Predation creates unique void layer in membrane-aerated biofilms.

The membrane-aerated biofilm reactor (MABR) is a novel wastewater treatment technology based on oxygen-supplying membranes. The counter diffusion of oxygen and electron donors in MABRs leads to unique behavior, and we hypothesized it also could impact predation. We used optical coherence tomography (OCT), microsensor analyses, and mathematical modeling to investigate predation in membrane-aerated biofilms (MABs). When protozoa were excluded from the inoculum, the MAB's OCT-observable void fraction was around 5%. When protozoa were included, the void fraction grew to nearly 50%, with large, continuous voids at the base of the biofilm. Real-time OCT imaging showed highly motile protozoa in the voids. MABs with protozoa and a high bulk COD (270 mg/L) only had 4% void fraction. DNA sequencing revealed a high relative abundance of amoeba in both high and low-COD MABs. Flagellates were only abundant in the low-COD MAB. Modeling also suggested a relationship between substrate concentrations, diffusion mode (co- or counter-diffusion), and biofilm void fraction. Results suggest that amoeba proliferate in the biofilm interior, especially in the aerobic zones. Voids form once COD limitation at the base of MABs allows predation rates to exceed microbial growth rates. Once formed, the voids provide a niche for motile protozoa, which expand the voids into a large, continuous gap. This increases the potential for biofilm sloughing, and may have detrimental effects on slow-growing, aerobic microorganisms such as nitrifying bacteria.

Periodic venting of MABR lumen allows high removal rates and high gas-transfer efficiencies.

The membrane-aerated biofilm reactor (MABR) is a novel treatment technology that employs gas-supplying membranes to deliver oxygen directly to a biofilm growing on the membrane surface. When operated with closed-end membranes, the MABR provides 100-percent oxygen transfer efficiencies (OTE), resulting in significant energy savings. However, closed-end MABRs are more sensitive to back-diffusion of inert gases, such as nitrogen. Back-diffusion reduces the average oxygen transfer rates (OTR), consequently decreasing the average contaminant removal fluxes (J). We hypothesized that venting the membrane lumen periodically would increase the OTR and J. Using an experimental flow cell and mathematical modeling, we showed that back-diffusion gas profiles developed over relatively long timescales. Thus, very short ventings could re-establish uniform gas profiles for relatively long time periods. Using modeling, we systematically explored the effect of the venting interval (time between ventings). At moderate venting intervals, opening the membrane for 20 s every 30 min, the venting significantly increased the average OTR and J without substantially impacting the OTEs. When the interval was short enough, in this case shorter than 20 min, the OTR was actually higher than for continuous open-end operation. Our results show that periodic venting is a promising strategy to combine the advantages of open-end and closed end operation, maximizing both the OTR and OTE.

A methodology to assess the effects of biofilm roughness on substrate fluxes using image analysis, substrate profiling, and mathematical modelling.

We present a novel approach, based on image analysis and modelling, to study the impact of morphological variability (roughness) and fluid dynamics on substrate mass fluxes in biofilms. Specifically, we used this method to assess substrate fluxes in counter-diffusional autotrophic biofilms in a hydrogen-based membrane biofilm reactor. The physical structure of the biofilm was determined in situ at the meso-scale using stereomicroscopy. Image analysis was used to characterize the biofilm structure, and substrate profiles were obtained using microsensors. A two-dimensional, continuum biofilm model including microbial reactions, mass transport, and fluid dynamics was developed to compute substrate conversion in irregularly shaped counter-diffusional biofilms. Experimental biofilm structures were reproduced in the model and simulated under the prevailing substrate and hydrodynamic conditions for flow velocities varied over three orders of magnitude. Model calculations were consistent with experimental results and showed enhanced conversion rates with increased roughness at higher flow velocities. Also, modelling showed that conversion rates in counter-diffusional biofilms were typically higher than in co-diffusional biofilms. This study highlights the potential to use a simple image acquisition approach coupled to a theoretical model, to evaluate biofilm overall substrate utilization related to biofilm morphological heterogeneity.

Energy-efficient wastewater treatment via the air-based, hybrid membrane biofilm reactor (hybrid MfBR).

