First Report of Geosmithia morbida on English Walnut and its Paradox Rootstock in California.

Geosmithia morbida, the causal agent of thousand cankers disease (TCD), is vectored by the walnut twig beetle (WTB), Pityophthorus juglandis, causing decline in eastern black walnut, Juglans nigra (4), and canker development on many Juglans species (5). In the summer of 2012, a survey for TCD incidence in English walnut, J. regia, in orchards in California identified many trees with WTB activity and characteristic TCD symptoms. Both the J. regia scion and its Paradox hybrid rootstock (J. hindsii× J. regia) were affected. In some cases, trees exhibited bleeding on the bark surface from WTB entrance holes. Removal of the outer bark revealed cankers in the phloem around the WTB galleries. Two samples were taken from scions and three samples were collected from rootstocks of trees in orchards in northern California. Pieces (~3 to 4 mm2) of symptomatic tissue were placed in acidified potato dextrose agar (APDA), and the plates were incubated for 4 to 5 days at 30°C. Samples exhibiting fungal growth similar in morphology to G. morbida were transferred to PDA plates to obtain pure cultures and then processed to obtain single-spore cultures. Culture morphology for five single-spore isolates (Gm103, Gm104, Gm105, Gm107, and Gm108) was similar to that described by Kolarík et al. (4) for G. morbida. Conidiophores were penicillate and verrucose. Conidia were narrowly cylindrical, 5.2 ± 0.06 × 2.2 ± 0.04 µm (n = 50). Single-spore isolates were then grown in 1% yeast extract glucose liquid culture for 7 to 10 days. DNA was extracted and the ITS region was amplified, including the 5.8S region by using primers ITS1F/ITS4. Sequences were assembled and deposited in GenBank under accessions KJ664793 to KJ664797. Sequences were compared to those in GenBank; all sequences matched (99 to 100% identity) the ITS sequences of G. morbida strain CBS 124663. Pathogenicity tests were performed on 28-cm-long detached branches of J. regia. Four branches per isolate were inoculated with a 5-mm-diameter mycelial plug from a 2-week-old culture. Branches were incubated at room temperature (23 ± 2°C) in a humidified container for 3 weeks, and then canker lengths were measured. Pieces of the cankered area were placed in APDA and incubated as described above with G. morbida re-isolated from the cankers for all of the isolates, completing Koch's postulates. Average canker lengths ranged from 48.6 ± 4.3 to 72.1 ± 7.1 mm. Re-isolated G. morbida exhibited the same growth and reproductive structure morphology in culture on PDA as the original cultures. TCD in association with WTB has been observed in California English walnut orchards since 2008 (1,2,3). However, this is the first report for completion of Koch's postulates and morphological and molecular confirmation of G. morbida in J. regia and the Paradox rootstock, the predominant rootstock used in commercial orchards. TCD is a concern to the walnut industry in California with over 245,000 bearing acres reported in 2012. References: (1) M. Flint et al. CAPCA Adviser 8:36, 2010. (2) A. D. Graves et al. Walnut Twig Beetle and Thousand Cankers Disease: Field Identification Guide. UC-IPM website publication, http://www.ipm.ucdavis.edu/PDF/MISC/thousand_cankers_field_guide.pdf , 2009. (3) J. Hasey et al. (Abstr.) Phytopathology 100:S48, 2010. (4) M. Kolarík et al. Mycologia 103:325, 2011. (5) C. Utley et al. Plant Dis. 97:601, 2013.

Application of a frequency distribution method for determining instars of the beet armyworm (Lepidoptera: Noctuidae) from widths of cast head capsules.

