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The disproportionate number of COVID-19 cases and deaths in the United States (US) requires understanding of health behaviors that contributed to low acceptance of public health guidelines. University students were at low risk for complications from COVID-19 infections but at high-risk for infecting others. It is important to understand which characteristics influenced willingness to receive the COVID-19 vaccine. The purpose of this cross-sectional study was to explore the social factors and identities that predicted university students' willingness to receive COVID-19 vaccines before one was available. This study used a stratified random sample from a Midwestern public university in 2020 ( N  = 614). Results suggest hesitancy toward childhood vaccinations, noncompliance with mask-wearing and large-group-avoidance guidelines, and conservative political ideology increased the odds of being unwilling to receive a COVID-19 vaccination. However, higher subjective social status and male gender increased the odds of being willing to be vaccinated.

Recent advances in electronics and microfluidics have enabled several research groups to develop fully integrated, sample-to-result isothermal nucleic acid amplification test (NAAT) platforms for the point of care. However, high component counts and costs have limited translation of these platforms beyond the clinic to low-resource settings-including homes. Many NAATs include complex, multi-component heater electronics based on flex circuits or multiple printed circuit boards (PCBs) to support essential NAAT steps such as lysis, sample deactivation, and nucleic acid amplification. In contrast, current commercial assays for home use, such as those for pregnancy or ovulation that include electronics, typically have just one onboard PCB. This work describes a generalizable strategy to integrate all heaters and the electronics needed to control them onto a single low-cost, USB-powered PCB. We built a multiplexable disposable NAAT ("MD NAAT") platform that applies these principles, integrating small-area heaters that heat small regions to near-boiling (for pathogen lysis and deactivation) and large-area heaters (for amplification) on the same PCB. We show that both classes of heaters have high intra-board and inter-device reproducibility despite only heating a NAAT cartridge from below. We validated the small-area heaters by lysing methicillin-resistant Staphylococcus aureus (MRSA) cells and the large-area heaters by performing two types of isothermal NAATs (isothermal strand displacement amplification (iSDA) and loop-mediated isothermal amplification (LAMP)). These results demonstrate the merit of integrating NAAT heaters and control electronics onto a single printed circuit board and are a step toward translating NAATs to the home., Competing Interests: The authors have read the journal’s policy and have the following competing interests: Paul Yager has a nonpaying appointment as CSO of the UbiDX corporation. This study was performed before his association with the company. There are no patents, products in development or marketed products associated with this research to declare. This does not alter our adherence to PLOS ONE policies on sharing data and materials., (Copyright: © 2023 Shah et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Of 1003 children with acute lymphoblastic leukaemia (ALL), 147 (14.7%) presented without peripheral blood blasts (PBB). While absence of PBB was not independently associated with survival outcomes when compared to those with PBB, patients without PBB had distinct genetic and clinical characteristics. Notably, we identified a novel genotype-phenotype relationship, in that the patients without PBB had a significantly higher incidence of hyperdiploid B-ALL, accounting for almost half of all patients without PBB (46.9% vs. 22.7%, p < 0.001). Further, absence of PBB was associated with decreased rates of leukaemia involvement of the central nervous system (p < 0.001)., (© 2022 British Society for Haematology and John Wiley & Sons Ltd.)

The lateral flow assay (LFA) is one of the most popular technologies on the point-of-care diagnostics market due to its low cost and ease of use, with applications ranging from pregnancy to environmental toxins to infectious disease. While the use of these tests is relatively straightforward, significant development time and effort are required to create tests that are both sensitive and specific. Workflows to guide the LFA development process exist but moving from target selection to an LFA that is ready for field testing can be labor intensive, resource heavy, and time consuming. To reduce the cost and the duration of the LFA development process, we introduce a novel development platform centered on the flexibility, speed, and throughput of an automated robotic liquid handling system. The system comprises LFA-specific hardware and software that enable large optimization experiments with discrete and continuous variables such as antibody pair selection or reagent concentration. Initial validation of the platform was demonstrated during development of a malaria LFA but was readily expanded to encompass development of SARS-CoV-2 and Mycobacterium tuberculosis LFAs. The validity of the platform, where optimization experiments are run directly on LFAs rather than in solution, was based on a direct comparison between the robotic system and a more traditional ELISA-like method. By minimizing hands-on time, maximizing experiment size, and enabling improved reproducibility, the robotic system improved the quality and quantity of LFA assay development efforts., (© 2022. The Author(s).)

The global COVID-19 pandemic has created an urgent demand for large numbers of inexpensive, accurate, rapid, point-of-care diagnostic tests. Analyte-based assays are suitably rapid and inexpensive and can be rapidly mass-produced, but for sufficiently accurate performance, they require highly optimized antibodies and assay conditions. We used an automated liquid handling system, customized to handle arrays of lateral flow (immuno)assays (LFAs) in a high-throughput screen, to identify anti-nucleocapsid antibodies that will perform optimally in an LFA. We tested 1021 anti-nucleocapsid antibody pairs as LFA capture and detection reagents with the goal of highlighting pairs that have the greatest affinity for the nucleocapsid protein of SARS-CoV-2 within the LFA format. In contrast to traditional antibody screening methods (e.g., ELISA, bio-layer interferometry), the method described here integrates real-time reaction kinetics with transport in, and immobilization directly onto, nitrocellulose. We have identified several candidate antibody pairs that are suitable for further development of an LFA for SARS-CoV-2., Competing Interests: The authors declare no competing financial interest., (© 2021 The Authors. Published by American Chemical Society.)

Nucleic acid amplification tests (NAATs) are common in laboratory and clinical settings because of their low time to result and exquisite sensitivity and specificity. Laboratory NAATs include onboard positive controls to reduce false negatives and specialized hardware to enable real-time fluorescence detection. Recent efforts to translate NAATs into at-home tests sacrifice one or more of the benefits of laboratory NAATs, such as sensitivity, internal amplification controls (IACs), or time to result. In this manuscript, we describe a mobile-phone-based strategy for real-time imaging of biplexed NAATs in paper. The strategy consisted of: (1) using mobile phones with multipass excitation and emission filters on the flash and camera to image the signal from distinct fluorophore-labeled probe types in a biplexed NAAT in a glass fiber membrane; and (2) analyzing the differential fluorescence signal between the red and green color channels of phone images to overcome a strong evaporation-induced optical artifact in heated glass fiber pads due to changes in the refractive index. We demonstrated that differential fluorescence imaging enabled low limits of detection (316 copies of methicillin-resistant Staphylococcus aureus DNA) in our lab's "MD NAAT" platform, even in biplexed isothermal strand displacement amplification reactions containing 100k copies of coamplifying IAC DNA templates. These results suggest that two-fluorophore mobile phone imaging may enable translating the benefits of extant laboratory-based, real-time NAATs to the point of care.

