Lernaeid parasites prevalence in commercial freshwater fish species at various fish farms in Pakistan

Abstract Reports abound on Lernaea parasitizing the brood stock, fingerlings, and marketable-sized culturable freshwater fish species in various parts of the world. We investigated seven small-scale aquaculture farms and how the prevailing Lernaea is impacting them. Randomly seven fish farms were selected to determine the prevalence percentage of lernaeid ectoparasites. Relevant information of the fishponds to estimate the various aspects such as effects of water source and quality, feed, stocking density, treatment used, and weight and length of fish, concerned with Lernaea infestation and prevalence was gathered. The results indicated that Catla catla (F. Hamilton, 1822) showed highest prevalence (41.7%) among the seven fish species, whereas Oreochromis niloticus showed zero. Other five fish species Ctenopharyngodon idella, Cirrhinus cirrhosis, Cyprinus carpio, Labeo rohita and Hypophthalmichthys molitrix showed 13.2%, 8.1%, 7.7%, 7.4%, 0.9% prevalence, respectively. In Royal Fish Farm 84.3% lernaeid infestation was observed, while no parasite was observed in the Vicents Chunnian fish farm. The water source, quality, feed, fertilizers, stocking density, water temperature, and potential treatment options displayed varying tendencies among fish farms and prevalence. Depending on the weight and length, the highest prevalence (56.7%, and 66.7%) was observed in 3501-4000 g and 81-90 cm groups. The infestation rate varied in various fish body parts with the dorsal fin the most vulnerable organ and showed 2.3% overall prevalence (while 18.4% contribution within total 12.6% infestation). Out of 147 infected fish samples, 45 were extensively contaminated by Lernaea spread. In conclusion, our findings confirm that Lernaea could pose a considerable threat to marketable fish, and various treatment options should be educated to the farmers to help mitigate the spread and potential losses. Furthermore, Catla catla is more vulnerable to Lernaea infestation (41.7%), so are the fish species being cultured at higher stocking densities.

Resumo Abundam os relatórios sobre Lernaea parasitando o estoque de cria, alevinos e espécies de peixes de água doce cultiváveis de tamanho comercial em várias partes do mundo. Investigamos sete fazendas de aquicultura de pequena escala e de que maneira a Lernaea predominante está impactando-as. Aleatoriamente, sete fazendas de peixes foram selecionadas para determinar a porcentagem de prevalência de ectoparasitas de Lernaea. Foram recolhidas informações relevantes sobre os viveiros de peixes para estimar os vários aspectos, tais como efeitos da fonte e qualidade da água, alimentação, densidade de povoamento, tratamento utilizado e peso e comprimento dos peixes, relacionados com a infestação e prevalência de Lernaea. Os resultados indicaram que Catla catla (F. Hamilton, 1822) apresentou maior prevalência (41,7%) entre as sete espécies de peixes, enquanto Oreochromis niloticus apresentou zero. Outras cinco espécies de peixes Ctenopharyngodon idella, Cirrhinus cirrhosis, Cyprinus carpio, Labeo rohita e Hypophthalmichthys molitrix apresentaram 13,2%, 8,1%, 7,7%, 7,4%, 0,9% de prevalência, respectivamente. Em Royal Fish Farm, 84,3% de infestação de Lernaea foi observada, enquanto não se observou nenhum parasita na fazenda de peixes Chunnian de Vicent. A fonte de água, qualidade, ração, fertilizantes, densidade de estocagem, temperatura da água e opções de tratamento potenciais exibiram tendências variadas entre as fazendas de peixes e prevalência. Dependendo do peso e comprimento, a maior prevalência (56,7% e 66,7%) foi observada nos grupos de 3501-4000 g e 81-90 cm. A taxa de infestação variou em várias partes do corpo dos peixes, sendo a nadadeira dorsal o órgão mais vulnerável e apresentou 2,3% de prevalência geral (enquanto 18,4% de contribuição dentro do total de 12,6% de infestação). Das 147 amostras de peixes infectados, 45 estavam amplamente contaminadas pela propagação de Lernaea. Em conclusão, nossos resultados confirmam que Lernaea pode representar uma ameaça considerável para peixes comercializáveis, e várias opções de tratamento devem ser educadas para os agricultores para ajudar a mitigar a propagação e as perdas potenciais. Além disso, Catla catla é mais vulnerável à infestação por Lernaea (41,7%), assim como as espécies de peixes sendo cultivadas em densidades de estocagem mais altas.

Lernaeid parasites prevalence in commercial freshwater fish species at various fish farms in Pakistan.

