Reviews in Aquaculture (2020) 12, 943–965 doi: 10.1111/raq.

12365

Sustainable aquaculture requires environmental-friendly

treatment strategies for fish diseases
Thora Lieke1,2 , Thomas Meinelt1, Seyed Hossein Hoseinifar3, Bo Pan4, David L. Straus5 and Christian
E. W. Steinberg2,4
1 Department of Ecophysiology and Aquaculture, Leibniz Institute of Freshwater Ecology and Inland Fisheries, Berlin, Germany
2 Faculty of Life Sciences, Freshwater and Stress Ecology, Humboldt University of Berlin, Berlin, Germany
3 Department of Fisheries, Faculty of Fisheries and Environmental Sciences, Gorgan University of Agricultural Sciences and Natural Resources,
Gorgan, Iran
4 Faculty of Environmental Science and Engineering, Kunming University of Science and Technology, Kunming, China
5 U.S. Department of Agriculture, Agricultural Research Service, Harry K. Dupree Stuttgart National Aquaculture Research Center, Stuttgart, AR,
USA

Correspondence Abstract
Thora Lieke, Leibniz Institute of Freshwater
Ecology and Inland Fisheries, Mu €ggelseedamm Many classical therapeutants are going to be banned in Europe, and an urgent
310, 12587 Berlin, Germany. Email: lieke@igb- need for alternatives is emerging. This issue can be exemplified by one major par-
berlin.de asitic disease in aquaculture and ornamental fish breeding: velvet disease. This
disease, caused by dinoflagellates of the genera Amyloodinium and Piscinoodinium,
Received 24 December 2018; accepted 5 June
is an important infection affecting cultured freshwater and marine ornamental
2019.
and food fish, and consistently causes great financial loss to the associated indus-
tries. Therapeutants available contain copper, malachite green, or methylene blue,
and which can be toxic to non-target organisms in the surrounding environment.
As a result, these chemicals are banned for use by the aquaculture industry in sev-
eral countries, and a prohibition for commercial ornamental fishkeeping is likely
to follow in most countries. Increasing development of resistance to therapeu-
tants, and growing public awareness for animal welfare and environmental pro-
tection, have prompted research in the areas of alternative treatment options and
immunostimulants. Hydrogen peroxide and peracetic acid are possible ‘green’
therapeutants which do not contribute residues to the environment. Natural feed
supplements such as pre- and probiotics can increase animal welfare and prevent
stress and/or infections. Humic substances are another promising, natural
immunostimulants which will be considered in depth. The aim of this review is to
provide an overview of risks and benefits of current treatment options and new
approaches to replace harmful therapeutants and minimize the number of toxic
residues discharged into the environment. Treatments will be discussed on vari-
ous parasitic infections and focus, where available, on Amyloodinium and Pisci-
noodinium.
Key words: biotics, copper sulphate, disease prevention, humic substances, immunostimulants,
Oodinium.

aquaculture). In aquaculture, up to 50% of production loss

Introduction
is caused by diseases (Assefa & Abunna 2018). High stock-
Finfish are hosts to a plethora of both ecto- and endopara- ing densities and poor water quality present optimal condi-
sites. These parasites are part of every ecosystem and they tions for the infestation and reproduction of parasites,
generally have limited effects on the fitness of healthy fish. leading quickly to pathogenic levels. Furthermore, spread-
However, parasites can become a problem under stressful ing of infectious pathogens is supported by the inevitable
conditions which can occur when fish are reared in captiv- transport of fish and equipment (Subasinghe et al. 2001).
ity (e.g., public aquariums, the ornamental fish trade and Parasitic infections can spread not only inside a system but

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This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and
distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.
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T. Lieke et al.

also to adjacent culture units via aerosol transfer (Paperna These supplements are used to increase of overall and
1980). These anthropogenic stressors can lead to acute and immune-related health of fish and thereby stimulation of
chronic stress, and the need to cope with the allostatic load host-specific defense mechanisms against pathogens. We
requires (energetic) metabolic reorganization, subsequently discuss the idea of whether or not the stimulation of the
decreasing growth performance, depressing immune system defense system increases overall protection and helps to
functions and leaving fish even more vulnerable to infec- prevent velvet disease as well as other pathogen-induced
tions (Bly et al. 1997; Tort 2011). For example, catfish diseases.
exposed to 30-min low-water stress had about 20% mortal-
ity when challenged with Edwardsiella ictaluri compared to
Velvet disease is a risk to both freshwater and
control fish (Small & Bilodeau 2005). This review focuses
marine finfish
on velvet disease, a major disease threat in warm water cul-
ture, as a foundation to review the transition to emerging Velvet disease (Noga 2011), also known as Rust or Gold
treatment approaches. Information on this disease is often Dust disease, is caused by dinoflagellates of the genera
distributed to different databases, paper, and books of Amyloodinium (Brown 1931; Brown & Hovasse 1946) and
research experts. In the first part we summarize informa- Piscinoodinium (Sch€aperclaus 1954; Lom 1981) and is a
tion on the different parasites, life cycles, and misinterpre- major threat in warm water fish culture facilities. It also
tations due to outdated taxonomical terms. poses a threat in coolwater facilities, although white spot
Current treatment options such as triphenylmethane disease, caused by Ichthyophthirius multifiliis in freshwater
dyes, copper, acriflavine and formalin, as well as their and Cryptocaryon irritans in marine environments salmon
effects on different life stages, are considered. Risks associ- lice (Lepeophtheirus salmonis), and species of the genus
ated with many of these ‘traditional’ chemical therapeu- Myxobolus are more are more of a threat. The name ‘Oodi-
tants to fish, to the environment and to consumers have led nium’, which can be found in old publications, is still used
to bans for their use in the aquaculture industry of many by ichthyopathologists and aquarium hobbyists but does
countries. The search for alternative environmentally not distinguish between the two genera (Lom & Dykova
friendly approaches for diseases has increased during the 1992). Taxonomically, the genus Oodinium Chatton, 1912,
last decade and shall be considered in detail with emphasis still exists and contains six species; however, their exact sys-
on velvet disease where available. tematic position is unclear (Guiry & Guiry 2018). These
Furthermore, we reviewed recent international literature, species are ectoparasitic dinoflagellates of marine inverte-
as well as ‘grey literature’ and included results from our brates. Species of the genus Crepidoodinium are also
own research. Application of hydrogen peroxide (H2O2) ectoparasites to marine fish but do not cause velvet disease.
and peracetic acid (PAA) as disinfectants to reduce the Figure 1 shows the systematic positions and relations of the
pathogenic load will be discussed. Supplementing food with different genera.
live cultures of microorganisms and their immunostimula- Piscinoodinium pillulare (Figure 2a) and P. limneticum
tory effects have been the subject of many studies in human are important pathogens of both, tropical and temperate
and fish. We will discuss the different types of “biotics” freshwater fishes, while Amyloodinium ocellatum (Fig-
including their interaction and different modes of action. ure 2b) is the marine analogue and is one of the most
Finally, supplementation of food and culture water with important parasites infesting warm water marine or brack-
different plant extracts and humic substances is presented. ish water fishes (Noga 2012). These two dinoflagellate

A. ocellatum
Thoracosphaerales Thoracosphaeraceae Amyloodonium
A. amylaceum

P. limnecticum
Dinophyceae Suessiales Suessiaceae Piscinoodinium
P. pillulare

Oodinium
Dinophyceae Oodiniaceae
ordo inc. sed.
Crepidoodinium

Figure 1 Systematic position and relation of Dinophycaea historically termed as “Oodinium”. Created, based on information from Guiry and Guiry
(2018) and Guillou and Not (2018).

