State University of Norte Fluminense

In 2008 we published the fi rst set of guidelines for standardiz- ing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the fi eld. Our knowledge base and relevant new tech- nologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in differ- ent organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between meas urements that monitor the num- bers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure fl ux through the autophagy pathway (i.e., the com- plete process including the amoun t and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accu mulation must be differentiated from stimuli that increas e autophagic activity, de fi ned as increased autophagy induction coupled with in creased delivery to, and degra- dation within, lysosomes (in most higher eukaryotes and some pro- tists such as Dictyostelium ) or the vacuole (in plants and fungi). In otherwords,itisespeciallyimportantthatinvestigatorsnewtothe fi eld understand that the appearance of more autophagosomes does not necessarily equate with mo re autophagy. In fact, in many cases, autophagosomes accumu late because of a block in traf fi cking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may re fl ect a reductionindegradativeactivity .Itisworthemphasizingherethat lysosomal digestion is a stage of au tophagy and evaluating its com- petence is a crucial part of the evaluation of autophagic fl ux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appro- priate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no indi- vidual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation, it is imperative to target by gene knockout or RNA interference more than one autophagy- related protein. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways implying that not all Atg proteins can be used as a speci fi c marker for an autophagic process. In these guidelines, we con- sider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular assays, we hope to encourage technical innovation in the fi eld

Introduction Recent decades have seen a major international effort to inventory tree communities in the Amazon Basin and Guiana Shield (Amazonia), but the vast extent and record diversity of these forests have hampered an understanding of basinwide patterns. To overcome this obstacle, we compiled and standardized species-level data on more than half a million trees in 1170 plots sampling all major lowland forest types to explore patterns of commonness, rarity, and richness. Methods The ~6-million-km 2 Amazonian lowlands were divided into 1° cells, and mean tree density was estimated for each cell by using a loess regression model that included no environmental data but had its basis exclusively in the geographic location of tree plots. A similar model, allied with a bootstrapping exercise to quantify sampling error, was used to generate estimated Amazon-wide abundances of the 4962 valid species in the data set. We estimated the total number of tree species in the Amazon by fitting the mean rank-abundance data to Fisher’s log-series distribution. Results Our analyses suggest that lowland Amazonia harbors 3.9 × 10 11 trees and ~16,000 tree species. We found 227 “hyperdominant” species (1.4% of the total) to be so common that together they account for half of all trees in Amazonia, whereas the rarest 11,000 species account for just 0.12% of trees. Most hyperdominants are habitat specialists that have large geographic ranges but are only dominant in one or two regions of the basin, and a median of 41% of trees in individual plots belong to hyperdominants. A disproportionate number of hyperdominants are palms, Myristicaceae, and Lecythidaceae. Discussion The finding that Amazonia is dominated by just 227 tree species implies that most biogeochemical cycling in the world’s largest tropical forest is performed by a tiny sliver of its diversity. The causes underlying hyperdominance in these species remain unknown. Both competitive superiority and widespread pre-1492 cultivation by humans are compelling hypotheses that deserve testing. Although the data suggest that spatial models can effectively forecast tree community composition and structure of unstudied sites in Amazonia, incorporating environmental data may yield substantial improvements. An appreciation of how thoroughly common species dominate the basin has the potential to simplify research in Amazonian biogeochemistry, ecology, and vegetation mapping. Such advances are urgently needed in light of the >10,000 rare, poorly known, and potentially threatened tree species in the Amazon.

Earthworms (Eisenia foetida) produce humic substances that can influence plant growth by mechanisms that are not yet clear. In this work, we investigated the effects of humic acids (HAs) isolated from cattle manure earthworm compost on the earliest stages of lateral root development and on the plasma membrane H(+)-ATPase activity. These HAs enhance the root growth of maize (Zea mays) seedlings in conjunction with a marked proliferation of sites of lateral root emergence. They also stimulate the plasma membrane H(+)-ATPase activity, apparently associated with an ability to promote expression of this enzyme. In addition, structural analysis reveals the presence of exchangeable auxin groups in the macrostructure of the earthworm compost HA. These results may shed light on the hormonal activity that has been postulated for these humic substances.

Humic substances (HS) have been widely recognized as a plant growth promoter mainly by changes on root architecture and growth dynamics, which result in increased root size, branching and/or greater density of root hair with larger surface area. Stimulation of the H + -ATPase activity in cell membrane suggests that modifications brought about by HS are not only restricted to root structure, but are also extended to the major biochemical pathways since the driving force for most nutrient uptake is the electrochemical gradient across the plasma membrane. Changes on root exudation profile, as well as primary and secondary metabolism were also observed, though strongly dependent on environment conditions, type of plant and its ontogeny. Proteomics and genomic approaches with diverse plant species subjected to HS treatment had often shown controversial patterns of protein and gene expression. This is a clear indication that HS effects of plants are complex and involve non-linear, cross-interrelated and dynamic processes that need be treated with an interdisciplinary view. Being the humic associations recalcitrant to microbiological attack, their use as vehicle to introduce beneficial selected microorganisms to crops has been proposed. This represents a perspective for a sort of new biofertilizer designed for a sustainable agriculture, whereby plants treated with HS become more susceptible to interact with bioinoculants, while HS may concomitantly modify the structure/activity of the microbial community in the rhizosphere compartment. An enhanced knowledge of the effects on plants physiology and biochemistry and interaction with rhizosphere and endophytic microbes should lead to achieve increased crop productivity through a better use of HS inputs in Agriculture.

