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Callosobruchus maculatus is a major agricultural pest of legume crops worldwide and an established model system in ecology and evolution. Yet, current molecular biological resources for this species are limited. Here, we employ Hi-C sequencing to generate a greatly improved genome assembly and we annotate its repetitive elements in a dedicated in-depth effort where we manually curate and classify the most abundant unclassified repeat subfamilies. We present a scaffolded chromosome-level assembly, which is 1.01 Gb in total length with 86% being contained within the 9 autosomes and the X chromosome. Repetitive sequences accounted for 70% of the total assembly. DNA transposons covered 18% of the genome, with the most abundant superfamily being Tc1-Mariner (9.75% of the genome). This new chromosome-level genome assembly of C. maculatus will enable future genetic and evolutionary studies not only of this important species but of beetles more generally.

Objective: B cell function and autoantibodies are important in SLE pathogenesis. In this work, we aimed to investigate the impact of cumulative SLE B cell genetics on SLE subphenotype and autoantibody profile. Methods: Female patients with SLE (n=1248) and healthy controls (n=400) were genotyped using Illumina's Global Screening Array. Two polygenic risk scores (PRSs), one representing B cell genes and the other B cell activation genes, were calculated for each individual using risk loci for SLE in genes assigned to B cell-related pathways according to the Kyoto Encyclopedia of Genes and Genomes, Gene Ontology and Reactome Databases. Results: Double-stranded DNA (dsDNA) antibodies were more prevalent among patients with a high compared with a low SLE B cell PRS (OR 1.47 (1.07 to 2.01), p=0.018), and effect sizes were augmented in patients with human leucocyte antigen (HLA) risk haplotypes HLA-DRB1*03:01 and HLA-DRB1*15:01 (DRB1*03/15 -/- (OR 0.99 (0.56 to 1.77), p=0.98; DRB1*03/15 +/- or -/+ (OR 1.64 (1.06 to 2.54), p=0.028; and DRB1*03/15 +/+ (OR 4.47 (1.21 to 16.47), p=0.024). Further, a high compared with a low B cell PRS was associated with low complement levels in DRB1*03/15 +/+ patients (OR 3.92 (1.22 to 12.64), p=0.022). The prevalence of lupus nephritis (LN) was higher in patients with a B cell activation PRS above the third quartile compared with patients below (OR 1.32 (1.00 to 1.74), p=0.048). Conclusions: High genetic burden related to B cell function is associated with dsDNA antibody development and LN. Assessing B cell PRSs may be important in order to determine immunological pathways influencing SLE and to predict clinical phenotype.

The patterns of reproductive timing and senescence vary within and across species owing to differences in reproductive strategies, but our understanding of the molecular underpinnings of such variation is incomplete. This is perhaps particularly true for sex differences. We investigated the evolution of sex-specific gene expression associated with life history divergence in replicated populations of the seed beetle Acanthoscelides obtectus, experimentally evolving under (E)arly or (L)ate life reproduction for >200 generations which has resulted in strongly divergent life histories. We detected 1,646 genes that were differentially expressed in E and L lines, consistent with a highly polygenic basis of life history evolution. Only 30% of differentially expressed genes were similarly affected in males and females. The evolution of long life was associated with significantly reduced sex differences in expression, especially in non-reproductive tissues. The expression differences were overall more pronounced in females, in accordance with their greater phenotypic divergence in lifespan. Functional enrichment analysis revealed differences between E and L beetles in gene categories previously implicated in aging, such as mitochondrial function and defense response. The results show that divergent life history evolution can be associated with profound changes in gene expression that alter the transcriptome in a sex-specific way, highlighting the importance of understanding the mechanisms of aging in each sex.

The aim of this research is to predict the mechanical properties along with the behaviors of standard insulating paper used in power transformers under thermal aging. This is conducted by applying an artificial neural network (ANN) trained with a multiple regression model and a particle swarm optimization (MR-PSO) model. The aging of the paper insulation is monitored directly by the tensile strength and the degree of polymerization of the solid insulation and indirectly by chemical markers using 2-furfuraldehyde compound content in oil (2-FAL). A mathematical model is then developed to simulate the mechanical properties (degree of polymerization (DPV) and tensile index (Tidx)) of the aged insulation paper. First, the datasets obtained from experimental results are used to create the MR model, and then the optimizer method PSO is used to optimize its coefficients in order to improve the MR model. Then, an ANN method is trained using the MR-PSO to create a nonlinear correlation between the DPV and the time, temperature, and 2-FAL values. The acquired results are assessed and compared with the experimental data. The model presents almost the same behavior. In particular, it has the capability to accurately simulate the nonlinear property behavior of insulation under thermal aging with an acceptable margin of error. Since the life expectancy of power transformers is directly related to that of the insulating paper, the proposed model can be useful to maintenance planners.

