Two distinct evolutionary conserved neural degeneration pathways characterized in a colonial chordate.

Colonial tunicates are marine organisms that possess multiple brains simultaneously during their colonial phase. While the cyclical processes of neurogenesis and neurodegeneration characterizing their life cycle have been documented previously, the cellular and molecular changes associated with such processes and their relationship with variation in brain morphology and individual (zooid) behavior throughout adult life remains unknown. Here, we introduce Botryllus schlosseri as an invertebrate model for neurogenesis, neural degeneration, and evolutionary neuroscience. Our analysis reveals that during the weekly colony budding (i.e., asexual reproduction), prior to programmed cell death and removal by phagocytes, decreases in the number of neurons in the adult brain are associated with reduced behavioral response and significant change in the expression of 73 mammalian homologous genes associated with neurodegenerative disease. Similarly, when comparing young colonies (1 to 2 y of age) to those reared in a laboratory for ∼20 y, we found that older colonies contained significantly fewer neurons and exhibited reduced behavioral response alongside changes in the expression of 148 such genes (35 of which were differentially expressed across both timescales). The existence of two distinct yet apparently related neurodegenerative pathways represents a novel platform to study the gene products governing the relationship between aging, neural regeneration and degeneration, and loss of nervous system function. Indeed, as a member of an evolutionary clade considered to be a sister group of vertebrates, this organism may be a fundamental resource in understanding how evolution has shaped these processes across phylogeny and obtaining mechanistic insight.

Sixty years of experimental studies on the blastogenesis of the colonial tunicate Botryllus schlosseri.

In the second half of the eighteenth century, Schlosser and Ellis described the colonial ascidian Botryllus schlosseri garnering the interest of scientists around the world. In the 1950's scientists began to study B. schlosseri and soon recognized it as an important model organism for the study of developmental biology and comparative immunology. In this review, we summarize the history of B. schlosseri studies and experiments performed to characterize the colony life cycle and bud development. We describe experiments performed to analyze variations in bud productivity, zooid growth and bilateral asymmetry (i.e., the situs viscerum), and discuss zooid and bud removal experiments that were used to study the cross-talk between consecutive blastogenetic generations and vascular budding. We also summarize experiments that demonstrated that the ability of two distinct colonies to fuse or reject is controlled by a single polymorphic gene locus (BHF) with multiple, codominantly expressed alleles. Finally, we describe how the ability to fuse and create chimeras was used to show that within a chimera somatic and germline stem cells compete to populate niches and regenerate tissue or germline organs. Starting from the results of these 60 years of study, we can now use new technological advances to expand the study of B. schlosseri traits and understand functional relationships between its genome and life history phenotypes.

Transcriptome dynamics in the asexual cycle of the chordate Botryllus schlosseri.

BACKGROUND: We performed an analysis of the transcriptome during the blastogenesis of the chordate Botryllus schlosseri, focusing in particular on genes involved in cell death by apoptosis. The tunicate B. schlosseri is an ascidian forming colonies characterized by the coexistence of three blastogenetic generations: filter-feeding adults, buds on adults, and budlets on buds. Cyclically, adult tissues undergo apoptosis and are progressively resorbed and replaced by their buds originated by asexual reproduction. This is a feature of colonial tunicates, the only known chordates that can reproduce asexually. RESULTS: Thanks to a newly developed web-based platform ( http://botryllus.cribi.unipd.it ), we compared the transcriptomes of the mid-cycle, the pre-take-over, and the take-over phases of the colonial blastogenetic cycle. The platform is equipped with programs for comparative analysis and allows to select the statistical stringency. We enriched the genome annotation with 11,337 new genes; 581 transcripts were resolved as complete open reading frames, translated in silico into amino acid sequences and then aligned onto the non-redundant sequence database. Significant differentially expressed genes were classified within the gene ontology categories. Among them, we recognized genes involved in apoptosis activation, de-activation, and regulation. CONCLUSIONS: With the current work, we contributed to the improvement of the first released B. schlosseri genome assembly and offer an overview of the transcriptome changes during the blastogenetic cycle, showing up- and down-regulated genes. These results are important for the comprehension of the events underlying colony growth and regression, cell proliferation, colony homeostasis, and competition among different generations.

Characterization of Ambra1 in asexual cycle of a non-vertebrate chordate, the colonial tunicate Botryllus schlosseri, and phylogenetic analysis of the protein group in Bilateria.

