Abstract The oligochaete Dero lutzi follows a life strategy that alternates between free-living periods in aquatic environments and endoparasitic phases. Most occurrences of D. lutzi in anurans are reported in species with arboreal habits, with studies limited to the recording of the oligochaete’s presence in the host. Our study recovered specimens of D. lutzi from the tree frogs Scinax fuscovarius and Scinax. nasicus. We performed a morphological assessment of the parasite using light microscopy, for the first time, scanning electron microscopy. Molecular characterization of D. lutzi was carried out using the mitochondrial gene 16S rRNA and the nuclear gene 28S rRNA. Additionally, a phylogenetic tree was constructed to assess the species´position in relation to other group members. In our results, we confirmed the phenotypic morphological characteristics of the endoparasitic phase of D. lutzi. We also presented its phylogenetic position with other oligochaetes in the group, demonstrating the proximity between the endoparasite D. lutzi and the free-living oligochaete D. superterrenus. freeliving free living phases D habits s host nasicus microscopy time S Additionally speciesposition members results superterrenus

Resumo O oligoqueta Dero lutzi possui uma estratégia de vida que alterna entre períodos de vida livre em ambientes aquáticos e fases endoparasitárias. A maioria das ocorrências de D. lutzi em anfíbios anuros é relatada em espécies com hábitos arborícolas, com estudos que se restringem a registros da presença do oligoqueta no hospedeiro. Este estudo recuperou espécimes de D. lutzi das pererecas Scinax fuscovarius e Scinax nasicus. Foi realizada uma avaliação morfológica do parasito por microscopia de luz e, pela primeira vez, por microscopia eletrônica de varredura. A caracterização molecular de D. lutzi foi realizada com o gene mitocondrial 16S rRNA e o gene nuclear 28S rRNA. Além disso, uma árvore filogenética foi construída para avaliar a posição da espécie em relação a outros membros do grupo. Os resultados deste estudo confirmaram as características morfológicas fenotípicas da fase endoparasitária de D. lutzi. Também foi apresentada sua posição filogenética com outros oligoquetas do grupo, o que permitiu demonstrar a proximidade entre o endoparasito D. lutzi e o oligoqueta de vida livre D. superterrenus. endoparasitárias D arborícolas hospedeiro nasicus vez varredura S disso grupo superterrenus

Abstract The skin of anuran species is a protective barrier against predators and pathogens, showing also chemical defense by substances that represent a potential source for bioactive substances. This review describes the current chemical and biological knowledge from the skin secretions of Leptodactylidae species, one of the most diverse neotropical frog families. These skin secretions reveal a variety of substances such as amines (12), neuropeptides (16), and antimicrobial peptides (72). The amines include histamine and its methylated derivatives, tryptamine derivatives and quaternary amines. The peptides of Leptodactylidae species show molecular weight up to 3364 Da and ocellatins are the most reported. The peptides exhibit commonly glycine (G) or glycine-valine (GV) as C-terminal amino acids, and the most common N-terminal amino acids are glutamic acid (E), lysine (K), and valine (V). The substances from Leptodactylidae species have been evaluated against pathogenic microorganisms, particularly Escherichia coli and Staphylococcus aureus, and the most active peptides showed MIC of 1-15 µM. Furthermore, some compounds showed also pharmacological properties such as immunomodulation, treatment of degenerative diseases, anticancer, and antioxidant. Currently, only 9% of the species in this family have been properly studied, highlighting a large number of unstudied species such as an entire subfamily (Paratelmatobiinae). The ecological context, functions, and evolution of peptides and amines in this family are poorly understood and represent a large field for further exploration. pathogens families 12, 12 , (12) 16, 16 (16) 72. 72 . (72) 336 reported G (G glycinevaline GV (GV Cterminal C terminal Nterminal N E, E (E) K, K (K) V. V (V) microorganisms aureus 115 1 15 1-1 µM Furthermore immunomodulation diseases anticancer antioxidant Currently 9 studied Paratelmatobiinae. Paratelmatobiinae (Paratelmatobiinae) context functions exploration (12 (16 7 (72 33 (E (K (V 11 1- (Paratelmatobiinae (1 (7 3 (

