Wound healing and Cadmium detoxification in the earthworm Lumbricus terrestris - a potential case for coelomocytes?

Earthworms are affected by physical stress, like injury, and by exposure to xenobiotics, such as the toxic metal cadmium (Cd), which enters the environment mainly through industry and agriculture. The stress response to the single and the combination of both stressors was examined in regenerative and unharmed tissue of Lumbricus terrestris to reveal if the stress response to a natural insult like injury (amputation) interferes with Cd detoxification mechanisms. We characterized the roles of metallothionein 1 (MT1) and MT2 isoforms, heat shock protein 70 as well as immune biomarkers such as the toll-like receptors (TLR) single cysteine cluster TLR and multiple cysteine cluster TLR. The role of the activated transcription factors (ATFs) ATF2, ATF7, and the cAMP responsive element binding protein as putative regulatory intersection as well as a stress-dependent change of the essential trace elements zinc and calcium was analyzed. Phosphorylated AMP activated protein kinase, the cellular energy sensor, was measured to explore the energy demand, while the energy status was determined by detecting carbohydrate and protein levels. Taken together, we were able to show that injury rather than Cd is the driving force that separates the four treatment groups - Control, Cd exposure, Injury, Cd exposure and injury. Interestingly, we found that gene expression differed regarding the tissue section that was analyzed and we hypothesize that this is due to the migration of coelomocytes, earthworm immune cells, that take over a key role in protecting the organism from a variety of environmental challenges. Surprisingly, we discovered a role for MT1 in the response to multiple stressors and an isoform-specific function for the two newly characterized TLRs. In conclusion, we gathered novel information on the relation of innate immunity, wound healing, and Cd detoxification mechanisms in earthworms.

One piece of the puzzle: Modeling vector presence and environment reveals seasonality, distribution, and prevalence of sandflies and Leishmania in an expansion area.

The recent geographic spread of Leishmania infantum along the borders of Argentina, Brazil and Paraguay has been highlighted. In our previous study, Lutzomyia longipalpis was found in 55 of 123 patches surveyed, and in some patches, sandflies were found at higher densities, forming hotspots. Based on the One Health approach, we investigated the seasonality of the vector, the presence of parasite DNA, and the environmental factors that contribute to vector and parasite dispersal in these previously described hotspots in Foz do Iguaçu, Brazil. Entomological surveys were conducted monthly for one year. Fourteen hotspots peridomicile and six intradomicile were sampled. PCR was used to assess the prevalence of Leishmania DNA in sandflies. Zero-inflated negative binomial regression was used to determine the association of micro- and mesoscale environmental variables with the occurrence and abundance of the three most abundant sandfly species sampled. A total of 3543 species were captured, with Lutzomyia longipalpis being the predominant species (71.78%) of the 13 species found. Evandromyia edwardsi, Expapillata firmatoi, Micropygomyia ferreirana and Pintomyia christenseni were reported for the first time in the region. NDVI, distance to water, precipitation, west-to-east wind, wind speed, maximum and minimum relative humidity, and sex were significant variables associated with vector presence/abundance in the environment. Vector presence/abundance in the peridomicile was associated with precipitation, altitude, maximum temperature, minimum and maximum relative humidity, west-to-east wind, wind speed, and sex. Leishmania DNA was detected in an average of 21% of Lu. longipalpis throughout the year. Vector abundance is concentrated in urban and peri-urban areas, with some specimens present in different parts of the city and some sites with high vector abundance. This distribution suggests that the risk of actual contact between humans and parasite vectors in urban areas during the epidemic period is associated with patches of peri-urban vegetation and then extends into urban areas.

