Isolation and characterization of 16 microsatellite loci in the mountain pine beetle, Dendroctonus ponderosae Hopkins (Coleoptera: Curculionidae: Scolytinae)

We isolated 16 polymorphic microsatellite loci in the mountain pine beetle (Dendroctonus ponderosae Hopkins) and developed conditions for amplifying these markers in four multiplex reactions. Three to 14 alleles were detected per locus across two sampled populations. Observed and expected heterozygosities ranged from 0.000 to 0.902 and from 0.100 to 0.830, respectively. Three loci deviated from Hardy-Weinberg equilibrium in one sampled population. One of these loci may be sex linked. These markers will be useful in the study of population structure in this important pest species.     

Areography of the genus Dendroctonus (Coleoptera: Curculionidae: Scolytinae) in Mexico

Objective To analyse whether the geographical ranges of Dendroctonus species are (1) associated with factors such as host species or elevation, and (2) in agreement with Halffter's Nearctic distribution pattern. (3) To identify and discuss the factors that are likely to act as barriers to the genus’ geographical distribution. (4) To explore whether there is an association between the size of the geographical ranges of Dendroctonus species and the number of Pinus host species used by each of them, and (5) to assess if these host species are most common at the elevations preferred by the individual Dendroctonus species. Site Mexico. Methods Records of 12 species of Dendroctonus were gathered from entomological collections in Mexico. Distribution ranges were defined by using the propinquity method ( Rapoport, 1975a ). Analysed parameters were: (1) geographical distribution of single species, (2) overlapping of species ranges, (3) disjunction patterns and barriers by means of isoprobabilistic lines, based on the morphotectonic subdivision of Mexico ( Ferrusquía‐Villafranca, 1998 ), (4) spatial variation in species richness with respect to latitude and altitude, (5) size of geographical ranges, and (6) host species for each Dendroctonus species. A correlation was determined between area size and number of pine host species. Results The species ranges varied in shape and size. Geographical ranges tend to be discontinuous in shape. Composite patterns showed that disjunctions among ranges do not closely follow Mexico's morphotectonic subdivision. There are repeated discontinuities among individual distributions, which define five areas: (1) Baja California Peninsula, (2) Sierra Madre Occidental (SMOC), (3) northern Sierra Madre Oriental (SMOR), (4) Sierra Madre de Chiapas, and (5) SMOR + Faja Volcanica Transmexicana (FVT) + Sierra Madre del Sur. The isoprobabilistic lines confirm that the inner part of SMOC provides an optimal environment for the genus, and the FVT province constitutes the broader corridor for it in the country. Richness does not directly decrease or increase with latitude. Richness behaviour of the insect is not associated with that of its host. Elevation distributions showed that most Dendroctonus species move within broad margins of tolerance and species richness is concentrated in the montane interval. Dendroctonus attack 24 of the 47 Pinus species distributed in Mexico. Preferred pine species belong predominantly to Leiophyllae, Ponderosae and Oocarpae subsections. The Spearman rank correlation between area size and number of pine host species was not significant. Dendroctonus clearly belongs to a Nearctic distribution pattern (sensu Halffter, 1987 ). Main conclusions Dendroctonus is present in all montane systems of Mexico and its species coexist within a high geographical sympatry. Overlapping of species distribution appears to be the result of two elements – generalized polyphagy inside Pinus and a wide elevation tolerance within mountainous environments. This behaviour, linked to a high vagility, has allowed the genus Dendroctonus to expand its distribution across Mexico and to employ mountainous systems as corridors separated by barriers that exert a low selective filter effect.     

Historical demography and phylogeography of a specialist bark beetle, Dendroctonus pseudotsugae Hopkins (Curculionidae: Scolytinae)

Contemporary distribution of North American species has been shaped by past glaciation events during the Quaternary period. However, their effects were not as severe in the southern Rocky Mountains and Northern Mexico as elsewhere in North America. In this context, we test hypotheses about the historical demography of Dendroctonus pseudotsugae, based on 136 haplotypes of mitochondrial cytochrome oxidase I. The phylogenetic analysis yielded four haplogroups corresponding to northwestern United States and southwestern Canada (NUS), southwestern United States (Arizona, SUS), northwestern Mexico (Sierra Madre Occidental, SMOC), and northeastern Mexico (Sierra Madre Oriental, SMOR). Predictions of demographic expansion were examined through neutrality tests against population growth and mismatch distribution. Results showed that the NUS and SMOC haplogroups have experienced demographic expansion events, whereas the SUS and SMOR haplogroups have not. Divergence times between pairs of haplogroups were estimated from early to middle Pleistocene. The longer divergence time between NUS and all other haplogroups could be the result of refugia within the Pacific Northwest and northern Rocky Mountains and long-term isolation from southernmost populations in Mexico. The results obtained in this study are in agreement with the evolutionary history of the host Douglas-fir, as the warmer climates of interglacial periods pushed conifers northward of Colorado, New Mexico, and Arizona, whereas environmental changes reduced the population size of Douglas-fir and forced fragmentation of distribution range southward into northern Mexico.     