Reports abound on Lernaea parasitizing the brood stock, fingerlings, and marketable-sized culturable freshwater fish species in various parts of the world. We investigated seven small-scale aquaculture farms and how the prevailing Lernaea is impacting them. Randomly seven fish farms were selected to determine the prevalence percentage of lernaeid ectoparasites. Relevant information of the fishponds to estimate the various aspects such as effects of water source and quality, feed, stocking density, treatment used, and weight and length of fish, concerned with Lernaea infestation and prevalence was gathered. The results indicated that Catla catla (F. Hamilton, 1822) showed highest prevalence (41.7%) among the seven fish species, whereas Oreochromis niloticus showed zero. Other five fish species Ctenopharyngodon idella, Cirrhinus cirrhosis, Cyprinus carpio, Labeo rohita and Hypophthalmichthys molitrix showed 13.2%, 8.1%, 7.7%, 7.4%, 0.9% prevalence, respectively. In Royal Fish Farm 84.3% lernaeid infestation was observed, while no parasite was observed in the Vicent's Chunnian fish farm. The water source, quality, feed, fertilizers, stocking density, water temperature, and potential treatment options displayed varying tendencies among fish farms and prevalence. Depending on the weight and length, the highest prevalence (56.7%, and 66.7%) was observed in 3501-4000 g and 81-90 cm groups. The infestation rate varied in various fish body parts with the dorsal fin the most vulnerable organ and showed 2.3% overall prevalence (while 18.4% contribution within total 12.6% infestation). Out of 147 infected fish samples, 45 were extensively contaminated by Lernaea spread. In conclusion, our findings confirm that Lernaea could pose a considerable threat to marketable fish, and various treatment options should be educated to the farmers to help mitigate the spread and potential losses. Furthermore, Catla catla is more vulnerable to Lernaea infestation (41.7%), so are the fish species being cultured at higher stocking densities.

Analysis of nuclear degradation during lens cell differentiation.

Lens cells demonstrate a terminal differentiation process with loss of their organelles including nuclei. Chromatin disappearance is characterised by the same changes as most apoptotic cells, i.e. condensation of chromatin and cleavage into high molecular weight fragments and oligonucleosomes. The endo-deoxyribonucleases (bicationic (Ca2+, Mg2+), mono-cationic (Ca2+ or Mg2+) and acidic non-cationic dependent nucleases) are present in lens fibre cells. Our results suggest that the acidic non-cationic nuclease (DNase II) plays a major role in chromatin cleavage. This nuclease, known to be lysosomal, is found in lens fibre nuclei and only an antibody directed against DNase II inhibits the acidic DNA cleavage of lens fibre nuclei. In addition, there must be another DNase implicated in the process which is not DNase I but appears to be a Ca2+, Mg2+ dependent molecule. Regulation of these DNase activities may be accomplished by the effect of post-translational modifications, acidic pH, mitochondrial release molecules, growth factors or oncogenes. Finally, fibre cells lose organelles without cytoplasmic elimination. The survival of these differentiated cells might be due to the action of survival factors such as FGF 1.

51Cr release and oxidative stress in the lens.

Examination of the opaque areas of human cortical cataracts has shown that a large portion of the opacity could be attributed to the globules found there. We tested models involving globule formation as a result of oxidative damage to rat lens cells in culture and whole chick embryo lenses. When cell monolayers from a lens cell line were exposed to oxidizing conditions they developed globules on the cell surface. The cells were protected from damage by the addition of glutathione and vitamin C. Thirteen-day chick embryo lenses were also incubated in oxidizing conditions and the amount of cellular damage was assessed using a chromium-51 release assay we have developed. After 24 hr the percent 51Cr in the medium increased by an average of 20% as a result of 10 mM hydrogen peroxide treatment. The addition of the 10 mM vitamin C to the hydrogen peroxide significantly reduced the 51Cr leakage to the control level. Light microscopy of sections of the lens showed a breakdown of the equatorial fibre arrangement in the presence of H2O2, while addition of vitamin C restored the fibre organization to almost normal. The findings suggest that oxidative stress is an important step in cataractogenesis and point towards the use of water soluble antioxidants as protective agents.

In vitro studies of microwave-induced cataract. II. Comparison of damage observed for continuous wave and pulsed microwaves.

