Characterisation of virulence factors, biofilm formation, and antimicrobial peptide gene (AMP) expression in the Indian white shrimp Penaeus indicus (H. Milne Edwards, 1837), following immersion challenge with Vibrio parahaemolyticus

Naveenkumar Radhakrishnan; Akshaya Panigrahi; Megha Kadam Bedekar; Saranya  Chakrapani; Arun  Nagarajan; Prasitha  Krishnakumar; Sathish Kumar Thangaraj; Siva C

doi:10.21077/

Authors

Naveenkumar Radhakrishnan PhD scholar, ICAR-CIFE https://orcid.org/0000-0002-0276-9257
Dr.Akshaya Panigrahi Principal Scientist, ICAR-CIBA
Dr. Megha Kadam Bedekar Principal Scientist, ICAR-CIFE
Dr.Saranya Chakrapani Research Associate
Arun Nagarajan Research Scholar
Prasitha Krishnakumar Research Associate
Dr. Sathish Kumar Thangaraj Senior Scientist, ICAR-CIBA
C. Siva Scientist, ICAR-CIBA

https://doi.org/10.21077/

Keywords:

Pathogenicity; Biofilm formation; Antimicrobial peptides; Penaeus indicus; Vibrio parahaemolyticus

Abstract

Vibrio parahaemolyticus is a major bacterial pathogen in shrimp aquaculture, possessing inherent virulence factors such as the haemolysin secretion system and biofilm forming ability, which enhance its pathogenic potential. In shrimp, antimicrobial defences play a central role in combating bacterial infections. This study investigated the pathogenicity factors of a Vibrio parahaemolyticus isolate from an infected early-larvae of Penaeus indicus (H. Milne Edwards, 1837). The virulence and biofilm-related genes were identified through PCR amplification, and biofilm formation ability was quantified using the crystal violet assay at different temperatures. Following immersion challenge with V. parahaemolyticus (1×10⁶ CFU ml-1), the relative expression of antimicrobial peptide (AMP) genes in circulating haemocytes of juvenile shrimp (2.0±0.5 g; n=30 per tank) was examined at 0, 12, 24, 48, 72, and 96 hours post-infection (hpi). Biofilm production was higher at 32°C than at 27°C. PCR analysis detected the presence of toxR, trh, aphA, motX, flig, mcp, and puvA, whereas tdh, tlh, luxO, and pomA genes were not detected. Enhanced expression of biofilm-related genes was expressed at levels of V. parahaemolyticus was observed at 32°C after 24 and 48 h. The relative expression of AMP genes, such as crustin, penaeidin, anti-lipopolysaccharide factor (ALF), and stylicin, showed significant upregulation (p<0.05) at 96 hpi. Notably, this study provides the first ecidence of stylicin expression in the haemocytes of P. indicus during V. parahaemolyticus infection, underscoring its potential role in the shrimp innate immune response and emphasising the importance of AMPs in host defence against the pathogen.

Keywords: Pathogenicity, Haemocytes, Host defence, Immersion challenge, Innate immunity virulence factors

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Author Biographies

Naveenkumar Radhakrishnan, PhD scholar, ICAR-CIFE

Department of Aquatic Animal Health Management, Aquatic Environment and Health Management Division, ICAR-Central Institute of Fisheries Education (CIFE), Mumbai, 400061, India.
Dr.Akshaya Panigrahi, Principal Scientist, ICAR-CIBA

Crustacean Culture Division, ICAR – Central Institute of Brackishwater Aquaculture, 75, Santhome High Road, R. A. Puram, Chennai, 600028, India.
Dr. Megha Kadam Bedekar, Principal Scientist, ICAR-CIFE

Department of Aquatic Animal Health Management, Aquatic Environment and Health Management Division, ICAR-Central Institute of Fisheries Education (CIFE), Mumbai, 400061, India.
Dr.Saranya Chakrapani, Research Associate

Crustacean Culture Division, ICAR – Central Institute of Brackishwater Aquaculture, 75, Santhome High Road, R. A. Puram, Chennai, 600028, India.
Arun Nagarajan, Research Scholar

