Landscape genetics of a tropical rescue pollinator

Pollination services are increasingly threatened by the loss and modification of natural habitats, posing a risk to the maintenance of both native plant biodiversity and agricultural production. In order to safeguard pollination services, it is essential to examine the impacts of habitat degradation on the population dynamics of key pollinators and identify potential “rescue pollinators” capable of persisting in these human-altered landscapes. Using a landscape genetic approach, we assessed the impact of landscape structure on genetic differentiation in the widely-distributed tropical stingless bee Trigona spinipes (Apidae: Meliponini) across agricultural landscape mosaics composed of coffee plantations and Atlantic forest fragments in southeastern Brazil. We genotyped 115 bees at 16 specific and highly polymorphic microsatellite loci, developed using next-generation sequencing. Our results reveal that T. spinipes is capable of dispersing across remarkably long distances, as we did not find genetic differentiation across a 200 km range, nor fine-scale spatial genetic structure. Furthermore, gene flow was not affected by forest cover, land cover, or elevation, indicating that reproductive individuals are able to disperse well through agricultural landscapes and across altitudinal gradients. We also found evidence of a recent population expansion, suggesting that this opportunistic stingless bee is capable of colonizing degraded habitats. Our results thus suggest that T. spinipes can persist in heavily-altered landscapes and can be regarded as a rescue pollinator, potentially compensating for the decline of other native pollinators in degraded tropical landscapes.     

Land Reform and Land-Use Changes in the Lower Amazon: Implications for Agricultural Intensification

Land tenure has been considered one of the key factors that define patterns and change in land-use systems. This paper examines the implications of land reform for household decisions regarding patterns of land use, agricultural intensification, and forest conservation. We look at an Amazonian caboclo settlement in the Lower Amazon that had experienced land reform by the end of the 1980s. Results show that defined land tenure is not enough to guarantee agricultural intensification and forest conservation. In fact, several factors working at different scales are affecting land-use change in the region. At the settlement level, privatization of upland forest has led to an overall increase in cultivated land—pasture and annual crops—and increasing deforestation rates. However, at the farm-property level, different systems of agricultural production—intensive, extensive, or abandonment of land—occur according to availability of labor, and capital, and access to different natural resources.     

Diffusible signals and fasciculated growth in reticulospinal axon pathfinding in the hindbrain

We have addressed the control of longitudinal axon pathfinding in the developing hindbrain, including the caudal projections of reticular and raphe neurons. To test potential sources of guidance signals, we assessed axon outgrowth from embryonic rat hindbrain explants cultured in collagen gels at a distance from explants of midbrain-hindbrain boundary (isthmus), caudal hindbrain, or cervical spinal cord. Our results showed that the isthmus inhibited caudally directed axon outgrowth by 80% relative to controls, whereas rostrally directed axon outgrowth was unaffected. Moreover, caudal hindbrain or cervical spinal cord explants did not inhibit caudal axons. Immunohistochemistry for reticular and raphe neuronal markers indicated that the caudal, but not the rostral projections of these neuronal subpopulations were inhibited by isthmic explants. Companion studies in chick embryos showed that, when the hindbrain was surgically separated from the isthmus, caudal reticulospinal axon projections failed to form and that descending pioneer axons of the medial longitudinal fasciculus (MLF) play an important role in the caudal reticulospinal projection. Taken together, these results suggest that diffusible chemorepellent or nonpermissive signals from the isthmus and substrate-anchored signals on the pioneer MLF axons are involved in the caudal direction of reticulospinal projections and might influence other longitudinal axon projections in the brainstem.     

High-level expression of murine terminal deoxynucleotidyl transferase inEscherichia coli grown at low temperature and overexpressingargU tRNA

Terminal deoxynucleotidyl transferase (TdT) is a highly conserved vertebrate enzyme that possesses the unique ability to catalyze the random addition of deoxynucleoside 5′-triphosphates onto the 3′-hydroxyl group of a single-stranded DNA. It plays an important role in the generation of immunoglobin and T-cell receptor diversity. TdT is usually obtained from animal thymus gland or produced in a baculovirus system, but both procedures are rather tedious, and proteolysis occurs during purification. Attempts to overexpress TdT in bacteria have been unsuccessful or have yielded an enzyme with a lower specific activity. A dearth of TdT has thus hampered detailed structural and functional studies. In the present study, we report that by lowering growth temperature and overexpressing a rare arginyl tRNA, it is possible to boost the production inEscherichia coli of murine TdT with minimal proteolysis and high specific activity.     

