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PubMed2002

Examination of the F2 screen for rare resistance alleles to Bacillus thuringiensis toxins in the diamondback moth (Lepidoptera: Plutellidae).

A synthetic laboratory population of the diamondback moth, Plutella xylostella (L.), was used to test the F2 screen developed for detecting the frequency of rare resistance alleles to Cry1Ac and Cry1C toxins of Bacillus thuringiensis (Bt). Of the 120 single-pair matings set up, 106 produced enough F2 families for screening of Cry1Ac or Cry1C resistance alleles using both transgenic broccoli and an artificial diet overlay assay with a diagnostic dose. When using Bt broccoli plants as the F2 screen method, only one F2 family was detected for Cry1Ac resistance and no family was detected for Cry1C resistance. Six families were detected for either Cry1Ac or Cry1C resistance using the diet assay. The survivors in the diagnostic diet assay were crossed with the resistant individuals to confirm their resistance genotypes. Four F2 families were confirmed to contain one copy of an allele resistant to Cry1Ac in the original single-pairs and four other F2 families contained an allele resistant to Cry1C. Our results suggest that using transgenic plants expressing a high level of a Bt toxin in an F2 screen may underestimate the frequency of resistance alleles with high false negatives, or fail to detect true resistance alleles. The diagnostic diet assay was a better F2 screen method to detect alleles, especially for the Cry1Ac resistance with monogenic inheritance in the diamondback moth. The estimated probabilities of false positives and false negatives were 33 and 1%, respectively, for detecting Cry1Ac resistance at the allele frequency of 0.012 using the diagnostic diet assay. Careful validation of the screening method for each insect-crop system is necessary before the F2 screen can be used to detect rare Bt resistance alleles in field populations.

Alleles↗
PubMed1997

Expression of a fungal cellobiohydrolase in insect cells.

The gene for Trichoderma reesei cellobiohydrolase I (CBHI) was expressed with a recombinant baculovirus and high levels of secreted protein were produced in Spodoptera frugiperda and Trichoplusia ni insect cells. Electrophoretic analysis indicated that the recombinant CBHI (rCBHI) was similar in apparent molecular weight to the native form and immunoblotting with anti-CBHI monoclonal antibodies confirmed its identity. The rCBHI was easily purified by affinity and hydrophobic interaction chromatography and demonstrated enzymatic activity on soluble substrate.

Amino Acid Sequence↗
PubMed1996

Characterization of canine herpesvirus glycoprotein C expressed by a recombinant baculovirus in insect cells.

The gene encoding the canine herpesvirus (CHV) glycoprotein C (gC) homologue has been identified by sequence homology analyses with other well studied herpesviruses. Previously, we have identified three CHV glycoproteins, gp145/112, gp80 and gp47 using a panel of monoclonal antibodies (MAbs). To determine which CHV glycoprotein corresponds to gC, a recombinant baculovirus which contains the putative CHV gC structural gene under the baculovirus polyhedrin promoter was constructed. The recombinant baculovirus expressed gC-related polypeptides (44-62 kDa), which reacted only with MAbs against CHV gp80, indicating that the previously identified CHV gp80 is the translation product of the gC gene. The baculovirus expressed gC was glycosylated and transported to the surface of infected cells. At least seven neutralizing epitopes were conserved on the gC produced in insect cells. It was found that the recombinant baculovirus infected cells adsorbed murine erythrocytes as is the case for CHV-infected cells. The hemadsorption activity was inhibited by heparin, indicating that the CHV gC binds to heparan sulfate on the surface of murine erythrocytes. Mice immunized with the recombinant gC produced strong neutralizing antibodies. Our results suggest that CHV gC produced in insect cells may be useful as a subunit vaccine to control CHV infections.

Animals↗
PubMed1999

Insect evolution: Redesigning the fruitfly.

Homeotic mutations in Drosophila can result in dramatic phenotypes that suggest the possibility for rapid morphological evolution, but dissection of the genetic pathway downstream of Ultrabithorax is beginning to reveal how wing morphology may have evolved by more gradual transformations.

