Fourth International Symposium on Molecular Insect Science

28 May to 2 June 2002, Tucson, Arizona, USA

Stuart E. Dryer (University of Houston), Peter D. Evans (Babraham, Cambridge, UK), Sarjeet S. Gill (University of California, Riverside), Lawrence G. Harshman (University of Nebraska), John G. Hildebrand (University of Arizona), Anthony A. James (University of California, Irvine), Michael R. Kanost (Kansas State University), Therese A Markow (University of Arizona), Nancy A. Moran (University of Arizona), Sharon D. Richards (University of Arizona), Lynn M. Riddiford (University of Washington, Seattle), Michael A. Wells (University of Arizona), Judith Willis (University of Georgia), Mariana F. Wolfner (Cornell University).

Abstracts are listed in alphabetical order by the last name of the senior author.

Analysis of Plasmodium gene expression during its development in the mosquito, Anophleles stephensi

Abraham EG ¹, Srinivasan P¹, Ghosh A¹, Islam S1, Valenzuela J², Ribeiro J², Jacobs-Lorena M¹.

¹Department of Genetics, School of Medicine, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, OH 44106-4955; ²NIH, NIAD, Bethesda, MD 20892

Plasmodium, the causative agent of malaria, has to complete a complex developmental program in its mosquito host for transmission to occur. However, little is known at the molecular level about mosquito-parasite interactions. By use of subtraction libraries, we have begun an analysis of mosquito and parasite genes expressed during Plasmodium berghei development in Anopheles stephensi. The libraries were obtained by subtracting mRNA of non-infected blood-fed mosquito guts and/or Plasmodium asexual stages from mRNA of P. berghei ookinetes or infected mosquito guts. Libraries enriched for genes preferentially expressed at 4 developmental stages were prepared: 1) in vitro cultured ookinetes, 2) early oocysts, 3) middle oocysts, and 4) late oocysts. A total of 1,657 unique sequences were identified among the 2182 clones that were sequenced. Of these, 1032 were of presumptive mosquito origin (<55% AT) and 625 of Plasmodium origin (>55% AT). Northern blot analysis of several randomly selected genes indicates upregulation at a time of development corresponding to the library of origin. A polyclonal antibody to a putative cell surface ookinete protein containing a von Willebrand type A adhesive domain inhibited oocyst formation by 60-79%, making this a candidate for transmission-blocking antigen. Clones encoding three different mosquito caspases were isolated. Interestingly, the three genes are induced in the midgut epithelium at the time of parasite invasion.

Reduced vector competence of transgenic mosquitoes expressing bee venom PLA2 from a gut-specific peritrophic matrix protein-1 (Ag-Aper1) promoter

Abraham EG, Donnelly-Doman M, Ghosh A, Moreira L, Jacobs-Lorena M.

Department of Genetics, School of Medicine, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, OH 44106-4955.

Ag-Aper1 encodes a major Anopheles gambiae peritrophic matrix (PM) protein. The PM is an acellular matrix secreted by the midgut epithelium in response to a blood meal. The Ag-Aper1 protein is stored in vesicles prior to the blood meal and is secreted immediately upon blood ingestion. This makes the Ag-Aper1 promoter an attractive candidate to drive the expression of molecules, such as the bee venom PLA2, which can interfere with Plasmodium differentiation (Zieler et al. 2001). To test this hypothesis, we transformed into the Anopheles stephensi germ line a piggyBac-based construct containing a 2.5 kb Ag-Aper1 promoter and signal sequence fused to the PLA2 coding sequence. Abundant gut-specific expression of PLA2 mRNA and protein was detected in four independent transgenic lines. As is the case for Ag-Aper1, the PLA2 protein was stored in the midgut epithelial cells of transgenic mosquitoes and secreted into the lumen upon blood ingestion. Significantly, formation of P. berghei oocysts was strongly (73-91%) inhibited in transgenic mosquitoes. These findings demonstrate the feasibility of genetic manipulation of mosquito vector competence.

Instability of the piggyBac element in transformed Aedes aegypti

Adelman ZN¹, Jasinskiene N¹, Peek C¹, Travanty EA², Olson KE², James AA¹.

¹Department of Molecular Biology and Biochemistry, University of California, Irvine, CA 926973900, ²Department of Microbiology, Colorado State University, Fort Collins, CO 80523 USA.

The piggyBac element has been used to transform a number of different insect species, including the mosquitoes Anopheles stephensi and Aedes aegypti. A piggyBac plasmid (p3xP3EGFPUbD2FB) was microinjected into Ae. aegypti embryos (Higgs white eye strain) along with a helper plasmid containing the piggyBac transposase gene. Five independent transpositions were identified as G, individual animals positive for the marker gene (EGFP+). Two of these individuals died before they could produce progeny, while two produced numerous progeny following mating, but none were EGFP+. The last family, # 104, was the only family to give EGFP+ progeny in the Gz. Surprisingly, only 10% of progeny from both an outcross and intercross were EGFP+. Gene amplification of pooled genomic DNA confirmed that the inserted piggyBac element was not present in EGFPindividuals, only in EGFP+ mosquitoes, ruling out a simple loss of EGFP protein expression. Southern analyses of line #104 showed multiple copies of the inserted piggyBac element in both Gz and G3 individuals. Genomic DNA fragments containing transposonchromosome junctions also were observed to contain plasmid DNA, while others did not, indicating multiple integrations, both precise and imprecise. Plasmid rescue experiments failed to detect episomal transposons, but full-length plasmid was easily recovered when genomic DNA was digested by a specific restriction endonuclease, religated and transformed into Escherichia coli. Line # 104 has been carried to GS by intercrossing and continues to produce 1030% EGFP+ individuals. Multiple outcrosses of this line have repeatedly led to complete loss of the inserted element. This work is supported in part by grant A148740 from NIAID.

RNAi based inhibition of salivary gland invasion by Plasmodium gallinaceum sporozoites using recombinant double subgenomic Sindbis viruses.

Adelman ZN, Coleman J, James AA

Department of Molecular Biology and Biochemistry, University of California, Irvine, CA, 926973900 USA.

RNA interference (RNAi) has been used in organisms such as plants, flies, and worms to generate loss of function mutant phenotypes. RNAi has also been shown to repress specific gene expression in the mosquito, Aedes aegypti, and in the protozoans, Trypanosoma and Paramecium. We have tested whether RNAi induced in Ae. aegypti could affect another protozoan, Plasmodium gallinaceum, the causative agent of avian malaria. Recombinant Sindbis viruses were generated that contained a portion of the P. gallinaceum TRAP or CSP gene in either sense or antisense orientation (TRS, TRAs, CSS, CSAs). Both TRAP and CSP are surface proteins involved in sporozoite mobility and salivary gland invasion. Recombinant viruses were injected into newly emerged A. aegypti female mosquitoes. At 710 days post injection, mosquitoes fed on P. gallinaceum infected chicks. After 13 days the number of sporozoites per pair of salivary glands (spz/gl) was determined. Mosquitoes injected with L15 medium or with a control virus contained an average of 10,000 spz/gl, as did uninjected mosquitoes. In contrast, 8/27 and 12/27 (30 and 44%) of TRS and TRAs injected mosquitoes contained between 1500 spz/gl. Similarly, 12/61 and 7/50 (20 and 14 %) of CSS or CSAs injected mosquitoes had 1500 spz/gl. These data show that RNAi induced in the mosquito host can affect development of P. gallinaceum. This work is supported in part by grant AI29746 from NIAID.

Characterization of the pyrokinin/pban receptor

Altstein M¹, Ben-Aziz O¹, Schafler I¹, Li Q-J², Martinz-Green M²

¹Institute of Plant Protection, The Voclani Center, Bet Dagan 50250, Israel; ²Department of Cell Biology and Neurosciences, University of California, Riverside, CA 92521, USA.

The pyrokinin (PK)/pheromone biosynthesis activating neuropeptide (PBAN) family of peptides is a multifunctional family that plays a major role in the physiology of insects. The presence of PK/PBAN peptides has been demonstrated in a variety of moths and in other non-lepidopteran species, and their mode of action has been studied extensively. Despite these studies, very little is known about the endogenous mechanism, and much remains to be determined concerning the structural, chemical and cellular basis of their activity. Most of these studies were performed with synthetic peptides and involved exogenous application (mainly by injection) of the tested compounds, which does not necessarily reflect the endogenous natural mechanisms. Currently, it is still not known which endogenous peptide(s) mediate(s) each of the in vivo functions, whether each function is mediated by a different peptide, and whether each peptide mediates one or several functions. It is also not clear whether these functions are mediated by the same receptor or by different receptors, or whether the receptors of the various PK/PBAN peptides share functional homologies. One way to obtain a better insight on the mode of activity of this family of peptides is by the characterization of their receptors. Currently very little is known on the receptors of the PK/PBAN family. Recently, we have developed a binding assay and characterized the properties of the PK/PBAN receptor using female moths (Heliothis peltigera) pheromone glands. A summary of our recent findings will be presented. A complementary approach for studying receptors is by means of receptor-selective agonists and antagonists. We have developed a novel approach, termed the backbone cyclic neuropeptide-based antagonist (BBC-NBA) for the discovery of neuropeptide antagonists. This approach has been applied to the insect PK/PBAN family and resulted in the discovery of highly potent, metabolically stable antagonists, devoid of agonistic activity. The application of the antagonists for the study of the functional diversity and characterization of the PK/PBAN receptors as well as their possible application as insect control agents will be discussed.

