Exposure to environmentally relevant concentrations of deltamethrin renders the Chinese rare minnow (Gobiocypris rarus) vulnerable to Pseudomonas fluorescens infection

Journal Pre-proof Exposure to environmentally relevant concentrations of deltamethrin renders the Chinese rare minnow (Gobiocypris rarus) vulnerable to Pseudomonas fluorescens infection

Le Zhang, Xiangsheng Hong, Xu Zhao, Saihong Yan, Xufa Ma, Jinmiao Zha PII:

S0048-9697(20)30453-8

DOI:

https://doi.org/10.1016/j.scitotenv.2020.136943

Reference:

STOTEN 136943

To appear in:

Science of the Total Environment

Received date:

25 October 2019

Revised date:

21 January 2020

Accepted date:

24 January 2020

Please cite this article as: L. Zhang, X. Hong, X. Zhao, et al., Exposure to environmentally relevant concentrations of deltamethrin renders the Chinese rare minnow (Gobiocypris rarus) vulnerable to Pseudomonas fluorescens infection, Science of the Total Environment (2020), https://doi.org/10.1016/j.scitotenv.2020.136943

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© 2020 Published by Elsevier.

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Exposure to environmentally relevant concentrations of deltamethrin renders the Chinese rare minnow (Gobiocypris rarus) vulnerable to Pseudomonas fluorescens infection Le Zhang1,2,3, Xiangsheng Hong1,2,3, Xu Zhao4, Saihong Yan1,2, Xufa Ma5, Jinmiao Zha1,2* Key Laboratory of Drinking Water Science and Technology, Research Center for

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Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China Beijing Key Laboratory of Industrial Wastewater Treatment and Reuse, Research

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Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing

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University of Chinese Academy of Sciences, Beijing 100049, China South China Institute of Environmental Sciences, Ministry of Environmental

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100085, China

Protection of the People's Republic of China, Guangzhou 510655, China Key Laboratory of Freshwater Animal Breeding, Ministry of Agriculture, College of

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Fisheries, Huazhong Agriculture University, Wuhan 430070, China *Address correspondence to

Jinmiao Zha, Ph.D./ Professor Key Laboratory of Drinking Water Science and Technology Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences 18 Shuangqing Road, Haidian District, Beijing, 100085 People’s Republic of China. Email: [email protected]; [email protected]

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Abstract In this study, to assess the immunotoxicity of deltamethrin on fish, adult Chinese rare minnows (Gobiocypris rarus) were exposed to 0.1, 0.3, and 1 μg/L deltamethrin for 28 d. Many immunological parameters and histopathological alterations were determined. The results showed that lymphocyte number was markedly decreased at 0.3 and 1 μg/L treatments, whereas the neutrophil number was strongly increased at 1

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μg/L treatments (p<0.05). Furthermore, lysozyme (LYS), immunoglobulin M (IgM),

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and complement component 3 (C3) levels at 0.3 and 1 μg/L treatments were markedly

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reduced, whereas alkaline phosphatase (ALP) activity were marked increased at 1

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μg/L treatments (p<0.05). The transcripts of almost all TLR (Toll-like receptor)

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signaling pathway-related genes were up-regulated. Histological lesions in the livers,

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intestines, and gills were observed at all treatments. Then, all remaining fish from controls and deltamethrin-exposed groups were injected with Pseudomonas

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fluorescens (P. fluorescens) for 48 h. At 24 and 48 h post-injection with P. fluorescens (hpi), the lymphocyte numbers were strongly reduced at 0.3 and 1 μg/L deltamethrin-exposed groups, whereas LYS and C3 levels were strongly reduced at 0.3 and 1 μg/L deltamethrin-exposed groups (p<0.05). Obvious reduces in IgM levels were also detected at 0.3 and 1 μg/L deltamethrin-exposed groups at 48 hpi (p<0.05). The transcripts of almost all TLR signaling pathway-related genes were significantly down-regulated, whereas the levels of related microRNAs (miRNAs) were markedly increased at all deltamethrin-exposed groups at 24 and 48 hpi. Moreover, the bacterial load in the liver and the mortality of fish were significantly increased at 1 μg/L 2

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deltamethrin-exposed groups at 24 and 48 hpi (p<0.05). Furthermore, obvious histological damage in the livers, intestines, and gills were observed at all deltamethrin-exposed fish at 48 hpi. Overall, our results demonstrated that environmentally relevant concentration deltamethrin suppressed immunity and rendered the fish vulnerable to P. fluorescens infection, subsequently inducing

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mortality. Keywords: Chinese rare minnow; Deltamethrin; Immunotoxicity; Immune responses;

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Pseudomonas fluorescens

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1. Introduction In the last few decades, due to their low persistence, high efficacy and low toxicity towards mammals and birds, pyrethroids have been developed as good substitutes for traditional pesticides such as organochlorines, organophosphates, and carbamates (Guardiola et al., 2014). As the usage of pyrethroids has steadily increased, they accounted for 38 percent of the worldwide pesticide market share in 2015 (Chen

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et al., 2016; Singh et al., 2016). Thereinto, deltamethrin, a α-cyano type 2 synthetic

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pyrethroid, is one of the most widely used pyrethroid insecticides in the world

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because of its high activity against insect pests and its photostability (Kumar et al.,

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2015; Lu et al., 2019; Ullah et al., 2019). Because of its extensive usage, deltamethrin

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has already been detected in the Ebro River Delta in the range of 2 ng/L to 58.8 ng/L

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(Feo et al., 2010) and in the Indus River Ravi in the range of 0.033 to 0.45 μg/L (Mahboob et al., 2015). Moreover, the detected concentration of deltamethrin reached

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4 μg/L in a stream in Cintra, São Paulo, Brazil (Belluta et al., 2010). Deltamethrin can be easily absorbed by biofilms and tissues and accumulate in organisms because of its lipophilic characteristics (Li et al., 2019; Zhang et al., 2016). In agricultural areas that exposure to pesticides, deltamethrin and its metabolites have been detected in urine of pregnant women and children (Qi et al., 2012). Therefore, because of its high environmental concentration and bioaccumulation in organisms, deltamethrin has attracted increasing attention. It is well known that deltamethrin is lowly toxic towards mammals and birds (Ullah et al., 2019), whereas fish are extremely sensitive to deltamethrin due to their 4

