Suppressors of cytokine signaling proteins as modulators of development and innate immunity of insects

Developmental and Comparative Immunology 104 (2020) 103561

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Suppressors of cytokine signaling proteins as modulators of development and innate immunity of insects

T

Muhammad Nadeem Abbasa,b,c,1, Saima Kausara,b,c,1, Erhu Zhaoa,b,c, Hongjuan Cuia,b,c,∗ a

State Key Laboratory of Silkworm Genome Biology, College of Biotechnology, Southwest University, Chongqing, 400715, China Key Laboratory of Sericulture Biology and Genetic Breeding, Ministry of Agricultural and Rural Affairs, Southwest University, Chongqing, 400715, China c Medical Research Institute, Southwest University, Chongqing, 400715, China b

A R T I C LE I N FO

A B S T R A C T

Keywords: Insects SOCS JAK-STAT pathway Development Negative regulators

The suppressors of cytokine signaling (SOCS) are a family of intracellular molecules. Many members of this family have been reported to be involved in various physiological processes in invertebrates and vertebrates (e.g., developmental process and immune response). The functions of SOCS molecules seem to remain conserved in animals throughout evolutionary history. The members of the SOCS family play vital roles in the physiological processes by regulating the extent and duration of signaling activities of both Janus Kinase-Signal Transducer and Activators of Transcription (JAK-STAT) and epidermal growth factor receptor (EGFR) pathways in vivo. So far, in different insect species, a variable number of SOCS and SOCS box domain-containing proteins have been identified. These proteins are categorized into different types based on their sequence diversification, leading to an alteration in structure and regulatory function. The biological roles of the many SOCS proteins have been established as a negative or positive regulator of the signaling pathways, as mentioned earlier. Here, we discussed the existing knowledge on the SOCS proteins and their involvement in different biological functions in insects, and future perspectives to further elucidate their physiological roles.

1. Introduction In multicellular organisms, several biological signaling pathways are indispensable for correct growth and development and to maintain homeostasis. However, dysregulation of the biological pathways is frequently related to growth abnormalities and a range of diseases (e.g., cancer, neoplasias). To avoid such events, numerous forms of molecular regulation have developed with essentially every level of the most signaling pathway being targeted for modulation. To assure precise control of signaling output, specialized proteins families have evolved that can play a crucial biological role through mechanisms, including the formation of inactive receptor complexes, sequestration of the signaling pathway ligands, suppression of kinases, or modulation of transcriptional activity (Stec and Zeidler, 2011; Liu et al., 2019). The suppressor of cytokine signaling (SOCS) is a family of intracellular proteins. The members of this family modulate various biological processes, including the JAK-STAT pathway and receptor tyrosine kinase signaling (e.g., EGFR pathway), and insulin receptors, etc. To date, eight family members (SOCS1–SOCS7 and CIS) have been described in mammals. These members comprise a SOCS box located in

the C-terminus and SH2 domain situated in the center. Additionally, SOCS 1–3 characterized by short an N-terminal region, which contains a kinase inhibitory region located next to the SH2 domain. Whereas, SOCS 4–7 proteins have an extended dissimilar N-terminal region, which lacks any distinct domains (Linossi and Nicholson, 2015; Jiang et al., 2017; Abbas et al., 2018). All of the SOCS family proteins bind to phosphorylated tyrosine residues by their SH2 domains. This association permits the SOCS molecules to bind to phosphorylated JAKs and transmembrane receptors. It may act as a direct steric inhibitor avoiding STAT molecules from associating with the stimulated receptor/JAK complex (Croker et al., 2008; Kausar et al., 2019). Furthermore, interactions through the SH2 domain also provide a substrate recognition function for the SOCS box associated Elongin-Cullin-SOCS (ECS) E3 ubiquitin ligase complex. In this scenario, the SOCS box domain interacts with Elongins B and C, which in turn recruit Cullin 5 and Roc/Rbx1 to generate a competent Ubiquitin E3 ligase complex. Docking of this complex allows the transfer of ubiquitin moieties onto the substrate molecule, targeting it for degradation (Stec and Zeidler, 2011). Insects are the most diverse and important group of animals on the

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Corresponding author. State Key Laboratory of Silkworm Genome Biology, College of Biotechnology, Southwest University, Chongqing, 400715, China. E-mail addresses: [email protected] (M.N. Abbas), [email protected] (S. Kausar), [email protected] (E. Zhao), [email protected] (H. Cui). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.dci.2019.103561 Received 13 September 2019; Received in revised form 21 November 2019; Accepted 22 November 2019 Available online 28 November 2019 0145-305X/ © 2019 Elsevier Ltd. All rights reserved.

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Fig. 1. Diversity of insect suppressor of cytokine signaling molecules: Phylogram of suppressor of cytokine signaling molecules from insect species (NCBI accession numbers given in Table 1) was generated by MEGA program (v5.05) and clustering was done using a neighbour-joining algorithm with 1000 bootstrap.

example, the functions of SOCS proteins and their biochemical interactions have progressively been elucidated in human; however, their physiological roles in vivo are less easily analyzed. Therefore, Drosophila is used as a model organism to determine the functions of the proteins (e.g., SOCS) in vivo. Here, we discuss the available knowledge on the SOCS proteins in insects, particularly their involvement in immune response and the regulation of EGFR and JAK-STAT signaling.

planet. Apart from the open ocean, they occupy approximately all habitats, e.g., forest, desert, swamps, and harsh environments (Abbas et al., 2012; Riaz et al., 2017). The majority of insects are directly important to human and the environment. For example, many insect species are parasitoids, predators on other harmful pests (Ruby et al., 2011; Inayat et al., 2011), and others are pollinators, decomposers of organic matter, or producers of valuable products, including honey or silk (Kausar et al., 2017a,b; Sun et al., 2018; Abbas et al., 2020). Many insect species have medical importance, e.g., mosquitoes, and some are model systems for human diseases, such as Drosophila. Additionally, silkworm species that are used as a model system for other lepidopteran insects (Abbas et al., 2013; Sun et al., 2017). Therefore, biochemical studies in Drosophila and other model systems are highly essential to understand different biological events in insects and humans. For

2. Discovery and structural properties of SOCS proteins The SOCS/CIS is a family of intracellular proteins. This family had initially identified in vertebrates nearly two decades ago when a first member (SOCS 1) was cloned using a functional screening for inhibitors of cytokine signaling (Starr et al., 1997; Dalpke et al., 2008). So far, 2

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Fig. 2. Suppressor of cytokine signaling box family protein. Domain structures of representative members from each of the three families of SOCS box family proteins. Abbreviations: ANK, ankyrin repeat; SH2, Src homology 2; SPRY, SPla/Ryanodine receptor domain.