In Campos dos Goytacazes, northern Rio de Janeiro state, Brazil, reports of uveitis consistent with toxoplasmosis led to a survey of the prevalence and risk factors for Toxoplasma gondii infection in 1997-1999. The survey population was selected randomly from schools, randomly chosen communities, and an army battalion. Serum samples from 1,436 persons were tested. With results adjusted for age, 84% of the population in the lower socioeconomic group was seropositive, compared with 62% and 23% of the middle and upper socioeconomic groups, respectively (p<0.001). When multivariate analysis was performed, drinking unfiltered water was found to increase the risk of seropositivity for the lower socioeconomic (odds ratio [OR]: 3.0, 95% confidence interval [CI] 1.3 to 6.9) and middle socioeconomic (OR: 1.7, 95% CI 1.2 to 2.3) populations. We also found a high T. gondii seroprevalence in this Brazilian community. Drinking unfiltered water increased the risk of T. gondii seropositivity, indicating the potential importance of oocyst transmission in water in this region.

Ecological intensification, or the improvement of crop yield through enhancement of biodiversity, may be a sustainable pathway toward greater food supplies. Such sustainable increases may be especially important for the 2 billion people reliant on small farms, many of which are undernourished, yet we know little about the efficacy of this approach. Using a coordinated protocol across regions and crops, we quantify to what degree enhancing pollinator density and richness can improve yields on 344 fields from 33 pollinator-dependent crop systems in small and large farms from Africa, Asia, and Latin America. For fields less than 2 hectares, we found that yield gaps could be closed by a median of 24% through higher flower-visitor density. For larger fields, such benefits only occurred at high flower-visitor richness. Worldwide, our study demonstrates that ecological intensification can create synchronous biodiversity and yield outcomes.

Abstract The impacts of the SAMARCO iron tailing spill along more than 650 km, between the dam and the plume of the Doce River in the Atlantic, were assessed by the determination of toxic metals. The tailing spill caused a substantial increase in suspended sediment loads (up to 33,000 mg L −1 ), in addition to large depositions of waste along the Doce basin. The highest estimated transport of dissolved metals was observed for Fe (58.8 μg s −1 ), Ba (37.9 μg s −1 ) and Al (25.0 μg s −1 ). Sediments reached the highest enrichment factors (EFs) for Hg (4,234), Co (133), Fe (43), and Ni (16), whereas As (55), Ba (64), Cr (16), Cu (17), Mn (41), Pb (38) and Zn (82) highest EFs were observed for suspended particulate matter (SPM). Iron, As, Hg, Mn exceeded sediment quality guidelines. Therefore, the risk of occurrence of adverse effects is highly possible, not only due to the dam failure, but also due to the Fe mining and the artisan Au mining. Heavy rain episodes will likely cause enhanced erosion, remobilization, and transport of contaminated particles, sustaining high inputs of SPM and metals for the years to come and threatening the ecosystem services.

