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Head smut of maize, caused by the fungus Sphacelotheca reiliana, is an economically important disease in all major maize-producing countries. Although seed treatments are commonly used for management purposes, evaluating these treatments for efficacy is both time consuming and inefficient. Therefore, in order to improve the capacity for evaluating seed treatment fungicides, we developed a real-time PCR-based seedling assay for S. reiliana infection. We optimized growth chamber conditions and inoculation methods to achieve infection incidence of 60 to 80% in inoculated, nontreated controls. The effects of five commercially available fungicidal seed treatment formulations on seedling infection incidence were compared by PCR analysis of root and mesocotyl tissues. Tebuconazole, fludioxonil, sedaxane, and Maxim Quattro (fludioxonil+mefenoxam+azoxystrobin+thiabendazole) reduced the incidence of infection (P < 0.05) compared with the control, but no difference was found between the azoxystrobin treatment and the control. All rates tested for both sedaxane and tebuconazole were equally effective for seeds coated with 106 teliospores∙seed–1. Sedaxane, at a rate of 0.1 mg/kernel, eliminated seedling infection if seeds were infested with a low inoculum concentration (101 teliospores∙seed–1). The assay developed here is a valuable tool not only for the detection of fungal infection at the seedling stage, but also for testing the relative efficacies of seed treatments for reducing incidence of infection.

Seed transmission of Zucchini yellow mosaic virus (ZYMV) has been demonstrated in a variety of cucurbits, including wild Cucurbita species, and pollen transmission of viruses is common in the family Potyviridae. Consequently, vertical transmission of ZYMV may occur via the pollen of infected wild squash as opposed to ovule transmission. To determine if vertical infection of ZYMV occurs via pollen transmission, we pollinated flowers on virus-free Cucurbita pepo ssp. texana plants with pollen from ZYMV infected plants. We found that seed infection of ZYMV via pollen occurs at a rate of 0.13 %. This rate is much lower than expected based on previously reported rates of seed transmission, which suggests that while seed transmission via pollen does occur in C. pepo, it is not the main mechanism for vertical infection. Because seed transmission of ZYMV has been shown in virus-resistant transgenic plants, it is possible that ZYMV infection of transgenic plants via pollen may contribute to the seed transmission and geographic distribution of this virus.

A real-time PCR assay was used to compare seedling infection by Sphacelotheca reiliana, the causal agent of head smut, among five inbred genotypes representing low, moderate, and high susceptibility to the disease. Seeds were coated with teliospores and planted in autoclaved field soil in a growth chamber. Incidence of seedling infection at growth stage V3 differed between an inbred genotype of low susceptibility and those of moderate and high susceptibility, but did not differ between the high and moderately susceptible groups (P < 0.05). The real-time PCR assay was also used to compare infection status at early and late vegetative stages with observable symptoms in the field. We detected infection via real-time PCR in maize at both growth stages during field trials conducted in Texas and California but observed no disease symptoms (smutted ears or tassels). Notably, the fungus was present in up to 31% of the ear shoots in plots without disease symptoms. The real-time assay can be a useful tool for screening seedling-stage host resistance, and for better understanding the progress of infection in different maize genotypes. The field data suggest that asymptomatic infection is much more common than previously thought, and may have important implications for the epidemiology of this fungus under diverse plant resistance and growing conditions. Accepted for publication 11 December 2015. Published 5 January 2016.

Zucchini yellow mosaic virus (ZYMV) is an economically important pathogen of cucurbits that is transmitted both horizontally and vertically. Although ZYMV is seed-transmitted in Cucurbita pepo, the potential for seed transmission in virus-resistant transgenic cultivars is not known. We crossed and backcrossed a transgenic squash cultivar with wild C. pepo, and determined whether seed-to-seedling transmission of ZYMV was possible in seeds harvested from transgenic backcrossed C. pepo. We then compared these transmission rates to those of non-transgenic (backcrossed and wild) C. pepo. The overall seed-to-seedling transmission rate in ZYMV was similar to those found in previous studies (1.37%), with no significant difference between transgenic backcrossed (2.48%) and non-transgenic (1.03%) backcrossed and wild squash. Fewer transgenic backcrossed plants had symptom development (7%) in comparison with all non-transgenic plants (26%) and may be instrumental in preventing yield reduction due to ZYMV. Our study shows that ZYMV is seed transmitted in transgenic backcrossed squash, which may affect the spread of ZYMV via the movement of ZYMV-infected seeds. Deep genome sequencing of the seed-transmitted viral populations revealed that 23% of the variants found in this study were present in other vertically transmitted ZYMV populations, suggesting that these variants may be necessary for seed transmission or are distributed geographically via seeds.

