Upregulation of torso-like protein (perforin) and granzymes B and G in non-adherent, lymphocyte-like haemocytes during a WSSV infection in shrimp. Issue 128 (September 2022)

• Record Type: Journal Article
• Authors: Zheng, Liping; Byadgi, Omkar; Rakhshaninejad, Mostafa; Nauwynck, Hans
• Is Part Of: Fish & shellfish immunology. Issue 128(2022)
• Page Start: 676
• Page End: 683
• Publication Date: 2022-09
• Subjects: Shrimp -- P. vannamei -- Haemocytes -- WSSV -- Torso-like protein -- Granzyme; Fishes -- Immunology -- Periodicals; Shellfish -- Immunology -- Periodicals; Poissons -- Immunologie -- Périodiques; Crustacés -- Immunologie -- Périodiques; 571.9617
• DOI: 10.1016/j.fsi.2022.08.021
• Languages: English
• ISSNs: 1050-4648

Abstract

Abstract: Invertebrates only have an innate immunity in which haemocytes play an important role. In our lab, 5 subpopulations of haemocytes were identified in the past by an iodixanol density gradient: hyalinocytes, granulocytes, semi-granulocytes and two subpopulations of non-phagocytic cells. For the two latter subpopulations, the haemocytes have small cytoplasm rims, do not adhere to the bottom of plastic cell-culture grade wells and present folds in the nucleus. These characteristics are similar to those of mammalian lymphocytes. Therefore, they were designated lymphocyte-like haemocytes. Although little is known about their function, we hypothesize, based on their morphology, that they may have a cytotoxic activity. First, a fast isolation technique was developed to separate the non-adherent haemocytes from the adherent haemocytes. After 60 min incubation on cell culture plates, the non-adherent haemocytes were collected. The purity reached 93% as demonstrated by flow cytometry and light microscopy upon a Hematoxylin and Eosin (H&E) staining. Cytotoxicity by lymphocytes is mediated by molecules such as perforin and granzymes and therefore, we searched for their genes in the shrimp genome. Genes coding for a torso-like protein, granzyme B and granzyme G were identified. Primers were designed and RT-PCR/RT-qPCR assays were developed. The results demonstrated that torso-like protein, granzyme B and granzyme G were mainly expressed in non-adherent haemocytes. The shrimp torso-like protein gene was most related to that of the crab torso-like protein; granzyme B gene was most related to that of mouse granzyme B and granzyme G gene was most related to that of zebrafish granzyme G. In a 72-hour in vivo WSSV infection challenge, the mRNA expression of shrimp torso-like protein, granzyme B and granzyme G in haemocytes was increasing over time, which indicated that torso-like protein, granzyme B and granzyme G of shrimp haemocytes are involved in the immune response during a viral infection. In the future, antibodies will be raised against these proteins for more in-depth functional analyses. Highlights: A novel technique was developed to isolate shrimp non-adherent and adherent haemocytes in a fast and efficient way. Shrimp haemocytes, mainly non-adherent haemocytes, express the cytotoxic molecules torso-like protein, granzymes B and G. During WSSV infection mRNA expression of torso-like protein, granzymes B and G in shrimp haemocytes increased significantly.