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lack of enzymes involved in pyrethroid hydrolyzation (Guardiola et al., 2014; Li et al., 2019). The LC50 of deltamethrin in fish ranges from 0.4 to 2 μg/L (WHO, 1990). A previous study reported that exposure to deltamethrin led to oxidative stress and metabolic effects in gilthead seabream (Guardiola et al., 2014). Deltamethrin exposure provoked histopathological alterations in different organs in fish, such as

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lamellar fusion and hyperplasia in the gills of Colossoma macropomum (Cunha et al., 2018) and induced necrosis in the livers and intestines of silver carp (Ullah et al.,

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2019). Deltamethrin exposure led to neurotoxic effects such as decreased

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acetylcholine esterase activity in Channa punctatus (Singh et al., 2018) and silver

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carp (Ullah et al., 2019) and significantly damaged the motoneurons of zebrafish (Liu

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et al., 2018). Moreover, deltamethrin mediated developmental toxicity in zebrafish,

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which led to abnormal embryo morphology (Li et al., 2019) and increased embryo mortality (Liu et al., 2018). Recently, Tewari et al. (2018) reported that chronic

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exposure to 0.1 and 0.05 mg/kg of deltamethrin could impair the immune responses of Swiss albino mice by altering cytokine/chemokine pathways. Kumar et al. (2018) observed a significant loss in thymocytes and splenocytes of mice following exposed to 5 mg/kg deltamethrin. Moreover, previous studies have reported that deltamethrin induces leukocytosis in Ancistrus multispinis (Pimpão et al., 2007) and alters hemolytic complement activity and the IgM (Immunoglobulin M) level in gilthead seabream (Guardiola et al., 2014). However, the immunotoxicity of environmentally relevant concentrations of deltamethrin towards fish is not well documented. The epithelial/mucosal barrier, humoral parameters and cellular components 5

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together constitute the non-specific immune system, its important for the immune defense of fish (Magnadottir, 2010). These immune defense components can rapidly respond to stress caused exogenous substances and are thus commonly used as important biomarkers for assessing the immune competence of fish (Dong et al., 2018; Magnadottir, 2010; Rehberger et al., 2017; Velmurugan et al., 2019). Additionally, the

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host resistance test has been considered to be the most optimal assay of immune function and is a good method for evaluating the immune toxicity of chemicals

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(Köllner et al., 2002; Soltanian and Fereidouni, 2017). Our work and previous studies

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have confirmed the feasibility and effectiveness of tests combining chemical exposure

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with experimental infection (Nakayama et al., 2017; Zhang et al., 2019).

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Chinese rare minnow (Gobiocypris rarus), a small endemic cyprinid species in

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China, it’s susceptible to environmental contaminants and has been widely used for aquatic toxicology research (Liang and Zha, 2016; Yuan et al., 2013). The Chinese

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rare minnow has also been used in immunological research because of its small size, short life cycle, and ease of husbandry and handling (Su et al., 2008; Zhang et al., 2019). Pseudomonas fluorescens (P. fluorescens) is an opportunistic pathogen in many kinds of fish, such as grass carp, Indian major carp, Japanese flounder, tilapia, and turbot (Wang et al., 2009). Infection with P. fluorescens can cause “red skin” disease, which leads to massive deaths in fish, and has been considered the pathogen that causing hemorrhagic septicemia (Sun and Sun, 2015; Swain et al., 2007). Therefore, the first objective of the present study was to assess the immunotoxicity of environmentally relevant concentration deltamethrin on fish. 6

Journal Pre-proof Chinese rare minnows (Gobiocypris rarus) were exposed to 0.1, 0.3, and 1 μg/L deltamethrin for 28 d and subsequently challenged with P. fluorescens for 48 h. Multiple endpoints including hematology, immune molecule levels, host resistance, and histomorphology were performed. The second objective was to discover the potential immunomodulatory mechanisms of environmentally relevant concentration deltamethrin on fish.

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2. Materials and methods

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2.1. Chemicals

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Deltamethrin (CAS No. 52918-63-5, ≥ 98% pure) was obtained from

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Sigma-Aldrich (Chemical Co., USA). It was dissolved in acetone (purity > 99.5%;

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Sinopharm, China) to prepare the deltamethrin stock solutions, which were stored in

0.01%.

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2.2. Fish maintenance

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brown bottles at 4 °C. The final acetone concentration in the exposure water was <

Chinese rare minnows were cultured in our laboratory for > 15 generations. The rare minnows were kept in a flow-through system with oxygenated water maintained with a constant dissolved oxygen concentration of 7.0-8.0 mg/L, a pH of 7.4 ± 0.2, a water hardness of 44.0-61.0 mg CaCO3/L, and a temperature of 25 ± 1 °C in 16 h of light and 8 h of darkness. The fish were fed commercial food pellets (Trea, Germany) once daily and fresh hatched brine shrimp (Artemia nauplii) twice daily. 2.3. Protocol for deltamethrin exposure All experimental procedures were approved by the Ethics Committee, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences (No. 7

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2018-025). Approximately 11-month-old adult healthy rare minnows (female:male = 1:1) with an average weight of 0.76 ± 0.13 g and a length of 39.68 ± 3.05 mm that were the offspring of the same pair of brood stock were used in the current study. The Chinese rare minnows were acclimated to the laboratory conditions for two weeks before experiments. The fish in the deltamethrin treatment groups were exposed to

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deltamethrin at nominal concentrations of 0.1, 0.3, and 1 µg/L for 28 d. In the control group, the fish were maintained in dechlorinated water for 28 d. Solvent control (<

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0.01% acetone) experiments were also conducted. Each test condition was conducted

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in quintuplicate containers, and each container (30-L) included 30 fishes. During the

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exposure period, the semistatic aqueous fish exposure test was performed, and the test

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solution, which was maintained at 25 ± 1 °C with a photoperiod of 16:8 h (light:dark),

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was renewed every 24 h. The fish were fed newly hatched brine shrimp (Artemia nauplii) two times each day.

