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)

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)

Comparative venomic profiles of three spiders of the genus Phoneutria

Abstract Background: 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.

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

Abstract 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.

Biodiversity of orb-web spiders (family: araneidae) of buner valley, Pakistan / Biodiversidade de aranhas construtoras de teia (família: araneidae) do vale buner, Paquistão

The present research was carried out to explore the spider fauna of Buner valley with taxonomic study from February 2018 to January 2019. For this purpose samples were collected, four times at each month from 4 tehsils: Daggar, Gagra, Mandan and Totalai. Two methods were used, hand picking and sweep net for collection of samples. During day and night, three habitats, arid area, agriculture land and building area were search for collection. A total of 534 samples of spider were collected from four sampling sites, in which 379 were belonging to family Araniedae. After confirmation, the identified species were belonging to 8 genera (Neoscona, Argiope, Cyclosa, Araneus, Cyrtophora, Larinia, Erivoxia and Poltys) and 19 species. 18 of them were identified to specie level while a single specie to its generic level. The genus Neoscona was the dominant genus 26.31% having 5 species while the genus Argiope 21.05% is the second dominant having 4 species followed by Cyclosa 15.78% having 3 species followed by Cyrtophora and Araneus 10.52% having two species both. The Poltys and Larinia 5.26% are the rarest genera represent single-single specie both. Statistical analysis show that specie richness (D) = 5.77, Simpson index (1-D) = 0.87, Shannon index (H) = 2.33. Diversity of spiders was evenly distributed and calculated Evenness value was H/InS = 0.5408. There is also few atypical species and Fisher alpha estimate high value (Fisher α) = 4.42. Chao-1 estimated we have reported 22 species.

A presente pesquisa foi realizada para explorar a fauna de aranhas do vale Buner, em um estudo taxonômico realizado de fevereiro de 2018 a janeiro de 2019. Para esse fim, foram coletadas amostras, quatro vezes por mês, a partir de 4 subdistritos: Daggar, Gagra, Mandan e Totalai. Foram utilizados dois métodos, coleta manual e rede de varredura, para a obtenção das amostras. Durante o dia e a noite, foram pesquisados três habitats: área árida, área agrícola e área de construção. Foram coletadas 534 amostras de aranha em quatro locais de amostragem, sendo que 379 pertenciam à família Araneidae. Após a confirmação, as espécies identificadas pertenciam a 8 gêneros (Neoscona, Argiope, Cyclosa, Araneus, Cyrtophora, Larinia, Erivoxia e Poltys) e a 19 espécies (18 delas foram identificados em seu nível de espécie, enquanto uma única foi identificada em seu nível genérico). O gênero Neoscona foi dominante (26,31%), com 5 espécies, seguido pelos gêneros Argiope (21,05%), com 4 espécies, Cyclosa (15,78%), com 3 espécies, e Cyrtophora e Araneus (10,52%), com 2 espécies. Poltys e Larinia (5,26%) são os gêneros mais raros, representando a mesma espécie. A análise estatística mostrou que a riqueza de espécies (D) foi de 5,77, o índice de Simpson (1-D) foi de 0,87 e o índice de Shannon (H) foi de 2,33. A diversidade de aranhas foi distribuída uniformemente, e o valor calculado da uniformidade foi H / InS = 0,5408. Também existem poucas espécies atípicas, e o alfa de Fisher (Fisher α) estimou o valor de 4,42. Já Chao-1 estimou 22 espécies.

