Effects of Green Alga, Chaetomorpha aerea Extract on Non-Specific Immune Responses and Disease Resistance against Edwardsiella tarda Infection in Labeo rohita

The current study evaluated the effects of a methanol extract from Chaetomorpha aerea (a green alga) on non-specific immune responses and resistance against Edwardsiella tarda infection in Labeo rohita. Different doses of the extract (5, 50 and 500 mg/kg of body weight) were injected into the fish intraperitoneally while a control group was injected with 0.2 mL of sterile physiological salt solution. Variations in several immunostimulatory parameters (i.e., neutrophil, serum lysozyme, myeloperoxidase, serum antiprotease, and ceruloplasmin activity), reactive oxygen species (ROS) and reactive nitrogen species (RNS) were assessed after 7, 14, 21, and 28 days of post stimulation. E. tarda culture was injected into the fish after 28 days of post stimulation to induce infection to monitor fish mortality within 14 days. Interestingly, all doses of methanolic extract enhanced neutrophil, lysozyme, and myeloperoxidase activity, ROS and RNS, while a dose of 50 mg/kg was the most effective. Fish injected with this optimal dose were also protected against infection with virulent strain of E. tarda. The results of the study suggest that C. aerea extract is a potential prophylactic agent against bacterial infections in finfish.


Introduction
Aquaculture has drawn a great deal of attention during the last few decades due to the recognition on its potent role in meeting nutritional requirements, providing employment, and generating foreign trade [1]. Over the years, aquaculture practices have expanded into more diverse ways all across the globe. As a result, the incidence of infectious diseases in farmed fish has also been increased. In line with such situation, research efforts on defense mechanisms against microbial attacks in fish are increasing [2]. Various prophylactic measures (i.e., immunostimulation, vaccination, genetic improvement) and therapies (disinfectants and antibiotics) are in use [3]. As the application of antibiotics has become limited due to widespread incidences of antibiotic resistance and environmental pollution [4], immunostimulants have become promising alternatives for disease management in farmed fish [5]. A chemical, drug or action may be considered as an immunostimulant if it enhances the defense mechanisms or immune responses that improve resistance to diseases [6]. Both specific and non-specific defense mechanisms play crucial roles in control

Experimental Animals
Ninety healthy L. rohita specimens with no sign of disease or previous history of parasitic infestation were obtained from a private fish farm, Sakkangudi (11.4201 • N, 79.6172 • E) in Chidambaram, Cuddalore District, Tamil Nadu, India. Fish were bathed with a 3% sodium chloride solution to eradicate ectoparasites. Fish were acclimatized to experimental conditions in 150 L fiber-reinforced plastic tanks for 15 days prior to experimentation. The tanks were filled with dechlorinated tap water. During acclimatization, the temperature of the tanks was maintained at 28.0 ± 2.0 • C. A natural photoperiod was maintained and tank water was replaced after every three days.
After acclimatization, fish were distributed into 15 tanks, each tank containing six fish specimens. The experiment was conducted in triplicate. During the course of the experiment, fish were fed a balanced diet ad libitum as described earlier [27]. Fish with a mean weight of 25.0 ± 0.23 g were reserved for serological assays and disease-resistance parameters. Fish with a mean weight of 50.0 ± 0.31 g were used for analysis of reactive oxygen species (ROS) and reactive nitrogen species (RNS).

Preparation of Extract
Methanol extract of C. aerea was prepared and employed according to a previously described protocol with slight modifications [28]. In brief, dried and coarse powdered algae were extracted by a cold maceration process that uses solvents with increasing polarity (i.e., petroleum ether, chloroform, ethyl acetate, methanol and 0.25% of chloroform:water [v/v]). Extracts were dried using a rotary vacuum evaporator (Buchi, Switzerland) and then stored at −20 • C until further use. Sterile water was used to reconstitute further dilutions of methanol extract.

