Assessment of Imidacloprid and Its Metabolites in Foliage of Eastern Hemlock Multiple Years Following Treatment for Hemlock Woolly Adelgid, Adelges tsugae (Hemiptera: Adelgidae), in Forested Conditions.

Widespread decline and mortality of eastern hemlock, Tsuga canadensis (L.) Carrière, have been caused by hemlock woolly adelgid, Adelges tsugae (Annand) (HWA) (Hemiptera: Adelgidae). The current study is a retrospective analysis conducted in collaboration with Great Smoky Mountains National Park (GRSM) to determine longevity of imidacloprid and its insecticidal metabolites (imidacloprid olefin, 5-hydroxy, and dihydroxy) in GRSM's HWA integrated pest management (IPM) program. Foliage samples were collected from three canopy strata of hemlocks that were given imidacloprid basal drench treatments 4-7 yr prior to sampling. Foliage was analyzed to assess concentrations in parts per billion (ppb) of imidacloprid and its metabolites. Imidacloprid and its olefin metabolite were present in most, 95 and 65%, respectively, branchlets 4-7 yr post-treatment, but the 5-hydroxy and dihydroxy metabolites were present in only 1.3 and 11.7%, respectively, of the branchlets. Imidacloprid and olefin concentrations significantly decreased between 4 and 7 yr post-treatment. Concentrations of both imidacloprid and olefin were below the LC50 for HWA 5-7 yr post-treatment. Knowledge of the longevity of imidacloprid treatments and its metabolite olefin can help maximize the use of imidacloprid in HWA IPM programs.

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.

Persistence and recovery of dinotefuran in eastern hemlock (Tsuga canadensis) foliage and twigs by UPLC-MS/MS and ELISA.

BACKGROUND: Dinotefuran, a systemic neonicotinoid insecticide, is approved for control of hemlock woolly adelgid (HWA) (Adelges tsugae Annand), an invasive sap-feeding insect that can kill eastern hemlocks (Tsuga canadensis). Dinotefuran is highly water soluble, facilitating more rapid translocation and HWA control than other neonicotinoids, but its persistence is not well-known. Samples of needles and twigs were collected in spring 2021 from 50 hemlocks treated with a dinotefuran basal trunk spray in 2018 or 2019 (131-145 weeks and 85-93 weeks before sampling, respectively). Processed samples were analyzed with ultra-performance liquid chromatography-tandem mass spectrometer (UPLC-MS/MS) and enzyme-linked immunosorbent assays (ELISA). RESULTS: Dinotefuran residues were 4.6-6.1 times higher in needles than in twigs collected from the same trees. Average (±SE) residues in foliage samples collected from trees treated in 2019 ranged from 0.663 ± 0.243 to 0.564 ± 0.119 mg kg-1 , compared with 0.213 ± 0.033 and 0.225 ± 0.132 mg kg-1 in foliage from trees treated in 2018. Foliage residues from UPLC-MS/MS were consistently lower but strongly related to those from ELISA. Matrix effects appeared to disrupt ELISA analysis of twigs. None of the 25 trees treated in 2019 had live HWA when samples were collected in 2021 while low densities of HWA were observed on 52% of trees treated in 2018. CONCLUSIONS: Dinotefuran was recovered from hemlock foliage, and to a lesser extent twigs, >2 years post-treatment. This, along with its relatively rapid translocation, suggests dinotefuran is a viable option for protecting declining or heavily infested hemlocks from HWA. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Hemlock woolly adelgid and elongate hemlock scale induce changes in foliar and twig volatiles of eastern hemlock.