Crustacean Culture Division, ICAR – Central Institute of Brackishwater Aquaculture, 75, Santhome High Road, R. A. Puram, Chennai, 600028, India.
Prasitha Krishnakumar, Research Associate

Crustacean Culture Division, ICAR – Central Institute of Brackishwater Aquaculture, 75, Santhome High Road, R. A. Puram, Chennai, 600028, India.
Dr. Sathish Kumar Thangaraj, Senior Scientist, ICAR-CIBA

Aquatic Animal Health & Environment Division, ICAR – Central Institute of Brackishwater Aquaculture, 75, Santhome High Road, R. A. Puram, Chennai, 600028, India.
C. Siva, Scientist, ICAR-CIBA

Crustacean Culture Division, ICAR – Central Institute of Brackishwater Aquaculture, 75, Santhome High Road, R. A. Puram, Chennai, 600028, India.

References

Reference

1. Afsal, V.V., Antony, S.P., Philip, R. and Bright Singh, I.S., 2016. Molecular characterization of a newly identified subfamily member of penaeidin from two penaeid shrimps, Fenneropenaeus indicus and Metapenaeus monoceros. Probiotics and antimicrobial proteins, 8, pp.46-52. https://doi.org/10.1007/s12602-015-9203-9

2. Ahmed, H.A., El Bayomi, R.M., Hussein, M.A., Khedr, M.H., Remela, E.M.A. and El-Ashram, A.M., 2018. Molecular characterization, antibiotic resistance pattern and biofilm formation of Vibrio parahaemolyticus and V. cholerae isolated from crustaceans and humans. International journal of food microbiology, 274, pp.31-37. https://doi.org/10.1016/j.ijfoodmicro.2018.03.013

3. Amparyup, P., Donpudsa, S. and Tassanakajon, A., 2008. Shrimp single WAP domain (SWD)-containing protein exhibits proteinase inhibitory and antimicrobial activities. Developmental & Comparative Immunology, 32(12), pp.1497-1509. https://doi.org/10.1016/j.dci.2008.06.005

4. Amparyup, P., Kondo, H., Hirono, I., Aoki, T. and Tassanakajon, A., 2008. Molecular cloning, genomic organization and recombinant expression of a crustin-like antimicrobial peptide from black tiger shrimp Penaeus monodon. Molecular Immunology, 45(4), pp.1085-1093. https://doi.org/10.1016/j.molimm.2007.07.031

5. An, M.Y., Gao, J., Zhao, X.F. and Wang, J.X., 2016. A new subfamily of penaeidin with an additional serine-rich region from kuruma shrimp (Marsupenaeus japonicus) contributes to antimicrobial and phagocytic activities. Developmental & Comparative Immunology, 59, pp.186-198. https://doi.org/10.1016/j.dci.2016.02.001

6. Antony, S.P., Bright Singh, I.S. and Philip, R., 2010. Molecular characterization of a crustin-like, putative antimicrobial peptide, Fi-crustin, from the Indian white shrimp, Fenneropenaeus indicus. Fish and Shellfish Immunology, 28(1), p.216.

7. Bej, A.K., Patterson, D.P., Brasher, C.W., Vickery, M.C., Jones, D.D. and Kaysner, C.A., 1999. Detection of total and hemolysin-producing Vibrio parahaemolyticus in shellfish using multiplex PCR amplification of tl, tdh and trh. Journal of microbiological methods, 36(3), pp.215-225. https://doi.org/10.1016/S0167-7012(99)00037-8

8. Benfield, A.H. and Henriques, S.T., 2020. Mode-of-action of antimicrobial peptides: membrane disruption vs. intracellular mechanisms. Frontiers in Medical Technology, 2, p.610997. https://doi.org/10.3389/fmedt.2020.610997

9. Chakrapani, S., Panigrahi, A., Palanichamy, E., Thangaraj, S.K., Radhakrishnan, N., Panigrahi, P. and Nagarathnam, R., 2024. Evaluation of Therapeutic Efficiency of Stylicin against Vibrio parahaemolyticus Infection in Shrimp Penaeus vannamei through Comparative Proteomic Approach. Probiotics and Antimicrobial Proteins, 16(1), pp.76-92. https://doi.org/10.1007/s12602-022-10006-w

