The Fifth International Symposium on Molecular Insect Science

Vitellogenesis in Dipetalogaster maxima, a vector of Chagas’ disease, Dipetalogaster maxima

S.A. Aguirre, L. Garcia, S. Frede, L. Sosa, L.E. Canavoso and E.R. Rubiolo.

Departamento de Bioquímica Clínica, CIBICI-Conicet. Facultad de Ciencias Químicas, Universidad Nacional de Córdoba. Córdoba, Argentina. CP 5000. lcanavo@mail.fcq.unc.edu.ar

Hematophagous insects, like vectors of Chagas’ disease, must surpass a threshold level in terms of the amount and quality of the blood meal in order to successfully produce eggs. During this process, the fat body synthesizes the main egg protein, vitellogenin (Vg), which will be taken up by the ovary to be stored as vitellin (Vt). Vg and Vt have been isolated from several species and their levels are indicative of the efficiency of the oogenesis process. However, information about their regulation in Chagas’ disease vectors is still scarce. In this work, therefore, we have analyzed in anautogenous and autogenous females of Dipetalogaster maxima (Hemiptera:Reduviidae): a] the kinetic of Vg synthesis in fat body, b] Vg levels in hemolymph, and c] the stores of Vt in ovary. Vg synthesis and its levels in hemolymph were measured by western-blot and enzyme-linked-immunoassay (ELISA), respectively. The stores of Vt in ovary were analyzed by immunohistochemistry. In anautogenous insects the study was performed between 2–8 days post-ecdysis and between 2– 20 days post-blood feeding. In addition, autogenous females were studied between 2–15 days post-ecdysis. During the post-ecdysis period, anautogenous D. maxima showed decreased synthesis of Vg and concomitantly, low levels of Vg in hemolymph (5.5x10⁻³ μg/μl at day 4). After a blood meal, Vg synthesis in fat body and its levels in hemolymph increased significantly, reaching at day 20 post-feeding approximately 19 μg/μl. Histological and immunohistochemical studies of the ovaries were in agreement with the biochemical findings, especially the development of the tissue from day 2 post-blood feeding as well as the accumulation of Vt in developing oocytes. Autogenous insects displayed a pattern for Vg and Vt quite different compared with anautogenous females, characterized by a decreased Vg synthesis and a poor development of the ovary. The levels of Vg in hemolymph during the period post-ecdysis fluctuated between 1.5– 4.0x10⁻³ μg/μl. This was sufficient however to produce at least one batch of eggs. Supported by Secyt-UNC (E.R.R) and CONICET-Argentina (L.E.C).

The arginine vasopressin-like peptide and its receptor in Tribolium castaneum

Michael J Aikins¹, Khurshida Begum¹, David A. Schooley², Yoonseong Park¹

¹ Entomology, Kansas State University, Manhattan, Kansas. ypark@ksu.edu

² Biochemistry and Molecular Biology, University of Nevada, Reno, Nevada.

Arginine vasopressin and its related peptides are well known for their pivotal function in vertebrates for diuresis, reproductive, and neural functions. The arginine vasopressin-like peptide (AVPL) in insect was originally described in the locust Locusta migratoria, whereas a similar sequence is absent in the genome of higher dipteran insects including Drosophila, Anopheles, Apis, and Bombyx. Analysis of the genome sequence of Tribolium castaneum (Coleoptera:Tenebrionidae) in BeetleBase (http://www.bioinformatics.ksu.edu/BeetleBase/) identified a putative avpl gene and a gene encoding a novel G protein-coupled receptor as a putative receptor for the AVPL. A pair of cells on the ventral surface of subesophageal ganglion expresses the AVPL. We found that the monomeric form of AVPL is highly potent on the receptor expressed in a heterologous expression system, suggesting an authentic ligand-receptor interaction. Bioactivity of the AVPL was tested for its diuretic activity. Injection of the monomeric AVPL in the adult Tribolium induced immediate diuresis implying that the AVPL functions as a diuretic peptide in Tribolium.

Proteomics to identify odorant binding proteins (OBPs) and chemosensory proteins (CSPs) from the antennae and tarsi of Tribolium brevicornis

Taofic Alabi ¹ Frederic Francis¹ Edwin De Paw² and Eric Haubruge²

¹Functional and Evolutionary Entomology, Gembloux Agricultural University, Passage des Deportes 2, 5030 Gembloux, Belgium

² Mass Spectrometry Laboratory, University of Liège, BAT. B6 Chimie physique, allee de la Chimie 3, 4000 Liege 1, Belgium, haubruge.e@fsagx.ac.be

Chemoreception in insects is mediated by small soluble proteins that are abundantly present in the aqueous lymph of chemosensilla and that interact with odorant molecules and pheromones on their way to and from olfactory receptor. Two major classes of such proteins have been described: odorant binding proteins (OBPs) and chemosensory proteins (CSPs). A proteomic approach based on two-dimensional polyacrylamide gel electrophoresis (2-D PAGE), in which proteins are separated according to charge (pI) by isoelectric focusing (IEF) and according to size (Mr) by SDS-PAGE, was performed for the resolution of complex mixtures of proteins from antennae and Tarsi of Tribolium brevicornis. The proteins were then silver-stained and analysed by Matrix assisted laser desorption time of flight MS (MALDI-TOF) or by Electrospray (ESI) coupled with tandem Mass Spectrometry (MS-MS). Proteins from this Tribolium species was found to present sequence similarities to OBPs and CSPs recently discovery in several other insect orders. Development of proteomic studies was discussed in term of efficiency in functional and evolutional entomology.

Application of insect genomics in the identification of resistance mechanisms and novel target sites

A. P. Alves¹, M. D. Lorenzen ², R. W. Beeman ², and B. D. Siegfried¹

¹Department of Entomology, University of Nebraska – Lincoln, Lincoln, NE, USA, 68583-0816. anaalves@unlserve.unl.edu

²Grain Marketing and Production Research Center, ARS-USDA, Manhattan, KS, USA, 66502

Bacillus thuringiensis (Bt) is a valuable source of insecticidal proteins for use in insect pest control either in conventional spray formulations or in transgenic crops. However, the evolution of insect resistance in field populations is an important threat to this technology especially with transgenic plants that express the Bt toxins. The western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte, one of the insect pests targeted with Bt transgenic plants, has displayed an amazing capacity to develop resistance to most management strategies, including soil insecticides, behavioral resistance to crop rotation, and foliar adulticides. Therefore, a critical need exists for new and effective management options. The objective of this project is to develop a system to identify genes and pathways important as possible target sites and in conferring insecticide resistance to WCR. To conduct this study, disruption of selected genes will be obtained through RNA interference (RNAi), based on the synthesis and injection of gene specific double stranded RNA. To validate the RNAi technique in WCR, silencing of WCR laccase, a gene involved in cuticle tanning, was successfully obtained in our lab. Gene silencing can be visualized by sustained lack of pigmentation of injected larvae after molting. This research will provide the basis for conducting large-scale identification of genes related to insect resistance and to vital pathways representing potential insecticide target sites.

Regulation of genetically-based variation in juvenile hormone esterase activity in cricket, Gryllus assimil

A.N. Anand, E.J. Crone, A.J. Zera

School of Biological Sciences, University of Nebraska, Lincoln, NE 68588-0118, USA. aanand2@unl.edu

A key step in understanding the evolution of hormonal control is the characterization of genetically-based variation and co-variation of endocrine traits. Previously, natural genetic variation was identified in an enzyme juvenile hormone esterase (JHE). JHE degrades and partially regulates the titer of the key insect developmental hormone, juvenile hormone (JH). Artificial selection produced replicated lines differing 8 fold in hemolymph JHE activity in Gryllus assimilis. Current study was undertaken to identify the variable molecular and physiological regulators responsible for JHE activity differences between the lines. Analysis of fat body and mid gut tissues, known to express Jhe gene, revealed that higher hemolymph JHE activity in high line was specifically correlated to increased fat body JHE activity. Also, no difference in juvenile hormone epoxide hydrolase (another JH degrading enzyme) activities was observed between the lines in either tissue. Age profile for Jhe gene transcript levels was determined for the tissues during the last larval stadium. Higher Jhe gene transcript levels in high selected line can partially explain the higher hemolymph JHE activity. Work is in progress to determine whether difference in Jhe gene transcript levels is due to cis-linked (e.g. promoter) vs. trans-unlinked (e.g. neurohormonal) regulators, using cross and intercrossed inbred lines. This is the first study to look at the molecular and physiological causes of genetically-based variation in an endocrine regulator in natural populations.

Specific interactions amongst classic and Plus-C odorant binding proteins of the African malaria vector Anopheles gambiae

Evi Andronopoulou¹, Vassiliki Labropoulou¹, Vassilis Douris¹, Daniel F. Woods², Harald Biessmann³ and Kostas Iatrou¹

¹Insect Molecular Genetics and Biotechnology Group, Institute of Biology, National Centre for Scientific Research “Demokritos”, 153 10 Aghia Paraskevi Attikis (Athens), Greece. iatrou@bio.demokritos.gr

²Inscent Inc., Irvine, CA 92614, USA; ³Developmental Biology Center, University of California, Irvine, CA 92697, USA.

We discuss results from a comprehensive study undertaken to deduce interactions between various antennal proteins of the African malaria vector Anopheles gambiae with a specific focus on the interactions among odorant binding proteins (OBPs). From an initial screen for proteins that interact with a member of the Plus-C group of odorant binding proteins, OBP48, which is primarily expressed in female antennae and down regulated after a blood meal, a number of interacting proteins were identified, which included five classic OBPs and OBP48 itself. The interacting OBPs as well a number of other classic and Plus-C group OBPs that were not identified in the initial screen, were expressed in lepidopteran cells and subsequently examined for in vitro interactions in the absence of exogenously added ligands. Co-immunoprecipitation and chemical cross-linking studies suggest that OBP48 is capable of homodimerizing, heterodimerizing and forming higher order complexes with those examined examples of classical OBPs identified in the initial screen but not with other classical or Plus-C group OBPs that failed to appear as interacting proteins in the screen. The latter OBPs are, however, also capable of forming homodimers in vitro and, at least in the case of two examined classic OBPs, heterodimers as well. These results suggest a previously unsuspected potential of non-random combinatorial complexity that may be crucial for odor discrimination by the mosquito.

An RNAi-based study of the metabolic pathways for cuticle sclerotization and pigmentation in Tribolium castaneum

Y. Arakane ¹, J. Lomakin ², S. Muthukrishnan ¹, R. W. Beeman ³, S. H. Gehrke ², M. R. Kanost ¹ and K. J. Kramer ^{1, 3}

¹ Department of Biochemistry, Kansas State University, Manhattan, KS 66506. arakane@gmprc.ksu.edu

² Department of Chemical and Petroleum Engineering, University of Kansas, Lawrence, KS 660452

³ Grain Marketing and Production Research Center, ARS-USDA, Manhattan, KS, 66502 USA

Insect cuticle tanning (sclerotization and pigmentation) is a complex process that involves the production of quinones and quinone methides from catechols, followed by their oxidative conjugation with cuticular proteins, a process that leads to cuticle hardening and darkening. Using RNA interference (RNAi) methodology, we previously demonstrated that laccase 2 is the enzyme catalyzing cuticle tanning in the red flour beetle, Tribolium castaneum. By tblastn analysis of the Tribolium genome, conducted through Beetlebase, we identified several genes probably involved in the synthesis of catechols that are potential laccase 2 substrates. These genes include dopa decarboxylase (DDC), dopamine N-acetyltransferase (NAT) and aspartate □-decarboxylase (black). To further clarify the metabolic pathways responsible for cuticle tanning and to determine the influence of these genes and different catechols on sclerotization and pigmentation, double stranded RNAs (dsRNAs) for DDC, NAT and black were injected into Tribolium larvae and the resulting changes in morphology, pigmentation, and mRNA levels were determined. Finally, dynamic mechanical analysis was conducted to measure physical properties of elytral cuticle obtained from body color-mutant strains and dsRNA-treated insects. A metabolic pathway for Tribolium cuticle sclerotization and pigmentation will be presented. Supported in part by the National Science Foundation (MCB-0236039 and IBN-0316963).

