Morphology and activities of cell populations of haemocytes in Oncomelania hupensis following Schistosoma japonicum infection.

Oncomelania hupensis is the only obligatory intermediate host of Schistosoma japonicum, the pathogen of zoonosis schistosomiasis. Haemocytes play a critical role in the cellular immune defence of O. hupensis against S. japonicum challenge. Here, the morphology and classification of haemocytes of O. hupensis were investigated by Giemsa staining and light microscopy, combining with the scanning and transmission electron microscopy and flow cytometry. Granulocytes and hyalinocytes were confirmed as two main types of haemocytes, account for ~ 10% and ~ 90% of all haemocytes, with size varying in 4.3-10.9 µm and 0.4-30.8 µm, respectively. Subpopulations can be identified further by granule feature, shape, size, and surface and inner structure of cells. The heterogeneity in morphology implied varied developmental process and function of haemocyte subpopulations. After the S. japonicum challenge, haemocytes of O. hupensis respond to S. japonicum invasion immediately. The dynamic change of haemocyte subpopulations indicates that the small hyalinocyte could differentiate into a larger one or granulocyte after S. japonicum challenge, and the granulocytes and larger hyalinocytes play leading roles in early defence reaction, but in different ways. Phagocytosis and apoptosis of haemocytes in O. hupensis were proved to be related to immune defence against S. japonicum, with the combined effect of granulocytes and larger hyalinocytes. However, the main pathway of each subpopulation to take effect in different periods need further investigation.

Functional shell matrix proteins tentatively identified by asymmetric snail shell morphology.

Molluscan shell matrix proteins (SMPs) are essential in biomineralization. Here, we identify potentially important SMPs by exploiting the asymmetric shell growth in snail, Lymnaea stagnalis. Asymmetric shells require bilaterally asymmetric expression of SMP genes. We examined expression levels of 35,951 transcripts expressed in the left and right sides of mantle tissue of the pond snail, Lymnaea stagnalis. This transcriptome dataset was used to identify 207 SMPs by LC-MS/MS. 32 of the 207 SMP genes show asymmetric expression patterns, which were further verified for 4 of the 32 SMPs using quantitative PCR analysis. Among asymmetrically expressed SMPs in dextral snails, those that are more highly expressed on the left side than the right side are 3 times more abundant than those that are more highly expressed on the right than the left, suggesting potentially inhibitory roles of SMPs in shell formation. The 32 SMPs thus identified have distinctive features, such as conserved domains and low complexity regions, which may be essential in biomineralization.

Roles of 5-HT on phase transition of neurite outgrowth in the identified serotoninergic neuron C1, Helisoma trivolvis.

Neurite outgrowth is a morphological marker of neuronal differentiation and neuroregeneration, and the process includes four essential phases, namely initiation, elongation, guidance and cessation. Intrinsic and extrinsic signaling molecules seem to involve morphological changes of neurite outgrowth via various cellular signaling cascades phase transition. Although mechanisms associated with neurite outgrowth have been studied extensively, little is known about how phase transition is regulated during neurite outgrowth. 5-HT has long been studied with regard to its relationship to neurite outgrowth in invertebrate and vertebrate culture systems, and many studies have suggested 5-HT inhibits neurite elongation and growth cone motility, in particular, at the growing parts of neurite such as growth cones and filopodia. However, the underlying mechanisms need to be investigated. In this study, we investigated roles of 5-HT on neurite outgrowth using single serotonergic neurons C1 isolated from Helisoma trivolvis. We observed that 5-HT delayed phase transitions from initiation to elongation of neurite outgrowth. This study for the first time demonstrated that 5-HT has a critical role in phase-controlling mechanisms of neurite outgrowth in neuronal cell cultures.

Cryptic diversity: Two morphologically similar species of invasive apple snail in Peninsular Malaysia.

