Nelson Mauricio Lopera-Barrero

Comparación de protocolos de extracción de ADN con muestras de aleta y larva de peces: extracción modificada con cloruro de sodio

N.M. Lopera-Barrero, J.A. Povh, R.P. Ribeiro, P.C. Gomes, C.B. Jacometo, and T. da Silva Lopes. Comparison of DNA extraction protocols of fi sh fi n and larvae samples: modifi ed salt (NaCl) extraction. 2008. Cien. Inv. Agr. 35(1):77-86. The use of appropriate sampling methods, the type of tissue or sample and the use of viable DNA extraction protocols are critical issues in studies based on PCR. In an attempt to identify a simple, reproducible, inexpensive and non-toxic method for obtaining high quality and quantity genomic DNA from fi sh fi n and larvae samples, modifi ed salt extraction protocol and modifi ed phenol-chloroform extraction protocol were compared. The samples obtained from different fi sh species (Brycon orbignyanus, Piaractus mesopotamicus, Oreochromis niloticus, Leporinus elongatus and Prochilodus lineatus) using salt extraction showed good DNA quality and quantity (DNA/RNA relationship 1.8-2.2). These DNA samples were successfully used in the amplifi cation of RAPD (Random Amplifi ed Polymorphic DNA) and microsatellite molecular markers, demonstrating the same effectiveness of this protocol in comparison with modifi ed phenol-chloroform extraction protocol. This DNA extraction procedure constitutes an alternative and effi cient replacement for previous protocols for improving fi sh molecular studies.

Conservation of Brycon orbignyanus natural populations and stocks for their reproductive, genetic, environmental sustainability: A model for species threatened with extinction

Several ecological and climatic factors, especially those related to human activities, have been contributing to the disappearance of natural populations of piracanjuba ( Brycon orbignyanus ). Due to the importance of these fi sh to the ecosystems in which they are located and their qualities of fast growth and ability to adapt to controlled environments, producers have expressed increased interest in recent years in using this species, with particular aims of increasing production, and participation in conservation programs. In this study, strategies for the sustainable management of B. orbignyanus are idealized and discussed, with the goals of perfecting the reproductive, genetic, environmental and sanitary management of this species and suggestions for improving monitoring stocks maintained in captivity and natural populations. These strategies can be used as models for other migratory species threatened with extinction. Diversos factores ecologicos y climaticos y especialmente aquellos relacionados con acciones humanas, han llevado a la desaparicion de poblaciones naturales de piracanjuba ( Brycon orbignyanus ). Por la importancia en los ecosistemas en los cuales esta ubicada y por sus cualidades de crecimiento rapido y adecuada adaptacion a ambientes controlados, en los ultimos anos el interes de los productores en la utilizacion de esa especie ha aumentado, con el objetivo de la produccion, la mejora economica y la participacion en programas de conservacion. Con el objetivo de perfeccionar el manejo e monitoreo reproductivo, genetico, ambiental y de sanidad de lotes mantenidos en cautiverio y de poblaciones naturales de B. orbignyanus, estrategias de manejo sustentable de esa especie son idealizados y discutidos, pudiendo ser usados como modelo para otras especies migratorias amenazadas de extincion.

Genetic diversity of piracanjuba used in stock enhancement programs with microsatellite markers

The objective of this work was to estimate the genetic diversity of a Brycon orbignyanus lot used in stock enhancement programs, using microsatellite markers. Samples of 44 broodstocks, 70 larvae and 69 fingerlings, were analyzed with amplification of five loci described for Brycon opalinus. The number of alleles, the observed (Ho) and expected (He) heterozygosity, Shannon index (IS), Neis genetic diversity (DGN), the inbreeding coefficient (Fis), distance (DG) and genetic identity (IG), the effective number of alleles, the test of Hardy-Weinberg equilibrium (EHW) and the linkage disequilibrium were calculated. Broodstocks and offspring had a similar number of alleles at the tested loci. Ho average, IS, DGN, DG and IG showed that there is less genetic distance between parental and larvae and a decrease of genetic variability in the fingerlings. Deviations in the EHW and disequilibrium ligation were observed in six pairs of loci. Inbreeding coefficient showed excess of heterozygotes in parental and larvae and heterozygote deficiencies in fingerlings. The broodstock is in process of allele losses and the genetic variability decreased between larva and fingerling stages.