Determining the extent and structure of intra-host genetic diversity and the magnitude and impact of population bottlenecks is central to understanding the mechanisms of viral evolution. To determine the nature of viral evolution following systemic movement through a plant, we performed deep sequencing of 23 leaves that grew sequentially along a single Cucurbita pepo vine that was infected with zucchini yellow mosaic virus (ZYMV), and on a leaf that grew in on a side branch. Strikingly, of 112 genetic (i.e. sub-consensus) variants observed in the data set as a whole, only 22 were found in multiple leaves. Similarly, only three of the 13 variants present in the inoculating population were found in the subsequent leaves on the vine. Hence, it appears that systemic movement is characterized by sequential population bottlenecks, although not sufficient to reduce the population to a single virion as multiple variants were consistently transmitted between leaves. In addition, the number of variants within a leaf increases as a function of distance from the inoculated (source) leaf, suggesting that the circulating sap may serve as a continual source of virus. Notably, multiple mutational variants were observed in the cylindrical inclusion (CI) protein (known to be involved in both cell-to-cell and systemic movement of the virus) that were present in multiple (19/24) leaf samples. These mutations resulted in a conformational change, suggesting that they might confer a selective advantage in systemic movement within the vine. Overall, these data reveal that bottlenecks occur during systemic movement, that variants circulate in the phloem sap throughout the infection process, and that important conformational changes in CI protein may arise during individual infections.

Fusarium oxysporum f. sp. phaseoli (Fop) is a devastating pathogen that can cause significant economic losses and can be introduced into fields through infested Phaseolus vulgaris (common bean) seeds. Efficient seed health testing methods can aid in preventing long-distance dissemination of this pathogen by contaminated seeds. In order to improve detection of Fop in seed, a rapid, accurate and sensitive real-time PCR assay (qPCR) protocol was developed for detection of Fop in common bean seeds. Seed lots of seven cultivars with infection incidence ranging from 0·25 to 20% were prepared by mixing known amounts of Fop-infected seeds with Fop-free seeds. Direct comparisons between SYBR Green and TaqMan qPCR methods were performed using primers based on the Fop virulence factor ftf1. The primers developed in this study produced a 63 bp product for highly virulent strains of Fop but did not produce an amplicon for nonpathogenic or weakly pathogenic isolates of F. oxysporum from P. vulgaris or other hosts. Under optimized conditions, both qPCR assays detected Fop infection at low levels (0·25%); however, the results suggest the TaqMan assay was more reliable at quantification than the SYBR Green assay. Linear regression models were fitted to the relationships between results of qPCR assays and infection incidence, but the models differed among cultivars. Fungal biomass per seed differed among cultivars and was related to seed size. The results indicate that the TaqMan assay developed in this study is a useful tool for the detection and quantification of Fop in bean seeds.

Sea urchins are important ecosystem engineers in subtidal ecosystems worldwide, providing biogenic structure and altering nutrient dynamics through intensive grazing and drift algal capture. The current work evaluates red urchin (Strongylocentrotus franciscanus) density on fixed transects through time, individual displacement, and urchin-associated benthic community composition using a field-based approach at multiple depths (in and outside of the macroalgal zone) and replicated across sites in the San Juan Archipelago, Washington. Urchins exhibited no large-scale, temporal or directional changes in density among depths. Furthermore, 87% of individual urchins observed in repeated small-scale surveys over 3 weeks exhibited no change in position. Individual displacement was negatively correlated to drift algal capture. Evidence of sedentary behavior from the displacement surveys was supported by the sessile and mobile community composition in areas directly under versus adjacent to (control) urchins. The benthos under urchins had a higher percentage of bare space, crustose coralline algae, and increased density of snails, crabs and shrimp relative to associated control plots. Abundance of mobile organisms associating with urchins increased relative to control plots at the deepest survey depth (30 m), indicating a greater strength of interaction with distance from macroalgal production. This work presents evidence of food availability-related behavior in red urchins and indicates that even when sedentary, urchins have a strong influence on ecosystem structure through increasing availability of shelter and macroalgal detritus to the benthos.

