Synthesis and characterization of delta-atracotoxin-ar1a, the lethal neurotoxin from venom of the Sydney funnel-web spider (Atrax robustus)

delta-Atracotoxin-Ar1a (delta-ACTX-Ar1a) is the major polypeptide neurotoxin isolated from the venom of the male Sydney funnel-web spider, Atrax robustus. This neurotoxin targets both insect and mammalian voltage-gated sodium channels, where it competes with scorpion alpha-toxins for neurotoxin receptor site-3 to slow sodium-channel inactivation. Progress in characterizing the structure and mechanism of action of this toxin has been hampered by the limited supply of pure toxin from natural sources. In this paper, we describe the first successful chemical synthesis and oxidative refolding of the four-disulfide bond containing delta-ACTX-Ar1a. This synthesis involved solid-phase Boc chemistry using double coupling, followed by oxidative folding of purified peptide using a buffer of 2 M GdnHCl and glutathione/glutathiol in a 1:1 mixture of 2-propanol (pH 8.5). Successful oxidation and refolding was confirmed using both chemical and pharmacological characterization. Ion spray mass spectrometry was employed to confirm the molecular weight. H-1 NMR analysis showed identical chemical shifts for native and synthetic toxins, indicating that the synthetic toxin adopts the native fold. Pharmacological studies employing whole-cell patch clamp recordings from rat dorsal root ganglion neurons confirmed that synthetic delta-ACTX-Ar1a produced a slowing of the sodium current inactivation and hyperpolarizing shifts in the voltage-dependence of activation and inactivation similar to native toxin. Under current clamp conditions, we show for the first time that delta-ACTX-Ar1a produces spontaneous repetitive plateau potentials underlying the clinical symptoms seen during envenomation. This successful oxidative refolding of synthetic delta-ACTX-Ar1a paves the way for future structure-activity studies to determine the toxin pharmacophore.

Faeces as a source of DNA for molecular studies in a threatened population of great bustards

With recent advances in molecular biology, it is now possible to use the trace amounts of DNA in faeces to non-invasively sample endangered species for genetic studies. A highly vulnerable population of approximately 100 great bustards (Otis tarda) exists in Morocco necessitating the use of non-invasive protocols to study their genetic structure. Here we report a reliable silica-based method to extract DNA from great bustard faeces. We found that successful extraction and amplification correlated strongly with faeces freshness and composition. We could not extract amplifiable DNA from 30% of our samples as they were dry or contained insect material. However 100% of our fresh faecal samples containing no obvious insect material worked, allowing us to assess the levels of genetic variation among 25 individuals using a 542 bp control region sequence. We were able to extract DNA from four out of five other avian species, demonstrating that faeces represents a suitable source of DNA for population genetics studies in a broad range of species.

Sensitivity of Helicoverpa armigera nucleopolyhedrovirus polyhedra to sodium dodecyl sulfate

Sodium dodecyl sulfate (SDS) is commonly used to extract polyhedra from infected cells and diseased dead larval tissues. It was found, however, that 80% of Helicoverpa armigera nucleopolyhedrovirus (HaSNPV) polyhedra produced via cell culture were damaged after 30 min of 0.5% SDS treatment whereas only 20% of in vivo produced polyhedra were damaged by the same treatment. Transmission and scanning electron microscopy revealed that the damaged polyhedra had lost their polyhedron envelopes and virions were dislodged from the polyhedrin matrix, leaving empty spaces that were previously occupied by the occluded virions. Up to 20% in vitro produced polyhedra were resistant to SDS and remained intact, even after a 24 h exposure to SDS. This sensitivity to SDS was observed across a range of cell culture media, including serum supplemented media. Electron microscopy also revealed that the inferior polyhedron envelope of in vitro produced polyhedra is likely due to poor interaction between the polyhedron envelope, polyhedron envelope protein (PEP), and polyhedrin matrix. The PEP gene was cloned and sequenced and mutations in this gene were ruled out as an explanation. In vitro produced polyhedra that were passed through insect larva once were resistant to SDS, indicating that a critical component is lacking in insect cell culture medium used for producing HaSNPV or the cells growing in culture are inefficient in some ways in relation to production of polyhedra. (C) 2002 Elsevier Science (USA). All rights reserved.

