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The close phylogenetic relationship between ascidians (Tunicata) and vertebrates makes them a powerful model for studying the innate immune system. To better understand the nature and dynamics of immune responses and the mechanisms through which bacterial infections are detected and translated into inflammation in Ciona robusta , we applied an approach combining in vivo lipopolysaccharide (LPS) stimulation, immune-labelling techniques and functional enzymatic analyses. The immunohistochemistry showed that Toll-like receptor 4 (TLR4) and nuclear factor kappa B (NFκB) were expressed during the inflammatory pharynx response 4 h post-LPS, with the formation of nodules in pharynx vessel lumen. Also, the endothelium vessels were involved in the inflammatory response. Observations of histological sections from naive and buffer-inoculated ascidians confirmed an immuno-positive response. Enzyme immune parameters-which included the activity of phenoloxidase, glutathione peroxidase, lysozyme, alkaline phosphatase and esterase-showed up-modulation 4 h after LPS injection, confirming their participation during ascidian inflammatory response. These findings provide new insights into the mechanisms underlying the LPS-induced C. robusta response and suggest that a broad innate immune mechanism, as in vertebrates, is involved in the regulation of inflammatory responses. Further findings in this direction are needed to cover knowledge gaps regarding the organized set of molecular and cellular networks involved in universal immune interactions with pathogens.

Allograft inflammatory factor-1 (AIF-1) is a calcium-binding scaffold/adaptor protein often associated with inflammatory diseases. Originally cloned from active macrophages in humans and rats, this gene has also been identified in other vertebrates and in several invertebrate species. Among metazoans, AIF-1 protein sequences remain relatively highly conserved. Generally, the highest expression levels of AIF-1 are observed in immunocytes, suggesting that it plays a key role in immunity. In mammals, the expression of AIF-1 has been reported in different cell types such as activated macrophages, microglial cells, and dendritic cells. Its main immunomodulatory role during the inflammatory response has been highlighted. Among invertebrates, AIF-1 is involved in innate immunity, being in many cases upregulated in response to biotic and physical challenges. AIF-1 transcripts result ubiquitously expressed in all examined tissues from invertebrates, suggesting its participation in a variety of biological processes, but its role remains largely unknown. This review aims to present current knowledge on the role and modulation of AIF-1 and to highlight its function along the evolutionary scale.

Microglia, the brain-resident immune cells, are critically involved in many physiological and pathological brain processes, including neurodegeneration. Here we characterize microglia morphology and transcriptional programs across ten species spanning more than 450 million years of evolution. We find that microglia express a conserved core gene program of orthologous genes from rodents to humans, including ligands and receptors associated with interactions between glia and neurons. In most species, microglia show a single dominant transcriptional state, whereas human microglia display significant heterogeneity. In addition, we observed notable differences in several gene modules of rodents compared with primate microglia, including complement, phagocytic, and susceptibility genes to neurodegeneration, such as Alzheimer's and Parkinson's disease. Our study provides an essential resource of conserved and divergent microglia pathways across evolution, with important implications for future development of microglia-based therapies in humans., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The functional preservation of the central nervous system (CNS) is based on the neuronal plasticity and survival. In this context, the neuroinflammatory state plays a key role and involves the microglial cells, the CNS-resident macrophages. In order to better understand the microglial contribution to the neuroprotection, microglia-derived extracellular vesicles (EVs) were isolated and molecularly characterized to be then studied in neurite outgrowth assays. The EVs, mainly composed of exosomes and microparticles, are an important cell-to-cell communication process as they exhibit different types of mediators (proteins, lipids, nucleic acids) to recipient cells. The medicinal leech CNS was initially used as an interesting model of microglia/neuron crosstalk due to their easy collection for primary cultures. After the microglia-derived EV isolation following successive methods, we developed their large-scale and non-targeted proteomic analysis to (i) detect as many EV protein markers as possible, (ii) better understand the biologically active proteins in EVs and (iii) evaluate the resulting protein signatures in EV-activated neurons. The EV functional properties were also evaluated in neurite outgrowth assays on rat primary neurons and the RNAseq analysis of the microglia-derived EVs was performed to propose the most representative miRNAs in microglia-derived EVs. This strategy allowed validating the EV isolation, identify major biological pathways in EVs and corroborate the regenerative process in EV-activated neurons. In parallel, six different miRNAs were originally identified in microglia-derived EVs including 3 which were only known in plants until now. The analysis of the neuronal proteins under the microglial EV activation suggested possible miRNA-dependent regulation mechanisms. Taken together, this combination of methodologies showed the leech microglial EVs as neuroprotective cargos across species and contributed to propose original EV-associated miRNAs whose functions will have to be evaluated in the EV-dependent dialog between microglia and neurons.