The role played by seed transmission in the evolution and epidemiology of viral crop pathogens remains unclear. We determined the seed infection and vertical transmission rates of zucchini yellow mosaic virus (ZYMV), in addition to undertaking Illumina sequencing of nine vertically transmitted ZYMV populations. We previously determined the seed-to-seedling transmission rate of ZYMV in Cucurbita pepo ssp. texana (a wild gourd) to be 1.6%, and herein observed a similar rate (1.8%) in the subsequent generation. We also observed that the seed infection rate is substantially higher (21.9%) than the seed-to-seedling transmission rate, suggesting that a major population bottleneck occurs during seed germination and seedling growth. In contrast, that two thirds of the variants present in the horizontally transmitted inoculant population were also present in the vertically transmitted populations implies that the bottleneck at vertical transmission may not be particularly severe. Strikingly, all of the vertically infected plants were symptomless in contrast to those infected horizontally, suggesting that vertical infection may be cryptic. Although no known virulence determining mutations were observed in the vertically infected samples, the 5’ untranslated region was highly variable, with at least 26 different major haplotypes in this region compared to the two major haplotypes observed in the horizontally transmitted population. That the regions necessary for vector transmission are retained in the vertically infected populations, combined with the cryptic nature of vertical infection, suggests that seed transmission may be a significant contributor to the spread of ZYMV.

Primary productivity by plants and algae is the fundamental source of energy in virtually all food webs. Furthermore, photosynthetic organisms are the sole source for ω-3 and ω-6 essential fatty acids (EFA) to upper trophic levels. Because animals cannot synthesize EFA, these molecules may be useful as trophic markers for tracking sources of primary production through food webs if different primary producer groups have different EFA signatures. We tested the hypothesis that different marine macrophyte groups have distinct fatty acid (FA) signatures by conducting a phylogenetic survey of 40 marine macrophytes (seaweeds and seagrasses) representing 36 families, 21 orders, and four phyla in the San Juan Archipelago, WA, USA. We used multivariate statistics to show that FA composition differed significantly (P

Within two decades of its discovery, Zucchini yellow mosaic virus (ZYMV) achieved a global distribution. However, whether or not seed transmission occurs in this economically significant crop pathogen is controversial, and the relative impact of seed transmission on the epidemiology of ZYMV remains unclear. Using reverse transcription–polymerase chain reaction, we observed a seed transmission rate of 1.6% in Cucurbita pepo subsp. texana and show that seed-infected C. pepo plants are capable of initiating horizontal ZYMV infections, both mechanically and via an aphid vector (Myzus persicae). We also provide evidence that ZYMV-infected seeds may act as effective viral reservoirs, partially accounting for the current geographic distribution of ZYMV. Finally, the observation that ZYMV infection of C. pepo seeds results in virtually symptomless infection, coupled with our finding that an antibody test failed to detect vertically transmitted ZYMV in infected seed, highlights the urgent need to standardize current detection methods for seed infection.

The role of native consumers in mediating biological invasions is poorly understood. In theory, there are reasons to expect both strong and weak effects of native consumers on non-native species. However, non-native ranges may include multiple regions or even continents, each with its own suite of consumers and invader–consumer interactions may play out differently in different places and times. In this Washington State (USA) study we found that the common herbivorous snail Lacuna vincta was 2–9 times more abundant on the non-native seaweed Sargassum muticum, compared to native kelps. Choice feeding trials with fresh tissue and artificial foods both suggest that S. muticum is a preferred food for Lacuna vincta. Lab experiments indicated that L. vincta did not experience diminished predation by two common predators on Sargassum muticum compared to native kelp hosts. Our results suggest that Sargassum experiences considerable herbivory by Lacuna vincta in our study region, a conclusion that is consistent with previous work and our own field observations. In our system, L. vincta and S. muticum have been coexisting in the same habitats for at least 50 years and available data suggest that it acquired a preference for S. muticum more than 30 years after the initial invasion. Comparison of our results to recent work on Sargassum–herbivore interactions in Europe suggests that the response of native consumer communities to S. muticum varies both within and among regions. Geographic and temporal variation in the response of native consumers are likely to be hallmarks of many large-scale invasions.

