Journal of Thermal Biology 45 (2014) 124–133

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Seasonal changes in the composition of storage and membrane lipids in overwintering larvae of the codling moth, Cydia pomonella Jan Rozsypal a, Vladimír Koštál a,b,n, Petra Berková a, Helena Zahradníčková a, Petr Šimek a a b

Institute of Entomology, Biology Centre of the Academy of Sciences of the Czech Republic, Branišovská 31, 370 05 České Budějovice, Czech Republic Faculty of Science, University of South Bohemia, České Budějovice, Czech Republic

art ic l e i nf o

a b s t r a c t

Article history: Received 8 July 2014 Received in revised form 26 August 2014 Accepted 26 August 2014 Available online 6 September 2014

The codling moth (Cydia pomonella) is a major insect pest of apples worldwide. It overwinters as a diapausing fifth instar larva. The overwintering is often a critical part of the insect life-cycle in temperate zone. This study brings detailed analysis of seasonal changes in lipid composition and fluidity in overwintering larvae sampled in the field. Fatty acid composition of triacylglycerol (TG) depots in the fat body and relative proportions of phospholipid (PL) molecular species in biological membranes were analyzed. In addition, temperature of melting (Tm) in TG depots was assessed by using differential scanning calorimetry and the conformational order (fluidity) of PL membranes was analyzed by measuring the anisotropy of fluorescence polarization of diphenylhexatriene probe in membrane vesicles. We observed a significant increase of relative proportion of linoleic acid (C18:2n6) at the expense of palmitic acid (C16:0) in TG depots during the larval transition to diapause accompanied with decreasing melting temperature of total lipids, which might increase the accessibility of depot fats for enzymatic breakdown during overwintering. The fluidity of membranes was maintained very high irrespective of developmental mode or seasonally changing acclimation status of larvae. The seasonal changes in PL composition were relatively small. We discuss these results in light of alternative survival strategies of codling moth larvae (supercooling vs. freezing), variability and low predictability of environmental conditions, and other cold tolerance mechanisms such as extending the supercooling capacity and massive accumulation of cryoprotective metabolites. & 2014 Elsevier Ltd. All rights reserved.

Keywords: Triacylglycerols Phospholipids Fatty acids Homeoviscous adaptation Membrane fluidity Cold tolerance

1. Introduction The codling moth (Cydia pomonella) is a major insect pest of apples in most apple producing areas situated in the temperate zones of both hemispheres and is currently spreading to subtropical and tropical countries (Barnes, 1991; Willett et al., 2009). The bionomy of codling moth is relatively well described (Audermard, 1991). In this paper, we focus on a Central European (South Bohemian) population, which overwinters as a diapausing fifth

Abbreviations: TG, triacylglycerol; PL, phospholipid; PE, phosphoethanolamine; PC, phosphocholine; PI, phosphoinositol; DSC, differential scanning calorimetry; HVA, homeoviscous adaptation; GC/MS, gas chromatography coupled to mass spectrometry; LC/ESI/MS, liquid chromatography combined with electrospray ionization mass spectrometry; FA, fatty acid; UFA/SFA, ratio of unsaturated/ saturated Fas; PUFA/MUFA, ratio of poly-unsaturated/mono-unsaturated Fas; UI, unsaturation index n Corresponding author at: Institute of Entomology, Biology Centre of the Academy of Sciences of the Czech Republic, Branišovská 31, 370 05 České Budějovice, Czech Republic. Fax: þ 420 385310354. E-mail address: [email protected] (V. Koštál). http://dx.doi.org/10.1016/j.jtherbio.2014.08.011 0306-4565/& 2014 Elsevier Ltd. All rights reserved.

instar larva in a loose cocoon in the litter near the base of apple trees (Miller, 1956; Rozsypal et al., 2013). The post-diapause larvae pupate inside their cocoons during April–June, and adults emerge from pupae during May–July (Miller, 1956). After mating, the females lay eggs on fruits, or on the leaves near the fruit, and the caterpillars develop inside the fruits. The codling moth has five larval instars (Williams and Macdonald, 1982) and most of the fully grown caterpillars directly enter diapause and overwinter (monovoltine life cycle) while only a small part may pupate and give rise to the summer generation (partially bivoltine life cycle) (Miller, 1956). The overwintering is often a critical part of insect life-cycles in the temperate zone with numerous abiotic (cold, frost, dehydration) and biotic (lack of food, predation, pathogens) stressors potentially threatening survival of individuals and, consequently, prosperity of the whole population (Leather et al., 1995; Bozinovic et al., 2011; Williams et al., 2014). We have reported on physiological mechanisms of cold tolerance in overwintering codling moth larvae in our earlier paper (Rozsypal et al., 2013). In accordance with other studies (Neven, 1999; Khani et al., 2007a; Khani and Moharramipour, 2007, 2010),