Abstract The distribution of amphibians is conditioned by historical factors and ecological drivers. Thus, Ecological Niche Models are important tools to provide information on the potential distribution of the species and determine where they will be requirements in future. Mainly, rare species or with restricted areas of occurrence, because this information are essential to understanding of their living area, microhabitat use, or natural history, serving as a basis for preservation actions. Xenohyla eugenioi is a poorly known species, restricted to transition areas of Caatinga and Atlantic Forest from Minas Gerais to Sergipe. This species exhibits little biological information available and few specimens housed in museums, which difficulty to plan conservation strategies. Here our aim was modeling the current and future distribution and discussing about conservation of Xenohyla eugenioi. We searched for occurrence records through literature and scientific collections data. For the future (2071-2100), we used ensemble models from three algorithms (CTA, GLM and ANN) to two shared socioeconomic pathways (SSP370 and SSP585 scenarios). This species has approximately 650 km in straight line between the extreme localities, occurring mainly in bromeliads near to water bodies. The Ensemble method indicates the most suitable areas of occurrence were over ecotonal range between Caatinga and Atlantic Forest and our projections have showed suitable conditions to highlands (up to 1.000 m). However, in the future, is expected total erosion of the X. eugenioi populations, due climatic changes, which reinforce the caution to conservation of this poorly known species and necessity of studies about its ecology, natural history and distribution. Moreover, we hope that this work can contribute to the discovery of new records, characterizing the narrower niche space than this species may actually inhabit. drivers Thus Mainly area use actions Sergipe museums strategies data 20712100, 20712100 2071 2100 , (2071-2100) CTA, CTA (CTA ANN SSP370 SSP (SSP37 SSP58 scenarios. scenarios . scenarios) 65 localities bodies up 1000 1 000 1.00 m. m m) However X populations changes ecology Moreover inhabit 2071210 207 210 (2071-2100 SSP37 (SSP3 SSP5 6 100 00 1.0 207121 20 21 (2071-210 SSP3 (SSP 10 0 1. 20712 2 (2071-21 (2071-2 (2071- (2071 (207 (20 (2 (

Abstract We studied the relationship between shape, size, and developmental time in the embryonic ontogeny of 15 species of the frog genus Physalaemus. As in other anuran exotrophic embryos, shape changes are correlated with size increase and mainly concern tail elongation, decrease in body height, and increase in fin height. Size ranges and developmental times vary interspecifically. Embryos of the P. signifer Clade and the P. gracilis Group are among the largest, are slightly peramorphic, and develop fast regarding congeneric species. Embryos of P. cicada combine the smallest sizes with fast development and the most peramorphic shapes. The paedomorphic shapes of embryos of P. biligonigerus and P. henselii groups are correlated with fast vs. slow developmental times respectively. Trajectories in the P. cuvieri Group are diverse and in general differ in size and developmental time. The embryos of P. cristinae and from the Argentinean lineage of P. cuvieri stand out with the longest development. Sequences of developmental events are overall conserved in the genus, and main differences concern mouthpart ontogeny. This study constitutes the first attempt to evaluate morphological, allometric, and heterochronic parameters of the early ontogeny of anurans and how these can vary and contribute to diversification in taxonomic groups. 1 Physalaemus elongation height interspecifically P largest vs respectively morphological allometric

Resumo A literatura científica sobre anomalias em anfíbios é vasta, mas casos registrados de heterocromia ocular em anfíbios estão quase ausentes, mesmo considerando registros anedóticos. No território brasileiro, apenas dois casos de heterocromia ocular foram registrados anteriormente para anfíbios, ambos para anuros, o primeiro para a Mata Atlântica do nordeste do Brasil e o segundo para a região amazônica do norte do Brasil. No presente relato, descrevemos dois casos de heterocromia ocular no hilídeo Boana albomarginata, ambos no bioma Mata Atlântica, estado da Bahia, nordeste do Brasil, representando o primeiro registro de heterocromia ocular para esta espécie e o terceiro caso registrado para uma espécie de anfíbio no Brasil. Um olho de cada exemplar observado apresentava atipicamente íris de cor azul chamativa e homogênea, preenchendo toda sua superfície ou apenas a metade inferior, representando casos de heterocromia completa e setorial, respectivamente. Além disso, a não-usual íris azul só esteve detectável sob a luz do dia, quando a cor de fundo da íris era branca. À noite, a cor de fundo padrão da íris cobreada ou dourada permaneceu inalterada e a cor azul não pôde ser detectada. Nenhuma outra anomalia detectável ou comportamento incomum foi observada para ambos os exemplares. Este é o primeiro relato de heterocromia ocular em anfíbios representado por uma cor azul bem-marcada e limitada a um padrão diurno de cor da íris. Mais estudos são necessários para determinar a natureza desta anomalia e suas potenciais consequências para o indivíduo e para a espécie. vasta ausentes anedóticos brasileiro anuros albomarginata Bahia homogênea inferior setorial respectivamente disso nãousual usual dia branca noite detectada exemplares bemmarcada bem marcada

