Appendix A. Color photos corresponding to Fig. 3.

Color photos corresponding to Fig. 3

Metabarcoding improves detection of eukaryotes from early biofouling communities: implications for pest monitoring and pathway management

<p>In this experimental study the patterns in early marine biofouling communities and possible implications for surveillance and environmental management were explored using metabarcoding, <i>viz.</i> 18S ribosomal RNA gene barcoding in combination with high-throughput sequencing. The community structure of eukaryotic assemblages and the patterns of initial succession were assessed from settlement plates deployed in a busy port for one, five and 15 days. The metabarcoding results were verified with traditional morphological identification of taxa from selected experimental plates. Metabarcoding analysis identified > 400 taxa at a comparatively low taxonomic level and morphological analysis resulted in the detection of 25 taxa at varying levels of resolution. Despite the differences in resolution, data from both methods were consistent at high taxonomic levels and similar patterns in community shifts were observed. A high percentage of sequences belonging to genera known to contain non-indigenous species (NIS) were detected after exposure for only one day.</p

Hydroids on net sample attached to a frame for high-pressure cleaning.

<p>Hydroids on net sample attached to a frame for high-pressure cleaning.</p

Effects of cnidarian biofouling on salmon gill health and development of amoebic gill disease

<div><p>This study examines the potential implications of biofouling management on the development of an infectious disease in Norwegian farmed salmon. The hydroid <i>Ectopleura larynx</i> frequently colonises cage nets at high densities (thousands of colonies per m²) and is released into the water during regular <i>in-situ</i> net cleaning. Contact with the hydroids’ nematocysts has the potential to cause irritation and pathological damage to salmon gills. Amoebic gill disease (AGD), caused by the amoeba <i>Paramoeba perurans</i>, is an increasingly international health challenge in Atlantic salmon farming. AGD often occurs concomitantly with other agents of gill disease. This study used laboratory challenge trials to: (1) characterise the gill pathology resulting from the exposure of salmon to hydroids, and (2) investigate if such exposure can predispose the fish to secondary infections–using <i>P</i>. <i>perurans</i> as an example. Salmon in tanks were exposed either to freshly ‘shredded’ hydroids resembling waste material from net cleaning, or to authentic concentrations of free-living <i>P</i>. <i>perurans</i>, or first to ‘shredded’ hydroids and then to <i>P</i>. <i>perurans</i>. Gill health (AGD gill scores, non-specific gill scores, lamellar thrombi, epithelial hyperplasia) was monitored over 5 weeks and compared to an untreated control group.</p><p>Nematocysts of <i>E</i>. <i>larynx</i> contained in cleaning waste remained active following high-pressure cleaning, resulting in higher non-specific gill scores in salmon up to 1 day after exposure to hydroids. Higher average numbers of gill lamellar thrombi occurred in fish up to 7 days after exposure to hydroids. However, gill lesions caused by hydroids did not affect the infection rates of <i>P</i>. <i>perurans</i> or the disease progression of AGD. This study discusses the negative impacts hydroids and current net cleaning practices can have on gill health and welfare of farmed salmon, highlights existing knowledge gaps and reiterates the need for alternative approaches to net cleaning.</p></div

Scanning electron microscope images of a hydroid tentacle and nematocysts.

<p>a) Close-up of a tentacle of <i>E</i>. <i>larynx</i>, showing cnidocilia of undischarged nematocysts protruding the surface, ready to discharge on contact. b) Two discharged stenotele nematocysts (identified according to Östmann et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0199842#pone.0199842.ref038" target="_blank">38</a>]) found after triggering release with acetic acid.</p

Descriptive and numeric scores corresponding to non-specific gill lesioning and AGD pathology, adapted from Taylor et al. [34].

<p>Descriptive and numeric scores corresponding to non-specific gill lesioning and AGD pathology, adapted from Taylor et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0199842#pone.0199842.ref034" target="_blank">34</a>].</p

Gill filament of Atlantic salmon showing a lamellar thrombus after exposure to hydroids (H&E and MSB stained).

<p>Gill filament of Atlantic salmon showing a lamellar thrombus after exposure to hydroids (H&E and MSB stained).</p

Experimental schedule showing sampling events (grey arrows) and numbers of sampled fish.

<p>Exposure to hydroids <i>Ectopleura larynx</i> ("H") took place one day post sampling of the naïve fish; exposure to <i>Paramoeba perurans</i> ("PP") took place one day post hydroid exposure (dphe). In addition, the distribution of the four treatment groups (C = Control, H = Hydroids, PP = <i>P</i>. <i>perurans</i>, H + PP = Hydroids + <i>P</i>. <i>perurans</i>) over the eight experimental tanks is shown. The numbers of sampled fish refer to each tank during each sampling event.</p