RNAi mediated knockout of a GATA type transcriptional factor in Aedes aegypti

Attardo G¹, Martin D², Higgs S³, Raikhel AS¹

¹Department of Entomology, University of California Riverside, Riverside CA 92521, ²Present address: Department of Physiology and Molecular Biodiversity, Institut de Biologia Molecular de Barcelona, CID, CSIC, Barcelona, Spain, ³Department of Pathology, University of Texas Medical Branch at Galveston, Galveston TX 77555-0609

In Aedes aegypti the reproductive cycle is tightly linked to the acquisition of a blood meal. Upon initiation of the reproductive cycle, tissue specific transcription, translation and secretion of yolk protein precursor genes (YPPs) from the fat body occurs on a massive scale. This process is called vitellogenesis. In the period prior to blood meal activation (previtellogenesis) YPP genes are repressed to prevent premature expression, this repressed state is called the state of arrest. Analysis of the promoter from the major yolk protein vitellogenin (Vg), revealed multiple binding sites for GATA type transcription factors. A fat body specific GATA factor (AaGATAr) was cloned and characterized. This factor is expressed during the previtellogenic period, and in cell transfection studies has been shown to have transcriptional repression activity. We hypothesize that this GATA factor maintains the previtellogenic state of arrest prior to blood feeding. To test this hypothesis we are using RNAi to knockout AaGATAr function. RNAi stimulates the degradation of a specific mRNA in response to the presence of a double stranded RNA homologus to the mRNA. Double stranded RNA was delivered by a recombinant Sindbis virus that expressed a single stranded transcript containing inverted repeats from AaGATAr. The inverted repeats cause the RNA to fold over into a double-stranded conformation thereby inducing the RNAi response. To test the effects of this knockout we used RT-PCR to measure levels of YPP transcripts in response to the knockout of this gene. Preliminary results indicate that knockout of AaGATAr caused a modest increase in expression of Vg indicating premature derepression of this gene in RNAi treated mosquitoes.

Initial characterization of a cDNA library from diapausing pupae in the fleshfly, Sarcophaga crassipalpis.

Ball E, Miller III H, Joplin KH

East Tennessee State University, Department of Biological Sciences, 70703. Johnson City TN 37614

Diapause is an optional, inducible developmental state in the flesh fly, Sarcophaga crassipalpis. We are interested in the molecular controls underlying the developmental control of the initation and maintenance of this state. A cDNA library was constructed from the brains of diapausing pupae of S. crassipalpis. Initial cDNA was constructed using RT-PCR and the cDNA inserts were cloned into bacteriophage vectors. Plasmids were rescued from the phage vector and initial characterization of the inserts has begun. We are attempting to assemble a set of representative genes for use in a microarray analysis of diapause.

Linkage mapping in a multiresistant strain of diamondback moth Plutella xylostella reveals separate genetic mechanisms of Cry1A and Cry1C resistance.

Baxter S¹, Zhou J², Shelton AM, Heckel D¹

¹Centre for Environmental Stress and Adaptation Research, Department of Genetics, University of Melbourne, Parkville, VIC 3010, AUSTRALIA; and ²Department of Entomology, New York State Agricultural Experiment Station, Cornell University ,Geneva, NY 14456, USA.

Linkage mapping with AFLPs was conducted in a backcross family originating from a cross between susceptible diamondback moths and a field-derived strain showing resistance to both Cry1Ac and Cryl1C. A single linkage group was responsible for the majority of the Cryl1Ac resistance. This is the same linkage group as previously identified in the NO-QA strain from Hawaii. Finer-scale mapping is underway to investigate whether the same chromosomal region is involved. Two separate linkage groups were associated with Cry1C resistance. Four aminopeptidases have been tested as candidate resistance genes and none of them map to the three resistance-associated linkage groups. We discuss the use of denaturing HPLC in rapidly mapping additional candidate loci.

Understanding the mechanism of juvenile hormone action in coleopterans

Bearfield J, Keeling CI, Eigenheer A, Blomquist GJ, Tittiger C

Department of Biochemistry, University of Nevada, Reno, NV 89557-0014

The mode of action of juvenile hormone (JH) is one of entomology's longest standing mysteries. In Dendroctonus jeffreyi and Ips pini, JH III induces an increase in pheromone biosynthetic gene expression, especially 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-R), in male midgut cells. These genes offer a new system to study JH regulation independent of complex processes involved in development and female reproductive maturation. To exploit the system, an EST library (currently 384 unique cDNAs) of male midgut cDNAs is being constructed using high-throughput technology. JH-responsive clones are being identified using microarrays. Preliminary northern blots prepared from beetles at various time points (within the first 8 hours) after JH treatment revealed some that are rapid, primary JH-responders, with transcripts visible after only 45 minutes. Curiously, HMG-R may be a secondary responder. The 5' flanking region of D. jeffreyi HMG-R has been isolated and sequenced. Important regions for induction will be mapped by transcriptional assays of reporter constructs transfected into isolated midgut cells. Tissue incubation studies are underway to determine the requirements for JH action on cells. Also, a genomic library is being prepared from I. pini in order to clone the 5' flanking regions from JH-inducible genes, including HMG-R. The isolation of JH-inducible genes and the promoter sequences that regulate them will provide important information about the mechanism of JH action in these two Coleoptera.

LaCrosse virus and Aedes triseriatus - molecular determinants of the most remarkable of arbovirus-vector interactions.

Beaty BJ¹, Bltivich BJ¹, Kempf BJ¹, Hughes MT¹, Black WC¹, Mackie RS², Rayms-Keller A², Blair CD¹

¹Arthropod-borne and Infectious Diseases Laboratory, Department of Microbiology, Colorado State University, Fort Collins, CO. 80523; ²Department of the Navy, Naval Surface Warfare Center, Dahlgren Division, Dahlgren, VA, 22448.

Transovarial transmission (TOT) of LaCrosse virus by Aedes triseriatus is very efficient. TOT maintains, amplifies, and promotes evolution of LAC virus in nature. The molecular determinants of this remarkable host-parasite relationship are being revealed. Co-regulation of host and virus transcription modulates viral virulence during ovarian quiescence and embryo diapause. Virus transcription, which requires scavenging of caps from host mRNAs, may target an inhibitor of apoptosis mRNA, thereby perturbing the apoptotic response and promoting viral persistence. LAC virus does not induce apoptosis in vector cells and eludes the host RNAi response, resulting in long term persistent infection of vectors. These long term infections and TOT greatly enhance the evolution of LAC and related viruses both by antigenic shift and drift, and may account for the amazing evolutionary potential of viruses in the family Bunyaviridae.

Immunocytochemical localization of ecdysis-triggering hormone in the epitracheal system and silk glands of the parasitic wasp Cotesia congregata

Beckage N, Foreman R, Park Y, Palmatier C, Surratt V, Marion K, Tan F

Departments of Entomology and Cell Biology and Neuroscience, 5429 Boyce Hall, University of California-Riverside, Riverside, CA 92521 USA

The release of ecdysis-triggering hormone (ETH) induces shedding of the cuticle during the final stages of a molt. In the present study we used antibodies directed against Drosophila melanogaster ETH to localize sites of synthesis of this peptide hormone in the epitracheal endocrine system of the parasitic wasp Cotesia congregata. This wasp undergoes its last larval ecdysis during emergence from the host larva. Pre-emergent wasps were found to have substantial amounts of ETH in both the epitracheal cells and silk glands, and the hormone appeared to be released during emergence of the wasps from the host. This report constitutes the first evidence that the hormone is present outside of the insect epitracheal system. Pre-absorption of antibody with D. melanogaster ETH abolished its binding to epitracheal cells and the silk gland lumen, indicating binding to ETH-containing cells was specific. The release of ETH appears to temporally correlated with wasp emergence, providing evidence for the hormonal regulation of the parasitoid's ecdysis and emergence behaviors. The release of ETH and emergence of the wasps is suppressed if the host is treated with the JH analogue methoprene, suggesting high levels of juvenile hormone have an inhibitory influence on the cascade of events triggering the wasp's emergence behavior.

A novel malaria gene, Pg4, is expressed preferentially in sporozoites isolated from mosquito salivary glands

Beerntsen BT¹, Ou R1, Coleman JL², James AA²

¹Veterinary Pathobiology, University of Missouri, Columbia, MO 65211, USA; ²Molecular Biology and Biochemistry, University of California, Irvine, CA 92697, USA

Malaria sporozoites exhibit infectivity for mosquito salivary glands and vertebrate host tissue, and are excellent targets for efforts designed to prevent malaria transmission. In this study, Plasmodium gallinaceum sporozoites were isolated from mosquito salivary glands and a cDNA library was constructed. Following a heterologous screening technique, a gene, designated, Pg4, was isolated from this sporozoite cDNA library. Pg4 is a novel gene as determined by BLAST analysis of its DNA sequence and translated protein sequence. It contains a signal peptide sequence that encodes a transmembrane domain as determined by PSORT, a protein sequence analysis program, and located near the carboxy terminus of the protein are ten six-amino acid tandem repeats. Transmembrane domains and repetitive sequences are characteristic of other Plasmodium surface antigens, suggesting that Pg4 may be a novel sporozoite surface antigen. Recombinant Pg4 protein (rPg4) was made and used to generate polyclonal antibodies that then were used in immuno-localization studies. Immuno-fluorescence assays suggest that Pg4 is a surface molecule and immuno-electron microscopy is being performed to confirm this cellular location. Interestingly, initial data suggest that Pg4 is preferentially expressed, at the mRNA and proteins levels, in the sporozoites isolated from mosquito salivary glands as compared to those isolated from oocysts located on the mosquito midgut. Future studies include the use of the anti-Pg4 polyclonal antibodies as well as rPg4 in in vivo blocking studies to determine if Pg4 may play a role in the invasion of mosquito salivary glands or vertebrate host tissue.

