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Spermatogenesis is a critical part of reproduction in insects; however, its molecular mechanism is still largely unknown. In this study, we identified a testis-specific gene CG3526 in Drosophila melanogaster. Bioinformatics analysis showed that CG3526 contains a zinc binding domain and 2 C 2 H 2 type zinc fingers, and it is clustered to the vertebrate really interesting new gene (RING) family E3 ubiquitin-protein ligases. When CG3526 was knocked down by RNA interference (RNAi), the testis became much smaller in size, and the apical tip exhibited a sharp and thin end instead of the blunt and round shape in the control testis. More importantly, compared to the control flies, only a few mature sperm were present in the seminal vesicle of C587-Gal4 > CG3526 RNAi flies. Immunofluorescence staining of the testis from CG3526 RNAi flies showed that the homeostasis of testis stem cell niche was disrupted, cell distribution in the apical tip was scattered, and the process of spermatogenesis was not completed. Furthermore, we found that the phenotype of CG3526 RNAi flies' testis was similar to that of testis of Stat92E RNAi flies, the expression level of CG3526 was significantly downregulated in the Stat92E F06346 mutant flies, and the promoter activity of CG3526 was upregulated by STAT92E. Taken together, our results indicated that CG3526 is a downstream effector gene in the JAK-STAT signaling pathway that plays a key role in the spermatogenesis of Drosophila., (© 2023 Institute of Zoology, Chinese Academy of Sciences.)

Wolbachia are Gram-negative endosymbionts that are known to cause embryonic lethality when infected male insects mate with uninfected females or with females carrying a different strain of Wolbachia, a situation characterized as cytoplasmic incompatibility (CI). However, the mechanism of CI is not yet fully understood, although recent studies on Drosophila melanogaster have achieved great progress. Here, we found that Wolbachia infection caused changes in the expressions of several immunity-related genes, including significant upregulation of kenny (key), in the testes of D. melanogaster. Overexpression of key in fly testes led to a significant decrease in egg hatch rates when these flies mate with wild-type females. Wolbachia-infected females could rescue this embryonic lethality. Furthermore, in key overexpressing testes terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling signal was significantly stronger than in the control testes, and the level of reactive oxygen species was significantly increased. Overexpression of key also resulted in alterations of some other immunity-related gene expressions, including the downregulation of Zn72D. Knockdown of Zn72D in fly testes also led to a significant decrease in egg hatch rates. These results suggest that Wolbachia might induce the defect in male host fertility by immunity-related pathways and thus cause an oxidative damage and cell death in male testes., (© 2019 Institute of Zoology, Chinese Academy of Sciences.)

Insects, which lack the adaptive immune system, have developed sophisticated innate immune system consisting of humoral and cellular immune responses to defend against invading microorganisms. Non-self recognition of microbes is the front line of the innate immune system. Repertoires of pattern recognition receptors (PRRs) recognize the conserved pathogen-associated molecular patterns (PAMPs) present in microbes, such as lipopolysaccharide (LPS), peptidoglycan (PGN), lipoteichoic acid (LTA) and β-1, 3-glucans, and induce innate immune responses. In this review, we summarize current knowledge of the structure, classification and roles of PRRs in innate immunity of the model organism Drosophila melanogaster, focusing mainly on the peptidoglycan recognition proteins (PGRPs), Gram-negative bacteria-binding proteins (GNBPs), scavenger receptors (SRs), thioester-containing proteins (TEPs), and lectins., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The Toll signaling pathway in Drosophila melanogaster regulates several immune-related functions, including the expression of antimicrobial peptide (AMP) genes. The canonical Toll receptor (Toll-1) is activated by the cytokine Spätzle (Spz-1), but Drosophila encodes eight other Toll genes and five other Spz genes whose interactions with one another and associated functions are less well-understood. Here, we conducted in vitro assays in the Drosophila S2 cell line with the Toll/interleukin-1 receptor (TIR) homology domains of each Toll family member to determine whether they can activate a known target of Toll-1, the promoter of the antifungal peptide gene drosomycin. All TIR family members activated the drosomycin promoter, with Toll-1 and Toll-7 TIRs producing the highest activation. We found that the Toll-1 and Toll-7 ectodomains bind Spz-1, -2, and -5, and also vesicular stomatitis virus (VSV) virions, and that Spz-1, -2, -5, and VSV all activated the promoters of drosomycin and several other AMP genes in S2 cells expressing full-length Toll-1 or Toll-7. In vivo experiments indicated that Toll-1 and Toll-7 mutants could be systemically infected with two bacterial species ( Enterococcus faecalis and Pseudomonas aeruginosa ), the opportunistic fungal pathogen Candida albicans , and VSV with different survival times in adult females and males compared with WT fly survival. Our results suggest that all Toll family members can activate several AMP genes. Our results further indicate that Toll-1 and Toll-7 bind multiple Spz proteins and also VSV, but they differentially affect adult survival after systemic infection, potentially because of sex-specific differences in Toll-1 and Toll-7 expression., (© 2019 Chowdhury et al.)

