Tetrahymena sp. infection in guppies, Poecilia reticulata Peters: parasite characterization and pathology of infected fish

Tetrahymena sp. infection was diagnosed in guppies imported from Singapore. The parasite was isolated (Tet-NI) and optimally cultured in vitro in RM-9 medium. Cytological analyses [silver-staining and scanning electron microscopy (SEM)] revealed a pyriform-shaped, 64 × 41-μm holotrich ciliate without caudal cilium, containing a macro-nucleus (18.25 × 16.83 μm) and micro-nucleus (5.73 × 5.40 μm). Wet-mount examination and histological analyses of fish exposed to the parasite by co-habitation, immersion and infection by i.p. (intra-peritoneal) and i.m. (intra-muscular) injection revealed numerous ciliates on the skin, and in the gill and caudal fin blood vessels. Ciliates surrounded internal organs, the peri-orbital region of the eye, and were observed inside developing guppy embryos. Some muscle necrosis was associated with infection, but little or no inflammatory response. Immersion, co-habitation and i.m. injection caused relatively high infection rates and levels in the skin and tail, and lower infection in the gill blood vessels and internal organs; i.p. injection caused higher infection in the gill blood vessels and internal organs. Co-habited fish had relatively high infection levels in the hind-gut sub-mucosa. This is the first report of controlled systemic infection by Tetrahymena sp.     

Treatment development for systemic Tetrahymena sp. infection in guppies,Poecilia reticulata Peters

Antibacterial and antiparasitic agents and a cysteine protease inhibitor (E‐64) were tested against Tetrahymena infection, a serious problem in guppy production worldwide. Chemicals were tested in vitro by a colorimetric assay for Tetrahymena survival. The most effective were niclosamide, albendazole and chloroquine, with 23%, 35% and 60% survival, respectively, following 2‐h exposure to 100 ppm. Longer incubation periods resulted in greater reductions in survival. Niclosamide was further studied in vivo at different dosages, administered orally to Tetrahymena‐infected guppies. Mortality rates were significantly lower in all treatment groups; in trial I, 30% and 33% mortality in 5 and 40 mg kg^?1 niclosamide‐fed fish vs. 59% mortality in controls; in trial II, 35%, 13% and 10% in 50, 100 and 200 mg kg^?1 niclosamide‐fed fish vs. 64% in controls. The effect of the cysteine protease inhibitor E64 was tested in tissue culture, by measuring histolytic activity of the parasite (Tet‐NI) on a guppy‐fin cell line, based on cell depletion. Tet‐NI feeding activity was significantly reduced following pretreatment with E‐64 relative to non‐treated Tet‐NI. E‐64‐pretreated Tet‐NI was injected i.p. into guppies: recorded mortality rates were significantly lower (35%) than that in non‐treated Tet‐NI (60%), suggesting inhibition of the parasite’s cysteine protease as a possible therapeutic approach.     

Effects of different commercial diets on growth performance,health and resistance to Tetrahymena sp. infection in guppies,Poecilia reticulata (Peters)

The effects of feeding guppy fry, Poecilia reticulata (Peters), different commercial diets from BernAqua [MeM (R = Regular, O = Ornamental and P = Premium) and experimental feed (EF] and Ocean Nutrition (ON) on growth was tested for 57 days. Health status and resistance to Tetrahymena infection were analysed at the end of the trial. The highest growth was obtained in fish fed MeM Regular and MeM Premium (mean final weights of 427 and 417 mg respectively). Fish fed EF had a significantly higher rate of spinal deformity (12.7%) than all other feeding groups (3.05% or less). Histological analysis revealed the accumulation of liver glycogen and/or lipid in fish from all feeding groups; fat accumulation in the abdomen was most pronounced in the EF and MeM Regular‐fed fish, and muscle dystrophy was observed in ca. 50% of the fish from all feeding groups except the MeM Ornamental‐fed group. The highest mortality following Tetrahymena infection occurred in fish fed EF (87%) and the lowest mortality in the MeM Ornamental‐fed fish (58%). Mortalities in all other feeding groups ranged between 69% and 76%. In summary, MeM Ornamental feed provided a moderate weight gain, no muscle dystrophy, negligible deformity and the greatest resistance to parasitic infection.     

