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Role of non-phagocytic, lymphocyte-like haemocytes in the antiviral and antibacterial defence of shrimp

Periodic Reporting for period 1 - ShrimpLLH (Role of non-phagocytic, lymphocyte-like haemocytes in the antiviral and antibacterial defence of shrimp)

Reporting period: 2020-10-01 to 2022-09-30

The general goal of ShrimpLLH project is to find better ways to combat infectious diseases in shrimp. One of the most important aims is to stimulate shrimp haemocytes in a direct way to enhance their immunological functions. However, to do so, the immunological role of the different haemocytes in shrimp immunity should be clarified. Therefore, in ShrimpLLH project, it is paramount to investigate the immunological function of the different haemocyte subpopulations in depth. This will be a valuable finding towards the development and screening of novel antiviral and antibacterial therapies in shrimp aquaculture. Also, the project will give rise to novel markers that can be used in shrimp research (quantification of the specific haemocyte populations and localization) to study the hostpathogen interaction.

As the haemocyte cells of the two new subpopulations do not show phagocytosis and have a large nucleus surrounded by only a small rim of cytoplasm, they resemble lymphocytes in mammals. Therefore, these cells could be called lymphocyte-like haemocytes (LLHs). The project has three main targets to address the research questions: (i) To isolate LLH. As a first goal, the novel in-house technique will be developed to separate the LLHs in a faster and more efficient manner. This will largely improve LLH viability. (ii) To characterise and subdivide the LLHs. To do so, monoclonal antibodies will be produced, and additional antibody-based separation techniques will be developed. With the use of monoclonal antibodies, LLHspecific
plasma membrane proteins will be identified and characterised. With new separation techniques, I will subdivide LLHs into new subpopulations. (iii) To analyse the immunological functions of haemocyte subpopulations. This will allow us to investigate the third research goal: to identify the LLH immunological functions in-vitro towards viruses and (pathogenic) bacteria such as Vibrio campbellii and Vibrio harveyi. ShrimpLLH project will fill the knowledge gap in shrimp immunity, by investigating the immunological role of the haemocyte subpopulations.
Fast isolation of non-adherent, non-phagocytic, lymphocyte-like haemocytes
Throughout the ShrimpLLH project, Specific pathogen-free (SPF) penaeid shrimp (Litopenaeus vannamei) were procurred from IMAQUA with a mean body weight of 10g. Haemolymph was collected from the ventral sinus of the second abdominal segment with a 1ml syringe and a 24G needle filled with marine anticoagulant (haemolymph: marine anticoagulant =1:2). Haemolymph was poured into 1.5 ml eppendorf microcentrifuge tubes and centrifuged on a table top (which company centrifuge?) at 250×g for 5min at 4oC. Later the supernatant was discarded and the pellet was resuspended in a haemocyte medium. Freshly collected haemocytes suspensions in medium were seeded into 5ml petri dish, incubated at 27 oC for 1h in order to allow the cell attachment. Further, agentle swirling the dish and the suspended cells were reseed into a new dish, and add another 1ml medium into the old dish to culture the adherent haemocytes. Throughout this process the cultures were observed by inverted microscope (specify microscope company and country). Flow cytometer for cell size and granularity and H and E staining was used to determine the cell populations.

Characterization of lymphocyte-like haemocytes
Because shrimp only possesses an innate immunity, we believe that LLHs might act like Natural Killer Cells (NKCs) in vertebrates. Therefore, at first we identified granzymes and torso-like proteins from shrimp genome database from NCBI. Bioinformatic analysis such as domain, length of the gene both mRNA and genomic DNA, molecular weight, alignment, coding region, ORF identification, number of introns and exons. Expression of Granzymes and Perforin in adherent and non-adherent cells by qPCR, calculation was set up. Subsequently, we determined the highly immunogenic peptides from the granzymes and torso like protein from shrimp. The production of monoclonal antibody for these molecules is under process and collaborating with DENOVO BIOLABS PVT LTD, India. Ones the antibody are available it will help us to study the immunological functions of LLH and also to study the LLH function towards virus-infected-cells, this will investigate wheather the LLHs will be able to recognize virus-infected-cells.
Identified new TLR6 and TLR13 from the genome of P. vannamei, Characterize their protein domain, phylogeny. Designed new primers to check their expression in healthy tissues and infected tissues.

Immunological functions of lymphocyte-like haemocytes

Main results
After 1h incubation at 27 oC, two different morphology and behaviour of haemocytes were observed under light microscope. One type is strongly adhered to the plate by pseudopod-like projections, which resulted in a high cell spreading, these cells were designated as adherent haemocytes. Another type of cell presented a very limited spreading and adherence, they are suspend in the supernatant, these cells were designated as non-adherent haemocytes (Fig. 1).
The H&E staining of haemocyte cytospins showed the morphological details of non-adherent haemocytes and adherent haemocytes. The average cell diameter of adherent haemocytes was bigger than non-adherent haemocytes, but the nucleus: cytoplasm ratio of non-adherent haemocytes was higher than adherent haemocytes (Fig. 1).
The forward scatter area (FSC-A) and side scatter area (SSC-A) values of flow cytometer were related with cell diameter and granularity. The result indicated that there are two populations of total haemocytes, by separating the non-adherent and adherent haemocytes can be known, the adherent haemocytes had a bigger size and higher granularity, whereas the non-adherent haemocytes were in a smaller size and lower granularity
ShrimpLLH project which is a based on understanding shellfish immunology has pushed forward in a numerous ways. The 2 special focus issues the fellow spearheaded have shed new light onto Shrimp immunology are fast isolation of haemocytes for immunological studies and their characterization.
The innovative technique for LLH isolation, shrimp TLR and the transcriptome database of adherent and non-adherent conducted by the Fellow are helping popularize the understanding of shrimp immunology. The potential publication in the journals, and the participation in several workshops and skills events by the Fellow will help cultivate a broader human capacity to produce quality research in the field of aquaculture. ShrimpLLH allowed the Fellow to develop research agility with many different research methodologies and promote best practices to the larger aquaculture community (e.g. in education and identifying how to report it for maximum impact).
ShrimpLLH allowed Fellow to guide PhD student, who is generating important new knowledge about Natural killer like cell processes and its importance in shrimp immunity. This will describe the new knowledge in shellfish aquaculture.
Impacts anticipated from the MSCA are increased and improved: Fellow has obtained faculty job and will be involved in teaching Fish and Shellfish Immunology to masters and PhD students. The experience and knowledge obtained from ShrimpLLH will be largely shared to the students. Importantly, a final overarching impact is enhanced public perception of shrimp immunity and shellfish immunology as a fun and innovative field to research on.
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