Low genetic diversity and lack of population structure in the endangered Galápagos penguin (<Emphasis Type="Italic">Spheniscus mendiculus</Emphasis>)

Long-term monitoring of the endangered Galápagos penguin (Spheniscus mendiculus) has indicated poor reproductive periods and severe population fluctuations in association with El Niño – Southern Oscillation events. An earlier mark and recapture study indicated that adults exhibit some degree of breeding-site and mate fidelity, and that juveniles potentially move more frequently than adults; however, the extent to which migrants and gene flow occur between islands within the Galápagos archipelago is largely unknown. This study tested the hypothesis that geographic isolation and adult breeding philopatry has led to a degree of genetic differentiation between island subpopulations within the archipelago. We examined the genetic diversity within and among different subpopulations and the extent to which gene flow occurs between island subpopulations. Estimates of allelic richness and gene diversity were not significantly different between subpopulations. Tests to detect genetic heterogeneity failed to reject the H ₀ of no difference in allele frequencies for chi-square (P = 0.28) and Fisher’s exact test (P = 0.19). All pairwise values of the F _ST variant θ were not significant, while a power analysis revealed a >99% probability of detecting a biologically true F _ST of 0.05. Migration estimates in BAYESASS+ suggest symmetrical gene flow throughout the species’ distribution. Our results indicate a low level of genetic diversity throughout the population and a seemingly high level of gene flow between subpopulations. We argue that the Galápagos penguin should be managed as one panmictic population and we discuss the risk of disease threats in the archipelago.     

Feral donkey (<Emphasis Type="Italic">Equus asinus</Emphasis>) eradications in the Galápagos

Introduced herbivores are major drivers of ecosystem change and biodiversity loss, particularly on islands. Tools and techniques now exist to routinely remove introduced herbivores from islands, providing a powerful conservation tool. Here, we summarize the few documented feral donkey removals on islands worldwide, and report on the removal of populations from the Galápagos archipelago, Ecuador. After decades of sporadic control programs on Santiago Island and Alcedo Volcano, Isabela Island, donkey populations were removed from both areas, concurrent with a goat eradication program. Both ground and aerial hunting programs were utilized. The latter method was highly efficient; donkeys were removed from Santiago Island with less than 80 h of aerial hunting. Given the clear impacts of introduced herbivores on islands worldwide, feral donkey populations should be routinely removed from islands.     

Pollination of the heterostylous Galápagos native, <Emphasis Type="Italic">Cordia lutea</Emphasis> (Boraginaceae)

The hypothesis of this study was that in the Galápagos Islands, fruit and seed set via nocturnal pollination would exceed that of diurnal pollination due to greater insect activity at night typical of hot, arid regions. Cordia lutea, a heterostylous member of the Galápagos flora, was submitted to pollination experiments, visitor observations, nectar sampling, pollen transfer studies, pollen-ovule ratio studies, and pollen measurements. Flowers set fruit and seed via open pollination, autonomous autogamy, facilitated autogamy, facilitated cross-pollination, diurnal pollination, and nocturnal pollination. There was a significant difference in fruit set between flowers experiencing legitimate cross-pollinations (pin × thrum) and those experiencing all other pollination treatments except facilitated autogamy. There was no significant difference in seed set among any of the treatments, but there was a trend toward greater seed set for flowers experiencing open-pollination, legitimate cross-pollination, and nocturnal pollination. There was no significant difference in fruit set or seed set between flowers experiencing diurnal pollination and nocturnal pollination, although there was a trend toward greater seed set resulting from nocturnal pollination. Carpenter bees were the most effective diurnal pollinators, whereas moths were the most effective nocturnal pollinators. Of the two, moths are more efficient at transporting pollen from plant to plant. Results indicate that an overall low productivity of this species is due to pollen limitation exacerbated by nectar robbing. Cordia lutea exhibits a mixed mating system, producing a relatively low level of fruits through a combination of self- and cross-pollination, facilitated by the relatively few insects that are available.     

Functional expression of the lipase gene Lip2 of <Emphasis Type="Italic">Pleurotus</Emphasis> <Emphasis Type="Italic">sapidus</Emphasis> in <Emphasis Type="Italic">Escherichia</Emphasis> <Emphasis Type="Italic">coli</Emphasis>

The lipase Lip2 of the edible basidiomycete, Pleurotus sapidus, is an extracellular enzyme capable of hydrolysing xanthophyll esters with high efficiency. The gene encoding Lip2 was expressed in Escherichia coli TOP10 using the gene III signal sequence to accumulate proteins in the periplasmatic space. The heterologous expression under control of the araBAD promoter led to the high level production of recombinant protein, mainly as inclusion bodies, but partially in a soluble and active form. A fusion with a C-terminal His tag was used for purification and immunochemical detection of the target protein. This is the first example of a heterologous expression and periplasmatic accumulation of a catalytically active lipase from a basidiomycete fungus.     