Invasive snails in the genus Pomacea have spread across Southeast Asia including Peninsular Malaysia. Their effects on natural and agricultural wetlands are appreciable, but species-specific effects are less clear because of morphological similarity among the species. Our objective was to establish diagnostic characteristics of Pomacea species in Malaysia using genetic and morphological criteria. The mitochondrial COI gene of 52 adult snails from eight localities in Peninsular Malaysia was amplified, sequenced, and analysed to verify species and phylogenetic relationships. Shells were compared using geometric morphometric and covariance analyses. Two monophyletic taxa, P. canaliculata and P. maculata, occurred in our samples. The mean ratio of shell height: aperture height (P = 0.042) and shell height: shell width (P = 0.007) was smaller in P. maculata. P. maculata co-occurred with P. canaliculata in five localities, but samples from three localities contained only P. canaliculata. This study is the first to confirm the presence of two of the most invasive species of Pomacea in Peninsular Malaysia using a molecular technique. P. canaliculata appears to be the more widespread species. Despite statistical differences, both quantitative and qualitative morphological characteristics demonstrated much interspecific overlap and intraspecific variability; thus, shell morphology alone cannot reliably verify species identity. Molecular techniques for distinguishing between these two highly invasive Pomacea species are needed to understand their specific ecological niches and to develop effective protocols for their management.

The structure of the cutaneous pedal glands in the banded snail Cepaea hortensis (Müller, 1774).

Although gastropods have been crawling through the ocean and on the land for 60 million years, we still know very little about the sticky mucus produced in their foot. Most research has been focused on marine species in particular and, to a lesser extent, on the well-known terrestrial species Arion vulgaris and Cornu aspersum. Within this study, we aim to characterize the foot anatomy of a smaller representative of the family Helicidae, the banded snail Cepaea hortensis. We are particularly interested in the microanatomy of the foot glands, their position, and the histochemical nature of their secretory content. Characterization of the dorsal foot region of Cepaea hortensis reveals four glands, differing in their size and in the granules produced. Histochemically, three of them react positively for sugars (PAS staining and lectin affinity tests for mannose, glucose and N-acetyl-d-glucosamine) and acidic proteins (positive Alcian blue and Toluidine blue staining), indicating the presence of acidic glycosaminoglycans. The fourth gland type does not react to any of these dyes. The ventral pedal region includes two different gland types, which are positive for the presence of acidic glycoproteins, with a lectin affinity for mannose only. A comparison with Helix pomatia indicates differences regarding the number of glands and their contents. In Helix, only three gland types are described in the dorsal region of the foot, which show a similar granular appearance but nevertheless differ in their chemical composition. Congruently, there are two gland types in the ventral region in both species, whereas in Helix an additional sugar moiety is found. This raises the question whether these differences between the pedal glandular systems of both helicid species are the result of protection or size-related adaptations, as they occur in the same habitat.

Morphogenesis along the animal-vegetal axis: fates of primary quartet micromere daughters in the gastropod Crepidula fornicata.

BACKGROUND: The Spiralia are a large, morphologically diverse group of protostomes (e.g. molluscs, annelids, nemerteans) that share a homologous mode of early development called spiral cleavage. One of the most highly-conserved features of spiralian development is the contribution of the primary quartet cells, 1a-1d, to the anterior region of the embryo (including the brain, eyes, and the anterior ciliary band, called the prototroch). Yet, very few studies have analyzed the ultimate fates of primary quartet sub-lineages, or examined the morphogenetic events that take place in the anterior region of the embryo. RESULTS: This study focuses on the caenogastropod slipper snail, Crepidula fornicata, a model for molluscan developmental biology. Through direct lineage tracing of primary quartet daughter cells, and examination of these cells during gastrulation and organogenesis stages, we uncovered behaviors never described before in a spiralian. For the first time, we show that the 1a2-1d2 cells do not contribute to the prototroch (as they do in other species) and are ultimately lost before hatching. During gastrulation and anterior-posterior axial elongation stages, these cells cleavage-arrest and spread dramatically, contributing to a thin provisional epidermis on the dorsal side of the embryo. This spreading is coupled with the displacement of the animal pole, and other pretrochal cells, closer to the ventrally-positioned mouth, and the vegetal pole. CONCLUSIONS: This is the first study to document the behavior and fate of primary quartet sub-lineages among molluscs. We speculate that the function of 1a2-1d2 cells (in addition to two cells derived from 1d12, and the 2b lineage) is to serve as a provisional epithelium that allows for anterior displacement of the other progeny of the primary quartet towards the anterior-ventral side of the embryo. These data support a new and novel mechanism for axial bending, distinct from canonical models in which axial bending is suggested to be driven primarily by differential proliferation of posterior dorsal cells. These data suggest also that examining sub-lineages in other spiralians will reveal greater variation than previously assumed.