We used modeling to predict the energy and cost savings associated with the air-based, hybrid membrane-biofilm reactor (hybrid MfBR). This process is obtained by replacing fine-bubble diffusers in conventional activated sludge with air-supplying, hollow-fiber membrane modules. Evaluated processes included removal of chemical oxygen demand (COD), combined COD and total nitrogen (TN) removal, and hybrid growth (biofilm and suspended). Target concentrations of COD and TN were based on high-stringency water reuse scenarios. Results showed reductions in power requirements as high as 86%. The decrease mainly resulted from the dramatically lower air flows for the MBfR, resulting from its higher oxygen-transfer efficiencies. When the MBfR was used for COD and TN removal, savings up to US$200/1,000 m(3) of treated water were predicted. Cost savings were highly sensitive to the costs of the membrane modules and electrical power. The costs were also very sensitive to membrane oxidation flux for ammonia, and the membrane life. These results suggest the hybrid MBfR may provide significant savings in energy and costs. Further research on the identified key parameters can help confirm these modeling predictions and facilitate scale-up.

Integrating fluorescent dye flow-curve testing and acoustic Doppler velocimetry profiling for in situ hydraulic evaluation and improvement of clarifier performance.

Enhancing the performance of clarifiers requires a thorough understanding of their hydraulics. Fluorescence spectroscopy and acoustic doppler velocimeter (ADV) profiling generally have been used separately to evaluate secondary settlers. We propose that simultaneous use of these techniques is needed to obtain a more reliable and useful evaluation. Experiments were performed on laboratory- and full-scale clarifiers. Factors affecting Fluorescein and Rhodamine 6G properties were identified. Underestimations up to 500% in fluorescence intensities may be derived from differential fluorescence quenching by oxygen. A careful control and interpretation of fluorescent dye experiments is needed to minimize artifacts in real settings. While flow-curve tests constructed under controlled conditions provided a more accurate overall quantitative estimation of the hydraulic performance, ADV velocity and turbulence profiling provided a detailed spatial understanding of flow patterns that was used to troubleshoot and fix the causes of hydraulic short-circuits.

Pie diabético: evolución en una serie de 121 pacientes / Evolution of diabetic foot in a series of 121 patients

Cohorte única de 121 pacientes portadores de pie diabético (PD) tratados entre 1998 y 2005. Se analiza la evolución, a través de incidencia de cicatrización, recidiva, amputación al año, a los tres años y letalidad. El origen del PDfue predominantemente neuropático en 52 pacientes (43 por ciento), predominantemente isquémico en 44 (36.4 por ciento) y mixto en 25 casos (20.6 por ciento). En 78 casos (64.5 por ciento) había infección asociada. Las lesiones mayores que Wagner 1 tuvieron cicatrización significativamente menor, p < 0.05. La media de antigüedad de la úlcera fue 10.8 semanas. La tasa de cicatrización, 31.4 por ciento y el tiempo medio de cicatrización, 6.2 semanas; la tasa de recidiva de la úlcera durante el primer año fue 52.1 por ciento. La media de tiempo para la recidiva de la úlcera fue 5.1 meses. La tasa de amputación al año de diagnosticada la úlcera fue 95.9 por ciento y 98.3 por ciento a los tres años. La tasa de reamputación de amputaciones menores fue 55.4 por ciento y de amputaciones mayores fue 24.4 por ciento, p < 0.05. La media entre el inicio del tratamiento y la amputación fue 11 semanas. La cirugía de rescate no modificó el desenlace de amputación (p =0.98). La letalidad fue 4.1 por ciento. Los grupos de riesgo de amputación fueron: diabéticos tipo 2 y lesiones Wagner 2 y superiores. Nuestro pobre desenlace del PD requiere implementar un plan de educación para reducir la aparición de úlcera y/ o infección local, disminuir la recidiva, mejorar la educación de los diabéticos respecto al cuidado de los pies y, replantear los niveles de amputaciones en el PD avanzado.

Background: Fifteen percent of diabetic patients will develop a foot ulcer during their lifetime. The predisposing conditions are neuropathy, ischemia and infections. Aim: To report the incidence of healing, relapse, amputation and mortality of diabetic foot in a period of eight years. Material and methods: A cohort of 134 patients hospitalized in a Surgical Service with the diagnosis of diabetic foot was studied; however in 13 patients, the clinical history was not available. Therefore the series included 81 males aged 68 + 11 years and 40 females aged 72 + 11 years. Results: Diabetic foot was predominantly neuropathic in 52 patients (43 percent), predominantly ischemic in 44 (36 percent) and mixed in 25 (21 percent). An associated infection was found in 78 patients (65 percent). The mean ulcer duration was 11 weeks. The rate of healing was 31 percent and the mean healing lapse was 6.2 weeks. The rate of relapse in the first year was 52 percent. The mean relapse time was 5.1 months. The amputation rate was 96 percent one year after diagnosis and 98 percent, three years after diagnosis. Minor and major amputation rates were 55 and 24 percent, respectively. The mean lapse between treatment onset and amputation was 11 weeks. Rescue surgical procedures did not modify the evolution of diabetic foot. Four percent of patients died. Conclusions: The overall results of diabetic foot treatment are poor. Therefore the efforts must be placed on prevention and education of patients to prevent the appearance of foot ulcers, local infections, diabetic foot relapse.