An important innate immune response in Drosophila melanogaster is the production of antimicrobial peptides (AMPs). Expression of AMP genes is mediated by the Toll and immune deficiency (IMD) pathways via NF-κB transcription factors Dorsal, DIF and Relish. Dorsal and DIF act downstream of the Toll pathway, whereas Relish acts in the IMD pathway. Dorsal and DIF are held inactive in the cytoplasm by the IκB protein Cactus, while Relish contains an IκB-like inhibitory domain at the C-terminus. NF-κB factors normally form homodimers and heterodimers to regulate gene expression, but formation of heterodimers between Relish and DIF or Dorsal and the specificity and activity of the three NF-κB homodimers and heterodimers are not well understood. In this study, we compared the activity of Rel homology domains (RHDs) of Dorsal, DIF and Relish in activation of Drosophila AMP gene promoters, demonstrated that Relish-RHD (Rel-RHD) interacted with both Dorsal-RHD and DIF-RHD, Relish-N interacted with DIF and Dorsal, and overexpression of individual RHD and co-expression of any two RHDs activated the activity of AMP gene promoters to various levels, suggesting formation of homodimers and heterodimers among Dorsal, DIF and Relish. Rel-RHD homodimers were stronger activators than heterodimers of Rel-RHD with either DIF-RHD or Dorsal-RHD, while DIF-RHD-Dorsal-RHD heterodimers were stronger activators than either DIF-RHD or Dorsal-RHD homodimers in activation of AMP gene promoters. We also identified the nucleotides at the 6th and 8th positions of the 3' half-sites of the κB motifs that are important for the specificity and activity of NF-κB transcription factors., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Drosophila melanogaster possesses a sophisticated and effective immune system composed of humoral and cellular immune responses, and production of antimicrobial peptides (AMPs) is an important defense mechanism. Expression of AMPs is regulated by the Toll and IMD (immune deficiency) pathways. Production of AMPs can be systemic in the fat body or a local event in the midgut and epithelium. So far, most studies focus on systemic septic infection in adult flies and little is known about AMP gene activation after ingestion of killed bacteria. In this study, we investigated activation of AMP genes in the wild-type w 1118 , MyD88 and Imd mutant flies after ingestion of heat-killed Escherichia coli and Staphylococcus aureus. We showed that ingestion of E. coli activated most AMP genes, including drosomycin and diptericin, in the first to third instar larvae and pupae, while ingestion of S. aureus induced only some AMP genes in some larval stages or in pupae. In adult flies, ingestion of killed bacteria activated AMP genes differently in males and females. Interestingly, ingestion of killed E. coli and S. aureus in females conferred resistance to septic infection by both live pathogenic Enterococcus faecalis and Pseudomonas aeruginosa, and ingestion of E. coli in males conferred resistance to P. aeruginosa infection. Our results indicated that E. coli and S. aureus can activate both the Toll and IMD pathways, and systemic and local immune responses work together to provide Drosophila more effective protection against infection., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Wolbachia are endosymbiotic bacteria present in a wide range of invertebrates. Although their dramatic effects on host reproductive biology have been well studied, little is known about the effects of Wolbachia on the learning and memory capacity (LMC) of hosts, despite their distribution in the host nervous system, including brain. In this study, we found that Wolbachia infection significantly enhanced LMC in both Drosophila melanogaster and D. simulans. Expression of LMC-related genes was significantly increased in the head of D. melanogaster infected with the wMel strain, and among these genes, crebA was up-regulated the most. Knockdown of crebA in Wolbachia-infected flies significantly decreased LMC, while overexpression of crebA in Wolbachia-free flies significantly enhanced the LMC of flies. More importantly, a microRNA (miRNA), dme-miR-92b, was identified to be complementary to the 3'UTR of crebA. Wolbachia infection was correlated with reduced expression of dme-miR-92b in D. melanogaster, and dme-miR-92b negatively regulated crebA through binding to its 3'UTR region. Overexpression of dme-miR-92b in Wolbachia-infected flies by microinjection of agomirs caused a significant decrease in crebA expression and