Ambra1 is a positive regulator of autophagy, a lysosome-mediated degradative process involved both in physiological and pathological conditions. Nowadays, Ambra1 has been characterized only in mammals and zebrafish. Through bioinformatics searches and targeted cloning, we report the identification of the complete Ambra1 transcript in a non-vertebrate chordate, the tunicate Botryllus schlosseri. Tunicata is the sister group of Vertebrata and the only chordate group possessing species that reproduce also by blastogenesis (asexual reproduction). B. schlosseri Ambra1 deduced amino acid sequence is shorter than vertebrate homologues but still contains the typical WD40 domain. qPCR analyses revealed that the level of B. schlosseri Ambra1 transcription is temporally regulated along the colonial blastogenetic cycle. By means of similarity searches we identified Wdr5 and Katnb1 as proteins evolutionarily associated to Ambra1. Phylogenetic analyses on Bilateria indicate that: (i) Wdr5 is the most related to Ambra1, so that they may derive from an ancestral gene, (ii) Ambra1 forms a group of ancient genes evolved before the radiation of the taxon, (iii) these orthologous Ambra1 share the two conserved WD40/YVTN repeat-like-containing domains, and (iv) they are characterized by ancient duplications of WD40 repeats within the N-terminal domain.

Sexual and asexual reproduction in the colonial ascidian Botryllus schlosseri.

The colonial tunicate Botryllus schlosseri is a widespread filter-feeding ascidian that lives in shallow waters and is easily reared in aquaria. Its peculiar blastogenetic cycle, characterized by the presence of three blastogenetic generations (filtering adults, buds, and budlets) and by recurrent generation changes, has resulted in over 60 years of studies aimed at understanding how sexual and asexual reproduction are coordinated and regulated in the colony. The possibility of using different methodological approaches, from classical genetics to cell transplantation, contributed to the development of this species as a valuable model organism for the study of a variety of biological processes. Here, we review the main studies detailing rearing, staging methods, reproduction and colony growth of this species, emphasizing the asymmetry in sexual and asexual reproduction potential, sexual reproduction in the field and the laboratory, and self- and cross-fertilization. These data, opportunely matched with recent tanscriptomic and genomic outcomes, can give a valuable help to the elucidation of some important steps in chordate evolution.

Evolutionary conservation of the placodal transcriptional network during sexual and asexual development in chordates.

BACKGROUND: An important question behind vertebrate evolution is whether the cranial placodes originated de novo, or if their precursors were present in the ancestor of chordates. In this respect, tunicates are of particular interest as they are considered the closest relatives to vertebrates. They are also the only chordate group possessing species that reproduce both sexually and asexually, allowing both types of development to be studied to address whether embryonic pathways have been co-opted during budding to build the same structures. RESULTS: We studied the expression of members of the transcriptional network associated with vertebrate placodal formation (Six, Eya, and FoxI) in the colonial tunicate Botryllus schlosseri. During both sexual and asexual development, each transcript is expressed in branchial fissures and in two discrete regions proposed to be homologues to groups of vertebrate placodes. DISCUSSION: Results reinforce the idea that placode origin predates the origin of vertebrates and that the molecular network involving these genes was co-opted in the evolution of asexual reproduction. Considering that gill slit formation in deuterostomes is based on similar expression patterns, we discuss possible alternative evolutionary scenarios depicting gene co-option as critical step in placode and pharynx evolution.

Evolutionary diversification of secondary mechanoreceptor cells in tunicata.