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Protection against pathogens – chemical treatments versus immunostimulants

Figure 2 Trophonts of a: Amyloodinium

ocellatum from Brown and Hovasse (1946)
with permission from John Wiley and Sons
and b: Piscinoodinium pillulare from Lom and
Schubert (1983) with permission from Folia
Parasitologica.

genera are morphologically very similar and range in size

from 80 to 150 lm. However, Piscinoodinium obtains
essential parts of its nutrition from photosynthesis,
although light is not crucial for survival, while Amylood-
inium lacks chloroplasts (Hoffman 1999; Abowei et al.
2011; Noga 2012). The life cycle of both genera is con-
trolled by temperature and consists of three stages. First,
free-swimming dinospores (also called gymnospores; Lom
and Dykova (1992) infect the host and penetrate the epithe-
lium (typically skin or gills) with an extension called a ’rhi-
zoid’. As Figure 2 indicates, the extension of
Piscinoodinium is nail-shaped (Abowei et al. 2011), while
that of Amyloodinium is root-shaped (Lom & Lawler 1973;
Cheung et al. 1981). Furthermore, Amyloodinium has a
special tentacle-like movable organ called a stomopode,
which Piscinoodinium lacks. The function may be food
ingestion and injection of lytic bodies into the host cells
(Guillou & Not 2018).
After attaching to the host, dinospores transform into
Figure 3 Life cycle of Amyloodinium ocellatum. Free swimming dinos-
trophonts which feed on the host’s cells. Finally, the para- pores embed in the host skin using rhizoids and ultimately turn into tro-
sites detach and transform into tomonts. This encapsulated phonts. Detaching trophonts encapsulate and divide into tomonts, the
form is the reproductive stage, and a single tomont pro- reproductive stage. After several cell divisions, dinospores are released
duces as many as 256 new dinospores that are ready to from the capsule and infest new hosts. From Francis-Floyd and Floyd
infect a new host. Tomont division occurs between 16 and (2011) and Noga (1987) reprinted with permission from Science.
30°C (Saraiva et al. 2011). At 25°C and under ideal repro-
duction conditions (e.g. salinity, host density), the entire host is very vulnerable to osmoregulatory impairment and
life cycle (Figure 3) takes about 4 to 5 days. secondary infections by other pathogens such as bacteria or
The high pathogenicity of these organisms is caused not fungi (Woo & Buchmann 2012).
only by extensive structural damage to the epithelium by
the rhizoid itself but also by histopathological changes such Diagnosis, current treatments, and related risks
as degeneration and necrosis in cells adjacent to the attach-
ment site. These changes are possibly induced by toxic sub- Velvet disease is highly contagious and considering its rapid
stances or irritants produced by the parasites (Lom & lifecycle, it is imperative to diagnose and treat as early as
Lawler 1973; Paperna 1980). Under such conditions, the possible. Visible symptoms of an infestation in fish are

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T. Lieke et al.

flashing, rapid gill movement, loss of appetite (and conse- crustaceans and gastropods (Hodson et al. 1979; Flemming
quently loss of weight), clamped fins and lethargy. The key & Trevors 1989). Not only the basic substance, but residues
symptom is a velvet film or ‘dusting’ on the skin ranging in and derivatives of these compounds can be unpredictably
colour from gold to brown to green. In an early stage of toxic to the environment and pose a high risk to fish, par-
infestation, symptoms may be weakly pronounced, if they ticularly when already weakened by infection (Alderman &
occur at all. Analyzing smears of mucus and skin or gills Clifton-Hadley 1993; Jung et al. 2001; Srivastava et al.
under the microscope can help to detect dinospores at an 2004).
early stage of infestation. Since dinospores detach after host Copper sulphate (CuSO4) is the most commonly used
death, fish have to be diagnosed while still living or directly therapeutant to treat Piscinoodinium and Amyloodinium
after death (Woo & Buchmann 2012). Levy et al. (2007) infestations. Concentrations of 0.5 to 10 mg L 1 CuSO4
developed a sensitive and specific PCR assay to detect Amy- were lethal to sporulating tomonts and dinospores of Amy-
loodinium using ribosomal DNA. They were able to detect a loodinium but did not interrupt the division process of the
single cell from each of the life cycle stages. An even more encapsulated tomont (Paperna 1984). Exposure to 1 mg
sensitive and specific approach is a loop-mediated isother- L 1 CuSO4 for 12 to 24 h reduced the reproductive success
mal amplification (LAMP) assay developed by Pic on- of tomonts, but dinospores, which could cause reinfesta-
Camacho et al. (2013) to detect all life stages of Amylood- tion, were still produced. Ideal treatment strategies would
inium. Using an enzyme-linked immunosorbent assay, either use concentrations high enough to ensure complete
Smith et al. (1992) showed the production of antibodies inhibition of the encapsulated tomont or continuous expo-
against Amyloodinium after infestation; this might also be sure with low concentrations to kill dinospores; this could
used to monitor pathogens. All these methods, however, be harmful or cause stress to fish. Continuous treatment
require specific laboratory equipment as well as qualified with a desired therapeutic concentration of 0.15 to 0.2 mg
personnel to perform the assays. Therefore, while they may L 1 free copper ion for 2 to 3 weeks helped to eliminate
have great importance for research purposes, microscopy is infection with Amyloodinium in marine systems (Francis-
still the standard diagnostic procedure for practical applica- Floyd & Floyd 2011). Concentrations need to be monitored
tion in aquaculture facilities. at least once a day as the free copper ion does not stay in
The infection is usually far advanced before being diag- solution for long and multiple treatments are often neces-
nosed, and by then, fish are already in a weakened condi- sary. Problems occur, however, as copper is highly toxic to
tion. At this point, treatment of the disease is often invertebrates and algae, even at low concentrations (Fran-
difficult. Prolonged immersion in common, non-iodized cis-Floyd & Floyd 2011; Noga 2012). For extremely sensi-
table salt at about 5 g L 1 (5 ppt or parts per thousand) is tive fish, copper is toxic at concentrations close to those
an effective treatment against Piscinoodinium infestations as required for treatment (Cardeilhac & Whitaker 1988). A
the trophonts dislodge (Puello-Cruz et al. 2010). Con- concentration of 10 lM CuSO4 (1.6 mg L 1 CuSO4) is
versely, short periods of hyposalinity (0 g L 1) dislodge used to induce leucocyte recruitment in zebrafish larvae
Amyloodinium trophonts from the skin and gills (Kingsford (d’Alencßon et al. 2010).
1975; Lawler 1977). After either treatment, fish need to be Depending on different factors such as alkalinity, organic
transferred to a clean environment to prevent a new infes- matter content, as well as adsorption to surfaces, copper
tation, and the treatment has to be repeated several times can quickly precipitate which reduces the amount of
with periods of a few days recovery between treatments to bioavailable copper (Smith et al. 2015). Furthermore, the
exclude recurrence. Multiple treatments are essential for an concentration of specific ions (e.g., Ca2+, Mg2+, Na+, H+)
effective regimen because the life cycles of individual organ- influence the active uptake of copper by Na+ transporters
isms are not synchronized; therefore, the parasite may be as they compete for the binding sides (Niyogi & Wood
present in several stages concurrently. Immersion treat- 2004). In freshwater ponds, risk of acute toxicity can be
ments may be employed on a small-scale, but are not logis- reduced if the concentration does not exceed 1% of the
tically practical for public aquaria, large professional total alkalinity (Boyd & Massaut 1999). McNevin and Boyd
aquaculture operations or wholesale of ornamental finfish. (2004) reported that 99% of the administered copper is
In addition, not all fish can tolerate the osmotic changes bound to pond sediments. To decrease the likelihood of
equally well and immersion treatment, as well as the neces- copper entrance into the effluents, water should not be dis-
sary transfer of fish, adds to increased stress levels. charged for at least 72 hours. When copper is used in the
Commercially available therapeutants for velvet disease marine environment, the concentration must be monitored
contain metals (mostly copper), triphenylmethane dyes, frequently because the free copper ion is unstable (Francis-
acriflavine, or formalin. These chemicals are effective at Floyd & Floyd 2011; Noga 2012). Different ion configura-
killing parasites, but are also harmful to non-target organ- tions of copper might have different specific toxicities;
isms, in particular plants and invertebrates such as however, the evidence is contradictory whether Cu2+ or the

