The Lyme disease agent co-opts adiponectin receptor-mediated signaling in its arthropod vector.

Adiponectin-mediated pathways contribute to mammalian homeostasis; however, little is known about adiponectin and adiponectin receptor signaling in arthropods. In this study, we demonstrate that Ixodes scapularis ticks have an adiponectin receptor-like protein (ISARL) but lack adiponectin, suggesting activation by alternative pathways. ISARL expression is significantly upregulated in the tick gut after Borrelia burgdorferi infection, suggesting that ISARL signaling may be co-opted by the Lyme disease agent. Consistent with this, RNA interference (RNAi)-mediated silencing of ISARL significantly reduced the B. burgdorferi burden in the tick. RNA-seq-based transcriptomics and RNAi assays demonstrate that ISARL-mediated phospholipid metabolism by phosphatidylserine synthase I is associated with B. burgdorferi survival. Furthermore, the tick complement C1q-like protein 3 interacts with ISARL, and B. burgdorferi facilitates this process. This study identifies a new tick metabolic pathway that is connected to the life cycle of the Lyme disease spirochete.

Many countries around the world are seeing an increase in the number of patients diagnosed with Lyme disease, with often serious joint, heart, and neurologic complications. This illness is caused by species of 'spirochete' bacteria that live and multiply inside black-legged ticks, and get injected into mammals upon a bite. Ticks are not simply 'syringes' however, and a complex relationship is established between spirochetes and their host. This is particularly true since Lyme disease-causing bacteria such as Borrelia burgdorferi rely on ticks to obtain energy and nutrients. Tang, Cao et al. delved into these complex interactions by focusing on the molecular cascades (or pathways) involving adiponectin, a hormone essential for regulating sugar levels and processing fats. Analyses of gene and protein databases highlighted that ticks carry a receptor-like protein for adiponectin but not the hormone itself, suggesting that an alternative pathway is at play. This may involve B. burgdorferi, which gets its fats and sugars from its host. And indeed, experiments showed that ticks produced more of the adiponectin receptor-like protein when they carried B. burgdorferi; conversely, silencing the receptor reduced the number of surviving spirochetes inside the tick. Further exploration showed that the receptor mediates molecular cascades that help to process fat molecules; these are associated with spirochete survival. In addition, the receptor-like protein was activated by C1QL3, a 'complement 1q domain-contained' molecule which might be part of the tick energy-making or immune systems. Larger quantities of C1QL3 were found in ticks upon B. burgdorferi infection, suggesting that the spirochete facilitates an interaction that boosts activity of the adiponectin receptor-like protein. Overall, the work by Tang and Cao et al. revealed a new pathway which B. burgdorferi takes advantage of to infect their host and multiply. Targeting this molecular cascade could help to interfere with the life cycle of the spirochete, as well as fight Lyme disease and other insect-borne conditions.

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.

G protein-coupled receptors in arthropod vectors: omics and pharmacological approaches to elucidate ligand-receptor interactions and novel organismal functions.

Regulation of many physiological processes in animals, certainly those controlled by neuropeptide hormones, involves G protein-coupled receptors (GPCRs). Our work focusing on endocrine regulation of diuresis and water balance in mosquitoes and ticks started in 1997 with the kinin receptor, at the dawn of the omics era. After the genomic revolution, we began work on the endocrinology of reproduction in the red imported fire ant. We will use the template of this comparative work to summarize key points about GPCRs and signaling, and emphasize the most recent developments in the pharmacology of arthropod neuropeptide GPCRs. We will discuss omics' contributions to the advancement of this field, and its influence on peptidomimetic design while emphasizing work on blood feeding arthropods.

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.

Identification and functional characterization of a pyrokinin neuropeptide receptor in the Lyme disease vector, Ixodes scapularis.