The methods of geometric morphometrics, which combine an explicitly geometric definition of shape with the flexible tools of multivariate statistics, have become established in the past decade as the “morphometric synthesis” (Bookstein, 1996, 1998). Considerable effort is now directed at applying these methods in diverse biological contexts. Therefore, the morphometric methods need to be linked to the experimental protocols and statistical frameworks that underlie diverse applications such as phylogenetic comparative analyses (Rohlf, 2001, 2002) or quantitative genetics (Klingenberg and Leamy, 2001; Monteiro et al., 2002). Morphometric analyses provide tools for answering specific questions that depend on the context of each particular study. Statistical tests and predictions of effects usually can make use of the existing theory for the application, whereas the interpretation and graphical display of effects are specific to geometric morphometrics. Precisely how the morphometric methods should be adapted to different biological contexts has provoked vigorous debate. An issue that has attracted particular attention is how to implement linear statistical models in geometric morphometrics. Should the inferred effects of experimental or observational factors on shape be considered exclusively in their absolute magnitude, using the Procrustes metric that is at the core of geometric morphometrics (e.g., Bookstein, 1996)? Or alternatively, is it legitimate, depending on context, to consider the effects considered relative to the observed variation as in standard multivariate analysis of variance and related statistical procedures? The controversy has focused on several specific issues that are aspects of this question. Most recently, the debate has been conducted on the Morphmet E-mail list (February 2004; archive available at http://www.mail-archive.com/morphmet@wfubmc.edu/) as well as in a published exchange on quantitative genetics of shape (Klingenberg, 2003b; Monteiro et al., 2003). One position is that some of the standard analyses of multivariate statistics are inherently incompatible with morphometrics. For example, Bookstein (1991: 114) stated “Discriminant-function analysis is not a morphometric technique …,” and added “Neither is the extension to multiple groups, canonical-variates analysis.” This position is controversial because canonical variate analysis is one of the most frequently used techniques in morphometrics (e.g., Rohlf et al., 1996; Duarte et al., 2000; Corti and Rohlf, 2001; Douglas et al., 2001; Rüber and Adams, 2001; Dobigny et al., 2002; Cardini, 2003; Debat et al., 2003; Harvati, 2003; Klingenberg et al., 2003a). Here we provide a general discussion of the problem, and we apply these considerations in two different examples of biological applications. In the first example, we extend and clarify an earlier debate about the application of geometric morphometrics to quantitative genetics (Klingenberg and Leamy, 2001; Monteiro et al., 2002; Klingenberg, 2003b; Monteiro et al., 2003). In the second example, we use the logic of transformed shape spaces in a different context to define a new measure of individual asymmetry of shape as an alternative to those suggested by Klingenberg and McIntyre (1998) and Palmer and Strobeck (2003). Shape is defined mathematically as all the geometric information about an object except for its size, position, and orientation or, in other words, all those properties that are invariant to scaling, translation, and rotation (e.g., Bookstein, 1991: 126, 180; Dryden and Mardia, 1998: 1). This definition is used throughout geometric morphometrics and is applicable both to configurations of landmarks and to outline contours of objects. Because multiple geometric features are required to characterize the shape even of very simple figures, shape is an inherently multidimensional phenotype. The shapes of landmark configurations can be represented as points in a shape tangent space (e.g., Dryden and Mardia, 1998; Rohlf, 1999), within which the methods of multivariate statistics can be used. Differences between pairs of shapes or deviations of individual shapes from the population average can be characterized by their magnitude, measured as a Procrustes distance, and their direction in the tangent space. As long as the complete information about shape is entered in the analysis, it does not matter whether the shape variables used are the complete set of partial warp scores and uniform components or the coordinates of Procrustes-aligned landmarks projected onto the tangent space. Both these sets of variables describe the same variation, and one can be transformed into the other by a rotation of the coordinate system (e.g., Rohlf, 1999: 214). A key characteristic of geometric morphometrics is that points in the shape tangent space can be related back to actual shapes in the original plane or three-dimensional space of the landmark coordinates. Likewise, vectors in the shape tangent space correspond to shape changes. This relationship between the shape tangent space and the physical space in which the configurations were digitized makes it possible to visualize directly the results of statistical analyses. Maintaining these relationships imposes some restrictions on the mathematical operations that can be used in morphometrics, because some transformations destroy the special properties of shape tangent space and the direct link to the original landmark configurations. A substantial body of mathematical theory has been developed for the special case when shape variation is isotropic, that is, when each landmark is equally variable, the variation at each landmark is the same in all directions, and variation is independent among landmarks (e.g., Goodall, 1991; Dryden and Mardia, 1998). This means that the scatter of landmark positions around the sample average is circular, with the same spread at each landmark. Although this model is convenient because it greatly simplifies the statistical analysis of shape variation, isotropic variation is rarely found in biological data sets. To the contrary, landmarks usually differ in their amounts of variation, they show a clear directionality of variation, and usually there exist associations between the landmarks. These deviations from the isotropic condition are often the very subject of interest, for instance, in studies of morphological integration (Klingenberg and McIntyre, 1998; Badyaev and Foresman, 2000; Debat et al., 2000; Klingenberg and Zaklan, 2000; Klingenberg et al., 2001a, 2003b, 2004; Bookstein et al., 2003). Accordingly, it is important to take into account the nonisotropic nature of shape variation. To adjust for this, many of the standard methods of multivariate statistics use transformations of the data space. The Procrustes approach has been criticized because it can produce estimates that are statistically inconsistent if variation is not isotropic (Lele, 1993; a critique repeated by Lele and Richtsmeier, 2001: 94). Kent and Mardia (1997) confirmed that statistical inconsistency can occur if the ratio of signal to noise goes toward if the is relative to the of the landmark positions in the that this does not apply if the data are around the shape and Mardia, as is the case in the of biological in studies at the or in among related Dryden and Mardia that for amounts of shape variation, the Procrustes methods and such as by Lele and Richtsmeier, produce shape coordinates that are linear transformations of each a of those spaces as we in this the different produce the same statistical and differ in the of Dryden and Mardia a of that if the Procrustes between the average shape and all the are about all methods very This is a of shape variation is usually found in morphometric data sets. a data set of in a space which be the shape tangent space for a of that the points to the means an these are at from each the between the in are not equally from each other because the variation within is not and for which the is with the of variation, are In is from one of the as the are to the of variation. This that the of not on the between on the direction of the relative to the directionality of variation within In if the variation within is isotropic, the of on the between means and of with nonisotropic variation. the between all means are means an the between and is clear between one of these and of with isotropic variation. are equally from each The of A with the space transformed that variation The are transformed from to The means an variation is the of between can be by the multidimensional space by the of the variation, which is to the multivariate space by and that variation within this transformed space for the in and that the has a on the between The in the transformed space measure the between relative to the variation, and are in multivariate statistics as the (e.g., Mardia et al., These directly the of between groups, and the direction of variation within not be considered in this space because it is isotropic by This is a for and for canonical variate analysis (e.g., and et al., The between two is as is the between the two means and is the of the The correspond to the pairs of means in and scores are by of the data points onto those canonical variate analysis is on a of the by with by a analysis of the and The canonical are those variables that account for the of relative to the variation. to the by the canonical or space is different from the space of the original variables to the that from to an The is if the of the variables greatly differ from each other or if the variables are with each the variation of shape is isotropic is to an the of the by some of and of the multivariate space and its that it does not the of a shape tangent space. Shape tangent spaces have special properties that link their to that of the plane or three-dimensional space in which the landmark has been digitized and in which shapes can be (Rohlf, that are in the original correspond to in shape tangent space. all the within each space are Therefore, two landmark in space that are in magnitude, which differ in their direction or occur at different produce of in shape tangent space amounts of shape These two properties are the for using Procrustes as a metric for shape and they that each in shape tangent space can be directly as a and by the with the can destroy these The apply a different for each landmark and even for different of variation for a landmark. that were in the original correspond to in the transformed shape tangent space. an of this in a of the of variation as it is found in many morphometric data shape variation in the is not isotropic, because the are equally in variation at different landmarks is which be in this A of and coordinate is at each and the have and for all landmarks The by has different effects on different as can be from the to the coordinate at the which differ in their and Because there is of the coordinates in the transformed we to with the landmark of the shape is even it the transformed coordinates are in of the of shape the with the original landmark in is The Procrustes metric and other features of shape tangent space not apply other by the there is direct or relationship from the transformed shape tangent space back to the original Shape variation in the and the by The of landmark variation in the The the of landmarks around the Procrustes average have all been by the same for that the landmarks differ in the and direction of variation. The and are at each landmark. The effects of the by the on the coordinate for the different landmarks. The coordinate from A are the the and of for some of the landmarks. are by the same they have been on the of the landmarks in the original the transformed there is for the and as there is in the The landmark and the transformed are not even in the same that this is to the of the transformed not as a suggested of this an important in applications of morphometric methods to a of different biological In this we on two quantitative the analysis of on and the of asymmetry in A context in which this of a is quantitative a for the and of shape 2002; et al., 2002). most quantitative studies of shape are analyses of shape variables from landmark or outline which use quantitative (e.g., and 1998; et al., 2000; et al., or for quantitative et al., 1996; et al., 2000; et al., 2003a). These analyses are a to a of shape in and they consider how different shape features be related to each In studies of the quantitative genetics of shape as a shape an explicitly multivariate methods have been for analyses (Klingenberg et al., 2004; et al., 2002) as