*3.2. Sea Trials*

3.2.1. *L. salmonis* Adult and Pre-Adult Sensory Setae Morphology

In adult and pre-adult specimens (length 9.9 ± 1; width 4.5 ± 0.2), the first antenna (Figure 12) presents a proximal segment with 27 setae (25 pinnate on ventral and 2 unramed on dorsal surface) and a distal segment bearing 1 seta on posterior margin and 11 setae and 2 aesthetes at apex. Some setae are visible on maxilas too. The five pairs of legs present unramed setae, pinnate setae, spines and rows of simple setules. Caudal ramus shows distal setae with distinctly inflated bases that are relatively short.

**Figure 12.** SEM. Adult and pre-adult *L. salmonis* morphology. Control animals: (**A**) Ventral view of the whole body of a pre-adult; (**B**) Ventral anterior view of an adult; (**C**) First antenna of an adult *L. salmonis*; (**D**) Mouth of a pre-adult; (**E**) Distal segment of the first antenna of *L. salmonis* adult; (**F**) Caudal ramus showing the distal setae; (**G**) Ventral arm showing the distribution of the pinnate setae. Scale bar: (**A**) = 2 mm; (**B**,**D**,**G**) = 500 μm; (**F**) = 300 μm; (**C**) = 100 μm; and (**E**) = 50 μm.

3.2.2. Ultrastructural Analysis of Pre-Adult and Adult First Antenna Setae after Noise Exposure

Ultrastructural changes took place on setae in adults and pre-adults of *L. salmonis* following acoustic exposure. Damaged setae were either extruded, completely missing, or presented flaccid, fused, or missing sensory hairs.

After two weeks of sound exposure (Figure 13B,D,E,G and Figure 14B–E) in comparison with the same tissues from control animals (Figures 13A and 14A), damage was observed on the first antenna setae. The setae of the proximal segment of the first antenna presented sensory hairs flaccid, fused or showed blebs. Some setae had lost part of the sensory hairs. Sometimes the setae had lost their rigidity and appeared unstructured and almost empty. Some animals presented setae partially or completely ejected above the antenna surface (Figures 13G and 14E).

**Figure 13.** SEM. Pre-adult *L-salmonis* setae on proximal segment of first antenna: (**A**,**C**,**H**) Control animals; (**B**,**D**–**G**,**I**) animals after 2 weeks of sound exposure on sea trials. (**A**) Image of healthy setae bearing organized sensory hairs (arrow). (**B**) Setae showing flaccid or fused sensory hairs. Some of them have almost entirely lost the sensory hairs (arrows). (**C**) Pinnate setae on ventral arms presenting normal aspect (arrow). Insert in (**C**), lateral fraction of ventral arms presenting sensory hairs with normal aspect (arrow). (**D**) Section of proximal segment of the first antenna showing all the setae bearing bend and flaccid sensory hairs (arrow). (**E**) Sensory hairs showing blebs (arrowheads). (**F**) Pinnate setae on ventral arms are fused. (**G**) Setae partially ejected (arrowheads) above the antenna surface. (**H**) Normal aspect of the sensory hairs in the distal setae on caudal ramus. (**I**) Caudal ramus has partially lost the sensory hairs. Scale bar: (**H**) = 300 μm; (**C**,**I**); Insert in (**C**) = 100 μm; (**F**) = 50 μm; (**A**) = 30 μm; (**B**,**D**,**G**) = 20 μm; and (**E**)=5 μm.

**Figure 14.** SEM. Adult *L. salmonis* setae on proximal segment of first antenna: (**A**) Control animals; (**B**–**F**): animals after two weeks of sound exposure on sea trials. (**A**) Image of healthy setae bearing organized sensory hairs (arrow). (**B**) Setae showing flaccid or fused sensory hairs. Some of them have almost lost totally the sensory hairs (arrows). (**C**) Some of the setae have almost lost totally the sensory hairs (arrows). (**D**) Sensory hairs showing blebs (arrowheads). (**E**) Two setae are partially (arrows) or totally ejected (arrowhead) above the antenna surface. (**F**) Pinnate setae on ventral arms are fused (arrows). Scale bar: (**A**) = 500 μm; (**B**,**C**,**E**,**F**) = 30 μm; and (**D**)=5 μm.

In some specimens, in addition to the lesions on the first antenna, we found some effects on setae located in other positions. The arms showed sections of different lengths of fused pinnate setae (Figures 13F and 14F). Some specimens presented loss of sensory hairs or broken bases of the distal setae on caudal ramus (Figure 13I).

