Evaluation of heterologous expression in Pichia pastoris of Pine Weevil TRPA1 by GFP and flow cytometry.

BACKGROUND: The wasabi receptor, also known as the Transient Receptor Potential Ankyrin 1 (TRPA1) ion channel, is a potential target for development of repellents for insects, like the pine weevil (Hylobius abietis) feeding on conifer seedlings and causing damage in forestry. Heterologous expression of TRPA1 from pine weevil in the yeast Pichia pastoris can potentially provide protein for structural and functional studies. Here we take advantage of the Green Fluorescent Protein (GFP) tag to examine the various steps of heterologous expression, to get more insight in clone selection, expression and isolation of the intact purified protein. RESULTS: The sequence of HaTRPA1 is reported and GFP-tagged constructs were made of the full-length protein and a truncated version (Δ1-708 HaTRPA1), lacking the N-terminal ankyrin repeat domain. Clones were screened on GFP expression plates, induced in small liquid cultures and in fed-batch cultures, and evaluated by flow cytometry and fluorescence microscopy. The screening on plates successfully identifies low-expression clones, but fails to predict the ranking of the best performing clones in small-scale liquid cultures. The two constructs differ in their cellular localization. Δ1-708 HaTRPA1 is found in a ring at the perimeter of cell, whereas HaTRPA1 is forming highly fluorescent speckles in interior regions of the cell. The pattern is consistent in different clones of the same construct and persists in fed-batch culture. The expression of Δ1-708 HaTRPA1 decreases the viability more than HaTRPA1, and in fed-batch culture it is clear that intact cells first express Δ1-708 HaTRPA1 and then become damaged. Purifications show that both constructs suffer from degradation of the expressed protein, but especially the HaTRPA1 construct. CONCLUSIONS: The GFP tag makes it possible to follow expression by flow cytometry and fluorescence microscopy. Analyses of localization, cell viability and expression show that the former two parameters are specific for each of the two evaluated constructs, whereas the relative expression of the constructs varies with the cultivation method. High expression is not all that matters, so taking damaged cells into account, something that may be linked to protein degradation, is important when picking the most suitable construct, clone, and expression scheme.

Characterization of olfactory sensory neurons in the striped ambrosia beetle Trypodendron lineatum.

Introduction: The striped ambrosia beetle Trypodendron lineatum (Coleoptera, Curculionidae, Scolytinae) is a major forest pest in the Holarctic region. It uses an aggregation pheromone and host and non-host volatiles to locate suitable host trees, primarily stressed or dying conifer trees. The beetles bore into the xylem and inoculate spores of their obligate fungal mutualist Phialophoropsis ferruginea inside their excavated egg galleries, with the fungus serving as the main food source for the developing larvae. Olfactory sensory neuron (OSN) responses to pheromones and host volatiles are poorly understood in T. lineatum and other ambrosia beetles, and nothing is known about potential responses to fungal volatiles. Methods: We screened responses of OSNs present in 170 antennal olfactory sensilla using single sensillum recordings (SSR) and 57 odor stimuli, including pheromones, host and non-host compounds, as well as volatiles produced by P. ferruginea and fungal symbionts of other scolytine beetles. Results and Discussion: Thirteen OSN classes were characterized based on their characteristic response profiles. An OSN class responding to the aggregation pheromone lineatin was clearly the most abundant on the antennae. In addition, four OSN classes responded specifically to volatile compounds originating from the obligate fungal mutualist and three responded to non-host plant volatiles. Our data also show that T. lineatum has OSN classes tuned to pheromones of other bark beetles. Several OSN classes showed similar response profiles to those previously described in the sympatric bark beetle Ips typographus, which may reflect their shared ancestry.

Addressing a century-old hypothesis - do pioneer beetles of Ips typographus use volatile cues to find suitable host trees?