well as for quantitative (Klingenberg and Leamy, 2001; Monteiro et al., 2002; et al., the applications have some debate (Klingenberg, 2003b; Monteiro et al., 2003). Klingenberg and the methods of geometric morphometrics with the standard multivariate theory of quantitative genetics (e.g., and 1998). In Monteiro et used Procrustes to define a new measure of for which is the ratio of the of shape variation to the of shape variation. Klingenberg of this it explicitly to the multivariate theory of quantitative and some of the In their Monteiro et questions about the sets of variables used to characterize and suggested that the direction and of shape variation can be as this and components of variation can be using the standard statistical tools of quantitative genetics and for the (Klingenberg and Leamy, or other of analyses of variation within and between of related et al., 2002; et al., 2003). These analyses estimates of the and the which are used in the analyses. For analyses of the and characterize the and shape variation, and directionality in all the of the shape tangent space. The and can be used to the complete both and with the multivariate of the In this is the to the in shape between the and and is the the between the shape in the and the shape of the to produce the the between shape and Both and are vectors in the shape tangent and as long as the complete shape information is in the and the analyses not restrictions on the a simple of this of analysis for two shape tangent space for a of that the direction of and not be the same and that the of on the specific used of in different in two (e.g., the tangent space for shape variation in a of the and directionality of variation in the and the used in this with two different In each is the and is the to of the of the as a of the direction of the of the in the direction of the The the of the by the multivariate which both the and direction of variation The the using the and the shape to Monteiro et The and the points to the examples in and that the coordinate system in the of is not the same as in A to which the shape tangent space. The geometric that the multivariate theory of quantitative genetics is to the transformations for The multivariate can be as of two in which the original is into the to In the first the is by the of the which it into the The by a new space in which the variation is isotropic, the effects of and the the direct effects of on the The is a of partial of relative on the variables and and The second this one using the the effects of to the in the In a the by is the of the by as it the of effects from the space of partial effects back to the original shape tangent it the because this is from the to the In the of both these as of the multivariate both the and a and they can produce results that at first (e.g., A key by Monteiro et is that the and direction of shape are questions that can be Accordingly, it should be possible to use the with the shape to the of the from the of the et al., or using the the the of the The shape of Monteiro et can be as the variance of by the variance of of the of by the of the of This approach from the one in the in that it does not consider the direction of the and the directionality of variation in and The shape et al., 2002) can be as the ratio of the of the and this and the the of the be the of the of the direction of et al., The with the multivariate are for two both of the same with different Both the and the of the to differ between the two repeated this for in all possible directions, and we the of the the direction of the as Because the same of is for the shape the is a In the for the of the by the multivariate is a shape a which that the of the to the direction of the As can be the two estimates of the of differ This that the on shape need not even provide a for the average of the with the multivariate For most of the the on shape the of the the direction of is for the of the This that and direction are not are and should be considered in quantitative studies of that attention should be to the in the shape of Monteiro et and that it should be used if variation is isotropic or if the and are (Klingenberg, to that a between the and as it can be by a et al., Klingenberg and McIntyre, does not the and are the be a multiple of the and all This is a to whether the and are for the use of the shape The two alternative of linear and differ in a of that are of the of by The is the of between and the shape or the of means in the and (e.g., and and The to the shape that can be by a partial analysis and of the between shape and the is a of the of it does not between direct and and does not provide information about the of the of on shape is of interest, the be the because it is the of the multiple of on For each shape variable, this the of all other aspects of shape on the direct of each on and for In the context of geometric morphometrics, these at a because they the for direct The is a shape the is not is in of the of The can be directly as the in shape and and or as the of shape with relative which is and are vectors in shape tangent space and can be directly with the graphical tools in geometric morphometrics (Klingenberg and Leamy, The of to the by the its that it is a in shape tangent space. the of interpretation apply to the with In should not be as shape the coordinate in and as this at first the for the shape features with should the for example, for canonical variate analysis et al., The of a multivariate of the shape variables or partial warp scores and uniform on the canonical variate which the shapes of with and canonical variate In the context of the of this approach is to visualize the shapes of with or The is to a for each individual by its shape variables by the The multivariate of shape on this a of that can be This is directly to the with a to the of the variance of the the multivariate has the from to An with a shape that is a of can be from a partial analysis of shape and and To it the in geometric morphometric studies of on shape is to display and not The about transformed spaces are applicable not in quantitative Here we use in a biological context to a shape that the of shape relative to the variation in a An application of this shape is which is to from in and and Klingenberg, 2003a). Because it is of an from have which is often of biological The of the is by the which in to factors such as or the and and Klingenberg and To these the of asymmetry is a measure of individual whereas the direction of individual asymmetry is not of For studies of asymmetry and other the direction of the effects in particular is of biological interest, we a measure that can provide a measure of the relative of shape into account that variation not be This measure is on the of standard and which is to the of the et al., that the shape be from such a measure and that as a shape is The of this a of the original space to a space in which the variation is isotropic The are as in the transformed space and can be to the of the original deviations The the of the both in of and in it the of each individual relative to the of sample variation in that Because the variation is the same in the information about can be and the can be directly A measure for shape for in the of nonisotropic variation. of shape deviations around their individual from the are as by the of the the variation around the is isotropic and the scatter of data points is transformed from an into a the the and of the deviations have each one has its relative to the variation in its particular The from the as a relative measure of shape Because variation is isotropic the relative can be directly among shape with different The in the transformed space can be used as a relative measure of shape are several methods for the of the shape deviations from the In we can with an that the for example, the shape that all the means are is the sample and is the of variables in the can be as the the The shape of each to the are found by the of the on the of the is the of An of these can be with standard statistics using the a analysis the of the for the of landmark and the scores for each the scores for each to variance the of these scores for and the of the for each the analysis is on partial scores and the uniform components of shape variation, all the variables should be in this if the coordinates of landmarks or Procrustes are the or three-dimensional components should be from to because they have to The can be used with data that are the of shape (Klingenberg and McIntyre, 1998; et al., Klingenberg et al., 2002). the components of individual asymmetry vectors from the in most statistics a for which is for shape data et al., Klingenberg et al., 1998). The asymmetry measure into account that asymmetry is usually nonisotropic (Klingenberg and McIntyre, 1998; Debat et al., 2000; Klingenberg and Zaklan, 2000; Klingenberg et al., and it is the individual asymmetry on Procrustes et al., Klingenberg and McIntyre, 1998). because this measure is and is to statistical it is the measure of asymmetry used by Klingenberg et this measure of shape with an of asymmetry in of from the and from a for the The data set the coordinates of landmarks on each The landmark configurations for the were and the shape information for all by Procrustes were as the coordinate of the and of each analyses suggested that relative to The variation among the of the of the asymmetry that the variation is not isotropic of the variation is in the first and the to The are because of the of in the Procrustes for variation in size, position, and For we show the for a data set with isotropic variation and the same sample in This a even of the variation the of the measure of relative shape to asymmetry of of the for asymmetry in to a data set from an isotropic of the scores for the first two components for with and of the asymmetry were using the measure of relative shape The of the first two scores of the in the scatter between the and that the clear of the data is not very from this which is a that a scatter of scores does not isotropic variation. To the of asymmetry between the two we a for the asymmetry of shape for each using the shape The of the asymmetry scores show a between the two with the scores for asymmetry in the in the The of the means of is statistically in a with This that this measure of asymmetry can in the amounts of This be in contexts such as studies of quantitative effects are often This measure of shape has been in a of the of asymmetry in et al., The use of this measure for individual asymmetry is possible by the of This is different from that in most other applications of geometric morphometrics. For instance, in quantitative genetics of the directionality does matter because to an from the population average by some in a particular This directionality is by the same at a and this directionality is for the of among and 1996; and 1998). Likewise, in phylogenetic comparative the deviations are from to and the directionality of the from the to be among related 2002). In both these it is this directionality that related or to each other in their morphological features and not in the of from the average and This has the of nonisotropic variation into account in geometric morphometrics. For instance, in phylogenetic comparative studies of shape (Rohlf, the nature of shape variation is often the very of morphometric studies that the shape features the most or the to characterize or and Likewise, in studies of morphological the of is the among landmarks (e.g., Badyaev and Foresman, 2000; Klingenberg and Zaklan, Here we have the of quantitative analyses of the direction of have an on both the and direction of the the and are (Klingenberg, and direction and of be as Because of nonisotropic variation the from the to the the of the multivariate space that it has the properties of a shape tangent space. As a the is not a shape and be of the the shapes to or should be used to visualize have a application, for asymmetry of the direction of shape deviations is not of biological This is because the that asymmetry are of The of the direction is that a measure of the of shape asymmetry can be which can be with methods and simplifies the analyses. Both these applications in very different the same of transformations of shape spaces and the relationships between Procrustes and This is by means to these specific should be equally applicable to a of morphometric analyses in different biological contexts. Leamy, and Rohlf for that to the of this and we are to Rohlf, and for on a of the The of is by and from a and and the of is by the and the the and the

