Genome-wide identification and characterization of ATP-binding cassette transporters in the silkworm, Bombyx mori

<p>Abstract</p> <p>Background</p> <p>The ATP-binding cassette (ABC) transporter superfamily is the largest transporter gene family responsible for transporting specific molecules across lipid membranes in all living organisms. In insects, ABC transporters not only have important functions in molecule transport, but also play roles in insecticide resistance, metabolism and development.</p> <p>Results</p> <p>From the genome of the silkworm, <it>Bombyx mori</it>, we have identified 51 putative ABC genes which are classified into eight subfamilies (A-H) by phylogenetic analysis. Gene duplication is very evident in the ABCC and ABCG subfamilies, whereas gene numbers and structures are well conserved in the ABCD, ABCE, ABCF, and ABCH subfamilies. Microarray analysis revealed that expression of 32 silkworm ABC genes can be detected in at least one tissue during different developmental stages, and the expression patterns of some of them were confirmed by quantitative real-time PCR. A large number of ABC genes were highly expressed in the testis compared to other tissues. One of the ABCG genes, <it>BmABC002712</it>, was exclusively and abundantly expressed in the Malpighian tubule implying that <it>BmABC002712 </it>plays a tissue-specific role. At least 5 ABCG genes, including <it>BmABC005226</it>, <it>BmABC005203</it>, <it>BmABC005202</it>, <it>BmABC010555</it>, and <it>BmABC010557</it>, were preferentially expressed in the midgut, showing similar developmental expression profiles to those of 20-hydroxyecdysone (20E)-response genes. 20E treatment induced the expression of these ABCG genes in the midgut and RNA interference-mediated knockdown of <it>USP</it>, a component of the 20E receptor, decreased their expression, indicating that these midgut-specific ABCG genes are 20E-responsive.</p> <p>Conclusion</p> <p>In this study, a genome-wide analysis of the silkworm ABC transporters has been conducted. A comparison of ABC transporters from 5 insect species provides an overview of this vital gene superfamily in insects. Moreover, tissue- and stage-specific expression data of the silkworm ABCG genes lay a foundation for future analysis of their physiological function and hormonal regulation.</p

20-Hydroxyecdysone (20E) Primary Response Gene \u3cem\u3eE93\u3c/em\u3e Modulates 20E Signaling to Promote \u3cem\u3eBombyx\u3c/em\u3e Larval-Pupal Metamorphosis

As revealed in a previous microarray study to identify genes regulated by 20-hydroxyecdysone (20E) and juvenile hormone (JH) in the silkworm, Bombyx mori, E93 expression in the fat body was markedly low prior to the wandering stage but abundant during larval-pupal metamorphosis. Induced by 20E and suppressed by JH, E93 expression follows this developmental profile in multiple silkworm alleles. The reduction of E93 expression by RNAi disrupted 20E signaling and the 20E-induced autophagy, caspase activity, and cell dissociation in the fat body. Reducing E93 expression also decreased the expression of the 20E-induced pupal-specific cuticle protein genes and prevented growth and differentiation of the wing discs. Importantly, the two HTH domains in E93 are critical for inducing the expression of a subset of 20E response genes, including EcR, USP, E74, Br-C, and Atg1. By contrast, the LLQHLL and PLDLSAK motifs in E93 inhibit its transcriptional activity. E93 binds to the EcR-USP complex via a physical association with USP through its LLQHLL motif; and this association is enhanced by 20E-induced EcR-USP interaction, which attenuates the transcriptional activity of E93. E93 acts through the two HTH domains to bind to GAGA-containing motifs present in the Atg1 promoter region for inducing gene expression. In conclusion, E93 transcriptionally modulates 20E signaling to promote Bombyx larval-pupal metamorphosis

Genome-wide regulation of innate immunity by juvenile hormone and 20-hydroxyecdysone in the Bombyx fat body

