CONICET | Buscador de Institutos y Recursos Humanos

Tropical diseases caused by gastropod-borne parasites affect millions of people worldwide and cause the loss of billions of dollars in developing countries every year. Gastropods are ideal hosts for helminth parasites, as trematodes and nematodes, because of their high adaptability to diverse ecosystems. However, little is known of the gastropod?helminth relationship and some authors have pointed to this issue [1]. In this context, the immune system of the apple snail Pomacea canaliculata has become the focus of international research over the past few years [2-7, e.g., 8] because of this species? role as an intermediate host of the parasitic nematode Angiostrongylus cantonensis, the primary aetiologic agent of eosinophilic meningitis [9]. This disease is often disabling and may even be lethal to humans [10]. While P. canaliculata, a native of the lower basin of Río de la Plata, has been invading many countries of South East Asia [11], A. cantonensis has spread throughout Brazil [12], i.e., it is on the verge to overlap the native range of P. canaliculata, which would facilitate the spread of the parasite. Over the past few years, we have been engaged in the understanding of the very many intriguing aspects of apple snail immunobiology. In particular, this project has been aimed at characterising the internal defence system of P. canaliculata, using both in vitro and in vivo approaches. Specifically, our studies have concerned the identification and the morphological/functional characterisation of (1) the immunocompetent cells (i.e., the haemocytes), (2) the organs that act as immune barriers, and (3) the haematopoietic cells and tissues.Our studies have shown that P. canaliculata has three distinct circulating haemocyte populations, namely hyalinocytes, agranulocytes and granulocytes (Figure 1). Hyalinocytes are the main phagocytes, although the other types also show some extent of phagocytic ability. Haemocytes have two acidic granule types (LysoTracker Red labelling) enabling them, to antigen destruction, as has been shown for other gastropods [13]. A notable feature of P. canaliculata?s haemocytes is their ability to form spheroidal aggregates in aseptic cultures (Figure 1). This intrinsic ability to form spheroids parallels that of many mammalian cancer cells, thus this evolutionary conserved behaviour may be significant in a much wider context.Since haemocyte?antigen encounters may occur into the systemic blood circulation we hypothesised that there are organs acting as immune barriers, whose characteristics facilitated those encounters. Such characteristics would be: (1) an adequate position of the organ in blood circulation, so as to prevent antigen dissemination, and/or (2) an intricate microcirculation, which would increase the likelihood of haemocyte-antigen contacts, and/or (3) a locally high haemocyte concentration. Here, we showed that both the kidney and the lung have suitable positions in the circulation and an intricate architecture of their blood spaces where the reactions of nodulation and proliferation are elicited in them after immune challenges (Figure 2), Using the 3D rendering of blood vessels we showed that blood flows in the kidney through a vascular bed in which haemocyte islets lie interposed. In addition, haemocyte nodules formed after the systemic injection of yeast cells. Moreover, the number of proliferating haemocytes increased 48 h after injection (BrdU incorporation). Nodules showed signs of regression (pigment deposition, apoptotic bodies) and a decrease in the number of dividing cells 96 h after injection. The lung also showed nodules 96 h after injection. The complexity of the vascular chambers that form the respiratory lamina in the lung would facilitate haemocyte?antigen contacts, which are required to provoke cellular aggregation, and hence, nodulation.We firstly chose a non-pathogenic microorganism (i.e. baker?s yeast) to observe the mere reaction to foreign cells, but in a preliminary study, Mycobacterium marinum-injected animals showed hypertrophy of the renal islets and nodules within them, and extensive loss of the renal epithelium. The gill showed numerous granulocytes and multinucleate cells infiltrating the gill epithelium, thickening of the underlying connective tissue, and obliteration of the blood spaces [14]. These reactions (hypertrophy, nodulation, multinucleate cell formation) are analogous to those of tuberculous granulomas [e.g., 15, 16], but lacking lymphocytes. P. canaliculata lacks an acquired immunity, but can develop nodules in response to immune challenges. In this context, zebrafish has been used as a model for tuberculosis, but only during a temporal window of development, when the adaptive system has not been formed yet [17]. Therefore, a potential development of a model for the experimental infection with M. marinum, in an animal that lacks an adaptive system, may be of high significance.Finally, we have explored the occurrence of haematopietic cells and tissues in this gastropod. It should be noted that haematopoiesis sensu lato involves two distinct processes, namely the production of haemocytes and the synthesis of the respiratory pigment (haemocyanin). Both in vitro and in vivo studies showed that progenitor cells occurred in the circulation of this species (Figure 3) and that may account for the haematopoietic response after metabolic impairment (5-fluorouracil administration). Regarding the second aspect of haematopoiesis, clusters of rhogocytes ? the haemocyanin-producing cells ? were found in the lung, thus suggesting this organ may also have a role in haematopoiesis. In addition, multimeric molecules of haemocyanin in the blood sinuses were found surrounding the renal haemocyte islets. Beyond the role of haemocyanin in oxygen transport, this notably large protein [7.6 MDa; 18] and its derived peptides are known to serve various roles in innate immunity [19]. Molluscan haemocyanins are known to stimulate the immune system of mammals when inoculated, eliciting strong cellular reactions and generating high levels of antibodies; these large proteins can also shift the immune response to a Th1 phenotype [20]. This has practical benefits; for example, purified haemocyanin from the gastropod Megathura crenulata is widely used in clinical trials of treatments for cancer and allergy [21].The recent development of -omics technologies and the growing available data for P. canaliculata [e.g., 22, 23-25] would prove helpful to definitely stablish this species as a model organism in which comparative and translational studies may be encouraged.