Effect of time of harvest of budded virus on the selection of baculovirus FP mutants in cell culture

Rapid formation and selection of FP (few polyhedra) mutants occurs during serial passaging of Helicoverpa armigera nucleopolyhedrovirus (HaSNPV) in insect cell culture. The production of HaSNPV for use as biopesticides requires the passaging of the virus over a number of passages to produce enough virus inoculum for large-scale fermentation. During serial passaging in cell culture, FP mutants were rapidly selected, resulting in declined productivity and reduced potency of virus. Budded virus (BV) is usually harvested between 72 and 96 h postinfection (hpi) in order to obtain a high titer virus stock. In this study, the effect of tine of harvest (TOH) for BV on the selection rate of HaSNPV FP mutants during serial passaging was investigated. BV were harvested at different times postinfection, and each series was serially passaged for six passages. The productivity and percentage of FP mutants at each passage were determined. It was found that the selection of FP mutants can he reduced by employing an earlier TOH for BV. Serial passaging with BV harvested at 48 hpi showed a slower accumulation of FP mutants compared to that of BV harvested after 48 hpi. Higher cell specific yields were also maintained when BV were harvested at 48 hpi. When BV that were formed between 48 and 96 hpi were harvested and serially passaged, FP mutants quickly dominated the virus population. This suggests that the V formed and released between 48 and 96 hpi are most likely from FP mutant infected cells.

Recombinant expression of Munc18c in a baculovirus system and interaction with syntaxin4

Two protein families that are critical for vesicle transport are the Syntaxin and Munc18/Sec1. families of proteins. These two molecules form a high affinity complex and play an essential role in vesicle docking and fusion. Munc18c was expressed as an N-terminally His-tagged fusion protein from recombinant baculovirus in Sf9 insect cells. His-tagged Munc18c was purified to homogeneity using both cobalt-chelating affinity chromatography and gel filtration chromatography. With this simple two-step protocol, 3.5 mg of purified Munc18c was obtained from a 1 L culture. Further, the N-terminal His-tag could be removed by thrombin cleavage while the tagged protein was bound to metal affinity resin. Recombinant Munc18c produced in this way is functional, in that it forms a stable complex with the SNARE interacting partner, syntaxin4. Thus we have developed a method for producing and purifying large amounts of functional Munc18c-both tagged and detagged-from a baculovirus expression system. We have also developed a method to purify the Munc18c:syntaxin4 complex. These methods will be employed for future functional and structural studies. Crown copyright (C) 2003 Published by Elsevier Inc. All rights reserved.

Ocorrência de Cephalonomia stephanoderis Betrem (Hymenoptera: Bethylidae) em cafezais da Amazônia Brasileira

Adultos de Cephalonomia stephanoderis Betrem foram detectados em novembro de 2003 durante amostragem de frutos de Coffea canephora Pierre ex A. Froehner danificados por Hypothenemus hampei Ferrari (Coleoptera: Scolytidae) em Ouro Preto D'Oeste, RO (10º45'S e 62º15'W). De janeiro a julho de 2004, o parasitóide foi amostrada mensalmente em 200 frutos danificados por H. hampei. Provavelmente, C. stephanoderis exerça alguma pressão de parasitismo sobre a população da broca-do-café. A ocorrência do parasitóide em condições naturais aponta para outra alternativa de controle biológico de H. hampei em Rondônia. Este é o primeiro registro de C. stephanoderis em plantações de café na Amazônia brasileira.

Ação transovariana de lufenuron (50 g/l) sobre adultos de Spodoptera Frugiperda (j. e. smith) (Lepidoptera: Noctuidae) e seu efeito sobre o parasitóide de ovos Trichogramma pretiosum Riley (Hymenoptera: Trichogrammatidae)

Avaliaram-se a ação transovariana do lufenuron em Spodoptera frugiperda e sua seletividade ao parasitóide de ovos Trichogramma pretiosum. Casais da praga foram isolados em gaiolas de PVC e alimentados com solução de mel a 10% na testemunha, e nos outros tratamentos, foi adicionado à solução de mel o regulador de crescimento de insetos Match® CE nas proporções de 12,5; 15,0 e 17,5 g i.a/l. Para verificação da ação transovariana, diariamente foram coletadas as posturas, contado o número de ovos e, posteriormente, o número de larvas eclodidas. Quarenta ovos provenientes de cada tratamento foram colados em cartelas de papel (cartolina) e expostos ao parasitismo, dentro de tubos de vidro de 1,0 x 3,5 cm, contendo uma fêmea de T. pretiosum no seu interior. Cartelas contendo 40 ovos de S. frugiperda foram imersas em soluções de lufenuron com a mesma concentração dos tratamentos anteriores e, posteriormente, expostas ao parasitismo por T. pretiosum. O lufenuron afetou consideravelmente a viabilidade dos ovos de S. frugiperda. Pelos resultados obtidos nos ensaios, relativos ao parasitóide, demonstram-se a seletividade do regulador de crescimento lufenuron e a possibilidade de sua utilização em programas de Manejo Integrado, juntamente com o parasitóide de ovos T. pretiosum.