<p>Abstract</p> <p>Background</p> <p>Insect innate immunity can be affected by juvenile hormone (JH) and 20-hydroxyecdysone (20E), but how innate immunity is developmentally regulated by these two hormones in insects has not yet been elucidated. In the silkworm, <it>Bombyx mori</it>, JH and 20E levels are high during the final larval molt (4 M) but absent during the feeding stage of 5^thinstar (5 F), while JH level is low and 20E level is high during the prepupal stage (PP). Fat body produces humoral response molecules and hence is considered as the major organ involved in innate immunity.</p> <p>Results</p> <p>A genome-wide microarray analysis of <it>Bombyx </it>fat body isolated from 4 M, 5 F and PP uncovered a large number of differentially-expressed genes. Most notably, 6 antimicrobial peptide (AMP) genes were up-regulated at 4 M versus PP suggesting that <it>Bombyx </it>innate immunity is developmentally regulated by the two hormones. First, JH treatment dramatically increased AMP mRNA levels and activities. Furthermore, 20E treatment exhibited inhibitory effects on AMP mRNA levels and activities, and RNA interference of the 20E receptor <it>EcR</it>-<it>USP </it>had the opposite effects to 20E treatment.</p> <p>Conclusion</p> <p>Taken together, we demonstrate that JH acts as an immune-activator while 20E inhibits innate immunity in the fat body during <it>Bombyx </it>postembryonic development.</p

An End-to-End Deep Fusion Model for Mapping Forests at Tree Species Levels with High Spatial Resolution Satellite Imagery

Mapping the distribution of forest resources at tree species levels is important due to their strong association with many quantitative and qualitative indicators. With the ongoing development of artificial intelligence technologies, the effectiveness of deep-learning classification models for high spatial resolution (HSR) remote sensing images has been proved. However, due to the poor statistical separability and complex scenarios, it is still challenging to realize fully automated and highly accurate forest types at tree species level mapping. To solve the problem, a novel end-to-end deep learning fusion method for HSR remote sensing images was developed by combining the advantageous properties of multi-modality representations and the powerful features of post-processing step to optimize the forest classification performance refined to the dominant tree species level in an automated way. The structure of the proposed model consisted of a two-branch fully convolutional network (dual-FCN8s) and a conditional random field as recurrent neural network (CRFasRNN), which named dual-FCN8s-CRFasRNN in the paper. By constructing a dual-FCN8s network, the dual-FCN8s-CRFasRNN extracted and fused multi-modality features to recover a high-resolution and strong semantic feature representation. By imbedding the CRFasRNN module into the network as post-processing step, the dual-FCN8s-CRFasRNN optimized the classification result in an automatic manner and generated the result with explicit category information. Quantitative evaluations on China&rsquo;s Gaofen-2 (GF-2) HSR satellite data showed that the dual-FCN8s-CRFasRNN provided a competitive performance with an overall classification accuracy (OA) of 90.10%, a Kappa coefficient of 0.8872 in the Wangyedian forest farm, and an OA of 74.39%, a Kappa coefficient of 0.6973 in the GaoFeng forest farm, respectively. Experiment results also showed that the proposed model got higher OA and Kappa coefficient metrics than other four recently developed deep learning methods and achieved a better trade-off between automaticity and accuracy, which further confirmed the applicability and superiority of the dual-FCN8s-CRFasRNN in forest types at tree species level mapping tasks

MET Is Required for the Maximal Action of 20-Hydroxyecdysone during <em>Bombyx</em> Metamorphosis

<div><p>Little is known about how the putative juvenile hormone (JH) receptor, the bHLH-PAS transcription factor MET, is involved in 20-hydroxyecdysone (20E; the molting hormone) action. Here we report that two MET proteins found in the silkworm, <em>Bombyx mori</em>, participate in 20E signal transduction. <em>Met</em> is 20E responsive and its expression peaks during molting and pupation, when the 20E titer is high. As found with results from RNAi knockdown of <em>EcR</em>-<em>USP</em> (the ecdysone receptor genes), RNAi knockdown of <em>Met</em> at the early wandering stage disrupts the 20E-triggered transcriptional cascade, preventing tissue remodeling (including autophagy, apoptosis and destruction of larval tissues and generation of adult structures) and causing lethality during the larval-pupal transition. MET physically interacts with EcR-USP. Moreover, MET, EcR-USP and the 20E-response element (EcRE) form a protein-DNA complex, implying that MET might modulate 20E-induced gene transcription by interacting with EcR-USP. In conclusion, the 20E induction of MET is required for the maximal action of 20E during <em>Bombyx</em> metamorphosis.</p> </div

