Authors: Tangxin Li, Yue Song, Quan Sun, Qiong Yang, Yunlin Tang, Zigang Shen, Zhangshuai He, Yabin Dou, Junzheng Zhang, Sheng Li, Tian Li, Xianzhi Meng, Zeyang Zhou, Jie Chen, Guoqing Pan
Categories: Research, Bombyx mori, Microsporidia, Nosema bombycis, Congenital infection, Immune response, Transcriptome
Source: BMC Genomics
Authors: Tangxin Li, Yue Song, Quan Sun, Qiong Yang, Yunlin Tang, Zigang Shen, Zhangshuai He, Yabin Dou, Junzheng Zhang, Sheng Li, Tian Li, Xianzhi Meng, Zeyang Zhou, Jie Chen, Guoqing Pan
Microsporidia are a group of intracellular and unicellular eukaryotic parasites, which can nearly infect all animals, including human beings. As the first identified microsporidia, Nosema bombycis is a world-wide threat for silkworm eggs production, it can cause the congenital infection via transovarial transmission. It is important for pathogenesis elucidation to unravel the molecular characteristics of N. bombycis proliferation and host immune responses to the congenital infection in embryo and larva stage. Here, we adopted dual RNA-seq approach to investigate and compare the dynamic molecular pattern of pathogen proliferation and host immune responses between diapause and non-diapause silkworm eggs. Our results showed the N. bombycis proliferation in non-diapause silkworm eggs is a continuous process, many parasites enter the sporogony stage at 2 days post-oviposition (dpo). For newly hatched larva (1 dph), the abundance of pathogen mRNA sequences is up to 2.32% in non-diapause strain, far higher than 0.34% of diapause strain, the main reason is the hot HCl bath treatment at 24 h post-oviposition for diapause silkworm eggs with the aim to free the egg diapause. As to immune responses, whatever for diapause strain or non-diapause strain, there is stronger immune responses to congenital infection in larva stage than that of embryo stage, however, the host immune responses to congenital infection are fairly different between non-diapause and diapause strains of silkworms, especially in embryo stage. We found the surprising “First day Chaos” that there are 6,071 differential expressed genes (DEGs) at 1 dpo for non-diapause strain between infection group and uninfected group, but decreases dramatically to 109 DEGs at 2 dpo. In non-diapause strain, the earliest DEGs of antimicrobial peptides were up-regulated at 1 dpo, then is 5 dpo with up-regulated lebocin, 7 dpo with morLP-B1,* morLP-B4.* For non-diapause strain, the well-established immune responses were observed in newly hatched larvae. On the contrast, for diapause strain, the earliest DEGs of AMPs appear at 5 dph, the mature immune responses are well established at 5 dph too. In non-diapause silkworms, we observed obvious pathogen’s regulation in the main immune pathways including Toll, IMD, JAK-STAT and melanization at the different steps such as immune recognition, signal modulation and transduction, effectors. Taken together, our results for the first time provide a global molecular view of microsporidia proliferation and innate immunity responses in a congenital infection system and provide some new insights into immune development and establishment in the embryo and early larva stage of Bombyx mori.
The online version contains supplementary material available at 10.1186/s12864-025-11762-z.
Regarded as “the Master Parasites” [1], Microsporidia are a group of fungi-related obligate intracellular and opportunistic parasites that infect a broad range of hosts, even immunocompromised humans [2–4]. To date, more than 1,700 species belonging to over 200 genera have been identified [5]. There are two modes of transmission of microsporidia, one is horizontal transmission and the other is vertical transmission [6, 7]. Vertical transmission is common in insect microsporidiosis. The insect microsporidia can reduce its virulence and can be transmitted to the next generation without affecting the ovarian development of the female host. Antonospora locustae infects embryonic tissue through vertical transmission, resulting in low hatching rate and high embryo mortality [7]. Vertical transmission of the Nosema fumiferanae can result in congenital infection that delay the hatching of Choristoneura fumiferana larvae [8]. As the first reported Microsporidia, Nosema bombycis is a major pathogen that causes a highly fatal silkworm disease, pébrine [9, 10]. The infection of N. bombycis in silkworm ovaries results in the parasite transovarial transmission, which causes congenital infection in silkworm embryos and larvae [11]. The congenital infection system composed of N. bombycis and silkworms becomes a good model to explore the characteristics of pathogen proliferation and host immune responses. In our previous work, Song et al. found that congenital N. bombycis proliferated mainly around yolk granules and in the intestinal lumen during the development of silkworm embryo, meanwhile, a small amount of infection presented in embryonic tissues [12]. Recently, Shen et al. investigated molecular proliferation in congenital infected embryo and larva of non-diapause silkworms, however, very limited data of only one time point (5 dpo) of embryo was obtained [13], the dynamic molecular proliferation characteristics of N. bombycis during the whole process of embryo development are not known yet.
In addition to N. bombycis proliferation pattern in congenital infection, the host immune responses are very vital to understand the pathogenesis of pébrine disease. Despite the lack of adaptive immunity, invertebrates have an efficient innate immunity to recognize and eliminate invading pathogens [14, 15]. Innate immunity, including humoral and cellular responses, is activated during pathogen invasion and infection inside the host [16–19]. Generally, innate immunity in insects mainly includes the Toll pathway, immune deficiency pathway (IMD pathway), Janus kinase-signal transducer and activator of transcription signaling pathway (JAK-STAT signaling pathway), and melanization cascade, etc [18]. Considering its economic value and the availability of genetic information [20], B. mori has become a valuable and well-characterized model system in Lepidoptera for studying insect innate immunity [21–23]. In 2013, Ma et al. found that N. bombycis oral infection induced a strong and complicated host response in silkworm larvae. Analysis of immune-related genes showed that the Toll pathway, JAK-STAT pathway, cellular immunity, and ROS response were induced, while the melanization of silkworm was inhibited [24]. For N. bombycis congenital infected silkworms, Shen et al. found that the expression of immune related genes, such as βGRP 2, Spz 3 and pro-phenol oxidase, decreased in 5-day embryos with N. bombycis congenital infection compared with uninfected samples. While most immune genes, such as peptidoglycan recognition protein like (PGRP-L), Toll-like receptor 3 and antimicrobial peptide genes, were up-regulated in larvae exposed to the congenital N. bombycis challenge compared with uninfected samples [25]. However, also because the limited data of only one time point (5 dpo) in the embryo immune responses in congenital infection are available, it is necessary to explore the dynamic host immune responses during the whole process of embryo development. What’s more, although studies on B. mori innate immunity have progressed dramatically in post-genomics era, it remains unclear when silkworm establishes an immunocompetent immune system during the embryo development and how it responds to pathogen congenital infection.
