High-throughput microsatellite isolation through 454 GS-FLX Titanium pyrosequencing of enriched DNA libraries

Microsatellites (or SSRs: simple sequence repeats) are among the most frequently used DNA markers in many areas of research. The use of microsatellite markers is limited by the difficulties involved in their de novo isolation from species for which no genomic resources are available. We describe here a high-throughput method for isolating microsatellite markers based on coupling multiplex microsatellite enrichment and next-generation sequencing on 454 GS-FLX Titanium platforms. The procedure was calibrated on a model species (Apis mellifera) and validated on 13 other species from various taxonomic groups (animals, plants and fungi), including taxa for which severe difficulties were previously encountered using traditional methods. We obtained from 11,497 to 34,483 sequences depending on the species and the number of detected microsatellite loci ranged from 199 to 5791. We thus demonstrated that this procedure can be readily and successfully applied to a large variety of taxonomic groups, at much lower cost than would have been possible with traditional protocols. This method is expected to speed up the acquisition of high-quality genetic markers for nonmodel organisms.     

Annual follow-up of the gastrointestinal parasitosis of white-tailed deer Odocoileus virginianus (Artiodactyla: Cervidae) in captivity in Yucatán, México]

Gastrointestinal parasites, and egg and oocyst output in the faeces of captive white-tailed deer (Odocoileus virginianus yucatanensis) were recorded in Yucatan, Mexico. Feces were obtained from from January through December 1995 (ten samples every two weeks per place). Samples were processed by flotation and the McMaster techniques. Faecal cultures for L3 larvae were made by the Corticelli-Lai technique. Oocysts in faeces were cultured in 2% potassium dicromate. Seven genera were determined (Haemonchus spp., Cooperia spp, Isospora spp., Eimeria spp., Trichuris spp., Strongyloides spp. and Moniezia spp.) which represent five orders. The most frequent genera were Haemonchus, Isospora and Eimeria. The genus Isospora is reported for the first time in deer of this region, although it was not possible to explain the source of this parasite. The frequency and level of faecal egg and oocyst outputs were variable during the year and increased during the rainy season. There was a positive correlation between relative humidity, environmental temperature and rainfall with the coccidia and strongylida orders. In the central zone of Yucatan the meteorological conditions during the rainy season are favourable for the development of gastrointestinal parasitism which enable an increased risk of infection for deer.     

To What did They Consent? Understanding Consent Among Low Literacy Participants in a Microbicide Feasibility Study in Mazabuka,Zambia

We conducted a study to review the consenting process in a vaginal Microbicide feasibility study conducted in Mazabuka, Zambia. Participants were drawn from those participating in the microbicide study. A questionnaire and focus group discussion were used to collect information on participants understanding of study aims, risks and benefits. Altogether, 200 participants took part in this study. The results of the study showed that while all participants signed or endorsed their thumbprints to the consent forms, full informed consent was not attained from most of the participants since 77% (n = 154) of the participants had numerous questions about the study and 34% (n = 68) did not know who to get in touch with concerning the study. Study objectives were not fully understood by over 61% of the participants. Sixty four percent of the participants were not sure of the risks of taking part in the microbicide study. A significant number thought the study was all about determining their HIV status. Some participants were concerned that their partners were not on the trial as they were convinced that being on the study meant that that they had a lifetime protection from HIV infection. The process of obtaining consent was inadequate as various phases of the study were not fully understood. We recommend the need for researchers to reinforce the consenting process in all studies and more so when studies are conducted in low literacy populations.     

When Anthropogenic River Disturbance Decreases Hybridisation between Non-Native and Endemic Cyprinids and Drives an Ecomorphological Displacement towards Juvenile State in Both Species

