Evolution of insect innate immunity through domestication of bacterial toxins.

Toxin cargo genes are often horizontally transferred by phages between bacterial species and are known to play an important role in the evolution of bacterial pathogenesis. Here, we show how these same genes have been horizontally transferred from phage or bacteria to animals and have resulted in novel adaptations. We discovered that two widespread bacterial genes encoding toxins of animal cells, cytolethal distending toxin subunit B (cdtB) and apoptosis-inducing protein of 56 kDa (aip56), were captured by insect genomes through horizontal gene transfer from bacteria or phages. To study the function of these genes in insects, we focused on Drosophila ananassae as a model. In the D. ananassae subgroup species, cdtB and aip56 are present as singular (cdtB) or fused copies (cdtB::aip56) on the second chromosome. We found that cdtB and aip56 genes and encoded proteins were expressed by immune cells, some proteins were localized to the wasp embryo's serosa, and their expression increased following parasitoid wasp infection. Species of the ananassae subgroup are highly resistant to parasitoid wasps, and we observed that D. ananassae lines carrying null mutations in cdtB and aip56 toxin genes were more susceptible to parasitoids than the wild type. We conclude that toxin cargo genes were captured by these insects millions of years ago and integrated as novel modules into their innate immune system. These modules now represent components of a heretofore undescribed defense response and are important for resistance to parasitoid wasps. Phage or bacterially derived eukaryotic toxin genes serve as macromutations that can spur the instantaneous evolution of novelty in animals.

Transdifferentiation and Proliferation in Two Distinct Hemocyte Lineages in Drosophila melanogaster Larvae after Wasp Infection.

Cellular immune responses require the generation and recruitment of diverse blood cell types that recognize and kill pathogens. In Drosophila melanogaster larvae, immune-inducible lamellocytes participate in recognizing and killing parasitoid wasp eggs. However, the sequence of events required for lamellocyte generation remains controversial. To study the cellular immune system, we developed a flow cytometry approach using in vivo reporters for lamellocytes as well as for plasmatocytes, the main hemocyte type in healthy larvae. We found that two different blood cell lineages, the plasmatocyte and lamellocyte lineages, contribute to the generation of lamellocytes in a demand-adapted hematopoietic process. Plasmatocytes transdifferentiate into lamellocyte-like cells in situ directly on the wasp egg. In parallel, a novel population of infection-induced cells, which we named lamelloblasts, appears in the circulation. Lamelloblasts proliferate vigorously and develop into the major class of circulating lamellocytes. Our data indicate that lamellocyte differentiation upon wasp parasitism is a plastic and dynamic process. Flow cytometry with in vivo hemocyte reporters can be used to study this phenomenon in detail.

Cellular Immunity of Drosophila willistoni Reveals Novel Complexity in Insect Anti-Parasitoid Defense.

Coevolution of hosts and their parasites has shaped heterogeneity of effector hemocyte types, providing immune defense reactions with variable effectiveness. In this work, we characterize hemocytes of Drosophila willistoni, a species that has evolved a cellular immune system with extensive variation and a high degree of plasticity. Monoclonal antibodies were raised and used in indirect immunofluorescence experiments to characterize hemocyte subpopulations, follow their functional features and differentiation. Pagocytosis and parasitization assays were used to determine the functional characteristics of hemocyte types. Samples were visualized using confocal and epifluorescence microscopy. We identified a new multinucleated giant hemocyte (MGH) type, which differentiates in the course of the cellular immune response to parasitoids. These cells differentiate in the circulation through nuclear division and cell fusion, and can also be derived from the central hematopoietic organ, the lymph gland. They have a binary function as they take up bacteria by phagocytosis and are involved in the encapsulation and elimination of the parasitoid. Here, we show that, in response to large foreign particles, such as parasitoids, MGHs differentiate, have a binary function and contribute to a highly effective cellular immune response, similar to the foreign body giant cells of vertebrates.

Distinctive features of Zaprionus indianus hemocyte differentiation and function revealed by transcriptomic analysis.

