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Short views on

Insect Molecular Biology

Short views on Insect Molecular Biology Edited by Raman Chandrasekar

Edited by

Raman Chandrasekar

UNIVERSITY OF KENTUCKY College of Agriculture Entomology S-225 Agricultural Science Centre Building-North Lexington, KY 40546-0091 USA.

Foreword Message

www.uky.edu Email: rpalli@uky.edu

Tel. 859-257-7450

Fax: 859-323-1120

Entomology as a science of interdepended branches like molecular entomology, insect biotechnology; has made rapid progress in its attributes in the light of modern discoveries.

This also implies that there is an urgent need to manage the available resources scientifically for the good of man. In the past five decades, entomology in the country has taken giant steps ahead. Continued research has evolved better pest management through molecular approaches.

The editor of the this book Dr. Raman Chandrasekar have vast experience in research. He is exposure to research in “Molecular Entomology” has added to his expertise for mature thinking to conceive the editorship of present venture “Short Views on Insect Molecular Biology”. The comprehensive review articles by the entomologist from around the world in various research Organizations/ Universities enhanced the book’s reach and appeal. I am quite confident that, this book will serve not only the needs of students of entomology but also useful as reference book to the researcher across the globe.

I heartily congratulate the editor for the high standard and competent manner in which he has achieved his goal.

globe. I heartily congratulate the editor for the hi gh standard and competent manner in which

Preface

The aim of this book Short Views on Insect Molecular Biologyis to integrate knowledge about molecular biology, biochemistry, physiology, genetics, developmental biology and reproductive biology of insects.

While earlier studies on insect endocrinology have focused on juvenile hormone and ecdysterioids, the focus during the last two decades shifted to physiological vital proteins, neuropeptides, pheromone-binding proteins, and G-protein coupled receptors. Genetically-engineered insects and microbial toxins are promising subjects for insect pest management. Increased use of beneficial insects also forms an important part of this biotechnological approach.

This book provides recent research from scientists around the world. The contributing authors are recognized experts in their field of molecular entomology.

I would like to express my heartfelt gratitude to my former teacher Prof. M. Krishnan and Prof. M. Kobayashi who supported me at the commencement of this “International Book Mission Program”. Others who encouraged me during the research are Prof. Immo A. Hansen, Prof. Seo Sook Jae, Dr. Emiel Janssen, Prof. M. Takeda, Prof. L.I. Gilbert, Prof. Sumio Tojo, Prof. Keun Woo Lee, Prof. Y.E. Park, Prof. Young-Chang Kim. I have no words to express my feelings for all those who provided valuable contributions and made the completion of this book possible. I thank the Geyongsang National University (GSNU), APSERI-08 Nagoya University, Japan, APMC9-South Korea and Department of Science and Technology (DST), New Delhi, for financial assistance in terms of travel grants and research fellowships. Further, I wish to recognize the emotional and moral support extended by my father, mother, sister and brothers and for providing a peaceful environment to pursue this work to the best of my abilities. My sister always told me what Thomas Edison had quoted “Genius is 99 percent perspiration and one percent inspiration”.

Last, but not least, I would like to thank all authors and the external reviewers for their contributions. They have devoted their time and careful efforts in submitting reports and reviews without any financial compensation. Their valuable evaluations have greatly contributed to the quality of this book. I hope that this volume will inspire interest on the diverse aspects of insect physiology and molecular biology in aspiring and established scientists.

of insect physiology and molecular biology in aspiring and established scientists. Raman Chandrasekar , Ph.D., Editor

Raman Chandrasekar, Ph.D., Editor

Dedicated

to

Dedicated to Prof. M. Krishnan

Prof. M. Krishnan

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Short Views on Insect Molecular Biology, Vol. (1), 2009

CONTENT

Page No.

1. Perspective of Molecular Approaches to Entomology Raman Chandrsekar

1

2. Molecular structure of insect clocks Le Thi Dieu Trang, Qi-Miao Shao and Makio Takeda

21

3.

Insect Hexamerin Storage Proteins: Biosynthesis, Utlization and Evolution Raman Chandrasekar, Muthu Meenakshi and Luiz Paulo MA.

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4. Structural characteristics/ biosynthesis mechanisms of insect Vitellogenin Tufail Muhammad and Makio Takeda

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5. Molecular mechanisms of insect Vitellogenin /lipophorin receptor

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Tufail Muhammad and

Makio Takeda

 

6. Nutritional signaling in adult mosquitoes

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Immo A. Hansen and Geoffrey M. Attardo

 

7. Pheromone Biosynthesis Activating Neuropetide (PBAN) and its G-protein coupled receptor Rachel Bober and Ada Rafaeli

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8. Molecular mechanisms of sex pheromone reception in Lepidoptera Emmanuelle Jacquin-Joly and Martine Maïbèche-Coisné

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9. Autophagic programmed cell death in the peripheral fat body tissues of the silkworm, Bombyx mori L. Sumithra P., Raman Chandrasekar and Krishnan M.

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10. Apoptosis Regulation in Mosquito and its Importance to Malaria Infection Sylvester SL. Lyantagaye

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11. Molecular mechanisms of cold hardiness in the European corn borer (Ostrinia nubilalis, Hubn.) Duško P. Blagojević, Mihajlo B. Spasić and Gordana N. Grubor – Lajšić

191

12. Molecular characteristics of the W chromosome, Z chromosome and Retro transposable elements of the silkworm, Bombyx mori Hiroaki Abe and Tsuguru Fujii

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Appendix - A (Glossary and Terms)

229

Appendix

Appendix

- B

(Author Index)

238

Contributing Authors Prof. Makio TAKEDA Kobe University Graduate School of Science & Technology Japan .
Contributing Authors
Contributing Authors

Prof. Makio TAKEDA Kobe University Graduate School of Science & Technology Japan.

Prof. Ada Rafaeli Department of Food Science & Stored Products Unit Agricultural Research Organization The volcani Center Israel.

Dr. Immo A. Hansen Department of Biology & Institute of Applied Biosciences New Mexico State
Dr. Immo A. Hansen
Department of Biology &
Institute of Applied Biosciences
New Mexico State University
USA.

Prof. Krishnan, M. Department of Environmental Biotechnology School of Environmental Sciences Bharathidasan University India.

Dr. Tufail Muhammad Kobe University, Graduate School of Science & Technology Japan.

Dr. Hiroaki ABE Department of Biological Production Faculty of Agriculture Tokyo University of Agriculture & Technology Tokyo, Japan.

Prof. Dusko Blagojevic Department of Physiology Institute for Biological Research Serbia, YU

Dr. Raman Chandrasekar College of Agriculture Entomology 2-255 Agricultural Science Centre Building – North, University of Kentucky, Lexington, KY USA.

Prof. Luiz Paulo Moura Andrioli Instituto de Biociencias Universidade de São Paulo. Cidade Universitária 05508-900 Sao Paulo, SP, Brazil.

Dr. Sylvester Lyantagaye Department of Molecular Biology and Biotechnology, University of Dar es Salaam P.O. Box 35179 - Dar es Salaam Tanzania.

Dr. Geoffrey M. Attardo Yale School of Public Health and Epidemiology 60 College Street, New Haven USA.

Prof. Emmanuelle Jacquin-Joly UMR PISC INRA-UPMC-AgroParisTech INRA centre de Versailles Route de Saint-Cyr F-78000 Versailles France.

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Short Views on Insect Molecular Biology, Vol.(1), 2009

External Supportive Members Prof. Marc J. Klowden Division of Entomology University of Idaho Moscow, ID
External Supportive Members
External Supportive Members
Prof. Marc J. Klowden Division of Entomology University of Idaho Moscow, ID 83844-2339 USA Prof.
Prof. Marc J. Klowden
Division of Entomology
University of Idaho
Moscow, ID 83844-2339
USA
Prof. Enoch Y. Park
Innovative Joint Research Center,
Laboratory of Biotechnology
Integrated Bioscience Section,
Graduate School of Science and Tech.
Shizuoka University,
Shizuoka, Japan.
Prof. Lawrence I. Gilbert
Department of Biology
The University of North Carolina
USA.
Prof. Michihiro KOBAYASHI
Division of Biodynamic
Graduate School of Biological Sciences
Nagoya University
Japan.
Prof. Seo Sook Jae
Division of Applied Life Science
Gyeongsang National University
South Korea.
Prof. Luiz Paulo Moura Andrioli
Instituto de Biociencias
Universidade de São Paulo.
Cidade Universitária 05508-900
Sao Paulo, SP, Brazil.

Prof. Frantisek Sehnal Entomological Institute Acad. Sci., Ceske Budejovice Czech Republic.

Prof. Reinhard F. Stocker Institute of Zoology, University of Fribourg Pérolles, 1700 Fribourg Switzerland.

Prof. Randolf Menzel Freie Universität Berlin Institut für Biologie - Neurobiologie Königin-Luise-Str. 28/30 D - 14195 Berlin Germany.

Prof. Shalom W. Applebaum Mauerberger Professor Emeritus of Entomology Department of Entomology Faculty of Agricultural, Food & Environmental Quality Sciences The Hebrew University – Rehovot Campus Isral.

Dr. Immo A. Hansen Department of Biology & Institute of Applied Biosciences New Mexico State University USA.

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Short Views on Insect Molecular Biology, Vol. (1), 2009

B O L O A K N M O I I S T A S
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Short Views on Insect Molecular Biology, Vol.(1), 2009

Short Views on Insect Molecular Biology

Lead Article

Vol. (1)., 00 – Vol. 00, 2009 (1), 1 – 20, 2009

Chapter – 1

Perspective of Molecular Approaches to Entomology

Raman Chandrasekar * Department of Environmental Biotechnology, Bharathidasan University, Tamilnadu, India.

Abstract

Entomology today is a super science, having deviated considerably from the morphological-taxonomical approaches of the first half of the century, embracing an interdisciplinary approach involving such aspects as biological diversity, chemical ecology, circadian rhythms, neuroendocrinology, medical-entomology, molecular biology and biotechnology. Molecular biology, integrated with genetics and biochemistry, has provided the necessary tools for transferring and evaluating genetic characteristics not only for a host of insects, but also for related host plants. The molecular approaches have enabled the study of physiological vital proteins and sensillar-neural complexes that are involved in pheromonal studies. Such knowledge is vital to devise safe and specific agents for disrupting insect life cycles, thus increasing the efficiency of efforts to manage agricultural pests and disease vectors.

Keywords: molecular biology, sensillomic, baculoviruses, display techniques, pheromone

*For Correspondence (email: koreanchandru@yahoo.com)

Overview

1. Introduction

2. Molecular and Biotechnology approaches

3. Physiological vital Proteins (3a). Hexamerin storage protein (3b). Vitellogenin

4. Trangenesis

5. Sensillomics

6. Hormonomimetic Compounds

7. Baculoviruses as Expression Vectors

8. Seribiotechnology

9. Medical Entomology

10. Integrated pest management

11. Concluding remarks

12. Reference

*Present Address:

S-225 Agricultural Science Center Building – North,

University of Kentucky, Lexington, KY, USA.

College of Agriculture Entomology,

1. Introduction

Insects are the most successful species on the planet. They have withstood the natural calamities because of their extraordinary diversity and adaptability. This vouches for the obstacles that are encountered in the development of a foolproof strategy for pest control. Apart from this, there are numerous other factors that determine the effectiveness of a pest control strategy. Most important of all is, to have an understanding of the significant ecological roles played by “pest” species in both unmanaged and agricultural environments and a thorough knowledge of the physiological, morphological and biochemical aspects of the insect, despite this being an arduous task.

Short Views on Insect Molecular Biology, Vol.(1), 2009

The biological knowledge will certainly provide the cue for developing a unique technology based on the exceptional characteristic of the pest species. In short, if either biological knowledge or technology is deficient, the control program will not be successfully accomplished (Pedigo, 1996).

Agricultural pest problems and action to alleviate them are nearly as old as the beginning of crop cultivation. The earliest record of pest technology seems to be the use of sulfur and thereafter, the advances made were tremendous. The, then popular insecticidal approach relied more on the ability to kill the pests than plant protection and environmental protection. The conventional synthetic organic pesticides are handicapped in the environmental context, by their long-term persistence, high toxicity and propensity for bioaccumulation. Further, as a result of development of pest resistances to organic pesticides, the concept of biological control increased. But, as natural enemies were non-dependable for satisfactory containment, the concept of integrated control was developed. Methods of natural chemical control to disrupt the pest's life cycle using semiochemicals or infochemicals (Dicke and Sabelis, 1988), which refers to chemical signals that mediate interspecific and intraspecific interactions between organisms, started gaining ground. This category includes pheromones and allelochemicals, which are used to attract insects into traps, or confuse them or block some of their essential functions. Pheromones mediate an interaction between organisms of the same species whereas allelochemicals mediate an interaction between individuals that belong to different species.

The identification of neuropeptides such as allatotropins and allatostatins that regulate juvenile hormone synthesis by corpus allatum has progressed considerably and promises new avenues of neuroendocrine manipulation for insect control, in particular their interaction with the receptors of target cells enabling hormonal imbalance in insects (Ananthakrishnan, 2001). Other insect hormones like juvenile hormone analogs and ecdysones have also been exploited extensively. Considerable research in the discovery and use of chemical inhibitors of juvenile hormone synthesis to cause chemical allatotectomy, juvenoids and juvenile hormone antagonists to trigger developmental disorders and ecdysone agonist/ antagonist to mediate insecticidal action, have been successful, but most of the time they also have an adverse effect on beneficial insects. In recent years, increasing research has focused on plant–derived insect antifeedants, which are non-pollutant, generally less toxic and easily biodegradable especially if used as total or enriched extractives (Babu et al, 1996). The Sterile Insect Technique (SIT) which involves the sterilization of the target pest by irradiation and sustained release of the large numbers of sterilized insects to reduce the native population through infertile mating has been further refined by recent progress in transformation systems that allow genetic engineering of diverse insects (Wimmer, 2005). The new trend involves the use of fluorescent transformation marker for identifying released insects, sex-separation methods based on female-specific expression of a conditional dominant lethal gene and a transgenetic system that causes embryo-specific lethality after transmission to the progeny, which could replace irradiation technique and produce more competitive sterile insects. There is also a demand for increasing use of baculovirus and NPV in pest management for some of the important agricultural pests.