Experimental Design
Reconstituted methanol extract (0.2 mL) was injected into fish in each experimental group (T1, T2 and T3) intraperitoneally with 10-fold increasing doses of 5, 50 and 500 mg/kg body weight, respectively. Controls were injected with 0.2 mL of sterile water. Blood was collected from each fish seven days before treatment and 7, 14, 21 and 28 days after treatment.

Blood Sampling and Serum Separation
Blood was collected using a 1 mL tuberculin syringe equipped with a 24-gauge needle through the common cardinal vein of fish [29]. To analyze serum, collected blood (200 µL) was stored in serological tubes at −4 • C overnight. Afterward, the blood was centrifuged at 400× g for 10 min. The resulting supernatant (serum) was transferred to sterilized Eppendorf tubes and stored at −20 • C until further analysis.
2.5. Immunological Parameters 2.5.1. Neutrophil Activity A nitro blue tetrazolium (NBT) assay was adapted to measure neutrophil activity following Stasiacket al. [30] protocol with little modifications. A 100 µL sample of blood was added to each well of a flat-bottom microplate. For adhesion of cells, the microplate was incubated for 1 h at 37 • C. The supernatant was discarded and loaded wells were washed three times using phosphate buffered saline (pH 7.5). Afterward, 0.2% NBT (100 µL) was added to each well and the plate was incubated again for1 h. Methanol (100%) was added to the cells and mixed for 2-3 min. Methanol (70%) was employed to wash the mixture three times and plates were air-dried. Next, 120 µL of 2 N KOH and 140 µL of dimethyl sulfoxide were poured into each well, resulting in blue precipitate of formazone. Optical density (OD) of the mixture was measured using a microplate reader (Systronics, Kolkata, India) at 620 nm.

Serum Lysozyme Activity
Turbidity method was used to estimate serum lysozyme activity [31]. In brief, 0.03% of lyophilized Micrococcus luteus was added to 0.05 mM sodium phosphate buffer (PBS) (pH 6.2) and the suspension was treated as substrate. Blood serum of fish (10 µL) was poured into 250 µL of microbial suspension in a microplate, which was incubated for 0.5 and 4.5 min at 22 • C. Absorbance at 490 nm was determined at both time points using a microplate reader. The reduction in absorbance was the difference in absorbance values of the two time points. One unit of lysozyme activity was considered equal to a decrease in absorbance of 0.001 per min.

Myeloperoxidase Activity
Total serum myeloperoxidase activity was assessed following Sahoo et al. protocol [32] with slight modifications. In brief, 10 mL of serum was added to 90 mL of Hank's balanced salt solution without Ca 2+ or Mg 2+ in a 96-well microplate to which 25 µL of 20 mM 3,3,5,5-tetra methyl benzidine hydrochloride (Genei, Bengaluru, India) and 5 mM H 2 O 2 were added in a 1:20 dilution and incubated for 2 min. Reaction was stopped by adding 50 µL of 4 M H 2 SO 4 . A microtiter plate reader (Cyper lab) at 450 nm was used to determine OD.

Ceruloplasmin Activity
To measure ceruloplasmin activity in serum, p-phenylene diamine oxidase activity was utilized as described by Dautremepuits et al. [33] with slight alterations. In brief, 0.5 µL of 1.2 M acetate buffer (pH 5.0) with 0.1% p-phenylene diamine as a substrate was added to 25 µL of serum and shaken well to mix the contents. Similarly, 0.5 mL of 0.5% sodium azide was added to 25 µL of the same serum sample to prepare a blank. The solutions were incubated at 30 • C for 30 min. To stop the reaction, 0.5 µL of 0.5% NaN 3 was added. The quantity of oxidase that catalyzed reduction in absorbance of 0.001 min −1 at 550 nm was considered one unit of ceruloplasmin.

