Diapause-Associated Changes in the Lipid and Metabolite Profiles of the Asian Tiger Mosquito, Aedes Albopictus Zachary A

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RESEARCH ARTICLE Diapause-associated changes in the lipid and metabolite profiles of the Asian tiger mosquito, Aedes albopictus Zachary A. Batz* and Peter A. Armbruster

ABSTRACT understood; diapause enhances overwinter survival via increased Diapause is an alternative life-history strategy that allows organisms to nutrient storage, reduced metabolic activity and elevated stress enter developmental arrest in anticipation of unfavorable conditions. tolerance (Hahn and Denlinger, 2011; Teets and Denlinger, 2013). Diapause is widespread among insects and plays a key role in Additionally, the prevalence and timing of diapause shape enhancing overwinter survival as well as defining the seasonal and the geographic distribution and seasonal abundance of insect geographic distributions of populations. Next-generation sequencing populations (Danks, 1987; Tauber and Tauber, 1976). Nevertheless, has greatly advanced our understanding of the transcriptional basis for despite the long-recognized ecological importance of diapause, this crucial adaptation but less is known about the regulation of until recently relatively little was known about the molecular embryonic diapause physiology at the metabolite level. Here, we mechanisms underpinning this alternative developmental strategy characterized the lipid and metabolite profiles of embryonic diapause (Denlinger, 2002; Ragland and Keep, 2017). in the Asian tiger mosquito, Aedes albopictus.Weusedanuntargeted Diapause is a complex, dynamic developmental program š approach to capture the relative abundance of 250 lipids and 241 consisting of several eco-physiological stages (Ko tál, 2006; š metabolites. We observed adjustments associated with increased Ko tál et al., 2017). First, in the induction phase, a token energy storage, including an accumulation of lipids, the formation of environmental cue is perceived during a sensitive period that may larger lipid droplets and increased lipogenesis, as well as metabolite occur far in advance of dormancy. The diapause-inducing signal, shifts suggesting reduced energy utilization. We also found changes in often a change in photoperiod, causes the organism to begin neuroregulatory- and insulin-associated metabolites with potential preparing for developmental arrest. In the preparation phase, the roles in diapause regulation. Finally, we detected a group of diapause-destined organism undergoes behavioral and/or unidentified, diapause-specific metabolites which have physical physiological adjustments to accommodate prolonged dormancy. properties similar to those of steroids/steroid derivatives and may be Then, in the initiation phase, the organism enters developmental associated with the ecdysteroidal regulation of embryonic diapause in arrest, thus marking the onset of diapause. This developmental A. albopictus. Together, these results deepen our understanding of the arrest can occur at any life stage, although typically a species will metabolic regulation of embryonic diapause and identify key targets for only arrest at a single life stage (Danks, 1987). In the maintenance future investigations. phase, diapause is sustained for some genetically determined duration, even if the organism is exposed to favorable conditions. KEY WORDS: Embryonic diapause, Developmental arrest, Finally, in the termination phase, the organism will gradually Untargeted metabolomics, Lipidomics regain sensitivity to environmental cues that permit continued development. If unfavorable conditions persist beyond the INTRODUCTION termination phase, development may remain environmentally The ability to exploit favorable seasonal conditions and survive suppressed in post-diapause quiescence, a dormant state that can stressful environments is a fundamental requirement for insects be terminated immediately upon exposure to favorable conditions. inhabiting temperate regions. To synchronize their life cycles with Over the past decade, transcriptomics has greatly advanced our recurring periods of seasonal variation, many insect taxa have understanding of the molecular regulation of diapause in many independently evolved diapause (Danks, 1987; Tauber et al., 1986). species (e.g. Gong et al., 2013; Huang et al., 2015; Kang et al., Diapause is an alternative life-history strategy defined by a 2016; Poelchau et al., 2013a,b; Poupardin et al., 2015; Qi et al., hormonally controlled developmental arrest that is initiated in 2015; Ragland et al., 2010; Yocum et al., 2015). Numerous studies anticipation of habitat deterioration (Denlinger et al., 2012; Lees, have identified significant diapause-associated changes in the 1956). The capacity to initiate diapause allows insects to regulate expression of thousands of genes including those involved in cell their energy utilization throughout the year, maximizing allocation proliferation, nutrient storage, metabolic activity, hormonal towards growth, development and reproduction during favorable signaling, circadian rhythms and stress resistance (see review in periods in the spring and summer, then minimizing such allocations Ragland and Keep, 2017). Metabolomic adjustments in diapause during the more inclement autumn and winter (Bradshaw et al., have also garnered significant attention primarily through narrowly 2004). The adaptive significance of insect diapause is well targeted assays for molecules expected a priori to contribute to the diapause phenotype. For example, many studies have measured changes in the abundance of molecules associated with nutrient Department of Biology, Georgetown University, 37th and O Streets NW, Washington, DC 20057, USA. storage, like glycogen and triacylglycerides (e.g. Goto et al., 1998; Shimizu, 1992; Wipking et al., 1995), or cryoprotective molecules *Author for correspondence ([email protected]) such as polyols and amino acids (e.g. Goto et al., 1998; Lafage et al., Z.A.B., 0000-0002-4483-2402 1974; Mohammadzadeh et al., 2017). Recently, emerging untargeted metabolomic technology has enabled simultaneous

Received 27 July 2018; Accepted 24 October 2018 assessment of a wide array of small molecules including amino Journal of Experimental Biology

