Thermal Discrimination and Transgenerational Temperature Response in Bemisia tabaci Mediterranean (Hemiptera: Aleyrodidae): Putative Involvement of the Thermo-Sensitive Receptor BtTRPA

Thermal Discrimination and Transgenerational Temperature Response in Bemisia tabaci Mediterranean... Abstract Anthropogenic climate change and global warming are expected to alter the geographic distribution and abundance of many ectothermic species, which will increase the invasion of new areas by exotic species. To survive in variable or fluctuating temperature conditions, insects require sensitive thermal sensory mechanisms to detect external thermal stimuli and induce the appropriate behavioral and physiological responses. TRPA, a thermal-activated transient receptor potential (TRP) family ion channel, is essential for thermotaxis in insects. Here, we investigated the potential role of BtTRPA in short-term and long-term thermal stress in Bemisia tabaci Mediterranean (Gennadius; Hemiptera: Aleyrodidae). We found that BtTRPA was mainly expressed in the head, where the antennae are located. Under short-term thermal stress, the BtTRPA gene was robustly expressed after exposure to acute low or high temperatures, BtTRPA expression reached the highest levels after exposure to 0°C for 3 h and 40°C for 5 h, but was relatively low after exposure to milder stimuli (12 and 35°C). These results demonstrated that BtTRPA could discriminate between innocuous and noxious temperature stimuli. Under long-term thermal stress, the highest expression level of BtTRPA occurred at G1 exposed to mild innocuous temperature of 21 and 31°C, along with BtTRPA sharply increased and peaked in adult females, implying that mild innocuous long-term thermal exposure could cause transgenerational expression effects to enhance the ability of offspring to cope with the same stress. This study demonstrates that the channel BtTRPA is important in temperature sensing and provides a molecular basis for thermosensation regulation in response to varied environmental temperature in B. tabaci Mediterranean. Global warming due to anthropogenic climate change has exposed ectothermic organisms to multifarious environmental stresses, resulting in the exploitation of various strategies to increase their adaptability in the face of stress (Walther et al. 2002, de Nadal et al. 2011). Invasive insects, which spread rapidly and colonize successfully after invading new geographical ranges, are becoming appropriate models for studying the patterns and mechanisms of rapid acclimation (Ward and Masters 2007, Richardson 2010). Theoretical and experimental studies have shown that the capacity to tolerate extreme and fluctuating temperatures is one of the foremost important abiotic factors determining the geographic distributions of most insects (Gutierrez et al. 2014, Lancaster 2016, Lancaster et al. 2016). As small poikilothermic organisms, insects are vulnerable to rapid body temperature changes in response to environmental temperature fluctuation. Thus, in order to survive, insects should execute appropriate physiological and behavioral responses, which must be regulated by proper thermosensation mechanisms (Hong et al. 2015). Transient receptor potential (TRP) channels have been implicated as key temperature-responsive ion channels in both mammals and insects (Rosenzweig et al. 2005, Wetsel 2011). TRPs are divided into seven different thermosensitive receptor subfamilies-namely, TRPC, TRPA, TRPV, TRPN, TRPM, TRPP, and TRPML-based on their sequence elements and six transmembrane domains (Matsuura et al. 2009, Fowler and Montell 2013). As previously reported, the members of the TRPM, TRPA, and TRPV subfamily are thought to mediate thermosensation (Dhaka et al. 2006, Wetsel 2011). Intriguingly, TRPAs in mammals are cold-activated channels (Story et al. 2003, Chen et al. 2011), whereas TRPAs in insects mediate the sensing of warm and high temperatures (Kang et al. 2011, Barbagallo and Garrity 2015). A recent study has shown that Bombyx mori (L.; Lepidoptera: Bombycidae) TRPA1 is activated at temperatures above ~21°C and affects the induction of diapause in progeny (Sato et al. 2014). In Drosophila melanogaster (Matsumura; Diptera: Drosophilidae), TRPA1 is critical for larvae to discriminate between the optimal temperature (18°C) and slightly higher temperatures (19~24°C) (Kwon et al. 2008, Fowler and Montell 2013), and it is also implicated in high-temperature nociception in adult flies (Neely et al. 2011). Helicoverpa armigera TRPA1 is activated by increasing the temperature from 20 to 45°C. Interestingly, Nasonia vitripennis TRPA appears to be activated as soon as temperature rises, regardless of the initial temperature, and this activation is highly dependent on the heating rate (Matsuura et al. 2009). Therefore, insect TRPA channels are part of the system of rapid response to heat and variability in temperature. Bemisia tabaci (Gennadius; Hemiptera: Aleyrodidae) is a complex species comprising at least 36 morphologically indistinguishable species, of which Mediterranean (MED, former biotype Q) and Middle East-Asia Minor 1 (MEAM 1, former biotype B) are the most common and cosmopolitan invasive (De Barro et al. 2011, Colvin and Barro 2012). MED, which originated in Europe, is now globally distributed and is found on all continents except Antarctica (Martin et al. 2000, Tay et al. 2012), possibly due to its high potential adaptability to various environmental temperatures (Cui et al. 2008, Elbaz et al. 2011, Yu et al. 2012). Previous studies have found that whiteflies can utilize not only physiological mechanisms such as sorbitol accumulation in response to elevated temperatures (Wolfe et al. 1998) but also heat shock proteins and other stress-related genes to guard against thermal stress (Mahadav et al. 2009, Lü and Wan 2011). However, the mechanism by which external temperature stimuli are transmitted to the neural network in whitefly remains unknown, and the ability of the neural signals to activate the expression of stress-related genes to increase thermal resistance has not been fully explored. Furthermore, as far as we know, previous reports on the thermosensation system of whitefly are rare. For example, study in MED has mainly focused on the discovery of TRP (Li et al. 2015), while Lü et al. (2014) found that MEAM1 BtTRPA is a key element in sensing high temperature and plays an essential role in B. tabaci MEAM1 heat tolerance ability. Our experimental system was specifically designed to provide insight into the temperature sensing mechanism and thermal plasticity role of the BtTRPA gene. We had two major objectives: first, we assessed the expression of BtTRPA throughout the course of development and among the tagmata, and second, we conducted a series of short-term and long-term experiments to quantify the expression patterns of BtTRPA in response to various temperatures in the invasive whitefly MED. Materials and Methods Insect Culture MED population used in this study was initially obtained from a tomato filed in Beijing in 2012 and have been reared in the laboratory for 50–60 generations. The species was reared on Lycopersicon esculentum Mill (Zhongza No. 9) in a climate room under a photoperiod of 14:10 (L:D) h at 26 ± 2°C and 50 ± 10.0% relative humidity (RH). Tagmata and Developmental Sample Collection For samples of different developmental stages, 2,000 egg, 1,000 1st–2nd nymph, 500 3rd–4th nymph, 500 pupae, 200 female adults, and 200 male adults were chosen. For samples of tagmatas, head, thorax, and abdomen were separately collected from 800 female and male adults. All the samples were frozen immediately in liquid nitrogen and stored at −80°C until further use. Short-term Temperature Stress Based