Pine Sawyer Research Articles

MiR-31-5p regulates cold acclimation of the wood-boring beetle Monochamus alternatus via ascaroside signaling

BackgroundSurvival to cold stress in insects living in temperate environments requires the deployment of strategies that lead to physiological changes involved in freeze tolerance or freeze avoidance. These strategies may consist of, for instance, the induction of metabolic depression, accumulation of cryoprotectants, or the production of antifreeze proteins, however, little is known about the way such mechanisms are regulated and the signals involved in their activation. Ascarosides are signaling molecules usually known to regulate nematode behavior and development, whose expression was recently found to relate to thermal plasticity in the Japanese pine sawyer beetle Monochamus alternatus. Accumulating evidence also points to miRNAs as another class of regulators differentially expressed in response to cold stress, which are predicted to target genes involved in cold adaptation of insects. Here, we demonstrate a novel pathway involved in insect cold acclimation, through miRNA-mediated regulation of ascaroside function.ResultsWe initially discovered that experimental cold acclimation can enhance the beetle’s cold hardiness. Through screening and functional verification, we found miR-31-5p, upregulated under cold stress, significantly contributes to this enhancement. Mechanistically, miR-31-5p promotes production of an ascaroside (asc-C9) in the beetle by negatively targeting the rate-limiting enzyme, acyl-CoA oxidase in peroxisomal β-oxidation cycles. Feeding experiments with synthetic asc-C9 suggests it may serve as a signal to promote cold acclimation through metabolic depression and accumulation of cryoprotectants with specific gene expression patterns.ConclusionsOur results point to important roles of miRNA-mediated regulation of ascaroside function in insect cold adaptation. This enhanced cold tolerance may allow higher survival of M. alternatus in winter and be pivotal in shaping its wide distribution range, greatly expanding the threat of pine wilt disease, and thus can also inspire the development of ascaroside-based pest management strategies.

Effect of aerial spraying of thiacloprid on pine sawyer beetles (Monochamus alternatus) and honey bees (Apis mellifera) in pine forests

AbstractMonochamus alternatus is a very important vector of the pine wood nematode (Bursaphelenchus xylophilus) which is the causal agent of pine wilt disease. To reduce population density of M. alternatus, thiacloprid has been sprayed by aircrafts on pine forests. Thus, we examined the effect of aerial spraying as contact toxicity and thiacloprid residues of pine branches as ingestion toxicity on the mortality of M. alternatus in pine forests in Yangsan, Korea. In addition, the effect of aerial spraying on honey bees, Apis mellifera, was tested in the same locality. Thiacloprid was sprayed once a month from June to August in 2016. To test the effect of thiacloprid, eight M. alternatus and ten honey bees were put into each small mesh cage, which were hung on pine trees in each study plot. Thiacloprid appeared to be effective for reducing longevity of M. alternatus, while mortality and abnormal behaviors were not found for honey bees. In addition, longevity of M. alternatus beetles was declined by thiacloprid residues of pine branches compared to the control group. However, we found that dead pine trees infected by pine wood nematodes in the next year similarly occurred in our study area whether thiacloprid‐aerial spraying occurred or not. Consequently, thiacloprid sprayed by aircraft may be an effective control agent for Monochamus beetle adults without negative effect on honey bees, but aerial application to prevent expansion of pine wilt disease should be reassessed.

A new direction to understand the life cycle of the Japanese pine sawyer considering the selection strategy of instar pathways

The Japanese pine sawyer, Monochamus alternatus Hope (Coleoptera: Cerambycidae), transfers the pine wood nematode, Bursaphelenchus xylophilus (Steiner and Buhrer) that causes pine wilt disease (PWD), especially in Asian countries. The key for the control of PWD is primarily focused on vector management. Thus, understanding the exact life history of M. alternatus is required. Since the late 1980s, the life cycle of M. alternatus has been accepted under the assumption that the final larvae pass four instars in the field. This study is revising the previous error for the life cycle hypothesis of M. alternatus by finding various instar pathways, which pathway is defined as the number of instars that larvae pass through prior to pupation. We confirm experimentally that the overwintered fourth or fifth instar larvae directly pupate to emerge as adults, indicating the presence of four and five instar pathways, respectively. The selection of instar pathway might be determined primarily by habitat temperature. This information will be useful to explain the variation of life history in M. alternatus populations worldwide based on the thermal environments, and also can be served to predict the northern distribution limit by applying the threshold degree-days for the completion of four instar pathway.