Thus, here we collected the silkworm eggs and larvae with Nosema bombycis congenital infection, dual RNA-seq was adopted to explore and compare the pathogen proliferation characteristics and host immune responses of the diapause and non-diapause silkworm embryos and larvae, we are also looking forward to investigate the temporal and spatial clues of innate immune system development and establishment in Bombyx mori.
N. bombycis isolate CQ1 (Chongqing, China) was purified form infected silkworms and conserved in the China Veterinary Culture Collection Center (CVCC No. 102059). Spores were isolated from silkworm pupae that were challenged at the fourth instar stage by oral infection (approximately 10^4^ spores per larvae) [26].
The silkworms of strain Chun 5 (diapause silkworm) and 305 (non-diapause silkworm), were reared at 26℃ under natural lighting in a dedicated room. Fifth instar larvae were challenged with mature spores by oral inoculation (approximately 10^6^ spores per larva). Surviving pupae closed at approximately 17 days post inoculation. Approximately 2 h post mating, the majority of moths completed oviposition and the eggs were collected and mixed immediately as pooled samples. Eggs from infected females were transferred to a sterile climate incubator at 28 ℃. Eggs were collected from uninfected moths and were prepared under the same conditions.
For diapause silkworm (strain: Chun 5), deep RNA sequencing from infected and uninfected eggs was performed at 1 day post-oviposition (dpo), 26 h post-oviposition (hpo), and 2, 4, 6, 8 dpo and 1 days post-hatched (dph) from the same pooled samples. Those above newly hatched larvae (1 dph) were collected directly without feeding mulberry leaves. Silkworm larvae at 5 dph and 10 dph from another batch of congenitally infected pooled samples were collected as larva stages samples. Approximately 24 hpo, eggs were treated with an HCl solution (specific gravity 1.075) at 46℃ for 5 min to prevent eggs from diapausing [27, 28]. In our study, samples at 1 dpo represented eggs without HCl solution treatment, and samples at 26 hpo represented eggs treated with HCl solution for 5 min at 24 h and collected 2 h later. Infected and uninfected samples were treated identically.
For non-diapause silkworm (strain: 305), the samples were collected at 1, 2, 3, 5, 7 dpo and 1, 2, 3 dph from the same pooled samples. Newly hatched larvae (1 dph) were collected immediately with no feeding of mulberry leaves. All samples were treated with liquid nitrogen and stored at −80℃. Three replicates were included at each time point, and each sample included 50 eggs or larvae.
Illumina RNA-seq for diapause silkworm was conducted by the Biomarker Technology Company, Beijing, China. And Illumina RNA-seq for non-diapause silkworm was conducted by the GeneDenovo Technology Company, Guangzhou, China. Total RNA was prepared from a mixture of 50 silkworm eggs or 50 larvae from each time point with three biological repeats using TRIzol^®^ reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. RNA purity and integrity, the generation of sequencing libraries and the clustering of index-coded samples were performed as previously described [29]. After total RNA was extracted, the host and parasite mRNA were enriched by Oligo(dT) beads. Then the enriched mRNAs were fragmented into short fragments and reverse transcribed into cDNAs with random primers. Subsequently, the cDNA fragments were purified, end repaired, poly(A) added, and ligated to Illumina sequencing adapters. The ligation products were sequenced using Illumina HiSeq2500 platform and paired-end reads were generated. The rRNA removed reads of each sample were then individually mapped to the reference genome by HISAT2 (version 2. 4) [30]. N. bombycis reference genome is download from https://silkpathdb.swu.edu.cn/ [20]. B. mori reference genome is download from https://silkdb.bioinfotoolkits.net [31]. The alignment parameters (1) Maximum read mismatch is 2; (2) Maximum distance between mate-pair reads is 50 bp; (3) The error of distance between mate-pair reads is ± 80 bp. Gene abundances were quantified by software StringTie (version 1.3.1) [32, 33] and normalized by using FPKM (Fragments Per Kilobase of transcript per Million mapped reads.\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ ::\text{F}\text{P}\text{K}\text{M}=\frac{10E6C}{NL/10E3}
The sequence data generated in this study have been submitted to the NCBI Sequence Read Archive ([https://www.ncbi.nlm.nih.gov/sra](https://www.ncbi.nlm.nih.gov/sra)) with the accession number PRJNA549766 (for diapause silkworms) and PRJNA1215458 (for non-diapause silkworms). The expression matrix data can be accessed with DOI 10.6084/m9.figshare.29126282.v1 (for diapause silkworm) and 10.6084/m9.figshare.29126300.v1 (for non-diapause silkworm) in figshare database. ### cDNA preparation and qRT-PCR detection of differential expression genes The RNA samples of non-diapause silkworm returned from the GeneDenovo Technology Company were reverse-transcribed by Hifair^®^II 1 st Strand cDNA Synthesis SuperMix for qRT-PCR (Yeasen Biotech Co., Ltd) to get the same concentration of cDNA. The obtained cDNA samples were used as a template for qRT-PCR. qRT-PCR were done by using Hieff^®^ qPCR SYBR^®^ Green Master Mix (Yeasen Biotech Co., Ltd), and the primers are listed in Table S1. ### Data analysis methods RNAs differential expression analysis was performed by DESeq2 [34] software between two different groups. The genes with the parameter of false discovery rate (FDR) below 0.05 and absolute fold change ≥ 2 were considered differentially expressed genes. Principal component analysis (PCA) was performed with R package gmodels ([http://www.r-project.org/](http://www.r-project.org/)) in this experience. PCA is a statistical procedure that converts hundreds of thousands of correlated variables (gene expression) into a set of values of linearly uncorrelated variables called principal components. PCA is largely used to reveal the relationship of the samples. All the heatmaps were done with TBtools [35], all phylogenetic tree were done with MEGA11. All the protein sequences of *N. bombycis* were downloaded from NCBI ([https://www.ncbi.nlm.nih.gov/datasets/taxonomy/27978/](https://www.ncbi.nlm.nih.gov/datasets/taxonomy/27978/)) and all the protein sequences of *B. mori* were downloaded from SilkDB 3.0 ([https://silkdb.bioinfotoolkits.net/main/species-info/−1](https://silkdb.bioinfotoolkits.net/main/species-info/-1)). Signal peptides prediction were all done by SignalP 6.0 ([https://services.healthtech.dtu.dk/services/SignalP-6.0/](https://services.healthtech.dtu.dk/services/SignalP-6.0/)). ## Results ### Molecular proliferation characteristics of *N. bombycis* during the development process of non-diapause silkworm eggs with congenital infection To characterize parasite proliferation in non-diapause silkworm embryos and larvae from a molecular viewpoint, we obtained 645.02 Gb Clean Data from the non-diapause silkworm eggs and larvae, with 0.51–5.20% of these reads mapped to the *N. bombycis* genome (Fig. 1A), totally, we get 4,413 genes expressed of the 4,486 annotated genes in *N. bombycis*. For diapause strain Chun 5, 300.42 Gb Clean Data from the infected eggs were obtained, with 0.07–0.89% of these reads mapped to the *N. bombycis* genome [12] (Fig. 1B). It is obvious that for the diapause eggs, the 24 hrs’ hot HCl treatment to lift the diapause has a big impact on the proliferation of *N. bombycis* inside the eggs, there was a sharp down at 2 dpo for the detected mRNA of *N. bombycis*. Hot HCl bath treatment killed many parasites in congenital infected eggs, finally contribute the big difference in pathogen load in newly hatched larva between the non-diapause strain 305 and the diapause strain Chun 5, similar results also were verified by qRT-PCR in 2019 [36]. Fig. 1The mapped rate of *N. bombycis* reads to its genome in the transcriptome data of non-diapause silkworms (305) and diapause silkworms (Chun 5). dpo, days post oviposition; hpo, hours post-oviposition; dph, days post-hatched Then, we performed principal component analysis (PCA) of *N. bombycis* in non-diapause silkworm eggs based on the original RNA-seq data (Fig. 2A). The samples of 2 dpo, 3 dpo, 5 dpo, 7 dpo and 1 dph gather into a big cluster, among them, samples of 5 dpo, 7 dpo and 1 dph form a small and closer cluster. The samples of 1 dpo, 2 dph and 3 dph are isolated from each other. *Histone H4* and *Spore wall protein 1* were the signature genes for merongony and sporogony stages respectively in the proliferation of *N. bombycis*, the expression level of *Histone H4* was relatively high and stable, which suggest the pathogen proliferation rate was rapid. The expression level of *SWP 1* increased sharply at 2 dpo, then decreased a little, and reached a peak at 3 dph (Fig. 2B), *SWP 1* become the top 1 highest transcribed gene except the 2 dph as top 2 gene, which suggest the more moronts start to enter the sporogony state from 2 dpo. In addition, we listed the number of DEG sets at each time point of *N. bombycis* (Fig. 2C). We found that there were more up-regulated genes at 2 dpo-vs-3 dpo and 3 dpo-vs-5 dpo, and more genes were down-regulated in the larval stage. Fig. 2Pathogenic transcripts dynamics in the transcriptome analysis of *N. bombycis* infected non-diapause silkworm embryos and larvae. **A** Principal component analysis of *N. bombycis* RNA-Seq data. **B** The transcription of *Histone H4* (lower blue line chart) and spore wall protein *SWP 1* (upper orange line chart) of *N. bombycis*. The microsporidium in silkworms began to develop spore walls and mature progressively from 2 dpo. **C** Numbers of significantly changed *N. bombycis* transcripts. dpo, days post oviposition; hpo, hours post-oviposition; dph, days post-hatched To explore transcriptional signatures of the life cycles of *N. bombycis*, we analyzed the top 100 highly expressed genes of *N. bombycis* from congenitally infected non-diapause silkworm samples (S1 Dataset). According to Venn diagrams from each time point, we identified 41 highly expressed core genes (Fig. 3A) including eleven ribosomal proteins, ten histone proteins, four hypothetical proteins, three tubulin proteins, three actin proteins and ten genes related to essential elements of cell biology, involved in transcription and translation, cellular component, protein transport. The identification of genes related to ribosome synthesis, DNA replication and translation factors showed *N. bombycis* rapid proliferation in the embryo. After removing *hypothetical proteins*,* ribosomal proteins* and *histone* related genes from the top 100 gene sets of all time points, we conducted continuous statistics on the transcription of *SWPs* (*spore wall proteins*) [37], *PTPs* (*polar tube proteins*) [38] and *tubulin proteins* in each gene set (Fig. 3B). At 1 dpo, cellular component *ycf1* (*yeast cadmium factor 1*) [39] was the highest transcribed gene, however *SWP 1* become the highest transcribed gene in the following time points except in 2 dph as the second highest transcribed gene. In the data of 1 dpo, we did not find *SWP 1* in Top 100 highly expressed genes, but ranked 1083 th. What’s more, we did not find *PTP 1*,* PTP 2*,* PTP 3* in 1 dpo data either. Above results suggest that most of parasites in 1 dpo just start their meront proliferation. However, from 2 dpo, more and more parasites enter into sporogony stage with the high expressed marker gene *SWP 1*. Fig. 3Consistently high expression genes in *N. bombycis* during proliferation in non-diapause silkworms. **A** The transcription profiles of 41 genes which were among the top 100 highly expressed gene sets at each time point. Each column corresponds to a time-point, while each row represents an individual gene. **B** Scatter plot showing the top 100 highly expressed gene sets of *N. bombycis*, excluding *histone proteins*, *ribosomal proteins* and *hypothetical proteins*. Red dots are *SWPs*, blue dots are *PTPs*, green dots are *Tubulins* and black dots are other genes in this sets For the sampling time between non-diapause silkworm 305 and diapause silkworm Chun 5, they share three common time points, 1 dpo, 2 dpo and 1 dph, which provide us an opportunity to compare *N. bombycis* proliferation tendency between them. For the 1 dpo, we did not observe the marker genes of sporogony, such as *SWP 1*,* PTP 1*,* PTP 2*,* PTP 3* in Top 100 highly expressed genes neither in diapause nor non-diapause samples, which suggest that for 1 dpo, the main parasites were in meront stage inside diapause and non-diapause eggs, which is in consistent with the IFA (indirect immunefluorescence assay) results. The IFA results revealed that only a few scattered early development stages *N. bombycis* were situated around the yolk granules and germ band at 1 dpo, with no mature spores being found [12]. For 2 dpo, in non-diapause samples, sporogony marker genes, *SWP 1*,* SWP 2*,* PTP 1*,* PTP 2* have been listed in Top 100 highly expressed genes, however for diapause samples, these genes were not listed in Top 100 highly expressed genes, for examples, *SWP 1* was ranked 709 th, two copies of *PTP 1*,* NBO_79 g0015*,* NBO_943 g004* are ranked 785 th and 1,861 th respectively. These above results suggest Hot HCl treatment at 24 h (1 dpo) killed many parasites in the diapause silkworm eggs, Wang et al. also verified the similar killing effect by qPCR method [36]. At 1 dph, for the *N. bombycis* Top 100 highly expressed genes, the diapause and non-diapause samples share 58 genes including *SWP 1*,* SWP 2*,* PTP 1*,* PTP 2*, which indicates that the parasite proliferation tendency is similar in newly hatched larvae of diapause and non-diapause silkworms. ### The expression characteristics of *N. bombycis* secreteome during the process of non-diapause silkworm egg development As the intracellular parasite, *N. bombycis* secreteome plays an important role in interaction between pathogen and host, especially some secreted effectors behaving in host manipulation. Here, a novel secreteome of *N. bombycis* was collected by SignalP6.0 prediction. Totally, 277 of 4,486 annotated proteins are predicted with signal peptides (Table S2). 