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Protection against pathogens – chemical treatments versus immunostimulants

reduced form Cu+ is more toxic (Fargasova 1998; Mathews 33.2 lg L 1. This discrepancy can be caused by different
et al. 2015). Chelated, or complexed, forms of copper are strains of a given fish species as well as differences in the
sometimes recommended as they reportedly reduce the condition of fish, water chemistry or compound quality
toxicity to fish. However, the bioavailable concentration is used for testing. It shows, however, that toxicity can vary
also reduced, which limits the effectiveness against patho- massively even in laboratory studies, and application in
gens (Keith 1981). While efforts are being made to obtain tanks or rearing facilities can be challenging. Furthermore,
United States (U.S.) Food and Drug Administration Van Duijn (1967) reported on fish sterility after application
(FDA)-approval for the use of copper sulphate as a thera- of acriflavine. This compound binds to DNA and inhibits
peutant to treat freshwater fish diseases in ponds and protein biosynthesis; the mutagenic potential was reported
hatcheries (Straus et al. 2016), it is not approved. Likewise, in different species such as Drosophila melanogaster and
environmental concerns in the European Union (EU) pro- Chlamydomonas reinhardtii (Tubbs et al. 1964; Xamena
hibit the use of copper compounds. et al. 1984; Dorthu et al. 1992). Obstoy et al. (2015)
The triphenylmethane dyes include malachite green, reported DNA damage in cultured human lymphocytes
methylene blue and crystal (gentian) violet. These have and lung cancer cells after exposure to 0.025% acriflavine.
been used since the mid-1930s against a plethora of patho- In summary, acriflavine is genotoxic and is banned for use
gens in fish culture. They are fungicides (especially effective in aquaculture in the EU (Penninks et al. 2017).
against Saprolegnia spp.), antiseptics and are effective treat- As mentioned previously, formalin is also used as a treat-
ments against internal and external parasites (Sudova et al. ment against ectoparasitic infections in cultured fish,
2007; Andersen et al. 2009). None of the tested concentra- including velvet disease. Amyloodinium infestation on the
tions of malachite green (0.1–100 lg L 1) had a lethal skin and gills of juvenile bullseye puffers (Sphoeroides annu-
effect on Amyloodinium, although concentrations of 0.5 lg latus) was significantly decreased after treatment with
L 1 suppressed sporulation (Paperna 1984). However, the 51 mg L 1 formalin for 1 h or 4 mg L 1 formalin for 7 h
compounds themselves, as well as their metabolites are 
(Fajer-Avila et al. 2003). Effectiveness however, is affected
toxic and carcinogenic. Malachite green and methylene by ambient temperature, water quality (i.e., salinity and
blue are toxic to channel catfish (Ictalurus punctatus) and content of dissolved organic carbon [DOC]), and storage
rainbow trout (Oncorhynchus mykiss) at concentrations of conditions (Abowei et al. 2011). During in vitro studies,
0.2 and 16 lg L 1, respectively (Willford 1966). Further- trophonts exposed to 25 to 200 mg L 1 formalin retracted
more, current studies showed malachite green and its their rhizoid and transformed into tomonts within 1 to 3 h
reduced form, leucomalachite green, to be in vivo mutagens (Paperna 1984), explaining the reduced infection load in
(Mitrowska et al. 2005, 2008; Culp et al. 2006). The same the studies mentioned above. Complete inhibition of
applies to crystal violet and brilliant green (Thomas & Mac- tomont division was observed only after 24 h exposure to
Phee 1984; Andersen et al. 2009), and highly concentrated 200 mg L 1 at 27–30°C. After 12 h exposure to 200 mg
methylene blue has been linked to neurotoxicity and L 1 formalin, cell division was only reduced by about 40%
encephalopathy (Sweet & Standiford 2007; Patel et al. leaving enough motile dinospores for recurrence. Formalin
2012). These substances persist in tissue for an extended (50 to 150 mg L 1) has been used successfully to reduce
period of time and therefore pose a risk not only to the fish infections with Saprolegnia parasitica in rainbow trout
but also to human health when fish are consumed. These (Gieseker et al. 2006). However, the 96-h-LC50 values of
chemicals are labeled as harmful to human health and the formalin at pH 7 on silver barb (Barbonymus gonionotus
environment, and also as toxic by EU regulation (EG1272 Bleeker), common carp (Cyprinus carpio L) and snakehead
2008) 1272/2008 (CLP); they are prohibited for use in (Channa striatus Flower) were 75, 107 and 150 mg L 1,
aquaculture within the EU. Malachite green and crystal vio- respectively (Chinabut et al. 1988), which is in the range of
let are also banned to use on fish intended for human con- therapeutic concentration. The parent compound of for-
sumption in the US and Canada (Culp & Beland 1996; malin, formaldehyde, is classified by the International
Srivastava et al. 2004; Andersen et al. 2009). Agency for Research on Cancer (IARC) as carcinogenic to
Acriflavine (acriflavinium chloride) is used to treat and humans (Group 1) (Cogliano et al. 2005; Bianchi et al.
prevent external infections in ornamental fish and has simi- 2007). It is currently approved for specific uses in aquacul-
lar antimicrobial and antifungal properties as the triphenyl- ture in the US, but not in the EU.
methane dyes (Plakas et al. 1999). Paperna (1984) reported Noga (2011) cites a personal communication mentioning
that total sporulation of Amyloodinium was only sup- chloroquine diphosphate as a treatment against Amylood-
pressed on incubation with 6 lg L 1 acriflavine. This con- inium. Treating false percula clownfish (Amphiprion ocel-
centration is in the range of the 48 h-LC50 value (6.8 lg laris) with 5 to 10 mg L 1 chloroquine diphosphate for
L 1) determined for channel catfish by Clemens and Sneed 10 days killed dinospores when they were exiting the cyst.
(1958), while Willford (1966) determined the LC50 value as Oral administration of chloroquine was used in cultured

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T. Lieke et al.

red drum (Sciaenops ocellata) as a chemotherapeutic agent organophosphates, commonly used against parasitic crus-
against amyloodinosis (Lewis et al. 1988). As chloroquine taceans and monogeneans. The use of drugs like trichlorfon
is used as an anti-malaria drug for humans, it is expensive. or praziquantel in bath treatments for ectoparasites also has
Furthermore, it is highly toxic to invertebrates, especially the disadvantage of resistance development (Umeda et al.
echinoderms and corals, and not approved for fish 2006). Reverter et al. (2014) report the resistance of platy-
intended for human consumption. In vitro studies using helminthes to praziquantel. There is limited available
the fish cell line PLHC-1 derived from topminnow (Poecil- knowledge about any resistances of Oodinium against
iopsis lucida) showed reduced lysosomal function, increased antiparasitic compounds, but the development of resistance
metallothionein and glucose-6P-dehydrogenase levels as must be expected.
well as increased activity of succinate dehydrogenase (Zur-
ita et al. 2005).
Alternative treatment strategies
Another major problem of many therapeutants used
against pathogens in fish is the development of resistance. The downside of many ‘traditional’ chemical therapeutants
Resistance can often be attributed to misuse or over use of as well as the lack of substances allowed in aquaculture,
precautionary treatments to prevent infections, or applica- require new strategies that have led to research focusing on
tion of low doses for treatments that only kill the weak alternative therapeutants. In general, there are two different
pathogens. Since therapeutants are applied to the water approaches: 1) treatment of the culture water and the
body, residues are being discharged into the natural envi- pathogen to reduce the overall load, and 2) treatment of
ronment. This dilution exerts a selection pressure and the fish to make them less susceptible against infections by
increases development of resistance even further, which not increasing the general stress resistance or by activating
only endangers fish and environment but also leads to defense systems (stimulation of immune response) that
human safety concerns, including human pathogen resis- protect against the pathogens.
tance and foodborne transmission of antimicrobial- and
multi-drug resistant microorganisms (White et al. 2002;
Hydrogen peroxide and peracetic acid as water
Hur et al. 2012). Multi-drug resistance (resistance to more
disinfectants
than two classes of antimicrobial agents) has been found
for example in Yersinia ruckeri, Aeromonas salmonicida and Research on alternative therapeutants for the treatment of
Piscirickettsia salmonis (Balta et al. 2010; Kim et al. 2011). velvet disease is rare and the few existing studies evaluate
Furthermore, resistance genes of important fish pathogen the effects on Amyloodinium. Montgomery-Brock et al.
species have been found to be commonly located on mobile (2001) studied the effect of H2O2 on trophonts of Amylood-
genetic elements such as the transferable plasmid IncA/C inium in cultured Pacific threadfin (Polydactylus sexfilis).
and Class 1 integrons, allowing easy transmission of resis- Application of 75 and 150 mg L 1 H2O2 for 30 min
tances by horizontal gene transfer, even between different resulted in a reduced number of parasites. However, treat-
bacterial species (Miller & Harbottle 2018). In contrast to ment with 300 mg L 1 H2O2 resulted in mortality of
the resistance of bacteria to antimicrobial substances, com- exposed fish. Treating European seabass (Dicentrarchus lab-
parably little is known about the resistance of parasites rax) with 100 and 200 mg L 1 H2O2 for 30 min signifi-
against chemical treatments. Most occurrences of the resis- cantly decreased the Amyloodinium load on gills (Seoud
tance of parasites usually originate from human or veteri- et al. 2017).
nary medicine. Ectoparasites of fish can be treated with An alternative to using H2O2 is the application of PAA
antiparasitic compounds which are routinely used in other (also known as peroxyacetic acid or acetylhydrogen perox-
higher vertebrates like birds and mammals. Livestock inten- ide), which is a stabilized mixture of acetic acid, H2O2, and
sification has led to an increased reliance on the use of water. It is used as a disinfectant for agriculture, food pro-
antiparasitic compounds to control parasites. cessing, and medical and veterinary facilities (Malchesky
In Norway, Scotland and other countries in recent years, 1993; EPA 2005). More recently, PAA has been used in
the patterns of sea lice (Lepeophtheirus salmonis) treatment wastewater treatment, commercial laundries and in aqua-
have changed as the resistance to traditional treatments has culture in several countries (Straus et al. 2017). Peracetic
increased (Sevatdal et al. 2005; Costello 2006; Lees et al. acid has proven to be an effective treatment against various
2008; Jones et al. 2013). The emergence of resistant para- aquatic pathogens including fungi and bacteria (Jussila
sites has limited the choice of effective compounds with a et al. 2011; Marchand et al. 2012). Straus et al. (2012)
resulting adverse effect on animal welfare and loss in ani- found that 2.5 mg L 1 PAA was effective at controlling
mal productivity (Taylor 1999). Resistance of parasites was saprolegniasis on channel catfish eggs, whereas Mitchell
further described by Roth et al. (1993), Burka et al. (1997) et al. (2009) reported that 250 mg L 1 H2O2 was the opti-
and Athanassopoulou et al. (2009) against mal treatment in an earlier study with Saprolegnia in the