Eastern hemlock (Tsuga canadensis) is in rapid decline because of infestation by the invasive hemlock woolly adelgid (Adelges tsugae; 'HWA') and, to a lesser extent, the invasive elongate hemlock scale (Fiorinia externa; 'EHS'). For many conifers, induced oleoresin-based defenses play a central role in their response to herbivorous insects; however, it is unknown whether eastern hemlock mobilizes these inducible defenses. We conducted a study to determine if feeding by HWA or EHS induced changes in the volatile resin compounds of eastern hemlock. Young trees were experimentally infested for 3 years with HWA, EHS, or neither insect. Twig and needle resin volatiles were identified and quantified by gas chromatography/mass spectrometry. We observed a suite of changes in eastern hemlock's volatile profile markedly different from the largely terpenoid-based defense response of similar conifers. Overall, both insects produced a similar effect: most twig volatiles decreased slightly, while most needle volatiles increased slightly. Only HWA feeding led to elevated levels of methyl salicylate, a signal for systemic acquired resistance in many plants, and benzyl alcohol, a strong antimicrobial and aphid deterrent. Green leaf volatiles, often induced in wounded plants, were increased by both insects, but more strongly by EHS. The array of phytochemical changes we observed may reflect manipulation of the tree's biochemistry by HWA, or simply the absence of functional defenses against piercing-sucking insects due to the lack of evolutionary contact with these species. Our findings verify that HWA and EHS both induce changes in eastern hemlock's resin chemistry, and represent the first important step toward understanding the effects of inducible chemical defenses on hemlock susceptibility to these exotic pests.

Variation in plant defense against invasive herbivores: evidence for a hypersensitive response in eastern hemlocks (Tsuga canadensis).

Herbivores can trigger a wide array of morphological and chemical changes in their host plants. Feeding by some insects induces a defensive hypersensitive response, a defense mechanism consisting of elevated H(2)O(2) levels and tissue death at the site of herbivore feeding. The invasive hemlock woolly adelgid Adelges tsugae ('HWA') and elongate hemlock scale Fiorinia externa ('EHS') feed on eastern hemlocks; although both are sessile sap feeders, HWA causes more damage than EHS. The rapid rate of tree death following HWA infestation has led to the suggestion that feeding induces a hypersensitive response in hemlock trees. We assessed the potential for an herbivore-induced hypersensitive response in eastern hemlocks by measuring H(2)O(2) levels in foliage from HWA-infested, EHS-infested, and uninfested trees. Needles with settled HWA or EHS had higher H(2)O(2) levels than control needles, suggesting a localized hypersensitive plant response. Needles with no direct contact to settled HWA also had high H(2)O(2) levels, suggesting that HWA infestation may induce a systemic defense response in eastern hemlocks. There was no similar systemic defensive response in the EHS treatment. Our results showed that two herbivores in the same feeding guild had dramatically different outcomes on the health of their shared host.

A comparative toxicity assessment of materials used in aquatic construction.

Comparative toxicity testing was performed on selected materials that may be used in aquatic construction projects. The tests were conducted on the following materials: (1) untreated wood species (hemlock [Tsuga ssp], Western red cedar (Thuja plicata), red oak [Quercus rubra], Douglas fir [Pseudotsuga menziesii], red pine [Pinus resinosa], and tamarack [Larix ssp]); (2) plastic wood; (3) Ecothermo wood hemlock stakes treated with preservatives (e.g., chromated copper arsenate [CCA], creosote, alkaline copper quaternary [ACQ], zinc naphthenate, copper naphthenate, and Lifetime Wood Treatment); (4) epoxy-coated steel; (5) hot-rolled steel; (6) zinc-coated steel; and (7) concrete. Those materials were used in acute lethality tests with rainbow trout, Daphnia magna, Vibrio fischeri and threespine stickleback. The results indicated the following general ranking of the materials (from the lowest to highest LC(50) values); ACQ > creosote > zinc naphthenate > copper naphthenate > CCA (treated at 22.4 kg/m(3)) > concrete > red pine > western red cedar > red oak > zinc-coated steel > epoxy-coated steel > CCA (6.4 kg/m(3)). Furthermore, the toxicity results indicated that plastic wood, certain untreated wood species (hemlock, tamarack, Douglas fir, and red oak), hot-rolled steel, Ecothermo wood, and wood treated with Lifetime Wood Treatment were generally nontoxic to the test species.

Distribution and abundance of Adelges tsugae (Hemiptera: Adelgidae) within hemlock trees.