Global warming and more frequent climate extremes have caused bark beetle outbreaks of unprecedented scale of these insects in many conifer forests world-wide. Conifers that have been weakened by drought and heat or damaged by storms are highly susceptible to bark beetle infestation. A large proportion of trees with impaired defences provides good conditions for beetle population build-up of beetles, but mechanisms driving host search of pioneer beetles are still uncertain in several species, including the Eurasian spruce bark beetle Ips typographus. Despite a two-century-long history of bark beetle research, we still lack a sufficient understanding of interactions between I. typographus and its host Norway spruce (Picea abies) to forecast future disturbance regimes and forest dynamics. Depending on the scale (habitat or patch) and beetle population state (endemic or epidemic), host selection is likely driven by a combination of pre and postlanding cues, including visual selection or olfactory detection (kairomones). Here, we discuss primary attraction mechanisms and how volatile emission profiles of Norway spruce may provide cues on tree vitality and suitability for attacks by I. typographus, in particular during the endemic phase. We identify several crucial knowledge gaps and provide a research agenda addressing the experimental challenges of such investigations.

Interactive effects of rising temperatures and urbanisation on birds across different climate zones: A mechanistic perspective.

Climate change and urbanisation are among the most pervasive and rapidly growing threats to biodiversity worldwide. However, their impacts are usually considered in isolation, and interactions are rarely examined. Predicting species' responses to the combined effects of climate change and urbanisation, therefore, represents a pressing challenge in global change biology. Birds are important model taxa for exploring the impacts of both climate change and urbanisation, and their behaviour and physiology have been well studied in urban and non-urban systems. This understanding should allow interactive effects of rising temperatures and urbanisation to be inferred, yet considerations of these interactions are almost entirely lacking from empirical research. Here, we synthesise our current understanding of the potential mechanisms that could affect how species respond to the combined effects of rising temperatures and urbanisation, with a focus on avian taxa. We discuss potential interactive effects to motivate future in-depth research on this critically important, yet overlooked, aspect of global change biology. Increased temperatures are a pronounced consequence of both urbanisation (through the urban heat island effect) and climate change. The biological impact of this warming in urban and non-urban systems will likely differ in magnitude and direction when interacting with other factors that typically vary between these habitats, such as resource availability (e.g. water, food and microsites) and pollution levels. Furthermore, the nature of such interactions may differ for cities situated in different climate types, for example, tropical, arid, temperate, continental and polar. Within this article, we highlight the potential for interactive effects of climate and urban drivers on the mechanistic responses of birds, identify knowledge gaps and propose promising future research avenues. A deeper understanding of the behavioural and physiological mechanisms mediating species' responses to urbanisation and rising temperatures will provide novel insights into ecology and evolution under global change and may help better predict future population responses.

Conifer-killing bark beetles locate fungal symbionts by detecting volatile fungal metabolites of host tree resin monoterpenes.

Outbreaks of the Eurasian spruce bark beetle (Ips typographus) have decimated millions of hectares of conifer forests in Europe in recent years. The ability of these 4.0 to 5.5 mm long insects to kill mature trees over a short period has been sometimes ascribed to two main factors: (1) mass attacks on the host tree to overcome tree defenses and (2) the presence of fungal symbionts that support successful beetle development in the tree. While the role of pheromones in coordinating mass attacks has been well studied, the role of chemical communication in maintaining the fungal symbiosis is poorly understood. Previous evidence indicates that I. typographus can distinguish fungal symbionts of the genera Grosmannia, Endoconidiophora, and Ophiostoma by their de novo synthesized volatile compounds. Here, we hypothesize that the fungal symbionts of this bark beetle species metabolize spruce resin monoterpenes of the beetle's host tree, Norway spruce (Picea abies), and that the volatile products are used as cues by beetles for locating breeding sites with beneficial symbionts. We show that Grosmannia penicillata and other fungal symbionts alter the profile of spruce bark volatiles by converting the major monoterpenes into an attractive blend of oxygenated derivatives. Bornyl acetate was metabolized to camphor, and α- and ß-pinene to trans-4-thujanol and other oxygenated products. Electrophysiological measurements showed that I. typographus possesses dedicated olfactory sensory neurons for oxygenated metabolites. Both camphor and trans-4-thujanol attracted beetles at specific doses in walking olfactometer experiments, and the presence of symbiotic fungi enhanced attraction of females to pheromones. Another co-occurring nonbeneficial fungus (Trichoderma sp.) also produced oxygenated monoterpenes, but these were not attractive to I. typographus. Finally, we show that colonization of fungal symbionts on spruce bark diet stimulated beetles to make tunnels into the diet. Collectively, our study suggests that the blends of oxygenated metabolites of conifer monoterpenes produced by fungal symbionts are used by walking bark beetles as attractive or repellent cues to locate breeding or feeding sites containing beneficial microbial symbionts. The oxygenated metabolites may aid beetles in assessing the presence of the fungus, the defense status of the host tree and the density of conspecifics at potential feeding and breeding sites.

