Molecular characterization of the fish-pathogenic fungus Aphanomyces invadans

Aphanomyces invadans (Saprolegniaceae) is a peronosporomycete fungus associated with the serious fish disease, epizootic ulcerative syndrome (EUS), also known as mycotic granulomatosis. In this study, interspecific relationships were examined between A. invadans isolates and other aquatic animal pathogenic Saprolegniaceae, and saprophytic Saprolegniaceae from EUS-affected areas. Restriction fragment length polymorphisms and sequences of ribosomal DNA confirmed that A. invadans is distinct from all other species studied. A sequence from the internal transcribed spacer region ITS1, unique to A. invadans, was used to design primers for a PCR-based diagnostic test. Intraspecific relationships were also examined by random amplification of polymorphic DNA using 20 isolates of A. invadans from six countries. The isolates showed a high degree of genetic homogeneity using 14 random ten-mer primers. This provides evidence that the fungus has spread across Asia in one relatively rapid episode, which is consistent with reports of outbreaks of EUS. Physiological distinctions between A. invadans and other Aphanomyces species based on a data set of 16 growth parameters showed remarkable taxonomic congruence with the molecular phylogeny.     

Infection experiments with Aphanomyces invadans in four species of estuarine fish

Along the eastern seaboard of the US, Atlantic menhaden, Brevoortia tyrannus, develop characteristic ulcerative lesions, a condition termed ulcerative mycosis. These lesions are identical to those seen across Asia in fish affected by epizootic ulcerative syndrome, a condition caused by the fungus-like oomycete Aphanomyces invadans. Young-of-the-year menhaden inhabiting estuarine environments are the primary species affected in the USA and little is known about the factors involved in the initiation of the lesions, or why menhaden are predominantly infected. Atlantic menhaden, hogchoker, Trinectus maculatus, striped killifish, Fundulus majalis, and mummichog, Fundulus heteroclitus, were inoculated with A. invadans (80 zoospores per fish) to explore species differences in infection and lesion development. All four species developed lesions. Killifish developed frank lesions similar to those observed in menhaden but the gross lesions occurred later, approximately 5-10 days after those on menhaden. Hogchoker and mummichog did not develop gross skin ulcers; rather, their lesions appeared as reddened areas under the epidermis. Mummichogs also showed evidence of significant healing with a well-developed granuloma and significant myocyte regeneration. These experiments show that species barriers as well as ecological barriers can explain some of the factors involved in the development of lesions in, and specificity of the water mould for, menhaden.     

鳢流行性溃疡综合征病原分离鉴定与病理形态学观察

为寻找冬春季鳢(Channa maculata)烂身病的病原,应用组织压片、组织切片、扫描电镜技术、HE 常规染色法和 Grocott 六胺银染色法、霉菌分离纯化及 ITS 的序列分析等对冬春季患疑似溃疡综合征的烂身病鳢进行了病原学与病理学研究。病变组织压片观察到大量直径 10~20 μm,分枝较少、纤细菌丝。扫描电镜观察到肌肉组织中延伸出大量的纤细的丝状真菌。患病鳢皮肤和肌肉表现为变性、坏死与炎性细胞浸润,溃疡灶肌肉内可见大量的慢性肉芽肿结节和炎性细胞浸润。结节基本结构由类上皮细胞和多核巨细胞、炎性细胞、霉菌菌丝(横断面呈圆形,斜断或纵断呈丝状)组成,霉菌位于结节中央。Grocott 六胺银染色观察到结节中有大量棕色菌体。无菌分离培养可观察到典型丝状霉菌;霉菌在灭菌池塘水 20℃过夜, 12 h 后可观察原代孢子群形成,真菌 ITS 序列分析表明其与侵袭丝囊菌(Aphanomyces invadans)同源性为 100%。侵袭丝囊霉菌(A. invadans)是杂交鳢溃疡综合症病的主要病原,为鱼类流行病学调查和疾病防治的深入研究奠定基础。     

Effect of plant active compounds on immune response and disease resistance in Cirrhina mrigala infected with fungal fish pathogen, Aphanomyces invadans