Molecular and evolutionary analysis of Juan and Juan: Two widely dispersed non-LTR retrotransposon families in mosquitoes

Biedler J¹, Tu Z¹

¹Department of Biochemistry, Virginia Tech, Blacksburg, VA 24061, USA

Juan,a retrotransposon originally discovered in Culex pipiens quinquefasciatus (Mouches et al. 1991), was found in 18 species of five genera. A new family related to Juan was found in eight species of three genera. This new family is named Juan while the original Juan is called Juan. Juan comprises divergent subfamilies, three of which were found by searching the sequenced Anopheles gambiae genome. However, Juan was not found in An. gambiae and appears to have been lost from this species. Phylogenetic analysis supports the grouping of Juan with Juan in the Jockey clade along with BS, Doc, and Jockey elements of Drosophila melanogaster. Tree topology of Juan has an overall congruence with mosquito phylogeny, supporting vertical transmission of these elements. No support was found for horizontal transfer of Juan among Aedes species as was previously proposed (Mouches et al. 1991). This demonstrates the first example of a non-LTR element without target site specificity that has been sustained in a major Dipteran lineage by vertical transmission. A conclusion regarding transmission cannot be made for Juan due to having sequences from few taxa and the presence of subfamilies. GC content analysis shows Juan sequences having an overall high GC content and G/C mutational bias in the 3^rd codon position, similar to that of host coding sequences. In contrast, Juan sequences are AT rich and have an A/T mutational bias in the 3^rd codon position. Also, a Juan copy was found inserted in close proximity to a histone gene in Culex quinqefasciatus. The apparent difference in mutational pressure between Juan and Juan is discussed. In a few species, a two to 100 fold higher copy number was determined for Juan by library screening. The differences described make for an interesting evolutionary study of two related retrotransposons in the mosquito. Finally, data reported here suggest recent activity of both Juan and Juan in some species. These include high sequence identity, intact open reading frames, a high ds/dn ratio among sequences, and ESTs that correspond to Juan in Aedes aegypti and Juan in An. gambiae. In addition, a RT-PCR product for Juan was found using An. gambiae cultured cells. With evidence of activity, these retrotransposons could contribute to the development of mutagenesis tools or transformation vectors for the study of medically important mosquitoes.

Using transposon display to identify polymorphic markers in the African malaria mosquito, Anopheles gambiae

Biedler J¹, Tu Z¹

¹Department of Biochemistry, Virginia Tech, Blacksburg, VA 24061, USA

Transposon display (TD), a modified form of amplified fragment length polymorphism (AFLP), has been successfully used to identify transposable element insertion polymorphisms (TEIPs) in Anopheles gambiae individual mosquitoes. TD differs from AFLP in that one of the primers is designed according to a transposable element. This technique was shown to be both specific and reproducible using several transposons in Anopheles gambiae. Re-amplification and sequencing of bands from a TD gel verified the presence of both transposon and genomic sequence, which allowed mapping of the transposon insertions onto the Anopheles gambiae genome. We also showed a relatively high level of insertion polymorphism of some of these transposons. With the completion of the An. gambiae genome sequence in the near future, the establishment of a robust TD assay for mosquitoes is invaluable. It will be a powerful new tool for the study of transposon activity and for population analysis of this medically important mosquito species.

cDNAs encoding putative odorant binding proteins and other ligand carrier proteins from the antennae of Anopheles gambiae.

Biessmann H¹, Walter MF¹, Justice R², Dimitratos S1, Woods DF¹

¹Devel. Biol Center, Univ. of California, Irvine, CA 92697 USA; ²Inscent, Inc., Irvine CA 92614, USA

To obtain a better understanding of the olfactory processes that allow mosquitoes to identify human hosts, a molecular study has been performed to identify and characterize molecules in the olfactory signaling pathway of the African malaria vector Anopheles gambiae. cDNA libraries from antennae of females and males were established and characterized. We report the isolation and preliminary characterization of eight cDNAs from these antennal libraries that encode putative odorant binding proteins (ODP). Their conceptual translation products show extensive sequence similarity to known insect OBP, especially to those of Drosophila melanogaster. The A. gambiae OBPs are expressed at different levels in the antennae of both genders. In addition, other cDNAs were found encoding small putative ligand carrier proteins. Two of these novel proteins show sequence similarity to the D. melanogaster protein TAKEOUT that is under control of a circadian clock and is linked to feeding behavior. A large-scale survey of 20,000 cDNA antennal clones on filter arrays revealed quantitative but not qualitative differences in antennal gene expression in males vs. females. OBPs are the most abundantly expressed genes in A. gambiae antennae, accounting for about15% of cDNAs.

Sphingomyelinase D from venoms of brown spiders: evolutionary insights from gene structure.

Binford GJ, Wells MA

Department of Biochemistry and Molecular Biophysics, University of Arizona, Tucson, AZ, USA

Loxosceles spider venoms are famous for causing dermonecrotic lesions in human tissues. The enzyme sphingomyelinase D (SMD) is a central causative agent for lesion formation. This enzyme is not known elsewhere in the animal kingdom, but is found in some pathogenic bacteria. We cloned a cDNA sequence from L. arizonica with homology only to previously reported N-terminal amino acid sequences for SMD from Loxosceles. We used this sequence to screen a L. arizonica genomic library. The gene spans at least 6,500 bp, contains 5 introns, and has at least one paralog. The presence of a signal sequence indicates SMD is expressed as a zymogen with a trypsin cleavage activation site. Weak similarity suggests SMD is a divergent member of the glycerophosphodiester phosphodiesterase family. These data suggest an evolutionary origin by gene duplication rather than horizontal transfer from bacteria.

The two Drosophila melanogaster receptors for tachykinin related peptides are differentially distributed in the brain

Birse RT, Muren JE, Nässel DR

Department of Zoology, Stockhohn University, SE 10691 Stockholm, Sweden

In Drosophila melanogaste two different G protein coupled receptors, NKD and DTKR, have been identified with tachykinin related peptides (TRPs) as likely ligands (Li et al., EMBO J. 10: 3221 322, 1991; Monnier et al., J. Biol. Chem. 267: 1298 1302, 1992). Transcripts of both receptor genes were shown to be expressed in the central nervous system. However, until now the distribution of the receptor proteins has not been studied. To identify possible sites of action of TRPs in the D. melanogaste brain we have raised antisera to portions of the NKD and DTKR proteins. As antigens for NKD we used two different peptides coupled to Keyhole limpet hemocyanin: (1) a portion of the 3^rd intracellular loop and (2) part of the C terminus. For DTKR we used a portion of the C terminus coupled to bovine serum albumin as antigen. The receptor antisera were used for westem blots on brain extracts. Both NKD antisera recognized protein bands of the same molecular weight (around 60 kDa) and the DTKR antiserum identified a band at about 65 kDa. The antisera were also employed for immunocytochemistry on sections of brains. The NKD protein was seen in neuronal varicosities in brain neuropils of the protocerebrum (central body, dorsal and lateral protocerebrum) and subesophageal ganglion, as well as in a small number of neuronal cell bodies. DTKR immunoreactivity was more abundant: labeled varicosities were detected in the central body, dorsal and lateral protocerebrum, the antennal lobes and part of optic lobe and cell bodies in the median neurosecretory cell group and a few other sites of the brain. Although the two receptor proteins in some cases are localized to the same general brain areas, it is clear that their neuronal distributions are mutually exclusive. Only DTKR was seen in antennal lobes and optic lobes and different layers of the central body contain NKD and DTKR immunoreactive varicosities. Taken together the distribution of the two receptors correlate well with the distribution of TRP containing neuronal processes. Thus the TRP signalling system appears to employ two distinct receptor types with a differential distribution. The functional consequences of the dual receptor system may in the future be approached in D. melanogaste by genetical disruption of each of the two receptors.

Patterning insect gnathal and thoracic appendages

Blachuta BJ¹, Cooley JR², Crance CE², Nagy LM²

¹Interdepartmental Program in Insect Science, University of Arizona, Tucson, AZ, USA 2Department of Molecular and Cellular Biology, University of Arizona, Tucson, AZ, USA

The development of the proximal distal axis of Drosophila melanogaste legs depends on the relative spatial amount of the morphogens wingless (wg) and decapentapiegic (dpp) in the early leg imaginal disc. High WG and DPP signals activate the expression of Distal-less (D11), which is necessary for the development of distal leg structures. While D11 and wg expression patterns are similar between the grasshopper Schistocerca americana leg and the D. melanogaste imaginal disc, expression patterns of dpp vary greatly between the two species. Although the dpp expression pattern differs in grasshoppers, high WG and DPP levels could nonetheless be required to set up the morphogenetic gradient. However, pSMAD, the downstream transducer of the DPP signal is localized in the same banding pattern as dpp in the grmhopper leg. Hence, it is more likely that D11 expression is not regulated exclusively by high levels of WG and DPP. In addition, the role of dpp, wg, and, D11 in the formation of branches was studied by looking at their expression in the ventral branches of the grasshopper mouthparts. The patterns of wg and dpp expression seen in the leg are similar in the grasshopper mouthpart during its patterning. wg is similarly expressed in the grasshopper mouthparts as it is in the leg, and there is no apparent modulation of wg expression during branch formation. pSMAD is expressed only in the distal tip of the mouthpart and not in the branches. These results suggest other factors likely establish branches in the grasshopper mouthpart. Thus although dpp, wg, and D11 are all involved in establishing the proximal distal axis during insect leg development, the specific roles of these gene products may vary.

Biochemistry and molecular biology of JH-regulated pheromone biosynthesis in pine bark beetles

Blomquist GJ¹, Tittiger C¹, Barkawi CL², Tillman JA², Seybold SJ²

¹Department of Biochemistry, University of Nevada, Reno, NV 89557-0014; ²Department of Entomology and Forest Resources, University of Minnesota, 219 Folwell Ave, St. Paul, MN 5108-6125

Important pheromone components of the pine bark beetles Ips pini and Dendroctonus jeffreyi are ipsdienol and frontalin, respectively. Bark beetle monoterpenoid pheromones have historically been thought to arise via simple modifications of host tree precursor molecules. A growing body of evidence now supports the de novo production of certain components. Since ipsdienol and frontalin synthesis in males is regulated by juvenile hormone (JH), we investigated the effects of JH on pheromone-related gene expression and metabolism in D. jeffreyi and I. pini. JH treatment induced a large increase in 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-R) gene expression, and a modest increase in HMG-CoA synthase mRNAs in both beetles, all of which correlate with increased pheromone production. HMG-R enzyme activity is also elevated by JH treatments, and radiolabeled mevalonate is incorporated into ipsdienol and frontalin in I. pini and D. jeffreyi, respectively. These data are strong evidence that the monoterpenoid pheromone components are derived de novo via the mevalonate pathway. They provide the basis for a paradigm shift of bark beetle pheromone production from the model in which host-tree precursors are modified to pheromone components to one where JH regulates de novo biosynthesis of key isoprenoid pheromone components.

