Introduction

Aquaculture traces its roots to the ancient water-oriented civilizations of the East, where fish served as a main part of people’s diets and the beginning of aquaculture can be dated to the period 2000–1000 bc in China where mainly common carp (Cyprinus carpio) were utilized. European aquaculture can be dated back to ancient Rome and Gaul (modern France), where oyster cultivation thrived. Like the Chinese, ancient Romans bred fish in ponds. In the United Kingdom, the first hatcheries for rainbow trout (Oncorhynchus mykiss Walbaum) and Atlantic salmon (Salmo salar L.) were established in the 1850s; but modern Atlantic salmon farming in Norway did not start until 1971, with larval Atlantic cod (Gadus morhua L.) production commencing in 1998.

It has been predicted that by 2050, the total population on the planet will be 9 billion and aquaculture will have an important role in catering to the increased demand for food (Godfray et al. 2010). Global aquaculture production reached 62.7 million tonnes in 2011 with an estimated production of 66.5 million tonnes in 2012 (FAO 2012). In comparison, global capture fisheries are estimated to be stable around 95 million tonnes, and are not likely to increase in the future (FAO 2012).

All animal production systems have challenges associated with disease and the best way to solve these is often through effective management practices, i.e. management of stock, soil, water, nutrition and environment. A number of approaches have been applied to address this problem, including sanitary prophylaxis, disinfection, and chemotherapy, with particular emphasis on the use of antibiotics. The application of antibiotics and other chemicals to aquaculture is quite expensive, undesirable due to contamination to the surrounding environment, and might lead to antibiotic resistance (Cabello 2006; Romero et al. 2012). According to Heuer et al. (2009) few countries monitor the use of antibiotics in aquaculture and large variations seem to occur between different countries. Smith (2008) estimate that antibiotic consumption ranges from 1 g per tonne production in Norway to 700 g per tonnes in Vietnam. The decreased use of antibiotics in indistrialised fisnfish farming is partly due to widespread use of vaccination against specific diseases. However, there are practical difficulties and undesirable consequences associated with some of these approaches. In spite of the relatively large amount of research performed, few DNA vaccines are commercialized and it has been suggested that DNA vaccines are third generation vaccines. If the gastrointestinal (GI) tract is involved in infection there are several alternative strategies to control pathogenic bacteria from adherence and colonization of the intestine; the probiotic, prebiotic and synbiotic concepts, as well as the use of immunostimulants and plant extracts.

From a global perspective, it is recognzed that pressure on natural marine resources should be lowered. For the preservation and optimal use of wild fish stocks and for the healthy development of aquaculture, research on alternative protein and oil sources is therefore essential (FAO 2003) and has gained momentum over the past decades. The main driving force is to meet the protein, amino acid and fatty acid requirements of farmed fish without relying too heavily on fish mean (FM) and fish oil (FO). As there will be a limitation in global supplies of FM and FO in the near future, sustainable alternatives have been explored (Gatlin III et al. 2007). Soybean meal (SBM) and soybean oil (SBO) are considered suitable alternatives for the partial replacement of FM and FO and are extensively utilzed in commercial aquafeeds. Given the predictable increase in the demand for aquafeed resources, the risk of deficits in these ingredients is real. Thus, the changes from FM and FO to soybean products present several metabolic and health challenges for the farmed fish. When using high dietary levels of plant derived materials, particularly those derived from soybean, it is important to consider the impacts on gut microbiota and gut histology (Merrifield et al. 2011a) as the GI tract can be one of the important infection routes for some pathogens in fish (Groff and LaPatra 2000; Birkbeck and Ringø 2005; Ringø et al. 2007, 2010).

Another aspect that has received attention is microalgae and their biotechnological potential as increasing knowledge regarding antibacterial activity of different extracts of microalgae has been reported (e.g. Day and Austin 1990; Alonso et al. 2012; Goecke et al. 2012). Even though some information is available on the use of microalgae in aquaculture, growth performance, feed utilization, immune system, gut morphology, gut microbiota and disease resistance of fish (Tulli et al. 2011; Cerezuela et al. 2012a, b, c) these topics merit further investigations.

This review provides and overview of vaccines and dietary supplements in aquaculture together with a critical evaluation of the results obtained so far. Finally, directions for further research are proposed.

Use of antibiotics

Due to intensive farming practices, infectious diseases are a major problem in finfish and shellfish aquaculture, causing heavy loss to farmers. In the 1970s and 1980s oxolinic acid, oxytetracycline (OTC), furazolidone, potential sulphonamides (sulphadiazine and trimethoprim) and amoxicillin were the most commonly used antibiotics in fish farming. However, the indiscriminate use of antibiotics in disease control has led to selective pressure of antibiotic resistance in bacteria, a property that may be readily transferred to other bacteria (Cabello 2006; Romero et al. 2012). Furthermore, use of antibiotics to control pathogenic bacteria can also reduce the numbers of non-pathogenic bacteria in the gut. On the other hand, it may be perceived that there is a problem associated with the release of antibiotic into the environment and the occurrence of antibiotic resistance bacteria in marine sediments near fish farms. Some research groups have addressed this topic and shown significant changes in the benthic bacterial community near fish farms with possible links to antibiotic susceptibility (Kerry et al. 1995; Chelossi et al. 2003).

More recently molecular tools such as PCR have been used in antibiotic resistance studies, with water and sediment from fish farms screened and tetR genes detected at significantly higher frequencies in water from farms with recent OTC use compared with water from farms without recent OTC use (Seyfried et al. 2010). However, OTC use was not correlated with the prevalence of tetR genes in sediment samples. A similar study using qPCR reported greater copy numbers of tetA, tetC, tetH, and tetM at the farms compared to pristine sites (Tamminen et al. 2011). However, no resistant genes were found in samples collected 200 m away from any of the farms. Furthermore, the analysis of tetracycline indicated that none of the samples contained therapeutic concentrations at any of the sampling times, suggesting that the prevalence of tetracycline-resistance genes may be caused by the persistence of these genes in the absence of selection pressure. An increase in antibiotic-resistance genes in the absence of the antibiotic itself has also been attributed to co-selection with other antibiotics.

