Examining the Impact of a Public Health Message on Fish Consumption in Bermuda

Background

In 2003 mean cord blood mercury concentrations in pregnant Bermudian women exceeded levels associated with adverse health outcomes in children. The principal mercury source was local fish species. Public health messages were developed suggesting pregnant women reduce consumption of fish species with higher mercury concentrations (e.g. swordfish), substituting species containing lower mercury concentrations, and elevated omega-3 fatty acids (e.g. anchovies). Recent evidence indicates mercury concentrations in Bermuda’s pregnant women have fallen five- fold.

Objectives

Assess whether changes in women’s fish eating patterns during pregnancy are consistent with the public health messaging. Determine who is making changes to their diet during pregnancy and why.

Methods

Mixed methods study with a cross-sectional survey of 121 pregnant women, including 13 opened-ended interviews. Health system, social vulnerability, public health messaging, and socio-demographic variables were characterized and related to changes in fish consumption during pregnancy. Qualitative data were coded according to nutritional advice messages, comprehension of communication strategies, and sources of information.

Results

95% of women surveyed encountered recommendations about fish consumption during pregnancy. 75% reported modifying fish eating behaviors because of recommendations. Principal sources of information about fish consumption in pregnancy were health care providers and the Internet. 71% of women reported reducing consumption of large fish species with greater mercury levels, but 60% reported reduced consumption of smaller, low mercury fish. No participant mentioned hearing about the benefits of fish consumption. More frequent exposure to public health messages during pregnancy was associated with lower reported consumption. Bermudian born women were less likely to reduce consumption of large fish species during pregnancy.

Conclusions

In Bermuda, public health messages advocating reduced consumption of larger, higher mercury-containing fish species appear effective, but masked the nutritional value message of small fish species, with low mercury concentration. Adjustment is needed to better balance the risk communication.     

Spatial Subsidies, Trophic State, and Community Structure: Examining the Effects of Leaf Litter Input on Ponds

In aquatic systems, light and subsidy input often co-vary along a canopy cover gradient. This creates systems where subsidies are more prevalent in areas with low primary productivity and less prevalent in areas with high primary productivity. We expect ecosystem processes and community structure to respond to these changes in resources. We examined the effects of light and subsidy input (leaf litter) on ponds by placing pond mesocosms along a canopy gradient and manipulating litter input. We then sampled mesocosms for ecosystem and community parameters for 2 years during April (pre-leaf out), July (full canopy), and November (start of leaf fall). Neither canopy cover nor litter input tended to dominate effects overall, highlighting the importance of the light-subsidy gradient combination that is frequently encountered in temperate aquatic systems. Ponds shifted from an autotrophy/heterotrophy balance to net heterotrophy with increasing canopy cover in concordance with our predictions. Although litter input affected dissolved oxygen, we did not detect an effect of litter on trophic state, primary production, or community respiration. We additionally found effects of both canopy cover and litter input on community composition, but very differently than that found in streams. In general, the grazer and shredder macroinvertebrate functional feeding groups were rare in the pond mesocosms, and thus did not respond to treatments. The collector proportion of the community, mainly chironomids, increased with canopy cover and was higher in mesocosms with litter input than those without. Contrary to predictions, there were few differences between subsidy input type (leaves or grass) despite differences in litter quality. This study, along with many others, highlights the importance of canopy gradients in determining ecosystem function and community composition.     

Impact of Mycobacterium Avium Infection on Macrophage-Liposome Interaction

Abstract

Mycobacterium avium (M. avium) can survive within macrophages, and alters various functions of its cellular host. Liposomes and other particulate drug carriers have been investigated as a means for enhancing drug delivery to the intracellular site of infection via the endocytic pathway. We have investigated the impact of M. avium infection on the endocytosis, retention, and intracellular disposition of liposomes, as these are essential macrophage functions that may determine the activity of liposome-encapsulated antibiotics. J774, a macrophage-like cell line, was infected with M. avium at various multiplicities. Infected cells accumulated and retained liposomes in a manner similar to uninfected cells, regardless of the time after infection. Moreover, the intracellular trafficking and disposition of liposomes was unaltered in J774 cells that contained live or heat-killed M. avium, as evidenced by the vesicular pH which liposomes encountered within cells. These results suggest that specific essential cellular functions involved in processing particulate drug carriers remain intact in macrophages infected with M. avium.     