on the occurrence time and the geographical distribution of MED invasion in fields in China, we designed four temperature treatments (0, 12, 35, and 40°C) as short-term thermal stress temperatures. MED adults were placed together in a 5 ml centrifuge tube covered with cotton. The tubes were then placed in a constant-temperature environment at 0, 12, 35, and 40°C, respectively. After 1, 3, and 5 h, the live adults were frozen instantaneously and stored at −80°C, respectively. We standardized adult age using newly emerged whitefly adults that were younger than 3 h and the sex of female and male is in a ratio of about 1:1 in each treatment. Adults maintained at 26°C were used as untreated controls. Each treatment was carried out with three replicates of 200 adults. Long-term Temperature Stress Simultaneously considering the fact that 17 and 35°C were close to the lower and upper thermal limits (Bonato et al. 2007), the need to obtain sufficient effective samples, and the developmental time, longevity and reproduction of adults in different temperature conditions, we finally chose 17, 21, 31, and 35°C as long-term thermal stress temperatures. Newly emerged MED adults were collected from tomato plants and placed into cages, The cages were then placed in climate-controlled chambers (Saifu, Ningbo, China) and were exposed to 17 and 21°C for 5, 10, 15, and 20 d and until G1, where G1 is the time by which G0 young adults exposed to thermal stress have spawned to produce G1 young adults. Adults maintained at 26°C were used as untreated controls. All samples were frozen instantaneously after treatment using liquid nitrogen. Each treatment was carried out with three replicates of 200 adults. Total RNA Extraction and Quantitative Real-time PCR Total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA) according to the instructions. The RNA concentration and quality were determined by NanoPhotometer P330 (Implen, Munich, Germany) and 1% agarose gel electrophoresis. The first-strand cDNA synthesis was carried out using 2.0 μg purified RNA using the Super Script First-Strand Synthesis System (Transgen, Beijing, China) following the manufacturer’s protocol and stored at −20°C. The primers used are based on references Lü et al. (2014) and Dai et al. (2017). The qRT-PCR reactions were performed in an ABI 7500 Real-time PCR system (Applied Biosystems, Foster, CA) with 20 μl PCR cocktail contained 1 μl of the cDNA, 10 μl of 2×TransStart Green qPCR SuperMix (Transgen), 0.5 μl of each 10 μM primer, and 0.4 µl of Passive Reference Dye. The amplification cycles were as follows: 30 s at 95°C, then 40 cycles of 5 s at 95°C, 15 s at 60°C, and 30 s at 72°C. β-tubulin and EF1-α (GenBank accession no.: EE600682) were used as reference genes, because they are constitutively expressed under various temperature stress conditions (Dai et al. 2017). All samples were run in triplicate to account for technical variation. The amplification efficiency (E) of all primer pairs was validated by constructing a standard curve using a twofold serial dilution of cDNA template using the equation: E= (10[−1/slope]−1) × 100 (Radonić et al. 2004). The main characteristics of the primers are listed in Supplementary Material. Statistical Analysis The expression of the TRPA gene was normalized using the 2−ΔΔCt method (Livak and Schmittgen 2001) after the threshold cycle (Ct) was normalized with the Ct of β-tubulin and EF1-α. Based on the equation ΔΔCt = (Cttarget − Ctreference) treatment − (Cttarget − Ctreference) control. The head sample and the pupae sample was used as the calibrator sample for tagmata treatments (expression = 1) and developmental treatments (expression = 1), respectively. A 26°C adults sample was used as the calibrator sample for thermal stress treatments (expression = 1). All expression data were log-transformed to ensure that they were normally distributed. Target gene mRNA expression was analyzed using a one-way ANOVA followed by Fisher’s least significant difference (LSD) test. The data were presented as the means ± standard error (mean ± SE). Differences were considered statistically significant at P ≤ 0.05. Results Expression Analysis of BtTRPA Gene Among Tagmata and During Development The BtTRPA gene was expressed in all three major tagmata examined at different levels, showing some tissue specificity (P < 0.05, Fig. 1). The results indicated that BtTRPA was expressed abundantly in the head and at relatively low levels in the thorax and abdomen. Experiments in H. armigera showed that TRPA1 was abundantly expressed in the antennae and moderately expressed in the labial palps and fairly low in the proboscises (Wei et al. 2015). Thus, the results indicated that BtTRPA may be highly expressed in antennae. Fig. 1. View largeDownload slide Tagmata expression of Bemisia tabaci MED BtTRPA. The results are expressed as the mean ± SEM after log-transformed. Error bars indicate the standard error of the calculated mean based on three biological replicates. Bars with a different lowercase letter at the top show a significant difference at P < 0.05. Fig. 1. View largeDownload slide Tagmata expression of Bemisia tabaci MED BtTRPA. The results are expressed as the mean ± SEM after log-transformed. Error bars indicate the standard error of the calculated mean based on three biological replicates. Bars with a different lowercase letter at the top show a significant difference at P < 0.05. BtTRPA mRNA was also present at all developmental stages (Fig. 2). However, the levels and pattern of expression appeared to change during the development of MED (P < 0.05, Fig. 2). BtTRPA expression in adult stage was significantly higher than the expression in the other stages, and the expression was extremely low and not significantly different in the egg, 1st–2nd nymph, 3rd–4th nymph, with the lowest expression in the pupae. However, at the adult stage, the BtTRPA transcript expression in females increased twofold, and was significantly higher than the level observed in males (Fig. 2). Fig. 2. View largeDownload slide Developmental expression of Bemisia tabaci MED BtTRPA. The results are expressed as the mean ± SEM after log-transformed. Error bars indicate the standard error of the calculated mean based on three biological replicates. Bars with a different lowercase letter at the top show a significant difference at P < 0.05. Fig. 2. View largeDownload slide Developmental expression of Bemisia tabaci MED BtTRPA. The results are expressed as the mean ± SEM after log-transformed. Error bars indicate the standard error of the calculated mean based on three biological replicates. Bars with a different lowercase letter at the top show a significant difference at P < 0.05. Short-term Cold Stress The expression levels of BtTRPA following short-term exposure to cold temperatures at 0°C from 1 to 5 h were significantly higher than at the control temperature of 26°C; the content of BtTRPA mRNA after exposure for 5 h was 8.0 and 2.02 times that after exposure for 1 and 3 h, respectively. The expression level of BtTRPA after 12°C exposure for 3 h was significantly higher than the levels after exposure for 1 and 5 h, with 3.39- and 2.15-fold increases, respectively (P < 0.05 in all cases; Fig. 3). Fig. 3. View largeDownload slide Relative expression levels of BtTRPA mRNA in Bemisia tabaci MED adults after short-term cold exposure. The results are expressed as the means ± SEM after log-transformed. Error bars indicate the standard error of the calculated mean based on three biological replicates. White, black, and gray bars represent the relative mean expression in the 26, 0, and 12°C exposed treatments, respectively. a, b and c at the top of the blue bars showed a significant difference at P < 0.05 in the 