Modified JcDNV somatic transformation vectors provide an efficient means of assessing promoter activity in insects

Bossin H, Shirk PD

USDA ARS Center for Medical, Agricultural and Veterinary Entomology, Gainesville, Florida

Plasmids containing a modified genome of the Junonia coenia lepidopteran densovirus (JcDNV) integrate into the genome of a host insect cell rather than becoming infectious. These plasmids have been utilized to achieve somatic transformation in Drosophila melanogaster that is stable from embryo to adult (Royer et al, 2001, Insect Molcular Biology 10, 275). We have assessed the effect of additional modifications including different expression cassettes on the efficiency of JcDNV plasmid somatic transformation activities in three orders of insects. The plasmid pJDsRed[3xP3EGFP]H, which contains the Densovirus P9 promoter controlling expression of DsRed fluorescent protein and the 3xP3EGFP expression cassette (Berghammer et al., 1999. Nature 402, p370), was injected into syncytial embryos of the fruitfly D. melanogaster w^m, the moths Plodia interpunctella and Ephestia kuehniella, and the beetle, Tribolium castaneum. Somatic transformation was observed on the basis of either DsRed and/or GFP fluorescence in G0 embryos and larvae of all four species at rates from 40-95%. Where comparisons could be made, the expression patterns were consistent with germ line transformed insects. Removal of the JcDNV coding sequences for nonstructural proteins or the right inverted terminal repeat had no effect on the rate of somatic transformation. Cloning of 3xP3EGFP in the unique PvuI site outside the JcDNV sequence did not affect the transformation rate either. When compared with observed production from the piggyBac gene vector, similar frequencies and patterns of DsRed and GFP expression were observed for pJDsRed[3xP3EGFP]H in D. melanogaster G0s. However, no detectable expression was observed from the piggyBac gene vector in P. interpunctella or E. kuehniella G0s while up to 90% of the insects expressed DsRed after somatic transformation with pJDsRed[3xP3EGFP]H. These experiments demonstrate the utility of JcDNV vectors in assessing promoter activity in insects.

piggyBac transposon-derived gene vector system activity in production of transgenic insects

Bossin H, Shirk PD

USDA ARS Center for Medical, Agricultural and Veterinary Entomology, Gainesville, Florida

The piggyBac transposon gene vector can be used to achieve germ line transformation in three insect orders. We have established protocols that result in consistently high transformation rates in Drosophila melanogaster by adjusting the timing of microinjection to a period of embryogenesis when there are few nuclei to transform. In addition, we have demonstrated that transformation with piggyBac is supported over a forty fold range of helper concentration in D. melanogaster w^[m] and is not subject to feedback inhibition at the highest concentrations of helper tested. We utilized the pB[3xP3EGFP]af (Berghammer et al., 1999, Nature 402, 370-371) transformation vector in combination with the phsp-pBac helper (Handler and Harrel, 1999, Insect Molcular Biology 8, 449-457) and varied the helper concentration from 0 to 600 ng per µl. A 20% transformation rate was maintained from 10 to 400 ng/µl. These observations suggest that the piggyBac gene vector can be used to achieve genetic transformation with a high probability of success because the transposition of the gene vector is not restricted by the mechanics of the transposase activity. In order to provide a more stable helper, the phsp-pBacwc helper was constructed by removal of both inverted terminal repeats. The phsp-pBacwc helper supports transformation rates identical to those of phsp-pBac. To establish the activity of an expression cassette containing a lepidopteran viral promoter IE1 (Jarvis et al., 1996, Protein Expr. Purif. 8:191-203), the vector pB[3xP3EGFP-IE1DsRed] was constructed and tested for activity in D. melanogaster. The IE1 promoter was active in the pupal and adult stages but not the embryonic or larval stages. The activity of this expression cassette is being assessed in lepidopteran and coleopteran species.

Forward genetic isolation of synaptic mutants in Drosophila melanogaster: slug-a-bed and rolling blackout in presynaptic mechanisms

Broadie K

Department of Biology, University of Utah, Salt Lake City UT 84112-0840. broadie@biology.utah.edu

Genetic screens in Drosophila melanogaster for paralyzed, or conditionally paralyzed, mutants have elucidated a number of fundamental neuronal mechanisms. Proteins identified in this way include founding members of ion channel families, e.g. paralytic (Na⁺ channel), and fundamental components of the synapse, e.g. shibire (Dynamin GTPase). In this seminar, I will present two new genes identified as paralytic mutants in flies: 1) slug-a-bed (slab) which encodes the sole ceramidase in the fly genome, and 2) rolling blackout (rbo) which encodes a novel 3-pass transmembrane protein. The slab gene was identified as an unconditional paralytic mutant with severely impaired presynaptic neurotransmitter release at the glutamatergic neuromuscular junction. Ultrastructural analyses reveal that synaptic vesicles in slab mutants are arrested in a tethered cytoplasmic state ("reserve pool") and lost from the docked state at presynaptic active zones ("readily releasable pool"). The function of slab ceramidase is to catalyze the conversion of ceramide to sphingosine. Thus, slab is required in the sphingosine lipid pathway for determining the ratio of reserve: docked vesicles at the synapse, and hence the efficacy of synaptic transmission. The rbo gene was identified as a temperature-sensitive paralytic mutant with a complete, reversible loss of both phototransduction in the eye and synaptic transmission at the nmj. Null mutants are embryonic lethal and nearly paralyzed. The expression of rbo is nervous system specific and the protein is present in the plasma membrane in most/all neurons (including motor neurons, interneurons and sensory neurons). In CNS neurons, the protein is restricted to axons and the synapse-rich neuropil; the protein also localizes to presynaptic neuromuscular junction boutons. In photoreceptors, rbo is acutely required for Ca²⁺ influx mediated by the transient receptor potential (trp) and trp-like (trpl) Ca²⁺ channels. We have hypothesized that rbo mediates Ca²⁺ influx through this class of Ca²⁺ channels (13 genes in flies) throughout the nervous system. Thus, rbo acts as a novel TM protein to acutely regulate Ca²⁺ channels essential for both sight and movement. On-going studies on slab and rbo, along with an extensive collection of other genes identified in the lab, will provide the foundation for dissecting mechanisms of synaptogenesis and neurotransmission.

Linkage analysis of resistance to Baciflus thuringknsis in cotton bollworm Heficoverpa armigera.

Brown S¹, Olsen K², Mahon R², Heckel DG¹

¹Centre for Environmental Stress and Adaptation Research, Department of Genetics, University of Melbourne, Parkville, VIC 3010, AUSTRALIA, ²CSIRO Entomology, Canberra, ACT 2601, AUSTRALIA.

The BX strain of H. arinigera is resistant to CrylA type Bt toxins and can complete development on transgenic cotton. To investigate the genetic basis of this resistance, a linkage map based on AFLPs was constructed from a backcross derived from a cross between BX and a susceptible strain. Only one linkage group showed an association with Bt resistance; an AFLP from this linkage group was cloned and sequenced for development as an STS marker. The H. armigera homologue of the 11 domain cadherin responsible for resistance in the YHD2 strain of the related species Heliothis virescens (Science 293: 856 (2001)) was mapped in this family, and found to reside on a different linkage group. This shows that BX does not have the YHD2 type of resistance mechanism. Several aminopeptidases were also mapped to separate linkage groups; and the search for a candidate resistance gene is still continuing.

Tribolium Hox genes repress antennal development in the gnathos and trunk.

Brown SJ

Division of Biology, Kansas State University, Manhattan, KS, USA

Homeotic genes determine developmental fate along the anterior-posterior axis in animals. Homeotic mutations alter developmental fate, and result in the misspecification of segmental identity in insects. Work in Drosophila melanogaster suggests that in their normal expression domains, some homeotic genes repress antennal development while directing segment-specific identity during adult development. Our embryonic analysis of Tribolium homeotic mutants indicates that repression of antennal development by homeotic genes is conserved and that it occurs throughout the gnathos and trunk. Antennae develop on posterior segments in the absence of homeotic gene function. In D melanogaster embryos, anterior transformation is most complete when other genes such as teashirt are also removed. However, our analysis of a putative Tribolium teashirt homolog suggests that while its role in leg development is conserved, it does not play a role in determining trunk identity. Therefore, Tribolium homeotic proteins may not require modifiers such as teashirt to determine developmental fate.

Odor and taste: Receptors, genes, and cells

Carlson JR

Dept. of Molecular, Cellular, and Developmental Biology, Yale University, New Haven CT, USA

We developed a computer algorithm to identify odor and taste receptors from the Drosophila melanogaster genome database. The algorithm identifies proteins with a particular structure, as opposed to a particular sequence. It maps the predicted products of open reading frames into an n-dimensional protein space and then determines which predicted proteins map into a portion of the space occupied by known G-protein-coupled receptors. We have isolated two large families of genes, the Or genes and the Gr genes, which are likely to encode odor and taste receptors. The molecular basis of odor coding has been studied by analyzing the expression and function of Or genes. The cellular basis of odor coding has been explored through physiological analysis of individual receptor neurons. We are now integrating the molecular and cellular maps of the olfactory system by recording from transgenic animals in which the promoters of individual Or genes are used to drive GFP expression. We record from neurons that are marked by GFP, thereby correlating particular Or genes with neurons exhibiting particular odor sensitivities. The goal is to understand the functional organization of the olfactory system at the molecular and cellular levels, and thereby to yield new insight into mechanisms of odor coding.

Factors controlling the retention or elimination of protein meals by the midgut of Aedes aegypti females.

Caroci AS¹, ², Noriega F¹

¹University of Arizona, Department of Biochemistry, Tucson, AZ, USA; ²Instituto de Ciencias Biomedicas, Universidade de Sao Paulo, Sao Paulo, SP, Brazil.

