Impact of graded doses of enrofloxacin on the safety and biological responses of Nile tilapia Oreochromis niloticus.

The cultivation of tilapias, the third most farmed fish group globally, has been rapidly growing, especially in Southeast Asia. This surge in tilapia farming intensification has led to increased use of antibiotics to control bacterial diseases. This study investigated the safety implications of administering graded doses of enrofloxacin (ENF) at 0 (control), 10, 30, 50 and 100 mg/kg biomass/day orally to Oreochromis niloticus. The 43-day study comprised 7 days of pre-dosing, 15 days of ENF-dosing, and a 21-day recovery period with a periodical assessment of the biological responses of fish. The results revealed that the overdosed groups experienced up to 21% reduction in feed consumption, 11% mortalities, and adverse impacts on hematology, including a decrease in erythrocytes, and monocytes and an increase in leukocytes, thrombocytes, lymphocytes, and neutrophils. Haematological indices like mean corpuscular volume and mean corpuscular hemoglobin decreased, while mean corpuscular hemoglobin concentration increased. The plasma biochemical parameters including glucose and liver and kidney enzymes unveiled a significant dose- and time-dependent increase, while calcium and chloride levels decreased. Erythrocytes displayed several erythrocyte cellular and nuclear abnormalities. The frequency of micronucleus increased with dose and time, suggesting potential genotoxicity of ENF. Additionally, a dose-dependent increase in residues in the tissues with the highest accumulation in muscle was documented. Nevertheless, the recovery of the measured parameters upon dose termination indicated that the ENF-induced alterations are reversible. The study affirmed the safety of ENF at the recommended dose (10 mg) in O. niloticus and their adoptive responses to higher doses.

Pharmacokinetics and residues of florfenicol in Nile tilapia (Oreochromis niloticus) post-oral gavage.

In the study on Oreochromis niloticus, singular oral gavage of florfenicol (FFC) at 15 mg/kg biomass/day was conducted, mimicking approved aquaculture dosing. Samples of plasma, bile, muscle, intestine, skin, liver, kidney, gill, and brain tissues were collected at 0, 2, 3, 4, 6, 8, 12, 16, 24, 32, 48, 64, 96, and 128 hours (h) after oral gavage. LC-MS/MS analysis revealed FFC concentrations peaked at 12.15 µg/mL in plasma and 77.92 µg/mL in bile, both at 24 hours. Elimination half-lives were 28.17 h (plasma) and 26.88 h (bile). The residues of FFC ranked muscle>intestine>skin>liver>kidney>gill. In contrast, the residues of florfenicol amine (FFA) ranked kidney>skin>liver>muscle>gill>intestine>brain, particularly notable in tropical summer conditions. The minimum inhibitory concentration of FFC was elucidated against several bacterial pathogens revealing its superior efficacy. Results highlight bile's crucial role in FFC elimination. Further investigation, especially during winter when fish susceptibility to infections rises, is warranted.

Intestinal Histopathological Aberrations in Oreochromis niloticus Juveniles upon Dietary Florfenicol Administration.

The aquaculture use of antibiotics can cause detrimental effects on fish organs and gut microbial dysbiosis. The impact of florfenicol (FFC) on fish intestinal histology, an approved antibiotic, remains unclear. This study aimed to investigate the effects of FFC on Oreochromis niloticus juveniles by administering FFC at 10 mg and 30 mg/kg biomass/day for 30 consecutive days to mimic long-term use. A dose-dependent reduction in feed intake, survival and biomass, with an upsurge in mortalities was observed. Even the therapeutic dose instigated mortalities on day 30 of FFC dosing (FD). Histopathological analysis revealed mild to moderate alterations, including loss of absorptive regions, epithelial degeneration, necrotized areas, intercellular enterocytic space and swollen laminar propria. Post-dosing, the observation of the detachment of lamina propria from the epithelium indicated imminent irritability. Goblet cells reduced drastically on day 30 FD, accompanied by an increase in intraepithelial lymphocytes. However, cessation of dosing for 13 days resulted in the reclamation of goblet cells and absorptive regions, indicating that the intestinal tissues underwent considerable repair after lifting antibiotic pressure. These findings suggested that O. niloticus can tolerate dietary FFC but emphasize the need for responsible use of antibiotics in aquaculture.