3.2.3. Ultrastructural Analysis of Chalimus, Pre-Adult and Adult *L. salmonis* Inner Tissues after Sound Exposure

Sound effects were observed in chalimus, pre-adult, and adult stages in comparison with the control animals. All noise-exposed individuals presented a similar variety of ultrastructural changes in cells of the inner tissues surrounding the eyes (in chalimus, specifically in cells involved in frontal filament production, namely A and B cells) and in axons of neurons around the eyes. In cell cytoplasm, ultrastructural changes were essentially a massive accumulation of dark inclusions (including ribosomes), the presence of numerous large double-membrane bounded autophagic vacuoles and of numerous lysosomes, and vacuolization of the cell cytoplasm (Figures 15–20). In addition, some B cells showed a deformed cell nucleus and chromatin compaction into the nucleus (Figure 15).

In the axons of neurons around the eyes, prominent ultrastructural changes were the presence of both abundant double-membrane-bounded autophagic vacuoles and myelinlike formations (Figures 15–18 and 21), and the accumulation of lysosomes at different stages of evolution (Figures 19 and 21). Large areas of empty cytoplasm (Figures 16–18, 20 and 21) and the accumulation of dark inclusions including ribosomes were also a common feature in these nervous cells (Figures 15, 17 and 18).

**Figure 15.** (**A**) Light microscopy; (**B**–**F**) TEM. Frontal medial section of a chalimus stage of sea lice; (**A**–**C**) Control specimens; (**D**–**F**) 3 weeks exposed specimens. (**A**) In light microscopy images of control animals there are not specially stained areas (corresponding to dark inclusions in TEM). Arrowhead shows the eyes. (**B**) Eyes of a control animal. The axons of the central nervous system surrounding the eye did not show dark inclusions (arrow). (**C**) Normal B cells nucleus (yellow arrowheads) in a control animal. (**D**) Dark inclusions in A cell cytoplasm are visible (arrows). (**E**) A large section of the cytoplasm of A cells are filled with ribosomes (dark inclusions, arrow). (**F**) By comparison to C, the B cell nuclei on exposed animals presented irregular shapes and chromatin compaction (yellow arrowheads). Scale bar: (**A**) = 200 μm; (**B**) = 20 μm; (**C**,**D**)=5 μm; (**E**,**F**)=2 μm.

**Figure 16.** TEM. Frontal medial section of a chalimus anterior cephalothorax: (**A**,**F**) Control specimens; (**B**–**E**,**G**) 3 weeks exposed specimens. (**A**) There are no dark inclusions visible in the central nervous system axons (yellow arrowhead) neighbours to the eye. (**B**) One axon of the central nervous system shows ribosome accumulation (arrow) and presence of double-membrane-bounded autophagic vacuoles (arrowheads). (**C**,**E**) "Myelin-like formations" (red arrowhead). (**D**) Double-membrane-bounded autophagic vacuoles (arrowheads). (**F**) Normal aspect of the tissue located next to the B cells. (**G**) the tissue shows a process of vacuolization (yellow arrowheads). Scale bar: (**B**,**G**) = 10 μm; (**F**)=5 μm; (**A**,**C**,**D**) = 2 μm; (**E**)=1 μm.

**Figure 17.** TEM. Frontal medial section of a chalimus anterior cephalothorax: (**A**,**E**) Control specimens; (**B**,**C**,**D**,**F**) Six weeks exposed specimens. (**A**) Normal aspect of the central nervous system between the two eyes. (**B**) By comparison to A, large dark inclusions (black arrowheads) are visible in the central nervous system between the two eyes. (**C**) "Myelin-like formations" (red arrowheads) and double-membrane-bounded autophagic vacuoles (yellow arrowheads) are present in axons. (**D**) Detail of "myelin-like formations" in an axon (red arrowhead). (**E**) Normal lysosomes (white asterisks) next to the B cell nuclei. (**F**) Some degraded lysosomes (white asterisks) are visible in a B cell of an exposed animal. "Myelin-like formations" (red arrowhead) and autophagic vacuoles (yellow arrowhead) are present. Note the empty cytoplasm in some areas of the tissue (black asterisks). Scale bar: (**A**–**C**,**E**,**F**)=2 μm; (**D**) = 1000 nm.

**Figure 18.** TEM. Frontal medial section of a pre-adult anterior cephalothorax showing tissues located around the eyes: (**A**) Control specimens; (**B**,**C**,**D**) Exposed specimens. (**A**) Normal aspect of cells in tissue surrounding the eyes. No dark inclusions are visible. (**B**) A large section of the cytoplasm is filled with ribosomes (dark inclusions, arrow). (**C**) One axon filled with dark inclusions (arrow) is adjacent located to one normal axon. (**D**) In the cytoplasm of an axon note the accumulation of ribosomes (dark inclusions, arrows) and the presence of autophagic vacuoles (arrowheads). Scale bar: (**C**) = 10 μm; (**A**)=2 μm; (**B**)=1 μm; (**D**) = 500 nm.