Background: Insects have specialized cell types that participate in the elimination of parasites, for instance, the lamellocytes of the broadly studied species Drosophila melanogaster. Other drosophilids, such as Drosophila ananassae and the invasive Zaprionus indianus, have multinucleated giant hemocytes, a syncytium of blood cells that participate in the encapsulation of the eggs or larvae of parasitoid wasps. These cells can be formed by the fusion of hemocytes in circulation or originate from the lymph gland. Their ultrastructure highly resembles that of the mammalian megakaryocytes. Methods: Morphological, protein expressional, and functional features of blood cells were revealed using epifluorescence and confocal microscopy. The respective hemocyte subpopulations were identified using monoclonal antibodies in indirect immunofluorescence assays. Fluorescein isothiocyanate (FITC)-labeled Escherichia coli bacteria were used in phagocytosis tests. Gene expression analysis was performed following mRNA sequencing of blood cells. Results: D. ananassae and Z. indianus encapsulate foreign particles with the involvement of multinucleated giant hemocytes and mount a highly efficient immune response against parasitoid wasps. Morphological, protein expressional, and functional assays of Z. indianus blood cells suggested that these cells could be derived from large plasmatocytes, a unique cell type developing specifically after parasitoid wasp infection. Transcriptomic analysis of blood cells, isolated from naïve and wasp-infected Z. indianus larvae, revealed several differentially expressed genes involved in signal transduction, cell movements, encapsulation of foreign targets, energy production, and melanization, suggesting their role in the anti-parasitoid response. A large number of genes that encode proteins associated with coagulation and wound healing, such as phenoloxidase activity factor-like proteins, fibrinogen-related proteins, lectins, and proteins involved in the differentiation and function of platelets, were constitutively expressed. The remarkable ultrastructural similarities between giant hemocytes and mammalian megakaryocytes, and presence of platelets, and giant cell-derived anucleated fragments at wound sites hint at the involvement of this cell subpopulation in wound healing processes, in addition to participation in the encapsulation reaction. Conclusion: Our observations provide insights into the broad repertoire of blood cell functions required for efficient defense reactions to maintain the homeostasis of the organism. The analysis of the differentiation and function of multinucleated giant hemocytes gives an insight into the diversification of the immune mechanisms.

Integrin ßν-mediated phagocytosis of apoptotic cells in Drosophila embryos.

To identify molecules that play roles in the clearance of apoptotic cells by Drosophila phagocytes, we examined a series of monoclonal antibodies raised against larval hemocytes for effects on phagocytosis in vitro. One antibody that inhibited phagocytosis recognized terribly reduced optic lobes (Trol), a core protein of the perlecan-type proteoglycan, and the level of phagocytosis in embryos of a Trol-lacking fly line was lower than in a control line. The treatment of a hemocyte cell line with a recombinant Trol protein containing the amino acid sequence RGD augmented the phosphorylation of focal adhesion kinase, a hallmark of integrin activation. A loss of integrin ßν, one of the two ß subunits of Drosophila integrin, brought about a reduction in the level of apoptotic cell clearance in embryos. The presence of integrin ßν at the surface of embryonic hemocytes was confirmed, and forced expression of integrin ßν in hemocytes of an integrin ßν-lacking fly line recovered the defective phenotype of phagocytosis. Finally, the level of phagocytosis in a fly line that lacks both integrin ßν and Draper, another receptor required for the phagocytosis of apoptotic cells, was lower than that in a fly line lacking either protein. We suggest that integrin ßν serves as a phagocytosis receptor responsible for the clearance of apoptotic cells in Drosophila, independent of Draper.

Sessile hemocytes as a hematopoietic compartment in Drosophila melanogaster.

The blood cells, or hemocytes, in Drosophila participate in the immune response through the production of antimicrobial peptides, the phagocytosis of bacteria, and the encapsulation of larger foreign particles such as parasitic eggs; these immune reactions are mediated by phylogenetically conserved mechanisms. The encapsulation reaction is analogous to the formation of granuloma in vertebrates, and is mediated by large specialized cells, the lamellocytes. The origin of the lamellocytes has not been formally established, although it has been suggested that they are derived from the lymph gland, which is generally considered to be the main hematopoietic organ in the Drosophila larva. However, it was recently observed that a subepidermal population of sessile blood cells is released into the circulation in response to a parasitoid wasp infection. We set out to analyze this phenomenon systematically. As a result, we define the sessile hemocytes as a novel hematopoietic compartment, and the main source of lamellocytes.

Hematopoietic plasticity mapped in Drosophila and other insects.

