Advertisement
Browse Subject Areas
?

Click through the PLOS taxonomy to find articles in your field.

For more information about PLOS Subject Areas, click here.

  • Loading metrics

Open Access

Peer-reviewed

Research Article

Protective effects of HSP Inducer on diazinon-exposed stellate sturgeon (Acipenser stellatus) fry: Insights on HSP70 gene expression, immune response, and enzyme indices

  • Leila Vahdatiraad,

    Roles Formal analysis, Investigation, Software, Writing – original draft

    Affiliation Faculty of Science, Department of Biology, University of Guilan, Rasht, Iran

    ⨯
  • Behrooz Heidari ,

    Roles Conceptualization, Data curation, Funding acquisition, Project administration, Supervision, Validation, Visualization, Writing – review & editing

    Bheidari@guilan.ac.ir (BH); t.sohrabi@areeo.ac.ir (TS)

    Affiliations Faculty of Science, Department of Biology, University of Guilan, Rasht, Iran, Department of Marine Sciences, The Caspian Sea Basin Research Center, University of Guilan, Rasht, Iran

    ⨯
  • Sevda Zarei,

    Roles Formal analysis, Investigation, Software

    Affiliation Faculty of Science, Department of Biology, University of Guilan, Rasht, Iran

    ⨯
  • Tooraj Sohrabi ,

    Roles Funding acquisition, Resources, Validation

    Bheidari@guilan.ac.ir (BH); t.sohrabi@areeo.ac.ir (TS)

    Affiliation International Caspian Sturgeon Research Institute, Agricultural Research, Education and Extension Organization (AREEO), Rasht, Iran

    ⨯
  • Hossein Ghafouri

    Roles Data curation, Project administration, Validation

    Affiliations Faculty of Science, Department of Biology, University of Guilan, Rasht, Iran, Department of Marine Sciences, The Caspian Sea Basin Research Center, University of Guilan, Rasht, Iran

    ⨯

Abstract

Aquatic environments face frequent exposure to organophosphate pesticides, such as diazinon, which are frequently utilized in agriculture. The goal of this study was to evaluate the effects of diazinon exposure on fish and to investigate the potential of the HSP inducer (HSPi) in developing a defense mechanism. To achieve this, several factors were analyzed, including the HSP70 gene expression, levels of immunity markers (lysozyme, IgM, and C3), antioxidant status, and the activity of acetylcholine esterase (AChE). Stellate sturgeon (Acipenser stellatus) fry, was exposed to diazinon (25, 50, and 75% of 96h-LC50) for 6 days after pre-treatment with an HSP inducer (HSPi), TEX-OE® (a prickly pear cactus extract), for 4 hours. Two HSPi concentrations, 100 and 200 mg.L-1, were used. Pre-treatment with HSPi significantly enhanced HSP70 gene expression in the gill and liver, as well as immune markers in the blood of Acipenser stellatus. Diazinon-treated groups exhibited higher antioxidant activities of SOD, CAT, and T-AOC. Increased activity also observed in control fish pre-treated with HSPi. However, stellate sturgeon receiving both diazinon and HSPi+diazinon experienced a significant decrease in AChE activity in comparison with control group. Cortisol levels were elevated in the fish that were subjected to diazinon. Those subjected to diazinon after receiving HSPi showed a significant decrease in cortisol levels. In conclusion, the study suggests that HSPi-mediated HSP70 induction may have a protective effect. The presence of an HSP inducer offers a potential strategy to mitigate the consequences of diazinon exposure in stellate sturgeon.

Citation: Vahdatiraad L, Heidari B, Zarei S, Sohrabi T, Ghafouri H (2023) Protective effects of HSP Inducer on diazinon-exposed stellate sturgeon (Acipenser stellatus) fry: Insights on HSP70 gene expression, immune response, and enzyme indices. PLoS ONE 18(11): e0294188. https://doi.org/10.1371/journal.pone.0294188

Editor: Sanjay Kumar Gupta, ICAR Indian Institute of Agricultural Biotechnology, INDIA

Received: July 26, 2023; Accepted: October 27, 2023; Published: November 13, 2023

Copyright: © 2023 Vahdatiraad et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the paper and its Supporting Information files.

Funding: This research has been financially supported by the Caspian Sea Basin Research Center (NO. 2978321), and the International Caspian Sturgeon Research Institute, Agricultural Research, Education and Extension Organization (AREEO) (NO. 132368/p15). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

1. Introduction

Nowadays, herbal extracts have emerged as safe, effective, and cost-effective alternatives to conventional drugs, chemicals, and antibiotics in the treatment of diseases and fish immunity enhancement [1]. Opuntia ficus indica, commonly called as the prickly pear or Nopal cactus, is a plant that is abundant in polyphenols, vitamins, polyunsaturated fatty acids, and amino acids. These components have antioxidant properties and can help reduce inflammation [2]. In recent years, there has been a growing body of research and industry reports that present substantiating evidence for the diverse health and medicinal advantages associated with this particular cactus [3–7]. TEX-OE®, a derivative obtained from Nopal cactus, has received approval to be used in fish and shellfish aquaculture due to its ability to naturally induce high amounts of endogenous or host-derived heat shock proteins (HSPs) [8,9].

Among the HSP family, HSP70 holds significant biological importance in fish. This includes its role in stress resilience, immunological activities, and the maintenance of protein integrity [8,10–13]. Many research studies indicate that being subjected to different stress factors, such as extreme temperatures, pollutants, anoxia, parasitism, predation, or competition, can cause a temporary rise in the expression of HSP70. This upregulation of HSP70 serves as a protective mechanism for organisms to prevent potential cellular harm [14–17]. As a result, HSP70 is commonly used as a stress biomarker. It provides valuable insights into the correlation between stress and biological reactions in fish.

Diazinon is categorized as a moderate persistence organophosphate pesticide (OP). It poses a threat to non-target aquatic organisms, including fish, as it can contaminate groundwater through drainage and surface runoff from cultivated lands [18–20]. In the northern provinces of Iran, situated along the southern coast of the Caspian Sea, diazinon is extensively used. It accounts for up to 60% of the total pesticide usage in the country [21]. The concentrations of diazinon have been measured in various rivers entering the Caspian Sea. For example, in Golestan province, the Qara-Su showed concentrations ranging from 18.6 to 22.4 mg.L-1, while the Gorgan-rud exhibited concentrations of 6.74 to 6.89 mg.L-1 [22]. Additionally, in Tonkabon city, the drainage of rice fields yielded concentrations of 93.1 to 0.98 mg.L-1 [23,24]. Moreover, the distribution and accumulation of diazinon in three important fish species from five estuaries along the Caspian Sea ranged from 0.01 to 0.15 mg.Kg-1 [25]. The presence of diazinon and its metabolites in fish can lead to various effects, including oxidative stress [26,27], alterations in hematological parameters [28], endocrine imbalances [18], and hyperactivity [29]. Numerous studies [30–33] have demonstrated that diazinon possesses the capacity to elicit profound transformations in liver enzymes and the corresponding biochemical indices. Consequently, the evaluation of fish health following exposure to acute or chronic diazinon toxicity can be performed by determining the activity of antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), total antioxidant (T-AOC), cortisol, as well as immune parameters including lysozyme, IgM, and complement component C3. Moreover, the inhibition of esterase activity, which results from the inhibition of acetylcholinesterase (AChE) activity by diazinon and its metabolites [34,35], represents a distinctive biomarker for fish exposed to diazinon, leading to disruptions in feeding, escape, and reproductive behaviors [36–38].

The stellate sturgeon (Acipenser stellatus) is one of five anadromous sturgeon species found in the Caspian Sea and relies on nearby rivers as significant natural spawning grounds [39,40]. Unfortunately, due to overfishing, habitat loss caused by river fragmentation, dam construction, and pollution, this species is critically endangered according to the latest assessment by the IUCN Red List [41]. To rehabilitate sturgeon populations, fish fry are released into rivers that flow into the Caspian Sea. In 2018, the Shahid Beheshti Sturgeon Restoration and Genetic Conservation Center (Rasht, Iran), released over 1.5 million sturgeon fry into these rivers [42]. However, post-release, there have been reports of considerable deaths and financial damages caused by environmental stresses [43,44]. Therefore, it is crucial to assess the fry’s ability to tolerate different stressors for a successful release into rivers.

This study focuses on exposing stellate sturgeon fry to an HSP inducer called TEX-OE® and sublethal concentrations (25%, 50%, and 75% of the LC50) of diazinon. The study assesses HSP70 gene expression levels in the liver and gills, measuring antioxidant activity (SOD, CAT, and T-AOC) in the liver. It also examines immunity parameters and cortisol levels in the blood, and evaluates the enzyme activity of AChE in the brain tissue.