Depth of damage caused by pulsed (PU) and continuous wave (CW) microwaves was estimated by scanning electron microscopy in rat lenses fixed immediately, after irradiation in vitro in circulating thermostatically controlled buffered saline. Pulses of 10 microseconds width and 24 kW peak power were delivered to the lens at different repetition rates in order to permit the same total energy to be delivered during 6, 20 or 60 min of irradiation at specific absorption rate (SAR) values of 0, 5.75, 11.5, 23, 69, 231 and 750 mW g-1; total energy [power (pow) x time] deposited in the lens was 0, 0.23, 0.46, 1.38, 4.6, and 15 W min g-1. Damage (granular degeneration of cells at the lens equator) was measured at the apex of penetration of the degeneration. The depth of degeneration (dep) of Pu or CW was compared either: (1) by a one-way analysis of variance (ANOVA) for the CW data alone and the 11 combinations of (pow x time); or (2) by using two alternative models to fit the data, to permit experimental distinguishment between: (a) reciprocal effects of pow x time; and (b) separate effects. Using the ANOVA analysis, the Pu mode of irradiation resulted in more damage at the same average power for every combination tested except one (23 mW g-1, 6 min). Although the separate-effects models explained more of the variation in depth of damage, the reciprocal effects model may provide an adequate fit for practical purposes and has the advantage of greater simplicity. For both models, the pulsed irradiation mode produced 4.7 times the depth of damage caused by CW irradiation. These results are discussed in relation to previous Pu-CW comparisons. It is proposed that this additional damage at the same average power is caused by thermoelastic expansion (TEE).

Chromatin condensation and terminal differentiation process in embryonic chicken lens in vivo and in vitro.

During embryonic chick lens differentiation, the epithelial cells become transformed into elongated fibres. Concomitantly, the fibre nuclei undergo degeneration and high molecular weight (HMW) DNA breaks down due to nuclear endodeoxyribonuclease activity. An electronmicroscopic study of lens epithelial and fibre nuclei was made at different stages of chick embryonic development, both in vivo and in vitro. The in vitro conditions are conducive to the expression of endogenous endodeoxyribonuclease activity in fibres. In both conditions we observed condensation of chromatin. The organization of some nuclear material into distinct linear arrays followed by streaming of nuclear material into the cytoplasm is recorded only in vitro. Such a condition may lead to acceleration of the process of aging in lens fibres.

Modelling cortical cataractogenesis VII: Effects of vitamin E treatment on galactose-induced cataracts.

The possibility that vitamin E or other antioxidants might prevent cataracts was tested by incubating rat lenses in vitro in galactose-enriched medium or by treating rats fed a diet containing 50% galactose (w/w). The vitamin E was added to the medium at 2.4 microM, and to the diet at a level of 5 g kg-1 diet. In vitro, lenses incubated with 55.6 mM galactose underwent globular degeneration, which was partially prevented by addition of vitamin E (2.4 microM). Even in such vitamin E-protected lenses, which appeared clear, many small globules could be seen in the region of interdigitation at the 'corners' where hexagonal cells intersected. In vivo, in dietary experiments, a dense nuclear opacity of the lens was observed after approximately 5 weeks; unlike diabetic cataracts, this was not prevented by the addition of vitamin E to the diet. The extensive globular degeneration observed was typical of that found in long-term (21-week diabetic) cataracts. Although no significant difference in cataract incidence was observed, the extent of damage in vitamin E-treated rat lenses appeared to be less. The difference in effectiveness of vitamin E in galactose-induced cataracts, as compared to diabetic cataracts, is tentatively ascribed to (1) the more severe osmotic stress expected from the products of the aldose reductase pathway for galactose and (2) the greater depletion of reduced pyridine nucleotides (NADPH + NADH) expected of galactose as compared to glucose.

In vitro studies of microwave-induced cataract: reciprocity between exposure duration and dose rate for pulsed microwaves.

Rat ocular lenses exposed to pulsed microwave irradiation were maintained at constant temperature by circulating phosphate buffered saline in a thermostatically-controlled chamber. Irradiations with pulsed radiation (10 musec, 24 kW pulses) of 918 MHz were done at several different specific absorption rates (SAR) for durations up to 1 hr in order to explore a possible reciprocal relationship. The extent of damage was measured by the maximum depth of granular degeneration in the equatorial region of lenses fixed immediately after irradiation. The parameters of the pulses were increased to 20 musec and 48 kW to explore the variation in the biological effects and threshold with respect to average power, as well as pulse parameters (pulse width, peak power and energy per pulse). A total of 47 lenses were used in 3 X 4 factorial experimental design to explore effects observed at different average powers and durations (6, 20 and 60 min). The results were statistically analyzed by ANOVA and multiple regression analysis with logarithmic transformation. The results are summarized as follows. This data showed clear trends towards increasing depth of granular degeneration with increasing duration of exposure and dose rate. There was considerable evidence to confirm such reciprocity suggesting that total dose is an important parameter. A model postulating reciprocity was shown to explain observed variation in depth of damage as well as one allowing for separate effects of duration and dose rate. Lens fibre cell effects were detected by scanning electron microscopy after 6 min irradiation at the SAR values of 40 and 20 mW g-1. Light microscopic evidence of lens fiber cell damage can be detected at an SAR of 10 mW g-1 after a 1 hr exposure.