In the past, singular strategies were used for the control of a pest species based on diverse aspects of either biological, physical, chemical. Maybe that was the reason behind the failure of these strategies! But now the trend is to integrate all individual techniques and tactics for insect pest management and use them in a system that combines the most appropriate characters of each to produce an ecologically sound and economically viable package called integrated pest management (IPM) (Ananthakrishnan, 2001). This strategy is based on a clear understanding of ecological functioning of the agroecosystems. An integration of pest monitoring and accurate timing of pesticide treatment, use of selective insecticides as well as combined use of semiochemicals, host plant resistance, trap crops, oviposition deterrents, antifeedents and of recent, biomimetics are important to manipulate the pest behaviour, effectively and efficiently. This lead to the metamorphosis of the term: stimulo-deterrent diversionary strategy (SDDS) (Ananthakrishnan, 2003).

Short Views on Insect Molecular Biology, Vol.(1), 2009

2. Molecular and Biotechnology approaches

Biochemistry, genetics and molecular biology have in recent years become essential ingredients in our understanding of entomological problems. With the molecular biology approach, a different emphasis has resulted from obtaining and handling DNA which has provided the necessary tools for transferring and evaluating genetic characters from a host of insects. Genomics is a broadly used term encompassing numerous scientific disciplines and technologies (Fig.1). These disciplines include: genome sequencing; assigning function to identified genes; determining genome architecture; studying gene expression at the transcription level (transcriptomics); studying protein expression at the proteome level

(proteomics); and investigating metabolite flux (metabolomics) (Fig.2). The polymerase chain reaching (PCR) enables repeated duplication of a trace amount of DNA resulting in sufficient amount for detailed DNA analysis.

ssyysstteemmss bbiioollooggyy systems biology molecular structures mmoolleeccuullaarr ssttrruuccttuurreess Application
ssyysstteemmss bbiioollooggyy
systems biology
molecular structures
mmoolleeccuullaarr ssttrruuccttuurreess
Application of “OMICS” technology to
Biotechnology
metabolomics
metabolomicsmetabolomics
transcriptomicstranscriptomics
transcriptomics
proteomics
proteomicsproteomics
genomicsgenomics
genomics
ggeennee nneettwwoorrkkss
gene networks

Fig.1 Schematic diagram showing the branches of molecular biology.

Schematic diagram showing the branches of molecular biology. Fig.2 The Molecular basis of life. The detection

Fig.2 The Molecular basis of life.

The detection of specific fragments of DNA is made possible through the restriction fragment length polymorphism (RFLP) technique. For identifying the degree of genetic variability of samples, the random amplified polymorphic (RAPD) technique is used, in particular to characterize genera, species and races, as in the studies of biotypes of mustard aphids and whiteflies from different regions. Reproducibility of RAPD-PCR results, when verified, showed that RAPD patterns were consistent for a given genotype (Dilawari and Gupta, 2004). Beside the routine DNA sequences, the use of microsatellite

Short Views on Insect Molecular Biology, Vol.(1), 2009

loci (MSL) as genetic markers has become an established technique. With the molecular biology approach, potentially resistant genes can be removed and transferred by a vector to a crop plant. A common example is the incorporation of the Bacillus thuringiness insecticidal crystal protein gene into host plants to have the toxic gene expressed in the host plant to give protection against several species of caterpillars. Informational molecules such as proteins and nucleic acids are now believed to have high taxonomic information, so that molecular approaches to systematics have become increasingly familiar

(Fig.3).

Nucleous DNA (structural genomics) Pre-mRNA cytoplasam mRNA mRNA (Transcriptiomics) Protein (Proteomics) Protein
Nucleous
DNA (structural genomics)
Pre-mRNA
cytoplasam
mRNA
mRNA (Transcriptiomics)
Protein (Proteomics)
Protein (Proteomics)
Functional
Genomics
Metabolites (Metabolomics)

Fig.3 Moving from targeted investigations of few genes to the investigation of many (all) genes in untargeted experiments that use highly parallel approaches in order to identify unknown genes.

3. Physiological vital Proteins

(3a) Hexamerin storage protein

In holometabolous insects, the larval period is an essential stage required for the accumulation of nutritional reserves for the non-feeding pupal stage. Essential nutrients obtained during the larval stage but needed by the non- feeding pupal stage must be sequestered and carried across stages until they are mobilized. Storage proteins (SP) appear to be a special adaptation in insect molting, metamorphosis, and cyclic reproduction, and have no known analogue in the vertebrates. The transformation from larval caterpillar to non-feeding pupal and adult moth involves a complete remodeling and restructuring of the insect, and its organs. The insect fat body is recognized as a complex multifunctional tissue: which participates in multiple biochemical and physiological functions, and it is a major site for the synthesis and storage of carbohydrates, lipids, proteins and nitrogenous components (Keeley, 1985; Haunerland et al.,1990). Due to the existence of fat body heterogeneity and by correlation of their structure and functions in Hyalophora cecropia (Tojo et al.,1978), G. mellonella (Bean and Silhacek, 1989), Heliothis zea (Wang and Haunerland, 1992) and B. mori (Vanishree et al., 2005), Amsacta albistriga (Chandrasekar et al., 2008a,b) these storage proteins are synthesized abundantly during the active feeding larval period in the peripheral fat body (PF) tissues and subsequently the proteins are released into the haemolymph. In a previous paper on the red hairy caterpillar, Amsacta albistriga we reported on regionally differentiated fat bodies (peripheral and perivisceral) associated with the segregation of synthetic and storage activities (Chandrasekar, et al.,2007; Chandrasekar, et al.,2008a). Additional

Short Views on Insect Molecular Biology, Vol.(1), 2009

evidence of ultra thin sections and immunogold labeling studies during the spinning stage provided direct evidence for the sequestering ability and huge accumulation of SP hexameric crystalline granules which were observed in perivisceral fat body tissue (PVF) of A. albistriga (Chandrasekar et al.,2008b)

(Fig.4).

The appearance of storage proteins in the haemolymph and fat body of one day old sixth instar larvae was in concert with the findings of Yoshiga et al. (1997). The storage proteins showed fluctuation patterns similar to that reported by many researchers (Tojo et al., 1981; Ryan et al., 1985; Nagata and Kobayashi, 1990; Yoshiga et al., 1997). Insects prepare for the synthetic demands of molting, metamorphosis and reproduction by accumulating storage proteins in their haemolymph and fat body (Pan and Telfer, 2001). The storage proteins are basic proteins, that are hexameric and have been best characterized with respect to their role in metamorphosis (Telfer and Kunkel, 1991; Pan

and Telfer, 2001). Most proteins destined for eggs must be obtained during the larval stage and stored until synthesis of yolk proteins begins. The storage proteins are obviously the storage reserve of amino acids (Wheeler et al., 2000, Chandrasekar, 2008b). Physiological links between the amino acids stored in storage proteins and those amino acids used in vitellogenesis may be common in insects. The uptake of hexamerins is an important process that allows holometabolous insects to survive during the non-feeding pupal period (Fig.5). In addition, the dynamics between sequestration and release of amino acids from storage proteins in the pupa/adult stage may be an important feature enabling many of their diverse strategies of reproduction.

enabling many of their diverse strategies of reproduction. Fig.4 Electron micrograph of perivisceral fat body (PVF)

Fig.4 Electron micrograph of perivisceral fat body (PVF) tissues from day 2 pupa of Amsacta albistriga showing abundance of protein granules. Scale bar - 3µm. PG – Crystalline protein granules; L – lipid droplet (From: Raman Chandrasekar, 2006).

Perivisceral fat body tissues Perivisceral fat body tissues
Perivisceral fat body tissues
Perivisceral fat body tissues

Peripheral fat body tissues

Peripheral fat body tissues

Fig.5 Schematic diagram showing the synthesis and uptake mechanism of hexamerin storage protein from PF to PVF tissue during larval- pupal transformation (From: Burmester and Scheller, 1999).

Short Views on Insect Molecular Biology, Vol.(1), 2009

(3b) Vitellogenin (Vg)

The developing embryo of oviparous animals and insects draws practically all of its requisite nutrients from a cache of proteins, lipids, and carbohydrates stored within the egg as yolk (Sappington et al., 1995, 1996). Yolk is the major internal food supply on which most embryos rely. Strict regulation of its utilization is essential to provide nutrients at the right time to the developing tissues, and to ensure survival of the embryo until it becomes a free-living organism, able to feed (Fagotto, 1995; Salerno et al., 2002).

Vitellogenin (Vg) is a multifaceted protein and so, analysis of this protein would serve diverse purpose. Many behavioral, ecological and physiological factors are known to induce a phenomenon called oosorption in various insects. When oosorption is induced, constituents that had accumulated in the oocytes are transported outside the oocytes, by a mechanism that has not been fully elucidated. Starvation induced oosorption in the females of Plautia crossota stali, elucidated that the Vg already accumulated in developing oocytes was released into the haemolymph (Kotaki, 2003). But the mechanism needs to be studied. Recently, fish Vg received attention as a biomarker for estrogenic compound in aquatic areas. Normally, Vg is observed in vitellogenic female serum however, male and juvenile fish can also synthesize Vg when exposed to exogenous estrogen (Specker and Sullivan, 1994) or to substance that mimic estrogens. Therefore, Vg is used as a biomarker for monitoring mimic estrogens in aquatic areas (Fakuda et al., 2003). Another alternative use of Vg had been reported by Amdam et al. (2003). Vg was found to be a source for the formation of the proteinaceous royal jelly that is produced by the hive bees which is used to feed larvae, queen, workers, and drones (Koeppe et al.,1985). This finding suggested that the evolution of a brood-rearing worker class and a specialized forager class in an advanced eusocial insect society has been directed by an alternative utilization of Vg (Amdam et al., 2003).

The Vgs of most insects are members of a conserved family of proteins that are present in organisms as diverse as nematodes and vertebrates (Sappington et al., 2002). The Vg primary product has been thoroughly characterized from 14 insect species, six of them belonging to hemimetabola and six belonging to holometabola (Sappington et al., 2002). The comparison of the protein primary structures of Vgs from different insect species has shown that they are highly conserved and form a gene superfamily (Chen et al., 1997; Lee et al., 2000; Sappington et al., 2002). The insect Vgs have been shown to be homologous with the Vgs of other invertebrates such as nematodes and those of oviparous vertebrates such as amphibians and birds (Chen et al., 1994). Similarly, insect Vg share homology with Vgs of other arthropods, such as millipedes (Prasath and Subramoniam, 1991), ticks (Chinzei et al., 1983; Rosell and Coons, 1991) and Crustaceans (Chen and Chen, 1993).

A A
A A
B B
B B
C C
C C
Mature Mature Mature D D Yolk body Yolk body Yolk body
Mature
Mature
Mature
D D
Yolk body
Yolk body
Yolk body

Fig. 6. Schematic diagram showing the vitellogenin (Vg) uptake by the ovary in insect.

A. Receptor-mediated endocytosis,

B. Accumulation of Vg

C. Formation of Transitional Yolk

Body, D. Mature Yolk Body (Courtesy: Prof. Keeley, L. L. , Texas

A&M University)

Short Views on Insect Molecular Biology, Vol.(1), 2009

The process of Vg uptake in the insect species present morphological peculiarities that are related to their life strategies. Specific binding of Vg to oocyte membrane preparations and solubilized membrane proteins has been demonstrated for insects, amphibians, fishes and birds (Opresko and Wiley, 1987; Stifani et al., 1990; Hiramatsu et al., 2002; Patino and Sullivan, 2002; Li et al., 2003). The initial steps in the yolk uptake pathway are similar to those described for general receptor-mediated endocytosis but in contrast to the degradation of the internalized ligands in lysosomes, the yolk proteins are stored as yolk granules for later use during embryogenesis (Fig.6). The yolk granules appear to be modified lysosomes with relatively high pH; during embryogenesis, the pH of the yolk granule drops to levels more typical of lysosomes (Fagotto, 1995). The single layer of follicle cells that surrounds oocytes constitutes an additional barrier to yolk precursors entering the oocyte. For invertebrates, the yolk protein receptor is described for the fruit fly, D. melanogaster (Schonbaum et al., 1995), and Vg receptors (VgRs) are described for the mosquito, A. aegypti (Sappington et al., 1996); the mosquito, Anopheles gambiae ; the American cockroach, P. americana; the silkworm B. mori (NCBI) and the nematode, Caenorhabditis elegans (Grant and Hirsh, 1999). In both insect and vertebrate VgRs, binding of Vg was saturable, ovary specific, showed high Vg affinity, was inhibited by suramin and was sensitive to changes in pH and Ca 2+ concentration (Konig and Lanzrein, 1985; Osir and Law, 1986; Konig et al., 1988; Dhadialla and Raikhel, 1991; Wang and Davey, 1992; Bujo et al., 1994). An integrative study on the hormonal influence on the process of receptor-mediated endocytosis is required to clearly portray the mechanism of regulation of this process so that it would provide a cue for the designing of a gene delivery system.