Lethal and defective phenotypes caused by <i>Met</i> RNAi in silkworms.

<p>dsRNA (10 µg per larva) was injected into selected larva during initiation of the early wandering stage. <i>egfp</i> dsRNA was used as a control. (A–C) Typical <i>Met1</i> RNAi and <i>Met2</i> RNAi treated silkworms died during the wandering stage (A) or during pupation (B), while some were arrested at the mid-pupal stage (C). The pictures (A–C) show the dying animals after <i>Met</i> RNAi. (D, E) <i>Met</i> RNAi affected adult structure formation. The surviving <i>Met1</i> RNAi and <i>Met2</i> RNAi treated pupae did not fully develop legs and wings during the late pupal stage (D). Many of the surviving <i>Met1</i> RNAi adults failed to shed the pupal cuticle attached to their head or abdomen, exhibiting shortened and distorted legs or unexpanded wings (E).</p

Physical interaction between MET and EcR-USP.

<p>(A) The CytoTrap yeast two-hybrid analyses revealed direct associations among MET1, MET2, EcR and USP. Strong associations between bait and prey proteins led to more yeast colonies. (B) When the <i>HA-EcR</i>, <i>FLAG-USP</i>, and <i>V5-Met1</i> constructs were co-transfected into human HEK 293 cells, 20E treatment for 6 hr at a final concentration of 1 µM had little or no stimulating effects on the physical interactions between MET and EcR-USP. In the immunoprecipation experiments, the bottom Western blot is input. IP, immunoprecipitate; Blot, Western blot. (C) The <i>HA-EcR</i>, <i>FLAG-USP</i>, <i>V5-Met1</i>, and <i>cMyc-Met2</i> constructs were co-transfected into the human HEK 293 cells. After nuclear extracts were bound with biotin-labeled EcRE, the protein-DNA complexes were separated on a 5% native PAGE gel followed by EMSA. Addition of the HA or FLAG antibody resulted in a shift of EcRE. In (C) and (D), the shift was indicated by a black arrow in comparison with a gray arrow. (D) The <i>HA-EcR</i>, <i>FLAG-USP</i>, <i>V5-Met1</i>, and <i>cMyc-Met2</i> constructs were co-transfected into human HEK 293 cells. After nuclear extracts were bound with biotin-labeled EcRE, the protein-DNA complexes were separated 5% native PAGE followed by EMSA. When the V5 or cMyc antibody was added, binding of EcR-USP-EcRE was shifted by MET1 and MET2 in EMSA showing that MET, EcR-USP and EcRE form a protein-DNA complex. (E) <i>Met1</i> RNAi and transfection were simultaneously conducted in <i>Bombyx</i> DZNU-Bm-12 cells for 48 hr, followed by 20E treatment for 6 hr at a final concentration of 1 µM, and measurements of EcRE-driven luciferase activity were done. MET is required for 20E function to induce gene expression via the ecdysone receptor and EcRE. The bars labeled with different lowercase letters are significantly different (P<0.05, ANOVA).</p

<i>Met</i> RNAi results in lethality during the larval-pupal-adult metamorphosis.

<p>dsRNA (10 µg per larva) was injected into selected larvae during initiation of the early wandering stage. Lethality was scored at the larval, prepupal, and pupal stages to compare the effects of <i>egfp</i>, <i>Met1</i>, <i>Met2</i>, and <i>Met1</i>+<i>2</i> dsRNAs.</p