Winter Ciliates in a British Columbian Fjord: Six New Species and an Analysis of Ciliate Putative Prey

ABSTRACT. This work provides the first study of North Pacific planktonic ciliates by quantitative protargol staining. Triplicate water bottle samples were collected at a depth of 2 m (above the shallow pycnocline) at six stations in Indian Arm, British Columbia, on February 15, 1990, and February 26, 1991. Thirty-six ciliate species were observed. Six new species are described from protargolstained specimens: Strombidium lynni n. sp., Strombidium taylori n. sp., Strombidium basimorphum n. sp., Slrombidiurn ventropinnum n. sp., Strobilidium undinum n. sp., and Urotricha cyrtonucleata n. sp.
Ciliate abundance varied significantly (ANOVA, α= 0.05) between sampling sites, ranging from 550 to 6,800 cells/liter in 1990 and from 1,800 to 7,900 cells/liter in 1991. Biomass also varied significantly (ANOVA, α= 0.05) ranging from 3.7 × 10⁵ to 3.3 × 10⁶ pg carbon/liter in 1990 and 3.04 × 10⁶− 6.97 × 10⁶ pg carbon/liter in 1991. Putative prey were enumerated in three size fractions (1.5–5 μm, 5–10 μm and 10–25 μm). The source of variation in ciliate abundance and biomass was not identified. Parameters of salinity, temperature, putative prey, chlorophyll a and pycnocline depth did not significantly correlate with ciliate biomass or abundance (α= 0.05).     

Low genetic diversity and population connectivity fuel vulnerability to climate change for the Tertiary relict pine Pinus bungeana

Endemic species are important components of regional biodiversity and hold the key to understanding local adaptation and evolutionary processes that shape species distributions. This study investigated the biogeographic history of a relict conifer Pinus bungeana Zucc. ex Endl. confined to central China. We examined genetic diversity in P. bungeana using genotyping-by-sequencing and chloroplast and mitochondrial DNA markers. We performed spatial and temporal inference of recent genetic and demographic changes, and dissected the impacts of geography and environmental gradients on population differentiation. We then projected P. bungeana's risk of decline under future climates. We found extremely low nucleotide diversity (average π 0.0014), and strong population structure (global F_ST 0.234) even at regional scales, reflecting long-term isolation in small populations. The species experienced severe bottlenecks in the early Pliocene and continued to decline in the Pleistocene in the western distribution, whereas the east expanded recently. Local adaptation played a small (8%) but significant role in population diversity. Low genetic diversity in fragmented populations makes the species highly vulnerable to climate change, particularly in marginal and relict populations. We suggest that conservation efforts should focus on enhancing gene pool and population growth through assisted migration within each genetic cluster to reduce the risk of further genetic drift and extinction.     

Systematics of Mukdenia and Oresitrophe (Saxifragaceae): Insights from genome skimming data

Oresitrophe and Mukdenia (Saxifragaceae) are epilithic sister genera used in traditional Chinese medicine. The taxonomy of Mukdenia, especially of M. acanthifolia, has been controversial. To address this, we produced plastid and mitochondrial data using genome skimming for Mukdenia acanthifolia and Mukdenia rossii, including three individuals of each species. We assembled complete plastomes, mitochondrial CDS and nuclear ribosomal ETS/ITS sequences using these data. Comparative analysis shows that the plastomes of Mukdenia and Oresitrophe are relatively conservative in terms of genome size, structure, gene content, RNA editing sites and codon usage. Five plastid regions that represent hotspots of change (trnH-psbA, psbC-trnS, trnM-atpE, petA-psbJ and ccsA-ndhD) are identified within Mukdenia, and six regions (trnH-psbA, petN-psbM, trnM-atpE, rps16-trnQ, ycf1 and ndhF) contain a higher number of species-specific parsimony-informative sites that may serve as potential DNA barcodes for species identification. To infer phylogenetic relationships between Mukdenia and Oresitrophe, we combined our data with published data based on three different datasets. The monophyly of each species (Oresitrophe rupifraga, M. acanthifolia and M. rossii) and the inferred topology ((M. rossii, M. acanthifolia), O. rupifraga) are well supported in trees reconstructed using the complete plastome sequences, but M. acanthifolia and M. rossii did not form a separate clade in the trees based on ETS + ITS data, while the mitochondrial CDS trees are not well-resolved. We found low recovery of genes in the Angiosperms353 target enrichment panel from our unenriched genome skimming data. Hybridization or incomplete lineage sorting may be the cause of discordance between trees reconstructed from organellar and nuclear data. Considering its morphological distinctiveness and our molecular phylogenetic results, we strongly recommend that M. acanthifolia be treated as a distinct species.     