10. De Lorgeril, J., Gueguen, Y., Goarant, C., Goyard, E., Mugnier, C., Fievet, J., Piquemal, D. and Bachère, E., 2008. A relationship between antimicrobial peptide gene expression and capacity of a selected shrimp line to survive a Vibrio infection. Molecular immunology, 45(12), pp.3438-3445. https://doi.org/10.1016/j.molimm.2008.04.002

11. de Souza Valente, C. and Wan, A.H., 2021. Vibrio and major commercially important vibriosis diseases in decapod crustaceans. Journal of Invertebrate Pathology, 181, p.107527. https://doi.org/10.1016/j.jip.2020.107527

12. Essig, A., Hofmann, D., Münch, D., Gayathri, S., Künzler, M., Kallio, P.T., Sahl, H.G., Wider, G., Schneider, T. and Aebi, M., 2014. Copsin, a novel peptide-based fungal antibiotic interfering with the peptidoglycan synthesis. Journal of Biological Chemistry, 289(50), pp.34953-34964. https://doi.org/10.1074/jbc.M114.599878

13. Farias, N.D., Falchetti, M., Matos, G.M., Schmitt, P., Barreto, C., Argenta, N., Rolland, J.L., Bachère, E., Perazzolo, L.M. and Rosa, R.D., 2019. Litopenaeus vannamei stylicins are constitutively produced by hemocytes and intestinal cells and are differentially modulated upon infections. Fish & shellfish immunology, 86, pp.82-92. https://doi.org/10.1016/j.fsi.2018.11.021

14. Gross, P.S., Bartlett, T.C., Browdy, C.L., Chapman, R.W. and Warr, G.W., 2001. Immune gene discovery by expressed sequence tag analysis of hemocytes and hepatopancreas in the Pacific White Shrimp, Litopenaeus vannamei, and the Atlantic White Shrimp, L. setiferus. Developmental & Comparative Immunology, 25(7), pp.565-577. https://doi.org/10.1016/S0145-305X(01)00018-0

15. Guo, D., Yang, Z., Zheng, X., Kang, S., Yang, Z., Xu, Y., Shi, C., Tian, H. and Xia, X., 2019. Thymoquinone inhibits biofilm formation and attachment-invasion in host cells of Vibrio parahaemolyticus. Foodborne pathogens and disease, 16(10), pp.671-678. https://doi.org/10.1089/fpd.2018.2591

16. Han, J.E., Tang, K.F., Tran, L.H. and Lightner, D.V., 2015. Photorhabdus insect-related (Pir) toxin-like genes in a plasmid of Vibrio parahaemolyticus, the causative agent of acute hepatopancreatic necrosis disease (AHPND) of shrimp. Diseases of aquatic organisms, 113(1), pp.33-40. https://doi.org/10.3354/dao02830

17. Han, N., Mizan, M.F.R., Jahid, I.K. and Ha, S.D., 2016. Biofilm formation by Vibrio parahaemolyticus on food and food contact surfaces increases with rise in temperature. Food Control, 70, pp.161-166. https://doi.org/10.1016/j.foodcont.2016.05.054

18. Jayasree, L., Janakiram, P. and Madhavi, R., 2006. Characterization of Vibrio spp. associated with diseased shrimp from culture ponds of Andhra Pradesh (India). Journal of the world aquaculture society, 37(4), pp.523-532. https://doi.org/10.1111/j.1749-7345.2006.00066.x

19. Jiang, H.S., Lv, L.X. and Wang, J.X., 2022. Anti-lipopolysaccharide factor D from kuruma shrimp exhibits antiviral activity. Marine Life Science & Technology, pp.1-10. https://doi.org/10.1007/s42995-021-00113-y

