Comment on "Global distribution of earthworm diversity".

Phillips et al (Reports, 25 October 2019, p. 480) incorrectly conclude that tropical earthworm communities are less diverse and abundant than temperate communities. This result is an artifact generated by some low-quality datasets, lower sampling intensity in the tropics, different patterns in richness-area relationships, the occurrence of invasive species in managed soils, and a focus on local rather than regional richness.

Effective size of a wild salmonid population is greatly reduced by hatchery supplementation.

Many declining and commercially important populations are supplemented with captive-born individuals that are intentionally released into the wild. These supplementation programs often create large numbers of offspring from relatively few breeding adults, which can have substantial population-level effects. We examined the genetic effects of supplementation on a wild population of steelhead (Oncorhynchus mykiss) from the Hood River, Oregon, by matching 12 run-years of hatchery steelhead back to their broodstock parents. We show that the effective number of breeders producing the hatchery fish (broodstock parents; N(b)) was quite small (harmonic mean N(b)=25 fish per brood-year vs 373 for wild fish), and was exacerbated by a high variance in broodstock reproductive success among individuals within years. The low N(b) caused hatchery fish to have decreased allelic richness, increased average relatedness, more loci in linkage disequilibrium and substantial levels of genetic drift in comparison with their wild-born counterparts. We also documented a substantial Ryman-Laikre effect whereby the additional hatchery fish doubled the total number of adult fish on the spawning grounds each year, but cut the effective population size of the total population (wild and hatchery fish combined) by nearly two-thirds. We further demonstrate that the Ryman-Laikre effect is most severe in this population when (1) >10% of fish allowed onto spawning grounds are from hatcheries and (2) the hatchery fish have high reproductive success in the wild. These results emphasize the trade-offs that arise when supplementation programs attempt to balance disparate goals (increasing production while maintaining genetic diversity and fitness).

Plant preference for ammonium versus nitrate: a neglected determinant of ecosystem functioning?

Although nitrogen (N) availability is a major determinant of ecosystem properties, little is known about the ecological importance of plants' preference for ammonium versus nitrate (ß) for ecosystem functioning and the structure of communities. We modeled this preference for two contrasting ecosystems and showed that ß significantly affects ecosystem properties such as biomass, productivity, and N losses. A particular intermediate value of ß maximizes the primary productivity and minimizes mineral N losses. In addition, contrasting ß values between two plant types allow their coexistence, and the ability of one type to control nitrification modifies the patterns of coexistence with the other. We also show that species replacement dynamics do not lead to the minimization of the total mineral N pool nor the maximization of plant productivity, and consequently do not respect Tilman's R* rule. Our results strongly suggest in the two contrasted ecosystems that ß has important consequences for ecosystem functioning and plant community structure.

Effects of laboratory culture on compatibility between snails and schistosomes.

The genetic control of compatibility between laboratory strains of schistosomes and their snail hosts has been studied intensively since the 1970s. These studies show (1) a bewildering array of genotype-by-genotype interactions - compatibility between one pair of strains rarely predicts compatibility with other strains, and (2) evidence for a variety of (sometimes conflicting) genetic mechanisms. Why do we observe such variable and conflicting results? One possibility is that it is partly an artifact of the use of laboratory strains that have been in culture for many years and are often inbred. Here we show that results of compatibility trials between snails and schistosomes - all derived from the same natural population - depend very much on whether one uses laboratory-cultured or field-collected individuals. Explanations include environmental effects of the lab on either host or parasite, and genetic changes in either host or parasite during laboratory culture. One intriguing possibility is that genetic bottlenecks during laboratory culture cause the random fixation of alleles at highly polymorphic loci that control the matched/mismatched status of hosts and parasites. We show that a simple model of phenotype matching could produce dose response curves that look very similar to empirical observations. Such a model would explain much of the genotype-by-genotype interaction in compatibility observed among strains.

A comparison between mitochondrial DNA and the ribosomal internal transcribed regions in prospecting for cryptic species of platyhelminth parasites.

We examined the relative merits of mitochondrial DNA loci and ribosomal DNA internal transcribed spacers for their use in prospecting for cryptic species of platyhelminth parasites. Sequence divergence at ITS1 and ITS2 was compared with divergence at 2 mtDNA loci (NADH dehydrogenase-1 and cytochrome c oxidase I) between closely related species of trematodes and cestodes. Both spacers accumulated substitutions substantially more slowly than mtDNA, which clearly shows a higher level of divergence among species relative to intra-specific variation. Besides a slow rate of substitution, other caveats that may be encountered when using ITS sequences as a prospecting marker are discussed. In particular, we note recent studies that suggest the existence of substantial levels of intra-individual variation in ITS sequences of flatworms. Because it is likely that closely related species share this phenomenon, it may confound the detection of cryptic species, especially if small sample sizes are studied. Although potential limitations of mtDNA are also recognized, the higher rate of evolution and smaller effective population size of this marker increases the probability of detecting diagnostic characters between cryptic species.

