Chalcone derivatives as promising antifoulants: Molecular optimization, bioactivity evaluation and performance in coatings.

Marine biofouling remains a huge concern for maritime industries and for environmental health. Although the current biocide-based antifouling coatings can prevent marine biofouling, their use has been associated with toxicity for the marine environment, being urgent to find sustainable alternatives. Previously, our research group has identified a prenylated chalcone (1) with promising antifouling activity against the settlement of larvae of the macrofouling species Mytilus galloprovincialis (EC50 = 16.48 µM and LC50 > 200 µM) and lower ecotoxicity when compared to Econea®, a commercial antifouling agent in use. Herein, a series of chalcone 1 analogues were designed and synthesized in order to obtain optimized antifouling compounds with improved potency while maintaining low ecotoxicity. Compounds 8, 15, 24, and 27 showed promising antifouling activity against the settlement of M. galloprovincialis larvae, being dihydrochalcone 27 the most potent. The effect of compound 24 was associated with the inhibition of acetylcholinesterase activity. Among the synthesized compounds, compound 24 also showed potent complementary activity against Navicula sp. (EC50 = 4.86 µM), similarly to the lead chalcone 1 (EC50 = 6.75 µM). Regarding the structure-activity relationship, the overall results demonstrate that the substitution of the chalcone of the lead compound 1 by a dihydrochalcone scaffold resulted in an optimized potency against the settlement of mussel larvae. Marine polyurethane (PU)-based coatings containing the best performed compound concerning anti-settlement activity (dihydrochalcone 27) were prepared, and mussel larvae adherence was reduced compared to control PU coatings.

Role of Organic Fertilizer in the Transfer of Lead to Vegetables Produced in Tropical Mountain Agroecosystems.

Understanding the relationship between the aerobic transformation of organic matter (OM) and the bioavailability of lead to plants may allow the safe application of organic fertilizers (OF) in agriculture. The present study aimed to elucidate the relationship of different OM structures with Pb, revealing the action of OF (poultry litter) on Pb dynamics, presenting the effects of OM transformations on bioavailability and transfer to vegetables produced in tropical mountain agroecosystems (TMA). The association of Pb with hydrophilic structures (CAlk-O and CAlk-di-O) during the aerobic transformation of poultry litter (PL) contributes to the increase in the water-soluble form of this metal (3.17-15.30%). The structural changes promoted by the transformation of OM, in addition to reducing the adsorption capacity of Pb in PL (Kd reduction from 1135.50 to 87.49), favor the formation of outer-sphere complexes. PL that have a more labile structure, i.e., those that are less humified, have greater affinity for Pb. The greater affinity of Pb for labile structures that are preserved in PL during OM transformations contributed to its increase and transport to edible plant parts. Considering the edible parts of vegetables grown in TMA and fertilized with fresh PL, 100% of broccoli, 91.78% of cabbage, 80.00% of tomato, 65.96% of parsley, 49.19% of lettuce, and 32.88% of cauliflower showed Pb contamination that exceeded the permitted level. Therefore, OF contributes to lead contamination of food produced in TMA, representing a risk to human health. Studies are needed to propose additional treatments for this residue before its use.

Spatial distribution and factors influencing the accumulation of toxic metals in soils in mountain agroecosystems, Rio De Janeiro, Brazil.

The concentrations of toxic metals (TM) were analyzed in 498 samples of agricultural soils used for intensive vegetable cultivation in the watershed of Barracão dos Mendes, Brazil. The goal of this study was to characterize the distribution of these elements and the main natural and anthropogenic factors affecting their accumulation. In general, the average concentrations of TM were higher than the reference quality values for cultivated soils in the region, with the exception of Cr, Co, Ni and Mn, and the average concentration of Cd was ten times greater. Three sources of variation in the distribution of TM concentrations were identified: one related to topographic relief, another related to lithology, and one related to the massive use of agrochemicals. These factors contributed to TM accumulation in the soil; moreover, the transport of toxic metal-enriched clay by runoff resulted in higher concentrations of these elements in the lower parts of the slope. The long-term application of massive amounts of fertilizers and pesticides resulted in the accumulation of Cr, Cu, Zn, Ni, Pb and Cd in the vegetable cultivation soils and promoted the enrichment of macronutrients, mainly P and K. Moreover, the spatial distribution of TM in the agricultural soils of this mountain agroecosystem was affected by intensive vegetable cultivation, which altered the natural TM distribution dynamics determined by variations in topographic relief and lithology. In intensive cultivation areas, the TM distribution was also influenced by soil management practices such as tillage along the slope direction and massive mineral and organic fertilization.