Safety, tolerability and biological responses of Oreochromis niloticus juveniles upon oral oxolinic acid administration.

In aquaculture, oxolinic acid (OA) is used as a second-line treatment at 12 mg/kg biomass/day for seven consecutive days. The present study evaluated the biosafety of 21 days of dietary administration of OA at 0, 12, 36, 60 and 120 mg by assessing the growth, biochemical, erythrocytic morphological and histopathological alterations and residue levels in Oreochromis niloticus. A significant dose-dependent reduction in feed intake and biomass and an increase in mortalities and erythrocytic cellular and nuclear changes were recorded. Significant elevations in plasma glucose, creatinine, alkaline phosphatase, alanine transaminase and aspartate transaminase and a decline in calcium and chloride levels were documented. The kidney, liver and intestine histoarchitecture showed mild to marked alterations. The edible tissue OA residues peaked on day 21 and decreased upon cessation of administration in all the dosing groups. The residue levels in the muscle of the recommended dose group were well within the maximum residue limit set by the European Medicines Evaluation Agency. Although the current study hinted at the safety and tolerability of OA even during long-term usage in O. niloticus in Indian conditions, care must be exercised for its aquacultural application because of its listing as a critically important medicine for humans.

In-feed emamectin benzoate abuse affects the biological responses and erythrocytes of Nile tilapia Oreochromis niloticus.

In aquaculture, drugs are often abused to accomplish disease control without considering the negative effects on fish health. This study aimed at elucidating the pernicious effects of in-feed antiparasitic drug emamectin benzoate (EB) abuse on the haemato-biochemistry and erythro-morphometry of healthy Nile tilapia Oreochromis niloticus. The fish were fed EB at 50 µg (1×) and 150 µg/kg biomass/d (3×) for 14 d as against the recommended 7 d and periodically assessed the blood parameters. A significant dose- and time-dependent reduction in feed intake, survival, total erythrocytes (TEC), monocytes (MC), hemoglobin (Hb), hematocrit (Ht) and mean corpuscular Hb concentration were noted. The total leukocytes (TLC), thrombocytes (TC), lymphocytes (LC) and neutrophils (NC) markedly augmented. The EB-dosing altered the fish physiology by enhancing the glucose, alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) and creatinine and reducing the calcium, chloride and acetylcholinesterase (AChE) levels dose-dependently. The fish recovered within 4 weeks in the 1× group post-dosing but persevered in the overdosed group. The erythro-cellular and nuclear dimensions were reduced with the increase in dose and normalized after the cessation of dosing, except for nuclear volume. The erythro-morphological alterations were more prominent in the overdosed group. The results implied the pernicious effect of oral EB medication on the biological responses of fish if abused.

Impacts of Oral Florfenicol Medication and Residues on the Kidney and Liver of Nile Tilapia Oreochromis niloticus (L.)

Florfenicol (FFC), an approved aquaculture antibiotic, is administered in feed at doses of 10-15 mg kg biomass-1 day-1 for 10 successive days. In this study, healthy Oreochromis niloticus were fed with 0-10 times the therapeutic dose of 15 mg kg biomass-1 day-1 for 10 days and tracked for 43 days post dosing. Assessments of residue accrual and depletion, oxidative stress, serum biochemistry, histopathology and extent of kidney and liver damages were made. FFC dosing reduced the feed intake significantly. The therapeutic dose produced no mortalities on day 10. Dose-dependent alterations in serum biochemistry were noted upon dosing. Several histopathological alterations were observed in the kidney and liver, which vindicated the toxic potentials of FFC. The residual FFC and florfenicol amine (FFA) accrual, depletion and oxidative stress responses, such as increased malondialdehyde, total nitric oxide, ferric reducing antioxidant power and reduced glutathione S-transferase activity, were documented. The dietary FFC persuaded the physiological state of O. niloticus, the effects of which normalized sparsely with time upon cessation of dosing at the higher doses. The study provided a brief outlook on the physiological responses upon oral FFC administration, which should be kept in mind during its application for fish health safety purposes.

Immune responses and protective immunity in Pangasius pangasius (Hamilton, 1822) as induced by outer membrane proteins of Edwardsiella tarda and aluminium hydroxide adjuvant complex.