There is a relationship between the progress of digestion and the retention or elimination of the meal by the midgut of female Aedes aegypti. We are trying to identify factors involved in the regulation of this physiological process. The addition of Soy trypsin inhibitor (STI) to a protein meal prevented digestion and resulted in a rapid elimination of the undigested protein from the midgut. SDS-PAGE analysis of feces collected at different times after feeding showed the presence of undigested protein. The largest amounts of proteins in the feces were detected as soon as 2-5 hours after feeding; with most of the protein eliminated before 10 hours. The addition of free amino acids to a protein meal together with STI results in a significant increase in the retention of the meal by the midgut during the first 10 hours after feeding. The undigested meals were ultimately eliminated, but the "peak of elimination" was shifted from 2-5 to 15-24 hours after feeding. If digestion is not preceding normally the mosquito needs to vacate the midgut contents to make room for a new blood meal. The presence of free amino acids in the midgut lumen seems to be an important signal used by the mosquito to regulate the retention or elimination of the meal. Decapitation of the females immediately after feeding changed the patterns of the retention or elimination of the meal.

Modulation of vertebrate immune responses by saliva of the mosquitoes Aedes aegypti and Anopheles stephensi

Champagne DE, Kumar S, Wasserman H, Ibrajim S, Silver M

Department of Entomology and Center for Tropical and Emerging Global Diseases,University of Georgia, Athens, GA

Successful blood feeding requires that mosquitoes inject saliva containing antihemostatic enzymes and other proteins. Exposure of these antigenic proteins to the host may be expected to elicit an immune response that could be deleterious to the mosquito. We have found that saliva of two important vector species, Aedes aegypti and Anopheles stephensi, inhibits several aspects of the host immune response, including T-cell and B-cell proliferation and production of TH1 and TH2-type cytokines from T-cells. Marked decreases in the proportions of both CD4 and CD8 positive T-cells were observed. In contrast there appears to be little effect on MHCII positive cells including professional antigen presenting cells. The activity appears to be due to a single component of the saliva, a large (280-300 kDa) glycoprotein. This component is present in saliva of females but not males, and it is depleted from the salivary glands following a blood meal, indicating a role in blood feeding. It is possible that by modulating the host response to salivary antigens, this protein protects the feeding mosquito from potentially harmful host immune responses.

Detection of carbohydrate-binding molecules implicated in the establishment of mosquito infection on Plasmodium berghei ookinete

Chen C, Billingsley PF,

Department of Zoology, University of Aberdeen, Tillydrone Avenue, Aberdeen AB24 2TZ, U.K.

Interactions between the invasive Plasmodium berghei ookinete and the midgut epithelium are believed to be critical for the establishment of infection of the parasite to the mosquito vector. Specific sugar inhibitors, such as N-acetyl-galactosamine (GalNac), incorporated in the mosquito blood meal containing in vitro cultured ookinetes significantly reduced the infectivity of the parasite to the midgut of mosquitoes, suggesting that carbohydrate-lectin interactions play important roles during infection. An in vitro binding assay and direct cell autoradiography with radiolabelled neoglycoproteins of known sugar moieties and the mosquito midgut microvillar protein extracts were developed to examine the nature of such carbohydrate-lectin interactions between the parasites and mosquitoes. Results showed that the radiolabelled neoglycoprotein, N-acetyl-galactosamine-BSA (GalNac-BSA), and the mosquito midgut microvilli (Mv) membrane proteins could specifically bind to all immature, retort ookinetes, but not to mature ookinetes and other parasite forms, such as merozoites and shizontes. Furthermore, the binding of the radiolabelled GalNac-BSA or midgut Mv membrane proteins to the ookinetes was inhibited with the addition of competitive sugar inhibitors, GalNac or mannose, in a dose-dependent manner, in both the in vitro binding assay and ookinete whole cell autoradiography. In contrast, radiolabelled N-acetyl-glucosamine-BSA (GlcNac-BSA) could bind to merozoites, shizontes and, to a less extent, to the immature ookinetes, but not to mature ookinetes. The binding of GlcNac-BSA to parasites was only inhibited by GlcNac itself, but not by either GalNac or mannose at 200 mM concentrations. In addition, deglycosylated midgut Mv proteins failed to bind to any parasite forms, as observed in ookinete cell autoradiography. We postulate that an N-acetyl-galactosamine/mannose-specific lectin is present on the immature transforming ookinetes, and is responsible for interactions with possible carbohydrate-receptor ligands from the mosquito midgut microvilli. Further experiments with plant lectins of known sugar-specificity support the presence of several unique glycoproteins between 25 and 32 kDa, as examined by autoradiography of isolated, radiolabelled Mv proteins in the SDS-PAGE under the reducing conditions, which contain GalNac and mannose moieties as potential ligands for the ookinete lectin.

Molecular characterization of bilin binding protein (BBP) from Hyphantria cunea

Cheon HM¹, Kong SY¹, Kim HJ¹, Yun CY², Seo SJ¹

¹Division of Life Science, College of Natural Sciences, Gyeongsang National University, Chinju, ²Department of Biology, Taejon University, Taejon, Korea.

The bilin-binding protein is a blue pigment protein binding heme-related compound in insects. The amino acid sequence from the bilin binding protein (BBP) of the fall webworm Hyphantria cunea has been determined. The cDNA has a length of 1333 bp coding for a 288 residue protein with a predicted molecular mass of 33kDa. This cDNA has a homology with human apolipoprotein D and other insecticyanin. Northern blot analysis clearly showed a ca 1.5kb transcript in the epidermis, brain, and fat body. Developmental expression profiles clearly showed that BBP transcripts are present in the whole body at only 4- and 6-day-old pupae. Computer searches of data banks yielded in a new member of this superfamily, lipocalin protein superfamily whose other members transport small hydrophobic ligands in a wide variety of biological context.

Very similar yolk protein genes in Hyphantria cunea

Cheon HM¹, Kim HJ¹, Yun CY², Lee HJ³, Seo SJ¹

¹Division of Life Science, College of Natural Sciences, Gyeongsang National University, Chinju, ²Department of Biology, Taejon University, Taejon, ³Department of Biology, Korea University, Seoul, Korea.

Yolk protein 1 and 2 in the fall webworm, Hyphantria cunea, were detected in large amounts from the ovaries of 10-day old pupae and accumulated in the eggs. We isolated and sequenced cDNA clones corresponding to the two yolk proteins. The cDNAs for YP1 (1.2 kb) and YP2 (1.1 kb) code for 290 residue proteins. The sequence identity between YP1 and YP2 was very high of 79.9%. Two H. cunea YPs were most closely related to the follicle specific yolk protein 4 from the moths, P. interpunctella and G. mellonella, but not related to Vg sharing similarity with vertebrate lipase. Northern blot analysis showed YP1 and YP2 transcripts were present in only female fat body and at trace level in the ovary. YP1 and YP2 cDNAs began to express from 10-day-old pupae and increased to adult stage. This result suggests that H. cunea YP1 and YP2 genes are expressed in sex-, tissue- and stage- specific way. The possibility of gene duplication and overlapping function for two very similar YPs in H. cunea will be discussed..

Pheromone Biosynthetic Pathways in the moths Helicoverpa zea and Helicoverpa assulta

Choi M-Y, Jurenka RA

Department of Entomology, Iowa State University, Ames IA 50011-3222 USA

Sex pheromones of many lepidopteran species have relatively simple structures consisting of a hydrocarbon chain with a functional group and usually one to several double bonds. The sex pheromones are usually derived from fatty acids through a specific biosynthetic pathways. We investigated the incorporation of deuterium-labeled palmitic and stearic acid precursors into pheromone components of Helicoverpa zea and Helicoverpa assulta. The major pheromone component for H. zea is (Z)11-hexadecenal (Z11-16:Ald) while H. assulta utilizes (Z)9-hexadecenal (Z9-16:Ald). We found that H. zea uses palmitic acid to form Z11-16:Ald via 11 desaturation and reduction, but also requires stearic acid to biosynthesize the minor pheromone components Z9-16:Ald and Z7-16:Ald. The Z9-16:Ald is produced by 11 desaturation of stearic acid followed by one round of chain-shortening and reduction to the aldehyde. The Z7-16:Ald is produced by 9 desaturation of stearic acid followed by one round of chain-shortening and reduction to the aldehyde. H. assulta uses palmitic acid as a substrate to form Z9-16:Ald, Z11-16:Ald and 16:Ald. The amount of labeling indicated that the 9 desaturase is the major desaturase present in the pheromone gland cells of H. assulta; whereas, the 11 desaturase is the major desaturase in pheromone glands of H. zea. It also appears that H. assulta lacks chain-shortening enzymes since stearic acid did not label any of the 16-carbon aldehydes. It will be of interest to determine the evolutionary relationship between the desaturases present in the Helicoverpa complex.

Comparative genomics of protein-coding genes: Anopheles gambiae vs. Drosophila melanogaster.

Clark AG, Sutton GG, Charlab R, Nusskern D, Lai J, Yandell M, Kraft C, Evans CA, Rusch DB, Subramanian G, Smith HO, Holt RA, and the Anopheles Consortium

Pennsylvania State University, 208 Mueller Laboratory, University Park, Pa 16802

The genome of the malaria vector, Anopheles gambiae, was submitted to a Whole Genome Shotgun procedure to establish an assembly of the complete euchromatic portion of the genome. Libraries were made from the inbred PEST strain provided by Frank Collins (Notre Dame). DNA was size fractionated to produce libraries of 2 kb, 10 kb and 50 kb inserts. 2,059,200 of these clones were sequenced at Celera, and an additional 195,345 were sequenced at Genoscope. In addition, a total of 27,324 BAC clones were end-sequenced at TIGR and Genoscope. Final sequence coverage was 10.15 X. At Celera Genomics these fragments were submitted to their whole genome assembly programs, which produced 8,997 scaffolds representing 278 Mbp of eukaryotic portion of the genome. The median scaffold size was 6.1 Mbp. These scaffolds were submitted to the Genome Crawler, a series of programs that perform automatic gene finding, megaBLAST pre-computes, and a variety of automated annotating functions. The starting point for the analysis presented in this talk is the set of inferred transcripts derived from Drosophila melanogaster and from Anopheles gambiae. These were submitted to an all-vs-all BLAST search, and the reciprocal best blast hits were then aligned. The resulting xxx alignments were examined for both qualitative assessment of presence/absence of gene sharing in pathways of interest, and for quantitative levels of divergence. Overall the nonsynonymous divergence rate was xxx, while the synonymous divergence was xxx, or essentially saturated. Some patterns of divergence between Drosophila and Anopheles in known developmental pathways will be illustrated. Single nucleotide polymorphisms (SNPs) were detected in the inbred strain in a highly clustered manner, suggesting heterozygosity for ancient polymorphic inversions and/or the possibility that there are segmental introgressions from other members of the species complex. SNPs were identified from alignments with a large collection of ESTs, and their distribution is also highly nonhomogeneous.