0°C exposed treatments. a′ and b′ at the top of the red bars showed a significant difference at P < 0.05 in the 12°C exposed treatments. No significant difference in the 26°C exposed treatment. Fig. 3. View largeDownload slide Relative expression levels of BtTRPA mRNA in Bemisia tabaci MED adults after short-term cold exposure. The results are expressed as the means ± SEM after log-transformed. Error bars indicate the standard error of the calculated mean based on three biological replicates. White, black, and gray bars represent the relative mean expression in the 26, 0, and 12°C exposed treatments, respectively. a, b and c at the top of the blue bars showed a significant difference at P < 0.05 in the 0°C exposed treatments. a′ and b′ at the top of the red bars showed a significant difference at P < 0.05 in the 12°C exposed treatments. No significant difference in the 26°C exposed treatment. Short-term Heat Stress Compared to level at the control temperature of 26°C, the expression level of BtTRPA following short-term exposure to heat at 35°C was higher at 5 h than at 1 and 3 h, with a 12.47- and 16.79-fold, increase, respectively. The expression level of BtTRPA at 40°C after 3 h of exposure was significantly higher than the levels after 1 and 5 h, with increases of 1.84- and 4.06-fold, respectively (P < 0.05 in all cases; Fig. 4). Fig. 4. View largeDownload slide Relative expression levels of BtTRPA mRNA in Bemisia tabaci MED adults after short-term heat exposure. The results are expressed as the means ± SEM after log-transformed. Error bars indicate the standard error of the calculated mean based on three biological replicates. White, black, and gray bars represent the relative mean expression in the 26, 35, and 40°C exposed treatments, respectively. a and b at the top of the blue bars showed a significant difference at P < 0.05 in the 35°C exposed treatments. a′, b′, and c′ at the top of the red bars showed a significant difference at P < 0.05 in the 40°C exposed treatments. No significant difference in the 26°C exposed treatment. Fig. 4. View largeDownload slide Relative expression levels of BtTRPA mRNA in Bemisia tabaci MED adults after short-term heat exposure. The results are expressed as the means ± SEM after log-transformed. Error bars indicate the standard error of the calculated mean based on three biological replicates. White, black, and gray bars represent the relative mean expression in the 26, 35, and 40°C exposed treatments, respectively. a and b at the top of the blue bars showed a significant difference at P < 0.05 in the 35°C exposed treatments. a′, b′, and c′ at the top of the red bars showed a significant difference at P < 0.05 in the 40°C exposed treatments. No significant difference in the 26°C exposed treatment. Long-term Cold Stress Compared to those of whiteflies grown at the control temperature of 26°C, the expression levels of BtTRPA declined significantly following long-term exposure to a low temperature of 17°C from 5 d to the G1 stage; the content of BtTRPA mRNA after exposure for 5 d was 4.13, 1.69, 3.25, and 6.25 times that after exposure for 10, 15, and 20 d and until G1, respectively. In contrast, the expression levels of BtTRPA at 21°C was significantly higher at G1 stage than other time-period, and with the lowest expression at 15 d; the content of BtTRPA after exposure until G1 was 25.35, 46.01, 221.84, and 45.51 times those after exposure for 5 , 10 , 15, and 20 d, respectively (P < 0.05 in all cases; Fig. 5). Fig. 5. View largeDownload slide Relative expression levels of BtTRPA mRNA in Bemisia tabaci MED adults after long-term cold exposure. The results are expressed as the mean ± SEM after log-transformed. Error bars indicate the standard error of the calculated mean based on three biological replicates. White, black, and gray bars represent the relative mean expression in the 26, 17, and 21°C exposed treatments, respectively. a, b, and c at the top of the blue bars showed a significant difference at P < 0.05 in the 17°C exposed treatments. a′, b′, c′ and d′ at the top of the red bars showed a significant difference at P < 0.05 in the 21°C exposed treatments. No significant difference in the 26°C exposed treatment. G1, the time by which G0 young adults exposed to thermal stress have spawned to produce G1 young adults. Fig. 5. View largeDownload slide Relative expression levels of BtTRPA mRNA in Bemisia tabaci MED adults after long-term cold exposure. The results are expressed as the mean ± SEM after log-transformed. Error bars indicate the standard error of the calculated mean based on three biological replicates. White, black, and gray bars represent the relative mean expression in the 26, 17, and 21°C exposed treatments, respectively. a, b, and c at the top of the blue bars showed a significant difference at P < 0.05 in the 17°C exposed treatments. a′, b′, c′ and d′ at the top of the red bars showed a significant difference at P < 0.05 in the 21°C exposed treatments. No significant difference in the 26°C exposed treatment. G1, the time by which G0 young adults exposed to thermal stress have spawned to produce G1 young adults. Long-term Heat Stress Compared to those of whiteflies grown at the control temperature of 26°C, the expression levels of BtTRPA increased significantly following long-term exposure to a high temperature of 31°C from 5 d to the G1 stage; the content of BtTRPA after exposure until G1 was 48.14, 49.78, 4.45, and 21.86 times that after exposure for 5, 10, 15, and 20 d, respectively. In contrast, the expression levels of BtTRPA at 35°C from 5 d until G1 declined significantly; the content of BtTRPA after exposure for 5 d was 25.21, 121.95, 97.11, and 32.17 times those after exposure for 10, 15, and 20 d and until G1, respectively (P < 0.05 in all cases; Fig. 6). Fig. 6. View largeDownload slide Relative expression levels of BtTRPA mRNA in Bemisia tabaci MED adults after long-term heat exposure. The results are expressed as the mean ± SEM after log-transformed. Error bars indicate the standard error of the calculated mean based on three biological replicates. White, black, and gray bars represent the relative mean expression in the 26, 31, and 35°C exposed treatments, respectively. a, b and c at the top of the blue bars showed a significant difference at P < 0.05 in the 31°C exposed treatments. a′, b′, and c′ at the top of the red bars showed a significant difference at P < 0.05 in the 35°C exposed treatments. No significant difference in the 26°C exposed treatment. G1, the time by which G0 young adults exposed to thermal stress have spawned to produce G1 young adults. Fig. 6. View largeDownload slide Relative expression levels of BtTRPA mRNA in Bemisia tabaci MED adults after long-term heat exposure. The results are expressed as the mean ± SEM after log-transformed. Error bars indicate the standard error of the calculated mean based on three biological replicates. White, black, and gray bars represent the relative mean expression in the 26, 31, and 35°C exposed treatments, respectively. a, b and c at the top of the blue bars showed a significant difference at P < 0.05 in the 31°C exposed treatments. a′, b′, and c′ at the top of the red bars showed a significant difference at P < 0.05 in the 35°C exposed treatments. No significant difference in the 26°C exposed treatment. G1, the time by which G0 young adults exposed to thermal stress have spawned to produce G1 young adults. Discussion The last few years have seen significant advances in our knowledge of the mechanisms of rapid adaptation to rising global temperature during biological invasions (Lee 2002, Yu et al. 2012). Insects, with their limited thermoregulation abilities and small size, are particularly susceptible to ambient