In the perpetual arms race against the insect pests of agricultural importance, it is imperative to develop novel strategies for pest control. But for this, it is indispensable to be equipped with a thorough understanding of the biological system of the insect pest at the cellular and molecular levels, which apart from opening new avenues in the development of novel strategies after molecular modeling of the SP- SPR, Vg-VgR, interactions and Vg/SP analog-designing, might certainly contribute to the success of it by overcoming possible loopholes. Nonetheless, such a basic yet in depth study will also find an application in enhancing the favorable characteristics of a beneficial insect. So, in this context the recent trend involves the identification and characterization of physiological essential proteins like apoLp-III, ferritin, storage protein, vitellogenin, lipophorin and their receptors, which can be subjected to various refinements and manipulations for their exploitation in combating the resistant pest species, in a target specific and eco-friendly manner.

4. Transgenesis

The first Bacillus thuringiness (Bt) toxin gene from Bacillus thuringiensis Berliner was cloned in 1981 and the first transgenic plants were produced by mid-1980s. Since then, several crop species have been genetically engineered to produce Bt toxins to control the target insect pests. Genes conferring resistance to insects have been inserted into crop plants such as maize, cotton, potato, tobacco, rice, broccoli, lettuce, walnuts, apples, alfalfa and soybean (Bennett, 1994; Federici, 1998; Griffiths, 1998). The first transgenic crop was grown in 1994 and large-scale cultivation was taken up in 1996 in USA (McLaren, 1998). Since then, there has been a rapid growth in the area under transgenic crops in USA, Australia and China. Transgenic plants, with insecticidal genes, are set to feature prominently in pest management in both developed and the developing world in the future. Among the developing countries; China, India, Argentina, Mexico, Brazil, Pakistan and South Africa are pursuing the research on transgenic crops vigorously. Entomologists, breeders and the molecular biologists need to determine how to deploy this technology for pest management, and at the same time avoid or reduce possible environmental risks. To achieve these objectives, it is necessary to have an appropriate understanding of the insect biology, behaviour, its response to the insecticidal proteins, temporal and spatial expression of insecticidal proteins in the plants, strategy for resistance management, impact of insecticidal proteins on natural enemies and non-target organisms. Equally important are the issues concerning the transfer of technology to the resource poor farmers. Development and deployment of transgenic plants with

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insecticidal genes for pest control will lead to: (a) reduction in insecticide sprays, (b) increased activity of natural enemies, and (c) IPM of secondary pests.

In recent years we have been witnessing a major revolution in entomological science, more especially with increased understanding of biological systems at the cellular and molecular levels. I am referring to the area of biotechnology relating to the fields of rDNA technology and genetic engineering and transgenics-areas which have great potential application in agriculture. Much of the current interest is centered on creation of pest resistant, herbicide resistant and disease resistant transgenic plants. The discovery that the aerobic, Gram-positive spore forming bacterium, Bacillus thuringiensis Berliner (Bt), which produces a toxic protein during sporulation, has introduced a new era of genetically engineered biocontrol agents.

The successful adoption of Bt crops by farmers has led some to muse on the possibility that the widespread use of Bt proteins in crops will lead to the development of insect populations that are resistant to these proteins, thus rendering Bt crops and Bt sprays less effective in controlling the destructive insects. The U.S. National Academy of Sciences recommended the development of insect resistance management (IRM) plans for both Bt crops and sprayable Bt pesticides. The goal of these plans is to ensure continued effectiveness of both the plant expressed and the sprayable formulations of this family of pesticidal proteins. A coordinated scientific approach is used to establish management practices that will minimize the risk of resistance and sustain the performance of Bt pesticidal proteins. Recently developed IRM plans for Bt crops couple Bt plants with a structured "refuge" of non-Bt plants. Refuge refers to a portion of the crop plants in or near a field that does not contain the Bt protein. A typical IRM plan in corn requires that at least 20% of the grower's corn acreage be planted to a non-Bt corn refuge within 1/2 mile of the Bt corn fields. The purpose of the refuge is to maintain a population of target insect pests that are susceptible to the Bt protein. Those susceptible insects can mate with rare resistant insects that may emerge from the Bt crop so that the resulting offspring will be susceptible to the Bt protein.

Moving Bt insecticide genes from the original bacterium to plants through the mediation of a vector, Psedomonus flourescens, accords crop protection from insect attack. By far the greatest research effort in developing pest resistant transgenic crops has gone into the expression of Bt toxins or delta endotoxins or crystalline proteins in different crop plants and tobacco and tomato provided the first examples of genetically engineered insect resistance. Being related proteins, several strains of this bacterium are known with distinct host ranges. At least ten genes encoding different Bt toxins such as cry1Aa, cry1Ab, cry1Ac etc., have been engineered into plants and the different toxins have different specifications for different orders of insects, notably Lepidopotera (Cry I), Coleoptera (Cry III) and Diptera (Cry II). The heterogeneity in toxin production is responsible for some of the diversity in the activity spectrum among strains (McGughey and Whalon, 1992). Currently there are other major groups of plant derived genes used to confer insect resistance in crops and which are inhibitors of digestive enzymes. These are the proteinase. Deciphering the chemical nature and information content of the molecules that transmit information between insects, plants and natural enemies. Volatile phytochemicals tend to promote or deter interactions between plants and phytophagous insects. Basic volatiles related from leaf surfaces form constitutive chemical defense which often include monoterpense, sequiterpenes and aromatics which accumulate considerably in plant parts. Chemical signals and signaling systems in insects hold great promise in the utilization of semiochemicals in insect control.

Recent research on induced defenses aims at generating intra- and inter-plant signals in plants due to insect damage. An induced defense is ‘a special mosaic of defensive chemistry’. Some of the best examples relate to the release ethylene, jasmonic acid and methyl jasmonate, salicylic acid and abscissic acid in insect damaged plants, notably jasmonic acid which can induce the expression of proteinase inhibitor gene even in nearby plants. Today we know that plants offer natural enemies with chemical information about the presence and densities of insect hosts. To cite an example, terpenes such as beta- ocimene, beta-farnescene, linanool and compounds like hexenyl acetate are released on damage and

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several parasitoids and predators are known to exploit insect induced signals to detect their hosts. The ability of host seeking insects to recognize and respond to such chemical cues is an essential aspect in titrophic interactions. Such is the dependence of parasitoids on plants signals that even a slight genetic change in plants may result in a decline of parasitism.

5. Sensillomics

The integration of the sensillar and neural complexes besides the biochemistry of chemoreception involving molecular components naturally calls for a new approach designated as “Sensillomics”. In order to fully integrate the sensillar and neural systems in chemoreception, a new approach designated as “sensillomics” is required in addition to a biochemical one. While the morphometric approach involving the ultrastructure of insect sensilla has been utilised to some extent, its functional counterpart, the electrophysiological recording has not received the attention it deserves. The behavioural significance of diverse chemical species that phytophagous insects encounter lies in the fact that neurons transmit information between each other and throughout the neuronal network, via signals called nerve impulses. These consist of spikes and action potentials. The ability to recognize specific molecules is inherent to the receptor sites, such as those of a sexual pheromone selectively binding proteins in the olfactory sensilla. Once the signal is detected it needs to be amplified in order to obtain a relevant response. The axon from the olfactory sense cells end in an area of the brain called the glomeruli, considered to be neuropile structures where integration of sensory inputs take place, thus enabling signal amplification. The insect antenna (Fig.7) serves as a molecular ‘sieve’. Only those molecules absorbed in the sensory hairs have a chance to cause a response in the dendrites of the receptor cells. Molecular absorption increases the efficiency of such molecular trap. As the primary sites where an extensive integration of olfactory inputs occurs, the glomeruli serve as centers in which inputs from the sensory cells with similar response characteristics merge. The adaptive nature of a specific olfactory sensitivity has enabled a better understanding of the response to chemical species. As a result, signals and signaling have become the essential components in behavioural studies. The direct pathways for transmitting information are located in the antennal lobe of the brain, while the general binding proteins occur in both sexes, glomeruli are species-specific in terms of number, size and arrangement. Glomeruli have been mapped in a number of insect species (Ryan, 1990). It is therefore in the understanding of the significance of the sensillar-neural complexes, involved in the recognition of molecules such as pheromone binding proteins that the composite field of sensillomics is concerned with. The cross-disciplinary field of sensillomics thus puts an emphasis on the sensillar-neural complexes that handle signals arising from the interaction between pheromones and specific binding proteins (Ananthakrishnan, 2003).

and specific binding proteins (Ananthakrishnan, 2003). Fig.7 A pair of pectinated antennae on the head of

Fig.7 A pair of pectinated antennae on the head of a male silkmoth. The antennal branches are carrying numerous cuticular expansions, the sensilla.

Further, our understanding of pheromone production has evolved from identifying biochemical pathways towards unraveling the molecular biology of key regulatory enzymes initiating a genomics approach. Today we know of the pheromone binding protein (PBPs) and pheromone degrading enzymes (PDEs) which inactivate pheromone signals within the sensilla. Biochemical transduction pathways are now identified including all important olfactory receptor proteins. A marriage of molecular genetics and genomics behaviour has resulted in a new understanding of how pheromones are detected. It is today well known that olfaction and taste work via sensory neurons and that odour and taste molecules stimulate these neurons by binding to receptor proteins.

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6. Hormonomimetic Compounds

It is interesting that plants are also known to synthesize compounds that distrupt normal development of the insects which feed on them. Examples of such compounds are phytoecdysone, juvabione, anti-juvanile hormones or precocenes, and juvocimenes that mimic the functions of insect hormones and are hence designated as hormonomimetic compounds. With advances in genetic engineering techniques, it has been possible through structural elucidation of polypeptides to not only identify the genes aiding in their synthesis, but also develop inhibitors of enzymes mobilizing such synthesis. Incorporation of such genetic factors into host plants, employing baculoviruses as carriers, may modulate production of hormones which can disrupt the physiology of the insect invader. Genetically engineered transgenic plants with the ability to synthesize defensive proteins such as phytoagglutinins, lectins and proteinase inhibitors are considered as alternate means to avoid excessive use of biocides that also tend to disrupt the natural enemy complexes. Evidence also point to insects’ ability to overcome resistance based on single gene expressions (Van Emden, 1991), and therefore emphasis is currently placed on the need to evolve multigene resistance mechanisms.

7. Baculoviruses as Expression Vectors

Baculoviruses are unique in that they form useful expression vectors. It has been suggested that by introducing appropriate foreign genes into the baculovirus genomes, pathogenicity and insecticidal effectiveness can be increased. In the formation of baculovirus insecticides, a recombinant baculovirus of increased toxicity is made by introducing appropriate foreign genes into highly expressed polyhedron gene sites. Infection with such recombinant viruses may cause direct toxicity, late behaviour or arrested development of insects. Recent advances in recombinant DNA technology and the genome engineering of insect baculoviruses have made possible the use of insect baculoviruses as expression vectors for neurohormone genes. Neurohormones, by virtue of their peptidic nature are amenable to applications using recombinant DNA and genetic engineering technology.

8. Seribiotechnology

Research on the silkworm Bombyx mori has a long history, but Seri-biotechnology is an emerging field of “Insect Biotechnology”. The potential of silkworms to produce high levels of exogenous proteins has attracted the attention of academic and industrial researchers. The silkworm is a useful model for genetic, biochemical and physiological studies, as an insect model system it occupies a position next to the fruit fly, Drosophila melanogaster, but is the system of choice among the Lepidoptera. Hence, recently we demonstrated that P-soyatose (hydrolyzed soy bean protein) supplementation is of importance in regulating the fibroin gene expression at transcriptional level (Chandrasekar et al., 2007b). This would be a practical contribution to sericulture for maximizing economic traits of economically important insect B. mori.

Insect offer many advantages for gene regulation studies compared to vertebrates or vertebrate cell culture. There is also a great

to vertebrates or vertebrate cell culture. There is also a great Fig.8. Life cycle of NPV

Fig.8. Life cycle of NPV baculovirus in nature.

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variety of applied problems with insect pests that need to be addressed. Differential gene expression underlies a range of biological processes, including development, reproduction, and behavior (Harshman and James, 1998). The silkworm has many advantages over other model organisms. However, in contrast to D. melanogaster, B. mori lacks an efficient germline transformation system. Plasmids and viruses are the contemporary vehicles for gene therapy and genetic vaccination and extremely promising results have been reported from in-vitro, in-vivo and clinical studies. At present many recombinant proteins are manufactured with a technology which has been directly transferred from laboratory to pilot scale without further engineering (Fig.8). This approach has been more widely adopted in Asian countries, including China, Japan and India, where silkworms are abundantly available and more laboratories have experience in growing and maintaining larvae. The silkworm, B. mori, is one of the most attractive hosts for large-scale production of eukaryotic proteins as well as recombinant baculoviruses for gene transfer to mammalian cells. Baculovirus expression systems (BESs) are widely used to express heterologous genes in cultured insect cells and insect larval hosts; gene expression is driven by the strong polyhedron promoter (Kato et al.,2005; Gubitosi-Klug et al.,2005; Smith et al.,2007). Conventional preparation of recombinant baculovirus requires at least 40 days. Now, it has been established that BmNPV bacmid system provides the rapid protein purification in silkworm (as long as 10 days), is free from hazard, and will be a powerful tool for the future production factory of recombinant eukaryotic proteins and baculoviruses (Motohashi et al.,2005). Results have shown that the earliest pupal stages are the most suitable for the production of viral proteins and probably for the expression of foreign genes under the control of late viral promoters (Mikhailov et al., 1992; Park et al.,2008). In addition to recombinant protein production, the surface display technology of recombinant protein on the surface of baculovirus has been developed (Grabherr et al.,2001; Oker-Blom et al.,2003) and baculovirus displaying foreign protein is used for monoclonal antibody production (Saitoh et al.,2007), subunit vaccines (Peralta et al.,2007) and construction and screening of eukaryotic epitope library.