Animals↗
PubMed2000

Molecular cloning and genomic organization of a second probable allatostatin receptor from Drosophila melanogaster.

We (C. Lenz et al. (2000) Biochem. Biophys. Res. Commun. 269, 91-96) and others (N. Birgül et al. (1999) EMBO J. 18, 5892-5900) have recently cloned a Drosophila receptor that was structurally related to the mammalian galanin receptors, but turned out to be a receptor for a Drosophila peptide belonging to the insect allatostatin neuropeptide family. In the present paper, we screened the Berkeley "Drosophila Genome Project" database with "electronic probes" corresponding to the conserved regions of the four rat (delta, kappa, mu, nociceptin/orphanin FQ) opioid receptors. This yielded alignment with a Drosophila genomic database clone that contained a DNA sequence coding for a protein having, again, structural similarities with the rat galanin receptors. Using PCR with primers coding for the presumed exons of this second Drosophila receptor gene, 5'- and 3'-RACE, and Drosophila cDNA as template, we subsequently cloned the cDNA of this receptor. The receptor cDNA codes for a protein that is strongly related to the first Drosophila receptor (60% amino acid sequence identity in the transmembrane region; 47% identity in the overall sequence) and that is, therefore, most likely to be a second Drosophila allatostatin receptor (named DAR-2). The DAR-2 gene has three introns and four exons. Two of these introns coincide with two introns in the first Drosophila receptor (DAR-1) gene, and have the same intron phasing, showing that the two receptor genes are clearly evolutionarily related. The DAR-2 gene is located at the right arm of the third chromosome, position 98 D-E. This is the first report on the existence of two different allatostatin receptors in an animal.

Amino Acid Sequence↗
PubMed2005

A molecular genetic approach to the biosynthesis of the insect steroid molting hormone.

Insect growth, development, and molting depend upon a critical titer of the principal molting hormone of arthropods, 20-hydroxyecdysone (20E). Although the structure of 20E as a polyhydroxylated steroid was determined more than five decades ago, the exact steps in its biosynthesis have eluded identification. Over the past several years, the use of the fly database and the techniques and paradigms of biochemistry, analytical chemistry, and molecular genetics have allowed the cloning and sequencing of four genes in the Halloween gene family of Drosophila melanogaster, all of them encoding cytochrome P450 (CYP) enzymes, each of which mediates one of the four terminal hydroxylation steps in 20E biosynthesis. Further, the sequence of these hydroxylations has been determined, and developmental alterations in the expression of each of these genes have been quantified during both embryonic and postembryonic life.

Animals↗
PubMed1992

Hepatitis B virus polymerase gene: expression of the long open reading frame using the baculovirus expression system.

A recombinant baculovirus was constructed containing a copy of the hepatitis B virus (HBV) genome which was inserted to produce an in-frame fusion of the precore (pre-C) coding region with the first 11 amino acids of the polyhedrin gene. The recombinant baculovirus expressed the 25K pre-C protein and two novel proteins, of approximately 93K and 72K. Both the 93K and 72K proteins are recognized by an anti-polymerase monoclonal antibody. Northern blot analysis of the mRNA produced during infection of Spodoptera frugiperda cells by the HBV recombinant baculovirus detected only one HBV mRNA species, suggesting that the three HBV-specific proteins expressed are translated from the same mRNA. No larger fusion proteins cross-reacting with either anti-core or polymerase antibodies were detected. These findings suggest that the two proteins encoded within the HBV polymerase gene are not produced via a core-polymerase fusion intermediate but by internal binding of ribosomes. These results are the first clear demonstration of efficient expression of two bona fide unprocessed polymerase proteins in a 1:1 ratio from an unspliced pre-C mRNA-like transcript. With the successful expression of the polymerase gene in insect cells it is now possible to produce large amounts of these proteins, allowing a more detailed structural and functional analysis of these proteins.

Baculoviridae↗
PubMed2004

Population genetics of autocidal control and strain replacement.