temperature changes. Thus, insects require the ability to sense environmental and internal temperatures in order to maintain the fundamental biochemistry of cellular metabolism and to avoid tissue-damaging noxious temperatures. TRPA is a cation channel with highly temperature-dependent conductance that participates in thermosensation in insects (Wetsel 2011). Therefore, investigating the function of TRPA in mediating thermosensation during biological invasions can reveal how organisms cope with rapidly changing environments in the wild. TRPA has been studied as an internal thermosensor; this gene is highly expressed in the olfactory organs and can be activated by warm temperatures in insects, including Anopheles gambiae (Wei et al. 2015) and H. armigera (Wang et al. 2009). In this study, BtTRPA was robustly expressed in the head, where the antennae are located, indicating that BtTRPA may also function as a thermoreceptor in MED. Furthermore, BtTRPA transcripts were present at all developmental stages in MED. BtTRPA expression increased sharply and peaked in adult females, indicating that the pattern of BtTRPA expression was sex-dependent during the adult stages. Determining whether a sensory stimulus is noxious or innocuous is critical for insect survival. The TRPA1 gene encodes multiple TRPA1 channel isoforms, which range from the highly warmth-responsive isoform to thermally insensitive isoforms (Kang et al. 2011, Zhong et al. 2012). For example, TRPA1 is a bona fide mediator of thermal nociception in Drosophila (Neely et al. 2011). Our data showed that the BtTRPA gene was robustly expressed after exposure to acute heat or cold, as the expression was the highest after 3 h at 0°C and 5 h at 40°C and relatively low after exposure to milder thermal stimuli of 12 and 35°C. This result was consistent with those of previous reports (Neely et al. 2011, Barbagallo and Garrity 2015). Simultaneously, based on these observations, short-term exposure to acute high or low temperatures significantly increased the expression of BtTRPA in MED, demonstrating that this rapid acclimation was probably mediated by thermosensory mechanisms. Thus, the data obtained in this study showed that BtTRPA could discriminate between innocuous and noxious temperature stimuli. Furthermore, short-term exposure was enough to trigger a transcriptional response to protect the organism from acute temperature stress. In the context of global climate warming, ectotherms continually encounter innocuous temperature variations that affect their body temperature and can have significant long-term physiological and ecological impacts (Dillon et al. 2010). In Drosophila, TRPA1 was initially found to be essential for avoidance of warm temperatures over ~30°C (Rosenzweig et al. 2005) and subsequently for sensing temperatures as low as ~20°C (Kwon et al. 2008). In this study, the expression of the BtTRPA gene increased rapidly after 5 d and was significantly higher than the subsequent treatments when exposed to the innocuous temperatures 17 and 35°C. These temperatures were close to the developmental extremes for MED, indicating that the BtTRPA gene could rapidly respond to temperature stress and then trigger related behavioral or physiological responses to survive (Klein et al. 2015). Intriguingly, the highest expression level of the BtTRPA gene was at the G1 stage under exposure to mild innocuous temperatures of 21 and 31°C, indicating that the BtTRPA gene had very high expression in the next generation. Although the above results showing that BtTRPA expression was significant higher in adult females than males, we used a 1: 1 ratio to avoid the possible differences in thermal response between sexes. Thus, we speculated that the highest expression of G1 might be cause by developmental cumulative effect, or because that the effects of mild innocuous temperature exposure could be transmitted to offspring via the parent to enhance the ability of the offspring to cope with similar temperature stress, or even a combined effect. Whereas, we could not ignore the significantly higher expression in adult females, as TRPA could be thermally activated during embryogenesis and acts as a molecular switch for the development of an alternative phenotype in B. mori (Sato et al. 2014). Thus, it is reasonable to speculate that the expression pattern of BtTRPA in response to thermal stress could be transferred to offspring through the maternal line. The diverse responses to thermal stress indicate that different regulatory mechanisms driven by different innocuous thermal exposures may exist. However, the exact mechanism remains elusive in MED. As MED is a worldwide invasive insect, and global warming is expected to accelerate its rate of invasion and expand its geographical distribution, sensitive thermosensory mechanisms for responding to hot and cold thermal stresses might be key mechanisms facilitating its rapid worldwide expansion. Further studies are needed to determine: 1) the mechanism of sex-dependent expression pattern of BtTRPA in MED and whether exist different thermal responses between sexes; 2) the molecular mechanism of the different BtTRPA expression of offspring; and 3) the precise function of BtTRPA in thermosensation in MED, e.g., by electrophysiological analysis or clustered regularly interspaced short palindromic repeats (CRISPR). Based on the results in this study, which show the involvement of TRPs in thermal acclimation at the gene expression level, we speculate that BtTRPA is mainly expressed in antennae in the head and functions as a peripheral thermal sensor in MED. Additionally, these data support the hypothesis that BtTRPA can discriminate between innocuous and noxious temperature stimuli. Furthermore, this study also shows that BtTRPA appears to be most strongly expressed in adult females and that mild innocuous thermal exposure can cause transgenerational expression effects to enhance the ability of offspring to cope with the same stresses. Therefore, flexible mechanisms of thermal stress response and the ability of the BtTRPA gene to shift its function between heat and cold stress may be important mechanisms facilitating the rapid worldwide expansion of the whitefly B. tabaci MED. Supplementary Material Supplementary data are available at Environmental Entomology online. Acknowledgments This work was supported by the National Key Research and Development Program (2016YFC1200600); the Ministry of Science and Technology, China; the National Natural Science Foundation of China (31672088); the International Science & Technology Cooperation Program of China (2015DFG32300); the Commonwealth Special Fund for the Agricultural Industry (201303019); the National Basic Research and Development Program (2013CB127605); and the Chinese Ministry of Agriculture, 948 Project (2016-X48). References Cited Barbagallo, B., and Garrity P. A.. 2015. Temperature sensation in Drosophila. Curr. Opin. Neurobiol . 34: 8– 13. Google Scholar CrossRef Search ADS PubMed  Bonato, O., Lurette A., Vidal C., and Fargues J.. 2007. Modelling temperature-dependent bionomics of Bemisia tabaci (Q-biotype). Physiol. Entomol . 32: 50– 55. Google Scholar CrossRef Search ADS   Chen, J., Kang D., Jing X., Marc L., Hong H. J. O. S. C. Karl, W. Betty, Y., and Donghee K.. 2011. 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Thermal Discrimination and Transgenerational Temperature Response in Bemisia tabaci Mediterranean (Hemiptera: Aleyrodidae): Putative Involvement of the Thermo-Sensitive Receptor BtTRPA

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Entomological Society of America
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© The Author(s) 2018. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.
