Perkinsus marinus suppresses in vitro eastern oyster apoptosis via IAP-dependent and caspase-independent pathways involving TNFR, NF-kB, and oxidative pathway crosstalk.

The protozoan parasite Perkinsus marinus causes Dermo disease in eastern oysters, Crassostrea virginica, and can suppress apoptosis of infected hemocytes using incompletely understood mechanisms. This study challenged hemocytes in vitro with P. marinus for 1 h in the presence or absence of caspase inhibitor Z-VAD-FMK or Inhibitor of Apoptosis protein (IAP) inhibitor GDC-0152. Hemocytes exposure to P. marinus significantly reduced granulocyte apoptosis, and pre-incubation with Z-VAD-FMK did not affect P. marinus-induced apoptosis suppression. Hemocyte pre-incubation with GDC-0152 prior to P. marinus challenge further reduced apoptosis of granulocytes with engulfed parasite, but not mitochondrial permeabilization. This suggests P. marinus-induced apoptosis suppression may be caspase-independent, affect an IAP-involved pathway, and occur downstream of mitochondrial permeabilization. P. marinus challenge stimulated hemocyte differential expression of oxidation-reduction, TNFR, and NF-kB pathways. WGCNA analysis of P. marinus expression in response to hemocyte exposure revealed correlated protease, kinase, and hydrolase expression that could contribute to P. marinus-induced apoptosis suppression.

An insight of anopheline larvicidal mechanism of Trichoderma asperellum (TaspSKGN2).

Anopheline larvicidal property of T. asperellum has been found recently in medical science. The mechanism of actions exhibited by T. asperellum to infect mosquito larvae is the pivotal context of our present study. To infect an insect, entomopathogens must undergo some events of pathogenesis. We performed some experiments to find out the mechanisms of action of T. asperellum against anopheline larvae and compared its actions with other two well recognized entomopathogens like Metarhizium anisopliae and Beauveria bassiana. The methodology adopted for this includes Compound light and SE Microscopic study of host-pathogen interaction, detection of fungal spore adhesion on larval surface (Mucilage assay), detection of cuticle degrading enzymes (Spore bound pr1, chitinase and protease) by spectro-photometric method, Quantitative estimation of chitinase and protease enzymes, and determination of nuclear degeneration of hemocyte cells of ME (methanolic extract) treated larvae by T. asperellum under fluorescence microscope. Compound light microscopic studies showed spore attachment, appressorium and germ tube formation, invasion and proliferated hyphal growth of T. asperellum on epicuticle and inside of dead larvae. SEM study also supported them. After 3 h of interaction, spores were found to be attached on larval surface exhibiting pink colored outer layer at the site of attachment indicating the presence of mucilage surrounding the attached spores. The enzymatic cleavage of the 4-nitroanilide substrate yields 4-nitroaniline which indicates the presence of spore-bound PR1 protein (Pathogenecity Related 1 Protein) and it was highest (absorbance 1.298 ± 0.002) for T. asperellum in comparison with control and other two entomopathogens. T. asperellum exhibited highest enzymatic index values for both chitinase (5.20) and protease (2.77) among three entomopathogens. Quantitative experiment showed that chitinase enzyme concentration of T. asperellum (245 µg mL-1) was better than other two M. anisopliae (134.59 µg mL-1) and B. bassiana (128.65 µg mL-1). Similarly protease enzyme concentration of this fungus was best (298.652 µg mL-1) among three entomopathogens. Here we have detected and estimated fragmentized nuclei of hemocyte cells by fluorescence microscopy in treated larvae with different ME doses of T. asperellum, and also observed that mosquito larvae exposed to 0.1 mg mL-1 dose of ME showed maximum (100%) nuclear fragmentations of hemocytes and while 20, 45, 70 and 85% of nuclear deformities were recorded at 0.02, 0.04, 0.06 and 0.08 mg mL-1 concentrations of ME. The knowledge of this work certainly will help in understanding of mechanism of action of T. asperellum for anopheline larval killing and consequently in eradication of malaria vector.

A Comprehensive Review on Crustaceans' Immune System With a Focus on Freshwater Crayfish in Relation to Crayfish Plague Disease.