Controlled infection of Poecilia reticulata Peters (guppy) with Tetrahymena by immersion and intraperitoneal injection

Tetrahymena is a protozoan parasite, which infects guppy, Poecilia reticulata Peters, and causes substantial economical losses in commercial farms worldwide. Studies of guppy infected by Tetrahymena require standardized infection protocols. The LD50 for Tetrahymena infection of guppies by intraperitoneal (IP) injection was calibrated, and the level obtained was 946 parasites per fish. Guppy infection with Tetrahymena by immersion, imitating the natural route of infection via the integument, was studied under normal or stress conditions. Exposure to cold and netting (CNI) and to cold only (CI) followed by immersion exposure to 10 000 Tetrahymena per mL resulted in 22.5% and 19.2% mortality, respectively, as compared to 14.2% and 10% in groups that were netted only (NI) or non‐stressed (I). Histopathology revealed that immersion infection resulted in a systemic infection. Lysozyme levels, measured 3 weeks after infection, were significantly higher in the CNI group (288 μg per mg protein) compared with CI‐, NI‐ and I‐treated groups (94.5, 64 and 62.3 μg mg^?1, respectively). There was no evident parasite immobilization activity in body homogenates, suggesting no development of acquired immunity. Re‐infection by IP injection revealed no increase in protection in any of the treatment groups, mortality range of 56.3–75%, higher than in the non‐exposed control (40.6% mortality).     

Experimental infections of a ciliate Tetrahymena pyriformis on ornamental fishes

SUMMARY: Experimental infection of Tetrahymena pyriformis was conducted on ornamental fishes with skin wounded by acetic acid treatment. Among fishes used in this experimental infection, guppy Poecilia reticulata , pristella Pristella maxillaris , neontetra Paracheirodon innesi and cherry barbs Puntius titteya proved to be sensitive to challenge, while medaka Oryzias latipes , dwarf gourami Colisa lalia , goldfish Carassius auratus , platy Xiphophorus maculatus and angelfish Pterophyllum scalare were resistant. In catfish Corydoras aeneus infection was unsuccessful. Exposure to Tetrahymena at ≥100 cells/mL at 25 to 30°C and at pH of 6.0 to 8.0 resulted in the successful infection of some sensitive fish species. Histological and bacteriological observations suggest that deeply destroyed skin tissues from the acid-treated method are the primary factors in successful infection.     

Effect of temperature on sex ratio in guppy Poecilia reticulata (Peters 1860)

In this study, the effect of temperature on survival and sex ratios of guppy Poecilia reticulata (Peters 1860) was investigated. Treatments of 19, 21 and 22.5°C low and 29, 32 and 35°C high water temperature were used on fry and gravid females for 11 days after parturitions and several days starting from the 16th day after first parturition until second parturition respectively. The high water temperature treatments caused the death of gravid females. Survival rates of heat‐treated fry were generally decreased with increasing and decreasing temperature. The sex ratios of the progenies from survived gravid females were not differed from a balanced sex ratio (P>0.05). The proportion of females increased gradually with decreasing temperature and the proportion of males increased gradually with increasing temperatures rather than having a threshold response in heat‐treated fry. However, mortality of heat‐treated fry was high; it is most likely that our results were biased by sex‐dependent mortality, which were also revealed by corrected sex ratios of the treatment groups and progeny testing of some individuals. These results suggest that a major gene linked to X‐chromosome could be responsible for resistance and sensitivity to both high and low temperature.     