20. Jiang, H.S., Zhang, Q., Zhao, Y.R., Jia, W.M., Zhao, X.F. and Wang, J.X., 2015. A new group of anti-lipopolysaccharide factors from Marsupenaeus japonicus functions in antibacterial response. Developmental & Comparative Immunology, 48(1), pp.33-42. https://doi.org/10.1016/j.dci.2014.09.001

21. Joshi, J., Srisala, J., Truong, V.H., Chen, I.T., Nuangsaeng, B., Suthienkul, O., Lo, C.F., Flegel, T.W., Sritunyalucksana, K. and Thitamadee, S., 2014. Variation in Vibrio parahaemolyticus isolates from a single Thai shrimp farm experiencing an outbreak of acute hepatopancreatic necrosis disease (AHPND). Aquaculture, 428, pp.297-302. https://doi.org/10.1016/j.aquaculture.2014.03.030

22. Kim, Y.B., Okuda, J.U.N., Matsumoto, C., Takahashi, N., Hashimoto, S. and Nishibuchi, M., 1999. Identification of Vibrio parahaemolyticus strains at the species level by PCR targeted to the toxR gene. Journal of Clinical Microbiology, 37(4), pp.1173-1177. https://doi.org/10.1128/jcm.37.4.1173-1177.1999

23. Kulkarni, A., Krishnan, S., Anand, D., Kokkattunivarthil Uthaman, S., Otta, S.K., Karunasagar, I. and Kooloth Valappil, R., 2021. Immune responses and immunoprotection in crustaceans with special reference to shrimp. Reviews in Aquaculture, 13(1), pp.431-459. https://doi.org/10.1111/raq.12482

24. Kumar, B.K., Deekshit, V.K., Raj, J.R.M., Rai, P., Shivanagowda, B.M., Karunasagar, I. and Karunasagar, I., 2014. Diversity of Vibrio parahaemolyticus associated with disease outbreak among cultured Litopenaeus vannamei (Pacific white shrimp) in India. Aquaculture, 433, pp.247-251. https://doi.org/10.1016/j.aquaculture.2014.06.016

25. Kumar, S., Kumar, C.B., Rajendran, V., Abishaw, N., Anand, P.S., Kannapan, S., Nagaleekar, V.K., Vijayan, K.K. and Alavandi, S.V., 2021. Delineating virulence of Vibrio campbellii: a predominant luminescent bacterial pathogen in Indian shrimp hatcheries. Scientific Reports, 11(1), p.15831. https://doi.org/10.1038/s41598-021-94961-4

26. Karunasagar, I., Pai, R., Malathi, G.R. and Karunasagar, I., 1994. Mass mortality of Penaeus monodon larvae due to antibiotic-resistant Vibrio harveyi infection. Aquaculture, 128(3-4), pp.203-209. https://doi.org/10.1016/0044-8486(94)90309-3

27. Li, L., Meng, H., Gu, D., Li, Y. and Jia, M., 2019a. Molecular mechanisms of Vibrio parahaemolyticus pathogenesis. Microbiological Research, 222, pp.43-51. https://doi.org/10.1016/j.micres.2019.03.003

28. Li, M., Ma, C., Zhu, P., Yang, Y., Lei, A., Chen, X., Liang, W., Chen, M., Xiong, J. and Li, C., 2019b. A new crustin is involved in the innate immune response of shrimp Litopenaeus vannamei. Fish & Shellfish Immunology, 94, pp.398-406. https://doi.org/10.1016/j.fsi.2019.09.028

29. Li, S. and Li, F., 2020. The anti-lipopolysaccharide factors in crustaceans. Vertebrate and invertebrate respiratory proteins, lipoproteins and other body fluid proteins, Subcellular Biochemistry, vol 94. Springer, Cham. pp.63-80. https://doi.org/10.1007/978-3-030-41769-7_3

30. Liu, H.T., Wang, J., Mao, Y., Liu, M., Niu, S.F., Qiao, Y., Su, Y.Q., Wang, C.Z. and Zheng, Z.P., 2015. Identification and expression analysis of a novel stylicin antimicrobial peptide from Kuruma shrimp (Marsupenaeus japonicus). Fish & Shellfish Immunology, 47(2), pp.817-823. https://doi.org/10.1016/j.fsi.2015.09.044

