Diverse Insect Orders Research Articles

The contribution of mitochondrial metagenomics to large-scale data mining and phylogenetic analysis of Coleoptera

A phylogenetic tree at the species level is still far off for highly diverse insect orders, including the Coleoptera, but the taxonomic breadth of public sequence databases is growing. In addition, new types of data may contribute to increasing taxon coverage, such as metagenomic shotgun sequencing for assembly of mitogenomes from bulk specimen samples. The current study explores the application of these techniques for large-scale efforts to build the tree of Coleoptera. We used shotgun data from 17 different ecological and taxonomic datasets (5 unpublished) to assemble a total of 1942 mitogenome contigs of >3000 bp. These sequences were combined into a single dataset together with all mitochondrial data available at GenBank, in addition to nuclear markers widely used in molecular phylogenetics. The resulting matrix of nearly 16,000 species with two or more loci produced trees (RAxML) showing overall congruence with the Linnaean taxonomy at hierarchical levels from suborders to genera. We tested the role of full-length mitogenomes in stabilizing the tree from GenBank data, as mitogenomes might link terminals with non-overlapping gene representation. However, the mitogenome data were only partly useful in this respect, presumably because of the purely automated approach to assembly and gene delimitation, but improvements in future may be possible by using multiple assemblers and manual curation. In conclusion, the combination of data mining and metagenomic sequencing of bulk samples provided the largest phylogenetic tree of Coleoptera to date, which represents a summary of existing phylogenetic knowledge and a defensible tree of great utility, in particular for studies at the intra-familial level, despite some shortcomings for resolving basal nodes.

Hyperactive piggyBac transposase improves transformation efficiency in diverse insect species

Even in times of advanced site-specific genome editing tools, the improvement of DNA transposases is still on high demand in the field of transgenesis: especially in emerging model systems where evaluated integrase landing sites have not yet been created and more importantly in non-model organisms such as agricultural pests and disease vectors, in which reliable sequence information and genome annotations are still pending. In fact, random insertional mutagenesis is essential to identify new genomic locations that are not influenced by position effects and thus can serve as future stable transgene integration sites. In this respect, a hyperactive version of the most widely used piggyBac transposase (PBase) has been engineered. The hyperactive version (hyPBase) is currently available with the original insect codon-based coding sequence (ihyPBase) as well as in a mammalian codon-optimized (mhyPBase) version. Both facilitate significantly higher rates of transposition when expressed in mammalian in vitro and in vivo systems compared to the classical PBase at similar protein levels. Here we demonstrate that the usage of helper plasmids encoding the hyPBase - irrespective of the codon-usage - also strikingly increases the rate of successful germline transformation in the Mediterranean fruit fly (Medfly) Ceratitis capitata, the red flour beetle Tribolium castaneum, and the vinegar fly Drosophila melanogaster. hyPBase-encoding helpers are therefore highly suitable for the generation of transgenic strains of diverse insect orders. Depending on the species, we achieved up to 15-fold higher germline transformation rates compared to PBase and generated hard to obtain transgenic T. castaneum strains that express constructs affecting fitness and viability. Moreover, previously reported high sterility rates supposedly caused by hyPBase (iPB7), encoded by ihyPBase, could not be confirmed by our study. Therefore, we value hyPBase as an effective genetic engineering tool that we highly recommend for insect transgenesis.

Diel behavior in moths and butterflies: a synthesis of data illuminates the evolution of temporal activity

Lepidoptera (butterflies and moths) are one of the most taxonomically diverse insect orders with nearly 160,000 described species. They have been studied extensively for centuries and are found on nearly all continents and in many environments. It is often assumed that adult butterflies are strictly diurnal and adult moths are strictly nocturnal, but there are many exceptions. Despite the broad interest in butterflies and moths, a comprehensive review of diel (day-night) activity has not been conducted. Here, we synthesize existing data on diel activity in Lepidoptera, trace its evolutionary history on a phylogeny, and show where gaps lie in our knowledge. Diurnality was likely the ancestral condition in Lepidoptera, the ancestral heteroneuran was likely nocturnal, and more than 40 transitions to diurnality subsequently occurred. Using species diversity estimates across the order, we predict that roughly 75-85% of Lepidoptera are nocturnal. We also define the three frequently used terms for activity in animals (diurnal, nocturnal, crepuscular), and show that literature on the activity of micro-moths is significantly lacking. Ecological factors leading to nocturnality/diurnality is a compelling area of research and should be the focus of future studies.