2. Materials and methods

2.1. Fish supply

Sturgeon fry weighing approximately 12.5 ± 2.5 g were provided from the Shahid Beheshti Sturgeon Breeding and Rearing Center in Guilan, Iran. It was transported to the Marine Biology laboratory at the University of Guilan. Prior to the experiment, the fish were provided with commercial sustenance (BioMar, France) at a dosage equivalent to 2% of their bodily mass twice a day. The fish were acclimated to a well-aerated fiberglass tank with a capacity of 2000 L for two weeks, with approximately 20% of tank water changed daily. Temperature (18 ± 1°C), dissolved oxygen (~8–9 mg.L-1), and pH (~7–8) were kept constant throughout the experiment. No mortality was observed among the specimens throughout the acclimation period.

2.2. Preparation of HSP inducer

To induce HSP expression, the fish fry were exposed to TEX-OE® Nopal endurance capsules (Source Naturals, INC, Box 2118, Santa Cruz, CA 95062) at two concentrations: 100 and 200 mg.L-1 [3,5,9]. The capsules were dissolved in sterilized distilled water to prepare the HSP inducer. The sturgeon fry were exposed to a duration of 4 hours in the TEX-OE® solution, following the procedural technique outlined by [45]. Following exposure, the fish were returned to the experimental tank and then treated with diazinon.

2.3. Experimental design

According to [46], the 96-hour LC50 of diazinon for stellate sturgeons was estimated to be 2.54 mg.L-1. In the experiment, fish fry were exposed to diazinon levels of 25% (D25), 50% (D50), and 75% (D75) of the diazinon LC50. A total of 360 fish were used in the study. Each treatment consisted of three repetitions and there were 10 fish in each aquarium. The treated groups were as follows:

  1. Control group (Control): Fish that were not exposed to HSPi or diazinon.
  2. Fish that received 100 and 200 mg.L-1 of HSPi: H100 and H200.
  3. Fish receiving sublethal diazinon doses: D25, D50, and D75.
  4. Fish that received a combination of both HSPi and diazinon (first HSPi and then diazinon): H100+D25, H100+D50, H100+D75, H200+D25, H200+D50, H200+D75.

The temperature was around 18 ± 1°C, dissolved oxygen levels were kept between 8–9 mg.L-1, and the pH was maintained around 7–8. Throughout the experimental period, the 40 L aquariums were aerated to ensure sufficient oxygen supply, but no water changes were made.

2.4. Blood and tissue sampling

9 fish were sampled from each group and time point after being exposed to diazinon for 1, 3, and 6 days. The fish were anesthetized with clove oil, and their blood was taken from the caudal vein via insulin syringes. After centrifugation at 3000 rpm for 5 minutes, the serum was stored at -80°C for subsequent analysis of immune parameters, including lysozyme, IgM, C3, and cortisol concentrations. The fish were euthanized with an overdose of the anesthetic. The gills and liver tissues were quickly frozen using liquid nitrogen and kept at a temperature of -80°C until the HSP70 gene expression analysis. The liver and brain tissues were manually homogenized, weighed, and placed in 1.0 mL of Triton X-100 buffer to measure antioxidant enzymes and AChE activity. The mixture was held on ice for 10 minutes. After centrifugation at 4°C, 15 min at 12,000 rpm, the supernatant was kept at -80°C until further evaluation.

Approval for the experimental methods utilized was given by the National Committee for Ethics in Biochemical Research, located in Tehran and functioning under Iran’s Ministry of Health and Medical Education (Reference Number 61530).

2.5. HSP70 gene expression

To extract total RNA, RNX-plus reagent (SINACLON, Iran) was used following the manufacturer’s instructions. In order to remove DNA contamination from total RNA before starting the cDNA synthesis, Rnase-free Dnase was used (After diluting extracted RNA, 1 μL enzyme, 1 μL buffer, and in the next step 1 μL of EDTA was added) (SINACLON, Iran). The RNA’s quality was evaluated by using electrophoresis on a 1.2% Agarose gel. The amount of RNA was measured using a NanoDrop spectrophotometer (NanoDrop one C, USA). The first strand of cDNA (SINACLON kit, Iran) was used for cDNA synthesis. The steps were performed according to the kit protocol. The total volume required for a cDNA synthesis reaction was 12 μL. In addition, the expression levels were standardized using the GAPDH gene as a reference gene. The real-time qRT-PCR primers for the HSP70 [47] and GAPDH [48] genes are shown in Table 1. To determine HSP70 gene expression in a 25 μL reaction volume, a Roche qPCR thermocycler (Roche light cycler 96 machines, Germany) was used. Each qRT-PCR reaction was conducted in triplicate with denaturation as a first step of 15 min at 95°C, followed by 40 cycles of amplification, 30 seconds at 63°C for primer annealing, and 30 seconds at 72°C for extension. The determination of quantification cycle (Ct) values was conducted by employing the light Cycler 961.1 application software (Roche, Germany). The relative changes in expression levels of the HSP70 and GAPDH gene were assessed using the 2-(ΔΔCt) technique.

thumbnail
Download:
Table 1. Specific primers used for real-time quantitative polymerase chain reaction analysis of HSP70 and GAPDH.

https://doi.org/10.1371/journal.pone.0294188.t001

2.6. Antioxidant enzymes

2.6.1. Superoxide Dismutase (SOD).

SOD activity was determined according to Bolann and Ulvik’s method [49], using a commercial kit (ZellBio GmbH (Germany) (CAT No. ZB-SOD-96A). All steps were followed according to the manufacturer’s protocol. Under enzymatic conditions, the superoxide anion was transformed into oxygen and hydrogen peroxide in this kit. At the end of the experiment, a colorimetric measurement at 420 nm was obtained for the chromogen.

2.6.2. Catalase (CAT).

In accordance with Goth’s method [50], CAT activity was identified by a commercial kit (ZellBio GmbH, Germany) (CAT No. ZB- CAT-96A). Next, this activity was determined calorimetrically at 405 nm.

2.6.3. Total Antioxidant Activity (T-AOC).

T-AOC was measured by [51]. The T-AOC level was estimated using an ELISA kit (ZellBio GmbH, Germany) (CAT NO. ZB- TAC -96A) at 490 nm.

2.7. Immune parameters

2.7.1. Lysozyme.

Lysozyme activity was determined by a turbidimetric method [17], using a bacterium (Micrococcus luteus) lysis ability. To perform the assay, 25 μL of serum and 175 μL of M. luteus (Sigma-Aldrich, St. Louis, USA) with concentration of 0.2 mg.L-1 were added to 0.5 M phosphate-buffered saline (PBS), pH 6.2, in triplicate wells of a 96-well plate, with PBS used as a negative control. The optical density (OD) was recorded at 530 nm after 1 hour and 5 hours at 22°C. In this study, a 0.001 U.min-1 decrease in absorbance was considered equivalent to one unit of lysozyme activity.

2.7.2 Complement Component C3.

The C3 assay was measured using an ELISA sandwich (enzyme-linked immunosorbent assay) with an ELISA fish kit (Hangzhou East Biopharm Co., Ltd) [5]. The supernatant consisted of a monoclonal antibody coated with a monoclonal C3 fish antibody. An immune complex was created by incubating the plate at 37°C. Next, the biotin-labeled C3 antibody was added and blended with streptavidin-horseradish peroxidase. The unmixed enzyme was removed by washing the plate. After adding sulfuric acid, the liquid turned yellow. The OD value was measured by a microplate reader at 450 nm and converted to C3 concentrations using calibration.

2.7.3. IgM.

A similar method was employed to measure the IgM levels. The IgM level in the supernatant was determined using a monoclonal antibody by an ELISA quantification kit (Hangzhou Eastbiopharm Co., Ltd) [52]. An ELISA reader was used to measure OD at 450 nm. The measurements were done in triplicates.

2.8. Cortisol

The levels of serum cortisol were assessed using commercial ELISA kits (Nanjing Jincheng Institute, Nanjing, China). Color changes (450 nm) were spectrophotometrically evaluated according to the kit instructions [53].