The Political Impact of Trade in Chiefdoms

Trade theories of state formation have failed to specify trade as market behavior, and to appreciate the political role of imported luxury goods in chiefdoms. When luxury goods and other valuable commodities become available through the market rather than through interpersonal, chiefly exchanges, the elite's power depends increasingly on economic exploitation and the control of arms. Archeological data from several locales illustrate the disruptive and formative effects of trade diasporas in chiefdoms.     

Investigating the Impact of Storage Conditions on Microbial Community Composition in Soil Samples

Recent advances in DNA sequencing technologies have allowed scientists to probe increasingly complex biological systems, including the diversity of bacteria in the environment. However, despite a multitude of recent studies incorporating these methods, many questions regarding how environmental samples should be collected and stored still persist. Here, we assess the impact of different soil storage conditions on microbial community composition using Illumina-based 16S rRNA V4 amplicon sequencing. Both storage time and temperature affected bacterial community composition and structure. Frozen samples maintained the highest alpha diversity and differed least in beta diversity, suggesting the utility of cold storage for maintaining consistent communities. Samples stored for intermediate times (three and seven days) had both the highest alpha diversity and the largest differences in overall beta diversity, showing the degree of community change after sample collection. These divergences notwithstanding, differences in neither storage time nor storage temperature substantially altered overall communities relative to more than 500 previously examined soil samples. These results systematically support previous studies and stress the importance of methodological consistency for accurate characterization and comparison of soil microbiological assemblages.     

Impact of Conjunctivochalasis on Visual Quality of Life: A Community Population Survey

Conjunctivochalasis (Cch) is a very common ocular disorder, which can cause an unstable tear film and ocular discomfort. The study of vision-related quality of life (VR-QoL) in a community population with Cch can provide a better understanding of the impact of Cch on common people than objective clinical examinations alone. This cross-sectional comparative study enrolled 360 participants ≥40 years old living in Sanle Community, Shanghai. In the study, 198 subjects were diagnosed with Cch and 86 with dry eye syndrome (DES) without Cch. The remaining 76 subjects were normal controls. Socio-demographical data were collected, and Cch and related ocular symptoms and signs were evaluated. In addition, all participants were required to complete the Chinese version of the 25-item National Eye Institute Visual Functioning Questionnaire (NEI VFQ-25) and Ocular Surface Disease Index Questionnaire (OSDI). Main outcome measures include the comparison on the OSDI score and VFQ-25 score among the subgroups, and the correlation of these scores with the socio-demographical and clinical data. The results revealed that subjects with Cch had significantly decreased tear film stability even compared with those with DES (P = 0.001). The participants with either Cch or DES reported significantly higher OSDI scores and lower VFQ-25 composite scores than the normal controls (P<0.001 and 0.007 respectively). Further comparisons among the subgroups of Cch revealed that the following factors were associated with higher OSDI scores and lower VFQ-25 composite scores: nasal-side Cch, chalasis folds higher than tear meniscus height, punctal occlusion, or increased extent of chalasis on digital pressure. In conclusion, Cch was associated with an adverse impact on VR-QoL in a community population, and the impairment in VR-QoL had a significant correlation with disease severity and tear film abnormalities.     

Orienteering in Knowledge Spaces: The Hyperbolic Geometry of Wikipedia Mathematics

In this paper we show how the coupling of the notion of a network with directions with the adaptation of the four-point probe from materials testing gives rise to a natural geometry on such networks. This four-point probe geometry shares many of the properties of hyperbolic geometry wherein the network directions take the place of the sphere at infinity, enabling a navigation of the network in terms of pairs of directions: the geodesic through a pair of points is oriented from one direction to another direction, the pair of which are uniquely determined. We illustrate this in the interesting example of the pages of Wikipedia devoted to Mathematics, or “The MathWiki.” The applicability of these ideas extends beyond Wikipedia to provide a natural framework for visual search and to prescribe a natural mode of navigation for any kind of “knowledge space” in which higher order concepts aggregate various instances of information. Other examples would include genre or author organization of cultural objects such as books, movies, documents or even merchandise in an online store.     