The first study on the bacterial flora of the european spruce bark beetle, Dendroctonus micans (Coleoptera: Scolytidae)

The European spruce bark beetle, Dendroctonus micans Kugelann (Coleoptera, Scolytidae), is one of the most serious pests of oriental spruce (Picea orientalis L.) in Turkey. In this study, we investigated bacterial flora of D. micans collected from different populations of the forests of Eastern Black Sea Region of Turkey from 2002 to 2004. Seven different bacteria were isolated from healthy, diseased and dead specimens based on the color of colony and morphology. According to morphological, physiological and biochemical properties, metobolic enyzme profile by BIOLOG microtiter plate system, and total cellular fatty acid profile by Microbial Identification System (MIS), isolates were identified as Micrococcus luteus, Bacillus thuringiensis subsp. morrisoni, Serratia grimesii, Enterobacter cloaceae, Enterobacter intermedius, Streptococcus sp. and Pseudomonas putida. This is the first study on the bacterial flora of D. micans.     

Helicosporidium infection of the great European spruce bark beetle, Dendroctonus micans (Coleoptera: Scolytidae)

An infection of the great European spruce bark beetle Dendroctonus micans (from Turkey) by the parasitic green alga Helicosporidium is described. This is the first time that Helicosporidium has been found to infect a bark beetle (Scolytidae) and the first time that a naturally infected beetle has been reported for the Eurasian continent. The typical cysts of Helicosporidium contain three ovoid cells and one helical, filamentous cell. The morphological characteristics are revealed by light and electron microscopy. Distinct electron-dense inclusions with a peculiar ultrastructure may represent pyrenoids. Since D. micans is an important pest, the discovery of a natural pathogen may offer a chance for biological control.     

Metabolism and cold tolerance of Chinese white pine beetle Dendroctonus armandi (Coleoptera: Curculionidae: Scolytinae) during the overwintering period

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Gut-associated yeast in bark beetles of the genus Dendroctonus Erichson (Coleoptera: Curculionidae: Scolytinae)

Scolytine bark beetles are the most destructive pests of conifers; they sometimes aggregate in such large numbers that they actually kill their hosts. They maintain close relationships with yeasts and fungi, in particular those that are assumed to aid in digestive, detoxification processes and pheromone production. In this study, 403 yeast strains were isolated from the guts, ovaries, eggs and frass of nine bark beetle species in the genus Dendroctonus Erichson. The beetles were collected from 10 conifer species at 34 locations in Mexico, Guatemala and the USA. Yeast identification was based on partial DNA sequences from 18S rDNA, 26S rDNA and internal transcribed spacer (ITS1), as well as morphological and physiological characteristics. A combined phylogenetic analysis delimited 11 clades with sequences similar to Candida arabinofermentans , C. ernobii , C. membranifaciens (including C. lessepsii , Pichia mexicana and P. scolyti ), C. oregonensis , C. piceae , Kuraishia capsulata (including K. capsulata and K. cf. molischiana ), Pichia americana , P. canadensis , P. glucozyma , P. guilliermondii and an undescribed species of Candida . Nucleotide divergences between the major clades were at least 5% while, with the exception of 30 isolates, yeasts within clades differed from named reference species at fewer than 1% of the nucleotide sites. There do not appear to be obligate relationships between particular yeasts and specific anatomical partitions, nor between particular yeasts and bark beetle species. Some yeasts do appear to be preferentially associated with bark beetles feeding on different conifer genera and therefore host plant defences may limit yeast community diversity in Dendroctonus .  © 2009 The Linnean Society of London, Biological Journal of the Linnean Society , 2009, 98 , 325–342.     