Identification of the gene encoding laccase of the silkworm, Bombyx mori: Purification, analyses of cDNA sequence, expression pattern and recombinant protein

T. Asano, H. Yamazaki, and S. Izumi

Department of Biological Sciences, Tokyo Metropollitan University, Minamiohsawa 1-1, Hachioji city, Tokyo, JAPAN. asano-tsunaki@comp.metro-u.ac.jp

The laccase-type phenoloxidase (laccase) that is present in the cuticle matrix has different enzymatic properties from tyrosinase type phenoloxidase for melanin synthesis. It is thought that the laccase plays an important role in cuticle formation, since it catalyzes the oxidation of phenolic compounds such as N-acetyl dopamine and N-□-alanyl dopamine to corresponding quinones, which is regarded as the key process of the quinone-tanning for cuticle sclerotization. Though insect laccases have been purified from several species, little is known about their structures. Recently, cDNA encoding a protein (laccase-like oxidase) that has the catalytic domain specific to laccases from other organisms such as bacteria or plants was cloned from the tobacco hornworm, Manduca sexta. Furthermore, the RNAi studies of the red flour beetle, Tribolium castaneum, revealed that laccase 2 functions in hardening and darkening of the cuticle. However, the properties of their gene products have not been characterized yet at the protein level. To clarify the relationship between laccase protein and laccase genes, we purified laccase from the pupal cuticles of the silkworm, Bombyx mori and investigated its partial amino acid sequences by mass spectrometry. The tryptic fragments were assigned to those predicted from the laccase-like oxidase gene of M. sexta, and finally an EST clone of B. mori laccase was found in a database. This clone encodes 91 kDa protein that shows high similarity (75 ~ 90 %) with proteins encoded in laccase 2 genes from other insects. The expression pattern was also similar to those of other insect laccase genes. The high level of expression was detected just before the ecdysis. Considering that the laccase activity is detected after ecdysis, this observation indicates that laccase accumulates in the new cuticle as an inactive precursor form and is activated after ecdysis. In order to test this hypothesis, we are currently undertaking the analysis of recombinant laccase protein.

Small heat shock proteins of the silkworm, Bombyx mori

Y. Aso ¹, D. Sakano ², B. Li ^{1, 2}, Q.-Y. Xia ^{1, 2}, and H. Fujii ¹

¹ Faculty of Agriculture, Kyushu University, Fukuoka 812-8581, Japan. yaso@agr.kyushu-u.ac.jp

² College of Sericulture and Biotechnology, Southwest University, Chongqing 400716, China.

Alpha-crystallin is one of lenticular proteins in mammals and has a C-terminal beta-strands-rich domain. Small heat shock protein (sHSP) is a ubiquitous family of 15–42 kDa polypeptides having a similar C-terminal domain to that of the alpha-crystallin. sHSP has been recognized to play important roles in a variety of physiological events, although details have not yet been clarified. The most documented sHSP of insects is that from the Drosophila melanogaster, but little information on lepidopteran sHSP’s are so far available. Results presented here were from studies on sHSP’s of the silkworm, Bombyx mori. shsp19.9, 20.1, 20.4, 20.8, 21.4, and 23.7 cDNA’s encoded sHSP’s having molecular sizes of 19.9, 20.1, 20.4, 20.8, 21.4, and 23.7 kDa, respectively. sHSP21.4 was notably different from other sHSP’s, based on all results from examinations so far done. Deduced amino acid sequence of sHSP21.4 was similar to that of the D. melanogaster CG14207-PA (DmCG), whereas the sequences of other five were quite similar to each other. sHSP20.8 was highly similar to sHSP from the Indianmeal moth, Plodia interpunctella (PI). The occurrence and alignment of Cys residue was characteristic. Each of sHSP20.8 and PI had a N-terminal Cys, and these overlapped. Each of sHSP19.9 and 20.1 also had a C-terminal Cys, and these also overlapped. sHSP23.7 had three Cys residues; two in a Cys-Pro-Cys might play a role in oxido-reduction reaction. Neither sHSP20.4, sHSP21.4, nor DmCG had any Cys residues. The transcriptions of all the B. mori shsp’s were constitutive, and transcripts were widely distributed in a variety of tissues, although their amounts were low. A heat shock triggered an increase in transcription of a shsp except shsp21.4. Results from phylogenetic analysis also suggested that the B. mori sHSP’s are grouped into at least two classes: sHSP21.4 and other five sHSP’s.

Molecular characterization of genes associated with oogenesis and milk production in the tsetse fly (Glossina morsitans morsitans)

Geoffrey M. Attardo¹, Nurper Guz¹, Patricia Strickler-Dinglasan², Serap Aksoy¹

¹ Yale School of Public Health and Epidemiology, 606 LEPH, 60 College Street, New Haven, CT 06520. geoffrey.attardo@yale.edu

² Center for Marine Biotechnology, University of Maryland Biotechnology Institute

Tsetse flies (Glossina sp.) vector African trypanosomiasis, a disease for which over 60 million people are at risk in sub-Saharan Africa. Tsetse flies are viviparous and are unique in that the adult flies carry their young in utero for the duration of their embryonic and larval development. Pregnant flies supply their larvae with nutrients in the form of a “milk” substance secreted from a modified accessory gland. Flies give birth to a fully developed third instar larvae which pupate shortly after birth. This line of research focuses on the dynamics of two gene products associated with reproduction during the first and second gonotrophic cycles of the tsetse fly. The gene products studied are a previously identified putative yolk protein (gmYp) and a gene bearing homology to a protein found in tsetse “milk” secretions (gmMGP). Stage and tissue specificity of gmYp expression shows that the transcripts for this gene are exclusively in the reproductive tract of the fly at the same time as oogenesis is occurring suggesting that this gene is acting as a vitellogenic protein. Expression analysis of gmMGP shows that transcripts for this gene become detectable in parallel with larvigenesis. Transcripts for gmMGP are specific to the fat body and milk gland tissues. GMMGP protein appears in the mother during larvigenesis and is transferred to the larvae over the duration of pregnancy. Immunohistochemical analysis of GMMGP shows it to be exclusively found in the milk gland of the mother. Staining is throughout the gland and leads directly to its entrance to the uterus where the mouthparts of the larvae are positioned. These results indicate that these gene products are involved in tsetse oogenesis (gmYp) and larvigenesis (gmMGP). These genes can be used as markers for further studies in tsetse reproduction in regard to the effects of symbiosis and trypanosome infection.

Sex-specific expression mediated by the cis-acting control elements of the AeAct-4 gene of the yellow fever mosquito, Aedes aegypti

Diane H. Aw¹, Nijole Jasinskiene¹, Rosemary S Burton², Matthew J Epton², Hoang Kim Phuc², Guoliang Fu³, Thomas U. Berendonk2*, Aurora Ashykian¹, Osvaldo Marinotti¹, Luke Alphey^{2, 3} and Anthony A. James^1,4

¹Department of Molecular Biology and Biochemistry, University of California, Irvine, California 92697-3900, USA. njasinsk@uci.edu

²Department of Zoology, University of Oxford, South Parks Road, Oxford OX1 3PS, UK

³Oxitec Ltd., 71 Milton Park, Abingdon, OX14 4RX, UK

⁴Department of Microbiology and Molecular Genetics, University of California, Irvine, California 92697-3900, USA

*Present address: Universität Leipzig, Institut für Zoologie Molekulare Evolution und Systematik der Tiere, Liebigstr.18, D-04103 Leipzig, Germany

Various means of reducing populations of vector insects have led to decreases in transmission of the pathogens that cause diseases such as dengue fever and malaria. Recent approaches include the development of transgenic mosquitoes to express dominant conditional lethal genes whose expression is driven by stage- and sex- specific DNA control elements. The Aedes Actin-4 gene (AeAct-4) was demonstrated previously to be expressed only in female pupae of the yellow fever mosquito, Aedes aegypti. The AeAct-4 cis-acting control DNA was cloned into the Mos I mariner transposable element and used to drive expression of the DsRed reporter gene. While both male and female pupae transcribe some mRNA, only adult females were found to accumulate DsRed protein. This transcriptional and translational regulation may be exploited to use the AeAct-4 control sequences to drive expression of a lethal gene in mosquitoes.

The frontal ganglion in insect ecdysis: A novel early role for Crustacean cardioactive peptide

Amir Ayali

Department of Zoology, Tel Aviv University, Tel Aviv 69978, Israel. ayali@post.tau.ac.il

Chemical modulation is well established as an important factor in the generation and control of motor patterns and behavior. Insect ecdysis offers an attractive model for the intricate interactions between endocrine and neural control. The molt-related behavior is composed of a series of motor patterns whose timing must be precisely coordinated to ensure the proper execution of its vital outcome. A considerable amount of knowledge has accumulated on the role of peptidergic modulators in inducing and controlling the different motor patterns. Yet, the picture in different insect groups is far from complete. We focus on the locust frontal ganglion (FG) and the neuronal circuit(s) within it, as a previously unexplored target for ecdysis peptides and molt-related modulation. Our recently reported results suggest that the FG is a key player in insect molting. In our current study we show that Crustacean cardioactive peptide (CCAP), previously reported to trigger ecdysis motor patterns, induce a dose-dependent increase in the frequency of the FG rhythmic activity. The modulatory effects of CCAP on the FG motor circuit are dependent on behavioral state and physiological context; The FG shows maximal competence to CCAP modulation during the molt. We suggest that this context dependency is the result of ETH acting on the FG prior to CCAP. Pre-treatment of an isolated ganglion (dissected from a non-molting animal) with ETH cause the CCAP-induced effects to be similar to those induced by CCAP alone during pre-ecdysis. The CCAP induced excitation can be also demonstrated in the intact animal, where it is accompanied by the appearance of air bubbles in the locust gut. Early low hemolymph concentrations of ETH are thought to precede CCAP release in vivo. Our in-vitro and in-vivo results indicate on a novel role for CCAP in generating air-swallowing behavior during the early stages of ecdysis. This is also supported by distinct changes in the profile of CCAP immunoreactivity in tritocerebral CCAP cells and their axonal arborisation in the FG neuropile. The CCAP increase in these cells precedes its peak in abdominal ganglia. Finally, it was previously observed in the abdominal ganglia of Manduca and elsewhere that molt-related CCAP release is cGMP dependent. No elevation of cGMP immunoreactivity is observed in the tritocerebrum CCAP cells innervating the FG at the different stages of the locust molt.

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Characterization of insect EcR and USP in a mammalian cell culture system

Josh Beatty¹, Jenna Callender¹, Torsten Fauth ^1,2, Cary Weinberger¹, Margarethe Spindler-Barth², and Vincent C. Henrich¹

¹Biotechnology and Genomic Research Center. University of North Carolina-Greensboro, Greensboro, NC 27402-6170. jmbeatt2@hotmail.com

²Dept. of General Zoology and Endocrinology, University of Ulm, Germany

The functional ecdysteroid receptor in Drosophila melanogaster and other insects is a heterodimer composed of the ecdysone receptor (EcR) and Ultraspiracle (USP). Combinations of three natural EcR isoforms and USP fusion proteins have been tested in a mammalian cell culture system (Chinese hamster ovary) for transcriptional activity, ligand-binding, and binding to the canonical inverted repeat ecdysone response element (EcRE), hsp27. In this system, the transcriptional activity of a luciferase reporter gene whose promoter contains tandem repeats of the hsp27 EcRE is elevated by 10-fold or more in the presence of micromolar amounts of muristerone A or 20-hydroxyecdysone. Both natural EcR isoforms and USP constructs dictate the level of transcriptional activity in both the absence and presence of ecdysteroids, indicating that both hterodimeric partners play a role in determining the level of transcriptional activity. For instance, an USP construct lacking its DNA-binding domain maintains a normal and high level of activity with EcRB1, but activity is drastically reduced with EcRA and EcRB2, suggesting strongly that the nature of the heterodimeric interaction varies among the isoforms. Juvenile hormone and other analogues fail to elevate transcriptional activity on the cell culture assay, but in conjunction with ecdysteroids, these compounds reduce the maximal dosage of ecdysteroids by about tenfold. The capacity for JH potentiation varies among the EcR isoforms and different USP construct. Site-directed mutagenesis further indicates that some amino acid substitutions in shared regions of both EcR and USP differentially affect heterodimeric transcriptional activity, presumably because isoform-specific transcriptional complexes involve different interactions with EcR and USP. The diversity of EcR/USP capabilities is further suggested by differences in transcriptional activity among receptors from different species. Collectively, the mutations provide an experimental basis for assessing specific receptor functions.