BACKGROUND: Hair cells are vertebrate secondary sensory cells located in the ear and in the lateral line organ. Until recently, these cells were considered to be mechanoreceptors exclusively found in vertebrates that evolved within this group. Evidence of secondary mechanoreceptors in some tunicates, the proposed sister group of vertebrates, has recently led to the hypothesis that vertebrate and tunicate secondary sensory cells share a common origin. Secondary sensory cells were described in detail in two tunicate groups, ascidians and thaliaceans, in which they constitute an oral sensory structure called the coronal organ. Among thaliaceans, the organ is absent in salps and it has been hypothesised that this condition is due to a different feeding system adopted by this group of animals. No information is available as to whether a comparable structure exists in the third group of tunicates, the appendicularians, although different sensory structures are known to be present in these animals. RESULTS: We studied the detailed morphology of appendicularian oral mechanoreceptors. Using light and electron microscopy we could demonstrate that the mechanosensory organ called the circumoral ring is composed of secondary sensory cells. We described the ultrastructure of the circumoral organ in two appendicularian species, Oikopleura dioica and Oikopleura albicans, and thus taxonomically completed the data collection of tunicate secondary sensory cells. To understand the evolution of secondary sensory cells in tunicates, we performed a cladistic analysis using morphological data. We constructed a matrix consisting of 19 characters derived from detailed ultrastructural studies in 16 tunicate species and used a cephalochordate and three vertebrate species as outgroups. CONCLUSIONS: Our study clearly shows that the circumoral ring is the appendicularian homologue of the coronal organ of other tunicate taxa. The cladistic analysis enabled us to reconstruct the features of the putative ancestral hair cell in tunicates, represented by a simple monociliated cell. This cell successively differentiated into the current variety of oral mechanoreceptors in the various tunicate lineages. Finally, we demonstrated that the inferred evolutionary changes coincide with major transitions in the feeding strategies in each respective lineage.

Studying Regeneration in Ascidians: An Historical Overview.

Ascidians are sessile tunicates, that is, marine animals belonging to the phylum Chordata and considered the sister group of vertebrates. They are widespread in all the seas, constituting abundant communities in various ecosystems. Among chordates, only tunicates are able to reproduce asexually, forming colonies. The high regenerative potentialities enabling tunicates to regenerate damaged body parts, or the whole body, represent a peculiarity of this taxon. Here we review the methodological approaches used in more than a century of biological studies to induce regeneration in both solitary and colonial species. For solitary species, we refer to the regeneration of single organs or body parts (e.g., siphon, brain, gonad, tunic, viscera). For colonial species, we review a plethora of experiments regarding the surgical manipulation of colonies, the regeneration of isolated colonial entities, such as single buds in the tunic, or part of tunic and its circulatory system.

Yamanaka Factors in the Budding Tunicate Botryllus schlosseri Show a Shared Spatio-Temporal Expression Pattern in Chordates.

In vertebrates, the four transcription factors Sox2, c-Myc, Pou5f1 and Klf4 are involved in the differentiation of several tissues during vertebrate embryogenesis; moreover, they are normally co-expressed in embryonic stem cells and play roles in pluripotency, self-renewal, and maintenance of the undifferentiated state in adult cells. The in vitro forced co-expression of these factors, named Yamanaka factors (YFs), induces pluripotency in human or mouse fibroblasts. Botryllus schlosseri is a colonial tunicate undergoing continuous stem cell-mediated asexual development, providing a valuable model system for the study of pluripotency in the closest living relatives of vertebrates. In this study, we identified B. schlosseri orthologs of human Sox2 and c-Myc genes, as well as the closest homologs of the vertebrate-specific Pou5f1 gene, through an in-depth evolutionary analysis of the YF gene families in tunicates and other deuterostomes. Then, we studied the expression of these genes during the asexual cycle of B. schlosseri using in situ hybridization in order to investigate their possible involvement in tissue differentiation and in pluripotency maintenance. Our results show a shared spatio-temporal expression pattern consistent with the reported functions of these genes in invertebrate and vertebrate embryogenesis. Moreover, Myc, SoxB1 and Pou3 were expressed in candidate stem cells residing in their niches, while Pou2 was found expressed exclusively in the immature previtellogenic oocytes, both in gonads and circulating in the colonial vascular system. Our data suggest that Myc, SoxB1 and Pou3 may be individually involved in the differentiation of the same territories seen in other chordates, and that, together, they may play a role in stemness even in this colonial ascidian.

Analysis of a botryllid enriched-full-length cDNA library: insight into the evolution of spliced leader trans-splicing in tunicates.

In some animals, mRNA may be modified after transcription by the addition of a 5' spliced leader sequence. This is known as spliced leader (SL) trans-splicing, and is of uncertain function and evolutionary origin. Here, we report the identification of SL trans-splicing in the colonial ascidian Botryllus schlosseri. Combining our own expressed sequence tag (EST) data with additional data from GenBank, we identify the dominant spliced leader sequence and show it to be similar to that of other ascidians and to that of Oikopleura dioica, a basally diverging tunicate. Gene Ontology analysis of B. schlosseri ESTs with and without a 5' spliced leader shows that genes encoding ribosomal proteins tend not to be trans-spliced, a character shared with the ascidian Ciona intestinalis. We also examine individual cases of genes that produce mRNAs that are SL trans-spliced in B. schlosseri but not in C. intestinalis. We conclude that SL trans-splicing evolved early in the tunicate lineage and shows stability over considerable evolutionary time. However, SL trans-splicing may be gained or lost in individual genes.