A draft Diabrotica virgifera virgifera genome: insights into control and host plant adaption by a major maize pest insect.

BACKGROUND: Adaptations by arthropod pests to host plant defenses of crops determine their impacts on agricultural production. The larval host range of western corn rootworm, Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae), is restricted to maize and a few grasses. Resistance of D. v. virgifera to crop rotation practices and multiple insecticides contributes to its status as the most damaging pest of cultivated maize in North America and Europe. The extent to which adaptations by this pest contributes to host plant specialization remains unknown. RESULTS: A 2.42 Gb draft D. v. virgifera genome, Dvir_v2.0, was assembled from short shotgun reads and scaffolded using long-insert mate-pair, transcriptome and linked read data. K-mer analysis predicted a repeat content of ≥ 61.5%. Ortholog assignments for Dvir_2.0 RefSeq models predict a greater number of species-specific gene duplications, including expansions in ATP binding cassette transporter and chemosensory gene families, than in other Coleoptera. A majority of annotated D. v. virgifera cytochrome P450s belong to CYP4, 6, and 9 clades. A total of 5,404 transcripts were differentially-expressed between D. v. virgifera larvae fed maize roots compared to alternative host (Miscanthus), a marginal host (Panicum virgatum), a poor host (Sorghum bicolor) and starvation treatments; Among differentially-expressed transcripts, 1,908 were shared across treatments and the least number were between Miscanthus compared to maize. Differentially-expressed transcripts were enriched for putative spliceosome, proteosome, and intracellular transport functions. General stress pathway functions were unique and enriched among up-regulated transcripts in marginal host, poor host, and starvation responses compared to responses on primary (maize) and alternate hosts. CONCLUSIONS: Manual annotation of D. v. virgifera Dvir_2.0 RefSeq models predicted expansion of paralogs with gene families putatively involved in insecticide resistance and chemosensory perception. Our study also suggests that adaptations of D. v. virgifera larvae to feeding on an alternate host plant invoke fewer transcriptional changes compared to marginal or poor hosts. The shared up-regulation of stress response pathways between marginal host and poor host, and starvation treatments may reflect nutrient deprivation. This study provides insight into transcriptomic responses of larval feeding on different host plants and resources for genomic research on this economically significant pest of maize.

Weak population genetic structure in Eurasian spruce bark beetle over large regional scales in Sweden.

The Eurasian spruce bark beetle, Ips typographus, is a major pest, capable of killing spruce forests during large population outbreaks. Recorded dispersal distances of individual beetles are typically within hundreds of meters or a few kilometers. However, the connectivity between populations at larger distances and longer time spans and how this is affected by the habitat is less studied, despite its importance for understanding at which distances local outbreaks may spread. Previous population genetic studies in I. typographus typically used low resolution markers. Here, we use genome-wide data to assess population structure and connectivity of I. typographus in Sweden. We used 152 individuals from 19 population samples, distributed over 830 km from Strömsund (63° 46' 8″ N) in the north to Nyteboda (56° 8' 50″ N) in the south, to capture processes at a large regional scale, and a transect sampling design adjacent to a recent outbreak to capture processes at a smaller scale (76 km). Using restriction site-associated DNA sequencing (RADseq) markers capturing 1409-1997 SNPs throughout the genome, we document a weak genetic structure over the large scale, potentially indicative of high connectivity with extensive gene flow. No differentiation was detected at the smaller scale. We find indications of isolation-by-distance both for relative (F ST) and absolute divergence (Dxy). The two northernmost populations are most differentiated from the remaining populations, and diverge in parallel to the southern populations for a set of outlier loci. In conclusion, the population structure of I. typographus in Sweden is weak, suggesting a high capacity to disperse and establish outbreak populations in new territories.

Odorant receptor orthologues in conifer-feeding beetles display conserved responses to ecologically relevant odours.

