Back to the future: Forgotten protocols for optimizing the isolation of arthropod haemocytes.

Consideration is given to previous and more recent protocols for harvesting arthropod haemocytes from Galleria, Drosophila, mosquitoes, Limulus and crustaceans. The optimal harvesting of these cells is essential for meaningful studies of invertebrate immunity in vitro. The results of such experiments, however, have often been flawed due to a lack of understanding of the fragile nature of arthropod haemocytes on exposure to bacterial lipopolysaccharides, resulting in the aggregation and loss of cell types during haemolymph clotting. This article emphasizes that although there are similarities between mammalian neutrophils and arthropod haemocytes, the protocols required for the successful harvesting of these cells vary significantly. The various stages for the successful harvesting of arthropod haemocytes are described in detail and should provide invaluable advice to those requiring both high cell viability and recovery of the different cell types for subsequent experimentation.

Dscam in arthropod immune priming: What is known and what remains unknown.

A popular view in the current academic circle is that invertebrates have no adaptive immunity. However, the immune memory and specificity of invertebrates pose a serious challenge to this view and constitute immune priming based on innate immunity. The Down syndrome cell adhesion molecule (Dscam) gene of invertebrates, with approximately 10,000 alternatively spliced isoforms, has a unique characteristic: it specifically binds to different types of bacteria and promotes cell phagocytosis; owing to its antibody-like function, Dscam is a key candidate protein for immune priming. However, the high molecular diversity of Dscam and the gaps and inconsistencies in the existing research make the study of regulation of immune priming by Dscam challenging. In recent years, significant research has been conducted on the Dscam-regulated immune functions in insects and crustaceans, providing preliminary results for Dscam-regulated innate immunity and immune priming, but some important questions remain unresolved. In this review, we summarize the existing knowledge about Dscam-regulated immunity and discuss three yet unanswered questions, the study of which may improve the understanding of the role of Dscam-regulated immune priming in invertebrates.

Allergens from Edible Insects: Cross-reactivity and Effects of Processing.

PURPOSE OF REVIEW: The recent introduction of edible insects in Western countries has raised concerns about their safety in terms of allergenic reactions. The characterization of insect allergens, the sensitization and cross-reactivity mechanisms, and the effects of food processing represent crucial information for risk assessment. RECENT FINDINGS: Allergic reactions to different insects and cross-reactivity with crustacean and inhalant allergens have been described, with the identification of new IgE-binding proteins besides well-known pan-allergens. Depending on the route of sensitization, different potential allergens seem to be involved. Food processing may affect the solubility and the immunoreactivity of insect allergens, with results depending on species and type of proteins. Chemical/enzymatic hydrolysis, in some cases, abolishes immunoreactivity. More studies based on subjects with a confirmed insect allergy are necessary to identify major and minor allergens and the role of the route of sensitization. The effects of processing need to be further investigated to assess the risk associated with the ingestion of insect-containing food products.

Immune properties of invertebrate phenoloxidases.

Melanin production from different types of phenoloxidases (POs) confers immunity from a variety of pathogens ranging from viruses and microorganisms to parasites. The arthropod proPO expresses a variety of activities including cytokine, opsonin and microbiocidal activities independent of and even without melanin production. Proteolytic processing of proPO and its activating enzyme gives rise to several peptide fragments with a variety of separate activities in a process reminiscent of vertebrate complement system activation although proPO bears no sequence similarity to vertebrate complement factors. Pathogens influence proPO activation and thereby what types of immune effects that will be produced. An increasing number of specialised pathogens - from parasites to viruses - have been identified who can synthesise compounds specifically aimed at the proPO-system. In invertebrates outside the arthropods phylogenetically unrelated POs are participating in melanization reactions obviously aimed at intruders and/or aberrant tissues.

Proteases and pseudoproteases in parasitic arthropods of clinical importance.

Parasitic arthropods feed on blood or skin tissue and share comparable repertoires of proteases involved in haematophagy, digestion, egg development and immunity. While proteolytically active proteases of multiple classes dominate, an increasing number of pseudoproteases have been discovered that have no proteolytic function but are pharmacologically active biomolecules, evolved to carry out alternative functions as regulatory, antihaemostatic, anti-inflammatory or immunomodulatory compounds. In this review, we provide an overview of proteases and pseudoproteases from clinically important arthropod parasites. Many of these act in central biological pathways of parasite survival and host-parasite interaction and may be potential targets for therapeutic interventions.