31. Livak, K.J. and Schmittgen, T.D., 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2− ΔΔCT method. methods, 25(4), pp.402-408. https://doi.org/10.1006/meth.2001.1262

32. Lu, R., Sun, J., Qiu, Y., Zhang, M., Xue, X., Li, X., Yang, W., Zhou, D., Hu, L. and Zhang, Y., 2021. The quorum sensing regulator OpaR is a repressor of polar flagellum genes in Vibrio parahaemolyticus. Journal of Microbiology, 59(7), pp.651-657. https://doi.org/10.1007/s12275-021-0629-3

33. Matos, G.M., Schmitt, P., Barreto, C., Farias, N.D., Toledo-Silva, G., Guzmán, F., Destoumieux-Garzón, D., Perazzolo, L.M. and Rosa, R.D., 2018. Massive gene expansion and sequence diversification is associated with diverse tissue distribution, regulation and antimicrobial properties of anti-lipopolysaccharide factors in shrimp. Marine drugs, 16(10), p.381. https://doi.org/10.3390/md16100381

34. Mizan, M.F.R., Jahid, I.K., Kim, M., Lee, K.H., Kim, T.J. and Ha, S.D., 2016. Variability in biofilm formation correlates with hydrophobicity and quorum sensing among Vibrio parahaemolyticus isolates from food contact surfaces and the distribution of the genes involved in biofilm formation. Biofouling, 32(4), pp.497-509. https://doi.org/10.1080/08927014.2016.1149571

35. Mookherjee, N., Anderson, M.A., Haagsman, H.P. and Davidson, D.J., 2020. Antimicrobial host defence peptides: functions and clinical potential. Nature reviews Drug discovery, 19(5), pp.311-332. https://doi.org/10.1038/s41573-019-0058-8

36. Munoz, M., Vandenbulcke, F., Gueguen, Y. and Bachère, E., 2003. Expression of penaeidin antimicrobial peptides in early larval stages of the shrimp Penaeus vannamei. Developmental & Comparative Immunology, 27(4), pp.283-289.https://doi.org/10.1016/S0145-305X(02)00102-7

37. Muñoz, M., Vandenbulcke, F., Saulnier, D. and Bachère, E., 2002. Expression and distribution of penaeidin antimicrobial peptides are regulated by haemocyte reactions in microbial challenged shrimp. European Journal of Biochemistry, 269(11), pp.2678-2689. https://doi.org/10.1046/j.1432-1033.2002.02934.x

38. Muñoz, M., Vandenbulcke, F., Gueguen, Y. and Bachère, E., 2003. Expression of penaeidin antimicrobial peptides in early larval stages of the shrimp Penaeus vannamei. Developmental & Comparative Immunology, 27(4), pp.283-289.https://doi.org/10.1016/S0145-305X(02)00102-7

39. Narayanan, S.V., Joseph, T.C., Peeralil, S., Koombankallil, R., Vaiyapuri, M., Mothadaka, M.P. and Lalitha, K.V., 2020. Tropical shrimp aquaculture farms harbour pathogenic Vibrio parahaemolyticus with high genetic diversity and Carbapenam resistance. Marine Pollution Bulletin, 160, p.111551. https://doi.org/10.1016/j.marpolbul.2020.111551

40. Navaneeth, K.A., Bhuvaneswari, T., Rajan, J.J.S., Alavandi, S.V., Vijayan, K.K. and Otta, S.K., 2020. Characterization of Vibrio parahaemolyticus isolates from shrimp farms of Southeast coast of India with special reference to Acute Hepatopancreatic Necrosis Disease (AHPND) status. Aquaculture, 518, p.734813. https://doi.org/10.1016/j.aquaculture.2019.734813

41. Okumura, T., 2007. Effects of lipopolysaccharide on gene expression of antimicrobial peptides (penaeidins and crustin), serine proteinase and prophenoloxidase in haemocytes of the Pacific white shrimp, Litopenaeus vannamei. Fish & shellfish immunology, 22(1-2), pp.68-76. https://doi.org/10.1016/j.fsi.2006.03.013

