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Evolution of Wolbachia cytoplasmic incompatibility types.

The success of obligate endosymbiotic Wolbachia infections in insects is due in part to cytoplasmic incompatibility (CI), whereby Wolbachia bacteria manipulate host reproduction to promote their invasion and persistence within insect populations. The observed diversity of CI types raises the question of what the evolutionary pathways are by which a new CI type can evolve from an ancestral type. Prior evolutionary models assume that Wolbachia exists within a host individual as a clonal infection. While endosymbiotic theory predicts a general trend toward clonality, Wolbachia provides an exception in which there is selection to maintain diversity. Here, evolutionary trajectories are discussed that assume that a novel Wolbachia variant will co-exist with the original infection type within a host individual as a superinfection. Relative to prior models, this assumption relaxes requirements and allows additional pathways for the evolution of novel CI types. In addition to describing changes in the Wolbachia infection frequency associated with the hypothesized evolutionary events, the predicted impact of novel CI variants on the host population is also described. This impact, resulting from discordant evolutionary interests of symbiont and host, is discussed as a possible cause of Wolbachia loss from the host population or host population extinction. The latter is also discussed as the basis for an applied strategy for the suppression of insect pest populations. Model predictions are discussed relative to a recently published Wolbachia genome sequence and prior characterization of CI in naturally and artificially infected insects.

Animals↗

Identification of a pentanucleotide telomeric sequence, (TTAGG)n, in the silkworm Bombyx mori and in other insects.

A pentanucleotide repetitive sequence, (TTAGG)n, has been isolated from a silkworm genomic library, using cross-hybridization with a (TTNGGG)5 sequence, which is conserved among most eukaryotic telomeres. Both fluorescent in situ hybridization and Bal 31 exonuclease experiments revealed major clusters of (TTAGG)n at the telomeres of all Bombyx chromosomes. To determine the evolutionary origin of this sequence, two types of telomeric sequence, (TTAGG)5 and a hexanucleotide repetitive sequence, (TTAGGG)4, which is conserved mainly among vertebrate and several invertebrate telomeres so far examined, were hybridized to DNAs from a wide variety of eukaryotic species under highly stringent hybridization conditions. The (TTAGGG)5 oligonucleotide hybridized to genomic DNAs from vertebrates and several nonvertebrate species, as has been reported so far, but not to any DNAs from insects. On the other hand, the Bombyx type of telomere sequence, (TTAGG)n, hybridized to DNAs from 8 of 11 orders of insect species tested but not to vertebrate DNAs, suggesting that this TTAGG repetitive sequence is conserved widely among insects.

Animals↗

Regulation of juvenile hormone esterase gene transcription by juvenile hormone.

Juvenile hormone (JH) is a major hormone regulating insect development. We have obtained a cDNA and a genomic clone for juvenile hormone esterase (JHE), the enzyme that is involved in the degradation of juvenile hormone and which is critical for insect development. Analysis of the regulation of JHE during the final larval stadium in the cabbage looper, Trichoplusia ni, showed that the JHE mRNA levels are maximal on days 2 and 4 of the final stadium. Nuclear run-on analyses demonstrated that changes in JHE mRNA levels are primarily due to changes in the transcription rate of the gene, which may be a single copy in the genome. Treatment with a JH analog resulted in induction of JHE gene transcription, which could be detected within three hours after treatment. Salient features present in the 5' flanking region of this JH-sensitive gene are presented, including the presence of sequences closely resembling binding sites for members of the family of nuclear receptors. This report is the first direct demonstration, by nuclear run-on analysis, of JH induction of gene transcription.

Amino Acid Sequence↗

A cluster of cuticle protein genes of Drosophila melanogaster at 65A: sequence, structure and evolution.

A 36-kb genomic DNA segment of the Drosophila melanogaster genome containing 12 clustered cuticle genes has been mapped and partially sequenced. The cluster maps at 65A 5-6 on the left arm of the third chromosome, in agreement with the previously determined location of a putative cluster encompassing the genes for the third instar larval cuticle proteins LCP5, LCP6 and LCP8. This cluster is the largest cuticle gene cluster discovered to date and shows a number of surprising features that explain in part the genetic complexity of the LCP5, LCP6 and LCP8 loci. The genes encoding LCP5 and LCP8 are multiple copy genes and the presence of extensive similarity in their coding regions gives the first evidence for gene conversion in cuticle genes. In addition, five genes in the cluster are intronless. Four of these five have arisen by retroposition. The other genes in the cluster have a single intron located at an unusual location for insect cuticle genes.

Animals↗

Evidence for the presence of a genome-linked protein in infectious flacherie virus.

