The immune strategies of mosquito Aedes aegypti against microbial infection

Developmental and Comparative Immunology xxx (2017) 1e10

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The immune strategies of mosquito Aedes aegypti against microbial infection Yan-Hong Wang a, Meng-Meng Chang a, b, Xue-Li Wang a, Ai-Hua Zheng a, Zhen Zou a, b, * a b

State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China University of Chinese Academy of Sciences, Beijing 100049, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 25 September 2017 Received in revised form 30 November 2017 Accepted 3 December 2017 Available online xxx

Yellow fever mosquito Aedes aegypti transmits many devastating arthropod-borne viruses (arboviruses), such as dengue virus, yellow fever virus, Chikungunya virus, and Zika virus, which cause great concern to human health. Mosquito control is an effective method to block the spread of infectious diseases. Ae. aegypti uses its innate immune system to fight against arboviruses, parasites, and fungi. In this review, we briefly summarize the recent findings in the immune response of Ae. aegypti against arboviral and entomopathogenic infections. This review enriches our understanding of the mosquito immune system and provides evidence to support the development of novel mosquito control strategies. © 2017 Elsevier Ltd. All rights reserved.

Keywords: Aedes aegypti Innate immunity Arbovirus Entomopathogenic fungus

1. Introduction Mosquitoes require repeated blood feeding to achieve their nutritional requirements for reproduction, and this makes them effective vectors of many dangerous human diseases (Attardo et al., 2005; Schnitger et al., 2009). For example, malaria, transmitted by the Anopheles genus, resulted in almost half a million deaths in 2015 (WHO, 2016). Another serious epidemic, dengue fever, which is mainly transmitted by the yellow fever mosquito Aedes aegypti, causes over a hundred million cases annually. Zika virus (ZIKV), also transmitted by Ae. aegypti, has recently become a significant global health concern due to its rapid geographical expansion in 2015 (Rajah et al., 2016). A number of factors contribute to this serious situation, including the lack of effective vaccines, rapid development of drug resistance, and socio-economic problems in endemic countries. Therefore, it is necessary to explore effective strategies to control mosquito-borne diseases. Vector insects control is the primary measure used to mitigate the spread of infectious diseases. However, insecticide resistance is one of the major obstacles to the control of insects pests (Wang et al., 2011, 2013). The long-term use of chemical insecticides in

* Corresponding author. State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China. E-mail address: [email protected] (Z. Zou).

the past decades has increased resistance in vector insects. Additionally, heavy use of chemical insecticides has also seriously polluted the environment. A new approach for mosquito control is the use of bio-insecticides, such as entomopathogenic fungi, which are considered as safe and green alternatives to most chemical insecticides (Read et al., 2009). However, fungal pesticides require improvements due to their relatively low virulence and the immune defense reaction from the hosts (Fang et al., 2012). These require a deep understanding of the molecular interactions between pathogens and host defense. Insects rely on their innate immune system to fight against invading bacteria, fungi, and parasites (Cirimotich et al., 2010; Hoffmann and Reichhart, 2002; Lemaitre and Hoffmann, 2007; Xing et al., 2017; Xiong et al., 2015). When microbes break through host's physical barriers (cuticle or epithelium of midgut) and reach the hemocoel, the host pattern recognition receptors (PRRs) identify pathogen-associated molecular patterns (PAMPs) located on the surface of microbes, which then trigger cellular and humoral responses (Kanost et al., 2004). The host's cellular responses are mediated by several types of immune cells-hemocytes (Hillyer et al., 2003a; Lavine and Strand, 2002). Humoral immunity mainly includes two major inducible responses, the Toll and immune deficiency (IMD) pathway, which produce antimicrobial peptides (AMPs) via a signal transduction cascade, melanin and reactive oxygen species (ROS) (Kanost et al., 2004; Kumar et al., 2010; Lemaitre and Hoffmann, 2007).

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Mosquitoes also use innate immune system to fight against microbes during their life cycles. As vectors of many diseases, mosquitoes are susceptible to pathogen infection and the innate immune system, including the production of AMPs and lysozymes, phagocytosis, and melanization plays an important role in limiting the virus to a non-lethal level (Xi et al., 2008). Recently, molecular biology and genomics tools have provided a direction for the study of mosquito immunity. As one of the best characterized mosquitoes, Ae. aegypti can be easily reared in the laboratory, and has provided much of the knowledge on physiology, reproductive biology, and vector competence (Severson et al., 2001). In addition, Ae. aegypti is considered as a model organism (Clemons et al., 2010) for studies on hormone regulated reproductive biology, carbohydrate and lipid metabolism, and to understand the genetic basis of vector competence particularly the interactions between filarial worms and mosquitoes (Attardo et al., 2005; Chen et al., 2008; Hou et al., 2015; Severson et al., 2004; Wang et al., 2017a). Here, we present a comprehensive review of the recent research advances on the immune responses of Ae. aegypti against viral and fungal pathogens. This review will improve our understanding of the interactions between mosquitoes and pathogens at the molecular level and will help develop highly effective and specific biopesticides. 2. The immune system of Ae. aegypti 2.1. Overview of the Ae. aegypti immune system Although lacking of acquired immunity, mosquitoes can kill a variety of prokaryotic and eukaryotic pathogens using their innate immune system (Dimopoulos, 2003). Insects have a powerful innate immune system, and whole genome sequencing provides an opportunity to study its origin and diversity. Genome sequencing of the most important vectors, Anopheles gambiae, Culex quinquefasciatus, and Ae. aegypti has allowed the systematic study of mosquito innate immune system and also the interaction between mosquitoes and pathogens (Bartholomay et al., 2010; Christophides et al., 2002; Dudchenko et al., 2017; Holt et al., 2002; Nene et al., 2007). Comparative analysis showed that there are 476 immune genes in Ae. aegypti, compared with 536, 400, and 345 immune genes in An. gambiae, C. quinquefasciatus, and D. melanogaster, respectively (Chen et al., 2015). Thus, the number of immune genes has undergone a major expansion in Ae. aegypti (Chen et al., 2015; Waterhouse et al., 2007). There is high conservation among the main immune pathways mosquitoes and other insects. However, due to their complex lifecycles, mosquitoes are exposed to a variety of pathogens and may have acquired certain features during evolution due to the stress of these pathogens. Among the immunityrelated molecules, the pattern recognition molecules, genes involved in the melanization pathway and effector molecules exhibit a major expansion, indicating that Ae. aegypti has a complex innate immune system. 2.2. Pattern recognition receptors of Ae. aegypti More than 100 immune recognition molecules have been identified in the Ae aegypti genome using bioinformatics analysis (Waterhouse et al., 2007). The main families of immune recognition molecules include C-type lectins (CTLs), peptidoglycan recognition proteins (PGRPs), and b-1,3- glucan binding proteins (b-1,3-GRPs). CTLs comprise a large family of PRRs and have the capability to bind carbohydrates in the presence of calcium, which help in the recognition of a broad spectrum of microbes (Fujita and Endo, 2004). An array of immune functions have been proposed for

