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.     

Effect of Neguvon® and Nuvan® treatment on crabs (Cancer pagurus,C. maenas), lobster (Homarus gammarus) and blue mussel (Mytilus edulis)

Survival tests were carried out with crabs, lobsters and blue mussels held in tanks of flowing seawater and subjected to Neguvon at 10, 1, 0.5 and 0.1 ppm and to Nuvan at 1, 0.01 ppm. Homarus gammarus was the most susceptible species; the animals died within 24 h at Neguvon levels of 0.5 ppm and higher and at Nuvan levels of 0.1 ppm and higher. The use of these chemicals in salmon farming appears to have deterious effects on marine invertebrate species.     

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 .     

Effects of container transparency and reflectivity upon oxygen consumption of hybrid lobsters

The effect of intra-specific visual communication on the rate of oxygen consumption of hybrid lobsters (Homarus americanus × H. gammarus) was tested in a partitioned metabolic chamber. Three partition linings were tested: opaque rubber, clear plexiglass, and glass mirror. Short-term mean oxygen comsumption rates observed for lobsters separated by rubber-lined partitions were 8% lower than those of lobsters separated by the clear partitions, and 29% lower than those of lobsters separated by mirrored partitions (P < 0.001).     

Factors influencing the efficacy of MS-222 to striped mullet (Mugil cephalus)

Some responses of juvenile striped mullet (Mugil cephalus) to the anesthetic MS-222 were investigated. Induction times to total loss of equilibrium decreased with increasing anesthetic concentration over the range of 40–120 ppm. Temperature, pH, and oxygen concentrations all significantly affected induction times to total loss of equilibrium in freshwater-adapted fish at a concentration of 80 ppm MS-222. Anesthetic toxicity was less for saltwater-acclimated fish than for freshwater-acclimated fish. The 24-h 50% mortality (LC₅₀) in fresh water was 33.1 ppm; in salt water it was 39.8 ppm. The results suggest that anesthetic action may be intensified in fish acclimated to conditions other than those near their physiological optima.     

Acute toxicity of emamectin benzoate (SLICE™) in fish feed to American lobster, Homarus americanus

SLICE? (active ingredient 0.2% emamectin benzoate (EMB)), a feed premix developed by Schering‐Plough Animal Health for the control of sea lice on cultured salmonids, is registered for use in several countries and is being prescribed on an emergency basis in Canada and the United States. The concentration of EMB in feed administered to farmed salmon ranges from 1 to 25 μg g^?1. To determine the acute toxicity of the compound to juvenile and adult American lobster (Homarus americanus), commercial salmon feed was coated with SLICE? at a range of concentrations and provided to the animals for 7 d in the laboratory. The LC₅₀ is estimated to be 644 μg g^?1 (95% CI=428, 1275) for adult lobsters and >589 μg g^?1 for stage V and VI juvenile lobsters. The consumption of medicated pellets by adult lobsters decreased significantly with increasing concentration of EMB. Adult lobsters that died during the study had a significantly greater concentration of emamectin B1a in their muscle tissue than those that survived. These results support the conclusion that salmon feed medicated with EMB at the concentrations used by the aquaculture industry is unlikely to pose an acute lethal threat to adult and small juvenile American lobsters.     

Effects of dietary carbohydrate on growth of juvenile New Zealand rock lobsters, Jasus edwardsii

The commercial aquaculture of spiny lobsters is impeded by the development of effective practical diets, which is partly due to a lack of knowledge of the digestive capabilities and nutritional needs of these species. Carbohydrates have the potential to provide a low cost energy source in practical diets for spiny lobsters. This research investigated the effect of algal carbohydrates, on the growth of juvenile rock lobsters. Juvenile lobsters, Jasus edwardsii, (10-12 mm carapace length) were provided with one of five diets; blue mussel, squid (Nototodarus gouldii), or a mussel diet partially substituted with algal carbohydrates, either agar, carrageenan or alginate over 80 days in culture. In all treatments, diet consumption and lobster growth were maintained or increased over time. Growth was highest for the mussel diet followed by the agar diet. Similar lobster growth occurred on the carrageenan and alginate diets, with the least on the squid diet. After 80 d there were significant differences in the percent lipid and glycogen stored in the mid-gut gland with highest levels of glycogen in lobsters fed the mussel only diet. Percent protein in the tail muscle was greatest for mussel fed lobsters. The mid-gut index (mid-gut weight/whole wet weight) was highest for the mussel only diet and this was similar to control lobsters at the start of the experiment. The results indicate that glycogen from mussels and agar could be used as carbohydrate source in the future development of a practical diet for spiny lobsters.     

