Comparative gene expression analyses reveal developmental stage dependent immune adaptations of Spodoptera frugiperda

doi:10.21203/rs.3.rs-4990584/v1

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Comparative gene expression analyses reveal developmental stage dependent immune adaptations of Spodoptera frugiperda

https://doi.org/10.21203/rs.3.rs-4990584/v1

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The fall armyworm, Spodoptera frugiperda, is a major agricultural pest responsible for substantial crop damage worldwide. Several critical physiological functions, including high reproductive and migratory abilities, a broad plant host range, coupled to the development of high resistance to various chemical pesticides, and a strong immune response to microbial pathogens, has made this insect particularly difficult to control. While various innate immune pathways are assumed to play crucial roles in microbial pathogen defense, a comprehensive analysis across different developmental stages has been lacking. This is particularly important as different developmental stages of S. frugiperda display differential sensitivity to chemical pesticides and/or microbial pathogens. Here, we performed a comparative transcriptomic analysis of S. frugiperda across ten developmental stages: egg, six larval instars (1–6), pupa, and adult (both female and male). We identified 56 gene families associated with insect immunity, with several exhibiting variable expression patterns across the developmental stages. Our findings provide new insights into the global gene expression landscape forming innate immune responses throughout development and between sexes in S. frugiperda. These results help explain the disparate innate immune responses of different developmental stages and provides clues for devising more effective pest control strategies.

Spodoptera frugiperda

cellular immunity

humoral immunity

developmental stage

transcriptomics

The immune system is critical for protecting organisms from potential microbial and parasitic pathogens. Host defense in insects relies on the innate immune response which is divided into two broad systems: humoral and cellular immunity. Humoral immunity operates through conserved signaling pathways involving recognition of the pathogen, typically resulting in the expression and production of effector molecules, e.g., antimicrobial peptides, whereas cellular immunity encompasses hemocyte and other cell type-mediated activities, resulting in phagocytosis, nodulation, encapsulation, and/or coagulation targeting in the invading pathogen [1]. Immune activity in insects is determined by several factors, including sex, mating status, social environment, feeding behavior, and/or locomotion [2, 3]. In vertebrates, immunity to pathogens is also known to be regulated with age [4]. However, for holometabolous insects that undergo complete metamorphosis, i.e., that develop through four distinct stages: egg, larva, pupa, and adult, in which significant rearrangement and shedding/molting of the body occurs, not only age, but these transitions influence resistance to microbial pathogens. As such, it has been widely reported that different stages, including different instars, show differential susceptibility to both chemical pesticides and microbial agents, including, regarding the latter, biological agents (viruses, bacteria, fungi) used for control of insect populations [5–7].

Insect humoral immunity involves recognition and transduction processes that lead to the production of immune factors that act to try to eliminate invading pathogens and/or prevent their proliferation and spread within the host. Recognition is canonically mediated by pattern recognition receptors (PRRs), which include cell membrane and soluble proteins. PRRs bind to pathogen-associated molecular patterns (PAMPs) found on pathogen surfaces, initiating innate immune signaling pathways. Two highly conserved immune signaling cascades are the nuclear-factor kappa B/Toll and Immune deficiency (Imd) pathways. The Toll pathway primarily addresses fungi and Gram-positive bacteria, while the Imd pathway primarily responds to Gram-negative bacteria. Both pathways also participate in insect antiviral responses [8]. The culmination of innate immune signaling results in the production of a wide range of antimicrobial peptides and effector molecules that seek to inhibit pathogen replication and eliminate invading cells from within the insect body [9–12]. Cellular immunity includes hemolymph coagulation, clot formation, and wound healing, as well as phagocytosis of foreign bodies. Typically, insect hemocytes, in the open circulatory system of the hemocoel, accumulate at pathogen invasion sites and/or any detected foreign cells to prevent the growth and spread of the pathogen. Detection of invading cells typically leads to rapid increases in hemocyte numbers and the activation of hemocyte-mediated immune responses cooperate with humoral reactions to clear the infection [13].

The differential expression of immune genes at various stages of the infection process has been reported in a variety of systems and can provide important insight into pathways activated during infection [14]. Recent findings indicate that the developmental stage of insect during infection can also play a significant role in regulating immune capacity and/or susceptibility to the infecting pathogen. For example, phenoloxidase enzyme activity, which is crucial for resisting infections, fluctuates as a function of age. This fluctuation is a strategic adaptation to extend survival and maximize offspring production. In beetles, for example, RNA interference (RNAi)-mediated silencing of phenoloxidase has been shown to prolong survival [15]. Immune responses can also vary at different time points within the same larval stage. In Manduca sexta, fifth instar larvae on day five exhibit fewer hemocytes and reduced capacities for nodulation, phagocytosis, and phenoloxidase activity compared to first-day fifth instar larvae [2]. Similarly, aging Apis mellifera honey bees demonstrate a decreased ability to resist pathogens and experience higher intensity of infection [16]. This suggests a complex interrelationship between development, age, and immunity.

