High level efficacy of lufenuron against sea lice (Lepeophtheirus salmonis) linked to rapid impact on moulting processes

Abstract

Drug resistance in the salmon louse Lepeophtheirus salmonis is a global issue for Atlantic salmon aquaculture. Multiple resistance has been described across most available compound classes with the exception of the benzoylureas. To target this gap in effective management of L. salmonis and other species of sea lice (e.g. Caligus spp.), Elanco Animal Health is developing an in-feed treatment containing lufenuron (a benzoylurea) to be administered prior to seawater transfer of salmon smolts and to provide long-term protection of salmon against sea lice infestations. Benzoylureas disrupt chitin synthesis, formation, and deposition during all moulting events. However, the mechanism(s) of action are not yet fully understood and most research completed to date has focused on insects. We exposed the first parasitic stage of L. salmonis to 700 ppb lufenuron for three hours and observed over 90% reduction in survival to the chalimus II life stage on the host, as compared to vehicle controls. This agrees with a follow up in vivo administration study on the host, which showed >95% reduction by the chalimus I stage. Transcriptomic responses of salmon lice exposed to lufenuron included genes related to moulting, epithelial differentiation, solute transport, and general developmental processes. Global metabolite profiles also suggest that membrane stability and fluidity is impacted in treated lice. These molecular signals are likely the underpinnings of an abnormal moulting process and cuticle formation observed ultrastructurally using transmission electron microscopy. Treated nauplii-staged lice exhibited multiple abnormalities in the integument, suggesting that the coordinated assembly of the epi- and procuticle is impaired. In all cases, treatment with lufenuron had rapid impacts on L. salmonis development. We describe multiple experiments to characterize the efficacy of lufenuron on eggs, larvae, and parasitic stages of L. salmonis, and provide the most comprehensive assessment of the physiological responses of a marine arthropod to a benzoylurea chemical.

Keywords: Aquaculture; Benzoylurea; Lufenuron; Moulting; Sea lice; Transcriptomics.

Graphical abstract

Fig. 1

Effects of lufenuron on L. salmonis infection. Lice counts were completed for two separate infection experiments between 100 and 150° days post lice infection (ddpi) of Atlantic salmon (Salmo salar). The first experiment (left plot; see B2), lice were pre-treated with 700 ppb lufenuron or acetone controls for 3 h before infecting salmon (n = 6) with 500 copepodids per fish. In the second experiment (right plot, see section 2.3) used salmon fed lufenuron at the recommended dose of 35 mg/kg or a control feed diet over 7 days. Five weeks post lufenuron cessation, fish (n = 640) were infected with 100 copepodids per fish. Letters denote significant differences in total live larval counts (t-test; p < 0.05).

Fig. 2

Development of Lepeophtheirus salmonis following eggstring/nauplii I treatment with seawater (SW), SW + acetone (Control), or seawater + acetone + 500 ppb lufenuron (Lufenuron; B7). Lice development was assessed at 7 days post hatch as an average lice number for each stage enumerated per 5 mL count (completed in triplicate per system). Mobile copepodid lice are represented in red while immobile nauplii I-staged lice are represented in yellow. Letters denote significant differences in total live larval counts using a one-way ANOVA (p < 0.05). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3

Principal Component Analysis (PCA) of 21 samples represented by the expression of 16,259 probes passing QC filters. Each square represents a pool of approximately 500 copepodids exposed to seawater alone, acetone alone, or acetone and lufenuron (30–1500 ppb) for 3 h before a 21 h holding period in seawater, at which time lice were flash frozen and stored for RNA extractions.

