Acute exposition to Roundup Transorb® induces systemic oxidative stress and alterations in the expression of newly sequenced genes in silverside fish (Odontesthes humensis)

Roundup Transorb® (RDT) is a glyphosate-based herbicide commonly used in agricultural practices worldwide. This herbicide exerts negative effects on the aquatic ecosystem and affects bioenergetic and detoxification pathways, oxidative stress, and cell damage in marine organisms. These effects might also occur at the transcriptional level; however, the expression of genes associated with oxidative stress has not been studied well. Odontesthes humensis is a native Brazilian aquatic species naturally distributed in the habitats affected by pesticides, including Roundup Transorb® (RDT). This study evaluated the toxic effects of short-term exposure to RDT on O. humensis. Moreover, the genes related to oxidative stress were sequenced and characterized, and their expressions in the gills, hepatopancreas, kidneys, and brain of the fish were quantified by quantitative reverse transcription-polymerase chain reaction. The animals were exposed to two environmentally relevant concentrations of RDT (2.07 and 3.68 mg L−1) for 24 h. Lipid peroxidation, reactive oxygen species (ROS), DNA damage, and apoptosis in erythrocytes were quantified by flow cytometry. The expression of the target genes was modulated in most tissues in the presence of the highest tested concentration of RDT. In erythrocytes, the levels of lipid peroxidation, ROS, and DNA damage were increased in the presence of both the concentrations of RDT, whereas cell apoptosis was increased in the group exposed to 3.68 mg L−1 RDT. In conclusion, acute exposure to RDT caused oxidative stress in the fish, induced negative effects on cells, and modulated the expression of genes related to the enzymatic antioxidant system in O. humensis.Fil: Martins, Amanda Weege S.. Universidade Federal de Pelotas; BrasilFil: Silveira, Tony L. R.. Universidade Federal de Pelotas; Brasil. Universidade Federal do Rio Grande; BrasilFil: Remião, Mariana H.. Universidade Federal de Pelotas; BrasilFil: Domingues, William Borges. Universidade Federal de Pelotas; BrasilFil: Dellagostin, Eduardo N.. Universidade Federal de Pelotas; BrasilFil: Varela Junior, Antônio Sergio. Universidade Federal do Rio Grande; Brasil. Universidade Federal de Pelotas; BrasilFil: Corcini, Carine D.. Universidade Federal de Pelotas; BrasilFil: Costa, Patrícia G.. Universidade Federal do Rio Grande; BrasilFil: Bianchini, Adalto. Universidade Federal do Rio Grande; BrasilFil: Somoza, Gustavo Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); ArgentinaFil: Robaldo, Ricardo B.. Universidade Federal de Pelotas; BrasilFil: Campos, Vinicius Farias. Universidade Federal de Pelotas; Brasi

Lactational transfer of PCBs and chlorinated pesticides in pups of southern elephant seals (Mirounga leonina) from Antarctica

a b s t r a c t Seven pairs of southern elephant seals (Mirounga leonina) pups and their dams were sampled during the late weaning season among a breeding population of seals on Elephant Island in Antarctica. The blubber of the pups and the milk and blubber of their dams were analyzed for lipid-normalized concentrations of PCBs and organochlorines compounds in order to evaluate the lactational transfer of these contaminants. The lipid-normalized concentrations in these tissues were in the ppb range (i.e., ng g À1 lipid). The levels of contaminants in southern elephant seals were low in comparison with residues that have been reported in pinnipeds from the northern hemisphere. The relative tissue concentrations of the analytes measured followed the pattern: RDDT > mirex > RPCB > Rchlordane > HCB > heptachlor epoxide > dieldrin > methoxychlor > RHCH > other organochlorines. The very high DDE/RDDT ratio (0.91) in the blubber of dams and pups was an indicative of long-term, extremely distant pollution. On the other hand, the relatively high levels of some other organochlorine pesticides (e.g. mirex, heptachlor epoxide, dieldrin, methoxychor) may reflect the continued use of these insecticides in developing countries located in the southern hemisphere. For most of the analytes measured, the lipid-normalized concentrations were lower in pup blubber and in the milk than in the maternal blubber. Lactational transfer rates were dependent on the log K ow (octanol/water partition coefficient) values of the analytes measured, less lipophilic compounds being more readily transferred to the pups by the lactational route

Gene and Blood Analysis Reveal That Transfer from Brackish Water to Freshwater Is Less Stressful to the Silverside Odontesthes humensis

