Effects of phosphate on arsenate and arsenite sensitivity in two rice (Oryza sativa L.) cultivars of different sensitivity

Arsenate and arsenite sensitivity and arsenate influx tests were conducted for two rice cultivars of different arsenic sensitivity. Azucena and Bala. These were to establish if the mechanism of reduced arsenic sensitivity is achieved through an altered phosphate uptake system, as shown for Holcus lanatus. High phosphate treatments (>= 50 mu M) provided protection against both arsenate and arsenite. Unlike the H. lanatus tolerance mechanism, in the less sensitive cultivar Bala, arsenate influx did not decrease with phosphate treatment and phosphate transporters appeared to be constitutively upregulated; V(max) for arsenate influx remain similar when Bala was grown in the presence or absence of phosphate (V(max) - 0.90 and 0.63 nmol g(-1) f.wt min(-1) respectively). Although mean K(m) appear different, Bala did not show lower affinity to arsenate than Azucena in the absence of phosphate (K(m) - Azucena, 0.30 mM and Bala, 0.18), while in phosphate treatment, Bala arsenate affinity was half that observed for Azucena (K(m) - Azucena, 0.14 and Bala, 0.36 mM). These were low compared to a 4 and 6 fold decrease seen for similar studies on H. lanatus in the absence and presence of phosphate. Phosphate-induced arsenic protection was observed but the mechanism does not resemble that of H. lanatus. Alternative mechanisms were discussed. (C) 2010 Elsevier B.V. All rights reserved.

Incorporating PGMs into a BDI Architecture

In this paper, we present a hybrid BDI-PGM framework, in which PGMs (Probabilistic Graphical Models) are incorporated into a BDI (belief-desire-intention) architecture. This work is motivated by the need to address the scalability and noisy sensing issues in SCADA (Supervisory Control And Data Acquisition) systems. Our approach uses the incorporated PGMs to model the uncertainty reasoning and decision making processes of agents situated in a stochastic environment. In particular, we use Bayesian networks to reason about an agent’s beliefs about the environment based on its sensory observations, and select optimal plans according to the utilities of actions defined in influence diagrams. This approach takes the advantage of the scalability of the BDI architecture and the uncertainty reasoning capability of PGMs. We present a prototype of the proposed approach using a transit scenario to validate its effectiveness.

Toluene dioxygenase-catalysed oxidation route to angular cis-monohydrodiols and other bioproducts from bacterial metabolism of 1,2-dihydrobenzocyclobutene and derivatives

A mutant strain (UV4) of the soil bacterium Pseudomonas putida, containing toluene dioxygenase, has been used in the metabolic oxidation of 1,2-dihydrobenzocyclobutene 12 dagger and the related substrates 1,2-dihydrobenzocyclobuten-1-ol 13 and biphenylene 33. Stable angular cis-monohydrodiol metabolites (1R,2S)-bicyclo[4.2.0]octa-3,5-diene-1,2 7, (1S,2S,8S)-bicyclo[4.2.0]octa-3,5-diene-1,2,8-triol 8 and biphenylene-cis-1,8b-diol 9, isolated from each of these substrates, have been structurally and stereochemically assigned. The structure, enantiopurity and absolute configuration of the other cis-diol metabolites, (2R,3S)-bicyclo[4.2.0]octa-1(6),4-diene-2,3-diol 14 and cis-1,2-dihydroxy-1,2-dihydrobenzocyclobutene 16, and the benzylic oxidation bioproducts, 1,2-dihydrobenzocyclobuten-1-ol 13, 1,2-dihydrobenzocyclobuten-1-one 15 and 2-hydroxy-1,2-dihydrobenzocyclobuten-1-one 17, obtained from 1,2-dihydrobenzocyclobutene and 1,2-dihydrobenzocyclobuten-1-ol, have been determined with the aid of chiral stationary-phase HPLC, NMR and CD spectroscopy, and stereochemical correlation. X-Ray crystallographic methods have been used in the determination of absolute configuration of the di-camphanates 27 (from diol 7) and 32 (from diol 9), and the di-MTPA ester 29 (from diol 14) of the corresponding cis-diol metabolites. The metabolic sequence involved in the formation of bioproducts derived from 1,2-dihydrobenzocyclobutene 12 has been investigated.

SN 2012ec: mass of the progenitor from PESSTO follow-up of the photospheric phase

We present the results of a photometric and spectroscopic monitoring campaign of SN 2012ec, which exploded in the spiral galaxy NGC 1084, during the photospheric phase. The photometric light curve exhibits a plateau with luminosity L = 0.9 x 10(42) erg s(-1) and duration similar to 90 d, which is somewhat shorter than standard Type II-P supernovae (SNe). We estimate the nickel mass M(Ni-56) = 0.040 +/- 0.015 M-circle dot from the luminosity at the beginning of the radioactive tail of the light curve. The explosion parameters of SN 2012ec were estimated from the comparison of the bolometric light curve and the observed temperature and velocity evolution of the ejecta with predictions from hydrodynamical models. We derived an envelope mass of 12.6 M-circle dot, an initial progenitor radius of 1.6 x 10(13) cm and an explosion energy of 1.2 foe. These estimates agree with an independent study of the progenitor star identified in pre-explosion images, for which an initial mass of M = 14-22 M-circle dot was determined. We have applied the same analysis to two other Type II-P SNe (SNe 2012aw and 2012A), and carried out a comparison with the properties of SN 2012ec derived in this paper. We find a reasonable agreement between the masses of the progenitors obtained from pre-explosion images and masses derived from hydrodynamical models. We estimate the distance to SN 2012ec with the standardized candle method (SCM) and compare it with other estimates based on other primary and secondary indicators. SNe 2012A, 2012aw and 2012ec all follow the standard relations for the SCM for the use of Type II-P SNe as distance indicators.