Insects are able to detect a plethora of olfactory cues using a divergent family of odorant receptors (ORs). Despite the divergent nature of this family, related species frequently express several evolutionarily conserved OR orthologues. In the largest order of insects, Coleoptera, it remains unknown whether OR orthologues have conserved or divergent functions in different species. Using HEK293 cells, we addressed this question through functional characterization of two groups of OR orthologues in three species of the Curculionidae (weevil) family, the conifer-feeding bark beetles Ips typographus L. ("Ityp") and Dendroctonus ponderosae Hopkins ("Dpon") (Scolytinae), and the pine weevil Hylobius abietis L. ("Habi"; Molytinae). The ORs of H. abietis were annotated from antennal transcriptomes. The results show highly conserved response specificities, with one group of orthologues (HabiOR3/DponOR8/ItypOR6) responding exclusively to 2-phenylethanol (2-PE), and the other group (HabiOR4/DponOR9/ItypOR5) responding to angiosperm green leaf volatiles (GLVs). Both groups of orthologues belong to the coleopteran OR subfamily 2B, and share a common ancestor with OR5 in the cerambycid Megacyllene caryae, also tuned to 2-PE, suggesting a shared evolutionary history of 2-PE receptors across two beetle superfamilies. The detected compounds are ecologically relevant for conifer-feeding curculionids, and are probably linked to fitness, with GLVs being used to avoid angiosperm nonhost plants, and 2-PE being important for intraspecific communication and/or playing a putative role in beetle-microbe symbioses. To our knowledge, this study is the first to reveal evolutionary conservation of OR functions across several beetle species and hence sheds new light on the functional evolution of insect ORs.

Ionotropic receptors in the turnip moth Agrotis segetum respond to repellent medium-chain fatty acids.

BACKGROUND: In insects, airborne chemical signals are mainly detected by two receptor families, odorant receptors (ORs) and ionotropic receptors (IRs). Functions of ORs have been intensively investigated in Diptera and Lepidoptera, while the functions and evolution of the more ancient IR family remain largely unexplored beyond Diptera. RESULTS: Here, we identified a repertoire of 26 IRs from transcriptomes of female and male antennae, and ovipositors in the moth Agrotis segetum. We observed that a large clade formed by IR75p and IR75q expansions is closely related to the acid-sensing IRs identified in Diptera. We functionally assayed each of the five AsegIRs from this clade using Xenopus oocytes and found that two receptors responded to the tested ligands. AsegIR75p.1 responded to several compounds but hexanoic acid was revealed to be the primary ligand, and AsegIR75q.1 responded primarily to octanoic acid, and less so to nonanoic acid. It has been reported that the C6-C10 medium-chain fatty acids repel various insects including many drosophilids and mosquitos. We show that the C6-C10 medium-chain fatty acids elicited antennal responses of both sexes of A. segetum, while only octanoic acid had repellent effect to the moths in a behavioral assay. In addition, using fluorescence in situ hybridization, we demonstrated that the five IRs and their co-receptor AsegIR8a are not located in coeloconic sensilla as found in Drosophila, but in basiconic or trichoid sensilla. CONCLUSIONS: Our results significantly expand the current knowledge of the insect IR family. Based on the functional data in combination with phylogenetic analysis, we propose that subfunctionalization after gene duplication plays an important role in the evolution of ligand specificities of the acid-sensing IRs in Lepidoptera.

Sex pheromone receptors of the light brown apple moth, Epiphyas postvittana, support a second major pheromone receptor clade within the Lepidoptera.

Sex pheromones facilitate species-specific sex communication within the Lepidoptera. They are detected by specialised pheromone receptors (PRs), most of which to date fall into a single monophyletic receptor lineage (frequently referred to as "the PR clade") within the odorant receptor (OR) family. Here we investigated PRs of the invasive horticultural pest, Epiphyas postvittana, commonly known as the light brown apple moth. Ten candidate PRs were selected, based on their male-biased expression in antennae or their relationship to the PR clade, for functional assessment in both HEK293 cells and Xenopus oocytes. Of these, six ORs responded to compounds that include components of the E. postvittana ('Epos') sex pheromone blend or compounds that antagonise sex pheromone attraction. In phylogenies, four of the characterised receptors (EposOR1, 6, 7 and 45) fall within the PR clade and two other male-biased receptors (EposOR30 and 34) group together well outside the PR clade. This new clade of pheromone receptors includes the receptor for (E)-11-tetradecenyl acetate (EposOR30), which is the main component of the sex pheromone blend for this species. Interestingly, receptors of the two clades do not segregate by preference for compounds associated with behavioural response (agonist or antagonist), isomer type (E or Z) or functional group (alcohol or acetate), with examples of each scattered across both clades. Phylogenetic comparison with PRs from other species supports the existence of a second major clade of lepidopteran ORs including, EposOR30 and 34, that has been co-opted into sex pheromone detection in the Lepidoptera. This second clade of sex pheromone receptors has an origin that likely predates the split between the major lepidopteran families.

