Beetle Host Research Articles

Circadian and seasonal flight activity differences between the sexes of the biocontrol agent Eadya daenerys (Hymenoptera: Braconidae) and the impact of host size on adult emergence

AbstractSince its accidental introduction into New Zealand in 1916, the invasive eucalypt leaf beetle Paropsis charybdis remains a pest of economic significance to Eucalyptus forestry. For this reason, the braconid parasitoid Eadya daenerys has been approved for release as a classical biological control agent. To aid in field monitoring using hand netting of biocontrol releases and laboratory rearing protocols, the flight activity over 6 a year field research program of E. daenerys and the relationships between the parasitoid and the size of its beetle hosts were evaluated in the country of origin. Wasps were observed flying over 32 days from mid‐November to mid‐December in Tasmania. E. daenerys was found to exhibit a morning‐based scramble competition mating system. Females flying in the early morning had lower mature egg loads (134.2 ± 27.6) than at other times of the day. In the first half of the season, male flight was most often seen in the early morning, whereas in the second half of the season, there was a 4.5‐fold decrease in any wasps flying. Female flight activity significantly increased to 70% female in the evening hours from 5:00 pm to 8:00 pm, which by the second half of the season was almost exclusively female. Wasps were caught flying across a wide range of air temperatures (10.7–23.8°C), humidity and light levels, but even light winds reduced flight when average wind speed exceeded 0.27 m/s for males and 0.73 m/s for females. Beetle prepupal weight was a predictor of E. daenerys parasitism success with mean beetle prepupal weights significantly higher for stung but unsuccessfully parasitised larva (62.09 mg) than those successfully parasitised (52.94 mg). The chance of an emerged wasp larva spinning a pupal cocoon was found to increase by 5% with every 1‐mg increase in its beetle prepupal weight. Heavier beetle prepupae produced bigger parasitoids. Field‐collected adult E. daenerys had larger (12%) head capsule widths (mean of 1.42 mm) than those reared in the laboratory (mean of 1.27 mm), suggesting that improving host nutrition and laboratory rearing conditions for increasing host size may help optimise E. daenerys rearing success.

Comparing the succession of microbial communities throughout development in field and laboratory nests of the ambrosia beetle Xyleborinus saxesenii.

Some fungus-farming ambrosia beetles rely on multiple nutritional cultivars (Ascomycota: Ophiostomatales and/or yeasts) that seem to change in relative abundance over time. The succession of these fungi could benefit beetle hosts by optimal consumption of the substrate and extended longevity of the nest. However, abundances of fungal cultivars and other symbionts are poorly known and their culture-independent quantification over development has been studied in only a single species. Here, for the first time, we compared the diversity and succession of both fungal and bacterial communities of fungus gardens in the fruit-tree pinhole borer, Xyleborinus saxesenii, from field and laboratory nests over time. By amplicon sequencing of probed fungus gardens of both nest types at three development phases we showed an extreme reduction of diversity in both bacterial and fungal symbionts in laboratory nests. Furthermore, we observed a general transition from nutritional to non-beneficial fungal symbionts during beetle development. While one known nutritional mutualist, Raffaelea canadensis, was occurring more or less stable over time, the second mutualist R. sulphurea was dominating young nests and decreased in abundance at the expense of other secondary fungi. The quicker the succession proceeded, the slower offspring beetles developed, suggesting a negative role of these secondary symbionts. Finally, we found signs of transgenerational costs of late dispersal for daughters, possibly as early dispersers transmitted and started their own nests with less of the non-beneficial taxa. Future studies should focus on the functional roles of the few bacterial taxa that were present in both field and laboratory nests.