Biodiversity of orb-web spiders (family: araneidae) of buner valley, Pakistan

Abstract The present research was carried out to explore the spider fauna of Buner valley with taxonomic study from February 2018 to January 2019. For this purpose samples were collected, four times at each month from 4 tehsils: Daggar, Gagra, Mandan and Totalai. Two methods were used, hand picking and sweep net for collection of samples. During day and night, three habitats, arid area, agriculture land and building area were search for collection. A total of 534 samples of spider were collected from four sampling sites, in which 379 were belonging to family Araniedae. After confirmation, the identified species were belonging to 8 genera (Neoscona, Argiope, Cyclosa, Araneus, Cyrtophora, Larinia, Erivoxia and Poltys) and 19 species. 18 of them were identified to specie level while a single specie to its generic level. The genus Neoscona was the dominant genus 26.31% having 5 species while the genus Argiope 21.05% is the second dominant having 4 species followed by Cyclosa 15.78% having 3 species followed by Cyrtophora and Araneus 10.52% having two species both. The Poltys and Larinia 5.26% are the rarest genera represent single-single specie both. Statistical analysis show that specie richness (D) = 5.77, Simpson index (1-D) = 0.87, Shannon index (H) = 2.33. Diversity of spiders was evenly distributed and calculated Evenness value was H/InS = 0.5408. There is also few atypical species and Fisher alpha estimate high value (Fisher ) = 4.42. Chao-1 estimated we have reported 22 species.

Resumo A presente pesquisa foi realizada para explorar a fauna de aranhas do vale Buner, em um estudo taxonômico realizado de fevereiro de 2018 a janeiro de 2019. Para esse fim, foram coletadas amostras, quatro vezes por mês, a partir de 4 subdistritos: Daggar, Gagra, Mandan e Totalai. Foram utilizados dois métodos, coleta manual e rede de varredura, para a obtenção das amostras. Durante o dia e a noite, foram pesquisados três habitats: área árida, área agrícola e área de construção. Foram coletadas 534 amostras de aranha em quatro locais de amostragem, sendo que 379 pertenciam à família Araneidae. Após a confirmação, as espécies identificadas pertenciam a 8 gêneros (Neoscona, Argiope, Cyclosa, Araneus, Cyrtophora, Larinia, Erivoxia e Poltys) e a 19 espécies (18 delas foram identificados em seu nível de espécie, enquanto uma única foi identificada em seu nível genérico). O gênero Neoscona foi dominante (26,31%), com 5 espécies, seguido pelos gêneros Argiope (21,05%), com 4 espécies, Cyclosa (15,78%), com 3 espécies, e Cyrtophora e Araneus (10,52%), com 2 espécies. Poltys e Larinia (5,26%) são os gêneros mais raros, representando a mesma espécie. A análise estatística mostrou que a riqueza de espécies (D) foi de 5,77, o índice de Simpson (1-D) foi de 0,87 e o índice de Shannon (H) foi de 2,33. A diversidade de aranhas foi distribuída uniformemente, e o valor calculado da uniformidade foi H / InS = 0,5408. Também existem poucas espécies atípicas, e o alfa de Fisher (Fisher ) estimou o valor de 4,42. Já Chao-1 estimou 22 espécies.

Biodiversity of orb-web spiders (family: araneidae) of buner valley, Pakistan / Biodiversidade de aranhas construtoras de teia (família: araneidae) do vale buner, Paquistão

The present research was carried out to explore the spider fauna of Buner valley with taxonomic study from February 2018 to January 2019. For this purpose samples were collected, four times at each month from 4 tehsils: Daggar, Gagra, Mandan and Totalai. Two methods were used, hand picking and sweep net for collection of samples. During day and night, three habitats, arid area, agriculture land and building area were search for collection. A total of 534 samples of spider were collected from four sampling sites, in which 379 were belonging to family Araniedae. After confirmation, the identified species were belonging to 8 genera (Neoscona, Argiope, Cyclosa, Araneus, Cyrtophora, Larinia, Erivoxia and Poltys) and 19 species. 18 of them were identified to specie level while a single specie to its generic level. The genus Neoscona was the dominant genus 26.31% having 5 species while the genus Argiope 21.05% is the second dominant having 4 species followed by Cyclosa 15.78% having 3 species followed by Cyrtophora and Araneus 10.52% having two species both. The Poltys and Larinia 5.26% are the rarest genera represent single-single specie both. Statistical analysis show that specie richness (D) = 5.77, Simpson index (1-D) = 0.87, Shannon index (H) = 2.33. Diversity of spiders was evenly distributed and calculated Evenness value was H/InS = 0.5408. There is also few atypical species and Fisher alpha estimate high value (Fisher α) = 4.42. Chao-1 estimated we have reported 22 species.

