Parallel barcoding of antibodies for DNA‐assisted proteomics

DNA‐assisted proteomics technologies enable ultra‐sensitive measurements in multiplex format using DNA‐barcoded affinity reagents. Although numerous antibodies are available, nowadays targeting nearly the complete human proteome, the majority is not accessible at the quantity, concentration, or purity recommended for most bio‐conjugation protocols. Here, we introduce a magnetic bead‐assisted DNA‐barcoding approach, applicable for several antibodies in parallel, as well as reducing required reagents quantities up to a thousand‐fold. The success of DNA‐barcoding and retained functionality of antibodies were demonstrated in sandwich immunoassays and standard quantitative Immuno‐PCR assays. Specific DNA‐barcoding of antibodies for multiplex applications was presented on suspension bead arrays with read‐out on a massively parallel sequencing platform in a procedure denoted Immuno‐Sequencing. Conclusively, human plasma samples were analyzed to indicate the functionality of barcoded antibodies in intended proteomics applications.     

Advanced procedures for labeling of antibodies with quantum dots

Semiconductor quantum dots (QDs) are proved to be unique fluorescent labels providing excellent possibilities for high-throughput detection and diagnostics. To explore in full QDs’ advantages in brightness, photostability, large Stokes shift, and tunability by size fluorescence emission, they should be rendered stable in biological fluids and tagged with the target-specific capture molecules. Ideal QD-based nanoprobes should not exceed 15 nm in diameter and should contain on their surface multiple copies of homogeneously oriented highly active affinity molecules, for example, antibodies (Abs). Direct conjugation of QDs with the Abs through cross-linking of QDs’ amines with the sulfhydryl groups issued from the reduced Abs’ disulfide bonds is the common technique. However, this procedure often generates conjugates in which the number of functionally active Abs on the surface of QDs does not always conform to expectations and is often low. Here we have developed an advanced procedure with the optimized critical steps of Ab reduction, affinity purification, and QD–Ab conjugation. We succeeded in reducing the Abs in such a way that the reduction reaction yields highly functional, partially cleaved, 75-kDa heavy–light Ab fragments. Affinity purification of these Ab fragments followed by their tagging with the QDs generates QD–Ab conjugates with largely improved functionality compared with those produced according to the standard procedures. The developed approach can be extended to conjugation of any type of Ab with different semiconductor, noble metal, or magnetic nanocrystals.     

Fluorescent-labeled antibodies: Balancing functionality and degree of labeling

A critical assumption in using labeled antibodies is that the conjugation reaction has no deleterious effects on antibody avidity. This study demonstrates that this assumption need not hold true and presents a methodology to quantitatively determine the degree of inactivation and/or changes in antibody-antigen binding that can occur with conjugation. Fluorescein isothiocyanate (FITC) was conjugated to a mouse monoclonal antibody, Fc125, against hemagglutinin (HA) using varying fluorophore/protein (F:P) labeling ratios. Antibody binding, as a function of the F:P labeling ratio, was evaluated using a kinetic enzyme-linked immunosorbent assay (ELISA) and analyzed using global fitting. A two-parameter adjustment of the antibody concentration and the maximum rate was sufficient to describe the rate changes. The concentration parameter dominated the rate changes, consistent with the hypothesis that the coupling reaction inactivated an increasing fraction of the antibody population with a smaller change (∼15% at the highest F:P ratio) in antibody-antigen binding. An optimal F:P ratio that minimized both inactivation and unlabeled antibody was calculated. This procedure can be used to prepare functional, labeled antibody reagents with defined activity and can aid in quantitative applications where the stoichiometry and functionality of the labeled antibody are critical.     

