An in vitro method for the analysis of hemocyte-derived extracellular traps in shrimp

The formation of extracellular traps (ETs) is a cell death mechanism relying on the release of nucleic acids in response to different stimuli. More recently, ETs have been recognized as an important cellular immune response since they are able to entrap and kill various microorganisms. The main goal was to describe a methodology to induce and visualize the in vitro formation of ETs by shrimp hemocytes. ETs formation was induced by the incubation of hemocyte monolayers from naïve shrimp (Penaeus vannamei) with a standard dose of Vibrio parahaemolyticus M0905. Following fixation, slides were stained with 4′,6-diamidino-2-phenylindole (DAPI) and imaged by fluorescence microscopy. The methodology proposed in this study successfully induced the formation and release of hemocyte-derived ETs in penaeid shrimp. The procedure described here can be used as a novel immune marker to assess shrimp health status.


a b s t r a c t
The formation of extracellular traps (ETs) is a cell death mechanism relying on the release of nucleic acids in response to different stimuli. More recently, ETs have been recognized as an important cellular immune response since they are able to entrap and kill various microorganisms. The main goal was to describe a methodology to induce and visualize the in vitro formation of ETs by shrimp hemocytes. ETs formation was induced by the incubation of hemocyte monolayers from naïve shrimp ( Penaeus vannamei ) with a standard dose of Vibrio parahaemolyticus M0905. Following fixation, slides were stained with 4 ′ ,6-diamidino-2-phenylindole (DAPI) and imaged by fluorescence microscopy. The methodology proposed in this study successfully induced the formation and release of hemocyte-derived ETs in penaeid shrimp. The procedure described here can be used as a novel immune marker to assess shrimp health status. We describe here a facile and low-cost procedure to induce the in vitro formation of ETs by shrimp hemocytes. In contact with a standard dose of the Gram-negative Vibrio parahaemolyticus M0905, circulating hemocytes are able to produce and release non-condensed DNA fibers that can be easily visualized by 4 ′ ,6-diamidino-2-phenylindole (DAPI) staining using a fluorescent microscope. Ethics:

Specifications
The shrimp for all the experimental work were used in accordance with the protocols of the Official Mexican Standard (NOM-062-ZOO-1999). Value of the Protocol: • A simple and reliable tool that requires few equipment resources to evaluate in short time (3 h) the formation and release of hemocyte-derived ETs in penaeid shrimp. • Compared to other methods for assessing shrimp health status, this low-cost procedure only requires DAPI staining.
• Shrimp do not need to be sacrificed for the evaluation of this cellular immune parameter.
Trial registration (if applicable):

Description of protocol
Infectious diseases caused by viruses and pathogenic bacteria are undoubtedly the main factors that limit the shrimp farming industry worldwide. To defend themselves from invading microorganisms, penaeid shrimp rely on the combination of cellular and humoral responses mediated mainly by hemocytes, the circulating immunocompetent cells [1] . Microbial recognition by pattern recognition receptors leads to hemocyte mediated reactions, such as phagocytosis, nodule and capsule formation, tissue infiltration and the production of reactive oxygen species (ROS) and antimicrobial host defense peptides (AMPs) [2] . Additionally, a new cellular immune reaction, named extracellular traps (ETs), was also described in crustaceans, in which DNA networks associated to histones are released by hemocytes to entrap and kill bacteria [ 3 , 4 ].
The formation and release of ETs is a conserved antimicrobial process present from protozoa to multicellular organisms, including plants and both vertebrates and invertebrates [5] . Actually, ETs is a cell death mechanism dependent on the generation of ROS by NADPH oxidase [6] , a phenomenon that was originally described in mammalian neutrophils [7] . ETs are mainly composed of chromatin-carrying histones associated with granular antimicrobial proteins and can be induced by either microbes (bacteria, fungi, parasites and viruses) or microbe-associated molecular patterns [8] . In penaeid shrimp, the extracellular DNA fibers released by circulating hemocytes are associated with the bactericidal c-type lysozyme protein and showed to be induced by phorbol myristate acetate (PMA), lipopolysaccharide (LPS) and peptidoglycan (PGN) as well as by live bacteria [ 3 , 9 ].
We describe here a facile and low-cost procedure to induce the in vitro formation of ETs by shrimp hemocytes. In contact with a standard dose of the Gram-negative Vibrio parahaemolyticus M0905, circulating hemocytes are able to produce and release noncondensed DNA fibers that can be easily visualized by 4 ′ ,6-diamidino-2-phenylindole (DAPI) staining using a fluorescent microscope. We propose the use of this technique as an additional cellular immune parameter in aquaculture to assess shrimp health status in different rearing or experimental conditions.

