Antiparasitic and Antibacterial Functionality of Essential Oils: An Alternative Approach for Sustainable Aquaculture

Using synthetic antibiotics/chemicals for infectious bacterial pathogens and parasitic disease control causes beneficial microbial killing, produces multi-drug resistant pathogens, and residual antibiotic impacts in humans are the major threats to aquaculture sustainability. Applications of herbal products to combat microbial and parasitic diseases are considered as alternative approaches for sustainable aquaculture. Essential oils (EOs) are the secondary metabolites of medicinal plants that possess bioactive compounds like terpens, terpenoids, phenylpropenes, and isothiocyanates with synergistic relationship among these compounds. The hydrophobic compounds of EOs can penetrate the bacterial and parasitic cells and cause cell deformities and organelles dysfunctions. Dietary supplementation of EOs also modulate growth, immunity, and infectious disease resistance in aquatic organisms. Published research reports also demonstrated EOs effectiveness against Ichthyophthirius multifiliis, Gyrodactylus sp., Euclinostomum heterostomum, and other parasites both in vivo and in vitro. Moreover, different infectious fish pathogenic bacteria like Aeromonas salmonicida, Vibrio harveyi, and Streptococcus agalactiae destruction was confirmed by plant originated EOs. However, no research was conducted to confirm the mechanism of action or pathway identification of EOs to combat aquatic parasites and disease-causing microbes. This review aims to explore the effectiveness of EOs against fish parasites and pathogenic bacteria as an environment-friendly phytotherapeutic in the aquaculture industry. Moreover, research gaps and future approaches to use EOs for sustainable aquaculture practice are also postulated.


