CryoLetters 37 (1), 10-18 (2016) © CryoLetters, [email protected]

HEAT INDUCIBLE EXPRESSION OF PROTEIN GENES FROM THE Tenebrio molitor AND Microdera punctipennis

Jieqiong Li, Wenjing Ma and Ji Ma*

Xinjiang Key Laboratory of Biological Resources and , College of Life Science and Technology, Xinjiang University, 14 Shengli Road, 830046 Urumqi, China *Corresponding author e-mail: [email protected]

Abstract

BACKGROUND: Antifreeze proteins (AFPs) play important roles in protecting poikilothermic organisms from cold damage. The expression of AFP genes (afps) is induced by low temperature. However, it is reported that heat can influence the expression of afps in the desert Microdera punctipennis. OBJECTIVE: To further detect whether heat also induce the expression of afps in other insects, and to determine the expression profiling of insect afps at different temperatures. MATERIALS AND METHODS: The expression of antifreeze protein genes in the two beetles, Microdera punctipennis and Tenebrio molitor that have quite different living environment, under different temperatures were studied by using real–time quantitative PCR. RESULTS: Mild low temperatures (5℃~15℃), high temperature (38℃~47℃ for M. punctipennis, or 37℃~42℃ for T. molitor) and temperature difference (10℃~30℃) all stimulated strongly to the expression of AFP genes (Mpafps) in M. punctipennis which lives in the wild filed in desert. The mRNA level of Mpafps after M. punctipennis were exposed to these temperatures for 1h~5h was at least 30-fold of the control at 25℃. For T. molitor which is breeding in door with wheat bran all these temperatures stimulated significantly to the expression of Tmafps, while the extent and degree of the temperature stimulation on Tmafps expression were much lower than on Mpafps. After T. molitor were exposed to 5℃ and 15℃ for 1h~5h, the mRNA level of Tmafps was over 6-fold and 45-fold of the control at 25℃. High temperature (37℃~42℃) for 1h~3h treatments increased Tmafps mRNA level 4.8-fold of the control. Temperature difference of 10℃ was effective in stimulating Tmafps expression. CONCLUSION: The expression of insect antifreeze protein genes both in M. punctipennis and T. molitor was induced by heat, suggesting that this phenomenon may be common in insects; the extent and degree of the influence differ in species that have different living conditions. The heat inducible expression of antifreeze protein genes hints that antifreeze proteins may involve in other functions except for antifreeze. Keywords: desert beetle; Microdera punctipennis; Tenebrio molitor; antifreeze protein; heat