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2.4. Infection studies

2.4.1. Bacterial preparation

P. fluorescens (CGMCC: 1.6279) was purchased from the China General Microbiological Culture Collection Center and stably cultured in Luria-Bertani (LB) broth supplemented with 20 μg/mL tetracycline and 50 μg/mL chloramphenicol at 28 °C. Prior to the bacterial challenge, the bacterial stock was thawed and spread on LB agar, which was incubated overnight at 28 °C. A single colony was selected and cultured in LB broth at 28 °C for 24 h. Then, the concentrations of bacterial solution were roughly estimated by optical density at 600 nm (OD600) using a UV-1800 8

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spectrophotometer (SHIMADZU, Japan), and the colony-forming units (CFU) were confirmed by counting the number of colonies after incubation of the diluted bacteria on LB agar. One day prior to bacterial injection, P. fluorescens was growth in LB broth overnight on an orbital shaker at 28 °C. At OD600 0.8, 1 mL of the P. fluorescens

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solution was removed and centrifuged at 5000 rpm at 4 °C for 5 min (Sun and Sun, 2015). The cells were then washed and re-suspended in 1 mL phosphate-buffered

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saline (PBS) (pH=7.4) to produce the bacterial injection solution containing 2×107

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CFU/mL.

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2.4.2. P. fluorescens infection

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After 28 d exposure, the Chinese rare minnow were randomly sacrificed and

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sampled. Then, all remaining fish from controls and deltamethrin treatments were intraperitoneally injected with 50 μL P. fluorescens solution according to a previous

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method with slight modifications (Kinkel et al., 2010). In brief, the rare minnows were anesthetized with a gradient decreasing of water temperature (17, 12, and 8 °C), then P. fluorescens solution was slowly injected intraperitoneally using a 100 µl micro-syringe (HAMILTON, Switzerland). Following the injection, the fish were recovered with a gradient ascending of water temperature (12, 17, and 25 °C) tank. Then the fish were immediately placed in 30-L containers with dechlorinated tap water and maintained at 25 ± 1 °C. Fish mortality was recorded until 48 h post-infection with P. fluorescens (hpi), and the dead fish were removed. The livers were collected at 24 and 48 hpi, weighed and homogenized, and then the 9

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homogenates were serially diluted and plated on LB plates supplemented with 20 μg/mL tetracycline and 50 μg/mL chloramphenicol to determine the CFUs (Huang et al., 2016). The colonies were counted to calculate the bacterial load and expressed as CFU/g of liver weight (n=10 per concentration group). 2.5. Blood and tissue sample collection

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On the 28th d of deltamethrin exposure and at 24 hpi and 48 hpi, the Chinese rare minnow were randomly anesthetized in 100 mg/L of MS-222 (3-aminobenzoic

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acid ethyl ester methane sulfonate salt; Sigma, USA) solution. Blood samples were

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collected with a heparinized microcapillary by tail ablation. Subsequently, the fish

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2.6.1. Hematological analysis

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2.6. Immunological parameters

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were dissected, and then the livers, spleens, intestines, and gills were excised on ice.

Blood smears were prepared on clean microscope slides and stained with

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Diff-Quik stain (Leagene, Beijing, China) according to the manufacturer’s instructions. The percentage of differentiated leukocytes in 100 white blood cells (WBCs) was determined by counting under oil immersion lenses (Olympus, Tokyo, Japan) (n=15 per concentration group). 2.6.2. Enzyme-linked immunosorbent assay (ELISA) The blood samples (a total of 15 fish per test condition) were centrifuged at 4000 rpm for 15 min at 4 °C, then collected the plasma and stored at -20 °C. The plasma samples were used for analyzing the levels of IgM and lysozyme (LYS). The livers were collected for measuring the activity of alkaline phosphatase (ALP) and the 10

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contents of complement component 3 (C3) and C-reactive protein (CRP). Plasma LYS activity, IgM, liver C3, CRP and ALP levels were determined by using enzyme-linked immunosorbent assays kits (Mlbio, Shanghai, China) according to the manufacturer’s protocols. 2.6.3 Histological analysis

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Liver, intestine and gill samples were fixed in paraformaldehyde solution (4%, w/v) for 48 h and then dehydrated with an ascending series of ethanol concentrations

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(70%-100%). The tissues were then rinsed in xylene and embedded in paraffin.

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Sections (3-4 μm) were cut with a microtome. The slides were dehydrated with an

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ascending series of ethanol concentrations, cleared in xylene, stained with

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(Olympus, Tokyo, Japan).

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hematoxylin and eosin (H&E) and visualized under a BX53 optical microscope

2.6.4. Analysis of gene expression

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2.6.4.1. Determination of immune-related gene expression by real-time PCR The spleens of Chinese rare minnows (a total of 15 fish per test condition) were homogenized in 1 mL Trizol reagent (Invitrogen, Madison, USA), and total RNA was extracted according to the manufacturer’s recommendations. The concentrations and qualities of the RNA were measured spectrophotometrically and confirmed by gel electrophoresis. Total RNA (1 μg) was used for reverse transcription to synthesize the first strand complementary DNA (cDNA) using HiScript II Q RT SuperMix (Vazyme, Nanjing, China). qPCR (Quantitative real-time polymerase chain reactions) were performed to quantify the expressions of ten immune-related genes (Table S1). The 11

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qPCR amplifications were performed in an Applied Biosystems Prism 7500 Real-time PCR system (Life Technologies, Carlsbad, CA, USA) using AceQ qPCR SYBR Green Master Mix (Vazyme, Nanjing, China). The thermal program used for each reaction was as follows: 95 °C for 8 min, 40 cycles of 95 °C for 30 s, 60 °C for 1 min, and 72 °C for 30 s. After the amplifications were completed, the melting curve was determined to identify the specificity of the amplified products. The relative

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expression levels were calculated with the 2-ΔΔCt method using the housekeeping gene

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β-actin as the reference gene (Schmittgen and Livak, 2008).

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2.6.4.2. Determination of microRNAs (miRNAs) expression by real-time PCR

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Total RNA was extracted from the spleen of Chinese rare minnows using a

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miRcute miRNA Isolation Kit (TIANGEN, Beijing, China) according to the

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manufacturer’s protocol. Two micrograms of total RNA was reverse-transcribed into cDNA using the miRcute miRNA First-Strand cDNA Synthesis Kit (TIANGEN,

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Beijing, China). Subsequently, qPCR was performed with a miRcute miRNA qPCR Detection Kit (TIANGEN, Beijing, China). The thermal program for each reaction was 94 °C for 2 min, 40 cycles of 94 °C for 20 s, and 60 °C for 34 s. After the amplifications were completed, a melting curve was determined to identify the specificity of the amplified products. All primers used in this study were shown in Table S1 and Table S2. The relative expression levels were calculated with the 2-ΔΔCt method using 5S rRNA as the reference gene (Hong et al., 2016). 2.7. Chemical concentration in the test solution Water samples were collected from the different exposure solutions 24 h after the 12

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test

was

started.

