Sequencing of the Lumbricus terrestris genome reveals degeneracy in its erythrocruorin genes.

The erythrocruorin of Lumbricus terrestris (LtEc) is a relatively large macromolecular assembly that consists of at least four different hemoglobin subunits (A, B, C, and D) and four linker subunits (L1, L2, L3, and L4). The complexity and stability of this large structure make LtEc an attractive hemoglobin-based oxygen carrier that could potentially be used as a substitute for donated red blood cells. However, the sequences of the LtEc subunit sequences must be determined before a scalable recombinant expression platform can be developed. The goal of this study was to sequence the L. terrestris genome to identify the complete sequences of the LtEc subunit genes. Our results revealed multiple homologous genes for each subunit (e.g., two homologous A globin genes; A1 and A2), with the exception of the L4 linker. Some of the homologous genes encoded identical peptide sequences (C1 and C2, L1a and L1b), while cDNA and mass spectrometry experiments revealed that some of the homologs are not expressed (e.g., A2). In contrast, multiple sequences for the B, D, L2, and L4 subunits were detected in LtEc samples. These observations reveal novel degeneracy in LtEc and other annelids, along with some new revisions to its previously published peptide sequences.

Bioavailable Sulforaphane Quantitation in Plasma by LC-MS/MS Is Enhanced by Blocking Thiols.

Quantifying sulforaphane (SFN) and its thiol metabolites in biological samples using liquid chromatography-tandem mass spectrometry is complicated by SFN's electrophilic nature and the facile dissociation of SFN-thiol conjugates. SFN can be lost during sample preparation due to conjugation with protein thiols, which are precipitated and discarded. We observe that only 32 ± 3% of SFN is recovered 2 h after spiking into fetal bovine serum. The SFN-glutathione conjugate prepared at 10 mM in 0.1% formic acid in water (pH 3) dissociated by approximately 95% to free SFN, highlighting the difficulty in preparing thiol metabolite standards. We used the alkylating agent iodoacetamide (IAA) to both release SFN from protein thiols and force the dissociation of SFN metabolites. This thiol-blocking method increased SFN percent recovery from serum from 32 to 94 ± 5%, with a 4.7 nM method limit of quantitation. Applying the method to clinical samples, SFN concentrations were on average 6 times greater than when IAA was omitted. The IAA thiol-blocking method streamlines the analysis of bioavailable SFN in plasma samples.

Crystal structures of the sulfones of 2,3-diphenyl-2,3-di-hydro-4H-1,3-benzo-thia-zin-4-one and 2,3-diphenyl-2,3-di-hydro-4H-pyrido[3,2-e][1,3]thia-zin-4-one.

The title sulfones, 2,3-diphenyl-2,3-di-hydro-4H-1,3-benzo-thia-zine-1,1,4-trione, C20H15NO3S, and 2,3-diphenyl-2,3-di-hydro-4H-pyrido[3,2-e][1,3]thia-zine-1,1,4-trione, C19H14N2O3S, crystallize in space group P21/n with two mol-ecules in each of the asymmetric units and have almost identical unit cells and extended structures. In both structures, the thia-zine rings exhibit a screw-boat pucker. The inter-molecular inter-actions observed are C-H⋯O-type hydrogen bonds and parallel partial π-π stacking between the fused aromatic rings (benzo- or pyrido-) of the core of the mol-ecules within each asymmetric unit, and also connecting to mol-ecules with translational periodicity in the a-axis direction in what can be described as columns (two per asymmetric unit) of stacked mol-ecules with alternating chirality. The pendant phenyl groups of both mol-ecules do not participate in aromatic ring inter-actions.

T3P-Promoted Synthesis of a Series of 2-Aryl-3-phenyl-2,3-dihydro-4H-pyrido[3,2-e][1,3]thiazin-4-ones and Their Activity against the Kinetoplastid Parasite Trypanosoma brucei.

A series of fourteen 2-aryl-3-phenyl-2,3-dihydro-4H-pyrido[3,2-e][1,3]thiazin-4-ones was prepared at room temperature by T3P-mediated cyclization of N-phenyl-C-aryl imines with thionicotinic acid, two difficult substrates. The reactions were operationally simple, did not require specialized equipment or anhydrous solvents, could be performed as either two or three component reactions, and gave moderate-good yields as high as 63%. This provides ready access to N-phenyl compounds in this family, which have been generally difficult to prepare. As part of the study, the first crystal structure of neutral thionicotinic acid is also reported, and showed the molecule to be in the form of the thione tautomer. Additionally, the synthesized compounds were tested against T. brucei, the causative agent of Human African Sleeping Sickness. Screening at 50 µM concentration showed that five of the compounds strongly inhibited growth and killed parasites.

