Phallus eversion sexing in Phrynops geoffroanus (Testudines: Chelidae): a new non-invasive approach / Sexagem por eversão do falo em Phrynops geoffroanus (Testudines: Chelidae): uma nova abordagem não invasiva

Abstract The sexing of species is an important tool for population management. In tortoises and turtles, sexing is usually done according to secondary characteristics, such as plastron morphology or phallus eversion techniques. In this paper, we present the phallus eversion method and compare its efficiency to other existing techniques. This new method aims at shortening the management time and reducing the physical aftereffects on the animals, fostering the clinical and reproductive management of Geoffroy's side-necked turtle (Phrynops geoffroanus) and related species. A sample of fifty individuals was taken in the central region of Piauí state, Northeastern Brazil. The proposed method consisted of moving the head from its resting position. The methods achieving a success rate higher than 80% were compared to each other as for the stimulation time required for phallus eversion. The method as here proposed achieved 100% efficiency in the sex determination of specimens with a linear carapace length between 10 and 34 cm. Also, when compared to the second most efficient method, it substantially reduced the time needed for managing the specimens (91%), avoiding possible sequelae in the individuals handled.

Resumo A sexagem das espécies é uma ferramenta importante para o manejo populacional. Em tartarugas, cágados e jabutis, a sexagem geralmente é feita de acordo com características secundárias, como morfologia do plastrão ou técnicas de eversão do falo. Neste artigo, apresentamos o método de eversão do falo e comparamos sua eficiência com outras técnicas existentes. Este novo método visa encurtar o tempo de manejo e reduzir os efeitos colaterais físicos nos animais, promovendo o manejo clínico e reprodutivo do cágado-de-barbicha (Phrynops geoffroanus) e espécies relacionadas. Uma amostra de cinquenta indivíduos foi coletada na região central do estado do Piauí, Nordeste do Brasil. O método proposto consiste em remover a cabeça de sua posição de repouso. Os métodos que obtiveram índice de sucesso superior a 80% foram comparados entre si quanto ao tempo de estimulação necessário para a eversão do falo. O método aqui proposto alcançou 100% de eficiência na determinação do sexo de espécimes com comprimento de carapaça linear entre 10 e 34 cm. Além disso, quando comparado ao segundo método mais eficiente, reduziu substancialmente o tempo necessário para o manejo dos espécimes (91%), evitando possíveis sequelas nos indivíduos manuseados.

Phallus eversion sexing in Phrynops geoffroanus (Testudines: Chelidae): a new non-invasive approach

Abstract The sexing of species is an important tool for population management. In tortoises and turtles, sexing is usually done according to secondary characteristics, such as plastron morphology or phallus eversion techniques. In this paper, we present the phallus eversion method and compare its efficiency to other existing techniques. This new method aims at shortening the management time and reducing the physical aftereffects on the animals, fostering the clinical and reproductive management of Geoffroys side-necked turtle (Phrynops geoffroanus) and related species. A sample of fifty individuals was taken in the central region of Piauí state, Northeastern Brazil. The proposed method consisted of moving the head from its resting position. The methods achieving a success rate higher than 80% were compared to each other as for the stimulation time required for phallus eversion. The method as here proposed achieved 100% efficiency in the sex determination of specimens with a linear carapace length between 10 and 34 cm. Also, when compared to the second most efficient method, it substantially reduced the time needed for managing the specimens (91%), avoiding possible sequelae in the individuals handled.

Resumo A sexagem das espécies é uma ferramenta importante para o manejo populacional. Em tartarugas, cágados e jabutis, a sexagem geralmente é feita de acordo com características secundárias, como morfologia do plastrão ou técnicas de eversão do falo. Neste artigo, apresentamos o método de eversão do falo e comparamos sua eficiência com outras técnicas existentes. Este novo método visa encurtar o tempo de manejo e reduzir os efeitos colaterais físicos nos animais, promovendo o manejo clínico e reprodutivo do cágado-de-barbicha (Phrynops geoffroanus) e espécies relacionadas. Uma amostra de cinquenta indivíduos foi coletada na região central do estado do Piauí, Nordeste do Brasil. O método proposto consiste em remover a cabeça de sua posição de repouso. Os métodos que obtiveram índice de sucesso superior a 80% foram comparados entre si quanto ao tempo de estimulação necessário para a eversão do falo. O método aqui proposto alcançou 100% de eficiência na determinação do sexo de espécimes com comprimento de carapaça linear entre 10 e 34 cm. Além disso, quando comparado ao segundo método mais eficiente, reduziu substancialmente o tempo necessário para o manejo dos espécimes (91%), evitando possíveis sequelas nos indivíduos manuseados.