insect CTLs, including activation of the melanization cascade (Yu and Kanost, 2000), encapsulation (Ling and Yu, 2006), nodule formation (Koizumi et al., 1999), and opsonization (Wilson et al., 1999). CTLs are both negative and positive regulators in the immune response of mosquitoes. In An. gambiae, two CTLs (CTL4 and CTLMA2) were identified to have protective effects during the invasion and development of Plasmodium parasites in the midgut (Osta et al., 2004). However, these two CTLs are indispensable for the clearance of Gram-negative bacteria in the hemolymph of An. gambiae (Schnitger et al., 2009). In Ae. aegypti, mosGCTL-1 was found to assist the infection of West Nile virus (Cheng et al., 2010). Moreover, a C-type lectin with a serine protease domain, CLSP2, in Ae. aegypti was reported to be involved in modulating the antifungal immunity as a negative regulator (Wang et al., 2015). PGRPs were first identified in silkworm Bombyx mori (Yoshida et al., 1996), followed by other insects. They can recognize peptidoglycan (PGN), a main molecule on the cell surface of pathogens (Royet and Dziarski, 2007). There are two types of PGN, Lys-type and DAP-type. Lys-type PGN is mainly located on the cell surface of Gram-positive bacteria, and DAP-type PGN is usually associated with both Gram-negative and -positive bacteria. PGRPs are classified into two forms, short (S) and long (L) form. In D. melanogaster and Tribolium castaneum, when PGRP-SA, -SC1, -SD recognize the Lys-type PGN, the Toll pathway and melanization pathway are activated (Michel et al., 2001; Zou et al., 2007). PGRP-LB and -LC are essential for the immune response against DAP-type PGN-containing bacteria through the IMD pathway (Zaidman-Remy et al., 2006). In Ae. aegypti, 9 PGRP genes have been identified, among which, PGRP-LC, -SC2 and -LB were thought to be involved in the immune response against Escherichia coli and Micrococcus luteus (Wang and Beerntsen, 2015). GNBPs were initially found in An. gambiae, and could mainly bind to b-1,3-glucan and lipopolysaccharide on the surface of pathogens (Dimopoulos et al., 1997). In D. melanogaster, GNBP3 binds to the cell components of fungi and activates the Toll pathway (Gottar et al., 2006). In Ae. aegypti, the expression of GNBP1 was up-regulated during fungal infection, indicating that GNBP1 may be involved in antifungal immune response (Wang et al., 2015). However, studies on the functions of Ae. aegypti PRRs is limited and more studies are needed in this area. 2.3. Immune signaling pathways deciphered in mosquito Ae. aegypti Studies have shown that in D. melanogaster the Toll, IMD, and Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathways constitute the major components of humoral immunity (Dostert et al., 2005; Ferrandon et al., 2007). When the Lys-type PGN from Gram-positive bacteria or b-1,3-glucan from fungi are recognized by host PRRs, the Toll pathway is triggered (Lemaitre and Hoffmann, 2007). Activation of an extracellular €etzle (Spz), a cysteine knot serine protease (SP) cascade cleaves Spa cytokine. Two clip domain SP (CLIPs)-Persephone and Sp€ aetzleprocessing enzyme (SPE) participated in this process (Jang et al., 2006; Ligoxygakis et al., 2002). The cleaved Spz binds to and activates the Toll receptor, trigerring the kinase cascade mediated by MyD88, Tube and Pelle. Activation of the intracellular signal cascade results in the phosphorylation and proteasomal degradation of Cactus, a negative regulator of the NF-кB transcription factorsdDorsal and Dif. Then, degradation of Cactus allows Dif and Dorsal translocation to the nucleus and leads to the expression of AMPs and other immune effectors (Lemaitre et al., 1996). When the DAP-type PGN is recognized by PGRP-LC, the IMD adaptor is activated, and the signal is transmitted into the cell vis the intracellular signal cascade that includs FADD, Dredd and Relish. The release of Relish leads to the production of AMPs (Ferrandon et al., 2007).

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Comparative genomics analysis has shown that the immune genes involved in the signaling pathways are rather conserved in D. melanogaster, An. gambiae and Ae. aegypti (Waterhouse et al., 2007). Similar to D. melanogaster, in mosquitoes, immune signaling pathways play key roles in their response to various pathogens (Dimopoulos, 2003). Previously, several D. melanogaster orthologs of the Toll pathway genes were identified in Ae. aegypti. For example, Spz1C, Toll5A, CLIPB5, CLIPB29, and GNBP1 were upregulated during fungal infection, indicating that these genes mediate the activation of Toll pathway (Shin et al., 2005; Wang et al., 2015; Zou et al., 2010). Moreover, the expression of these genes was regulated by CLSP2, suggesting that CLSP2 inhibits the activation of Toll pathway (Wang et al., 2015). Although the Toll pathway is conserved in Ae. aegypti (Fig. 1), more details of Ae. aegypti Toll pathway are needed for a comprehensive comparison with the well-characterized D. melanogaster Toll pathway. Toll and IMD are major innate immune pathways responsible for the production of AMPs and other effectors (Hoffmann and Reichhart, 2002). In D. melanogaster, there are seven AMP families with distinct roles against different pathogens (Lemaitre and Hoffmann, 2007). In Ae. aegypti, the activation of genes encoding AMPs and other immune effectors is achieved by the release of the NF-kB transcription factors REL1 and REL2, the orthologs of D. melanogaster Dorsal and Relish, respectively (Shin et al., 2002, 2005, 2003). In mosquitoes, the AMPs are mainly represented by defensins

Fig. 1. Ae. aegypti Toll pathway and melanization pathway. In the Toll signaling pathway, when Lys-type PGN is recognized by PRRs such as GNBP1, the extracellular serine protease (SP) cascade, negatively regulated by CLSP2, is €etzle (Spz1C) binds to the Toll receptor (Toll5A). This triggered and the cleaved Spa tranmits the signals on to the intracellular siganl cascade mediated by MyD88, Tube and Pelle, resulting in the phosphorylation and proteasomal degradation of Cactus, a negtive regulator of the NF-кB transcription factor, Rel1. The degradation of Cactus allows Rel1 translocation to the nucleus and leads the expression of AMPs, TEPs, Lysozymes, and other immune effectors. In the tissue melanization pathway, TMP, IMP-1 and Serpin-2 mediate the cleavage of PPOs to produce POs, resulting in the formation of melanotic pseudotumors. In immune melanization pathway, detection of Lys-type PGN by PGRPs or GNBPs triggers the activation of IMP-1 and IMP-2, which mediate the proteolytic cleavage of PPO (PPO3). The cleavege of PPO is negatively regulated by Serpin-1 and CLSP2.

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and cecropins (Waterhouse et al., 2007). Two transgenic strains of mosquitoes were constructed to overexpress NF-kB transcription factors containing the Vg promoter, which is activated by blood meal (Kokoza et al., 2000). It was shown that, the cooverexpression of two AMPs, DefensinA and CecropinA in transgenic Ae. aegypti mosquitoes increased the resistance to bacteria and avian malaria parasite Plasmodium gallinaveum (Kokoza et al., 2010). Moreover, a transcriptomics-based analysis of transgenic mosquitoes indicated that a number of immune genes are induced in the Plasmodium infected mosquito fat body. These NF-кB transcription factors synergistically activiate the expression of immune genes in the hybrid strain which ectopically expresses both Rel1 and Rel2. Compared to the well-characterized Toll and IMD pathways, the knowledge on the JAK-STAT signaling pathway is limited and focuses specifically on antiviral responses (Dostert et al., 2005). The antiviral role of JAK-STAT pathway is conserved in Ae. aegypti during dengue virus (DENV) infection (Souza-Neto et al., 2009). In Ae. aegypti transgenic mosquitoes, silencing PIAS, the main inhibitor gene of JAK-STAT, can reduce the proliferation of Plasmodium, suggesting that the JAK-STAT pathway is involved in the antiPlasmodium defense (Zou et al., 2011). 2.4. The melanization pathway of Ae. aegypti Melanization is an essential component of the invertebrate defense system and plays an important role in multiple physiological reactions, including wound healing and innate immunity (Cerenius et al., 2008; Soderhall and Ajaxon, 1982). Previous studies have shown that melanization participated in immune response against pathogens in different species, such as Penaeus monodon, Manduca sexta and D. melanogaster (Sutthangkul et al., 2015; Tang et al., 2006; Zhao et al., 2007). Melanization is triggered by the recognition of conserved molecular patterns on the surface of pathogens by PRRs, such as PGRPs and bGRPs. Then, an enzyme cascade composed of SPs containing regulatory clip domains (CLIPs) is activated to amplify the signal and convert prophenoloxidase (PPO) to phenoloxidase (PO) (Takahashi et al., 2014). PO, the key enzyme in melanization reactions, catalyzes monophenols to the toxic melanin, which then deposits around the wound or invading pathogens (Nappi and Christensen, 2005). CLIPs that directly cleave PPO to produce PO are designated as melanization proteases (MPs). In the final steps of the proteolytic activation cascade, PPO is cleaved and activated by MPs in the presence of clip domain serine protease homologs (SPHs) (Ashida and Brey, 1998; Cerenius and Soderhall, 2004). Moreover, the SP cascade is strictly regulated by SP inhibitors (Serpins) to ensure that the cascade is activated only at a specific time (Kanost, 1999). Mosquitoes also use melanization to fight invading pathogens. For example, in An. gambiae, melanization is involved in defense against the fungus, Beauveria bassiana (Yassine et al., 2012). Furthermore, the mosquito Armigeres subalbatus, employs melanization in antibacterial immune responses (Hillyer et al., 2003a). Similarly, in Ae. aegypti, melanization is involved in immune response (Zou et al., 2008). Analysis of mosquito genomes has shown a major expansion in the number of genes involved in the melanization pathway. For instance, there are 10 PPO genes in Ae. aegypti, compared with 3 PPOs in D. melanogaster (Waterhouse et al., 2007). Insect SPs and SPHs participate in a variety of physiological processes, such as digestion, development, and immune defenses (Krem and Di Cera, 2002). As non-digestive SPs, CLIP proteases are present in the hemolymph of insects and other arthropods, and their major function is to act as immune factors (Kanost and Jiang, 2015). They are involved in the melanization and