that were deforested. Forest degradation is a separate and increasing form of forest disturbance, and the area affected is now greater than that due to deforestation.

Large rivers create major gaps in reef distribution along tropical shelves. The Amazon River represents 20% of the global riverine discharge to the ocean, generating up to a 1.3 × 10(6)-km(2) plume, and extensive muddy bottoms in the equatorial margin of South America. As a result, a wide area of the tropical North Atlantic is heavily affected in terms of salinity, pH, light penetration, and sedimentation. Such unfavorable conditions were thought to imprint a major gap in Western Atlantic reefs. We present an extensive carbonate system off the Amazon mouth, underneath the river plume. Significant carbonate sedimentation occurred during lowstand sea level, and still occurs in the outer shelf, resulting in complex hard-bottom topography. A permanent near-bottom wedge of ocean water, together with the seasonal nature of the plume's eastward retroflection, conditions the existence of this extensive (~9500 km(2)) hard-bottom mosaic. The Amazon reefs transition from accretive to erosional structures and encompass extensive rhodolith beds. Carbonate structures function as a connectivity corridor for wide depth-ranging reef-associated species, being heavily colonized by large sponges and other structure-forming filter feeders that dwell under low light and high levels of particulates. The oxycline between the plume and subplume is associated with chemoautotrophic and anaerobic microbial metabolisms. The system described here provides several insights about the responses of tropical reefs to suboptimal and marginal reef-building conditions, which are accelerating worldwide due to global changes.