Deltamethrin

concentrations

were

analyzed

using

gas

chromatography techniques according to previously described protocols (Bhanu et al., 2011). A gas chromatography instrument equipped with an ion-trap mass spectrometer (FOCUS-ITQ 700 Thermo Scientific Inc.) and an electron capture detector (ECD) was used for analysis in the experiment. The deltamethrin concentrations in the test

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solutions were determined by triplicate measurements. No deltamethrin was detected in the controls. The actual concentrations (mean ± S.E.M.) of deltamethrin in the

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deltamethrin-exposed groups were 0.06±0.05, 0.17±0.14 and 0.70±0.21, respectively.

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Subsequent experiments using the nominal concentrations of deltamethrin.

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2.8. Statistical analysis

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All data was analyzed using GraphPad Prism 7.0 (GraphPad Software, USA) and

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SPSS 22.0 (SPSS 22, Chicago, IL). Differences between the control and deltamethrin treatments were evaluated by one-way analysis of variance (ANOVA) followed by

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Dunnett’s multiple comparisons test. A value of P <0.05 was considered statistically significant. All data values were reported as the mean ± standard error of the mean (S.E.M.). 3．Results 3.1. The deltamethrin exposure experiment 3.1.1. Differential counts of leukocytes The lymphocytes possessed a roughly spherical nucleus with a notch and a thin blue thin cytoplasm (Fig. 1A). Monocytes were round or irregularly shaped; the nuclei were usually eccentric and occupied approximately half of the cells, and they had a 13

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strongly basophilic and vacuolated cytoplasm (Fig. 1A). Neutrophils were roughly round, with elliptical, horseshoe-shaped, eccentric or multilobulated segmented nuclei that usually stained purple, and the cytoplasm was colorless or pale pink and contained very small pale blue or pink granules (Fig. 1A). There was no obvious differences between the solvent control and the controls for all measured endpoints (data not shown). Compared with the controls, the

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lymphocyte ratios were dramatically reduced at 0.3 and 1 μg/L deltamethrin-exposed

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deltamethrin-exposed group (Fig. 1B, p<0.05).

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groups, whereas the neutrophil ratio was markedly increased at 1 μg/L

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3.1.2. Plasma IgM and LYS levels and liver C3, CRP and ALP levels

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In plasma, the LYS activity was strongly decreased at all deltamethrin-exposed

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groups (Table 1, p<0.05), whereas the IgM content was greatly decreased at 0.3 and 1 μg/L deltamethrin-exposed groups (Table 1, p<0.05).

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Obvious reduces in the C3 level was detected in the livers of rare minnows following exposure to 0.3 and 1 μg/L deltamethrin (Table 1, p<0.05). Inversely, the ALP activity greatly increased in livers of rare minnow after exposure to 1 μg/L deltamethrin (Table 1, p<0.05). However, no obvious differences in the CRP content were observed between any of the deltamethrin-treated groups and controls were detected. 3.1.3. Histopathological observations The normal gills were composed of an intact primary lamellae and secondary lamellae, and the surface of the gill lamella was covered with simple squamous 14

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epithelial cells (Fig. 2A). Compared with that in the controls, the increased proliferation of epithelial cells at the base of secondary lamellae was observed in gills from fish in any of the treatment groups (Fig. 2B-D). Moreover, hypertrophy of the epithelial cells occurred in the gills from fish exposed to 1 μg/L deltamethrin (Fig. 2D).

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The intestine showed a compact intestinal epithelium with an intact edge of villi and cell boundaries in the controls (Fig. 2E). Cellular vacuolization was observed in

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intestines from fish in all deltamethrin treatment group (Fig. 2F-H), whereas the

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disintegration of cell boundaries and the edges of villi were found in intestines from

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fish exposed to 1 μg/L deltamethrin (Fig. 2H).

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The normal livers were composed of a continuous mass of hepatocytes located

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between the blood sinusoids, with hepatocytes that were tightly arranged and showed a normal, clear, spherical nucleus (Fig. 2I). Vacuolation and the loose arrangement of

2L).

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hepatocytes were observed in livers from fish exposed to 1 μg/L deltamethrin (Fig.

3.1.4. Transcriptional levels of immune-related genes As seen in Fig. 3 and Table S3, the ap-1 and il-6 gene expressions were markedly up-regulated at all deltamethrin-exposed groups (p<0.05). Moreover, pronounced up-regulation of tlr5, il-1β, irak1, and traf6 gene expressions were observed at 0.3 and 1 μg/L deltamethrin-exposed groups (p<0.05), whereas the il-1β level was dramatically reduced in spleens following treatment with 0.1 μg/L deltamethrin (p<0.05). In addition, exposure to 0.1 and 0.3 μg/L deltamethrin greatly 15

Journal Pre-proof up-regulated the tlr2, trif, and traf3 mRNA levels, whereas 1 μg/L deltamethrin treatment markedly down-regulated the tlr2 transcriptional level (p<0.05). The expressions of tlr4 and tnfα genes were dramatically induced at 0.3 µg/L deltamethrin-exposed group, whereas a large decrease was observed at 1 µg/L deltamethrin-exposed group (p<0.05). Additionally, exposure to 1 µg/L deltamethrin

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greatly down-regulated the irf3 mRNA levels (p<0.05). Meanwhile, a striking down-regulation of nf-kb level was observed only at 0.3 µg/L deltamethrin-exposed

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group, whereas dramatical up-regulation of myd88, irak4, and il-12 gene was

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observed at 1 µg/L deltamethrin-exposed group (p<0.05).