Short-term dietary supplementation with the microalga Parietochloris incisa enhances stress resistance in guppies Poecilia reticulata

Two trials were conducted to determine the effects of dietary enrichments with the microalga Parietochloris incisa , rich in arachidonic acid (ARA), on stress resistance in guppies Poecilia reticulata . The microalga was added to commercial diets as a neutral lipid (NL) extract and its fractions or as broken cells. Experimental diets were applied for a period of 14 days. In trial 1, commercial diets were supplemented with NL (containing 25 mg ARA and 0.11 mg β-carotene g⁻¹ feed), its triacylglycerol (TAG) fraction (containing 25 mg ARA g⁻¹ feed and no β-carotene) and the β-carotene fraction (containing 0.11 mg carotenoid g⁻¹ feed and minute amounts of ARA). Neutral lipid-fed fish demonstrated the highest resistance ( P <0.05) to osmotic stress (32-ppt NaCl), followed by fish fed with diets supplemented with TAG and β-carotene alone, which were more resistant than control ( P <0.05). In trial 2, fish fed diets supplemented with higher levels of broken alga (26.1 mg ARA g⁻¹ feed) were more resistant ( P <0.05) to stress as compared with fish fed lower ARA (16.3 mg g g⁻¹) or an unsupplemented control diet. We suggest a dietary supplementation with broken P. incisa cells to enhance stress resistance in guppies before a stressful event.     

Simultaneous effect of sex and dose on efficacy of clove oil,tricaine methanesulfonate, 2‐phenoxyethanol and propofol as anaesthetics in guppies,Poecilia reticulata (Peters)

We studied the simultaneous effect of sex and dose on anaesthesia efficacy to estimate, if possible, the lowest effective dose (LED) for clove oil, tricaine methanesulphonate (MS‐222), 2‐phenoxyethanol (2‐PE) and propofol in mature guppies. LED is the lowest dose needed to reach A5 stage in a mean time of 3 min, with mean recovery (R5) time of 5 min. We used four doses/anaesthetic: 25, 50, 75 and 100 mg/L for clove oil; 120,140,160 and 180 mg/L for MS‐222; 800, 1,000, 1,200 and 1,400 mg/L for 2‐PE, and 7.50, 8.25, 10.00 and 11.25 mg/L for propofol. Each dose was tested on 10 females and 10 males. Morbidity, mortality and behavioural changes were checked on two control groups (10 males and 10 females/group). Sedation (A3), A5 and R5 times were recorded. Significant interactive effect dose*sex on A5 time was found for each anaesthetic agent (p_dose*sex < .05). Except for MS‐222 (p_dose*sex = .284), significant interactive effect dose * sex on R5 time was found (p_dose*sex < .05). A5 time in females tended to be greater than in males, but, in general, R5 times were longer in males. Body size differences between males and females could explain these differences in MS‐222 on A5 time and for clove oil, 2‐PE and propofol on R5 time. No dose simultaneous meet LED′s conditions relating to both A5 and R5 times; therefore the lowest doses inducing A5 in a mean time of 3 min could be a safe guideline for anaesthetic procedure in both male and females.     

Using attraction to light to decrease cannibalism and increase fry production in guppy (Poecilia reticulata Peters) hatcheries. II: the effects of light and cannibalistic adults

Predation of newborn fish by cannibalistic adults is a major cause of fry loss in guppy breeding tanks. The efficacy of using light to attract newborn guppies into a safe area, thereby reducing the cannibalism rate, was evaluated in aquaria. The aquaria were divided using small‐mesh netting into a section with adult fish and a safe section without adults, above which a light source was installed to attract the newborns. Both, the presence of cannibalistic adults and the light, increased the number of newborns in the safe section. The effect of the cannibals was pronounced during the day, and the effect of light seemed to be more pronounced at night. The presence of light significantly decreased cannibalism rate after 30 min, but not 16 h after the introduction of newborns. When tested with cannibalistic adults that have not been fed for 5 days prior to testing, cannibalism rate was higher and the effect of light on reducing cannibalism was evident both 30 min and 16 h after the introduction of newborns. The results demonstrated the potential efficacy of using white light in reducing cannibalism and increasing fry yields in guppy hatcheries.     