1 RESEARCH ARTICLE Journal of Experimental Biology (2018) 221, jeb189480. doi:10.1242/jeb.189480

established that many metabolic genes, including those involved in List of abbreviations gluconeogenesis and lipid metabolism, are differentially expressed CPT carnitine palmitoyltransferase throughout the course of embryonic diapause relative to expression DG diacylglyceride in non-diapausing embryos (Poelchau et al., 2013a,b). Additionally, dpo days post-oviposition multiple stress-tolerance mechanisms are enhanced at the FDR false discovery rate transcriptional level in A. albopictus diapause. For example, a FOXO forkhead transcription factor HMDB human metabolome database greater abundance of long-chain hydrocarbons is deposited on the JH3 juvenile hormone III chorion of the egg, significantly increasing desiccation resistance LC liquid chromatography (Urbanski et al., 2010). The complex molecular changes observed LD long day (16 h light: 8 h dark) during diapause imply that evidence from multiple regulatory levels m/z mass-to-charge ratio will enhance our understanding of the physiological basis of this MS mass spectrometry crucial adaptation, though only a few studies have attempted to do PC phosphatidylcholine PCA principal component analysis so in any species (e.g. Colinet et al., 2012; Zhang et al., 2012, 2013). PEA pathway enrichment analysis In this study, we investigated the metabolome of embryonic plasmenyl-PE plasmenyl-phosphatidylethanolamine diapause in A. albopictus. We performed shotgun lipidomics and QC quality control untargeted metabolomics on A. albopictus eggs in early diapause QEA quantitative enrichment analysis maintenance (11 days post-oviposition, dpo) and on age-matched, RH relative humidity non-diapause eggs to characterize diapause-associated changes in RSD relative standard deviation RT retention time the relative abundance of lipids and metabolites. We identified SD short day (8 h light: 16 h dark) specific metabolic pathways and individual metabolites that were TG triacylglyceride significantly altered during early diapause. We draw upon the extensive transcriptomic data available in this species (Batz et al., 2017; Huang et al., 2015; Poelchau et al., 2011, 2013a,b; Reynolds acids, lipids, polyols, fatty acids and metabolic intermediates. The et al., 2012) to inform the interpretation of our metabolomic data advantage of an untargeted metabolomics approach is that it and provide novel insights regarding the molecular basis of provides an unbiased snapshot of organismal physiology, embryonic diapause. potentially identifying both expected and unexpected metabolic adjustments and informing future targeted assessments (Macel MATERIALS AND METHODS et al., 2010). The disadvantage of this approach is that it lacks the Mosquito rearing and tissue generation specificity and complete pathway coverage of targeted analyses. All experiments were completed with an F3 laboratory colony of Furthermore, which metabolites are detected depends on the A. albopictus. This colony was originally established in August extraction and separation methodologies utilized (Cajka and 2015 from over 200 larvae collected from 15 tires at a recycling Fiehn, 2016; Patti, 2011; Yanes et al., 2011). Finally, center in Manassas, VA, USA. Prior to the start of this experiment, metabolomic approaches cannot assess the regulation of enzymes the laboratory colony was maintained under a long-day such as allosteric control or covalent modification. A small number photoperiod (LD; 16 h light:8 h dark) at 21°C and 80% relative of studies have applied untargeted analyses to characterize humidity (RH) as described previously (Armbruster and Conn, the metabolome of diapause at the larval, pupal and adult 2006; Armbruster and Hutchinson, 2002). To generate samples for stages (Khodayari et al., 2013; Li et al., 2015; Lu et al., 2014), this experiment, larvae were reared under a LD photoperiod at 21°C but to date no study has investigated the metabolomic profile of and 80% RH and fed a near-optimal diet consisting of a slurry of embryonic diapause. dog food and brine shrimp (Armbruster and Conn, 2006). Upon The Asian tiger mosquito, Aedes albopictus (Skuse), is an pupation, pupae were divided evenly into two treatments: four emerging model system for studying embryonic diapause biological replicates were established under an unambiguous short- (Denlinger and Armbruster, 2014, 2016). Temperate populations dayphotoperiod(SD;8hlight:16hdark)toinduceproductionof of A. albopictus undergo a facultative embryonic diapause diapause eggs, and four biological replicates were established modulated by the photoperiod experienced by female pupae and under a LD photoperiod to induce production of direct developing adults in the previous generation (Hawley, 1988; Pumpuni, 1989). (non-diapause) eggs. Each SD and LD replicate was established Under long-day photoperiods (e.g. 16 h light:8 h dark), females with at least 150 pupae. produce eggs that complete embryonic development and are then Adult females were provided a human blood meal 7–14 days immediately responsive to hatching stimuli. In contrast, under short- post-eclosion. The Georgetown University Institutional Review day photoperiods (e.g. 8 h light:16 h dark), females produce eggs Board (IRB) has determined that mosquito blood feeding is not that complete embryonic development, enter diapause as pharate human research and thus does not require IRB approval; however, larvae within the chorion of the egg, and then undergo a genetically the blood feeding protocol has been approved by the Georgetown controlled period in which they are not responsive to hatching University Occupational Health and Safety Committee. Beginning stimuli (Denlinger and Armbruster, 2014). 4 days post-blood meal, females were provided with an oviposition Transcriptional changes throughout the eco-physiological cup lined with an unbleached paper towel and half-filled with trajectory of diapause have been extensively characterized in deionized water. Eggs were collected daily, maintained on a wet A. albopictus. Previous studies have identified thousands of paper towel for approximately 48 h, then gently air dried and stored differentially expressed genes across multiple developmental time in a Tupperware container under a SD photoperiod at 21°C and 80% points, including during diapause induction (Huang et al., 2015), RH. A subset of eggs from each biological replicate was used to throughout diapause preparation (Batz et al., 2017; Poelchau et al., confirm diapause incidence (see below). At 11 dpo, eggs were 2011, 2013a; Reynolds et al., 2012) and in diapause maintenance gently brushed from the unbleached paper towel onto a weigh boat

(Batz et al., 2017; Poelchau et al., 2013b). These studies have and weighed to the nearest 1 μg using a Mettler-Toledo AX5 Journal of Experimental Biology