Freshwater crayfish immunity has received great attention due to the need for urgent conservation. This concern has increased the understanding of the cellular and humoral defense systems, although the regulatory mechanisms involved in these processes need updating. There are, however, aspects of the immune response that require clarification and integration. The particular issues addressed in this review include an overall description of the oomycete Aphanomyces astaci, the causative agent of the pandemic plague disease, which affects freshwater crayfish, and an overview of crustaceans' immunity with a focus on freshwater crayfish. It includes a classification system of hemocyte sub-types, the molecular factors involved in hematopoiesis and the differential role of the hemocyte subpopulations in cell-mediated responses, including hemocyte infiltration, inflammation, encapsulation and the link with the extracellular trap cell death pathway (ETosis). In addition, other topics discussed include the identity and functions of hyaline cells, the generation of neoplasia, and the emerging topic of the role of sessile hemocytes in peripheral immunity. Finally, attention is paid to the molecular execution of the immune response, from recognition by the pattern recognition receptors (PRRs), the role of the signaling network in propagating and maintaining the immune signals, to the effector elements such as the putative function of the Down syndrome adhesion molecules (Dscam) in innate immune memory.

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.

Cellular and molecular complementary immune stress markers for the model species Dreissena polymorpha.

This study aimed to combine cellular and molecular analyses for better detail the effects of various stresses on a sentinel species of freshwater invertebrate. For this purpose, the hemocytes of the zebra mussel, Dreissena polymorpha, were exposed to different stresses at two different intensities, high or low: chemical (cadmium and ionomycin), physical (ultraviolet B), or biological ones (Cryptosporidium parvum and Toxoplasma gondii). After exposure, flow cytometry and droplet digital PCR analyses were performed on the same pools of hemocytes. Several responses related to necrosis, apoptosis, phagocytosis, production of nitric oxide and expression level of several genes related to the antioxidant, detoxification and immune systems were evaluated. Results showed that hemocyte integrity was compromised by both chemical and physical stress, and cellular markers of phagocytosis reacted to ionomycin and protozoa. While cadmium induced oxidative stress and necrosis, ionomycin tends to modulate the immune response of hemocytes. Although both biological stresses led to a similar immune response, C. parvum oocysts induced more effects than T. gondii, notably through the expression of effector caspases gene and an increase in hemocyte necrosis. This suggests different management of the two protozoa by the cell. This work provides new knowledge of biomarkers in the zebra mussel, at both cellular and molecular levels, and contributes to elucidate the mechanisms of action of different kinds of stress in this species.

A CD63 Homolog Specially Recruited to the Fungi-Contained Phagosomes Is Involved in the Cellular Immune Response of Oyster Crassostrea gigas.

Cluster of differentiation 63 (CD63), a four-transmembrane glycoprotein in the subfamily of tetraspanin, has been widely recognized as a gateway from the infection of foreign invaders to the immune defense of hosts. Its role in Pacific oyster Crassostrea gigas is, however, yet to be discovered. This work makes contributions by identifying CgCD63H, a CD63 homolog with four transmembrane domains and one conservative CCG motif, and establishing its role as a receptor that participates in immune recognition and hemocyte phagocytosis. The presence of CgCD63H messenger RNA (mRNA) in hepatopancreas, labial palps, gill, and hemocytes is confirmed. The expression level of mRNA in hemocytes is found significantly (p < 0.01) upregulated after the injection of Vibrio splendidus. CgCD63H protein, typically distributed over the plasma membrane of oyster hemocytes, is recruited to the Yarrowia lipolytica-containing phagosomes after the stimulation of Y. lipolytica. The recombinant CgCD63H protein expresses binding capacity to glucan (GLU), peptidoglycan (PGN), and lipopolysaccharide (LPS) in the presence of lyophilized hemolymph. The phagocytic rate of hemocytes toward V. splendidus and Y. lipolytica is significantly inhibited (p < 0.01) after incubation with anti-CgCD63H antibody. Our work further suggests that CgCD63H functions as a receptor involved in the immune recognition and hemocyte phagocytosis against invading pathogen, which can be a marker candidate for the hemocyte typing in C. gigas.

Hemocyte-specific FREP13 abrogates the exogenous bacterial population in the hemolymph and promotes midgut endosymbionts in Anopheles stephensi.