Introduction
Farming of aquatic plants and animals is generally known as aquaculture, and the annual growth of this rapidly expanding food industry is 4.5%, accounting for a value of 243.26 billion USD [1] to meet up the protein demand of ever increasing world population. This important industry is also generating jobs, income, and providing 50% of global fish consumption [2,3]. Due to the increase of consumer demand, aquaculture technique has been shifted from extensive to super-intensive; intensification of aquaculture needs a higher amount of artificial feed supply, water treatment and reuse, and high stocking density resulting in aquatic environmental degradation [4][5][6]. Mounting of stress and quality deterioration of living environment increases the activity and virulence of infectious and opportunistic microbial pathogens [7], decrease immunity and immune-related gene transcription of aquatic animals [8], and elevate uni and multicellular parasitic infestation [9]; finally, initiate infectious diseases outbreak along with the death of cultured species. Gonzales, et al. [10] reported global aquaculture loss of 1.05 to 9.58 billion USD/year due to infectious diseases and parasitic attacks.
To eliminate diseases and parasitic attacks in the aquaculture industry, different synthetic antibiotics, chemical drugs, vaccines, and chemotherapeutics are being used at high rates from year after year [11,12]. Using of these chemical substances cause mass killing of beneficial aquatic bacteria [13], produce multi-drugs resistant pathogens [14], and leaving residues in fish which can be transmitted to human [15,16]. These problems are the most concerning aquaculture sustainability [17,18], and infectious diseases and parasitic infestation treatment with natural substances/compounds are the demanding sustainable aquaculture features [19].
The use of medicinal plants and their derivatives in aquaculture is increasing day by day all over the world because of having biodegradable properties [20][21][22][23][24], availability and ease to cultivate, and do not accumulate in animal tissues as a residue [25,26]. Essential oils (EOs) are the secondary metabolites of medicinal plants and possess bioactive properties to be used as a phytotherapeutic agent for sustainable aquaculture [27,28]. Terpens, terpenoids, phenylpropenes, and isothiocyanates are the key chemical groups identified in EOs [29]. EOs mainly penetrate and act upon the membrane and cytoplasm of bacteria to inhibit their action mechanisms by altering cell morphology and organelles deformities [30,31]. Generally, Gram-positive bacteria are more sensitive to EOs than Gramnegative due to lipoteichoic acids in cell membranes that might facilitate the penetration of EOs hydrophobic compounds [32]. According to Carson, et al. [33], EO comprises different compounds that have no specific cellular target in parasites. Monoterpenes α-pinene and sabinene of EOs have proved mentionable antiprotozoal activity. Moreover, synergistic effects of different compounds in EOs are another key feature that showed a higher mode of action relative to individual compounds. EOs cause leakage of potassium ions and cytoplasmic content of parasitic cells due to hydrophobicity and cell permeability, which cause cell morphology alteration and cessation of parasitic activity [34]. Staining with fluorocromes SYBR-14 and propidium iodide confirmand the plasma membrane damage in Ichthyophthirius multifiliis by the action of Varronia curassavica derived EOs [35].
Different microbial and parasitic diseases are the major threats to the aquaculture industry. Application of nanoemulsions EOs or other herbal products to combat microbial [36,37] and parasitic [9,25] diseases is considered a new alternative approach for sustainable aquaculture. Extensive research activities were performed for the identification and characterization of EOs effects for the fish and shellfish preservation and shelf life elongation [38,39], modulation of growth, immunity, and infectious disease resistance in commercially cultured fish species [35,40,41], against different pathogenic microbial activity [42,43] and destruction and retardation of fish parasitic activity [9,10]. In the fisheries and aquaculture sector, EOs act as a natural preservative [44], stress-reducing agent [45], herbal anesthetics [46], and oregano herb and medicinal plant as immunomodulators [26] and immunostimulants [47]. However, no study was conducted to identify EOs antiparasitic and antimicrobial properties for sustainable aquaculture.
Although natural EOs have enough potential for sustainable aquaculture, EOs have high volatility and can be decomposed by exposure to heat, humidity, light, and oxygen to lose effectiveness [48]. Application to the EOs in their oil form render it subjected to degradation during processing, storage, and handling [49]. The use of nano-encapsulated EOs becomes a promising trend in the field of EOs applications [50], especially in the aquaculture sectors [51], protecting the volatilization, low stability, low solubility in water, and associated problems of using EOs [52]. Nanoemulsion technology is currently solving the effectiveness disruption problems of EOs in aquaculture. This technology also protects EOs from the digestive enzyme's actions in the intestine.
The main focus of this article is to identify EOs antimicrobial and antiparasitic properties that can be used for sustainable aquaculture practices. Moreover, EOs effects for aquaculture species growth, immunomodulation, and infection resistances were also postulated. In addition, research gaps and tentative future research activities are also mentioned to effectively use EOs in sustainable fish culture.