The concept that an insect species' genome could be altered in a manner that would result in the control of that species (i.e., autocidal control) or in the replacement of a pestiferous strain of the species with a more benign genotype was first proposed in the mid-twentieth century. A major research effort in population genetics and ecology followed and led to the development of a set of classical genetic control approaches that included use of sterile males, conditional lethal genes, translocations, compound chromosomes, and microbe-mediated infertility. Although there have been a number of major successes in application of classical genetic control, research in this area has declined in the past 20 years for technical and societal reasons. Recent advances in molecular biology and transgenesis research have renewed interest in genetically based control methods because these advances may remove some major technical problems that have constrained effective genetic manipulation of pest species. Population genetic analyses suggest that transgenic manipulations may enable development of strains that would be 10 to over 100 times more efficient than strains developed by classical methods. Some of the proposed molecular approaches to genetic control involve modifications of classical approaches such as conditional lethality, whereas others are novel. Experience from the classical era of genetic control research indicates that the population structure and population dynamics of the target population will determine which, if any, genetic control approaches would be appropriate for addressing a specific problem. As such, there continues to be a need for ongoing communication between scientists who are developing strains and those who study the native pest populations.

Animals↗
PubMed1995

Isolation and sequence determination of the cDNA encoding DNA polymerase delta from Drosophila melanogaster.

The cDNA encoding the catalytic subunit of Drosophila melanogaster (Dm) DNA polymerase delta (Pol delta) was isolated by a combination of PCR amplification and cDNA library screening. The cDNA is 3457 nucleotides in length and contains an open reading frame (ORF) that encodes a protein of 1092 amino acids (124,799 Da). The ORF contains the sequence that was determined for a peptide from the purified catalytic subunit of Dm Pol delta. Polyclonal antibodies raised against Dm Pol delta specifically recognize a protein of the expected size when the cDNA is expressed in either Escherichia coli or insect cells. Comparison of the deduced aa sequence with other Pol delta sequences demonstrates that Pol delta is one of the most highly conserved of the DNA polymerases.

Amino Acid Sequence↗
PubMed1995

Biochemical analysis of the N-glycosylation pathway in baculovirus-infected lepidopteran insect cells.

The baculovirus-insect cell system is used routinely for foreign glycoprotein production, but the precise nature of the N-glycosylation pathway in this system remains unclear. Some studies indicate that these cells cannot process N-linked oligosaccharides to complex forms containing outer-chain galactose and sialic acid, while others indicate that they can. In this study, we used the major virion envelope glycoprotein of the baculovirus Autographa california multicapsid nuclear polyhedrosis virus (AcMNPV) to probe the N-glycosylation pathway in baculovirus-infected lepidopteran insect cells. The results showed that gp64 contained mannose, fucose, and probably N-acetylglucosamine, but no detectable galactose or sialic acid. These same results were observed with gp64 produced in any one of three different lepidopteran insect cell lines derived from Spodoptera frugiperda, Trichoplusia ni, or Estigmene acrea, whether it was produced at relatively earlier or later times after infection. These results indicated that the gp64 produced in AcMNPV-infected lepidopteran insect cells lacks complex N-linked oligosaccharides containing outer-chain galactose and sialic acid. By contrast, gp64 produced in mammalian cells contained both galactose and sialic acid, and endoglycosidase digestions revealed that these sugars were constituents of N-linked, not O-linked, oligosaccharides. This showed that at least one N-linked side chain on gp64 has the potential to be processed to a complex form. Together, these results suggest either that AcMNPV-infected lepidopteran insect cells are unable to convert any of the N-linked side chains on gp64 to complex structures or that outer-chain galactose and sialic acid residues are added to gp64 and then removed by cellular or viral exoglycosidases.

Acetylglucosamine↗
PubMed1998

Drosophila metamorphosis: the only way is USP?

The steroid insect molting hormone 20-hydroxyecdysone is believed to control critical aspects of development and reproduction through a heterodimeric receptor comprising the Ecdysone Receptor and the Ultraspiracle proteins. Recent findings suggest that other hormones and receptors might also be involved.

Animals↗
PubMed2004

Isolation and characterization of a novel rice gene encoding a putative insect-inducible protein homologous to wheat Wir1.

