
==== Front
J Microbiol Biotechnol
J Microbiol Biotechnol
Journal of Microbiology and Biotechnology
1017-7825
1738-8872
The Korean Society for Microbiology and Biotechnology

39081252
10.4014/jmb.2402.02001
jmb-34-8-1705
Research article
Biotechnology and Bioengineering (BB)
Host-Microbe Interactions and Pathogenesis
Effects of Microplastic Exposure against White Spot Syndrome Virus Infection in Pacific White Shrimp (Penaeus vannamei)
Jeon Hye Jin 1†
Seo Sangsu 1†
Lee Chorong 1
Kim Bumkeun 1
Piamsomboon Patharapol 23
Kim Ji Hyung 4*
Han Jee Eun 15*
1 Laboratory of Aquatic Biomedicine, College of Veterinary Medicine, Kyungpook National University, Daegu 41566, Republic of Korea
2 Department of Veterinary Medicine, Faculty of Veterinary Science, Chulalongkorn University, Bangkok, Thailand
3 Veterinary Medical Aquatic Animal Research Center of Excellence, Chulalongkorn University, Bangkok, Thailand
4 Department of Food Science and Biotechnology, Gachon University, Seongnam 13120, Republic of Korea
5 Institute for Veterinary Biomedical Science, Kyungpook National University, Daegu 41566, Republic of Korea
* Corresponding authors JH Kim Tel: +82-31-750-5383 Fax: +82-31-750-5389 E-mail: kzh81@gacheon.ac.kr
JE Han Tel: +82-5395-05972 Fax: +82-5395-05951 E-mail: jehan@knu.ac.kr
† These authors contributed equally to this work.

28 8 2024
22 3 2024
22 3 2024
34 8 17051710
1 2 2024
5 3 2024
10 3 2024
Copyright © 2024 by the authors. Licensee KMB
2024
https://creativecommons.org/licenses/by/4.0/ This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license
Plastic waste has emerged as a major environmental concern in recent years. As plastic waste discharged into the marine environment, it undergoes a breakdown process, eventually accumulating in aquatic organisms in the form of microplastics (MPs). To date, reduced food intake, nutritional absorption, and impaired immune system are known adverse effects of MPs-exposed aquatic organisms. This study aims to investigate whether MP exposure accelerated white spot syndrome virus (WSSV) infection in Pacific white shrimp (Penaeus vannamei) via laboratory tests. Briefly, experimental shrimp were divided into four groups; WSSV (group 1); MP (group 2); WSSV + MP (group 3); and Control (group 4). No mortality was observed in group 2, group 4, and even in group 1. However, group 3 showed a cumulative mortality of 50% during the experimental period. The PCR assay results showed no WSSV in the other three groups (groups 1, 2, and 4), but the dead and alive shrimp collected from group 3 were confirmed to be infected with the virus. Histopathological examination revealed normal structures in the hepatopancreas, gill, and muscle tissues of group 4, whereas numerous abnormally shaped nuclei were detected in the gill tissue of group 2. Moreover, group 1 showed minor WSSV-related lesions with few basophilic inclusion bodies in the gills, interestingly, group 3 exhibited severe lesions with numerous basophilic inclusion bodies in the gills. In conclusion, this study confirmed the correlation between the viral disease of shrimp and MPs, which can cause significant economic losses to the shrimp aquaculture industry.

Aquaculture
histopathology
microplastics
marine pollution
shrimp
==== Body
pmcIntroduction

Crustaceans are among the most popular seafoods worldwide and contain beneficial nutrients for human health [1-3]. Shrimp are the most widely farmed crustaceans [4]. However, for several decades, shrimp farming has been threatened by viral diseases, which have caused significant economic losses worldwide [5-7]. White spot syndrome virus (WSSV) is one of the most severe viral pathogens affecting shrimp [8-10]. Its main symptom consists of distinct white spots (0.5–3.0 mm in diameter) under the shrimp’s carapace, and it has a high mortality of up to 100% within days [11]. Previous studies have confirmed that WSSV replication can be accelerated by various stress factors (e.g., physical and chemical changes in water), increasing infection rates [12, 13].

