Sphingomonas SaMR12, isolated from Sedum alfredii Hance, appears to increase plant biomass and zinc-extraction from contaminated soil; however, the mechanism by which this occurs is not clear. Here, the ability of SaMR12 to promote zinc extraction and its effects on root morphology and exudation were examined in hydroponics. Zinc treatment increased shoot biomass by 30 to 45%, and by a further 10 to 19% when combined with SaMR12 inoculation. Zinc treatment also increased zinc accumulation modestly and this too was enhanced with SaMR12. Both biomass and zinc levels increased in a dose-dependent manner with significant effects seen at 50 µM zinc and apparent saturation at 500 µM. Zinc and the endophyte also increased levels of auxin but not at 50 µM and zinc increased levels of superoxide and hydrogen peroxide but mainly at 500 µM. As for root morphology, SaMR12 increased root branching, the number of root tips, and surface area. Zinc and SaMR12 also increased the exudation of oxalic acid. For most assays the effects of the endophyte and zinc were additive, with the notable exception of SaMR12 strongly reducing the production of reactive oxygen species at 500 µM zinc. Taken together, these results suggest that the promotion of growth and zinc uptake by exposure to zinc and to SaMR12 are independent of reactive oxygen and do not involve increases in auxin.The endophytic bacterium,  Zinc is an essential trace element that can have toxic effects on organisms at millimolar levels through soil or water contamination. Excessive zinc is toxic to plants and negatively affects human health For successful phytoremediation, to overcome the shortcomings of low biomass and low growth rate, various approaches have been examined, such as identifying hyperaccumulators, fertilizing soil to improve plant biomass, and applying organic acid or chelators to increase metal bioavailability Sedum alfredii is a plant found in an ancient, Chinese mining region near Quzhou (Zhejiang province) and has a strong ability to extract heavy metals from soil. In previous studies S. alfredii was found to show heavy metal resistance and accumulation, as well as the ability to efficiently transport heavy metals from the root to the shoot. Roots of S. alfredii mainly produce malic acid, oxalic acid, and tartaric acid, presumably contributing to enhanced bioavailability of heavy metals A member of the Crassulaceae, However, root exudates not only improve access to metals they also significantly affect microbial growth and community structure within the rhizoshpere S. alfredii roots are host to at least four species of endophytic bacteria, and when this plant is inoculated with one of them, Sphingomonas SaMR12, plant biomass and heavy metal absorption are both significantly increased SaMR12 on heavy metal bioavailability and root exudation. According to Přikryl and Vančura Pseudomonas putida enhanced the release of root exudates. Therefore, here, we hypothesize that, in Sedum alfredii, SaMR12 affects root morphology and organic acid composition of root exudates, thereby altering zinc bioavailability and rhizosphere conditions, and leading to larger plant biomass and higher zinc extraction.According to a previous study, Sedum alfredii Hance, a zinc/cadmium hyperaccumulator identified previously Sphingomonas SaMR12, previously isolated from S. alfrediiThe endophytic bacterium S. alfredii were collected in the field and cultured in Hoagland nutrient solution for 2 months in 2.5 L (10.2-cm top diameter) standard round green plastic pots, aerated continuously and renewed every week. Single shoot tips were excised and grown as described above for another month to remove heavy metals. Plants showing uniform growth were treated with the indicated concentrations of zinc, with 6 plants per pot for hydroponic experiments. Supplemental zinc was supplied as ZnSO4·7H2O and added to maintain the zinc concentration every three days. Unsupplemented Hoagland's contains 5 µM zinc (considered here as the ‘control’ level of zinc). A second group of plants was subjected to the same treatment and also inoculated with SaMR12 at densities of 104 to 105 CFU/mL. Each treatment was repeated three times and the experiment lasted for one month.Healthy shoots of At the end of the one-month experiment, roots were washed with distilled water three times, photographed by a digital camera , scanned using an automatic root scanner , and analyzed using WinRHIZO software . Additionally, 3 cm apical root segments were imaged through a light microscope , from which root hair morphology was assessed.3-HClO4  for 8 h at 180°C before subjecting the samples to inductively coupled plasma-mass spectroscopy . To ensure accuracy of determination, a standard zinc solution was run first to calibrate the instrument.At the end of the experiment, shoots and roots were separated and weighed. The roots were washed in several changes of 5 mM Tris-HCl pH 6.0 containing 5 mM EDTA, and were then washed with distilled water to remove non-specifically bound zinc. Shoots and roots were oven-dried at 80°C to a consistent weight. For subsequent analysis, oven-dried samples were ground using a stainless steel mill  to an average particle size of 0.5 mm. Next, the ground material was digested using acid HNO2 for 4 h. The solution was passed through a resin column [Amberlite IR–120 (H+ form)] and then through another resin column [Dowex 1 × 8 100–200 (Cl− form)]. Exudates were dried using a rotary evaporator and resolved in methanol before high-pressure liquid chromatography (HPLC) analysis . Organic acids were detected at 210 nm by UV detection by comparing retention times and absorption spectra with organic acid standards.Root exudates were collected according to the method of Ma et al. g for 10 min and extracted two additional times with a 10 mL extraction solution for 1 h. After concentration, liquid partition, column separation, and elution, the eluent was passed through a sterile 0.2 µm filter prior to injection into the HPLC.IAA concentration in the plant was determined according to the method described by Hou et al. 2O2 as described by Tang et al. The generation of intracellular reactive oxygen species (ROS) was analyzed by measuring the superoxide anion as described by Nag et al. SaMR12 inoculation (S), zinc concentration (Zn), and inoculation × zinc concentration (S × Zn) on root morphology using the SPSS software . All figures were produced using Origin .Data were compared by least significant difference (LSD) tests at a 5% significance level. Two-way analysis of variance was performed to determine the effects of SaMR12 inoculation significantly stimulated plant growth  but at the higher concentrations of zinc, lateral root formation was not stimulated and root hairs were more numerous and only slightly longer than that of the respective non-inoculated treatment control.Regarding root hairs, zinc treatment alone caused little conspicuous alteration except that on 500 µM zinc they appeared to be less numerous . After iOrganic acids in root exudates were detected by HPLC. Absolute values and significance are shown in Solanum nigrum biomass and enhanced cadmium extraction from the soil. In a previous study, the endophytic bacterium SaMR12 was shown to be effective for promoting growth of the host plant SaMR12 affects plant growth, root morphology, oxidative stress, and root exudates.During phytoremediation, it is an important for plants to continually absorb heavy metals from the soil and transport them from the root to the shoot. Three factors are necessary for this process to be effective: (1) the plants must be resistant to heavy metal; (2) the plants must absorb metals from soil; (3) the metal should be transported from the root to the shoot The biomass of a plant is a primary factor limiting successful remediation of heavy metal contamination. In sugar beet, above-ground, endophytic bacteria increased photosynthetic efficiency by increasing chlorophyll content, leading to increased carbohydrate synthesis P. putida GR12-2 compared with non-inoculated control, while the IAA-deficient mutant did not show this increase Tuber melanopsorum, induces alterations in both host (Cistus incanus) and non-host  roots, such as promoting root hair elongation, that are reminiscent of altered auxin levels SaMR12 increase auxin levels, the concentration dependence differed from the promotion of biomass and zinc accumulation. Both of the latter were clearly stimulated on 50 µM zinc whereas there was little increase in IAA. Furthermore, root hairs responded vigorously to the presence of endophyte but this response seemed more or less independent of the level of zinc  have been reported S. alfredii, it remains unclear whether the observed alleviation is a consequence of better nutrition or a direct action of the endophyte, such as the production of anti-oxidative enzymes or siderophore.Zinc is typically not considered to induce reactive oxygen in plants, however, various studies have demonstrated that zinc can activate copper/zinc superoxide dismutase and proline production to alleviate metal-induced oxidative stress or enhance the activities of antioxidant enzymes d plants  and 6. SRoot exudates comprise a complex set of compounds, which help mobilize mineral nutrients and condition the rhizosphere SaMR12. Higher zinc (500 µM) treatment stimulated citric acid secretion, which agreed with the results of Saber et al. Pinus sylvestris, oxalic acid was stimulated by exposure to elevated aluminum and was found to be significantly higher in mycorrhizal-treated plants than in non-mycorrhizal controls In a previous study, the exogenous addition of citric or tartaric acid significantly increased the availability and absorption of cadmium Brassica juncea).The activating effect of organic acid on heavy metal uptake is commonly related to pH. Succinate enhanced the mobilization of arsenic, copper, lead, and zinc under neutral pH, but inhibited this mobilization under acidic conditions SaMR12, on plant growth, root morphology, and root exudates, a study that might facilitate the understanding of mechanisms of microbe-assisted phytoremediation. Through hydroponic experiments, SaMR12 inoculation significantly enhanced the efficiency of zinc phyto-extraction by increasing S. alfredii biomass, promoting zinc absorption, improving root morphology, and enhancing root exudates. Further we could show that although IAA levels and reactive oxygen species rose, both could be plausibly disconnected from the promoting action of both zinc itself and the endophyte. The application of endophytes to phytoremediation not only strengthens the ability of phyto-accumulators to acclimate to the contaminated environment, but also expands the application range of phytoremediation.This is apparently the first study to investigate the effects of inoculation with the endophytic bacterium,