Spodoptera frugiperda (J. E. Smith) is a noctuid pest native to the Americas, known for causing significant economic damage to a wide variety of crops and exhibiting remarkable resistance (to environmental stress and chemical pesticides) and high adaptability, which has facilitated its global spread [17]. Although previous research in S. frugiperda has identified and characterized some aspects of immunity, global studies of immune pathway gene expression as a function of developmental stage has been lacking. This information is critical in developing models to understand and predict immune responses in wide array of contexts. Here, we use high-throughput RNA sequencing (RNA-Seq) to perform a systematic analysis of immune pathway gene expression in different developmental stages and adult male and females of S. frugiperda. These data establish a foundation for studying insect immune mechanisms and comparative analyses in relation to insect development, leading to increased insights into fundamental aspects of immunity and targeting these pathways for improving pest control strategies.

Insect rearing and sample collection

Spodoptera frugiperda were reared on maize leaves in a climate chamber at Guizhou University. The chamber conditions were maintained at a temperature of 25 ± 1°C, with a light photoperiod of 14:10 hours and a relative humidity of 70 ± 5%.

Sample collection, RNA extraction and sequencing

Spodoptera frugiperda samples were collected at different developmental stages, including: egg (E), 1st instar (L1), 2nd instar (L2), 3rd instar (L3), 4th instar (L4), 5th instar (L5), 6th instar (L6), pupae (P), and adult males (AM) and adult females (AF). Approximately 300 eggs, 200 1st instar larvae, 100 2nd instar larvae, 50 3rd instar larvae, 10 larvae from the 4th instar, 5 larvae from the 5th and 6th instars, 5 pupae, and 5 adult males and females were collected for each replicate, with three biological replicates performed for each stage. Samples were collected in centrifuge tubes, immediately frozen in liquid nitrogen, and stored at -80°C for later use.

Total RNA was extracted from samples using Trizol reagent. Briefly, the samples were placed in sterile mortars pre-cooled with liquid nitrogen and ground using a pestle. Approximately 100 mg of each sample were collected into an RNA-free 2 mL tube, and 500 µL of Trizol lysate were added to each tube for total RNA extraction according to the manufacturer's instructions. RNA integrity was evaluated using agarose gel electrophoresis and samples were stored at -80°C. The quality and quantity of RNA samples was determined using Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). cDNA libraries were prepared using TruSeq RNA Sample Prep Kit v2 (Illumina, USA) according to the manufacturer's instructions, and Illumina sequencing was performed on the Illumina Hiseq2000 sequencing system at the Beijing Genomics Institute (BGI-Shenzhen, China).

Transcriptome assembly, annotation and analysis

Low-quality reads, defined as those with more than 20% of bases having a quality score less than 10, reads containing adapters, and reads with more than 5% unknown bases (N bases), were removed using FASTQC and Trimmomatic. Paired-end clean reads were mapped to the S. frugiperda genome (http://v2.insect-genome.com/Organism/715) using HISAT2. The assembled unigene sequences of S. frugiperda were then compared to several databases, including Nr (non-redundant protein databases), SwissProt, COG (Cluster of Orthologous Groups), and KEGG (Kyoto Encyclopedia of Genes and Genomes), using the BLASTx algorithm (http://www.ncbi.nlm.nih.gov/) with a cut-off E-value of 10^− 5.

Differential gene expression analyses were conducted using the DGESeq R package, with P-values adjusted by the Benjamini & Hochberg method [18]. Significant differential expression was determined with a P-value threshold of 0.05 and a log2(fold change) of 1. Immune-related genes (IRG) were identified based on previous descriptions. The heatmaps of IRG in each stage were generated with log₂(IRG_FPKM).

Quantitative Real-Time PCR (RT-qPCR)

Ten differentially expressed genes across ten developmental stages were selected for verification by RT-qPCR. Primers for RT-qPCR (Supplemental Table 1) were designed using Beacon Designer 8 software (Primer Biosoft International, Palo Alto, CA, USA). RT-qPCR was performed using the SYBR Premix Ex TaqTM Kit (Takara, China) with 20 µL reaction volumes. The PCR protocol was as follows: initial denaturation at 95°C for 5 s, followed by 40 cycles of 60°C for 10 s, and 72°C for 10 s. Ct values were calculated relative to Rpl32 using the 2^−△Ct method. Among which, Rpl32 was used as the reference gene to normalize gene expression levels.