The fate of antibodies and their radiolabels bound to tumor cells in vitro: The effect of cross-linking at the cell surface and of anti-idiotype antibodies

Summary In order to obtain rapid blood clearance of circulating antibodies (Ab) at a desired time, cross-linking reagents such as second Ab are often employed. Such reagents will generally bind to Ab located at the tumor site as well as free Ab, and we therefore investigated whether the cross-linking of Ab bound to the surface of tumor cells affects the processing of those Ab. Cross-linking was induced in various ways: a polyclonal second Ab [rabbit anti-(mouse IgG)], a monoclonal rat anti-(mouse IgG constant region) Ab, and streptavidin used in conjunction with a biotinylated first Ab. Processing was followed for 3 days, to allow nearly all of the bound Ab to reach its ultimate fate. Results depended strongly on the particular first Ab used. Two basic effects were observed. First, the second Ab efficiently prevented the early dissociation of intact Ab from the cell; once the second Ab bound, there was virtually no dissociation of the primary Ab bound to the cells. For most Ab, where only a small proportion of bound Ab dissociated intact, this effect was relatively small. However, for an unusual Ab, where the majority dissociated intact (L6) the effect of a second Ab in prolonging Ab retention by the cell was dramatic. Second, cross-linking sometimes resulted in markedly accelerated internalization and degradation of the bound Ab, coupled with the release of degradation products into the medium. This process resulted in much shorter retention of the radioisotope by the cell. If a residualizing radiolabel was used,¹²⁵I-dilactitoltyramine, which is probably trapped within lysosomes after Ab catabolism, the effect of the second Ab in accelerating loss from the cell was largely prevented. We also tested anti-idiotype Ab as cross-linking reagents. In addition to testing anti-idiotype Ab known to react with the cell-bound primary Ab, we also tested antiidiotype Ab not expected to bind to cell-bound Ab, initially as a negative control. Unexpectedly, all anti-indiotype Ab tested induced rapid release of the primary Ab from the cell. This effect was similar to the effect of a large excess of unlabeled Ab, and we attribute it to the blocking of the free binding site of a wobbling Ab, which prevents its rebinding to a second antigen molecule. We conclude that the use of selected anti-idiotype Ab to clear circulating Ab, while not reacting with cell-bound Ab, must be done cautiously. These effects must be taken into consideration in developing procedures that utilize second Ab or other crosslinking agents.     

Bromodomains shake the hegemony of pan‐acetyl antibodies

Acetylation signaling pathways are involved in numerous cellular processes and are used as therapeutic targets in several disease contexts. However, acetylated proteins only represent a minor fraction of the full proteome, and the identification and quantification of acetylated sites remain a technological challenge. Currently, pan‐acetyl antibodies are used to increase the abundance of acetylated peptides through affinity purification before MS analysis. These antibodies are powerful reagents, but they are hampered by a lack of specificity, affinity, and batch‐to‐batch reproducibility. In this issue, Bryson et al. (Proteomics 2015 15, 1470–1475) present an interesting alternative to these antibodies, in the form of bromodomains. These domains specifically recognize acetylated lysines, and were successfully used in this study to enrich for acetylated peptides before MS analysis. Future development of this pioneering approach could help overcome this limiting step in the characterization of acetylproteomes.     

Pilot scale recovery of monoclonal antibodies by expanded bed ion exchange adsorption

