Molecular mechanisms and energetic costs of long-term recovery from freezing in the polyextremophile midge Belgica antarctica Jacobs (Diptera: Chironomidae)

For the select ectotherms that can survive internal ice formation, the ability to recover from freezing, reestablish homeostasis, and resume essential ecological functions after a freezing event is critical. In this study, we tracked changes in gene expression and energy stores in the Antarctic midge Belgica antarctica following recovery from an acute nonlethal freezing event to test the hypothesis that recovery from freezing would demand large-scale changes in gene expression, resulting in a depletion of energy stores. To address this hypothesis, B. antarctica larvae were divided into two experimental groups (frozen and control), from which RNA and energy reserves were extracted. RNA-seq protocol: Frozen groups were submitted -5C for 24 h, while control groups were maintained at 4C for 24 h. Frozen and Control were sampled immediately after thawing and at 1, 3, 7, and 15 days of recovery for whole-body RNA sequencing. The resulting transcriptomic samples were then mapped to B. antarctica's genome. Genome assembly protocol: High molecular weight (HMW) DNA was extracted from one individual B. antarctica larva using the Qiagen genomic-tip DNA extraction kit. Because the amount of purified HMW DNA from a single individual was not enough for a standard PacBio SMRT bell library prep, we followed the PacBio Ultra-low library prep protocol using the SMRTbell Express Template Prep Kit 2.0 (PacBio, Menlo Park, CA, USA). Briefly, HMW DNA was sheared to 10 kbp fragments using a Diagenode Megaruptor followed by whole genome amplification before standard library preparation. The ultra low library was then sequenced on a PacBio Revio flow cell at the Brigham Young University DNA Sequencing Center. To prepare the CCS reads for assembly, we first trimmed PCR adapters and then removed PCR duplicates. We then assembled the trimmed and de-duplicated HiFi reads using hifiasm v0.9.14. Genome completeness was evaluated with compleasm v0.2.6 using the insecta_odb10 BUSCO gene set (1332 genes): S: 97.4, D: 1.2, F: 0.29, M: 1.02%.