Histology and ultrastructure of hepatic nodular alterations in Clarias gariepinus (Burchell, 1822) 非洲鱘魚（Burchell, 1822）肝結節變化的組織學和超微結構
Refilwe Lukhwareni | Cobus van Dyk (B)
Department of Zoology, Faculty of Science, University of Johannesburg, Auckland Park, South Africa 約翰尧斯堡大學科學學院動物學系，南非奧克蘭公園
Correspondence 通信
Cobus van Dyk, Department of Zoology, Faculty of Science, University of Johannesburg, Auckland Park, South Africa. Email: cvandyk@uj.ac.za 南非約翰尼斯堡大學科學學院動物學系 Cobus van Dyk 教授。聯繫郵箱：cvandyk@uj.ac.za
Funding information 資助資訊
National Research Foundation (NRF) 國家研究基金會（NRF）
Abstract 摘要
This study aimed to assess the macroscopic appearance, histology and ultrastructure of liver nodular alterations in sharptooth catfish Clarias gariepinus (Burchell, 1822) from a hyper-eutrophic freshwater impoundment. The livers of 25 fish were sampled for microscopy analyses, otoliths were collected for age estimation, and the hepatosomatic index (HSI) was calculated. The light microscopic analysis of the nodular tissue revealed hepatocellular and nuclear pleomorphism, pyknotic nuclei, variation in cytoplasmic appearance and focal areas of cellular alterations (FCAs). An increase in the size and number of melano-macrophage centres, intracellular deposits, steatosis and giant cells was also identified. The ultrastructural alterations identified included cell membrane disruption, irregular shaped nuclei with augmentation of the nucleolus and deformed nuclear envelopes. In addition, we found dilation, proliferation, fragmentation and vesiculation of the cisternae of the rough endoplasmic reticulum (RER). Furthermore, there was an increase in degenerative mitochondria (transparent matrix), swelling of mitochondria and proliferation of both peroxisomes and lysosomes. The histological and ultrastructural alterations identified did not explain the variation in macroscopic colour of the nodules. We did not find a strong positive correlation between age and HSI; however, the oldest fish (18 years) did present the most nodules. 本研究旨在評估鋸齒鯰（Clarias gariepinus）在一個高度富養分的淡水水庫中的肝臟結節性變化的巨觀外觀、組織學和超微結構。對 25 條魚的肝臟進行了顯微鏡分析，收集了耳石進行年齡估計，並計算了肝臟體指數（HSI）。結節組織的光學顯微分析顯示肝細胞和核的多形性，核小體，細胞質外觀的變化和細胞變化的局部區域（FCAs）。還識別出了墨黑-巨噬細胞中心的大小和數量增加，細胞內沉積物，脂肪肝和巨細胞。識別出的超微結構變化包括細胞膜破裂，形狀不規則的核，核仁增大和變形的核膜。此外，我們發現粗面內質網（RER）囊泡的擴張、增殖、碎裂和囊泡化。此外，還有退化的線粒體增加（透明基質），線粒體腫脹和過氧化物酶體和溶酶體的增殖。 組織學和超微結構的變化並不能解釋結節的宏觀顏色變異。我們沒有發現年齡和 HSI 之間存在強烈的正相關；然而，最年長的魚（18 歲）確實出現了最多的結節。
KEYWORDS 關鍵詞
age, hepatosomatic index, histopathology, liver, Roodeplaat Dam 年齡、肝臟重指數、組織病理學、肝臟、Roodeplaat 水壩
1 | INTRODUCTION 1 | 簡介
The Roodeplaat Dam is a hyper-eutrophic freshwater system located in the north-eastern parts of South Africa (Van Ginkel, Hohls, Belcher, Vermaak, & Gerber, 2001). Due to its polluted status, we are currently conducting research to investigate the health of fish inhabiting this system. The primary aim is to investigate liver health responses, motivated by the presence of hepatotoxins released by the seasonal cyanobacterial algal blooms. One of the objectives is to investigate seasonal and species differences with regard to liver pathology. To date, six sampling surveys conducted over a three-year period (2013 to 2015), have consistently shown the presence of macroscopically visible hepatic nodular alterations in sharptooth catfish Clarias gariepinus (Burchell, 1822). The respective necropsy results revealed that the severity of occurrence of these hepatic nodules differs between individual fish and that these nodules also vary in terms of size, morphological location on the liver and colour. We do not suspect the difference in size or morphological location to be determining factors of the underlying hepatocellular alterations. However, we do hypothesize that the variation in macroscopic colour might be indicative of specific histological and ultrastructural changes. Roodeplaat 水壩是位於南非東北部的一個高度富養分的淡水系統（Van Ginkel, Hohls, Belcher, Vermaak, & Gerber, 2001）。由於其受污染的狀態，我們目前正在進行研究，以調查棲息在這個系統中的魚類的健康狀況。主要目的是調查肝臟對健康的反應，這是由季節性藍綠藻水華釋放的肝毒素所驅使的。其中一個目標是調查在肝臟病理方面的季節性和物種差異。到目前為止，在三年的時間內進行了六次採樣調查（2013 年至 2015 年），一致顯示了鋸齒鯰（Clarias gariepinus）的肝臟中存在著肉眼可見的肝腫瘤變化（Burchell, 1822）。相應的解剖結果顯示，這些肝腫瘤的發生嚴重程度在個別魚類之間存在差異，這些腫瘤在肝臟上的大小、形態位置和顏色也有所不同。我們不懷疑大小或形態位置的差異是潛在肝細胞變化的決定因素。 然而，我們假設宏觀顏色的變化可能暗示特定的組織學和超微結構變化。
Limited research is available on the microscopic structure of macroscopically visible nodular growths in freshwater fish, especially in Africa, and to the best of our knowledge, there have been no published reports on the ultrastructure of such macroscopic nodules 有關淡水魚宏觀可見結節性生長的微觀結構的研究有限，尤其是在非洲地區，據我們所知，尚未有關於這種宏觀結節的超微結構的發表報告。
in C. gariepinus. There is, however, reports on nodular growths in marine species, for example flounder Platichthys flesus (Linnaeus, 1758). (Lang, Mellergaard, Wosniok, Kadakas, & Neumann, 1999; Bogovski, Lang, & Mellergaard, 1999) and the common dab Limanda limanda (Linnaeus, 1758). (ICES, 2004). It has been shown in certain marine species that the colour of nodules is often associated with specific alterations, for example, opaque and white nodules are linked to acidophilic and clear cell hepatocytes, while more yellow nodules can be associated with basophilic hepatocellular lesions. Green-coloured nodules may be associated with malignant and benign tumours of the bile ducts (cholangiocarcinoma and cholangioma) (ICES, 2004). Dark-red nodules can show neoplasms involving vascular structures (i.e., hemangioma and hemangiosarcoma) (ICES, 2004). Associations between the macroscopic colour of liver nodules and specific microscopic alterations could be extremely useful in the screening process for neoplastic growths during necropsy procedures. Therefore, our aim was to assess whether the hepatic nodules observed in the freshwater sharptooth catfish represent different and/or specific histological and ultrastructural alterations. However, the exact cause of these hepatic nodules is still under investigation. 在C. gariepinus中。然而，有關海洋物種結節性生長的報告，例如比目魚Platichthys flesus（Linnaeus，1758年）。 （Lang，Mellergaard，Wosniok，Kadakas和Neumann，1999年；Bogovski，Lang和Mellergaard，1999年）和常見的鰈魚Limanda limanda（Linnaeus，1758年）。 （ICES，2004年）。已經在某些海洋物種中顯示，結節的顏色通常與特定的變化相關，例如，不透明和白色的結節與嗜酸性和清晰細胞肝細胞病變相關，而更黃色的結節可能與嗜鹼性肝細胞病變相關。綠色結節可能與膽管的惡性和良性腫瘤有關（膽管癌和膽管瘤）（ICES，2004年）。深紅色結節可能顯示涉及血管結構的新生物（即血管瘤和血管肉瘤）（ICES，2004年）。肝臟結節的宏觀顏色與特定微觀變化之間的關聯可能在屍檢程序中對腫瘤生長的篩查過程中非常有用。 因此，我們的目標是評估淡水鋸齒鯰魚觀察到的肝結節是否代表不同和/或特定的組織學和超微結構變化。然而，這些肝結節的確切原因仍在調查中。
Previous studies have confirmed the presence of hepatotoxins such as microcystins in the Roodeplaat Dam (Van Ginkel, 2008; Conradie & Barnard, 2012). It has been suggested that these hepatotoxins are the most likely cause, of these nodules (Van Dyk, Cochrane, & Wagenaar, 2012; Marchand, Van Dyk, Barnhoorn, & Wagenaar, 2012). A number of studies are available that specifically show ultrastructural alterations in fish liver following exposure to microcystins (Li, Xie, & Chen, 2005; Qiu, Xie, Ke, Li, & Guo, 2007; Atencio et al., 2008; Branka et al., 2011; Mitsoura et al., 2013). These alterations include highly vacuolated cytoplasm and pyknotic nuclei, containing micro-vesicular steatosis with glycogen present. Moreover, dilated rough endoplasmic reticulum (RER), lysosomes and swollen mitochondria were present throughout the cytoplasm and fewer microvilli in some cells of the bile canaliculi (Li et al., 2005; Qiu et al., 2007). One objective would therefore be to assess whether the above-mentioned alterations are associated with any of the macroscopically visible nodular alterations in fish from Roodeplaat Dam. 先前的研究已確認Roodeplaat水壩中存在肝毒素，如微囊藻毒素（Van Ginkel, 2008; Conradie & Barnard, 2012）。有人提出這些肝毒素很可能是這些結節的主要原因（Van Dyk, Cochrane, & Wagenaar, 2012; Marchand, Van Dyk, Barnhoorn, & Wagenaar, 2012）。有許多研究特別顯示暴露於微囊藻毒素後，魚肝臟出現超微結構變化（Li, Xie, & Chen, 2005; Qiu, Xie, Ke, Li, & Guo, 2007; Atencio et al., 2008; Branka et al., 2011; Mitsoura et al., 2013）。這些變化包括高度空泡化的細胞質和核小體，含有微小泡性脂肪肝和存在葡萄糖原。此外，擴張的粗面內質網（RER）、溶酶體和腫脹的粒線體分佈於整個細胞質，有些膽小管細胞的微絨毛較少（Li等，2005年; Qiu等，2007年）。因此，一個目標是評估上述變化是否與Roodeplaat水壩魚類中任何宏觀可見的結節變化有關。