Microsatellite analysis of the parental contribution of Piaractus mesopotamicus to the production of offspring in the semi-natural system of reproduction

The objective of this study was to evaluate the genetic diversity and the parental contribution of Piaractus mesopotamicus in the production of offspring in the semi-natural system of reproduction. Twenty parental fishes (eleven males and nine females) and the total of 100 larvae were evaluated by microsatellite marker. The parents and offspring had thirty-one alleles and heterozygosity of 0.550 and 0.563, respectively. The females were fertilised by two up to six males while the males fertilised three up to five females. The contribution of the females and males to the offspring were 66.6 and 58%, respectively. Such results indicated no loss in the genetic variability in the offspring, and the parents had multiple paternity and reasonable contribution to the offspring production.

Monitoramento da variabilidade genética de pacu, Piaractus mesopotamicus, do programa de aumento de estoque do rio Paranapanema

Avaliou-se a variabilidade genetica dos estoques de reprodutores e dos peixes jovens de Piaractus mesopotamicus de tres pisciculturas do estado do Parana, utilizadas no programa de aumento de estoque de peixes no rio Paranapanema. Foi utilizado o marcador RAPD para avaliar as amostras do estoque de reprodutores e dos peixes jovens das pisciculturas de Palotina, Cambara e Andira. A porcentagem de fragmentos polimorficos e o indice de diversidade genetica de Shannon dos estoques de reprodutores variaram de 75,0% a 71,4% e de 0,434 a 0,376, respectivamente. Os peixes jovens das pisciculturas apresentaram valores mais elevados para ambos os parâmetros, com excecao da piscicultura de Palotina, na qual o indice de diversidade genetica de Shannon foi semelhante. Os estoques de reprodutores apresentaram alta variabilidade genetica, e esta foi mantida nos peixes jovens.

Microsatellite analysis of pacu broodstocks used in the stocking program of Paranapanema River, Brazil

Monitoring the genetic diversity has fundamental importance for fish stocking programs. This experiment aims to evaluate the genetic diversity in two hatchery stations (A and B) with pacu Piaractus mesopotamicus (Holmberg, 1887) in Andira, state of Parana, Brazil used in stocking programs of Paranapanema River. Six microsatellite loci were amplified using DNA extracted from 60 fin-clipping samples. The broodstock B had the average number of alleles and the mean heterozygosity (alleles: 3.7 and HO: 0.628) higher than the broodstock A (alleles: 3.5 and HO: 0.600). Alleles with low frequency levels were observed in the both broodstocks. The positive coefficients of endogamy in the locus Pme2 (broodstock A: FIS = 0.30 and broodstock B: FIS = 0.20), Pme5 (broodstock B: FIS = 0.15), Pme14 (broodstock A: FIS = 0.07) and Pme28 (broodstock A: FIS = 0.24 and broodstock B: FIS = 0.20) indicated deficiency of heterozygotes. Presence of null allele in the locus Pme2 was detected. The negative estimates in loci Pme4 (broodstock A: FIS = - 0.43 and broodstock B: FIS = - 0.37), Pme5 (broodstock A: FIS= - 0.11), Pme14 (broodstock B: FIS= - 0.15) and Pme32 (broodstock A: FIS = - 0.93 and broodstock B: FIS = - 0.60) were indicating the excess of heterozygotes. Evidence of linkage disequilibrium and lower allelic richness was found only in the broodstock A. Neis gene diversity was high in both broodstocks. The genetic distance (0.085) and identity (0.918) showed similarity between broodstocks, which reflects a possible common origin. 6.05% of the total genetic variance was due to differences among broodstocks. Recent bottleneck effect in two broodstocks was observed. The results indicated a higher genetic diversity in the two broodstocks and they presented low genetic difference. This was caused by the reproductive management in both hatchery stations, reduction of population size and genetic exchange between the hatchery stations.

Ovopel® and carp pituitary extract for induction of reproduction in Colossoma macropomum females

The aim of this study was to evaluate the efficiency of the hormonal inducers Ovopel® and carp pituitary extract (CPE) for induction of reproduction in Colossoma macropomum females. The treatments were CPE at the dose of 5.5 mg/kg divided into two applications (10%; and 90% after 12 h) and Ovopel® at doses of 0.2 and 0.4 pellet/kg body weight in a single application. Eight replicates were used in each of the three treatments, totaling 24 experimental units. The females spawned when treated with the 0.2 pellet of Ovopel® (100.0%), 0.4 pellet of Ovopel® (62.5%), and CPE (87.5%), but there were no significant differences among the treatment groups in spawning rate. When there was treatment with Ovopel® spawning occurred with greater (P < 0.05) degree-hours (average water temperature × number of hours until spawning; 0.2 pellet: 417.7; 0.4 pellet: 412.3) in relation to the CPE treatment (268.9). The total oocyte weight was similar when there was treatment with Ovopel® (0.2 pellet: 832.3 g; 0.4 pellet: 798.9 g) and CPE (688.3 g). By contrast, the production index was greater (P < 0.05) with the Ovopel® treatments (0.2 pellet: 8.8%; 0.4 pellet: 9.0%) as compared with CPE (6.7%). Fertility and hatching rates were similar among the treatment groups. Ovopel® and CPE are efficient in induction of reproduction in C. macropomum females. Of the two Ovopel® treatments assessed in this study, the dose of 0.2 pellet/kg body weight is sufficient for effective induction of reproductive processes.