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0046-225X
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1938-2936
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10.1093/ee/nvx202
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Abstract

Abstract Anthropogenic climate change and global warming are expected to alter the geographic distribution and abundance of many ectothermic species, which will increase the invasion of new areas by exotic species. To survive in variable or fluctuating temperature conditions, insects require sensitive thermal sensory mechanisms to detect external thermal stimuli and induce the appropriate behavioral and physiological responses. TRPA, a thermal-activated transient receptor potential (TRP) family ion channel, is essential for thermotaxis in insects. Here, we investigated the potential role of BtTRPA in short-term and long-term thermal stress in Bemisia tabaci Mediterranean (Gennadius; Hemiptera: Aleyrodidae). We found that BtTRPA was mainly expressed in the head, where the antennae are located. Under short-term thermal stress, the BtTRPA gene was robustly expressed after exposure to acute low or high temperatures, BtTRPA expression reached the highest levels after exposure to 0°C for 3 h and 40°C for 5 h, but was relatively low after exposure to milder stimuli (12 and 35°C). These results demonstrated that BtTRPA could discriminate between innocuous and noxious temperature stimuli. Under long-term thermal stress, the highest expression level of BtTRPA occurred at G1 exposed to mild innocuous temperature of 21 and 31°C, along with BtTRPA sharply increased and peaked in adult females, implying that mild innocuous long-term thermal exposure could cause transgenerational expression effects to enhance the ability of offspring to cope with the same stress. This study demonstrates that the channel BtTRPA is important in temperature sensing and provides a molecular basis for thermosensation regulation in response to varied environmental temperature in B. tabaci Mediterranean. Global warming due to anthropogenic climate change has exposed ectothermic organisms to multifarious environmental stresses, resulting in the exploitation of various strategies to increase their adaptability in the face of stress (Walther et al. 2002, de Nadal et al. 2011). Invasive insects, which spread rapidly and colonize successfully after invading new geographical ranges, are becoming appropriate models for studying the patterns and mechanisms of rapid acclimation (Ward and Masters 2007, Richardson 2010). Theoretical and experimental studies have shown that the capacity to tolerate extreme and fluctuating temperatures is one of the foremost important abiotic factors determining the geographic distributions of most insects (Gutierrez et al. 2014, Lancaster 2016, Lancaster et al. 2016). As small poikilothermic organisms, insects are vulnerable to rapid body temperature changes in response to environmental temperature fluctuation. Thus, in order to survive, insects should execute appropriate physiological and behavioral responses, which must be regulated by proper thermosensation mechanisms (Hong et al. 2015). Transient receptor potential (TRP) channels have been implicated as key temperature-responsive ion channels in both mammals and insects (Rosenzweig et al. 2005, Wetsel 2011). TRPs are divided into seven different thermosensitive receptor subfamilies-namely, TRPC, TRPA, TRPV, TRPN, TRPM, TRPP, and TRPML-based on their sequence elements and six transmembrane domains (Matsuura et al. 2009, Fowler and Montell 2013). As previously reported, the members of the TRPM, TRPA, and TRPV subfamily are thought to mediate thermosensation (Dhaka et al. 2006, Wetsel 2011). Intriguingly, TRPAs in mammals are cold-activated channels (Story et al. 2003, Chen et al. 2011), whereas TRPAs in insects mediate the sensing of warm and high temperatures (Kang et al. 2011, Barbagallo and Garrity 2015). A recent study has shown that Bombyx mori (L.; Lepidoptera: Bombycidae) TRPA1 is activated at temperatures above ~21°C and affects the induction of diapause in progeny (Sato et al. 2014). In Drosophila melanogaster (Matsumura; Diptera: Drosophilidae), TRPA1 is critical for larvae to discriminate between the optimal temperature (18°C) and slightly higher temperatures (19~24°C) (Kwon et al. 2008, Fowler and Montell 2013), and it is also implicated in high-temperature nociception in adult flies (Neely et al. 2011). Helicoverpa armigera TRPA1 is activated by increasing the temperature from 20 to 45°C. Interestingly, Nasonia vitripennis TRPA appears to be activated as soon as temperature rises, regardless of the initial temperature, and this activation is highly dependent on the heating rate (Matsuura et al. 2009). Therefore, insect TRPA channels are part of the system of rapid response to heat and variability in temperature. Bemisia tabaci (Gennadius; Hemiptera: Aleyrodidae) is a complex species comprising at least 36 morphologically indistinguishable species, of which Mediterranean (MED, former biotype Q) and Middle East-Asia Minor 1 (MEAM 1, former biotype B) are the most common and cosmopolitan invasive (De Barro et al. 2011, Colvin and Barro 2012). MED, which originated in Europe, is now globally distributed and is found on all continents except Antarctica (Martin et al. 2000, Tay et al. 2012), possibly due to its high potential adaptability to various environmental temperatures (Cui et al. 2008, Elbaz et al. 2011, Yu et al. 2012). Previous studies have found that whiteflies can utilize not only physiological mechanisms such as sorbitol accumulation in response to elevated temperatures (Wolfe et al. 1998) but also heat shock proteins and other stress-related genes to guard against thermal stress (Mahadav et al. 2009, Lü and Wan 2011). However, the mechanism by which external temperature stimuli are transmitted to the neural network in whitefly remains unknown, and the ability of the neural signals to activate the expression of stress-related genes to increase thermal resistance has not been fully explored. Furthermore, as far as we know, previous reports on the thermosensation system of whitefly are rare. For example, study in MED has mainly focused on the discovery of TRP (Li et al. 2015), while Lü et al. (2014) found that MEAM1 BtTRPA is a key element in sensing high temperature and plays an essential role in B. tabaci MEAM1 heat tolerance ability. Our experimental system was specifically designed to provide insight into the temperature sensing mechanism and thermal plasticity role of the BtTRPA gene. We had two major objectives: first, we assessed the expression of BtTRPA throughout the course of development and among the tagmata, and second, we conducted a series of short-term and long-term experiments to quantify the expression patterns of BtTRPA in response to various temperatures in the invasive whitefly MED. Materials and Methods Insect Culture MED population used in this study was initially obtained from a tomato filed in Beijing in 2012 and have been reared in the laboratory for 50–60 generations. The species was reared on Lycopersicon esculentum Mill (Zhongza No. 9) in a climate room under a photoperiod of 14:10 (L:D) h at 26 ± 2°C and 50 ± 10.0% relative humidity (RH). Tagmata and Developmental Sample Collection For samples of different developmental stages, 2,000 egg, 1,000 1st–2nd nymph, 500 3rd–4th nymph, 500 pupae, 200 