Pyrokinin-related peptides are pleiotropic factors that are defined by their conserved C-terminal sequence FXPRL-NH2. The pyrokinin nomenclature derives from their originally identified myotropic actions and, as seen in some family members, a blocked amino terminus with pyroglutamate. The black-legged tick, Ixodes scapularis, is well known as a vector of Lyme disease and various other illnesses; however, in comparison to blood-feeding insects, knowledge on its physiology (along with other Ixodid ticks) is rather limited. In this study, we have isolated, examined the expression profile, and functionally deorphanized the pyrokinin peptide receptor in the medically important tick, I. scapularis. Phylogenetic analysis supports that the cloned receptor is indeed a bona fide member of the pyrokinin-related peptide receptor family. The tick pyrokinin receptor transcript expression is most abundant in the central nervous system (i.e. synganglion), but is also detected in trachea, female reproductive tissues, and in a pooled sample comprised of Malpighian (renal) tubules and the hindgut. Finally, functional characterization of the identified receptor confirmed it as a pyrokinin peptide receptor as it was activated equally by four endogenous pyrokinin-related peptides. The receptor was slightly promiscuous as it was also activated by a peptide sharing some structural similarity, namely the CAPA-periviserokinin (CAPA-PVK) peptide. Nonetheless, the I. scapularis pyrokinin receptor required a CAPA-PVK peptide concentration of well over three orders of magnitude to achieve a comparable receptor activation response, which indicates it is quite selective for its native pyrokinin peptide ligands. This study sets the stage for future research to examine the prospective tissue targets identified in order to resolve the physiological roles of this family of peptides in Ixodid ticks.

Grandeur Alliances: Symbiont Metabolic Integration and Obligate Arthropod Hematophagy.

Several arthropod taxa live exclusively on vertebrate blood. This food source lacks essential metabolites required for the maintenance of metabolic homeostasis, and as such, these arthropods have formed symbioses with nutrient-supplementing microbes that facilitate their host's 'hematophagous' feeding ecology. Herein we highlight metabolic contributions of bacterial symbionts that reside within tsetse flies, bed bugs, lice, reduviid bugs, and ticks, with specific emphasis on B vitamin and cofactor biosynthesis. Importantly, these arthropods can transmit pathogens of medical and veterinary relevance and/or cause infestations that induce psychological and dermatological distress. Microbial metabolites, and the biochemical pathways that generate them, can serve as specific targets of novel control mechanisms aimed at disrupting the metabolism of hematophagous arthropods, thus combatting pest invasion and vector-borne pathogen transmission.

Proteomics characterization of tick-host-pathogen interactions.

Ticks are blood-feeding arthropod ectoparasites of wild and domestic animals that transmit disease-causing pathogens to humans and animals worldwide and a good model for the characterization of tick-host-pathogen interactions. Tick-host-pathogen interactions consist of dynamic processes involving genetic traits of hosts, pathogens, and ticks that mediate their development and survival. Proteomics provides information on the protein content of cells and tissues that may differ from results at the transcriptomics level and may be relevant for basic biological studies and vaccine antigen discovery. In this chapter, we describe various methods for protein extraction and for proteomics analysis in ticks based on one-dimensional gel electrophoresis to characterize tick-host-pathogen interactions. Particularly relevant for this characterization is the use of blood-fed ticks. Therefore, we put special emphasis on working with replete ticks collected after feeding on vertebrate hosts.

Novel identification of Dermacentor variabilis Arp2/3 complex and its role in rickettsial infection of the arthropod vector.

Tick-borne spotted fever group (SFG) Rickettsia species must be able to infect both vertebrate and arthropod host cells. The host actin-related protein 2/3 (Arp2/3) complex is important in the invasion process and actin-based motility for several intracellular bacteria, including SFG Rickettsia in Drosophila and mammalian cells. To investigate the role of the tick Arp2/3 complex in tick-Rickettsia interactions, open reading frames of all subunits of the protein including Arp2, Arp3, ARPC1, ARPC2, ARPC3, ARPC4, and ARPC5 were identified from Dermacentor variabilis. Amino acid sequence analysis showed variation (ranging from 25-88%) in percent identity compared to the corresponding subunits of the complex from Drosophila melanogaster, Mus musculus, Homo sapiens, and Saccharomyces cerevisiae. Potential ATP binding sites were identified in D. variabilis (Dv) Arp2 and Arp3 subunits as well as five putative WD (Trp-Asp) motifs which were observed in DvARPC1. Transcriptional profiles of all subunits of the DvArp2/3 complex revealed greater mRNA expression in both Rickettsia-infected and -uninfected ovary compared to midgut and salivary glands. In response to R. montanensis infection of the tick ovary, the mRNA level of only DvARPC4 was significantly upregulated compared to uninfected tissues. Arp2/3 complex inhibition bioassays resulted in a decrease in the ability of R. montanensis to invade tick tissues with a significant difference in the tick ovary, indicating a role for the Arp2/3 complex in rickettsial invasion of tick cells. Characterization of tick-derived molecules associated with rickettsial infection is imperative in order to better comprehend the ecology of tick-borne rickettsial diseases.

