Repetitive sequences of the sea urchin genome: Distribution of members of specific repetitive families

Three repetitive sequence families from the sea urchin genome were studied, each defined by homology with a specific cloned probe one to a few hundred nucleotides long. Recombinant λ-sea urchin DNA libraries were screened with these probes, and individual recombinants were selected that include genomic members of these families. Restriction mapping, gel blot, and kinetic analyses were carried out to determine the organization of each repeat family. Sequence elements belonging to the first of the three repeat families were found to be embedded in longer repeat sequences. These repeat sequences frequently occur in small clusters. Members of the second repeat family are also found in a long repetitive sequence environment, but these repeats usually occur singly in any given region of the DNA. The sequences of the third repeat are only 200 to 300 nucleotides long, and are generally terminated by single copy DNA, though a few examples were found associated with other repeats. These three repeat sequence families constitute sets of homologous sequence elements that relate distant regions of the DNA.     

Intensive RNAi with lentiviral vectors in mammalian cells

RNAi is a powerful technology for analyzing gene function in human cells. However, its utility can be compromised by inadequate knockdown of the target mRNA or by interpretation of effects without rigorous controls. We review lentiviral vector-based methods that enable transient or stable knockdowns to trace mRNA levels in human CD4+ T cell lines and other targets. Critical controls are reviewed, including rescue of the pre-knockdown phenotype by re-expression of the targeted gene. The time from thinking about a potential knockdown target to analysis of phenotypes can be as short as a few weeks.     

An order-specific monoclonal antibody to Diptera reveals the impact of alternative prey on spider feeding behavior in a complex food web

Generalist predators have the capacity to restrict pest population growth, especially early in the season before densities increase. However, their polyphagous feeding habits sometimes translate into reduced pest consumption when they target alternative prey. An order-specific monoclonal antibody was developed to examine the strength of trophic connections between Diptera, a major category of non-pest prey, and linyphiid spiders in alfalfa. We report the development and characterization of a monoclonal antibody with order-level specificity to Diptera. This antibody elicited strong absorbance to 22 Diptera from 13 families, no false-positive reactivity to non-dipteran invertebrates, and antigen detection periods following prey consumption that were comparable between spiders. Over 900 field-collected females of the linyphiid spiders Erigone autumnalis and Bathyphantes pallidus were screened for Diptera antigen. Significantly more B. pallidus screened positive for Diptera (40%) compared to E. autumnalis (16%), indicating differential reliance on these prey. In parallel with the collection of spiders for gut-content analysis, prey availability was estimated at web sites. The two spiders exhibited different feeding responses to prey availability. Consumption of Diptera by B. pallidus was strongly correlated with Diptera abundance whilst the availability of other potential prey did not influence predation rates. Conversely, E. autumnalis did not prey upon Diptera in proportion to availability, but increased Collembola activity-density reduced dipteran consumption. Integration of molecular gut-content analysis with precise sampling of prey demonstrated how two closely related linyphiid spiders exhibit different feeding responses to the availability of prey under natural field conditions. Elucidating the feeding preferences of natural enemies is critical to effective incorporation of biological control by generalist predators in the management of agricultural pests.     

Diversification of Hawaiian Cyrtandra (Gesneriaceae) under the influence of incomplete lineage sorting and hybridization

Cyrtandra (Gesneriaceae) is a genus of flowering plants with over 800 species distributed throughout Southeast Asia and the Pacific Islands. On the Hawaiian Islands, 60 named species and over 89 putative hybrids exist, most of which are identified on the basis of morphology. Despite many previous studies on the Hawaiian Cyrtandra lineage, questions regarding the reconciliation of morphology and genetics remain, many of which can be attributed to the relatively young age and evidence of hybridization between species. We utilized targeted enrichment, high‐throughput sequencing, and modern phylogenomics tools to test 31 Hawaiian Cyrtandra samples (22 species, two putative hybrids, four species with two samples each, one species with four samples) and two outgroups for species relationships and hybridization in the presence of incomplete lineage sorting (ILS). Both concatenated and species‐tree methods were used to reconstruct species relationships, and network analyses were conducted to test for hybridization. We expected to see high levels of ILS and putative hybrids intermediate to their parent species. Phylogenies reconstructed from the concatenated and species‐tree methods were highly incongruent, most likely due to high levels of incomplete lineage sorting. Network analyses inferred gene flow within this lineage, but not always between taxa that we expected. Multiple hybridizations were inferred, but many were on deeper branches of the island lineages suggesting a long history of hybridization. We demonstrated the utility of high‐throughput sequencing and a phylogenomic approach using 569 loci to understanding species relationships and gene flow in the presence of ILS.