Portal de Periódicos da CAPES

Identification of a Gene, Desiccate, Contributing to Desiccation Resistance in Drosophila melanogaster

2010; Elsevier BV; Volume: 285; Issue: 50 Linguagem: Inglês

10.1074/jbc.m110.168864

1083-351X

Takeshi Kawano, Masami Shimoda, Hitoshi Matsumoto, Masasuke Ryuda, Seiji Tsuzuki, Yoichi Hayakawa,

Heat shock proteins research

Suitable alterations in gene expression are believed to allow animals to survive drastic changes in environmental conditions. Drosophila melanogaster larvae cease eating and exit moist food to search for dry pupation sites after the foraging stage in what is known as the wandering stage. Although the behavioral change from foraging to wandering causes desiccation stress, the mechanism by which Drosophila larvae protect themselves from desiccation remains obscure. Here, we identified a gene, CG14686 (designated as Desiccate (Desi)), whose expression was elevated during the wandering stage. The Desi expression level was reversibly decreased by transferring wandering larvae to wet conditions and increased again by transferring them to dry conditions. Elevation of Desi expression was also observed in foraging larvae when they were placed in dry conditions. Desi encoded a 261-amino acid single-pass transmembrane protein with notable motifs, such as SH2 and PDZ domain-binding motifs and a cAMP-dependent protein kinase phosphorylation motif, in the cytoplasmic region, and its expression was observed mainly in the epidermal cells of the larval integuments. Overexpression of Desi slightly increased the larval resistance to desiccation stress during the second instar. Furthermore, Desi RNAi larvae lost more weight under dry conditions, and subsequently, their mortalities significantly increased compared with control larvae. Under dry conditions, consumption of carbohydrate was much higher in Desi RNAi larvae than control larvae. Based on these results, it is reasonable to conclude that Desi contributes to the resistance of Drosophila larvae to desiccation stress. Suitable alterations in gene expression are believed to allow animals to survive drastic changes in environmental conditions. Drosophila melanogaster larvae cease eating and exit moist food to search for dry pupation sites after the foraging stage in what is known as the wandering stage. Although the behavioral change from foraging to wandering causes desiccation stress, the mechanism by which Drosophila larvae protect themselves from desiccation remains obscure. Here, we identified a gene, CG14686 (designated as Desiccate (Desi)), whose expression was elevated during the wandering stage. The Desi expression level was reversibly decreased by transferring wandering larvae to wet conditions and increased again by transferring them to dry conditions. Elevation of Desi expression was also observed in foraging larvae when they were placed in dry conditions. Desi encoded a 261-amino acid single-pass transmembrane protein with notable motifs, such as SH2 and PDZ domain-binding motifs and a cAMP-dependent protein kinase phosphorylation motif, in the cytoplasmic region, and its expression was observed mainly in the epidermal cells of the larval integuments. Overexpression of Desi slightly increased the larval resistance to desiccation stress during the second instar. Furthermore, Desi RNAi larvae lost more weight under dry conditions, and subsequently, their mortalities significantly increased compared with control larvae. Under dry conditions, consumption of carbohydrate was much higher in Desi RNAi larvae than control larvae. Based on these results, it is reasonable to conclude that Desi contributes to the resistance of Drosophila larvae to desiccation stress. IntroductionA wide variety of stressful stimuli change patterns of gene expression, which enables animals to adapt to stress, and such changes in gene expression are believed to allow them to survive drastic environmental changes. Activation of heat shock protein genes (hsp) is a typical example; all organisms express a particular set of hsp genes in response to stressors such as temperature extremes, aversive chemical application, anoxia, and many other environmental injuries (1Feder M.E. Hofmann G.E. Annu. Rev. Physiol. 