Phoretic mite assemblage of the pinyon pine beetle, Ips confusus (Curculionidae: Scolytinae), in Arizona

Mites are among the most common associates of bark beetles and they can influence the ecology and microbial composition within bark beetle-colonized trees. The pinyon pine engraver, Ips confusus is a common beetle in pinyon trees in the southwestern United States, but the mite composition associated with this beetle has been little studied. In this study, we quantify the abundance, diversity, and attachment locations of phoretic mites on Ips confusus that emerge from naturally infested trees. In total, we observed 342 beetles for mites, 95% of which had at least one mite. We collected a total of 5842 mites, representing seven families and seven species: Cercoleipus coelonotus (Cercomegistidae) Dendrolaelaps quadrisetosimilis (Digamasellidae), Ereynetes propescutulis (Ereynetidae), Iponemus confusus confusus (Tarsonemidae), Mexecheles cf. virginiensis (Cheyletidae), Proctolaelaps subcorticalis (Melicharidae), and Trichouropoda californica (Trematuridae). We calculated the average number of mites per beetle to be 18, with a maximum of 147 mites on a single beetle. The vast majority of mites (98% of total abundance) was represented by three species (I. c. confusus, D. quadrisetosimilis, and T. californica). Attachment locations on the beetle varied across mite species, with mite species exhibiting a preference for specific locations. Mite abundances on emerging host beetles varied over time, with some species occurring mostly on early-emerging beetles, while others occurred on later emerging beetles.

Strong effects of food quality on host life history do not scale to impact parasitoid efficacy or life history

Parasitoids are small insects, (e.g., small wasps or flies) that reproduce by laying eggs on or within host arthropods. Parasitoids make up a large proportion of the world’s biodiversity and are popular agents of biological control. Idiobiont parasitoids paralyze their hosts upon attack and thus are expected to only target hosts large enough to support offspring development. Host resources generally impact host attributes and life histories including size, development, and life span. Some argue slow host development in response to resource quality increases parasitoid efficacy (i.e., a parasitoid’s ability to successfully reproduce on or within a host) due to longer host exposure to parasitoids. However, this hypothesis is not always supported and does not consider variation in other host traits in response to resources that may be important for parasitoids (e.g., variation in host size is known to impact parasitoid efficacy). In this study we test whether trait variation within host developmental stages in response to host resources is more important for parasitoid efficacy and life histories than trait variation across host developmental stages. We exposed seed beetle hosts raised on a food quality gradient to mated female parasitoids and measured the number of hosts parasitized and parasitoid life history traits at the scale of host stage- and age-structure. Our results suggest host food quality does not cascade to impact idiobiont parasitoid life histories despite large food quality effects on host life history. Instead, variation in host life histories across host developmental stages better predicts parasitoid efficacy and life histories, suggesting finding a host in a specific instar is more important for idiobiont parasitoids than finding hosts on or within higher quality resources.

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 β-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.

Symbiosis: Partners in crime

Defensive symbionts protect their hosts against imminent threats. A new study uncovers a symbiosis whereby a fungus safeguards its beetle host from predation, but also exploits the beetle as a vector to help it attack plants and cause disease.

Native European ants can discourage host colonization and reduce reproductive success of the invasive ambrosia beetle Xylosandrus compactus

Due to their ubiquity and their nature as generalist predators, ants have long been used as biological control agents in forest and agricultural systems. Several exotic ambrosia beetles (Coleoptera: Curculionidae: Scolytinae, Platypodinae) are considered emerging widespread pests of various trees and shrubs growing in forests, nurseries, orchards, and urban areas. Among them, the Scolytinae Xylosandrus compactus (Eichhoff) is an invasive fungus-farming species native to Asia and able to cause serious damage to a broad range of natural and cultivated plants worldwide, exerting significant ecological and economic costs. Its biology makes conventional control strategies often ineffective, while little is known about natural enemies. We explored the potential of native European predators as natural enemy of this pest, conducting laboratory tests with four widespread ant species using chestnut and laurel as beetle hosts. In particular, we evaluated the interactions between X. compactus and four species of native Euro-Mediterranean ants that usually forage on plants: Crematogaster scutellaris (Olivier), Tapinoma magnum Mayr, Temnothorax affinis (Mayr), and Temnothorax mediterraneus Ward, Brady, Fisher & Schultz. Results indicate that ants may significantly limit the reproductive success of X. compactus, increasing the mortality of the beetle foundresses and reducing their offspring. Smaller ant species may also invade X. compactus nests, killing larvae, pupae and adults, while female beetles avoid nesting in twigs previously visited by ants. These results encourage to explore possible applications of ants in the biological control of X. compactus and the ecological implications of these interactions in the field.