Novel Microsatellite Markers for the Invasive Golden Mussel Limnoperna fortunei

ABSTRACT The golden mussel (Limnoperna fortunei), a mollusc originating from Asia, has caused considerable environmental and economic damage in various Brazilian river basins and reservoirs. Genetic studies that focus on characterizing its invasive potential and the feasibility of control measures are required. The present study identified and characterized 13 new microsatellite markers (of which eight were polymorphic) in L. fortunei using enriched genetic libraries. The analysis performed on 48 individuals from three populations identified 32 alleles, ranging from one to seven alleles per locus. The size of the fragments ranged from 172 to 381 bp. The polymorphic information content values ranged from 0.114 to 0.768, with observed and expected heterozygosity values ranging from 0.043–0.938 to 0.123–0.805, respectively. Six loci showed significant deviation from the Hardy–Weinberg equilibrium, possibly influenced by the heterozygous deficit. The microsatellite primers developed will be useful in studies of the genetic diversity and population structure of L. fortunei and will also contribute to the adoption of control measures for this invasive species in Brazilian and other South American basins.

Parental contribution of Curimba offspring in different reproductive system

The knowledge of appropriate reproductive managements is fundamental for the management and orientation of activities in restocking centers. The aim of the present study was to evaluate the influence of artificial and semi-natural reproductive systems and sex ratios on parental contribution progeny of Prochilodus lineatus. Samples of caudal fins from 25 breeders subjected to the artificial reproductive system and 10 breeders subjected to the semi-natural reproductive system were collected in different sex ratios (1♂:1♀ and 2♂:1♀, and 1♂:1♀, respectively). Two hundred and eight fingerlings from these matings were evaluated. Breeding mortality was recorded shortly after reproductive procedures. For paternity analysis, 10 microsatellite loci were amplified. Three breeders died in the artificial reproductive system (two in the 1:1 ratio and one in the 2:1 ratio). In the 1:1 ratio in the artificial reproductive system, one male (M1.4) had a higher contribution in the progeny formation (41.8%), whereas in the 2:1 ratio, four males contributed from 14.5 to 21.7%. Lower reproductive dominance was identified in the semi-natural reproductive system, with values ranging from 22.86 to 25.71% for four males. In the formation of families, both systems demonstrated a reproductive dominance of some couples. So, the semi-natural reproductive system allowed greater homogeneity in the contribution of progeny without losses due to mortality.

A comparative study of four DNA extraction protocols from adductor muscle in golden mussel (Limnoperna fortunei)

The golden mussel, Limnoperna fortunei, is a mollusk native to Southeast Asia and a highly invasive species in South American countries such as Brazil, Uruguay, and Argentina. In order to better understand the biological behavior of the species and develop alternative control methods, genetic studies involving the optimization of DNA isolation procedures are of utmost importance. The objective of the present study was to develop a simple, reproducible, free of contaminants, and cheap protocol to extract DNA from L. fortunei using the adductor muscle of the mussel as the source. Four DNA extraction protocols were compared: extraction with SDS and proteinase K (P1); extraction with SDS, proteinase K and phenol (P2); TRIzol extraction (P3); and NaCl, SDS and RNase extraction (P4). DNA concentration (ng μL-1) and purity (at 260/280 nm) were measured using a spectrophotometer. DNA purity and amplification were verified by electrophoresis and PCR, respectively. P1 resulted in samples with low DNA concentrations or without any DNA, as revealed by the quantification and purity analysis; P2 had low efficiency, given the absence of DNA in most of the samples subjected to electrophoresis. On the other hand, P3 showed contamination with proteins, as indicated by an absorbance of <1.8 and by the low-quality electrophoresis results. Finally, P4 resulted in well-defined bands, absorbance between 1.8 and 2.0, and successful amplification by PCR. In conclusion, the extraction protocol P4 is a practical, fast, free of contaminants, and efficient method for the isolation of L. fortunei DNA.