56 SEPTEMBER 2016 • WORLD AQUACULTURE • WWW.WAS.ORG of dark organic material under the shell with an unpleasant aspect (Fig. 5), signs that usually suggest the presence of burrowing worms and opportunistic bacteria. To identify these worms and bacteria, conventional taxonomy and DNA analyses were carried out. Study Methods Thirty-three red abalone with external shell lesions were collected from an aquaculture facility in spring 2015. Soft tissues were detached from the shell and the conchioline layer was carefully removed with a needle (Fig. 6). Dark organic material and detritus behind this layer was observed and several worms remained among this material (Fig. 7). Worms were carefully collected with dissection clips and a needle (Fig. 8). Twelve of the best preserved worms were identified with specialized taxonomic keys. A fraction of worm tissue was collected for DNA analysis using the 18S rDNA gene sequence. In parallel, under aseptic conditions, samples of organic deposits were collected and transferred to culture media for bacteria growth (Marine Agar and Thiosulfate Citrate Bile Salts Sucrose [TCBS]). Bacteria were cultured and isolated for identification using conventional bacteriological procedures and analysis of the 16S rDNA gene sequence. Mud Worms in Abalone Culture In high-density abalone aquaculture, infestations by burrowing worms can reach lethal levels. There are examples of high mortalities of abalone associated with burrowing worms in Australia and South Africa. Between 1995 and 1997 producers of Tasmanian blacklip abalone Haliotis rubra reported cumulative mortality of more than 50 percent related to the burrowing worms Boccardia knoxi and Polydora hoplura (Lleonart et al. 2003). In South Africa, infestations of the burrowing worms Polydora hoplura and Boccardia sp. have had a negative impact on the culture of abalone Haliotis midae (Simon et al. 2006). To date, no records of burrowing worms in cultured red abalone Haliotis rufescens in Mexico have been published. However, in recent years, producers have occasionally noted that some juvenile abalones show unusual perforations and scars over the shell and some organisms easily lose their shell (Fig. 1). Perforations and scars can be observed all over the shell but most are located close to the spire (Fig. 2). Extensions of the damage are more clearly observed on the inner surface of the shell (Fig. 3). In severe cases, perforations reach the border of the shell (Fig. 4). These animals showed a reduction and darkening of the mantle, foot muscle and accumulation FIGURE 2. The most frequent affected area of the shell was close to the spire and in severe cases the perforation may form a channel or rupture that reaches the shell border. FIGURE 3. The extent of the damage is more clearly observed on the inner surface of the shell. Note that the lesion in the last shell is covered by calcite. FIGURE 4. Severe damage in the shell of a juvenile red abalone caused by burrowing worms. Note the wide rupture of the shell reaching the border and the chitinolytic brown deposit. FIGURE 5. Reduction of the mantle and foot muscle, darkened soft body and the organic brown deposit is evident in a juvenile red abalone severely affected by the burrowing worm.
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