42. Panigrahi, A., Das, R.R., Sundaram, M., Sivakumar, M.R., Jannathulla, R., Lalramchhani, C., Antony, J., Anand, P.S., Kumar, K.V., Jayanthi, M. and Dayal, J.S., 2021. Cellular and molecular immune response and production performance of Indian white shrimp Penaeus indicus (H. Milne-Edwards, 1837), reared in a biofloc-based system with different protein levels of feed. Fish & Shellfish Immunology, 119, pp.31-41. https://doi.org/10.1016/j.fsi.2021.09.002

43. Panigrahi, A., Vinay, T.N., Patil, P.K., Nithin, M.S. and Diwan, A.D., 2023. Shellfish Microbiome and Its Significant Role in Immune Response and Health Management. In Microbiome of Finfish and Shellfish (pp. 165-179). Singapore: Springer Nature Singapore. https://doi.org/10.1007/978-981-99-0852-3_8

44. Paria, P., Kunal, S.P., Behera, B.K., Mohapatra, P.K.D., Das, A., Parida, P.K. and Das, B.K., 2019. Molecular characterization and genetic diversity study of Vibrio parahaemolyticus isolated from aquaculture farms in India. Aquaculture, 509, pp.104-111. https://doi.org/10.1016/j.aquaculture.2019.04.076

45. Ponprateep, S., Tharntada, S., Somboonwiwat, K. and Tassanakajon, A., 2012. Gene silencing reveals a crucial role for anti-lipopolysaccharide factors from Penaeus monodon in the protection against microbial infections. Fish & shellfish immunology, 32(1), pp.26-34. https://doi.org/10.1016/j.fsi.2011.10.010

46. Raheem, N. and Straus, S.K., 2019. Mechanisms of action for antimicrobial peptides with antibacterial and antibiofilm functions. Frontiers in microbiology, 10, p.2866. https://doi.org/10.3389/fmicb.2019.02866

47. Raja, R.A., Sridhar, R., Balachandran, C., Palanisammi, A., Ramesh, S. and Nagarajan, K., 2017. Pathogenicity profile of Vibrio parahaemolyticus in farmed Pacific white shrimp, Penaeus vannamei. Fish & Shellfish Immunology, 67, pp.368-381. https://doi.org/10.1016/j.fsi.2017.06.020

48. Rolland, J.L., Abdelouahab, M., Dupont, J., Lefevre, F., Bachere, E. and Romestand, B., 2010. Stylicins, a new family of antimicrobial peptides from the Pacific blue shrimp Litopenaeus stylirostris. Molecular immunology, 47(6), pp.1269-1277. https://doi.org/10.1016/j.molimm.2009.12.007

49. Rosa, R.D., Vergnes, A., de Lorgeril, J., Goncalves, P., Perazzolo, L.M., Saune, L., Romestand, B., Fievet, J., Gueguen, Y., Bachere, E. and Destoumieux-Garzon, D., 2013. Functional divergence in shrimp anti-lipopolysaccharide factors (ALFs): from recognition of cell wall components to antimicrobial activity. PLoS One, 8(7), p.e67937. https://doi.org/10.1371/journal.pone.0067937

50. Sato, K. and Homma, M., 2000. Multimeric structure of PomA, a component of the Na+-driven polar flagellar motor of Vibrio alginolyticus. Journal of Biological Chemistry, 275(26), pp.20223-20228. https://doi.org/10.1074/jbc.M002236200

51. Saucedo-Vázquez, J.P., Gushque, F., Vispo, N.S., Rodriguez, J., Gudiño-Gomezjurado, M.E., Albericio, F., Tellkamp, M.P. and Alexis, F., 2022. Marine arthropods as a source of antimicrobial peptides. Marine drugs, 20(8), p.501. https://doi.org/10.3390/md20080501

52. Saulnier, D., Haffner, P., Goarant, C., Levy, P. and Ansquer, D., 2000. Experimental infection models for shrimp vibriosis studies: a review. Aquaculture, 191(1-3), pp.133-144. https://doi.org/10.1016/S0044-8486(00)00423-3