Expression of a Musashi-like gene in sexual and asexual development of the colonial chordate Botryllus schlosseri and phylogenetic analysis of the protein group.

Tunicates are the unique chordates to possess species reproducing sexually and asexually. Among them, the colonial ascidian Botryllus schlosseri is a reference model for the study of similarities and differences in these two developmental pathways. We here illustrate the characterization and expression pattern during both pathways of a transcript for a gene orthologous to Dazap1. Dazap1 genes encode for RNA-binding proteins and fall into the Musashi-like (Msi-like) group. Our phylogenetic analysis shows that these are related to other RNA-binding proteins (Tardbp and several heterogeneous nuclear ribonucleoproteins types) that share the same modular domain structure of conserved tandem RNA Recognition Motifs (RRMs). We also classify the whole group as derived from a single ancient duplication of the RRM. Our results also show that Dazap1 is expressed with discrete spatiotemporal pattern during embryogenesis and blastogenesis of B. schlosseri. It is never expressed in wholly differentiated tissues, but it is located in all bud tissues and in different spatiotemporally defined territories of embryos and larva. These expression patterns could indicate different roles in the two processes, but an intriguing relationship appears if aspects of cell division dynamics are taken into account, suggesting that it is related to the proliferative phases in all tissues, and raising a similarity with known Dazap1 orthologs in other metazoans.

An elongated COI fragment to discriminate botryllid species and as an improved ascidian DNA barcode.

Botryllids are colonial ascidians widely studied for their potential invasiveness and as model organisms, however the morphological description and discrimination of these species is very problematic, leading to frequent specimen misidentifications. To facilitate species discrimination and detection of cryptic/new species, we developed new barcoding primers for the amplification of a COI fragment of about 860 bp (860-COI), which is an extension of the common Folmer's barcode region. Our 860-COI was successfully amplified in 177 worldwide-sampled botryllid colonies. Combined with morphological analyses, 860-COI allowed not only discriminating known species, but also identifying undescribed and cryptic species, resurrecting old species currently in synonymy, and proposing the assignment of clade D of the model organism Botryllus schlosseri to Botryllus renierii. Importantly, within clade A of B. schlosseri, 860-COI recognized at least two candidate species against only one recognized by the Folmer's fragment, underlining the need of further genetic investigations on this clade. This result also suggests that the 860-COI could have a greater ability to diagnose cryptic/new species than the Folmer's fragment at very short evolutionary distances, such as those observed within clade A. Finally, our new primers simplify the amplification of 860-COI even in non-botryllid ascidians, suggesting their wider usefulness in ascidians.

Muscle differentiation in a colonial ascidian: organisation, gene expression and evolutionary considerations.

BACKGROUND: Ascidians are tunicates, the taxon recently proposed as sister group to the vertebrates. They possess a chordate-like swimming larva, which metamorphoses into a sessile adult. Several ascidian species form colonies of clonal individuals by asexual reproduction. During their life cycle, ascidians present three muscle types: striated in larval tail, striated in the heart, and unstriated in the adult body-wall. RESULTS: In the colonial ascidian Botryllus schlosseri, we investigated organisation, differentiation and gene expression of muscle beginning from early buds to adults and during zooid regression. We characterised transcripts for troponin T (BsTnT-c), adult muscle-type (BsMA2) and cytoplasmic-type (BsCA1) actins, followed by in situ hybridisation (ISH) on sections to establish the spatio-temporal expression of BsTnT-c and BsMA2 during asexual reproduction and in the larva. Moreover, we characterised actin genomic sequences, which by comparison with other metazoans revealed conserved intron patterns. CONCLUSION: Integration of data from ISH, phalloidin staining and TEM allowed us to follow the phases of differentiation of the three muscle kinds, which differ in expression pattern of the two transcripts. Moreover, phylogenetic analyses provided evidence for the close relationship between tunicate and vertebrate muscle genes. The characteristics and plasticity of muscles in tunicates are discussed.

Vascular regeneration and angiogenic-like sprouting mechanism in a compound ascidian is similar to vertebrates.