Neuronal activity is closely influenced by glia, especially microglia which are the resident immune cells in the central nervous system (CNS). Microglia in medicinal leech are the only cells able to migrate to the injury site within the 24 hours post-lesion. The microglia-neuron interactions constitute an important mechanism as there is neither astrocyte nor oligodendrocyte in the leech CNS. Given that axonal sprouting is impaired when microglia recruitment is inhibited, the crosstalk between microglia and neurons plays a crucial role in neuroprotection. The present results show that neurons and microglia both use ALK4/5 (a type of TGF-β receptor) signaling in order to maintain mutual exchanges in an adult brain following an axonal injury. Indeed, a TGF-β family member (nGDF) is immediately released by injured axons contributing to the early recruitment of ALK4/5 + microglia to the lesion site. Surprisingly, within the following hours, nGDF from microglia activates ALK4/5 + neurons to maintain a later microglia accumulation in lesion. Taken together, the results demonstrate that ALK4/5 signaling is essential throughout the response to the lesion in the leech CNS and gives a new insight in the understanding of this pathway. This latter is an important signal contributing to a correct sequential mobilization over time of microglia recruitment leading to axon regeneration.

In Mammals, microglial cells are considered as the resident immune cells in central nervous system (CNS). Many studies demonstrated that, after injury, these cells are activated and recruited at the lesion site. Leech microglia present a similar pattern of microglial activation and migration upon experimental lesion of CNS. This activation is associated with the release of a large amount of extracellular vesicles (EVs). We collected EVs released by microglia primary culture and compared two different protocols of isolation: one with differential ultracentrifugation (UC) and one using an additional Optiprep ™ Density Gradient (ODG) ultracentrifugation. Nanoparticles tracking analysis (NTA) and transmission electron microscopy (TEM) were used to assess vesicles size and morphology. The protein content of isolated EVs was assessed by mass spectrometry approaches. Results showed the presence of EV-specific proteins in both procedures. The extensive proteomic analysis of each single ODG fractions confirmed the efficiency of this protocol in limiting the presence of co-isolated proteins aggregates and other membranous particles during vesicles isolation. The present study permitted for the first time the characterisation of microglial EV protein content in an annelid model. Interestingly, an important amount of proteins found in leech vesicles was previously described in EV-specific databases. Finally, purified EVs were assessed for neurotrophic activity and promote neurites outgrowth on primary cultured neurons.

Recent studies demonstrated that allograft inflammatory factor-1 (AIF-1) and RNASET2 act as chemoattractants for macrophages and modulate the inflammatory processes in both vertebrates and invertebrates. The expression of these proteins significantly increases after bacterial infection; however, the mechanisms by which they regulate the innate immune response are still poorly defined. Here, we evaluate the effect of bacterial lipopolysaccharide injection on the expression pattern of these genes and the interrelation between them during innate immune response in the medicinal leech, an invertebrate model with a simple anatomy and a marked similarity with vertebrates in inflammatory processes. Collectively, prokaryotic-eukaryotic co-cultures and in vivo infection assays suggest that RNASET2 and AIF-1 play a crucial role in orchestrating a functional cross-talk between granulocytes and macrophages in leeches, resulting in the activation of an effective response against pathogen infection. RNASET2, firstly released by granulocytes, likely plays an early antibacterial role. Subsequently, AIF-1+ RNASET2-recruited macrophages further recruit other macrophages to potentiate the antibacterial inflammatory response. These experimental data are in keeping with the notion of RNA-SET2 acting as an alarmin-like molecule whose role is to locally transmit a "danger" signal (such as a bacterial infection) to the innate immune system in order to trigger an appropriate host response., (© 2018 The Author(s) Published by S. Karger AG, Basel.)