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As seen in Fig. 3 and Table S3, 0.3 µg/L deltamethrin treatment greatly inhibited

up-regulated

the

miR-146a

transcriptional

levels

and

markedly

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strongly

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the expressions of miR-146a and miR-146b, whereas 1 µg/L deltamethrin treatment

down-regulated the miR-21 level (p<0.05). Additionally, the miR-155 gene at all

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deltamethrin treatment groups showed a significant down-regulation, whereas the transcriptional level of miR-143 was strongly reduced following 0.3 and 1 µg/L deltamethrin treatments (p<0.05). 3.2. P. fluorescens infection test 3.2.1. Disease resistance The

bacterial

load

was

strongly

increased

in

livers

at

1

µg/L

deltamethrin-exposed group at 24 hpi and at 0.3 and 1 µg/L deltamethrin-exposed groups at 48 hpi (Fig. 4A, p<0.05). Furthermore, higher mortality was recorded in the deltamethrin-exposed fish after injection with P. fluorescens, and the mortality was 16

Journal Pre-proof significantly increased in 1 μg/L deltamethrin-treated fish at 24 and 48 hpi (Fig. 4B, p<0.05). 3.2.2. Differential counts of leukocytes The neutrophil ratio was markedly increased at all deltamethrin treatment groups at 24 hpi (Fig. 1C, p<0.05). Additionally, the monocyte count was greatly increased at

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0.3 μg/L deltamethrin-exposed group at 24 hpi but markedly decreased at 1 μg/L deltamethrin-exposed group (Fig. 1C, p<0.05). Moreover, significant reduces in

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lymphocyte ratios were observed in fish exposed 0.3 and 1 μg/L deltamethrin at 24

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and 48 hpi (Fig. 1C, D, p<0.05), whereas the neutrophil ratios were significantly

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increased at all deltamethrin treatments at 24 hpi and at 0.3 and 1 μg/L

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deltamethrin-exposed groups at 48 hpi (Fig. 1C, D, p<0.05).

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3.2.3. Plasma IgM and LYS levels and liver C3, CRP and ALP levels As shown in Table 1, significant inhibitions of plasma LYS activity was

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observed in groups with 0.3 and 1 μg/L deltamethrin treatments at 24 and 48 hpi (p<0.05), whereas 0.3 and 1 µg/L deltamethrin treatments strongly reduced the plasma IgM content at 48 hpi (p<0.05). Furthermore, liver ALP level was markedly decreased at all deltamethrin treatment groups at 24 hpi, whereas significant decreases were observed in groups with 0.3 and 1 μg/L deltamethrin treatments at 48 hpi (p<0.05). Moreover, the liver C3 and CRP levels were dramatically reduced at 0.3 and 1 µg/L deltamethrin treatments at 24 hpi, whereas the CRP contents at 1 µg/L deltamethrin-exposed group and the C3 contents at all deltamethrin-exposed groups were strongly decreased at 48 hpi (p<0.05). 17

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3.2.3. Histopathological observations At 48 hpi, no obvious histopathological characteristics were observed in the livers, gills, and intestines from fish in the controls. The increased proliferation of epithelial cells at the base of the secondary lamellae in gills was observed at all deltamethrin-exposed groups (Fig. 5B-D), and the hypertrophy of epithelial cells

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occurred in the gills from fish exposure to 1 μg/L deltamethrin (Fig. 5D). The intestine showed a cellular vacuolation in all deltamethrin treatment groups (Fig.

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5F-H), and disintegration of cell boundaries and the edges of villi were also showed in

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the 0.3 and 1 μg/L deltamethrin-exposed fish (Fig. 5G, H). Moreover, following

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exposed to 0.3 and 1 μg/L deltamethrin, rare minnow showed a vacuolation and loose

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arrangement of hepatocytes in the liver (Fig. 5K, L).

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3.2.4. Transcriptional levels of immune-related genes As seen in Fig. 3 and Table S4, at 24 hpi, the mRNA levels of tlr2, myd88, irak1,

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irak4, traf6, tak1, nf-kb, trif, traf3, irf3, il-1β, and tnfa were markedly decreased at all deltamethrin-exposed groups (p<0.05), whereas 1 μg/L deltamethrin treatment greatly down-regulated the ap-1, tlr4, tlr5, and il-12 mRNA levels (p<0.05). On the contrary, the expression of il-6 gene were strongly induced following 0.3 and 1 μg/L deltamethrin treatments (p<0.05). As shown in Fig. 3 and Table S5, at 48 hpi, a significant down-regulation of tlr2, tlr4, tlr5, myd88, irak1, irak4, traf3, and ap-1 mRNA expressions were detected at all deltamethrin-exposed groups (p<0.05). Exposure to 0.1 μg/L deltamethrin greatly up-regulated the expressions of tak1, il-6, traf6, and il-12, whereas 0.3 and 1 μg/L 18

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deltamethrin treatments strongly down-regulated the tak1, il-6, and l-1β mRNA levels (p<0.05). Moreover, no obvious alterations in trif, irf3, and tnfα mRNA expression were detected at any of deltamethrin-exposed groups and the controls. In addition, the expressions of miR-21, miR-143, miR-146a, miR-146b, and miR-155 were strikingly increased at all deltamethrin-exposed groups at 24 hpi (Fig. 3

of

and Table S4, p<0.05). Moreover, transcripts of miR-21, miR-146a, and miR-146b were strongly elevated at all deltamethrin-exposed at 48 hpi (Fig. 3 and Table S5,

ro

p<0.05). Additionally, a striking up-regulation of miR-143 gene expression were

-p

detected in groups with 0.1 and 0.3 μg/L deltamethrin treatments at 48 hpi (p<0.05),

re

whereas transcription of miR-155 was strongly up-regulated following exposure to 0.3

na

4. Discussion

lP

and 1 μg/L deltamethrin at 48 hpi (Fig. 3 and Table S5, p<0.05).

