New qPCR protocol to detect Diplodia corticola shows phoretic association with the oak pinhole borer Platypus cylindrus.

BACKGROUND: Botryosphaeria canker (causal agent: Diplodia corticola) is considered one of the most important diseases of cork oak (Quercus suber) stands since it necrotizes the subero-phellodermic layer preventing cork regeneration after harvesting. One of the most intriguing etiological issues of this disease is its plausible spreading through trunk borer insects. In this study, we highlight the phoretic relationship between D. corticola and the oak pinhole borer Platypus cylindrus (Coleoptera, Curculionidae) which massively colonizes debarked cork oaks in southern Europe. We extracted DNA from 154 adults of P. cylindrus collected in six cork oak stands in north-eastern Spain during the summer of 2021. We developed a new nested quantitative PCR-based protocol for quick detection of D. corticola carried by insects. RESULTS: The use of real time amplification of a highly specific mitochondrial marker allowed us to detect spore loads down to a single conidium within the first 29 cycles of qPCR. The 29.62% of insect pools (corresponding to 31.16% of studied insects) resulted in an estimated spore load higher than one conidium/insect, with a moderate value of mean spore load for the whole dataset (~33 conidia/insect). Estimated spore load was significantly higher in May and August, regardless of insect abundance in the field. CONCLUSION: This study provides new tools for diagnosis of this emergent pathogen that would be useful for developing monitoring strategies and epidemiological studies. © 2022 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Succession of Ambrosia Beetles Colonizing the Logs of Fallen Alder and Birch Trees.

Ambrosia beetles bore into the xylem of woody plants, reduce timber quality, and can sometimes cause devastating damage to forest ecosystems. The colonization by different beetle species is dependent on host status, from healthy trees to the early stages of wood decay, although the precise factors influencing their host selection are not well known. Classic studies on plant ecology have determined the niches of different plant species in vegetation succession, based on comparisons of successions in different locations using ordination analyses, although the factors influencing the colonization of each species are largely undetermined. In this study, to characterize the succession of ambrosia beetles after tree felling, two Betulaceae tree species, an alder (Alnus hirsuta), and a white birch (Betula platyphylla var. japonica) were felled as bait logs in central Hokkaido, Japan, in 2016. From 2016 to 2018, the bait logs were dissected late in each flying season, and ambrosia beetles were collected from the logs. During the period of monitoring, the beetle colonization in both tree species was mostly concentrated in the first 2 years. We observed similarities in the beetle faunas colonizing the two plant species, and that individual species appeared in the same sequence in the logs of the two plant species, although the temporal patterns of colonization differed. Consequently, significant differences in beetle community compositions in the two host species were detected in each of the first 2 years of the study, whereas the difference in the overall composition of beetle assemblages (=pooled over 3 years) between the two plant species was smaller than that in either 2016 or 2017. We speculated that the differences in the temporal pattern of colonization could be attributable to differences in the rates at which the wood of the two tree species deteriorated. Treptoplatypus severini and Xylosandrus crassiusculus were considered to be early-successional species that commenced log colonization soon after felling, although T. severini has a wide niche and was collected during all 3 years of the study. Conversely, Xyleborinus attenuatus and Heteroborips seriatus were identified as probable late-successional species that showed a preference for older logs.

Microbiome Associated with the Mycangia of Female and Male Adults of the Ambrosia Beetle Platypus cylindrus Fab. (Coleoptera: Curculionidae).

The ambrosia beetle Platypus cylindrus Fab. (Coleoptera: Curculionidae) is a major cork oak pest in Portugal. Female and male beetles have different roles in host tree colonization and are both equipped with prothoracic mycangia for fungal transportation. Despite a known beneficial role of bacteria in ambrosia beetles, information on bacterial composition associated with prothoracic mycangia structures is scarce. Bacterial community from mycangia of P. cylindrus male and female beetles collected from cork oak galleries was investigated by means of 16S metagenomics. Mycangia anatomical structure was also explored with histological techniques and X-ray computed microtomography to highlight evidence supporting biological sexual dimorphism. A bacterial community with highly diverse bacterial taxa with low abundances at the genus level was revealed. Lactobacillales, Leptotrichia, Neisseria, Rothia, and Sphingomonadaceae were significantly more abundant in males, while Acinetobacter, Chitinophagaceae, Enterobacteriaceae, Erwiniaceae, Microbacteriaceae, and Pseudoclavibacter were more abundant in females. Additionally, a core bacteriome of five genera was shared by both sexes. Histological examination revealed visible connections linking external and internal tissues in females, but none in males. Overall, these results provide the first insights into sexual differentiation for bacteria in a Platypodinae beetle species, identifying key patterns of bacteria distribution in the context of beetle ecology and functional behavior.