2.9. Acetylcholinesterase (AchE)

AchE activity was evaluated based on [54] method. Brain tissue was weighted and suspended in 10 mL of sodium phosphate 0.1 M (Triton X-100) with pH 7.5. After homogenization on ice, the samples were centrifuged at 12,000 rpm for 15 min at 4°C. The supernatant was then removed and kept at 4°C. AchE activity was measured by determining the hydrolysis rate of 40 μL supernatant, using 40 μL of acetylthiocholine iodide 2.5 mM (Sigma-Aldrich, USA) and 140 μL phosphate buffer 0.1 M, and 20 μL of the 5, 5′-Dithio-bis-(2-nitrobenzoesAure) (DTNB, Sigma-Aldrich, USA) dissolved in 10 mL Tris-HCl buffer and 15.2 mg of NaHCO3. The final volume was 240 μL. The addition of acethylthiocholine initiated the reaction. A microplate reader at 415 nm was used to detect hydrolysis of DTNB, over 5 min (in intervals of 1 min). To determine the protein concentration, the Bradford method was used with bovine plasma albumin (Sigma-Aldrich, USA) [55].

2.10. Statistical analysis

The normality of the data was examined using the Kolmogorov-Smirnov test. The data were analyzed by applying one-way analysis of variance (ANOVA) within the SPSS software (ver. 19.0) to compare group means. Two-way ANOVA was applied to identify significant differences in the interactive effects of treatments and time. Furthermore, Duncan’s multiple range tests were carried out. To perform principal component analysis (PCA) between the studied parameters and different treatments in diazinon stress, the PCA test was used in R studio (ver. 1. 3. 1093). A statistical significance was determined using a significance level of 0.05. GraphPad Prism 8 was used to create the graphs. All data were stated as mean ± standard deviation.

3. Results

3.1. HSP70 gene expression

In the liver, on the first day after pre-treatment with 100 and 200 mg.L-1 HSPi, the expression of the HSP70 gene exhibited a statistically significant rise in comparison to the control group and D75 groups (P < 0.05) (Fig 1A). Furthermore, in fish pre-treated with HSPi and then exposed to diazinon, the HSP70 gene expression under 200 mg.L-1 of HSPi, demonstrated a significant increase in comparison to the control, D75, and H100+D75 treatments (P < 0.05) (Fig 1A).

thumbnail
Download:
Fig 1.

Induction of HSP70 gene expression in the liver (A) and gill (B) of Acipenser stellatus exposed to sublethal concentration of diazinon (75% of LC50) for six days; Control, fish that were not exposed to any HSPi or diazinon stress; H100 and H200, fish that received 100 and 200 mg.L-1 of HSPi for four hours; D75, fish that received 75% of the concentration of LC50 diazinon; H100+D75 and H200+D75, samples pre-treated with HSPi compound for 4 hours and then subjected to diazinon. Standard error bars are based on three biological replicates. Different letters are used to indicate the statistical differences between treatment groups. The significant stars denote the mean time comparisons.

https://doi.org/10.1371/journal.pone.0294188.g001

On the third day, there was a noteworthy decrease observed in the expression of the HSP70 gene across all experimental groups in comparison to the control group (P < 0.05). Meanwhile, on the sixth day, the HSP70 gene expression level was remarkably more than that of the control (P < 0.05). However, the expression of gene did not show any noticeable disparity among the H100, D75, H100+D75, and H200+D75 treatments (P > 0.05). The highest level of expression was observed in the H200 treatment on the sixth day (Fig 1A).

The two-way ANOVA indicated that there was a noteworthy interaction effect between the treatment and time (Table 2). There was no significant time difference in the average time pairs between days 1 and 6 (P > 0.05). Nevertheless, a significant difference was observed between days 1 with 3 and days 3 with 6 (P < 0.05) (Fig 1A).

thumbnail
Download:
Table 2. Two-way ANOVA on the effect of treatment and time and their interactions on studied parameters.

https://doi.org/10.1371/journal.pone.0294188.t002

There was a notable rise in the expression of the HSP70 gene in the gill of sturgeon fry that were pre-treated with HSPi, particularly on the first and sixth days of sampling. This increase was significant when compared to both the control group and the D75 treatment group (P < 0.05) (Fig 1B). On the first day, fish pre-treated and subjected to diazinon had significantly higher gene expression than control and D75. The maximum expression of HSP70 was recorded in fish receiving HSPi and H100+D75 on the first day (P < 0.05) (Fig 1B).

On the third day after the stress, HSP70 gene expression decreased in all groups. However, it was significantly higher in H100+D75, and H200+D75 treatments compared to the control and D75 groups (Fig 1B). On the sixth day, HSP70 gene expression significantly reduced after pre-treatment with HSPi and diazinon exposure than the control and D75 (P < 0.05) (Fig 1B).

The findings of two-way ANOVA revealed that the main effect of time on HSP70 gene expression was significant and their interaction effects were also significant (P < 0.05) (Table 2).

3.2. Activity of antioxidant enzymes

During the entire experimental period, fish that were only exposed to diazinon (i.e., D25, D50, and D75) showed the highest SOD activity (P < 0.05) (Fig 2A). In fish pre-treated with HSPi and subjected to diazinon, SOD activity decreased significantly on days 1, 3, and 6. In addition, SOD activity in fish pre-treatment with 100 mg.L-1 of HSPi was significantly lower than in fish pre-treatment with 200 mg.L-1 (P < 0.05) (Fig 2A). The comparison between fish pre-treated with HSPi and control groups indicated that SOD activity in pre-treated fish increased significantly for three days of sampling. The highest and lowest levels of SOD activity were observed in D50 and control groups, respectively.

thumbnail
Download:
Fig 2.

Antioxidant activity, (A) SOD, (B) CAT, and (C) T-AOC, exposed to diazinon for six days in Acipenser stellatus fry. Control, fish that were not exposed to any HSPi or diazinon stress; H100 and H200, fish that received 100 and 200 mg.L-1 of HSPi for four hours; D25, D50, and D75 groups, fish that received 25%, 50%, and 75% of the concentration of LC50 diazinon; H100+D and H200+D, samples were pre-treated with HSPi for 4 hours and then subjected to diazinon. Standard error bars are based on three biological replicates. Different letters are used to indicate the statistical differences between treatment groups. Significant asterisks are related to time averages comparison.

https://doi.org/10.1371/journal.pone.0294188.g002

As shown in Fig 2B, CAT activity in the liver increased in all groups one day after applying diazinon. The highest activity of CAT was recorded in fish exposed to different diazinon concentrations (D25, D50, and D75) for all time points (P < 0.05) (Fig 2B). CAT activity in H100 was significantly different from the controls and slightly increased from day 1 to day 3. However, a significant reduction was observed in H100 treatment on day 6 (P < 0.05). For H200, compared to the control group, CAT activity was significantly lower on the first and sixth days (P > 0.05). But it significantly increased on the third day (P < 0.05) (Fig 2B). The high activity of CAT was observed on the third day in the D75 treatment and the lowest level was observed in the control group.

For total antioxidant (T-AOC) on the first day, a lower level was observed in control, H100, and H200 groups compared to diazinon-only treatments (P < 0.05) (Fig 2C). The highest amount of T-AOC was detected in diazinon treatments (D25, D50, and D75) during the experimental period (P < 0.05) (Fig 2C). The same T-AOC trend was repeated on the third and sixth days of the experiment. Comparison between the HSPi pre-treatments and control demonstrated a significant difference in T-AOC activity on all three days of sampling (P < 0.05). In fish that underwent pre-treatment with a concentration of 100 mg L-1 of HSPi, the activity of T-AOC was found to be significantly decreased compared to that of the group treated with a concentration of 200 mg.L-1 (P < 0.05) (Fig 2C).

In addition, the two-way ANOVA showed that the interaction effects of time and treatment had a significant effect on SOD, CAT, and T-AOC activity level (Table 2).

3.3. Immune parameters

On the first day, lysozyme activity in the HSPi+diazinon groups significantly increased compared to the control and diazinon-only treatments (P < 0.05). Thus, the highest lysozyme activity was observed in the H100+diazinon and H200+diazinon groups (Fig 3A). On the third and sixth days, the activity of lysozyme in the fish receiving HSPi and then diazinon exhibited a significant increase when compared to the other treatments (P < 0.05). In comparison, the lowest lysozyme activity was observed in diazinon-only treatments (D25, D50, and D75). On the first and third days of sampling, the lysozyme activity in H100+diazinon treatments was higher than in the H200+diazinon group (Fig 3A).

thumbnail
Download:
Fig 3.

