Dietary Lactobacillus sp. mitigates Deltamethrin-induced toxic and immune-suppression impacts in Nile Tilapia (Oreochromis niloticus)

doi:10.21203/rs.3.rs-3969700/v1

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Dietary Lactobacillus sp. mitigates Deltamethrin-induced toxic and immune-suppression impacts in Nile Tilapia (Oreochromis niloticus)

https://doi.org/10.21203/rs.3.rs-3969700/v1

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Both organic and inorganic contaimanants in the aquatic environment seriously affect marine organisms, including fish. In this study we aim to isolate diatry Lactobacillus sp. from both sea bass (Dicentrarchus labrax) and sea bream (Sparus aurata) fish and evaluate their probiotic impact and ability to mitigate the toxic impact of the existing insecticide deltamethrin (DLM) residuals in fish farming water resources. Out of the 40 isolated Lactobacillus sp., 22 were gram positive, catalase-negative and non-hemolytic in the γ hemolysis reaction. One out of the above mentioned 22 isolates (denoted as SB8) were antagonistically reacted with Aeromonas hydrophilic ss. hydrophila (ATCC 130 37) and Pseudomonas fluorescens (ATCC13525).

The probiotic candidate SB8 was resistant to acidic conditions, grow well in 0.3% bile salt and 0.4% phenol. Morover, an in-vivo study was conducted on Nile tilapia to evaluate the impact of using the SB8 as feed supplement on haematological profile, oxidative stress enzymatic activities, protein content, micronucleus formation, and IL-1 & IL-6 expression.

The obtained results indicated that 1) white blood cells (WBC), neutrophils (Neu), lymphocytes (Lum), monocytes and eosinophils (Mon and Eos, 2) malondialdehyde (MDA), superoxide dismutase (SOD), catalase and glutathione peroxidase (MDA, SOD, CAT and GPx, 3) Protein content (total protein, albumin (A), globulin (B) and albumin/globulin (A/G), 4) micronucleus formation, and 5) Interleukin-1 and Interleukin-6 (IL-1 and IL-6 expression in the DLM-exposed/ SB8-supplemented and in the control fish are reasonably similar in comparison with the DLM-exposed fish. In conclusion, the probiotic candidtae SB8 has a potential to mitigate the DLM-induced deleterious oxidative stress impact in Nile tilapia fish.

Biological sciences/Biotechnology

Biological sciences/Immunology

Biological sciences/Microbiology

Sea bass and sea bream

Deltamethrin (DLM)

Probiotic Lactobacillus

Nile tilapia

Oxidative stress

In Egypt, Aquaculture has been an important sector in the agricultural economy for decades. Today, Egypt is one of the top aquaculture producers in Africa with an estimated annual production of over 1 million metric tons of fish and other seafood products [1]. This sector is subjected to agriculture drainage water containing pesticides and other toxic pollutants [2]. Pesticides present in water used in fish farming can cause many health problems in fish. Deltamethrin (DLM) is a widely used synthetic insecticide that belongs to the class of pyrethroids. It is extensively employed in agricultural practices to control a broad range of pests, including insects, mites, and ticks. However, the increased use of DLM has raised concerns about its potential negative impact on the environment, particularly its presence in water bodies negatively affects the aquaculture systems [3]. Such pesticides can be present in water used in fish farming and cause many adverse effects.

The residual of DLM derivatives may accumulate inside fish tissues, impede their growth performance, and deteriorate their health [4]. Moreover, the accumulation of DLM in aquaculture products can pose risks to human health through consumption [5]. Therefore, understanding the fate and behavior of DLM in water bodies and its impacts on aquaculture systems is crucial for ensuring sustainable aquaculture practices and protecting the aquatic ecosystems and human health.

The rapid expansion in aquaculture, intensive farming systems, and many related infections and illnesses motivate researchers to develop alternative means for mitigating both biotic and abiotic stresses in the surrounding ecosystem. The use of probiotics in controlling pathogenic bacteria and improving overall fish health has a wide encuragment within the aquaculture industry [6, 7, 8]. Originally, probiotics were developed to benefit human and animal livestock and the first suggestion for their application in the aquaculture sector was by Yasuda and Taga [9]. However, Kosaza, [10] and Gatesoupe et al. [11] were among the first researchers who published preliminary studies on this issue.

