Effects of simulated gut pH environment on bacterial composition and pheromone production of Dendroctonus valens.

Bark beetles are an economically and ecologically important insect group, with aggregation behavior and thus host colonization success depends on pheromone-mediated communication. For some species, such as the major invasive forest pest in China, red turpentine beetle (Dendroctonus valens), gut microbiota participates in pheromone production by converting tree monoterpenes into pheromone products. However, how variation in gut microenvironment, such as pH, affects the gut microbial composition, and consequently pheromone production, is unknown. In this study, we fed wild caught D. valens with 3 different pH media (main host diet with natural pH of 4.7; a mildly acidic diet with pH 6 mimicking the beetle gut pH; and highly acidic diet with pH 4), and measured their effects on the gut pH, bacterial community and production of the main aggregation and anti-aggregation pheromone (verbenone). We further tested the verbenone production capacity of 2 gut bacterial isolates in different pH environments (pH 6 and 4). Compared to natural state or main host diet, feeding on less acidic diet (pH 6) diluted the acidity of the gut, whereas feeding on highly acidic diet (pH 4) enhanced it. Both changes in gut pH reduced the abundance of dominant bacterial genera, resulting in decreased verbenone production. Similarly, the highest pheromone conversion rate of the bacterial isolates was observed in pH mimicking the acidity in beetle gut. Taken together, these results indicate that changes in gut pH can affect gut microbiota composition and pheromone production, and may therefore have the potential to affect host colonization behavior.

Emerald Ash Borer Management and Research: Decades of Damage and Still Expanding.

Since the discovery of the ash tree (Fraxinus spp.) killer emerald ash borer (EAB; Agrilus planipennis) in the United States in 2002 and Moscow, Russia in 2003, substantial detection and management efforts have been applied to contain and monitor its spread and mitigate impacts. Despite these efforts, the pest continues to spread within North America. It has spread to European Russia and Ukraine and is causing sporadic outbreaks in its native range in China. The dynamics of EAB's range expansion events appear to be linked to the lack of resistant ash trees in invaded ranges, facilitated by the abundance of native or planted North American susceptible ash species. We review recently gained knowledge of the range expansion of EAB; its ecological, economic, and social impacts; and past management efforts with their successes and limitations. We also highlight advances in biological control, mechanisms of ash resistance, and new detection and management approaches under development, with the aim of guiding more effective management.

Gut Bacteria of Wood-Boring Streltzoviella insularis and Their Involvement in Pinoresinol Degradation of Ash Tree (Fraxinus sp.) Host.

Insect gut microbiota have been widely reported to help the insects to overcome host tree defense. Streltzoviella insularis (Lepidoptera: Cossidae) is one of the most common wood borers in China, attacking various hosts, including ash trees (Fraxinus sp.), but little is known about its gut microbial associates and their involvement in host tree defense. We isolated gut bacteria of S. insularis larvae, analyzed their ability to degrade pinoresinol (a defense compound of ash trees) and cellulose, and identified pinoresinol degradation products. Larval mortality increased with increasing pinoresinol concentration (reflecting natural variation observed in the host trees). All the five detected gut bacteria isolates were able to degrade pinoresinol, two of which were also capable of cellulose degradation. Furthermore, gut bacteria were also shown to degrade pinoresinol via the gluconeogenesis pathway. These results suggest that S. insularis-associated microorganisms help to overcome host pinoresinol defense and possibly contribute to insects or gut microbial nutrition via carbohydrate synthesis.

Hypoxia-induced tracheal elasticity in vector beetle facilitates the loading of pinewood nematode.

Many pathogens rely on their insect vectors for transmission. Such pathogens are under selection to improve vector competence for their transmission by employing various tissue or cellular responses of vectors. However, whether pathogens can actively cause hypoxia in vectors and exploit hypoxia responses to promote their vector competence is still unknown. Fast dispersal of pinewood nematode (PWN), the causal agent for the destructive pine wilt disease and subsequent infection of pine trees, is characterized by the high vector competence of pine sawyer beetles (Monochamus spp.), and a single beetle can harbor over 200,000 PWNs in its tracheal system. Here, we demonstrate that PWN loading activates hypoxia in tracheal system of the vector beetles. Both PWN loading and hypoxia enhanced tracheal elasticity and thickened the apical extracellular matrix (aECM) of the tracheal tubes while a notable upregulated expression of a resilin-like mucin protein Muc91C was observed at the aECM layer of PWN-loaded and hypoxic tracheal tubes. RNAi knockdown of Muc91C reduced tracheal elasticity and aECM thickness under hypoxia conditions and thus decreasing PWN loading. Our study suggests a crucial role of hypoxia-induced developmental responses in shaping vector tolerance to the pathogen and provides clues for potential molecular targets to control pathogen dissemination.