Influence of nutrient, toxic metal and herbicide contents on the soil bacterial communities in tropical vegetable growing areas.

The relationship between bacterial diversity and the bioavailability of nutrients, toxic metals and the herbicide oxyfluorfen in a tropical vegetable growing area was evaluated. The study was conducted in a vegetable growing area located in the mountainous region of Rio de Janeiro (Brazil), and samples were collected in areas of vegetable cultivation and areas of environmental reserve. Fertility analyses and determination of the pseudototal levels of toxic metals in the soil samples were performed. The profile of the soil bacterial community was determined by amplification of the 16S rRNA gene and separation by DGGE. The results showed that the levels of toxic metals and elements associated with soil fertility were higher in vegetable production areas. These differences in the physical and chemical characteristics of the soil favored the presence of a greater number of OTUs in the cultivation areas (17.3-27 OTUs) than in the areas of environmental reserve (13-22 OTUs). Therefore, this study demonstrates that the presence of toxic metals and the herbicide oxyfluorfen and the increase in fertility in soils in areas with intensive vegetable cultivation resulting from the intensive management adopted in these areas promotes a differentiation of the bacterial profiles in soils in tropical vegetable growing areas.

CCR5∆32 and SDF1 3'A: Gene Variants, Expression and Influence on Biological Markers for the Clinical Progression to AIDS among HIV-1 Virus Controllers in a Mixed Population of the Amazon Region of Brazil.

CCR5Δ32 and SDF1-3'A polymorphisms were investigated in a cohort of viremia controllers, without the use of therapy, along with their influence on CD4+ T lymphocytes (TLs), CD8+ TLs, and plasma viral load (VL). The samples were analyzed from 32 HIV-1-infected individuals classified as viremia controllers 1 and 2 and viremia non-controllers, from both sexes, mostly heterosexuals, paired with 300 individuals from a control group. CCR5∆32 polymorphism was identified by PCR amplification of a fragment of 189 bp for the wild-type allele and 157 bp for the allele with the ∆32 deletion. SDF1-3'A polymorphism was identified by PCR, followed by enzymatic digestion (restriction fragment length polymorphism) with the Msp I enzyme. The relative quantification of gene expression was performed by real-time PCR. The distribution of allele and genotype frequencies did not show significant differences between the groups. The gene expression of CCR5 and SDF1 was not different between the profiles of AIDS progression. There was no significant correlation between the progression markers (CD4+ TL/CD8+ TL and VL) and the CCR5∆32 polymorphism carrier status. The 3'A allele variant was associated with a marked loss of CD4+ TLs and a higher plasma VL. Neither CCR5∆32 nor SDF1-3'A was associated with viremia control or the controlling phenotype.

Occurrence, molecular diversity and pathogenicity of Acanthamoeba spp. isolated from aquatic environments of Northeastern Brazil.

Acanthamoeba is a free-living amoeba (FLA) that is ubiquitous in nature and can cause serious pathologies in humans. This protozoan has been detected in several environmental sources, such as soil, water, and swimming pools. The aim of this study was to evaluate the occurrence and molecular diversity of Acanthamoeba spp. in aquatic environments of the state of Sergipe, northeastern Brazil, and to determine the pathogenic potential of the isolated samples. A total of 138 samples were collected from 69 aquatic environments and, after cell culture, 74% of the samples were positive for FLA, 47% belonging to the genus Acanthamoeba. Genotypic analysis was performed using the primers JDP1 and JDP2, confirming distinct Acanthamoeba genotypes: 18 (75%) isolates belonging to genotype T4, two (8%) to T3, and one isolate (4%) to genotype T5. Tests carried out to analyze the pathogenic potential showed that 11 isolates could grow at 0.5 M mannitol concentration and seven isolates supported hyperosmolarity. In the thermotolerance test, two isolates grew at 37°C. These results confirm the presence and the pathogenic potential of FLA of the genus Acanthamoeba in aquatic environments of the municipalities of Sergipe.