Hydrogen-Poor Superluminous Supernovae and Long-Duration Gamma-Ray Bursts Have Similar Host Galaxies

We present optical spectroscopy and optical/near-IR photometry of 31 host galaxies of hydrogen-poor superluminous supernovae (SLSNe), including 15 events from the Pan-STARRS1 Medium Deep Survey. Our sample spans the redshift range 0.1 ≲ z ≲ 1.6, and is the first comprehensive host galaxy study of this specific subclass of cosmic explosions. Combining the multi-band photometry and emission-line measurements, we determine the luminosities, stellar masses, star formation rates, and metallicities. We find that, as a whole, the hosts of SLSNe are a low-luminosity (〈M_B 〉 ≈ -17.3 mag), low stellar mass (〈M〉 ≈ 2 × 10⁸ M_⊙) population, with a high median specific star formation rate (〈sSFR〉 ≈ 2 Gyr^-1). The median metallicity of our spectroscopic sample is low, 12 + log (O/H) ≈ 8.35 ≈ 0.45 Z_⊙, although at least one host galaxy has solar metallicity. The host galaxies of H-poor SLSNe are statistically distinct from the hosts of GOODS core-collapse SNe (which cover a similar redshift range), but resemble the host galaxies of long-duration gamma-ray bursts (LGRBs) in terms of stellar mass, SFR, sSFR, and metallicity. This result indicates that the environmental causes leading to massive stars forming either SLSNe or LGRBs are similar, and in particular that SLSNe are more effectively formed in low metallicity environments. We speculate that the key ingredient is large core angular momentum, leading to a rapidly spinning magnetar in SLSNe and an accreting black hole in LGRBs.

Effects of arsenic-contaminated irrigation water on growth, yield, and nutrient concentration in rice

A study was undertaken to determine the effects of different concentrations of arsenic (As) in irrigation water on Boro (dry-season) rice (Oryza sativa) and their residual effects on the following Aman (wet-season) rice. There were six treatments, with 0, 0.1, 0.25, 0.5, 1, and 2 mg As L-1 applied as disodium hydrogen arsenate. All the growth and yield parameters of Boro rice responded positively at lower concentrations of up to 0.25 mg As L-1 in irrigation water but decreased sharply at concentrations more than 0.5 mg As L-1. Arsenic concentrations in grain and straw of Boro rice increased significantly with increasing concentration of As in irrigation water. The grain As concentration was in the range of 0.25 to 0.97 μg g-1 and its concentration in rice straw varied from 2.4 to 9.6 μg g-1 over the treatments. Residual As from previous Boro rice showed a very similar pattern in the following Aman rice, although As concentration in Aman rice grain and straw over the treatments was almost half of the As levels in Boro rice grain. Arsenic concentrations in both grain and straw of Boro and Aman rice were found to correlate with iron and be antagonistic with phosphorus. Copyright © Taylor & Francis Group, LLC.

Response of soil microbial communities to single and multiple doses of an organic pollutant

The effect of 100 μg 1,2-dichlorobenzene (1,2-DCB) g^-1 dry weight (dw) of soil introduced either as a single dose or multiple (10 fortnightly) doses of 10 μg g^-1 dw, on the microbial biomass, diversity of culturable bacterial community and the rate of 1,2-DCB mineralisation, were compared. After 22 weeks exposure both application regimes significantly reduced total bacterial counts and viable fungal hyphal length. The single dose had the greatest overall inhibitory effect, although the extent of inhibition varied throughout the study. Total culturable bacterial counts, determined after 22 weeks exposure showed little response to 1,2-DCB, but pseudomonad counts in single and multiple treatments were reduced to 9.7 and 0.147%, respectively, of the numbers detected in the control soil. The effect of 1,2-DCB application on the taxonomic composition of the culturable bacteria community was determined by fatty acid methyl ester (FAME) analysis. Compared to control soils, the single dose treatment had a lower percentage of Arthrobacter and Micrococcus. Multiple applications had a significant effect upon pseudomonad abundance, which represented only 2% of the identified community, compared to 45.6% in the control. The multi-dosed soils contained a high percentage of bacilli (> 25%). The effects of 1,2-DCB applications on the metabolic potential of the soil microbial community was determined by BIOLOG profiling. The number of carbon compounds utilised by the community in the multi-dosed soils (49 positives) was significantly less (P < 0.05) than detected in the single dose treatment (76) and control (66). The rate of 1,2-DCB mineralisation, determined by ¹⁴CO₂ production from radiolabelled [UL-¹⁴C] 1,2-DCB, declined throughout the study, and after 22 weeks was slightly but significantly (P < 0.05) lower in the multiply- than the singly-dosed soils. The differential response to 1,2-DCB treatments was attributed to its reduced bioavailability in soils after a single exposure, compared to multiple applications.