Edwardsiella tarda is considered one of the important bacterial fish pathogens. The outer membrane proteins (OMPs) of E. tarda are structurally and functionally conserved, and immunogenic. This study assessed the effects of the OMPs of E. tarda CGH9 as a vaccine without aluminium hydroxide [AH] (T1) and with AH adjuvant (T2) on the respiratory burst (ROB) activity, lymphocyte proliferation of head kidney (HK) leukocytes, and serum antibody production in pangas catfish Pangasius pangasius. The ROB activity and lymphocyte proliferation of HK leukocytes increased in both vaccinated groups compared to the control. Nonetheless, the T2 group showed a gradual increase in ROB activity and lymphocyte proliferation of HK leukocytes up to 3-weeks post-vaccination (wpv). The serum antibody production in the T1 group decreased initially for up to 2-wpv and increased from 3-wpv; whereas, in the T2 group, the serum-specific antibody levels were significantly high from 1-wpv compared to control. Simultaneously, the protective efficacy in terms of relative percentage survival in the T2 group after injecting with a lethal dose of E. tarda CGH9 was high (89.00±15.56) compared to the T1 group (78.00±0.00). Furthermore, the catfish administered with a booster dose of E. tarda OMPs with or without AH adjuvant showed no additional increase in immune response or protective immunity. These results suggested that E. tarda OMPs and AH adjuvant complex has a higher potential to induce protective immunity, which may be a good choice as a vaccine to combat E. tarda infection in catfish.

Histopathological changes and tissue residue concentrations of monosex Nile tilapia (Oreochromis niloticus, L) fries exposed to oxytetracycline.

The current study evaluated the biosafety of oxytetracycline (OTC) exposure for 30 days in monosex Oreochromis niloticus fries. The fries were exposed to OTC for 3 h/day for 30 days at 350 (0.5X), 700 (1X), 2100 (3X), 3500 (5X), and 7000 (10X) mg/L and compared with control (0X). The OTC exposure at 5X and 10X concentrations caused 100% mortality within 4 days and 5 min, respectively. The mortalities recorded in 0.5X, 1X, and 3X groups were 3.33 ± 1.15%, 14.67 ± 1.15%, and 47.33 ± 11.37% on day 30, respectively. The feed intake was decreased up to 23.33% in the 3X group during the exposure period. The OTC residue levels on 30-day exposure were 216.53 ± 14.71, 450.56 ± 44.31, and 1141.26 ± 63.64 µg/kg, which reduced to 40.40 ± 3.25, 76.68 ± 2.77, and 95.61 ± 5.13 µg/kg after 15 days of termination of exposure in the 0.5X, 1X, and 3X groups, respectively. The histopathological changes observed in the 1X group were epithelial detachment, desquamation of secondary lamellar epithelium, lamellar fusion, and inflamed cartilaginous core in the gills, alteration in the integrity of gut mucosa, degeneration of muscularis mucosae and necrosis in the intestine, the disintegration of the nephritic tubule, necrosis, and glomerulopathy in the kidney, and dilated vascular duct, necrotized hepatic tissue, diffused hepatic parenchyma, vacuolation, and fatty changes in the liver. The OTC exposure induced marked tissue changes histologically in a dose- and time-dependent manner, which undoubtedly reduced the growth of tilapia. Supplementary Information: The online version contains supplementary material available at 10.1007/s10499-022-00892-w.

The effects of extended feeding of florfenicol coated medicated diets on the safety, serum biomarkers and blood cells morphology of Nile tilapia Oreochromis niloticus (L.).