Viable populations of the cheilostome bryozoan Cribrilina mutabilis Ito, Onishi & Dick exist in the NW Pacific (Russian Far East and northern Japan), NE Atlantic (Scandinavia and Scotland), and NW Atlantic (Maine, USA). The first NE and NW Atlantic records are from Norway (2008) and Casco Bay, Maine, USA (2018), respectively, indicating a relatively recent introduction to the region. Mitochondrial COI gene sequences from North Atlantic populations (Sweden, Norway, and Maine) showed two haplotypes differing by one substitution, but differed from two haplotypes from Akkeshi, northern Japan, by 6-8 substitutions. North Atlantic populations differed morphologically from the Akkeshi population in that some zooids formed a suboral projection, and frontal zooids were more common. While C. mutabilis in northern Japan has been found only on natural or artificial eelgrass ( Zostera marina ), across its range it has been found on several species of algae, plastic panels and strips, several species of Zostera , and mollusc shells. Similar frequencies of heteromorphic zooids with differing degree of frontal wall calcification, i.e., R (rib)-, I (intermediate)-, and S (shield)-type zooids, in colonies on eelgrass at comparable times of the season and across populations suggest an innate response to seasonal environmental fluctuations, although zooid frequencies were different on non-eelgrass substrates. The increase in trans-Arctic shipping along the Northern Sea Route in recent decades, and previous documentation of C. mutabilis on ship hulls in the Sea of Japan, indicate a clear mechanism for anthropogenic introduction from the Far East to Europe in recent decades.

Lateral flow assays (LFAs) are rapid, inexpensive, easy-to-manufacture and -use tests widely employed in medical and environmental applications, particularly in low resource settings. Historically, LFAs have been stigmatized as having limited sensitivity. However, as their global usage expands, extensive research has demonstrated that it is possible to substantially improve LFA sensitivity without sacrificing their advantages. In this critical review, we have compiled state-of-the-art approaches to LFA sensitivity enhancement. Moreover, we have organized and evaluated these approaches from a system-level perspective, as we have observed that the advantages and disadvantages of each approach have arisen from the integrated and tightly interconnected chemical, physical, and optical properties of LFAs.

Foster care alumni lag behind the general population in post-secondary academic achievement. This cross-sectional study uses a random sample of forty-six foster care alumni from a Midwestern public university to explore the relationship between exposure to trauma and post-secondary academic achievement (14.5% response rate; age range 17-24). While no significant relationship was found between trauma exposure and GPA or failing classes, participants who experienced increased exposure to trauma were more likely to have withdrawn from a class because it was too difficult or the semester was too overwhelming. More research is needed to further understand these findings., Competing Interests: Conflict of InterestThe authors declare they have no conflict of interest., (© Springer Nature Switzerland AG 2018.)

While the UK has committed to reduce CO 2 emissions to 80% of 1990 levels by 2050, transport accounts for nearly a fourth of all emissions and the degree to which decarbonization can occur is highly uncertain. We present a new methodology using vehicle and powertrain parameters within a Bayesian framework to determine the impact of engineering vehicle improvements on fuel consumption and CO 2 emissions. Our results show how design changes in vehicle parameters (e.g., mass, engine size, and compression ratio) result in fuel consumption improvements from a fleet-wide mean of 5.6 L/100 km in 2014 to 3.0 L/100 km by 2030. The change in vehicle efficiency coupled with increases in vehicle numbers and fleet-wide activity result in a total fleet-wide reduction of 41 ± 10% in 2030, relative to 2012. Concerted internal combustion engine improvements result in a 48 ± 10% reduction of CO 2 emissions, while efforts to increase the number of diesel vehicles within the fleet had little additional effect. Increasing plug-in and all-electric vehicles reduced CO 2 emissions by less (42 ± 10% reduction) than concerted internal combustion engines improvements. However, if the grid decarbonizes, electric vehicles reduce emissions by 45 ± 9% with further reduction potential to 2050.

Polyembryony-the production of multiple cloned embryos from a single fertilised egg-is a seemingly paradoxical combination of reproductive modes that nevertheless persists in diverse taxa. We document features of polyembryony in the Cyclostomata (Bryozoa)-an ancient order of modular colonial marine invertebrates-that suggest a substantial reduction in the paradoxical nature of this enigmatic reproductive mode. Firstly, we provide molecular evidence for polyembryony in three exemplar species, supporting the widely cited inference that polyembryony characterises the entire order. Secondly, genotyping demonstrates protracted release of cloned offspring from the primary embryo in a given gonozooid (chamber for embryonic incubation), thus exposing the same genotype to changing environmental conditions over time. Finally, we confirm that each gonozooid produces a distinct genotype, with each primary embryo being the result of a separate fertilisation event. We hypothesise that the sustained release of one or a few genotypes against varying environmental conditions achieves levels of risk-spreading similar to those in organisms that release multiple, unique genotypes at a single time. We argue that polyembryony, specifically with the production of a large number of progeny per fertilisation event, has been favoured in the Cyclostomata over long geological periods., Competing Interests: The authors have declared that no competing interests exist.

The prototype demonstrated here is the first fully integrated sample-to-result diagnostic platform for performing nucleic acid amplification tests that requires no permanent instrument or manual sample processing. The multiplexable autonomous disposable nucleic acid amplification test (MAD NAAT) is based on two-dimensional paper networks, which enable sensitive chemical detection normally reserved for laboratories to be carried out anywhere by untrained users. All reagents are stored dry in the disposable test device and are rehydrated by stored buffer. The paper network is physically multiplexed to allow independent isothermal amplification of multiple targets; each amplification reaction is also chemically multiplexed with an internal amplification control. The total test time is less than one hour. The MAD NAAT prototype was used to characterize a set of human nasal swab specimens pre-screened for methicillin-resistant Staphylococcus aureus (MRSA) bacteria. With qPCR as the quantitative reference method, the lowest input copy number in the range where the MAD NAAT prototype consistently detected MRSA in these specimens was ∼5 × 10(3) genomic copies (∼600 genomic copies per biplexed amplification reaction).