264 of 277 secreteome genes were detected with transcription, 194 of 264 coding genes were annotated as hypothetical proteins (Fig. 4A). 66 annotated genes were detected expression at each time-points (Fig. 4B). We also listed the number of DEG sets of secreteome genes between two adjacent time points (Fig. 4C). Among these genes with signal peptides, a part of genes are coding some structural proteins, such as *PTP 1 ~ 3*,* SWP 1*,* SWP 5*,* ycf1*, most of them are belonging to highly expressed genes. What’ more, there are some enzymes with signal peptides, such as *peptidase* (NBO_65 g0001), *threonyl-tRNA synthetase* (NBO_76 gi004), *Isoleucyl-tRNA synthetase* (NBO_71 g0001), *Hexokinase-2* (NBO_1320 g0001), *Trehalase* (NBO_10 g0113), *polysaccharide deactylase* (NBO_53 g0005), *Acidic endochitinase sp2* (NBO_41 g0043), *Thioredoxin* (NBO_10 g0070). Here we carried out comparative genomics of *Thioredoxin* in microsporidia and found that *Thioredoxin* (with the signal peptide) is conserved in nearly all microsporidia, all orthologs in other microsporidia are with the predicted signal peptides (Fig. S1). Specifically, the thioredoxin in *N. bombycis* may be secreted and modulate the host’s redox balance, and create favorable conditions for the pathogen’s survival and proliferation [40, 41]. Thus, microsporidia thioredoxin’s localization and function are worthy of being investigated further. Now, we have confirmed that the *N. bombycis* secreted effectors, such as serpin 6 involved in host immunity regulation, serpin 14 inhibiting host cell apoptosis [42, 43]. It is believed that there are more secreted effectors in the secreteome that would be the arsenals of *N. bombycis*. Fig. 4Secretome characteristics of *N. bombycis* in non-diapause silkworms. **A** The KEGG pathway of 264 *N. bombycis* secreted protein genes predicted by SignalP 6.0 The global and overview maps contain a high-level comprehensive presentation of key metabolic pathways and functions in living organisms, including antibiotics biosynthesis, secondary metabolites biosynthesis, carbon metabolism, general metabolic processes and microbial metabolism across diverse environments. **B** Clustering analysis of 66 core annotation genes with predicted signal peptide throughout the entire embryonic development. Each column corresponds to a time-point, and each row represents an individual gene. Gene expression variations at different time points are quantified relative to the 1 day post-oviposition (dpo) using log2 fold change (Log2 FC). **C** The numbers of DEGs which were predicted with signal peptide. dpo, days post oviposition; hpo, hours post-oviposition; dph, days post-hatch. **D** Clustering analysis of *N. bombycis* serine protease inhibitors *(Nbserpins)*. Genes highlighted in red are predicted to encode signal peptides. The color gradient transitioning from white to red signifies a progressive increase in gene transcript levels. Each column represents a time-point, and each row represents a gene Serpin family is very unique in *Nosema* genus, 19 *NbSerpin* members were annotated in the *N. bombycis* genome [44]. Among them, ten were predicted with the signal peptide. Here, the transcription of *NbSerpins* were analyzed (Fig. 4D, Table S6). On the whole, the expression of *NbSerpins* in silkworm eggs were relatively low, and the transcription of *serpin 9* and *serpin 14* were not detected. *Serpin 2*, *serpin 3*, *serpin 4*, *serpin 6* and *serpin 10* were expressed relatively higher than other *serpins*, coincidentally, these five *serpins* are all with predicted signal peptides. Meanwhile, these results are very similar to the *Nbserpins* expressions in diapause silkworm samples [12]. At 1 dpo, the transcription of *Serpin 2*, *serpin 8*, *serpin 12*,* serpin 15*,* serpin 16*, *serpin 18* and *serpin 19* was not detected, *serpin 4*,* serpin 5*,* serpin 6*,* serpin 10* were expressed relatively higher. For the newly hatched larva at 1 dph (9 dpo), only *serpin 7* transcription was not detected. The function of *NbSerpins* family during the embryo stage still need more verification in future. ### Differential expression genes screening in host responses to microsporidia congenital infection In this study, we extracted total RNA from three replicates of uninfected and infected silkworm eggs or larvae at different time points. For diapause silkworm strain, nine time-points including 1 dpo, 26 hpo, 2, 4, 6, 8 dpo in embryo stages, 1 dph (9 dpo) and 5 dph, 10 dph of larva stages were covered. For non-diapause silkworm eggs or larvae, eight time-points (1, 2, 3, 5, 7 dpo and 1, 2, 3 dph) were designed. Then we utilized the HiSeq Illumina platform to obtain a global and continuous profile of transcriptome related to embryo and larva response to *N. bombycis* congenital infection. After the sequence analysis, we obtained 684.03 Gb Clean data from diapause silkworm and 645.02 Gb Clean Data from non-diapause silkworm. All of the clean reads were mapped with *B. mori* reference genome, the individual mapped rate from all samples was ranged from 54.11 to 86.05% in diapause silkworms (Table S3) and from 79.43 to 87.72% in non-diapause silkworms (Table S4). In addition, we also obtained information for 13,727 transcribed genes (93.8%) in diapause silkworm and 14,372 transcribed genes (98.28%) in non-diapause silkworm, compared with the total 14,623 annotated genes in *B. mori*. Pairwise correlation coefficients (Fig. 5A) and principal component analysis (Fig. 5B) of transcripts across all diapause silkworm egg samples illustrated that the reproducibility between samples was high. For both diapause silkworms and