Lifelong neurogenesis in the cerebral ganglion of the Chinese mud snail, Cipangopaludina chinensis.

INTRODUCTION: A small group of Gastropods possessing giant neurons have long been used to study a wide variety of fundamental neurophysiological phenomena. However, the majority of gastropods do not have large neurons but instead have large numbers of small neurons and remain largely unstudied. We explored neuron size and rate of increase in neuron numbers in the Chinese mud snail, Cipangopaludina chinensis. METHODS: Using histological sections and whole mounts of the cerebral ganglia, we collected cross-sectional data on neuron number and size across the lifespan of this animal. Neurogenesis was verified using Click-it EdU staining. RESULTS: We found that total neuron number in the cerebral ganglia increases throughout the lifespan of this species at a constant rate. New neurons arise primarily near the nerve roots. Females live longer (up to 7 years) than males (up to 5 years) and thus achieve larger numbers of neurons in the cerebral ganglion. Neuron size is consistently small (<10 µm) in the cerebral ganglia at all ages, however, cells in the posterior section of the cerebral ganglia are modestly but significantly larger than cells at the anterior. CONCLUSIONS: These features suggest that C. chinensis and similar species of Caenogastropoda are good candidates for studying gastropod neurogenesis, senescence, and sex differences in the nervous system.

Gastropod-derived haemocyte extracellular traps entrap metastrongyloid larval stages of Angiostrongylus vasorum, Aelurostrongylus abstrusus and Troglostrongylus brevior.

BACKGROUND: Phagocyte-derived extracellular traps (ETs) were recently demonstrated mainly in vertebrate hosts as an important effector mechanism against invading parasites. In the present study we aimed to characterize gastropod-derived invertebrate extracellular phagocyte trap (InEPT) formation in response to larval stages of important canine and feline metastrongyloid lungworms. Gastropod haemocytes were isolated from the slug species Arion lusitanicus and Limax maximus, and the snail Achatina fulica, and exposed to larval stages of Angiostrongylus vasorum, Aelurostrongylus abstrusus and Troglostrongylus brevior and investigated for gastropod-derived InEPT formation. RESULTS: Phase contrast as well as scanning electron microscopy (SEM) analyses of lungworm larvae-exposed haemocytes revealed ET-like structures to be extruded by haemocytes thereby contacting and ensnaring the parasites. Co-localization studies of haemocyte-derived extracellular DNA with histones and myeloperoxidase in larvae-entrapping structures confirmed classical characteristics of ETs. In vivo exposure of slugs to A. vasorum larvae resulted in InEPTs being extruded from haemocytes in the slug mucous extrapallial space emphasizing the pivotal role of this effector mechanism against invasive larvae. Functional larval entrapment assays demonstrated that almost half of the haemocyte-exposed larvae were contacted or even immobilized by released InEPTs. Overall, as reported for mammalian-derived ETs, different types of InEPTs were here observed, i.e. aggregated, spread and diffused InEPTs. CONCLUSIONS: To our knowledge, this study represents the first report on metastrongyloid lungworm-triggered ETosis in gastropods thereby providing evidence of early mollusc host innate immune reactions against invading larvae. These findings will contribute to the better understanding on complex parasite-intermediate host interactions since different gastropod species bear different transmitting capacities for metastrongyloid infections.