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.

To facilitate analysis, humoral immune-related genes were divided into six categories: (i) Recognition, including X, Y, and Z processes, (ii) Toll pathway components, (iii) Imd pathogen components, (iv) Effector genes, (v) SP pathways genes, and (vi) SPI pathways genes. These were then comprehensively analyzed with respect to the eight developmental stages: egg, 1st – 6th instars, and pupae, and two adult types; male and female. Total humoral immune index analyses showed that the Toll and Imd pathways were expressed in all developmental stages, and that expression levels of these systems decreased gradually from the egg to the larval stage, while increasing in the pupal and adult stages. Not too surprisingly expression level of genes in Recognition, Effector, SP, and SPI categories was low in the egg stage. However, expression of SP genes was substantially low also in the pupal, and adult stages, indicating that these systems may mainly function during the larval stages. In general, the expression of humoral immunity genes increased in the early larval stages and then decreased in the late larval stages. These data indicate that different larval stages possess different susceptibility to pathogens, consistent bioassays studies that have shown similar outcomes. Humoral immune genes were also expressed at higher levels in females than in males, indicating a great allocation of resources and resistance in the former likely linked to egg laying/generation of progeny, leading to evolutionary preferences for reproduction potential in males that can be short term, and immune function in females that may be required for longer time periods and/or for successful egg laying [19, 20].

The gradual increase of Recognition gene expression across development and peaking in female adults may be due to less exposure to the external environment during the egg stage, and the presence of serosa in the ectodermal epithelium surrounding the egg yolk and embryo, which provides protection against microbial invaders which may minimize the need for “recognition” systems [21]. In contrast, immune recognition is indispensable in the adult stage to deal with a range of microbes. Recognition is mediated by two main protein families; peptidoglycan recognition proteins (PGRPs) and (Gram-negative bacteria-binding proteins) GNBPs [22]. Our data show that most GNBPs and PGRPs are highly expressed in the adult stage, with two PGRPs (Sfru001290 and Sfru122870) particularly highly expressed in adults, and two others, PGRP_Sfru183090 and PGRP_Sfru184410, showing higher expressed in female adults than males. In addition, GNBP_Sfru123480 and PGRP_Sfru182360 were highly expressed in larval stages, suggesting specific functions for these related to developmental humoral immune recognition [23].

Expression of many antimicrobial peptide genes are regulated by the Toll and Imd signaling pathways. The Toll pathway is activated by Gram-positive bacteria, yeast, and/or fungi, while the Imd pathway is mainly triggered by many Gram-negative bacteria and some Gram-positive bacteria containing DAP-type peptidoglycan (PGN) [23]. Most Toll and Imd genes were highly expressed in the egg and pupal stages, which was consistent with previous results in P. xylostella [24]. However, our data show that a subset of Toll (TOLLIP_Sfru194190, Cactus_Sfru062560, Dl/Dif_Sfru194500) and Imd pathway genes (FADD_Sfru102350, Jra_Sfru212830, Ird5_Sfru098380) were highly expressed at the female adult stage, which may be related to the immune response after mating. In Drosophila, sex peptides have been shown to activate immune functioning in female flies after mating by stimulating the Toll and Imd pathways [25].

The “Effector” set of immune genes includes the anionic antimicrobial peptide/protein (AMP) genes found in invertebrates, vertebrates, and plants [26]. These peptides are active against bacteria, fungi, and viruses. Here we found that AMP Sfru123110 was highly expressed in all developmental stages except for the first instar and reached its highest expression the pupal and adult stages. This suggests that AAMP_sfru123110 may be broadly targeting potential pathogens during most of development. Interestingly, previous studies have shown that the bacterial pathogen Campylobacter jejuni can be transmitted through fly, Musca domestica, life stages, however, during pupal development the bacterial titers decrease substantially, which coincides with increased expression of AMP-encoding genes [27].