Tilapia is one of the most consumed farmed fish, which requires the use of antibiotics in certain phases of its production. This study assessed the safety of 30 days of oral florfenicol (FFC) dosing at 0-10 times the therapeutic dose (1 × : 10 mg/kg biomass/day) in Oreochromis niloticus juveniles. Behavioural changes, feed consumption, mortality and biomass were evaluated. Besides, the levels of serum glucose, calcium, chloride, creatinine, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase and blood cell morphology were determined at scheduled intervals. The 30 days of oral FFC dosing caused 3.33% (1 ×) to 18.33% (10 ×) mortalities, reduced feed intake and biomass in a dose-dependent manner. The fish fed the therapeutic dose recorded 1.25-fold increase in biomass, while the control group recorded 1.45-fold increase in 30 days. No significant erythrocyte morphological alterations were observed in the 1 × group compared to the control. However, marked morphological alterations like tear-shaped, spindle-shaped and degenerative erythrocytes in higher dosing groups indicated FFC cytotoxicity. All the serum biomarkers of O. niloticus increased significantly on day 10 and day 30 FFC dosing in a dose-dependent manner, except for calcium and chloride, which reduced significantly during the dosing period. Within 2 weeks of suspension of FFC dosing, the serum biomarker levels became normal except for alkaline phosphatase and creatinine. The recovery of biomass, feed intake, serum biomarker levels and erythrocyte morphological changes suggested that the FFC-induced changes are reversible. This study has, thus, proclaimed the safety of FFC at the therapeutic dose in O. niloticus.

Influence of dietary emamectin benzoate on the biological responses of monosex (all-male) Oreochromis niloticus (L.) fries.

The application of antiparasitic drugs plays a crucial role in the removal of infectious parasites in aquaculture. Emamectin benzoate (EB) is predominantly used as a feed premix against ectoparasites on temperate fish. This study evaluated the influence of 14 days of EB-dosing at 0-10 times the recommended dose (1X: 50 µg/kg biomass/day) on the biological responses and accrual/depletion of EB-residues in a tropical fish monosex Oreochromis niloticus fries. A significant dose-dependent reduction in feed intake by 3.50% in 1X and 43.00% in 10X groups, and an increase in mortalities from 2.92% (1X) to 11.25% (10X) during the EB-dosing period was noted. A significant increase in glucose and alkaline phosphatase and reduction in calcium and chloride ions, superoxide dismutase (SOD) and acetylcholinesterase levels in the muscle and/or brain tissue was observed. On day 21 post-EB-dosing, the levels of muscle glucose and SOD reached normalcy in the 1X group, while the levels of other biomarkers failed to recuperate. The EB-residue levels peaked on day 14 EB-dosing (2.77 ng/g) in the 1X group and decreased later with detectable levels (0.03 ng/g) even on day 21 post-EB-dosing. The EB-residue levels were within the permissible limits of the Canadian Food Inspection Agency and the European Commission. The EB-dosing negatively influenced the health of O. niloticus by altering the physiological state in a dose- and time-dependent way. The results suggested that the use of EB might be plausibly risky in tropical aquaculture.

Accumulation and clearance of tissue residues and health status of Nile tilapia Oreochromis niloticus (L.) juveniles as influenced by the extended oral oxytetracycline-dosing.

Antibiotics are considered an important primary therapy for bacterial diseases in aquaculture. This study evaluated the influence of oral administration of oxytetracycline (OTC) on feed intake, growth, mortality, residue accumulation and clearance, and histopathological changes in the vital organs of six groups of Nile tilapia Oreochromis niloticus when fed at 0-10 times the therapeutic dose (1×: 80 mg/kg biomass/day) for 10 and 20 consecutive days. The feed intake was reduced only slightly, viz., 2% in 10-day and 4.25% in 20-day dosing trials at 1× dose compared to control. While in other groups, an OTC-dose-dependent reduction in feed intake up to 31.25% was noted. The fish of the 0.5× and 1× groups recorded significantly high biomass, while the other OTC-dosed groups recorded significantly lower biomass than the control. The fold change in biomass between the control and 1× groups was insignificant. Dose-dependent mortalities were recorded in OTC-dosed fish in 10-day (1.67-6.67%) and 20-day (3.33-8.33%) trials. The OTC concentration in fish muscle established a dose- and time-response relationship. The OTC residue levels in muscle even on day 20 OTC-dosing were lower than the maximum residue limit (MRL) permitted by Codex Alimentarius (200 ng/g). On day 23 post OTC-dosing, the residue levels were traces to <10 µg/g in all groups, except the 10× group. The OTC-dosing caused mild to moderate pathological changes in the gills, liver and kidney of O. niloticus and the fish were able to mount adaptive biological responses to overcome the stress with time.

Dietary therapeutic dose of oxytetracycline negatively influences the antioxidant capacity and immune-related genes expression in Nile tilapia Oreochromis niloticus (L.).