Immune parameters activity (A) lysozyme, (B) IgM, and (C) complement C3, in the serum of Acipenser stellatus fry exposed to sublethal concentrations of diazinon (25%, 50%, and 75% of LC50) for six days. Control, fish that were not exposed to any HSPi or diazinon stress; H100 and H200 fish that received 100 and 200 mg.L-1 of HSPi for four hours; D25, D50, and D75 groups fish that received 25%, 50%, and 75% of the concentration of LC50 diazinon; H100+D and H200+D samples were pre-treated with HSPi for 4 hours and then subjected to diazinon. Standard error bars are based on three biological replicates. Different letters are used to indicate the statistical differences between treatment groups.

https://doi.org/10.1371/journal.pone.0294188.g003

The pattern of IgM and C3 in the serum of Acipenser stellatus during the experiment was similar (Fig 3B and 3C). The IgM and C3 concentrations showed a significant increase in stellate fry receiving HSPi and HSPi+diazinon treatments during the entire experimental period. Here, H100+D treatments had the highest amount of IgM and C3 (P < 0.05) (Fig 3B). Fish exposed to diazinon after HSPi pre-treatment showed a significant increase in IgM and C3 concentrations on days 1, 3, and 6 (P < 0.05). The lowest IgM and C3 levels were observed in the fish receiving diazinon. Furthermore, it was observed that the H200 group exhibited a significant decrease in both IgM and C3 levels compared to the H100 group (P < 0.05) (Fig 3B).

According to the two-way ANOVA, there was a statistically significant interaction between the variables of time and treatment in relation to the levels of lysozyme, IgM, and C3 activity (P < 0.05) (Table 2). There was a notable discrepancy between the means of the time pairs and treatment pairs, as determined by Duncan’s test.

3.4. Cortisol

The highest amount of cortisol was observed in diazinon treatments (D25, D50, and D75) throughout the experiment (P < 0.05) (Fig 4). The level of cortisol in the fish subjected to diazinon after pre-treatment with HSPi was significantly decreased (P < 0.05) (Fig 4). Comparing the control and HSPi pre-treatments indicated that H200 remarkably increased the serum cortisol (P < 0.05), although, not as much as diazinon. Based on the two-way ANOVA analysis, there was a considerable interaction observed between time and treatment on cortisol level (P < 0.05) (Table 2). Average cortisol levels were significantly different between all treatment pairs. However, for time pairs, on day 3, cortisol levels were different from day 1 and day 6.

thumbnail
Download:
Fig 4. Cortisol levels of stellate sturgeon fry exposed for 6 days to different diazinon concentrations.

Control, fish that were not exposed to any HSPi or diazinon stress; H100 and H200 fish that received 100 and 200 mg.L-1 of HSPi for four hours; D25, D50, and D75 groups, fish that received 25%, 50%, and 75% of the concentration of LC50 diazinon; H100+D and H200+D samples pretreated with HSPi for 4 hours and then exposed to diazinon. Standard error bars are based on three biological replicates. Different letters are used to indicate the statistical differences between treatment groups. Significant asterisks are related to time averages comparison.

https://doi.org/10.1371/journal.pone.0294188.g004

3.5. Acetylcholinesterase (AchE)

Throughout the experiment period, the activity of AchE in the brain showed a significant increase in the control and the fish received HSPi compared to other treated groups (P < 0.05) (Fig 5). The alterations in AchE followed a consistent pattern across various treatments and on different days (1st, 3rd, and 6th) (Fig 5). However, there was no significant difference among fish receiving diazinon and HSPi+diazinon. The outcomes of the two-way ANOVA revealed no substantial interaction between time and treatment on the activity of AchE (P > 0.05), whereas treatment and time separately had a significant effect (P < 0.05) (Table 2).

thumbnail
Download:
Fig 5. AChE activity in Acipenser stellatus fry exposed to different concentrations of diazinon for six days.

Control, fish that were not exposed to any HSPi or diazinon stress; H100 and H200, fish that received 100 and 200 mg.L-1 of HSPi for four hours; D25, D50 and D75 groups, fish that received 25%, 50% and 75% of the concentration of LC50 diazinon; H100+D and H200+D samples were pre-treated with HSPi for 4 hours and then subjected to diazinon. Standard error bars are based on three biological replicates. Different letters are used to indicate the statistical differences between treatment groups. The significant stars are related to the comparison of time averages.

https://doi.org/10.1371/journal.pone.0294188.g005

3.6. Principal component analysis (PCA)

The principal component analysis was performed based on 10 variables and 36 treatments. The first and second principal components account for 49.25% and 23.68% of the variation, respectively (Table 3). Considering a score higher than 0.7, lysozyme, IgM, C3, SOD, CAT, T-AOC, and cortisol variables were important factors in determining the relationship between treatments and tested variables in the first component (Table 4). The results of this analysis were separated into two main groups (Fig 6). The first group consisted of treatments D25, D50, and D75, while the second group included the remaining treatments. The first group showed the highest correlation with cortisol, CAT, SOD, and T-AOC variables, while the HSPi+diazinon treated groups had the greatest impact on immune parameters (Fig 6).

thumbnail
Download:
Fig 6. Biplot graph resulting from principal component analysis (PCA) for diazinon stress.

1, 2, and 3 represent sampling times.

https://doi.org/10.1371/journal.pone.0294188.g006

thumbnail
Download:
Table 3. Eigenvalue, percentage of variance and cumulative percentage of variance related to all three components.

https://doi.org/10.1371/journal.pone.0294188.t003

thumbnail
Download:
Table 4. Scores of each variable in the main components.

https://doi.org/10.1371/journal.pone.0294188.t004

4. Discussion

This study aimed to investigate the protective effects of a heat shock protein (HSP) inducer (HSPi) on stellate sturgeon (Acipenser stellatus) fry against diazinon-induced stress. The HSP70 family is the most extensively studied HSP in aquatic organisms [56]. HSP70 plays a crucial role in protecting cellular components from damage caused by stress, as it effectively prevents protein aggregation while also facilitating proper protein folding [57]. TEX-OE® is a patented prickly pear cactus extract (Opuntia ficus indica) and an alternative method to enhance HSP70 in the body through HSP induction [58]. Several studies have shown that TEX-OE® accelerates HSP70 synthesis in fish [3,5] and shellfish [8,58,59], enhancing their resistance to various stressors. In a different study, the effect of HSPi on HSP70 modulation was analyzed in Artemia franciscana nauplii, which showed that HSPi induced HSP70 expression both in a concentration and time-dependent approach [60]. Another evaluation of the Tex-OE® compound on Artemia franciscana’s interaction with pathogenic Vibrios demonstrated increased protection against Vibrio campbellii, although long-term exposure decreased their survival. Likewise, in a study evaluating the effect of HSPi (TEX-OE®) on the HSP70 gene expression in the gill, intestine, and liver of Acipenser persicus during the 7-day post-injection period with the bacteria Aeromonas hydrophila, HSPi significantly increased HSP70 expression in a dose-dependent manner [9]. In another study, Tex-OE® rapidly increased the levels of HSPs in fingerlings of common carp before acute ammonia stress [58]. In the present study, 4 hours of pre-exposure to Tex-OE® increased HSP70 gene expression in both liver and gills of stellate sturgeon fry, especially on the first day. The upregulation of HSP70 expression in fish subjected to diazinon suggests that the pesticide acts as a stressor, triggering the cellular stress response pathway. This upregulation is believed to be a protective mechanism to mitigate the potential damage caused by diazinon-induced stress. Furthermore, the effectiveness of Tex-OE® in increasing HSP70 gene expression in liver tissues was higher than that in the gills on the sixth day, this may be attributed to the direct exposure of the gills to the surrounding environment, leading to a rapid rise in expression of the HSP70 on the first day. It is significant to note that persistent stress might lessen the capacity of the stress response, which includes the generation of HSP70. In cases of chronic stress, even an increased level of HSP70 may not be enough to protect fish from the detrimental consequences of stress, thereby making them more prone to stress-related complications.

Oxidative stress can occur when there is an imbalance between the generation of reactive oxygen species (ROS) and the quantities of antioxidants, ultimately leading to cellular harm [60,61]. According to a study by [62], crucian carp exposure to 0.3 mg.L-1 of diazinon per day for 21 days induced oxidative stress. In one experiment, larvae of zebrafish subjected to diazinon concentrations reaching from 0.002 to 0.02 mg.L-1 showed differences in the activity of oxidative enzymes and gene expression, with no detectable CAT activity and significantly increased expression of oxidative enzymes [26,63]. Another study evaluated the effects of sublethal doses of diazinon (0, 0.8, 1.6, and 3.2 mg.L-1) on zebrafish after 30 days of treatment, and found significant increases in the expression of immune and antioxidant (SOD and CAT) genes [18]. In this study, we found that the fish treated with all three sublethal concentrations of diazinon had higher antioxidant enzyme activity, but this activity decreased when the fish were first treated with HSPi and then exposed to diazinon. This suggests that the increase in HSP70 due to Tex-OE® treatment may play an important role in controlling ROS production and regulating the activity of antioxidant enzymes. Similarly, in Labio rohita fingerlings subjected to starvation stress, HSP70 gene expression and antioxidant enzyme activity increased. This implies that HSP70 could aid in preserving the stability of cells by reducing the impact of oxidative stress caused by starvation [64].