A full-length cDNA, designated BpHi008A, was cloned representing a rice (Oryza sativa) mRNA that accumulates after brown planthopper (BPH) Nilapar vata lugens Stål feeding. The cDNA encodes a putative 82 amino acid protein (BpHi008A) exhibiting about 37% amino acid sequence identity to Wir1 family of proteins that are encoded by pathogen-induced transcripts in wheat. Like Wir1 proteins, it consists of a hydrophobic N-terminal half and a hydrophilic C-terminal half relatively rich in glycine and proline. These proteins are predicted to be integrated into the membrane, with the C-terminus being extracytoplastic. Genomic Southern analysis indicated that the BpHi008A gene was present as a single-copy sequence in the rice genome. Temporal and spatial studies showed that BpHi008A were systemically induced in rice when 2nd and 3rd-instars were feeding. The BpHi008A transcripts level was also increased in seedlings damaged by mechanical wounding. These data indicated that BphHi008A was implicated in the response of rice plants to BPH feeding and wounding.

Amino Acid Sequence↗
PubMed2000

Cloning of vitellogenin cDNA of the American cockroach, Periplaneta americana (Dictyoptera), and its structural and expression analyses.

A cDNA expression library constructed from poly (A)(+) RNA prepared from vitellogenic female fat body cells of the American cockroach, Periplaneta americana (Dictyoptera) was screened using a polyclonal antiserum against the 100-kD polypeptide(s) from the egg extract. A partial Vg cDNA clone was obtained and sequenced. The 5' end portion of the cDNA was then obtained by the RACE method, cloned, and sequenced. The combined complete Vg cDNA was 5,854 bp long and contained a single ORF encoding 1,896 amino acids. The entire deduced amino acid sequence was aligned confidently with those of the known insect Vgs. A GL/ICG motif, a number of cysteines at conserved locations following this motif, and a DGXR motif upstream of the GL/ICG motif were present near the C-terminal. The chemically determined N-terminal amino acid sequence of the 170-kD polypeptide from the egg extract completely matched the deduced sequence starting from just after one of the consensus (RXXR) cleavage sites, indicating the occurrence of post-translational cleavage in the fat body cells. The Vg gene begins to be expressed in the 2-day-old adult female fat body cells but is never expressed in ovaries or in male fat body cells. Hemolymph Vg was first detected by immunoblotting in 4-day-old adult females, 2 days after the beginning of gene expression. Western blot analysis of major yolk polypeptides in nine cockroach species belonging to the two superfamilies, Blattoidea and Blaberoidea, using the antisera against P. americana major yolk polypeptides showed that the similarities in Vn antigenicity are basically limited to within a superfamily.

Amino Acid Sequence↗
PubMed2003

The molecular biology of knockdown resistance to pyrethroid insecticides.

The term "knockdown resistance" is used to describe cases of resistance to diphenylethane (e.g. DDT) and pyrethroid insecticides in insects and other arthropods that result from reduced sensitivity of the nervous system. Knockdown resistance, first identified and characterized in the house fly (Musca domestica) in the 1950's, remains a threat to the continued usefulness of pyrethroids in the control of many pest species. Research since 1990 has provided a wealth of new information on the molecular basis of knockdown resistance. This paper reviews these recent developments with emphasis on the results of genetic linkage analyses, the identification of gene mutations associated with knockdown resistance, and the functional characterization of resistance-associated mutations. Results of these studies identify voltage-sensitive sodium channel genes orthologous to the para gene of Drosophila melanogaster as the site of multiple knockdown resistance mutations and define the molecular mechanisms by which these mutations cause pyrethroid resistance. These results also provide new insight into the mechanisms by which pyrethroids modify the function of voltage-sensitive sodium channels.

Amino Acid Substitution↗
PubMed2001

The invertebrate odorant-binding protein LUSH is required for normal olfactory behavior in Drosophila.