Towards a benchmarking model for winery wastewater treatment and disposal.

We propose a benchmarking model for winery wastewater treatment systems and use it to quantitatively compare the performance of Chilean wine-making operations. The benchmarking model integrates three components: the influent characteristics, the wastewater treatment alternatives, and the location constraints. Four performance levels may be defined when plotting the available data of the wine production versus the ratio of wastewater to wine, for the French, US, and Chilean industries. Knowing where a certain system lies in this diagram helps to quantify the gap between the current and a target performance, and to set performance goals for planned expansions. The analysis of construction and operating costs of treatment systems currently in operation in Chile shows that similar compliance levels can be achieved at remarkably different costs. A steep decrease in the unitary cost is observed as wastewater flow increases; yet, the treatment alternative for achieving that cost may change. Further selection is obtained when location constraints are considered, including stringent discharge standards and proximity to urban settlements. The application of this simple benchmark model to three Chilean winery facilities shows how it produces meaningful quantitative and qualitative results. However, there is still ample room to improve this benchmarking model by considering additional complexity, including technical detail in the treatment options and costs related to technology conversion.

Antibacterial activity of lactose-binding lectins from Bufo arenarum skin

Amphibians respond to microbial infection through cellular and humoral defense mechanisms such as antimicrobial protein secretion. Most humoral defense proteins are synthetized in the skin. In this study we isolated two beta-galactoside-binding lectins with molecular weights of 50 and 56 KDa from the skin of Bufo arenarum. These lectins have significant hemagglutination activity against trypsinized rabbit erythrocytes, which was inhibited by galactose-containing saccharides. They are water-soluble and independent of the presence of calcium. The antimicrobial analysis for each lectin was performed. At mumolar concentration lectins show strong bacteriostatic activity against Gram negative bacteria (Escherichia coli K12 4100 and wild strains of Escherichia coli and Proteus morganii) and Gram positive bacteria (Enterococcus faecalis). The antibacterial activity of these lectins may provide an effective defense against invading microbes in the amphibian Bufo arenarum.

Antibacterial activity of lactose-binding lectins from Bufo arenarum skin

Amphibians respond to microbial infection through cellular and humoral defense mechanisms such as antimicrobial protein secretion. Most humoral defense proteins are synthetized in the skin. In this study we isolated two beta-galactoside-binding lectins with molecular weights of 50 and 56 KDa from the skin of Bufo arenarum. These lectins have significant hemagglutination activity against trypsinized rabbit erythrocytes, which was inhibited by galactose-containing saccharides. They are water-soluble and independent of the presence of calcium. The antimicrobial analysis for each lectin was performed. At mumolar concentration lectins show strong bacteriostatic activity against Gram negative bacteria (Escherichia coli K12 4100 and wild strains of Escherichia coli and Proteus morganii) and Gram positive bacteria (Enterococcus faecalis). The antibacterial activity of these lectins may provide an effective defense against invading microbes in the amphibian Bufo arenarum. (AU)

Renal extracellular matrix alterations in lead-treated rats.

Although the nephrotoxic effects of lead are well documented, the subcellular mechanisms of its action on the kidney remain unclear. The aim of the present work was to investigate the effects of chronic lead exposure on the expression of laminin-1 and fibronectin in the kidney of lead-treated rats. Western immunoblotting of the kidney extracts revealed that experimental exposure to lead resulted in a marked decrease in the intensity of the bands corresponding to laminin-1 and an increase in the intensity of the band corresponding to fibronectin. Immunohistochemical studies demonstrated a weak labelling to laminin-1 and a strong labelling to fibronectin in all renal basement membranes together with a decrease in their thickness. Other ultrastructural alterations found were a diminution in the amount of endothelial fenestrae, an increased fusion of foot processes in epithelial cells of the glomerulus and the presence of intranuclear inclusion bodies in the proximal tubule cells. Lead intoxication might be responsible for the above alterations in the renal extracellular matrix that could play an important role in the pathogenesis of lead nephropathy.

