In Vitro Cytokine Production by Dengue-Infected Human Monocyte-Derived Dendritic Cells.

Despite many advances on the understanding of dengue pathogenesis in the last decades, some questions remained to be clarified. The virulence of the pathogen and the host immune response are the main factors involved in pathogenesis of dengue infection. In addition, skin dendritic cells (DCs) are one of the primary targets for dengue virus infection. After infection, DCs process and present antigens to T cells and also secrete cytokines that shape the immune response. Although relevant for the development of antiviral immune response, an imbalance in the cytokine production by immune cells could lead to cytokine storm observed in severe dengue fever cases. Therefore, this chapter will describe the protocols for the in vitro differentiation of human monocytes into human monocyte-derived dendritic cells (mdDCs), followed by dengue virus infection, as well as the cytokine quantification produced by mdDCs using a cytometric bead array method.

In Vitro Generation of Human Antibody-Secreting Cells Through the Stimulation of PBMCs with Dengue Virus Particles.

The Dengue pathophysiology has had several aspects determined over the years. However, some points remain elusive, such as the metabolic factors that regulate the massive B cell differentiation into antibody-secreting cells observed in Dengue patients. In this chapter, we describe an in vitro method capable of mimicking this Dengue-induced cell expansion. More specifically, this approach allows dengue virus-stimulated peripheral blood mononuclear cells (PBMCs) from healthy individuals to enhance the frequency of phenotypical and functional antibody-secreting cells (ASCs) after 7 days of culture. A manuscript recently published by Bonezi and colleagues displays results generated through this methodology.

Flavonoids as Molecules With Anti-Zika virus Activity.

Zika virus (ZIKV) is an arthropod-born virus that is mainly transmitted to humans by mosquitoes of the genus Aedes spp. Since its first isolation in 1947, only a few human cases had been described until large outbreaks occurred on Yap Island (2007), French Polynesia (2013), and Brazil (2015). Most ZIKV-infected individuals are asymptomatic or present with a self-limiting disease and nonspecific symptoms such as fever, myalgia, and headache. However, in French Polynesia and Brazil, ZIKV outbreaks led to the diagnosis of congenital malformations and microcephaly in newborns and Guillain-Barré syndrome (GBS) in adults. These new clinical presentations raised concern from public health authorities and highlighted the need for anti-Zika treatments and vaccines to control the neurological damage caused by the virus. Despite many efforts in the search for an effective treatment, neither vaccines nor antiviral drugs have become available to control ZIKV infection and/or replication. Flavonoids, a class of natural compounds that are well-known for possessing several biological properties, have shown activity against different viruses. Additionally, the use of flavonoids in some countries as food supplements indicates that these molecules are nontoxic to humans. Thus, here, we summarize knowledge on the use of flavonoids as a source of anti-ZIKV molecules and discuss the gaps and challenges in this area before these compounds can be considered for further preclinical and clinical trials.

Impact of Toxoplasma gondii infection on TM3 Leydig cells: Alterations in testosterone and cytokines levels.

Leydig cells play pivotal roles in eliciting male characteristics by producing testosterone and any damage to these cells can compromise male fertility Toxoplasma gondii (T. gondii) is an intracellular parasite capable to invade any nucleated cell, including cells from male reproductive system. Herein, we evaluated the capacity of RH strain of T. gondii to infect TM3 Leydig cells and the impact of this infection on testosterone and inflammatory mediators production. We first, by performing adherence, infection, and intracellular proliferation assays, we found a significant increase in the number of infected Leydig cells, peaking 48 h after the infection with T. gondii. Supernatants of TM3 infected cells exhibited, in a time-dependent manner, increased levels of testosterone as well as monocyte chemoattractant protein-1 (MCP-1) and interferon-γ (IFN-γ), which is correlated with the robust T. gondii infection. In conclusion, our study provides new insights regarding the harmful effects of T. gondii infection on male reproductive system.

Pirahy virus: Identification of a new and potential emerging arbovirus in South Brazil.