Silversides are fish that inhabit marine coastal waters, coastal lagoons, and estuarine regions in southern South America. The freshwater (FW) silversides have the ability to tolerate salinity variations. Odontesthes humensis have similar habitats and biological characteristics of congeneric O. bonariensis, the most studied silverside species and with great economic importance. Studies revealed that O. bonariensis is not fully adapted to FW, despite inhabiting hyposmotic environments in nature. However, there is little information about stressful environments for cultivation of silverside O. humensis. Thus, the aim of this study was to evaluate the stress and osmoregulation responses triggered by the osmotic transfers on silverside O. humensis. Silversides were acclimated to FW (0 ppt) and to brackish water (BW, 10 ppt) and then they were exposed to opposite salinity treatment. Silverside gills and blood were sampled on pre-transfer (D0) and 1, 7, and 15 days (D1, D7, and D15) after changes in environmental salinity, the expression levels of genes atp1a3a, slc12a2b, kcnh1, and hspa1a were determined by quantitative reverse transcription-PCR for evaluation of osmoregulatory and stress responses. Furthermore, glycemia, hematocrit, and osmolality were also evaluated. The expression of atp1a3a was up- and down-regulated at D1 after the FW–BW and BW–FW transfers, respectively. Slc12a2b was up-regulated after FW–BW transfer. Similarly, kcnh1 and hspa1a were up-regulated at D1 after the BW–FW transfer. O. humensis blood osmolality decreased after the exposure to FW. It remained stable after exposure to BW, indicating an efficient hyposmoregulation. The glycemia had a peak at D1 after BW–FW transfer. No changes were observed in hematocrit. The return to the pre-transfer levels at D7 after the significant increases in responses of almost all evaluated molecular and blood parameters indicated that this period is enough for acclimation to the experimental conditions. In conclusion, our results suggest that BW–FW transfer is more stressful to O. humensis than FW–BW transfer and the physiology of O. humensis is only partially adapted to FW

Evaluation of Reference Genes to Analyze Gene Expression in Silverside Odontesthes humensis Under Different Environmental Conditions

Some mammalian reference genes, which are widely used to normalize the qRT-PCR, could not be used for this purpose due to its high expression variation. The normalization with false reference genes leads to misinterpretation of results. The silversides (Odontesthes spp.) has been used as models for evolutionary, osmoregulatory and environmental pollution studies but, up to now, there are no studies about reference genes in any Odontesthes species. Furthermore, many studies on silversides have used reference genes without previous validations. Thus, present study aimed to was to clone and sequence potential reference genes, thereby identifying the best ones in Odontesthes humensis considering different tissues, ages and conditions. For this purpose, animals belonging to three ages (adults, juveniles, and immature) were exposed to control, Roundup®, and seawater treatments for 24 h. Blood samples were subjected to flow-cytometry and other collected tissues to RNA extraction; cDNA synthesis; molecular cloning; DNA sequencing; and qRT-PCR. The candidate genes tested included 18s, actb, ef1a, eif3g, gapdh, h3a, atp1a, and tuba. Gene expression results were analyzed using five algorithms that ranked the candidate genes. The flow-cytometry data showed that the environmental challenges could trigger a systemic response in the treated fish. Even during this systemic physiological disorder, the consensus analysis of gene expression revealed h3a to be the most stable gene expression when only the treatments were considered. On the other hand, tuba was the least stable gene in the control and gapdh was the least stable in both Roundup® and seawater groups. In conclusion, the consensus analyses of different tissues, ages, and treatments groups revealed that h3a is the most stable gene whereas gapdh and tuba are the least stable genes, even being considered two constitutive genes

Le Peuple : organe quotidien du syndicalisme

21 août 19321932/08/21 (A12,N4236)-1932/08/21

Table1.docx

<p>Some mammalian reference genes, which are widely used to normalize the qRT-PCR, could not be used for this purpose due to its high expression variation. The normalization with false reference genes leads to misinterpretation of results. The silversides (Odontesthes spp.) has been used as models for evolutionary, osmoregulatory and environmental pollution studies but, up to now, there are no studies about reference genes in any Odontesthes species. Furthermore, many studies on silversides have used reference genes without previous validations. Thus, present study aimed to was to clone and sequence potential reference genes, thereby identifying the best ones in Odontesthes humensis considering different tissues, ages and conditions. For this purpose, animals belonging to three ages (adults, juveniles, and immature) were exposed to control, Roundup®, and seawater treatments for 24 h. Blood samples were subjected to flow-cytometry and other collected tissues to RNA extraction; cDNA synthesis; molecular cloning; DNA sequencing; and qRT-PCR. The candidate genes tested included 18s, actb, ef1a, eif3g, gapdh, h3a, atp1a, and tuba. Gene expression results were analyzed using five algorithms that ranked the candidate genes. The flow-cytometry data showed that the environmental challenges could trigger a systemic response in the treated fish. Even during this systemic physiological disorder, the consensus analysis of gene expression revealed h3a to be the most stable gene expression when only the treatments were considered. On the other hand, tuba was the least stable gene in the control and gapdh was the least stable in both Roundup® and seawater groups. In conclusion, the consensus analyses of different tissues, ages, and treatments groups revealed that h3a is the most stable gene whereas gapdh and tuba are the least stable genes, even being considered two constitutive genes.</p

