Effects of chitin synthesis inhibitor treatment on Lepeophtheirus salmonis (Copepoda, Caligidae) larvae

The salmon louse (Lepeophtheirus salmonis) is an ectoparasite infecting Atlantic salmon (Salmo salar), which causes substantial problems to the salmon aquaculture and threatens wild salmon. Chitin synthesis inhibitors (CSIs) are used to control L. salmonis in aquaculture. CSIs act by interfering with chitin formation and molting. In the present study, we investigated the action of four CSIs: diflubenzuron (DFB), hexaflumuron (HX), lufenuron (LF), and teflubenzuron (TFB) on larval molt. As the mode of action of CSIs remains unknown, we selected key enzymes in chitin metabolism and investigated if CSI treatment influenced the transcriptional level of these genes. All four CSIs interfered with the nauplius II molt to copepodids in a dose-dependent manner. The EC50 values were 93.2 nM for diflubenzuron, 1.2 nM for hexaflumuron, 22.4 nM for lufenuron, and 11.7 nM for teflubenzuron. Of the investigated genes, only the transcriptional level of L. salmonis chitin synthase 1 decreased significantly in hexaflumuron and diflubenzuron-treated larvae. All the tested CSIs affected the molt of nauplius II L. salmonis larvae but at different concentrations. The larvae were most sensitive to hexaflumuron and less sensitive to diflubenzuron. None of the CSIs applied had a strong impact on the transcriptional level of chitin synthesis or chitinases genes in L. salmonis. Further research is necessary to get more knowledge of the nature of the inhibition of CSI and may require methods such as studies of protein structure and enzymological studies.publishedVersio

Chitin synthases are critical for reproduction, molting, and digestion in the salmon louse (Lepeophtheirus salmonis)

Chitin synthase (CHS) is a large transmembrane enzyme that polymerizes Uridine diphosphate N-acetylglucosamine into chitin. The genomes of insects often encode two chitin synthases, CHS1 and CHS2. Their functional roles have been investigated in several insects: CHS1 is mainly responsible for synthesizing chitin in the cuticle and CHS2 in the midgut. Lepeophtheirus salmonis is an ectoparasitic copepod on salmonid fish, which causes significant economic losses in aquaculture. In the present study, the tissue-specific localization, expression, and functional role of L. salmonis chitin synthases, LsCHS1 and LsCHS2, were investigated. The expressions of LsCHS1 and LsCHS2 were found in oocytes, ovaries, intestine, and integument. Wheat germ agglutinin (WGA) chitin staining signals were detected in ovaries, oocytes, intestine, cuticle, and intestine in adult female L. salmonis. The functional roles of the LsCHSs were investigated using RNA interference (RNAi) to silence the expression of LsCHS1 and LsCHS2. Knockdown of LsCHS1 in pre-adult I lice resulted in lethal phenotypes with cuticle deformation and deformation of ovaries and oocytes in adult lice. RNAi knockdown of LsCHS2 in adult female L. salmonis affected digestion, damaged the gut microvilli, reduced muscular tissues around the gut, and affected offspring. The results demonstrate that both LsCHS1 and LsCHS2 are important for the survival and reproduction in L. salmonis.publishedVersio

A comparison of two ketamine doses for field anaesthesia in horses undergoing castration

Background: Ketamine at 2.2 mg/kg given i.v. is often used to induce anaesthesia for surgical procedures in horses under field conditions. Commonly, additional doses are needed to complete the surgery. We hypothesised that surgical conditions would be improved when 5 mg/kg of ketamine was used to induce anaesthesia, while induction and recovery qualities would not differ from those when 2.2 mg/kg ketamine was used. Objective: To compare the anaesthetic effects of two ketamine doses (5 and 2.2 mg/kg) during field anaesthesia for castration of horses. Study design: Prospective, randomised, blinded, clinical study. Method: Seventy-seven client-owned Icelandic horses presented for castration under field conditions were studied. Pre-anaesthetic medication was xylazine (0.7 mg/kg) butorphanol (25 μg/kg) and acepromazine (50 μg/kg) injected i.v. Anaesthesia was induced with either 2.2 mg/kg (K2.2) or 5 mg/kg (K5) i.v. of ketamine mixed with diazepam (30 μg/kg). The quality of induction, surgical conditions and recovery were compared using subjective and objective measures, and the number of additional ketamine doses recorded. Results: Ketamine 5 mg/kg provided better surgical conditions and a more rapid induction. Recovery quality was subjectively better in K2.2. Five horses in K2.2 and two in K5 required additional ketamine doses. Main limitations: While the pre-anaesthetic sedation and benzodiazepine doses were consistent among horses, the level of sedation and muscle relaxation achieved differed. Conclusion: A ketamine dose of 5 mg/kg can be used to improve the quality of field anaesthesia for castration in Icelandic horses. Although recovery quality is subjectively better when using 2.2 mg/kg, no adverse events were observed during recovery with either dos

Acute phase inflammation is characterized by rapid changes in plasma/peritoneal fluid N-glycosylation in mice.