2 RESEARCH ARTICLE Journal of Experimental Biology (2018) 221, jeb189480. doi:10.1242/jeb.189480 microbalance (Mettler-Toledo, Columbus, OH, USA). The median Lipidome data generation sample mass was 5.014 mg (range: 3.329–9.009 mg). Finally, the To generate the lipidome data, the eight lipid extracts were analyzed eggs were transferred to a 1.5 ml Eppendorf tube and snap frozen in in a random order using an LC-MS/MS system. LC separation was liquid nitrogen. Snap freezing occurred between 12:00 h and performed on a Shimadzu UFLC XR system (Shimadzu, Kyoto, 13:00 h each day (Zeitgeber time 4–5 h) and all egg samples Japan) with a Waters Acquity HSS T3 column (1.8 μm particle size, collected on a single day were snap frozen simultaneously to limit 50 mm×2.1 mm i.d.; Waters, Milford, MA, USA). MS/MS analysis non-biological sources of variation. Snap frozen samples were was performed on an AB-SCIEX 5600 tripleTOF analyzer with a immediately stored at −80°C. DuoSpray ion source (AB-SCIEX, Redwood City, CA, USA). Each of the eight experimental samples was assessed once in positive ion Assessing diapause incidence mode and once in negative ion mode using MS/MS with dynamic To ensure that adults under a SD photoperiod produced diapause mass exclusion. The QC sample was run four times in each mode eggs and adults under a LD photoperiod produced non-diapause to monitor machine drift and assess the reliability of feature eggs, we quantified diapause incidence with a subset of eggs quantification. In both positive and negative modes, mobile phase A collected from each biological replicate as previously described was acetonitrile:water (40:60) with 10 mmol l−1 ammonium (Urbanski et al., 2012). Briefly, 11 dpo eggs from each biological acetate and mobile phase B was acetonitrile:water:isopropanol replicate (four diapause, four non-diapause) were stimulated to (10:5:85) with 10 mmol l−1 ammonium acetate. The column was hatch by submersion in water and larval food slurry. The mean programmed to perform a gradient elution from 40% to 100% number of eggs assessed per replicate was 444 (range: 167–1047 mobile phase B over 10 min. Mobile phase B was maintained at eggs). We recorded the number of larvae that hatched in each 100% for 2 min, then decreased to 40% over 0.1 min. Prior to the biological replicate, air dried the remaining eggs, and then repeated next sample injection, the column was held at 40% for 3 min. Flow the process 1 week later. After the second attempt to stimulate rate was 0.4 ml min−1 and the column temperature was maintained hatching, the remaining unhatched eggs were bleached (Trpiš, at 55°C. 1970) and the number of embryonated, unhatched eggs was counted (i.e. the number of diapause eggs). Diapause incidence was Metabolome data generation calculated for each replicate according to the following formula: To generate the untargeted metabolomic data, eight metabolite (number of embryonated unhatched eggs)/(number of hatched eggs extracts were analyzed in a random order on an LC-MS system. LC +number of embryonated unhatched eggs). separation was performed on an Agilent 1290 Infinity Binary UHPLC system (Agilent, Santa Clara, CA, USA) with a Waters Sample preparation Acquity HSS T3 column (1.8 μm particle size, 100 mm×2.1 mm Lipidomic and metabolomic sample preparation and data generation i.d.). MS analysis was performed on an Agilent 6530 quadrupole were performed at the University of Michigan Metabolomics TOF system with a Jetstream ion source. Full mass spectra were Resource Core. All samples were shipped overnight on dry ice, acquired over the mass-to-charge ratio (m/z) range of 50–1000 Da. then stored at −80°C until extraction. All reagents were liquid Each of the eight experimental samples was analyzed once in chromatography (LC)-mass spectrometry (MS) grade and were positive ion mode and once in negative ion mode. The QC sample purchased from Sigma-Aldrich (St Louis, MO, USA). was run three times in each ion mode to assess machine drift and For the shotgun lipidomics, eight egg samples (four diapause, reliability of feature quantification. Mobile phase A was 0.1% four non-diapause) collected from separate biological replicates on a formic acid in water and mobile phase B was 0.1% formic acid in single day were thawed on ice then extracted using a modified methanol. The column was programmed to perform a gradient Bligh-Dyer method (Bligh and Dyer, 1959). The extraction was elution from 2% mobile phase B to 75% over 20 min. Next, mobile carried out at room temperature using 400 µl of water:methanol: phase B was increased to 98% over 2 min, held at this level for dichloromethane (1:1:1) and 12 internal standards from a range of 8 min, then reset to 2% in 0.1 min. Prior to the next sample lipid classes were added (Table S1). Next, the organic layer was injection, the column was re-equilibrated for 7 min. Throughout the collected and dried under a stream of nitrogen gas then re-suspended runs, flow rate was 0.35 ml min−1 and the column was maintained in 100 µl of acetonitrile:water:isopropanol (10:5:85) with at 55°C. 10 mmol l−1 ammonium acetate. Additionally, a pooled quality control (QC) sample was prepared by combining aliquots of each Feature identification and quantification experimental sample. Chromatographic peaks (i.e. features) detected during lipidomics For the untargeted metabolomics, eight additional egg samples and untargeted metabolomics were identified using the LipidBlast (four diapause, four non-diapause) collected from separate package and Agilent’s MassHunter software, respectively. Feature biological replicates on a single day were thawed on ice and alignment between samples was performed allowing up to 0.5 min combined with 400 µl of methanol:acetonitrile:acetone (1:1:1) and retention time (RT) shift and 20 ppm mass shift between sample five internal standards (Table S1). Next, tubes were sonicated for runs; these shifts were verified using the RTs of known internal 5 min in a water bath to settle the eggs before each sample was probe standards. Lipid features were identified by comparing MS/MS sonicated at 40% power for 10 s. Post-sonication, egg disruption spectra with in silico spectra in the LipidBlast database (Kind et al., was confirmed under a dissecting microscope. To complete 2013). Metabolite features were identified by recursively searching metabolite extraction, samples were further sonicated in a water the dataset using Agilent’s ‘Find by Feature’ algorithm. Metabolite bath for 60 min then centrifuged (14,000 g for 20 min at 4°C). features were then annotated by matching feature m/z and RT to Finally, 400 μl of the supernatant was transferred to a clean vial and an in-house library maintained by the University of Michigan dried under a stream of nitrogen gas. The extract was reconstituted in Metabolomics Resource Core containing known standards obtained 50 μl methanol:water (1:1) containing 1 μl zeatin, an instrument under identical analytical conditions (Table S2). Relative performance standard. A pooled QC sample was also prepared by quantification of lipids and metabolites was carried out using the combining aliquots of each experimental sample. AB-SCIEX Multiquant and Agilent MassProfiler Pro programs, Journal of Experimental Biology