Effect of short-term pig slurry amendment of soil on humified organic matter and its relationship with the dynamics of heavy metals and metals uptake by plants.

The impact of pig slurry (PS) application on the structural dynamics of humic substances (HS) and on the mobility of Cu, Zn, Ni, and Pb in a dystrophic Red Nitosol planted with winter forage grasses was evaluated. After four PS applications, the humic acids (HA) and fulvic acids (FA) were characterized by spectroscopy techniques allied to chemometrics methods. The metals contents in soil, in HS and in the tissues of plant were quantified. PS application increases the total organic carbon, especially the nonhumic carbon, which contribute to increase FA content. The carbon in FA and HA increases with the highest PS dose applied, especially aliphatic structures in FA and aromatic structures in HA. The amount of Pb and Cu in FA and HA increases respectively, as well as Cu, Zn, Ni, and Pb bioavailable. PS applications increase the biomass production in grasses and the metals content accumulated in the tissues. Our study shows that the PS application modifies the structure of SOM, incorporating fragments, and modifying its dynamics, which regulates the dynamics and the accumulation of metals in soils and plants. The association of metals with soluble structures seems to inactivate their toxicity and does not affect plant growth.

Cadmium and lead transfer factors to kale plants (Brassica oleracea var. acephala) grown in mountain agroecosystem and its risk to human health.

The aim of the present study is to assess soil Cd and Pb contamination in kale (Brassica oleracea var. acephala) production sites in mountain agroecosystem, as well as its potential risk to human health. The study was carried out within 24 kale-production sites located in mountainous region of Rio de Janeiro (Brazil). Soil, plant, and fertilizer samples were collected in all assessed properties. Pseudo-total and bioavailable soil Cd and Pb content and their concentration and amount accumulated in plants were analyzed. The recorded results were used to calculate the pollution index. Risks to human health associated with kale consumption were assessed by comparing the limits set by the Brazilian regulating agency and by calculating estimated daily intake (EDI), noncarcinogenic target hazard quotient (THQ), and carcinogenic risk factor (CRF). Soil Cd and Pb enrichment was observed. Inappropriate management adopted in the assessed sites and terrain slope and the intensive use of mineral (phosphate and potassium) and organic (poultry litter) fertilizers were the main factors contributing to increase soil Cd and Pb pseudo-total and bioavailability contents. Most Cd and Pb contents in leaves, in natura, exceed the maximum values set by the Brazilian regulating agency. The present EDI, THQ, and CRF results recorded for these metals were within the tolerable ranges.

Variability factors of heavy metals in soils and transfer to pasture plants of Mayabeque in Cuba.

Studies to assess variability factors of heavy metals in soils are essential to understand their behavior in the environment and for the assessment of contaminated areas. In this context, this research aimed to study the factors that influence the variability of heavy metal levels and their bioavailability in pasture areas in the Alturas de Nazareno region, Mayabeque, Cuba, as well as the transfer to plants. Forty-five points were distributed in a natural pasture and at two depths (0-20 and 20-40 cm) as a function of relief and soil type. Pseudo-total heavy metal contents were determined according to method 3051A of USEPA, geochemical fractionation according to the Community Bureau of Reference method, and plant digestion according to method 3052 of USEPA. Soil samples were separated into two groups, according to cluster analysis. The type of soil associated with the pH and Ca attributes were the factors that most influenced the variability of the total and bioavailable levels of metals in natural pasture soils in the region of Alturas de Nazareno. Group 1 showed the lowest heavy metal contents and the highest pH and calcium values, which were predominantly associated with carbonate soils. Group 2 had the highest contents of heavy metals and Fe, corresponding mainly to the soil Ferralítico Rojo. The concentration of metals in plants was directly related to their bioavailable content in the soil, where G2 represents the group with the highest risk of transferring metals to the food chain.

Potential of water lettuce (Pistia stratiotes L.) for phytoremediation: physiological responses and kinetics of zinc uptake.