female adults, and 200 male adults were chosen. For samples of tagmatas, head, thorax, and abdomen were separately collected from 800 female and male adults. All the samples were frozen immediately in liquid nitrogen and stored at −80°C until further use. Short-term Temperature Stress Based on the occurrence time and the geographical distribution of MED invasion in fields in China, we designed four temperature treatments (0, 12, 35, and 40°C) as short-term thermal stress temperatures. MED adults were placed together in a 5 ml centrifuge tube covered with cotton. The tubes were then placed in a constant-temperature environment at 0, 12, 35, and 40°C, respectively. After 1, 3, and 5 h, the live adults were frozen instantaneously and stored at −80°C, respectively. We standardized adult age using newly emerged whitefly adults that were younger than 3 h and the sex of female and male is in a ratio of about 1:1 in each treatment. Adults maintained at 26°C were used as untreated controls. Each treatment was carried out with three replicates of 200 adults. Long-term Temperature Stress Simultaneously considering the fact that 17 and 35°C were close to the lower and upper thermal limits (Bonato et al. 2007), the need to obtain sufficient effective samples, and the developmental time, longevity and reproduction of adults in different temperature conditions, we finally chose 17, 21, 31, and 35°C as long-term thermal stress temperatures. Newly emerged MED adults were collected from tomato plants and placed into cages, The cages were then placed in climate-controlled chambers (Saifu, Ningbo, China) and were exposed to 17 and 21°C for 5, 10, 15, and 20 d and until G1, where G1 is the time by which G0 young adults exposed to thermal stress have spawned to produce G1 young adults. Adults maintained at 26°C were used as untreated controls. All samples were frozen instantaneously after treatment using liquid nitrogen. Each treatment was carried out with three replicates of 200 adults. Total RNA Extraction and Quantitative Real-time PCR Total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA) according to the instructions. The RNA concentration and quality were determined by NanoPhotometer P330 (Implen, Munich, Germany) and 1% agarose gel electrophoresis. The first-strand cDNA synthesis was carried out using 2.0 μg purified RNA using the Super Script First-Strand Synthesis System (Transgen, Beijing, China) following the manufacturer’s protocol and stored at −20°C. The primers used are based on references Lü et al. (2014) and Dai et al. (2017). The qRT-PCR reactions were performed in an ABI 7500 Real-time PCR system (Applied Biosystems, Foster, CA) with 20 μl PCR cocktail contained 1 μl of the cDNA, 10 μl of 2×TransStart Green qPCR SuperMix (Transgen), 0.5 μl of each 10 μM primer, and 0.4 µl of Passive Reference Dye. The amplification cycles were as follows: 30 s at 95°C, then 40 cycles of 5 s at 95°C, 15 s at 60°C, and 30 s at 72°C. β-tubulin and EF1-α (GenBank accession no.: EE600682) were used as reference genes, because they are constitutively expressed under various temperature stress conditions (Dai et al. 2017). All samples were run in triplicate to account for technical variation. The amplification efficiency (E) of all primer pairs was validated by constructing a standard curve using a twofold serial dilution of cDNA template using the equation: E= (10[−1/slope]−1) × 100 (Radonić et al. 2004). The main characteristics of the primers are listed in Supplementary Material. Statistical Analysis The expression of the TRPA gene was normalized using the 2−ΔΔCt method (Livak and Schmittgen 2001) after the threshold cycle (Ct) was normalized with the Ct of β-tubulin and EF1-α. Based on the equation ΔΔCt = (Cttarget − Ctreference) treatment − (Cttarget − Ctreference) control. The head sample and the pupae sample was used as the calibrator sample for tagmata treatments (expression = 1) and developmental treatments (expression = 1), respectively. A 26°C adults sample was used as the calibrator sample for thermal stress treatments (expression = 1). All expression data were log-transformed to ensure that they were normally distributed. Target gene mRNA expression was analyzed using a one-way ANOVA followed by Fisher’s least significant difference (LSD) test. The data were presented as the means ± standard error (mean ± SE). Differences were considered statistically significant at P ≤ 0.05. Results Expression Analysis of BtTRPA Gene Among Tagmata and During Development The BtTRPA gene was expressed in all three major tagmata examined at different levels, showing some tissue specificity (P < 0.05, Fig. 1). The results indicated that BtTRPA was expressed abundantly in the head and at relatively low levels in the thorax and abdomen. Experiments in H. armigera showed that TRPA1 was abundantly expressed in the antennae and moderately expressed in the labial palps and fairly low in the proboscises (Wei et al. 2015). Thus, the results indicated that BtTRPA may be highly expressed in antennae. Fig. 1. View largeDownload slide Tagmata expression of Bemisia tabaci MED BtTRPA. The results are expressed as the mean ± SEM after log-transformed. Error bars indicate the standard error of the calculated mean based on three biological replicates. Bars with a different lowercase letter at the top show a significant difference at P < 0.05. Fig. 1. View largeDownload slide Tagmata expression of Bemisia tabaci MED BtTRPA. The results are expressed as the mean ± SEM after log-transformed. Error bars indicate the standard error of the calculated mean based on three biological replicates. Bars with a different lowercase letter at the top show a significant difference at P < 0.05. BtTRPA mRNA was also present at all developmental stages (Fig. 2). However, the levels and pattern of expression appeared to change during the development of MED (P < 0.05, Fig. 2). BtTRPA expression in adult stage was significantly higher than the expression in the other stages, and the expression was extremely low and not significantly different in the egg, 1st–2nd nymph, 3rd–4th nymph, with the lowest expression in the pupae. However, at the adult stage, the BtTRPA transcript expression in females increased twofold, and was significantly higher than the level observed in males (Fig. 2). Fig. 2. View largeDownload slide Developmental expression of Bemisia tabaci MED BtTRPA. The results are expressed as the mean ± SEM after log-transformed. Error bars indicate the standard error of the calculated mean based on three biological replicates. Bars with a different lowercase letter at the top show a significant difference at P < 0.05. Fig. 2. View largeDownload slide Developmental expression of Bemisia tabaci MED BtTRPA. The results are expressed as the mean ± SEM after log-transformed. Error bars indicate the standard error of the calculated mean based on three biological replicates. Bars with a different lowercase letter at the top show a significant difference at P < 0.05. Short-term Cold Stress The expression levels of BtTRPA following short-term exposure to cold temperatures at 0°C from 1 to 5 h were significantly higher than at the control temperature of 26°C; the content of BtTRPA mRNA after exposure for 5 h was 8.0 and 2.02 times that after exposure for 1 and 3 h, respectively. The expression level of BtTRPA after 12°C exposure for 3 h was significantly higher than the levels after exposure