Furthermore, it is currently unknown whether the nodular alterations are more prevalent in older fish. It is assumed that fish exposed for longer periods to hepatotoxins might be more prone to develop liver abnormalities. According to Bernet, Schmidt, Meier, Burkhardt-Holm, and Wahli (1999), age determination is vital to better understand pathologies found during histological assessment of fish tissue as neoplasia has been found to occur more in older fish. Also, Stentiford, Bignell, Lyons, Thain, and Feist (2010) have demonstrated that liver tumour occurrence increases with age and age onset also differed for pre-neoplastic and neoplastic lesions in marine fish. 此外，目前尚不清楚結節性變化在年長魚類中是否更普遍。人們認為，長時間暴露於肝毒素的魚類可能更容易發展肝臟異常。根據Bernet、Schmidt、Meier、Burkhardt-Holm和Wahli（1999）的研究，年齡確定對於更好地理解魚類組織組織學評估中發現的病理變化至關重要，因為已發現腫瘤在年長魚類中更常見。此外，Stentiford、Bignell、Lyons、Thain和Feist（2010）已證明肝腫瘤發生率隨年齡增加，並且早期和腫瘤病變的年齡起始時間也有所不同。
Subsequent to the 2013 and 2015 surveys, as mentioned above, we did a follow-up fish health survey at the Roodeplaat Dam in 2016, with the aim to assess whether the variation in colour of the macroscopically visible hepatic nodules is associated with different or specific histological and ultrastructural alterations. We also hypothesized that older fish will be more affected in terms of hepatic nodular growths. We further hypothesized that the presence of hepatic nodular growths will result in organ mass increase and subsequently an increase in the HSI. Therefore, age should positively correlate with the HSI of the affected fish. 繼 2013 年和 2015 年的調查之後，如上所述，我們於 2016 年在 Roodeplaat 水壩進行了一次後續魚類健康調查，旨在評估肝臟結節的顏色變化是否與不同或特定的組織學和超微結構變化相關。我們還假設年長的魚類在肝臟結節生長方面受到更多影響。我們進一步假設肝臟結節的存在將導致器官質量增加，隨後導致 HSI 增加。因此，年齡應與受影響魚類的 HSI 呈正相關。
2 | MATERIALS AND METHODS 2 | 材料和方法
2.1 Fish collection 2.1 魚類收集
Clarias gariepinus ( ) were collected over a three-day period in June 2016 in the western side of Roodeplaat Dam (25.37'15"S . Fish were collected with gill nets with mesh sizes of 9 to [Permit no: CPE 2-160]. Fish were placed in aerated water baths and transported to a close-by field laboratory for tissue processing. As a reference for the descriptive histology and ultrastructure of fish collected from the Roodeplaat Dam, two reference fish (male and female) from the same species were included in the study. These fish were kept under controlled conditions in tap water in the aquarium research facilities at the University of Johannesburg. 2016 年 6 月，在 Roodeplaat 水壩西側的三天內收集了非洲鱧魚（ ）。魚類是用網眼大小為 9 至 的鰓網收集的[許可證號：CPE 2-160]。魚類被放置在通氣的水浴中，並被運送到附近的野外實驗室進行組織處理。作為對從 Roodeplaat 水壩收集的魚類的描述性組織學和超微結構的參考，研究中還包括了來自同一物種的兩條參考魚（雄性和雌性）。這些魚類在約翰內斯堡大學的水族館研究設施中以自來水在受控條件下飼養。
2.2 Necropsy and tissue sampling 2.2 屍檢和組織取樣
Each fish was weighed , and its total length ) was measured. Fish were sedated by placing a damp cloth over the eyes before severing the spinal cord anterior to the dorsal fin (Van Dyk, Marchand, Smit, & Pieterse, 2009). Ethical approval was obtained from the Ethics Committee of the Faculty of Science, University of Johannesburg. Each specimen was macroscopically examined to identify any internal and/or external abnormalities or injuries. The liver was excised, and its mass was recorded to calculate the hepatosomatic index (liver mass/body mass * 100). Each liver was carefully examined to identify any hepatic nodular alterations. Nodules were categorized based on macroscopic colour, and to standardize the method for categorization, we made use of a colour chart (President Paint). A digital photograph of each liver was taken (Kodak 26X), and tissue samples of each nodule category were taken for respective light and electron microscopy analyses. Tissue samples of non-nodular liver tissue from the affected fish were also sampled for comparison. For the non-nodular tissue, samples were taken from both liver lobes, including the right anterior (RA), left anterior (LA), right posterior (RP) and left posterior (LP) regions. For age estimation, the brain cavity was dorsally exposed, and the left and right otoliths were collected. 每條魚都被稱重，並測量其總長。在切斷背鰭前的脊髓之前，魚被用濕布蓋住眼睛使其鎮靜（Van Dyk, Marchand, Smit, & Pieterse, 2009）。我們從約翰尧斯堡大學科學學院的倫理委員會獲得了道德批准。每個標本都被用肉眼檢查以確定是否存在任何內部和/或外部異常或傷害。肝臟被切除，其質量被記錄以計算肝臟指數（肝臟質量/體重*100）。每個肝臟都被仔細檢查以確定是否存在任何肝臟結節性變化。結節根據肉眼顏色進行分類，為了標準化分類方法，我們使用了一個顏色圖表（President Paint）。每個肝臟的數碼照片被拍攝（Kodak 26X），並且對每個結節類別的組織樣本進行了相應的光學和電子顯微鏡分析。同時也對受影響魚類的非結節性肝組織進行了採樣以進行比較。 對於非結節性組織，樣本來自於肝葉的兩側，包括右前部（RA）、左前部（LA）、右後部（RP）和左後部（LP）區域。 針對年齡估計，腦腔背面暴露，並收集左右耳石。
2.3 Tissue processing and microscopic analyses 2.3 組織處理和顯微分析
Liver samples for light microscopy were immediately fixed in neutrally buffered formalin (Sigma, SA), embedded in paraffin wax and sectioned at . Sections were prepared for light microscopy using a standard protocol for haematoxylin and eosin (H&E) staining (Sigma, SA). Tissue sections were analysed on a multi-headed light microscope by two assessors for increased accuracy. 用於光學顯微鏡檢視的肝臟樣本立即固定在中性緩衝福馬林（Sigma, SA）中，埋入石蠟並切割至。 採用血酮和伊紅（H&E）染色（Sigma, SA）的標準方案，為肝臟切片準備光學顯微鏡的標本。 兩名評估者使用多頭光學顯微鏡分析組織切片，以提高準確性。
For transmission electron microscopy, liver samples were immediately fixed in glutaraldehyde (Sigma, SA) prepared in sodium cacodylate buffer (Sigma, SA) and post-fixed in osmium tetroxide (Sigma, SA). Samples were processed following the manufacturer's protocol (JEOL). Resin blocks were sectioned at 50-90 nm, and the ultrathin sections were stained with lead citrate and uranyl acetate (Sigma, SA) (Reynolds, 1963). A quality control evaluation on tissue sections was done, and sections of three fish were excluded due to artefactual changes observed. Electron micrographs were taken using a Philips CM10 electron microscope at the Electron Microscopy Unit of the University of Pretoria, Onderstepoort Campus. Ultrastructural alterations were identified and described by two assessors based on the ultrastructural alterations as presented by Grund et al. (2010). 進行傳輸電子顯微鏡檢查時，肝臟樣本立即在鈉卡比二胺緩衝液（Sigma, SA）中準備的戊二醛（Sigma, SA）中固定，並在四氧化二鎝（Sigma, SA）中後固定。樣本按照製造商的方案（JEOL）進行處理。樹脂塊切割成 50-90 納米厚的薄片，超薄切片用檸檬酸鉛和醋酸鈉（Sigma, SA）染色（Reynolds, 1963）。對組織切片進行質量控制評估，由於觀察到的人工變化，三條魚的切片被排除。電子顯微鏡圖像是在普利茲大學 Onderstepoort 校區的電子顯微鏡單位使用飛利浦 CM10 電子顯微鏡拍攝的。基於 Grund 等人（2010 年）提出的超微結構變化，兩名評估者識別並描述了超微結構變化。
2.4 Age estimation 2.4 年齡估計
Otoliths were prepared following the technique by Wischniowski and Bobko (1998). Embedded otoliths were transversely sectioned using a double-bladed diamond-edged otolith saw, and sections were then analysed independently by counting the growth rings (annuli) for each otolith under a light microscope. This was done by two assessors to increase the objectivity of the estimation. To ensure accurate results, the estimation was done three times by the same assessors, each seven days apart (Campana, 2001). A mean value was then calculated if three different values were recorded, or, if the same value was recorded during two of the three readings, that value was used as the estimated age. 依照 Wischniowski 和 Bobko（1998 年）的技術，製備即棘骨。使用一把雙刃鑽石邊緣的即棘骨鋸進行橫向切片，然後使用光學顯微鏡獨立地計數每個即棘骨的生長環（年輪）。這是由兩名評估者完成的，以增加估計的客觀性。為了確保準確的結果，評估是由同一名評估者進行三次，每次相隔七天進行（Campana，2001 年）。如果記錄了三個不同的值，則計算平均值；如果在三次讀數中有兩次記錄了相同的值，則使用該值作為估計年齡。
