Mosquito Gap Junctions - American Chemical Society

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Mosquito Gap Junctions: Molecular Biology, Physiology, and Potential for Insecticide Development T. L. Calkins and P. M. Piermarini* Department of Entomology, Ohio Agricultural Research and Development Center, The Ohio State University, 1680 Madison Avenue, Wooster, Ohio 44691, United States *E-mail: [email protected].

Mosquitoes are the most dangerous animals on the planet due to the pathogens they transmit to humans. For many mosquito-borne diseases we do not have effective vaccines or therapeutics and instead must control the mosquito vectors to prevent transmission of the diseases. However, mosquitoes are developing resistance to insecticides currently used in vector control (e.g., pyrethroids), making the development of new insecticides a necessity. Here we review the molecular biology and physiology of intercellular channels, known as gap junctions, in dipteran insects and discuss their potential as targets for mosquitocide development. Gap junctions allow for direct communication between adjacent cells. All animals possess gap junctions, but they are comprised of evolutionarily distinct families of proteins in vertebrates (connexins) vs. invertebrates (innexins). Gap junctions play key roles in a diverse range of physiological functions ranging from embryogenesis to reproduction. In Drosophila melanogaster, gap junctions are integral to development, allow for cell coupling in electrical synapses, and are involved in functional gonad formation and gamete production. Recent work by our group and others in mosquitoes has found key roles of gap junctions in reproduction, immunity, excretion, and larval and adult survival. Thus, gap junctions play integral roles in

© 2017 American Chemical Society Gross et al.; Advances in Agrochemicals: Ion Channels and G Protein-Coupled Receptors (GPCRs) as Targets for Pest ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.

mosquito biology and if their function can be disrupted with selective chemical and/or genetic inhibitors then they may provide excellent targets for mosquitocide development.

1. Introduction


1.1. Control of Mosquito Borne Disease There are over 3,500 species of mosquitoes around the world, which are found on every continent except Antarctica (1). Only a handful of these species represent a threat to human and/or animal health, yet they are considered the most dangerous animals on Earth (2). Mosquitoes of several genera (e.g., Anopheles, Aedes, and Culex) transmit parasitic and/or viral pathogens, including those that cause malaria, West Nile, dengue fever, and Zika in human hosts. The burden of these diseases is severe, with malaria alone causing over a half million deaths every year, dengue afflicting hundreds of millions of people annually, and Zika being linked to serious birth defects when infecting pregnant women (3–6). Moreover, the burden of mosquito-borne diseases extends to animals, with mosquitoes transmitting pathogens that cause heartworm in canines, encephalitis in equines, and Rift Valley fever in livestock (7). Unfortunately, many of these diseases lack vaccines and therapeutics; thus, control of the mosquito vectors is typically the primary strategy to control their transmission. Mosquito control often relies on the use of insecticides that target the nervous system, such as pyrethroids that modulate voltage-gated sodium channels and carbamates that inhibit acetylcholineesterase. Although these compounds are highly effective at killing mosquitoes, the overuse of a limited diversity of active compounds has exerted a strong selective pressure for the evolution of resistance in the form of target-site resistance (e.g., point mutations in voltage-gated Na+-channels) and/or metabolic resistance. (e.g., up-regulation of cytochrome P450 detoxification enzymes; (8, 9)). Thus, to replenish and diversify our chemical arsenal and facilitate the control of ‘resistant’ mosquitoes, it is necessary to modify existing compounds (10), and/or identify new molecular and physiological targets for developing insecticides with novel mechanisms of action. Here, we explore the latter possibility by reviewing the molecular biology and physiology of gap junctions in dipterans within the context of potentially exploiting them to develop insecticides for mosquito control. 1.2. A Brief History of Gap Junctions Gap junctions are intercellular channels that mediate direct communication between adjacent cells via the transfer of small molecules and/or ions. They have been identified in nearly all animals from hydra to humans (11–13). In vertebrates, ultrastructural evidence for gap junctions—hexagonal arrays of proteins in adjacent cell membranes—was first observed in the goldfish (Carassius auratus) brain in what was described as an electrical synapse (14). It was not until similar structures were discovered in the heart and liver cells of mice that these formations were recognized as new intercellular junctions and the term ‘gap 92 Gross et al.; Advances in Agrochemicals: Ion Channels and G Protein-Coupled Receptors (GPCRs) as Targets for Pest ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


