Overview of immune related genes and their expression trends
A total of 157 genes involved in the Toll and Imd signaling pathways and 185 genes involved in cellular immunity were differentially expressed in S. frugiperda between any two developmental stages or sexes (p < 0.05). To reveal the overall humoral immune capacity in each developmental stage and sex of S. frugiperda, the sum Log2(FPKM) of all humoral immune related genes (IRGs) in various classifications, including genes classified into immune recognition, modulation (serine protease and serine protease inhibitor), Toll and Imd transduction, and effectors pathways, were used to define an age/development-dependent humoral immune index. The results showed that the total immunity indices for L3 and L5 stages were the highest, reaching 233.64 and 210.14, respectively (Fig. 1A). These were followed by 4th instar (L4), 2nd instar (L2), and adult females (AF) with 186.28, 172.35 and 163.93, respectively (Fig. 1A). The overall humoral immunity index for 1st instar (L1), 6th instar (L6), and adult males (AM) was similar, 131.04 ~ 155.89, whereas for pupal stage (P) was 2.32, and for eggs was the lowest (-304.07) (Fig. 1A).
Cellular immunity involves coagulation, hematopoiesis, and hemocyte-mediated immune responses. The sum of all related genes in these functional categories was calculated to reveal the development-dependent cellular immunity index. The total index showed a progressive upward trend from the egg stage to the L4 stage, with the number increasing from 23.0 to 145.3, with a minimum of -77 at the L6 stage, followed by peaks at the pupal (295.7) and adult female stages (271.0) (Fig. 1B). Notably, the total cellular immunity index was very low in adult males (-7.1) compared to adult females (Fig. 1B). The trend of the hemocyte-mediated immunity index was similar to the total cellular immunity index, but the values were much lower, ranging from − 121.4 to 45.3 (Fig. 1B).
Humoral immunity
Recognition
Pathogen recognition modules relevant to the determined immune gene index included five identified Gram-negative bacteria-binding proteins (GNBPs) and eight peptidoglycan recognition proteins (PGRPs). This set showed a gradual increase in expression from egg to adult stages (-45.24 to 62.21) (Fig. 2A), with GNBP_Sfru123480 showing the highest expressed, and PGRP_Sfru122870 the lowest (Fig. 2B). Overall, the GNBP family was commonly expressed throughout all developmental stages and sexes. The expression levels of two β-1,3-glucan-binding protein-like genes (GNBP_Sfru064880 and GNBP_Sfru196670) were moderate in all developmental stages (Fig. 2B). Two additional β-1,3-glucan-binding protein-like genes (GNBP_Sfru064870 and GNBP_Sfru196660) were expressed at lower levels in the egg, L1, and L2 stages than in the other developmental stages examined, and their expression levels peaked at the pupal stage (Fig. 2B). In contrast, the four members of the PGRP family (PGRP_Sfru001280, PGRP_Sfru001270, PGRP_Sfru001290, and PGRP_Sfru122870), were expressed at low levels in the egg stage, but increased significantly (P < 0.001) at the other developmental stages (Fig. 2B), although expression of each PGRP differed between developmental stages, e.g., the PGRP-like isoform X2 gene (Sfru182360) and the PGRP-like gene (Sfru122850) showed very high expression from the L2 larval stage to the adult (Fig. 2B). The former gene showed low expression in the egg stage and moderate levels in the L1 stage, whereas the latter gene showed low transcript abundance in both the egg and L1 stages (Fig. 2B). The expression level of the PGRP LB-like gene, Sfru001280, was low in the egg and L1 stages, but very high in the L2 to adult female stages, but moderate in male adults (Fig. 2B). Transcript abundance of the PGRP LB-like gene, (Sfru001270, was high from L2 to the adult stage in both males and females and low in the egg and L1 stages, except for the pupal stage (Fig. 2B). Another PGRP LB-like gene (Sfru001290) was highly expressed in female and male adults, but had moderate to low expression in other stages, with the lowest transcript abundance in eggs (Fig. 2B). The PGRP-like isoform X2 (Sfru122870) showed high expression in nymphs and female adults and low expression levels in the other eight stages (Fig. 2B). In contrast to the first six genes of the PGRP gene family, which showed very low expression levels during the egg stage, the transcript abundance of the remaining two PGRP genes, PGRP SB2-like isoform X2 (Sfru184410) and PGRP LC-like isoform X1 (Sfru183090), was high during the egg stage and differed in all the other stages (Fig. 2B).
