德國蜚蠊昇海藻糖激素於生殖與抗氧化逆境反應之角色

國立臺灣大學生物資源暨農學院昆蟲學系 博士論文

Department of Entomology

College of Bioresources and Agriculture National Taiwan University

Doctoral Dissertation

德國蜚蠊昇海藻糖激素於生殖與抗氧化逆境反應之角色 Functional Roles of Hypertrehalosemic Hormone Related to Reproduction and Antioxidative Stress Responses in the

German cockroach Blattella germanica (L.)

黃佳欣 Jia-Hsin Huang

指導教授：李後晶 博士 Advisor: How-Jing Lee, Ph.D.

中華民國一百年十二月

December 2011

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誌 謝

這本博士論文我想獻給我親愛的家人。從幼稚園一路念到博士班畢業是漫長 的時間，我由衷地感謝你們默默支持，不論是在經濟上或是精神上，都是我最大 的支柱。本論文能順利的完成，衷心感謝李後晶教授對我在研究路上的栽培，給 我很大的空間讓我能夠去做我想研究的主題，其中有很多都是我自己天馬行空的 想法，但是李老師仍然支持我去檢驗自己粗淺的假說，讓我能夠學習與成長。

在這段研究所的學習之旅，要感謝很多前輩的指導。感謝楊永裕博士在我念 研究所的時間，教導我許多研究工作的經驗與想法，對於一個正在研究路上起步 的我，受惠良多。感謝陳美娥博士啟蒙了我對研究工作的認識，將近一年的近身 學習，她不藏私的教我分子生物學與研究的「眉角」。同時感謝鐘芳珠學姐細心 的教導我做實驗的細節與步驟，讓我建立起好的基礎做往後的研究。感謝陳淇釧 博士鼓勵我直攻博士班發展我自己人生的興趣與志向，現在我已經爬上了最小的 一座山頂，往後還要繼續爬向更高的山，欣賞不同的美景！

研究所的期間兩次前往國外的短期進修，讓我獲得許多成長。感謝捷克國家 科學院的 Prof. Dalibor Kodrík，很榮幸有機會近身學習到昆蟲神經胜肽的知識。感 謝西班牙科學委員會演化生物系的 Dr. Xavier Bellés 及 Dr. Maria Dolors Piulachs 在 巴塞隆納一年期間的教導，開啟我對研究工作的熱誠與感動！

感謝論文口試委員楊恩誠博士、張俊哲博士、路光暉博士、陳美娥博士、楊 永裕博士、 Dr. Xavier Bellé 對論文的指正與建議，不僅讓論文的內容更臻完備，

也啟發我對於往後的研究之路有更多想法。

研究所的期間，要感謝很多的同好與朋友對我的關心與支持。謝謝我最好的 同學斯佳，讓我在苦悶的研究時間外跟我聊天南地北。還有昆蟲行為研究室的伙 伴們：杏惠學姊、書綺學姊、琦玫學姊、宛恬學姊、淑君學姊、兆晉學長、志仁 學姊、秀慧學姊、易呈學長、稑焜學長、鼎鉉學長、佳恩學姊、耘姐、玟欣、文 馨、維倫、慧妍、威翰、雁婷、Fabien、子鈞、育賢、Pierre、思淵、黃(素華) 姐的幫忙，你們都是讓我能夠完成論文的重要助力！另外，我還要感謝我的女朋 友，逸如，感謝你經常的陪伴及包容，這本論文是在你的支持下才能夠完成。

感謝所有幫助過我的人。最後，僅將此份榮耀與喜悅與你們分享！

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摘 要

昇海藻糖激素是一種胜肽荷爾蒙，屬於降脂激素家族，其主要功能是促使昆 蟲體內海藻糖的生成。本論文研究是在探討德國蜚蠊昇海藻糖激素於生殖與抗氧 化逆境反應的功能。該研究分別選殖出昇海藻糖激素與其受器基因，接著利用核 醣核酸干擾技術來靜默基因表現以研究其功能。本研究首先證實昇海藻糖激素調 控昆蟲體內海藻糖的恆定性，當昇海藻糖激素基因或其受器基因受到核糖核酸干 擾而抑制後，德國蜚蠊血淋巴中海藻糖濃度顯著地降低。此外，處女雌蟲血淋巴 海藻糖濃度在每個生殖週期有逐漸上升的趨勢，而且與卵巢發育具有正相關性；

經實驗驗證，昇海藻糖激素具有調節生殖週期內海藻糖濃度上升的腳色。昇海藻 糖激素也會影響雌蟲排出卵鞘的行為，因為處女雌蟲體內的昇海藻糖激素或其受 器基因受到靜默後，卵鞘排出的時間出現顯著性的延遲。

另一方面，該論文研究尚且探討德國蜚蠊昇海藻糖激素具有對抗氧化逆境的 功能。該研究使用殺草劑巴拉刈注射到 10 日齡的雄蟲體內，不僅會造成雄蟲體內 的氧化壓力上升並且後來會促使雄蟲死亡。當正常的雄蟲在接受固定濃度的巴拉 刈注射後，過半的致死蟲數發生於第 4 天；然而，額外添加不同濃度的昇海藻糖 激素 (10, 40, 80 pmole) 都能夠顯著地延後死亡率的發生，過半的致死蟲數則 達第 7 天以後。此外，巴拉刈的致死作用對於昇海藻糖激素及其受器基因受到靜 默而降低表現的雄蟲反而有更劇烈的影響，處理藥劑後的第二天就有半數以上的 雄蟲死亡。接下來我進行血淋巴中脂質代謝分析作為昆蟲體內氧化壓力的一項指 標。分析的研究結果顯示，將昇海藻糖激素與巴拉刈同時注射到雄蟲體內能夠維 持丙二醛 (malondialdehyde) 濃度在正常量的水準；但是對於昇海藻糖激素與其 受器基因受到靜默的雄蟲，外加注射的昇海藻糖激素並無法降低氧化逆境的傷害 產生，其丙二醛的總量則呈現顯著性地升高。最後，本研究還發現昇海藻糖激素 能夠在外加注射巴拉刈的狀態下提高粒線體細胞色素氧化酶第一次單元基因 (cytochrome c oxidase subunit I) 與細胞色素基因 (CYP4G19) 的表現量。上述的兩 個基因曾經被指出與昆蟲抗氧化能力反應相關，關於昇海藻糖激素抵抗氧化壓力 的機制亦在本論文研究中進一步討論。

關鍵詞：降脂激素，海藻糖，排卵，氧化壓力，昇海藻糖激素受器，核糖核酸干 擾，巴拉刈

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Abstract

A hypertrehalosemic hormone (HTH) peptide has been identified in the adipokinetic hormone (AKH) family to induce mobilization of carbohydrate in many insects. The aim of this study was to investigate the functions of hypertrehalosemic hormone in relation to reproduction and antioxidative stress response in the German cockroach Blattella germanica. I have isolated the Blage-HTH and its receptor (Blage-HTHR) genes, and thus studied their function through RNA interference (RNAi) to knockdown their expressions. The conventional role of HTH mediating trehalose homeostasis was observed in both sexes, in which the hemolymph trehalose was significantly reduced by RNAi-mediated knockdown of either Blage-HTH or

Blage-HTHR genes. Especially, Blage-HTH regulated the increase of hemolymph

trehalose in parallel with the ovarian development in the virgin females. Furthermore, Blage-HTH would assist the oviposition process because the ootheca production was delayed in the virgin females with either Blage-HTH or Blage-HTHR silenced.