Content Volatility of Scientific Topics in Wikipedia: A Cautionary Tale

Wikipedia has quickly become one of the most frequently accessed encyclopedic references, despite the ease with which content can be changed and the potential for ‘edit wars’ surrounding controversial topics. Little is known about how this potential for controversy affects the accuracy and stability of information on scientific topics, especially those with associated political controversy. Here we present an analysis of the Wikipedia edit histories for seven scientific articles and show that topics we consider politically but not scientifically “controversial” (such as evolution and global warming) experience more frequent edits with more words changed per day than pages we consider “noncontroversial” (such as the standard model in physics or heliocentrism). For example, over the period we analyzed, the global warming page was edited on average (geometric mean ±SD) 1.9±2.7 times resulting in 110.9±10.3 words changed per day, while the standard model in physics was only edited 0.2±1.4 times resulting in 9.4±5.0 words changed per day. The high rate of change observed in these pages makes it difficult for experts to monitor accuracy and contribute time-consuming corrections, to the possible detriment of scientific accuracy. As our society turns to Wikipedia as a primary source of scientific information, it is vital we read it critically and with the understanding that the content is dynamic and vulnerable to vandalism and other shenanigans.     

Impact of Nutrient Composition on a Degradative Biofilm Community

A microbial community was cultivated in flow cells with 2,4,6-trichlorobenzoic acid (2,4,6-TCB) as sole carbon and energy source and was examined with scanning confocal laser microscopy and fluorescent molecular probes. The biofilm community which developed under these conditions exhibited a characteristic architecture, including a basal cell layer and conspicuous mounds of bacterial cells and polymer (approximately 20 to 30 (mu)m high and 25 to 40 (mu)m in diameter) occurring at 20- to 200-(mu)m intervals. When biofilms grown on 2,4,6-TCB were shifted to a labile, nonchlorinated carbon source (Trypticase soy broth), the biofilms underwent an architectural change which included the loss of mound structures and the formation of a more homogeneous biofilm. Neutrally charged fluorescent dextrans, which upon hydration become cationic, were observed to bind to mounds, as well as to the basal cell layer, in 14-day biofilms. In contrast, polyanionic dextrans bound only to the basal cell layer, indicating that this material incorporated sites with both positive and negative charge. The results from this study indicate that nutrient composition has a significant impact on both the architecture and the physicochemistry of degradative biofilm communities.     

Examining the Impact of the National Institutes of Health Public Access Policy on the Citation Rates of Journal Articles

PurposeTo examine whether National Institutes of Health (NIH) funded articles that were archived in PubMed Central (PMC) after the release of the 2008 NIH Public Access Policy show greater scholarly impact than comparable articles not archived in PMC.MethodsA list of journals across several subject areas was developed from which to collect article citation data. Citation information and cited reference counts of the articles published in 2006 and 2009 from 122 journals were obtained from the Scopus database. The articles were separated into categories of NIH funded, non-NIH funded and whether they were deposited in PubMed Central. An analysis of citation data across a five-year timespan was performed on this set of articles.ResultsA total of 45,716 articles were examined, including 7,960 with NIH-funding. An analysis of the number of times these articles were cited found that NIH-funded 2006 articles in PMC were not cited significantly more than NIH-funded non-PMC articles. However, 2009 NIH funded articles in PMC were cited 26% more than 2009 NIH funded articles not in PMC, 5 years after publication. This result is highly significant even after controlling for journal (as a proxy of article quality and topic).ConclusionOur analysis suggests that factors occurring between 2006 and 2009 produced a subsequent boost in scholarly impact of PubMed Central. The 2008 Public Access Policy is likely to be one such factor, but others may have contributed as well (e.g., growing size and visibility of PMC, increasing availability of full-text linkouts from PubMed, and indexing of PMC articles by Google Scholar).     

