A new Leptographium species associated with the northern spruce engraver, Ips perturbatus, in western Canada

An undescribed Leptographium species was isolated from the spruce-infesting bark beetle Ips perturbatus collected from felled spruce trees and logs in northern British Columbia and Yukon Territory. Morphologically, this fungus is similar to L. abietinum and L. hughesii but differed in a number of characteristics (e.g. the arrangement of its conidiophores). The fungus grew optimally at 25 C on 2% malt-extract agar and showed a high level of tolerance to cycloheximide. Comparison of rDNA and beta-tubulin gene sequences also confirmed that this Leptographium species represents an undescribed taxon. Thus we described it as a new species, Leptographium fruticetum sp. nov.     

Survey and phylogenetic analysis of culturable microbes in the oral secretions of three bark beetle species

In a recent study, we reported a previously undescribed behavior in which a bark beetle exuded oral secretions containing bacteria that have antifungal properties, and hence defend their galleries against pervasive antagonistic Hyphomycete fungi. Actinobacteria, a group known for their antibiotic properties, were the most effective against fungi that invade the spruce beetle galleries. In the present study, we describe the isolation and identification of microorganisms from oral secretions of three bark beetles (Coleoptera: Curculionidae: Scolytinae): the spruce beetle, Dendroctonus rufipennis Kirby, the mountain pine beetle, Dendroctonus ponderosae Hopkins, and the pine engraver, Ips pini Say. Bacteria isolated from these three species span the major bacterial classes α-, β-, and γ-Proteobacteria, Firmicutes, Bacteroidetes, and Actinobacteria, except for D. ponderosae , which yielded no α-proteobacteria or Bacteroidetes isolates. Spruce beetles and pine engraver beetles had similar numbers of α-proteobacteria isolates, but pine engravers yielded twice as many Bacteroidetes isolates as spruce beetles. In contrast, mountain pine beetles yielded more isolates in the β- and γ-proteobacteria than spruce beetles and pine engravers. The highest percentage of Actinobacteria was obtained from spruce beetles, followed by pine engravers and mountain pine beetles. All of the fungal isolates obtained from the three beetle species were Ascomycetes. The greatest fungal diversity was obtained in spruce beetles, which had nine species, followed by pine engravers with five, and mountain pine beetles with one.     

Bacteria in oral secretions of an endophytic insect inhibit antagonistic fungi

Abstract.  1. Colonisation of host trees by an endophytic herbivore, the spruce beetle, Dendroctonus rufipennis , is accompanied by invasion of its galleries by a number of fungal species. Four of these associated species were identified as Leptographium abietinum , Aspergillus fumigatus , Aspergillus nomius , and Trichoderma harzianum .
2.  Trichoderma and Aspergillus significantly reduced spruce beetle survival and reproduction in controlled assays.
3. A previously undescribed behaviour was observed, in which spruce beetle adults exuded oral secretions, especially within fungus-pervaded galleries.
4. These oral secretions inhibited the growth of fungi except A. nomius , and disrupted the morphology of the latter. Administration of these secretions indicated a dose-dependent inhibitory effect.
5. Oral secretions cultured on microbiological media yielded substantial bacterial growth.
6. Filter-sterilised secretions failed to inhibit fungal growth, evidence that the bacteria are responsible for the antifungal activity.
7. Nine bacterial isolates belonging to the Actinobacteria, Firmicutes, Gammaproteobacteria, and Betaproteobacteria taxa were obtained from the secretions.
8. Bacterial isolates showed species-specific inhibitory activity against the four fungi antagonistic to spruce beetle. The bacterium with the strongest fungal inhibition activity was the actinomycete Micrococcus luteus .
9. The production of bark beetle secretions containing bacteria that inhibit fungal growth is a novel finding. This suggests an additional level of complexity to ecological associations among bark beetles, conifers, and microorganisms, and an important adaptation for colonising subcortical tissue.     