2,3-Diphenyl-2,3-dihydro-4H-1,3-thiaza-4-one heterocycles inhibit growth and block completion of cytokinesis in kinetoplastid parasites.

Kinetoplastid parasites are model eukaryotes with a complex cell cycle that is highly regulated both spatially and temporally. In addition, diseases caused by these parasites continue to have a significant impact on human and animal health worldwide. While there have been advancements in chemotherapy for these diseases, there is a continual need for an arsenal of compounds that have robust anti-parasite activity with minimal impact on the human host. While investigating a series of 2,3-diphenyl-2,3-dihydro-4H-1,3-thiaza-4-one heterocycles with potential activity against these parasites, we found a pyridothiazinone that inhibits growth of the monoxenous parasite Crithidia fasciculata and two life cycle stages of Trypanosoma brucei. This inhibition is more pronounced in T. brucei and is associated with an unusual pre-abscission cell cycle arrest. Exploring the mode of action for these and related compounds in kinetoplastids may provide tools with which to explore cell cycle regulation in these important organisms.

HPLC-MS detection of pyrrolizidine alkaloids and their N-oxides in herbarium specimens dating back to the 1850s.

PREMISE OF THE STUDY: Understanding the phylogenetic distribution of defensive plant secondary metabolites is essential to the macroevolutionary study of chemically mediated plant-animal interactions. The chemical ecology of pyrrolizidine alkaloids (PAs) has been extensively studied in a number of plant-herbivore systems, including Apocynaceae (the milkweed and dogbane family) and Danainae (the milkweed and clearwing butterflies). A systematic survey is necessary to establish a detailed understanding of their occurrence across Apocynaceae. A survey of this species-rich, mainly tropical and subtropical family will rely heavily on small tissue samples removed from herbarium specimens, some of which will be very old and/or preserved with alcohols or mercuric chloride. METHODS: We optimized PA extraction methods from small leaf fragments of recently collected silica-dried leaves of the PA-positive Echites umbellatus, varying crushing and extraction time. We then applied our optimized method to leaf fragments from 70-167-year-old herbarium specimens of E. umbellatus. To simulate the effect of alcohol treatment on PA detectability in herbarium specimens, we incubated freshly collected leaves of the PA-positive Parsonsia alboflavescens in three different alcohols before drying and compared PA recovery to freshly dried controls. PAs were quantified using high-performance liquid chromatography-mass spectrometry analysis. X-ray fluorescence was used to identify mercury-containing specimens. RESULTS: Fifteen seconds of leaf crushing followed by 24 h of extraction were optimal for PA free-base and N-oxide recovery. This method yielded ~50-fold greater PA recovery than prior methods. Half of the herbarium specimens (13 of 23), including the oldest, tested positive for PAs; leaf age did not correlate with success in PA extraction. Treatment of fresh leaves with alcohol before drying did not diminish PA recovery; mercury was observed in both PA-positive and PA-negative specimens. CONCLUSIONS: PAs can be reliably detected in small tissue samples from herbarium specimens up to 167 years old, including specimens that had been treated with alcohol or mercury salts. The variability of PA presence among herbarium specimens of E. umbellatus indicates that multiple specimens will need to be tested before a particular species is determined to lack PAs.

Method development for binding media analysis in painting cross-sections by desorption electrospray ionization mass spectrometry.

RATIONALE: Art conservation science is in need of a relatively nondestructive way of rapidly identifying the binding media within a painting cross-section and isolating binding media to specific layers within the cross-section. Knowledge of the stratigraphy of cross-sections can be helpful for removing possible unoriginal paint layers on the artistic work. METHODS: Desorption electrospray ionization mass spectrometry (DESI-MS) was used in ambient mode to study cross-sections from mock-up layered paint samples and samples from a 17th century baroque painting. The DESI spray was raster scanned perpendicular to the cross-sectional layers to maximize lateral resolution then analyzed with a triple quadrupole mass analyzer in linear ion trap mode. From these scans, isobaric mass maps were created to map the locations of masses indicative of particular binding media onto the cross-sections. RESULTS: Line paint-outs of pigments in different binding media showed specific and unique ions to distinguish between the modern acrylic media and the lipid-containing binding media. This included: OP (EO)9 surfactant in positive ESI mode for acrylic (m/z 621), and oleic (m/z 281), stearic (m/z 283), and azelaic (m/z 187) acids in negative ESI mode for oil and egg tempera. DESI-MS maps of mock-up cross-sections of layered pigmented binding media showed correlation between these ions and the layers with a spatial resolution of 100 µm. CONCLUSIONS: DESI-MS is effective in monitoring binding media within an intact painting cross-section via mass spectrometric methods. This includes distinguishing between lipid-containing and modern binding materials present in a known mock-up cross-section matrix as well as identifying lipid-binding media in a 17th century baroque era painting.