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Toll pathways signal cascade. Besides these CLIPs, many other SPs with special domain structures are also involved in immune response. A previous study showed the presence of putative regulatory domains (e. g. LDLa, CUB, Gd) in 16 SPs and 2 SPHs in An. gambiae. (Cao et al., 2017). In Ae. aegypti, CLIPs are classified into five clades (CLIPA, CLIPB, CLIPC, CLIPD, CLIPE) (Waterhouse et al., 2007). Recent examination identified more CLIPs (Table 1). 62 out of 82 belong to CLIPB, CLIPC, and CLIPD, which are expected to have SP activity. 17 members belonging to CLIPA and CLIPE clades, which are composed of SPHs. Besides, 15 SPs and 2 SPHs with special domain structures were identified (Table 1) and were presumed to play important regulatory roles in immune responses. For example, Ae. aegypti AaHP14, a SP with 2 Sushi domains, is orthologous to M. sexta HP14, which could be an initial protease in the melanization pathway. These results suggest the high complexity of SP cascades involved in the antipathogen immune responses in these insects. There are at least two melanization pathways in Ae. aegypti (Fig. 1). First is immune melanization, which is activated by the recognition of Lys-PGN, and regulated by SP cascades, such as immune melanization proteases (IMP-1 and IMP-2) and Serpin-1, mediating the hemolymph PPO (PPO3) cleavage and melanin formation. Cleavage of PPO3 is negatively regulated by CLSP2 (Wang et al., 2015). The second is tissue melanization, which is mediated by tissue melanization protease (TMP), IMP-1, and Serpin-2, represented by melanin tumors often associated with the damage of host tissues (Zou et al., 2010). 2.5. Cellular immunity of Ae. aegypti Cellular immunity is an important part of insect innate immune system, including hemocyte-mediated immune responses such as phagocytosis, nodulation and encapsulation (Strand and Pech, 1995). Although humoral immunity plays a critical role in antimicrobial immune responses, hemocytes circulating in the hemolymph are responsible for this response by secreting humoral immune factors, such as AMPs, opsonins and components of the melanization pathway. For example, transcription of PPO was upregulated in the hemocytes of mosquito Armigeres subalbatus infected with filariasis nematode Brugia malayi (Huang et al., 2001). In addition, it was demonstrated that hemocytes were involved in the synthesis of thioester-containing protein 1 (TEP1), a key component of the complement-like system in mosquitoes (Blandin et al., 2004). Moreover, hemocytes-derived microvesicles may transmit some critical factor(s) that promote the activation of TEP1 during antiplasmodial responses in mosquitoes (Castillo et al., 2017). By means of morphology, lectin binding, and enzyme activity assays, the hemocytes from adult Ae. aegypti were classified into four types: granulocytes, oenocytoids, adipohemocytes, and thrombocytoids. (Hillyer and Christensen, 2002). Granulocytes and oenocytoids are the major hemocytes circulating in Ae. aegypti hemolymph (Hillyer and Christensen, 2002). Granulocytes are the most abundant cell type in Ae. aegypti and can phagocytose bacteria and Plasmodium sporozoites (Hillyer et al., 2003b). Furthermore, PPO, the key enzyme of melanization, was detected inside the oenocytoids (Hillyer and Christensen, 2002). Previous studies indicated that hemocytes comprise an important part of the mosquito immune system. For example, in Ae. aegypti, hemocytes were present at the encapsulation site during the immune response to Dirofilaria immitis microfilariae (Christensen and Forton, 1986). In mosquito Ar. suballbatus, hemocytes were involved in phagocytosis and melanization against bacterial infection (Hillyer et al., 2003a). However, current knowledge about the types and functions of hemocytes in mosquitoes is limited. Characterization of the

hemocytes and identifying their functions in immunity of Ae. aegypti will enrich the understanding of the mosquito immune system. 3. The immune responses of Ae. aegypti against fungi Increased resistance to chemical insecticides compromise the effort to control vector-borne diseases. One of the new approaches for mosquito control is the use of entomopathogenic fungi, which can be a safe and green alternative to chemical insecticides. Insect pathogenic fungi, such as B. bassiana and Metarhizium anisophliae, which infect insects by penetrating directly into the cuticle, have been considered as potential biological control agents (Glare et al., 2012; Ortiz-Urquiza et al., 2015). However, for practical application, it is necessary to improve their genetic characteristics to overcome the relatively low virulence to boost the host immune system. A recent study showed that exposure of Ae. aegypti to fungal conidia can cause infection and result in the death of mosquitoes. Moreover, the fungus can be transmitted between mosquitoes through mating (Garcia-Munguia et al., 2011). However, the mechanism of interaction between the mosquito immune system and fungi is not well understood. Therefore, detailed studies are needed to understand the antifungal immunity in mosquitoes to improve fungi as biocontrol agents. In insects, Toll pathway is the major immune pathway responsible for the antifungal response (Lemaitre and Hoffmann, 2007; Roh et al., 2009). In Drosophila, antifungal defense is dependent on the Toll pathway (Matskevich et al., 2010). During fungal infection, Dif, one of the NF-кB transcription factors, translocates to the nucleus and leads to the production of the antifungal peptide, Drosomycin (Lemaitre et al., 1997). In Ae. aegypti, the antifungal immune response involving the Toll pathway is transduced through Rel1. AaRel1 affects the expression of Serpin-2, and regulates the antifungal immune response against B. bassiana, indicating that AaRel1 is a key regulator of the Toll antifungal immune pathway (Shin et al., 2005). One of the Toll receptors and its ligand, Toll5A and Spz1C, have been identified to mediate the Toll pathway in response to fungal infection (Shin et al., 2006). In addition, two CLIP proteases, CLIPB5 and CLIPB29, were involved in the activation of Toll pathway during fungal infection (Zou et al., 2010). All these studies indicate that Toll pathway plays an important role in antifungal immune response of Ae. aegypti. Melanization is another important mechanism involved in systemic antifungal immune responses (Kanost et al., 2004). In the melanization pathway, the proteolytic cleavage of PPO into PO is the key reaction. Biochemical analysis shows that the cleavage patterns of PPO differ in insects. There are 10 PPOs in Ae. aegypti, and a comparison of their amino acid sequences show that some but not all mosquito PPOs have a typical cleavage site for activation (Zou et al., 2008). The cleavage site is present in the majority of biochemically characterized PPOs from moths, beetles, flies, and mosquitoes. For example, in M. sexta and B. mori, PPO is activated by cleaving Arg51 from the N-terminus (Jiang et al., 1998; Satoh et al., 1999). However, in the crayfish, the cleavage site of PPO is located in Arg176 (Cerenius and Soderhall, 2004). In the scarab beetle Holotrichia diamphalia, PPO is cleaved at a second cleavage site, Arg162, leading to an active PO (Kim et al., 2002). In Ae. aegypti, infection of B. bassiana, an entomopathogenic fungus, causes the cleavage of hemolymph PPOs, generating two bands, a long 50 kDa C-terminal PO and a short 18e20 kDa band, respectively. This result indicates that the cleavage of PPOs inactivates the enzymes and eventually destroys the humoral immunity (Zou et al., 2010). Moreover, six PPOs (PPO1, PPO2, PPO3, PPO4, PPO5, and PPO8) are expressed in response to fungal challenge, and the expression of PPOs is regulated by CLSP2, a SP with a

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Y.-H. Wang et al. / Developmental and Comparative Immunology xxx (2017) 1e10 Table 1 Serine proteases (SPs) and serine proteinase homologs (SPHs) in Ae. aegypti.

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a

CLIP, clip domain; SP, serine proteinase catalytic domain; SPH, serine proteinase-like domain CUB, a domain identified in Complement 1r/s, Uegf, and Bmp1; LDLA, lowdensity lipoprotein receptor class A domain; CB, chitin-binding domain; SRCR, scavenger receptor cysteine-rich domain; ZnF, zinc finger domain; Ig, immuno-globulin; LamG, laminin G; EGF, Ca2þ-binding EGF domain; Sushi, Sushi domain, also known as CCP or SCR; SEA, a ~120-residue domain in Sperm protein; Fz, frizzled; CTL, C-type lectin domain; TSP1, thrombospondin type I repeat. b Enzyme specificity predicted based on Perona and Craik (1995). T, trypsin; C, chymotrypsin; E, elastase; ?: not predictable. Letters in parentheses: amino acid residues determining the primary specificity of a serine proteinase. c Red, putative activation cleavage site; ?, not predicted.