Instar determination of field-collected insect larvae has generally been based on the analysis of head capsule width frequency distributions or bivariate plotting, but few studies have tested the validity of such methods. We used head capsules from exuviae of known instars of the beet armyworm, Spodoptera exigua (Hübner) (Lepidoptera: Noctuidae), to determine the larval instars with the frequency distribution method and Dyar's rule. Head capsule widths of S. exigua ranged from 0.313 to 1.446 mm. The number of instars from the analytical method matched that from the observed data. Based on misclassification rules derived from nonlinear least square fitting of the head capsule width data, the theoretical misclassification rates ranged from 0.62 to 1.92%. Comparing the theoretical distribution to the observed data, the observational misclassification probabilities ranged from 1.18 to 3.03%. There were also 10 head capsules, eight third instars and two fourth instars, not classified into any of the known instars based on the theoretical distributions. Dyar's growth ratios of successive instars ranged from 1.41 to 1.65, and those based on the observed data and theoretical distribution were similar. Both approaches yielded a linear relationship between the natural logarithm of the mean head capsule width and the instar number, which indicates full representation of the larval instars. The results demonstrated that the frequency distribution-based method was robust, although we recommend caution when using such methods to classify head capsules into specific instar classes. Application of computer algorithms should also be accompanied by visual inspection to determine instars from the frequency distribution.

Isolation and extreme sex-specific expression of cytochrome P450 genes in the bark beetle, Ips paraconfusus, following feeding on the phloem of host ponderosa pine, Pinus ponderosa.

We have identified cDNAs and characterized the expression of 13 novel cytochrome P450 genes of potential importance in host colonization and reproduction by the California fivespined ips, Ips paraconfusus. Twelve are of the Cyp4 family and one is of the Cyp9 family. Following feeding on host Pinus ponderosa phloem, bark beetle transcript levels of several of the Cyp4 genes increased or decreased in males only or in both sexes. In one instance (IparaCyp4A5) transcript accumulated significantly in females, but declined significantly in males. The Cyp9 gene (Cyp9T1) transcript levels in males were > 85 000 x higher at 8 h and > 25 000 x higher at 24 h after feeding compared with nonfed controls. Transcript levels in females were approximately 150 x higher at 24 h compared with nonfed controls. Cyp4G27 transcript was present constitutively regardless of sex or feeding and served as a better housekeeping gene than beta-actin or 18S rRNA for the real-time TaqMan polymerase chain reaction analysis. The expression patterns of Cyp4AY1, Cyp4BG1, and, especially, Cyp9T1 in males suggest roles for these genes in male-specific aggregation pheromone production. The differential transcript accumulation patterns of these bark beetle P450s provide insight into ecological interactions of I. paraconfusus with its host pines.

Male jeffrey pine beetle, Dendroctonus jeffreyi, synthesizes the pheromone component frontalin in anterior midgut tissue.

The male Jeffrey pine beetle, Dendroctonus jeffreyi Hopkins (Coleoptera: Scolytidae), produces the bicyclic ketal frontalin as part of a complex semiochemical blend. A key regulated enzyme in the mevalonate pathway, 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-R), showed high transcript levels in the anterior midgut of male Jeffrey pine beetles by in situ hybridization. HMG-R expression in this area of the alimentary canal was related to male emergence, where emerged males demonstrated significant up-regulation of HMG-R transcript and pre-emerged males showed only basal levels. Pre-emerged males were induced to express high levels of HMG-R transcript by treatment with juvenile hormone (JH) III. Additionally, isolated anterior midgut tissue from JH III-treated males converted radiolabeled acetate to frontalin, as assayed by radio-HPLC, providing strong evidence that this is the site of frontalin production in male beetles.

Isolation and endocrine regulation of an HMG-CoA synthase cDNA from the male Jeffrey pine beetle, Dendroctonus jeffreyi (Coleoptera: Scolytidae).

We have isolated a full length 3-hydroxy-3-methylglutaryl coenzyme A synthase (HMG-S) cDNA from the male Jeffrey pine beetle, Dendroctonus jeffreyi Hopkins, and studied the effects of topical applications of juvenile hormone III (JH III) on its expression. The predicted translation product of this apparently single copy gene has 63% and 58% identity with HMG-S1 and HMG-S2 from Blattella germanica (L.), and 61% identity with Drosophila melanogaster Hmgs. HMG-S transcript levels remain uniformly low in JH III-treated and control D. jeffreyi females, but are induced approximately 2.5- to 5-fold in JH III-treated males. JH III causes a dose- and time-dependent increase in HMG-S transcripts in the male metathoracic-abdominal region. Since monoterpenoid pheromone precursor synthesis and HMG-CoA reductase expression are under the control of JH III in the metathorax of Ips bark beetles, the observed HMG-S expression pattern suggests that the isoprenoid pathway is similarly important for semiochemical production in D. jeffreyi.