DNA markers for estimation of inbreeding depression and heterosis in the guppy Poecilia reticulata

Molecular markers have significant potential for use in precise breeding programmes in aquaculture. This paper reviews the use of DNA markers to estimate inbreeding depression and heterosis in the guppy Poecilia reticulata. Full‐sib matings revealed that inbreeding causes declines in survival and salinity tolerance, but not in undwarfism, growth and high water temperature tolerance, indicating the effects of inbreeding differ among fitness‐related traits. Salinity tolerance was used to quantify the level of inbreeding depression and heterosis because the trait is strongly sensitive to inbreeding and shows a linear decrease with an increase in inbreeding coefficient. A positive correlation was observed between heterozygosity at microsatellite loci and salinity tolerance among 17 guppy populations. This indicates that heterozygosity estimated from microsatellites is a useful indicator for the estimation of inbreeding depression, suggesting that overall heterozygosity is important for fitness‐related traits that show inbreeding depression. Use of DNA markers to estimate the amount of heterosis in various strain combinations was examined using diallele and reciprocal crosses among four domestic strains. The amount of heterosis differed among the strain combinations and correlated with Nei's genetic distance measured by microsatellites and also by dissimilarity using random amplified polymorphic DNA (RAPD) analysis. This indicates that microsatellite and RAPD markers are useful for estimating the amount of heterosis in various strain combinations, further suggesting that the amount of heterosis depends on the genetic differences between the strains. The present study showed that DNA markers are useful tools for estimating inbreeding depression and heterosis in guppy breeding.     

Rhabdomyosarcoma in the Japanese medaka, Oryzia latipes (Temminck & Schlegel) and guppy, Poecilia reticulata Peters

Abstract. Three cases of skeletal muscle neoplasms occurring in two small fish species used in carcinogen tests are reported. The cases illustrated a wide range of histologic patterns, and consisted of a well-differentiated juvenile type, a well-differentiated pleomorphic type and a poorly differentiated pleomorphic type. A rhabdomyosarcoma with juvenile type features developed in the Japanese medaka, Oryzias latipes , exposed to 0·5 ml/1 of the solvent dimethylformamide. A pleomorphic type rhabdomyosarcoma occurred in one medaka and in one guppy, Poecilia reticulata , exposed to the carcinogen methylazoxymethanol acetate. These cases indicate the potential for skeletal muscle cells in medaka and guppy to become neoplastic but, because the tumours occurred at a low frequency, there may not necessarily be a chemical aetiology.     

Dietary supplementation with the microalgae Parietochloris incisa increases survival and stress resistance in guppy (Poecilia reticulata) fry

Dietary enrichments with the arachidonic acid (ARA)‐rich microalga, Parietochloris incisa, on the survival of guppy (Poecilia reticulata) fry were examined. Diets were applied via Artemia enrichment to fish from two commercial farms for 34 and 36 days of experimental period (trials 1 and 2, respectively). In trial 1, Artemia nauplii were enriched with dry biomass of whole algal cells at 0 (control), 0.1, 0.2 and 0.4 mg mL^?1. Fry fed with Artemia enriched with 0.4 mg mL^?1 demonstrated the lowest mortality rates (24% and 1% in farms 1 and 2, respectively) compared with controls (36% and 13% in farms 1 and 2, respectively). In trial 2, fry were fed with Artemia, enriched with whole algal cells (0.4 mg ml^?1), algal hexane extract (HE; containing primarily ARA‐rich triacylglycerols and β‐carotene; 0.19 mg ml^?1) or the extraction residue (0.28 mg ml^?1). Acute stress (5 min air exposure) was applied after 18 days. The lowest mortality was recorded in the whole alga‐fed group (av. 26% and 2.6% in farms 1 and 2, respectively), with a slightly, but not significantly higher mortality in the HE‐fed group (av. 29% and 6.2% in farms 1 and 2, respectively). Elevated lysozyme was associated with the reduced mortality. Overall, the use of P. incisa as a dietary supplement for guppy fry during their first month of life enhanced their survival and stress resistance.     