Screening of entomopathogenic fungi against the European spruce bark beetle,Dendroctonus micans (Coleoptera: Scolytidae)

The European spruce bark beetle, Dendroctonus micans Kugelann (Coleoptera: Scolytidae), is one of the most serious destructive pests of oriental spruce (Picea orientalis L.). In order to find an effective biocontrol agent against this pest, we determined the effectiveness of entomopathogenic fungi against D. micans. Virulence of nine highly pathogenic strains including Beauveria bassiana (2), Beauveria cf. bassiana (2), Metarhizium anisopliae (2), Metarhizium sp. (1), Isaria fumosorosea (1) and Evlachovaea sp. (1) was evaluated on D. micans larvae and adults under laboratory conditions. Mortality values for both larvae and adults ranged from 83 to 100% and from 23 to 100%, respectively. Larvae were more susceptible to fungi than adults (P<0.05). Based on screening tests, B. cf. bassiana isolate KTU-53 was found the most effective isolate. LC₅₀ values were calculated as 1.77×10⁴ and 2.65×10⁴ conidia mL^?1 for isolate KTU-53 against larvae and adults, respectively. Consequently, B. cf. bassiana isolate KTU-53 appears to be the most promising microbial control agent for biocontrol of D. micans.     

Phylogeography of spruce beetles (Dendroctonus rufipennis Kirby) (Curculionidae: Scolytinae) in North America

Tree-feeding insects that are widespread in north temperate regions are excellent models for studying how past glaciations have impacted differentiation and speciation. We used mitochondrial DNA (mtDNA) sequences and allele frequencies at nine microsatellite loci to examine genetic population structure across the current range of the spruce beetle (Dendroctonus rufipennis), an economically important insect in North America. Two major haplotype groups occur across northern North America, from Newfoundland to Alaska, on white spruce (Picea glauca), and a third distinctive haplotype group occurs throughout the Rocky Mountains on Engelmann spruce (Picea engelmannii). The two mtDNA lineages found in northern populations are 3-4% divergent from each other and from the lineages found in the Rocky Mountains. Analyses of microsatellite data also suggest the existence of major population groupings associated with different geographical regions. In the Pacific Northwest, concordant contact zones for genetically distinct populations of spruce beetles and their principal hosts appear to reflect recent secondary contact. Although we could detect no evidence of historical mtDNA gene flow between allopatric population groups, patterns of variation in the Pacific Northwest suggest recent hybridization and introgression. Together with the pollen record for spruce, they also suggest that beetles have spread from at least three glacial refugia. A minimum estimate of divergence time between the Rocky Mountain and northern populations was 1.7 Myr (million years), presumably reflecting the combined effects of isolation during multiple glacial cycles.     

Temperature‐dependent development of the great European spruce bark beetle Dendroctonus micans (Kug.) (Coleoptera: Curculionidae: Scolytinae) and its predator Rhizophagus grandis Gyll. (Coleoptera: Monotomidae: Rhizophaginae)

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Phylogeny of Dendroctonus bark beetles (Coleoptera: Curculionidae: Scolytinae) inferred from morphological and molecular data

Bark beetles in the genus Dendroctonus may attack and kill several species of coniferous trees, some of them causing major economic losses in temperate forests throughout North and Central America. For this reason, they have been widely studied. However, various aspects of the taxonomy and evolutionary history of the group remain contentious. The genus has been subdivided in species groups according to morphological, biological, karyological or molecular attributes, but the evolutionary affinities among species and species groups within the genus remain uncertain. In this study, phylogenetic relationships among Dendroctonus species were reassessed through parsimony‐based cladistic analysis of morphological and DNA sequence data. Phylogenetic inference was based on 36 morphological characters and on mitochondrial DNA sequences of the cytochrome oxidase I (COI) gene. Analyses were carried out for each dataset, as well as for the combined data analysed simultaneously, under equal and implied weights. According to the combined analysis, the genus Dendroctonus is a monophyletic group defined by at least three synapomorphic characters and there are four main lineages of varied composition and diversity within the genus. Within these lineages, several monophyletic groups match, to some extent, species groups defined by previous authors, but certain groups proposed by those authors are polyphyletic or paraphyletic.     