Insect pheromones--an overview of biosynthesis and endocrine regulation.

This overview describes, compares, and attempts to unify major themes related to the biosynthetic pathways and endocrine regulation of insect pheromone production. Rather than developing and dedicating an entirely unique set of enzymes for pheromone biosynthesis, insects appear to have evolved to add one or a few tissue-specific auxiliary or modified enzymes that transform the products of "normal" metabolism to pheromone compounds of high stereochemical and quantitative specificity. This general understanding is derived from research on model species from one exopterygote insect order (Blattodea) and three endopterygote insect orders (Coleoptera, Diptera, and Lepidoptera). For instance, the ketone hydrocarbon contact sex pheromone of the female German cockroach, Blattella germanica, derives its origins from fatty acid biosynthesis, arising from elongation of a methyl-branched fatty acyl-CoA moiety followed by decarboxylation, hydroxylation, and oxidation. Coleopteran sex and aggregation pheromones also arise from modifications of fatty acid biosynthesis or other biosynthetic pathways, such as the isoprenoid pathway (e.g. Cucujidae, Curculionidae, and Scolytidae), or from simple transformations of amino acids or other highly elaborated host precursors (e.g. Scarabaeidae and Scolytidae). Like the sex pheromone of B. germanica, female-produced dipteran (e.g. Drosophilidae and Muscidae) sex pheromone components originate from elongation of fatty acyl-CoA moieties followed by loss of the carbonyl carbon and the formation of the corresponding hydrocarbon. Female-produced lepidopteran sex pheromones are also derived from fatty acids, but many moths utilize a species-specific combination of desaturation and chain-shortening reactions followed by reductive modification of the carbonyl carbon. Carbon skeletons derived from amino acids can also be used as chain initiating units and elongated to lepidopteran pheromones by this pathway (e.g. Arctiidae and Noctuidae). Insects utilize at least three hormonal messengers to regulate pheromone biosynthesis. Blattodean and coleopteran pheromone production is induced by juvenile hormone III (JH III). In the female common house fly, Musca domestica, and possibly other species of Diptera, it appears that during hydrocarbon sex pheromone biosynthesis, ovarian-produced ecdysteroids regulate synthesis by affecting the activities of one or more fatty acyl-CoA elongation enzyme(s) (elongases). Lepidopteran sex pheromone biosynthesis is often mediated by a 33 or 34 amino acid pheromone biosynthesis activating neuropeptide (PBAN) through alteration of enzyme activities at one or more steps prior to or during fatty acid synthesis or during modification of the carbonyl group. Although a molecular level understanding of the regulation of insect pheromone biosynthesis is in its infancy, in the male California fivespined ips, Ips paraconfusus (Coleoptera: Scolytidae), JH III acts at the transcriptional level by increasing the abundance of mRNA for 3-hydroxy-3-methylglutaryl-CoA reductase, a key enzyme in de novo isoprenoid aggregation pheromone biosynthesis.

Juvenile hormone regulation of HMG-R gene expression in the bark beetle Ips paraconfusus (Coleoptera: Scolytidae): implications for male aggregation pheromone biosynthesis.