Abundance and diversity of flower visitors on wild and cultivated cacao (Theobroma cacao L.) in Bolivia

Despite the economic importance of Theobroma cacao, surprisingly little is known about its pollination ecology. Ceratopogonid midges are considered to be the main pollinators, but the limited available evidence on the sexual reproduction is based almost exclusively on cultivated cacao and knowledge is nonexistent for wild populations. We documented flower visitors in wild and cultivated plants by applying glue on 2237 flowers of wild and cultivated cacao trees in Bolivia to trap floral visitors. We caught 631 insects belonging to seven orders, corresponding to a mean capture rate of 0.3 insects per flower. The most abundant and diverse insect order on both cacao types was Hymenoptera, represented mainly by small parasitoids. Hymenoptera were more abundant on wild cacao, whereas species richness was higher on cultivated cacao. The abundance and species richness of Diptera were not significantly different between wild and cultivated cacao. However, species composition and proportion of Diptera species differed between both wild and cultivated cacao. Ceratopogonidae were only represented by 13 individuals belonging to seven species. Cacao pollen was carried by only a single specimen of Encyrtidae. We were thus unable to identify actual pollinators. We found significant differences among the visitor assemblages between wild and cultivated cacao, which suggest that midges alone were probably too rare to act as main or even sole pollinators of cacao in our study region. Potential additional pollinators would be small Diptera (e.g., Chloropidae and Phoridae) and Hymenoptera (e.g., Eulophidae and Platygastridae).

The unity, diversity and conformity of bugs (Hemiptera) through time

ABSTRACTThis paper outlines and discusses the fossil record of the Hemiptera – the fifth most diverse insect order. The diversity of these insects in comparison with the “Big Four” group is given, together with a short history of its classification. Updated information is presented about the fossil record of particular families, with a brief analysis. The main evolutionary traits of the major Hemiptera lineages are briefly described. The influence of biotic interactions with endosymbionts, shaping the evolution of the hemipterans as well as abiotic events and major global changes, is disputed. The innovations and perils of the evolutionary history of the Hemiptera are presented.

Evidence for common horizontal transmission of Wolbachia among butterflies and moths.

BackgroundWolbachia is one of the most widespread bacteria on Earth. Previous research on Wolbachia-host interactions indicates that the bacterium is typically transferred vertically, from mother to offspring, through the egg cytoplasm. Although horizontal transmission of Wolbachia from one species to another is reported to be common in arthropods, limited direct ecological evidence is available. In this study, we examine horizontal transmission of Wolbachia using a multilocus sequence typing (MLST) strains dataset and used Wolbachia and Lepidoptera genomes to search for evidence for lateral gene transfer (LGT) in Lepidoptera, one of the most diverse cosmopolitan insect orders. We constructed a phylogeny of arthropod-associated MLST Wolbachia strains and calibrated the age of Wolbachia strains associated with lepidopteran species.ResultsOur results reveal inter-specific, inter-generic, inter-familial, and inter-ordinal horizontal transmission of Wolbachia strains, without discernible geographic patterns. We found at least seven probable cases of horizontal transmission among 31 species within Lepidoptera and between Lepidoptera and other arthropod hosts. The divergence time analysis revealed that Wolbachia is recently (22.6–4.7 mya, 95 % HPD) introduced in Lepidoptera. Analysis of nine Lepidoptera genomes (Bombyx mori, Danaus plexippus, Heliconius melpomene, Manduca sexta, Melitaea cinxia, Papilio glaucus, P. polytes, P. xuthus and Plutella xylostella) yielded one possible instance of Wolbachia LGT.ConclusionsOur results provide evidence of high incidence of identical and multiple strains of Wolbachia among butterflies and moths, adding Lepidoptera to the growing body of evidence for common horizontal transmission of Wolbachia. This study demonstrates interesting dynamics of this remarkable and influential microorganism.Electronic supplementary materialThe online version of this article (doi:10.1186/s12862-016-0660-x) contains supplementary material, which is available to authorized users.