Wireless electrochemical modification of catalytic activity on a mixed protonic-electronic conductor

A novel approach to electrochemical modification of catalytic activity using a wireless configuration has been undertaken. This paper presents preliminary results on the modification of a platinum catalyst film supported on a pellet of Sr_0.97Ce_0.9Yb_0.1O_3-δ (SCYb), considered to be a mixed protonic-electronic conductor under reducing conditions. The wireless configuration utilises the mixed ionic and electronic conductivity of the supporting membrane to supply an ionic promoting species to the catalyst surface. Control of the flux of this species is achieved by adjusting the effective hydrogen chemical potential difference across the membrane in a dual-chamber reactor with one chamber acting as the "reaction side" and the other as the "sweep side". The reaction rate can be promoted by up to a factor of 1.6, for temperatures around 500 °C and low reactant concentrations, when hydrogen is introduced on the sweep side of the membrane reactor. The use of helium, moist helium and oxygen in helium as sweep gases did not modify the reaction rate. © 2007 Elsevier B.V. All rights reserved.

Stoichiometric Molecularly Imprinted Polymers for the Recognition of Anti-Cancer Pro-drug Tegafur

Molecularly Imprinted Polymers (MIPs) targeting tegafur, an anti-cancer 5-fluorouracil pro-drug, have been prepared by stoichiometric imprinting using 2,6-bis(acrylamido)pyridine (BAAPy) as the functional monomer. Solution association between tegafur and BAAPy was studied by 1H NMR titration, which confirmed the formation of 1:1 complexes with an affinity constant of 574±15 M-1 ¬in CDCl3. Evaluation of the synthesised materials by HPLC and equilibrium rebinding experiments revealed high selectivity of the imprinted polymer for the pro-drug versus 5-fluorouracil and other competing analytes, with maximum imprinting factors of 25.3 and a binding capacity of 45.1 μmol g-1. The synthesised imprinted polymer was employed in solid-phase extraction of the pro-drug using an optimised protocol that included a simple wash with the porogen used in the preparation of the material. Tegafur recoveries of up to 96% were achieved from aqueous samples and 92% from urine samples spiked with the template and three competing analytes. The results demonstrate the potential of the prepared polymers in the pre-concentration of tegafur from biological samples, which could be an invaluable tool in the monitoring of patient compliance and drug uptake and excretion.

Acute Cocoa Supplementation Increases Postprandial HDL Cholesterol and Insulin in Obese Adults with Type 2 Diabetes after Consumption of a High-Fat Breakfast

BACKGROUND: Dietary cocoa is an important source of flavonoids and is associated with favorable cardiovascular disease effects, such as improvements in vascular function and lipid profiles, in nondiabetic adults. Type 2 diabetes (T2D) is associated with adverse effects on postprandial serum glucose, lipids, inflammation, and vascular function.

OBJECTIVE: We examined the hypothesis that cocoa reduces metabolic stress in obese T2D adults after a high-fat fast-food-style meal.

METHODS: Adults with T2D [n = 18; age (means ± SEs): 56 ± 3 y; BMI (in kg/m(2)): 35.3 ± 2.0; 14 women; 4 men) were randomly assigned to receive cocoa beverage (960 mg total polyphenols; 480 mg flavanols) or flavanol-free placebo (110 mg total polyphenols; <0.1 mg flavanols) with a high-fat fast-food-style breakfast [766 kcal, 50 g fat (59% energy)] in a crossover trial. After an overnight fast (10-12 h), participants consumed the breakfast with cocoa or placebo, and blood sample collection [glucose, insulin, lipids, and high-sensitivity C-reactive protein (hsCRP)] and vascular measurements were conducted at 0.5, 1, 2, 4, and 6 h postprandially on each study day. Insulin resistance was evaluated by homeostasis model assessment.

RESULTS: Over the 6-h study, and specifically at 1 and 4 h, cocoa increased HDL cholesterol vs. placebo (overall Δ: 1.5 ± 0.8 mg/dL; P ≤ 0.01) but had no effect on total and LDL cholesterol, triglycerides, glucose, and hsCRP. Cocoa increased serum insulin concentrations overall (Δ: 5.2 ± 3.2 mU/L; P < 0.05) and specifically at 4 h but had no overall effects on insulin resistance (except at 4 h, P < 0.05), systolic or diastolic blood pressure, or small artery elasticity. However, large artery elasticity was overall lower after cocoa vs. placebo (Δ: -1.6 ± 0.7 mL/mm Hg; P < 0.05), with the difference significant only at 2 h.

