Latitudinal variation in cold tolerance and the role of vrille and Yolk protein 3 in enhancing cold adaptation in highly cold-tolerant insect species

20 Identifying elements that enhance insect adaptation into changing environmental conditions is 21 challenging, especially for traits affected by multiple factors. We studied latitudinal variation 22 in the basal cold tolerance and body colour of two highly cold-tolerant Drosophila virilis group 23 species, D. montana and D. flavomontana, in climatically diverse locations in North America. 24 D. montana, which is generally found on higher latitudes and altitudes than D. flavomontana, 25 was darker and more cold-tolerant than D. flavomontana. In D. montana, only fly cold tolerance 26 showed latitudinal variation, while in D. flavomontana both traits varied according to latitude 27 and local climatic conditions, but showed no correlation with each other. We also examined the 28 role of a circadian clock gene vrille and an insulin-signalling pathway gene vitellogenin in the 29 basal cold tolerance and cold acclimation ability of D. montana females using RNA 30 interference. Silencing of vrille induced expression changes in the period, but not clock gene, 31 and decreased flies’ basal cold tolerance and cold acclimation ability, while silencing of 32 vitellogenin affected only cold acclimation. Our study demonstrates that the dependence of 33 insect cold tolerance on latitudinally varying factors and local climatic conditions may vary 34 even between closely-related species. Furthermore, we propose that a functional circadian clock 35 system plays an essential role both in insect basal cold tolerance and cold acclimation ability, 36 and that vitellogenin affects cold acclimation likely through its interactions with other genes in 37 the insulin-signalling pathway. 38

in ion transport, membrane restructuring, calcium signalling and the synthesis of 91 cryoprotectants and antifreeze proteins, which can evoke cold acclimation (Teets & Denlinger,  cycle further activate an interlocked feedback loop that includes vrille (vri) and Par domein 111 protein 1 (Pdp1), which repress and activate clock transcription, respectively. vrille also 112 regulates neuropeptide Pigment-dispersing factor (PDF) expression in clock neurons, which 113 makes it a key regulator of circadian behavioural rhythms (Gunawardhana & Hardin, 2017). 114 Interestingly, vrille expression has been found to increase during cold acclimation e.g. in 115 Drosophila montana (Parker et al., 2015;Vesala, Salminen, Laiho, et al., 2012). The insulin- 116 signalling pathway, on the other hand, is known to have widespread effects e.g. on insect 117 development, immune function and stress resistance (reviewed in Flatt, Tu, & Tatar, 2005). 118 This pathway involves a major endocrine regulator, juvenile hormone (JH), for which effects 119 are mediated, among other genes, by vitellogenin genes that code for a phospholipoglycoprotein   Throckmorton, 1982). Body colour of D. montana is almost black, while that of D. 137 flavomontana varies from light to dark brown (Patterson, 1952). America, Asia and Europe (Throckmorton, 1982), while the distribution of D. flavomontana is 156 restricted to North America (Patterson, 1952;Throckmorton, 1982). In the central Rocky probably invaded only during the last decades, both species live at much lower altitudes (see 160 Patterson, 1952;Poikela et al., 2019). 161 We performed phenotypic assays on the females from 23 D. montana and 20 D. flavomontana 162 isofemale strains, which were established from the progenies of fertilized females collected 163 from several sites in North America between 2013 and 2015 (Fig. 1). Each site was represented 164 by three isofemale strains per population per species, when possible ( Fig. 1 177 We investigated latitudinal variation in the cold tolerance of D. montana and D. flavomontana 178 females using two well-defined and ecologically relevant methods: chill coma temperature 179 (CTmin; also called critical thermal minimum) and chill coma recovery time (CCRT). CTmin 180 corresponds to the temperature, at which the fly loses all neurophysiological activity and 181 coordination and falls into a chill coma. In this test, we placed the females individually in glass 182 vials, which were submerged into a 30 % glycol bath. We then decreased the bath temperature   194 We analysed the body colour of the same flies that had been phenotyped in CTmin or CCRT   206 We used different statistical models to investigate whether variation in fly cold tolerance or 207 body colour was associated with latitude and/or local climatic factors, and whether these traits 208 showed correlation with each other. In these models, we used either CTmin data (chill coma   (Table S3)    Here we used cold tolerance measures as response variables (as explained above) and body 233 colour (divided by 100 to scale the variables) as an explanatory variable. All the analyses were 234 conducted in R (v1.2.1335-1) and R studio (v3.6.1). 237 We performed RNAi studies in D. montana, which has become a model species for studying 238 adaption to seasonally varying environments at phenotypic (e.g. Tyukmaeva, Lankinen,    Table S4). qPCR mix contained 10 µl of 2x Power SYBR Green  Table S4.  293 We generated fragments of vrille, vitellogenin and LacZ genes, with the length of 347, 419 and 294 529 bp respectively, with PCR (primer information given in Table S4). LacZ, which codes a 295 part of a bacterial gene, was used here as a control for dsRNA injections. PCR products were 296 purified with GeneJET Gel Extraction kit (Thermo Fisher Scientific) and cloned using bacterial solutions were analysed for the size of the products in the second PCR, which was 303 carried out with pJET primers from the cloning kit, followed by agarose gel runs. We then 304 selected the initial samples with the right size products for the third PCR using pJET primers, 305 where the R primer contained T7 promoter sequence at the 5' end of the primer (primer 306 sequences given in Table S4). PCR products were first purified with GeneJet Gel Extraction kit 307 and then used in transcription synthesis of the double-stranded RNA (dsRNA), using the 308 TranscriptAid T7 High Yield Transcription kit (Thermo Fisher Scientific). Finally, we purified 309 and precipitated the synthesized products with ethanol, suspended them into a salt buffer and 310 quantified them using NanoDrop and agarose gel.  compared to controls 48h after injections (Fig. S3). Thus, we used this time point to find out 341 whether the silencing of vrille or vitellogenin had changed also the expression of two circadian 342 clock genes, clock and period (primer sequences given in Table S4). We synthesized cDNA 343 using equal quantities of RNA (150 ng) and quantified the expression levels of all four genes 344 as described above, using ∆∆(Ct) normalisation method (Livak & Schmittgen, 2001) with Tub2 345 as a reference gene.  Table S6).   (Table  401 S2). Direction of the arrows indicate which bioclimatic variables contribute to PC1 and which to PC2. 402