Phallus eversion sexing in Phrynops geoffroanus (Testudines: Chelidae): a new non-invasive approach.

The sexing of species is an important tool for population management. In tortoises and turtles, sexing is usually done according to secondary characteristics, such as plastron morphology or phallus eversion techniques. In this paper, we present the phallus eversion method and compare its efficiency to other existing techniques. This new method aims at shortening the management time and reducing the physical aftereffects on the animals, fostering the clinical and reproductive management of Geoffroy's side-necked turtle (Phrynops geoffroanus) and related species. A sample of fifty individuals was taken in the central region of Piauí state, Northeastern Brazil. The proposed method consisted of moving the head from its resting position. The methods achieving a success rate higher than 80% were compared to each other as for the stimulation time required for phallus eversion. The method as here proposed achieved 100% efficiency in the sex determination of specimens with a linear carapace length between 10 and 34 cm. Also, when compared to the second most efficient method, it substantially reduced the time needed for managing the specimens (91%), avoiding possible sequelae in the individuals handled.

Acoustic niche partitioning in an anuran community from the municipality of Floriano, Piauí, Brazil / Partição de nicho acústico em uma comunidade de anuros do município de Floriano, Piauí, Brasil

Abstract Vocalizations are an important trait for the identification of cryptic and/or closely related amphibian species. Different vocalizations also contribute to partitioning of the acoustic space by sympatric species. This study aimed to describe the advertisement calls of anurans in a pond of the municipality of Floriano, State of Piauí, Brazil, and infer the acoustic niche partitioning of amphibians. Euclidean distance was used in a cluster analysis approach to infer the acoustic similarities among species. Thirteen species were analysed: Boana raniceps, Dendropsophus nanus, D. rubicundulus , D. minutus, Leptodactylus fuscus, L. troglodytes, L. vastus, Pithecopus nordestinus , Physalaemus cuvieri, P. nattereri, Pleurodema diplolister, Proceratophrys cristiceps and Scinax ruber. From these, six showed more than 90% of acoustic overlap: P. nattereri , P. cuvieri, L. fuscus and L. vastus (Leptodactylidae); and, D. nanus and D. rubicundulus (Hylidae). Despite the acoustic similarities among these six species, the acoustic interference was reduced due to the small number of sympatric species and to distinct features on carrier frequency such as dominant frequency and the degree of modulation in the frequency. Environmental factors limit the periods and sites of reproduction respectively, which may maintain the low anuran diversity and consequently reduce acoustic overlap.

Resumo A vocalização é um componente importante na identificação de espécies crípticas e/ou proximamente relacionadas de anfíbios anuros. Diferenças na bioacústica também contribuem para o particionamento do espaço acústico entre espécies simpátricas. Este estudo descreve o canto de anúncio de anuros em uma lagoa temporária do município de Floriano, Estado do Piauí, Brasil, além de buscar inferir o particionamento do nicho acústico entre essas espécies. Para tanto, foi utilizada a distância Euclidiana para compor uma análise de conglomerados e avaliar a similaridade/dissimilaridade da bioacústica da anurofauna. Treze espécies foram amostradas: Boana raniceps, Dendropsophus nanus, D. rubicundulus , D. minutus, Leptodactylus fuscus, L. troglodytes, L. vastus, Pithecopus nordestinus , Physalaemus cuvieri, P. nattereri, Pleurodema diplolister, Proceratophrys cristiceps e Scinax ruber. Destas, seis apresentaram mais de 90% de sobreposição acústica: P. nattereri, P. cuvieri, L. fuscus e L. vastus (Leptodactylidae); e, D. nanus e D. rubicundulus (Hylidae). Apesar da similaridade acústica entre essas seis espécies, a interferência é reduzida devido ao pequeno número de espécies simpátricas e certas características distintas na frequência do canto, tais como: frequência dominante e sua modulação. Fatores ambientais específicos do semiárido limitam os períodos e sítios reprodutivos, o que mantém baixa diversidade de espécies e consequentemente reduz a sobreposição no nicho acústico.

Acoustic niche partitioning in an anuran community from the municipality of Floriano, Piauí, Brazil.

