Brief Review of the Associations of Xylobiont Nematodes with Bark Beetles (Coleoptera,Curculionidae: Scolytinae)

Nematode pathogens cause wilt diseases in conifers and deciduous trees. The longhorn beetles (Coleoptera: Cerambycidae) and bark beetles (Coleoptera: Curculionidae: Scolytinae) act as nematode vectors spreading the invasive juvenile stages during their maturation feeding or during oviposition on the plant hosts. There are numerous reviews of nematodes associated with bark beetles on conifers, while little attention has been paid to the nematodes of deciduous trees. The development of Dutch elm disease and ash dieback is mainly caused by fungal pathogens transmitted by bark beetles; the latter act as vectors of not only fungi but also nematodes enclosed in nematangia under their elytra, and also in the tracheae and Malpighian canals. Apart from phytopathogenic nematodes, bark beetles transmit mycophagous and bacterivorous nematodes and own parasites of bark beetles. The ecological groups of nematodes associated with Scolytinae are reviewed; the known records of associations of nematodes with bark beetles are listed for coniferous host plants of Russia and neighboring countries; the world-wide list of these associations for deciduous plant hosts is given.

     

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.     

Pathogens of bark beetles (Curculionidae: Scolytinae) and other beetles in Bulgaria

The presence and diversity of beetle pathogens associated with different tree species in Bulgaria was investigated. In total, 818 specimens belonging to 22 beetle species were examined. Pathogens occurred in 9 host species. The infections were found in the gut (virus, nematodes, protozoans, microsporidia) and haemolymph (nematodes) of the infected insects. The following pathogen species: ItEPV, Beauveria bassiana, B. brongniartii, Isaria farinosa, Gregarina typographi, Gregarina spp., Chytridiopsis typographi, Chytridiopsis sp., and nematodes were revealed. Insects with mycoses were mummified and filled up with fungal structures, formed pseudosclerotium. Most pathogens were established in bark beetles (Scolytinae). For the first time, the fungus B. bassiana was reported in Tomicus piniperda, Orthotomicus longicollis, O. erosus, X. spinole, Taphrorychus villifrons and Phylobius sp. in Bulgaria. We also present the first records of gregarines in O. longicollis, Acanthocinus aedilis, Rhagium inquisitor, Pyrochroa coccinea, and of the microsporidium Chytridiopsis sp. in O. longicollis.     

Steinernema feltiae (DD-136) and S. glaseri: Persistence in Soil and Bark Compost and Their Influence on Native Nematodes

Infective juveniles (J3) of the entomogenous nematodes Steinernema feltiae DD-136 (ca. 10,000 J3/100 ml) and S. glaseri (ca. 2,500 J3/100 ml) were incubated in steam-sterilized and nonsterilized sandy soil and bark compost for 8 weeks at 25 C. The nematodes were recovered by a two-step extraction procedure at 1-week intervals, and their infectivity to lepidopterous larvae (Spodoptera litura and Galleria mellonella) and their effect on the population and community of native nematodes in soil were determined. Survival of inoculated nematodes and mortality of insects were enhanced in sterilized media. Nonsterilized bark compost proved to be equally as suitable a medium as sterilized compost. In nonsterilized soil, the survival curve of S.feltiae declined more rapidly than that or S. glaseri which was less infective to insects despite its greater persistence even in nonsterilized soil. Soon after the addition of steinernematids to soil, the population of native nematodes showed a fluctuation with an increase in rhabditids and a decrease in other kinds of nematodes.     

Association of Geosmithia fungi (Ascomycota: Hypocreales) with pine- and spruce-infesting bark beetles in Poland

Bark beetles (Coleoptera, Scolytinae) are known to be associated with fungi, particularly species of the orders Ophiostomatales and Microascales. However, very little is known about other ectosymbionts of phloeophagous bark beetles on Pinaceae. In this study, we examined the Geosmithia species associated with eight bark beetle species infesting Picea abies and Pinus sylvestris branches in Poland. Fungi were isolated from 1 731 samples collected from 14 study sites. We identified a total of 653 isolates that were sorted into nine taxa based on their phenotypic similarity and phylogeny of their ITS-LSU regions of rDNA, β-tubulin, elongation factor 1α and the second-largest subunit of the RNA polymerase II gene. They represented nine species without formal names. There were large quantitative and qualitative differences in the composition of Geosmithia communities between P. sylvestris and P. abies trees. The proportion of samples infested with Geosmithia species suggests that this association is more widespread among bark beetles infesting branches of P. sylvestris. In addition, these beetles were vectors of different Geosmithia species compared with than the beetles that colonize P. abies. In mixed-conifer forests, the Geosmithia communities were more diverse and richer than in pure spruce or pine stands, where the insects Pityogenes chalcographus and Pityophthorus pityographus with low host-specificity play a distributing role for various Geosmithia species. Among eight bark beetle species examined, only P. bidentatus, P. pityographus, P. chalcographus and Polygraphus poligraphus acted as effective vectors for Geosmithia species. The following hypothesis emerges from these studies: changes in the composition of ectosymbionts of pine- and spruce-infesting bark beetles in Central Europe run along a gradient of thickness of the wood substrata preferred by insects.     