Assay procedure
Preparation of bacterial inoculum 1. Inoculate a bacterial colony in 10 mL of Tryptic Soy Broth (TSB) supplemented with 2% of NaCl (TSB + Bioxon) in a 15 mL culture tube and shake at 30 °C for 24 h. In this study, we used the Vibrio parahaemolyticu s strain M0904 causing shrimp Acute Hepatopancreatic Necrosis Disease (AHPND), which was isolated from a shrimp farm affect by AHPND in northwestern Mexico. 4. Mix hemolymph samples by gently pipetting up-and-down in order to prevent cell clumping. 5. Transfer 10 μL of the hemolymph sample onto a hemocytometer. 6. Using a light microscope, count and record the number of hemocytes in each one of the four outer squares of the hemocytometer (Neubauer chamber). 7. Each square in a hemocytometer with a coverslip placed represents 10 − 4 cm 3 and 1 mL is equivalent to 1 cm 3 , thus the number of hemocytes/mL of hemolymph = average number of cells in all four squares x 2 dilution factor (1 MAS: 1 hemolymph) x 10 4 . 8. Adjust the dilution with MAS to 1 × 10 6 cells/mL.

Monolayers preparation and bacterial stimulation
1. For hemocyte monolayer preparation, pipette 100 μL of the hemocyte suspension containing 1 × 10 6 cells/mL on a conventional uncoated glass slide and spread the cells uniformly using a pipette tip.

Method validation
We described here a methodology to induce the in vitro formation and release of ETs by the circulating hemocytes of penaeid shrimp ( P. vannamei ). The formation of ETs is an evolutionarily conserved cellular immune response relying on the release of nucleic acids in response to different stimuli [5] . In penaeid shrimp, the formation and release of DNA fibers by hemocytes (the immune cells) showed to be induced by microbial-associated molecular patterns (LPS, PGN) and by a non-pathogenic bacterium for crustaceans, Escherichia coli [ 3 , 9 ]. Here, we successfully induced the in vitro formation of hemocyte-derived ETs using the shrimp pathogen V. parahaemolyticus M0905. During the first hour of stimulation (100 CFU: 1 hemocyte), cells formed highly decondensed chromatin structures revealed by DAPI staining (Figs. 1 and S1). After 2 and 3 h, hemocyte-derived ETs showed the form of flexible fibers and meshes (Figs. 1 and S1). Besides, the released ETs were able to entrap the Vibrio cells within their fibers ( Fig. 1 ).
Hemocyte-derived ETs appear to be a previous step for nodule and capsule formation. In this process, different hemocyte subpopulations would take part [ 4 , 10 ]. It has been pointed out that hyaline and semi-granular cells release chromatin [4] , and suffer lysis and nuclear alterations [10] . In this context, hyaline hemocytes could start the ET process due to its ability to undergo respiratory bursts by producing ROS [4] , while the presence of melanin in the capsules indicates that there is also the degranulation of granular hemocytes [11] . Additionally to melanin, this cellular immune response showed to be also associated with the c-type lysozyme [9] , a bactericidal protein involved in shrimp immunity [12] . Since the c-type lysozyme is produced by granule-containing hemocytes [ 11 , 13 ], it is likely that those cell subsets are also involved in the formation and release of ETs.
This easy-to-use protocol can find applications in aquaculture from the perspectives of assessing shrimp health status. Indeed, the analysis of cellular (total and differential hemocyte counts, hemocyte phagocytic activity, anion superoxide production, percentage of apoptotic hemocytes) and humoral (total serum protein concentration, coagulation-time of the hemolymph, phenoloxidase activity, serum agglutination titer, serum antimicrobial activity) immune parameters and gene expression analysis has been extensively used to assess the immunocompetence of farmed species in different conditions [14][15][16] . For instance, the ability of circulating hemocytes to trigger this cellular response can be compared between animals submitted to an experimental condition ( e.g. , biotic and/or abiotic stressors) and to a control condition [ 15 , 16 ]. In this case, the percentage of hemocyte-derived ETs can be calculated based on the count of a minimum of 100 DAPI-stained nuclei. Hemocyte monolayers incubated with 2% NaCl can be used as technical control ( Fig. 1 ). From another point of view, many immunostimulants are offered in the aquaculture market, and their ability to induce ETs can be used as an extra parameter for evaluation of their possible in vitro efficacy, before performing in vivo tests. In conclusion, the evaluation of hemocyte-derived ETs emerges as a new immune marker associated to shrimp cellular immune defenses.

Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability
Data will be made available on request.