We studied the distribution of hemlock woolly adelgid, Adelges tsugae Annand (Hemiptera: Adelgidae), within hemlock trees for three summer (progrediens) and two winter (sistens) generations in northern Georgia. Eastern hemlock, Tsuga canadensis (L.) Carrière, trees were treated with 0, 10, or 25% of 1.5 g of imidacloprid per 2.5 cm of tree diameter at breast height and fertilized or not in a factorial design. Adelgid ovisacs per centimeter of branch were more abundant from June 2007 to June 2008 in the upper tree crown of insecticide untreated trees and when all trees were combined and that was the general trend for most comparisons. However, ovisacs were more abundant in the lower crown of insecticide treated trees in June 2008. More sistens nymphs settled on the upper crown branches than on the lower branches in summers 2007 and 2008. Higher eggs per ovisac were observed in the upper crown in February 2008 and in both the winter and summer 2009. In contrast, adelgids were more fecund in the lower crown in June 2008. On fertilized trees, eggs laid per adult were higher in the upper crown in February 2008. In summer 2008, eggs per ovisac were higher in the lower crown, but this reversed again to the upper crown by summer 2009. New growth of branches also varied among sample dates. These data demonstrate the variable distribution of adelgid and hemlock growth within trees over time and suggest that sampling only one crown area will not provide accurate estimates of adelgid densities.

High temperature and fire behavior of hydrothermally modified wood impregnated with carbon nanomaterials.

Wood is one of the most widely used construction materials but it is thermally degradable and combustible, which poses serious safety concerns. In this research, the high temperature and fire behavior of hydrothermally modified western hemlock, impregnated with carbon nanomaterials pre-adsorbed with alkali lignin, was examined by cone calorimetry, scanning electron microscopy, thermal gravimetric analysis, and Fourier transform infrared spectroscopy. The hydrothermal treatment made the wood less hydrophilic, allowing the formation of a dense protective layer of carbon-rich additives on the external wood surface at low loading (5 wt%) after aqueous-phase vacuum impregnation. Results revealed that the unique combination of these two processes reduced the total heat release by up to 32%, diminished flame spread by 31%, decreased the average carbon dioxide yield by 12%, lowered the total mass loss by 10%, and significantly slowed the pyrolytic reactions of wood. This research has important implications for the development of valued-added wood products with superior fire safety from relatively low cost timbers, such as western hemlock.

Evaluation of ELISA for imidacloprid detection in eastern hemlock (Tsuga canadensis) wood and needle tissues.

BACKGROUND: Imidacloprid is the primary insecticide used against the exotic invasive insect hemlock woolly adelgid, Adelges tsugae Annand, a pest of eastern hemlock [Tsuga canadensis (L.) Carrière] trees in the eastern United States. A competitive enzyme-linked immunosorbent assay (ELISA) was evaluated for quantification of imidacloprid in eastern hemlock wood and needle tissues. RESULTS: Matrix effects in the form of false positives and overestimated imidacloprid concentrations were observed in both wood and needle extracts. Tissues required a 100-1000-fold dilution with water in order to reduce matrix effects. Standard curves in 1% wood or needle extract were not significantly different from standard curves prepared in water. Matrix effects were more pronounced at concentrations in the lower working range of the kit, with recovery of 5 microg L(-1) imidacloprid more accurate than recovery of 0.2 microg L(-1). CONCLUSION: ELISA remains a valuable tool for semi-quantitative imidacloprid detection within the hemlock system because of its sensitivity, cost and ease of use. However, a 1000-fold dilution of hemlock tissue extract is recommended to ensure accurate imidacloprid determinations.

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.

Quantitative determination of benzalkonium chloride in treated wood by solid-phase extraction followed by liquid chromatography with ultraviolet detection.

Ammoniacal copper quat (ACQ) compound wood preservative is comprised of copper and quaternary ammonium compounds with benzalkonium chloride (BAC) as the active ingredient. Solid-phase extraction (SPE) followed by liquid chromatography with ultraviolet detection (LC-UV) was developed for quantitative determination of BAC in treated wood. Five species of wood were used, Japanese cedar (Cryptomeria japonica), Japanese larch (Larix leptolepis), Yezo spruce (Picea jezoensis), Sakhalin fir (Abies sachalinensis), and western hemlock (Tsuga heterophylla). BAC used in the present study was composed of 66% C12, 33% C14 and less than 1% C16. BAC was added to each wood species (500 mg) then extracted with HCl-ethanol (20 ml) and quantitatively determined with LC-UV (262 nm). Wood extractives from the heartwood of each species, except western hemlock, interfered with quantitative determination of BAC, but SPE with an Oasis MCX cartridge was effective in preventing this. Using the present methods, BAC homologue peaks were clearly confirmed without interference. Recoveries from wood ranged from 92 to 101% and the limit of quantitation was approximately 240 microg/g wood for the C12 and C14 homologues.

