Ultrastructural, molecular and haemato-immunological changes: multifaceted toxicological effects of microcystin-LR in rohu, Labeo rohita.

No water body is resilient to afflicts of algal bloom, if goes unmanaged. With the increasing trend of intensification, eutrophication and climate change, Labeo rohita (rohu) is highly anticipated to suffer from the deleterious effects of bloom and eventually its toxins. A comprehensive study was conducted to understand the toxicopathological effects of microcystin-LR (MC-LR) in rohu following intraperitoneal injection of 96 h-LD50 dose i.e., 713 µg kg-1. Substantial changes in micro- and ultrastructural level were evident in histopathology and transmission electron microscope (TEM) study. The haematological, biochemical, cellular and humoral innate immune biomarkers were significantly altered (p<0.05) in MC-LR treated fish. The mRNA transcript levels of IL-1ß, IL-10, IgM and IgZ in liver and kidney tissues were significantly up-regulated in 12 hpi and declined in 96 hpi MC-LR exposed fish. The relative mRNA expression of caspase 9 in the liver and kidney indicates mitochondrial-mediated apoptosis which was strongly supported by TEM study. In a nutshell, our study illustrates for the first time MC-LR induced toxicological implications in rohu displaying immunosuppression, enhanced oxidative stress, pathophysiology, modulation in mRNA transcription, genotoxicity, structural and ultrastructural alterations signifying it as a vulnerable species for MC-LR intoxication.

Effect of azadirachtin on haematological and biochemical parameters of Argulus-infested goldfish Carassius auratus (Linn. 1758).

Argulosis hampers aquaculture production and alters the host physiology and growth. Azadirachtin is recognized as a potential antiparasitic agent against Argulus sp. The present study aimed to investigate the effect of different concentration of azadirachtin solution on haematological and serum biochemical parameters of Argulus-infested goldfish Carassius auratus. Ninety Argulus-infested goldfish were randomly divided into six equal groups. Fish of group 1-5 were treated with azadirachtin solution through bath of 1, 5, 10, 15 and 20 mg L(-1) as T1, T2, T3, T4 and T5, respectively, and group 6 was exposed to 2% DMSO solution without azadirachtin and considered as negative control T0(-). Along with six treatment groups, a positive control T0(+) of healthy goldfish free from Argulus infestation was also maintained. Parasitic mortality was evaluated after 3 days of consecutive bath treatment. After 7 days of post-treatment, the blood and serum were drawn from each of the treatment groups and haematological and serum biochemical parameters were evaluated. Total leucocyte count (TLC), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), blood glucose, total protein (TP), globulin, serum glutamate oxaloacetate transaminase (SGOT) and serum glutamate pyruvate transaminase (SGPT) were significantly (p < 0.05) high in negative control group when compared with positive control group. It could be concluded that Argulus infestation altered marked haematological and serum biochemical parameters. However, in treated groups complete elimination of Argulus was found in T4 and T5 groups. Also significant (p < 0.05) reduction in haematological and serum biochemical parameters of all the treatment groups were recorded in comparison with negative control group. In addition, T4 and T5 groups showed significantly (p < 0.05) high superoxide dismutase (SOD), catalase, total erythrocyte count (TEC) and haemoglobin (Hb). However, higher mean corpuscular haemoglobin concentration (MCHC), blood glucose and lactate dehydrogenase (LDH) levels in T5 group revealed that higher concentration of azadirachtin have notable effects on activity of vital tissues function and physiology of the host. Argulus spp. from infested goldfish could be eliminated using bath treatment with solution of azadirachtin having concentration of 15 mg L(-1) and that also shifted haematological and serum biochemical parameters towards homeostasis.

Effect of orally administered azadirachtin on non-specific immune parameters of goldfish Carassius auratus (Linn. 1758) and resistance against Aeromonas hydrophila.