To access publisher's full text version of this article, please click on the hyperlink in Additional Links field or click on the hyperlink at the top of the page marked Files. This article is open access.Murine zymosan-induced peritonitis is a widely used model for studying the molecular and cellular events responsible for the initiation, persistence and/or resolution of inflammation. Among these events, it is becoming increasingly evident that changes in glycosylation of proteins, especially in the plasma and at the site of inflammation, play an important role in the inflammatory response. Using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS)-based glycosylation profiling, we investigated the qualitative and quantitative effect of zymosan-induced peritonitis on N-glycosylation in mouse plasma and peritoneal fluid. Our results show that both N-glycomes exhibit highly similar glycosylation patterns, consisting mainly of diantennary and triantennary complex type N-glycans with high levels (>95 %) of galactosylation and sialylation (mostly NeuGc) and a medium degree of core fucosylation (30 %). Moreover, MS/MS structural analysis, assisted by linkage-specific derivatization of sialic acids, revealed the presence of O-acetylated sialic acids as well as disialylated antennae ("branching sialylation") characterized by the presence of α2-6-linked NeuGc on the GlcNAc of the NeuGcα2-3-Galβ1-3-GlcNAc terminal motif. A significant decrease of (core) fucosylation together with an increase of both α2-3-linked NeuGc and "branching sialylation" were observed in N-glycomes of mice challenged with zymosan, but not in control mice injected with PBS. Importantly, substantial changes in glycosylation were already observed 12 h after induction of peritonitis, thereby demonstrating an unexpected velocity of the biological mechanisms involved.Dutch Arthritis Association (Reumafonds) LLP-24 Innovative Medicines Initiative Joint Undertaking (IMI JU)/ 115142-2 Netherlands Genomic Initiative/93511033 info:eu-repo/grantAgreement/EC/FP7/278535info:eu-repo/grantAgreement/EC/FP7/27853

Chitin synthesis in the salmon louse Lepeophtheirus salmonis

The salmon louse Lepeophtheirus salmonis is a parasitic copepod that lives and feeds on Atlantic salmon (Salmo salmar). At present, this parasite is the number one threat to the Norwegian aquaculture. The density of salmons living together in aquaculture sea cages gives the salmon lice optimal conditions for infecting and reproducing. Many delousing methods, both non-chemical and chemical, are available for the treatments of salmon lice. In salmon aquaculture, four benzoylureas-derived chitin synthesis inhibitors (CSI) are used: Hexaflumuron, diflubenzuron, teflubenzuron, and lufenuron. In-feed treatments based on diflubenzuron or teflubenzuron are used by Norwegian farmers, while hexaflumuron- and lufenuron-based treatments are known to be available in Chile. Despite many options for treating lice, current treatments are not optimal because 1) resistance can develop, 2) there are environmental issues associated with chemical treatments, and 2) non-chemical treatments can inflict stress-related damage on the fish. Therefore, new treatments are needed. By investigating the molecular biology of L. salmonis, it is hoped that new candidates can be discovered. Salmon lice have a rigid exoskeleton around the body, which must be removed for future growth to occur. During the development, the salmon louse molt to get to the next life stage. Molt, or ecdysis, is an essential process in all arthropods, and the salmon louse molts eight times to reach the adult life stage. Chitin is one of the main components of the exoskeleton, and the chitin synthesis pathway is very conserved in chitin-synthesizing organisms. The key chitin synthesis transcripts (LsGFAT, LsGNA1, LsAGM, LsUAP, and LsCHS1/2) were cloned and their sequences were analyzed to understand chitin synthesis in L. salmonis better. The results showed that L. salmonis has enzymes similar to other arthropods and these are most similar to other crustaceans' sequences, especially other copepods. The expression patterns of these chitin synthesis genes and chitinases (LsChi1, LsChi2, and LsChi4) were analyzed during the synthesis of a new exoskeleton. The formation of a new exoskeleton was analyzed in pre-adult I males. LsGFAT, LsCHS1, LsCHS2, LsChi1, and LsChi2 changed significantly during the synthesis of a new exoskeleton. The expression patterns of these genes were also analyzed in the nauplii stages. LsGFAT, LsCHS1, LsCHS2, and LsChi2 increased significantly from postmolt to premolt in nauplii II larvae, while in nauplius I, no molting-related expression patterns were observed. CSIs interfere with the chitin formation, but their exact mode of action is not known. Arthropods treated with CSIs have arrested molt, incomplete exoskeleton structure, and fertility reductions in adult females. Chitin synthesis related transcripts have been shown to be little affected by CSIs treatments, and different CSIs have been shown to require different concentrations to have the same effect on the same species. To find out if L. salmonis would react similarly to CSIs, four bioassays using hexaflumuron, diflubenzuron, lufenuron, and teflubenzuron, were performed on the nauplius I stage of L. salmonis. Bioassays revealed that the effective concentration needed to affect half of the animals tested was lowest for hexaflumuron and highest for diflubenzuron. The four chemicals had similar modes of action on the phenotype (molting arrest and abnormal copepodid with bloated appearances). The CSIs had negligible or no effects on the chitin synthesis (LsGFAT, LsGNA1, LsAGM, LsUAP, and LsCHS1/2) and chitinases (LsChi1, LsChi2, and LsChi4) transcripts. In the insects, Culex pipiens and Plutella xylostella, and in a gene-modified Drosophila melanogaster, the molecular mode of action of CSIs has been shown to target chitin synthase (CHS), the last enzyme in the chitin synthesis pathway. LsCHS1 and LsCHS2 were shown to have similar functions to insect CHS1 and CHS2, respectively. LsCHS1 was the main enzyme synthesizing chitin into the exoskeleton, and it also has a role in the development of oocytes. LsCHS2 was found to have a role in digestion and perhaps also in offspring formation. In summary, we demonstrated that the key chitin synthesis enzymes in L. salmonis are similar to homologous enzymes in other crustaceans. CSIs, do not affect key chitin synthesis or chitinase transcripts, and the phenotypes obtained in CSIs-treated larvae resemble phenotypes obtained after LsCHS1 and LsCHS2 knockdowns. This study revealed that the mode of action (here defined as functional or anatomical changes on the phenotype) of CSIs in L. salmonis larvae is similar to CSI action against other arthropods. This study contributes to the fundamental knowledge of the chitin synthesis pathway