The antibacterial activity of individual and mixed medicinal plant compounds, azadirachtin (Az), camphor (Ca) and curcumin (Cu), was tested at 100, 200, 300, 400, 500, 600 and 700 ppm (mg L⁻¹) against fungal fish pathogen, Aphanomyces invadans, in vitro . At the lower concentrations between 100 and 300 ppm, the mixture of the tri-herbal (Az+Ca+Cu) compound yielded a higher ( P >0.05) zone of inhibition (ZI) of 7 mm than the positive control; the maximum ZI values (8–15 mm) were realized between 400 and 700 ppm ( P <0.05). At the lowest minimum inhibitory concentration (MIC) the tri-herbal compound (100 ppm) yielded 13 colony-forming units; hence, this dose can be effectively used at the lowest concentration of 100 mg L⁻¹ to ward off the growth of A. invadans in vitro . In Cirrhina mrigala , intramuscular administration (100 μL) of the selected doses of 100, 400 and 700 ppm (mg L⁻¹) significantly enhanced ( P <0.05) the serum lysozyme activity (Ly), production of reactive oxygen species and reactive nitrogen species (RNS or NO) by peripheral blood leucocytes on the 10th, 20th and 30th day. A priori administration of the compound in the fish (100 ppm on 30th day) decreased the percentage mortality when challenged with the pathogen while in the untreated group the mortality increased ( P <0.05). This study indicates that intramuscular administration of the tri-herbal compound Az+Ca+Cu at a concentration of 100 ppm could augment the immune response in C. mrigala against A. invadans .     

First record of epizootic ulcerative syndrome from the Upper Congo catchment: An outbreak in the Bangweulu swamps,Zambia

We report on the first outbreak of epizootic ulcerative syndrome (EUS) amongst wild fish populations in the Bangweulu swamps, an inland delta, in the north of Zambia during 2014. The area supports a large and diverse fish fauna related to, but distinct from, that of the Zambezi River system where EUS outbreaks have occurred since 2006. A sizeable artisanal fishery, based on extensive fish weirs, is sustained by the annual flooding of the swamps, and observations of the disease outbreak by fishermen were recorded. Signs typical of infection with Aphanomyces invadans were observed in a number of species. Clinical observations, histology and molecular diagnostic methods were used to confirm infection with A. invadans in two of the most commonly and severely affected species. Several features of the wetland may have contributed to the outbreak and the annual recurrence of the disease. Modes by which the disease may have been introduced into the swamps are discussed. The outbreak is of great significance as the Bangweulu swamps drain into the Congo River in neighbouring Democratic Republic of Congo, Africa's largest drainage system with an extensive and diverse fish fauna previously unaffected by EUS.     

A fluorescence‐based assay for in vitro screening of Saprolegnia inhibitors

The incidence of fish pathogenic oomycetes, Saprolegnia, has increased significantly in aquaculture since the ban of malachite green. For the efficient characterization of anti‐Saprolegnia therapeutics, simple accurate methods are required. However, the current screening methods are limited by time, and none of them are confirming the viability of treated spores or hyphae. In this study, a modified fluorescence‐based assay for the in vitro screening of Saprolegnia inhibitors has been developed. This method involves the use of FUN‐1 viability dye combined with calcofluor white M2R, and is based on the formation of orange‐red cylindrical intravacuolar structures (CIVS) in metabolically active spores, hyphae and biofilms. Heat‐killed and bronopol‐treated Saprolegnia spores, hyphae and biofilms exhibited diffuse bright green fluorescence which confirms complete loss of viability. For boric acid‐treated spores, no germination was observed. However, tiny CIVS were observed in 50% of treated spores which indicated reduction in their viability. Our results proved that FUN‐1 dye is an efficient tool to distinguish between live and dead Saprolegnia spores, hyphae and biofilms and to monitor the change in Saprolegnia viability during qualitative evaluation of potential anti‐Saprolegnia compounds.     

Development of real-time PCR assays for the detection and differentiation of Australian and European ranaviruses

Serious systemic disease in fish and amphibians is associated with the ranaviruses, epizootic haematopoietic necrosis virus (EHNV) and Bohle iridovirus (BIV) in Australia, and European sheatfish virus (ESV) and European catfish virus (ECV) in Europe. EHNV, ESV and ECV are recognized causative agents of the OIE (Office International des Epizooties) notifiable systemic necrotizing iridovirus syndrome and are currently identified by protein-based assays, none of which are able to rapidly identify the specific agents. The aim of this study was to develop TaqMan real-time PCR assays that differentiated these viruses using nucleotide sequence variation in two ranavirus genes. A conserved probe representing 100% sequence homology was used as a reference for virus-specific probes. The virus-specific probes produced a similar signal level to the conserved probe while those probes binding to non-target viral DNA produced an altered fluorescent curve. The pattern of probe binding was characteristic for each virus. Sensitivity, specificity and dynamic range of the assay were assessed. The test is currently useful as a research and initial screening tool, with the potential to become a sensitive and specific method for detection and differentiation of ranaviruses with further development.     