El modelo de ovario artificial como prueba celular para evaluar la seguridad de los extractos de Spirulina maxima y Kéfir: un estudio preliminar / The model of artificial ovary as a cellular test to evaluate the safety of Spirulina máxima and Kefir extracts: a preliminary study

RESUMEN S. maxima y Kefir son conocidos y utilizados por sus propiedades antioxidantes e inmunoestimulantes. El objetivo en este estudio fue evaluar los extractos naturales de estos dos agentes con la técnica de manipulación de ovocitos incluidos en folículos preantrales (MFP), porque la técnica puede reemplazar el uso de animales de laboratorio y también podría armonizar las leyes que intentan reducir y mejorar el uso de animales para el estudio de nuevos fármacos y para integrar a las buenas prácticas de laboratorio. Los extractos naturales estudiados fueron obtenidos a partir de S. maxima y Kéfir, las dos sustancias son conocidas en el mercado por su actividad antioxidante e inmunoestimulante. Los dos extractos fueron evaluados en suspensiones de 10, 50, 100 and 200 µg.mL-1. Los resultados muestran que Spirulina produjo disminución en la sobrevivencia, el desarrollo y el diámetro folicular. Mientras que Kéfir no mostró influencias positivas o negativas sobre el crecimiento y desarrollo de los folículos preantrales, sólo la concentración de 200 µg.mL-1 disminuyó la sobrevivencia folicular. La técnica MFP demostró encajar en la política de las 3R (reemplazo, reducción y refinamiento) y permitió evaluar la citotoxicidad mostrando que la técnica puede ser usada como una prueba de seguridad en extractos naturales.

SUMMARY S. maxima and Kéfir are known and used for their antioxidant and immunostimulant properties. This study aimed to evaluate the natural extracts of these two agents with the technique of manipulation of oocytes included in preantral follicles (MFP), because the technique can replace the use of laboratory animals and could also harmonize the laws that try to reduce and improve the use of animals for the study of new drugs and to integrate good laboratory practices. The natural extracts studied were obtained from S. maxima and Kefir, both substances are known in the market for their antioxidant and immunostimulating activity. Both were evaluated in suspensions of 10, 50, 100 and 200 µg.mL-1. The results show that Spirulina produced a decrease in survival, development, and follicular diameter. While Kefir did not show positive or negative influences on the growth and development of preantral follicles, only the concentration of 200 µg.mL-1 decreased follicular survival. The MFP technique proved to fit into the 3R policy (replacement, reduction, and refinement) and allowed to evaluate cytotoxicity, showing that the technique can be used as a safety test in natural extracts.

Cadmium-Related Effects on Cellular Immunity Comprises Altered Metabolism in Earthworm Coelomocytes.

The heavy metal cadmium (Cd) is known to modulate the immune system, challenging soil-dwelling organisms where environmental Cd pollution is high. Since earthworms lack adaptive immunity, we determined Cd-related effects on coelomocytes, the cellular part of innate immunity, which is also the site of detoxification processes. A proteomics approach revealed a set of immunity-related proteins as well as gene products involved in energy metabolism changing in earthworms in response to Cd exposure. Based on these results, we conducted extracellular flux measurements of oxygen and acidification to reveal the effect of Cd on coelomocyte metabolism. We observed a significantly changing oxygen consumption rate, extracellular acidification, as well as metabolic potential, which can be defined as the response to an induced energy demand. Acute changes in intracellular calcium levels were also observed, indicating impaired coelomocyte activation. Lysosomes, the cell protein recycling center, and mitochondrial parameters did not change. Taken together, we were able to characterize coelomocyte metabolism to reveal a potential link to an impaired immune system upon Cd exposure.

Effect of long-term thermal challenge on the Antarctic notothenioid Notothenia rossii.