Toll signaling pathway genes
28 differentially expressed genes involved in Toll signaling were identified, representing 8 gene families. The total expression of genes in the Toll signaling pathway showed a gradual decrease from the egg and L1-L6 stages, reaching a minimum value of 4.14 at the L6 stage, and then fluctuated from the L6 stage to the adult stage, reaching eventually a maximum value of 97.37 at the pupal stage (Fig. 3A). A total of 14 Toll receptor-like genes were identified in S. frugiperda, most of which showed similar expression patterns in the egg, L1, L6, and pupal stages. Seven protein Toll genes (Toll_Sfru156630, Sfru078860, Sfru078650, Sfru078750, Sfru191410 Sfru156680, and Sfru087470) were more highly expressed in the egg and pupal stages than in the other eight stages, while the opposite was true for genes Sfru052470, Sfru191290, Sfru078770, and Sfru073570 (Fig. 3B). High expression levels were observed in all developmental stages except for egg and L6. The remaining three Toll genes had high transcript abundance in the L2-L5, pupal, and adult stages, with different expression patterns in the remaining stages (Fig. 3B). Two genes coding for Toll-interacting proteins (TOLLIP_Sfru194190, Sfru085820) and one interleukin-1 receptor-associated kinase (Tube_Sfru151100) had similar expression profiles and were widely expressed in 10 stages (Fig. 3B). A total of five Spaetzle (Spz) genes were differentially expressed during development. One of the Spz genes (Sfru176170) was highly expressed throughout the developmental stages (Fig. 3B). All five Spz genes were highly expressed at the pupal stage, but four of them (Sfru052300, Sfru163900, Sfru203820, and Sfru073580) were expressed at low levels at the L6 stage (Fig. 3B). Five Spz genes had different expression patterns at the other eight stages. The death domain containing myeloid differentiation factor 88 (MyD88_Sfru103100), NF-kappa-B inhibitor alpha (cactus_Sfru062560), and two c-Rel proto-oncogene proteins (DI/Dif_Sfru194500 and Sfru114500) had similar expression profiles with high transcript abundance at all developmental stages (Fig. 3B). Two TrCPs (F-box and WD-40 domain protein) (Sfru170750 and Sfru021590) were expressed at moderate to high levels in egg, pupal and female adult stages and at low levels in the other stages (Fig. 3B).
Imd signaling pathway genes
17 differentially expressed genes involved in IMD signaling were identified, representing 10 gene families. The total expression of genes in the Imd pathway decreases during development from the egg to the L6 stage and gradually increases from the L6 stage, peaking at the pupal stage (57.15) and the female adult stage (55.94) (Fig. 4A). A similar expression pattern was found for the ubiquitin-binding enzyme E2 variant 2 (Sfru208330), fas-related death domain gene (FADD_Sfru102350), Ird5 (Sfru098380) and transforming growth factor β-activated kinase-1 (TAK1_Sfru107230) genes, which were moderately expressed at all 10 developmental stages (Fig. 4B). Two of the three transcription factor AP (Jra) genes had opposite expression profiles, with one Jra (Sfru028390) showing high levels at all 10 stages and the other (Sfru199080) showing low levels throughout (Fig. 4B). The third Jra (Sfru212830) gene, showed moderate to high expression levels in most developmental stages except L5 (Fig. 4B). Ankyrin-1-like isoform X5 (Sfru165290) showed low levels before the pupal stage and high expression in the pupal and adult stages (Fig. 4B). Three FAS-related factor genes (Sfru023860, Sfru082950, and Sfru174210) showed similar expression profiles, with high levels in 9 of the 10 developmental stages (except the L5 stage) and highest expression levels in the egg stage (Fig. 4B). MEKK1 (Sfru128270) was expressed at high levels in the egg, pupal, and adult stages, but low in the other stages (Fig. 4B). The cyclic adenosine-dependent transcription factor ATF-6α isoform X2 (Sfru058210) showed moderate to high expression levels throughout development (Fig. 4B). The dual oxidase isoform X1 (Sfru190250) showed high expression in the L3, L4, L5 larval, pupal, and female adult stages, and relatively low expression in the other stages (Fig. 4B).