Assessing hemlock decline using visible and near-infrared spectroscopy: indices comparison and algorithm development.

Near-infrared reflectance spectroscopy was evaluated for its effectiveness at predicting pre-visual decline in eastern hemlock trees. An ASD FieldSpec Pro FR field spectroradiometer measuring 2100 contiguous 1-nm-wide channels from 350 nm to 2500 nm was used to collect spectra from fresh hemlock foliage. Full spectrum partial least squares (PLS) regression equations and reduced stepwise linear regression equations were compared. The best decline predictive model was a 6-term linear regression equation (R2= 0.71, RMSE = 0.591) based on: Carter Miller Stress Index (R694/R760), Derivative Chlorophyll Index (FD705/FD723), Normalized Difference Vegetation Index ((R800 - R680)/(R800 + R680)), R950, R1922, and FD1388. Accuracy assessment showed that this equation predicted an 11-class decline rating with a 1-class tolerance accuracy of 96% and differentiated healthy trees from those in very early decline with 72% accuracy. These results indicate that narrow-band sensors could be developed to detect very early stages of hemlock decline, before visual symptoms are apparent. This capability would enable land managers to identify early hemlock woolly adelgid infestations and monitor forest health over large areas of the landscape.

Effects of Light and Water Availability on the Performance of Hemlock Woolly Adelgid (Hemiptera: Adelgidae).

Eastern hemlock (Tsuga canadensis (L.) Carriere) is a dominant shade-tolerant tree in northeastern United States that has been declining since the arrival of the hemlock woolly adelgid (Adelges tsugae Annand). Determining where A. tsugae settles under different abiotic conditions is important in understanding the insect's expansion. Resource availability such as light and water can affect herbivore selectivity and damage. We examined how A. tsugae settlement and survival were affected by differences in light intensity and water availability, and how adelgid affected tree performance growing in these different abiotic treatments. In a greenhouse at the University of Rhode Island, we conducted an experiment in which the factors light (full-sun, shaded), water (water-stressed, watered), and adelgid (infested, insect-free) were fully crossed for a total of eight treatments (20 two-year-old hemlock saplings per treatment). We measured photosynthesis, transpiration, water potential, relative water content, adelgid density, and survival throughout the experiment. Adelgid settlement was higher on the old-growth foliage of shaded and water-stressed trees, but their survival was not altered by foliage age or either abiotic factor. The trees responded more to the light treatments than the water treatments. Light treatments caused a difference in relative water content, photosynthetic rate, transpiration, and water potential; however, water availability did not alter these variables. Adelgid did not enhance the impact of these abiotic treatments. Further studies are needed to get a better understanding of how these abiotic factors impact adelgid densities and tree health, and to determine why adelgid settlement was higher in the shaded treatments.

Volatile emissions of eastern hemlock, Tsuga canadensis, and the influence of hemlock woolly adelgid.

The volatile emissions of eastern hemlock, Tsuga canadensis Carriere, were identified and quantified using standard and chiral gas chromatography and mass spectrometry. All of the identified compounds were monoterpenes, and included alpha-pinene, myrcene, tricyclene, camphene, alpha-phellandrene, beta-pinene, limonene, beta-phellandrene, terpinolene, and bornyl acetate. alpha-Pinene, myrcene, and camphene comprised greater than 75% by mass of the total release. Infestation by the exotic insect, hemlock woolly adelgid (HWA, Adelges tsugae Annand), resulted in an increased release rate of monoterpenes from branch tips. Release rate was negatively correlated to the amount of the branch tip sample that was new growth, suggesting that release rate is greater from previous-year foliage. Additionally the percent composition of the volatile profile is slightly altered by infestation, with alpha-pinene comprising 57% of volatiles from infested foliage and 66% from uninfested foliage.