Recently, there has been a noticeable surge in the utilization of plastic, and as a result, the amount of plastic waste has also increased. Such waste is released into the environment, where it breaks down due to various chemical and physical processes [14], resulting in particles of different sizes, i.e., nanoplastics, microplastics (MPs), mesoplastics, and macroplastics [15]. Unfortunately, a considerable amount of plastic waste is discharged into the marine environment [16], after it undergoes multiple stages of degradation it is ingested and accumulated in marine organisms in the form of MPs [14, 17]. MP particles have a wide range of sizes (<5 mm) and a variety of shapes and colors due to differences in sources and production [15]. Once they have entered the food chain, MPs eventually bioaccumulate in species at higher trophic levels, such as mammalians [18, 19] or even humans [20]. As MPs degrade into smaller particles, they are more likely to be ingested by animals [17, 21, 22], resulting in a more severe impact [23, 24]. Moreover, MPs contribute significantly to environmental pollution, as they can adsorb pollutants such as antibiotics, heavy metals, and organic compounds in the water [25]. The adsorptive nature of MPs is a cause of even greater concern. The pollution of aquatic environments with plastic particles induces stress in aquatic organisms [26-28], which can disrupt their immune defense system [28, 29].

Numerous studies have investigated the tissue damage caused by MP accumulation in several aquatic organisms. For example, histopathological studies have examined the intestine of zebrafish [30], the brain tissue structure of crucian carp [31], the gills, hepatopancreas, and muscle of shrimp [32], and the liver and intestine of European sea bass [33] after exposure to MPs. In Penaeus vannamei (P. vannamei), known as Pacific white shrimp, histopathological changes in various organs, such as muscle, midgut gland, hepatopancreas, and gills, were observed after exposure to MPs at varying concentrations [32]. Recently, Shan et al. [34] have also reported a correlation between viral disease and MP exposure in shrimp; however, this correlation has not been yet confirmed based on histopathological examination. In this study, we hypothesized that MPs could accelerate viral disease in shrimp and tested this hypothesis by conducting a laboratory test. The experimental shrimp were infected with WSSV and then exposed to MPs. The correlation between the virus and MP exposure was then examined by determining mortality rates as well as by conducting a PCR assay and histopathological analysis.

Materials and Methods

Preparation for the Shrimp Test (Shrimp, WSSV Stock, and MPs)

Pacific white shrimp (P. vannamei) at the post-larval stage (approximately 0.6 g) were purchased from a local shrimp farm (Republic of Korea) and transported to the Laboratory of Aquatic Biomedicine, College of Veterinary Medicine, Kyungpook National University (Republic of Korea). The shrimp were reared to an average weight of 1.5 ± 0.05 g in 700-L maintenance tanks filled with aerated artificial seawater (25°C–28°C, 25 ppt salinity).

WSSV-tissue homogenates (WSSV stock, 1.62 × 108 copies/μl), which had also been previously used in Han et al. [35], were quantified by qPCR analysis [9] and frozen at −80°C until use.

Fluorescent polystyrene microspheres (FSFR005, concentration: 1%) in a liquid state, purchased from Bangs Laboratories, Inc. (USA), were used as MPs in this study. The particles were colorless and had a mean diameter of 2.07 μm.

Shrimp Test (WSSV and MP Exposure)

The experimental shrimp (average 1.5 ± 0.05 g, N = 32) were divided into four groups: group 1, WSSV; group 2, MP; group 3, WSSV + MP; and group 4, Control. The shrimp in each group (N = 8) were further divided into duplicates of four shrimp each and placed in different 12-L tanks (working water volume: 8 L). The artificial seawater in each tank was maintained as aforementioned rearing conditions. All tested shrimp were subjected to a 24-h fast before experimental infection.

The experimental shrimp (N = 8) of group 1 (WSSV) were exposed to WSSV once via a single feeding with the WSSV stock at 3% of the shrimp's average body weight on day 0. On day 2, 100 μl of 1× PBS was orally administered to each shrimp.

The experimental shrimp (N = 8) of group 2 (MP) were pre-exposed to the muscle of specific pathogen-free (SPF) shrimp once via a single feeding at 3% of the shrimp's average body weight on day 0. On day 2, 100 μl of MP solution was orally administered to each shrimp. MPs were previously diluted 20 times in 1× PBS (final concentration: 0.05%) and then mixed with yellow food dye (CHUN-WOO Co., Ltd, Republic of Korea) in a 1:1 v/v (yellow dye: MP) before administration.

The experimental shrimp (N = 8) of group 3 (WSSV + MP) were exposed to WSSV once via a single feeding with the WSSV stock at 3% of the shrimp's average body weight on day 0. On day 2, 100 μl of MP solution was orally administered to each shrimp.

