Annotation and visualization of parasite, fungi and arthropod genomes with Companion.

As sequencing genomes has become increasingly popular, the need for annotation of the resulting assemblies is growing. Structural and functional annotation is still challenging as it includes finding the correct gene sequences, annotating other elements such as RNA and being able to submit those data to databases to share it with the community. Compared to de novo assembly where contiguous chromosomes are a sign of high quality, it is difficult to visualize and assess the quality of annotation. We developed the Companion web server to allow non-experts to annotate their genome using a reference-based method, enabling them to assess the output before submitting to public databases. In this update paper, we describe how we have included novel methods for gene finding and made the Companion server more efficient for annotation of genomes of up to 1 Gb in size. The reference set was increased to include genomes of interest for human and animal health from the fungi and arthropod kingdoms. We show that Companion outperforms existing comparable tools where closely related references are available.

The Patterns of Codon Usage between Chordates and Arthropods are Different but Co-evolving with Mutational Biases.

Different frequencies amongst codons that encode the same amino acid (i.e. synonymous codons) have been observed in multiple species. Studies focused on uncovering the forces that drive such codon usage showed that a combined effect of mutational biases and translational selection works to produce different frequencies of synonymous codons. However, only few have been able to measure and distinguish between these forces that may leave similar traces on the coding regions. Here, we have developed a codon model that allows the disentangling of mutation, selection on amino acids and synonymous codons, and GC-biased gene conversion (gBGC) which we employed on an extensive dataset of 415 chordates and 191 arthropods. We found that chordates need 15 more synonymous codon categories than arthropods to explain the empirical codon frequencies, which suggests that the extent of codon usage can vary greatly between animal phyla. Moreover, methylation at CpG sites seems to partially explain these patterns of codon usage in chordates but not in arthropods. Despite the differences between the two phyla, our findings demonstrate that in both, GC-rich codons are disfavored when mutations are GC-biased, and the opposite is true when mutations are AT-biased. This indicates that selection on the genomic coding regions might act primarily to stabilize its GC/AT content on a genome-wide level. Our study shows that the degree of synonymous codon usage varies considerably among animals, but is likely governed by a common underlying dynamic.

Selection Across the Three-Dimensional Structure of Venom Proteins from North American Scolopendromorph Centipedes.

Gene duplication followed by nucleotide differentiation is one of the simplest mechanisms to develop new functions for genes. However, the evolutionary processes underlying the divergence of multigene families remain controversial. We used multigene families found within the diversity of toxic proteins in centipede venom to test two hypotheses related to venom evolution: the two-speed mode of venom evolution and the rapid accumulation of variation in exposed residues (RAVER) model. The two-speed mode of venom evolution proposes that different types of selection impact ancient and younger venomous lineages with negative selection being the predominant form in ancient lineages and positive selection being the dominant form in younger lineages. The RAVER hypothesis proposes that, instead of different types of selection acting on different ages of venomous lineages, the different types of selection will selectively contribute to amino acid variation based on whether the residue is exposed to the solvent where it can potentially interact directly with toxin targets. This hypothesis parallels the longstanding understanding of protein evolution that suggests that residues found within the structural or active regions of the protein will be under negative or purifying selection, and residues that do not form part of these areas will be more prone to positive selection. To test these two hypotheses, we compared the venom of 26 centipedes from the order Scolopendromorpha from six currently recognized species from across North America using both transcriptomics and proteomics. We first estimated their phylogenetic relationships and uncovered paraphyly among the genus Scolopendra and evidence for cryptic diversity among currently recognized species. Using our phylogeny, we then characterized the diverse venom components from across the identified clades using a combination of transcriptomics and proteomics. We conducted selection-based analyses in the context of predicted three-dimensional properties of the venom proteins and found support for both hypotheses. Consistent with the two-speed hypothesis, we found a prevalence of negative selection across all proteins. Consistent with the RAVER hypothesis, we found evidence of positive selection on solvent-exposed residues, with structural and less-exposed residues showing stronger signal for negative selection. Through the use of phylogenetics, transcriptomics, proteomics, and selection-based analyses, we were able to describe the evolution of venom from an ancient venomous lineage and support principles of protein evolution that directly relate to multigene family evolution.