non-diapause silkworms, both uninfected groups and infected groups at the same time-point were clustered together (Fig. 5C), suggesting that the congenital infection of *N. bombycis* did not significantly impact embryonic development in silkworm. For non-diapause silkworm data, we chose 32 differentially expressed genes randomly for relative qRT-PCR to verify the accuracy of data. The qRT-PCR results of 25 genes were consistent with the trend of transcriptome differences (Fig. 5D), which proved that the differential genes screened by RNA-seq data were reliable. For the accuracy of diapause silkworm data, it had been verified by our previous work [12]. Fig. 5Transcriptome characterization of silkworm congenitally infected with *N. bombycis*. **A**, **B** Pairwise correlation coefficients (A) and principal component analysis (B) of diapause silkworm embryos with *N. bombycis* congenital infection. **C** Principal component analysis of non-diapause silkworm embryos and larvae with *N. bombycis* congenital infection. Un, healthy silkworm; In, *N. bombycis* infected silkworm. dpo, days post oviposition; hpo, hours post-oviposition; dph, days post-hatched. **D** Comparison of qRT-PCR results with transcriptome expression trends Analysis of DEGs (FDR ≤ 0.05, Fold change ≥ 2, S2 Dataset) at different host stages provides a number of potential clues to host stage-specific response against the *N. bombycis* congenital infection (Table 1, Fig. S2). In general, the congenital infection of *N. bombycis* in diapause silkworm induces a weak embryo response with a total of 527 DEGs, including 342 up-regulated and 145 down-regulated in embryo stages (6 time-points), compared to a strong larva response with a total of 2,671 DEGs including 1,171 up-regulated and 1,500 down-regulated in larva stages (3 time-points) (Database S3). For non-diapause silkworm, we found 7,311 DEGs from eggs and 7,431 DEGs from larvae (Database S4). The surprising result is that there were so many DEGs (3,447 up-regulated and 2,624 down-regulated) at 1 dpo, we call it “First day Chaos”, the reason is not still known. We repeated the RNA-seq with same samples of 1 dpo, including the infected and uninfected sample, we obtained similar results. In the remaining seven time points of the DEGs, 372 genes were up-regulated and 868 genes were down-regulated in all four time points of embryo stages, 1,827 genes up-regulated and 5,604 genes were down-regulated in all three time-points of larva stages. Table 1The number of DEGs in diapause silkworms and non-diapause silkwormsA. The number of DEGs in diapause silkworms (FDR ≤ 0.05, Fold change ≥ 2)1 dpo26 hpo2 dpo4 dpo6 dpo8 dpo1 dph5 dph10 dphUp172535261423979169Down2661142831317907576Total4331419306417401886745B. The number of DEGs in non-diapause silkworms (FDR ≤ 0.05, Fold change ≥ 2)1 dpo2 dpo3 dpo5 dpo7 dpo1 dph2 dph3 dphUp3,447117151191214172341,176Down2,624721262743964061,6523,546Total6,0711891413935178231,8864,722 Due to limited length of this paper, it is difficult to cover all host dynamic responses such as cell division, tissue and organ development, host metabolism and other responses according to the DEGs collected. Here we mainly analyze the host immune responses to the congenital infection in diapause and non-diapause silkworms. Besides the analysis of DEGs, we have screened most innate immunity-related genes involved in pathogen recognition (Fig. 6), classic immune pathway (Figs. 7, 8, 9 and 10) and AMPs (Fig. 11) in embryo and larva stages. The numbers of DEGs related to immune response in diapause silkworms and non-diapause silkworms were listed in Table 2, IDs of genes involved in immune response were listed in Table S5. Fig. 6The transcriptional dynamics and differential expression of recognition protein genes were investigated in non-diapause (left) and diapause (right) silkworms. A positive sign (+) indicates significant up-regulation, whereas a negative sign (-) indicates significant down-regulation. Each column in the heatmap corresponds to a time-point, and each row represents a gene. dpo, days post oviposition; hpo, hours post-oviposition; dph, days post-hatched Fig. 7The transcriptional dynamics and differential expression analysis of Toll pathway related genes in non-diapause (left) and diapause (right) silkworms. The positive sign (+) means up-regulated significantly and the negative sign (-) means down-regulated significantly. Each column represents a time-point, each row represents a gene. dpo, days post oviposition; hpo, hours post-oviposition; dph, days post-hatched Fig. 8The transcriptional dynamics and differential expression analysis of IMD pathway related genes in non-diapause (left) and diapause (right) silkworms. The positive sign (+) means up-regulated significantly and the negative sign (-) means down-regulated significantly. Each column represents a time-point, each row represents a gene. dpo, days post oviposition; hpo, hours post-oviposition; dph, days post-hatched Fig. 9The transcriptional dynamics and differential expression analysis of JAK-STAT related pathway genes in non-diapause (left) and diapause (right) silkworms. The positive sign (+) means up-regulated significantly and the negative sign (-) means down-regulated significantly. Each column represents a time-point, each row represents a gene. dpo, days post oviposition; hpo, hours post-oviposition; dph, days post-hatched Fig. 10The transcriptional dynamics and differential expression analysis of melanization related genes in non-diapause (left) and diapause (right) silkworms. The positive sign (+) means up-regulated significantly and the negative sign (-) means down-regulated significantly. Each column represents a time-point, each row represents a gene. dpo, days post oviposition; hpo, hours post-oviposition; dph, days post-hatched Fig. 11The transcriptional dynamics and differential expression analysis of antimicrobial peptides genes in non-diapause (left) and diapause (right) silkworms. The positive sign (+) means up-regulated significantly and the negative sign (-) means down-regulated significantly. Each column represents a time-point, each row represents a gene. dpo, days post oviposition; hpo, hours post-oviposition; dph, days post-hatched Table 2The number of DEGs involved in immune response in diapause and non-diapause silkwormsA. The number of DEGs involved in immune response in diapause silkworms (FDR ≤ 0.05, Fold change ≥ 2)1 dpo26 hpo2 dpo4 dpo6 dpo8 dpo1 dph5 dph10 dphUp0500001382Down23000101717Total28000115519B. The number of DEGs involved in immune response in non-diapause silkworms (FDR ≤ 0.05, Fold change ≥ 2)1 dpo2 dpo3 dpo5 