The draft genome of the pest tephritid fruit fly Bactrocera tryoni: resources for the genomic analysis of hybridising species

Background

The tephritid fruit flies include a number of economically important pests of horticulture, with a large accumulated body of research on their biology and control. Amongst the Tephritidae, the genus Bactrocera, containing over 400 species, presents various species groups of potential utility for genetic studies of speciation, behaviour or pest control. In Australia, there exists a triad of closely-related, sympatric Bactrocera species which do not mate in the wild but which, despite distinct morphologies and behaviours, can be force-mated in the laboratory to produce fertile hybrid offspring. To exploit the opportunities offered by genomics, such as the efficient identification of genetic loci central to pest behaviour and to the earliest stages of speciation, investigators require genomic resources for future investigations.

Results

We produced a draft de novo genome assembly of Australia’s major tephritid pest species, Bactrocera tryoni. The male genome (650 -700 Mbp) includes approximately 150Mb of interspersed repetitive DNA sequences and 60Mb of satellite DNA. Assessment using conserved core eukaryotic sequences indicated 98% completeness. Over 16,000 MAKER-derived gene models showed a large degree of overlap with other Dipteran reference genomes. The sequence of the ribosomal RNA transcribed unit was also determined. Unscaffolded assemblies of B. neohumeralis and B. jarvisi were then produced; comparison with B. tryoni showed that the species are more closely related than any Drosophila species pair. The similarity of the genomes was exploited to identify 4924 potentially diagnostic indels between the species, all of which occur in non-coding regions.

Conclusions

This first draft B. tryoni genome resembles other dipteran genomes in terms of size and putative coding sequences. For all three species included in this study, we have identified a comprehensive set of non-redundant repetitive sequences, including the ribosomal RNA unit, and have quantified the major satellite DNA families. These genetic resources will facilitate the further investigations of genetic mechanisms responsible for the behavioural and morphological differences between these three species and other tephritids. We have also shown how whole genome sequence data can be used to generate simple diagnostic tests between very closely-related species where only one of the species is scaffolded.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1153) contains supplementary material, which is available to authorized users.     

Drosophila APC2 is a cytoskeletally-associated protein that regulates wingless signaling in the embryonic epidermis.

The tumor suppressor adenomatous polyposis coli (APC) negatively regulates Wingless (Wg)/Wnt signal transduction by helping target the Wnt effector beta-catenin or its Drosophila homologue Armadillo (Arm) for destruction. In cultured mammalian cells, APC localizes to the cell cortex near the ends of microtubules. Drosophila APC (dAPC) negatively regulates Arm signaling, but only in a limited set of tissues. We describe a second fly APC, dAPC2, which binds Arm and is expressed in a broad spectrum of tissues. dAPC2's subcellular localization revealed colocalization with actin in many but not all cellular contexts, and also suggested a possible interaction with astral microtubules. For example, dAPC2 has a striking asymmetric distribution in neuroblasts, and dAPC2 colocalizes with assembling actin filaments at the base of developing larval denticles. We identified a dAPC2 mutation, revealing that dAPC2 is a negative regulator of Wg signaling in the embryonic epidermis. This allele acts genetically downstream of wg, and upstream of arm, dTCF, and, surprisingly, dishevelled. We discuss the implications of our results for Wg signaling, and suggest a role for dAPC2 as a mediator of Wg effects on the cytoskeleton. We also speculate on more general roles that APCs may play in cytoskeletal dynamics.     

Acute vascular responses to isometric handgrip exercise and effects of training in persons medicated for hypertension

Previous work from our laboratory demonstrated that isometric handgrip (IHG) training improved local, endothelium-dependent vasodilation in medicated hypertensives [McGowan CL (PhD Thesis), 2006; McGowan et al. Physiologist 47: 285, 2004]. We investigated whether changes in the capacity of smooth muscle to dilate (regardless of endothelial factors) influenced this training-induced change, and we examined the acute vascular responses to a single bout of IHG. Seventeen subjects performed four 2-min unilateral IHG contractions at 30% of maximal voluntary effort, three times a week for 8 wk. Pre- and posttraining, brachial artery flow-mediated dilation (FMD, an index of endothelium-dependent vasodilation) and nitroglycerin-mediated maximal vasodilation (an index of endothelium-independent vasodilation) were measured in the exercised arm by using ultrasound before and immediately after acute IHG exercise. IHG training resulted in improved resting brachial FMD (P < 0.01) and no change in nitroglycerin-mediated maximal vasodilation. Pre- and posttraining, brachial artery FMD decreased following an acute bout of IHG exercise (normalized to peak shear rate, pre-, before IHG exercise: 0.01 +/- 0.002, after IHG exercise: 0.008 +/- 0.002%/s(-1); post-, before IHG exercise: 0.020 +/- 0.003, after IHG exercise: 0.010 +/- 0.003%/s(-1); P < 0.01). Posttraining, resting brachial artery FMD improved yet nitroglycerin-mediated maximal vasodilation was unchanged in persons medicated for hypertension. This suggests that the training-induced improvements in the resting brachial artery FMD were not due to underlying changes in the forearm vasculature. Acute IHG exercise attenuated brachial artery FMD, and although this impairment may be interpreted as hazardous to medicated hypertensives with already dysfunctional endothelium, the effects appear transient as repeated exposure to the IHG stimulus improved resting endothelium-dependent vasodilation.     