3. Sequence and structural diversity of SOCS

mammals contain eight members, including CIS and SOCS 1 to SOCS 7 of this family. The functions of these proteins have been well established in vertebrates (Alexander, 2002; Greenhalgh et al., 2002). In insects, the identification and characterization of SOCS family members is still incomplete, as only three members (SOCS 36E, SOCS 44A, and SOCS 16D) described from Drosophila and (Stec and Zeidler, 2011), and four (SOCS 2–12, SOCS 5A, SOCS 5B, and SOCS 6) from B. mori (Abbas et al., 2017, 2018). These proteins have also been reported from many other insect species such as mosquitos, beetles, Manduca sexta, etc., suggesting their broad distribution in them (Gupta et al., 2009; Zuo, 2012; Abbas et al., 2017; Patnaik et al., 2019). Furthermore, many SOCS family members remain unidentified and uncharacterized in several insect species, for example, 14 SOCS members identified in Locuta migratoria, but their functions remain to elucidate (Zhang et al., 2015). All of the CIS/SOCS family members share a central SH2 domain, SOCS box, a conserved C-terminal, and an amino-terminal domain differs considerably in sequence and number of base pairs between different family members. The SH2 domain is required for the interaction with phosphorylated JAKs and receptors in tyrosine-based interaction. Many other proteins also contain the C-terminal SOCS box. The SOCS box associates with elongin B and C, Cullins, and the RING finger-domain-only protein RBX2. The C-terminal SOCS box mainly functions in the recruitment of ubiquitin transferase system that may work like E3 ubiquitin ligase, and thus trigger the degradation of a protein associated with N-terminal region of SOCS proteins (Kamura et al., 1998; Abbas et al., 2017; Wang et al., 2018). Besides, the SOCS proteins, many other SOCS box containing protein sequences, are also available on the NCBI database; however, all of these proteins remain uncharacterized in insects (Table 2). Collectively, SOCS proteins are present in both invertebrates and vertebrates, suggesting the functional importance of these proteins in the animals. The number of SOCS family members seems to vary in vertebrates and invertebrates (e.g., insects); however, still, it is preliminary as most of the SOCS family members in insects are uncharacterized and many of them are not available on the NCBI database. Therefore, more work is required to understand the comprehensive picture of SOCS proteins in insects.

Although numerous SOCS family members have been reported, however still there is a lack of a complete record in insects. The rapid advancements in genome sequencing technology have significantly improved sequence information. To date, many SOCS family members have been reported from different insect species, e.g., agriculturally, medically important species. Some of these species are used as model systems such as B. mori, A. perenyi, D. melanogaster, etc. (Abbas et al., 2018; Kausar et al., 2017a,b; Zeidler et al., 2000). Amino acid sequence-based phylogenetic analysis has shown that SOCS genes remain evolutionarily conserved in various insect species (Fig. 1). Besides the functional importance of SOCS proteins, our knowledge is limited in insects. Furthermore, a complete record of SOCS gene sequences from various species is still unavailable in public repository (NCBI). As shown in Fig. 1, within the clades, different insect species comprise related SOCS proteins, which could be indicating an evolutionary divergence among them. However, Some SOCS proteins are distantly related to the other associated types; this may be because of their low amino acid sequence similarity with other related types of SOCS members. For example, some SOCS 6 proteins formed a clade with un-related proteins, as shown in Fig. 1. Human SOCS proteins consist of the central SH2 domain, a carboxyterminal 40-amino-acid module that is known as the SOCS box, and an amino-terminal domain of variable length and divergent sequence (Yoshimura et al., 2007a,b). The sequence analysis revealed that all of the insect SOCS proteins comprise these conserved domains, suggesting could be similar functions to that of their mammalian counterpart (Fig. 2).

4. The SOCS box containing proteins in insects The SOCS box domain-containing proteins were initially identified as the members of the SOCS family. A human's genome includes 30 different kinds of SOCS box domain-containing proteins. These proteins are classified into six subgroups based on their additional domains responsible for controlling localization and function (Durham et al., 2019). In insects, there is no complete record of SOCS Box domaincontaining proteins, and only incomplete information is available on 3

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different physiological activities. The knowledge of these proteins will help to understand various physiological events better and also will help develop strategies for the management of insect species, especially economically important insects.

the public repository (NCBI). However, SOCS box domain-containing proteins are broadly distributed in insects, and till now, only some members have been described (Table 2). This section provides an overview of other SOCS box containing families that have been identified in insects.

5. Distribution of SOCS proteins in tissues and developmental stages

4.1. The SOCS proteins SOCS proteins, since their discovery, have been broadly studied in vertebrates, and so far, eight members have been identified in them. SOCS proteins have been shown to play multiple biological roles, and mainly they regulate immune and developmental activities by regulation of different signaling pathways (Yoshimura et al., 2007a,b; Wilson, 2014). However, the functional importance of SOCS proteins has not been well described in insects, as still there is incomplete knowledge in them (Rawlings et al., 2004; Abbas et al., 2018). Therefore, more studies are required to identify and characterize SOCS proteins to understand the different physiological processes of insects.

In insects, the SOCS molecules have been shown to regulate various physiological functions such as growth and development, immunological responses, etc. The SOCS being multifunctional proteins are widely distributed in different developmental stages and multiple tissues of insects. For example, these proteins have been shown to produce in the various tissue of insects (e.g., mosquitoes); however, their concentration increases on crucial developmental stages (Noh et al., 2006; Dhawan et al., 2015). Recently, Abbas et al. (2017, 2018) also reported similar trends for SOCS2-12 and SOCS6 in different developmental stages of the silkworm, B. mori. Additionally, SOCS5, SOCS6, and SOCS7 are also expressed throughout the life Tenebrio molitor (Patnaik et al., 2019). It seems that SOCS proteins are involved in diverse physiological functions throughout animal life, but they change their concentration with physiological requirements of the developmental stage. Furthermore, the SOCS members also have broad distribution patterns in different tissues of insects. Abbas et al. (2017, 2018) noted a high expression of SOCS proteins in fat body and hemocytes. This trend seems to conserve among insects as the expression of SOCS5, SOCS6, and SOCS7 has been reported high in hemocytes Tenebrio molitor (Patnaik et al., 2019). Hemocytes and fat bodies are the primary immune tissues in insects (Tzou et al., 2002; Ramet, et al., 2002). The hemocytes play an essential role in cellular defense, including phagocytosis and wound repair. Whereas, fat bodes are the key source of antimicrobial peptides. In some insect species, hemocytes also produce these immune peptides in small quantities (Meister, 2004; Kausar et al., 2017a,b; Abbas et al., 2019). Taken together, this high expression level of the SOCS proteins in the immune tissues suggests that they might play a critical role in the innate immune responses of insects.

4.2. The sp1A/ryanodine receptor (SPRY) domain-containing SOCS box protein (SPSB) To date, three SPSB proteins (SPSB1, SPSB3, SPSB 3-like) reported from insects, each of which contains an N terminal SPRY domain responsible for target protein interaction linked to a C terminal SOCS box domain (Table 2, Fig. 2). Four SPRY domain-containing protein (SPSB14) identified from vertebrates, most of them have been well characterized. These proteins are involved in immune responses by degrading nitric oxide synthase protein, thereby prevent the accumulation of cytotoxic levels of nitric oxide that could damage host cells/tissue (Nishiya et al., 2011; Wang et al., 2018; Durham et al., 2019). A recent study suggested that SPSB proteins are multifunctional and are involved in the development, cell growth, innate immune signaling, and cytokine signaling suppression. Further, this study showed that the expression SPSB-3 gene is increased after bacterial and viral infections, suggesting the involvement of this gene to limit infection. Besides, SPSB-3 is mainly distributed in the cytoplasm and plays a critical biological role in the regulation of cytokines production in granulocytes (Wang et al., 2019). This study failed to elaborate on the regulatory mechanism of SPSB-3 gene. Apart from the functional importance of this gene family, so far, no one explored the biological roles of these genes in insects. Therefore, studies on these proteins will help us to understand their cellular growth, development, and immune functions in insects.