The experimental shrimp (N = 8) of group 4 (Control) were exposed to the muscle of SPF shrimp once via a single feeding at 3% of the shrimp's average body weight on day 0. On day 2, 100 μl of 1× PBS was orally administered to each shrimp.

After exposure, the shrimp were fed three times a day with commercial shrimp feed containing 30% of crude protein at a total feed of 5% of their body weight for shrimp maintenance [36] and monitored every 12 h for 6 days. During the experiment, dead shrimp (on day 1 and day 3) and live shrimp (on day 6) were collected, and 30 mg of their gills were used for DNA extraction using the DNeasy Blood & Tissue Kit (Qiagen, Germany). The WSSV PCR assay was conducted as described in Nunan and Lightner [37].

Histological Examination

At the end of the experiment (day 6), four moribund shrimp, one from each group, were fixed in Davidson’s AFA fixative (pH 3.0–4.0) [38] for 24 h and then transferred to 70% ethanol for histological examination. After dehydration in a graded ethanol series to absolute ethanol, the samples were embedded in paraffin and each tissue (hepatopancreas, gill, and muscle) was sectioned (4 μm thickness) following standard methods [39]. After staining with hematoxylin and eosin (H&E staining), the sections were analyzed by light microscopy. The severity of WSSV infection was determined on a scale from grade 1 (G1) to grade 4 (G4) [39, 40].

Results

Shrimp Test (WSSV and MP Exposure)

During the experiment, no mortality was observed in group 1 (WSSV), group 2 (MP), and group 4 (Control) until the last day (day 6). However, in group 3, shrimp mortality was observed within 24 h, cumulatively reaching 50% during the entire experimental period (6 days). The PCR assay confirmed WSSV infection in the dead shrimp collected from group 3 (WSSV + MP). We also analyzed the live shrimp collected from each group on day 6, and interestingly, WSSV was detected in the individuals from group 3 (WSSV + MP) but not in those from group 1 (WSSV) (Table 1 and Fig. 1).

Histological Examination

We observed the histopathological effects of exposure to WSSV and MPs in the hepatopancreas, gill, and muscle tissues of the experimental shrimp. Normal structures of the hepatopancreas (Fig. 2A), gills (Fig. 2B), and muscle fibers (Fig. 2C) were observed in group 4 (Control). However, compared with the control, the individuals in group 2 (MP) exhibited collapsed tubular structures and B-cell losses (large vacuole in tubules; white arrow) in the hepatopancreas (Fig. 2D). Also, cytoplasmic effusion was observed in the gill tissue (Fig. 2E), and many nuclei showing hemocytic infiltration (red arrow) and slight lysis (yellow arrow) were observed in the muscle fibers (Fig. 2F).

The typical WSSV lesions were not observed in group 2 (MP) but were observed in group 1 (WSSV) and group 3 (WSSV + MP). In group 1 (WSSV), histopathological changes, including hypertrophied nuclei, were observed in the hepatopancreas (black arrowheads; Fig. 2G), and a few basophilic inclusion bodies in the gill tissue (black arrowheads; Fig. 2H), and along with few nuclei showing hemocytic infiltration (red arrow; Fig. 2I) were observed in the muscle fibers. However, in group 3 (WSSV + MP), hypertrophied nuclei (black arrowheads), and lumen separate from the basement membrane (red arrowheads) were observed in the hepatopancreas (Fig. 2J), and several basophilic inclusion bodies were observed in the gill tissue (black arrowheads; Fig. 2K). In addition, many nuclei showing hemocytic infiltration (red arrows), as well as infiltrated and dissolved muscle fibers (yellow arrows) were observed in the individuals of group 3 (Fig. 2L), but not in those of group 1 (WSSV). The histopathological examination revealed more severe WSSV lesions in group 3 (WSSV + MP) (G4) than in group 1 (WSSV) (G1).

Discussion

Several studies have been conducted on the negative impact of plastics on various aquatic crustaceans, including crab, langoustine, and shrimp species. For example, MPs were shown to have negative effects on the growth of the Chinese mitten crab (Eriocheir sinensis) by inducing oxidative stress in the liver [41]. In the Norway lobster (Nephrops norvegicus), the feeding rate, body mass, and metabolic rate as well as the catabolism of stored lipids were shown to be reduced after exposure to MPs [42]. In Pacific white shrimp (P. vannamei), after exposure to nanoplastics, there were changes in nutritional compositions including a decrease in the levels of some essential amino acids and fatty acids, and alterations in intestine microbial activity were observed [43]. After exposure to MPs, the promotion of stress and immune responses was confirmed in P. vannamei [27]. In addition, the accumulation of plastic debris in shrimp was shown to make them more susceptible to various disease and stress factors [44], which could eventually lead to reduced production. However, the correlation between MPs and infectious viral diseases has been rarely studied in crustaceans.

