Multiple hybridization events and repeated evolution of homoeologue expression bias in parthenogenetic, polyploid New Zealand stick insects.

During hybrid speciation, homoeologues combine in a single genome. Homoeologue expression bias (HEB) occurs when one homoeologue has higher gene expression than another. HEB has been well characterized in plants but rarely investigated in animals, especially invertebrates. Consequently, we have little idea as to the role that HEB plays in allopolyploid invertebrate genomes. If HEB is constrained by features of the parental genomes, then we predict repeated evolution of similar HEB patterns among hybrid genomes formed from the same parental lineages. To address this, we reconstructed the history of hybridization between the New Zealand stick insect genera Acanthoxyla and Clitarchus using a high-quality genome assembly from Clitarchus hookeri to call variants and phase alleles. These analyses revealed the formation of three independent diploid and triploid hybrid lineages between these genera. RNA sequencing revealed a similar magnitude and direction of HEB among these hybrid lineages, and we observed that many enriched functions and pathways were also shared among lineages, consistent with repeated evolution due to parental genome constraints. In most hybrid lineages, a slight majority of the genes involved in mitochondrial function showed HEB towards the maternal homoeologues, consistent with only weak effects of mitonuclear incompatibility. We also observed a proteasome functional enrichment in most lineages and hypothesize this may result from the need to maintain proteostasis in hybrid genomes. Reference bias was a pervasive problem, and we caution against relying on HEB estimates from a single parental reference genome.

Gripping performance in the stick insect Sungaya inexpectata in dependence on the pretarsal architecture.

Insect attachment devices and capabilities have been subject to research efforts for decades, and even though during that time considerable progress has been made, numerous questions remain. Different types of attachment devices are known, alongside most of their working principles, however, some details have yet to be understood. For instance, it is not clear why insects for the most part developed pairs of claws, instead of either three or a single one. In this paper, we investigated the gripping forces generated by the stick insect Sungaya inexpectata, in dependence on the number of available claws. The gripping force experiments were carried out on multiple, standardized substrates of known roughness, and conducted in directions both perpendicular and parallel to the substrate. This was repeated two times: first with a single claw being amputated from each of the animals' legs, then with both claws removed, prior to the measurement. The adhesive pads (arolia) and frictional pads (euplantulae) remained intact. It was discovered that the removal of claws had a detractive effect on the gripping forces in both directions, and on all substrates. Notably, this also included the control of smooth surfaces on which the claws were unable to find any asperities to grip on. The results show that there is a direct connection between the adhesive performance of the distal adhesive pad (arolium) and the presence of intact claws. These observations show collective effects between different attachment devices that work in concert during locomotion, and grant insight into why most insects possess two claws.

A Hybrid Parallel Balanced Phasmatodea Population Evolution Algorithm and Its Application in Workshop Material Scheduling.

The phasmatodea population evolution algorithm (PPE) is a recently proposed meta-heuristic algorithm based on the evolutionary characteristics of the stick insect population. The algorithm simulates the features of convergent evolution, population competition, and population growth in the evolution process of the stick insect population in nature and realizes the above process through the population competition and growth model. Since the algorithm has a slow convergence speed and falls easily into local optimality, in this paper, it is mixed with the equilibrium optimization algorithm to make it easier to avoid the local optimum. Based on the hybrid algorithm, the population is grouped and processed in parallel to accelerate the algorithm's convergence speed and achieve better convergence accuracy. On this basis, we propose the hybrid parallel balanced phasmatodea population evolution algorithm (HP_PPE), and this algorithm is compared and tested on the CEC2017, a novel benchmark function suite. The results show that the performance of HP_PPE is better than that of similar algorithms. Finally, this paper applies HP_PPE to solve the AGV workshop material scheduling problem. Experimental results show that HP_PPE can achieve better scheduling results than other algorithms.

Phylomitogenomics provides new perspectives on the Euphasmatodea radiation (Insecta: Phasmatodea).