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C-type lectin domain in the C-terminus (Wang et al., 2015). Our further studies showed that PPO3 is cleaved in the hemolymph after B. bassiana infection, and this is regulated by CLSP2 (Wang et al., 2015). Recombinant PPO3 (rPPO3) was also cleaved in mosquito after fungal challenge, and the survival rate of Ae. aegypti infected with fungus increased after the injection of rPPO3, indicating that proteolytic cleavage of the mosquito PPO3 plays an important role in antifungal immune response of mosquitoes (Wang et al., 2017b). These studies suggest that melanization plays important roles in the antifungal immunity of Ae. aegypti. 4. The immune responses of Ae. aegypti against arboviruses In the past decades, efforts have been taken towards the prevention and control of vector-borne diseases. However, arboviruses remain an important public health concern in many regions of the world. The majority of highly pathogenic arboviruses belong to the families of Flaviviridae, Togaviridae, and Bunyaviridae are mostly transmitted by mosquitoes (Blair et al., 2000). Most arboviruses are transmitted by Aedes and Culex mosquitoes, among which Ae. aegypti is the major vector of DENV, ZIKV, and Chikungunya virus causing global epidemic. Female mosquitoes transmit viruses during repeated blood feeding on the host. When mosquitoes feed in an arbovirus infected blood meal, the virus first infects the mosquito's midgut, replicates and then spreads to other tissues including the hemolymph, fat body, and salivary glands, where they further replicate in order to be transmitted to human host (Franz et al., 2015). During these processes, the arboviruses are exposed to the mosquito immune system, while the viruses defend against the attack from the immune system, the mosquitoes use efficient strategies to restrict the replication of viruses to nonpathogenic levels. Sequencing of the Ae. aegypti genome (Nene et al., 2007) uncovered the pathways related to its immune system, among which the Toll, IMD, and JAK-STAT pathways play key roles in antiviral defense. Upon DENV infection, several immune genes, such as Spz, Toll, and Rel1A involved in the Toll pathway are up-regulated, while the negative regulator of Toll pathway is down-regulated, indicating that the Toll signaling pathway is activated. The extent of dengue infection in the midgut reduced 4-fold after silencing Cactus by RNAi, indicating that the infection of DENV triggers the Toll pathway, which then induces the anti-dengue effect (Xi et al., 2008). In Ae. aegypti, the antiviral role of JAK-STAT pathway is

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conserved. Domeless (Dome), one of the key components of the JAK-STAT pathway, is strongly induced during dengue infection. The expression of DVRF-1 and -2, STAT-regulated anti-dengue restriction factors were also up-regulated, indicating that the JAKSTAT pathway was activated (Souza-Neto et al., 2009; Xi et al., 2008). Fungal infection activates Toll and JAK-STAT pathways, and induces the expression of various DENV restriction factor genes, which restrict the replication of DENV in the Ae. aegypti midgut (Dong et al., 2012). Reactive oxygen species (ROS) have also been reported to modulate the competence of Ae. aegypti to DENV infections (Oliveira et al., 2017). Wolbachia pipientis, a maternally transmitted endosymbiotic bacterium, infects approximately 40% of all known arthropod species (Zug and Hammerstein, 2012). The use of Wolbachia has become a biocontrol strategy against DENV (McGraw and O'Neill, 2013). Recent studies showed that Wolbachia can block the transmission of DENV in Ae. aegypti (Pan et al., 2012; Ye et al., 2013). An immune priming model showed that Wolbachia infections can activate the innate immune response, which helps mosquitoes fight DENV infection (Pan et al., 2012; Rances et al., 2012). However, little is known about the mechanism of Wolbachia-mediated virus blocking in Ae. aegypti. Sindbis virus (SINV, Family Togaviridae) can infect laboratory reared Ae. aegypti, and in the early stage of infection, this virus activates the Toll pathway (Sanders et al., 2005). The IMD pathway, which is well known to play essential roles in insect defense against Gram-negative bacteria (Kaneko and Silverman, 2005) is also involved in the anti-viral defense of mosquitoes. In Ae. aegypti, the microbiota present in the midgut activates the IMD pathway, which impairs the replication of SINV (Barletta et al., 2017). The mechanism of RNA interference (RNAi) in antiviral response is not a classical immune response. RNAi pathway has been reported to influence the RNA virus replication and pathogenicity in D. melanogaster (Zambon et al., 2006). In Ae. aegypti, RNAi has also been found to modulate the vector competence for SINV in the midgut (Khoo et al., 2010). ZIKV, a member of the Flaviviridae family, is transmitted primarily by Ae. aegypti. Since its first isolation in 1947 in Uganda, the transmission of ZIKV was limited to the tropical area of Africa and Asia until recently when it spread globally. In 2015, ZIKV emerged for the first time in the Americas (Brazil in March), and by the end of January 2016, ZIKV has been found in more than 20 countries or territories (Campos et al., 2015; Musso and Gubler, 2016; Zanluca

Table 2 Immunity-related genes of Ae. aegypti response to pathogens. Gene family/Pathway Recognition C-type lectins (CTLs) peptidoglycan recognition proteins (PGRPs) beta-1,3- glucan binding proteins (b-1,3GRPs) Signaling

Effectors

Function

Name

activation of the melanization cascade, encapsulation, nodule formation, opsonization recognize peptidoglycan (PGN)

eg:mosGCTL-1 CLSP2 eg: PGRP-LC, -SC2 and -LB

bind to b-1,3-glucan and lipopolysaccharide

eg:GNBP1

activates the downstreamed receptor

eg: Spz1C eg: Toll5A

negative regulator of the immune pathway NF-кB transcription factors

Cactus, PIAS Rel1A eg: CLIPB5, CLIPB29 eg: Serpin-1, Serpin-2

€etzle (Spz) Spa Toll-like receptor MyD88, Tube, Pelle, IMD, FADD, Dredd, Domeless Cactus, PIAS REL1, REL2 CLIP SP/SPH SP inhibitors (Serpins)

regulated the SP cascade

AMPs phenoloxidase (PO)

fight against microbes key enzyme for melanization reaction

thioester-containing protein

eg: DefensinA, CecropinA eg: PPO1, PPO2, PPO3, PPO4, PPO5, PPO8 TEP22

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et al., 2015). However, little is known about the interactions between ZIKV and its vector. A recent study showed that ZIKV induced RNAi response in Ae. aegypti. After injection of ZIKV, the virusderived short interfering RNAs and PIWI-interacting RNAs were drastically increased (Saldana et al., 2017). However, our knowledge about Ae. aegypti antiviral immune pathway is still limited, and the antiviral mechanisms need further investigation. 5. Conclusions In the past decade, many researchers paid attention to mosquito immunity. The whole genome sequencing of medically important mosquito species such as An. gambiae (Holt et al., 2002), C. quinquefasciatus (Arensburger et al., 2010), and Ae. aegypti (Nene et al., 2007) increased the resource necessary to systematically identify putative immune genes. A number of immune genes are likely involved in the immune responses of Ae. aegypti (Table 2). Our understanding of mosquito antimicrobial immunity has also expanded. Several studies showed that the classical immune signaling pathways are involved in antiviral immune responses of mosquitoes. However, there are still several questions that need to be answered. For example, the molecular mechanisms involved in the activation and function of these immune signaling pathways during antiviral immunity are unknown. Elucidating the molecular mechanism of the antiviral immunity and identifying new antiviral immune genes will be an important area for future research. Entomopathogenic fungi have been used as pesticides to control mosquitoes. However, for the industrial application of fungi it is necessary to improve their genetic characteristics to increase the speed of killing and reduce the high dose of treatment by unraveling the molecular mechanism of fungal pathogenicity. In Ae. aegypti, B. bassiana causes the cleavage of hemolymph PPOs (Zou et al., 2010), indicating that this cleavage could inactivate the enzymes and eventually destroy humoral immunity. Characterizing the key role of melanization against fungal pathogens and revealing the suppression mechanism of host melanization by pathogenic fungi will improve our understanding of the interaction between mosquitoes and fungi at the molecular level. Furthermore, it will also provide valuable information for developing highly effective and specific biopesticides. Acknowledgments This work was supported by the National Key Plan for Scientific Research and Development of China (No. 2017YFC1201004, 2016YFD0500300, 2016YFC1200603), Strategic Priority Research Program of the CAS (No. XDB11030600), National Science Foundation of China (No. 31402013, 31472008, 31672291). Appendix A. Supplementary data Supplementary data related to this article can be found at https://doi.org/10.1016/j.dci.2017.12.001. References Arensburger, P., Megy, K., Waterhouse, R.M., Abrudan, J., Amedeo, P., Antelo, B., Bartholomay, L., Bidwell, S., Caler, E., Camara, F., Campbell, C.L., Campbell, K.S., Casola, C., Castro, M.T., Chandramouliswaran, I., Chapman, S.B., Christley, S., Costas, J., Eisenstadt, E., Feschotte, C., Fraser-Liggett, C., Guigo, R., Haas, B., Hammond, M., Hansson, B.S., Hemingway, J., Hill, S.R., Howarth, C., Ignell, R., Kennedy, R.C., Kodira, C.D., Lobo, N.F., Mao, C., Mayhew, G., Michel, K., Mori, A., Liu, N., Naveira, H., Nene, V., Nguyen, N., Pearson, M.D., Pritham, E.J., Puiu, D., Qi, Y., Ranson, H., Ribeiro, J.M., Roberston, H.M., Severson, D.W., Shumway, M., Stanke, M., Strausberg, R.L., Sun, C., Sutton, G., Tu, Z.J., Tubio, J.M., Unger, M.F., Vanlandingham, D.L., Vilella, A.J., White, O., White, J.R., Wondji, C.S., Wortman, J., Zdobnov, E.M., Birren, B., Christensen, B.M., Collins, F.H., Cornel, A.,