A highly-contiguous genome assembly of the Eurasian spruce bark beetle, Ips typographus, provides insight into a major forest pest.

Conifer-feeding bark beetles are important herbivores and decomposers in forest ecosystems. These species complete their life cycle in nutritionally poor substrates and some can kill enormous numbers of trees during population outbreaks. The Eurasian spruce bark beetle (Ips typographus) can destroy >100 million m3 of spruce in a single year. We report a 236.8 Mb I. typographus genome assembly using PacBio long-read sequencing. The final phased assembly has a contig N50 of 6.65 Mb in 272 contigs and is predicted to contain 23,923 protein-coding genes. We reveal expanded gene families associated with plant cell wall degradation, including pectinases, aspartyl proteases, and glycosyl hydrolases. This genome sequence from the genus Ips provides timely resources to address questions about the evolutionary biology of the true weevils (Curculionidae), one of the most species-rich animal families. In forests of today, increasingly stressed by global warming, this draft genome may assist in developing pest control strategies to mitigate outbreaks.

Codon Optimization of Insect Odorant Receptor Genes May Increase Their Stable Expression for Functional Characterization in HEK293 Cells.

Insect odorant receptor (OR) genes are routinely expressed in Human Embryonic Kidney (HEK) 293 cells for functional characterization ("de-orphanization") using transient or stable expression. However, progress in this research field has been hampered because some insect ORs are not functional in this system, which may be due to insufficient protein levels. We investigated whether codon optimization of insect OR sequences for expression in human cells could facilitate their functional characterization in HEK293 cells with stable and inducible expression. We tested the olfactory receptor co-receptor (Orco) proteins from the bark beetles Ips typographus ("Ityp") and Dendroctonus ponderosae ("Dpon"), and six ItypORs previously characterized in Xenopus laevis oocytes and/or HEK cells. Western blot analysis indicated that codon optimization yielded increased cellular protein levels for seven of the eight receptors. Our experimental assays demonstrated that codon optimization enabled functional characterization of two ORs (ItypOR25 and ItypOR29) which are unresponsive when expressed from wildtype (non-codon optimized) genes. Similar to previous Xenopus oocyte recordings, ItypOR25 responded primarily to the host/conifer monoterpene (+)-3-carene. ItypOR29 responded primarily to (+)-isopinochamphone and similar ketones produced by fungal symbionts and trees. Codon optimization also resulted in significantly increased responses in ItypOR49 to its pheromone ligand (R)-(-)-ipsdienol, and improved responses to the Orco agonist VUAA1 in ItypOrco. However, codon optimization did not result in functional expression of DponOrco, ItypOR23, ItypOR27, and ItypOR28 despite higher protein levels as indicated by Western blots. We conclude that codon optimization may enable or improve the functional characterization of insect ORs in HEK cells, although this method is not sufficient for all ORs that are not functionally expressed from wildtype genes.

Natural variation in yolk fatty acids, but not androgens, predicts offspring fitness in a wild bird.

BACKGROUND: In egg-laying animals, mothers can influence the developmental environment and thus the phenotype of their offspring by secreting various substances into the egg yolk. In birds, recent studies have demonstrated that different yolk substances can interactively affect offspring phenotype, but the implications of such effects for offspring fitness and phenotype in natural populations have remained unclear. We measured natural variation in the content of 31 yolk components known to shape offspring phenotypes including steroid hormones, antioxidants and fatty acids in eggs of free-living great tits (Parus major) during two breeding seasons. We tested for relationships between yolk component groupings and offspring fitness and phenotypes. RESULTS: Variation in hatchling and fledgling numbers was primarily explained by yolk fatty acids (including saturated, mono- and polyunsaturated fatty acids) - but not by androgen hormones and carotenoids, components previously considered to be major determinants of offspring phenotype. Fatty acids were also better predictors of variation in nestling oxidative status and size than androgens and carotenoids. CONCLUSIONS: Our results suggest that fatty acids are important yolk substances that contribute to shaping offspring fitness and phenotype in free-living populations. Since polyunsaturated fatty acids cannot be produced de novo by the mother, but have to be obtained from the diet, these findings highlight potential mechanisms (e.g., weather, habitat quality, foraging ability) through which environmental variation may shape maternal effects and consequences for offspring. Our study represents an important first step towards unraveling interactive effects of multiple yolk substances on offspring fitness and phenotypes in free-living populations. It provides the basis for future experiments that will establish the pathways by which yolk components, singly and/or interactively, mediate maternal effects in natural populations.