dpo7 dpo1 dph2 dph3 dphUp410079221520Down26432631851Total6743915253371 ### Comparison of immune responses in embryo stage between diapause and non-diapause silkworms Based on the heat map, most of the immune-related genes in the diapause silkworms were expressed at a low level in embryo stages, among them, only a few genes that belonged to DEGs were significantly changed. For immune recognition genes, *βGRP 1* (*β*-*Glucan recognition protein 1*) [21, 45] was down-regulated at 1 dpo, *CTL 16* (*C-type lectin 16*) [46] and *SCRC* (*Scavenger receptor-C*) [21] were down-regulated at 26 hpo. *CTL 15* was up-regulated at 8 dpo in diapause silkworm eggs (Fig. 6). For non-diapause silkworms (Fig. 6), a number of recognition genes were up-regulated at 1 dpo, such as *PGRP-L5* (*peptidoglycan recognition protein-L5*) [47], *PGRP-S2*,* CTL 2/7/9/10/13/15/16/19*,* SCRB 1/5/6/8/9/13* and *SCRC*. Meanwhile, the *βGRP 1/3*,* PGRP-L6*,* PGRP-S1*,*CTL 4/5*,* SCRAC 1*,* SCRB 3* and *SCRB 12* were down-regulated at 1 dpo. Different from diapause silkworms, more recognition genes were significantly differentially expressed in the embryonic stage of non-diapause silkworms. However, at 2 dpo, only three genes were down-regulated, respectively were *βGRP 2*, *CTL 5*, *SCRB 7*. At 3 dpo, *CTL 5*, *SCRB 8* were down-regulated. There were no up-regulated DEGs observed at 2 dpo and 3 dpo. At 5 dpo, *PGRP-L1*, *SCRB 13* were up-regulated while *PGRP-S2* and *CTL 20* were down-regulated. In silkworm, PGRP-S2 had been reported to be involved in the activation of IMD pathway [48], and PGRP-L1 has no interaction with IMD [49]. At 7 dpo, *PGRP-S1*was up-regulated, it had been reported that PGRP-S1 can bind to PGN resulting the activation of the PPO cascade [47]. More analyses of *PGRPs*,* CTLs* and *SCRs* with signal peptides and domain information were showed in Fig. S3, S4 and S5. After the pathogens were recognized by pattern recognition receptors (PRRs), it would be presented to the signaling molecules of the immune pathways. For diapause silkworms (Fig. 7), there were no significantly expressed genes in Toll pathway in the embryonic stage. Notably, a universal adapter protein used by almost all *tolls*, *Myd88*, was not expressed at embryonic stages. For non-diapause silkworms (Fig. 7), at 1 dpo, *Spz 1* (*Spätzle 1*) [50], *Spz 2*,* Toll receptor 8/9 − 2/10 − 2/10 − 3* [23, 50] and *Pelle* were up-regulated, only *Toll 3–3* and *TRAF 2* were down-regulated. For the rest time points of the embryonic stage, only *Spz 3* was significantly down-regulated at 7 dpo. It is interesting that in non-diapause silkworms, *Myd88* expression was detected in all time points but at a relatively low level. In diapause silkworms, genes of IMD pathway were not induced significantly yet in the embryonic stage (Fig. 8). In non-diapause silkworms (Fig. 8), there were five DEGs (*Fadd* was up-regulated, *IAP 2*,* Tak 1*,* ikk β* and *Relish* were down-regulated) in the embryonic stage, they were all belong to 1 dpo DEGs sets. For JAK-STAT pathway (Fig. 9), there were no DEGs in diapause silkworm in the embryos, and two down-regulated DEGs (*Socs* and *Fos*) in non-diapause silkworms at 1 dpo, however, no DEG in the following time points in embryonic stage. As to the PPO pathway, because now most CLIPs and SPNs function are not identified with detail, here we temporarily classify all *CLIPs* and *SPNs* into PPO pathway though some CLIPs are also behave in Toll and other pathways [51]. For diapause silkworms, only *SPN 24* was down-regulated at 1 dpo and *SPN 20* was down-regulated at 2 dpo, *CLIP 2/4/8/9* were down-regulated at 2 dpo (Fig. 10). In non-diapause silkworm (Fig. 10), at 1 dpo, *CLIP 1/3/5/15* were up-regulated, *CLIP 2/4/9* were down regulated. *SPN 1/18/20/21/24* were up-regulated, *SPN 3/5/6/7/22/26* were down regulated. We also found *PPAE* was up-regulated in 1 dpo. A putative defense protein precursor named *Reeler 1* (BGIBMGA014360) was listed as up-regulated DEG at 1 dpo, which was verified as the reeler protein involved in regulating the PPO activity in PPO pathway [52]. After 1 dpo, DEGs decreased a lot, there was only one DEG, *SPN 9* (down-regulated) at 2 dpo, *CLIP 4* was down regulated at 3 dpo, *CLIP 7*, *CLIP 15*, *Lysozyme* and *Reeler 1* were up-regulated at 5 dpo. At 7 dpo, there are three up-regulated gene (*SPN 22*,* PPO 1*,* PPO 2*) and five down-regulated genes (*CLIP 5/6/14*,* SPN 18/21*). More analyses of *CLIPs* and *SPNs* with signal peptides and domain information were showed in Fig. S6 and S7. As to the antimicrobial peptides (Fig. 11), there was no AMP gene listed as DEG in diapause silkworms. On the contrary, in non-diapause silkworms, the earliest AMPs listed in DEGs were up-regulated *gloverin 2*,* gloverin 3*,* gloverin 4* and *defensin* at 1 dpo. *Lebocin* was up-regulated at 5 dpo. At 7 dpo, *morLP-B1*, *morLP-B4* were up-regulated. Although some genes exhibited with big changes, but not listed as DEGs because FDR value was not ≤ 0.01. ### Comparison of immune responses in larva stage between diapause and non-diapause silkworms #### Immune responses to congenital infection in larva stage of diapause silkworms For diapause silkworms, compared with embryonic stage, it was evident that immune responses of larvae were induced strongly by *N. bombycis* congenital infection, mainly reflected by the significant expression changes of host immune-related genes. At 1 dph, there was only one DEG, *Reeler 1* involved in regulating the PPO activity in PPO pathway. Although the expression of *cecropin A1*/*3*, *cecropin B3*, and *gloverin 2* were higher in the infected group than the expression in the uninfected group but not significantly different (Fig. 11), suggesting the congenital infection of *N. bombycis* induced the innate immunity. By contrast, as shown in the heat map at 5 dph, a dozen of recognition related genes such as *βGRP 2*, *PGRP-S1*/*S3*/*S5*/*S6*, *CTL 11*, *CTL 19*,* SCRAL1*,* SCRB 6*/*7*/*10*/*13* were up-regulated, but *CTL 1*/*2*/*4*/*12*/*16*/*18* and *SCRASP 4* were down-regulated (Fig. 6). In the Toll pathway (Fig. 7), *Spz 2/3/4/6* were down-regulated but only *Spz 4/6* were listed as DEGs, *Toll 9 − 1* and *TRAF 3* were up-regulated significantly. Interestingly, the *Cactus*, a negative regulatory factor in the Toll pathway, was significantly up-regulated. In addition, there was no DEG in IMD pathway (Fig. 8) and two up-regulated DEGs (*Dome* and *Fos*) in JAK-STAT pathway (Fig. 9). However, the PPO pathway was another sight (Fig. 10), *CLIP 4/11*/*12*, *SPN 5*/*7*/*/19*/*22* and *Reeler 1*were up-regulated; however, *CLIP 5*/*14*, *SPN 