Fig. 4

Chitin synthesis pathway derived from Drosophila melanogaster (KEGG: dme00520) showing genes are conserved across insects and copepods. The gene name provided by KEGG is on top followed by the gene name for sea lice (taken from microarray annotation) and the abbreviated gene name on bottom with the corresponding e-value derived from a BLASTx search using the D. melanogaster sequence against the TSA database (NCBI) limited to L. salmonis sequences. Those sequences highlighted in red were differentially expressed in at least one condition. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 5

Lepeophtheirus salmonis expression (calibrated normalized relative quantity – CNRQ) of phosphoacetylglucosamine mutase showing a concentration-dependent induction under lufenuron exposure (see B3). CNRQ calculated against ef1α and eif4 reference genes. Letters denote significant differences in expression between conditions (p < 0.05)?

Fig. 6

Heatmap of chitin/cuticle-related transcripts differentially expressed by at least one concentration of lufenuron. Hierarchical clustering (Euclidian distance metric; Ward's linkage rule) clustered transcripts based on averaged expression profiles in each lufenuron treatment. Expression is represented as Log2 Cy5/Cy3 ratios.

Fig. 7

Heatmap of correlation coefficients (Pearson's r) derived from a pairwise comparison (all against all) of cuticle/chitin-related transcripts differentially expressed by at least one concentration of lufenuron. Each transcript, represented by a probe ID (see Additional File 1), is plotted once on the x-axis and y-axis. An example of a negative (red) and positive (green) correlation is represented in the scatterplots on the right (blue and pink, respectively). All correlation coefficients from this analysis can be viewed in Additional File 4. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 8

Images of control Lepeophtheirus salmonis nauplii II integument and underlying tissue during apolysis using electron microscopy. A-B) 10,000× magnification with scale bar = 2 μm. C-D) 60,000× magnification with scale bar = 500 nm. Abbreviations: Old cuticle (Oc), new procuticle (P), epidermis (Ep), vesicle aggregates (V), old epicuticle (Oe), old procuticle (Op), exuvial cleft (Ec), new epicuticle (E), transitional procuticle (Tp), apical membrane (A), and diffuse substance beneath Op in the Ec (black star).

Fig. 9

Images of Lepeophtheirus salmonis nauplii II integument and underlying tissue during apolysis 24 h post lufenuron treatment using electron microscopy. A) 10,000× magnification with scale bar = 2 μm, B) 20,000× magnification with scale bar = 2 μm C) 60,000× magnification with scale bar = 500 nm, D) 120,000× magnification with scale bar = 100 nm. Abbreviations: Old cuticle (Oc), epithelium (Ep), vesicle (V), secondary membrane separating electron dense epithelium (white arrow heads), exuvial cleft (Ec), procuticle (P), poorly defined epicuticle and transitional procuticle (E/Tp), old procuticle (Op), diffuse substance beneath Op in the Ec (black star), electron-dense focii (white stars), apical membrane (A), and old epicuticle (Oe).

Fig. 10

Images of control Lepeophtheirus salmonis copepodid integument using electron microscopy. A) 10,000× magnification with scale bar = 2 μm B) 60,000× magnification with scale bar = 500 nm, C) 30,000× magnification with scale bar = 500 nm. Abbreviations: Epicuticle (E), procuticle (P), epithelium (Ep), electron-dense band separating cuticle and Ep (black arrow heads), exocuticle (Exo), endocuticle (Endo), and vesicles (V).

Fig. 11

Images of Lepeophtheirus salmonis copepodid integument 48 h post lufenuron treatment using electron microscopy. A) Dead, deformed copepodid at 80,000× magnification with scale bar = 500 nm. An ecdysial membrane (Em) was observed in treated lice that had arrested moulting. The epicuticle (E) often had several microridges, B) Mobile copepodid with region of completed moult at 10,000× magnification with scale bar = 2 μm. The procuticle (P) is well adhered to the epithelium (Ep), C) 120,000× magnification with scale bar = 100 nm showing mobile treated copepodid integument with an electron-lucid endocuticle (Endo), an unorganized epicuticle (E), and a poorly defined band between the cuticle and epidermis (black arrow head). The exocuticle (Exo) appeared normal.