Antibacterial benzaldehydes produced by seaweed-derived Streptomyces atrovirens PK288-21

Marine-derived actinomycetes (87 strains isolated from seaweed and 98 strains isolated from marine sediment) were screened for antimicrobial activity against bacterial fish pathogens. The most potent active strain (PK288-21) isolated from the rhizosphere of the marine seaweed Undaria pinnatifida was identified as Streptomyces atrovirens by 16S rDNA sequence analysis. Two active compounds were isolated from a culture extract of strain PK288-21 by silica-gel flash chromatography and high-performance liquid chromatography. The structures of the two compounds were identified as 2-hydroxy-5-(3-methylbut-2-enyl)benzaldehyde (B1) and 2-hepta-1,5-dienyl-3,6-dihydroxy-5-(3-methylbut-2-enyl) benzaldehyde (B2) by nuclear magnetic resonance and high-resolution fast atomic bombardment mass spectroscopy. The antimicrobial activities of the two compounds were tested against bacterial fish pathogens and expressed in terms of the minimum inhibitory concentration (MIC). Metabolite production was found to be optimized in A1BFe medium following the screening of eight different media. The two compounds killed Edwardsiella tarda after 12?h and Streptococcus iniae after 16?h at the MIC. Compound B1, 2-hydroxy-5-(3-methylbut-2-enyl)benzaldehyde, is a new benzaldehyde derivative, and this is the first time that either of these compounds have been reported in the genus Streptomyces.     

Chemotherapy of Cytophaga/Flexibacter-like bacteria (CFLB) infections in fish: studies validating clinical efficacies of selected antimicrobials

Abstract. In vitro and in vivo efficacies of commonly used chemotherapeutants were determined for Cytophaga johnsonae, Cytophaga psychrophila, Flexibacter colutnnaris and Flexibacter maritimus. Treatmeat of barramundi, Lutes calcarifer (Bloch), with oxolinic acid (OA) as a bath (50 ppm) or by mouth (10 mg kg⁻¹ body weight) resulted in serum levels above the minimum inhibitory concentration (MIC) for F. columnaris and produced significant clinical efficacy ( P < 0.05). Amoxycillin (AM) was found to produce adequate serum levels against F. maritimus when used as a bath (200 ppm) or given orally (80 mg kg⁻¹ body weight) to Atlantic salmon, Salmo salar L. and rainbow trout, Oncorhynchus mykiss (Walbaum), respectively. At these dose rates, this antibiotic was also clinically efficacious against F. maritimus ( P <0.05). Trimethoprim produced more than adequate serum levels for the control of F. maritimus when given as a bath (50 ppm) or orally (10 mg kg⁻¹ body weight). Trimethoprim was significantly more protective than AM when tested in vivo ( P < 0.05). For C. johnsonae and C. psychrophila , the MIC values for OA and oxytetracycline were low, whereas that for TMP was high. Also, based on MIC values, C. psychrophila strains were more sensitive to AM and norfloxacin than C. johnsonae.     

Prevention of Saprolegniasis in rainbow trout (Oncorhynchus mykiss) eggs using oregano (Origanum onites) and laurel (Laurus nobilis) essential oils

The present study investigated the antifungal effects of essential oils of oregano (Origanum onites) and laurel (Laurus nobilis) on Saprolegniasis, a disease that occurs in rainbow trout eggs during the incubation period. Oregano and laurel were ground after drying, and essential oils were obtained by water distillation method using a Clevenger apparatus. The essential oils were added to potato dextrose agar (PDA) at the rates of 1–1000 ppm, and the minimum inhibitory concentration (MIC) was determined as 250 ppm whereas the minimum lethal concentration (MLC) was determined to be 500 ppm for both plants. In the in vivo trials, fertilized eggs were treated with predetermined doses either by bathing during water hardening and incubation period or only during incubation period, and death rates were monitored during embryological development. The best larvae hatching rate was determined in 500 ppm oregano and 500 ppm laurel groups treated during water hardening plus daily as 82.11% and 79.87%, respectively. According to the results, it was determined that oregano and laurel essential oils exhibited better results in all doses compared with the negative control group, and 500 ppm dose had a better effect than the positive control group treated with formalin.     