Impact of Neonatal Antibiotic Treatment on the Biodiversity of the Murine Intestinal Lactobacillus Community

In this study, we investigated the impact of neonatal amoxicillin treatment on the development of the murine intestinal Lactobacillus community. Suckling BALB/c mice received a daily intragastric gavage of amoxicillin or saline from postnatal day 7 (PND 7) to PND 20. Just after the treatment (PND 21) and 5 weeks later (PND 56), the colon digesta samples were analyzed by Lactobacillus-specific quantitative real-time PCR analysis and PCR-denaturing gradient gel electrophoresis (DGGE) technique. Real-time PCR results showed that the levels of lactobacilli in the treatment group were similar to those in the control at PND 56. However, in DGGE analysis the number of DGGE bands and Shannon index were decreased significantly in comparison with control (P < 0.05). The dominant Lactobacillus strain in the murine colon changed from L. johnsonii to L. murinus. These results demonstrated that neonatal amoxicillin treatment led to a significant impact on the biodiversity of the murine intestinal Lactobacillus community within a long-time period.     

Identification of the Mind Bomb1 Interaction Domain in Zebrafish DeltaD

Ubiquitylation promotes endocytosis of the Notch ligands like Delta and Serrate and is essential for them to effectively activate Notch in a neighboring cell. The RING E3 ligase Mind bomb1 (Mib1) ubiquitylates DeltaD to facilitate Notch signaling in zebrafish. We have identified a domain in the intracellular part of the zebrafish Notch ligand DeltaD that is essential for effective interactions with Mib1. We show that elimination of the Mind bomb1 Interaction Domain (MID) or mutation of specific conserved motifs in this domain prevents effective Mib1-mediated ubiquitylation and internalization of DeltaD. Lateral inhibition mediated by Notch signaling regulates early neurogenesis in zebrafish. In this context, Notch activation suppresses neurogenesis, while loss of Notch-mediated lateral inhibition results in a neurogenic phenotype, where too many cells are allowed to become neurons. While Mib1-mediated endocytosis of DeltaD is essential for effective activation of Notch in a neighboring cell (in trans) it is not required for DeltaD to inhibit function of Notch receptors in the same cell (in cis). As a result, forms of DeltaD that have the MID can activate Notch in trans and suppress early neurogenesis when mRNA encoding it is ectopically expressed in zebrafish embryos. On the other hand, when the MID is eliminated/mutated in DeltaD, its ability to activate Notch in trans fails but ability to inhibit in cis is retained. As a result, ectopic expression of DeltaD lacking an effective MID results in a failure of Notch-mediated lateral inhibition and a neurogenic phenotype.     

Rice to Vegetables: Short- Versus Long-Term Impact of Land-Use Change on the Indigenous Soil Microbial Community

Land-use change is known to have a significant effect on the indigenous soil microbial community, but it is unknown if there are any general trends regarding how this effect varies over time. Here, we describe a comparative analysis of microbial communities from three adjacent agricultural fields: one-century-old paddy field (OP) and two vegetable fields (new vegetable field (NV) and old vegetable field (OV)) that were established on traditional paddy fields 10 and 100 years ago, respectively. Soil chemical and physical analysis showed that both vegetable fields were more nutrient rich than the paddy field in terms of organic C, total N, total P, and available K. The vegetable fields possessed relatively higher abundance of culturable bacteria, fungi, and specific groups of bacteria (Actinomyces, nitrifying bacteria, and cellulose-decomposing bacteria) but lower levels of microbial biomass C and N. Notably, the decrease of biomass was further confirmed by analysis of seven additional soils in chronosequence sampled from the same area. Next we examined the metabolic diversity of the microbial community using the EcoPlate^TM system from Biolog Inc. (Hayward, CA, USA). The utilization patterns of 31 unique C substrates (i.e., community-level physiological profile) showed that microorganisms in vegetable soil and paddy soil prefer to use different C substrates (polymeric compounds for NV and OV soils, phenolic acids for OP soil). Principal component analysis and the average well color development data showed that the NV is metabolically more distinct from the OV and OP. The effect was likely attributable to the elevated soil pH in NV soil. Furthermore, we assessed the diversity of soil bacterial populations using the cultivation-independent technology of amplified ribosomal DNA restriction analysis (ARDRA). Results showed that levels of bacterial diversity in OP and NV soils were similar (Shannon’s diversity index H = 4.83 and 4.79, respectively), whereas bacteria in OV soil have the lowest score of diversity (H = 3.48). The low level of bacterial diversity in OV soil was supported by sequencing of ten randomly selected 16S rDNA clones from each of the three rDNA libraries. Phylogenetic analysis showed that all the ten OV clones belonged to Proteobacteria with eight in the gamma-subdivision and two in the alpha-subdivision. In contrast, the ten clones from NV and OP soils were classified into four and eight bacterial classes or unclassified groups, respectively. Taken together, our data suggest that land-use change from rice to vegetables resulted in a decrease of bacterial diversity and soil biomass despite an increase in the abundance of culturable microorganisms and, moreover, the decrease of bacterial diversity occurred during long-term rather than short-term vegetable cultivation.     