Induction and development of neural crest in Xenopus laevis.

Neural crest cells are a migratory embryonic cell population that form at the border between the neural plate and the future epidermis. This border, the neural plate border, corresponds to the neural fold. The neural fold surrounds the entire neural plate, but only the lateral and posterior portions of the fold give rise to neural crest cells, while the anterior neural fold differentiates as forebrain. This review focuses on neural crest development in Xenopus laevis embryos, and analyzes aspects of the induction of the neural crest in Xenopus, summarizing available information relating to the expression of several genes in the neural crest. Two models for neural crest induction are discussed. In the first model, the neural crest is induced by the interaction between the neural plate and the epidermis. In the second, the specification of the neural plate border arises as a consequence of a gradient of BMP activity. The role of posteriorizing signals on neural crest specification is also discussed. Finally, we propose that the specification and differentiation of the neural crest is controlled by a cascade of transcription factors, encoded and expressed from a hierarchy of genes. A set of extracellular signals establishes the positional information in the ectoderm, which activates Prepattern genes (Gli, Xiro, Zic, Dlx, etc.) across extended and overlapping domains. A local combination of these genes at the neural plate border activates the cascade of neural crest specification, while different sets of genes are activated at both sides of the neural folds (in the epidermis and the neural plate). The genes activated in regions adjacent to the neural plate border have an inhibitory effect on the neural crest transcription program.

Fracturas expuestas, clasificación y fijación externa: opción del tercer mundo / Open fractures, classification and external fixation: a proposal for third world countries

Antecedentes. En materia de clasificaciones de fracturas, cualquier modelo debe ser seguro, confiable y reproducible. Para el trabajo clínico cotidiano, además debe ser de simple aplicación. En relación con estas recomendaciones la clasificación de Gustilo sigue siendo cuestionada. Objetivos. El propósito del presente estudio es mostrar una alternativa de clasificación de fracturas abiertas que apuntan a los conceptos arriba recomendados y mostrar igualmente, una propuesta de fijación externa ®desechable¼ como método sencillo, seguro y barato, para todas las opciones de fracturas abiertas y para cualquier procedimiento que requiere de fijación externa. Métodos. Se consideraron por un lado, las características del daño de los tejidos blandos tomando en cuenta fundamentalmente su dimensión por tercios, en tres grados. Por otro lado, las características de la morfología fracturaria según el examen radiográfico divididas en cuatro grupos, de menos a más, según su complejidad. Ambas características fueron entrecruzadas. Además se consideró el momento del tratamiento inicial, en tres momentos. Todas estas características se graficaron en una plantilla portátil con la cual se registraron, sin exclusiones, durante cuatro años seguidos, 125 fracturas expuestas de la diáfisis tibial. Todas fueron inmovilizadas por fijación externa ®desechable¼. Durante el tratamiento fueron registrados el número de actos operatorios hasta su curación, las dificultades, las complicaciones, el tiempo de consolidación y sus secuelas. El rango de seguimiento postoperatorio fue de 11 meses a 10 años. Resultados. Todas las fracturas pudieron ser claramente diagnosticadas. Las dificultades en el logro de la reducción y en el logro de la inmovilización así como los tiempos de consolidación fueron proporcionales con la escala clasificatoria. La técnica operatoria y el material de implante para la fijación externa ®desechable¼ estuvo al alcance de todas las opciones de fracturas, y al alcance de cualquier paciente, tanto en el medio rural como en la gran ciudad. Conclusiones. El diagnóstico fue fácil y rápido, y fue ubicado dentro de 15 probabilidades con características bien definidas que apuntan a lo exigido en clasificaciones. En la práctica clínica, esta alternativa de clasificación así como la técnica de fijación externa desechable demostraron ser funcionales.

Hypoglycemic effect of the water extract of Smallantus sonchifolius (yacon) leaves in normal and diabetic rats.