Functional Evolution of a Bark Beetle Odorant Receptor Clade Detecting Monoterpenoids of Different Ecological Origins.

Insects detect odors using an array of odorant receptors (ORs), which may expand through gene duplication. How and which new functions may evolve among related ORs within a species remain poorly investigated. We addressed this question by functionally characterizing ORs from the Eurasian spruce bark beetle Ips typographus, in which physiological and behavioral responses to pheromones, volatiles from host and nonhost trees, and fungal symbionts are well described. In contrast, knowledge of OR function is restricted to two receptors detecting the pheromone compounds (S)-(-)-ipsenol (ItypOR46) and (R)-(-)-ipsdienol (ItypOR49). These receptors belong to an Ips-specific OR-lineage comprising seven ItypORs. To gain insight into the functional evolution of related ORs, we characterized the five remaining ORs in this clade using Xenopus oocytes. Two receptors responded primarily to the host tree monoterpenes (+)-3-carene (ItypOR25) and p-cymene (ItypOR27). Two receptors responded to oxygenated monoterpenoids produced in larger relative amounts by the beetle-associated fungi, with ItypOR23 specific for (+)-trans-(1R, 4S)-4-thujanol, and ItypOR29 responding to (+)-isopinocamphone and similar ketones. ItypOR28 responded to the pheromone E-myrcenol from the competitor Ips duplicatus. Overall, the OR responses match well with those of previously characterized olfactory sensory neuron classes except that neurons detecting E-myrcenol have not been identified. The characterized ORs are under strong purifying selection and demonstrate a shared functional property in that they all primarily respond to monoterpenoids. The variation in functional groups among OR ligands and their diverse ecological origins suggest that neofunctionalization has occurred early in the evolution of this OR-lineage following gene duplication.

Putative ligand binding sites of two functionally characterized bark beetle odorant receptors.

BACKGROUND: Bark beetles are major pests of conifer forests, and their behavior is primarily mediated via olfaction. Targeting the odorant receptors (ORs) may thus provide avenues towards improved pest control. Such an approach requires information on the function of ORs and their interactions with ligands, which is also essential for understanding the functional evolution of these receptors. Hence, we aimed to identify a high-quality complement of ORs from the destructive spruce bark beetle Ips typographus (Coleoptera, Curculionidae, Scolytinae) and analyze their antennal expression and phylogenetic relationships with ORs from other beetles. Using 68 biologically relevant test compounds, we next aimed to functionally characterize ecologically important ORs, using two systems for heterologous expression. Our final aim was to gain insight into the ligand-OR interaction of the functionally characterized ORs, using a combination of computational and experimental methods. RESULTS: We annotated 73 ORs from an antennal transcriptome of I. typographus and report the functional characterization of two ORs (ItypOR46 and ItypOR49), which are responsive to single enantiomers of the common bark beetle pheromone compounds ipsenol and ipsdienol, respectively. Their responses and antennal expression correlate with the specificities, localizations, and/or abundances of olfactory sensory neurons detecting these enantiomers. We use homology modeling and molecular docking to predict their binding sites. Our models reveal a likely binding cleft lined with residues that previously have been shown to affect the responses of insect ORs. Within this cleft, the active ligands are predicted to specifically interact with residues Tyr84 and Thr205 in ItypOR46. The suggested importance of these residues in the activation by ipsenol is experimentally supported through site-directed mutagenesis and functional testing, and hydrogen bonding appears key in pheromone binding. CONCLUSIONS: The emerging insight into ligand binding in the two characterized ItypORs has a general importance for our understanding of the molecular and functional evolution of the insect OR gene family. Due to the ecological importance of the characterized receptors and widespread use of ipsenol and ipsdienol in bark beetle chemical communication, these ORs should be evaluated for their potential use in pest control and biosensors to detect bark beetle infestations.