EOs as Growth, Immunity, and Disease Resistance Enhancer
Several studies have been conducted to identify EOs growth and immunity elevation property; however, no specific research was conducted to identify the action mechanism of EOs for the alteration of these properties [28,[53][54][55]. Jang, et al. [56] mentioned the possible reason for growth and feed utilization parameters modulation by EOs is due to elevation of digestive enzymes in the intestines. Moreover, EOs increased the appetite of aquaculture species [57] may be another reason. Antioxidant activity increased due to aromatic rings and the position of hydroxyl ion in EOs [58]. Modulation of the intestinal microbiome by EOs can be considered one of the possible reasons for the modulation of immune-related genes [59]. Significantly, phenolic compounds like thymol and carvacrol modulate innate immunity through two possible ways i) direct action on host tissue ii) influence on the intestinal microbial community [60].
A 60-day experiment was conducted with dietary supplementation with bitter lemon (Citrus limon) [61], and sweet orange peels (C. sinensis) [62] originated EOs in Mozambique tilapia (Oreochromis mossambicus). In both cases, EOs elevated innate immune parameters (NBT, WBCs, lysozyme, and myeloperoxidase activity) and decreased serum/blood glucose, cholesterol, and triglycerides. C. limon and C. sinensis EOs administrated tilapia demonstrated resistance against Streptococcus iniae and Edwardsiella tarda, respectively. In addition, a similar type of immunomodulation and infection protection of tilapia were also found after C. limon peel EOs supplementation at (1, 2, 5, and 8%) in Labeo victorianus for 28 days [63]. However, growth (WG% and SGR) and feed conversion ratio (FCR) modulation in the former study remained unchanged but in the latter two experiments increased significantly (Table 1). The authors claim active compound of EOs (limonene) concentration in the former experiment was 54.4%, whereas later studies were 94.74 and 81.40, respectively, may be the causal factors of these differences. In Nile tilapia (O. niloticus), lemongrass (Cymbopogon citratus) and geranium (Pelargonium graveolens) [40], and Oregano (Origanum vulgare) [64], supplementation increased growth and feed utilization, and resistance against the action of Aeromonas hydrophila and Vibrio alginolyticus, respectively. C. citratus and P. graveolens supplemented fishes not only improved immunity but also decreased the concentration levels of intestinal coliforms, Escherichia coli, and Aeromonas spp. Moreover, origanum EOs (1 g/kg) improved immunity and vibriosis protection in Tilapia zillii [65].
Eight weeks feeding trial with 0.05% of Oregano (O. heracleoticum) originated EOs showed better growth, body indices (VSI, HSI, and CF), and antioxidant property (SOD and CAT) in channel catfish (Ictalurus punctatus) [66]. Carvacrol and thymol are the active substances of oregano EOs; however, in this fish species, O. vulgare originated commercial EOs showed inferior results relative to O. heracleoticum. Silver catfish (Rhamdia quelen) was dietary administrated (2 mL/Kg) with Aloysia triphylla EOs [41] and bath treatment (5 and 10 mg/L) with EOs compound, eugenol [67]. Bath treatment was unable to upregulate hematological and immunological parameters, but dietary administration improved healthy blood cells (leukocyte, lymphocyte, and neutrophil) and protein levels. Most importantly, these two catfish species had increased tolerance against A. hydrophila infection protection after feeding or bath treatment with plant originated EOs.
Eight weeks of feeding with O. vulgare EOs increased both immune and antioxidant properties and resistance against A. hydrophila in Cyprinus carpio [60,64]. EOs increased transcription levels of interleukin (IL)-1β and IL-10 and down-regulated tumor necrosis factor (TNF)-α and transforming growth factor (TGF)-β. Moreover, the increment of digestive enzyme activities and enrichment of beneficial bacterial genera in the intestinal microbial community were also found after EOs supplementation (Table 1). Feeding with O. onites instead of O. vulgare, similarly positive immunity and anti-oxidant activity modulation, and infectious disease protection was found in rainbow trout (Oncorhynchus mykiss) [68]. Futher, water extract of Ocimum sanctum leaves increased total RBC, WBC, hemoglobin, and other immune and anti-oxidant parameters in L. rohita [69].  Table 1. Effects of herbal essential oils on growth, immunity, and infectious diseases protection in commercial fish species.   Ngugi, et al. [63] ), no significant change.

Acanthocephalas Neoechinorhynchus buttnerae
Neoechinorhynchus buttnerae is an acanthocephalan parasite causing significant economic losses in Colossoma macropomum fish in the region of Amazon [71,72]. It was reported that Mentha piperita, Lippia alba, and Zingiber officinale [73] and Piper hispidinervum, Piper hispidum, Piper marginatum, and Piper callosum [74] essential oils showed 100% anthelmintic effect on N. buttnerae. When EO of piper hispidinervum was applied on N. buttnerae parasite in 0.78 mg/L concentration for 15 min, it gave the most effective result in terms of dose and time [74] (Table 2).

