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.

Correlated expression patterns of microRNA genes with age-dependent behavioural changes in honeybee.

The hive-living honeybees (Apis mellifera) show age-dependent behavioural changes; young bees usually nurse the broods in the colony and the older bees engage in foraging activities. These developmentally regulated behavioural changes were previously shown to be correlated with genome-wide transcriptional changes in the honeybee brain. The indigenous small regulatory RNA molecules, known as microRNAs (miRNAs), are potent regulators of gene expression and also are developmentally regulated. Thus, we wanted to study if there might be correlation of differential expression of miRNA genes in the brain with age-dependent behavioural changes of the bees. We determined expression patterns of a set (n= 20) of predicted miRNA genes, by quantitative real-time PCR assays, in the brains of young and old bees that were engaged in nursing or foraging activities in the colony, respectively. Our data show correlated up-regulation of miRNA-124, miRNA-14, miRNA-276, miRNA-13b, let-7 and miRNA-13a in the young nurse bees. miRNA-12, miRNA-9, miRNA-219, miRNA-210, miRNA-263, miRNA-92 and miRNA-283 showed correlated expression patterns in the old forager bees. The modular changes of miRNA genes in the young nurse and old forager bees suggest possible roles of miRNAs in age-dependent behavioural changes in bees. The correlated expression of intronic miRNA genes and their host genes as well as of miRNA genes physically clustered in the genome are also observed.

Basolateral localization of the Caenorhabditis elegans epidermal growth factor receptor in epithelial cells by the PDZ protein LIN-10.

In Caenorhabditis elegans, the EGF receptor (encoded by let-23) is localized to the basolateral membrane domain of the epithelial vulval precursor cells, where it acts through a conserved Ras/MAP kinase signaling pathway to induce vulval differentiation. lin-10 acts in LET-23 receptor tyrosine kinase basolateral localization, because lin-10 mutations result in mislocalization of LET-23 to the apical membrane domain and cause a signaling defective (vulvaless) phenotype. We demonstrate that the previous molecular identification of lin-10 was incorrect, and we identify a new gene corresponding to the lin-10 genetic locus. lin-10 encodes a protein with regions of similarity to mammalian X11/mint proteins, containing a phosphotyrosine-binding and two PDZ domains. A nonsense lin-10 allele that truncates both PDZ domains only partially reduces lin-10 gene activity, suggesting that these protein interaction domains are not essential for LIN-10 function in vulval induction. Immunocytochemical experiments show that LIN-10 is expressed in vulval epithelial cells and in neurons. LIN-10 is present at low levels in the cytoplasm and at the plasma membrane and at high levels at or near the Golgi. LIN-10 may function in secretion of LET-23 to the basolateral membrane domain, or it may be involved in tethering LET-23 at the basolateral plasma membrane once it is secreted.

Behavior and the limits of genomic plasticity: power and replicability in microarray analysis of honeybee brains.

Transcription is slow relative to many post-transcriptional processes in the brain. Using the rich system of division of labor in the honeybee (Apis mellifera), we found extreme differences in the extent to which behavioral occupations of different durations were associated with gene-expression differences in the brain. Nursing and foraging, occupations lasting > 1 week, were associated with significant expression differences for nearly one-quarter of the genes tested (1208 of 5563 cDNAs tested; P < 0.01, anova), consistent with previous results. In contrast, transitional occupations, performed for 1-2 days after nursing and before the onset of foraging, were associated with either no differences (guards vs. undertakers; 19 cDNAs, fewer than the expectation of 56 false-positives) or few differences (comb builders vs. guards and undertakers; 248 cDNAs), but extensive differences relative to both nursing and foraging (> 500 cDNAs, all contrasts). Statistical power analysis indicated that expression differences of two-, 1.5- and 1.25-fold should have been detected in 100, 92 and 37% of cases, respectively. Replication of previous results at these magnitudes was 95, 71 and 51%, with no genes showing differences in the opposite direction. These results indicate that behavioral plasticity over different time-scales may be associated with substantial differences in the extent of genomic plasticity in the brain.

Inhibition of Caenorhabditis elegans vulval induction by gap-1 and by let-23 receptor tyrosine kinase.

During induction of the Caenorhabditis elegans hermaphrodite vulva, a signal from the anchor cell activates the LET-23 epidermal growth factor receptor (EGFR)/LET-60 Ras/MPK-1 MAP kinase signaling pathway in the vulval precursor cells. We have characterized two mechanisms that limit the extent of vulval induction. First, we found that gap-1 may directly inhibit the LET-60 Ras signaling pathway. We identified the gap-1 gene in a genetic screen for inhibitors of vulval induction. gap-1 is predicted to encode a protein similar to GTPase-activating proteins that likely functions to inhibit the signaling activity of LET-60 Ras. A loss-of-function mutation in gap-1 suppresses the vulvaless phenotype of mutations in the let-60 ras signaling pathway, but a gap-1 single mutant does not exhibit excess vulval induction. Second, we found that let-23 EGFR prevents vulval induction in a cell-nonautonomous manner, in addition to its cell-autonomous role in activating the let-60 ras/mpk-1 signaling pathway. Using genetic mosaic analysis, we show that let-23 activity in the vulval precursor cell closest to the anchor cell (P6.p) prevents induction of vulval precursor cells further away from the anchor cell (P3.p, P4.p, and P8.p). This result suggests that LET-23 in proximal vulval precursor cells might bind and sequester the inductive signal LIN-3 EGF, thereby preventing diffusion of the inductive signal to distal vulval precursor cells.

The LIN-2/LIN-7/LIN-10 complex mediates basolateral membrane localization of the C. elegans EGF receptor LET-23 in vulval epithelial cells.

In C. elegans, the LET-23 receptor tyrosine kinase is localized to the basolateral membranes of polarized vulval epithelial cells. lin-2, lin-7, and lin-10 are required for basolateral localization of LET-23, since LET-23 is mislocalized to the apical membrane in lin-2, lin-7, and lin-10 mutants. Yeast two-hybrid, in vitro binding, and in vivo coimmunoprecipitation experiments show that LIN-2, LIN-7, and LIN-10 form a protein complex. Furthermore, compensatory mutations in lin-7 and let-23 exhibit allele-specific suppression of apical mislocalization and signaling-defective phenotypes. These results present a mechanism for basolateral localization of LET-23 receptor tyrosine kinase by direct binding to the LIN-2/LIN-7/LIN-10 complex. Each of the binding interactions within this complex is conserved, suggesting that this complex may also mediate basolateral localization in mammals.

LIN-10 is a shared component of the polarized protein localization pathways in neurons and epithelia.

We tested the model that neurons and epithelial cells use a shared mechanism for polarized protein sorting by comparing the pathways for localizing basolateral and postsynaptic proteins in C. elegans. GLR-1 glutamate receptors are localized to postsynaptic elements of central synapses and, when ectopically expressed, to basolateral membranes of epithelial cells. Proper localization of GLR-1 in both neurons and epithelia requires the PDZ protein LIN-10, defining LIN-10 as a shared component of the basolateral and postsynaptic localization pathways. Changing the GLR-1 carboxy-terminal sequence from a group I PDZ-binding consensus (-TAV) to a group II consensus (-FYV) restores GLR-1 synaptic localization in lin-10 mutants. Thus, these interneurons utilize at least two separate postsynaptic localization pathways.