A presente pesquisa foi realizada para explorar a fauna de aranhas do vale Buner, em um estudo taxonômico realizado de fevereiro de 2018 a janeiro de 2019. Para esse fim, foram coletadas amostras, quatro vezes por mês, a partir de 4 subdistritos: Daggar, Gagra, Mandan e Totalai. Foram utilizados dois métodos, coleta manual e rede de varredura, para a obtenção das amostras. Durante o dia e a noite, foram pesquisados três habitats: área árida, área agrícola e área de construção. Foram coletadas 534 amostras de aranha em quatro locais de amostragem, sendo que 379 pertenciam à família Araneidae. Após a confirmação, as espécies identificadas pertenciam a 8 gêneros (Neoscona, Argiope, Cyclosa, Araneus, Cyrtophora, Larinia, Erivoxia e Poltys) e a 19 espécies (18 delas foram identificados em seu nível de espécie, enquanto uma única foi identificada em seu nível genérico). O gênero Neoscona foi dominante (26,31%), com 5 espécies, seguido pelos gêneros Argiope (21,05%), com 4 espécies, Cyclosa (15,78%), com 3 espécies, e Cyrtophora e Araneus (10,52%), com 2 espécies. Poltys e Larinia (5,26%) são os gêneros mais raros, representando a mesma espécie. A análise estatística mostrou que a riqueza de espécies (D) foi de 5,77, o índice de Simpson (1-D) foi de 0,87 e o índice de Shannon (H) foi de 2,33. A diversidade de aranhas foi distribuída uniformemente, e o valor calculado da uniformidade foi H / InS = 0,5408. Também existem poucas espécies atípicas, e o alfa de Fisher (Fisher α) estimou o valor de 4,42. Já Chao-1 estimou 22 espécies.

Evaluation of mesenchymal cells and dapsone for the treatment of dermonecrotic wounds caused by Loxosceles laeta venom in rabbits / Avaliação de células mesenquimais e dapsona para o tratamento de feridas dermonecróticas causadas pelo veneno de Loxosceles laeta em coelhos

This study aimed to evaluate the efficacy of mesenchymal stem cells (MSC), alone or associated with dapsone (DAP), in treating dermonecrotic wounds caused by Loxosceles laeta venom. Twenty-five male rabbits were distributed into five groups. Negative control received ultrapure water (C-), whilst all other groups were injected with 20 μg of L. laeta venom. After 4 hours, each group received one of the following treatments: PBS (C+), DAP, MSC, and DAP+MSC. Animals were evaluated daily and photographic records made for analysis of wound area. Twelve days after, animals were euthanized and skin samples removed for histological analysis. We observed that DAP showed the best percentage of wound contraction at day 3. In the treatments using MSCs, a negative value of wound contraction was observed for the isolated MSCs, as well as a lower contraction value for the association of the MSC + DAP when compared to PBS, probably, by the increase in initial infammation after the application of stem cells, due to the fact that MSCs secrete a broad spectrum of bioactive molecules such as cytokines and growth factors that favor regeneration. Histologically, it was observed that animals of C+ showed extensive areas of necrosis, ulcers, neutrophilic infiltrate, and mineralization. Collagen deposition showed increase in MSC+DAP treatment, however vascularization remained unchanged. This is the first report using MSC and MSC+DAP as a treatment for cutaneous loxoscelism and more studies are needed to determine its use as an alternative therapy for dermonecrotic lesions caused by Loxosceles spider.

Este estudo teve como objetivo avaliar a eficácia das células-tronco mesenquimais (CTMs), isoladas ou associadas à dapsona (DAP), no tratamento de feridas dermonecróticas causadas pelo veneno de Loxosceles laeta. Vinte e cinco coelhos machos foram distribuídos em cinco grupos. O controle negativo recebeu água ultrapura (C-), enquanto todos os outros grupos foram injetados com 20 μg de veneno de L. laeta. Após 4 horas, cada grupo recebeu um dos seguintes tratamentos: PBS (C+), DAP, CTMs e DAP + CTMs. Os animais foram avaliados diariamente durante 12 dias, e feitos registros fotográficos para análise da ferida e no 12º dia, foram eutanasiados e, retiradas amostras de pele para análise histológica. Observou-se que a DAP apresentou o melhor percentual de contração da ferida no terceiro dia. Nos tratamentos com CTMs, observou-se uma contração negativa da ferida tanto para as CTMs isoladas, bem como a associação CTMs + DAP em relação ao PBS, possivelmente, pelo aumento da infamação inicial após a aplicação de células-tronco. Isso é devido ao fato de que as CTMs secretam um amplo espectro de moléculas bioativas como citocinas e fatores de crescimento que favorecem a regeneração. Histologicamente, observou-se que os animais de C+ apresentaram extensas áreas de necrose, úlceras, infiltrado neutrofílico, além de mineralização. Houve aumento de deposição de colágeno no tratamento CTMs + DAP, no entanto, a vascularização permaneceu inalterada. Este é o primeiro relato usando CTMs e CTMs + DAP como tratamento para loxoscelismo cutâneo e mais estudos são necessários para determinar seu uso como terapia alternativa para lesões demonecróticas causadas pela aranha Loxosceles.