Analysis of terpenoids from hemlock (Tsuga) species by solid-phase microextraction/gas chromatography/ion-trap mass spectrometry.

A sampling method for determining the volatile terpenoid composition from single needles of seven Tsuga species was developed using headspace solid-phase microextraction (SPME). A reproducible sampling method for the volatile components was generated by examination of sample storage, method of needle cutting, and headspace sampling duration. Following SPME collection of the volatile compounds from the seven Tsuga species, gas chromatography/ion-trap mass spectrometry was used to identify 51 terpenoids present in the needle headspace. A semiquantitative method was devised to express individual terpenoid amounts as a percentage of all of the identified peaks in the chromatogram. The semiquantitative results permitted facile interspecies comparison using principal component analysis. Two components were able to account for 90% of the variance and were interpreted as a "species" component and a "resistance/susceptibility" component. Three interspecies groupings were evident from the principal component analysis: (1) Tsuga canadensis and Tsuga caroliniana; (2) Tsuga chinesnsis, Tsuga diversifolia, Tsuga heterophylla, and Tsuga sieboldii; and (3) Tsuga mertensiana. The finding that T. mertensiana was grouped alone and far removed from the other species adds to the morphological evidence that this species should be segregated from other Tsuga.

Abietoid seed fatty acid compositions--a review of the genera Abies, Cedrus, Hesperopeuce, Keteleeria, Pseudolarix, and Tsuga and preliminary inferences on the taxonomy of Pinaceae.

The seed fatty acid (FA) compositions of Abietoids (Abies, Cedrus, Hesperopeuce, Keteleeria, Pseudolarix, and Tsuga) are reviewed in the present study in conclusion to our survey of Pinaceae seed FA compositions. Many unpublished data are given. Abietoids and Pinoids (Pinus, Larix, Picea, and Pseudotsuga)-constituting the family Pinaceae-are united by the presence of several delta5-olefinic acids, taxoleic (5,9-18:2), pinolenic (5,9,12-18:3), coniferonic (5,9,12,15-1 8:4), keteleeronic (5,11-20:2), and sciadonic (5,11,14-20:3) acids, and of 14-methyl hexadecanoic (anteiso-17:0) acid. These acids seldom occur in angiosperm seeds. The proportions of individual delta5-olefinic acids, however, differ between Pinoids and Abietoids. In the first group, pinolenic acid is much greater than taxoleic acid, whereas in the second group, pinolenic acid is greater than or equal to taxoleic acid. Moreover, taxoleic acid in Abietoids is much greater than taxoleic acid in Pinoids, an apparent limit between the two subfamilies being about 4.5% of that acid relative to total FA. Tsuga spp. appear to be a major exception, as their seed FA compositions are much like those of species from the Pinoid group. In this respect, Hesperopeuce mertensiana, also known as Tsuga mertensiana, has little in common with Abietoids and fits the general FA pattern of Pinoids well. Tsuga spp. and H. mertensiana, from their seed FA compositions, should perhaps be separated from the Abietoid group and their taxonomic position revised. It is suggested that a "Tsugoid" subfamily be created, with seed FA in compliance with the Pinoid pattern and other botanical and immunological criteria of the Abietoid type. All Pinaceae genera, with the exception of Pinus, are quite homogeneous when considering their overall seed FA compositions, including delta5-olefinic acids. In all cases but one (Pinus), variations from one species to another inside a given genus are of small amplitude. Pinus spp., on the other hand, have highly variable levels of delta5-olefinic acids in their FA compositions, particularly when sections (e.g., Cembroides vs. Pinus sections) or subsections (e.g., Flexiles and Cembrae subsections from the section Strobus) are compared, although they show qualitatively the same FA patterns characteristic of Pinoids. Multicomponent analysis of Abietoid seed FA allowed grouping of individual species into genera that coincide with the same genera otherwise characterized by more classical botanical criteria. Our studies exemplify how seed FA compositions, particularly owing to the presence of delta5-olefinic acids, may be useful in sustaining and adding some precision to existing taxonomy of the major family of gymnosperms, Pinaceae.