Mechanisms of Insecticidal Action of Metarhizium anisopliae on Adult Japanese Pine Sawyer Beetles (Monochamus alternatus).

Pine wilt disease, caused by Bursaphelenchus xylophilus (pine wood nematode), leads to severe environmental and economic damage. Here, we report the results of experiments on the biological control of pine wilt disease through termination of the insect vector of the nematode and the mechanism of the insecticidal action of Metarhizium anisopliae JEF-279 against Monochamus alternatus (Japanese pine sawyer). A combined treatment with a fungal conidia suspension and a fungal protease-containing culture filtrate caused 75.8% mortality of the insect vector. Additionally, the presence of destruxins was confirmed in the dead Japanese pine sawyer adults, and half of the 10 protein spots in proteomic analysis were identified as an actin related to muscle contraction. Based on proteomic and microscopic analyses, the infection cycle of the Japanese pine sawyer by M. anisopliae JEF-279 was inferred to proceed in the following sequence: (1) host adhesion and germination, (2) epicuticle degradation, (3) growth as blastospore, (4) killing by various fungal toxins (insecticidal metabolites), (5) immune response as defense mechanism, and (6) hyphal extrusion and conidiation. Consequently, the combined fungal conidia suspension and protease-containing culture filtrate treatment may be applied as an insecticidal agent, and flaccid paralysis is likely a major mechanism underlying the insecticidal action of M. anisopliae JEF-279 on host insects.

Monitoring of Nitrophenols and Insecticides in the Daecheong Lake, Served as Resources of Drinking Water

Objectives:This study aimed to investigate distributions of three nitrophenols (4-nitrophenol, 2,4-dinitrophenol, 2-methyl-4,6-dinitrophenol) and five insecticides (acetamiprid, dinotefuran, thiacloprid, triflumuron, chlorfluazuron) in the Daecheong lake that is primary water resources for the central area in Korea including Daejeon. Furthermore, the effect of land use type and monthly precipitation on the concentration trends of nitrophenols and insecticides were assessed.Methods:River water samples were obtained for once in a month for five months (June to October in 2016) in seven sites of the Daecheong lake. All residues were analyzed by LC-ESI/MSMS after extraction using solid phase extraction cartridge. Method detection limit (MDL) of nitrophenols and insecticides were 0.4-1.2 ng/L and 0.2-5.0 ng/L, respectively. Average recoveries were 93.1% for nitrophenols (2,4,6-Tribromophenol), 68.7% and 41.3% for insecticides (Acetamiprid-d3, Thiacloprid-d4, respectively).Results and Discussion:Detection frequencies (DF) and total concentrations of three nitrophenols were 71.4 to 100%, and not detected (ND) to 487 ng/L, respectively. Among insecticides, DF of neonicotinoids (acetamiprid, dinotefuran, thiacloprid) and benzoylureas (triflumuron, chlorfluazuron) ranged 28.6 to 100%, and 0 to 28.6%. Total concentrations of insecticides were ND to 253 ng/L. Total concentrations of all target compounds showed a significant difference between upstream and downstream, and adjacent lake by Mann-whitney U test. According to raising precipitation, an increasing pattern of three nitrophenols was revealed in tributaries and lakes but not observed three neonicotinoids in three lake sites. In June, dinotefuran was not detected at the Juwon upstream site with a forest land coverage, however the concentrations rapidly increased from July to August, followed by a sharp decrease in September. For the other four sites (Daecheong dam, Munui, Chudong, and Pumgok streams), dinotefuran concentrations increased by over 20 times between July and October.Conclusions:In the Daecheong lake, which is used as a water supply source for over 3,470,000 personnel, three nitrophenol levels indicated 1,000 times lower than US EPA water quality criteria (WQC) unless high detection frequency (up to 70%). Similarly, there is only WQC among insecticides, acetamiprid showed under 1% of those of guideline. These results are acceptable as a drinking water supply source. To assess the correlation between characteristics of potential origin sources by land coverage and pollutant distributions, and relative monthly precipitation, it is indicated to be increased levels along with upstream surrounding with shrubland to downstream and adjacent lake covered with mixed land coverage (i.g. shrub, and human activities such as rice paddy, farm, transportation, and living). Besides, precipitation was higher as much as increasing concentration. It might be assumed that nitrophenols and insecticides spilled as non-target contamination sources with rainfall. Dinotefuran concentrations in Juwon upstream and three-site of lakes were massively increased in summer and autumn season considered as a pesticide spraying season for bug control such as Japanese pine sawyer, fruit moth, lace bug, and mites. These results implicated the possibility of using insecticides in a sanctuary where use of pesticides is prohibited.