海水养殖水体中的危害性纤虫2新种——小冠须虫和拟迈氏冠须虫↑（*）

报道了采自青岛海区贝类育苗和养成池中的２ 种原纤类纤毛虫———小冠须虫Ｓｔｅｐｈａｎｏｐｏｇｏｎ ｍｉｎｕｔａ ｎ ．ｓｐ ．和拟迈氏冠须虫Ｓ．ｐａａｒａｍｅｓｎｉｌｉ ｎ ．ｓｐ．。依据蛋白银染色并结合活体观察,对其形态学做了综合描述,并就其主要鉴定特征与相近种进行了比较。     

Using attraction to light to decrease cannibalism and increase fry production in guppy (Poecilia reticulata Peters) hatcheries. I: phototactic reaction and light colour preference

A substantial proportion of newborns are lost in guppy hatcheries to cannibalism by the broodfish although small‐mesh baskets, which allow passage to small fry only and provide a predation‐free area, are used in breeding tanks. If newborn guppies are attracted by light, illuminating the small‐mesh baskets might enhance aggregation of newborns in the mesh basket and thus mitigate cannibalism effect. In this study, light attraction and light colour preference in newborn guppies were investigated. Testing the attraction to white, blue, green and red LED lights revealed attraction to each of these light types in newborns, but no significant attraction in adult females. Both newborns and adult females exhibited reduced mobility under red LED light. In a preference experiment in which all light colours were presented simultaneously, newborns were most attracted to the white light (52.0% of the newborns) and least attracted to the red light and darkness (6.8% and 6.7% of the newborns respectively). These results support the potential efficacy of using white light to attract newborns into the baskets in guppy breeding tanks and reduce cannibalism effects.     

Pathogenicity of Pasteurella sp. in lumpsuckers (Cyclopterus lumpus L.)

The incidence of disease caused by Pasteurella sp. in farmed lumpsuckers in Norway has been steadily increasing in recent years, causing significant economic losses and fish welfare issues. The disease affects all life stages, both in hatcheries and after release into salmon cages. Therefore, it is important to establish robust challenge models, to be used for vaccine development. Exposure experiments via intramuscular and intraperitoneal injection underlined the high virulence of the bacteria, whereas the cohabitation and bath models allowed the chronic symptoms of the disease to be studied more accurately. Skin lesions and haemorrhage at the base of fins were observed in the more acute cases of the disease. Symptoms including white spots over the skin, especially around the eyes, characterized the chronic cases. The latter were most prominent from the bath challenge model. Histopathology indicated a systemic pattern of disease, whereas qPCR analysis from head kidney showed that bacteria may be present in survivor fish at the end of the challenges. In all the challenge models investigated, Pasteurella sp. was re‐isolated from the fish, thus fulfilling Koch's postulates. These findings highlight the importance of screening of lumpsuckers prior to transfer to minimize the risks of carrying over asymptomatic carriers.     

Cell types and structures involved in tench,Tinca tinca (L.), defence mechanisms against a systemic digenean infection