Energy use by the mountain pine beetle (Coleoptera: Curculionidae: Scolytinae) for dispersal by flight

The mountain pine beetle Dendroctonus ponderosae Hopkins is a major native pest of Pinus Linnaeus (Pinaceae) in western North America. Host colonization by the mountain pine beetle is associated with an obligatory dispersal phase, during which beetles fly in search of a suitable host. Mountain pine beetles use stored energy from feeding in the natal habitat to power flight before host colonization and brood production. Lipids fuel mountain pine beetle flight, although it is not known whether other energy sources are also used during flight. In the present study, we compare the level of energy substrates, proteins, carbohydrates and lipids of individual mountain pine beetles flown on flight mills with unflown control beetles. We use a colorimetric method to measure the entire metabolite content of each individual beetle. The present study reveals that mountain pine beetles are composed of more protein and lipid than carbohydrate. Both female and male mountain pine beetles use lipids and carbohydrates as energy sources during flight. There is variation between sexes, however, in the energy substrates used for flight. Male mountain pine beetles use protein, in addition to lipids and carbohydrates, to fuel flight, whereas protein content is not different between flown and control females.     

Artificial diet and rearing methods for Thanasimus dubius (Coleoptera: Cleridae), a predator of bark beetles (Coleoptera: Scolytidae)

Clerid beetles are common natural enemies of bark beetles, and could potentially be used as biological control agents if they could be reared in sufficient numbers. We developed an artificial diet devoid of insect components for rearing Thanasimus dubius (Fabricius), a clerid that attacks several economically important bark beetles in eastern North America. We reared larvae of this predator using the artificial diet, and then used either natural or factitious prey to feed the adults so produced. Two different methods of presenting the diet were also examined. We then compared the performance of T. dubius reared on the artificial diet with newly-emerged wild individuals collected from the field. Our results suggest that adult predators reared on the diet are near in quality to wild ones, and high R₀ values can be obtained. No difference in prey preference was found between wild and diet-reared individuals after five generations in the laboratory. Sufficient numbers of predators could be generated using these techniques to permit limited field trials of augmentative biological control.     

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.     

Testing phylogeographic predictions on an active volcanic island: Brachyderes rugatus (Coleoptera: Curculionidae) on La Palma (Canary Islands)

Volcanic islands with well-characterized geological histories can provide ideal templates for generating and testing phylogeographic predictions. Many studies have sought to utilize these to investigate patterns of colonization and speciation within groups of closely related species across a number of islands. Here we focus attention within a single volcanic island with a well-characterized geological history to develop and test phylogeographic predictions. We develop phylogeographic predictions within the island of La Palma of the Canary Islands and test these using 69 haplotypes from 570 base pairs of mitochondrial DNA cytochrome oxidase II sequence data for 138 individuals of Brachyderes rugatus rugatus, a local endemic subspecies of curculionid beetle occurring throughout the island in the forests of Pinus canariensis. Although geological data do provide some explanatory power for the phylogeographic patterns found, our network-based analyses reveal a more complicated phylogeographic history than initial predictions generated from data on the geological history of the island. Reciprocal illumination of geological and phylogeographic history is also demonstrated with previous geological speculation gaining phylogeographic corroboration from our analyses.     

Isolation and virulence of entomopathogenic fungi against the great spruce bark beetle,Dendroctonus micans (Kugelann) (Coleoptera: Scolytidae)

Twelve fungal strains including Lecanicillium muscarium (Petch.) Zare and Gams, Isaria farinosa (Holmsk.) Fr., Fusarium sp., Beauveria bassiana Sensu Lato and Beauveria sp. were isolated from larvae and adults of D. micans. In addition, virulence of these isolates against this pest was determined. Conidia suspensions of 1×10⁶ conidia mL^–1 were applied to larvae and adults. The highest mortality and mycosis for larvae were obtained from isolate ARSEF 9271 (Beauveria bassiana) with 90% mortality and mycosis within 10 days. ARSEF 9271 also produced 93% mortality and mycosis in adults. On the other hand, the highest mortality and mycosis for adults were obtained with isolate ARSEF 9272 (Beauveria sp.), with 100% mortality and 80% mycosis within 10 days. These results indicate that isolates ARSEF 9271 and ARSEF 9272 seem to be the most promising potential fungal biocontrol agents against D. micans.     

The role of lignin as a defence against the spruce bark beetle Dendroctonus micans: effect on larvae and adults

Summary The role of lignin as a physical defence against Dendroctonus micans was investigated in laboratory feeding experiments. The effect of lignin is dose-dependent, reducing larval survival, growth rate, and weight, as well as affecting gallery construction. Adults lay fewer eggs in lignified bark and also tend to construct abnormal galleries. The distribution of lignin in trees suggests a role in defence against bark beetles that feed in the thicker bark on the lower bole.     