Modelling the dynamics of Pine Wilt Disease with asymptomatic carriers and optimal control

Pine wilt disease is a lethal tree disease caused by nematodes carried by pine sawyer beetles. Once affected, the trees are destroyed within a few months, resulting in significant environmental and economic losses. The role of asymptomatic carrier trees in the disease dynamics remains unclear. We developed a mathematical model to investigate the effect of asymptomatic carriers on the long-term outcome of the disease. We performed a stability and sensitivity analysis to identify key parameters and used optimal control to examine several intervention options. Our model shows that, with the application of suitable controls, the disease can be eliminated in the vector population and all tree populations except for asymptomatic carriers. Of the possible controls (tree injection, elimination of infected trees, insecticide spraying), we determined that elimination of infected trees is crucial. However, if the costs of insecticide spraying increase, it can be supplemented (although not replaced entirely) by tree injection, so long as some spraying is still undertaken.

Identification of putative olfactory genes in newly hatched larvae of a Coleopteran ectoparasitoid Dastarcus helophoroides (Fairmaire) (Coleoptera: Bothrideridae) by transcriptome analysis

AbstractDastarcus helophoroides (Fairmaire) (Coleoptera: Bothrideridae) is an effective ectoparasitoid of pine sawyer beetle Monochamus alternatus Hope (Coleoptera: Cerambycidae) which is the major median insect for transmission of pine wood nematode, Bursaphelenchus xylophilus (Steiner et Buhrer) Nickle (Aphelenchida: Parasitaphelenchidae). A sensitive and acute olfactory system is extremely essential for newly hatched larva of D. helophoroides to forage for a host. Therefore, we established the transcriptome of newly hatched D. helophoroides larvae and characterized the expression profiles of olfactory genes. We identified 20 transcripts encoding putative odorant binding proteins (OBPs), 11 putative chemosensory proteins (CSPs), 9 putative odorant receptors (ORs), 11 putative ionotropic receptors (IRs), 7 putative gustatory receptors (GRs) and 4 putative sensory neuron membrane proteins (SNMPs) olfactory genes from the transcriptome of newly hatched D. helophoroides larvae. According to the phylogenetic analysis, 3 OBPs (DhelOBP6, DhelOBP14 and DhelOBP19), 1 CSP (DhelCSP11), 1 OR (DhelOrco1), 3 IRs (DhelIR3, DhelIR6 and DhelIR8), 3 GRs (DhelGR2, DhelGR4 and DhelGR5) and 2 SNMPs (DhelSNMP1 and DhelSNMP3) had high similarities with reported Coleopteran species. The highest expressed olfactory gene was DhelCSP3, followed by DhelOBP8 and DhelCSP2. The expression levels of the majority of OR, IR, GR and SNMP genes were low. These results indicate that CSPs and OBPs may play important roles in the olfactory recognition process in newly hatched D. helophoroides larvae. The findings from the present study are helpful in unraveling the profound mystery between D. helophoroides and M. alternatus, as well as improving the parasitism efficiency of D. helophoroides larvae.

Characterization of MaltOBP1, a Minus-C Odorant-Binding Protein, From the Japanese Pine Sawyer Beetle, Monochamus alternatus Hope (Coleoptera: Cerambycidae).