Migrant blackbirds, Turdus merula, have higher plasma levels of polyunsaturated fatty acids compared to residents, but not enhanced fatty acid unsaturation index.

Birds have been observed to have dietary preferences for unsaturated fatty acids during migration. Polyunsaturated fatty acids (PUFAs) may increase the exercise performance of migrant birds; however, PUFAs are also peroxidation prone and might therefore incur increased costs in terms of enhanced oxidative damage in migratory individuals. To shed light on this potential constraint, we analyzed plasma fatty acid (FA) composition and estimated the unsaturation index as a proxy for susceptibility to lipid peroxidation of migrants and residents of the partially migratory common blackbird (Turdus merula) at a stopover site during autumn migration. As predicted, migrant birds had higher relative and absolute levels of PUFAs compared to resident birds. This included the strictly dietary ω-3 PUFA α-linolenic acid, suggesting a dietary and/or storage preference for these FAs in migrants. Interestingly, the FA unsaturation index did not differ between migrants and residents. These findings suggest a mechanism where birds alter their levels of metabolic substrate without simultaneously increasing the susceptibility of the substrate to lipid peroxidation. In summary, our results are in line with the hypothesis that increased exercise performance during migration might be constrained by oxidative stress, which is manifested in changes in the composition of key FAs to retain the unsaturation index constant despite the increased levels of peroxidizable PUFAs.

Functional characterization of odorant receptors from the moth Eriocrania semipurpurella: A comparison of results in the Xenopus oocyte and HEK cell systems.

The Xenopus oocyte and the Human Embryonic Kidney (HEK) 293 cell expression systems are frequently used for functional characterization (deorphanization) of insect odorant receptors (ORs). However, the inherent characteristics of these heterologous systems differ in several aspects, which raises the question of whether the two systems provide comparable results, and how well the results correspond to the responses obtained from olfactory sensory neurons in vivo. Five candidate pheromone receptors were previously identified in the primitive moth Eriocrania semipurpurella (Esem) and their responses were characterized in HEK cells. We re-examined the responses of these five EsemORs in Xenopus oocytes. We showed that in both systems, EsemOR1 specifically responded to the plant volatile ß-caryophyllene. EsemOR3 responded stronger to the pheromone component (S,Z)-6-nonen-2-ol than to its enantiomer (R,Z)-6-nonen-2-ol, the second pheromone component. However, EsemOR3 also responded secondarily to the plant volatile ß-caryophyllene in the oocyte system, but not in the HEK cell system. EsemOR4 was unresponsive in the HEK cells, but responded primarily to (R,Z)-6-nonen-2-ol followed by (S,Z)-6-nonen-2-ol in the oocytes, representing a discovery of a new pheromone receptor in this species. EsemOR5 was broadly tuned in both systems, but the rank order among the most active pheromone compounds and antagonists was different. EsemOR6 showed no response to any compound in either system. We compared the results obtained in the two different heterologous systems with the activity previously recorded in vivo, and performed in situ hybridization to localize the expression of these OR genes in the antennae. In spite of similar results overall, differences in OR responses between heterologous expression systems suggest that conclusions about the function of individual ORs may differ depending on the system used for deorphanization.

Characterization of olfactory sensory neurons in the red clover seed weevil, Protapion trifolii (Coleoptera: Brentidae) and comparison to the closely related species P. fulvipes.

Protapion trifolii and P. fulvipes (Coleoptera: Brentidae) are major pests in European clover seed production. Previous studies have reported a high host plant fidelity of these weevils for red and white clover species, respectively, driven by host plant olfactory cues. Given the specific host preferences observed in these weevils, we aimed to elucidate to which extent such selectivity is reflected in their peripheral olfactory systems. Using an electrophysiological approach, we performed the first functional characterisation of olfactory sensory neurons (OSNs) in P. trifolii to a panel of volatile compounds emitted by red clover plants, and compared the results with the reported OSN types of P. fulvipes. Nineteen OSN classes were characterized in P. trifolii, with the majority of these neurons responding strongly to common volatiles released by the host plant. Based on response profiles, eight of these OSN classes have clear matches to OSN classes in P. fulvipes. The OSN colocalisation patterns and antennal frequency of these classes are similar in the two species. Additionally, the responses of these OSNs are generally highly conserved in the two species, with clear response shifts only revealed for two of the OSN classes. These response shifts in combination with additional response dissimilarities for compounds that vary in abundance between red and white clover plants may underlie the species-specific host preferences. Further behavioural and field experiments should focus on these differentially detected compounds to elucidate their potential role in host selection and use in semiochemical-based control of these pests.