Effectors
Five differential expressed antimicrobial peptide coding genes were identified in S. frugiperda. The sum of their gene expression levels was similar throughout development except for L6 (19.93) in which expression showed a gradual increase across developmental stages, reaching a maximum in the pupal stage (36.85) and the female adult stage (37.95) (Fig. 5A). Two defensin precursor genes (Sfru120890 and Sfru018720) and the anionic antimicrobial peptide 2-like gene (Sfru123110) showed high expression levels throughout the examined developmental stages, except for a relatively lower level at the L1 stage (Fig. 5B). One cecropin A2 (Sfru063640) presented low expression level in the egg and L1 stages and higher levels in the other 8 stages (Fig. 5B). The transcript abundance of another cecropin A2 (Sfru009390) was low in the egg, L1 and L6 stages but high in the other 7 stages (Fig. 5B).
Serine proteases
A total of 70 serine protease (SP) genes were found to be expressed. 11 SP genes (Sfru157760, Sfru002110, Sfru183050, Sfru139950, Sfru147560, Sfru224700, Sfru094840, Sfru054250, Sfru149890, Sfru149310 and Sfru085130) showed similar expression profiles, with moderate to high levels of expression in all 10 developmental stages and higher expression in the egg stage (Fig. 6B). In contrast the SP genes: Sfru124810, Sfru147650, Sfru105980, Sfru064550, Sfru140070, Sfru064580 Sfru026440, Sfru106420, Sfru199970, Sfru159340, and Sfru213980, were expressed at lower levels in the egg stage compared to the other nine developmental stages (Fig. 6B). Gene Sfru029070 was expressed at low levels in the egg, pupal, and adult stages and at moderate levels in the other six stages (Fig. 6B). Of four SP genes (Sfru147840, Sfru139640, Sfru147830, and Sfru139800), Sfru147840 and Sfru139640 exhibited low expression in the L3 and L5 larval stages, with higher expression levels in the other stages (Fig. 6B). Sfru147830 showed low expression levels at the L2, L5, L6, and adult stages, with higher expression levels in the remaining five stages (Fig. 6B). Sfru139800 expression was high in L1, L2, and male adults, but low in the other developmental stages (Fig. 6B). Seven SP genes (Sfru044380, Sfru001750, Sfru153450, Sfru057200, Sfru045930, Sfru219640 and Sfru057340) showed high transcript abundance in the L1-L6 larval stages and low transcript abundance in the egg, pupal and adult stages (Fig. 6B). Whereas nine SP genes (Sfru7Sfru001730, Sfru157770, Sfru092830, Sfru057190, Sfru140040, Sfru141420, Sfru094510, Sfru194460 and Sfru147640) showed low transcript abundance in the egg, pupal and adult stages, and moderate transcript abundance in the remaining six stages (Fig. 6B). Seven SP genes (Sfru147660, Sfru113960, Sfru171370, Sfru139820, Sfru064540, Sfru147600 and Sfru020980) were mostly expressed at low levels throughout development, with very few periods of higher expression (Fig. 6B). Additionally, Sfru113960 showed high expression levels in L1 stage, while Sfru139820 exhibited high expression levels in L5 stage (Fig. 6B). Eight SP genes (Sfru139760, Sfru120120, Sfru213680, Sfru213660, Sfru147680, Sfru057330, Sfru108000 and Sfru027460) were highly expressed in the adult stage, except for genes Sfru120120 and Sfru057330, which were expressed at higher level in the male adult stage and L1 stage, respectively (Fig. 6B). Finally, gene Sfru139940 was highly expressed in the egg stage and the female adult stage and at lower levels in the remaining eight developmental stages (Fig. 6B).