Dimopoulos, G., Hannick, L.I., Higgs, S., Lanzaro, G.C., Lawson, D., Lee, N.H., Muskavitch, M.A., Raikhel, A.S., Atkinson, P.W., 2010. Sequencing of Culex quinquefasciatus establishes a platform for mosquito comparative genomics. Science 330, 86e88. Ashida, M., Brey, P.T., 1998. Recent Advances on the Research of the Insect Prophenoloxidase Cascade. In: Brey, P.T., Hultmark, D. (Eds.), Molecular Mechanisms of Immune Responses in Insects Chapman and Hall, London, pp. 135e172. Attardo, G.M., Hansen, I.A., Raikhel, A.S., 2005. Nutritional regulation of vitellogenesis in mosquitoes: implications for anautogeny. Insect biochem. Mol. Biol. 35, 661e675. Barletta, A.B., Nascimento-Silva, M.C., Talyuli, O.A., Oliveira, J.H., Pereira, L.O., Oliveira, P.L., Sorgine, M.H., 2017. Microbiota activates IMD pathway and limits Sindbis infection in Aedes aegypti. Parasit. Vectors 10, 103. Bartholomay, L.C., Waterhouse, R.M., Mayhew, G.F., Campbell, C.L., Michel, K., Zou, Z., Ramirez, J.L., Das, S., Alvarez, K., Arensburger, P., Bryant, B., Chapman, S.B., Dong, Y., Erickson, S.M., Karunaratne, S.H., Kokoza, V., Kodira, C.D., Pignatelli, P., Shin, S.W., Vanlandingham, D.L., Atkinson, P.W., Birren, B., Christophides, G.K., Clem, R.J., Hemingway, J., Higgs, S., Megy, K., Ranson, H., Zdobnov, E.M., Raikhel, A.S., Christensen, B.M., Dimopoulos, G., Muskavitch, M.A., 2010. Pathogenomics of Culex quinquefasciatus and metaanalysis of infection responses to diverse pathogens. Science 330, 88e90. Blair, C.D., Adelman, Z.N., Olson, K.E., 2000. Molecular strategies for interrupting arthropod-borne virus transmission by mosquitoes. Clin. Microbiol. Rev. 13, 651e661. Blandin, S., Shiao, S.-H., Moita, L.F., Janse, C.J., Waters, A.P., Kafatos, F.C., Levashina, E.A., 2004. Complement-like protein TEP1 is a determinant of vectorial capacity in the malaria vector Anopheles gambiae. Cell 116, 661e670. Campos, G.S., Bandeira, A.C., Sardi, S.I., 2015. Zika virus outbreak, Bahia, Brazil. Emerg. Infect. Dis. 21, 1885e1886. Cao, X., Gulati, M., Jiang, H., 2017. Serine protease-related proteins in the malaria mosquito, Anopheles gambiae. Insect biochem. Mol. Biol. 88, 48e62. Castillo, J.C., Ferreira, A.B.B., Trisnadi, N., Barillas-Mury, C., 2017. Activation of mosquito complement antiplasmodial response requires cellular immunity. Sci. Immunol. 2. Cerenius, L., Lee, B.L., Soderhall, K., 2008. The proPO-system: pros and cons for its role in invertebrate immunity. Trends Immunol. 29, 263e271. Cerenius, L., Soderhall, K., 2004. The prophenoloxidase-activating system in invertebrates. Immunol. Rev. 198, 116e126. Chen, X.G., Jiang, X., Gu, J., Xu, M., Wu, Y., Deng, Y., Zhang, C., Bonizzoni, M., Dermauw, W., Vontas, J., Armbruster, P., Huang, X., Yang, Y., Zhang, H., He, W., Peng, H., Liu, Y., Wu, K., Chen, J., Lirakis, M., Topalis, P., Van Leeuwen, T., Hall, A.B., Jiang, X., Thorpe, C., Mueller, R.L., Sun, C., Waterhouse, R.M., Yan, G., Tu, Z.J., Fang, X., James, A.A., 2015. Genome sequence of the Asian Tiger mosquito, Aedes albopictus, reveals insights into its biology, genetics, and evolution. Proc. Natl. Acad. Sci. U. S. A. 112, E5907eE5915. Chen, X.G., Mathur, G., James, A.A., 2008. Gene expression studies in mosquitoes. Adv. Genet. 64, 19e50. Cheng, G., Cox, J., Wang, P., Krishnan, M.N., Dai, J., Qian, F., Anderson, J.F., Fikrig, E., 2010. A C-type lectin collaborates with a CD45 phosphatase homolog to facilitate West Nile virus infection of mosquitoes. Cell 142, 714e725. Christensen, B.M., Forton, K.F., 1986. Hemocyte-mediated melanization of microfilariae in Aedes aegypti. J. Parasitol. 72, 220e225. Christophides, G.K., Zdobnov, E., Barillas-Mury, C., Birney, E., Blandin, S., Blass, C., Brey, P.T., Collins, F.H., Danielli, A., Dimopoulos, G., Hetru, C., Hoa, N.T., Hoffmann, J.A., Kanzok, S.M., Letunic, I., Levashina, E.A., Loukeris, T.G., Lycett, G., Meister, S., Michel, K., Moita, L.F., Muller, H.M., Osta, M.A., Paskewitz, S.M., Reichhart, J.M., Rzhetsky, A., Troxler, L., Vernick, K.D., Vlachou, D., Volz, J., von Mering, C., Xu, J., Zheng, L., Bork, P., Kafatos, F.C., 2002. Immunity-related genes and gene families in Anopheles gambiae. Science 298, 159e165. Cirimotich, C.M., Dong, Y., Garver, L.S., Sim, S., Dimopoulos, G., 2010. Mosquito immune defenses against Plasmodium infection. Dev. Comp. Immunol. 34, 387e395. Clemons, A., Haugen, M., Flannery, E., Tomchaney, M., Kast, K., Jacowski, C., Le, C., Mori, A., Simanton Holland, W., Sarro, J., Severson, D.W., Duman-Scheel, M., 2010. Aedes aegypti: an Emerging Model for Vector Mosquito Development. Cold Spring Harbor Protocols 2010 pdb emo141. Dimopoulos, G., 2003. Insect immunity and its implication in mosquito-malaria interactions. Cell Microbiol. 5, 3e14. Dimopoulos, G., Richman, A., Muller, H.M., Kafatos, F.C., 1997. Molecular immune responses of the mosquito Anopheles gambiae to bacteria and malaria parasites. Proc. Natl. Acad. Sci. U. S. A. 94, 11508e11513. Dong, Y., Morton Jr., J.C., Ramirez, J.L., Souza-Neto, J.A., Dimopoulos, G., 2012. The entomopathogenic fungus Beauveria bassiana activate toll and JAK-STAT pathway-controlled effector genes and anti-dengue activity in Aedes aegypti. Insect biochem. Mol. Biol. 42, 126e132. Dostert, C., Jouanguy, E., Irving, P., Troxler, L., Galiana-Arnoux, D., Hetru, C., Hoffmann, J.A., Imler, J.L., 2005. The Jak-STAT signaling pathway is required but not sufficient for the antiviral response of Drosophila. Nat. Immunol. 6, 946e953. Dudchenko, O., Batra, S.S., Omer, A.D., Nyquist, S.K., Hoeger, M., Durand, N.C., Shamim, M.S., Machol, I., Lander, E.S., Aiden, A.P., Aiden, E.L., 2017. De novo assembly of the Aedes aegypti genome using Hi-C yields chromosome-length scaffolds. Science 356, 92e95. Fang, W., Azimzadeh, P., St Leger, R.J., 2012. Strain improvement of fungal insecticides for controlling insect pests and vector-borne diseases. Curr. Opin.