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Protection against pathogens – chemical treatments versus immunostimulants

same system. Furthermore, PAA can be used as a treatment evaluated carefully (Cobo et al. 2013; Cobo Labarca et al.
against the fish parasite Ichthyophthirius multifiliis, which 2015). A major problem with oral vaccination is that doses
causes Ich, or white spot disease in freshwater fish. This cil- are not distributed equally among all fish but differ based
iated protozoan has a triphasic life cycle similar to Piscinoo- on feeding behavior. Furthermore, the harsh gastric envi-
dinium and Amyloodinium. Meinelt et al. (2009) reported ronment often breaks down the antigen, making it ineffec-
that different developmental stages of I. multifiliis have tive (Embregts & Forlenza 2016). Encapsulation with
varying degrees of sensitivity to PAA. Application of 0.6 to microalgae, alginate particles, nanoparticles or biofilms for
0.9 mg L 1 PAA killed up to 82% of newly released example could help reduce this breakdown (Mutoloki et al.
tomonts, but none of the tested concentrations (0.5 to 2015; Embregts et al. 2019). Injecting the fish is the most
3.0 mg L 1) halted theront production of encysted effective way of protection. Viji et al. (2013) treated orna-
tomonts. Pic on Camacho (2010) reported that treatment mental goldfish with whole cell, extracellular products,
with 8 to 15 mg L 1 PAA for 1 h was effective at killing all outer membrane proteins and biofilm vaccines from Aero-
developmental stages, including cysts. monas hydrophila which decreased mortality when the fish
Similar to H2O2, PAA produces hydroxyl radicals that were challenged. Furthermore, they supplemented the vac-
oxidize enzymes and proteins, and increase the permeabil- cination with extracts from Asparagus racemosus which
ity of cell walls by destruction of sulfhydryl and sulfur com- increased phagocytosis, bactericidal activity of the serum
pounds. But is more potent than H2O2 because of its fat and albumin:globulin ratio compared to the group without
solubility; in contrast to the microbial breakdown of H2O2 immunoadjuvant.
by catalase activity, PAA is primarily degraded by chemical In order to produce vaccination against parasites, those
oxidation (Block 1991, 2001; Kitis 2004). This non-specific parasites have to be cultivated. In some cases, rearing can
mode of action prevents adaptation of microorganisms, be done in vitro; A. occelatum can be propagated using G1B
therefore preventing the development of resistance. The cells (Noga 1987; Cobb et al. 1998). Often a cultivation of
advantages of H2O2 and PAA over conventional therapeu- parasites requires a fish host population making the vacci-
tants become even more apparent when considering envi- nation expensive and laborious. Recent advances include
ronmental impact. While H2O2 degrades to water and the development of multivalent vaccines, synthetic pep-
oxygen, PAA degrades to water, oxygen and acetic acid, tides, and subunit vaccines that use immunogenic parts
which is quickly metabolized by microorganisms. In con- (Plant & LaPatra 2011; Dadar et al. 2017). However, injec-
trast to conventional therapeutants, no toxic residues are tion vaccination requires separate handling of each fish
discharged into the environment. Thus, H2O2 and PAA resulting in stress and subsequent feed intake reduction or
have great potential to be effective against Piscinoodinium injuries; fish also have to be of sufficient size (excluding
and to replace conventional therapeutants. Future research young fingerling) to be eligible for injection vaccination.
determining effective concentrations to treat the different For more details about the present status of fish vaccina-
lifecycle stages and the effects of both substances on the tion, we recommend the reviews by Muktar et al. (2016)
hosts would be extremely valuable. and Assefa and Abunna (2018).

Vaccination to enhance the immune system Immunomodulatory feed supplements as an approach to

increase animal welfare
As in terrestrial vertebrates, vaccination is a method to pro-
tect against infections using the adaptive immune system. Chronic stress and infection lead to immune system sup-
Exposition to an immunogen primes the immune system pression of the host (Tort 2011; Zanuzzo et al. 2019). As a
by forming T- and B-cells. If challenged again later, these result, the defense systems are weakened and susceptibility
primed cells recognize specific antigens, starting defense to disease is increased. Subsequently, a treatment against an
mechanisms and antibody production. infestation is inevitable; however, as explained above, many
Oral vaccination with antigens was tested, but gave con- conventional therapeutants can be toxic and cause addi-
troversial results as antigens are often degraded by the gut tional stress which may result in loss of fish. Furthermore,
biota (Hart et al. 1988; Irie et al. 2005). Most commonly, inside the EU, many of the traditional chemical therapeu-
fish are vaccinated by dip or bath immersion, oral vaccina- tants are already banned1 . An alternative approach is to
tion or by intramuscular or intraperitoneal injection (Som- prophylactically stimulate overall fish health and immune-
merset et al. 2005). Immersion is an easy way to vaccinate related health to prevent diseases (Dawood et al. 2018).
and can be enhanced by low frequency sonophoresis to
increase skin permeability during the bath (Fernandez- 1
Chemicals banned in the European Union can be found in the ‘List of
Alonso et al. 2001; Zhou et al. 2002). As gills are very sensi- chemicals: Annex I’ (https://echa.europa.eu/regulations/prior-informed-
tive to sonication, intensities to be used have to be consent/list-chemicals)

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T. Lieke et al.

