Impact of nitrogen and phosphorus fertilizers on Cry1Ac protein contents in transgenic cotton

Abstract Application of different fertilizers to check the efficiency of expression of Bt (Bacillus thuringiensis) gene in one of the leading commercialized crops (cotton) against Lepidopteran species is of great concern. The expression of Cry protein level can be controlled by the improvement of nutrients levels. Therefore, the myth of response of Cry toxin to different combinations of NP fertilizers was explored in three Bt cotton cultivars. Combinations include three levels of nitrogen and three levels of phosphorus fertilizers. Immunostrips and Cry gene(s) specific primer based PCR (Polymerase Chain Reaction) analysis were used for the presence of Bt gene that unveiled the presence of Cry1Ac gene only. Further, the ELISA (enzyme-linked immunosorbent assay) kit was used to quantify the expression of Cry1Ac protein. Under various NP fertilizers rates, the level of toxin protein exhibited highly significant differences. The highest toxin level mean was found to be 2.3740 and 2.1732 µg/g under the treatment of N150P75 kg ha-1 combination while the lowest toxin level mean was found to be 0.9158 and 0.7641 µg/g at the N50P25 kg ha-1 level at 80 and 120 DAS (Days After Sowing), respectively. It was concluded from the research that the usage of NP fertilizers has a positive relation with the expression of Cry1Ac toxin in Bt cotton. We recommend using the N150P50 kg ha-1 level as the most economical and practicable fertilizer instead of the standard dose N100P50 kg ha-1 to get the desired level of Cry1Ac level for long lasting plant resistance ( 1.5). The revised dose of fertilizer may help farmers to avoid the cross-resistance development in contradiction of insect pests.

Resumo A aplicação de diferentes fertilizantes para verificar a eficiência da expressão do gene Bt (Bacillus thuringiensis) em uma das principais culturas comercializadas (algodão) contra espécies de lepidópteros é uma grande preocupação. A expressão do nível de proteína Cry pode ser controlada pela melhoria dos níveis de nutrientes. Portanto, o mito da resposta da toxina Cry a diferentes combinações de fertilizantes NP foi explorado em três cultivares de algodão Bt. As combinações incluem três níveis de nitrogênio e três níveis de fertilizantes de fósforo. A análise de PCR (reação em cadeia da polimerase) específica para o gene (s) Immunostrips e Cry (s) foi usada para a presença do gene Bt que revelou a presença do gene Cry1Ac apenas. Além disso, o kit ELISA (ensaio de imunoabsorção enzimática) foi usado para quantificar a expressão da proteína Cry1Ac. Sob várias taxas de fertilizantes NP, o nível de proteína de toxina exibiu diferenças altamente significativas. A média do nível mais alto de toxina foi de 2,3740 e 2,1732 µg / g sob o tratamento da combinação N150P75 kg ha-1, enquanto a média do nível mais baixo de toxina foi de 0,9158 e 0,7641 µg / g no nível de N50P25 kg ha-1 em 80 e 120 DAS (dias após a semeadura), respectivamente. Concluiu-se com a pesquisa que o uso de fertilizantes NP tem relação positiva com a expressão da toxina Cry1Ac no algodão Bt. Recomendamos o uso do nível de N150P50 kg ha-1 como o fertilizante mais econômico e praticável em vez da dose padrão N100P50 kg ha-1 para obter o nível desejado de nível de Cry1Ac para resistência de planta de longa duração ( 1,5). A dose revisada de fertilizante pode ajudar os agricultores a evitar o desenvolvimento de resistência cruzada em contradição com as pragas de insetos.

Transcriptome analysis of the spider Phonotimpus pennimani reveals novel toxin transcripts

Background: Phonotimpus pennimani (Araneae, Phrurolithidae) is a small-sized (3-5 mm) spider endemic to the Tacaná volcano in Chiapas, Mexico, where it is found in soil litter of cloud forests and coffee plantations. Its venom composition has so far not been investigated, partly because it is not a species of medical significance. However, it does have an important impact on the arthropod populations of its natural habitat. Methods: Specimens were collected in Southeastern Mexico (Chiapas) and identified taxonomically by morphological characteristics. A partial sequence from the mitochondrial gene coxI was amplified. Sequencing on the Illumina platform of a transcriptome library constructed from 12 adult specimens revealed 25 toxin or toxinlike genes. Transcripts were validated (RT-qPCR) by assessing the differential expression of the toxin-like PpenTox1 transcript and normalising with housekeeping genes. Results: Analysis of the coxI-gene revealed a similarity to other species of the family Phrurolithidae. Transcriptome analysis also revealed similarity with venom components of species from the families Ctenidae, Lycosidae, and Sicariidae. Expression of the toxinlike PpenTox1 gene was different for each developmental stage (juvenile or adult) and also for both sexes (female or male). Additionally, a partial sequence was obtained for the toxin-like PpenTox1 from DNA. Conclusion: Data from the amplification of the mitochondrial coxI gene confirmed that P. pennimani belongs to the family Phrurolithidae. New genes and transcripts coding for venom components were identified.(AU)

Impact of nitrogen and phosphorus fertilizers on Cry1Ac protein contents in transgenic cotton / Impacto de fertilizantes de nitrogênio e fósforo nos conteúdos de proteína Cry1Ac em algodão transgênico