2.5 | Data analyses 2.5 | 數據分析
The results of this study were primarily descriptive in nature. However, the percentage prevalence was calculated for the number of fish presenting a specific nodule type, based on colour, as well as the number of fish presenting a specific histological or ultrastructural alteration. Quantitative data, that is HSI and age estimation results, were analysed using Spearman correlation analysis (IBM SPSS) to determine a possible correlation between liver mass and fish age. 本研究的結果主要具有描述性。然而，根據顏色對呈現特定結節類型的魚的數量以及對呈現特定組織學或超微結構變化的魚的數量，計算了百分比的盛行率。使用 Spearman 相關分析（IBM SPSS）對定量數據，即 HSI 和年齡估計結果進行分析，以確定肝臟質量和魚的年齡之間是否存在可能的相關性。
3 RESULTS 3 個結果
The sample group had a mean body mass of and a mean total length of . The mean HSI was . For the two reference specimens, the mean body mass was and mean total length was . The mean HSI for the reference specimens was . The external examination of the fish from Roodeplaat Dam showed no abnormalities. The internal macroscopic examination of the livers, however, showed the presence of hepatic nodular alterations in of the fish. These nodules varied in terms of colour, size and morphological distribution (Figure 1b). These observations corresponded with observations of the same species during previous surveys (2013 and 2015) where seasonal variation in occurrence of liver nodules was investigated. The nodules were mostly observed on the surface of the livers but also, in some cases, embedded in the liver tissue. Overall, nodules of 16 different colours were identified based on the colour chart including four shades of brown, five shades of orange, two shades of purple, two shades of cream and a single shade of yellow, green and red, respectively (Table 1). Interestingly, of affected fish had more than one type of nodule (based on colour), and this observation was more pronounced in male specimens. The reference fish had no macroscopically visible hepatic nodules (Figure 1a). The liver of . gariepinus is normally a bilaterally symmetrical organ composed of a left and right lobe (Figure 1a). As seen in Figure 1b, this symmetry was distorted in some livers due to the nodular growths. 樣本組 的平均體重為 ，平均總長為 。平均 HSI 為 。對於兩個參考標本，平均體重為 ，平均總長為 。參考標本的平均 HSI 為 。羅德普拉特水壩魚類的外部檢查未顯示異常。然而，肝臟的內部巨觀檢查顯示 的魚類存在肝臟結節性變化。這些結節在顏色、大小和形態分佈方面各不相同（圖 1b）。這些觀察結果與之前調查（2013 年和 2015 年）中對同一物種的觀察相符，當時調查了肝臟結節的季節變化。結節主要觀察在肝臟表面，但在某些情況下也嵌入在肝臟組織中。總的來說，根據顏色圖表識別了 16 種不同顏色的結節，包括四種棕色調、五種橙色調、兩種紫色調、兩種奶油色調，以及單一的黃色、綠色和紅色調（表 1）。 有趣的是，受影響的魚中有超過一種類型的結節（基於顏色），這種觀察在雄性標本中更為明顯。參考魚的肝臟沒有肉眼可見的肝結節（圖 1a）。非洲鱷魚的肝臟通常是一個由左葉和右葉組成的對稱器官（圖 1a）。如圖 1b 所示，由於結節性生長，一些肝臟的對稱性被扭曲。
The histological assessment of the livers of the reference fish showed no histological alterations. Hepatocytes exhibited a clear to granular cytoplasm, and the cells were arranged in a cord-like pattern. Hepatocytes were mainly polygonal in shape and similar in size with a centrally located nucleus and a single nucleolus. Few melanomacrophage centres (MMCs) and intracellular deposits were observed in the reference fish. 參考魚肝臟的組織學評估顯示沒有組織學改變。肝細胞呈現清晰至粒狀的細胞質，細胞呈線狀排列。肝細胞主要呈多邊形，大小相似，核心位於中央，有一個單一核仁。在參考魚中觀察到少量黑色巨噬細胞中心（MMCs）和細胞內沉積物。
The histological assessment of the nodular livers showed several alterations associated with the nodular tissue as well as the nonnodular tissue. The percentage prevalence of histological alterations per nodule type (or colour) is summarized in Table 1. Most of the histological alterations could not exclusively be associated with a specific nodule type, and tissue samples from most nodules presented a list of the same alterations. These included hepatocellular and nuclear pleomorphism, pyknotic nuclei, variation in cytoplasmic appearance and focal areas of cellular alteration (FCA). An increase in the number and size of MMCs, intracellular deposits, steatosis and giant cells were also identified. Inflammation was not a common occurrence, but a few livers did show perivascular inflammatory infiltration of lymphocytes. MMCs varied in staining intensity ranging from light brown to light gold, and its occurrence was also not exclusive to a specific colour nodule. MMCs were mostly associated with tissue regions surrounding FCAs within a nodule (Figure ). 對結節性肝臟的組織學評估顯示了與結節組織以及非結節組織相關的幾種變化。每種結節類型（或顏色）的組織學變化發生率百分比摘要如表1所示。大多數組織學變化無法專屬於特定的結節類型，大多數結節的組織樣本呈現相同的變化清單。這些包括肝細胞和核多形性、核固縮、細胞質外觀的變化以及細胞變化的局部區域（FCA）。還發現了MMC的數量和大小增加、細胞內沉積物、脂肪肝和巨細胞。炎症並不常見，但有幾個肝臟顯示淋巴細胞周圍的血管周圍炎性浸潤。MMC的染色強度有所不同，從淺褐色到淺金色不等，它的出現也不專屬於特定顏色的結節。MMC主要與結節內部FCA周圍的組織區域相關。
The cytoplasm of hepatocytes within nodules varied between a smooth, granular or clear appearance, or, in some cases, a single tissue section presented a mixture of these cytoplasmic characteristics. Cytoplasmic appearance was also not found to be a determining factor of a specific nodule colour. However, most of the nodular types were characterized by hepatocytes with a granular appearance, and of the different nodule types showed an exclusivity towards a specific cytoplasmic appearance. Diffuse alterations (Table 1), which included macro- and micro-vesicular steatosis, intracellular deposits and MMCs, were prevalent in most of the nodule types with the lighter-coloured nodules (cream and yellow) showing no intracellular deposits and MMCs. The purple and green nodules had no evidence of micro-vesicular steatosis. Furthermore, it was also observed that the cream and yellow nodules had no FCAs, while the other nodule types had one or more type of FCA. These included basophilic, eosinophilic, clear cell, steatosis and hypertrophic focal areas (Figure 1c). The FCAs varied in size, some consisting of only a few cells to larger 肝細胞核內質變化在結節內呈現平滑、粒狀或清晰外觀，有些情況下，單一組織切片呈現這些細胞質特徵的混合。細胞質外觀也不是特定結節顏色的決定因素。然而，大多數結節類型的特徵是具有粒狀外觀的肝細胞，不同結節類型中的大部分對特定細胞質外觀呈現排他性。擴散性變化（表1），包括大、小泡脂肪變性、細胞內沉積物和MMC，在大多數結節類型中普遍存在，較淺色結節（奶油和黃色）不顯示細胞內沉積物和MMC。紫色和綠色結節沒有微小泡脂肪變性的證據。此外，觀察到奶油和黃色結節沒有FCAs，而其他結節類型有一種或多種類型的FCA。這些包括嗜鹼性、嗜酸性、清晰細胞、脂肪變性和肥大灶（圖1c）。FCAs的大小不等，有些只由幾個細胞組成，也有些更大。
FIGURE 1 Photographs presenting the macroscopic appearance of the livers of the reference fish (a) and the fish with nodular alterations ( ) from Roodeplaat Dam (b); light micrographs of liver tissue showing (H&E) (c) hypertrophic focal area of cellular alteration (1), intercellular inflammatory infiltration (2), diffused macro- and micro-vesicular steatosis (3), eosinophilic FCA and (4) melanomacrophage centre 圖 1 照片展示參考魚的肝臟的肉眼外觀（a）和具有結節性變化的魚（ ）來自 Roodeplaat 水壩（b）；肝組織的光學顯微鏡圖像顯示（H＆E）（c）細胞變化的肥大局部區域（1），細胞間炎症浸潤（2），弥漫性的大、小泡脂肪生成（3），嗜酸性 FCA 和（4）黑色素巨噬細胞中心
FCAs consisting of multiple cells. The FCAs had a random distribution throughout the nodular tissue. 由多個細胞組成的 FCA。 FCA 在結節組織中隨機分佈。
The ultrastructural alterations identified are listed in Table 2. The ultrastructural organization of the reference fish's hepatocytes (Figure 3a) confirmed a clear cell cytoplasm with intact cell membranes. In addition to the hepatocytes, endothelial and stellate cells, sinusoids and the space of Disse were clearly visible. The bile canaliculi were identified at the junction of two hepatocytes with intact microvilli covering the lumen (Figure 3a). Nuclei were mostly centrally located with stacked cisternae of RER surrounding it. The cytoplasm was characterized by different organelles which included elongated and oval mitochondria, peroxisomes, Golgi fields consisting of cisternae and smooth endoplasmic reticulum. Few MMCs, macrophages, lysosomal elements and lipid droplets were observed in the reference fish. 在表2中列出了識別出的超微結構變化。參考魚的肝細胞的超微結構組織（圖3a）確認了細胞質清晰，細胞膜完整。除了肝細胞外，內皮細胞和星狀細胞、血窦和Disse空間也清晰可見。膽小管位於兩個肝細胞交界處，具有完整的微絨毛覆蓋腔道（圖3a）。細胞核大多位於中央，周圍環繞著堆積的RER囊泡。細胞質以不同的細胞器為特徵，包括細長和橢圓形的粒線體、過氧化物酶體、高爾基體區域由囊泡和平滑內質網組成。在參考魚中觀察到少量MMC、巨噬細胞、溶酶體元素和脂滴。
