Multiple Paternity in a Reintroduced Population of the Orinoco Crocodile (Crocodylus intermedius) at the El Frío Biological Station,Venezuela

The success of a reintroduction program is determined by the ability of individuals to reproduce and thrive. Hence, an understanding of the mating system and breeding strategies of reintroduced species can be critical to the success, evaluation and effective management of reintroduction programs. As one of the most threatened crocodile species in the world, the Orinoco crocodile (Crocodylus intermedius) has been reduced to only a few wild populations in the Llanos of Venezuela and Colombia. One of these populations was founded by reintroduction at Caño Macanillal and La Ramera lagoon within the El Frío Biological Station, Venezuela. Twenty egg clutches of C. intermedius were collected at the El Frío Biological Station for incubation in the lab and release of juveniles after one year. Analyzing 17 polymorphic microsatellite loci from 335 hatchlings we found multiple paternity in C. intermedius, with half of the 20 clutches fathered by two or three males. Sixteen mothers and 14 fathers were inferred by reconstruction of multilocus parental genotypes. Our findings showed skewed paternal contributions to multiple-sired clutches in four of the clutches (40%), leading to an overall unequal contribution of offspring among fathers with six of the 14 inferred males fathering 90% of the total offspring, and three of those six males fathering more than 70% of the total offspring. Our results provide the first evidence of multiple paternity occurring in the Orinoco crocodile and confirm the success of reintroduction efforts of this critically endangered species in the El Frío Biological Station, Venezuela.     

Distribution of actin in spreading macrophages: a comparative study on living and fixed cells

The distribution of actin in proteose peptone-elicited murine peritoneal macrophages is examined with fluorescent analog cytochemistry (FAC), immunofluorescence, and electron microscopy (EM). Living adherent macrophages, microinjected with 5- iodoacetamidofluorescence-labeled actin, show a rather uniform distribution of actin with punctuate and linear fluorescence in the thin peripheral areas of the cell. Apparent incorporation of a portion of linear fluorescence in the thin peripheral areas of the cell. Apparent incorporation of a portion of the microinjected actin into the cell’s actin cytoskeleton is also demonstrated when microinjected cells are subsequently examined for fluorescein fluorescence after fixation and extraction. However, a substantial perinuclear pool of actin, observed with FAC, is lost when microinjected cells are prepared for immunofluorescence using standard fixation methods. These results suggest that part of the cellular actin, possibly nonfilamentous or oligomeric, can be extracted during the normal preparative steps for immunofluorescence. When the dynamic distributin of actin structures is examined in living cells, extension of the cell’s periphery is associated with the formation of punctuate structures. The distribution of the most stable, nonextractable actin structures in fixed cells at different stages of spreading is quantified using rhodamine-labeled phalloidin and antiactin indirect immunofluorescence. At early stages, the rounded cells show cortical bands of fluorescence surrounding the nuclear region with punctuate structures directly above the plane of the attached plasma membrane. At later time periods, fully spread cells contain both punctuate and linear fluorescent structures. Adherent macrophage membranes, a preparation in which the attached membrane and membrane-cortex are isolated by shearing away the unattached plasma membrane and underlying cytoplasm, show punctuate and linear fluorescence when stained with rhodamine-labeled phalloidin. When the same cell remnant is negatively stained and examined with EM, the fluorescent punctuate structures coincide with electron-dense foci and associated radiating thin filaments. We suggest that the optimal approach for elucidating the distribution of cytoskeletal and contractile proteins involved in motile processes is a combined approach using all three techniques. Although each technique is subject to potential artifacts and limitations, the use of FAC can permit the visualization of both the soluble and stabilized components of the cytoskeleton in living, functional cells. A qualitative method for determining differences in local concentrations of proteins is also presented.     

Population genetics of Drosophila amylase. V. Genetic background and selection on different carbohydrates

Frequency changes in amylase allozymes and patterns of tissue-specific expression of amylase have been monitored in laboratory populations of Drosophila pseudoobscura maintained on media in which the only carbohydrate source was maltose or starch. Nonrandom changes occurred in patterns of expression, whereas no patterns in allozyme frequency changes were discernible. The nature of the pattern changes was similar to an identical study done on populations derived from a natural population several hundred miles from the population used in the present experiments. However, in the previous study nonrandom changes in allozyme frequencies were also noted. Evidently, selection on the Drosophila amylase system differs depending upon the genetic background of the population. Furthermore, the evolutionary dynamics of structural gene variants and those regions controlling its expression may be independent, a result consistent with DNA sequence data.     