The genomic RNA of infectious flacherie virus (IFV), an insect picornavirus, was shown to contain a covalently attached substance which can be labeled with 125I-Bolton and Hunter reagent. This substance was found to be a protein covalently linked to the virus genome, because it cosedimented and was copurified with the viral RNA and was degraded by proteinase K but not by RNases. The molecular weight of the protein was found to be 11,500 in the sodium dodecyl sulfate polyacrylamide gel electrophoresis. This protein apparently corresponds to viral protein genome-linked (VPg) detected in mammalian picornaviruses, because it was not required for the synthesis of viral proteins in a cell-free translation system of a rabbit reticulocyte lysate and because it was not immunochemically related to the virus capsid proteins. The results presented here leave a possibility that insect picornavirus also replicates its RNA by using VPg as a primer as reported for mammalian picornaviruses. These observations on VPg of an insect picornavirus are discussed in the view point of comparative virology.

Bombyx↗

A family of genes clustered at the Triplo-lethal locus of Drosophila melanogaster has an unusual evolutionary history and significant synteny with Anopheles gambiae.

Within the unique Triplo-lethal region (Tpl) of the Drosophila melanogaster genome we have found a cluster of 20 genes encoding a novel family of proteins. This family is also present in the Anopheles gambiae genome and displays remarkable synteny and sequence conservation with the Drosophila cluster. The family is also present in the sequenced genome of D. pseudoobscura, and homologs have been found in Aedes aegypti mosquitoes and in four other insect orders, but it is not present in the sequenced genome of any noninsect species. Phylogenetic analysis suggests that the cluster evolved prior to the divergence of Drosophila and Anopheles (250 MYA) and has been highly conserved since. The ratio of synonymous to nonsynonymous substitutions and the high codon bias suggest that there has been selection on this family both for expression level and function. We hypothesize that this gene family is Tpl, name it the Osiris family, and consider possible functions. We also predict that this family of proteins, due to the unique dosage sensitivity and the lack of homologs in noninsect species, would be a good target for genetic engineering or novel insecticides.

Amino Acid Sequence↗

Rice stripe virus 23.9 K protein aggregates and forms inclusion bodies in cultured insect cells and virus-infected plant cells.

The genome of Rice stripe virus (RSV, genus Tenuivirus) contains seven open reading frames (ORFs). Little is known about the products of four of these ORFs, including the 23.9 K protein encoded by the virus-sense ORF of RNA3. Western blotting revealed that the 23.9 K protein was synthesized in the host plant and also in the planthopper vector of RSV. Using a baculovirus vector, the 23.9 K protein was expressed, both unfused and fused with red-shifted green fluorescent protein, in Spodoptera frugiperda cells. Inclusion bodies were observed by light microscope in cells expressing fused or unfused proteins. Inclusion bodies in cells expressing the fused protein fluoresced under blue light. By immunoelectron microscopy, electron-dense inclusion bodies in cells expressing the unfused protein were specifically labeled with 23.9 K protein antiserum. Moreover, electron-dense masses labeled with 23.9 K protein antiserum were observed in virus-infected wheat tissue by electron microscopy. This paper thus demonstrates that RSV 23.9 K protein can aggregate in vivo and form inclusion bodies in infected plant tissue.

Animals↗

Genetic engineering of Periplaneta fuliginosa densovirus as an improved biopesticide.

The smoky-brown cockroach (Periplaneta fuliginosa) densovirus (PfDNV) has previously shown potential in urban pest control. To improve its efficacy as a biopesticide, the genome of PfDNV was engineered by inserting the insect-specific toxin gene BmKIT1 in the open reading frame encoding the major structural proteins. A green fluorescent protein (GFP) marker was tagged to the BmKIT1 at its C-terminus for in vivo imaging using Confocal laser scanning microscopy (CFSM). Using a virion rescue strategy, the genomes of recombinant and wild-type (wt) PfDNV were then cotransfected in P. fuliginosa nymphs. Reverse transcription PCR (RT-PCR) showed that the inserted BmkIT1 genes were expressed in the P. fuliginosa nymphs 48 h after cotransfection. CFSM and transmission electron microscopy also confirmed the generation of virus particles and expression of BmKIT1-GFP fusion protein in the cotransfected nymphs. The recombinant viruses remained infective to P. fuliginosa nymphs in feeding tests. Using the LT(50) bioassay method, the coninfection of the recombinant and wt PfDNV killed the P. fuliginosa nymphs approximate 32% faster than wt PfDNV only. This is the first report showing the improvement of engineered densovirus for the potential application of biopesticide.

Animals↗

Molecular identification of a myosuppressin receptor from the malaria mosquito Anopheles gambiae.