Abstract Application of different fertilizers to check the efficiency of expression of Bt (Bacillus thuringiensis) gene in one of the leading commercialized crops (cotton) against Lepidopteran species is of great concern. The expression of Cry protein level can be controlled by the improvement of nutrients levels. Therefore, the myth of response of Cry toxin to different combinations of NP fertilizers was explored in three Bt cotton cultivars. Combinations include three levels of nitrogen and three levels of phosphorus fertilizers. Immunostrips and Cry gene(s) specific primer based PCR (Polymerase Chain Reaction) analysis were used for the presence of Bt gene that unveiled the presence of Cry1Ac gene only. Further, the ELISA (enzyme-linked immunosorbent assay) kit was used to quantify the expression of Cry1Ac protein. Under various NP fertilizers rates, the level of toxin protein exhibited highly significant differences. The highest toxin level mean was found to be 2.3740 and 2.1732 µg/g under the treatment of N150P75 kg ha-1 combination while the lowest toxin level mean was found to be 0.9158 and 0.7641 µg/g at the N50P25 kg ha-1 level at 80 and 120 DAS (Days After Sowing), respectively. It was concluded from the research that the usage of NP fertilizers has a positive relation with the expression of Cry1Ac toxin in Bt cotton. We recommend using the N150P50 kg ha-1 level as the most economical and practicable fertilizer instead of the standard dose N100P50 kg ha-1 to get the desired level of Cry1Ac level for long lasting plant resistance (<1.5). The revised dose of fertilizer may help farmers to avoid the cross-resistance development in contradiction of insect pests.

Resumo A aplicação de diferentes fertilizantes para verificar a eficiência da expressão do gene Bt (Bacillus thuringiensis) em uma das principais culturas comercializadas (algodão) contra espécies de lepidópteros é uma grande preocupação. A expressão do nível de proteína Cry pode ser controlada pela melhoria dos níveis de nutrientes. Portanto, o mito da resposta da toxina Cry a diferentes combinações de fertilizantes NP foi explorado em três cultivares de algodão Bt. As combinações incluem três níveis de nitrogênio e três níveis de fertilizantes de fósforo. A análise de PCR (reação em cadeia da polimerase) específica para o gene (s) Immunostrips e Cry (s) foi usada para a presença do gene Bt que revelou a presença do gene Cry1Ac apenas. Além disso, o kit ELISA (ensaio de imunoabsorção enzimática) foi usado para quantificar a expressão da proteína Cry1Ac. Sob várias taxas de fertilizantes NP, o nível de proteína de toxina exibiu diferenças altamente significativas. A média do nível mais alto de toxina foi de 2,3740 e 2,1732 µg / g sob o tratamento da combinação N150P75 kg ha-1, enquanto a média do nível mais baixo de toxina foi de 0,9158 e 0,7641 µg / g no nível de N50P25 kg ha-1 em 80 e 120 DAS (dias após a semeadura), respectivamente. Concluiu-se com a pesquisa que o uso de fertilizantes NP tem relação positiva com a expressão da toxina Cry1Ac no algodão Bt. Recomendamos o uso do nível de N150P50 kg ha-1 como o fertilizante mais econômico e praticável em vez da dose padrão N100P50 kg ha-1 para obter o nível desejado de nível de Cry1Ac para resistência de planta de longa duração (<1,5). A dose revisada de fertilizante pode ajudar os agricultores a evitar o desenvolvimento de resistência cruzada em contradição com as pragas de insetos.

Impact of nitrogen and phosphorus fertilizers on Cry1Ac protein contents in transgenic cotton / Impacto de fertilizantes de nitrogênio e fósforo nos conteúdos de proteína Cry1Ac em algodão transgênico

Application of different fertilizers to check the efficiency of expression of Bt (Bacillus thuringiensis) gene in one of the leading commercialized crops (cotton) against Lepidopteran species is of great concern. The expression of Cry protein level can be controlled by the improvement of nutrients levels. Therefore, the myth of response of Cry toxin to different combinations of NP fertilizers was explored in three Bt cotton cultivars. Combinations include three levels of nitrogen and three levels of phosphorus fertilizers. Immunostrips and Cry gene(s) specific primer based PCR (Polymerase Chain Reaction) analysis were used for the presence of Bt gene that unveiled the presence of Cry1Ac gene only. Further, the ELISA (enzyme-linked immunosorbent assay) kit was used to quantify the expression of Cry1Ac protein. Under various NP fertilizers rates, the level of toxin protein exhibited highly significant differences. The highest toxin level mean was found to be 2.3740 and 2.1732 µg/g under the treatment of N150P75 kg ha-¹ combination while the lowest toxin level mean was found to be 0.9158 and 0.7641 µg/g at the N50P25 kg ha-¹ level at 80 and 120 DAS (Days After Sowing), respectively. It was concluded from the research that the usage of NP fertilizers has a positive relation with the expression of Cry1Ac toxin in Bt cotton. We recommend using the N150P50 kg ha-1 level as the most economical and practicable fertilizer instead of the standard dose N100P50 kg ha-¹ to get the desired level of Cry1Ac level for long lasting plant resistance (<1.5). The revised dose of fertilizer may help farmers to avoid the cross-resistance development in contradiction of insect pests.