Deltamethrin has been used as a insecticide and acaricide worldwide due to its

Jo ur

short biodegradation period and low toxicity towards mammals and birds (Lu et al., 2019; Sands et al., 2018). Evidence has indicated that deltamethrin is highly toxic to nontarget aquatic fish organisms (Ullah et al., 2019). In current study, rare minnows were exposed to environmentally relevant concentrations of deltamethrin for 28 d and then challenged with P. fluorescens for 48 h. The susceptibility of the fish to bacterial infection was increased, and mortality was increased in deltamethrin-exposed fish. Our findings indicated that deltamethrin could induce immunotoxicity in Chinese rare minnows, predisposing them to microbial infection. Histopathological inspection is a useful tool to assess the toxic effects of 19

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contiminants on organisms (Ullah et al., 2019). In present study, histopathological alterations included vacuolation, the loose arrangement of hepatic or intestinal epithelial cells, the disintegration of cell boundary and edges of villi in intestines, the detachment and proliferation of epithelial cells and the expansion of secondary lamellae in gills in fish subjected to deltamethrin treatments. Moreover, similar

of

histopathological changes were also observed in livers, intestines, and gills in the deltamethrin treatment groups following 48 h challenged with P. fluorescens, and

ro

these pathological characters were more obvious than fish exposed to deltamethrin

-p

alone. Similarly, lamellar fusion and hyperplasia in gills and pyknotic nuclei in livers

re

were observed after Colossoma macropomum was exposed to 64.4 μg/L deltamethrin

lP

for 96 h (Cunha et al., 2018). Moreover, vacuolation of hepatocytes and epithelial

na

hyperplasia of secondary lamellae were observed in Nile tilapia following 1.46 μg/L deltamethrin exposure for 7 d and 21 d (El-Sayed and Saad, 2008). Additionally, after

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exposure of mosquitofish to deltamethrin for 30 d, nuclear pyknosis in livers and epithelial hyperplasia and fusion of the secondary lamellae in gills were observed at 0.25 μg/L, and necrosis in the gut was observed at 0.5 μg/L (Cengiz and Unlu, 2006). The destructive effects on these tissues could result in severe physiological and biochemical responses, which are mainly related to the immunological system (El-Sayed and Saad, 2008). Hematological and biochemical parameters are important indicators to determine the health of organisms (Rao et al., 2006). White blood cells plays a vital role in cellular and humoral immunity and defense against infectious diseases (Vetvicka et al., 20

Journal Pre-proof 2013; Whyte, 2007). In this study, exposure to 0.3 and 1 μg/L deltamethrin significantly decreased the lymphocyte ratio, whereas 1 μg/L deltamethrin treatment strongly increased the neutrophil ratio (p<0.05). Similarly, a significant reduce in the lymphocyte ratio and an increase in the neutrophil ratio were detected in common carp after treatment with 0.17 μg/L cypermethrin (Soltanian and Fereidouni, 2017).

of

Furthermore, the lymphocyte ratio was strikingly reduced after exposure of Nile tilapia to 0.89 mg/L penoxsulam for 15 and 30 d (Galal et al., 2018). Therefore, the

ro

results of previous studies and our results suggested that the decreased production of

-p

leukocytes may be due to deltamethrin-induced tissue damage and severe disturbance

including

immunoglobulin,

LYS,

complement

components,

and

lP

proteins,

re

of the nonspecific immune system (El-Sayed and Saad, 2008). It is known that serum

na

bacteriocidal substances are mainly produced by leukocytes (Misra et al., 2006 a, b). In this study, exposure of rare minnow to 0.3 and 1 μg/L deltamethrin dramatically

Jo ur

decreased the plasma LYS, IgM, and liver C3 levels. Similarly, Soltanian and Fereidouni (2017) found that exposure of common carp to 0.085 and 0.17 μg/L cypermethrin strongly reduced the IgM and LYS levels. Furthermore, levels of plasma LYS and IgM were strongly reduced in rainbow trout after treatement with 2 and 4 μg/L deltamethrin for 30 min (Siwicki et al., 2010) and in gilthead seabream after treatment with 0.1 μg/L deltamethrin for 14 d (Guardiola et al., 2014). The LYS and C3 are crutial defensive component of the non-specific immunity in fish, whereas the IgM is a common molecule of the specific immune system (Li et al., 2013). They plays a vital role in protecting fish against pathogen infections (Li et al., 2013). Taken 21

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together, the results of previous studies and our findings suggested that deltamethrin exposure caused tissue damage and disrupted the innate and specific immunity in fish. The non-specific immune system plays a predominant role in the immune defense of fish and mainly relies on signal transduction by the TLR (Toll-like receptor) signaling pathway to detect and respond to invading pathogens (Dong et al., 2018).

of

Activation of the TLR signaling pathway could promote the expression of inflammatory cytokine, result in inflammatory responses (Liew et al., 2005). In

ro

current study, significant up-regulation of the expression levels of il-1β, il-6, and il-12

-p

were detected at 1 μg/L deltamethrin-exposed group (p<0.05). Similarly, the

re

expression of il-1β and il-6 gene was enhanced in common carp following exposure to

lP

1.6 mg/L paraquat for 7 d (Ma et al., 2018). Furthermore, the transcripts of

na

inflammatory cytokines (il-1β, il-6, il-8, and tnf-α) were also increased in zebrafish larvae after treatment with 300 μg/L atrazine for 96-h (Liu et al., 2017). These

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cytokines are acute phase response factors and are rapidly produced by macrophages in responses to tissue damage (Jia et al., 2014). MiRNAs plays a vital role in the regulation of TLR signaling (Garg et al., 2013). In this study, significant inhibition of the transcription of miR-21, miR-143, and miR-155 was observed at 1 μg/L deltamethrin-exposed group (p<0.05). Evidence has indicated that the expression of TLR pathway-related genes (myd88, irak1, irak4, and traf6) was increased by miR-21 inhibition and decreased by miR-21 over-expression (Chen et al., 2013). Moreover, He et al. (2014) found that miR-155 could negatively regulate TLR signaling pathways by combining with myd88. In inflammatory responses, the high expression 22

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of immune response activator genes must be balanced by the high expression of regulatory miRNAs to balance the effects of the immune system activators; if there is a decrease or no change in the expression of such miRNAs, this may result in pathological inflammation and increased mortality (Andreassen and Høyheim, 2017). Immunity plays a crucial role in protecting animals from infection and

of

maintaining internal homeostasis (Dong et al., 2018). Teleost fish primarily relies on the non-specific immune system to recognize and defense against pathogen invasion

ro

(Uribe et al., 2011). In the current study, a striking increase in the neutrophil ratio and

-p

a great reduce in the lymphocyte ratio and the levels of LYS, C3, and CRP were

re

observed at 0.3 and 1 μg/L deltamethrin treatments after challenged with P.