Stimulation of the fish’s immune system and other defense incidence of bone deformities. This shows that despite the
mechanisms would have an effect primarily on the free-swim- beneficial effects on fish, similar to ‘traditional’ therapeu-
ming dinospores. Increased mucus, for example, can prevent tants, vitamin supplementation can have its downsides and
the dinospores from attaching to the skin and inhibiting the needs to be used and monitored carefully.
pathogen from completing its life cycle. Enzymes and other
defense mechanisms in gill and skin epithelial cells could affect
Prebiotics, probiotics, postbiotics, parabiotics,
dinospores attached to the fish. This mucosal immunity will
symbiotics
be discussed in later. Research on the effects of immune stim-
ulatory substances against Amyloodinium or Piscinoodinium is Vitamins are not the only group of dietary supplements that
scarce or even nonexistent. The following section will, there- have been studied for their health benefit in aquatic animals.
fore, focus on research with regard to increasing overall health There are numerous available studies on the effect of food-
and defense mechanisms with a focus on velvet disease were supplementation with nutrients (‘functional feeds’) such as
information is available. However, even studies about defense amino acids (Tesser et al. 2005; Peres & Oliva-Teles 2008),
mechanisms against other pathogens give important informa- polyunsaturated fatty acids (Martinez-Rubio et al. 2012,
tion and may be worthwhile avenues to investigate for pre- 2013), short chain fatty acids (Hoseinifar et al. 2017b) and
venting outbreaks of velvet disease. We recommend the book different kinds of ‘biotics’ have been evaluated in a plethora
entitled ‘Aquatic Animal Nutrition’ (Steinberg 2018) that of studies (Hoseinifar et al. 2018; Van Doan et al. 2019).
explains how diets define the digestive tract and its micro- The following section will discuss the different kinds of bio-
biota, which in turn influences life history and behavioral tics, their effects on the immune status and pathogen resis-
traits of the host. tance of fish and effects against aquatic parasites.
Gibson and Roberfroid (1995) define prebiotics as in the
following: ‘[Prebiotics] are nondigestible food ingredients
Vitamins
that beneficially affect the host by selectively stimulating
A body of evidence exists illustrating the effects of dietary the growth and/or activity of one or a limited number of
vitamin supplementation on health benefits. Vitamins are bacterial species already resident in the colon, and thus
widely known to improve growth and survival and stimu- attempt to improve host health’. Main targets are either the
late the immune response. Lim et al. (2002) reported that increase in potentially beneficial bacteria proliferation or
feeding 1,000 to 2,000 mg kg 1 vitamin C increased resis- counteracting the negative ones (Encarnacß~ao 2016). Benefi-
tance against osmotic stress in guppy (Poecilia reticulata). cial bacteria, such as Lactobacillus and Bifidobacterium,
Furthermore, vitamin C enhanced resistance to infections hydrolyse the prebiotics which results in production of var-
in various fishes (Li & Lovell 1985; Navarre & Halver 1989; ious bio-active compounds such as short chain fatty acids
Hardie et al. 1991) and vitamin E supplements decreased (scFA), vitamins and peptides. These are capable of increas-
stress caused by the fungicide copper oxychloride in Nile ing fitness over less beneficial or pathogenic bacteria,
tilapia (Oreochromis niloticus) (Hassaan et al. 2014). As thereby indirectly improving fish health (Hoseinifar et al.
mentioned previously, reduction of stress will make fish 2015a; Hoseinifar et al. 2017c; Jung-Schroers et al. 2016).
less vulnerable to infections. Although supplementing fish Furthermore, prebiotics can directly improve fish health by
feed with vitamins is beneficial, and lack of these sub- increasing the immune response (Ringø et al. 2010; Song
stances leads to deficiency symptoms, there is also the risk et al. 2014; Torrecillas et al. 2014). Acting as “risk” signals,
of over-supplementation. Dietary oversupply (hypervita- ß-glucans activate defense mechanisms that are also trig-
minosis) of vitamin C and D leads to decreased growth, gered after cell damage including antigen presenting cells
elevated levels of alkaline phosphatase and lethargy, and and increased production of Th1 and Th2 cells (Dalmo &
this can cause slow wound repair and depressed immunity Bøgwald 2008). Information on combating challenges by
(Merchie et al. 1996; Darias et al. 2011). In addition, skele- parasites is scarce and often inconsistent. Feeding rainbow
tal abnormalities, jaw malformations, albinism and melan- trout with 0.2% ß-glucan Lauridsen and Buchmann (2010)
ism caused by high concentrations of vitamin C have been decreased susceptibility to I. multifiliis while plasma lyso-
reported in olive flounder (Paralichthys olivaceus) and in zyme activity was increased. Feeding common carp with
striped trumpeter (Latris lineata) (Negm et al. 2014; 3% b-1,3/1,6-glucan increased the relative gene expression
Takeuchi 2014). Overdose by supplementing with high of interleukin 1-b; however, susceptibility against I. multi-
concentrations of vitamins is particularly risky in the case filiis was not significantly altered (Herczeg et al. 2017). Pro-
of vitamin A. Dedi et al. (1995) demonstrated that feeding tection against the marine parasitic flatworm Neobenedenia
olive flounder with Artemia enriched with more than girellae was gained after feeding greater amberjack (Seriola
40 mg vitamin A palmitate produced a negative effect on dumerili) with different forms of mannan oligosaccharides
growth (body weight and total length) and a high (Fernandez-Montero et al. 2019). A promising study for

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Protection against pathogens – chemical treatments versus immunostimulants

the use of prebiotics against velvet disease was conducted 3a (c3a), and genes of interleukins, tumor necrosis factors,
by Buentello et al. (2010); in this research red drum feed toll-like receptors and lysozyme. At the same time, relative
was supplemented with several prebiotics at 10 g kg 1 expression of genes involved in oxidative stress such as super-
which resulted in increased serum lysozyme activity and oxide dismutase (SOD), glutathione peroxidase 1a (Gpx1a),
survival after infection with A. ocellatum. Recently, Guer- nitric oxide synthase 2a (NOS2a) and heat shock protein 70
reiro et al. (2018) gave an overview about currently used (Hsp70) was decreased (Qin et al. 2018; Lin et al. 2019). In
prebiotics. They point out the importance of different fac- addition, mortality was decreased when challenged with A.
tors that can influence effects such as fish age and size, envi- hydrophilia and S. agalactiae. The few reported trials with
ronmental factors as temperature and give advises on dietary pre- and probiotics as therapeutic strategy against
timing and duration of administration as misuse of prebi- Amyloodinium are mixed. Reyes-Becerril et al. (2008) investi-
otics can also cause adverse effects such as alterations in gut gated the effects of the dietary intake of live yeast Debary-
histomorphology. omyces hansenii on the immune response of juvenile leopard
The term postbiotics evolved in the last years of research groupers (Mycteroperca rosacea) and on their resistance to
of functional foods, referring to “soluble factors (products infection by A. ocellatum; only fish with yeast-supplemented
or metabolic byproducts) secreted by live bacteria or diet recovered from the parasitic challenge. The underlying
released after bacterial lysis” (Aguilar-Toala et al. 2018). stimulated immune response was based on a significant
These include short-chain fatty acids (scFA), enzymes, pep- increase in immunoglobulin M content as well as the activity
tides, cell surface proteins, and vitamins (Tsilingiri & of superoxide dismutase (Reyes-Becerril et al. 2008). The sec-
Rescigno 2012; Konstantinov et al. 2013). Although ond attempt we are aware of failed; Li et al. (2005) evaluated
immune response promotion was found, the exact mecha- the effects of dietary supplementation with brewer’s yeast and
nisms of postbiotic modes of action have not yet been fully nucleotides on juvenile red drum’s resistance to A. ocellatum
elucidated (Kareem et al. 2016; Nawaz et al. 2018). Recent infection and found that in situ challenge by this parasite
studies shed light on the mode of action of prebiotic on caused 100% mortality regardless of dietary treatment within
host immune response. It has been shown that dietary a 48 h period. Feeding rainbow trout for 14 days with
administration of prebiotics and their fermentation by ben- 108CFU g 1 Aeromonas sobria (GC2) resulted in 100% sur-
eficial communities of gut microbiota drastically increase vival after infection with I. multifiliis (0% in control) while,
the levels of scFAs, including acetate, propionate and buty- using 1010 CFU g 1 Brochothrix thermosphacta BA211 did
rate (Hoseinifar et al. 2017a). These scFAs are capable of not reduce the mortality (Pieters et al. 2008). As mentioned,
modulating immune response through binding to G pro- I. multifiliisand Amyloodinium/Piscinoodinium have similar
tein coupled receptor, GPR43, which is expressed mainly live cycles; therefore, A. sobria (GC2) might be a treatment
on innate immune response and inflammatory cells option against velvet disease. However, BA211 significantly
(Maslowski & Mackay 2011). However, such scFAs-recep- increased survival against Aeromonas bestiarum (Pieters et al.
tors have not been reported in fish. 2008). These results show that one probiotic strain can have
The FAO/WHO defined probiotics as ‘live microorgan- different effects on different challenges, and that effects of
isms which, when administered in adequate amounts, confer timing, age and species of fish, as well as effect against specific
health benefits on hosts’ (FAO 2001). This includes host-as- pathogen or parasite has to be evaluated carefully prior to
sociated microorganisms, such as Lactobacillus, Bifidobac- being recommended for application in aquaculture facilities.
terium, Vibrio, and Aeromonas, but also yeast (Saccharomyces Concerns have been raised about the functionality and
cerevisiae and Saccharomyces exiguous) and microalgae (Tet- applicability of live microbes including the viability in the
raselmis suecica). Although immunostimulatory effects of supplementing product, persistence in the gut, horizontal
probiotics have been analysed in several studies, there are gene transfer from pathogenic bacteria, and enhanced
limited studies available about effects of probiotics on resis- inflammatory responses (Kataria et al. 2009; van Reenen &
tance against parasites. Going into detail would go beyond Dicks 2011; Taverniti & Guglielmetti 2011). Inactivated
the scope of this review; we therefore focus on a few selected (non-viable) preparations of probiotics (the so-called para-
examples were bacterial and parasitic challenges have been probiotics) have also shown to be effective immune stimu-
studied and recommend the reviews from Zorriehzahra et al. lants (Rampengan et al. 2010). Immunostimulatory effects
(2016) and Hoseinifar et al. (2018). Bacillus strains were (respiratory burst activity, increases of plasma lysozyme
screened for their anti-Vibrio properties and the effective and myeloperoxidase activity) of heat-inactivated Bacillus
mechanism was cell lysis by disruption of cell membranes amyloliquefaciens FPTB16 were found in the South Asian
(Gao et al. 2017a). Zebrafish (Danio rerio) treated with dif- carp Gibelion catla (Hamilton, 1822) (Singh et al. 2017).
ferent strains of Lactobacillus and Bacillus amyloliquefaciens In rainbow trout, Enterobacter sp. was used as effective
increased transcription of immune related genes, such as supplementation against Flavobacterium psychrophilum and
insulin-like growth factor-1 (igf-1), complement component inactivated Enterococcus faecalis significantly decreased

