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Abstract Background The lower Dipteran fungus fly, Sciara coprophila, has many unique biological features that challenge the rule of genome DNA constancy. For example, Sciara undergoes paternal chromosome elimination and maternal X chromosome nondisjunction during spermatogenesis, paternal X elimination during embryogenesis, intrachromosomal DNA amplification of DNA puff loci during larval development, and germline-limited chromosome elimination from all somatic cells. Paternal chromosome elimination in Sciara was the first observation of imprinting, though the mechanism remains a mystery. Here, we present the first draft genome sequence for Sciara coprophila to take a large step forward in addressing these features. Results We assembled the Sciara genome using PacBio, Nanopore, and Illumina sequencing. To find an optimal assembly using these datasets, we generated 44 short-read and 50 long-read assemblies. We ranked assemblies using 27 metrics assessing contiguity, gene content, and dataset concordance. The highest-ranking assemblies were scaffolded using BioNano optical maps. RNA-seq datasets from multiple life stages and both sexes facilitated genome annotation. A set of 66 metrics was used to select the first draft assembly for Sciara. Nearly half of the Sciara genome sequence was anchored into chromosomes, and all scaffolds were classified as X-linked or autosomal by coverage. Conclusions We determined that X-linked genes in Sciara males undergo dosage compensation. An entire bacterial genome from the Rickettsia genus, a group known to be endosymbionts in insects, was co-assembled with the Sciara genome, opening the possibility that Rickettsia may function in sex determination in Sciara. Finally, the signal level of the PacBio and Nanopore data support the presence of cytosine and adenine modifications in the Sciara genome, consistent with a possible role in imprinting.

Huanglongbing (HLB), also known as citrus greening disease, is caused by the bacterium Candidatus Liberibacter asiaticus (CLas). It is a serious threat to global citrus production. This bacterium is transmitted by the Asian citrus psyllid, Diaphorina citri (Hemiptera). There are no effective in planta treatments for CLas. Therefore, one strategy is to manage the psyllid population. Manual annotation of the D. citri genome can identify and characterize gene families that could be novel targets for psyllid control. The yellow gene family is an excellent target because yellow genes, which have roles in melanization, are linked to development and immunity. Combined analysis of the genome with RNA-seq datasets, sequence homology, and phylogenetic trees were used to identify and annotate nine yellow genes in the D. citri genome. Manual curation of genes in D. citri provided in-depth analysis of the yellow family among hemipteran insects and provides new targets for molecular control of this psyllid pest. Manual annotation was done as part of a collaborative Citrus Greening community annotation project.

Hox genes and their cofactors are essential developmental genes specifying regional identity in animals. Hox genes have a conserved arrangement in clusters in the same order in which they specify identity along the anterior–posterior axis. A few insect species have breaks in the cluster, but these are exceptions. We annotated the 10 Hox genes of the Asian citrus psyllid Diaphorina citri, and found a split in its Hox cluster between the Deformed and Sex combs reduced genes – the first time a break at this position has been observed in an insect Hox cluster. We also annotated D. citri orthologs of the Hox cofactor genes homothorax, PKNOX and extradenticle and found an additional copy of extradenticle in D. citri that appears to be a retrogene. Expression data and sequence conservation suggest that the extradenticle retrogene may have retained the original extradenticle function and allowed divergence of the parental extradenticle gene.

The circadian rhythm involves multiple genes that generate an internal molecular clock, allowing organisms to anticipate environmental conditions produced by the Earth’s rotation on its axis. Here, we present the results of the manual curation of 27 genes that are associated with circadian rhythm in the genome of Diaphorina citri, the Asian citrus psyllid. This insect is the vector for the bacterial pathogen Candidatus Liberibacter asiaticus (CLas), the causal agent of citrus greening disease (Huanglongbing). This disease severely affects citrus industries and has drastically decreased crop yields worldwide. Based on cry1 and cry2 identified in the psyllid genome, D. citri likely possesses a circadian model similar to the lepidopteran butterfly, Danaus plexippus. Manual annotation will improve the quality of circadian rhythm gene models, allowing the future development of molecular therapeutics, such as RNA interference or antisense technologies, to target these genes to disrupt the psyllid biology.

Ubiquitination is an ATP-dependent process that targets proteins for degradation by the proteasome. Here, we annotated 15 genes from the ubiquitin-proteasome pathway in the Asian citrus psyllid, Diaphorina citri. This psyllid vector has come to prominence in the last decade owing to its role in the transmission of the devastating bacterial pathogen, Candidatus Liberibacter asiaticus (CLas). Infection of citrus crops by this pathogen causes Huanglongbing (HLB), or citrus greening disease, and results in the eventual death of citrus trees. The identification and correct annotation of these genes in D. citri will be useful for functional genomic studies to aid the development of RNAi-based management strategies aimed at reducing the spread of HLB. Investigating the effects of CLas infection on the expression of ubiquitin-proteasome pathway genes may provide new information about the role these genes play in the acquisition and transmission of CLas by D. citri.