Immunometabolism in Arthropod Vectors: Redefining Interspecies Relationships.

Metabolism influences biochemical networks, and arthropod vectors are endowed with an immune system that affects microbial acquisition, persistence, and transmission to humans and other animals. Here, we aim to persuade the scientific community to expand their interests in immunometabolism beyond mammalian hosts and towards arthropod vectors. Immunometabolism investigates the interplay of metabolism and immunology. We provide a conceptual framework for investigators from diverse disciplines and indicate that relationships between microbes, mammalian hosts and their hematophagous arthropods may result in cost-effective (mutualism) or energetically expensive (parasitism) interactions. We argue that disparate resource allocations between species may partially explain why some microbes act as pathogens when infecting humans and behave as mutualistic or commensal organisms when colonizing arthropod vectors.

Multifunctional Roles of Hemocyanins.

The copper-containing hemocyanins are proteins responsible for the binding, transportation and storage of dioxygen within the blood (hemolymph) of many invertebrates. Several additional functions have been attributed to both arthropod and molluscan hemocyanins, including (but not limited to) enzymatic activity (namely phenoloxidase), hormone transport, homeostasis (ecdysis) and hemostasis (clot formation). An important secondary function of hemocyanin involves aspects of innate immunity-such as acting as a precursor of broad-spectrum antimicrobial peptides and microbial/viral agglutination. In this chapter, we present the reader with an up-to-date synthesis of the known functions of hemocyanins and the structural features that facilitate such activities.

Development of a comprehensive protein microarray for immunoglobulin E profiling in horses with severe asthma.

BACKGROUND: Severe asthma in horses, known as severe equine asthma (SEA), is a prevalent, performance-limiting disease associated with increased allergen-specific immunoglobulin E (IgE) against a range of environmental aeroallergens. OBJECTIVE: To develop a protein microarray platform to profile IgE against a range of proven and novel environmental proteins in SEA-affected horses. ANIMALS: Six SEA-affected and 6 clinically healthy Warmblood performance horses. METHODS: Developed a protein microarray (n = 384) using protein extracts and purified proteins from a large number of families including pollen, bacteria, fungi, and arthropods associated with the horses, environment. Conditions were optimized and assessed for printing, incubation, immunolabeling, biological fluid source, concentration techniques, reproducibility, and specificity. RESULTS: This method identified a number of novel allergens, while also identifying an association between SEA and pollen sensitization. Immunolabeling methods confirmed the accuracy of a commercially available mouse anti-horse IgE 3H10 source (R2 = 0.91). Biological fluid source evaluation indicated that sera and bronchoalveolar lavage fluid (BALF) yielded the same specific IgE profile (average R2 = 0.75). Amicon centrifugal filters were found to be the most efficient technique for concentrating BALF for IgE analysis at 40-fold. Overnight incubation maintained the same sensitization profile while increasing sensitivity. Reproducibility was demonstrated (R2 = 0.97), as was specificity using protein inhibition assays. Arthropods, fungi, and pollens showed the greatest discrimination for SEA. CONCLUSIONS AND CLINICAL IMPORTANCE: We have established that protein microarrays can be used for large-scale IgE mapping of allergens associated with the environment of horses. This technology provides a sound platform for specific diagnosis, management, and treatment of SEA.

Cellular and Humoral Responses to Cte f 2, a Cat Flea Allergen, in Children with Papular Urticaria.

BACKGROUND: Papular urticaria (PU) is a common insect bite skin hypersensitivity in tropical countries. In order to gain insight into its causal allergens, we aimed to evaluate cellular and humoral immune responses to the recombinant salivary antigen Cte f 2 from the cat flea Ctenocephalides felis. METHOD: Sixty patients with PU and 27 healthy controls were included in this study. Specific IgE, IgG, IgG1, and IgG4 against Cte f 2 and C. felis extract were determined by ELISA. The T-cell response was analyzed using a carboxyfluorescein succinimidyl ester (CFSE)-based dilution assay and Th1/Th2/Th17 cytokine measurements. In addition, a proteomic analysis of IgG and IgE reactive spots of C. felis extract was performed. RESULTS: The frequency of IgE sensitization to Cte f 2 was similar between patients (36.7%) and controls (40.7%). The specific IgE, IgG1, and IgG4 responses to Cte f 2 and C. felis extract were not significantly different between patients and controls. Among the 3 conditions (i.e., Cte f 2, C. felis extract, and only medium) Cte f 2 was the strongest inducer of CD3+CD4+ proliferation in the patients; however, the mean response was not significantly different from those in controls (Cte f 2: 4.5 vs. 2.5%; p = 0.46). No salivary proteins were identified in C. felis, and most of the spots were identified as muscle-skeletal components (tropomyosin, actin, myosin, and ankirin). CONCLUSIONS: Cte f 2 induces IgE and IgG production as well as T-cell proliferation in children living in a geographical area where PU induced by a flea bite is common. The use of C. felis extract is not recommended for the study of bite-induced hypersensitivity disease since salivary antigens are not well represented.