Abstract The scientific literature on amphibian anomalies is large, but registered cases of amphibians’ ocular heterochromia are almost absent, even considering anecdotal records. In the Brazilian territory, only two cases of ocular heterochromia were formerly registered for amphibians, both for anurans, the first from the Atlantic Forest of northeastern Brazil and the second from the Amazonian region of northern Brazil. In the present account, we report two cases of ocular heterochromia in the hylid treefrog Boana albomarginata, both from the Atlantic Forest biome, Bahia state, northeastern Brazil, representing the first record of ocular heterochromia for this species and the third case registered for an amphibian’s species in Brazil. One eye of each observed specimen had an unusually flashy and homogeneous blue-colored iris, filling its entire surface or only its lower half, representing cases of complete and sectoral heterochromia, respectively. Furthermore, the unusual flashy blue iris was only detectable during daylight, when the iris background color was white. At night, the standard copper to golden iris background color remains unchanged, and the blue color cannot be detected. No other detectable anomalies or unusual behaviors were observed for both specimens. This is the first report of amphibian ocular heterochromia represented by a flashy blue color iris and limited to a daylight iris color pattern. Further studies are necessary to determine the nature of this anomaly and its potential consequences for the specimen and the species. large amphibians absent records territory anurans account albomarginata biome state s bluecolored colored half respectively Furthermore white night unchanged detected specimens pattern

Abstract Land cover changes resulting from agricultural expansion and cattle ranching are the primary factors influencing the diversity of amphibian and reptile communities. In this study, we assessed the variation in the diversity of amphibians and reptiles across four land-cover types (ponds, grasslands, temporary crops, and secondary vegetation) in a rural landscape in the Colombian Caribbean region. Amphibians and reptiles were sampled through visual encounter surveys between April and August 2018. A total of 19 species of amphibians (frogs and toads) and 23 species of reptiles (14 lizards, 7 snakes, 1 turtle, and 1 alligator) were recorded. Species diversity differed among land covers, for both amphibians and reptiles. Amphibian diversity was higher in ponds and lower in grasslands, whereas reptile diversity was higher in secondary vegetation and lower in temporary crops. Our results suggest that the maintenance of ponds and secondary vegetation could be important for the conservation of herpetofauna in rural landscapes where anthropogenic activities such as agriculture and cattle ranching predominate.

Resumen Los cambios en la cobertura del suelo debido a la expansión agrícola y ganadera son las principales causas que influyen en la diversidad de las comunidades de anfibios y reptiles. En este estudio, evaluamos la variación de la diversidad de anfibios y reptiles en cuatro tipos de cobertura del suelo (jagüeyes, pastizales, cultivos transitorios y vegetación secundaria) en un paisaje rural de la región Caribe colombiana. Los anfibios y reptiles fueron muestreados a través del método de relevamiento por encuentros visuales entre abril y agosto de 2018. Se registró un total de 19 especies de anfibios (ranas y sapos) y 23 especies de reptiles (14 lagartos, 7 serpientes, 1 tortuga y 1 caimán). La diversidad de especies difirió entre las coberturas del suelo, tanto para anfibios como para reptiles. La diversidad de anfibios fue mayor en los jagüeyes y menor en los pastizales, mientras que la de reptiles fue mayor en la vegetación secundaria y menor en los cultivos transitorios. Nuestros resultados sugieren que el mantenimiento de jagüeyes y vegetación secundaria podría ser importante para la conservación de la herpetofauna en paisajes rurales donde predominan actividades antropogénicas como la agricultura y la ganadería.

