Histological and three dimensional organizations of lymphoid tubules in normal lymphoid organ of Penaeus monodon

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Abstract

The normal lymphoid organ of Penaeus monodon (which tested negative for WSSV and YHV) was composed of two parts: lymphoid tubules and interstitial spaces, which were permeated with haemal sinuses filled with large numbers of haemocytes. There were three permanent types of cells present in the wall of lymphoid tubules: endothelial, stromal and capsular cells. Haemocytes penetrated the endothelium of the lymphoid tubule's wall to reside among the fixed cells. The outermost layer of the lymphoid tubule was covered by a network of fibers embedded in a PAS-positive extracellular matrix, which corresponded to a basket-like network that covered all the lymphoid tubules as visualized by a scanning electron microscope (SEM). Argyrophilic reticular fibers surrounded haemal sinuses and lymphoid tubules. Together they formed the scaffold that supported the lymphoid tubule. Using vascular cast and SEM, the three dimensional structure of the subgastric artery that supplies each lobe of the lymphoid organ was reconstructed. This artery branched into highly convoluted and blind-ending terminal capillaries, each forming the lumen of a lymphoid tubule around which haemocytes and other cells aggregated to form a cuff-like wall. Stromal cells which form part of the tubular scaffold were immunostained for vimentin. Examination of the whole-mounted lymphoid organ, immunostained for vimentin, by confocal microscopy exhibited the highly branching and convoluted lymphoid tubules matching the pattern of the vascular cast observed in SEM.

Introduction

The lymphoid or Oka organ was first described in Penaeus orientalis by Oka [1], and later in Sicyonia ingentis by Martin et al. [2] who incorrectly described it as haematopoietic nodules. The lymphoid organ of penaeid shrimp consists of two lobes situated ventro-anterior to the hepatopancreas [3]. Each lobe receives the haemolymph from the anterior aorta via the subgastric artery [3], [4], which is further branched several times in each lobe of the lymphoid organ. At the light microscope level, the lymphoid organ is composed of two parts: lymphoid tubules and the spaces in between which are occupied by haemal sinuses. Lymphoid tubules consist of the central lumen lined by flattened endothelial cells. Next to the endothelial lining are two types of stromal cells. Cells in the inner layer have a clear, unstained cytoplasm, whereas those in the outer layer have a more darkly stained cytoplasm with H and E staining [4]. It is believed that haemocytes penetrate the endothelial cells into the space between stromal cells, where they settle and later migrate into the haemal sinuses which form part of the open circulatory system [3]. Apart from cellular constituents, there is a connective tissue scaffold that provides support for the lymphoid tubules. In penaeid shrimp, there is no detailed description of the fibrous scaffold of the lymphoid organ. Furthermore, the three dimensional structure of lymphoid tubules and their relationship with the branching subgastric artery have never been demonstrated. In this study the normal lymphoid organ of Penaeus monodon, which tested negative for white spot syndrome virus (WSSV) and yellow head virus (YHV), was studied by light microscopy using special staining and transmission electron microscopy in order to demonstrate the histological organization, cellular components, and the connective tissue scaffold, and by a vascular cast technique and SEM to visualize the three dimensional structure of the tubules.

Section snippets

Shrimps

Wild caught fully mature P. monodon were obtained from the Gulf of Thailand. The shrimps were confirmed to be WSSV-negative by PCR and YHV-negative by RT-PCR using the IQ 2000 detection kit (Farming IntelliGene Technology Corporation, Taipei, Taiwan).

Tissue preparation for light microscopic observation

Dissected lymphoid organs were fixed in Davidson's fixative, dehydrated through ascending concentrations of ethanol, cleared with dioxane, and embedded in paraffin wax. Four to six micron thick sections were cut and stained with hematoxylin–eosin

Gross anatomy of lymphoid organ

The lymphoid organs were paired and lay ventral to the stomach and dorso-anterior to the ventral hepatopancreas (Fig. 1A). Each lymphoid organ had an ovoid shape and was connected to the heart via the subgastric artery. In the fully mature shrimp (with the body length of 18–20 cm from head to telson, and body weight of 150–170 g), lymphoid organs were about 3–4 mm long, 2–3 mm wide, and 1–2 mm thick.

General histology of lymphoid organ

Each lymphoid organ was surrounded by a connective tissue capsule (Fig. 1B). The capsule invaginated

Discussion

The structural organizations of lymphoid organs of penaeid shrimp have been studied by many authors, i.e., in P. orientalis by Oka [1], in S. ingentis by Martin et al. [2], and in Penaeus stylirostris by Bell and Lightner [4]. These authors described the lymphoid organ as being made up of lymphoid tubules which are the basic unit. It is believed that this organ plays a very important role against infection especially by viruses, as it is essentially a filtering organ that clears the haemolymph

Acknowledgements

This research was supported by the Commission on Higher Education (PhD. Scholarship to P. Duangsuwan and Research Group Grant to P. Sobhon) and TRF (Senior Research Scholar Fellowship to P. Sobhon).

References (18)

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