The ultrastructural alterations in both the nodular and the nonnodular tissue of the fish from Roodeplaat Dam included cell membrane disruption, irregular shape of nuclei with augmentation of the nucleolus, deformed nuclear envelopes as well as dilation, proliferation, fragmentation and vesiculation of the cisternae of the RER. Furthermore, there was an increase in degenerative mitochondria (transparent matrix) (Figure 3f) and swelling of mitochondria and proliferation of both peroxisomes and lysosomes. 羅德普拉特水壩魚類的結節性和非結節性組織中的超微結構變化包括細胞膜破壞，核的形狀不規則，核仁增大，變形的核膜以及 RER 囊泡的擴張，增生，碎裂和囊泡化。此外，退化的線粒體（透明基質）增加（圖 3f），線粒體腫脹，過氧化物酶體和溶酶體增生。
The different cytoplasmic appearances of hepatocytes observed during light microscopy were characterized by specific ultrastructural changes: Granular cytoplasm on light microscopy showed proliferation and swelling of mitochondria on electron microscopy, while the smooth cytoplasm on light microscopy had a high prevalence of proliferated, dilated RER and proliferated mitochondria on electron microscopy. Granular to clear cytoplasm showed swelled-up degenerative mitochondria (transparent matrix) throughout the cytoplasm. 在光學顯微鏡下觀察到的肝細胞不同的細胞質外觀特徵化為特定的超微結構變化：光學顯微鏡下的顆粒狀細胞質顯示出電子顯微鏡下線粒體增生和腫脹，而光學顯微鏡下的光滑細胞質在電子顯微鏡下顯示出增生，擴張的 RER 和增生的線粒體。從顆粒狀到清晰的細胞質顯示出整個細胞質中腫脹的退化線粒體（透明基質）。
Overall, the number of organelles in the hepatocytes from the nodular tissue appeared to have increased with an irregular distribution throughout the cytoplasm. The MMCs (as seen on light microscopy) and lipid droplets increased in number and size compared to the reference fish. In addition, lysosomal elements (myelin bodies, secondary lysosomes and vacuoles) also increased in size and numbers in the nodular tissue. The MMCs, mast cells and fibrillar inclusions were also found to be more prevalent in non-nodular tissue regions as compared to nodular tissue. 整體而言，從結節組織中的肝細胞中的器官數量似乎增加，並且在整個細胞質中呈不規則分佈。與參考魚相比，MMC（在光學顯微鏡下看到）和脂滴的數量和大小增加。此外，溶酶體元素（髓鞘體、次級溶酶體和囊泡）在結節組織中的大小和數量也增加。與結節組織相比，非結節組織區域中的 MMC、肥大細胞和纖維包涵物也更普遍。
Similarly, to the light microscopy assessment, most of the ultrastructural alterations were found in all nodule types. of the nodule types showed no alterations associated with hepatocyte structure while of the nodule types had ultrastructural alterations associated with the nuclei and mitochondria. Only of the 類似於光學顯微鏡評估，大多數超微結構變化均發現在所有結節類型中。其中 結節類型未顯示與肝細胞結構相關的變化，而 結節類型具有與細胞核和粒線體相關的超微結構變化。只有 的
FIGURE 2 Light micrographs of liver tissue showing (H&E) showing examples of histological alterations identified in the nodular livers of fish from Roodeplaat Dam: (a) intracellular deposits (arrows) ; (b) perivascular inflammatory infiltration (arrow) and melano-macrophage centre (arrowhead) (×200); (c) focal area of steatosis characterized by large, round vacuoles (arrow) ( ); (d) eosinophilic focal area of cellular alteration with surrounding melano-macrophage centre ; (d) nuclear pleomorphism showing large nuclei (arrows) and small nuclei (arrowheads) ( ; and a random distribution of melano-macrophage centres (arrows) and various focal areas of cellular alterations (1 and 2) ( ) nodule types had no alterations to the RER and peroxisomes, with showing the presence of myelin bodies, lipid droplets and macrophages (Figure 4d,e). 圖2 肝組織的光學顯微圖（H&E染色），顯示在Roodeplaat水壩魚類的結節性肝臟中識別出的組織學改變示例：(a) 細胞內沉積（箭頭）; (b) 血管周圍的炎症浸潤（箭頭）和黑色素-巨噬細胞中心（箭頭）（×200）; (c) 以大而圓的空泡為特徵的脂肪肝局部區域（箭頭）; (d) 周圍黑色素-巨噬細胞中心的嗜酸性細胞變化局部區域; (d) 核多形性顯示大核（箭頭）和小核（箭頭）; 以及隨機分佈的黑色素-巨噬細胞中心（箭頭）和各種細胞變化的局部區域（1和2）結節類型未對RER和過氧化物酶體進行改變，其中顯示出髓鞘體、脂滴和巨噬細胞的存在（圖4d，e）。
It was also found that and of the nodule types presented mast cells and collagen fibrillar inclusions, respectively (Figure 4b). The latter were unevenly distributed in the extracellular space. Steatosis was a prevalent alteration observed in the nodular livers during light microscopy, and the electron microscopy confirmed both macro- and micro-vesicular steatosis (Figures 1c and 3b). Some of the hepatocytes were bi-nucleated with augmentation of the nucleoli observed (Figure 3e) and RER had proliferated (Figure 3d) with fragmentation, dilation and vesiculation observed (Figure ). 發現 和 的結節型態分別呈現肥大細胞和膠原纖維包含物（見圖4b）。後者在細胞外空間中分佈不均。脂肪肝是光學顯微鏡下觀察到的結節性肝臟的常見變化，電子顯微鏡確認了宏觀和微觀脂肪肝（見圖1c和3b）。一些肝細胞是雙核的，觀察到核仁增大（見圖3e），RER增生（見圖3d），並觀察到碎裂、擴張和囊泡化（見圖 ）。
An unexpected and interesting finding in the non-nodular tissue of affected livers was the presence of rhomboid intracellular crystalline structures with black pigments (Figure 4a). These crystals of varying sizes were found in one of the four non-nodular sampled regions of two different fish in close proximity of the nucleus, with the RER in some cases encircling the small crystals. The ultrastructural assessment also clearly showed the engulfed content by macrophages which included, for the most part, pigments, myelin bodies and whorl-like structures, lysosomes and vacuoles (Figure 4c). The microvilli of the bile canaliculi between two hepatocytes were reduced in number as compared to the reference fish with myelin present in some bile canaliculi. 受影響肝臟非結節組織中一個意外且有趣的發現是存在帶有黑色顆粒的菱形細胞內結晶結構（圖4a）。這些大小不一的結晶在兩條不同魚類的四個非結節採樣區域中的一個區域中發現，靠近細胞核，有時RER會環繞小結晶。超微結構評估還清楚地顯示了被巨噬細胞吞噬的內容，其中包括大部分的顆粒、髓鞘體和螺旋狀結構、溶酶體和囊泡（圖4c）。與參考魚相比，兩個肝細胞之間的膽小管微絨毛數量減少，有些膽小管中存在髓鞘。
Age estimation was achieved for of the sampled fish. Fish age ranged from 2 to 18 years with a mean age for the sample group of 6 years . The liver of the oldest fish presented the highest number of different types of nodules. However, even though nodular growths were postulated to result in increased liver mass, the mean HSI did not show a positive correlation with age. Interestingly, the results showed that male fish were on average older compared to female fish. Also, female fish presented either one or two hepatic nodule types while the male fish presented three to four types of hepatic nodules. 估計了樣本魚的年齡。魚的年齡範圍從 2 到 18 歲，樣本組的平均年齡為 6 歲。最老的魚的肝臟呈現出最多不同類型的結節。然而，即使認為結節性生長會導致肝臟增重，平均 HSI 並未顯示出與年齡的正相關。有趣的是，結果顯示，雄性魚平均年齡比雌性魚大。此外，雌性魚呈現出一種或兩種肝結節類型，而雄性魚呈現出三到四種肝結節類型。
4 DISCUSSION 4 討論
The studies by Marchand et al. (2012) and Van Dyk et al. (2012) were the first reports of macroscopically visible hepatic nodular alterations in C. gariepinus from Roodeplaat Dam. However, although these reports included a histological assessment of the liver tissue, it Marchand 等人（2012 年）和 Van Dyk 等人（2012 年）的研究是首次報告在 Roodeplaat 水壩的 C. gariepinus 中出現肝臟結節性變化的宏觀可見報告。然而，儘管這些報告包括對肝組織的組織學評估，它們
did not investigate the nodules with regard to the difference in their macroscopic appearance, nor did they investigate the possibility of specific histological and/or ultrastructural alterations to be associated with the different nodule types, based on colour. This information could be important and very useful in future with regard to fish necropsy procedures and observations. 未對結節的巨觀外觀差異進行研究，也未對不同結節類型的可能具有特定組織和/或超微結構變化進行研究，基於顏色的關聯性。這些資訊在未來對於魚類屍檢程序和觀察可能非常重要和有用。