Accelerated degradation of the wetlands and fragmentation of surrounding vegetation in the Andean–Orinoco Piedmont are the main threats to diversity and ecological integrity of these ecosystems; however, information on this topic is of limited availability. In this region, we evaluated the value of 37 lentic wetlands as reservoirs of woody and aquatic plants and analyzed diversity and changes in species composition within and among groups defined according to management given by: (1) type  and (2) origins . A total of 506 plant species were recorded: 80% woody and 20% aquatic. Of these, 411 species (81%) were considered species typical of the area (Meta Piedmont distribution). Diversity patterns seem to be driven by high landscape heterogeneity and wetland management. The fish farms presented the highest diversity of woody plants, while swamps ranked highest for aquatic plant diversity. Regarding wetland origin, the artificial systems were the most diverse, but natural wetlands presented the highest diversity of typical species and can therefore be considered representative ecosystems at the regional scale. Our results suggest that lentic wetlands act as refuges for native vegetation of Meta Piedmont forest, hosting 55% of the woody of Piedmont species and 29% of the aquatic species of Orinoco basin. The wetlands showed a high species turnover and the results indicated that small wetlands (mean ± SD: size = 11 ± 18.7 ha), with a small area of surrounding forest (10 ± 8.6 ha) supported high local and regional plant diversity. To ensure long-term conservation of lentic wetlands, it is necessary to develop management and conservation strategies that take both natural and created wetlands into account. The vegetation associated with wetlands is one of the most distinctive characteristics of aquatic ecosystems . Plants The Andean Piedmont of the Colombian Orinoco river basin once formed a continuous corridor of vegetation between the Eastern cordillera of the Andes, and the savannah ecosystems of the Llanos Orientales . This waWetlands are a conservation priority at regional , nationaThe importance of type and origin of wetlands as drivers of biotic homogenization and differentiation of plant diversity is poorly known. In highly modified landscapes as in Andean Piedmont, vegetation is reduced to small fragments, many of them associated with lentic wetlands. Several studies highlight the role that these remnants have for the maintenance of plant diversity regardless whether these are natural  or creatIn this study we evaluate the role of natural and created lentic wetlands as a refuge of plant diversity of the Meta Piedmont biome, and the influence of the wetland management in the diversity of woody and aquatic plants, focusing on two questions: (1) are lentic wetlands, despite its size, refuges of plant diversity; and, (2) how do the diversity and composition of plants associated with lentic wetlands change according to their management given by type and origin? We expect that wetland type has a strong influence on local and regional plant diversity, that natural wetlands retain the greatest plant diversity of Andean Piedmont and that lentic wetlands are potential reservoirs of plant diversity. This study presents the first estimates of plant diversity in lentic wetlands in the region, and aims to be a scientific baseline for the sustainable management and conservation of these wetlands and their associated vegetation.Andean Piedmont is a subregion of the Colombian Orinoco river basin, located in the East Andes in a transitional confluence area between the Andean mountains, and the Amazon and Savannah ecosystems, located within the physiographic unit known as Piedmont . Our stu′′N–73°42′56.8′′W and 3°59′41.1′′N–73°5′12.2′′W)  . SamplinWetland management was studied in six types of wetlands: (1) swamps (SW) are natural forest wetlands with emergent woody vegetation, they are flooded lands without a defined open body of water, associated with sources of other wetlands like streams or rivers; (2) heronries (HC) are natural swamplands with flooded areas and are characterized by the congregation of mixed species of breeding aquatic birds, specially herons and egrets; (3) rice fields (RF) are farms of irrigated rice crops that are mixed with natural water sources, channels and sometimes small dams, also they have fencerows with vegetation; (4) semi-natural lakes (SNL) were created after some management on natural wetlands that retain features of them and have tourist and recreational purposes; (5) constructed lakes (CL) are artificial wetlands that were built in its entirety and are used as recreational lakes and dams; (6) fish farms (FF) are generally rectangular shaped ponds, created for aquaculture of freshwater fish production (see We classified wetland types into three categories according to their origins: (1) natural: those that retain their original natural characteristics and where human intervention does not affect its natural dynamics (SW and HC); (2) mixed: those that have a natural origin but were transformed through human actions with extensive modification, so despite they can retain their natural features, they depend heavily on the constructed sections and facilities that regulate their hydric cycle (SNL and RF), and (3) artificial: wetlands that are completely human-made in places where no previous lentic wetland existed (CL and FF).We studied plant formations of woody and aquatic species. The sampling was carried out through standardized methods in each wetland. Woody plants  were sampled in four linear 50 × 2 m transects (0.04 ha), located as close as possible to the wetland shore, within a 200 m buffer around, and with a minimum distance of 100 m between transects. All individuals with a diameter at breast height (DBH) ≥2.5 cm were recorded. Aquatic plants were sampled using ten 1 × 1 m plots placed in the flooded area of the wetland shore, where emergent vegetation was present  with a minimum distance of 5 m between plots. In each plot, the percent cover of every species was measured. Only flowering plants (Angiosperms) and vascular cryptogams (Pteridophytes) were considered in this study. Species categorized as ‘typical’ were those having a distribution in the Meta Piedmont biome, and we refer to introduced exotic (from foreign countries) and native exotic species (with distribution in others native Colombian ecosystems) as ‘introduced’ plants. All plants were identified in the field by a specialist. The taxonomic species list is based on n is the total number of plants, f1 and f2 are the numbers of singletons and doubletons of the sample. We used the sample coverage estimator proposed by leteness .qD  which is not sensitive to abundances , and can be interpreted as the number of ‘dominant’ species , diversity (α-diversity) was calculated in three ways: local alpha diversity in each wetland (αw), total alpha diversity corresponding to pooled diversity of each type and origin category (α), and mean alpha diversity (γ-diversity) as the total diversity of all wetlands.For both orders (q = 0 and 2) were compared directly and also using the percentage of species diversity difference magnitude (%MD) between types and origins of wetlands: qDsample > S is the sample with more diversity and qDsample < S is the sample of less diversity . When RLIq0, is 0, it has maximum equality, when it is 1, it indicates maximum inequality , which is the logarithmic transformation of the inequality factor IF0,q : (3)\docqCS , and N is the number of samples; qCS = 0 when all samples are completely distinct, and qCS = 1 when all are identical. When q = 0, and N = 2, it equals the Jaccard index, and for q = 2 the Morisita–Horn–Index .We recorded a total of 506 vascular plant species from 85 families and 324 genera in the 37 lentic wetlands. 407 species were woody plants and 99 aquatic species. There were 411 (81%) typical species of Meta Piedmont and of the remaining 95 introduced species (19%), 43 corresponded to introduced exotic species and 52 to native exotic ones. Additionally, some of the species are categorized as threatened in red lists: lnerable , and alsThe sampling coverage in each wetland was higher than 74%. Taking into account the wetlands according to the type and origin, completeness was greater than 92% . Given t0D) and dominant species (2D) (α) showed differences among wetland types for both woody and aquatics plants (0D) was highest in FF (212 ± 12) and SNL (201 ± 13), and it was lowest in HC (78 ± 9) and RF (68 ± 6), presenting large richness difference magnitude between FF and RF (212%) as well as between SNL and RF (196%) (2D), especially in HC, which showed the highest inequality   –S2, beinies (2D) . There wd origin . Total as plants . Woody sF (196%) . The pat = 0.52) , being t0D and 2D), especially between SW and HC with a difference magnitude of 167% for species richness and 177% in terms of dominant species , mixed wetlands were more diverse regarding dominant species (2D), and natural wetlands were the least diverse . Species richness difference magnitude was 31% between artificial and natural wetlands, and dominant species difference magnitude was 101% between mixed and natural wetlands. In aquatic plants, the differences in diversity were low between origins . However, natural wetlands had greater species richness (0D) and 14 species were exclusive in these.With regard to wetland origin, woody plants in artificial wetlands had higher species richness , and heronries (HC) for dominant species (2CS = 0.01), while rice fields (RF) showed higher similarity, which increased as q increased . All other wetland types had values lower than 0.17. Regarding dominant species, SW showed the lowest and HC the highest values   was low, but it showed a high variability within wetland types . For botg groups  and 4B. , 0,2CS) .0CS = 0.16 and 2CS = 0.27) (qCS < 0.16) and intermediate among them (qCS = 0.5) for both richness (0CS) and dominant species (2CS)  and intermediate among them. Natural wetlands had the highest similarity, especially in dominant species  = 0.27) . In the es (2CS) .This is the first comparative study on the diversity of plants in freshwater inland wetlands under different regimes of management in the Neotropical region. Although the total area of the 37 wetlands studied (424 ha) and their forest area (435 ha) associated represent less than 1% of wetland coverage in the Orinoco region and 1% of the Meta Piedmont tropical humid zonobiome , our resContrary to our expectation, the origin of natural wetlands had low diversity both in richness and dominant species, which is associated to the fact that the heronries (HC) presented the lowest diversity between wetland types. This is probably due to the accumulation of guano caused by the congregation of birds, which may have led to a naturally caused impoverishment of diversity due to the high nitrogen content of excrements , and it The finding that artificial wetlands have a greater diversity of woody plants can be explained by the fact that both fish farms (FF) and constructed lakes (CL) are created by private enterprises, where owners have particular interests. Most of these wetlands were built on relict forest. Meanwhile, mixed wetlands retain a high diversity of species typical of Piedmont because they were developed on natural wetlands for recreational  and agricultural (rice fields—RF) purposes, and its management allows to keep a portion of the diversity of native plants. The use of agrochemicals is often associated with the decline of plant diversity ; althougOur results show that the diversity patterns of plant species in and around wetlands are the result of both the heterogeneity of these wetlands at the landscape scale and human activities related with their management. Although wetlands are isolated systems, their heterogeneity and diversity have been attributed largely to the interaction with adjacent terrestrial and other aquatic ecosystems such as flooded and riparian forests , elementThe results of this study support the importance of small forest fragments to conservation in human-dominated landscapes e.g., . DespiteIn our study compositional similarity was low (<0.5) compared to other studies in small forest patches in human modified landscapes, where higher CS-values were related to homogenization, and lower values to differentiation processes  diversity is high because of the landscape heterogeneity, similar to the natural diversity pattern before fragmentation, and patches are sufficiently connected in order to maintain diversity in the long term; and (2) the low compositional similarity is a sign of independent dynamics of the wetland communities, hence plant populations are not sufficiently connected, increasing the probability that rare species may disappear from most wetlands . This coOur results suggest that created lentic wetlands  can have a key role in plant diversity conservation by increasing the available habitat in the region for a variety of typical species. Nevertheless, the plant diversity in natural wetlands may not be replaced or equaled by artificial wetlands, yet the human-made wetlands are a way of safeguarding biodiversity and partially compensate against rapid loss and transformation of natural wetlands . CreatedCurrently the Piedmont is a highly fragmented landscape  and cons10.7717/peerj.2267/supp-1Table S1(Click here for additional data file.10.7717/peerj.2267/supp-2Table S2Click here for additional data file.10.7717/peerj.2267/supp-3Table S3Click here for additional data file.10.7717/peerj.2267/supp-4Table S4Click here for additional data file.