Coaxial Electrospray Ionization for the Study of Rapid In-source Chemistry.

Coaxial electrospray has been used effectively for several dual-emitter applications, but has not been utilized for the study of rapid in-source chemistry. In this paper, we report the fabrication of a coaxial, micro-volume dual-emitter through the modification of a manufacturer's standard electrospray probe. This modification creates rapid mixing inside the Taylor cone and the ability to manipulate fast reactions using a variety of solvents and analytes. We demonstrate its potential as a low-cost, dual-emitter assembly for diverse applications through three examples: relative ionization in a biphasic electrospray, hydrogen-deuterium exchange, and protein supercharging. Graphical Abstract.

A comparison of electrospray ionization, atmospheric pressure chemical ionization, and atmospheric pressure photoionization for the liquid chromatography/tandem mass spectrometric analysis of bisphenols. Application to bisphenols in thermal paper receipts and U.S. currency notes.

RATIONALE: Human health concerns have arisen for common bisphenols (BPA and BPS) as well as new structural analogs. While native sulfone bisphenols and sulfone-derivatized bisphenols are amenable to electrospray ionization (ESI), alternative ionization methods such as atmospheric pressure chemical ionization (APCI) and APPI were investigated to analyze non-derivatized, non-sulfone-containing bisphenols. METHODS: Ionization of bisphenols using negative ion APPI was compared to negative ion ESI and negative ion APCI for their relative abilities to produce [M-H]- precursor ions for subsequent collision-induced dissociation (CID) in a triple quadrupole mass analyzer. Multiple reaction monitoring (MRM) transitions were optimized using the most sensitive ion transitions and coupled with high-performance liquid chromatographic (HPLC) separation to detect and quantify BPA, BPB, BPF, BPZ, BPAP, BPS, and BPSIP. RESULTS: APPI provides a more universal sensitivity over ESI toward the classes of bisphenols studied with detection limits of 20-50 pg on-column. The LC/APPI-MS/MS method was used to examine the levels of these seven bisphenols in thermal paper receipts and in U.S. paper currency. In both receipts and currency, BPA and BPS were the dominant bisphenols found in the paper. CONCLUSIONS: The measurement of BPA and bisphenol analogs in thermal paper receipts and transfer of bisphenols from receipts to currency notes is of increasing importance to assess human dermal exposure routes to this class of compounds. Equivalent sensitivity between sulfone- and non-sulfone-containing bisphenols is achievable through the use of alternate ionization sources such as APCI and APPI that circumvents tedious and time-consuming derivatization procedures to render analytical sensitivity by ESI.

Spatial and Temporal Distribution of Imidacloprid Within the Crown of Eastern Hemlock.

Systemic imidacloprid is the most widely used insecticide to control the hemlock woolly adelgid, Adelges tsugae Annand (Hemiptera: Adelgidae), an exotic pest of eastern hemlock, Tsuga canadensis (L.) Carriére in the United States. This study was conducted to 1) determine the effect of treatment timing (spring vs. fall) and application method (trunk injection vs. soil injection) on the spatial and temporal distribution of imidacloprid within the crown of A. tsugae-free eastern hemlock using a competitive enzyme-linked immunosorbent assay (ELISA), 2) compare ELISA to gas chromatography-mass spectrometry (GC/MS) for the detection of imidacloprid in xylem fluid, and 3) determine the concentration of imidacloprid in leaf tissue using high performance liquid chromatography with tandem mass spectrometric (LC/MS/MS) detection methods. Xylem fluid concentrations of imidacloprid were found to be significantly higher for spring applications than for fall applications and for trunk injections than soil injections in the first year posttreatment. A total of 69% of samples analyzed by ELISA gave 1.8 times higher concentrations of imidacloprid than those found by GC/MS, leading to evidence of a matrix effect and overestimation of imidacloprid in xylem fluid by ELISA. A comparison of the presence of imidacloprid with xylem fluid and in leaf tissue on the same branch showed significant differences, suggesting that imidacloprid moved intermittently within the crown of eastern hemlock.

