Does Host Plant Drive Variation in Microbial Gut Communities in a Recently Shifted Pest?

Biotic interactions can modulate the responses of organisms to environmental stresses, including diet changes. Gut microbes have substantial effects on diverse ecological and evolutionary traits of their hosts, and microbial communities can be highly dynamic within and between individuals in space and time. Modulations of the gut microbiome composition and their potential role in the success of a species to maintain itself in a new environment have been poorly studied to date. Here we examine this question in a large wood-boring beetle Cacosceles newmannii (Cerambycidae), that was recently found thriving on a newly colonized host plant. Using 16S metabarcoding, we assessed the gut bacterial community composition of larvae collected in an infested field and in "common garden" conditions, fed under laboratory-controlled conditions on four either suspected or known hosts (sugarcane, tea tree, wattle, and eucalyptus). We analysed microbiome variation (i.e. diversity and differentiation), measured fitness-related larval growth, and studied host plant lignin and cellulose contents, since their degradation is especially challenging for wood-boring insects. We show that sugarcane seems to be a much more favourable host for larval growth. Bacterial diversity level was the highest in field-collected larvae, whereas lab-reared larvae fed on sugarcane showed a relatively low level of diversity but very specific bacterial variants. Bacterial communities were mainly dominated by Proteobacteria, but were significantly different between sugarcane-fed lab-reared larvae and any other hosts or field-collected larvae. We identified changes in the gut microbiome associated with different hosts over a short time frame, which support the hypothesis of a role of the microbiome in host switches.

Genome-scale phylogeography resolves the native population structure of the Asian longhorned beetle, Anoplophora glabripennis (Motschulsky).

Human-assisted movement has allowed the Asian longhorned beetle (ALB, Anoplophora glabripennis (Motschulsky)) to spread beyond its native range and become a globally regulated invasive pest. Within its native range of China and the Korean peninsula, human-mediated dispersal has also caused cryptic translocation of insects, resulting in population structure complexity. Previous studies used genetic methods to detangle this complexity but were unable to clearly delimit native populations which is needed to develop downstream biosurveillance tools. We used genome-wide markers to define historical population structure in native ALB populations and contemporary movement between regions. We used genotyping-by-sequencing to generate 6102 single-nucleotide polymorphisms (SNPs) and amplicon sequencing to genotype 53 microsatellites. In total, we genotyped 712 individuals from ALB's native distribution. We observed six distinct population clusters among native ALB populations, with a clear delineation between northern and southern groups. Most of the individuals from South Korea were distinct from populations in China. Our results also indicate historical divergence among populations and suggest limited large-scale admixture, but we did identify a restricted number of cases of contemporary movement between regions. We identified SNPs under selection and describe a clinal allele frequency pattern in a missense variant associated with glycerol kinase, an important enzyme in the utilization of an insect cryoprotectant. We further demonstrate that small numbers of SNPs can assign individuals to geographic regions with high probability, paving the way for novel ALB biosurveillance tools.

DNA barcoding for bio-surveillance of emerging pests and species identification in Afrotropical Prioninae (Coleoptera, Cerambycidae).

DNA barcoding has been succesfully used for bio-surveillance of forest and agricultural pests in temperate areas, but has few applications in the tropics and particulary in Africa. Cacosceles newmannii (Coleoptera: Cerambycidae) is a Prioninae species that is locally causing extensive damage in commercially-grown sugarcane in the KwaZulu-Natal Province in South Africa. Due to the risk of spread of this species to the rest of southern Africa and to other sugarcane growing regions, clear and easy identification of this pest is critical for monitoring and for phytosanitary services. The genus Cacosceles Newman, 1838 includes four species, most being very similar in morphology. The damaging stage of the species is the larva, which is inherently difficult to distinguish morphologically from other Cerambycidae species. A tool for rapid and reliable identification of this species was needed by plant protection and quarantine agencies to monitor its potential abundance and spread. Here, we provide newly-generated barcodes for C. newmannii that can be used to reliably identify any life stage, even by non-trained taxonomists. In addition, we compiled a curated DNA barcoding reference library for 70 specimens of 20 named species of Afrotropical Prioninae to evaluate DNA barcoding as a valid tool to identify them. We also assessed the level of deeply conspecific mitochondrial lineages. Sequences were assigned to 42 different Barcode Index Numbers (BINs), 28 of which were new to BOLD. Out of the 20 named species barcoded, 11 (52.4%) had their own unique Barcode Index Number (BIN). Eight species (38.1%) showed multiple BINs with no morphological differentiation. Amongst them, C. newmannii showed two highly divergent genetic clusters which co-occur sympatrically, but further investigation is required to test whether they could represent new cryptic species.