Phasmatodea species diversity lies almost entirely within its suborder Euphasmatodea, which exhibits a pantropical distribution and is considered to derive from a recent and rapid evolutionary radiation. To shed light on Euphasmatodea origins and diversification, we assembled the mitogenomes of 17 species from transcriptomic sequencing data and analysed them along with 22 already available Phasmatodea mitogenomes and 33 mitogenomes representing most of the Polyneoptera lineages. Maximum Likelihood and Bayesian Inference approaches retrieved consistent topologies, both showing the widespread conflict between phylogenetic approaches and traditional systematics. We performed a divergence time analysis leveraging ten fossil specimens representative of most polyneopteran lineages: the time tree obtained supports an older radiation of the clade with respect to previous hypotheses. Euphasmatodea diversification is inferred to have started ~ 187 million years ago, suggesting that the Triassic-Jurassic mass extinction and the breakup of Pangea could have contributed to the process. We also investigated Euphasmatodea mitogenomes patterns of dN, dS and dN/dS ratio throughout our time-tree, trying to characterize the selective regime which may have shaped the clade evolution.

Assembling large genomes: analysis of the stick insect (Clitarchus hookeri) genome reveals a high repeat content and sex-biased genes associated with reproduction.

BACKGROUND: Stick insects (Phasmatodea) have a high incidence of parthenogenesis and other alternative reproductive strategies, yet the genetic basis of reproduction is poorly understood. Phasmatodea includes nearly 3000 species, yet only the genome of Timema cristinae has been published to date. Clitarchus hookeri is a geographical parthenogenetic stick insect distributed across New Zealand. Sexual reproduction dominates in northern habitats but is replaced by parthenogenesis in the south. Here, we present a de novo genome assembly of a female C. hookeri and use it to detect candidate genes associated with gamete production and development in females and males. We also explore the factors underlying large genome size in stick insects. RESULTS: The C. hookeri genome assembly was 4.2 Gb, similar to the flow cytometry estimate, making it the second largest insect genome sequenced and assembled to date. Like the large genome of Locusta migratoria, the genome of C. hookeri is also highly repetitive and the predicted gene models are much longer than those from most other sequenced insect genomes, largely due to longer introns. Miniature inverted repeat transposable elements (MITEs), absent in the much smaller T. cristinae genome, is the most abundant repeat type in the C. hookeri genome assembly. Mapping RNA-Seq reads from female and male gonadal transcriptomes onto the genome assembly resulted in the identification of 39,940 gene loci, 15.8% and 37.6% of which showed female-biased and male-biased expression, respectively. The genes that were over-expressed in females were mostly associated with molecular transportation, developmental process, oocyte growth and reproductive process; whereas, the male-biased genes were enriched in rhythmic process, molecular transducer activity and synapse. Several genes involved in the juvenile hormone synthesis pathway were also identified. CONCLUSIONS: The evolution of large insect genomes such as L. migratoria and C. hookeri genomes is most likely due to the accumulation of repetitive regions and intron elongation. MITEs contributed significantly to the growth of C. hookeri genome size yet are surprisingly absent from the T. cristinae genome. Sex-biased genes identified from gonadal tissues, including genes involved in juvenile hormone synthesis, provide interesting candidates for the further study of flexible reproduction in stick insects.

Comparative whole-body kinematics of closely related insect species with different body morphology.

Legged locomotion through natural environments is very complex and variable. For example, leg kinematics may differ strongly between species, but even within the same species it is adaptive and context-dependent. Inter-species differences in locomotion are often difficult to interpret, because both morphological and ecological differences among species may be strong and, as a consequence, confound each other's effects. In order to understand better how body morphology affects legged locomotion, we compare unrestrained whole-body kinematics of three stick insect species with different body proportions, but similar feeding ecology: Carausius morosus, Aretaon asperrimus and Medauroidea extradentata (=Cuniculina impigra). In order to co-vary locomotory context, we introduced a gradually increasing demand for climbing by varying the height of stairs in the setup. The species were similar in many aspects, for example in using distinct classes of steps, with minor differences concerning the spread of corrective short steps. Major differences were related to antenna length, segment lengths of thorax and head, and the ratio of leg length to body length. Whereas all species continuously moved their antennae, only Medauroidea executed high swing movements with its front legs to search for obstacles in the near-range environment. Although all species adjusted their body inclination, the range in which body segments moved differed considerably, with longer thorax segments tending to be moved more. Finally, leg posture, time courses of leg joint angles and intra-leg coordination differed most strongly in long-legged Medauroidea.