Background: Prognosis for patients infected with human immunodeficiency virus (HIV) correlates with levels of CD4+ T cells. Initiation of antiretroviral therapy (ART) interrupts multiple points in the virus life cycle, causing an increase in CD4 cells. The rate at which the CD4 count recovers is highly variable and subject to influence by many factors., Methods: We performed a deidentified data review to determine factors influencing the rate of CD4 count recovery after ART initiation. The associations between the changes in CD4 count from baseline at 5 time points, and factors including age, race, weight, baseline CD4 count, baseline viral load, specific ART medications and various comorbidities, were evaluated with univariate and multivariate analyses, using t-test, analysis of variance and multiple regressions., Results: CD4 count continued to rise even up to 10 years after ART initiation, with the steepest increase in the first 3 months. High baseline viral load and low baseline CD4 count had the most consistent positive influence on CD4 count recovery rate across the 5 measured time points. Other factors that were significantly positively associated with CD4 recovery rate included younger baseline age, higher baseline weight and female gender., Conclusions: CD4 counts in HIV positive patients who consistently take ART continue to increase out to at least 10 years. Patients with a more advanced HIV infection at baseline, as indicated by high viral loads or low CD4 counts, have a greater rate of CD4 count recovery after starting ART, possibly because their CD4 counts have more room for improvement., (Copyright © 2016 Southern Society for Clinical Investigation. Published by Elsevier Inc. All rights reserved.)

Human-mediated dispersal interplays with natural processes and complicates understanding of the biogeographical history of species. This is exemplified by two invasive tunicates, Ciona robusta (formerly Ciona intestinalis type A) and C. intestinalis (formerly Ciona intestinalis type B), globally distributed and sympatric in Europe. By gathering new mitochondrial sequences that were merged with published datasets, we analysed genetic patterns in different regions, with a focus on 1) their sympatric range and 2) allopatric populations in N and S America and southern Europe. In the sympatric range, the two species display contrasting genetic diversity patterns, with low polymorphism in C. robusta supporting the prevalent view of its recent introduction. In the E Pacific, several genetic traits support the non-native status of C. robusta. However, in the NE Pacific, this appraisal requires a complex scenario of introduction and should be further examined supported by extensive sampling efforts in the NW Pacific (putative native range). For C. intestinalis, Bayesian analysis suggested a natural amphi-North Atlantic distribution, casting doubt on its non-native status in the NW Atlantic. This study shows that both natural and human-mediated dispersal have influenced genetic patterns at broad scales; this interaction lessens our ability to confidently ascertain native vs. non-native status of populations, particularly of those species that are globally distributed.

Sample preparation, including bacterial lysis, remains a hurdle in the realization of complete point-of-care tests for many pathogens. Here, we developed a sample preparation methodology for enzymatic lysis and sample heating for low-resource, point-of-care applications. We show an instrument-free chemical heater system for rapid lysis of a gram-positive bacterium ( Staphylococcus aureus ) and an RNA virus (human respiratory syncytial virus) using a dried lysis enzyme mixture (achromopeptidase) for S. aureus . After a lysis step (<1 minute), lysis enzymes are heat deactivated (<5 minutes) using a simple disposable chemical heater. We demonstrated that both DNA and RNA in the heat-treated sample could be directly amplified without purification, even in the presence of a clinically-obtained human nasal sample. This simple approach to dry enzyme storage and sample heating is adaptable to many applications where samples need to be lysed, including use in low-resource laboratories and in single-use or cartridge-based point-of-care diagnostic devices.

Dendritic cells (DCs) are key regulators of host immunity that are capable of inducing either immune tolerance or activation. In addition to their well-characterized role in shaping immune responses to foreign pathogens, DCs are also known to be critical for the induction and maintenance of anti-tumor immune responses. Therefore, it is important to understand how tumors influence the function of DCs and the quality of immune responses they elicit. Although the majority of studies in this field to date have utilized either immortalized DC lines or DC populations that have been generated under artificial conditions from hematopoietic precursors in vitro, we wished to investigate how tumors impact the function of already differentiated, tissue-resident DCs. Therefore, we used both an ex vivo and in vivo model system to assess the influence of melanoma-derived factors on DC maturation and activation. In ex vivo studies with freshly isolated splenic DCs, we demonstrate that the extent to which DC maturation and activation are altered by these factors correlates with melanoma tumorigenicity, and we identify partial roles for tumor-derived transforming growth factor (TGF)β1 and vascular endothelial growth factor (VEGF)-A in the altered functionality of DCs. In vivo studies using a lung metastasis model of melanoma also demonstrate tumorigenicity-dependent alterations to the function of lung-resident DCs, and skewed production of proinflammatory cytokines and chemokines by these tumor-altered cells is associated with recruitment of an immune infiltrate that may ultimately favor tumor immune escape and outgrowth.

Decoupling nucleic acid amplification assays from infrastructure requirements such as grid electricity is critical for providing effective diagnosis and treatment at the point of care in low-resource settings. Here, we outline a complete strategy for the design of electricity-free precision heaters compatible with medical diagnostic applications requiring isothermal conditions, including nucleic acid amplification and lysis. Low-cost, highly energy dense components with better end-of-life disposal options than conventional batteries are proposed as an alternative to conventional heating methods to satisfy the unique needs of point of care use.

Rapid assessment surveys of non-indigenous species (NIS) of sessile invertebrates were made at seven marinas in NW France and 10 marinas in SW England in 2010, and repeated in 2013. Fourteen NIS were recorded, 12 of which were seen on both coasts. Site occupancy differed between the opposite sides of the western English Channel. In Brittany, most species occurred at most sites in both 2010 and 2013. In 2010, site occupancy in Devon & Cornwall was distinctly lower; by 2013, the difference compared to Brittany had narrowed considerably, largely because of rapid colonisation of additional sites by species that were infrequent in 2010. Three more of the recent NIS are present in Devon & Cornwall but have still not become widespread. It is concluded that the recently introduced fouling animals studied here are longer established in NW France than in SW England, and have probably spread northwards across the Channel., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The emergence of rapid, user-friendly, point-of-care (POC) diagnostic systems is paving the way for better disease diagnosis and control. Lately, there has been a strong emphasis on developing molecular-based diagnostics due to their potential for greatly increased sensitivity and specificity. One of the most critical steps in developing practical diagnostic systems is the ability to perform sample preparation, especially the purification of nucleic acids (NA), at the POC. As such, we have developed a simple-to-use, inexpensive, and disposable sample preparation system for in-membrane purification and concentration of NAs. This system couples lateral flow in a porous membrane with chitosan, a linear polysaccharide that captures NAs via anion exchange chromatography. The system can also substantially concentrate the NAs. The combination of these capabilities can be used on a wide range of sample types, which are prepared for use in downstream processes, such as qPCR, without further purification.