Environmental Determinants of and Impact on Childhood Asthma by the Bacterial Community in Household Dust

Asthma increased dramatically in the last decades of the 20th century and is representative of chronic diseases that have been linked to altered microbial exposure and immune responses. Here we evaluate the effects of environmental exposures typically associated with asthma protection or risk on the microbial community structure of household dust (dogs, cats, and day care). PCR-denaturing gradient gel analysis (PCR-DGGE) demonstrated that the bacterial community structure in house dust is significantly impacted by the presence of dogs or cats in the home (P = 0.0190 and 0.0029, respectively) and by whether or not children attend day care (P = 0.0037). In addition, significant differences in the dust bacterial community were associated with asthma outcomes in young children, including wheezing (P = 0.0103) and specific IgE (P = 0.0184). Our findings suggest that specific bacterial populations within the community are associated with either risk or protection from asthma.Recent studies have begun to explore the microbial composition of house dust, finding great diversity and a high abundance of Gram-positive organisms (18, 24). Yet this transient community remains relatively unexplored despite increasing evidence of an association between microbial exposure and human health, in particular, the development of chronic diseases such as asthma. Settings with high levels of microbial exposure, such as farms and day care centers, have been associated with protection from asthma (3, 23), while interventions that reduce home microbial exposures have been related to higher rates of allergic sensitization (29). Moreover, variability in home microbial exposures has been linked to differences in immune response and asthma risk in childhood (1, 4). Finally, genes involved in innate immune responses to microbes have been found to interact with microbial exposures, resulting in altered risks for asthma (10). Microbial exposure has primarily been assessed in these studies through measurement of surrogates, such as lipopolysaccharide (LPS, or endotoxin), a component of the outer membrane of Gram-negative bacteria (16), and more recently N-acetyl muramic acid for Gram-positive bacteria (28) and ergosterol for fungi (25). While the use of such surrogates has confirmed a negative relationship between levels of microbial exposure and the development of asthma, exploration is needed beyond a handful of surrogates to more completely represent the complexity of the actual microbial communities present in homes.A preliminary experiment was performed to characterize the bacterial communities of dust from homes in Tucson, AZ. First, general bacterial community diversity was examined in house dust samples by using a PhyloChip microarray as described by Brodie et al. (5). These samples were obtained from homes of four volunteers in the Tucson area. Array results revealed an average of 295 ± 16 (mean ± standard deviation) unique operational taxonomic units (OTUs) per sample, where a taxon was considered present in the sample when more than 92% of the assigned probe pairs for its corresponding probe set were positive. Of these OTUs, 85% belonged to the same 13 divisions in each dust sample, with the 5 most dominant divisions including the Alphaproteobacteria (17.6 ± 2.6%), Firmicutes (16.3 ± 4.1%), Actinobacteria (13.4 ± 2.2%), Gammaproteobacteria (11.8 ± 1.1%), and Deltaproteobacteria (8.7 ± 0.4%) (see Fig. S1 in the supplemental material). Distributions among the 13 major divisions were fairly consistent but not identical among the