Annotation of the Tribolium genome: A progress report

R.W. Beeman¹, Marcé D. Lorenzen¹, S. Richards², G. Weinstock², R. Gibbs² and S.J. Brown³

¹USDA-ARS-GMPRC, 1515 College Ave., Manhattan, KS, 66502, USA. richard.beeman@gmprc.ksu.edu

²Human Genome Sequencing Center, Baylor College of Medicine, 1 Baylor Plaza, Houston, TX, 77030, USA

³Division of Biology, Kansas State University, Manhattan, KS, 66506, USA.

The 165 Mb euchromatic genome sequence of Tribolium castaneum consists of 420 scaffolds, containing >95% of a sample of known genes. We have anchored 80% of the sequenced genome to the linkage groups at ~1 cM resolution using SSCP-based recombinational mapping. Alternative sets of gene predictions derived from Genscan, Augustus, FgenesH, Geneid, NCBI, and Ensembl have been compiled at the Human Genome Sequencing Center, and a Glean statistical analysis and aggregation of these sets has been conducted (Aaron J. Mackey, U. Penn.). Currently the gene count is approximately 16,000 genes, of which ~ 9,500 have supporting evidence from both the Ensembl (run at the HGSC) and NCBI Gnomen annotation pipelines. The genome sequence and associated annotation tracks can be viewed in the Genboree genome browser (www.genboree.org) developed at the Baylor College of Medicine, and any desired sequences can be retrieved either directly or via links to NCBI. These computer predictions form a preliminary annotation that will be further amended and refined manually. Annotation teams have been organized for several gene families. Genome tiling and gene transcript arrays are being developed at several locations to facilitate the genome annotation and to support gene expression studies. RNA interference and transposon-mediated mutagenesis will help elucidate functions of predicted genes. BeetleBase (http://www.bioinformatics.ksu.edu/BeetleBase/) will be the final repository

Cytochrome P450 genes in insects–an embarrassment of riches

May Berenbaum

University of Illinois at Urbana-Champaign. maybe@uiuc.edu

The genes encoding cytochrome P450 monooxygenases, heme-thiolate enzymes that catalyze the NADPH- associated reductive cleavage of oxygen to produce a functionalized product and water, constitute one of the largest superfamilies known, with over 5000 members identified and named (http://drnelson.utmem.edu/CytochromeP450.html). Although some P450 families are found in a wide variety of taxa, others are more restricted in distribution and accordingly are thought to have more specialized functions. The CYP6 family is unique to the Class Insecta and together with the CYP4 family represents half of the 90 P450s characterized in the genome of Drosophila melanogaster (Tijet et al. 2000). To date, CYP6 genes in Lepidoptera have been implicated exclusively in metabolism of xenobiotics and are thought to be particularly important in metabolism of plant chemicals in this almost exclusively phytophagous group. How they have evolved and diversified, however, remains an open question. Possible mechanisms underlying this diversification are described for the subfamily CYP6B within the genus Papilio, the swallowtail butterflies, which radiated in the context of the toxic furanocoumarin-containing host families Rutaceae and Apiaceae.

Gene expression in sensory tissues of the malaria mosquito, Anopheles gambiae

H. Biessmann¹, Q. K. Nguyen² D. Le¹, R. Justice³ and M. F. Walter¹

¹Developmental Biology Center, University of California, Irvine, California 92697, USA. hbiessma@uci.edu

²Department of Biological Chemistry, University of California, Irvine, California 92697, USA

³Inscent, Inc, 17905 Sky Park Circle, Irvine, California 92614, USA

Female Anopheles gambiae mosquitoes respond to odors emitted from humans to find a blood meal, while males are nectar feeders. This complex behavior is controlled at several levels, but is probably initiated by the interaction of various molecules in the antennal sensilla. Important molecules in the early odor recognition events include odorant binding proteins (OBPs), which may be involved in odor molecule transport, odorant receptors (ORs) that are expressed in the chemosensory neurons and odor degrading enzymes (ODEs). To obtain a better understanding of the expression patterns of genes that may be involved in host odor perception in females, we have generated a custom microarray to study their steady state mRNA levels in chemosensory tissues, antennae and palps. Microarray results were validated by quantitative RT PCR. Our study identified several OBPs that are expressed at significantly higher levels in antennae and palps of females vs. males, while others showed the opposite expression pattern. Most OBPs are slightly down-regulated 24 hrs after blood feeding, but some, especially those with higher expression levels in males, are up-regulated in blood fed females, suggesting a shift in blood-fed females from human host seeking to nectar feeding. qRT PCR determinations of OBP mRNA levels in early and late pupae as well as in heads of 1 day-old and 4 day-old females and males also detected significant shifts in developmental expression profiles. Polyclonal antibodies against some OBPs have been generated that recognize the native proteins in mosquito heads. Supported by the U.S. Public Health Service grant AI051485 and by Inscent, Inc, Irvine, CA.

A cell culture system and infectious clone for the study of Rhopalosiphum padi virus

S. Boyapalle¹, R. Beckett², N. Pal¹, W.A. Miller², and B.C. Bonning¹

¹Department of Entomology Iowa State University, Ames, IA, USA. bonning@iastate.edu

²Department of Plant Pathology, Iowa State University, Ames, IA, USA

Rhopalosiphum padi virus (Dicistroviridae) (RhPV) is an icosahedral aphid virus with a 10 kb positive-sense RNA genome. We screened lepidopteran, dipteran and homopteran cell lines for susceptibility to RhPV following RNA transfection and observed CPE in homopteran cell lines derived from the glassy winged sharp shooter, Homalodisca coagulata, and the corn leaf hopper, Dalbulus maidis. Infection, viral replication and production of virions was confirmed by northern blot hybridization, RT-PCR, western blot analysis and immuno-electron microscopy. Full-length cDNA clones of RhPV were synthesized. RNA transcripts produced from one of the clones were infectious following transfection of the susceptible cell lines. Infection was confirmed by CPE and immuno-electron microscopy. Virions were purified from infected cells and fed to bird cherry-oat aphids, Rhopalosiphum padi. Aphids tested positive for infection by the RhPV clone by RT-PCR, western blot analysis and immuno-localization by light microscopy, two weeks after acquisition in three replicate experiments. The cDNA clone of the RhPV genome was inserted into the genome of Autographa californica multiple nucleopolyhedrovirus to create the recombinant baculovirus AcRhPV6. Expression of the RhPV genome in Sf21 cells resulted in formation of RhPV virus-like particles (VLPs) whose capsids are structurally and immunologically indistinguishable from the native virions. The presence of genomic RhPV RNA in recombinant baculovirus infected cells and in VLPs was confirmed by RT-PCR. Assembly of RhPV VLPs in the nucleus of baculovirus infected cells suggests that in Sf21 cells (1) both the 5′ and IGR IRES of RhPV are active, (2) the virus encoded protease is functional for processing of RhPV polyproteins, and (3) replication of RhPV is not required for encapsidation of RNA. For analysis of the infectivity of baculovirus expressed RhPV6, virions purified from baculovirus-infected Sf21 cells were fed to R. padi. Aphids were tested for infection by the baculovirus-produced RhPV clone by RT-PCR and western blot analysis, four weeks after acquisition. Baculovirus expression of RhPV in lepidopteran cell lines that do not support replication of RhPV provides a potential alternative approach for in vitro production of clones of this virus.

Important interactions of Bacillus thuringiensis toxins with membrane receptors and their role in insect resistance

A.Bravo, I.Gomez, L.Pardo, C.Muñoz, C.Pérez, L.Fernandez and M.Soberón

Departamento de Microbiología Molecular, Instituto de Biotecnología Universidad Nacional Autónoma de México, Cuernavaca Morelos, Mexico. bravo@ibt.unam.mx

The insecticidal proteins produced by Bacillus thuringiensis (Bt), Cry toxins, are used to control insect pests. The primary action of Cry toxins is to lyse midgut epithelial cells by forming lytic pores in the apical membrane. Cry toxins are modular proteins comprised of three domains, domain I is involved in pore formation and domains II and III in receptor interactions. Here we summarize recent findings on the Cry-receptor interactions and their role in toxin action. Cry toxins interact sequentially with multiple receptors. In lepidopteran insects, Cry1A toxins interact initially with a cadherin-receptor. This interaction involves three different epitopes and promotes a final proteolytic processing of the toxin that induces the formation of a 250 kDa pre-pore structure, which has been suggested to be the responsible for the ionic pore-formation. The pre-pore showed 200 fold higher affinity to the second receptor, a glucosyl-phosphatidil-ionositol (GPI)-anchored Aminopeptidase N, this interaction leads to the insertion of oligomeric toxin into membrane microdomains (or lipid rafts) inducing cell swelling and insect death. Some authors suggested that a GPI-anchored Alkaline phosphatase could also participate in driving the pre-pore to lipid rafts. In addition, recent data shows that not only protein receptors are involved in Cry toxin interaction with membrane of susceptible organisms since certain glycolipids have a role in toxin action. These glycolipids are specific for insects or nematodes. The role of all these interactions in promoting insect resistance is of interest. In the case of mosquitocidal Cry toxins, we found that GPI-anchored proteins are also involved in binding Cry11Aa toxin. The case of mosquitocidal Bt toxins is very interesting since Bt subsp. israelensis (Bti) produces two different type of toxins, Cry and Cyt proteins, which together show a synergistic effect in their toxicity. In addition, no-resistance to Cry toxins has been selected in the presence of Cyt1A and Cyt1A overcomes insect resistance to different Cry toxins. We found that the molecular mechanism of synergism involves interaction of these two toxins and we identified the specific epitopes involved in this interaction. We will present data that show that Cyt1A synergizes or suppresses resistance to Cry toxins by functioning as membrane bound receptor. Bti is a highly effective pathogenic bacterium as it produces a toxin and also its functional receptor in the same crystal inclusion, promoting toxin binding to the membrane and avoiding the generation of insect resistance.

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Function and evolution of a mosquito salivary protein family

E. Calvo, B.J. Mans, J.F. Andersen and J.M. Ribeiro

Section of Vector Biology, Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases/NIH, 12735 Twinbrook Parkway, Rockville, MD 20852, USA. ecalvo@niaid.nih.gov

Saliva of blood-sucking arthropods contains a complex and diverse mixture of antihemostatic, antiinflammatory, and immunomodulatory compounds. The D7 salivary family of proteins is abundantly expressed in blood-feeding Diptera and is distantly related to the odorant-binding protein super family. In mosquitoes, two subfamilies exist, the long and short D7 proteins. Ticks and kissing bugs evolved salivary lipocalins that act as efficient scavengers of biogenic amines, and a similar function was postulated for the D7 proteins. Accordingly, we expressed the five members of the small D7 family of the African malaria vector Anopheles gambiae and a D7 long form from Aedes aegypti and showed by isothermal microcalorimetry, a modified and very sensitive non-equilibrium chromatography/spectrum distortion method, and by smooth muscle bioassay that four of these five short D7 proteins and the D7 long form bind serotonin with high affinity, as well as histamine and norepinephrine. The nonbinding D7 protein is poorly expressed in the salivary glands and appears to be on the path to becoming a pseudogene. Scavenging of host amines would antagonize their vasoconstrictor, platelet-aggregating, and pain-inducing properties. It appears that counteracting biogenic amines is of strong adaptive value in the convergent evolution of arthropods to hematophagy. This adaptation has been solved independently in ticks, bugs, and mosquitoes by co-option of either member of the lipocalin or, as shown here, by the odorant-binding protein families. This work was supported by the Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health.