Anaesthesia management of a pug (in late stage pregnancy) with lung lobe torsion.

Anaesthetic management can be challenging when patients have multiple comorbidities. This report presents one such case: a pug with brachycephalic airway syndrome, in late gestation, presented with a lung lobe torsion. Under general anaesthesia the dog initially had low PaO2, with severe ventilation perfusion mismatch and pulmonary shunt; this was mildly improved with the patient positioned on a head-up tilted table in right lateral recumbency. Tension pneumothorax developed after initiation of positive pressure ventilation and was resolved with rapid entry into the thoracic cavity. Ventilation was adjusted with the aid of spirometry, blood gases, capnography and direct visualisation of the lung tissue. The dog made a full recovery and was discharged from hospital five days postoperatively

Effects of chitin synthesis inhibitor treatment on Lepeophtheirus salmonis (Copepoda, Caligidae) larvae

The salmon louse (Lepeophtheirus salmonis) is an ectoparasite infecting Atlantic salmon (Salmo salar), which causes substantial problems to the salmon aquaculture and threatens wild salmon. Chitin synthesis inhibitors (CSIs) are used to control L. salmonis in aquaculture. CSIs act by interfering with chitin formation and molting. In the present study, we investigated the action of four CSIs: diflubenzuron (DFB), hexaflumuron (HX), lufenuron (LF), and teflubenzuron (TFB) on larval molt. As the mode of action of CSIs remains unknown, we selected key enzymes in chitin metabolism and investigated if CSI treatment influenced the transcriptional level of these genes. All four CSIs interfered with the nauplius II molt to copepodids in a dose-dependent manner. The EC50 values were 93.2 nM for diflubenzuron, 1.2 nM for hexaflumuron, 22.4 nM for lufenuron, and 11.7 nM for teflubenzuron. Of the investigated genes, only the transcriptional level of L. salmonis chitin synthase 1 decreased significantly in hexaflumuron and diflubenzuron-treated larvae. All the tested CSIs affected the molt of nauplius II L. salmonis larvae but at different concentrations. The larvae were most sensitive to hexaflumuron and less sensitive to diflubenzuron. None of the CSIs applied had a strong impact on the transcriptional level of chitin synthesis or chitinases genes in L. salmonis. Further research is necessary to get more knowledge of the nature of the inhibition of CSI and may require methods such as studies of protein structure and enzymological studies

Effects of chitin synthesis inhibitor treatment on Lepeophtheirus salmonis (Copepoda, Caligidae) larvae

The salmon louse (Lepeophtheirus salmonis) is an ectoparasite infecting Atlantic salmon (Salmo salar), which causes substantial problems to the salmon aquaculture and threatens wild salmon. Chitin synthesis inhibitors (CSIs) are used to control L. salmonis in aquaculture. CSIs act by interfering with chitin formation and molting. In the present study, we investigated the action of four CSIs: diflubenzuron (DFB), hexaflumuron (HX), lufenuron (LF), and teflubenzuron (TFB) on larval molt. As the mode of action of CSIs remains unknown, we selected key enzymes in chitin metabolism and investigated if CSI treatment influenced the transcriptional level of these genes. All four CSIs interfered with the nauplius II molt to copepodids in a dose-dependent manner. The EC50 values were 93.2 nM for diflubenzuron, 1.2 nM for hexaflumuron, 22.4 nM for lufenuron, and 11.7 nM for teflubenzuron. Of the investigated genes, only the transcriptional level of L. salmonis chitin synthase 1 decreased significantly in hexaflumuron and diflubenzuron-treated larvae. All the tested CSIs affected the molt of nauplius II L. salmonis larvae but at different concentrations. The larvae were most sensitive to hexaflumuron and less sensitive to diflubenzuron. None of the CSIs applied had a strong impact on the transcriptional level of chitin synthesis or chitinases genes in L. salmonis. Further research is necessary to get more knowledge of the nature of the inhibition of CSI and may require methods such as studies of protein structure and enzymological studies