Bacterial vaccines

Compared with human vaccine history, fish vaccine development has a very short history starting in the 1970s with the first licensed fish vaccine made commercially available in 1976 (Evelyn 1997). In fish vaccination, three main delivery approaches are used: injection, oral delivery and immersion; bath and spray vaccination. Vaccination plays an important role in large-scale commercial fish farming and has been a key reason for the success of salmon cultivation. In addition to Atlantic salmon and rainbow trout, commercial vaccines are available for channel catfish (Ictalurus punctatus), European sea bass (Dicentrarchus labrax), sea bream (Sparus aurata), Japanese amberjack (Seriola quinquerdiata), tilapia (Oreochromis niloticus), Atlantic cod, barramundi (Lates calcarifer), tilapia (Tilapia spp.), turbot (Scophthalmus maximus L.), yellowtail (Seriola quinqueradiata), purplish and gold-striped amberjack (Seriola dumereli) and striped jack (Pseudocaranx dentex). The range of bacterial infections for which vaccines are commercially available now comprises classical vibriosis (Vibrio (Listonella) anguillarum), cold-water vibriosis (Aliivibrio (Vibrio) salmonicida), Vibrio ordalii, furunculosis (Aeromonas salmonicida subsp. salmonicida), yersiniosis (Yersinia ruckeri), pasteurellosis (Photobacterium damselae subsp. piscicida), edwardsiellosis (Edwardsiella ictaluri), winter ulcer (Moritella viscosa), and streptococcosis/lactococcosis (Streptococcus iniae/Lactococcus garvieae). Furthermore, experimental vaccines are used against infections caused by Vibrio harveyi and Photobacterium damselae subsp. damselae in barramundi, piscirickettsiosis and bacterial kidney disease in salmonids, as well as infection with Flexibacter maritimus in turbot. However, vaccination has both advantages and drawbacks and readers with special interest are referred to the reviews of Sommerset et al. (2005), Plant and LaPatra (2011) and Clarke et al. (2013).

A brief overview of the developments in fish vaccinology is presented in Table 1. In general, empirically developed vaccines based on inactivated bacterial pathogens have proven to be very efficacious in fish. Substantial efficacy data is available for new fish vaccines and advanced technology has been implemented. However, before such vaccines can be successfully commercialized, several hurdles have to be overcome regarding the production of cheap but effective antigens and adjuvants, while bearing in mind environmental and associated regulatory concerns (e.g., those that limit the use of live vaccines). Pharmaceutical companies have performed a considerable amount of research on fish vaccines; however, limited information is available in scientific publications. In addition, salmonids dominate both the literature and commercial focus, despite their relatively small contribution to the total volume of farmed fish in the world.

Table 1 A brief overview of the developments in fish vaccinology
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Salmonids are usually immunized with multivalent vaccines by intraperitoneal injection. In marine fish species vaccination is generally performed by immersion, but use of injection vaccination is increasing, particularly in the Mediterranean region. Only limited use of orally administered fish vaccines is reported. In general, vaccines against bacterial diseases provide good protection (Plant and LaPatra 2011). The best protection is obtained with injectable, adjuvanted vaccines (Brudeseth et al. 2013). However, injection-site adverse reactions often occur when such products are used (Mutoloki et al. 2006; Brudeseth et al. 2013).

DNA vaccines

To limit the impact of infectious diseases a continuous effort to improve vaccine strategies for fish is required. One of the vaccine strategies that has been tested recently is DNA vaccination. A DNA vaccine is composed of the DNA sequence encoding a protective antigen inserted into a small circular piece of DNA, a plasmid expression vector. A strong viral promoter is present in the plasmid to drive the in vivo expression of the antigen. The plasmid can easily be amplified and purified from bacterial cultures and subsequently used for vaccination. It is possible to make plasmids encode more than one antigen and also to incorporate sequences for immunostimulatory purposes (adjuvants).

DNA vaccines have several advantages over more traditional vaccines. The vaccine antigen is produced inside the cells of the host, which ensures correct protein folding. Also, this intracellular protein production mimics a natural infection with an intracellular pathogen and both the humoral and cellular arms of the immune system are activated. The production of DNA vaccines is rather easy and does not require purification of protein or of the pathogen, as with subunit or whole pathogen vaccines. Compared to attenuated vaccines there is no risk for reversion to virulence as only one gene from the pathogen is present in the DNA vaccine.

DNA vaccination has been experimentally tested in various fish species against mainly viral pathogens, but also against bacteria and parasites (Tonheim et al. 2008; Gomez-Casado et al. 2011; Liu et al. 2011; von Gersdorff Jorgensen et al. 2012). DNA vaccines to the rhabdoviruses infectious haematopoietic necrosis virus (IHNV) and viral haemorrhagic septicemia virus (VHSV) in rainbow trout have shown very good protective effects. These DNA vaccines are based on the gene encoding the viral surface glycoprotein (G protein). The G protein is responsible for viral cell attachment and the neutralizing antibody response is directed against this protein. DNA vaccines, based on other rhabdoviral proteins, do not provide the same levels of protection (Corbeil et al. 1999). The protective mechanisms provided by the G gene vaccines are based on a rapid and transient innate immune response involving activation of the antiviral interferon system followed by long-term specific immunity (Kim et al. 2000; Lorenzen et al. 2002). Long-term protection may last up to two years after vaccination (Kurath et al. 2006). To optimize efficacy of the G gene vaccines the effects of fish size, vaccine dose and administration routes have been investigated (Corbeil et al. 2000). Intramuscular injection of the vaccines gives good protection. No oil-adjuvants are needed, which for other fish vaccines are known to cause serious side effects. A DNA vaccine based on the IHNV G gene is in commercial use for Atlantic salmon in Canada (Apex-IHN, Novartis) (Salonius et al. 2007; Alonso and Leong 2013). However, in the US and Europe this vaccine has not been approved for commercial use due to safety concerns. Recently an oral DNA vaccine with PLGA (Poly (d,l-lactic-co-glycolic acid)) nanoparticles containing the IHNV G gene plasmid was tested (Adomako et al. 2012). The prevalence of fish expressing the G gene after receiving the feed coated with the vaccine was very low and only a minor increase in survival was recorded after virus challenge. However, the data suggests that it might be possible to deliver a DNA vaccine orally, although major improvements of the technology are required.

For fish pathogenic viruses other than rhabdoviruses moderate to low protective effects have been observed after DNA vaccination (Tonheim et al. 2008; Gomez-Casado et al. 2011). In two recent studies the protective effects of different types of vaccines against infectious pancreatic necrosis virus (IPNV) and salmonid alphavirus (SAV) were compared. In both cases the vaccine based on inactivated whole virus provided better protection after challenge than DNA vaccination (Munang’andu et al. 2012; Xu et al. 2012).

Although the G gene vaccines to VHSV and IHNV have proven to be very efficient, it is only in Canada that such a vaccine has been licensed for commercial use (Salonius et al. 2007). There are uncertainties as to how long plasmid DNA can remain intact in fish tissues and whether there is a risk for integration of plasmid DNA into the genome (Gillund et al. 2008; Tonheim et al. 2008). Different approaches have been used to try to develop plasmid DNA that is considered safer and more acceptable to use as vaccines. Plasmids where viral regulatory sequences have been replaced by regulatory sequences from fish have been developed (Martinez-Lopez et al. 2013). Also to reduce possible homologous recombination between all-fish plasmids with the fish genome, core and enhancer sequences from fish origin have been combined with those of cytomegalovirus (CMV) to design alternative hybrid promoters (Martinez-Lopez et al. 2012). To limit the long-term persistence of plasmid DNA in cells after vaccination a suicidal DNA vaccine construct was developed. After inducing protective immunity the cells harboring the plasmid are killed by apoptosis (Alonso et al. 2011).