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.     

Transmission of Bursaphelenchus xylophilus through Oviposition Wounds of Monochamm carolinensis (Coleoptera: Cerambycidae)

Transmission of pinewood nematode through Monochamus carolinensis oviposition wounds was documented. Nematode transmission was measured as the average number of nematodes isolated per oviposition wound excavated and also as the percentage of oviposition wounds from which nematodes were isolated. The influence of three factors that might affect nematode transmission was investigated: age of the beetle vector, number of nematodes carried per beetle, and egg deposition in the oviposition wound. Only the number of nematodes carried by the beetle was found to have a significant effect on transmission. Nematodes were transmitted more frequently and in slightly greater numbers by beetles carrying more nematodes. The influence of pinewood on nematode exit from beetles were investigated by comparing nematode exit from beetles placed over pine chips with those placed over distilled water. Nematodes exited in greater numbers and at a higher frequency from beetles over pine chips than from beetles over distilled water. Apparently, the nematodes are able to detect a factor from the pine chips that promotes their exit from the beetles.     

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.     

Bark and ambrosia beetles (Curculionidae: Scolytinae), their phoretic mites (Acari) and associated Geosmithia species (Ascomycota: Hypocreales) from Virgilia trees in South Africa

Bark and ambrosia beetles are ecologically and economically important phloeophagous insects that often have complex symbiotic relationships with fungi and mites. These systems are greatly understudied in Africa. In the present study we identified bark and ambrosia beetles, their phoretic mites and their main fungal associates from native Virgilia trees in the Cape Floristic Region (CFR) of South Africa. In addition, we tested the ability of mites to feed on the associated fungi. Four species of scolytine beetles were collected from various Virgilia hosts and from across the CFR. All were consistently associated with various Geosmithia species, fungi known from phloeophagous beetles in many parts of the world, but not yet reported as Scolytinae associates in South Africa. Four beetle species, a single mite species and five Geosmithia species were recovered. The beetles, Hapalogenius fuscipennis, Cryphalini sp. 1, and Scolytoplatypus fasciatus were associated with a single species of Elattoma phoretic mite that commonly carried spores of Geosmithia species. Liparthrum sp. 1 did not carry phoretic mites. Similar to European studies, Geosmithia associates of beetles from Virgilia were constant over extended geographic ranges, and species that share the same host plant individual had similar Geosmithia communities. Phoretic mites were unable to feed on their Geosmithia associates, but were observed to feed on bark beetle larvae within tunnels. This study forms the first African-centred base for ongoing global studies on the associations between arthropods and Geosmithia species. It strengthens hypotheses that the association between Scolytinae beetles and dry-spored Geosmithia species may be more ubiquitous than commonly recognised.     

Number of gallery arms of the tree-killing bark beetle Polygraphus proximus (Coleoptera: Curculionidae: Scolytinae)

Polygraphus proximus Blandford (Coleoptera: Curculionidae: Scolytinae) is a non-aggressive monogynous bark beetle that has caused mass mortality in fir (Abies spp.) forests in the last decade. Although gallery structures made by polygynous beetles may influence their reproductive success, the gallery structure and the number of eggs laid by the monogynous tree-killing bark beetle P. proximus has not been investigated in detail in the natural setting of their native range. We, therefore, investigated the length of mother galleries of P. proximus and the number of eggs oviposited by the beetles in relation to gallery systems with different numbers of arms. The number of eggs oviposited in two-armed galleries was significantly greater than that observed in one- and three-armed galleries. Additionally, the length of one- or two-armed galleries with no oviposition was significantly shorter than that in galleries consisting of more than two arms. Our data may suggest that the optimal number of arms in a gallery system (in terms of the number of eggs oviposited) was two.     

