Transcriptome analysis and identification of chemosensory genes in the larvae of Plagiodera versicolora.

BACKGROUND: In insects, the chemosensory system is crucial in guiding their behaviors for survival. Plagiodera versicolora (Coleoptera: Chrysomelidae), is a worldwide leaf-eating forest pest in salicaceous trees. There is little known about the chemosensory genes in P. versicolora. Here, we conducted a transcriptome analysis of larvae heads in P. versicolora. RESULTS: In this study, 29 odorant binding proteins (OBPs), 6 chemosensory proteins (CSPs), 14 odorant receptors (ORs), 13 gustatory receptors (GRs), 8 ionotropic receptors (IRs) and 4 sensory neuron membrane proteins (SNMPs) were identified by transcriptome analysis. Compared to the previous antennae and foreleg transcriptome data in adults, 12 OBPs, 2 CSPs, 5 ORs, 4 IRs, and 7 GRs were newly identified in the larvae. Phylogenetic analyses were conducted and found a new candidate CO2 receptor (PverGR18) and a new sugar receptor (PverGR23) in the tree of GRs. Subsequently, the dynamic expression profiles of various genes were analyzed by quantitative real-time PCR. The results showed that PverOBP31, OBP34, OBP35, OBP38, and OBP40 were highly expressed in larvae, PverOBP33 and OBP37 were highly expressed in pupae, and PverCSP13 was highly expressed in eggs, respectively. CONCLUSIONS: We identified a total of 74 putative chemosensory genes based on a transcriptome analysis of larvae heads in P. versicolora. This work provides new information for functional studies on the chemoreception mechanism in P. versicolora.

Isolation, Behavioral Identification, and Pathogenicity Assessment of Entomopathogenic Fungi from a Forest Wood Borer.

Forest wood borers (FWB) cause severe tree damage and economic losses worldwide. The release of entomopathogenic fungi (EPF) during the FWB emergence period is considered an acceptable alternative to chemical control. However, EPF resources have been significantly less explored for FWBs, in contrast to agricultural insect pests. This paper presents a protocol for exploring EPF resources from FWBs using wild Monochamus alternatus populations as an example. In this protocol, the assignment of traps baited with M. alternatus attractants to different populations guaranteed the collection of adequate samples with natural infection symptoms, during the emergence periods of the beetle. Following finely dissecting integuments and placing them onto a selective medium, fungal species were isolated from each part of beetle bodies and identified based on both molecular and morphological traits. Several fungal species were certified as parasitic EPFs via re-infection of healthy M. alternatus with spore suspensions. Their behavioral phenotypes on M. alternatus were observed using scanning electron microscopy and further compared with those on the Coleopteran model insect Tribolium castaneum. For EPFs that present consistent parasitism phenotypes on both beetle species, evaluation of their activities on T. castaneum provided valuable information on lethality for future study on M. alternatus. This protocol helped the discovery of EPF newly reported on M. alternatus populations in China, which could be applied as an efficient approach to explore more EPF resources from other FWBs.

Adaptive Reproductive Strategies of an Ectoparasitoid Sclerodermus guani under the Stress of Its Entomopathogenic Fungus Beauveria bassiana.

Complex interspecific relationships between parasites and their insect hosts involve multiple factors and are affected by their ecological and evolutionary context. A parasitoid Sclerodermus guani (Hymenoptera: Bethylidae) and an entomopathogenic fungus Beauveria bassiana (Hypocreales: Cordycipitaceae) shared the same host in nature, Monochamus alternatus (Coleoptera: Cerambycidae). They often encountered the semi-enclosed microhabitat of the host larvae or pupae. We tested the survival and reproduction of the parasitoid's parent and its offspring fitness under different concentrations of B. bassiana suspension. The results show that S. guani parent females carrying higher concentrations of the pathogen shorten the pre-reproductive time and regulate their own fertility and their offspring's survival and development. This minimal model of the interspecific interactions contains three dimensionless parameters, vulnerability (θ), dilution ratio (δ), and PR, which were used to evaluate the mortality effect of the parasitoid S. guani on its host M. alternatus under the stress of the entomopathogenic fungus B. bassiana. We compared the infection and lethal effect of the fungus B. bassiana with different concentrations to the parasitoid S. guani and the host larvae M. alternatus. At higher concentrations of the pathogen, the parasitoid parent females shorten the pre-reproductive time and regulate their own fertility and their offspring's survival and development. At moderate concentrations of the pathogen, however, the ability of the parasitoid to exploit the host is more flexible and efficient, possibly reflecting the potential interspecific interactions between the two parasites which were able to coexist and communicate with their hosts in ecological contexts (with a high overlap in time and space) and cause interspecific competition and intraguild predation.

Electrophysiological and Behavioral Responses of Batocera horsfieldi Hope to Volatiles from Pistacia chinensis Bunge.