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we observed that the winter larvae extend their supercooling capacity and increase their chill-tolerance, which means that larvae mostly rely on a strategy of freeze-avoidance. Their whole-body supercooling point decreases from approximately 15.3 °C during summer to  26.3 °C during winter. Seasonal extension of supercooling capacity is assisted by partial dehydration, increasing osmolality of body fluids, and the accumulation of a complex mixture of winter specific metabolites. Glycogen and glutamine reserves are depleted, while fructose, alanine and some other sugars, polyols and free amino acids are accumulated during winter. The concentrations of trehalose and proline remain high and relatively constant in all seasons, and may contribute to the stabilization of proteins and membranes at subzero temperatures (Rozsypal et al., 2013). In parallel to the extension of supercooling capacity, winter larvae also acquire a good ability to survive in a frozen state (freeze-tolerance) after inoculation with external ice crystals (Rozsypal et al., 2013). Some aspects of cold tolerance physiology, especially the seasonal restructuring of lipid composition, have been insufficiently explored in codling moth larvae. There is only a single report by Khani et al. (2007b) who observed increasing/decreasing relative contents of linoleic (C18:2n6)/palmitic (C16:0) fatty acids, respectively, in total lipids extracted from codling moth larvae during their transition from direct development (non-diapause) to diapause state. The restructuring of lipid composition is intimately linked to changing ambient temperature in various poikilotherms including insects (for reviews, see Cossins and Sinensky, 1984; Hazel, 1989; Koštál, 2010). The adaptive meaning of lipid restructuring is often explained in terms of maintaining sufficient/ optimal phase and fluidity of either triacylglycerol (TG) depots (ensuring availability of energy stores for enzymatic breakdown) or phospholipid (PL) membranes (maintaining the membrane functionality and/or structural integrity) at low body temperatures (Irving et al., 1957; Cossins, 1994; Marshall et al., 2014). This adaptive explanation is generally known as “homeoviscous adaptation” (HVA, Sinensky, 1974) and its validity has been supported by indirect and direct pieces of evidence in different ectotherms (Browse et al., 1994; Allakhverdiev et al., 1999; Murray et al., 2007; Holmstrup et al., 2014). The main purpose of this paper was to bring detailed analysis of seasonal changes of lipid composition and fluidity in overwintering larvae of C. pomonella. Such data supplement our previous study (Rozsypal et al., 2013) and extend the insight into the physiology of overwintering and cold tolerance in codling moth larvae. The larvae were regularly sampled in the field throughout the overwintering period 2010/2011 in parallel with the samples taken for our earlier study (Rozsypal et al., 2013). Here, we focused on analysis of storage lipid depots in the fat body and membraneforming PLs in the whole body of codling moth larvae. In addition to detailed biochemical characterization of lipid composition, we also measured biophysical features that are related to lipid phase behavior and fluidity. The temperature of melting (Tm) in TG depots was assessed by using differential scanning calorimetry (DSC) and the conformational order of PL membranes was analyzed by measuring the anisotropy of fluorescence polarization of diphenylhexatriene probe in artificial membrane vesicles prepared from PLs that were extracted from larvae. Our data allowed us to test some of the basic predictions of HVA theory on mutual associations between ambient temperature, lipid composition, lipid fluidity and cold tolerance in a special case of codling moth larva.

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2. Materials and methods 2.1. Insects and chemicals Fully grown caterpillars of the last instar of the codling moth, C. pomonella (Walsingham, 1897) [synonym: Phalaena Tinea pomonella (Linnaeus, 1758)] (Lepidoptera: Tortricidae) were collected from apple tree alleys in the vicinity of České Budějovice (48°59′ NW, 14°29′ EL) in South Bohemia, Czech Republic. Approximately 200 circular bands (width 30 cm) made of corrugated cardboard were mounted on the apple tree trunks at the height of approximately 1.5 m above ground during May 2010. The fully grown caterpillars were “trapped” inside the bands during their wandering from the tree crown down to the soil. The cocooned caterpillars were collected from the bands on six sampling occasions during 2010/2011: 20 July 2010, non-diapause larvae; 6 September 2010, diapause maintenance; 11 November 2010, diapause termination; 10 January 2011, just terminated diapause; 8 March 2011, post-diapause low temperature quiescence; 11 April 2011, post-diapause resumption of developmental activity. The stages of diapause development (sensu Koštál, 2006) in different sampling dates were estimated based on our observations on photoperiodic sensitivity and rate of developmental resumption (time to pupation at constant 25 °C) measured in parallel samples of larvae (Rozsypal and Koštál, unpublished data). All larvae were transported to České Budějovice, stored outdoors overnight and processed the next morning. If not stated otherwise, all chemicals used in this study were purchased from Sigma-Aldrich (St. Louis, MO, USA). 2.2. Extraction and GC/MS analysis of fat-body storage lipids Fat bodies of three larvae were dissected and pooled. Three biological replicates (9 larvae in total) were taken on each date. The total lipids were extracted twice in 400 ml of chloroform: methanol solution (2/1, v/v) (Folch et al., 1957), evaporated to dryness and later reconstituted in 50 ml hexane. In a preliminary experiment, we found that the total fat-body lipids contain predominantly (98.2%) the non-polar lipids (mainly TGs, but also diacylglycerols, free and esterified fatty acids), while the polar lipids (mainly PLs) represent only 1.8% of the total fat-body lipids (Rozsypal and Koštál, unpublished data). This result means that total fat-body lipids nicely represent the storage form of lipids and we took them as a source material for biochemical analysis of storage lipid fatty acid composition and also for biophysical measurements of lipid melting temperature. To 2.5 ml of each hexane-reconstituted extract, 10 nmol of tetracosane diluted in hexane was added as an internal standard. The sample was then evaporated to dryness under nitrogen and the lipids were transesterified with 100 ml of 2 M sodium methanolate solution in dried methanol and 100 ml of hexane under extensive mixing (Zahradníčková et al., 2014). The reaction was stopped by adding 60 ml of 3 M HCl and the arising fatty acid methyl esters (FAMEs) were extracted twice with 200 ml of hexane. Finally, the sample extract was evaporated to dryness under nitrogen and then dissolved in 100 ml of isooctane and 1.5 ml aliquot was injected into a gas chromatograph GC-2014 equipped with an AOC-20 s autosampler and flame ionization detector, all controlled by a GC Solution software (all from Shimadzu, Kyoto, Japan). Separation and quantification of FAMEs were accomplished on a 30 m BPX-70 fused silica capillary column, 0.25 mm I.D.,