Insect Odorant-Binding Proteins (OBPs) play crucial roles in the discrimination, binding and transportation of odorants. Herein, the full-length cDNA sequence of Minus-C OBP1 (MaltOBP1) from the Japanese pine sawyer beetle, Monochamus alternatus, was cloned by 3′ and 5′ RACE-PCR and analyzed. The results showed that MaltOBP1 contains a 435 bp open reading frame (ORF) that encodes 144 amino acids, including a 21-amino acid signal peptide at the N-terminus. The matured MaltOBP1 protein possesses a predicted molecular weight of about 14 kDa and consists of six α-helices, creating an open binding pocket, and two disulfide bridges. Immunoblotting results showed that MaltOBP1 was most highly expressed in antennae in both sexes, followed by wings and legs. Fluorescence assays demonstrated that MaltOBP1 protein exhibited high binding affinity with (R)-(+)-α-pinene, (−)-β-pinene, trans-caryophyllene, (R)-(+)-limonene and (–)-verbenone, which are the main volatile compounds of the pine tree. Our combined results suggest that MaltOBP1 plays a role in host seeking behavior in M. alternatus.

Yearly changes in dispersal and life‐history traits of Monochamus alternatus Hope with reference to its outbreak

AbstractThe Japanese pine sawyer, Monochamus alternatus Hope, is the primary vector of the pinewood nematode, Bursaphelenchus xylophilus (Steiner et Buhrer) Nickle, the causative agent of pine wilt disease in East Asia. The range of B. xylophilus expands through the dispersal capability of its vectors and transport of host trees infested with the pathogenic nematode and its vector. Outbreaks of M. alternatus populations occur together with the epidemics of pine wilt disease, because the insect reproduces on host trees recently killed by the disease. We measured some dispersal and life‐history traits of adults for four years to determine the change in flight capability and life history of a field population of beetles in relation to an outbreak. The population monitored exhibited an outbreak and subsequent collapse. The greatest mean body mass, largest area of hind wings, smallest wing load, and shortest preoviposition period were observed in the year of outbreak. By contrast, there was no difference in the ovariole number between pre‐outbreak (latent) and outbreak years. The greatest mean hind wing area and smallest wing load suggest likely result in greater flight performance. As other studies showed, adult body mass is related positively to the flight performance and oviposition rate. Moreover, a shortened preoviposition period leads to a high reproduction rate. Thus, adults in outbreak populations are “superdispersers” because they are likely to have enhanced flight capability and reproduction power. This suggests that M. alternatus populations at the onset of a population outbreak enhance the expansion rate of B. xylophilus range more than those during the latent and pre‐outbreak periods.

Comparison of methods to obtain and maintain cultures of the pinewood nematode, Bursaphelenchus xylophilus

ABSTRACTThe pinewood nematode Bursaphelenchus xylophilus, native from North America, is the causal agent of the pine wilt disease, being considered one of the most important invasive pests in Eurasian pine forests. The establishment of laboratory isolates of B. xylophilus has been crucial to research on this important pathogen of pine trees. However, studies on the effects of in vitro serial passage on the pathogen evolution, and suitability of standard culture media to the wild populations have been surprisingly few. In the present work, we tested the suitability of different culture media for obtaining B. xylophilus lab populations from dauer larvae that have been isolated from pine sawyer beetles (Monochamus spp.), which serve as their vector. We conducted studies in North America and Portugal. Results showed that adding groundwood (xylem from pine trees) to standard agar medium dramatically improves the survival and growth of wild populations of B. xylophilus in the laboratory. Further, malt extract agar proved to be more suitable than potato dextrose agar. Once in culture, B. xylophilus grew well feeding on either of two fungus species: Botrytis cinerea or Ophiostoma minus. Growth rates were highest on B. cinerea, the most widely used fungus to maintain lab cultures of B. xylophilus, but O. minus has the advantage of being a natural associate and putative diet of wild populations. There remains a need for better understanding of how to obtain and maintain laboratory populations of this pathogen such that their natural characteristics are maintained.

Application of harmonic radar to analyze dispersal behavior of the Japanese pine sawyer beetle, Monochamus alternatus (Coleoptera: Cerambycidae)