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T. Lieke et al.

cumulative mortality and frequency of infection with A. secondary metabolites (PSM), on health benefits. Often
salmonicida (Rodriguez-Estrada et al. 2013; LaPatra et al. inspired by traditional Chinese medicine, a plethora of fish
2014). Application in aquaculture is still limited and so far and aquatic invertebrates have been treated with extracts
no studies exist evaluating the potential protective effect from medicinal plants (i.e., herbal drugs). In addition to
against parasites. Choudhury and Kamilya () presented a the direct promotion of health, such as benefits in growth
good comprehensive review about current studies and the and food conversion ratio, many PSM isolates increase
role of paraprobiotics in modifying responses in fish. specific and non-specific immunity, and thereby resistance
Often a combination of different treatment strategies is against stress and various pathogens. One example is the
more successful. Over the past year, an updated idea was change in the internal cell oxidative status (mitohormesis).
suggested regarding modulation of intestinal microbiota Increasing evidence indicates that oxidative burst-released
aimed at improved host health, the so-called ‘Synbiotic’ ROS not only cause oxidative stress, but rather may func-
(Gibson & Roberfroid 1995). The idea refers to combined tion as signaling molecules that promote health by prevent-
administration of a probiotic bacterium with an optimum ing or delaying a number of chronic diseases, and
prebiotic as substrate. Using mannan oligosaccharide com- ultimately extend lifespan (Ristow & Schmeisser 2014).
bined with E. faecalis decreased the frequency of A. Apart from increasing the specific growth rate, very low
salmonicida even more than compared to application of concentrations of anthraquinone extract from rhubarb
each supplement alone (Rodriguez-Estrada et al. 2013). (Rheum officinale) increased the activities of blood lyso-
Feeding angelfish (Pterophyllum scalare) with symbiotic zyme and resistance against crowding stress or artificial A.
(Pediococcus acidilactici plus fructooligosaccharide) hydrophila infection in common carp and prevented high
enriched Artemia was more effective in increasing skin temperature stress in giant river prawn (Macrobrachium
mucus lysozyme, protease activity, and immunoglobulin rosenbergii) (Xie et al. 2008; Liu et al. 2010). After treat-
concentration than singular enrichment with probiotics or ment with an extract of Siberian ginseng (Eleutherococcus
prebiotics alone (Azimirad et al. 2016). Similar results were senticosus), olive flounder demonstrated increased non-
found after treating rainbow trout for 8 weeks with galac- specific immunity and resistance against infection with
tooligosaccharides (GOS), P. acidilactici and the combina- Edwardsiella tarda and Vibrio anguillarum (Won et al.
tion (Hoseinifar et al. 2015b); activity of serum lysozyme, 2008). Anusha et al. (2014) reported that extracts from
respiratory burst, and alternative complement was signifi- jungle geranium (Ixora coccinea) protected goldfish
cantly increased compared to singular enrichment.The (Carassius auratus) against A. hydrophila infection. Resver-
highest resistance against Streptococcus iniae was observed atrol, a phytoalexin found in grapes and several berries
in the synbiotic group. Several combinations have been can extend lifespan, protect the nervous system, increase
tested for their stimulatory potential (Cerezuela et al. 2011; cognitive abilities and retard ageing-related histological
Hoseinifar et al. 2016a; Huynh et al. 2017). markers in redtail notho (Nothobranchius guentheri) (Yu &
Dietary ‘biotics’ exhibit optima with regard to doses and Li 2012; Genade & Lang 2013). Although, many studies
frequency of application, and can exert adverse effects when focus on anti-bacterial effects of PSMs, there is evidence
applied above their optimum concentration or for too long. of anti-parasitic and anti-fungal effects as well. Applying
Feeding tiger shrimp (Penaeus monodon) with 1000 mg kg 1 extracts of garlic (Allium sativum) and Indian almond
of the immunostimulant alginic acid for 35 days, Ojerio (Terminalia catappa) to rearing water of tilapia fingerlings,
et al. (2018) found an enhanced resistance against white spot Chitmanat et al. (2005) were able to completely remove a
syndrome virus (WSSV), while groups fed higher concentra- Trichodina sp. infection After a 2-day treatment. Garlic
tions had reduced survival rates. Apart from the beneficial was also effective at killing theronts (63.5 mg L 1) and
effects of the feed supplementation on growth and immune tomocyts (570 mg L 1) of I. multifiliis (Buchmann et al.
parameters, few studies with real challenges against patho- 2003). Magnolia (Magnolia officinalis), Euphorbiaceae
gens or parasites have been conducted yet. Molecular studies (Euphorbia fischeriana Steud.), black tyme (Thymbra spi-
are also required to assess the mechanisms behind the stimu- cata L.), oregano (Origanum onites L.), and savory
latory effects, and further understanding of the pathways of (Satureja tymbra L.) were used to control Saprolegnia sp.
these compounds will be a crucial step towards their broad (Gormez & Diler 2014; Huang et al. 2015). Extracts from
application in aquaculture and the replacement of antibiotics different medicinal herbs, e.g. cinnamon (Cinnamomum
and traditional therapeutants. cassia), golden larch (Pseudolarix amabilis), juanilama
(Lippia alba) applied either as bath or orally, were effective
anthelminthics (Ji et al. 2012; Reverter et al. 2014; Soares
Plant secondary metabolites
et al. 2016). These are only a few examples of studies ana-
A growing field of research is focusing on evaluating the lyzing the effects of medicinal plants and secondary
effect of natural organic matter (NOM), including plant metabolites. Stratev et al. (2018) presented a

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Protection against pathogens – chemical treatments versus immunostimulants

comprehensive overview about current treatments using and immunity, and reduce diseases in poultry and rats
medicinal plants as well as the mechanisms of action. (Yasar et al. 2002; TeraVita 2004, Islam et al. 2005). Vet-
vicka et al. (2010) tested two different types of humic acids
on humoral and cellular immune reactions of mice; evi-
Humic substances: rediscovered remedy
dence of immunomodulating activity was demonstrated
Major components of Natural Organic Matter (NOM) are through increased phagocytosis by neutrophils and macro-
humic substances. These ubiquitous, organic compounds phages, as well as stimulated secretion of IL-2 by spleen
arise from the physical, chemical and microbiological trans- cells.
formation (humification) of biomolecules and occur in Increased vigor, growth, lifespan and increased stress
decaying debris and soil. Up to 95% of dissolved organic resistance have also been reported after application of 5 to
matter (DOM) found in aquatic ecosystems are humic sub- 180 mg L 1 DOC of an artificial humic substance (HS
stances (Thurman 1985; Haitzer et al. 1998; Steinberg 1500) for 21 weeks to culture water of swordtail (Xiphopho-
2003). Humic substances can be divided into three main rus helleri) (Meinelt et al. 2004). In the cladoceran Moina
fractions: humic acids, fulvic acids and humins (Stevenson macrocopa, osmotic stress was reduced in the presence of
1982). These subdivisions are arbitrarily based on the solu- 10 mg L 1 DOC of a natural humic substance from a
bility of each fraction in water at different pH levels as well Brazilian polyhumic coastal lagoon (Suhett et al. 2011);
as their molecular weight (Pettit 2004). stress resistance was inherited by the succeeding generation,
Figure 4 shows a schematic structure of a humic acid most likely via DNA methylation (Menzel et al. 2011). Sup-
with various functional groups which are important for plementing feed with 2 g kg 1 of a fulvic acid significantly
interactions with exposed organisms. Most effects of humic increased survival of white shrimp (Litopenaeus vannamei)
substances have been evaluated in terrestrial animals so far. challenged with Vibrio parahaemolyticus (Fierro-Coronado
Administration of humic substances as food supplements et al. 2018). In this research, TCTP (translationally con-
has been shown to increase vitality traits such as growth trolled tumor protein), which is involved in the regulation

Figure 4 Structure of a soil humic substance. Major functional groups are indicated. Redrawn after Kleinhempel (1970) with permission from Taylor
& Francis.