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0.25 mm film thicknesses (SGE, Canberra, Australia) by external calibration method by means of the authentic FAMEs standards. Operation conditions were: hydrogen carrier gas, 45 cm/s: injector temperature, 240 °C; split 9:1; detector, 260 °C; temperature program, 140 °C, 6 °C min  1 to 230 °C. Identity of the detected FAMEs in samples was further confirmed by GC/MS analysis using the same BPX-70 column installed in a Trace Ultra gas chromatograph coupled with a DSQ quadrupole mass spectrometer controlled by an Xcalibur 2.1 software (all from Thermo Scientific, San Jose, CA, USA) operated in EI mode, 70 eV, mass range, 40–400. The relative molar concentrations of individual FAs were expressed in percentages assuming that the total concentration is 100%. 2.3. DSC analysis of storage lipid melting temperatures Thermal analyzes of storage lipids were conducted using a differential scanning calorimeter DSC4000 (Perkin Elmer, Waltham, MA, USA). The heat flow and the temperature scale of the instrument were calibrated using mercury, indium and zinc and were verified by measuring the areas under the melting endotherms of known masses of ice and tripalmitoyl–acylglycerol standard. The chloroform solution of storage lipids was pipetted into the 50 ml aluminum pan and the solvent was allowed to evaporate under nitrogen at room temperature. The netto total mass of each lipid sample (approximately 2 mg) was measured as a difference between brutto and tara readings on NewClassic MF balance with sensitivity 0.01 mg (type MS 105DU, Mettler Toledo, Greifensee, Switzerland). Each lipid sample was hermetically sealed in the pan and subjected to a program consisting of 6 steps: (1) hold for 1 min at 30 °C; (2) warm to 40 °C at a rate of 1 °C min  1; (3) hold for 1 min at 40 °C; (4) cool to 70 °C at a rate of 1 °C min  1; (5) hold for 5 min at  70 °C; and (6) warm to 40 °C at a rate of 1 °C min  1. The melt endotherms on thermal curves were analyzed using Pyris Software (v. 10.1.0.0412, Perkin Elmer, Waltham, MA, USA). The peak (temperature) and area under each melt endotherm (enthalpy change during depot fat melting) were calculated (Caffrey et al., 1991). 2.4. Extraction and LC/ESI/MS analysis of phospholipid composition of membrane lipids Total lipids were extracted as described above from five larvae individually on each sampling date. Total lipid extract was then separated into polar and non-polar classes by dissolution and liquid–liquid extraction between 2 ml acetonitrile in water (80:20) and 2 ml hexane. The lower aqueous acetonitrile phase contained polar lipids (Koštál et al., 2013) and it was used as a source of PLs. The samples were analyzed using high performance LC combined with electrospray ionization mass spectrometry (ESI/MS) as described in previous studies (Tomčala et al., 2006; Overgaard et al., 2008; Koštál et al., 2013). Briefly, an LTQ-XL mass spectrometer (Thermo Fisher Scientific) equipped with ESI, Accela 600 pump HPLC system, and Accela AS autosampler (Thermo Fisher Scientific, San Jose, CA, USA) were used. A volume of 200 μl was collected from the PL fractions, solvent was evaporated to dryness and the residue dissolved in 300 μl of methanol. The aliquots of 5 μl were injected into a Gemini C18 HPLC column (150  2 mm2 ID, 3 μm) (Phenomenex, Torrance, CA, USA) thermostated at 35 °C. The mobile phase flow rate was 250 μl min  1 with gradient elution of A:B:C (A¼10 mM ammonium acetate in methanol with ammonia (0.025%), B ¼10 mM ammonium acetate in water, C ¼isopropanol–MeOH 8:2) – 0 min: 92:8:0, 7 min: 97:3:0, 12 min: 100:0:0, 19 min: 93:0:7, 20–23 min: 90:0:10, 24 min: 100:0:0 and for equilibration of column 26–45 min: 92:8:0. The ESI/MS was carried out either in the positive or the negative ion

detection mode at potential þ3 kV or 2.5 kV, with capillary temperature 200 °C. Eluting ions were detected with full scan mode from 200 to 1000 Da with the collisionally induced MS2 fragmentations (collision energy 35%). Data were acquired and processed by means of Xcalibur 2.1 software (Thermo Fisher Scientific). The responses of analyzed phospholipids were corrected by comparison to the signals of internal lipid standards that were obtained from Avanti Polar Lipids (Alabaster, AL, USA). The corrected areas under individual analytical peaks were expressed in percentages assuming that the total area is 100%. 2.5. Analysis of membrane lipid fluidity Total contents of phospholipids in the polar fractions of total lipids were measured using assay of phosphorus that was released by mineralization of the dry sample for 20 min at 180 °C in 125 ml of 70% perchloric acid (Rouser et al., 1970; Koštál et al., 2013). All samples were then levelled to exactly 50 mM concentration of PLs in phosphate buffered saline (PBS, amounts of solutes per 1 l: NaCl, 8 g; KCl, 0.2 g; Na2 HPO4  2H2O, 1.8 g; KH2PO4, 0.24 g; pH 7.4). The lipid samples were dried under the stream of nitrogen for approximately 10 min while constantly rotating the glass vial in order to create a uniform and thin film of lipids on its wall. Dry lipids were then dissolved in 2.5 mL of PBS and the solution was incubated at 65 °C for 2 h to allow hydration of lipids (the samples were Vortexed every 5 min during the second hour of hydration). The flocks and large multilamellar vesicles (LMVs) were formed during hydration. In order to increase the abundance of small unilamellar vesicles (SUVs), we sonicated the samples in a water bath sonicator (type UC 003 BS1, Tesla, Pardubice, Czech Republic) for 1 h at room temperature and 300 W. Next, we mixed PL solutions with 1,6-diphenyl-1,3,5-hexatriene (DPH) probe dissolved in tetrahydrofuran so that the ratio of final concentrations of lipids:probe was 100:1 (50:0.5 mM). The samples were then loaded into the MicroWell 96-Well Optical-Bottom Plates with Polymer Base (Thermo Scientific Nunc, Rochester, NY, USA) and analyzed by using the microplate reader Infinite 200Pro (Tecan, Grödig/Salzburg, Austria). Each sample was loaded in three technical replicates (the mean of which was taken for calculations) and the blank samples (no lipids) were also included. The excitation/ emission wavelengths were 360/430 nm. The anisotropy (A) of fluorescence polarization (FP) of the probe penetrating into the lipid bilayers of SUVs was measured at three different temperatures: 20 °C, 30 °C and 40 °C successively, with 30 min incubation at each temperature. The anisotropy was calculated using the following formula:

A = (G*Ipar − Icross )/(G*Ipar − 2*Icross ) where G is a correction factor of 1.198 based on calibration of our device using 1 nM fluorescein; Ipar is an emission intensity of polarized light parallel to the plane of excitation with blank values subtracted, and Icross is an emission intensity of polarized light perpendicular to the plane of excitation with blank values subtracted. The A is correlated with membrane conformational order (or inversely correlated with membrane fluidity) (Behan-Martin et al., 1993; Hazel, 1995). 2.6. Statistics Three different indices of the degree of FA unsaturation were calculated from the GC/MS or LC/ESI/MS analytical data. The UFA/ SFA ratio is a cumulative percent of all unsaturated fatty acids divided by the cumulative percent of all saturated fatty acids. The PUFA/MUFA ratio is an analogy for polyunsaturated vs. monounsaturated fatty acids. The unsaturation index (UI) gives the sum of

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the percent unsaturated FAs multiplied by their number of double bonds. All datasets (parameters) were tested for deviation from Gaussian distribution using the Kolmogorov–Smirnov (KS, alpha ¼0.05) test prior to statistical analysis. Since all datasets passed the KS test, we used parametric statistical methods. The differences in parameters between non-diapause (collected in July) and diapause (September) generations of larvae were tested using Student's unpaired two-tailed t-tests (alpha ¼ 0.05). The influence of the sampling date on parameters' development in overwintering generation of diapausing larvae (from September to April) was analyzed using one-way ANOVA (with confidence intervals set to 95%) followed by Bonferroni's multiple comparison tests to find the differences among individual sampling dates. These statistical calculations were performed using Prism v.4 (GraphPad Software, San Diego, CA, USA). The complex association of phospholipid composition as it related to the calendar season (sampling date) was determined by Principal Component Analysis (PCA) using Canoco v. 4.52 for Windows (Biometris-Plant Research International).

3. Results 3.1. Storage fat The GC/MS analysis detected seven dominant fatty acids in storage fat of C. pomonella larvae (Table S1). The seasonal changes in the degree of FA unsaturation and in the relative proportions of three most abundant species, namely palmitic acid (C16:0), oleic acid (C18:1n9), and linoleic acid (C18:2n6), are shown in Fig. 1. The changes in relative proportions of the three dominant FAs were mostly responsible for the changes in different indices describing the degree of FA unsaturation. All three descriptors, UFA/SFA, UI and PUFA/MUFA, confirmed that the degree of FA unsaturation was significantly higher in the diapausing larvae (September) than in the non-diapausing larvae (July). Relatively low degree of FA unsaturation in non-diapausing group was caused mainly by their relatively high content of C16:0 (25.5%) countered by low content of C18:2n6 (12.5%). Within the group of overwintering larvae (September–April), ANOVA detected a significant increase of UFA/SFA ratio in three samples taken during the coldest part of overwintering (November–March), while the samples taken during warmer parts (September and April) showed relatively lower UFA/SFA. The UI index gradually increased with time in overwintering larvae, which was caused mainly by steeply increasing PUFA/MUFA, which, in turn, was driven by the gradual increases of relative proportions of C18:2n6 and C18:3n3 (linolenic acid) countered by gradual decrease of C18:1n9 (Fig. 1 and Table S1). The seasonal change in degree of FA unsaturation was reflected in the change of lipid melting temperature. The DSC thermal analysis detected two relatively broad Tm transitions (melting endotherms) in each storage lipid sample (Fig. 2). The peaks of both endotherms shifted significantly toward left between July (non-diapausing larvae) and September (diapausing larvae), which means that the Tm1 dropped from  12.3 °C to  20.1 °C, while Tm2 dropped from þ7.5 °C to þ3.2 °C. Detailed analysis of melt endotherms is presented in Fig. S1, which documents that the decrease of Tm between July and September was accompanied with an alteration in the change of enthalpy (ΔH) of the endothermic event reflecting the quantitative change in lipid composition. During the overwintering period (September–April), the Tm2 remained almost constant (it fluctuated from þ2.2 to þ3.2 °C), while Tm1 continued in a decreasing trend and reached a minimum of  27.1 °C during April (Fig. S1).

Fig. 1. Results of GC/MS analysis of fatty acids (FAs) in storage lipids extracted from fat bodies of Cydia pomonella larvae sampled in the field on different months of 2010/2011. (A) the seasonal development of the indices of overall degree of unsaturation are shown: unsaturation ratio (UFA/SFA) and unsaturation index (UI). (B) the relative proportions of molar concentrations of three most abundant FAs (palmitic, C16:0; oleic, C18:1; and linoleic, C18:2) and the ratio PUFA/MUFA are shown. Each point or column is a mean 7SD of three biological replications. The differences between non-diapause (July) and diapause (September) generations were tested using Student's unpaired two-tailed t-tests (*, P o0.05; **, Po 0.01; ***, Po 0.001). The influence of sampling date on overwintering generation (from September to April) was analyzed using one-way ANOVA followed by Bonferroni's multiple comparison tests (means flanked by different letters are significantly different).