Genomic insights into the chromosomal elongation in a family of Collembola.

Collembola is a highly diverse and abundant group of soil arthropods with chromosome numbers ranging from 5 to 11. Previous karyotype studies indicated that the Tomoceridae family possesses an exceptionally long chromosome. To better understand chromosome size evolution in Collembola, we obtained a chromosome-level genome of Yoshiicerus persimilis with a size of 334.44 Mb and BUSCO completeness of 97.0% (n = 1013). Both genomes of Y. persimilis and Tomocerus qinae (recently published) have an exceptionally large chromosome (ElChr greater than 100 Mb), accounting for nearly one-third of the genome. Comparative genomic analyses suggest that chromosomal elongation occurred independently in the two species approximately 10 million years ago, rather than in the ancestor of the Tomoceridae family. The ElChr elongation was caused by large tandem and segmental duplications, as well as transposon proliferation, with genes in these regions experiencing weaker purifying selection (higher dN/dS) than conserved regions. Moreover, inter-genomic synteny analyses indicated that chromosomal fission/fusion events played a crucial role in the evolution of chromosome numbers (ranging from 5 to 7) within Entomobryomorpha. This study provides a valuable resource for investigating the chromosome evolution of Collembola.

Genetic underpinnings of arthropod community distributions in Populus trichocarpa.

Community genetics seeks to understand the mechanisms by which natural genetic variation in heritable host phenotypes can encompass assemblages of organisms such as bacteria, fungi, and many animals including arthropods. Prior studies that focused on plant genotypes have been unable to identify genes controlling community composition, a necessary step to predict ecosystem structure and function as underlying genes shift within plant populations. We surveyed arthropods within an association population of Populus trichocarpa in three common gardens to discover plant genes that contributed to arthropod community composition. We analyzed our surveys with traditional single-trait genome-wide association analysis (GWAS), multitrait GWAS, and functional networks built from a diverse set of plant phenotypes. Plant genotype was influential in structuring arthropod community composition among several garden sites. Candidate genes important for higher level organization of arthropod communities had broadly applicable functions, such as terpenoid biosynthesis and production of dsRNA binding proteins and protein kinases, which may be capable of targeting multiple arthropod species. We have demonstrated the ability to detect, in an uncontrolled environment, individual genes that are associated with the community assemblage of arthropods on a host plant, further enhancing our understanding of genetic mechanisms that impact ecosystem structure.

Comparative genomic analysis of chemosensory-related gene families in gastropods.

Chemoreception is critical for the survival and reproduction of animals. Except for a reduced group of insects and chelicerates, the molecular identity of chemosensory proteins is poorly understood in invertebrates. Gastropoda is the extant mollusk class with the greatest species richness, including marine, freshwater, and terrestrial lineages, and likely, highly diverse chemoreception systems. Here, we performed a comprehensive comparative genome analysis taking advantage of the chromosome-level information of two Gastropoda species, one of which belongs to a lineage that underwent a whole genome duplication event. We identified thousands of previously uncharacterized chemosensory-related genes, the majority of them encoding G protein-coupled receptors (GPCR), mostly organized into clusters distributed across all chromosomes. We also detected gene families encoding degenerin epithelial sodium channels (DEG-ENaC), ionotropic receptors (IR), sensory neuron membrane proteins (SNMP), Niemann-Pick type C2 (NPC2) proteins, and lipocalins, although with a lower number of members. Our phylogenetic analysis of the GPCR gene family across protostomes revealed: (i) remarkable gene family expansions in Gastropoda; (ii) clades including members from all protostomes; and (iii) species-specific clades with a substantial number of receptors. For the first time, we provide new and valuable knowledge into the evolution of the chemosensory gene families in invertebrates other than arthropods.

The first record of the giant pill-millipede genus Prionobelum Verhoeff, 1924 from Thailand, with the integrative description of two species (Diplopoda, Sphaerotheriida, Zephroniidae).