53. Seyfi, R., Kahaki, F.A., Ebrahimi, T., Montazersaheb, S., Eyvazi, S., Babaeipour, V. and Tarhriz, V., 2020. Antimicrobial peptides (AMPs): roles, functions and mechanism of action. International Journal of Peptide Research and Therapeutics, 26, pp.1451-1463. https://doi.org/10.1007/s10989-019-09946-9

54. Silvester, R., Alexander, D. and Ammanamveetil, M.H.A., 2015. Prevalence, antibiotic resistance, virulence and plasmid profiles of Vibrio parahaemolyticus from a tropical estuary and adjoining traditional prawn farm along the southwest coast of India. Annals of microbiology, 65(4), pp.2141-2149. https://doi.org/10.1007/s13213-015-1053-x

55. Song, X., Ma, Y., Fu, J., Zhao, A., Guo, Z., Malakar, P.K., Pan, Y. and Zhao, Y., 2017. Effect of temperature on pathogenic and non-pathogenic Vibrio parahaemolyticus biofilm formation. Food control, 73, pp.485-491. https://doi.org/10.1016/j.foodcont.2016.08.041

56. Soonthornchai, W., Rungrassamee, W., Karoonuthaisiri, N., Jarayabhand, P., Klinbunga, S., Soderhall, K. and Jiravanichpaisal, P., 2010. Expression of immune-related genes in the digestive organ of shrimp, Penaeus monodon, after an oral infection by Vibrio harveyi. Developmental & Comparative Immunology, 34(1), pp.19-28. https://doi.org/10.1016/j.dci.2009.07.007

57. Sruthy, K.S., Nair, A., Puthumana, J., Antony, S.P., Singh, I.B. and Philip, R., 2017. Molecular cloning, recombinant expression and functional characterization of an antimicrobial peptide, Crustin from the Indian white shrimp, Fenneropenaeus indicus. Fish & Shellfish Immunology, 71, pp.83-94. https://doi.org/10.1016/j.fsi.2017.09.071

58. Stewart, B.J. and McCarter, L.L., 2003. Lateral flagellar gene system of Vibrio parahaemolyticus. Journal of bacteriology, 185(15), pp.4508-4518. https://doi.org/10.1128/jb.185.15.4508-4518.2003

59. Sun, F., Zhang, Y., Wang, L., Yan, X., Tan, Y., Guo, Z., Qiu, J., Yang, R., Xia, P. and Zhou, D., 2012. Molecular characterization of direct target genes and cis-acting consensus recognized by quorum-sensing regulator AphA in Vibrio parahaemolyticus. https://doi.org/10.1371/journal.pone.0044210

60. Sun, B., Wang, Z. and Zhu, F., 2017. The crustin-like peptide plays opposite role in shrimp immune response to Vibrio alginolyticus and white spot syndrome virus (WSSV) infection. Fish & Shellfish Immunology, 66, pp.487-496. https://doi.org/10.1016/j.fsi.2017.05.055

61. Supungul, P., Klinbunga, S., Pichyangkura, R., Jitrapakdee, S., Hirono, I., Aoki, T. and Tassanakajon, A., 2002. Identification of immune-related genes in hemocytes of black tiger shrimp (Penaeus monodon). Marine Biotechnology, 4, pp.487-494. https://doi.org/10.1007/s10126-002-0043-8

62. Talapko, J., Meštrović, T., Juzbašić, M., Tomas, M., Erić, S., Horvat Aleksijević, L., Bekić, S., Schwarz, D., Matić, S., Neuberg, M. and Škrlec, I., 2022. Antimicrobial peptides—Mechanisms of action, antimicrobial effects and clinical applications. Antibiotics, 11(10), p.1417. https://doi.org/10.3390/antibiotics11101417

63. Tamura, K., Stecher, G. and Kumar, S., 2021. MEGA11: molecular evolutionary genetics analysis version 11. Molecular biology and evolution, 38(7), pp.3022-3027.