Genomic and epidemiological surveillance are paramount for the discovery of new viruses with the potential to cross species barriers. Here, we present a new member of the genus Alphavirus found in Trichoprosopon and Wyeomia mosquitoes, tentatively named Pirahy virus (PIRAV). PIRAV was isolated from mosquito pools collected in a rural area of Piraí do Sul, South Brazil. In vitro assays revealed that PIRAV replicates and causes cytopathic effects in vertebrate cell lines such as Vero E6, SH-SY5Y, BHK-21 and UMNSAH/DF-1. Genomic signature analysis supports these results showing a dinucleotide and codon usage balance compatible with several hosts. Phylogenetic analyses placed PIRAV basal to the Venezuelan equine encephalitis complex. Genome analyses, electron microscopy, and biological characterization show findings that may alert for the emergence of a new arbovirus in South America.

The citrus flavonoid naringenin impairs the in vitro infection of human cells by Zika virus.

The Zika virus (ZIKV) is an arthropod-borne virus that belongs to the Flaviviridae family. The ZIKV infection is usually asymptomatic or is associated with mild clinical manifestations; however, increased numbers of cases of microcephaly and birth defects have been recently reported. To date, neither a vaccine nor an antiviral treatment has become available to control ZIKV replication. Among the natural compounds recognized for their medical properties, flavonoids, which can be found in fruits and vegetables, have been found to possess biological activity against a variety of viruses. Here, we demonstrate that the citrus flavanone naringenin (NAR) prevented ZIKV infection in human A549 cells in a concentration-dependent and ZIKV-lineage independent manner. NAR antiviral activity was also observed when primary human monocyte-derived dendritic cells were infected by ZIKV. NAR displayed its antiviral activity when the cells were treated after infection, suggesting that NAR acts on the viral replication or assembly of viral particles. Moreover, a molecular docking analysis suggests a potential interaction between NAR and the protease domain of the NS2B-NS3 protein of ZIKV which could explain the anti-ZIKV activity of NAR. Finally, the results support the potential of NAR as a suitable candidate molecule for developing anti-ZIKV treatments.

Genetic and biological characterisation of Zika virus isolates from different Brazilian regions.

BACKGROUND: Zika virus (ZIKV) infections reported in recent epidemics have been linked to clinical complications that had never been associated with ZIKV before. Adaptive mutations could have contributed to the successful emergence of ZIKV as a global health threat to a nonimmune population. However, the causal relationships between the ZIKV genetic determinants, the pathogenesis and the rapid spread in Latin America and in the Caribbean remain widely unknown. OBJECTIVES: The aim of this study was to characterise three ZIKV isolates obtained from patient samples during the 2015/2016 Brazilian epidemics. METHODS: The ZIKV genomes of these strains were completely sequenced and in vitro infection kinetics experiments were carried out in cell lines and human primary cells. FINDINGS: Eight nonsynonymous substitutions throughout the viral genome of the three Brazilian isolates were identified. Infection kinetics experiments were carried out with mammalian cell lines A549, Huh7.5, Vero E6 and human monocyte-derived dendritic cells (mdDCs) and insect cells (Aag2, C6/36 and AP61) and suggest that some of these mutations might be associated with distinct viral fitness. The clinical isolates also presented differences in their infectivity rates when compared to the well-established ZIKV strains (MR766 and PE243), especially in their abilities to infect mammalian cells. MAIN CONCLUSIONS: Genomic analysis of three recent ZIKV isolates revealed some nonsynonymous substitutions, which could have an impact on the viral fitness in mammalian and insect cells.

Quercetin promotes antipromastigote effect by increasing the ROS production and anti-amastigote by upregulating Nrf2/HO-1 expression, affecting iron availability.

American cutaneous leishmaniasis is a zoonotic disease caused by protozoans of the genus Leishmania. The treatment of cutaneous leishmaniasis is unsatisfactory, thus, much research effort has been focused on investigating new compounds with lower collateral effects to the patients and derived from low-cost sources, such as natural products. In the present study, we evaluated the in vitro directly effect of the flavonoid quercetin against Leishmania (Viannia) braziliensis. Quercetin inhibited the proliferation of promastigote forms at all tested concentrations, these effect were due to increasing the reactive oxygen species (ROS) production, phosphatidylserine exposure and loss of plasma membrane integrity. Moreover, quercetin reduced the number of parasites in L. braziliensis-infected macrophages, reducing the levels of TNF-α and increasing IL-10 synthesis without modulate nitric oxide (NO) production. In addition, quercetin upregulated Nrf2/HO-1 expression and modulated the labile iron pool in infected macrophages, culminating in a depletion of available iron for L. braziliensis replication.