10*/*12*/*15*/*17*/*20*/*21* were down-regulated. *Lysozyme* and *Lysozyme like protein 2* were significantly up-regulated. Effectors related genes including *Cec B1 ~ B6*, *gloverin 2*, *defensin*, *moricin*, *morLP-B2*, and *morLP-B5* were all up-regulated (Fig. 11). All results above showed that the innate immune system has been fully initiated by the congenital infection, and the pathogens also behave a lot to manipulate the host immunity through regulating the immune recognition and classic immune pathways. At 10 dph, the congenital infection became more serious, the infected silkworms were very weak and near to die. At this time point, recognition related genes such as *CTL 1*/*2*/*5*/*9* and *SCRB 8* were significantly down-regulated, *βGRP 1*/*3*/*4* and *PGRP-L2*/*S3*/*S5*/*S6*, *CTL 3* were up-regulated but not significant. In modulation related genes, *CLIP 5*/*9*/*10*/*14* and *SPN 6*/*20* were significantly down-regulated. At this time, most of Toll pathway genes did not show significant differences in expression, only *Spz 2*, *Spz 4* and *Spz 6* were significantly down-regulated, *Spz 3* was also down-regulated but not significantly (Fig. 7). In the PPO pathway (Fig. 10), only *Reeler 1* was significantly up-regulated, the other genes listed as DEGs were all down-regulated, they were *CLIP 5/9/10*, *SPN 6/20* and *PPO 1/2*, suggesting that the PPO pathway was inhibited by pathogens at this time. Moreover, effectors related genes such as *enb 1*,* enb 2*,* gloverin 1*,* gloverin 2*,* leb* and *morLP-B1* were up-regulated but only *gloverin 3* was significant. Notably, although the expression of *Cec B1 ~ B6* were down-regulated, but not significantly, which formed an obvious contrast with the data of 5 dph. DEGs aforementioned implied that the host (larva stage) significantly enhanced the immunity against *N. bombycis* infection. Simultaneously, the pathogen may evade or manipulate the host’s innate immunity supported by these regulated genes. #### Immune responses to congenital infection in larva stage of non-diapause silkworms Similar to dispause silkworms, the immune response in the larva stage of non-diapause silkworm was stronger than embryo stage. At 1 dph, immune recognition genes (Fig. 6), *PGRP-L1/S1/S3* and *SCRB 13* were up-regulated, *PGRP-L4*, *SCRB 10* and *SCRB 11* were down-regulated. The signal transduction genes in Toll pathway (Fig. 7), IMD pathway (Fig. 8) and JAK-STAT pathway (Fig. 9) had no DEG at 1 dph. In PPO pathway (Fig. 10), there seven genes up-regulated but only three genes (*SPN 17*, *Lysozyme* and *Reeler 1*) were significant. Most of antimicrobial peptides genes were up-regulated, *cec A1*, *cec B1 ~ B6*, *gloverin 2/4* and *morLP-B1 ~ B5* significantly up-regulated with big changes (Fig. 11). At 2 dph, there were two up-regulation DEGs (*CTL 7* and *SCRC*) and six down-regulation DEGs (*PGRP-S4*, *SCRAC 2*, *SCRASP 2*, *SCRB 7*, *SCRB 8* and *SCRB 13*) listed in the recognition related genes (Fig. 6). There were six *Tolls* (*Toll 6/7 − 2/7 − 3/8/10 − 2/12*) were down-regulated while *Toll 3–3* was up-regulated, implied that the Toll pathway was inhibited at this time. In JAK-STAT pathway (Fig. 9), it was the similar case, two genes (*Dome* and *Hem*) were significantly down-regulated. Inhibition of JAK-STAT pathway resulted in decreased survival rate and antibacterial activity of silkworm, we also observed that the larvae began to die at 2 dph. According to the transcriptome data, the surviving larvae at this time were also weak and the function of the immune system was deteriorated. Although the positive regulator of PPO pathway *PGRP-S4* was down-regulated at this time point, the PPO pathway seemed as activated (Fig. 10). *CLIP 3/13*, *SPN 16 PPO 1/2* and *Reeler 1* were up-regulated, *CLIP 8* and *SPN 10/20/21* were down-regulated. In addition, *SPN 15* was up-regulated which was the negative regulator of PPO pathway. At this time point, it was interesting that the *morLP-B1 ~ B5* were up-regulated, which same as 1 dph, while the other *AMPs* were down-regulated (Fig. 11). At 3 dph, most of immune-related DEGs were down-regulation. The recognition related genes (Fig. 6), only four DEGs (*PGRP-L4*,* PGRP-S5*,* CTL 11* and *SCRAL 1*) were up-regulated while ten DEGs (PGRP-S3, CTL 5/7/9/10/12, SCRAC 1/2, SCRB 7/9/13) were down-regulated. PGRP-S5 has been reported as the negative regulator of IMD pathway [53]. As expected, the IMD pathway was inhibited (Fig. 8), five genes (*IMD*,* Dredd*, Table 2, *ikk β* and *Relish*) were down-regulated. The upstream of Toll pathway was up-regulation and the downstream was down-regulated (Fig. 7). *Spz 1/3*,* Toll 3 − 2/3–3* and *TRAF 3* were significantly up-regulated, *Spz 2*,* Toll 6/9 − 2/10 − 2/10 − 3*,* Tube* and *TRAF 2* were significantly down-regulated. At this time, The *Socs* and *Fos* in JAK-STAT pathway were up-regulated, and *Hem* was down-regulated (Fig. 9).The PPO pathway at 3 dph was regulated by pathogens (Fig. 10). *CLIP 5*,* SPN 8/10/12/15/16/18/21* and *PPAE* were significant down-regulated and *CLIP 12/13*,* SPN 22* and *Reeler 1* were up-regulated significantly. The *Lysozyme* was also significantly down-regulated. Similar to 2 dph, almost all *AMPs* were significant down-regulation but *morLP-B1 ~ B5* were significant up-regulation at 3 dph. In general, compared with diapause silkworms, the immune-related genes, especially the *AMPs* of non-diapause silkworm larvae showed a fully induced immune responses in newly hatched larvae, and a lot of immune related genes in different steps were down-regulated, indicating that pathogens could regulate host immune pathways. ## Discussion *N. bombycis* has a complex and unique route to enter into the eggs and lead to congenital infection [54]. After that, *N. bombycis* starts to proliferate inside the eggs with embryo development. The histopathogical characteristics of *N. bombycis* proliferation in non-diapause silkworms had been investigated [12], here we provide the molecular proliferation characteristics of *N. bombycis* by Dual RNA-seq, our result is consistent with histopathological characteristics. For non-diapause silkworms, most of parasites were in meront stage at 1 dpo. From 2 dpo, many meronts enter into the sporogony stage, the corresponding molecular marker is *SWP 1* ranking as the Top 1 expressed gene. We discovered that the big difference of the molecular proliferation characteristics between non-diapause silkworm and diapause silkworm resulted from the hot HCl treatment for diapause silkworm eggs, which can kill many parasites inside the eggs [36]. The hot HCl bath treatment