Spheniscus demersus has decreased from more than 1.5 million individuals in the early 1900s to c.a. 23 000 pairs in 2013. Determining the trophic interactions of species, especially those of conservation concern, is important when declining numbers are thought to be driven by food limitation. By and large, African penguin dietary studies have relied on the identification of prey remains from stomach contents. Despite all the advantages of this method, it has well known biases. We therefore assessed the African penguin’s diet, using stable isotopes, at two colonies in Algoa Bay (south-east coast of South Africa). These represent over 50% of the world population. Various samples  were collected for carbon and nitrogen stable isotope analyses. Results indicate that the trophic ecology of African penguins is influenced by colony, season and age class, but not adult sex. Isotopic niches identified by standard Bayesian ellipse areas and convex hulls, highlighted differences among groups and variability among individual penguins. Using Bayesian mixing models it was for the first time shown that adults target chokka squid Loligo reynaudii for self-provisioning during particular stages of their annual cycle, while concurrently feeding their chicks primarily with small pelagic fish. This has important ramifications and means that not only pelagic fish, but also squid stocks, need to be carefully managed in order to allow population recovery of African penguin.Many species of seabirds, including the only penguin species breeding on the African continent, are threatened with extinction. The world population of the endangered African penguin  The marine environment has in recent years been profoundly altered by anthropogenic activities. Although all trophic levels have been affected, directly or indirectly, seabirds as a group have been particularly vulnerable and are among the most threatened taxonomic groups . For manSpheniscus demersus is endemic to the southern African coast and the only penguin species breeding on the African continent. Like some of its congeners in South America, the population of African penguins has declined sharply over the last century. Estimated between 1.5 and 3 million individuals in the early 1900s )Given that the bulk of the African penguin population is now situated in Algoa Bay, and the need to understand how this species will adapt to continuing climate change and anthropogenic influences, a reassessment of their trophic ecology in the Eastern Cape is essential and timely. In this study, we used stable isotopes to investigate the trophic ecology of African penguins from St Croix and Bird islands in Algoa Bay. Due to varying energetic demands, this was done in relation to breeding stage, sex and age class. In addition to blood, feathers and egg membranes were therefore also collected to obtain information on the little known but nevertheless crucial pre-moulting and pre-laying periods.All fieldwork and data collection were undertaken under the research permit RES2013/18 issued by the Department of Environmental Affairs and the ethics clearance reference A13-SCI-ZOO-008 issued by the Research Ethics Committee at the Nelson Mandela Metropolitan University. Access to study sites (Bird Island and St Croix Island) and permission to conduct the research were granted by South African National Parks (SANParks), the managing authority of the islands. No penguins were injured as a result of handling and sampling for the present study. Sampling procedures were carried out briefly (<10 min of handling) by experienced researchers to minimize disturbance.Geographic variation in the timing of the annual cycle has been observed in African penguins . The onlrd July and 4th August 2013. Adults were also caught outside their breeding season on the 19th and 20th January 2013 on Bird Island only, due to logistical constraints. To minimise disturbance at the nests ).Differences in stable isotope values can potentially be influenced by size and the physical condition of the African penguins. To control for this, two indices were estimated: a structural size index and a body condition index.2 = 0.26, P < 0.001). Similarly, an index of structural size was defined for blues: Principal Component 1 = 0.60 * bill length + 0.54 * bill depth + 0.59 * flipper length. Their body condition was defined as the residuals of a regression of body mass on the index of structural size .A number of measurements were taken from each adult and blue including bill length and depth for sexing in the field , flipperAs one of the aims of the study was to examine whether differences in diet existed between sexes, assigning sex was crucial. Using length and depth measurements of the bill to build a discriminant function, >90% of the adults can be sexed accurately . This le® extraction method [® Spectrophotometer (Thermo Scientific), and the supernatant stored at -20°C. DNA fragments of the sex-linked CHD-1 gene  were then amplified as in [™ Nucleic Acid Gel stain and bands were visualized under ultraviolet light.Whole genomic DNA was extracted from the feather roots using a Chelexn method . The DNAed as in . The onl2, respectively. Internal laboratory standards were calibrated against reference materials from the International Atomic Energy Agency  and run throughout all runs, typically 2 standards for every 10–12 samples. Within and among run measurement errors are detailed in Prior to analysis, egg membranes were carefully brushed in distilled water before being dried at 50°C for 24 hours. Feathers were initially washed in chloroform-methanol (2 parts to 1) in an ultrasonic bath for 2 minutes, rinsed in successive baths of methanol and distilled water, and then dried at 50°C for 24 hours. Each feather and piece of membrane were then cut into small pieces and homogenized. For whole blood sampled from birds, it is usually assumed that no pre-treatment is necessary . Homogen13C and exhibit more negative δ13C values than proteins and carbohydrates [13C were therefore reduced by mathematically normalizing the carbon ratios using an equation developed for aquatic animals δ13C = δ13C − 3.32 + (0.99 x C:N) [Within an organism, lipids are typically depleted in hydrates , 40. A shydrates . Accordi9 x C:N) .-1), and the Bayesian estimate of the standard ellipse area (SEAB) [After verification of the multivariate normality assumptions , stable isotope Bayesian ellipses were generated in R  .Etrumeus whiteheadi, chub mackerel Scomber japonicus and squid Loligo reynaudii. A muscle section was sampled from each individual, dried at 50°C for 24 h, ground to powder and delipidated using cyclohexane  previously identified for African penguins in Algoa Bay , were coailed in ). CarbonAccurate diet-tissue discrimination factors are also essential in mixing models . A feediThe adequacy of the food sources and discrimination factors was checked using simulated mixing polygons  before r13C and δ15N values were therefore checked within each group  by comparing values for individuals exhibiting a lower (negative) and higher (positive) body condition index than average. The comparison of stable isotope values between sexes, age classes and islands required the use of various parametric and non-parametric analyses depending on whether normality and homoscedasticity hypotheses were verified.All statistical analyses were conducted using R v3.2.3  and Past13C in adults or in blues . Similarly, no statistical differences were found in the δ15N values between birds with different body conditions  with the exception of adults sampled outside the breeding season on Bird Island. In the latter case, birds with a lower body condition than average exhibited statistically higher δ15N values .Overall, males exhibited statistically larger features and were heavier than females . Importantly, these carbon values were correlated with high C:N ratios , while C:N values were all lower than 3.5 for all the other samples. Consequently, non-breeding female blood δ6,70 = 17.73, P < 0.001, pairwise comparison all P < 0.004 with the exception of breeding season samples where no difference existed between male and female samples P = 0.391; 15N values , and during non-breeding males were separated from females based on their δ13C values . The overall isotopic spaces of all four groups were similar .No seasonal or sex related variation in δisland = 2.63, P = 0.077; Fsex = 2.02, P = 0.116; Fisland*sex = -2.71, P = 0.736). Univariate analysis however showed that carbon stable isotope values of adult blood samples were significantly different between the two islands with samples from St Croix being depleted in 13C . None of these factors were significant when considering δ15N values . When adding the samples for blues, the island still had a significant effect on δ13C values , and neither did the interaction of these two factors , and the interaction island/age class had a significant impact on δ15N values . When considering samples from adults and blues, no statistical difference in the stable isotope values was highlighted between the two islands, but age class was a significant factor  with δ13C and δ15N values being significantly lower in blues than in adults  nor for δ15N . Theref13C and elevated C:N ratios observed in female blood at that time of the year were therefore possibly related to higher proportions of circulating lipids in their blood due to egg formation [Spheniscus magellanicus [Eudyptes chrysolophus [The breeding cycle of African penguins is protracted and on Bird Island egg laying mainly takes place from February to July, sometimes followed by second clutches for both successful and failed breeders , 63. Wheormation . Elevatellanicus ) and Macsolophus ) penguinsolophus , 67, butsolophus  and obseMegadyptes antipodes [One of our most important results is that parents are selectively favouring small pelagics when catching food for their chicks, but targeting squid for self-provisioning, as indicated by blood and feather stable isotope data. Such selective foraging has also been observed in the Yellow-eyed penguin ntipodes .15N than birds with a higher body condition at the same period. This effect of physiology on δ15N values [13C and δ15N values, we sampled chicks as closely as possible to fledging . The weights recorded (~3000 g) for Bird and St Croix island blues suggest that they were not experiencing nutritional stress as these approached the weight of hand-reared chicks [13C and δ15N values. The geographical difference observed, with St Croix chicks being fed a larger proportion of higher trophic level prey such as squid than their counterparts on Bird Island, may be explained by the greater availability of squid in the south west area of the bay [Our results from feathers indicate that adults from both islands preyed upon a mix of small pelagics and squid prior to moulting. Blood samples suggested that, thereafter, the adults maintained a similar diet prior to breeding. Birds with a lower body condition than average in January exhibited higher δN values  may artiN values , were faN values , the chiN values . Growth N values , 58 . To redd chicks . It is t the bay  where br the bay .The availability of the prey species in the environment was likely to also have played a role in the seasonality that we observed in adult diet. Penguins that have been tracked exclusively during chick-rearing on both islands remained in an area within 44 km of their breeding colony . Therefo13C and δ15N values. The non-convergence could be explained by a number of factors including (i) the use of an erroneous trophic discrimination factor, and/or (ii) the absence of the main prey species in the database targeted by females prior to laying. Only one study has determined the diet-egg membrane trophic discrimination factor in penguins . A studBreeding colonies of African penguins are located both in the warm Agulhas current bioregion on the east coast of South Africa, and the cold Benguela current on the west . These cSufflogobius bibarbatus after the collapse of small pelagic stocks due to overfishing in the 1970s . Overall values among all runs are presented as mean ± SD (n = number of standard duplicates). -: values not used in calibration.(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Tablen: number of samples.(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.