AbstractMonochamus alternatus (Hope) is a severe wood‐boring pest in coniferous forests and a major vector of pine wilt disease in East Asia. Harmonic radar is a powerful tool for studying the dispersal behavior of insects and it could be applied to control pine wilt disease. In this study, we validated the application of harmonic radar for analyzing the dispersal behavior of M. alternatus beetles in a field environment. We determined the wing capacities of the beetles and the effects of electronic tagging and marking on their movement, flight ability, survivorship, and food consumption in the laboratory to confirm the suitability of this technique. The detection rate and recovery rate for beetles were analyzed separately using radar on caged pine stands and in the field environment. The results showed that the minimum wing capacity of the Japanese pine sawyer was 24.9 mg, which was seven times the weight of the electronic tag (3.5 mg). Marking and tagging the beetles had no significant adverse effects on their movement, flight capacity, food consumption, and survivorship. The detection rate using the radar system and recovery rate based on visual observations of the beetles in caged pines were both 95.6%. However, in the field environment, the detection and recovery rates were only 55.6% and 37.8% after one week, respectively, and 33.3% and 7.8% after two weeks. Harmonic radar is a promising technique for studying the dispersal behavior of the Japanese pine sawyer, but its performance is not satisfactory and major improvements are required for both the radar system and electronic tags.

Management of pine wilt disease vectoring Monochamus alternatus adults using spray and soil application of Metarhizium anisopliae JEF isolates

Abstract Chemical control is widely used to control the Japanese pine sawyer beetle, Monochamus alternatus, but strong chemical regulations require an environmentally sound management strategy. In this work, we investigated the use of entomopathogenic fungi and their application as a means of practical pest management. Thirty-two diverse species of fungal isolates were assayed against adult pine sawyer beetles using a contact method under laboratory conditions, and four isolates showed over 70% virulence consequently. These isolates, two each of Beauveria bassiana and Metarhizium anisopliae were sprayed on the adult beetles at 1 × 107 conidia/ml in plastic containers, respectively. The M. anisopliae-treated adult beetles showed 67% mortality. M. anisopliae isolates JEF-197 and JEF-279 demonstrated dosage-dependent insecticidal activity. Following the laboratory experiments, semi-field trials were conducted in young pine trees under high (RH 94%) and low (RH 35%) humidity conditions. In the high humidity conditions, most of the adult beetles stayed on the top of the branches. When the two M. anisopliae isolates were sprayed on the beetles, they showed ca. 50–70% insecticidal activity 11 days after application. In contrast, in low humidity conditions, the adult beetles tried to move off the branches and onto the soil. When the beetles reached the JEF-197 and JEF-279-treated soil, we measured >90% insecticidal activity. This work suggests that M. anisopliae was the most virulent entomopathogenic fungus against adult Japanese pine sawyer beetles, and this forest insect could be ecologically controlled by the spray and soil application of the M. anisopliae isolates.

Colonization of Metarhizium anisopliae on the surface of pine tree logs: A promising biocontrol strategy for the Japanese pine sawyer, Monochamus alternatus

We investigated the colonization potential of five Metarhizium anisopliae isolates on pine tree surfaces under laboratory conditions, determined the influence of the pine bark extract on fungal growth and evaluated the insecticidal activity following colonization on the Japanese pine sawyer. Finally, the effect of colonization on adults pine sawyer was evaluated using the top three performing isolates (JEF-197, JEF-271 and JEF-279) under laboratory and field conditions. As a result, isolate JEF-197 showed the highest conidial production on the pine surfaces, and five isolates, including JEF-197, showed higher hyphal growth on autoclaved pine bark extract agar, compared to a water agar. Pine bark treated with the isolates showed 40–70 % mortality of adults pine sawyer. Under mimicked overwintering conditions, in the JEF-197 treatment group, 40 % of the inserted larvae became adults and all were dead after 59 d. In a field test, colonized isolate JEF-197 also showed 37 % insecticidal activity against emerged adults from the pine logs as overwintering sites. This work suggests that M. anisopliae isolate JEF-197 possibly colonized the pine surface and application of a conidial suspension on the pine logs as overwintering sites could be an effective strategy to control the pine sawyer.

Bidens pilosa Extract Effects on Pine Wilt: Causal Agents and Their Natural Enemies

Abstract We investigated the nematicidal and insecticidal activities of the aqueous extract from Bidens pilosa var. radiata on pine-wood nematode (Bursaphelenchus xylophilus) and Japanese pine sawyer (Monochamus alternatus) larvae and adults, the causal agent and vector of pine wilt, respectively. The aqueous extract killed the pine-wood nematode, adults, and larvae of the vector in vitro at all concentrations tested, and the effect decreased significantly with increasing extract dilution. Repellent activity was observed on the nematode and the vector adults as well, although the activity decreased with time in case of the vector. Furthermore, the aqueous extract of B. pilosa effectively promoted approximately 3.0–9.0 mm of hyphal growth in Beauveria spp., when compared with the control treatment. A minor insecticidal effect was also observed on two species of click beetle (Cryptalaus larvatus pini and Paracalais berus), which are natural predators of the Japanese pine-sawyer larvae. Pesticidal and repellent activities of the aqueous extract observed on several organisms related to pine wilt suggest that a multifactorial approach may effectively control this devastating disease.