Chitinases are enzymes that digest the polysaccharide polymer chitin. During insect development, breakdown of chitin is an essential step in molting of the exoskeleton. Knockdown of chitinases required for molting is lethal to insects, making chitinase genes an interesting target for RNAi-based pest control methods. The Asian citrus psyllid, Diaphorina citri, carries the bacterium causing Huanglongbing, or citrus greening disease, a devastating citrus disease. We identified and annotated 12 chitinase family genes from D. citri as part of a community effort to create high-quality gene models to aid the design of interdictory molecules for pest control. We categorized the D. citri chitinases according to an established classification scheme and re-evaluated the classification of chitinases in other hemipterans. In addition to chitinases from known groups, we identified a novel class of chitinases present in D. citri and several related hemipterans that appears to be the result of horizontal gene transfer.

The hemipteran insect Diaphorina citri, or Asian citrus psyllid, is a vector for Candidatus Liberibacter asiaticus (CLas), the bacterium causing citrus greening disease, or Huanglongbing (HLB). Millions of citrus trees have been destroyed, and every grove in Florida, USA, has been directly affected by this disease. In eukaryotes, vacuolar-type ATP synthase (V-ATPase) is an abundant heterodimeric enzyme that serves the cell with essential compartment acidification through the active processes that transport protons across the membrane. Fifteen putative V-ATPase genes in the D. citri genome were manually curated. Comparative genomic analysis revealed that D. citri V-ATPase subunits share domains and motifs with other insects, including the V-ATPase-A superfamily domain. Phylogenetic analysis separates D. citri V-ATPase subunits into expected clades with orthologous sequences. Annotation of the D. citri genome is a critical step towards developing directed pest management strategies to reduce the spread of HLB throughout the citrus industry.

Citrus greening disease is caused by the pathogen Candidatus Liberibacter asiaticus and transmitted by the Asian citrus psyllid, Diaphorina citri. No curative treatment or significant prevention mechanism exists for this disease, which causes economic losses from reduced citrus production. A high-quality genome of D. citri is being manually annotated to provide accurate gene models to identify novel control targets and increase understanding of this pest. Here, we annotated 25 D. citri genes involved in glycolysis and gluconeogenesis, and seven in trehaloneogenesis. Comparative analysis showed that glycolysis genes in D. citri are highly conserved but copy numbers vary. Analysis of expression levels revealed upregulation of several enzymes in the glycolysis pathway in the thorax, consistent with the primary use of glucose by thoracic flight muscles. Manually annotating these core metabolic pathways provides accurate genomic foundation for developing gene-targeting therapeutics to control D. citri.

Insects have a segmented body plan that is established during embryogenesis when the anterior–posterior (A–P) axis is divided into repeated units by a cascade of gene expression. The cascade is initiated by protein gradients created by translation of maternally provided mRNAs, localized at the anterior and posterior poles of the embryo. Combinations of these proteins activate specific gap genes to divide the embryo into distinct regions along the anterior–posterior axis. Gap genes then activate pair-rule genes, which are usually expressed in parts of every other segment. The pair-rule genes, in turn, activate expression of segment polarity genes in a portion of each segment. The segmentation genes are generally conserved among insects, although there is considerable variation in how they are deployed. We annotated 25 segmentation gene homologs in the Asian citrus psyllid, Diaphorina citri. Most of the genes expected to be present in D. citri based on their phylogenetic distribution in other insects were identified and annotated. Two exceptions were eagle and invected, which are present in at least some hemipterans, but were not found in D. citri. Many of the segmentation pathway genes are likely to be essential for D. citri development, and thus they may be useful targets for gene-based pest control methods.

The polysaccharide chitin is critical for the formation of many insect structures, including the exoskeleton, and is required for normal development. Here we report the annotation of three genes from the chitin synthesis pathway in the Asian citrus psyllid, Diaphorina citri (Hemiptera: Liviidae), the vector of Huanglongbing (citrus greening disease). Most insects have two chitin synthase (CHS) genes but, like other hemipterans, D. citri has only one. In contrast, D. citri is unusual among insects in having two UDP-N-acetylglucosamine pyrophosphorylase (UAP) genes. One of the D. citri UAP genes is broadly expressed, while the other is expressed predominantly in males. Our work helps pave the way for potential utilization of these genes as pest control targets to reduce the spread of Huanglongbing.