Lactobacillus species are renowned for their capacity to produce lactic acid, which aids in maintaining pH levels in water and inhibiting the proliferation of harmful pathogens [12]. Moreover, these bacteria can enhance nutrient digestion and absorption in fish, resulting in enhanced feed utilization and growth performance [13]. Many studies have demonstrated that supplementing aquaculture with Lactobacillus can reduce disease occurrence, boost immune response, and improve overall fish health [13]. Therefore, this study aims to isolate and select lactic acid bacteria from both sea bass (Dicentrarchus labrax) and sea bream (Sparus aurata) fish intestine and assess impact of their use as food supplement on mitigating the toxic effect of the insecticide DLM which is widely contaminating the fish farming water resources.

Ex-vivo studies

Fish sampling and preparing of the intestine homogenate

Ten seabass (Dicentrarchus labrax) and 10 seabream (Sparus aurata) fish were collected from the Gulf of Suez, Egypt. The fish were transported in an icebox to the Biotechnology and Biodiversity Conservation Laboratory, Centre of Excellence for Advanced Sciences, National Research Center. Upon arrival at the laboratory, the fish were dissected under aseptic conditions and skins were sterilized with 70% ethanol. Fish intestines were excised using sterile scissors and forceps. 10 grams of the excised intestine was homogenized in a 90 ml saline solution (0.85% NaCl). Serial dilutions 10¹ to 10³ were prepared to isolate probiotic bacteria from the prepared suspension.

Isolation of Lactobacillus sp./probiotic candidates

Probiotic/Lactobacillus sp. were isolated using the pour-plate technique and the modified De Man Rogose Sharpe (MRS) agar. The MRS agar was modified by reducing the pH to 2.5 (oxgal, Sigma-Aldrich, St. Louis, Mo, USA) and the prepared plates were incubated at 37°C for 24–48 h according to Vinderola and Reinheimer [14]. For purification, the typical lactic acid bacterial colonies were picked up and subcultured on MRS agar plates for 48h. The suspected isolates were morphologically and biochemically invisitgated by applying Gram staining and catalase test, respectively. The γ hemolysis test of gram-positive and catalase-negative isolates were assessed using blood agar plates according to Gerhardt et al. [15]. We gave the intention only to the Gram-positive, catalase–negative and γ-hemolytic (non-hemolytic) isolates where they were preserved in tryptic soy broth supplemented with 30% glycerol (glycerol stock) at -80^oC as a master cell bank for the further evaluation.

Antimicrobial activity of presumptive probiotic isolates

The antimicrobial activity was evaluated using the well diffusion test, as described by Bauer et al. [16]. The targeted pathogenic bacterial were standard Aeromonas hydrophila ss. hydrophila and Pseudomonas fluorescens obtained from American Type Culture Collection, USA (ATCC 13037 and ATCC13525, respectively). The probiotic isolates were grown in MRS broth at 37°C for 24 h, followed by harvesting the cells by centrifugation at 4000 rpm for 15 min at 4°C. The melted Mueller-Hinton agar (Himedia, India) was inoculated with 10% freshly prepared broth culture of each pathogenic strain and then poured into petri plates for the well diffusion assay.

Once solidified, three wells with a diameter of 6 mm were punctured, and 50 µl of each prepared cell-free supernatant was placed in the wells. After incubation at 37 ^oC for 24h, the resulted inhibition zones around the wells were measured.

Acid and bile salt tolerance assays

Acid tolerance of the bacterial isolates was evaluated by inoculating the prepared cell pellets in MRS broth with pH ranginges of 2.0 to 4.0 as descrioed by Verdenelli et al. [17]. The bacterial cell count was adjusted for each cell suspension to have 10⁸ CFU/mL using the 0.5 McFarland turbidity standard. The viable residual cells were calculated using the plate count method on MRS agar after incubation at 37°C for 2 and 5h. The survival rate was calculated according to the following formula: Survival rate (%) = (log cfu N_t / log cfu N₀) × 100 where N_t and N₀ represent surviving bacterial count after incubation time and the initial count at zero time, respectively.

Bile salt tolerance was evaluated according to Gilliland et al. [18]. The extracted pellet was cultured in MRS broth supplemented with 0.3% of oxgall bile (Sigma-Aldrich, St. Louis, Mo, USA) at 37°C for 2 and 5h. Residual viable cells and survival rates were determined as described above.

Phenol tolerance

The ability of probiotic isolates to tolerate phenol was determined according to Xanthopoulos et al. [19]. Two milliliters of standardized probiotic isolates (10⁸ cfu/mL) were cultured at 37°C in 8 ml of MRS broth supplemented with phenol at 0.4% concentration for 24 h. The bacterial enumeration was conducted using the plate count method on MRS agar at 0 h and 24 h incubation and the residual viable cells and survival rate were estimated as aforementioned.