First Report of Bursaphelenchus xylophilus (Nematoda: Aphelenchoididae) on Monochamus galloprovincialis (Coleoptera: Cerambycidae) in Spain

The pinewood nematode (PWN), Bursaphelenchus xylophilus (Steiner & Buhrer) Nickle, was found to be the causal agent of pine wilt disease in Pinus in Japan in 1971 (Mamiya 1987). In Europe, the PWN was first detected in Portugal in 1999 (Mota et al. 1999) and then in Spain in 2008 (Robertson et al. 2011). In 2001, pine sawyer beetle (Monochamus galloprovincialis Olivier) was associated with the PWN in Portugal (Sousa et al. 2001). In As Neves (Galicia, northwest Spain), the PWN was detected in 2010 (Abelleira et al. 2011), resulting in the establishment of a demarcated zone (DZ) 20 km wide in all directions. By 2018, the PWN was detected from four more municipalities (Salvaterra de Mino, Ponteareas, Tui, and Porrino), resulting in further expansion of the size of the DZ. In an attempt to detect M. galloprovincialis as a possible vector of the PWN in the DZ, surveys were conducted during spring and summer of 2018. Fifty multiple-funnel traps baited with aggregation pheromone were hung on a 2-m-high iron structure at the edge of adult pine stands to determine the distribution of M. galloprovincialis and maximize their capture. The traps were checked weekly from May to October, and the insects inside the traps were collected and visually identified to the species level. A total of 265 M. galloprovincialis were captured from the traps. Each insect was placed in an individual Petri dish containing sterile distilled water, cut into small pieces, and kept at room temperature (18 to 22 C°) for 24 h to extract dauer juveniles of the nematode. The dauer juveniles obtained from the insect were identified morphologically and further substantiated by DNA-based methods. For morphological study, 100 dauer juveniles were mounted on temporary slides in Amman’s lactophenol and observed under a light microscope (Eclipse 80i, Nikon Instruments, Tokyo, Japan) at different magnifications (200× to 1,000×). The dauer juveniles had a dome-shaped head, weak stylet, poorly defined median bulb, and an elongated conoid tail, with the mucronate terminus similar to that described by Penas et al. (2008). The population density of the dauer juveniles ranged from 1 to 33,000 individuals per plate. The total percentage of M. galloprovincialis carrying dauer juveniles of the PWN was 28%. To study morphological characteristics of the adults, dauer juveniles were reared on Botrytis cinerea Pers. in potato dextrose agar plates for 15 days at 25°C. All morphological characters of adults (n = 50) corresponded with B. xylophilus (Penas et al. 2008). The DNA was extracted from dauer juveniles and adults using a QIAamp DNA Micro Kit (Qiagen, Germany). A region of ribosomal DNA (rDNA), containing the internal transcribed spacer regions ITS1 and ITS2 were amplified using the method described by Burgermeister et al. (2009). Half of the PCR-amplified product was used for sequencing in our laboratory, and the other half was subjected to restriction enzyme digestion using AluI, HaeIII, HinfI, MspI, and RsaI. The restriction patterns corresponded to those described for B. xylophilus by Burgermeister et al. (2009). The nucleotide sequences generated in this study were compared with nonredundant sequences in the GenBank using the MegaBLAST algorithm in the BLASTn program and had 99 to 100% homology with B. xylophilus (accession nos. JQ288086 and KF025322). Two representative sequences were submitted to GenBank (accession nos. MK789297 and MK789298). This is the first report of the presence of dauer juveniles of PWN on M. galloprovincialis in Spain. Our study further substantiates the importance of M. galloprovincialis as a vector of B. xylophilus in Europe.