Amblyomma sculptum Salivary PGE2 Modulates the Dendritic Cell-Rickettsia rickettsii Interactions in vitro and in vivo.

Amblyomma sculptum is an important vector of Rickettsia rickettsii, causative agent of Rocky Mountain spotted fever and the most lethal tick-borne pathogen affecting humans. To feed on the vertebrate host's blood, A. sculptum secretes a salivary mixture, which may interact with skin resident dendritic cells (DCs) and modulate their function. The present work was aimed at depicting the A. sculptum saliva-host DC network and the biochemical nature of the immunomodulatory component(s) involved in this interface. A. sculptum saliva inhibits the production of inflammatory cytokines by murine DCs stimulated with LPS. The fractionation of the low molecular weight salivary content by reversed-phase chromatography revealed active fractions eluting from 49 to 55% of the acetonitrile gradient. Previous studies suggested that this pattern of elution matches with that observed for prostaglandin E2 (PGE2) and the molecular identity of this lipid mediator was unambiguously confirmed by a new high-resolution mass spectrometry methodology. A productive infection of murine DCs by R. rickettsii was demonstrated for the first time leading to proinflammatory cytokine production that was inhibited by both A. sculptum saliva and PGE2, a result also achieved with human DCs. The adoptive transfer of murine DCs incubated with R. rickettsii followed by treatment with A. sculptum saliva or PGE2 did not change the cytokine profile associated to cellular recall responses while IgG2a-specific antibodies were decreased in the serum of these mice. Together, these findings emphasize the role of PGE2 as a universal immunomodulator of tick saliva. In addition, it contributes to new approaches to explore R. rickettsii-DC interactions both in vitro and in vivo.

Detection and clearance of a mosquito densovirus contaminant from laboratory stocks of Zika virus.

BACKGROUND: The Zika virus (ZIKV) epidemics that affected South America in 2016 raised several research questions and prompted an increase in studies in the field. The transient and low viraemia observed in the course of ZIKV infection is a challenge for viral isolation from patient serum, which leads to many laboratories around the world sharing viral strains for their studies. C6/36 cells derived from Aedes albopictus larvae are commonly used for arbovirus isolation from clinical samples and for the preparation of viral stocks. OBJECTIVES: Here, we report the contamination of two widely used ZIKV strains by Brevidensovirus, here designated as mosquito densovirus (MDV). METHODS: Molecular and immunological techniques were used to analyse the MDV contamination of ZIKV stocks. Also, virus passages in mammalian cell line and infecting susceptible mice were used to MDV clearance from ZIKV stocks. FINDINGS: MDV contamination was confirmed by molecular and immunological techniques and likely originated from C6/36 cultures commonly used to grow viral stocks. We applied two protocols that successfully eliminated MDV contamination from ZIKV stocks, and these protocols can be widely applied in the field. As MDV does not infect vertebrate cells, we performed serial passages of contaminated stocks using a mammalian cell line and infecting susceptible mice prior to re-isolating ZIKV from the animals' blood serum. MDV elimination was confirmed with immunostaining, polymerase chain reaction (PCR), and analysis of the mosquitoes that were allowed to feed on the infected mice. MAIN CONCLUSIONS: Since the putative impact of viral contaminants in ZIKV strains generally used for research purposes is unknown, researchers working in the field must be aware of potential contaminants and test viral stocks to certify sample purity.

Amblyomma sculptum salivary PGE2 modulates the dendritic cell-Rickettsia rickettsii interactions in vitro and in vivo

Amblyomma sculptum is an important vector of Rickettsia rickettsii, causative agent of Rocky Mountain spotted fever and the most lethal tick-borne pathogen affecting humans. To feed on the vertebrate host’s blood, A. sculptum secretes a salivary mixture, which may interact with skin resident dendritic cells (DCs) and modulate their function. The present work was aimed at depicting the A. sculptum saliva-host DC network and the biochemical nature of the immunomodulatory component(s) involved in this interface. A. sculptum saliva inhibits the production of inflammatory cytokines by murine DCs stimulated with LPS. The fractionation of the low molecular weight salivary content by reversed-phase chromatography revealed active fractions eluting from 49 to 55% of the acetonitrile gradient. Previous studies suggested that this pattern of elution matches with that observed for prostaglandin E2 (PGE2) and the molecular identity of this lipid mediator was unambiguously confirmed by a new high-resolution mass spectrometry methodology. A productive infection of murine DCs by R. rickettsii was demonstrated for the first time leading to proinflammatory cytokine production that was inhibited by both A. sculptum saliva and PGE2, a result also achieved with human DCs. The adoptive transfer of murine DCs incubated with R. rickettsii followed by treatment with A. sculptum saliva or PGE2 did not change the cytokine profile associated to cellular recall responses while IgG2a-specific antibodies were decreased in the serum of these mice. Together, these findings emphasize the role of PGE2 as a universal immunomodulator of tick saliva. In addition, it contributes to new approaches to explore R. rickettsii-DC interactions both in vitro and in vivo.