CONCLUSION: Acute cocoa supplementation showed no clear overall benefit in T2D patients after a high-fat fast-food-style meal challenge. Although HDL cholesterol and insulin remained higher throughout the 6-h postprandial period, an overall decrease in large artery elasticity was found after cocoa consumption. This trial was registered at clinicaltrials.gov as NCT01886989.

A survey of perceived training differences between ophthalmology residents in Hong Kong and China.

Background: To study the differences in ophthalmology resident training between China and the Hong Kong Special Administrative Region (HKSAR).Methods: Training programs were selected from among the largest and best-known teaching hospitals. Ophthalmology residents were sent an anonymous 48-item questionnaire by mail. Work satisfaction, time allocation between training activities and volume of surgery performed were determined.Results: 50/75 residents (66.7 %) from China and 20/26 (76.9 %) from HKSAR completed the survey. Age (28.9 ± 2.5 vs. 30.2 ± 2.9 years, p = 0.15) and number of years in training (3.4 ± 1.6 vs. 2.8 ± 1.5, p = 0.19) were comparable between groups. The number of cataract procedures performed by HKSAR trainees (extra-capsular, median 80.0, quartile range: 30.0, 100.0; phacoemulsification, median: 20.0, quartile range: 0.0, 100.0) exceeded that for Chinese residents (extra-capsular: median = 0, p < 0.0001; phacoemulsification: median = 0, p < 0.0001). Chinese trainees spent more time completing medical charts (>50 % of time on charts: 62.5 % versus 5.3 %, p < 0.0001) and received less supervision (≥90 % of training supervised: 4.4 % versus 65 %, p < 0.0001). Chinese residents were more likely to feel underpaid (96.0 % vs. 31.6 %, p < 0.0001) and hoped their children would not practice medicine (69.4 % vs. 5.0 %, p = 0.0001) compared HKSAR residents.Conclusions: In this study, ophthalmology residents in China report strikingly less surgical experience and supervision, and lower satisfaction than HKSAR residents. The HKSAR model of hands-on resident training might be useful in improving the low cataract surgical rate in China.

The impact of an educational DVD on pregnant women with gestational diabetes mellitus: a randomised controlled trial

Background The diagnosis of gestational diabetes (GDM) during pregnancy can lead to anxiety. Little research has focused on the education these women receive and how this is best delivered in a busy clinic. Aim This study evaluated the impact of an innovative patient-centred educational DVD on anxiety and glycaemic control and in newly diagnosed women with GDM. Method 150 multi-ethnic women, aged 19-44 years, from three UK hospitals were randomised to either standard care plus DVD (DVD group, n=77) or standard care alone (control group, n=73) at GDM diagnosis. Women were followed up at their next clinic visit at a mean ± SD of 2.5 ± 1.6 weeks later. Primary outcomes were anxiety (State-Trait Anxiety Inventory) and mean 1-hour postprandial capillary self-monitored blood glucose for all meals, on day prior to follow-up. Secondary outcomes included pregnancy specific stress (Pregnancy Distress Questionnaire), emotional adjustment to diabetes (Appraisal of Diabetes Scale), self-efficacy (Diabetes Empowerment Scale) and GDM knowledge (non-validated questionnaire). Other outcomes included mean fasting and 1-hour postprandial blood glucose at each meal, on day prior to follow-up. Women in the DVD group completed a feedback questionnaire on the resource. Results No significant difference between the DVD and control group were reported, for anxiety (37.7 ± 11.7 vs 36.2 ± 10.9; mean difference after adjustment for covariates (95%CI) 2.5 (-0.8, 5.9) or for mean 1-hour postprandial glucose (6.9 ± 0.9 vs 7.0 ± 1.2 mmol/L; -0.2 (-0.5, 0.2). Similarly, no significant differences in the other psychosocial variables were identified between the groups. However, the DVD group had significantly lower postprandial breakfast glucose compared to the control group (6.8 ± 1.2 vs 7.4 ± 1.9 mmol/L; -0.5 (-1.1, -<0.1; p=0.04). Using a scale of 0-10, 84% rated the DVD 7 or above for usefulness (10 being very useful), and 88% rated it 7 or above when asked if they would recommend to a friend (10 being very strongly recommend). Women described the DVD as ‘reassuring’, ‘a fantastic tool’, that ‘provided a lot of information in a quick and easy way’ and ‘helped reinforce all the information from clinic’. Discussion While no significant change was observed in anxiety or mean postprandial glucose, the DVD was rated highly by women with GDM and may be a useful resource to assist with educating newly diagnosed women. This project is supported by BRIDGES, an IDF programme supported by an educational grant from Lilly Diabetes.

Visual morbidity due to inaccurate spectacles among school children in rural China: the See Well to Learn Well Project, report 1.