four different dust samples. The remaining 15% of OTUs belonged to 33 other bacterial divisions but were present at frequencies of less than 1% each. Thus, the number of OTUs and the phylogenetic distributions of these OTUs among the four households were similar.A second experiment was performed to see how the structure of house dust communities varies with environmental parameters and asthma outcomes. The dust samples, examined using denaturing gradient gel electrophoresis (DGGE), were obtained from homes of infants enrolled in the Infant Immune Study (IIS), a nonselected birth cohort (12). Dust samples were collected when the child was approximately 3 months old, at which time the home condition was evaluated. Other information available on this cohort includes data on (i) early life exposures potentially related to asthma risk (the presence of a dog or cat in the home and day care attendance in the first months of life) and (ii) asthma-related outcomes of wheezing and aeroallergen sensitization (specific IgE [Sp-IgE]) (7). A subset (n = 44) of the IIS dust samples was used in this analysis. Based upon the premise that health outcomes will be influenced by the most abundant members of the dust bacterial community (in terms of numbers), DGGE of 16S rRNA gene PCR amplicons was selected as the profile technique to compare the microbial community structures of dust samples from the 44 homes. PCR-DGGE profiles evaluate 16S rRNA genes from all bacterial populations whose initial template concentrations represent >1% of the total community DNA (6, 13, 17). PhyloChip analysis was not used for this experiment because the cost would have been prohibitive for the number of households analyzed.DNA extraction was performed on the IIS dust samples by direct lysis using the Fast DNA spin kit for soil (MP Biomedicals, Solon, OH) as described previously (9). The amount of dust used for extraction ranged from 21 to 35 mg. For each set of extractions a DNA blank (no dust) was included to monitor potential contamination during the extraction process. The extracted DNA was labeled with the PicoGreen double-stranded DNA (dsDNA) quantitation reagent (Molecular Probes, Eugene, OR) and quantified using the Turner Biosystems TBS 380 fluorometer. DNA yields ranged from 0.2 to 41.2 ng DNA/mg of dust.For DGGE analysis, the community DNA obtained from the dust samples was PCR amplified (25 cycles) using the V7/V8 variable region of the 16S rRNA gene and primers 1070f and 1392R with a 40-bp GC clamp (11), following the protocol of Colores et al. (8). Unacetylated bovine serum albumin (Sigma, St. Louis, MO) was added to the PCR mixture at a concentration of 0.4 g liter⁻¹ to relieve PCR inhibition. Amplicons were separated on DGGE gels by using the Bio-Rad Laboratories system (D-Code; Hercules, CA) with a 6% acrylamide gel and a 45% to 65% urea-formamide denaturing gradient. Each DGGE gel run in this study examined either an environmental exposure or an asthma-related outcome variable, as detailed in Tables ​Tables11 and ​and2.2. For example, children reported to have actively wheezed on at least two out of three questionnaires obtained at 1, 2, and 3 years were compared with children for whom all three questionnaires were completed but for whom no wheezing was reported. Fourteen samples were analyzed on each gel, including seven different households from the negative group and seven different households from the positive group. Two lanes of the gel were also loaded with negative controls. DGGE gels were run for 15.5 h at a constant voltage of 50 V, stained with 3× SYBR green I (Molecular Probes, Eugene, OR) for 40 min, and then visualized.