A aplicação de diferentes fertilizantes para verificar a eficiência da expressão do gene Bt (Bacillus thuringiensis) em uma das principais culturas comercializadas (algodão) contra espécies de lepidópteros é uma grande preocupação. A expressão do nível de proteína Cry pode ser controlada pela melhoria dos níveis de nutrientes. Portanto, o mito da resposta da toxina Cry a diferentes combinações de fertilizantes NP foi explorado em três cultivares de algodão Bt. As combinações incluem três níveis de nitrogênio e três níveis de fertilizantes de fósforo. A análise de PCR (reação em cadeia da polimerase) específica para o gene (s) Immunostrips e Cry (s) foi usada para a presença do gene Bt que revelou a presença do gene Cry1Ac apenas. Além disso, o kit ELISA (ensaio de imunoabsorção enzimática) foi usado para quantificar a expressão da proteína Cry1Ac. Sob várias taxas de fertilizantes NP, o nível de proteína de toxina exibiu diferenças altamente significativas. A média do nível mais alto de toxina foi de 2,3740 e 2,1732 µg / g sob o tratamento da combinação N150P75 kg ha-¹, enquanto a média do nível mais baixo de toxina foi de 0,9158 e 0,7641 µg / g no nível de N50P25 kg ha-¹ em 80 e 120 DAS (dias após a semeadura), respectivamente. Concluiu-se com a pesquisa que o uso de fertilizantes NP tem relação positiva com a expressão da toxina Cry1Ac no algodão Bt. Recomendamos o uso do nível de N150P50 kg ha-¹ como o fertilizante mais econômico e praticável em vez da dose padrão N100P50 kg ha-¹ para obter o nível desejado de nível de Cry1Ac para resistência de planta de longa duração (<1,5). A dose revisada de fertilizante pode ajudar os agricultores a evitar o desenvolvimento de resistência cruzada em contradição com as pragas de insetos.

Impact of nitrogen and phosphorus fertilizers on Cry1Ac protein contents in transgenic cotton / Impacto de fertilizantes de nitrogênio e fósforo nos conteúdos de proteína Cry1Ac em algodão transgênico

Application of different fertilizers to check the efficiency of expression of Bt (Bacillus thuringiensis) gene in one of the leading commercialized crops (cotton) against Lepidopteran species is of great concern. The expression of Cry protein level can be controlled by the improvement of nutrients levels. Therefore, the myth of response of Cry toxin to different combinations of NP fertilizers was explored in three Bt cotton cultivars. Combinations include three levels of nitrogen and three levels of phosphorus fertilizers. Immunostrips and Cry gene(s) specific primer based PCR (Polymerase Chain Reaction) analysis were used for the presence of Bt gene that unveiled the presence of Cry1Ac gene only. Further, the ELISA (enzyme-linked immunosorbent assay) kit was used to quantify the expression of Cry1Ac protein. Under various NP fertilizers rates, the level of toxin protein exhibited highly significant differences. The highest toxin level mean was found to be 2.3740 and 2.1732 µg/g under the treatment of N150P75 kg ha-¹ combination while the lowest toxin level mean was found to be 0.9158 and 0.7641 µg/g at the N50P25 kg ha-¹ level at 80 and 120 DAS (Days After Sowing), respectively. It was concluded from the research that the usage of NP fertilizers has a positive relation with the expression of Cry1Ac toxin in Bt cotton. We recommend using the N150P50 kg ha-1 level as the most economical and practicable fertilizer instead of the standard dose N100P50 kg ha-¹ to get the desired level of Cry1Ac level for long lasting plant resistance (<1.5). The revised dose of fertilizer may help farmers to avoid the cross-resistance development in contradiction of insect pests.(AU)

A aplicação de diferentes fertilizantes para verificar a eficiência da expressão do gene Bt (Bacillus thuringiensis) em uma das principais culturas comercializadas (algodão) contra espécies de lepidópteros é uma grande preocupação. A expressão do nível de proteína Cry pode ser controlada pela melhoria dos níveis de nutrientes. Portanto, o mito da resposta da toxina Cry a diferentes combinações de fertilizantes NP foi explorado em três cultivares de algodão Bt. As combinações incluem três níveis de nitrogênio e três níveis de fertilizantes de fósforo. A análise de PCR (reação em cadeia da polimerase) específica para o gene (s) Immunostrips e Cry (s) foi usada para a presença do gene Bt que revelou a presença do gene Cry1Ac apenas. Além disso, o kit ELISA (ensaio de imunoabsorção enzimática) foi usado para quantificar a expressão da proteína Cry1Ac. Sob várias taxas de fertilizantes NP, o nível de proteína de toxina exibiu diferenças altamente significativas. A média do nível mais alto de toxina foi de 2,3740 e 2,1732 µg / g sob o tratamento da combinação N150P75 kg ha-¹, enquanto a média do nível mais baixo de toxina foi de 0,9158 e 0,7641 µg / g no nível de N50P25 kg ha-¹ em 80 e 120 DAS (dias após a semeadura), respectivamente. Concluiu-se com a pesquisa que o uso de fertilizantes NP tem relação positiva com a expressão da toxina Cry1Ac no algodão Bt. Recomendamos o uso do nível de N150P50 kg ha-¹ como o fertilizante mais econômico e praticável em vez da dose padrão N100P50 kg ha-¹ para obter o nível desejado de nível de Cry1Ac para resistência de planta de longa duração (<1,5). A dose revisada de fertilizante pode ajudar os agricultores a evitar o desenvolvimento de resistência cruzada em contradição com as pragas de insetos.(AU)