Hemocytes, similar to vertebrate blood cells, play important roles in insect development and immunity, but it is not well understood how they perform their tasks. New technology, in particular single-cell transcriptomic analysis in combination with Drosophila genetics, may now change this picture. This review aims to make sense of recently published data, focusing on Drosophila melanogaster and comparing to data from other drosophilids, the malaria mosquito, Anopheles gambiae, and the silkworm, Bombyx mori. Basically, the new data support the presence of a few major classes of hemocytes: (1) a highly heterogenous and plastic class of professional phagocytes with many functions, called plasmatocytes in Drosophila and granular cells in other insects. (2) A conserved class of cells that control melanin deposition around parasites and wounds, called crystal cells in D. melanogaster, and oenocytoids in other insects. (3) A new class of cells, the primocytes, so far only identified in D. melanogaster. They are related to cells of the so-called posterior signaling center of the larval hematopoietic organ, which controls the hematopoiesis of other hemocytes. (4) Different kinds of specialized cells, like the lamellocytes in D. melanogaster, for the encapsulation of parasites. These cells undergo rapid evolution, and the homology relationships between such cells in different insects are uncertain. Lists of genes expressed in the different hemocyte classes now provide a solid ground for further investigation of function.

The Effect of Feeding with Central European Local Mulberry Genotypes on the Development and Health Status of Silkworms and Quality Parameters of Raw Silk.

Silkworm rearing activities ceased in the 1970's in several European countries. Attempts on the re-establishment of ecological and sustainable sericulture in Slovenia and Hungary are ongoing. The aim of the study was to assess the usability of locally adapted mulberry genotypes for sericulture and to estimate connections between leaf compound and silkworm performance parameters. A controlled feeding experiment of silkworms was performed to test the influence of leaves from selected trees on the growth of larvae, the health and microbiological status of larvae (e.g., gut bacterial microbiome, Bombyx mori nucleopolyhedrovirus infection), weight of cocoons and raw silk parameters. The Slovenian and Hungarian mulberry genotypes had significantly higher total protein contents, and lower total phenolic contents and differed significantly in some individual phenolics compared to the reference sericultural and fruit varieties. Significant differences were found in the contents of the macro- and microelements, namely S, Mn, Fe, and Sr. Based on correlative statistics and multivariate analysis, a combined positive influence of proteins, specific phenolics, and microelements on larval growth and silk thread parameters was predicted. The results of the study indicate that selected local Slovenian and Hungarian mulberry varieties are suitable for high-quality silk cocoon and raw silk production.

Broad Ultrastructural and Transcriptomic Changes Underlie the Multinucleated Giant Hemocyte Mediated Innate Immune Response against Parasitoids.

Multinucleated giant hemocytes (MGHs) represent a novel type of blood cell in insects that participate in a highly efficient immune response against parasitoid wasps involving isolation and killing of the parasite. Previously, we showed that circulating MGHs have high motility and the interaction with the parasitoid rapidly triggers encapsulation. However, structural and molecular mechanisms behind these processes remained elusive. Here, we used detailed ultrastructural analysis and live cell imaging of MGHs to study encapsulation in Drosophila ananassae after parasitoid wasp infection. We found dynamic structural changes, mainly driven by the formation of diverse vesicular systems and newly developed complex intracytoplasmic membrane structures, and abundant generation of giant cell exosomes in MGHs. In addition, we used RNA sequencing to study the transcriptomic profile of MGHs and activated plasmatocytes 72 h after infection, as well as the uninduced blood cells. This revealed that differentiation of MGHs was accompanied by broad changes in gene expression. Consistent with the observed structural changes, transcripts related to vesicular function, cytoskeletal organization, and adhesion were enriched in MGHs. In addition, several orphan genes encoding for hemolysin-like proteins, pore-forming toxins of prokaryotic origin, were expressed at high level, which may be important for parasitoid elimination. Our results reveal coordinated molecular and structural changes in the course of MGH differentiation and parasitoid encapsulation, providing a mechanistic model for a powerful innate immune response.

Immunoprofiling of Drosophila Hemocytes by Single-cell Mass Cytometry.