On the color pattern variation in the horse-fly <Emphasis Type="Italic">Chrysops</Emphasis> (<Emphasis Type="Italic">Heterochrysops</Emphasis>) <Emphasis Type="Italic">vanderwulpi</Emphasis> Kröber (Diptera,Tabanidae)

A dark form of the female horsefly Chrysops (Heterochrysops) vanderwulpi Kröber is described from Primorskii Territory of Russia and illustrated with a figure of the abdominal pattern.     

<Emphasis Type="Italic">Carex</Emphasis> × <Emphasis Type="Italic">involuta</Emphasis> and <Emphasis Type="Italic">Carex juncella</Emphasis> in the flora of Slovakia

The paper brings information on an isolated occurrence and morphological characters of Carex × involuta and C. juncella populations in the Vel’ká Fatra Mts. Their presence has been known neither from the territory of Slovakia nor from the whole Western Carpathians till now.     

Molecular variation of the testes-specific β<Emphasis Type="Italic">NACtes</Emphasis> genes in the <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> genome

The βNACtes gene family of the Drosophila melanogaster genome provides a model for investigating the mechanisms of the molecular evolution of recently evolved genes. The βNACtes genes code for proteins that are homologous to the subunit of the nascent polypeptide-associated complex (NAC), are expressed exclusively in the testis, and are localized on the X chromosome as two-gene clusters and one separate copy. Population polymorphism of the βNACtes genes was studied using several wild-type D. melanogaster stocks, and βNACtes paralogs were compared with each other. A heterogeneous pattern was observed for βNACtes polymorphism: the 3′ genes of the two-gene clusters were low polymorphic, whereas, separate, the βNACtes1 gene was the most variable. The 5′ βNACtes copies of the two-gene tandems were practically identical, whereas the 3′ βNACtes copies were highly diverged. Hence, local gene conversion was assumed to provide for the selective homogenization of the 5′ genes. A comparison of the βNACtes paralogs showed that the majority of amino acid differences were in the N-terminal region, containing the βNAC domain. The McDonald-Kreitman test was used to analyze the divergence of βNACtes paralogs and implicated positive selection in the evolution of the βNACtes gene family.     

Brood cell size of <Emphasis Type="Italic">Apis</Emphasis> <Emphasis Type="Italic">mellifera</Emphasis> modifies the reproductive behavior of <Emphasis Type="Italic">Varroa</Emphasis> <Emphasis Type="Italic">destructor</Emphasis>

We undertook a field study to determine whether comb cell size affects the reproductive behavior of Varroa destructor under natural conditions. We examined the effect of brood cell width on the reproductive behavior of V. destructor in honey bee colonies, under natural conditions. Drone and worker brood combs were sampled from 11 colonies of Apis mellifera. A Pearson correlation test and a Tukey test were used to determine whether mite reproduction rate varied with brood cell width. Generalized additive model analysis showed that infestation rate increased positively and linearly with the width of worker and drone cells. The reproduction rate for viable mother mites was 0.96 viable female descendants per original invading female. No significant correlation was observed between brood cell width and number of offspring of V. destructor. Infertile mother mites were more frequent in narrower brood cells.     

Floreana Island re-colonization potential of the Galápagos short-eared owl (<Emphasis Type="Italic">Asio flammeus galapagoensis</Emphasis>)

Non-native invasive species threaten Galápagos’ endemic biodiversity, and increasing efforts are underway to protect its species from further harm. One such project is focused on the eradication of invasive rodents using rodenticide bait on Floreana, the archipelago’s sixth largest island. Short-eared owls (Asio flammeus galapagoensis) that consume poisoned rodents will, therefore, be at risk of secondary poisoning. If negatively impacted, it is not known to what degree the Floreana Island short-earned owl population is isolated, and whether potential re-colonization exists from its closest neighboring large population on Santa Cruz. Based on eight microsatellite loci and mtDNA control region sequence data from museum and contemporary samples, the short-eared owl populations on Floreana and Santa Cruz are not isolated from each other. However, gene flow is asymmetric from Floreana to Santa Cruz and not in the opposite direction. Morphometric data, including tarsus and bill size, and behavioral observations corroborate the genetic results and suggest that the Floreana population may possess unique traits compared to neighboring populations. For example, Floreana short-eared owls are more crepuscular than neighboring islands, which are predominately nocturnal, and were also non-responsive to inter-island call back recordings. Therefore, these results have important management implications concerning short-eared owl persistence on Floreana following rodenticide application. We recommend that managers implement additional precautions to protect the short-eared owl population until the risk of secondary poisoning has passed such as maintaining individuals in captivity. This study provides no evidence to suggest that short-eared owls are likely to disperse from Floreana’s closest large population on Santa Cruz if the local population is negatively impacted by rodenticide exposure, and the observed morphological and behavioral traits argue against translocating owls between islands.     