Phoretic mites and nematode associates of Scolytus multistriatus and Scolytus pygmaeus (Coleoptera: Scolytidae) in Austria

Abstract 1 The species assemblages and abundance of phoretic mites and nematodes associated with the elm bark beetles, Scolytus multistriatus and Scolytus pygmaeus, were studied in Austria. 2 A total of 3922 individual mites were recorded from 144 adults of S. multistriatus and 178 adults of S. pygmaeus. The species spectrum was identical and the relative abundance of mites was very similar for both species of scolytids. Nine mite species, Pyemotes scolyti, Pseudotarsonemoides eccoptogasteri, Trichouropoda bipilis, Tarsonemus crassus, Proctolaelaps eccoptogasteris, Proctolaelaps scolyti, Chelacheles michalskii, nr. Eueremaeus sp. and Elattoma sp. were detected. Two of the nine species, nr. Eueremaeus sp. and Elattoma sp., are documented here as new associates of Scolytus spp. 3 Pyemotes scolyti was the most frequent mite species, and Ps. eccoptogasteri and T. bipilis were relatively common, whereas the other mites occurred occasionally or were rare. 4 The trophic roles of most of the mites associated with S. multistriatus and S. pygmaeus are poorly known, but they may include fungivores, parasitoids of bark beetle broods, predators of bark beetle broods and/or mites and/or nematodes. 5 Besides phoretic mites, two nematode associates were seen on the investigated insects. A species of Cryptaphelenchus occurred under the elytra of both scolytid species, whereas the adults of a Neoparasitylenchus sp. were present inside abdomens of S. multistriatus, but absent from S. pygmaeus.     

Transmission of the Pinewood Nematode, Bursaphelenchus xylophilus,to Slash Pine Trees and Log Bolts by a Cerambycid Beetle,Monochamus titillator, in Florida

Field-collected adults of the southern pine sawyer, Monochamus titillator (F.) (Coleoptera: Cerambycidae), naturally infested with fourth-stage juveniles (dauerlarvae) of the pinewood nematode, Bursaphelenchus xylophilus (Steiner and Buhrer, 1934) Nickle, 1970, were maturation fed on excised shoots of typical slash pine, Pinus elliottii Engelm. var elliottii, for 21 days. During August 1981, a male and female adult beetle were held in a sleeve cage placed on the terminal of a side branch of each of seven replicate, healthy 10-year-old slash pine trees. All seven branch terminals showed evidence of beetle feeding on the bark after 1 week, and pinewood nematodes were present in wood samples taken near these feeding sites. Four of the seven trees showed wilt symptoms in 4-6 weeks and died about 9 weeks after beetle feeding. Pinewood nematodes were recovered from the roots and trunks of the dead trees. Each of seven replicate slash pine log bolts was enclosed in a jar with a pair of the same beetles used in the sleeve cages. After 1 week, wood underlying beetle oviposition sites in the bark of all replicate log bolts was infested with the pinewood nematode.     

Nematodes associated with Ips cembrae (Coleoptera: Curculionidae): comparison of generations,sexes and sampling methods

A study of nematodes associated with the large larch bark beetle Ips cembrae (Heer 1836) was carried out at three locations in the Czech Republic. The proportion of beetles infested by endoparasitic nematodes (representatives of genera Contortylenchus, Parasitylenchus, Cryptaphelenchus and Parasitorhabditis) ranged from 29.9 to 50.9%. Significant differences were determined in nematode infestation levels among locations, generations and sampling methods. No differences were found in infestation rates between males and females. The percentage of bark beetles with phoretic nematodes ranged from 18 to 42.9%. Phoretic nematodes directly found under elytra, on wings and between body segments of the bark beetles belong to the genus Micoletzkya. However, adults and juveniles of other two phoretic species Laimaphelenchus penardi and Bursaphelenchus sp. were found in the gallery frass of I. cembrae. Infestation by phoretic nematodes positively correlated with the presence of mites under elytra.     