Various parasites, bacteria and other disease-causing pathogens are transmitted by insects. A tiny worm called the pine wood nematode, for example, is spread by pine sawyer beetles which can carry up to 280,000 worms in their trachea, the network of tubes they use to breathe. This has resulted in millions of hectares of pine forests in Asia and Europe becoming infected with the deadly disease caused by the nematodes. Pine wood nematodes, as well as other pathogens, can exploit the biological processes of the insects carrying them to make the insects transmit them more effectively. Precisely how nematodes and other disease-causing agents do this is unclear. One possibility is that they reduce the amount of oxygen being supplied to the trachea ­ a phenomenon known as hypoxia ­ which occurs naturally at specific stages in the life of an insect, and during infections. To test this theory, Tang, Zhou, Koski et al. used genetics and imaging approaches to study how pine wood nematodes affect the trachea of pine sawyer beetles. The experiments found that when the nematodes infected the beetles, their trachea did indeed develop hypoxia. This, in turn, made the beetles' airways more elastic and made the layer of structure lining the trachea, known as the apical extracellular matrix, thicker. These changes increased the amount of pinewood nematodes the trachea could hold, allowing the beetle to spread more worms from tree to tree. Further experiments revealed that hypoxia in the trachea increased the levels of a protein called Muc91C in the apical extracellular matrix. When the levels of Muc91C were artificially decreased in the beetles, this made their airways less elastic and the apical extracellular matrix thinner. This work suggests that pine wood nematodes exploit the beetles' normal responses to loss of oxygen supply to make the beetles more effective at transmitting the nematodes between pine trees. Other pathogens carried by insects may also use this strategy to help increase their transmission. Further studies on the Muc91C protein may provide clues for potential drug targets to control pine wood nematodes and protect pine trees from disease.

The effect of inactivation of aldehyde dehydrogenase on pheromone production by a gut bacterium of an invasive bark beetle, Dendroctonus valens.

Semiochemical-based management strategies are important for controlling bark beetles, such as invasive Red Turpentine Beetle (Denroctonus valens), the causal agent for mass mortality of pine trees (Pinus spp.) in China. It has been previously shown that the pheromone verbenone regulates the attack density of this beetle in a dose-dependent manner and that the gut bacteria of D. valens are involved in verbenone production. However, molecular functional verification of the role of gut bacteria in the pheromone production of D. valens is still lacking. To better understand the molecular function of gut bacterial verbenone production, we chose a facultative anaerobic gut bacterium (Enterobacter xiangfangensis) of D. valens based on its strong ability to convert cis-verbenol to verbenone, as shown in our previous study, and investigated its transcriptomics in the presence or absence of cis-verbenol under anaerobic conditions (simulating the anoxic environment in the beetle's gut). Based on this transcriptome analysis, aldehyde dehydrogenase (ALDH1) was identified as a putative key gene responsible for verbenone production and was knocked-down by homologous recombination to obtain a mutant E. xiangfangensis strain. Our results show that these mutants had significantly decreased the ability to convert the monoterpene precursor to verbenone compared with the wild-type bacteria, indicating that ALDH1 is primarily responsible for verbenone conversion for this bacterium species. These findings provide further mechanistic evidence of bacterially mediated pheromone production by D. valens, add new perspective for functional studies of gut bacteria in general, and may aid the development of new gene silencing-based pest management strategies.

Invasion History of the Pinewood Nematode Bursaphelenchus xylophilus Influences the Abundance of Serratia sp. in Pupal Chambers and Tracheae of Insect-Vector Monochamus alternatus.

Pine wilt disease (PWD) has caused extensive mortality in pine forests worldwide. This disease is a result of a multi-species interaction among an invasive pinewood nematode (PWN) Bursaphelenchus xylophilus, its vector Monochamus sp. beetle, and the host pine tree (Pinus sp.). In other systems, microbes have been shown to attenuate negative impacts on invasive species after the invasion has reached a certain time point. Despite that the role of PWD associated microbes involved in the PWD system has been widely studied, it is not known whether similar antagonistic "hidden microbial players" exist in this system due to the lack of knowledge about the potential temporal changes in the composition of associated microbiota. In this study, we investigated the bacteria-to-fungi ratio and isolated culturable bacterial isolates from pupal chambers and vector beetle tracheae across five sampling sites in China differing in the duration of PWN invasion. We also tested the pathogenicity of two candidate bacteria strains against the PWN-vector beetle complex. A total of 118 bacterial species belonging to 4 phyla, 30 families, and 54 genera were classified based on 16S sequencing. The relative abundance of the genus Serratia was lower in pupal chambers and tracheae in newly PWN invaded sites (<10 years) compared to the sites that had been invaded for more than 20 years. Serratia marcescens strain AHPC29 was widely distributed across all sites and showed nematicidal activity against PWN. The insecticidal activity of this strain was dependent on the life stage of the vector beetle Monochamus alternatus: no insecticidal activity was observed against final-instar larvae, whereas S. marcescens was highly virulent against pupae. Our findings improved the understanding of the temporal variation in the microbial community associated with the PWN-vector beetle complex and the progress of PWD and can therefore facilitate the development of biological control agents against PWN and its vector beetle.