White spot disease (WSD), which is caused by WSSV, is a notable viral infection primarily affecting farmed shrimp. When infected with this virus, shrimp develop white spots on the exoskeleton, which is unsightly, and the disease itself is highly infectious. WSD is recognized for its severity and is listed in the World Organization for Animal Health list of aquatic animal diseases [45, 46]. Moreover, WSD caused economic losses after the first outbreak in 1992, resulting in a production loss of over US$ 2 billion in China in three years [11]. Ecuador, the outbreak occurred in 1999 and resulted in losses of over US$ 1 billion from 1998 to 2001 [47], and over 100 million US$ in Panama, over 70 million US$ in Peru over 3 years [11]. The present study was conducted to evaluate whether MPs accelerate WSSV infection or mortality in juvenile P. vannamei.

Shan et al. [34] revealed that shrimp (larvae and adults) exposed to MP exhibited an increased susceptibility to WSSV and increased mortality; however, the study did not involve any histopathological examination. A previous study by Hsieh et al. [32], described histopathological examination of the shrimp tissues after MP exposure. However, shrimp were exposed to a small amount of MP using intramuscular injection, which is an unnatural exposure route for shrimp. The results of the histopathological examination conducted in the present study confirmed an accelerated WSSV infection in juvenile shrimp (1.5 ± 0.05 g) as well as increased mortality after MP exposure by oral administration. In line with Mohan et al. [48], the extent of tissue lesions is related to mortality data. In the present study, group 1 (WSSV) showed suspected WSSV-related lesions, but group 3 (WSSV + MP) showed serious WSSV-related lesions, and a higher mortality rate was observed in group 3 with severe WSSV-related lesions. The same WSSV stock with the same viral concentration (1.62 × 108 copies/μl) was used to induce infection in both group 1 and group 3. However, group 1 did not show clinical symptoms or mortality, whereas group 3 exhibited a cumulative mortality of 50% by the 6th day after infection. This indicated that MP exposure amplified WSSV infection.

The results of this study highlighted the increased risk of infection that may result from exposure to MPs in shrimp and the consequent potentially elevated risk of significant economic losses in this aquaculture industry. The present study may contribute to research on the correlation between MP exposure and diseases in shrimp, as well as to raising awareness about protecting the marine environment. In addition, further studies will be necessary to obtain reliable data through long-term experiments based on the observation of histopathological changes due to MP exposure. Follow-up research is expected to confirm the correlation between MPs and other major diseases in shrimp farms as a potential factor for disease exacerbation.

Acknowledgments

This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF2019R1C1C1006212 and NRF2022R1I1A3066435). The work was also supported by the Development of technology for biomaterialization of marine fisheries by-products of the Korea Institute of Marine Science & Technology Promotion (KIMST) funded by the Ministry of Oceans and Fisheries (KIMST-20220128).

Fig. 1 Result of WSSV PCR assay in dead shrimp during the experiment and live shrimp in each group on the last day (day 6).

Lane M: 100-bp ladder; Lane 1: Dead shrimp in group 3 (WSSV+MP) within 24 h after MP exposure; Lane 2: Dead shrimp in group 3 (WSSV+MP) within 96 h after MP exposure; Lane 3: Live shrimp in group 1 (WSSV) on day 6; Lane 4: Live shrimp in group 2 (MP) on day 6; Lane 5: Live shrimp in group 3 (WSSV+MP) on day 6; Lane 6: Live shrimp in group 4 (Control) on day 6; Lane N: Negative control (DEPC-water); Lane P: WSSV positive control (941-bp).

Fig. 2 Photomicrographs of sections of shrimp tissues (hepatopancreas, gill, and muscle) for each experimental group.

(A), (B), and (C): hepatopancreas, gill, and muscle of shrimp in group 4 (Control), respectively; (D), (E), and (F): hepatopancreas, gill, and muscle of shrimp in group 2 (MP); (G), (H), and (I): hepatopancreas, gill, and muscle of shrimp in group 1 (WSSV); (J), (K), and (L): hepatopancreas, gill, and muscle of shrimp in group 3 (WSSV+MP). All sections were stained with H&E. Scale bars = 50 μm.