Effects of the dietary therapeutic dose of oxytetracycline (OTC) at 80 mg/kg biomass/day for consecutive 10 days on the behaviour, feed intake, mortality, residue accumulation and depletion, antioxidant capacity and immune-related genes expression in juvenile Nile tilapia Oreochromis niloticus were evaluated. OTC-dosing caused mortalities, reduced feed intake, and biomass reduction at 24.5-28.5 °C. OTC residues recorded on day 10 (161.40 ± 11.10 ng/g) were within the maximum residue limits of the Codex Alimentarius. The withdrawal period was 7 days as per the European Commission's regulation. Traces of residues were present even on day 42 post-OTC-dosing. Dietary OTC reduced the antioxidant capacity of the liver and muscle tissues and down-regulated the expression of tumour necrosis factor-α, interleukin-1ß, and heat shock protein-70 genes in the liver significantly during the dosing period. The data generated on the biosafety of OTC-dosing may offer inputs for the development of management strategies in maintaining fish health and food safety.

Effects of dietary supplementation of vitamin-E and commercial probiotics on the innate immunity of Labeo rohita against Aeromonas hydrophila infection.

Immunomodulation is one of the useful tools to prevent diseases in aquaculture. In this study, the immunomodulatory effects of vitamin-E (100 mg/kg feed) and commercial probiotic consortia, Rhodomax™ (5 g/kg feed) on the innate immunity of Labeo rohita and their protective effect against Aeromonas hydrophila infection were evaluated and compared. Three groups of fish at 30 numbers/tank were fed with vitamin-E, probiotic and control diets at 3% body weight for 30 days, in triplicate. Following this, the fish of all groups were injected intramuscularly with A. hydrophila N10P at 2.40 × 107 cells/fish. The growth indices like specific growth rate (SGR) and feed conversion ratio (FCR) during the feeding and the non-specific immune responses during the feeding and post-challenge regimen were recorded. The dietary supplementation of vitamin-E and/or commercial probiotics caused significant improvements in the innate immunity of L. rohita compared to control. Nevertheless, the vitamin-E diet offered markedly better results in terms of SGR, FCR, ceruloplasmin, antiprotease, myeloperoxidase and phagocytic activities of L. rohita during the feeding and post-challenge regimen. While the respiratory oxidative burst activity was enhanced in probiotic diet-fed L. rohita only during the feeding regimen. All the immune parameters reached normalcy on day 15 post-injection with A. hydrophila. These findings revealed that supplementation vitamin-E at 100 mg/kg feed may improve the growth indices, prime the non-specific immune responses of L. rohita against A. hydrophila infection and enhance the overall health status than the tested commercial probiotics.

Safety of emamectin benzoate administered in feed to Nile tilapia Oreochromis niloticus (L.).

Emamectin benzoate (EB) premix top-coated onto feed is extensively used to treat ectoparasitic crustacean infestations in aquaculture. This study evaluated the safety of EB-dosing in Nile tilapia Oreochromis niloticus at the recommended dose and dosage of 50 µg/kg biomass/day for 7 consecutive days (1X) and compared with control and 10 times the recommended dose (10X). Depletion of EB-residues in the edible muscle of 1X-dosed Nile tilapia was also studied. Mortality, behavioural changes, feed consumption, biomass, EB-residue depletion, and histopathological alterations in the kidney, liver and intestine were determined at slated intervals. Significant dose-dependent reduction in feed intake and biomass and insignificant mortalities were noted in 1X and 10X EB-dosed fish. In 1X EB-dosed fish muscle, the residues peaked on day 7 EB-dosing (9.72 ng/g) and decreased subsequently. Nevertheless, the residue levels were within the acceptable limit of the European Commission and the Canadian Food Inspection Agency even during the EB-dosing period. Histologically, tubule degeneration in the kidney, mild glycogen vacuolation in the liver, and loss of absorptive vacuoles, inflammation and disintegration of the epithelial layer in the intestine of Nile tilapia fed the 1X EB-diet were observed. The fish reverted back to their normal functions with time upon termination of oral-EB-dosing. This work contributed scientific data on the safety of EB particularly on the feed intake, growth reduction, mortality, histopathological alterations, and EB-residue levels in the edible tissues of Nile tilapia fed at the recommended dose and dosage, which suggested that EB-therapy might be reasonably risky in a tropical climate.

