Ultrastructure of the mycangium of the southern pine beetle,Dendroctonus frontalis (Coleoptera: Curculionidae,Scolytinae): complex morphology for complex interactions

Yuceer, C, Hsu, C.‐Y., Erbilgin, N and Klepzig, K.D. 2011. Ultrastructure of the mycangium of the southern pine beetle, Dendroctonus frontalis (Coleoptera: Curculionidae, Scolytinae): complex morphology for complex interactions. —Acta Zoologica (Stockholm) 92 : 216–224. The southern pine beetle (SPB) (Dendroctonus frontalis Zimmermann) is the most economically important pest of southern pine forests. Beetles carry fungal cells within specialised cuticular structures, called mycangia. Little is known about the mycangia ultrastructure or function. We used cryo‐fracturing and scanning electron microscopy to examine the ultrastructural features of SPB mycangia and surrounding tissues. Mycangia, one on each side of anterior portion of the prothorax, are terminated on the dorsal side at a ‘mycangial bridge’. This sclerotised mycangial bridge does not appear to provide a passage between the two mycangia, suggesting that each mycangium functions independently. Mycangia are surrounded by abundant tracheoles connecting the structures to the outside via openings within the prothorax. Previously unknown pits overlying the mycangial gland cells were also observed in both the inner wall and anterior fold of prothorax. We hypothesise that these openings and pits may play roles in determining which fungi enter, and grow within, the mycangium.     

Nonhost angiosperm volatiles and verbenone disrupt response of western pine beetle, Dendroctonus brevicomis (Coleoptera: Scolytidae), to attractant-baited traps

Nonhost angiosperm volatiles and verbenone were tested for their ability to disrupt the response of western pine beetle, Dendroctonus brevicomis LeConte (Coleoptera: Scolytidae), to attractant-baited multiple funnel traps. Verbenone significantly reduced attraction; however, no difference was observed between 4 and 50 mg/24-h release rates. Combinations of six bark volatiles (benzyl alcohol, benzaldehyde, trans-conophthorin, guaiacol, nonanal, and salicylaldehyde), three green leaf volatiles [(E)-2-hexenal, (E)-2-hexen-1-ol, and (Z)-2-hexen-1-ol], and the nine compounds combined did not significantly reduce D. brevicomis response to attractant-baited traps. However, a significant effect was observed when the bark and green leaf volatiles were combined with verbenone. The nine nonhost angiosperm volatiles (NAVs) significantly augmented the effect of both release rates of verbenone, reducing trap catches to levels significantly below that of either release rate of verbenone alone. trans-Conophthorin, a compound reported to have behavioral activity in a number of other scolytids, was not critical to the efficacy of our NAV blend. Our results suggest that the addition of nonhost angiosperm volatiles to verbenone could be important for developing successful semiochemical-based management techniques for D. brevicomis.     

Scramble competition in the southern pine beetle, Dendroctonus frontalis

Abstract .1. The nature of intraspecific competition was investigated in the southern pine beetle, Dendroctonus frontalis , a highly destructive pest of pine forests in the southern U.S.A. Data were analysed from an observational study of naturally-attacked trees, and from field experiments where attack density was manipulated by adding different numbers of beetles to caged trees.
2. The effect of attack density on gallery construction, oviposition, brood survival, and the overall rate of increase was examined, and a flexible model of intraspecific competition used to classify the type of competition (contest or scramble) at different points in the life cycle.
3. The results of these analyses suggest that contest competition occurs during gallery construction and oviposition, in accord with previous work on D. frontalis . Strong scramble competition occurs later on in development, however, and the overall competitive process is better characterized as scramble competition, similar to other bark beetles. Trees with attack densities sufficiently high to produce significant competition are common in the field.     

Semiochemical emission from individual galleries of the southern pine beetle, (Coleoptera: Curculionidae: Scolytinae), attacking standing trees