Single-cell mass cytometry (SCMC) combines features of traditional flow cytometry (i.e., fluorescence-activated cell sorting) with mass spectrometry, making it possible to measure several parameters at the single-cell level for a complex analysis of biological regulatory mechanisms. In this study, weoptimizedSCMC to analyze hemocytes of the Drosophila innate immune system. We used metal-conjugated antibodies (against cell surface antigens H2, H3, H18, L1, L4, and P1, and intracellular antigens 3A5 and L2) and anti-IgM (against cell surface antigen L6) to detect the levels of antigens, while anti-GFP was used to detect crystal cells in the immune-induced samples. We investigated the antigen expression profile of single cells and hemocyte populations in naive states, in immune-induced states, in tumorous mutants bearing a driver mutation in the Drosophila homologue of Janus kinase (hopTum) and carrying a deficiency of the tumor suppressor gene lethal(3)malignant blood neoplasm-1  [l(3)mbn1], as well as in stem cell maintenance-defective hdcΔ84 mutant larvae. Multidimensional analysis enabled the discrimination of the functionally different major hemocyte subsets for lamellocytes, plasmatocytes, and crystal cells, anddelineated the unique immunophenotype of Drosophila mutants. We have identified subpopulations of L2+/P1+ and L2+/L4+/P1+ transitional phenotype cells in the tumorous strains l(3)mbn1 and hopTum, respectively, and a subpopulation of L4+/P1+ cells upon immune induction. Our results demonstrated for the first time that SCMC, combined with multidimensional bioinformatic analysis, represents a versatile and powerful tool to deeply analyze the regulation of cell-mediated immunity of Drosophila.

Regression plane concept for analysing continuous cellular processes with machine learning.

Biological processes are inherently continuous, and the chance of phenotypic discovery is significantly restricted by discretising them. Using multi-parametric active regression we introduce the Regression Plane (RP), a user-friendly discovery tool enabling class-free phenotypic supervised machine learning, to describe and explore biological data in a continuous manner. First, we compare traditional classification with regression in a simulated experimental setup. Second, we use our framework to identify genes involved in regulating triglyceride levels in human cells. Subsequently, we analyse a time-lapse dataset on mitosis to demonstrate that the proposed methodology is capable of modelling complex processes at infinite resolution. Finally, we show that hemocyte differentiation in Drosophila melanogaster has continuous characteristics.

Nimrod, a putative phagocytosis receptor with EGF repeats in Drosophila plasmatocytes.

The hemocytes, the blood cells of Drosophila, participate in the humoral and cellular immune defense reactions against microbes and parasites [1-8]. The plasmatocytes, one class of hemocytes, are phagocytically active and play an important role in immunity and development by removing microorganisms as well as apoptotic cells. On the surface of circulating and sessile plasmatocytes, we have now identified a protein, Nimrod C1 (NimC1), which is involved in the phagocytosis of bacteria. Suppression of NimC1 expression in plasmatocytes inhibited the phagocytosis of Staphylococcus aureus. Conversely, overexpression of NimC1 in S2 cells stimulated the phagocytosis of both S. aureus and Escherichia coli. NimC1 is a 90-100 kDa single-pass transmembrane protein with ten characteristic EGF-like repeats (NIM repeats). The nimC1 gene is part of a cluster of ten related nimrod genes at 34E on chromosome 2, and similar clusters of nimrod-like genes are conserved in other insects such as Anopheles and Apis. The Nimrod proteins are related to other putative phagocytosis receptors such as Eater and Draper from D. melanogaster and CED-1 from C. elegans. Together, they form a superfamily that also includes proteins that are encoded in the human genome.

Identification of reference markers for characterizing honey bee (Apis mellifera) hemocyte classes.

Cell mediated immunity of the honey bee (Apis mellifera) involves the activity of several hemocyte populations, currently defined by morphological features and lectin binding characteristics. The objective of the present study was to identify molecular markers capable of characterizing subsets of honey bee hemocytes. We developed and employed monoclonal antibodies with restricted reactions to functionally distinct hemocyte subpopulations. Melanizing cells, known as oenocytoids, were defined by an antibody to prophenoloxidase, aggregating cells were identified by the expression of Hemolectin, and phagocytic cells were identified by a marker expressed on granulocytes. We anticipate that this combination of antibodies not only allows for the detection of functionally distinct hemocyte subtypes, but will help to further the exploration of hematopoietic compartments, as well as reveal details of the honey bee cellular immune defense against parasites and microbes.

Cellular Immune Response Involving Multinucleated Giant Hemocytes with Two-Step Genome Amplification in the Drosophilid Zaprionus indianus.