The Coleoptera of the Province of Prince Edward Island, Canada: 295 new records from Lindgren funnel traps and a checklist to species.

The Coleoptera fauna of the province of Prince Edward Island has long been one of the most poorly known jurisdictions in Canada, with fewer than half the number of species recorded in the neighbouring provinces of New Brunswick and Nova Scotia. If much of the difference in species richness was due to less intensive sampling of the province compared to other parts of Atlantic Canada it was predicted that surveys with semiochemical-baited traps would detect many previously undetected species. Lindgren funnel traps were baited with longhorn beetle pheromones and host volatiles and placed in the canopy and understory of coniferous and deciduous trees at the Valleyfield, New Harmony, Auburn, and Brookvale Demonstration Woodlots during the summers of 2018 and 2019. Two hundred and ninety-five species of Coleoptera are newly recorded from Prince Edward Island from 53 families. One of these, the Palaearctic Pityophagusferrugineus (Linnaeus, 1760) is reported for the first time from North America and Canada. The families Lycidae, Derodontidae, Lymexylidae, Sphindidae, Cucujidae, Ripiphoridae, Salpingidae, and Nemonychidae are newly recorded for the province. A checklist of the Coleoptera of Prince Edward Island is provided.

Evolutionary and ecological signals in Wolbachia-beetle relationships: A review

Intracellular bacteria of the genus Wolbachia are the most abundant endosymbionts infecting many arthropods, with Coleoptera being the most diverse hosts in terms of taxonomy and ecology. There has been great progress in studies on the relations between Wolbachia and beetles, however, only some of the research details the consequences of infection. In this review, I summarise the knowledge on the evolutionary relations or ecological associations between Wolbachia and its beetle hosts. These bacteria often cause cytoplasmic incompatibility in the infected hosts and are responsible for a selective sweep of the mitochondrial genomes in some beetles. Wolbachia can manipulate the sex ratio or reproduction of some species of beetles, however, it does not induce parthenogenesis, with the possible rare exception of some Naupactini. Proof of the co-evolution of Wolbachia with beetles is missing, but some aquatic groups seem to be prone to co-speciation, unlike terrestrial taxa. On the other hand, there is a growing number of studies indicating or proving horizontal transmission of Wolbachia among beetle hosts, mostly via common host plants or the foraging substrate (such as dung). Wolbachia is not alone in infecting beetles as other endosymbiotic bacteria occur in beetles (Rickettsia, Spiroplasma, Cardinium, Arsenophorus), which have often been reported as interchangeable, suggesting the infection by various bacteria is dynamic. Nonetheless, there are still many issues associated with Wolbachia that are not yet been described in beetles (like the provision of nutrition or protection against pathogens) and high-throughput sequencing should be used to improve our understanding of Wolbachia-Coleoptera relations.

Diverse Heat Tolerance of the Yeast Symbionts of Platycerus Stag Beetles in Japan.