Acaricidal activity of the aggregation pheromone of Japanese pine sawyer against two-spotted spider mite

Abstract Japanese pine sawyer (Monochamus alternatus) (Coleoptera: Cerambycidae), a vector of pine wilt disease, produces male-produced aggregation pheromone called monochamol, 2-(1-undecyloxy)-1-ethanol (C11OEtOH). After the identification of unique nematicidal activity of the synthetic monochamol and its homologues (ROEtOH: R = C7-C11) against Bursaphelenchus xylophilus nematode, their acaricidal activities were evaluated against two spotted spider mite (TSSM, Tetranychus urticae Koch) (Acari: Tetranychidae). In a leaf disc bioassay method, 100 mgL−1 of monochamol and 2-(1-decyloxy)-1-ethanol (C10OEtOH) exhibited 100% adult mortality and zero fecundity. Along with C11OEtOH and C10OEtOH, 2-(1-octyloxy)-1-ethanol (C8OEtOH) and 2-(1-nonyloxy)-1-ethanol (C9OEtOH) were also 100% acaricidal with complete oviposition inhibition at increased concentration of 1000 mgL−1. When the TSSM eggs on leaf discs were immersed in 1000 mgL−1 solutions, none of the components were ovicidal. Egg hatching was completely inhibited by three components (C9OEtOH, C10OEtOH and C11OEtOH) at 6000 mgL−1 and by four components (C8OEtOH, C9OEtOH, C10OEtOH and C11OEtOH) at 8000 mgL−1. However, none of the compounds showed acaricidal effects in 0.5 h and 24 h residual toxicity tests at 1000 mgL−1. This study highlights the potential acaricidal activity of monochamol and its homologues. Notably, the monochamol and its C10-homologue could be listed as efficient acaricidal agents.

Morphological and optical features of the apposition compound eye of Monochamus alternatus Hope (Coleoptera: Cerambycidae)

The Japanese pine sawyer beetle, Monochamus alternatus Hope (Coleoptera: Cerambycidae) is currently the most destructive forest pest as it transmits the pine wilt nematode Bursaphelenchus xylophilus. Morphological, optical features and dark/light adaptational changes of the compound eyes of M. alternatus adults were examined by light, scanning and transmission electron microscopy. The eye of M. alternatus is apposition type and contains 489–712 ommatidia, depending on the beetle’s body size. Each ommatidium features a large corneal lens, composed of a thick inner lens (ILU) and a thin outer lens unit (OLU); an acone-type of cone of four cone cells, a semi-fused type of rhabdom formed by eight retinular cells (two central cells: R7-R8 surrounded by six peripheral cells: R1-R6). Dark/light adaptational changes affect size and shape of the cones as well as the rhabdom’s cross-sectional area and outline, to optimize the amount of light that reaches the photopigment. The compound eyes of M. alternatus have an F-number of 0.94, an interommatidial angle of 5.34°, an eye parameter P of 4.98 μm rad and a ratio of acceptance to interommatidial angle of 0.45. The eye is characterized by relatively poor spatial resolution, but can be expected to exhibit high absolute sensitivity and contrast in dim light.

Effect of Monochamus galloprovincialis feeding on Pinus pinaster and Pinus pinea, oleoresin and insect volatiles