The aim of the investigations was to estimate the scale up properties of an efficient chromatographic first capture step for the recovery of murine IgG₁ from undiluted and unclarified hybridoma cell culture broth using an ion exchange matrix in expanded bed mode. The tested new sulfopropyl-based ion exchange matrix (Streamline^TM SP XL, Amersham Pharmacia Biotech) stands out due to its enhanced capacity compared to its precursor (Streamline^TM SP). Defining the working pH in preliminary electrophoretic analyses (titration curve, SDS-PAGE) and small-scaled chromatographic binding studies showed, that the optimal value for the IgG purification was pH 4.6, where a co-chromatography of the medium supplement albumin (500 mg l^-1, pI = 4.8) could not be avoided. Further scouting experiments dealt with the dynamic capacity of the matrix, which was evaluated by frontal adsorption analysis. In packed bed mode no break-through of the target protein was achieved even after 6.5 mg IgG per ml matrix were applied. These results could not be reproduced in expanded bed mode with cell-free supernatant, where the dynamic capacity was found to be only 1.5 mg IgG/ml SP XL. Processing cell-containing broth resulted in an additional decrease of the value down to 0.5 mg ml^-1, presumably caused by the remarkable biomass adsorption to the matrix. The search for the reasons led to the examination of the hydrodynamic conditions. Buffer experiments with a tracer substance (acetone) pointed out, that the flow in expanded bed was significantly more influenced by back-mixing effects and channel formations than in packed bed. These effects could be compensated with an enhanced viscosity of the liquid phase, which was achieved by the addition of glucose. As a result of the improved hydrodynamic conditions in the expanded bed, the dynamic capacity could be increased from 0.5 to more than 4.5 mg IgG/ml matrix for the processing of cell culture broth with 400 mM glucose. Finally, the scale up from a Streamline^TM 25 to a Streamline^TM 200 column was performed under conditions, which proved to be optimal: 100 L of unclarified hybridoma broth were concentrated with a binding rate of 95% in less than 3.5 hours. Loading the column no break-through of the target protein was achieved. However, the eluate still contained debris and cells, which points out the major disadvantage of the method: the biomass attachment to the matrix.     

固沙区时间尺度上生态健康状况分析

以干旱区沙坡头不同时间尺度上人工植被防护体系为研究对象,初步研究了在区域荒漠化的治理过程中,生态环境的演变对生态健康的影响。结果表明：土壤水分和土壤结皮是影响固沙区生态健康的主要因子,老固沙区的健康状况趋于衰退,而新固沙区在演变过程中逐渐达到一种健康稳定状态。这对今后如何对老固沙区进行生态恢复和人工建设、对新固沙区进行调节和维持,使其发挥最优防护效益具有重要的参考价值。     

Phosphorus in agricultural soils: drivers of its distribution at the global scale

Phosphorus (P) availability in soils limits crop yields in many regions of the World, while excess of soil P triggers aquatic eutrophication in other regions. Numerous processes drive the global spatial distribution of P in agricultural soils, but their relative roles remain unclear. Here, we combined several global data sets describing these drivers with a soil P dynamics model to simulate the distribution of P in agricultural soils and to assess the contributions of the different drivers at the global scale. We analysed both the labile inorganic P (P_ILAB), a proxy of the pool involved in plant nutrition and the total soil P (P_TOT). We found that the soil biogeochemical background corresponding to P inherited from natural soils at the conversion to agriculture (BIOG) and farming practices (FARM) were the main drivers of the spatial variability in cropland soil P content but that their contribution varied between P_TOT vs. P_ILAB. When the spatial variability was computed between grid cells at half‐degree resolution, we found that almost all of the P_TOT spatial variability could be explained by BIOG, while BIOG and FARM explained 38% and 63% of P_ILAB spatial variability, respectively. Our work also showed that the driver contribution was sensitive to the spatial scale characterizing the variability (grid cell vs. continent) and to the region of interest (global vs. tropics for instance). In particular, the heterogeneity of farming practices between continents was large enough to make FARM contribute to the variability in P_TOT at that scale. We thus demonstrated how the different drivers were combined to explain the global distribution of agricultural soil P. Our study is also a promising approach to investigate the potential effect of P as a limiting factor for agroecosystems at the global scale.     