The immune response in fish can be deregulated by pollutants like diazinon existing in aquatic ecosystems [35,65,66]. Sharifpour et al., [67] found that the decreased immunity observed in the fish that were exposed to diazinon confirmed the immunosuppressive effects of this substance. In the current study, the immunity status, such as lysozyme, IgM, and C3 was reduced in the diazinon groups (D25, D50, and D75) compared to control. It is believed that these immunosuppressive effects occur primarily by inhibiting the synthesis of key components of the immune system [19,66,68]. Diazinon has a suppressive effect on fish cellular immunity, which is achieved by reducing lymphocyte proliferation. The decrease in lymphocytes may be due to the release of cortisol in reaction to stress, which causes lymphocyte apoptosis, or their redistribution from the blood to the tissues [69]. The reported reduction in IgM levels in response to diazinon may be due to diazinon inhibitory effects on lymphocyte proliferation, which is responsible for IgM production in hematopoietic tissues [70]. On the first, third, and sixth days of the experiment, it was observed that the H100+D groups showed the most elevated levels of lysozyme, IgM, and C3. Recent studies have shown that HSPs stimulators or inducers have a significant impact on both innate and acquired immunity, and possess immuno-enhancing properties [8,13]. Notably, HSP70 plays a critical role in the immune function of fish. Studies have reported that HSP70 can stimulate white blood cells and antibodies, leading to improved infection-fighting abilities. The active expulsion of HSP70 to the outside of the cells or the induction of HSP70 can activate both the innate and adaptive immune systems [71]. Additionally, HSPs have been shown to generate pro-oxidant activity and induce protective immune responses in crustaceans [8]. Fish with higher HSP70 expression levels showed enhanced activity of immune parameters such as serum complement and immunoglobulin levels, as well as antioxidant enzyme activities [72]. Studies have also demonstrated that the concentration of IgM, complement C3, and lysozyme can rise after exposure to HSP inducer in Persian sturgeon fry [5]. Moreover, the innate immune system genes of prophenoloxidase and transglutaminase can be up-regulated in species like Artemia franciscana when responding to HSP70 induction [60]. HSP70 expression was found to interact with other immune parameters and antioxidant enzymes, improving the fish’s ability to adapt to the environment [73]. It seems that in fish, HSP70 has the potential to improve the functioning of the immune system through triggering the release of cytokines via signal transduction when recognized by immune cells [13]. However, the extent of the benefit relies on different factors, including chronic exposure to stress, which can impair the immune response and reduce the amount of HSP70 produced in the organism. Therefore, if stress persists, the protective effect of high levels of HSP70 might not sufficiently protect the fish from damage caused by stress, leaving it vulnerable to illnesses and other complications associated with stress.

To respond efficiently to stressors, cortisol is the most common corticosteroid generated by the hypothalamus-pituitary-adrenal/internal (HPI) axis in humans and fish [74]. In our study, the significant reduction of cortisol levels in the HSPi+diazinon groups supports the anti-stress role of HSPi in dealing with diazinon stress, resulting in a return of cortisol levels to normal condition in fish that received HSPi before diazinon stress. Cortisol may lead to changes in HSP70 levels in fish tissues during periods of physiological stress, suggesting a correlation between the stress reactions in the nervous and endocrine systems with those at the cellular level [10]. The synthesis of HSPs is controlled by an intricate system of communication pathways, such as the HPI axis, which regulates the release of cortisol [75]. When the functioning of the HPI axis decreases, it results in a cycle of negative feedback that impacts the production of cortisol [76]. There is evidence that suggests HSP70 and the glucocorticoid receptor (GR) connect and produce a stable HSP70-GR-HSP90 molecular structure in the cytosol [75,77], which finally inhibits cortisol production. Our research indicates that HSPs and cortisol interact as chaperone and client, respectively, meaning that HSPs help to modulate the activity of cortisol during periods of stress. The fish that were treated with HSPi+diazinon exhibited less cortisol activity compared to diazinon (D25, D50 and D75) treated groups.

Measuring AChE activity in fish brains is a method of diagnosing pesticide exposure, particularly organophosphates that inhibit anticholinesterase enzymes in aquatic environments [21,65]. The inhibition of AChE activity suggests that these pollutants can interfere with crucial processes such as nerve cell energy metabolism. Furthermore, diazinon inhibition of AChE can affect other metabolic activities in animals, resulting in death [78]. Evaluating HSP70 and AChE in a molluscan species (Geukensia demissa) over time has revealed a relationship between chaperone and client proteins of the HSP70/AChE system, demonstrating various adaptive mechanisms used by organisms to tolerate habitats subjected to environmental change [79]. Some researchers have linked organophosphates such as diazinon to changes in neurotransmission in fish [66,80,81]. Chronic exposure to a non-lethal concentration of diazinon significantly inhibits AChE activity in the brain of Oreochromis niloticus [82]. In Oreochromis niloticus, acute exposure to diazinon significantly decreases AChE activity [83]. According to the current study, diazinon significantly decreases AChE levels in stellate sturgeon fry brain. Pre-exposure to HSPi did not restore enzyme activity to normal levels. Overall, it appears that HSPi at concentrations of 100 and 200 mg.L-1 are insufficient to affect AChE activity or moderate the neurotoxic effects of diazinon. However, a higher concentration of HSPi may increase AChE activity in fish under diazinon-induced stress.

5. Conclusion

The findings of this study indicate that prior exposure to Tex-OE® can result in the upregulation of the HSP70 gene in stellate sturgeon fry. This increase in expression offers improved protection against pesticide exposure and boosts their biochemical and immunological defense mechanisms. The application of an HSP inducer, such as Tex-OE®, can potentially enhance the overall health of valuable sturgeon and aid in their ability to cope with environmental stressors like diazinon, a pesticide. Given the high mortality rates observed when sturgeon fry are released into the river from artificial breeding centers and exposed to environmental stressors, it is recommended to expose them to HSP-inducing compounds before releasing them into the natural environment. This pre-exposure can offer optimal conditions for their survival and improve their chances of adapting to the challenges they may face in the wild.

Acknowledgments

We extend our heartfelt thanks to Mrs. Dr. Mahvash Hadavi from the University of Guilan for her invaluable assistance in our laboratory work. Furthermore, we would like to express my deepest appreciation to Professor Hassan Ebrahimzadeh for his unwavering support throughout this research project.