Cichlidogyrus spp.
Cichlidogyrus is the parasite genus that occurs naturally in cichlid fish and has the most species among gill parasites, with its 131 different species known [80]. Scutogyrus species can also be dominant in the winter season among fish belonging to the Cichlidae family [81]. de Oliveira Hashimoto, et al. [82] reported that Lippia sidoides EO had 100% efficacy against Cichlidogyrus spp. and Scutogyrus longicornis when applied as 160 mg/L for 1 min 58 s while Mentha piperita EO had 100% efficacy when applied as 320 mg/L for 8 min 11 s (Table 2).

Dawestrema spp.
Dawestrema cycloancistrium and Dawestrema cycloancistrioides are two of the most significant parasite types causing death and economic losses in Arapaima gigas fish, which are cultured in the region of Amazon [83,84]. Application of M. piperita EO as 160 and 320 mg/L for 30 min showed 100% efficacy on D. cycloancistrium and D. cycloancistrioides parasites [85] (

Trepomonadea Hexamita inflata
Hexamita inflate is a flagellated anaerobic protozoan and free-living in fresh and seawater. Moon, et al. [87] reported that L. angustifolia and L. intermedia EOs as 1 and 0.5% for 30 min exhibited 100% efficacy on H. inflate (Table 2).

Clinostomidae Euclinostomum heterostomum
Euclinostomum heterostomum is parasitic trematodes and very common in Europe, Asia, and Africa [88]. It infects muscular tissues and kidneys of freshwater fish [88,89]. Verbesina alternifolia and Mentha piperita EOs could act on E. Heterostomum in high doses and for a long time [90] (Table 2).

Oligohymenophorea Ichthyophthirius multifiliis
Ichthyophthirius multifiliis is the most famous virulent ciliated protozoan ectoparasite that invades the skin, fins, and gills of fish. de Castro Nizio, et al. [35] indicated that Varronia curassavica EO showed 100% efficacy against I. multifiliis trophont and tomont when applied as 10 mg/L and 50 mg/L for one h, respectively. Hyptis mutabilis (10 mg/L for 30 min) [91] and Melaleuca alternifolia, Lavandula angustifolia, and Mentha piperita (455 µL/L for 1 h) [92] EOs applications were also found to be effective on I. multifiliis (Table 2).  Aeromonas salmonicida has been known as the causative agent of furunculosis [94]. Aeromonas hydrophila, Aeromonas sobria, and Aeromonas veronii are among the most common bacteria that cause motile Aeromonas septicemia in fish [94,95]. In addition, it is known that many different Aeromonas species cause disease in fish.
The antimicrobial effects of essential oils of some herbs on Aeromonas salmonicida subsp. Salmonicida has been investigated (Table 3). Hayatgheib, et al. [96] found that MIC and MBC values of essential oils (EOs) of different herbs on different A. salmonicida subsp. Salmonicida isolates were in the range of 113 to ≥3628 µg/mL, and the most effective (MIC and MBC: ≤520 µg/mL) herb species were Cinnamomum zeylanicum/verum, Origanum vulgare, Origanum compactum, Origanum heracleoticum, Eugenia caryophyllata, and Thymol rich Thyme vulgaris.
In a different study, the antimicrobial effects of Origanum onites, O. vulgare, and Thymbra spicata EOs on 18 different A. salmonicida isolates, and it was reported that EOs of these herbs formed 10 to 30 mm zone depending on the disc diffusion test, and they had moderate inhibitory depending on MIC values (800 µg/mL) [97]. Among Thymus vulgaris, Laurus nobilis, Rosmarinus officinalis, Petroselinum crispum, and Thymus vulgaris EOs showed the highest zone diameter with 30 mm on A. salmonicida [98], while Azadirachta indica nanoemulsion also exhibited similar results [99]. Cinnamomum cassia EO was reported to have a very high inhibitory effect on A. salmonicida subsp. with a 56 mm zone diameter [100].
Tural, et al. [98] reported that among T. vulgaris, L. nobilis, R. officinalis, and P. crispum EOs, T. vulgaris EO had the highest zone diameter on Aeromonas sobria and Aeromonas veronii with 31.5 mm and 36 mm, respectively. It was determined that Origanum acutidens EO formed a zone diameter of 32.7 mm on Aeromonas hydrophila [101].
A strong inhibitory effect of Ocimum basilicum EO with 3 µL/mL and 9 µL/mL MIC values was reported on A. hydrophila and A. veronii, respectively [105]. Among nine different herb EOs, Conobea scoparioides and Lippia origanoides EOs had remarkable activity against A. hydrophila with the low respective MIC and MBC values of 200 µg/mL [106].
It was reported that Eucalyptus globulus, Lavendula angustifolia, Origanum vulgare, and Melaleuca alternifolia nanoemulsions were more effective on A. hydrophila than their EOs, and among four different herbs, O. vulgare essential oil was found as the most effective with 25 µg/mL MIC and MBC, and the nano-emulsion was also found as the most effective with 3.12 µg/mL MIC and 12.5 µg/mL MBC [51]. However, generally moderate and weak inhibitory effects of Ocimum americanum [86], Hesperozygis ringens and Ocimum gratissimum [107], and Lippia alba [108] EOs on different A. hydrophila isolates were also reported.