Histopathological and ultrastructural investigations were conducted on 36 tench, Tinca tinca (L.), from Lake Trasimeno (Italy). The gills, intestine, liver, spleen, kidney and heart of 21 individuals were found to harbour an extensive infection of larvae of an unidentified digenean trematode. The eyes, gonads, swim bladder and muscles were uninfected. The parasites in each tissue type were embedded in a granulomatous proliferation of tissue, forming a reactive fibroconnective capsule around each larva. Most of the encysted larvae were metacercariae, in a degenerative state, but on occasion some cercariae were found. Many of the granulomas were either necrotic or had a calcified core. Within the granuloma of each, the occurrence of granulocytes, macrophages, rodlet cells and pigment‐bearing macrophage aggregates was observed. Hearts bore the highest parasitic infection. Whilst the presence of metacercariae within the intestine was found positioned between the submucosa and muscle layers, metacercariae in the liver were commonly found encysted on its surface where the hepatocytes in close contact with the granuloma were observed to have electron‐lucent vesicles within their cytoplasm. Metacercariae encysting adjacent to the cartilaginous rods of gill filaments were seen to elicit a proliferation of the cartilage from the perichondrium. Rodlet cells, neutrophils and mast cells were frequently observed in close proximity to, and within, infected gill capillaries. Given the degenerated state of most granulomas, a morphology‐based identification of the enclosed digeneans was not possible.     

寄生海水养殖鱼类的拟格拉夫涡虫的流行、危害与防治

自2000年10月中旬,在罗源湾发现一种罕见的能够寄生鱼类的涡虫,对其流行病学、形态学以及进化地位进行了大量的研究,结果表明：这种涡虫是一种未被报道过的新种,在福建省的各海水养殖区广泛流行,能够寄生于眼斑拟石首鱼（Sciaenops ocellatus）、大黄鱼（Pseudosciaena crocea）、鮸鱼（Miichthys miiuy）、牙鲆（Paralichthys olivaceus）、黑鲷（Sparus macrocephalus）和横纹东方鲍（Takifugu oblongus）等海水养殖鱼类的鳃、鳍及体表,造成寄生部位的严重损伤,鱼因呼吸困难或细菌的继发性感染而造成大量死亡,死亡率可达20％-60％;流行季节主要为秋季（8-11月）;水温22-26℃,盐度25-30。此外,提出了使用地下水,降低盐度、温度,合理用药等切实可行的防治本涡虫病的建议。     

First report of Shewanella sp. and Listonella sp. infection in freshwater cultured loach,Misgurnus anguillicaudatus

A novel disease outbreak occurred among cultured loach, Misgurnus anguillicaudatus in China. The diseased fish displayed lethargy, loss of appetite, black discolouration, anasarca and ventral skin hyperaemia. Clinical signs were observed in most of the fish examined, which consisted of discoloured liver, swollen spleen and petechial haemorrhage in the intestine and peritoneal membranes. The causative agent was found to be two species of bacteria by experimental infection, identified as Shewanella sp. and Listonella sp. The result showed that these were likely to be Shewanella putrefaciens and Listonella anguillarum respectively. Bacterial identification consisted of physiological and biochemical tests as well as 16S rRNA sequence analysis. In addition, antibiotic susceptibility testing was performed, revealing that Shewanella putrefaciens and Listonella anguillarum were susceptible to enrofloxacin, ciprofloxacin, levofloxacin, pipemidicacid and norfloxacin. This is the first report of S. putrefaciens and L. anguillarum from cultured loach.     

密斯特黄彩鲶仔、稚、幼鱼的生长与发育研究

在水温22～24℃,pH7.0左右的人工培育条件下,对密斯特黄彩鲶(Mystus sp.)仔、稚、幼鱼的形态特征和生长发育进行了观察与研究。密斯特黄彩鲶的鱼苗发育分为卵黄囊期仔鱼期(初孵至第5天)、晚期仔鱼期(第6～10天)、稚鱼期(第11～20天)和幼鱼期(第20天以后)。初孵仔鱼平均全长5.12 mm,卵黄囊体积1.63 mm3;仔鱼第3天开始摄食,第5天卵黄囊被完全吸收,鱼苗主要外部器官主要集中在晚期仔鱼阶期和稚鱼期内形成。仔鱼在饥饿状态下最高初次摄食率出现在出膜后第6天,为84.0%,其饥饿的不可逆点则为出膜后第8天,第8天以后即进入不可逆PNR期,至第11天绝大部分饥饿仔鱼死亡。