The thermal stability of the Antarctic Ocean raises questions concerning the metabolic plasticity of Antarctic notothenioids to changes in the environmental temperature. In this study, Notothenia rossii survived 90 days at 8 °C, and their condition factor level was maintained. However, their hepatosomatic (0.29×) index decreased, indicating a decrease in nutrient storage as a result of changes in the energy demands to support survival. At 8 °C, the plasma calcium, magnesium, cholesterol, and triglyceride concentrations decreased, whereas the glucose (1.91×) and albumin (1.26×) concentrations increased. The main energy substrate of the fish changed from lipids to glucose due to a marked increase in lactate dehydrogenase activity, as demonstrated by an increase in anaerobic metabolism. Moreover, malate dehydrogenase activity increased in all tissues, suggesting that fish acclimated at 8 °C exhibit enhanced gluconeogenesis. The aerobic demand increased only in the liver due to an increase (2.23×) in citrate synthase activity. Decreases in the activities of superoxide dismutase, catalase, and glutathione-S-transferase to levels that are most likely sufficient at 8 °C were observed, establishing a new physiological activity range for antioxidant defense. Our findings indicate that N. rossii has some compensatory mechanisms that enabled its long-term survival at 8 °C.

Hidden danger: Unexpected scenario in the vector-parasite dynamics of leishmaniases in the Brazil side of triple border (Argentina, Brazil and Paraguay).

Every year about 3 million tourists from around the world visit Brazil, Argentina and Paraguay´s triple border region where the Iguaçu Falls are located. Unfortunately, in recent years an increasing number of autochthonous canine and human visceral leishmaniasis (VL) cases have been reported. The parasite is Leishmania (Leishmania) infantum and it is transmitted by sand flies (Phlebotominae). To assess the risk factors favorable for the establishment and spread of potential vectors the Centers for Disease Control and Prevention light trap (CDC-light trap) collections were made in the Foz do Iguaçu (FI) and Santa Terezinha de Itaipu (STI) townships and along two transects between them. Our study determined the Phlebotominae fauna, the factors that affect the presence and abundance of Lutzomyia longipalpis and Nyssomyia whitmani, the presence of L. infantum in different sand fly species and which Leishmania species are present in this region. Lutzomyia longipalpis was the prevalent species and its distribution was related to the abundance of dogs. Leishmania infantum was found in Lu. longipalpis, Ny. whitmani, Ny. neivai and a Lutzomyia sp. All the results are discussed within the Stockholm Paradigm and focus on their importance in the elaboration of public health policies in international border areas. This region has all the properties of stable VL endemic foci that can serve as a source of the disease for neighboring municipalities, states and countries. Most of the urban areas of tropical America are propitious for Lu. longipalpis establishment and have large dog populations. Pan American Health Organization´s initiative in supporting the public health policies in the border areas of this study is crucial and laudable. However, if stakeholders do not act quickly in controlling VL in this region, the scenario will inevitable become worse. Moreover, L. (Viannia) braziliensis found in this study supports the need to develop public health policies to avoid the spread of cutaneous leishmaniasis. The consequences of socioeconomic attributes, boundaries and frontiers on the spread of diseases cannot be neglected. For an efficient control, it is essential that urban planning is articulated with the neighboring cities.

Heat stress in the heart and muscle of the Antarctic fishes Notothenia rossii and Notothenia coriiceps: Carbohydrate metabolism and antioxidant defence.

Carbohydrate metabolism and the antioxidant defence system of heart and muscle of the Antarctic notothenioids Notothenia rossii and Notothenia coriiceps were evaluated in response to heat stress (8 °C) over 144 h. N. rossii heart exhibited decreased glycolysis and aerobic metabolism after up to 12 h of exposure to 8 °C, and anaerobiosis was inhibited within 24 h. However, these pathways were stimulated after 72 h at 8 °C. The consumption of glucose-6-phosphate, derived from hexokinase (HK), by glucose-6-phosphate dehydrogenase (G6PDH) decreased in N. rossii heart within 6 h at 8 °C, with a subsequent increase at 72 h. In N. rossii muscle at 8 °C, glycolysis was stimulated within 2 h by an increase in pyruvate kinase (PK), and aerobic metabolism was stimulated at 144 h, together with anaerobiosis. In N. coriiceps heart at 8 °C, glucose break down by HK decreased within 2 h and subsequently increased at 12 and 24 h. Increased glucose-6-phosphate consumption by G6PDH occurred within 6 h at 8 °C. In N. coriiceps muscle at 8 °C, glycolysis was stimulated at 2 and 6 h, with subsequent inhibition within 24 h, as indicated by HK activity. Aerobic metabolism was inhibited at 72 and 144 h at 8 °C through the inhibition of citrate synthase (CS). Heat stress caused responses were only occasional and transient in antioxidant defence system of both species in the heart and muscle, leading to increased glutathione (GSH) and decreased levels of lipoperoxidation in the heart of both species. The results obtained in this study in the heart and muscles indicate that under heat stress at 8 °C, N. rossii is more responsive than N. coriiceps with respect to carbohydrate metabolism.

qPCR for the detection of foodborne Trypanosoma cruzi.

