Climate, host and geography shape insect and fungal communities of trees.

Non-native pests, climate change, and their interactions are likely to alter relationships between trees and tree-associated organisms with consequences for forest health. To understand and predict such changes, factors structuring tree-associated communities need to be determined. Here, we analysed the data consisting of records of insects and fungi collected from dormant twigs from 155 tree species at 51 botanical gardens or arboreta in 32 countries. Generalized dissimilarity models revealed similar relative importance of studied climatic, host-related and geographic factors on differences in tree-associated communities. Mean annual temperature, phylogenetic distance between hosts and geographic distance between locations were the major drivers of dissimilarities. The increasing importance of high temperatures on differences in studied communities indicate that climate change could affect tree-associated organisms directly and indirectly through host range shifts. Insect and fungal communities were more similar between closely related vs. distant hosts suggesting that host range shifts may facilitate the emergence of new pests. Moreover, dissimilarities among tree-associated communities increased with geographic distance indicating that human-mediated transport may serve as a pathway of the introductions of new pests. The results of this study highlight the need to limit the establishment of tree pests and increase the resilience of forest ecosystems to changes in climate.

Worldwide diversity of endophytic fungi and insects associated with dormant tree twigs.

International trade in plants and climate change are two of the main factors causing damaging tree pests (i.e. fungi and insects) to spread into new areas. To mitigate these risks, a large-scale assessment of tree-associated fungi and insects is needed. We present records of endophytic fungi and insects in twigs of 17 angiosperm and gymnosperm genera, from 51 locations in 32 countries worldwide. Endophytic fungi were characterized by high-throughput sequencing of 352 samples from 145 tree species in 28 countries. Insects were reared from 227 samples of 109 tree species in 18 countries and sorted into taxonomic orders and feeding guilds. Herbivorous insects were grouped into morphospecies and were identified using molecular and morphological approaches. This dataset reveals the diversity of tree-associated taxa, as it contains 12,721 fungal Amplicon Sequence Variants and 208 herbivorous insect morphospecies, sampled across broad geographic and climatic gradients and for many tree species. This dataset will facilitate applied and fundamental studies on the distribution of fungal endophytes and insects in trees.

Plant diversity drives global patterns of insect invasions.

During the last two centuries, thousands of insect species have been transported (largely inadvertently) and established outside of their native ranges worldwide, some with catastrophic ecological and economic impacts. Global variation in numbers of invading species depends on geographic variation in propagule pressure and heterogeneity of environmental resistance to invasions. Elton's diversity-invasibility hypothesis, proposed over sixty years ago, has been widely explored for plants but little is known on how biodiversity affects insect invasions. Here we use species inventories from 44 land areas, ranging from small oceanic islands to entire continents in various world regions, to show that numbers of established insect species are primarily driven by diversity of plants, with both native and non-native plant species richness being the strongest predictor of insect invasions. We find that at large spatial scales, plant diversity directly explains variation in non-native insect species richness among world regions, while geographic factors such as land area, climate and insularity largely affect insect invasions indirectly via their effects on local plant richness.

From east to west across the Palearctic: Phylogeography of the invasive lime leaf miner Phyllonorycter issikii (Lepidoptera: Gracillariidae) and discovery of a putative new cryptic species in East Asia.