The probiotic concept

Probiotics, generally defined as live microorganisms with different beneficial characteristics, are increasingly becoming accepted as an alternative prophylactic treatment for humans and animals to either treat pathogen-related diseases or to be used in preventive treatments. Probiotic research has mainly focused on the host’s GI tract, while applications to skin or gill surfaces have been less investigated.

Several reviews have published varied opinions on what are considered to be important characteristics for the selection of probionts for applications in aquaculture (e.g. Gatesoupe 1999; Gram and Ringø 2005; Balcázar et al. 2006; Gómez and Balcázar 2008; Lamari et al. 2013; Lauzon et al. 2014a). Merrifield et al. (2010a) collated such characteristics and extended them to produce the following comprehensive list of criteria (Table 2).

Table 2 Probiotic selection criteria
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Even though fish microbiologists have gained some knowledge about adherence of probiotic bacteria in the GI tract of fish during the last two decades, there is a long way to go compared to the information available from non-aquaculture studies. For example, in a study using crude mucus from small intestine of a 23-day-old healthy piglet, Macías-Rodríguez et al. (2009) demonstrated that adhesion of the potential probiotic Lactobacillus fermentum originally isolated from faeces of a piglet involved two adhesion-associated proteins with a relative molecular weight of 29 and 32 kDa that are attached non-covalently to the cell surface. In a study with Lactobacillus rhamnosus a piliated bacterium, von Ossowski et al. (2010) reported that 2 pilin subunits (SpaB and SpaC) in the SpaCBA pilus fiber are involved in binding to intestinal mucus. Moreover, Huang et al. (2013) evaluated the relationship between adhesive ability of probiotic bacteria and soluable acid residues in the human colonic mucin (sHCM). Based on their results using a Biacore binding assay the authors concluded that there was a strong relationship between probiotic adhesion and acid residues of sHCM. As no fish studies have been carried out on cell surface components of marine probiotic bacteria responsible for mucosal adhesion we recommend that this topic merits further investigations especially related to the discussion of whether colonzation of probiotic bacteria to intestinal mucus is a favorable or essential criterion.

In their search for good probiotics to use in aquaculture some authors have hinted on the use of lactic acid bacteria (LAB) isolated from sources other than aquatic animals (El-Haroun et al. 2006; Bagheri et al. 2008; Salinas et al. 2008a; Merrifield et al. 2010a; Salma et al. 2011; Zhou et al. 2012; Ren et al. 2013). This selection criterion is mainly based on their proven efficiency and safety in humans and livestock (Azad and Al-Marzouk 2008). However, efficacy in aquatic environments and safety to new hosts must be demonstrated. Several of the reported probiotic studies conducted in vivo evaluated allochthonous LAB strains (Lauzon and Ringø 2012). It is interesting to consider the application spectrum of allochthonous LAB, their adhesion capacity and/or colonization as well as the reproducibility of beneficial effects towards different hosts.

The use of allochthonous LAB strains in aquaculture has been shown to provide beneficial effects in various aquatic animals, and mainly consists of lactobacilli species, and to a lesser extent carnobacteria, enterococci, Lactococcus lactis subsp. lactis, Leuconostoc mesenteroides and Pediococcus acidilactici strains. Most of these rearing trials involved the use of monospecies (Table 3), but multispecies applications have also been successful and even complementary (Lauzon and Ringø 2012). Comparing the beneficial effects in gilthead sea bream observed during the application of closely related strains (Diaz-Rosales et al. 2006) compared with more distant ones (Salinas et al. 2005) enhanced immunomodulation was detected. In this regard, a question has risen whether the systematic relationship of multispecies probiotics is an influencing factor (Dimitroglou et al. 2011).

Table 3 Application of LAB as probiotics in aquaculture by monospecies treatments and main beneficial effects observed
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Adhesion capacity and/or colonization of allochthonous LAB after fish treatment is not always verified or successfully confirmed. Detection of lactobacilli in the gut of fish a few days post-treatment has been reported (Nikoskelainen et al. 2003; Panigrahi et al. 2005; Iehata et al. 2009; Son et al. 2009), for carnobacteria (Robertson et al. 2000; Irianto and Austin 2002), enterococci (Lauzon et al. 2010a, b) and Ped. acidilactici (Villamil et al. 2010). Analysis of mucosal samples obtained from treated fish has also demonstrated the ability of allochthonous LAB to colonize the gut of rainbow trout (Merrifield et al. 2010b, 2011b). Indeed, LAB have in general a good ability to adhere to different cell types (Rinkinen et al. 2003; Lauzon et al. 2008). It is noteworthy that competition of allochthonous LAB with autochthonous bacteria added at high levels in rearing trials have shown that autochthonous bacteria isolated from larvae will more rapidly colonize the gut at an early developmental stage (Ringø 1999). In contrast autochthonous bacteria from adult fish seem to colonize the gut only after fish metamorphosis, allowing allochthonous LAB to colonize at earlier stages but displacing them at a later stage (Carnevali et al. 2004). The possible influence of the fish developmental stage on probiont colonization may explain the decreasing colonization of probiotic strains observed from larval to juvenile cod stages (Lauzon et al. 2010a, b, c). These findings should be considered in the selection of probionts for multispecies probiotics.

Another important matter to reflect on during the selection of autochthonous LAB relates to reproducibility of the beneficial effects produced by a LAB species towards different hosts. Due to different experimental design and parameters analyzed during probiotic application, very few trials can be compared. Nevertheless, disease control has often resulted in the presence of lactobacilli species to combat different fish pathogens; Aeromonas hydrophila in carp (Harikrishnan et al. 2010b), LCDV virus in olive flounder (Harikrishnan et al. 2010a), Streptococcus spp. in groupers (Son et al. 2009; Harikrishnan et al. 2010c), Edwardsiella tarda in tilapia (Pirarat et al. 2006), and A. salmonicida subsp. salmonicida in rainbow trout (Nikoskelainen et al. 2001; Balcázar et al. 2007a). C. divergens from salmon provided short term protection against V. (L.) anguillarum during rearing of cod juveniles (Gildberg and Mikkelsen 1998), while another Carnobacterium strain reduced the effect in rainbow trout (Robertson et al. 2000). Lac. lactis subsp. lactis and Leu. mesenteroides also controlled the A. salmonicida subsp. salmonicida infection in rainbow trout (Balcázar et al. 2007a). Finally, Ped. acidilactici also showed promising results in combatting a pathogenic Vibrio in shrimp (Castex et al. 2010).

