Comparative Proteomic Analysis Provides New Insights into the Molecular Basis of Thermal-Induced Parthenogenesis in Silkworm (<i>Bombyx mori)</i>

Artificial parthenogenetic induction via thermal stimuli in silkworm is an important technique that has been used in sericultural production. However, the molecular mechanism underlying it remains largely unknown. We have created a fully parthenogenetic line (PL) with more than 85% occurrence and 80% hatching rate via hot water treatment and genetic selection, while the parent amphigenetic line (AL) has less than 30% pigmentation rate and less than 1% hatching rate when undergoing the same treatment. Here, isobaric tags for relative and absolute quantitation (iTRAQ)-based analysis were used to investigate the key proteins and pathways associated with silkworm parthenogenesis. We uncovered the unique proteomic features of unfertilized eggs in PL. In total, 274 increased abundance proteins and 211 decreased abundance proteins were identified relative to AL before thermal induction. Function analysis displayed an increased level of translation and metabolism in PL. After thermal induction, 97 increased abundance proteins and 187 decreased abundance proteins were identified. An increase in stress response-related proteins and decrease in energy metabolism suggested that PL has a more effective response to buffer the thermal stress than AL. Cell cycle-related proteins, including histones, and spindle-related proteins were decreased in PL, indicating an important role of this decrease in the process of ameiotic parthenogenesis

Transcriptome Analysis of Thermal Parthenogenesis of the Domesticated Silkworm

<div><p>Thermal induction of parthenogenesis (also known as thermal parthenogenesis) in silkworms is an important technique that has been used in artificial insemination, expansion of hybridization, transgenesis and sericultural production; however, the exact mechanisms of this induction remain unclear. This study aimed to investigate the gene expression profile in silkworms undergoing thermal parthenogenesis using RNA-seq analysis. The transcriptome profiles indicated that in non-induced and induced eggs, the numbers of differentially expressed genes (DEGs) for the parthenogenetic line (PL) and amphigenetic line (AL) were 538 and 545, respectively, as determined by fold-change ≥ 2. Gene ontology (GO) analysis showed that DEGs between two lines were mainly involved in reproduction, formation of chorion, female gamete generation and cell development pathways. Upregulation of many chorion genes in AL suggests that the maturation rate of AL eggs was slower than PL eggs. Some DEGs related to reactive oxygen species removal, DNA repair and heat shock response were differentially expressed between the two lines, such as <i>MPV-17</i>, <i>REV1</i> and <i>HSP68</i>. These results supported the view that a large fraction of genes are differentially expressed between PL and AL, which offers a new approach to identifying the molecular mechanism of silkworm thermal parthenogenesis.</p></div

Bioinformatic analyses of RNA-seq data.

<p>(A) Pearson correlation between four sets of egg samples. (B) Reproducing kernel particle method (RKPM) distribution of four sets of egg samples. (C) RPKM density distribution of four sets of egg samples.</p

Statistics of genes regulated between two lines.

<p>Note: Compare 1: PLUI_eggs <i>vs</i>. ALUI_eggs, Compare 2: PLHI_eggs <i>vs</i>. ALHI_eggs.</p><p>Statistics of genes regulated between two lines.</p

Bioinformatic analyses of RNA-seq data.

<p>(A) Pearson correlation between four sets of egg samples. (B) Reproducing kernel particle method (RKPM) distribution of four sets of egg samples. (C) RPKM density distribution of four sets of egg samples.</p

Comparison of parthenogenetic ability and fertilization between PL and AL.

<p>(A) and (B) The virgin female moths of 54A and wu 14, respectively. (C) Selfing by mating of 54A. (D) and (E) Non-thermally induced eggs of 54A and wu 14 dissected from the virgin female moths, respectively. (F), (G) and (H) Pigmentation rate of 54A (selfing), 54A (parthenogenetic induction) and wu 14 (parthenogenetic induction), respectively. (I), (J) and (K) Hatching rate of 54A (selfing), 54A (parthenogenetic induction) and wu 14 (parthenogenetic induction), respectively. (L) Selfing through mating. (M) Parthenogenetic induction.</p

GO enrichment analysis for DEGs between two lines.

<p>(A) Biological process for DEGs before thermal induction. (B) Cellular component for DEGs before thermal induction. (C) Molecular function for DEGs before thermal induction. (D) Biological process for DEGs after thermal induction. (E) Cellular component for DEGs after thermal induction. (F) Molecular function for DEGs after thermal induction. The sizes of the circles are proportional to the number of genes associated with the GO term. The arrows represent the relationship between parent-child terms. The color scale indicates the corrected P-value of the enrichment analysis.</p

Parthenogenetic ability between PL and AL.

<p>Notes: The data of the pigmentation rate and hatching rate are the average value of 20 sets of eggs (one set of eggs laid by one moth). The survival rate of parthenogenetic offspring was obtained from 500 individuals, and the abnormal rate of parthenogenetic offspring was obtained from 300 individuals (AL of parthenogenetic induction only have 297 individuals). The pigmentation rate data were obtained in October 2013 and the hatching rate and abnormal rate data were obtained in May 2014, respectively.</p><p>^a Pigmentation rate is the ratio of the number of pigmented eggs to the total number of eggs treated.</p><p>^b Hatching rate is the ratio of the number of eggs that hatched into silkworms to the total number of eggs treated.</p><p>^c Survival rate is the ratio of developed complete silkworms (egg to moth) to the total number of parthenogenetic offspring.</p><p>^d Abnormal rate is the ratio of abnormal individuals to the total number of parthenogenetic offspring; the number of abnormal individuals was obtained on the 3^rd day of the 5^th instar of the larvae.</p><p>Parthenogenetic ability between PL and AL.</p

GO bar chart of DEGs between PL and AL.

<p>(A) The most enriched GO terms for DEGs between two the lines in non-thermally induced eggs. (B) The most enriched GO terms for DEGs between two lines in thermally induced eggs.</p

Statistics for filtering and mapping reads.

<p>Statistics for filtering and mapping reads.</p