Quantitative trait locus mapping and functional genomics of an organophosphate resistance trait in the western corn rootworm, Diabrotica virgifera virgifera.

The western corn rootworm, Diabrotica virgifera virgifera, is an insect pest of corn and population suppression with chemical insecticides is an important management tool. Traits conferring organophosphate insecticide resistance have increased in frequency amongst D. v. virgifera populations, resulting in the reduced efficacy in many corn-growing regions of the USA. We used comparative functional genomic and quantitative trait locus (QTL) mapping approaches to investigate the genetic basis of D. v. virgifera resistance to the organophosphate methyl-parathion. RNA from adult methyl-parathion resistant and susceptible adults was hybridized to 8331 microarray probes. The results predicted that 11 transcripts were significantly up-regulated in resistant phenotypes, with the most significant (fold increases ≥ 2.43) being an α-esterase-like transcript. Differential expression was validated only for the α-esterase (ST020027A20C03), with 11- to 13-fold greater expression in methyl-parathion resistant adults (P < 0.05). Progeny with a segregating methyl-parathion resistance trait were obtained from a reciprocal backcross design. QTL analyses of high-throughput single nucleotide polymorphism genotype data predicted involvement of a single genome interval. These data suggest that a specific carboyxesterase may function in field-evolved corn rootworm resistance to organophosphates, even though direct linkage between the QTL and this locus could not be established.

Expression of lacunin, a large multidomain extracellular matrix protein, accompanies morphogenesis of epithelial monolayers in Manduca sexta.

Morphogenesis is a complex process operating at several levels of organization--organism, tissues, cells, and molecules. Complex interactions occur between and within these levels. Many of the molecules that mediate these interactions are predictably turning out to be large multidomain proteins. Here we describe one such novel protein associated with remodeling of epithelial monolayers in embryos and developing wings of the moth Manduca sexta. On the basis of its sequence and its expression pattern along lacunae of developing wings, we propose the name lacunin for this extracellular matrix protein that contains nine different types of domains, most of which are present in multiple copies. These include domains of various types: Kunitz proteinase inhibitors, thrombospondin type I, immunoglobulin-like, and several newly defined domains of unknown function (PAL, PLAC, and lagrin domains). This rich patchwork of distinct domains probably exerts multiple effects on a variety of cell behaviors associated with the complex phenomenon of epithelial morphogenesis.

Bmmar6, a second mori subfamily mariner transposon from the silkworm moth Bombyx mori.

A second member of the divergent mori subfamily of mariner transposons, Bmmar6, is described from the silkworm moth Bombyx mori genome. A confident consensus sequence for Bmmar6 was obtained from a single genomic copy, 17 EST sequences, and the direct sequencing of a 'family' sequence from an amplification of all full-length genomic copies. Bmmar6 is most similar to Bmmar1 in the mori subfamily, which now also includes several fly and nematode transposons. These might be viewed as a discrete family of transposons within the IS630-Tc1-mariner superfamily with a distinctive D,D37D catalytic motif, and another small divergent D,D41D clade is recognized as their sister group of transposons.

The genomes of most animals have multiple members of the Tc1 family of transposable elements.

A PCR assay was employed to detect sequence homologous to the transposase gene of the Tc1 family of transposable elements in a wide variety of animals. Amplification products of the appropriate size were obtained from most insects (92 of 108 examined; 85%), most other invertebrates (33 of 43; 77%), and many vertebrates (18 of 36; 50%). Sequencing a sample of cloned PCR products from eight insects, one hydra, and two frogs revealed that each had multiple distinct members of the family in their genomes. In the most extreme case, the horn fly Haematobia irritans yielded evidence of seventeen distinct types of Tc1 family elements. Most of the sequences obtained indicate that the elements are within the range of variation already known from fungi, nematodes, flies, fish and frogs. Some, however, had novel length variants or divergent sequences, indicating that they represent new subfamilies of these transposons. These results indicate that this family of transposons is extremely common in animal genomes, with multiple representatives in most genomes.

Loss of transposase-DNA interaction may underlie the divergence of mariner family transposable elements and the ability of more than one mariner to occupy the same genome.

Mariners are a large family of eukaryotic DNA-mediated transposable elements that move via a cut-and-paste mechanism. Several features of the evolutionary history of mariners are unusual. First, they appear to undergo horizontal transfer commonly between species on an evolutionary timescale. They can do this because they are able to transpose using only their own self-encoded transposase and not host-specific factors. One consequence of this phenomenon is that more than one kind of mariner can be present in the same genome. We hypothesized that two mariners occupying the same genome would not interact. We tested the limits of mariner interactions using an in vitro transposition system, purified mariner transposases, and DNAse I footprinting. Only mariner elements that were very closely related to each other (ca. 84% identity) cross-mobilized, and then inefficiently. Because of the dramatic suppression of transposition between closely related elements, we propose that to isolate elements functionally, only minor changes might be necessary between elements, in both inverted terminal repeat and amino acid sequence. We further propose a mechanism to explain mariner diversification based on this phenomenon.

Diversity of odourant binding proteins revealed by an expressed sequence tag project on male Manduca sexta moth antennae.

A small expressed sequence tag (EST) project generating 506 ESTs from 375 cDNAs was undertaken on the antennae of male Manduca sexta moths in an effort to discover olfactory receptor proteins. We encountered several clones that encode apparent transmembrane proteins; however, none is a clear candidate for an olfactory receptor. Instead we found a greater diversity of odourant binding proteins (OBPs) than previously known in moth antennae, raising the number known for M. sexta from three to seven. Together with evidence of seventeen members of the family from the Drosophila melanogaster genome project, our results suggest that insects may have many tens of OBPs expressed in subsets of the chemosensory sensilla on their antennae. These results support a model for insect olfaction in which OBPs selectively transport and present odourants to transmembrane olfactory receptors. We also found five members of a family of shorter proteins, named sensory appendage proteins (SAPs), that might also be involved in odourant transport. This small EST project also revealed several candidate odourant degrading enzymes including three P450 cytochromes, a glutathione S-transferase and a uridine diphosphate (UDP) glucosyltransferase. Several first insect homologues of proteins known from vertebrates, the nematode Caenorhabditis elegans, yeast and bacteria were encountered, and most have now also been detected by the large D. melanogaster EST project. Only thriteen entirely novel proteins were encountered, some of which are likely to be cuticle proteins.