Modelling cortical cataractogenesis: VI. Induction by glucose in vitro or in diabetic rats: prevention and reversal by glutathione.

Cataractogenesis can be induced by glucose in the rat lens in vitro and in vivo. In vitro, this is done by increasing the amount of glucose in the medium surrounding the isolated lens; within 48 hr considerable globular degeneration is seen subcapsularly, deeper in the equatorial region. In vivo, it is achieved by making the rat diabetic by injecting streptozotocin i.v., which selectively destroys the beta-cells of the pancreas; the blood serum glucose level increases markedly, and thence the aqueous humour level and, in turn, the lens concentration. Globular degeneration occurs as in vitro, but not until 6 weeks is a degree of damage observed comparable to that seen in the lenses incubated in vitro for only 48 hr. Lenticular sorbitol and fructose are also markedly elevated as a result of the high glucose levels. If glutathione (GSH) is present in the medium (0.1 mM) or injected s.c. daily into the diabetic rats, there is no evidence of subcapsular globular degeneration of the cortical fiber cells, even though the lenticular levels of glucose, sorbitol and fructose are the same as when GSH was not given; this is true for either the in vitro or in vivo situation, although individual values in the two situations do differ somewhat from one another. When rats were given GSH beginning several weeks after the diabetic state had been induced, the damage subsequently observed was much less than if the rats had been diabetic without GSH for the same total length of time; it was also much less than damage which should have occurred by the time GSH treatment was instituted. It would thus appear that a certain amount of reversal of the globular degeneration is possible, although damage in the equatorial region (wedge-shaped) seems less amenable to rescue by glutathione. The data indicate that glutathione can prevent or diminish the severity of sugar cataractogenesis, and that there would appear to be more steps in sugar cataractogenesis than simply osmotic damage, although this may be the primary event.

Modeling cortical cataractogenesis. V. Steroid cataracts induced by solumedrol partially prevented by vitamin E in vitro.

Rat lenses incubated in tissue culture medium (M 199) maintain their transparency for a long period of time. The soluble corticosteroid, solumedrol (methyl prednisolone sodium succinate) was added to the medium, at concentrations including the range expected during rejection episodes following organ transplantation (3.8 X 10(-9) M-3.8 X 10(-6) M). At the lowest level used (3.8 X 10(-9) M), five lenses of 12 became opaque following a 48 hr incubation, while at higher concentrations of solumedrol almost all lenses developed opacities. Addition of vitamin E to the medium resulted in partial prevention of the cataract as judged by the smaller proportion of lenses becoming opaque. Examination of the lenses by scanning and transmission electron microscopy (SEM and TEM, respectively), indicated that in untreated lenses the initial location of the cataract is at the anterior pole of the lens where a deepening area of degeneration formed, followed by a uniform subcapsular layer of degeneration spreading over the remainder of the lens. Damage at this location is not typical of most in vitro cortical cataracts. In the presence of vitamin E the extent of damage was less, involving, initially, an equatorial wedge of globular degeneration and spreading anteriorly and posteriorly in a thinner subcapsular layer. This type of damage was more typical of that seen previously for cataracts induced by cytochalasin D, elevated glucose and hygromycin B.

Modelling cortical cataractogenesis: 3. In vivo effects of vitamin E on cataractogenesis in diabetic rats.