Vocalizations are an important trait for the identification of cryptic and/or closely related amphibian species. Different vocalizations also contribute to partitioning of the acoustic space by sympatric species. This study aimed to describe the advertisement calls of anurans in a pond of the municipality of Floriano, State of Piauí, Brazil, and infer the acoustic niche partitioning of amphibians. Euclidean distance was used in a cluster analysis approach to infer the acoustic similarities among species. Thirteen species were analysed: Boana raniceps, Dendropsophus nanus, D. rubicundulus , D. minutus, Leptodactylus fuscus, L. troglodytes, L. vastus, Pithecopus nordestinus , Physalaemus cuvieri, P. nattereri, Pleurodema diplolister, Proceratophrys cristiceps and Scinax ruber. From these, six showed more than 90% of acoustic overlap: P. nattereri , P. cuvieri, L. fuscus and L. vastus (Leptodactylidae); and, D. nanus and D. rubicundulus (Hylidae). Despite the acoustic similarities among these six species, the acoustic interference was reduced due to the small number of sympatric species and to distinct features on carrier frequency such as dominant frequency and the degree of modulation in the frequency. Environmental factors limit the periods and sites of reproduction respectively, which may maintain the low anuran diversity and consequently reduce acoustic overlap.

Acoustic niche partitioning in an anuran community from the municipality of Floriano, Piauí, Brazil / Partição de nicho acústico em uma comunidade de anuros do município de Floriano, Piauí, Brasil

Vocalizations are an important trait for the identification of cryptic and/or closely related amphibian species. Different vocalizations also contribute to partitioning of the acoustic space by sympatric species. This study aimed to describe the advertisement calls of anurans in a pond of the municipality of Floriano, State of Piauí, Brazil, and infer the acoustic niche partitioning of amphibians. Euclidean distance was used in a cluster analysis approach to infer the acoustic similarities among species. Thirteen species were analysed: Boana raniceps, Dendropsophus nanus, D. rubicundulus , D. minutus, Leptodactylus fuscus, L. troglodytes, L. vastus, Pithecopus nordestinus , Physalaemus cuvieri, P. nattereri, Pleurodema diplolister, Proceratophrys cristiceps and Scinax ruber. From these, six showed more than 90% of acoustic overlap: P. nattereri , P. cuvieri, L. fuscus and L. vastus (Leptodactylidae); and, D. nanus and D. rubicundulus (Hylidae). Despite the acoustic similarities among these six species, the acoustic interference was reduced due to the small number of sympatric species and to distinct features on carrier frequency such as dominant frequency and the degree of modulation in the frequency. Environmental factors limit the periods and sites of reproduction respectively, which may maintain the low anuran diversity and consequently reduce acoustic overlap.(AU)

A vocalização é um componente importante na identificação de espécies crípticas e/ou proximamente relacionadas de anfíbios anuros. Diferenças na bioacústica também contribuem para o particionamento do espaço acústico entre espécies simpátricas. Este estudo descreve o canto de anúncio de anuros em uma lagoa temporária do município de Floriano, Estado do Piauí, Brasil, além de buscar inferir o particionamento do nicho acústico entre essas espécies. Para tanto, foi utilizada a distância Euclidiana para compor uma análise de conglomerados e avaliar a similaridade/dissimilaridade da bioacústica da anurofauna. Treze espécies foram amostradas: Boana raniceps, Dendropsophus nanus, D. rubicundulus , D. minutus, Leptodactylus fuscus, L. troglodytes, L. vastus, Pithecopus nordestinus , Physalaemus cuvieri, P. nattereri, Pleurodema diplolister, Proceratophrys cristiceps e Scinax ruber. Destas, seis apresentaram mais de 90% de sobreposição acústica: P. nattereri, P. cuvieri, L. fuscus e L. vastus (Leptodactylidae); e, D. nanus e D. rubicundulus (Hylidae). Apesar da similaridade acústica entre essas seis espécies, a interferência é reduzida devido ao pequeno número de espécies simpátricas e certas características distintas na frequência do canto, tais como: frequência dominante e sua modulação. Fatores ambientais específicos do semiárido limitam os períodos e sítios reprodutivos, o que mantém baixa diversidade de espécies e consequentemente reduz a sobreposição no nicho acústico.(AU)

Antifungal activity of tyrosol and farnesol used in combination against Candida species in the planktonic state or forming biofilms.

AIM: This study examined the antifungal activity of the combination of tyrosol and farnesol against Candida albicans and Candida glabrata in the planktonic state or forming biofilms. METHODS AND RESULTS: The effect of drug association against Candida planktonic cells was assessed by the fractional inhibitory concentration index. Mono- and dual-species biofilms were developed during 24 h and then treated with the compounds for 3 days, with two daily treatments of 1 min each. After, the total biomass, metabolic activity and the number of cultivable cells were quantified. Planktonic cells of the two species showed a similar susceptibility to the drug combination, however, a synergistic effect was only verified for C. glabrata. Regarding biofilm susceptibility, significant reductions in C. glabrata biomass, metabolism of C. albicans and mixed biofilms, and cultivable cells of single biofilms were verified for the drug combination, indicating an additive effect. For all other experiments, the effects were classified as indifferent. CONCLUSION: The combined use of tyrosol and farnesol was advantageous for some of the analysed parameters against Candida species. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings may contribute to the development of oral care products containing tyrosol and farnesol to combat oral infections caused by Candida species.