Juvenile hormone III (JH III) induces acyclic isoprenoid pheromone production in male Ips paraconfusus. A likely regulatory enzyme in this process is 3-hydroxy-e-methylglutaryl-CoA reductase (HMG-R). To begin molecular studies on pheromone production, a 1.16-kb complementary DNA representing approximately one-third of I. paraconfusus HMG-R was isolated by polymerase chain reaction and sequenced. The predicted translation product is 59% and 75% identical to the corresponding portion of HMG-R from the fruit fly, Drosophila melanogaster, and the German cockroach, Blattella germanica, respectively. Northern blots show that topical application of JH III increases HMG-R transcript levels in male thoraces in an apparent dose- and time-dependent manner. These data support the model that JH III raises HMG-R transcript levels, resulting in increased activity of the isoprenoid pathway and de novo pheromone production.

Olfactory responses ofIps plastographus maritimus lanier (coleoptera: Scolytidae) to insect and host-associated volatiles in the laboratory.

Attraction of both sexes ofIps plastographus maritimus Lanier to bark-phloem-xylem discs of Monterey pine,Pinus radiata D. Don, was demonstrated in the laboratory. Increasing concentrations of male and female volatiles trapped separately and released in a one-to-one ratio decreased attraction for both sexes combined. Attraction of both sexes to volatiles derived from males and females tunneling together in a one-to-one ratio increased with increasing concentration of extract. Attraction of males and females to male-infested discs and to trapped male volatiles increased with increasing dose of males or male extract. Attraction of males and females to female-infested discs and to trapped female volatiles was also demonstrated. The presence of females in male galleries reduced the attractiveness of infested disks to both sexes combined. Increasing numbers of females, tunneling separately from males in the same disc, reduced attraction of males, but not females. When a constant attractive dose of male volatiles was released with increasing doses of female volatiles, there was no difference in response of either sex when female volatiles were present compared with the response to male volatiles alone. When a constant attractive dose of male volatiles was released with increasing concentrations of volatiles derived from males and females tunneling together in a one-to-one ratio, attraction ofI. p. maritimus decreased. Response of females was frequently higher than that of males to the same attractant source. Hence, both sexes produce an attractant, and both sexes tunneling together in the same gallery reduce attraction of males and females to an attractive dose of male attractant.

De novo biosynthesis of the aggregation pheromone components ipsenol and ipsdienol by the pine bark beetles Ips paraconfusus Lanier and Ips pini (Say) (Coleoptera: Scolytidae).

The California five-spined ips, Ips paraconfusus Lanier, produces the myrcene-derived acyclic monoterpene alcohols ipsenol (2-methyl-6-methylene-7-octen-4-ol) and ipsdienol (2-methyl-6-methylene-2,7-octadien-4-ol) as components of its aggregation pheromone. The pine engraver beetle, Ips pini (Say), produces only ipsdienol. Previous studies have shown that myrcene, a monoterpene in the pines colonized by these beetles, is a direct precursor to these pheromone components. In vivo radiolabeling studies reported here showed that male I. paraconfusus incorporated [1-14C]acetate into ipsenol, ipsdienol, and amitinol (trans-2-methyl-6-methylene-3,7-octadien-2-ol), while male I. pini incorporated [1-14C]acetate into ipsdienol and amitinol. Females of these species produced neither labeled nor unlabeled pheromone components. The purified radiolabeled monoterpene alcohols from-males were identified by comparison of their HPLC and GC retention times with those of unlabeled standards. HPLC-purified fractions containing the individual radiolabeled components were analyzed by GC-MS and were shown to include only the pure alcohols. To further confirm that ipsdienol and ipsenol were radiolabeled, diastereomeric ester derivatives of the isolated alcohols were synthesized and analyzed by HPLC and GC-MS. After derivatization of the radiolabeled alcohols, the HPLC analysis demonstrated expected shifts in retention times with conservation of naturally occurring stereochemistry. The results provide direct evidence for de novo biosynthesis of ipsenol, ipsdienol, and amitinol by bark beetles.

Enantiomeric composition of ipsdienol: A chemotaxonomic character for north American populations ofIps spp. in thepini subgeneric group (coleoptera: Scolytidae).