Adult rats, some pretreated for 2 weeks with daily injections of vitamin E (961 IU/kg) were made diabetic by an intravenous injection of streptozotocin. The glucose levels in the serum rose to about five times normal in both groups. In the animals that had not been treated with vitamin E, changes appeared in the lenses that worsened as the hyperglycemia continued: by 4 days, irregular structure of the fibre cells at the equator; by 1 week, twisted ends and irregular undulations of these cells; by 2 weeks, jagged protrusions from the cell surfaces and twisting of the entire cell; by 3 weeks, flakiness of the surfaces of the equatorial epithelial cells, and disorganized and jagged fibre cells in a large area at the equator; and by 6 weeks, extensive subcapsular globular degeneration of the cortical fibre cells. In contrast, the lenses of the diabetic animals that continued to receive vitamin E showed minimal changes: after 6 weeks the equatorial fibre cells displayed the structural irregularities found after 4 days in the diabetic control animals. The protective effect of vitamin E could not be ascribed to the effect on aldose reductase, for at 6 weeks the levels of fructose and glucose in the lenses of the diabetic animals treated with the vitamin, although increased, were not significantly different from those in the lenses of the untreated diabetic rats, and the sorbitol levels were significantly higher in the treated animals. These results are discussed in terms of a multistep mechanism of cortical cataractogenesis.

Modelling cortical cataractogenesis: 1. In vitro effects of glucose, sorbitol and fructose on intact rat lenses in medium 199.

After 2 days, lenses with intact capsules from adults rats remained clear when incubated in medium 199 with a normal glucose concentration but showed opacities when the level of glucose was increased up to 10 times normal. Scanning electron microscopy of the fixed lenses demonstrate globular degeneration in the visibly opaque lenses. The depth of degeneration increased as a function of the glucose concentration in the medium, as did the opacity. Thus, in glucose-induced cataracts, as in other types of cortical cataracts, opacity and globular degeneration of the lens appear to be associated. Sorbitol (but not fructose) also induced globular degeneration and lens opacities, indicating that there may be a mechanism of cataractogenesis other than osmosis. The high risk of cataracts in persons with controlled diabetes may be related to the observation that, in vitro, a lower concentration of glucose in medium 199, 8.9 mM (1.6 times the normal level), with 96 hours of incubation was as effective as the minimum concentration capable of causing cataracts, 11.1 mM (2 times the normal level), with 24 hours of incubation.

Scanning electron microscopy of normal and lased rabbit pigment epithelium.

Scanning electron microscopic (SEM) observations of the rabbit retinal pigment epithelium (RPE) are reported both for normal tissue and for tissue subjected to laser radiation. In normal RPE, cells were observed to be closely packed and to have the characteristic hexagonal shape. The microvilli were abundant and of two types. Suprathreshold lesions were produced with 0.4 musec pulses of high-intensity yellow light (585 nm.) from a dye laser. In the lased RPE, cells were denuded of their microvilli, and a number of cells with single discrete holes were observed. Hypertrophied cells were always present. Within 5 days of irradiation groups of small proliferating RPE cells were found in the lesion area.

Scanning electron microscopy of normal and lased rabbit retina.

This study presents the topography as seen by scanning electron microscopy of the rabbit retina in general and the photoreceptors in particular; and of large laser lesions in the retina.

Isolated nerve endings (neurosecretosomes) from the posterior pituitary. Partial separation of vasopressin and oxytocin and the isolation of microvesicles.

Subcellular fractions of the bovine posterior pituitary, including one composed almost exclusively of pinched-off nerve endings (neurosecretosomes), were characterized electron microscopically, hormonally, and enzymically. 15% of the nerve terminals in the gland were isolated as neurosecretosomes, as estimated from determinations of lactic dehydrogenase, a soluble, cytoplasmic enzyme. Neurosecretosomes were subdivided into three fractions by density-gradient centrifugation. The three subfractions, each shown to be nearly homogeneous populations of neurosecretosomes by means of electron microscopic and enzymic criteria, differed from each other in their vasopressin/oxytocin (VP/OT) ratios. The VP/OT ratio increased from the lightest to the densest fraction, indicating that VP is localized to denser and OT to lighter neurosecretosomes; similar results have been obtained previously for subfractions of neurosecretory granules (NSG). No morphological differences were apparent in neurosecretosomes among the three subfractions. Although complete separation of VP and OT was not achieved, the findings suggest that VP and OT are each stored in a different species of nerve ending and support the hypothesis that a given neurosecretory cell synthesizes, stores, and secretes only one of the peptide hormones. Microvesicles, 40-80 mmicro diameter and contained in typical neurosecretory cell terminals, are believed to be degradation products of membrane ghosts of depleted NSG; electron micrographs indicative of this transformation are presented. A fraction rich in microvesicles, but containing some NSG membranes, was prepared by density-gradient centrifugation of an osmolysate of neurosecretosomes. Smaller, apparently nonneurosecretory nerve endings, lacking NSG but filled with small vesicles, are occasionally seen in sections from whole gland. The vesicles in these atypical posterior pituitary nerve endings may be true neurohumor-containing, "synaptic" vesicles.