The display of proteins on the surface of bacteria requires a mechanism by which a desired, recombinant polypeptide: (a) is exported from the cytoplasm, a process typically involving the participation of the protein secretory apparatus of the cell, (b) is targeted to the outer membrane, and (c) can transverse the outer membrane so that it anchors to the external surface. Unfortunately, the mechanisms that dictate targeting and insertion of proteins within the outer membrane are not well understood. Moreover, the incorporation of aberrant proteins within the outer membrane can be toxic to the cell. Poor exposure of surface anchored heterologous polypeptide sequences, typically resulting from steric effects caused by the lipopolysaccharide layer on the outer membrane, can prevent the interaction of surface-displayed heterologous proteins with antibodies and small molecule ligands.

Baculovirus display strategies have also been used for modification of the viral surface to influence baculovirus-mediated transduction of mammalian cells (Kost et al.,2005). It has also been shown that membrane proteins produced in infected

also been shown that membrane proteins produced in infected Fig.9. Schematic diagram for the preparation of

Fig.9. Schematic diagram for the preparation of functional bacterial magnetic particles (BacMP) using the protein display technique. To express a functional protein onto BacMPs, the functional protein gene was fused to an anchor protein gene. A plasmid containing the fusion gene was transformed into M. magneticum AMB-1. Expression efficiency is strongly dependent on the expression level of the anchor protein. The functional BacMPs produced were extracted by disrupting cells. (From: Matsunaga et al. (2007) in TRENDS in Biotech

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insect cells can be incorporated into baculovirus particles in a functional form. This was first observed for the β-2-adrenergic receptor, which was recover in a functional form complexed with heterotrimeric G-proteins (Loisel et al.,1997) and more recently for the human leukotriene B4 receptor (Masuda et al.,2003). This approach has been used successfully to produce a functional γ-secretase complex on the surface of baculovirus particles (Hayashi et al.,2004). Co-infection of Sf9 cells with viruses produced virus particles with γ-secretase activity that was concentrated ~2.5-fold higher in the budded virus particles as compared to Sf9 cell membranes. These studies show that baculovirus particles can provide a unique scaffold for the assembly and enrichment of functional membrane bound protein complexes. Current trends in the development of nanoparticles such as quantum dots, gold particles, and magnetic particles recently have attracted research interest because of the uniqueness of their physical properties, and they have been used a immobilization support (Phadtare et al.,2004; Yang et al.,2004), probes (Akerman et al.,2002; Gao et al.,2004) or catalysts (Samuelson et al.,2002). These techniques have been achieved using anchor molecules to display foreign proteins on the surface of microorganisms. Foreign proteins also have been displayed on the surface of magnetic nanoparticles (Fig.9). Protein display on bacterial magnetic particles was realized by the development of a fusion technique involving anchor protein isolated from magnetic bacteria (Okamura et al., 2000). Recently, novel proteins tightly bound to Bacterial Magnetic Particles (BMPs) were discovered in T. Matsunaga’s laboratory (Yoshino and Matsunaga, 2006). These proteins were highly expressed in the lipid bilayer membrane covering the BMPs.

In addition very high levels of expression of luciferase through recombinant BmNPV in the larval caterpillar (10 mg recombinant protein per larva) were achieved, which resulted in the generation of ‘Glowing silkworms’ emanating significant luminescence on administration of the substrate luciferin (Motohashi et al.,2005). A number of studies have documented enhanced protein production following cotranfection with baculoviruses expressing chaperone proteins, which are know to aid in the folding and modification of newly synthesized proteins. The expression of correctly assembled shaker potassium channels in Sf9 cells was enhanced by coexpression of the calcium-binding, lectin-chaperone calnexin together with substitution of the polyhedron promoter with the weaker basic protein promoter to derive expression of the ion channel. Recent studies showed that co-transfected 3GnT2 of human origin fused with GFP (green fluorescent protein) marker protein as a model protein and hCRT (human calreticulin) as a model chaperone to assist in folding in endoplasmic reticulum (Park et al., 2008). These studies demonstrate potential value of co-expressing chaperones to enhance functional protein production.

Furthermore, the identification of new higher value functional nanoparticles displayed antibody using BmNPV bacmid bioproducts is a chance for short term successes in industrial biotechnology. Enzyme and protein engineering has the potential to create new biomolecules, metabolic engineering can contribute to develop new metabolic pathways, may be even for unnatural compounds. However, the efficiency and stability of proteins displayed on functional proteins have been limited. Hence the overall study required for development of strategies for displaying foreign peptides and proteins on virus particles and the insertion of mammalian cell active expression cassettes in baculoviruses to express genes efficiently into many different mammalian cell types (Sf-9 and Tn-5B1-4 cells). BmNPV engineered to display foreign peptides and functional proteins on the viral surface have been proven particularly useful as immunogens and surface nanoparticles display of fusion protein. The ability to observe and count the antigen-bound gold nanoparticles is a novel method and suggests that antibody- antigen binding was successfully observed at the molecular level, leading to high sensitivity. Protein- based nanoparticles conjugated with an antibody against a specific cellular antigen hold promise as selective drug delivery systems for specific cell types.

9. Medical Entomology

Applied entomology encompasses a broad range of research areas (agricultural, forestry, domestic and medicolegal, etc.). Medical and veterinary entomology (MVE) is a key eld that involves: (a)

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communicable diseases, including parasitic (malaria, trypanosomosis, leishmaniosis, lariosis, etc.), bacterial (plague, typhus, Lyme disease, etc.) and viral (yellow fever, dengue, hemorrhagic fever, West Nile, etc.) forms; (b) allergic phenomena; (c) pests; (d) envenomation protection. Entomology also deals with some domestic damage caused by insect pests (Dominique and Jean-Antoine 2004).

Arthropods—which are responsible for transmitting a broad range of diseases—have innitely varied behavioural patterns: blood feeding (by vectors and pests), inoculation of venomous toxins (envenomation by stinging by black ies, hymenopterans, ticks, scorpions or spiders), and passive transport of pathogens (anthrax, trachoma, salmonellosis). Moreover, epidemiological systems (metazoonosis) and intravectorial cycles (anterograde or posterograde digestive infestations in vectors of trypanosomosis, transovarian transmission of viruses and rickettsia) are highly complex.

In this context, MVE specialists require ‘broad-ranging scientic knowledge and extensive eld experience.’ Besides basic entomological training, they must have a full overall understanding of agents that are transmitted (parasites, bacteria, viruses), epidemiological cycles (pathogen systems) and concerned environments, in both natural ecosystems and ones changed by man (e.g. agrosystems). Medical and veterinary entomologists are specialised to deal with this extraordinary pathogenic and eco- epidemiological complexity (Geong, 2001; Mouchet and Bellec, 1990).

Chikungunya virus is geographically distributed in Africa (Jawetz and Adelberg’s, 2004; Power et al., 2007), Southeast Asia (Lam et al., 2001; Laras et al., 2005; Parola, et al., 2006) and India (Chandrakant and Pradhan, 2006; WHO Report, 2006). Sporadic cases are regularly reported from different countries in the affected regions (Power et al., 2007; Laras et al., 2005; Chandrakant and Pradhan, 2006; Halstead et al., 1969; Warner et al., 2006). Aedes albopictus (Fig.10) was implicated as a vector of the chikungunya virus (CHIK) disease of humans, the hallmark feature of CHIKV disease is a debilitating and prolonged arthralgic syndrome that primarily affects the peripheral small joints. The pain

associated with CHIKV infection of the joints typically persists for weeks or months causing serious economic and social impact on both the individual and the affected communities. The most recent epidemic re-emergences were documented by International Society for Infectious Disease’s Program for Monitoring Emerging Diseases and WHO in Kinshasa (1999–2000), Indonesia (2001–2003), the Indian Ocean islands of Mayotte, Mauritius, Re´union, and the (2005–2006), Singapore (2006), Hong Kong (2006), India (almost 1.4 to 6.5 million estimated cases in 2006–2007), Japan (2007) and Europe Spain, Gabon, Italy (Ravi, 2006; WHO Wkly Epidem Rec 2000; Shailendra, 2007; Queyriaux, et al., 2008). Globalisation, and the increased travel and trade that it brings, is also quickening the pace at which diseases can spread to new areas. In addition to the influence of increasing temperatures (Global warming) and rainfall through warming of the oceans, and alteration of the natural cycles that stabilise climate, one is inevitably drawn to the conclusion that Chikungunya virus will continue to emerge in new regions (Power et al., 2007; Shailendra, 2007; Tyagi, 2007).

(Power et al ., 2007; Shailendra, 2007; Tyagi, 2007). Fig.10 Aedes albopictus Scientifically, the large scope

Fig.10

Aedes albopictus

Scientifically, the large scope of the outbreaks has provided opportunities to accurately document transmission and epidemiological patterns associated with movement of the virus an interest in expanding basic and applied scientific knowledge. But, there is a critical lack of knowledge on the biology of CHIKV, contrasting with related model alphaviruses like Sindbis virus (SINV), Semliki Forest virus (SFV), and Ross River virus (RRV); this probably reflects the fact that CHIKV has mostly afflicted persons in developing countries (Marion Sourisseau et al., 2007). Currently, no effective therapeutic drugs or licensed vaccine is available for prevention against this pathogenic virus. Therefore, there is an immediate need for the research on CHIKV cirus, for an effective vaccine besides strengthening the existing diagnostic laboratory facilities.

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The genome of CHIKV consists of a linear, positive-sense, single-stranded RNA of approximately 11.8 kb, and contains structural genes that encode three structural proteins; E1 and E2 of envelope, and nucleocapsid protein (Samuell and Diamond, 2006). Viruses have developed amazing strategies to exploit cellular machineries like vesicular and protein trafficking, biosynthesis and sorting machineries. Alphaviruses attach to poorly characterized receptors on many different cell types in various species. Individual viruses have different, but wide tropism, accounting somewhat for different disease patterns. Recently, Cheu et al. (2006) has also dealt extensively with molecular characterization and analysis of the endocytic pathway mediating the infectious entry of mosquito-borne flavivirus West Nile Virus (WNV) in A. albopictus. Very interestingly, they have identified αVβ3 integrin served as the functional receptor for West Nile virus (WNV) entry into cells. Domain III (DIII) of WNV envelope protein (E) was postulated to mediate virus binding to the cellular receptor (Lee et al., 2006). Further, the results indicate that the WNV enters into the mammalian and mosquito cells C6/36 through clathrin-dependent endocytosis and viral particles colocalize with both early and late endosomes (Chu and Nag, 2004a; Chu and Nag, 2004b; Chu et al., 2006a,b).

This critical membrane fusion reaction is mediated by a virus transmembrane protein known as the fusion protein, which inserts its hydrophobic fusion peptide into the cell membrane and refolds to drive the fusion reaction (reviewed more extensively in Margaret Kielian, 2006). Interestingly CHIKV virus,

E1 envelope protein is a class II fusion protein that mediates low pH-triggered membrane fusion during

virus infection. The E2 envelope protein is a type I transmembrane glycoprotein and has been known to

be responsible for receptor biding during the course of alphavirus cycle (Byungki Cho et al., 2008;

Seema and Jain, 2005). So far, alphaviruses have been mostly studied in murine and other animal cells.

In particular, the interaction of CHIKV with mammalian and mosquito cells C6/36 cells has not been

extensively characterized. Further molecular characterization of receptor and ligand-receptor binding affinity by using modern-day techniques. The modulation of receptor gene(s) and/or protein(s) can be used as a method for interfering with virus entry and can thus become a new method for disease prevention. The importance of understanding the details of CHIKV virus entry-pathway by host cells

lies in its potential for exploitation in novel vector control strategies, and the molecular characterization

of (specific domain) the proteins involved has made the development of target oriented specific drug/

peptide for anti-vrial strategies a realistic possibility.

10. Integrated pest management

We are now at a stage where all individual techniques and tactics for insect pest management can be integrated and used in a system that combines the most appropriate characters of each to produce an ecologically sound and economically viable package, IPM. While several

definitions exist for IPM, to succeed it

is

critical to have detailed information

on

the biology of the pest and how it

interacts with the environment, natural enemies and the crop itself. IPM has been misconstrued for reducing pesticide inputs, but should be based on

a clear understanding of ecological

functioning of the agro-ecosystems (Fig.9). What is important is pest monitoring and accurate timing of pesticide treatment, use of selective insecticides as well as combined use of

Indian Council of Agricl. Research Indian Council of Agricl. Research Indian Institute of Agril. Science
Indian Council of Agricl. Research
Indian Council of Agricl. Research
Indian Institute of Agril. Science
Indian Institute of Agril. Science
Intl. Center for Semi-Tropical Research
Intl. Center for Semi-Tropical Research
DST, New Delhi
DST, New Delhi
CSIR, New Delhi
CSIR, New Delhi
DBT, New Delhi
DBT, New Delhi
Integrated Pest Management
Integrated Pest Management
Pheromone Pheromone Technology Technology Sterile Technology Sterile Technology Biological method Biological method
Pheromone
Pheromone
Technology
Technology
Sterile Technology
Sterile Technology
Biological method
Biological method
Molecular Target Orientated
Molecular Target Orientated
Pesticidal Method
Pesticidal Method
Insect Physiology
Insect Physiology
Insect Molecular Biology
Insect Molecular Biology
Developmental Biology
Developmental Biology

Fig.11. Schematic diagram showing the current scenario of Integrated Pest Management approaches.

