Morphological and functional characterization of the hemocytes from the pearl oyster Pteria hirundo and their immune responses against Vibrio infections

Highlights

• Three hemocyte types were identified: hyalinocytes, granulocytes and blast-like cells.

• Hemocytes showed phagocytic capacity and were able to produce reactive oxygen species.

• Specific Vibrio agglutination were increased in hemolymph after cognate challenge.

Abstract

Hemocyte populations of the pearl oyster Pteria hirundo were characterized at morphological, ultrastructural and functional levels. Three main hemocyte populations were identified: hyalinocytes, granulocytes and blast-like cells. Hyalinocytes were the most abundant population (88.2%) characterized by the presence of few or no granules in the cytoplasm and composed by two subpopulations, large and small hyalinocytes. Comparatively, granulocytes represented 2.2% of the hemocyte population and were characterized by the presence of numerous large electron-lucid granules in the cytoplasm. Finally, the blast-like cells (9.5%) were the smallest hemocytes, showing spherical shape and a high nucleus/cytoplasm ratio. Hemocytes exhibited a significant phagocytic capacity for inert particles (38.5%) and showed to be able to produce microbicidal molecules, such as reactive oxygen species (ROS) (ex vivo assays). The immune role of hemocytes was further investigated in the P. hirundo defense against the Gram-negative Vibrio alginolyticus. A significant decrease in the total number of hemocytes was observed at 24 h following injection of V. alginolyticus or sterile seawater (injury control) when compared to naïve (unchallenged) animals, indicating the migration of circulating hemocytes to the sites of infection and tissue damage. Bacterial agglutination was only observed against Gram-negative bacteria (Vibrio) but not against to marine Gram-positive-bacteria. Besides, an increase in the agglutination titer was observed against V. alginolyticus only in animals previously infected with this same bacterial strain. These results suggest that agglutinins or lectin-like molecules may have been produced in response to this particular microorganism promoting a specific recognition. The ultrastructural and functional characterization of P. hirundo hemocytes constitutes a new important piece of the molluscan immunity puzzle that can also contribute for the improvement of bivalve production sustainability.

Introduction

The pearl oyster Pteria hirundo has a natural occurrence on the Brazilian coast with a great potential to aquaculture due to its gastronomic appeal and to its capacity to produce pearls [1]. Despite of its potential commercial importance, little is known about the physiology and immune system of this bivalve species. The impact of infectious diseases on aquaculture production has required efforts on culture management, as well as on the improvement of knowledge on immune responses triggered by animals during infections. Nowadays, eight infectious diseases impacting mollusks are listed by the Office International des Epizooties (OIE, the World Organization for Animal Health), comprising five protistan infections, two viral infections and one bacterial infection. To note, bacterial pathogens from genus Vibrio can have a relevant impact on bivalve culture infecting larvae, juveniles and adults from oysters, mussels, abalones, clams, and scallops (see review of [2]).

In bivalves, hemocytes represent the immunocompetent cells related to the hemolymph [3]. These cells participate in different defense responses against pathogens, including chemotaxis, phagolysosomal activity, encapsulation/nodulation, production and/or release of a wide range of antimicrobial compounds, antiviral defense, apoptosis and expression of several pattern-recognition receptors/proteins [4], [5]. In addition to the hemolymph-mediated reactions, bivalves account with immune effectors produced by epithelial cells from various organs (gills, mantle, digestive gland and intestine), which participate in local antimicrobial defenses (see review of [6]. In addition, recent reports have shown that invertebrates, including mollusks, can trigger complex and specific immune responses against pathogens, suggesting the existence of an innate immune memory or priming in these animals [7], [8], [9].

Bivalve hemocytes comprise heterogeneous cell populations and their identification and characterization are often contradictory and depends on the techniques used. In order to identify the hemocyte types, different approaches have been used to access their morphology, cytochemistry and immunological function [10], [11], [12], [13], [14], [15], [16], [17], [18], [19]. According to techniques, hemocytes of different species of bivalves have been classified into two [20], three or more morphotypes [21]. However, recent reports agree on the existence of three main hemocyte types, named granulocytes, hyalinocytes and blast-like cells [14], [15], [17], [22]. Granulocytes are the most abundant cell type found in bivalve hemolymph that contain many cytoplasmic granules rich in different immuneffector molecules [23], whereas few or no cytoplasmic granules are characteristic of hyalinocytes. Usually, hyalinocytes are smaller and less abundant than granulocytes. Blast-like cells are undifferentiated and scarce in the hemolymph, presenting a high nucleus/cytoplasm ratio.

To date, hemocytes from pearl oysters were identified only in two species, Pinctada fucata [18], [24] and P. imbricata [25], and although three types were shown to be present in both species, authors classified the blast-like cells as small agranulocytes or small hyalinocytes. However, there are no reports to date on immunological mechanisms triggered by pearl oysters during microbial infections. In order to achieve this unexplored aim, the present study focused on the characterization of the hemocyte populations of the pearl oyster P. hirundo and on some cellular and humoral immune responses triggered by a Vibrio pathogen. Ours results bring new insights on pathogen-host interaction occurring in bivalves.