Sequence analysis of candidate genes associated with longevity in lines of Drosophila melanogaster.

Clark C, Keim P, Service PM

School of Biological Sciences, Northern Arizona University, Flagstaff, AZ 86011-5640

In recent years, sequencing analysis has become an important tool in molecular biology. Additionally, the completion of the Drosophila melanogaster genome has also become a valuable resource for researchers. The information that is available regarding the nucleotide sequences of genes that are associated with longevity has provided us with an excellent tool for comparing genetic differences. A long-standing selection experiment (Rose, 1984) has produced populations of flies with divergent life spans. From these populations, we produced a series of long and short-lived inbred lines. The present study entails comparing the genetic sequences among those inbred lines and the comparison of our lines with the published sequence for several candidate genes. If a candidate gene affects longevity differences in our experimental populations, then we would expect to see nucleotide and amino acid differences when we compare long and short-lived inbred lines. Any differences observed between the published sequence and those lines with increased longevity are subject to further investigation. A significant difference may be defined as a change in amino acid sequence that results in a possible change of function at the protein level. All changes that occur at the level of the amino acid do not result in altered function. The purpose of this study is to aid in the identification of new candidate genes for longevity and to provide additional evidence for the existence of current candidate genes associated with longevity.

The insect cytokine plasmatocyte spreading peptide (PSP) interacts with its receptor through highly specific domains

Clark KD¹, Arora A², Volkman BF³, Strand MR¹

¹Department of Entomology, University of Georgia, Athens GA; ²Department of Entomology, University of Wisconsin-Madison, Madison, WI; ³Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI

Plasmatocyte Spreading Peptide (PSP) is a 23 amino acid cytokine that induces a class of insect immune cells called plasmatocytes to spread on foreign surfaces. PSP consists of an unstructured N-terminus (residues 1-6) and a well-defined core (residues 7-23) stabilized by a disulfide bond and a short -hairpin. Mutagenesis studies have identified several domains critical for activity. These include the cysteines used to form the disulfide bond, charged residues within the -hairpin and, most importantly, residues Glu¹ and Phe³. The structure of Phe³ is essential, as adding a hydroxyl (Tyr), or altering the chirality (D-Phe), or replacing the aromatic ring with a branched aliphatic chain (Val) each destroyed the activity. Activity was partially restored by the addition of a methylene group to Val (to make Leu), whereas removal of a methylene group from Phe³ (phenyl-Gly) destroyed activity, indicating a branched carbon chain with a methylene spacer as the minimum functional structural motif. We also tested alterations of Glu¹ and Asn². Ala substitutions at Glu¹ or Asn² both increased activity, but a Glu¹ deletion eliminated activity, indicating that only the charged N-terminal amine is essential. Mutant peptides lacking activity were assayed for antagonism of wild-type PSP activity. Phe³ substitutions were unable to antagonize PSP, whereas mutants lacking the charged N-terminal amine were effective. These data suggest that Phe³ is essential for PSP/receptor binding. This Phe³-mediated interaction likely serves to stabilize the normally unstructured N-terminus and therefore facilitates binding and activation of the receptor by the N-terminal amine.

Race to death: The encapsulation response by insect hemocytes is mediated by the surface coat proteins and cuticular proteins of Heterorhabditis bacteriophora.

Cox-Foster DL, Kazi A, Miller K.

Department of Entomology, Penn State University, University Park, PA, USA.

Entomopathogenic nematodes, like Heterorhabditis bacteriophora, have evolved a lifecycle in which survival depends upon overcoming insect immunity for survival and reproduction. After the nematode invades the insect hemocoel and before an extensive cellular immune response by the insect, the symbiotic bacterium Photorhabdus luminescens must be released from the nematode gut and established for nematode reproduction. The underlying mechanisms used by the nematode to evade the insect immune response and the countermeasures used by the insect present a unique system for study of host/pathogen coevolution and for discovery of key regulators of cellular immunity. Evidence is presented that the initial, immediate insect immune response is critical and that the nematode itself is contributing to overcoming this defense. The interactions of the nematodes and the hemocytes from a series of resistant and susceptible hosts (Manduca sexta, Galleria mellonella, Popilla japonica, and Acheta domesticus) were visualized by light microscopy in sterile, in vitro cultures, and captured with time-lapse computer-generated movies (available via web-site access). In addition, the cellular interactions were examined using scanning electron microscopy. Initial recognition of the nematode by the hemocytes determines success of nematode. In the resistant host M. sexta, hemocytes rapidly recognize the nematode ends and encapsulate the entire nematode, while producing reactive oxygen species. In a semi-permissive host (P. japonica), recognition is also rapid but directed first at the middle of the nematode and then the ends, permitting release of the bacteria. In the susceptible host G. mellonella, hemocyte recognition is weak, allowing release of the bacteria and survival of the nematode. Preliminary data suggest that less than 15 major proteins are present in the surface coat proteins, and that these can disrupt melanization and coagulation by M. sexta hemocytes. The nematode/bacterium produce factors eliminating reactive oxygen species that underlie the killing of invaders by insect hemocytes. In a semi-permissive host like P. japonica these factors may permit the nematode to survive until the bacterium can act. Thus, a triad of interactions governs the fate of the nematode/bacterium versus insect.

Immune mechanisms in mosquitoes: Cells, gene expression and melanin

Christensen BM¹, Li J², Chen CC³

¹Department of Animal Health and Biomedical Sciences, University of Wisconsin, Madison, WI 53706, USA; ²Department of Pathobiology, University of Illinois, Urbana, IL 61801, USA; ³Department of Parasitology, National Yang-Ming University, Taipei, Taiwan.

The innate immune response of insects is receiving considerable attention from the general immunology community, primarily because of the striking similarity with acute phase responses of vertebrates. The primary emphasis in recent years has centered on the insect humoral response and the regulation of immune peptide production. Recently we have begun studies of the cellular response of mosquitoes and the role hemocytes play in phagocytosis and melanotic encapsulation reactions against malaria parasites and filarial worms. Circulating hemocytes of Aedes aegypti and Armigeres subalbatus consist of granulocytes and oenocytoids, with granulocytes being the predominant cell type and also the hemocyte responsible for phagocytosis. Oenocytoids are less numerous, but these hemocytes are responsible for the production of key enzymes involved in the production of melanin. Transmission electron microscopy and colloidal gold labeling studies determined that two of the rate-limiting enzymes, phenoloxidase and phenylalanine hydroxylase, are produced in oenocytoids. We have been able to use the Sindbis virus transducing system in gene silencing experiments to verify the role specific genes play in melanotic encapsulation. It is anticipated that our successful construction of cDNA libraries from immune-activated hemocytes from these two mosquito species will greatly enhance our ability to better understand gene expression involved in the cellular immune response of mosquitoes against the parasites they transmit. Supported by NIH grant AI 19769 and AI 46032.

A type III secretion system facilitates the conversion to mutualism in a lineage of insect endosymbionts.

Dale C¹, Plague GR², ³, Wang B², Ochman H¹, Moran N²

¹Department of Biochemistry and Molecular Biophysics, ²Department of Ecology and Evolutionary Biology and ³Center for Insect Science, University of Arizona, Tucson, Arizona 85721

The view that parasites can develop cooperative symbiotic relationships with their hosts is both appealing and widely held, however, there is no molecular genetic evidence of such a transition. Here we demonstrate that a mutualistic bacterial endosymbiont of grain weevils maintains and expresses inv/spa genes encoding a type III secretion system (TTSS) homologous to that used for invasion by bacterial pathogens. Phylogenetic analyses indicate that the inv/spa genes were present in a pre-symbiotic ancestor of the weevil endosymbionts, occuring at least 50 million years ago. The function of the inv/spa genes in maintaining symbiosis is demonstrated by the up-regulation of their expression under both in vivo and in vitro conditions that coincide with host cell invasion.

The epiproctodeal glands and their apparent release of an ecdysterostatic peptide in Manduca sexta.

Davis NT¹, Blackburn MB¹, Golubev EG², Hildebrand JG¹

¹Division of Neurobiology, Arizona Research Laboratories, University of Arizona, Tucson, AZ, 85721 0077, USA; ²Insect Biocontrol Laboratory, USDA, ARS, PSI, BARC West, Beltsville, MD, 20705, USA

In immature insects the release prothoracicotropic hormone (PTTH) from the brain activates the prothoracic glands to cause a rapid increase in ecdysone titer that initiates the molting process. For normal development to continue, the ecdysteroid titer must then decline rapidly. Recently, it has been shown that a myoinhibitory peptide, first identified in Manduca sexta, acts as an ecdysterostatic factor to block PTTH stimulated secretion of ecdysone in Bombyx mori. Using an antiserum to M. sexta myoinhibitory peptide (MIP), we have found a pair of epiproctodeal glands, that are MIP immunoreactive. In 4th instar larvae these glands apparently release MIP into the hemolymph at a time that corresponds to the decline in ecdysteroid titer in M sexta. The structure of these gland cells was described in a previous study, and their cells were shown to be multinucleate, and the gland is located on the proctodeal nerve at the junction of the hindgut and rectum. We have demonstrated that the glands have a very extensive array of varicose, neurohemal processes extending on the surface of branches of the proctodeal nerve. The glands exhibit a distinct secretory cycle that is correlated with the molting cycle. In the fourth larval instar, the glands become depleted at about the time of the ecdysone peak, and this depletion is followed by a phase of synthesis of MIP for release in the next instar. These glands are also found in pupae and adults, and we have found similar glands in B. mori.