宏生态尺度上景观破碎化对物种丰富度的影响

生物多样性的地理格局及其形成机制是宏生态学与生物地理学的研究热点。大量研究表明,景观尺度上的生境破碎化对物种多样性的分布格局具有重要作用,但目前尚不清楚这种作用是否足以在宏生态尺度上对生物多样性地理格局产生显著影响。利用中国大陆鸟类和哺乳动物的物种分布数据,在100 km×100 km网格的基础上生成了这两个类群生物的物种丰富度地理格局,进一步利用普通最小二乘法模型和空间自回归模型研究了物种丰富度与气候、生境异质性、景观破碎化的相关关系。结果表明,景观破碎化因子与鸟类和哺乳动物的物种丰富度都具有显著的关联关系,其方差贡献率可达约30%—50%(非空间模型)和60%—80%(空间模型),略低于或接近于气候和生境异质性因子。方差分解结果显示,景观破碎化因子与气候和生境异质性因子的方差贡献率的重叠部分达20%—40%。相对鸟类而言,景观破碎化对哺乳动物物种丰富度的地理格局具有更高的解释率。     

Use of frozen bagged seed inoculum for secondary metabolite and bioconversion processes at the pilot scale

Frozen bagged seed inoculum was prepared, thawed and tested for seven cultures. Thawing techniques were developed and other key influences on thawing rate were quantified; seed bag thawing without a water bath rarely required more than 4 to 5 h and was as short as 0.5 to 1 h for lower fill volume bags. Testing included growth of bagged seed as a function of bag fill volume (0.5, 1.0, 2.0, and 3.5 L), comparison of culture age at time of bagging, growth of bagged versus laboratory-prepared seed, productivity of production cultures derived from bagged versus laboratory-prepared seed, growth of bagged seed as a function of volume percent glycerol added at time of bagging, and growth of bagged seed as a function of frozen storage time and temperature. For each culture tested, conditions were developed such that seed tanks inoculated with bagged seed showed only minimal delay in attaining the target oxygen uptake rate (OUR) relative to seed tanks inoculated with laboratory-prepared inoculum. Although the bag fill volume did influence culture growth in some cases, bag fill volumes required were reasonable (typically 2.0 to 3.5 L) compared with laboratory seed inoculum volumes of 2.0 L. In the most remarkable example, frozen bagged seed was prepared from a second-stage seed-tank cultivation of Glarca lozoyensis, then thawed and inoculated into first-stage seed medium. It grew to the desired OUR in a similar timeframe as laboratory-prepared inoculum inoculated into first-stage seed medium. Thus, the frozen bagged seed replaced an existing laboratory inoculum preparation period of 7 days without an appreciable delay in either of the two subsequent seed-tank growth stages. Furthermore, productivities were found to be comparable for bagged-seed-derived and laboratory-seed-derived production cultivations for four different fermentation processes.     

流域尺度土地利用与土壤类型空间分布的相关性研究

随着人类活动日益加强,土地利用变化及其驱动力研究已经成为国际地理学界研究的热点。但目前更多的工作侧重于研究人为因子,如人口增长、政策变化等因子对土地利用变化的影响,其实土地利用在宏观尺度上的变化一定程度上取决于自然环境背景,因此研究土地利用变化的自然环境背景特征对于进一步探讨土地利用变化的自然驱动力具有重要意义。土壤类型的空间分布在一定程度将影响土地利用的空间分布格局和变化过程。本文利用遥感、地理信息系统,通过选取景观格局指标,以于桥水库流域为例,研究了土地利用与土壤类型空间分布之间的相互关系。结果表明：(1)受人类活动干扰较弱的土地利用类型,如有林地、稀疏林地、灌木林地、其他林地和草地,主要分布在淋溶褐土、褐土性土以及棕壤地区;而与人类活动密切的土地利用类型,如山区旱耕地、丘陵区水田、丘陵区旱耕地和平原区旱耕地主要分布在淋溶褐土、褐土性土和潮土地区;(2)同类地区土地利用的多样性指数要比土壤的低。并且土地利用和土壤类型多样性均表现出明显的地形梯度效应,从中低山区、低山丘陵区、丘陵平原区到山间盆地区,土壤多样性指数从小到大有规律地变化。而土地利用类型多样性变化较为复杂,反映出人类活动对土地利用格局的影响较强,而对土壤类型的影响相对较小;(3)随着平均斑块面积的增加,流域多样性指数将呈下降趋势。土地利用多样性指数与流域总面积之间的相关性较差,但土壤多样性指数与流域总面积表现出的相关性较好;(4)土地利用类型与土壤类型之间,平均斑块面积上没有明显的相关关系,但在多样性指数之间存在着较好的相关性。土壤类型丰富多样的地区,土地利用的多样性指数也相对较高。     