In most of these studies, the immune response was enhanced by the probiotics applied. LAB species affecting immunomodulation include Lb. rhamnosus in rainbow trout (Nikoskelainen et al. 2003; Panigrahi et al. 2004, 2005, 2007) and tilapia (Pirarat et al. 2006); Lb. sakei in kelp grouper (Harikrishnan et al. 2010b), rainbow trout (Balcázar et al. 2007b) and brown trout (Balcázar et al. 2007a); Lb. delbrüeckii subsp. lactis in gilthead sea bream (Salinas et al. 2005; 2008b) and Atlantic salmon (Salinas et al. 2008a); Lac. lactis subsp. lactis in rainbow trout (Balcázar et al. 2007b), brown trout (Balcázar et al. 2007a) and turbot (Villamil et al. 2002); Enterococcus faecium in tilapia (Wang et al. 2008) and rainbow trout (Panigrahi et al. 2007); and Ped. acidilactici (Bactocell®) in red tilapia (Ferguson et al. 2010) and rainbow trout (Merrifield et al. 2011a). Enhanced growth is commonly reported during probiotic treatments, where various strains affect different aquatic species (Table 3).

Finally, an important characteristic of probiotics is their safety to the host. Integrity of gut mucosa supports the safety of probiotic administration, which has been demonstrated upon use of Lb. delbrüeckii subsp. lactis (Salinas et al. 2008a, b), Lb. plantarum and Lb. fructivorans (Picchietti et al. 2007). In contrast to these results, Salma et al. (2011) noticed severe cell damage when distal intestine of beluga (Huso huso) was exposed to Lb. plantarum originally isolated from traditional Sabalan Iranian cheese prepared from raw sheep milk. Based on the latter results, we therefore recommend the use of light—and electron microscopy investigations of the intestine when evaluating the potential of probiotic bacteria in fish.

During the last two decades several comprehensive reviews have reflected on the promising use of probiotics in aquaculture. The use of probiotics has also opened a new era of health management strategies in aquaculture; immunity. Readers with special interest in probiotics and immunity in fish are referred to the comprehensive review of Nayak (2010).

The prebiotic concept

The use of probiotics is generally difficult in the feed production industry because of the low viability of the bacteria after pelleting and storage, as well as problems with feed handling and preparation. In addition, there is the possibility of probiotics entering into the environment. As an alternative, prebiotics have been assessed in an attempt to overcome these issues. Rather than introducing probiotic bacteria, the aim of prebiotics is to stimulate selected beneficial indigenous microbiota populations. In order for a food ingredient to be classified as a prebiotics, Gibson and Roberfroid (1995) suggested that prebiotics should; (1) be neither hydrolyzed nor absorbed in the upper part of the GI tract, (2) be a selective substrate for one or a limited number of beneficial bacteria commensal to the colon, which are stimulated to grow and/or are metabolically activated, (3) consequently, be able to alter the colonic flora in favor of a healthier composition and (4) induce luminal or systemic effects that are beneficial to the host health.

The initial research with prebiotics dates back to the end of the 1970s when Japanese scientists showed that bifidobacteria selectively fermented several carbohydrates (especially fructooligosaccharides; FOS). Prebiotics consist mainly of oligosaccharides; mannan oligosaccharides (MOS), fructooligosaccharides (FOS, including short chain-fructooligosaccharides; sc-FOS), glucooligosaccharides (GOS) and trans-galactooligosaccharides (TOS; galactooligosaccharides are also included). According to Lauzon et al. (2014b) inulin, a fructan polysaccharide, also has documented prebiotic qualities.

Readers with special interest in the use of prebiotics in aquaculture are referred to the reviews of Merrifield et al. (2010a), Ringø et al. (2010, 2014), Ganguly et al. (2013), Daniels and Hoseinifar (2014) and Torrecillas et al. (2014), and the recent research papers of Lokesh et al. (2012), Zhang et al. (2012a, b), Anguiano et al. (2013), Liu et al. (2013), Hoseinifar et al. (2013), Torrecillas et al. (2013), Wu et al. (2013a) and Zadeh et al. (2014).

The synbiotic concept

Synbiotic refers to nutritional supplements combining a mixture of probiotics and prebiotics in a form of synergism. The idea is that prebiotics will improve the survival of the live microbial supplements in the GI tract of the host (Gibson and Roberfroid 1995). Since the first fish study on synbiotics was published in 2009 (Rodriguez-Estrada et al. 2009) there has been a growing interest in the use of synbiotics in aquaculture (Cerezuela et al. 2011). However, since this review was published several synbiotic studies have emerged (Table 4). The focus of these studies have spanned from growth performance, feed utilization, digestive enzyme activities, body composition, immunological responses, haematological/serum biochemical parameters, disease resistance, survival rate and gut microbiota of synbiotic fed finfish, shellfish and echinoderms. To avoid duplication, fish studies reviewed by Cerezuela et al. (2011) are not discussed in this sub-section and readers with special interest are referred to the original review.

Table 4 Synbiotic applications in finfish
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The effect of Biomin IMBO (Enterococcus faecium and FOS; 0.5, 1, 1.5 g kg−1) on rainbow trout’s specific growth rate (SGR), feed conversion ratio (FCR), feed conversion efficiency (FCE), survival and disease resistance towards Saprolegnia parasitica was evaluated by Firouzbakhsh et al. (2012). All inclusion levels significantly improved SGR, FCE, survival and resistance against S. parasitica while FCR and condition factor (CF) were decreased. Similar improvements on growth, and in some cases survival, have been observed with the application of commercial synbiotic Biomin IMBO to kutum (Rutilus frisii Nordmann, 1840) (Haghighi et al. 2010), angelfish (Pterophyllum scalare) and zebrafish (Danio rerio) (Nekoubin et al. 2012b).