In the Northeast Pacific the nonnative seagrass Zostera japonica frequently exists at the same sites as the native seagrass Zostera marina. Although at some sites their vertical distributions overlap, at most sites in the Pacific Northwest there is a distinctive unvegetated zone between them. The objective of this study was to better understand why a gap between the lower limit of Z. japonica and the upper limit of Z. marina exists. To address this issue we carried out transplant experiments, conducted in situ monitoring of existing Z. japonica patches, and collected sediment samples at South Beach on Shaw Island, Washington, during the spring and summer of 2006. Transplant and in situ monitoring data indicate that survival and performance of Z. japonica are reduced lower in the intertidal zone. In addition, Z. japonica patches tended to be smaller and more spaced out at lower tidal heights. Although we found no Z. japonica seeds within or outside extant Z. japonica patches, high transplant mortality indicates that Z. japonica dispersal limitation is an unlikely cause of the unvegetated gap zone. Our field observations further suggest that herbivory, bioturbation, and epiphytes are unlikely causes of the gap pattern at our study site. Instead, we hypothesize that light limitation prevents Z. japonica from occurring lower in the intertidal. A review of published vertical distribution data for both Zostera species indicates that the lower limit of Z. japonica is relatively invariant among sites. In contrast, the upper limit of Z. marina is highly variable, ranging by more than 4 m within some subregions in Washington State. Consequently we hypothesize that intersite variability in the vertical distribution of Z. marina is the primary driver of spatial variability in the presence of the unvegetated gap.

Spatial subsidies link adjacent habitats and can have strong effects on recipient systems. Within temperate nearshore marine ecosystems, algae dominate rocky benthic communities in the shallow subtidal but are supplanted by invertebrates at deeper depths where light becomes limiting. Detrital macrophytes transported into deep subtidal habitats by hydrodynamic forces may subsidize deep organisms, but little is known about the availability or utilization of this organic mate- rial. We used descriptive field studies to study drift macrophyte utilization by the red urchin Strongy- locentrotus franciscanus, a commercially harvested species found in both shallow and deep habitats in Washington, USA. Our primary goals were to evaluate urchin diet, urchin fecundity, and drift macrophyte abundance in the deep subtidal zone (>20 m depth). Although seasonally variable, macrophyte detritus was present year-round in the deep subtidal (23 m depth) and diet data suggest it is the primary food resource for deep subtidal red urchins. Perennial kelps were the main contrib- utors to the detrital pool (54% of biomass), particularly the shallow subtidal species Saccharina sub- simplex (37% of biomass). At 4 of our 5 study sites, red urchins from deep habitats had gonad indices as large as sea urchins from shallow habitats, suggesting that drift algae are an abundant food resource in the deep subtidal zone of our study region. A comparison of our results with previous studies indicates that the availability of macroalgal detritus in the deep subtidal varies considerably in space and may be influenced by the hydrodynamic context. We suggest that successful manage- ment of subsidized species may require understanding subsidy sources and fluxes because changes in the production or delivery of organic material can have important consequences for recipient populations.

Aerial or overhead photography is a technique commonly used to measure spatiotemporal variability in surface canopy kelps. To evaluate potential biases in this technique, we used elevated (shore-based) oblique angle photography coupled with in situ measurements of currents and tides to sample 4 independent Nereocystis luetkeana beds at sites adjacent to San Juan Island (Washington State, USA) in 2004 and 2005. Data were collected at high frequency (every 10 min) to quantify the effects of tidal height and tidal currents on the amount of N. luetkeana visible at the surface. Effects of tidal height were highly significant in all 6 time series. Current significantly affected apparent bed size in 5 of the 6 time series, although the magnitude of the effect varied among sites. N. luetkeana beds decrease in apparent size as current velocity increases and as tidal level increases. Currents can fluctuate rapidly, changing by many 10s of cm s–1 over periods as short as 10 min, due in large part to interactions of tides with local topography and bathymetry close to shore, where kelp beds are typically found. As a result, currents can introduce significant variability to estimates of kelp population size collected by remote sensing techniques.

Summary 1. Invading species typically need to overcome multiple limiting factors simultaneously in order to become established, and understanding how such factors interact to regulate the invasion process remains a major challenge in ecology. 2. We used the invasion of marine algal communities by the seaweed Sargassum muticum as a study system to experimentally investigate the independent and interactive effects of disturbance and propagule pressure in the short term. Based on our experimental results, we parameterized an integrodifference equation model, which we used to examine how disturbances created by different benthic herbivores influence the longer term invasion success of S. muticum . 3. Our experimental results demonstrate that in this system neither disturbance nor propagule input alone was sufficient to maximize invasion success. Rather, the interaction between these processes was critical for understanding how the S. muticum invasion is regulated in the short term. 4. The model showed that both the size and spatial arrangement of herbivore disturbances had a major impact on how disturbance facilitated the invasion, by jointly determining how much space-limitation was alleviated and how readily disturbed areas could be reached by dispersing propagules. 5. Synthesis. Both the short-term experiment and the long-term model show that S. muticum invasion success is co-regulated by disturbance and propagule pressure. Our results underscore the importance of considering interactive effects when making predictions about invasion success.