Impacts of trunk and soil injections of low rates of imidacloprid on hemlock woolly adelgid (Hemiptera: Adelgidae) and eastern hemlock (Pinales: Pinaceae) health.

Eastern hemlocks (Tsuga canadensis Carrière) at two sites in southwestern Virginia were treated by trunk and soil injections of imidacloprid to determine the insecticide's impact on hemlock woolly adelgid, Adelges tsugae Annand. Treatments were 25, 50, and 100% of the highest labeled dosage rates for both stem and soil injection. Three and 4 yr after treatment, the half and full rates had significantly reduced A. tsugae populations, which were accompanied by increased new hemlock shoot growth and higher hemlock health scores on a visual rating of tree appearance. Imidacloprid and metabolite concentrations in tissue of treated trees were determined by liquid chromatography-tandem mass spectrometry, and A. tsugae density decreased as imidacloprid concentrations increased in wood tissue. There were no observed A. tsugae populations in all trees with imidacloprid tissue concentrations >413 ppb. Olefin, di-hydroxy, and 6-chloro-nicotinic-acid metabolites were the imidacloprid metabolites recovered in the highest concentrations. This suggests that hemlock metabolism of imidacloprid may increase efficacy of the parent compound. Stem and soil treatments of low rates of systemic imidacloprid reduce adelgid populations and promote hemlock health, but still may provide a remnant food source for beneficial predators.

Levels of polybrominated diphenyl ethers (PBDEs) in dust from personal automobiles in conjunction with studies on the photochemical degradation of decabromodiphenyl ether (BDE-209).

The levels of 21 PBDE congeners were detemined in the dust sampled from 66 personal automobiles. The dominant congener in automobile dust was BDE-209 with a median level of 8.12 µg g⁻¹. Personal vehicle dust samples contained the characteristic profile of the PBDE congeners that comprise the PentaBDE and DecaBDE commercial formulations. Levels of PBDEs in personal automobiles are generally reduced in comparison to our previously reported levels in resale vehicles on dealership lots presumably due to a dilution effect introduced by dust or debris that does not originate from the vehicle. Laboratory photochemical studies were conducted on both automobile dust collected from personal vehicles as well as BDE-209 adsorbed to sodium sulfate. No significant degradation occurred in the personal vehicle dust after 56 days of constant UVA irradiation while significant degradation did occur with BDE-209 adsorbed to sodium sulfate. PBDEs from the degradation of BDE-209 were identified and potential degradation pathways elucidated. Human exposure potential to PBDEs from automobile dust ingestion remains a serious concern in the U.S. population.

Lethal and sublethal effects of imidacloprid on hemlock woolly adelgid (Hemiptera: Adelgidae) and two introduced predator species.

Eastern hemlock, Tsuga canadensis (L.) Carrière, branchlets were systemically treated with imidacloprid and bioassayed with hemlock woolly adelgid, Adelges tsugae (Annand) (Hemiptera: Adelgidae), and predators. After 10 d, two hemlock woolly adelgid predators, Laricobius nigrinus Fender (Coleoptera: Derodontidae) and Sasajiscymnus tsugae Sasaji & McClure (Coleoptera: Coccinellidae), were allowed to feed on remaining hemlock woolly adelgid for 20 d on branches systemically treated with 1, 10, or 100 ppm imidacloprid. Every 5 d, mortality, mobility (measured as flip time), number of hemlock woolly adelgid consumed, and degree of intoxication of each individual beetle were recorded. Liquid chromatography-mass spectrometry was used to quantify imidacloprid and some of its major metabolites in hemlock wood tissues and in the predator beetles postmortem. Probit analysis of hemlock woolly adelgid mortality and imidacloprid concentrations recovered from branch wood tissues determined the 30 d LC50 to be 242 ppb. A topical application of imidacloprid to the ventral abdomen of individual beetles resulted in a 6 d LD50 value of 1.8 and 0.71 ng imidacloprid per beetle for L. nigrinus and S. tsugae, respectively. In no-choice tests, L. nigrinus mortality was significantly higher on hemlock branchlets treated with 100 ppm imidacloprid than on controls, but S. tsugae mortality was not. S. tsugae consumed the same number of adelgids on treated branchlets as on controls, but L. nigrinus consumed fewer adelgids from the 100 ppm branchlets than on controls. In choice tests, beetle mortality and flip times were generally not significantly different from controls. At times, both beetle species displayed intoxication symptoms after feeding on adelgids from treated branchlets and imidacloprid was recovered from both beetle species postmortem. These results suggest that systemic imidacloprid displayed both lethal and sublethal effects on these two nontarget predators of the hemlock woolly adelgid.