Spatial sampling to detect slug abundance in an arable field

Overuse of molluscicides by farmers in arable systems can lead to environmental and product contamination. Here we assess a simple and inexpensive surface trapping method for monitoring populations of slugs (Deroceras reticulatum and Arion intermedius). This method was biased against small slugs, and against A. intermedius, when compared to direct soil sampling. Regression was used to model the relationship between the results of surface trapping and soil sampling methods. Spatial Analysis by Distance IndicEs (SADIE) algorithms were used to describe the spatial relationships between the two sets of samples. Using both traditional statistical methods and spatial statistics, the spatial information collected from surface traps was sufficient to identify patches and gaps in slug numbers and possibly to allow the spot application of slug control, and thus provide land managers who experience slug damage with a way of reducing molluscicides use, whilst maintaining slug control. Further improvements and applications of the model are discussed.     

First chemical synthesis of a scorpion alpha-toxin affecting sodium channels: the Aah I toxin of Androctonus australis hector.

Aah I is a 63-residue alpha-toxin isolated from the venom of the Buthidae scorpion Androctonus australis hector, which is considered to be the most dangerous species. We report here the first chemical synthesis of Aah I by the solid-phase method, using a Fmoc strategy. The synthetic toxin I (sAah I) was renatured in DMSO-Tris buffer, purified and subjected to thorough analysis and comparison with the natural toxin. The sAah I showed physico-chemical (CD spectrum, molecular mass, HPLC elution), biochemical (amino-acid composition, sequence), immunochemical and pharmacological properties similar to those of the natural toxin. The synthetic toxin was recognized by a conformation-dependent monoclonal anti-Aah I antibody, with an IC50 value close to that for the natural toxin. Following intracerebroventricular injection, the synthetic and the natural toxins were similarly lethal to mice. In voltage-clamp experiments, Na(v) 1.2 sodium channel inactivation was inhibited by the application of sAah I or of the natural toxin in a similar way. This work describes a simple protocol for the chemical synthesis of a scorpion alpha-toxin, making it possible to produce structural analogues in time.     

Preservation of in situ reef framework in regions of low hurricane frequency: Pleistocene of Curaçao and Bonaire, southern Caribbean

Luxuriant fringing reefs along the southwestern shores of the Caribbean islands of Curaçao and Bonaire (12°N), located outside the most frequent hurricane tracks, are rarely affected by major storms. Consequently, reef growth and long-term preservation are potentially optimal and distinct from reefs experiencing greater hurricane frequency. Hurricane Lenny (November 1999) took an unusual west-to-east track, bisecting the Caribbean Basin north of these islands, but generated heavy waves (3-6  m) that severely damaged reefs along the normally leeward shores. Massive coral colonies >100 years old were toppled, but even at the most severely damaged sites, 82–85% of colonies remained in growth position. Late Pleistocene (125  ka) elevated reefs in the Lower Terrace of Curaçao record even higher proportions of corals in growth position (93%), possibly reflecting a low hurricane frequency during the Pleistocene highstand. In comparison, coeval Pleistocene reefs in regions that today experience a high hurricane frequency (Great Inagua Island and San Salvador, Bahamas) have lower proportions of corals preserved in growth position (79% and 38%, respectively). These results are consistent with the hypothesis that reefs in regions experiencing very low hurricane frequency, like the southern Caribbean, are more likely to be preserved with corals in primary growth position in comparison to regions with higher hurricane frequency.     

Human Epidermal Melanocyte and Keratinocyte Melanocortin Receptors: Visualization by Melanotropic Peptide Conjugated Microspheres (Latex Beads)

The objectives of this research were to determine whether melanocortin receptors are characteristic (constant) membrane markers of human epidermal melanocytes. Methodologies were developed to visualize melanotropin receptors by scanning electron microscopy (SEM). Multiple copies (up to a hundred) of [Nle⁴,D-Phe⁷]α-MSH, a superpotent analog of α-melanocyte stimulating hormone (α-MSH), were conjugated to a macromo-lecular carrier (latex beads: microspheres). Incubation in the presence of the melanotropin-conjugated microspheres resulted in binding of human normal epidermal melanocytes to the beads. Almost every (possibly all) melanocyte possesses melanocortin receptors as visualized by SEM. Specificity of binding of the macromolecular conjugate was demonstrated by several studies: 1) Binding of melanocytes to the microspheres was specific since it could be blocked by prior incubation of the cells in the presence of the unconjugated hormone analog; 2) microspheres lacking bound ligand did not bind to the melanocytes; 3) micro-spheres that were first treated with reducing agents (e.g., dithiothreitol) did not subsequently bind to melanocytes; 4) another peptide hormone ligand (e.g., a substance-P analog) attached to the latex beads failed to bind to the cells; 5) B16/F10 mouse melanoma cells known to express melanocortin receptors bound to the microspheres; and 6) cells of nonmelanocyte origin (e.g., mammary cancer cells, small-cell lung cancer cells, fibroblasts) did not bind to the macromolecular conjugate. One exception was that human epidermal keratinocytes also expressed melanocortin receptors as determined by all the criteria established above for epidermal melanocytes. Thus, cell specific melanocortin receptors appear to be characteristic cell surface markers of epidermal melanocytes and keratinocytes.