We collected, identified, and quantified volatiles arising from individual gallery entrances of the monogamous bark beetle Dendroctonus frontalis Zimmermann. Samples were collected while the insects were mass attacking mature loblolly pines (Pinus taeda L.) in an established infestation in western Mississippi, 1 August through 3 October 2005. Following volatile sample collection, the entrances were dissected and categorized according to those that 1) contained a solitary female (the gallery initiating sex), 2) contained a pair that had not yet produced an egg gallery, 3) led to an egg gallery with niches and/or eggs, or 4) represented failed attacks (either abandoned or containing dead beetles). The greatest mean release rate of the female-produced aggregation pheromone components frontalin (74 ng/h) and trans-verbenol (0.35 microg/h) was detected from entrances of solitary females, whereas the highest mean quantities of the male-produced multifunctional pheromone components endo-brevicomin (18 ng/h) and verbenone (0.15 microg/h) were detected from entrances of preoviposition beetle pairs. Alpha-pinene, a host-produced monoterpene that functions as a synergist for the aggregation attractant for D. frontalis, was detected from entrances of solitary females and preoviposition pairs at a rate of 0.6 mg/h, or 3-4 orders of magnitude greater than the insect-produced components of the attractant. Our results indicate that the release rates of pheromone components used in published field studies of the chemical ecology of D. frontalis (generally > 0.1 mg/h) represent thousands of 'attack equivalents' or production rates on the scale of a beetle mass attack on a single host. Additionally, our data suggest that the loss in attractiveness of host tissue fully colonized by D. frontalis is because of the disappearance of attractants rather than an increase in inhibitors.     

Phylogeography of the bark beetle Dendroctonus mexicanus Hopkins (Coleoptera: Curculionidae: Scolytinae)

Dendroctonus mexicanus is polyphagous within the Pinus genus and has a wide geographical distribution in Mexico and Guatemala. We examined the pattern of genetic variation across the range of this species to explore its demographic history and its phylogeographic pattern. Analysis of the mtDNA sequences of 173 individuals from 25 Mexican populations allowed to us identify 53 geographically structured haplotypes. High haplotype and low nucleotide diversities and Tajima’s D indicate that D. mexicanus experienced rapid population expansion during its dispersal across mountain systems within its current range. The nested clade phylogeographic analysis indicates that the phylogeographic pattern of D. mexicanus is explained by continuous dispersion among lineages from the Sierra Madre Occidental, the Sierra Madre Oriental and the Trans-Mexican Volcanic Belt. However, we also observed isolation events among haplotypes from the Cofre de Perote/Trans-Mexican Volcanic Belt/Sierra Madre Oriental and the Trans-Mexican Volcanic Belt/Sierra Madre del Sur, which is consistent with the present conformation of mountain systems in Mexico and the emergence of geographical barriers during the Pleistocene.     

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.     

Adult parasitoids of the southern pine beetle, Dendroctonus frontalis Zimmermann (Coleoptera: Scolytidae), feed on artificial diet on pine boles, pine canopy foliage and understory hardwood foliage

This study examined adult parasitoids of Dendroctonus frontalis Zimmermann (Coleoptera: Scolytidae) utilizing an artificial parasitoid food on locations with and without hosts. Eliminade™ was applied to bark on boles of infested pines, to pine canopy foliage and to understory hardwood foliage. Adult parasitoids were collected, identified and dissected to determine the presence of Eliminade™ within their guts. Individuals from eight parasitoid species were collected on pine boles within D. frontalis infestations in the Talladega National Forest (Alabama) in the summer of 1999 and 2000. Mean percentage of parasitoids feeding on Eliminade™ was 62±10% during both summers. Percentage of parasitoids feeding was the same 24 h after Eliminade™ application, regardless of food location or parasitoid species. Seven days later, level of parasitoid feeding on Eliminade™ was insignificant in the pine canopy and hardwood understory. In contrast, percent of parasitoid feeding on Eliminade™ applied to pine boles remained the same between 1 and 7 days of the application. Therefore, not only will adult parasitoids visit and feed on artificial food on pine boles with hosts, but also non-host locations such as pine canopy and understory hardwood foliage. These results have implications for potential sources of adult nutrition for parasitoids of D. frontalis.     

Global and comparative proteomic profiling of overwintering and developing mountain pine beetle,Dendroctonus ponderosae (Coleoptera: Curculionidae), larvae

BackgroundMountain pine beetles, Dendroctonus ponderosae Hopkins (Coleoptera: Curculionidae), are native to western North America, but have recently begun to expand their range across the Canadian Rocky Mountains. The requirement for larvae to withstand extremely cold winter temperatures and potentially toxic host secondary metabolites in the midst of their ongoing development makes this a critical period of their lives.ResultsWe have uncovered global protein profiles for overwintering mountain pine beetle larvae. We have also quantitatively compared the proteomes for overwintering larvae sampled during autumn cooling and spring warming using iTRAQ methods. We identified 1507 unique proteins across all samples. In total, 33 proteins exhibited differential expression (FDR < 0.05) when compared between larvae before and after a cold snap in the autumn; and 473 proteins exhibited differential expression in the spring when measured before and after a steady incline in mean daily temperature. Eighteen proteins showed significant changes in both autumn and spring samples.ConclusionsThese first proteomic data for mountain pine beetle larvae show evidence of the involvement of trehalose, 2-deoxyglucose, and antioxidant enzymes in overwintering physiology; confirm and expand upon previous work implicating glycerol in cold tolerance in this insect; and provide new, detailed information on developmental processes in beetles. These results and associated data will be an invaluable resource for future targeted research on cold tolerance mechanisms in the mountain pine beetle and developmental biology in coleopterans.     