Comparison of naturally and synthetically baited spruce beetle trapping systems in the central Rocky Mountains

We compared naturally baited trapping systems to synthetically baited funnel traps and fallen trap trees for suppressing preoutbreak spruce beetle, Dendroctonus rufipennis Kirby, populations. Lures for the traps were fresh spruce (Picea spp.) bolts or bark sections, augmented by adding female spruce beetles to create secondary attraction. In 2003, we compared a naturally baited system ("bolt trap") with fallen trap trees and with synthetically baited funnel traps. Trap performance was evaluated by comparing total beetle captures and spillover of attacks into nearby host trees. Overall, the trap systems did not significantly differ in spruce beetle captures, although bolt traps caught 6 to 7 times more beetles than funnel traps during the first 4 wk of testing. Funnel traps with synthetic lures had significantly more spillover than either trap trees or bolt traps. The study was repeated in 2004 with modifications including an enhanced blend synthetic lure. Again, trap captures were generally similar among naturally and synthetically baited traps, but naturally baited traps had significantly less spillover. Although relatively labor-intensive, the bolt trap could be used to suppress preoutbreak beetle populations, especially when spillover is undesirable. Our work provides additional avenues for management of spruce beetles and suggests that currently used synthetic lures can be improved.     

First report of parthenogenesis in the genus Bursaphelenchus Fuchs, 1937: a description of Bursaphelenchus okinawaensis sp. nov. isolated from Monochamus maruokai (Coleoptera: Cerambycidae)

An evolutionarily interesting nematode, Bursaphelenchus okinawaensis sp. nov., is described and illustrated. The new species has several characteristic morphological traits, i.e., four lateral lines in both sexes, lack of a vulval flap in females, and a triangular spicule shape and stout P4 caudal papillae in males, and characteristic biological traits, including phoretic association with Monochamus maruokai, a species of longhorn beetle, parthenogenetic reproduction, and a high frequency of dauer production. Bursaphelenchus okinawaensis sp. nov. shares several important traits with various phylogenetic groups within the genus. The new species shares its spicule shape with B. hellenicus and B. hofmanni. It shares four lateral lines, P4 caudal papillar structure (size and position), and female vulval shape with the 'hunti' group, although it was molecularly inferred to be phylogenetically closer to the 'xylophilus' group and B. africanus. The autapomorphic traits of B. okinawaensis sp. nov. are parthenogenetic reproduction and high frequency of dauer production. All other nominal Bursaphelenchus nematodes have bisexual reproduction and tightly synchronized dauer production. The unique morphological and biological traits of B. okinawaensis sp. nov. suggest genetic flexibility within the genus. The importance of the morphology and arrangement of the caudal papillae is discussed relative to the phylogeny of the genus.     

The bacterial community of entomophilic nematodes and host beetles

Insects form the most species‐rich lineage of Eukaryotes and each is a potential host for organisms from multiple phyla, including fungi, protozoa, mites, bacteria and nematodes. In particular, beetles are known to be associated with distinct bacterial communities and entomophilic nematodes. While entomopathogenic nematodes require symbiotic bacteria to kill and reproduce inside their insect hosts, the microbial ecology that facilitates other types of nematode–insect associations is largely unknown. To illuminate detailed patterns of the tritrophic beetle–nematode–bacteria relationship, we surveyed the nematode infestation profiles of scarab beetles in the greater Los Angeles area over a five‐year period and found distinct nematode infestation patterns for certain beetle hosts. Over a single season, we characterized the bacterial communities of beetles and their associated nematodes using high‐throughput sequencing of the 16S rRNA gene. We found significant differences in bacterial community composition among the five prevalent beetle host species, independent of geographical origin. Anaerobes Synergistaceae and sulphate‐reducing Desulfovibrionaceae were most abundant in Amblonoxia beetles, while Enterobacteriaceae and Lachnospiraceae were common in Cyclocephala beetles. Unlike entomopathogenic nematodes that carry bacterial symbionts, insect‐associated nematodes do not alter the beetles' native bacterial communities, nor do their microbiomes differ according to nematode or beetle host species. The conservation of Diplogastrid nematodes associations with Melolonthinae beetles and sulphate‐reducing bacteria suggests a possible link between beetle–bacterial communities and their associated nematodes. Our results establish a starting point towards understanding the dynamic interactions between soil macroinvertebrates and their microbiota in a highly accessible urban environment.     