The sea squirt Ciona intestinalis species complex is a widely used model system for genomics and developmental biology, as well as ecology. Contrary to previous reports, here we show no difference in the success of development and hatching between hybrid and conspecific crosses between the two species within this complex known as types A and B, from a region in the English Channel where they are sympatric. We grew laboratory hybrids in the field for three months, and successfully obtained reproductive adults. In back-crosses of F1 laboratory hybrids to parental types, normal larvae were obtained. We conclude that hybrid crosses generate viable offspring and the resulting hybrids are interfertile with types A and B. However we also show that introgression in the natural sympatric population remains low. We discuss possible pre-zygotic and post-zygotic mechanisms which reproductively isolate these species.

The solitary ascidian Ascidiella aspersa (Müller, 1776) has sometimes been regarded as conspecific with A. scabra (Müller, 1776), although previous detailed morphological comparisons have indicated that the two are distinguishable by internal structures. Resolution of this taxonomic issue is important because A. aspersa has been known as a notoriously invasive ascidian, doing much damage to aquaculture e.g. in Hokkaido, Japan. We collected many specimens from European waters (including the Swedish coast, near the type localities of these two species) and Hokkaido, Japan (as an alien population) and made molecular phylogenetic analyses using the mitochondrial cytochrome c oxidase subunit I (COI) gene, and found that in terms of COI sequences all the analyzed specimens were clustered into two distinct groups, one of which is morphologically referable to A. aspersa and the other to A. scabra. Thus, these two species should be regarded as distinct from each other.

Background. Prior studies have shown earlier recurrence and decreased survival in patients with head and neck cancer who smoked while undergoing radiation therapy. The purpose of the current study was to determine whether smoking status at the time of partial mastectomy and radiation therapy for breast cancer affected recurrence or survival. Method. A single institution retrospective chart review was performed to correlate smoking status with patient demographics, tumor characteristics, and outcomes for patients undergoing partial mastectomy and radiation therapy. Results. There were 624 patients who underwent breast conservation surgery between 2002 and 2010 for whom smoking history and follow-up data were available. Smoking status was associated with race, patient age, and tumor stage, but not with grade, histology, or receptor status. African American women were more likely to be current smokers (22% versus 7%, P < 0.001). With a mean follow-up of 45 months, recurrence was significantly higher in current smokers compared to former or never smokers (P = 0.039). In a multivariate model adjusted for race and tumor stage, recurrence among current smokers was 6.7 times that of never smokers (CI 2.0-22.4). Conclusions. Although the numbers are small, this study suggests that smoking may negatively influence recurrence rates after partial mastectomy and radiation therapy. A larger study is needed to confirm these observations.

Boat harbours are an increasingly common form of artificial habitat. This paper presents a comparative study of contaminants and foulers of a habitat-forming native kelp (Saccharina latissima) in four marinas and four reference locations along the south-west coast of the UK. Fouling of algal laminae was light (<2% cover) in reference locations, while epibiota cover ranged from 25% to 80% of laminae in marinas. Metals associated with antifouling paints were up to six times more concentrated in algal tissues from marinas than from the reference locations. Marinas also carried the greatest cover and diversity of non-indigenous epibiota on the kelp laminae. This indicates not only a potential stress to kelps in these environments, but also the possibility that detached laminae will act as vectors for the dispersal of non-indigenous species. The development of boat harbours creates habitats that are high risk source localities for pollution-tolerant fouling organisms., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Many invertebrates reproduce asexually by budding, but morphogenesis and the role of cell proliferation in this diverse and nonconserved regeneration-like process are generally poorly understood and particularly little investigated in didemnid ascidians. We here analyzed cell proliferation patterns and telomerase activity during budding in the colonial didemnid ascidian Diplosoma listerianum, with special focus on the thoracic bud where a new brain develops de novo. To help define developmental stages of the thoracic bud, the distribution of acetylated tubulin was also examined. We found extensive cell proliferation in both the thoracic and abdominal buds of D. listerianum as well as higher telomerase activity in bud tissue compared to adult tissues. In the parent adult, proliferation was found in various tissues, but was especially intense in the adult esophagus and epicardial structures that protrude into the proliferating and developing buds, confirming these tissues as the primary source of the cells that form the buds. The neural complex in the thoracic bud forms from a hollow tube that appears to separate into the neural gland and the cerebral ganglion. Whereas most of the bud undergoes proliferation, including the hollow tube and the neural gland, the cerebral ganglion shows little or no proliferation. Pulse-chase labeling experiments indicate that the ganglion, as well as the myocardium, in adult zooids are instead composed of postmitotic cells.

In organisms that propagate by agametic cloning, the parental body is the reproductive unit and fitness increases with clonal size, so that colonial metazoans, despite lack of experimental data, have been considered potentially immortal. Using asexual propagation rate as a measure of somatic performance, and telomerase activity and relative telomere length as molecular markers of senescence, old (7-12 years) asexual strains of a colonial ascidian, Diplosoma listerianum, were compared with their recent sexually produced progeny. We report for the first time evidence for long-term molecular senescence in asexual lineages of a metazoan, and that only passage between sexual generations provides total rejuvenation permitting indefinite propagation and growth. Thus, this colonial ascidian has not fully escaped ageing. The possibility of somatic replicative senescence also potentially helps to explain why metazoans, with the capacity for asexual propagation through agametic cloning, commonly undergo cycles of sexual reproduction in the wild., (Copyright © 2011 Wiley-Liss, Inc., A Wiley Company.)