Reduced mammary gland carcinogenesis in transgenic mice expressing a growth hormone antagonist.

Several reports have provided evidence that body size early in life is positively correlated with risk of subsequent breast cancer, but the biological basis for this relationship is unclear. We examined tumour incidence in transgenic mice expressing a growth hormone (GH) antagonist and in non-transgenic littermates following exposure to dimethylbenz[ a ]anthracene (DMBA), a well characterized murine mammary gland carcinogen. The transgenic animals had lower IGF-I levels, were smaller in terms of body size and weight, and exhibited decreased tumour incidence relative to controls. The demonstration that both body size early in life and breast cancer incidence are influenced by experimental perturbation of the GH-IGF-I axis in a transgenic model provides evidence that variability between individuals with respect to these hormones underlies the relationship between body size early in life and breast cancer risk observed in epidemiological studies.

Genetic structure of populations of the human hookworm, Necator americanus, in China.

Twenty-one to 58 individual Necator americanus were sampled from each of four villages in south-western China. Each nematode was sequenced for 588 bp of the mitochondrial cytochrome oxidase I gene. Allelic and nucleotide diversity varied two-fold among villages. Overall FST among populations was approximately 0.28, but this large value resulted from one low-diversity population that had a large genetic distance to the other three populations (F(ST) = 0.10 without that population). There was no correlation between geographical and genetic distance among sites. Thus, the genetic structure of this species in China may be characterized by variable effective sizes and uneven movement among sites. We discuss the implications of this genetic structure for vaccine development and the spread of drug resistance in human hookworms, and compare the genetic structure of hookworms with that of other nematodes.

Identification of symbiotic bacteria (Photorhabdus and Xenorhabdus) from the entomopathogenic nematodes Heterorhabditis marelatus and Steinernema oregonense based on 16S rDNA sequence.

Two species of entomopathogenic nematodes, Heterorhabditis marelatus and Steinernema oregonense, were described recently from the west coast of North America. It is not known whether the bacterial symbionts of these nematodes are also unique. Here we compared partial 16S rRNA sequences from the symbiotic bacteria of these two nematodes with sequence from previously described Photorhabdus and Xenorhabdus species. The 16S sequence from the new Xenorhabdus isolate appears very similar to, although not identical to, that of X. bovienii, the common symbiont of S. feltiae. The new Photorhabdus isolate appears to be very distinct from other known Photorhabdus species, although its closest affinities are with the P. temperata group. We also verified a monoxenic association between each isolate and its nematode by amplifying and sequencing bacterial 16S sequence from crushed adult and juvenile nematodes and from bacterial cultures isolated from infected hosts.

Neutrality tests on mtDNA: unusual results from nematodes.

McDonald-Kreitman tests of neutrality on mitochondrial DNA (mtDNA) of butterflies, Drosophila, and a variety of vertebrates usually show excess (over the neutral expectation) intraspecific polymorphism at nonsilent sites. These results are of great interest because they are the opposite of what is usually found for nuclear genes, in which the neutral pattern or evidence of adaptive divergence between species is usually observed. However, only vertebrates and insects have been tested so far, so it is not clear whether this intriguing pattern is typical for mtDNA in all taxa. Here I tested three pairs of nematode species and found that they all show a deficit of replacement polymorphism. Taken at face value, this result suggests that adaptive evolution proceeds more efficiently in nematode mtDNA than in mtDNA of vertebrates or insects. An alternate explanation is that the nematode pattern is an artifact of silent-site saturation that results from the rapid and composition-biased way in which nematode mtDNA evolves. Further studies are needed to distinguish between these two hypotheses.

Genetic correction of sickle cell disease: insights using transgenic mouse models.

Sickle cell disease is a hereditary disorder characterized by erythrocyte deformity due to hemoglobin polymerization. We assessed in vivo the potential curative threshold of fetal hemoglobin in the SAD transgenic mouse model of sickle cell disease using mating with mice expressing the human fetal Agamma-globin gene. With increasing levels of HbF, AgammaSAD mice showed considerable improvement in all hematologic parameters, morphopathologic features and life span/survival. We established the direct therapeutic effect of fetal hemoglobin on sickle cell disease and demonstrated correction by increasing fetal hemoglobin to about 9-16% in this mouse model. This in vivo study emphasizes the potential of the SAD mouse models for quantitative analysis of gene therapy approaches.