A dityrosine network mediated by dual oxidase and peroxidase influences the persistence of Lyme disease pathogens within the vector.

Ixodes scapularis ticks transmit a wide array of human and animal pathogens including Borrelia burgdorferi; however, how tick immune components influence the persistence of invading pathogens remains unknown. As originally demonstrated in Caenorhabditis elegans and later in Anopheles gambiae, we show here that an acellular gut barrier, resulting from the tyrosine cross-linking of the extracellular matrix, also exists in I. scapularis ticks. This dityrosine network (DTN) is dependent upon a dual oxidase (Duox), which is a member of the NADPH oxidase family. The Ixodes genome encodes for a single Duox and at least 16 potential peroxidase proteins, one of which, annotated as ISCW017368, together with Duox has been found to be indispensible for DTN formation. This barrier influences pathogen survival in the gut, as an impaired DTN in Doux knockdown or in specific peroxidase knockdown ticks, results in reduced levels of B. burgdorferi persistence within ticks. Absence of a complete DTN formation in knockdown ticks leads to the activation of specific tick innate immune pathway genes that potentially resulted in the reduction of spirochete levels. Together, these results highlighted the evolution of the DTN in a diverse set of arthropod vectors, including ticks, and its role in protecting invading pathogens like B. burgdorferi. Further understanding of the molecular basis of tick innate immune responses, vector-pathogen interaction, and their contributions in microbial persistence may help the development of new targets for disrupting the pathogen life cycle.

Decoding the ubiquitin-mediated pathway of arthropod disease vectors.

Protein regulation by ubiquitin has been extensively described in model organisms. However, characterization of the ubiquitin machinery in disease vectors remains mostly unknown. This fundamental gap in knowledge presents a concern because new therapeutics are needed to control vector-borne diseases, and targeting the ubiquitin machinery as a means for disease intervention has been already adopted in the clinic. In this study, we employed a bioinformatics approach to uncover the ubiquitin-mediated pathway in the genomes of Anopheles gambiae, Aedes aegypti, Culex quinquefasciatus, Ixodes scapularis, Pediculus humanus and Rhodnius prolixus. We observed that (1) disease vectors encode a lower percentage of ubiquitin-related genes when compared to Drosophila melanogaster, Mus musculus and Homo sapiens but not Saccharomyces cerevisiae; (2) overall, there are more proteins categorized as E3 ubiquitin ligases when compared to E2-conjugating or E1-activating enzymes; (3) the ubiquitin machinery within the three mosquito genomes is highly similar; (4) ubiquitin genes are more than doubled in the Chagas disease vector (R. prolixus) when compared to other arthropod vectors; (5) the deer tick I. scapularis and the body louse (P. humanus) genomes carry low numbers of E1-activating enzymes and HECT-type E3 ubiquitin ligases; (6) R. prolixus have low numbers of RING-type E3 ubiquitin ligases; and (7) C. quinquefasciatus present elevated numbers of predicted F-box E3 ubiquitin ligases, JAB and UCH deubiquitinases. Taken together, these findings provide novel opportunities to study the interaction between a pathogen and an arthropod vector.

Functional insights into recombinant TROSPA protein from Ixodes ricinus.