In-vivo studies

Fish

Animal ethics. Animal experiments were performed in accordance with the protocols approved by of the National Research Centre–Medical Research Ethics Committee (NRC-MREC) for the use of animal subjects (Approval no. 06410923) and in accordance with the ARRIVE guidelines and regulations.

Nile Tilapia have been brought from EL-Noubarya, Egypt, and transported to the biotechnology and biodiversity conservation laboratory, Centre of Excellence for Advanced Sciences, National Research Centre, Cairo, Egypt, in large plastic water containers supplied with battery-operated aerators as a source of oxygen. Fish were acclimatized for 10 days in glass aquaria (80× 40×30 cm) filled with 65 L of dechlorinated tap water. Aquaria glass (n-4) was stacked with 10 Tilapia fish (40.2 ± 5.5 g) and fed a basal fish feed with no additives. During the experiment, the water parameters (temperature, dissolved oxygen, ammonia, and nitrite) were measured and maintained within the recommended range according to APHA [20]. The water temperature, pH, and dissolved oxygen ranged between 26.7 ± 2.1°C, pH 7.2–8.2 and 6.9 ± 0.5 mg/l, respectively. The mean concentrations of ammonia and nitrite were recorded as 0.040 ± 0.005 and 0.06 ± 0.013 mg/l, respectively.

Fish feed preparation

The selected SB8 Lactobacillus candidate was grown for 24 h at 30°C in MRS broth in a shaking incubator. After incubation, the cells were harvested by centrifugation (4000 g), washed twice with phosphate-buffered saline (pH 7.2), and resuspended in the same buffer. The absorbance at 600 nm was adjusted to 0.25 ± 0.05 to standardize the number of bacteria (10⁶ to 10⁷ CFU mL^− 1). Dilution plating was conducted to verify the relationship between absorbance at 600 nm and CFU/ milliliter. Commercial basal fish feed (Zoo Control, Egypt) was used as the main diet and the SB8 Lactobacillus candidate was used as supplement to reach to a final concentration of 10⁶ to 10⁷ CFU g^− 1 feed. The count/bacterial conentration was determined using the standard plate count method on MRS agar [21], and the bacterial suspensions were slowly sprayed onto the feed with a contentious mixing.

Experimental design

Fish were randomly divided into four groups. Each group contained 30 fish in triplicates. Fish in the negative control group were fed a basal diet and not exposed DLM. Fish in the second group were fed a basal diet SB8-supplemented. Third group was a positive control where it fed a basal diet and was DLM-exposed (14.9 µg/L) [22]. Fish in the fourth (test ) group fed a basal diet supplemented with the SB8 and DLM-exposed (14.9 µg/l). Fish diets were provided twice daily at of 3% of fish live body weight rate for 30 days. At the end of the experiment, whole blood, serum, and liver samples were collected, immediately tested and/or stored in the proper storage condition until being analyzed.

Haematological investigation

Blood samples were collected from three anesthetized fish per aquarium. The blood has been withdrown from the caudal vein using a heparinized hypodermic syringe. The collected blood was mixed with a phosphate buffer saline solution and some hematological parameters such as total WBC, Neu, Lym, Mon, and Eos (neutrophils, lymphocytes, monocytes, and eosinophils) were measured according to Gabriel et al. [23]. where The WBC count was determined by diluting the blood sample 1:100 in PBS. And finally, the differential leukocytes count was determined indirectly by staining a blood smear with May-Grunwald-Giemsa.

Oxidative stress enzymatic activities

Liver samples were homogenized in an ice bath (homogenization ratio of 1g tissue/ 9mL physiologic saline. The homogenate was centrifuged at 3000g for 10 min to collect the supernatant which was stored at -80°C until being used to assess the oxidative stress enzymatic activities as described by Peña-Llopis et al. [24]. In brief the total protein level in the supernatant was measured according to the manufacturer’s instructions, by using the whole protein assay kit (BCA method, Jiancheng Bioengineering Institute) [24]. The malondialdehyde (MDA) level and superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) activities were measured with the respective commercial ELISA kits according to the manufacturer’s instructions (Jiancheng Bioengineering Institute, Nanjing, China) [24].