The solitary ascidian Styela clava Herdman, 1882 is considered to be native to Japan, Korea, northern China and the Russian Federation in the NW Pacific, but it has spread globally over the last 80 years and is now established as an introduced species on the east and west coasts of North America, Europe, Australia and New Zealand. In eastern Canada it reaches sufficient density to be a serious pest to aquaculture concerns. We sequenced a fragment of the cytochrome oxidase subunit I mitochondrial gene (COI) from a total of 554 individuals to examine the genetic relationships of 20 S. clava populations sampled throughout the introduced and native ranges, in order to investigate invasive population characteristics. The data presented here show a moderate level of genetic diversity throughout the northern hemisphere. The southern hemisphere (particularly New Zealand) displays a greater amount of haplotype and nucleotide diversity in comparison. This species, like many other invasive species, shows a range of genetic diversities among introduced populations independent of the age of incursion. The successful establishment of this species appears to be associated with multiple incursions in many locations, while other locations appear to have experienced rapid expansion from a potentially small population with reduced genetic diversity. These contrasting patterns create difficulties when attempting to manage and mitigate a species that continues to spread among ports and marinas around the world.

Background: Despite the impressive growth of sequence databases, the limited availability of nuclear markers that are sufficiently polymorphic for population genetics and phylogeography and applicable across various phyla restricts many potential studies, particularly in non-model organisms. Numerous introns have invariant positions among kingdoms, providing a potential source for such markers. Unfortunately, most of the few known EPIC (Exon Primed Intron Crossing) loci are restricted to vertebrates or belong to multigenic families., Results: In order to develop markers with broad applicability, we designed a bioinformatic approach aimed at avoiding multigenic families while identifying intron positions conserved across metazoan phyla. We developed a program facilitating the identification of EPIC loci which allowed slight variation in intron position. From the Homolens databases we selected 29 gene families which contained 52 promising introns for which we designed 93 primer pairs. PCR tests were performed on several ascidians, echinoderms, bivalves and cnidarians. On average, 24 different introns per genus were amplified in bilaterians. Remarkably, five of the introns successfully amplified in all of the metazoan genera tested (a dozen genera, including cnidarians). The influence of several factors on amplification success was investigated. Success rate was not related to the phylogenetic relatedness of a taxon to the groups that most influenced primer design, showing that these EPIC markers are extremely conserved in animals., Conclusions: Our new method now makes it possible to (i) rapidly isolate a set of EPIC markers for any phylum, even outside the animal kingdom, and thus, (ii) compare genetic diversity at potentially homologous polymorphic loci between divergent taxa.

The Order Stolidobranchiata comprises the families Pyuridae, Styelidae and Molgulidae. Early molecular data was consistent with monophyly of the Stolidobranchiata and also the Molgulidae. Internal phylogeny and relationships between Styelidae and Pyuridae were inconclusive however. In order to clarify these points we used mitochondrial and nuclear sequences from 31 species of Styelidae and 25 of Pyuridae. Phylogenetic trees recovered the Pyuridae as a monophyletic clade, and their genera appeared as monophyletic with the exception of Pyura. The Styelidae, on the other hand, appeared as a paraphyletic group split into several clades. One of them was formed by solitary oviparous species, of which the Pyuridae were a sister group. A second clade included the colonial genera Botryllus, Botrylloides and Symplegma. The remaining colonial and solitary genera formed several poorly resolved clades. One of the more species genus, Polycarpa, was shown to be polyphyletic, and the species Styela plicata grouped into two genetically distant clades suggesting the existence of two cryptic species. The internal phylogeny of Styelidae has bearings on the origin of coloniality in this family. We suggest to abandon the traditional division of colonial forms into social and compound species and use instead the categories of aggregated colonies that do not have common vascular systems, and integrated colonies, that do possess such systems. Our molecular results indicate that there have been several independent acquisitions of coloniality in the Styelidae, and that viviparity may be a pre-adaptation for a colonial life-style.

Styela clava, an ascidian native to the northwest Pacific, was first recorded in the Atlantic at Plymouth, southwest England, in 1953. It now ranges in the northeast Atlantic from Portugal to northern Denmark, and has colonized the east coast of North America. Within the region of first introduction, we aimed to characterize current genetic diversity in the species, elucidate the respective roles of human-aided vs. natural dispersal, and assess the extent of larval dispersal by looking for genetic differentiation at very small scales. Eight sites, mostly marinas, were studied along c. 200 km of coast in southwest England encompassing Plymouth. Five microsatellite loci were genotyped in 303 individuals to analyse gene flow at regional (among sites) and fine (within sites) scales. F-statistics and assignment tests were used to investigate regional genetic structure. At the fine scale, deviation from mutation-drift equilibrium was tested, and isolation by distance and genetic clustering analyses were undertaken. Significant genetic differentiation existed between sites, unrelated to geographical separation; migration between geographically distant marinas was inferred, highlighting the likely importance of human-mediated dispersal in range expansion and occupancy by S. clava. Fine-scale population structure was present within at least four sites, which may be explained by the limited dispersal ability of this ascidian and recruitment from differentiated pools of larvae. Populations in enclosed marinas had higher self-recruitment rates than those in open sites. Some marinas might therefore function as reservoirs of propagules for subsequent spread, whereas others might be sinks for migrants.

Hemichordates, like echinoderms and chordates, are deuterostomes, and study of their developmental biology could shed light on chordate origins. To date, molecular developmental studies in hemichordates have been confined to the enteropneusts or acorn worms. Here, we introduce the developmental biology of the other group of hemichordate, the pterobranchs. Pterobranchs generally live in cold, deep waters; this has hampered studies of this group. However, about 40 years ago, the colonial pterobranchs Rhabdopleura compacta and R. normani were discovered from shallow water, which has facilitated their study. Using Rhabdopleura compacta from south-west England, we have initiated molecular developmental studies in pterobranchs. Here, we outline methods for collecting adults, larvae, and embryos and demonstrate culturing of larvae under laboratory conditions. Given that the larval and adult forms differ from enteropneusts, we suggest that molecular developmental studies of pterobranchs may offer new insights into chordate origins., (Copyright 2008 Wiley-Liss, Inc.)