Lyme disease (also called borreliosis) is a prevalent chronic disease transmitted by ticks and caused by Borrelia burgdorferi s. l. spirochete. At least one tick protein, namely TROSPA from I. scapularis, commonly occurring in the USA, was shown to be required for colonization of the vector by bacteria. Located in the tick gut, TROSPA interacts with the spirochete outer surface protein A (OspA) and initiates the tick colonization. Ixodes ricinus is a primary vector involved in B. burgdorferi s. l. transmission in most European countries. In this study, we characterized the capacities of recombinant TROSPA protein from I. ricinus to interact with OspA from different Borrelia species and to induce an immune response in animals. We also showed that the N-terminal part of TROSPA (a putative transmembrane domain) is not involved in the interaction with OspA and that reduction of the total negative charge on the TROSPA protein impaired TROSPA-OspA binding. In general, the data presented in this paper indicate that recombinant TROSPA protein retains the capacity to form a complex with OspA and induces a significant level of IgG in orally immunized rats. Thus, I. ricinus TROSPA may be considered a good candidate component for an animal vaccine against Borrelia.

Reciprocal regulation of NF-kB (Relish) and Subolesin in the tick vector, Ixodes scapularis.

BACKGROUND: Tick Subolesin and its ortholog in insects and vertebrates, Akirin, have been suggested to play a role in the immune response through regulation of nuclear factor-kappa B (NF-kB)-dependent and independent gene expression via interaction with intermediate proteins that interact with NF-kB and other regulatory proteins, bind DNA or remodel chromatin to regulate gene expression. The objective of this study was to characterize the structure and regulation of subolesin in Ixodes scapularis. I. scapularis is a vector of emerging pathogens such as Borrelia burgdorferi, Anaplasma phagocytophilum and Babesia microti that cause in humans Lyme disease, anaplasmosis and babesiosis, respectively. The genome of I. scapularis was recently sequenced, and this tick serves as a model organism for the study of vector-host-pathogen interactions. However, basic biological questions such as gene organization and regulation are largely unknown in ticks and other arthropod vectors. PRINCIPAL FINDINGS: The results presented here provide evidence that subolesin/akirin are evolutionarily conserved at several levels (primary sequence, gene organization and function), thus supporting their crucial biological function in metazoans. These results showed that NF-kB (Relish) is involved in the regulation of subolesin expression in ticks, suggesting that as in other organisms, different NF-kB integral subunits and/or unknown interacting proteins regulate the specificity of the NF-kB-mediated gene expression. These results suggested a regulatory network involving cross-regulation between NF-kB (Relish) and Subolesin and Subolesin auto-regulation with possible implications in tick immune response to bacterial infection. SIGNIFICANCE: These results advance our understanding of gene organization and regulation in I. scapularis and have important implications for arthropod vectors genetics and immunology highlighting the possible role of NF-kB and Subolesin/Akirin in vector-pathogen interactions and for designing new strategies for the control of vector infestations and pathogen transmission.

Tubular structure induced by a plant virus facilitates viral spread in its vector insect.

Rice dwarf virus (RDV) replicates in and is transmitted by a leafhopper vector in a persistent-propagative manner. Previous cytopathologic and genetic data revealed that tubular structures, constructed by the nonstructural viral protein Pns10, contain viral particles and are directly involved in the intercellular spread of RDV among cultured leafhopper cells. Here, we demonstrated that RDV exploited these virus-containing tubules to move along actin-based microvilli of the epithelial cells and muscle fibers of visceral muscle tissues in the alimentary canal, facilitating the spread of virus in the body of its insect vector leafhoppers. In cultured leafhopper cells, the knockdown of Pns10 expression due to RNA interference (RNAi) induced by synthesized dsRNA from Pns10 gene strongly inhibited tubule formation and prevented the spread of virus among insect vector cells. RNAi induced after ingestion of dsRNA from Pns10 gene strongly inhibited formation of tubules, preventing intercellular spread and transmission of the virus by the leafhopper. All these results, for the first time, show that a persistent-propagative virus exploits virus-containing tubules composed of a nonstructural viral protein to traffic along actin-based cellular protrusions, facilitating the intercellular spread of the virus in the vector insect. The RNAi strategy and the insect vector cell culture provide useful tools to investigate the molecular mechanisms enabling efficient transmission of persistent-propagative plant viruses by vector insects.