Thailand hosts a very rich but underexplored giant pill-millipede (Sphaerotheriida) fauna, with 11 of its 13 species described in the last three years. Currently, all known Thai giant pill-millipedes belong to the genera Zephronia Gray, 1832 (nine species) and Sphaerobelum Verhoeff, 1924 (four species). Here we describe the first two species of the genus Prionobelum Verhoeff, 1924 (previously restricted to Vietnam and China), Prionobelum inthanonense n. sp. and P. naevium n. sp. from Thailand. The species occur at Thailands highest mountain (2500 m) Doi Inthanon and the lowland rainforests at Bang Lang National Park touching the border with Malaysia. Both species are described integratively, utilizing light microscopy, scanning electron microscopy as well as DNA barcoding. Both new species of Prionobelum differ from other Zephroniidae species, as well as from one another, by more than 20% p-distance in the COI barcoding gene suggesting that potential closer related species are still awaiting discovery.

A network analysis of early arthropod evolution and the potential of the primitive.

It is often thought that the primitive is simpler, and that the complex is generated from the simple by some process of self-assembly or self-organization, which ultimately consists of the spontaneous and fortuitous collision of elementary units. This idea is included in the Darwinian theory of evolution, to which is added the competitive mechanism of natural selection. To test this view, we studied the early evolution of arthropods. Twelve groups of arthropods belonging to the Burgess Shale, Orsten Lagerstätte, and extant primitive groups were selected, their external morphology abstracted and codified in the language of network theory. The analysis of these networks through different network measures (network parameters, topological descriptors, complexity measures) was used to carry out a Principal Component Analysis (PCA) and a Hierarchical Cluster Analysis (HCA), which allowed us to obtain an evolutionary tree with distinctive/novel features. The analysis of centrality measures revealed that these measures decreased throughout the evolutionary process, and led to the creation of the concept of evolutionary developmental potential. This potential, which measures the capacity of a morphological unit to generate changes in its surroundings, is concomitantly reduced throughout the evolutionary process, and demonstrates that the primitive is not simple but has a potential that unfolds during this process. This means for us the first empirical evolutionary evidence of our theory of evolution as a process of unfolding.

SYNCAS: Efficient CRISPR/Cas9 gene-editing in difficult to transform arthropods.

The genome editing technique CRISPR/Cas9 has led to major advancements in many research fields and this state-of-the-art tool has proven its use in genetic studies for various arthropods. However, most transformation protocols rely on microinjection of CRISPR/Cas9 components into embryos, a method which is challenging for many species. Alternatively, injections can be performed on adult females, but transformation efficiencies can be very low as was shown for the two-spotted spider mite, Tetranychus urticae, a minute but important chelicerate pest on many crops. In this study, we explored different CRISPR/Cas9 formulations to optimize a maternal injection protocol for T. urticae. We observed a strong synergy between branched amphipathic peptide capsules and saponins, resulting in a significant increase of CRISPR/Cas9 knock-out efficiency, exceeding 20%. This CRISPR/Cas9 formulation, termed SYNCAS, was used to knock-out different T. urticae genes - phytoene desaturase, CYP384A1 and Antennapedia - but also allowed to develop a co-CRISPR strategy and facilitated the generation of T. urticae knock-in mutants. In addition, SYNCAS was successfully applied to knock-out white and white-like genes in the western flower thrips, Frankliniella occidentalis. The SYNCAS method allows routine genome editing in these species and can be a game changer for genetic research in other hard to transform arthropods.

Assessing arthropod biodiversity with DNA barcoding in Jinnah Garden, Lahore, Pakistan.

Previous difficulties in arthropod taxonomy (such as limitations in conventional morphological approaches, the possibility of cryptic species and a shortage of knowledgeable taxonomists) has been overcome by the powerful tool of DNA barcoding. This study presents a thorough analysis of DNA barcoding in regards to Pakistani arthropods, which were collected from Lahore's Jinnah Garden. The 88 % (9,451) of the 10,792 specimens that were examined were able to generate DNA barcodes and 83% (8,974) of specimens were assigned 1,361 barcode index numbers (BINs). However, the success rate differed significantly between the orders of arthropods, from 77% for Thysanoptera to an astounding 93% for Diptera. Through morphological exams, DNA barcoding, and cross-referencing with the Barcode of Life Data system (BOLD), the Barcode Index Numbers (BINs) were assigned with a high degree of accuracy, both at the order (100%) and family (98%) levels. Though, identifications at the genus (37%) and species (15%) levels showed room for improvement. This underscores the ongoing need for enhancing and expanding the DNA barcode reference library. This study identified 324 genera and 191 species, underscoring the advantages of DNA barcoding over traditional morphological identification methods. Among the 17 arthropod orders identified, Coleoptera, Diptera, Hemiptera, Hymenoptera, and Lepidoptera from the class Insecta dominated, collectively constituting 94% of BINs. Expected malaise trap Arthropod fauna in Jinnah Garden could contain approximately 2,785 BINs according to Preston log-normal species distribution, yet the Chao-1 Index predicts 2,389.74 BINs. The Simpson Index of Diversity (1-D) is 0.989, signaling high species diversity, while the Shannon Index is 5.77, indicating significant species richness and evenness. These results demonstrated that in Pakistani arthropods, DNA barcoding and BOLD are an invaluable tool for improving taxonomic understanding and biodiversity assessment, opening the door for further eDNA and metabarcoding research.