Dietary influences of oxytetracycline on the growth and serum biomarkers of Oreochromis niloticus (L.).

Tilapias are cultured globally and are rising in acceptance as the most important freshwater aquaculture species. Monitoring of serum biomarkers is a promising tool in aquaculture to screen the health status as they are virtuous indicators of extreme stress and organ dysfunction in fish. The present study examined the serum biomarkers of oxytetracycline (OTC)-dosed Nile tilapia Oreochromis niloticus at 0, 80 and 800 mg/kg biomass/day, i.e., 0X, 1X, and 10X the approved dose (X = 80 mg OTC/kg biomass/day) for 10 consecutive days. The fish biomass and levels of serum glucose, aspartate aminotransferase, alanine aminotransferase, creatinine and C-reactive protein (CRP) were determined at scheduled intervals. A significant dose-dependent reduction in fish biomass during the OTC-dosing (5.84%) and post-OTC dosing (8.16%) periods was observed. All the serum biomarkers of Nile tilapia increased significantly on day 10 OTC-dosing. Though their levels reduced significantly, normalcy was not achieved even after 42 days of cessation of OTC-dosing, except CRP. The CRP reached the normal level on day 25 post-OTC dosing in the 1X group. The results, thus, demonstrated that the oral OTC-dosing influences the physiological state of apparently healthy Nile tilapia in a dose-dependent manner. These changes were, however, reversible upon discontinuation of OTC-dosing. The set of data observed on growth reduction and elevated serum biomarker levels even after 42 days of cessation of OTC-dosing, thus, raises questions on the utility of oral OTC-dosing.

Molecular characterization of Myxoboluscatmrigalae (Myxosporea: Myxobolidae) infecting the gill lamellae of carp Cirrhinusmrigala (Hamilton).

The present study attempted sequencing the 18S rRNA gene of Myxoboluscatmrigalae infecting the gill lamellae of carp, Cirrhinusmrigala and compared its genetic homology and phylogenetic characteristics with 18S rRNA genes of other Myxobolus spp. The infected fish had up to 3 small, creamy white plasmodia per gill filament with 30-50 spores each. The spore size was 17.90 ± 0.70 × 7.40 ± 0.40 µm. The sporoplasm contained two large nuclei of size 0.57 ± 0.09 µm and no iodinophilous vacuole. The DNA sequence of M.catmrigalae was clustered phylogenetically with other Myxobolus spp. infecting the gills of cyprinids available in GenBank, which showed 77-87 % homogeneity. On the phylogenetic tree, M.catmrigalae (KC933944) was clustered with M.pavlovskii (HM991164) infecting the gill lamellae of silver carp, Hypophthalmichthysmolitrix. The species most closely related to M.catmrigalae in GenBank was M.pavlovskii (AF507973) infecting the gill lamellae of big head carp, Aristichthysnobilis with 87 % homogeneity. This is the first report on molecular characterization of gill lamellae infecting M. catmrigalae.

Detection of goldfish haematopoietic necrosis herpes virus (Cyprinid herpesvirus-2) with multi-drug resistant Aeromonas hydrophila infection in goldfish: First evidence of any viral disease outbreak in ornamental freshwater aquaculture farms in India.

This outbreak report details of a mortality event where Cyprinid herpes virus-2 (CyHV-2) was detected in association with multidrug-resistant Aeromonas hydrophila infection in goldfish, Carassius auratus, from commercial farms. The goldfish exhibited large scale haemorrhages on the body, fins and gills, lepidorthosis, necrosed gills, protruded anus and shrunken eyes. White nodular necrotic foci in spleen and kidneys were noticed, along with necrosis and fusion of gill lamellae. Transmission electron microscopy of affected tissues revealed the presence of mature virus particles. Involvement of CyHV-2 was confirmed by PCR, sequencing and observed cytopathic effect in koi carp fin cell line along with experimental infection study. A bacterium isolated from the internal organs of affected fish was found to be pathogenic Aeromonas hydrophila having resistance to more than 10 classes of antibiotics. We postulate that CyHV-2 was the primary etiological agent responsible for this outbreak with secondary infection by A. hydrophila. The experimental infection trials in Labeo rohita and koi carp by intraperitoneal challenge with CyHV-2 tissue homogenates failed to reproduce the disease in those co-cultured fish species. This is the first report of a viral disease outbreak in organised earthen ornamental fish farms in India and bears further investigation.