Tapia-Paniagua et al. (2011) evaluated modulation of the intestinal allochthonous microbiota of gilthead sea bream (~ 80 g) by administration of Debaryomyces hansenii in combination with inulin. Experimental fish were fed either a commercial diet (control diet), or diet supplemented with D. hansenii strain L2 (106 CFU g−1) plus 3 % inulin (experimental diet II) for 4 weeks. After 2 and 4 weeks of feeding, samples of the whole intestine were aseptically removed for allochthonous microbiota analysis using PCR-DGGE and sequence analysis. Additionally, the expression of 12 selected genes related to the immune response (IgM, MHCIα, MHCIIα, C3, IL-1β, TLR9A, TNFα, CSF-1R, NCCRP-1, Hep, TCRβ and CD8) from the skin, intestine, liver and HK tissue was analyzed by real-time PCR. Samples of blood and HK were obtained for the determination of humoral and cellular immune parameters. The results revealed that fish fed the experimental diet had lower intestinal microbial species richness and greater similarity indices compared with fish fed the control diet for 4 weeks, but Pseudomonas spp. dominated the intestinal microbiota in both experimental groups. Peroxidase activity was the only haematological parameter that was significantly increased in fish fed the synbiotic diet. RT-PCR revealed that several immune-related genes were up-regulated in the skin and intestine after 2 weeks of feeding. The maximum intestinal transcript levels for the major histocompatibility complex (MHC) genes MHCI and MHCII were significantly up-regulated. After 4 weeks of feeding, relatively lower gene transcript levels were recorded in the skin and intestine, but higher levels of complement 3, the pro-inflammatory cytokine TNFα and colony stimulating factor 1 receptor (CSF-1R), a receptor for a cytokine which controls macrophages production, differentiation and function, were observed in the intestine. In addition, at week 4 a greater effect was observed in the HK than week 2. This was especially prominent in the up-regulation of C3, the pro-inflammatory cytokine IL-1β, CSF-1R and non-specific cytotoxic cell receptor protein 1 (NCCRP-1; a surface protein which functions in target cell recognition and cytotoxicity) and the potential for improved disease resistance. Indeed, Lin et al. (2012) reported elevated peripheral total leucocyte counts, respiratory burst–, lysozyme—and superoxide activities, which afforded increased protection against Aeromonas veronii infection in koi fed a synbiotic application of Bacillus coagulans and COS. In addition, the inclusion of the synbiotic significantly improved SGR and FCR.

In three recent studies using gilthead sea bream, Cerezuela and colleagues evaluated the effect of Bacillus subtilis and inulin on immune-related gene expression and disease resistance against P. damselae subsp. piscicida (Cerezuela et al. 2012d), gut microbiota and gut histology (Cerezuela et al. 2013a), as well as the expression of different genes in the anterior intestine (Cerezuela et al. (2013b). Synbiotic administration significantly increased complement activity following four weeks of feeding, but not after two weeks of feeding. Respiratory burst activity was not affected. Serum IgM level was significantly higher after 2 weeks of feeding but not after four weeks. The expression of immune-related genes in HK of fish fed synbiotic for two weeks displayed no significant effect. Surprisingly, the cumulative mortality after challenge with P. damselae subsp. piscicida (i.p) was significant higher in the synbiotic group compared to the control group. In the study of Cerezuela et al. (2013a), the synbiotic group revealed signs of damage in the anterior intestine; similar to that reported in Arctic charr (Salvelinus alpinus L.) fed inulin (Olsen et al. 2001). Synbiotic administration also significantly increased villi height and intestinal diameter, but reduced the number of goblet cells and microvilli height. Gut microbiota, evaluated by DGGE, revealed that number of OTUs in fish fed synbiotic was significantly lower (6.0 ± 0.0) than that of the control fish (17.3 ± 0.9). Cerezuela et al. (2013b) investigated the effect of synbiotic administration on intestinal gene expression in gilthead sea bream, and revealed that only β-actin and occludin were significantly affected by synbiotic supplementation. The conclusions of these studies are that the synbiotic application of B. subtilis and inulin increases some immune parameters, but has a negative effect on gut morphology and gut microbiota, with a lesser effect on intestinal gene expression in the anterior intestine and a negative effect on disease resistance towards P. damselae subsp. piscicida. Further investigations are warranted to ascertain if benefits can be achieved with optimized inclusion levels.

Immunostimulants

The use of immunostimulants offers a unique approach for fish culturists to control disease losses in their facilities. Numerous polysaccharides from a variety of sources have the ability to stimulate the immune system, and thus behave as immunostimulants (Raa 1996; Vadstein 1997; Sakai 1999; Bricknell and Dalmo 2005; Soltanian et al. 2009; Ringø et al. 2012; Meena et al. 2013). The biological effects of immunostimulants are highly dependent on the receptors on the target cells recognizing them as potential high-risk molecules thus triggering various defense pathways. Thus, it is also important to increase knowledge of whether receptor specificity and the inflammatory processes are induced with each potential immunostimulant. However, many mammalian receptors reported to bind immunostimulants such as NLR (NOD-like receptors) have yet to be reported in fish. Nevertheless, assuming that fish and mammalian cells share many similar receptors, one may predict the biological outcome of immunostimulants in fish.

β-glucan

Immunostimulants have been used as feed additives for several years in aquaculture, and yeast β-glucan may be the one with the longest track record. In nature, β-glucans are widespread and have been characterized in microorganisms, algae, fungi and plants (Volman et al. 2008). The chemical structure of β-glucan varies with respect to molecular weight and degree of branching. For example, β-glucan from yeast contains a particular carbohydrate consisting of glucose and mannose residues and is a major constituent in the cell membrane. In aquaculture, glucans have been successfully used to enhance the resistance of finfish and crustaceans against bacterial and viral infections. Readers are referred to the reviews of Soltanian et al. (2009), Ringø et al. (2012) and Meena et al. (2013) for detailed overview of studies on glucans as immunostimulants in aquaculture.

The second major by-product from the brewing industry is baker’s yeast (Saccharomyces cerevisiae) which contains various immunostimulating compounds such as β-glucans (the cell walls are constructed almost entirely from β-1,3-d-glucan, β-1,6-d-glucan, mannoproteins and chitin bound together by covalent linkages), nucleic acids and oligosaccharides (Ferreira et al. 2010). Bakers yeast has the capacity to enhance growth and increase both humoral (myeloperoxidase and antibody titer) and cellular (phagocytosis, respiratory burst and cytotoxicity) immune responses, and to increase or confer resistance against pathogenic bacteria in various fish species (Soltanian et al. 2009; Ringø et al. 2012).

MacroGard®

According to Biorigin, MacroGard® is a source of highly purified, exposed, and preserved β 1,3/1,6 glucans produced from a specially-selected strain of the yeast Saccharomyces cerevisiae (http://www.biorigin.net). It is an environmentally sound alternative to antibiotics and the compound has been in use worldwide for almost 25 years as an immune modulating agent in animal husbandry and aquaculture (e.g. Sealey et al. 2008; Soltanian et al. 2009; Ringø et al. 2012; Meena et al. 2013).

Alginate

The adaptive immune system is poorly developed in the early developmental stages of fish, and in this respect, alginate has been proposed as a potential immune stimulator candidate. Alginate is a polysaccharide composed of β-1,4-d-mannuronic acid (M) and C5-epimer α-L-glucuronic acid (G) (Remminghorst and Rehm 2006).

Commercially available alginates have M-content ranging between 30 and 70 %. Alginates with up to 80 % M-content have also been shown to be potent stimulators of immune cells such as human monocytes (Skjåk-Brak et al. 2000). High-M alginate has also been used as an immunostimulant for enhancement of innate immune resistance in fish larvae and fry (Vadstein 1997; Skjermo and Vadstein 1999; Vollstad et al. 2006; Ringø et al. 2012).