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semiochemicals, host plant resistance, trap crops, oviposition deterrents and antifeedents to manipulate pest behaviour. Today the term stimulodeterrent diversionary strategy is used.

11. Concluding remarks

The ideal transgenic technology should be commercially feasible, environmentally benign (biodegradable), and easy to use in diverse agroecosystems as well as show a wide-spectrum of activity against the crop pests. It should also be harmless to the natural enemies, target the sites in insects that have developed resistance to the conventional pesticides, flexible enough to allow ready deployment of alternatives (if and when the resistance is developed by the pest), and preferably produce acute rather than chronic effects on the target insects (Sharma et al., 2000).

Transgenic crops have many of these requirements. Some of the criteria can be achieved by exploiting genes that are based on antibody technology (Hilder and Boulter, 1999). Single chain antibodies (ScFvs) can be used to block the function of essential pest proteins. The potential of plant expressed antibodies or antibody fragments to serve as insect control agents against nematodes, pathogens and viruses has also been described (Atkinson, 1993; van Engelen et al., 1994; Rosso et al., 1996). This approach of controlling insects would offer the advantage of allowing some degree of selection for specificity effects, so that pests, but not the beneficial organisms, are targeted. The development of a delivery system from transgenic plants to the insect haemolymph will remove a key constraint in the transgenic approach to crop protection. Incorporation of Bt genes will have a tremendous effect on pest management. We need to pursue the management strategy that reflects the pest biology, insect plant interactions and their influence on the natural enemies to prolong the life span of the transgenics. Refugia can play an important role in resistance management and should take into account the pest complex, the insect hosts and the environment. Expression of more than one gene (gene pyramiding) and single chain antibody genes, which would be compatible with the likely trends in pesticide discovery using biology derived target based methods. Emphasis should be placed on combining exotic genes with conventional host plant resistance, and also with traits conferring resistance to other insect pests and diseases of importance in the target region. Several genes conferring resistance to insects can also be deployed as multilines or synthetics.

We have only touched upon the fringes of various aspects of research and it is needless to emphasize that phenomenal progress has been made in all aspects of Entomology and with every passing day new techniques in molecular biology are emerging to improve crop resistance to pests. What holds greater promise for the millennium is the possibility of modifying pathways leading to semiochemical production, enhancing the possibility of biotechnological production of man made “smart molecules” preventing them from reaching their respective target tissues. This novel strategy can be applied to arrest the insect pest’s physiology and development, thereby controlling the pest population in a natural economical and eco-friendly way without using toxic pesticides. I may herein mention that one of the most controversial and interesting topics of today pertains to insects and climate change, with the assumption that global warming is centered on the increase of concentration of CO 2 which influences phytophagous pests. Rising levels of carbon dioxide have a potential to alter this state of terrestrial communities, changing the relative abundance of species and in the long run, the species complexes of local communities.

12. Acknowledgement

I would like to express my sincere thanks to Prof. Ada Rafaeli, Institute for Technology & Storage of Agricultural Products, ARO, Israel, for a critical reading of this article. The author also thanks to Prof. Justin Chu, NUS, Singapore and Prof. Y.E. Park, Shizouka University, Japan for their encouragement and updating my knowledge in the field of medical and applied entomology.

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Invited Review

Invited Review

Vol. (1), 00 – 00, 2009

Vol. (1), 21 – 48, 2009

ChapterChapterChapterChapter –––– 2222

Molecular Structure of Insect Circadian Clocks

Le Thi Dieu Trang, Qi-Miao Shao, and Makio Takeda* Graduate School of Agricultural Science, Kobe University, 1-1 Rokkodai, Kobe 657-8501, Japan.

Abstract

Circadian rhythms are observed in a wide range of organisms from cyanobacteria to insects as daily cycles of behavioral, developmental, physiological, or biochemical events that are controlled by endogenous oscillators. Drosophila melanogaster has contributed to molecular elucidation of circadian mechanism because of an ease for forward genetics approaches. Many clock genes have been identified and cloned from D. melanogaster. Basically, the circadian oscillation is regulated by interlocked feedback loops. Studies in other insects have shown that genes involved in circadian oscillation are conserved but they are not always shared the same structures or functions. Interestingly, some components are more closely related to those of mammals. In this review, we describe molecular structures of Drosophila circadian clock system in comparison to that of other insect species in detail. The description is not only on genetic aspects but also in sub-cellular localization. Similarities and discrepancies between Drosophila and other insects cast a new light for a diversity in the mode of the circadian oscillation among organisms. A diversity was found also among closely related species in neurological structure.

Key words: circadian clock; Drosophila; insects; oscillator system; input pathway; output pathway

*For Correspondence (email: mtakeda@kobe-u.ac.jp)

Overview

1. Introduction

2. Molecular structure of Drosophila circadian clock

(2a)

Molecular components of circadian pacemaker

(2b)

Molecular circuitry of circadian oscillator and

(2b.1)

roles of post-translational mediators Entrainment of two interlocked feedback loops in

(2b.2)

Drosophila Post-transcriptional regulation in the core

(2c).

mechanism of circadian oscillation Circadian clock input pathway

(2d).

Circadian clock output pathway

3. Non-drosophilid insects and divergent models of circadian system

(3a).

Core oscillator system

(3b).

Input pathway

(3c).

Output pathway

4. Localization of insect circadian clock components: all in their head?

5. Acknowledgements

6. References

Ths article has been scientifically edited by Chandrasekar R.

1. Introduction

Organisms living on this planet must cope with daily and seasonal changes in environment such as UV light, high or low temperature, high or low humidity and abundance or scarcity of food and natural enemies. To respond to these changes, a wide range of organisms employ endogenous clocks for temporal synchronization of life processes to environmental changes. The rhythms driven by these clocks free-run in constant environmental conditions with a period about 24 hours (Aschoff, 1960; DeCoursey, 1983; Pittendrigh, 1981; Saunders, 2002). The endogenous oscillation underlying these rhythms is thus called circadian oscillation; circa = about and dian = a day. The circadian oscillation has three fundamental features: (1) it free-runs in constant temperature, light or dark with a circadian period (τ); (2) it has a temperature compensation in τ at a wide range of temperatures; and (3) it can be entrained by Zeitgebers (Johnson et al., 2004). Functionally,

21

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Invited Review

the circadian systems consist of a central circadian pacemaker (CPM), an input and output pathways (Fig. 1). The input pathway transmits environmental signals to the CPM. Driven by the CPM, the output pathway affects developmental, physiological or behavioral events of the organisms. Several model systems have contributed to understanding of molecular structure of circadian oscillation such as the blue green alga Synechococcus, the bread mold Neurospora crassa, the higher plant Arabidopsis thaliana, the fruit fly Drosophila melanogaster and the mouse Mus musculus (Dunlap, 1999; Edery, 2000; Young and Kay, 2001). Although there are substantial differences among organisms, the central mechanism of circadian system is supported in most cases by a negative auto-regulatory feedback loop. Such a loop is highly conserved between mice and fruit fly (Allada et al., 2001; Stanewsky, 2002, 2003). However, recent studies on cyanobacteria demonstrated that phosphorylation-dephosphorylation of clock protein without transcription and translation processes could provide an alternative mechanism generating circadian rhythms (Tomita et al., 2005; Nakajima et al., 2005).

rhythms (Tomita et al., 2005; Nakajima et al., 2005). Fig.1 Three functional subunits of the circadian

Fig.1

Three functional subunits of the circadian clock system.

In the negative feedback loop, the transcription of clock genes is inhibited by their protein products interfering the transcription regulators that bind a transcription enhancer element. In N. crassa, the first cloned and best understood circadian clock gene is frequency (frq) (Feldman and Hoyle, 1973). Frq is the central component in circadian clock of N. crassa (Aronson et al., 1994a, 1994b), encoding two forms of FRQ proteins. white collar-1 (wc-1) and 2 (wc-2) encode the proteins that form a mono- or heterodimer to activate transcription of frq (Crosthwaite et al., 1997).

Clock components of insects may be conserved but the mechanisms of circadian oscillation may differ in each insect species. The PER/TIM complex translocates into the nucleus from the cytoplasm to function as transcriptional mediators during night in D. melanogaster (Saez and Young, 1996). Conversely, Antheraea pernyi PER-like signal was not strong in the nucleus of pacemaker cells throughout the night due to masking of PER nucleic degradation and /or nuclear export and/or cytoplasmic sequestration processes (Sauman and Reppert, 1996; Chang et al., 2003). Bombyx mori shows a clear circadian rhythm in hatching (Tanaka, 1966; Niino and Yoshitake, 1982), eclosion behavior (Shimizu, 1989) and photoperiodic response for diapause during egg and early larval stages

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(Shimizu, 1982; Sakamoto and Shimizu, 1994). Several clock components such as Bmper, Bmtim, Bmcyc, Bmdbt, BmCKs, Bmlark, and Bmcry were cloned and analyzed their expression (Markova et al., 2003; Takeda et al., 2004; Iwai et al., 2006; 2007, 2008; Trang et al., 2006).

2. Molecular structure of Drosophila circadian clock

(2a). Molecular components of circadian pacemaker

D. melanogaster has been a leading edge for a forward genetic approach to molecular mechanism of circadian oscillation. Many clock genes have been identified and cloned in this system and genetically analyzed for their functions (a review by Hall, 2003) (Table 1). period (per) is the first clock gene identified in D. melanogaster by Konopka and Benzer (1971) and the first cloned clock gene a decade later of all the organisms. Subsequently, other genes were discovered and they were grouped according

to the molecular nature of their products as transcriptional activators, transcriptional repressors, protein

stabilizers or subcellular localizers, or protein degradators (Hardin, 2005).

Circadian oscillation is a product of two interlocked feedback loops. CLOCK (CLK) and CYCLE (CYC) are the transcriptional activators of the first loop that contain basic-helix-loop-helix/ 2 PAS domains. They are critical for circadian rhythmicity and transcription of per and timeless (tim) (Rutila et al., 1998; Allada et al., 1998; Bae et al., 1998; Darlington et al., 1998). In both clk and cyc mutant flies, per transcription is severely reduced. CLK and CYC form a heterodimer and bind to a cis-regulatory sequence termed E-box to activate per and tim transcription. clk mRNA level cycles (peak at ZT23-4) in anti-phase to those of per and tim (ZT13-16) while cyc mRNA level is constantly expressed (Bae et al., 1998). PER/TIM represses their own transcription by interfering CLK/CYC. Furthermore, a basic-leucine zipper transcription factor, Par domain protein 1ε (Pdp1ε) that activates clk transcription by binding V/P box of Pdp1ε gene regulatory region (Cyran et al., 2003). CLK/CYC activates Pdp1ε transcription.

VRILLE (VRI) is a basic-leucine zipper transcriptional repressor (Cyran et al., 2003; Blau and Young, 1999; Glossop et al., 2003). vri has an E-box in its regulatory region and is transcriptionally activated by CLK/CYC. VRI represses clk transcription by binding V/P box of clk regulatory region. This is the second loop. PER contains PAS domains that mediates homodimerization (Huang et al., 1993) and heterodimerization with TIM (Gekakis et al., 1995). per mRNA is rhythmically expressed with an early peak at ZT13-16), while its product PER displays an peak lagging behind the mRNA cycle by 6-8h (ZT19-21) (Hardin et al., 1990; Zeer et al., 1990).

The second clock gene identified in D. melanogaster is called timeless. A null mutation of tim resulted in arrhythmic behavior as with per o (Sehgal et al., 1994). TIM is rhythmically expressed with a peak around ZT17-ZT19. TIM physically binds to PER forming a heterodimer to inhibit CLK-CYC function. Both PER and TIM contain a nuclear localization signal (NLS) required for nuclear entry and cytoplasmic localization domain (CLD) that promotes cytoplasmic retention. Remarkably, TIM also binds to the CLD region of PER (Saez and Young, 1996). A new clock component, clockwork orange (cwo) encodes a transcription factor synergizing with PER and inhibiting CLK-mediated transcription (Kadener et al., 2007; Lim et al., 2007).

DOUBLETIME (DBT) is a protein stabilizer or subcellular localizer that destabilizes PER. This is

a Drosophila homolog to a mammalian casein kinase Iε (CKIε), destabilizing PER and affecting its

subcellular localization (Price et al., 1998; Kloss et al., 1998; Cyran et al., 2005). CASEIN KINASE II (CKII) also destabilizes PER and makes a nuclear localization of PER (Lin et al., 2002; Akten et al., 2003). Another kinase is the glucose synthase kinase 3 (GSK3) homolog SHAGGY (SGG) that phosphorylates TIM to promote its degradation and the nuclear entry of PER/TIM (Martinek et al., 2001). In contrast to the destabilization effects of protein kinases, protein phosphatase 2a (PP2a) stabilizes PER via dephosphorylation (Sathyanarayanan et al., 2004), and PP1 stabilizes TIM from proteasome-mediated degradation (Fang et al., 2007). The protein degrader group includes the

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F-box/WD40 protein SLIMB (SLMB) which targets phosphorylated PER for degradation in the proteasome (Ko et al., 2002; Grima et al., 2002). A further study showed that Cryptochrome (CRY) not only functions as a photoreceptor for TIM degradation but also a transcription repressor for the oscillation of peripheral circadian clocks in Drosophila (Collins et al., 2006). Recently, the phosphorylation condition and stability of CLK were demonstrated to be controlled by DBT and protein phosphatase (Kim and Edery, 2007).