for 1 and 5 h, with 3.39- and 2.15-fold increases, respectively (P < 0.05 in all cases; Fig. 3). Fig. 3. View largeDownload slide Relative expression levels of BtTRPA mRNA in Bemisia tabaci MED adults after short-term cold exposure. The results are expressed as the means ± SEM after log-transformed. Error bars indicate the standard error of the calculated mean based on three biological replicates. White, black, and gray bars represent the relative mean expression in the 26, 0, and 12°C exposed treatments, respectively. a, b and c at the top of the blue bars showed a significant difference at P < 0.05 in the 0°C exposed treatments. a′ and b′ at the top of the red bars showed a significant difference at P < 0.05 in the 12°C exposed treatments. No significant difference in the 26°C exposed treatment. Fig. 3. View largeDownload slide Relative expression levels of BtTRPA mRNA in Bemisia tabaci MED adults after short-term cold exposure. The results are expressed as the means ± SEM after log-transformed. Error bars indicate the standard error of the calculated mean based on three biological replicates. White, black, and gray bars represent the relative mean expression in the 26, 0, and 12°C exposed treatments, respectively. a, b and c at the top of the blue bars showed a significant difference at P < 0.05 in the 0°C exposed treatments. a′ and b′ at the top of the red bars showed a significant difference at P < 0.05 in the 12°C exposed treatments. No significant difference in the 26°C exposed treatment. Short-term Heat Stress Compared to level at the control temperature of 26°C, the expression level of BtTRPA following short-term exposure to heat at 35°C was higher at 5 h than at 1 and 3 h, with a 12.47- and 16.79-fold, increase, respectively. The expression level of BtTRPA at 40°C after 3 h of exposure was significantly higher than the levels after 1 and 5 h, with increases of 1.84- and 4.06-fold, respectively (P < 0.05 in all cases; Fig. 4). Fig. 4. View largeDownload slide Relative expression levels of BtTRPA mRNA in Bemisia tabaci MED adults after short-term heat exposure. The results are expressed as the means ± SEM after log-transformed. Error bars indicate the standard error of the calculated mean based on three biological replicates. White, black, and gray bars represent the relative mean expression in the 26, 35, and 40°C exposed treatments, respectively. a and b at the top of the blue bars showed a significant difference at P < 0.05 in the 35°C exposed treatments. a′, b′, and c′ at the top of the red bars showed a significant difference at P < 0.05 in the 40°C exposed treatments. No significant difference in the 26°C exposed treatment. Fig. 4. View largeDownload slide Relative expression levels of BtTRPA mRNA in Bemisia tabaci MED adults after short-term heat exposure. The results are expressed as the means ± SEM after log-transformed. Error bars indicate the standard error of the calculated mean based on three biological replicates. White, black, and gray bars represent the relative mean expression in the 26, 35, and 40°C exposed treatments, respectively. a and b at the top of the blue bars showed a significant difference at P < 0.05 in the 35°C exposed treatments. a′, b′, and c′ at the top of the red bars showed a significant difference at P < 0.05 in the 40°C exposed treatments. No significant difference in the 26°C exposed treatment. Long-term Cold Stress Compared to those of whiteflies grown at the control temperature of 26°C, the expression levels of BtTRPA declined significantly following long-term exposure to a low temperature of 17°C from 5 d to the G1 stage; the content of BtTRPA mRNA after exposure for 5 d was 4.13, 1.69, 3.25, and 6.25 times that after exposure for 10, 15, and 20 d and until G1, respectively. In contrast, the expression levels of BtTRPA at 21°C was significantly higher at G1 stage than other time-period, and with the lowest expression at 15 d; the content of BtTRPA after exposure until G1 was 25.35, 46.01, 221.84, and 45.51 times those after exposure for 5 , 10 , 15, and 20 d, respectively (P < 0.05 in all cases; Fig. 5). Fig. 5. View largeDownload slide Relative expression levels of BtTRPA mRNA in Bemisia tabaci MED adults after long-term cold exposure. The results are expressed as the mean ± SEM after log-transformed. Error bars indicate the standard error of the calculated mean based on three biological replicates. White, black, and gray bars represent the relative mean expression in the 26, 17, and 21°C exposed treatments, respectively. a, b, and c at the top of the blue bars showed a significant difference at P < 0.05 in the 17°C exposed treatments. a′, b′, c′ and d′ at the top of the red bars showed a significant difference at P < 0.05 in the 21°C exposed treatments. No significant difference in the 26°C exposed treatment. G1, the time by which G0 young adults exposed to thermal stress have spawned to produce G1 young adults. Fig. 5. View largeDownload slide Relative expression levels of BtTRPA mRNA in Bemisia tabaci MED adults after long-term cold exposure. The results are expressed as the mean ± SEM after log-transformed. Error bars indicate the standard error of the calculated mean based on three biological replicates. White, black, and gray bars represent the relative mean expression in the 26, 17, and 21°C exposed treatments, respectively. a, b, and c at the top of the blue bars showed a significant difference at P < 0.05 in the 17°C exposed treatments. a′, b′, c′ and d′ at the top of the red bars showed a significant difference at P < 0.05 in the 21°C exposed treatments. No significant difference in the 26°C exposed treatment. G1, the time by which G0 young adults exposed to thermal stress have spawned to produce G1 young adults. Long-term Heat Stress Compared to those of whiteflies grown at the control temperature of 26°C, the expression levels of BtTRPA increased significantly following long-term exposure to a high temperature of 31°C from 5 d to the G1 stage; the content of BtTRPA after exposure until G1 was 48.14, 49.78, 4.45, and 21.86 times that after exposure for 5, 10, 15, and 20 d, respectively. In contrast, the expression levels of BtTRPA at 35°C from 5 d until G1 declined significantly; the content of BtTRPA after exposure for 5 d was 25.21, 121.95, 97.11, and 32.17 times those after exposure for 10, 15, and 20 d and until G1, respectively (P < 0.05 in all cases; Fig. 6). Fig. 6. View largeDownload slide Relative expression levels of BtTRPA mRNA in Bemisia tabaci MED adults after long-term heat exposure. The results are expressed as the mean ± SEM after log-transformed. Error bars indicate the standard error of the calculated mean based on three biological replicates. White, black, and gray bars represent the relative mean expression in the 26, 31, and 35°C exposed treatments, respectively. a, b and c at the top of the blue bars showed a significant difference at P < 0.05 in the 31°C exposed treatments. a′, b′, and c′ at the top of the red bars showed a significant difference at P < 0.05 in the 35°C exposed treatments. No significant difference in the 26°C exposed treatment. G1, the time by which G0 young adults exposed to thermal stress have spawned to produce G1 young adults. Fig. 6. View largeDownload slide Relative expression levels of BtTRPA mRNA in Bemisia tabaci MED adults after long-term heat exposure. The results are expressed as the mean ± SEM after log-transformed. Error