Following infestation by phytophagous insects, changes in the composition and relative proportion of volatile components emitted by plants may be observed. Some phytophagous insects can accurately identify these compounds to locate suitable host plants. We investigated whether herbivore-induced plant volatiles (HIPVs) generated by herbivory on Pistacia chinensis Bunge (Sapindales: Aceraceae) might be semiochemicals for the host location of Batocera horsfieldi Hope (Coleoptera: Cerambycidae). We performed two-choice bioassays (indoor darkroom, inside cages) on plants damaged by adult feeding and intact control plants. Volatiles from these plants were then collected and identified, and the response of adult antennae to these compounds was tested via electroantennography (EAG). The behavioral responses of B. horsfieldi to these compounds were finally assessed using a Y-tube olfactometer. Host plant choice tests show that B. horsfieldi prefers feeding-damaged P. chinensis over healthy trees. In total, 15 compounds were collected from healthy and feeding-damaged P. chinensis, 10 of which were shared in both healthy and feeding-damaged P. chinensis, among which there were significant differences in the quantities of five terpenes, including α-pinene, ß-pinene, α-phellandrene, D-limonene, and ß-ocimene. In EAG assays, the antennae of B. horsfieldi adults responded strongly to (Z)-3-hexen-1-ol, ß-ocimene, 3-carene, γ-terpinene, D-limonene, myrcene, and α-phellandrene. The antennae of B. horsfieldi adults responded in a dose-response manner to these compounds. Y-tube behavioral experiments showed that four compounds attracted mated females ((Z)-3-hexen-1-ol, ß-ocimene, 3-carene, and α-phellandrene), two compounds ((Z)-3-hexen-1-ol and α-phellandrene) attracted males, and adults of both sexes avoided D-limonene. Feeding bioassays showed that (Z)-3-hexen-1-ol and ß-ocimene could promote the feeding of B. horsfieldi and that D-limonene inhibited this response. These results could provide a theoretical basis for developing attractants or repellents for B. horsfieldi.

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.

Hot Air Treatment Elicits Disease Resistance against Colletotrichum gloeosporioides and Improves the Quality of Papaya by Metabolomic Profiling.

Forced air heat treatment could induce defenses to protect fruit from pathogen attacks and has been applied as an alternative to methyl bromide for phytosanitary treatment before exportation. However, few studies were reported on the regulation mechanism of antifungal effect and delayed physiological disorders of papaya by heat treatment. Therefore, we aim to explore the fruit's resistance to pathogens and the inhibition of physiological disorders by metabolomic profiling. In our study, papaya fruits were treated with 47.2°C for 30, 60, and 90 min by forced hot air treatment. The disease resistance against Colletotrichum gloeosporioides, quality parameters, and metabolites of papaya fruits were measured during 10 days of storage after heat treatment by metabolomic profiling. Papaya fruits after 30 and 60 min heat treatment had higher firmness, a delayed degreening and yellowing (lower a value) process, and a higher lightness (L) and hue angle (h) during storage. Heat treatment also delayed ripening, inhibiting the growth of C. gloeosporioides and softening of papaya. Metabolites and enzymes inhibited ROS scavenging, depressed ABA-regulated respiratory, and activated phenylpropanoid metabolism. Our study provides a broad picture of fruit resistance to pathogens and the inhibition of physiological disorders by metabolomic profiling, which is induced by heat treatment.

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.

Sentinel nurseries to assess the phytosanitary risks from insect pests on importations of live plants.

Ornamental plants represent a major pathway of invasion for alien pests worldwide. Commodity risk analyses are carried out to assess the risk posed by a new trade in a commodity, but they are restricted by our limited knowledge of the pests carried by traded plants. We used the sentinel nursery technique to identify insects attacking woody plants imported into Europe. We established two sentinel nurseries in China, with five traded Asian plants. These nurseries were monitored for two years to obtain lists of insects that can be expected on these commodities. These records were compared with those obtained from literature surveys, which are usually the sources of information available to pest risk assessors. At each site, 105 insect species and host associations were found on sentinel plants and 90% of these associations had not been found in a previous literature survey of insect pests of the five plants. Nearly 80% of these associations were not found in an a posteriori literature survey. An assessment classified 9%, 7% and 84% of the insect records as presenting a high, moderate and low likelihood of introduction, respectively. These results show the benefit of sentinel nurseries to identify potential infestation of plant commodity imports.

Role of host volatiles in mate location by the Japanese pine sawyer, Monochamus alternatus hope (Coleoptera: Cerambycidae).