The immune blood cells "hemocytes" of mosquitoes impart a highly selective immune response against various microorganisms/pathogens. Among several immune effectors, fibrinogen-related proteins (FREPs) have been recognized as key modulators of cellular immune responses; however, their physiological relevance has not been investigated in detail. Our ongoing comparative RNA-sequencing analysis identified a total of 13 FREPs originating from naïve sugar-fed, blood-fed, bacterial challenged and Plasmodium vivax-infected hemocytes in Anopheles stephensi. Transcriptional profiling of the selected seven FREP transcripts showed distinct responses against different pathophysiological conditions, where an exclusive induction of FREP12 after 10 days of P. vivax infection was observed. This represents a possible role of FREP12 in immunity against free circulating sporozoites and needs to be explored in the future. When challenged with live bacterial injection in the thorax, we observed a higher affinity of FREP13 and FREP65 toward Gram-negative and Gram-positive bacteria in the mosquito hemocytes, respectively. Furthermore, we observed increased bacterial survival and proliferation, which is likely compromised by the downregulation of TEP1, in FREP13 messenger RNA-depleted mosquito hemolymph. In contrast, after blood-feeding, we also noticed a significant delay of 24 h in the enrichment of gut endosymbionts in the FREP13-silenced mosquitoes. Taken together, we conclude that hemocyte-specific FREP13 carries the unique ability of tissue-specific regulation, having an antagonistic antibacterial role in the hemolymph, and an agonistic role against gut endosymbionts.

Temporal dynamics of infection of cockles Cerastoderma edule with the protistan parasite Minchinia tapetis (Rhizaria: Haplosporida) in Galicia (NW Spain).

Uninucleate and binucleate cells and multinucleate plasmodia of a haplosporidan-like protist associated with heavy haemocytic infiltration were observed in histological sections of cockles, Cerastoderma edule, from the Ría de Noia (Galicia, NW Spain) in the course of a cockle health surveillance programme. Molecular assays provided identification of this protist as Minchinia tapetis, which we thus record from a new host. Prevalence of M. tapetis as high as 93% was recorded but infection intensity was low to moderate, never heavy, and abnormally high cockle mortality was not observed in the ria by shellfishers. A significant positive correlation was found between M. tapetis prevalence and sea water temperature. Sea water temperature increase associated with climate change might contribute to increase the prevalence of this infection in cockles and, as a consequence, this parasite may be considered a threat for cockle production.

Recent advances on the immunobiology of Bithynia spp. hosts of Opisthorchis viverrini.

This article reviews the past and present scientific reports regarding Bithynia spp. focusing on the biology, ecology and life cycle of Bithynia snails and their responses to Opisthorchis viverrini infection. Moreover, new data regarding comparative molecular genomics and proteomic approaches have recently revealed novel molecular components involved in the immune defence responses from Bithynia spp., providing additional perspectives for future studies. Studies on the specific interaction between Bithynia snails and their trematodes will contribute to further understanding the snail-parasite relationship with regards to epidemiology and control of Opisthorchiasis and broaden the scope on comparative immunology of gastropod snails.

Snail defence responses to parasite infection: The Lymnaea stagnalis-Trichobilharzia szidati model.

Lymnaea stagnalis is a common freshwater gastropod. Importantly, the snail serves as the intermediate host for more than one hundred species of digenetic trematodes, including the avian schistosome Trichobilharzia szidati, a causative agent of cercarial dermatitis in humans. Infection of L. stagnalis by T. szidati initiates a dynamic confrontation between the host and the parasite that culminates in immunocompatibility ensuring survival and development of larvae. Unfortunately, the molecular mechanisms determining this immunocompatibility remain poorly characterised. By employing a variety of immune elicitors, including chemical compounds, PAMPs and bacteria, research in the last two decades has elucidated some of the molecular processes that regulate the snail internal defence response such as haemocyte signalling pathways. These discoveries provide a framework for future studies of molecular interactions between T. szidati and L. stagnalis to help elucidate factors and mechanisms enabling transmission of schistosome parasites. Moreover, support from recently available next generation sequence data and CRISPR-enabled functional genomics should further enable L. stagnalis as an important model for comparative immunology and contribute to a more comprehensive understanding of immune functions in gastropod molluscs.

The immunobiological interplay between Pseudosuccinea columella resistant/susceptible snails with Fasciola hepatica: Hemocytes in the spotlight.