Total protein analysis

Blood samples were withdrawn from the caudal vein of the remaining anesthetized fish and then collected in untreated anticoagulant eppendorf tubes. The tubes were kept at 4°C for 10 h and then centrifuged at 3000 g for 15 min, and the supernatant serum was collected. The serum was immediately stored at -80°C for further biochemical analyses. All collected serum samples were analyzed for total protein using the Bradford method [25] and albumin content according to Doumas et al. [26]. Addtionally, the globulin content (subtracting albumin from total protein) and albumin/globulin ratio (A/G) were measured using commercial kits (Nanjing Jiancheng Bioengineering Institute, China) following the instructions provided.

Analysis of micronucleus formation

Micronucleus and erythrocyte alteration test was carried out relying on caudal incision to obtain blood smears on clean grease-free microscopic slides. After drying at room temperature, the slides were fixed in absolute methanol for 10 minutes. The slides were stained with hematoxylin and eosin. They were then dehydrated in ascending concentrations of alcohol (30, 50, 70, and 90%). Finally, the slides were cleared in xylene and permanently mounted by DPX [27]. Slides were selected based on the staining quality, then coded, randomized, and scored blindly. In each group, 10,000 cells (a minimum of 1000 per slide) were examined using 40× objective lens and 15× eyepiece for micronucleated and morphologically altered erythrocytes identification. We followed the well established criteria for identifying MN according to Shimdt [28] to ensure authentic scoring. The morphologically altered erythrocytes were examined according to Mekkawy et al. [29].

Expression analysis of immune genens

RNA isolation and RT-PCR Reaction

TRIzol® Reagent (Invitrogen, Germany) was utilized to extract total RNA from the liver tissues of Nile tilapia following the instructions provided by the manufacturer. The resulting total RNA was then treated with one unit of RQ1 RNAse-free DNAse (Invitrogen, Germany) to digest DNA residues. Subsequently, it was suspended in DEPC-treated water, and quantified spectrophotometrically at 260 nm. The purity of the total RNA was between 1.8 and 2.1 as recognized by the 260/280 nm ratio. Aliquots of the RNA were immediately used for reverse transcription (RT) or stored at -80°C for future use. Complete Poly (A)⁺ RNA isolated from liver tissues was reverse transcribed into cDNA in a total volume of 20 µl using RevertAid^™ First Strand cDNA Synthesis Kit (Fermentas, Germany). The RT reaction conditions were 10 min at 25°C, followed by 1 h at 42°C, and the denaturation step was 5 min at 99°C [30]. Afterward, the reaction tubes containing RT preparations were flash-cooled in an ice chamber until being used for cDNA amplification via quantitative Real-Time- polymerase chain reaction (qRT-PCR).

Real-Time- PCR (qrt-PCR)

StepOne Real-Time PCR system (Applied Biosystems, USA) was used to determine the liver cDNA copy number of IL-1 and IL-6. The PCR reaction mixrures were 25 µl containing 12.5 µl 1× SYBR^® Premix Ex Taq TM (TaKaRa, Biotech. Co. Ltd.), 0.5 µL 0.2 µM sense primer, 0.5 µL 0.2 µM antisense primer, 6.5 µl distilled water, and 5 µl of cDNA template. Each experiment included a distilled water control. The sequences of the used specific primers are listed in Table 1 [31]. After each qPCR, a melting curve analysis was performed at 95.0°C to assess the effectiveness of the primers used. The relative quantification of the target to reference gene was calculated according to the 2^−ΔΔCT method.

Table 1

Primers sequences used for qRT-PCR
Genes	Primer sequence ^a	References (Accession no.)
IL-1	F: 5`- caa gga tga cga caa gcc ag -3`	JF957370.1
IL-1	R: 5`- ggt agc gga cag aca tga ga -3`	JF957370.1
IL-6	F: 5`- aat tcc ttc tgg ccc tga ca -3`	XM_019350387.2
IL-6	R: 5`- tcc tct gtc tcc tca cct ga -3`	XM_019350387.2
β-Action	F: 5`- cca gcc ttc ctt cct tgg ta -3`	KJ126772.1
β-Action	R: 5`- agg tgg ggc aat gat ctt ga -3`	KJ126772.1
^a F: forward primer; R: a reverse primer. Tm: temperature

Statistical analysis

The statistical software Statistical Analysis System (SAS) [32] was used to analyze the data of this study. The General Linear Models (GLM) procedure was applied, followed by the Scheffé-test to identify significant differences among the fish groups. The values were presented as mean±SEM. The differences were considered significant if the P < 0.05.