Amblyomma sculptum salivary PGE2 modulates the dendritic cell-Rickettsia rickettsii interactions in vitro and in vivo

Amblyomma sculptum is an important vector of Rickettsia rickettsii, causative agent of Rocky Mountain spotted fever and the most lethal tick-borne pathogen affecting humans. To feed on the vertebrate host’s blood, A. sculptum secretes a salivary mixture, which may interact with skin resident dendritic cells (DCs) and modulate their function. The present work was aimed at depicting the A. sculptum saliva-host DC network and the biochemical nature of the immunomodulatory component(s) involved in this interface. A. sculptum saliva inhibits the production of inflammatory cytokines by murine DCs stimulated with LPS. The fractionation of the low molecular weight salivary content by reversed-phase chromatography revealed active fractions eluting from 49 to 55% of the acetonitrile gradient. Previous studies suggested that this pattern of elution matches with that observed for prostaglandin E2 (PGE2) and the molecular identity of this lipid mediator was unambiguously confirmed by a new high-resolution mass spectrometry methodology. A productive infection of murine DCs by R. rickettsii was demonstrated for the first time leading to proinflammatory cytokine production that was inhibited by both A. sculptum saliva and PGE2, a result also achieved with human DCs. The adoptive transfer of murine DCs incubated with R. rickettsii followed by treatment with A. sculptum saliva or PGE2 did not change the cytokine profile associated to cellular recall responses while IgG2a-specific antibodies were decreased in the serum of these mice. Together, these findings emphasize the role of PGE2 as a universal immunomodulator of tick saliva. In addition, it contributes to new approaches to explore R. rickettsii-DC interactions both in vitro and in vivo.

Genetic and biological characterisation of Zika virus isolates from different Brazilian regions

BACKGROUND Zika virus (ZIKV) infections reported in recent epidemics have been linked to clinical complications that had never been associated with ZIKV before. Adaptive mutations could have contributed to the successful emergence of ZIKV as a global health threat to a nonimmune population. However, the causal relationships between the ZIKV genetic determinants, the pathogenesis and the rapid spread in Latin America and in the Caribbean remain widely unknown. OBJECTIVES The aim of this study was to characterise three ZIKV isolates obtained from patient samples during the 2015/2016 Brazilian epidemics. METHODS The ZIKV genomes of these strains were completely sequenced and in vitro infection kinetics experiments were carried out in cell lines and human primary cells. FINDINGS Eight nonsynonymous substitutions throughout the viral genome of the three Brazilian isolates were identified. Infection kinetics experiments were carried out with mammalian cell lines A549, Huh7.5, Vero E6 and human monocyte-derived dendritic cells (mdDCs) and insect cells (Aag2, C6/36 and AP61) and suggest that some of these mutations might be associated with distinct viral fitness. The clinical isolates also presented differences in their infectivity rates when compared to the well-established ZIKV strains (MR766 and PE243), especially in their abilities to infect mammalian cells. MAIN CONCLUSIONS Genomic analysis of three recent ZIKV isolates revealed some nonsynonymous substitutions, which could have an impact on the viral fitness in mammalian and insect cells.