Increased efficacy of HIV-1 neutralization by antibodies at low CCR5 surface concentration

It has been observed that some antibodies, including the CD4-induced (CD4i) antibody IgG X5 and the gp41-specific antibody IgG 2F5, exhibit higher neutralizing activity in PBMC-based assays than in cell line based assays [J.M. Binley, T. Wrin, B. Korber, M.B. Zwick, M. Wang, C. Chappey, G. Stiegler, R. Kunert, S. Zolla-Pazner, H. Katinger, C.J. Petropoulos, D.R. Burton, Comprehensive cross-clade neutralization analysis of a panel of anti-human immunodeficiency virus type 1 monoclonal antibodies, J. Virol. 78 (2004) 13232-13252]. It has been hypothesized that the lower CCR5 concentration on the surface of the CD4 T lymphocytes compared to that on cell lines used for the neutralization assays could be a contributing factor to the observed differences in neutralizing activity. To test this hypothesis and to further elucidate the contribution of CCR5 concentration differences on antibody neutralizing activity, we used a panel of HeLa cell lines with well-defined and differential surface concentrations of CCR5 and CD4 in a pseudovirus-based assay. We observed that the CCR5 cell surface concentration but not the CD4 concentration had a significant effect on the inhibitory activity of X5 and several other CD4i antibodies including 17b and m9, as well as that of the gp41-specifc antibodies 2F5 and 4E10 but not on that of the CD4 binding site antibody (CD4bs), b12. The 50% inhibitory concentration (IC50) decreased up to two orders of magnitude in cell lines with low CCR5 concentration corresponding to that in CD4 T cells used in PBMC-based assays (about 10(3) per cell) compared to cell lines with high CCR5 concentration (about 10(4) or more). Our results suggest that the CCR5 cell surface concentration could be a contributing factor to the high neutralizing activities of some antibodies in PBMC-based-assays but other factors could also play an important role. These findings could have implications for development of vaccine immunogens based on the epitopes of X5 and other CD4i antibodies, for elucidation of the mechanisms of HIV-1 neutralization by antibodies, and for design of novel therapeutic approaches.     

基于斑块尺度的云南省景观生态安全时空演变及归因

随着人类活动的需求增强和范围扩张,建设开发、农林种植等行为不断侵占和切分原有土地系统,导致区域景观斑块小型化和破碎化,产生了诸多生态问题。云南既是我国西南生态安全屏障,同时也是生态环境比较脆弱敏感的地区,研究云南省景观生态安全及其影响因素,对区域、国家乃至国际生态安全具有重要意义。以云南省为研究对象,提出基于斑块尺度的景观生态安全评价方法评估云南省1990-2018年近三十年的景观生态安全,采用空间自相关方法分析其时空演变规律,并通过地理探测器识别影响景观生态安全的主要驱动因子。结果表明：（1）与传统生态安全评价模型相比,斑块尺度的景观生态安全模型对区域内部景观生态安全的变化更敏感,能够反映区域内部多年景观生态安全状况的细微变化。（2）云南省1990-2018年整体生态安全成本成波动上升趋势,其中"三屏两带"地区的景观生态安全状况提升明显,而滇中城市群外围以及滇东南喀斯特地带的部分区域有所下降。云南省景观生态安全的Global Moran''s I指数平均值为-0.293,区域内部斑块破碎,空间分布上具有离散性。（3）云南省景观生态安全状况目前主要受到人口密度、年平均温度和海拔高度的影响;从交互驱动方面看,云南省景观生态安全空间分布是自然和人文因子共同作用的结果,其中自然因子的交互驱动作用更明显。研究结果可为云南省景观生态安全格局优化提供理论支撑与科学依据。     