Chitin deacetylases (CDAs) are one of the least understood components of insect chitin metabolism. The partial deacetylation of chitin polymers appears to be important for the proper formation of higher order chitin structures, such as long fibers and bundles, which contribute to the integrity of the insect exoskeleton and other structures. Some CDAs may also be involved in bacterial defense. Here, we report the manual annotation of four CDA genes from the Asian citrus psyllid, Diaphorina citri, laying the groundwork for future study of these genes.

The Asian citrus psyllid, Diaphorina citri, is an insect vector that transmits Candidatus Liberibacter asiaticus, the causal agent of the Huanglongbing (HLB), or citrus greening disease. This disease has devastated Florida’s citrus industry, and threatens California’s industry as well as other citrus producing regions around the world. To find novel solutions to the disease, a better understanding of the vector is needed. The D. citri genome has been used to identify and characterize genes involved in Wnt signaling pathways. Wnt signaling is utilized for many important biological processes in metazoans, such as patterning and tissue generation. Curation based on RNA sequencing data and sequence homology confirms 24 Wnt signaling genes within the D. citri genome, including homologs for beta-catenin, Frizzled receptors, and seven Wnt-ligands. Through phylogenetic analysis, we classify D. citri Wnt ligands as Wg/Wnt1, Wnt5, Wnt6, Wnt7, Wnt10, Wnt11, and WntA. The D. citri version 3.0 genome with chromosomal length scaffolds reveals a conserved Wnt1-Wnt6-Wnt10 gene cluster with a gene configuration like that in Drosophila melanogaster. These findings provide greater insight into the evolutionary history of D. citri and Wnt signaling in this important hemipteran vector. Manual annotation was essential for identifying high quality gene models. These gene models can be used to develop molecular systems, such as CRISPR and RNAi, which target and control psyllid populations to manage the spread of HLB. Manual annotation of Wnt signaling pathways was done as part of a collaborative community annotation project.

Artificial sweeteners are ubiquitous constituents of sanitary wastewater and are not completely attenuated by wastewater treatment processes. Consequently, artificial sweeteners are increasingly employed as a tool to detect wastewater and help evaluate its impact on aquatic environments. In rural areas, septic systems are known point sources of artificial sweeteners to groundwater, however the potential contribution of artificial sweeteners to streams via this pathway is unknown. We analyzed 294 samples from 173 stream sites in Southern Ontario, Canada, for acesulfame, saccharin, cyclamate, and sucralose, and found that 91% had one or more of these compounds present. The stream sites sampled did not have municipal wastewater treatment plants upstream and therefore septic system effluent was the most likely source of the artificial sweeteners. Acesulfame, which is the most recalcitrant of the four artificial sweeteners, was by far the most ubiquitous in streams, with a 91% detection frequency, compared to 27, 8, and 3% for saccharin, cyclamate, and sucralose, respectively. Stream concentrations ranged from non-detectable to maximum values of 225, 380, 204, and 291 ng/L for acesulfame, saccharin, cyclamate, and sucralose, respectively. We calculated that water from septic effluent contributed from

Abstract Background The de novo assembly of repeat-rich mammalian genomes using only high-throughput short read sequencing data typically results in highly fragmented genome assemblies that limit downstream applications. Here, we present an iterative approach to hybrid de novo genome assembly that incorporates datasets stemming from multiple genomic technologies and methods. We used this approach to improve the gray mouse lemur (Microcebus murinus) genome from early draft status to a near chromosome-scale assembly. Methods We used a combination of advanced genomic technologies to iteratively resolve conflicts and super-scaffold the M. murinus genome. Results We improved the M. murinus genome assembly to a scaffold N50 of 93.32 Mb. Whole genome alignments between our primary super-scaffolds and 23 human chromosomes revealed patterns that are congruent with historical comparative cytogenetic data, thus demonstrating the accuracy of our de novo scaffolding approach and allowing assignment of scaffolds to M. murinus chromosomes. Moreover, we utilized our independent datasets to discover and characterize sequences associated with centromeres across the mouse lemur genome. Quality assessment of the final assembly found 96% of mouse lemur canonical transcripts nearly complete, comparable to other published high-quality reference genome assemblies. Conclusions We describe a new assembly of the gray mouse lemur (Microcebus murinus) genome with chromosome-scale scaffolds produced using a hybrid bioinformatic and sequencing approach. The approach is cost effective and produces superior results based on metrics of contiguity and completeness. Our results show that emerging genomic technologies can be used in combination to characterize centromeres of non-model species and to produce accurate de novo chromosome-scale genome assemblies of complex mammalian genomes.