3.2. Membrane phospholipids The HPLC/ESI/MS analysis revealed 97 different molecular species of PLs in tissues of C. pomonella larvae (Table S2). The oleyl–linoleyl–phosphatidylcholine (PC 18:1/18:2, compound no. 51 in Table S2) was the most abundant PL species, which means that its relative proportions ranged between 5.3% and 9.6% of total PLs. The relative proportions of PC 18:1/18:2 reached a broad maximum during September–January, while the minima were observed during July and April. The complex association of membrane lipid composition with seasonal time was analyzed using PCA (Fig. S2). Thus, the sample taken during July was most characteristically associated with PE 18:0/18:1 (25); September with PC 18:2/18:2 (49); November and March with PI 18:1/18:2 (81); January with PC 18:1/18:2 (51); and April with PC 16:1/18:3 (40) or PI 18:0/18:3 (83) (numbers in brackets refer to the compound numbering in Table S2). Overall, the seasonal changes in relative proportions were small and the associations of individual molecular species with particular season were relatively weak (only seldom the eigenvectors were extending over 80% fit of the PCA model). For instance, the PE 18:2/18:2 (20) displayed the best fit to the PCA model among all PL species (87% fit, longest eigenvector), which, in practice, means that a broad peak of its relative proportion (1.8–2.5%) coincided with samples taken during cold period (September–March), while relatively low proportions (1.1% and 1.2%) were found in samples taken during warm periods (July and April, respectively). The PE 16:0/18:2 (14) also showed a weak association with cold season, with a broad peak of

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Fig. 3. Results of LC/ESI/MS analysis of molecular phospholipid in polar fraction of total lipids extracted from whole bodies of Cydia pomonella larvae sampled in the field on different months of 2010/2011. The relative proportions of major phospholipid classes were calculated from analytical data (see Tables S2 and S3 for details). (A) PE, phosphoethanolamines; PC, phosphocholines; PE/PC, ratio of PE to PC. (B) lyso-PL, lyso-phospholipids; PS, phosphoserines; PI, phosphoinositols; SM, sphingomyelins. Each point or column is a mean 7 SD of five biological replications (larvae). The differences between non-diapause (July) and diapause (September) generations were tested using Student's unpaired two-tailed t-tests (*, P o0.05; **, Po 0.01; ***, P o 0.001). The influence of sampling date on overwintering generation (from September to April) was analyzed using one-way ANOVA followed by Bonferroni's multiple comparison tests (means flanked by different letters are significantly different).

Fig. 2. Examples of melt endotherms obtained by thermal analysis (differential scanning calorimetry, DSC) of storage lipids extracted from fat bodies of Cydia pomonella larvae sampled in the field on different months of 2010/2011. The samples were warmed at a rate of 10 °C min  1. The heat flows in individual samples corresponded to the rates of electric energy conversion (mW, see segment) in the DSC device. The starting points on y-axis are arbitrary. The peaks of two melt endotherms were taken to represent major melting temperatures (Tm1 and Tm2) in a complex mixture of lipids and the areas under melt endotherms (highlighted) were integrated (mJ) to calculate the change of enthalpy (ΔH) of each endothermic event. Data analysis is corroborated in Fig. S1.

2.7–3.1% during September–March and two minima of 1.8% and 2.3% during July and April, respectively. In contrast, some other species, such as PE 18:0/18:1 (25), PC 16:1/18:3 (40), or PI 18:0/ 18:3 (83), showed a weak tendency to reach maximal relative proportions during warm periods of July and April (for details, see Fig. S2 and Table S2). The seasonal changes in relative proportion of different PL classes are summarized in Fig. 3 and Table S3. Phosphocholines (PCs) were the most abundant PL class and represented 46.5– 52.2% of total PLs. Phosphoethanolamines (PEs) were the second most abundant class (26.4–30.3%). The seasonal changes in PL classes were small but statistically significant in some cases. Thus, relative proportion of PCs slightly increased, while that of PEs slightly decreased during the transition to diapause between July and September. Among the samples of overwintering larvae,

ANOVA followed with post hoc test showed that the total PCs were highest in early diapausing larvae (September), while the total PEs were most abundant during cold months of January and March. The relative proportions of PL fatty acyls were calculated from HPLS/ESI/MS data and the most important results are summarized in Fig. 4 (for more details, see Table S3). The C. pomonella larval PLs were composed of 10 different fatty acyls. Two FAs, C18:1 and C18:2, clearly dominated and, together, represented more than half of total FA pool. In July sample, oleic acid was higher (32.8%) than linoleic acid (23.4%), while in September their relative proportions switched to 25.0% and 32.4%, respectively. This was by far the largest observed seasonal change, which was mostly responsible for the significant increase of the ratio of PUFA/MUFA (Fig. 4). Within the group of overwintering larvae (September– April), ANOVA detected a broad statistically significant peak of UFA/SFA ratio in two samples taken during the coldest part of overwintering (January–March), while the UI was not significantly affected by sampling date (Fig. 4). The fluidity of membrane vesicles prepared from total PLs extracted from tissues of codling moth larvae were measured as anisotropy of the fluorescence polarization of DPH probe at three different temperatures (Fig. 5). There was no difference in anisotropy between the samples of non-diapausing (July) and diapausing (September) larvae. A statistically significant increase of anisotropy was detected to occur between November and January.

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Fig. 4. Results of LC/ESI/MS analysis of molecular phospholipid in polar fraction of total lipids extracted from whole bodies of Cydia pomonella larvae sampled in the field on different months of 2010/2011. (A) The seasonal development of the indices of overall degree of unsaturation were calculated from analytical data (see Tables S2 and S3 for details): unsaturation ratio (UFA/SFA) and unsaturation index (UI). (B) the relative proportions of molar concentrations of two most abundant FAs (oleic, C18:1; and linoleic, C18:2) and the ratio PUFA/MUFA are shown. Each point or column is a mean 7 SD of five biological replications (larvae). The differences between non-diapause (July) and diapause (September) generations were tested using Student's unpaired two-tailed t-tests (*, Po 0.05; **, Po 0.01; ***, Po 0.001). The influence of sampling date on overwintering generation (from September to April) was analyzed using one-way ANOVA followed by Bonferroni's multiple comparison tests (means flanked by different letters are significantly different).