Toxinology in the proteomics era: a review on arachnid venom proteomics

The word venomics was coined to acknowledge the studies that use omics to investigate venom proteins and peptides. Venomics has evolved considerably over the last 20 years. The first works on scorpion or spider venomics were published in the early 2000's. Such studies relied on peptide mass fingerprinting (PMF) to characterize venom complexity. After the introduction of new mass spectrometers with higher resolution, sensitivity and mass accuracy, and the next-generation nucleotide sequencing, the complexity of data reported in research on scorpion and spider venomics increased exponentially, which allowed more comprehensive studies. In the present review article, we covered key publications on scorpion venomics and spider venomics, presenting historical grounds and implemented technologies over the last years. The literature presented in this review was selected after searching the PubMed database using the terms "(scorpion venom) AND (proteome)" for scorpion venomics, and "(spider venom) AND (proteome)" for publications on spider venomics. We presented the key aspects related to proteomics in the covered papers including, but not restricted to, the employed proteomic strategy (i.e., PMF, two-dimensional gel electrophoresis, shotgun/bottom-up and/or top-down/peptidome), and the type of mass spectrometer used. Some conclusions can be drawn from the present study. For example, the scorpion genus Tityus is the most studied concerning venomics, followed by Centruroides; whereas for spiders the studied genera were found more equally distributed. Another interesting conclusion is the lack of high throughput studies on post-translational modifications (PTMs) of scorpion and spider proteins. In our opinion, PTMs should be more studied as they can modulate the activity of scorpion and spider toxins.(AU)

Recombinant expression and antigenicity of two peptide families of neurotoxins from Androctonus sp

Background: Scorpion neurotoxins such as those that modify the mammalian voltagegated sodium ion channels (Nav) are the main responsible for scorpion envenomation. Their neutralization is crucial in the production of antivenoms against scorpion stings. Methods: In the present study, two in silico designed genes ­ one that codes for a native neurotoxin from the venom of the Anatolian scorpion Androctonus crassicauda, named Acra 4 ­ and another non-native toxin ­ named consensus scorpion toxin (SccTx) obtained from the alignment of the primary structures of the most toxic neurotoxins from the Middle Eastern and North African scorpions ­ were recombinantly expressed in E. coli Origami. Results: Following bacterial expression, the two expressed neurotoxins, hereafter named HisrAcra4 and HisrSccTx, were obtained from inclusion bodies. Both recombinant neurotoxins were obtained in multiple Cys-Cys isoforms. After refolding, the active protein fractions were identified with molecular masses of 8,947.6 and 9,989.1 Da for HisrAcra4 and HisrSccTx, respectively, which agreed with their expected theoretical masses. HisrAcra4 and HisrSccTx were used as antigens to immunize two groups of rabbits, to produce either anti-HisrAcra4 or anti-HisrSccTx serum antibodies, which in turn could recognize and neutralize neurotoxins from venoms of scorpion species from the Middle East and North Africa. The antibodies obtained from rabbits neutralized the 3LD50 of Androctonus australis, Leiurus quinquestriatus hebraeus and Buthus occitanus venoms, but they did not neutralize A. crassicauda and A. mauritanicus venoms. In addition, the anti-HisrAcra4 antibodies did not neutralize any of the five scorpion venoms tested. However, an antibody blend of anti-HisrAcra4 and anti-HisrSccTx was able to neutralize A. crassicauda and A. mauritanicus venoms. Conclusions: Two recombinant Nav neurotoxins, from different peptide families, were used as antigens to generate IgGs for neutralizing scorpion venoms of species from the Middle East and North Africa.(AU)

Voracity, reaction to stings, and survival of domestic hens when feeding on the yellow scorpion (Tityus serrulatus)

Scorpionism is a worldwide problem that has already made thousands of victims, and multi-disciplinary approaches for controlling their populations are to be more successful. Hens are often mentioned as tools for controlling scorpions; however, systematic/experimental behavioral studies are not available. Moreover, there is no systematic information on the effect of scorpion venoms on hens. Using the venomous yellow scorpion Tityus serrulatus, the present study aimed to clarify the following aspects: (1) voracity of hens, (2) how hens react when stung, (3) the effect of scorpion stings on hen behavior during attacks, and (4) hen survivorship after feeding on scorpions. Methods: We attracted hens with corn powder, offered them scorpions and then recorded the hen-scorpion interaction. To test the effects of the sting we manually removed the scorpion's telson. Results: We found that some hens ate up to six scorpions within minutes. By means of an ethogram and drawings, we showed that they exhibited several aversive behaviors when capturing scorpions. Removal of the scorpion telson stopped the aversive reactions, which was not observed in the control group. Finally, hens did not exhibit atypical behaviors after 1, 7 and 30 days and were all alive after 30 days. Conclusion: This is the first empirical and video recorded study providing evidence that hens are clearly affected by scorpion venom but do not die. Therefore, they may have potential to be used in biological control of these arthropods.(AU)

Proteinograma de ovelhas imunizadas com veneno detoxificado do escorpião Tityus serrulatus / Proteinogram of immunized sheep with detoxified Tityus serrulatus scorpion venom

Background: Scorpionism is a worldwide medical issue, especially relevant in the tropical and subtropical countries. Tityusserrulatus is the species responsible for most cases in Brazil. Antivenom administration to victims is the sole specific therapyobtained from donor animals. Most of these donors suffer with symptoms of the poisoning, debilitating their health andreducing their life expectancy. The aim of the present research was to evaluate whether the immunogens prepared fromthe crude and detoxified venom of T. serrulatus promoted different changes in fractionated sheep plasma proteins, duringa scorpion antivenom serum production.Materials, Methods & Results: Twelve sheep, healthy, mean weight of 30 kg, were distributed into 3 groups (n = 4): G1(control), G2 (crude venom) and G3 (detoxified venom). The adopted immunization protocol (first cycle) had 6 doses, 3using Freund’s adjuvant, with a 21-day interval between each one (day 0, 22 and 43), and 3 doses with no adjuvant (booster)and 0.2 mg of antigen (reinforcement), spaced 3 days between each other (day 50, 53 and 56). Group control (G1) received6 immunizations with phosphate buffered saline (PBS) associated with Freund’s adjuvant (1:1), while the other 2 groupsreceived 0.5 mg of venom (G2) and detoxified venom (G3), respectively, diluted in PBS, associated with the Freund adjuvant. The boosters were 1/3 of the initial dose, diluted only PBS. At baseline (T0) and at 24 and 48 h after immunization,all animals underwent clinical examinations. Blood samples were collected at day 0, 22, 43, 53 and 56 for proteinogramanalysis. Total protein, albumin and globulins fractions were measured. Plasma albumin concentration at T0 ranged from3.41-4.86 g/dL, with a mean value of 4.12 g/dL. There was no statistical difference between...