Ergosan

This is an algal based product that contains 1 % alginic acid extracted from Laminaria digitata. To the author’s knowledge, the first study on Ergosan in aquaculture was reported by Miles et al. (2001) on striped snakehead (Channa striata). Ergosan was injected intraperitoneally and improved the ability of macrophages to inhibit growth and the ability of serum to inhibit growth and germination of Aphanomyces invadans.

In order to present an acceptable overview of the information available on Ergosan, general information is presented here.

A single intraperitoneal (i.p.) injection of 1 mg of Ergosan significantly stimulated the non-specific immune system of rainbow trout, augmented the proportion of neutrophils in the peritoneal wall, increased the degree of phagocytosis, respiratory burst activity and expression of interleukin-1β (IL-1β), interleukin-8 (IL-8) and one of the two known isoforms of tumor necrosis factor-alpha (TNF-α) in peritoneal leucocytes one day post-injection (Peddie et al. 2002). However, humoral immune parameters were less responsive to intraperitoneal alginate administration with complement stimulation only evident in the 1 mg-treated group at 2 days post-injection.

An evaluation of the effect of Ergosan (5 g kg−1) in prevention of columnaris disease (Flavobacterium columnare) reported that supplementation with Ergosan had no effect on cumulative mortality of 1.2 g rainbow trout, but a small non-significant improvement was noticed when 5 g fish were used (Suomalainen et al. 2009).

Sheikhzadeh et al. (2010) evaluated the effect of Ergosan (6 and 20 mg kg−1) on semen quality (spermatocrit, sperm concentrations, sperm motility and seminal plasma compositions) of rainbow trout (~2,300 g). In fish receiving 20 mg Ergosan kg−1; a significant increase of spermatocrit and sperm count and Ca2+ compared to the control group was observed. The aspartate aminotransferase and lactate dehydrogenase significantly decreased in both Ergosan groups, while no effect on sperm motility, K+, K+/Na+ ratio, total protein, glucose and triglycerides compared to the control group were observed. Ergosan exerts positive effects in male trout broodstock, but further studies are warranted with regards to mechanisms (Sheikhzadeh et al. 2010).

Ergosan (5 g kg−1) also significantly elevated SGR and feed intake (136.8 vs. 111 g fish−1), but reduced FCR (1.43 vs. 2.0) in rainbow trout (~110 g) (Heidarieh et al. 2012). Furthermore, lipase activity and leukocyte and erythrocyte counts also increased in Ergosan fed fish, but trypsin and amylase activities were not affected. Gut morphology evaluation of pyloric caeca and proximal intestine by light microscopy displayed normal appearance in both dietary groups, but a higher percentage of goblet cells (mucus producing cells) were seen in pyloric caeca and proximal intestine of the Ergosan fed fish.

Dietary Ergosan (5 g kg−1) significantly increased growth performance, lysozyme, protease, alkaline phosphatase and esterase activities in rainbow trout (≈110 g) compared to the control group where skin mucus agglutination of enterocytes was not observed (Sheikhzadeh et al. 2012). However, agglutination was observed in Ergosan fed fish. Moreover, the antibacterial activity of skin mucus towards Yersinia ruckeri was significantly higher in Ergosan fed fish after 50 days.

Merrifield et al. (2011c) investigated the effect of 5 g Ergosan kg−1 on growth performance, intestinal microbiota and gut histology of tilapia; for 9 weeks. Dietary Ergosan did not affect growth performance and intestinal microbiota (allochthonous and autochthonous, and species diversity and richness). No signs of cell or tissue damage, evaluated by light and electron microscopy, were seen in the Ergosan group compared to the control group. Trends towards elevated survival and body protein content, and a lower microvilli density in the posterior intestine were also reported. As dietary Ergosan did not affect the gut health status, a critical question arises. Does Ergosan reach the intestine and is it fermented in the stomach? This topic merits further investigation.

In a study evaluating the immunomodulatory activity of Ergosan (0.5 % supplementation) in sea bass, significant elevation in serum complement activity was reported after 15 days treatment, while significant increases were noticed in serum lysozyme, gill and liver heat shock protein (HSP) after 30 days (Bagni et al. 2005). However at the end of the experiment (45 days), no significant differences were noticed along with no effect on growth performance and FCR. A dramatic decrease in both innate and acquired immune parameters during the winter season was observed, but a partial recovery was noticed when the rearing temperature increased.

A 60-day study on beluga juveniles (~42 g) investigating the effect of different inclusion level of Ergosan (0, 2, 4 and 6 g kg−1) revealed a significant elevation in growth rate, FCR and body protein when beluga were fed at the two highest inclusion levels (Jalali et al. 2009). Generally, supplementation of Ergosan did not alter haematological parameters, except for lymphocyte count and survival rate was not different among the dietary treatments. In a more recent study, Heidarieh et al. (2011) evaluated whether Ergosan, 5 g kg−1 affected growth performance, immunocompetent cell population and plasma lysozyme content of beluga (~110 g). A significant increase was noticed in growth performance, lymphocyte count and lysozyme activity in plasma of fish fed Ergosan compared to the control group.

An evaluation of the effect of Ergosan on immune stimulation of white shrimp reported no obvious differences of haemolymph proteins and total haemocyte counts (~105 cells ml−1) (Montero-Rocha et al. 2006). However, a detailed analysis of the haemotocyte population showed significant changes in the relative levels of hyaline, semi-granular- and granular haemocytes. In vitro antibacterial activity of haemolymph towards two shrimp pathogens, V. harveyi and Vibrio parahaemolyticus revealed enhanced activity of the Ergosan treated shrimps. It is worth noting that the enhancement was greatest against V. parahaemolyticus. Furthermore, a significant improvement in growth and length of the shrimp was seen when they were fed Ergosan. Although Ergosan revealed positive effects to physiological and immunological parameters, further studies are recommended to elucidate optimum timing and concentration to ensure maximum benefit (Montero-Rocha et al. 2006).