The effects of temperature on the gas exchange cycle in Agathemera crassa.

Insects exhibit three patterns of gas exchange: continuous (CoGE), cyclic (CGE) and discontinuous (DGE). In this work, we present the first record of a DGE in Phasmatodea and its transition to CGE and to CoGE through a thermal gradient. The rate of CO2 production (VCO2) at 10, 20 and 30°C was examined in adults of Agathemera crassa, a high-Andean phasmid of central Chile. Carbon dioxide release was recorded during 24 h with L:D cycle of 12:12 h in order to record both rest and activity periods. At rest, A. crassa showed three patterns of gas exchange, highlighting the use of DGE preferably at 10°C. As the temperature increased, the CoGE pattern was more frequent being the only pattern observed in all individuals at 30°C. During activity, patterns changed to CoGE with a significant increase in VCO2. Our results support the idea that gas exchange patterns in insects are not distinct but correspond to a continuum of responses addressed by metabolic demand and where DGE can be expressed only under an absolute state of rest. Our results support the idea that the presence of the DGE may be underestimated in other insect taxa because they may have been measured under conditions where this pattern not necessarily can be expressed.

Complete mitochondrial genome of the introduced Indian walking stick Carausius morosus (Lonchodidae, Insecta) from California.

We present the complete mitochondrial genome of Carausius morosus from Salinas, CA. The mitochondrial genome of C. morosus is circular, AT rich (78.1%), and 16,671 bp in length. It consists of 13 protein-coding, 22 transfer RNA, and 2 ribosomal RNA genes and is identical in gene content to Carausius sp.

Evolution of alternative reproductive systems in Bacillus stick insects.

Reproduction is a key feature of all organisms, yet the way in which it is achieved varies greatly across the tree of life. One striking example of this variation is the stick insect genus Bacillus, in which five different reproductive modes have been described: sex, facultative and obligate parthenogenesis, and two highly unusual reproductive modes: hybridogenesis and androgenesis. Under hybridogenesis, the entire genome from the paternal species is eliminated and replaced each generation by mating with the corresponding species. Under androgenesis, an egg is fertilized, but the developing diploid offspring bear two paternal genomes and no maternal genome, as a consequence of unknown mechanisms. Here, we reevaluate the previous descriptions of Bacillus lineages and the proposed F1 hybrid ancestries of the hybridogenetic and obligately parthenogenetic lineages (based on allozymes and karyotypes) from Sicily, where all these reproductive modes are found. We generate a chromosome-level genome assembly for a facultative parthenogenetic species (B. rossius) and combine extensive field sampling with RADseq and mtDNA data. We identify and genetically corroborate all previously described species and confirm the ancestry of hybrid lineages. All hybrid lineages have fully retained their F1 hybrid constitution throughout the genome, indicating that the elimination of the paternal genome in hybridogens is always complete and that obligate parthenogenesis in Bacillus hybrid species is not associated with an erosion of heterozygosity as known in other hybrid asexuals. Our results provide a stepping stone toward understanding the transitions between reproductive modes and the proximate mechanisms of genome elimination.

Resource allocation strategies and mechanical constraints drive the diversification of stick and leaf insect eggs.

The diversity of insect eggs is astounding but still largely unexplained. Here, we apply phylogenetic analyses to 208 species of stick and leaf insects, coupled with physiological measurements of metabolic rate and water loss on five species, to evaluate classes of factors that may drive egg morphological diversification: life history constraints, material costs, mechanical constraints, and ecological circumstances. We show support for all three classes, but egg size is primarily influenced by female body size and strongly trades off with egg number. Females that lay relatively fewer but larger eggs, which develop more slowly because of disproportionately low metabolic rates, also tend to bury or glue them in specific locations instead of simply dropping them from the foliage (ancestral state). This form of parental care then directly favors relatively elongated eggs, which may facilitate their placement and allow easier passage through the oviducts in slender species. In addition, flightless females display a higher reproductive output and consequently lay relatively more and larger eggs compared with flight-capable females. Surprisingly, local climatic conditions had only weak effects on egg traits. Overall, our results suggest that morphological diversification of stick insect eggs is driven by a complex web of causal relationships among traits, with dominant effects of resource allocation and oviposition strategies, and of mechanical constraints.