ß-micrustoxin (Mlx-9), a PLA2 from Micrurus lemniscatus snake venom: biochemical characterization and anti-proliferative effect mediated by p53

Background: Endogenous phospholipases A2 (PLA2 ) play a fundamental role in inflammation, neurodegenerative diseases, apoptosis and cellular senescence. Neurotoxins with PLA2 activity are found in snake venoms from the Elapidae and Viperidae families. The mechanism of action of these neurotoxins have been studied using hippocampal and cerebellar neuronal cultures showing [Ca2+]i increase, mitochondrial depolarization and cell death. Astrocytes are rarely used as a model, despite being modulators at the synapses and responsible for homeostasis and defense in the central nervous system. Preserving the cell division ability, they can be utilized to study the cell proliferation process. In the present work cultured astrocytes and glioblastoma cells were employed to characterize the action of ß-micrustoxin (previously named Mlx-9), a PLA2 isolated from Micrurus lemniscatus snake venom. The ß-micrustoxin structure was determined and the cell proliferation, cell cycle phases and the regulatory proteins p53, p21 and p27 were investigated. Methods: ß-micrustoxin was characterized biochemically by a proteomic approach. Astrocytes were obtained by dissociation of pineal glands from Wistar rats; glioblastoma tumor cells were purchased from ATCC and Sigma and cultured in DMEM médium. Cell viability was evaluated by MTT assay; cell proliferation and cell cycle phases were analyzed by flow cytometry; p53, p21 and p27 proteins were studied by western blotting and immunocytochemistry. Results: Proteomic analysis revealed fragments on ß-micrustoxin that aligned with a PLA2 from Micrurus lemniscatus lemniscatus previously identified as transcript ID DN112835_C3_g9_i1/m.9019. ß-micrustoxin impaired the viability of astrocytes and glioblastoma tumor cells. There was a reduction in cell proliferation, an increase in G2/M phase and activation of p53, p21 and p27 proteins in astrocytes. Conclusion: These findings indicate that ß-micrustoxin from Micrurus lemniscatus venom could inhibit cell proliferation through p53, p21 and p27 activation thus imposing cell cycle arrest at the checkpoint G2/M.(AU)

Comparative venomic profiles of three spiders of the genus Phoneutria

Spider venoms induce different physio-pharmacological effects by binding with high affinity on molecular targets, therefore being of biotechnological interest. Some of these toxins, acting on different types of ion channels, have been identified in the venom of spiders of the genus Phoneutria, mainly from P. nigriventer. In spite of the pharmaceutical potential demonstrated by P. nigriventer toxins, there is limited information on molecules from venoms of the same genus, as their toxins remain poorly characterized. Understanding this diversity and clarifying the differences in the mechanisms of action of spider toxins is of great importance for establishing their true biotechnological potential. This prompted us to compare three different venoms of the Phoneutria genus: P. nigriventer (Pn-V), P. eickstedtae (Pe-V) and P. pertyi (Pp-V). Methods: Biochemical and functional comparison of the venoms were carried out by SDS-PAGE, HPLC, mass spectrometry, enzymatic activities and electrophysiological assays (whole-cell patch clamp). Results: The employed approach revealed that all three venoms had an overall similarity in their components, with only minor differences. The presence of a high number of similar proteins was evident, particularly toxins in the mass range of ~6.0 kDa. Hyaluronidase and proteolytic activities were detected in all venoms, in addition to isoforms of the toxins Tx1 and Tx2-6. All Tx1 isoforms blocked Nav1.6 ion currents, with slight differences. Conclusion: Our findings showed that Pn-V, Pe-V and Pp-V are highly similar concerning protein composition and enzymatic activities, containing isoforms of the same toxins sharing high sequence homology, with minor modifications. However, these structural and functional variations are very important for venom diversity. In addition, our findings will contribute to the comprehension of the molecular diversity of the venoms of the other species from Phoneutria genus, exposing their biotechnological potential as a source for searching for new active molecules.(AU)

Antimicrobial activity and partial chemical structure of acylpolyamines isolated from the venom of the spider Acanthoscurria natalensis