Draft genome sequence of Bacillus thuringiensis 147, a Brazilian strain with high insecticidal activity.

Bacillus thuringiensis is a ubiquitous Gram-positive and sporulating bacterium. Its crystals and secreted toxins are useful tools against larvae of diverse insect orders and, as a consequence, an alternative to recalcitrant chemical insecticides. We report here the draft genome sequence ofB. thuringiensis 147, a strain isolated from Brazil and with high insecticidal activity. The assembled genome contained 6,167,994 bp and was distributed in seven replicons (a chromosome and 6 plasmids). We identified 12 coding regions, located in two plasmids, which encode insecticidal proteins.

Wolbachia in butterflies and moths: geographic structure in infection frequency.

IntroductionButterflies and moths (Lepidoptera) constitute one of the most diverse insect orders, and play an important role in ecosystem function. However, little is known in terms of their bacterial communities. Wolbachia, perhaps the most common and widespread intracellular bacterium on Earth, can manipulate the physiology and reproduction of its hosts, and is transmitted vertically from mother to offspring, or sometimes horizontally between species. While its role in some hosts has been studied extensively, its incidence across Lepidoptera is poorly understood. A recent analysis using a beta-binomial model to infer the between-species distribution of prevalence estimated that approximately 40 % of arthropod species are infected with Wolbachia, but particular taxonomic groups and ecological niches seem to display substantially higher or lower incidences. In this study, we took an initial step and applied a similar, maximum likelihood approach to 300 species of Lepidoptera (7604 individuals from 660 populations) belonging to 17 families and 10 superfamilies, and sampled from 36 countries, representing all continents excluding Antarctica.ResultsApproximately a quarter to a third of individuals appear to be infected with Wolbachia, and around 80 % of Lepidoptera species are infected at a non-negligible frequency. This incidence estimate is very high compared to arthropods in general. Wolbachia infection in Lepidoptera is shown to vary between families, but there is no evidence for closely related groups to show similar infection levels. True butterflies (Papilionoidea) are overrepresented in our data, however, our estimates show this group can be taken as a representative for the other major lepidopteran superfamilies. We also show substantial variation in infection level according to geography – closer locations tend to show similar infection levels. We further show that variation in geography is due to a latitudinal gradient in Wolbachia infection, with lower frequencies towards higher latitudes.ConclusionsOur comprehensive survey of Wolbachia infection in Lepidoptera suggests that infection incidence is very high, and provides evidence that climate and geography are strong predictors of infection frequency.Electronic supplementary materialThe online version of this article (doi:10.1186/s12983-015-0107-z) contains supplementary material, which is available to authorized users.

When caterpillars attack: biogeography and life history evolution of the Miletinae (Lepidoptera: Lycaenidae).

Of the four most diverse insect orders, Lepidoptera contains remarkably few predatory and parasitic species. Although species with these habits have evolved multiple times in moths and butterflies, they have rarely been associated with diversification. The wholly aphytophagous subfamily Miletinae (Lycaenidae) is an exception, consisting of nearly 190 species distributed primarily throughout the Old World tropics and subtropics. Most miletines eat Hemiptera, although some consume ant brood or are fed by ant trophallaxis. A well-resolved phylogeny inferred using 4915 bp from seven markers sampled from representatives of all genera and nearly one-third the described species was used to examine the biogeography and evolution of biotic associations in this group. Biogeographic analyses indicate that Miletinae likely diverged from an African ancestor near the start of the Eocene, and four lineages dispersed between Africa and Asia. Phylogenetic constraint in prey selection is apparent at two levels: related miletine species are more likely to feed on related Hemiptera, and related miletines are more likely to associate with related ants, either directly by eating the ants, or indirectly by eating hemipteran prey that are attended by those ants. These results suggest that adaptations for host ant location by ovipositing female miletines may have been retained from phytophagous ancestors that associated with ants mutualistically.