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INTRODUCTION test whether the heat inducible expression of insect afps is common in other beetles. The Insects survive cold environment mainly by multi-factor inductivity of the expression of producing antifreeze proteins (AFPs), which can insect antifreeze proteins suggests that antifreeze lower the non-colligative point of water protein may confer insect environmental without depressing the melting point, thus adaptability. The microhabitat of an insect may generate a temperature difference between the strongly influence its behavior due to the usually hysteretic freezing point and the equilibrium small body size and the ectotherm nature of melting point, termed thermal hysteresis activity insect. This may be reflected by the sequence (THA) (3). Antifreeze protein was firstly variability of antifreeze proteins of the insects discovered in Antarctic marine fishes in that have different microhabitats (17, 33), and by 1971 (4), since then, AFPs have been described the differential expression of antifreeze protein in diverse taxa including terrestrial arthropods genes in these insects under the same conditions. (6, 11, 21), and fungi (8, 32) and In this study, two tenebrionids, Microdera (14, 15). The structure of beetle AFP is punctipennis and Tenebrio Molitor, that have characterized by the most regular -helical quite different living habitat were determined by structure composed with different numbers of real time quantitative PCR for the expression repeat unit TCTXSXXCXXAX (where, X pattern of antifreeze protein genes under represents for any amino acids) (19). different temperatures. Both of them produce In general, the expression of antifreeze numerous isoforms of antifreeze protein genes proteins corresponds to the yearly temperature (13, 17, 18, 20, 24, 26). The common yellow changes (1-2, 9-10, 15-16), in that the mealworm T. molitor is a pest of stored grain expression level of antifreeze proteins began to products in cold temperate regions. The survival increase in early autumn, with peaks in winter, of T. molitor in winter is facilitated by and a decline in spring. This yearly expression accumulation of antifreeze proteins (27, 28). T. pattern of antifreeze proteins is consistent with molitor has a relatively stable microhabitat in its function of protecting organisms from cold terms of temperature, humidity and light length damage. However, apparent expression of afps due to living in door. It is relatively easy to and AFPs in summer were also found in beetles maintain cultures of them under laboratory Dendroides canadensis (1), Tenebrio molitor conditions (25˚, 60-70% RH, 16:8 L:D cycle). M. (22), Choristoneura fumiferana (5), Rhagium punctipennis is an endemic species found in mordax (30) and Microdera punctipennis (16, Gurbantonggut desert, northwest of China, 25), though the thermal hysteresis value was low where the climate is characterized by very rare in summer insects (7). Numerous cDNAs of afps rainfall, large temperature difference between have been cloned from M. punctipennis in day and night, and extremely hot in summer. M. summer beetles (17). Most of these sequences punctipennis has well adapted to the harsh desert have one or two of the repeat unit shorter than environment (29) and overwinters in adult (16). those cloned from the overwinter ones, and the In this study we aimed at discovering (1) the amino acid sequences at the C-terminal ends are heat inductivity of the expression of antifreeze also different from each other. protein genes from these two beetle species; (2) In addition to low temperature, several the influence of different temperatures on the environmental factors could induce the expression of antifreeze protein genes to each expression of antifreeze protein genes, such as beetle. Our results showed that the mRNA levels dry and hungry (12), and even heavy metal (22), of the afps from the two species were which suggests that insect antifreeze proteins significantly increased in response to low and may possess other functions except for high temperatures and to temperature antifreeze (1, 24, 25). Interestingly, it was found differences. The effective temperatures for each that the expression of antifreeze protein genes in of them in stimulating the expression of afps the desert beetle M. punctipennis was induced were quite different, suggesting that antifreeze by high temperature, which strongly suggested proteins may confer insects of different that insect antifreeze proteins have other microhabitat different environmental functions besides antifreeze, such as thermal adaptability. Our results will help to further protection (24, 25). At a glance, it is hard to extend the current knowledge on insect predict that the expression of antifreeze protein antifreeze proteins. genes could be induced by heat. So it is worth to

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MATERIALS AND METHODS refrigerator for RNA extraction. Insect collection Extraction of total RNA and cDNA synthesis M. punctipennis adults were collected from The frozen insect was homogenized in Fukang (N 44˚24, E 087˚51’, 444 m), which is liquid nitrogen in a pre-cold mortar by mixing about 100km north of the geographical center of with 1ml TRIzol reagent (Invitrogen, CA, USA). Asia, and is located at the southern edge of RNA extraction was performed according to the Gurbantonggut Desert in Xinjiang, the northwest manufacturer’s instructions. Each sample was of China. Part of the freshly field-collected treated at 37˚ for 25min with 10 units of RNase- adults were immediately kept in liquid nitrogen, free DNase I (TaKaRa, China) to eliminate any and later were transferred into -80° refrigerator contamination of genomic DNA, then total RNA for latter RNA extraction; the others were was combined with diethyl pyrocarbonate carried back to laboratory for later research. T. (DEPC)-treated water. RNA concentration was molitor were purchased from Hualing market in quantified and quality-checked by using Urumqi, China. NanoDrop ND1000 spectrophotometry (NanoDrop Technologies, USA). The quality of Insect treatment with different temperatures each RNA sample was checked by gel After one week of rearing in laboratory at electrophoresis prior to cDNA synthesis. Total room temperature (about 25℃) adults were RNA (1.0 µg) was reverse transcribed into cDNA with M-MLV Reverse Transcriptase and randomly chosen for temperature treatment. (1) OligodT-Adaptor Primer (3'-Full RACE Core Mild low temperature treatment: insects were Set, TaKaRa, China) with PCR reaction of 70˚ treated at 5˚, 10˚ and 15˚ for 1h, 3h and 5h, for 10min, 42˚ for 60min, 70˚ for 15min. The respectively. (2) High temperature treatment: cDNA was stored at -20˚ until use. insects were treated at 37˚, 42˚ and 47˚ for 1h, 3h and 5h, respectively; T. molitor adults were Primers designed for real-time quantitative treated at 37˚, 40˚ and 42˚ for 1h, 3h and 5h, PCR respectively. (3) Temperature difference Primers for real-time quantitative PCR treatment: insects were randomly divided into (qRT-PCR) to detect the expression of antifreeze three groups, and all were firstly incubated at protein genes in M. punctipennis (Mpafps) and T. 10˚ for 5h, and then at 20˚, 30˚ and 40˚ molitor (Tmafps) after temperature treatment respectively for 5h to generate 10˚, 20˚ and 30˚ were designed according to the sequences of the temperature differences. Insects at room conserved region of each of them by using temperature about 25℃ without any treatment DNAMAN6.0 software. Due to beetle afps exist in many numbers of isorforms which are were used as control. Four individuals in each of relatively conserved except for the variation in the above treatment were frozen in liquid the number of the 12-amino acid repeat units, it nitrogen, and later were transferred into -80˚ is hard to detect one single isoform of Mpafps Table 1 Primers for real time quantitative PCR Primer Size of amplification Primer sequence name (bp) Mpafp F 118 5′-AACTGCAATAGAGCGATGACGTG-3′