Serine protease inhibitors
Seventeen genes were identified related to immune modulation acting as serine protease inhibitors, (SPIs) along with 7 serpin genes were found to be differentially expressed between stages. Among the SPI genes, seven (Sfru118470, Sfru064460, Sfru118730, Sfru064440, Sfru105540, Sfru055080, and Sfru155870) were expressed at high levels throughout the examined developmental stages, with the exception of the last three genes, which were expressed at low levels in the egg stage (Fig. 7B). Two SPI genes (Sfru105400 and Sfru055210) were also expressed at low levels in the egg and L1 stages and then highly expressed in the remaining eight stages (Fig. 7B). One SPI gene (Sfru064470) was expressed at low levels in the pupal stage and moderately expressed throughout development, while gene Sfru094100 was expressed at the highest level in the pupal stage and the lowest level in the L6 stage (Fig. 7B). Five SPI genes (Sfru034470, Sfru108630, Sfru055090, Sfru185160, and Sfru105520) had similar expression profiles, and genes Sfru055090, Sfru185160, and Sfru105520 were all moderately-highly expressed in the adult male and female stages, and lowly expressed in all other stages (Fig. 7B). Three serpin genes (Sfru205920, Sfru212840, and Sfru155860) were moderately expressed in the egg stage and moderately-highly expressed in all other stages, while three other serpin genes (Sfru009670, Sfru098660, and Sfru009680) showed medium expression in L1, L2, L3, L5, and L6 stages and low expression in the remaining five stages (Fig. 7B). Another serpin gene (Sfru009640) was highly expressed in the egg stage and then reached minimum expression in the L4 larval stage (Fig. 7B).
Cellular immune gene expression patterns: Hemolymph coagulation, clot formation and wound healing
Regarding the expression of hemolymph clotting related genes (22 genes in 3 families), the total gene expression index gradually increased from the egg stage to the L4 larval stage, and then decreased from L4-L6 before it gradually increased again reaching the maximum values in L4 and pupal stages, which were 94.97 and 94.06, respectively, while the lowest value was 30.95 in the L6 stage (Fig. 8A). Two apolipophorin (ApoLp), six gelsolins, and 14 lipophorin (LP) genes were expressed in different developmental stages and both sexes, and genes from the same gene family were expressed at various levels in the same developmental stage (Fig. 8B). The two ApoLp genes (Sfru104770 and Sfru014290) were moderately expressed in the L1 stage and at high levels in the rest of the developmental stages (Fig. 8B). Four genes (Sfru215420, Sfru086370, Sfru086380, and Sfru131920) were expressed at intermediate levels throughout development (Fig. 8B). Two genes (Sfru154490 and Sfru151730) were expressed at higher levels in the L2-L5 stage than in the remaining six stages (Fig. 8B). Expression of the 14 LP genes varied widely throughout development, with five genes (Sfru192180, Sfr u103510, Sfru101150, Sfru215410, and Sfru101140) having low transcript abundance in the egg stage and higher in the other nine stages (Fig. 8B). Gene Sfru056250 was highly expressed in the L2-L4 stage and showed the lowest expression level in the male adult stage (Fig. 8B). Five LP genes (Sfru216220, Sfru216200, Sfru216230, Sfru164010, and Sfru116370) showed low or no expression levels throughout development, with higher expression in the pupal stage than in the other stages (Fig. 8B). Genes Sfru164010 and Sfru116370 were expressed at higher levels in the egg and pupal stages, respectively, and showed low or no expression in the other developmental stages (Fig. 8B). Gene Sfru156990 had low transcript abundance in all developmental stages compared to the other 13 genes in the LP gene family (Fig. 8B).