Microhabitat Governs the Microbiota of the Pinewood Nematode and Its Vector Beetle: Implication for the Prevalence of Pine Wilt Disease.

Our understanding of environmental acquisition of microbes and migration-related alteration of microbiota across habitats has rapidly increased. However, in complex life cycles, such as for many parasites, exactly how these microbes are transmitted across multiple environments, such as hosts and habitats, is unknown. Pinewood nematode, the causal agent of the globally devastating pine wilt disease, provides an ideal model to study the role of microbiota in multispecies interactions because its successful host invasion depends on the interactions among its vector insects, pine hosts, and associated microbes. Here, we studied the role of bacterial and fungal communities involved in the nematode's life cycle across different micro- (pupal chamber, vector beetle, and dispersal nematodes) and macrohabitats (geographical locations). We identified the potential sources, selection processes, and keystone taxa involved in the host pine-nematode-vector beetle microbiota interactions. Nearly 50% of the microbiota in vector beetle tracheae and ~60% that of third-stage dispersal juveniles were derived from the host pine (pupal chambers), whereas 90% of bacteria of fourth-stage dispersal juveniles originated from vector beetle tracheae. Our results also suggest that vector beetles' tracheae selectively acquire some key taxa from the microbial community of the pupal chambers. These taxa will be then enriched in the dispersal nematodes traveling in the tracheae and hence likely transported to new host trees. Taken together, our findings contribute to the critical information toward a better understanding of the role of microbiota in pine wilt disease, therefore aiding the knowledge for the development of future biological control agents. IMPORTANCE Our understanding of animal microbiota acquisition and dispersal-mediated variation has rapidly increased. In this study, using the model of host pine-pinewood nematode-vector beetle (Monochamus sp.) complex, we disentangled the routes of microbial community assembly and transmission mechanisms among these different participants responsible for highly destructive pine wilt disease. We provide evidence that the microhabitat is the driving force shaping the microbial community of these participants. The microbiota of third-stage dispersal juveniles (LIII) of the nematodes collected around pupal chambers and of vector beetles were mainly derived from the host pine (pupal chambers), whereas the vector-entering fourth-stage dispersal juveniles (LIV) of the nematodes had the simplest microbiota community, not influencing vector's microbiota. These findings enhanced our understanding of the variation in the microbiota of plants and animals and shed light on microbiota acquisition in complex life cycles.

'Resistance Mixtures' Reduce Insect Herbivory in Strawberry (Fragaria vesca) Plantations.

The transition toward more sustainable plant protection with reduced pesticide use is difficult, because there is no "silver bullet" available among nonchemical tools. Integrating several plant protection approaches may thus be needed for efficient pest management. Recently, increasing the genetic diversity of plantations via cultivar mixing has been proposed as a possible method to reduce pest damage. However, previous studies have not addressed either the relative efficiency of exploiting cultivar mixing and intrinsic plant herbivore resistance or the potential utility of combining these approaches to increase cropping security. Here, using a full factorial experiment with 60 woodland strawberry plots, we tested for the relative and combined effect of cultivar mixing and intrinsic plant resistance on herbivore damage and yield. The experiment comprised two levels of diversity ("high" with 10 varieties and "low" with two varieties) and three levels of resistance ("resistant" comprising only varieties intrinsically resistant against strawberry leaf beetle Galerucella tenella; "susceptible" with susceptible varieties only; and "resistance mixtures" with 50:50 mixtures of resistant and susceptible varieties). The experiment was carried out over two growing seasons. Use of resistant varieties either alone or intermixed with susceptible varieties in "resistance mixtures" reduced insect herbivory. Interestingly, resistant varieties not only reduced the mean damage in "resistance mixtures" by themselves being less damaged, but also protected intermixed susceptible varieties via associational resistance. The effect of higher genetic diversity was less evident, reducing herbivory only at the highest level of herbivore damage. In general, herbivory was lowest in plots with high diversity that included at least some resistant varieties and highest in low diversity plots consisting only of susceptible varieties. Despite this, no significant difference in yield (fruit biomass) was found, indicating that strawberry may be relatively tolerant. Our results demonstrate that combined use of high genetic diversity and resistant varieties can help reduce pest damage and provide a useful tool for sustainable food production. "Resistance mixtures" may be particularly useful for sensitive food crops where susceptible varieties are high yielding that could not be completely replaced by resistant ones.