Nerve-granular cell communication in the atrium of the snail Achatina achatina occurs via the cardioexcitatory transmitters serotonin and FMRFamide.

In the present study, the anatomical association and functional interaction between nerve fibres and granular cells in the atrium of the snail Achatina achatina are investigated using a combination of scanning electron microscopy (SEM), pharmacological and immunofluorescence techniques. The SEM studies support a close anatomical association of axons with granular cells and new features of surface morphology are revealed. Pharmacological experiments showed that both serotonin and FMRFamide were able to induce degranulation of granular cells and the release of cysteine-rich atrial secretory protein. Serotonin- and FMRFamide-immunoreactive nerve fibres were observed at variable distances from granular cells, ranging from close contact to distances as far as the diameter of a muscle bundle. These results suggest that serotonin and FMRFamide play a role as paracrine excitatory transmitters in nerve-to-granular cell communication.

Breeding system, shell size and age at sexual maturity affect sperm length in stylommatophoran gastropods.

BACKGROUND: Sperm size and quality are key factors for fertilization success. There is increasing empirical evidence demonstrating that sperm form and function are influenced by selective pressures. Theoretical models predict that sperm competition could favour the evolution of longer sperm. In hermaphrodites, self-fertilizing species are expected to have shorter sperm than cross-fertilizing species, which use sperm stored from several mating partners for the fertilization of their eggs and thus are exposed to intense sperm competition. We tested this hypothesis by comparing original data on sperm length in 57 species of simultaneously hermaphroditic stylommatophoran gastropods from Europe and South America with respect to the species' breeding system. We used 28S rRNA nuclear and COI mitochondrial sequence data to construct a molecular phylogeny. Phylogenetic generalized linear models were applied to examine the potential influence of morphological and life-history characters. RESULTS: The best-fit model revealed that the breeding system and age at sexual maturity influence sperm length in gastropods. In general, species with predominant cross-fertilization had longer sperm than species with predominant self-fertilization or a mixed breeding system. Across species with shells (snails), sperm length also increased with shell size. CONCLUSIONS: Our study provides evidence that sperm length in stylommatophoran gastropods is influenced by the risk of sperm competition, as well as by age at sexual maturity and shell size. This finding extends present knowledge of sperm evolution to a group of so far poorly studied simultaneous hermaphrodites.

Endogenous growth factor stimulation of hemocyte proliferation induces resistance to Schistosoma mansoni challenge in the snail host.

Digenean trematodes are a large, complex group of parasitic flatworms that infect an incredible diversity of organisms, including humans. Larval development of most digeneans takes place within a snail (Gastropoda). Compatibility between snails and digeneans is often very specific, such that suitable snail hosts define the geographical ranges of diseases caused by these worms. The immune cells (hemocytes) of a snail are sentinels that act as a crucial barrier to infection by larval digeneans. Hemocytes coordinate a robust and specific immunological response, participating directly in parasite killing by encapsulating and clearing the infection. Hemocyte proliferation and differentiation are influenced by unknown digenean-specific exogenous factors. However, we know nothing about the endogenous control of hemocyte development in any gastropod model. Here, we identify and functionally characterize a progranulin [Biomphalaria glabrata granulin (BgGRN)] from the snail B. glabrata, a natural host for the human blood fluke Schistosoma mansoni Granulins are growth factors that drive proliferation of immune cells in organisms, spanning the animal kingdom. We demonstrate that BgGRN induces proliferation of B. glabrata hemocytes, and specifically drives the production of an adherent hemocyte subset that participates centrally in the anti-digenean defense response. Additionally, we demonstrate that susceptible B. glabrata snails can be made resistant to infection with S. mansoni by first inducing hemocyte proliferation with BgGRN. This marks the functional characterization of an endogenous growth factor of a gastropod mollusc, and provides direct evidence of gain of resistance in a snail-digenean infection model using a defined factor to induce snail resistance to infection.