Isolation and characterization of probiotic Lactobacillus sp. candidates

A total of 40 bacterial isolates were collected from the intestine of the obtained Seabass and Seabream fish from Suez Gulf, Egypt. Gram staining and catalase test revealed that 22 isolates were Gram-positive and catalase-negative.

Testing the ability of catalase-negative isolates to induce blood hemolysis indicated that 4 isolates denoted as SB7, SB8, SB10, and SB14 were negative to γ hemolytic test .

Antibacterial activity of the only selected SB8 probiotic candidate was evaluated against Aeromonas hydrophila ss. hydrophila (ATCC 13037) and Pseudomonas fluorescens, (ATCC13525) through determining the inhibition zone of cell-free supernatant (CFS). The obtained results revealed that cell-free supernatant of SSB8 has antagonistic activity against the used two pathogenic bacterial starins. The inhibition zone ranged from 12.2–14.5, and 14.6–16.4 mm for the ATCC 13037 and ATCC13525, respectively. The impact of 2–4 acidic pH range and 0.3% bile salt concentration on the SB8 survival was assessed. The SB8 was able to survive the applied acidic conditions (Table 2).

Table 2

Acid tolerance of isolated probiotic bacteria (SB8)
	Survival rate %
	pH tolerance
pH	T0	T2	survival %	T5	survival%
2	8.9 ± 0.05	6.2 ± 0.11	69.6	4.2 ± 0.11	47.7
3	8.4 ± 0.2	7.7 ± 0.05	89.1	6.2 ± 0.32	73.5
4	9.2 ± 0.11	8.2 ± 0.11	91.3	7.5 ± 0.10	81.5

Incubated SB8 at pH 2, 3, and 4 for 2 h showed 69.6%, 89.1%, and 91.3% survival rates, respectively whereas the survival rates were 47.7%, 73.5%, and 81.5% at pH 2, 3, and 4, respectively for the 5 h incubation. The strongest tolerance of the SB8 to 0.3% bile salts (Oxgall) after 2 h resulted in 106.9% survival rate wherease after 5 h, the strongest tolerance revealed 118.05% survival rate (Table 3). Additionally, the SB8 was able to grow in 0.4% phenol after 24 h incubation (Table 3).

Table 3

Bile salt and phenol tolerance of isolated probiotic bacteria (SB8)
Bile salt tolerance (0.3%)					Phenol tolerance (0.4%)
T0	T2	survival%	T5	survival%	T0	T24
7.2 ± 0.11	7.7 ± 0.11	106.9	8.5 ± 0.12	118.05	5.2 ± 0.48	2.6 ± 0.24

Comparing the haematological parameters among the studied Nile Tlipia groups

The percentages of WBC, Neu, Lum, Mon, and Eos in the studied fish groups are presented in Table 4. The results showed 1) significant reduction in both WBC and Lum levels, 2) significant increase in Neu, Mon, and Eos levels in the DLM-exposed fish compared to the negative control ones. In the DLM-exposed and SB8-suppleminted fish, enhanced percentages of WBC, Neu, Lum, Mon, and Eos were noticed where they are reasonably similar to the negative control fish on comparison with the positive control Ones.

Table 4

Haematological parameters of the studied Tilapia groups
Treatments	WBC (×10⁹/L)	Neu (%)	Lym (%)	Mon (%)	Eos (%)
Negative Control	152.40 ± 8.21^a	55.94 ± 0.62^b	36.33 ± 0.74^a	5.10 ± 0.91^c	4.15 ± 0.35^ab
SB8	147.16 ± 9.32^a	54.25 ± 0.81^b	37.28 ± 1.04^a	4.86 ± 0.58 ^c	3.85 ± 0.60^b
DLM	112.81 ± 10.26^c	60.72 ± 1.12^a	22.50 ± 1.12^c	13.47 ± 1.13^a	5.17 ± 0.42^a
SB8 + DLM	134.52 ± 7.61^b	57.66 ± 1.24^ab	31.25 ± 0.92^b	8.29 ± 0.81^b	3.80 ± 0.38^b
Negative control: fed with basal diet only, SB8: fed with basal diet and SB8 supplemented, DLM: exposed to DLM and fed with basal diet, SB8 + DLM: exposed to DLM and fed with basal diet and SB8 supplemented.The data are presented as mean±SEM of at least ten samples per group. ^a,b,c,d Mean values with not similar superscript letters are significantly different (P < 0.05).