(2b). Molecular circuitry of circadian oscillator and roles of post-translational mediators

Circadian oscillator consists of mutual coupled autoregulatory loops by which rhythmic transcription of particular clock genes is controlled via feed backs from their own protein products. To synchronize transcriptional feedback, the levels and subcellular localization of clock proteins are regulated by post-transcriptional mechanisms.

(2b.1). Entrainment of two interlocked feedback loops in Drosophila

The molecular nature of the Drosophila clock was illustrated in Fig. 2. In the late morning, transcription of per and tim genes is activated by the CLK/CYC proteins heterodimer which binds to an E-box of per and tim genes to activate their transcription. Transcriptional activation results in increased per and tim mRNA with their peaks at the beginning of the night (Allada et al., 1998; Rutila et al., 1998; So and Rosbash, 1997). However, TIM reaches a maximum level at around midnight since it undergoes photodegradation by CRY-mediated ubiquination and proteasome while accumulation of PER in cytoplasm proceeds more slowly with the peak in the second half of the night (Marrus et al., 1996). This delay in PER accumulation is due to destabilization by DBT and CKII activities which phosphorylate PER. Subsequently, TIM stabilizes phosphorylated PER by forming PER/TIM heterodimer (Kloss et al., 1998; Price et al., 1998; Akten et al., 2003; Nawathean and Rosbash, 2004). PER is also stabilized by PP2a, as suggested to remove the phosphates added by DBT and CKII (Sathyanarayanan et al., 2004). The DBT/PER/TIM complex enters the nucleus upon SGG-dependent phosphorylation and DBT/CKII-dependent PER phosphorylation (Lin et al., 2002; Akten et al., 2003; Martinek et al., 2001; Kloss et al., 2001; Ashmore et al., 2003; Shafer et al., 2002; Cyran et al., 2005). Once in the nucleus, the complex binds to a CLK/CYC dimer that removes CLK/CYC from the E-box and inhibits per and tim transcriptions (Lee et al., 1998, 1999; Nawathean and Rosbash, 2004). PER appears to be a more effective inhibitor of CLK/CYC dependent transcription than PER/TIM dimmer (Ashmore et al., 2003; Shafer et al., 2002) consistent with the fact that TIM is degraded shortly after nuclear entry of the DBT/PER/TIM complex probably through the action of SGG and possibly by other tyrosine kinases (Zheng et al., 1996, Naidoo et al., 1999; Martinek et al., 2001; Hardin, 2005). The association between DBT/PER/TIM and CLK/CYC complexes probably directs and facilitates phosphorylation of CLK by DBT. PER and CLK are then destabilized via continuous DBT phosphorylation and degraded that forces the cycle to start all over again (Kloss et al., 2001; Ashmore et al., 2003; Shafer et al., 2002; Cyran et al., 2005; Kim et al., 2007; Nawathean et al., 2007).

This negative feedback loop is interlocked by another loop, in which the VRI regulates rhythmically the transcription of clk gene. In this loop, the CLK/CYC heterodimer binds to an E-box to activate transcription of vri and pdp1ε during the late day and early night (Cyran et al., 2003; Blau and Young, 1999; Glossop et al., 2003). VRI accumulates and binds to VRI/PDP1ε box (V/P box) to inhibit transcription of clk gene. Consequently, PDP1ε accumulates to high levels during the mid-to-late evening and activates clk transcription. Thus, clk mRNA cycles in anti-phase to per and tim transcrips. In contrast to the rhythmically regulated clk gene, cyc gene is constitutively expressed and CYC is abundant throughout the day. Therefore, CLK is the limiting factor for CLK/CYC heterodimer and dictates the numbers of dimers available for activating per and tim transcriptions. A similar feature of the per/tim and clk feedback loops is required for the CLK/CYC activation. Since rhythmic transcription of CLK/CYC activated genes requires a feedback by PER/TIM, the per/tim feedback loop is also needed for clk loop (Hardin, 2005). The clk loop also controls rhythmic transcription of cry, which encodes a circadian photoreceptor that functions as a clock component in some tissues (Stanewsky et al., 1998; Emery et al., 1998; Krishnan et al., 2001).

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Table 1.

Comparison of Drosophila and mammalian core clock components

 

Drosophila

Mammals

Gene name

Protein type

Function

Gene name

Protein type

Function

period (per)

Circadian, PAS protein. Transcriptional repressor.

Binds to TIM and inhibits CLK/CYC transcriptional activation.

period 1 (per1) period 2 (per2)

Circadian protein. PAS domain

Physical associates with CRY and inhibits CLK/BMAL1 –dependent transcription activation. Not binding to DNA, may be an indirect inhibitor. Possible clock output pathway Not clear in clock function

 

period 3 (per3)

Cyclic protein. PAS domain

timeless (tim)

Circadian protein. Transcriptional repressor.

Binds to PER to stabilize PER and inhibits CLK/CYC transcriptional activation. Binds to CYC to activate other clock gene transcription.

timeless (tim)

Constitutively expressed.

clock (clk)

Circadian, bHLH-PAS protein. Transcriptional activator. Q-rich

clock (clk)

Constitutively expressed.

Heterodimerizes with BMAL1 to activate other clock gene expression. Heterodimerizes with CLK to activate other clock gene expression. Associate with PER to stabilize PER and inhibit CLK/BMAL1 dependent transcription activation. Represses per1 transcriptional via DBP-reponse element. Activates per1 transcription via DBP-response element.

Transcription activator.

cycle (cyc)

Non-cyclic, bHLH-PAS protein. Transcriptional activator.

Binds to CLK to activate other clock gene transcription.

bmal1

Cyclic RNA. bHLH-PAS protein. Transcriptional activator. Cyclic RNA. Flavoprotein

crypochrome (cry)

Cyclic protein. Circadian photoreceptor. Flavin binding domain

Promote light-dependent degradation of TIM.

cryptochrome 1

(cry1)

 

cryptochrome 2

 

(cry2)

vrille (vri)

Cyclic protein. bZIP transcriptional repressor. Cyclic protein. bZIP-PAR transcriptional activator. Cyclic protein. Transcriptional repressor.

Represses clk transcription.

E4bp4

Cyclic RNA. bZIP transcriptional repressor. Cyclic protein. bZIP-PAR transcriptional activator.

PAR domain protein

Activates clk transcription.

d-element binding

1ε (pdp lε)

protein (dbp)

Clockwork orange

Synergizes PER to inhibit CLK/CYC transcriptional activation.

 

(cwo)

doubletime (dbt)

Non-Cyclic protein. Ser/Thr kinase.

Promotes degradation of PER and CLK via phosphorylation. Affects subcellular localization of PER and CLK.

Casein kinase 1ε

Non-Cyclic protein.

Affects PER stability via phosphorylation.

Ser/Thr kinase

 

(CKI ε)

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Casein kinase II α (CKIIα)

Non-Cyclic protein. Ser/Thr kinase.

Promotes degradation and affects subcellular localization of PER.

Casein kinase Iiβ

(CKII β)

Shaggy (sgg)

Non-Cyclic protein. Ser/Thr kinase

Phosphorylates TIM and enhances PER/TIM subcellular localization.

Protein

Protein phosphatase

Stabilizes TIM via dephosphorylation

phosphatase 1

(PP1)

Protein

Protein phosphatase.

Stabilize PER via dephosphorylation

phosphatase 2A

Cyclic RNA in twins (tws) and

(PP2A)

widerborst (wdb) regulatory subunits.

Glycogen

Drosophila sgg homolog

Not clear in clock function

synthase kinase

3β(SGK3β)

REV-ERBα

Nuclear receptor

Inhibitor of BMAL1 in nucleus. Links feedback loop.

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on Insect Molecular Biology, Vol.(1), 2009 Invited Review Fig.2 A schematic illustration of interlocked feedback loops

Fig.2 A schematic illustration of interlocked feedback loops in Drosophila. CLK/CYC heterodimer binds to E-box and activates transcription of per, and tim. PER is phosphorylated by DBT and CK2, leading to its degradation. TIM binds to, and stabilizes phosphorylated PER. PER is also stabilized by PP2A. TIM is phosphorylated by SGG and the TIM/PER/DBT complex enters the nucleus where the complex binds to CLK/CYC and removes them from E-box. It releases the transcription inhibition of clock genes. PER and CLK are then destabilized by DBT-mediated phosphorylation. Unphosphorylated CLK forms heterodimer with CYC and another cycle of per and tim transcription begins. Similarly, CLK/CYC binds to E-box of vri and Pdp1εto activate their transcription. Accumulated VRI binds to V/P box on the promoter region of clk gene to inhibit clk transcription. PDP1εis accumulated later that replaces VRI from V/P box and activates clk transcription. Solid lines with arrow: sequential steps in the feedback loops; solid lines with bold bar: inhibition steps in the feedback loops; wave line: per, tim, vri, Pdp1, or clk mRNA; dash lines: nuclear membrane.

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(2b.2). Post-transcriptional regulation in the core mechanism of circadian oscillation

In addition to transcription regulation, the post-transcriptional modifications contribute to stabilization of the circadian system (Curtin et al., 1995; Dembinska et al., 1997; Rosbash, 1997, Kim et al., 2002; and Shafer et al., 2002). The best-studied case is the temporally gated phosphorylation of PER and TIM (Kloss et al., 1998, 2001; Price et al., 1998; Scully and Kay, 2000; Martinek et al., 2001; Lin et al., 2002; Nawathean and Rosbash, 2004) and a light-dependent clearance of TIM by the CRY that synchronizes the clock with LD cycle (Stanewsky et al., 1998). SGG appears to phosphorylate TIM and accelerates the nuclear accumulation of PER (Martinek et al., 2001; Cyran et al., 2005) while two casein kinases, DBT and CKII, modify PER (Lin et al., 2002; Akten et al., 2003; Nawathean and Rosbash, 2004). Although data indicates that DBT as well as CKII directly phosphorylates PER (Kloss et al., 1998; Price et al., 1998; Lin et al., 2002), the possibility that one kinase regulates the activity of the other cannot be ruled out. At least in vitro experiments show that period-altering dbt mutations decrease the kinase activity of CKIs (Suri et al., 2000; Preuss et al., 2004). Phosphorylation targets cytoplasmic as well as the nuclear monomeric PER in the absence of TIM and influences its accumulation and degradation (Kloss et al., 1998; Price et al., 1998). Degradation proceeds through the ubiquitin-proteasome pathway and is stimulated by Slimb that interacts preferentially with phosphorylated PER (Grima et al., 2002; Ko et al., 2002). The PP2A keeps PER from DBT-mediated degradation by dephosphorylating PER (Sathyanarayanan et al., 2004). It is considered that the kinase activity delays the accumulation of cytoplasmic PER until lights turn off. Increased TIM levels results in stable DBT-resistant PER/TIM/DBT complex, which is translocated to the nucleus, where continued association of PER and TIM extends the cycle by interacting with CLK/CYC and repressing it for transcriptional activation. Following light-dependent degradation of TIM, the nuclear level of PER falls during the day that is most likely in response to phosphorylation-mediated degradation. Therefore, combined activity of protein kinases delays the nuclear accumulation of PER and TIM until their mRNAs reached the maximum levels. Moreover, the degradation of nuclear PER allows a resumption of per and tim transcription (Price et al., 1998; Rothenfluh et al., 2000; Suri et al., 2000; Kloss et al., 2001).

However, this model is revised by recent studies. PER was detected in the nucleus of lateral neurons at time when TIM was only cytoplasmatic (Shafer et al., 2002; 2004). Experiments using the D. melanogaster S2 cells have indicated that PER can translocate to the nucleus without TIM and repress transcription (Chang and Reppert, 2003; Weber and Kay, 2003; Nawathean and Rosbash, 2004; Cyran et al., 2005). In S2 cells, DBT together with CKII phosphorylate PER and therefore directly increase transcriptional repression activity of PER. The PER nuclear localization was suggested as an indirect effect by the association of phosphorylated PER with DNA or chromatin (Nawathean and Rosbash, 2004). The latest in vivo study in D. melanogaster confirmed that PER was translocated to the nucleus and repressed transcription in tim 01 null mutants only if DBT kinase activity was inhibited. Thus, DBT does not seem essential for transcriptional repression by PER in vivo. DBT not only stabilizes PER but also regulates subcellular localization of PER. Phosphorylated PER was retained in the cytoplasm, whereas unphosphorylated PER was accumulated in the nucleus (Cyran et al., 2005). Nuclear entry of monomeric PER was prevented by DBT-mediated phosphorylation. TIM tended to stay in the cytoplasm but SGG made TIM enter the nucleus and/or remain there. Although unphosphorylated PER does not required TIM for the nuclear entry, TIM inhibits DBT-dependent phosphorylation of PER (Kloss et al., 2001; Ko et al., 2002) and co-production of PER and TIM promotes nuclear accumulation of both proteins, that is supported by SGG-dependent phosphorylation of TIM (Cyran et al., 2005).

Not only PER, CLK was shown existing as differentially phosphorylated proteins (Bae et al., 2000; Kim et al., 2002; Lee et al., 1998). Recent studies suggested that PER brought DBT to the CLK/CYC complex, which led to CLK phosphorylation and suppression of transcriptional activation potential (Kim and Edery, 2007; Yu et al., 2006). The latest studies identified a conserved domain in PER that is important for in vivo interaction with DBT. This domain is absolutely required for PER hyperphosphorylation, nuclear localization and transcriptional repression (Kim et al., 2007; Nawathean et al., 2007). After nuclear entry of phosphorylated PER, CLK phosphorylation was accelerated by

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interactions between PER/DBT/CKII and CLK/CYC complexes (Nawathean et al., 2007). However, this domain could also act as a docking site for other factors beside DBT for inhibiting CLK/CYC dependent transactivation (Kim et al., 2007).