Uncovering the cultivable microbial diversity of costa rican beetles and its ability to break down plant cell wall components.

Coleopterans are the most diverse insect order described to date. These organisms have acquired an array of survival mechanisms through their evolution, including highly efficient digestive systems. Therefore, the coleopteran intestinal microbiota constitutes an important source of novel plant cell wall-degrading enzymes with potential biotechnological applications. We isolated and described the cultivable fungi, actinomycetes and aerobic eubacteria associated with the gut of larvae and adults from six different beetle families colonizing decomposing logs in protected Costa Rican ecosystems. We obtained 611 isolates and performed phylogenetic analyses using the ITS region (fungi) and 16S rDNA (bacteria). The majority of fungal isolates belonged to the order Hypocreales (26% of 169 total), while the majority of actinomycetes belonged to the genus Streptomyces (86% of 241 total). Finally, we isolated 201 bacteria spanning 19 different families belonging into four phyla: Firmicutes, α, β and γ-proteobacteria. Subsequently, we focused on microbes isolated from Passalid beetles to test their ability to degrade plant cell wall polymers. Highest scores in these assays were achieved by a fungal isolate (Anthostomella sp.), two Streptomyces and one Bacillus bacterial isolates. Our study demonstrates that Costa Rican beetles harbor several types of cultivable microbes, some of which may be involved in symbiotic relationships that enable the insect to digest complex polymers such as lignocellulose.

Phylogenomics provides strong evidence for relationships of butterflies and moths.

Butterflies and moths constitute some of the most popular and charismatic insects. Lepidoptera include approximately 160 000 described species, many of which are important model organisms. Previous studies on the evolution of Lepidoptera did not confidently place butterflies, and many relationships among superfamilies in the megadiverse clade Ditrysia remain largely uncertain. We generated a molecular dataset with 46 taxa, combining 33 new transcriptomes with 13 available genomes, transcriptomes and expressed sequence tags (ESTs). Using HaMStR with a Lepidoptera-specific core-orthologue set of single copy loci, we identified 2696 genes for inclusion into the phylogenomic analysis. Nucleotides and amino acids of the all-gene, all-taxon dataset yielded nearly identical, well-supported trees. Monophyly of butterflies (Papilionoidea) was strongly supported, and the group included skippers (Hesperiidae) and the enigmatic butterfly-moths (Hedylidae). Butterflies were placed sister to the remaining obtectomeran Lepidoptera, and the latter was grouped with greater than or equal to 87% bootstrap support. Establishing confident relationships among the four most diverse macroheteroceran superfamilies was previously challenging, but we recovered 100% bootstrap support for the following relationships: ((Geometroidea, Noctuoidea), (Bombycoidea, Lasiocampoidea)). We present the first robust, transcriptome-based tree of Lepidoptera that strongly contradicts historical placement of butterflies, and provide an evolutionary framework for genomic, developmental and ecological studies on this diverse insect order.

The phylogeny of Sodalis-like symbionts as reconstructed using surface-encoding loci

Phylogenetic analyses of 16S rRNA support close relationships between the Gammaproteobacteria Sodalis glossinidius, a tsetse (Diptera: Glossinidae) symbiont, and bacteria infecting diverse insect orders. To further examine the evolutionary relationships of these Sodalis-like symbionts, phylogenetic trees were constructed for a subset of putative surface-encoding genes (i.e. ompA, spr, slyB, rcsF, ycfM, and ompC). The ompA and ompC loci were used toward examining the intra- and interspecific diversity of Sodalis within tsetse, respectively. Intraspecific analyses of ompA support elevated nonsynonymous (dN) polymorphism with an excess of singletons, indicating diversifying selection, specifically within the tsetse Glossina morsitans. Additionally, interspecific ompC comparisons between Sodalis and Escherichia coli demonstrate deviation from neutrality, with higher fixed dN observed at sites associated with extracellular loops. Surface-encoding genes varied in their phylogenetic resolution of Sodalis and related bacteria, suggesting conserved vs. host-specific roles. Moreover, Sodalis and its close relatives exhibit genetic divergence at the rcsF, ompA, and ompC loci, indicative of initial molecular divergence. The application of outer membrane genes as markers for further delineating the systematics of recently diverged bacteria is discussed. These results increase our understanding of insect symbiont evolution, while also identifying early genome alterations occurring upon integration of microorganisms with eukaryotic hosts.