Differential effects of the combination of tyrosol with chlorhexidine gluconate on oral biofilms.

OBJECTIVE: This study assessed the effect of tyrosol and chlorhexidine gluconate in combination against Candida albicans, Candida glabrata, and Streptococcus mutans in the planktonic state or forming biofilms in vitro. MATERIALS AND METHODS: Checkerboard assays were performed for determination of minimum inhibitory concentration. Biofilms were cultivated during 24 h on specimens of acrylic resin and hydroxyapatite and treated with the drugs alone or in combination twice a day for 1 min, during 3 days. The antibiofilm effect was determined by quantification of the metabolic activity and cultivable cells. The drug combination was also applied on C. albicans to investigate its action on the number of hyphae. Data were statistically examined by two-way ANOVA and Holm-Sidak test (P < 0.05). RESULTS: The effect of drug combination on planktonic cells was classified as antagonistic for C. albicans and indifferent for the other strains. Also, the drugs were ineffective against the tested biofilms. However, the drug combination showed a synergistic effect in reducing the number of hyphae by C. albicans. CONCLUSION: The combination of tyrosol with chlorhexidine gluconate was only effective in reducing the number of hyphae by C. albicans, a relevant virulence factor of this species.

Activity of tyrosol against single and mixed-species oral biofilms.

AIM: This study aimed to evaluate the effect of tyrosol on the formation of single and mixed biofilms of Candida albicans ATCC 10231, Candida glabrata ATCC 90030 and Streptococcus mutans ATCC 25175 formed on acrylic resin (AR) and hydroxyapatite (HA) surfaces. METHODS AND RESULTS: Single and mixed biofilms were formed on AR and HA in the presence of tyrosol at 50, 100 and 200 mmol l(-1), during 48 h. Next, antimicrobial activity was assessed through metabolic activity (XTT reduction assay) and the number of colony-forming units (CFUs). Scanning electron microscopy observations were performed in order to analyse biofilm structure. Tyrosol, mainly at 200 mmol l(-1), significantly decreased the metabolic activity and number of CFUs for all single and mixed-species biofilms formed on both surfaces. SEM images suggested cell damage caused by tyrosol. CONCLUSION: Tyrosol showed inhibitory effects against biofilms formed by important oral pathogens. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study showing the antibiofilm effect of tyrosol on Candida species and Strep. mutans in single and mixed cultures. These results may be useful in the development of topical therapies focused on preventing biofilm-associated oral diseases, such as denture stomatitis and dental caries.

Silver colloidal nanoparticles: effect on matrix composition and structure of Candida albicans and Candida glabrata biofilms.

AIM: The aim of this study was to assess the effect of different silver nanoparticles (SN) concentrations on the matrix composition and structure of Candida albicans and Candida glabrata biofilms. METHODS AND RESULTS: Candida biofilms were developed in 6-well microtiter plates during 48 h. After, these biofilms were exposed to 13.5 or 54 µg SN ml(-1) for 24 h. Then, extracellular matrices were extracted from biofilms and analysed chemically in terms of proteins, carbohydrates and DNA. To investigate the biofilm structure, scanning electron microscopy (SEM) and epifluorescence microscopy were used. SN interfered with the matrix composition of Candida biofilms tested in terms of protein, carbohydrate and DNA, except for the protein content of C. albicans biofilm. By SEM, Candida biofilms treated with SN revealed structural differences, when compared with the control groups. Further, SN showed a trend of agglomeration within the biofilms. Epifluorescence microscopy images suggest that SN induced damage on cell walls of the Candida isolates tested. CONCLUSIONS: In general, irrespective of concentration, SN affected the matrix composition and structure of Candida biofilms and these findings may be related to the mechanisms of biocide action of SN. SIGNIFICANCE AND IMPACT OF THE STUDY: This study reveals new insights about the behaviour of SN when in contact with Candida biofilms. SN may contribute to the development of therapies to prevent or control Candida infections.

Silver nanoparticles: influence of stabilizing agent and diameter on antifungal activity against Candida albicans and Candida glabrata biofilms.