Knowing the phylogeographic structure of invasive species is important for understanding the underlying processes of invasion. The micromoth Phyllonorycter issikii, whose larvae damage leaves of lime trees Tilia spp., was only known from East Asia. In the last three decades, it has been recorded in most of Europe, Western Russia and Siberia. We used the mitochondrial cytochrome c oxidase subunit I (COI) gene region to compare the genetic variability of P. issikii populations between these different regions. Additionally, we sequenced two nuclear genes (28S rRNA and Histone 3) and run morphometric analysis of male genitalia to probe for the existence of cryptic species. The analysis of COI data of 377 insect specimens collected in 16 countries across the Palearctic revealed the presence of two different lineages: P. issikii and a putative new cryptic Phyllonorycter species distributed in the Russian Far East and Japan. In P. issikii, we identified 31 haplotypes among which 23 were detected in the invaded area (Europe) and 10 were found in its putative native range in East Asia (Russian Far East, Japan, South Korea and China), with only two common haplotypes. The high number of haplotypes found in the invaded area suggest a possible scenario of multiple introductions. One haplotype H1 was dominant (119 individuals, 67.2%), not only throughout its expanding range in Europe and Siberia but, intriguingly, also in 96% of individuals originating from Japan. We detected eight unique haplotypes of P. issikii in East Asia. Five of them were exclusively found in the Russian Far East representing 95% of individuals from that area. The putative new cryptic Phyllonorycter species showed differences from P. issikii for the three studied genes. However, both species are morphologically undistinguishable. They occur in sympatry on the same host plants in Japan (Sendai) and the Russian Far East (Primorsky krai) without evidence of admixture.

A P-Glycoprotein Is Linked to Resistance to the Bacillus thuringiensis Cry3Aa Toxin in a Leaf Beetle.

Chrysomela tremula is a polyvoltine oligophagous leaf beetle responsible for massive attacks on poplar trees. This beetle is an important model for understanding mechanisms of resistance to Bacillus thuringiensis (Bt) insecticidal toxins, because a resistant C. tremula strain has been found that can survive and reproduce on transgenic poplar trees expressing high levels of the Cry3Aa Bt toxin. Resistance to Cry3Aa in this strain is recessive and is controlled by a single autosomal locus. We used a larval midgut transcriptome for C. tremula to search for candidate resistance genes. We discovered a mutation in an ABC protein, member of the B subfamily homologous to P-glycoprotein, which is genetically linked to Cry3Aa resistance in C. tremula. Cultured insect cells heterologously expressing this ABC protein swell and lyse when incubated with Cry3Aa toxin. In light of previous findings in Lepidoptera implicating A subfamily ABC proteins as receptors for Cry2A toxins and C subfamily proteins as receptors for Cry1A and Cry1C toxins, this result suggests that ABC proteins may be targets of insecticidal three-domain Bt toxins in Coleoptera as well.

Host tracking or cryptic adaptation? Phylogeography of Pediobius saulius (Hymenoptera, Eulophidae), a parasitoid of the highly invasive horse-chestnut leafminer.

Classical biological control is often advocated as a tool for managing invasive species. However, accurate evaluations of parasitoid species complexes and assessment of host specificity are impeded by the lack of morphological variation. Here, we study the possibility of host races/species within the eulophid wasp Pediobius saulius, a pupal generalist parasitoid that parasitize the highly invasive horse-chestnut leaf-mining moth Cameraria ohridella. We analysed the population genetic structure, host associations and phylogeographic patterns of P. saulius in Europe using the COI mitochondrial gene. This marker strongly supports a division into at least five highly differentiated parasitoid complexes, within two of which clades with differing degrees of host specialization were found: a Balkan clade that mainly (but not only) attacks C. ohridella and a more generalist European group that attacks many hosts, including C. ohridella. The divergence in COI (up to 7.6%) suggests the existence of cryptic species, although this is neither confirmed by nuclear divergence nor morphology. We do not find evidence of host tracking. The higher parasitism rates observed in the Balkans and the scarcity of the Balkan-Cameraria haplotypes out of the Balkans open the possibility of using these Balkan haplotypes as biological control agents of C. ohridella elsewhere in Europe.

Pyrosequencing of the midgut transcriptome of the poplar leaf beetle Chrysomela tremulae reveals new gene families in Coleoptera.