Effects of hemlock woolly adelgid and elongate hemlock scale on eastern hemlock growth and foliar chemistry.

In the eastern United States, two invasive specialist insects share a native host plant, Eastern hemlock, Tsuga canadensis. In recent years, much research has focused on the impacts of the hemlock woolly adelgid (Adelges tsugae) because of the detrimental effects it has on hemlock growth and survival. In contrast, the invasive elongate hemlock scale (Fiorinia externa) is thought to have only minor impacts on hemlock. We infested hemlock saplings with each insect and compared them with control (i.e., neither insect herbivore) saplings to assess how early infestations impact Eastern hemlock health (measured using new branch growth, foliar %N, and C:N ratio). Our study showed that, at equal densities, the two insects differed in their effect on Eastern hemlock. F. externa did not impact plant growth or foliar chemistry over the course of the 2-yr experiment. A. tsugae significantly reduced plant growth and caused a reduction of %N in the first year of the experiment. By the end of the experiment, A. tsugae trees had the same %N in their foliage as control and F. externa trees but drastically reduced growth patterns. The most likely explanation for this result is the greater growth in control and F. externa saplings during the second year resulted in the dilution of available foliar N over a larger amount of newly produced plant tissue. For early infestations of both insects, our study suggests that management plans should focus on the more detrimental A. tsugae.

Characterization of pollen by vibrational spectroscopy.

Classification, discrimination, and biochemical assignment of vibrational spectra of pollen samples belonging to 43 different species of the order Pinales has been made using three different vibrational techniques. The comparative study of transmission (KBr pellet) and attenuated total reflection (ATR) Fourier transform infrared (FT-IR) and FT-Raman spectroscopies was based on substantial variability of pollen grain size, shape, and relative biochemical composition. Depending on the penetration depth of the probe light, vibrational techniques acquire predominant information either on pollen grain walls (FT-Raman and ATR-FT-IR) or intracellular material (transmission FT-IR). Compared with the other two methods, transmission FT-IR obtains more comprehensive information and as a result achieves superior spectral identification and discrimination of pollen. The results strongly indicate that biochemical similarities of pollen grains belonging to the same plant genus or family lead to similar features in corresponding vibrational spectra. The exploitation of that property in aerobiological monitoring was demonstrated by simple and rapid pollen identification based on relatively small spectral libraries, with the same (or better) taxonomic resolution as that provided by optical microscopy. Therefore, the clear correlation between vibrational spectra and pollen grain morphology, biochemistry, and taxonomy is obtained, while successful pollen identification illustrates the practicability of such an approach in environmental studies.

Are histones the targets for flavan-3-ols (catechins) in nuclei?

Binding of flavan-3-ols to nuclei is characteristic of Tsuga canadensis (coniferous tree). This is achieved with the DMACA reagent (p-dimethylamino-cinnamaldehyde), which stains almost exclusively monomeric and oligomeric flavan-3-ols with an intense blue colour. Deep flavanol staining also occurred when calf cells of small intestine were enriched with added catechins. In order to detect the components of nuclei that may associate with catechins, the principal components of chromatin (DNA, histones) were subjected to UV-VIS spectroscopic titration. DNA or histone sulphate containing the histones H1, H2A, H2B, H3 and H4 were dissolved in cationic and anionic buffers (Tris, phosphate) at different pH values (pH 8.0, 7.4 and 7.0) and titrated with EGCG (epigallocatechin gallate) or catechin. The results show that DNA of calf thymus and the catechins investigated form no spectroscopically detectable association equilibria. However, strong association complexes are found between histone sulphate and EGCG or catechin by means of the Mauser diagrams (A and AD diagrams). The association equilibria can be accompanied by aggregation (precipitation) of histone proteins, especially initiated by EGCG. The titration equilibria are spectroscopically more pronounced in Tris buffers at higher pH values than at lower values, whereas in phosphate buffers the opposite trend is found. Surprisingly, catechin shows nearly no interactions with histone sulphate in phosphate buffers in the pH range 7.0-8.0, which is in contrast to EGCG. Fundamentally, the targets of chromosomes for catechins seem to be the histone proteins of chromatin.