Honeybees and bumblebees share similar bacterial symbionts

Associations with symbiotic microorganisms are a major source for evolutionary innovation in eukaryotes. Arthropods have long served as model systems to study such associations, especially since Paul Buchner’s (1965) seminal work that beautifully illustrated the enormous diversity of microorganisms associated with insects. Particularly high taxonomic and functional diversities of microbial symbionts have been found in the guts and gut-associated organs of insects. These microorganisms play important roles in the digestion, nutrition and defence of the host. However, most studies of gut microorganisms have focused on single host taxa, limiting the ability to draw general conclusions on composition and functional roles of the insect gut microbiota. This is especially true for the diverse and important insect order Hymenoptera that comprises the bees, wasps and ants. Recently, Russell et al. (2009) analysed the bacterial community associated with diverse ant species and found evidence for changes in the microbial gut community coinciding with the evolution of herbivory. In this issue of Molecular Ecology, Martinson et al. (2011) provide the first broad-scale bacterial survey for bees. Their findings substantiate earlier evidence for a surprisingly simple gut microbiota in honeybees (Apis mellifera) that is composed of only six to ten major phylotypes. Importantly, Martinson et al. demonstrate for the first time that the same bacterial phylotypes are major constituents of other Apis as well as Bombus species, but not of any other bees and wasps outside of the corbiculate bees, a clade of four tribes within the subfamily Apinae. These results indicate that corbiculate bees harbour a specific and possibly co-evolved bacterial community in their digestive tract. Furthermore, the comparison with other bees and wasps suggests that changes in social lifestyle may have had a stronger effect on the evolution of the gut microbiota than the dietary shift from predatory ancestors to pollen-feeding (i.e. herbivorous) species. These findings have far-reaching implications for research on the microbial symbionts of insects as well as on the nutritional physiology of the ecologically and economically important group of corbiculate bees.

Identification of an atypical insect olfactory receptor subtype highly conserved within noctuids

Olfaction is primarily mediated by the large family of olfactory receptors. Although all insect olfactory receptors share the same structure with seven transmembrane domains, they present poor sequence homologies within and between species. As the only exception, Drosophila melanogaster OR83b and its orthologues define a receptor subtype singularly conserved between insect species. In this article, we report the identification of a new subtype of putative olfactory receptors exceptionally conserved within noctuids, a taxonomic group that includes crop pest insects. Through homology-based molecular cloning, homologues of the previously identified OR18 from Heliothis virescens were identified in the antennae of six noctuid species from various genera, presenting an average of 88% sequence identity. No orthologues were found in genomes available from diverse insect orders and selection pressure analysis revealed that the noctuid OR18s are under purifying selection. The OR18 gene was studied in details in the cotton leafworm, Spodoptera littoralis, where it presented all the characteristic features of an olfactory receptor encoding gene: its expression was restricted to the antennae, with expression in both sexes; its developmental expression pattern was reminiscent of that from other olfactory genes; and in situ hybridization experiments within the antennae revealed that the receptor-expressing cells were closely associated with the olfactory structures, including pheromone- and non-pheromone-sensitive structures. Taken together, our data suggest that we have identified a new original subtype of olfactory receptors that are extremely conserved within noctuids and that might fulfil a critical function in male and female noctuid chemosensory neurones.

