Image3C, a multimodal image-based and label-independent integrative method for single-cell analysis.

Image-based cell classification has become a common tool to identify phenotypic changes in cell populations. However, this methodology is limited to organisms possessing well-characterized species-specific reagents (e.g., antibodies) that allow cell identification, clustering, and convolutional neural network (CNN) training. In the absence of such reagents, the power of image-based classification has remained mostly off-limits to many research organisms. We have developed an image-based classification methodology we named Image3C (Image-Cytometry Cell Classification) that does not require species-specific reagents nor pre-existing knowledge about the sample. Image3C combines image-based flow cytometry with an unbiased, high-throughput cell clustering pipeline and CNN integration. Image3C exploits intrinsic cellular features and non-species-specific dyes to perform de novo cell composition analysis and detect changes between different conditions. Therefore, Image3C expands the use of image-based analyses of cell population composition to research organisms in which detailed cellular phenotypes are unknown or for which species-specific reagents are not available.

Cells are the building blocks of all living organisms. They come in many types, each with a different role. Understanding the composition of cells, i.e., how many cells and which types of cells are present inside an organ can indicate what that organ does. It can also reveal how that organ changes under different conditions, like during an infection or treatment. The most powerful methods for studying cells work well for species researchers already know a lot about, such as mice, zebrafish or humans, but not for less studied animals. To change this Accorsi, Box, Peuß et al. created a new tool called Image3C to be used for studying the composition of cells in less researched organisms. Instead of using reagents that only work for specific species, the tool uses molecules that work across many species, like dyes that stain the cell nucleus. A cell-sorting machine, known as a flow cytometer, connected to a microscope then takes pictures of hundreds of stained cells each second and Image3C groups them based on their appearance, without the need for any prior knowledge about the cell types. Accorsi et al. then tested Image3C on immune system cells of zebrafish, a well-studied animal, and apple snails, an under-studied animal. For both species, the tool was able to sort cells into groups representing different parts of the immune system. Image3C speeds up the grouping process and reduces the need for user intervention and time. This lowers the risk of bias compared to manual counting of cells. It can sort cells even when the types of cells in an organism are unknown and even when specialized reagents for an organism do not exist. This means that it could characterise the cell make-up of new tissues coming from organisms never studied before. Access to this uncharted world of cells stands to reveal previously inaccessible clues about how organs behave and evolve and allow researchers to investigate the impact of environmental changes on these cells.

Signatures of Divergence, Invasiveness, and Terrestrialization Revealed by Four Apple Snail Genomes.

The family Ampullariidae includes both aquatic and amphibious apple snails. They are an emerging model for evolutionary studies due to the high diversity, ancient history, and wide geographical distribution. Insight into drivers of ampullariid evolution is hampered, however, by the lack of genomic resources. Here, we report the genomes of four ampullariids spanning the Old World (Lanistes nyassanus) and New World (Pomacea canaliculata, P. maculata, and Marisa cornuarietis) clades. The ampullariid genomes have conserved ancient bilaterial karyotype features and a novel Hox gene cluster rearrangement, making them valuable in comparative genomic studies. They have expanded gene families related to environmental sensing and cellulose digestion, which may have facilitated some ampullarids to become notorious invasive pests. In the amphibious Pomacea, novel acquisition of an egg neurotoxin and a protein for making the calcareous eggshell may have been key adaptations enabling their transition from underwater to terrestrial egg deposition.

Toward the Molecular Deciphering of Pomacea canaliculata Immunity: First Proteomic Analysis of Circulating Hemocytes.

Pomacea canaliculata is a freshwater snail with interesting biological features that include invasiveness, human parasite hosting, and adult regeneration. Its immune system may represent the target for strategies aimed at controlling the spread of the snail population and its hosting of the human parasite Angiostrongylus cantonensis. Moreover, immune functions likely have a role in the snail's ability to wound heal and regenerate. Despite its importance in multiple processes, very little is known about the molecular basis of P. canaliculata immunity. Aiming to contribute to filling this gap, the ultrastructure of circulating hemocytes in healthy snails is studied and the first proteomic analysis of these cells is performed, evidencing 83 unique proteins, 96% of which have identifiable homologs in other species. Fifteen proteins are retrieved as potentially involved in immune-related signaling pathways, such as hemocyanin, C1q-like protein, and HSP90 together with cytoskeleton and cytoskeleton-related proteins involved in cell motility and membrane dynamics. This first proteome study on non-stimulated hemocytes provides a valid reference for future investigations on the molecular changes under stressful circumstances, like pathogen exposure, wounding, or environmental changes.