Detection and clearance of a mosquito densovirus contaminant from laboratory stocks of Zika virus

BACKGROUND The Zika virus (ZIKV) epidemics that affected South America in 2016 raised several research questions and prompted an increase in studies in the field. The transient and low viraemia observed in the course of ZIKV infection is a challenge for viral isolation from patient serum, which leads to many laboratories around the world sharing viral strains for their studies. C6/36 cells derived from Aedes albopictus larvae are commonly used for arbovirus isolation from clinical samples and for the preparation of viral stocks. OBJECTIVES Here, we report the contamination of two widely used ZIKV strains by Brevidensovirus, here designated as mosquito densovirus (MDV). METHODS Molecular and immunological techniques were used to analyse the MDV contamination of ZIKV stocks. Also, virus passages in mammalian cell line and infecting susceptible mice were used to MDV clearance from ZIKV stocks. FINDINGS MDV contamination was confirmed by molecular and immunological techniques and likely originated from C6/36 cultures commonly used to grow viral stocks. We applied two protocols that successfully eliminated MDV contamination from ZIKV stocks, and these protocols can be widely applied in the field. As MDV does not infect vertebrate cells, we performed serial passages of contaminated stocks using a mammalian cell line and infecting susceptible mice prior to re-isolating ZIKV from the animals' blood serum. MDV elimination was confirmed with immunostaining, polymerase chain reaction (PCR), and analysis of the mosquitoes that were allowed to feed on the infected mice. MAIN CONCLUSIONS Since the putative impact of viral contaminants in ZIKV strains generally used for research purposes is unknown, researchers working in the field must be aware of potential contaminants and test viral stocks to certify sample purity.

Concanavalin-A displays leishmanicidal activity by inducing ROS production in human peripheral blood mononuclear cells.

The context of the article: Leishmania amazonensis has a wide geographical distribution throughout South American countries and can cause self-healing to severe cases as mucocutaneous or visceral forms. Leishmaniasis presents a balance of inflammatory and anti-inflammatory cytokines which is responsible for promoting the activation of phagocytes, essential to control the infection and lead to tissue repair/resolution of the disease, respectively. Results and discussion: Our model revealed that the treatment with Con-A was capable to stimulate human PBMC cells by increasing the phagocytic capacity and promoting parasite elimination. The pretreatment with Con-A promoted inflammatory (IFN-γ, TNF-α, IL-2 and IL-6) and anti-inflammatory (IL-4 and IL-10) cytokines production, increased the reactive oxygen species (ROS) sinthesys as well as the expression and presence of iNOS enzyme, but not nitric oxide production. Conclusion: Based on the data obtained, it was possible to infer that Con-A induces the ROS production, responsible for eliminating parasites in addition to regulatory cytokines synthesis which are important for disease resolution.

Caryocar coriaceum extracts exert leishmanicidal effect acting in promastigote forms by apoptosis-like mechanism and intracellular amastigotes by Nrf2/HO-1/ferritin dependent response and iron depletion: Leishmanicidal effect of Caryocar coriaceum leaf exracts.

Leishmania (L.) amazonensis is the American Cutaneous Leishmaniasis-causing agents, and the available drugs for this disease present toxicity, low efficiency and difficulty of administration. Plants belong23ing to the Caryocar genus are found in Brazilian Cerrado, where fruits are used as food and in folk medicine, and previous studies showed several biological effects of extracts of this plant. The present work evaluated the leishmanicidal and immunomodulatory activity of ethyl acetate (EAC) and methanol (MET) C. coriaceum leaf extracts EAC and MET showed an antipromastigote effect after 24, 48 and 72 h. The extracts also induced loss of mitochondrial membrane potential, reactive oxygen species (ROS) production, damage to the plasma membrane, and phosphatidylserine exposure on promastigote forms, and most parasites were going through a late apoptosis-like process. The range of concentrations used did not alter the viability of peritoneal macrophages of BALB/c mice; therefore, we observed that the treatment with extracts was able to reduce the infection of this cells. Thereafter, the extracts were able to significantly improve the levels of TNFα, IL-6, MCP-1, and IL-10, and reduced the levels of MDA and ROS without interfering on NO levels released by infected macrophages. In addition, both EAC and MET up-regulated Nrf2/HO-1/Ferritin expression and reduced the labile iron pool in infected macrophages. Based on the data obtained, it is possible to infer that different solvent extracts of the C. coriaceum leaves exert leishmanicidal effect, acting on promastigote forms through apoptosis-like mechanisms and intracellular amastigote forms involving a Nrf2/HO-1 dependent antioxidant response, which culminates in a depletion of available iron for L. amazonensis replication.

Biogenic silver nanoparticles inducing Leishmania amazonensis promastigote and amastigote death in vitro.