junction’ was coined (15, 16). In the early 1980’s, partial protein sequences were elucidated from the rat liver (17). However, it was not until the late 1980s, that the genes encoding vertebrate gap junctional subunits, named connexins, were first described in humans and rats (11, 18). In invertebrates, ultrastructural evidence of gap junctions and their subsequent electrophysiological verification were first described in the crayfish (Astacus fluviatilis) (19, 20). Despite this evidence, immunochemical attempts to identify connexin-like molecules in crayfish failed (21). Furthermore, the genomes of both Drosophila melanogaster and Caenorhabditis elegans yielded no homologous connexin genes, indicating that a separate gene family must be responsible for encoding gap junctional subunits in invertebrates (22, 23). In the late 1990’s, a family of genes unrelated to the connexins was discovered in D. melanogaster and C. elegans, and demonstrated to form gap junctions in vitro when expressed heterologously in paired Xenopus oocytes; these genes were named ‘innexins’ (invertebrate connexins) (24–27). Intriguingly, vertebrate genomes possess innexin homologues known as pannexins (28). However, pannexins only appear to form hemichannels in the plasma membrane of vertebrate cells and not gap junctions between cells. Pannexins play important functional roles in vertebrate cells, such as mediating the transport of small molecules (e.g. ATP) across the plasma membrane (29, 30). Physiological evidence suggests that some innexins are also able to form functional hemichannels in the plasma membrane (31, 32). In addition, a viral-based group of innexins (vinnexins) has recently been identified in polydnaviruses. The vinnexins are homologous to insect innexins and can form gap junctions when expressed heterologously in Xenopus oocytes (33). Thus, the innexins are part of the proposed ‘pannexin’ gene family (34) that also includes vertebrate pannexins and viral vinnexins. 1.3. A General Comparison of Connexins and Innexins Connexins and innexins are evolutionarily distinct protein families that have convergently evolved to form gap junctions. Despite minimal similarity between the amino-acid sequences of connexins and innexin, the resulting monomeric subunits are predicted to share a similar membrane topology that includes four transmembrane domains, 2 extracellular loops, one intracellular loop, and intracellular NH2- and COOH- termini (Figure 1A). The monomeric subunits form oligomeric hemichannels, which consist of identical (homotypic) or different (heterotypic) connexin/innexin subunits. Each hemichannel reaches the membrane via vesicular transport where it docks with a complementary hemichannel in the plasma membrane of an adjacent cell to form a pore between the cells, thereby allowing for direct intercellular communication (Figure 1B). The amino-acid sequences of the transmembrane domains are the most highly conserved within connexins or innexins; these domains form the intercellular pore of the gap junction (35). On the other hand, the amino-acid sequences of the extracellular loops and particularly the intracellular termini are relatively less conserved (36, 37), with the intracellular termini playing a role in modulating gating of the channel (38). Notably, the extracellular loops of connexins and 93 Gross et al.; Advances in Agrochemicals: Ion Channels and G Protein-Coupled Receptors (GPCRs) as Targets for Pest ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


innexins possess conserved cysteine residues (Figure 1A), which are thought to mediate docking between hemichannels in opposing membranes (39). If the molecular composition of the docked hemichannels is identical then the resulting gap junctions are considered homotypic, but if they are different then they are considered heterotypic. In addition to their similar topology and structure, the factors influencing the opening and closing of gap junctions (i.e., gating) are often similar. For example, a rapid decrease in intracellular pH or a rapid increase of intracellular Ca2+ typically results in the closure of gap junctions (40, 41). Likewise, most gap junctions are similarly gated by electophysiological parameters of the plasma membrane. For example, changes of the membrane potential or increased junctional voltage conductance can lead to closure of gap junctions (42). The subunit composition (i.e. the specific innexins/connexins forming a gap junction) also influences the gating of the gap junctional channels and how they respond to changes in membrane potential (38, 43). The gating of gap junctions is also regulated by cell signaling factors. For example, intracellular cAMP can open gap junctions (44), presumably through activation of protein kinase A and the phosphorylation of amino acid residues on the intracellular termini of connexins/innexins (45).