Characterization of the complete mitochondrial genome of Spirobolus grahami (Diplopoda: Spirobolidae) with phylogenetic analysis.

Diplopoda is one of the most diverse and important groups of soil arthropods, but little research has been done on their phylogenetic relationship and evolution. Here, we sequenced and annotated the complete mitochondrial genomes of Spirobolus grahami. The total mitogenome of S. grahami was typical circular, double-stranded molecules, with 14,875 bp in length, including 13 protein-coding genes, 22 tRNAs, two rRNAs, and one control region. Base composition analysis suggested that the mitochondrial sequences were biased toward A and T, with A + T content of 58.68%. The mitogenomes of S. grahami exhibited negative AT and positive GC skews. Most of the 13 PCGs had ATN as the start codon, except COX1 start with CGA, and most PCGs ended with the T stop codon. The dN/dS values for most PCGs were lower than 1, suggesting that purifying selection was likely the main driver of mitochondrial PCG evolution. Phylogenetic analyses based on 13 PCGs using BI and ML methods support the classification of genus Spirobolus and Tropostreptus. Glomeridesmus spelaeus is distantly related to the other Diplopoda species.

The First Complete Mitochondrial Genome of the Genus Litostrophus: Insights into the Rearrangement and Evolution of Mitochondrial Genomes in Diplopoda.

This study presents the complete mitochondrial genome (mitogenome) of Litostrophus scaber, which is the first mitogenome of the genus Litostrophus. The mitogenome is a circular molecule with a length of 15,081 bp. The proportion of adenine and thymine (A + T) was 69.25%. The gene ND4L used TGA as the initiation codon, while the other PCGs utilized ATN (A, T, G, C) as the initiation codons. More than half of the PCGs used T as an incomplete termination codon. The transcription direction of the L. scaber mitogenome matched Spirobolus bungii, in contrast to most millipedes. Novel rearrangements were found in the L. scaber mitogenome: trnQ -trnC and trnL1- trnP underwent short-distance translocations and the gene block rrnS-rrnL-ND1 moved to a position between ND4 and ND5, resulting in the formation of a novel gene order. The phylogenetic analysis showed that L. scaber is most closely related to S. bungii, followed by Narceus magnum. These findings enhance our understanding of the rearrangement and evolution of Diplopoda mitogenomes.

A long-read draft assembly of the Chinese mantis (Mantodea: Mantidae: Tenodera sinensis) genome reveals patterns of ion channel gain and loss across Arthropoda.

Praying mantids (Mantodea: Mantidae) are iconic insects that have captivated biologists for decades, especially the species with cannibalistic copulatory behavior. This behavior has been cited as evidence that insects lack nociceptive capacities and cannot feel pain; however, this behaviorally driven hypothesis has never been rigorously tested at the genetic or functional level. To enable future studies of nociceptive capabilities in mantids, we sequenced and assembled a draft genome of the Chinese praying mantis (Tenodera sinensis) and identified multiple classes of nociceptive ion channels by comparison to orthologous gene families in Arthropoda. Our assembly-produced using PacBio HiFi reads-is fragmented (total size = 3.03 Gb; N50 = 1.8 Mb; 4,966 contigs), but is highly complete with respect to gene content (BUSCO complete = 98.7% [odb10_insecta]). The size of our assembly is substantially larger than that of most other insects, but is consistent with the size of other mantid genomes. We found that most families of nociceptive ion channels are present in the T. sinensis genome; that they are most closely related to those found in the damp-wood termite (Zootermopsis nevadensis); and that some families have expanded in T. sinensis while others have contracted relative to nearby lineages. Our findings suggest that mantids are likely to possess nociceptive capabilities and provide a foundation for future experimentation regarding ion channel functions and their consequences for insect behavior.