The Fasciola hepatica/Pseudosuccinea columella interaction in Cuba involves a unique pattern of phenotypes; while most snails are susceptible, some field populations are naturally resistant to infection and parasites are encapsulated by snail hemocytes. Thus, we investigated the hemocytes of resistant (R) and susceptible (S) P. columella, in particular morphology, abundance, proliferation and in vitro encapsulation activity following exposure to F. hepatica. Compared to susceptible P. columella, hemocytes from exposed resistant snails showed increased levels of spreading and aggregation (large adherent cells), proliferation of circulating blast-like cells and encapsulation activity of the hemocytes, along with a higher expression of the cytokine granulin. By contrast, there was evidence of a putative F. hepatica-driven inhibition of host immunity, only in susceptible snails. Additionally, (pre-)incubation of naïve hemocytes from P. columella (R and S) with different monosaccharides was associated with lower encapsulation activity of F. hepatica larvae. This suggests the involvement in this host-parasite interaction of lectins and lectins receptors (particularly related to mannose and fucose sensing) in association with hemocyte activation and/or binding to F. hepatica.

Comparison of viability and phagocytic responses of hemocytes withdrawn from the bivalves Mytilus edulis and Dreissena polymorpha, and exposed to human parasitic protozoa.

Bivalve molluscs are now considered indicator species of aquatic contamination by human parasitic protozoa. Nonetheless, the possible effects of these protozoa on the immune system of their paratenic hosts are poorly documented. The aim of this study was to evaluate the effects of two protozoa on hemocyte viability and phagocytosis from two mussels, the zebra mussel (freshwater habitat) and the blue mussel (seawater habitat). For these purposes, viability and phagocytic markers have been analysed on hemocytes from mussels without biological stress (control hemocytes), and on hemocytes exposed to a biological stress (Toxoplasma gondii and Cryptosporidium parvum oocysts). We report, for the first known time, the interactions between protozoa and hemocytes of mussels from different aquatic environments. Zebra mussel hemocytes showed a decrease in phagocytosis of fluorescent microbeads after exposure to both protozoa, while blue mussel hemocytes reacted only to T. gondii oocysts. These decreases in the ingestion of microbeads can be caused by competition between beads and oocysts and can be influenced by the size of the oocysts. New characterisations of their immune capacities, including aggregation, remain to be developed to understand the specificities of both mussels.

Independent effects on cellular and humoral immune responses underlie genotype-by-genotype interactions between Drosophila and parasitoids.

It is common to find abundant genetic variation in host resistance and parasite infectivity within populations, with the outcome of infection frequently depending on genotype-specific interactions. Underlying these effects are complex immune defenses that are under the control of both host and parasite genes. We have found extensive variation in Drosophila melanogaster's immune response against the parasitoid wasp Leptopilina boulardi. Some aspects of the immune response, such as phenoloxidase activity, are predominantly affected by the host genotype. Some, such as upregulation of the complement-like protein Tep1, are controlled by the parasite genotype. Others, like the differentiation of immune cells called lamellocytes, depend on the specific combination of host and parasite genotypes. These observations illustrate how the outcome of infection depends on independent genetic effects on different aspects of host immunity. As parasite-killing results from the concerted action of different components of the immune response, these observations provide a physiological mechanism to generate phenomena like epistasis and genotype-interactions that underlie models of coevolution.

Encapsulation of the Acanthocephalan Corynosoma strumosum (Rudolphi, 1802) LÜHE, 1904, in the Intermediate Host Spinulogammarus ochotensis.

We describe the thin and ultra-thin structures of the envelopes surrounding the cystacanth of Corynosoma strumosum (Rudolphi, 1802) Lühe, 1904, in its intermediate host. A total of 4,357 amphipods from 2 species were examined: Locustogammarus locustoides (Brandt, 1851) and Spinulogammarus ochotensis (Brandt, 1851). Eleven corynosome cystacanths were found in 6 S. ochotensis specimens. Three were enclosed in acellular cysts originating from the parasite. Three other cystacanths were also encysted and were surrounded by a lighter capsule consisting of the host's hemocytes. Five cystacanths were enclosed in a cyst and a darker capsule, in which both the acanthocephalans and their surrounding envelopes were destroyed. We suggest that the cystacanth's cyst is a protective barrier against the host's cellular response, while the lighter and darker capsules represent different stages of parasite degeneration.