Rapid induction of <Emphasis Type="Italic">Agrobacterium</Emphasis> <Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transgenic roots directly from adventitious roots in <Emphasis Type="Italic">Panax</Emphasis> <Emphasis Type="Italic">ginseng</Emphasis>

Root segments from seedlings of Panax ginseng produced adventitious roots directly when cultured on 1/2 MS solid medium lacking NH₄NO₃ and containing 3.0 mg l⁻¹ IBA. Using this adventitious root formation, we developed rapid and efficient transgenic root formation directly from adventitious root segments in P. ginseng. Root segments were co-cultivated with Agrobacterium tumefaciens (GV3101) caring β-glucuronidase (GUS) gene. Putative transgenic adventitious roots were formed directly from root segments on medium with 400 mg l⁻¹ cefotaxime and 50 mg l⁻¹ kanamycin. Kanamycin resistant adventitious roots were selected and proliferated as individual lines by subculturing on medium with 300 mg l⁻¹ cefotaxime and 50 mg l⁻¹ kanamycin at two weeks subculture interval. Frequency of transient and stable expression of GUS gene was enhanced by acetosyringon (50 mg l⁻¹) treatment. Integration of transgene into the plants was confirmed by the X-gluc reaction, PCR and Southern analysis. Production of transgenic plants was achieved via somatic embryogenesis from the embryogenic callus derived from independent lines of adventitious roots. The protocol for rapid induction of transgenic adventitious roots directly from adventitious roots can be applied for a new Agrobacterium tumefaciens-mediated genetic transformation protocol in P. ginseng.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of <Emphasis Type="Italic">Codonopsis lanceolata</Emphasis> using the <Emphasis Type="Italic">γ-TMT</Emphasis> gene

Efficient transformation of leaf disc-derived callus of Codonopsis lanceolata was obtained using Agrobacterium tumefaciens strain LBA4404 harboring a binary vector, pYBI121, that carries the neomycin phosphotransferase (npt II) gene as a selectable marker. The green shoots recovered from agroinfected explants on selection medium (containing 0.1 mg/l α-naphthaleneacetic acid (NAA), 1 mg/l 6-benzylaminopurine (BAP), 100 mg/l kanamycin, and 250 mg/l cefotaxime) were rooted on Murashige and Skoog (MS) medium supplemented with 2 mg/l IBA and 10 mg/l kanamycin. To optimize the transformation conditions, several factors were assessed, including the co-cultivation period, the duration of pre- and post-culture in darkness and light, the kanamycin concentration, and the Agrobacterium densities. We produced transgenic Codonopsis lanceolata overexpressing γ-tocopherol methyltransferase (γ-TMT) by this protocol. Moreover, the α-tocopherol content of the plants was enhanced by the overexpression of this gene. Bimal Kumar Ghimire and Eun Soo Seong contributed equally to this work.     

Mating system and seed variation of <Emphasis Type="Italic">Acacia</Emphasis> hybrid (<Emphasis Type="Italic">A. mangium</Emphasis> × <Emphasis Type="Italic">A. auriculiformis</Emphasis>)

The mating system and seed variation of Acacia hybrid (A. mangium × A. auriculiformis) were studied using allozymes and random amplified polymorphic DNA (RAPD) markers, respectively. Multi-locus outcrossing rate estimations indicated that the hybrid was predominantly outcrossed (mean±s.e. t _m = 0.86±0.01). Seed variation was investigated using 35 polymorphic RAPD fragments. An analysis of molecular variance (AMOVA) revealed the highest genetic variation among seeds within a pod (66%–70%), followed by among pods within inflorescence (29%–37%), and the least variation among inflorescences within tree (<1%). In addition, two to four RAPD profiles could be detected among seeds within pod. Therefore, the results suggest that a maximum of four seeds per pod could be sampled for the establishment of a mapping population for further studies.     

Production of a Soluble Disulfide Bond-Linked TCR in the Cytoplasm of <Emphasis Type="Italic">Escherichia</Emphasis> <Emphasis Type="Italic">coli</Emphasis><Emphasis Type="Italic">trx</Emphasis>B <Emphasis Type="Italic">gor</Emphasis> Mutants

Previously, we have described the use of phage display to generate high affinity disulfide bond-linked T cell receptors (TCRs). The affinities of the mutant TCRs were analysed after refolding of separately expressed α and β chains from Escherichia coli inclusion bodies. This approach is only suitable for the analysis of small numbers of TCR variants. An attractive alternative would be soluble expression within the bacterial periplasm, but the generic production of TCRs within the E. coli periplasm has so far not proved successful. Here we show that functional, soluble TCR can be produced within the cytoplasm of trxB gor mutant E. coli strains, with maximum yields of 3.4 mg/l. We also investigated the effect of coexpressing the folding modulators Skp and DsbC finding that the TCR expression levels were largely unaffected by these chaperones. Importantly, we demonstrated that the amount of protein purified from 50 ml starter cultures was sufficient to show functionality of the TCR by specific antigen binding in both ELISA and surface plasmon resonance (SPR) assays. This TCR production method has the potential to allow rapid and medium throughput analysis of affinity-matured TCRs selected from TCR phage display libraries.     