In order to evaluate whether immunostimulants may act in synergy with pro– and prebiotics additional research is required. Probiotics appear to modulate immunity of the host by improving the barrier properties of mucosa and modulating production of cytokines (protein mediators produced by immune cells) and contribute to cell growth, differentiation and defense mechanisms of the host (Nayak 2010). Viable live probionts are better than the non-viable heat-killed probionts in inducing higher immune responses in rainbow trout, especially enhancing head kidney leucocyte phagocytosis, serum complement activity etc. In recent years, several in vivo and in vitro studies have investigated the interaction between dietary probiotics and immunocompetence in humans as well as in fish and aquatic animals (Gómez and Balcázar 2008; Dimitroglou et al. 2011; Ganguly et al. 2010; Nayak 2010). By increasing the host’s adaptive and innate immune mechanisms, LAB can protect the host against infection by enteric pathogens and tumor development. Immunological and other mechanisms behind the probiotic action may include; simulation of antibody secreting cell response (Kaila et al. 1992), enhancement of phagocytosis of pathogens (Panigrahi et al. 2004; 2005), modification/enhancement of cytokine production/natural complement activity (Panigrahi et al. 2007; Salinas et al. 2008b) and improvement of the host innate or acquired immune responses, direct effect on other microorganisms in the digestive tract, adhesion sites, microbial action or response stemming from microbial products, host products or food components (Oelschlaeger 2010). Consequently, probiotic bacteria may influence both adaptive and innate immune responses, and may reverse the increased intestinal permeability induced by antigens, but no information is available about long-term effects.

Nucleotide-supplemented diets are not strictly immunostimulants by definition but provide a dietary supplement that allows improved resistance to a pathogen insult. Readers with special interest on the use of nucleotide-supplementations in finfish and shellfish aquaculture are referred to the reviews of Li and Gatlin (2006) and Ringø et al. (2012).

Plant extracts

Some immunostimulants cannot be used because of various disadvantages, such as high production cost or limited effectiveness upon administration. Accordingly, numerous investigations have evaluated the effect of plant products on innate and adaptive immune response and their ability to control fish and shellfish diseases. To avoid duplication, studies on the topic; effect of plant products on disease resistance, innate and adaptive immune response of fish and shellfish reviewed by Dügenci et al. (2003), Galina et al. (2009), Harikrishnan et al. (2011) and Ringø et al. (2012) are not discussed in this sub-section and readers with special interest are referred to the original reviews. Recent research on the use of plant products in aquaculture is displayed in Table 5.

Table 5 Recent use of plant extract in aquaculture
Full size table

Nootash et al. (2013) investigated oral administration of green tea (Camellia sinensis) on expression of immune relevant genes and biochemical parameters in rainbow trout (~23.5 g) concluding that dietary supplementation, especially at an inclusion level of 100 mg kg−1, enhanced the antioxidant system and augmented the investigated immune parameters including immune-related gene expression. However, further investigations into different gene expressions, gut morphology, gut microbiota and challenge studies are waranted.

Chakrabarti and Srivastava (2012) evaluated the effect of prickly chaff-flower (Achyranthes aspera) on rohu (Labeo rohita) larvae and concluded that administration of 5 g kg−1 prevented tissue damage and provided protection against oxidative stress. Furthermore, prickly chaff-flower improved disease resistance against A. hydrophila when injected intraperitoneally (i.p.).

The effects of ginger (Zingiber officinale Roscoe) on growth performance, haematological and biochemical parameters, immune response and disease response against V. harveyi of Asian sea bass (Lates calcarifer Bloch) were investigated by Talpur et al. (2013). Growth performance was improved and blood parameters; glucose, lipid, triglyceride and cholesterol levels, were lower by dietary ginger. Moreover, ginger strengthened the non-specific immunity and protection against V. harveyi.

Wu et al. (2013b) investigated the effect of polypore mushroom (Coriolus versicolor) polysaccharides (CVP) on haematological, biochemical parameters as well as disease resistance against A. hydrophila; injected i.p. in allogynogenetic crucian carp. At an inclusion level of 0.5 and 1 g CVP kg−1 affected haematological and biochemical parameters, in contrast to a low inclusion level (0.25 g kg−1; no effect) and high inclusion (2 and 4 g kg−1; negative effect). Furthermore, fish fed 1 g CVP kg−1 prevented the experimental infection by A. hydrophila.

Wu et al. (2013) tested the effect of Sophora flavescens on the non-specific humoral responses (lysozyme, antiprotease and complement) and cellular immune responses (reactive oxygen species and nitrogen species and myeloperoxidase) and disease protection against i.p. injection of Streptococcus agalactiae in tilapia. Supplementation of S. flavescens, at all inclusion levels, significantly enhanced non-specific humoral responses and myeloperoxidase activity. Cumulative mortality in the challenge experiment was significantly reduced in all groups fed S. flavescens, but inclusion level at 1 g gave the best protection. Based on their results, the authors suggested that S. flavescens is a promising immunostimulant in tilapia aquaculture.

Macro- and microalgae and biotechnology potential

The generas Gracilaria (red algae; Rhodophyta) and Ulva (sea lettuces a group of edible green algae), have fast growth and low-cost production (Viera et al. 2005). In addition to their successful use in bioremediation of aquaculture effluent (Marinho-Soriano et al. 2012), they may also be used as feed additives in aquaculture, replacing FM. Positive properties of Gracilaria and Ulva are that they are biocompatible, biodegradable and safe for the environment and human health (Viera et al. 2005). However, prior to use as feed additives it is of importance to evaluate their effect on fish health; gut microbiota, gut morphology, immune stimulation and disease resistance.

Mass-cultured microalgae are the main component of the first tropic level in the aquatic food chain and they are the source of indispensable nutrients for larval and juvenile bivalves, and for the larvae of some crustacean and multiple fish species in mariculture (Brown et al. 1997). During the last decade, microalgae production and its use in aquaculture has been extended and optimized in hatcheries. Most algae species in aquaculture have been selected on the basis of their mass-cultured potential, cellular size and overall nutritional value (Brown et al. 1997; Alonso et al. 2012). The most frequently used microalgae species in aquaculture are; Skeletonema costatum, Thalassiosira pseudonana, Chaetoceros gracilis, Chaetoceros calcitrans, Isochrysis galbana, Tetraselmis suecica and Chlorella spp. (Coutteau 1996). These species can be produced industrially (Spolaore et al. 2006) or at a small scale in batches or in continuous in hatchery installations (Jorquera et al. 2010), where the combination of different microalgae species is optimized to provide a well-balanced diet and improve larval development (Benemann 1992).

Although the progress has been slow in the genetic engineering of microalgae, Walker et al. (2005) demonstrated the potential for genetic modification of Phaeodactylum spp. as well as the application of transgenic microalgae in aquaculture (Sayre et al. 2001). These investigations open the possibility of genetic transformation of microalgae, but the topic merits further investigation.

The wide variety of species and the morphological similarity between some algae species, make it necessary to use a combination of biochemical, physiological and morphological characters to correctly understand the taxonomic classifications. Molecular characterization with 18S rRNA and 16S rRNA has been used in the classification of 18 species of microalgae used in aquaculture (Alonso et al. 2012). Even though the molecular markers used in this study allowed optimal classification to genus level, the authors concluded that that other conserved markers should be evaluated in further studies.