In addition, I have unveiled another function of Blage-HTH against oxidative stress in B. germanica. Paraquat (PQ), a bipyridilium herbicide used in this study to elicit oxidative stress, causes a detrimental effect on the lifespan of 10-day-old adult males. Co-injection of synthetic HTH peptides at 10, 40, and 80 pmol concentrations significantly reduced the speed of mortality increase, with median survival time attained about 7 days in contrast to 4 days without HTH injection. However, PQ treatment caused a rapid mortality increase, with median survival time attained on 2 days, in the male adults whose Blage-HTH and Blage-HTHR were silenced simultaneously. I further examined the lipid peroxidation as an indicator of oxidative stress in the hemolymph after PQ treatment. Results showed that co-injeciton of HTH peptide maintained a normal level of lipid peroxidation, but it was not rescued by HTH in Blage-HTH and

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Blage-HTHR knockdown specimens. Finally, Blage-HTH also stimulated the

expression of COXI (cytochrome c oxidase subunit I) and CYP4G19 (cytochrome P4504G19) in the fat body under oxidative stress elicited by PQ. The mechanism how Blage-HTH potentiates antioxidant protection has been discussed in the present study.

Keywords: Adipokinetic hormone, Trehalose, Oviposition, Oxidative stress,

Hypertrehalosemic hormone receptor, RNA interference, Paraquat

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Table of contents

口試委員會審定書 --- 誌謝 --- 摘要 --- Abstract --- Table of contents --- Chapter I. General introduction --- References --- Chapter II. RNA interference unveils functions of the hypertrehalosemic hormone

on cyclic fluctuation of hemolymph trehalose and oviposition in the virgin female Blattella germanica --- Chapter III. Functional characterization of hypertrehalosemic hormone receptor in

relation to hemolymph trehalose and to oxidative stress in the cockroach Blattella germanica --- Chapter IV. General discussion and conclusion --- References --- Appendices ---

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Chapter I.

General introduction

One of the notable issues in insect endocrinology is on the neuropeptide research because neuropeptides mediate a variety of physiological and behavioral events in insects (Gade and Hoffman, 2005; Mercier et al., 2006; De Loof, 2008). Neuropeptides in the adipokinetic hormone (AKH) family, which are synthesized, stored and released by corpora cardiaca (CC) (Goldsworthy et al., 1972), have been intensively isolated nearly 50 different forms and characterized their function in relation to energy metabolism in insects for five decades (Gade, 2009). Common structural characteristics of AKH peptides are 8-10 amino acids long and start with a pyroglutamate residue blocking the N-terminus, an aromatic amino acid (phenylalanine or tyrosine) at position 4, a tryptophan at position 8, a glycine at position 9 (if present), and end by a amide group blocking the C-terminus. Peptides that can stimulate lipid mobilization are given the name of adipokinetic hormone, while peptides in AKH family which increase the level of hemolymph trehalose in the species in which they were isolated are referred to as hypertrehalosemic hormone (HTH). The principle action of AKH peptides on energy substrate released into the hemolymph, either lipids, trehalose, or a combination, depends on the insect species and the times during the metabolic needs (Schooley et al., 2005).

The action of AKH peptide is through a G protein-coupled membrane receptor with seven transmembrane segments. The identification of AKH receptors (AKHR) in insects has remarkably lagged behind. The insect AKHR was first indentified from the fruit fly Drosophila melanogaster and the silkworm Bombyx mori (Staubli et al., 2002).

The obese phenotype of Akhr mutant of D. melanogaster that shows the metabolic

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abnormality in accumulation of energy reserves in the fat body reveals the important function of AKH on regulation of energy homeostasis (Gronke et al., 2007; Bharucha et

al., 2008). Recently, the advance of whole genome sequencing provides more sequence

information of AKHR in insects. In Africa malaria mosquito

Anopheles gambiae, the supreme action of AKH is required a high expression level of its receptor because the mosquitoes whose AKHR expression is silenced fail to mobilize the glycogen reserves in the fat body after injection of Anoga-AKH-I (Kaufmann and Brown, 2008).

Furthermore, the expression of AKHR in the dorsal unpaired median neurons has been demonstrated to mediate the action of Peram-AKH-I on stimulating locomotion in Periplaneta americana (Wicher et al. 2006). On the cellular mechanisms of AKH signaling, the in vitro study reports that the Bombyx AKH brings about a dose- and time-dependent internalization of AKHR via the clathrin-coated pit pathway (Huang et al., 2011).

More recently, the insect AKH peptides have been proposed to share a common ancestral peptide with the gonadotropin-releasing hormone (GnRH) in other metazoans (Lindemans et al., 2009). The same authors demonstrated a delay in time of egg-lying process and a decrease in progeny in the nematodes Caenorhabditis elegans after RNAi-mediated knockdown of the Ce-AKH-GnRH and Ce-GnRHR transcripts. GnRH is a pivotal peptide hormone to govern the reproductive process such as release of gonadotropins and behavior via the control of both endocrine and neural pathways in vertebrates (Okubo and Nagahama, 2008). The finding of Bombyx AKH activating one of the GnRH receptor of the amphioxus, a most basal chordate, also supports a common ancestor between GnRH and AKH signaling systems (Tello and Sherwood, 2009). In addition, the two AKH receptors of D. melanogaster and B. mori are regarded to be structurally and evolutionarily related to vertebrate GnRH receptors (Staubli et al.,

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2002). However, the canonical role of insect AKHs is the energy mobilization but only few studies suggest the involvement of AKHs on reproduction by finding the expression of AKH mRNA (Kaufmann and Brown, 2006; Abdel-Latief and Hoffmann, 2007) and

Aedae-AKHR mRNA (Kaufmann et al., 2009) in the insect ovary. So far, the functional

examination of AKH peptides in reproductive processes remains an uncertain conclusion in insects.

The female German cockroach Blattella germanica has a cyclic reproduction to produce an ootheca in every cycle regardless a successful mating or not (Roth and Stay, 1962). During each reproductive cycle, the terminal oocytes become mature by uptake of vitellogenin which is an energy consuming process since less growing of oocytes after injection of trehalase inhibitor in the cockroaches (Kono et al., 2001). In addition, the virgin female B. germanica displays a mate-finding behavior to increase locomotor activity which masks the locomotor circadian rhythm in a few days before oviposition (Lee and Wu, 1994; Lin and Lee, 1996). Therefore, I presume that the virgin female B.

germanica would require a higher energy demand during a reproductive cycle for

physiological and behavioral needs. Trehalose is the major sugar in the hemolymph of most insects that is used as energy source by tissues like muscles, ovaries, etc.