Previously, a novel cell type, the multinucleated giant hemocyte (MGH) was identified in the ananassae subgroup of Drosophilidae. These cells share several features with mammalian multinucleated giant cells, a syncytium of macrophages formed during granulomatous inflammation. We were able to show that MGHs also differentiate in Zaprionus indianus, an invasive species belonging to the vittiger subgroup of the family, highly resistant to a large number of parasitoid wasp species. We have classified the MGHs of Z. indianusas giant hemocytes belonging to a class of cells which also include elongated blood cells carrying a single nucleus and anuclear structures. They are involved in encapsulating parasites, originate from the lymph gland, can develop by cell fusion, and generally carry many nuclei, while possessing an elaborated system of canals and sinuses, resulting in a spongiform appearance. Their nuclei are all transcriptionally active and show accretion of genetic material. Multinucleation and accumulation of the genetic material in the giant hemocytes represents a two-stage amplification of the genome, while their spongy ultrastructure substantially increases the contact surface with the extracellular space. These features may furnish the giant hemocytes with a considerable metabolic advantage, hence contributing to the mechanism of the effective immune response.

Evolution of genes and repeats in the Nimrod superfamily.

The recently identified Nimrod superfamily is characterized by the presence of a special type of EGF repeat, the NIM repeat, located right after a typical CCXGY/W amino acid motif. On the basis of structural features, nimrod genes can be divided into three types. The proteins encoded by Draper-type genes have an EMI domain at the N-terminal part and only one copy of the NIM motif, followed by a variable number of EGF-like repeats. The products of Nimrod B-type and Nimrod C-type genes (including the eater gene) have different kinds of N-terminal domains, and lack EGF-like repeats but contain a variable number of NIM repeats. Draper and Nimrod C-type (but not Nimrod B-type) proteins carry a transmembrane domain. Several members of the superfamily were claimed to function as receptors in phagocytosis and/or binding of bacteria, which indicates an important role in the cellular immunity and the elimination of apoptotic cells. In this paper, the evolution of the Nimrod superfamily is studied with various methods on the level of genes and repeats. A hypothesis is presented in which the NIM repeat, along with the EMI domain, emerged by structural reorganizations at the end of an EGF-like repeat chain, suggesting a mechanism for the formation of novel types of repeats. The analyses revealed diverse evolutionary patterns in the sequences containing multiple NIM repeats. Although in the Nimrod B and Nimrod C proteins show characteristics of independent evolution, many internal NIM repeats in Eater sequences seem to have undergone concerted evolution. An analysis of the nimrod genes has been performed using phylogenetic and other methods and an evolutionary scenario of the origin and diversification of the Nimrod superfamily is proposed. Our study presents an intriguing example how the evolution of multigene families may contribute to the complexity of the innate immune response.

Headcase is a Repressor of Lamellocyte Fate in Drosophila melanogaster.

Due to the evolutionary conservation of the regulation of hematopoiesis, Drosophila provides an excellent model organism to study blood cell differentiation and hematopoietic stem cell (HSC) maintenance. The larvae of Drosophila melanogaster respond to immune induction with the production of special effector blood cells, the lamellocytes, which encapsulate and subsequently kill the invader. Lamellocytes differentiate as a result of a concerted action of all three hematopoietic compartments of the larva: the lymph gland, the circulating hemocytes, and the sessile tissue. Within the lymph gland, the communication of the functional zones, the maintenance of HSC fate, and the differentiation of effector blood cells are regulated by a complex network of signaling pathways. Applying gene conversion, mutational analysis, and a candidate based genetic interaction screen, we investigated the role of Headcase (Hdc), the homolog of the tumor suppressor HECA in the hematopoiesis of Drosophila. We found that naive loss-of-function hdc mutant larvae produce lamellocytes, showing that Hdc has a repressive role in effector blood cell differentiation. We demonstrate that hdc genetically interacts with the Hedgehog and the Decapentaplegic pathways in the hematopoietic niche of the lymph gland. By adding further details to the model of blood cell fate regulation in the lymph gland of the larva, our findings contribute to the better understanding of HSC maintenance.

Two Nimrod receptors, NimC1 and Eater, synergistically contribute to bacterial phagocytosis in Drosophila melanogaster.