In healthy or pathological brains, the neuroinflammatory state is supported by a strong communication involving microglia and neurons. Recent studies indicate that extracellular vesicles (EVs), including exosomes and microvesicles, play a key role in the physiological interactions between cells allowing central nervous system (CNS) development and/or integrity. The present report used medicinal leech CNS to investigate microglia/neuron crosstalk from ex vivo approaches as well as primary cultures. The results demonstrated a large production of exosomes from microglia. Their incubation to primary neuronal cultures showed a strong interaction with neurites. In addition, neurite outgrowth assays demonstrated microglia exosomes to exhibit significant neurotrophic activities using at least a Transforming Growth Factor beta (TGF-β) family member, called nGDF (nervous Growth/Differentiation Factor). Of interest, the results also showed an EV-mediated dialog between leech microglia and rat cells highlighting this communication to be more a matter of molecules than of species. Taken together, the present report brings a new insight into the microglia/neuron crosstalk in CNS and would help deciphering the molecular evolution of such a cell communication in brain.

Extracellular ATP is among molecules promoting microglia activation and inducing the release of extracellular vesicles (EVs), which are potent mediators of intercellular communication between microglia and the microenvironment. We previously showed that EVs produced under ATP stimulation (ATP-EVs) propagate a robust inflammatory reaction among astrocytes and microglia in vitro and in mice with subclinical neuroinflammation (Verderio et al., 2012). However, the proteome of EVs released upon ATP stimulation has not yet been elucidated. In this study we applied a label free proteomic approach to characterize the proteome of EVs released constitutively and during microglia activation with ATP. We show that ATP drives sorting in EVs of a set of proteins implicated in cell adhesion/extracellular matrix organization, autophagy-lysosomal pathway and cellular metabolism, that may influence the response of recipient astrocytes to EVs. These data provide new clues to molecular mechanisms involved in microglia response to ATP and in microglia signaling to the environment via EVs.

Allograft inflammatory factor 1 (AIF-1) is a highly conserved gene involved in inflammation, cloned and characterized in several evolutionary distant animal species. Here, we report the molecular identification, characterization and expression of AIF-1 from the common sea urchin Paracentrotus lividus. In this species, AIF-1 encodes a predicted 151 amino acid protein with high similarity to vertebrate AIF-1 proteins. Immunocytochemical analyses on coelomocytes reveal localization of the AIF-1 protein in amoebocytes (perinuclear cytoplasmic zone) and red sphaerulocytes (inside granules), but not in vibratile cells and colorless sphaerula cells. The significant increase of AIF-1 expression (mRNA and protein) found in the coelomocytes of the sea urchin after Gram + bacterial challenge suggests the involvement of AIF-1 in the inflammatory response. Our analysis on P. lividus AIF-1 contributes to elucidate AIF-1 function along the evolutionary scale and consolidate the key evolutionary position of echinoderms throughout metazoans with respect to the common immune paths., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Allograft inflammatory factor-1 (AIF-1) is a 17-kDa cytokine-inducible calcium-binding protein that, in vertebrates, plays an important role in the allograft immune response. Its expression is mostly limited to the monocyte/macrophage lineage. Until recently, AIF-1 was assumed to be a novel molecule involved in inflammatory responses. To clarify this aspect, we have investigated the expression of AIF-1 after bacterial challenge and its potential role in regulating the innate immune response in an invertebrate model, the medicinal leech (Hirudo medicinalis). Analysis of an expressed sequence tag library from the central nervous system of Hirudo revealed the presence of the gene Hmaif-1/alias Hmiba1, showing high homology with vertebrate aif-1. Immunohistochemistry with an anti-HmAIF-1 polyclonal antibody revealed the constitutive presence of this protein in spread CD68(+) macrophage-like cells. A few hours after pathogen (bacterial) injection into the body wall, the amount of these immunopositive cells co-expressing HmAIF-1 and the common leucocyte marker CD45 increased at the injected site. Moreover, the recombinant protein HmAIF-1 induced massive angiogenesis and was a potent chemoattractant for macrophages. Following rHmAIF-1 stimulation, macrophage-like cells co-expressed the macrophage marker CD68 and the surface glycoprotein CD45, which, in vertebrates, seems to have a role in the integrin-mediated adhesion of macrophages and in the regulation of the functional responsiveness of cells to chemoattractants. CD45 is therefore probably involved in leech macrophage-like cell activation and migration towards an inflammation site. We have also examined its potential effect on HmAIF-1-induced signalling.