An adaptable chromosome preparation methodology for use in invertebrate research organisms.

BACKGROUND: The ability to efficiently visualize and manipulate chromosomes is fundamental to understanding the genome architecture of organisms. Conventional chromosome preparation protocols developed for mammalian cells and those relying on species-specific conditions are not suitable for many invertebrates. Hence, a simple and inexpensive chromosome preparation protocol, adaptable to multiple invertebrate species, is needed. RESULTS: We optimized a chromosome preparation protocol and applied it to several planarian species (phylum Platyhelminthes), the freshwater apple snail Pomacea canaliculata (phylum Mollusca), and the starlet sea anemone Nematostella vectensis (phylum Cnidaria). We demonstrated that both mitotically active adult tissues and embryos can be used as sources of metaphase chromosomes, expanding the potential use of this technique to invertebrates lacking cell lines and/or with limited access to the complete life cycle. Simple hypotonic treatment with deionized water was sufficient for karyotyping; growing cells in culture was not necessary. The obtained karyotypes allowed the identification of differences in ploidy and chromosome architecture among otherwise morphologically indistinguishable organisms, as in the case of a mixed population of planarians collected in the wild. Furthermore, we showed that in all tested organisms representing three different phyla this protocol could be effectively coupled with downstream applications, such as chromosome fluorescent in situ hybridization. CONCLUSIONS: Our simple and inexpensive chromosome preparation protocol can be readily adapted to new invertebrate research organisms to accelerate the discovery of novel genomic patterns across the branches of the tree of life.

A prokineticin-like protein responds to immune challenges in the gastropod pest Pomacea canaliculata.

The golden apple snail Pomacea canaliculata is an invasive pest originating from South America. It has already been found in Asia, the southern United States and more recently in the EU. Aiming to target the immune system of the snail as a way to control its spreading, we have developed organ-specific transcriptomes and looked for molecules controlling replication and differentiation of snail hemocytes. The prokineticin domain-containing protein Astakine 1 is the only cytokine known thus far capable of regulating invertebrate hematopoiesis, and we analyzed the transcriptomes looking for molecules containing a prokineticin domain. We have identified a prokineticin-like protein (PlP), that we called Pc-plp and we analyzed by real-time PCR (qPCR) its expression. In control snails, highest levels of Pc-plp were detected in the digestive gland, the ampulla (i.e., a hemocyte reservoir) and the pericardial fluid (i.e., the hematopoietic district). We tested Pc-plp expression after triggering hematopoiesis via multiple hemolymph withdrawals, or during bacterial challenge through LPS injection. In both cases a reduction of Pc-plp mRNA was observed. The multiple hemolymph withdrawals caused a significant decrease of Pc-plp mRNA in pericardial fluid and circulating hemocytes, while the LPS injection promoted the Pc-plp mRNA drop in anterior kidney, mantle and gills, organs that may act as immune barrier in molluscs. Our data indicate an important role for prokineticin domain-containing proteins as immunomodulators also in gastropods and their dynamic expression may serve as a biosensor to gauge the effectiveness of immunological interventions aimed at curtailing the spreading of the gastropod pest P. canaliculata.

Molluscs as models for translational medicine.

This paper describes the advantages of adopting a molluscan model for studying the biological basis of some central nervous system pathologies affecting humans. In particular, we will focus on the freshwater snail Lymnaea stagnalis, which is already the subject of electrophysiological studies related to learning and memory, as well as ecotoxicological studies. The genome of L. stagnalis has been sequenced and annotated but the gene characterization has not yet been performed. We consider the characterization of the gene networks that play crucial roles in development and functioning of the central nervous system in L. stagnalis, an important scientific development that comparative biologists should pursue. This important effort would add a new experimental model to the limited number of invertebrates already used in studies of translational medicine, the discipline that seeks to improve human health by taking advantage of knowledge collected at the molecular and cellular levels in non-human organisms.

Microglia of medicinal leech (Hirudo medicinalis) express a specific activation marker homologous to vertebrate ionized calcium-binding adapter molecule 1 (Iba1/alias aif-1).