In Portugal, the pine black sawyer Monochamus galloprovincialis is the principal vector of the pinewood nematode, Bursaphelenchus xylophilus, the causal agent of pine wilt disease (PWD), a lethal phyopathogen with major ecological and economic consequences to European forestry. The aim of this study was to determine the influence of M. galloprovincialis feeding on the volatiles emitted by pine trees. This study focused on the pine species which are most relevant to Portugal, that is, Pinus pinaster (maritime pine) and Pinus pinea (stone or umbrella pine), assessing to what extent pine chemotypes might influence feeding by the insect vector. Preliminary evaluation of each maritime pine essential oil allowed recognizing the existence of two main chemotypes (C1 and C2) and absence of chemical variability in P. pinea. Emission of volatiles from pine trees was evaluated before and during 24 h of feeding by a mixed-sex pair of newly emerged, unfed M. galloprovincialis. Volatiles were also collected from the oleoresin released from the feeding wounds as well as from the insects after feeding. Pine volatiles were collected by solid phase microextraction (SPME) and insect volatiles extracted with pentane, and all analysed by gas chromatography-mass spectrometry (GC-MS) and by GC for component identification and quantification, respectively. Of the seventeen emitted volatiles detected in SPME analyses of P. pinaster, β-pinene, α-pinene, β-caryophyllene, and germacrene D showed the highest average fold increases as a result of M. galloprovincialis feeding. When grouped by P. pinaster chemotype, C1 and C2 groups of trees showed different patterns of responses. β-Caryophyllene and germacrene D showed the highest fold increase in C1 trees, whereas β-pinene and α-pinene clearly dominated in C2 trees. Likewise, the oleoresin volatiles from C1 trees were dominated by δ-3-carene and/or β-pinene, whereas α-pinene and β-pinene were the main volatile components from oleoresin of C2 trees. Nine components were detected in P. pinea volatiles, of which limonene showed the highest fold increase as a result of insect feeding. The volatiles collected from the insects after they had fed on P. pinaster included α-pinene, β-pinene, and abietic acid, and by the straight-chain n-alkanes n-C27, n-C29, and n-C25, together with the methyl-branched hydrocarbons 3-meC29, 2-meC28, and 3-meC27. A better understanding of the responses of different P. pinaster chemotypes to feeding by M. galloprovincialis may be helpful in the development of new lures to improve pine sawyer trapping in integrated pest management for control of PWD.

Advanced strategy to produce insecticidal destruxins from lignocellulosic biomass Miscanthus

BackgroundBiorefineries are widely recognized as the most feasible solution to the problem of achieving environmental sustainability along with economic growth. Furthermore, pine wilt disease has caused severe environmental and economic damage worldwide to date. Herein, a highly efficient, advanced process for producing destruxins (DTXs) from Miscanthus (MCT) is reported, along with an application strategy.ResultsThe acetic acid–sodium chlorite pretreatment of MCT (AASC-MCT) is found to improve the monosaccharide production. Through biocatalytic conversion processes (simultaneous saccharification and cultivation), Metarhizium anisopliae JEF-279 can efficiently produce DTXs from 1% (w/v) AASC-MCT, i.e., DTX E (334.8 mg/L), A (288.8 mg/L), and B (48.6 mg/L). Monochamus alternatus (MA, Japanese pine sawyer) is known to act as a mediator transferring Bursaphelenchus xylophilus to pinewood. As B. xylophilus is associated with the occurrence of pine wilt disease, biological control of MA is a major strategy or controlling this disease. In this study, upon the application of a mixture of DTXs and protease-containing culture filtrate (PCF), complete mortality of MA is observed after a 5-day incubation. The MA immune system response is believed to cause an overexpression of actin and tropomyosin as a defense mechanism against the flaccid paralysis induced by the DTXs and PCF treatment.ConclusionsThese results suggest that MCT can be used as a major feedstock in the biorefinery industry and that DTXs can be applied as an insecticide for biological control of pine wilt disease via MA termination.

Crowd surveillance: estimating citizen science reporting probabilities for insects of biosecurity concern

Data streams arising from citizen reporting activities continue to grow, yet the information content within these streams remains unclear, and methods for addressing the inherent reporting biases little developed. Here, we quantify the major influence of physical insect features (colour, size, morphology, pattern) on the propensity of citizens to upload photographic sightings to online portals, and hence to contribute to biosecurity surveillance. After correcting for species availability, we show that physical features and pestiness are major predictors of reporting probability. The more distinctive the visual features, the higher the reporting probabilities—potentially providing useful surveillance should the species be an unwanted exotic. Conversely, the reporting probability for many small, nondescript high priority pest species is unlikely to be sufficient to contribute meaningfully to biosecurity surveillance, unless they are causing major harm. The lack of citizen reporting of recent incursions of small, nondescript exotic pests supports the model. By examining the types of insects of concern, industries or environmental managers can assess to what extent they can rely on citizen reporting for their surveillance needs. The citrus industry, for example, probably cannot rely on passive unstructured citizen data streams for surveillance of the Asian citrus psyllid (Diaphorina citri). In contrast, the forestry industry may consider that citizen detection and reporting of species of the large and colourful insects such as pine sawyers (Monochamus spp.) may be sufficient for their needs. Incorporating citizen surveillance into the general surveillance framework is an area for further research.