Modulation of the immune responses using active bio-ingredients as a possible prophylaxis measure has been novel prospect for aquaculture. The present study evaluated the effects of azadirachtin EC 25% on non-specific immune responses in goldfish Carassius auratus and resistance against pathogenic bacteria Aeromonas hydrophila. The experimental trial for effects of azadirachtin on immuno-haematoloical parameters in goldfish was conducted by feeding the various levels of azadirachtin as control T(0) (without azadirachtin), T(1) (0.1%), T(2) (0.2%), T(3) (0.4%), T(4) (0.8%) and T(5) (1.6%) for a period of 28 days. Fishes were challenged with A. hydrophila 28 days post feeding and relative percentage survival (%) was recorded over 14 days post infection. Immuno-haematoloical (total erythrocyte count, total leukocyte count, haemoglobin, packed cell volume, NBT activity, phagocytic activity, serum lysozyme activity, myeloperoxidase activity, total immunoglobulin) and serum biochemical parameters (serum glutamate oxaloacetate transaminase (SGOT), serum glutamate pyruvate transaminase (SGPT) and blood glucose) of fishes were examined at 14 and 28 days of feedings. Fish fed with azadirachtin, showed significantly (p < 0.05) enhanced TEC, TLC, Total Ig, total protein, NBT activity, serum lysozyme activity and myeloperoxidase level in different treatment groups in comparison with control group. Similarly, SGOT, SGPT and blood glucose level were found to be significantly (p < 0.05) high but PCV and Hb did not differ significantly (p > 0.05) in the treatment groups compared to control groups. Azadirachtin at the concentration of 4 g kg(-1) showed significantly (p < 0.05) higher relative percentage survival (42.60%) when compared with the control against A. hydrophila infection. This study indicated that azadirachtin EC 25% (4 g kg(-1)) showed higher NBT activity, serum lysozyme, protein profiles, leukocyte counts and resistance against A. hydrophila infection and thus, can be used as a potential immunostimulant in aquaculture.

Antiparasitic efficacy of piperine against Argulus spp. on Carassius auratus (Linn. 1758): in vitro and in vivo study.

Argulus are common aquatic ectoparasites that create one of the major threats to aquaculture due to absence of suitable therapy. Piperine, a bioactive component of Piper longum, has medicinal properties and acts as anti-inflammatory, antibacterial, and antifungal, considering eco-friendliness and cost-effectiveness. The present study aimed to evaluate antiparasitic effect of piperine against an ectoparasite Argulus spp. on Carassius auratus. Artificial Argulus infection was carried out by cohabitation method, and the fishes were selected for in vivo study when intensity of Argulus infestation was observed to be 15-20 Argulus per fish. In vitro and in vivo studies were performed at different concentration 1.0 (T (1)), 3.0 (T (2)), 5.0 (T (3)), 7.0 (T (4)), and 9.0 mg l(-1) (T (5)) of piperine solution to treat Argulus for 3 and 72 h, respectively. The acute toxicity test for piperine EC 97 % against goldfish was performed for 96 h. The 96 h median lethal concentration (LC(50)) for piperine was found to be 52.64 mg l(-1). In vitro effect of piperine solution led to 100 % mortality of Argulus at 9.0 mg l(-1) in 3 h whereas, under in vivo test, the 100 % antiparasitic efficacy of piperine solution was found at 9.0 mg l(-1) in 48 h. The EC(50) for 48 h was 9.0 mg l(-1), and thus, therapeutic index is 5.8. The results revealed that piperine at a concentration of 9.0 mg l(-1) can be used as a potential natural agent for controlling Argulus parasite.

In vitro and in vivo antiparasitic activity of Azadirachtin against Argulus spp. in Carassius auratus (Linn. 1758).

Argulus is one of the most common and predominant ectoparasites which cause serious parasitic disease and is a potent carrier of viruses and bacteria in the ornamental fish industry. In recent years, organic (herbs)-based medicines are widely used to cure the disease, and neem (Sarbaroganibarini) medicine is very popular and effective throughout the world. The present study was conducted to find the effects of Azadirachtin against Argulus spp. on Carassius auratus under in vitro and in vivo conditions. The 96-h median lethal concentration (LC(50)) for Azadirachtin EC 25% against Carassius auratus was found to be 82.115 mg L(-1). The antiparasitic activity test under in vitro and in vivo was evaluated at 1 (T1), 5 (T2), 10 (T3), 15 (T4) and 20 mg L(-1) (T5) to treat Argulus for 3 h and 72 h, respectively. In vitro effect of Azadirachtin solution led to 100% mortality of Argulus at 20 and 15 mg L(-1) for 2.5 and 3 h, respectively. Whereas, under in vivo test, the 100% antiparasitic efficacy of Azadirachtin solution was found at 15 and 20 mg L(-1) for 72 and 48 h, respectively. The EC(50) for 48 h was 20 mg L(-1), and thus, therapeutic index is 4.10. The results provided evidence that Azadirachtin can be used as a potential agent for controlling Argulus.