Prepupal diapause and instar IV developmental rates of the spruce beetle, Dendroctonus rufipennis (Coleoptera: Curculionidae, Scolytinae)

The spruce beetle, Dendroctonus rufipennis (Kirby), is an important mortality agent of native spruces throughout North America. The life-cycle duration of this species varies from 1 to 3 years depending temperature. The univoltine cycle (one generation per year) is thought to maximize outbreak risk and accelerate host mortality in established outbreaks. Prepupal diapause is associated with the semivoltine cycle (one generation per 2 years) and we investigated thermal conditions that result in diapause induction. Preliminary experiments used respirometry in an attempt to distinguish the diapause state of experimental insects but the technique was apparently confounded by low respiration before and during pupation, regardless of diapause status. Therefore, diapause induction was deduced using developmental delays. The observed developmental response was not a “switch”, with developmental delay either present or absent, but instead varied continuously. We found that temperatures <15 °C from instar III through mid-instar IV were associated with developmental delays beyond that expected from cool temperatures. Moreover, the duration of exposure to cool temperatures was important in determining the degree of developmental delay. Small, if any, delays were observed if the cumulative exposure to <15 °C was <20 d whereas >40 d cumulative exposure was associated with distinct developmental suppression. Intermediate exposure to cool temperatures resulted in minor developmental delays. We used our results to parameterize a maximum likelihood estimation model of temperature-dependent instar IV developmental rates, including the effect of diapause. This model can be included as part of a spruce beetle phenology model for predicting population dynamics.     

DNA条形码试剂盒检测技术在大小蠹属种类鉴定中的应用

[目的]DNA条形码技术已成为生物分类鉴定的有力工具.DNA条形码技术的相关问题,如物种种内和种间的遗传距离出现重叠区域,将直接影响到物种鉴定的准确性.我们应用DNA条形码试剂盒检测技术来快速、准确地鉴定口岸截获的检疫性大小蠹属种类.[方法]针对大小蠹昆虫设计引物以提高PCR扩增效率.运用自主研发的基因条码分析软件找出基因片段上区分每个物种的多态位点规律,作为该物种的鉴定特征并建立数据库,应用于物种鉴定.[结果]使用针对大小蠹属昆虫设计的引物成功扩增出325 bp的COI基因片段.将大小蠹属12种昆虫的COI基因片段上的核苷酸诊断位点的组合作为物种的鉴定特征,可以准确地区分近似种.通过比对植物检疫鉴定系统数据库里的鉴定特征,将6个大小蠹属的未知样品成功鉴定到种(核苷酸序列一致性为100％),与形态鉴定结果一致.[结论]结果表明DNA条形码试剂盒检测技术可以准确鉴定大小蠹属的种类.该检测技术可以应用于其他经济重要性有害生物的检测鉴定.     

Effect of associated fungi on the immunocompetence of red turpentine beetle larvae,Dendroctonus valens (Coleoptera: Curculionidae: Scolytinae)

Abstract Dendroctonus–fungus symbioses are often considered as the ideal model systems to study the development and maintenance of ectosymbioses, and diverse interactions, including antagonism, commensalism and mutualism, have been documented between these organisms. The red turpentine beetle, Dendroctonus valens LeConte (Coleoptera: Curculionidae: Scolytinae) is a pine‐killing invasive beetle in northern China. Fungi species Ophiostoma minus, Leptographium sinoprocerum, L. terebrantis and L. procerum were associated with this bark beetle. Antagonistic interactions between D. valens and its associated fungi, such as O. minus and L. sinoprocerum, have been demonstrated, but the underlying causes of this phenomenon are unknown. Here, we first found the two tested fungi species retarded the net weight gain of D. valens larvae after completing 3‐day feeding on their media. Furthermore, we provide direct evidence indicating the effect of associated fungi on the immunocompetence of D. valens larvae to explain the documented antagonism. Our results showed that the activity of phenoloxidase and total phenoloxidase in D. valens larvae were significantly upregulated by two strains of associated fungi, O. minus and L. sinoprocerum as compared with the controls. The phenoloxidase ratio increased significantly in the larvae which had fed for 3 days on media inoculated with O. minus. Because insect immune defenses are costly to be deployed, these results could be explored as one of the underlying mechanisms of the documented antagonism.