PURPOSE: Inadequately corrected refractive error is the leading cause of visual disability among children in China; inaccurate spectacles are a potential cause. The prevalence and visual impact of spectacle inaccuracy were studied among rural, secondary-school children, to determine the optimal timing for updating of refraction. METHODS: A random sample of children from years 1 and 2 in all junior and senior high schools in Fuyang Township, Guangdong Province, underwent ocular examination. All children who reported wearing glasses received cycloplegic refraction, vision assessment, and measurement of current spectacles. RESULTS: Among 3226 examined children, 733 (22.7%) reported owning spectacles. Refractive error and spectacle power were assessed for 588 (80.2%) children. They had a mean age of 15.0 +/- 1.6 years; 70.2% were girls, 83.3% had more than -1.5 D of myopia, and 17.9% had presenting vision < or = 6/12 in the better eye. The glasses of 48.8% of children were inaccurate by > or = 1 D; inaccuracy was > or = 2 D in 17.7%. Children with inaccurate glasses (> or = 1 D) had presenting vision in the better eye significantly (P < 0.001) worse than that of children with accurate glasses, and 30.3% had presenting acuity < or = 6/12. In multivariate models, younger age (P = 0.004), more myopic refractive error (P < 0.001), and having glasses > or = 1 year old (P = 0.04) were associated with inaccurate spectacles. DISCUSSION: Inaccurate spectacles are common and are associated with significant visual impairment among children in rural China. Reducing outdated glasses could lessen the visual burden, although refractive services may have to be offered on an annual basis for optimal benefit.

Pine wilt disease: global issues, trade and economic impact

Pine wilt disease (PWD) is perhaps the most serious threat to pine forests worldwide. Since it´s discovery in the early XXth century by Japanese forest researchers, and the relationship with its causative agent, the pinewood nematode (PWN) Bursaphelenchus xylophilus, in the 1970s, PWD has wreaked havoc wherever it appears. Firstly in the Far East (Japan, China and Korea) and now, more recently in 1999, in the EU (Portugal). The forest sector in Portugal plays a major role in the Portuguese economy with a 12% contribution to the industrial gross domestic product, 3.2% of the gross domestic product, 10% of foreign trade and 5% of national employment. Maritime pine (Pinus pinaster) is one of the most important pine productions, and industrial activity, such as the production of wood and resin, as well as coastal protection associated with sand dunes. Also, stone pine (Pinus pinea) plays an important role in the economy with a share derived from the exports of high-quality pineon seed. Thus, the tremendous economical and ecological impact of the introduction of a pest and pathogen such as the PWN, although as far as is known, the only species susceptible to the nematode is maritime pine. Immediately following detection, the research team involved (Univ. Évora, INIAP) informed the national plant quarantine and forest authorities, which relayed the information to Brussels and the appropriate EU authorities. A task force (GANP), followed by a national program (PROLUNP) was established. Since then, national surveys have been taking place, involving MADRP (Ministry of Agriculture), the University of Évora and several private corporations (e.g. UNAC). Forest growers in the area are particularly interested and involved since the area owned by the growers organizations totals 700 000 ha, largely affected by PWD. Detection of the disease has led to serious consequences and restrictions regarding exploration and commercialization of wood. A precautionary phytosanitary strip, 3 km-wide, has been recently (2007) established surrounding the affected area. The Portuguese government, through its national program PROLUNP, has been deeply involved since 1999, and in conjunction with the EU (Permanent Phytosanitary Committee, and FVO) and committed to controlling this nematode and the potential spread to the rest of the country and to the rest of the EU. The global impact of the presence of Bursaphelenchus xylophilus or the threat of its introduction and the resulting pine wilt disease in forested areas in different parts of the world is of increasing concern economically. The concern is exacerbated by the prevailing debate on climate change and the putative impact this could have on the vulnerability of the world’s pine forests to this disease. The scientific and regulatory approach taken in different jurisdictions to the threat of pine wilt disease varies from country to country depending on the perceived vulnerability of their pine forests to the disease and/or to the economic cost due to lost trade in wood products. Much of the research surrounding pine wilt disease has been located in the northern hemisphere, especially in southern Europe and in the warmer, coastal, Asian countries. However, there is an increased focus on this problem also in those countries in the southern hemisphere where plantations of susceptible pine have been established over the years. The forestry sector in Australia and New Zealand are