The short neuropeptide F-like receptor from the red imported fire ant, Solenopsis invicta

Mei-Er Chen and Patricia V. Pietrantonio

Department of Entomology, Texas Agricultural Experiment Station, Texas A&M University, College Station, TX 77843-2475, USA. p-pietrantonio@tamu.edu

In invertebrates, neuropeptide F (NPF) peptides share structural similarity with vertebrate neuropeptide Y, which regulates food consumption, circadian rhythms, anxiety and other physiological processes. The insect neuropeptide F receptors belong to the G protein-coupled receptor (GPCR) rhodopsin family. We have cloned the fire ant, Solenopsis invicta Buren (Hymenoptera: Formicidae), putative short NPF receptor using PCR and RACE methods. The complete 2185 bp cDNA encodes a 387-residue protein with a predicted GPCR seven transmembrane region structure. We propose that the sequence of the honey bee short NPF receptor, that has not yet been annotated, encodes a protein of 393 residues. In fire ant mated queens, receptor transcripts were detected in the brain, midgut, hindgut, Malpighian tubules, fat body and ovaries. The highest transcriptional expression was found in the brain. The downregulation of the fire ant short NPF receptor transcriptional expression in the brain with starvation suggests that the short NPF signal transduction cascade may play a role in feeding regulation in fire ant mated queens. Arch. Insect Biochem. Physiol. 61: 195-208, 2006.

Eph-ephrin interactions establish a midline boundary for migrating neurons during the formation of the enteric nervous system in Manduca sexta

T.M. Coate¹, T.L. Swanson¹, A. Nighorn², and P.F. Copenhaver¹

¹Dept. Cell & Developmental Biology L-215, Oregon Health & Science University, Portland, OR 97239. coatet@ohsu.edu

²Program in Neuroscience and Arizona Research Laboratories, Div. of Neurobiology, University of Arizona, Tucson, AZ 85721

Eph receptor tyrosine kinases and their ephrin ligands modulate the guidance of many developing neurons in the vertebrate nervous system, but overlapping expression patterns and promiscuous interactions among multiple ephrins and Ephs have hindered a functional analysis of specific ligand-receptor pairs in vivo. As an alternative strategy, we have investigated the role of ephrin-Eph interactions in the control of neuronal migration within the developing enteric nervous system (ENS) of the moth, Manduca sexta. During the formation of the ENS, a population of ~300 neurons (EP cells) migrates along a set of identified muscle band pathways on the midgut while avoiding adjacent interband regions. We have shown that the EP cells express a single GPI-linked ephrin (MsEphrin), which can be detected in their filopodial processes as they explore the midgut surface. Concurrently, the midline interband regions of the midgut (which are inhibitory to migration) express MsEph, the sole Eph receptor homologue in Manduca. Blocking endogenous MsEph receptors in cultured embryos with soluble MsEphrin-Fc fusion proteins induced abnormal midline crossing by the neurons and their axons. In contrast, treating the EP cells with soluble MsEph-Fc proteins inhibited their migration and outgrowth without inducing midline crossing. These results indicate that the expression of MsEph by the midline cells of the midgut normally prevents ectopic growth by the migratory EP cells across this interband boundary. They also suggest a novel role for reverse signaling via a GPI-linked ephrin ligand in the control of neuronal guidance. Previous in vitro studies have suggested that several different non-receptor tyrosine kinases (NRTKs) may be activated during reverse signaling by GPI-linked ephrins, but validation of these observations in vivo has been lacking. We are currently investigating the extent to which Src-family kinases and other NRTKs are coupled to MsEphrin-mediated reverse signaling in the EP cells as a mechanism for controlling the motile behavior and guidance of these neurons within the developing ENS.

Paratransgenesis: constructing the enemy within

R. E. Collier¹, C.Husseneder¹, L. Foil¹, R. Cooper² and F. Enright²

¹Department of Entomology, Louisiana State University Agricultural Center, Baton Rouge, LA 70803. rcollier@agcenter.lsu.edu

²Veterinary Science, Louisiana State University Agricultural Center, Baton Rouge, LA 70803

Paratransgenesis is the genetic manipulation of a host’s symbiotic microorganisms to achieve an array of objectives, ranging from disease eradication to control of the host organism. The application of paratransgenesis is promising in social insects because social interactions promote the exchange of microbes between colony members. Within the social insects, termites are known not only for their ecological and economical importance but for their close relationship with microbial symbionts. The hind gut of the Formosan subterranean termite (FST) provides a refuge for an array of protozoa and bacteria that fulfill important functions in the survival of their hosts, such as cellulose digestion. These symbionts are excellent tools and targets for paratransgenesis, which this study aims to employ in the control of FST. The goal of this study is to genetically engineer termite specific indigenous gut bacteria to secrete peptide toxins to target and kill the termite’s protozoa, which are responsible for the digestion of cellulose into metabolites subsequently utilized by the termite host. Without their protozoa and a supply of nutrients the termite dies. This study has shown that defaunation of termite guts using Metronidazole is followed by starvation and death of the termite host within six weeks. Peptide toxins were screened in vitro and in vivo for protozoicidal activity. In vitro tests on anaerobic cultures of protozoa confirmed activity of the peptides against the termite protozoa targets. In vivo tests using microinjection resulted in defaunation of the termite gut within 72hr of treatment with the selected toxins. The genes of the detrimental toxins have now been synthesized and a gene-shuttle system is presently being constructed to deliver, express and secrete the selected genes into the termite hind gut. The gene-shuttle is currently being optimized for inducible expression within the recently characterized termite specific anaerobic, gram positive bacterium Pilibacter termitis. The optimization of the gene-shuttle will ensure that environmental impacts are minimized and efficient colony level control of FST is achieved.

A normal role for a nasty protein: the insect homologue of the Amyloid Precursor Protein (APP) regulates neuronal migration during embryonic development

P. F. Copenhaver, T. L. Swanson, L. M. Knittel, T. M. Coate, S. M. Farley, and M. A. Snyder

Department of Cell & Developmental Biology L-215, Oregon Health & Science University, Portland, OR 97239. copenhav@ohsu.edu

APP-like protein (APPL) is the insect homologue of vertebrate APP, a transmembrane protein that is aberrantly cleaved to produce the amyloid peptides associated with Alzheimer’s Disease. While both APP and APPL are abundantly expressed during neural development, their normal functions remain controversial. We have investigated the potential role of APPL as a neuronal guidance receptor in the enteric nervous system (ENS) of Manduca sexta, a preparation that permits manipulations of developing neurons in vivo. During the formation of the ENS, an identified set of neurons (EP cells) migrates along pre-formed muscle band pathways before extending axons and terminal synapses onto the gut musculature. We found that Manduca APPL (MsAPPL) is strongly expressed by the EP cells during their migration, while post-translational processing and trafficking of the holoprotein coincides with specific phases of neuronal differentiation. Provocative work has shown that human APP can regulate the heterotrimeric G protein Go□ in cell culture, suggesting that it may act as an unconventional G protein-coupled receptor. We found that Manduca Go□ co-immunoprecipitates with MsAPPL; the two proteins also co-localize within the leading processes of the EP cells, suggesting that they may directly interact. Activating Go□ within the EP cells inhibited their motility in a calcium-dependent manner, supporting the model that Go□-coupled receptors may mediate the response of migrating EP cells to inhibitory guidance cues. To test whether MsAPPL might act as such a receptor, we inhibited its expression in the developing ENS with antisense morpholinos; this treatment resulted in aberrant, ectopic migration of the EP cells onto the interband regions of the gut (normally inhibitory to migration). Treating the ENS with synthetic ectodomain fragments of MsAPPL (designed to interfere with its endogenous ligands) caused similar effects. Together, these results suggest that MsAPPL may act as a Go□-coupled receptor for one or more guidance cues that normally prevent the EP cells from migrating into inappropriate regions. We are currently testing whether MsAPPL regulates Go□ activation in the EP cells, and we are using an expression cloning strategy to screen for potential ligands of MsAPPL in the ENS. Supported by NIH R56 AG025525 and a grant from the Oregon Partnership for Alzheimer’s Research.

Microevolution of endocrine regulation: Jhe transcript abundance underlies genetic variation in JHE activity

Erica J. Crone, Anurag Anand and Anthony, J. Zera

School of Biological Sciences, University of Nebraska at Lincoln, 1104 T St, Lincoln, NE 68588. ecrone2@unl.edu

Juvenile hormone acting in concert with the steroid hormone 20-hydroxy ecdysone is responsible for many essential developmental processes in insects. Many studies have shown that juvenile hormone levels in the hemolymph are under tight control, both by biosynthesis of the hormone and degradation of free hormone by the specific esterase juvenile hormone esterase (JHE). Hemolymph JHE activity from a species of field cricket, Gryllus assimilis, has been used as a model to study the microevolution of an endocrine trait. Selection for elevated or decreased hemolymph JHE activity showed that hemolymph JHE activity differences are heritable in this species. However, this and other work on the evolution of endocrine traits has primarily focused on the study of biochemical and physiological aspects and not the underlying molecular mechanism(s) controlling the microevolution of these traits. This study is attempting to address this deficit using hemolymph JHE activity in G. assimilis as a model. We have begun by looking for evidence that the heritable differences in hemolymph JHE activity are due to differential transcription of the Jhe gene. Evidence documents that transcript levels in the fat body and mid gut, tissue and hemolymph enzyme activity and an internal Jhe gene marker are correlated on one developmental day in two separately selected blocks of lines. This study shows that differences in Jhe transcript abundance are responsible for the heritable differences in hemolymph JHE activity in the selected lines. It is the first study to document the molecular basis for naturally occurring genetic variation in a hormonal regulator. Heritable differences in hemolymph JHE activity are also seen in a wing polymorphic species, Gryllus firmus, the molecular basis for these differences are also under investigation.

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The cloning of one putative octopamine receptor and two putative serotonin receptors from the tobacco hawkmoth, Manduca sexta

Andrew M. Dacks¹. Joel B. Dacks², Thomas A. Christensen¹, Alan J. Nighorn¹ 1

¹Arizona Research Laboratories, Division of Neurobiology, The University of Arizona, Tucson, Arizona 85721, U.S.A. adacks@email.arizona.edu

²Department of Biological Sciences, The University of Calgary, Calgary, Alberta, T2N 1N4 Canada.

Serotonin (5HT) and octopamine (OA) are biogenic amines that are active throughout the nervous systems of insects, affecting sensory processing, information coding and behavior. As an initial step towards understanding the modulatory roles of these amines in olfactory processing we cloned one putative OA (MsOAR) and two 5HT receptors (Ms5HT1,R and Ms5HT1ijR) from the moth Manduca sexta. Phylogenetic analysis confirmed that MsOAR shares significant sequence homology with OA receptors previously characterized in the cockroach and the bee, but shows much less similarity to putative OA/tyramine receptors from the moths Bombyx mori and Heliothis virescens. Using the MsOAR sequence, fragments encoding putative OA receptors were obtained from 6. mori and H. virescens, and this analysis grouped these receptors in a separate clade with other identified tyramine receptors. Collectively, our results indicate that MsOAR is likely the first OA receptor cloned from a lepidopteran species. Ms5HT1jR and Ms5HT1jiR were both similar to 5HT1-type receptors from other invertebrate and vertebrate species but differed from each other in their N-terminus and 3^rd cytoplasmic loop. Ms5HT1,R was nearly identical to a 5HT receptor from H. virescens and MsSHTInR was almost identical to a 5HT receptor from B. mori. The sequences for homologs of Ms5HT1,R from B. mori and Ms5HT1,iR from H. virescens were also obtained and phylogenetic analysis of these data confirmed that the Lepidoptera likely have at least two 5HT1-type receptors.