Table2.docx

<p>Some mammalian reference genes, which are widely used to normalize the qRT-PCR, could not be used for this purpose due to its high expression variation. The normalization with false reference genes leads to misinterpretation of results. The silversides (Odontesthes spp.) has been used as models for evolutionary, osmoregulatory and environmental pollution studies but, up to now, there are no studies about reference genes in any Odontesthes species. Furthermore, many studies on silversides have used reference genes without previous validations. Thus, present study aimed to was to clone and sequence potential reference genes, thereby identifying the best ones in Odontesthes humensis considering different tissues, ages and conditions. For this purpose, animals belonging to three ages (adults, juveniles, and immature) were exposed to control, Roundup®, and seawater treatments for 24 h. Blood samples were subjected to flow-cytometry and other collected tissues to RNA extraction; cDNA synthesis; molecular cloning; DNA sequencing; and qRT-PCR. The candidate genes tested included 18s, actb, ef1a, eif3g, gapdh, h3a, atp1a, and tuba. Gene expression results were analyzed using five algorithms that ranked the candidate genes. The flow-cytometry data showed that the environmental challenges could trigger a systemic response in the treated fish. Even during this systemic physiological disorder, the consensus analysis of gene expression revealed h3a to be the most stable gene expression when only the treatments were considered. On the other hand, tuba was the least stable gene in the control and gapdh was the least stable in both Roundup® and seawater groups. In conclusion, the consensus analyses of different tissues, ages, and treatments groups revealed that h3a is the most stable gene whereas gapdh and tuba are the least stable genes, even being considered two constitutive genes.</p

Npy and sbgnrh gene expression in juvenile and adult male brazilian flounder paralichthys orbignyanus

The objective of this study was to evaluate neuropeptide Y (NPY) and sea bream gonadotropin-release hormone (sbGnRH) gene expression in juvenile and adult males of Brazilian flounder. Hypothalamuses from fish were sampled for total RNA extraction. After cDNA synthesis, real-time PCR was used to measure gene expression. NPY showed approximately 2-fold increases in their mRNA levels while sbGnRH showed 3-fold increases in adult fish. These results suggest that these peptides could be involved on hypothalamic regulation of Brazilian flounder sexual maturation.O objetivo deste estudo foi avaliar a expressão gênica do neuropeptídeo Y (NPY) e da variante sea bream do hormônio liberador de gonadotrofinas (sbGnRH) em linguados machos juvenis e adultos. O hipotálamo foi isolado para a extração de RNA total. Após a síntese de cDNA, a PCR em tempo real foi usada para avaliar a expressão gênica. Foi observado um aumento de aproximadamente duas vezes nos níveis de NPY e de aproximadamente três vezes nos níveis de sbGnRH nos peixes adultos. Esses resultados demonstram que estes peptídeos podem estar envolvidos na regulação, via hipotálamo, da maturação sexual no linguado

Movement of Southern Elephant Seals (Mirounga leonina L.) from Elephant Is. South Shetlands, Antarctica

In 1999, at-sea activity of two young southern elephant seal males (Mirounga leonina) from Elephant Is.(61°13’S,55°23’W), Antarctica, was monitored and tracked for 9 months. The individuals were randomly selected, captured,sedated (Zoletil 50®- 1mg/kg), weighed, measured, bled, paint-marked and fitted with satellite tags (STDR - ST- 6PPT, Telonics®, USA). Deployment of the STDR took about 45 min since each animal had a lower incisor tooth extracted for age determination. The seals exhibited individual behaviors. Seal “V”-23842 (BM ~ 801kg) moved from Elephant Is. (61.2ºS 55.3ºW) in Jan. 1999 to King George Is. (62.2ºS 58.1ºW) in Feb. 1999 when the tag stopped signaling. Seal “T”-23843 (BM ~ 656 kg) was restricted to the area around Elephant Is. (61.2ºS 54.4ºW -61.6ºS 55.4ºW) from January to May 1999, when it started to move south-eastwards. Although the age of these individuals was not yet determined it was likely to explain the difference in the two patterns of movement reported here. The temporal and spatial association of these movements with areas of high productivity is being investigated to assess whether the observed distribution reflects foraging activity