[Individual Growth of the Great Ramshorn Snail Planorbarius corneus (Gastropoda, Planorbidae) Embryos].

Individual growth of the great ramshorn snail Planorbarius corneus has been studied by intravital video imaging. As has been observed, the types of growth change over the embryogenesis. The linear dimensions slightly but in a statistically significant manner decrease during the stages of cleavage to blastula. Starting from the stage of blastula to trochophore, the embryo diameter remains constant to commence increasing at the stage of middle trochophore. During the larval stages (trochophore and veliger), the growth is synchronous (in Dettlaffs, biological time units) for the embryos in both the same clutch and different clutches. The growth at that time is exponential but later desynchronizes in individual clutches. The embryos in eight clutches grew and developed slower and hatched later as compared with the remaining five egg clutches. An accelerated growth follows an asymptomatic pattern according to the von Bertalanffy equation. A retarded growth is describable with a linear equation. The observed differences are likely to be associated with the number of embryos in a clutch. All types of changes in the linear dimensions observed in the great ramshorn snail embryogenesis can be described with the same united equation.

Anti-oxidative cellular protection effect of fasting-induced autophagy as a mechanism for hormesis.

The aim of this investigation was to test the hypothesis that fasting-induced augmented lysosomal autophagic turnover of cellular proteins and organelles will reduce potentially harmful lipofuscin (age-pigment) formation in cells by more effectively removing oxidatively damaged proteins. An animal model (marine snail--common periwinkle, Littorina littorea) was used to experimentally test this hypothesis. Snails were deprived of algal food for 7 days to induce an augmented autophagic response in their hepatopancreatic digestive cells (hepatocyte analogues). This treatment resulted in a 25% reduction in the cellular content of lipofuscin in the digestive cells of the fasting animals in comparison with snails fed ad libitum on green alga (Ulva lactuca). Similar findings have previously been observed in the digestive cells of marine mussels subjected to copper-induced oxidative stress. Additional measurements showed that fasting significantly increased cellular health based on lysosomal membrane stability, and reduced lipid peroxidation and lysosomal/cellular triglyceride. These findings support the hypothesis that fasting-induced augmented autophagic turnover of cellular proteins has an anti-oxidative cytoprotective effect by more effectively removing damaged proteins, resulting in a reduction in the formation of potentially harmful proteinaceous aggregates such as lipofuscin. The inference from this study is that autophagy is important in mediating hormesis. An increase was demonstrated in physiological complexity with fasting, using graph theory in a directed cell physiology network (digraph) model to integrate the various biomarkers. This was commensurate with increased health status, and supportive of the hormesis hypothesis. The potential role of enhanced autophagic lysosomal removal of damaged proteins in the evolutionary acquisition of stress tolerance in intertidal molluscs is discussed and parallels are drawn with the growing evidence for the involvement of autophagy in hormesis and anti-ageing processes.

[The Anatomical Method of Isolating Central Ganglia from Oncomelania hupensis].

In this experiment the soft tissue of Oncomelania hupensis was obtained by breaking the shell with a hemostat. The central ganglia of 0. hupensis were then collected from the fresh soft tissue under a dissecting microscope. This method lays a base for studying the effects of molluscicides or various biological and physicochemical factors on the central ganglia of 0. hupensis.

Development of blastomere clones in the Ilyanassa embryo: transformation of the spiralian blastula into the larval body plan.