**Figure 19.** TEM. Frontal medial section of an exposed pre-adult anterior cephalothorax. Exposed animal: (**A**) General view of a section of tissue showing different features on sound exposed cells (Details in **B**–**D**); (**B**) Two adjacent cells are visible. On the right a normal cell shows its nucleus with inner nucleolus. On the left (arrow) a sound affected cell shows organelles destroyed by an enzymatic process; (**C**) Detail from (**B**,**D**), presence of very dark lysosomes (asterisks). Scale bar: (**A**) = 10 μm; (**D**)=5 μm; and (**B**,**C**)=2 μm.

**Figure 20.** TEM. Sagittal section of adult sea lice anterior cephalothorax: (**A**–**C**) control animals; (**D**,**E**) Exposed animals; (**A**–**C**) No abnormal features are visible. (**D**) Cells present large empty areas in the cytoplasm (asterisks). Red triangles point to "myelin-like formations". (**E**) Detail from D. Note the double-membrane-bounded autophagic vacuoles (yellow arrowheads), the empty areas of cytoplasm (asterisk), the "myelin-like formations" (red arrowheads) and ribosome accumulation (arrow). Scale bar: (**A**) = 10 μm; (**B**–**D**)=2 μm; (**E**)=1 μm.

**Figure 21.** TEM. Frontal medial section of exposed adult anterior cephalothorax showing tissues located around the eye. (**A**) Arrows point to cells of the central nervous tissue filled with dark inclusions (ribosomes). (**B**) Detail from the optic nerves (ON) showing ribosome accumulation (arrows). (**C**) The axons of the central nervous system between the two eyes present some large dark inclusions (arrowheads). (**D**,**E**) Cells of the central nervous system shows autophagic vacuoles (yellow arrowheads), empty areas of cytoplasm (asterisks), "myelin-like formations" (red triangles) and ribosome accumulation (arrows). (**F**) The large amount of lysosomes type 3 suggest the evolution sequence of lysosomes going from the darkest (1) to lightest (3) appearance probably linked to sustained autophagy. Yellow arrowhead points to a type 3 lysosome releasing their inner content to a next autophagosoma. Scale bar: (**C**,**F**) = 10 μm; (**A**,**B**,**D**,**E**)=2 μm.

#### **4. Discussion**

The sea louse *L. salmonis* causes millions of dollars in commercial losses to the salmon aquaculture industry globally. It reduces the productivity at fish farms through either low feed efficiency or growth reduction of the fish. In addition to such an industrial problem, it was recently shown that lice from salmon farms can have an adverse impact on wild migratory salmonids by increasing the abundance of this parasite in bays and estuaries adjacent to the farms [1].

Different methods have been used in the fight against the sea lice infestation. In-feed treatments and usage of skirts (sheets hung around the salmon cages to prevent sea lice from entering) are very expensive methods. Skirts have low impact on the salmon welfare and the environment but reduce oxygen flow, which may cause a detrimental effect on fish respiratory functions [18].

Other methods such as cleaner fish, fresh water, physical removal measures, and veterinary medicines (sea lice have built up resistance to most of the chemicals that are used in medicines [19]) have environmental, health, and welfare impacts [19]. Less costeffective methods include the use of hydrogen peroxide baths that, in addition, have effects on fish welfare and, consequently, on environmental and human health. From this perspective, the complexity of sea lice control requires a global holistic approach [18].

In this context, sound exposure methods can constitute an effective, innovative, and promising technology to address sea lice infestation. Our results showed the first ultrastructural images that characterize pathological changes in copepodids, chalimus, adult and pre-adults *L. salmonis* sensory first antenna setae after sound exposure. Essentially, the lesions were partial or complete fusions of the setae irregular branching tips of the first antenna. Fusion of fine sensory structures is typically the result of mechanical constraints due loud sound vibrations. Fusion of stereocilia has been shown to occur in statocysts sensory epithelium and lateral line systems of cephalopods [17,20] and in statocyst of cnidarians [14] after underwater noise exposure. Moreover, stereocilia fusion on auditory hair cells is a morphological characteristic of acoustic trauma in terrestrial animals [21,22]. Such pathological changes that directly affect the main *L. salmonis* sensory organ could make difficult finding of a host for a copepodid [8,9]. Additionally, exposed sea lice showed lesions on some distal pinnate setae on the caudal ramus and ventral arms. These abnormalities could provoke difficulties for the sea lice to move around the fish, which could also contribute to the decrease in the number of attachments to the salmons.

Moreover, to the best of our knowledge, this study shows the first published ultrastructural images of sea lice inner tissues affected by sound. The exposure affected the central nervous system of all analysed stages, likely altering their normal behaviour and challenging their survival as has been shown in other invertebrates [10,14]. In the copepodids and chalimus stages the A/B cells, which are responsible for the secretion of the precursor of frontal filament [7], were affected, thus probably challenging a correct anchoring to the fish. Similar lesions were shown on the tissues located in the same regions in both adults and pre-adults. In all of the stages, those lesions appear to be produced by autophagic and apoptotic processes [23–25].