Background: Acylpolyamines are one of the main non-peptide compounds present in spider venom and represent a promising alternative in the search for new molecules with antimicrobial action. Methods: The venom of Acanthoscurria natalensis spider was fractionated by reverse-phase liquid chromatography (RP-HPLC) and the antimicrobial activity of the fractions was tested using a liquid growth inhibition assay. The main antimicrobial fraction containing acylpolyamines (ApAn) was submitted to two additional chromatographic steps and analyzed by MALDI-TOF. Fractions of interest were accumulated for ultraviolet (UV) spectroscopy and ESI-MS/MS analysis and for minimum inhibitory concentration (MIC) and hemolytic activity determination. Results: Five acylpolyamines were isolated from the venom with molecular masses between 614 Da and 756 Da, being named ApAn728, ApAn614a, ApAn614b, ApAn742 and ApAn756. The analysis of UV absorption profile of each ApAn and the fragmentation pattern obtained by ESI-MS/MS suggested the presence of a tyrosyl unit as chromophore and a terminal polyamine chain consistent with structural units PA43 or PA53. ApAn presented MIC between 128 µM and 256 µM against Escherichia coli and Staphylococcus aureus, without causing hemolysis against mouse erythrocytes. Conclusion: The antimicrobial and non-hemolytic properties of the analyzed ApAn may be relevant for their application as possible therapeutic agents and the identification of an unconventional chromophore for spider acylpolyamines suggests an even greater chemical diversity.(AU)

Type C botulism in dogs from rural properties located in Goiânia, Brazil

Botulism is a disease usually fatal, caused by the ingestion of neurotoxins produced by Clostridium botulinum. In dogs, intoxication is caused by the ingestion of botulinum toxin type C, and animals often recover spontaneously. The present study describes the occurrence of type C botulism in two dogs domiciled on neighboring rural properties in the municipality of Goiânia, state of Goiás, Brazil, probably associated with ingestion of decomposing bovine carcass. Upon clinical evaluation, the dogs were alert in the lateral decubitus position with ascending flaccid paralysis, absence of eyelid reflexes, and reduced muscle tone. Due to their worsening clinical symptoms, the animals died within 12 h and 3 days after supportive treatment. Botulinum toxin type C was identified, in the serum and feces of both dogs, by seroneutralization in mice with homologous monovalent antitoxin. The results of the high-throughput gene sequencing showed that the abundance of C. botulinum in the fecal microbiota of one of the affected dogs was low (0.53%). In this way, the present study highlights the need of sanitary practices related to the appropriate collection and disposal of bovine carcasses in rural areas since they represent a risk factor for the occurrence of botulism in dogs domiciled on rural properties.

Antimicrobial resistance of Shiga toxin­producing Escherichia coliisolated from sheep / Escherichia coli produtora de toxina Shiga resistentes a antimicrobianos isoladas de ovinos

The presence of Shiga toxin-producing Escherichia coli (STEC) and resistance to beta-lactams in healthy sheep represents a potential public health risk. This study aimed to characterize STEC isolates in sheep feces for toxin production and resistance to beta-lactam antibiotics. In the present study, among the 40 isolates, we found a predominance of subtype Stx1 (22/40), followed by subtype Stx1 + Stx2 (11/40), while the less prevalent group was Stx2 (7/40). Also, we found phenotypical resistance to beta-lactam antibiotics in 50% (20/40) of the strains analyzed, forming two groups, one with resistant isolates and the other with non-resistant isolates. The cytotoxicity of the isolates did not vary among the groups. In addition to having this characteristic, some multiresistant isolates produced significant amounts of toxins. This leads to the conclusion that the mechanisms of antimicrobial resistance via beta-lactamases are present in sheep STEC and that the cytotoxicity of those isolates is variable regarding such resistance.(AU)

A presença de Escherichia coli produtora de toxina Shiga (STEC) e resistente a beta-lactâmicos em ovinos saudáveis, representa um risco potencial para a saúde pública. O objetivo deste estudo foi caracterizar isolados STEC presentes em fezes de ovinos, quanto a produção de toxina, bem como a resistência aos antibióticos beta-lactâmicos. No presente estudo, dentre os quarenta isolados, foi observada a predominância do subtipo Stx1 (22/40), seguido do subtipo Stx1+ Stx2 (11/40), enquanto o grupo menos prevalente foi Stx2 (7/40). A resistência fenotípica aos antibióticos beta-lactâmicos foi observada em 50% (20/40) das cepas analisadas, formando dois grupos, um com isolados resistentes e outro de isolados não resistentes. A citotoxicidade dos isolados não variou entre os grupos. Alguns isolados multirresistentes, além de possuírem essa característica, produziram quantidades significativas de toxinas. Isto conclui, que os mecanismos de resistencia antimicrobiana, por meio de beta-lactamases estão presentes em STEC de ovinos, e que a citotoxicidade desses isolados é variável com relação a esta resistência.(AU)

Compositional and toxicological investigation of pooled venom from farm-raised Naja atra

Background: Naja atra is a venomous snake species medically relevant in China. In the current study, we evaluated the composition and toxicological profile of venom collected from farm-raised N. atra. Methods: Venom was collected from third-generation captive bred N. atra on a snake farm in Hunan Province, China. The venom was analyzed using sodium dodecyl sulfate polyacrylamide gel electrophoresis and nano-liquid chromatography with electrospray ionization tandem mass spectrometry. In addition, hemolytic activity, median lethal dose, serum biochemical and histopathological parameters were accessed. Results: N. atra venom proteome was dominated by phospholipase A2 (46.5%) and three-finger toxins (41.4 %), and a set of common low relative abundance proteins, including cysteine-rich secretory proteins (4.7%), NGF-beta (2.4%), snake venom metalloproteinase (1.5%), glutathione peroxidase (0.6%), vespryn (0.3%), and 5ʹ-nucleotidases (0.2%) were also found. Furthermore, the venom exhibited direct hemolytic activity, neurotoxicity, myotoxicity, and high lethal potency in mice, with a subcutaneous median lethal dose of 1.02 mg/kg. Histopathological analysis and serum biochemical tests revealed that venom caused acute hepatic, pulmonary and renal injury in mice. Conclusion: This study revealed the composition and toxicity of venom collected from farm-raised N. atra, thereby providing a reference for the analysis of venom samples collected from captive-born venomous snakes in the future.(AU)