DNA nanomotors are synthetic biochemical devices whose motion can be controlled at the molecular scale. Some DNA devices require several exogenous additions of different types of fuel to operate, which limits their potential uses. However, several devices that operate autonomously have recently been described. One such DNA nanomotor, based on a 10-23 DNA enzyme (DNAzyme), was introduced by Chen, Wang, and Mao (Angew. Chem., Int. Ed. 2004, 43, 3554). Although this DNAzyme nanomotor operates autonomously, its performance degrades over time in experiments. In this paper, we describe a mathematical model that predicts this degradation by accounting for the gradual accumulation of waste in the system. We also introduce and experimentally demonstrate two improved versions of the DNAzyme nanomotor. In particular, the new nanomotor systems use the enzyme ribonuclease H to selectively digest waste, resulting in nanomotors whose performance does not degrade significantly over time.

Marine invertebrates belonging to a broad range of taxa disperse aquatic spermatozoa to fertilize eggs that are retained rather than spawned. We outline the occurrence of this mechanism, which we refer to as spermcast mating, and identify tentative generalizations relating to it. Contrasts are drawn where appropriate with broadcast spawning of both eggs and sperm for external fertilization, and with copulation or pseudocopulation. Spermcast mating may involve the gradual accumulation of long-lived spermatozoa from dilute suspension, probably during suspension feeding, and the subsequent storage of spermatozoa by the recipient (acting female) prior to fertilization. This process may involve extensive contact between spermatozoa and recipient (maternal) tissue. Mating may be influenced by compatibility systems, and receipt of compatible allosperm may trigger female investment, giving apparent scope for sexual conflict over levels of maternal investment. External fertilization of cohesive egg masses remaining close to the acting female may appear somewhat intermediate between spermcast mating and broadcast spawning but, while it may be possible to envisage a continuum between the 2 modes, the end points are distinct, commonplace, and involve contrasting reproductive characteristics. Three variants of the typical pattern of spermcast mating are briefly discussed: the spawning of zygotes (rather than the more usual brooding of progeny), polyembryony, and the dispersal of spermatophores rather than individual spermatozoa.

Mitogens activate the mammalian target-of-rapamycin (mTOR) pathway through phosphatidylinositol 3-kinase (PI3K). The activated mTOR kinase phosphorylates/ activates ribosomal protein S6 kinase (p70S6K) and phosphorylates/inactivates eukaryotic initiation factor 4E-binding protein-1 (4E-BP1), resulting in the initiation of translation and cell-cycle progression. The prolactin receptor signaling cascade has been implicated in crosstalk with the mTOR pathway, but whether prolactin (PRL) directly activates mTOR is not known. This study showed that PRL stimulated the phosphorylation of mTOR, p70S6K, Akt, and Jak2 kinases in a dose- and time-dependent manner in PRL-dependent rat Nb2 lymphoma cells. PRL-stimulated phosphorylation of mTOR was detected as early as 10 min, closely following the phosphorylation of Akt (upstream of mTOR), but preceding that of the downstream p70S6K. PRL activation of mTOR was inhibited by rapamycin (mTOR inhibitor), LY249002, and wortmannin (P13K inhibitors), but not by AG490 (Jak2 inhibitor), indicating that it was mediated by the P13K/Akt, but not Jak2, pathway. PRL also stimulated phosphorylation of 4E-BP1 in Nb2 cells. PRL-induced phosphorylation of p70S6K and 4E-BP1 was inhibited by rapamycin, but not by okadaic acid (inhibitor of protein phosphatase, PP2A). PRL induced a transient interaction between p70S6K and the catalytic subunit of PP2A (PP2Ac) in 1 and 2 h, whereas a PP2Ac-4E-BP1 complex was constitutively present in quiescent and PRL-treated Nb2 cells. These results suggested that p70S6K and 4E-BP1 were substrates of PP2A and the inhibition of mTOR promoted their dephosphorylation by PP2A. In summary, PRL-stimulated phosphorylation of mTOR is mediated by PI3K. PRL-activated mTOR may phosphorylate p70S6K and 4E-BP1 by restraining PP2A.

Prolific polyembryony is reported in few major taxa, but its occurrence has generated theoretical debate on potential conflict between sexual and asexual reproduction. It is, therefore, important to genetically confirm a widely cited inference, based on microscopy, that polyembryony characterizes marine bryozoans of the order Cyclostomata. Microsatellite genotyping of brooded embryos and maternal colonies conclusively demonstrated polyembryony, while genetic variation among broods within colonies indicated outcrossing via water-borne sperm, in the rocky-shore species Crisia denticulata. The characteristically voluminous brood chamber of cyclostomes is judged to be an adaptation linked to larval cloning and hence an indicator of polyembryony. We speculate that although the almost universal occurrence of polyembryony among crown-group Cyclostomata is probably attributable to phylogenetic constraint, adaptive consequences are likely to be significant.

BimC kinesins are required for mitotic spindle assembly in a variety of organisms. These proteins are localized to centrosomes, spindle microtubules, and the spindle midzone. We have previously shown that the Caenorhabditis elegans Aurora B kinase AIR-2 is required for the localization of the ZEN-4 kinesin protein to midzone microtubules. To determine whether the association of BimC kinesins with spindle microtubules is also dependent on AIR-2, we examined the expression pattern of BMK-1, a C. elegans BimC kinesin, in wild-type and AIR-2-deficient embryos. BMK-1 is highly expressed in the hermaphrodite gonad and is localized to meiotic spindle microtubules in the newly fertilized embryo. In mitotic embryos, BMK-1 is associated with spindle microtubules from prophase through anaphase and is concentrated at the spindle midzone during anaphase and telophase. In the absence of AIR-2, BMK-1 localization to meiotic and mitotic spindles is greatly reduced. This is not a consequence of loss of ZEN-4 localization because BMK-1 is appropriately localized in ZEN-4-deficient embryos. Furthermore, AIR-2 and BMK-1 directly interact with one another and the C-terminal tail domain of BMK-1 is specifically phosphorylated by AIR-2 in vitro. Together with our previous data, these results suggest that at least one function of the Aurora B kinases is to recruit spindle-associated motor proteins to their sites of action.