Mpafp R 118 5′-TTGGACATCCTGTTGAATGAGTGC-3′

TmafpF 124 5′-AGCCACAACATGTACTGGGTCTACA-3′

TmafpR 124 5′-GCAGTGTAACAGTTGCTTGAGCCA-3′

β-actin F 120 5′-TACTCCGTATGGATCGGTGGATC-3′

β-actin R 120 5′-TTAGAAGCACTTGCGGTGGAC-3′

12 and Tmafps. Thus, primers for detecting Mpafps Reactions were monitored by melting curve and Tmafps are designed for detecting the total analysis after the whole cycling was complete to expression of Mpafps and Tmafps. β-actin genes ensure that the observed fluorescent signal was were used as reference genes. Primers for due solely to the specific amplification. The detecting β-actin genes were designed based on values were calculated using the 9600-sequence the sequences of the conserved region of β-actin detection software. The triplicate averages for genes from all insects in NCBI. All of the Mpafps and Tmafps were subtracted from Mpβ- primers for qRT-PCR are listed in table 1, and actin and Tmβ-actin values respectively for each were synthesized by Sangong Company sample to normalize the data. Relative (Shanghai, China). Real-time quantitative PCR quantitative values were expressed using the E- was performed in three duplicates with the ΔΔCt method as fold changes in the target gene Platinum® SYBR® Green qPCR Super Mix- normalized to the reference gene and related to UDG kit (Invitrogen, CA, USA) according to the the expression of the control. manufacturer’s instructions on a GeneAmp To verify the amplification of Mpafp and Thermal Cycler 9600. Tmafp transcripts, the RT-PCR products of these fragments were detected on a 1.5% agarose gel Real-time quantitative PCR and then sequenced. The reaction was carried out in a tube containing 2μl cDNAs, 0.25 mol/L forward and Statistical analysis reverse primers, respectively, 10μl 2×Real The difference of the relative expression MasterMix/20×SYBR Solution, and DNase/ fold changes in each gene under different RNase free ddH2O was added to a final volume treatment were analyzed with one-way analysis of 20 μl. The PCR reaction conditions for afps of variation (ANOVA) and Turkey’s multiple and β-actins was 50℃ for 2min, 95℃ for 5min; comparison tests by using GraphPad Prism 5 45 repeats of 95℃ for 30s, 60℃ for 30s, 72℃ for software. 30s followed by a standard melting curve.

Figure 1. Relative mRNA level of afps in M. punctipennis (A) and T.molitor (B) under mild low temperatures. Control means 25˚. Different letters above the bars indicate significant difference (P<0.05).