Insights into sequence characteristics and evolutionary history of DGATs in arthropods.

Triacylglycerol (TAG) is crucial in animal energy storage and membrane biogenesis. The conversion of diacylglycerol (DAG) to triacylglycerol (TAG) is catalyzed by diacylglycerol acyltransferase enzymes (DGATs), which are encoded by genes belonging to two distinct gene families. Although arthropods are known to possess DGATs activities and utilize the glycerol-3-phosphate pathway and MAG pathway for TAG biosynthesis, the sequence characterization and evolutionary history of DGATs in arthropods remains unclear. This study aimed to comparatively evaluate genomic analyses of DGATs in 13 arthropod species and 14 outgroup species. We found that arthropods lack SOAT2 genes within the DGAT1 family, while DGAT2, MOGAT3, AWAT1, and AWAT2 were absent from in DGAT2 family. Gene structure and phylogenetic analyses revealed that DGAT1 and DGAT2 genes come from different gene families. The expression patterns of these genes were further analyzed in crustaceans, demonstrating the importance of DGAT1 in TAG biosynthesis. Additionally, we identified the DGAT1 gene in Swimming crab (P. trituberculatus) undergoes a mutually exclusive alternative splicing event in the molt stages. Our newly determined DGAT inventory data provide a more complete scenario and insights into the evolutionary dynamics and functional diversification of DGATs in arthropods.

Lower Ordovician synziphosurine reveals early euchelicerate diversity and evolution.

Euchelicerata is a clade of arthropods comprising horseshoe crabs, scorpions, spiders, mites and ticks, as well as the extinct eurypterids (sea scorpions) and chasmataspidids. The understanding of the ground plans and relationships between these crown-group euchelicerates has benefited from the discovery of numerous fossils. However, little is known regarding the origin and early evolution of the euchelicerate body plan because the relationships between their Cambrian sister taxa and synziphosurines, a group of Silurian to Carboniferous stem euchelicerates with chelicerae and an unfused opisthosoma, remain poorly understood owing to the scarce fossil record of appendages. Here we describe a synziphosurine from the Lower Ordovician (ca. 478 Ma) Fezouata Shale of Morocco. This species possesses five biramous appendages with stenopodous exopods bearing setae in the prosoma and a fully expressed first tergite in the opisthosoma illuminating the ancestral anatomy of the group. Phylogenetic analyses recover this fossil as a member of the stem euchelicerate family Offacolidae, which is characterized by biramous prosomal appendages. Moreover, it also shares anatomical features with the Cambrian euarthropod Habelia optata, filling the anatomical gap between euchelicerates and Cambrian stem taxa, while also contributing to our understanding of the evolution of euchelicerate uniramous prosomal appendages and tagmosis.

Functional similarity, despite taxonomical divergence in the millipede gut microbiota, points to a common trophic strategy.