Differences in incubation behaviour and niche separation of two competing flycatcher species.

ABSTRACT: Food availability sets the stage for incubation behaviour of a female bird and thereby indirectly determines the nest temperature, which in turn affects development and metabolism of avian embryos. Changes in development and metabolism in turn are known to influence offspring's ability to adjust to environmental changes later in life. However, few studies have investigated the role of interspecific differences in incubation behaviour in relation to niche separation between competing sibling species. We studied the effects of habitat quality (in terms of caterpillar availability) on incubation behaviour of two ecologically similar and closely related species, collared and pied flycatchers (Ficedula albicollis and F. hypoleuca), in their hybrid zone on the island of Öland, Sweden. Even though both species prefer caterpillar-rich deciduous forests as nesting sites, collared flycatchers, whose nestlings have higher energetic demands, are able to nest only in deciduous forests, whereas pied flycatchers have more flexible habitat requirements. Overall, higher food availability was associated with increased nest attendance, higher incubation temperature and a lower number of foraging trips across species. In addition, collared flycatchers had more frequent and shorter foraging trips across habitat types, allocated more heat to eggs and therefore maintained higher nest temperatures compared to pied flycatchers. We argue that the higher heat allocation or the need to maintain a higher nest temperature for embryo development may constrain collared flycatchers to focus on relatively more profitable prey. Our results highlight the importance of considering incubation behaviour in the context of understanding species differences in niche use. SIGNIFICANCE STATEMENT: Niche separation plays an important role in mitigating effects of competition between closely related species. Whether species differences in incubation behaviour relate to differences in niche use remains unknown. We compared incubation behaviour of two sympatric flycatcher species that differ in sensitivity to food availability. The competitively more dominant and larger species, the collared flycatcher, whose nestlings are more sensitive to food shortages, made more frequent foraging trips but allocated more heat to eggs, leading to higher nest temperature despite lower nest attendance, compared to pied flycatchers. These interspecific differences may be a result of differences in embryo sensitivity or female physiology and contribute to the niche separation between the species, which in turn can facilitate coexistence.

Effects of Insect Herbivory on Bilberry Production and Removal of Berries by Frugivores.

The evolutionary purpose of a fleshy fruit is to attract seed dispersers and get the seeds dispersed by frugivorous animals. For this reason, fruits should be highly rewarding to these mutualists. However, insect herbivory can alter plant reproductive success e.g. by decreasing fruit yield or affecting the attractiveness of the fruits to mutualistic seed dispersers. Under natural conditions, we tested the effects of experimental larval-defoliation on berry ripening and consumption of a non-cultivated dwarf shrub, the bilberry (Vaccinium myrtillus L.), which produces animal-dispersed berries with high sugar and anthocyanin concentration. Bilberry ramets with high fruit yield were most likely to have their berries foraged, indicating that frugivores made foraging choices based on the abundance of berries. Moreover, the probability for berries being foraged was the lowest for non-defoliated ramets that grew adjacent to larval-defoliated ramets, even though larval-defoliation did not affect the biochemical composition (total concentrations of anthocyanins, sugars and organic acids) or the probability of ripening of berries. We hypothesise that the lower probability for berries being foraged in these ramets may be a consequence of rhizome- or volatile-mediated communication between ramets, resulting in a priming effect of the herbivore defence and lower attractiveness of the non-defoliated ramets.

A role for volatiles in intra- and inter-plant interactions in birch.

One of the first observations that plants might utilize cues released by damaged neighbors under natural conditions was made in birch forests in 1985. However, the mechanisms underlying the observations were not determined, and birch (Betula spp.) has been neglected as a study system for inter-plant interaction ever since. Volatiles released by vegetative plant parts in response to herbivore damage play important roles as signals in plant-to-plant interactions in a range of woody and herbaceous plant species, and also have been shown to mediate signaling between branches of the same plant that have limited vascular connection. We established greenhouse experiments to assess: 1) whether exposure to plant volatiles from herbivore-damaged birches primes defense responses in undamaged neighbors; and 2) whether defenses also are primed in undamaged parts of the same plants with limited vascular connection. We observed a priming of defense responses, which were manifested in an augmented emission of terpenes and aromatic compounds in undamaged conspecific neighbors, and also an augmented emission of green leaf volatiles in systemic branches. Our work provides strong evidence of inter-plant signaling by volatiles, and an intra-plant systemic response in birch. However, the responses are specific, with emissions of different groups of plant volatiles typifying the primed response. This work complements and extends the previous work conducted with a natural population of birches.