Resumen Introducción: El uso frecuente de plaguicidas es considerado actualmente una causa de contaminación ambiental debido a las altas tasas de ingreso de estas sustancias a los agroecosistemas. Esta situación es un riesgo para las especies que habitan en estos ecosistemas, en particular los anuros cuyas características los hacen propensos a la exposición e interacción con contaminantes ambientales. Objetivo: Informar la presencia de anormalidades en larvas del sapo común Rhinella arenarum que habitan en estanques rodeados por un agroecosistema. Métodos: En dos primaveras consecutivas (2015 y 2016), se monitorearon los eventos reproductivos del sapo común proveniente de sistemas de estanques temporales ubicados en zonas agrícolas y no agrícolas, cerca de la ciudad de La Plata (Buenos Aires, Argentina). Se midieron los parámetros fisicoquímicos de los estanques y se registraron las etapas de cada evento reproductivo como el número de sapos adultos, amplexos y nidadas. En el laboratorio, las larvas fueron medidas y fotografiadas, se registró su estado de desarrollo y se examinó la morfología de cada una bajo microscopio estereoscópico. Se procesaron muestras representativas (normales y anormales) de cada estanque estudiado para análisis histopatológico. Resultados: En la población de R. arenarum que vive dentro de un agroecosistema, se observó un menor número de adultos reproductores y puestas en relación con la del estanque en la zona no agrícola. Se recolectaron un total de 1 910 larvas: 529 y 1 381 larvas de estanques ubicados en zonas no agrícolas y agrícolas, respectivamente. Las larvas del agroecosistema mostraron dos tipos de anormalidades: severa flexión de la cola y distensión abdominal. Además, se pudo determinar cinco grados de gravedad en relación con la distensión abdominal. Conclusiones: Una alta frecuencia y severidad de anormalidades en los estadios tempranos de larvas de R. arenarum que viven dentro de un agroecosistema proporciona evidencia del impacto negativo que las actividades agrícolas causan en los ecosistemas acuáticos rodeados por áreas de cultivo.

Abstract Introduction: The frequent use of pesticides is currently considered a cause of environmental pollution due to the high rate of entry of these substances into agroecosystems. This constitutes a risk for the species that inhabit these ecosystems, in particular anurans whose characteristics make them prone to exposure to and interaction with environmental pollutants. Objective: To report the occurrence of abnormalities in larvae of the common toad Rhinella arenarum inhabiting ponds surrounded by agroecosystems. Methods: In two consecutive springs (2015 and 2016), reproductive events of common toads were monitored in temporary pond systems in agricultural and non-agricultural areas, located near the city of La Plata (Buenos Aires, Argentina). The physicochemical parameters of the ponds were measured, and the stage of each reproductive event was recorded, such as the numbers of adult toads, amplexus and clutches. In the laboratory, the larvae were measured and photographed, their stage of development was recorded, and their morphology was examined under a stereomicroscope. Representative samples (normal and abnormal) from each pond studied were processed for histopathological analysis. Results: In the field studies carried out on a population of R. arenarum collected in an agroecosystem, a lower number of reproductive adults and clutches were observed in relation to the population of a non-agricultural pond. A total of 1 910 larvae were collected: 529 and 1 381 larvae from ponds located in non-agricultural and agricultural areas, respectively. Larvae from the agroecosystem showed two types of abnormalities: severe tail flexure and abdominal bloating. In addition, five degrees of severity could be determined in relation to abdominal bloating. Conclusions: This work reports the high frequency and severity of abnormalities observed in the early stages of R. arenarum larvae living within an agroecosystem, providing evidence of the negative impact that agricultural activities cause on aquatic ecosystems surrounded by farming areas.

Resumen En Colombia, los anfibios se posicionan como uno de los grupos vertebrados con mayor riqueza taxonómica, y una alta representatividad de especies endémicas. En los últimos años, las colecciones regionales han contribuido a llenar vacíos de información sobre la biogeografía, la variación intra e interespecífica y la sistemática de los anfibios del país. En este trabajo presentamos la información de 1533 registros depositados en la Colección de Anfibios del Museo de Historia Natural de la Universidad de Caldas, los cuales representan 143 especies distribuidas en 49 géneros y 19 familias. La mayoría de los especímenes (n = 1194) proceden del departamento de Caldas, sin embargo, se cuenta con registros (n = 339) de otros 16 departamentos del país. El orden más representado es Anura (n = 1487 registros), seguido por Caudata (n = 27 registros) y Gymnophiona (n = 19 registros). La colección alberga 152 especímenes que han sido referenciados en 13 publicaciones nacionales y extranjeras.