Molecular Identification of bursicon neurons in central nervous system of the tobacco hawkmoth, Manduca sexta

Li Dai¹, Lisa Pitts², Hans-Willi Honegger², and Michael E. Adams¹

¹Departments of Entomology and Cell Biology/Neuroscience, University of California, Riverside, CA 92521, USA. lidai@ucr.edu

²Department of Biological Sciences, Vanderbilt University, Nashville, TN 37235-1634, USA.

During post-eclosion, adult insects undergo sequential processes of wing expansion, sclerotization and melanization under hormonal control. Bursicon, a key neurohormonal regulator of these behaviors, is highly conserved in the Insecta. Recent reports characterize bursicon as a pburs/burs heterodimeric cysteine knot protein in Drosophila melanogaster. We show the presence of two predicted proteins encoded by genes Mas-burs and Mas-pburs in Manduca sexta. in situ hybridization with Mas-burs and Mas-pburs DNA probes and immunohistochemistry with bursicon antibodies were used to label neurons, which express bursicon in the CNS of pharate larvae, pupae and adults. During development, the morphology and number of bursicon-expressed neurons in ventral ganglia changes during transitions through larva to pupa to adult stages. A cluster of intrinsic cells was identified in corpora cardiaca labeled only by pburs-specific DNA and antibody probes, and an additional pair of lateral cells in several abdominal ganglia were labeled only by a burs antibody probe. Using a recombinant bursicon protein, we observed that the pure hormone has dual functional roles in both wing expansion and tanning in Manduca sexta.

Strategic expression of conserved ion transport peptide gene products in central and peripheral neurons of insects

Li Dai¹, Dusan Zitnan², and Mike E. Adams¹

¹Depts. of Entomology and Cell Biology/Neuroscience, University of California, Riverside, CA 92521, USA. lidai@ucr.edu

²Institute of Zoology, Slovak Academy of Sciences, Dúbravská cesta 9, 84506 Bratislava, Slovakia.

Structurally related ion transport peptides (ITP) and crustacean hyperglycemic hormones are increasingly implicated in diverse metabolic and developmental functions in arthropods. We have identified a conserved ITP gene encoding two peptides in Manduca sexta, Bombyx mori and Aedes aegypti: A C-terminally amidated ion transport peptide (ITP) and C-terminally unblocked ITP-like peptide (ITPL). In silico genomic DNA analysis indicates the ITP gene is conserved in other insect species. These peptides are expressed in two, regionally distinct neuronal populations. Mas-ITP expression is confined to the brain in five pairs of lateral neurosecretory cells (Type Ia2) projecting ipsilateral axons into the retrocerebral complex and 3-4 pairs of adjacent small lateral cells with extensive arborizations within the brain. Expression of Mas-ITPL is comparatively weak in the brain, but strong in the ventral ganglia and peripheral nervous system, where MasITP is absent. Mas-ITPL occurs in multiple bilaterally paired cells in the thoracic ganglia and one bilateral pair in each abdominal ganglion (AG1-9). In the peripheral nervous system, we find strong Mas-ITPL expression co-localized with crustacean cardioactive peptide (CCAP) in peripheral abdominal neurons (L1) of abdominal segments 2–7, which project axons into the transverse neurohemal nerves. Expanded cellular expression of MasITPL during metamorphosis consists of two additional pairs of small lateral neurons in the brain, and one pair of medial cells in each abdominal ganglion AG3-6. A similar pattern of ITP and ITPL expression was observed in the central and peripheral nervous systems of Bombyx mori and Schistocerca americana. These distinctive cellular expression patterns suggest that ITP and ITPL have evolved specialized physiological functions in arthropods.

Isolation and characterization of a bombyxin-Like cDNA from Manduca sexta

L.J. D’Amico^{1, 2}, H.F. Nijhout¹

¹Dept. of Biology, Duke University, Box 90338, Durham, NC 27708, USA

²Dept. of Biology, Northeastern University, 360 Huntington Ave. Boston, MA 02115, USA. l.damico@neu.edu

To fully understand the regulation of growth and size in an organism requires knowledge of the cellular mechanisms regulating growth processes and how those mechanisms are integrated at the organismal level with nutritional, metabolic, and endocrine factors. The insulin signaling pathway has been implicated as an important regulator of the growth of developing tissues as well as the overall size of an organism. Understanding the interaction between insulin signaling and the systemic regulation of growth requires a model organism amenable to the molecular study of insulin signaling, as well as characterization of the systemic regulation of body size. Here we describe the isolation of a cDNA from the tobacco hornworm, Manduca sexta, encoding a protein with similarity to the insect insulin-like neurohormone, bombyxin. A combination of degenerate PCR and 3′ and 5′ RACE yielded a 300bp amplicon sharing 58% identity at the protein level with a previously identified Agrius bombyxin-related peptide. Subsequent real-time PCR showed that transcript abundance increased in response to carbohydrate injection. Comparative homology modeling is used to generate a predictive 3D structure of our Manduca bombyxin. The work presented here is an important first step in developing tools for the molecular analysis of insulin signaling in an ideal lepidopteran research model. Research supported by a NSF Graduate Research Fellowship to LJD, and NSF award IBN0315897 to HFN.

Temperature mediated modulation of vector competence of Aedes aegypti by influencing gut flora and intrinsic factors

Dileep N. Deobagkar^1,2 Anjali D. Apte-Deshpande², Mandar S. Paingankar¹

¹Department of Zoology, University of Pune, Pune -411007, INDIA. dndeo@unipune.ernet.in

²Institute of Bioinformatics and Biotechnology, University of Pune, Pune -411007, INDIA

Serratia sp. was found to be the common gut bacterium of Aedes aegypti larvae isolated from different natural environments and laboratory reared colonies. It was also retained in the guts of emerging adults by transtadial transmission. When Serratia was incorporated in blood meal along with dengue virus, there was statistically significant increase in number of susceptible Ae. aegypti females. We observed that Serratia specific 40 KDa polypeptide (P40) interacted with gut brush border membrane fraction containing dengue viral receptor component of Ae. aegypti. These interactions were further confirmed using dissected gut from Ae. aegypti females. P40 identified to be a periplasmic membrane component of Serratia thus seems to enhance the dengue viral interaction in the midgut of mosquito. We are attempting to use this microbe as a vehicle for paratransgenesis. Interestingly, the unique feature of this bacterium was its survival in the midgut of Aedes at higher ambient temperature. Expression of P40 was also enhanced at higher temperature. Higher temperature thus could assist Serratia mediated enhancement of viral susceptibility and therefore, vector competence of Ae. aegypti. Temperature is also known to modulate EIP as well as vector gene expressions. Using Ae. aegypti and Ae. albopictus derived cell lines we found that dengue-2 virus binding was enhanced on exposure to higher temperature. Temperature as an extrinsic factor thus can enhance the vector competence by modulating intrinsic factors including receptor gene regulation and also gut bacterium like Serratia sp.

Anopheles anti-Plasmodium defense responses

George Dimopoulos

Dept. Molecular Microbiology and Immunology, Johns Hopkins School of Public Health, Baltimore, MD 21231, USA. gdimopou@jhsph.edu

Transmission of malaria requires successful completion of complex interactions between the Anopheles vector and the Plasmodium parasite. These interactions involve mosquito immune and other physiological responses to the invading ookinetes and other components of infected blood, and accurate execution of Plasmodium’s gene expression program that directs its developmental transitions and interactions with the vector. Major obstacles are encountered in the midgut tissue, where most parasites are killed by the mosquito’s immune system. Understanding the molecular interactions taking place between the malaria parasite and mosquito vector is essential for the development of malaria control strategies based o Plasmodium blocking in the mosquito. So far, most studies have focused on the rodent parasite model, Plasmodium berghei, which is more amenable to experimental procedures. A comparative analysis of A. gambiae transcript responses to midgut invasion of P. berghei and P. falciparum ookinetes showed broad variations and have identified factors that can modulate infection levels of both or only one of the two parasite species. Invasion by P. berghei had a more profound impact on the mosquito transcriptome, including a variety of functional gene classes, while P. falciparum elicited a broader immune response at the gene transcript level. Ingestion of human malaria-infected blood lacking invasive ookinetes also induced a variety of immune genes, including several anti-Plasmodium factors. Seven of 12 tested genes were found to influence mosquito resistance to both parasite species. An MD2-like receptor, AgMDL1, and an immunolectin, FBN39, showed specificity in regulating only resistance to P. falciparum, while the antimicrobial peptide Gambicin and a novel putative short secreted peptide, IRSP5, were more specific for defense against the rodent parasite P. berghei. While all the genes that affected Plasmodium development also influenced mosquito resistance to bacterial infection, four of the antimicrobial genes had no effect on Plasmodium development. The defense against the two Plasmodium species is mediated by antimicrobial factors with both universal and Plasmodium-species specific activities. The mosquito is capable of sensing infected blood constituents in the absence of invading ookinetes, thereby inducing anti-Plasmodium immune responses.

angaGEDUCI: Anopheles gambiae gene expression database with integrated comparative algorithms for identifying conserved DNA motifs in promoter sequences

Sumudu Dissanayake¹, Osvaldo Marinotti¹, Jose Marcos C. Ribeiro² and Anthony A. James^1,3

¹Department of Molecular Biology and Biochemistry, University of California, Irvine, CA 92697. omarinot@uci.edu

²Laboratory of Malaria and Vector Research (NIH/NIAID), Rockville, MD 20852

³Department of Microbiology and Molecular Genetics, University of California, Irvine, CA 2697

The completed sequence of the Anopheles gambiae genome has enabled genome-wide analyses of gene expression and regulation in this principal vector of human malaria. These investigations have created a demand for efficient methods of cataloguing and analyzing the large quantities of data that have been subsequently produced. The organization of genome-wide data into one unified database makes possible the efficient identification of spatial and temporal patterns of gene expression, and by pairing these findings with comparative algorithms, may offer a tool to gain insight into the molecular mechanisms that facilitate such expression patterns. We developed a publicly-accessible database and integrated data-mining tool, angaGEDUCI, that unifies 1) stage- and tissue-specific microarray analyses of gene expression in An. gambiae at different developmental stages, and temporal separations following a bloodmeal, 2) functional gene annotation, and 3) promoter sequence comparison algorithms. The database can be used to study genes expressed in particular stages, tissues, and patterns of interest, and to identify conserved promoter sequence motifs that may play a role in the regulation of such expression. The database is accessible from the address http://www.angaged.bio.uci.edu. The combination of gene expression, function, and sequence data in the angaGEDUCI database streamlines spatial and temporal pattern-finding and produces a straightforward means of developing predictions and designing experiments to assess how gene expression may be controlled at the molecular level.

Caste-based differences in gene expression in the polyembryonic wasp Copidosoma floridanum

David M. Donnell and Michael R. Strand

Department of Entomology, University of Georgia, Athens, GA 30602 USA. donnell@bugs.ent.uga.edu

The polyembryonic parasitoid Copidosoma floridanum produces two morphologically and behaviorally distinct larval castes, soldiers and reproductives, during development within its host. Soldier larvae defend the brood against competitors while reproductive larvae develop into adult wasps. In this study we used a bi-directional suppression subtractive hybridization (SSH) approach to isolate differentially expressed genes of the two larval castes of C. floridanum. We isolated 230 novel expressed sequence tags (ESTs) from the two subtractions (114 soldier / 116 reproductive ESTs). Among these ESTs were sequences with significant similarity to serine proteinases, proteinase inhibitors, odorant and chemosensory binding proteins, and cuticular proteins. RT-PCR analysis of ESTs from each of these categories indicated that 85% were differentially expressed in one caste or the other. We conclude that our SSH strategy was effective in identifying a number of genes differentially expressed in each of the larval castes and suggest several of these differentially expressed genes will be useful in characterizing caste-specific gene networks in C. floridanum.

