383 384
RNAi-mediated depletion of Blage-HTHR mRNA was found to decrease 385
hemolymph trehalose levels in the adult of both sexes (Figure 3C and 3D), although the 386
female displays fluctuations of trehalose related to the reproductive cycle. Our present 387
results of Blage-HTHR RNAi (Figure 3C) supports our previous hypothesis postulating 388
that HTH induces hemolymph trehalose mobilization during the reproductive cycle in 389
virgin females (Huang and Lee, 2011). The results suggest that HTH plays a critical role 390
in carbohydrate homeostasis, as occurs in AKH-cell-deficient D. melanogaster that has 391
relatively low hemolymph trehalose levels (Lee and Park, 2004).
392 393
The involvement of AKH peptides in anti-oxidant protection in insects was inferred 394
from the increase in AKH titer after exposure to oxidative stress (Kodrik et al., 2007, 395
Vecera et al., 2007). The same authors showed that exogenous AKH application enhanced 396
the efflux of reduced glutathione to the hemolymph and maintained the normal levels of 397
protein carbonyl, a marker of oxidative stress. In the present study, we have fully 398
demonstrated the protective role of HTH upon oxidative stress under different 399
experimental approaches, first, through experiments of co-injection of PQ and HTH on 400
control specimens (Figure 4A), and secondly, in specimens with the expression levels of 401
Blage-HTH and Blage-HTHR depleted by RNAi (Figure 5B). These results provide a new
402and relevant information supporting that Blage-HTH potentiates a stress hormone on 403
anti-oxidative stress response in B. germanica. The promoter of oxidative stress used in 404
our experiments has been PQ, which acts on the redox-cycling reactions, thus eliciting 405
free radicals as well as causes oxidative modification of macromolecules, such as lipid 406
peroxidation (Hassan, 1984). Accordingly, we examined lipid peroxidation in the 407
hemolymph 4 h after PQ treatment (Figure 5C). Levels of lipid peroxidation in the 408
hemolymph increased significantly after PQ injection in dsEGFP and dsHTH/R groups.
409
However, the co-injection of HTH maintained the lipid peroxidation at a normal level in 410
controls but not in the Blage-HTH and Blage-HTHR knockdowns, which suggests that 411
HTH lowers the oxidative damage in B. germanica. A likely mechanism might be that 412
HTH induces the efflux of glutathione into the hemolymph, as demonstrated in other 413
insect AKHs (Kodrik et al., 2007, Vecera et al., 2007). However, the significant reduction 414
of Blage-HTHR expression after PQ treatment (Figure 4B), suggests that the oxidative 415
stress directly impairs its expression. The less severe reduction of Blage-HTHR 416
expression observed after co-injecting PQ and HTH (Figure 4B) might be the outcome of 417
downstream response induced by HTH to relieve the oxidative damage. Moreover, 418
genome-wide analysis in D. melanogaster after PQ treatment have shown that 419
cytochrome P450 genes become up- and down-regulated (Girardot et al., 2004), and it has 420
been additionally reported that HTH stimulates cytochrome P450 expression in the 421
cockroach Blaberus discoidalis (Lu et al., 1995). Therefore, we further speculate that 422
HTH might induce the expression of P450 genes, thus contributing to decrease the 423
oxidative stress in B. germanica.
424 425
Acknowledgements
426We are grateful to Chin-Cheng Yang for his help with the phylogenetic analyses. Support 427
for this research was provided by National Science Council of Taiwan (NSC 428
97-2313-B-002-031 and NSC 100-2923-B-002-002 to H.-J.L; NSC 98-2917-I-002-147 429
to J.-H.H. for working one year in Barcelona), and the Spanish MICINN (grant 430
CGL2008-03517/BOS to X.B.) and by the CSIC (grant 2010TW0019, from the Formosa 431
Belles, X. (2010). Beyond Drosophila: RNAi in vivo and functional genomics in insects.
435
Annu. Rev. Entomol. 55, 111-128.