High quality gene models are necessary to expand the molecular and genetic tools available for a target organism, but these are available for only a handful of model organisms that have undergone extensive curation and experimental validation over the course of many years. The majority of gene models present in biological databases today have been identified in draft genome assemblies using automated annotation pipelines that are frequently based on orthologs from distantly related model organisms and usually have minor or major errors. Manual curation is time consuming and often requires substantial expertise, but is instrumental in improving gene model structure and identification. Manual annotation may seem to be a daunting and cost-prohibitive task for small research communities but involving undergraduates in community genome annotation consortiums can be mutually beneficial for both education and improved genomic resources. We outline a workflow for efficient manual annotation driven by a team of primarily undergraduate annotators. This model can be scaled to large teams and includes quality control processes through incremental evaluation. Moreover, it gives students an opportunity to increase their understanding of genome biology and to participate in scientific research in collaboration with peers and senior researchers at multiple institutions.

In the short-germ beetle Tribolium castaneum, waves of pair-rule gene expression propagate from the posterior end of the embryo towards the anterior and eventually freeze into stable stripes, partitioning the anterior-posterior axis into segments. Similar waves in vertebrates are assumed to arise due to the modulation of a molecular clock by a posterior-to-anterior frequency gradient. However, neither a molecular candidate nor a functional role has been identified to date for such a frequency gradient, either in vertebrates or elsewhere. Here we provide evidence that the posterior gradient of Tc-caudal expression regulates the oscillation frequency of pair-rule gene expression in Tribolium. We show this by analyzing the spatiotemporal dynamics of Tc-even-skipped expression in strong and mild knockdown of Tc-caudal, and by correlating the extension, level and slope of the Tc-caudal expression gradient to the spatiotemporal dynamics of Tc-even-skipped expression in wild type as well as in different RNAi knockdowns of Tc-caudal regulators. Further, we show that besides its absolute importance for stripe generation in the static phase of the Tribolium blastoderm, a frequency gradient might serve as a buffer against noise during axis elongation phase in Tribolium as well as vertebrates. Our results highlight the role of frequency gradients in pattern formation.

Artificial sweeteners have been widely incorporated in human food products for aid in weight loss regimes, dental health protection and dietary control of diabetes. Some of these widely used compounds can pass non-degraded through wastewater treatment systems and are subsequently discharged to groundwater and surface waters. Measurements of artificial sweeteners in rivers used for drinking water production are scarce. In order to determine the riverine concentrations of artificial sweeteners and their usefulness as a tracer of wastewater at the scale of an entire watershed, we analyzed samples from 23 sites along the entire length of the Grand River, a large river in Southern Ontario, Canada, that is impacted by agricultural activities and urban centres. Municipal water from household taps was also sampled from several cities within the Grand River Watershed. Cyclamate, saccharin, sucralose, and acesulfame were found in elevated concentrations despite high rates of biological activity, large daily cycles in dissolved oxygen and shallow river depth. The maximum concentrations that we measured for sucralose (21 µg/L), cyclamate (2.4 µg/L) [corrected], and saccharin (7.2 µg/L) are the highest reported concentrations of these compounds in surface waters to date anywhere in the world. Acesulfame persists at concentrations that are up to several orders of magnitude above the detection limit over a distance of 300 km and it behaves conservatively in the river, recording the wastewater contribution from the cumulative population in the basin. Acesulfame is a reliable wastewater effluent tracer in rivers. Furthermore, it can be used to assess rates of nutrient assimilation, track wastewater plume dilution, separate human and animal waste contributions and determine the relative persistence of emerging contaminants in impacted watersheds where multiple sources confound the usefulness of other tracers. The effects of artificial sweeteners on aquatic biota in rivers and in the downstream Great Lakes are largely unknown.

The phenomenon of RNAi, in which the introduction of dsRNA into a cell triggers the destruction of the corresponding mRNA resulting in a gene silencing effect, is conserved across a wide array of plant and animal phyla. However, the mechanism by which the dsRNA enters a cell, allowing the RNAi effect to occur throughout a multicellular organism (systemic RNAi), has only been studied extensively in certain plants and the nematode Caenorhabditis elegans. In recent years, RNAi has become a popular reverse genetic technique for gene silencing in many organisms. Although many RNAi techniques in non-traditional model organisms rely on the systemic nature of RNAi, little has been done to analyze the parameters required to obtain a robust systemic RNAi response. The data provided here show that the concentration and length of dsRNA have profound effects on the efficacy of the RNAi response both in regard to initial efficiency and duration of the effect in Tribolium castaneum. In addition, our analyses using a series of short dsRNAs and chimeric dsRNA provide evidence that dsRNA cellular uptake (and not the RNAi response itself) is the major step affected by dsRNA size in Tribolium. We also demonstrate that competitive inhibition of dsRNA can occur when multiple dsRNAs are injected together, influencing the effectiveness of RNAi. These data provide specific information essential to the design and implementation of RNAi based studies, and may provide insight into the molecular basis of the systemic RNAi response in insects.