Potato spindle tuber viroid (PSTVd) is an emerging pathogen that primarily affects members of the Solanaceae. The distribution of this pathogen is expanding rapidly with several countries reporting new infections in recent years. This pathogen is of phytosanitary concern because infected seed is thought to be one of the factors contributing to the spread of this disease. Thus we developed a qRT-PCR method to determine the seed infection frequency of PSTVd in tomato, as well as a RT-PCR method to determine the frequency of transmission of this pathogen to tomato seedlings. Infection frequency of PSTVd in tomato seeds from infected plants (cv. ‘Beefsteak’) ranged from 62.3-69% and frequency of PSTVd transmission to tomato seedlings was 50.9%. Germination of the infected seed was significantly lower (53%) than the parent seed (98%), and we also determined that viroid titers in the infected seeds were significantly lower (average 173 ng/μl) than in the infected seedlings (785 ng/μl). Interestingly ~60% of the infected seedlings did not display any observable symptoms of PSTVd until the second or third week; however, plants that were grown to maturity developed severe symptoms, suggesting that a grow-out assay might not be an accurate method for detection of this pathogen.

Although many studies have investigated how community characteristics such as diversity and disturbance relate to invasibility, the mechanisms underlying biotic resistance to introduced species are not well understood. I manipulated the functional group composition of native algal communities and invaded them with the introduced, Japanese seaweed Sargassum muticum to understand how individual functional groups contributed to overall invasion resistance. The results suggested that space preemption by crustose and turfy algae inhibited S. muticum recruitment and that light preemption by canopy and understory algae reduced S. muticum survivorship. However, other mechanisms I did not investigate could have contributed to these two results. In this marine community the sequential preemption of key resources by different functional groups in different stages of the invasion generated resistance to invasion by S. muticum. Rather than acting collectively on a single resource the functional groups in this system were important for preempting either space or light, but not both resources. My experiment has important implications for diversity-invasibility studies, which typically look for an effect of diversity on individual resources. Overall invasion resistance will be due to the additive effects of individual functional groups (or species) summed over an invader's life cycle. Therefore, the cumulative effect of multiple functional groups (or species) acting on multiple resources is an alternative mechanism that could generate negative relationships between diversity and invasibility in a variety of biological systems.

Introduced algae have become a prominent component of the marine flora in many regions worldwide. In the NE Pacific, the introduced Japanese alga Sargassum muticum (Yendo) Fensholt is common and abundant in shallow, subtidal, rocky habitats, but its effects on subtidal, ben- thic communities in this region have not previously been studied. I measured the response of native species to experimental manipulation of S. muticum in field experiments in the San Juan Islands of Washington State. Native canopy (brown) and understory (red) algae were more abundant in plots from which S. muticum had been removed, and the native kelp Laminaria bongardiana (the most abundant species of brown alga in the absence of S. muticum) grew more than twice as fast in plots where S. muticum was absent. The negative effects of S. muticum on native algae appear to be a re- sult of shading, rather than changes in water flow, sedimentation, or nutrient availability. S. muticum also had a strongly negative indirect effect on the native sea urchin Stronglyocentrotus droebachi- ensis by reducing abundances of the native kelp species on which it prefers to feed. My results indicate that S. muticum has a substantial impact on native communities in this region, including effects at multiple trophic levels. Because of their worldwide distribution and capacity to alter native communities, non-indigenous algae are potentially important agents of global ecological change.