Role of histidine-50, glutamic acid-96, and histidine-137 in the ribonucleolytic mechanism of the ribotoxin alpha-sarcin

alpha-Sarcin is a ribotoxin secreted by the mold Aspergillus giganteus that degrades the ribosomal RNA by acting as a cyclizing ribonuclease. Three residues potentially involved in the mechanism of catalysis--histidine-50, glutamic acid-96, and histidine-137--were changed to glutamine. Three different single mutation variants (H50Q, E96Q, H137Q) as well as a double variant (H50/137Q) and a triple variant (H50/137Q/E96Q) were prepared and isolated to homogeneity. These variants were spectroscopically (circular dichroism, fluorescence emission, and proton nuclear magnetic resonance) characterized. According to these results, the three-dimensional structure of these variants of alpha-sarcin was preserved; only very minor local changes were detected. All the variants were inactive when assayed against either intact ribosomes or poly(A). The effect of pH on the ribonucleolytic activity of alpha-sarcin was evaluated against the ApA dinucleotide. This assay revealed that only the H50Q variant still retained its ability to cleave a phosphodiester bond, but it did so to a lesser extent than did wild-type alpha-sarcin. The results obtained are interpreted in terms of His137 and Glu96 as essential residues for the catalytic activity of alpha-sarcin (His137 as the general acid and Glu96 as the general base) and His50 stabilizing the transition state of the reaction catalyzed by alpha-sarcin.     

Site-specific labeling of cytoplasmic peptide:N-glycanase by N,N'-diacetylchitobiose-related compounds

Peptide:N-glycanase (PNGase) is the deglycosylating enzyme, which releases N-linked glycan chains from N-linked glycopeptides and glycoproteins. Recent studies have revealed that the cytoplasmic PNGase is involved in the degradation of misfolded/unassembled glycoproteins. This enzyme has a Cys, His, and Asp catalytic triad, which is required for its enzymatic activity and can be inhibited by "free" N-linked glycans. These observations prompted us to investigate the possible use of haloacetamidyl derivatives of N-glycans as potent inhibitors and labeling reagents of this enzyme. Using a cytoplasmic PNGase from budding yeast (Png1), Man9GlcNAc2-iodoacetoamide was shown to be a strong inhibitor of this enzyme. The inhibition was found to be through covalent binding of the carbohydrate to a single Cys residue on Png1, and the binding was highly selective. The mutant enzyme in which Cys191 of the catalytic triad was changed to Ala did not bind to the carbohydrate probe, suggesting that the catalytic Cys is the binding site for this compound. Precise determination of the carbohydrate attachment site by mass spectrometry clearly identified Cys191 as the site of covalent attachment. Molecular modeling of N,N'-diacetylchitobiose (chitobiose) binding to the protein suggests that the carbohydrate binding site is distinct from but adjacent to that of Z-VAD-fmk, a peptide-based inhibitor of this enzyme. These results suggest that cytoplasmic PNGase has a separate binding site for chitobiose and other carbohydrates, and haloacetamide derivatives can irreversibly inhibit that catalytic Cys in a highly specific manner.     

Association of CAPN1 316, CAPN1 4751 and TG5 markers with bovine meat quality traits in Mexico

We examined allele and genotype frequencies for the molecular markers CAPN1 316, CAPN1 4751 and TG5, and determined whether they are associated with beef quality traits in Mexican cattle. One hundred and twenty-four longissimus dorsi muscle samples were collected from cattle from north, central and southern Mexico. CAPN1 316 and CAPN1 4751 frequencies were determined using the allelic discrimination assay and the TG5 marker was typed by PCR-RFLP. Meat quality traits included intramuscular fat content (IMF) and tenderness determined by Warner-Bratzler shear force (WBSF) at 24 h postmortem. The association test was made using a mixed model, including genotypes, genetic group, and sampling location as fixed effects. Least squares means and significant interactions were compared using least significant differences based on the mixed procedure. CAPN1 316 CC was found at a low frequency (0.03) and has been reported as a favorable genotype associated with tenderness meat. Genotype frequencies for CAPN1 4751 were similar in favorable (CC) and unfavorable (TT) genotypes (0.26 and 0.28, respectively). The TG5 CC genotype had a frequency of 0.73, while the TT genotype frequency was 0.01. The means for WBSF and IMF were 4.08 ± 1.35 kg and 5.23 ± 2.14%, respectively. Sampling site and the CAPN1 316 genotypes significantly affected WBSF (P < 0.05). Samples collected from Hermosillo, Sonora, had the lowest WBSF (P < 0.05), while those collected in Veracruz were toughest (WBSF = 5.267 kg). The effect of GG and TG5 genotypes on IMF was significant (P < 0.05). CAPN1 316 and TG5 markers were found to be significantly associated with beef quality traits and thus will be useful for Mexican beef characterization.     

Abnormal Pelage Color in an Isolated Population of <Emphasis Type="Italic">Alouatta guariba clamitans</Emphasis> Cabrera, 1940 in South Brazil

We located 4 brown howlers (1 adult male, 2 adult females, and 1 juvenile male) showing abnormally lighter pelage in 3 social groups comprising 5, 6, and 9 individuals in a 20 ha-forest fragment in the State of Rio Grande do Sul, Brazil. Two additional groups composed only of normally colored individuals also live in the fragment, which is isolated from nearby fragments by 267–1009 m. They were the only brown howlers with abnormal pelage color out of a total of 386 individuals belonging to 67 groups in 21 fragments in the 5876-ha study area. The isolation of the forest fragment, its high howler density (2.2 individuals⁄ha), and large group size (8.8 ± 2.4 individuals) may decrease the likelihood of successful immigration into the population, leading to an increased probability of inbreeding that may facilitate the expression of rare alleles.