RNAi-Mediated Functional Analysis of Bursicon Genes Related to Adult Cuticle Formation and Tanning in the Honeybee, <i>Apis mellifera</i>

<div><p>Bursicon is a heterodimeric neurohormone that acts through a G protein-coupled receptor named rickets (rk), thus inducing an increase in cAMP and the activation of tyrosine hydroxylase, the rate-limiting enzyme in the cuticular tanning pathway. In insects, the role of bursicon in the post-ecdysial tanning of the adult cuticle and wing expansion is well characterized. Here we investigated the roles of the genes encoding the bursicon subunits during the adult cuticle development in the honeybee, <i>Apis mellifera</i>. RNAi-mediated knockdown of <i>AmBurs α</i> and <i>AmBurs β</i> bursicon genes prevented the complete formation and tanning (melanization/sclerotization) of the adult cuticle. A thinner, much less tanned cuticle was produced, and ecdysis toward adult stage was impaired. Consistent with these results, the knockdown of bursicon transcripts also interfered in the expression of genes encoding its receptor, AmRk, structural cuticular proteins, and enzymes in the melanization/sclerotization pathway, thus evidencing roles for bursicon in adult cuticle formation and tanning. Moreover, the expression of <i>AmBurs α</i>, <i>AmBurs β</i> and <i>AmRk</i> is contingent on the declining ecdysteroid titer that triggers the onset of adult cuticle synthesis and deposition. The search for transcripts of <i>AmBurs α</i>, <i>AmBurs β</i> and candidate targets in RNA-seq libraries prepared with brains and integuments strengthened our data on transcript quantification through RT-qPCR. Together, our results support our premise that bursicon has roles in adult cuticle formation and tanning, and are in agreement with other recent studies pointing for roles during the pharate-adult stage, in addition to the classical post-ecdysial ones.</p></div

Expression of <i>AmBurs α and AmBurs β</i> in the brain, and expression of <i>AmRk</i> in the integument during adult cuticle formation.

<p><b>(A)</b> The main molting events (pupal ecdysis, apolysis, synthesis and differentiation of the adult cuticle, adult ecdysis) are indicated in relation to the ecdysteroid titer variation and adult cuticle phenotype. Pupa (Pw), pharate-adults (Pp, Pdp, Pb, Pbl, Pbm and Pbd) and newly-emerged adults (NE) are the successive phases of the honeybee development. Relative quantification of <b>(B)</b> <i>AmBurs α</i> and <b>(C)</b> <i>AmBurs β</i> mRNAs in the brain of pupae (Pw), pharate adults (Pb, Pbl) and newly emerged-adults (NE). <b>(D)</b> Relative quantification of bursicon receptor (<i>AmRk</i>) transcripts in the integument of the same developmental phases. Transcript levels determined through RT-qPCR. Bars represent mean ± standard error (se) of three samples, each prepared with seven brains or seven thoracic/abdominal integuments. The asterisk indicates statistically significant increase of transcripts in pharate adults in relation to the pupal (Pw) phase (Student's t-test, p<0.05). <b>(E-G)</b> mRNA levels (FPKM+1) in the brain <b>(E, F)</b> and in the integument <b>(G)</b> of pharate adults (Pbm phase) and newly-ecdysed adults (NE phase) determined through RNA-seq. Levels of <i>AmBurs α</i> and <i>AmBurs β</i> were identified in two brain libraries, each prepared with five pooled brains extracted from the Pbm or NE phases. Levels of <i>AmRk</i> were identified in six integument libraries (three from Pbm and three from NE phases), each prepared with five integuments. The asterisk in <b>G</b> indicates a statistically significant difference (adjusted p value = 0.001).</p

20E-dependent expression of <i>AmBurs α</i>, <i>AmBurs β</i> and <i>AmRk</i>.

<p><b>(A)</b> Representation of the moment of 20E injection (Pw phase) and collection of brain and integument samples at the Pb and Pbd pharate adult phases. External aspect of the developing honeybees injected with 20E and (C) controls. <i>AmBurs α</i> transcript levels in the brain of <b>(B)</b> Pb and <b>(C)</b> Pbd phases. <i>AmBurs β</i> transcript levels in the brain of <b>(D)</b> Pb and <b>(E)</b> Pbd phases. <i>AmRk</i> transcript levels in the integument of <b>(F)</b> Pb and <b>(G)</b> Pbd phases. Transcript levels quantified through RT-qPCR. Bars represent mean ± se of three samples, each prepared with two brains or two integuments. The asterisks indicate statistically significant differences (Student's t-test, p<0,05).</p

The expression of genes encoding structural cuticular proteins was up- or downregulated by <i>AmBurs α</i> and <i>AmBurs β</i> knockdown.