(Thompson, 2004). A question that arises is whether the level of hemolymph trehalose is increased for energy needs during reproductive cycle in the virgin female B.

germanica. And next question is whether HTH peptide regulates the changes of

hemolymph trehalose in related to those reproductive processes.

RNA interference (RNAi), the biological mechanism by which a introduced double-stranded RNA (dsRNA) is diced into a pool of 21-nucleotide small interfering RNA (siRNA) duplexes, induces gene silencing by targeting complementary mRNA for degradation, is one of the milestone discoveries in biological sciences for the past 15

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years (Ketting, 2011; Meister and Tuschl, 2004). Systemic RNAi has become the most potent tool to unveil the gene functions in non-model insects because of its advantages including fast and maneuverable approach, higher versatility of silencing, stage-specific survey and so on (Belles, 2010). Among the non-model insects studied, the German cockroach B. germanica is particularly sensitive toward RNAi and has been studied many gene functions in diverse biological levels in vivo using this tool successfully (Belles, 2010). Consequently, RNAi provides a great opportunity to examine the roles of HTH in B. germanica through a reverse functional approach to silence its expression.

In order to examine the question if HTH mediates level of hemolymph trehalose during reproductive cycle in female B. germanica or not, I approached it using the molecular method by cloning of Blage-HTH and its receptor (Blage-HTHR) genes, thereby through RNAi to study their function. In chapter II, a sexual dimorphism in the changes of hemolymph trehalose were examined in B. germanica and the fluctuation of hemolymph trehalose showed a positive correlation with the terminal oocyte length in the virgin females. The further RNAi experiments of Blage-HTH and Blage-HTHR have demonstrated the critical role of Blage-HTH on the regulation of carbohydrate homeostasis. Additionally, silencing either Blage-HTH or Blage-HTHR resulted in a delay of ootheca production in the virgin females. These results revealed the roles of Blage-HTH involving in the reproductive processes in the female B. germanica.

AKH peptides exert numerous biological activities in addition to their well-known metabolic role (Kodrik, 2008). Generally, AKHs stimulate most catabolic reactions such as mobilization of energy reserves in the fat body while inhibit the anabolic reactions including RNA and protein synthesis at the same time. Furthermore, AKHs are considered as stress response hormones because some of their functions are associated to the stress conditions such as starvation (Lee and Park, 2004), pathogen (Goldsworthy

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et al., 2002, 2003), oxidative stress (Kodrik et al., 2007, Vecera et al., 2007), insecticide treatment (Candy, 2002; Kodrik and Socha, 2005) and photophase interruption (Kodrik et al., 2005). However, the mechanism how AKH peptides exert their action in those stress conditions remains an open question even though some information about the signaling cascade of AKHs inducing energy mobilization in the fat body has been reviewed (Van der Horst et al., 2001; Gade and Auerswald, 2003).

Although a wealth of information has been available about the action of AKHs in relation to stress response at biochemical levels, information about molecular support has not been approached in this field yet. In Chapter III, I focused on the detailed examination of Blage-HTH against oxidative stress using B. germanica as a model through a forward method by injection of HTH peptide as well as a reversed method through interrupting Blage-HTH signaling pathway using RNAi under oxidative stress.

These results unveil the role of Blage-HTH against oxidative stress at both biochemical and molecular levels. Moreover, I examined the other gene expressions related to oxidative stress response with the combination of paraquat, HTH, and RNAi treatments.

Finally, a further discussion underlying the molecular mechanism how Blage-HTH potentiates an antioxidant protection in the cockroach B. germanica has been approached in this study. The goals of this study were to provide an experimental paradigm in molecular approach for the functional study of peptide hormones and provide the fundamental research for comprehensively understanding the pleiotropic roles of HTH peptides in this cockroach. The findings from these studies uncover the Blage-HTH functions in relation to reproduction and oxidative stress response and provide a possibility of application for pest control in the future.

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Tello, J. A., and Sherwood, N. M., 2009. Amphioxus: Beginning of Vertebrate and end of invertebrate type GnRH Receptor lineage. Endocrinol. 150, 2847-2856.

Thompson, S. N., 2004. Trehalose: the insect „blood‟ sugar. Adv. Insect Physiol. 31, 205-285.

Vecera, J., Krishnan, N., Alquicer, G., Kodrik, D., and Socha, R., 2007. Adipokinetic hormone-induced enhancement of antioxidant capacity of Pyrrhocoris apterus hemolymph in response to oxidative stress. Comp. Biochem. Physiol. C Toxicol.

Pharmacol. 146, 336-342.

Van der Horst, D. J., Van Marrewijk, W. J. A., and Diederen, J. H. B., 2001.

"Adipokinetic hormones of insect: Release, signal transduction, and responses," in

International Review of Cytology - a Survey of Cell Biology, ed. K.W. Jeon

10

(Elsevier), 179-240.

Wicher, D., Agricola, H. J., Sohler, S., Gundel, M., Heinemann, S. H., Wollweber, L., Stengl, M., and Derst, C., 2006. Differential receptor activation by cockroach adipokinetic hormones produces differential effects on ion currents, neuronal activity, and locomotion. J. Neurophysiol. 95, 2314-2325.

11

Chapter II.

RNA interference unveils functions of the hypertrehalosemic hormone on cyclic fluctuation of hemolymph trehalose and oviposition in the virgin female Blattella germanica

The main focus of this chapter is to investigate the functions of hypertrehalosemic hormone (Blage-HTH) in relation to reproductive processes in the virgin female

Blattella germanica. Blage-HTH mediates increase of hemolymph trehalose in parallel

to ovarian development as well as ootheca extruded at the time of oviposition during a reproductive cycle in the virgin females. These results provide a first functional examination of the insect AKHs involving in the reproductive processes to support a recent hypothesis about the evolution of AKHs from a superfamily of GnRH neuropeptides.

The question that arose at the beginning of project was whether the energy demand increases in a reproductive cycle of the virgin female B. germanica because the virgin females display a robust ovarian development and locomotion. The preliminary results showed a positive correlation between the fluctuations of hemolymph trehalose and the terminal oocytes length in the virgin females. Next, I have isolated Blage-HTH gene and performed RNAi to study its function. The virgin females whose Blage-HTH were silenced showed a delayed increase of hemolymph trehalose in contrast to dsEGFP-treated control. The potential role of hemolymph trehalose increase in assistance to vitellogenin uptake into growing oocytes during a reproductive cycle was discussed (Appendix 1). Of note, those dsHTH-treated females also delayed the time of ootheca production, and a few (10%) specimens produced a malformed ootheca. The unexpected finding of Blage-HTH RNAi in delay of ootheca production is

12

corresponding to another study of Ce-AKH-GnRH-like peptide mediating egg-laying behavior in C. elegans in 2009. Accordingly, the present study unveils a novel function of Blage-HTH in the reproductive processes of the female German cockroach.