bars indicate the standard error of the calculated mean based on three biological replicates. White, black, and gray bars represent the relative mean expression in the 26, 31, and 35°C exposed treatments, respectively. a, b and c at the top of the blue bars showed a significant difference at P < 0.05 in the 31°C exposed treatments. a′, b′, and c′ at the top of the red bars showed a significant difference at P < 0.05 in the 35°C exposed treatments. No significant difference in the 26°C exposed treatment. G1, the time by which G0 young adults exposed to thermal stress have spawned to produce G1 young adults. Discussion The last few years have seen significant advances in our knowledge of the mechanisms of rapid adaptation to rising global temperature during biological invasions (Lee 2002, Yu et al. 2012). Insects, with their limited thermoregulation abilities and small size, are particularly susceptible to ambient temperature changes. Thus, insects require the ability to sense environmental and internal temperatures in order to maintain the fundamental biochemistry of cellular metabolism and to avoid tissue-damaging noxious temperatures. TRPA is a cation channel with highly temperature-dependent conductance that participates in thermosensation in insects (Wetsel 2011). Therefore, investigating the function of TRPA in mediating thermosensation during biological invasions can reveal how organisms cope with rapidly changing environments in the wild. TRPA has been studied as an internal thermosensor; this gene is highly expressed in the olfactory organs and can be activated by warm temperatures in insects, including Anopheles gambiae (Wei et al. 2015) and H. armigera (Wang et al. 2009). In this study, BtTRPA was robustly expressed in the head, where the antennae are located, indicating that BtTRPA may also function as a thermoreceptor in MED. Furthermore, BtTRPA transcripts were present at all developmental stages in MED. BtTRPA expression increased sharply and peaked in adult females, indicating that the pattern of BtTRPA expression was sex-dependent during the adult stages. Determining whether a sensory stimulus is noxious or innocuous is critical for insect survival. The TRPA1 gene encodes multiple TRPA1 channel isoforms, which range from the highly warmth-responsive isoform to thermally insensitive isoforms (Kang et al. 2011, Zhong et al. 2012). For example, TRPA1 is a bona fide mediator of thermal nociception in Drosophila (Neely et al. 2011). Our data showed that the BtTRPA gene was robustly expressed after exposure to acute heat or cold, as the expression was the highest after 3 h at 0°C and 5 h at 40°C and relatively low after exposure to milder thermal stimuli of 12 and 35°C. This result was consistent with those of previous reports (Neely et al. 2011, Barbagallo and Garrity 2015). Simultaneously, based on these observations, short-term exposure to acute high or low temperatures significantly increased the expression of BtTRPA in MED, demonstrating that this rapid acclimation was probably mediated by thermosensory mechanisms. Thus, the data obtained in this study showed that BtTRPA could discriminate between innocuous and noxious temperature stimuli. Furthermore, short-term exposure was enough to trigger a transcriptional response to protect the organism from acute temperature stress. In the context of global climate warming, ectotherms continually encounter innocuous temperature variations that affect their body temperature and can have significant long-term physiological and ecological impacts (Dillon et al. 2010). In Drosophila, TRPA1 was initially found to be essential for avoidance of warm temperatures over ~30°C (Rosenzweig et al. 2005) and subsequently for sensing temperatures as low as ~20°C (Kwon et al. 2008). In this study, the expression of the BtTRPA gene increased rapidly after 5 d and was significantly higher than the subsequent treatments when exposed to the innocuous temperatures 17 and 35°C. These temperatures were close to the developmental extremes for MED, indicating that the BtTRPA gene could rapidly respond to temperature stress and then trigger related behavioral or physiological responses to survive (Klein et al. 2015). Intriguingly, the highest expression level of the BtTRPA gene was at the G1 stage under exposure to mild innocuous temperatures of 21 and 31°C, indicating that the BtTRPA gene had very high expression in the next generation. Although the above results showing that BtTRPA expression was significant higher in adult females than males, we used a 1: 1 ratio to avoid the possible differences in thermal response between sexes. Thus, we speculated that the highest expression of G1 might be cause by developmental cumulative effect, or because that the effects of mild innocuous temperature exposure could be transmitted to offspring via the parent to enhance the ability of the offspring to cope with similar temperature stress, or even a combined effect. Whereas, we could not ignore the significantly higher expression in adult females, as TRPA could be thermally activated during embryogenesis and acts as a molecular switch for the development of an alternative phenotype in B. mori (Sato et al. 2014). Thus, it is reasonable to speculate that the expression pattern of BtTRPA in response to thermal stress could be transferred to offspring through the maternal line. The diverse responses to thermal stress indicate that different regulatory mechanisms driven by different innocuous thermal exposures may exist. However, the exact mechanism remains elusive in MED. As MED is a worldwide invasive insect, and global warming is expected to accelerate its rate of invasion and expand its geographical distribution, sensitive thermosensory mechanisms for responding to hot and cold thermal stresses might be key mechanisms facilitating its rapid worldwide expansion. Further studies are needed to determine: 1) the mechanism of sex-dependent expression pattern of BtTRPA in MED and whether exist different thermal responses between sexes; 2) the molecular mechanism of the different BtTRPA expression of offspring; and 3) the precise function of BtTRPA in thermosensation in MED, e.g., by electrophysiological analysis or clustered regularly interspaced short palindromic repeats (CRISPR). Based on the results in this study, which show the involvement of TRPs in thermal acclimation at the gene expression level, we speculate that BtTRPA is mainly expressed in antennae in the head and functions as a peripheral thermal sensor in MED. Additionally, these data support the hypothesis that BtTRPA can discriminate between innocuous and noxious temperature stimuli. Furthermore, this study also shows that BtTRPA appears to be most strongly expressed in adult females and that mild innocuous thermal exposure can cause transgenerational expression effects to enhance the ability of offspring to cope with the same stresses. Therefore, flexible mechanisms of thermal stress response and the ability of the BtTRPA gene to shift its function between heat and cold stress may be important mechanisms facilitating the rapid worldwide expansion of the whitefly B. tabaci MED. Supplementary Material Supplementary data are available at Environmental Entomology online. 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Environmental EntomologyOxford University Press

Published: Feb 1, 2018

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