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