Comparing the oxidative stress enzymatic activities among the studied Nile Tilapia groups

The MDA, SOD, CAT, and GPx oxidative stress enzymatic activities in the studied Nile Tilapia groups are presented in Fig. 1. The results showed that 1) DLM-exposed fish showed significant reduction in the activity of SOD, CAT, and GPx, 2) significant increase of MDA activity compared to the negative control fish, 3) The MDA, SOD, CAT, and GPx activities were relatively similar in the SB8-supplemented group, 4) DLM-exposed and SB8-supplemented fish showed improved MDA, SOD, CAT, and GPx reactivities compared to the DLM-exposed one.

Comparing protein contenet levels among the studied Nile Tilapia groups

Total protein content, albumin (A), globulin (B), and A/G levels in the studied Nile Tilapia groups are presented in Table 5. The DLM-exposed fish showed significant reduction in the total protein, albumin, and globulin levels compared to the negative control ones.

Table 5

Protein contenet levels among the studied Nile Tilapia groups
Treatments	Total protein (g/L)	Albumin (g/L)	Globulin (g/L)	A/G ratio
Negative control	39.41 ± 2.74^a	4.15 ± 0.50 ^a	39.60 ± 3.11^a	0.104 ± 0.01^a
SB8	41.49 ± 4.13^a	4.87 ± 0.48^a	44.19 ± 4.62^a	0.089 ± 0.01^a
DLM	23.54 ± 3.15^c	2.11 ± 0.87^c	21.71 ± 2.18 ^c	0.097 ± 0.01^a
SB8 + DLM	36.72 ± 5.17^b	3.81 ± 0.63^b	34.85 ± 1.91^b	0.109 ± 0.01^a
Negative control: fed only with basal diet, SB-8: fed with basal diet and SB8 supplemented, DLM: exposed to DLM and fed with basal diet, SB8 + DLM: exposed to DLM, fed with basal diet, and SB8 supplemented.
The data are presented as mean±SEM of at least ten samples per group. ^a,b,c,d Mean values with not similar superscript letters are significantly different (P < 0.05).

Total protein, albumin, and globulin contents in the SB8-supplemented fish were relatively similar to the negative control ones. Moreover, the DLM-exposed and SB8-supplemented fish showed enhanced total protein, albumin, and globulin contents compared to the DLM-exposed ones.

Comparing the incidence of micronucleus (Mn) formation among the studied Nile Tilapia groups

Table 6 represent the incidence of Mn formation in the thestudied Nile Tilapia groups. The DLM-exposed fish showed significant increase in the Mn formation in comparison with negative control fish. The incidence of Mn formation in SB8-supplemented fish was relatively similar to the negative control ones. Moreover, the incidence of Mn in the DLM-exposed/SB8-supplemented fish showed significant decrease compared to the DLM-exposed ones.

Table 6

**The incidence of micronucleus (Mn) formation among the studied Nile Tilapia groups**
Treatment	MnPCEs / PCEs
Negative Control	8.6± 0.9^c
SB8	8.2± 0.4^c
DLM	29.5± 1.6^a
SB8 + DLM	16.7± 1.2^b
Negative control: fed only with basal diet, SB-8: fed with basal diet and SB8 supplemented, DLM: exposed to DLM and fed with basal diet, SB8 + DLM: exposed to DLM, fed with basal diet, and SB8 supplemented. The data are presented as mean±SEM of at least ten samples per group. ^a,b,c,d Mean values with not similar superscript letters are significantly different (P < 0.05) While, ^c,c Mean values with similar superscript letters are of not significant differences (P > 0.05).
Figure legands

Comparing of IL-1 and IL-6 expression in the liver among the studied Nile Tilapia groups

Expression of IL-1 and IL-6 in the liver of the studied Nile Tilapia groups were pesented in Fig. 2. The DLM-exposed fish showed significant increase in IL-1 and IL-6 expression compared to the negative control ones. Expression of IL-1 and IL-6 were relatively similar in the DLM-exposed and in the SB8-suppleminted fish. Moreover, DLM-exposed/SB8-supplemented fish showed significant decrease in the IL-1 and IL-6 expression compared to the DLM-exposed ones.

Recently, many studies have addressed probiotic properties of the existing lactic acid bacteria within the intestinal microbiota of terrestrial animals [33]. The obtained results of the biochemical assays (gram staining and catalase test are in accordance with the described morphological/biochemical characteristics of lactic acid bacteria by Sonomoto [34]. The results of blood hemolysis assay suggest isolation of a probiotic bacterial candidate denoted as SB8 which was γ hemolytic negative. Blood hemolysis assay has been used guarantee the bacterial disability to cause any diseases [35] .