Spiralian embryogenesis is deeply conserved and seems to have been in place in the last common ancestor of the large assemblage of protostome phyla known as the Lophotrochozoa. While the blastula fate maps of several spiralian embryos have been determined, little is known about the events that link the early embryo and the larva. For all cells in the Ilyanassa blastula, we determined the clonal morphology at four time points between the blastula and veliger stages. We found that ectomesoderm comes mostly from 3a and 3b, but also from 2c and 2b. We also observed the ingression and early proliferation of 3a- and 3b-derived ectomesoderm. We found cells in the 2b clone that marked the anterior edge of the blastopore and later the mouth and cells in the 3c/3d clones that marked the posterior edges of these structures. This demonstrates directly that the mouth forms in the same location as the blastopore. In the development of the shell field, we observed dramatic cell migration events that invert the positions of the 2b and 2d clones that contribute to the shell. Using time-lapse imaging, we followed and described the cleavage pattern of the conserved endomesodermal blast cell, 4d, up to 4d + 45 h, when there were 52 cells in the clone. Our results show the growth and movement of clones derived from cells of the spiralian blastula as they transform into the trochophore-like and veliger stages. They have implications for the evolution of the shell in gastropods, the origins of mesoderm in spiralians, and the evolution of mouth formation in metazoans.

Pedal sole immunoreactive axons in terrestrial pulmonates: Limax, Arion, and Helix.

A century ago histological techniques such as formic acid-gold chloride showed the nerve morphology of the pedal sole in Limax and Helix. There have been no similar descriptions since then of the central nervous system relevant to locomotory pedal waves in the foot of slugs and snails. Topical application of 5-HT affects locomotory waves, but the innervation of the pedal sole with 5-HT axons is not known. Three-dimensional morphology of pedal axons in terrestrial pulmonate embryos is shown herein with modern histological techniques using antibodies and the confocal microscope. In Limax maximus, pedal ganglia are shown with Tritonia pedal peptide (TPep) antibodies. Ladder-like cross bridges in the pedal sole are shown with antibodies to both TPep and 5-HT. In Arion ater, pedal ganglia neurons and their axons that form a plexus in the pedal sole are shown with 5-HT antibodies. In Helix aspersa, 5-HT immunoreactive pedal ganglia neurons and a developing pedal sole axon plexus are seen as in A. ater. Axons in this plexus that grow across the pedal sole can be seen growing into pre-existing nerves. No peripheral 5-HT neurons were identified in these three species. This immunoreactive plexus to 5-HT antibodies in A. ater and H. aspersa spreads over the pedal sole epithelium. Axons immunoreactive to 5-HT antibodies in A. ater and H. aspersa extend the length of the foot, primarily in the rim, so that activity in these axons cannot provide local patterned input to produce locomotory waves, but may provide modulatory input to pedal sole muscles.

Effects of repeated hemolymph withdrawals on the hemocyte populations and hematopoiesis in Pomacea canaliculata.

Pomacea canaliculata is a freshwater gastropod considered an invasive pest by several European, North American and Asiatic countries. This snail presents a considerable resistance to pollutants and may successfully face stressful events. Thanks to the unusual possibility to perform several hemolymph collections without affecting its survival, P. canaliculata is a good model to study the hematopoietic process and the hemocyte turnover in molluscs. Here we have analyzed the effects of repeated hemolymph withdrawals on circulating hemocyte populations and pericardial organs, i.e., the heart, the main vessels entering and leaving the heart and the ampulla, of P. canaliculata. Our experiments revealed that the circulating hemocyte populations were maintained constant after 3 collections performed in 48 h. The tissue organization of the heart and the vessels remained unaltered, whereas the ampulla buffered the effects of hemolymph collections acting as hemocyte reservoir, and its original organization was progressively lost by the repeated hemolymph withdrawals. The hematopoietic tissue of P. canaliculata was evidenced here for the first time. It is positioned within the pericardial cavity, in correspondence of the principle veins. Mitoses within the hematopoietic tissue were not influenced by hemolymph collections, and circulating hemocytes never presented mitotic activity.

