Whole-animal accumulation, oxidative stress, transcriptomic and metabolomic responses in the pink shrimp (Pandalus montagui) exposed to teflubenzuron.

The benzoylurea chitin synthesis inhibitor teflubenzuron, widely used against sea lice in North Atlantic aquaculture, may pose an environmental threat to non-targeted crustaceans. In this experiment, laboratory acclimated pink shrimp (Pandalus montagui), a species found in fjords with Atlantic salmon farming, were exposed to dietary teflubenzuron for 46 days (control; low dose: 0.01 µg/g; high dose: 0.1 µg/g). The exposure doses represent 0.1% and 1% of a standard treatment dose for Atlantic salmon. Mortality and prevalence of deformities, pharmacokinetics, oxidative stress and transcriptomic and metabolomic profiling were used to assess the response to teflubenzuron exposure. Mortality in the high-dose group was 25% (five of 20 individuals). No control or low-dose group shrimps died. Phenotypic responses,i.e., leg deformities (0 control, 6 low, 8 high) and cloudy eyes (0 control, 3 low, 7 high), were observed in some surviving shrimps (control n = 15, low n = 17, high n = 15). Accumulated levels of teflubenzuron in shrimps from the high-dose group ranged from 4.7 to 369 ng/g wet weight. Transcriptomic profiling showed very few significantly altered genes in the exposed shrimps. Teflubenzuron-induced changes to the metabolome pointed to well-known effects of benzoylurea agents, with reduced levels of N-acetylglucosamine indicating an effect on chitin synthesis. The metabolomic profiling showed that teflubenzuron exposure was associated with reduced energy metabolism. Some metabolites pointed to increased necrosis and/or bacterial overgrowth in the teflubenzuron-exposed shrimps. In conclusion, this study shows that teflubenzuron causes phenotypic effects in P. montagui exposed to 0.1% of the treatment dose given to Atlantic salmon.

Introducing a Novel Media to Improve the Recovery of Culturable Bacteria from the Fish Parasite Anisakis spp. larvae (Nematoda: Anisakidae).

This paper describes a cultivation method to increase the recovery of bacteria from the marine muscle-invading parasitic nematode larvae of Anisakis spp. These larvae hold a high and complex population of accumulated bacteria, originating from seawater, crustaceans, fish, and marine mammals, all involved in the lifecycle of Anisakis. Two in-house agars based on fish juice prepared by either mechanical or enzymatic degradation of the fish tissue, were made. The Anisakis larvae were homogenised prior to cultivation on the in-house fish juice agars and the bacterial numbers and diversity were compared to those obtained applying the commercially available Marine Agar and Iron Agar Lyngby. Bacterial colonies of unique appearance were subcultured and identified by 16S rRNA gene sequencing. Totally three of twenty identified taxa were found on the in-house fish juice agars only. Fish juice agar prepared enzymatically would be the best supplementary agar, as this agar gave significantly higher heterotrophic plate counts, compared to mechanical preparation. The enzymatically prepared fish juice gave more suitable agar quality, was more resource efficient, and had apparently increased nutrient density and availability.

Transcriptional responses to teflubenzuron exposure in European lobster (Homarus gammarus).

Increasing use of pharmaceutical drugs to delouse farmed salmon raises environmental concerns. This study describes an experiment carried out to elucidate the molecular mechanisms of the antiparasitic drug teflubenzuron on a non-target species, the European lobster. Juvenile lobsters (10.3±0.9 mm carapace length) were fed two environmentally relevant doses of teflubenzuron, corresponding to 5 and 20% of a standard salmon medication (10 mg/kg day), termed low and high dose in this study. After 114 days of dietary exposure, whole-animal accumulation of teflubenzuron was determined. One claw from each animal was collected for transcriptional analysis. Overall, exposed animals showed low cumulative mortality. Six animals, two from the low dose treatment and four from the high dose, showed exoskeletal abnormalities (claw deformities or stiff walking legs). Residual levels of teflubenzuron in juvenile lobster were 2.7-fold higher in the high dose (282 ng/g) compared to the low dose treatment (103 ng/g). The transcriptional examination showed significant effects of teflubenzuron on 21 out of 39 studied genes. At the transcriptional level, environmentally relevant levels of the anti-salmon lice drug impacted genes linked to drug detoxification (cyp3a, cyp6a2, cyp302a, sult1b1, abcc4), cellular stress (hsp70, hsp90, chh), oxidative stress (cat, gpx3) and DNA damage (p53), as well as molting and exoskeleton regulation (chi3l1, ecr, jhl1, chs1, ctbs, gap65, jhel-ces1) in claw tissue (muscle and exoskeleton). In conclusion, teflubenzuron at sub-lethal levels can affect many molecular mechanisms in European lobster claws.

Mortality and deformities in European lobster (Homarus gammarus) juveniles exposed to the anti-parasitic drug teflubenzuron.

This study describes experiments carried out to examine effects of the antiparasitic drug teflubenzuron, used in delousing farmed salmon, on a non-target species, the European lobster (Homarus gammarus). Juvenile lobsters were fed two doses of teflubenzuron, 10 and 20mg/kg successively for 7 days corresponding to a standard medication of the fish (10mg/kg day) and twice the standard dose (20mg/kg day). Monitoring lasted 3 months to include at least one moulting period for all individuals. Cumulative mortality was higher in all replicates given medicated feed compared with the control group. Mean cumulative mortality for each dosing was 41 ± 13% for 10mg/kg and 38 ± 8% for 20mg/kg, i.e. no difference. Drug residue was analysed in all juveniles that died, in addition to 12 juveniles at day 8 and the first 12 surviving lobsters. A decline in concentration of teflubenzuron from over 8,000 ng/g (day 5) to 14 ng/g (day 70) was observed in the juveniles that died during the experiment. Twelve individuals that died contained 82 ng/g or less whereas the mean concentration in the first 12 lobsters that survived moulting was 152 ng/g. Following a single oral administration, the half-life of teflubenzuron in lobster was estimated to 3.4 days and the initial concentration (C0) to 515 ng/g at time t0. At the end of the study a considerable number of juvenile lobsters were observed with deformities in various organs; carapace, walking legs, cheliped, tail fan, abdomen and antenna. The occurrence of observed deformities varied from 0 to 15% in treated replicates and will most likely affect ability to locate and consume food (antenna, claw and walking legs), respiration (carapace) and ability to move/swim (walking legs, tail fan and abdomen). In total, the mortality and senescent damages were close to 50% in all replicates. Juveniles that survived medication without deformities however, moulted and increased in size at each moult equally well as the unmedicated controls.