The invertebrate odorant-binding proteins consist of a large family of low-molecular-weight, highly divergent proteins expressed exclusively in the chemosensory sensilla of insects. Each member of this family studied to date is secreted into the sensillum lymph of a small subset of sensilla by non-neuronal support cells. These expression patterns suggests an odor-specific function for these proteins as opposed to a general role in sensillum biology. Consistent with this notion, mutants defective for LUSH, a Drosophila member of this family, have odor-specific defects in olfactory behavior. The Drosophila genome contains at least 32 members of this gene family, rivaling the number of odorant receptors in this species. The relationship between these two protein families and how they act to determine odor specificity of olfactory neurons will be the topic of future studies.

Animals↗
PubMed1994

Transposable elements as population drive mechanisms: specification of critical parameter values.

With a view to the possible use of transposable elements (TEs) as a mechanism to drive genes into insect vector populations, we used a three-parameter density dependent growth equation to examine the critical parameter values that determine whether or not a mobile element will spread and become fixed in a finite diploid vector population. Populations were simulated with parameter values affecting size, reproductive rate, density-dependence, and transposition efficiency of the mobile element. Simulations indicated that an equilibrium was reached quickly, typically in < 50 generations. Even when initially present at < or = 1% of a large population, the mobile element spread quickly and became fixed if transposition efficiency was equal to unity and infertility caused by the element decreased reproductive capacity by as much as 45%. These results were insensitive to the values of basic wild type reproductive rates and density dependence, but population size, transposition efficiency of the element, reproductive rate individuals bearing TEs and initial ratio of TE-bearing to wild individuals modified the outcome. As population size and transposition efficiency decreased in value, TEs became fixed less easily. However, even in populations as small as n = 100, an element with a transposition efficiency > 0.75 that reduces fertility < 25% will become fixed when introduced at a frequency as low as 1% of the total population. These results are consistent with previously reported population genetics models. They suggest that engineered transposons with a wide range of properties may be used to drive genes, such as those for parasite resistance, into wild vector populations.

Animals↗
PubMed2000

The molecular basis of melanism and mimicry in a swallowtail butterfly.

Melanism in Lepidoptera, either industrial or in mimicry, is one of the most commonly cited examples of natural selection [1] [2]. Despite extensive studies of the frequency and maintenance of melanic genes in insect populations [1] [2], there has been little work on the underlying molecular mechanisms. Nowhere is butterfly melanism more striking than in the Eastern Tiger Swallowtail (Papilio glaucus) of North America [3] [4] [5]. In this species, females can be either yellow (wild type) or black (melanic). The melanic form is a Batesian mimic of the distasteful Pipevine Swallowtail (Battus philenor), which is also black in overall color. Melanism in P. glaucus is controlled by a single Y-linked (female) black gene [6]. Melanic females, therefore, always have melanic daughters. Black melanin replaces the background yellow in melanic females. Here, we show that the key enzyme involved is N-beta-alanyl-dopamine-synthase (BAS), which shunts dopamine from the melanin pathway into the production of the yellow color pigment papiliochrome and also provides products for cuticle sclerotization. In melanic females, this enzyme is suppressed, leading to abnormal melanization of a formerly yellow area, and wing scale maturation is also delayed in the same area. This raises the possibility that either reduced BAS activity itself is preventing scale sclerotization (maturation) or, in contrast, that the delay in scale maturation precludes expression of BAS at the correct stage. Together, these data show how changes in expression of a single gene product could result in multiple wing color phenotypes. The implications for the genetic control of mimicry in other Lepidoptera are discussed.

Animals↗
PubMed2002

The many selves of social insects.

Social insects show multiple levels of self identity. Most individuals are sterile workers who selflessly labor for their colony, which is often viewed as a superorganism. The superorganism protects itself with colony recognition systems based on learned odors, typically cuticular hydrocarbons. Transfer of these odors within the colony obscures separate clan identities. Residual individual interests do appear to cause conflicts within colonies over sex ratio, male production, caste, and reproductive dominance. However, genomic imprinting theory predicts that the individual's maternal and paternal genes will evolve separate infraorganismal identities, perhaps leaving virtually no coherent individual identity.

Animals↗