3 RESEARCH ARTICLE Journal of Experimental Biology (2018) 221, jeb189480. doi:10.1242/jeb.189480 respectively. Lipidome and metabolome data are available via Hochberg, 1995); only functional groups and pathways containing Metabolomics Workbench, study accession ST000722. at least two metabolites annotated in our study were retained in either analysis. Lipidome and metabolome data analyses To ensure that our datasets contained only features that could be Characterization of diapause-exclusive features reliably quantified, we removed features with >30% relative Features that were detected in all four diapause samples but not standard deviation (RSD) calculated across replicate detected in any non-diapause samples were further investigated. All measurements of QC samples (Liu et al., 2014). Both datasets features exhibiting this pattern were unannotated metabolites. To were then scaled by pre-extraction egg mass to correct for variation better understand the potential diapause-associated functions of in input amount and the data were filtered by interquartile range to these unannotated metabolites, we examined all annotated remove low-information features (i.e. those features with low metabolites detected with similar RTs in our experiment. LC variability across all experimental samples in both treatment groups; separates molecules based on physical properties such that Gentlemen et al., 2005). Finally, data from both positive and molecules that elute with similar RTs share similar characteristics. negative ion modes were combined to produce a single lipid set and For each diapause-exclusive feature, we identified all annotated a single metabolite set. metabolites within ±0.5 min RT. Next, we determined the super- To visualize the overall similarity of lipidomic or metabolomic class, class and sub-class categorizations for each annotated profiles between treatment groups, we generated principal metabolite based on the molecular taxonomy in the Human component analysis (PCA) plots using data from all features Metabolome Database (HMDB; Wishart et al., 2007, 2018). (including unannotated features) via the MetaboAnalyst online toolkit (Xia and Wishart, 2011a; Xia et al., 2015). These analyses RESULTS were first performed with the pooled samples included to allow for Diapause incidence visual assessment of technical variation due to machine drift and Under a diapause-inducing SD photoperiod, the mean diapause then performed again with only the experimental samples included. incidence across biological replicates was 99.6% (range 99.2–100%), Next, we used MetaboAnalyst to calculate the log2 fold-change in while under a diapause-averting LD photoperiod, the mean diapause abundance between diapause and non-diapause eggs for each lipid incidence was 10.2% (range 6.3–14.0%; Table S3). Thus, our and metabolite (annotated and unannotated). experimental conditions consistently induced the desired phenotypes. For further analysis of lipid and metabolite shifts during diapause, only annotated lipids and metabolites were considered. For features LC-MS performance and data preparation identified by multiple adduct ions (e.g. cystine+H+ and cystine+Na+), Across four repeat measurements of our lipidomic QC sample, the adduct ion measured with the lowest RSD across technical the median RSD was 9.6%, while across three repeat measurements replicates of the QC sample was retained. The annotated data were of our metabolomics QC sample, the median RSD was 4.6%. then auto-scaled, such that metabolites were given equal weight in Additionally, PCA plots for both lipidomic and untargeted analysis and calculations of significance were decoupled from the metabolomic datasets showed tight clustering of QC measured concentration (van den Berg et al., 2006). Next, the fold- measurements indicating low machine drift throughout the change in relative abundance between diapause and non-diapause experimental runs (Fig. S1). Together, these results demonstrate a groups was calculated for each lipid and metabolite. Significant high level of consistency for the analytical platform used throughout differences in either lipids or metabolites between treatments were our experiments. identified via t-test followed by a Benjamini–Hochberg false In the lipid dataset, we annotated 265 lipids in positive mode and discovery rate (FDR) correction to account for multiple 202 lipids in negative mode (467 total lipids). From this set we comparisons (Benjamini and Hochberg, 1995). The top 50 lipids, retained 400 lipids with RSD <30% across repeated QC sample as ranked by P-value, were further analyzed by generating a heat measurements. We then removed duplicate adducts to produce a map using MetaboAnalyst. For each lipid class, we calculated the final dataset that contained 250 lipids. A PCA plot of these lipid unsaturation index, which quantifies the average number of features showed clear clustering of diapause and non-diapause unsaturated bonds per fatty acid (Lehmann et al., 2018). samples (Fig. 1A) with 56.4% of the variation explained by the first Significant differences in unsaturation index by lipid class two principal component axes. between diapause and non-diapause eggs were identified using In the untargeted metabolomics dataset, we identified 4511 t-tests followed by a Benjamini–Hochberg FDR correction features in positive mode and 1157 features in negative mode (5668 (Benjamini and Hochberg, 1995). total features). From this set, we retained 5216 features with To identify functionally related sets of metabolites affected RSD <30% across QC sample measurements. We generated a PCA by diapause state, we performed both quantitative enrichment plot of these features and found a clear separation of diapause and analysis (QEA) and pathway enrichment analysis (PEA). QEA non-diapause samples (Fig. 1B), with 87.9% of the variation is derived from the gene set enrichment analysis originally explained by the first two principal component axes. developed for transcriptomics and identifies coordinated changes Degradation is a consistent concern in metabolomic studies due within metabolic pathways (Xia and Wishart, 2010). PEA is to thermal instability (Lerma-Ortiz et al., 2016) and/or enzymatic conceptually similar to QEA, but further integrates topological conversions of short half-life molecules during sample processing information such that metabolites occupying more central positions (Vuckovic, 2012). In our study, we did not identify ATP or ADP, but in a pathway are weighted more heavily in the analysis than we did detect AMP and adenosine, consistent with enzymatic metabolites on the pathway periphery (Xia and Wishart, 2011b). For dephosphorylation during processing. However, we successfully both QEA and PEA, the complete in-house library of identifiable annotated several molecules known to be sensitive to degradation metabolites was used as the background reference set (Table S2). In during LC-MS sample preparation including idinosine, insosine both analyses, we performed a global test for significant enrichment monophosphate and arginine, suggesting acceptable metabolite followed by a Benjamini–Hochberg FDR correction (Benjamini and recovery (Fang et al., 2015; Lerma-Ortiz et al., 2016). Journal of Experimental Biology

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AB 15 +2e+07 ND4

ND1 ND2 D1 D2 ND4 ND3 ND1 ND2 0 ND3 0 D3 D4 D2 PC axis 2 (14.8%) PC axis 2 (22.2%) D3 D4 D1

–15 –3e+07

–20 0 30 –4e+07 0 4e+07 PC axis 1 (34.2%) PC axis 1 (73.1%)

Fig. 1. Overall metabolomic and lipidomic profiles differ for diapause and non-diapause samples. Principal component analysis (PCA) plot of (A) lipidomic and (B) untargeted metabolomic datasets. Purple triangles represent biological replicates of diapause (D) eggs. Green crosses represent biological replicates of non-diapause (ND) eggs. Purple and green shaded regions surrounding the points represent the 95% confidence interval for diapause and non-diapause conditions, respectively. The percentage variance explained by each of the first two principal components is listed parenthetically.