According to the ICES (2004), liver nodule macroscopic colour can be an indication of the histological nature of the nodule, primarily based on work done on marine species. In this study, we identified 16 different coloured nodules macroscopically with of the fish sampled presenting some form of macroscopic nodular alterations and presenting more than one type of nodule in the same organ. Based on work done on marine species, that is flatfish dab and flounder, white and opaque nodules were found to be associated with acidophilic and clear cell hepatocytes, while a yellow colour is associated with basophilic hepatocellular lesions (ICES, 2004). Green nodules were linked to malignant and benign tumours of the bile ducts (cholangiocarcinoma and cholangioma), and dark-red nodules can show neoplasia involving vascular structures (ICES, 2004). However, the authors also indicated that the macroscopic appearance of nodules alone cannot be used for diagnoses. 根據ICES（2004）的說法，肝結節的巨觀顏色可能是結節的組織性質的指示，主要基於對海洋物種的研究。在這項研究中，我們巨觀識別了16種不同顏色的結節，樣本魚中有 呈現一些形式的巨觀結節變化，並且 在同一器官中呈現了多種結節類型。根據對海洋物種（比如比目魚和鰈魚）的研究，白色和不透明的結節與嗜酸性和清晰細胞肝細胞病變有關，而黃色與嗜鹼性肝細胞損傷相關（ICES，2004）。綠色結節與膽管的惡性和良性腫瘤（膽管癌和膽管瘤）有關，而深紅色結節可能顯示涉及血管結構的新生物（ICES，2004）。但作者也指出，僅憑結節的巨觀外觀不能用於診斷。
Unexpectedly, our result did not show histological alterations that were exclusively associated with a specific nodule colour. We rather observed the same spectrum of alterations across most of the nodule types, with more than one type of alteration observed in a specific type of nodule. It was expected that the dark, browncoloured nodules might be linked to an increase in pigments, that is more MMCs (which might contain melanin), yellow or cream nodules would show increased steatosis or bile-related alterations (as the bile colour of C. gariepinus is yellow), and that red-coloured nodules would be associated with circulatory alterations. We rather found that diffuse steatosis was present at a high prevalence in all nodule types, not just the light-coloured nodules. Also, the cream and yellow-coloured nodules were unique in the fact that these tissue sections had no FCAs, including steatosis. 出乎意料的是，我們的結果並未顯示與特定結節顏色專屬相關的組織學變化。我們反而觀察到大多數結節類型之間出現相同的變化範圍，在特定結節類型中觀察到了多種類型的變化。一般預期深色的棕色結節可能與色素增加有關，即可能含有黑色素的多色素細胞（MMC），黃色或奶油色結節可能會顯示脂肪堆積或與膽汁相關的變化（因為C. gariepinus的膽汁顏色是黃色），紅色結節可能與循環變化有關。我們卻發現所有結節類型中都普遍存在弥漫性脂肪肝變化，而不僅僅是淺色結節。此外，奶油色和黃色結節在這些組織切片中的特點是沒有纖維膠原增生（FCA），包括脂肪堆積。
Similarly to the light microscopy analysis, a number of different ultrastructural alterations were identified in the nodular tissue. These included fragmentation, dilation and vesiculation of RER, fibrillar inclusion, degenerative mitochondria (transparent matrix) and the presence of myelin bodies, lipid droplets, mast cells and secondary lysosomes. According to , Zhou, Xu, Schramm, and Kettrup (1999) and Grund et al. (2010), fragmentation, vesiculation and proliferation of the RER are normally seen as a result of xenobiotic stress, and proliferation of myelin bodies and lysosomes is believed to be an adaptation mechanism in response to cell organelle degeneration. In a study on C. gariepinus from a polluted system in Egypt, Abdel-Moneim and Abdel-Mohsen (2010) also indicated that myelinlike figures could be an indication of autophagy of degenerative membranous components and a high turnover of cell organelles. According to Crivellato and Ribatti (2010), mast cells in fish are involved in the defence mechanism and could be associated with parasitic infections. Even though we did not observe any intrahepatic parasitic infections, we did observe overall inflammatory 與光學顯微鏡分析類似，結節組織中也發現了許多不同的超微結構變化。這些包括RER的碎裂、擴張和囊泡化、纖維包涵體、變性的粒線體（透明基質）以及髓鞘體、脂滴、肥大細胞和次級溶酶體的存在。根據Zhou、Xu、Schramm和Kettrup（1999年）以及Grund等人（2010年）的研究，RER的碎裂、囊泡化和增生通常被視為外源性壓力的結果，而髓鞘體和溶酶體的增生被認為是對細胞器官變性的適應機制。在一項關於埃及受污染系統中C. gariepinus的研究中，Abdel-Moneim和Abdel-Mohsen（2010年）還指出，類似髓鞘體的結構可能表明膜結構成分的自噬和細胞器官的高周轉。根據Crivellato和Ribatti（2010年）的研究，魚類中的肥大細胞參與防禦機制，可能與寄生蟲感染有關。儘管我們沒有觀察到任何肝內寄生蟲感染，但我們確實觀察到總體上的炎症。
FIGURE 3 Transmission electron micrographs showing ultrastructural alterations of the livers of the reference fish and the fish with nodular alterations. 圖 3 顯示參考魚和具有結節性變化的魚肝臟的超微結構變化。
(a) Cell membrane between two hepatocytes from reference fish (bar ); (b) micro- and macrovesicular steatosis (MacS; MicS) (bar ); (c) fragmented rough endoplasmic reticulum (RER) and augmentation of heterochromatin [LDlipid droplets] (bar ; (d) proliferated and dilated RER (bar ); (e) binucleated hepatocyte (bar ; and degenerative mitochondria and nucleolar augmentation (bar ) (a) 來自參考魚的兩個肝細胞之間的細胞膜（標尺）; (b) 微小和巨大脂肪肝（MacS; MicS）（標尺）; (c) 碎片化的粗面內質網（RER）和異染色質增多[LD 脂滴]（標尺）; (d) 增生和擴張的 RER（標尺）; (e) 雙核肝細胞（標尺）; 和變性的線粒體和核仁增多（標尺）
responses in the form of perivascular and intercellular lymphocytic infiltrations during light microscopy. 在光學顯微鏡下以血管周圍和細胞間淋巴細胞浸潤的形式出現的反應。
Based on the categorization by Lang et al. (2006), adapted from ICES (2004), the histological alterations we identified could be categorized mostly as early toxicopathic, non-neoplastic lesions. Also, according to Myers et al. (1998), FCAs and hepatocellular and nuclei pleomorphism (HNP) are caused by exposure to contaminants with FCAs known as a precursor of benign or malignant liver tumours. These alterations are possibly as a result of exposure to specific toxicants or bio-toxins, or a mixture of these, present within the system. A possible cause might be microcystins, a hepatotoxin that has been reported in the Roodeplaat Dam before (Van Ginkel, 2008; Conradie & Barnard, 2012). 根據 Lang 等人（2006）的分類，改編自 ICES（2004），我們識別的組織學變化主要可歸類為早期毒理病理性、非腫瘤性病變。此外，根據 Myers 等人（1998）的說法，FCAs 和肝細胞及核變異性（HNP）是由暴露於含有 FCAs 的污染物引起的，而 FCAs 被認為是良性或惡性肝腫瘤的前體。這些變化可能是由於暴露於特定毒素或生物毒素，或這些物質的混合物，存在於系統中。可能的原因可能是微囊藻毒素，這是一種已報告在 Roodeplaat 水壩中存在的肝毒素（Van Ginkel，2008；Conradie＆Barnard，2012）。
There is also much evidence of the ultrastructural and biochemical alterations in different tissues of fish following laboratory exposures to cyanobacteria, and specifically microcystins, but limited information has been reported on the effect in wild fish exposed under natural conditions (Qiu et al., 2007; Clemente et al., 2010). 也有許多證據表明，在實驗室暴露於藍藻，特別是微囊藻後，魚類不同組織的超微結構和生化變化，但有關在自然條件下暴露於野生魚類的影響的信息有限（Qiu 等，2007；Clemente 等，2010）。
Previous studies by Branka et al. (2011) and Mitsoura et al. (2013) showed that microcystin exposure does cause ultrastructural alterations (cell membrane disruption, vesiculation and dilation of RER, proliferation of mitochondria, secondary lysosomes and myelin bodies) in fish liver following both natural and laboratory exposures. Therefore, we suspect that microcystins might be a possible causative or contributing factor for the development of the nodular alterations identified. Irrespective of the macroscopic appearance, most of the above-mentioned histological and ultrastructural alterations had a high prevalence in most nodule types. As a result, a specific alteration could not be associated with a specific nodule colour. Branka 等人（2011 年）和 Mitsoura 等人（2013 年）的先前研究顯示，微囊藻毒素暴露確實會導致魚肝發生超微結構變化（細胞膜破裂、囊泡化和 RER 擴張、粒線體增生、次級溶酶體和髓鞘體），無論是在自然環境還是實驗室暴露後。因此，我們懷疑微囊藻毒素可能是導致或促成結節性變化發展的可能因素。儘管在宏觀外觀上有所不同，但大多數上述組織學和超微結構變化在大多數結節類型中普遍存在。因此，特定的變化無法與特定的結節顏色相關聯。
However, the transmission electron microscopy analysis did provide some clarity on the cytoplasmic appearance of the hepatocytes observed during light microscopy. A distinct observation was the variation in cytoplasmic appearance (with H&E staining), which primarily included granular, smooth and clear cells, and in some cases, tissue section presented a mixture of these different cytoplasmic 然而，透射電子顯微鏡分析確實為光學顯微鏡觀察到的肝細胞質外觀提供了一些澄清。一個明顯的觀察是細胞質外觀的變化（使用 H&E 染色），主要包括顆粒狀、光滑和清晰細胞，有時，組織切片呈現這些不同細胞質的混合。