Morphology and Description of Bursaphelenchus platzeri n. sp. (Nematoda: Parasitaphelenchidae), an Associate of Nitidulid Beetles

Bursaphelenchus platzeri n. sp., an associate of nitidulid beetles in southern California, is described and illustrated. Adult males and females of B. platzeri n. sp. were examined by scanning electron microscopy for ultrastructural comparisons with other members of the genus. Bursaphelenchus cocophilus (red ring nematode) appears to be the closest related taxon to B. platzeri n. sp. based upon shared morphological features of the fused spicules, female tail shape, phoresy with non-scolytid beetles, and molecular analysis of the near full-length small subunit (SSU) rDNA. Unfortunately, sequence data from the D2D3 expansion segments of the large subunit (LSU) rDNA and partial mitochondrial DNA COI did not help resolve the relationship of nearest relative. In addition to significant molecular sequence differences in SSU, LSU, and COI, B. platzeri n. sp., which is an obligate fungal feeder, can be differentiated from B. cocophilus because it is an obligate parasite of palms. Bursaphelenchus platzeri n. sp. can be differentiated from all other species of Bursaphelenchus by the length and shape of the female tail and spicule morphology. The spicules are fused along the ventral midline and possess unfused cucullae; the fused unit appears to function as a conduit for sperm. Population growth of B. platzeri n. sp. was measured in a time-course experiment at 25°C in the laboratory on cultures of the fungus Monilinia fructicola grown on 5% glycerol-supplemented potato dextrose agar (GPDA). Nematode population densities rapidly increased from 25 to approximately 200,000/culture within 14 d and then plateaued for up to 28 d.     

Taxonomic re-evaluation of three related species of Graphium, based on morphology, ecology and phylogeny

Two fungi associated with bark beetles, Graphium pseudormiticum (described in 1994) and Rhexographium fimbriisporum (described in 1995), have two micromorphological characters in common. Both species produce conidia with conspicuous basal frills, and the conidia align in chains, despite being produced in slime. The association of G. pseudormiticum with the pine bark beetle, Orthotomicus erosus, and the association of R. fimbriisporum with the spruce bark beetle, Ips typographus, suggest ecological differences between the two fungal species. Analyses of micromorphology and phylogenetic analyses of aligned 18S and ITS sequences suggest that these two species are congeneric and should be classified in Graphium but that they represent distinct species. A collection of strains tentatively identified as Graphium spp., isolated from Ips typographus on Picea abies, Ips cembrae on Larix decidua and Tomicus minor on Pinus sylvestris in Austria share the same unusual basal conidial frills and conidial chains. Isolates from spruce were identified as G. fimbriisporum and those from pine as G. pseudormiticum. The strains from Ips cembrae on Larix decidua, distinguished by the reddish color of their colonies, microscopic structures and molecular characteristics, are described as the new species Graphium laricis sp. nov., and the close relationship of this species with the other two species is confirmed.     

Transmission of Pinewood Nematode Through Feeding Wounds of Monochamus carolinensis (Coleoptera: Cerambycidae)

Transmission of pinewood nematode, Bursaphelenchus xylophilus, to mature, field grown Scots pines through feeding wounds of Monochamus carolinensis was investigated by caging nematode-infested beetles on pine branches for 24 hours. Nematodes were transmitted to 31 of 64 branches. Frequency of successful transmission was independent of the sex of the beetle but dependent upon beetle age. Transmission frequencies were highest for beetles 2, 3, and 4 weeks after emergence as adults. The number of nematodes transmitted per branch was low and did not differ between beetle sexes or among beetle age categories. The number of nematodes extracted per branch was correlated with the number of nematodes carried per beetle but was not correlated with the feeding area on the branch.     

Threshold facilitations of interacting species

The dynamics of species interactions are of central importance for the understanding of ecological coexistence, community structure and the effects of biological invasions. Using bark beetles that colonize the same habitat as an example, we explore species interactions in a resource-based model system with positive feedback between insect abundance and resource availability. The net interspecies interaction was found to be highly dynamic and may alternate in time between competition and mutualism. When both bark beetle species were able to kill trees (“aggressive”), our simulations showed strong facilitations between beetle species. This may lead to escape from control by competition, and increase the frequency of outbreaks of tree-killing. The frequency of net positive interactions varied with interaction strengths and the relative aggressiveness of the species and was highest when both species were strongly aggressive; which predicts disastrous outbreaks if, e.g., the European spruce bark beetle Ips typographus and the North American spruce beetle Dendroctonus rufipennis should become interacting species due to introductions. In imbalanced pairs, the relatively less aggressive species was facilitated more often than the aggressive species. Net positive interactions did not occur for strongly inferior species, but their survival was an increasing function of interaction strength with aggressive species and availability of resources. The benefits for the inferior species in the model are consistent with the structure of one aggressive and several less aggressive or non-aggressive species, which is common in bark beetle communities in many parts of the world.     