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RESULTS punctipennis was induced by high temperature (25), to detect whether this heat-inductivity is afp mRNAs level under low temperatures common for the expression of Tmafps, and to To detect the influence of mild low compare the influence extent and degree of high temperature to the expression of antifreeze temperature on the expression of Mpafps and protein genes in the insects, the beetles were Tmafps, M. punctipennis and T. molitor were exposed at 5˚, 10˚ and 15˚ for 1~5h, exposed to high temperatures for various times. respectively. In general, the mRNA levels of the The results showed that the expression of both afps in both insects were significantly increased Mpafps and Tmafps were induced by high by the stimulation of these mild low temperatures. temperatures, but the responsive pattern of them For M. punctipennis, the Mpafps mRNA was quite different from each other. For M. level was very significantly increased (F3,12 punctipennis after expose at 5˚, 10˚and 15˚ for =8.11, P<0.001) after the insects were exposed 1h the mRNA level of Mpafps drastically at 37˚, 42˚ and 47˚ for 1h~5h (Fig.2A), which increased by 80-fold, 56-fold and 40-fold of the was at least 20-fold of the control at 25˚, and control at 25˚, respectively, and kept the high these high mRNA levels were kept during the level thereafter (Fig.1A). For T. molitor, the tested 5h. There was no significant difference expression of Tmafps gradually increased from among the effects of these high temperatures. 8-fold at 5˚ for 1h to 47.5-fold at 15˚ for 5h of For T. molitor, the highest temperature the control. At each temperature, with the time treatment was 42˚ instead of 47˚ for M. prolonged, the mRNA level of Tmafps increased punctipennis, because 47˚ is intolerant for T. significantly (F3,12=94.74, P<0.0001), except for molitor. The expression pattern of Tmafps a short decrease at 5˚ for 3h (Fig.1B). influenced by high temperature was quite afp mRNAs level under high temperatures different from those of Mpafps (Fig.2B). Firstly, In previous work we found that the the response of the expression of Tmafps to high expression of antifreeze protein gene in M. temperature was not as strong as that of Mpafps.

Figure 2. Relative mRNA level of afps in M. punctipennis (A) and T. molitor (B) under high temperatures. Control means 25 ℃.Different letters indicate significant difference (P<0.05).

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The highest mRNA level in Tmafps stimulated difference to the expression of afps, three groups by 37˚ for 1h was 13-fold of the control, much of insects for each species were treated with 10˚, lower than that of Mpafps which was 46.3-fold 20˚ and 30˚ temperature difference, respectively, at the same temperature and time length. by firstly incubating the insects at 10˚ for 5 h, Secondly, with temperature elevated the and then at 20˚, 30˚ and 40˚ for 5 h, respectively, response of the expression of Tmafps to high so as to generate 10˚, 20˚ and 30˚ temperature temperature decreased; when the insect was differences. For M. punctipennis, the expression exposed at 42˚ for 1h Tmafps mRNA level was of Mpafps was greatly up-regulated by all three only 6-fold of the control at 25˚. In addition, the temperature differences (Fig.3A) in that the response duration of the expression of Tmafps to relative mRNA level of Mpafps in M. high temperature was not as long as that of punctipennis adults treated by 10˚, 20˚ and 30˚ Mpafps. When exposed at 37˚, 40˚and 42˚ for 3h temperature difference was 31.2-, 66.9- and the Tmafps mRNA levels almost fell back to the 58.9-fold of the control, respectively level of the control (Fig.2B). (F3,8=64.18, P<0.0001). For T. molitor, the expression of Tmafps afp mRNAs level under temperature difference was also stimulated by these temperature To detect the influence of temperature differences (Fig.3B) in that the relative mRNA

Figure 3. Relative mRNA level of the afps in M. punctipennis (A) and T. molitor (B) under temperature differences. Control means 25 ℃.Different letters indicate significant difference (P<0.05).