PHYSIOLOGICAL PRINCIPLES MEET INFORMATION EXPLOSION

![Figure][1] J. L. Nation's 2nd edition of `Insect Physiology and Biochemistry', which builds on the 1st edition published in 2002, provides a timely, updated snapshot of modern insect physiology. The book strives to be comprehensive, and the array of topics covered is impressive – from

Evaluating nuclear protein-coding genes for phylogenetic utility in beetles

Although nuclear protein-coding genes have proven broadly useful for phylogenetic inference, relatively few such genes are regularly employed in studies of Coleoptera, the most diverse insect order. We increase the number of loci available for beetle systematics by developing protocols for three genes previously unused in beetles (alpha-spectrin, RNA polymerase II and topoisomerase I) and by refining protocols for five genes already in use (arginine kinase, CAD, enolase, PEPCK and wingless). We evaluate the phylogenetic performance of each gene in a Bayesian framework against a presumably known test phylogeny. The test phylogeny covers 31 beetle specimens and two outgroup taxa of varying age, including three of the four extant beetle suborders and a denser sampling in Adephaga and in the carabid genus Bembidion. All eight genes perform well for Cenozoic divergences and accurately separate closely related species within Bembidion, but individual genes differ markedly in accuracy over the older Mesozoic and Permian divergences. The concatenated data reconstruct the test phylogeny with high support in both Bayesian and parsimony analyses, indicating that combining data from multiple nuclear loci will be a fruitful approach for assembling the beetle tree of life.

Aquatic insect diversity of Costa Rica: state of knowledge

Costa Rica hosts an extraordinarily high biodiversity and is among the best studied neotropical countries. Insects represent the most diverse group of organisms, not only in terrestrial but also in aquatic, especially freshwater, habitats. Among the most diverse aquatic insect orders are the Trichoptera, Diptera and Coleoptera; although Ephemeroptera can locally also be very abundant and diverse. In Costa Rica, the taxonomically best known orders of aquatic insects are the caddisflies (Trichoptera), dragonflies (Odonata) and stoneflies (Plecoptera) and within the Dipterans, groups of medical importance have received special attention. The interest in aquatic insects has been constantly growing in Costa Rica over the past 10 years, but scientific publications are widely dispersed and often difficult to locate. Due to the importance of aquatic organisms in environmental impact studies and biomonitoring of freshwater habitats, there is an urgent need for comprehensive studies and publications that are locally available. In this sense, the present paper tries to give an overview on the state of knowledge and the literature published to date on the aquatic insects of Costa Rica, taking in account taxonomic, biological and ecological studies. Rev. Biol. Trop. 56 (Suppl. 4): 273-295. Epub 2009 June 30.

Up-regulation of heat shock proteins is essential for cold survival during insect diapause

Diapause, the dormancy common to overwintering insects, evokes a unique pattern of gene expression. In the flesh fly, most, but not all, of the fly's heat shock proteins (Hsps) are up-regulated. The diapause up-regulated Hsps include two members of the Hsp70 family, one member of the Hsp60 family (TCP-1), at least four members of the small Hsp family, and a small Hsp pseudogene. Expression of an Hsp70 cognate, Hsc70, is uninfluenced by diapause, and Hsp90 is actually down-regulated during diapause, thus diapause differs from common stress responses that elicit synchronous up-regulation of all Hsps. Up-regulation of the Hsps begins at the onset of diapause, persists throughout the overwintering period, and ceases within hours after the fly receives the signal to reinitiate development. The up-regulation of Hsps appears to be common to diapause in species representing diverse insect orders including Diptera, Lepidoptera, Coleoptera, and Hymenoptera as well as in diapauses that occur in different developmental stages (embryo, larva, pupa, adult). Suppressing expression of Hsp23 and Hsp70 in flies by using RNAi did not alter the decision to enter diapause or the duration of diapause, but it had a profound effect on the pupa's ability to survive low temperatures. We thus propose that up-regulation of Hsps during diapause is a major factor contributing to cold-hardiness of overwintering insects.

Web-Spinning Caterpillar Stalks Snails

Moths and butterflies compose one of the most diverse insect orders, but they are overwhelmingly herbivorous. Less than 0.2% are specialized predators, indicating that lepidopteran feeding habits are highly constrained. We report a Hawaiian caterpillar that specializes on snails, a unique food source requiring an unusual feeding strategy. The caterpillar uses silk to restrain live prey. All caterpillars have silk glands, but none are known to use silk in this spiderlike fashion. Considering the canalization of caterpillar diets, evolution to attack and feed on snails is an anomaly. Hawaii s isolation and consequently disharmonic biota likely promote evolutionary experiments that occur nowhere else.