**2. Microbiological threats facing Chilean aquaculture**

As stated earlier, Chilean aquaculture is a successful industry. In fact, Chile is the first world producer of rainbow trout and coho salmon, is the second world producer of Atlantic salmon, and, recently, has become the world fourth producer of the Chilean mussel (*Mytilus chilensis*) [2, 8]. This level of production is achieved by the natural condition of the water present in the south of Chile and by the intensive farming strategy used. In fact, using less water surface, Chile competes to Norway in salmon production [12]. As consequence, Chilean aquaculture has faced several sanitary problems, related with outbreaks of viruses such as infectious pancreatic necrosis virus (IPNV) [13] and infectious salmon anemia virus (ISAV) [14]; bacterial pathogens such as *Piscirickettsia salmonis* [15], *Flavobacterium psychrophilum* [16], and *Renibacterium salmoninarum* [17]; and parasites such as *Caligus rogercresseyi* [15]*.* Diseases caused by viruses are one of the great challenges of Chilean salmon farming industry; the first virus being identified in Chile was IPNV, which is highly persistent and causes severe mortality. However, the introduction of individuals with QTL related to the resistance to the disease and the administration of vaccines has partially reduced mortalities in Atlantic salmon [15, 18, 19]. Due to its prevalence and because IPNV has been found in healthy fishes, it is now considered endemic in

the Chilean Coast [13]. The ISAV, an orthomyxovirus related with influenza, caused several outbreaks between 2007 and 2008 forcing to close about 90% of the Atlantic salmon hatcheries located in Region X [14, 20]. To say the least, the industry almost collapsed. New outbreaks of ISAV have been detected in the last years; however, its pathogenicity is not comparable to the 2007–2008 outbreaks [15]. Recently, piscine reovirus (PRV) has been found in some Atlantic salmon with the so-called heart and skeletal muscle inflammation (HSMI) and also rainbow trout [21], although this virus has not been related to outbreaks with important losses [22].

Regarding bacterial pathogens, *P. salmonis* accounts for about 15% of production loss of post-smolt Atlantic salmon [15], while *F. psychrophilum* produces mortalities between 20 and 80% in rainbow trout and Atlantic salmon during the freshwater phase [23]. *R. salmoninarum* is the etiological agent of the bacterial kidney disease (BKD) and is responsible of the systemic and chronic infection in rainbow trout, Atlantic salmon, and coho salmon, with about 40% of prevalence in the latter species [24]. Several types of vaccines have been produced to control viral- and bacterial-caused diseases that, however, have proved inefficient as disease outbreaks continue to appear. This is the reason why antibiotics are still the main therapeutic strategy against bacterial pathogens. More than 186 tons were used only in the first semester of 2018 [25, 26].

Regarding farmed mussels (*Mytilus chilensis*) in Chile, there are no serious disease outbreaks reported so far, as it has occurred in major producer centers in Spain where congeneric species are intensively cultivated (*M. galloprovincialis* and *M. edulis*). Most of the registered mussel centers [8] located in the De Los Lagos Region (Lake District), southern Chile, cultivate mussels from ropes hanging from long lines. Spats are obtained either by the existing natural beds or by natural larva settlement. A recent review on bacteria associated to mollusk farming concentrated in the Chilean scallop (*Argopecten purpuratus*), a species cultivated in the north that showed potential aquaculture relevance prior to mussels [27]. This study also reports bacteria with probiotic effect on the pathogens listed. More recently, Lohrmann et al. [28] provided baseline information on the symbionts and other conditions of cultivated mussels from the Lake District, such as parasites (protozoan *Marteilia* sp., coccidian, and gregarines), intestinal copepods, castrating trematodes (*Proctoeces* sp.), intracellular bacteria in gills and digestive glands, ciliates on gills, microsporidian and metazoan parasites, and other conditions.

Currently, the factors that preclude bacterial and viral outbreaks remain unknown. With no doubt, the intensive culture strategy used by the industry accounts for disease prevalence and economic losses. But still, the cost–benefit ratio is extremely positive for the industry. Very likely, several unknown factors affect the host-pathogen-environment relationship. While the physicochemical factors enhancing disease outbreaks are relatively easy to determine, either by direct or satellite monitoring, the analysis of the microbiological factor is still biased to cultivable bacteria (see below), which means that more than 99% of the all microorganism present in the environment are left out. Metagenomics provides a more holistic view of the microbial ecosystem and their interactions, and so it is expected to shed light on this complex problem for the industry, for the environment, and also to humans that eat fish with antibiotic traces.