Phasmid species that inhabit colder environments are less likely to have the ability to fly.

A vast majority of insects can fly, but some cannot. Flight generally increases how far an individual can travel to access mates, enables the exploitation of additional food resources, and aids in predator avoidance. Despite its functional significance, much remains unknown about the factors that influence the evolution of flight. Here, I use phylogenetic comparative methods to investigate whether average annual temperature or wind speed, two components of the flying environment, is correlated with the evolution of flight using data from 107 species of stick and leaf insects (Insecta: Phasmatodea). I find no association between wind speed and flying ability in this clade. However, I find that colder temperatures are associated with the lack of flying ability. This pattern may be explained by the additional metabolic costs required for insects to fly when it is cold. This finding contradicts previous patterns observed in other insect groups and supports the hypothesis that cold temperatures can influence the evolution of flight.

Nine Mitochondrial Genomes of Phasmatodea with Two Novel Mitochondrial Gene Rearrangements and Phylogeny.

The classification of stick and leaf insects (Order Phasmatodea) is flawed at various taxonomic ranks due to a lack of robust phylogenetic relationships and convergent morphological characteristics. In this study, we sequenced nine new mitogenomes that ranged from 15,011 bp to 17,761 bp in length. In the mitogenome of Carausis sp., we found a translocation of trnR and trnA, which can be explained by the tandem duplication/random loss (TDRL) model. In the Stheneboea repudiosa Brunner von Wattenwyl, 1907, a novel mitochondrial structure of 12S rRNA-CR1-trnI-CR2-trnQ-trnM was found for the first time in Phasmatodea. Due to the low homology of CR1 and CR2, we hypothesized that trnI was inverted through recombination and then translocated into the middle of the control region. Control region repeats were frequently detected in the newly sequenced mitogenomes. To explore phylogenetic relationships in Phasmatodea, mtPCGs from 56 Phasmatodean species (composed of 9 stick insects from this study, 31 GenBank data, and 16 data derived from transcriptome splicing) were used for Bayesian inference (BI), and maximum likelihood (ML) analyses. Both analyses supported the monophyly of Lonchodinae and Necrosciinae, but Lonchodidae was polyphyletic. Phasmatidae was monophyletic, and Clitumninae was paraphyletic. Phyllidae was located at the base of Neophasmatodea and formed a sister group with the remaining Neophasmatodea. Bacillidae and Pseudophasmatidae were recovered as a sister group. Heteroptergidae was monophyletic, and the Heteropteryginae sister to the clade (Obriminae + Dataminae) was supported by BI analysis and ML analysis.

Influence of native and exotic plant diet on the gut microbiome of the Gray's Malayan stick insect, Lonchodes brevipes.

Herbivorous insects require an active lignocellulolytic microbiome to process their diet. Stick insects (phasmids) are common in the tropics and display a cosmopolitan host plant feeding preference. The microbiomes of social insects are vertically transmitted to offspring, while for solitary species, such as phasmids, it has been assumed that microbiomes are acquired from their diet. This study reports the characterization of the gut microbiome for the Gray's Malayan stick insect, Lonchodes brevipes, reared on native and introduced species of host plants and compared to the microbiome of the host plant and surrounding soil to gain insight into possible sources of recruitment. Clear differences in the gut microbiome occurred between insects fed on native and exotic plant diets, and the native diet displayed a more species-rich fungal microbiome. While the findings suggest that phasmids may be capable of adapting their gut microbiome to changing diets, it is uncertain whether this may lead to any change in dietary efficiency or organismal fitness. Further insight in this regard may assist conservation and management decision-making.

Characterization of Morphologically Distinct Components in the Tarsal Secretion of Medauroidea extradentata (Phasmatodea) Using Cryo-Scanning Electron Microscopy.