The genus Platycerus (Coleoptera: Lucanidae) is a small stag beetle group, which is adapted to cool-temperate deciduous broad-leaved forests in East Asia. Ten Platycerus species in Japan form a monophyletic clade endemic to Japan and inhabit species-specific climatic zones. They are reported to have co-evolutionary associations with their yeast symbionts of the genus Sheffersomyces based on host cytochrome oxidase subunit I (COI) and yeast intergenic spacer (IGS) phylogenies. Here we examined the heat tolerances of the yeast colonies isolated from the mycangia of 37 females belonging ten Japanese Platycerus species. The upper limits of growth and survival temperatures of each colony were decided by cultivating it at ten temperature levels between 17.5 and 40°C. Although both temperatures varied during 25.0–31.25°C, the maximum survival temperatures (MSTs) were a little higher than the maximum growth temperatures (MGTs) in 16 colonies. Pearson’s correlations between these temperatures and environmental factors (elevation and 19 bioclimatic variables from Worldclim database) of host beetle collection sites were calculated. These temperatures were significantly correlated with elevation negatively, the maximum temperature of the warmest month (Bio5) positively, and some precipitative variables, especially in the warm season (Bio12, 13, 16, 18) negatively. Sympatric Platycerus kawadai and Platycerus albisomni share the same lineage of yeast symbionts that exhibit the same heat tolerance, but the elevational lower range limit of P. kawadai is higher than that of P. albisomni. Based on the field survey in their sympatric site, the maximum temperature of host wood of P. kawadai larvae is higher about 2–3°C than that of P. albisomni larvae in the summer, which may restrict the elevational range of P. kawadai to higher area. In conclusion, it is suggested that the heat tolerance of yeast symbionts restricts the habitat range of their host Platycerus species or/and that the environmental condition that host Platycerus species prefers affect the heat tolerance of its yeast symbionts.

Validation of reference genes for quantitative PCR in the forest pest, Ips calligraphus

The six-spined ips, Ips calligraphus, is a North American bark beetle that can exploit most eastern North American Pinus species and can cause mortality. Biotic and abiotic disturbances weaken trees, creating breeding substrate that promotes rapid population growth. Management historically relied on silvicultural practices, but as forests become increasingly stressed, innovative management is needed. Manipulation of the cellular RNA interference (RNAi) pathway to induce gene silencing is an emerging means of insect suppression, and is effective for some bark beetles. Quantitative PCR (qPCR) is a powerful tool for analysis of gene expression, and is essential for examining RNAi. To compare gene expression among individuals, stably expressed reference genes must be validated for qPCR. We evaluated six candidate reference genes (18s, 16s, 28s, ef1a, cad, coi) for stability under biotic (beetle sex, developmental stage, and host plant), and abiotic (temperature, photoperiod, and dsRNA exposure) conditions. We used the comprehensive RefFinder tool to compare stability rankings across four algorithms. These algorithms identified 18s, 16s, and 28s as the most stably expressed. Overall, 16s and 28s were selected as reference genes due to their stability and moderate expression levels, and can be used for I. calligraphus gene expression studies using qPCR, including those evaluating RNAi.

Changes in tree drought sensitivity provided early warning signals to the California drought and forest mortality event.

Climate warming in recent decades has negatively impacted forest health in the western United States. Here, we report on potential early warning signals (EWS) for drought-related mortality derived from measurements of tree-ring growth (ring width index; RWI) and carbon isotope discrimination (∆13 C), primarily focused on ponderosa pine (Pinus ponderosa). Sampling was conducted in the southern Sierra Nevada Mountains, near the epicenter of drought severity and mortality associated with the 2012-2015 California drought and concurrent outbreak of western pine beetle (Dendroctonus brevicomis). At this site, we found that widespread mortality was presaged by five decades of increasing sensitivity (i.e., increased explained variation) of both tree growth and ∆13 C to Palmer Drought Severity Index (PDSI). We hypothesized that increasing sensitivity of tree growth and ∆13 C to hydroclimate constitute EWS that indicate an increased likelihood of widespread forest mortality caused by direct and indirect effects of drought. We then tested these EWS in additional ponderosa pine-dominated forests that experienced varying mortality rates associated with the same California drought event. In general, drier sites showed increasing sensitivity of RWI to PDSI over the last century, as well as higher mortality following the California drought event compared to wetter sites. Two sites displayed evidence that thinning or fire events that reduced stand basal area effectively reversed the trend of increasing hydroclimate sensitivity. These comparisons indicate that reducing competition for soil water and/or decreasing bark beetle host tree density via forest management-particularly in drier regions-may buffer these forests against drought stress and associated mortality risk. EWS such as these could provide land managers more time to mitigate the extent or severity of forest mortality in advance of droughts. Substantial efforts at deploying additional dendrochronological research in concert with remote sensing and forest modeling will aid in forecasting of forest responses to continued climate warming.

