Clathrin-dependent pathways and the cytoskeleton network are involved in ceramide endocytosis by a parasitic protozoan, Giardia lamblia

Although identified as an early-diverged protozoan, Giardia lamblia shares many similarities with higher eukaryotic cells, including an internal membrane system and cytoskeleton, as well as secretory pathways. However, unlike many other eukaryotes, Giardia does not synthesize lipids de novo, but rather depends on exogenous sources for both energy production and organelle or membrane biogenesis. It is not known how lipid molecules are taken up by this parasite and if endocytic pathways are involved in this process. In this investigation, we tested the hypothesis that highly regulated and selective lipid transport machinery is present in Giardia and necessary for the efficient internalization and intracellular targeting of ceramide molecules, the major sphingolipid precursor. Using metabolic and pathway inhibitors, we demonstrate that ceramide is internalized through endocytic pathways and is primarily targeted into perinuclear/endoplasmic reticulum membranes. Further investigations suggested that Giardia uses both clathrin-dependent pathways and the actin cytoskeleton for ceramide uptake, as well as microtubule filaments for intracellular localization and targeting. We speculate that this parasitic protozoan has evolved cytoskeletal and clathrin-dependent endocytic mechanisms for importing ceramide molecules from the cell exterior for the synthesis of membranes and vesicles during growth and differentiation.     

Diverse viscerotropic isolates of Leishmania all express a highly conserved secretory nuclease during human infections

Previously, we characterized a gene encoding the unique nuclease (LdNuc(s)) from a Sudanese isolate of the human pathogen Leishmania donovani. This parasite secretory enzyme is involved in the salvage of host-derived purines and is constitutively expressed by both developmental forms of the parasite. Currently, we assessed whether an LdNuc(s)-like nuclease was conserved among other geographically disparate isolates of L. donovani and whether this enzyme was produced by intracellular amastigotes during human infections. Using RT-PCR and Southern blotting, we showed that LdNuc(s) gene homologs were present in each of the viscerotropic Leishmania tested (i.e., L. donovani isolates from the Sudan, Ethiopia and India as well as L. infantum). Further results of in situ enzyme activity gel analyses showed that each of these parasite isolates also expressed a released/secreted LdNuc(s)-like nuclease activity. In Western blots, our anti-LdNuc(s) (Sudan) peptide-specific antibody reacted with only a single ~35 kDa protein in each of the viscerotropic Leishmania isolates. Further, the ~35 kDa nuclease secreted by each of these isolates was specifically immunoprecipitated by the anti-LdNuc(s) antibody above. In situ gel analyses showed that each of these immunoprecipitates had LdNuc(s)-like nuclease activity. Moreover, sera from acute visceral leishmaniasis patients from India, Sudan and Brazil all immunoprecipitated an LdNuc(s)-HA expressed nuclease demonstrating, that these patients possessed antibodies against this parasite secretory enzyme. Cumulatively, these results showed that the LdNuc(s) homologs were functionally conserved among geographically disparate visceral Leishmania spp. and that amastigotes of these parasites must produce this nuclease enzyme during the course of human disease.     

Biological and genetic aspects of crosses between species of the genus Meccus (Hemiptera: Reduviidae Triatominae)

The degree of reproductive isolation between Meccus phyllosomus and the remaining five species of the genus Meccus, as well as between Meccus bassolsae and Meccus pallidipennis, Meccus longipennis and Meccus picturatus, was examined. Fertility and the segregation of morphological characteristics were examined in two generations of hybrids from crosses between these species. The percentage of couples with offspring (fertile) was high in the vast majority of sets of crosses, with the exception of that between ♀M. phyllosomus and ♂Meccus mazzottii. In sets of crosses involving M. bassolsae specimens, no first-generation (F1) individuals were morphologically similar to M. bassolsae, but instead shared the morphology of the other parental species. A similar phenomenon was observed in most sets of crosses involving M. phyllosomus. These results indicated that different degrees of reproductive isolation exist among the species of Meccus involved in this study. The biological evidence obtained in this study does not support the proposal that M. bassolsae is a full species. It could indicate that, on the contrary, it should be considered a subspecies of a single polytypic species. The biological evidence does support the proposal that M. phyllosomus is a full species.     