A beta-glucoronidase (GUS)-marked strain of Herbaspirillum seropedicae Z67 was inoculated onto rice seedling cvs. IR42 and IR72. Internal populations peaked at over 10(6) log CFU per gram of fresh weight by 5 to 7 days after inoculation (DAI) but declined to 10(3) to 10(4) log CFU per gram of fresh weight by 28 DAI. GUS staining was most intense on coleoptiles, lateral roots, and at the junctions of some of the main and lateral roots. Bacteria entered the roots via cracks at the points of lateral root emergence, with cv. IR72 appearing to be more aggressively infected than cv. IR42. H. seropedicae subsequently colonized the root intercellular spaces, aerenchyma, and cortical cells, with a few penetrating the stele to enter the vascular tissue. Xylem vessels in leaves and stems were extensively colonized at 2 DAI but, in later harvests (7 and 13 DAI), a host defense reaction was often observed. Dense colonies of H. seropedicae with some bacteria expressing nitrogenase Fe-protein were seen within leaf and stem epidermal cells, intercellular spaces, and substomatal cavities up until 28 DAI. Epiphytic bacteria were also seen. Both varieties showed nitrogenase activity but only with added C, and the dry weights of the inoculated plants were significantly increased. Only cv. IR42 showed a significant (approximately 30%) increase in N content above that of the uninoculated controls, and it also incorporated a significant amount of 15N2.

(Madagascar periwinkle) in small quantities, and heterologous reconstitution of vinblastine biosynthesis could provide an additional source of this drug. However, the chemistry underlying vinblastine synthesis makes identification of the biosynthetic genes challenging. Here we identify the two missing enzymes necessary for vinblastine biosynthesis in this plant: an oxidase and a reductase that isomerize stemmadenine acetate into dihydroprecondylocarpine acetate, which is then deacetoxylated and cyclized to either catharanthine or tabersonine via two hydrolases characterized herein. The pathways show how plants create chemical diversity and also enable development of heterologous platforms for generation of stemmadenine-derived bioactive compounds.