The process of granule exocytosis in non-stimulated atrial granular cells of the snail, Achatina achatina: an ultrastructural, histochemical and immunocytochemical study.

Abundant secretory granular cells (GCs) in the Giant African land snail atrium harbor a range of bioactive substances and undergo rapid total degranulation in response to stimulation of the cardiac nerve or stressful influences. Here we have analyzed exocytotic events in the non-stimulated GCs. It was shown that the GCs contain three major distinct types of granules that differ histochemically, immunocytochemically and ultrastructurally, each performing specific functions. The type I granules characteristically filled with electron-lucent homogeneous materials exhibit intense immunoreactivity for bioactive proteins and therefore are considered to be storage granules. Histochemistry using vital staining with Acridine Orange and Gomori acid phosphatase technique has revealed lysosomal-related nature of the electron-dense type II granules. Digestion remnants appearing as fine filamentous materials fill the type III granules. Only the type III granules fuse together and with the plasma membrane form degranulation channels and surface pores, through which the debris is removed from the cell. The finding of granules exhibiting intermediate ultrastructural, histochemical and immunocytochemical features suggests that the major granule types represent most stable states along a granule empting continuum. Thus, under physiological conditions, the GCs continuously produce secretory proteins and so maintain readiness for stress-response, but use protein degradation machinery to prevent massive release of these bioactive substances into hemolymph.

[Aqueous and salt solutions of quinine of low concentrations: self-organization, physicochemical properties and actions on the electrical characteristics of neurons].

Self-organization, the physicochemical properties of aqueous and salt solutions of quinine and the effects of salt quinine solutions in a wide range of concentrations (1 x 10(-22) - 1 x 10(-3) M) on the electrical characteristics of the edible snail's identified neurons were studied. Similar non-monotonic concentration dependencies of physicochemical properties of aqueous and salt quinine solutions at low concentrations are obtained. This allows of predicting the occurrence of biological effects at low concentrations of quinine solutions. Intrinsic (within 5% of the interval) changes in membrane potential, the amplitude and duration of the neuron action potential under the influence of quinine salt solutions at concentrations of quinine of 1 x 10(-20), 1 x 10(-18), 1 x 10(-10) M are found. For these concentrations the extreme values of specific conductivity and pH are shown.

Comparative analysis of circulating hemocytes of the freshwater snail Pomacea canaliculata.

Molluscs are invertebrates of great relevance for economy, environment and public health. The numerous studies on molluscan immunity and physiology registered an impressive variability of circulating hemocytes. This study is focused on the first characterization of the circulating hemocytes of the freshwater gastropod Pomacea canaliculata, a model for several eco-toxicological and parasitological researches. Flow cytometry analysis identified two populations of hemocytes on the basis of differences in size and internal organization. The first population contains small and agranular cells. The second one displays major size and a more articulated internal organization. Light microscopy evidenced two principal morphologies, categorized as Group I (small) and II (large) hemocytes. Group I hemocytes present the characteristics of blast-like cells, with an agranular and basophilic cytoplasm. Group I hemocytes can adhere onto a glass surface but seem unable to phagocytize heat-inactivated Escherichia coli. The majority of Group II hemocytes displays an agranular cytoplasm, while a minority presents numerous granules. Agranular cytoplasm may be basophilic or acidophilic. Granules are positive to neutral red staining and therefore acidic. Independently from their morphology, Group II hemocytes are able to adhere and to engulf heat-inactivated E. coli. Transmission electron microscopy analysis clearly distinguished between agranular and granular hemocytes and highlighted the electron dense content of the granules. After hemolymph collection, time-course analysis indicated that the Group II hemocytes are subjected to an evident dynamism with changes in the percentage of agranular and granular hemocytes. The ability of circulating hemocytes to quickly modify their morphology and stainability suggests that P. canaliculata is endowed with highly dynamic hemocyte populations able to cope with rapid environmental changes as well as fast growing pathogens.