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.     

Microsatellite loci for the southern pine beetle (Dendroctonus frontalis) and cross‐species amplification in Dendroctonus

We developed microsatellite loci for the southern pine beetle (Dendroctonus frontalis). Twelve microsatellite loci were identified. Eight loci were polymorphic and sufficiently variable in 62 individuals (expected heterozygosity ranged from 0.707 to 0.880) to investigate population structure. All loci conformed to HWE except Dfr‐14, which showed heterozygote excess, and no two loci deviated from linkage equilibrium. The loci were tested for cross‐species amplification in four species of Dendroctonus (D. valens, D. terebrans, D. brevicomis, and D. ponderosae). Seven loci were polymorphic in at least one of the species tested.     

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.     

Fine-scale genetic population structure of southern pine beetle (Coleoptera: Curculionidae) in Mississippi forests

The southern pine beetle, Dendroctonus frontalis Zimmerman, is the most destructive insect pest of pine forests in the southeastern United States, Mexico, and Central America. Southern pine beetle aggressively attacks pine trees, and when in epidemic stages, they are capable of killing even the most healthy pine trees in a short period of time. Despite the amount of destruction caused by the southern pine beetle and the amount of monetary loss faced by the timber industry and recreation, the population genetics of this species has been limited to comparisons among distant geographic locations. This study investigates the fine-scale genetic population structure of the southern pine beetle in Mississippi. Very little genetic differentiation was observed among samples. Bayesian assignment testing failed to detect multiple groups within all samples; estimates of genetic differentiation and genetic distance were very low in magnitude; and a Mantel test did not reveal a significant relationship between genetic distance and geographic distance. These results suggest that management of the southern pine beetle needs to consider the potential movements of individuals within and among national forests and should be focused on a large scale, at least as big as continuously forested areas and possibly even multiple forests. These results further suggest that removal of beetle-infested trees is important.     

Colonization patterns of the red turpentine beetle, Dendroctonus valens (Coleoptera: Curculionidae), in the Luliang Mountains, China

The alien red turpentine beetle (RTB), Dendroctonus valens LeConte, is one of the most economically destructive forest pests in China, having killed more than 6 million pines in recent years. There is a need to understand the basic biology and ecology of the beetle in order to develop an effective monitoring and management strategy. In this study, the effects of hillside exposure (south- and north-facing), host-tree locations according to relief (valley, mid-slope, and ridge-top) and tree diameters on RTB colonization were investigated in one valley (3 sites). The results showed that (i) RTB clearly preferred colonizing pines growing on south-facing hillsides, especially in the valley; (ii) RTB preferred to colonize the pines growing at the valley rather than pines growing at mid-slope or on ridge-top; (iii) RTB preferred to colonize trees with large diameter over small and medium-sized pines; (iv) the attack density of RTBs (measured by pitch tubes/pine) was obviously higher on larger trees standing in the valley than other trees standing at other places. We conclude from RTB colonization patterns, that RTB prefers to attack large trees in the valley, which may be useful in developing a pest-management strategy.     

Fitness consequences of pheromone production and host selection strategies in a tree-killing bark beetle (Coleoptera: Curculionidae: Scolytinae)

Timing of arrival at a resource often determines an individual’s reproductive success. Tree-killing bark beetles can reproduce in healthy trees by attacking in adequate numbers to overcome host defences that could otherwise be lethal. This process is mediated by aggregation and antiaggregation pheromones. Beetles that arrive early in such a “mass attack” must contend with undiminished tree defences, and produce enough pheromones to attract more beetles, but have a head start on gallery construction and egg-laying. Beetles that arrive late may be impeded by competition and diminishing availability of phloem, but should experience fewer costs associated with pheromone production and battling tree defences. We investigated relationships between timing of arrival, body size, pheromone production and fitness in the southern pine beetle, Dendroctonus frontalis. In field experiments, we captured beetles that arrived early (pioneers) and late on slash pine trees, Pinus elliottii, and measured pheromone amounts in their hindguts. We marked gallery entrances of beetles as they landed on a tree and measured their reproductive success after the attack terminated. We found no difference in body size or pheromone amounts between early and late arrivers. Most beetles arrived at the middle of the attack sequence, and excavated longer galleries per day than early arrivers. The number of offspring produced per day by beetles that established galleries midway through mass attack was higher than those that arrived early or very late in the sequence. Our results suggest that beetles do not exhibit adaptive phenotypic plasticity in pre-landing pheromone production, depending on the extent of previous colonisation of a host. Rather, it appears that stabilising selection favours beetles that attack in the middle of the sequence, and contributes to attack synchrony. Synchronous attack on trees is essential before population booms characteristic of tree-killing bark beetles can occur in nature.     