Please cite this article in press as: Wang, Y.-H., et al., The immune strategies of mosquito Aedes aegypti against microbial infection, Developmental and Comparative Immunology (2017), https://doi.org/10.1016/j.dci.2017.12.001

Y.-H. Wang et al. / Developmental and Comparative Immunology xxx (2017) 1e10 Microbiol. 15, 232e238. Ferrandon, D., Imler, J.L., Hetru, C., Hoffmann, J.A., 2007. The Drosophila systemic immune response: sensing and signalling during bacterial and fungal infections. Nat. Rev. Immunol. 7, 862e874. Franz, A.W., Kantor, A.M., Passarelli, A.L., Clem, R.J., 2015. Tissue barriers to arbovirus infection in mosquitoes. Viruses 7, 3741e3767. Fujita, T.M.M., Endo, Y., 2004. The lectin-complement pathwaydits role in innate immunity and evolution. Immunol. Rev. 198, 185e202. Garcia-Munguia, A.M., Garza-Hernandez, J.A., Rebollar-Tellez, E.A., RodriguezPerez, M.A., Reyes-Villanueva, F., 2011. Transmission of Beauveria bassiana from male to female Aedes aegypti mosquitoes. Parasit. Vectors 4, 24. Glare, T.C.J., Gelernter, W., Jackson, T., Keyhani, N., Kohl, J., et al., 2012. Have biopesticides come of age? Trends Biotechnol. 30 (5), 250e258. Gottar, M., Gobert, V., Matskevich, A.A., Reichhart, J.M., Wang, C., Butt, T.M., Belvin, M., Hoffmann, J.A., Ferrandon, D., 2006. Dual detection of fungal infections in Drosophila via recognition of glucans and sensing of virulence factors. Cell 127, 1425e1437. Hillyer, J.F., Christensen, B.M., 2002. Characterization of hemocytes from the yellow fever mosquito, Aedes aegypti. Histochem. Cell Biol. 117, 431e440. Hillyer, J.F., Schmidt, S.L., Christensen, B.M., 2003a. Hemocyte-mediated phagocytosis and melanization in the mosquito Armigeres subalbatus following immune challenge by bacteria. Cell Tissue Res. 313, 117e127. Hillyer, J.F., Schmidt, S.L., Christensen, B.M., 2003b. Rapid phagocytosis and melanization of bacteria and Plasmodium sporozoites by hemocytes of the mosquito Aedes aegypti. J. Parasitol. 89, 62e69. Hoffmann, J.A., Reichhart, J.M., 2002. Drosophila innate immunity: an evolutionary perspective. Nat. Immunol. 3, 121e126. Holt, R.A., Subramanian, G.M., Halpern, A., Sutton, G.G., Charlab, R., Nusskern, D.R., Wincker, P., Clark, A.G., Ribeiro, J.M., Wides, R., Salzberg, S.L., Loftus, B., Yandell, M., Majoros, W.H., Rusch, D.B., Lai, Z., Kraft, C.L., Abril, J.F., Anthouard, V., Arensburger, P., Atkinson, P.W., Baden, H., de Berardinis, V., Baldwin, D., Benes, V., Biedler, J., Blass, C., Bolanos, R., Boscus, D., Barnstead, M., Cai, S., Center, A., Chaturverdi, K., Christophides, G.K., Chrystal, M.A., Clamp, M., Cravchik, A., Curwen, V., Dana, A., Delcher, A., Dew, I., Evans, C.A., Flanigan, M., Grundschober-Freimoser, A., Friedli, L., Gu, Z., Guan, P., Guigo, R., Hillenmeyer, M.E., Hladun, S.L., Hogan, J.R., Hong, Y.S., Hoover, J., Jaillon, O., Ke, Z., Kodira, C., Kokoza, E., Koutsos, A., Letunic, I., Levitsky, A., Liang, Y., Lin, J.J., Lobo, N.F., Lopez, J.R., Malek, J.A., McIntosh, T.C., Meister, S., Miller, J., Mobarry, C., Mongin, E., Murphy, S.D., O'Brochta, D.A., Pfannkoch, C., Qi, R., Regier, M.A., Remington, K., Shao, H., Sharakhova, M.V., Sitter, C.D., Shetty, J., Smith, T.J., Strong, R., Sun, J., Thomasova, D., Ton, L.Q., Topalis, P., Tu, Z., Unger, M.F., Walenz, B., Wang, A., Wang, J., Wang, M., Wang, X., Woodford, K.J., Wortman, J.R., Wu, M., Yao, A., Zdobnov, E.M., Zhang, H., Zhao, Q., Zhao, S., Zhu, S.C., Zhimulev, I., Coluzzi, M., della Torre, A., Roth, C.W., Louis, C., Kalush, F., Mural, R.J., Myers, E.W., Adams, M.D., Smith, H.O., Broder, S., Gardner, M.J., Fraser, C.M., Birney, E., Bork, P., Brey, P.T., Venter, J.C., Weissenbach, J., Kafatos, F.C., Collins, F.H., Hoffman, S.L., 2002. The genome sequence of the malaria mosquito Anopheles gambiae. Science 298, 129e149. Hou, Y., Wang, X.L., Saha, T.T., Roy, S., Zhao, B., Raikhel, A.S., Zou, Z., 2015. Temporal coordination of carbohydrate metabolism during mosquito reproduction. PLoS Genet. 11, e1005309. Huang, L.H., Christensen, B.M., Chen, C.C., 2001. Molecular cloning of a second prophenoloxidase cDNA from the mosquito Armigeres subalbatus: prophenoloxidase expression in blood-fed and microfilariae-inoculated mosquitoes. Insect Mol. Biol. 10, 87e96. Jang, I.H., Chosa, N., Kim, S.H., Nam, H.J., Lemaitre, B., Ochiai, M., Kambris, Z., Brun, S., Hashimoto, C., Ashida, M., Brey, P.T., Lee, W.J., 2006. A Spatzle-processing enzyme required for toll signaling activation in Drosophila innate immunity. Dev. Cell 10, 45e55. Jiang, H., Wang, Y., Kanost, M.R., 1998. Pro-phenol oxidase activating proteinase from an insect, Manduca sexta: a bacteria-inducible protein similar to Drosophila easter. Proc. Natl. Acad. Sci. U. S. A. 95, 12220e12225. Kaneko, T., Silverman, N., 2005. Bacterial recognition and signalling by the Drosophila IMD pathway. Cell Microbiol. 7, 461e469. Kanost, M.R., 1999. Serine proteinase inhibitors in arthropod immunity. Dev. Comp. Immunol. 23, 291e301. Kanost, M.R., Jiang, H., 2015. Clip-domain serine proteases as immune factors in insect hemolymph. Curr. Opin. Insect Sci. 11, 47e55. Kanost, M.R., Jiang, H., Yu, X.Q., 2004. Innate immune responses of a lepidopteran insect, Manduca sexta. Immunol. Rev. 198, 97e105. Khoo, C.C., Piper, J., Sanchez-Vargas, I., Olson, K.E., Franz, A.W., 2010. The RNA interference pathway affects midgut infection- and escape barriers for Sindbis virus in Aedes aegypti. BMC Microbiol. 10, 130. Kim, M.S., Baek, M.J., Lee, M.H., Park, J.W., Lee, S.Y., Soderhall, K., Lee, B.L., 2002. A new easter-type serine protease cleaves a masquerade-like protein during prophenoloxidase activation in Holotrichia diomphalia larvae. J. Biol. Chem. 277, 39999e40004. Koizumi, N., Imai, Y., Morozumi, A., Imamura, M., Kadotani, T., Yaoi, K., Iwahana, H., Sato, R., 1999. Lipopolysaccharide-binding protein of Bombyx mori participates in a hemocyte-mediated defense reaction against gram-negative bacteria. J. Insect Physiol. 45, 853e859. Kokoza, V., Ahmed, A., Cho, W.L., Jasinskiene, N., James, A.A., Raikhel, A., 2000. Engineering blood meal-activated systemic immunity in the yellow fever mosquito, Aedes aegypti. Proc. Natl. Acad. Sci. U. S. A. 97, 9144e9149. Kokoza, V., Ahmed, A., Woon Shin, S., Okafor, N., Zou, Z., Raikhel, A.S., 2010. Blocking

