Modulation of surface charge, particle size and morphological properties of chitosan-TPP nanoparticles intended for gene delivery

This work investigates the polyanion initiated gelation process in fabricating chitosan-TPP (tripolyphosphate) nanoparticles in the size range of 100-250 nm intended to be used as carriers for the delivery of gene or protein macromolecules. It demonstrates that ionic gelation of cationic chitosan molecules offers a flexible and easily controllable process for systematically and predictably manipulating particle size and surface charge which are important properties in determining gene transfection efficacy if the nanoparticles are used as non-viral vectors for gene delivery, or as delivery carriers for protein molecules. Variations in chitosan molecular weight, chitosan concentration, chitosan to TPP weight ratio and solution pH value were examined systematically for their effects on nanoparticle size, intensity of surface charge, and tendency of particle aggregation so as to enable speedy fabrication of chitosan nanoparticles with predetermined properties. The chitosan-TPP nanoparticles exhibited a high positive surface charge across a wide pH range, and the isoelectric point (IEP) of the nanoparticles was found to be at pH 9.0. Detailed imaging analysis of the particle morphology revealed that the nanoparticles possess typical shapes of polyhedrons (e.g., pentagon and hexagon), indicating a similar crystallisation mechanism during the particle formation and growth process. This study demonstrates that systematic design and modulation of the surface charge and particle size of chitosan-TPP nanoparticles can be readily achieved with the right control of critical processing parameters, especially the chitosan to TPP weight ratio. (c) 2005 Elsevier B.V. All rights reserved.

Comparison of genetic and morphological methods to detect the presence of American lobsters, Homarus americanus H. Milne Edwards, 1837 (Astacidea: Nephropidae) in Norwegian waters

American lobsters (Homarus americanus H. Milne Edwards, 1837) are imported live to Europe and should according regulations be kept in land-based tanks until sold. In spite of the strict regulations aimed specifically at preventing the introduction of this species into the NE Atlantic, several specimens of H. americanus have been captured in the wild, especially in Oslofjord, Norway since 1999. One of the great concerns is interbreeding between the introduced American species and the local European lobster, H. gammarus (Linnaeus, 1758). For this reason an awareness campaign was launched in 2000 focusing on morphologically "unusual" lobsters caught in local waters. Morphological characters have been based on colour and sub-ventral spines on the rostrum. Two samples of H. americanus were used for comparisons, as well as samples of European lobster from Oslofjord collected in 1992. Previous genetic analyses (allozymes, mtDNA and microsatellite DNA) have demonstrated that the American lobster is distinct from its European counterpart, with several additional alleles at many loci in addition to different allelic frequency distribution of alleles of "shared" alleles. During the present study, thirteen microsatellite loci were tested in the initial screening, and the three most discriminating loci (Hgam98, Hgam197b and Hgam47b) were used in a detailed comparison between the two species. A total of 45 unusual lobsters were reported captured from Ålesund (west) to Oslofjord (southeast) from 2001 to 2005 and these were analysed for the three microsatellite loci. Nine specimens were identified as American lobsters. Comparisons between morphological and genetic characteristics revealed that morphological differences are not reliable in discrimination the two species, or to identify hybrids. Further, some loci display almost no overlapping in allele frequency distribution for the reference samples analysed, thus providing a reliable tool to identify hybrids.

A new species of Pareledone (Cephalopoda : Octopodidae) from Antarctic Peninsula Waters

During recent cruises aboard RV Polarstern in the Antarctic Peninsula region, a new species of benthic octopodid was discovered whose generic affinities based on morphological characteristics were uncertain. Molecular sequence analysis of six mitochondrial and nuclear genes allows this species to be placed with confidence within the genus Pareledone. The species is described herein and morphological diagnostic characters are provided for its identification.