1999; 61: 243-282Crossref PubMed Scopus (3129) Google Scholar, 2Sun Y. MacRae T.H. FEBS J. 2005; 272: 2613-2627Crossref PubMed Scopus (285) Google Scholar). Hsp proteins are generally divided into three families: the 90-kDa, 70-kDa, and small heat shock proteins (3Parsell D.A. Lindquist S. Annu. Rev. Genet. 1993; 27: 437-496Crossref PubMed Scopus (1855) Google Scholar). It has been reported that a nonlethal desiccation at 0% relative humidity (RH) 2The abbreviation used is: RHrelative humidity. enhanced transcriptional levels of the two hsp genes, hsp70 and hsp23, in pupae of the flesh fly Sarcophaga crassipalpis (4Tammariello S.P. Rinehart J.P. Denlinger D.L. J. Insect Physiol. 1999; 45: 933-938Crossref PubMed Scopus (72) Google Scholar). Although the two hsp transcripts were up-regulated in response to desiccation, the up-regulation was less dramatic than that elicited by heat shock, and desiccation failed to generate tolerance to high or low temperatures. Recently, it has been also reported that dehydration elicited expression of hsp70 in three mosquito species, Aedes aegypti, Anopheles gambiae, and Culex pipiens, but hsp90 expression levels remained fairly constant. Furthermore, injection of dsRNA to knock down expression of hsp70 and hsp90 significantly decreased survival rates of A. aegypti under dehydration (5Benoit J.B. Lopez-Martinez G. Phillips Z.P. Patrick K.R. Denlinger D.L. J. Insect Physiol. 2010; 56: 151-156Crossref PubMed Scopus (86) Google Scholar). Exposure of adult male Drosophila melanogaster to desiccation enhanced transcriptional levels of Frost and senescence marker protein-30 (smp-30) but did not change those of hsp70Aa and hsp23 (6Sinclair B.J. Gibbs A.G. Roberts S.P. Insect Mol. Biol. 2007; 16: 435-443Crossref PubMed Scopus (162) Google Scholar). Although these previous studies demonstrated desiccation-induced gene expression, we do not know whether the up-regulation of the gene expression levels confers desiccation resistance on animals.Drosophila melanogaster, like all holometabolous insects, undergoes complete metamorphosis to reach adulthood. Each phase of the life cycle is characterized by a coordinated program of developmental events and behavioral transitions that have evolved to promote fitness and survival (7Riddiford L.M. Bate M. Martinez-Arias A. The Development of Drosophila melanogaster. Cold Spring Harbor Laboratory Press, Plainview, NY1993: 899-939Google Scholar, 8Thummel C.S. Dev. Cell. 2001; 1: 453-465Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar). In Drosophila larvae, the essential midthird instar transition from foraging (feeding) to wandering (nonfeeding) behavior occurs prior to pupariation and metamorphosis. Although this behavioral transition imposes desiccation stress on the larvae, it is unknown not only whether there is a specific mechanism responsible for providing the larvae with desiccation tolerance but also how the tolerance of the larvae is enhanced during the wandering stage. In this study, we sought a gene whose expression was elevated by desiccation stress and identified Desiccate (Desi). Desi expression in the larvae is dependent on relative humidity; desiccation enhanced its expression, and conversely, humidification repressed its expression. Desi encoded a single-pass transmembrane protein and expressed its transcripts actively in epidermal cells of the integument. Furthermore, Desi expression specifically increased during the wandering stage and the survival rates of Desi RNAi larvae significantly declined under the dry condition.DISCUSSIONWater conservation is especially essential for the survival of holometabolous insect larvae because they have less lipidic cuticles than pupae and adults. Despite this, Drosophila larvae change their habitat from wet to arid environments during the midthird instar for normal metamorphosis. Although it was recently reported that the sensory neuron of the degenerin/epithelial sodium channel subunit, Pickpocket1, contributes to regulation of the behavioral transition of Drosophila larvae from foraging to wandering stages (18Ainsley J.A. Kim M.J. Wegman L.J. Pettus J.M. Johnson W.A. Dev. Biol. 2008; 322: 46-55Crossref PubMed Scopus (34) Google Scholar), we do not know how they adapt to the rapid change of habitat from wet to arid conditions. To