Viruses mimic host short linear motifs (SLiMs) to hijack and deregulate cellular functions. Studies of motif-mediated interactions therefore provide insight into virus-host dependencies, and reveal targets for therapeutic intervention. Here, we describe the pan-viral discovery of 1,712 SLiM-based virus-host interactions using a phage peptidome tiling the intrinsically disordered protein regions of 229 RNA viruses. We find mimicry of host SLiMs to be a ubiquitous viral strategy, reveal novel host proteins hijacked by viruses, and identify cellular pathways frequently deregulated by viral motif mimicry. Using structural and biophysical analyses, we show that viral mimicry-based interactions have similar binding strength and bound conformations as endogenous interactions. Finally, we establish polyadenylate-binding protein 1 as a potential target for broad-spectrum antiviral agent development. Our platform enables rapid discovery of mechanisms of viral interference and the identification of potential therapeutic targets which can aid in combating future epidemics and pandemics.

Oxygen (O2) plays an essential role in aerobic organisms including terrestrial insects. Under hypoxic stress, the cowpea bruchid (Callosobruchus maculatus) ceases feeding and growth. However, larvae, particularly 4th instar larvae exhibit very high tolerance to hypoxia and can recover normal growth once brought to normoxia. To better understand the molecular mechanism that enables insects to cope with low O2 stress, we performed RNA-seq to distinguish hypoxia-responsive genes in midguts and subsequently identified potential common cis-elements in promoters of hypoxia-induced and -repressed genes, respectively. Selected elements were subjected to gel-shift and transient transfection assays to confirm their cis-regulatory function. Of these putative common cis-elements, AREB6 appeared to regulate the expression of CmLPCAT and CmScylla, two hypoxia-induced genes. CmZFH, the putative AREB6-binding protein, was hypoxia-inducible. Transient expression of CmZFH in Drosophila S2 cells activated CmLPCAT and CmScylla, and their induction was likely through interaction of CmZFH with AREB6. Binding to AREB6 was further confirmed by bacterially expressed CmZFH recombinant protein. Deletion analyses indicated that the N-terminal zinc-finger cluster of CmZFH was the key AREB6-binding domain. Through in silico and experimental exploration, we discovered novel transcriptional regulatory components associated with gene expression dynamics under hypoxia that facilitated insect survival.

Efforts to unravel the genomic basis of incipient speciation are hampered by a mismatch between our toolkit and our understanding of the ecology and genetics of adaptation. While the former is focused on detecting selective sweeps involving few independently acting or linked speciation genes, the latter states that divergence typically occurs in polygenic traits under stabilizing selection. Here, we ask whether a role of stabilizing selection on polygenic traits in population divergence may be unveiled by using a phenotypically informed integrative approach, based on genome-wide variation segregating in divergent populations. We compare three divergent populations of seed beetles (Callosobruchus maculatus) where previous work has demonstrated a prominent role for stabilizing selection on, and population divergence in, key life history traits that reflect rate-dependent metabolic processes. We derive and assess predictions regarding the expected pattern of covariation between genetic variation segregating within populations and genetic differentiation between populations. Population differentiation was considerable (mean F-ST = 0.23-0.26) and was primarily built by genes showing high selective constraints and an imbalance in inferred selection in different populations (positive Tajima's D-NS in one and negative in one), and this set of genes was enriched with genes with a metabolic function. Repeatability of relative population differentiation was low at the level of individual genes but higher at the level of broad functional classes, again spotlighting metabolic genes. Absolute differentiation (d(XY)) showed a very different general pattern at this scale of divergence, more consistent with an important role for genetic drift. Although our exploration is consistent with stabilizing selection on polygenic metabolic phenotypes as an important engine of genome-wide relative population divergence and incipient speciation in our study system, we note that it is exceeding

Background: Understanding the forces that maintain diversity across a range of scales is at the very heart of biology. Frequency-dependent processes are generally recognized as the most central process for the maintenance of ecological diversity. The same is, however, not generally true for genetic diversity. Negative frequency dependent selection, where rare genotypes have an advantage, is often regarded as a relatively weak force in maintaining genetic variation in life history traits because recombination disassociates alleles across many genes. Yet, many regions of the genome show low rates of recombination and genetic variation in such regions (i.e., supergenes) may in theory be upheld by frequency dependent selection. Results: We studied what is essentially a ubiquitous life history supergene (i.e., mitochondrial DNA) in the fruit fly Drosophila subobscura, showing sympatric polymorphism with two main mtDNA genotypes co-occurring in populations world-wide. Using an experimental evolution approach involving manipulations of genotype starting frequencies, we show that negative frequency dependent selection indeed acts to maintain genetic variation in this region. Moreover, the strength of selection was affected by food resource conditions. Conclusions: Our work provides novel experimental support for the view that balancing selection through negative frequency dependency acts to maintain genetic variation in life history genes. We suggest that the emergence of negative frequency dependent selection on mtDNA is symptomatic of the fundamental link between ecological processes related to resource use and the maintenance of genetic variation.