4. Discussion Compositional remodeling of lipids has been well documented as a widespread seasonal response to a decrease of body temperature in many different poikilothermic or heterothermic animals (Hazel, 1989; Frank, 1991; Cossins, 1994; Koštál, 2010). Nevertheless, recurrent discussions occur on the adaptive meaning of the observed seasonal changes. The question remains on whether, how and how much the lipid restructuring can contribute to the winter increase of cold tolerance. In this paper, we indirectly address this question by bringing detailed description of seasonal restructuring of storage and membrane lipids in overwintering larvae of codling moth and correlating these results with previously measured cold tolerance in the same species (Rozsypal et al., 2013). We know that the codling moth larvae rely on extensive supercooling (or freeze-avoidance) for survival of the winter cold (Neven, 1999; Khani and Moharramipour, 2010), but they can also survive in frozen state (Rozsypal et al., 2013). The non-diapause larvae collected in July showed relatively weak cold tolerance (no survival at  15 °C in supercooled state, no ability to tolerate freezing at  5 °C for 1 h), while the cold tolerance increased gradually in diapausing larvae and reached a broad plateau between November and April (30–40% survival at  15 °C/ 7 d in supercooled state; or 75–100% survival at  5 °C for 1 h in frozen state) (Rozsypal et al., 2013). These earlier data on survival at low temperatures will be now correlated with seasonal restructuring of lipids. Though the HVA theory (Sinensky, 1974) may serve as a common explanatory paradigm, we will discuss the seasonal changes in storage lipids and membrane lipids separately.

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Fig. 5. Anisotropy of fluorescence polarization of diphenylhexatriene probe in artificial membrane vesicles prepared from total phospholipids (PLs) that were extracted from larvae of Cydia pomonella sampled in the field on different months of 2010/2011. Anisotropy is directly related to conformational order (or indirectly related to fluidity) of artificial PL-bilayers. (A) Results of anisotropy analysis at three different temperatures. (B) Linear extrapolation of the data from panel A to lower temperatures. Each point is a mean7 SD of five biological replications (larvae). The differences between non-diapause (July) and diapause (September) generations were tested using Student's unpaired two-tailed t-tests (P 40.05, not significant). The influence of sampling date on overwintering generation (from September to April) was analyzed using one-way ANOVA followed by Bonferroni's multiple comparison tests (means flanked by different letters are significantly different).

4.1. Storage fat It has been well documented that various animals increase the relative proportion of unsaturated FAs in their storage fat either prior to or upon experiencing a decrease of body temperature linked to overwintering. This is true not only for insects (Harwood and Takata, 1965; Keith, 1966; Baldus and Mutchmor, 1988; Ohtsu et al., 1993; Joanisse and Storey, 1996; Buckner et al., 2004; Atapour et al., 2007) but also for vertebrate poikilotherms (Geiser et al., 1992; Shen et al., 2005) or heterothermic mammalian hibernators (Fawcett and Lyman, 1954; Frank, 1991; Geiser, 1993; Munro and Thomas, 2004). The seasonal increase in unsaturation is believed to be adaptive, aimed at preventing solidification/maintaining fluidity of depot fats which serve as substrates to fuel metabolism during period of dormancy at low body temperatures (Frank, 1991; Joanisse and Storey, 1996). Our analysis of FA composition in fat body lipid depots in larvae of C. pomonella of Central European population almost perfectly matches the results obtained earlier by Khani et al. (2007b) for the Middle East population. Not only the FA compositions, but also the seasonal responses, were closely similar in both populations. Considerable increase of relative proportion of linoleic acid (C18:2n6) at the expense of palmitic acid (C16:0) during the larval transition to diapause from summer to autumn was observed in both populations. It is important to note that the non-diapausing (June) and early diapausing (September) larvae did not differ in