Figure 1. Structure of gap junctions. A) Illustration of the membrane topology of an innexin/connexin gap junctional subunit. The purple cysteines residues are found in both innexins and connexins, whereas the green cysteine residues are found only in connexins; they are thought to modulate the docking of opposing hemichannels. B) Illustration of how innexin or connexin subunits combine to form hemichannels and gap junctions, allowing for intercellular communication.

In general, the physiological roles of connexins and innexins are remarkably similar. Both are required during embryonic development for proper tissue formation and cell migration (46–49), as well as for post-embryonic tissue development (50–52). Moreover, connexins and innexins are critical to the functions of excitable and epithelial tissues (19, 53–55). Below, we focus on the specific molecular and functional properties of innexins, as well as their physiological roles, in dipteran insects. 94 Gross et al.; Advances in Agrochemicals: Ion Channels and G Protein-Coupled Receptors (GPCRs) as Targets for Pest ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


1.4. Molecular and Functional Properties of Dipteran Innexins In dipteran insects, there are 6 phylogenetic clades of innexins: inx1, inx2, inx3, inx4, inx7, and inx8 (Figure 2). Fruit flies (D. melanogaster), mosquitoes, and tsetse flies (Glossina austeni) each possess a single gene representative of each clade, with the exception of the inx4 clade, which has diversified via apparent gene duplications to form inx5 and inx6 in D. melanogaster and inx5 in G. austeni (Figure 2). Mosquito genomes typically possess six innexins, which are named after their homologues in D. melanogaster: inx1, inx2, inx3, inx4, inx7, and inx8. Thus, dipterans appear to have evolved with a core set of at least 6 innexins. The molecular diversity of dipteran innexins can be further enhanced by alternative splicing of mRNA transcripts. In D. melanogaster, inx8 is demonstrated to have at least 3 splice variants resulting in novel predicted amino-acid sequences in the NH2-terminal domain; these splices show differential expression in the giant fiber system (56). Additionally, inx2 in D. melanogaster is expressed as two splice variants, but the predicted amino acid sequences are identical given that the splicing occurs in the 5’ or 3’ untranslated regions (57). In the Malpighian tubules of mosquitoes, we have cloned two splice variants of inx1 and inx3, which result in predicted proteins with distinct COOH-termini (36). To date, the functional characterization of dipteran innexins in heterologous expression systems has been limited to a few genes in D. melanogaster. Inx8 of D. melanogaster (DmInx8; also known as Shaking-B, ShakB, and passover) was the first insect innexin shown to form functional homotypic gap junctions (58). That is, when expressed heterologously in Xenopus oocytes, inx8 mediated the transport of electrical current between paired cells (Figure 3; (58)). Moreover, two of the aforementioned differential splice forms of inx8 in D. melanogaster form heterotypic gap junctions with novel gating and rectification properties when expressed heterologously in Xenopus oocytes (38, 59). Thus, alternative splicing of innexins can influence the functional properties of the formed gap junctions. In contrast to DmInx8, homomeric hemichannels of DmInx2 or DmInx3 do not consistently form functional homotypic gap junctions when expressed in paired Xenopus oocytes (57). However, if both DmInx2 and DmInx3 are expressed in the same cells, then they form functional gap junctions that mediate the transport of electrical current between paired oocytes (57). Thus, DmInx2 and DmInx3 subunits likely oligomerize to generate heteromeric hemichannels that form homotypic gap junctions between cells. Specific functional properties of the other innexin members in heterologous expression systems remain to be elucidated, but as described in the following section, molecular expression, reverse genetic, and immunochemical studies have revealed insights into the broader physiological roles innexins are likely playing in dipteran insects.

95 Gross et al.; Advances in Agrochemicals: Ion Channels and G Protein-Coupled Receptors (GPCRs) as Targets for Pest ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


Figure 2. Phylogenetic tree of innexin proteins of Ae. aegypti (AaInx), An. gambiae (AgInx), Culex quinquefasciatus (CqInx), D. melanogaster (DmInx), G. austeni (GaInx). The tree is rooted to a human pannexin (HumanPANX1). All innexin proteins cluster into 6 general clades: inx1, inx2, inx3, inx4, inx7, and inx8. Although, inx1 is not annotated in the C. quinquefasciatus genome and is not included in this figure, a BLAST of AaInx1 against the C. quinquefasciatus genome reveals high homology in an unannotated region (5 potential exons located on supercontig3.776 from 71,153 to 131,352 bp).