Viral pathogens comprise approximately half of the emerging diseases in plants, and plant introductions (including the international movement of seed) are considered to be one of the most important contributing factors to the emergence of these pathogens. For the most part plant viruses are incapable of surviving outside of host tissue making their long-term propagation dependent on their hosts. Thus infected seeds are an effective strategy that not only allows for pathogen survival from one season to the next, but also for their dispersal. The Potyviridae, as the largest plant virus family, is often considered to be the most economically important and its members rank among the most successful plant pathogens. Seed transmission within the Potyviridae family is not uncommon, however the exact mechanism of viral entry into the germ line is currently unknown, and the genetic basis of seed transmission has yet to be completely elucidated. Seed transmission rates are influenced by complex interactions among a variety of factors including the host cultivar, the virus isolate, environmental conditions, the timing of infection, vector characteristics, and viral synergism. Seed transmission can have an enormous effect on the epidemiology of crop pathogens due in part to the ecology of plant viruses which are often secondarily disseminated via insect vectors with the effect that extremely low frequencies of seed transmission can result in devastating epidemics. This is compounded by the fact that vertically infected seedlings often do not exhibit symptoms of viral infection. Given the potential for seed transmitted viral pathogens to initiate epidemics, it is vital to understand how seed transmission rates translate into epidemics. In addition, as seed transmission is a means of dispersal for these viral pathogens, effective phytosanitary measures to control the spread of these pathogens are crucial.

To determine the primary excretory by-products of testosterone (T), 85 microCi [3H]T was administered i.v. to two adult Eld's deer stags. Blood (10 ml) was collected by jugular venipuncture at 0, 5, 10, 15, 30, 45, 60, 90, 120, 150, 180, 240, and 480 min after isotope infusion, and all urine and feces were collected for 96 h after injection. Seventy percent of labeled circulating steroid was conjugated by 30 min postinfusion. The majority (80.4 +/- 3.2%) of T metabolites were excreted into urine, and 95.0 +/- 0.9% of these were conjugated, 95.8 +/- 0.2% being hydrolyzable with glucuronidase. Seven urinary androgen metabolites, including androstanediol (5 alpha-androstan-3 alpha-17 beta-diol and 5 beta-androstan-3 alpha-17 beta-diol), were identified in glucoronidase-hydrolyzed, ether-extracted Eld's deer urine pools after gas chromatography/mass spectrometry. A double-antibody 125I RIA for 5 alpha-androstanediol-3 alpha, 17 beta-diol,17-glucuronide (3 alpha-diol-G) was validated for unprocessed urine. Longitudinal assessments of urine samples collected from 13 stages for 3 yr revealed biological concordance between fluctuations in urinary 3 alpha-diol-G and serum T, as well as seasonal changes in secondary sexual characteristics. Overall correlation between "same-day" matched serum T and urinary 3 alpha-diol-G was 0.58, (n = 6; p < 0.001). Thus, monitoring urinary 3 alpha-diol-G provides a noninvasive, alternative method for characterizing male endocrine interrelationships in an endangered ungulate species.

The genetic diversity present in populations of RNA viruses is likely to be strongly modulated by aspects of their life history, including mode of transmission. However, how transmission mode shapes patterns of intra- and inter-host genetic diversity, particularly when acting in combination with de novo mutation, population bottlenecks and the selection of advantageous mutations, is poorly understood. To address these issues, this study performed ultradeep sequencing of zucchini yellow mosaic virus in a wild gourd, Cucurbita pepo ssp. texana, under two infection conditions: aphid vectored and mechanically inoculated, achieving a mean coverage of approximately 10 000×. It was shown that mutations persisted during inter-host transmission events in both the aphid vectored and mechanically inoculated populations, suggesting that the vector-imposed transmission bottleneck is not as extreme as previously supposed. Similarly, mutations were found to persist within individual hosts, arguing against strong systemic bottlenecks. Strikingly, mutations were seen to go to fixation in the aphid-vectored plants, suggestive of a major fitness advantage, but remained at low frequency in the mechanically inoculated plants. Overall, this study highlights the utility of ultradeep sequencing in providing high-resolution data capable of revealing the nature of virus evolution, particularly as the full spectrum of genetic diversity within a population may not be uncovered without sequence coverage of at least 2500-fold.