ANKK1, DRD2, DRD3, DRD4, SLC6A3/DAT1, MAOA, MAOB, COMT, CPE) for associations with percent weight change at 12 months post-transplantation. Seventy kidney transplant recipients had demographic and clinical data collected as a part of a larger observational study. DNA was extracted from repository buffy coat samples taken at the time of transplant, and genotyped using Taqman and PCR based methods. Three SNPs were independently associated with percent weight change: ANKK1 rs1800497 , SLC6A3/DAT1 rs6347 (p = 0.046), and CPE rs1946816 (p = 0.028). Stepwise regression modelling confirmed the combined associations of age (p = 0.0021), DRD4 VNTR 4/5 genotype (p = 0.0074), and SLC6A3/DAT1 rs6347 CC genotype (p = 0.0009) and TT genotype (p = 0.0004) with percent weight change in a smaller sample (n = 35) of these kidney transplant recipients that had complete genotyping. These associations indicate that there may be a genetic, and an age component to weight changes post transplantation.Kidney transplant recipients often experience a significant amount of weight gain in the first year post-transplantation. While demographic factors such as age, race, and sex have been associated with weight gain in this population, these factors do not explain all of the variability seen. A number of studies have suggested that genetics also plays a critical role in weight changes. Recently, alterations in the activity of the neurotransmitter dopamine have been associated with weight change, and gene expression studies in kidney transplant recipients have supported this association. The purpose of this pilot study is to first examine the feasibility and methodology, and then to examine the associations of age, race, sex, and genotype for 13 SNPs and 3 VNTRs in 9 dopaminergic pathway genes ( Approximately one third of all kidney transplant recipients will gain a significant amount of weight (6–13 kilograms) during the first year after transplantation . This exA small amount of the variance in weight change can be explained by certain demographic characteristics , with older African American women being the most likely to gain weight . HoweverDRD2, DRD3, and DRD4), the dopamine transporter gene (SLC6A3/DAT1), the dopamine degradation enzymes monoamine oxidase A and B , and catechol-O-methyltransferase (COMT) [CPE) was also differentially expressed [CPE is not typically placed within the dopaminergic pathway, there is some evidence that it could affect the dopamine transporter’s efficiency [Dopamine is a neurotransmitter that has previously been implicated in substance addiction , 11, bute (COMT) . The enzxpressed . Althougficiency .All of the genes in the dopaminergic pathway have polymorphisms (such as single nucleotide polymorphisms (SNPs) or variable number tandem repeats (VNTRs)) that could cause gene activity to be altered and subsequently place individuals at a greater risk for gaining weight. Identification of individuals as being at a genetically or demographically higher risk for weight gain would enable implementation of personalized lifestyle interventions aimed at reducing environmental contributions to weight gain. The aims of this study are to 1) evaluate the ability of selected dopaminergic polymorphisms to predict percent weight change in kidney transplant recipients and 2) determine the relationship of polymorphisms predictive of percent weight change with demographic characteristics  and their combined ability to predict percent weight change in a kidney transplant population. This pilot study will examine the feasibility and methodology of this technique in order to replicate the results in a larger cohort in the future.A candidate gene association approach was used to study percent weight change in a subset of kidney transplant recipients with repository samples obtained from participants in a larger prospective study. Subjects were voluntarily recruited and consented from all patients at a midsouth regional transplant center awaiting kidney transplantation and meeting inclusion/exclusion criteria from August of 2007 until March of 2011.2). Percent weight change at 12 months was calculated as a percentage of total pretransplant body weight. These variables were shared in a deidentified format. Additionally, participants in the larger study had blood taken at the time of kidney transplant surgery. Buffy coat samples were frozen and stored in a -80°C freezer. The participants in this study also had to sign a repository consent form to allow their biospecimens and genetic data to be used in future work. This protocol was approved by the Institutional Review Board.This study represents repository subjects from a larger observational study on demographic and genetic factors related to weight after kidney transplantation. The larger study included men and women between 18 and 70 years old, receiving their first transplant, and who were able to read and write in English. Exclusion criteria for the larger study were multiple organ transplantation, previous kidney transplantation, or a nonfunctioning graft during any point in the study period. Participants in the larger study had age, race, and sex information collected, as well as marital status information. Baseline weight and height and 12 month post-transplant weight were also collected. Height data allowed the calculation of body mass index (BMI), as well as changes in BMI at one year post-transplantation  . All TaqMan® assays were conducted on the ABI Prism 7000 Sequence Detection System. VNTRs were genotyped using polymerase chain reaction (PCR) based methods and gel electrophoresis  . The resDemographic variables were collected from clinic records. Age was recorded in years and fractional months at time of transplantation. The outcome of percent weight change was calculated by the formula ((12 month weight-baseline weight)/baseline weight)*100). Descriptive data were presented as means, standard deviations, and ranges. The same data were also examined with appropriate correlations, t tests, chi-squares, and ANOVA statistical tests using R statistical software package, version 2.14.1 .To evaluate the ability of dopaminergic polymorphisms to predict percent weight change in kidney transplant recipients (aim 1), each polymorphism was analyzed using ANOVA, chi squares, and point biserial correlations as appropriate. All analyses were conducted initially using a dose dependent approach. In a dose dependent approach, the assumption is made that a genotype AA is different from Aa, and each of those genotypes is different from aa when considering the outcome of percent weight change post kidney transplant. Therefore, 3 groups  are used to compare the impact of genotype on percent weight change.Next, analyses were conducted in a risk allele fashion. In this type of analysis, those individuals either homozygous (AA) or heterozygous (Aa) for the risk allele for increased percent weight change post kidney transplant (A) are analyzed together as one group and compared to those not carrying a risk allele . Genetic variants identified as approaching significance (using the standard p<0.20 ) were chIn regression modelling, the variables were first assessed for possible multicollinearity. Any variables that were found to be highly correlated (by variation inflation factor (vif)>10) were removed from the model . Then a p = 0.006). A one tailed t-test found no difference in percent weight change by sex (p = 0.23). Likewise, no difference was found for percent weight change by race (p = 0.77).Demographic characteristics for the 70 participants in this study are listed in ANKK1 rs1800497 , and MAOB rs1799836 SNPs . Additionally, SLC6A3/DAT1 rs6347 and CPE rs1946816 were significantly  associated with percent weight change. Lastly, SLC6A3/DAT1 VNTR met criteria for inclusion in a regression analysis , as did ANKK1 rs1800497 .The first analysis of genotype data was conducted in a dose dependent manner, which assumes that the homozygous risk allele genotype is different from either the heterozygous or the homozygous non-risk allele genotype. Thus, if found to be significant this means that having the risk allele for increased percent weight change in either one or two doses makes a difference in the overall outcome of percent weight change . PercentSLC6A3/DAT1 rs6347 (p = 0.0003), and for the C risk allele of CPE rs1946816 (p = 0.00004) . Several other variants approached a level of significance that warrants inclusion in a regression model .Risk allele analyses increases power by collapsing categories based upon the presence of the risk allele, regardless of whether it is present in either one or two doses. For instance, if the risk allele is A, then heterozygous and homozygous individuals carrying the A allele are treated as one group and compared to those individuals homozygous for the other alleles. Risk allele chi-squares showed a significant effect for the A risk allele 0.00004) . The C aDRD4 VNTR, SLC6A3/DAT1 VNTR, rs1799836, rs6347, and rs1946816. These variables, along with the demographic characteristics age, race, and sex, were first assessed for possible multicollinerarity using the criterion of vif>10 [DRD4 VNTR (4/4 genotype), and SLC6A3/DAT1 VNTR .Only polymorphisms with a p≤0.20 in previous statistical analyses were included in the regression model . Based uf vif>10 . This anDRD4 VNTR  SLC6A3/DAT1 VNTR (9/10 and 10/11 genotypes), rs1799836, rs6347, and rs1946816). For this model, sample size was reduced to 35 from the total N of 70 subjects due to incomplete genotyping for some variants. The model was significant (p = 0.0001), with the variables age (p = 0.0021), DAT1 9/10 genotype (p = 0.0199), DRD4 4/5 genotype (p = 0.0074), rs6347 CC genotype (p = 0.0009), and rs6347 TT genotype (p = 0.0004) all being important contributors to percent weight change , t change .This pilot study was conducted to evaluate the feasibility and methodology to uncover the associations of dopaminergic polymorphisms and demographic characteristics with 12 month percent weight change in kidney transplant recipients. Kidney transplant recipients are an ideal model population for studies of weight change as they are likely to gain a substantial amount of weight in the first year after transplantation. However, since not all kidney transplant recipients gain weight, they provide the desired phenotypic  variability for genetic and demographic association studies. In our sample, 25 people gained greater than 5% of their pretransplant body weight, 20 people stayed within ±5% of their pretransplant body weight, and 25 people lost greater than 5% of their pretransplant body weight. This supports previously findings that a minimum of 1/3 of all kidney transplant recipients gain a significant amount of weight within the first year following transplantation , 20.It is important to note that an increase in percent weight change does not necessarily signify that an individual became overweight or obese by BMI classification. Many individuals stayed in the same weight classification, while others lost weight. Only four individuals gained enough weight to become newly classified as obese. Unless the subject was severely underweight at the time of transplant, any increase in percent weight could potentially become problematic and over time lead to obesity. No individual included in