Factors influencing parasitism of adult Japanese beetles,Polillia japonica (Col.: Scarabaeidae) by entomopathogenic nematodes

Several factors that influence the activity of steinernematid and heterorhabditid nematodes against adult Japanese beetles were examined in the laboratory. The effect of nematode concentration on mortality of adult beetles was evaluated using a Petri plate bioassay. The adults were exposed to 1,000 to 10,000 infective stage juveniles (J3) ofSteinernema glaseri per 10 beetles with or without food for 24 hr after which they were held with food for an additional 6 days. The LC50s for males with and without food during exposure were 3,435 and 2,854 J3s/10 adults, respectively. The LC50s for mixtures of males and females with and without food were 5,228 and 1,762 J3s/10 adults respectively. Although mortality occurred during and shortly after exposure, significant additional mortality was observed 1–4 days following exposure. Exposure of males and females with food to 10,000 J3s/10 adults for 6, 12, 18 or 24 hr resulted in 47, 58, 72 and 77% mortality, respectively. Comparative activity ofS. glaseri, S. carpocapsae (All strain),S. feltiae (Biosys experimental cold adapted strain=bibionis),S. feltiae (Biosys experimental strain 27),Heterorhabditis bacteriophora, andHeterorhabditis sp. (Terceiran isolate) was evaluated against adult Japanese beetles using a 24 hr exposure to 8,000 J3s/10 adults. The most virulent species wereS. glaseri, S. feltiae (=bibionis), the Terceiran isolate ofHeterorhabditis andS. carpocapsae producing 55, 44, 36 and 34% mortality respectively. Our results indicate that adult Japanese beetles infected with entomopathogenic nematodes could serve as a mechanism for nematode dispersal.     

New species of bark beetles from the Rovno amber (Insecta: Coleoptera: Scolytidae)

Two new species of bark beetles are described from the Late Eocene Rovno amber. Xylechinus mozolevskae sp. nov. (Hylesininae: Tomicini) is the first fossil representative of this genus, differing from recent species in having large triangular scales along the suture. Taphramites rovnoensis sp. nov. (Scolytinae: Dryocoetini) differs from the closest species, T. gnathotrichus from Baltic amber, in the presence of short thin hairs on the elytra.     

Nitrogen-Fixing and Uricolytic Bacteria Associated with the Gut of Dendroctonus rhizophagus and Dendroctonus valens (Curculionidae: Scolytinae)

The bark beetles of the genus Dendroctonus feed on phloem that is a nitrogen-limited source. Nitrogen fixation and nitrogen recycling may compensate or alleviate such a limitation, and beetle-associated bacteria capable of such processes were identified. Raoultella terrigena, a diazotrophic bacteria present in the gut of Dendroctonus rhizophagus and D. valens, exhibited high acetylene reduction activity in vitro with different carbon sources, and its nifH and nifD genes were sequenced. Bacteria able to recycle uric acid were Pseudomonas fluorescens DVL3A that used it as carbon and nitrogen source, Serratia proteomaculans 2A CDF and Rahnella aquatilis 6-DR that used uric acid as sole nitrogen source. Also, this is the first report about the uric acid content in whole eggs, larvae, and adults (male and female) samples of the red turpentine beetle (Dendroctonus valens). Our results suggest that the gut bacteria of these bark beetles could contribute to insect N balance.     

Interactions between the phloem-feeding speciesTomicus piniperda (Col.: Scolytidae) andAcanthocinus aedilis (Col.: Cerambycidae), and the predatorThanasimus formicarius (Col.: Cleridae) with special reference to brood production

Interactions betweenTomicus piniperda (L.) (Col.: Scolytidae),Acanthocinus aedilis (L.) (Col.: Cerambycidae) andThanasimus formicarius (L.) (Col.: Cleridae) were investigated in caged pine bolts. The treatments wereT. piniperda alone,A. aedilis alone,T. piniperda together withA. aedilis, T. piniperda together withT. formicarius and all three species together. T. piniperda offspring production per m² was reduced by 92% when reared withT. formicarius, by 78% when reared withA. aedilis, and by 94% when all three species were reared together, compared withT. piniperda reared alone.A. aedilis had a negative influence on the offspring production ofT. formicarius and vice versa. When both species were present in the same bolt (together withT. piniperda) offspring production was reduced by 74% forA. aedilis and by 42% forT. formicarius compared with their respective production values when each species was present alone with the bark beetle. The new generation ofT. formicarius emerged as larvae from June to August while most of theA. aedilis offspring emerged as adults from September to October, leaving only a few larvae in the bolts to hibernate.A. aedilis only reproduced in a small part of one of the bolts without bark beetles.     

Preferential host switching and codivergence shaped radiation of bark beetle symbionts,nematodes of Micoletzkya (Nematoda: Diplogastridae)

Host‐symbiont systems are of particular interest to evolutionary biology because they allow testable inferences of diversification processes while also providing both a historical basis and an ecological context for studies of adaptation. Our investigations of bark beetle symbionts, predatory nematodes of the genus Micoletzkya, have revealed remarkable diversity of the group along with a high level of host specificity. Cophylogenetic analyses suggest that evolution of the nematodes was largely influenced by the evolutionary history of beetles. The diversification of the symbionts, however, could not be attributed to parallel divergence alone; our results indicate that adaptive radiation of the nematodes was shaped by preferential host shifts among closely related beetles along with codivergence. Whereas ecological and geographic isolation have played a major role in the diversification of Micoletzkya at shallow phylogenetic depths, adaptations towards related hosts have played a role in shaping cophylogenetic structure at a larger evolutionary scale.     