We estimated fusion frequency with respect to coancestry in the bryozoan Celleporella hyalina, whose briefly planktonic sexually produced larvae settle on algal substrata and proceed to form encrusting colonies by iterative budding. Frequency of fusion between paired colonies growing on an artificial substratum was positively correlated with coefficient of relatedness, with allorecognition ability increasing during the early stages of colonial growth after larval settlement. Parents repressed the growth of F1 progeny with which they had fused. The results are concordant with the Feldgarden-Yund model of selection for self-recognition, which regards fusion with kin as an inevitable source of error whose cost diminishes with increasing relatedness. Contrary to fusion compatibility, gametic compatibility is negatively correlated with coancestry, indicating a need for further research on the possibility of common or linked genetic control that has opposite effect at somatic and gametic levels.

The importance of sexual compatibility between mates has only recently been realized in zoological research into sexual selection, yet its study has been central to botanical research for many decades. The reproductive characteristics of remote mating, an absence of precopulatory mate screening, internal fertilization and embryonic brooding are shared between passively pollinated plants and a phylogenetically diverse group of sessile aquatic invertebrates. Here, we further characterize the sexual compatibility system of one such invertebrate, the colonial ascidian Diplosoma listerianum. All 66 reciprocal pairings of 12 genetic individuals were carried out. Fecundities of crosses varied widely and suggested a continuous scale of sexual compatibility. Of the 11 animals from the same population c. 40% of crosses were completely incompatible with a further c. 20% having obvious partial compatibility (reduced fecundity). We are unaware of other studies documenting such high levels of sexual incompatibility in unrelated individuals. RAPD fingerprinting was used to estimate relatedness among the 12 individuals after a known pedigree was successfully reconstructed to validate the technique. In contrast to previous results, no correlation between genetic similarity and sexual compatibility was detected. The blocking of many genotypes of sperm is expected to severely modify realized paternity away from 'fair raffle' expectations and probably reduce levels of intra-brood genetic diversity in this obligatorily promiscuous mating system. One adaptive benefit may be to reduce the bombardment of the female reproductive system by outcrossed sperm with conflicting evolutionary interests, so as to maintain female control of somatic : gametic investment.

The life cycles of sexually reproducing animals and flowering plants begin with male and female gametes and their fusion to form a zygote. Selection at this earliest stage is crucial for offspring quality and raises similar evolutionary issues, yet zoology and botany use dissimilar approaches. There are striking parallels in the role of prezygotic competition for sexual selection on males, cryptic female choice, sexual conflict, and against selfish genetic elements and genetic incompatibility. In both groups, understanding the evolution of sex-specific and reproductive traits will require an appreciation of the effects of prezygotic competition on fitness.

Fertilization success may be severely limited in marine invertebrates that spawn both male and female gametes. In a diverse group of aquatic organisms only sperm are released, with sperm-egg fusion occurring at the mother. Here, we report fertilization kinetics data for two such 'brooding' or 'spermcast' species--representing each major clade of the animal kingdom. High levels of fertilization were achieved at sperm concentrations of two or three orders of magnitude lower than is common with broadcast spawning species. At a concentration of 100 sperm ml(-1), fertilization rates of a bryozoan and colonial ascidian were near maximum, whereas most broadcast spawners would have displayed near complete reproductive failure. A further experiment looked at the rate of uptake of sperm under natural conditions. Results suggested that sperm released at ca. 0.9 m from an acting female could be collected at a rate of 3-12 times greater than the minimum required simply to avoid sperm limitation. Thus, evolutionary pressures on gametic and other reproductive characteristics of many species that release sperm but retain eggs may be quite different from those of broadcast spawners and may confer on the former an enhanced scope for sperm competition and female choice.

Sex-allocation theory developed for hermaphroditic plants predicts that impaired phenotype or reduced parental survivorship caused by environmental stress should induce relatively greater allocation to the male function. We provide experimental evidence of stress-induced maleness, already well documented in flowering plants, in a modular animal. By using cloned copies of replicate genotypes, we show that the marine bryozoan Celleporella hyalina increases the ratio of male to female modules in response to diverse environmental stressors. Mating trials confirmed that paternity is determined by fair-raffle sperm competition, which should obviate local mate competition at characteristic population density and promote the advantage of increased male allocation. The demonstrated similarity to plants transcends specific physiological pathways and suggests that stress-induced bias toward male function is a general response of hermaphroditic modular organisms to impaired prospects for parental productivity or survival.

Negative frequency-dependent mating success--the rare male effect--is a potentially powerful evolutionary force, but disagreement exists as to whether previous work, focusing on copulating species, has robustly demonstrated this phenomenon. Noncopulating sessile organisms that release male gametes into the environment but retain their eggs for fertilization may routinely receive unequal mixtures of sperm. Although promiscuity seems unavoidable it does not follow that the resulting paternity obeys 'fair raffle' expectations. This study investigates frequency dependence in the mating of one such species, the colonial ascidian Diplosoma listerianum. In competition with an alternative sperm source males fathered more progeny if previously mated to a particular female than if no mating history existed. This suggests positive frequency-dependent selection, but may simply result from a mate order effect involving sperm storage. With fewer acclimation matings, separated by longer intervals, this pattern was not found. When, in a different experimental design, virgin females were given simultaneous mixtures of gametes at widely divergent concentrations, sperm at the lower frequency consistently achieved a greater than expected share of paternity--a rare male effect. A convincing argument as to why D. listerianum should favour rare sperm has not been identified, as sperm rarity is expected to correlate very poorly with ecological or genetic male characteristics in this pattern of mating. The existence of nongenetic female preferences at the level of colony modules, analogous in effect to fixed female preferences, is proposed. If visible to selection, indirect benefits from increasing the genetic diversity of a sibship appear the only likely explanation of the rare male effect in this system as the life history presents virtually no costs to multiple mating, and a near absence of direct (resource) benefits, whereas less controversial hypotheses of female promiscuity (e.g. trade up, genetic incompatibility) do not seem appropriate.

Young colonies of the bryozoan Celleporella hyalina are capable of acquiring water-borne allosperm and of using it to fertilize ova for a period of 3-6 weeks after reaching female sexual maturity. In these simultaneous hermaphrodites, early allocation to female modules, but not male, is greatly enhanced by the acquisition of allosperm. The degree of enhancement is inversely proportional to coancestry of the recipient and donor colonies, thus promoting outcrossing. This apparently novel mechanism of adjusting operational sex ratio depends on the uptake and storage of sperm by nonreproductive (somatic) modules and subsequent translocation to females.