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the total content of lipids which was 13.3% or 13.7% of fresh mass, respectively (Rozsypal et al., 2013). That is because final instar larvae of both developmental modes accumulate lipid depots in order to secure the upcoming metamorphosis during pupal stage. The main difference between two developmental modes might consist in higher activity of Δ12 FA-desaturase in diapausedestined larvae. This enzyme introduces a double bond into the FA chain beyond the Δ9 double bond position produced previously by Δ9 FA-desaturase. While the Δ9 FA-desaturase is ubiquitously found in all animals and plants, only plants and some invertebrates, including insects, are able to introduce a second double bond between the existing double bond and the terminal methyl group (Vance and Vance, 2002). The activity of Δ12 FAdesaturase has been confirmed in several insect species (Batcabe et al., 2000; Blomquist et al., 1982; Buckner and Hagen, 2003) and it is widespread in moths (Knipple et al., 2002). Thus, the Δ12 FAdesaturase might be the tool that is used by diapause-destined larvae of C. pomonella to seasonally enrich their depot fats by C18:2n6. In accordance with the change in depot fat composition, we observed that the enthalpy (ΔH) of melt transition changes, and mainly, the melting temperature (Tm) of total lipids dramatically decreases in parallel with the seasonal increase of relative proportion of C18:2n6. This means that the range of temperatures at which depot fat remains fluid significantly shifts toward lower temperatures, which seems to conform well to the adaptive explanation that the lipid depots must remain fluid to be accessible as energy substrate for metabolism during winter (Frank, 1991; Joanisse and Storey, 1996; Munro and Thomas, 2004; Marshall et al., 2014). Indeed, diapausing larvae of C. pomonella utilize their lipid depots to fuel metabolism during overwintering. We found that 46% of initial lipid depots were depleted in overwintering larvae between November and April (Rozsypal et al., 2013). Some authors questioned the adaptive meaning of maintaining high fluidity of lipid depots in overwintering insects as many species show the high rates of metabolism only when they are exposed to warm temperatures in the autumn and spring (Kemp et al., 2004; Buckner et al., 2004). We also observed highest rates of fresh mass loss in codling moth larvae during warm parts of overwintering (Rozsypal et al., 2013). But, it is also true that: (1) the ambient temperatures fluctuate widely and may often fall below zero even in autumn and spring, and (2) even the deeply supercooled insect maintains slow but active metabolism to support basal homeostatic functions such as maintenance of transmembrane electrochemical ion potentials, which in turn, requires continuous access to energy substrates (Koštál et al., 2004; Zachariassen et al., 2004). Glycogen, another potential energy substrate, is almost completely converted to sugars and polyols in overwintering codling moth larvae (Rozsypal et al., 2013) as well as in many other insect species (Storey and Storey, 1991). Accumulated hexameric proteins in hemolymph probably do not serve as metabolic fuels (Telfer and Kunkel, 1991) and remain practically constant in overwintering larvae from September to April (Rozsypal and Koštál, unpublished data; see also, Brown, 1980). TG depots in fat body thus seem to serve as major energy substrate during winter and, therefore, our results indirectly suggest a linkage between the high relative proportion of C18:2n6, the low melting temperature of total lipids, the accessibility of depot fats for enzymatic breakdown, and, consequently, successful winter survival in overwintering larvae of C. pomonella. We suggest that sufficient fluidity of lipid depots might be particularly important for long-term survival in chilled or supercooled state at moderate body temperatures around zero when a slow supply of energy is needed. In fact, moderate temperatures prevail during larval overwintering in the leaf litter layer (Rozsypal et al., 2013). Nevertheless, the outermost limits of larval survival

over brief episodes of extremely cold spells, or during partial freezing, are probably set by other adaptive mechanisms such as supercooling point depression and high concentrations of compounds such as trehalose, proline, fructose or alanine, which may protect protein and membrane structures during extreme cold or during freeze-dehydration (Rozsypal et al., 2013). 4.2. Membrane phospholipids Low temperature reduces the molecular motion and increases the conformational order of membrane lipids (decreases membrane fluidity). At a specific temperature, the membrane, or a part of it, may undergo a thermotropic phase transition from liquid crystalline phase (Lα) to highly ordered lamellar gel phase (Lβ), where the lipid molecules are closely packed and their lateral diffusion is prevented (Chapman, 1975). Additional risk linked to low temperatures occurs in the case of freezing of body water when the cells gradually dehydrate as more and more water molecules become trapped in ice crystals. The efflux of water from cells renders the membrane bilayers less hydrated which can cause their lyotropic transition from Lα phase to an inverted hexagonal phase (HII), in which the membranes form cylinders with inwardly oriented PL headgroups surrounding a central column of remaining liquid water (Kirk et al., 1984). When occurring in unregulated manner, both types of phase transition (fluid/gel or fluid/hexagonal) are considered incompatible with numerous vital processes of a functional biological membrane (for review, see McElhaney, 1984; Hazel and Williams, 1990; Hazel, 1995). The actual temperature of fluid/gel (Tm) and fluid/hexagonal (Th) transitions depend on hydration and pressure but, primarily, they are dictated by chemical composition of a membrane (Chapman, 1975). Many organisms from bacteria to vertebrates are known to undergo temperature-induced compositional change of membrane lipids with the probable adaptive aim to maintain a specific optimal level of membrane fluidity (homeoviscous adaptation sensu Sinensky, 1974), and/or to prevent occurrence of phase transitions (homeophasic adaptation sensu McElhaney, 1984), and/ or to prevent/ to allow occurrence of the unregulated/regulated phase transitions (dynamic phase behaviour sensu Hazel, 1995). Seasonal restructuring of membrane lipids was also reported in many insects during their preparation for overwintering (for review, see Koštál, 2010). The overwintering larvae of codling moth are exposed in leaf litter to moderately low temperatures fluctuating from þ 10 °C to  5 °C. Some larvae may overwinter under the bark on tree trunks where they experience much higher fluctuations of ambient temperature (approximately from þ 20 °C to  15 °C) and spells of extreme cold (far below  20 °C) may occur as well (Rozsypal et al., 2013). Moreover, at any given temperature below the equilibrium freezing point, the larvae can exist in either supercooled or frozen state depending on the moisture of their habitat and the presence/absence of external ice (Rozsypal et al., 2013). The lowest temperature limits of larval survival are approximately  26 °C for supercooled state and  15 °C for frozen state (Rozsypal et al., 2013). The large variations in ambient temperature and alternative survival strategies should be taken into account when considering potential adaptive meaning of membrane restructuring. The fluctuations of ambient temperature are unpredictable, which requires adjusting the composition of membranes in such a way that the membrane remains functional (fluid) over relatively wide span of ambient/body temperatures. Moreover, occasional freezing may cause removal of liquid water from PL headgroups, which would make the bilayer much more prone to the fluid/hexagonal transition (Yeagle and Sen, 1986; Webb et al., 1993). Thus, there is an adaptive tradeoff in membrane restructuring in codling moth larvae, which stems from the fact that some of