responses

to

eliminate

pathogens,

including

the

production

of

na

immune

lP

fluorescens (p<0.05). In fact, following pathogen entry, the host mounted a series of

broad-spectrum antimicrobial substances and acute phase proteins, nonclassical

Jo ur

complement activation, the release of cytokines, inflammation and phagocytosis (Ellis et al., 2001). Previous studies have reported that bacterial infection or lipopolysaccharides could induce the production of immune components in fish (Ellis et al., 2001; Saurabh and Sahoo, 2008). Conversely, a decrease in immune substances indicated that the inhibition of fish immunity resulted in the reduced resistance of fish to bacterial infections (Hart et al., 2016). In general, TLRs were highly expressed in fish, and they in charge of recognizing and responding to bacteria (Li et al., 2017). In current study, exposure to deltamethrin strongly reduced the levels of tlr2 and tlr5 at 24 and 48 hpi, suggesting deltamethrin suppressed the recognition to bacteria in fish. 23

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Furthermore, the down-regulation of myd88 and trif genes were also down-regulated. The myd88 and trif are important non-specific immune signaling molecules downstream of TLRs. The knockdown of myd88 gene could disrupt the elimination of bacteria (van der Sar et al., 2006), and the deficient in trif led to increasing host susceptibility to virus (Totura et al., 2015). After recognition of bacteria, various host

of

defense genes downstream of TLR signaling pathway were induced (Zhang et al., 2014). In this study, the down-regulation of almost all genes downstream of TLR

ro

signaling pathway and the over-expression of miRNAs were observed in

-p

deltamethrin-treated fish at 24 and 48 hpi. Similarly, the transcrips of TLR signaling

re

pathway-related genes were decreased in F1 zebrafish larvae with bacterial and virus-

lP

mimicking treatments after parental exposure to bisphenols (Dong et al., 2018). These

na

findings demonstrated that exposure to deltamethrin inhibited innate immune defense via decreasing immune molecule and disturbing the TLR signaling pathway. In

Jo ur

addition to the innate immune defense, the adaptive immune response also plays a vital role in resisting pathogen invasion (Uribe et al., 2011). The IgM is the most crucial humoral molecule of the adaptive immune system in fish (Li et al., 2013). In current study, the contents of plasma IgM were greatly reduced at 0.3 and 1 μg/L deltamethrin treatmens after challenge with P. fluorescens (p<0.05). This may be due to the decreases in the number of leukocytes, which are the primary source of immunoglobulin (Misra et al., 2006 a, b). The changes in IgM levels suggested that deltamethrin caused a negative effect on the non-specific immune response of fish. Previous study suggested that the reduced immune responses to pathogens 24

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infection may result in the increasing susceptibility of fish to pathogens (Soltanian and Fereidouni, 2017). In this study, a significantly increased bacterial load and greater mortality were observed in 1 μg/L deltamethrin-exposed fish at 24 and 48 hpi. Likewise, after challenge with Aeromonas hydrophila (A. hydrophila), effective A. hydrophila infection persistence and significant mortality in fluoride-exposed zebrafish were observed (Singh et al., 2017). Soltanian and Fereidouni (2017) found

of

that the mortality rate was strongly increased in 0.17 μg/L cypermethrin-exposed

ro

common carp following challenged with A. hydrophila. Moreover, our previous study

-p

also observed a large number of deaths in 1.5 μg/L cypermethrin-exposed rare

re

minnows at 48 hpi (Zhang et al., 2019). Therefore, our findings further demonstrated

lP

that deltamethrin exposure reduced the immunity of fish and inhibited the immune

causing fish death.

Jo ur

5. Conclusion

na

responses of fish to pathogens, predisposing them to bacterial infection and ultimately

In this study, alterations in the differential leukocyte ratio, a reduction in immune molecule levels, disturbances of the TLR pathway, and histological changes suggested that environmentally relevant concentrations deltamethrin induced immunotoxicity in the Chinese rare minnow. In addition, challenge with P. fluorescens increased bacterial load and mortality in deltamethrin-exposed fish, suggesting that deltamethrin exposure interfered with the recognition and clearance of fish immune system to pathogens by damaging tissues and reducing the levels of immune molecules, and this indicated that deltamethrin inhibited the immunity of rare minnow and rendered them 25

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susceptible to P. fluorescens infection, which subsequently resulted in mortality. Acknowledgments This work was supported by the Major International Joint Research Project of the National Natural Science Foundation of China (51420105012) and the National Natural Science Foundation of China (21677165).

of

Abbreviations Used Pseudomonas fluorescens, P. fluorescens; Alkaline phosphatase, ALP; Aeromonas

ro

hydrophila, A. hydrophila; complementary DNA, (cDNA); Optical density at 600 nm,

-p

OD600; Immunoglobulin M, IgM; Colony-forming units, CFU; Quantitative real-time

re

polymerase chain reactions, qPCR; MicroRNAs, MiRNAs; Lysozyme, LYS; h

lP

postinjection with P. fluorescens, hpi; C-reactive protein, CRP; Luria-Bertani, LB;

na

Phosphate buffer saline, PBS; Complement component 3, C3; Enzyme-linked immunosorbent assays, ELISA; 3-aminobenzoic acid ethyl ester methane sulfonate

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of rare minnow CYP1s and CYP2s mRNA exposed to the AHR agonist

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benzo[a]pyrene. Chemosphere 93, 209-216.

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Zhang, L., Zhao, X., Yan, S.H., Zha, J.M., Ma, X.F., 2019. The immune responses of

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the Chinese rare minnow (Gobiocypris rarus) exposed to environmentally

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relevant concentrations of cypermethrin and subsequently infected by the bacteria Pseudomonas fluorescens. Environ. Pollut. 250, 990-997.

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Zhang, J., Kong, X., Zhou, C., Li, L., Nie, G., Li, X., 2014. Toll-like receptor recognition of bacteria in fish: ligand specificity and signal pathways. Fish Shellfish Immunol. 41, 380-388. Zhang, X., Yang, H., Ren, Z., Cui. Z., 2016. The toxic effects of deltamethrin on Danio rerio: the correlation among behavior response, physiological damage and AChE. RSC Advances 6, 109826-109833.

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Legend of Figures and Tables Fig. 1. Light micrographs of peripheral blood cells and differential counts of leukocytes from Chinese rare minnow. (A) Photomicrograph of a peripheral blood smear showing a sequence of images corresponding to: erythrocyte (black arrows), lymphocyte (blue arrows), monocyte (green arrows), and neutrophil (red arrows) were

exposure

to

deltamethrin;

(C)

differential

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stained with Diff-Quik; Bar=5 μm. (B) Differential counts of leukocytes after 28 d of counts

of

leukocytes

in

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deltamethrin-exposed Chinese rare minnows at 24 h after challenged with P.

