Transcriptome Analysis of Neonatal Larvae after Hyperthermia-Induced Seizures in the Contractile Silkworm, Bombyx mori

Abstract

The ability to respond quickly and efficiently to transient extreme environmental conditions is an important property of all biota. However, the physiological basis of thermotolerance in different species is still unclear. Here, we found that the cot mutant showed a seizure phenotype including contraction of the body, rolling, vomiting gut juice and a momentary cessation of movement, and the heartbeat rhythm of the dorsal vessel significantly increases after hyperthermia. To comprehensively understand this process at the molecular level, the transcriptomic profile of cot mutant, which is a behavior mutant that exhibits a seizure phenotype, was investigated after hyperthermia (42°C) that was induced for 5 min. By digital gene expression profiling, we determined the gene expression profile of three strains (cot/cot ok/ok, +/+ ok/ok and +/+ +/+) under hyperthermia (42°C) and normal (25°C) conditions. A Venn diagram showed that the most common differentially expressed genes (DEGs, FDR<0.01 and log2 Ratio≥1) were up-regulated and annotated with the heat shock proteins (HSPs) in 3 strains after treatment with hyperthermia, suggesting that HSPs rapidly increased in response to high temperature; 110 unique DEGs, could be identified in the cot mutant after inducing hyperthermia when compared to the control strains. Of these 110 unique DEGs, 98.18% (108 genes) were up-regulated and 1.82% (two genes) were down-regulated in the cot mutant. KEGG pathways analysis of these unique DEGs suggested that the top three KEGG pathways were “Biotin metabolism,” “Fatty acid biosynthesis” and “Purine metabolism,” implying that diverse metabolic processes are active in cot mutant induced-hyperthermia. Unique DEGs of interest were mainly involved in the ubiquitin system, nicotinic acetylcholine receptor genes, cardiac excitation–contraction coupling or the Notch signaling pathway. Insights into hyperthermia-induced alterations in gene expression and related pathways could yield hints for understanding the relationship between behaviors and environmental stimuli (hyperthermia) in insects.

Functional Loss of Bmsei Causes Thermosensitive Epilepsy in Contractile Mutant Silkworm, Bombyx mori

“…The heat shock proteins (Hsps) are crucial in the response to high temperature 5 7 . In our previous study, most Hsps were significantly upregulated in both the cot mutant strains and control strains (Dazao and ok ) when exposed to hyperthermia for 5 min 30 . In the silkworm, Hsp-1 is up-regulated in the fat body after treatment with constant high temperature 37 .…”

“…However, because the periods and strength of rubbing differ, it was difficult to discriminate the cot/cot and cot/+ genotypes in backcrossed larvae (BC 1 ) with this method. Recently, we found that cot is a temperature-sensitive mutant 30 . To determine the appropriate temperature at which to distinguish the cot/cot and cot/+ genotypes, larvae of different genotypes ( cot/cot , cot /+ and +/+) were subjected to different temperatures, ranging from 25 °C to 48 °C.…”

“…In 2012, Zamorano-Leon found that the hERG gene is a potential link between epilepsy and LQT2 syndrome 46 . In our previous study, we showed that the cot mutant not only manifests the epilepsy phenotype, but importantly, also displays an increased heartbeat when exposed to high temperature 30 . An LC–MS–MS analysis of neonatal larvae demonstrated that the level of BGIBMGA001031 (troponin I-like isoform X3) was also significantly up-regulated in the cot mutant.…”

“…For positional cloning, we used single-pair backcrossed (BC 1 ) individuals from the cot/cot ♀ × F 1 ♂ cross. Individuals with genotypes ok/ok , cot/cot or ok/ok , +/+ were obtained with a previously described method 30 , and used for subsequent experiments.…”

“…The recessive mutant cot is controlled by a single gene located at locus 25.0 centimorgan (cM) on silkworm genetic linkage group 15 29 . In our previous study, we showed that cot is a thermo-sensitive paralysis mutant, which manifests when it is exposed to 35 °C, at which temperature it displays strong contractions, rolling, vomiting, and a temporary lack of movement after manual stimulation, followed by slow recovery 30 . However, the corresponding mutant gene was not isolated.…”