On the pathogens of the spruce bark beetle Ips typographus (Coleoptera: Scolytinae) in the Western Carpathians

Pathogens of spruce bark beetle Ips typographus were compared at eleven sites in Slovakia (the Western Carpathians). Seven different pathogen species were recorded: protozoan species Chytridiopsis typographi, Gregarina typographi, Malamoeba scolyti, nematodes of the genus Neoparasitylenchus and one unidentified nematode species. Presence of the entomopathogenic fungus Beauveria bassiana was also observed as well as parasitization by hymenopteran parasitoids. High infection rate of G. typographi and the nematode Neoparasitylenchus was detected in almost all sites and lethal impact of M. scolyti by massive infections was observed. The beetles from seven sites were screened for endosymbiotic bacteria Wolbachia, however, with no positive result.     

Newly Discovered Transmission Pathway of Bursaphelenchus xylophilus from Males of the Beetle Monochamus alternatus to Pinus densiflora Trees via Oviposition Wounds

The transmission of Bursaphelenchus xylophilus from Monochamus alternatus males to Pinus densiflora trees via oviposition wounds has been determined. Nematode-infested males, with mandibles fixed experimentally to prevent feeding, were placed for 48 hours with pine bolts containing oviposition wounds that had been made by nematode-free females. After removal of the nematode-infested males, the pine bolts were held for 1 month and then examined for the presence of nematodes. Reproducing nematode populations were recovered from pine bolts that were exposed to male beetles carrying a high number of nematodes. No reproducing nematode population could be recovered from pine bolts exposed to beetles with a small number of nematodes. Nematode reproduction in the pine bolts was not related to the number of oviposition wounds per bolt. Fourth-stage dispersal B. xylophilus juveniles, collected from beetle body surfaces, were inoculated on pine bolt bark 0, 5, 10, and 15 cm away from a single artificial, small hole. These dauer juveniles successfully entered some bolts. The probability of successful nematode reproduction decreased with increased distance between inoculation point and artificial hole. The results indicated that B. xylophilus can move a significant distance to oviposition wounds along the bark surface and enter a tree via the wounds. The new transmission pathway is considered important for the nematode to persist in pine forests such as in North America where pine wilt disease does not occur.     

Horizontal Transmission of Bursaphelenchus xylophilus between Sexes of Monochamus alternatus

Four experiments were conducted using nematode-infested and nematode-free adults of the cerambycid beetle, Monochamus alternatus, to determine horizontal transmission pathways of Bursaphelenchus xylophilus. When nematode-infested beetles of one sex and nematode-free beetles of the opposite sex were paired in containers for 48 or 72 hours, the number of nematodes carried by nematode-free beetles tended to increase with increased number of nematodes carried by nematode-infested beetles. The nematodes acquired by "nematode-free" beetles could be transmitted to pine. A female beetle that received 13 nematodes from a male transmitted one nematode to a Pinus densiflora bolt via an oviposition wound. When the nematode-infested and nematode-free beetles were observed continuously, it was observed that the number of nematodes carried by nematode-free beetles at the end of the first sexual mounting increased as the number of nematodes carried by nematode-infested beetles just before mounting increased. The number of nematodes transferred to nematode-free beetles was positively related to duration time of mounting. There was no difference in transmission efficacy between male-to-female transmission and female-to-male transmission. The horizontal transmission pathways are discussed relative to the persistence of B. xylophilus in resistant pine forests and the control of pine wilt disease.     