Metabolite annotation is another significant challenge for Pathway enrichment analysis identified tyrosine metabolism and untargeted metabolomics (Aksenov, et al., 2017; Dunn et al., 2013; tryptophan metabolism as significantly enriched in diapause eggs Johnson and Gonzalez, 2012). Untargeted LC-MS datasets contain (Fig. S2). features representing various isotopes, alternative adducts, experimental artifacts and metabolites without commercially Diapause-exclusive features available standards for comparison (Mahieu and Patti, 2017). Thus, Among the detected features, 36 were found in all four diapause egg untargeted studies typically unambiguously annotate 2–10% of samples but were undetected in all four non-diapause samples. In detected features (Aksenov et al., 2017). In our study, 389 features contrast, only one feature was detected in all four non-diapause (7.5%) were annotated as specific metabolites with high confidence samples but undetected in all four diapause samples. None of the 36 (Table S2). Removing duplicate adduct ions from this annotated set diapause-exclusive features could be annotated but the composite produced a final dataset that contained 241 metabolites. spectra, masses and RTs for these unknown features are listed in Table S5. All 36 features were detected in positive ion mode and 31 Differentially abundant lipids and metabolites of these features had RTs between 21.3 and 24.2 min. Annotated We identified nine lipids, two diacylglycerides (DGs) and seven metabolites eluting within 0.5 min of the unknown, diapause- triacylglycerides (TGs; Fig. 2A), that were significantly more abundant exclusive features were nearly all in the ‘lipid and lipid-like in diapause eggs than in non-diapause eggs. A heat map of the top 50 molecule’ HMDB super class (84.7% of metabolites). Further, the lipids, as ranked by P-value (Fig. 2B; Table S4), revealed that the top three HMDB molecule classes in the co-eluting annotated distinct lipidome of diapause is due to lipid accumulation, primarily in dataset were ‘steroids and steroid derivatives’ (32.8%), ‘fatty acyls’ the form of TGs (30 lipids, 60%) and DGs (11 lipids, 22%). There were (30.0%) and ‘glycerophospholipids’ (14.3%). Within the steroid no differences in saturation levels of these lipids, except for plasmenyl and steroid derivative class, most annotated metabolites were phosphatidylethanolamines (plasmenyl-PEs), which were more classified as ‘bile acids and bile acid derivatives’ (52.2%). saturated in diapause (Table 1; t=4.13, d.f.=6, P=0.006). Our untargeted metabolomics dataset (Fig. 3A; Table S4) DISCUSSION included 296 features that were less abundant (≤−1 fold-change) Diapause is a widespread adaptation that is crucial for overwinter and 102 features that were more abundant (≥+1 fold-change) in survival as well as defining the distribution and abundance of many diapause eggs relative to non-diapause eggs (Fig. 3A). Further insect populations (Bale and Hayward, 2010; Bradshaw, 1993; Lees, analysis including only annotated metabolites identified 49 1956; Tauber et al., 1986). Despite the ecological significance of metabolites with fold-change values ≥|1|, 47 of which were less diapause, relatively little was known about the molecular regulation abundant in diapause eggs (Fig. 3B). We identified 17 annotated of this adaptation until recently (Denlinger, 2002). In the past decade, metabolites with significant differences in abundance (Table 2). high-throughput RNA sequencing has rapidly advanced our Eleven of these metabolites had a fold-change >|1|, all of which understanding of the transcriptional basis of diapause (Koštál et al., were less abundant in diapause eggs. 2017; Ragland and Keep, 2017). However, fewer studies have investigated regulatory mechanisms at the metabolite level, especially Metabolite set enrichment analyses in the case of embryonic diapause. In this paper, we addressed this Quantitative enrichment analysis identified five metabolic pathways gap by identifying shifts in lipids, metabolites and metabolic that were significantly enriched in diapause eggs (Table 3; Table S6). pathways associated with embryonic diapause in A. albopictus. Journal of Experimental Biology

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5 A

3 -value) P ( 10 2 –log

0 CE (6) LysoPC (1) LysoPE (11) Plama-PE (15) CL (8) DG (14) FFA (14) PC (37) PE (41) PG (15) PI (12) PS (16) SM (4) TG (56)

B D1 D2 D3 D4 ND1 ND2 ND3 ND4 DG 28:1 DG 30:1 DG 30:2 DG 32:1 DG 32:2 DG 33:2 DG 34:2 DG 34:3 DG 34:4 DG 34:5 DG 36:4 PE 31:1 PE 35:3 Pl 34:2 Plasm-PE 34:2 PS 32:1 2 PS 32:2 PS 34:1 PS 34:2 PS 36:3 TG 49:2 1 TG 49:3 TG 50:4 -score TG 50:5 Z TG 51:1 0 TG 51:2 TG 51:3 TG 51:4 TG 52:1 TG 52:2

Row-wise –1 TG 52:3 TG 52:4 TG 52:5 –2 TG 52:6 TG 52:7 TG 53:2 TG 53:3 TG 54:0 TG 54:1 TG 54:2 TG 54:3 TG 54:4 TG 54:5 TG 54:7 TG 54:8 TG 56:1 TG 56:3 TG 56:4 TG 56:7 TG 56:8

Fig. 2. Diapause eggs have increased lipid abundance, primarily in the form of diacylglycerides and triacylglycerides. (A) log10 P-value of t-test for differences in lipid abundance between biological replicates of diapause (n=4) and non-diapause (n=4) eggs. Lipids are sorted and color-coded by class; the number of measured lipids within each class is identified parenthetically. The dashed line indicates a false discovery rate (FDR)-corrected P-value of 0.05; the solid line indicates the top 50 lipids by P-value. (B) The relative abundance for each of the top 50 lipids according to P-value in A displayed as a heat map. Each row in the heat map has been normalized such that the color corresponds to the Z-score for that lipid. Red cells represent higher relative abundance (Z-score) in diapause eggs and blue cells represent lower relative abundance in diapause eggs. Colored blocks to the right of the heat map match the lipid class colors used in A. Lipid abbreviations are as follows: CE, ceramide; CL, cardiolipin; DG, diacylglyceride; FFA, free fatty acid; lysoPC, lyso-phosphatidylcholine; lysoPE, lyso-phosphatidylethanolamine; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PI, phosphatidylinositol; Plasm-PE plasmenyl-phosphoethanolamine; PS, phosphatidylserine; SM, sphingomyelin; TG, triacylglyceride.

Lipidomics storage molecule in insects and accumulation of lipids, We identified distinct lipid profiles for eggs in early diapause particularly TGs, is a common hallmark of diapause (Hahn and maintenance (11 dpo) compared with those in age-matched, Denlinger, 2007; Hahn and Denlinger, 2011). Blood-fed non-diapause eggs (Fig. 1A). Lipids are the primary energy A. albopictus adult females overexpress the gene encoding fatty Journal of Experimental Biology

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Table 1. Unsaturation index change in diapause eggs versus Table 2. Overview of top 20 differences by P-value in relative metabolite non-diapause eggs by lipid class abundance between diapause and non-diapause eggs