We evaluated the responses of male and female Monochamus alternatus Hope (Coleoptera: Cerambycidae) to various terpenes commonly associated with host trees. Electroantennogram (EAG) tests were conducted with 12 plant volatile compounds and ethanol. Antennae of both sexes were highly sensitive to (R)-(+)-alpha-pinene, (+)-3-carene, (-)-beta-pinene, and terpinolene. Both sexes of M. alternatus were attracted by traps baited with (+)-alpha-pinene, (-)-beta-pinene, (+)-3-carene, or terpinolene. Our results support the first of the three-stage hypothesis posed by Ginzel and Hanks that suggests that location of stressed trees by cerambycids involves three stages: (1) both sexes locate larval hosts by using plant volatiles as kairomones; (2) males produces sex pheromones to attract females after both sexes land on the larval hosts; (3) males and female recognize each other by contract pheromones in their epicuticular wax layer. Males and females showed differences in their EAG responses to several compounds, including (R)-(+)-alpha-pinene, (-)-beta-pinene, myrcene, (+)-3-carene, (R)-(+)-limonene, terpinolene, and trans-caryophyllene. In all cases, males exhibited greater sensitivity than females. In laboratory assays, male M. alternatus showed strong preference for 1% (+)-alpha-pinene and 1% (-)-beta-pinene over other compounds. In field assays, traps baited with (+)-alpha-pinene, (-)-beta-pinene, (+)-3-carene, or terpinolene caught more beetles than control traps. We found strong male bias in beetle catches in baited traps and those captured on the stem of stressed trees despite a strong female bias in emerging beetles in 2004. We hypothesize that male M. alternatus are more responsive than females to plant volatiles and that males have more capacity than females in finding mating locations.

Summer diapause induced by high temperatures in the oriental tobacco budworm: ecological adaptation to hot summers.

Summer diapause in Helicoverpa assulta (Hübner), which prolongs the pupal stage, particularly in males, is induced by high temperatures. In the laboratory, 3(rd)-, 4(th)-, 6(th)-instar and prepupal larvae were exposed to high temperatures - 33 and 35 °C with a photoperiod of LD16:8 - until pupation to induce summer diapause. The results showed that the incidence of summer diapause was influenced by temperature, stage exposed, and sex. The higher the temperature, the more often summer diapause was attained. Sixth-instar and prepupal larvae were the sensitive stages for summer diapause induction. H. assulta summer-diapausing pupae needed diapause development to resume development when temperatures became favorable. Furthermore, both body mass and energy storage capacity (lipid and glycogen) were significantly affected by diapause rather than sex, and were significantly higher in summer-diapausing pupae than in non-diapausing pupae. In addition, the body mass loss and respiration rate showed that the rate of metabolism in the summer-diapausing pupae was consistently lower than in non-diapausing pupae, which were significantly affected by diapause and pupal age. We conclude that summer diapause in H. assulta is a true diapause, and H. assulta has evolved mechanisms to accumulate energy storage and to lower its metabolism to adapt to hot summers.

Sentinel trees as a tool to forecast invasions of alien plant pathogens.

Recent disease outbreaks caused by alien invasive pathogens into European forests posed a serious threat to forest sustainability with relevant environmental and economic effects. Many of the alien tree pathogens recently introduced into Europe were not previously included on any quarantine lists, thus they were not subject to phytosanitary inspections. The identification and description of alien fungi potentially pathogenic to native European flora before their introduction in Europe, is a paramount need in order to limit the risk of invasion and the impact to forest ecosystems. To determine the potential invasive fungi, a sentinel trees plot was established in Fuyang, China, using healthy seedlings of European tree species including Quercus petreae, Q. suber, and Q. ilex. The fungal assemblage associated with symptomatic specimens was studied using the tag-encoded 454 pyrosequencing of the nuclear ribosomal internal transcribed spacer-1 (ITS 1). Taxa with probable Asiatic origin were identified and included plant pathogenic genera. These results indicate that sentinel plants may be a strategic tool to improve the prevention of bioinvasions.

Planting sentinel European trees in eastern Asia as a novel method to identify potential insect pest invaders.

Quarantine measures to prevent insect invasions tend to focus on well-known pests but a large proportion of the recent invaders were not known to cause significant damage in their native range, or were not even known to science before their introduction. A novel method is proposed to detect new potential pests of woody plants in their region of origin before they are introduced to a new continent. Since Asia is currently considered to be the main supplier of insect invaders to Europe, sentinel trees were planted in China during 2007-2011 as an early warning tool to identify the potential for additional Asian insect species to colonize European trees. Seedlings (1-1.5 m tall) of five broadleaved (Quercus petraea, Q. suber, Q. ilex, Fagus sylvatica, and Carpinus betulus) and two conifer species (Abies alba and Cupressus sempervirens) were planted in blocks of 100 seedlings at two widely separated sites (one in a nursery near Beijing and the other in a forest environment near Fuyang in eastern China), and then regularly surveyed for colonization by insects. A total of 104 insect species, mostly defoliators, were observed on these new hosts, and at least six species were capable of larval development. Although a number of the insects observed were probably incidental feeders, 38 species had more than five colonization events, mostly infesting Q. petraea, and could be considered as being capable of switching to European trees if introduced to Europe. Three years was shown to be an appropriate duration for the experiment, since the rate of colonization then tended to plateau. A majority of the identified species appeared to have switched from agricultural crops and fruit trees rather than from forest trees. Although these results are promising, the method is not appropriate for xylophagous pests and other groups developing on larger trees. Apart from the logistical problems, the identification to species level of the specimens collected was a major difficulty. This situation could be improved by the development of molecular databases.