Hematopoiesis
A total of 79 hematopoiesis related genes distributed within 12 families that showed differential expression in the 10 developmental stages tested, with the total immunity index highest in the egg, pupal, and female adult stages, with a downward trend from the egg stage to the lowest value in the L6 larval stage (-16.77) (Fig. 9A). Among them, 27 Notch genes (Sfru025660, Sfru063940, Sfru059120, Sfru046440, Sfru015630, Sfru209530, Sfru095710, Sfru059830, sfru210130, sfru116070, sfru185390, sfru120730, sfru183570, sfru091010, sfru104240, sfru067090, sfru096690, sfru030500, sfru104290, Sfru091000, Sfru033630, Sfru085450, Sfru033640, Sfru162900, Sfru218740, Sfru162060 and Sfru058540) had similar expression profiles and they were widely expressed throughout development (Fig. 9B). These genes were expressed at higher levels in the egg, pupal and adult stages. Two genes (Sfru210920 and Sfru210940), in contrast, were expressed at low levels in the egg and adult stages and at high levels in the remaining developmental stages (Fig. 9B). Gene sfru098270 was widely expressed in most stages, with medium expression in the L2-L5 larval stages, high expression in the pupal stage, and low expression in the other stages (Fig. 9B). Two Notch genes (Sfru019950, Sfru020670) exhibited high expression levels only in the egg stage, with relatively low expression levels in the other nine developmental stages (Fig. 9B). Among eight genes, (Serrate_Sfru113650, Serrate_Sfru070960, Rac1_Sfru021980, Rac1_Sfru111260, Rac1_Sfru185850, Ras_Sfru069260, Ras_Sfru023500, and Ras_Sfru204090), except for Ras_Sfru023500 and Ras_Sfru204090, where two genes had low expression levels in the egg stage, the other six genes exhibited high expression levels at each developmental stage (Fig. 9B). The three Dorsal/Dif genes (Sfru104280, Sfru035450, Sfru120270) were moderately to highly expressed in all stages (Fig. 9B). Two JAK/STAT genes (Sfru102000 and Sfru053630) had high expression levels in the former except for the egg and L6 stages with the highest expression in the adult stage, while the latter had low expression in the L1-L6 stages and high expression in all other stages (Fig. 9B). Genes encoding wntD (Sfru125280, Sfru133410 and Sfru009780) were expressed throughout (Fig. 9B). Sfru178200 was expressed at low levels in the egg and pupal stages, while expression was high in the rest of the stages (Fig. 9B). In contrast, genes (Sfru133430 and Sfru125260) were expressed at low levels in the L1-L6 larval stage and at high levels in all the other stages. Genes Sfru178200 and Sfru050390 were highly expressed in the L2-L6 stages and at low levels in the egg, L1, and pupal stages (Fig. 9B). Four genes (Sfru133450, Sfru133440, Sfru225820, and Sfru064030) and the Dorsal gene (Sfru047810) were transcriptionally abundant in most developmental stages (Fig. 9B). The two Combs genes (Sfru048480 and Sfru048470) were expressed at intermediate levels throughout development, while gene Sfru048490 was highly expressed in the egg and adult female stages, with the remaining eight periods showing low expression (Fig. 9B). Gcm2 genes (Sfru068620 and Sfru075630) and Col genes (Sfru141830) showed low expression levels in eight developmental stages except for the egg and pupal stages in which they were expressed at high levels (Fig. 9B).
Hemocyte-mediated immune responses
A total of 84 genes, representing 15 gene families, were identified as being involved in the hemocyte-mediated immune response. During the S. frugiperda development, the total immune index of this set of genes fluctuated showing low values in the egg, L6, and male adult stages, and high values in the female adult (45.31) (Fig. 10A). Eight immunoglobulin-like cell adhesion molecules (Sfru005140, Sfru003210, Sfru005100, Sfru084590, Sfru064160, Sfru005070, Sfru005080, and Sfru005090) had similar expression patterns, with higher expression in the L1 and pupal stages and lower expression in the rest of the stages (Fig. 10B). Among them, genes Sfru141380, Sfru005150, Sfru172210, Sfru005170, and Sfru064510 as well as Dscam_Sfru064520 had the lowest transcript abundance in all stages (Fig. 10B). PLA2 genes (Sfru023640, Sfru023080, and Sfru032090) were widely expressed throughout all developmental stages (Fig. 10B). Genes Sfru133580, Sfru022540 and Sfru198580 were lowly expressed in all stages (Fig. 10B). Three (Sfru133540, Sfru068390, and Sfru046280) and