Supercritical fluid extraction of polybrominated diphenyl ethers (PBDEs) from house dust with supercritical 1,1,1,2-tetrafluoroethane (R134a).

The extraction of polybrominated diphenyl ethers (PBDEs) from SRM 2585 (Organic Contaminants in House Dust) was investigated using supercritical fluid R134a as an extraction solvent. Three methods of dust extraction were studied: (1) extraction of dry dust, (2) extraction of dry dust dispersed on Ottawa sand and (3) extraction of dust wet with dichloromethane. For each of the three sample preparation methods, extracts at three temperatures (110, 150, and 200 degrees C) above the critical temperature of R134a were performed. Eight PBDE congeners (BDE-28, -47, -99, 100, -153, -154, -183, and -209) in the SFE extracts were analyzed by liquid chromatography negative-ion atmospheric pressure photoionization tandem mass spectrometry (LC/NI-APPI/MS/MS). The optimum extraction of PBDEs from house dust using supercritical R134a is obtained when the dust is pre-wet with dichloromethane prior to extraction to swell the dust. For all sample preparation methods, higher temperatures afforded higher percent recoveries of the eight PBDE congeners. Only a combination of high-temperature (200 degrees C) and pre-wetting the dust with dichloromethane produced high recovery of the environmentally important, fully brominated PBDE congener, BDE-209.

Polybrominated diphenyl ether (PBDE) levels in dust from previously owned automobiles at United States dealerships.

The levels of BDE-28, BDE-47, BDE-99, BDE-100, BDE-153, BDE-154, BDE-183, and BDE-209 were determined in the dust sampled from 60 automobiles that were available for resale at U.S. dealerships. The dominant congener in automobile dust was BDE-209 comprising 95% of the total PBDE levels with a median level of 48.1 microg g(-1). Statistical analysis of the vehicle attributes indicates that the BDE-209 levels are different (p<0.05) with respect to groupings by vehicle model year, vehicle manufacturer, and the country of manufacture. Vehicle dust samples contained the characteristic profile of the PBDE congeners that comprise the PentaBDE formulation. While DecaBDE use is banned in Maine and Washington and is targeted for restriction in the near future by six U.S. states, vehicles and airplanes are exempt from the ban. It is anticipated that the human exposure potential to PBDEs from automobile dust ingestion will continue for an indefinite future period in the U.S. population.

Analysis of polybrominated diphenyl ethers (PBDEs) by liquid chromatography with negative-ion atmospheric pressure photoionization tandem mass spectrometry (LC/NI-APPI/MS/MS): application to house dust.

Eight polybrominated diphenyl ether (PBDE) congeners of primary interest to the US EPA were separated using reverse-phase liquid chromatography on an octadecylsilane column. BDE-28, BDE-47, BDE-99, BDE-100, BDE-153, BDE-154, BDE-183, and BDE-209 were baseline-resolved under isocratic conditions in 92:8 methanol/water (v/v). Negative-ion atmospheric pressure photoionization (NI-APPI) with a toluene dopant produced precursor ions corresponding to [M-Br+O](-) for the eight congeners studied. Each congener was quantified by tandem mass spectrometry through a unique multiple reaction monitoring (MRM) transition. On-column limits of detection were between 2.4 and 27.8 pg for the eight congeners studied, with an intra-day method precision of 9%. The LC/NI-APPI/MS/MS method was validated for the analysis of the eight PBDE congeners in NIST SRM 2585 (Organics in House Dust). Pressurized liquid extraction (PLE) with subsequent LC/NI-APPI/MS/MS analysis afforded quantitative recovery for all eight PBDE congeners with recoveries ranging from 92.7 to 113%. The liquid-phase separation of the LC/NI-APPI/MS/MS method is not prone to the thermal degradation issues that plague splitless GC based analyses of highly brominated PBDEs such as BDE-209.

Characterization of terpenoid volatiles from cultivars of eastern hemlock (Tsuga canadensis).