Figure 3. A) Illustration of a dual voltage clamp paired oocyte assay, one of several approaches used to measure the coupling mediated by innexins or connexins. The image is modified from (24) with permission. Oocytes are injected with capped RNA encoding an innexin or connexin subunit, and after a few days are placed in direct contact with each other, allowing gap junctions to potentially form between the cells. Each oocyte is pierced with two microelectrodes to respectively measure the membrane voltage (Vm) of the cells and inject current (Im). Both cells are initially clamped to the same membrane potential before initiating a series of voltage steps in cell 1 to generate a transjunctional voltage (Vj) between the cells (panel B). The current injected into cell 2 to maintain the indicated Vj is the junctional current (Ij) and is indicative of electrical coupling via gap junctions (panel C). The Ij in paired control (H2O-injected) oocytes would be nominal. Reproduced with permission from reference (58). 1998 Macmillan Publishers Ltd.

2. Physiological Roles of Dipteran Innexins In flies, innexins are integral to numerous physiological processes, including embryonic development, nervous system function, reproduction, immune responses, and excretion; all are of great potential use for exploitation in mosquito control. The subsections below highlight the roles of innexins in these physiological processes of dipterans. 97 Gross et al.; Advances in Agrochemicals: Ion Channels and G Protein-Coupled Receptors (GPCRs) as Targets for Pest ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


2.1. Embryonic Development Throughout embryogenesis flies develop from a single cell to a multicellular organism with differentiated cells and tissues. This process involves coordination between cells for proper tissue differentiation, orientation, and development. As such, gap junctional communication between developing cells is expected to be critical for embryogenesis. Consistent with this notion, in situ hybridization studies indicate that most innexins undergo differential expression during early embryonic development (60). Specific roles of innexins in development have been elucidated utilizing reverse genetic techniques. For example, RNAi-driven depletion of inx3 mRNA in D. melanogaster led to a failure to complete dorsal closure (49). Moreover, in D. melanogaster, loss of function mutants of inx2 exhibit defective epithelial cell migration and morphogenesis (61, 62). In the central nervous system (CNS; giant fiber system and ganglia) of D. melanogaster, inx8 isoforms are differentially expressed throughout embryonic development (63). Moreover, mutations of the inx8 locus lead to impaired CNS development in flies (64). Innexins are also important in the development of peripheral nerves. Notably, loss of functional inx1 expression via mutation in the gene locus results in a severely impaired optical nerve development, inspiring its original name ogre (optical ganglion reduced) (50, 65). Furthermore, RNAimediated knockdown of inx7 results in a severe reduction of peripheral nervous system development (48).

2.2. Nervous System Function Innexins remain integral during larval and adult development, especially in the nervous system. Larvae of D. melanogaster require inx1 for proper cell proliferation in the CNS (66). Inx1 and inx2 are also required in glial cells for normal neuronal development into adulthood (51). Moreover, inx1 and inx8 are required for pre- and post- synaptic development of photoreceptor neurons during pupation and for proper neuronal development (67). As adults, D. melanogaster require gap junctions for functional electrical synapses. Like the giant motor neuron of the crayfish and its role in the tail flip escape response (19, 68), D. melanogaster possesses a giant fiber system (GFS) that utilizes electrical synapses for its jump-flight escape response (69). In the GFS, loss of function mutations in inx8 abolish electrical synapses in the adult fly and the jump response (26). Moreover, splice variants of inx8 that exhibit unique voltage-gating properties in electrical synapses are expressed throughout the GFS, likely allowing for differential rectification of the electrical synapses (38, 63). In D. melanogaster inx8 and other innexins are involved in connecting the neuronal circuitry of the Johnston’s organ to that of the GFS (70, 71). Thus, innexins, especially inx8, are critical to proper development and functioning of the dipteran nervous system.