Table 1 Mortality rate (%), result of the white spot syndrome virus (WSSV) PCR assay, and observation of typical WSSV lesion by histological examination after exposure to WSSV and MPs.

Group	Mortality (%)	WSSV detection	
PCR (dead)	PCR (live)	WSSV histology	
Group 1 (WSSV)	0	NAa	-	+ (G1)b	
Group 2 (MP)	0	NA	-	-	
Group 3 (WSSV+MP)	50	+	+	+ (G4)	
Group 4 (Control)	0	NA	-	-	
aNA: not applicable. bClassified to grades by histopathological lesion [38, 39].

Ethics Approval

The animal experiment was approved by the Ethics Committee of Kyungpook National University (KNU 2021‐0050).

Author Contributions

Hye Jin Jeon: Writing – original draft, Sangsu Seo: Data curation, Chorong Lee: Data curation, Bumkeun Kim: Formal analysis, Patharapol Piamsomboon: Supervision, Ji Hyung Kim: Supervision, and Jee Eun Han: Supervision.

Conflict of Interest

The authors have no financial conflicts of interest to declare.
==== Refs
References

1 Gunalan BSNT Nina TS Soundarapandian P Anand T 2013 Nutritive value of cultured white leg shrimp Litopenaeus vannamei Int. J. Fish. Aquacult. 5 166 171
2 Venugopal V Mérillon JM Ramawat K 2019 Nutrients and Nutraceuticals from Seafood Bioactive Molecules in Food. Reference Series in Phytochemistry Springer, Cham
3 FAO 2020 The state of world fisheries and aquaculture. Sustainability in action Rome, Italy FAO
4 FAO 2022 The State of World Fisheries and Aquaculture 2022. Towards Blue Transformation Rome FAO
5 Chen SN Chang PS Kou GH 1989 Observation on pathogenicity and epizootiology of Penaeus monodon baculovirus (MBV) in cultured shrimp in Taiwan Fish Pathol. 24 189 195 10.3147/jsfp.24.189
6 Lightner DV Redman RM Poulos BT Nunan LM Mari JL Hasson KW 1997 Risk of spread of penaeid shrimp viruses in the Rev. Sci. Tech. Off. Int. Epiz 16 146 160 10.20506/rst.16.1.1010 9329114
7 Stentiford GD Neil DM Peeler EJ Shields JD Small HJ Flegel TW 2012 Disease will limit future food supply from the global crustacean fishery and aquaculture sectors J. Invertebr. Pathol. 110 141 157 10.1016/j.jip.2012.03.013 22434002
8 Otta SK Shubha G Joseph B Chakraborty A Karunasagar I 1999 Polymerase chain reaction (PCR) detection of white spot syndrome virus (WSSV) in cultured and wild crustaceans in India Dis. Aquat. Org. 38 67 70 10.3354/dao038067
9 Durand SV Lightner DV 2002 Quantitative real time PCR for the measurement of white spot syndrome virus in shrimp J. Fish Dis. 25 381 389 10.1046/j.1365-2761.2002.00367.x
10 Tsai JM Wang HC Leu JH Wang AHJ Zhuang Y Walker PJ 2006 Identification of the nucleocapsid, tegument, and envelope proteins of the shrimp white spot syndrome virus virion J. Virol. 80 3021 3029 10.1128/JVI.80.6.3021-3029.2006 16501111
11 Bir J Howlader P Ray S Sultana S Khalil SI Banu GR 2017 A critical review on white spot syndrome virus (WSSV): a potential threat to shrimp farming in Bangladesh and some Asian countries Int. J. Microbiol. Mycol. 6 39 48
12 Lo CF Ho CH Peng SE Chen CH Hsu HC Chiu YL 1996 White spot syndrome baculovirus (WSBV) detected in cultured and captured shrimp, crabs and other arthropods Dis. Aquat. Org. 27 215 225 10.3354/dao027215
13 Lo CF Kou GH 1998 Virus-associated white spot syndrome of shrimp in Taiwan: a review Fish Pathol. 33 365 371 10.3147/jsfp.33.365
14 Xu S Ma J Ji R Pan K Miao AJ 2020 Microplastics in aquatic environments: occurrence, accumulation, and biological effects Sci. Total Environ. 703 134699 10.1016/j.scitotenv.2019.134699 31726297