Sulfate reduction and S-oxidation in a moorland pool sediment

In an oligotrophic moorland pool in The Netherlands, S cycling near the sediment/water boundary was investigated by measuring (1) SO₄ ^2– reduction rates in the sediment, (2) depletion of SO₄ ^2– in the overlying water column and (3) release of³⁵S from the sediment into the water column. Two locations differing in sediment type (highly organic and sandy) were compared, with respect to reduction rates and depletion of SO₄ ^2– in the overlying water.Sulfate reduction rates in sediments of an oligotrophic moorland pool were estimated by diagenetic modelling and whole core³⁵SO₄ ^2– injection. Rates of SO₄ ^2– consumption in the overlying water were estimated by changes in SO₄ ^2– concentration over time in in situ enclosures. Reduction rates ranged from 0.27–11.2 mmol m^–2 d^–1. Rates of SO₄ ^2– uptake from the enclosed water column varied from –0.5, –0.3 mmol m^–2 d^–1 (November) to 0.43–1.81 mmol m^–2 d^–1 (July, August and April). Maximum rates of oxidation to SO₄ ^2– in July 1990 estimated by combination of SO₄ ^2– reduction rates and rates of in situ SO₄ ^2– uptake in the enclosed water column were 10.3 and 10.5 mmol m^–2 d^–1 at an organic rich and at a sandy site respectively.Experiments with³⁵S^2– and³⁵SO₄ ^2– tracer suggested (1) a rapid formation of organically bound S from dissimilatory reduced SO₄ ^2– and (2) the presence of mainly non SO₄ ^2–-S derived from reduced S transported from the sediment into the overlying water. A³⁵S^2– tracer experiment showed that about 7% of³⁵S^2– injected at 1 cm depth in a sediment core was recovered in the overlying water column.Sulfate reduction rates in sediments with higher volumetric mass fraction of organic matter did not significantly differ from those in sediments with a lower mass fraction of organic matter.Corresponding author     

Carcinogenic N-hydroxylaminopurine derivatives do not act as base analog mutagens in Salmonella typhimurium

N-Hydroxylaminopurines are highly mutagenic for growing as well as resting Salmonella typhimurium strain TA100 and to a lesser extent for strain TA98. Aminopurines, under similar conditions, are not mutagenic. N-Methylhydroxylaminopurine, under similar conditions, exhibits only minimal activity. The results are taken to indicate that unlike non-hydroxylated aminopurines, N-hydroxylaminopurines exert their mutagenicity not by acting as base analogs but by direct covalent binding with DNA-guanine.     

ConA激活小鼠胸腺T淋巴细胞增殖过程中c-myc与核骨架蛋白的结合

采用DNA-蛋白质体外吸附的方法研究伴刀豆球蛋白激活小鼠胸腺T淋巴细胞增殖过程中c-myc与核骨架蛋白的结合.实验结果显示,c-myc与核骨架蛋白的结合具有特异性,在淋巴细胞激活过程中c-myc与P₃₄/P₃₆核骨架蛋白及核纤层蛋白结合,并发生动态变化.     

Deposition of Erysiphe graminis Conidia on a Barley Crop

Naturally released Erysiphe graminis conidia were trapped (on horizontal slides, on vertical sticky cylinders and in suction traps) in a barley crop infected with powdery mildew and the numbers of single spores and of clumps of different sizes deposited on the traps were counted. The efficiencies of impaction calculated from deposits and wind speed measurements were higher than expected from mean wind speed measurements. The values were consistent with the hypothesis that spores were removedpredominantly in gusts. More than half the conidia were removed in clumps of two or more spores. The measurements suggest that clumps were more effectively deposited than single spores. The measurements demonstrate that spore release mechanisms can influence spore deposition significantly, especially close to the source.     