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fluorescens; (D) differential counts of leukocytes in deltamethrin-exposed Chinese

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rare minnows at 48 h after challenged with P. fluorescens. Data expressed as means ±

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(p<0.05).

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S.E.M. (n=15). Asterisks (*) represents significant differences from the controls

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Fig. 2. Light micrographs of gill, intestinal and liver tissues in Chinese rare minnow after exposure to deltamethrin for 28 d, stained with hematoxylin and eosin (H&E). (A) Normal gill tissue from control fish; (B-D) gill tissue from fish in the 0.1, 0.3, and 1 μg/L deltamethrin-treated groups; (E) normal intestinal tissue from control fish; (F-H) intestinal tissue from fish in the 0.1, 0.3, and 1 μg/L deltamethrin-treated groups; (I) normal liver tissue from control fish; (J-L) liver tissue from fish in the 0.1, 0.3, and 1 μg/L deltamethrin-treated groups. Pr indicates proliferation of epithelial cells; Ep: epithelial hypertrophy; Va: vacuolation; Di: disintegration of cell boundaries and the edges of villi. 36

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Fig. 3. Transcript expressions of the selected genes of TLR signaling pathway and miRNA following Chinese rare minnows were exposed to deltamethrin for 28 d and subsequently challenged with P. fluorescens (n=3 replicates, 5 fish per replicate).

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Fig. 4. The effect of deltamethrin on disease resistance in the Chinese rare minnow. (A) The bacterial load in the livers of P. fluorescens (Pf) infected Chinese rare

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minnows was assessed at 24 h and 48 h, and the mean CFU/g liver is shown with the

-p

means ± S.E.M. (n=10 per time point, p<0.05); (B) mortality percentages in

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deltamethrin-exposed Chinese rare minnows at 24 and 48 h after challenged with P.

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fluorescens (Pf). Data expressed as means ± S.E.M.. Asterisks (*) indicate significant

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difference from the controls (p<0.05).

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Fig. 5. Light micrographs of gill, intestinal and liver tissues in deltamethrin-exposed Chinese rare minnows at 48 h following challenged with P. fluorescens, stained with hematoxylin and eosin (H&E). (A) Gill tissue from control fish; (B-D) gill tissue from fish in the 0.1, 0.3, and 1 μg/L deltamethrin-treated groups; (E) intestinal tissue from control fish; (F-H) intestinal tissue from fish in the 0.1, 0.3, and 1 μg/L deltamethrin-treated groups; (I) liver tissue from control fish; (J-L) liver tissue from fish in the 0.1, 0.3, and 1 μg/L deltamethrin-treated groups. Pr indicates proliferation of epithelial cells; Ep: epithelial hypertrophy; Va: vacuolation; Di: disintegration of cell boundaries and the edges of villi. 37

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Table 1. Alterations in plasma LYS and IgM and liver C3, CRP and ALP levels following Chinese rare minnows were exposure to deltamethrin for 28 d and subsequently challenged with P. fluorescens (Pf) for 24 and 48 h. Data showed as the means ± S.E.M. (n=3 replicates, 5 fish per replicate). * Value in

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rows indicates the significant differences between deltamethrin-exposed groups and control (p <0.05). **Value in rows indicates the significant difference between

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deltamethrin-exposed groups and control (p <0.01). ***Value in rows indicates the

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significant difference between deltamethrin-exposed groups and control (p <0.001).

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Journal Pre-proof Table 1. Alterations in plasma LYS and IgM and liver C3, CRP and ALP levels after Chinese rare minnows were exposed to deltamethrin for 28 d and subsequently 24 and 48 h challenged with P. fluorescens (Pf). Concentrations of deltamethrin (μg/L) 1

IgM (μg/mL)

371.11±7.48

345.41±40.61

306.74±20.6**

271.47±33.69**

LYS (U/L)

72.19±1.71

63.80±4.38*

61.70±0.27*

46.15±5.81**

C3 (mg/L)

55.65±4.10

59.53±3.57

48.29±1.13*

32.62±1.32**

CRP (mg/L)

8.89±0.88

9.41±0.94

7.62±0.56

8.88±1.32

ALP (U/L)

25.90±1.77

23.39±3.15

24.68±3.37

31.69±1.89*

329.4±29.65

326.73±19.1

280.92±38.7

302.92±26.29

LYS (U/L )

57.80±5.05

50.85±4.72

46.92±4.42*

46.18±3.59*

C3 (mg/L)

68.86±3.63

68.83±1.6

57.41±3.22**

44.67±4.32***

CRP ( mg/L)

10.74±0.54

10.47±0.70

7.03±0.91**

6.52±0.72**

ALP ( U/L)

39.44±2.64

31.73±2.86*

18.53±4.54***

28.92±2.76*

315.05±15.77

308.79±3.96

244.46±20.67*

241.37±25.87*

65.59±4.07

57.82±7.72*

52.47±2.07*

IgM ( μg/mL)

IgM ( μg/mL)

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0.3

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Pf-48h

0.1

LYS (U/L)

63.15±4.38

C3 (mg/L)

69.83±3.41

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Pf-24h

CK

59.21±6.92*

58.19±1.24*

35.07±3.92***

CRP ( mg/L)

10.99±1.01

10.67±0.24

9.89±0.48

7.99±0.43*

ALP ( U/L)

16.15±0.86

16.67±2.63

7.77±0.58***

9.23±0.96***

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28d

Parameters

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Times

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Data expressed as the means ± S.E.M. (n=3 replicates, 5 fish per replicate). *Value in rows indicates the significant difference between deltamethrin-exposed groups and control (p <0.05). **Value in rows indicates the significant difference between deltamethrin-exposed groups and control (p <0.01). ***Value in rows indicates the significant difference between deltamethrin-exposed groups and control (p <0.001).

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Journal Pre-proof Declaration of competing interests

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The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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Graphical abstract Highlights: 

Deltamethrin induced immunotoxicity in Chinese rare minnow at environmentally relevant concentrations.



Deltamethrin caused histopathological damage to liver, intestine, and gill of Chinese rare minnows.



Deltamethrin exposure immunosuppression.



Deltamethrin exposure rendered fish vulnerable to P. fluorescens infection.

fish

immune

system

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destroyed

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and

induced

fish

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5