Anabolism,catabolism and proteomic analysis in the slow‐twitch muscle of pacu (Piaractus mesopotamicus) submitted to prolonged fasting followed by refeeding

Here, we aimed to study the slow muscle of the fish Piaractus mesopotamicus submitted to 30 days of fasting (D30) followed by 1 day (D31) or 30 days of refeeding (D60). Histological analysis of fibre diameter was performed in D30 and D60. The gene expression of parvalbumin (pvalb), atrogenes (murf1a, murf1b, mafbx) and anabolic genes (igf‐1, mtor) was analysed using RT‐qPCR in D30, D31 and D60. The proteome was obtained by shotgun proteomics at D30 and D60, and the set of differentially expressed proteins was analysed by bioinformatics. In all experiments, the control was regularly fed fish. The histological analysis showed no changes in muscle fibre diameter. The expression of catabolic and anabolic genes was not changed, except for the downregulation of igf‐1 in D30 and of mafbx in D31. The expression of pvalb was not changed in D30 and D60 but was decreased in D31. The proteomic analysis identified 169 proteins in D30 (24 upregulated and 18 downregulated) and 170 proteins in D60 (17 upregulated and 21 downregulated); many of them were related to energetic metabolism and intracellular Ca²⁺ homoeostasis. Overall, our results indicate that the slow‐twitch muscle presented few changes upon prolonged fasting and refeeding condition.     

Citral and linalool chemotypes of <Emphasis Type="Italic">Lippia alba</Emphasis> essential oil as anesthetics for fish: a detailed physiological analysis of side effects during anesthetic recovery in silver catfish (<Emphasis Type="Italic">Rhamdia quelen</Emphasis>)

The viability using Lippia alba essential oil as an anesthetic for fish was studied, particularly with respect to physiological effects during recovery. Anesthesia of silver catfish (Rhamdia quelen) using 100 and 300 μL L^?1 of two different chemotypes of L. alba essential oil (citral EO-C and linalool EO-L) prevented the increase of plasma cortisol levels caused by handling, but did not avoid alterations in energetic metabolism. Silver catfish did not have increased the levels of thiobarbituric acid reactive species in the kidney and liver during recovery after anesthesia with either EO, avoiding lipid damage. On the other hand, fish anesthetized with EO-C showed higher protein carbonylation levels, superoxide dismutase, catalase, and glutathione S-transferase activities and non-protein thiol group levels in both tissues compared to controls. Our results suggest that both oils show antioxidant capacity, but anesthesia with EO-L does not cause damage to lipids or proteins, only temporary changes, typical of physiological adjustments during recovery from anesthesia. Therefore, EO-L is an effective anesthetic for silver catfish with fewer side effects than EO-C.     

Pigmentation of gilthead seabream, Sparus aurata (L. 1875), using Chlorella vulgaris (Chlorophyta, Volvocales) microalga

A feeding experiment was conducted over 9 weeks with seven groups of 30 (fish per group) unpigmented gilthead seabream, Sparus aurata (L. 1875) (initial mean weight = 145.2 ± 12.3 g). Three experimental diets were prepared by adding to a basal diet free of carotenoid (final pigment content of around 40 mg per kg feed): (i) a biomass of the carotenogenic Chlorella vulgaris (Chlorophyta, Volvocales); (ii) a synthetic astaxanthin; and (iii) a mixture (1:1) of microalgal biomass and synthetic astaxanthin. At 3‐week intervals, five fish were sampled from each tank for total carotenoids analysis in skin and muscle. The carotenoid pigments (total amount = 0.4%) identified in the carotenogenic alga were lutein (0.3%), β‐carotene (1.2%), canthaxanthin (36.2%), astaxanthin, free and esterified forms (55.0%), and other pigments (7.3%). Carotenoid pigments were significantly deposited in the four skin zones studied during the feeding trial: the forefront between the eyes, the opercule, along the dorsal fin and in the abdominal area. In the muscle, regardless of the astaxanthin source, the amount of carotenoids measured was very low (less than 1 mg kg^?1) and differences not significant. Moreover, no muscle pigmentation was evident, and there was no variation in the amount of carotenoid analysed in skin tissue, through the trial, for each treatment. It was concluded that supplementing the feed with C. vulgaris would be an acceptable practice in aquaculture to improve the market appeal of the gilthead seabream.     

Seroprevalence of Toxoplasma gondii antibodies from wild canids from Brazil

The prevalence of anti-Toxoplasma gondii antibodies was evaluated by the indirect immunofluorescent-antibody test in serum of 57 wild canids from three different species: Lycalopex gymnocercus, Cerdocyon thous and Dusicyon vetulus from the northeast, southeast and southern regions of Brazil. The prevalence was 35.1%, with 20 of the 57 canids demonstrating antibodies anti-T. gondii at dilutions of 1:16 in 2, 1:32 in 4, 1:64 in 2, 1:128 in 2, 1:256 in 6, 1:512 in 2 and 1:2048 in 2 animals. None of the D. vetulus were positive. Among the L. gymnocercus 11 (91.7%) of the 12 samples were positive and among C. thous 9 (60%) of the 15 had antibodies anti-T. gondii.     