interrupts the proliferation cycle of *N. bombycis*, and reduces the pathogen load in host. It has been reported that spore formation and parasite loads of *Nosema muscidifuracis* were reduced because of heat shock treatments in *Musidifurax raptor* eggs [55]. The pathogen load in non-diapause silkworm eggs and larvae was much higher than that of diapause silkworm eggs and larvae, which far influence the host immune responses in embryos and larvae. Compared previous results of larvae immune responses to *N. bombycis* infection [24, 56] with our current RNA-Seq analysis, immune responses to congenital infection in embryo stages were weaker than that of larva stages both in diapause and non-diapause silkworms. Four reasonable explanations were put forward. Firstly, the embryo gradually mounts a systemic and positive immunity and acquires its immune competence after or near hatching. Secondly, the proliferation of *N. bombycis* in embryos primarily centered around yolk granules and intestinal lumen [12]. The strategy adopted by *N. bombycis* may reduce the parasite’s direct stimulation on the embryo and weaken the priming of the host immune system. On the contrary, the parasites can cause a systemic and serious infection in larvae [57]. Thirdly, for diapause silkworm, the unique sequence mapped rates of *N. bombycis* were between 0.07% and 0.89% at embryo stages but increased to 3.35%~13.89% at larva stages. As a result, the parasite load difference between embryo and larva stages is another candidate factor. Finally, *N. bombycis* may actively regulate or escape the host immune response through its secreted proteins such as Serpin 6 [58], or abundant highly expressed genes while the function of these secretions is poorly understood [12, 59, 60]. It also has been reported that *Encephalitozoon intestinalis* may escape from host immune recognition through the modulation of dendritic cell differentiation and maturation [61]. The systematic immune response of *B. mori* to pathogen infection has been extensively analyzed [21, 22, 53]. However, there is limited knowledge regarding the equipping time of *B. mori* innate immunity. Here, we found the transcription of *AMPs* in infected groups significantly up-regulated at the 5 dpo in non-diapause silkworm embryo if we ignore the *AMPs* DEGs in “First day chaos”. At 7 dpo, the significant up-regulation of PPO 1 and PPO 2 marks the establishment of melanization pathway. In conclusion, for non-diapause silkworm, the differential expression of host genes proved that the congenital infection of *N. bombycis* in the embryo may lead to an earlier establishment of the host positive immune system. However, to elucidate the equipping time of silkworm innate immunity is a challenging task, only considering the transcription of immune-related genes is not enough to answer this question. We have analyzed the proteome composition in silkworm eggs with *N. bombycis* congenital infection and found there are several different AMPs and a lot of different immune related proteins, such as PRRs, CTL, SPNs, Spz 1, etc. (our unpublished work), which suggest that maternal immunity may be also existed in insect eggs, especially the eggs with congenital infection [62]. In the larva stage, with the establishment of the immune system, the host immune response was more intense, and a large number of AMP genes were significantly up-regulated. However, at 2 dph, the upstream genes of PPO cascade was inhibited, the expression of intracellular signaling molecules in Toll pathway and IMD pathway were down-regulated, and the JAK-STAT pathway, which was related to survival and antibacterial activity, was also inhibited. On the whole, although the immune system of the host was completely established in the larva stage, the pathogen load increased and the physiological state of the host continued to deteriorate, the main antimicrobial peptides also showed a dynamic trend of up-regulation and then down-regulation, except for *Moricins*, which is the unique AMP family to Lepidoptera. *Moricins* maintained a very high expression level through the larva stage, its role in resistance to microsporidia infection needs further study. We obtained a large number (6,071) DEGs at 1 dpo for non-diapause silkworms, but at the following time points in embryo, the number of DEGs decreased to a lot, we call this phenomenon as “First Day Chaos” in non-diapause silkworm eggs. Why are there so many DEGs obtained at 1 dpo? It is an intrigued question for future verification, we are wondering whether there is connection with different strains of silkworms. There may be different pathogen tolerance existed between the non-diapause silkworms and diapause silkworms [63, 64]. Notably, there are some limitations in our work. Although we found some genes with different expression patterns in immune responses to pathogen infection between the diapause silkworms and non-diapause silkworms, we should be cautious to face these differences, because their genetic backgrounds are not identical, some of our findings are just based on transcriptomic analyses and provide a clue for further research. It is necessary to adopt careful wet-lab work for future confirmation and verification. In summary, our study using deep transcriptional profiling of the silkworm hosts illustrates that gene expression characteristics of pathogen and host different immune responses in diapause and non-diapause silkworm embryo and larvae with *N. bombycis* congenital infection. Rapid proliferation of pathogen in host, weak immune responses in embryo stage and strong immune responses in larvae stage are the main findings here. Different pathogen loads in diapause and non-diapause silkworm are results from the hot HCL bath treatment for diapause silkworm eggs at 24 h post oviposition, which has the strong killing effect for *N. bombycis*. In addition, the low pathogen load in diapause silkworm embryo and larvae is also connected with the weaker immune responses compared with non-diapause silkworm group. In general, here we not only developed a microsporidia-host system for analysis of microsporidia congenital infection and the host response, but also provide abundant molecular data for silkworm embryo innate immunity development anda sound basis for the complicated host-pathogen interactions. ## Supplementary Information Supplementary Material 1. Supplementary Material 2. Supplementary Material 3. Supplementary Material 4. Supplementary Material 5. Supplementary Material 6. Supplementary Material 7. Supplementary Material 8. Supplementary Material 9. Supplementary Material 10. Supplementary Material 11.