6. The biological functions of SOCS proteins in the regulation of insect immunity In eukaryotes, the innate immune responses to microbial pathogens are initiated after recognition of these pathogens by Toll-like receptors (TLRs), which are highly specific in recognition of the pathogens: TLR 3, 5, and 9 recognize Gram-negative bacterial and viral components, and TLR2 recognizes Gram-positive bacterial components (Medzhitov, 2001). It was believed that insects utilize Toll and immune deficiency (Imd) pathways for immune responses (Lemaitre and Hoffmann, 2007; SouzaNeto et al., 2009; Cheng et al., 2016a,b; Zhu et al., 2019). Later it was discovered that Janus kinase signal transduction and activators of transcription (JAK-STAT) pathway is also an important contributor in the innate immune responses (Myllymäki and Rämet, 2014). The involvement of this pathway in immune responses was first reported in the mosquito, Anopheles gambiae (Barillas-Mury et al., 1999). The Imd, Toll, and JAK-STAT pathways use different receptors to recognize Gram-positive, Gram-negative bacteria, fungi, yeasts, and viruses (Souza-Neto et al., 2009; Cheng et al., 2016a,b; Liu et al., 2019). In this section, we describe the JAK-STAT pathway involvement in different physiological processes and its regulation by SOCS molecules in insects.

4.3. Ankyrin repeat and SOCS box (ASB) There is a lack of knowledge on ankyrin repeat SOCS box proteins in invertebrates, particularly in insects. The sequence of only five members is available in the public repository database (NCBI), which contains a variable number of ankyrin repeats upstream of a C terminal SOCS box (Table 2, Fig. 2). Contrary to invertebrates, 18 members (ASB1–18) of this family have been identified from vertebrates, and ankyrin repeats vary from 1 to 12 in them (Durham et al., 2019). In human, Key substrates of ASB proteins are signaling molecules including, insulin receptor substrate 4, an adaptor molecule important for leptin and insulin receptor signaling in the brain, and tumor necrosis factor receptor, which is targeted for degradation by ASB3 protein, thereby preventing TNF-mediated apoptotic responses in Jurkat cells (Chung et al., 2005; Durham et al., 2019). Collectively SOCS box domain-containing proteins are multifunctional and have been shown to involve in different crucial physiological activities in animals. In particular, they regulate developmental events and innate immune functions. Despite the importance of these proteins, only a few studies are available in invertebrates, especially in insects. Thus, future studies should focus on discovering the more members of this family, further determining their involvement in

6.1. The JAK-STAT pathway in insects The JAK-STAT signaling pathway remained conserved throughout evolutionary history, and structural and functional components homologues observed in the vertebrate system also exist in insects (Zeidler 4

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Fig. 3. Interaction of suppressor of cytokine signaling (SOCS) molecules with different physiological pathways. The SOCS molecules negatively modulate the JAK-STAT signaling pathway (A). The SOCS proteins positively and negatively control the EGFR signaling pathway (B).

domains of STAT molecules. The STAT molecules are generally present in the cytoplasm as inactive monomers before recruitment to the JAK/ receptor complex. Though, it has also been reported that STAT molecules constitutively shuttle between the cytoplasm and nucleus before being retained in the nucleus after activation (Vinkemeier, 2004). STATs are themselves phosphorylated, thereby forming homo or heterodimeric complex following attachment with the receptor/JAK complex. A dimer complex is stabilized by the interaction between the SH2 domain of one molecule and the phospho-Tyr of the other molecule and then translocate to the cell nucleus where they bind to a palindromic sequence of DNA in the promoters of pathway target genes to stimulate transcription (Fig. 1). Finally, the dimerization of STATs via an N-terminal domain interaction can occur before stimulation of pathway, only complexes activated by Tyrphosphorylation seem to stimulate expression of the target gene (Braunstein et al., 2003).

et al., 2000). This pathway contains a diverse family of extracellular ligands that bind to transmembrane receptors, and the intracellular part of these receptors is associated with JAK kinases (Schindler, 2002; Kisseleva et al., 2002). The JAK-STAT pathway mechanism of activation has been well established in both vertebrates and invertebrates, including insects, e.g., Drosophila (Fig. 3A). Comparative genomic analysis on different insect species identified orthologous for core components of the JAK-STAT signaling pathway (Domeless, hsp70/ hsp90 organizing protein (Hop), STAT, SOCS and protein inhibitor of activated STAT (PIAS)) in them (Waterhouse et al., 2007; Souza-Neto et al., 2009). In a typical model of the JAK-STAT pathway, following stimulation, an extracellular ligand binds to an extracellular part of a transmembrane protein, leading to the activation of the intracellular part of a transmembrane protein-associated JAKs. The JAK tyrosine kinases then phosphorylate themselves and their associated intracellular part of the protein to create docking sites for the SH2 5

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2001; Tulina and Matunis, 2001). Spermatogenesis is a well-studied example of the JAK-STAT pathway involvement in this process. M. sexta testis at larval stages comprises hub-like structures surrounded by mitotically active cells that are capable of self-renewal stem cell population. The expression of SOCS is increased in hub-adjacent cells, along with the enhanced activity of the JAK-STAT pathway. The enhanced expression of SOCS along with the JAK-STAT pathway suggesting the strong regulatory (negative) role of SOCS in testis stem cell maintenance (Elliott and Zeidle, 2008). Furthermore, The JAK-STAT pathway signaling has been demonstrated to play an essential role in the maintenance of stem cells in the testis stem cell niche (Kiger et al., 2001; Leatherman and Dinardo, 2008). Investigation of interactions between various components of the niche has also exhibited a biological role for the SOCS 36E gene in the maintenance of different populations of stem cells in the niche. Germline stem cell loss has also been observed in SOCS 36E mutant testis compared with somatic stem cells. In addition, following SOCS 36E depletion STAT 92E transcription level is increased in somatic stem cells and hub cells. Whereas, SOCS 36E overexpression in the testis leads to loss of somatic cells but not germline stem cells, indicating that the SOCS 36E protein negatively modulates self-renewal and maintenance of somatic stem cells, and allowing for germline stem cells selfrenewal (Singh et al., 2010; Amoyel et al., 2016). The involvement of this pathway in the Drosophila oogenesis process is another example of the participation of the JAK-STAT pathway in development. This pathway maintains the stem cell balance in the ovary niche in a manner analogous to the testis (Decotto and Spradling, 2005). It also modulates the migration of the border cells in the developing egg (Ghiglione et al., 2002). Transcription of the Upd gene in the paired polar cells resided at the posterior and anterior tips of the follicle causes recruitment of the adjacent follicular cells to form a cluster of presumptive border cells. Approximately eight to ten follicular cells migrate along the midline of the egg chamber to meet the oocyte and form the micropyle (Montell, 2003). SOCS 36E overexpression in the border cells causes impairs in migration and recruitment consistent with a decrease in the activity of the JAK-STAT pathway (Silver et al., 2005). During development, cell differentiation is also considered an essential developmental process, and a recent study suggested that InRAkt-TORC1 signaling attenuates border cell fate by SOCS 36E protein (Kang et al., 2018). It has been shown that SOCS 36E expression is by STAT and controls JAK-STAT activities to restrict the high level of STAT activity in the border cells (Yoon et al., 2011; Monahan and StarzGaiano, 2013). However, functional loss of SOCS 36E results in the differentiation of extra border cells. A recent study demonstrated that InR, Akt, or Raptor's inhibition of border cell fate was specifically mediated by SOCS 36E (Kang et al., 2018). In vitro analysis exhibited that the SOCS 36E protein level is greatly decreased in the InR, Akt, or Tor mutant follicle cell clones, accompanied by a rise of STAT activity in clones near to polar cells. In contrast, SOCS 36E overexpression rescue the increase in border cell number but not the migration abnormality caused by Akt, InR, or Raptor suppression, suggesting their role in the earlier cell fate determination process, but not in the later migratory process are mediated by SOCS36. While, depression of SOCS 36E protein rescued a decrease in border cell number in Pten and Tsc1 depleted cells (Kang et al., 2018). Moreover, the JAK-STAT pathway participation in the development of Drosophila wings and their venation is also providing clues about the function of this biological pathway (Yan et al., 1996; Callus and Mathey-Prevot, 2002). Ectopic expression of SOCS 36E in the developing wing is responsible for a stretched-out wing phenotype, similar to that observed in regulating upd mutants (Callus and Mathey-Prevot, 2002). Ectopic expression of SOCS44A also produces defects in wing venation that do not completely phenocopy those attained by SOCS 36E misexpression, suggesting that the two proteins may have different biological functions (Rawlings et al., 2004). Genetic interaction