中草药对刺参腐皮综合征病原菌的体外抑菌试验

用改良二倍稀释法和抑菌圈法测定了24种单方中草药和9种复方中草药对刺参腐皮综合征4种重要病原菌灿烂弧菌、假交替单胞菌、哈维氏弧菌和恶臭假单胞菌的体外抑菌活性。结果显示,不同中草药对特定病原菌抑菌效果差异较大,单方中草药最低抑菌浓度（MIC）为1.56mg/ml,中草药水煎剂H-5和H-9对假交替单胞菌的抑菌圈最大,达22mm。单方中草药中穿心莲、大青叶、金银花和川芎4种中草药对4株病原菌抑菌效果最好,其水煎剂对4株病原菌平均抑菌浓度分别为6.25、7.81、5.86和3.52mg/ml;平均抑菌圈直径分别是17.5、11.3、14.0和11.5mm。将筛选的4种单方中草药组成9种复方,复方MIC和抑菌圈均显著好于单方,最低MIC降低到0.20mg/ml。其中复方HC-G和HC-D抑菌浓度最低,效果最好,平均抑菌浓度分别降低为0.54和0.64mg/ml;平均抑菌圈直径分别为15.3mm和16.3mm。试验结果显示,复方最佳配比为穿心莲、大青叶、金银花和川芎=2∶1∶3∶2。该复方可为生产高效专用中草药,以替代抗生素,为刺参健康养殖提供技术保障。     

In vitro survival and the effect of water chemistry and oxidative chemical treatments on isolated gill amoebae from AGD-affected Atlantic salmon

Amoebic gill disease (AGD) of cultured salmonids in Tasmania is caused by the amphizoic parasitic amoeba Neoparamoeba pemaquidensis. The freshwater tolerance of amoebae isolated from the gills of AGD-affected salmon (predominantly N. pemaquidensis) was tested in vitro using a trypan blue exclusion assay. Amoebae exposed to water containing high concentrations of Ca²⁺ or Mg²⁺ (200 mg l⁻¹) showed high levels of survival up to 3 h of exposure. Exposure to water containing elevated Na⁺, choline chloride or water at different pH all had no significant survival of amoebae. Exposure of amoebae to different concentrations of chlorine dioxide, chloramine-T or hydrogen peroxide in artificially hard water demonstrated that chloramine-T and hydrogen peroxide were the most efficacious at killing amoebae in vitro. This work suggests that the hardness of freshwater may be an important factor for the survival of marine amoebae (predominantly N. pemaquidensis) on the gills of AGD-affected salmon and have significant implications with regard to the efficacy of freshwater bathing practices for the control of AGD on farms. Additionally, chloramine-T and hydrogen peroxide appear to be efficacious at killing marine gill amoebae in vitro and may be useful for the control of AGD in farmed Atlantic salmon.     

In vitro activity of chemicals and commercial products against Saprolegnia parasitica and Saprolegnia delica strains

Oomycetes of the genus Saprolegnia are responsible for severe economic losses in freshwater aquaculture. Following the ban of malachite green in food fish production, the demand for new treatments pushes towards the selection of more safe and environment‐friendly products. In the present work, in vitro activity of ten chemicals and three commercial products was tested on different strains of Saprolegnia, using malachite green as reference compound. The compounds were screened in agar and in water to assess the minimum inhibitory concentration (MIC) and the minimum lethal concentration (MLC), respectively. Two strains of Saprolegnia parasitica and one isolate of Saprolegnia delica were tested in triplicate per each concentration. Among tested chemicals, benzoic acid showed the lowest MIC (100 ppm) followed by acetic acid, iodoacetic acid and copper sulphate (250 ppm). Sodium percarbonate was not effective at any tested concentration. Among commercial products, Virkon^?S was effective in inhibiting the growth of the mycelium (MIC = MLC = 1,000 ppm). Actidrox® and Detarox® AP showed MIC = 5,000 and 1,000 ppm, respectively, while MLCs were 10‐fold lower than MICs, possibly due to a higher activity of these products in water. Similarly, a higher effectiveness in water was observed also for iodoacetic acid.     