Two greenhouse experiments were carried out to evaluate the phytoremediation potential, physiological responses and zinc (Zn) uptake kinetics of water lettuce (Pistia stratiotes L.). The phytoextraction experiment evaluated four doses of Zn (0.7 mg L-1 - represented the Zn in the nutrient solution, 1.8, 18 and 180 mg L-1 - corresponded to ten, hundred and a thousand times, respectively, the maximum permitted content for fresh water) at four different culture times (24, 48, 72 and 168 h). The Zn uptake kinetics of water lettuce were evaluated at two concentrations of Zn (1.8 and 18 mg L-1). The water lettuce attained the highest percentage removal at the lowest evaluated doses (0.7 and 1.8 mg L-1), reaching a maximum value of approximately 72% removal (when cultivated in 1.8 mg L-1 of Zn after 168 h of culture). The Zn uptake increased with culture time, increasing the synthesis of carotenoids at all doses evaluated. The highest doses of Zn resulted in a reduction in photosynthetic efficiency. The results showed a high potential of water lettuce to absorb and tolerate Zn, accumulating preferably in the roots, demonstrating that these plants are able to absorb large quantities of Zn in contaminated solution.

Lead and Cadmium Transfer Factors and the Contamination of Tomato Fruits (Solanum lycopersicum) in a Tropical Mountain Agroecosystem.

The objectives of this study was to diagnose Cd and Pb contamination in soil and to identify the main factors that contribute to the transfer of these elements to tomato plants and fruits and contamination levels of the fruits in tropical mountain conditions. Contamination of the study area soils by Cd and Pb was verified. This contamination stemmed from the intensive use of agricultural inputs, mainly organic fertilizers and soluble mineral fertilizers. The relief of the terrain and inadequate soil management influenced the spatial distribution of these two metals. The Cd concentration in tomato fruits was very low, but the Pb contamination detected in approximately 80% of the fruit samples was considerably higher than the limits that pose a danger to human health. The translocation of Pb to the tomato fruits was associated with the use of organic fertilizer, mainly poultry litter.

Relation between changes in organic matter structure of poultry litter and heavy metals solubility during composting.

Poultry litter (PL) is widely used as fertilizer because of its rich N, P and Ca content. When PL is applied to previously untreated soil, it is a potential contaminant. Composting is an alternative for stabilizing organic and mineral components. This study aimed to elucidate the structural changes and its influence on the solubility of heavy metals in poultry litter during the first 30 d of composting, which is the period when the most intense transformations occur. For this analysis, the transformation dynamics of the organic structures and the availability of mineral elements were studied via spectroscopic characterization, total heavy metal content determination and chemical fractionation at three composting times (0, 15, and 30 d). During composting, the material's aromaticity increased, while its aliphaticity decreased, and the hydrophobicity index increased as the polarity decreased. These results indicate that during the first 30 d, PL composting occurs via transformation of the most labile structures (carbohydrate, peptide and fatty acid fragments), thereby preserving the most stable and least functionalized structures. Composting increased the concentrations of Cu, Cr, Pb and Zn and the transformation of CAlk-O and CAlk-di-O associated with peptides and carbohydrates and favored solubilizing and leaching a water-soluble fraction rich in these compounds. The labile fractions of Fe and the humified organic matter fractions of Cu, Fe, Mn, Zn, and Al increased. The structural changes reduced heavy metal solubility, thus indicating that after 30 d of composting, the heavy metal contamination risk is low.

Spatial variability and solubility of barium in a petroleum well-drilling waste disposal area.

The petroleum industry generates a range of wastes which is often are disposed in soil close to the well location, negatively affecting soil and water quality. The objective of this study was to evaluate the solubility and map the spatial variability of barium in a potentially contaminated area. The study area consisted of a petroleum well-drilling waste disposal site located in the municipality of Mato Rico-PR. A large georeferenced sampling grid was organized. Soil samples were collected at depths of 30, 60, 90, and 120 cm for determination of the "pseudo-total" concentrations and geochemical fractionation of barium. The barium concentrations showed spatial dependence, which permitted the use of geostatistical interpolators. Regarding depth, the depth of 0-30 cm showed the largest contaminated area; however, higher concentrations of barium were found at the depth of 60-90 cm. The results of geochemical fractionation showed that the analyzed samples contained percentages higher than 99% in the non-labile fraction (residual). These results indicate clearly that the barium was in a condition of low solubility, even for samples that had the highest concentrations, presenting low-environmental risk.

Soil heavy metal content on the hillslope region of Rio de Janeiro, Brazil: reference values.