<p><b>(A)</b> Pupae (Pw) were injected in the brain with ds<i>AmBurs α</i> or ds<i>AmBurs β</i>, and integument samples were collected when bees reached the Pbd and NE phases. Transcript levels of <b>(B)</b> <i>AmCPR14</i>, <b>(C)</b> <i>AmCPR3</i>, <b>(D)</b> <i>AmTwdl1</i>, <b>(E)</b> <i>AmTwdl2</i>, <b>(F)</b> <i>Amapd2</i> and <b>(G)</b> <i>Amapd3</i> were quantified in the integument through RT-qPCR using <i>AmRP49</i> as reference gene. Bars represent mean ± se of four samples, each prepared with a single integument. Different letters on the bars indicate statistically significant differences (Student's t-test, p<0,05). RNA-seq using integument samples support the presence of <b>(H)</b> <i>AmCPR14</i>, <b>(I)</b> <i>AmCPR3</i>, <b>(J)</b> <i>AmTwdl1</i>, <b>(K)</b> <i>AmTwdl2</i>, <b>(L)</b> <i>Amapd2</i> and <b>(M)</b> <i>Amapd3</i> transcripts during adult cuticle formation in pharate-adults (Pbm phase) and show that, except for <i>Amapd2</i> <b>(L)</b> (adjusted p-value = 0.226), there is a significant decrease in transcript levels after ecdysis (NE phase) (adjusted p-values ≤ 0.001).</p

Effect of <i>AmBurs α</i> and <i>AmBurs β</i> knockdown on the expression of genes encoding enzymes with roles in the melanization/sclerotization pathway, and on the expression of the bursicon receptor.

<p><b>(A)</b> Pupae (Pw) were injected in the brain with ds<i>AmBurs α</i> or ds<i>AmBurs β</i>, and integument samples were collected when bees reached the Pbl pharate-adult phase and after ecdysis (NE phase). Transcript levels of <b>(B)</b> <i>Amddc</i>, <b>(C)</b> <i>Amth</i>, <b>(D)</b> <i>Ampxd</i> and <b>(E</b>) <i>AmRk</i> were quantified in the integument through RT-qPCR using <i>AmRP49</i> as reference gene. Bars represent mean ± se of four samples, each prepared with the integument of a single bee. Different letters on the bars indicate statistically significant differences (Student's t-test, p<0,05). RNA-seq using integument samples support the presence of <b>(F)</b> <i>Amddc</i>, <b>(G)</b> <i>Amth</i>, <b>(H)</b> <i>Ampxd</i> and <b>(I)</b> <i>AmRk</i> transcripts during adult cuticle formation in pharate-adults (Pbm phase) and show that, except for <i>Amth</i> <b>(B)</b>, which was up-regulated, there is a significant decrease in transcript levels after ecdysis (NE phase) (adjusted p-values ≤ 0.001).</p

<i>AmBurs α</i> and <i>AmBurs β</i> knockdown mediated by dsRNA delivery into the abdominal cavity impairs the complete formation of adult cuticle, tanning and ecdysis.

<p><b>(A)</b> Pupae (Pw) were injected with ds<i>AmBurs α</i> or ds<i>AmBurs β</i>; brain and integument samples were collected after ecdysis of the controls (NE phase). Bee phenotypes and brain transcript levels after injection of 0.3, 1 or 3 μg of <b>(B)</b> ds<i>AmBurs α</i> and <b>(C)</b> ds<i>AmBurs β</i> in comparison with untreated control, C. Transcript levels determined through RT-sqPCR using <i>AmRP49</i> as reference gene. <b>(D)</b> The thoracic and abdominal integuments were dissected from the dsRNA-treated and untreated bees and stained with methylene blue and basic fuchsin for microscopic examination. Cuticle (Ct), epidermis (Ep), thoracic musculature (Mc) and fat body (Fb) are identified in the histological sections. Note the melanized cuticle in the thoracic integument section of a control bee in contrast to the incompletely differentiated cuticle of the dsRNA-treated bees. The control abdominal cuticle shows a dark and toothed superficial layer, which was much less evident in the dsRNA-treated bees.</p

The genomes of three Bradyrhizobium sp. isolated from root nodules of Lupinus albescens grown in extremely poor soils display important genes for resistance to environmental stress

<div><p>Abstract Lupinus albescens is a resistant cover plant that establishes symbiotic relationships with bacteria belonging to the Bradyrhizobium genus. This symbiosis helps the development of these plants in adverse environmental conditions, such as the ones found in arenized areas of Southern Brazil. This work studied three Bradyrhizobium sp. (AS23, NAS80 and NAS96) isolated from L. albescens plants that grow in extremely poor soils (arenized areas and adjacent grasslands). The genomes of these three strains were sequenced in the Ion Torrent platform using the IonXpress library preparation kit, and presented a total number of bases of 1,230,460,823 for AS23, 1,320,104,022 for NAS80, and 1,236,105,093 for NAS96. The genome comparison with closest strains Bradyrhizobium japonicum USDA6 and Bradyrhizobium diazoefficiens USDA110 showed important variable regions (with less than 80% of similarity). Genes encoding for factors for resistance/tolerance to heavy metal, flagellar motility, response to osmotic and oxidative stresses, heat shock proteins (present only in the three sequenced genomes) could be responsible for the ability of these microorganisms to survive in inhospitable environments. Knowledge about these genomes will provide a foundation for future development of an inoculant bioproduct that should optimize the recovery of degraded soils using cover crops.</p></div

RAD tag (SgrAI) derived SNPs from Bombus impatiens

RAD tag (SgrAI) derived SNPs from Bombus impatiens from Sadd et al. (2015) "The genomes of two key bumblebee species with primitive eusocial organisation