LMC, while inhibition of dme-miR-92b in Wolbachia-free flies by microinjection of antagomirs resulted in a significant increase in crebA expression and LMC. These results suggest that Wolbachia may improve LMC in Drosophila by altering host gene expression through a miRNA-target pathway. Our findings help better understand the host-endosymbiont interactions and, in particular, the impact of Wolbachia on cognitive processes in invertebrate hosts., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Dynamitin (Dmn) is a major component of dynactin, a multiprotein complex playing important roles in a variety of intracellular motile events. We previously found that Wolbachia bacterial infection resulted in a reduction of Dmn protein. As Wolbachia may modify sperm in male hosts, we speculate that Dmn may have a function in male fertility. Here we used nosGal4 to drive Dmn knock down in testes of Drosophila melanogaster to investigate the functions of Dmn in spermatogenesis. We found that knockdown of Dmn in testes dramatically decreased male fertility, overexpression of Dmn in Wolbachia-infected males significantly rescued male fertility, indicating an important role of Dmn in inducing male fertility defects following Wolbachia infection. Some scattered immature sperm with late canoe-shaped head distributed in the end of Dmn knockdown testis and only about half mature sperm were observed in the Dmn knockdown testis relative to those in the control. Transmission electron microscopy (TEM) exhibited fused spermatids in cysts and abnormal mitochondrial derivatives. Immunofluorescence staining showed significantly less abundance of tubulin around the nucleus of spermatid and scattered F-actin cones to different extents in the individualization complex (IC) during spermiogenesis in Dmn knockdown testes, which may disrupt the nuclear condensation and sperm individualization. Since dynein-dynactin complex has been shown to mediate transport of many cellular components, including mRNAs and organelles, these results suggest that Dmn may play an important role in Drosophila spermiogenesis by affecting transport of many important cytoplasmic materials., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Wolbachia are endosymbionts that infect many insect species. They can manipulate the host's reproduction to increase their own maternal transmission. Cytoplasmic incompatibility (CI) is one such manipulation, which is expressed as embryonic lethality when Wolbachia-infected males mate with uninfected females. However, matings between males and females carrying the same Wolbachia strain result in viable progeny. The molecular mechanisms of CI are currently not clear. We have previously reported that the gene Juvenile hormone-inducible protein 26 (JhI-26) exhibited the highest upregulation in the 3rd instar larval testes of Drosophila melanogaster when infected by Wolbachia. This is reminiscent of an interaction between Wolbachia and juvenile hormone (JH) pathway in flies. Considering that Jhamt gene encodes JH acid methyltransferase, a key regulatory enzyme of JH biosynthesis, and that methoprene-tolerant (Met) has been regarded as the best JH receptor candidate, we first compared the expression of Jhamt and Met between Wolbachia-infected and uninfected fly testes to investigate whether Wolbachia infection influence the JH signaling pathway. We found that the expressions of Jhamt and Met were significantly increased in the presence of Wolbachia, suggesting an interaction of Wolbachia with the JH signaling pathway. Then, we found that overexpression of JhI-26 in Wolbachia-free transgenic male flies caused paternal-effect lethality that mimics the defects associated with CI. JhI-26 overexpressing males resulted in significantly decrease in hatch rate. Surprisingly, Wolbachia-infected females could rescue the egg hatch. In addition, we showed that overexpression of JhI-26 caused upregulation of the male accessory gland protein (Acp) gene CG10433, but not vice versa. This result suggests that JhI-26 may function at the upstream of CG10433. Likewise, overexpression of CG10433 also resulted in paternal-effect lethality. Both JhI-26 and CG10433 overexpressing males resulted in nuclear division defects in the early embryos. Finally, we found that Wolbachia-infected males decreased the propensity of the mated females to remating, a phenotype also caused by both JhI-26 and CG10433 overexpressing males. Taken together, our results provide a working hypothesis whereby Wolbachia induce paternal defects in Drosophila probably by interaction with the JH pathway via JH response genes JhI-26 and CG10433., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