Sensitivity of juvenile lobsters, Homarus americanus L., to different concentrations of malachite green during various states of the intermoult cycle

Abstract. Survival of the early stages (egg, larva and juvenile) of the American lobster, Homarus americanus L., reared under controlled conditions has often been threatened by a variety of fungi and microbial epibionts. A preliminary study was conducted for the purpose of defining toxicity levels of malachite green for 5th stage juveniles during each of the following intermoult (or fused intermoult) states: A-B, C₄, D₀ and D₂-D₃ Irrespective of exposure time or concentration, all the lobsters treated during intermoult state A-B died. This contrasts with seemingly good survival of lobsters in intermoult states C₄, D₀ and D₂- D₃ treated at concentrations of 900 mg/1 malachite green for exposure periods of 10 min. The findings suggest that an effective treatment combination for lobsters immersed in malachite green may be one which is employed during intermoult state C₄, for about 6min and at concentrations of about 700–800 mg/l.     

Bait-subsidized diets and their effects on ovigerous North American lobsters (<Emphasis Type="Italic">Homarus americanus</Emphasis>)

Ovigerous American lobsters (Homarus americanus) display a protracted period of ovary maturation and maternal care when incubating their eggs, potentially influencing offspring fitness. Lobsters consume a wide range of food items; however, trap bait may comprise a larger proportion of their diet in some fished areas compared to non-fished areas, and the long-term consequences of a bait-based diet remain largely unexplored in lobsters. We tested the hypothesis that disproportionate amounts of bait in the diets of pre-ovigerous females affect the quality of their ovaries and eggs. We held pre-ovigerous lobsters (n?=?29) over a period of ~?300 days (range?=?270–378) and fed them diets of herring bait, natural prey items (crab, mussel, urchin, macroalgae), or a combination of both diet types. Nutritional status, measured as biweekly blood indices and total glucose levels, suggest differences between lobsters fed a natural or combination diet and lobsters fed a bait-based diet (ANOVA; P?<?0.05). We found that bait diets contained more protein (58.5%) and lipids (31.6%) compared to natural diets (34.5 and 13.2%, respectively) and lipid levels in ovaries and eggs significantly correlated with each other for all treatments (r?=?0.76, n?=?15, P?=?0.028). Histopathological analysis indicates that ovaries contained more variable maturation in starved lobsters or those fed with bait, with some animals showing delayed gonad maturation. Results suggest that a varied diet promotes the overall fitness of ovigerous lobsters and the associated reserves that are used for ovarian development and subsequent oocyte formation.     

Effect of seaweed epibiotic bacterium Streptomyces violaceoruber SCH-09 on marine fouling organisms

Seaweed-associated bacteria play a role in helping to protect host organisms from biofouling by producing anti-fouling compounds. In a first step to develop such anti-fouling compounds, we isolated epibiotic bacteria from seaweed and subsequently screen culture extracts for anti-fouling activities. The active epibiotic bacterium studied was isolated from Undaria pinnatifida and identified as Streptomyces violaceoruber based on 16S rDNA sequence analysis. Two active compounds were isolated from the culture extracts by silica gel chromatography and high-performance liquid chromatography, and their structures were analyzed by nuclear magnetic resonance and high-resolution electron ionization mass spectrometry. The compounds were identified as furanone derivatives: 3-octa-1′,3′-dienyl-4-methylfuran-2(5H)-one and 3-octa-1′-enyl-4-methylfuran-2(5H)-one, respectively. These compounds showed anti-fouling activities against the following fouling organisms: zoospores of Ulva pertusa, the diatom Navicula annexa, and the mussel Mytilus edulis, with an EC₅₀ (minimum concentration for 50 % spore inhibition) range of 0.02–0.1 μg/ml. In the acute toxicity tests on juvenile rockfish Shebastes shlegelli, brine shrimp Artemia salina, and microalga Tetraselmis suecica, the two compounds showed LC₅₀ (concentration lethal to 50 % of the spores) ranges of 13.9–118.9 and 14.8–81.9 μg/ml, respectively.     