Genomic content of chemosensory genes correlates with host range in wood-boring beetles (Dendroctonus ponderosae, Agrilus planipennis, and Anoplophora glabripennis).

BACKGROUND: Olfaction and gustation underlie behaviors that are crucial for insect fitness, such as host and mate selection. The detection of semiochemicals is mediated via proteins from large and rapidly evolving chemosensory gene families; however, the links between a species' ecology and the diversification of these genes remain poorly understood. Hence, we annotated the chemosensory genes from genomes of select wood-boring coleopterans, and compared the gene repertoires from stenophagous species with those from polyphagous species. RESULTS: We annotated 86 odorant receptors (ORs), 60 gustatory receptors (GRs), 57 ionotropic receptors (IRs), 4 sensory neuron membrane proteins (SNMPs), 36 odorant binding proteins (OBPs), and 11 chemosensory proteins (CSPs) in the mountain pine beetle (Dendroctonus ponderosae), and 47 ORs, 30 GRs, 31 IRs, 4 SNMPs, 12 OBPs, and 14 CSPs in the emerald ash borer (Agrilus planipennis). Four SNMPs and 17 CSPs were annotated in the polyphagous wood-borer Anoplophora glabripennis. The gene repertoires in the stenophagous D. ponderosae and A. planipennis are reduced compared with those in the polyphagous A. glabripennis and T. castaneum, which is largely manifested through small gene lineage expansions and entire lineage losses. Alternative splicing of GR genes was limited in D. ponderosae and apparently absent in A. planipennis, which also seems to have lost one carbon dioxide receptor (GR1). A. planipennis has two SNMPs, which are related to SNMP3 in T. castaneum. D. ponderosae has two alternatively spliced OBP genes, a novel OBP "tetramer", and as many as eleven IR75 members. Simple orthology was generally rare in beetles; however, we found one clade with orthologues of putative bitter-taste GRs (named the "GR215 clade"), and conservation of IR60a from Drosophila melanogaster. CONCLUSIONS: Our genome annotations represent important quantitative and qualitative improvements of the original datasets derived from transcriptomes of D. ponderosae and A. planipennis, facilitating evolutionary analysis of chemosensory genes in the Coleoptera where only a few genomes were previously annotated. Our analysis suggests a correlation between chemosensory gene content and host specificity in beetles. Future studies should include additional species to consolidate this correlation, and functionally characterize identified proteins as an important step towards improved control of these pests.

Volatile organic compounds influence the interaction of the Eurasian spruce bark beetle (Ips typographus) with its fungal symbionts.

Insects have mutualistic symbioses with a variety of microorganisms. However, the chemical signals that maintain these insect-microbe relationships are poorly known compared to those from insect-plant symbioses. The spruce bark beetle, Ips typographus, the most destructive forest pest in Europe, has a symbiotic relationship with several fungi that are believed to contribute to its successful invasion of Norway spruce. Here we tested the hypothesis that volatile organic compounds (VOCs) emitted from fungal symbionts could be cues for bark beetles to recognize and distinguish among members of its microbial community. Behavioral experiments with fungi showed that immature adults of I. typographus are attracted to food sources colonized by their fungal symbionts but not to saprophytic fungi and that this attraction is mediated by volatile cues. GC-MS measurements revealed that the symbionts emitted VOCs. Testing the activity of these compounds on beetle antennae using single sensillum recordings showed that beetles detect many fungal volatiles and possess olfactory sensory neurons specialized for these compounds. Finally, synthetic blends of fungal volatiles attracted beetles in olfactometer experiments. These findings indicate that volatile compounds produced by fungi may act as recognition signals for bark beetles to maintain specific microbial communities that might have impact on their fitness.