The cuticular protein genes of Anopheles gambiae and Apis mellifera: Annotation and insights

W.A. Dunn, T. Togawa, J.H. Willis

Dept. of Cellular Biology, University of Georgia, Athens, GA 30602, USA. wadunn83@mac.com

Sequence and electrophoretic analyses revealed that the cuticle has many distinct cuticular proteins (CP), with the majority sharing a conserved sequence, the Rebers and Riddiford (RR) Consensus that has chitin-binding capability. We have annotated the CP genes in the RR family in Anopheles gambiae, using the conserved consensus as our guide and the genomic sequence data from the PEST strain. Over 130 RR genes have been identified, indicating that Anopheles devotes close to 1% of its genes to coding for this one class of cuticular proteins. Almost three quarters of the RR genes were found in just 5 clusters ranging in size from 9–35 members, with both plus and minus orientations. Within each cluster there may be several genes with virtually identical coding sequences, but with different 5′ and 3′ UTRs. Nearest neighbor analyses of all the RR-2 proteins revealed that sequences within a cluster formed distinct groupings; this was not found with the RR-1 proteins that are more divergent outside the strict consensus. In contrast with A. gambiae, only 28 members of the RR family were identified in Apis mellifera. The biological questions posed by these findings are provocative. At the sequence level alone, important questions abound about the origin of multigene families and the roles of gene conversion and purifying selection. Speculation is tempered by concerns about the accuracy of the Anopheles genome assembly. In addition to the RR family, Anopheles also has four members of each of two far smaller families, CPF and CPTC as well as another moderate size family that codes for CP with low sequence complexity caused by a high proportion of glycine or alanine residues.

The ligand-bond X-ray structure of Aedes aegpti sterol carrier protein-2 like-2 at 1.7-angstrom resolution

David H. Dyer¹, Katrina Forest¹, Que Lan²

¹Department of Bacteriology, University of Wisconsin-Madison, Madison, WI 53706 USA. dyer_dave@hotmail.com

²Department of Entomology, University of Wisconsin-Madison, Madison, WI 53706 USA

Sterol carrier protein-2-like-2 (SCP-2-L2) gene from the yellow fever mosquito, Aedes aegypti, is a member of the SCP2 gene family. The protein fold of SCP2-L2 is very similar to other members of the SCP2 structural family. However in the SCP-2-L2 structure, along with a similar position for the previously seen fatty acid in SCP-2, the formation of a dimer occurs revealing a large internal cavity spanning the two monomers. Within the cavity is a bound fatty acid which is in an orientation similar to the Triton X-100 seen in the human SCP2 domain from the peroxisomal multifunctional enzyme.

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Analysis of Aedes aegypti mosquito vitelline envelope gene promoters

Marten J. Edwards

Muhlenberg College, Biology Department, 2400 Chew Street, Allentown, PA 18104, USA. mailto: edwards@muhlenberg.edu

Three ecdysone-responsive Ae. aegypti vitelline envelope genes (15a-1, 15a-2 and 15a-3) are strongly expressed in the follicular epithelium of the ovaries following a blood meal. To assess whether vitelline envelope gene promoters can drive ectopic gene expression in transgenic Ae. aegypti mosquitoes, ~2.0 kb sequences upstream of the three genes were linked to a red fluorescent protein (RFP) reporter gene and inserted into a piggyBac transformation vector (3XP3-EGFP). These construct are being microinjected into Ae. aegypti embryos. D. melanogaster were transformed with the 15a-2.RFP.pBac construct. In four independent transgenic lines, ovarian RFP expression was not observed. Transformation of D. melanogaster with 15a-1.RFP and 15a-3.RFP piggyBac constructs is in progress. The three upstream sequences have also been linked to a human antiviral gene (MxA) and inserted into the piggyBac transformation vector. Driving MxA expression in the follicular epithelium of mosquitoes following a blood meal may allow us to assess whether infection of the follicular epithelium is a pre-requisite for the transovarial transmission of LaCrosse virus in Oc. triseriatus mosquitoes.

RNAi suppression of recognition protein mediated immune responses in the tobacco hornworm Manduca sexta causes increased susceptibility to the insect pathogen Photorhabdus

I. Eleftherianos, P.J. Millichap, R.H. ffrench-Constant and S.E. Reynolds

Department of Biology and Biochemistry, University of Bath, Bath BA2 7AY, UK. bssie@bath.ac.uk

Bacterial pathogens either hide from or overcome the immune response of their hosts. Here we show that two different species of insect pathogenic bacteria, Photorhabdus luminescens TT01 and Photorhabdus asymbiotica ATCC43949 were both recognised by the immune system of its host Manduca sexta, as indicated by a rapid increase in the levels of mRNAs encoding three different inducible microbial recognition proteins, Hemolin, Immulectin-2 and Peptidoglycan Recognition Protein. RNAi mediated inhibition of expression (“knock-down”) of each of these genes at the level of both mRNA and protein was achieved through injection of double-stranded RNA (dsRNA). Knock-down of any one of these genes markedly decreased the ability of the insects to withstand infection when exposed to either species of Photorhabdus, as measured by the rate at which infected insects died. RNAi against immulectin-2 caused the greatest reduction in host resistance to infection. The decreased resistance to infection was associated with reduced hemolymph phenoloxidase activity. These results show not only that Photorhabdus is recognised by the M. sexta immune system but also that the insect’s immune system plays an active, but ultimately ineffective, role in countering infection.

A gene operon that enables the insect-pathogenic bacterium Photorhabdus asymbiotica to survive within phagocytic hemocytes of the insect Manduca sexta

I. Eleftherianos, N.R. Waterfield, R.H. ffrench-Constant, and S.E. Reynolds

Department of Biology and Biochemistry, University of Bath, Bath BA2 7AY, UK. bssie@bath.ac.uk

Photorhabdus bacteria are lethal pathogens of insects. Like all pathogens, the bacteria must evade or overcome host immune defences in order to survive and proliferate. Photorhabdus produces several lethal toxins (eg Tc toxins and Mcf-1), but other virulence genes are likely to contribute to pathogenesis by enabling Photorhabdus to persist and multiply within the insect until bacteria are present in sufficient numbers to be able to kill the host. Here we describe a screen of a fully sequenced Photorhabdus genome that aimed to discover such persistence genes. We screened cosmids to find those that allowed a cloning strain of E. coli to persist within and ultimately kill Manduca sexta caterpillars. In this way we identified a Photorhabdus operon kdp that encodes the protein subunits of a bacterial K⁺ ion transporter and the two-component regulator that governs their expression. Expression of these Photorhabdus genes allows E. coli to persist within phagocytic hemocytes; disruption of any one of the operon’s genes prevents persistence. Since expression of the two-component regulator genes alone is sufficient for persistence, we conjectured that the distinctive feature of Photorhabdus kdp is the sensor-regulator pair, which can also regulate expression of the E. coli kdp genes to the detriment of the host insect. We confirmed this hypothesis by showing that whereas E. coli kdp genes are not expressed after phagocytosis, the kdp genes of Photorhabdus are strongly expressed within Manduca phagocytes.

Genes for honey bee heat shock proteins: Description and comparison

Michelle M. Elekonich

Department of Biological Sciences, University of Nevada Las Vegas, 4505 Maryland Parkway, Las Vegas, Nevada 89154, USA. michelle.elekonich@unlv.edu

Hsps and their encoding genes (hsps) are nearly universal in organisms, highly-conserved, and assigned to families on the basis of sequence homology and typical molecular weight. Members of several Hsp families differ in inducibility by stressors, intracellular localization, and function. Hsps interact with other proteins that are in non-native conformations (whether due to protein-denaturing stress or because the peptides they comprise are not fully mature) to promote refolding, minimize their aggregation or target them for degradation and removal from the cell. As part of a larger annotation effort following the sequencing of the honey bee genome, we identified heat shock protein genes from the hsp70, hsp90, and hsp 40 families. Despite being endothermic insects and exhibiting extreme heat tolerance (up to 50°C for 1 hour) there has not been a large increase in the number honey bee hsp70s compared to those in other insect genomes. Comparisons between the honey bee, fly, and mosquito sequences suggest that the fly model may not represent the ancestral situation.

A stage-specific ovarian factor with stable stimulation of juvenile hormone synthesis by corpora allata of the cockroach Diploptera punctata

K.L. Elliott, A.P. Woodhead, B. Stay

Department of Biological Sciences, University of Iowa, Iowa City, IA 52242, USA. barbara-stay@uiowa.edu

In vivo studies of the cockroach Diploptera punctata have shown stage-specific ovarian stimulation of juvenile hormone (JH) synthesis by corpora allata (CA) (Rankin and Stay, 1984. Gen. and Comp. Endocrinology. 54: 382–388). Using ovary-conditioned medium (OCM) to treat CA in vitro, a non-stage-specific stimulatory factor was found to be released by all stages of ovaries in the 8 days of the first ovarian cycle and this factor was recovered in the flow-through after solid-phase extraction of the OCM (Unnithan et al., 1998. J. Insect Physiol. 44: 1027–1037). The present study provides evidence for a different ovarian factor that stimulates JH synthesis and is stage-specific. The stage-specific factor was found by conditioning medium with ovaries from different stages in the reproductive cycle (pre-vitellogenic, day 1; rapid vitellogenesis, days 2–4; and post-oviposition, day 8). One member of a pair of CA from day 3 mated females was treated with OCM and the other with the flow-through of that OCM after solid-phase extraction on a C₁₈ reverse phase Sep-Pak cartridge. CA were then transferred to new medium and rates of JH synthesis were measured by an in vitro radiochemical assay. Only CA conditioned with day 2 and 3 OCM had significantly higher rates of JH synthesis than CA conditioned with flow-through. The CA responded to this factor in a dose-dependent manner and the factor was shown to be sensitive to trypsin but not to freezing. These results indicate that the increasing rates of JH synthesis that accompany rapid growth of the basal ooctyes of the ovary results from the release of a stage-specific peptidergic ovarian factor that acts directly on the CA to induce a stable stimulation of JH synthesis.

Distribution of ionotropic (RDL) and metabotropic (GABA_B) receptors for GABA in the brain of Drosophila

L. E. Enell, Y. Hamasaka, D.R. Nässel

Department of Zoology, Stockholm University, SE10691 Stockholm, Sweden. lina.enell@zoologi.su.se

Gamma-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in insects, including Drosophila. GABA is produced in a large number of interneurons throughout the CNS of Drosophila; no GABAergic sensory or motorneurons have been detected. Two major types of GABA receptors are known: (1) ligand-gated ion channel-type GABA_A receptors formed as multimers of different subunits and (2) metabotropic GABA_B receptors (GABA_BRs) which are G-protein-coupled receptors (GPCRs). The Drosophila GABA_BR1 and R2 subunits form functional heterodimers. We raised specific antisera to the GABA_BR2 and to the GABA_AR subunit RDL (Resistance to Dieldrin) of Drosophila to map receptor distribution in the CNS. The receptor distribution was compared to that of GABAergic neurons identified by immunocytochemistry or by driving green fluorescent protein in GAL4 lines specific for GABA signaling or specific interneurons. We find an abundant, but selective, distribution of both RDL and GABA_BR2 immunoreactivity in the brain. The two types of receptors display similar general distribution in some, but not all, neuropil areas. Most prominently labeled with antisera to both receptors were neuropils of the antennal lobes, optic lobes and the calyces of the mushroom bodies. Here, we especially investigated the antennal lobes. There is a close match between distribution of GABA and the two receptor types. The difference in detailed distribution of GABA_A and GABA_B receptors may reflect their postulated functional properties: GABA_A units form a postsynaptic receptor mediating fast inhibition and GABA_B units form pre- and/or postsynaptic receptors mediating slow inhibition. Supported by the Swedish Research Council and the Swedish Animal Welfare Agency.