The insect midgut is the primary target site for Bt-derived insecticides and Bt alternatives. However, despite extensive recent study, the precise role and nature of different Bt receptors remains a subject of considerable debate. This problem is fuelled by a lack of understanding of the genes expressed in the insect midgut and their physiological roles. The poplar leaf beetle, Chrysomela tremulae, is an important model for understanding the mode of action of, and resistance to, coleopteran-specific Bt toxins and currently shows the only known naturally occurring case of resistance to Cry3A toxins. Moreover it belongs to the same family as the corn rootworm, Diabrotica virgifera, an economically important beetle pest and target of hybrid corn expressing Cry3 toxins. Pyrosequencing is a fast and efficient way of defining the transcriptome of specific insect tissues such as the larval midgut. Here we use 454 based pyrosequencing to sample the larval midgut transcriptome of C. tremulae. We identify candidate genes of putative Bt receptors including transcripts encoding cadherin-like proteins, aminopeptidase N and alkaline phosphatase. We also describe a wealth of new transcripts predicting rapidly evolving gene families involved in plant tissue digestion, which have no homologs in the genome of the stored product pest the Red Flour beetle, Tribolium castaneum.

Genetics of resistance to transgenic Bacillus thuringiensis poplars in Chrysomela tremulae (Coleoptera: Chrysomelidae).

The area under genetically engineered plants producing Bacillus thuringiensis (Bt) toxins is steadily increasing. This increase has magnified the risk of alleles conferring resistance to these toxins being selected in natural populations of target insect pests. The speed at which this selection is likely to occur depends on the genetic characteristics of Bt resistance. We selected a strain of the beetle Chrysomela tremulae Fabricius on a transgenic Bt poplar clone Populus tremula L. x Populus tremuloides Michx producing high levels of B. thuringiensis Cry3Aa toxin. This strain was derived from an isofemale line that generated some F2 offspring that actively fed on this Bt poplar clone. The resistance ratio of the strain was >6400. Susceptibility had decreased to such an extent that the mortality of beetles of the strain fed Bt poplar leaves was similar to that of beetles fed nontransgenic poplar leaves. Genetic crosses between susceptible, resistant, and F1 hybrids showed that resistance to the Cry3Aa toxin was almost completely recessive (D(LC) = 0.07) and conferred by a single autosomal gene. The concentration of Cry3Aa produced in the transgenic Bt poplar used in this study was 6.34 times higher than the LC99 of the F1 hybrids, accounting for the complete recessivity (D(ML) = 0) of survival on Bt poplar leaves. Overall, the genetic characteristics of the resistance of C. tremulae to the Cry3Aa toxin are consistent with the assumptions underlying the high-dose refuge strategy, which aims to decrease the selection of Bt resistance alleles in natural target pest populations.

Initial frequency of alleles conferring resistance to Bacillus thuringiensis poplar in a field population of Chrysomela tremulae.

Globally, the estimated total area planted with transgenic plants producing Bacillus thuringiensis (Bt) toxins was 12 million hectares in 2001. The risk of target pests becoming resistant to these toxins has led to the implementation of resistance-management strategies. The efficiency and sustainability of these strategies, including the high-dose plus refuge strategy currently recommended for North American maize, depend on the initial frequency of resistance alleles. In this study, we estimated the initial frequencies of alleles conferring resistance to transgenic Bt poplars producing Cry3A in a natural population of the poplar pest Chrysomela tremulae (Coleoptera: Chrysomelidae). We used the F(2) screen method developed for detecting resistance alleles in natural pest populations. At least three parents of the 270 lines tested were heterozygous for a major Bt resistance allele. We estimated mean resistance-allele frequency for the period 1999-2001 at 0.0037 (95% confidence interval = 0.00045-0.0080) with a detection probability of 90%. These results demonstrate that (i) the F(2) screen method can be used to detect major alleles conferring resistance to Bt-producing plants in insects and (ii) the initial frequency of alleles conferring resistance to Bt toxin can be close to the highest theoretical values that are expected prior to the use of Bt plants if considering fitness costs and typical mutation rates.