Serine proteases (SPs) regulate the biological activity of target proteins through proteolytic cleavage. When these serine proteases are no longer needed, they are inactivated by serine protease inhibitors/serpins (SPIs), which play key roles in insect digestion, metamorphosis, and development as well as immunity [28–30]. The expression pattern of SP genes was different in different developmental stages, with a subset highly expressed in the larval stage, whereas others were highly expressed in egg stage or adult stage. These data suggest discrete developmental roles for SP/SPI genes that may help explain their apparent genetic redundancy. SPI activity in many Lepidoptera species has been shown to be controlled by hormone levels and fluctuated greatly during development [31]. In G. mellonella, SPI activity is up-regulated during pupation, consistent with regulation of protease activity during metamorphosis, similar to expression seen for SPI_Sfru094100, SPI_Sfru118730 and SPI_Sfru064460 in our study [32]. Some SPI genes were also seen highly expressed in the larval stage, e.g., SPI_Sfru070490, SPI_Sfru155870, SPI_Sfru105540 and SPI_Sfru055080.

Gelsolins regulates cell growth and apoptosis by modulating the actin cytoskeleton, and expression has been associated with development and age-related apoptosis. In our study, the gelsolin gene family (Sfru101140, Sfru101150, Sfru215410, and Sfru215420) genes were expressed at low levels except for the egg stage, with Sfru154490 and Sfru151730 were expressed at low levels from the larval to the adult stage, suggesting that in S. frugiperda gelsolins may have some function during initial stages of egg development into (1st instar) larvae [33]. In hemolymph coagulation, clot formation, and wound healing, lipoproteins are responsible for lipid transport in insect hemolymph. Apolp III (apolipophorin-III) is involved in the translocation of lipids, pathogen recognition, nodulation, and antimicrobial activity. The Apolp III gene of S. frugiperda was highly expressed throughout development, consistent with reports in the beet armyworm, S. exigua [34]

Notch signaling regulates larval hematopoiesis and Serrate, the receptor for Notch, is expressed in plasmatocytes. In our data, Notch and Serrate family genes were expressed at higher levels in S. frugiperda eggs than in other stages, similar to what has been reported for honeybee embryo [35, 36]. Ras (ras-specific guanine nucleotide-releasing factor) is involved in phagocytosis. In S. exigua, Ras genes are constitutively expressed from egg to adult [37], In the present study, most of the Ras genes of Spodoptera frugiperda were also expressed constitutively from egg to adult. Pvr (Vegfr receptor) is involved in the regulation of hemocyte proliferation and identified as an important regulator of embryonic hematopoiesis [38], In Spodoptera frugiperda, moderate to high levels of expression were found throughout the developmental stage. Dorsal/Dif (Dorsal-related immunity factor) has a role as an initiator of effectors (e.g., AMPs) and in the regulation of plasma cell production in hematopoiesis. Rac1 (ras-related protein Rac1) regulate the cytoskeleton, encapsulation and hemocyte recruitment [39]. Previous work has shown constitutive expression of Rac1_Sfru111260 and Rac1_Sfru185850 in S. frugiperda [40, 41]. WntD (Wnt inhibitor of Dorsal) is a cytokine that regulates the NF-κB pathways as well as other signaling pathways depending on the developmental stage [42]. JAK/STAT (Janus kinase/signal transducers and activators of transcription) controls hemocyte proliferation and limits tumor growth during trauma. Our data show that JAK/STAT_Sfru211330 is expressed at low levels in the egg and L6, despite hemocyte proliferation being rapid in the egg stage [41]. Our data also show that the Gcm2 (transcription factor, glial cells missing 2-like) genes Gcm2_Sfru075630 and Gcm2_Sfru068620 are expressed in the egg and pupal stages, where cell division is rapid or metamorphosis is in progress, consistent with previous reports [41, 43]. PLA2 (phospholipase A2) is involved in activating the Toll pathway and affecting the expression of immune genes such as MyD88, with highest expression observed in fat bodies, and in the egg and adult stages [44]. In our study, PLA2 genes (Sfru023640, Sfru023080, and Sfru032090) were widely expressed throughout all developmental stages. The expression of PLA2_Sfru023640 is highest in the adult stage, PLA2_Sfru032080 has a higher expression level during the pupal stage compared to the larval stage, and PLA2_Sfru133580 shows the highest expression level during the egg stage.