This work has been published in the Journal of Insect Physiology (see Appendix 1 for more details) and could be accessed via the following citation:

Huang, J. H., and Lee, H. J., 2011. RNA interference unveils functions of the

hypertrehalosemic hormone on cyclic fluctuation of hemolymph trehalose and oviposition in the virgin female Blattella germanica. J. Insect Physiol. 57, 858-864.

13

Chapter III.

Functional characterization of hypertrehalosemic hormone receptor in relation to hemolymph trehalose and to oxidative stress in the cockroach Blattella germanica

Nowadays, the advent of genomic era, many tools of molecular biology provide much assistance to understand the molecular mechanisms of biological events. Insect AKHs have been proposed as a stress hormone through the biochemical methodology previously. Here, I have conducted one of the most breakthrough tool, RNAi, to study the function of Blage-HTH against oxidative stress.

In the present study, I first isolated a hypertrehalosemic hormone receptor (Blage-HTHR) cDNA in B. germanica. Comparing the protein sequences of known AKHRs in insects, those receptors are likely to evolve fast within each insect order because the intracellular C-terminal region shows a distinct variety of sequences shared in the same order (Appedix 2, supplementary Figure S2). For verifying this novel sequence as the receptor of Blage-HTH functionally, I conducted RNAi to silence its expression and thereby examine the hypertrehalosemia of exogenous Blage-HTH in the specimens. Injection of 10 pmol Blage-HTH could induce a 113% increase of hemolymph trehalose 2 hr after treatment in dsEGFP controls but a significantly less level (49%) in dsHTHR-treated cockroaches. In addition, the levels of the hemolymph trehalose were significantly lower in the both sexes of B. germanica whose HTHR was silenced. These results confirm that Blage-HTH mediates the carbohydrate homeostatsis in B. germanica through its receptor obtained in the present study.

Next, I investigated the question whether Blage-HTH potentiates an antioxidant protection in B. germanica through forward method by injection of Blage-HTH and

14

reversed method using RNAi to silence Blage-HTH and Blage-HTHR simultaneously.

Paraquat (PQ), a bipyridium herbicide, was injected into male B. germanica to elicit the lethal oxidative stress in the present study. Co-injection of varied concentration of Blage-HTH with PQ resulted in a delay of mortality increase for 3 days at least.

However, the males with a disrupting Blage-HTH singling by RNAi-silencing both Blage-HTH and Blage-HTHR showed a short median survival time (MST) attained on day 2 after PQ treatment in contrast to the control‟s MST attained on day 5.

Furthermore, the lipid peroxidation, an indicator of oxidative stress, was increased significantly 4 hr after PQ treatment. However, lipid peroxidation was maintained in a normal level in the dsEGFP-treated males but increased in the dsHTH and dsHTHR-treated males after co-injection of Blage-HTH and PQ. These results proves the role of Blage-HTH against oxidative stress through reducing the oxidative damages such as lipid peroxidation.

This work has been accepted in Frontiers in Experimental Endocrinology, a specialty of Frontiers in Endocrinology, in participation of the research topic

“Molecular mechanisms underlying insect behaviors: receptors, peptides, &

biosynthetic pathways” and be accessed via the following citation:

Huang, J. -H., Belles, X., and Lee, H. -J., 2011. Functional characterization of

hypertrehalosemic hormone receptor in relation to hemolymph trehalose and to oxidative stress in the cockroach Blattella germanica. Front. Endocrin. 2, 114. doi:

10.3389/fendo.2011.00114.

15

Chapter IV.

General discussion and conclusion

This study unveils that Blage-HTH plays two roles on reproduction in female B.

germanica. One is to regulate the elevation of hemolymph trehalose, which is used for

ovarian development and locomotion. Another is to promote oviposition process. In addition, Blage-HTH behaves as a stress hormone, which is released at the period of high stress, to relieve oxidative stress in particular.

AKH peptides are considered as the general endocrine regulator of energy homeostasis in insects (Lorenz and Gade, 2009). In chapter II, we have discussed the possible role of Blage-HTH on daily orchestrated regulation of anabolic and catabolic reactions during reproductive cycle in the female B. germanica because Blage-HTH displays a contradictory effect on the syntheses of trehalose and vitellogenin.

Additionally, the hemolymph trehalose fluctuated in the 6-day-old females treated with dsEGFP, but the level of hemolymph was not fluctuated and significant lower in the females treated with dsHTHR (Appendix 3). These results of Blage-HTHR RNAi support our previous hypothesis postulating that Blage-HTH mediates daily fluctuation of hemolymph trehalose. The endogenous circadian clock mediating rhythmic AKH release in the hemolymph is also inferred from the daily changes of AKH titer in fire bug Pyrrhocoris apterus under both light-dark photoperiod and constant darkness (Kodrik et al., 2005) as well as higher AKH titer in cricket Gryllus bimaculatus, during early scotophase than during early photophase (Lorenz and Gade 2009). Therefore, Blage-HTH is most likely to release at different time within a day to regulate the allocation of energy metabolism during reproduction in female B. germanica.

16

More recently, insect AKH peptides are proposed in the superfamily of gonadotropin releasing hormone (GnRH) which is a critical regulator of reproduction in vertebrates and their receptors could share a common ancestral origin in the deep of Bilaterian animals (Roch et al., 2011). Equivalently, Blage-HTH regulating reproductive events was found in parallel since Blage-HTH RNAi resulted in a delay of oviposition in virgin female B. germanica in chapter II. Furthermore, similar effect on delaying oviposition was observed in Blage-HTHR RNAi (Appendix 4) and possible action of Blage-HTH was proposed to stimulate the contraction of oviduct muscles with

Blage-HTHR mRNA expression (in chapter III). In addition, the involvement of AKH

peptides on reproductive processes was presumed according to the mRNA of AKHR gene in the ovary of mosquito Anopheles gambiae (Kaufmann and Brown, 2006) and

Aedes agypti (Kaufmann et al., 2009). In summary, those results suggest that insect

AKHs mediates the reproduction and are functionally related to the GnRH in other metazoa.

In chapter III, the involvement of Blage-HTH in antioxidant capability was demonstrated through injection of HTH peptides and interruption of Blage-HTH signaling by RNAi in male B. germanica under oxidative stress (injection of paraquat, PQ). The antioxidant capability of Blage-HTH is likely a quick response because the response of signaling cascades to this peptide by cockroach trophocytes is demonstrated within few minutes (Steele et al., 2001) and the induction of locomotion is about 1 hr after AKH I injection in P. americana (Wicher et al., 2006). Accordingly, the levels of lipid peroxidation in the hemolymph maintained 4 h post co-injection PQ and HTH in dsEGFP controls revealed the quick response of HTH against oxidative stress. Similar effect of AKH on glutathione efflux is reported 4 hr post oxidative stress treatment in P.

apterus (Vecera et al., 2007). However, PQ could exert detrimental effect on survival in

17

both normal and RNAi-treated cockroaches. Oxidative stress might cause some degrees of non-reversible damage to cockroaches.