2.3. Reproduction In D. melanogaster and An. gambiae, inx4 (also known as zero population growth or zpg) is necessary for gonad development and the proper differentiation and survival of developing gametes in both males and females (72–74). Moreover, RNAi-induced gene silencing of inx4 in embryos of An. gambiae or larvae and pupae of Ae. aegypti results in a sterile (spermless) male phenotype (75, 76). Although the sterile males still mate with females, the resulting eggs laid are not viable (75, 76). In D. melanogaster, inx4 mRNA expression and protein localization occurs in the germline cells of the gonads, which are adjacent to somatic cells that express inx2. This potentially allows for the formation of heterotypic gap junctions between somatic and germline cells, which may be necessary for signaling involved with oogenesis and spermatogenesis (77–79). Inx2 also co-localizes with inx3 in the intercellular membranes of somatic cells in the ovaries of D. melanogaster, where it is considered necessary for formation of the egg chamber (79, 80). In adult Ae. aegypti, we have shown that inx1, inx2, inx3, and inx4 mRNAs are expressed in both the testes and ovaries (36). Moreover, inx3 immunoreactivity localizes to the intercellular boundaries between epithelial cells of the follicle in the ovaries of Ae. aegypti (Figure 4), suggesting a potential role of inx3 in coordinating secretion of the chorion. Furthermore, gap junctions mediate coupling between follicle cells and the developing oocyte in dipterans, allowing the follicle cells to provision the developing oocyte properly. Calmodulin is one such molecule required by the developing oocyte for induction of vitellogenin (major yolk protein) uptake (81, 82). In D. melanogaster, calmodulin is transferred from the follicle cells to the oocyte via gap junctions (83). Additionally, in mosquitoes, gap junctions between the oocyte and follicle cells have been observed (84). Thus, gap junctions are integral to fly reproduction from gamete production through vitellogenesis.

2.4. Immune Responses In cockroaches and lepidopterans, gap junctions form between hemocytes during the encapsulation response (85, 86), which is a component of the cellular immune response to parasites that enter the hemolymph. Moreover, inx2 and inx3 cDNAs have been cloned from the hemocytes of a lepidopteran (87). Intriguingly, polydnaviruses associated with parasitic wasps possess vinnexins (the viral orthologs of innexins) that may disrupt the encapsulation response of the wasps’ lepidopteran hosts by interacting with inx2 in the hemocytes of the host (32, 88, 89). Although hemocytes of D. melanogaster have a similar encapsulation response, gap junctions have not yet been implicated (90).



Figure 4. Inx3 immunolabeling in an ovary of Ae. aegypti. Inx3 localizes to the intercellular boundaries between the follicle epithelial cells that surround each oocyte resulting in a honeycomb-like pattern of immunolabeling. In color version, inx3 labeling is green and DAPI nuclear staining is cyan.

In An. gambiae, inx1 is integral to the up-regulation of thioester-containing protein 1 (TEP1) after ingesting blood meals containing malarial parasites (Plasmodium falciparum) (91). TEP1 is a complement-like protein that targets pathogens for phagocytosis by hemocytes (92). As such, knockdown of inx1 mRNA expression via RNAi leads to increased invasion and infection of the mosquito by malarial parasites (91). Thus, innexins could potentially play an important role in modulating the vector competence of mosquitoes. 2.5. Excretion The renal (Malpighian) tubules of mosquitoes are essential for diuresis after engorging on a blood meal. In the Malpighian tubules of adult female Ae. aegypti, gap junctions are present between principal cells and allow for electrical coupling within the epithelium (54), which expresses as many as 4 innexin mRNAs: inx1, inx2, inx3, and inx7 (36, 54). Notably, in the Malpighian tubules of Ae. aegypti, Inx3 immunoreactivity localizes to the intercellular boundaries between principal cells (Figure 5) (36), suggesting that it may be playing a role in their electrical coupling. Furthermore, gap junctions are likely 100 Gross et al.; Advances in Agrochemicals: Ion Channels and G Protein-Coupled Receptors (GPCRs) as Targets for Pest ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


involved with intercellular signaling in Malpighian tubules and coordinating their rapid physiological responses to diuretic neuropeptides. That is, the receptors for kinin and calcitonin-like peptides, key neuroendocrine factors that regulate the post-prandial diuresis, do not occur in every cell along the length of the Malpighian tubules (93, 94). However, upon treatment with either of these hormones, the epithelium generates a rapid, coordinated physiological response, suggesting robust intercellular communication mediated by gap junctions (95). Consistent with an important role of gap junctions in the post-prandial diuresis, we have shown that the diuretic capacity of adult female mosquitoes is suppressed when gap junctions are pharmacologically inhibited in vivo (96).