References

  1. 1. Hajirezaee Saeed, Rafieepour Ahmad, Shafiei Shafigh, and Rahimi Ruhollah. "Immunostimulating Effects of Ginkgo Biloba Extract against Toxicity Induced by Organophosphate Pesticide, Diazinon in Rainbow Trout, Oncorhynchus Mykiss: Innate Immunity Components and Immune-Related Genes." Environmental Science and Pollution Research 26, no. 9 (2019): 8798–807.
  2. 2. El-Mostafa Karym, Youssef El Kharrassi Asmaa Badreddine, Andreoletti Pierre, Vamecq Joseph, et al. "Nopal Cactus (Opuntia Ficus-Indica) as a Source of Bioactive Compounds for Nutrition, Health and Disease." Molecules 19, no. 9 (2014): 14879–901. pmid:25232708
  3. 3. Vahdatiraad Leila, Heidari Behrooz, Zarei Sevda, Sohrabi Tooraj, and Ghafouri Hossein. "Biological Responses of Stellate Sturgeon Fingerlings (Acipenser Stellatus) Immersed in Hsp Inducer to Salinity Changes." Marine Environmental Research 191 (2023): 106145. pmid:37595360
  4. 4. Alimi Hichem, Hfaiedh Najla, Bouoni Zouhour, Hfaiedh Mbarka, Sakly Mohsen, et al. "Antioxidant and Antiulcerogenic Activities of Opuntia Ficus Indica F. Inermis Root Extract in Rats." Phytomedicine 17, no. 14 (2010): 1120–26.
  5. 5. Baharloei Mina, Heidari Behrooz, Zamani Hojjatolah, and Hadavi Mahvash. "Effects of Pro‐Tex® on the Expression of Hsp70 Gene and Immune Response Parameters in the Persian Sturgeon Fingerlings, Acipenser persicus, Infected with Aeromonas hydrophila." Journal of Applied Ichthyology 36, no. 4 (2020): 393–401.
  6. 6. Morales Patricia, Esther Ramírez-Moreno, María de Cortes Sanchez-Mata, Ana Maria Carvalho, and Isabel CFR Ferreira. "Nutritional and Antioxidant Properties of Pulp and Seeds of Two Xoconostle Cultivars (Opuntia Joconostle Fac Weber Ex Diguet and Opuntia Matudae Scheinvar) of High Consumption in Mexico." Food Research International 46, no. 1 (2012): 279–85.
  7. 7. Valente Ligia MM, Djavan da Paixão, Do Adriana CNascimento , Priscila FP dos Santos, Scheinvar Leia A, et al. "Antiradical Activity, Nutritional Potential and Flavonoids of the Cladodes of Opuntia Monacantha (Cactaceae)." Food Chemistry 123, no. 4 (2010): 1127–31.
  8. 8. Kumar Vikash, Roy Suvra, Bijay Kumar Behera, and Basanta Kumar Das. "Heat Shock Proteins (Hsps) in Cellular Homeostasis: A Promising Tool for Health Management in Crustacean Aquaculture." Life 12, no. 11 (2022): 1777. pmid:36362932
  9. 9. Baharloei Mina, Heidari Behrooz, Zamani Hojjatolah, Ghafouri Hossein, and Hadavi Mahvash. "Effects of Heat Shock Protein Inducer on Hsp70 Gene Expression and Immune Parameters During Streptococcus Iniae Infection in a Persian Sturgeon Fry. " Veterinary Research Forum 12, no. 4 (2021): 473.
  10. 10. Basu N, T Nakano EG Grau , and Iwama GK. "The Effects of Cortisol on Heat Shock Protein 70 Levels in Two Fish Species." General and Comparative Endocrinology 124, no. 1 (2001): 97–105. pmid:11703075
  11. 11. Deane , Eddie E, and Norman YS Woo. "Advances and Perspectives on the Regulation and Expression of Piscine Heat Shock Proteins." Reviews in Fish Biology and Fisheries 21 (2011): 153–85.
  12. 12. Yabu Takeshi, Imamura Shintaro, Manush S Mohammed Ken Touhata, Minami Takayuki, et al. "Differential Gene Expression of Hsc70/Hsp70 in Yellowtail Cells in Response to Chaperone‐Mediated Autophagy." The FEBS Journal 278, no. 4 (2011): 673–85. pmid:21205201
  13. 13. Zininga Tawanda, Ramatsui Lebogang, and Shonhai Addmore. "Heat Shock Proteins as Immunomodulants." Molecules 23, no. 11 (2018): 2846. pmid:30388847
  14. 14. Taleb Mona, Carlene S Brandon Fu-Shing Lee, Lomax Margaret I, Dillmann Wolfgang H, et al. "Hsp70 Inhibits Aminoglycoside-Induced Hair Cell Death and Is Necessary for the Protective Effect of Heat Shock." Journal of the Association for Research in Otolaryngology 9 (2008): 277–89. pmid:18512096
  15. 15. Padmini Ekambaram, and Munuswamy Usha Rani. "Impact of Seasonal Variation on Hsp70 Expression Quantitated in Stressed Fish Hepatocytes." Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology 151, no. 3 (2008): 278–85. pmid:18694842
  16. 16. Radons Jürgen. "The Human Hsp70 Family of Chaperones: Where Do We Stand?". Cell Stress and Chaperones 21, no. 3 (2016): 379–404. pmid:26865365
  17. 17. Kim Jong Youl, Barua Sumit, Mei Ying Huang Joohyun Park, Yenari Midori A, et al. "Heat Shock Protein 70 (Hsp70) Induction: Chaperonotherapy for Neuroprotection after Brain Injury." Cells 9, no. 9 (2020): 2020. pmid:32887360
  18. 18. Darvishi Masoumeh, Safari Roghieh, Seyed Hossein Hoseinifar Ali Shabani, Dadar Maryam, et al. "Sublethal Doses of Diazinon Affected Reproductive, Immune, and Oxidative Status in Female Zebrafish (Danio Rerio)." Aquaculture Reports 22 (2022): 100944.
  19. 19. Banaee M, A Sureda AR Mirvaghefi , and Ahmadi K. "Effects of Diazinon on Biochemical Parameters of Blood in Rainbow Trout (Oncorhynchus Mykiss)." Pesticide Biochemistry and Physiology 99, no. 1 (2011): 1–6.
  20. 20. Wu Xiaozhen, Li Jiayi, Zhou Zhe, Lin Ziqiu, Pang Shimei, et al. "Environmental Occurrence, Toxicity Concerns, and Degradation of Diazinon Using a Microbial System." Frontiers in Microbiology 12 (2021): 717286. pmid:34790174
  21. 21. Behrooz Dahmardeh, Reza Abbas Esmaili-sari, Urbaniak Magdalena, and Chakraborty Paromita. "Assessing Diazinon Pollution in the Three Major Rivers Flowing into the Caspian Sea (Iran)." Water 13, no. 3 (2021): 335.
  22. 22. Shayeghi M, M Khobdel F Bagheri , and Abtai M"Azynfosmethyl and Diazinon Residues in Qarasu River and Gorganrood in Golestan Province." J Pub Heal and Health Res Instit 6 (2008): 75–82.
  23. 23. Shayeghi Mansoureh, and Javadian Ezataldin. "Study of the Residue of Lindane and Diazinon in the Rice Fields in Tonekabon City (Mazandaran Province) 1999. Journal of Environmental Science and Technology 9:51–58." (2001).
  24. 24. Banaee Mahdi, Sureda Antoni, Ali Reza Mirvaghefi, and Kamal Ahmadi. "Biochemical and Histological Changes in the Liver Tissue of Rainbow Trout (Oncorhynchus Mykiss) Exposed to Sub-Lethal Concentrations of Diazinon." Fish Physiology and Biochemistry 39 (2013): 489–501. pmid:22972375
  25. 25. Golshani Reza, Ali Mashinchian Moradi, Nodoshan R Mosavi, Fatemi SM, and Pargol GHavam Mostafavi. "Organophosphorus Pesticides (Diazinon, Malathion and Azinfos Methyl) Accumulation in Three Fish Species, in South Coasts of the Caspian Sea, Iran." Iranian Journal of Fisheries Sciences 19, no. 6 (2020): 3050–62.
  26. 26. Bharti Sandhya, and Rasool Fazle. "Analysis of the Biochemical and Histopathological Impact of a Mild Dose of Commercial Malathion on Channa Punctatus (Bloch) Fish." Toxicology Reports 8 (2021): 443–55. pmid:33717997
  27. 27. Sule , Rasheed O, Liam Condon, and Gomes Aldrin V. "A Common Feature of Pesticides: Oxidative Stress-the Role of Oxidative Stress in Pesticide-Induced Toxicity." Oxidative Medicine and Cellular Longevity 2022 (2022). pmid:35096268
  28. 28. Köprücü Sibel Şimşek, Kenan Köprücü Mevlüt Şener Ural, İspir Ünal, and Pala Murat. "Acute Toxicity of Organophosphorous Pesticide Diazinon and Its Effects on Behavior and Some Hematological Parameters of Fingerling European Catfish (Silurus Glanis L.)." Pesticide Biochemistry and Physiology 86, no. 2 (2006): 99–105.