Extratos vegetais e Bacillus thuringiensis visando o manejo de Duponchelia fovealis Zeller (Lepidoptera: Crambidae)

A lagarta Duponchelia fovealis Zeller (Lepidoptera: Crambidae) foi relatada em plantios comerciais do morangueiro no estado do Espírito Santo ocasionando grandes problemas à cultura. Porém, por ser uma praga recente, não há registro de produtos para o seu controle. A cada dia aumenta a demanda por alimentos e outros produtos livres de resíduos, além da necessidade de uma agricultura mais desenvolvida e sustentável. Pesquisas com agentes de controle biológico e extratos vegetais surgem como alternativa para o manejo desse inseto-praga. Este trabalho teve como objetivo avaliar a eficiência de formulados comerciais à base de Bacillus thuringiensis e a atividade do uso dos extratos aquosos de alho e fumo, visando sua adoção como métodos alternativos de controle de D. fovealis. Nos bioensaios para avaliar a patogenicidade e virulência de duas formulações comerciais à base de B. thuringiensis, Agree® e Dipel WP®, sobre a dieta artificial adaptada à base de farelo de soja, germe de trigo e açúcar, proposta por King e Hartley (1985) para Diatraea saccharalis (Lepidoptera: Crambidae), foram inoculados 70 μL de cada formulado comercial, na concentração 1 x 108 esporos·mL-1. Em seguida, avaliou-se a virulência dos respectivos formulados, isso através da estimativa da concentração letal (CL50) para o estádio de maior suscetibilidade. Em virtude dos resultados encontrados, observou-se que o estágio 1 de desenvolvimento apresentou 95,88% e 86,76% de mortalidades para os produtos Agree® e Dipel WP®, respectivamente, demonstrando patogenicidade e virulência à D. fovealis. No bioensaio para avaliar a atividade dos extratos aquosos de alho e fumo, estes foram aplicados na concentração 10% (m/v). Todos os tratamentos foram pulverizados com Torre de Potter, calibrada a pressão de 15 lb/pol². Posteriormente estimou-se a concentração letal (CL50) do extrato aquoso de fumo, o qual apresentou 95% de mortalidade no teste de suscetibilidade. Desta forma, com os resultados obtidos na presente pesquisa, concluiu-se que a utilização de formulados comerciais à base de B. thuringiensis e extrato de fumo podem ser uma alternativa no manejo fitossanitário de D. fovealis.

Evaluating the Guimarães 2012 European Capital of Culture (ECOC): a Tourist Perception Approach

Guimarães has hosted, during the year of 2012, one of the European Capital of Culture (ECOC). The evaluation of the event is needed as public, private and community funds are involved. The approach considers the tourists (domestic and international) as external and independent stakeholders who assessed the cultural activities developed during the event and the attributes of the city. The results of the survey show that hosting the 2012 ECOC was a major contribution for attracting new visitors to the city, although many of them just for a short period of time. The main source of general information collected by tourists was the Internet, and the traditional media. Only a small amount of respondents demonstrated a specialized knowledge of the cultural program. The most cited and appreciated performances came from the areas of music, exhibitions, and theatre. According to the perceptions of tourists, the tangible assets were clearly detached from the set of attributes of Guimarães, including buildings, churches and chapels, whereas intangible assets were less noted. Overall, Guimarães received a very positive evaluation related to city image and stay and is highly recommended by tourists to friends and relatives.

Does gender affect visiting a world heritage site?

The city of Guimarães in Portugal is a place of strong symbolic and cultural significance, and the nomination of its historical center as a World Heritage Site in 2001 enhanced its tourism potential. This study presents the results of a survey conducted in 2010 and 2011 to capture the profile and motivations of tourists visiting Guimarães as a cultural tourism destination. The study addressed two main issues: whether males and females have similar or different preferences in choosing the city as their destination, and whether there are gender differences in the perception of the attributes of Guimarães. A better understanding of the gendered nature of the destination is a valuable cue for shaping products and services according to visitors’ preferences. The results suggest that both men and women are aware of the main elements responsible for the city’s World Heritage status. That the destination is a Heritage Site that also offers the opportunity to tour the region has a significant positive effect on male tourists’ choice of Guimarães. Regarding the perceived attributes of the city, results indicate minor gender differences with one exception: Women expressed more apprehension than men regarding the perceived security of the destination.