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T. Lieke et al.

of the host defense response against bacterial and viral radicals were the most efficient in reducing fungal growth
infections, was significantly increased, while Hsp70 was sig- and caused sporangia formation.Steinberg et al. (2007)
nificantly decreased 72 h after feeding; this indicates a identified that those humic substances effective with Sapro-
modulatory effect of the fulvic acid on the immune and legnia also triggered longevity in the nematode Caenorhab-
stress response. Adding 1% of humus extract to the feed of ditis elegans. On the other hand, humic substances with
ayu fish (Plecoglossus altivelis) decreased the development high shares of aliphatic structures, such as carbohydrates,
of skin lesions during infection with F. psychrophilum tended to support Saprolegnia growth and did not extend
(Nakagawa et al. 2009). A direct effect on the pathogen was lifespan in C. elegans (Steinberg et al. 2007). These exam-
excluded, as the pathogenic load in the water did not ples show that structure matters and structural aspects of
decrease due to the treatment. Supplementing feed of juve- humic substances have to be considered and evaluated
nile loach (Paramisgurnus dabryanus (Sauvage)) with 1.5. when applying humic substances as treatments against vel-
% fulvic acid for 60 days resulted not only in elevated vet disease and similar pathogens.
growth performance but in an increase in intestinal pro- Increased animal welfare, as well as enhanced
tease activity, antioxidant activity, lysozyme activity immune-related and overall health, may help prevent
(LZM), complement 3 (C3) content, immune globulin M diseases or help fish recover from infections easier.
(IgM) content, acid phosphatase activity (ACP), and alka- Whether an increase in mechanical and chemical defense
line phosphatase activity (AKP) (Gao et al. 2017b). Intesti- mechanisms by application of humic substances helps to
nal health also increased as shown by elevated abundance decrease susceptibility to Piscinoodinium and Amylood-
of Lactobacillus and decreased amounts of Serratia, Acineto- inium should be evaluated in future research and may
bacter, Aeromonas and Edwardsiella. provide an alternative to conventional therapeutants.
Studies using fish eggs and larvae demonstrated that However, when considering humic substances as a treat-
humic substances increased defense against pathogenic ment or preventative against fish pathogens, one must
fungi (Saprolegnia spp.) and different microorganisms keep in mind that the very supplements that improve
(Aeromonas spp.) (Schreckenbach et al. 1994; Meinelt et al. fish health may also promote the growth of dinoflagel-
2008). Common carp had significantly reduced infection lates. Low concentrations (6 to 32 lg mL 1) of humic
rates when challenged with A. salmonicida subsp. salmoni- substances exerted a stimulatory effect that was reflected
cida after oral application of humic-rich sludge from a by increased yield, growth rate and 14C uptake in mar-
recirculating aquaculture system, a synthetic humic acid, ine dinoflagellates of the genus Gonyaulax (Prakash &
and an extract derived from Leonardite (Yamin et al. Rashid 1968). At higher concentrations (over 35 lg
2017). Similar results were found by Kodama et al. (2007) mL 1), growth rate and yield significantly decreased.
after oral administration of humus extracts to carp infected Effects of humic substances on hosts and parasites
with A. salmonicida. However, research of whether humic should be monitored and evaluated carefully to ensure
substances can be a suitable supplement to reduce stress in proper treatment. Apart from that, humic substances
finfish caused by treatment against, or the infection itself, is have the potential to reduce susceptibility to infections
still new and only few current studies have examined this and pathogens by increasing animal welfare and stimu-
topic yet. lating host-specific defense mechanisms.
Only a few studies have documented the rare phe-
nomenon of bio-toxicity of humic substances (Gau et al.
Defense mechanisms
2000; Chen et al. 2002; Hseu et al. 2008), yet the effect was
found in vitro using different cell cultures. Bernacchi et al. The defense system of vertebrates is organized in two levels
(1996) examined the in vivo effect by feeding mice a single 1) the innate and 2) the adaptive defense system. Innate
dose of an aqueous solution containing 4 g L 1 of humic protection has a general character and protects against
acids (100 mg kg 1 body weight). Orally administrated unspecific pathogens without recognition and is a fast act-
humic acids induced structural and numerical chromoso- ing defense system. The adaptive protection in contrast
mal abnormalities in intestinal cells. However, the concen- depends on recognition of distinct molecular structures of
tration used during these experiments was 10 to 1000 times the invading organisms and memory development.
higher than those that are environmentally realistic. Response is slow (weeks to month) (Van Muiswinkel
As mentioned previously, the structure of humic sub- 1995). Innate defense mechanisms of fish include epithelial
stances is very diverse and different humic substances can barriers, lysozyme, phagocytic cells, and interferons and are
therefore provoke contrasting effects. Combating the water similar to that of mammals and birds. However, due to the
mold Saprolegnia parasitica with 20 humic preparations, intimate contact with their environment, fish have estab-
Meinelt et al. (2007) found that humic preparations with lished some special defense mechanisms which are less pro-
high aromaticity which contain a high number of organic nounced in other vertebrates.

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 17535131, 2020, 2, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/raq.12365 by BINGOL UNIVERSITY, Wiley Online Library on [12/05/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Protection against pathogens – chemical treatments versus immunostimulants

(i.e., epidermis and gills) and are a first line of defense

Mucosal immunity
against pathogens. The AMPs consist of up to 100 amino
Beside skin, there are other mucosal surfaces in fish which acids and most demonstrate an amphipathic design with
have important barrier function (Salinas 2015; Hoseinifar clusters of hydrophobic and cationic amino acids (Schulze
et al. 2019). These barriers are composed of the epithelia 2010). The basis of specificity of AMPs arises from differ-
and their mucus secretions, is called mucosa-associated ent physicochemical properties of the outer membranes.
lymphoid tissue (MALT). The MALT is presented in gut The outer leaflet of the bilayer of bacteria consists of phos-
(gut-associated lymphoid tissue or GALT), gills (gill-associ- pholipids with negatively charged headgroups, while that
ated lymphoid tissue or GIALT), skin (skin-associated lym- of plants and animals has no net charge (Op den Kamp
phoid tissue or SALT) and as recently discovered in nose 1979; Zasloff 2002). The mode of action of AMPs is not
(nasopharynx-associated lymphoid tissue (NALT)) (Lazado completely understood. According to the Shai–Matsuzaki–
& Caipang 2014). The skin mucus can be considered a first Huang–model (Matsuzaki 1999; Shai 1999; Yang et al.
line of defense against attacks of pathogens and parasites. 2000), accumulation of AMPs at the outer layer is aided
Consequently, fish have developed means to improve the by electrostatic interactions, followed by formation of a-
mucosal immunity. Feeding Persian hogweed (Heracleum helices and insertion of the hydrophobic clusters leading
persicum) powder to common carp, Hoseinifar et al. to membrane permeation. Inhibition of target organisms
(2016b) found significantly elevated immunological factors can be different, including the disruption of membrane
such as immunoglobulins, lysozyme, protease and alterna- integrity and arising plasmolysis, inhibition of proteins,
tive complement activities in carp mucus but not in serum. DNA and RNA synthesis and interaction with intracellular
Feeding Caspian white fish (Rutilus frisii kutum) and Cas- targets (Bahar & Ren 2013). Because of the physical dis-
pian brown trout (Salmo trutta caspius) with dietary pep- ruption of the cell membrane, resistance against AMPs will
permint extracts increased skin mucus defense (protein not develop. Due to the lack of net charge and the high
concentration, alkaline phosphatase and antimicrobial content of cholesterol, AMPs do not accumulate at mem-
activity) (Adel et al. 2015). Furthermore, immune genes in branes of multicellular organisms (Matsuzaki et al. 1995;
mucosal tissues were upregulated and resistance against Toke 2005), preventing impairment of non-target organ-
parasites was increased after feeding Greater amberjack isms. Therefore, they have been analysed as a replacement
(Seriola dumerili) with cMOS supplemented diets (Fernan- for traditional therapeutants (Hancock & Patrzykat 2002;
dez-Montero et al. 2019). These studies indicate that nutri- Finlay & Hancock 2004) and could be a useful tool to pre-
tion plays one central role in this defense process. Addition vent velvet disease.
of a humic substance to culture water reduced attachment Noga et al. (2002, 2003) reported negative effects of
and penetration of the fish skin by pathogens (Schrecken- HLP-1, which has high sequence homology to histone
bach et al. 1996). Furthermore, increased mucosal lyso- H2B, on the infection intensity and propagation of A. occe-
zyme activity was found when fish were exposed to a fulvic latum. Addition of 100 lg mL 1 HLP-1, isolated from
acid (which is also a humic substance) while serum lyso- rainbow trout (Oncorhynchus mykiss) and hybrid striped
zyme was unaffected (Lieke, et al., in preparation). This bass (female Morone chrysops x male M. saxatilis), inhibited
indicates that apart from nutrition, other pathways seem to young and mature trophonts in vitro. HLP-1 caused
be involved in regulation of mucosal immunity and the delayed or irregular development of the parasites and led to
mode of action has not been determined and merit further less aggressive feeding behavior and slower monolayer
research. destruction (Noga et al. 2002, 2003). In vitro upregulation
of the AMPs results in expression concentrations, especially
in skin and gill epithelia, which are in the range of the anti-
Antimicrobial peptides are natural defense
parasitic concentrations, measured in vitro (Ullal et al.
mechanisms against pathogens
2008).
Antimicrobial peptides (AMPs), such as histone-like pro- Stimulation of these host-specific defensive mechanisms
teins (HLPs) and piscidines, are a diverse group of mole- could help protect fish against velvet disease, as parasites
cules that are widespread in the animal and plant are challenged at an early stage of infection and immune
kingdoms and are important parts of the innate immune system functions attempt to destroy them. Consequently,
system (Schr€ oder 2013). They were discovered when an these host-specific defense mechanisms decrease the
extract from a soil Bacillus strain was used to protect mice chances of parasite propagation, prevent mass infections
from pneumococci infection (Dubos 1939a, b). More than and prevent excessively weakened health conditions of fish.
5,500 AMPs have been discovered or synthesized to date As humic substances are immunostimulatory, they may
(Zhao et al. 2013). In animals, AMPs are located in differ- induce expression of AMPs, but there is presently no
ent tissues; especially those exposed to the environment research about this.