Increasing knowledge regarding the antibacterial activity of different extracts of microalgae has evolved as new sources of specific antibacterial compounds have been reported. To the author’s knowledge, the first studies using microalgae in this respect were carried out by Austin and Day (1990) and Austin et al. (1992). Heterotrophically grown, spray-dried T. suecica used as feed for penaeids was observed to rapidly inhibit growth of prawn pathogenic strains of Vibrio (Austin and Day 1990) and when used as a feed additive for Atlantic salmon, the algal cells led to a reduction in the level of bacterial diseases (Austin et al. 1992). In a more recent study, pressurized lipid extracts from Dunaliella salina had an antimicrobial effect against several microorganisms of importance for the food industry (Escherichia coli, Staphylococcus aureus, Candida albicans and Aspergillus niger) (Herrero et al. 2006). When discussing marine bioactives it is also of importance to note that extracts from marine phytoplankton and macroalgae exhibit antibacterial activities (del Pilar Sánchez-Saavedra et al. 2010; Goecke et al. 2012).

Information is available on the use of Chlorella minutissima and Tetraselmis chuii bioencapsulated in Artemia during weaning of Senegalese sole (Solea senegalensis Kaup; Makridis et al. 2009) and the effect of T. suecica on growth, feed utilization and fillet composition of European sea bass (Tulli et al. 2012). Recently, several papers have investigated the effect of microalgae inclusion in gilthead seabream (Sparus aurata L.) diets on; the immune system (Cerezuela et al. 2012a), immune system and disease resistance (Cerezuela et al. 2012b), intestinal ultrastructure and gut microbiota (Cerezuela et al. 2012c), and in combination with synbiotics (inulin and Bacillus subtilis) on intestinal gene expression (Cerezuela et al. 2013b).

It is well known that quorum sensing, bacterial cell-to-cell communication with small signal molecules such acyl-homoserine lactones, regulates the virulence of many pathogenic bacteria. In a study with 19 micro-algal strains, Natrah et al. (2011) investigated the effect of the acyl-homoserine lactones, and reported that extracts of the most promising micro-algal strain; Chlorella saccharophilia inhibited quorum sensing regulated gene expression in all three reporter strains, Chromobacterium violaceum, Escherichia coli and V. harveyi, tested. These results are of high interest for future aquaculture and the topic merits further investigation.

The concept of functional food as a method to protect or improve consumer health was introduced in Japan at the beginning of the 1980s, based on several studies demonstrating the connection between diet and possible health effect (e.g. Salminen et al. 1998; Saulnier et al. 2009; Lordan et al. 2011). Ibañez and Cifuentes (2013) discussed the benefits of using algae as natural sources of functional ingredients. Even though there are beneficial effects for one or more functions of the human organism, the authors suggested that more research is needed for a comprehensive screening of bioactive metabolites produced by different marine organisms and that biomass production, recovery of bioactives and further processing must be optimized. These arguments are also valid for the aquaculture industry and deserve further attention.

Conclusions and further perspectives

The present study addressed key issues of importance in finfish and shellfish aquaculture. However, there are several related issues that also deserve attention. We therefore recommend readers to have a closer look at the review papers of Defoirdt et al. (2011; alternative to antibiotics for the control of bacterial disease in aquaculture), Crab et al. (2012; biofloc technology in aquaculture), Raina et al. (2009; quorum sensing), Galloway et al. (2012; inhibitors of quorum sensing in Gram-negative bacteria), Kalia (2013; quorum sensing inhibitors), Beaz-Hidalgo and Figueras (2013; Aeromonas—secretion systems, iron acquisition and quorum sensing mechanisms) and Cabrita et al. (2010; cryopreservation of fish sperm). Furthermore, the research papers of Dr. Martins’s group (Tacchi et al. 2011, 2012; transcriptomic responses to functional feeds and FM substitution), professor Zhou’s group (Chen et al. 2010; Cao et al. 2012; N-acetyl homoserine lactones as sisnal molecule), seaweeds as potential ingredient in aquafeed (Henry 2012; Saez et al. 2013), seafood biopreservation by LAB (Ghanbari et al. 2013) and the untapped source of novel compounds in the marine environment and their potential as novel drugs, personal care products and antimicrobial peptides (Kim et al. 2008; Pan et al. 2008; Wijffels 2008; Sperstad 2009; Schumacher et al. 2011) also focus on important issues that merit further investigation.

Recently, rapid genetic sequencing methods have become available, and these could be important tools to elucidate the diversity of antibiotic-resistance genes present in the fish gut and aquaculture environments. These approaches should allow the diversity of antibiotic resistance genes in the gut to be analysed, even when antibiotics are not used, and allow appropriate therapies to be proposed based on the presence of any resistant genes.

DNA vaccines are promising candidates for future disease control in aquaculture. DNA vaccines against Rhabdoviruses have proven to be highly efficacious which has resulted in commercialisation of a vaccine against IHNV. For other pathogens these vaccines have shown variable effects and investigations to improve vaccine potency are being undertaken. Aspects related to the safety of DNA vaccines have to be addressed to make the use of these vaccines more acceptable.

During the last two decades several comprehensive reviews have reflected on the promising use of probiotics in aquaculture. This paper emphasizes the wide application spectrum of allochthonous LAB, which should stimulate further developments in the field. Research in aquaculture probiotics is still at its infancy, and emphasis should be towards topics dealing with probiotic adhesion and mechanisms, among others. Importantly, host safety must be considered as well as the early application of probiotics, which has been shown to provide enhancement of beneficial effects. Even though numerous studies have investigated the effect of immunostimulants on the immune system of finfish and crustaceans the issue still merits further investigation as innate immune response is biologically linked to gut health. There is also a need to emphasise the effect of immunostimulants on adherece and colonization of potential probiotics to the intestinal muscus, ligand-receptor interaction, involved signal transduction pathways, and expression of pro-inflamatory and anti-inflamatory cytokines. Although our understanding of microalgae and their biotechnology potential has grown during the last decade, additional knowledge is needed especially on their antibacterial potential and their potential as functional dietary ingredients.

Furthermore, with the increased inclusion of plant-based feedstuffs in diets, the intake of antinutritional factors (ANFs) will increase. The effects of different ANFs on digestive physiology and ultimately on metabolism will change utilization of specific nutrients (Francis et al. 2001; Krogdahl et al. 2010). This will change the dietary levels of specific nutrients needed to meet nutritional requirements. Such adjustments require extensive research in addition to the research needed to adjust recommended nutrient requirements for today’s farmed fish. Furthermore, the gut microbiota, which may be influenced by various dietary nutrients, non-nutrients and ANFs, is also of importance for the host’s gut and general health (Bauer et al. 2006). In their review devoted to important ANFs, Krogdahl et al. (2010) speculated that the intestinal microbiota may modify the ANFs and hence influence their interactions and biological effects. However, to the authors’ knowledge no information is available on this topic in relation to finfish and shellfish and merits further investigation.