Biodiversity of orb-web spiders (family: araneidae) of buner valley, Pakistan / Biodiversidade de aranhas construtoras de teia (família: araneidae) do vale buner, Paquistão

The present research was carried out to explore the spider fauna of Buner valley with taxonomic study from February 2018 to January 2019. For this purpose samples were collected, four times at each month from 4 tehsils: Daggar, Gagra, Mandan and Totalai. Two methods were used, hand picking and sweep net for collection of samples. During day and night, three habitats, arid area, agriculture land and building area were search for collection. A total of 534 samples of spider were collected from four sampling sites, in which 379 were belonging to family Araniedae. After confirmation, the identified species were belonging to 8 genera (Neoscona, Argiope, Cyclosa, Araneus, Cyrtophora, Larinia, Erivoxia and Poltys) and 19 species. 18 of them were identified to specie level while a single specie to its generic level. The genus Neoscona was the dominant genus 26.31% having 5 species while the genus Argiope 21.05% is the second dominant having 4 species followed by Cyclosa 15.78% having 3 species followed by Cyrtophora and Araneus 10.52% having two species both. The Poltys and Larinia 5.26% are the rarest genera represent single-single specie both. Statistical analysis show that specie richness (D) = 5.77, Simpson index (1-D) = 0.87, Shannon index (H) = 2.33. Diversity of spiders was evenly distributed and calculated Evenness value was H/InS = 0.5408. There is also few atypical species and Fisher alpha estimate high value (Fisher α) = 4.42. Chao-1 estimated we have reported 22 species.(AU)

A presente pesquisa foi realizada para explorar a fauna de aranhas do vale Buner, em um estudo taxonômico realizado de fevereiro de 2018 a janeiro de 2019. Para esse fim, foram coletadas amostras, quatro vezes por mês, a partir de 4 subdistritos: Daggar, Gagra, Mandan e Totalai. Foram utilizados dois métodos, coleta manual e rede de varredura, para a obtenção das amostras. Durante o dia e a noite, foram pesquisados três habitats: área árida, área agrícola e área de construção. Foram coletadas 534 amostras de aranha em quatro locais de amostragem, sendo que 379 pertenciam à família Araneidae. Após a confirmação, as espécies identificadas pertenciam a 8 gêneros (Neoscona, Argiope, Cyclosa, Araneus, Cyrtophora, Larinia, Erivoxia e Poltys) e a 19 espécies (18 delas foram identificados em seu nível de espécie, enquanto uma única foi identificada em seu nível genérico). O gênero Neoscona foi dominante (26,31%), com 5 espécies, seguido pelos gêneros Argiope (21,05%), com 4 espécies, Cyclosa (15,78%), com 3 espécies, e Cyrtophora e Araneus (10,52%), com 2 espécies. Poltys e Larinia (5,26%) são os gêneros mais raros, representando a mesma espécie. A análise estatística mostrou que a riqueza de espécies (D) foi de 5,77, o índice de Simpson (1-D) foi de 0,87 e o índice de Shannon (H) foi de 2,33. A diversidade de aranhas foi distribuída uniformemente, e o valor calculado da uniformidade foi H / InS = 0,5408. Também existem poucas espécies atípicas, e o alfa de Fisher (Fisher α) estimou o valor de 4,42. Já Chao-1 estimou 22 espécies.(AU)