The increasing concern regarding the preservation of soil quality suggests the adoption of quality reference values as a first step in environmental monitoring actions. In this context, the objective of this study was to establish soil quality reference values for Al, Zn, Mn, Cd, Fe, Pb, Ni, Cr, and Cu and to correlate several soil properties with the occurrence of these elements in the hillslope region of Rio de Janeiro State. Sixty-two samples were collected at depths of 0-20 and 20-40 cm and analyzed using the USEPA method 3051A. After clustering analysis, the samples were separated into three groups. Classification functions were obtained based on the variables pH - Mg - Fe - Mn, i.e., the variables that were best related to heavy metal content. We determined to establish the quality reference values for the 75th percentile of the three groups. The relief forms of the area, which facilitate the transport of soil fine-particle fractions and accumulation in lowlands, were revealed to be one of most important factors in understanding the distribution of soil heavy metals.

Soil Factors Related to the Severity of Clubroot in Rio de Janeiro, Brazil.

Soil pH and calcium levels are determining factors in the success or failure of managing clubroot during the cultivation of Brassica spp. The aim of the present study was to evaluate the influence of soil attributes in tropical regions on the development of roots and clubroot and the accumulation of biomass and nutrients in cauliflower. One hundred and fifty-one samples of soil and plants were collected from 16 family farms that have a history of more than 50 years of regular cauliflower cultivation in Nova Friburgo, Rio de Janeiro, Brazil. Chemical and physical analyses were performed on the soil samples, and the severity of clubroot and the accumulation of biomass and macronutrients in individual plants and plant tissues. Clustering and main principal component analyses were performed on the data. The disease occurred on all farms, but with different intensities. A direct relationship was observed for the soil attributes (acidity and exchangeable aluminum content in particular) with the percentage of roots with clubroot and with the accumulation of biomass and macronutrients in the different plant organs. To reduce losses from clubroot in weathered soils, practices should aim to reduce the pathogen's inoculum potential and improve the physical and chemical conditions of the soil, which would favor root development of the plants.

Quality reference values of trace elements in Brazilian organosols.

Determination of the natural background levels of trace elements in organosols and the proposal of quality reference values (QRVs) for these elements are essential for monitoring these soils because they are fragile and subject to change more intensely and rapidly than other soil classes. Given the above information, the objectives of this study were to determine the QRVs of trace elements for organosols and to correlate some soil properties with the occurrence of these elements. Forty organic soil horizon samples from different regions of Brazil were selected to determine the pseudo-total content of trace elements. The samples were separated into three groups according to a cluster analysis. The soil variable Fe and C contents had the strongest influence on the trace element contents in the organosols and were therefore used in the group classification functions. QRVs were proposed according to the 75th percentiles of the groups. The classification functions are a suitable tool for the allocation of new samples into previously established groups and may potentially be used to estimate the degree of organosol degradation.

Growth of the tropical zoanthid Palythoa caribaeorum (Cnidaria: Anthozoa) on reefs in northeastern Brazil.

In Brazilian reefs, zoanthids, especially Palythoa caribaeorum are fundamental for structuring the local benthic community. The objective of this study was to determine the growth rate of P. caribaeorum, and to assess the influence of the site (different beaches), season (dry and wet), location (intertidal or infralittoral zones), and human pressure associated with tourism. For one year we monitored the cover of P. caribaeorum in transects and focused on 20 colonies. We cut off a square (100 cm2) from the central part of the colony and monitored the bare area for four months in each season. The average growth rates varied from 0.015 and 0.021 cm.day(-1). The rate was homogeneous in all localities, and there was no influence from colony site, location, or touristic visitation, showing that the growth velocity may be an intrinsic characteristic of the species, with a strong genetic component. The growth rate of P. caribaeorum differed among months, and peaked in the first month after injury. The average cover varied from 6.2 to 22.9% and was lower on the reef visited by tourists. The present study corroborates the hypothesis that P. caribaeorum is important for coastal reef dynamics due to its fast and continuous growth.

Preliminary hazard assessment of a new nature-inspired antifouling (NIAF) agent.