Update on the proteomics of major arthropod vectors of human and animal pathogens.

Vector-borne diseases (VBDs) are defined as infectious diseases of humans and animals caused by pathogenic agents such as viruses, protists, bacteria, and helminths transmitted by the bite of blood-feeding arthropod (BFA) vectors. VBDs represent a major public health threat in endemic areas, generally subtropical zones, and many are considered to be neglected diseases. Genome sequencing of some arthropod vectors as well as modern proteomic and genomic technologies are expanding our knowledge of arthropod-pathogen interactions. This review describes the proteomic approaches that have been used to investigate diverse biological questions about arthropod vectors, including the interplay between vectors and pathogens. Proteomic studies have identified proteins and biochemical pathways that may be involved in molecular crosstalk in BFA-pathogen associations. Future work can build upon this promising start and functional analyses coupled with interactome bioassays will be carried out to investigate the role of candidate peptides and proteins in BFA-human pathogen associations. Dissection of the host-pathogen interactome will be key to understanding the strategies and biochemical pathways used by BFAs to cope with pathogens.

Molecular characterization and tissue-specific gene expression of Dermacentor variabilis α-catenin in response to rickettsial infection.

Alpha catenin is a cytoskeleton protein that acts as a regulator of actin rearrangement by forming an E-cadherin adhesion complex. In Dermacentor variabilis, a putative α-catenin (Dvα-catenin) was previously identified as differentially regulated in ovaries of ticks chronically infected with Rickettsia montanensis. To begin characterizing the role(s) of Dvα-catenin during rickettsial infection, the full-length Dvα-catenin cDNA was cloned and analysed. Comparative sequence analysis demonstrates a 3069-bp cDNA with a 2718-bp open reading frame with a sequence similar to Ixodes scapularisα-catenin. A portion of Dvα-catenin is homologous to the vinculin-conserved domain containing a putative actin-binding region and ß-catenin-binding and -dimerization regions. Quantitative reverse-transcription PCR analysis demonstrated that Dvα-catenin is predominantly expressed in tick ovaries and is responsive to tick feeding. The tissue-specific gene expression analysis of ticks exposed to Rickettsia demonstrates that Dvα-catenin expression was significantly downregulated 12 h after exposure to R. montanensis, but not in Rickettsia amblyommii-exposed ovaries, compared with Rickettsia-unexposed ticks. Studying tick-derived molecules associated with rickettsial infection will provide a better understanding of the transmission dynamics of tick-borne rickettsial diseases.

Molecular identification of Salp15, a key salivary gland protein in the transmission of lyme disease spirochetes, from Ixodes persulcatus and Ixodes pacificus (Acari: Ixodidae).

Salp15 is a multifunctional protein, vital to the tick in its need to obtain vertebrate host blood without stimulating a host inflammatory and immune response. The Salpl5 protein from both Ixodes scapularis Say and Ixodes ricinus (L.), the principal vectors of the Lyme disease spirochete in eastern North America and Europe, respectively, have been well characterized and found to bind the murine CD4 receptor, DC-SIGN, and the OspC protein of Borrelia burgdorferi. In the current study, we characterized the full salp15 gene in Ixodes pacificus Cooley & Kohls and Ixodes persulcatus Schulze, the principal vectors of Lyme disease spirochetes in western North America and Asia, respectively. In comparing the Salp15 protein of all four principal vector ticks of public health importance for the transmission of Lyme disease spirochetes, we find the 53 C-terminal amino acids to have a high degree of similarity. There are at least three clades in the tree of Salp15 and its homologues, probably representing a multigene family.

The crystal structure of D7r4, a salivary biogenic amine-binding protein from the malaria mosquito Anopheles gambiae.