Mitochondrial DNA (mtDNA) consists of few but vital maternally inherited genes that interact closely with nuclear genes to produce cellular energy. How important mtDNA polymorphism is for adaptation is still unclear. The assumption in population genetic studies is often that segregating mtDNA variation is selectively neutral. This contrasts with empirical observations of mtDNA haplotypes affecting fitness-related traits and thermal sensitivity, and latitudinal clines in mtDNA haplotype frequencies. Here, we experimentally test whether ambient temperature affects selection on mtDNA variation, and whether such thermal effects are influenced by intergenomic epistasis due to interactions between mitochondrial and nuclear genes, using replicated experimental evolution inCallosobruchus maculatusseed beetle populations seeded with a mixture of different mtDNA haplotypes. We also test for sex-specific consequences of mtDNA evolution on reproductive success, given that mtDNA mutations can have sexually antagonistic fitness effects. Our results demonstrate natural selection on mtDNA haplotypes, with some support for thermal environment influencing mtDNA evolution through mitonuclear epistasis. The changes in male and female reproductive fitness were both aligned with changes in mtDNA haplotype frequencies, suggesting that natural selection on mtDNA is sexually concordant in stressful thermal environments. We discuss the implications of our findings for the evolution of mtDNA.

The male ejaculate contains a multitude of seminal fluid proteins (SFPs), many of which are key reproductive molecules, as well as sperm. However, the identification of SFPs is notoriously difficult and a detailed understanding of this complex phenotype has only been achieved in a few model species. We employed a recently developed proteomic method involving whole-organism stable isotope labelling coupled with proteomic and transcriptomic analyses to characterize ejaculate proteins in the seed beetle Callosobruchus maculatus. We identified 317 proteins that were transferred to females at mating, and a great majority of these showed signals of secretion and were highly male-biased in expression in the abdomen. These male-derived proteins were enriched with proteins involved in general metabolic and catabolic processes but also with proteolytic enzymes and proteins involved in protection against oxidative stress. Thirty-seven proteins showed significant homology with SFPs previously identified in other insects. However, no less than 92 C. maculatus ejaculate proteins were entirely novel, receiving no significant blast hits and lacking homologs in extant data bases, consistent with a rapid and divergent evolution of SFPs. We used 3D micro-tomography in conjunction with proteomic methods to identify 5 distinct pairs of male accessory reproductive glands and to show that certain ejaculate proteins were only recovered in certain male glands. Finally, we provide a tentative list of 231 candidate female-derived reproductive proteins, some of which are likely important in ejaculate processing and/or sperm storage.

Animal mitogenomes are generally thought of as being economic and optimized for rapid replication and transcription. We use long-read sequencing technology to assemble the remarkable mitogenomes of four species of seed beetles. These are the largest circular mitogenomes ever assembled in insects, ranging from 24,496 to 26,613 bp in total length, and are exceptional in that some 40% consists of non-coding DNA. The size expansion is due to two very long intergenic spacers (LIGSs), rich in tandem repeats. The two LIGSs are present in all species but vary greatly in length (114-10,408 bp), show very low sequence similarity, divergent tandem repeat motifs, a very high AT content and concerted length evolution. The LIGSs have been retained for at least some 45 my but must have undergone repeated reductions and expansions, despite strong purifying selection on protein coding mtDNA genes. The LIGSs are located in two intergenic sites where a few recent studies of insects have also reported shorter LIGSs (>200 bp). These sites may represent spaces that tolerate neutral repeat array expansions or, alternatively, the LIGSs may function to allow a more economic translational machinery. Mitochondrial respiration in adult seed beetles is based almost exclusively on fatty acids, which reduces the need for building complex I of the oxidative phosphorylation pathway (NADH dehydrogenase). One possibility is thus that the LIGSs may allow depressed transcription of NAD genes. RNA sequencing showed that LIGSs are partly transcribed and transcriptional profiling suggested that all seven mtDNA NAD genes indeed show low levels of transcription and co-regulation of transcription across sexes and tissues.

The seed beetle Callosobruchus maculatus is a significant agricultural pest and increasingly studied model of sexual conflict. Males possess genital spines that increase the transfer of seminal fluid proteins (SFPs) into the female body. As SFPs alter female behaviour and physiology, they are likely to modulate reproduction and sexual conflict in this species. Here, we identified SFPs using proteomics combined with a de novo transcriptome. A prior 2D-sodium dodecyl sulphate polyacrylamide gel electrophoresis analysis identified male accessory gland protein spots that were probably transferred to the female at mating. Proteomic analysis of these spots identified 98 proteins, a majority of which were also present within ejaculates collected from females. Standard annotation workflows revealed common functional groups for SFPs, including proteases and metabolic proteins. Transcriptomic analysis found 84 transcripts differentially expressed between the sexes. Notably, genes encoding 15 proteins were highly expressed in male abdomens and only negligibly expressed within females. Most of these sequences corresponded to 'unknown' proteins (nine of 15) and may represent rapidly evolving SFPs novel to seed beetles. Our combined analyses highlight 44 proteins for which there is strong evidence that they are SFPs. These results can inform further investigation, to better understand the molecular mechanisms of sexual conflict in seed beetles.