ML (MD-2 (myeloid differentiation factor-2)-related Lipid-recognition) is a conserved domain identified in MD-2, MD-1, NPC2 (Niemann-Pick disease type C2), and mite major allergen protein from animals, plants, and fungi. Vertebrate members of the ML family proteins, such as NPC2 and MD-2, play important roles in lipid metabolism and immune signaling pathway. MD-2 is an essential co-receptor in the lipopolysaccharide (LPS)/Toll-like receptor 4 (TLR4) signaling pathway. Insects contain multiple ML genes, arbitrarily named md-2- or npc2-like genes. However, whether insect ML genes have functions similar to vertebrate md-2 is unknown. In Drosophila melanogaster, there are eight npc2 genes (npc2a-h), and they can be further divided into three subgroups based on the numbers of cysteine residues (6, 7 and 8 Cys) in the mature proteins. The purpose of this study is to investigate whether any Drosophila npc2 genes may have functions in immune signaling pathways. We chose npc2a, npc2e and npc2h genes representing the three subgroups for this study. We showed that recombinant NPC2a, NPC2e and NPC2h not only bound to LPS and lipid A, but also bound to peptidoglycan (PG) and lipoteichoic acid (LTA), a property that has not been reported previously for vertebrate NPC2 or MD-2. More importantly, we showed that over-expression of NPC2a and NPC2e activated diptericin promoter reporter in S2 cells stimulated by PG, suggesting that NPC2e and NPC2a may play a role in the immune deficiency (Imd) pathway. This is the first in vitro study about NPC2 proteins in innate immunity of D. melanogaster., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Insect prophenoloxidases (PPOs) are a group of important innate immunity proteins. Although there have been numerous studies dealing with the PPO activation cascade, the detailed biochemical behaviors of the PPO family proteins remain to be clearly established. This is due primarily to the difficulty in obtaining adequate amounts of PPO proteins for comprehensive characterization. In this study, we expressed three Drosophila melanogaster PPO genes in Escherichia coli, and extensively evaluated expression conditions for obtaining soluble proteins. Through the manipulation of expression conditions, particularly the culture temperature of PPO-transformed E. coli cells, we were able to obtain large quantities of soluble recombinant PPO proteins. Additional Cu(2+), either added into the culture medium during PPO induction or directly mixed with the purified rPPO preparations, was necessary to produce Cu(2+) associated proenzymes. Cu(2+) associated PPOs showed obvious enzyme activities after activation by either ethanol or cetylpyridinium chloride, or by AMM1 (a pupal protein fraction containing native serine proteases for PPO activation). Dose responses for association of individual purified Drosophila rPPOs with Cu(2+) showed that Drosophila rPPO1 and rPPO3 had relatively higher affinity for Cu(2+) than rPPO2 did. Surprisingly, however, high concentration of Cu(2+) (2 mM) completely inhibited PPO activity. Each rPPO had similar activity when dopamine or l-DOPA was the substrate. However, rPPO1 alone had very high activity if l-tyrosine was used as a substrate. After activation by ethanol or 2-propanol, Km and Vmax of the three rPPOs changed as shown in the following: rPPO2

Dipteran insects, like mosquitoes, possess more than two prophenoloxidase (PPO) genes, but it is unclear whether their gene products differ in biochemical properties and physiological functions. Here, we used three Drosophila melanogaster PPOs as models to study their properties through expression in S2 cells. Our data revealed that the PPOs were expressed in the ethanol-activatable conformation: rPPO1 and rPPO2 needed additional Cu(2+) in the medium, but rPPO3 did not. rPPO1 bound Cu(2+) within minutes; rPPO2 did that in hours when Cu(2+) were present at a higher concentration. Thus, rPPO1 and rPPO2 were expressed as apo-rPPO and became holo-PPO upon Cu(2+) binding; rPPO3 was holo-PPO immediately after expression. Surprisingly, in the absence of ethanol, the apparently intact rPPO3 catalyzed dopamine oxidation and melanization. The successful method for rPPO expression in S2 cells described in this paper will provide us with an opportunity to study the properties of a specific PPO gene in a small insect like mosquitoes in the future., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Insects produce a variety of antimicrobial peptides (AMPs). Induction of insect AMP genes is regulated by the Toll and IMD (immune deficiency) pathways via NF-κB and GATA factors. Little is known about species-specific regulation of AMP genes. In this report, we showed that activities of most Manduca sexta and Drosophila melanogaster AMP gene promoters were regulated in a species-specific manner in Drosophila (Dipteran) S2 cells and Spodoptera frugiperda (Lepidopteran) Sf9 cells. A κB-GATA element (22 bp) from M. sexta moricin (MsMoricin) promoter could significantly increase activities of Drosophila AMP gene promoters in S2 cells, and an MsMoricin promoter activating element (MPAE) (140 bp) could increase activity of drosomycin promoter specifically in Sf9 cells. However, κB and GATA factors alone were not sufficient for MsMoricin gene activation, suggesting that other co-regulators may be required to fully activate AMP genes. Our results suggest that induction of insect AMP genes may require a transcription complex composed of common nuclear factors (such as NF-κB and GATA factors) and species-related co-regulators, and it is the co-regulators that may confer species-specific regulation of AMP genes. In addition, we showed that activity of Drosophila drosomycin promoter could be activated cooperatively by the inserted exogenous κB-GATA element and the endogenous κB element. These findings revealed an approach of engineering AMP genes with enhanced activities, which may lead to broad applications., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Ubiquitin carboxyl-terminal hydrolases (UCHs) are implicated in the proteolytic processing of polymeric ubiquitin. The high specificity for the recognition site makes UCHs useful enzymes for in vitro cleavage of ubiquitin fusion proteins. In this work, an active C-terminal His-tagged UCH from Drosophila melanogaster (DmUCH) was produced as a secretory form in a recombinant strain of the methylotrophic yeast Pichia pastoris. The production of recombinant DmUCH by Mut(s) strain was much higher than that by Mut(+) strain, which was confirmed by Western blot analysis. When expression was induced at pH 6.0 in a BMMY/methanol medium, the concentration of recombinant DmUCH reached 210 mg l(-1). With the (His)(6)-tag, the recombinant DmUCH was easily purified by Ni-NTA chromatography and 18 mg pure active DmUCH were obtained from 100ml culture broth supernatant. Ubiquitin-magainin fusion protein was efficiently cleaved by DmUCH, yielding recombinant magainin with high antimicrobial activity. After removing the contaminants by Ni-NTA chromatography, recombinant magainin was purified to homogeneity easily by reversed-phase HPLC. Analysis of the recombinant magainin by ESI-MS showed that the molecular weight of the purified recombinant magainin was 2465 Da, which perfectly matches the mass calculated from the amino acid sequence. The result of mass spectrometry confirmed that the purified His-tagged DmUCH can recognize the ubiquitin-magainin fusion protein and cleave it at the carboxyl terminus of ubiquitin precisely. Our results showed that P. pastoris is a robust system to express the secreted form of DmUCH.

C-type lectins are calcium-dependent carbohydrate binding proteins, and animal C-type lectins participate in innate immunity and cell-cell interactions. In the fruit fly Drosophila melanogaster, more than 30 genes encode C-type lectin domains. However, functions of Drosophila C-type lectins in innate immunity are not well understood. This study is to investigate whether two Drosophila C-type lectins, CG33532 and CG33533 (designated as DL2 and DL3, respectively), are involved in innate immune responses. Recombinant DL2 and DL3 were expressed and purified. Both DL2 and DL3 agglutinated Gram-negative Escherichia coli in a calcium-dependent manner. Though DL2 and DL3 are predicted to be secreted proteins, they were detected on the surface of Drosophila hemocytes, and recombinant DL2 and DL3 also directly bound to hemocytes. Coating of agarose beads with recombinant DL2 and DL3 enhanced their encapsulation and melanization by Drosophila hemocytes in vitro. However, hemocyte encapsulation was blocked when the lectin-coated beads were pre-incubated with rat polyclonal antibody specific for DL2 or DL3. Our results suggest that DL2 and DL3 may act as pattern recognition receptors to mediate hemocyte encapsulation and melanization by directly recruiting hemocytes to the lectin-coated surface.