| Synthesis of double-stranded RNA for RNAi
The longer the arrow is, the greater its contribution is. 403  Table S7).

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The best-fit model for CTmin, CCRT and body colour of D. montana, judged from their Akaike 413 information criterion (AIC) and Akaike weight values, included only latitude (Table S7). CTmin 414 and body colour of this species showed only minor variation, which was not significantly 415 associated with latitude (Fig. 3A, 3C; Table S8), while CCRT showed the fly cold tolerance to 416 improve towards northern latitudes ( Fig. 3B; Table S8). In D. flavomontana, best-fit model for 417 418 body colour latitude and PC1 (Table S7). CTmin of this species showed only minor variation 419 and no significant association with latitude ( Fig. 3A; Table S8). However, CCRT values 420 showed the cold tolerance of D. flavomontana to improve towards North and to be strongly  Table S8). On these latitudes, fly cold tolerance was higher in the humid, low-altitude western  Table S8).  Table S9). that of vitellogenin showed less variation but was also highest at 22 pm (Fig. 5). Accordingly, 451 all cold tolerance experiments were run between 22 pm and 00 am.   Table S10). Expression of the clock gene showed no significant changes in either 467 of the RNAi treatments ( Fig. 6; Table S10).    Table S11). In CCRT tests, cold acclimation improved the cold tolerance of no-injection 489 females, had no effect on the cold tolerance of LacZor vitellogenin-injected females, and 490 decreased that of vrille-injected females ( Fig. 7B; Table S11).

491
Next, we compared the basal cold tolerance of vrilleand vitellogenin-injected females with 492 that of the LacZ controls. Here the only significant differences were detected between vrille and 493 LacZ-injected females in CTmin test ( Fig. 7A; Table S12) and between LacZ and no-injection 494 females in CCRT tests ( Fig. 7B; Table S12). The first difference suggests that the RNAi  Table S12). The same phenomenon was also detected 501 in the CCRT test, but only for vrille ( Fig. 7B; Table S12). In the CCRT test, a significant 502 difference between LacZ and no-injection control implicates physical damage or immune 503 response induced by injection ( Fig. 7B; Table S12). cold tolerance within each group, and solid lines significant differences between the LacZ control and 509 other groups among non-acclimated or cold-acclimated females. Significance levels were obtained from 510 GLMMs and only significant observations are shown: * P < 0.05, ** P < 0.01 and *** P < 0.001. 511

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Studies on insect cold adaptation in a wide range of environments, combined with information 513 on the geographical location of study populations and the climatic conditions prevailing on 514 these sites, offer a good possibility to predict species potential to survive in changing 515 environmental conditions. We performed this kind of study on two highly cold tolerant species,  Myrmica rubra and Myrmica ruginodis, and he argues that CCRT is a more useful indicator of 539 adaptation than CTmin due to its higher sensitivity to photoperiod and/or climatic conditions.  (Stevenson, 1985). This kind of behaviour has been detected in the Colorado     582 Cold acclimation has been found to induce shifts e.g. in insect metabolic profile and in the 583 production of cryoprotectants, which helps to maintain osmotic balance and stabilise cell

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Understanding the mechanisms that generate variation in species stress tolerance is a key 643 component for predicting their adaptation ability in the face of global warming. Species, whose 644 cold or heat tolerance is tightly linked with latitude, may encounter more difficulties in adapting 645 to changing environmental conditions than species whose tolerances are also affected by local 646 climatic conditions. This study deepens our understanding on how latitudinally varying factors 647 and local climatic conditions shape the evolution of cold tolerance. We show that cold tolerance

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The authors have declared no conflicting interests.