Vector Immunity and Evolutionary Ecology: The Harmonious Dissonance.

Recent scientific breakthroughs have significantly expanded our understanding of arthropod vector immunity. Insights in the laboratory have demonstrated how the immune system provides resistance to infection, and in what manner innate defenses protect against a microbial assault. Less understood, however, is the effect of biotic and abiotic factors on microbial-vector interactions and the impact of the immune system on arthropod populations in nature. Furthermore, the influence of genetic plasticity on the immune response against vector-borne pathogens remains mostly elusive. Herein, we discuss evolutionary forces that shape arthropod vector immunity. We focus on resistance, pathogenicity and tolerance to infection. We posit that novel scientific paradigms should emerge when molecular immunologists and evolutionary ecologists work together.

Eosinophil-Related Disease and the Skin.

Eosinophils are bone marrow-derived cells that infiltrate skin and mucous membrane in a broad spectrum of primary and reactive inflammatory diseases and malignancies. The eosinophil has potent proinflammatory activities, particularly, through the effects of its toxic granule proteins. In addition, eosinophils have prothrombotic and profibrotic activities. Eosinophil participation in the pathogenesis of certain diseases without identifiable intact eosinophil infiltration may not be recognized because eosinophil degranulation is poorly visualized on hematoxylin-and-eosin-stained histopathology sections. Eosinophil-related pathophysiology can involve virtually every component of skin. Commonly recognized dermatoses associated with eosinophils are arthropod bite and sting reactions and drug eruptions, "bugs and drugs." Skin involvement is common in eosinophil-related systemic diseases including the hypereosinophilic syndromes. Eosinophil-related pathophysiology may play a key role in numerous disorders that, therefore, may benefit from therapies targeted to reduce or eliminate eosinophils.

Metazoan Parasite Vaccines: Present Status and Future Prospects.

Eukaryotic parasites and pathogens continue to cause some of the most detrimental and difficult to treat diseases (or disease states) in both humans and animals, while also continuously expanding into non-endemic countries. Combined with the ever growing number of reports on drug-resistance and the lack of effective treatment programs for many metazoan diseases, the impact that these organisms will have on quality of life remain a global challenge. Vaccination as an effective prophylactic treatment has been demonstrated for well over 200 years for bacterial and viral diseases. From the earliest variolation procedures to the cutting edge technologies employed today, many protective preparations have been successfully developed for use in both medical and veterinary applications. In spite of the successes of these applications in the discovery of subunit vaccines against prokaryotic pathogens, not many targets have been successfully developed into vaccines directed against metazoan parasites. With the current increase in -omics technologies and metadata for eukaryotic parasites, target discovery for vaccine development can be expedited. However, a good understanding of the host/vector/pathogen interface is needed to understand the underlying biological, biochemical and immunological components that will confer a protective response in the host animal. Therefore, systems biology is rapidly coming of age in the pursuit of effective parasite vaccines. Despite the difficulties, a number of approaches have been developed and applied to parasitic helminths and arthropods. This review will focus on key aspects of vaccine development that require attention in the battle against these metazoan parasites, as well as successes in the field of vaccine development for helminthiases and ectoparasites. Lastly, we propose future direction of applying successes in pursuit of next generation vaccines.

A systematic investigation on the composition, evolution and expression characteristics of chemokine superfamily in grass carp Ctenopharyngodon idella.