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.     

Electrophysiological and behavioral responses of Dendroctonus frontalis (Coleoptera: Curculionidae) to volatiles isolated from conspecifics

Olfactory sensitivity of the southern pine beetle, Dendroctonus frontalis Zimmermann, to compounds isolated from the mid/hindguts of newly emerged conspecific adults was assayed with coupled gas chromatography-electroantennographic detection. All previously reported pheromones for D. frontalis plus eight additional compounds (fenchyl alcohol, myrtenal, cis-verbenol, trans-pinocarveol, acetophenone, trans-myrtanol, cis-myrtanol, and 2-phenylethanol) consistently elicited antennal responses from at least one sex. The eight additional compounds were assayed individually at three release rates (0.4-0.8, 3-9, and 25-100 mg/d) for the ability to alter D. frontalis responses to traps baited with D. frontalis attractant (4 mg/d frontalin and 17 mg/d alpha-pinene). At the high release rate, cis-verbenol enhanced attraction of D. frontalis females, whereas the other seven compounds significantly reduced attraction of one or both sexes. Acetophenone significantly reduced attraction of male D. frontalis at the low release rate, and five compounds (fenchyl alcohol, trans-pinocarveol, acetophenone, cis-myrtanol, and 2-phenylethanol) reduced attraction of one or both sexes at the intermediate rate. Only acetophenone significantly altered the sex ratio of beetles trapped, decreasing the proportion of males. Attraction of predatory checkered beetles (Cleridae) was enhanced by cis-verbenol released at the high rate but was not altered by any compound inhibitory to D. frontalis. Analyses of volatiles from individual D. frontalis indicated that the majority of the eight compounds were produced in greater quantities by newly emerged beetles than ones attacking pine bolts. Five of the compounds were associated predominantly with one sex. Possible ecological roles of these compounds in the biology of D. frontalis are discussed.     

Temperature extremes, density dependence, and southern pine beetle (Coleoptera: Curculionidae) population dynamics in east Texas

Previous studies of the southern pine beetle, Dendroctonus frontalis Zimm., established that its population in east Texas responds to a delayed density-dependent process, whereas no clear role of climate has been determined. We tested two biological hypotheses for the influence of extreme temperatures on annual southern pine beetle population growth in the context of four alternative hypotheses for density-dependent population regulation. The significance of climate variables and their interaction with population regulation depended on the model of density dependence. The best model included both direct and delayed density dependence of a cubic rather than linear form. Population growth declined with the number of days exceeding 32 degrees C, temperatures previously reported to reduce brood survival. Density dependence also changed with the number of hot days. Growth was highest in years with average minimum winter temperatures. Severely cold winters may reduce survival, whereas warm winters may reduce the efficiency of spring infestation formation. Whereas most previous studies have incorporated climate as an additive effect on growth, we found that the form of delayed density dependence changed with the number of days >32 degrees C. The interaction between temperature and regulation, a potentially common phenomenon in ecology, may explain why southern pine beetle outbreaks do not occur at perfectly regular intervals. Factors other than climate, such as forest management and direct suppression, may have contributed significantly to the timing, severity, and eventual cessation of outbreaks since the mid-1950s.     

Predicting Dendroctonus pseudotsugae (Coleoptera: Curculionidae) antiaggregation pheromone concentrations using an instantaneous puff dispersion model

An instantaneous puff dispersion model was used to assess concentration fields of the Douglas-fir beetle, Dendroctonus pseudotsugae Hopkins, antiaggregation pheromone, 3-methylcyclohex-2-en-1-one (MCH), within a 1-ha circular plot. Several combinations of MCH release rate and releaser spacing were modeled to theoretically analyze optimal deployment strategies. The combinations of MCH release rate and releaser spacing used in the modeling exercise were based on results of previous field studies of treatment efficacy. Analyses of model results suggest that a release rate up to six times the initial standard, at a correspondingly wider spacing to keep the total amount of pheromone dispersed per unit area constant, may be effective at preventing Douglas-fir beetle infestation. The model outputs also provide a visual representation of pheromone dispersion patterns that can occur after deployment of release devices in the field. These results will help researchers and practitioners design more effective deployment strategies.