Influence of the color of semi-funnel traps on Xylophagous coleoptera capture efficiency in forest fragments

Bark and ambrosia beetles, mainly the ones belonging to groups Scolytinae, Bostrichidae and Platypodinae, can kill trees from reforestation areas or native forests and damage the wood. Population monitoring and the identification of quarantine species are carried out by assembling ethanol-baited traps. The aims of the current study are to evaluate the influence of the color of ethanol-baited traps on the efficient capture of these insects, as well as to measure changes in colorimetric variables based on trap exposure in the field and to investigate whether these changes affect capture efficiency. Eight ethanolic traps (red, yellow, black and transparent traps - two of each color) were installed in a forest fragment in the first experimental stage ­ samples were collected on a weekly basis, for 11 months. New and used transparent traps were installed in the field in the second experimental stage - samples were collected for additional 11 months. A portable spectrophotometer was used to measure the colorimetric variables in these traps. The mean number of Scolytinae individuals (± SD) captured in transparent traps (48±50) was significantly higher than that of individuals captured in black (24±25), yellow (23±21) and red (22±21) traps. However, transparent traps subjected to field conditions were colonized by such as fungi, bacteria and mosses, which changed the transparent state of the traps into a darkened color and significantly affected their capture efficiency. The total number of 6,268 Scolytinae individuals were collected at this experimental stage: 4,977 of them were captured in new traps, whereas 1,291 were captured in the old ones. Based on the herein measured colorimetric variables, such color change got significantly intensified as transparent traps remained under field conditions. In conclusion, transparent traps were more efficient in capturing Scolytinae individuals than the black, yellow and red traps. In addition, the exposure to field conditions has progressively changed equipment color and decreased its capture efficiency.

Relationships among wood-boring beetles, fungi, and the decomposition of forest biomass.

A prevailing paradigm in forest ecology is that wood-boring beetles facilitate wood decay and carbon cycling, but empirical tests have yielded mixed results. We experimentally determined the effects of wood borers on fungal community assembly and wood decay within pine trunks in the southeastern United States. Pine trunks were made either beetle-accessible or inaccessible. Fungal communities were compared using culturing and high-throughput amplicon sequencing (HTAS) of DNA and RNA. Prior to beetle infestation, living pines had diverse fungal endophyte communities. Endophytes were displaced by beetle-associated fungi in beetle-accessible trees, whereas some endophytes persisted as saprotrophs in beetle-excluded trees. Beetles increased fungal diversity several fold. Over forty taxa of Ascomycota were significantly associated with beetles, but beetles were not consistently associated with any known wood-decaying fungi. Instead, increasing ambrosia beetle infestations caused reduced decay, consistent with previous in vitro experiments that showed beetle-associated fungi reduce decay rates by competing with decay fungi. No effect of bark-inhabiting beetles on decay was detected. Platypodines carried significantly more fungal taxa than scolytines. Molecular results were validated by synthetic and biological mock communities and were consistent across methodologies. RNA sequencing confirmed that beetle-associated fungi were biologically active in the wood. Metabarcode sequencing of the LSU/28S marker recovered important fungal symbionts that were missed by ITS2, though community-level effects were similar between markers. In contrast to the current paradigm, our results indicate ambrosia beetles introduce diverse fungal communities that do not extensively decay wood, but instead reduce decay rates by competing with wood decay fungi.

A mid-Cretaceous ambrosia fungus, Paleoambrosia entomophila gen. nov. et sp. nov. (Ascomycota: Ophiostomatales) in Burmese (Myanmar) amber, and evidence for a femoral mycangium.

An ambrosia fungus is described from filamentous sporodochia adjacent to a wood-boring ambrosia beetle (Coleoptera: Curculionidae: Platypodinae) in mid-Cretaceous Burmese amber. Yeast-like propagules and hyphal fragments of Paleoambrosia entomophila gen. nov. et sp. nov. occur in glandular sac mycangia located inside the femur of the beetle. This is the first record of a fossil ambrosia fungus, showing that symbiotic associations between wood-boring insects and ectosymbiotic fungi date back some 100 million years ago. The present finding moves the origin of fungus-growing by insects from the Oligocene to the mid-Cretaceous and suggests a Gondwanan origin.