Serum Antiprotease Activity
Serum antiprotease activity was measured by employing an assay as described by Rao and Chakrabarti [34]. In brief, 10 µL of fish serum was mixed with 100 mL of bovine trypsin type I and incubated at 25 • C for 30 min. Two blanksamplescontaining 110 µL PBS and three reference samples containing 10 mL PBS/100 mL trypsin were also prepared. The reaction mixtures were incubated for 15 min with 1 µL of casein and the reaction was terminated by adding 500 µL of 10% trichloroacetic acid to each mixture. The mixtures were centrifuged at 10,551× g for 5 min and the OD of the supernatants at 280 nm was measured. The following formula was used to calculate percent inhibition:

Preparation of Viable Leukocytes from Peripheral Blood
To separate peripheral blood leukocytes, a 10 mL syringe with 2 mL of a bloodcollecting medium (RPMI 1640 supplemented with 50,000 IU/L sodium heparin, 100,000 IU/L penicillin, and 100 mg/L streptomycin) was used. An equal volume of lymphocyte separation medium (Lymphosep, ICN Biomedicals, Inc., Irvine, CA, USA) was mixed with diluted blood. The cells were centrifuged at 800× g for 20 min. Leukocytes collected from the interface were washed twice using a wash medium (RPMI 1640 supplemented with 50,000 IU/L sodium heparin, 100,000 IU/L penicillin, and 100 mg/L streptomycin). The washed leukocytes were re-suspended in culture medium (RPMI 1640 supplemented with 3% [v/v] of pooled tilapia serum, 100,000 IU/L penicillin, 100 mg/L streptomycin and 4 mM/L glutamine, Biochrom AG, Berlin, Germany). The number of viable cells was estimated following the trypan blue exclusion method [35] and adjusted to 4 × 10 7 cells/mL using a culture medium.

Reactive Oxygen Species Production
For measurement of intracellular respiratory burst activity, a previously published method [36] with minor modifications was used. 25 µL of NBT (1 g/L) was added to 175 µL of culture medium and incubated at 28 • C with a fixed concentration (1 × 10 6 cells/well) of peripheral blood leukocytes for 2 h. The supernatants were then discarded and the remaining material with cells was mixed in absolute methanol for 5 min. Wells were washed twice with 125 µL of 70% methanol and the wells were air-dried overnight. Finally, 150 µL of dimethyl sulfoxide and 125 µL of 2 N KOH in each well were used to dissolve reduced NBT (in the form of formazan). OD was found using a microplate reader (at 650 nm).

Reactive Nitrogen Species Production
Leukocytes in peripheral blood generate NO, which is instantly converted into nitrite. Concentration of this nitrite extract can be estimated calorimetrically by adding a Griess reagent [37]. Briefly, peripheral blood leukocytes were added to 175 µL of culture medium and incubated for 96 hat 28 • C. A solution of 1% copper sulfate was used to maintain moisture during incubation. 50 µL of culture supernatant was shifted to wells of a new microplate and 50 µL of Griess reagent (1% sulfanilamide, 2.5% phosphoric acid, and 0.1% N-naphthul-ethylenediamine) was added to each well with culture supernatant. After an incubation time of 10 min, the NO 2 concentration (in moles) was calculated following a pre-generated standard curve (formed from a graded series of NaNO 2 concentrations in the same culture medium).

Disease Resistance
To determine disease-resistance capability, E. tarda MTCC 2400 (IMTECH, Chandigarh, India) was used. Before initiation of the trial, LD 50 of the pathogen was determined as 1.2 × 10 8 colony forming units (cfu) /fish employing a separate fish group. Brain-heart infusion broth was used to culture E. tarda for a period of 24 h at 28 • C, and then centrifuged at 3000× g for 10 min. The supernatant was discarded and the pellets were re-suspended in sterile PBS. After administration of methanol extracts, the fish were injected with 0.1 mL of E. tarda (1.2 × 10 8 cfu/mL) suspended in PBS intraperitoneally. OD of bacterial culture suspension absorbance was adjusted to 1.2 as it was previously calculated that 0.1 mL of the bacterial culture with an OD of 1.2 at 600 nm has a bacterial count of 1.2 × 10 8 cfu/mL. Ten fish from each tank of experimental and control groups were used to test resistance against E. tarda infection. Mortality of the fish was observed for 14 days. Disease resistance was calculated as relative percent survival (RPS) based on recorded mortality data, using the following formula [38]: where, n = total number of fish.