9

of Plasmodium transmission by cooperative action of Cecropin A and Defensin A in transgenic Aedes aegypti mosquitoes. Proc. Natl. Acad. Sci. U. S. A. 107, 8111e8116. Krem, M.M., Di Cera, E., 2002. Evolution of enzyme cascades from embryonic development to blood coagulation. Trends biochem. Sci. 27, 67e74. Kumar, S., Molina-Cruz, A., Gupta, L., Rodrigues, J., Barillas-Mury, C., 2010. A peroxidase/dual oxidase system modulates midgut epithelial immunity in Anopheles gambiae. Science 327, 1644e1648. Lavine, M.D., Strand, M.R., 2002. Insect hemocytes and their role in immunity. Insect biochem. Mol. Biol. 32, 1295e1309. Lemaitre, B., Hoffmann, J., 2007. The host defense of Drosophila melanogaster. Annu. Rev. Entomol. 25, 697e743. Lemaitre, B., Nicolas, E., Michaut, L., Reichhart, J.M., Hoffmann, J.A., 1996. The dorsoventral regulatory gene cassette spatzle/Toll/cactus controls the potent antifungal response in Drosophila adults. Cell 86, 973e983. Lemaitre, B., Reichhart, J.M., Hoffmann, J.A., 1997. Drosophila host defense: differential induction of antimicrobial peptide genes after infection by various classes of microorganisms. Proc. Natl. Acad. Sci. U. S. A. 94, 14614e14619. Ligoxygakis, P., Pelte, N., Hoffmann, J.A., Reichhart, J.M., 2002. Activation of Drosophila Toll during fungal infection by a blood serine protease. Science 297, 114e116. Ling, E., Yu, X.Q., 2006. Cellular encapsulation and melanization are enhanced by immulectins, pattern recognition receptors from the tobacco hornworm Manduca sexta. Dev. Comp. Immunol. 30, 289e299. Matskevich, A.A., Quintin, J., Ferrandon, D., 2010. The Drosophila PRR GNBP3 assembles effector complexes involved in antifungal defenses independently of its Toll-pathway activation function. Eur. J. Immunol. 40, 1244e1254. McGraw, E.A., O'Neill, S.L., 2013. Beyond insecticides: new thinking on an ancient problem. Nat. Rev. Microbiol. 11, 181e193. Michel, T., Reichhart, J.M., Hoffmann, J.A., Royet, J., 2001. Drosophila Toll is activated by gram-positive bacteria through a circulating peptidoglycan recognition protein. Nature 414, 756e759. Musso, D., Gubler, D.J., 2016. Zika virus. Clin. Microbiol. Rev. 29, 487e524. Nappi, A.J., Christensen, B.M., 2005. Melanogenesis and associated cytotoxic reactions: applications to insect innate immunity. Insect biochem. Mol. Biol. 35, 443e459. Nene, V., Wortman, J.R., Lawson, D., Haas, B., Kodira, C., Tu, Z.J., Loftus, B., Xi, Z., Megy, K., Grabherr, M., Ren, Q., Zdobnov, E.M., Lobo, N.F., Campbell, K.S., Brown, S.E., Bonaldo, M.F., Zhu, J., Sinkins, S.P., Hogenkamp, D.G., Amedeo, P., Arensburger, P., Atkinson, P.W., Bidwell, S., Biedler, J., Birney, E., Bruggner, R.V., Costas, J., Coy, M.R., Crabtree, J., Crawford, M., Debruyn, B., Decaprio, D., Eiglmeier, K., Eisenstadt, E., El-Dorry, H., Gelbart, W.M., Gomes, S.L., Hammond, M., Hannick, L.I., Hogan, J.R., Holmes, M.H., Jaffe, D., Johnston, J.S., Kennedy, R.C., Koo, H., Kravitz, S., Kriventseva, E.V., Kulp, D., Labutti, K., Lee, E., Li, S., Lovin, D.D., Mao, C., Mauceli, E., Menck, C.F., Miller, J.R., Montgomery, P., Mori, A., Nascimento, A.L., Naveira, H.F., Nusbaum, C., O'Leary, S., Orvis, J., Pertea, M., Quesneville, H., Reidenbach, K.R., Rogers, Y.H., Roth, C.W., Schneider, J.R., Schatz, M., Shumway, M., Stanke, M., Stinson, E.O., Tubio, J.M., Vanzee, J.P., Verjovski-Almeida, S., Werner, D., White, O., Wyder, S., Zeng, Q., Zhao, Q., Zhao, Y., Hill, C.A., Raikhel, A.S., Soares, M.B., Knudson, D.L., Lee, N.H., Galagan, J., Salzberg, S.L., Paulsen, I.T., Dimopoulos, G., Collins, F.H., Birren, B., Fraser-Liggett, C.M., Severson, D.W., 2007. Genome sequence of Aedes aegypti, a major arbovirus vector. Science 316, 1718e1723. Oliveira, J.H.M., Talyuli, O.A.C., Goncalves, R.L.S., Paiva-Silva, G.O., Sorgine, M.H.F., Alvarenga, P.H., Oliveira, P.L., 2017. Catalase protects Aedes aegypti from oxidative stress and increases midgut infection prevalence of Dengue but not Zika. PLoS Negl. Trop. Dis. 11, e0005525. Ortiz-Urquiza, A., Luo, Z., Keyhani, N.O., 2015. Improving mycoinsecticides for insect biological control. Appl. Microbiol. Biotechnol. 99, 1057e1068. Osta, M.A., Christophides, G.K., Kafatos, F.C., 2004. Effects of mosquito genes on Plasmodium development. Science 303, 2030e2032. Pan, X., Zhou, G., Wu, J., Bian, G., Lu, P., Raikhel, A.S., Xi, Z., 2012. Wolbachia induces reactive oxygen species (ROS)-dependent activation of the Toll pathway to control dengue virus in the mosquito Aedes aegypti. Proc. Natl. Acad. Sci. U. S. A. 109, E23eE31. Perona, J.J., Craik, C.S., 1995. Structural basis of substrate specificity in the serine proteases. Protein Sci. 4, 337e360. Rajah, M.M., Pardy, R.D., Condotta, S.A., Richer, M.J., Sagan, S.M., 2016. Zika Virus: emergence, phylogenetics, challenges, and opportunities. ACS Infect. Dis. 2, 763e772. Rances, E., Ye, Y.H., Woolfit, M., McGraw, E.A., O'Neill, S.L., 2012. The relative importance of innate immune priming in Wolbachia-mediated dengue interference. PLoS Pathog. 8, e1002548. Read, A.F., Lynch, P.A., Thomas, M.B., 2009. How to make evolution-proof insecticides for malaria control. PLoS Biol. 7, e1000058. Roh, K.B., Kim, C.H., Lee, H., Kwon, H.M., Park, J.W., Ryu, J.H., Kurokawa, K., Ha, N.C., Lee, W.J., Lemaitre, B., Soderhall, K., Lee, B.L., 2009. Proteolytic cascade for the activation of the insect toll pathway induced by the fungal cell wall component. J. Biol. Chem. 284, 19474e19481. Royet, J., Dziarski, R., 2007. Peptidoglycan recognition proteins: pleiotropic sensors and effectors of antimicrobial defences. Nat. Rev. Microbiol. 5, 264e277. Saldana, M.A., Etebari, K., Hart, C.E., Widen, S.G., Wood, T.G., Thangamani, S., Asgari, S., Hughes, G.L., 2017. Zika virus alters the microRNA expression profile and elicits an RNAi response in Aedes aegypti mosquitoes. PLoS Negl. Trop. Dis. 11, e0005760.

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Y.-H. Wang et al. / Developmental and Comparative Immunology xxx (2017) 1e10