Attachment to the substrate is an important phenomenon that determines the survival of many organisms. Most insects utilize wet adhesion to support attachment, which is characterized by fluids that are secreted into the interface between the tarsus and the substrates. Previous research has investigated the composition and function of tarsal secretions of different insect groups, showing that the secretions are likely viscous emulsions that contribute to attachment by generating capillary and viscous adhesion, leveling surface roughness and providing self-cleaning of the adhesive systems. Details of the structural organization of these secretions are, however, largely unknown. Here, we analyzed footprints originating from the arolium and euplantulae of the stick insect Medauroidea extradentata using cryo-scanning electron microscopy (cryo-SEM) and white light interferometry (WLI). The secretion was investigated with cryo-SEM, revealing four morphologically distinguishable components. The 3D WLI measurements of the droplet shapes and volumes over time revealed distinctly different evaporation rates for different types of droplets. Our results indicate that the subfunctionalization of the tarsal secretion is facilitated by morphologically distinct components, which are likely a result of different proportions of components within the emulsion. Understanding these components and their functions may aid in gaining insights for developing adaptive and multifunctional biomimetic adhesive systems.

Multi-modal locomotor costs favor smaller males in a sexually dimorphic leaf-mimicking insect.

BACKGROUND: In most arthropods, adult females are larger than males, and male competition is a race to quickly locate and mate with scattered females (scramble competition polygyny). Variation in body size among males may confer advantages that depend on context. Smaller males may be favored due to more efficient locomotion leading to higher mobility during mate searching. Alternatively, larger males may benefit from increased speed and higher survivorship. While the relationship between male body size and mobility has been investigated in several systems, how different aspects of male body morphology specifically affect their locomotor performance in different contexts is often unclear. RESULTS: Using a combination of empirical measures of flight performance and modelling of body aerodynamics, we show that large body size impairs flight performance in male leaf insects (Phyllium philippinicum), a species where relatively small and skinny males fly through the canopy in search of large sedentary females. Smaller males were more agile in the air and ascended more rapidly during flight. Our models further predicted that variation in body shape would affect body lift and drag but suggested that flight costs may not explain the evolution of strong sexual dimorphism in body shape in this species. Finally, empirical measurements of substrate adhesion and subsequent modelling of landing impact forces suggested that smaller males had a lower risk of detaching from the substrates on which they walk and land. CONCLUSIONS: By showing that male body size impairs their flight and substrate adhesion performance, we provide support to the hypothesis that smaller scrambling males benefit from an increased locomotor performance and shed light on the evolution of sexual dimorphism in scramble competition mating systems.

Cladomorphus petropolisensis, a New Species of Stick Insect from the Atlantic Forest, Rio de Janeiro, Brazil.

Cladomorphus petropolisensis sp. nov., a new species of stick insect from Petrópolis, Rio de Janeiro, Brazil, is herein described and compared to the other sympatric species, C. phyllinus Gray, 1835 (Phasmatidae, Cladomorphinae). The description of the new species is supported by morphological and molecular evidence. Kimura-2-parameter (K2P) intraspecific COI divergences among the holotype of C. petropolisensis sp. nov. and C. phyllinus individuals ranged from 2.9% to 4.4%, which are suggestive of distinct species, especially when considering that all Cladomorphus individuals studied were collected in the Petrópolis municipality. The new species can be distinguished from C. phyllinus Gray, 1835 by several characteristics: smaller size, the presence of two spines on the hind femora, the relative longer length of the ovipositor, and spiny tegument, especially in the mesonotum, sculpturing of the operculum of the egg.

Cytochrome P450 Genes Expressed in Phasmatodea Midguts.

Cytochrome P450s (CYPs) are xenobiotic detoxification genes found in most eukaryotes, and linked in insects to the tolerance of plant secondary chemicals and insecticide resistance. The number and diversity of CYP clans, families, and subfamilies that an organism produces could correlate with its dietary breadth or specialization. This study examined the CYP diversity expressed in the midguts of six species of folivorous stick insects (Phasmatodea), to identify their CYP complement and see if any CYPs correlate with diet toxicity or specialization, and see what factors influenced their evolution in this insect order. CYP genes were mined from six published Phasmatodea transcriptomes and analyzed phylogenetically. The Phasmatodea CYP complement resembles that of other insects, though with relatively low numbers, and with significant expansions in the CYP clades 6J1, 6A13/14, 4C1, and 15A1. The CYP6 group is known to be the dominant CYP family in insects, but most insects have no more than one CYP15 gene, so the function of the multiple CYP15A1 genes in Phasmatodea is unknown, with neofunctionalization following gene duplication hypothesized. No correlation was found between CYPs and diet specialization or toxicity, with some CYP clades expanding within the Phasmatodea and others likely inherited from a common ancestor.