Spatial heterogeneity of soil respiration and related properties at the plant scale

Geostatistical techniques were used to quantify the scale and degree of soil heterogeneity in 2 m² plots around 9-year-old poplar trees and within a wheat field. Samples were taken during two years, on an unaligned grid, for analysis of soil respiration, C and N content, available P, gravimetric moisture, pH, nitrification potential, and root biomass. Kriged maps of soil respiration, moisture, and C content showed strong spatial structure associated with poplar trees but not with wheat rows. All soil properties showed higher autocorrelation in June than in April. Isopleth patchiness for all variates was less in June. This was associated with lower respiration rates due to lower litter decomposition. From the degree and scale of heterogeneity seen in this study, we conclude that the main causes of soil heterogeneity at this scale (2 m²) are likely to be found at micro scales controlled in part by plant root and plant residue patterns. These must be understood in the evaluation of ecosystem processes. This revised version was published online in June 2006 with corrections to the Cover Date.     

冬小麦生育期农田尺度下土壤硝态氮淋失动态的数值模拟

在北京通州区永乐店田间试验的基础上 ,假设土壤由一系列不发生相互作用的一维土柱组成 ,根据实测的土壤有机质含量 ,假定土壤有机氮的矿化作用速率常数 (零级动力学 )和有机质含量成正比 ,运用 HYDRUS- 1D软件 ,分别就考虑和不考虑土壤有机氮的矿化速率的空间变异性这两种方案 ,对 2 0 0 0～ 2 0 0 1年冬小麦生长条件下农田尺度土壤氮素转化和硝态氮淋失规律进行了数值分析。两种方案的模拟结果表明 :考虑和不考虑土壤有机氮矿化速率的空间变异性对剖面 2 5 0 cm埋深处硝态氮淋失量的影响很小 ,其差异主要在于前者对土壤氮素的矿化量、固持及反硝化量、作物吸氮量的影响更大 ,其空间变异性高于不考虑矿化速率时的结果。剖面 2 5 0 cm埋深处平均的土壤水渗透量和累积硝态氮淋失量分别为 2 .2 5 mm、0 .0 0 984 m g/cm2 ,变异系数大于 1.4 6 ,属于强变异性。对模拟结果进行地统计学分析 ,结果表明 :剖面 2 5 0 cm埋深处的土壤水渗透量和硝态氮淋失量的半方差函数为纯块金形式 ,在空间上表现为相互独立。考虑有机氮矿化速率空间变异性时的土壤氮素净转化量、吸氮量均可用球状模型描述 ,其变程与土壤有机质含量的变程接近 ,约为 4 .7m;而不考虑有机氮矿化速率空间变异性时的土壤氮素净转化量用线性无基台值     

The proportion of impervious surfaces at the landscape scale structures wild bee assemblages in a densely populated region

Given the predicted expansion of cities throughout the world, understanding the effect of urbanization on bee fauna is a major issue for the conservation of bees. The aim of this study was to understand how urbanization affects wild bee assemblages along a gradient of impervious surfaces and to determine the influence of landscape composition and floral resource availability on these assemblages. We chose 12 sites with a proportion of impervious surfaces (soil covered by parking, roads, and buildings) ranging from 0.06% to 64.31% within a 500 m radius. We collected using pan trapping and estimated the landscape composition of the sites within a 500 m radius and the species richness of plant assemblages within a 200 m radius. We collected 1104 bees from 74 species. The proportion of impervious surfaces at the landscape scale had a negative effect on wild bee abundance and species richness, whereas local flower composition had no effect. Ground‐nesting bees were particularly sensitive to the urbanization gradient. This study provides new evidences of the impact of urbanization on bee assemblages and the proportion of impervious surfaces at the landscape scale emerged as a key factor that drives those assemblages.     