Sanders, H.R., Foy, B.D., Evans, A.M., Ross, L.S., Beaty, B.J., Olson, K.E., Gill, S.S., 2005. Sindbis virus induces transport processes and alters expression of innate immunity pathway genes in the midgut of the disease vector, Aedes aegypti. Insect biochem. Mol. Biol. 35, 1293e1307. Satoh, D., Horii, A., Ochiai, M., Ashida, M., 1999. Prophenoloxidase-activating enzyme of the silkworm, Bombyx mori. purification, characterization, and cDNA cloning. J. Biol. Chem. 274, 7441e7453. Schnitger, A.K., Yassine, H., Kafatos, F.C., Osta, M.A., 2009. Two C-type lectins cooperate to defend Anopheles gambiae against Gram-negative bacteria. J. Biol. Chem. 284, 17616e17624. Severson, D.W., Brown, S.E., Knudson, D.L., 2001. Genetic and physical mapping in mosquitoes: molecular approaches. Annu. Rev. Entomol. 46, 183e219. Severson, D.W., Knudson, D.L., Soares, M.B., Loftus, B.J., 2004. Aedes aegypti genomics. Insect biochem. Mol. Biol. 34, 715e721. Shin, S.W., Bian, G., Raikhel, A.S., 2006. A toll receptor and a cytokine, Toll5A and Spz1C, are involved in toll antifungal immune signaling in the mosquito Aedes aegypti. J. Biol. Chem. 281, 39388e39395. Shin, S.W., Kokoza, V., Ahmed, A., Raikhel, A.S., 2002. Characterization of three alternatively spliced isoforms of the Rel/NF-kappa B transcription factor Relish from the mosquito Aedes aegypti. Proc. Natl. Acad. Sci. U. S. A. 99, 9978e9983. Shin, S.W., Kokoza, V., Bian, G., Cheon, H.M., Kim, Y.J., Raikhel, A.S., 2005. REL1, a homologue of Drosophila dorsal, regulates toll antifungal immune pathway in the female mosquito Aedes aegypti. J. Biol. Chem. 280, 16499e16507. Shin, S.W., Kokoza, V., Lobkov, I., Raikhel, A.S., 2003. Relish-mediated immune deficiency in the transgenic mosquito Aedes aegypti. Proc. Natl. Acad. Sci. U. S. A. 100, 2616e2621. Soderhall, K., Ajaxon, R., 1982. Effect of quinones and melanin on mycelial growth of aphanomyces spp. and extracellular protease of Aphanomyces astaci, a parasite on crafish. J. Invertebr. Pathol. 39, 105e109. Souza-Neto, J.A., Sim, S., Dimopoulos, G., 2009. An evolutionary conserved function of the JAK-STAT pathway in anti-dengue defense. Proc. Natl. Acad. Sci. U. S. A. 106, 17841e17846. Strand, M.R., Pech, L.L., 1995. Immunological basis for compatibility in parasitoidhost relationships. Annu. Rev. Entomol. 40, 31e56. Sutthangkul, J., Amparyup, P., Charoensapsri, W., Senapin, S., Phiwsaiya, K., Tassanakajon, A., 2015. Suppression of shrimp melanization during white spot syndrome virus infection. J. Biol. Chem. 290, 6470e6481. Takahashi, D., Dai, H., Hiromasa, Y., Krishnamoorthi, R., Kanost, M.R., 2014. Selfassociation of an insect beta-1,3-glucan recognition protein upon binding laminarin stimulates prophenoloxidase activation as an innate immune response. J. Biol. Chem. 289, 28399e28410. Tang, H., Kambris, Z., Lemaitre, B., Hashimoto, C., 2006. Two proteases defining a melanization cascade in the immune system of Drosophila. J. Biol. Chem. 281, 28097e28104. Wang, S., Beerntsen, B.T., 2015. Functional implications of the peptidoglycan recognition proteins in the immunity of the yellow fever mosquito, Aedes aegypti. Insect Mol. Biol. 24, 293e310. Wang, X., Hou, Y., Saha, T.T., Pei, G., Raikhel, A.S., Zou, Z., 2017a. Hormone and receptor interplay in the regulation of mosquito lipid metabolism. Proc. Natl. Acad. Sci. U. S. A. 114, E2709eE2718. Wang, Y., Jiang, H., Cheng, Y., An, C., Chu, Y., Raikhel, A.S., Zou, Z., 2017b. Activation of Aedes aegypti prophenoloxidase-3 and its role in the immune response against entomopathogenic fungi. Insect Mol. Biol. 26, 552e563. Wang, Y.H., Gu, Z.Y., Wang, J.M., Sun, S.S., Wang, B.B., Jin, Y.Q., Shen, W.D., Li, B., 2013. Changes in the activity and the expression of detoxification enzymes in silkworms (Bombyx mori) after phoxim feeding. Pestic. Biochem. Physiol. 105, 13e17. Wang, Y.H., Hu, Y., Xing, L.S., Jiang, H., Hu, S.N., Raikhel, A.S., Zou, Z., 2015. A critical role for CLSP2 in the modulation of antifungal immune response in mosquitoes. PLoS Pathog. 11, e1004931. Wang, Y.H., Wang, J.M., Peng, G.D., Sun, B.X., Li, B., Shen, W.D., 2011. Gene expression analysis from phoxim-induced domesticated silkworm (Bombyx mori) by

whole-genome oligonucleotide microarray. Pestic. Biochem. Phys. 101, 48e52. Waterhouse, R.M., Kriventseva, E.V., Meister, S., Xi, Z., Alvarez, K.S., Bartholomay, L.C., Barillas-Mury, C., Bian, G., Blandin, S., Christensen, B.M., Dong, Y., Jiang, H., Kanost, M.R., Koutsos, A.C., Levashina, E.A., Li, J., Ligoxygakis, P., Maccallum, R.M., Mayhew, G.F., Mendes, A., Michel, K., Osta, M.A., Paskewitz, S., Shin, S.W., Vlachou, D., Wang, L., Wei, W., Zheng, L., Zou, Z., Severson, D.W., Raikhel, A.S., Kafatos, F.C., Dimopoulos, G., Zdobnov, E.M., Christophides, G.K., 2007. Evolutionary dynamics of immunerelated genes and pathways in disease-vector mosquitoes. Science 316, 1738e1743. WHO, 2016. Malaria. World Health Organization, Geneva. Wilson, R., Chen, C., Ratcliffe, N.A., 1999. Innate immunity in insects: the role of multiple, endogenous serum lectins in the recognition of foreign invaders in the cockroach, Blaberus discoidalis. J. Immunol. 162, 1590e1596. Xi, Z., Ramirez, J.L., Dimopoulos, G., 2008. The Aedes aegypti toll pathway controls dengue virus infection. PLoS Pathog. 4, e1000098. Xing, L., Yuan, C., Wang, M., Lin, Z., Shen, B., Hu, Z., Zou, Z., 2017. Dynamics of the interaction between cotton bollworm helicoverpa armigera and nucleopolyhedrovirus as revealed by integrated transcriptomic and proteomic analyses. Mol. Cell. proteomics MCP 16, 1009e1028. Xiong, G.H., Xing, L.S., Lin, Z., Saha, T.T., Wang, C., Jiang, H., Zou, Z., 2015. High throughput profiling of the cotton bollworm Helicoverpa armigera immunotranscriptome during the fungal and bacterial infections. BMC genomics 16, 321. Yassine, H., Kamareddine, L., Osta, M.A., 2012. The mosquito melanization response is implicated in defense against the entomopathogenic fungus Beauveria bassiana. PLoS Pathog. 8, e1003029. Ye, Y.H., Woolfit, M., Rances, E., O'Neill, S.L., McGraw, E.A., 2013. Wolbachia-associated bacterial protection in the mosquito Aedes aegypti. PLoS Negl. Trop. Dis. 7, e2362. Yoshida, H., Kinoshita, K., Ashida, M., 1996. Purification of a peptidoglycan recognition protein from hemolymph of the silkworm, Bombyx mori. J. Biol. Chem. 271, 13854e13860. Yu, X.Q., Kanost, M.R., 2000. Immulectin-2, a lipopolysaccharide-specific lectin from an insect, Manduca sexta, is induced in response to gram-negative bacteria. J. Biol. Chem. 275, 37373e37381. Zaidman-Remy, A., Herve, M., Poidevin, M., Pili-Floury, S., Kim, M.S., Blanot, D., Oh, B.H., Ueda, R., Mengin-Lecreulx, D., Lemaitre, B., 2006. The Drosophila amidase PGRP-LB modulates the immune response to bacterial infection. Immunity 24, 463e473. Zambon, R.A., Vakharia, V.N., Wu, L.P., 2006. RNAi is an antiviral immune response against a dsRNA virus in Drosophila melanogaster. Cell Microbiol. 8, 880e889. Zanluca, C., Melo, V.C., Mosimann, A.L., Santos, G.I., Santos, C.N., Luz, K., 2015. First report of autochthonous transmission of Zika virus in Brazil. Memorias do Inst. Oswaldo Cruz 110, 569e572. Zhao, P., Li, J., Wang, Y., Jiang, H., 2007. Broad-spectrum antimicrobial activity of the reactive compounds generated in vitro by Manduca sexta phenoloxidase. Insect biochem. Mol. Biol. 37, 952e959. Zou, Z., Evans, J.D., Lu, Z., Zhao, P., Williams, M., Sumathipala, N., Hetru, C., Hultmark, D., Jiang, H., 2007. Comparative genomic analysis of the Tribolium immune system. Genome Biol. 8, R177. Zou, Z., Shin, S.W., Alvarez, K.S., Bian, G., Kokoza, V., Raikhel, A.S., 2008. Mosquito RUNX4 in the immune regulation of PPO gene expression and its effect on avian malaria parasite infection. Proc. Natl. Acad. Sci. U. S. A. 105, 18454e18459. Zou, Z., Shin, S.W., Alvarez, K.S., Kokoza, V., Raikhel, A.S., 2010. Distinct melanization pathways in the mosquito Aedes aegypti. Immunity 32, 41e53. Zou, Z., Souza-Neto, J., Xi, Z., Kokoza, V., Shin, S.W., Dimopoulos, G., Raikhel, A., 2011. Transcriptome analysis of Aedes aegypti transgenic mosquitoes with altered immunity. PLoS Pathog. 7, e1002394. Zug, R., Hammerstein, P., 2012. Still a host of hosts for Wolbachia: analysis of recent data suggests that 40% of terrestrial arthropod species are infected. PloS one 7, e38544.

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