Spiroplasma eriocheiris Invasion Into Macrobrachium rosenbergii Hemocytes Is Mediated by Pathogen Enolase and Host Lipopolysaccharide and ß-1, 3-Glucan Binding Protein.

Spiroplasma eriocheiris is a crustacean pathogen, without a cell wall, that causes enormous economic loss. Macrobrachium rosenbergii hemocytes are the major targets during S. eriocheiris infection. As wall-less bacteria, S. eriocheiris, its membrane protein should interact with host membrane protein directly and firstly when invaded in host cell. In this investigation, six potential hemocyte receptor proteins were identified firstly that mediate interaction between S. eriocheiris and M. rosenbergii. Among these proteins, lipopolysaccharide and ß-1, 3-glucan binding protein (MrLGBP) demonstrated to bind to S. eriocheiris using bacterial binding assays and confocal microscopy. Four spiroplasma ligand proteins for MrLGBP were isolated and identified. But, competitive assessment demonstrated that only enolase of S. eriocheiris (SeEnolase) could be a candidate ligand for MrLGBP. Subsequently, the interaction between MrLGBP and SeEnolase was confirmed by co-immunoprecipitation and co-localization in vitro. After the interaction between MrLGBP and SeEnolase was inhibited by antibody neutralization test, the virulence ability of S. eriocheiris was effectively reduced. The quantity of S. eriocheiris decreased in Drosophila S2 cells after overexpression of MrLGBP, compared with the controls. In addition, RNA interference (RNAi) knockdown of MrLGBP made M. rosenbergii more sensitive to S. eriocheiris infection. Further studies found that the immune genes, including MrLGBP and prophenoloxidase (MrproPO), MrRab7A, and Mrintegrin α1 were significantly up-regulated by SeEnolase stimulation. After SeEnolase pre-stimulation, the ability of M. rosenbergii resistance to S. eriocheiris was significantly improved. Collectively, this investigation demonstrated that MrLGBP and pathogen SeEnolase involved in mediating S. eriocheiris invasion into M. rosenbergii hemocytes.

Chemical depletion of phagocytic immune cells in Anopheles gambiae reveals dual roles of mosquito hemocytes in anti-Plasmodium immunity.

Mosquito immunity is composed of both cellular and humoral factors that provide protection from invading pathogens. Immune cells known as hemocytes, have been intricately associated with phagocytosis and innate immune signaling. However, the lack of genetic tools has limited hemocyte study despite their importance in mosquito anti-Plasmodium immunity. To address these limitations, we employ the use of a chemical-based treatment to deplete phagocytic immune cells in Anopheles gambiae, demonstrating the role of phagocytes in complement recognition and prophenoloxidase production that limit the ookinete and oocyst stages of malaria parasite development, respectively. Through these experiments, we also define specific subtypes of phagocytic immune cells in An. gambiae, providing insights beyond the morphological characteristics that traditionally define mosquito hemocyte populations. Together, this study represents a significant advancement in our understanding of the roles of mosquito phagocytes in mosquito vector competence and demonstrates the utility of clodronate liposomes as an important tool in the study of invertebrate immunity.

Spodoptera litura cyclophilin A is required for Microplitis bicoloratus bracovirus-induced apoptosis during insect cellular immune response.

Microplitis bicoloratus bracovirus (MbBV) is a polydnavirus found in the parasitic wasp M. bicoloratus. Although MbBV is a known inducer of apoptosis in host hemocytes, the mechanism by which this occurs remains elusive. In this study, we found that expression of cyclophilin A (CypA) was significantly upregulated in Spodoptera litura hemocytes at 6-day post-parasitization. Similar results were reported in High Five cells (Hi5 cells) infected by MbBV, suggesting that the upregulation of CypA is linked to MbBV infection in insect cells. cDNA encoding CypA was cloned from parasitized hemocytes of S. litura, and bioinformatic analyses showed that S. litura CypA belongs to the cyclophilin family of proteins. Overexpression of S. litura CypA in Hi5 cells revealed that the protein promotes MbBV-induced apoptosis in vitro. Conversely, suppression of the expression and activity of CypA protein significantly rescued the apoptotic phenotype observed in MbBV-infected Hi5 cells, suggesting that it plays a key role in this process. MbBV infection also promoted the cytoplasmic-nuclear translocation of CypA in Hi5 cells. Taken together, these results suggest that MbBV infection upregulates the expression of CypA, which is required for MbBV-mediated apoptosis. Our findings provide insight into the role that CypA plays in insect cellular immune response.

