The appendage role of insect disco genes and possible implications on the evolution of the maggot larval form

AbstractThough initially identified as necessary for neural migration, Disconnected and its partially redundant paralog, Disco-related, are required for proper head segment identity during Drosophila embryogenesis. Here, we present evidence that these genes are also required for proper ventral appendage development during development of the adult fly, where they specify medial to distal appendage development. Cells lacking the disco genes cannot contribute to the medial and distal portions of ventral appendages. Further, ectopic disco transforms dorsal appendages toward ventral fates; in wing discs, the medial and distal leg development pathways are activated. Interestingly, this appendage role is conserved in the red flour beetle, Tribolium (where legs develop during embryogenesis), yet in the beetle we found no evidence for a head segmentation role. The lack of an embryonic head specification role in Tribolium could be interpreted as a loss of the head segmentation function in Tribolium or gain of this function during evolution of flies. However, we suggest an alternative explanation. We propose that the disco genes always function as appendage factors, but their appendage nature is masked during Drosophila embryogenesis due to the reduction of limb fields in the maggot style Drosophila larva

New components of Drosophila leg development identified through genome wide association studies.

The adult Drosophila melanogaster body develops from imaginal discs, groups of cells set-aside during embryogenesis and expanded in number during larval stages. Specification and development of Drosophila imaginal discs have been studied for many years as models of morphogenesis. These studies are often based on mutations with large developmental effects, mutations that are often lethal in embryos when homozygous. Such forward genetic screens can be limited by factors such as early lethality and genetic redundancy. To identify additional genes and genetic pathways involved in leg imaginal disc development, we employed a Genome Wide Association Study utilizing the natural genetic variation in leg proportionality found in the Drosophila Genetic Reference Panel fly lines. In addition to identifying genes already known to be involved in leg development, we identified several genes involved in pathways that had not previously been linked with leg development. Several of the genes appear to be involved in signaling activities, while others have no known roles at this time. Many of these uncharacterized genes are conserved in mammals, so we can now begin to place these genes into developmental contexts. Interestingly, we identified five genes which, when their function is reduced by RNAi, cause an antenna-to-leg transformation. Our results demonstrate the utility of this approach, integrating the tools of quantitative and molecular genetics to study developmental processes, and provide new insights into the pathways and networks involved in Drosophila leg development

An Optimized Small-Scale Rearing System to Support Embryonic Microinjection Protocols for Western Corn Rootworm, <i>Diabrotica virgifera virgifera</i>

Western corn rootworm (WCR), a major pest of corn, has been reared in laboratories since the 1960s. While established rearing methods are appropriate for maintaining WCR colonies, they are not optimal for performing germline transformation or CRISPR/Cas9-based genome editing. Here we report the development of an optimized rearing system for use in WCR functional genomics research, specifically the development of a system that facilitates the collection of preblastoderm embryos for microinjection as well as gathering large larvae and pupae for downstream phenotypic screening. Further, transgenic-based experiments require stable and well-defined survival rates and the ability to manipulate insects at every life stage. In our system, the WCR life cycle (egg to adult) takes approximately 42 days, with most individuals eclosing between 41 and 45 days post oviposition. Over the course of one year, our overall survival rate was 67%. We used this data to establish a quality control system for more accurately monitoring colony health. Herein, we also offer detailed descriptions for setting up single-pair crosses and conducting phenotypic screens to identify transgenic progeny. This study provides a model for the development of new rearing systems and the establishment of highly controlled processes for specialized purposes

DroID analyses identifying connections between candidates and canonical leg development genes.

<p>Genes in orange connect directly to the candidate (Green), and may be linked directly or through genes in blue to the canonical appendage cascade genes (red). Yellow identifies those of our candidate genes that are linked to the test gene. A) Analysis of PI31. Links shared with Cka are shown in dark blue. B) Analysis of Cka. Links shared with PI31 are shown in dark orange and dark blue. C) Analysis of CG6841, including a link to disco in pink.</p

<i>Dll</i>-Gal4 driving RNAi phenocopies in legs.

<p>Second thoracic legs from female flies with <i>Dll</i>-Gal4 driving UAS-RNAi insertions. All flies were grown at room temperature. A) Wild-type. B) RNAi of <i>CSN1b</i> resulted in reduced leg size, but did not alter shape. C) RNAi of <i>PI31</i> reduced length of tarsal segments, with occasional loss of a single distal segment. D) RNAi of <i>eIF6</i> caused deletions and fusions of tarsal segments. E) RNAi of <i>CG6841</i> deleted most of the tarsal segments and terminal structures. F) RNAi of <i>Cka</i> was similar to that of <i>CG6841</i>, but more severe. Abbreviations: fe, femur; ti, tibia; ta, tarsal segments; cl, claw.</p

Summary of protein functions.