Tunicates are useful models for comparing differing developmental processes such as embryogenesis, asexual reproduction, and regeneration, because they are the closest relatives to vertebrates and are the only chordates to reproduce both sexually and asexually. Among them, the ascidian Botryllus schlosseri displays high regenerative potential of the colonial circulatory system (CCS). The CCS runs in the common tunic, forming an anastomized network of vessels defined by simple epithelia and connected to the open circulatory system of the zooids. During asexual propagation, new vessels form by means of a tubular-sprouting mechanism, resembling that occurring in other metazoans, particularly during vertebrate angiogenesis. We studied the regeneration of experimentally ablated CCS by analyzing the general dynamics of reorganization of vessels and tunic, their ultrastructure, cell proliferation, and the immunohistology of regenerating structures using antibodies against vertebrate angiogenic factors-vascular endothelial growth factor (VEGF), fibroblast growth factor-2 (FGF-2), epidermal growth factor (EGF), and receptors: VEGFR-1, VEGFR-2, and EGFR. Results show that the regenerative process of CCS occurs by a sprouting mechanism, with participation of angiogenic factors. They also show correspondence between the CCS sprouting of B. schlosseri and angiogenic sprouting in vertebrates, during both normal development and regeneration, and support the idea that this morphogenetic mechanism was co-opted during the evolution of various developmental processes in different taxa.

Novel rhamnose-binding lectins from the colonial ascidian Botryllus schlosseri.

In a full-length cDNA library from the compound ascidian Botryllus schlosseri, we identified, by BLAST search against UniProt database, five transcripts, each with complete coding sequence, homologous to known rhamnose-binding lectins (RBLs). Comparisons of the predicted amino acid sequences suggest that they represent different isoforms of a novel RBL, called BsRBL-1-5. Four of these isolectins were found in Botryllus homogenate after purification by affinity chromatography on acid-treated Sepharose, analysis by reverse-phase HPLC and mass spectrometry. Analysis of both molecular masses and tryptic digests of BsRBLs indicated that the N-terminal sequence of the purified proteins starts from residue 22 of the putative amino acid sequence, and residues 1-21 represent a signal peptide. Analysis by mass spectrometry of V8-protease digests confirmed the presence and alignments of the eight cysteines involved in the disulphide bridges that characterise RBLs. Functional studies proved the enhancing effect on phagocytosis of the affinity-purified material. Results are discussed in terms of phylogenetic relationships of BsRBLs with orthologous molecules from protostomes and deuterostomes.

Does hair cell differentiation predate the vertebrate appearance?

It is generally accepted that the three main chordate groups (tunicates, cephalochordates and vertebrates) originated from a common ancestor having the basic features of the chordate body plan, i.e. a neural tube and a notochord flanked by striated musculature. There is now increasing evidence that tunicates, rather than cephalochordates, are the vertebrate sister-group. Correlated with this, tunicates have sensory structures similar to those derived from placodes or neural crest in vertebrates. In this context, we discuss here whether the precursors of vertebrate hair cells, which are placodal in origin, were present in ancestral chordates. The ascidian tunicates possess a coronal organ, consisting of a row of mechanosensory cells that runs around the base of the oral siphon. Its function is to monitor the incoming water flow. The cells are secondary sensory cells, i.e. they lack axons and synapse with neurons whose somata lie in the cerebral ganglion. They are accompanied by supporting cells and, as in vertebrates, have varying morphologies in the species so far examined: in one order (Enterogona), they are multiciliate; in the other (Pleurogona), they may possess an apical apparatus, consisting of one or two cilia accompanied by stereovilli, that are graded in length. Coronal cells thus resemble vertebrate hair cells closely in their morphology, embryonic origin and arrangement, which suggests they originated early in ancestral chordates. We are continuing our study of the coronal organ in other ascidian species, and report new data here on Botrylloides leachi, which conforms with the pattern of Pleurogona and, in particular, with previously published results on other botryllid ascidians.

Differentiation and Induced Sensorial Alteration of the Coronal Organ in the Asexual Life of a Tunicate.