SUMMARY This work was motivated by subtidal observations of red urchins (Strongylocentrotus franciscanus) moving their spines into streamlined positions as water current increased in the field. Trials in a flume across flow speeds from 0 to 65 cm s–1 enabled us to observe the change in overall shape of the urchins and quantify the decrease in spine angle that occurred as flow speeds increased. The effect of this behaviour on drag and lift was measured with physical models made from urchin tests with spines in the `up' position (typical in stagnant and slow velocities) and in the `down' position (typical of posture in high velocities). Streamlining spines decreased the drag, but increased the lift experienced by urchin models at flow speeds between 10 and 40 cm s–1, current velocities that are commonly encountered by these animals in the field in Washington, USA. Total force (combination of drag and lift) was similar for `up' and `down' models at all flow speeds, lift comprising the majority of the force for `down' models, and drag slightly higher for `up' models. Live urchins in the field routinely adopt a streamlined `down' posture in flow, suggesting that they may be better able to cope with lift than drag. This behaviour, although affecting hydrodynamic forces and enabling S. franciscanus to remain attached to the substratum in high currents, may lead to reduced capture of drift kelp, which is entrapped on upright spines and then eaten, delivery of which is positively related to flow speed. Urchins living in deep subtidal habitats rely on drift kelp capture but must stay attached to the substratum to be successful in a habitat. Therefore, this streamlining behaviour may be an important factor enabling S. franciscanus to persist in deep, high-current areas.

Genetic diversity in RNA viruses is shaped by a variety of evolutionary processes, including the bottlenecks that may occur at inter-host transmission. However, how these processes structure genetic variation at the scale of individual hosts is only partly understood. We obtained intra-host sequence data for the coat protein (CP) gene of Zucchini yellow mosaic virus (ZYMV) from two horizontally transmitted populations – one via aphid, the other without – and with multiple samples from individual plants. We show that although mutations are generated relatively frequently within infected plants, attaining similar levels of genetic diversity to that seen in some animal RNA viruses (mean intra-sample diversity of 0.02%), most mutations are likely to be transient, deleterious, and purged rapidly. We also observed more population structure in the aphid transmitted viral population, including the same mutations in multiple clones, the presence of a sub-lineage, and evidence for the short-term complementation of defective genomes.

Zucchini yellow mosaic virus (ZYMV) is an economically important virus of cucurbit crops. However, little is known about the rate at which this virus has evolved within members of the familyCucurbitaceae, or the timescale of its epidemiological history. Herein, we present the first analysis of the evolutionary dynamics of ZYMV. Using a Bayesian coalescent approach we show that the coat protein of ZYMV has evolved at a mean rate of 5.0×10−4nucleotide substitutions per site, per year. Notably, this rate is equivalent to those observed in animal RNA viruses. Using the same approach we show that the lineages of ZYMV sampled here have an ancestry that dates back no more than 800 years, suggesting that human activities have played a central role in the dispersal of ZYMV. Finally, an analysis of phylogeographical structure provides strong evidence for thein situevolution of ZYMV within individual countries.

This object of this review is to consider whether mycoplasmas, particularly those that are important pathogens in cattle, sheep, goats and pigs, are likely to be transmitted by semen and embryo transfer techniques, and if so, what can be done to prevent such transmission. The first part gives a brief overview of semen and embryo transfer techniques, focusing on general risks of disease transmission and on routine disease control strategies. Subsequent parts provide information about mycoplasmas, including their classification, pathogenesis, diagnosis and control. Details are then given of the specific mycoplasmas of cattle, sheep, goats and pigs, and the seriously pathogenic species are distinguished from the opportunist pathogens and harmless commensals. It is emphasized, however, that within species the virulence of different strains can vary widely. An important feature of the mycoplasmas is that they often reside in the reproductive tracts of their hosts and therefore tend to pose a higher risk of being transmitted by semen and embryos than do many other micro-organisms. They also tend to adhere to cells and tissues, including semen and embryos, and can be difficult to remove. Some mycoplasmas can also invade and reside within non-phagocytic cells. Their resistance to routine disease control procedures such as semen and embryo washing, and to the inclusion of antibiotics in semen diluents and in embryo media, adds further challenges. It remains true, nevertheless, that semen and embryo transfer techniques offer great advantages over the movement of live animals for transferring genetic material, and will minimize the risks of disease transmission between different farms or countries. The review concludes by recommending control strategies, uniquely rigorous ones in this case, to avoid the transfer of mycoplasmas via semen and embryos.