福建海区网箱养殖刺参“腐皮综合症”病原分析与鉴定

2011年冬季,福建省宁德市霞浦县海区网箱养殖刺参发生"腐皮综合症",并伴有死亡现象出现。刺参的发病症状表现为:厌食、排脏、身体萎缩、体表局部溃烂乃至大面积溃烂。从患病刺参病灶处分离得到两种优势细菌CS1和CS2。经人工回接感染实验证明两种菌对健康刺参都具有较强的感染性,且感染病参的症状与自然发病刺参的症状相同。通过生理生化试验、16S rDNA序列分析及系统进化树分析,结果表明两株菌分别与灿烂弧菌(Vibrio splendidus)和假交替单胞菌(Pseudoalteromonas sp.)相似。菌株CS1与灿烂弧菌的亲缘关系最近,相似率达到99%,菌株CS2与假交替单胞菌的亲缘关系最近,相似率达到95%。菌株CS1可鉴定为灿烂弧菌,菌株CS2可鉴定为假交替单胞菌。另外,在患病刺参呼吸树膜和腔体内发现大量后口虫(Boveria sp.)。所以导致本次刺参"腐皮综合症"的原因可能是致病性细菌和寄生虫共同作用的结果。本文首次揭示了该地区"腐皮综合症"导致养成刺参大规模死亡的致病原因,对刺参病害防治和健康养殖具有重要的指导意义。     

刺参(Apostichopus japonicus)大水面养殖池塘环境中优势益生菌筛选及其特性分析

2014年12月-2015年12月,在大连地区刺参(Apostichopus japonicus)大水面养殖池塘进行了春、夏、秋、冬四季有益菌分离筛选,从其水体和底泥中共分离得到66株细菌.以刺参“腐皮综合征”主要病原菌——灿烂弧菌(Vibrio splendidus)和假交替单胞菌(Pseudoalteromonas nigrifaciens)为指示菌进行拮抗作用实验,利用选择培养基对菌株产淀粉酶和蛋白酶的能力进行测定,最后通过安全性实验得到潜在益生菌株YQ-2.结果显示,该菌株对灿烂弧菌和假交替单胞菌有较强的抑制作用,抑菌圈分别达到22 mm和24 mm;对淀粉和蛋白选择培养基水解圈的直径达到22 mm和36mm.安全性实验显示,该菌株无论是在108 CFU/ml浸浴还是投喂108 CFU/g的粉末饲料感染,30 d内供试刺参没有发病和死亡现象,健康程度好,且相对于对照组的体重明显增长,108 CFU/g粉末饲料投喂组的相对增长率达到39.31％.此外,本研究对YQ-2菌株的生理生化指标、16S rDNA序列进行了分析,其同源性与枯草芽孢杆菌Bacillus subtilis strain KLP2015相似度达99％,故将该菌株鉴定为枯草芽孢杆菌.该株枯草芽孢杆菌在大水面刺参池塘四季水体中数量为140-280 CFU/ml,高于其他菌株;同时,该菌株在水体中还具有较高的优势度,优势度分别为4.2％、3.5％、2.6％、4.6％,冬、春季节的优势度明显高于夏、秋季节;它属于土著分离菌株,对引起刺参腐皮综合征的2株病原菌具有较强的抑制作用,这对刺参大水面生态养殖具有较大的应用潜力.     

Clinical assessment of chloramine-T and freshwater as treatments for the control of gill amoebae in Atlantic salmon, Salmo salar L.

Infections of gill amoebae that manifest as amoebic gill disease (AGD) occur in Atlantic salmon in Tasmania. The treatment of choice is freshwater bathing; however, the effectiveness of this treatment has declined over time. In this experiment, cage trials of chloramine‐T (Cl‐T) to treat AGD in Atlantic salmon were conducted over 3 months, and involved an initial bath in either freshwater or seawater with Cl‐T, followed by a second bath 6 weeks later. Amoeba densities were reduced to 50–80% of original values for both treatments. Neoparamoeba sp. density was not affected by bathing, and was not significantly different over the course of the experiment. Lesion prevalence was higher for Cl‐T‐treated fish than for freshwater‐treated fish, with overall prevalence levels of 14.30±1.00% and 8.03±0.57% respectively. This was also seen for gross gill scores. In the fortnight after each of the two baths, Cl‐T‐treated fish had significantly higher lesion levels, although this difference was then resolved by 4 weeks post bathing. The use of Cl‐T in seawater is at least as effective as freshwater at reducing amoebae density, and may be a more practical alternative when freshwater is in short supply.     