The D7-related (D7r) proteins of the malaria vector Anopheles gambiae have been shown to bind the biogenic amines serotonin, norepinephrine, and histamine with high affinity. One member of the group (D7r1 or hamadarin) has also been shown to have an anticoagulant/antikinin activity. To understand the mechanistic details of its antihemostatic/anti-inflammatory effects, we have determined the crystal structure of one member of this group, D7r4, along with the structures of ligand complexes with serotonin, tryptamine, histamine, and norepinephrine. The D7 fold consists of an arrangement of eight alpha-helices stabilized by three disulfide bonds. The structure is similar to those of the arthropod odorant-binding proteins, a relationship that had been predicted based on sequence comparisons. Although odorant-binding proteins commonly have six alpha-helices, D7r4 has eight, resulting in significantly different positioning and structure of the ligand binding pocket. The pocket itself is lined by hydrophobic side chains along with polar and charged groups oriented to form hydrogen bonds with the aliphatic amino group and with groups on the aromatic portions of the ligands. These structures, along with accompanying mutagenesis studies, have allowed us to identify critical residues for biogenic amine binding and to predict which members of the large D7 protein family found in blood-feeding nematocerous Diptera will function as biogenic amine-binding proteins.

Babesial vector tick defensin against Babesia sp. parasites.

Antimicrobial peptides are major components of host innate immunity, a well-conserved, evolutionarily ancient defensive mechanism. Infectious disease-bearing vector ticks are thought to possess specific defense molecules against the transmitted pathogens that have been acquired during their evolution. We found in the tick Haemaphysalis longicornis a novel parasiticidal peptide named longicin that may have evolved from a common ancestral peptide resembling spider and scorpion toxins. H. longicornis is the primary vector for Babesia sp. parasites in Japan. Longicin also displayed bactericidal and fungicidal properties that resemble those of defensin homologues from invertebrates and vertebrates. Longicin showed a remarkable ability to inhibit the proliferation of merozoites, an erythrocyte blood stage of equine Babesia equi, by killing the parasites. Longicin was localized at the surface of the Babesia sp. parasites, as demonstrated by confocal microscopic analysis. In an in vivo experiment, longicin induced significant reduction of parasitemia in animals infected with the zoonotic and murine B. microti. Moreover, RNA interference data demonstrated that endogenous longicin is able to directly kill the canine B. gibsoni, thus indicating that it may play a role in regulating the vectorial capacity in the vector tick H. longicornis. Theoretically, longicin may serve as a model for the development of chemotherapeutic compounds against tick-borne disease organisms.

Comparative structure-activity analysis of insect kinin core analogs on recombinant kinin receptors from Southern cattle tick Boophilus microplus (Acari: Ixodidae) and mosquito Aedes aegypti (Diptera: Culicidae).

The systematic analysis of structure-activity relationships of insect kinins on two heterologous receptor-expressing systems is described. Previously, kinin receptors from the southern cattle tick, Boophilus microplus (Canestrini), and the dengue vector, the mosquito Aedes aegypti (L.), were functionally and stably expressed in CHO-K1 cells. In order to determine which kinin residues are critical for the peptide-receptor interaction, kinin core analogs were synthesized as an Ala-replacement series of the peptide FFSWGa and tested by a calcium bioluminescence plate assay. The amino acids Phe(1) and Trp(4) were essential for activity of the insect kinins in both receptors. It was confirmed that the pentapeptide kinin core is the minimum sequence required for activity and that the C-terminal amide is also essential. In contrast to the tick receptor, a large increase in efficacy is observed in the mosquito receptor when the C-terminal pentapeptide is N-terminally extended to a hexapeptide. The aminoisobutyric acid (Aib)-containing analog, FF[Aib]WGa, was as active as superagonist FFFSWGa on the mosquito receptor in contrast to the tick receptor where it was statistically more active than FFFSWGa by an order of magnitude. This restricted conformation Aib analog provides information on the conformation associated with the interaction of the insect kinins with these two receptors. Furthermore, the analog FF[Aib]WGa has been previously shown to resist degradation by the peptidases ACE and nephrilysin and represents an important lead in the development of biostable insect kinin analogs that ticks and mosquitoes cannot readily deactivate.