BACKGROUND: Many arthropods rely on their gut microbiome to digest plant material, which is often low in nitrogen but high in complex polysaccharides. Detritivores, such as millipedes, live on a particularly poor diet, but the identity and nutritional contribution of their microbiome are largely unknown. In this study, the hindgut microbiota of the tropical millipede Epibolus pulchripes (large, methane emitting) and the temperate millipede Glomeris connexa (small, non-methane emitting), fed on an identical diet, were studied using comparative metagenomics and metatranscriptomics. RESULTS: The results showed that the microbial load in E. pulchripes is much higher and more diverse than in G. connexa. The microbial communities of the two species differed significantly, with Bacteroidota dominating the hindguts of E. pulchripes and Proteobacteria (Pseudomonadota) in G. connexa. Despite equal sequencing effort, de novo assembly and binning recovered 282 metagenome-assembled genomes (MAGs) from E. pulchripes and 33 from G. connexa, including 90 novel bacterial taxa (81 in E. pulchripes and 9 in G. connexa). However, despite this taxonomic divergence, most of the functions, including carbohydrate hydrolysis, sulfate reduction, and nitrogen cycling, were common to the two species. Members of the Bacteroidota (Bacteroidetes) were the primary agents of complex carbon degradation in E. pulchripes, while members of Proteobacteria dominated in G. connexa. Members of Desulfobacterota were the potential sulfate-reducing bacteria in E. pulchripes. The capacity for dissimilatory nitrate reduction was found in Actinobacteriota (E. pulchripes) and Proteobacteria (both species), but only Proteobacteria possessed the capacity for denitrification (both species). In contrast, some functions were only found in E. pulchripes. These include reductive acetogenesis, found in members of Desulfobacterota and Firmicutes (Bacillota) in E. pulchripes. Also, diazotrophs were only found in E. pulchripes, with a few members of the Firmicutes and Proteobacteria expressing the nifH gene. Interestingly, fungal-cell-wall-degrading glycoside hydrolases (GHs) were among the most abundant carbohydrate-active enzymes (CAZymes) expressed in both millipede species, suggesting that fungal biomass plays an important role in the millipede diet. CONCLUSIONS: Overall, these results provide detailed insights into the genomic capabilities of the microbial community in the hindgut of millipedes and shed light on the ecophysiology of these essential detritivores. Video Abstract.

The role of ATP-binding cassette transporters in arthropod pesticide toxicity and resistance.

Pesticide resistance in arthropods threatens agricultural productivity and the control of vector-borne diseases. The ATP-binding cassette (ABC) transporters have emerged as important factors in the toxicity of synthetic pesticides, as well as for Bacillus thuringiensis insecticidal Cry protein binding. Depending on the localization of expression, both higher and lower expression of ABCs have been linked with pesticide resistance. The recent development of genetic-based approaches such as RNAi and CRISPR/Cas9 gene editing in nonmodel species, has greatly contributed to unveil their functional importance in pesticide toxicity and resistance. Using these tools, we are now poised to further unravel the molecular genetic mechanisms of gene regulation uncovering more elusive regulatory resistance genes.

Composition of constitutive heterochromatin of Pseudonannolene strinatii Mauriès, 1974 (Diplopoda, Spirostreptida) analyzed by AT/CG specific fluorochromes

Triple staining with fluorochromes (DA/DAPI/CMA) and C-banding were used to characterize the composition of Pseudonannolene strinatii heterochromatin. C-banding showed C+ bands of different labeling intensity on chromosomes 1 and 2 in some cells. Fluorochrome staining revealed DAPI+ regions corresponding to the C-banding pattern, indicating that the heterochromatin of this species is abundant in AT-rich sequences.

Applicability of RAPD markers on silver-stained polyacrylamide gels to ascertain genetic diversity in Peripatus acacioi (Peripatidae; Onychophora)

RAPD (random amplification of polymorphic DNA) molecular markers can be utilized for analyzing genetic variability in populations for which only a few or no molecular markers are available. They were used in a study of an endangered species, Peripatus acacioi, found in the Tripuí Ecological Station, in Ouro Preto, MG, Brazil. The ecological station was specifically created to protect this velvet worm species, the first of this group found in Brazil. For an initial evaluation of the genetic diversity of this species, DNA samples from the lobopods of four individuals, collected at random, were analyzed using RAPD. Each reaction was run with a different primer (Operon RAPD 10-mer Kits), totaling 13 primers (OPC2, OPC3, OPC4, OPC6, OPC8, OPC10, OPC11, OPL2, OPL7, OPL11, OPL13, OPL18, and OPL19). Due to the low amplification yield, RAPD fragments were separated in polyacrylamide gels and stained with silver nitrate. Numerous bands were observed. Fifty-five of the amplified bands proved to be reproducible, both in terms of presence and intensity. Among these, 27 were variable and 28 were constant. The average number of bands per gel was 4.2. Nine of the 13 primers tested allowed the identification of constant and variable bands among these four individuals. RAPD analysis of genetic variation using silver-stained polyacrylamide gel electrophoresis provided measures of band sharing among the individuals, and therefore could be used in population genetics studies of P. acacioi.