this study was underweight at the time of transplant. One subject was categorized as normal weight at the time of transplant, but lost weight and then became classified as underweight at 12 months post-transplant .The demographic characteristics evaluated in this study were age, race, and sex. In our sample, race and sex were not found to be associated with percent weight change at 12 months. In earlier work, it has been suggested that older African American women were the most likely to gain weight after transplantation . But in p = 0.05). This SNP lies in the ANKK1 gene, however it is known to impact density of the dopamine receptor, type 2. The association found in this study occurred in a dose dependent manner, indicating that the genotype TT is different than either the TC or CC genotype in the effect on percent weight change of kidney transplant recipients at 12 months. This finding is consistent with previous findings that the C allele is associated with higher numbers of D2 receptors in the brain [Three of the SNPs  tested in this study were independently associated with percent weight change at twelve months post-transplantation. The first is a SNP rs1800497 , this SNP showed a dose dependent effect in this sample, and also an effect for the risk allele A. The results found in this study agree with previous associations between the A allele and overweight/obesity [The second SNP independently associated with percent weight change was the /obesity . It is pCPE. While CPE has not commonly been considered a dopaminergic pathway gene, there is growing evidence that it could interact to change the rate of dopamine reuptake via the dopamine transporter [CPE in adipose tissue of kidney transplant recipients was found to be significantly associated with weight change [The last SNP independently associated with percent weight change at twelve months was rs1946816 in nsporter . This rst change .DRD2 SNP rs6277 has a minor C allele frequency of 0.396 in Caucasian populations. This means that 39.6% of the alleles carried by Caucasian individuals are minor C alleles. In contrast, African American populations have a minor allele frequency of 0.913 .While these three SNPs hold promise for future work, these independent associations in such a small sample could be the result of population stratification. This sample was heterogeneous in racial background, making it likely there is a representation of several different ancestral lines containing different minor allele frequencies. For example, the Another explanation for potential population stratification could lie in the characteristics of individuals who may be more prone to become a candidate for kidney transplantation. To the best of our knowledge, there is no known relationship between dopamine and kidney transplantation. However, it is possible that some inherent dopaminergic related characteristics are present in individuals with kidney disease and these increase the likelihood that they will require a transplant. Perhaps a reduction in dopaminergic reward from certain behaviors could be related to a reduction in health promoting behaviors. Over time, this could lead to an increased likelihood of kidney failure and the need for transplantation, thereby resulting in an over-selection for this genotype when sampling kidney transplant recipients.SLC6A3/DAT1 VNTR, DRD4 VNTR, rs1799836, rs6347, and rs1946816). Although sample size was greatly reduced (n = 35) and thus the model is underpowered, the model was predictive of percent weight change  and the seven polymorphisms meeting the p≤0.20 criterion  were not considered in this pilot work. Many of these values could hint to future comorbidities for those individuals gaining excessive weight in the first 12 months post transplantation. These values should be collected and examined along with demographic and genotype information in future work. Exclusion criteria related to edema should also be added in these future studies.A further limitation for this study is that within gene variation was not completely sampled. While many of the most widely studied polymorphisms were tested, this study did not include an exhaustive list. Additionally, there could be other regions of variation within these genes that have not yet been identified, and hence were not sampled in this study. The genotype of the donor kidney was also not considered for this study, which has been shown to impact blood pressure in animal models .DRD4 VNTR, and the SLC6A3/DAT1 SNP rs6347 also have predictive value for percent weight change. Replications of this study with a larger sample size should be done to fully assess the contributions of these polymorphisms to prediction of post transplant weight gain. Expansions and replications of this study may help to develop future genetic testing to better identify kidney transplant recipients most at risk for weight gain. In the meantime, medical professionals should continue to educate all kidney transplant recipients, but most especially younger patients, about the possibility of substantial weight gain in the first year post-transplantation. These patients should be directed to individualized intervention therapies aimed at reducing the amount of weight gained after surgery.This pilot study was conducted to assess the value of dopaminergic polymorphisms and demographic characteristics  for predicting percent weight change at 12 months’ post kidney transplantation. Although the results are somewhat tempered by the small sample size that was used for this study, age at time of transplantation was an important factor in determining the amount of weight change at one year. Genotype for the S1 TableMAOA, SLC6A3/DAT1, DRD4).VNTRs were genotyped using polymerase chain reaction (PCR) based methods and gel electrophoresis ((DOCX)Click here for additional data file.


The independent skeletal effect of thyrotropin  has been suggested in animal studies. However, clinical data on the association between bone loss and variations in TSH levels is inconsistent. This study aimed to investigate the relationship between TSH levels and bone mineral density (BMD).We conducted a cross-sectional study with 37,431 subjects  aged over 35 years. We performed thyroid function tests and measured BMD at the lumbar spine, femur neck, and total hip.Levels of TSH and T3 were positively correlated in women  and uncorrelated in men. In both men and women, TSH levels correlated positively and T3 levels correlated negatively with BMD at all skeletal sites in age and body mass index adjusted analyses. BMD increased steadily with TSH levels from the subclinical hyperthyroid to subclinical hypothyroid range in subjects with T3 levels in the highest tertile (119.5–200.0 ng/dL), but was no longer significant in subjects with lower plasma T3 levels.The variations in TSH levels within the euthyroid and subclinical range were positively correlated with BMD in healthy men and women. The negative effect of T3 on BMD appears to be compensated for by increased TSH in subjects with plasma T3 levels in the upper normal range. Overt hyperthyroidism is an established risk factor for low bone mineral density (BMD) . Excess Rodent model studies suggest that low or elevated thyrotropin  levels may play an important role in bone remodeling and homeostasis –8. This In the present study, we sought to investigate the relationship between TSH and BMD in a large institutional cohort. We also examined whether plasma triiodothyronine (T3), the active form of thyroid hormone that may vary independently of TSH levels, affects the relationship between TSH and BMD.These cross-sectional analyses were based on the database of the institutional cohort of Health Promotion Center of the Samsung Medical Center in Seoul, Korea. From January 2007 to December 2013, 62,304 adults aged 35 years or older visited the center for routine health check-ups and underwent BMD and thyroid function tests (TFTs). After exclusion of 16 subjects due to invalid scans, we excluded 8,049 subjects with a history of cancer, diabetes mellitus, hyperlipidemia, or renal disease. We also excluded 3,995 subjects who were current smokers, as cigarette smoking is known to alter TSH level and bone metabolism , 11. In Each subject completed a health-related questionnaire addressing history of thyroid disease, other comorbidities, smoking habits and medication information at the visit. Current smoking was defined as smoking regularly for at least the past 12 months. Height and body weight were measured using a digital scale, and body mass index (BMI) was calculated. Blood samples were collected from each subject after an 8-hour overnight fast. The concentrations of TSH, total T3, and free T4 were measured at the central laboratory of Samsung Medical Center using an immunoradiometric assay kit  for measurement of TSH and radioimmunoassay kits (Beckman Coulter) for measurement of total T3 and free T4. Coefficients of variation were as follows: TSH, ≤3.7%; total T3, ≤6.3%; and free T4, ≤10.3%.The subjects were placed into three groups based on the results of their TFTs : 1) subcBMD of the lumbar spine (L1-L4), femur neck, and total hip were measured via dual X-ray absorptiometry using Lunar Prodigy Advance  according to the manufacturer’s protocol. The left hip was scanned routinely and, in the case of a left hip fracture or device, the right hip was scanned. For all subjects in this study, BMD was measured with the same instruments. Data from our center showed that the coefficients of variation for duplicate measurement in 30 adults were 0.95%, 0.95%, and 1.35% at the lumbar spine, femur neck, and total hip, respectively.We stratified subjects with subclinical thyroid dysfunction and euthyroid status. Data from women and men were analyzed separately. The values of TSH were normalized by base-10 logarithmic transformation or categorized as a group variable for further analyses. Possible associations among various parameters of TFT and BMD were tested using Pearson’s partial correlation by adjusting for age and BMI. In the euthyroid range of TSH level (0.51 to 5.00 mIU/L), a further subdivision was made into tertile categories so that all subjects were divided into five different TSH groups as follows : subclinP value of <0.05 was considered statistically significant.Differences in BMD among TSH groups were identified by analysis of covariance (ANCOVA) by adjusting for age and BMI and expressed as estimated marginal means and SEM. Plasma T3 levels were grouped into tertiles as follows so that each group contains a third of the population: 119.5–200.0 ng/dL, 104.1–119.4 ng/dL, and 60.0–104.0 ng/dL. ANCOVA analyses with the covariates of age and BMI were used to examine whether TSH levels were related to BMD in each T3 group. Statistical analyses were performed using SAS version 9.4  software. A Overall, 37,431 adult subjects were analyzed for associations between thyroid function and BMD . The meaFrom the correlation analysis adjusted for age and BMI, there was a robust negative relationship between TSH and free T4 level , except for BMD at lumbar spine in those with T3 levels in the middle tertile (P = 0.037). In men, significant positive relationships between TSH and BMD at all sites were observed only in groups with T3 levels in the highest tertile. In subjects with the highest