Using µCT in live larvae of a large wood-boring beetle to study tracheal oxygen supply during development.

How respiratory structures vary with, or are constrained by, an animal's environment is of central importance to diverse evolutionary and comparative physiology hypotheses. To date, quantifying insect respiratory structures and their variation has remained challenging due to their microscopic size, hence only a handful of species have been examined. Several methods for imaging insect respiratory systems are available, in many cases however, the analytical process is lethal, destructive, time consuming and labour intensive. Here, we explore and test a different approach to measuring tracheal volume using X-ray micro-tomography (µCT) scanning (at 15 µm resolution) on living, sedated larvae of the cerambycid beetle Cacosceles newmannii across a range of body sizes at two points in development. We provide novel data on resistance of the larvae to the radiation dose absorbed during µCT scanning, repeatability of imaging analyses both within and between time-points and, structural tracheal trait differences provided by different image segmentation methods. By comparing how tracheal dimension (reflecting metabolic supply) and basal metabolic rate (reflecting metabolic demand) increase with mass, we show that tracheal oxygen supply capacity increases during development at a comparable, or even higher rate than metabolic demand. Given that abundant gas delivery capacity in the insect respiratory system may be costly (due to e.g. oxygen toxicity or space restrictions), there are probably balancing factors requiring such a capacity that are not linked to direct tissue oxygen demand and that have not been thoroughly elucidated to date, including CO2 efflux. Our study provides methodological insights and novel biological data on key issues in rapidly quantifying insect respiratory anatomy on live insects.

X-ray micro-tomographic data of live larvae of the beetle Cacosceles newmannii.

Quantifying insect respiratory structures and their variation has remained challenging due to their microscopic size. Here we measure insect tracheal volume using X-ray micro-tomography (µCT) scanning (at 15 µm resolution) on living, sedated larvae of the cerambycid beetle Cacosceles newmannii across a range of body sizes. In this paper we provide the full volumetric data and 3D models for 12 scans, providing novel data on repeatability of imaging analyses and structural tracheal trait differences provided by different image segmentation methods. The volume data is provided here with segmented tracheal regions as 3D models.

Metabolic responses to starvation and feeding contribute to the invasiveness of an emerging pest insect.

Metabolic rate, and the flexibility thereof, is a complex trait involving several inter-linked variables that can influence animal energetics, behavior, and ultimately, fitness. Metabolic traits respond readily to ambient temperature variation, in some cases increasing relative or absolute energetic costs, while in other cases, depending on the organism's metabolic and behavioral responses to changing conditions, resulting in substantial energy savings. To gain insight into the rapid recent emergence of the indigenous South African longhorn beetle Cacosceles newmannii as a crop pest in sugarcane, a better understanding of its metabolic rate, feeding response, digestion times, and aerobic scope is required, in conjunction with any behavioral responses to food availability or limitation thereof. Here, we therefore experimentally determined metabolic rate, estimated indirectly as CO2 production using flow-through respirometry, in starved, fasted, and fed C. newmannii larvae, at 20 °C and 30 °C. We estimated multiple parameters of metabolic rate (starved, standard, active, and maximum metabolic rates) as well as aerobic scope (AS), specific dynamic action (SDA), and the percentage time active during respirometry trials. Additionally, in individuals that showed cyclic or discontinuous gas exchange patterns, we compared rate, volume, and duration of cycles, and how these were influenced by temperature. Standard and active metabolic rate, and AS and SDA were significantly higher in the larvae measured at 30 °C than those measured at 20 °C. By contrast, starved and maximum metabolic rates and percentage time active were unaffected by temperature. At rest and after digestion was complete, 35% of larvae showed cyclic gas exchange at both temperatures; 5% and 15% showed continuous gas exchange at 20 °C and 30 °C respectively, and 10% and 0% showed discontinuous gas exchange at 20 °C and 30 °C respectively. We propose that the ability of C. newmannii larvae to survive extended periods of resource limitation, combined with a rapid ability to process food upon securing resources, even at cooler conditions that would normally suppress digestion in tropical insects, may have contributed to their ability to feed on diverse low energy resources typical of their host plants, and become pests of, and thrive on, a high energy host plant like sugarcane.