We further to examine the possible mechanism of Blage-HTH against oxidative stress by verifying gene expression in the fat body of B. germanica. Results showed that co-injection of HTH peptide resulted in a up-regulation of cytochrome c oxidase subunit I (CoxI) and a cytocrhome P450 (CYP4G19) genes 4 hr after PQ treatment (Appendix 5). RNAi mutant of D. melanogaster with inactivated mitochondrial respiratory comlexe IV including CoxI shows a decrease in PQ resistance (Copeland, et al., 2009).

Moreover, the cytochrome P450 genes become up- and down-regulated in response to PQ treatment in D. melanogaster (Girardot et al., 2004). Subsequently, these results suggest that the molecular mechanism of Blage-HTH against oxidative stress could induce or maintain the expression of CoxI and CYP4G19 genes in the fat body of B.

germanica.

Why does Blage-HTH have an antioxidant capability? The intrinsic effect of Blage-HTH elevates the synthesis of trehalose in the fat body. The synthesis of trehalose is an energy consuming process (Becker et al., 1996). The reactive oxygen species (ROS) generated in the mitochondrial electron chain during aerobic respiration is well known in mammals as well as discovered in D. melanogaster recently (Sanz et al., 2010). It appears that higher mitochondrial respiration would produce more endogenous free radicals to cause oxidative stress. In addition, AKH peptides also stimulate the locomotor activity in many insects (Kodrik et al. , 2000; Lorenz et al., 2004; Wicher et al., 2006). Higher locomotor activity requires more energy expenditure in the muscles, which could counter more endogenous free radical produced by mitochondria at the mean time. In order to relieve the damage of endogenous ROS, the signaling cascades of HTH peptide might evolve to potentiate antioxidant response in

18

insects.

In summary, this study has revealed the pleiotropic function of Blage-HTH with respect to reproduction and oxidative stress responses in the German cockroach B.

germanica through experimental demonstration at molecular level. Meanwhile, this

study provides an open window to study the molecular mechanism how AKH peptides exert a pleiotropic function in insects as well as the basic information for potential applications in insect pharmacology such as development of peptide mimics as insecticides.

19

References

Becker, A., Schloder, P., Steele, J. E., and Wegener, G., 1996. The regulation of trehalose metabolism in insects. Experientia 52, 433-439.

Copeland, J. M., Cho, J., Lo, T. Jr., Hur, J. H., Bahadorani, S., Arabyan, T., Rabie, J., Soh, J., and Walker, D. W., 2009. Extension of Drosophila life span by RNAi of the mitochondrial respiratory chain. Curr. Biol. 19, 1591-1598.

Girardot, F., Monnier, V., and Tricoire, H., 2004. Genome wide analysis of common and specific stress responses in adult Drosophila melanogaster. BMC Genomics 5, 74.

Kodrik, D., Socha, R., Simek, P., Zemek, R., and Goldsworthy, G. J., 2000. A new member of the AKH/RPCH family that stimulates locomotory activity in the firebug, Pyrrhocoris apterus (Heteroptera). Insect Biochem. Mol. Biol. 30, 489-98.

Kodrik, D., Socha, R., Syrová, Z. and Zemek, R., 2005. The effect of constant darkness on the content of adipokinetic hormone, adipokinetic response and walking activity in macropterous females of Pyrrhocoris apterus (L.). Physiol. Entomol. 30, 248- 255

Kaufmann, C., and Brown, M. R., 2006. Adipokinetic hormones in the African malaria mosquito, Anopheles gambiae: Identification and expression of genes of two peptides and a putative receptor. Insect Biochem. Mol. Biol. 36, 466-481.

Kaufmann, C., Merzendorfer, H., and Gade, G., 2009. The adipokinetic hormone system in Culicinae (Diptera: Culicidae): molecular identification and characterization of two adipokinetic hormone (AKH) precursors from Aedes

aegypti and Culex pipiens and two putative AKH receptor variants from Ae.

aegypti. Insect Biochem. Mol. Biol. 39, 770-781.

Lorenz, M. W., and Gade, G., 2009. Hormonal regulation of energy metabolism in

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insects as a driving force for performance. Integr. Comp. Biol. 49, 380-392.

Lorenz, M. W., Zemek, R., Kodrik, D., and Socha, R., 2004. Lipid mobilization and locomotor stimulation in Gryllus bimaculatus by topically applied adipokinetic hormone. Physiol. Entomol. 29, 146-151.

Roch, G. J., Busby, E. R., and Sherwood, N. M., 2011. Evolution of GnRH: diving deeper. Gen. Comp. Endocrinol. 171, 1-16.

Sanz, A., Stefanatos, R., and McIlroy G., 2010. Production of reactive oxygen species by the mitochondrial electron transport chain in Drosophila melanogaster. J.

Bioenerg. Biomembr. 42, 135-142.

Steele, J. E., Garcha, K., and Sun, D., 2001. Inositol trisphosphate mediates the action of hypertrehalosemic hormone on fat body of the American cockroach, Periplaneta

americana. Comp. Biochem. Physiol. B 130, 537-545.

Vecera, J., Krishnan, N., Alquicer, G., Kodrik, D., and Socha, R. 2007. Adipokinetic hormone-induced enhancement of antioxidant capacity of Pyrrhocoris apterus hemolymph in response to oxidative stress. Comp. Biochem. Physiol. C Toxicol.

Pharmacol. 146, 336-342.

Wicher, D., Agricola, H. J., Sohler, S., Gundel, M., Heinemann, S. H., Wollweber, L., Stengl, M., and Derst, C., 2006. Differential receptor activation by cockroach adipokinetic hormones produces differential effects on ion currents, neuronal activity, and locomotion. J. Neurophysiol. 95, 2314-2325.

21

Appendices

The appendix section includes one published paper (Appendix 1), one accepted manuscript (Appendix 2), and three unpublished data (Appendices 3 - 5).

Author's personal copy

RNA interference unveils functions of the hypertrehalosemic hormone on cyclic fluctuation of hemolymph trehalose and oviposition in the virgin female Blattella germanica

Jia-Hsin Huang, How-Jing Lee *

DepartmentofEntomology,NationalTaiwanUniversity,Taipei106,Taiwan

1. Introduction

Hypertrehalosemic hormone (HTH) is a neuropeptide that belongstotheadipokinetichormone/redpigment-concentrating hormone(AKH/RPCH)family,whichregulatesenergymetabolism bystimulatingthemobilizationofenergysubstrateinthefatbody (Ga¨deetal.,1997).Theinitialdiscoveryofthesehormonesinthe corporacardiaca(CC)showedthattheyservedashyperglycemic factors,byinducingtrehaloseincreaseinthehemolymphofthe Americancockroach,Periplanetaamericana(Steele,1961).Peptides oftheAKH/RPCHfamilyareusuallysynthesizedandstoredinthe CC(VanderHorstetal.,2001), andnearly50 differentpeptide variantshavebeenidentifiedindiversenumbersofinsectspecies (Ga¨de, 2009). AKHpeptidesare 8–10-amino acids long,with a pyroglutamateblockedN-terminusandanamidatedC-terminus, aromaticresiduesatpositions4and8,and aglycineresidueat position9,whenpresent(Ga¨de,2009).

