Insights to micropsoridia Nosema bombycis congenital infection and host immune responses in the embryo and larva stages of silkworms

Abstract

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.

Supplementary Information: The online version contains supplementary material available at 10.1186/s12864-025-11762-z.

Keywords: Bombyx mori; Nosema bombycis; Congenital infection; Immune response; Microsporidia; Transcriptome.

Fig. 1

The 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

Fig. 2

Pathogenic 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

Fig. 3

Consistently 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

Fig. 4

Secretome 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

Fig. 5

Transcriptome 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

Fig. 6

The 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. 7

The 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. 8

The 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. 9

The 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. 10

The 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. 11

The 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