(2c). Circadian clock input pathway

As Zeitgebers, light intensity, temperature, food abundance, and social interactions could give a temporal cue to a circadian system. Light must be perceived by the photoreceptor system, which then alters the level of a cog of clock mechanism prone to photodegradation. This causes a phase-shift. If an animal is transferred from an LD cycle to constant darkness (DD), light pulses given during the subjective day cause little or no effect on phase, but those falling at early subjective night cause phase-delays and those falling at late subjective night cause phase advance (Yang et al., 1998; Suri et al., 1998; Konopka et al., 1989). The plot of a series of phase-shifts is called a phase response curve (PRC). PRC shape is conserved in almost all organisms either diurnal, nocturnal or crepuscular (Johnson et al.,

2004).

In Drosophila, several photoreceptor molecules are known, one of which could trigger the ubiquitin-proteasome dependent degradation of TIM via tyrosine phosphorylation, i.e., Crytochrome (CRY) (Naidoo et al., 1999). Depending on tim mRNA levels, photo-degradation restores different levels of TIM that affects PER level (Hunter-Ensor et al., 1996; Myers et al., 1996; Zheng et al., 1996; Lee et al., 1996). Altered PER/TIM levels may release the suppression of transcription activators, CLK/CYC. Late at night, light accelerates degradation of nuclear TIM, resulting in the release of CLK/CYC repression by the PER/TIM dimmer, so that the per and tim can be transcribed again. Then, translation follows, i.e., phase advance. In early evening, light reduces TIM accumulation, but tim mRNA levels are high so that TIM protein level is rapidly restored by translation, resulting in phase delays. During the subjective day, no phase shifting occurs. In Drosophila, at least three photoreceptor organs are known to affect the light dependent phase shift in behavioral system. They are (1) external photoreceptors in the compound eyes, (2) those of the ocelli, and (3) internal photoreceptors in the Hofbauer-Buchner eyelet. The blue light photoreceptors, CRYs are responsible photopigment proteins (Stanewsky et al., 1998; Emery et al., 1998). Unlike mammals, Drosophila has only one CRY species, CRY b that is not involved in rhythm generation per se, at least in the pacemaker neurons. CRY b is expressed in oscillator cells throughout the head (Klarsfeld et al., 2004) and plays an important role in TIM degradation in response to light. CRY b possesses a highly conserved photolyase domain and a unique carboxy-terminal domain (Cashmore et al., 1999). When activated by light, this C-terminal domain is suggested to alter position or release an inhibitor for exposing a TIM binding site (Rosato et al., 2001). It then binds TIM, and triggers its tyrosine phosphorylation and degradation by the proteasome (Naidoo et al., 1999). CRY is also degraded by light, albeit more slowly. However, the light-dependent TIM degradation mechanism of external and internal photoreceptors in neurons that control behavioral rhythms has not been elucidated yet.

A recent study suggests that internal photoreceptors which express Rhodopsin Rh6 (extraretinal

H-B cluster) and the ventral lateral neuron photoreceptor (circadian pacemaker) are not required for Drosophila larval response to light but have an exclusive role as a photoreceptor for circadian input

pathway (Hassan et al., 2005).

(2d). Circadian clock output pathway

In addition to the central mechanism based on transcriptional feedback loops, output pathway is

likely be regulated by clock dependent transcription of genes.

In Drosophila, pupal eclosion and adult locomotor activity depend on the lateral neurons (LNs).

The neuropeptide, pigment-dispersing factor (PDF) expressed within LNs of larvae and the small ventral lateral neurons (sLN v s) regulate the rhythmicity of both events (review by Stanewsky, 2002).

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Furthermore, a loss of function mutation in pdf gene results in short-period locomotor activity rhythms that gradually become arrhythmic in Drosophila adult (Renn et al., 1999). In the larval LNs and adult sLNv, cyc 01 and clk Jrk mutation affected pdf mRNA levels, suggesting a transcriptional regulation of the gene by CLK and CYC (Park et al., 2000a, Blau and Young, 1999). However, this regulation is probably indirect, since neither pdf mRNA levels are daily oscillating (Park and Hall, 1998) nor the E-box for expression is required in promoter region of pdf mRNA (Park et al., 2000a). In addition, PDF expression is post-transcriptionally regulated by PER, TIM and VRI. Thus, expression of pdf is both transcriptionally and post-transcriptionally regulated by clock genes (reviewed by Stanewsky, 2002).

PDF expressed in LNs must regulate the eclosion or/and locomotor centers, though so far, the mechanism remains to be understood. A potential downstream pathway, dependent on pdf and clock gene function is the Ras/MAP-kinase pathway via Neurofibromatosis-1 (Nf1) that encodes a Ras-GTPase activating protein. Mutation of Nf1 results in arrhythmic locomotor activity and an overactivation of MAP-kinase signaling (William et al., 2001). In pdf 01 mutations, levels of activated MAP-kinase are downregulated, suggesting a role of this signaling pathway in the output of circadian clock.

Another regulator of behavior rhythms is cAMP-dependent protein kinase A (PKA). Several studies show the arrhythmic behavior in PKA mutated flies in catalytic and regulatory subunits (Park et al., 2000b), and mutations in dunce, a gene encoding a cAMP specific phosphodiesterase, affect the cAMP signaling in the input and output pathway of neuronal pacemaker in various organisms, including mammals, probably due to an altered cAMP metabolism (Levine et al., 1995; Reppert and Weaver, 2002). The function of PKA in the output pathway seems specific for locomotor activity rhythms (Majercak et al., 1997).

takeout (to) provides a link between feeding behavior and the circadian clock. It is a member of a lipid-binding protein family including one that binds juvenile hormone (So et al., 2000; Claridge-Chang et al., 2001; McDonald and Robash, 2001). Like pdf, to is indirectly regulated by CLK and CYC. to requires no E-box for rhythmic expression, suggesting that other transcription factors, regulated by CLK and CYC, mediate its transcriptional regulation (So et al., 2000). A 3’-UTR deleted mutation of to probably produces an unstable transcript that leads to low TO protein levels (Sarov-Blat et al., 2000). These mutations show abnormal locomotor activity rhythms and die earlier than wild type flies under starvation.

Another clock output factor identified in D. melanogaster is lark. The null alleles of the lark gene had dominant effects on pupal eclosion rhythm but did not affect circadian period or the clock regulation of a distinct circadian rhythm in adult activity. However, increased lark gene dosage led to a late eclosion phenotype (Newby and Jackson, 1993; 1996). Molecular analysis of LARK indicates that it is an RNA-binding protein, which possesses two RNA recognition motif (RRM) along with a retroviral-type zinc finger (RTZF) (Newby and Jackson, 1996). This protein did oscillate in abundance at late pupal stage, even though its mRNA level did not (McNeil et al., 1998). Expression of LARK oscillated in crustacean cardioactive peptide (CCAP)-containing neurons within the subesophageal ganglion and the ventral nerve cord (VNC) of the third instar larvae and pharate adults (McNeil et al., 1998; Zhang et al., 2000). LARK/CCAP brain neurons were shown to project from larval and pharate adult brains to VNC; other neuritis terminated on esophageal muscles and in the prothoracic gland of late pupae; VNC neurons contain both LARK and CCAP that send projections posteriorly to gut muscles (Zhang et al., 2000).

In vertebrates, melatonin is widely accepted as a time-keeping hormone and an endogenous mediator of photoperiodic information (Reiter, 1993). Melatonin level of Drosophila exhibited a rhythmicity with a peak during day time (Hintermanm et al., 1996).

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3. Non-drosophilid insects and divergent models of circadian system

(3a) Core oscillator system

A vast and still growing knowledge on Drosophila genetics, anatomy, physiology and behavior has been great assets for biological clock research in other species.

In the Chinese oak silkmoth, Antheraea pernyi, per, tim, clk and cyc homologs have been cloned. The per rescued an egg hatching rhythm in the per 0 mutant of Drosophila. Based on S2 cell culture assays, a circadian feedback loop model was supported in which per transcript translocates to cytoplasm where it is translated into ApPER. ApPER enters the nucleus and inhibits ApCLK/CYC, thereby shutting off its own transcription (Reppert et al., 1994, Levine et al., 1005; Chang et al., 2003). However, ApPER and ApTIM were immunohistochemically detected primarily in the cytoplasm at all times of the day. It may be due to a high rate degradation in the nucleus and/or nuclear export and/or cytoplasmic sequestration of these proteins. In addition, antisense per RNA and perW, a homolog of about a half size of Apper were expressed in Antheraea female (Sauman and Reppert, 1996; Gotter et al., 1999; Chang et al., 2003).

Several homologues to Drosophila clock genes were identified in the commercial silkmoth

Bombyx mori such as Bmcyc, Bmper, Bmtim, Bmcry, Bmdbt, BmCKIα, BmCKIIα, BmCKIIβ , and Bmlark (Iwai et al., 2006; 2007, 2008; Markova et al., 2003, Takeda et al., 2004; Trang et al., 2006). At least three Bmper isoforms were isolated in B. mori genome with deletion/insertion sequence transcribed from one locus has been reported in PER of Apis cerana also (Shimizu et al., 2001). Bmper mRNA and BmPER showed a rhythmic expression in the brain (Takeda et al., 2004; Sehadova et al., 2004). Two casein kinase I, (BmCKIα and Bmdbt) were cloned from B. mori brain and analysed. Their putative proteins showed highly homologous to insect and vertebrate homologs of CKIε especially at ATP binding, catalytic domain and nuclear localization signal (NLS) (Trang et al., 2006). In situ hybridization revealed an identical localization of Bmdbt, bmCKIα, Bmper transcripts in the putative clock neurons identified by antibodies against CRY, PER, CKIα, CYC and arylalkylamine N-acetyltransferase (aaNAT) (Sehadova et al., 2004; Trang et al., 2006). Interestingly, the same cell that expressed high level of dbt, CKIα and per mRNAs also expressed high level of dbt, CKIα, and per antisense transcripts (Fig.3) (Trang et al., 2006). Following this study, two casein kinases II, CKIIα and CKIIβ, were cloned and analyzed for their expression (Iwai et al., 2008). In situ hybridization analyses indicated that the subunits were expressed in brain neurons expressing PER-like protein and surprisingly, antisense RNAs of CKIIα and CKIIβ were also detected in the putative clock neurons (Fig.3). The lateral brain neurons in

A. pernyi female also co-expressed per sense and antisense transcripts which are derived from a per

locus on the chromosome W and therefore presumably attributed to the output mechanisms controlling female-specific behaviors (Sauman and Reppert, 1996; Gotter et al., 1999). Antisense transcripts of unknown function have been described for the Neurospora clock gene, frq (Dunlap et al., 1995). A unique feature in B. mori is that the antisense signal was detected in both sexes and in the retina cells as well. The functional meaning of antisense signals is important for understanding circadian system but currently it is difficult even to speculate.

Levels of dbt mRNAs in the brain of B. mori showed no daily oscillation while CKIα mRNAs significantly decreased at ZT12 in both transcripts. The constant accumulation of CKI-like antigen in the cytoplasm of the clock neurons (Sehadova et al., 2004) may be caused by constitutive expression of DBT. Steady levels of dbt mRNA and DBT protein were observed also in D. melanogaster, but subcellular localization of Drosophila DBT oscillated (Kloss et al., 1998; 2001).

The honey bee, Apis mellifera is an interested organism for molecular analysis of circadian rhythms because its clock system is implicated in a set of complex behaviors included time memory, sun compass navigation, and dance language communication (von Frisch 1967; Moore-Ede et al., 1982). In

A. mellifera, putative homologs of per, tim, cry, cyc, clk, vri, and Pdp1 were cloned, and characterized

(Rubin et al., 2006). The structure of AmCYC and AmCLK is different from Drosophila but similar to

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the mouse and A. pernyi in which the main transactivation domain of the CLK/CYC complex is in the C terminal end of CYC. Amper and Amcry mRNA levels oscillate strongly with a similar phase, whereas in Drosophila they are almost in anti-phase (Rubin et al., 2006; Emery et al., 1998; Glossop et al., 2003). AmCRY and AmTIM lack domains and motifs thought to be essential for TIM and CRY function in the Drosophila clock. Furthermore, AmCRY is structurally similar to mammalian CRY and includes domains and motifs implicated in the transcription repressive function of these proteins (Rubin et al.,

2006).

repressive function of these proteins (Rubin et al., 2006). Fig.3. An illustration for expression of clock

Fig.3. An illustration for expression of clock genes and their proteins in the brain of B. mori (holometabola).

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(A). Co-localization of B. mori clock proteins. anti-sense signals.