FIGURE 4 Transmission electron micrographs showing ultrastructural alterations of the livers of fish with nodular alterations. (a) Crystals (C) in nonnodular tissue (bar ); (b) fibrillar inclusions (F) and mast cells (Mc) in the extracellular space (bar ); (c) melano-macrophage centres (MMCs) (bar ); (d) secondary lysosomes (Lys) filled with myelin bodies ; (e) macrophage (Mac) (bar ); and (f) mast cells (bar ) characteristics. This variation was also reported in C. gariepinus from Roodeplaat Dam sampled more than 10 years ago (Van Dyk et al., 2012). A suggested reason for the difference in cytoplasmic appearance is the proliferation of RER (ICES, 2004; Van Dyk et al., 2012), Our results did show that granular cytoplasm was associated with the proliferation and swelling of mitochondria while smooth cytoplasm showed a high prevalence of the swelling of mitochondria and proliferation of RER. Clear cells showed degenerative mitochondria (transparent matrix) or little to no mitochondria. 圖4 顯示具有結節性變化的魚肝臟的超微結構變化的傳遞電子顯微圖。 (a) 結晶體（C）在非結節組織中（橫條 ）; (b) 纖維包涵體（F）和肥大細胞（Mc）在細胞外空間中（橫條 ）; (c) 黑色素-巨噬細胞中心（MMCs）（橫條 ）; (d) 充滿髓鞘體的次級溶酶體（Lys） ; (e) 巨噬細胞（Mac）（橫條 ）; 和（f）肥大細胞（橫條 ）的特徵。這種變化也在超過10年前從Roodeplaat水壩採樣的C. gariepinus中報告過（Van Dyk等，2012年）。對細胞質外觀差異的建議原因是RER的增殖（ICES，2004年；Van Dyk等，2012年），我們的結果確實顯示，顆粒狀細胞質與線粒體的增殖和腫脹有關，而光滑細胞質則表明線粒體的腫脹和RER的增殖普遍存在。清晰細胞顯示變性的線粒體（透明基質）或幾乎沒有線粒體。
A common observation in the liver tissue was the presence of MMCs. An increase in size or number of MMCs has been shown as a good indicator of environmental stress or pollution (Herraez & Zapata, 1986; Agius & Roberts, 2003; Fafioye, Adebisi, & Fagade, 2004; Sayed & Younes, 2017). However, Van Dyk et al. (2012) observed that MMCs were present in C. gariepinus from both polluted (e.g., Roodeplaat Dam) and reference sites. In the current study, we did observe a higher incidence of MMCs in the nodular livers compared to the reference fish. The MMCs varied in colour from gold to light and dark brown (with H&E staining). Interestingly, all the MMCs identified were located in tissue regions surrounding FCAs. The absence of MMCs in FCAs was also reported by Van Dyk et al. (2012) and ICES (2004). Based on special stains done on liver tissue of . gariepinus from Roodeplaat Dam in a previous study, it was found that these structures consisted of hemosiderin (Perls' Prussain Blue), lipofuscin (Schmorl's) and melanin (Masson-Fontana), whereas the intracellular deposits, which had a similar appearance after H&E preparation, were primarily made up of hemosiderin (Van Dyk et al., 2012). 在肝組織中普遍觀察到MMC的存在。MMC的大小或數量增加被證明是環境壓力或污染的良好指標（Herraez＆Zapata，1986; Agius＆Roberts，2003; Fafioye，Adebisi＆Fagade，2004; Sayed＆Younes，2017）。然而，Van Dyk等人（2012年）觀察到MMC存在於受污染（例如Roodeplaat水壩）和參考站點的C. gariepinus中。在本研究中，我們觀察到與參考魚相比，結節性肝臟中MMC的發生率較高。MMC的顏色從金色到淺褐色和深褐色（使用H＆E染色）不等。有趣的是，所有識別出的MMC都位於周圍組織區域，這些區域周圍是FCAs。Van Dyk等人（2012年）和ICES（2004年）也報告了FCAs中MMC的缺乏。根據對Roodeplaat水壩中C. gariepinus的肝組織進行的特殊染色，在先前的研究中發現，這些結構由含鐵血黃素（Perls' Prussain Blue）、脂褐素（Schmorl's）和黑色素（Masson-Fontana）組成，而細胞內沉積物，在H＆E處理後具有相似的外觀，主要由含鐵血黃素組成（Van Dyk等人，2012年）。
Other observations included rhomboid cytoplasmic crystalline structures, specifically identified in the non-nodular liver tissue. These were abundant in the cytoplasmic matrix, characterized by black pigments. These structures also differed in size. Crystalline inclusions are usually associated with protein (Ghadially, 1985). According to Ghadially (1985), crystalline inclusions of a protein nature have been identified in different cell structures including the 其他觀察包括菱形細胞質結晶結構，特別是在非結節性肝組織中明確識別。這些結構在細胞質基質中豐富，以黑色色素為特徵。這些結構在大小上也有所不同。結晶包含物通常與蛋白質有關（Ghadially，1985年）。根據Ghadially（1985年）的說法，蛋白質性結晶包含物已在不同的細胞結構中被識別出。
nucleus, mitochondria, RER and the cytoplasmic matrix of cells. Such crystalline inclusions have also previously been identified in different tumours, for example alveolar soft part sarcoma (Shipkey, Liberman, Foote, & Stewart, 1964). A study by Marquet and Sobel (1969) also found proteinaceous rhomboid crystals in the granular ER and nuclear envelope of the spinal cord oligodendroglia of the guppy Poecilia reticulata Peters, 1859 and the lungfish Polypterus endlicheri Heckel, 1847 while Hamilton, Fawcett, and Christensen (1966) identified crystals in the liver of the slender salamander Batrachoseps attenuatus (Eschscholtz, 1833) related to reserve product utilized during hibernation. Although it was clear from our analysis that these crystalline structures were not present in nodular tissue but rather the surrounding non-nodular tissue, the exact cause is still unclear and needs further investigation. 細胞核、粒線體、粗面內質網和細胞質基質。這樣的結晶包含物也曾在不同的腫瘤中被鑑定出現，例如肺泡軟部肉瘤（Shipkey, Liberman, Foote, & Stewart, 1964）。Marquet和Sobel（1969）的研究還發現了蛋白質菱形結晶存在於孔雀魚Poecilia reticulata Peters, 1859和肺魚Polypterus endlicheri Heckel, 1847的脊髓寡突細胞的顆粒內質網和細胞核膜，而Hamilton, Fawcett和Christensen（1966）識別出了細胞核膜中的結晶在細長蠑螈Batrachoseps attenuatus（Eschscholtz, 1833）的肝臟中，這些結晶與冬眠期間利用的儲備產物有關。儘管從我們的分析中清楚地看出這些結晶結構並不存在於結節組織中，而是存在於周圍的非結節組織中，但確切原因仍不清楚，需要進一步調查。
The overall results were thus unexpected as it was clear that the histological and ultrastructural alterations identified could not explain the variation in macroscopic colour of the nodules. The number of alterations was more prevalent in the nodular tissue compared to non-nodular tissue with steatosis found to be the most prevalent. Macro- and micro-vesicular steatosis has been found to occur in fish with the decreased function of catalase that results in the build-up of lipids in the liver (Braunbeck, Storch, & Bresch, 1990). Furthermore, in comparison with non-nodular regions, the liver nodules had less MMCs, fewer mast cells and fibrillar inclusion, which might be indicative of the regenerative nature of the nodules. 總體結果出乎意料，因為明顯可見，組織學和超微結構的變化無法解釋結節的巨觀顏色變化。與非結節組織相比，結節組織中的變化更為普遍，其中脂肪肝變化最為普遍。已發現在魚類中發生宏觀和微觀脂肪肝變化，這是由於過氧化氫酶功能下降導致肝臟中脂質積聚（Braunbeck, Storch, & Bresch, 1990）。此外，與非結節區域相比，肝臟結節中的MMC數量較少，肥大細胞和纖維包涵體也較少，這可能表明結節的再生特性。
We further hypothesized that HSI would be higher in older fish from Roodeplaat Dam, as we suspected that they would present more macroscopic nodules (resulting in hypertrophy or hyperplasia of liver cells) due to a longer exposure time to the polluted water. We did not find a strong positive correlation with the age of the fish and the ; however, the oldest fish (18 years) did present the most nodules. We also could not identify a relationship between the fish age and the specific types of nodules present. Sample size should be considered here. Although 25 fish are sufficient to conduct a histological and ultrastructural descriptive study, correlation analysis will become more conclusive as we investigate this aspect over the coming years as part of this ongoing research. 我們進一步假設，Roodeplaat水壩的年長魚類的HSI會較高，因為我們懷疑它們會因長時間暴露於受污染的水中而出現更多的巨觀結節（導致肝細胞肥大或增生）。我們並未發現魚類年齡與 之間存在較強的正相關性；然而，最年長的魚類（18歲）確實出現了最多的結節。我們也無法確定魚類年齡與出現的特定結節類型之間的關係。這裡應該考慮樣本大小。儘管25條魚足以進行組織學和超微結構描述性研究，但隨著我們在未來幾年中對這一方面進行調查，相關性分析將變得更具有說服力，作為這項持續研究的一部分。