6.2. Participation of SOCS proteins in the regulation JAK-STAT signaling pathway All of the insects SOCS proteins contain SOCS and SH domains, suggesting these molecules may have similar functions to that of their counterpart, vertebrates. Despite insects, SOCS are less explored proteins, and the available information indicates that they are an essential regulator of the JAK-STAT pathway (Fig. 3A). For example, SOCS2-12 (B. mori), SOCS (Anopheles dirus) SOCS2 (A. pernyi: unpublished), SOCS 36E and SOCS44A (Drosophila) have been reported to control the activity of this pathway (Rawlings et al., 2004; Abbas et al., 2017; Liew et al., 2018). Growing evidence suggest the involvement of the JAKSTAT pathway in development and immune responses in insects. It has been shown that microbial infection (dengue virus) can stimulate the JAK-STAT signaling pathway in Aedes aegypti. Further, the authors suggested that the knockdown of SOCS and protein inhibitors of activated STAT (PIAS) induce resistance in mosquitoes to the dengue virus. While, silencing of the JAK-STAT pathway and its receptor Domeless (Dome) and the heat shock protein 70/90 organizing protein (Hop) by RNAi can enhance susceptibility to the virus Souza-Neto et al., 2009). Contrary to this study, recently, Liew and his co-workers (2018) suggested that the JAK-STAT pathway-related molecules (e.g., SOCS, STAT, PIAS) are not significantly changed after P. berghei infection. However, the authors observed that this pathway is activated following ookinete invasion in the midgut, and the repressor proteins (SOCS, PIAS) are produced to counter the activated pathway as a negative regulator (Dhawan et al., 2015). A hallmark of JAK-STAT pathway activation is the translocation of activator of transcription STAT to the nucleus, where it initiates the transcription of target genes. Further, the activation of this pathway can be determined by immunochemistry analysis of Anopheles STAT (a STAT) and cellular localization (BarillasMury et al., 1999). In Drosophila fat body cells, STAT92E translocates into the nucleus after immune challenge. This translocation is generally halted with reduced JAK activity (hopM38/hopmsv1) (Agaisse et al., 2003). Conversely, robust staining was detected both in the cytoplasm and in the nucleus of flies carrying a Drosophila JAK gain-of-function mutation [Tumorous-lethal (Tum-l)]. These results establish the existence of a JAK-dependent activation of STAT in the Drosophila fat body in response to septic injury (Agaisse et al., 2003). In un-infected mosquitoes, a STAT protein resides both in the nucleus and in the cytoplasm. Whereas in infected mosquitoes, a STAT considerably translocates from the cytoplasm and accumulates in the nucleus. Collectively, the JAK-STAT pathway seems to be involved in immune functions, and SOCS proteins strongly regulate its immune activity in insects. A recent study suggested the enhancement of antimicrobial peptides in response to microbial infection, when the SOCS2-12, a JAK-STAT negative regulator, is silenced. The authors further proposed that the JAK-STAT pathway regulates antimicrobial peptides production. Although, this study provided multiple evidence for the increase of antimicrobial peptides by using various techniques, e.g., qRT-PCR analysis, bacterial clearance assay, etc. However, this study failed to clarify whether SOCS protein responsive antimicrobial peptides are the sole factor to clear pathogens from plasma, as it may also have other antimicrobial factors that contribute to this process. Additionally, the authors reported the stimulation of cecropin expression by Gram-positive bacteria following SOCS 2–12 suppression, which is usually controlled by the Imd pathway and is produced following Gram-negative bacteria infection. This interesting phenomenon suggests that SOCS proteins may govern the various physiological process by a complex mechanism in insects (Abbas et al., 2017). Besides the participation of the JAK-STAT pathway in immune function, there are multiple lines of evidence suggesting its involvement in the developmental process of insects. The JAK-STAT signaling pathway reported playing a crucial biological role in the maintenance of stem cells (testis) in Manduca sexta and D. melanogaster (Kiger et al., 6

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JAK-STAT cascade during microbial infection. The SOCS 2–12 deficient plasma of B. mori (Dazao) has been reported to be higher resistance to bacterial growth. Additionally, microbial infection increases antimicrobial peptides production in the SOCS 2–12 depleted organisms. This study further demonstrated that different microbial pathogens (e.g., bacteria, viruses, and fungus) significantly induced SOCS 2–12; however, the time of the induced expression varies with various types of pathogens (Abbas et al., 2017). In Drosophila, SOCS 36E can downregulate the Upd3 and STAT signaling in the gut and seems to be associated with the innate immune system. As oral microbial infection in flies can induce the production of immune responsive elements such as antimicrobial peptides and their regulators. However, the expression of SOCS 36E protein and antibacterial peptides (e.g., drosomycin) production is reduced in STAT and Upd3 deficient flies, even after Erwinia carotovora carotovora 15 infection (Buchon et al., 2009), suggesting the interaction of STAT and Upd3 molecules with SOCS protein and the immune effectors. Another study showed that SOCS deficient mosquitoes (Anopheles gambiae) are more resistant against microbial infection e.g., plasmodium infection. In A. gambiae, silencing of SOCS reduce the number of plasmodium oocyst in the midgut and thereby to increase the survival of this species (A. gambiae). The lack of SOCS following infection induces the STAT molecule, which participates in the production of nitric oxide synthase (NOS) when the insect is exposed to plasmodial or bacterial infection. The STAT pathway mediates the late-phase antiplasmodial response that reduces oocyst survival in A. gambie (Gupta et al., 2009). Later, Dhawan et al. (2015) identified SOCS from Anopheles culicifacies that share a strikingly high level of amino acid sequence similarity with the reported SOCS of other mosquitoes. The authors found that SOCS expression significantly increased in the midgut following plasmodial infection. Furthermore, A recent study suggested the enhancement of three SOCS molecules (SOCS 5, 6, 7) in the fat body, gut, and hemocyte of Tenebrio molitor after fungus Candida albicans exposure (Patnaik et al., 2019). Contrary to these studies, Liew et al. (2018) demonstrated no significant variation in SOCS mRNA expression in Anopheles dirus at different time points following Plasmodium berghei infection (Liew et al., 2018). There are multiple lines of evidence suggesting the microbial infection can change the expression of SOCS genes in insects. For example, SOCS 6 expression is enhanced in the immune tissues (fat body and hemocyte) of B. mori when its larvae are exposed to microbial pathogens such bacteria, fungi, and viruses. (Abbas et al., 2018). This preliminary study did not explore the molecular mechanism underlying this phenomenon. Concurrently, a study on Eriocheir sinensis suggested a possible mechanism for SOCS 6 enhancement in response to microbial infection. The SOCS6 stimulates the production of Akt and Relish, thereby activating the NF-κB signaling pathway for the protection against pathogen infection (Qu et al., 2018). Although SOCS6 involvement in the immune functions has been established, however, the detailed molecular mechanism needs to be explored in invertebrates, including insects. Taken together, the immunological roles of SOCS proteins are still infancy, and these studies provide a piece of preliminary information on the immune responses of SOCS molecules against microbial infection in insects. On the whole, SOCS proteins in various species probably exhibit similar physiological functions in different species and their diverse tissue/organs, suggesting the biological role of SOCS proteins remained conserved throughout evolutionary history. Although many studies elaborated on the functions of these proteins, however their detailed mechanism in most of the insect species is still unknown or. Additionally, due to the lack of genome sequence in many insect species, the complete record of this family is not available.