  29. 29. Abtahi B, H Nabavi V Jafari-Shamushaki , Gorbani R, and Kasumyan AO. "Influence of Insecticides Diazinon and Endosulfan on Taste Reception in Persian Sturgeon Acipenser Persicus (Acipenseridae)." Journal of Ichthyology 58, no. 2 (2018): 248–54.
  30. 30. Kalender Suna, Ogutcu Ayse, Uzunhisarcikli Meltem, Fatma Açikgoz Dilek Durak, et al. "Diazinon-Induced Hepatotoxicity and Protective Effect of Vitamin E on Some Biochemical Indices and Ultrastructural Changes." Toxicology 211, no. 3 (2005): 197–206. pmid:15925023
  31. 31. Vali Sara, Majidiyan Nava, Azadikhah Dariush, Varcheh Matin, Tresnakova Nikola, et al. "Effects of Diazinon on the Survival, Blood Parameters, Gills, and Liver of Grass Carp (Ctenopharyngodon Idella Valenciennes, 1844; Teleostei: Cyprinidae)." Water 14, no. 9 (2022): 1357.
  32. 32. Karatas Tayfun, and Albayrak Mevlut. "Determination of Diazinon Effects on Some Haematological Parameters in Rainbow Trout, Oncorhynchus Mykiss." Indian Journal of Animal Research 52, no. 3 (2018): 413–15.
  33. 33. Banaee Mahdi, Impellitteri Federica, Cristiana Roberta Multisanti Antoni Sureda, Arfuso Francesca, et al. "Evaluating Silymarin Extract as a Potent Antioxidant Supplement in Diazinon-Exposed Rainbow Trout: Oxidative Stress and Biochemical Parameter Analysis." Toxics 11, no. 9 (2023): 737. pmid:37755747
  34. 34. Zeinali Majid, Shafaei Azam, Rafatpanah Houshang, Mosavat Arman, Naser Tayebi-Meybodi, et al. "Potential Protective Effects of Chrysin against Immunotoxicity Induced by Diazinon." Scientific Reports 12, no. 1 (2022): 15578. pmid:36114367
  35. 35. Díaz-Resendiz KJG, PC Ortiz-Lazareno CE Covantes-Rosales AMTrujillo-Lepe, GA Toledo-Ibarra , et al. "Effect of Diazinon, an Organophosphate Pesticide, on Signal Transduction and Death Induction in Mononuclear Cells of Nile Tilapia Fish (Oreochromis Niloticus)." Fish & Shellfish Immunology 89 (2019): 12–17.
  36. 36. Cong Van, Nguyen Nguyen Thanh Phuong, and Bayley Mark. "Effects of Repeated Exposure of Diazinon on Cholinesterase Activity and Growth in Snakehead Fish (Channa Striata)." Ecotoxicology and Environmental Safety 72, no. 3 (2009): 699–703. pmid:19054558
  37. 37. Camacho-Pérez Milton Rafael, Carlos Eduardo Covantes-Rosales Gladys Alejandra Toledo-Ibarra, Ulises Mercado-Salgado María Dolores Ponce-Regalado, et al. "Organophosphorus Pesticides as Modulating Substances of Inflammation through the Cholinergic Pathway." International Journal of Molecular Sciences 23, no. 9 (2022): 4523.
  38. 38. Sandoval-Herrera Natalia, Mena Freylan, Espinoza Mario, and Romero Adarli. "Neurotoxicity of Organophosphate Pesticides Could Reduce the Ability of Fish to Escape Predation under Low Doses of Exposure." Scientific Reports 9, no. 1 (2019): 10530.
  39. 39. Ovissipour M, and Rasco B. "Sturgeon: Conservation of Caspian Sea Stocks." Journal of Aquaculture Research & Development 3, no. 7 (2012): 2.
  40. 40. Fazli H, M Tavakoli MR Behrouz Khoshghalb, and Moghim M. "Population Dynamics of Stellate Sturgeon Acipenser Stellatus Pallas, 1771 in the Southern Caspian Sea." Iranian Journal of Fisheries Sciences 20, no. 1 (2021): 45–61.
  41. 41. Mugue N., Friedrich T., Chebanov M., and Ruban G. "Acipenser Stellatus." The IUCN Red List of Threatened Species 2022 e.T229A135062806. (2022).
  42. 42. Abdolmalaki Shahram, Jalil Jalilpour Roudkoli, and Ali Hallajian. "Quantitative and Qualitative Study of Sturgeon Fish until Release in the Sefidroud River." Iranian Journal of Fisheries Science 30, no. 2 (2021): 93–102.
  43. 43. Khodabandeh Saber, Mosafer Saeedeh, and Khoshnood Zahra. "Effects of Cortisol and Salinity Acclimation on Na+/K+/2cl–-Cotransporter Gene Expression and Na+, K+-Atpase Activity in the Gill of Persian Sturgeon, Acipenser Persicus, Fry." Scientia Marina 73, no. S1 (2009): 111–16.
  44. 44. Bakhshalizadeh Shima, Leonid Alexandrovich Zykov Botagoz Murasovna Nasibulina, Tatyana Fedorovna Kurochkina, and Francesco Fazio. "Influence of Growth on the Biological and Commercial Productivity of Caspian Great Sturgeon Huso Huso." Aquaculture 533 (2021): 736139.
  45. 45. Baruah Kartik, Norouzitallab Parisa, Ronald J Roberts Patrick Sorgeloos, and Bossier Peter. "A Novel Heat-Shock Protein Inducer Triggers Heat Shock Protein 70 Production and Protects Artemia Franciscana Nauplii against Abiotic Stressors." Aquaculture 334 (2012): 152–58.
  46. 46. Pazhand Z., Esmaeili S. A., and Piri Z. M. "Effect of Diazinonon the Persian Sturgeon and Stellate Sturgeon Fingerlings.". Journal of Pajouhesh-va-Sazandegi 158, no. 16 (2003): 64–67.
  47. 47. Safari R, A Shabani S Ramezanpour, MR Imanpour, and S Rezvani. "Alternations of Heat Shock Proteins (Hsp70) Gene Expression in Liver and Gill of Persian Sturgeon (Acipenser Persicus Borodin, 1987) Exposed to Cadmium Chloride." Iranian Journal of Fisheries Sciences 13, no. 4 (2014): 979–97.
  48. 48. Akbarzadeh Arash, Farahmand Hamid, Mahjoubi Frouzandeh, Mohammad Ali Nematollahi Piia Leskinen, et al. "The Transcription of L-Gulono-Gamma-Lactone Oxidase, a Key Enzyme for Biosynthesis of Ascorbate, During Development of Persian Sturgeon Acipenser Persicus." Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology 158, no. 4 (2011): 282–88.
  49. 49. Bolann BJ, and Ulvik RJ"Improvement of a Direct Spectrophotometric Assay for Routine Determination of Superoxide Dismutase Activity." Clinical Chemistry 37, no. 11 (1991): 1993–99. pmid:1657455
  50. 50. Goth L. "A Simple Method for Determination of Serum Catalase Activity and Revision of Reference Range." Clinica Chimica Acta 196, no. 2–3 (1991): 143–51. pmid:2029780
  51. 51. Li Xiaoyu, Liu Li, Zhang Yaning, Fang Qian, Li Yuanyuan, et al. "Toxic Effects of Chlorpyrifos on Lysozyme Activities, the Contents of Complement C3 and Igm, and Igm and Complement C3 Expressions in Common Carp (Cyprinus Carpio L.)." Chemosphere 93, no. 2 (2013): 428–33. pmid:23769463
  52. 52. Valipour Abdolmajid, Heidari Behrooz, Hadavi Mahvash, and Yousefi Ayub. "Changes in Immune Parameters (Lysozyme, Igm, C3) in Early Life Stages and Broodstock of Siberian Sturgeon, Acipenser Baerii." Fisheries & Aquatic Life 26, no. 1 (2018): 21–30.
  53. 53. Long Lina, Zhang Haigeng, Ni Qi, Liu Huang, Wu Fan, and Wang Xiaodong. "Effects of Stocking Density on Growth, Stress, and Immune Responses of Juvenile Chinese Sturgeon (Acipenser Sinensis) in a Recirculating Aquaculture System." Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology 219 (2019): 25–34.
  54. 54. Ellman , George L, Courtney K Diane, Valentino Andres Jr, and Featherstone Robert M. "A New and Rapid Colorimetric Determination of Acetylcholinesterase Activity." Biochemical Pharmacology 7, no. 2 (1961): 88–95. pmid:13726518
  55. 55. Kruger Nicholas J. "The Bradford Method for Protein Quantitation." The Protein Protocols Handbook (2009): 17–24.
  56. 56. Valenzuela-Castillo Adán, Arturo Sánchez-Paz Reina Castro-Longoria, Marco Antonio López-Torres, and José Manuel Grijalva-Chon. "Hsp70 Function and Polymorphism, Its Implications for Mollusk Aquaculture: A Review." Latin American Journal of Aquatic Research 47, no. 2 (2019): 224–31.
  57. 57. Liu Ju-Fang, Chen Po-Chun, Ling Thai-Yen, and Hou Chun-Han. "Hyperthermia Increases Hsp Production in Human Pdmcs by Stimulating Ros Formation, P38 Mapk and Akt Signaling, and Increasing Hsf1 Activity." Stem Cell Research & Therapy 13, no. 1 (2022): 1–11. pmid:35659731