The antimicrobial/bacterial activity results suggested ability of the SB8 propiotic bacterial candidate to inhibit the growth of the both used bacterial pathogens. Many studies have demonstrated the antagonistic activity of lactic acid bacteria against Aeromonas hydrophila and Pseudomonas fluorescens where the L. acidophilus, L. casei, L. reuteri, L. rhamnosus, L. paracasei, L. plantarum, L. bulgaricus, and L. fermentum, Propionibacterium acnes, and Lactococcus lactis were the most active strains of the Lactobacillus species [36, 37, 38]. The antagonistic effect of the Lactobacillus species were attributed to the competitive exclusion and released acids or bacteriocin-like substances [39, 40, 41]. Additionally, fish diet supplemented with Lactobacillus sp. protected Nile tilapia from toxic compounds i.e.: ammonium chloride, and pathogenic bacterial infection i.e: Streptococcus agalactiae [ 42].

The obtained results from acid, bile salt and phenol tolerance assays are very essential for survival and colonization of probiotic bacteria in the fish gastrointestinal tract [43]. Therfore, SB8 probiotic bacterial candidate was tolerant to acidic conditions, bile salt, and phenol and showed antagonistic chracteristics against the used bacterial pathogens. Altogether, our results confirmed ability of the SB8 probiotic bacterial candidate to survive in the fish gastrointestinal tract which has been previously documented [44, 45].

It has been reported that, harsh environmental conditions in closed/indoor aquaculture systems e.g., water temperature variation, excessive stocking rate, fish malnutrition, and infectious diseases debilitate the organism’s immune system, increase the susceptabilty to infection by the invaders, and endanger fish stock [46]. As well, it has been documented that the used agricultural drainage water in the aquatic animal farming is polluted by pesticide and insecticide derivatives [47, 48]. The residual derivatives accumulate inside the fish body and hinder their growth and overall health condition.

The DLM is a common pyrethroid insecticide regularly used in agriculture-related activities [4]. In the present study, the DLM has induced severe toxicity in Nile tilapia. This is indicated by a significant reduction of WBC and Lum levels/ percenages and significant increase of the Neu, Mon, and Eos percentages. Moreover, the DLM-exposed Nile Tilapia to showed significant reductions in SOD, CAT, and GPx activities and a significant increase in MDA level. The ROS overproduction due to the DLM exposure could be the reason behind the previously reported impairment of fish tissues [49]. The increased MDA level in the DLM-exposed fish may be a resulted from inhibiting the production of antioxidant enzymes by the formed free radicals by the pesticides [50].

Additionally, a significant decrease in total protein levels, albumin, and globulin, an increase in the incidence of Mn formation and increase of IL-1 and IL-6 expression were reported in Nile Tilapia exposed to DLM at varience to Nile Tilapia fed with SB-8. It has been reported that DLM weakens the fish's immune system and makes them vulnerable to bacterial pathogens [49].

Also, the results of IL-1 and IL-6 expression are similar to those of Zahran et al. [51] as IL-1β and IL-8 were significantly up-regulated in Nile Tilapia exposed to chlorpyrifos (pesticide found in aquaculture water). Aquatic toxins like DLM are primarily absorbed by gills, and body's skin of fish and then, reach to remaining tissues [52]. Once toxins attack the tissues,they might lose their function and the local immunity could be suppressed [53, 54].

Utilizing functional additives in the fish diet has been widley used to improve aquatic animal overall health conditions [55, 56]. Lactobacillus spp. have impelled great attention as probiotics in feed industries due to their safeness (non-toxic) and beneficial impact on the host animals [42], remarkable stress resistance [57], and steadiness under processing conditions [58]. Adding Lactobacillus sp. into Nile tilapia diet improved immune and antioxidant defense mechanisms to ambient DLM toxicity through their anti-inflammatory impact in several tissues [59].

In the current work, dietary Lactobacillus sp. allowed Nile tilapia to overcome the negative impacts induced by DLM toxicity. The current findings exhibited that suppleminting the DLM-exposed fish diet with Lactobacillus sp induced a significant improvement of WBC, Lum, Neu, Mon, and Eos levels compared with the positive control fish (exposed to DLM alone).

Addtionally, fish diet supplementation with Lactobacillus sp induced a significant increase in SOD, CAT, and GPx activities and a significant reduction in the MDA levels compared to positive control fish. Suppleminting with Lactobacillus sp. help in protecting the fish liver tissues by neutralizing the ROS generation through enhancement of the antioxidative response. The SOD, CAT, and GPx genes refer to capacity of fish to scavenge the excessive ROS, generated during stress, toxicity, and outbreaks [60, 61]. Therefore, obtained results in the current study are in agreement with the previously published work where, the Nile tilapia diet supplemented with Lactobacillus sp enhanced both the immunity and antioxidant capacity to cope the DLM toxicity [60].