InsectAKHshavepleiotropicfunctionsinvariousinsectspecies (Schooleyetal.,2005;Kodrik,2008).Besidestheprimaryroleof inducing mobilization of energetic compounds, insect AKHs possessmyotropiceffects,thustheyaccelerateheartbeatrhythms incockroaches(Scarboroughetal.,1984;Keeleyetal.,1991)and

thecontractionrateoflegmusclesinlocusts(O’Sheaetal.,1984)as wellasgenerallocomotioninseveralspecies(Kodriketal.,2000;

Lorenzetal.,2004;Wicheretal.,2006).Experimentsofgenetic manipulationofAKHgeneexpressioninDrosophilamelanogaster pointtorolesrelatedtolocomotionandhemolymphtrehalosetiter inresponsetostarvationstress(LeeandPark,2004;Isabeletal., 2005).

LorenzandGa¨de(2009)regardtheAKHsasgeneralregulators of energy homeostasis, which not only control the energy metabolismunderlyinglocomotion,butalsoparticipateinother energy-demandingprocesses,suchasdevelopmentandreproduc- tion.Inthecricket,Gryllusbimaculatus,forexamples,thereisan orchestrationofanabolicandcatabolicreactionsinfatbodyduring oogenesis (Lorenz and Anand, 2004), and AKHs have been proposed as the triggers of catabolic reactions in the fat body that produce energetic substrates to be incorporated into the growingoocytesofG.bimaculatus(LorenzandGa¨de,2009).Onthe otherhand,many studiesindicatethat theinvolvementofAKH peptidesinreproductiveprocessesistheinhibitionofvitellogene- siswhichisahugeanabolicprocessinthefatbody(Carlisleand Loughton,1979,1986;MoshitzkyandApplebaum,1990;Comas etal.,2001;Lorenz,2003).Somepreviousstudiesalsosuggesta roleofAKHsrelatedtoreproductionbyfindingtheexpressionof AKH genes (Kaufmann and Brown, 2006; Abdel-Latief and Hoffmann, 2007) and Aedae-AKH receptor mRNA (Kaufmann etal.,2009)intheovary.Furthermore,AKH-likepeptidesinthe JournalofInsectPhysiology57(2011)858–864

ARTICLE INFO

Articlehistory:

Received16November2010 Receivedinrevisedform15March2011 Accepted15March2011

Keywords:

Adipokinetichormone Trehalose

RNAinterference Ootheca Reproductivecycle

ABSTRACT

Hypertrehalosemichormone(HTH)isaneuropeptidewithintheadipokinetichormone(AKH)family thatinducesareleaseoftrehalosefromfatbodyintohemolymphinanumberofinsects.Inthisstudy,we firstshowedthatfemaleadultGermancockroach,Blattellagermanica,displayedacyclicfluctuationof hemolymphtrehaloselevelscorrelatedtothematurationofoocytesinthereproductivecycle.After cloningtheHTHcDNAfromtheGermancockroach(Blage-HTH),expressionstudiesindicatedthatBlage- HTHmRNAshowedthecyclicchangesduringthefirstreproductivecycle,wherepeakvaluesoccurredin 8-day-oldvirginfemalecockroaches,whichweregoingtoproduceoothecae.ThefunctionsofBlage-HTH werestudiedusingRNAinterference(RNAi)toknockdownitsexpression.AdultvirginfemalesofB.

germanicainjectedwithBlage-HTHdsRNAincreasedhemolymphtrehaloselevelsinthelateperiodof vitellogenesismoreslowlythancontrol.Furthermore,RNAiofBlage-HTHdelayedovipositiontimeand some(10%)individualsdidnotproducethefirstoothecauntil15daysaftereclosion,whereasthecontrol groupproducedoothecabefore9daysinallcases.

CrownCopyrightß2011PublishedbyElsevierLtd.Allrightsreserved.

* Correspondingauthor.Tel.:+886223636581;fax:+886223636581.

E-mailaddress:m480@ntu.edu.tw(H.-J.Lee).

ContentslistsavailableatScienceDirect

Journal of Insect Physiology

j our na l ho me pa ge : w ww . e l se v i e r . com / l oca t e / j i ns phy s

0022-1910/$–seefrontmatter .CrownCopyrightß2011PublishedbyElsevierLtd.Allrightsreserved.

doi:10.1016/j.jinsphys.2011.03.012

Appendix 1

Author's personal copy

nematode Caenorhabditis elegans are involved in egg-laying behavior through a structurally vertebrate-like gonadotropin- releasinghormone(GnRH)receptor(Lindemansetal.,2009).

FemalesoftheGermancockroach,Blattellagermanica,follow several reproductive cycles in their adult life and produce an oothecaenclosingthematureeggs,eitherfertilizedorunfertilized, ineachreproductivecycle(RothandStay,1962).Virginfemalesof B.germanicadisplaybothcallingbehaviorandrobustlocomotor activityforseveraldaysbeforeoviposition(LeeandWu,1994;Lin andLee,1996).Increasedlocomotionisrelatedtomate-searching behavior, since the females of B. germanica reduce locomotion immediatelyafterasuccessfulmating(LinandLee,1998).Wecan presumethatahighenergydemandisneededtosupportrobust mate-searchingbehavior,andtrehaloseisthemajorsugarinthe hemolymph that is used as an energy source by tissues like muscles and ovaries(Thompson, 2004).Moreover, theinvolve- mentoftrehaloseinvitellogeninintakebydevelopingoocyteshas beendemonstratedincockroaches(Konoetal.,2001).Therefore, mobilizationoftrehaloseshouldbeexpectedtoincreasebefore ovipositioninthefemaleB.germanica.

In the present study, the hemolymph trehalose titers of B.

germanicaweredeterminedduringreproductivecycles,andresults showthatthefluctuationsofhemolymphtrehaloselevelsparallthe ovarian growth. Then, the Blage-HTH gene was cloned in B.

germanica,anditsphysiologicalfunctionwasinvestigatedbyRNA interference(RNAi)towhichthiscockroachspeciesisparticularly sensitive(Belle´s,2010).RNAi-mediatedknockdownofBlage-HTH geneexpressiondelayedtheincreaseofhemolymphtrehaloseand theproductionofthefirstoothecainvirginfemaleB.germanica.