DNA polymorphisms detected in a population of glassy-winged sharpshooters (Homalodisca coagulata) from Weslaco, Texas by PCR-based DNA fingerprinting methods

de León JH

Beneficial Insects Research Unit, Subtropical Agricultural Research Center, Agricultural Research Service, United States Department of Agriculture, Weslaco, Texas 78596

The glassy-winged sharpshooter is a large and slender xylem feeding leafhopper measuring about half an inch in length that was native to the southeastern United States, including Texas. Recently, this sharpshooter has become a concern in California because it vectors a strain of Xylella fastidiosa, a bacterium that causes Pierce's Disease in grapevines. This disease poses a potentially serious threat to the wine and table grape industry in California. The current work was undertaken to develop molecular genetic makers for the glassy-winged sharpshooter by various PCR-based DNA fingerprinting methods for the purpose of estimating the level of genetic variation within and among populations with the aim of achieving genetic information useful for improving biological control of this leafhopper. These fingerprinting methods included RAPD (Random Amplification of Polymorphic DNA), RAMP (Randomly Amplified Microsatellite Loci), and SAMPL (Selective Amplification of Microsatellite Polymorphic Loci). A combined total of about 204 polymorphic bands were detected with the three methods and three insects per primer combination (77 total), specifically 73, 79, and 52 polymorphic bands were generated by RAPD, RAMP, and SAMPL, respectively. We than applied and compared these PCR-based DNA fingerprinting methods to a local population of thirty glassy-winged sharpshooters and demonstrated extensive genetic variation within the population.

Peritrophic matrix proteins of the mosquitoes Anopheles gambiae and Aedes aegypti.

Devenport M¹, Shao L¹, Fujioka H², Jacobs-Lorena M¹.

¹Department of Genetics and ²Department of Pathology, Case Western Reserve University, Cleveland, OH.

The PM is a thick extra cellular structure secreted by the mosquito midgut epithelial cells, which completely surrounds the blood meal and is a potential barrier to malaria parasite development. Antisera were raised against newly identified PM proteins from Aedes aegypti and Anopheles gambiae and used to study their secretion and localization by immunofluorescence and immunoelectron microscopy. In Ae. aegypti midgut epithelial cells, RER cisternae are often assembled into characteristic whorls that unfold after a blood meal, correlating with activation of protein synthesis. Thus mRNAs encoding PM proteins (rather than the proteins themselves) are thought to be stored in midgut epithelial cells and their translation induced by blood feeding. Consistent with this view, mRNAs encoding the Ae. aegypti PM proteins AEIMUC and Aa-Aper50 are present before and after blood feeding, while the corresponding proteins can only be detected after blood feeding. In contrast, midgut epithelial cells of anophelines contain a large number of apical secretion granules before the blood meal. Upon blood feeding, the apical granules disappear and their contents are presumably released into the lumen. Thus, at least some of the PM proteins of anophelines are thought to be stored prior to blood feeding. In agreement with this model, two cloned An. gambiae genes, Ag-Aper1 and Ag-Aper14, are both expressed and translated prior to blood feeding and co-localize to secretory vesicles lying beneath the epithelial cell apical region. After blood feeding both Ag-Aper1 and Ag-Aper14 localize to the PM and are depleted from the epithelial cells.

Molecular identification of bursicon, the insect cuticle sclerotizing hormone

Dewey IL, Honegger H-W

Dept. of Biological Sciences, Vanderbilt University, Nashville, TN 37235, USA

Bursicon is the key hormone responsible for triggering the darkening and hardening (sclerotization) of new cuticle after the insect has shed its old one during ecdysis. Its actions and timed release are of critical importance to the survival of all insects and, most likely, all arthropods. We obtained four partial amino acid sequences of bursicon from the cockroach Periplaneta americana by microsequencing protein from one of four bursiconbioactive spots in two dimensional gels. All of the P. americana peptide sequences align with the D. melanogaster CG13419 gene product. Evidence supporting the hypothesis that CG13419 codes for bursicon will be presented. The CG13419 gene is found on the third chromosome at position 93F. Its gene product is predicted to be a 19 kDa protein and has a C terminal cystine knot like domain (CTCK), which is predicted for formation of homodimers. Bursicon functions as a dimer. We have identified partial sequences from Anopheles gambide (mosquito) and Apis mellifera (honey bee), which are 92% and 83% identical, respectively, to the CG 13419 gene product. We identified the cellular localization of CG.13419 in the ventral nervous system of third larval instar D. melanogaster using in situ hybridization analysis. The CG]3419 labeled neurons were also crustacean cardioactive peptide (CCAP) immunoreactive. We are currently producing recombinant CG13419, which we expect will show bioactivity in the ligated fly bioassay, a well known assay for bursicon activity. A positive result will provide additional evidence that the sequence CG13419 is the bursicon sequence of D. melanogaster. Elucidation of the sequence and structure of this hormone will finally resolve a major unsolved problem in insect endocrinology.

Spatial and temporal binding sites of sex-peptide pheromones in Drosophila melanogaster females.

Ding Z, Ottiger M, Kubli E

Zoologisches Institut der Universitaet, Zuerich-Irchel, Winterthurerstrasse 190, CH-8057 Zuerich, Switzerland. E-mail: dingzb@zool.unizh.ch

Mating in Drosophila melanogaster, as in many other insects, significantly affects female reproductive behavior and physiology. Two conspicuous changes are 1) elevation of egg laying and 2) decreased receptivity. Sex-peptide and DUP99B, the peptides of the Sex-peptide pheromone family, elicit these two post mating responses. To elucidate the mechanism of action, we identified the target sites and compared the binding properties of the peptides. Cryostat sections of adult animals were incubated with alkaline phosphatase labelled peptides. In virgin females, the peptides have widespread but specific target sites located in the nervous system and in the genital tract. Incubation of sections of mated females with AP-peptides showed that some of these target sites are blocked by the peptides transferred during copulation. These results suggest that the binding sites characterized in the virgin females are indeed the in vivo targets of the two peptides. Neuronal binding is dependent on the C-terminal parts of the peptides, binding in the genital tract is less specific in terms of peptide sequence. On affinity blots AP-peptides bind to proteins extracted from abdomen and head+thorax, respectively. These proteins are membrane bound and are separable on PAGE. Taken together, we conclude that the binding proteins of the neuronal system and the genital tract differ in their molecular properties. Calculation of Kd's and the minimum concentrations necessary for binding, suggest that SP is the key player. DUP99B may act synergistically with SP to produce a maximal post mating response.

Cloning and developmental expression of cDNAs encoding putative laccases from Manduca sexta and Anopheles gambiae.

Dittmer NT¹, Suderman RJ¹, Jiang H¹, Zhu Y-C², Gorman MJ¹, Kramer KJ², Kanost MR¹

¹Department of Biochemistry, Kansas State University, Manhattan, KS 66506, and ²Grain Marketing and Production Research Center, ARS-USDA, 1515 College Avenue, Manhattan, KS 66502.

We have cloned 2 cDNAs for the phenoloxidase laccase from Manduca sexta and 1 from Anopheles gambiae by a combination of RT-PCR, RACE-PCR, and library screening methods. The M. sexta laccase 1 and 2 cDNAs encode 801 amino acid (aa) and 760 aa proteins respectively, while the A gambiae laccase cDNA encodes a 1009 aa protein. All 3 cDNAs contain putative secretion signal sequences and the 10 His and 1 Cys residues shown to be necessary for copper binding in fungal laccases. Novel to the insect laccases is a larger amino terminal sequence, with the A. gambiae laccase nearly 350 aa longer and the M sexta laccases 200 aa longer than the fungal proteins in this region. These extensions appear to be unique as they share no sequence similarity to each other. Northern blot analysis has identified a single transcript of ~4.4 kb for the A. gambiae laccase, and ~3.6 kb for both M. sexta laccase 1 and 2. Northern blot analysis has also shown the A. gambiae laccase to be expressed in all life stages. RT-PCR was used to examine the tissue distribution and developmental profile of laccase gene expression during the last larval stage of M. sexta. Laccase 1 was most abundant in the midgut, Malphigian tubules and epidermis, with reduced expression in the fat body and very low expression in hemocytes. Laccase 1 was constitutively expressed but showed enhanced expression in the pharate pupal and pupal epidermis, consistent with its presumed role in cuticle sclerotization. Laccase 2 was most abundant in the epidermis, followed by low expression in the midgut and Malphigian tubules, and very low expression in the fat body and hemocytes. Similar to laccase 1, laccase 2 is also constitutively expressed with enhanced expression in the epidermis during periods of molting. The expression of laccase in the fat body led us to examine if either gene was stimulated upon microbial challenge. Preliminary experiments indicate that laccase 1 is up regulated by the gram-negative bacteria Escherichia coli, but not by the gram-positive bacteria M. luteus or by yeast. In contrast, laccase 2 was induced by all 3 microbial challenges. Future experiments are planed to further investigate the potential role of laccase in the immune response.

Comparison of two cloned monoamine transporters from the CNS of the cabbage looper moth Trichoplusia ni

Donly BC¹, Gallant P¹,², Malutan T¹,², McLean H², Verellen L¹, Caveney S²

¹Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada, London, Ontario, Canada N5V 4T3; ²Department of Zoology, The University of Western Ontario, London, Ontario, Canada N6A 5B7

The Na⁺/Cl^- dependent family of neurotransmitter transporters includes those responsible for the uptake of monoamines. We recently characterized a novel phenolamine transporter from the nervous system of the cabbage looper (Trichoplusia ni) that takes up octopamine with high affinity. To demonstrate that this lepidopteran octopamine transporter (OAT) is distinct from known dopamine transporters characterized in other organisms, we have also cloned the T. ni dopamine transporter (DAT) for comparison. Here we show that the T. ni DAT has greater similarity to other known DAT's and has a different pattern of expression in caterpillar nervous system compared to that of T. ni OAT. We also compare the function and pharmacology of the two transporters expressed in cultured insect cells and show that they have different affinities for various biogenic amines as well as distinctive pharmacological properties.