Abstract In Colombia, amphibians are a vertebrate group with a high taxonomic richness, and an elevated representativity of species with restricted distributions. In recent years, regional collections have assisted to fill information gaps on the biogeography, intra- and interspecific variation, and systematics of the amphibians of the country. In this work we present the information of 1533 records deposited in the Amphibian Collection of the Museo de Historia Natural of the Universidad de Caldas, represented by 143 species, belonging to 49 genera and 19 families. Most of the records (n = 1194) come from the Department of Caldas, but there are records for 16 additional departments. The most representative order is Anura (n = 1487 records) followed by Caudata (n = 27 records), and Gymnophiona (n = 19 records). The collection houses 152 specimens that have been referenced in 13 national and foreign publications.

Resumen En este trabajo reanalizamos especímenes de Paraguay asignados a la especie Rhinella icterica provenientes de los departamentos de Canindeyú y Amambay ubicados en la ecorregión del Cerrado. En base a la evaluación de caracteres morfológicos externos concluimos que los mismos deben ser asignados a la especie Rhinella cerradensis. Por otro lado, consideramos que no hay evidencia de que la especie R. icterica se encuentre en el país.

Abstract In this work, we reanalyzed specimens from Paraguay assigned to the species Rhinella icterica from the departments of Canindeyú and Amambay located in the Cerrado ecoregion. Based on the evaluation of external morphological characters, we conclude that they should be assigned to the species Rhinella cerradensis. On the other hand, we consider that there is no evidence that R. icterica is present in the country.

Resumo Neste trabalho reanalisamos espécimes do Paraguai atribuídos à espécie Rhinella icterica dos departamentos de Canindeyú e Amambay localizados na ecorregião do Cerrado. Com base na avaliação dos caracteres morfológicos externos, concluímos que eles devem ser atribuídos à espécie Rhinella cerradensis. Por outro lado, consideramos que não há evidências de que a espécie R. icterica seja encontrada no país.

Abstract Ectothermic animals present melanin-containing cells in their integument and viscera. Besides cutaneous melanophores, amphibians have melanomacrophages in the hepatic parenchyma and melanocytes in the viscera, which are also present in their testicular stroma. The native melanocyte stimulating hormone (α-MSH) is the main hormone that modulates the color change in melanophores. However, we still know too little about how the α-MSH acts in vivo on visceral melanin-containing cells. In this study, we collected 30 adult males of Physalaemus nattereri (Anura, Leptodactylidae) to evaluate the short-term effects of α-MSH on melanophores, melanocytes and melanomacrophages under light microscopy. For this, we injected 0.05 ml of a single intraperitoneal dose containing 2.5x10-7 mmol/10g of α-MSH, diluted in ringer solution, in five experimental groups with five individuals each one. The different groups were analyzed after 1, 3, 6, 12 and 24h. The control group with five other individuals received only 0.05 ml of ringer solution. The skin pigmentation increased quickly after animals received the hormone α-MSH with the consequent darkening of the body (body darkness). Melanophores, melanocytes and melanomacrophages responded similarly to the test, with an increase in the area containing melanin. However, melanophores and melanomacrophages reached their darkest pigmentation in a shorter period of time in comparison to the testicular melanocytes, probably due to specific metabolic characteristics of each organ. Thus, we verified that the three types of cells, although present in different organs, are responsive to the native hormone α-MSH, which enables us to treat them as a pigmentary system. melanincontaining melanin viscera stroma αMSH α MSH (α-MSH However study 3 Anura, Anura (Anura Leptodactylidae shortterm short term microscopy 005 0 05 0.0 2.5x107 25x107 x 2.5x10 7 2 5x10 2.5x10- mmol10g mmolg mmol 10g g αMSH, MSH, solution one 1 6 24h h darkness. darkness . darkness) Melanophores test organ Thus organs system 00 0. 5x107 25x10 2.5x1 5x1 25x1 2.5x 5x 25x