Acute columnaris infection in channel catfish, Ictalurus punctatus (Rafinesque): efficacy of practical treatments for warmwater aquaculture ponds

Columnaris disease was induced in channel catfish, Ictalurus punctatus (Rafinesque), by bath exposure to four highly virulent isolates of Flavobacterium columnare. In untreated controls, mortality began 20 h after exposure and reached 100% by 48 h. Mortality in channel catfish given antibiotic treatments with oxytetracycline or a combination of sulphadimethoxine and ormetoprim in feed prior to bacterial challenge was zero with all four strains of F. columnare. Diquat (Zeneca Agricultural Products, Wilmington, DE, USA) was the most effective bath treatment; mortality with all four strains was zero. With potassium permanganate, chloramine-T, hydrogen peroxide and copper sulphate, bath treatment efficacy varied significantly among strains (P = 0.0346) and among treatments (P = 0.0033). Bath treatments with chloramine-T and potassium permanganate significantly reduced (P < 0.05) mortality from 100 to 75 and 69%, respectively, but copper sulphate and hydrogen peroxide treatments were not effective. Based on our results, oral antibiotics prevented columnaris disease but, of the bath treatments, only Diquat produced a dramatic reduction in the mortality of acutely infected fish. Diquat is labelled for aquatic use as an herbicide in the USA but in large ponds it is prohibitively expensive.     

In situ hypochlorous acid generation for the treatment of brackish shrimp aquaculture wastewater

This study presents an unconventional framework for treating shrimp aquaculture wastewater based on in situ hypochlorous acid (HOCl) oxidation. The in situ oxidation process makes use of the salinity present in aquaculture wastewater to generate HOCl. The undivided electrolytic cell consisted of two sets of graphite as the anode and stainless sheets as the cathode. The electrochemical oxidation of shrimp aquaculture wastewater was carried out for an influent COD concentration of 1730 mg L^?1 at current densities of 37.2 and 74.5 mA cm^?2. The results showed that in order to achieve a residual COD concentration of 50±5 mg L^?1 at current densities of 37.2 and 74.5 mA cm^?2, electrolysis periods of 60 and 30 min are required respectively. Hence, for the above‐mentioned current densities, the corresponding energy requirements were found to be 19.4 and 13.3 W h L^?1. The cost incurred in treating 1 m³ of shrimp aquaculture wastewater was found to be RM 4 and 3 when the electrolytic reactor was operated at a current density of 37.2 and 74.5 mA cm^?2 with a salinity of 23‰. The foregoing study highlights the potential of in situ HOCl oxidation in treating brackish shrimp aquaculture wastewater.     

溶壁酶体外抗同丝水霉活性研究

为探究溶壁酶对水产动物水霉病的预防和治疗效果,以同丝水霉(Saprolegnia monoica)为研究对象,溶壁酶设0、5、10、20、40、80 mg/m L共计6个浓度处理组,取浓度为103个/m L的水霉孢子菌悬液0.1 m L,涂布于不同溶壁酶浓度的马铃薯葡萄糖琼脂培养基(PDA)上,25℃培养5 d,以最低抑菌浓度为判定指标,研究溶壁酶溶解同丝水霉孢子的抑菌活性;在此基础上,以溶壁酶最低抑菌浓度30℃分别水浴2 h和4 h处理同丝水霉菌丝块,PDA培养基上25℃培养5 d,通过用体外抑菌试验、菌丝显微结构观察等方法,研究溶壁酶最低抑菌浓度对同丝水霉菌丝的抑菌活性,为进一步开发水产动物抗真菌(替代孔雀石绿)药物提供科学依据。结果显示,溶壁酶对同丝水霉孢子的最低抑菌浓度为20 mg/m L,通过间隔添加溶壁酶该浓度,72 h能够有效抑制同丝水霉菌丝的生长;溶壁酶30℃水浴处理同丝水霉菌丝块的时间(2 h和4 h)与溶壁酶溶解菌丝效果呈正相关。     

In vitro and in vivo efficacy of drugs against the protozoan parasite Azumiobodo hoyamushi that causes soft tunic syndrome in the edible ascidian Halocynthia roretzi (Drasche)