Δ Unsaturation index log2(fold-change FDR-corrected Lipid class (D–ND) P-value Metabolite D–ND) P-value Plasmenyl-phosphatidylethanolamines −0.06 0.01 N-Acetylserotonin −1.07 0.032 Cardiolipins +0.01 0.06 Octopamine −2.53 0.032 Lyso-phosphatidylethanolamines −0.02 0.06 Hippuric acid −1.02 0.032 Triacylglycerols +0.01 0.07 Phosphocholine −1.30 0.032 Lyso-phosphatidylcholines −0.03 0.17 Glycero-3-phosphocholine −0.77 0.032 Phosphatidylinositols −0.02 0.18 Inosine −0.45 0.032 Ceramides −0.04 0.23 Dodecanoylcarnitine −1.72 0.032 Diacylglycerols +0.01 0.37 Nicotinic acid −0.82 0.032 Phosphatidylserines −0.04 0.43 Nicotinuric acid −1.23 0.032 Phosphatidylcholines −0.01 0.48 Azelaic acid −1.14 0.032 Sphingomyelins −0.01 0.54 Guanosine −0.48 0.042 Free fatty acids −0.01 0.68 Carnitine −0.30 0.042 Phosphatidylglycerols +0.01 0.77 Tetradecanoyl-L-carnitine −1.55 0.042 Phosphatidylethanolamines −0.01 0.92 Oleoyl glycine −1.80 0.042 − D, diapause; ND, non-diapause. Deoxycytidine 0.74 0.042 Dopamine −3.29 0.046 Allantoin −1.05 0.046 acid synthase when induced to produce diapause eggs compared Biotin +1.23 0.052 with adult females induced to produce non-diapause eggs; this Histamine −0.97 0.059 upregulation is consistent with greater maternal provisioning of lipids Xanthine −1.98 0.060 to diapause eggs (Huang et al., 2015). Additionally, A. albopictus A complete list of metabolites along with associated fold-changes and diapause eggs upregulate the gene encoding lipid storage droplet P-values is provided in Table S4. protein 2 during diapause preparation (3 dpo; Poelchau et al., 2013a), FDR, false discovery rate. differentially express 23 genes related to lipid metabolism during early diapause maintenance (11 dpo; Poelchau et al., 2013b) and have A. albopictus, three genes encoding fatty acid desaturases are greater overall lipid abundance (Reynolds et al., 2012). Our results upregulated in blood-fed adult females under diapause-inducing further reveal that the increased lipid abundance in A. albopictus conditions, consistent with increased maternal provisioning of diapause is primarily due to an accumulation of DGs and TGs unsaturated fatty acids (Huang et al., 2015). Furthermore, the gene (Fig. 2B). We identified nine lipid features (2 DGs, 7 TGs) that were encoding Δ(9)-desaturase, a rate-limiting enzyme in unsaturated significantly more abundant during diapause (Fig. 2A), although we fatty acid synthesis, is significantly upregulated during diapause lacked the resolution necessary to determine the specific fatty acid preparation (6 dpo; Reynolds et al., 2012). In this species, diapause composition of these differentially abundant lipids. moderately improves cold tolerance but this effect is greatly We assessed changes in saturation across lipid classes during enhanced by a period of cold acclimation (i.e. extended exposure to diapause but detected only minor changes to plasmenyl-PEs sub-lethal low temperatures; Hanson and Craig, 1994). Thus, it is (Table 1). This result was contrary to our expectations, because possible that in A. albopictus, changes in lipid unsaturation may changes in lipid unsaturation are frequently observed during occur following exposure to low temperatures (i.e. cold diapause in other species (Bashan and Cakmak, 2005; Bennett acclimation), rather than diapause per se. et al., 1997; Izumi et al., 2009; Khani et al., 2007; Shimizu, 1992). Increased abundance of unsaturated fatty acids promotes lipid Metabolomics mobility and membrane fluidity at low temperatures, contributing Sample clustering in our metabolite PCA plot (Fig. 1B) indicated to cold tolerance (Clark and Worland, 2008; Hazel, 1995). In that the overall metabolite profile differed between diapause and

ABFig. 3. Metabolites tend to exhibit +4 +4 lower abundance in diapause eggs. Data are log2 fold-change in relative abundance of untargeted metabolites +2 +2 in biological replicates of diapause eggs (n=4) relative to non-diapause 0 0 eggs (n=4) for (A) all features with relative standard deviation (RSD)<30% and (B) only annotated metabolites. –2 –2 (fold-change D–ND) Dashed lines indicate a log2 fold- 2 change of |1|. Purple dots indicate log –4 –4 metabolites with >|1| log2 fold-change and green dots represent metabolites

with >|2| log2 fold-change; counts of the number of features at each log (fold-change D–ND) No. of features log (fold-change D–ND) No. of features 2 2 fold-change cutoff are provided in the >2 18 >2 0 tables below each figure. A complete >1 84 >1 2 list of log2 fold-change values for all <–1 268 <–1 39 metabolites in these figures is provided in Table S4.