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., (© 2014 Wiley Periodicals, Inc.)

Background: The medicinal leech is considered as a complementary and appropriate model to study immune functions in the central nervous system (CNS). In a context in which an injured leech's CNS can naturally restore normal synaptic connections, the accumulation of microglia (immune cells of the CNS that are exclusively resident in leeches) has been shown to be essential at the lesion to engage the axonal sprouting. HmC1q (Hm for Hirudo medicinalis) possesses chemotactic properties that are important in the microglial cell recruitment by recognizing at least a C1q binding protein (HmC1qBP alias gC1qR)., Material and Methods: Recombinant forms of C1q were used in affinity purification and in vitro chemotaxis assays. Anti-calreticulin antibodies were used to neutralize C1q-mediated chemotaxis and locate the production of calreticulin in leech CNS., Results: A newly characterized leech calreticulin (HmCalR) has been shown to interact with C1q and participate to the HmC1q-dependent microglia accumulation. HmCalR, which has been detected in only some microglial cells, is consequently a second binding protein for HmC1q, allowing the chemoattraction of resident microglia in the nerve repair process., Conclusions: These data give new insight into calreticulin/C1q interaction in an immune function of neuroprotection, suggesting another molecular target to use in investigation of microglia reactivity in a model of CNS injury.

The medicinal leech is notable for its capacity to regenerate its central nervous system (CNS) following mechanical trauma. Using an electrochemical nitric oxide (NO)-selective electrode to measure NO levels, we found that the time course of NO release in the injured leech CNS is partially under the control of endocannabinoids, namely, N-arachidonyl ethanolamide (AEA) and 2-arachidonyl glycerol (2-AG). Relative quantification of these endocannabinoids was performed by stable isotope dilution (2AGd8 and AAEd8) coupled to mass spectrometry in course of regeneration process or adenosine triphosphate (ATP) treatment. Data show that 2-AG levels rose to a maximum about 30 min after injury or ATP treatment, and returned to baseline levels 4 h after injury. In same conditions, AEA levels also rapidly (within 5 min) dropped after injury or ATP treatment to the nerve cord, but did not fully return to baseline levels within 4 h of injury. In correlation with these data, chemoattraction activities of endocannabinoids on isolated leech microglial cells have been shown in vitro and in vivo reflecting that control over NO production is accompanied by the controlled chemoattraction of microglia directed from the periphery to the lesion site for neuronal repair purposes. Taken together, our results show that in the leech, after injury concurrent with ATP production, purinergic receptor activation, NO production, microglia recruitment, and accumulation to lesion site, a fine imbalance occurs in the endocannabinoid system. These events can bring explanations about the ability of the leech CNS to regenerate after a trauma and the key role of endocannabinoids in this phenomenon., (Copyright © 2013 Wiley Periodicals, Inc.)

Microglia are intrinsic components of the central nervous system (CNS). During pathologies in mammals, inflammatory processes implicate the resident microglia and the infiltration of blood cells including macrophages. Functions of microglia appear to be complex as they exhibit both neuroprotective and neurotoxic effects during neuropathological conditions in vivo and in vitro. The medicinal leech Hirudo medicinalis is a well-known model in neurobiology due to its ability to naturally repair its CNS following injury. Considering the low infiltration of blood cells in this process, the leech CNS is studied to specify the activation mechanisms of only resident microglial cells. The microglia recruitment is known to be essential for the usual sprouting of injured axons and does not require any other glial cells. The present review will describe the questions which are addressed to understand the nerve repair. They will discuss the implication of leech factors in the microglial accumulation, the identification of nerve cells producing these molecules, and the study of different microglial subsets. Those questions aim to better understand the mechanisms of microglial cell recruitment and their crosstalk with damaged neurons. The study of this dialog is necessary to elucidate the balance of the inflammation leading to the leech CNS repair.