Plants represent the main source of molecules for the development of new drugs, which intensifies the interest of transnational industries in searching for substances obtained from plant sources, especially since the vast majority of species have not yet been studied chemically or biologically, particularly concerning anti-inflammatory action. Anti-inflammatory drugs can interfere in the pathophysiological process of inflammation, to minimize tissue damage and provide greater comfort to the patient. Therefore, it is important to note that due to the existence of a large number of species available for research, the successful development of new naturally occurring anti-inflammatory drugs depends mainly on a multidisciplinary effort to find new molecules. Although many review articles have been published in this regard, the majority presented the subject from a limited regional perspective. Thus, the current article presents highlights from the published literature on plants as sources of anti-inflammatory agents.

Rhodnius prolixus not only has served as a model organism for the study of insect physiology, but also is a major vector of Chagas disease, an illness that affects approximately seven million people worldwide. We sequenced the genome of R. prolixus, generated assembled sequences covering 95% of the genome (∼ 702 Mb), including 15,456 putative protein-coding genes, and completed comprehensive genomic analyses of this obligate blood-feeding insect. Although immune-deficiency (IMD)-mediated immune responses were observed, R. prolixus putatively lacks key components of the IMD pathway, suggesting a reorganization of the canonical immune signaling network. Although both Toll and IMD effectors controlled intestinal microbiota, neither affected Trypanosoma cruzi, the causal agent of Chagas disease, implying the existence of evasion or tolerance mechanisms. R. prolixus has experienced an extensive loss of selenoprotein genes, with its repertoire reduced to only two proteins, one of which is a selenocysteine-based glutathione peroxidase, the first found in insects. The genome contained actively transcribed, horizontally transferred genes from Wolbachia sp., which showed evidence of codon use evolution toward the insect use pattern. Comparative protein analyses revealed many lineage-specific expansions and putative gene absences in R. prolixus, including tandem expansions of genes related to chemoreception, feeding, and digestion that possibly contributed to the evolution of a blood-feeding lifestyle. The genome assembly and these associated analyses provide critical information on the physiology and evolution of this important vector species and should be instrumental for the development of innovative disease control methods.

BACKGROUND: The insect gut harbors a variety of microorganisms that probably exceed the number of cells in insects themselves. These microorganisms can live and multiply in the insect, contributing to digestion, nutrition, and development of their host.Recent studies have shown that midgut bacteria appear to strengthen the mosquito's immune system and indirectly enhance protection from invading pathogens. Nevertheless, the physiological significance of these bacteria for mosquitoes has not been established to date. In this study, oral administration of antibiotics was employed in order to examine the contribution of gut bacteria to blood digestion and fecundity in Aedes aegypti. RESULTS: The antibiotics carbenicillin, tetracycline, spectinomycin, gentamycin and kanamycin, were individually offered to female mosquitoes. Treatment of female mosquitoes with antibiotics affected the lysis of red blood cells (RBCs), retarded the digestion of blood proteins and reduced egg production. In addition, antibiotics did not affect the survival of mosquitoes. Mosquito fertility was restored in the second gonotrophic cycle after suspension of the antibiotic treatment, showing that the negative effects of antibiotics in blood digestion and egg production in the first gonotrophic cycle were reversible. CONCLUSIONS: The reduction of bacteria affected RBC lysis, subsequently retarded protein digestion, deprived mosquito from essential nutrients and, finally, oocyte maturation was affected, resulting in the production of fewer viable eggs. These results indicate that Ae. aegypti and its midgut bacteria work in synergism to digest a blood meal.Our findings open new possibilities to investigate Ae. aegypti-associated bacteria as targets for mosquito control strategies.

Abstract Aim To investigate how reef fish trophic structure responds to latitudinal changes, using a simple model: the extensive Brazilian coast. Location Six Brazilian tropical and subtropical coral and rocky coastal reefs, and the oceanic island of Atol das Rocas, between latitudes 0° and 27° S. Methods Underwater visual census data collected by the authors (five locations) or obtained from the literature (two locations) were used to estimate the relative abundance of 123 fish species belonging to 33 reef‐associated families. Cryptic species were excluded from the analysis. Fishes were grouped in eight trophic categories: roving herbivores, territorial herbivores, mobile invertebrate feeders, sessile invertebrate feeders, omnivores, planktivores, piscivores and carnivores. After a series of detailed predictions based on phylogeny, physiological constraints and anthropogenic impacts was established, the community trophic structure was analysed along a latitudinal gradient and among coastal, mid‐shore and oceanic sites. Results The trophic structure of Brazilian reef fish assemblages clearly changed with latitude. Roving herbivores such as scarids and acanthurids were proportionally more abundant at low latitudes. The browsing herbivores kyphosids followed an opposite latitudinal pattern. The parrotfish genus Sparisoma , more plastic in its feeding habits than Scarus , presented wider distribution. The relative abundance of territorial herbivores did not decrease towards higher latitudes. Mobile invertebrate feeders were the most important (in low latitudes) or the second most important trophic guild (in high latitudes) at all coastal sites. Sessile invertebrate feeders did not show any clear latitudinal trend, despite an expected increase in abundance towards low latitudes. Omnivores dominated high latitude reefs (27° S) and planktivores the oceanic island Atol das Rocas. Piscivores and carnivores were proportionally better represented in high latitudes. Main conclusions Latitudinal patterns seem to be influenced by phylogeny, physiological constraints (mainly related to temperature), and also by anthropogenic impacts. Grazing scarids and acanthurids are largely restricted to tropical reefs and show an abrupt decline beyond 23° S. This does not reflect the amount of algae present, but probably temperature‐dependent physiological constraints. Other herbivores seem to overcome this through symbiotic microbial digestive processes (kyphosids), manipulating the structure of algal turfs or increasing animal protein from within the territory (pomacentrids). Omnivores dominate the southern sites Arraial do Cabo and Arvoredo, being more adapted to environment constraints related to seasonal and/or stochastic shifts. Large carnivores (including piscivores) extend farther into high‐latitude habitats, apparently not constrained by thermal thresholds that limit the herbivores. Overfishing and/or ornamental harvesting certainly has been modifying local fish communities, but could not be detected properly at the large‐scale patterns found in this study. The data presented put in evidence for the first time how reef fish trophic structure behave in the extensive south‐western Atlantic latitudinal gradient.