Total protein, albumin and globulin were higher in fish fed with Lactobacillus sp. compared with the other groups. Interestingly, in the DLM-exposed/SB8-supplemented fish, total protein, albumin and globulin were realtively similar to the negative control ones. In accordance with the present study Gewaily et al. [22] stated that the LP20 (dietary supplement) supported the DLM-exposed Nile tilapia by reducing the oxidative stress, the induced inflammation, and by increasing its ability to resist the hemolysis.

Furthermore, the DLM-exposed/SB8 supplemented fish showed significant increase in IL-1 and IL-6 expression compared to the control fish. Similarly, Dawood et al. [60] reported upregulated expression of the IFN-g in fish that were fed probiotics and exposed to DLM, suggesting that LP20, a type of Lactobacillus sp, has the potential to alleviate the caused impairments by the DLM and A. hydrophila through the induction of pro-inflammatory response. The immunostimulant properties of Lactobacillus sp. are likely due to its high content of peptidoglycan and lipopolysaccharides, which can promote the activation of T lymphocytes [62]. Therefore, the reduced mortality rates observed in the DLM-exposed/SB8-suppleminted fish can be attributed to the enhanced immunity via upregulation of pro-inflammatory cytokines production.

The finding of this study indicate that Nile tilapia exposed to DLM toxicity had experienced a decrease in their immune response and antioxidant capacity. However, when Nile tilapia fish were fed a diet containing Lactobacillus sp., the negative effects of DLM toxicity on inflammation were mitigated. This was achieved through the modulation of reactive oxygen species (ROS) production, enhancement of antioxidant capacity, and regulation of immune-related genes. Finally, an additional course of work will be conducted to identify the specific species of Lactobacillus and fully characterize its probiotic properties. This will involve evaluating its susceptibility to different antibiotics, and its tolerance to simulated gastrointestinal conditions.

DLM: Deltamethrin; WBC: White blood cells; PBS: phosphate buffered saline; Neu: Neutrophils; Lum: Lymphocytes; Mon: Monocytes; Eos: Eosinophiles; MDA: Malondialdehyde; SOD: Superoxide dismutase; CAT: Catalase; GPx: Glutathione peroxidase; IL-1: Interleukin-1; IL-6: Interleukin-6; MN: Micronucleus; MnPCEs: micronucleated polychromatic erythrocytes; ROS: reactive oxygen species.

Statements and Declarations

Ethics approval and consent to participate

The research described herein was performed on Nile tilapia (Oreochromis niloticus). This study was conducted in strict accordance with the guidelines of the Ethical Committee, National Research Centre, Egypt, on the care and use of animals for scientific purposes.

Consent for publication

Not applicable.

Availability of data and materials

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Competing interests

The authors declare that they have no competing interests.

Funding

There was no external support for this study.

Acknowledgements

This work was supported by the Biotechnology and Genetic Conservation group laboratory, Centre of Research and Applied Studies for Climate Change and Sustainable Development, Water Pollution Research Department, CEAS, National Research Centre (NRC), Egypt.

Author Contribution

All authors have been contributed to the conception and study design and gave their approval to the final version of the manuscript.

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No competing interests reported.

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Dietary Lactobacillus sp. mitigates Deltamethrin-induced toxic and immune-suppression impacts in Nile Tilapia (Oreochromis niloticus)

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Abstract

Figures

Introduction

Materials and methods

Antimicrobial activity of presumptive probiotic isolates

Acid and bile salt tolerance assays

Phenol tolerance

In-vivo studies

Fish feed preparation

Experimental design

Haematological investigation

Oxidative stress enzymatic activities

Total protein analysis

Analysis of micronucleus formation

Statistical analysis

Results

Comparing the haematological parameters among the studied Nile Tlipia groups

Comparing the oxidative stress enzymatic activities among the studied Nile Tilapia groups

Comparing protein contenet levels among the studied Nile Tilapia groups

Comparing the incidence of micronucleus (Mn) formation among the studied Nile Tilapia groups

Comparing of IL-1 and IL-6 expression in the liver among the studied Nile Tilapia groups

Discussion

Conclusion

Abbreviations

Declarations

References

Additional Declarations

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