Myers et al. (1998) showed that age is a significant risk factor when investigating different types of lesions in bottom fish species, as the latency period from exposure to development (neoplasia, preneoplastic FCA and non-neoplastic proliferative lesion) increases the risk of lesion occurrence each year. Furthermore, studies by Stentiford et al. (2010) demonstrated that age plays a role in disease pattern, especially in older fish, although disease prevalence of specific age classes differs between sites. All of these aspects will have to be considered in future. 1998 年，Myers 等人表明年齡是在研究底棲魚類不同類型病變時的一個重要風險因素，由於從暴露到發展（新形成物、前新形成的 FCA 和非新形成的增生病變）的潛伏期每年都會增加病變發生的風險。此外，Stentiford 等人（2010 年）的研究表明年齡在疾病模式中發揮作用，特別是在老年魚中，儘管特定年齡類別的疾病患病率在不同地點之間存在差異。所有這些方面將在未來得到考慮。
ACKNOWLEDGEMENTS 致謝詞
The authors would like to acknowledge the National Research Foundation (NRF) of South Africa for financial assistance; Prof IEJ Barnhoorn for assistance during the field sampling; and the Spectrum facilities at the University of Johannesburg for the digital imagery. 作者們要感謝南非國家研究基金會（NRF）提供的財務援助；IEJ Barnhoorn 教授在野外採樣期間的幫助；以及約翰內斯堡大學的 Spectrum 設施提供的數字影像。
CONFLICT OF INTEREST 利益衝突
The authors have no conflict of interests to declare. 作者沒有利益衝突需要宣布。
Abdel-Moneim, A. M., & Abdel-Mohsen, H. A. (2010). Ultrastructure changes in hepatocytes of catfish Clarias gariepinus from Lake Maruit, Egypt. Journal of Environmental Biology, 31, 715-720. Abdel-Moneim, A. M., & Abdel-Mohsen, H. A. (2010). 埃及馬魯伊特湖克拉里斯魚肝細胞的超微結構變化。《環境生物學期刊》，31, 715-720。
Agius, C., & Roberts, R. J. (2003). Melano-macrophage centres and their role in fish pathology. Journal of Fish Diseases, 26, 499-509. https://doi.org/10.1046/j.1365-2761.2003. 00485.x Agius, C., & Roberts, R. J. (2003). 魚類病理學中的黑色巨噬細胞中心及其作用。《魚類疾病期刊》，26，499-509。https://doi.org/10.1046/j.1365-2761.2003.00485.x
Atencio, L., Moreno, I., Prieto, A. I., Moyano, R., Molina, A. M., & Camea, A. M. (2008). Acute effects of microcystins MC-LR and MC-RR on acid and alkaline phosphatase activities and pathological changes in intraperitoneally exposed Tilapia fish (Oreochromis sp.). Toxicologic Pathology, 36, 449-458. https://doi.org/10.1177/0192623308315356 Atencio, L., Moreno, I., Prieto, A. I., Moyano, R., Molina, A. M., & Camea, A. M. (2008). 微囊藻毒素 MC-LR 和 MC-RR 對腹腔內暴露的吉利波魚（Oreochromis sp.）的酸性和鹼性磷酸酶活性和病理變化的急性影響。《毒理病理學》，36，449-458。https://doi.org/10.1177/0192623308315356
Bernet, D., Schmidt, H., Meier, W., Burkhardt-Holm, P., & Wahli, T. (1999). Histopathology in fish: Proposal for a protocol to assess aquatic pollution. Journal of Fish Diseases, 22, 25-34. https://doi.org/ 10.1046/j.1365-2761.1999.00134.x Bernet, D., Schmidt, H., Meier, W., Burkhardt-Holm, P., & Wahli, T. (1999). 魚類組織病理學：提出一個評估水體污染的方案。《魚類疾病期刊》，22，25-34。https://doi.org/10.1046/j.1365-2761.1999.00134.x
Bogovski, S., Lang, T., & Mellergaard, S. (1999). Histopathological examinations of liver nodules in flounder (Platichthys flesus) from the Baltic Sea. ICES Journal of Marine Science, 56, 148-151. https://doi.org/10. 1006/jmsc. 1999.0461
Branka, R. P., Svetlana, G. D., Tijana, B. R., Jelena, P. G., Slavica, S. B., Pavlovic, S. Z., ... Zorica, S. S. (2011). Biochemical and ultrastructural changes in the liver of European perch (Perca Fluviatilis) in response to cyanobacterial bloom in the Gruža Reservoir. Archives of Biological Sciences, 63, 979-989. https://doi.org/10.2298/ABS1104979P
Braunbeck, T., Storch, V., & Bresch, H. (1990). Species-specific reaction of liver ultrastructure in zebra fish (Brachydanio rerio) and trout (Salmo guirdneri) after prolonged exposure to 4-chloroaniline. Review of Environmental Contamination and Toxicology, 19, 405-418. https://doi. org/10.1007/BF01054986
Campana, S. E. (2001). Accuracy, precision and quality control in age determination, including a review of the use and abuse of age validation methods. Journal of Fish Biology, 59, 197-242. https://doi.org/ 10.1006/jfbi.2001.1668 Campana, S. E. (2001). 準確性、精確性和年齡確定的質量控制，包括對年齡驗證方法的使用和濫用的回顧。《魚類生物學雜誌》，59，197-242。https://doi.org/10.1006/jfbi.2001.1668
Clemente, Z., Busato, R. H., Ribeiro, C. A. O., Cestari, M. M., Ramsdorf, W. A., Magalhaes, V. F., ... Assis, H. C. S. (2010). Analyses of paralytic shellfish toxins and biomarkers in a southern Brazilian reservoir. Toxicon, 55, 369-406. https://doi.org/10.1016/j.toxicon.2009.09.003 Clemente, Z., Busato, R. H., Ribeiro, C. A. O., Cestari, M. M., Ramsdorf, W. A., Magalhaes, V. F., ... Assis, H. C. S. (2010). 南巴西水庫對麻痺性貝毒和生物標誌的分析。《毒素學》，55，369-406。https://doi.org/10.1016/j.toxicon.2009.09.003
Conradie, K. R., & Barnard, S. (2012). The dynamics of toxic Microcystis strains and microcystin production in two hypertrophic South African reservoirs. Harmful Algae, 20, 1-10. https://doi.org/10.1016/j.hal. 2012.03.006 Conradie, K. R., & Barnard, S. (2012). 兩個南非高營養水庫中有毒微囊藻株和微囊藻毒素生產的動態。《有害藻類》，20，1-10。https://doi.org/10.1016/j.hal. 2012.03.006
Crivellato, E., & Ribatti, D. (2010). The mast cell: An evolutionary perspective. Biological Reviews, 85, 347-360. https://doi.org/10.1111/j. 1469-185X.2009.00105. x Crivellato, E., & Ribatti, D. (2010). 肥大細胞：一個演化的觀點。生物評論, 85, 347-360。https://doi.org/10.1111/j. 1469-185X.2009.00105. x
Fafioye, O. O., Adebisi, A. A., & Fagade, S. O. (2004). Toxicity of Parkia biglobosa and Raphia vinifera extracts on Clarias gariepinus juveniles. African Journal of Biotechnology, 3, 627-630. Fafioye, O. O., Adebisi, A. A., & Fagade, S. O. (2004). Parkia biglobosa 和 Raphia vinifera 提取物對 Clarias gariepinus 仔魚的毒性。非洲生物技術期刊, 3, 627-630。
Ghadially, F. N. (1985). Diagnostic electron microscopy of tumours, 2nd edn (pp. 394-397). London, UK: Butterworth. Ghadially, F. N. (1985). 腫瘤的診斷電子顯微鏡學, 第 2 版 (頁 394-397)。英國倫敦：巴特沃斯。
Grund, S., Keiter, S., Bottcher, M., Seitz, N., Wurm, K., Manz, W., ... Braunbeck, T. (2010). Assessment of fish health status in the Upper Danube River by investigation of ultrastructural alterations in the Grund, S., Keiter, S., Bottcher, M., Seitz, N., Wurm, K., Manz, W., ... Braunbeck, T. (2010). 通過對多瑙河上游魚類健康狀況的評估，通過對超微結構變化的研究來評估
liver of barbel Barbus barbus. Diseases of Aquatic Organisms, 88, 235248. https://doi.org/10.3354/dao02159 鯉魚 Barbus barbus 的肝臟。水生生物疾病，88，235248。https://doi.org/10.3354/dao02159
Hamilton, D. W., Fawcett, D. W., & Christensen, A. K. C. (1966). The liver of the slender salamander Batrachoseps attenuatus I. The structure of its crystalline inclusion. Microscopic Anatomy, 70, 347. Hamilton, D. W., Fawcett, D. W., & Christensen, A. K. C. (1966). 細長蠑螈 Batrachoseps attenuatus 的肝臟 I. 其結晶包涵物的結構。顯微解剖，70，347。
Herraez, M. P., & Zapata, A. G. (1986). Structure and function of the melanomacrophage centres of the goldfish Carassius auratus. Veterinary Immunology and Immunopathology, 12, 117-126. https://doi.org/10. 1016/0165-2427(86)90116-9 Herraez, M. P., & Zapata, A. G. (1986). 金魚 Carassius auratus 的黑色巨噬細胞中心的結構和功能。獸醫免疫學和免疫病理學, 12, 117-126。https://doi.org/10.1016/0165-2427(86)90116-9
ICES (2004). Biological effects of contaminants: Use of liver pathology of the European flatfish dab (Limanda limanda L.) and flounder (Platichthys flesus L.) for monitoring. In S. W. Feist, T. Lang, G. D. Stentiford & A. Kohler (Eds.), ICES techniques in marine environmental sciences 38, 1-11. No. 38, 42, Denmark: ICES. ICES (2004). 污染物的生物效應: 使用歐洲比目魚 Limanda limanda L. 和牙鮃 Platichthys flesus L. 的肝臟病理學進行監測。在 S. W. Feist, T. Lang, G. D. Stentiford 和 A. Kohler (Eds.) 的 ICES 海洋環境科學技術 38, 1-11。第 38, 42 號, 丹麥: ICES。
Lang, T., Mellergaard, S., Wosniok, W., Kadakas, V., & Neumann, K. (1999). Spatial distribution of grossly visible diseases and parasites in flounder (Platichthys flesus) from the Baltic Sea: A synoptic study. ICES Journal of Marine Science, 56, 138-147. https://doi.org/10.1006/ jmsc. 1999.0465 Lang, T., Mellergaard, S., Wosniok, W., Kadakas, V., & Neumann, K. (1999). 波羅的海比目魚 Platichthys flesus 中明顯可見的疾病和寄生蟲的空間分佈: 一項綜合研究。ICES 海洋科學期刊, 56, 138-147。https://doi.org/10.1006/jmsc.1999.0465