436
Candy, D. J. (2002). Adipokinetic hormones concentrations in the haemolymph of 437
Schistocerca gregaria, measured by radioimmunoassay. Insect Biochem. Mol.
438
Drosophila parkinsonism model. J. Neurosci. 27, 2457-2467.
442
Gäde, G. (2009). Peptides of the adipokinetic hormone/red pigment-concentrating 443
hormone family: a new take on biodiversity. Ann. NY Acad. Sci. 1163, 125-136.
444
Gäde, G., and Auerswald, L. (2003). Mode of action of neuropeptides from the 445
adipokinetic hormone family. Gen. Comp. Endocrinol. 132, 10-20.
446
Girardot, F., Monnier, V., and Tricoire, H. (2004). Genome wide analysis of common and 447
specific stress responses in adult Drosophila melanogaster. BMC Genomics 5, 74.
448
Granett, J., and Leeling, N. C. (1972). A hyperglycemic agent in the serum of 449
DDT-prostrate American cockroaches, Periplaneta americana. Ann. Entomol.
450
Soc. Am. 65, 299-302.
451
Halliwell, B., and Gutteridge, J. M. C. (2007). Free Radicals in Biology and Medicine.
452
4th ed. Oxford: Clarendon Press.
453
Hansen, K. K., Hauser, F., Cazzamali, G., Williamson, M., and Grimmelikhuijzen, C. J. P.
454
(2006). Cloning and characterization of the adipokinetic hormone receptor from 455
the cockroach Periplaneta americana. Biochem. Bioph. Res. Co. 343, 638-643.
456
adipokinetic hormone receptor that are essential for receptor export, 461
phosphorylation and internalization. Cell Signal 23, 1455-1465.
462
Huang, J. -H., and Lee, H. -J. (2011). RNA interference unveils functions of the 463
hypertrehalosemic hormone on cyclic fluctuation of hemolymph trehalose and 464
flight performance by a hypertrehalosaemic hormone in the mosquito Anopheles 468
gambiae. J. Insect Physiol. 54, 367-377.
469
Kodrik, D. (2008). Adipokinetic hormone functions that are not associated with insect 470
flight. Physiol. Entomol. 33, 171-180.
471
Kodrik, D., Krishnan, N., and Habustova, O. (2007). Is the titer of adipokinetic peptides 472
in Leptinotarsa decemlineata fed on genetically modified potatoes increased by 473
oxidative stress? Peptides 28, 974-980.
474
Kodrik, D., and Socha, R. (2005). The effect of insecticide on adipokinetic hormone titre 475
in the insect body. Pest Manag. Sci. 61, 1077-1082.
476
Lee, H. J., and Wu, Y. L. (1994). Mating effects on the feeding and locomotion of the 477
German cockroach, Blattella germanica. Physiol. Entomol. 19, 39-45.
478
Lee, G., and Park, J. H. (2004). Hemolymph sugar homeostasis and starvation-induced 479
hyperactivity affected by genetic manipulations of the adipokinetic 480
hormone-encoding gene in Drosophila melanogaster. Genetics 167, 311-323.
481
Lu, K. -H., Bradfield, J. Y., and Keeley, L. L. (1995). Hypertrehalosemic 482
hormone-regulated gene expression for cytochrome P4504C1 in the fat body of 483
the cockroach Blaberus discoidalis. Arch. Insect Biochem.Physiol. 28, 79–90.
484
Nicholas, K. B., Nicholas, H. B., and Deerfield, D. W. (1997). GeneDoc: analysis and 485
visualization of genetic variation. EMBNEW NEWS 4, 14.
486
Pfaffl, M. W. (2001). A new mathematical model for relative quantification in real-time 487
RT-PCR. Nucleic Acids Res. 29, e45.
488
Samaranayaka, M. (1974). Insecticide-induced release of hyperglycaemic and 489
adipokinetic hormones of Schistocerca gregaria. Gen. Comp. Endocrinol. 24, 490
424-436.