15 Hartmann NB Huffer T Thompson RC Hassello?v M Verschoor A Daugaard AE 2019 Are we speaking the same language? Recommendations for a definition and categorization framework for plastic debris Environ. Sci. Technol. 53 1039 1047 10.1021/acs.est.8b05297 30608663
16 Moore CJ 2008 Synthetic polymers in the marine environment: a rapidly increasing, long-term threat Environ. Res. 108 131 139 10.1016/j.envres.2008.07.025 18949831
17 Andrady AL 2011 Microplastics in the marine environment Mar. Pollut. Bull. 62 1596 1605 10.1016/j.marpolbul.2011.05.030 21742351
18 Nelms SE Galloway TS Godley BJ Jarvis DS Lindeque PK 2018 Investigating microplastic trophic transfer in marine top predators Environ. Pollut. 238 999 1007 10.1016/j.envpol.2018.02.016 29477242
19 Scherer C Weber A Lambert S Wagner M 2018 Interactions of microplastics with freshwater biota. Freshwater Microplastics Springer, Cham 153 180
20 Wang W Gao H Jin S Li R Na G 2019 The ecotoxicological effects of microplastics on aquatic food web, from primary producer to human: a review Ecotoxicol. Environ. Saf. 173 110 117 10.1016/j.ecoenv.2019.01.113 30771654
21 Browne MA Niven SJ Galloway TS Rowland SJ Thompson RC 2013 Microplastic moves pollutants and additives to worms, reducing functions linked to health and biodiversity Curr. Biol. 23 2388 2392 10.1016/j.cub.2013.10.012 24309271
22 Sharma VK Ma X Lichtfouse E Robert D 2022 Nanoplastics are potentially more dangerous than microplastics Environ. Chem. Lett. 1 4 10.1007/s10311-022-01539-1 36593978
23 Karbalaei S Hanachi P Walker TR Cole M 2018 Occurrence, sources, human health impacts and mitigation of microplastic pollution Environ. Sci. Pollut. Res. 25 36046 36063 10.1007/s11356-018-3508-7 30382517
24 Kögel T Bjorøy Toto B Bienfait AM Sanden M 2020 Micro-and nanoplastic toxicity on aquatic life: determining factors Sci. Total Environ. 709 136050 10.1016/j.scitotenv.2019.136050 31887526
25 Tang Y Liu Y Chen Y Zhang W Zhao J He S 2021 A review: research progress on microplastic pollutants in aquatic environments Sci. Total Environ. 766 142572 10.1016/j.scitotenv.2020.142572 33183825
26 Dhawan A Pandey A Sharma V 2011 Toxicity assessment of engineered nanomaterials: resolving the challenges J. Biomed Nanotechnol. 7 6 7 10.1166/jbn.2011.1173 21485775
27 Han JE Choi SK Jeon HJ Park JK Han SH Jeong J 2021 Transcriptional response in the whiteleg shrimp (Penaeus vannamei) to short-term microplastic exposure Aquac. Rep. 20 100713 10.1016/j.aqrep.2021.100713
28 Mao X Xu Y Cheng Z Yang Y Guan Z Jiang L Tang K 2022 The impact of microplastic pollution on ecological environment: a review Front. Biosci. 27 46 10.31083/j.fbl2702046 35226989
29 Greven AC Merk T Karagöz F Mohr K Klapper M Jovanović B 2016 Polycarbonate and polystyrene nanoplastic particles act as stressors to the innate immune system of fathead minnow (Pimephales promelas) Environ. Toxicol. Chem. 35 3093 3100 10.1002/etc.3501 27207313
30 Lei L Wu S Lu S Liu M Song Y Fu Z 2018 Microplastic particles cause intestinal damage and other adverse effects in zebrafish Danio rerio and nematode Caenorhabditis elegans Sci. Total Environ. 619 1 8 10.1016/j.scitotenv.2017.11.103 29136530
31 Franzellitti S Canesi L Auguste M Wathsala RH Fabbri E 2019 Microplastic exposure and effects in aquatic organisms: a physiological perspective Environ. Toxicol. Pharmacol. 68 37 51 10.1016/j.etap.2019.03.009 30870694
32 Hsieh SL Wu YC Xu RQ Chen YT Chen CW Singhania RR 2021 Effect of polyethylene microplastics on oxidative stress and histopathology damages in Litopenaeus vannamei Environ. Pollut. 288 117800 10.1016/j.envpol.2021.117800 34329062