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the typical changes aimed to prevent thermotropic solidification (fluid/gel transition) of a hydrated (supercooled) membrane do, at the same time, increase the probability of lyotropic hyperfluidization (fluid/hexagonal transition) of a dehydrated (frozen) membrane (Pruitt and Lu, 2008). For instance, the increased FA unsaturation and increased relative proportion of PEs make the geometry of PL molecules less cylindrical and more conical, which means less efficiently packing into bilayer and more prone to undergo a transition into inverted hexagonal phase. Therefore, the insects overwintering in microhabitats exposed to rapid temperature fluctuations, especially those species having two alternative survival strategies (supercooling vs. freezing), should not be expected to tune their membranes very finely to specific (stable) conditions but rather should make the adjustments that allow them to survive unpredictable variation temperature and hydration state variation. We found that the membrane PLs of C. pomonella larvae are composed of only 10 major FAs but, in combination with different PL headgroups, they form at least 97 different molecular species. The ratio of PE/PC was relatively low and stable (0.51–0.62) in all seasons. Most insect species studied so far exhibited slightly higher PE/PC ratios ranging from 0.7 to 2.2 and some species exhibited increasing PE/PC ratio during the transition to winter diapause and/or in response to cold-acclimation (Koštál, 2010). Interestingly, all species that showed an increase of PE/PC ratio were those which use a strategy of supercooling (Koštál, 2010). This observation is in a good accordance with adaptive explanation that elevated PEs help to prevent solidification of hydrated membrane. In contrast, no seasonal change of PE/PC ratio, or even a slight decrease of it, was observed in two other insects that use a strategy of freeze-tolerance (Koštál et al., 2003; Pruitt and Lu, 2008), similarly as the larvae of C. pomonella. It seems that freezetolerant species must carefully control the relative proportion of non-bilayer forming PL classes (such as PEs) because the membranes rich in cone-shaped PEs would be prone to fluid-hexagonal lyotropic transition upon cell dehydration, which always accompanies partial extracellular freezing (Pruitt and Lu, 2008). Looking at the individual PL fatty acids, there was a clear increase in relative proportion of C18:2 at the expense of C18:1 during the transition to diapause (July vs. September), which again suggests elevated activity of Δ12 FA-desaturase in early diapausing larvae, similarly as discussed above (see Section 4.1) for the case of storage lipids. We assessed the conformational order/fluidity of codling moth larvae membranes by measuring the anisotropy of fluorescence polarization of diphenylhexatriene probe in membrane vesicles prepared from PLs that were extracted from larvae. The values of anisotropy measured at 20 °C were relatively low, ranging between 0.10 and 0.12 in all samples. In fact, values higher than that were detected even in Antarctic fish, Notothenia neglecta (0.16). Other animals, including trout, perch, cichlid, rat and pigeon, showed much higher values of anisotropy at 20 °C, ranging from 0.17 to 0.28 (Behan-Martin et al., 1993). At  1.8 °C, which is a typical body temperature of Antarctic fish, the anisotropy increases from 0.16 to approximately 0.24 (Behan-Martin et al., 1993). Our device does not allow measuring the anisotropy at low temperatures but using the data extrapolation, we estimated the anisotropy to be somewhere between 0.13 and 0.15 at 0 °C. The anisotropy values might be slightly underestimated because the PL vesicles, that were prepared from total PLs extracted from C. pomonella larvae, did not contain any sterols and tocopherols. These compounds were excluded during our extraction steps. Nevertheless, we know that muscle tissue of C. pomonella larva contains approximately 3.4 mol% of cholesterol, traces of phytosterols, 0.2 mol% of α- and 0.2 mol% of β-tocopherol (Koštál et al., 2013). Sterols and tocopherols are known to organize lipid bilayer

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and increase its conformational order or decrease its fluidity (Urano et al., 1988; Stillwell et al., 1996). Collectively, it seems that the fluidity of membranes is maintained very high in larvae of C. pomonella irrespective of their developmental mode or acclimation status. No acclimatory adaptation is therefore needed to increase the fluidity further during the cold season. This can also explain why seasonal changes in PL composition are relatively small. Moreover, the membranes might be protected by high concentrations of trehalose and proline that can significantly modulate membrane lipid phase behaviour (Tsvetkova and Quinn, 1994). As in the case of fluidity of storage lipids, we do not assume that fluidity of membranes sets the outermost limits for cold survival in overwintering codling moth larvae. Such limits are most probably set by supercooling capacity (in the case of survival in supercooled state) or by a limit of ice fraction, which determines the level of cell dehydration (in the case of survival in partially frozen state). Due to variable and unpredictable environmental conditions and also regarding the alternative overwintering strategies, the membranes of C. pomonella larvae are probably designed to (1) function at widely fluctuating subzero temperatures, which is guaranteed by constitutively high membrane fluidity, (2) avoid transition to hexagonal phase at relatively high temperatures and/or withstand potential freeze dehydration at subzero temperatures, which is assisted by relatively low levels of non-bilayer forming PEs and high levels of low molecular mass cryoprotectants. In conclusion, this paper brings an indirect, correlative piece of evidence for participation of seasonal restructuring of storage and membrane lipids in the seasonal increase of cold tolerance in overwintering larvae of codling moth. The triacylglycerol storage fat is significantly enriched by linoleic acid (C18:2n6) at the expense of palmitic acid (C16:0) during the larval transition to winter diapause. Consequently, the melting temperature of total lipids decreases, which might increase the accessibility of depot fats for enzymatic breakdown during overwintering. In membrane lipids, the fluidity is maintained constitutively very high (probably to avoid the unregulated transition to the gel phase during cold spells) while the levels of phosphoethanolamines are maintained relatively low (probably to avoid the unregulated transition to the hexagonal phase at warm spells or during freeze dehydration).

Acknowledgements We thank Irena Vacková and Anna Heydová (Biology Centre, ASCR, České Budějovice) for their assistance with sample processing, extractions, derivatizations and biochemical analyzes. This work was supported by the Ministry of Education, Youth and Sports of the Czech Republic Grant no. Kontakt LH12103 (to V.K) and the Czech Science Foundation grant no. 13-18509S (to P.Š). We acknowledge the use of polarizing fluorimeter that has been purchased thanks to funding received from the European Union Seventh Framework Programme (FP7/2007-2013) under Grant agreement no. 316304.

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