Genome-wide analysis and identification of the <Emphasis Type="Italic">SMXL</Emphasis> gene family in apple (<Emphasis Type="Italic">Malus</Emphasis> × <Emphasis Type="Italic">domestica</Emphasis>)

Strigolactones (SLs) are a recently discovered type of plant hormone that controls various developmental processes. The DWARF53 (D53) protein in rice and the SMAX1-LIKE (SMXL) family in Arabidopsis repress SL signaling. In this study, bioinformatics analyses were performed, and 236 SMXL proteins were identified in 28 sequenced plants. A phylogenetic analysis indicated that all potential SMXL proteins could be divided into three groups and that the SMXL proteins may have originated in Bryophytes. An analysis of the SMXL chromosomal locations suggested that gene duplication events at different times led to expansion of the SMXL family members in Angiospermae. Subsequently, the gene structure and protein modeling of MdSMXLs showed that they are highly conserved. The expression patterns of MdSMXLs indicated that they were expressed in different organs of apple (stems, roots, leaves, flowers, and fruits) at varying levels and that MdSMXLs may participate in the SL signaling pathway and the response to abiotic stress. This study provides a valuable foundation for additional investigations into the function of the SMXL gene family in plants.     

Sequence Analysis of the <Emphasis Type="Italic">Lactoferrin</Emphasis> Gene and Variation of <Emphasis Type="Italic">g</Emphasis>.<Emphasis Type="Italic">7605C</Emphasis>→<Emphasis Type="Italic">T</Emphasis> in 10 Chinese Indigenous Goat Breeds

Much attention has been focused on the study of lactoferrin at the protein or nucleotide level in mice, humans, and cattle, but little is known about it in goats. The goat LF gene from 5' UTR to exon 17 was amplified, and the variation of g.7605C→T in 10 Chinese indigenous goat breeds was analyzed. Among the three ruminant species (cattle, sheep, and goats), the intron-exon distribution pattern was similar, and all the exons had the same length, but the length of introns varied greatly due to insertions or deletions. The frequency of allele T at g.7605C→T (50.12%) was a little higher than that of allele C (49.88%), and the genotype distribution differed greatly between goat populations. The g.7605C→T site showed higher genetic diversity in goat populations. The genetic differentiation was 0.0783, and gene flow was 2.9433 among the 10 Chinese indigenous goat populations.     

Conjugal transfer using the bacteriophage ϕC31 <Emphasis Type="Italic">att</Emphasis>/<Emphasis Type="Italic">int</Emphasis> system and properties of the <Emphasis Type="Italic">attB</Emphasis> site in <Emphasis Type="Italic">Streptomyces ambofaciens</Emphasis>

To facilitate molecular genetic studies of Streptomyces ambofaciens that produces spiramycin, a commercially important macrolide antibiotic used in human medicine against Gram-positive pathogenic bacteria, the conditions for the conjugal transfer of DNA from E. coli to S. ambofaciens were established using a bacteriophage ϕC31 att/int system. The transconjugation efficiency of S. ambofaciens varied with the medium used; the highest frequency was obtained on AS-1 medium containing 10 mM MgCl₂ without heat treatment of the spores. In addition, by cloning and sequencing the attB site, we identified that S. ambofaciens contains a single attB site within an ORF coding for a pirin homolog, and its attB site sequence shows 100% nt identity to the sequence of S. coelicolor and S. lividans, which have the highest efficiency in transconjugation using the ϕC31 att/int system.     

Characteristics of β-lactamases and their genes (<Emphasis Type="Italic">blaA</Emphasis> and <Emphasis Type="Italic">blaB</Emphasis>) in <Emphasis Type="Italic">Yersinia intermedia</Emphasis> and <Emphasis Type="Italic">Y. frederiksenii</Emphasis>

Background  

The presence of β-lactamases in Y. enterocolitica has been reported to vary with serovars, biovars and geographical origin of the isolates. An understanding of the β-lactamases in other related species is important for an overall perception of antibiotic resistance in yersiniae. The objective of this work was to study the characteristics of β-lactamases and their genes in strains of Y. intermedia and Y. frederiksenii, isolated from clinical and non-clinical sources in India.