experiments also indicate different physiological roles for SOCS44A and SOCS 36E. Increased SOCS44A dosage in Drosophila carrying combinations of weak loss-of-function Hop alleles causes raised lethality, whereas ectopic Hop expression leads to lethality that can be rescued by SOCS 36E (Callus and Mathey-Prevot, 2002). Another study suggested the proliferative role of the JAK-STAT pathway in the Drosophila gut and observed that proliferative activity is reduced when JAK-STAT activity and SOCS 36E expression is decreased (Sun et al., 2018). Collectively, SOCS 36E protein is a critical negative regulator of the JAKSTAT pathway, whereas SOCS44A can reduce this pathway activity to a weaker extent. The biochemical analysis of SOCS 36E highlighted the regulatory mechanism of this gene in Drosophila. The SOCS 36E regulates the JAKSTAT pathway activity via two independent mechanisms. (i) Drosophila Cullin-5 and Elongin B/C act through the SOCS box of SOCS 36E to suppress the pathway activity, especially in response to ligand stimulation, a process that involves endocytic trafficking and lysosomal degradation of the Domeless receptor. (ii) SOCS 36E also reduces both basal and stimulated pathway activity through an Elongin/Cullin independent mechanism that is mediated by the N-terminus of SOCS 36E. The SOCS 36E requires this terminal region for the physical interaction with a Domeless receptor (Stec et al., 2013). Later, another study suggested that SOCS 36E regulates JAK-STAT signaling through a Cul2dependent mechanism, as well as by a Cullin independent manner, in vivo. SOCS 36E genetically interacts with the Cullin 2 scaffolding protein and involves in specification of motile border cells in Drosophila oogenesis. Like SOCS 36E, Cul 2 is required to limit the number of motile cells in egg chambers. The authors further suggested that loss of Cul 2 in the follicle cells remarkably enhances levels of nuclear STAT protein that causes additional cells acquiring invasive properties. Further, the reduction of Cul 2 suppressed border cell migration defects. The SOCS 36E can also attenuate STAT activity in the egg chamber even without a functional SOCS box (Monahan and Starz-Gaiano, 2015). It has also been established that the JAK-STAT pathway controls many aging-related cellular functions (Doles and Olwin, 2014). Moskalev and his co-workers (2019) demonstrated that conditional overexpression of upd1 in various tissues of Drosophila imago induce pro-aging or pro-longevity effects in a tissue-dependent manner. The impact of upd1 on life span is accompanied by the transcription activation of the JAK-STAT pathway target genes such as domeless and SOCS 36E. The enhanced SOCS 36E expression, along with the JAKSTAT pathway, suggest controlling the robustness of this pathway. Overall, the JAK-STAT is an essential biological pathway that governs both the innate immune responses and developmental activities in animals. For example, it controls various developmental processes, including proliferation and maintenance of germline cells, wing development in Drosophila, etc. Besides, the regulation of developmental activities also modulates the innate immune responses, e.g., involves in the production of antimicrobial peptides to limit microbial infection. However, the robustness of this pathway is controlled by SOCS proteins (Fig. 3A). Further, it seems to remain to conserve throughout the evolutionary period. To further deepen insight into the insect SOCS protein, more studies are required on different insect species. 7. SOCS proteins and microbial infection Bacteria, fungi, and viruses are important groups of microbial pathogens, which are responsible for various insect diseases. The innate immune system of insects detects them by recognizing their specific pathogen-associated molecular patterns (PAMPs) and induce a robust immune response by changing the expression of immune genes, which usually govern signaling pathways. Many studies suggested that various microbial pathogens affect SOCS genes expression in insects (Table 1). For example, the SOCS 2 protein, a negative regulator of the JAK-STAT pathway, plays a crucial role in maintaining a balance of antimicrobial and proinflammatory effects of ligand signaling associated with the 7

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Table 1 SOCS proteins from insects. Genes Name

Species name

Accession number

Number of amino acids

Immune/Developmental function

Participation Pathway

Reference

SOCS 2-12 SOCS 6 SOCS 5 SOCS

Bombyx mori Bombyx mori Bombyx mori Anopheles culicifacies

NP_001243921 ADO51636 AFI61406 AII72405

263 305 174 325

Bacteria, fungus, and virus ↑ Bacteria, fungus, and virus ↑ – Plasmodium infection ↑

JAK-STAT EGFR –

Abbas et al. (2017) Abbas et al. (2018) – Dhawan et al. (2015)

SOCS

Anopheles dirus

KX870847

394

P. berghei infection: No significant change in expression

JAK-STAT

Liew et al. (2018)

SOCS

Anopheles gambiae

ABV01933

396

Plasmodium infection ↑

JAK-STAT

Gupta et al. (2009); Smitha et al. (2015)

SOCS SOCS

Anopheles stephensi Anopheles sinensis

477

Kajla et al. (2016) Noh et al. (2006)

SOCS SOCS6 SOCS7 OCS36E

Aedes aegypti Aedes aegypti Aedes aegypti Drosophila melanogaster Drosophila melanogaster Drosophila melanogaster Tenebrio tribolium Tenebrio molitor

Escherichia coli and Micrococcus luteus ↑ Bacteria, and immune elicitor (lipoteicpic acid, CpG-DNA, laminarin) ↑ – Anti-dengue defense – – –

JAK-STAT

KFB35251

– JAK-STAT – – EGFR and JAK-STAT

– Souza-Neto et al. (2009) – – Callus and MatheyPrevot (2002) Rawlings et al. (2004)

SOCS44A SOCS16D SOCS SOCS5, SOCS6, SOCS7 SOCS2 SOCS6 SOCS7 SOCS6 SOCS6 SOCS5 SOCS4 SOCS7 SOCS5 SOCS7 SOCS5 SOCS6 SOCS5 SOCS16D SOCS44A SOCS2 SOCS5 SOCS6 SOCS7 SOCS SOCS2-12 SOCS6 SOCS7 SOCS2 SOCS5 SOCS6 SOCS2 SOCS7 SOCS6 SOCS5 SOCS SOCS SOCS5 SOCS5 SOCS2 SOCS5 SOCS6 SOCS7