Feeding, defecation, and development times of Meccus longipennis Usinger, 1939 (Hemiptera: Reduviidae: Triatominae) under laboratory conditions

Aspects related to hatching, time-lapse between presenting the blood-meal and beginning of feeding, feeding time, postfeed defecation delay, mortality, and fecundity for each stage of Meccus longipennis life-cycle were evaluated. The bugs were maintained in a dark incubator at 27 1 C and 80 5% rh, were fed weekly and checked daily for ecdysis or death. The hatching rate observed for 300 eggs was 76.7% and the average time of hatching was 19.8 days. Mean time-lapse between presentation of the blood meal and the beginning of feeding was under 5 min in nymphal stages and postfeed defecation delay was under 10 min in most stages, except in fourth and fifth stages. Mean feeding time was longer than 10 min in most stages, except in fourth stage. One hundred thirty-one nymphs (N) (65.5%) completed the cycle and the average time from NI to adult was 192.6 34.8 days. The average span in days for each stage was 18.1 for NI, 21.4 for NII, 29.5 for NIII, 45.5 for NIV and 55.9 for NV. The number of bloodmeals at each nymphal stage varied from 1 to 5. The mortality rate was 3.29 for NI, 6.8 for NII, 2.92 for NIII 3.76 for NIV, and 10.16 for NV nymphs. The average number of eggs laid per female in a 9-month period was 615.6. Based on our results, we conclude that M. longipennis has some biological and behavioral characteristics which influence its capacity of becoming infected and transmitting Trypanosoma cruzi to human populations in those areas of Mexico where it is currently present.     

Impact of a hurricane on the herpetofaunal assemblages of a successional chronosequence in a tropical dry forest

Land‐use change is the main cause of deforestation and degradation of tropical forest in Mexico. Frequently, these lands are abandoned leading to a mosaic of natural vegetation in secondary succession. Further degradation of the natural vegetation in these lands could be exacerbated by stochastic catastrophic events such as hurricanes. Information on the impact of human disturbance parallel to natural disturbance has not yet been evaluated for faunal assemblages in tropical dry forests. To evaluate the response of herpetofaunal assemblages to the interaction of human and natural disturbances, we used information of pre‐ and post‐hurricane herpetofaunal assemblages inhabiting different successional stages (pasture, early forest, young forest, intermediate forest, and old growth forest) of dry forest. Herpetofaunal assemblages were surveyed in all successional stages two years before and two years after the hurricane Jova that hit the Pacific Coast of Mexico on October 2011. We registered 4093 individuals of 61 species. Overall, there were only slight effects of successional stage, hurricane Jova or the interaction between them on abundance, observed species richness and diversity of the herpetofauna. However, we found marked changes in estimated richness and composition of frogs, lizards, and snakes among successional stages in response to hurricane Jova. Modifications in vegetation structure as result of hurricane pass promoted particular changes in each successional stage and taxonomic group (anurans, lizards, and snakes). Secondary forests at different stages of succession may attenuate the negative effects of an intense, short‐duration, and low‐frequency natural disturbance such as hurricane Jova on successional herpetofaunal trajectories and species turnover.     

The ESCRT-III machinery participates in the production of extracellular vesicles and protein export during Plasmodium falciparum infection