Abstract: Insects synthesize a battery of antimicrobial peptides (AMPs) and expression of AMP genes is regulated by the Toll and Imd (immune deficiency) pathways in Drosophila melanogaster. Drosophila Toll pathway is activated after Spätzle (Spz) is cleaved by Spätzle processing enzyme (SPE) to release the active C-terminal C106 domain (DmSpz-C106), which then binds to the Toll receptor to initiate the signaling pathway and regulate expression of AMP genes such as drosomycin. Toll and Spz genes have been identified in other insects, but interaction between Toll and Spz and direct evidence for a Toll-Spz pathway in other insect species have not been demonstrated. Our aim is to investigate a Toll-Spz pathway in Manduca sexta, and compare M. sexta and D. melanogaster Toll-Spz pathways. Co-immunoprecipitation (Co-IP) assays showed that MsTollecto (the ecto-domain of M. sexta Toll) could interact with MsSpz-C108 (the active C-terminal C108 domain of M. sexta Spz) but not with full-length MsSpz, and DmTollecto could interact with DmSpz-C106 but not DmSpz, suggesting that Toll receptor only binds to the active C-terminal domain of Spz. Co-expression of MsToll-MsSpz-C108, but not MsToll-MsSpz, could up-regulate expression of drosomycin gene in Drosophila S2 cells, indicating that MsToll-MsSpz-C108 complex can activate the Toll signaling pathway. In vivo assays showed that activation of AMP genes, including cecropin, attacin, moricin and lebocin, in M. sexta larvae by purified recombinant MsSpz-C108 could be blocked by pre-injection of antibody to MsToll, further confirming a Toll-Spz pathway in M. sexta, a lepidopteran insect. [Copyright &y& Elsevier]

Abstract: Dipteran insects, like mosquitoes, possess more than two prophenoloxidase (PPO) genes, but it is unclear whether their gene products differ in biochemical properties and physiological functions. Here, we used three Drosophila melanogaster PPOs as models to study their properties through expression in S2 cells. Our data revealed that the PPOs were expressed in the ethanol-activatable conformation: rPPO1 and rPPO2 needed additional Cu2+ in the medium, but rPPO3 did not. rPPO1 bound Cu2+ within minutes; rPPO2 did that in hours when Cu2+ were present at a higher concentration. Thus, rPPO1 and rPPO2 were expressed as apo-rPPO and became holo-PPO upon Cu2+ binding; rPPO3 was holo-PPO immediately after expression. Surprisingly, in the absence of ethanol, the apparently intact rPPO3 catalyzed dopamine oxidation and melanization. The successful method for rPPO expression in S2 cells described in this paper will provide us with an opportunity to study the properties of a specific PPO gene in a small insect like mosquitoes in the future. [Copyright &y& Elsevier]

C-type lectins (CTLs) recognize various glycoconjugates through carbohydrate recognition domains (CRDs) and they play important roles in immune responses. In this study, comparative genomic analysis of CTLs were performed in 7 holometabolous species. CTL-S1 to S8 and CTL-X1 to X4 orthologous groups existed in the 7 species, while CTL-X5 group with dual-CRD, CTL-S11 group with triple-CRD, CTL-S9 group with a long C-terminus and Lepidopteran specific CTL-S10 group were not conserved. SliCTL-S12 to S14 cluster was only present in Spodoptera litura , and CTL-S genes were expanded on chromosomes 2 L and 2 R in Drosophila melanogaster. Most IMLs were clustered into three groups and the numbers of IMLs vary among species due to gene duplications. D. melanogaster specific CTLs and Lepidopteran IMLs within each of the three groups evolved more rapidly with higher dN/dS ratios. Two CRDs in IMLs clustered into two clades, with conserved Cys4-Cys5 and Cys1-Cys2 bonds in the first and second CRDs, respectively. The CTL-S and CTL-X family members in S. litura were mainly expressed in the fat body of 5th but not 6th instar larvae, and responded differently to S. litura nucleopolyhedrovirus (SpltNPV) and Nomuraea rileyi infection. The transcription levels of SliCTLs that expressed in fat body but not highly expressed in hemocytes were decreased at the middle and late stages of SpltNPV infection, and the mRNA levels of SliCTLs highly or specifically expressed in hemocytes were mainly decreased by SpltlNPV, N. rileyi and Bacillus thuringiensis infection. These results provide valuable information for further exploration of CTL functions in host-pathogen interaction. Image 1 • CTLs were identified in S. litura and H. armigera , and CTLs from 7 holometabolous species were classified and analyzed. • CTL-S1 to S8, CTL-X1 to X4 groups were conserved in the 7 species, CTL-S10 group only presented in Lepidopteran species. • Most IMLs containing tandemly arranged in-paralogs in four Lepidopteran species were clustered into three groups. • D. melanogaster specific CTL-S members and IMLs within the three groups evolved more rapidly with higher dN/dS ratios. • The expressions of S. litura CTLs in different tissues after NPV, N. rileyi and B. thuringiensis infection were analyzed. [ABSTRACT FROM AUTHOR]