Eater and NimC1 are transmembrane receptors of the Drosophila Nimrod family, specifically expressed in haemocytes, the insect blood cells. Previous ex vivo and in vivoRNAi studies have pointed to their role in the phagocytosis of bacteria. Here, we have created a novel NimC1 null mutant to re-evaluate the role of NimC1, alone or in combination with Eater, in the cellular immune response. We show that NimC1 functions as an adhesion molecule ex vivo, but in contrast to Eater it is not required for haemocyte sessility in vivo. Ex vivo phagocytosis assays and electron microscopy experiments confirmed that Eater is the main phagocytic receptor for Gram-positive, but not Gram-negative bacteria, and contributes to microbe tethering to haemocytes. Surprisingly, NimC1 deletion did not impair phagocytosis of bacteria, nor their adhesion to the haemocytes. However, phagocytosis of both types of bacteria was almost abolished in NimC11 ;eater1 haemocytes. This indicates that both receptors contribute synergistically to the phagocytosis of bacteria, but that Eater can bypass the requirement for NimC1. Finally, we uncovered that NimC1, but not Eater, is essential for uptake of latex beads and zymosan particles. We conclude that Eater and NimC1 are the two main receptors for phagocytosis of bacteria in Drosophila, and that each receptor likely plays distinct roles in microbial uptake.

T2prhd: a tool to study the patterns of repeat evolution.

BACKGROUND: The models developed to characterize the evolution of multigene families (such as the birth-and-death and the concerted models) have also been applied on the level of sequence repeats inside a gene/protein. Phylogenetic reconstruction is the method of choice to study the evolution of gene families and also sequence repeats in the light of these models. The characterization of the gene family evolution in view of the evolutionary models is done by the evaluation of the clustering of the sequences with the originating loci in mind. As the locus represents positional information, it is straightforward that in the case of the repeats the exact position in the sequence should be used, as the simple numbering according to repeat order can be misleading. RESULTS: We have developed a novel rapid visual approach to study repeat evolution, that takes into account the exact repeat position in a sequence. The "pairwise repeat homology diagram" visualizes sequence repeats detected by a profile HMM in a pair of sequences and highlights their homology relations inferred by a phylogenetic tree. The method is implemented in a Perl script (t2prhd) available for downloading at http://t2prhd.sourceforge.net and is also accessible as an online tool at http://t2prhd.brc.hu. The power of the method is demonstrated on the EGF-like and fibronectin-III-like (Fn-III) domain repeats of three selected mammalian Tenascin sequences. CONCLUSION: Although pairwise repeat homology diagrams do not carry all the information provided by the phylogenetic tree, they allow a rapid and intuitive assessment of repeat evolution. We believe, that t2prhd is a helpful tool with which to study the pattern of repeat evolution. This method can be particularly useful in cases of large datasets (such as large gene families), as the command line interface makes it possible to automate the generation of pairwise repeat homology diagrams with the aid of scripts.

Hemolectin expression reveals functional heterogeneity in honey bee (Apis mellifera) hemocytes.

The identification of molecular markers considerably facilitated the classification and functional analysis of blood cell types. Apis mellifera hemocytes have been classified by morphological criteria and lectin binding properties; however, the use of molecular markers has been minimal. Here we describe a monoclonal antibody to a non-phagocytic subpopulation of A. mellifera hemocytes and to a constituent of the hemolymph clot. We demonstrate that the antibody identifies the A. mellifera hemolectin, a protein carrying human von Willebrand factor homology domains, characteristic of proteins involved in blood coagulation and platelet aggregation in mammals. Hemolectin expressing A. mellifera hemocytes contain the protein as cytoplasmic granules and contribute to the formation of a protein matrix, building up around foreign particles. Consequently, hemolectin as a marker molecule reveals a clear functional heterogeneity of hemocytes, allowing for the analytical separation of hemocyte classes, and could promote the molecular identification of hemocyte lineages in A. mellifera.

Genes encoding cuticular proteins are components of the Nimrod gene cluster in Drosophila.

The Nimrod gene cluster, located on the second chromosome of Drosophila melanogaster, is the largest synthenic unit of the Drosophila genome. Nimrod genes show blood cell specific expression and code for phagocytosis receptors that play a major role in fruit fly innate immune functions. We previously identified three homologous genes (vajk-1, vajk-2 and vajk-3) located within the Nimrod cluster, which are unrelated to the Nimrod genes, but are homologous to a fourth gene (vajk-4) located outside the cluster. Here we show that, unlike the Nimrod candidates, the Vajk proteins are expressed in cuticular structures of the late embryo and the late pupa, indicating that they contribute to cuticular barrier functions.