Structure, biodiversity, and historical biogeography of nematode faunas in holarctic ruminants: morphological and molecular diagnoses for Teladorsagia boreoarcticus n. sp. (Nematoda: Ostertagiinae), a dimorphic cryptic species in muskoxen (Ovibos moschatus).

Discovery of the ostertagiine nematode Teladorsagia boreoarcticus n. sp. in muskoxen, Ovibos moschatus, from the central Canadian Arctic highlights the paucity of knowledge about the genealogical and numerical diversity of nematode faunas characteristic of artiodactyls at high latitudes across the Holarctic. Teladorsagia boreoarcticus is a dimorphic cryptic species distinguished from Teladorsagia circumcincta/Teladorsagia trifurcata in domestic sheep by a 13% divergence in the ND4 region of mitochondrial DNA, constant differences in the synlophe, and significantly longer esophageal valve, spicules, gubernaculum, and bursa. Teladorsagia boreoarcticus represents an archaic component of the North American fauna and may have a Holarctic distribution in muskoxen and caribou. Recognition of T. boreoarcticus in muskoxen, in part, corroborates hypotheses for the existence of a cryptic species complex of Teladorsagia spp. among Caprinae and Cervidae at high latitudes and indicates the importance of climatological determinants during the late Tertiary and Pleistocene on diversification of the fauna. Also reinforced is the concept of the North American fauna as a mosaic of endemic and introduced species. Discovery of a previously unrecognized species of Teladorsagia has additional implications and clearly indicates that (1) our knowledge is incomplete relative to potentially pathogenic nematodes that could be exchanged among domestic and wild caprines; (2) we do not have sufficient knowledge of the fauna to understand the ecological control mechanisms (limitations) on dissemination and host range; and (3) an understanding of historical and geographical influences on the genealogical diversity and distribution of nematode faunas in domestic and wild ruminants is requisite to define the interface between agricultural and natural ecosystems across the Holarctic.

Life cycle variation and the genetic structure of nematode populations.

Few data are available on population genetic structure in nematode species, and little of the available data allows direct comparison of the genetic structures of species having different life cycles. Here we use mtDNA sequence data to describe the genetic structure of a heterorhabditid nematode, and compare results to published data on other nematode species. Heterorhabditis marelatus is a parasite of soil-dwelling insects. Its life cycle and local ecology should result in small effective population sizes and restricted gene flow. As predicted, H. marelatus shows much lower mtDNA diversity within populations and over the species as a whole, and has a much more strongly subdivided population structure, than parasites of mobile vertebrate hosts. From data such as these we can begin to generalize about the effects of life cycle variation on genetic structure in different nematode species.

Altered hematopoiesis in murine sickle cell disease.

We investigated the mechanisms of sickle cell disease (SCD) hematopoietic/erythropoietic defects using bone marrow, spleen, and/or peripheral blood from the transgenic SAD mouse model, which closely reproduces the biochemical and physiological disorders observed in human SCD. First, the erythropoietic lineage late precursors (polychromatophilic normoblasts to the intramedullary reticulocytes) of SAD mouse bone marrow were significantly altered morphologically. These anomalies resulted from high levels of hemoglobin polymers and were associated with increased cell fragmentation occurring during medullary endothelial migration of reticulocytes. Secondly, analysis of bone marrow erythropoiesis in earlier stages showed a marked depletion in SAD erythroid burst-forming units (BFU-E; of approximately 42%) and erythroid colony-forming units (CFU-E; of approximately 23%) progenitors, despite a significant increase in their proliferation, suggesting a compensatory mechanism. In contrast to the bone marrow progenitor depletion, we observed (1) a high mobilization/relocation of BFU-E early progenitors (approximately 4-fold increase) in peripheral blood of SAD mice as well as of colony-forming units-granulocyte-macrophage (CFU-GM) and (2) a 7-fold increase of SAD CFU-E in the spleen. Third, and most importantly, SAD bone marrow multipotent cells (spleen colony-forming units [CFU-S], granulocyte-erythroid-macrophage-megakaryocyte colony-forming units [CFU-GEMM], and Sca(+)Lin(-)) were highly mobilized to the peripheral blood (approximately 4-fold increase), suggesting that peripheral multipotent cells could serve as proliferative and autologous vehicles for gene therapy. Therefore, we conclude the following. (1) The abnormal differentiation and morphology of late nucleated erythroid precursors result in an ineffective sickle erythropoiesis and likely contribute to the pathophysiology of sickle cell disorders; this suggests that transfer of normal or modified SCD bone marrow cells may have a selective advantage in vivo. (2) A hematopoietic compensatory mechanism exists in SAD/SCD pathology and consists of mobilization of multipotent cells from the bone marrow to the peripheral blood and their subsequent uptake into the spleen, an extramedullary hematopoietic site for immediate differentiation. Altogether, these results corroborate the strong potential effectiveness of both autologous and allogeneic bone marrow transplantation for SCD hematopoietic therapy.

