Chemotaxis Assay for Marsupenaeus japonicas Hemocytes and Application for the Development of an Oral Immunostimulant Against White Spot Syndrome Virus

Abstract

The chemotactic activity of vertebrate leukocytes is an important host-defense mechanism. However, chemotaxis of invertebrate immune cells, particularly those of shrimp species, is incompletely understood and critically understudied. In this study, we aimed to optimize the conditions for a Boyden chamber chemotaxis assay using hemocytes (granulocytes) from cultured kuruma shrimp, Marsupenaeus japonicas (Mj) and the optimal conditions were: 5 µm-pore-size Polyvinylpyrrolidone membrane; culture buffer at pH 7.0; and chemotactic factor N-formyl-methionyl-leucylphenylalanine (fMLP) 10 −8 mol/L; 4 h incubation time. We then applied the chemotaxis assay to develop an oral immunostimulant against white spot syndrome virus (WSSV), which results in high mortality rates in several shrimp species worldwide. We focused on the kelp Laminaria japonica, as this species contains immunostimulative molecules such as β-glucan. We prepared Heat Extracts (HE) and Crude Laminarans (CL) from kelp using hot water and hydrochloric acid extraction methods, respectively. HE and CL ware mixed with normal shrimp feed. Kelp extracts were orally administered for 7 days, and hematocyte chemotaxis toward fMLP was compared. No difference was detected between control and kelp extracts on day 3, but HE stimulated chemotaxis 2-fold and CL stimulated chemotaxis 3-fold relative to control on day 7 after initiating administration. Kelp extract administration protected against WSSV exposure. Finally, we identified that Kelp extracts stimulated hematocyte superoxide production on days 3 and 7, and increased hematocyte phagocytosis and phenol oxidase activity on day 7 after administration. We concluded that the chemotaxis assay is informative in assessment of shrimp hemocyte immunological activity, and is applicable to the development of immunostimulants against shrimp infectious diseases.

Brown Seaweed Food Supplementation: Effects on Allergy and Inflammation and Its Consequences

“…A study using a phylogenetically more distinct organism, kuruma shrimp, indicated that oral supplementation with Laminaria japonica significantly increased survival upon White Spot Syndrome virus infection [ 32 ]. This was accompanied by enhancement of chemotaxis as well as other defense mechanisms by hemolymph leukocytes (hemocytes), including superoxide production and antioxidative phenoloxidase activity upon appropriate stimulation.…”

“…effects) [ 30 ] GFS = hot water extract with galactofucan sulfate therapy: 4 × 560 mg/day, 10 days, 1–24 mo. maintenance: 2 × 560 mg/day Undaria pinnatifida human; response in patients with active or latent Herpes infection 2 15/15 patients with active disease: ↓ symptoms or full clearance of infection no side effects inhibition of relapse in pts with latent disease [ 31 ] hot water extract, and HCl-EtOH extract 100 mg/kg/day (heat extract), 10 mg/kg/day (HCl-EtOH extract), for 3–7 days Laminaria japonica kuruma shrimp ( Marsupenaeus japonicas ) in vivo WSSV infection in vitro hemocyte analysis 1,2 ↑ survival upon WSSV infectionhemocyte fMet-Leu-Phe stimulation: ↑ chemotaxis ↑ superoxide production ↑ phenol oxidase activity ↑ phagocytosis [ 32 ] hot water-EtOH extract 20–200 mg/kg; 7 weeks Hizikia. = Sargassum fusiforme male SD rat; ligature-induced periodontitis 3 ↓ alveolar bone loss due to inflammation [ 33 ] hot water extract of 10 different herbs incl.…”

Expression profile of heat shock protein 70 in lymphoid organs of Penaeus monodon in response to white spot syndrome virus infection

“…Although hyperthermia protects shrimp from viruses, maintaining warmer water in farming systems is not practical and economical. Oral administration of β‐glucan from the kelp species Laminaria japonica to Marsupenaeus japonicus improved their survival upon challenge with WSSV as compared to the control shrimp, suggesting the efficacy of kelp extracts as an immunostimulant for shrimp against WSSV (Imai & Takahashi, 2020). All these studies demonstrate the usefulness of immunostimulating the animals to boost the innate immune system to protect against viral infection.…”