Atg (Autophagy-related genes) exhibited constitutive medium to high expression at the different developmental stages of S. frugiperda examined, consistent with the expression profile of Atg genes in Tenebrio molitor [45]. Integrins transmits signals to promote cell adhesion and spreading and regulates cellular immunity by regulating phagocytosis and encapsulation. Expression of most integrin genes increases with developmental stage in S. frugiperda [46], consistent with our data. NOS (nitric oxide synthase-like), which regulates NO synthesis, acts as a vasodilator, by inducing immunity through the activation of signaling pathways, it also induces high expression of effectors, and leads to increase in NOS expression following pathogen infestation [47],so our study shows NOS synthase genes (Sfru019260, Sfru092590, and Sfru096580) showed moderate-high transcript abundance throughout all stages. SOD (peroxidase-like) regulates the concentration and detoxification of reactive oxygen species (ROS). SOD_Sfru103720, SOD_Sfru208820, and SOD_Sfru189680 were found to be highly expressed throughout development, which may be related to the high survival ability and resistance to stress seen for S. frugiperda. Insect PO (phenoloxidase) plays an immune role against entomopathogens and promotes melanin synthesis [48]. PO gene expression was high in older larval stages and in the female adult stage. The high expression of PO in adult females contributes to resistant to pathogens, increasing survival likely for successful reproduction. Clip domain serine proteases (CLIPs), characterized by one or more conserved clip domains, are essential components of extracellular signaling cascades in various biological processes, especially in innate immunity and the embryonic development of insects[49]. In both the Cigarette beetle and the silkworm, Clip domain serine proteases are expressed at different developmental stages, with the highest expression during the pupal stage [49, 50]. However, in our study, both CLIP_Sfru162580 and CLIP_Sfru162500 show no expression, while CLIP_Sfru074510 was expressed at low levels in all developmental stages. The Draper gene is involved in the phagocytosis of apoptotic cells and is expressed in hemocytes, the gene is expressed at all developmental stages, reaching its peak during the pupal or adult stage [51]. Similarly, in the fall armyworm, Draper_Sfru041420 and Draper_Sfru008020 are expressed at all developmental stages, with the highest expression levels during the adult stage. dSR-CI (Drosophila Scavenger receptor class C, type I) is a PRR (pattern recognition receptor) that recognizes Gram-negative bacteria; Dscam (Down syndrome cell adhesion molecule-like protein) also recognizes pathogens and mediates phagocytosis, and the gene Pvf affects hemocyte proliferation, CRQ (protein croquemort-like), PER (peroxidase-like), BH4, and other genes have been less studied regarding immunity and development. Our data show that the dSR-CI _Sfru120000, BH4_Sfru093120, Pvf_Sfru003450, Pvf_Sfru068840, CRQ_Sfru117630 and CRQ_Sfru120400 are expressed at moderate to high levels at all developmental stages. Dscam (Down syndrome cell adhesion molecule-like protein) and PER (peroxidase-like) are expressed at relatively low levels at all developmental stages.

As the above discussion shows, although significant effort has been made to examine and understand insect immune responses, most studies have focused on a small subset of pathways components or even a single gene/gene family and/or a limited range of developmental stages. Here, we provide a comprehensive global analysis of immune system gene expression across all of the major developmental stages of S. frugiperda including males and females. These data help explain the remarkable versatility and adaptability of this pest to both climactic/abiotic stress and to attempts to control this pest via chemical and/or biological control methods. Our data highlight that S. frugiperda utilizes discrete but overlapping immune mechanisms across developmental stages, and, in particular, invests significantly more resources in these systems in females as compared to males. Such variation should be taken into account and can be exploited for developing more effective approaches at controlling this insect pest.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Funding

This research was supported by National Natural Science Foundation of China (32160666, 32472644) and the Guizhou Province Science and Technology Support Project ([2022] General 239).

Author Contribution

GW, JR, CJ, LZ prepared the samples, performed the transcriptomic experiment, validation of the data with qRT-PCR, and constructed the figures. AM, WZ, NOK, IE supervised the project, interpreted the results and wrote the manuscript. All authors read and approved the final manuscript.

Data Availability

Sequence data that support the findings of this study have been deposited to NCBI under the bioproject number PRJNA1145502.

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Comparative gene expression analyses reveal developmental stage dependent immune adaptations of Spodoptera frugiperda

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Abstract

Figures

Introduction

Materials and methods

Insect rearing and sample collection

Sample collection, RNA extraction and sequencing

Transcriptome assembly, annotation and analysis

Quantitative Real-Time PCR (RT-qPCR)

Results

Overview of immune related genes and their expression trends

Humoral immunity

Recognition

Toll signaling pathway genes

Imd signaling pathway genes

Effectors

Serine proteases

Serine protease inhibitors

Cellular immune gene expression patterns: Hemolymph coagulation, clot formation and wound healing

Hematopoiesis

Hemocyte-mediated immune responses

qRT-PCR gene validation

Discussion

Declarations

Funding

Author Contribution

Data Availability

References

Additional Declarations

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