(B). Co-localization of B. mori clock genes mRNA and

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In the migratory monarch butterflies Danaus plexippus, transcription of the per gene is under circadian control (Froy et al., 2003), probably via the activity of CLK/CYC heterodimer (Zhu et al., 2005). PER-ir exhibited a robust 24 hr rhythm that is under circadian control in the putative clock neurons and that is abolished by constant light (Sauman et al., 2005). Two CRYs were discovered in the monarch butterfly, one is Drosophila-type CRY and the other is mammalian-type CRY (Zhu et al., 2005). However, PER is not detected in the nucleus of clock cells in the monarch brain. It is therefore possible that the second CRY is an important transcriptional repressor for the central clockwork of monarch butterflies, as well as in other nondrosophilid insects (Reppert, 2006). The second CRY discovered recently is light insensitive and shares a common ancestor with the two mammalian CRYs that have potent repressive activity on CLK/CYC-mediated transcription (Zhu et al., 2005). The discovery of two distinct cry genes in the butterfly led to the recognition of the existence of two cry genes in several non-drosophilid insects. A Drosophila-type CRY was co-localized with PER and TIM in the dorsal lateral region and large neurosecretory cells in the pars intercerebralis (PI) of the monarch butterfly brain, suggesting this CRY acts as a blue-light photoreceptor for the circadian clock of monarch butterflies, as CRY does in Drosophila (Sauman et al., 2005; Stanewsky, 2003). However, the CRY-ir cells in the lobula region of the optic lobe do not contain detectable PER.

Molecular analyses of structures of circadian clock have been accumulated gradually in various insect species. per, tim, cyc, and cry have been cloned from the flesh fly, Scrcophaga crassipalpis (Goto and Denlinger, 2002); tim from the mosquito Aedes aegypti (Gentile et al., 2006); cyc from the saw fly Athalia rosae (Bembenek et al., 2007) and from the cricket Dianemobius nigrofasciatus (Shao et al., 2008). per from P. americana, and so on. Several other studies have demonstrated expression of clock proteins in brain of numerous insects included Periplaneta americana, M. sexta, D. nigrofasciatus, Allonemobius allardi which are described below.

(3b). Input pathway

Light-dependent input systems to circadian oscillator in most animals are distinct from the visual system and thus referred to as extraocular photoreceptors. In insects other than D. melanogaster, extraocular photoreceptors have been postulated for the regulation of the circadian rhythms as well as for photoperiodism (Truman 1976). The previous studies in A. pernyi (Williams et al., 1964, 1965; Williams 1969), the aphid, Megoura viciae (Lees, 1964; Steel and Lees 1977) and in the blowfly, Calliphora vicina (Cymborowski and Korf 1995) indicated dorsal protocerebrum as a region essential for photoperiodic reception. The most of CRY-positive cells in the cephalic ganglia of B. mori shows extensive overlap with the staining for several components of the core oscillator, suggesting for interactions of CRY with other clock proteins (Sehadova et al., 2004) as in D. melanogaster. Recently the cerebral opsin-photoreceptor has been cloned in B. mori and designated as a Boceropsin. Immunohistochemical localization of Boceropsin revealed several restricted clusters of cells in the central brain of Bombyx larvae (Shimizu et al., 2001). Besides this extraocular opsin, two additional opsins were isolated from the Bombyx compound eyes (Shimizu et al., 1998).

In Danaus butterflies, a Drosophila-type CRY was co-localized with PER and TIM in the dorso-lateral region and large neurosecretory cells in the pars intercerebralis (PI), suggesting this CRY acts as a blue-light photoreceptor for the circadian clock of monarch butterflies, as CRY does in Drosophila (Sauman et al., 2005; Stanewsky, 2003). However, the CRY-ir cells in the lobula region of the optic lobe do not contain detectable PER (Sauman et al., 2005). Ultraviolet (UV)-, blue- and extraretinal long-wave-length (LW)-sensitive opsins were also immunohistochemically examined in hawkmoths Manduca sexta, Acherotina atropos, Agrius convolvuli, and Hyppotion celerio. UV and blue opsins were expressed in the adult retina and stemata while LW opsin was distinctly expressed in dorsal and ventral neurons of the optic lobes. Together with adult stemata, the lamina, medulla, lobula and lobula plate, accessory medulla and adjacent neurons that exhibited strong LW opsin immunoreactivity were considered as extraretinal photoreceptors for detection of changes in ambient light (Lampel et al.,

2005).

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(3c). Output pathway

A homolog of Drosophila output factor, lark was identified in the head and other peripheral tissues

of B. mori. Bmlark had two types of transcripts. However, Bmlark mRNA failed to show its rhythmic

expression both in circadian timing and developmental stages (Iwai et al., 2007).

As in vertebrates, melatonin and aaNAT activity were detected in numerous insects and crustaceans. Melatonin level of the damselfly, Enallagma civile (Tilden et al., 1994) lacks rhythmicity, while that of prawn, Macrobrachium rosenbergii (Withyachumnarnkul et al., 1992) exhibits a peak during day. The compound eyes and brain of the locus Locusta migratoria (Viven-Roels et al., 1984),

the dragon fly, Ischnura verticalis (Tilden et al., 1994), moths, Trichoplucia ni (Linn et al., 1995), and

B. mori (Itoh et al., 1995) showed a peak of melatonin in the dark phase. P. americana also exhibits

melatonin-like labeling in several brain regions (Takeda et al., 1988) and NAT-like immunoreactivity were detected in some neuronsecretory neurons in A. pernyi (Takeda et al., 1999). L’Helias group (1995)

has shown a circadian rhythm of NAT in the brain and stemata of of Pieris brassicae. Melatonin content and aaNAT activity showed day/night fluctuation in P. americana in the brain, retina, hemolymph and peripheral tissues (Bembenek et al., 2005), though melatonin affects several physiological and

behavioral responses in insects. Melatonin administered in drinking water affected the locomotor activity of Acheta domesticus (Yamano et al., 2001). Both BmNAT and melatonin receptor signals were detected in the clock neurons including the bilaterally coupling system of optic lobe CPM neurons in

P. americana (Hiragaki et al.; Bembenek et al., in prep.)

4. Localization of insect circadian clock components: all in their head?

In Drosophila, many organs express circadian clock proteins to regulate specific physiological processes. Brain clocks are particularly important because they determine when animals rest or become active. PER-expressing neurons in Drosophila brain can be roughly classified into 3 groups, that is, dorsal neurons (DNs) located in the dorsal part of the protocerebrum, lateral neuron (LNs) located near the optic lobe, and a group of neurons located in the lateral posterior protocerebrum (LPNs) (Kaneko and Hall, 2000; Yoshii et al., 2005). The LNs are further classified into 4 categories according to their locations and molecular and cellular characteristics, that is 4 PDF-positive s-LNvs and a single PDF-negative s-LNvs (5 th s-LNv) with small cells located ventrally, about 5 l-LNvs with larger PDF-positive cell bodies located ventrally, and about 6 PDF-negative LNds located rather dorsally. The DNs are classified into 3 categories according to their location, that is, most dorsally located DN1s, 2 DN2s and dorsolaterally located DN3s. Molecular and cellular study studies revealed that the ventrally located LNs are responsible for driving the morning and a part of the evening peak while LNs plus the PDF-negative 5 th s-LNv are responsible for driving the rest of the evening peak (Rieger et al., 2006).

In D. melanogaster PER-, TIM-, CLK- and CYC-positive neurons are located in the LN as well as

in the DN (Helfrich-Forster, 2004). Two ventral groups of the LNs co-express PDF (Helfrich-Forster, 1995). The PDF-neurons show a wide fiber network on the surface of the optic lobe, connect both optic lobes and run into the dorsal protocerebrum. This arborization pattern makes them suited to transfer rhythmic signals to other brain areas, as well as to synchronize the cycling of all clock neurons.

The existence of a dorsal brain clock controlling rhythmic eclosion and flight activity was shown for the silk moths, Antherea pernyi and Hyalophora cecropia (Truman, 1972). Sauman and Reppert (1996) verified that PER-ir and per mRNA were both expressed in the cytoplasm of 8 neurons in the dorsolateral region of the protocerebrum. Both PER and per mRNA exhibits prominent circadian rhythms. PER protein peaked between ZT16 to ZT22 and disappeared between ZT 4 to ZT8. per mRNA level peaked between ZT14 to ZT22 and disappeared between ZT4 and ZT10. Sehadova and coworkers (2004) also showed that PER-ir, CYC-ir and DBT-ir were co-expressed in the 8 big neurons and some small neurons of the dorsolateral region of the protocerebrum of B. mori (Fig. 3). PER protein showed a robust oscillation with a peak level between ZT 8 to ZT16 and a low level between ZT 20 to ZT4.

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on Insect Molecular Biology, Vol.(1), 2009 Invited Review Fig.4 Distribution of clock proteins in the brain

Fig.4

Distribution of clock proteins in the brain of two related crickets (hemimetabola).

(A).

D. nigrofasciatus. (B).

A. allardi.

In crickets, the positions of the master clock are more divergent. Teleogryllus commodus has the CPM in the proximal optic lobe, while Gryllus bimaculatus the master clock may reside in the distal optic lobe (reviewed by Tomioka and Abdelsalam, 2004). On the other hand in A. domesticus, the pars intercerebralis was reported to be the locus of the circadian clock. The neurosecretory cells in this area

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show circadian rhythm in RNA and protein synthesis (Cymborowski and Dutkowski, 1968, 1969). Moreover, careful examination of the role of PDF neurons in G. bimaculatus in combination of partial removal of optic lobe with immunohistochemistry using anti-PDF antibody revealed that the PDFMe neurons located near the accessory medulla are not the clock neurons (Okamoto et al., 2001): The destruction of the distal area of the optic lobe without affecting PDFMe neurons eliminated the circadian locomotor rhythm. We have reported that the immunoreactivities to three clock proteins, Cryptochrome (CRY), PER, and Doubletime (DBT) differed substantially between two closely related cricket species, D. nigrofasciatus, and A. allardi. In D. nigrofasciatus, the CPM is located in the aMe, whereas in A. allardi the central brain and the suboesophageal ganglion (SOG) are the most likely loci of the CPM (Shao et al., 2006) (Fig. 4). While immunohistochemical results of two transcriptional factors CYC and CLK showed that SOG may play an important role in both the cricket species since they were colocalized in the SOG (Shao et al., 2008).

In Leucophaea maderae, transplantation of the optic lobe to an animal that had been arrhythmic by bilateral optic lobe removal restored circadian rhythm with a period close to that of the donor (Page, 1982). Stengl and her coworkers suggested that the accessory medulla area containing PDF -immunoreactive neurons lying at the ventromedial edge is the most likely locus of the cockroach circadian clock. The most convincing evidence for this notion is that the transplantation of the tissue into optic lobeless cockroach restored the locomotor rhythms in L. maderae (Reischig and Stengl, 2003). There are three clusters of PDF-immunoreactive neurons in the optic lobe of crickets and cockroaches, i.e., (1) near proximal medulla with innervation of accessory medulla (PDFMe), (2) ventrally and (3) dorsally near the outer chiasma (PDFLa). Stengl and coworkers stress that the PDFMe is the likely locus of the circadian clock (Stengl and Homberg, 1994; Reichig and Stengl, 2003). Rather indirect evidence includes that the rhythm reappeared in the optic stalk severed cockroaches in correlation with the regeneration of PDF-immunoreactive fibers from the accessory medulla to median protocerebrum (Stengl and Homberg, 1994). A similar correlation between the rhythm restoration and the PDF innervations was found when an AMe from a rhythmic donor was transplanted to an arrhythmic host cockroach (Reischig and Stengl, 2003). However, immunostainings with anti-Periplaneta PER antibody in P. americana were not perfectly consistent with this pattern: PER-immunoreactivity was observed in two clusters of small sized cells located dorsal and ventral to the outer optic chiasma in addition to the neurons in the pars intercerebralis and pars lateralis in the central brain (Takeda et al., 2000).

The locomotor activity rhythm of L. maderae is controlled by bilaterally symmetric circadian packmakers in the optic lobes (Nishiitsutsuji-Uwo and Pittendrigh, 1968a; Roberts, 1974; Sokolove, 1975; Page, 1982; reviews by Page, 1984; Chiba and Tomioka, 1987). In contrast to crickets, the cockroach pacemakers are more strongly coupled (Page et al., 1977; Wiedenmann, 1984; Ushirogawa et al., 1997). There is strong evidence that the accessory medulla (AMe), a small neuropil at the ventromedial edge of the medulla is the circadian pacemaker (Stengl and Homberg, 1994; Reischig and Stengl, 1996; Petri and Stengl, 1997). The AMe is densely innervated by neurites from PDH-ir neurons, which are circadian pacemaker candidates in insects (Homberg et al., 1991; Helfrich-Förster, 1995; reviewed by Helfrich-Forster et al., 1998). PDH-ir neurons contain circadian clock proteins, such as PER and TIM in D. melanogaster (Helfrich-Förster, 1995; Helfrich-Förster et al., 1998). A comparison of the morphology of PDH-ir neurons in crickets and cockroaches revealed that the numbers of PDH-ir commissures were correlated with the length of the period, and the strength of bilateral coupling via posterior optic commisure (Homberg et al., 1991; Stengl and Homberg, 1994). PDH-ir neurons mediate a coupling pathway between bilateral pacemakers in the cockroach. Recently, a backfill experiment by injecting HRP and dextran into one AMe, seven systems of commissures connecting both optic lobes were found; three connected both AMe and two matched the arborization pattern of PDH-ir neurons.

Various other insects including Archeognatha (bristletails), Zygentoma (firebrats), Ephemeroptera

(mayflies), Odonata (damselflies), Plecoptera (stoneflies), Caelifera (locusts), Blattaria (cockroaches),

Lepidoptera (butterflies), Trichoptera

(caddisflies) and other Diptera (flies) have been analyzed for localization of their clock neurons with

antibodies against PER and PDF (Frich et al. 1996; Zavodska et al., 2003b; Bloch et al., 2003). Other

Heteroptera (bugs), Coleoptera (beetles), Hymenoptera (bees),

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insects were examined exclusively for PER-positive neurons (Wise