The volatile terpenoid fraction from needles in 13 cultivars of Tsuga canadensis L. (Carriere) was analyzed by gas chromatography with mass spectrometry (GC-MS). The results of this study are considered along with previously reported results for foliar terpenoid levels of the Asian (T. sieboldii, T. chinensis, T. diversifolia), western North American (T. mertensiana, T. heterophylla), and eastern North American species (T. canadensis, T. caroliniana) of hemlock to draw conclusions about the potential of cultivar host resistance to the hemlock woolly adelgid (Adelges tsugae Annand). It is suggested that hemlocks in eastern North America have adapted their terpenoid chemistry for protection against endemic defoliators and that this has made them vulnerable to non-native, sucking pests such as adelgids and scales. Some cultivars of T. canadensis have a terpenoid profile that resembles that of the resistant noneastern North American species and are candidates for biological screening for resistance. Among the cultivars, the variation in terpenoid chemistry did not absolutely correspond with the considerable differences in morphological characters observed, indicating that the terpenoid chemistry is not definitively coupled with hemlock morphology.

Flow injection analysis of imidacloprid in natural waters and agricultural matrixes by photochemical dissociation, chemical reduction, and nitric oxide chemiluminescence detection.

A flow injection analysis (FIA) to quantify imidacloprid was developed based upon (1) ultraviolet (254 nm) photochemical dissociation of imidacloprid to produce nitrite, (2) chemical reduction of the nitrite to nitric oxide by iodide in acid, (3) removal of gas-phase nitric oxide from the aqueous stream using a membrane separator, and (4) detection of the nitric oxide by chemiluminescent reaction with ozone. The cross-reactivity of imidacloprid with eight metabolites of imidacloprid was determined using a commercial ELISA kit and the FIA method. While the ELISA kit demonstrated varying degrees of cross-reactivity, cross-reactivity in the FIA method was observed for only the N-nitro and N-nitroso metabolites. The optimized analytical FIA method, FIA provides a linear response in imidacloprid concentration over four orders of magnitude, has a limit of detection of 5.6 pmol (1.5 ng) of imidacloprid, and exhibits an inter-day precision of 0.4%. Spike-recoveries by FIA demonstrated excellent recovery of imidacloprid in natural waters, hemlock xylem fluid, honey, and grapes with little to no interference from the matrix.

A colorimetric assay for the determination of polyhexamethylene biguanide in pool and spa water using nickel-nioxime.

A novel nickel-nioxime analytical method to measure polyhexamethylene biguanide (PHMB) in swimming pools and spas was developed. This method utilizes nickel(II) chloride and 1,2-cyclohexanedionedioxime (nioxime) chemistry. In the method, nickel ions bind and neutralize PHMB in the solution. Excess, un-reacted nickel ions react with nioxime and the resulting colored solution is measured at 550nm using a colorimetric assay. Currently, the colorimetric method to measure PHMB uses bromophenol blue (BPB). However, high levels of quaternary ammonium based algaecides and surfactant based products interfere with this colorimetric method. A time-consuming and expensive high-performance liquid chromatographic (HPLC) analysis can be used for samples with high levels of quaternary ammonium based algaecides or surfactants. The proposed nickel-nioxime detection method achieves comparable PHMB results to HPLC in about 5min and is a very economical and simple method to perform.

Temporal and spatial variation of terpenoids in eastern hemlock (Tsuga canadensis) in relation to feeding by Adelges tsugae.

The terpenoid content of eastern hemlock (Tsuga canadensis) foliage was measured over an annual cycle of development from bud opening, shoot elongation, shoot maturation, to bud-break at the start of the next growing season. The objective was to determine if variation in terpenoid composition is linked with spatial and temporal feeding preferences of the hemlock woolly adelgid (HWA; Adelges tsugae). The HWA has two periods of feeding over the course of 1 yr spanning two complete generations. There are two periods of feeding separated by a nonfeeding period where the adelgid estivates. HWA prefers to feed on mature, rather than young, expanding tissue. Feeding occurs in the leaf cushion at the base of the needle. The needle is the only tissue in hemlock with resin canals that store terpenoids. The needle and leaf cushion of both the current and previous years' growth were analyzed separately over a 1-yr period to examine the variation of terpenoid composition in space and time. Terpenoids were quantified by using headspace solid-phase microextraction/gas chromatography/mass spectrometry (SPME/GC/MS). New growth needles and leaf cushions do not resemble the previous year's growth either visually or in chemical composition until October/November, when the adelgid breaks estivation and begins feeding. Nearly all of the 23 terpenoids present exceeding 0.1% varied significantly either temporally or spatially, usually with complex interactions. Ordination and factor analysis revealed that terpenoids are less variable in mature leaf cushions than in young tissue. By entering a nonfeeding diapause during the late spring and summer, HWA avoids the unstable, variable levels of terpenoids in the immature leaf cushion and needles.