Background: In invertebrates, the medicinal leech is considered to be an interesting and appropriate model to study neuroimmune mechanisms. Indeed, this non-vertebrate animal can restore normal function of its central nervous system (CNS) after injury. Microglia accumulation at the damage site has been shown to be required for axon sprouting and for efficient regeneration. We characterized HmC1q as a novel chemotactic factor for leech microglial cell recruitment. In mammals, a C1q-binding protein (C1qBP alias gC1qR), which interacts with the globular head of C1q, has been reported to participate in C1q-mediated chemotaxis of blood immune cells. In this study, we evaluated the chemotactic activities of a recombinant form of HmC1q and its interaction with a newly characterized leech C1qBP that acts as its potential ligand., Methods: Recombinant HmC1q (rHmC1q) was produced in the yeast Pichia pastoris. Chemotaxis assays were performed to investigate rHmC1q-dependent microglia migration. The involvement of a C1qBP-related molecule in this chemotaxis mechanism was assessed by flow cytometry and with affinity purification experiments. The cellular localization of C1qBP mRNA and protein in leech was investigated using immunohistochemistry and in situ hybridization techniques., Results: rHmC1q-stimulated microglia migrate in a dose-dependent manner. This rHmC1q-induced chemotaxis was reduced when cells were preincubated with either anti-HmC1q or anti-human C1qBP antibodies. A C1qBP-related molecule was characterized in leech microglia., Conclusions: A previous study showed that recruitment of microglia is observed after HmC1q release at the cut end of axons. Here, we demonstrate that rHmC1q-dependent chemotaxis might be driven via a HmC1q-binding protein located on the microglial cell surface. Taken together, these results highlight the importance of the interaction between C1q and C1qBP in microglial activation leading to nerve repair in the medicinal leech.

The biopolymer matrigel loaded with cytokine can be used for the recruitment in vivo of specific cell populations and as a vector for the preparation of cell cultures. Data demonstrate that the injection of the matrigel biopolymer supplemented with interleukin-8 (IL-8) in the leech Hirudo medicinalis can be used to purify cell populations showing the same morphofunctional and molecular mechanisms of specific populations of vertebrate hematopoietic precursor cells involved in tissue repair. These cells spontaneously differentiated into myofibroblasts. This approach highlights how the innovative use of a cytokine-loaded biopolymer for an in vivo cell sorting method, applied to a simple invertebrate model, can be a tool for studying myofibroblast cell biology and its regulation, step by step., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

In contrast to mammals, the medicinal leech Hirudo medicinalis can completely repair its central nervous system (CNS) after injury. This invertebrate model offers unique opportunities to study the molecular and cellular basis of the CNS repair processes. When the leech CNS is injured, microglial cells migrate and accumulate at the site of lesion, a phenomenon known to be essential for the usual sprouting of injured axons. In the present study, we demonstrate that a new molecule, designated HmIL-16, having functional homologies with human interleukin-16 (IL-16), has chemotactic activity on leech microglial cells as observed using a gradient of human IL-16. Preincubation of microglial cells either with an anti-human IL-16 antibody or with anti-HmIL-16 antibody significantly reduced microglia migration induced by leech-conditioned medium. Functional homology was demonstrated further by the ability of HmIL-16 to promote human CD4+ T cell migration which was inhibited by antibody against human IL-16, an IL-16 antagonist peptide or soluble CD4. Immunohistochemistry of leech CNS indicates that HmIL-16 protein present in the neurons is rapidly transported and stored along the axonal processes to promote the recruitment of microglial cells to the injured axons. To our knowledge, this is the first identification of a functional interleukin-16 homologue in invertebrate CNS. The ability of HmIL-16 to recruit microglial cells to sites of CNS injury suggests a role for HmIL-16 in the crosstalk between neurons and microglia in the leech CNS repair.