Differential Roles of the Glycogen-Binding Domains of β Subunits in Regulation of the Snf1 Kinase Complex

Members of the AMP-activated protein kinase family, including the Snf1 kinase of Saccharomyces cerevisiae, are activated under conditions of nutrient stress. AMP-activated protein kinases are heterotrimeric complexes composed of a catalytic α subunit and regulatory β and γ subunits. In this study, the role of the β subunits in the regulation of Snf1 activity was examined. Yeasts express three isoforms of the AMP-activated protein kinase consisting of Snf1 (α), Snf4 (γ), and one of three alternative β subunits, either Sip1, Sip2, or Gal83. The Gal83 isoform of the Snf1 complex is the most abundant and was analyzed in the greatest detail. All three β subunits contain a conserved domain referred to as the glycogen-binding domain. The deletion of this domain from Gal83 results in a deregulation of the Snf1 kinase, as judged by a constitutive activity independent of glucose availability. In contrast, the deletion of this homologous domain from the Sip1 and Sip2 subunits had little effect on Snf1 kinase regulation. Therefore, the different Snf1 kinase isoforms are regulated through distinct mechanisms, which may contribute to their specialized roles in different stress response pathways. In addition, the β subunits are subjected to phosphorylation. The responsible kinases were identified as being Snf1 and casein kinase II. The significance of the phosphorylation is unclear since the deletion of the region containing the phosphorylation sites in Gal83 had little effect on the regulation of Snf1 in response to glucose limitation.The Snf1 protein kinase of Saccharomyces cerevisiae is the yeast ortholog of the AMP-activated protein kinase (AMPK) found in mammals and other eukaryotes. AMPK acts as a nutrient and energy sensor, becoming activated under conditions of nutrient and energy depletion (6). In mammals, AMPK plays a key role in glucose homeostasis and is a target for drugs used to treat metabolic syndrome and type 2 diabetes (34). In yeast, the Snf1 kinase plays an essential role during aerobic growth and fermentative growth on alternative carbon sources. Cells lacking Snf1 kinase activity are viable but display numerous phenotypes including poor or no growth on alternative carbon sources, defects in meiosis and sporulation, defects in response to ion stress, and defects in pseudohyphal growth (7).The Snf1 kinase and all members of the AMPK family function as heterotrimers composed of a catalytic α subunit complexed with regulatory β and γ subunits (2). The γ subunit in mammalian enzymes directly binds three molecules of AMP (26, 33), which stimulates enzyme activity by inhibiting the dephosphorylation of the conserved threonine residue in the kinase activation loop (23). In yeast, there is no evidence that the γ subunit binds AMP; however, similar to mammals, the key glucose-regulated step is the dephosphorylation of the kinase activation loop (22).In this study, we examine the role of the β subunits in the regulation of the Snf1 kinase activity. Yeasts express three isoforms of the Snf1 kinase that differ depending on which of the three distinct β subunits, Sip1, Sip2, and Gal83, is incorporated into the enzyme. Previous studies have shown that the Snf1 isoforms have distinct substrate preferences (24), subcellular localizations (32), and stress response capacities (9). Only the Snf1 isoform containing Gal83 as the β subunit is able to localize to the cell nucleus in a process that requires Sak1, one of the three Snf1-activating protein kinases. Since all three of the Snf1-activating kinases (SAKs) are capable of phosphorylating Snf1 on its activation loop (3), it has remained a mystery as to why the Sak1 kinase is specifically required for Snf1 nuclear localization.The β subunits of Snf1 as well as mammalian AMPK contain a domain that is referred to as either a carbohydrate-binding module (CBM) (11) or a glycogen-binding domain (GBD) (19). The structure of this domain has been solved (20), and it was previously shown that this domain binds most tightly to branched oligosaccharides like glycogen that contain α1→6 branches (12). The binding of glycogen to the β subunit causes an allosteric inhibition of AMPK activity and inhibits phosphorylation by the upstream activating kinase. The β subunits of yeast contain the GBDs, but the importance of binding glycogen is questionable since cells that lack the ability to make glycogen show a normal regulation of Snf1 kinase in response to glucose limitation (15). Nonetheless, the deletion of the GBD from the Gal83 protein caused an increased activity of the Snf1 enzyme and release from glucose repression. Therefore, the GBD acts as a negative regulator of kinase activity in both mammalian and fungal cells.In this study we examine the role of the GBD present in the Sip2 and Sip1 proteins. We also extend the characterization of the Gal83 GBD by determining what connection this domain has with the regulated dephosphorylation of the Snf1 kinase. Finally, we have characterized other N-terminal domains in the β subunits that control accumulation and phosphorylation.