TABLE 1.

Gel comparison groups for early life environmental exposures

Impact of an 8-Year-Old Transgenic Poplar Plantation on the Ectomycorrhizal Fungal Community

The long-term impact of field-deployed genetically modified trees on soil mutualistic organisms is not well known. This study aimed at evaluating the impact of poplars transformed with a binary vector containing the selectable nptII marker and β-glucuronidase reporter genes on ectomycorrhizal (EM) fungi 8 years after field deployment. We generated 2,229 fungal internal transcribed spacer (ITS) PCR products from 1,150 EM root tips and 1,079 fungal soil clones obtained from the organic and mineral soil horizons within the rhizosphere of three control and three transformed poplars. Fifty EM fungal operational taxonomic units were identified from the 1,706 EM fungal ITS amplicons retrieved. Rarefaction curves from both the root tips and soil clones were close to saturation, indicating that most of the EM species present were recovered. Based on qualitative and/or quantitative α- and β-diversity measurements, statistical analyses did not reveal significant differences between EM fungal communities associated with transformed poplars and the untransformed controls. However, EM communities recovered from the root tips and soil cloning analyses differed significantly from each other. We found no evidence of difference in the EM fungal community structure linked to the long-term presence of the transgenic poplars studied, and we showed that coupling root tip analysis with a soil DNA cloning strategy is a complementary approach to better document EM fungal diversity.The poplar has become a model tree species in genetic engineering as it can easily be transformed and clonally propagated and has a small genome size (7, 77, 80). Tree growth, agronomic traits, and timber quality can be improved through genetic engineering (61), thereby avoiding the long reproductive cycles of conventional breeding (47, 59, 83). However, concerns have arisen about the potential impact of genetically modified (GM) trees on the environment (10). The potential environmental hazards linked to GM trees differ from those associated with transgenic crop plants at both spatial and temporal scales (84) because trees are long-lived perennials, unlike annual crop plants. They display several biotic interactions with soil microbial communities such as bacteria and fungi. Interactions between GM trees and these communities could result in exposure to the expression of new traits over several decades, a period longer than those for GM crop plants.Impact studies of GM plants on nontarget organisms usually focus on the potential risk linked to transgene expression (expected effects) that confers a genetic advantage to the transformed plant rather than on unforeseen (pleiotropic) effects from transgene insertion or the expression of other transgene components such as selection markers or reporter genes. The nptII gene, encoding neomycin phosphotransferase II (EC 2.7.1.95), and the GUS gene, encoding β-glucuronidase (GUS; EC 3.2.1.31), are frequently used for genetic selection of transformed cells and for monitoring transgene presence and expression during transgenic plant lifetime (76). The products of the nptII and GUS genes have been subjected to safety assessment studies and were shown to be nondeleterious to human and animal health (21, 23, 27, 51). Nevertheless, pleiotropic effects in crop plants transformed with the nptII and GUS genes have been observed (2, 15, 17, 43). Pleiotropic effects from GM trees coexpressing such selectable markers have also been recorded. For example, Pasonen et al. (56) showed a significant decrease in the number of root tips colonized by Paxillus involutus associated with a line of chitinase-transformed silver birch in vitro. Similar results have been observed in vivo with P. involutus associated with a line of lignin-modified silver birches (72).Many trees in temperate, boreal, tropical, and subtropical forests establish mutualistic interactions with ectomycorrhizal (EM) fungi (42, 66, 67, 68). EM fungi are a polyphyletic group comprising over 5,000 species (49) that play key roles in biogeochemical soil processes and plant health. They represent one-third of the total microbial biomass in the soil of boreal forests (32). Fine roots colonized by EM fungi, also called EM root tips or ectomycorrhizae, display a fungal mantle from which extends the extraradical mycelium to prospect the soil for nutrient uptake. These two anatomical parts can be sampled for EM fungus molecular identification, but some studies have highlighted dissimilarities between the EM fungal diversity recorded in root tip sampling and that recorded in extraradical mycelium sampling (26, 37, 39).Given the potential cumulative effects caused by the presence and stable constitutive expression of transgenes over years on GM tree fitness and on the environment, impact studies of GM trees require long-term field trials (5, 72, 84). In this study, we investigated the potential long-term impact on the EM fungal community of hybrid poplars transformed with the binary vector containing the selectable nptII marker and GUS reporter genes, field deployed for 8 years. This plantation was part of the first confined field trial of transgenic trees in Canada. Hybrid poplars constitutively expressed the nptII gene for kanamycin resistance driven by the NOS promoter (30). The activity of the NOS promoter has been shown to increase in the lower part of transgenic tobacco plants (4). Such a vertical gradient has also been observed in transgenic hybrid poplars, where the NOS promoter activity was 2.4-fold higher in roots than in leaves (87).As no direct negative impact of nptII or GUS gene expression on fungal organisms has been reported in the literature, we first tested the null hypothesis (H₀) that the EM fungal community recorded from transgenic poplars was similar to that from untransformed poplars. Second, since the EM fungal diversity picture can be influenced by the sampling method, we contrasted the EM fungal community recovered from root tips with that recorded in soil cloning analyses. Internal transcribed spacer (ITS) sequences from the nuclear rRNA were produced from both EM root tips and extraradical mycelia to compare the EM fungal communities associated with three control and three transgenic poplars. EM fungal communities were characterized by measuring the usual qualitative and quantitative EM species diversity within each community (α-diversity) and then estimating the nucleotide diversity between EM communities in relation to EM phylotype relative abundances (quantitative β-diversity).