It was discovered recently that infection by a protozoan parasite, Azumiobodo hoyamushi, is the most probable cause for soft tunic syndrome in an edible ascidian, Halocynthia roretzi (Drasche). In an attempt to develop measures to eradicate the causative parasite, various drugs were tested for efficacy in vitro and in vivo. Of the 20 antiprotozoal drugs having different action mechanisms, five were found potent (24‐h EC₅₀ < 10 mg L^?1) in their parasite‐killing effects: formalin, H₂O₂, bithionol, ClO₂ and bronopol. Moderately potent drugs (10 < 24‐h EC₅₀ < 100 mg L^?1) were quinine, fumagillin, amphotericin B, ketoconazole, povidone‐iodine, chloramine‐T and benzalkonium chloride. Seven compounds, metronidazole, albendazole, paromomycin, nalidixic acid, sulfamonomethoxine, KMnO₄, potassium monopersulphate and citric acid, exhibited EC₅₀ > 100 mg L^?1. When ascidians were artificially infected with A. hoyamushi, treated using 40 mg L^?1 formalin, bronopol, ClO₂, or H₂O₂ for 1 h and then monitored for 24 h, very low mortality was observed. However, the number of surviving parasite cells in the ascidian tunic tissues was significantly reduced by treating with 40 mg L^?1 formalin or ClO₂ for 1 h. The data suggest that we might be able to develop a disinfection measure using a treatment regimen involving commonly available drugs.     

In vitro and in vivo selection of probiotic purple nonsulphur bacteria with an ability to inhibit shrimp pathogens: acute hepatopancreatic necrosis disease‐causing Vibrio parahaemolyticus and other vibrios

Shrimp cultivation has been faced with huge losses in productivity caused by infectious shrimp pathogenic vibrios, especially Vibrio parahaemolyticus that causes acute hepatopancreatic necrosis disease (AHPND). Hence, purple nonsulphur bacteria (PNSB) were isolated from shrimp ponds for investigating their abilities to control shrimp pathogenic Vibrio spp. and their use as probiotics for sustainable shrimp cultivation. Based on their probiotic properties, strains S3W10 and SS15 were selected because of their strong abilities to produce amylase, gelatinase and vitamin B12. However, only three PNSB strains (SS15, TKW17 and STW181) strongly inhibited V. harveyi_KSAAHRC and V. vulnificus_KSAAHRC including V. parahaemolyticusAHPND strains by secreting antivibrio compounds. Four selected PNSB also grew in the presence of pancreatic enzymes, and they were identified as Rhodobacter sphaeroides for strains S3W10, SS15 and TKW17 and Afifella marina for strain STW181. The effects of a mixed culture were also investigated as follows: T1 (S3W10 + SS15), T2 (S3W10 + TKW17) and T3 (S3W10 + STW181) on postlarval white shrimp (Litopenaeus vannamei) for 60 days by comparison with a control. All three probiotic PNSB sets significantly improved the digestive enzyme activities and shrimp growth with their proliferation in shrimp gastrointestinal tract although the shrimp survival was not significantly different. They also significantly reduced the cumulative mortality of shrimp exposed to a virulent AHPND strain (V. parahaemolyticusSR2). This is the first to conclude that selected probiotic PNSB strains have great potential to be used for shrimp cultivation to control vibrios including AHPND strains.     

Pigment deposition and in vitro screening of natural pigment sources for enhancing pigmentation in male Siamese fighting fish (Betta splendens Regan, 1910)

Distribution in pigment deposition was studied in male Siamese fighting fish (Betta splendens Regan, 1910) of solid Blue and solid Red phenotypes. Growths were similar, and also indicated by similar activity ratio of trypsin to chymotrypsin. Growth strategy was different at maturing stage, whereas trypsin expression decreased in solid Blue while chymotrypsin expression decreased in solid Red (P ≤ 0.05). Ranking of total pigment contents was muscle > skin > caudal fin > anal fin > dorsal fin > pelvic fin > pectoral fin. Immature fish had similar total contents and concentrations of pigments, except solid Blue had higher total contents in dorsal fin than solid Red. At maturing stage, solid Blue showed higher total pigment contents than solid Red due to higher contents in the fins. Solid blue had higher concentration in pelvic fin, while solid Red had higher in pectoral and dorsal fins. Age, phenotype and their interaction influenced pigment deposition. Determinations of pigment levels, high temperature tolerant of pigments and in vitro digestibilities of pigments, protein and carbohydrate from twenty natural sources indicated that fermented red rice was the most suitable pigment source, following with Chlorococcum sp.