Overview of protein posttranslational modifications in Arthropoda venoms

Accidents with venomous animals are a public health issue worldwide. Among the species involved in these accidents are scorpions, spiders, bees, wasps, and other members of the phylum Arthropoda. The knowledge of the function of proteins present in these venoms is important to guide diagnosis, therapeutics, besides being a source of a large variety of biotechnological active molecules. Although our understanding about the characteristics and function of arthropod venoms has been evolving in the last decades, a major aspect crucial for the function of these proteins remains poorly studied, the posttranslational modifications (PTMs). Comprehension of such modifications can contribute to better understanding the basis of envenomation, leading to improvements in the specificities of potential therapeutic toxins. Therefore, in this review, we bring to light protein/toxin PTMs in arthropod venoms by accessing the information present in the UniProtKB/Swiss-Prot database, including experimental and putative inferences. Then, we concentrate our discussion on the current knowledge on protein phosphorylation and glycosylation, highlighting the potential functionality of these modifications in arthropod venom. We also briefly describe general approaches to study "PTM-functional-venomics", herein referred to the integration of PTM-venomics with a functional investigation of PTM impact on venom biology. Furthermore, we discuss the bottlenecks in toxinology studies covering PTM investigation. In conclusion, through the mining of PTMs in arthropod venoms, we observed a large gap in this field that limits our understanding on the biology of these venoms, affecting the diagnosis and therapeutics development. Hence, we encourage community efforts to draw attention to a better understanding of PTM in arthropod venom toxins.(AU)

S- and P-type cobra venom cardiotoxins differ in their action on isolated rat heart

Background: The cardiovascular system is one of the first systems to be affected by snake toxins; but not many toxins exert a direct effect on the heart. Cobra venom cardiotoxins are among those few toxins that attack the heart. Although the two cardiotoxin types (S and P) differ in their central-loop structure, it is not known whether they differ in their effect on the mammalian heart. We compared the effects of S- and P-type cardiotoxins, CTÐ¥-1 and CTÐ¥-2, respectively, from the cobra Naja oxiana, on the isolated rat heart. Methods: An isolated rat heart perfused according to the Langendorff technique was used in this study to investigate the activity of cardiotoxins CTX-1 and CTX-2. The following parameters were registered: the left ventricular developed pressure, calculated as the difference between systolic and diastolic pressure in the left ventricle, the end-diastolic pressure, the heart rate, time to maximal end-diastolic pressure (heart contracture), and time to depression of the heart contraction. Results: Both cardiotoxins at the concentration of 5 µg/mL initially produce a slight increase in systolic intraventricular pressure, followed by its rapid decrease with a simultaneous increase in diastolic intraventricular pressure until reaching contracture. CTX-2 blocks cardiac contractions faster than CTX-1; in its presence the maximum diastolic pressure is reached faster and the magnitude of the developed contracture is higher. Conclusion: The P-type cardiotoxin CTX-2 more strongly impairs rat heart functional activity than the S-type cardiotoxin CTX-1, as expressed in its faster blockage of cardiac contractions as well as in more rapid development and greater magnitude of contracture in its presence.(AU)

Antimicrobial susceptibility of Salmonella spp and Staphylococcus aureus isolated from beef sold in Campo Grande, Mato Grosso do Sul, Brazil / Suscetibilidade antimicrobiana de Salmonella spp e Staphylococcus aureus isolados de carnes bovinas comercializadas em Campo Grande, Mato Grosso do Sul, Brasil

Hygiene failures in meat can be identified based on the evaluation of pathogenic microorganisms, which compromise the microbiological quality of food and can transmit food-borne diseases. The aim of the present study was to evaluate the hygienic quality of beef sold at supermarkets, butcher shops and public markets in the city of Campo Grande, state of Mato Grosso do Sul, Brazil, through the phenotypic and genotypic characterization of Salmonella spp. and Shiga toxin-producing Escherichia coli (STEC) as well as the investigation and quantification of Staphylococcus aureus. Seventy-one samples of beef from 17 commercial establishments were evaluated. Isolates were tested for antimicrobial susceptibility using the disk diffusion method recommended by the Clinical & Laboratory Standards Institute. Salmonella was found in 7.04% of the samples and 70.0% of the isolates were sensitive to the antimicrobials tested. A total of 25.35% of the samples were positive for Staphylococcus aureus, with counts ranging from 1.0 x 102 to 4.3 x 104 CFU/g; these isolates exhibited resistance to penicillin (87.5%), tetracycline (18.75%) and chloramphenicol (6.25%). None of the samples was positive for STEC. The detection of these pathogens in food poses a danger to public health, mainly due to the presence of antimicrobial-resistant isolates. These findings underscore the need for good hygiene and manufacturing practices at retail establishments.