Abstract In this study we investigate the niche interaction between the frogs Physalameus cuvieri and Physalaemus kroyeri that occur in syntopy in water bodies of the Atlantic Forest located in the eastern Bahia State, Brazil. We investigated the niche width and overlap in the calling activity time, microhabitat use, diet composition, advertisement call, and body size. Both species preferred the same substrate and calling substrate categories, and showed low niche width values and high spatial niche overlap values. The pseudocommunity analysis revealed the absence of competition in space use. Ants and termites were the most important items in the diet of both species with the pseudocommunity analysis indicating an absence of competition in the diet. The two species demonstrate a greater similarity in body proportions and a high overlap in the calling activity time. However, they showed divergence in acoustic parameters, especially in the dominant frequency and call duration. Our results reinforce the role of advertisement call as a relevant attribute for anuran coexistence and highlight the importance of analyzing the various axes of the multidimensional niche for the most reliable description of the existence and magnitude of niche partitioning. State Brazil time use composition size categories However parameters duration partitioning

Abstract Faunal inventories contribute to our understanding of regional diversity, and are fundamental for policy and decision-making regarding the management and conservation of large natural areas. This study aimed to inventory and compile information on amphibian species occurring in the North Pantanal region, in the municipal limits of Cáceres, Mato Grosso, Brazil. We used three methods to inventory amphibian species: (1) fieldwork, (2) analysis of specimens deposited in scientific collections, and (3) literature reviews. We registered 49 amphibian species in Cáceres. Of them, 48 species belonged to the Anura order and were distributed across eight families and 20 genera, and one species belonged to the Gymnophiona order (Siphonops paulensis). The families Leptodactylidae (20 spp.) and Hylidae (17 spp.) were dominant in terms of richness, accounting for 75.5% of all species found in Cáceres. The remaining families had between four and one species each. The municipality is strongly influenced by non-forested formations (e.g., the Cerrado and Pantanal) and presents a high species richness for a non-forested location in Brazil. Our findings highlight Cáceres as one of the richest areas in amphibian species in the North Pantanal region, expanding our knowledge regarding frog diversity. This study provides a foundation for future conservation strategies and additional assessments of amphibian species in light of potential population declines and other emerging threats. diversity decisionmaking decision making region Grosso Brazil 1 (1 fieldwork 2 (2 collections 3 (3 reviews 4 them genera Siphonops paulensis. paulensis . paulensis) spp. spp 17 755 75 5 75.5 each nonforested non forested e.g., eg e g (e.g. threats ( 7 75. e.g. (e.g e.g

ABSTRACT Measuring cardiorespiratory variables can be challenging in developing animals, especially when they use bimodal gas exchange to maintain metabolic activity. In tadpoles, gas exchange may occur through the integument and gills when breathing in the water and through the lungs when breathing air, with varying contributions of each respiratory structure during development. The interaction between aquatic and air breathing results in a complex physiological response that may affect the cardiac cycle. Measuring the heart rate (fH) together with aquatic and aerial gas exchange in anurans during their development can be challenging, since it may involve handling small animals and/or a certain degree of invasiveness (i.e., surgery to implant electrodes). Here, we evaluated concomitantly aquatic and aerial gas exchange, lung ventilation, and fH in three stages of development of the bullfrog Lithobates catesbeianus (Shaw, 1802). We built a novel, noninvasive, closed respirometry system capable of measuring fH, aerial and aquatic gas exchange simultaneously in animals of different sizes. Our integrative analysis revealed a decrease in the heart rate and an increase in oxygen consumption during the developmental stages of the bullfrog, but there was no adjustment of heart rate after or during air breathing. Moreover, tadpoles in metamorphosis showed higher oxygen consumption in air than in water, while aquatic breathing was responsible for releasing CO2. Our results are consistent with those found in the literature, yet our study represents the first non-invasive investigation to evaluate bimodal gas exchange and heart rate simultaneously. Moreover, our setup holds potential for further advancements that would allow for controlled water and air composition. This tool could greatly facilitate the investigation of how cardiorespiratory physiology responds to varying environmental conditions. activity cycle (fH andor i.e., ie i e (i.e. electrodes. electrodes . electrodes) Here ventilation Shaw, Shaw (Shaw 1802. 1802 1802) novel noninvasive sizes Moreover CO2 CO literature non invasive composition conditions i.e. (i.e 180 i.e 18 1