Organization of the mitochondrial genome of Ramulus irregulatiter dentatus (Phasmatidae: Phasmatidae).

Recently, the species of the insect order Phasmatodea, have attracted the interest of more and more enthusiasts. Here, we obtained the complete mitochondrial genome of Ramulus irregulatiter dentatus (R. irregulatiter dentatus), which belongs to the subfamily of Phasmatidae, detected by Illumina next-generation sequencing. The entire mitochondrial genome is 16,060 bp in length and contains a standard set of 13 protein-coding genes, 22 transfer RNA genes (tRNAs), 2 ribosomal RNA genes (rRNAs), and a putative A + T-rich region. The base composition and codon usage were typical of Phasmatodea species. The mitochondrial gene organization (37 genes) was consistent with that of other Phasmatidae. A phylogenetic tree was built from the sequence information of the 13 protein-coding genes by Bayesian analyses. The newly sequenced R. irregulatiter dentatus was most closely related to the family Phasmatidae. The complete mitochondrial genome of R. irregulatiter dentatus also provides valuable molecular information for future studies on Phasmatidae insect taxonomy and a framework to unveil more of their cryptic and unknown diversity, so that it can be used to control forest pests and protect crops.

The tracheal system in the stick insect prothorax and prothoracic legs: Homologies to Orthoptera and relations to mechanosensory functions.

Arthropod respiration depends on the tracheal system running from spiracles at the body surface through the body and appendages. Here, three species of stick insects (Carausius morosus, Ramulus artemis, Sipyloidea sipylus) are investigated for the tracheae in the prothorax and foreleg. The origin of the tracheae from the mesothoracic spiracle that enter the foreleg is identified: five tracheae originate from the mesothoracic spiracle, of which two enter the foreleg (supraventral trachea, trachea pedalis anterior). These two tracheae run separately through the leg to the femur-tibia joint where they fuse, but in the proximal tibia split again into two tracheae. The leg tracheae in stick insects are homologous to those in Tettigoniidae (bushcrickets). Stick insects have two chordotonal organs in the proximal tibia (subgenual organ and distal organ) which locate dorsally of the leg trachea. The tracheal system shows no adaptation specific to the propagation of airborne sound, like enlarged spiracles or tracheal volumes. Tracheal vesicles form in the tibia proximally to the mechanosensory organs, but no tracheal sacks or expansions occur at the level of the sensory organs that could mediate the detection of airborne sound or amplify substrate vibrations transmitted in the hemolymph fluid. Rather, the morphological characteristics indicate a respiratory function.

Divergent Gene Expression Following Duplication of Meiotic Genes in the Stick Insect Clitarchus hookeri.

Some animal groups, such as stick insects (Phasmatodea), have repeatedly evolved alternative reproductive strategies, including parthenogenesis. Genomic studies have found modification of the genes underlying meiosis exists in some of these animals. Here we examine the evolution of copy number, evolutionary rate, and gene expression in candidate meiotic genes of the New Zealand geographic parthenogenetic stick insect Clitarchus hookeri. We characterized 101 genes from a de novo transcriptome assembly from female and male gonads that have homology with meiotic genes from other arthropods. For each gene we determined copy number, the pattern of gene duplication relative to other arthropod orthologs, and the potential for meiosis-specific expression. There are five genes duplicated in C. hookeri, including one also duplicated in the stick insect Timema cristinae, that are not or are uncommonly duplicated in other arthropods. These included two sister chromatid cohesion associated genes (SA2 and SCC2), a recombination gene (HOP1), an RNA-silencing gene (AGO2) and a cell-cycle regulation gene (WEE1). Interestingly, WEE1 and SA2 are also duplicated in the cyclical parthenogenetic aphid Acyrthosiphon pisum and Daphnia duplex, respectively, indicating possible roles in the evolution of reproductive mode. Three of these genes (SA2, SCC2, and WEE1) have one copy displaying gonad-specific expression. All genes, with the exception of WEE1, have significantly different nonsynonymous/synonymous ratios between the gene duplicates, indicative of a shift in evolutionary constraints following duplication. These results suggest that stick insects may have evolved genes with novel functions in gamete production by gene duplication.