The hypoglycemic effect of the water extract of the leaves of Smallantus sonchifolius (yacon) was examined in normal, transiently hyperglycemic and streptozotocin (STZ)-induced diabetic rats. Ten-percent yacon decoction produced a significant decrease in plasma glucose levels in normal rats when administered by intraperitoneal injection or gastric tube. In a glucose tolerance test, a single administration of 10% yacon decoction lowered the plasma glucose levels in normal rats. In contrast, a single oral or intraperitoneal administration of yacon decoction produced no effect on the plasma glucose levels of STZ-induced diabetic rats. However, the administration of 2% yacon tea ad libitum instead of water for 30 days produced a significant hypoglycemic effect on STZ-induced diabetic rats. After 30 days of tea administration, diabetic rats showed improved body (plasma glucose, plasma insulin levels, body weight) and renal parameters (kidney weight, kidney to body weight ratio, creatinine clearance, urinary albumin excretion) in comparison with the diabetic controls. Our results suggest that yacon water extract produces an increase in plasma insulin concentration.

Changes in the expression of small intestine extracellular matrix proteins in streptozotocin-induced diabetic rats.

Diabetes mellitus is characterized by anatomical and functional alterations of the intestinal tract. However, the aetiology of these disturbances remains unclear. The aim of the present work was to investigate the effects of diabetes on the expression of laminin-1 and fibronectin in the small intestine of Streptozotocin (STZ)-induced diabetic rats. The Western immunoblotting of the extracts from the small intestine revealed that experimental diabetes resulted in a marked increase in the intensity of the bands corresponding to laminin-1 and fibronectin. Immunohistochemical studies demonstrated a strong labelling to these two extracellular matrix (ECM) proteins in the small intestine of diabetic rats, mainly localized in the smooth muscle layer. These results occur together with a thickening of the basement membrane (BM) of the smooth muscle cells, demonstrated by transmission electron microscopy (TEM). We propose that the accumulation of ECM proteins in the smooth muscle layer may be an effect mediated by hyperglycaemia, since insulin treatment of diabetic rats reversed this accumulation. These results could provide information on the potential role of the ECM in the intestine, an organ which is known to exhibit important alterations in diabetes.

Changes in liver gangliosides in streptozotocin-induced diabetic rats.

Diabetes mellitus is associated with various structural and functional liver abnormalities that affect the glycogen and lipid metabolisms. The effects of streptozotocin-induced diabetes and of insulin supplementation to Sprague-Dawley diabetic rats on ganglioside patterns in liver were determined. Diabetic livers showed a tendency to hepatomegaly 3 weeks after STZ-induction of diabetes. The concentration of total gangliosides in diabetic and non-diabetic livers was similar, but the concentration of total gangliosides in the liver of insulin-stabilized rats was slightly increased. Bidimensional TLC chromatographic analysis of gangliosides isolated from normal diabetic and insulin-stabilized diabetic livers showed quantitative and qualitative changes. In comparison with normal controls, the densitometric analyses of diabetic liver ganglioside patterns had increased amounts of GM3, GM1, GD1b, and GT1b gangliosides, while GM2 could not be detected. The hepatic ganglioside pattern of insulin-stabilized diabetic rats was partially restored, resembling the profile of normal rats. The activity of GalNAcT, GalT-2 and SialT-4 transferases was measured in liver microsomal fractions of the different groups of animals. Diabetic rats showed an increased activity of GalNAcT and a decrease in the activity of GalT-2 and SialT-4 compared with the controls. The enzymatic activities found in insulin-treated rats showed a tendency to return to the values observed in normal control animals. The results evidenced that streptozotocin-induced diabetes affects the liver ganglioside pattern and the ganglioside synthesis enzyme activity. The alterations found in ganglioside metabolism could represent one of the earliest changes associated with the diabetic pathology.

Inhibition of the synthesis of glycosphingolipid by a ceramide analogue (PPMP) in the gastrulation of Bufo arenarum.

In the present study the role of glycosphingolipids (GSL) in amphibian development was investigated. We analysed the de novo synthesis of neutral GSL and gangliosides through the initial stages of Bufo arenarum embryo development and their participation during gastrulation using 1-phenyl-2-palmitoyl-3-morpholino-1-propanol (PPMP), a potent inhibitor of glucosylceramide synthase. Ganglioside synthesis began at the blastula stage and reached a maximum during gastrulation (stages 10-12) while neutral GSL synthesis showed a slight gradual increase, the former being quantitatively more significant than the latter. Ganglioside synthesis was reduced by 90% while neutral GSL synthesis was inhibited by 65% when embryos at blastula stage were cultured for 24 h in 20 microM PPMP. The depletion of GSL from amphibian embryos induced an abnormal gastrulation in a dose-dependent manner. We found that PPMP had a pronounced effect on development since no embryos exhibited normal gastrulation; their developmental rate either slowed down or, more often, became totally arrested. Morphological analysis of arrested embryos revealed inhibition of the gastrulation morphogenetic movements. Analysis of mesodermal cell morphology in those embryos showed a severe decrease in the number and complexity of cellular extensions such as filopodia and lamellipodia. Mesodermal cells isolated from PPMP-treated embryos had very low adhesion percentages. Our results suggest that glycosphingolipids participate in Bufo arenarum gastrulation, probably through their involvement in cell adhesion events.