tertile of T3 levels, the differences in BMD between subjects with subclinical hyperthyroidism and subclinical hypothyroidism were 51, 40, and 33 mg/cm2 at the lumbar spine, femur neck, and total hip, respectively.Differences in BMD between TSH groups were evident after classifying subjects according to plasma T3 levels. In women, significant positive relationships between TSH and BMD at all skeletal sites were observed when T3 levels were in the highest tertile (119.5–200.0 ng/dL) . In thisIn this large cross-sectional study, we demonstrated that variations in TSH levels within subclinical range were related to BMD in a healthy adult population with normal thyroid hormone levels. Lower TSH and higher T3 levels were associated with reduced BMD at all skeletal sites in both sexes. The negative impact of T3 on BMD appeared to be compensated for by increased TSH only in subjects with T3 levels in the upper normal range.To the best of our knowledge, this is the first clinical study to show the protective effect of TSH on bone loss independent of the effects of active thyroid hormone, T3. Based on the distinct distribution between plasma T3 and TSH levels unrelated to simple feedback inhibitory mechanisms, we performed stratified analysis to explore the relationships between TSH, T3 and BMD in a large population of healthy men and women. We excluded patients with various chronic conditions and medication history that may act as confounders in the relationship between thyroid function and bone mass. Clinicians should consider the modest protective effect of TSH against bone loss when treating biochemically defined subclinical hyper- or hypothyroidism, particularly when it is in combination with upper normal range plasma T3 levels.Plasma levels of active thyroid hormone T3 are remarkably stable in healthy adult individuals  as a resPrevious studies investigating the risk of bone loss and fractures in combination with abnormal TSH levels have been inconsistent. In a study of postmenopausal women, TSH levels above the reference range were associated with a 35% reduced risk of nonvertebral fractures, although TSH was not related to statistically significant BMD change . ConversGiven that TSH can either indirectly promote or directly suppress bone turnover according to local T3 availability, it is not surprising that previous clinical studies, which did not take into account plasma T3 status, have produced inconsistent results regarding the effect of TSH on bone remodeling. We observed a significant dose-dependent trend between TSH levels and BMD exclusively in men and women in the highest tertile T3 level. The findings from our large population study are consistent with basic mechanistic studies as mentioned above.There are several limitations to this study. This was a cross-sectional observational study, and therefore the causal relationship between TSH and T3 and BMD cannot be affirmed by this data. We cannot address whether the treatment of subclinical thyroid dysfunction could lead to reciprocal changes in BMD, although nonrandomized trials have suggested that normalization of suppressed TSH increases BMD in postmenopausal women with subclinical hyperthyroidism , 27. InfIn conclusion, in a large cohort of health-screening recipients, we demonstrated that variations in TSH levels even within the subclinical range were positively correlated with BMD in healthy men and women with normal thyroid hormone levels. Furthermore, plasma T3 status may affect the relationship between TSH and BMD. Thus the protective effect of TSH against bone loss should be taken into consideration during treatment decisions regarding subclinical thyroid dysfunction, particularly when it presents in combination with upper normal range plasma T3 levels.S1 FigData are shown as mean and SEM (error bar). P values for trends across strata of TSH were obtained using ANOVA.(TIF)Click here for additional data file.S1 Table(DOC)Click here for additional data file.


Echium plantagineum.Napthoquinones, antimicrobial and phytotoxic metabolites that are involved in plant defence, are produced and released into the rhizosphere by root hairs and root periderm tissue of  Echium plantagineum, an invasive endemic weed in Australia. Roots of E. plantagineum produced red-coloured NQs in the periderm of primary and secondary roots, while seedling root hairs exuded NQs in copious quantities. Confocal imaging and microspectrofluorimetry confirmed that bioactive NQs were deposited in the outer layer of periderm cells in mature roots, resulting in red colouration. Intracellular examination revealed that periderm cells contained numerous small red vesicles for storage and intracellular transport of shikonins, followed by subsequent extracellular deposition. Periderm and root hair extracts of field- and phytotron-grown plants were analysed by UHPLC/Q-ToF MS (ultra high pressure liquid chromatography coupled to quadrupole time of flight mass spectrometry) and contained more than nine individual NQs, with dimethylacrylshikonin, and phytotoxic shikonin, deoxyshikonin and acetylshikonin predominating. In seedlings, shikonins were first found 48h following germination in the root-hypocotyl junction, as well as in root hair exudates. In contrast, the root cortices of both seedling and mature root tissues were devoid of NQs. SPRE (solid phase root zone extraction) microprobes strategically placed in soil surrounding living E. plantagineum plants successfully extracted significant levels of bioactive shikonins from living roots, rhizosphere and bulk soil surrounding roots. These findings suggest important roles for accumulation of shikonins in the root periderm and subsequent rhizodeposition in plant defence, interference, and invasion success.Bioactive plant secondary products are frequently the drivers of complex rhizosphere interactions, including those with other plants, herbivores and microbiota. These chemically diverse molecules typically accumulate in a highly regulated manner in specialized plant tissues and organelles. We studied the production and localization of bioactive naphthoquinones (NQs) in the roots of  Although our knowledge of root structure and function has improved in recent years, the complex activities and interactions of roots in the soil rhizosphere and at the soil-root interface are often poorly described, and this is particularly true for invasive weeds. It has become increasingly evident that both root exudation and rhizodeposition in plants are responsive to biotic and abiotic stressors and are clearly important for the protection of sessile terrestrial plants, particularly in the immediate area surrounding living roots, otherwise known as the rhizosphere .et al. first described the role and mode of action of phytotoxic sorgoleone and related long chain hydroquinones produced by living sorghum root hairs as plant growth inhibitors, and also described localization and release of sorgoleone by living root hairs  in its roots. Analysis of many field-collected roots of E. plantagineum by the authors revealed that the outer layers of root tissue in the primary taproot or smaller fibrous secondary roots are very often pink or red due to the production of a mixture of bioactive, brightly coloured NQs known as shikonins  at low elevations produced significantly (3–5-fold) higher concentrations of NQs than plants collected from similar sites with cooler average temperatures or higher elevations  to perform metabolic profiling of root, root hair and soil extracts. We also utilized SPRE (solid phase root zone extraction) microprobes in the soil rhizosphere to profile PSPs of interest in the rhizosphere and bulk soil surrounding living plant roots to further define the role of PSPs as potential drivers of plant/plant and plant/organismal interactions in the rhizosphere.In order to gain a more fundamental understanding of the ecological role of NQs in Echium plantagineum root tissues were collected from densely populated local field stands in Wagga Wagga, NSW, Australia  in 2014. At least five mature flowering specimens were collected for microscopic evaluation of NQs at various intervals from August to October 2014. On all occasions following collection, plants were placed in wet paper towelling to prevent dehydration and maintained at a temperature of 4 °C until evaluation under a microscope, which was typically performed within 1h of collection. E. plantagineum seed was also collected from the same field location in Wagga Wagga, NSW, in 2013 and used to generate seedlings for time-course experimentation of NQ production under controlled environmental conditions. Seeds were germinated in June 2014 on Whatman No. 1 filter paper moistened with 5ml sterile deionized water in sterile 9-cm plastic Petri dishes containing 20 seeds per dish, with three replicates for each harvest time interval (n=60 seedlings). Harvest times were 12, 24, 48, 72, 96, 128 and 144h after experimental initiation. During incubation, dishes were sealed with parafilm and placed in a lighted incubator at 25/18 °C day/night temperatures with a 12-h photoperiod; microscopic evaluation was performed immediately following each harvest.E. plantagineum fresh root periderm spectra with those generated with an ethanolic solution of pure shikonin  at a concentration of 1mg ml−1. For spectral analyses, images were scanned sequentially with excitation wavelength of 405 and 488nm, with a wavelength interval of 6nm and emission range from 405–550 and 550–740nm, respectively. The two spectral scans were then automatically combined by Nikon confocal NIS ver 4.10 to create the emission spectrum presented. For seedlings generated during the time course experiment, stereoscopic light microscopy using both a Nikon SMZ25 and a Leica M205FA was performed at each time interval with numerous sampled seedlings.Mature roots and seedling root and hypocotyl tissues were hand sectioned for examination using confocal microscopy . In most cases, sections were directly examined without staining, however calcofluor white (1% aqueous solution)  was used on a few occasions for more pronounced staining of the cell wall. The fluorescence from calcofluor white was detected with an excitation of 405nm and emission from 425 to 475nm. The same confocal microscope was used for both hyperspectral analyses and imaging to compare 2 gas for approximately 20min. The resulting dried extract was weighed and resuspended in ethanol, filtered through a 22 μm Millex syringe filter and subjected to UHPLC/Q-ToF MS analysis as described below.Solid phase root zone extraction (SPRE) microprobes (5cm) were prepared as per Greenhouse-grown plants were propagated for 16 weeks in a soil mix containing 6:4 peat potting mix:sand . Seedlings were pre-germinated by imbibing in sterile water for 1 week using field-collected seed as described above in NSW, Australia; after 7 d, seedlings were transplanted into 1.5-l pots and were maintained in the glasshouse at 25/18 °C day/night temperatures at ~55% relative humidity. Plants were watered every other day by subirrigation and fertilized once per fortnight using a commercial liquid fertilizer . At 14 weeks of age, as plants began to flower, plants were sampled for rhizosphere-produced NQs using 5cm microprobes, as described above. Eight probes were placed equidistantly