基于乡镇尺度的土地生态安全时空格局评价研究——以博尔塔拉蒙古自治州为例

近年来,国内外学者已做了大量区域土地生态安全评价的研究,但大多针对流域或县级以上行政单元,乡镇尺度的土地生态安全评价研究甚少。因此,选择地处我国西北干旱生态脆弱区的博尔塔拉蒙古自治州为研究区,以"3S"技术和数学统计方法为支撑,结合研究区自然地理特征和社会经济状况,构建博尔塔拉蒙古自治州乡镇级压力-状态-响应(Pressure-StateResponses,P-S-R)土地生态安全评价模型,并对其土地生态安全空间格局特征及成因进行诊断性分析。结果表明:(1)2014年博州土地生态安全较2011年有所恶化,Ⅲ级向Ⅳ级转化的面积最大,高达2555.33 km~2,主要在温泉县境内,其次是Ⅴ级区域向Ⅳ级转化1356.53 km~2,主要发生在精河县的茫丁乡和托托乡;(2)博州大部分乡镇的土地生态安全状况处于Ⅲ级水平,区域生态结构不稳定,亟待调整;(3)博州土地生态安全状况具有明显地域性分布的特征,低值区多集中于博州东部荒漠地区,高值区多集中在博州中部绿洲区域和湖区。研究结果可为乡镇级土地生态安全评价提供参考,为区域生态保护协调推进与乡镇可持续发展提供理论指导。     

Abundance estimation of rocky shore invertebrates at small spatial scale by high-resolution digital photography and digital image analysis

We have tested both the usefulness of high-resolution digital photography for data acquisition and digital image analysis, by non-supervised classification and high pass filter, for recognition and abundance estimation of benthic intertidal organisms. These digital tools were compared with visual scan and photo quadrat conventional methods. The comparison was done using 40 quadrats (10×5 cm) randomly selected along a 5-m transect on the rocky shore of the Pemaquid Point, Maine, USA. ANOVA for repeated measures was used to test differences among methods. Monte Carlo simulation analysis was used to explore differences among methods over a large set of data (n=100, 500, 1000 quadrats). Differences among methods were observed when 40 quadrats were used. Tukey multiple comparison test showed that abundance estimation from visual scan, photo quadrat and digital image analysis by high pass filter do not differ significantly among them but differ from non-supervised classification results. Due to its accurate estimation, high pass filter (Prewitt) method was chosen as the most reliable digital method to estimate species abundance. Monte Carlo simulation of visual scan, photo quadrat and high pass filter results showed significant differences when the number of quadrats was larger. These results showed that the combined use of digital photography and digital image analysis techniques for the acquisition and analysis of recorded data is a powerful method for the study of intertidal benthic organisms. Results produced using these techniques were similar than those produced by conventional methods but were obtained in a much-reduced time.     

Determinants of bird species richness: role of climate and vegetation structure at a regional scale

We examined the respective roles of climate and vegetation structure on geographical variation in bird species richness. The Province of Buenos Aires (central-eastern Argentina) was divided into 146 squares of 50 km on a side. For each square we evaluated the number of bird species, the value of thirteen climatic variables, and the value of a vegetation strata index. The climatic matrix was analyzed by Principal Component Analysis (PCA), and the first factors resulting from PCA were considered as multifactorial climatic gradients. Simple and Partial Correlation Analysis among bird species richness, vegetation strata, and the first two factors derived from PCA (65% of total variation) indicated that bird richness distribution was determined by the availability of vegetation strata, associated with different vegetation types that, at the same time, were influenced by the climatic conditions summarized in the first climatic factor (a gradient of precipitation, relative humidity, annual termical amplitude, and frost occurrence). This relationships reflect the complexity of factors that can act directly as well as indirectly on the geographical patterns in species richness. Also, we evaluated the importance of study scale comparing our results with previous studies at macrogeographic and local scales, found out that the vegetation structure was the principal determinant of bird species richness at this three geographical scales.