Infection with Plasmodium falciparum enhances extracellular vesicle (EV) production in parasitized red blood cells (pRBCs), an important mechanism for parasite-to-parasite communication during the asexual intraerythrocytic life cycle. The endosomal sorting complex required for transport (ESCRT), and in particular the ESCRT-III sub-complex, participates in the formation of EVs in higher eukaryotes. However, RBCs have lost the majority of their organelles through the maturation process, including an important reduction in their vesicular network. Therefore, the mechanism of EV production in P. falciparum-infected RBCs remains to be elucidated. Here we demonstrate that P. falciparum possesses a functional ESCRT-III machinery activated by an alternative recruitment pathway involving the action of PfBro1 and PfVps32/PfVps60 proteins. Additionally, multivesicular body formation and membrane shedding, both reported mechanisms of EV production, were reconstituted in the membrane model of giant unilamellar vesicles using the purified recombinant proteins. Moreover, the presence of PfVps32, PfVps60 and PfBro1 in EVs purified from a pRBC culture was confirmed by super-resolution microscopy and dot blot assays. Finally, disruption of the PfVps60 gene led to a reduction in the number of the produced EVs in the KO strain and affected the distribution of other ESCRT-III components. Overall, our results increase the knowledge on the underlying molecular mechanisms during malaria pathogenesis and demonstrate that ESCRT-III P. falciparum proteins participate in EV production.     

Influence of the blood meal source on the biology of Meccus picturatus Usinger 1939 (Hemiptera: Reduviidae: Triatominae) under laboratory conditions

Aspects related to hatching, time-lapse between presenting the blood meal and beginning of feeding, feeding time, postfeed defecation delay,life time, mortality and fecundity for each stage of Meccus picturatus, life-cycle were evaluated and compared in two cohorts of M. picturatus fed on hens or rabbits. The hatching rate observed for each of the two studied groups of eggs was 78.1% (n = 2298) on the group fed on hens and 82.1% (n = 2704) on that fed on rabbits, and the average time of hatching was 20 days. Mean time-lapse for beginning feeding was under 3 min in nymphal stages and postfeed defecation delay was under 10 min in all stages, in both cohorts. Mean feeding time was significantly (P < 0.05) shorter in triatomines fed on hens than on rabbits. A similar number of nymphs of each cohort, 69 fed on hens (34.5%) and 68 fed on rabbits (34%), completed the cycle. No significantly (P > 0.05) differences were recorded among the average times from NI to adult in the cohort fed on hens (196.8 15.8 days) and the average time in the cohort fed on rabbits (189.5 22.9). The average span in days for each stage fed on hens was not significantly different to the average span for each stage fed on rabbits. The number of blood meals at each nymphal stage varied from 1 to 6 in both cohorts. The mortality rates were higher on fifth nymphal stage, in both cohorts. No significant (P > 0.05) differences were recorded on mortality rates on most nymphal stages of both cohorts. The average number of eggs laid per female from the cohort fed on hens in a 9-month period was 791.1, whereas the average number of eggs in the cohort fed on rabbits was 928.3.     

Biodiesel synthesis from Chlorella vulgaris under effect of nitrogen limitation,intensity and quality light: estimation on the based fatty acids profiles

Cultures under nitrogen limitation for Chlorella vulgaris were kept under different light quality (white, blue, yellow and violet) at 70 and 140 µE m^?2 s^?1; to evaluate the effect on fatty acids profiles and biodiesel quality. The results showed a maximum biomass and cell density at 140 µE m^?2 s^?1 of: white light (0.69 g L^?1 and 6.5?×?10⁶ cells mL^?1, respectively) and blue light (0.65 g L^?1 and 8.0?×?10⁶ cells mL^?1, respectively); compared to violet and yellow light. The chlorophyll concentration (µg mg^?1 biomass dry weight) at 70 µE m^?2 s^?1 were in the order of light: white (25.61)?>?violet (17.10)?>?yellow (11.68)?>?blue (11.40) and, at 140 µE m^?2 s^?1 were: violet (23.64)?>?white (10.20)?>?yellow (9.66)?>?blue (7.99), suggesting the violet light stimulates the increase of chlorophyll a at higher intensity. The maximum lipid content (% w/w) were present under blue light (43.11), yellow (70.92) and violet (83.87) at 140 µE m^?2 s^?1. The different wavelengths did not have a negative effect on the quality of the biodiesel, however; violet light presented greater productivity and the indicators such as CFPP were related to the oxidative stability value and low PUFA content, leading biodiesel to good oxidative stability.