Chemokines are a superfamily of small cytokines and characterized based on their ability to induce directional migration of cells along a concentration gradient by binding to chemokine receptors, which have important roles in immunology and development. Due to the numerous and diverse members, systematic identifications of chemokine superfamily genes are difficult in many species. To that end, a comprehensive analysis of BLAST and scripting language was conducted to systematically identify and characterize chemokine system in grass carp (Ctenopharyngodon idella). Our results showed that C. idella chemokine superfamily consists of 81 chemokines and 37 receptors, in which, most genes possess typical structural features of the chemokine superfamily. Phylogenetic analyses confirmed the existence of three chemokine subfamilies (CC, CXC and XC) in C. idella and revealed their homologous relationships with other species. Chemokine receptors are transmembrane receptors and contains CCR, CXCR, XCR and ACKR subfamilies. mRNA expression analyses of chemokine superfamily genes indicated that many members are sustainably expressed in multiple tissues before and after grass carp reovirus (GCRV) or Aeromonas hydrophila infection, which provides in vivo evidence for the response patterns after viral or bacterial infection. Meanwhile, this study also explored the evolution of chemokine system from arthropod to higher vertebrates and then investigated the changes in gene number/diversification, gene organization and encoded proteins during vertebrate evolution. These results will serve the further functional and evolutional studies on chemokine superfamily.

Assessment of the ability of V920 recombinant vesicular stomatitis-Zaire ebolavirus vaccine to replicate in relevant arthropod cell cultures and vector species.

V920, rVSVΔG-ZEBOV-GP, is a recombinant vesicular stomatitis-Zaire ebolavirus vaccine which has shown an acceptable safety profile and provides a protective immune response against Ebola virus disease (EVD) induced by Zaire ebolavirus in humans. The purpose of this study was to determine whether the V920 vaccine is capable of replicating in arthropod cell cultures of relevant vector species and of replicating in live mosquitoes. While the V920 vaccine replicated well in Vero cells, no replication was observed in Anopheles or Aedes mosquito, Culicoides biting midge, or Lutzomyia sand fly cells, nor in live Culex or Aedes mosquitoes following exposure through intrathoracic inoculation or feeding on a high-titer infectious blood meal. The insect taxa selected for use in this study represent actual and potential epidemic vectors of VSV. V920 vaccine inoculated into Cx. quinquefasciatus and Ae. aegypti mosquitoes demonstrated persistence of replication-competent virus following inoculation, consistent with the recognized biological stability of the vaccine, but no evidence for active virus replication in live mosquitoes was observed. Following administration of an infectious blood meal to Ae. aegypti and Cx. quinquefasciatus mosquitoes at a titer several log10 PFU more concentrated than would be observed in vaccinated individuals, no infection or dissemination of V920 was observed in either mosquito species. In vitro and in vivo data gathered during this study support minimal risk of the vector-borne potential of the V920 vaccine.

The Dose Makes the Poison: Nutritional Overload Determines the Life Traits of Blood-Feeding Arthropods.

Vertebrate blood composition is heavily biased towards proteins, and hemoglobin, which is a hemeprotein, is by far the most abundant protein. Typically, hematophagous insects ingest blood volumes several times their weight before the blood meal. This barbarian feast offers an abundance of nutrients, but the degradation of blood proteins generates toxic concentrations of amino acids and heme, along with unparalleled microbiota growth. Despite this challenge, hematophagous arthropods have successfully developed mechanisms that bypass the toxicity of these molecules. While these adaptations allow hematophagous arthropods to tolerate their diet, they also constitute a unique mode of operation for cell signaling, immunity, and metabolism, the study of which may offer insights into the biology of disease vectors and may lead to novel vector-specific control methods.

Allergens involved in the cross-reactivity of Aedes aegypti with other arthropods.