Lang, T., Wosniok, W., Baršiene, J., Broeg, K., Kopecka, J., & Parkkonen, J. (2006). Liver histopathology in Baltic flounder (Platichthys flesus) as indicator of biological effects of contaminants. Marine Pollution Bulletin, 53, 488-496. https://doi.org/10.1016/j.marpolbul.2005.11.008 Lang, T., Wosniok, W., Baršiene, J., Broeg, K., Kopecka, J., & Parkkonen, J. (2006). 立陶宛比目魚（Platichthys flesus）的肝臟組織病理學作為污染物生物效應的指標。 海洋污染公報，53，488-496。 https://doi.org/10.1016/j.marpolbul.2005.11.008
Li, L., Xie, P., & Chen, J. (2005). In vivo studies on toxin accumulation in liver and ultrastructural changes of hepatocyte of the phytoplanktivorous bigheard carp i.p.-injected with extracted microcystin. Toxicon, 46, 533-545. https://doi.org/10.1016/j.toxicon.2005.06.025 Li, L., Xie, P., & Chen, J. (2005). 對注射提取的微囊藻毒素的大頭鯉肝臟中毒素積累和肝細胞超微結構變化的體內研究。 毒素，46，533-545。 https://doi.org/10.1016/j.toxicon.2005.06.025
Marchand, M. J., Van Dyk, J. C., Barnhoorn, I. E. J., & Wagenaar, G. M. (2012). Histopathological changes in two indicator fish species inhabiting a hyper-eutrophic freshwater ecosystem: A baseline study. African Journal of Aquatic Science, 37, 39-48. https://doi.org/10.2989/ 16085914.2011.636902 Marchand, M. J., Van Dyk, J. C., Barnhoorn, I. E. J., & Wagenaar, G. M. (2012). 生活在高度富養分淡水生態系統中的兩種指示性魚類的組織病理學變化：基線研究。非洲水產科學雜誌，37，39-48。 https://doi.org/10.2989/16085914.2011.636902
Marquet, E., & Sobel, H. J. (1969). Crystalline inclusions in the nuclear envelope and granular endoplasmic reticulum of the fish spinal cord. Journal of Cell Biology, 41, 774-785. https://doi.org/10.1083/jcb.41.3.77 Marquet, E., & Sobel, H. J. (1969). 魚類脊髓核膜和顆粒內質網中的結晶包含物。細胞生物學雜誌, 41, 774-785。https://doi.org/10.1083/jcb.41.3.77
Mitsoura, A., Kagalou, I., Papaioannou, N., Berillis, P., Mente, E., & Papadimitriou, T. (2013). The presence of microcystin in fish Cyprinus carpio tissues: A histopathological study. International Aquatic Research, 5, 8. https://doi.org/10.1186/2008-6970-5-8 Mitsoura, A., Kagalou, I., Papaioannou, N., Berillis, P., Mente, E., & Papadimitriou, T. (2013). Cyprinus carpio 魚組織中微囊藻毒素的存在：組織病理學研究。國際水產研究, 5, 8。https://doi.org/10.1186/2008-6970-5-8
Myers, M. S., Johnson, L. L., Hom, T., Collier, T. K., Stein, J. E., & Varanasi, U. (1998). Toxicopathic lesions in sub-adult English sole (Pleuronectes vetulus) from Puget Sound, Washington, USA: Relationships with other biomarkers of contaminant exposure. Marine Environmental Perspectives, 45, 47-67. https://doi.org/10.1016/S0141-1136(97) 00021-4 Myers, M. S., Johnson, L. L., Hom, T., Collier, T. K., Stein, J. E., & Varanasi, U. (1998). 華盛頓州普吉特灣亞成年英國比目魚的毒理病變：與其他污染物暴露生物標誌物的關係。海洋環境透視, 45, 47-67。https://doi.org/10.1016/S0141-1136(97) 00021-4
Qiu, T., Xie, P., Ke, Z., Li, L., & Guo, L. (2007). In situ studies on physiological and biochemical responses of four fishes with different trophic levels to toxic cyanobacterial blooms in a large Chinese lake. Toxicon, 50, 365-376. https://doi.org/10.1016/j.toxicon.2007.04.006 Qiu, T., Xie, P., Ke, Z., Li, L., & Guo, L. (2007). 在一個中國大湖中對具有不同營養級別的四種魚類對有毒藍藻水華的生理和生化反應的原位研究。毒素, 50, 365-376。https://doi.org/10.1016/j.toxicon.2007.04.006
Reynolds, E. S. (1963). The use of lead citrate at high as an electronopaque stain in electron microscopy. Journal of Cell Biology, 17, 208212. https://doi.org/10.1083/jcb.17.1.208 Reynolds, E. S. (1963). 在電子顯微鏡中作為電子不透明染劑的高濃度檸檬酸鉛的使用。細胞生物學雜誌, 17, 208-212。https://doi.org/10.1083/jcb.17.1.208
Sayed, A. H., & Younes, H. A. M. (2017). Melanomacrophage centers in Clarias gariepinus as an immunological biomarker for toxicity of silver nanoparticles. Journal of Microscopy and Ultrastructure, 5, 97-104. https://doi.org/10.1016/j.jmau.2016.07.003 Sayed, A. H., & Younes, H. A. M. (2017). Clarias gariepinus 中的黑色巨噬細胞中心作為銀納米粒子毒性的免疫生物標記。顯微鏡和超結構學雜誌, 5, 97-104。https://doi.org/10.1016/j.jmau.2016.07.003
Shipkey, H. F., Liberman, P. H., Foote, F. W., & Stewart, F. W. (1964). Ultrastructure of alveolar soft part sarcoma. Cancer, 17, 821-830. https://doi.org/10.1002/(ISSN)1097-0142 Shipkey, H. F., Liberman, P. H., Foote, F. W., & Stewart, F. W. (1964). 隔膜軟組織肉瘤的超微結構。癌症，17，821-830。https://doi.org/10.1002/(ISSN)1097-0142
Stentiford, G. D., Bignell, J. P., Lyons, B. P., Thain, J. E., & Feist, S. W. (2010). Age at onset of fish diseases: Application to assessment of marine ecological health status. Marine Ecology Progress Series, 411, 215-223. https://doi.org/10.3354/meps08693 Stentiford, G. D., Bignell, J. P., Lyons, B. P., Thain, J. E., & Feist, S. W. (2010). 魚類疾病發病年齡：應用於評估海洋生態健康狀態。海洋生態進展，411，215-223。https://doi.org/10.3354/meps08693
Van Dyk, J. C., Cochrane, M. J., & Wagenaar, G. M. (2012). Liver histopathology of the sharptooth catfish Clarias gariepinus as a biomarker of aquatic health. Chemosphere, 87, 304-311. https://doi.org/ 10.1016/j.chemosphere.2011.12.002 Van Dyk, J. C., Cochrane, M. J., & Wagenaar, G. M. (2012). 利用刺嘴鯰 Clarias gariepinus 的肝臟組織病理學作為水生生態健康的生物標記。環境圈，87，304-311。https://doi.org/10.1016/j.chemosphere.2011.12.002
Van Dyk, J. C., Marchand, M. J., Smit, N. J., & Pieterse, G. M. (2009). A histology-based fish health assessment of four commercially and ecologically important species from the Okavango Delta panhandle, Botswana. African Journal of Aquatic Science, 34, 273-282. https://doi.org/10.2989/AJAS.2009.34.3.9985 Van Dyk, J. C., Marchand, M. J., Smit, N. J., & Pieterse, G. M. (2009). 一項基於組織學的魚類健康評估，來自博茨瓦納奧卡萬戈三角洲沿岸四個商業和生態重要物種。非洲水產科學期刊，34，273-282。https://doi.org/10.2989/AJAS.2009.34.3.9985
Van Ginkel, C. E. (2008). Investigating the applicability of ecological informatics modelling techniques for predicting harmful algal blooms in hypertrophic reservoirs of South Africa. PhD thesis. North-West University, Potchefstroom, South Africa, 193. Van Ginkel, C. E. (2008). 探討生態信息學建模技術在預測南非富營養水庫有害藻類水華中的應用性。博士論文。西北大學，南非波特赫夫斯布魯姆，193。
Van Ginkel, C. E., Hohls, B. C., Belcher, A., Vermaak, E., & Gerber, A. (2001). Assessment of the Trophic Status Project. Internal Report No. N/0000/00/DEQ/1799. Institute for Water Quality Studies. Department of Water Affairs and Forestry. Pretoria. Van Ginkel, C. E., Hohls, B. C., Belcher, A., Vermaak, E., & Gerber, A. (2001). 營養狀態評估項目。內部報告編號 N/0000/00/DEQ/1799。水質研究所。水務和林業部。比勒陀利亞。
Wischniowski, S., & Bobko, S. (1998). Age and growth laboratory manual. Norfolk, USA: Old Dominion University, Virginia Marine Resources Commission. Wischniowski, S., & Bobko, S. (1998). 年齡和生長實驗室手冊。美國諾福克: 維吉尼亞州老多明尼恩大學，維吉尼亞州海洋資源委員會。
Wu, W. Z., Zhou, B. S., Xu, Y., Schramm, K., & Kettrup, A. (1999). Cytological and biochemical alterations in Carassius auratus hepatocytes from exposure to sediment containing dioxins and related compounds. Archives of Environmental Contamination and Toxicology, 37, 358-363. https://doi.org/10.1007/s002449900525 吳維中，周保生，徐洋，施拉姆，凱特魯普。 (1999). 金魚肝細胞在含有二恶英和相關化合物的沉積物中暴露後的細胞學和生化變化。環境污染與毒理學檔案，37，358-363。https://doi.org/10.1007/s002449900525
How to cite this article: Lukhwareni R, van Dyk C. Histology and ultrastructure of hepatic nodular alterations in Clarias gariepinus (Burchell, 1822). J Fish Dis. 2018;00:1-12. https://doi.org/10.1111/jfd. 12900 如何引用本文: Lukhwareni R, van Dyk C. 非洲鱸（Burchell, 1822）肝臟結節性變化的組織學和超微結構。J Fish Dis. 2018;00:1-12。https://doi.org/10.1111/jfd. 12900