Tenebrio molitor Zeugodacus cucurbitae Zeugodacus cucurbitae Frankliniella occidentalis Leptinotarsa decemlineata Leptinotarsa decemlineata Ceratitis capitate Ceratitis capitate Drosophila guanche Drosophila guanche Halyomorpha halys Halyomorpha halys Cryptotermes secundus Drosophila busckii Drosophila busckii Lasius niger Lasius niger Lasius niger Lasius niger Danaus plexippus Danaus plexippus Danaus plexippus Danaus plexippus Eumeta japonica Eumeta japonica Eumeta japonica Harpegnathos saltator Harpegnathos saltator Harpegnathos saltator Harpegnathos saltator Ooceraea biroi Ooceraea biroi Ooceraea biroi Pediculus humanus corporis Pediculus humanus corporis Temnothorax longispinosus Temnothorax longispinosus Temnothorax longispinosus

XP_001660156 XP_021707736 NM_078869

312 260 736

NM_078935

342

NM_001272733

1021

EGFR/MAPK

Oogenesis and embryogenesis E. coli, S. aureus and C. albicans ↑

JAK-STAT

Bäumer et al. (2011) Patnaik et al. (2019)

XP_011182900 JAD11819 XP_026279961

372 911 268

E. coli ↑ – – -

– – –

Patnaik et al. (2019) – – –

XP_023024218

345

–

–

–

XP_023024843

494

–

–

–

XP_004535944 JAC02138 SPP79984 SPP78288 XP_014279177 XP_014279265 XP_023715051 ALC49865 ALC42463 KMQ89956 KMQ91264 KMQ97455 KMQ86622 OWR51232 OWR41390 OWR49085 OWR46815 GBP47348 GBP10163 GBP66459 EFN78171 EFN83798 EFN81362 EFN79604 EZA60425 EZA56456 XP_011336667 XP_002424953

378 912 668 1121 457 280 536 1065 360 672 482 277 833 349 329 307 685 299 378 192 627 907 323 515 854 615 398 526

–

–

–

– – – – – – – – – – – – – – – – – – – – – – – – – –

– – – – – – – – – – – – – – – – – – – – – – – – – –

– – – – – – – – – – – – – – – – – – – – – – – – – –

XP_002425560

613

–

–

–

TGZ54263

562

–

–

–

TGZ49887

402

–

–

–

TGZ32731

832

–

–

–

(continued on next page) 8

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Table 1 (continued) Genes Name

Species name

Accession number

Number of amino acids

Immune/Developmental function

Participation Pathway

Reference

SOCS2

Melipona quadrifasciata Melipona quadrifasciata Melipona quadrifasciata Melipona quadrifasciata Bombus terrestris Diaphorina citri Eufriesea Mexicana Eufriesea Mexicana Eufriesea Mexicana Eufriesea Mexicana Apis mellifera Acyrthosiphon pisum Asbolus verrucosus Orchesella cincta Orchesella cincta Orchesella cincta Onthophagus taurus

KOX74779

566

–

–

–

KOX76219

516

–

–

–

KOX77407

413

–

–

–

KOX78292

722

–

–

–

XP_003393038 XP_008478830 OAD55668 OAD52276 OAD52403 OAD58426 ABV01936 XP_003245252 RZC42709 ODM98547 ODM98322 ODN00090 XP_022915889

516 395 494 305 421 767 305 482 268 418 877 199 448

– – – – – – – – – – – – –

– – – – – – – – – – – – –

– – – – – – – – – – – – –

SOCS5 SOCS6 SOCS7 SOCS5 SOCS5 SOCS2 SOCS5 SOCS6 SOCS7 SOCS5 SOCS5 SOCS5 SOCS4 SOCS7 SOCS2 SOCS5

glycoprotein, belongs to the tyrosine kinase receptor family. The developmental role of EGFR has been well established both in invertebrates and vertebrates. In vertebrates, EGFR mutation is considered to be responsible for different organs abnormality, such as skin, lung, brain, eye, heart, and even cause death (Bogdan and Klambt, 2001). While, experimental studies on many invertebrate species (e.g., Caenorhabditis elegans,Gryllus bimaculatus, and Drosophila melanogaster) suggest that EGFR is an essential pathway for the regulation of dorsoventral development, spermatogenesis, oogenesis, eye development, growth, etc. (Chang and Sternberg, 1999; Shilo, 2003; Mutti et al., 2011; Ventura et al., 2011; Dabour et al., 2011). Among insects, the developmental role of the EGFR pathway has been reported in different species; however, it explored thoroughly in Drosophila. In Drosophila, the EGFR comprises Gurken, Spitz, Argos, and Boss ligands, which bind to different receptors such as Torso, DER, and Sevenless, result in stimulation of the RAS-RAF-MAPK pathway (Simon, 2000). The components of this pathway remain extremely conserved across the evolutionary period. The pathway activity has been shown to govern by different SOCS molecules for example, SOCS 36E has been shown to negatively regulate this pathway, while B. mori SOCS6 and Drosophila SOCS44A are capable of enhancing its activity (Callus and Mathey-Prevot, 2002; Kang et al., 2018; Abbas et al., 2018). Furthermore, Janeh et al. (2017) identified orthologous of the SOCS 36E in mosquitoes (Aedes albopictus) and found be involved in the negative regulation of the EGFR pathway (Fig. 3B). The wing and eye development of Drosophila provided evidence about the negative regulation of EGFR by SOCS proteins. It has been shown that SOCS 36E gene ectopic expression within the developing wing of Drosophila generates defects in the adult wing venation that partially phenocopies loss of DER receptor and suppression of EGFR signaling (Callus and Mathey-Prevot, 2002). Another study on the Drosophila eye development further supported the involvement of SOCS 36E in the negative regulation of EGFR signaling. During eye development, intracellular signaling, which is governed by EGFR, is required for the specification of eight photoreceptors (R1–R8) that occur within each ommatidial cluster (Freeman, 1996). Further, the differentiation of the R7 receptor requires an additional burst of signal in the form of Sevenless (Sev) activation (Simon et al., 1991; Freeman, 1996). EGFR receptor transcription localizes to R1, R3, R4, R6, R7, and four ancillary cone cells, while SOCS 36E is transcribed in all the cells except for R2, R5, and R7 (Almudi et al., 2009). In a SOCS 36E mutant extra R7 receptors are recruited, whereas the upregulation of this gene is sufficient

Table 2 Other SOCS box domain containing proteins from insects. Gene name

Species name

Accession number

Number of amino acids

SPSB3 SPSB 3-like SPSB3 SPSB 3-like SPSB1 SPSB 3 ASB 3-like

Bombyx mori Bombyx mori Aedes aegypti Aedes aegypti Zeugodacus cucurbitae Zeugodacus cucurbitae Frankliniella occidentalis Frankliniella occidentalis Frankliniella occidentalis Leptinotarsa decemlineata Ceratitis capitata Ceratitis capitata Drosophila guanche Halyomorpha halys Halyomorpha halys Cryptotermes secundus Cryptotermes secundus Lasius niger Lasius niger Danaus plexippus Danaus plexippus Danaus plexippus Danaus plexippus Eumeta japonica Harpegnathos saltator Harpegnathos saltator Harpegnathos saltator Ooceraea biroi Melipona quadrifasciata Melipona quadrifasciata Bombus terrestris Diaphorina citri

XP_021205021 XP_021201962 XP_021702004 XP_021708110 JAD00221 XP_011187836 XP_026293908

291 316 377 362 350 364 543

XP_026278250

465

XP_026282475

298

XP_023015083

213

XP_020716089 JAC04843 SPP76787 XP_014278739 XP_014292039 XP_023706317 XP_023711130 KMQ88818 KMR04892 OWR45322 OWR45386 OWR52763 OWR45903 GBP81083 EFN87895 EFN82355 EFN86298 XP_011339511 KOX76621 KOX67195 XP_003399872 XP_008473208

299 364 348 248 464 673 241 195 465 331 309 527 462 648 460 244 449 270 449 446 469 241

ASB 14-like SPSB 3 SPSB 3 SPSB 3 SPSB 1 SPSB 3 SPSB 3 ASB -3-like ASB -3 SPSB 3 SPSB 3-like ASB 17 SPSB 1 SPSB 3 ASB 3 ASB 17 SPSB 3 ASB 17 SPSB 3 SPSB 1 SPSB 3 SPSB 1 Ankyrin-2 ASB 16 SPSB 3-like

ASB: Ankyrin Repeat and SOCS Box, SPSB: SPRY Domain-Containing SOCS Box Protein.