Water was the suspected vehicle of Toxoplasma gondii dissemination in a toxoplasmosis outbreak in Brazil. A case-control study and geographic mapping of cases were performed. T. gondii was isolated directly from the implicated water and genotyped as SAG 2 type I.

The role of interleukin 10 (IL-10) and gamma interferon (IFN-gamma) on the development of pathology in human Chagas' disease was investigated. Two categories of patients, low and high producers of IFN-gamma, were identified based on the levels of secretion of this cytokine in the supernatant of peripheral blood mononuclear cell (PBMC) cultures. Eighty-three percent of the patients presenting with cardiac disease (CARD) of different degrees and 59% of the patients with the indeterminate form of disease (IND) were identified as high IFN-gamma producers. PBMC from IND patients classified as low IFN-gamma producers secreted significantly higher amounts of IL-10 than did those from other groups. Flow cytometry analysis demonstrated that in PBMC from the IND group, the majority of the IL-10-producing cells were monocytes (CD14(High+) cells), whereas in the CARD group, the major sources of IFN-gamma were T lymphocytes (CD3(+) CD4(+) cells). These results suggest an association between the production of IFN-gamma by CD3(+) CD4(+) cells and morbidity in Chagas' disease, whereas the production of IL-10 by macrophages/monocytes leads to regulation of the immune response in IND patients. We hypothesize that an exacerbated production of IFN-gamma against Trypanosoma cruzi antigens favors the development of a strong Th1 response in CARD patients, which leads to progression of heart disease.

Summary Anthropogenic global change compromises forest resilience, with profound impacts to ecosystem functions and services. This synthesis paper reflects on the current understanding of forest resilience and potential tipping points under environmental change and explores challenges to assessing responses using experiments, observations and models. Forests are changing over a wide range of spatio‐temporal scales, but it is often unclear whether these changes reduce resilience or represent a tipping point. Tipping points may arise from interactions across scales, as processes such as climate change, land‐use change, invasive species or deforestation gradually erode resilience and increase vulnerability to extreme events. Studies covering interactions across different spatio‐temporal scales are needed to further our understanding. Combinations of experiments, observations and process‐based models could improve our ability to project forest resilience and tipping points under global change. We discuss uncertainties in changing CO 2 concentration and quantifying tree mortality as examples. Synthesis . As forests change at various scales, it is increasingly important to understand whether and how such changes lead to reduced resilience and potential tipping points. Understanding the mechanisms underlying forest resilience and tipping points would help in assessing risks to ecosystems and presents opportunities for ecosystem restoration and sustainable forest management.

AIM: The accurate mapping of forest carbon stocks is essential for understanding the global carbon cycle, for assessing emissions from deforestation, and for rational land-use planning. Remote sensing (RS) is currently the key tool for this purpose, but RS does not estimate vegetation biomass directly, and thus may miss significant spatial variations in forest structure. We test the stated accuracy of pantropical carbon maps using a large independent field dataset. LOCATION: Tropical forests of the Amazon basin. The permanent archive of the field plot data can be accessed at: http://dx.doi.org/10.5521/FORESTPLOTS.NET/2014_1. METHODS: Two recent pantropical RS maps of vegetation carbon are compared to a unique ground-plot dataset, involving tree measurements in 413 large inventory plots located in nine countries. The RS maps were compared directly to field plots, and kriging of the field data was used to allow area-based comparisons. RESULTS: The two RS carbon maps fail to capture the main gradient in Amazon forest carbon detected using 413 ground plots, from the densely wooded tall forests of the north-east, to the light-wooded, shorter forests of the south-west. The differences between plots and RS maps far exceed the uncertainties given in these studies, with whole regions over- or under-estimated by > 25%, whereas regional uncertainties for the maps were reported to be < 5%. MAIN CONCLUSIONS: Pantropical biomass maps are widely used by governments and by projects aiming to reduce deforestation using carbon offsets, but may have significant regional biases. Carbon-mapping techniques must be revised to account for the known ecological variation in tree wood density and allometry to create maps suitable for carbon accounting. The use of single relationships between tree canopy height and above-ground biomass inevitably yields large, spatially correlated errors. This presents a significant challenge to both the forest conservation and remote sensing communities, because neither wood density nor species assemblages can be reliably mapped from space.