Vibrio spp., Listonella anguillarum, and Photobacterium damselae
Historically, vibrionaceae family members are the most severe infectious diseases in marine fish species [109]. The antimicrobial effects of O. vulgare, M. alternifolia, C. citratus, C. verum, and T. vulgaris EOs on Vibrio campbellii, Vibrio harveyi, Vibrio vulnificus, and Vibrio parahaemolyticus have been researched, and it was reported that generally moderate and weak inhibitory effects of these EOs on Vibrio spp [110]. Wei and Wee [102] indicated that Cymbopogon nardus EO showed potent inhibitory effects with 0.244 µg/mL and 0.488 µg/mL MIC values on Vibrio spp. and Vibrio damsela, respectively. Similarly, a strong inhibitory effect of Thymus vulgaris EO was reported, respectively, with 320 µg/mL MIC for Vibrio ordalii and Vibrio anguillarum and 80 µg/mL MIC for Vibrio parahaemolyticus [111]. A marked activity of Syzygium aromaticum EO with 0.015 µg/mL MIC values was reported on six different isolates of Vibrio spp. [103].
It was reported that E. globulus, L. angustifolia, O. vulgare, and M. alternifolia nanoemulsions were more effective on Photobacterium damselae than their EOs, and among these herbs, O. vulgare EO and nano-emulsion were found as the most effective [51].

Pseudomonas fluorescens
Pseudomonas fluorescens is a harmful pathogen in a variety of farmed fish. It was reported that Ocimum basilicum EO exhibited a potent inhibitory with 9 µL/mL MIC value on P. fluorescens [105]. C. Nardus [102] and S. aromaticum [103] EOs showed marked activity on Pseudomonas spp. and P. Aeruginosa. Thymus vulgaris EO had a moderate inhibitory effect on Pseudomonas sp. with 640 µg/mL MIC value [111].
Among T. vulgaris, L. nobilis, R. officinalis, and P. crispum EOs, T. vulgaris EO exhibited the highest zone diameter with 26.5 mm on P. fluorescens [98]. T. vulgaris was also found as the most effective with a 13 mm zone diameter on P. Aeruginosa [114].
It was determined that C. freundii showed susceptibility towards the Argania spinosa EO with a zone diameter of 15 mm [113], and C. nardus EO with a MIC value of 0.244 µg/mL [102].

Raoultella ornithinolytica
Raoultella ornithinolytica was isolated from kidneys and skin lesions of naturally diseased silver catfish (Rhamdia quelen), and Ocimum gratissimum EO showed a moderate inhibitory effect on this pathogen [37].