Cryptic diversity within the Poecilochirus carabi mite species complex phoretic on Nicrophorus burying beetles: Phylogeny, biogeography, and host specificity.

Coevolution is often considered a major driver of speciation, but evidence for this claim is not always found because diversity might be cryptic. When morphological divergence is low, molecular data are needed to uncover diversity. This is often the case in mites, which are known for their extensive and often cryptic diversity. We studied mites of the genus Poecilochirus that are phoretic on burying beetles (Silphidae: Nicrophorus). Poecilochirus taxonomy is poorly understood. Most studies on this genus focus on the evolutionary ecology of Poecilochirus carabi sensu lato, a complex of at least two biological species. Based on molecular data of 230specimens from 43locations worldwide, we identified 24genetic clusters that may represent species. We estimate that these mites began to diversify during the Paleogene, when the clade containing P. subterraneus branched off and the remaining mites diverged into two further clades. One clade resembles P. monospinosus. The other clade contains 17genetic clusters resembling P. carabi s.l.. Among these are P. carabi sensu stricto, P. necrophori, and potentially many additional cryptic species. Our analyses suggest that these clades were formed in the Miocene by large-scale geographic separation; co-speciation of mites with the host beetles can be largely ruled out. Diversification also seems to have happened on a smaller scale, potentially due to adaptation to specific hosts or local abiotic conditions, causing some clusters to specialize on certain beetle species. Our results suggest that biodiversity in this genus was generated by multiple interacting forces shaping the tangled webs of life.

Warming increased bark beetle-induced tree mortality by 30% during an extreme drought in California.

Quantifying the responses of forest disturbances to climate warming is critical to our understanding of carbon cycles and energy balances of the Earth system. The impact of warming on bark beetle outbreaks is complex as multiple drivers of these events may respond differently to warming. Using a novel model of bark beetle biology and host tree interactions, we assessed how contemporary warming affected western pine beetle (Dendroctonus brevicomis) populations and mortality of its host, ponderosa pine (Pinus ponderosa), during an extreme drought in the Sierra Nevada, California, United States. When compared with the field data, our model captured the western pine beetle flight timing and rates of ponderosa pine mortality observed during the drought. In assessing the influence of temperature on western pine beetles, we found that contemporary warming increased the development rate of the western pine beetle and decreased the overwinter mortality rate of western pine beetle larvae leading to increased population growth during periods of lowered tree defense. We attribute a 29.9% (95% CI: 29.4%-30.2%) increase in ponderosa pine mortality during drought directly to increases in western pine beetle voltinism (i.e., associated with increased development rates of western pine beetle) and, to a much lesser extent, reductions in overwintering mortality. These findings, along with other studies, suggest each degree (°C) increase in temperature may have increased the number of ponderosa pine killed by upwards of 35%-40% °C-1 if the effects of compromised tree defenses (15%-20%) and increased western pine beetle populations (20%) are additive. Due to the warming ability to considerably increase mortality through the mechanism of bark beetle populations, models need to consider climate's influence on both host tree stress and the bark beetle population dynamics when determining future levels of tree mortality.

Nematode Interactions on Beetle Hosts Indicate a Role of Mouth-Form Plasticity in Resource Competition

Competition is rampant across kingdoms, arising over potential mates, food resources, and space availability. When faced with opponents, phenotypic plasticity proffers organisms indispensable advantageous strategies to outcompete rivals. This tactic is especially crucial on decaying insect hosts as myriad microbes and numerous nematodes struggle to establish thriving populations and ensure resource availability for future generations. Scarab beetles and their associated nematode symbionts on La Réunion Island have provided exceptional systems to study complicated cross-phylum interactions in soil, and recently we have identified a previously unexplored beetle host, Gymnogaster bupthalma, to be reliably co-infested with diplogastrids Pristionchus mayeri and Acrostichus spp. These nematodes maintain the capacity to plastically respond to environmental conditions by developing disparate mouth forms, a strict bacterial-feeding morph or an omnivorous morph that enables predation on other nematodes. In addition, under stressful settings these worms can enter an arrested development stage called dauer, non-feeding dispersal larvae that resume development into reproducing adults when conditions improve. By investigating this beetle-nematode system in a natural context, we uncovered a novel Pristionchus strategy, wherein dauer dispersal from the carcass is gradual and a reproducing population is sustained. Remarkably, usually preferential-bacterial morph P. mayeri develop as predators in populations dense with competitors.