Statistical Analyses
The significance of differences between means was evaluated by one-way analysis of variance following Duncan's multiple-range test (at 5% probability level) and using the statistical package SPSS, version 16.

Results and Discussion
Fish treated with methanolic extract of C. aerea at various doses showed significant (p < 0.05) differences for non-specific immune responses by the 7th, 14th, 21st and 28th day after treatment. Findings of the present study indicated that all doses of methanolic extract significantly increased neutrophil, lysozyme, and myeloperoxidase activity, as well as ROS and RNS. Injections of 50 mg/kg of methanolic extract were the most effective in increasing non-specific immune responses in L. rohita (Table 1).

Neutrophil Activity
The neutrophil activity (NBT activity; OD 620 nm) of the treated groups was higher (p < 0.05) than that of the controls. It was highest in the T2 group on the 14th and 21st days after treatment. Moreover, neutrophil activity showed a noticeably increasing trend from 7 to 14 days of post-treatment ( Figure 1). Similarly, neutrophil activity has been reported to be significantly different in treated groups of L. rohita compared with controls, after treatment with methanolic extract of C. antennina. The NBT values of 75 mg/kg exhibited significantly higher values compared with controls (Sattanathan, 2020a). Gora, AH et al. (2018), has also reported that the highest and significantly different NBT activity was found in L. rohita treated with 2% dietary level of Fucoidan-rich seaweed (Sargassum wightii) extract [39].

Lysozyme and Myeloperoxidase Activity
In the present study, serum lysozyme activity in fish treated with C. aerea extract significantly increased from 7 to 21 days (p < 0.05) post treatment. Maximum lysozyme activity was found in the T2 group on the 28th day after treatment compared with controls ( Figure 2). Similar findings have also been reported in other studies. In one of our previous studies, treatment with methanolic extract of C. antennina significantly increased serum lysozyme level in L. rohita [24]. It has been reported that Fucoidan-rich seaweed (Sargassum wightii) extract at 2% dietary level significantly increased serum lysozyme activity in L. rohita [39]. Lysozymes constitute an important part of various defense molecules of the immune system. They mediate resistance against the microbial attack of gram-positive bacteria [40] as well as gram-negative bacteria [41] by destroying cellular walls of bacteria. Lysozymes split the linkages between the β (1→4) linking N-acetyl muramic acid and N-acetyl glucosamine in gram-positive bacteria's cell walls. In the case of gram-negative bacteria, the lysozymes mediate other enzymes to interrupt the outer cell wall, thus exposing the inner peptidoglycan layer [40,41]. Lysozyme also plays a role to regulate immune cell differentiation and proliferation, and stimulates phagocytic activity [42]. Methanolic extracts of various herbs (i.e., Ocimum sanctum, Myristica fragrans, and Withania somnifera have significantly improved several immune parameters, including serum bactericidal and phagocytic activity, leukocrit and the albumin-globulin ratio, against bacterial infection induced by Vibrio harveyi in larvicultured juvenile Epinephelus tauvina, a grouper fish species [43]. It has been shown that L. rohita cultured on a diet with 0.5% Achyranthes aspera seeds exhibit enhanced lysozyme activity [44]. In addition, serum lysozyme activity increased significantly in rainbow trout (Oncorhynchus mykiss) cultured on food containing a marine algae, Dunaliella salina [45]. Divyagnaneswari and colleagues (2007) used various doses of water extract of Solanum trilobatum leaves (4, 40 and 400 mg/kg body weight) intraperitoneally to tilapia (Oreochromis mossambicus) and found that a dose of 40 mg/kg significantly increased lysozyme activity on days 4, 6, and 8 post-treatment [11].  In the current study, myeloperoxidase activity of the experimental groups varied significantly (p < 0.05) compared with the controls (Figure 3). The enhanced myeloperoxidase (MPO) activity was observed in groups treated with 5 mg/kg (T1) and 50 mg/kg (T2) of C. aerea extract with the highest in T2 group (Figure 3). Myeloperoxidase has been reported to oxidize macrophages and neutrophil cells after invasion in the fish body [46]. In the study performed by Sattanathan, G 2020a [21] on the effect of methanolic extract of Chaetomorpha antennina on non-specific immune response in Labeo rohita, MPO activity increased in both 25 and 150 mg/kg of body weight. Previously, it was observed that rainbow trout (O. mykiss) treated with methanolic extract of nettle showed greater MPO activity [47]. In the current study, fish receiving methanolic extract of C. aerea showed significantly increased respiratory burst activity compared with the control group during the whole trial period. Rao et al. [44] reported an increase in superoxide anion production in leucocytes in L. rohita given feed fortified with Allium sativum and Achyranthus aspera. Production of superoxide anions during the respiratory burst of phagocytes is considered an important factor for limiting fish pathogens growth [48].