elucidate the key physiological mechanisms underlying the desiccation resistance that they must acquire around the time of initiation of the wandering stage, we conducted differential display RT-PCR to seek a gene whose expression is enhanced by desiccation. One gene, Desiccate (Desi), that we identified in this study encoded a 261-amino acid single transmembrane protein. The cytoplasmic tail of Desi contains several notable motifs such as SH2 (Src homology 2) domain-binding motifs, a cAMP-dependent protein kinase (protein kinase A) phosphorylation site, and PDZ domain-binding motifs. One of the SH2 domain-binding motifs is a GRB2-like SH2 domain-binding motif (19Chardin P. Cussac D. Maignan S. Ducruix A. FEBS Lett. 1995; 369: 47-51Crossref PubMed Scopus (124) Google Scholar) and the other is a STAT5-family SH2 domain-binding motif (20Beisenherz-Huss C. Mundt M. Herrala A. Vihko P. Schubert A. Groner B. Mol. Cell Endocrinol. 2001; 183: 101-112Crossref PubMed Scopus (8) Google Scholar). The PDZ domain-binding motifs in Desi are categorized as a class III PDZ domain-binding motif (21Fanning A.S. Anderson J.M. Curr. Biol. 1996; 6: 1385-1388Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar). Both binding motifs for SH2 domains and PDZ domains are found in signaling molecules through binding with other component proteins of signal transductions. Furthermore, it has been reported that protein kinase A-induced phosphorylation regulates activities of some receptors, including acetylcholine receptors (22Nishizaki T. Sumikawa K. Brain Res. 1998; 812: 242-245Crossref PubMed Scopus (23) Google Scholar) and N-methyl-d-aspartate receptors (23Crump F.T. Dillman K.S. Craig A.M. J. Neurosci. 2001; 21: 5079-5088Crossref PubMed Google Scholar). Therefore, the presence of the conserved motifs in the Desi cytoplasmic tail suggested the possibility that Desi transmits signals of extracellular desiccation stress to the intracellular signaling pathway. It is worth emphasizing the fact that most of these motifs are conserved in the orthologous genes identified in the EST data bases of mosquitoes, bees, and beetles.To demonstrate the biological significance of this gene, we established transgenic Drosophila larvae whose Desi expression was either up-regulated by expressing a functional Desi cDNA (UAS-Desi) or down-regulated by expressing Desi dsRNA (UAS-dsDesi). Although a significantly positive effect on desiccation-resistance by overexpression of Desi during the third larval instar was not apparent, the same treatment slightly but significantly increased the survival rates of the second instar larvae under the desiccated condition. This stage-dependent difference in the effect on stress resistance must be due to the difference in the endogenous expression levels of Desi; Desi expression is basically higher in third instar larvae than second instar larvae. Furthermore, it is often observed that larvae repeatedly crawl up and down the food especially at the end of the foraging third instar. Because elevation of Desi expression seemed to have begun already during this stage, it was difficult to detect the positive effect on desiccation-resistance by Desi overexpression in the third instar larvae. Therefore, the overexpression-dependent increase in the survival rates under the desiccated condition occurred only in second instar larvae. Desi knockdown, in contrast, significantly decreased desiccation resistance even during the third larval instar, indicating the contribution of Desi to the protection of Drosophila larvae from desiccation stress. The high mortality rate in Desi RNAi larvae was likely due to increased water loss because the RNAi larvae were the only ones that lost significantly more weight than control larvae after 5 h of desiccation treatment (Fig. 5C). Sørensen et al. (24S⊘rensen J.G. Nielsen M.M. Loeschcke V. J. Evol. Biol. 2007; 20: 1624-1636Crossref PubMed Scopus (115) Google Scholar) reported changes in gene expression in D. melanogaster selected for ecologically relevant environmental stress resistance traits including desiccation, starvation, and cold resistance. However, CG14686 (Desi) was not on the list of genes whose expression was changed in such stress-resistant flies. Furthermore, Telonis-Scott and Hoffmann (25Telonis-Scott M. Hoffmann A.A. J. Insect Physiol. 