Nematodes associated with the double‐spined bark beetle Ips duplicatus (Coleoptera: Curculionidae) in central Europe

Ips duplicatus (Sahlberg) is an important forest pest in central Europe, but its nematode associates have seldom been studied. Therefore, nematodes associated with I. duplicatus were determined at three localities in the Czech Republic. The percentage of beetles with phoretic nematodes ranged from 18 to 65%. Micoletzkya buetschlii and other phoretic nematodes were found under elytra, on wings, and between body segments. The percentage of beetles with nematodes in the haemocoel ranged from 3 to 30%, and the nematodes included Contortylenchus diplogaster and Parasitylenchus cf. aculeatus. Juveniles of Parasitorhabditis obtusa (Fuchs 1915) were found in the intestines of 0–16% of the beetles. The most abundant species in I. duplicatus galleries were P. obtusa and M. buetschlii. Cryptaphelenchus sp., Parasitaphelenchus sp. and unidentified tylenchid juveniles also were found in the galleries. The percentage of beetles with nematodes was greater in the overwintering than in the offspring generation, and numbers of nematodes per gallery increased with gallery development.     

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.     

Descriptions of Deladenus albizicus n. sp. and D. processus n. sp. (Nematoda: Hexatylina) from Haryana,India

Two different nematodes were isolated from the bark of Albizia lebbeck trees; one from insect infested and another from noninfested, healthy tree. Based on the biological, morphological, and molecular evidences, the nematodes are described as Deladenus albizicus n. sp. and D. processus n. sp. (Nematoda: Hexatylina). Deladenus albizicus n. sp., isolated from insect-infested tree, multiplied on the fungus Nigrospora oryzae. Myceliophagous females of this nematode reproduced by parthenogenesis and spermathecae were indistinct. Infective females, readily produced in the cultures, are dorsally curved. Only one type of males containing small-sized sperms in their genital tracts were produced in the culture. Myceliophagous females: L = 0.75 to 1.71 mm, a = 32.3 to 50.8, b = 9.3 to 11.2, b’ = 5.2 to 7.3, c = 27.2 to 35.6, V = 91.0 to 93.3, c’ = 2.0 to 2.9, stylet = 11 to 12 µm, excretory pore in the region of median pharyngeal bulb, 43 to 47 µm anterior to hemizonid. Deladenus processus n. sp., isolated from bark of healthy A. lebbeck tree, was cultured on Alternaria alternata. Myceliophagous females reproduced by amphimixis and their spermathecae contained rounded sperms. Infective females were never produced, even in old cultures. Myceliophagous females: L = 0.76 to 0.99 mm, a = 34 to 49, b = 13.3 to 17.7, b’ = 3.8 to 5.8, c = 19.6 to 22.8, V = 92.2 to 93.5, c’ = 2.7 to 3.5, stylet = 6 to 7 µm, excretory pore in the proximity of hemizonid, tail conoid, tapering from both sides to a long pointed central process. It is proposed to classify Deladenus species in three groups: durus, siricidicola, and laricis groups based on female and spermatogonia dimorphism, mode of reproduction, and insect parasitism.     

Maturation feeding and transmission of Bursaphelenchus xylophilus (Nematoda: Parasitaphelenchidae) by Monochamus alternatus (Coleoptera: Cerambycidae) inoculated with Beauveria bassiana (Deuteromycotina: Hyphomycetes)

We examined the amount of maturation feeding and transmission of pinewood nematodes, Bursaphelenchus xylophilus (Steiner et Buhrer) Nickle (Nematoda: Parasitaphelenchidae), to healthy pine (Pinus spp.) trees by pine sawyer Monochamus alternatus Hope (Coleoptera: Cerambycidae) adults infected with Beauveria bassiana (Balsamo) Vuill. (Deuteromycotina: Hyphomycetes). Inoculated beetles fed less than noninoculated beetles, probably because feeding by inoculated beetles began to decrease at about 4 d postinoculation and inoculated beetles ceased to feed for several days before their death. In inoculated beetles carrying >1,000 nematodes, some beetles died before nematode departure. The remaining heavily nematode-infested beetles lived until the beginning of nematode departure, but they had stopped feeding, preventing the nematodes from entering pine twigs. We suggest that microbial control of pine sawyer adults by B. bassiana may be effective in preventing transmission of pine wilt disease to healthy pine trees.