A novel gene, clone 50, expressed at specific stage of Maduca sexta is down-regulated by juvenile hormone and 20E

Du J, Hiruma K, Riddiford LM

Department of Zoology, University of Washington, Box 351800, Seattle, WA 98195-1800, USA

A novel gene, clone 50, was cloned from the epidermis of tobacco hornworm, Manduca sexta, using PCR-based Suppression Subtractive Hybridization. It belongs to a gene family that includes takeout of Drosohila melanogaster and several JH-binding proteins. Clone 50 mRNA first appears in the epidermis on day 0 of the 5^th instar and rises to its peak expression by mid-day 2, then declines rapidly on day 3 and is gone by the onset of wandering. No expression was seen in the fat body, muscle, eye and wing discs. After allatectomy early in the 4^th instar that induced precocious metamorphosis, clone 50 mRNA increased to high levels by 33h, then disappeared by the onset of wandering. Allatectomy after the critical period for juvenile hormone (JH) in the larval molt caused a more rapid upregulation on day 1 of the 5^th instar. The JH mimic pyriproxifen suppressed most expression both in normal and allatectomized larvae, indicating that the appearance of clone 50 mRNA is initiated by the decline in JH in the final larval instar. Infusion of 20-hydroxyecdysone (20E) into ligated abdomens of day 2, 5^th instar larvae and culture of day 2, 5^th abdominal epidermis with 20E in vitro caused a rapid decline of clone 50 mRNA. A slower and variable decline also occurred in controls treated with hormone-free media, indicating that another factor may be present in the larva that sustains high expression. Thus, clone 50 mRNA is expressed only in epidermis of the final instar larva and only when JH declines. Its expression is terminated by the commitment peak of ecdysone as metamorphosis begins. The role of the protein is currently under study. Supported by NIH.

Characterization of heme oxygenase and biliverdin reductase homologues from Drosophila melanogaster and Anopheles gambiae

Dunkov BC, Georgieva T, Georgiev I

Department of Biochemistry and Molecular Biophysics and Center for Insect Science, University of Arizona, Tucson, AZ, USA.

In vivo heme is degraded by heme oxygenases (HOs) yielding biliverdin, carbon monoxide and iron. The released biliverdin can be further converted to bilirubin by biliverdin reductases (BVRs). Oxidative degradation of heme is essential for iron reutilization and the cleavage products of the porphyrin ring play important physiological roles as antioxidants and/or signaling molecules. While heme catabolism in vertebrates is well understood, little is known about how insects deal with endogenous and exogenous heme. In an attempt to investigate insect heme degradation, we have identified genes and cDNAs encoding homologues of the vertebrate heme oxygenase and biliverdin IX reductase in the genome sequences and EST collections of Drosophila melanogaster and Anopheles gambiae. The genomes of both dipterans encode single HO homologues, in contrast to mammalian genomes, which have three HO genes. Conservation of sequence motifs and catalytically important residues in the deduced proteins suggest possible conservation of function between the insect and vertebrate enzymes. Northern blotting experiments were employed to study the developmental and organ specific expression patterns of HO and BVR messages. In D. melanogaster both messages are better represented in the larval midgut than in the fat body and are abundant in the testes and ovaries of adult flies. Iron and/or heme enrichment of the diet leads to increase in the message abundance. In female A. gambiae mosquitoes BVR messages are detected only in the midgut, Malpighian tubules and ovaries. The abundance of BVR messages increases in the midgut 24h after blood feeding. We also report the production of recombinant HO and BVR from D. melanogaster and BVR from A. gambiae. The recombinant BVR enzymes effectively converted , ,and , but not biliverdin isomers into the corresponding bilirubins and also displayed flavin and ferric reductase activities, much like the vertebrate biliverdin reductases. In addition, we found that insecticyanin, a biliverdin IX??chromoprotein from the hemolymph of Manduca sexta, is a good substrate for these dipteran BVRs. The results of these studies are discussed in the light of the possible functions of these enzymes in insect iron and heme homeostasis as compared to their vertebrate counterparts.

Expression studies on Drosophila melanogaster G-protein coupled receptors

Evans PD, Reale V, Chatwin H, Yu E

The Laboratory of Receptor Signalling, The Babraham Institute, Babraham, Cambridge, CB2 4AT, UK

A wide range of seven transmembrane spanning (7-TM) G-protein coupled receptors have been cloned and expressed from the fruitfly, Drosophila melanogaster. Many of them can be specifically activated by either biogenic amines or neuropeptides and some show a high degree of structural conservation with their vertebrate counterparts. Multiple receptor subtypes have been demonstrated for particular receptor classes. The power of D. melanogaster genetics should allow the identification of the functional roles of such receptors. However, it is clear that functional expression studies on such receptors in heterologous expression systems, such as insect or vertebrate clonal cell lines or Xenopus oocytes, can provide much information about their potential endogenous ligands, pharmacology and potential coupling to second messenger systems. A number of examples of such studies will be discussed to illustrate some of the general phenomena that have been discovered using this approach. The concept of the agonist-specific coupling of specific receptors to different second messenger pathways will be discussed in relation to studies on a cloned octopamine/tyramine receptor (Arakawa et al., 1990, Neuron 2:343-354; Robb et al., 1994, EMBO, J., 13:1325-1330) and on a novel cloned Neuropeptide F-like receptor (Feng et al., 1999, Soc Neurosci. Abstr., 25:183). Recent expression studies on a novel cloned D. melanogaste -adrenergic-like receptor (Yu et al., 2000, Society Neuroscience Abstracts, 26:916) will also be discussed. The completion of the sequencing of the D. melanogaster genome makes the identification of potential 7-TM receptor sequences much easier, but functional genomic studies will also be required to assess the physiological roles of these receptors.

Plodia interpunctella 0-1,3-glucan recognition protein Properties of the N terminal carbohydrate binding domain

Fabrick JA¹,², Baker JE¹, Kanost MR²

¹USDA ARS Grain Marketing and Production Research Center, 1515 College Ave., Manhattan, KS 66502, USA; ²Department of Biochemistry, Kansas State University, Manhattan, KS 66506, USA.

Bacteria and fungi possess specific molecular pattern molecules that form essential structural scaffolding. Typical molecular patterns associated with Gram negative bacteria, Gram positive bacteria, and fungi include lipopolysaccharide, peptidoglycan, and 0-1,3-glucan, respectively. Invertebrate pattern recognition molecules function to stimulate the host innate immune response after encountering such nonself moieties. The pyralid moth, Plodia interpunctella, possesses a 0-1,3-glucan recognition molecule (PiPGRP) that may function to continuously survey the hemolymph for the presence of foreign fungal cells. The PiPGRP possesses two putative domains consisting of a novel N terminal carbohydrate recognitioni'domain and a C terminal glucanase like domain, which can be separated by an in vitro proteinase treatment. The PiPGRP C terminal domain sequence shares similarity with other recognition proteins and 0-1,3- glucanases from bacteria and a sea urchin, whereas the N terminal sequence is unique to members of the arthropod GRP family and lacks sequence similarity with the 0- 1,3glucanases. The full length PiPGRP and constructs corresponding to 118- and 18 I-residues from the N-terminus and 290-residues from the C-terminus of PiPGRP were expressed as recombinant proteins using an Escherichia coli heterologous expression system. Circular dichroisin (CD) analysis of the 118-and 18 1 -residue N-terminal constructs indicate that the recombinant proteins are folded and possess primarily a helical secondary structure. The full-length protein binds to insoluble P-1,3-glucan and causes in vitro aggregation of Gram positive and Gram negative bacteria, as well as yeast. The 181-residue N-terminal construct causes significant aggregation of yeast cells but no aggregation of bacteria. These data suggest that the PiPGRP functions as a pattern recognition molecule in the innate immune system of P. interpunctella and that the N-terminal domain possesses a carbohydrate binding site necessary for recognition of non-self.

Analysis of dietary proteins derived from prey eggs and an embryonic cell line and their effects on the fecundity of Orius insidiosus

Ferkovich SM, Shapiro JP

USDA-ARS Center for Medical, Agricultural, and Veterinary Entomology, 1700 SW 23rd Dr., Gainesville, FL 32604. smferkovich@gainesville.usda.ufl.edu

The fecundity of the insidious flower bug, Orius insidiosus (Hemiptera: Anthocoridae), was poor when reared on a minimal artificial diet (control diet) composed of brewers yeast, soy protein hydrolysate and chicken yolk. Consequently, we supplemented test diets with homogenates of eggs from the Indian meal moth (Plodia interpunctella), proteins or lipids extracted from Plodia eggs, or an embryonic cell line (PIE) derived from Plodia eggs. Test diets were also supplemented with each of three fatty acids identified to be predominant in prey eggs (palmitic, linoleic and oleic acid), bovine serum albumin (BSA), chicken liver, beef liver, or chicken egg white albumin. Diets were compared against an optimal standard, Plodia eggs, and the control diet on the basis of the average total number of eggs a female oviposited during her lifetime. Only proteins derived from Plodia eggs and the cell line produced significant improvements in fecundity over the control diet at relatively low concentration of protein, indicating the quality of protein is important in selecting supplements. Proteins extracted from prey eggs and the cell line were further separated by preparative isoelectric focusing and are being evaluated in the artificial diet.

Genomic approaches to the evolution and function of a large multigene family

Feyereisen R¹, Tijet N², Helvig C², Murataliev M², Koener JF¹, LeGoff G¹, Sabourault C¹

¹Unité Mixte de Recherche 1112 INRA-Université de Nice-Sophia Antipolis, Centre de Recherches d'Antibes, France, ²Department of Entomology, The University of Arizona, Tucson, Arizona.

The cytochrome P450 gene family is represented by 90 sequences in the Drosophila melanogaster genome. Clues to the evolution of this large multigene family are provided by an analysis of the sequences, of their organization on the chromosomes and by comparisons to other species. Strategies used to unravel the functions of these multiple genes in the fruit fly and in other insect species includethe production of functional proteins in heterologous systems and DNA microarray analysis of expression patterns.

Molecular characterization of a family of candidate odorant receptors from the malaria vector mosquito Anopheles gambiae</