How does gender affect visiting a world heritage site?: the case study of Guimarães

To date few studies have been undertaken in Portugal dealing with the attitudes, motivations, and profile of tourists who visit World Heritage Sites. Also, few studies have dealt with destination image (e.g., Agapito, Mendes & Valle, 2010; Lopes, 2011). As far as it is known, none have approached the issue of gender differences in the choice of a Portuguese heritage destination. Since cultural tourism destinations need to differentiate themselves from each other, appropriate market segmentation must be based on a deep understanding of the customers’ motivations and preferences. Keeping in mind results from empirical literature (e.g., Silberberg, 1995; Beerli & Martin, 2004; Richards, 2004; Pérez, 2009; Sheng, Shen, & Chen, 2008), gender seems to be a possible approach to market segmentation, whether for Guimarães or for other cultural tourism destinations around the world. Located in the north-western region of Portugal, Guimarães is a city of strong symbolic and cultural significance, and the nomination of its historical centre as a World Heritage Site in 2001 enhanced its tourism potential. This study analyses the possible relation between gender and attitudes and motivations towards a World Heritage Site, such as Guimarães. Additionally, the empirical approach used in the study tries to capture differences in the perceived attributes of the city. Commonalities and distinctions within and between groups of tourists, by focusing on the specific characteristic of gender, were analysed. The study addressed two main questions: first, whether males and females have similar or different preferences in choosing the city as their destination; and, second, whether there are gender differences in the perception of the attributes of Guimarães. A better understanding of the gendered nature of the destination is a valuable cue for shaping products and services according to visitors’ preferences.

Does Gender Affect Visiting a World Heritage Site?

The city of Guimar˜aes in Portugal is a place of strong symbolic and cultural significance, and the nomination of its historical center as a World Heritage Site in 2001 enhanced its tourism potential. This study presents the results of a survey conducted in 2010 and 2011 to capture the profile and motivations of tourists visiting Guimar˜aes as a cultural tourism destination. The study addressed two main issues: whether males and females have similar or different preferences in choosing the city as their destination, and whether there are gender differences in the perception of the attributes of Guimar˜aes. A better understanding of the gendered nature of the destination is a valuable cue for shaping products and services according to visitors’ preferences. The results suggest that both men and women are aware of the main elements responsible for the city’s World Heritage status. That the destination is a Heritage Site that also offers the opportunity to tour the region has a significant positive effect on male tourists’ choice of Guimar˜aes. Regarding the perceived attributes of the city, results indicate minor gender differences with one exception: Women expressed more apprehension than men regarding the perceived security of the destination.

Evaluating the Guimarães 2012 European Capital of Culture: National and International Tourists’ Behaviors and Perceptions

In 2012, Guimarães hosted the European Capital of Culture (ECOC). An evaluation of this event was needed because public, private, and community funds were involved. This analysis considers tourists as external and independent stakeholders who assessed the cultural activities developed during the event as well as the attributes of the city. The main objectives of the research conducted were to assess the visitors` motivations during the hosting of the Guimarães ECOC 2012, their perceptions towards the city and if national and international visitors kept different perceptions of it. For two months, in the summer of 2012, a survey was applied to 390 visitors. The results revealed that hosting the 2012 ECOC was a major contribution towards attracting new visitors to the city (though many of the visitors stayed only for a short period of time). Based on tourists’ perceptions, the tangible heritage was clearly detached from the set of attributes associated to Guimarães, whereas the intangible heritage was less noted. The Portuguese tourists seem to be more prone to value the tangible heritage than the foreign tourists. Overall, Guimarães received a very positive evaluation relating to the city’s image and, as stated by tourists, visiting it was declared to be highly recommended. Following the obtained empirical results, the need for changing the city’s promoted image emerges, which has been too centered on its tangible heritage. In doing so, it is believed that there will be longer overnight stays by visitors.

A new species of Culex (Melanoconion) from Southern Brazil (Diptera: Culicidae)

A new species from Southern Brazil, named Culex (Melanoconion) ribeirensis is described. The description includes adults, pupal and larval stages, illustrating the morphological aspects and a picture of a breeding place. Some data about known distribution and bionomics are presented.

Vector competence experiments with Rocio virus and three mosquito species from the epidemic zone in Brazil

First-generation progeny of field-collected Psorophora ferox, Aedes scapularis, and Aedes serratus from the Rocio encephalitis epidemic zone in S.Paulo State, Brazil, were tested for vector competency in the laboratory. Psorophora ferox and Ae. scapularis are susceptible to per os infection with Rocio virus and can transmit the virus by bite following a suitable incubation period. Oral ID50S for the two species (10(4.1) and 10(4.3) Vero cell plaque forming units, respectively) did not differ significantly. Infection rates in Ae. serratus never exceeded 36%, and, consequently, an ID50 could not be calculated for this species. It is unlikely that Ae. serratus is an epidemiologically important vector of Rocio virus. The utility of an in vitro feeding technique for demonstrating virus transmission by infected mosquitoes and difficulties encountered in working with uncolonized progeny of field-collected mosquitoes are discussed.