Tunicates, the sister group of vertebrates, possess a mechanoreceptor organ, the coronal organ, which is considered the best candidate to address the controversial issue of vertebrate hair cell evolution. The organ, located at the base of the oral siphon, controls the flow of seawater into the organism and can drive the "squirting" reaction, i.e., the rapid body muscle contraction used to eject dangerous particles during filtration. Coronal sensory cells are secondary mechanoreceptors and share morphological, developmental, and molecular traits with vertebrate hair cells. In the colonial tunicate Botryllus schlosseri, we described coronal organ differentiation during asexual development. Moreover, we showed that the ototoxic aminoglycoside gentamicin caused morphological and mechanosensorial impairment in coronal cells. Finally, fenofibrate had a strong protective effect on coronal sensory cells due to gentamicin-induced toxicity, as occurs in vertebrate hair cells. Our results reinforce the hypothesis of homology between vertebrate hair cells and tunicate coronal sensory cells.

Developmental signature, synaptic connectivity and neurotransmission are conserved between vertebrate hair cells and tunicate coronal cells.

In tunicates, the coronal organ represents a sentinel checking particle entrance into the pharynx. The organ differentiates from an anterior embryonic area considered a proto-placode. For their embryonic origin, morphological features and function, coronal sensory cells have been hypothesized to be homologues to vertebrate hair cells. However, vertebrate hair cells derive from a posterior placode. This contradicts one of the principle historical criteria for homology, similarity of position, which could be taken as evidence against coronal cells/hair cells homology. In the tunicates Ciona intestinalis and C. robusta, we found that the coronal organ expresses genes (Atoh, Notch, Delta-like, Hairy-b, and Musashi) characterizing vertebrate neural and hair cell development. Moreover, coronal cells exhibit a complex synaptic connectivity pattern, and express neurotransmitters (Glu, ACh, GABA, 5-HT, and catecholamines), or enzymes for their synthetic machinery, involved in hair cell activity. Lastly, coronal cells express the Trpa gene, which encodes an ion channel expressed in hair cells. These data lead us to hypothesize a model in which competence to make secondary mechanoreceptors was initially broadly distributed through placode territories, but has become confined to different placodes during the evolution of the vertebrate and tunicate lineages.

Fixation, description and DNA barcode of a neotype for Botryllus schlosseri (Pallas, 1766) (Tunicata, Ascidiacea).

Botryllus schlosseri is a widespread colonial ascidian commonly considered cosmopolitan and amply used as model for researches ranging from developmental biology to immunobiology. Recently, molecular data lead to hypothesize that the species named B. schlosseri may consist of more than a single taxon. Indeed, five highly divergent clades, named A-E, have been genetically identified and are referred as cryptic species. In this context, and lacking both a type and a detailed morphological description, we believe that it is necessary, as a taxonomic reference point, to designate a neotype and re-describe the species. Therefore, a sample from the Lagoon of Venice (Adriatic Sea, Italy) was deposited as neotype in the Natural History Museum of Venice (Italy), preserved both in formalin and in 90% ethanol. Here we provide a morphological description of the suggested neotype of B. schlosseri that takes into account several developmental stages (oozooid, zooid of first blastogenic generations, and mature zooid) and is carefully compared with the previous descriptions of samples coming from other European and non-European localities. Finally, we associate our morphological description to a "DNA barcode", consisting in a long fragment of the mitochondrial COI gene. Our description is associated to clade A, although at now we cannot guarantee that this association is univocal.

External amebocytes guard the pharynx entry in a tunicate (Ascidiacea).

In the present report, we describe the identification of unusual free amebocytes, completely exposed to seawater, which inhabit the inner surface of the oral and atrial siphons of the compound ascidian Botryllus schlosseri (Urochordata). The origin and biological role of these cells were studied by cytochemical and ultrastructural analysis. These amebocytes are mononucleate cells, with numerous round granules, varying in content, and long filopodia, which contact the cuticle protrusions of the tunic in the siphon. Histochemical, histoenzymatic and immunohistochemical assays were carried out under light microscopy on sections and on living and fixed cultured hemocytes. Results showed that the phagocytic blood cells and the free amebocytes of the siphons shared: (i) affinity for the alpha-mannose specific agglutinin of Narcissus pseudonarcissus (NPA), (ii) occurrence of hydrolytic activities of acid phosphatase and non-specific esterases inside lysosomal vesicles and large vacuoles, (iii) membrane labeling with the lipophilic dye PKH26 specific for phagocytic cells, (iv) anti-CD39 immunocytochemical labeling specific for lysosomes of mammalian macrophages. All histochemical data support the hypothesis that these cells are 'sentinel cells' belonging to the hyaline amebocyte population of the phagocytic differentiation line of the immunocytes, since they can also recognize and phagocytize carmine experimentally administered as target particles.