Diapause is a programmed dormancy that allows organisms to tolerate predictable periods of unfavorable conditions by temporarily halting development and reducing metabolism. Diapause is widespread among insects and is crucial for allowing organisms to coordinate their growth and reproduction with favorable environmental conditions. While the adaptive significance of diapause is well-understood, the molecular mechanisms underpinning diapause remain unresolved. We performed high-throughput sequencing to investigate the role of microRNAs in the diapause of the Asian tiger mosquito, Aedes albopictus. We first investigate miRNAs in diapause induction by characterizing maternally-provisioned miRNAs in mature oocytes of Ae. albopictus under diapause-inducing and diapause-averting conditions. Second, we investigate miRNAs in diapause maintenance by characterizing miRNAs in diapause and non-diapause pharate larvae. We identified 162 miRNAs, 152 previously known and 10 putatively novel. We identified no differentially abundant miRNAs in mature oocytes and seven differentially abundant miRNAs in pharate larvae. The predicted targets of differentially abundant miRNAs include genes affecting several processes related to diapause maintenance including ecdysone regulation, immune response, lipid metabolism, and regulation of development. Our results suggest that Ae. albopictus does not maternally-provision a unique set of miRNAs during diapause induction but miRNAs are a component of diapause maintenance in this species.

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Premenstrual Dysphoric Disorder (PMDD) is characterized by debilitating mood symptoms in the luteal phase of the menstrual cycle. Prior studies of affected women have implicated a differential response to ovarian steroids. However, the molecular basis of these patients’ differential response to hormone remains poorly understood. We performed transcriptomic analyses of lymphoblastoid cell lines (LCLs) derived from women with PMDD and asymptomatic controls cultured under untreated (steroid-free), estradiol-treated (E2), and progesterone-treated (P4) conditions. Weighted gene correlation network analysis (WGCNA) of transcriptomes identified four gene modules with significant diagnosis x hormone interactions, including one enriched for neuronal functions. Next, in a gene-level analysis comparing transcriptional response to hormone across diagnoses, a generalized linear model identified 1522 genes differentially responsive to E2 (E2-DRGs). Among the top 10 E2-DRGs was a physically interacting network (NUCB1, DST, GCC2, GOLGB1) involved in endoplasmic reticulum (ER)-Golgi function. qRT-PCR validation reproduced a diagnosis x E2 interaction (F(1,24)=7.01, p = 0.014) for NUCB1, a regulator of cellular Ca2+ and ER stress. Finally, we used a thapsigargin (Tg) challenge assay to test whether E2 induces differences in Ca2+ homeostasis and ER stress response in PMDD. PMDD LCLs had a 1.36-fold decrease in Tg-induced XBP1 splicing response compared to controls, and a 1.62-fold decreased response (p = 0.005), with a diagnosis x treatment interaction (F(3,33)=3.51, p = 0.026) in the E2-exposed condition. Altered hormone-dependent in cellular Ca2+ dynamics and ER stress may contribute to the pathophysiology of PMDD.

Photoperiodic diapause is an important adaptation that allows individuals to escape harsh seasonal environments via a series of physiological changes, most notably developmental arrest and reduced metabolism. Global gene expression profiling via RNA-Seq can provide important insights into the transcriptional mechanisms of photoperiodic diapause. The Asian tiger mosquito, Aedes albopictus, is an outstanding organism for studying the transcriptional bases of diapause due to its ease of rearing, easily induced diapause, and the genomic resources available. This manuscript presents a general experimental workflow for identifying diapause-induced transcriptional differences in A. albopictus. Rearing techniques, conditions necessary to induce diapause and non-diapause development, methods to estimate percent diapause in a population, and RNA extraction and integrity assessment for mosquitoes are documented. A workflow to process RNA-Seq data from Illumina sequencers culminates in a list of differentially expressed genes. The representative results demonstrate that this protocol can be used to effectively identify genes differentially regulated at the transcriptional level in A. albopictus due to photoperiodic differences. With modest adjustments, this workflow can be readily adapted to study the transcriptional bases of diapause or other important life history traits in other mosquitoes. Video LinkThe video component of this article can be found at