two (Sfru079350, and Sfru119270) Atg genes showed medium-high and medium expression levels, respectively, throughout all developmental stages (Fig. 10B). Three integrin genes (Sfru190220, Sfru164770, and Sfru210440) showed medium-high expression levels during development, and three Integrin genes (Sfru125340, Sfru125350, and Sfru107170) showing low expression in the egg stage and medium expression in all the other stages (Fig. 10B). One Integrin gene (Sfru210170) was highly expressed in all stages, except for the egg stage with the highest expression in the adult stage (Fig. 10B). In contrast, one Integrin gene (Sfru125360) was expressed at low levels in all 10 stages (Fig. 10B). Two Integrin genes (Sfru080300 and Sfru080330) were expressed at low levels in the egg stage and at high levels in the remaining stages (Fig. 10B). One Integrin gene (sfru029980) was expressed at low levels in the egg and L1 stages and then highly expressed throughout development (Fig. 10B). One Integrin gene (sfru125910) was not expressed in the L6 stage and was expressed at low levels in all the other stages (Fig. 10B). Two DSR-CI genes (Sfru104410 and Sfru126570) were lowly expressed in the L1-L6 larval stages with higher transcript abundance than in the remaining four stages (Fig. 10B). One DSR-CI (Sfru120000) and one BH4 (Sfru093120) as well as three NOS synthase genes (Sfru019260, Sfru092590, and Sfru096580) showed moderate-high transcript abundance throughout all stages, except for Sfru096580 which was expressed at a lower level in the egg stage than in the rest of the stages (Fig. 10B). Four SOD genes (Sfru154600, Sfru103720, Sfru208820, and Sfru189680) were widely expressed at high levels throughout the growth stages (Fig. 10B). One SOD gene (Sfru103700) was expressed at high levels with the exception of the egg stage, with the highest expression in the L1 and adult stages (Fig. 10B). The Pvf genes (Sfru003450 and Sfru068840) had similar moderate expression profiles, and CRQ (Sfru117630 and Sfru120400) showed moderate to high expression throughout development, with the highest expression in female adults (Fig. 10B). Two PO genes (Sfru000420 and Sfru105150) were highly expressed in the L3-L6 and adult female stages (Fig. 10B). Two PER genes (Sfru169930 and Sfru206280) showed medium expression in most developmental stages except for the egg and L6 stages, and gene Sfru047110 showed the lowest expression in the pupal stage (Fig. 10B). Gene PER_sfru178350 showed high expression in all stages, except for male adults where it was expressed at low levels (Fig. 10B). Three PER Genes (Sfru065640, Sfru178370, and Sfru047130) had medium-high expression in the L1-L6 stages and the lowest expression in the pupal and adult stages (Fig. 10B). Gene PER_Sfru143000 had high expression in the egg stage and gene PER_Sfru080530 had high expression in the L1-L5 stage and low expression in the egg, L6, and adult stages (Fig. 10B). Gene PER_Sfru127290 showed lower expression in L1, L5, L6, and pupal stages than the rest of the stages (Fig. 10B). Gene PER_Sfu178380 showed higher expression in the L2-L4 stages and lower expression in the other stages, and gene PER_Sfru080720 showed higher expression in the egg and pupal stages (Fig. 10B). Two PER genes (Sfru127900 and Sfru12890) had similar expression profiles, being mainly expressed in the egg and female adult stages and at low levels in the remaining stages (Fig. 10B). The expression profile of PER genes Sfru178340 and Sfru1783300 was similar in all developmental stages except for the former where transcript abundance was moderate-high in the L1 stage (Fig. 10B). An NADPH gene Sfru096530 was expressed at moderate levels everywhere apart from the egg stage, whereas NADPH_sfru119860 was expressed in the egg, L1, L2, L6, male and female adult stages and at low levels in the rest (Fig. 10B). Two clip domain serine proteases (CLIPs) genes (Sfru162500 and Sfru162580) showed low expression throughout the whole period (Fig. 10B).
qRT-PCR gene validation
To validate the RNA-seq results, RT-qPCR experiments were performed to monitor the expression of a subset of the identified humoral immune-related genes and cellular immune-related genes (Fig. 11). The results showed that the overall expression level of most selected genes had similar expression trends as presented in the RNA-seq data. Discrepancies between the two datasets (RNA-seq/Rt-qPCR) were minor.