into each of six plant pots and were fully inserted into the soil surrounding the living plant, ~8cm from the centre of the plant rosette. The probes were placed equidistantly around the plant at a distance of ~5cm from the taproot, and were not in contact with the foliage of the plant.In addition, soil media was collected separately from the rhizosphere (soil not adhering to plant roots but located around living plant root system) of each pot, thoroughly filtered to remove any small residual root pieces using a fine wire mesh screen (<1mm mesh holes), air-dried and extracted in 100% ethanol, filtered using a 22 μm Millex filter and subjected to analysis using an ion trap mass spectrometer for NQ detection.E. plantagineum were collected in the field from 21 locations across NSW and Australian Capital Territory, Australia, in 2013 and 2014. At each collection site, GPS coordinates were recorded and five or more intact root specimens were collected from mature, flowering plants. Roots were carefully collected from field sites using a mattock to remove the majority of the root system without excessive damage to the taproot and main secondary roots. Excess soil was removed and roots were placed in moist paper towelling and stored at 4 °C for 24h prior to extraction. Root periderm extracts were prepared by thinly peeling the coloured outer periderm layer only from taproots or primary roots using a sharp scalpel blade. To minimize the impact of plant-to-plant variation of extracts to be subjected to metabolic profiling, composite samples were prepared from 5–6 individual plant roots collected at each of 21 locations using ~0.2g of fresh periderm per plant to generate 1g total fresh weight of periderm tissue. Periderm peels (1g) were then extracted in 10ml of 100% HPLC grade ethanol  for 14h in the dark, at room temperature, after placement on a slow orbital shaker at 120rpm. Following extraction, samples were filtered using a 22 μm Millex syringe filter, and 1ml of each extract was transferred into individual HPLC vials with duplicate samples available for replicated UHPLC/Q-ToF MS profiling  at 25 °C equipped with an SB-C8 guard column   and a flow rate of 0.5ml min−1. The column was equilibrated for 30min prior to analysis. Solvents used for extraction and UHPLC/MS were HPLC grade. Acetonitrile was obtained from Hipersolv , formic acid (>99% purity) from Sigma  and LC-MS water from Merck . Separation of NQs was achieved using a gradient of mobile phase A (water+0.1% formic acid) and mobile phase B (95% acetonitrile+0.1% formic acid), starting with 50% B for 1min and reaching 100% B over 7min, continuing at 100% B until 10.50min, returning to 50% B over 0.1min and held at 50% B from 10.60–17.00min. (−1. Data were collected in negative ion mode using an extended dynamic range (2 GHz). Additional LC/MS-MS experimentation was performed for selected molecules of interest  mass spectrometer with Dual Agilent Jet Stream Electrospray Ionisation Source    column with a gradient of solvent A  and solvent B . The gradient was initiated with 40% B for 1min and reached 98% B over 9min, continued at 100% B until 13.0min and returned to 40% B over 0.1min and held at 40% B from 13.1–18.50min. Optimization of the MRM transitions for purified standards was performed using standards formulated at 5ppm (in 50:50 methanol:water) that were injected into the MS/MS interface using syringe infusion at 10 µl min−1. To determine specific precursor and product ions for each standard, MS/MS was performed, and after optimization shikonins were evaluated in negative ion mode  and using analytical standards (as described in the previous section) and HPLC grade solvents including methanol from Rathburn  and formic acid from Merck . Separation was achieved using Kinetex XB-Cion mode . Data weAbundance of deoxyshikonin, shikonin, acetylshikonin and dimethylacrylshikonin was analysed in 21 samples, representing 21 populations. Analysis of variance was performed on log transformed data in IBM SPSS statistics software . Homogeneity of variances was assessed using Levene’s test prior to further analysis. Tukey HSD was used as a post hoc test to evaluate differences among metabolite levels averaged over populations.E. plantagineum roots, shikonins were clearly identified in the outer one to two cell layers of newly formed periderm in mature and seedling taproots as well as secondary roots  both in their unique colouration and their autofluorescence  in all field periderm extracts, while deoxyshikonin was present in significantly lower abundance (P<0.001) in all samples. Deoxyshikonin is thought to be the precursor of shikonin and is therefore likely rapidly converted to shikonin and also potentially numerous higher molecular weight derivatives, particularly dimethylacrylshikonin  showed greatly enhanced production of shikonins, specifically dimethylacrylshikonin, in contrast to the other 20 populations evaluated. Interestingly, plants in White Cliffs are typically exposed to high UV and summer temperatures and low average rainfall events, as this location borders the Australian outback, a large inland desert. The evaluation of gene expression underlying biosynthesis of dimethylacrylshikonin and other NQs in this population is now underway.Despite variation among samples, we found a consistent pattern of compound abundance among root periderm extracts from mature field-collected plant populations . DimethyE. plantagineum roots or placed in soil of glasshouse-grown potted plants was acetylshikonin, observed in probe extracts at levels of ~0.9ppm and in ethanolic extracts of soil at levels of 2ppm using ethanolic extraction. Our limit of detection (LOD) of acetylshikonin in soil and microprobe samples subjected to potted soils and roots was ~0.3ppm. In all probe and soil extracts collected from potted soil rhizospheres surrounding mature E. plantagineum, shikonin was the next most abundant compound  method that allowed detection of trace levels of selected shikonins in both soil and microprobe extracts. The main constituent found in microprobes brought into contact manually with living compound . Deoxyshcompound . Interescompound , and acecompound  of livinE. plantagineum plants produced in several climatic zones in Spain contained >2-fold lower concentrations of shikonins than plants produced in field conditions across southern Australia  suggest that shikonins play an important role in plant protection against microbial invaders, insect herbivores and germinating plants in the rhizosphere of E. plantagineum. These studies utilized a combination of intricate approaches to study the localization and distribution of bioactive shikonins in the plant and rhizosphere directly surrounding living E. plantagineum. The reported biological activity and observed accumulation of shikonins and other NQ derivatives in the periderm and their subsequent deposition in the rhizosphere through exudation or tissue degradation are all suggestive of their potential importance as bioactive novel weapons of significance. This is likely to be of particular importance in monocultural stands under warm and dry conditions in Australia where shikonin production was noted to be enhanced. Studies are now underway to further evaluate and compare NQ and pyrrolizidine alkaloid production and genetics of E. plantagineum and other related species in Australia and their native range in the Iberian Peninsula.The role of various root-produced naphthoquinones such as shikonins and their derivatives has been little described in the literature of higher plants under natural field settings, particularly those that are classified as invasive weeds of significance. Interestingly, we have found that


Ceratitis capitata (Wiedemann): (1) wild males, (2) irradiated with Co-60 (steriles), and (3) irradiated (steriles) and treated with ginger oil. The canonical variate analysis discriminated two groups , based on the morphological shape of the wings. Among males that emit buzz signals, wild males obtained copulation more frequently than males in Groups 2 and 3. The individuals of Group 3 achieved more matings than those in Group 2. Wild males displayed lower pulse duration, higher intervals between pulses, and higher dominant frequency. Regarding the reproductive success, the morphological differences in the wings' shape between accepted and nonaccepted males are higher in wild males than in the irradiated ones. The present results can be useful in programs using the sterile insect technique for biological control of C. capitata.The sterile insect technique (SIT) is widely utilized in the biological control of fruit flies of the family Tephritidae, particularly against the Mediterranean fruit fly. This study investigated the interaction between mating success and morphometric variation in the wings and the production of acoustic signals among three male groups of  Ceratitis capitata (Wiedemann) is considered one of the most important pests of fruit crops worldwide. Currently, one of the most widely used techniques in controlling C. capitata is the sterile insect technique (SIT), considered a type of autocidal or genetic control, in which the pest is utilized for its own control. Sterile males are released in production areas where they will compete with the wild males to copulate with wild females [True fruit flies (Diptera: Tephritidae) are well known worldwide because they infest economically important fruits. In the family, the Mediterranean fruit fly females . C. capitata, the mating compatibility between the sterile male and the wild female is problematic, because the species displays a lek mating system characterized by active female choice. In male aggregations, at least five males compete for copulations [In tephritids, such asulations , 3. Whenulations , 5 at fuulations . During ulations . Males e C. capitata should be capable of joining or forming leks in the host plant, attracting wild females and copulating with them [If the female is receptive, the male rides it and tries to assure copulation. Females are sometimes receptive to some males' cohort; however, they accept only one sexual partner . On the ith them . C. capitata are more receptive to wild males than to sterile ones or males originated from mass rearing [ C. capitata males to induce wild females to copulate [Participation in leks is the first step that the released males must successfully accomplish in order to have the chance to obtain mating success. These males must exhibit a behavioral repertoire that closely matches that of wild individuals, because wild females of rearing , 10. Thecopulate . Therefocopulate –16. C. capitata detect some characteristics in a male that make it attractive as a sexual partner? This issue was highlighted by several authors who sought an answer to this question by analyzing behavioral and morphometric characters in males [ C. capitata, there is a positive correlation between mating success and symmetry of frontal supraorbital bristles. It is also known that the stress suffered during development due to the high larval density on the process of C. capitata mass production can contribute to increasing morphological asymmetry in structures with bilateral symmetry [ Anastrepha zenildae that males with symmetrical distribution of the bristles on the front plate achieved