Here we presented a potential real-time PCR (qPCR) method with public health importance and relevance for detection of Trypanosoma cruzi in açai pulp. There is not a current process to identify T. cruzi in açai, that ensures innocuity of this food concerning oral transmission. First, six new primers were designed using the DNA sequences of T. cruzi y152 and Emerald strains obtained from GenBank. For primers evaluation and titration they were validated regarding the amplification and not with the fluorophore chosen 1ngµL-1 of the T. cruzi DNA as target. For determination of the ideal concentration the titration of the primers drawn in this study showed T. cruzi DNA amplification in five primer pairs at concentrations 100, 200 and 300nM and DNA fixed concentrations at 1ngµL-1. For standardization all reactions were performed in triplicate with 5.0µL and positives and negatives controls were included in every run. As positive control DNA from two genotypes TcI and TcII were used. As negative control the reaction product without DNA of the parasite was used. The best primer concentration, for the expected fragments, was 300nM. From six primers improved the Ep1F/Ep1R primer detected 1×10-4ngµL-1 for both genotype of the parasite. The Bp1F/Bp1R showed amplification for 1.70.10-7ngµL-1 for TcI and 4.31.10-8ngµL-1 for TcII, based on the standard curve. The last step we tested the selected primers in qPCR for monitoring T. cruzi in açai pulp experimentally contaminated. The recovery rate for the TcII was 71%, whereas in açai samples contaminated with TcI it was 76%.

Time course of lead induced proteomic changes in gill of the Antarctic limpet Nacella Concinna (Gastropoda: Patellidae).

The effect of increasing levels of metals from anthropogenic sources on Antarctic invertebrates is poorly understood. Here we exposed limpets (Nacella concinna) to 0, 0.12 and 0.25 µg L− 1 lead for 12, 24, 48 and 168 h. We subsequently quantified the changes in protein abundance from gill, using 2D gel electrophoresis and mass spectrometry. We identified several antioxidant proteins, including the metal binding Mn-superoxide dismutase and ferritin, increasing abundances early on. Chaperones involved in the redox-dependent maturation of proteins in the endoplasmic reticulum (ER) showed higher abundance with lead at 48 h. Lead also increased the abundance of Zn-binding carbonic anhydrase at 12 h, suggesting a challenge to acid-base balance. Metabolic proteins increased abundance at 168 h, suggesting a greater ATP demand during prolonged exposure. Changes in abundance of the small G-protein cdc42, a signaling protein modifying cytoskeleton, increased early and subsequently reversed during prolonged exposure, possibly leading to the modification of thick filament structure and function. We hypothesize that the replacement of metals initially affected antioxidant proteins and increased the production of reactive oxygen species. This disrupted the redox-sensitive maturation of proteins in the ER and caused increased ATP demand later on, accompanied by changes in cytoskeleton. SIGNIFICANCE: Proteomic analysis of gill tissue in Antarctic limpets exposed to different concentrations of lead (Pb) over a 168 h time period showed that proteomic changes vary with time. These changes included an increase in the demand of scavenging reactive oxygen species, acid-base balance and a challenge to protein homeostasis in the endoplasmic reticulum early on and subsequently an increase in energy metabolism, cellular signaling, and cytoskeletal modifications. Based on this time course, we hypothesize that the main mode of action of lead is a replacement of metal-cofactors of key enzymes involved in the scavenging of reactive oxygen species and the regulation of acid-base balance.