A recently synthesized aminated 3,4-dioxygenated xanthone (Xantifoul2) was found to have promising antifouling (AF) effects against the settlement of the macrofouler Mytilus galloprovincialis larvae. Preliminary assessment indicated that Xantifoul2 has reduced ecotoxicological impacts: e.g., being non-toxic to the marine crustacea Artemia salina (<10 % mortality at 50 µM) and showing low bioconcentration factor in marine organisms. In order to meet the EU Biocidal Product Regulation, a preliminary hazard assessment of this new nature-inspired antifouling (NIAF) agent was conducted in this work. Xantifoul2 did not affect the swimming ability of the planktonic crustacean Daphnia magna, the growth of the diatom Phaeodactylum tricornutum, and the cellular respiration of luminescent Gram-negative bacteria Vibrio fischeri, supporting the low toxicity towards several non-target marine species. Regarding human cytotoxicity, Xantifoul2 did not affect the cell viability of retinal human cells (hTERT-RPE-1) and lipidomic studies revealed depletion of lipids involved in cell death, membrane modeling, lipid storage, and oxidative stress only at a high concentration (10 µM). Accelerated degradation studies in water were conducted under simulated sunlight to allow the understanding of putative transformation products (TPs) that could be generated in the aquatic ecosystems. Both Xantifoul2 and photolytic-treated Xantifoul2 in the aqueous matrix were therefore evaluated on several nuclear receptors (NRs). The results of this preliminary hazard assessment of Xantifoul2, combined with the high degradation rates in water, provide strong evidence of the safety of this AF agent under the evaluated conditions, and provide the support for future validation studies before this compound can be introduced in the market.

Expression of MRJP3 and HSP70 mRNA Levels in Apis mellifera L. Workers after Dietary Supplementation with Proteins, Prebiotics, and Probiotics.

Royal jelly is an essential substance for the development of bees from larval to adult stages. Studies have identified a group of key proteins in royal jelly, denominated major royal jelly proteins (MRJPs). The group currently consists of nine proteins (MRJP1-MRJP9), with MRJP1 being the most abundant and MRJP3 being used as a microsatellite marker for the selection of queens with a greater production of royal jelly. The diet of bees is mostly composed of proteins, and supplementing this intake to encourage a higher production of their primary product is important for producers. It is estimated that, by adding probiotic and prebiotic organisms to their diets, the benefits to bees will be even greater, both for their immune systems and primary responses to stress. Circumstances that are adverse compared to those of the natural habitat of bees eventually substantially interfere with bee behavior. Stress situations are modulated by proteins termed heat shock proteins (HSPs). Among these, HSP70 has been shown to exhibit abundance changes whenever bees experience unusual situations of stress. Thus, we sought to supplement A. mellifera bee colony diets with proteins and prebiotic and probiotic components, and to evaluate the expression levels of MRJP3 and HSP70 mRNAs using qRT-PCR. The results revealed that differences in the expression of MRJP3 can be attributed to the different types of feed offered. Significant differences were evident when comparing the expression levels of MRJP3 and HSP70, suggesting that protein supplementation with pre/probiotics promotes positive results in royal jelly synthesis carried out by honey bee nurses.

The Transcriptome of the Zoanthid Protopalythoa variabilis (Cnidaria, Anthozoa) Predicts a Basal Repertoire of Toxin-like and Venom-Auxiliary Polypeptides.

Protopalythoa is a zoanthid that, together with thousands of predominantly marine species, such as hydra, jellyfish, and sea anemones, composes the oldest eumetazoan phylum, i.e., the Cnidaria. Some of these species, such as sea wasps and sea anemones, are highly venomous organisms that can produce deadly toxins for preying, for defense or for territorial disputes. Despite the fact that hundreds of organic and polypeptide toxins have been characterized from sea anemones and jellyfish, practically nothing is known about the toxin repertoire in zoanthids. Here, based on a transcriptome analysis of the zoanthid Protopalythoa variabilis, numerous predicted polypeptides with canonical venom protein features are identified. These polypeptides comprise putative proteins from different toxin families: neurotoxic peptides, hemostatic and hemorrhagic toxins, membrane-active (pore-forming) proteins, protease inhibitors, mixed-function venom enzymes, and venom auxiliary proteins. The synthesis and functional analysis of two of these predicted toxin products, one related to the ShK/Aurelin family and the other to a recently discovered anthozoan toxin, displayed potent in vivo neurotoxicity that impaired swimming in larval zebrafish. Altogether, the complex array of venom-related transcripts that are identified in P. variabilis, some of which are first reported in Cnidaria, provides novel insight into the toxin distribution among species and might contribute to the understanding of composition and evolution of venom polypeptides in toxiferous animals.