Potential vector switching in the evolution of Bursaphelenchus xylophilus group nematodes (Nematoda: Aphelenchoididae)

To show the importance of vector switching of nematodes in the evolution of the Bursaphelenchus xylophilus group, we tested a hypothesis that “Bursaphelenchus doui (or its ancestor) was transferred by Acalolepta fraudatrix, Acalolepta sejuncta, and/or Monochamus subfasciatus (or their ancestral species) from broad‐leaved trees to conifers, switched vectors from these cerambycid beetles to Monochamus beetles in conifers, and then evolved into the common ancestor of Bursaphelenchus mucronatus and B. xylophilus.” We used a simple nematode‐loading method to beetles and produced 20 binary combinations of five B. xylophilus group species and four cerambycid beetle species in the tribe Lamiini. The affinity of the nematodes for the beetles was examined based on phoretic stage formation of the nematodes. Phoretic stages of B. doui appeared in all beetle species examined, namely Acalolepta luxuriosa, Psacothea hilaris, A. fraudatrix, and Monochamus alternatus, although the affinity of the nematode for M. alternatus was weak. This finding indicates that B. doui could switch vectors to conifer‐using Monochamus beetles after transfer by A. fraudatrix from broad‐leaved trees to conifers. We conclude that vector switching of nematodes could have potentially happened during the evolutionary history of the B. xylophilus group.     

Susceptibility of lady beetles (Coleoptera: Coccinellidae) to entomopathogenic nematodes

We investigated differential susceptibility of lady beetles to entomopathogenic nematodes, for two reasons: (1) to estimate potential nontarget effects on natural lady beetle populations, (2) to compare the susceptibility of exotic versus native lady beetle species. We hypothesize that successful establishment of some exotically introduced arthropods may be due, in part, to a lower susceptibility relative to competing native species. In laboratory studies, we compared the pathogenicity, virulence, and reproductive capacity of Heterorhabditis bacteriophora and Steinernema carpocapsae among two native (Coleomegilla maculata and Olla v-nigrum) and two successfully established exotic (Harmonia axyridis and Coccinella septempunctata) lady beetles, and a known susceptible lepidopteran host, Agrotis ipsilon. After 1 and 2 days of exposure to either nematode species, mortality of A. ipsilon was higher than in all lady beetles. Thus, we predict that nematode field applications would have significantly less impact on lady beetle populations than on a susceptible target pest. Additionally, the impact of soil-applied nematodes may be lower on lady beetles than on soil-dwelling hosts because the former spends relatively less time on the soil. Exotic lady beetles were less susceptible to nematode infection than native species. Reproductive capacity data also indicated lower host suitability in H. axyridis, but not in C. septempunctata. Overall, the hypothesis that low susceptibility to pathogens in certain exotic lady beetles may have contributed to competitive establishment was supported (especially for H. axyridis). Additional studies incorporating different hosts and pathogens from various geographic locations will be required to further address the hypothesis.     

Attack density and breeding success of bark beetles (Coleoptera, Scolytidae) at different distances from forest-clearcut edge

1 Attack density and breeding success of bark beetles (Coleoptera: Scolytidae) and the abundance of their predators were studied at forest-clearcut edges in southern Finland on Norway spruce, Picea abies, bolts placed at different distances from a forest-clearcut edge. 2 Attack densities of two species, Hylurgops palliatus and H. glabratus, increased markedly towards the forest interior. The breeding success of H. palliatus was increased with increasing distance from the stand edge. None of the recorded 10 bark beetle species was distinctly abundant near the edge, but Pityogenes chalcographus appeared to prefer this zone. 3 There were no significant differences in numbers of bark beetle predators between the forest interior and the edge. Forest-clearcut edge had a significant effect on the desiccation of sample bolts. 4 Thus, forest edge is likely to affect the colonization behaviour and breeding success of bark beetles via host material properties.     

Species of Ophiostomataceae isolated from Picea abies infested by the bark beetle Ips typographic

Isolations of microorganisms were made from the sapwood of Norway spruce ( Picea abies ) (L.) Karst. infested by the spruce bark beetle Ips typographus L. The most important group of fungi isolated, the species of Ophiostomataceae, is treated. Ten species were isolated, of which Ophiostoma polonicum Siem. appears to be the most active invader of the sapwood. Three new species are described, Ophiostoma ainoae sp. nov., O. flexuosum sp. nov. and O. cucullatum sp. nov. and three new combinations are proposed, O. davidsonii (Olchow. & Reid.) comb. nov., O. europhioides (Wright & Cain) comb. nov. and O. sagmatospora (Wright & Cain) comb. nov.