AIM: The purpose of this work was to evaluate the size-dependent antifungal activity of different silver nanoparticles (SN) colloidal suspensions against Candida albicans and Candida glabrata mature biofilms. METHODS AND RESULTS: The research presented herein used SN of three different average sizes (5, 10 and 60 nm), which were synthesized by the reduction of silver nitrate through sodium citrate and which were stabilized with ammonia or polyvinylpyrrolidone. Minimal inhibitory concentration (MIC) assays were performed using the microdilution methodology. The antibiofilm activity of SN was determined by total biomass quantification (by crystal violet staining) and colony forming units enumeration. MIC results showed that all SN colloidal suspensions were fungicidal against the tested strains at very low concentrations (0·4-3·3 µg ml(-1) ). With regard to biomass quantification, SN colloidal suspensions were very effective only against C. glabrata biofilms, achieving biomass reductions around 90% at a silver concentration of 108 µg ml(-1) . In general, all SN suspensions promoted significant log(10) reduction of the mean number of cultivable biofilm cells after exposure to silver concentrations at or higher than 108 µg ml(-1) . Moreover, the results showed that the particle size and the type of stabilizing agent used did not interfere in the antifungal activity of SN against Candida biofilms. CONCLUSIONS: This study suggests that SN have antifungal therapeutic potential, but further studies are still required namely regarding formulation and delivery means. SIGNIFICANCE AND IMPACT OF THE STUDY: SN may contribute to the development of new strategies for the improvement of oral health and quality of life particularly of the complete denture wearers.

Silver colloidal nanoparticles: antifungal effect against adhered cells and biofilms of Candida albicans and Candida glabrata.

The aim of this study was to evaluate the effect of silver nanoparticles (SN) against Candida albicans and Candida glabrata adhered cells and biofilms. SN (average diameter 5 nm) were synthesized by silver nitrate reduction with sodium citrate and stabilized with ammonia. Minimal inhibitory concentration (MIC) tests were performed for C. albicans (n = 2) and C. glabrata (n = 2) grown in suspension following the Clinical Laboratory Standards Institute microbroth dilution method. SN were applied to adhered cells (2 h) or biofilms (48 h) and after 24 h of contact their effect was assessed by enumeration of colony forming units (CFUs) and quantification of total biomass (by crystal violet staining). The MIC results showed that SN were fungicidal against all strains tested at very low concentrations (0.4-3.3 µg ml(-1)). Furthermore, SN were more effective in reducing biofilm biomass when applied to adhered cells (2 h) than to pre-formed biofilms (48 h), with the exception of C. glabrata ATCC, which in both cases showed a reduction ∼90%. Regarding cell viability, SN were highly effective on adhered C. glabrata and respective biofilms. On C. albicans the effect was not so evident but there was also a reduction in the number of viable biofilm cells. In summary, SN may have the potential to be an effective alternative to conventional antifungal agents for future therapies in Candida-associated denture stomatitis.

Relationship between Candida and nocturnal denture wear: quantitative study.

The purpose of this study was to evaluate the relationship between Candida and denture wear during the night. Twenty-four edentulous volunteers were randomly divided into two groups. Group I (GI, n = 11) was composed of volunteers who wore their complete dentures day and night and Group II (GII, n = 13) was composed of volunteers who wore their complete dentures only during the day. Three examination periods were performed for both groups. In GI, the first examination (A) was carried out after overnight denture wearing. Subsequent examinations were conducted after one (B) and seven nights (C) without denture use during sleep. In GII, the first (A) was done without previous use during sleep, and the following were carried out after one (B) and seven nights (C) of overnight denture wearing. Total unstimulated saliva was collected in a sterile container and cultured in duplicate inside Petri dishes. The values of colony forming units (CFU mL(-1) +/- s.d.) were obtained: GI A - 10.1 x 10(3) +/- 1.2 x 10(4), B - 2.0 x 10(3) +/- 2.6 x 10(3), and C - 2.6 x 10(3) +/- 5.9 x 10(3) and GII: A - 0.4 x 10(3) +/- 0.6 x 10(3), B - 9.4 x 10(3) +/- 17.7 x 10(3) and C - 6.3 x 10(3) +/- 15.3 x 10(3). The mean counts for Candida sp. were expressed as log (CFU + 1) mL(-1) and statistical significance of differences among groups was tested by anova (alpha = 0.05). Multiple comparisons were performed according to Bonferroni test and indicated significant differences between A-B and A-C, but not between B and C for both groups. It was concluded that there is a significant relationship between continuous denture wear and Candida sp.