The metamorphosis-regulators, E75 and Broad play conserved and divergent roles in the direct-developing milkweed bug, Oncopeltus fasciatus

Erezyilmaz, D.F., Kelstrup, H., Truman, J.W. and Riddiford, L.M.

Department of Biology, University of Washington, Box 351800, Seattle, WA. USA. denizere@u.washington.edu

The transcription factors Broad (Br) and E75A play well-established roles in regulating molting and metamorphosis in holometabolous insects. br is required for the larval-pupal transition, while E75A acts to couple the molting cycle with metamorphosis. To shed light upon the genetic basis for the evolution of complete metamorphosis, we have examined the function of these two transcription factors in the milkweed bug, Oncopeltus fasciatus. We find that the postembryonic roles of the two genes are comparable with their functions during metamorphosis of holometabolous insects. During the nymphal stages, E75A is required for molting, as E75A dsRNA-injected nymphs fail to molt to the next stage. Unlike its postembryonic expression during holometabolous development, where br is restricted to the larval-pupal transition, we find that br is expressed at each nymphal molt, but not at the molt to the adult. Injection of br RNAi into nymphs led to a repeat of the stage at the next molt. This stasis involved both the pigmentation pattern and the wing pad proportions that normally characterize a given stage. In contrast to their postembryonic roles, the effects of E75 and br kock-down were entirely unexpected. We find that br is expressed during segmentation, which occurs in Oncopeltus in the context of germ band invagination. Loss of br through maternal RNAi results in posterior truncations. Interestingly, E75 also plays a role in segmentation. In this case, however, loss of E75 results in loss of the labial, T2 and T3 segments through fusion with more posterior segments. In addition, loss of abdominal segments may occur through fusion. We are currently trying to determine the relationship between these transcription factors and canonical patterning genes in early embryonic development. This work was supported by NIH RO1GM-10166.

Pathway and transcriptional insights into honey bee immunity from the Honey Bee Genome Project

J.D. Evans, and the Honey Bee Genome Sequencing Consortium

Bee Research Laboratory, USDA-ARS Beltsville, MD 20705. evansj@ba.ars.usda.gov

Honey bees combat disease through both social, or group-level, mechanisms and individual defenses. Bees face significant parasites and pathogens across many taxonomic groups, including bacteria, viruses, protists, and fungi, and a wealth of pathology data for honey bees can be used to explore host-parasite interactions. Like other insects, honey bees use components of the innate immune response to defend against pathogens, and recent work has explored the mechanisms¹ and efficacy² of this response. With the sequencing, assembly, and annotation of the honey bee genome it is possible to propose and test hypothetical immune-pathway models for bees. Honey bees show likely orthologs for nearly all members of the canonical insect innate immune pathways (Toll, Imd, JNK, and Jak/STAT). Surprisingly, honey bees have sharply lower immune-pathway redundancy when compared to other insects, implying decreased flexibility in the immune responses of bees toward pathogens. Of 17 immune gene families implicated in recognition, signaling, and effecting an immune response, bees have approximately 1/3 the gene diversity found in the Drosophila melanogaster and Anopheles gambiae genomes. This reduction could reflect the protective strength of behavioral and environmental barriers to honey bee disease, a tendency of bees to be attacked by a limited set of coevolved pathogens, or novel, parallel mechanisms with which bees achieve immune response flexibility. Transcript abundance levels for pathway members and effectors are presented in order to describe the covariance and heritability of immune responses in bees. ¹K. Aronstein, E. Saldivar, Apidologie 36, 3–14 (2005), ²J. D. Evans, J. S. Pettis, Evolution 59, 2270–2274 (2005)

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Bt toxin binding domains in pink bollworm (Pectinophora gossypiella) cadherin

Jeff A. Fabrick¹ and Bruce E. Tabashnik²

¹USDA-ARS, U.S. Arid Land Agricultural Research Center, 21881 N. Cardon Lane, Maricopa, AZ 85239, USA. jfabrick@wurl.ars.usda.gov

²Department of Entomology, University of Arizona, Tucson, AZ 85721, USA.

Transgenic crops producing toxins from Bacillus thuringiensis (Bt) are widely used for pest control, including cotton that produces Bt toxin Cry1Ac and kills key lepidopteran pests. Both cadherin and aminopeptidase have been implicated as Bt toxin receptors. Although field-evolved resistance to Bt crops has not yet occurred, laboratory selection results show that many pests can evolve resistance to Bt toxins. The most common mechanism of resistance is reduced binding of toxin to midgut receptors. Resistance to Cry1Ac in several lab-selected strains of the global cotton pest, pink bollworm (Pectinophora gossypiella), is tightly linked to a cadherin gene (Morin et al., 2003. PNAS 100, 5004–5009 and Tabashnik et al., 2005. J. Econ. Entomol. 98, 635–644). We report that Cry1Ac binds to recombinant peptides corresponding to extracellular regions of the pink bollworm cadherin (BtR). Similar to other lepidopteran cadherin receptors, pink bollworm BtR has at least two binding domains, each adjacent to the membrane proximal region. However, unlike cadherins from Manduca sexta and Bombyx mori, toxin binding was not observed in regions more distally located from the membrane proximal region. We also report that both the protoxin and activated toxin forms of Cry1Ac bound to recombinant pink bollworm BtR fragments, suggesting that Cry1Ac activation may occur either before or after receptor binding. The results support the hypothesis that cadherin is a receptor for Cry1Ac in pink bollworm.

Effects of Wolbachia infection on metabolic processes in cultured mosquito cells

Ann M Fallon

Department of Entomology, University of Minnesota, 1980 Folwell Ave, St. Paul, MN 55108. fallo002@umn.edu

In mosquitoes, the intracellular bacterium known as Wolbachia pipientis causes a reproductive distortion known as cytoplasmic incompatibility, which favors production of progeny by infected females. Because the infection is transmitted to mosquito offspring, Wolbachia facilitates its own spread through uninfected populations. Mathematical models indicate that Wolbachia provides a potentially effective agent for introducing, into vector populations, transgenes designed to reduce disease transmission. In the laboratory, Wolbachia can be transferred between insect species, and can be introduced into cultured cells. Alternatively, infected cell lines, such as the Aedes albopictus Aa23 line (O’Neill et al., 1997; Insect Molecular Biology 6, 33–39), can be derived from infected insect embryos. We are standardizing the in vitro production of Wolbachia in Aedes albopictus mosquito cell lines by comparing infected Aa23 cells to the uninfected C7-10 Aedes albopictus cell line, and to C7-10 cells infected with Wolbachia from Aa23 cells. Wolbachia-infected cells grow more slowly than uninfected cultures, have reduced ability to incorporate tritiated thymidine into DNA, and undergo apoptosis as the abundance of Wolbachia increases. In Aa23 cells, the secretion of immune-induced proteins into the cell culture medium is substantially reduced, relative to that in C7-10 cells. We hypothesize that in infected cell cultures, modulation of the immune response contributes to Wolbachia survival and replication.

Proteomic approach to investigate aphid - plant interactions

Frédéric Francis¹, Nicolas Harmel¹, Edwin De Pauw² and Eric Haubruge¹

¹ Functional and Evolutionary Entomology, Gembloux Agricultural University, Passage des Deportes 2, 5030 Gembloux, Belgium. francis.f@fsagx.ac.be

²Mass Spectrometry Laboratory, University of Liège, BAT. B6 Chimie physique, allee de la Chimie 3, 4000 Liege 1, Belgium

Plant-insect relations are mainly regulated by the evolution of the defense mechanisms developed by plants and the ways herbivore insects adapt themselves to these defensive systems. Plant defense can be direct or indirect, localised or systemic. A common property of these mechanisms is the broad range of phytophagous agents, including insect pests, which are efficiently controlled by the defensive produced molecules. To cope with the induction of several direct defense molecules, herbivores developed several detoxification enzymatic systems such as the gluthathione S-transferases and monooxygenases. Here we studied the chemical ecology of aphid (such as M. persicae)–plant relations using a proteomic approach. The aphid switch from one host plant to others within the Solanaceae and Brassicaceae family was first investigated to assess the metabolic changes and potential adaptations in aphids according to particular host plant species. Specific associations between aphids and their host plants were previously shown to be related to the presence of particular bacterial symbionts. The respective role of the aphid and their related symbionts in the adaptation to the host plant was also investigated considering the proteome variations of aphids in presence or absence of endosymbionts. Finally, the particular role of aphids in plant defensive responses due to its sucking feeding behavior was investigated focusing on the protein composition of aphid saliva. The complex protein mixtures from different aphid materials were separated by two dimension electrophoresis methods and the related spots of proteins significantly varying were selected and identified by mass spectrometry (ESI-MS-MS and Maldi-Tof-MS-MS) coupled with data bank investigations. The impact of the down regulated or over expressed aphid proteins involved in different metabolic pathways was discussed. This broad proteomic approach is a very reliable tool to study the biologically involved proteins from aphids in response to several environmental changes, and particularly the insect-host plant interactions.

Mapping of hemoglobin proteolysis in the hard tick Ixodes ricinus

Z. Franta¹, M. Horn², D.Sojka¹, M. Mares², and P. Kopácek¹

¹Institute of Parasitology, Biological Centre Academy of Sciences of the Czech Republic and Faculty of Biological Sciences University of South Bohemia, Ceské Budejovice. zdeny@paru.cas.cz

²Institute of Organic Chemistry ad Biochemistry, Academy of Sciences of the Czech Republic, Prague.

Ticks differ from other hemaptophagous parasites in the intracellular localization of hemoglobin proteolysis. Hemoglobin digestion in ticks is a critical process for two reasons: It provides primary energy resources, and the generated hemoglobin fragments function as antimicrobial peptides. Hemoglobin digestion in ticks is still poorly understood at the molecular level. We have analyzed the peptidase spectrum in the gut of the hard tick Ixodes ricinus, a vector of Lyme disease and tick-borne encephalitis. Substrate/inhibitor-based profiling demonstrated endo- and exopeptidases of cysteine and aspartic class in the tick gut homogenate. The screening of gut-specific cDNA by PCR amplification was performed with primers derived from the conserved regions of the detected peptidases. It resulted in identification of genes coding for (i) cysteine peptidases: asparaginyl endopeptidase (legumain), cathepsin B1, B2 and L, and dipeptidyl peptidase I (cathepsin C), and (ii) aspartic peptidase cathepsin D. Tissue expression analysis by RT PCR revealed that all peptidases (with exception of cathepsin L) are expressed specifically in the tick gut. Taken together, the proteolytic machinery in the tick gut closely resembles the digestive system of Schistosoma blood flukes but differs substantially from hematophagous insects relying mainly on serine peptidases. This work was supported by Grant Agency of the Czech Republic No. 206/06/0865, and research projects Nos. Z60220518, Z40550506 and MSMT 6007665801.