on “high alert” for this disease and are practicing strict quarantine procedures at all ports of entry for wood products. As well, there is heightened awareness, as there is worldwide, for the need to monitor wood packaging materials for all imported goods. In carrying out the necessary monitoring and assessment of products for B. xylophilus and its vectors substantial costs are incurred especially when decisions have to be made rapidly and regardless of whether the outcome is positive or negative. Australia’s response recently to the appearance of some dying pines in a plantation illustrated the high sensitivity of some countries to this disease. Some $200,000 was spent on the assessment in order to save a potential loss of millions of dollars to the disease. This rapid, co-ordinated response to the report was for naught, because once identified it was found not to be B. xylophilus. This illustrates the particular importance of taking the responsibility at all levels of management to secure the site and the need of a rapid, reliable diagnostic method for small nematode samples for use in the field. Australia is particularly concerned about the vulnerability of its 1million hectares of planted forests, 80% of which are Pinus species, to attack from incursions of one or more species of the insect vector. Monochamus alternatus incursions in wood pallets have been reported from Brisbane, Queensland. The climate of this part of Australia is such that the Pinus plantations are particularly vulnerable to the potential outcome of such incursions, and the state of Queensland is developing a risk management strategy and a proactive breeding programme in response to this putative threat. New Zealand has 1.6 million hectares of planted forests and 89% of the commercial forest is Pinus radiata. Although the climate where these forests are located tends to be somewhat cooler than that in Australia the potential for establishment and development of the disease in that country is believed to be high. The passage alone of 200,000 m³/year of wood packaging through New Zealand ports is itself sufficient to require response. The potential incursion of insect vectors of pinewood nematode through the port system is regarded as high and is monitored carefully. The enormous expansion of global trade and the continued use of unprocessed/inadequately-processed wood for packaging purposes is a challenge for all trading nations as such wood packaging material often harbours disease or pest species. The extent of this problem is readily illustrated by the expanding economies and exports of countries in south-east Asia. China. Japan and Korea have significant areas of forestland infested with B. xylophilus. These countries too are among the largest exporting countries of manufactured goods. Despite the attempts of authorities to ensure that only properly treated wood is used in the crating and packaging of goods B. xylophilus and/or its insect vector infested materials is being recorded at ports worldwide. This reminds us, therefore, of the ease with which this nematode pest can gain access to forest lands in new geographic locations through inappropriate use, treatment or monitoring of wood products. It especially highlights the necessity to find an alternative to using low-grade lumber for packaging purposes. Lest we should believe that all wood products are always carriers of B. xylophilus and its vectors, it should be remembered that international trade of all kinds has occurred for thousands of years and that lumber-born pests and diseases do not have worldwide distribution. Other physico-biological factors have a significant role in the occurrence, establishment and sustainability of a disease. The question is often raised as to why the whole of southern Europe doesn’t already have B. xylophilus and pine wilt disease. European countries have traded with countries that are infested with B. xylophilus for hundreds of years. Turkey is an example of a country that appears to be highly vulnerable to pine wilt disease due to its extensive forests in the warm, southern region where the vector, Monochamus galloprovincialis, occurs. However, there is no record of the presence of B. xylophilus occurring there despite the importation of substantial quantities of wood from several countries In many respects, Portugal illustrates both the challenge and the dilemma. In recent times B. xylophilus was discovered there in the warm coastal region. The research, administrative and quarantine authorities responded rapidly and B. xylophilus appears to have been confined to the region in which it was found. The rapid response would seem to have “saved the day” for Portugal. Nevertheless, it raises again the long-standing questions, how long had B. xylophilus been in Portugal before it was found? If Lisbon was the port of entry, which seems very likely, why had B. xylophilus not entered Lisbon many years earlier and established populations and the pine wilt disease? Will the infestation in Portugal be sustainable and will it spread or will it die out within a few years? We still do not have sufficient understanding of the biology of this pest to know the answers to these questions.