491
Scarborough, R. M., Jamieson, G. C., Kalish, F., Kramer, S. J., McEnroe, G. A., Miller, C.
492
A., and Schooley, D. A. (1984). Isolation and primary structure of two peptides 493
with cardioacceleratory and hyperglycemic activity from the corpora cardiaca of 494
Periplaneta americana. Proc. Natl. Acad. Sci. USA 81, 5575-5579.
495
Schooley, D. A., Horodyski, F. M., and Coast, G. M. (2005). "Hormones controlling 496
homeostasis in insects," in Comprehensive Molecular Insect Science, ed. L. I.
497
Gilbert, K. Iatrou, S. S. Gill ( Elsevier Press), 493-550.
498
Staubli, F., Jorgensen, T. J. D., Cazzamali, G., Williamson, M., Lenz, C., Sondergaard, L., 499
Roepstorff, P., and Grimmelikhuijzen, C. J. P. (2002). Molecular identification of
Steele, J. E. (1961). Occurrence of a hyperglycaemic factor in the corpus cardiacum of an 503
insect. Nature 192, 680-681.
504
Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M., and Kumar, S. (2011).
505
MEGA5: Molecular Evolutionary Genetics Analysis using Maximum Likelihood, 506
Evolutionary Distance, and Maximum Parsimony Methods. Mol. Biol. Evol. 28, 507
2731-2739.
508
Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F., and Higgins, D. G. (1997).
509
The CLUSTAL_X windows interface: flexible strategies for multiple sequence 510
alignment aided by quality analysis tools. Nucleic Acids Res. 25, 4876-4882.
511
Van der Horst, D. J., Van Marrewijk, W. J. A., and Diederen, J. H. B. (2001).
512
"Adipokinetic hormones of insect: Release, signal transduction, and responses,"
513
in International Review of Cytology - a Survey of Cell Biology, ed. K.W. Jeon 514
(Elsevier), 179-240.
515
Vecera, J., Krishnan, N., Alquicer, G., Kodrik, D., and Socha, R. (2007). Adipokinetic 516
hormone-induced enhancement of antioxidant capacity of Pyrrhocoris apterus 517
hemolymph in response to oxidative stress. Comp. Biochem. Physiol. C Toxicol.
518
Pharmacol. 146, 336-342.
519
Velki, M., Kodrik, D., Vecera, J., Hackenberger, B. K., and Socha, R. (2011). Oxidative 520
stress elicited by insecticides: a role for the adipokinetic hormone. Gen. Comp.
521
Endocrinol. 172, 77-84.
522
Valko, M., Leibfritz, D., Moncol, J., Cronin. M. T, Mazur, M., and Telser, J. (2007). Free 523
radicals and antioxidants in normal physiological functions and human disease.
524
Int. J. Biochem. Cell Biol.39, 44-84.
525
Wicher, D., Agricola, H. J., Sohler, S., Gundel, M., Heinemann, S. H., Wollweber, L., 526
Stengl, M., and Derst, C. (2006). Differential receptor activation by cockroach 527
adipokinetic hormones produces differential effects on ion currents, neuronal 528
activity, and locomotion. J. Neurophysiol. 95, 2314-2325.
529
534
Figure legends
535536
Figure 1. (A) Alignment of the deduced amino acid sequences of the Blattella germanica 537
HTH receptor (Blage-HTHR, GenBank GU591493) with the Periplaneta americana 538
AKH receptor (Peram-AKHR, GenBank ABB20590). Identical amino acid residues are 539
highlighted in grey. The seven-transmembrane α-helices are indicated by TM1-7. The 540
dotted-line rectangle indicates an insertion present in both cockroach species. (B) 541
Neighbor joining tree of AKH receptor insect sequences. The tree was generated using 542
MEGA 5 with 1000 bootstrap replicates. The evolutionary distance is given in units of the 543
number of amino acid substitutions per site. The abbreviated names of insect AKHR are:
544
Acypi-AKHR (Acyrthosiphon pisum), Aedae-AKHR-1 and Aedae-AKHR2 (Aedes 545