Molecular differentiation and phylogeny of entomopathogenic nematodes (rhabditida: heterorhabditidae) based on ND4 gene sequences of Mitochondrial DNA.

We determined partial ND4 gene sequences of mitochondrial DNA from 15 heterorhabditid nematode isolates, representing 5 species collected from different regions of the world, by using polymerase chain reaction (PCR) and direct-sequencing of PCR products. Aligned nucleotide as well as amino acid sequences were used to differentiate nematode species by comparing sequence divergence and to infer phylogeny of the nematodes by using maximum parsimony and likelihood methods. Robustness of our phylogenetic trees was checked by bootstrap tests. The 15 nematode isolates can be divided into 7 haplotypes based on DNA sequences. On a larger scale, the sequence divergence revealed 4 distinct groups corresponding to 4 described species. No sequence divergence was detected from 5 isolates of Heterorhabditis bacteriophora or between Heterorhabditis marelatus to Heterorhabditis hepialius. Our sequence data yielded phylogenetic trees with identical topologies when different tree-building methods were used. Most relationships were also confirmed by using amino acid sequences in maximum parsimony analysis. Our molecular phylogeny of Heterorhabditis species support an existing taxonomy that is based largely on morphology and the sequence divergence of the ND4 gene permits species identification.

Substitution bias, rapid saturation, and the use of mtDNA for nematode systematics.

Only relatively recently have researchers turned to molecular methods for nematode phylogeny reconstruction. Thus, we lack the extensive literature on evolutionary patterns and phylogenetic usefulness of different DNA regions for nematodes that exists for other taxa. Here, we examine the usefulness of mtDNA for nematode phylogeny reconstruction and provide data that can be used for a priori character weighting or for parameter specification in models of sequence evolution. We estimated the substitution pattern for the mitochondrial ND4 gene from intraspecific comparisons in four species of parasitic nematodes from the family Trichostrongylidae (38-50 sequences per species). The resulting pattern suggests a strong mutational bias toward A and T, and a lower transition/transversion ratio than is typically observed in other taxa. We also present information on the relative rates of substitution at first, second, and third codon positions and on relative rates of saturation of different types of substitutions in comparisons ranging from intraspecific to interordinal. Silent sites saturate extremely quickly, presumably owing to the substitution bias and, perhaps, to an accelerated mutation rate. Results emphasize the importance of using only the most closely related sequences in order to infer patterns of substitution accurately for nematodes or for other taxa having strongly composition-biased DNA. ND4 also shows high amino acid polymorphism at both the intra- and interspecific levels, and in higher level comparisons, there is evidence of saturation at variable amino acid sites. In general, we recommend using mtDNA coding genes only for phylogenetics of relatively closely related nematode species and, even then, using only nonsynonymous substitutions and the more conserved mitochondrial genes (e.g., cytochrome oxidases). On the other hand, the high substitution rate in genes such as ND4 should make them excellent for population genetics studies, identifying cryptic species, and resolving relationships among closely related congeners when other markers show insufficient variation.

Mitochondrial DNA diversity in nematodes.

The relatively small literature on mitochondrial DNA (mtDNA) diversity in nematode species is summarized here. Nematodes show a wide range of overall genetic diversities and population genetic structures. Species-wide levels of diversity correlate strongly with the breeding system and other life cycle features that control effective population size. Obligate outcrossers that parasitize mobile vertebrate hosts are the most diverse, species having hermaphroditic stages are less so, and species having asexual reproductive stages appear even less diverse. Nevertheless, these conclusions are preliminary because there exist so few data on DNA diversity in nematodes. What is needed are more comparative studies using similar sampling designs and the same DNA markers, including nuclear loci and further work with mtDNA.

Quinolines as potent 5-lipoxygenase inhibitors: synthesis and biological profile of L-746,530.

Leukotriene biosynthesis inhibitors have potential as new therapeutic agents for asthma and inflammatory diseases. A series of novel substituted 2-cyanoquinolines have been synthesized and the structure activity relationships were evaluated with respect to their ability to inhibit the formation of leukotrienes via the 5-lipoxygenase enzyme. [1S,5R]-2-Cyano-4-(3-furyl)-7-¿3-fluoro-5-[3-(3 alpha-hydroxy-6,8-dioxabicyclo[3.2.1]-octanyl)]phenoxymethyl ¿quinoline (L-746,530) 3 represents a distinct class of inhibitors and possesses in vitro and in vivo potency comparable or superior to naphthalenic analog (L-739,010) 2.