2003; 49: 1013-1020Crossref PubMed Scopus (6) Google Scholar) have reported a desiccation-resistant mutant of D. melanogaster that showed 2-fold higher resistance than control flies when flies were placed under desiccation stress. They had a higher water concentration after desiccation treatment due to decreased rates of water loss but showed no change in body mass, glycogen concentration, hemolymph volume, or the water concentration at death from desiccation. Although the mutant allele is mapped to chromosome 2, specific gene(s) attributed to desiccation resistance have not been identified yet. Although we cannot deny certain functional similarities between the gene(s) mutated in this line and Desi, we recognize a clear difference between the two mutant lines. Although the desiccation resistance of this mutant line was demonstrated using adults, the repressed resistance of Desi transgenic lines to desiccation stresses was demonstrated only in its RNAi larvae. Because there was no significant difference in desiccation resistance between the control and Desi RNAi adults, it is reasonable to expect that the prompt adaptation to desiccation stress in the integument via Desi is more important in larvae than adults.The proximal causes of mortality from desiccation stress are not well understood. It has been suggested that hemolymph volume and hemolymph solute concentrations are important for the enhancement of desiccation resistance in D. melanogaster (26Folk D.G. Han C. Bradley T.J. J. Exp. Biol. 2001; 204: 3323-3331Crossref PubMed Google Scholar). Furthermore, Marron et al. (27Marron M.T. Markow T.A. Kain K.J. Gibbs A.G. J. Insect Physiol. 2003; 49: 261-270Crossref PubMed Scopus (165) Google Scholar) reported that the rates of lipid and protein metabolism in Drosophila flies were similar during starvation and desiccation but that carbohydrate metabolism was several times higher during desiccation. This observation is consistent with the previous report that concluded that lower overall rates of water loss in the flies are achieved by reduction of respiratory losses (28Gibbs A.G. Fukuzato F. Matzkin L.M. J. Exp. Biol. 2003; 206: 1183-1192Crossref PubMed Scopus (202) Google Scholar). The present study indicated a remarkable decrease in internal water and carbohydrates in the Desi RNAi larvae under a dry condition compared with control larvae. In contrast, ATP concentrations in both the RNAi and control larvae were increased under a dry condition, although no significant differences in ATP concentrations were determined between RNAi and control larvae either before or after desiccation. It is possible to interpret these results to mean that the Desi RNAi larvae lost excess amounts of the internal water needed to enhance the metabolic rate by producing ATP molecules that are used by these animals to maintain homeostasis. To evaluate the desiccation resistance of Drosophila, the importance of recovery processes from desiccation stress was also pointed out. Folk and Bradley (29Folk D.G. Bradley T.J. J. Exp. Biol. 2004; 207: 2671-2678Crossref PubMed Scopus (18) Google Scholar) showed that the greatest desiccation resistance in a Drosophila population with enhanced desiccation resistance is associated with the restoration of all tested somatic components, whole-body water, dry mass, and sodium concentrations, suggesting the importance of nutrition during rehydration in determining recovery time. Sinclair et al. (6Sinclair B.J. Gibbs A.G. Roberts S.P. Insect Mol. Biol. 2007; 16: 435-443Crossref PubMed Scopus (162) Google Scholar) indicated that up-regulated expression of Frost, a Drosophila stress-responsive gene, was more obvious during recovery from desiccation than during desiccation. These results indicate that temporal factors are involved in desiccation resistance. Although the functional role of Frost was characterized during the adult stage, Desi contributed to enhancing desiccation resistance during the larval stage. To clarify the mechanism underlying the desiccation resistance of insects, it is important to consider the different life stages.Analyses aimed at revealing the molecular function of Desi will lead to an enhanced understanding of desiccation resistance