In vertebrates, central nervous system (CNS) protection is dependent on many immune cells including microglial cells. Indeed, activated microglial cells are involved in neuroinflammation mechanisms by interacting with numerous immune factors. Unlike vertebrates, some lophotrochozoan invertebrates can fully repair their CNS following injury. In the medicinal leech Hirudo medicinalis, the recruitment of microglial cells at the lesion site is essential for sprouting of injured axons. Interestingly, a new molecule homologous to vertebrate C1q was characterized in leech, named HmC1q (for H. medicinalis) and detected in neurons and glial cells. In chemotaxis assays, leech microglial cells were demonstrated to respond to human C1q. The chemotactic activity was reduced when microglia was preincubated with signaling pathway inhibitors (Pertussis Toxin or wortmannin) or anti-human gC1qR antibody suggesting the involvement of gC1qR in C1q-mediated migration in leech. Assays using cells preincubated with NO chelator (cPTIO) showed that C1q-mediated migration was associated to NO production. Of interest, by using anti-HmC1q antibodies, HmC1q released in the culture medium was shown to exhibit a similar chemotactic effect on microglial cells as human C1q. In summary, we have identified, for the first time, a molecule homologous to mammalian C1q in leech CNS. Its chemoattractant activity on microglia highlights a new investigation field leading to better understand leech CNS repair mechanisms.

Genetically altering the disease vector status of insects using recombinant DNA technologies is being considered as an alternative to eradication efforts. Manipulating the endogenous immune response of mosquitoes such as the temporal and special expression of antimicrobial peptides like cecropin may result in a refractory phenotype. Using transgenic technology a unique pattern of expression of cecropin A (cecA) in Anopheles gambiae was created such that cecA was expressed beginning 24 h after a blood meal in the posterior midgut. Two independent lines of transgenic An. gambiae were created using a piggyBac gene vector containing the An. gambiae cecA cDNA under the regulatory control of the Aedes aegypti carboxypeptidase promoter. Infection with Plasmodium berghei resulted in a 60% reduction in the number of oocysts in transgenic mosquitoes compared with nontransgenic mosquitoes. Manipulating the innate immune system of mosquitoes can negatively affect their capacity to serve as hosts for the development of disease-causing microbes.

A novel mosquito antimicrobial peptide, gambicin, and the corresponding gene were isolated in parallel through differential display-PCR, an expressed sequence tag (EST) project, and characterization of an antimicrobial activity in a mosquito cell line by reverse-phase chromatography. The 616-bp gambicin ORF encodes an 81-residue protein that is processed and secreted as a 61-aa mature peptide containing eight cysteines engaged in four disulfide bridges. Gambicin lacks sequence homology with other known proteins. Like other Anopheles gambiae antimicrobial peptide genes, gambicin is induced by natural or experimental infection in the midgut, fatbody, and hemocyte-like cell lines. Within the midgut, gambicin is predominantly expressed in the anterior part. Both local and systemic gambicin expression is induced during early and late stages of natural malaria infection. In vitro experiments showed that the 6.8-kDa mature peptide can kill both Gram-positive and Gram-negative bacteria, has a morphogenic effect on a filamentous fungus, and is marginally lethal to Plasmodium berghei ookinetes. An oxidized form of gambicin isolated from the cell line medium was more active against bacteria than the nonoxidized form from the same medium.

The elucidation of digestive processes in the Anopheles gambiae gut leading to the utilization of the blood meal will result in a deeper understanding of the physiology of blood digestion and its impact on parasite-vector interactions. Accordingly, the identification of digestive serine proteases in A. gambiae has implications for the development of alternative strategies for the control of mosquito-borne diseases. We report here on the cDNA and genomic cloning and on the expression analysis of two closely related chymotrypsin genes, Anchym1 and Anchym2. Genomic cloning revealed that Anchym1 and Anchym2, which map on chromosomal division 25D, are clustered in tandem within 6 kb, both genes being interrupted by two short introns. After blood feeding, transcription of Anchym1 and Anchym2 is induced in the midgut epithelium, followed by secretion of the translated products into the midgut lumen where the Anchym1 and Anchym2 zymogens are activated by partial tryptic digestion. The amino-acid residues forming the substrate pocket of Anchym1 and Anchym2 suggested chymotryptic cleavage specificity. This was confirmed by mass spectrometry analysis and Edman degradation sequencing of proteolytic products generated by the recombinant, trypsin-activated Anchym1.