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 17535131, 2020, 2, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/raq.12365 by BINGOL UNIVERSITY, Wiley Online Library on [12/05/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
T. Lieke et al.

they could add to the stress level of the infected fish due to
Conclusion
their high oxidative capacity and are toxic when applied at
Diseases cause significant financial losses to public aquari- high concentrations. Conditioning the fish prior to treat-
ums, ornamental enthusiasts and aquaculture operations. ment potentially helps decrease this stress.
Few studies exist evaluating effects of environmental Supplementing food with vitamins or biotics has been
friendly therapeutants to diseases, such as velvet disease, demonstrated to reduce stress-levels and susceptibility to
maybe due to its lower infectivity at low temperatures. infections; however, there is a risk of over-supplementa-
Household remedies such as saline baths are often used by tion. Dose/response curve of immunostimulants does not
hobby aquarists but are unsuitable for commercial usage. follow a linear relationship (Bliznakov & Adler 1972). It is
Commercially available therapeutants are toxic to non-tar- often a hormetic one, with beneficial effects increasing up
get organisms and can increase stress levels, lower health to a certain concentration and then decreasing if that con-
conditions, and potentially cause death of treated fish. Dis- centration is exceeded. In addition, life time, duration, and
carded residues of these therapeutants can be harmful to frequency of application can play a tremendous part. After
the environment or lead to the development of resistance continuous feeding for 40 days with glucan at a concentra-
in microorganisms, which can pose a subsequent risk to tion of 2 g glucan kg 1 feed, which represents optimal con-
human safety as well. Especially inside the EU regulations centration for Asian tiger shrimp (Penaeus monodon),
are strict and EU aquaculture is renowned for its high qual- immunity was reduced (Chang et al. 2000). Indian white
ity sustainability and consumer protection standards. These shrimp (Fenneropenaeus indicus) had highest survival rate
include regulations of environmental impact assessment, when challenged with white spot syndrome virus (WSSV)
water use, food and safety hygiene, animal welfare, and ani- when 2 g glucan kg 1 was added once a week as feed sup-
mal health. The European Commission is helping and plement. Higher intervals decreased survival (Sajeevan
demanding to increase a sustainable aquaculture that is safe et al. 2009). These results indicate that continuous use of
but also competitive with other countries. This shows the immunostimulants even at an optimal dose may suppress
high demand of replacing chemical therapeutants not only the immunity and administration at definite intervals
by the government but by end consumers as well, and has would give better performance resulting in enhanced sur-
fueled research approaches on alternative strategies such as vival. Clearly, more research is needed in order to identify
disinfection of water, immunostimulatory feed ingredients, optimal application strategies, particularly studies on fish
and water supplements. Figure 5 summarizes current are, to the best of our knowledge, not available.
knowledge on these three topics and how they can work to Health benefits of natural organic matter have been pro-
prevent disease outbreaks. ven in a plethora of different animals. Many plant extracts
Two compounds, H2O2 and PAA degrade into harmless not only have general beneficial effects, such as enhanced
residues and are therefore more environmental friendly growth and food conversion rate, but also increase specific
than other compounds. They have been proven to be effec- and non-specific immunity. The effects of humic sub-
tive against a variety of aquatic pathogens and represent stances have recently been rediscovered and scientifically
promising approaches to treat velvet disease. Nevertheless, proven; used as feed additives, they can increase vitality

Figure 5 Scheme of environmental friendly

treatments used to prevent or treat diseases.
?: stimulation; ┤: inhibition; ?: proposed
pathway.

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 17535131, 2020, 2, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/raq.12365 by BINGOL UNIVERSITY, Wiley Online Library on [12/05/2025]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Protection against pathogens – chemical treatments versus immunostimulants

and lifespan, stimulate growth, accelerate wound healing and the microorganisms. A favourable concept for environ-
and increase resistance to pathogenic bacterial loads. The mentally friendly treatments against velvet disease and
immune system and host-specific defense mechanisms in other fish diseases is to concentrate on two aspects: 1) treat-
land vertebrates were stimulated by humic substances and ment of the invading parasites using residue-free or natu-
they reduced susceptibility to stress and microorganisms. rally occurring substances, and 2) fortifying the fish to
Conditioning fish using natural organic matter is a promis- reduce susceptibility to parasites and to allow significantly
ing approach to enhance overall and immune-related reduced use of antiparasitic therapeutants. In this way, the
health, making fish less susceptible to velvet disease. Ongo- most sustainable outcome for the environment as well as
ing research in our laboratory suggests that humic sub- for the farmer can be achieved.
stances can increase the MALT system and thereby help in
preventing diseases. Humic substances are therefore Acknowledgements
another promising approach of immunostimulation. Natu-
ral organic matter can increase host defense systems in The present study was financially supported by the AiF Pro-
order to decrease or even replace use of chemical therapeu- jekt GmbH (Federal Ministry for Economic Affairs and
tants. Furthermore, humic substances are part of the natu- Energy) under project No. ZF4240502SK7. The authors
ral environment of fish. In contrast to plant extracts, fish thank Dr. Chuck Weirich for proofreading and his com-
defense systems are ‘accustomed’ to these natural xenobi- ments and suggestions to improve the manuscript. Men-
otics, making them ideal candidates for use in culture tion of trade names or commercial products in this article
water. is solely for providing specific information and does not
Stimulation of immune response does not necessarily imply recommendation or endorsement by the Leibniz-
reflect an increased protection against pathogens. Studies Institute of Freshwater Ecology and Inland Fisheries, Hum-
where fish are challenged with pathogens after administra- boldt University, Federal Ministry for Economic Affairs
tion of immunostimulants are scarce and most focus on and Energy, or the U.S. Department of Agriculture.
bacterial infections due to easier implementation of the
experiment. Also, the higher energy demand needed to acti- Compliances with ethical standards
vate the immune system might even be accompanied by
adverse effects such as impairment of growth. It is therefore The authors declare that there are no conflicts of interest.
important to monitor parameters from different regulatory The founding sponsors had no role in the design, content
pathways (metabolism, stress response, growth) in addition or decision to publish the review.
to the immune parameters in order to ensure safe applica-
tion of new immunostimulants. Nevertheless, they have References
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