2. Materialsandmethods 2.1. Insects

TheGermancockroach,B.germanica(L.),colonywasrearedin environmentalchambersunder288CandL:D=16:8hconditions.

Dogchowand waterwereprovidedad libitum.Newlyemerged adultswerecollectedwithin24handseparatedbysex.Detailed informationaboutrearinghasbeengivenpreviously(LeeandWu, 1994).

2.2. CloningofBlage-HTHinB.germanica

Total RNA was isolated with TRIzol¹ reagent (Invitrogen, Carlsbad,CA)frombrain–corporacardiaca–corporaallata(Br–CC–

CA) complexes of adult B. germanica females, following the manufacture’sprotocol.WeusedSuperscriptIIIreversetranscrip- tase (Invitrogen)and theoligo(dT)20primer togeneratecDNAs.

ThefirstprimersetwhichwasdesignedbasedontheknownAKH sequencesofotherinsects,includingBlaberusdiscoidalis(U35277), P.americana(AY622321),andBombyxmori(AB298930),andwas asfollows:forward,5⁰-TGTGTGAGGCTCAGGTGAACTT-3⁰;and reverse, 5⁰-TGA GAA TTT TTC ACA TTC CA-3⁰. The amplified fragment (159bp) was subcloned into the pGEMT-easy vector (Promega) and sequenced. To complete the Blage-HTH cDNA sequence, GeneRacerTMKit (Invitrogen)wasappliedtoaccom- plishrapidamplificationofcDNAends(RACE),accordingtothe manufacturer’sprotocol.For5⁰-RACE,thereverseprimerwas5⁰- CAC ATTCCAGCAGTTTCTGA-3⁰,and for3⁰-RACE,theforward primerwas5⁰-GTACATGTACAGTGCAATGC-3⁰.AllPCRproducts were subcloned into pGEMT-easy vector (Promega) and se- quenced. BLAST analyses indicated that the sequence obtained in B. germanica was B. discoidalis prepro-hypertrehalosemic hormonehomologue(GenBank:U35277.1)andwecalleditasB.

germanica prepro-hypertrehalosemichormone(Blage-HTH,Gen- Bankaccessionno.FJ943774).

2.3. QuantitativerealtimePCRanalysis

To quantify the mRNA levels of Blage-HTH, we followed a quantitative real-time PCR (qRT-PCR) approach. TotalRNA was extractedfrom3adultfemalebrain–CC–CAcomplexeswithTRIzol¹ reagent(Invitrogen,Carlsbad,CA).DNaseI(Promega)wasusedto removethegenomicDNAcontamination.About1

m

goftotalRNAof eachsamplewasreverse transcribedintocDNAwith oligo(dT)20

primer using Superscript III reverse transcriptase (Invitrogen).

Expressionofthehousekeepinggene,actin(AJ862721),wasusedas areference,andtheprimerpairswereasfollows:forward,5⁰-TTG TGCTGGACTCTGGTGAC-3⁰,andreverse,5⁰-ACGATTTCTCGCTCT GCTGT-3⁰.TheprimerstostudytheBlage-HTHgenewere:forward, 5⁰-TTGGTAGTTGTGGTGGCTCTAGCA-3⁰,andreverse,5⁰-CCAGCA GTTTCTGAGCTTCACTCT-3⁰.Theefficiencyofeachprimersetwas firstvalidatedbyconstructingastandardcurvethroughfourserial dilutions.TheqRT-PCRreactionswerecarriedoutintriplicateina Bio-RadiCycleriQ5Real-TimePCRdetectionsystem(Bio-Rad),using SYBR Green Realtime PCR Master Mix (#QPK-201; Toyobo Co., Osaka, Japan). A control without template was included in all batches.ThePCRprogrambeganwith asinglecycleat958Cfor 3min,40cyclesat958Cfor15sand608Cfor60s.Afterwards,the PCRproductswereheatedto958Cfor15s,cooledto558Cfor15s and increasing temperature 0.58C/min in order tomeasure the dissociationcurvesanddetermineauniquePCRproductforeach gene. We followed a method based on Ct (threshold-cycle) to measure the gene expression levels according to the Pfaffl mathematicalmodel(Pfaffl,2001).Theresultswereanalyzedusing the software associated with the instrument. The values were normalizedwiththevaluesofactinincontrasttothevaluesof1-day- oldsamples.Resultsof6biologicalindependentsampleswereused tocalculatethemeanSEM.

2.4. RNAiandsemiquantitativeRT-PCRanalysis

The Blage-HTH double-stranded RNA fragment(dsRNA) was preparedfollowingthePCR-templatemethodusingMEGAscript RNAi Kit (Ambion, Inc.). The Blage-HTH cDNA of the German cockroachwasfirstclonedintothepGEMTeasyvector(Promega, Madison,WI),thena330basepair(bp)fragmenttargetingtothe wholeORFofBlage-HTHmRNA(Fig.2A)wasamplifiedbyPCR.The primers, which contained theT7 promoter sequence (TAATAC- GACTCACTATAGGGAGACCAC),usedinthisPCRreactionwereas follows:forwardprimer,5⁰-AGCTCCTACATCCCACGTGTT-3⁰,and reverse primer, 5⁰-TGT ACA TGT ACT GTG CAA TGC A-3⁰. The MEGAscriptRNAiKit(Ambion)wasusedtogeneratethedsRNA following themanufacturer’sprotocol.The dsRNAsolution was storedat808Cuntiluse.Adoseof 3

m

gofthe332-bpdsRNA targetingtheBlage-HTHgenewasinjectedintotheabdomenofthe newlyemergedfemaleadults.AsthedsRNAcontrol,weusedthe enhanced green fluorescence proteingene applied of thesame dose.TheBlage-HTHgeneexpressioninthebrain–CC–CAcomplex wasdeterminedbysemiquantativeRT-PCRwithinthefollowing daysafterdsRNAtreatment.

ToinvestigatetheeffectofRNAi,Blage-HTHexpressioninthe cockroacheswasdeterminedbysemiquantativeRT-PCRbasedon theORFoftheBlage-HTHgene.Thus,the25

m

lPCRmixturefor amplifying the Blage-HTH fragment included 1

m

l of cDNA, 10pmolofforwardprimer(5⁰-CTGCCATTCAACTGGAAGACG A-3⁰),10pmolofreverseprimer(5⁰-AGCATTCCCCACTCATAC ATACAAAATC-3⁰),and5

m

lofTaqpolymerasemixture(Protech).

ThePCR(30 cycles)wasperformedasfollows:denaturationat 948Cfor30s,annealingat588Cfor30s,extensionat728Cfor 30s. In PCRs for amplifying the actin gene fragment were performed in parallel. The PCR products were separated and visualizedona1.5%agarosegel.