8. Participation of SOCS molecules in the regulation of epidermal growth factor receptor pathway Epidermal growth factor receptor (EGFR), a transmembrane 9

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invading pathogens acting as a physical barrier, activating local defenses, and triggering the systemic immune response. Microbial infection can change the physiology of the gut that included regulation of stress response and enhance cell proliferation and epithelial renewal (Buchon et al., 2009; Janeh et al., 2017). In Aedes albopictus, oral bacterial or chemical treatment, cell proliferation in the digestive tract and midgut is increased to maintain homeostasis. Analysis of signaling pathways suggested the activation of the EGFR pathway; further, the authors also observed the enhancement of SOCS molecules, suggests that SOCS molecules strongly regulate the activity of the EGFR pathway (Janeh et al., 2017). In honeybees, EGFR signaling has been reported to be involved in queen development (Kamakura, 2011). Additionally, EGFR signaling also controls reproduction in honeybee workers, as EGFR suppression analysis suggested the loss of functional ovaries in a queenless environment (FormesynaCardoenb et al., 2014). Furthermore, microarray (Cardoen et al., 2011; Grozinger et al., 2007) and proteomic analyses (Cardoen et al., 2011) suggested neither EGFR or its gurken, ligand, are differentially transcribed between nonreproductive and reproductive workers. The EGFR inhibitors (e.g., Argos, Sprouty, and Cbl) have also been shown to increase in sterile workers (Cardoen et al., 2011). The depletion of EGFR signaling might involve in the suppression of worker ovary activation, whereas its upregulation might initiate worker egglaying in queenless colonies. In addition, downstream factors, including Raf kinase and SOCS 5 (negative regulation of EGFR signaling), are also upregulated among sterile workers (Cardoen et al., 2011). It seems that EGFR signaling is crucial for queen determination at the larval stage (Kamakura, 2011), and to control reproduction in adult honeybee workers. The SOCS 5 protein act as a potent negative regulator of the EGFR pathway. Collectively, the EGFR pathway is essential in the modulation of developmental activities, and dysregulation of this pathway may cause adverse developmental abnormalities in animals (Bogdan and Klambt, 2001). The SOCS proteins monitor the EGFR pathway by inducing or suppressing its activity. The Regulatory activity of some SOCS proteins (SOCS6) may depend on tissues or receptors. Future studies should aim to determine the SOCS regulatory mechanism in different insect species, which will help to draw a comparative picture of their molecular mechanism, and also improve our understanding of the SOCS family.

to inhibit differentiation of R7 cell. This phenomenon describes a requirement for SOCS 36E in the modulation of fate determination in the developing eye, fate decision of a cell, which does not involve the JAKSTAT signaling pathway (Zeidler et al., 1999). Moreover, misexpression of the EGFR pathway downstream components together with SOCS 36E also resulted in the recruitment of extra R7 cells, suggesting a specific and direct interaction between Sev and SOCS 36E. However, it has been reported that SOCS 36E is only a weak inhibitor of Sev as high levels of Sevenless signaling are capable of repressing the phenotypes caused by expression of SOCS 36E (Almudi et al., 2009). Overall, these studies suggest that SOCS 36E protein is capable of weakly prevent the EGFR signaling pathway in these and other tissues, further that demonstrating a conserved function of this gene across species. A recent study on mosquitoes (Aedes albopictus) suggested the involvement of the EGFR pathway in the proliferation of gut cells following chemical and bacterial damage. The authors further noted the upregulation of orthologous of SOCS 36E in the gut and suggested that this increase in expression may control the extensive activities of the EGFR pathway (Janeh et al., 2017). Furthermore, in insects, SOCS proteins have a stimulatory effect on the activity of the EGFR pathway. For example, SOCS 44A seems to be essential during the wing development of Drosophila since the SOCS 44A misexpression in the developing wing generates defects in venation similar to functional loss of JAK-STAT and EGFR gain of function. The phenotypes are characteristic for heterozygous mutations in EGFR, and ras85D were rescued upon over-expression of SOCS 44A and increased by argos loss, a negative regulator of the EGFR signaling pathway. Based on these observations, the authors suggested that SOCS 44A can enhance EGFR signaling in the Drosophila wing (Rawlings et al., 2004). However, later studies on the developing eye failed to provide evidence in favor of SOCS 44A as a regulator of the EGFR signaling pathway (Almudi et al., 2009). These studies suggest that there are different types of EGF-like receptors in both tissues, and SOCS 44A may exhibit specificity to a specific receptor. However, studies in mammalian systems suggest a different function for the SOCS 44A homologue, SOCS 6, which downregulates the EGFR receptor c-KIT by targeting it for degradation (Zadjali et al., 2011). A recent study on silkworm Bombyx mori showed that SOCS6 upregulates the EGFR signaling pathway and may play a crucial role in the growth process of this species. This study employed an RNA interference technique and the recombinant protein to determine the SOCS 6 regulatory role of EGFR signaling. However, this failed to explain the molecular mechanism of the SOCS 6 molecule (Abbas et al., 2018). Thus, future studies should focus on the regulatory role of SOCS proteins in both EGFR and JAK-STAT signaling pathways at both the genetic and biochemical levels. Niche competition in the Drosophila testis is also an example of MAPK-EGFR modulation by SOCS 36E. In testis, the niche supports somatic stem cells and germline stem cells (Amoyel et al., 2014). It has been shown that somatic stem cells compete with each other and with also germline stem cells for niche access, and mutations have been identified that confer increased competitiveness to somatic stem cells, resulting in the mutant stem cell and its descendants outcompeting wild type resident stem cells (Morrison and Spradling, 2008; Amoyel et al., 2014). A recent study suggested that SOCS 36E is an important modulator of niche competition by governing the activity of unbridled MAPK signaling. The competitive behavior of SOCS 36E mutant somatic stem cells is due in large part to unbridled MAPK signaling, and in SOCS 36E mutant clones, MAPK activity is increased. This enhancement of MAPK activity in somatic stem cells leads to their out competition of wild type somatic stem cells and of germline stem cells, recapitulating the SOCS 36E mutant phenotype. Whereas, suppression of MAPK activity from SOCS 36E mutant clones results in loss of their competitiveness (Amoyel et al., 2016). In mosquitoes, pathogens enter mainly through the oral route, which places the insects’ gut at the front line of the battle. Indeed, the gut epithelium of the mosquito plays crucial biological roles against

9. Conclusion The regulation of biological signaling pathways is crucial to maintain normal physiological processes in living organisms. So far, several studies on the SOCS family members and their physiological role in different signaling pathways have been carried out on model insect species (e.g., Drosophila, silkworm, M. sexta) and some other insect species etc. These studies provide insight into the biological role of SOCS protein in the innate immunity of other insect species, growth, and development. Analysis of SOCS proteins in the insect's development and innate immunity exhibited that these proteins regulate different signaling pathways, particularly the EGFR and JAK-STAT signaling pathways. Most of the SOCS family members generally function to finetune the signal adding to the robustness of the signal transduction pathways. Moreover, from the perspective of systems biology, SOCS proteins can be viewed as indispensable components of the developmental machinery, allowing for precise modulation of cell fate specification, survival, and death, among many other consequences. Despite the immense progress that has been made since the discovery of SOCS proteins, still much remains to be elucidated. Firstly, only a few members of the SOCS family members reported from insects, and their exact physiological role still needs to be clarified. Secondly, although the sequences of many SOCS box-containing proteins are available on the NCBI database, their functional role remains to explore. Thirdly, many studies have shown that some SOCS proteins are implicated in the EGFR and JAK-STAT pathways; many of the SOCS 10

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family members functional role in the physiological processes is unknown. Finally, in vivo and invitro interpretation of the insect SOCS proteins might facilitate our understanding of the complex interactions of these proteins in insects and other animals.