<–2 28 <–2 8 Journal of Experimental Biology

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Table 3. Overview of top 10 pathways identified by quantitative were significantly less abundant (Table 2) and the remaining nine enrichment analysis showed a downward trend (Table S4). This consistent pattern of Metabolites reduced acyl-carnitine abundance supports the hypothesis that the carnitine shuttle is suppressed independent of CPT expression in Pathway N Δ Abundance P-value early diapause, likely contributing to lipid conservation during Tryptophan metabolism 9 2 Up 7 Down 0.026 diapause via reduced β-oxidation. Catecholamine biosynthesis 3 0 Up 3 Down 0.029 We also found that phosphocholine was less abundant in Tyrosine metabolism 7 1 Up 6 Down 0.029 Bile acid biosynthesis 5 0 Up 5 Down 0.030 diapause eggs (Table 2). Phosphocholine is the metabolite utilized Biotin metabolism 2 1 Up 1 Down 0.039 in the rate-limiting step of phosphatidylcholine (PC) synthesis via Nicotinate and nicotinamide metabolism 3 0 Up 3 Down 0.053 the Kennedy pathway (Pol et al., 2014; Kent, 1997). PCs are a major Histidine metabolism 4 0 Up 4 Down 0.083 component of membranes and in D. melanogaster they comprise Insulin-like signaling 4 0 Up 4 Down 0.083 approximately 25% of the surface of lipid droplets (Pol et al., 2014). Pyrimidine metabolism 13 2 Up 11 Down 0.083 – Such droplets are the primary intracellular reservoir for neutral Glucose alanine cycle 6 1 Up 5 Down 0.084 lipids like TGs (Suzuki et al., 2011) and thus play a critical role in N, number measured; Δ Abundance, abundance change in diapause eggs. A long-term energy storage for insects in diapause (Arrese and complete list of altered metablotes by pathway is provided in Table S6. Soulages, 2010). Greater TG abundance activates the Kennedy pathway, which produces additional surface PCs to prevent lipid non-diapause eggs. Distinct metabolome profiles for diapause and droplet coalescence (Krahmer et al., 2011). However, when the non-diapause samples have been consistently observed across insect Kennedy pathway is experimentally suppressed in D. melanogaster taxa in the limited metabolomic studies to date (Dean et al., 2016; cells, PC production is hindered, resulting in larger lipid droplets Khodayari et al., 2013; Lehmann et al., 2018; Li et al., 2015; Lu that are more resistant to lipolysis (Krahmer et al., 2011). Increased et al., 2014; Michaud and Denlinger, 2007; Puraćet al., 2015; Teets lipid accumulation and reduced lipolysis are key facets of the et al., 2012; Vesala et al., 2012; Wang et al., 2017). In our samples, diapause phenotype across diverse insect taxa (Hahn and Denlinger, most features were less abundant (Fig. 3A) and this difference was 2011). Aedes albopictus diapause eggs have increased neutral lipid more pronounced among our annotated metabolites (Fig. 3B). abundance (Fig. 2) but we hypothesize that reduced phosphocholine Lower metabolite abundance has also been observed during abundance hinders PC production, thereby increasing the abundance diapause in larvae of Calliphora vicina (Johnson, 2013), and of larger, coalescent lipid droplets. Furthermore, in A. albopictus,two adults of Tetranychus urticea (Khodayari et al., 2013), Drosophila genes encoding enzymes on the Kennedy pathway are downregulated montana and Oncopeltus fasciatus (Dean et al., 2016; Vesala et al., during diapause preparation, consistent with reduced PC abundance 2012). However, this pattern is not universal. The majority of altered and the production of larger lipid droplets (Poelchau et al., 2013b; metabolites are more abundant in diapausing larvae of Nasonia Reynolds et al., 2012). Together, these data imply that adjustments in vitripennis (Li et al., 2015), and in diapausing pupae of Pieris napi the Kennedy pathway may be an important component of lipolysis (Lehmann et al., 2018) and Helicoverpa armigera (Lu et al., 2014). suppression in A. albopictus diapause. For the remainder of the Discussion, we focus on specific metabolites and metabolic pathways that are significantly altered in Regulation of developmental arrest diapause. We suggest potential diapause-associated roles for these Two catecholamines, dopamine and octopamine, were significantly metabolites and pathways. Where possible, we interpret our less abundant in diapause (Table 2). Furthermore, the tyrosine metabolome data with orthogonal evidence from previous metabolism pathway containing these molecules was significantly transcriptional analyses in this species. We note that some altered in diapause, as was catecholamine biosynthesis, a sub- pathways that were expected a priori to change under diapause pathway wholly contained within the tyrosine metabolism pathway conditions, such as glycolysis and the TCA cycle (Ragland et al., (Table 3; Fig. S2). Dopamine and octopamine can act as 2010), were not identified, likely because of limited annotation of neuromodulators regulating insect hormonal activity (Gilbert key metabolites in these pathways. et al., 2000). However, their specific role can vary from promoter to inhibitor, depending on species, age and sex (e.g. Granger et al., Alterations in lipid metabolism pathways 1996; Gruntenko et al., 2007; Kaatz et al., 1994; Pastor et al., 1991; As anticipated based on the lipidomic results (Figs 1A and 2), the Woodring and Hoffmann, 1994). The role of juvenile hormone III abundance of several metabolites relevant to lipid metabolism was (JH3) in regulating diapause is similarly difficult to generalize. For altered in diapause consistent with decreased lipid catabolism and example, repression of JH3 synthesis maintains the adult diapause increased lipid storage. For example, carnitine was significantly less of Culex pipiens (Sim and Denlinger, 2008; Spielman, 1974) but abundant in diapause eggs (Table 2). Carnitine is bound to fatty elevated JH3 abundance initiates and maintains larval diapause in acyl-CoA molecules via the enzymatic action of carnitine Diatraea grandiosella (Yin and Chippendale, 1973). Given the palmitoyltransferase (CPT), to produce acyl-carnitines that can be variable roles played by these molecules and the lack of an shuttled into the mitochondrial matrix where β-oxidation occurs identified hormonal mechanism to initiate and maintain diapause in (Bremer, 1983). The formation of acyl-carnitines is the rate-limiting A. albopictus (Denlinger and Armbruster, 2016), it is difficult to step in β-oxidation and thus a critical regulatory point in the predict how repression of catecholamines and alterations in their generation of energy from lipid stores (Foster, 2004). The gene associated metabolic pathway might contribute to diapause encoding the CPT enzyme is significantly downregulated during regulation in this species. However, treating A. albopictus diapause preparation (6 dpo) in A. albopictus, suggesting diapause eggs with a JH3 analog terminates diapause (Suman transcriptional repression of the carnitine shuttle system (Poelchau et al., 2015) and expression of a gene encoding a JH3-degrading et al., 2013a). In contrast, CPT expression is not altered in early esterase is upregulated throughout diapause maintenance (Poelchau diapause (11 dpo; Reynolds et al., 2012; Poelchau et al., 2013b). et al., 2013b). Together, these data suggest that low levels of JH3

However, of the 11 acyl-carnitines detected in early diapause, two may help maintain diapause in A. albopictus. Potential upstream Journal of Experimental Biology