Figure 5. Inx3 immunolabeling in a Malpighian tubule of an adult female Ae. aegypti. Inx3 localizes to the intercellular boundaries between principal cells (e.g., short arrow), but not those between principal and stellate cells (long arrow indicates a stellate cell nucleus), suggesting a potentially important role of inx3 in the coupling of principal cells. Strong circular labeling is DAPI nuclear staining. In color version, inx3 labeling is green and DAPI staining is cyan.

3. Gap Junctions as Molecular Targets for Mosquito Control The diverse and critical physiological roles of innexins/gap junctions in dipterans reviewed above suggests that inhibiting gap junctions could have drastic consequences on development, nervous function, immune function, reproduction, and excretion in mosquitoes. Thus, gap junctions may be intriguing targets for insecticide development. We have recently tested the hypothesis that the 101 Gross et al.; Advances in Agrochemicals: Ion Channels and G Protein-Coupled Receptors (GPCRs) as Targets for Pest ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


inhibition of gap junctions would impair the survival of Ae. aegypti by using parallel pharmacological and reverse genetic approaches. Notably, the addition of chemical inhibitors of gap junctions to the larval rearing water kills 1st instar larvae within 24 hr (Figure 6) (96). Likewise, the hemolymph injection of gap junction inhibitors is toxic to adult female mosquitoes within 24 hr (Figure 6) (96). Consistent with these results, the simultaneous knockdown of several innexin mRNAs (inx1, inx2, inx3, inx4, inx7) in adult female mosquitoes via RNA interference significantly shortens their life span (96). Thus, our data suggest that the inhibition of gap junctions is a potentially viable mode of action for killing mosquitoes.

Figure 6. Toxicity of gap junction inhibitors in A) larvae (via addition to rearing water) and B) adult females (via hemolymph injection) of Ae. aegypti. Mortality or efficacy (i.e., death or inability to fly) was assessed 24 h after treatment. Reproduced with permission from reference (96). Copyright 2015 Calkins, Piermarini.

Before gap junctions can be fully validated as potential mosquitocide targets, it must first be demonstrated that mosquito- selective chemical and/or genetic inhibitors of innexins can be developed to minimize effects on non-target species. RNAi-based insecticides targeting mosquito innexins appear to offer the best chance for species selectivity. However, our recent development of a mosquito-selective, small-molecule inhibitor of inward rectifier K+ (Kir) channels that is topically toxic to mosquitoes and not honey bees (97) suggests that chemical-based mosquitocides targeting innexins should not be ruled out as a possibility. In addition to insecticide targets, gap junctions could be considered as potential targets for developing sterile male mosquitoes for utilization in biological control. Notably, RNAi- induced gene silencing of inx4 in An. gambiae and Ae. aegypti results in sterile males (75, 76). Thus, if the pharmacological or genetic inhibition of inx4 can be induced in mosquito production facilities, then it may offer a mechanism for producing sterile males sans radiation. 102 Gross et al.; Advances in Agrochemicals: Ion Channels and G Protein-Coupled Receptors (GPCRs) as Targets for Pest ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.

4. Conclusion


Mosquito-borne diseases are emerging and reemerging globally, threatening human health. Moreover, mosquito populations are evolving resistance to currently used insecticides for vector control, adding urgency for the development of insecticides with new mechanisms of action for controlling resistant mosquitoes. The vital and diverse physiological roles of innexins/gap junctions in fruit flies and mosquitoes suggests that they offer viable targets for insecticide development. It remains to be determined whether highly specific inhibitors of innexins/gap junctions can be developed that would not have toxic effects on beneficial insects, such as pollinators, and vertebrates.

Acknowledgments The authors are grateful for funding from the National Institutes of Health (R03DK090186), Mosquito Research Foundation (2014–03), OARDC SEEDS program (Grant# 2014– 078; oardc.osu.edu/seeds), Ohio Mosquito Control Association Grant-In-Aid, Sigma Xi Grants-in-Aid-of Research, and state and federal funds appropriated to the OARDC of The Ohio State University, which have supported their work on mosquito gap junctions over the past 6 years.

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