As falhas na qualidade higiênico-sanitária da carne podem ser identificadas a partir da avaliação de microrganismos patogênicos que comprometem a qualidade microbiológica do alimento e podem veicular doenças de origem alimentar. O presente estudo objetivou avaliar a qualidade higiênica-sanitária de carnes bovinas comercializadas em supermercados, açougues e mercados públicos da cidade de Campo Grande (Mato Grosso do Sul, Brasil) por meio da pesquisa e caracterização fenotípica e genotípica de Salmonella spp. e Escherichia coli produtora de toxina Shiga (STEC) e pesquisa e contagem de Staphylococcus aureus. Foram avaliadas 71 amostras de carne bovina de 17 estabelecimentos comerciais que foram submetidas a pesquisa de detecção de Salmonella spp., Escherichia coli produtora de toxina Shiga (STEC) e pesquisa e contagem de Staphylococcus aureus. Os isolados obtidos foram submetidos ao perfil de sensibilidade aos antimicrobianos pelo teste de difusão em disco, de acordo com o Clinical & Laboratory Standards Institute (CLSI). Constatou-se a presença de Salmonella em 7,04% das amostras avaliadas, sendo que 70,0% dos isolados foram sensíveis aos antimicrobianos testados. Em relação ao Staphylococcus aureus, 25,35% das amostras foram positivas com contagens variando entre 1,0 x 102 a 4,3 x 104 UFC/g, sendo que os isolados apresentaram resistência para penicilina (62,5%), tetraciclina (18,75%) e cloranfenicol (6,25%). Nenhuma amostra apresentou-se positiva para STEC. A detecção desses patógenos em alimentos representa um perigo a saúde pública, principalmente, devido a presença de isolados resistentes a antimicrobianos. Além disso, ressalta-se a necessidade do emprego das boas práticas de higiene e fabricação nos estabelecimentos varejistas.

Spodoptera frugiperda (Noctuidae) fed on transgenic maize can transfer Bt proteins to Podisus nigrispinus (Pentatomidae)

An important concern with the use of genetically modified (GM) plants expressing Bacillus thuringiensis (Bt) insecticidal toxins is the deleterious effect on non-target organisms. The predatory stink bug Podisus nigrispinus (Dallas) (Hemiptera: Pentatomidae) is used in biological control programs and may be exposed to Bt toxins. This study evaluated the indirect effects of different Cry proteins on P. nigrispinus with the prey Spodoptera frugiperda (J.E. Smith, 1797) (Lepidoptera: Noctuidae), fed on simple or pyramided Bt maize genotypes. The experiment was carried out in a completely randomized design with three treatments: i) Isohybrid (not Bt), ii) Herculex® (transgenic maize encoding Cry1F protein) and iii) PowerCore® (pyramidal transgenic maize encoding the Cry1F, Cry1A.105, and Cry2Ab2 proteins), which were used to feed S. frugiperda for 48 h. The caterpillars were used as prey by P. nigrispinus females. We evaluated the presence of Cry proteins, consumed prey biomass (predation), oviposition period, number of postures, number of eggs, number of eggs per posture, number of nymphs, egg viability, embryonic period, female longevity and development, and survival rates of immature. The results show that different Cry proteins move through the food chain of P. nigrispinus and provide evidence that the ingestion of three different proteins does not lead to unexpected synergistic effects. However, Cry toxins promoted histopathological changes in midgut cells of P. nigrispinus.

Achados post mortem em cães intoxicados por picadas de abelhas / Post mortem findings in dogs intoxicated by bee stings

As intoxicações por picadas de abelhas possuem grande relevância na medicina humana e veterinária. Os componentes da toxina apresentam ações lesivas aos tecidos, principalmente aos rins, e podem culminar com a morte, mesmo quando a dose inoculada é pequena. A identificação precoce desse tipo de intoxicação permite a implementação de medidas terapêuticas adequadas e a melhoria do prognóstico. Desta forma, o presente estudo foi desenvolvido com o objetivo de relatar os achados anatomopatológicos observados em dois cães, sem raça definida, os quais foram vítimas fatais de picadas de abelhas. Os animais eram irmãos de ninhada, um macho e uma fêmea, com três anos de idade e com cerca de 30kg. Os cães foram encontrados mortos no período da noite, já em rigor mortis, o que conduziu à suspeita de que a morte havia ocorrido há, no máximo, cinco horas. As principais lesões macroscópicas observadas foram: petéquias cutâneas, algumas associadas à presença de ferrões; congestão generalizada; hemorragia; necrose e edema; assim como insetos com morfologia compatível com Apis mellifera dispersos no trato gastrointestinal. Microscopicamente, degeneração, necrose e hemorragias renais constituíram os achados de maior importância, além de acentuado edema pulmonar, ao qual foi atribuída a causa mortis. Assim, as alterações mais importantes neste tipo de intoxicação são necrose, hemorragia, edema e congestão. Além disso, o óbito pode ocorrer de forma rápida, mesmo com baixas doses da toxina.

Bee sting poisonings have great relevance for both human and veterinary medicine. The toxin components present harmful actions on tissues, especially on kidneys, and can lead to death, even when the inoculated dose is small. The early identification of this type of intoxication allows the implementation of appropriate therapeutic measures and improvement of the prognosis. Thus, the present study aimed to report the anatomopathological findings of two mixed-breed dogs, which were fatal victims of bee stings. The animals were litter brothers, one male and one female, with three years old and weighing about 30kg. The dogs were found dead at night, already in rigor mortis, which led to the suspicion that the death had occurred no more than five hours ago. The main macroscopic lesions were: cutaneous petechiae, some of them associated with bee stingers, generalized congestion, haemorrhage, necrosis, and oedema, as well as insects with morphology compatible with Apis mellifera dispersed in the gastrointestinal tract. Microscopically, degeneration, necrosis, and renal haemorrhages were the most relevant findings, in addition to marked pulmonary oedema, which was considered the causa mortis. Thus, the most important alterations in this type of intoxication are necrosis, haemorrhage, oedema, and congestion. Moreover, death can occur quickly, even with low doses of the toxin.