Species-specific DNA markers for identification of two root-knot nematodes of coffee: Meloidogyne arabicida and M. izalcoensis

Seven root-knot nematodes (RKN), including Meloidogyne exigua, M. incognita, M. paranaensis, M. enterolobii, M. arabicida, M. izalcoensis and M. arenaria are major pathogens of coffee crop in the Americas. Species-specific primers for their identification have been developed for five of them and constitute a fast and reliable method of identification. Here we report a PCR-based assay for specific detection of M. arabicida and M. izalcoensis. Random Amplified Polymorphic DNA fragments specific for these two species were converted into sequence characterized amplified region (SCAR) markers. PCR amplification using the SCAR primers produced a specific fragment of 300 bp and 670 bp for M. arabicida and M. izalcoensis, respectively, which were absent in other coffee-associated Meloidogyne spp. tested. SCAR primers also allowed successful amplification of DNA from single second-stage juveniles (J2), males and females. In addition, these primers were able to unambiguously detect the target species in nematode suspensions extracted from soil and roots samples, in different isolates of the same species or when used in multiplex PCR reactions containing mixtures of species. These results demonstrated the effectiveness of these SCAR markers and their multiplex use with those previously developed for M. exigua, M. incognita, M. paranaensis, M. enterolobii and M. arenaria constitute an essential detection tool. This diagnostic kit will contribute for specific J2 identification of the major RKN infecting coffee from field samples in the Americas.     

Guava decline: effect of root exudates from Meloidogyne enterolobii-parasitized plants on Fusarium solani in vitro and on growth and development of guava seedlings under controlled conditions

In guava decline, Fusarium solani-immune guava trees become susceptible to extensive root rot caused by this fungus after parasitism by Meloidogyne enterolobii. To understand the mechanisms involved in this disease, root exudates were collected from nematode-inoculated (NI) or uninoculated (UN) guava plants cultivated in sand. After filtration through a Millipore® membrane, NI and UN exudates were used: i) to prepare media to assess their effect on mycelial growth and production of propagules of F. solani isolate UENF/CF 163, and ii) to incubate macro- and microconidia to assess their effect on germination. NI exudates promoted (P?<?0.05) more mycelial growth and production of propagules than UN exudates or water. NI and UN exudates were used to water guava seedlings laid over seed germination paper inside plastic boxes. Half of the seedlings had an agar plug colonized by the fungus positioned in the collar region. Upon watering with NI exudates the fungus caused (P?<?0.05) extensive rotting of the seedlings’ rootlets. NI and UN exudates, either unlyophilized or lyophilized and re-suspended to the original concentration, were used to water guava seedlings grown in sterile sand before being inoculated (or left uninoculated) as described before. Solely upon watering with NI exudates, in its unlyophilized form or after lyophilization, the fungus caused a reduction (P?<?0.05) of shoot and root biomass associated with rotting of roots. These results suggest that M. enterolobii induces chemical changes in the root exudates of guava trees, which are necessary for root invasion causing root rotting by F. solani.     

Methods for Cercospora coffeicola protoplast isolation and genetic transformation with the green fluorescent protein

Cercospora coffeicola is the causal agent of brown eye spot on coffee leaves. Although the disease has significant importance, few molecular studies have been done with C. coffeicola. Here we report a protocol for isolating protoplasts as well as development of a genetic transformation system using Green Fluorescent Protein. High yields of protoplasts (≈10⁸/ml) were obtained from mycelial cultures from five isolates of C. coffeicola. One isolate was transformed with a vector encoding hygromycin resistance and Green Fluorescent Protein. Out of 43 hygromycin-resistant transformants obtained, Green Fluorescent Protein was highly expressed in one (2.3 %).     

Characterization of Phytophthora infestans populations of southern Brazil in 2004 and 2005

The populations of Phytophthora infestans (Pi) in southern Brazil in 2004 and 2005 are characterized herein. The isolates were collected from potato and tomato plants in the states of Paraná (PR), Santa Catarina (SC), and Rio Grande do Sul (RS). The mating type of 131 potato and 32 tomato isolates was determined. Forty-nine isolates from potatoes and 11 from tomatoes were analyzed for their Gpi phenotype. A subset of 35 isolates was evaluated for mitochondrial (mtDNA) polymorphisms. A sample of 146 isolates was tested for sensitivity to the fungicide metalaxyl, and most isolates (64%) were moderately sensitive. Fifty-nine isolates were classified as A1 mating type and 103 as A2. One isolate behaved as both A1 and A2 mating type. All tomato isolates were A1 mating type and presented the 86/100 pattern for the enzyme GPI and mtDNA Ib, indicating that these isolates belong to the US-1 clonal lineage. Of the 131 potato isolates, 103 were A2, 27 were A1 and one was A1/A2 mating type. Among the potato isolates 27 exhibited the Gpi phenotype 100/100, the same as BR-1, and 20 were 86/100, the same as US-1. Potato isolates presented the mitochondrial haplotypes Ia (74%) and IIa (26%). The data suggest the presence of only the BR-1 clonal lineage on potatoes in the states of PR and SC. However, in the state of RS, more than one clonal lineage was observed infecting potatoes, and there may be sexual reproduction between the lineages.