Gene-sized DNA molecules of the macronuclei in three species of hypotrichs: Size distributions and absence of nicks

Macronuclear DNAs from three related hypotrichous ciliated protozoans were compared by agarose gel electrophoresis. Each was shown to be composed of DNA duplexes that yielded a unique pattern of bands overlying a continuous distribution of DNA sizes ranging from 400 bp to 20,000 bp. By EM, the number average molecular sizes for doublestranded DNA were 2,200 bp for Oxytricha sp., 2,514 bp for Stylonychia pustulata and 1,836 bp for Euplotes aediculatus. Contrary to previous reports we present evidence that the macronuclear DNAs in each of these three organisms lack single-stranded interruptions.     

Hemocyanin gene family evolution in spiders (Araneae), with implications for phylogenetic relationships and divergence times in the infraorder Mygalomorphae

Hemocyanins are multimeric copper-containing hemolymph proteins involved in oxygen binding and transport in all major arthropod lineages. Most arachnids have seven primary subunits (encoded by paralogous genes a–g), which combine to form a 24-mer (4 × 6) quaternary structure. Within some spider lineages, however, hemocyanin evolution has been a dynamic process with extensive paralog duplication and loss. We have obtained hemocyanin gene sequences from numerous representatives of the spider infraorders Mygalomorphae and Araneomorphae in order to infer the evolution of the hemocyanin gene family and estimate spider relationships using these conserved loci. Our hemocyanin gene tree is largely consistent with the previous hypotheses of paralog relationships based on immunological studies, but reveals some discrepancies in which paralog types have been lost or duplicated in specific spider lineages. Analyses of concatenated hemocyanin sequences resolved deep nodes in the spider phylogeny and recovered a number of clades that are supported by other molecular studies, particularly for mygalomorph taxa. The concatenated data set is also used to estimate dates of higher-level spider divergences and suggests that the diversification of extant mygalomorphs preceded that of extant araneomorphs. Spiders are diverse in behavior and respiratory morphology, and our results are beneficial for comparative analyses of spider respiration. Lastly, the conserved hemocyanin sequences allow for the inference of spider relationships and ancient divergence dates.     

Thermal performance of quartz capillaries for vitrification

In this paper we report the thermal behavior of a new approach for vitrification. Thermal performance of traditional open pulled straws is compared with a new technique based on the combined use of quartz capillaries with slush nitrogen. This new method of vitrification achieved ultrafast cooling rates of 250,000 °C/min. As a result, a much lower concentration of cryoprotectant was needed to reach vitrification. In fact, a cryoprotectant solution typically used in oocyte slow freezing protocols was shown to remain transparent after cooling to liquid nitrogen temperatures indicating apparent “vitrification”. This approach offers a new and very promising technique for vitrification of cells using low levels of cryoprotectants.     

Development of a molecular diagnostic system to discriminate Dreissena polymorpha (zebra mussel) and Dreissena bugensis (quagga mussel)

A 3-primer PCR system was developed to discriminate invasive zebra (Dreissena polymorpha) and quagga (Dreissena bugensis) mussel. The system is based on: 1) universal primers that amplifies a region of the nuclear 28s rDNA gene from both species and 2) a species-specific primer complementary to either zebra or quagga mussel. The species-specific primers bind to sequences between the binding sites for the universal primers resulting in the amplification of two products from the target species and one product from the nontarget species. Therefore, nontarget products are positive amplification controls. The 3-primer system accurately discriminated zebra and quagga mussels from seven geographically distinct populations.     

Utility of the nuclear protein-coding gene, elongation factor-1 gamma (EF-1gamma), for spider systematics, emphasizing family level relationships of tarantulas and their kin (Araneae: Mygalomorphae)

Spider systematics has overwhelmingly relied on morphological characters to resolve higher-level phylogenetic questions. Molecular phylogenetic studies of spiders above the genus level have been rare, partly because of a paucity of characterized genes available for amplification and sequencing. Here we show the phylogenetic utility of a new molecular marker, elongation factor-1 gamma (EF-1gamma) for discerning family level relationships in the spider infraorder, Mygalomorphae. We included genomic sequences from 26 mygalomorph genera in 14 families as well as cDNA sequences from 10 families in the infraorder Araneomorphae. We found strong support for the traditional split of mygalomorphs into atypoids (Antrodiaetidae, Atypidae, and Mecicobothriidae) and non-atypoids (all other families). Some families with multiple generic representatives were found to be polyphyletic or paraphyletic, such as the Nemesiidae, Ctenizidae, and Hexathelidae. A small portion of genomic EF-1gamma that could be amplified from araneomorphs contained a short intron, suggesting that longer genomic sequences could not be amplified due to the presence of introns. This intron may be useful for intra-familial araneomorph relationships. A tentative timeline for spider evolution is proposed using the evolutionary rate of EF-1gamma, estimated to be approximately 0.22% pairwise divergence per million years based on a non-parametric smoothing method (NPRS) and fossil constraints.     

A total evidence assessment of the phylogeny of North American euctenizine trapdoor spiders (Araneae, Mygalomorphae, Cyrtaucheniidae) using Bayesian inference

North American trapdoor spiders of the subfamily Euctenizinae (Cyrtaucheniidae) are among the most diverse mygalomorph spiders (trapdoor spiders, tarantulas, and their relatives) on the continent in terms of species numbers and ecological habits. We present a generic level phylogenetic study of the subfamily based on a total evidence approach. Our dataset comprises approximately 3.7 kb of molecular characters (18S and 28S rRNA gene sequences) and 71 morphological characters scored for 32 taxa. When analyzed independently, these data sets, particularly the morphology, depict very different views of mygalomorph and euctenizine relationships, albeit with weak support. However, when these data are combined we recover a tree topology that is supported by high posterior probability for most nodes. The combined data recover a phylogenetic pattern for euctenizines different than previously published and indicate the presence of a narrowly endemic new genus from central California. While euctenizine monophyly is unequivocal, the monophyly of a number of other mygalomorph groups is questionable (e.g., Cyrtaucheniidae, Mecicobothriodina, Rastelloidina). This non-monophyly is noteworthy, as our analysis represents the first employing a total evidence approach for mygalomorphs, a group known to be morphologically conservative.     

Beta-hydroxybutyrate and response to hypoxia in the ground squirrel, Spermophilus tridecimlineatus

1. Previous studies have suggested that elevated ketone levels are associated with increased survival time in rodents exposed to hypoxia. In this study the association between whole blood BHB (beta-hydroxybutyrate) and hypoxic survival time was investigated in hibernating and non-hibernating ground squirrels and in rats. 2. Non-hibernating ground squirrels and rats were exposed to hypoxia (4.5% O2). One hundred per cent of ground squirrels survived 1 hr of hypoxia vs 20% of rats. 3. Ketone levels were significantly higher in ground squirrels than rats during hypoxia, and rats surviving the longest had the highest ketone levels. 4. When hibernation was induced in ground squirrels there was a significant increase in beta-hydroxy-butyrate from 0.45 to 1.6 mM (P = 0.0005). 5. Ground squirrel heart mitochondrial respiratory control ratios and ATP synthesis rates indicated no preferential ketone utilization which might suggest a possible extramitochondrial role of BHB during hypoxia. 6. We conclude that elevated blood BHB levels are associated with increased hypoxic survival and they may have evolved in response to life-threatening hypoxia as experienced during hibernation.