J.-H.Huang,H.-J.Lee/JournalofInsectPhysiology57(2011)858–864 859

Author's personal copy

2.5. Trehalosedetermination

Trehaloseinthehemolymphwasdeterminedwiththemethod ofParrouandFranc¸ois(1997),slightlymodified.Hemolymphwas obtained fromthe hind leg coxa and a 5

m

l volume from two individuals was collected using a pipetman. The hemolymph samplewasplacedinapolyethylenecentrifugetubecontaining 200

m

l0.25MNa2CO3buffer,itwasthenvortexedfor1min,and incubatedat968Cfor2htoinactivateallenzymesandtoconvert glucoseintoitsreductiveform.Then,120

m

lof1Maceticacidand 480

m

l0.25MNa-acetate(pH5.2)wereadded,andthesolution wascentrifugedat 12,000rpm, 248Cfor10min.Anamountof 100

m

lofsupernatantwasincubatedovernightat378Cwith2

m

l porcine kidney trehalase (Sigma, #T8778) in order to convert trehaloseintoglucose.Theamountofglucosein50

m

loftheabove solutionwasmeasuredusingtheGlucoseAssayKit(Sigma,GAGO- 20).Glucoseconcentrationwascorrectedbydeductingtheglucose amount of the same supernatant prepared under the same conditionsintheabsenceoftrehalase.

2.6. Statisticalanalysis

The results in the graphs represent the means of measur- ementsSEM.Statisticalanalysiswasperformedusingthecomput- ing environment R (The R Foundation for Statistical Computing, 2009).Significancesoftheresultswereevaluatedusingunpairedt- test(Figs.1Aand4B),one-wayANOVA(a=0.05)withFisher’sLSD multiplecomparisonspost-test(Fig.3B),andWilcoxontest(Fig.4C).

Correlation betweenthe hemolymph trehalose andbasal oocytes lengthinvirginfemaleB.germanicawasanalyzedbylinearregression usingbasicregressioncommand,GLM,inR.

3. Results

3.1. Dailyfluctuationofhemolymphtrehalose

FluctuationofthetrehalosetiterinthehemolymphinadultB.

germanicaafterimaginalmoltisshowninFig.1A.Trehalosetiter inthevirginfemaleswasabout4

m

g/

m

lafterimaginalmoltand steadilyincreasedalmost2-fold(7.5

m

g/

m

l)onday8,whenthe oothecawasformed.Whentheunviableoothecaeweredropped onday9,trehalosetiterbecamesimilartothatofindividualsat the early stage of the reproductive cycle. Then, hemolymph trehalosetiterofvirginfemalesincreasedagainwhilethenext reproductive cycle proceeded. Regression analysis showed a positive correlation between hemolymph trehalose and basal oocyte length (Fig.1B) in virgin femaleB. germanica(r=0.98, P<0.0001). Interestingly, a successful mating advanced the decreased response of the trehalose titer in the hemolymph (Fig. 1A). When virgin females were mated on day 5, the hemolymphtrehalosetitershowedsignificantdifferenceswith thatof virgin females onday7 (t-test, P<0.05)and8 (t-test, P<0.001).Moreover,thehemolymphtrehaloseof thefemales carrying an ootheca did not increase, and showed significant differenceswiththatofvirginfemaleswhowereintothenext reproductive cycle on days 17 and 19 (t-test, P<0.001). In contrast,theadultvirginmaleB.germanicadisplayedarelative lowandconstanttrehaloselevelafteremergenceinthefirstdays ofadultlife(Fig.1A).

3.2. CloningandcharacterizationofBlage-HTH

Blage-HTH cDNA (GenBank accession no. FJ943774) of B.

germanica was cloned and sequenced following a two-steps PCRstrategy,firstamplifyingafragmentofthespecificsequence with primers based on the conserved motifs of other HTH

sequences,andthenusing5⁰-and3⁰-RACEapproachtocomplete the sequence (Fig. 2A). The Blage-HTH mRNA contained 77 nucleotidesinthe5⁰untranslatedregion(UTR)beforetheopen readingframe(ORF),whichextendsfromthestartcodonATGat position78tothestopcodonTGAatposition296.TheORFwas followedbya3⁰-UTRof189nucleotidesincludingthepotential polyadenylationsignalAATAAA(Beaudoingetal.,2000)foundat position439, and a 20-nucleotide poly (A)tail. The predicted aminoacidsequenceofBlage-HTHpeptidewasQVNFSPGWGT, whichwasidenticaltotheHTHpeptidepreviouslyisolatedfrom the cockroach(Ga¨deandRinehart, 1990).The HTH-precursor- related-peptide (HPRP) for the putativeBlage-HTH C-terminal peptide hadtwo cysteine residues(Fig. 2B) predictedto form disulfidebondsforhomodimerizationduringstorageintheCC (O’SheaandRayne,1992).Inaddition,alignmentamongknown AKHprepropeptidesequencesintheOrderBlattariashowedthat Blage-HTHsharedahighpercentageofidenticalresidueswithB.

discoidalisHTH(72.2%;U35277)andP.americanaAKH-II(57.7%;

AY622321)(Fig.2B).

3.3. ExpressionofBlage-HTH

TheexpressionofBlage-HTHwasstudiedindifferenttissues includingbrain–CC–CAcomplex,ventralnervecord,midgut,fat body,ovary, oviduct,andaccessory gland of 6-and 7-day-old adultvirginfemaleB.germanica.Thebrain–CC–CAcomplexwas the only tissue expressing the Blage-HTH gene (Fig. 3A). The expressionpatternoftheBlage-HTHgeneinvirginfemalesofB.

germanicaafter imaginal molt is shown in Fig. 3B.Expression graduallyincreasedafteremergenceandreachedatapeakonday 8(one-wayANOVA,F_66,4.606=6.0588,P=0.01646;LSDposthoc test).Whentheunfertilizedootheca wasformedby thevirgin female, the highest expression level of the Blage-HTH gene immediately dropped to the same level as the beginning of reproductivecycle.

Fig.1.(A)DailychangesofhemolymphtrehaloselevelsinadultsofBlattella germanica.Thecurvewithsolidsquaresrepresentsthehemolymphtrehalose concentrationofvirginfemales(n=10–16)andthesolidarrowindicatesthe timeofovipositionformorethan50%ofthevirginfemales.Thecurvewithopen trianglesrepresentsthehemolymphtrehaloseconcentrationsofmatedfemales whomatedwithsexuallymaturemaleadults(10-day-old)onday5(marked withagraytriangle),andtheopenarrowindicatesthetimeofovipositionfor more than 50%of the matedfemales (n=12). Thecurve withopen circles representsthehemolymphtrehaloseconcentrationofmales(n=10).(B)The growthofbasaloocytelengthinvirginfemalesofBlattellagermanica(n=10).All valuesrepresentthemeanSEM;significantdifferencesofhemolymphtrehalose titerbetweenvirginandmatedfemalesareindicatedbyasterisks(t-test,*P<0.05;

**P<0.001).

J.-H.Huang,H.-J.Lee/JournalofInsectPhysiology57(2011)858–864 860