Understanding the impact of non-native species on native species is a major challenge in molecular ecology, particularly for genetically compatible fish species. Invasions are generally difficult to study because their effects may be confused with those of environmental or human disturbances. Colonized ecosystems are differently impacted by human activities, resulting in diverse responses and interactions between native and non-native species. We studied the dynamics between two Cyprinids species (invasive Chondrostoma nasus and endemic Parachondrostoma toxostoma) and their hybrids in 16 populations (from allopatric to sympatric situations and from little to highly fragmented areas) corresponding to 2,256 specimens. Each specimen was assigned to a particular species or to a hybrid pool using molecular identification (cytochrome b and 41 microsatellites). We carried out an ecomorphological analysis based on size, age, body shape, and diet (gut vacuity and molecular fecal contents). Our results contradicted our initial assumptions on the pattern of invasion and the rate of introgression. There was no sign of underperformance for the endemic species in areas where hybridisation occurred. In the unfragmented zone, the introduced species was found mostly downstream, with body shapes similar to those in allopatric populations while both species were found to be more insectivorous than the reference populations. However, high level of hybridisation was detected, suggesting interactions between the two species during spawning and/or the existence of hybrid swarm. In the disturbed zone, introgression was less frequent and slender body shape was associated with diatomivorous behaviour, smaller size (juvenile characteristics) and greater gut vacuity. Results suggested that habitat degradation induced similar ecomorphological trait changes in the two species and their hybrids (i.e. a transition towards a pedomorphic state) where the invasive species is more affected than the native species. Therefore, this study reveals a diversity of relationships between two genetically compatible species and emphasizes constraints on the invasion process in disturbed areas.     

Estimation of heterozygosity for single-probe multilocus DNA fingerprints

In spite of the increasing application of DNA fingerprinting to natural populations and to the genetic identification of humans, explicit methods for estimation of basic population genetic parameters from DNA fingerprinting data have not been developed. Contributing to this omission is the inability to determine, for multilocus fingerprinting probes, relatively important genetic information, such as the number of loci, the number of alleles, and the distribution of these alleles into specific loci. One of the most useful genetic parameters that could be derived from such data would be the average heterozygosity, which has traditionally been employed to measure the level of genetic variation within populations and to compare genetic variation among different loci. We derive here explicit formulas for both the estimation of average heterozygosity at multiple hypervariable loci and a maximum value for this estimate. These estimates are based upon the DNA restriction-pattern matrices that are typical for fingerprinting studies of humans and natural populations. For several empirical data sets from our laboratory, estimates of average and maximal heterozygosity are shown to be relatively close to each other. Furthermore, variances of these statistics based on simulation studies are relatively small. These observations, as well as consideration of the effect of missing alleles and alternate numbers of loci, suggest that the average heterozygosity can be accurately estimated using phenotypic DNA fingerprint patterns, because this parameter is relatively insensitive to the lack of certain genetic information.      

Structure and development of the metapleural gland in Technomyrmex vitiensis

Insectes Sociaux - We studied the internal and external appearance of the metapleural gland in the five castes of Technomyrmex vitiensis. All castes do possess the gland, but with a clear gradient...     

Characterization of ripening-regulated cDNAs and their expression in ethylene-suppressed charentais melon fruit

Charentais melons (Cucumis melo cv Reticulatus) are climacteric and undergo extremely rapid ripening. Sixteen cDNAs corresponding to mRNAs whose abundance is ripening regulated were isolated to characterize the changes in gene expression that accompany this very rapid ripening process. Sequence comparisons indicated that eight of these cDNA clones encoded proteins that have been previously characterized, with one corresponding to ACC (1-aminocyclopropane-1-carboxylic acid) oxidase, three to proteins associated with pathogen responses, two to proteins involved in sulfur amino acid biosynthesis, and two having significant homology to a seed storage protein or a yeast secretory protein. The remaining eight cDNA sequences did not reveal significant sequence similarities to previously characterized proteins. The majority of the 16 ripening-regulated cDNAs corresponded to mRNAs that were fruit specific, although three were expressed at low levels in vegetative tissues. When examined in transgenic antisense ACC oxidase melon fruit, three distinct patterns of mRNA accumulation were observed. One group of cDNAs corresponded to mRNAs whose abundance was reduced in transgenic fruit but inducible by ethylene treatment, indicating that these genes are directly regulated by ethylene. A second group of mRNAs was not significantly altered in the transgenic fruit and was unaffected by treatment with ethylene, indicating that these genes are regulated by ethylene-independent developmental cues. The third and largest group of cDNAs showed an unexpected pattern of expression, with levels of mRNA reduced in transgenic fruit and remaining low after exposure to ethylene. Regulation of this third group of genes thus appears to ethylene independent, but may be regulated by developmental cues that require ethylene at a certain stage in fruit development. The results confirm that both ethylene-dependent and ethylene-independent pathways of gene regulation coexist in climacteric fruit.