The Effect of Oxygen Limitation on a Xylophagous Insect's Heat Tolerance Is Influenced by Life-Stage Through Variation in Aerobic Scope and Respiratory Anatomy.

Temperature has a profound impact on insect fitness and performance via metabolic, enzymatic or chemical reaction rate effects. However, oxygen availability can interact with these thermal responses in complex and often poorly understood ways, especially in hypoxia-adapted species. Here we test the hypothesis that thermal limits are reduced under low oxygen availability - such as might happen when key life-stages reside within plants - but also extend this test to attempt to explain that the magnitude of the effect of hypoxia depends on variation in key respiration-related parameters such as aerobic scope and respiratory morphology. Using two life-stages of a xylophagous cerambycid beetle, Cacosceles (Zelogenes) newmannii we assessed oxygen-limitation effects on metabolic performance and thermal limits. We complement these physiological assessments with high-resolution 3D (micro-computed tomography scan) morphometry in both life-stages. Results showed that although larvae and adults have similar critical thermal maxima (CTmax) under normoxia, hypoxia reduces metabolic rate in adults to a greater extent than it does in larvae, thus reducing aerobic scope in the former far more markedly. In separate experiments, we also show that adults defend a tracheal oxygen (critical) setpoint more consistently than do larvae, indicated by switching between discontinuous gas exchange cycles (DGC) and continuous respiratory patterns under experimentally manipulated oxygen levels. These effects can be explained by the fact that the volume of respiratory anatomy is positively correlated with body mass in adults but is apparently size-invariant in larvae. Thus, the two life-stages of C. newmannii display key differences in respiratory structure and function that can explain the magnitude of the effect of hypoxia on upper thermal limits.

Fine-scale invasion genetics of the quarantine pest, Anoplophora glabripennis, reconstructed in single outbreaks.

The xylophagous cerambycid Anoplophora glabripennis, the Asian long-horned beetle (ALB), is highly polyphagous and can colonize a wide range of broadleaved host trees causing significant economic damage. For this reason, it is considered a quarantine pest in Europe and North America. Although the global spread of ALB has been depicted recently, no comprehensive studies exist on the genetic pattern of populations' establishment and dynamics at fine-scale (i.e. within invasive outbreaks), before eradication measures are applied. This information may, however, be particularly important for an efficient management and control of invasive pests. Here, we characterized population genetic diversity and patterns of spread of ALB within and among the four outbreaks detected in Switzerland between 2011 and 2015. For this, we genotyped 223 specimens at 15 nuclear microsatellite loci and conducted specific population-based analyses. Our study shows: (1) At least three independent introductions and a, human-mediated, secondary dispersal event leading to the four outbreaks in the country; (2) An overall low intra-population genetic diversity in the viable and several years active invasive populations; (3) A colonization of single trees by homogeneous ALB genotypes; And (4) an establishment of populations several generations prior to its official discovery.

First Screening of Entomopathogenic Nematodes and Fungus as Biocontrol Agents against an Emerging Pest of Sugarcane, Cacosceles newmannii (Coleoptera: Cerambycidae).

Cacosceles newmannii (Coleoptera: Cerambycidae) is an emerging pest of sugarcane in South Africa. The larvae of this cerambycid beetle live within the sugarcane stalk and drill galleries that considerably reduce sugar production. To provide an alternative to chemical control, entomopathogenic nematodes and fungus were investigated as potential biological control agents to be used in an integrated pest management system. The nematodes Steinernema yirgalemense, S. jeffreyense, Heterorhabditis indica, and different concentrations of the fungus Metarhizium pinghaense were screened for efficacy (i.e., mortality rate) against larvae of C. newmannii. The different biocontrol agents used, revealed a low level of pathogenicity to C. newmannii larvae, when compared to control treatments.

Oxygen limitation is not the cause of death during lethal heat exposure in an insect.