The insect myosuppressins (X1DVX2HX3FLRFamide) are neuropeptides that generally block insect muscle activities. We have used the genomic sequence information from the malaria mosquito Anopheles gambiae Genome Project to clone a G protein-coupled receptor that was closely related to the two previously cloned and characterized myosuppressin receptors from Drosophila [Proc. Natl. Acad. Sci. USA 100 (2003) 9808]. The mosquito receptor cDNA was expressed in Chinese hamster ovary cells and was found to be activated by low concentrations of Anopheles myosuppressin (TDVDHVFLRFamide; EC50, 1.6 x 10(-8)M). The receptor was not activated by a library of 35 other insect neuropeptides and monoamines, including neuropeptides that resembled myosuppressin in their C-terminal moiety, such as PDRNFLRFamide (Anopheles FMRFamide-3), other Anopheles FMRFamide peptides, or neuropeptide F-like peptides, showing that the receptor was quite selective for myosuppressin. These results also showed that the myosuppressin receptor needs a much larger portion than the C-terminal FLRFamide sequence for its activation. The insect myosuppressins are often grouped together with the insect FMRFamides under the name FaRPs (FMRFamide-related peptides). However, this is not justified anymore, because the insect myosuppressin receptor/ligand couple is both functionally and evolutionarily fully unrelated to the insect FMRFamide receptor/ligand couple. To our knowledge, this is the first report on the molecular identification of a mosquito neuropeptide receptor.

Amino Acid Sequence↗

Bacteriophage WO and virus-like particles in Wolbachia, an endosymbiont of arthropods.

Wolbachia are intracellular symbionts mainly found in arthropods, causing various sexual alterations on their hosts by unknown mechanisms. Here we report the results that strongly suggest that Wolbachia have virus-like particles of phage WO, which was previously identified as a prophage-like element in the Wolbachia genome. Wolbachia (strain wTai) infection in an insect was detected with the antibody against Wsp, an outer surface protein of Wolbachia, by fluorescence microscopy and immunoelectron-microscopy for the first time. Virus-like particles in Wolbachia were observed by electron-microscopy. The 0.22-microm filtrate of insect ovary contained DAPI-positive particles, and PCR analysis demonstrated that a phage WO DNA passed through the filter while Wolbachia DNA were eliminated, suggesting that the DAPI-positive particles were phage WO.

Animals↗

Taxonomic characteristics of fijiviruses based on nucleotide sequences of the oat sterile dwarf virus genome.

Sequence determination of full-length cDNA clones of genomic segments 7-10 (S7-S10) of oat sterile dwarf fijivirus (OSDV) revealed that the 5' and 3' ends of the plus strands of these segments had the same conserved terminal sequences, 5' AACGAAAAA and UUUUUUUAGUC 3'. These sequences are similar, but not identical, to the conserved terminal nucleotide sequences of the genomic segments of rice black streaked dwarf fijivirus (RBSDV) and maize rough dwarf fijivirus (MRDV). The coding strands of S7 and S10 each contained two large nonoverlapping open reading frames (ORFs), as do RBSDV S7 and S9, MRDV S6 and S8 and Nilaparvata lugens reovirus (NLRV; a putative member of Fijivirus) S9. These results strongly suggest that the dicistronic nature of certain genomic segments is characteristic of fijiviruses. Computer analyses revealed sequence homology between RBSDV S7 ORF2, MRDV S6 ORF2 and OSDV S7 ORF2, suggesting that this protein is conserved among plant fijiviruses. No counterparts were found in the genome of NLRV, which is a nonphytopathogenic insect reovirus. Furthermore, phylogenetic trees derived from multiple sequence alignments of each of the homologous proteins from OSDV, RBSDV, MRDV and NLRV suggest that NLRV did not evolve from either Fijivirus group 2 (RBSDV and MRDV) or group 3 (OSDV).

Amino Acid Sequence↗

Malaria parasite and vector genomes: partners in crime.

The publication of the genome sequences of the malaria parasite Plasmodium falciparum and the insect vector Anopheles gambiae paves the way for scientists to study these organisms by using technologies developed to observe global changes in transcription and translation, as well as computational tools. Researchers are now able to investigate complex changes involved in development, growth and reaction to external factors. Given the medical importance of these organisms, much of this work is targeted on drug or insecticide discovery (including mechanisms of resistance to existing treatments), but the genome information also provides the opportunity to develop novel therapies.

Animals↗

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↗

Separation of the plus and minus strands of cytoplasmic polyhedrosis virus and human reovirus double-stranded genome RNAs by gel electrophoresis.