8 RESEARCH ARTICLE Journal of Experimental Biology (2018) 221, jeb189480. doi:10.1242/jeb.189480 regulation of JH3 by dopamine and octopamine merits further Supplementary information investigation. Supplementary information available online at Oleoyl glycine was significantly less abundant in diapause eggs http://jeb.biologists.org/lookup/doi/10.1242/jeb.189480.supplemental (Table 2). This metabolite is an N-acyl amino acid that, in rats, References increases insulin-mediated inactivation of forkhead transcription Aksenov, A. A., da Silva, R., Knight, R., Lopes, N. P. and Dorrestein, P. C. factor (FOXO) via the Akt pathway (Wang et al., 2015). Oleoyl (2017). Global chemical analysis of biology by mass spectrometry. Nat. Rev. glycine has not been studied in insects, but because many Chem. 1, 0054. Armbruster, P. and Conn, J. E. (2006). Geographic variation of larval growth in components of the insulin signaling pathway are conserved in North American Aedes albopictus (Diptera: Culicidae). Ann. Entomol. Soc. Am. insects and mammals (Claeys et al., 2002), a significant reduction of 99, 1234-1243. this metabolite in diapause A. albopictus eggs could increase FOXO Armbruster, P. and Hutchinson, R. A. (2002). Pupal mass and wing length as activity as a result of reduced insulin-mediated phosphorylation. indicators of fecundity in Aedes albopictus and Aedes geniculatus (Diptera: Culicidae). J. Med. Entomol. 39, 699-704. Such FOXO activation should result in greater fat accumulation and Arrese, E. L. and Soulages, J. L. (2010). Insect fat body: energy, metabolism, and improved resistance to oxidative stress, as occurs in the adult regulation. Annu. Rev. Entomol. 55, 207-225. diapause of C. pipiens and D. melanogaster, as well as during dauer Bale, J. S. and Hayward, S. A. L. (2010). Insect overwintering in a changing climate. formation in Caenorhabditis elegans, a diapause-like state in worms J. Exp. Biol. 213, 980-994. Barthel, A., Schmoll, D. and Unterman, T. G. (2005). FoxO proteins in insulin (Barthel et al., 2005; Sim and Denlinger, 2008). For A. albopictus, action and metabolism. Trends Endocrinol. Metab. 16, 183-189. no Akt pathway genes are differentially expressed during diapause Bashan, M. and Cakmak, O. (2005). Changes in composition of phospholipid and maintenance (Poelchau et al., 2013b) but the Akt gene is triacylglycerol fatty acids prepared from prediapausing and diapausing individuals downregulated in diapause preparation (6 dpo; Poelchau et al., of Dolycoris baccarum and Piezodorus lituratus (Heteroptera: Pentatomidae). Ann. Entomol. Soc. Am. 98, 575-579. 2013a). Additionally, the insulin-suppressed microRNA bantam-5p Batz, Z. A., Goff, A. C. and Armbruster, P. A. (2017). MicroRNAs are differentially is significantly more abundant in diapause eggs (Batz et al., 2017) abundant during Aedes albopictus diapause maintenance but not diapause consistent with reduced insulin. In our data, metabolite changes in induction. Insect Mol. Biol. 26, 721-733. the insulin-like signaling pathway approached significance Benjamini, Y. and Hochberg, Y. (1995). Controlling the false discovery rate : a practical and powerful approach to multiple testing. J. R. Stat. Soc. B 57, 289-300. (Table 3). Together, these data suggest that insulin-mediated Bennett, V. A., Pruitt, N. L. and Lee, R. E., Jr. (1997). Seasonal changes in fatty regulation of FOXO via oleoyl glycine could contribute to acid composition associated with cold-hardening in third instar larvae of Eurosta diapause regulation in A. albopictus. solidaginis. J. Comp. Physiol. B Biochem. Syst. Environ. Physiol. 167, 249-255. Finally, among unannotated metabolites (Table S5), 36 features Bligh, E. G. and Dyer, W. J. (1959). A rapid method of total lipid extraction and purification. Can. J. Biochem. Phys. 37, 911-917. were exclusively identified in all four diapause samples. Annotated Bradshaw, W. E. (1993). Evolution in seasonal environments. In Seasonal metabolites co-eluting with these unknown features were primarily Adaptation and Diapause in Insects (ed. M. Takeda and S. Tanaka), classified as steroids/steroid derivatives (Table S5). Ecdysteroids, a pp. 121-133. Tokyo: Bun-ichi SôgôShuppan. class of steroid hormones not present in our annotation database, are Bradshaw, W. E., Zani, P. A. and Holzapfel, C. M. (2004). Adaptation to temperate climates. Evolution 58, 1748-1762. known to regulate larval and pupal diapause in numerous insect Bremer, J. (1983). Carnitine – metabolism and functions. Physiol. Rev. 63, species (Denlinger, 2002) and high ecdysteroid abundance appears 1420-1480. to maintain embryonic diapause in Lymantria dispar (Suzuki et al., Cajka, T. and Fiehn, O. (2016). Toward merging untargeted and targeted methods 1993; Lee et al., 1997). In A. albopictus, both microRNAs (Batz in mass spectrometry-based metabolomics and lipidomics. Anal. Chem. 88, 524-545. et al., 2017) and mRNAs (Poelchau et al., 2011; Poelchau et al., Claeys, I., Simonet, G., Poels, J., Van Loy, T., Vercammen, L., De Loof, A. and 2013a,b) involved in ecdysone signaling are differentially abundant Vanden Broeck, J. (2002). Insulin-related peptides and their conserved signal in diapause. Thus, the consistent observation of alterations related to transduction pathway. Peptides 23, 807-816. ecdysone signaling at the transcriptional level throughout diapause, Clark, M. S. and Worland, M. R. (2008). How insects survive the cold: molecular mechanisms - a review. J. Comp. Physiol. B Biochem. Syst. Environ. Physiol. 178, combined with this intriguing pattern of diapause-specific, steroid- 917-933. like features detected in our dataset, suggest ecdysone signaling may Colinet, H., Renault, D., Charoy-Guével, B. and Com, E. (2012). Metabolic and contribute to diapause regulation in A. albopictus. proteomic profiling of diapause in the aphid parasitoid Praon volucre. PLoS ONE 7, e32606. Acknowledgements Danks, H. V. (1987). Insect Dormancy: An Ecological Perspective. Ottawa: We thank the University of Michigan Metabolomic Resource Core for performing Biological Survey of Canada (Terrestrial Arthropods). ̌ ́ ̌ the lipidomic and untargeted metabolomic data collection. We also thank Dean, C. A. E., Teets, N. M., Kostal, V., Simek, P. and Denlinger, D. L. (2016). Drs Maureen Kachman and Maeva Millan for technical assistance. Finally, we thank Enhanced stress responses and metabolic adjustments linked to diapause and Drs Steven Cook, Leslie Ries, Anne Rosenwald, Vladimir Koštál and Colin Brent and onset of migration in the large milkweed bug Oncopeltus fasciatus. Physiol. two anonymous reviewers for valuable feedback on earlier versions of this Entomol. 41, 152-161. Denlinger, D. L. (2002). Regulation of diapause. Annu. Rev. Entomol. 47, 93-122. manuscript. Denlinger, D. L. and Armbruster, P. A. (2014). Mosquito diapause. Annu. Rev. Entomol. 59, 73-93. Competing interests Denlinger, D. L. and Armbruster, P. A. (2016). Molecular physiology of mosquito The authors declare no competing or financial interests. diapause. In Advances in Insect Physiology, Vol. 51 (ed. A. S. Raikhel), pp. 329-361. Cambridge, MA: Academic Press. Author contributions Denlinger, D. L., Yocum, G. D. and Rinehart, J. P. (2012). Hormonal control of Conceptualization: Z.A.B., P.A.A.; Methodology: Z.A.B.; Formal analysis: Z.A.B.; diapause. In Insect Endocrinology (ed. L. I. Gilbert), pp. 430-463. Cambridge, MA: Investigation: Z.A.B.; Writing - original draft: Z.A.B., P.A.A.; Writing - review & editing: Academic Press. Z.A.B., P.A.A.; Visualization: Z.A.B.; Funding acquisition: P.A.A. Dunn, W. B., Erban, A., Weber, R. J. M., Creek, D. J., Brown, M., Breitling, R., Hankemeier, T., Goodacre, R., Neumann, S., Kopka, J. et al. (2013). Mass Funding appeal: metabolite identification in mass spectrometry-focused untargeted This work was supported by the National Institutes of Health (grant no. R15 metabolomics. Metabolomics 9, S44-S66. AI111328 to P.A.A.). Deposited in PMC for release after 12 months. Fang, M., Ivanisevic, J., Benton, H. P., Johnson, C. H., Patti, G. J., Hoang, L. T., Uritboonthai, W., Kurczy, M. E., Siuzdak, G. (2015). 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