Proteomic Analysis of Biomphalaria glabrata Hemocytes During in vitro Encapsulation of Schistosoma mansoni Sporocysts.

Circulating hemocytes of the snail Biomphalaria glabrata, a major intermediate host for the blood fluke Schistosoma mansoni, represent the primary immune effector cells comprising the host's internal defense system. Within hours of miracidial entry into resistant B. glabrata strains, hemocytes infiltrate around developing sporocysts forming multi-layered cellular capsules that results in larval death, typically within 24-48 h post-infection. Using an in vitro model of hemocyte-sporocyst encapsulation that recapitulates in vivo events, we conducted a comparative proteomic analysis on the responses of hemocytes from inbred B. glabrata strains during the encapsulation of S. mansoni primary sporocysts. This was accomplished by a combination of Laser-capture microdissection (LCM) to isolate sections of hemocyte capsules both in the presence and absence of sporocysts, in conjunction with mass spectrometric analyses to establish protein expression profiles. Comparison of susceptible NMRI snail hemocytes in the presence and absence of sporocysts revealed a dramatic downregulation of proteins in during larval encapsulation, especially those involved in protein/CHO metabolism, immune-related, redox and signaling pathways. One of 4 upregulated proteins was arginase, competitor of nitric oxide synthetase and inhibitor of larval-killing NO production. By contrast, when compared to control capsules, sporocyst-encapsulating hemocytes of resistant BS-90 B. glabrata exhibited a more balanced profile with enhanced expression of shared proteins involved in protein synthesis/processing, immunity, and redox, and unique expression of anti-microbial/anti-parasite proteins. A final comparison of NMRI and BS-90 host hemocyte responses to co-cultured sporocysts demonstrated a decrease or downregulation of 77% of shared proteins by NMRI cells during encapsulation compared to those of the BS-90 strain, including lipopolysaccharide-binding protein, thioredoxin reductase 1 and hemoglobins 1 and 2. Overall, using this in vitro model, results of our proteomic analyses demonstrate striking differences in proteins expressed by susceptible NMRI and resistant BS-90 snail hemocytes to S. mansoni sporocysts during active encapsulation, with NMRI hemocytes exhibiting extensive downregulation of protein expression and a lower level of constitutively expressed immune-relevant proteins (e.g., FREP2) compared to BS-90. Our data suggest that snail strain differences in hemocyte protein expression during the encapsulation process account for observed differences in their cytotoxic capacity to interact with and kill sporocysts.

Assessing the cellular and humoral immune response in Rhipicephalus sanguineus sensu lato (Acari: Ixodidae) infected with Leishmania infantum (Nicolle, 1908).

The aim of this study was to evaluate aspects of the innate cellular and humoral immune response by evaluating hemocyte dynamics, phagocytosis, phenoloxidase (PO) activity and nitric oxide (NO) production in Rhipicephalus sanguineus sensu lato (s.l.) (Acari: Ixodidae) infected with Leishmania infantum and to assess the persistence of parasites at time 0 and 1, 2, 5, and 7 days post-infection (dpi). The total and differential count of the five types of hemocytes circulating in the hemolymph of R. sanguineus s.l. females showed the average total number of hemocytes in the group infected with L. infantum to be significantly higher (p < 0.05) on the 1st and 2nd dpi compared to the control group. The hemocyte differential count showed that the average number of plasmatocytes and granulocytes increased significantly on the 1st, 2nd, and 5th dpi with L. infantum compared to the control group (p < 0.001). Phagocytosis assays revealed that plasmatocytes and granulocytes were able to perform phagocytosis of latex beads and L. infantum on the 1st and 2nd dpi, respectively. NO production was significantly increased (p < 0.001) on the 1st, 2nd, and 5th dpi with L. infantum and PO activity increased significantly (p < 0.05) only on the 5th dpi. L. infantum DNA was significantly increased (p < 0.001) on the 5th and 7th dpi compared to time 0. Although there are no studies describing the response of R. sanguineus s.l. to an infection with L. infantum, these results suggest that R. sanguineus s.l. activates the cellular and humoral immune response after infection with L. infantum. Further studies are however, needed to assess the impact of such a response on fighting infection.