0.549 0.931 0.558 0.974 0.726 0.908 0.483 0.376 0.851 0.307 0.737 0.791
0.951 0.920 2.750 1.054
2 2 3 2
0.622 0.631 0.432 0.590
1.538 3.802
2 3
0.463 0.284
Overall, the characterisation of P. bassensis melanised muscle, confirmed the presence of localised deposition of melanin, and melanin association with either muscle fibres or blood vessels, sometimes in the same individual. These findings therefore justify future research into specific factors that are reported to influence melanogenesis (melanin synthesis), including; heavy metals (Bowness and Morton, 1953; McGraw, 2003), ultraviolet radiation (Adachi et al., 2005) and dietary imbalances (Adachi et al., 2005). Increased sampling of P. bassensis from the Tamar estuary could provide further insights on these findings. This study has confirmed site specificity within the Tamar Estuary. Deceitful Cove had both a higher proportion of affected P. bassensis and a significant difference in the percentage surface area of melanised muscle, when compared to Inspection Head, West Arm and Middle Island. This is consistent with previous P. bassensis muscle melanisation research (Ooi et al., 2019; Stocker et al., 2019) and indicates that the phenomenon may be caused by site-specific factors. A state-wide questionnaire-based survey of recreational fishers proposed that in addition to the Tamar Estuary, there are potentially other melanisation ‘hotspots’ around Tasmania (Stocker et al., 2019). The verification of site specificity within the Tamar Estuary emphasises the need for further sampling across Tasmania, to compare and confirm site specificity in other locations and identify similar factors that are responsible for the increased occurrence and severity of P. bassensis muscle melanisation. The presence of muscle melanisation in P. bassensis was unrelated to condition index, age, sex, maturation stage, fish weight, fish length and MMCs in the liver and spleen. The P. bassensis sampled in this study did not vary substantially in age, maturation stage, fish length or splenic MMCs (Table 1). Sampling of P. bassensis with
Please cite this article as: Stocker, C.W et al., Muscle melanisation of southern sand flathead (Platycephalus bassensis) in the Tamar Estuary, Tasmania, Australia, Environmental Pollution, https://doi.org/10.1016/j.envpol.2019.113452
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Fig. 3. Melanin association with muscle fibres (A, B): Black dashed arrows demonstrate the location where melanin is positive in Fontana Masson (A) and absent in H&E (B).
broader ranges for those variables would allow for a clearer evaluation of these relationships. Melanogenesis has been reported to be influenced by factors that adversely affect the health of the fish, including; injury or infection (Fagerland et al., 2013; Larsen et al., 2012), heavy metals (Bowness and Morton, 1953; McGraw, 2003), ultraviolet radiation (Adachi et al., 2005) and dietary imbalances (Adachi et al., 2005). The condition index (a function of fish weight and fish length) is commonly implemented as a biomarker for fish health (Nash et al., 2006). In P. bassensis specifically, sub-optimal health has been reported to increase the size and abundance of MMCs in the liver and spleen (Fu et al., 2017; Langdon, 1986). MMCs are capable of melanogenesis in fish (Agius and Roberts, 2003;
Kumar et al., 2016). Age (Agius, 1981; Passantino et al., 2014) and sex (Coffey et al., 2009; Ferreira, 2011) are reported factors that alter the size and prevalence of MMCs. Variables that are reported to either directly or indirectly influence melanogenesis were found unrelated to P. bassensis muscle melanisation. Therefore, the origin of the phenomenon remains unknown and further studies on causation are required. The gross melanisation scoring system was successful in identifying the presence or absence of P. bassensis muscle melanisation (significant difference between score 0 and scores 1, 2 and 3). However, the system does not accurately allocate P. bassensis into different gross scores (no significant difference between scores 1, 2
Please cite this article as: Stocker, C.W et al., Muscle melanisation of southern sand flathead (Platycephalus bassensis) in the Tamar Estuary, Tasmania, Australia, Environmental Pollution, https://doi.org/10.1016/j.envpol.2019.113452
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Fig. 4. Melanin association with blood vessels (A, B): Black dashed arrows demonstrate the location where melanin is positive in Fontana Masson (A) and absent in H&E (B).
and 3). A comparison of the statistical analysis results of both methods of quantifying muscle melanisations (gross melanisation scoring system and the percentage surface area of melanised muscle), suggests they are equally accurate in testing associations with the phenomenon. This signifies that although a more complex method is needed to precisely quantify the severity of muscle melanisation (percentage surface area of melanised muscle), the gross melanisation scoring system is a more efficient and equally statistically accurate method. This study has indicated the limitations and strengths of the gross melanisation scoring system, enabling the application of the appropriate methodology in future muscle melanisation research.
5. Conclusion Eumelanin and pheomelanin were both most likely involved in the occurrence of muscle melanisation in P. bassensis. Site specificity within the Tamar Estuary was confirmed and variables that were reported to directly or indirectly influence levels of melanin in fish, were unrelated to the occurrence of P. bassensis muscle melanisation. The successful development of an image analysis method for quantifying the severity of muscle melanisation in P. bassensis, and the resulting validation of a gross melanisation scoring system will allow for accurate quantification in future muscle melanisation studies.
Please cite this article as: Stocker, C.W et al., Muscle melanisation of southern sand flathead (Platycephalus bassensis) in the Tamar Estuary, Tasmania, Australia, Environmental Pollution, https://doi.org/10.1016/j.envpol.2019.113452
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Please cite this article as: Stocker, C.W et al., Muscle melanisation of southern sand flathead (Platycephalus bassensis) in the Tamar Estuary, Tasmania, Australia, Environmental Pollution, https://doi.org/10.1016/j.envpol.2019.113452