BACKGROUND: Mycobacterium tuberculosis complex (MTBC) isolates are typically stored at −70 °C in cryovials containing 1 mL aliquots of a liquid medium, with or without 50% glycerol. Multiple uses of the culture stock may decrease the strain viability while increasing the risk of culture contamination. Small culture aliquots may be more practical; however, storage capacity remains challenging. MicrobankTM beads (25 beads/vial) for the long-term storage of fungal cultures is well documented, but their use for storing MTBC isolates is uninvestigated OBJECTIVE: The study aimed to determine the feasibility of using MicrobankTM beads for long-term storage of MTBC isolates at a laboratory in South Africa. METHODS: In February 2020, 20 isolates in liquid culture were stored in MicrobankTM beads, following an in-house developed protocol, at −70 °C. At defined time points (16 months [15 June 2021] and 21 months [18 November 2021]), two beads were retrieved from each storage vial and assessed for viability and level of contamination. RESULTS: Stored liquid isolates demonstrated MTBC growth within an average time-to-detection of 18 days following retrieval, even at 21 months post storage. Contaminating organisms were detected in 2 of 80 (2.5%) culture isolates. CONCLUSION: MicrobankTM beads will allow for the reculture of up to 25 culture isolates using a reduced culture volume compared to current storage methods. MicrobankTM beads represent a storage solution for the medium-term storage of MTBC isolates. What this study adds: This study evaluated the use of MicrobankTM beads as an alternate method for storing MTBC culture isolates at −70 °C and provided a suitable option for medium-term storage of MTBC.

Resumen Se estudió la comunidad de helmintos de Lithobates spectabilis (n =77) de Las Pilas (altitud baja) y Metznoxtla (altitud alta), Reserva de la Biosfera Barranca de Metztitlán, Hidalgo, México. Se comparó la diversidad de especies mediante los números de Hill a nivel de la infracomunidad y del componente de comunidad, y se calculó la diversidad beta. Se encontraron 5 especies de Digenea, 5 de Nematoda y 2 de Acanthocephala en las ranas de las 2 localidades. La riqueza de especies de parásitos en Las Pilas (9 especies) fue mayor que la de Metznoxtla (7 especies). La comunidad de helmintos en Las Pilas estuvo dominada por nemátodos y en Metznoxtla por digéneos. La especie más prevalente en Las Pilas fue Foleyellides cf. flexicauda (65% de las ranas estaban infectadas) y en Metznoxtla fue Megalodiscus americanus (76% estaban infectadas). Glypthelmins quieta fue la especie con mayor abundancia media en ambas localidades (Las Pilas = 4.5; Metznoxtla = 11.0). La diferencia en la composición de especies entre las 2 localidades fue significativa (Anosim; R = 0.54, p > 0.05). Las diferencias en la composición de especies de helmintos entre las 2 localidades sugieren que las condiciones ambientales locales son diferentes, lo que puede reflejar diferencias en la dieta de los hospederos a nivel local.

Abstract The helminth community of Lithobates spectabilis (n =77) was studied from Las Pilas (low altitude) and Metznoxtla (high altitude), Biosphere Reserve Barranca de Metztitlán, Hidalgo, Mexico. Species diversity was compared at the infracommunity and the component community level using Hill numbers, and the beta diversity was calculated. Five species of Digenea, 5 of Nematoda, and 2 of Acanthocephala were found in the frogs from the 2 localities. The species richness of parasites in Las Pilas (9 species) was higher than that of Metznoxtla (7 species). The helminth community in Las Pilas was dominated by nematodes and, in Metznoxtla, it was dominated by digeneans. The most prevalent species in Las Pilas was Foleyellides cf. flexicauda (65% of frogs were infected) and, in Metznoxtla, it was Megalodiscus americanus (76% were infected). Glypthelmins quieta was the species with the highest mean abundance in both localities (Las Pilas = 4.5; Metznoxtla = 11.0). The difference in species composition between the 2 localities was significant (Anosim; R = 0.54, p < 0.05). The differences in the composition of species of helminths between the 2 localities suggest that local environmental conditions are different, which may reflect differences in local host diet.