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Funding This study was funded by the National Key Research and Development Program of China (grant number 2016YFC1302204 and 2017YFC1308600) and by the National Natural Science Foundation of China (grant number 81672502 to H. Cui). Declaration of competing interest Authors declare that they have no conflict of interest. References Abbas, M.N., Liang, H., Kausar, S., Dong, Z., Cui, H., 2020. Zinc finger protein RP-8, the Bombyx mori ortholog of programmed cell death 2, regulates cell proliferation. Dev. Comp. Immunol. 104, 103542. Abbas, M.N., Kausar, S., Cui, H., 2019. The biological functions of peroxiredoxins in innate immune responses of aquatic invertebrates. Fish Shellfish Immunol. 89, 91–97. Abbas, M.N., Kausar, S., Sun, Y.X., Tian, J.W., Zhu, B.J., Liu, C.L., 2018. Suppressor of cytokine signaling 6 can enhance epidermal growth factor receptor signaling pathway in Bombyx mori (Dazao). Dev. Comp. Immunol. 81, 187–192. Abbas, M.N., Zhu, B.J., Kausar, S., Dai, L.S., Sun, Y.X., Tian, J.W., Liu, C.L., 2017. Suppressor of cytokine signaling 2-12 regulates antimicrobial peptides and ecdysteroid signaling pathways in B. mori (Dazao). J. Insect Physiol. 103, 47–56. Abbas, M.N., Rana, S.A., Khan, H.A., Rehman, Khalil-ur, 2012. Status of trophic guild of invertebrates utilizing weeds of wheat and sugarcane fields of Faisalabad. Pak. J. Agric. Sci. 49, 189–198. Abbas, M.N., Sajeel, M., Kausar, S., 2013. House fly (Musca domestica), a challenging pest; biology, management and control strategies. Elixir Entomol. 64, 19333–19338. Agaisse, H., Petersen, U.M., Boutros, M., Mathey-Prevot, B., Perrimon, N., 2003. Signaling role of hemocytes in Drosophila JAK-STAT dependent response to septic injury. Dev. Cell 5, 441–450. Alexander, W.S., 2002. Suppressors of cytokine signaling (SOCS) in the immune system. Nat. Rev. Immunol. 2, 410–416. Almudi, I., Stocker, H., Hafen, E., Corominas, M., Serras, F., 2009. SOCS 36E specifically interferes with sevenless signaling during Drosophila eye development. Dev. Biol. 326, 212–223. Amoyel, M., Anderson, J., Suisse, A., Glasner, J., Bach, E.A., 2016. SOCS 36E controls niche competition by repressing MAPK signaling in the Drosophila Testis. PLoS Genet. 12, e1005815. Amoyel, M., Simons, B.D., Bach, E.A., 2014. Neutral competition of stem cells is skewed by proliferative changes downstream of Hh and Hpo. EMBO J. 33, 20. Barillas-Mury, C., Han, Y.S., Seeley, D., Kafatos, F.C., 1999. Anopheles gambiae Ag-STAT, a new insect member of the STAT family, is activated in response to bacterial infection. EMBO J. 18, 959–967. Bäumer, D., Trauner, J., Hollfelder, D., Cerny, A., Schoppmeier, M., 2011. JAK-STAT signaling is required throughout telotrophic oogenesis and short-germ embryogenesis of the beetle Tribolium. Dev. Biol. 350, 169–182. Bogdan, S., Klambt, C., 2001. Epidermal growth factor receptor signaling. Curr. Biol. 11, 292–295. Braunstein, J., Brutsaert, S., Olson, R., Schindler, C., 2003. STATs dimerize in the absence of phosphorylation. J. Biol. Chem. 278, 34133–34140. Buchon, N., Broderick, N.A., Poidevin, M., Pradervand, S., Lemaitre, B., 2009. Drosophila intestinal response to bacterial infection: activation of host defense and stem cell proliferation. Cell Host Microbe 5, 200–211. Callus, B.A., Mathey-Prevot, B., 2002. SOCS 36E, a novel Drosophila SOCS protein, suppresses JAK-STAT and EGF-R signaling in the imaginal wing disc. Oncogene 21, 4812–4821. Cardoen, D., Wenseleers, T., Ernst, U.R., Danneels, E.L., Laget, D., de Graaf, D.C., Schoofs, L., Verleyen, P., 2011. Genome-wide analysis of alternative reproductive phenotypes in honeybee workers. Mol. Ecol. 20, 4070–4084. Chang, C., Sternberg, P.W., 1999. C elegans vulval development as a model system to study the cancer biology of EGFR signaling. Cancer Metastasis Rev. 18, 203–213. Cheng, G., Liu, Y., Wang, P., Xiao, X., 2016a. Mosquito defense strategies against viral infection. Trends Parasitol. 32, 177–186. Cheng, G., Liu, Y., Wang, P., Xiao, X., 2016b. Mosquito defense strategies against viral infection. Trends Parasitol. 32, 177–186. Chung, A.S., Guan, Y.J., Yuan, Z.L., Albina, J.E., Chin, Y.E., 2005. Ankyrin repeat and SOCS box 3 (ASB3) mediates ubiquitination and degradation of tumor necrosis factor receptor II. Mol. Cell. Biol. 25, 4716–4726. Croker, B.A., Kiu, H., Nicholson, S.E., 2008. SOCS regulation of the JAK-STAT signaling pathway. Semin. Cell Dev. Biol. 19, 414–422. Dabour, N., Bando, T., Nakamura, T., Miyawaki, K., Mito, T., Ohuchi, H., Noji, S., 2011. Cricket body size is altered by systemic RNAi against insulin signaling components and epidermal growth factor receptor. Dev. Growth Differ. 53, 857–869.

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Glossary PIAS: Protein inhibitors of activated STAT Dome: Domeless Hop: Heat shock protein 70/90 organizing protein NF-κB: Nuclear factor kappa light chain enhancer of activated B cells SOCS: Suppressors of cytokine signaling NOS: Nitric oxide synthase JAK-STAT: Janus Kinase-Signal Transducer and Activators of Transcription EGFR: Epidermal growth factor receptor NCBI: National Center for Biotechnology Information SPRY: sp1A/ryanodine receptor SPSB: sp1A/ryanodine receptor domain-containing SOCS box protein Sev: Sevenless MAPK: Mitogen-activated Protein Kinase ECS: Elongin-Cullin-SOCS

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