The complementary strands of most of the genome double-stranded RNA segments of insect cytoplasmic polyhedrosis virus (CPV) and human reovirus are separated for the first time by agarose gel electrophoresis in in the presence of 7 M urea. CPV (+) strands and most reovirus (-) strands migrate faster than the corresponding strands of opposite polarity. Glyoxal treatment, which modifies guanine residues and prevents G-C basepairing, results in a loss of strand resolution and concomitantly a significant decrease in electrophoretic mobilities. Reovirus mRNAs synthesized in vitro with ITP substituted for GTP show similar decreased electrophoretic mobilities as the glyoxalated mRNAs. These results clearly indicate that the basis for (+) and (-) strand resolution is the presence of secondary structure formed mainly by G-C(U) base-pairs that are maintained during gel electrophoresis in the presence of 7 M urea. When the plus and minus strands of CPV genomes were separated and compared for protein synthesizing activity, it was found that only the plus strands were able to form stable 80S ribosome-RNA initiation complexes in wheat germ cell-free extracts.

Base Composition↗

Insect densoviruses may be widespread in mosquito cell lines.

A diagnostic PCR assay was designed based on conserved regions of previously sequenced densovirus genomic DNA isolated from mosquitoes. Application of this assay to different insect cell lines resulted in a number of cases of consistent positive amplification of the predicted size fragment. Positive PCR results were subsequently confirmed to correlate with densovirus infection by both electron microscopy and indirect fluorescent antibody test. In each case the nucleotide sequence of the amplified PCR fragments showed high identity to previously reported densoviruses isolated from mosquitoes. Phylogenetic analysis based on these sequences showed that two of these isolates were examples of new densoviruses. These viruses could infect and replicate in mosquitoes when administered orally or parenterally and these infections were largely avirulent. In one virus/mosquito combination vertical transmission to progeny was observed. The frequency with which these viruses were detected would suggest that they may be quite common in insect cell lines.

Animals↗

A member of the reoviridae (DpRV) has a ploidy-specific genomic segment in the wasp Diadromus pulchellus (Hymenoptera).

Wild and laboratory populations of the parasitoid wasp Diadromus pulchellus are infected with a new member of the reoviridae (DpRV) described in this paper. The particles of this virus possess two capsid shells (diameters: 35 and 70 nm) made up of 11 proteins. The virus is present mainly in the gut of the wasp, with smaller quantities in its venom gland. The genome of virus particles purified from haploid insects (functional males) contains 10 segments, whereas virus from diploid insects (females and sterile diploid males) contains a supernumerary 3.33-kb segment. The sequence of this dsRNA segment revealed that it is a triplicated 1050-bp motif which is 97.5% similar to the 5' region of one of the 10 basic segments, the 3.80-kb segment.

Amino Acid Sequence↗

Functional roles of neuropeptides in the insect central nervous system.

With the completion of the Drosophila genome sequencing project we can begin to appreciate the extent of the complexity in the components involved in signal transfer and modulation in the nervous system of an animal with reasonably complex behavior. Of all the different classes of signaling substances utilized by the nervous system, the neuropeptides are the most diverse structurally and functionally. Thus peptidergic mechanisms of action in the central nervous system need to be analyzed in the context of the neuronal circuits in which they act and generalized traits cannot be established. By taking advantage of Drosophila molecular genetics and the presence of identifiable neurons, it has been possible to interfere with peptidergic signaling in small populations of central neurons and monitor the consequences on behavior. These studies and experiments on other insects with large identifiable neurons, permitting cellular analysis of signaling mechanisms, have outlined important principles for temporal and spatial action of neuropeptides in outputs of the circadian clock and in orchestrating molting behavior. Considering the large number of neuropeptides available in each insect species and their diverse distribution patterns, it is to be expected that different neuropeptides play roles in most aspects of insect physiology and behavior.

Animals↗

Transcriptional interference gates monogenic odorant receptor expression in ants.

Communication is crucial to social life, and in ants, it is mediated primarily through olfaction. Ants have more odorant receptor (OR) genes than any other group of insects, generated through tandem duplications that produce large genomic arrays of related genes. The mechanism by which olfactory sensory neurons (OSNs) produce a single functional OR from these arrays remains unclear. In ant OSNs, only mRNA from one OR in an array is exported into the cytoplasm, while upstream genes are silent and transcripts from downstream genes remain nuclear. Here, we show that readthrough transcription in the downstream direction generates non-translated transcripts. We also find that OR promoters are bidirectional, producing antisense long non-coding RNAs. We suspect that neither readthrough nor antisense transcription produces functional RNA but that bidirectional transcription alone is critical to suppressing the expression of all other OR genes in a tandem array. Finally, we present evidence that this regulatory architecture is conserved across ants and bees, suggesting that this mechanism for functionally monogenic OR expression is widespread in insects with expanded OR repertoires.

Animals↗