Determinant factors for the use of imposex in TBT pollution monitoring

Os compostos de tributilestanho (TBT) foram utilizados como biocidas em tintas antivegetativas (AV) e amplamente aplicados, durante décadas, de forma a evitar a bioincrustação em superfícies submersas, principalmente em cascos de embarcações. Os seus efeitos deletérios em organismos não-alvo tornaram-se evidentes após o aparecimento de gastrópodes prosobrâquios com imposex – sobreposição de caracteres sexuais masculinos sobre o tracto reprodutivo das fêmeas. Desde então, a expressão do imposex em prosobrânquios tem sido amplamente utilizada como biomarcador da poluição por TBT. Um dos objectivos da presente tese é avaliar se as mais recentes restrições legais na utilização das tintas AV com compostos organoestânicos (OTs) resultaram numa redução da poluição pelos mesmos na costa continental Portuguesa. Para tal, foi levada a cabo a análise da variação temporal do imposex como biomarcador dos níveis ambientais de TBT e a validação de procedimentos, de modo a seguir de forma precisa a evolução da intensidade deste fenómeno ao longo do tempo. O trabalho de investigação teve início no momento em que a ineficácia da legislação anterior (Directiva 89/677/CEE) na redução da poluição por TBT no litoral Português era reportada na literatura e quando estavam já agendados instrumentos decisivos para a diminuição definitiva deste tipo de poluição: o Regulamento (CE) N.º 782/2003 do Parlamento e do Conselho da União Europeia (UE) que bania as tintas AV baseadas em TBT na sua frota a partir de 1 de Julho de 2003; a "Convenção Internacional sobre o Controlo de Sistemas Antivegetativos Nocivos em Navios” (Convenção AFS), adoptada em 2001 pela Organização Marítima Internacional (OMI), que procurava erradicar os OTs da frota mundial até 2008. Os níveis de imposex e as concentrações de OTs nos tecidos de fêmeas de Nucella lapillus foram medidos em 17 locais de amostragem ao longo da costa Portuguesa em 2003, a fim de se avaliarem os impactos do TBT nas populações desta espécie e de se criar uma base de dados para seguir a sua evolução futura. O índice da sequência do vaso deferente (VDSI), o índice do tamanho relativo do pénis (RPSI) e a percentagem de fêmeas afectadas por imposex (%I) foram utilizados na medição da intensidade deste fenómeno em cada local de amostragem e os seus valores variaram entre 0,20-4,04, 0,0- 42,2% e 16,7-100,0%, respectivamente. Foram encontradas fêmeas estéreis em 3 locais de amostragem, com percentagens a variar entre 4,0 e 6,2%. As concentrações de TBT e dibutilestanho (DBT) nas fêmeas variaram respectivamente entre 23-138 e <10-62 ng Sn.g-1 de peso seco, e o conteúdo em TBT nos tecidos revelou-se significativamente correlacionado com o imposex (nomeadamente com o RPSI e o VDSI). Os níveis de expressão do fenómeno e o conteúdo em OTs nos tecidos, foram superiores na proximidade de portos, confirmando as conclusões obtidas anteriormente por outros autores de que os navios e a actividade dos estaleiros são as principais fontes destes compostos no litoral Português. As infra-estruturas associadas ás principais fontes de OTs – portos, estaleiros e marinas – estão geralmente localizadas no interior de estuários, motivo pelo qual estas áreas têm vindo a ser descritas como as mais afectadas por estes poluentes. Por esta razão, foi levado a cabo o estudo pormenorizado da poluição por TBT na Ria de Aveiro, como caso de estudo representativo da poluição por estes compostos num sistema estuarino em Portugal continental. N. lapillus foi usada como bioindicador para avaliar a tendência temporal da poluição por TBT nesta área entre 1997 e 2007. Foi registada uma diminuição da intensidade do imposex após 2003, embora as melhorias mais evidentes tenham sido observadas entre 2005 e 2007, provavelmente devido à implementação do Regulamento (CE) N.º 782/2003 que proibiu a aplicação de tintas AV com TBT em navios com a bandeira da UE. Apesar desses progressos, as análises ao conteúdo em OTs nos tecidos de fêmeas de N. lapillus e em amostras de água colhidas em 2006 indicaram contaminação recente por TBT na área de estudo, evidenciando assim a permanência de fontes de poluição. A utilização de N. lapillus como bioindicador da poluição por TBT na Ria de Aveiro apresenta algumas limitações uma vez que a espécie não ocorre nas áreas mais interiores da Ria e não vive em contacto com sedimentos. Assim, a informação obtida a partir da sua utilização como bioindicador é fundamentalmente relativa aos níveis de TBT na coluna de água. Foi então necessário recorrer a um bioindicador complementar – Hydrobia ulvae – para melhor avaliar a evolução temporal da poluição por TBT no interior deste sistema estuarino e estudar a persistência de TBT nos sedimentos. Não foi registada uma diminuição dos níveis de imposex em H. ulvae na Ria de Aveiro entre 1998 e 2007, apesar da aplicação do Regulamento (CE) N.º 782/2003. Pelo contrário, houve um aumento global significativo da percentagem de fêmeas afectadas por imposex e um ligeiro aumento do VDSI, contrastando com o que tem sido descrito para outros bioindicadores na Ria de Aveiro no mesmo período. Estes resultados mostram que a diferente biologia/ecologia das espécies indicadoras determina vias distintas de acumulação de TBT, apontando a importância da escolha do bioindicador dependendo do compartimento a ser monitorizado (sedimento versus água). A ingestão de sedimento como hábito alimentar em H. ulvae foi discutida como sendo a razão para a escolha da espécie como indicadora da contaminação dos sedimentos por TBT. Foram também estudados os métodos mais fiáveis para reduzir a influência de variáveis críticas na medição dos níveis de imposex em H. ulvae. As comparações de parâmetros do imposex baseados em medições do pénis devem ser sempre realizadas sob condições de narcotização bem standardizadas uma vez que este procedimento provoca um aumento significativo do comprimento do pénis (PL) em ambos os sexos. O VDSI, a % e o PL em ambos os sexos revelaram ser fortemente influenciados pelo tamanho dos espécimes: a utilização de fêmeas mais pequenas conduz à subestimação do VDSI, da %I e do PL, enquanto que diferenças no tamanho dos machos provocam variações no índice do comprimento relativo do pénis (RPLI), independentemente dos níveis de poluição por TBT. Existe, portanto, a necessidade de controlar algumas variáveis envolvidas na análise do imposex que mostraram afectar a fiabilidade dos resultados. Uma vez que N. lapillus é o principal bioindicador dos efeitos biológicos específicos do TBT para a área da OSPAR, foi também estudada a influência de algumas variáveis na avaliação dos níveis de imposex nesta espécie, especificamente as relacionadas com o ciclo reprodutor e o tamanho dos espécimes. O estudo do ciclo reprodutor e a variação sazonal/espacial do comprimento do pénis do macho (MPL) incidiu num único local no litoral Português (Areão – região de Aveiro) de forma a avaliar se o RPSI varia sazonal e espacialmente na mesma estação de amostragem e se tais variações influenciam os resultados obtidos em programas de monitorização do imposex. Nos meses de Dezembro de 2005 a Junho de 2007, foram encontrados espécimes de N. lapillus sexualmente maturos e potencialmente aptos para a reprodução. Contudo, foi também evidente um padrão sazonal do ciclo reprodutor – o estado de desenvolvimento da gametogénese nas fêmeas variou sazonalmente e ocorreu uma diminuição do volume da glândula da cápsula e do factor de condição no final do Verão / início do Outono. Contrariamente, a gametogénese nos machos não apresentou variação sazonal significativa, embora os valores mais baixos do factor de condição, do comprimento do pénis e dos volumes de esperma e da próstata tenham também sido registados no final do Verão / início do Outono. Além disso, o MPL mostrou variar, no mesmo local de amostragem, inversamente com a distância aos ninhos de cápsulas ovígeras; um aumento dos valores do MPL foi também observado em espécimes de maior tamanho. Todas estas variações no MPL introduzem desvios nos resultados da avaliação do imposex quando é usado o RPSI. A magnitude do erro envolvido foi quantificada e revelou-se superior em locais com níveis mais elevados de poluição por TBT. Apesar do RPSI ser um índice que fornece informação importante sobre os níveis de poluição por TBT, a sua interpretação deve ser cuidadosa e realizada complementarmente com os outros índices, destacando-se o VDSI como índice mais fiável e com significado biológico mais expressivo. Novas campanhas de monitorização do imposex em N. lapillus foram realizadas ao longo da costa Portuguesa em 2006 e 2008, e os resultados subsequentemente comparados com a base de dados criada em 2003,de forma a avaliar a evolução da poluição por TBT no litoral Português naquele período. Nestes estudos foram aplicados novos procedimentos na monitorização e tratamento dos dados, de forma a minimizar a variação nos índices de avaliação do imposex induzida pelos factores acima descritos, para seguir com maior consistência a tendência da poluição por TBT entre 2003 e 2008. Foi observado um declínio global significativo na intensidade de imposex na área de estudo durante o referido período e a qualidade ecológica da costa Portuguesa, segundo os termos definidos pela Comissão OSPAR, revelou melhorias notáveis após 2003 confirmando a eficácia do Regulamento (CE) N.º 782/2003. Não obstante, as populações de N. lapillus revelaram-se ainda amplamente afectadas por imposex, tendo sido detectadas emissões de TBT na água do mar ao longo da costa em 2006, apesar da restrição anteriormente referida. Estes inputs foram atribuídos principalmente aos navios que à data ainda circulavam com tintas AV com TBT aplicadas antes de 2003, uma vez que a sua utilização nas embarcações foi apenas proibida em 2008. Considerando que o Regulamento (CE) N.º 782/2003 constitui uma antecipação da proibição global da OMI que entrou em vigor em Setembro de 2008, prevê-se, por analogia, que haja uma rápida diminuição da poluição por TBT à escala mundial num futuro próximo. Na sequência desta previsão, é apresentada uma discussão teórica preliminar relativamente ás possíveis estratégias usadas por N. lapillus na recolonização de locais onde, no passado, as populações terão aparentemente sido extintas devido a níveis extremamente elevados de TBT. Estes locais são tipicamente zonas abrigadas junto de infra-estruturas portuárias, cuja recolonização por esta espécie será provavelmente muito lenta dada a mobilidade reduzida dos adultos e o ciclo de vida não apresentar fase larvar pelágica. Foram então equacionadas duas hipotéticas vias de recolonização de zonas abrigadas por espécimes provenientes de populações de costa aberta/exposta: a migração de adultos e/ou a dispersão de juvenis. No entanto, em ambos os casos, estaria implicada a transposição de um problema amplamente descrito na bibliografia: as populações de N. lapillus de costa aberta podem apresentar um fenótipo muito diferente das de zonas abrigadas, podendo inclusivamente variar no número de cromossomas. A recolonização pode portanto não ter sucesso pelo simples facto dos novos recrutas não estarem bem adaptados aos locais a recolonizar. Para analisar este problema, foram estudados tanto a forma da concha de espécimes de N. lapillus ao longo da costa Portuguesa como o respectivo número de cromossomas. Embora a forma da concha tenha revelado diferenças, de acordo com o grau de hidrodinamismo entre as populações de N. lapillus avaliadas, o cariótipo 2n = 26, típico de zonas expostas, foi registado em todos os locais amostrados. Por outro lado, foi também testada em laboratório a possível mortalidade de juvenis em dispersão no interior menos salino dos estuários. Foi então verificada a ocorrência de mortalidade elevada de juvenis expostos a salinidades baixas (=100% após 1 hora a salinidades ≤9 psu), o que também pode comprometer a recolonização de zonas estuarinas menos salinas por esta via. Mesmo assim, os juvenis mostraram um comportamento de flutuação à superfície da água em condições laboratoriais, que pode ser considerado um benefício específico na colonização de áreas mais internas dos estuários, se tal facto vier a ser confirmado em estudos de campo. As conclusões deste estudo contribuem certamente para a descrição do final da “história do TBT” dado que, uma vez controlados alguns factores determinantes no uso do imposex como biomarcador, a avaliação do declínio da poluição por estes compostos, agora esperado à escala global, se torna mais rigorosa.