The Ionized calcium-Binding Adapter molecule 1 (Iba1), also known as Allograft Inflammatory Factor 1 (AIF-1), is a 17 kDa cytokine-inducible protein, produced by activated macrophages during chronic transplant rejection and inflammatory reactions in Vertebrates. In mammalian central nervous system (CNS), Iba1 is a sensitive marker associated with activated macrophages/microglia and is upregulated following neuronal death or brain lesions. The medicinal leech Hirudo medicinalis is able to regenerate its CNS after injury, leading to a complete functional repair. Similar to Vertebrates, leech neuroinflammatory processes are linked to microglia activation and recruitment at the lesion site. We identified a gene, named Hmiba1, coding a 17.8 kDa protein showing high similarity with Vertebrate AIF-1. The present work constitutes the first report on an Iba1 protein in the nervous system of an invertebrate. Immunochemistry and gene expression analyses showed that HmIba1, like its mammalian counterpart, is modulated in leech CNS by mechanical injury or chemical stimuli (ATP). We presently demonstrate that most of leech microglial cells migrating and accumulating at the lesion site specifically expressed the activation marker HmIba1. While the functional role of Iba1, whatever species, is still unclear in reactive microglia, this molecule appeared as a good selective marker of activated cells in leech and presents an interesting tool to investigate the functions of these cells during nerve repair events.

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.

Epigenetic modification in neurons of the mollusc Pomacea canaliculata after immune challenge.

In human and rodents, the transcriptional response of neurons to stress is related to epigenetic modifications of both DNA and histone proteins. To assess the suitability of simple invertebrate models in studying the basic mechanisms of stress-related epigenetic modifications, we analyzed epigenetic modifications in neurons of the freshwater snail Pomacea canaliculata after the injection of Escherichia coli-derived lipopolysaccharide (LPS). The phospho-acetylation of histone H3, together with the induction of stress-related factors, c-Fos and HSP70, were evaluated in large and small neurons of the pedal ganglia of sham- and LPS-injected snails. Immunocytochemical investigations showed that after LPS injection, the immunopositivity towards phospho (Ser10)-acetyl (Lys14)-histone H3 and c-Fos increases in the nuclei of small gangliar neurons. Western blot analysis confirmed a significant increase of phospho (Ser10)-acetyl (Lys14)-histone H3 in nuclear extracts from 2h LPS-injected animals. c-Fos protein levels were significantly augmented 6h after LPS injection. Immunocytochemistry and western blot indicated that no changes occurred in HSP70 distribution and protein levels. To our knowledge this is the first demonstration of epigenetic changes in molluscan neurons after an immune challenge and indicate the gastropod P. canaliculata as a suitable model for evolutionary and translational studies on stress-related epigenetic modifications.

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.

Drosophila Helical factor is an inducible protein acting as an immune-regulated cytokine in S2 cells.

The innate immunity of Drosophila melanogaster is based on cellular and humoral components. Drosophila Helical factor (Hf), is a molecule previously discovered using an in silico approach and whose expression is controlled by the immune deficiency (Imd) pathway. Here we present evidence demonstrating that Hf is an inducible protein constitutively produced by the S2 hemocyte-derived cell line. Hf expression is stimulated by bacterial extracts that specifically trigger the Imd pathway. In absence of any bacterial challenge, the recombinant form of Hf can influence the expression of the antimicrobial peptides (AMPs) defensin but not drosomycin. These data suggest that in vitro Hf is an inducible and immune-regulated factor, with functions comparable to those of secreted vertebrate cytokines.

The evolution of pro-opiomelanocortin: looking for the invertebrate fingerprints.

The presence and role of the pro-opiomelanocortin (POMC) gene and encoded peptides in invertebrates are here summarized and discussed. Some of the POMC-derived peptides show a significant similarity regarding their functions, suggesting their appearance before the split of protostomian-deuterostomian lineages and their maintenance during evolution. The basic mechanisms that govern the exchange of information between cells are usually well conserved, and this could have also been for POMC-derived peptides, that are mainly involved in fundamental functions such as immune and neuroendocrine responses. However, the presence and functions that POMC-derived peptides exhibit in taxonomically distant models, are not always reflected by the expected gene homology, leaving the problem of POMC evolution in invertebrates in need of additional study.