Lipid metabolism in the hematophagous Panstrongylus megistus: Interaction lipophorin-midgut membrane

L.L. Fruttero¹, R. Stariolo², E.R. Rubiolo¹ and L.E. Canavoso¹

¹Departamento de Bioquímica Clínica, CIBICI-Conicet. Facultad de Ciencias Químicas, Universidad Nacional de Córdoba. Córdoba, Argentina. lcanavo@mail.fcq.unc.edu.ar

²Coordinacion Nacional de Control de Vectores. Córdoba, Argentina, CP 5000

In insects, the transfer of midgut lipids to the hemolymph is a remarkable event mediated by lipophorin (Lp), the main insect lipoprotein. In order to understand the regulation of this process in hematophagous insects we have analyzed the transfer of diacylglycerol into circulation, and the interaction Lp with the midgut membrane using a solid-phase binding assay. In addition, the sites of interaction of Lp with the midgut cells were localized by immunofluorescence assays. This study was performed employing Panstrongylus megistus (Hemiptera:Reduviidae), an important vector of Chagas’ disease. This insect takes large blood meals, containing a substantial amount of lipid. Lp was isolated from hemolymph of fifth instar nymphs by a KBr gradient and the membranes were obtained by ultracentrifugation of midgut homogenates. Thereafter, the membranes were suspended by mild sonification, adsorbed in plates, and the amount of bound Lp was quantified by ELISA. The factors analyzed included the effect of ionic strength on binding, the effect of pH, the requirement of divalent cations, and the effect of suramin. The saturation kinetics most likely fit a ligand-binding model for a single binding site. The interaction between Lp and the membrane showed a strong dependence with the pH and, in contrast with LDL receptor family, did not required Ca⁺² or Mg⁺². Like other lipoprotein receptors, suramin significantly inhibited the interaction between Lp and the membrane. The effect of ionic strength suggested that Lp binding is optimal at low NaCl concentration. In addition, the partial effect on binding after membranes were treated with proteases or changes in temperature strongly indicated that such interaction needs proteins in addition to other components of the membrane. Finally, immunofluorescence assays showed that the interaction of Lp with midgut cells mainly occurred at the basolateral region of the membrane cells. Supported by Secyt-UNC (E.R.R) and CONICET-Argentina (L.E.C). L.L. Fruttero is a research fellow from CONICET-Argentina.

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Role of the transcriptional repressor mdGfi-1 in CYP6D1v1-mediated insecticide resistance in the house fly, Musca domestica

Jianwei Gao, Jeffrey G. Scott

Department of Entomology, Comstock Hall, Cornell University, Ithaca, NY 14853-0901, USA. jgs5@cornell.edu

Gfi-1 is a C₂H₂-type zinc finger protein that is a transcriptional repressor in vertebrates and has been implicated in control of CYP6D1 expression in house flies (Musca domestica). A 15 bp insert which disrupts a putative mdGfi-1 binding site in the CYP6D1v1 promoter has been implicated as a cause of increased expression of CYP6D1, and thus insecticide resistance. Using electrophoretic mobility shift assays we demonstrate that the CYP6D1 promoter from susceptible strains binds mdGfi-1. The 15 bp insert that interrupts the mdGfi-1 binding site in insecticide resistant strains reduces the amount of mdGfi-1 binding by 9- to 20-fold, consistent with the role of mdGfi-1 in resistance. Partial sequences of mdGfi-1 (spanning the first intron) from individual house flies from 11 different strains revealed the presence of 23 alleles. There was no consistent difference in the mdGfi-1 alleles between susceptible and CYP6D1-mediated insecticide resistant strains, indicating that mdGfi-1 alleles were not likely involved in resistance. Polymorphisms were used to map mdGfi-1 to autosome 1. Quantitative real time PCR (qRT-PCR) revealed Gfi-1 expression was higher in the thorax compared to the head and abdomen, and varied between life stages and between strains. However, similar levels of mdGfi-1 were detected in susceptible and resistant adults suggesting that altered levels of mdGfi-1 were not likely a cause of insecticide resistance. The significance of these results to understanding insecticide resistance is discussed.

Molecular mechanisms of resistance to Bacillus thuringiensis and variation in the magnitude of fitness costs

Aaron J. Gassmann, Yves Carrière, and Bruce E. Tabashnik

Department of Entomology, The University of Arizona, Tucson, AZ, 85721. gassman@ag.arizona.edu

During the past decade, 35 studies have tested for fitness costs associated with resistance to the insecticidal toxins produced by the bacterium Bacillus thuringiensis. These studies demonstrate a high level of variation in the extent to which resistance to Bt is associated with fitness trade-offs. More recently, the molecular basis of resistance has come to light for several insect species. Although resistance is often associated with mutations at a caderhin locus, alternative resistance mechanisms exist. We review the literature on fitness costs of resistance to Bt and on the molecular basis of resistance. We consider whether differences in the magnitude or manifestation of fitness costs arise between insect species with contrasting molecular mechanisms of resistance, and whether the fitness costs of resistance differ among insect species that have cadherin-based resistance to Bt.

The regulation by iron of a putative divalent metal transporter and ferritin in Anopheles gambiae 4a3b cells

D. L. Geiser¹, J. S. D. Bridgers² and J. J. Winzerling¹

¹Nutritional Sciences, The University of Arizona, 1177 E. 4^th St., Shantz Bldg. Rm. 405, Tucson, AZ, USA. dlgeiser@email.arizona.edu

²Biochemistry and Molecular Biophysics, The University of Arizona, Tucson, AZ, USA

Forty-one percent of the world’s population lives in areas where malaria is transmitted and an estimated 700,000-2.7 million persons die of malaria each year. Female mosquitoes such as Anopheles gambiae (African malaria mosquito, Diptera) require a blood meal for oogenesis (egg laying). It is during blood-feeding that these mosquitoes transmit diseases and receive a potentially toxic level of iron in both heme and non-heme forms. Heme is incorporated into the peritrophic matrix in the midgut of Aedes aegypti (yellow fever mosquito) during digestion and is potentially the primary mechanism of heme detoxification. It is not clear how non-heme iron is transported. In mammals, the divalent metal transporter, DMT1 (NRAMP2/ SLC11A2), is the primary importer of dietary non-heme iron into intestinal epithelial cells. DMT1 also transports iron from endosomes into the cytoplasm in somatic cells. We previously demonstrated that cultured A. aegypti larval cells will take up iron from culture medium by as yet unknown mechanisms. Iron inductively coupled plasma-mass spectrometry (ICP-MS) shows a dose dependent increase in iron concentration in the cytoplasm and membrane extracts of iron treated cells. Mosquito ferritin is the chief iron storage protein for these animals which is composed of heavy chain (HCH) and light chain (LCH) subunits that are homologues of the vertebrate ferritin subunits. Electromobility shift assays for iron regulatory protein 1 (IRP1) binding activity and immunoblot analysis for ferritin also supports the entry of iron into these mosquito cells. As part of a pilot study, we identified a putative DMT1 (pDMT1) from the A. gambiae protein database (NCBI). This pDMT1 was amplified, cloned and sequenced from A. gambiae larval cells, MOS55. Our preliminary experiments demonstrated that pDMT1 message in MOS55 cells has a biphasic response to increasing concentrations of ferric ammonium citrate (FAC). Recently, we successfully obtained the cDNA sequences for A. gambiae HCH and LCH. We have evaluated the mRNA expression of pDMT1, HCH, LCH as well as IRP1 under varying concentrations of iron excess, iron deprivation and a ferric rescue in A. gambiae hemocyte-like cells, 4a3b. Ultimately, studies will be conducted to determine the importance of these proteins in iron transport and storage by co-immunofluorescence, EM and ICP-MS.

Activation of the EGF receptor influences extension and fasciculation of receptor axons in the developing olfactory system of the moth Manduca sexta

N.J.Gibson and L.P.Tolbert

Arizona Research Laboratories, Division of Neurobiology, University of Arizona, Tucson, AZ, USA. njgibson@neurobio.arizona.edu

Developing olfactory systems provide models for exploring mechanisms that influence axons as they seek their targets. During development of the adult antennal (olfactory) system of Manduca sexta, axons of olfactory receptor neurons (ORNs) interact with each other and with glial cells in a specialized "sorting zone" (SZ) as they sort by odor specificity. Once sorted, they seek the correct target sites in the antennal lobe (AL) of the brain and terminate in protoglomeruli. We reported previously that as they extend through the SZ and form protoglomeruli, ORN axons can be labeled with antibodies against human EGF receptors (EGFRs), and that treatment with the EGFR kinase blocker PD168393 results in failure of the axons to extend and sort properly in the SZ and in the nerve layer. We also reported that neuroglian, an IgCAM that can activate EGFRs through homophilic interactions, is present transiently on ORN axons and glia in the SZ and AL during the same period. Now we know that 9 of 10 amino acids through which PD168393 binds to human EGFR are conserved in insects. Blast analysis of Manduca ESTs indicates 67% homology to the peptide sequence used to produce the anti-human EGFR antibodies, and blocking with the corresponding moth peptide eliminates labeling. We have used Triton extraction tests to determine whether neuroglian binding may be occurring in the SZ, as a prerequisite to potentially activating EGFRs. We find that neuroglian is resistant to Triton extraction, indicating attachment to the cytoskeleton in response to homo- or heterophilic binding, on ORN axons only in the SZ and in the nerve layer of the AL. This supports the hypothesis that homophilic interactions between neuroglian molecules in these regions causes activation of EGFRs, which is necessary for further axonal extension and for sorting. In addition to blocking activation of EGFRs, PD168393 treatment results in a reduction of immunolabeling for neuroglian on ORN axons, suggesting that EGFR function and neuroglian expression are coupled. Support Contributed By: NIH DC004598

Structural and functional characterization of a Campoletis sonorensis ichnovirus (CsIV) Cys-motif protein (WHv1.6)

Torrence A. Gill, and Bruce A Webb

Department of Entomology, University of Kentucky. gilltorrence@hotmail.com

Campoletis sonorensis is an endoparasitoid that of lepidopteran larvae. C. sonorensis has a symbiotic mutualism with a polydnavirus (CsIV) which is integrated in the genome of the wasp. During oviposition of endoparasitoid egg into the lepidopteran host, the wasp also injects venoms, ovarian proteins and CsIV. The ovarian and venom proteins transiently inhibit lepidopteran immune response over the initial 24 hours. Viral protein titers become high enough over this period to induce pathologic effects on the host, notably the viral proteins suppress cellular and humoral immunity, arrest host development, and suppress synthesis of some host proteins. CsIV has a double stranded, segmented DNA genome, that contains multiple segments present in different molar ratios. The CsIV genome also encodes multiple gene families. The CsIV cys-motif gene family has 10 family members, all of which appear to be expressed. Although some of these genes have previously been investigated the Whv1.6 gene is little studied. Here we report studies of this gene family member and show that is has the highest expression level. Whv1.6 is encoded on a hypermolar segment in the CsIV genome, Segment W, which is also a nested segment. To study the function of the encoded protein, the Whv1.6 gene was cloned into a baculovirus expression vector and the expressed protein purified. The purified protein was shown to inhibit insect growth when fed to larvae. The protein was then used to produce an antibody with the protein detected in parasitized plasma 6 hours post-parasitization, and throughout infection the normal period of parasitization. WHv1.6 was detected by Western blots in several host tissues, notably fat body and hemocytes, at two and seven days post infection, and this finding was corraborated by immunofluorescence detection assays. These functional studies suggest that this protein is involved in suppression of host immune and developmental systems in parasitized larvae.

Endocrine regulation of pheromone production in the pinyon Ips, Ips confusus

Matthew D. Ginzel¹. Christopher I. Keeling^1’2, Claus Tittiger¹ and Gary J. Blomquist¹

¹Department of Biochemistry and Molecular Biology, University of Nevada, Reno, NV 89523, USA. ginzel@unr.edu

²Michael Smith Laboratories, University of British Columbia, Vancouver BC V6T 1Z4, CANADA

Bark beetles are among the most economically important forest pests in the northern hemisphere, and rely on monoterpenoid aggregation pheromones to coordinate host colonization and mating. In this study, we investigate the interplay between feeding on host phloem and the induction of de novo pheromone biosynthesis in the pinyon Ips, Ips confuses (Coleoptera: Scolytidae). I. confusus has become a major pest in the southwestern Unit