Hiding among holes: mechanisms underlying the evolution of masquerade in flea beetles (Chrysomelidae)

1. The herbivorous flea beetles (Chrysomelidae: Alticini) have evolved a masquerading strategy by hiding among their own feeding damage. Similarities between beetle bodies and their feeding damage may act as a defence that reduces detection by visual predators.2. We analyse the evolutionary origin of flea beetles' camouflage using the French fauna (284 species) as a case study. We assess two competing hypotheses: (H1) the type of leaf tissue is the primary determinant of the colour and size of beetle species feeding on them, because each type of leaf tissue may have a tendency to result in a particular type of damage or, (H2) the morphological characteristics of the beetle explain the damage pattern, because beetle species evolve strategies to produce feeding damage that matches its own characteristics.3. We assessed bipartite networks of feeding interactions (beetle species and host plants). Beetle‐host interactions were non‐randomly distributed and highly modular, with more than 25% of the network modules showing beetle colour and size distributions significantly different from null expectations.4. Our results suggest that the evolutionary origin of flea beetles masquerading can be partially explained by both hypotheses. Some host plants seem to restrict feeding damage appearance on their leaves, favouring the survival of specific beetles with matching colour and size (H1). However, in most plant taxa, it is suggested, the existence of beetle‐associated constraints exert a selective pressure for the beetle to damage leaves in a particular way, similar to its own colour and size (H2).

Transmission of Bacterial Symbionts With and Without Genome Erosion Between a Beetle Host and the Plant Environment.

Many phytophagous insects harbor symbiotic bacteria that can be transmitted vertically from parents to offspring, or acquired horizontally from unrelated hosts or the environment. In the latter case, plants are a potential route for symbiont transfer and can thus foster a tripartite interaction between microbe, insect, and plant. Here, we focus on two bacterial symbionts of the darkling beetle Lagria villosa that belong to the genus Burkholderia; the culturable strain B. gladioli Lv-StA and the reduced-genome strain Burkholderia Lv-StB. The strains can be transmitted vertically and confer protection to the beetle’s eggs, but Lv-StA can also proliferate in plants, and both symbiont strains have presumably evolved from plant pathogens. Notably, little is known about the role of the environment for the transmission dynamics and the maintenance of the symbionts. Through manipulative assays, we demonstrate the transfer of the symbionts from the beetle to wheat, rice and soybean plants, as well as leaf litter. In addition, we confirm that aposymbiotic larvae can pick up Lv-StA from dry leaves and the symbiont can successfully establish in the beetle’s symbiotic organs. Also, we show that the presence of plants and soil in the environment improves symbiont maintenance. These results indicate that the symbionts of L. villosa beetles are still capable of interacting with plants despite signatures of genome erosion and suggest that a mixed-mode of bacterial transmission is likely key for the persistence of the symbiosis.

Transposon-insertion Sequencing as a Tool to Elucidate Bacterial Colonization Factors in a Burkholderia gladioli Symbiont of Lagria villosa Beetles.