in adults as well as larvae of Drosophila. The outcome of this type of study can develop a better knowledge of the effects of desiccation stress not only on insects but also on vertebrates. IntroductionA wide variety of stressful stimuli change patterns of gene expression, which enables animals to adapt to stress, and such changes in gene expression are believed to allow them to survive drastic environmental changes. Activation of heat shock protein genes (hsp) is a typical example; all organisms express a particular set of hsp genes in response to stressors such as temperature extremes, aversive chemical application, anoxia, and many other environmental injuries (1Feder M.E. Hofmann G.E. Annu. Rev. Physiol. 1999; 61: 243-282Crossref PubMed Scopus (3129) Google Scholar, 2Sun Y. MacRae T.H. FEBS J. 2005; 272: 2613-2627Crossref PubMed Scopus (285) Google Scholar). Hsp proteins are generally divided into three families: the 90-kDa, 70-kDa, and small heat shock proteins (3Parsell D.A. Lindquist S. Annu. Rev. Genet. 1993; 27: 437-496Crossref PubMed Scopus (1855) Google Scholar). It has been reported that a nonlethal desiccation at 0% relative humidity (RH) 2The abbreviation used is: RHrelative humidity. enhanced transcriptional levels of the two hsp genes, hsp70 and hsp23, in pupae of the flesh fly Sarcophaga crassipalpis (4Tammariello S.P. Rinehart J.P. Denlinger D.L. J. Insect Physiol. 1999; 45: 933-938Crossref PubMed Scopus (72) Google Scholar). Although the two hsp transcripts were up-regulated in response to desiccation, the up-regulation was less dramatic than that elicited by heat shock, and desiccation failed to generate tolerance to high or low temperatures. Recently, it has been also reported that dehydration elicited expression of hsp70 in three mosquito species, Aedes aegypti, Anopheles gambiae, and Culex pipiens, but hsp90 expression levels remained fairly constant. Furthermore, injection of dsRNA to knock down expression of hsp70 and hsp90 significantly decreased survival rates of A. aegypti under dehydration (5Benoit J.B. Lopez-Martinez G. Phillips Z.P. Patrick K.R. Denlinger D.L. J. Insect Physiol. 2010; 56: 151-156Crossref PubMed Scopus (86) Google Scholar). Exposure of adult male Drosophila melanogaster to desiccation enhanced transcriptional levels of Frost and senescence marker protein-30 (smp-30) but did not change those of hsp70Aa and hsp23 (6Sinclair B.J. Gibbs A.G. Roberts S.P. Insect Mol. Biol. 2007; 16: 435-443Crossref PubMed Scopus (162) Google Scholar). Although these previous studies demonstrated desiccation-induced gene expression, we do not know whether the up-regulation of the gene expression levels confers desiccation resistance on animals.Drosophila melanogaster, like all holometabolous insects, undergoes complete metamorphosis to reach adulthood. Each phase of the life cycle is characterized by a coordinated program of developmental events and behavioral transitions that have evolved to promote fitness and survival (7Riddiford L.M. Bate M. Martinez-Arias A. The Development of Drosophila melanogaster. Cold Spring Harbor Laboratory Press, Plainview, NY1993: 899-939Google Scholar, 8Thummel C.S. Dev. Cell. 2001; 1: 453-465Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar). In Drosophila larvae, the essential midthird instar transition from foraging (feeding) to wandering (nonfeeding) behavior occurs prior to pupariation and metamorphosis. Although this behavioral transition imposes desiccation stress on the larvae, it is unknown not only whether there is a specific mechanism responsible for providing the larvae with desiccation tolerance but also how the tolerance of the larvae is enhanced during the wandering stage. In this study, we sought a gene whose expression was elevated by desiccation stress and identified Desiccate (Desi). Desi expression in the larvae is dependent on relative humidity; desiccation enhanced its expression, and conversely, humidification repressed its expression. Desi encoded a single-pass transmembrane protein and expressed its transcripts actively in epidermal cells of the integument. Furthermore, Desi expression specifically increased during the wandering stage and the survival rates of Desi RNAi larvae significantly declined under the dry condition.