greater success in mating than asymmetrical males.Can the female ofin males , 17, 18.symmetry . Souza esymmetry  observedGeometric morphometrics is a relatively new technique that is employed for the analysis of the variation in shape , 21. The C. capitata males and those irradiated with gamma radiation and its relationship with the production of acoustic signals in order to define whether possible morphological alterations may influence the acoustic communication and male mating success.By using the technique of geometric morphometrics, this study investigated the shape variation of wings between the groups of wild C. capitata were obtained from infested fruits of guava (Psidium guajava), which were collected in orchards located in the region of Petrolina, São Francisco River Valley, in Northeastern Brazil . After sampling, fruits were taken to the Semi-arid Entomology Laboratory of Embrapa and placed in plastic trays containing vermiculite as substrate and remained there until specimens of C. capitata completed their larval development. After 10–15 days, the vermiculite was sifted to obtain the pupae which were later transferred and kept in acrylic boxes (30 cm cubes) until the emergence of adults. Then, the flies were separated by sex and kept separately in net cages (30 cm cubes), under natural conditions, with temperature ranging between 27–31°C and relative humidity between 10–80%, with water and an ad libitum diet  until they reached sexual maturity, which occurred between 8–10 days after adult emergence.Adult wild individuals of tsl-Vienna 8, were supplied by Biofábrica Moscamed Brasil located in Juazeiro, Bahia state, Northeastern Brazil. Males were irradiated during the pupal stage, with a dose of 95 Gy of gamma radiation (Co-60), two days before the emergence of adults. The irradiated pupae were then divided into two net cages (30 cm cubes), each containing 40 mL of pupae. Both cages were provided with artificial diet (3 sugar : 1 hydrolyzed protein) and water ad libitum and kept on a controlled room . In the 4th to 5th day after emergence, the flies from one of the cages were exposed to 1.5 mL of ginger root oil of Zingiber officinale Roscoe (Zingiberaceae) (GRO) in a small room (27 m3) for 20 hours, with 25–27°C, 50–60% RH, and 12 h photoperiod (6:30–18:30 h), where the GRO aroma was dispersed by air fans [Sterile males, mutant strainair fans . The cagOne day before the beginning of sexual compatibility tests, wild males and females (8–15 days old) and the GRO-treated and nontreated sterile males (5–7 days old) were marked on the thorax with a small drop of nontoxic and water-soluble white ink (Faber Castell), by alternating the strain that was painted, in order to distinguish between them. To this end, ten individuals at a time were placed inside tulle bags, as they were immobilized between two fingers, one by one, in order to make the painting easier. Ficus spp. plant inside, measuring 1.80 m (height), with enough foliage to house groups of calling males. In each cage, ten wild males and ten sterile males were released at 7:30 a.m. Wild males and sterile males were released into two cages and wild and sterile GRO-treated males were released into two other cages. After males formed leks and initiated calling behavior, 10 virgin wild females were released into each cage. The mating couples were collected from 7:45 a.m. until 12:30 p.m. and kept separately in transparent acrylic tubes until the end of copulation. Then, males, whether they are successful or not in mating, were individualized in Eppendorf tubes (2 mL), forming six groups: accepted and not accepted wild males for copulation, accepted and not accepted sterile males, and accepted and not accepted sterile males treated with GRO.Mating tests were conducted in four field cages measuring 2 × 2 × 2 m, located in the shade under tree canopies. Each field cage contained aD2), and Canonical Variables Analysis (CVA) were performed in order to detect variables capable of distinguishing individuals, and from this analysis, MANOVA, comparative deformation grids were obtained from the comparison of CV1. Warped outlines were generated from the landmark coordinates to more clearly demonstrate the vector variations of deformation grids related to the analysis of each group of C. capitata which consisted of wild males (Group 1), irradiated sterile males (Group 2), and irradiated sterile males aromatically treated with ginger oil (GRO) (Group 3), with and without successful copulation.In order to perform the analysis by geometric morphometrics, 50 males free of injury, from each category of males selected in the experiment described above, had their left wing gently removed and then mounted between a slide and a coverslip with the use of cedar oil. The scanned images of these structures were obtained from a digital camera Motic 5.0 attached to a stereomicroscope. The TpUtil software was used to randomize the images and subsequently Tps Dig  was usedAn experimental arena was constructed with a cylindrical plastic cage (18 cm diameter × 5 cm height). The microphone used for recording was inserted through a small hole (3.5 cm diameter) on the top of the cage.Males  and females with 10–20 days old were introduced into the arena. In general, the experiment was initiated with 3–5 males and 1 female, and more insects were introduced to a maximum of 10 males and 6 females. If the insects were nonresponsive after 10 minutes of observation, they were replaced by new ones.Experiments were conducted at Embrapa Genetic Resources and Biotechnology  in a sound-insulated room on a shock-proof table in order to decrease environmental noise. Observations and recordings were performed between 09:00 and 17:30 hrs. The insects were observed and their behavior was recorded until the first copulation or for 30 minutes if no copulations were registered. Songs were characterized and named according to the behavioral context in which they were emitted and to the previous nomenclature –28: contThe acoustical signals were recorded with a professional microphone  measuring 3.3 × 22 cm (diameter × height) and response range of 50 Hz to 20 KHz. The signals captured by the microphone were amplified by a professional amplifier Hot Sound SPA4300 , digitized by USB audio-captures UA-25EX (Edirol-Roland-24 bits-96 KHZ), and stored to the computer using Cool EditPro  at sample rate of 46 bits and 44.100. Further analyses were done using Sound Forge 4.5 (Sonic Foundry).n = 5–10 for each treatment). A pulse was defined as a homogeneous unitary parcel of vibration of finite duration and pulse trains as repeatable and temporally distinct groups of pulses and song as a sequence of pulses and/or pulse trains with distinct beginning and end [Signals were characterized by calculating their temporal and spectral parameters using 30 to 50 pulses of each individual . All analyses were performed using the statistical program R 3.0.1 [In order to compare the proportion of tested insects in each group  that emit buzz signals and the proportion of insects that successfully copulated (in relation to those that emit buzz),  R 3.0.1 . C. capitata revealed significant morphometric divergences (P < 0.01) when compared with the pattern for sterile males (P < 0.01). Individuals with and without successful copulation were discriminated by the medial shape of their wings in relation to CV1 axis, which explains 70.04% of the observed variation, as well as in relation to CV2, which explains 15.79% , which was greater among those who succeeded in copulation. Similarly, when comparing the wing shape of wild males to that of sterile males, there was a difference related to the width and the length of the wings; that is, the sterile males had elongated and narrow wings relative to the wild ones.Individuals with and without successful copulation were discriminated by the medial shape of their wings in relation to CV2 . Morpholχ2 = 1.3; P = 0.52) . A higher proportion of wild males that emit buzz signals achieved copulation  . When thpulation  relativepulation . Buzz sipulation .In tephritids, wings play an important role in cohort behavior. In lek, for example, males produce, release, and disperse pheromone through the vibration of wings to attract virgin females . In addiThe success of sterile insect release programs is directly related to the success of copulation between sterile males and wild females. Changes in certain morphological characters have been frequently used to detect environmental stress in insects. The mass production of sterile insects, for many generations, can cause different types of deformations  that areP < 0.01). The variation detected in the wing shape of wild males that succeeded or not in copula (MANOVA) was enough to distinguish the two groups , does not affect the copulation index with wild females compared to nonirradiated males from laboratory [ C. capitata per se may affect individuals presenting malformations/morphological differentiation or even behavioral changes, making them less sexually competitive than wild males when released in the field. This can be mainly influenced by factors such as genetic drift and artificial selection in the environment where they are bred [ C. capitata to aromatization with ginger oil, which contains the attractive compound α-copaene, has increased the reproductive success of these males compared to nonirradiated sterile males, a fact that had been previously demonstrated by other authors [Changes in wing shape resulting from artificial selection under mass-rearing may affect the sexual success of the sterile males, since the results presented here show that temporal and spectral parameters of the beating of wings feature different patterns between wild and sterile males. Studies have shown that sterilization ofboratory . The proare bred . On the  authors , 33–35.  C. capitata from sterile males. Thus, the improvement and development of new procedures and handling for colonization and management of creations are required to minimize genetic, physiological, morphological, and behavioral wear caused by the process of sterile males' mass production. The patterns of wings obtained in this study of wild males accepted for copulation could be used in the selection of parental males of mutant strain tsl Vienna 8, so they were used in industrial production in biofactories for being released in the field, thus obtaining greater success in programs using the sterile insect technique for C. capitata.Additionally, the results suggest that the first canonical variable scores (CV1) can be employed to distinguish wild males of tsl Vienna 8, with and without treatment with the aromatic ginger oil. Data suggest the usefulness of CV1 scores on morphological distinction of wild and sterile males of C. capitata based on the wings. The noninterference of the irradiation process as well as the aromatic treatment with GRO on the emission of buzz signals by males is clear. On the other hand, successful copulation could preferably be related to the duration and pulse interval and to the dominant frequency of the signals produced by the wings, which are probably associated to morphological changes of the wings.The conjunction of the analyzed factors allowed a better understanding of the factors influencing the reproductive success of sterile males,