Gaffkemia in the California spiny lobster, Panulirus interruptus: Infection and immunization

Infection (gaffkemia) can be induced in the California spiny lobster (P. interruptus) with injections of Pediococcus homari (formerly Gaffkya homari). An LD_{5 0} of 10³ microbes per ml hemolymph is observed at 17°C. Prior immunization of lobsters (P. interruptus) with an avirulent strain of P. homari protects lobsters against 100 × LD_{5 0}. This is the first report of acquired immunity to P. homari in a susceptible species. The high incidence of gaffkemia in American lobsters (Homarus americanus) held in live holding facilities makes prophylactic measures essential in regions outside the enzootic area of the North Atlantic to avoid inadvertent transplantation of the disease to other susceptible populations of animals.     

Toxicity of malachite green to cultured american lobster larvae

Malachite green has been used to control fungal and epibiotic infestations of larval lobsters. The concentration of malachite green and the exposure period are critical to survival. The effects of increasing concentrations and exposure periods show survival to be adversely effected with treatments above 8 ppm for 16 min and above 20 ppm for 8 min administered every other day throughout the larval period. Development of the larvae is retarded with increasing treatment levels. Survivors of high treatment levels show no immediate post-treatment effects.     

Toxic levels of mercury for sequential larval stages of Macrobrachium rosenbergii (de Man)

The toxicity of mercury for sequential stages of Macrobrachium rosenbergii (de Man) larvae was studied under static water conditions at a salinity of 12‰ and temperature of 25.5 – 27.0°C. The results revealed that stage 1 larvae had the lowest threshold lethal concentration (TLC) of mercury, 0.041 ppm Hg, while the post-larvae had a TLC of 0.325 ppm Hg. There was an abrupt increase in the TLC of mercury after the larvae had reached stage 5. The lower TLC prior to stage 5 is most probably due to the more frequent moulting and the relatively thinner cuticle of the larvae.     

Does Morning as Opposed to Night‐time Feeding Affect Growth in Juvenile Spiny Lobsters,Jasus edwardsh?

Previous research on juvenile lobsters has shown that they display a nocturnal rhythm in their oxygen consumption and ammonia excretion, such that lobsters that are fed in the morning have shorter specific dynamic action (SDA) response than those lobsters that are fed at night. It was therefore suggested that juvenile lobsters should be fed in the morning to maximize energy utilization. This research tested the hypothesis that the growth of juvenile rock lobsters would be improved by feeding in the morning rather at night. Juvenile spiny lobsters Jasus edwardsii were fed a squid meal (5% body weight) either in the morning or at night over a period of 80 d. Lobsters fed in the morning had a 108% weight gain, an increase of 4.6 mm in carapace length and a growth rate (SGR) of 0.92%. These were significantly higher than the 78% weight gain, carapace length increase of 3.8 mm, and growth rate of 0.76% recorded for lobsters fed at night. Abdomen muscle protein and mid-gut gland lipid and glycogen content were similar after 80 d. The digestive gland index (DGI) was the same in lobsters exposed to the different feeding regime but had decreased compared with control lobsters at the start of the experiment. Morning-fed lobsters outperformed night-fed lobsters in all aspects of growth and survival, and it is suggested that commercial farmers feed their lobsters in the morning rather than at night. Because these growth differences were predicted from specific dynamic action measurements, it is concluded here that this technique would be useful for the rapid evaluation of the effectiveness of natural and artificial diets on lobster growth.     

A comparison of techniques for the measurement of growth in adult lobsters

Four non-destructive measures of growth (wet weight in air, wet weight in water, displacement volume and carapace length) were correlated to two destructive measures of growth (dry weight and total body protein level) in adult American lobsters (Homarus americanus). All non-destructive parameters were significantly (P < 0.05) correlated to dry weight and protein for female lobsters. Only wet weight in water was significantly correlated to dry weight and total body protein level in male lobsters.     

Antibacterial properties of anthraquinones extracted from rhubarb against Aeromonas hydrophila

Antibacterial properties of crude extract from rhubarb and its major bioactive compounds against Aeromonas hydrophila were assayed. Major bioactive compounds (anthraquinone derivatives) in rhubarb collected from different cultivation areas were determined by ultra-performance liquid chromatography (UPLC); the antibacterial activity [minimum inhibitory concentration (MIC)] of rhubarb was positively related to the anthraquinone content (r = 0.9306, P < 0.01). The MIC values of five anthraquinones against A. hydrophila were found to be in the range 50–200 μg/ml. Action-mode studies showed that anthraquinones (emodin) inhibits cellular functions by binding to cell DNA after penetrating the cell membrane, resulting in cell death. The present study suggests that anthraquinones extracted from rhubarb have potential use as antimicrobials for control of A. hydrophila.