BACKGROUND: Cross-reactivity between Aedes aegypti and mites, cockroaches, and shrimp has been previously suggested, but the involved molecular components have not been fully described. OBJECTIVE: To evaluate the cross-reactivity between A aegypti and other arthropods. METHODS: Thirty-four serum samples from patients with asthma and/or allergic rhinitis were selected, and specific IgE to A aegypti, Dermatophagoides pteronyssinus, Dermatophagoides farinae, Blomia tropicalis, Periplaneta americana. and Litopenaeus vannamei was measured by enzyme-linked immunosorbent assay. Cross-reactivity was investigated using pooled serum samples from allergic patients, allergenic extracts, and the recombinant tropomyosins (Aed a 10.0201, Der p 10, Blo t 10, Lit v 1, and Per a 7). Four IgE reactive bands were further characterized by matrix-assisted laser desorption/ionization tandem time of flight. RESULTS: Frequency of positive IgE reactivity was 82.35% to at least one mite species, 64.7% to A aegypti, 29.4% to P americana, and 23.5% to L vannamei. The highest IgE cross-reactivity was seen between A aegypti and D pteronyssinus (96.6%) followed by L vannamei (95.4%), B tropicalis (84.4%), and P americana (75.4%). Recombinant tropomyosins from mites, cockroach, or shrimp inhibited the IgE reactivity to the mosquito at a lower extent than the extracts from these arthropods. Several bands of A aegypti cross-reacted with arthropod extracts, and 4 of them were identified as odorant binding protein, mitochondrial cytochrome C, peptidyl-prolyl cis-trans isomerase, and protein with hypothetical magnesium ion binding function. CONCLUSION: We identified 4 novel cross-reactive allergens in A aegypti allergenic extract. These molecules could influence the manifestation of allergy to environmental allergens in the tropics.

Infection-derived lipids elicit an immune deficiency circuit in arthropods.

The insect immune deficiency (IMD) pathway resembles the tumour necrosis factor receptor network in mammals and senses diaminopimelic-type peptidoglycans present in Gram-negative bacteria. Whether unidentified chemical moieties activate the IMD signalling cascade remains unknown. Here, we show that infection-derived lipids 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) and 1-palmitoyl-2-oleoyl diacylglycerol (PODAG) stimulate the IMD pathway of ticks. The tick IMD network protects against colonization by three distinct bacteria, that is the Lyme disease spirochete Borrelia burgdorferi and the rickettsial agents Anaplasma phagocytophilum and A. marginale. Cell signalling ensues in the absence of transmembrane peptidoglycan recognition proteins and the adaptor molecules Fas-associated protein with a death domain (FADD) and IMD. Conversely, biochemical interactions occur between x-linked inhibitor of apoptosis protein (XIAP), an E3 ubiquitin ligase, and the E2 conjugating enzyme Bendless. We propose the existence of two functionally distinct IMD networks, one in insects and another in ticks.

Traces of an ancient immune system - how an injured arthropod survived 465 million years ago.

This report of a severely injured trilobite from the Middle Ordovician (~465 Ma) accords with a number of similar observations of healed lesions observed in trilobites. The uniqueness of the specimen described here is that the character of the repair-mechanisms is reflected by the secondarily built structures, which form the new surface of the ruptured compound eye. Smooth, repaired areas inside the visual surface advert to a clotting principle, rather similar to those of today, and the way in which broken parts of the exoskeleton fused during restoration seem to simulate modern samples. The irregularity and variance of newly inserted visual units indicate the severity of the injury, which, most probably, was caused by a predatory attack, presumably by a cephalopod; these were most likely, the top predators of the Ordovician. Furthermore, the state of the moulted cephalon tells the dramatic struggle of an organism that lived in the Palaeozoic, to survive. In sum the specimen analysed here is evidence of an ancient clotting mechanism not dissimilar to those of today, rapidly preventing any exsanguination and the breakdown of osmoregulation of this marine arthropod.

Arthropod Innate Immune Systems and Vector-Borne Diseases.

Arthropods, especially ticks and mosquitoes, are the vectors for a number of parasitic and viral human diseases, including malaria, sleeping sickness, Dengue, and Zika, yet arthropods show tremendous individual variation in their capacity to transmit disease. A key factor in this capacity is the group of genetically encoded immune factors that counteract infection by the pathogen. Arthropod-specific pattern recognition receptors and protease cascades detect and respond to infection. Proteins such as antimicrobial peptides, thioester-containing proteins, and transglutaminases effect responses such as lysis, phagocytosis, melanization, and agglutination. Effector responses are initiated by damage signals such as reactive oxygen species signaling from epithelial cells and recognized by cell surface receptors on hemocytes. Antiviral immunity is primarily mediated by siRNA pathways but coupled with interferon-like signaling, antimicrobial peptides, and thioester-containing proteins. Molecular mechanisms of immunity are closely linked to related traits of longevity and fertility, and arthropods have the capacity for innate immunological memory. Advances in understanding vector immunity can be leveraged to develop novel control strategies for reducing the rate of transmission of both ancient and emerging threats to global health.