Nocardia seriolae
Nocardia seriolae is the causative agent of nocardiosis in cultured fish species [115]. Ismail and Yoshida [116] reported that MIC values of C. Zeylanicum, Thymus vulgaris, Cymbopogon flexuosus, and Melaleuca alternifolia EOs on 80 Nocardia seriolae isolates were in the range of 5 to >5120 µg/mL, and the most effective herb species were C. zeylanicum and T. vulgaris with MICs 5-160 µg/mL, respectively.

Flavobacterium spp.
Flavobacterium species are widespread in soil habitats and fresh and marine waters and cause economic losses in cultured fish. T. vulgaris EO exhibited a potent inhibitory with 320 µg/mL MIC value on F. psychrophilum [111].
Previous studies have reported that Flavobacterium spp. showed high susceptibility towards the S. aromaticum EO with a MIC value of 0.031 µg/mL [103], and C. nardus EO with a MIC value of 0.977 µg/mL [102]. R. officinalis EO showed a moderate zone diameter with >~18 mm on F. psychrophilum [117]. A remarkable activity of Allium tuberosum EO with 20 µg/mL to 80 µg/mL MIC values was reported on six different isolates of Flavobacterium columnare [118].
Gholipourkanani, et al. [51] determined that among E. globulus, L. angustifolia, O. vulgare, and M. alternifolia nano-emulsions and EOs, O. vulgare EO and/or nano-emulsion were found as the most effective on Streptococcus iniae. Oliveria decumbens EO had a zone of inhibition of 69 mm, and MIC and MBC values of 0.5 mg/mL and 2 mg/mL, respectively, on S. iniae [120].
A remarkable activity of Z. multiflora and R. officinalis EOs were reported, respectively, with 0.06 µL/mL and 0.5 µL/mL MIC, and 0.12 µL/mL and 0.25 µL/mL MBC for S. iniae [121]. Similarly, R. Officinalis, Z. Multiflora, A. Graveolens, and E. Globulus EOs exhibited potent inhibitory effects on S. iniae, and R. Officinalis showed the highest inhibition with a zone of 45 mm, and MIC value of 3.9 µg/mL, and MBC value of 7.8 µg/mL [122].
It was reported that Streptococcus spp. showed high susceptibility towards the S. aromaticum EO with a MIC value of 0.062 [103] and C. nardus EO with a MIC value of 0.488 [102].
Thymus vulgaris EO had marked activity with a zone diameter of 36.7 mm on L. Garvieae [101]. Among T. vulgaris, L. nobilis, R. officinalis, and P. crispum EOs, T. vulgaris EO exhibited the highest zone diameter with 29.5 mm on L. Garvieae [98].

Research Gaps and Concluding Remarks
Using of herbal compounds in aquaculture is increasing day by day as a means of aquaculture sustainability. Essential oils (EOs) show beneficial effects on growth, immunity, antibacterial and antiparasitic activities in fish culture and are used as anesthetic compounds during fish handling and transportation. The efficiency of EOs depends on plant variables, chemical compositions of bioactive compounds, environmental characteristics of plant origin, and parts of plants from which EOs is extracted. Sometimes plant originated EOs possess a mixture of different compounds, which may produce undesirable side effects on fish and shellfish. Commercial pharmaceutical companies might play significant roles in refining the desirable and undesirable compounds of EOs to achieve better effects in fish culture.
Importantly, EOs molecular mechanisms for fish immunity increment, bacteria, and parasite destruction are also questionable. Future research through cell culture and in vitro identification and characterization of EOs action pathways may solve these questions. In the upcoming days, EOs optimum doses against infectious bacteria and parasites for worldwide commercial fish species should be extensively studied.
Lastly, the synergistic relationship between/among the bioactive compounds of EOs also opens a new research area. Before applying EOs in aquaculture from any new plants, local and international drug regulating agencies (FDA or EU) permission or guidelines should be needed or followed.
Author Contributions: Authors shared equally in this work. All authors have read and agreed to the published version of the manuscript.