Prostaglandin F in the fallopian tube secretion of the ewe

Oviducal secretions were obtained from conscious unrestrained ewes throughout the oestrous cycle via indwelling cannulae and the content of prostaglandin F (PGF) was determined by radioimmunoassay. Levels of PGF of up to 230 ng/ml were found in oviducal fluids obtained from ewes showing regular patterns of secretion and normal cyclical ovarian function as indicated by plasma progesterone measurement. Relatively large day to day fluctuations in content were evident, but there was no consistent relationship between concentration and stage of the oestrous cycle. Concentrations of PGF in excess of 100 ng/ml were common in preparations where autopsy later revealed infection or tissue irritation, and the concentration of PGF invariably exceeded 75 ng/ml when the concentration of protein in the oviducal fluid was abnormally high.     

Isolation and characterization of microsatellites in the bryozoan Crisia denticulata

Cyclostomata bryozoa are thought to reproduce via polyembryony, a clonal replication of a fertilized egg. To test this hypothesis and to assess the impact of their reproductive strategy on the structure of populations, we isolated microsatellite markers in Crisia denticulata (Cyclostomata, Stenolemata), using an initial enrichment step with random amplified polymorphic DNA (RAPD) primers. A total of nine microsatellites, one tetra‐ and eight dinucleotides repeats were isolated; seven were found to be polymorphic in a test sample of 30 individuals, with allele numbers/locus varying from 2 to 6. The tetranucleotide locus showed heterozygote deficiency. These primers did not amplify the DNA of Crisia eburnea.     

Cestode parasites in Potamotrygon motoro (Natterer) (Chondrichthyes: Potamotrygonidae) from southwestern Brazil, including Rhinebothroides mclennanae n. sp. (Tetraphyllidea: Phyllobothriidae), and a revised host-parasite checklist for helminths inhabiting neotropical freshwater stingrays.

Specimens of 5 species of cestodes were collected in 6 specimens of the freshwater stingray species Potamotrygon motoro (Natterer), collected in the vicinity of Corumba, Mato Grosso do Sul, Brazil. Acanthobothrium regoi, Potamotrygonocestus orinocoensis, Rhinebothroides venezuelensis, and Rhinebothrium paratrygoni are reported from P. motoro and from southwestern Brazil for the first time. Rhinebothroides mclennanae n. sp. appears to be the sister species of Rhinebothroides glandularis, the only other member of the genus exhibiting darkly staining glandular cells lying free in the parenchyma surrounding the terminal genitalia. The new species resembles Rhinebothroides glandularis, Rhinebothroides freitasi, and Rhinebothroides scorzai by having poral ovarian arms that extend anteriorly beyond the posterior margin of the cirrus sac, coiled vaginae, and vitelline follicles not interrupted on the poral side in the vicinity of the genital pore. It differs from all 6 previously described members of the genus by possessing an average of 31 testes per proglottid, compared with an average of 45 for R. glandularis, 55 for R. freitasi and R. venezuelensis, 77 for Rhinebothroides circularisi and Rhinebothroides moralarai, and 80 for R. scorzai. An updated phylogenetic tree for Rhinebothroides is presented.     

Long Term Storage of Dry versus Frozen RNA for Next Generation Molecular Studies

The standard method for the storage and preservation of RNA has been at ultra-low temperatures. However, reliance on liquid nitrogen and freezers for storage of RNA has multiple downsides. Recently new techniques have been developed for storing RNA at room temperature utilizing desiccation and are reported to be an effective alternative for preserving RNA integrity. In this study we compared frozen RNA samples stored for up to one year to those which had been desiccated using RNAstable (Biomatrica, Inc., San Diego, CA) and stored at room temperature. RNA samples were placed in aliquots and stored after desiccation or frozen (at −80°C), and were analyzed for RNA Integrity Number (RIN), and by qPCR, and RNA sequencing. Our study shows that RNAstable is able to preserve desiccated RNA samples at room temperature for up to one year, and that RNA preserved by desiccation is comparable to cryopreserved RNA for downstream analyses including real-time-PCR and RNA sequencing.