Thirty-five populations ofIps pini (Say) and one population each ofIps avulsus (Eichhoff) andIps bonanseai (Hopkins) were analyzed for the enantiomeric composition of ipsdienol (2-methyl-6-methylene-2,7-octadien-4-ol). Populations ofI. pini occur as at least two distinct regional pheromone variants: New York type [32%-(-) to 56%-(-)-ipsdienol] and California type [94%-(-) to 98%-(-)-ipsdienol]. A third phenotype may occur in southeastern British Columbia, Idaho, and Montana [91%-(-) to 95%-(-)], possibly indicating a zone of hybridization. Populations of the New York type occur in southwestern British Columbia, Alberta, Saskatchewan, Minnesota, and Wisconsin suggesting a continuum through the Canadian provinces and Lake States. The presence of the New York type in western Canada is likely linked to the Quaternary history of the transcontinentally distributed host,Pinus banksiana Lamb. MaleI. avulsus [∼25%-(-)] and maleI. bonanseai [-29%-(-)] both produce ipsdienol, but not ipsenol. Production of ipsdienol by maleI. pini was evaluated in six differentPinus spp. hosts. Following transfer of maleI. pini to hosts other than the host of origin, the percentage of the (-)-enantiomer of ipsdienol declined when compared to production in the host of origin.

Role of chirality in olfactory-directed behavior: Aggregation of pine engraver beetles in the genusIps (Coleoptera: Scolytidae).

There has been a renaissance of interest in the significance of enantiomeric composition in biological systems. Three chiral monoterpene alcohol aggregation pheromone components (ipsenol, ipsdienol, andcis-verbenol) commonly isolated from engraver beetles (Ips spp.) provide a paradigm for this theme as it relates to olfactory-guided insect behavior. The literature pertaining to this system is reviewed and the effects of the enantiomeric composition of these semiochemicals on theIps spp. community is explored on two trophic levels. Hypotheses generated from the well-studied aggregation pheromone production and response patterns forI. paraconfusus Lanier andI. pini (Say) are generalized to the North American species in the genus. Despite the progress withI. paraconfusus andI. pini, substantial deficiencies exist in our understanding of the role of enantiomeric composition in pheromonal/allomonal effects in different subgeneric groups, in the regulation and mechanisms of stereoselective biosynthesis of the monoterpene alcohols, and in the benefits derived by individual insects that produce relatively large proportions of inactive or interruptive enantiomers with attractive enantiomers.

The role of lanierone in the chemical ecology ofIps pini (Coleoptera: Scolytidae) in California.

Five doses of lanierone (2-hydroxy-4,4,6-trimethyl-2,5-cyclohexadien-1-one) were tested with one dose of enantiomerically pure [99.4% (4R)-(-)] ipsdienol (2-methyl-6-methylene-2,7-octadien-4-ol) for activity as an aggregation pheromone ofIps pini (Say) in California. The response ofI. pini to 1 mg/day ipsdienol + 20 µg/day lanierone was significantly greater than the response to ipsdienol alone, but the response pattern did not demonstrate a clear dose-response relationship. The response to the highest dose of lanierone (2 mg/day) was significantly lower than the response to ipsdienol alone. Ipsdienol attracted significantly moreI. pini than a male-infested log. Lanierone did not alter the percentage of maleI. pini responding to ipsdienol alone. Neither sex ofI. pini orDendroctonus brevicomis LeConte from California produced detectable amounts of lanierone, but myrcene-aerated maleD. brevicomis produced 97.8%-(4S)-(+)-ipsdienol. The black-bellied clerid,Enoclerus lecontei (Wolcott) (Coleoptera: Cleridae) was attracted to lanierone when released with ipsdienol. Neither compound was attractive when released alone, proving synergism for the kairomone of this predator. Lanierone did not influence the response of the predatorsTemnochila chlorodia (Mannerheim) (Coleoptera: Trogositidae) andEnoclerus sphegeus (F.) (Coleoptera: Cleridae), which were attracted to all treatments containing ipsdienol.Tomicobia tibialis Ashmead (Hymenoptera: Pteromalidae) responded in significantly greater numbers to the male-infested log than it did to ipsdienol or ipsdienol + 20 µg/day lanierone.