Detecting Symbioses in Complex Communities: the Fungal Symbionts of Bark and Ambrosia Beetles Within Asian Pines.

Separating symbioses from incidental associations is a major obstacle in symbiosis research. In this survey of fungi associated with Asian bark and ambrosia beetles, we used quantitative culture and DNA barcode identification to characterize fungal communities associated with co-infesting beetle species in pines (Pinus) of China and Vietnam. To quantitatively discern likely symbioses from coincidental associations, we used multivariate analysis and multilevel pattern analysis (a type of indicator species analysis). Nearly half of the variation in fungal community composition in beetle galleries and on beetle bodies was explained by beetle species. We inferred a spectrum of ecological strategies among beetle-associated fungi: from generalist multispecies associates to highly specialized single-host symbionts that were consistently dominant within the mycangia of their hosts. Statistically significant fungal associates of ambrosia beetles were typically only found with one beetle species. In contrast, bark beetle-associated fungi were often associated with multiple beetle species. Ambrosia beetles and their galleries were frequently colonized by low-prevalence ambrosia fungi, suggesting that facultative ambrosial associations are commonplace, and ecological mechanisms such as specialization and competition may be important in these dynamic associations. The approach used here could effectively delimit symbiotic interactions in any system where symbioses are obscured by frequent incidental associations. It has multiple advantages including (1) powerful statistical tests for non-random associations among potential symbionts, (2) simultaneous evaluation of multiple co-occurring host and symbiont associations, and (3) identifying symbionts that are significantly associated with multiple host species.

Spatio-Temporal Distribution of Bark and Ambrosia Beetles in a Brazilian Tropical Dry Forest.

Bark and the ambrosia beetles dig into host plants and live most of their lives in concealed tunnels. We assessed beetle community dynamics in tropical dry forest sites in early, intermediate, and late successional stages, evaluating the influence of resource availability and seasonal variations in guild structure. We collected a total of 763 beetles from 23 species, including 14 bark beetle species, and 9 ambrosia beetle species. Local richness of bark and ambrosia beetles was estimated at 31 species. Bark and ambrosia composition was similar over the successional stages gradient, and beta diversity among sites was primarily determined by species turnover, mainly in the bark beetle community. Bark beetle richness and abundance were higher at intermediate stages; availability of wood was the main spatial mechanism. Climate factors were effectively non-seasonal. Ambrosia beetles were not influenced by successional stages, however the increase in wood resulted in increased abundance. We found higher richness at the end of the dry and wet seasons, and abundance increased with air moisture and decreased with higher temperatures and greater rainfall. In summary, bark beetle species accumulation was higher at sites with better wood production, while the needs of fungi (host and air moisture), resulted in a favorable conditions for species accumulation of ambrosia. The overall biological pattern among guilds differed from tropical rain forests, showing patterns similar to dry forest areas.

Classification of weevils as a data-driven science: leaving opinion behind.

Data and explicit taxonomic ranking criteria, which minimize taxonomic change, provide a scientific approach to modern taxonomy and classification. However, traditional practices of opinion-based taxonomy (i.e., mid-20(th) century evolutionary systematics), which lack explicit ranking and naming criteria, are still in practice despite phylogenetic evidence. This paper discusses a recent proposed reclassification of weevils that elevates bark and ambrosia beetles (Scolytinae and Platypodinae) to the ranks of Family. We demonstrate that the proposed reclassification 1) is not supported by an evolutionary systematic justification because the apparently unique morphology of bark and ambrosia beetles is shared with other unrelated wood-boring weevil taxa; 2) introduces obvious paraphyly in weevil classification and hence violates good practices on maintaining an economy of taxonomic change; 3) is not supported by other taxonomic naming criteria, such as time banding. We recommend the abandonment of traditional practices of an opinion-based taxonomy, especially in light of available data and resulting phylogenies.

The external morphology of Austroplatypus incompertus (Schedl) (Coleoptera, Curculionidae, Platypodinae).

Previous descriptions of adult Austroplatypus incompertus (Schedl) are completed by the addition of descriptions and illustrations of the adults and, in particular, their maxillary palps. I describe and illustrate the non-adult phases of the life cycle and provide a key to the larval instars. The sexual dimorphism of Austroplatypus incompertus is atypical and includes a latitudinal cline which obeys Bergmann's rule. The taxonomic position of the genus within the Platypodinae is clarified. Platypus incostatus Schedl is recognised as the male of the species, and hence a new synonym of Austroplatypus incompertus.