American Cutaneous Leishmaniasis (ACL) is a zoonosis caused by Leishmania protozoa. The ACL chemotherapy available is unsatisfactory motivating researches to seek alternative treatments. In this study, we investigated the action of biogenic silver nanoparticle (AgNp-bio) obtained from Fusarium oxysporium, against Leishmania amazonensis promastigote and amastigote forms. The AgNp-bio promastigote treatment results in promastigote death leading to apoptosis-like events due an increased production of reactive oxygen species (ROS), loss of mitochondrial integrity, phosphatidylserine exposure and damage on promastigotes membrane. In L. amazonensis infected macrophages, AgNp-bio treatment was still able to reduce the percentage of infected macrophages and the amount of amastigotes per macrophage, consequently, the amount of promastigotes recovered. This leishmanicidal effect was also accompanied by a decrease in the levels of ROS and nitric oxide. By observing the ultrastructural integrity of the intracellular amastigotes, we found that the AgNp-bio treatment made a significant damage, suggesting that the compound has a direct effect on intracellular amastigotes. These results demonstrated that AgNp-bio had a direct effect against L. amazonensis forms and acted on immunomodulatory ability of infected macrophages, reducing the infection without inducing the synthesis of inflammatory mediators, which continuous stimulation can generate and aggravate leishmaniotic lesions. Overall, our findings suggest that the use of AgNp-bio stands out as a new therapeutic option to be considered for further in vivo investigations representing a possible treatment for ACL.

Brazilian propolis promotes immunomodulation on human cells from American Tegumentar Leishmaniasis patients and healthy donors infected with L. braziliensis.

American Tegumentar Leishmaniasis (ATL) is an infectious disease caused by Leishmania parasites with ineffective treatment. The properties of propolis have been studied in different experimental studies, however, few works have investigated the effects of propolis on human-derived peripheral blood mononuclear cells (PBMC) in leishmaniasis models. Thus, we investigate the immunomodulatory effects of propolis treatment on PBMC from ATL patients and on PBMC from healthy donors infected with Leishmania braziliensis. Our data demonstrate that propolis pretreatment shows immunomodulatory effects on both healthy donors and ATL patients adherent cells, increasing IL-4 and IL-17 and decreasing IL-10, in either the presence or absence of the L. braziliensis infection, demonstrating that propolis contributes with the decrease of the inflammation and could also contribute with parasite control.

Antileishmanial Activity and Inducible Nitric Oxide Synthase Activation by RuNO Complex.

Parasites of the genus Leishmania are capable of inhibiting effector functions of macrophages. These parasites have developed the adaptive ability to escape host defenses; for example, they inactivate the NF-κB complex and suppress iNOS expression in infected macrophages, which are responsible for the production of the major antileishmanial substance nitric oxide (NO), favoring then its replication and successful infection. Metal complexes with NO have been studied as potential compounds for the treatment of certain tropical diseases, such as ruthenium compounds, known to be exogenous NO donors. In the present work, the compound cis-[Ru(bpy)2SO3(NO)]PF6, or RuNO, showed leishmanicidal activity directly and indirectly on promastigote forms of Leishmania (Leishmania) amazonensis. In addition, treatment with RuNO increased NO production by reversing the depletion of NO caused by Leishmania. We also found increased expression of Akt, iNOS, and NF-κB in infected and treated macrophages. These results demonstrated that RuNO was able to kill the parasite by NO release and modulate the transcriptional capacity of the cell.

Galleria mellonella hemocytes: A novel phagocytic assay for Leishmania (Viannia) braziliensis.

Galleria mellonella is an excellent invertebrate model for the study of diseases that involve interactions with cells from the innate immune system, since they have an innate immune system capable of recognizing the pathogens. Here we present for the first time, an alternative model for an in vitro phagocytic assay using hemocytes of G. mellonella larvae to study infection by Leishmania (Viannia) braziliensis. We showed that the insect phagocytic cells were able to engulf promastigotes. Furthermore, this infective form differentiated into the amastigote form inside those cells. However, the cells in this model seem resistant to the parasite, since amastigotes were depleted after 24h and NO levels were maintained after infection. Our model opens an avenue of possibilities for new investigations regarding other Leishmania species, mechanisms of invasion and evasion, receptors involved, release of signaling molecules and, above all, it is a novel infection model using invertebrate animals.