Molecular characterization of Argulus bengalensis and Argulus siamensis (Crustacea: Argulidae) infecting the cultured carps in West Bengal, India using 18S rRNA gene sequences.

The present study characterized Argulus spp. infecting the cultured carps using 18S rRNA gene sequences, estimated the genetic similarity among Argulus spp. and established their phylogenetic relationship. Of the 320 fish samples screened, 34 fish (10.6%) had Argulus infection. The parasitic frequency index (PFI) was observed to be high (20%) in Hypophthalmichthys molitrix and Labeo bata. The frequency of infection was high in September (PFI: 17%) and October (PFI: 12.9%). The 18S rRNA sequences of five A. bengalensis (KF583878, KF192316, KM016968, KM016969, and KM016970) and one A. siamensis (KF583879) of this study showed genetic heterogeneity and exhibited 77-99% homology among the 18S rRNA gene sequences of Argulus spp. of NCBI GenBank database. Among the Indian Argulus spp. the sequence homology was 87-100%. Evolutionary pair-wise distances between Indian Argulus spp. and other Argulus spp. ranged from 0 to 20.20%. In the phylogenetic tree, all the crustaceans were clustered together as a separate clade with two distinct lineages. The lineage-1 comprised exclusive of Branchiura (Argulus spp.). All Argulus bengalensis clustered together and A. siamensis (KF583879) was closely related to Argulus sp. JN558648. The results of the present study provided baseline data for future work on population structure analysis of Indian Argulus species.

Molecular phylogeny of Myxobolus orissae (Myxosporea: Myxobolidae) infecting the gill lamellae of mrigal carp Cirrhinus mrigala (Actinopterygii: Cyprinidae).

Myxosporeans are best known for the diseases they cause in commercially important fish species. Identification of myxosporeans at the species-level is mainly based on conventional methods. The 18S rRNA gene sequence of morphologically identified Myxobolus orissae infecting the gill lamellae of mrigal carp Cirrhinus mrigala was characterized in the present study. The plasmodia of M.orissae were small, elongated and white to pale in colour. Phylogenetically, the 18S rDNA nucleotide sequence of M.orissae was clustered with other gill-infecting Myxobolus spp. of cyprinids. The species closely related to M. orissae was M. koi (FJ841887) infecting the gill lamellae of Cyprinus carpio with 96% similarity. The carp fin-infecting Thelohanelluscaudatus (KC865607) from India exhibited only 78% DNA sequence similarity with M. orissae. Low level of M. orissae infection on gill caused thickening of epithelial cells surrounding the plasmodium. Under stressful conditions, it is likely that such infection can easily spread in confined fish and may cause serious disease outbreaks and economical losses.

Molecular and phylogenetic characterization of Thelohanellus qadrii (Myxozoa, Myxosporea, Bivalvulida) infecting the secondary gill epithelium of Indian major carp, Catla catla (Hamilton, 1822).

Myxosporean taxonomy which is traditionally based on the morphology of the myxospore stage, is in a state of flux given new insights provided by the expanding dataset of DNA sequences. To date, more than 40 species of Thelohanellus from India have been described according to morphometric characteristics. Nevertheless, molecular data on these histozoic myxosporean parasites of freshwater fish are scarce. In the present study, molecular characterizations of Thelohanellus qadrii infecting the secondary gill epithelium of Indian major carp Catla catla (Hamilton, 1822) and its phylogenetic relationship is reported. The sub-adult cultured catla were observed to have low to moderate gill myxosporean infections. The morphometry of mature spores was in compliance with original descriptions of T. qadrii. Based on the analysis of 18S rRNA gene, phylogenetic clusters which were established according to a consensus sequence, illustrated the taxonomic placement of a series of myxobolids. The DNA sequence homogeneity of T. qadrii (KF170928) with other Thelohanllus spp. ranged from 78% to 95% and formed a dichotomy with cyprinid gill lamellae infecting T. toyamai (HQ338729). Distance matrix results indicated a high genetic diversity among myxosporeans. The present report is the first on the molecular and phylogenetic characterizations of T. qadrii.