Tracing early stages of species differentiation: Ecological, morphological and genetic divergence of Galapagos sea lion populations

Background: Oceans are high gene flow environments that are traditionally believed to hamper the build-up of genetic divergence. Despite this, divergence appears to occur occasionally at surprisingly small scales. The Galápagos archipelago provides an ideal opportunity to examine the evolutionary processes of local divergence in an isolated marine environment. Galápagos sea lions (Zalophus wollebaeki) are top predators in this unique setting and have an essentially unlimited dispersal capacity across the entire species range. In theory, this should oppose any genetic differentiation.
Results: We find significant ecological, morphological and genetic divergence between the western colonies and colonies from the central region of the archipelago that are exposed to different ecological conditions. Stable isotope analyses indicate that western animals use different food sources than those from the central area. This is likely due to niche partitioning with the second Galápagos eared seal species, the Galápagos fur seal (Arctocephalus galapagoensis) that exclusively dwells in the west. Stable isotope patterns correlate with significant differences in foraging-related skull morphology. Analyses of mitochondrial sequences as well as microsatellites reveal signs of initial genetic differentiation.
Conclusion: Our results suggest a key role of intra- as well as inter-specific niche segregation in the evolution of genetic structure among populations of a highly mobile species under conditions of free movement. Given the monophyletic arrival of the sea lions on the archipelago, our study challenges the view that geographical barriers are strictly needed for the build-up of genetic divergence. The study further raises the interesting prospect that in social, colonially breeding mammals additional forces, such as social structure or feeding traditions, might bear on the genetic partitioning of populations.

The diet and landclass affinities of the Irish hare Lepus timidus hibernicus

The diet of the Irish hare Lepus timidus hibernicus was investigated in Northern Ireland. Faecal pellets were collected at regular intervals from three contrasting study sites and from a number of randomly selected sites from known land classes. Microhistological techniques were employed to analyse the pellets. Grass species occurred in high percentage frequencies in all samples of faecal pellets and a wide variety of plant species were represented (up to 26 species in one study site). In a stratified, random survey of the presence of hares, areas of lowland intensive agriculture had fewer positive records than expected. It is contended that L. t. hibernicus may not be able to sustain viable populations in areas of intensive agriculture, which are almost exclusively composed of ryegrass Lolium spp.

Morphological and molecular characters reveal differentiation in a Neotropical social bee, Melipona beecheii (Apidae : Meliponini)

Morphometrics and DNA microsatellites were used to analyse the genetic structure of populations of the stingless bee M. beecheii from two extremes of its geographic range. The results showed that populations from Costa Rica and Yucatan exhibit substantial phenotypic and molecular differentiation. Bees from Yucatan were smaller and paler than those from Costa Rica. The value of multilocus F-ST = 0.280 (P <0.001) confirmed that there were significant molecular genetic differences between the two populations. Populations showed significant deviation from Hardy Weinberg equilibrium and the values of FIS (the inbreeding coefficient) were positive for Costa Rica = 0.416 and the Yucatan Peninsula = 0.193, indicating a lack of heterozygotes in both populations possibly due to inbreeding. The DNA sequence of 678 bp of the mitochondrial gene COI differed between populations by 1.2%. The results of this study should be considered in conservation programmes, particularly with regard to the movement of colonies between regions.

Gayliella gen. nov in the tribe Ceramieae (Ceramiaceae, Rhodophyta) based on molecular and morphological evidence

On the basis of comparative morphology and phylogenetic analyses of rbcL and LSU rDNA sequence data, a new genus, Gayliella gen. nov., is proposed to accommodate the Ceramium flaccidum complex (C. flaccidum, C. byssoideum, C. gracillimum var. byssoideum, and C. taylorii), C. fimbriatum, and a previously undescribed species from Australia. C. transversale is reinstated and recognized as a distinct species. Through this study, G. flaccida (Kutzing) comb. nov., G. transversalis (Collins et Hervey) comb. nov., G. fimbriata (Setchell et N. L. Gardner) comb. nov., G. taylorii comb. nov., G. mazoyerae sp. nov., and G. womersleyi sp. nov. are based on detailed comparative morphology. The species referred to as C. flaccidum and C. dawsonii from Brazil also belong to the new genus. Comparison of Gayliella with Ceramium shows that it differs from the latter by having an alternate branching pattern; three cortical initials per periaxial cell, of which the third is directed basipetally and divides horizontally; and unicellular rhizoids produced from periaxial cells. Our phylogenetic analyses of rbcL and LSU rDNA gene sequence data confirm that Gayliella gen. nov. represents a monophyletic clade distinct from most Ceramium species including the type species, C. virgatum. We also transfer C. recticorticum to the new genus Gayliella.