<p>Known and predicted functions of proteins were obtained from Flybase and are provided only for genes with phenocopy (<i>boi</i> was included because of its known redundancy with <i>ihog</i>). The five candidates that gave antenna-to-leg transformation with the <i>Dll</i> driver are marked with an asterisk.</p

Effect in antennae of <i>Dll</i>-Gal4 driving RNAi.

<p>Heads from flies with both <i>Dll</i>-Gal4 and UAS-RNAi insertions that disrupted antennal development. Arrows with numbers indicate the antennal segments. A) Wild-type. B) RNAi of <i>CSN1b</i> resulted in loss of arista, and appearance of sclerotized structure C). RNAi of <i>PI31</i> resulted in loss of arista while the remaining structures appear normal. D) RNAi of <i>CG6841</i> resulted in loss of arista, and drastic changes to the appearance of antennal segment 3, and some reduction of A2. E) RNAi of <i>Cka</i> resulted in loss of all but proximal structures. F) RNAi of <i>CG9129</i> is characteristic of the five lines that resulted in transformation of antennae toward leg identity beginning in distal A2, though slight differences were noted between the five lines that caused these transformations.</p

Model for larval leg disc patterning, proportionality and adult leg structure.

<p>A) Leg imaginal disc patterning begins (left) when En, present in the posterior (yellow) portion of the disc, activates <i>hh</i> expression. The Hh ligand diffuses into the anterior portion of the disc. There it activates the expression of <i>dpp</i> (purple) dorsally and <i>wg</i> (green) ventrally. Dpp and Wg, in turn, diffuse throughout the dorsal and ventral portions, respectively, of the disc, creating a gradient of their mutual presence. This gradient is responsible for the pattern of expression of transcription factors (middle) that establish the proximal-distal axis of the leg (right). In the center of the disc, mutual Dpp and Wg is highest, activating the expression of <i>Dll</i>, which is responsible for patterning the most distal structures of the leg, including the terminal structures (claw and pulvillus). Reduced mutual Dpp and Wg results in the activation of <i>dac</i>, responsible for the patterning of the middle portions of the leg. Where there is almost no mutual Dpp or Wg, Hth and Exd are active, patterning the proximal leg portions and the junction with the rest of the body. B–C illustrate how proportions might change without affecting over-all length. B) Enhanced Hh signal (dark yellow) could result in an expansion of Wg and Dpp, causing a broader domain of <i>Dll</i> expression, at the expense of <i>dac</i> (middle). This could, in turn, cause the tarsal segments to make up a larger portion of the leg without changing leg length (right). C) Similarly, the proportion of the femur could be expanded (right) if <i>dac</i> expression were expanded (middle). This might result from a decrease in expression or distribution of Dpp and Wg (left), which could, in turn, be caused by reduced Hh signal (light yellow).</p

Distribution of candidate gene mRNA in imaginal discs.

<p>Wild-type third instar imaginal discs stained to detect mRNAs from several of our candidate genes. Examples with several different mRNA distribution patterns are shown. There are several that are of note, the cross-like pattern of <i>boi</i> in the wing disc that borders <i>wg</i> and <i>dpp</i> expression, the narrow tarsal staining of <i>eIF6</i>. We also note that <i>disco-r</i> is expressed in the ventral edge of the wing which had not been reported previously.</p

Genome of the small hive beetle (Aethina tumida , Coleoptera: Nitidulidae), a worldwide parasite of social bee colonies, provides insights into detoxification and herbivory

Background: The small hive beetle (Aethina tumida; ATUMI) is an invasive parasite of bee colonies. ATUMI feeds on both fruits and bee nest products, facilitating its spread and increasing its impact on honey bees and other pollinators. We have sequenced and annotated the ATUMI genome, providing the first genomic resources for this species and for the Nitidulidae, a beetle family that is closely related to the extraordinarily species-rich clade of beetles known as the Phytophaga. ATUMI thus provides a contrasting view as a neighbor for one of the most successful known animal groups. Results: We present a robust genome assembly and a gene set possessing 97.5% of the core proteins known from the holometabolous insects. The ATUMI genome encodes fewer enzymes for plant digestion than the genomes of wood-feeding beetles but nonetheless shows signs of broad metabolic plasticity. Gustatory receptors are few in number compared to other beetles, especially receptors with known sensitivity (in other beetles) to bitter substances. In contrast, several gene families implicated in detoxification of insecticides and adaptation to diverse dietary resources show increased copy numbers. The presence and diversity of homologs involved in detoxification differ substantially from the bee hosts of ATUMI. Conclusions: Our results provide new insights into the genomic basis for local adaption and invasiveness in ATUMI and a blueprint for control strategies that target this pest without harming their honey bee hosts. A minimal set of gustatory receptors is consistent with the observation that, once a host colony is invaded, food resources are predictable. Unique detoxification pathways and pathway members can help identify which treatments might control this species even in the presence of honey bees, which are notoriously sensitive to pesticides