Inferring the function of genes by manipulating their activity is an essential tool for understanding the genetic underpinnings of most biological processes. Advances in molecular microbiology have seen the emergence of diverse mutagenesis techniques for the manipulation of genes. Among them, transposon-insertion sequencing (Tn-seq) is a valuable tool to simultaneously assess the functionality of many candidate genes in an untargeted way. The technique has been key to identifymolecular mechanisms for the colonization of eukaryotic hosts in several pathogenic microbes and a few beneficial symbionts. Here, Tn-seq is established as a method to identify colonization factors in a mutualistic Burkholderia gladioli symbiont of the beetle Lagria villosa. By conjugation, Tn5 transposon-mediated insertion of an antibiotic-resistance cassette iscarried out at random genomic locations in B. gladioli. To identify the effect of gene disruptions on the ability of the bacteria to colonize the beetle host, the generated B. gladioli transposon-mutant library is inoculated on the beetle eggs, while a control is grown in vitro in a liquid culture medium. After allowing sufficient time for colonization, DNA is extracted from the in vivo and in vitro grown libraries. Following a DNA library preparation protocol, the DNA samples are prepared for transposon-insertion sequencing. DNA fragments that contain the transposon-insert edge and flanking bacterial DNA are selected, and the mutation sites are determined by sequencing away from the transposon-insert edge. Finally, by analyzing and comparing the frequencies of each mutant between the in vivo and in vitro libraries, the importance of specific symbiont genes during beetle colonization can be predicted.

Transposon-insertion Sequencing as a Tool to Elucidate Bacterial Colonization Factors in a <em>Burkholderia gladioli</em> Symbiont of <em>Lagria villosa</em> Beetles

Inferring the function of genes by manipulating their activity is an essential tool for understanding the genetic underpinnings of most biological processes. Advances in molecular microbiology have seen the emergence of diverse mutagenesis techniques for the manipulation of genes. Among them, transposon-insertion sequencing (Tn-seq) is a valuable tool to simultaneously assess the functionality of many candidate genes in an untargeted way. The technique has been key to identify molecular mechanisms for the colonization of eukaryotic hosts in several pathogenic microbes and a few beneficial symbionts. Here, Tn-seq is established as a method to identify colonization factors in a mutualistic Burkholderia gladioli symbiont of the beetle Lagria villosa. By conjugation, Tn5 transposon-mediated insertion of an antibiotic-resistance cassette is carried out at random genomic locations in B. gladioli. To identify the effect of gene disruptions on the ability of the bacteria to colonize the beetle host, the generated B. gladioli transposon-mutant library is inoculated on the beetle eggs, while a control is grown in vitro in a liquid culture medium. After allowing sufficient time for colonization, DNA is extracted from the in vivo and in vitro grown libraries. Following a DNA library preparation protocol, the DNA samples are prepared for transposon-insertion sequencing. DNA fragments that contain the transposon-insert edge and flanking bacterial DNA are selected, and the mutation sites are determined by sequencing away from the transposon-insert edge. Finally, by analyzing and comparing the frequencies of each mutant between the in vivo and in vitro libraries, the importance of specific symbiont genes during beetle colonization can be predicted.

Diversity and prevalence of entomopathogenic fungi (Ascomycota, Hypocreales) in epidemic populations of bark beetles (Coleoptera, Scolytinae) in spruce forests of the Tatra National Park in Slovakia

Bark beetles are serious forest pests in Slovakia. Their outbreaks may have significant ecological and economic impacts on spruce forests. There is a variety of natural enemies that activate themselves during population outbreaks of insects and entomopathogenic fungi belong to important antagonists with a potential to regulate populations of their hosts. In 2014–2016, species richness and prevalence of entomopathogenic fungi were evaluated during the bark beetle outbreaks in spruce forests affected by windstorms in the Tatra National Park in Slovakia. Three Beauveria species, B. bassiana, B. caledonica and B. pseudobassiana, with Metapochonia bulbillosa were identified from 271 specimens of three bark beetle species, Ips typographus, Ips amitinus and Pityogenes chalcographus. Beauveria bassiana was the dominant pathogen and infected all three bark beetle species. Phylogenetic analysis identified three phylogenetic groups of B. bassiana in the evaluated host populations. M. bulbillosa was reported for the first time from bark beetle hosts and Slovakia. The prevalence of fungal infection in natural populations of I. typographus was low, varied between 0.07 and 0.72%, and have little influence on the bark beetle abundance.