64. Tassanakajon, A., Amparyup, P., Somboonwiwat, K. and Supungul, P., 2011. Cationic antimicrobial peptides in penaeid shrimp. Marine Biotechnology, 13, pp.639-657. https://doi.org/10.1007/s10126-011-9381-8

65. Tassanakajon, A., Somboonwiwat, K., Supungul, P. and Tang, S., 2013. Discovery of immune molecules and their crucial functions in shrimp immunity. Fish & shellfish immunology, 34(4), pp.954-967. https://doi.org/10.1016/j.fsi.2012.09.021

66. Tharntada, S., Somboonwiwat, K., Rimphanitchayakit, V. and Tassanakajon, A., 2008. Anti-lipopolysaccharide factors from the black tiger shrimp, Penaeus monodon, are encoded by two genomic loci. Fish & shellfish immunology, 24(1), pp.46-54. https://doi.org/10.1016/j.fsi.2007.07.010

67. Thompson, F.L., 2006. FABIANO L. THOMPSON, BRIAN AUSTIN, AND JEAN SWINGS. The BIOLOGY of VIBRIOS, p.409.

68. Tran, L., Nunan, L., Redman, R.M., Mohney, L.L., Pantoja, C.R., Fitzsimmons, K. and Lightner, D.V., 2013. Determination of the infectious nature of the agent of acute hepatopancreatic necrosis syndrome affecting penaeid shrimp. Diseases of aquatic organisms, 105(1), pp.45-55.

69. Vargas-Albores, F., Yepiz-Plascencia, G., Jiménez-Vega, F. and Ávila-Villa, A., 2004. Structural and functional differences of Litopenaeus vannamei crustins. Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology, 138(4), pp.415-422. https://doi.org/10.1016/j.cbpc.2004.05.007

70. Wang, L., Ling, Y., Jiang, H., Qiu, Y., Qiu, J., Chen, H., Yang, R. and Zhou, D., 2013. AphA is required for biofilm formation, motility, and virulence in pandemic Vibrio parahaemolyticus. International journal of food microbiology, 160(3), pp.245-251. https://doi.org/10.1016/j.ijfoodmicro.2012.11.004

71. Wong, H.C., Chung, Y.C. and Yu, J.A., 2002. Attachment and inactivation of Vibrio parahaemolyticus on stainless steel and glass surface. Food Microbiology, 19(4), pp.341-350. https://doi.org/10.1006/fmic.2002.0478

72. Wu, B., Zhang, C., Qin, X., Shi, L. and Zhao, M., 2019. Identification and function of penaeidin 3 and penaeidin 5 in Fenneropenaeus merguiensis. Fish & Shellfish Immunology, 89, pp.623-631. https://doi.org/10.1016/j.fsi.2019.04.032

73. Xiao, B., Liao, X., Wang, H., He, J. and Li, C., 2021. BigPEN, an antimicrobial peptide of penaeidin family from shrimp Litopenaeus vannamei with membrane permeable and DNA binding activity. Fish and Shellfish Immunology Reports, 2, p.100034. https://doi.org/10.1016/j.fsirep.2021.100034

74. Zhang, Y., Qiu, Y., Tan, Y., Guo, Z., Yang, R. and Zhou, D., 2012. Transcriptional regulation of opaR, qrr2–4 and aphA by the master quorum-sensing regulator OpaR in Vibrio parahaemolyticus. PLoS One, 7(4), p.e34622. https://doi.org/10.1371/journal.pone.0034622

75. Zhou, L., Li, G., Li, A., Jiao, Y., Li, S., Huang, J., Yang, L. and Wang, C., 2019. Characterization of a group D anti-lipopolysaccharide factor (ALF) involved in anti-Vibrio response in Penaeus monodon. Fish & Shellfish Immunology, 89, pp.384-392. https://doi.org/10.1016/j.fsi.2019.03.049

76. Zulkifli, Y., Alitheen, N.B., Son, R., Yeap, S.K., Lesley, M.B. and Raha, A.R., 2009. Identification of Vibrio parahaemolyticus isolates by PCR targeted to the toxR gene and detection of virulence genes. International Food Research Journal, 16(3), pp.291-298.