Ceruloplasmin and Antiprotease Activity
Fourteen days after treatment, all groups showed relatively higher ceruloplasmin activity compared with the control. T2 group showed the highest ceruloplasmin activity (p < 0.05) among all groups. However, on the 7th day after treatment, ceruloplasmin activity was higher in T1 and T2 and lower in T3 (Figure 4). In another similar study, methanolic extract of C. antennina also significantly increased ceruloplasmin activity in treatment groups of L. rohita after induction of E. tarda infection [21]. In a previous study, a significantly positive (0.51) correlation was observed with all the studied immune parameters and survival in L rohita against aeromoniasis [49]. The plasma concentrations of several acute phase proteins including ceruloplasmin show a rapid and sharp rise against infections induced by bacterial pathogens. Such non-specific acute phase reactions are considered first-line defenses against pathogenic attack [49]. Ceruloplasmin performs its function in different ways depending on its activities like revamping tissue damage, preventing spread of infectious microbes, and invading pathogens [40].
All groups of fish exposed to C. aerea extract showed significantly higher (p < 0.05) antiprotease activity relative to controls on the 14th day after treatment. Antiprotease activity was highest in T2 compared with controls ( Figure 5). Similarly, in another study, highest level of antiprotease activity, such as trypsin inhibition, was observed in T2 group (75 mg/kg of body weight) of L. rohita compared to controls after treatment with methanolic extract of C. antennina [21]. Several antiproteases, including a1 antiprotease, a2 antiplasmin, and a2 macroglobulin, have been documented in fish plasma. These antiproteases have been shown to restrict bacterial invasion and growth in vivo [50]. We found significantly higher antiprotease activity in treatment groups. A higher inhibition (%) due to antiprotease activity was observed in the susceptible line compared with a resistant line, although this difference was not statistically significant [51]. In another study, serum antiprotease activity varied widely in several species of rohu. A negative correlation was found with survival against Edwardsiellosis [52].

Reactive Oxygen Species (ROS) and Reactive Nitrogen Species (RNS) Production
ROS production was higher (p < 0.05) in C. aerea-treated groups compared with controls ( Figure 6). T2 fish treated with 50 mg/kg dose of methanolic extract of C. aerea showed the highest ROS production at all post-treatment time points, followed by T1 and T3. Similarly, RNS production also showed an increasing trend during the post-treatment period (Figure 7). Increased superoxide anion production in blood leukocytes has been observed in L. rohita fed with food containing 0.5% Achyranthes [44].  Respiratory burst, reflected by enhanced oxidation levels of phagocytes after stimulation with foreign agents, is regarded as an important indicator of non-specific immune mechanisms in fish [53]. In vitro treatment of rainbow trout with glycyrrhizin extracted from Glycyrrhiza glabra has been reported to increase macrophage levels. This stimulatory effect may be the result of the proliferative responses of leukocytes [54]. In Ergosan-injected rainbow trout, immunostimulatory effects had been suggested due to an increase in respiratory burst activity, the degree of phagocytosis and interleukin expression [55]. The reduction in NBT by intracellular superoxide radicals (produced by leukocytes) can be used to measure respiratory burst activity. It has been reported that treatment with low concentrations of immunostimulants yields higher respiratory burst activity of phagocytes in fish, while higher doses of immunostimulants yield lower NBT values, suppressing defensive mechanisms [56].