The t(7;12)(q36;p13) is a recurrent abnormality in acute myeloid leukemia (AML) of childhood. The involved gene on chromosome 12 is TEL; the 7q36 partner gene has not been identified. We describe morphologic, molecular and cytogenetic characterization of two cases of 7q36/12p13-associated AML that provide important insights regarding the consequences of this rearrangement. First, the molecular organization of the breakpoint regions differ significantly: one case is a reciprocal 7;12 translocation (RTR); the other has an insertion of 7q into 12p (INS). While 12p13 breakpoints in both patients interrupt TEL intron 1, the centromere to telomere orientation of the 7q36 sequences relative to the TEL sequences are inverted in INS compared to RTR. This difference makes it difficult to postulate a mechanism whereby both patients could produce a common fusion transcript. Further, no evidence was obtained for any TEL-containing fusion transcripts. Finally, we report the first cloning of a 7;12 genomic breakpoint (from RTR) and find that it maps to a site 30 kbp proximal to the HLXB9 gene in 7q36. Together, these data suggest that, unlike most leukemia-associated chromosomal rearrangements, the important consequence of the t(7;12) is likely not the generation of a novel fusion transcript, but instead the inactivation of TEL and/or a gene at 7q36.

Pupae of the onion maggot, Hylemya antiqua (Meigen), did not complete their development when irradiated with 3.0 kiloroentgens within five days of pupal formation. Older pupae were not adversely affected. Females from treated pupae were sterile and laid no eggs when caged with normal males. The males from treated pupae were sterilized to a high degree, yet normal females mated with them and laid normal complements of eggs. Less than 0.2% of 10,616 eggs laid by normal females caged with sterile males hatched. A 6:1 ratio of sterile to normal flies reduced egg hatch to 1.7%.

1. The production of propionate in the caecum of the horse has been measured in two Shetland-type ponies fitted with caecal and colonic cannulas and fed on hay or on hay and wheat bran. A continuous intracaecal infusion of14C-labelled sodium propionate was used and samples were obtained from a cannula at the origin of the right ventral colon. A simultaneous intravenous infusion of [2-3H]glucose was used to measure total glucose entry.2. On a hay diet which provided 177 kJ/kg body-weight per d, mean caecal propionate production was 19.6 (range 17.2–21.2) mg/h per kg body-weight and on a hay and wheat bran diet, which provided 187 KJ/kg body-weight per d, mean caecal propionate production was 34.0 (range 28.9–38.3) mg/h per kg body-weight.3. Mean total glucose production (mg/h per kg body-weight) in one pony was 104 (range 100–110) and in the other 135 (range 123–153). Rates were not influenced by diet.4. About 7% of total glucose production was derived from propionate produced in the caecum and this percentage was unaffected by diet or by individual animals.

Many factors, such as interleukin 1, TGF alpha, tumor necrosis factor alpha and beta, and PGs, have been implicated in etiological roles in HHM (Martin and Mundy, 1987). Much interest in the past has also centered upon the likelihood of ectopic secretion of PTH in this condition. We have purified a protein (PTHrP) implicated in HHM from a human lung cancer cell line (BEN). Full-length cDNA clones have been isolated and were found to encode a prepropeptide of 36 amino acids and a mature protein of 141 amino acids. Eight of the first 13 amino acids were identical with human PTH, although antisera directed to the NH2 terminus of PTHrP do not recognize PTH; this homology is not maintained in the remainder of the molecule. PTHrP therefore represents a previously unrecognized hormone, possibly related to the PTH gene by a gene duplication mechanism. In support of this notion, the PTHrP gene has been localized to the short arm of chromosome 12; it is believed that chromosome 11, containing the PTH gene, and chromosome 12 are evolutionarily related. In addition, the human PTHrP gene has been isolated, characterized, and shown to have a similar intron--exon organization as the PTH gene. It is possible that the original ancestral gene is indeed the PTHrP gene; resolution of this question awaits studies in lower species. Peptides synthesized to the predicted protein sequence have enabled detailed structure-function studies that have identified NH 2-terminal sequences to be responsible for the biological effects of the molecule. Antibodies raised against the various synthetic peptides have led to the immunohistochemical localization of PTHrP in many human squamous cell carcinomas as well as in a subpopulation of keratinocytes of normal skin. The availability of these antibodies has opened the way for the development of a radioimmunoassay to detect PTHrP in the sera of cancer patients at risk of developing hypercalcemia. The recent characterization of PTHrP-like activity in the ovine fetus suggests some physiological function for PTHrP. It is possible that PTHrP, as the fetal counterpart of PTH, has the role of maintaining the maternal-fetal calcium gradient. The isolation and characterization of PTHrP have added to our understanding of the mechanisms of hypercalcemia and may contribute to the understanding of other metabolic bone diseases, such as osteoporosis and Paget's disease. Finally, and perhaps most importantly, PTHrP may play a hitherto unrecognized role in normal cell physiology.