Oxygen- and capacity-limited thermal tolerance (OCLTT) is a controversial hypothesis claiming to explain variation in, and mechanistically determine, animal thermal limits. The lack of support from Insecta is typically argued to be a consequence of their high-performance respiratory systems. However, no studies have reported internal body oxygen levels during thermal ramping so it is unclear if changes in ambient gas are partially or fully offset by a compensatory respiratory system. Here we provide such an assessment by simultaneously recording haemolymph oxygen (pO2) levels-as an approximation of tissue oxygenation-while experimentally manipulating ambient oxygen and subjecting organisms to thermal extremes in a series of thermolimit respirometry experiments using pupae of the butterfly Pieris napi. The main results are that while P. napi undergo large changes in haemolymph pO2 that are positively correlated with experimental oxygen levels, haemolymph pO2 is similar pre- and post-death during thermal assays. OCLTT predicts that reduction in body oxygen level should lead to a reduction in CTmax. Despite finding the former, there was no change in CTmax across a wide range of body oxygen levels. Thus, we argue that oxygen availability is not a functional determinant of the upper thermal limits in pupae of P. napi.

Deciphering the worldwide invasion of the Asian long-horned beetle: A recurrent invasion process from the native area together with a bridgehead effect.

Retracing introduction routes is crucial for understanding the evolutionary processes involved in an invasion, as well as for highlighting the invasion history of a species at the global scale. The Asian long-horned beetle (ALB) Anoplophora glabripennis is a xylophagous pest native to Asia and invasive in North America and Europe. It is responsible for severe losses of urban trees, in both its native and invaded ranges. Based on historical and genetic data, several hypotheses have been formulated concerning its invasion history, including the possibility of multiple introductions from the native zone and secondary dispersal within the invaded areas, but none have been formally tested. In this study, we characterized the genetic structure of ALB in both its native and invaded ranges using microsatellites. In order to test different invasion scenarios, we used an approximate Bayesian "random forest" algorithm together with traditional population genetics approaches. The strong population differentiation observed in the native area was not geographically structured, suggesting complex migration events that were probably human-mediated. Both native and invasive populations had low genetic diversity, but this characteristic did not prevent the success of the ALB invasions. Our results highlight the complexity of invasion pathways for insect pests. Specifically, our findings indicate that invasive species might be repeatedly introduced from their native range, and they emphasize the importance of multiple, human-mediated introductions in successful invasions. Finally, our results demonstrate that invasive species can spread across continents following a bridgehead path, in which an invasive population may have acted as a source for another invasion.

Uncovering the benefits of fluctuating thermal regimes on cold tolerance of drosophila flies by combined metabolomic and lipidomic approach.

When exposed to constant low temperatures (CLTs), insects often suffer from cumulative physiological injuries that can severely compromise their fitness and survival. Yet, mortality can be considerably lowered when the cold stress period is interrupted by periodic warm interruption(s), referred to as fluctuating thermal regimes, FTRs. In this study, we have shown that FTRs strongly promoted cold tolerance of Drosophila melanogaster adults. We then assessed whether this marked phenotypic shift was associated with detectable physiological changes, such as synthesis of cryoprotectants and/or membrane remodeling. To test these hypotheses, we conducted two different time-series Omics analyzes in adult flies submitted to CLTs vs. FTRs: metabolomics (GC/MS) and lipidomics (LC/ESI/MS) targeting membrane phospholipids. We observed increasing levels in several polyhydric alcohols (arabitol, erythritol, sorbitol, mannitol, glycerol), sugars (fructose, mannose) and amino acids (serine, alanine, glutamine) in flies under CLT. Prolonged exposure to low temperature was also associated with a marked deviation of metabolic homeostasis and warm interruptions as short as 2h were sufficient to periodically return the metabolic system to functionality. Lipidomics revealed an increased relative proportion of phosphatidylethanolamines and a shortening of fatty acyl chains in flies exposed to cold, likely to compensate for the ordering effect of low temperature on membranes. We found a remarkable correspondence in the time-course of changes between the metabolic and phospholipids networks, both suggesting a fast homeostatic regeneration during warm intervals under FTRs. In consequence, we suggest that periodic opportunities to restore system-wide homeostasis contribute to promote cold tolerance under FTRs.