15 level of Tmafps in the beetles exposed to 10˚, strong influence on the expression of Mpafps by 20˚and 30˚temperature differences was 4.8-, 3.5- causing 30- to 80-fold increase in the Mpafps and 2.2-fold of the control, respectively mRNA level, suggesting these low temperatures (F3,8=8.07, P<0.01), though 30˚ temperature were very effective in stimulating Mpafps difference showed no significant effect on the expression. M. punctipennis lives in the desert mRNA level of Tmafps compared to the control. field, it has adapted well to the extreme temperatures and temperature differences. The DISCUSSION drastic increase in Mpafps mRNA level indicated that M. punctipennis reacts more Many over wintering insects produce rapidly to changes in ambient temperatures. In antifreeze proteins as their adaptive strategies to contrast, the raring temperature for T. molitor is survive low temperature. In general, the 24˚~27˚(31), 5˚~15˚ caused a gradual increase in expression of antifreeze protein gene is Tmafps mRNA level. This is in accord with the regulated by seasonal temperatures, but other cold acclimation of T. molitor to low environmental factors also have influence on the temperatures (23). In addition, 15˚ was more expression of antifreeze protein genes (12, 22, prominent in stimulating Tmafps mRNA level 25). In this study, two beetles that have different than was 5˚ and 10˚., so 15˚might be less living habitats were determined and compared stressful than 5˚ and 10˚ for T. molitor living. for the influence of different temperatures on the Similar discrepancy in the influence of transcriptional expression of their antifreeze temperature difference on the expression of afps protein genes. M.punctipennis lives in the field between these two beetles could be explained in Gurbantonggut desert, northwest of China, with the same reason. Exposure to temperature where the air temperature ranges from about difference of 10˚~30˚ caused at least 30-fold 40℃ in summer to about -40℃ in winter. While increase in the Mpafps mRNAs level, this may account for the adaptation of M. punctipennis to T. molitor lives in barn and has been cultured the desert environment of Gurbantonggut, where world wide. The rearing conditions is 25℃, 60- temperature difference varied greatly between 70% RH, 16:8 L:D cycle. day and night during summer, and the average Exposure to low temperature is a primary air temperature difference was around 30˚, even trigger for insect raising its cold resistance, mild up to 40˚ (16). While for T. molitor, the mRNA low temperature 5˚, 10˚ and 15˚ all could level of Tmafps only increased by 4.4- to 2.5- stimulate the expression of afps in both insects, fold of the control under the same conditions. though the expression patterns were different Moreover, with the value of temperature from each other. The expression of Mpafps and difference increased, its influence on the Tmafps induced by mild low temperatures for expression of Tmafps decreased. Temperature short time could interpret cold acclimation in the difference of 30˚ did not stimulate Tmafps molecular perspective. The induction of 5˚and mRNA level. This may suggest that large 10˚ to the expression of Mpafps is in accord with temperature difference might be harmful to T. the field results when the average daily air molitor which usually lives in constant temperature in August was over 10˚, the mRNA temperatures around 25˚. Long-term breeding level of Mpafps began to increase significantly makes T. molitor less sensitive to temperature (16). difference than M. punctipennis that lives in the Exposure of T. molitor large larvae to 4˚ for desert. four weeks caused 12-fold increase in the Interestingly, the influence of high mRNA level of Tmafps (12), while in this study temperature on the expression of afps was only one hour of exposure of T. molitor adults to significant in both beetle species. High 5˚ caused 7.6-fold increase in Tmafps mRNA temperatures from 37˚ to 47˚ caused an average level. This discrepancy may account for the increase of the Mpafps mRNAs level by 30-fold dependency of insect cold hardiness to of the control, which is consistent with the developmental stage, as all environmental results we previously reported (25). The heat conditions that caused increased thermal inducible expression of antifreeze protein genes hysteresis also inhibited growth (12). was further confirmed by Tmafps in T. molitor In comparing the expression pattern of in this study, suggesting that the expression of Mpafps and Tmafps in response to the mild low antifreeze protein genes stimulated by heat may temperatures, we could see that 5˚ to 15˚ showed be common in insects. In Oodescelis chinensis,

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