Relative Percent Survival
No mortality was observed up to 24 h after exposing fish to E. tarda infection. Fish treated with varied concentrations of methanolic extract of C. aerea exhibited higher survival (p < 0.05) compared with the control. The highest relative survival (83.5%) was shown in the T2 group, which received 50 mg/kg of algal extract, while it was lowest in the controls ( Figure 8). This may have resulted from an increase in non-specific immune responses. In several previous reports, various herbal medicinal plant extracts (Cyanodon dactylon, Tinospora cordifolia, Aegle marmelos, Eclipta alba, and Picrorhiza kurooa) exhibited a positive influence on survival rate (74%), and were associated with a decrease in viral load in shrimps infected with the white spot syndrome virus [57]. The results of the study demonstrated that the RPS was strongly correlated with functional assays as well as hematological parameters. An inverse relationship was found between the amount of extract administered and the mortality rate. In another similar study, methanolic extract of C. antennina significantly increased survival percentage in L. rohita after induction of E. tarda infection [21]. It has been found that various doses of fucoidan-rich extract of a seaweed (Sargassum wightii, a brown algae) has also reduced mortality in infected L. rohita compared to controls [39]. The low mortality rate in fish with algal extract treatment may also be attributed to antimicrobial properties. It has also been reported that intraperitoneal injections of protein-bound polysaccharides from Coriolus versicolor reduced mortality rate in Oreochromis niloticus against E. tarda infection [58]. Screening of antimicrobial activities of extracts from various algal species including Chnoosporabic analiculata, Ulva fasciata, Cheilosporum spectabile, Bryopsisplumosa, Grateloupia filicina, C. antennina, Hypneapannosa, Centroceras clavulatum, Portieria hornemannii, Gracilaria corticata, Sargassum wightii, A. orientalis, Stocheospermum marginatum, and Padinate trastromatica has been performed.It has been demonstrated that antimicrobial compounds extracted from fresh algae using methanol: toluene (3:1) solvents are more effective due to maintenance of lipophilic nature over wide temperature range (30-60 • C), whereas the extracts from dry algal species showed decreased level of bioactive compounds. Several of the extracts from algae like Acrosiphonia orientalis exhibited antibacterial properties against 70% of the microbes analyzed. The extracts from several algal species like Stocheospermum marginatum and Gracilaria corticata were found to be more effective against Gram-negative bacteria compared to Gram-positive bacteria [59]. The findings of the current study suggest a novel insight into the immunostimulation capacity of the methanolic extract of C. aerea in L. rohita to reduce mortality caused by bacterial infection. Thus, the health of the fish can be improved by the use of ethnoveterinary therapeutic agents like algal extracts which boost the non-specific immune response. The immunostimulants obtained from algal extracts may replace the use of antibiotics in aquaculture. However, as a future perspective, antimicrobial activity of C. aerea extract against E. tarda needs to be assessed in vitro.

Conclusions
Outcomes of the study suggest that the methanolic extract of C. aerea exerts a protective effect in L. rohita against E. tarda infection. This protective effect could be mediated through non-specific immune mechanisms, as evidenced by the enhanced activity of the several nonspecific immunostimulatory parameters, including neutrophil, lysozyme, ceruloplasmin, and serum antiprotease activity level, as well as elevated ROS and RNS levels. The results of our study also give a comparative insight into immunomodulatory effects of various doses of C. aerea in L. rohita. The 50 mg/kg body weight dose of C. aerea extract may be optimal, as indicated by increased levels of non-specific immune-response parameters and high survival rates.
The results of the present study suggest that methanolic extract of C. aerea administered as an IP treatment to fish could replace antibiotics. The immunostimulants present in C. aerea extract have a positive effect on fish health and therefore reduce aquaculture costs.