33 Espinosa C Esteban MÁ Cuesta A 2019 Dietary administration of PVC and PE microplastics produces histological damage, oxidative stress and immunoregulation in European sea bass (Dicentrarchus labrax L.) Fish Shellfish Immunol. 95 574 583 10.1016/j.fsi.2019.10.072 31683003
34 Shan LP Hu Y Hu L Liu HW Liu L Chen J 2023 Involvement of microplastics in the conflict between host immunity defense and viral virulence: promoting the susceptibility of shrimp to WSSV infection Environ. Sci. Technol. 57 11634 11642 10.1021/acs.est.3c01566 37498082
35 Han JE Kim JE Jo H Eun JS Lee C Kim JH 2019 Increased susceptibility of white spot syndrome virus exposed Penaeus vannamei to Vibrio- parahaemolyticus causing acute hepatopancreatic necrosis disease Aquaculture 512 734333 10.1016/j.aquaculture.2019.734333
36 Kureshy N Davis DA 2002 Protein requirement for maintenance and maximum weight gain for the Pacific white shrimp, Litopenaeus vannamei Aquaculture 204 125 143 10.1016/S0044-8486(01)00649-4
37 Nunan LM Lightner DV 2011 Optimized PCR assay for detection of white spot syndrome virus (WSSV) J. Virol. Methods 171 318 321 10.1016/j.jviromet.2010.11.015 21111001
38 Bell TA Lightner DV 1988 A handbook of normal penaeid shrimp histology World Aquaculture Society Baton Rouge, LA 114
39 Lightner DV 1996 A handbook of shrimp pathology and diagnostic procedures for diseases of cultured penaeid shrimp
40 Durand SV Redman RM Mohney LL Tang-Nelson K Bonami JR Lightner DV 2003 Qualitative and quantitative studies on the relative virus load of tails and heads of shrimp acutely infected with WSSV Aquaculture 216 9 18 10.1016/S0044-8486(02)00230-2
41 Yu P Liu Z Wu D Chen M Lv W Zhao Y 2018 Accumulation of polystyrene microplastics in juvenile Eriocheir sinensis and oxidative stress effects in the liver Aquat. Toxicol. 200 28 36 10.1016/j.aquatox.2018.04.015 29709883
42 Welden NA Cowie PR 2016 Long-term microplastic retention causes reduced body condition in the langoustine, Nephrops norvegicus Environ. Pollut. 218 895 900 10.1016/j.envpol.2016.08.020 27524255
43 Chae Y Kim D Choi MJ Cho Y An YJ 2019 Impact of nano-sized plastic on the nutritional value and gut microbiota of whiteleg shrimp Litopenaeus vannamei via dietary exposure Environ Int. 130 104848 10.1016/j.envint.2019.05.042 31325904
44 Timilsina A Adhikari K Yadav AK Joshi P Ramena G Bohara K 2023 Effects of microplastics and nanoplastics in shrimp: mechanisms of plastic particle and contaminant distribution and subsequent effects after uptake Sci. Total Environ. 894 164999 10.1016/j.scitotenv.2023.164999 37348723
45 Lightner DV Redman RM Pantoja CR Tang KFJ Noble BL Schofield P 2012 Historic emergence, impact and current status of shrimp pathogens in the Americas J. Invertebr. Pathol. 110 174 183 10.1016/j.jip.2012.03.006 22434000
46 Patil PK Geetha R Ravisankar T Avunje S Solanki HG Abraham TJ 2021 Economic loss due to diseases in Indian shrimp farming with special reference to Enterocytozoon hepatopenaei (EHP) and white spot syndrome virus (WSSV) Aquaculture 533 736231 10.1016/j.aquaculture.2020.736231
47 Chakraborty S Ghosh U 2014 White spot syndrome virus (WSSV) in crustaceans: an overview of host-pathogen interaction J. Mar. Oceanogr. Mar. Biol. 31 doi:10.4172/2324-8661.1000121 10.4172/2324-8661.1000121
48 Mohan CV Corsin F Thakur PC Padiyar PA Madhusudan M Turnbull JF 2002 Usefulness of dead shrimp specimens in studying the epidemiology of white spot syndrome virus (WSSV) and chronic bacterial infection Dis. Aquat. Org. 50 1 8 10.3354/dao050001 12152899