Effect of gap junction uncoupling in full-grown Bufo arenarum ovarian follicles: participation of cAMP in meiotic arrest.

The aim of the present study was to determine the presence of the connexins Cx43, Cx32 and Cx26 in Bufo arenarum ovarian follicles during the breeding season as well as to analyse the possible alterations in the meiotic process when connexins are blocked by specific antibodies. Western blot analysis revealed that the Cx43 and Cx32 proteins were present but not Cx26. We demonstrated that the anti-Cx43 and anti-Cx32 antibodies produced the uncoupling of the gap junctions. When these junctions are blocked the maturation process is triggered in the oocytes. We determined that dbcAMP exerts an inhibitory effect on the maturation induced by the uncoupling of the gap junctions when the oocytes are injected or pretreated with this metabolite. We propose the idea that cAMP is the regulatory molecule in meiotic arrest in this amphibian species.

Participation of the GM1 ganglioside in the gastrulation of anuran amphibian Bufo arenarum.

In the present paper we established the ganglioside composition of the blastula and gastrula stages of the anuran amphibian Bufo arenarum, two relevant stages characterized by dynamic changes in morphology and cellular rearrangements. Densitometric studies evidenced that GD1a and GT1b were the more abundant gangliosides of the blastula embryos whereas GM1 and GM2 were the predominant species in gastrula embryos. Analysis of ganglioside abundance indicates that the "a" and "b" synthesis pathways perform similar biosynthetic activities in the blastula stage, in contrast to the gastrula stage in which a marked predominance of the "a" pathway occurred. The spatio-temporal expression of GM1 and of polygangliotetraosyl ceramides (pGTC) was investigated by wholemount immunocytochemistry using cholera toxin B subunit (CTB) and an affinity purified human anti-GM1 antibody. The pGTC were detected as GM1 after treatment with neuraminidase. Blastomeres from the inner surface of the blastocoelic roof (BCR) of blastula embryos were GM1 and pGTC positive. At midgastrula stage, embryos showed an increased labeling on the inner surface of BCR. To establish whether the GM1 ganglioside was involved in the gastrulation processes, CTB, anti-GM1 antibodies and anti-GM1 Fab' fragments were microinjected into the blastocoel cavity of blastula embryos. Treatment with the probes blocked gastrulation. Scanning electron microscopy analysis of blocked embryos revealed that mesodermal cell migration, radial interdigitation, and convergent extension movements were affected. The blocking of gastrulation was correlated with the absence of fibronectin and EP3/EP4 on the inner surface of blastocoelic roof of CTB- or anti-GM1 treated embryos. Results show that the GM1 ganglioside is differentially expressed by embryonic cells and participates in the morphogenetic processes of amphibian gastrulation. J. Exp. Zool. 286:457-472, 2000.

Comparative study of vitellogenesis in the anuran amphibians Ceratophrys cranwelli (Leptodactilidae) and Bufo arenarum (Bufonidae).

The present study analyses, by transmission electron microscopy, vitellogenesis in two anuran amphibian families: Leptodactilidae (Ceratophrys cranwelli) and Bufonidae (Bufo arenarum). These differ in the type of stimulus that sets off their reproductive period, pluvial changes being the trigger in C. cranwelli and temperature increase in B. arenarum. We found that vitellogenesis follows an endocytic pathway that involves membranous structures (coated pits, coated vesicles, endosomes and multivesicular bodies). This process results in a fully grown yolk platelet of similar structure in both species. Despite the above similarity, a distinctive feature in B. arenarum was that the multivesicular bodies exhibited condensed proteins together with lipid droplets, the latter remaining as such even in the primordial yolk platelet. In C. cranwelli, however, lipids droplets were only found attached to the primordial yolk platelet. The coexistence of lipid droplets together with proteins in the nascent precursor yolk platelets observed in B. arenarum is similar to that found in B. marinus. This fact might constitute a characteristic feature of the Bufonidae family.