  58. 58. Sung YY, Roberts RJ, and Peter Bossier. "Enhancement of Hsp70 Synthesis Protects Common Carp, Cyprinus Carpio L., against Lethal Ammonia Toxicity." Journal of Fish Diseases 35, no. 8 (2012): 563–68.
  59. 59. Niu Yufeng, Norouzitallab Parisa, Baruah Kartik, Dong Shuanglin, and Bossier Peter. "A Plant-Based Heat Shock Protein Inducing Compound Modulates Host–Pathogen Interactions between Artemia Franciscana and Vibrio Campbellii." Aquaculture 430 (2014): 120–27.
  60. 60. Baruah Kartik, Norouzitallab Parisa, Linayati Linayati, Sorgeloos Patrick, and Bossier Peter. "Reactive Oxygen Species Generated by a Heat Shock Protein (Hsp) Inducing Product Contributes to Hsp70 Production and Hsp70-Mediated Protective Immunity in Artemia Franciscana against Pathogenic Vibrios." Developmental & Comparative Immunology 46, no. 2 (2014): 470–79. pmid:24950414
  61. 61. Fittipaldi S, I Dimauro N Mercatelli , and Caporossi D. "Role of Exercise-Induced Reactive Oxygen Species in the Modulation of Heat Shock Protein Response." Free Radical Research 48, no. 1 (2014): 52–70. pmid:23957557
  62. 62. Tang Jiali, Wang Wenqian, Jiang Yuanhe, and Chu Weihua. "Diazinon Exposure Produces Histological Damage, Oxidative Stress, Immune Disorders and Gut Microbiota Dysbiosis in Crucian Carp (Carassius Auratus Gibelio)." Environmental Pollution 269 (2021): 116129. pmid:33261961
  63. 63. Velki Mirna, Henriette Meyer-Alert Thomas-Benjamin Seiler, and Hollert Henner. "Enzymatic Activity and Gene Expression Changes in Zebrafish Embryos and Larvae Exposed to Pesticides Diazinon and Diuron." Aquatic Toxicology 193 (2017): 187–200. pmid:29096092
  64. 64. Dar Showkat Ahmad, Prem Prakash Srivastava Tincy Varghese, Mir Ishfaq Nazir Subodh Gupta, and Krishna Gopal. "Temporal Changes in Superoxide Dismutase, Catalase, and Heat Shock Protein 70 Gene Expression, Cortisol and Antioxidant Enzymes Activity of Labeo Rohita Fingerlings Subjected to Starvation and Refeeding." Gene 692 (2019): 94–101.
  65. 65. Díaz-Resendiz KJG, JA Bernal-Ortega CE Covantes-Rosales Ortiz-Lazareno PC, Toledo-Ibarra GA, Ventura-Ramon GH, et al. "In-Vitro Effect of Diazoxon, a Metabolite of Diazinon, on Proliferation, Signal Transduction, and Death Induction in Mononuclear Cells of Nile Tilapia Fish (Oreochromis Niloticus)." Fish & Shellfish Immunology 105 (2020): 8–15. pmid:32629105
  66. 66. Khoshbavar-Rostami HA, Soltani M, and Hassan HMD. "Immune Response of Great Sturgeon (Huso Huso) Subjected to Long-Term Exposure to Sublethal Concentration of the Organophosphate, Diazinon." Aquaculture 256, no. 1–4 (2006): 88–94.
  67. 67. Sharifpour I, HA Khosbavar Rostami, and Mokarami-Rostam A. "Sublethal Toxicity of Organophosphate, Diazinon on Some Organs of Great Sturgeon (Huso Huso)." Sustainable Aquaculture and Health Management Journal 1, no. 1 (2015): 27–35.
  68. 68. Girón-Pérez Manuel Iván, Santerre Anne, Fabiola Gonzalez-Jaime Josefina Casas-Solis, Marcela Hernández-Coronado, et al. "Immunotoxicity and Hepatic Function Evaluation in Nile Tilapia (Oreochromis Niloticus) Exposed to Diazinon." Fish & Shellfish Immunology 23, no. 4 (2007): 760–69. pmid:17478099
  69. 69. Mozanzadeh Mansour Torfi, Safari Omid, Oosooli Rahim, Mehrjooyan Shapour, Mojtaba Zabayeh Najafabadi Seyed Javad Hoseini, Saghavi Hamid, and Monem Javad. "The Effect of Salinity on Growth Performance, Digestive and Antioxidant Enzymes, Humoral Immunity and Stress Indices in Two Euryhaline Fish Species: Yellowfin Seabream (Acanthopagrus Latus) and Asian Seabass (Lates Calcarifer)." Aquaculture 534 (2021): 736329.
  70. 70. Sharifian Iman, Rastiannasab Abulhasan, and Gandomkar Habibollah. "Effects of Diazinon on Some Immunological Components of Common Carp, Cyprinus Carpio Juveniles." Comparative Clinical Pathology 24, no. 6 (2015): 1339–41.
  71. 71. Mansilla M José , Carme Costa, Herena x, Vanja Tepavcevic, Mireia Castillo, et al. "Hsp70 Regulates Immune Response in Experimental Autoimmune Encephalomyelitis." PLoS One 9, no. 8 (2014): e105737. pmid:25153885
  72. 72. Yu Huanhuan, Zhang Chongying, Xing Wei, Li Tieliang, Xu Guanling, et al. "Comparative Study on the Non-Specific Immune Response and Hsp70 Gene Expression among Three Strains of Koi (Cyprinus Carpio) under Acute Heat Stress." Aquaculture Reports 18 (2020): 100461.
  73. 73. Conde-Guerrero Priscila, Tania Zenteno-Savín Crisalejandra Rivera-Pérez, Fausto Valenzuela-Quiñonez, and Juan Antonio De Anda-Montañez. "Diurnal and Seasonal Hsp70 Gene Expression in a Cryptic Reef Fish, the Bluebanded Goby Lythrypnus Dalli (Gilbert 1890)." Biotecnia 23, no. 2 (2021): 81–88.
  74. 74. Aerts Johan, Juriaan Rogier Metz Bart Ampe, Decostere Annemie, Flik Gert, and Sarah De Saeger. "Scales Tell a Story on the Stress History of Fish." PLoS One 10, no. 4 (2015): e0123411.
  75. 75. Baker Jeremy D, Ilayda Ozsan, Santiago Rodriguez Ospina, Danielle Gulick, and Blair Laura J. "Hsp90 Heterocomplexes Regulate Steroid Hormone Receptors: From Stress Response to Psychiatric Disease." International Journal of Molecular Sciences 20, no. 1 (2018): 79. pmid:30585227
  76. 76. Thau Lauren, Gandhi Jayashree, and Sharma Sandeep. "Physiology, Cortisol." In Statpearls [Internet]. StatPearls Publishing, 2021.
  77. 77. Morishima Yoshihiro, Ranjit K Mehta Miyako Yoshimura, Lau Miranda, Southworth Daniel Ret al. "Chaperone Activity and Dimerization Properties of Hsp90α and Hsp90β in Glucocorticoid Receptor Activation by the Multiprotein Hsp90/Hsp70-Dependent Chaperone Machinery." Molecular Pharmacology 94, no. 3 (2018): 984–91.
  78. 78. Deidda Irene, Russo Roberta, Bonaventura Rosa, Costa Caterina, Zito Francesca, et al. "Neurotoxicity in Marine Invertebrates: An Update." Biology 10, no. 2 (2021): 161. pmid:33670451
  79. 79. Ravaschiere Andrew, Cutler Caroline, Edleson Kristi, Halem Zachery, Magun Henry, et al. "Quantification of Heat Shock Protein 70 and Acetylcholinesterase over a Time Course Suggests Environmental Adaptation in a Foundational Molluscan Species." Ecotoxicology and Environmental Safety 142 (2017): 222–29.
  80. 80. Kim Jun-Hwan, Su Kyoung Kim, and Young Baek Hur"Temperature-Mediated Changes in Stress Responses, Acetylcholinesterase, and Immune Responses of Juvenile Olive Flounder Paralichthys Olivaceus in a Bio-Floc Environment." Aquaculture 506 (2019): 453–58.
  81. 81. Ahmadi Kamal, Ali Reza Mirvaghefei, Mahdi Banaee, and Abdol Rahim Vosoghei. "Effects of Long-Term Diazinon Exposure on Some Immunological and Haematological Parameters in Rainbow Trout Oncorhynchus Mykiss (Walbaum, 1792)." Toxicology and Environmental Health Sciences 6, no. 1 (2014): 1–7.
  82. 82. Üner Nevin, Elif Özcan Oruç Yusuf Sevgiler, Şahin Nesli, Durmaz Hülya, et al. "Effects of Diazinon on Acetylcholinesterase Activity and Lipid Peroxidation in the Brain of Oreochromis Niloticus." Environmental Toxicology and Pharmacology 21, no. 3 (2006): 241–45.
  83. 83. Toledo-Ibarra GA, KJG Díaz-Resendiz L Pavón-Romero , Rojas-García AE, Medina-Díaz IM, and Girón-Pérez MI. "Effects of Diazinon on the Lymphocytic Cholinergic System of Nile Tilapia Fish (Oreochromis Niloticus)." Veterinary Immunology and Immunopathology 176 (2016): 58–63. pmid:27260186