Typical features of autophagic processes include the presence of numerous lysosomes and double-membrane-bounded vacuoles, "myelin-like formations" resulting from cell membrane destruction, large aggregates of dark material, and massive accumulation of ribosomes in the cell cytoplasm. Interestingly, in our exposed tissues we could follow the normal evolution sequence from primary to mature secondary lysosome with decreasing activity, which posteriorly released its content to an adjacent autophagosome [26,27]. The presence of a large amount of secondary lysosomes and residual bodies in the exposed animals is a clear sign of the extreme functioning of the cytological mechanisms caused by the stress situation originated after sound exposure. Moreover, the frequent presence of large areas of empty cytoplasm was the hallmark of advanced stages of cell degeneration through autophagy. Beside autophagy, pathological features such as deformed cell nuclei, chromatin compaction into the nuclei, and cytoplasm condensation strongly suggest the occurrence of apoptotic processes.

The mechanisms by which sound induced massive autophagy and apoptosis in the present specimens, have yet to be precisely determined. One possible hypothesis is that acoustic exposure primarily induced an oxidative stress that is known to regulate the expression of both autophagy and apoptosis [28]. In support of the oxidative stress hypothesis, the accumulation of dark inclusions around the eye could correspond to mitochondrial autophagic profiles. Mitochondria in fact play a key role in oxidative stress [29–31], and autophagy-damaged mitochondria has been described as the effect of sound exposure in the optic nerve [22]. Another possibility is that part of the dark inclusions was due to hyperpigmentation. Hyperpigmentation actually may occur as a consequence of an oxidative stress following acoustic trauma in several tissues, including the eyes [32].

Altogether, the present study indicates that the central nervous system in all stages and the A/B cells (responsible for the secretion of the precursor of frontal filament) [7] in copepodids and chalimus stages were affected by sound exposure. These encouraging findings therefore indicate that sound exposure can lead to severe consequences on the capacity of the sea lice to infest its host. The present results were completed by an exhaustive health status analysis of the exposed salmons [33] that showed that the use of these frequency combinations did not affect the fish. In this assessment through gross pathology and histopathological analysis, salmons didn't show any lesion that could be related to sound exposure. In addition, the analysis of the otolith's organs did not show any effects on the auditory organs of the fish. Although some consequences of the sound-induced lesions found in lice remain to be further studied, this method constitutes a promising approach to address lice plagues while also reducing the need for chemical treatments of the fish.

#### **5. Patents**

André M., Solé M., Van der Schaar, De Vreese S (International Patent WO 2018/167003 A1). 20-09-2018. A method for inducing lethal lesions in sensory organs of undesirable aquatic organisms by use of sound. Licensed to SEASEL SOLUTIONS AS [NO/NO]; P.O. BOX 93 N-6282 BRATTVÅG (NO).

André M., Solé M., Van der Schaar (Norwegian patent WO/2020/048945). 03-09-2019. System and method for reducing sea lice exposure in marine fish farming. Licensed to SEASEL SOLUTIONS AS [NO/NO]; P.O. BOX 93 N-6282 BRATTVÅG (NO).

**Author Contributions:** M.S. and M.A. planned the research and designed the study; M.S., M.A., M.v.d.S. and S.D.V. conducted experimental/lab work; M.S. and M.A. conducted sea trial work; M.S. and J.-M.F. performed SEM analysis; M.S. performed TEM analysis; M.S., M.L. and M.A. analysed the data; M.S. and M.A. prepared the figures; M.S. and M.A. wrote the article. All authors reviewed the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** Funding for this project was provided by SEASEL SOLUTIONS AS. Project: *An acoustic and Bioacoustic solution to sea lice infestation on salmon* P.O. BOX 93 N-6282 BRATTVÅG. Norway.

**Institutional Review Board Statement:** Although there are no legal requirements for studies involving crustaceans in Spain, the experimental protocol strictly followed the necessary precautions to comply with current ethical and welfare considerations when dealing with animals in scientific experimentation (Royal Decree 1386/2018, of 19 November). This process was also carefully analysed and approved by the Ethical Committee for Scientific Research of the Technical University of Catalonia, BarcelonaTech (UPC) (approval code B9900085).

**Informed Consent Statement:** Not applicable.

**Acknowledgments:** We would like to thank the staff of Mowi Fish Feed A/S Avd Averøy for their assistance and helpful cooperation during the experiments at sea. Special thanks to Josep M. Rebled, Eva Prats, Adriana Martínez, and Rosa Rivera (Unitat de microscòpia electrònica, Hospital Clínic, Universitat de Barcelona) for assistance and advice in obtaining TEM images.

**Conflicts of Interest:** The authors declare no conflict of interest.