Uridine diphosphoglucose pyrophosphorylase (UDPGP) is a developmentally regulated enzyme in Dictyostelium discoideum, which is involved in trehalose, cellulose, and glycogen synthesis. Two independent UDPGP proteins are believed to be responsible for this activity. To determine the relative contributions of each protein, the genes encoding them were disrupted individually. Cells lacking the udpgp1 gene exhibit normal growth and development and make normal levels of cellulose. In agreement with these phenotypes, udpgp1(-) cells still have UDPGP activity, although at a reduced level. This supports the importance of the second UDPGP gene. This newly identified gene, ugpB, encodes an active UDPGP as determined by complementation in Escherichia coli. When this gene is disrupted, cells undergo aberrant differentiation and development ending with small, gnarled fruiting bodies. These cells also have decreased spore viability and decreased levels of glycogen, whose production requires UDPGP activity. These phenotypes suggest that UgpB constitutes the major UDPGP activity produced during development. Sequence analysis of the two UDPGP genes shows that UgpB has higher homology to other eukaryotic UDPGPs than does UDPGP1. This includes the presence of 5 conserved lysine residues. Udpgp1 only has 1 of these lysines.

How the events of mitosis are coordinated is not well understood. Intriguing mitotic regulators include the chromosomal passenger proteins. Loss of either of the passengers inner centromere protein (INCENP) or the Aurora B kinase results in chromosome segregation defects and failures in cytokinesis. Furthermore, INCENP and Aurora B have identical localization patterns during mitosis and directly bind each other in vitro. These results led to the hypothesis that INCENP is a direct substrate of Aurora B. Here we show that the Caenorhabditis elegans Aurora B kinase AIR-2 specifically phosphorylated the C. elegans INCENP ICP-1 at two adjacent serines within the carboxyl terminus. Furthermore, the full length and a carboxyl-terminal fragment of ICP-1 stimulated AIR-2 kinase activity. This increase in AIR-2 activity required that AIR-2 phosphorylate ICP-1 because mutation of both serines in the AIR-2 phosphorylation site of ICP-1 abolished the potentiation of AIR-2 kinase activity by ICP-1. Thus, ICP-1 is directly phosphorylated by AIR-2 and functions in a positive feedback loop that regulates AIR-2 kinase activity. Since the Aurora B phosphorylation site within INCENP and the functions of INCENP and Aurora B have been conserved among eukaryotes, the feedback loop we have identified is also likely to be evolutionarily conserved.

Accurate chromosome segregation during cell division requires not only the establishment, but also the precise, regulated release of chromosome cohesion. Chromosome dynamics during meiosis are more complicated, because homologues separate at anaphase I whereas sister chromatids remain attached until anaphase II. How the selective release of chromosome cohesion is regulated during meiosis remains unclear. We show that the aurora-B kinase AIR-2 regulates the selective release of chromosome cohesion during Caenorhabditis elegans meiosis. AIR-2 localizes to subchromosomal regions corresponding to last points of contact between homologues in metaphase I and between sister chromatids in metaphase II. Depletion of AIR-2 by RNA interference (RNAi) prevents chromosome separation at both anaphases, with concomitant prevention of meiotic cohesin REC-8 release from meiotic chromosomes. We show that AIR-2 phosphorylates REC-8 at a major amino acid in vitro. Interestingly, depletion of two PP1 phosphatases, CeGLC-7alpha and CeGLC-7beta, abolishes the restricted localization pattern of AIR-2. In Ceglc-7alpha/beta(RNAi) embryos, AIR-2 is detected on the entire bivalent. Concurrently, chromosomal REC-8 is dramatically reduced and sister chromatids are separated precociously at anaphase I in Ceglc-7alpha/beta(RNAi) embryos. We propose that AIR-2 promotes the release of chromosome cohesion via phosphorylation of REC-8 at specific chromosomal locations and that CeGLC-7alpha/beta, directly or indirectly, antagonize AIR-2 activity.

A diverse array of sessile marine invertebrates mate by passive dispersal of sperm which fertilize the brooded eggs of neighbours. In two such species, a sea-mat (phylum Bryozoa) and an ascidian (phylum Chordata), vitellogenic egg growth is absent in reproductively isolated specimens, but is triggered by a water-borne factor released by conspecifics. In both of these colonial, hermaphroditic species, the active factor can be removed from water by filtration. The effect involves self-/non-self-recognition: water conditioned by a separate subcolony of the same genetic individual does not prompt oocyte growth. In each species, allosperm move from the surrounding water to the ovary and are then stored in close association with the growing oocytes. We concluded that sperm themselves are the water-borne factor that triggers the major phase of female reproductive investment. This mechanism is, to our knowledge, previously undescribed in animals, but has parallels with the initiation of maternal investment in flowering plants following the receipt of compatible pollen. The species studied may be representative of many other aquatic invertebrates which mate in a similar way. The stimulation of egg growth by allosperm could lead to intersexual conflict during oogenesis.

The compound ascidian Diplosoma listerianum releases aquatic sperm which are dispersed passively to potential mates as individual gametes prior to storage of sperm, internal fertilization and brooding of embryos. The storage of exogenous sperm enables D. listerianum to produce a lengthy series of progeny following a brief period of mating. Molecular paternity analysis following sequential mating of colonies in laboratory culture revealed a consistent pattern with a clear initial bias in paternity towards the first of two acting males. The sites of sperm storage and fertilization and the morphology of the ovary in D. listerianum suggest that this bias reflects first-in-first-out use of individual stored gametes. The proportion of second-male paternity subsequently increased with time within the progeny arrays. This may have reflected the ageing or passive loss of first-male sperm. It is also possible that the modular nature of the organism contributed to this temporal trend: any recently budded colony modules maturing in the interval between matings would have been available exclusively to second-male sperm as virgin zooids. Two sets of mating trials were run. In the first, the collection of progeny suffered an interruption of 13 days and each male gained a larger proportion of recorded paternity within the progeny analysed when mating first rather than when mating second. In one mating combination, the first male obtained almost 100% of recorded paternity. In the second set of trials, with different clonal combinations, the complete sequence of progeny was collected and the estimated overall proportion of second-male paternity (P2) was consistently > 0.5. Taken as a whole, the results suggest that the overall P2-value can vary widely within the population studied. Proposed mechanisms of mating-order effects in species with copulatory mating include several which can have no counterpart in indirect aquatic mating since they involve the active removal, sealing off, volumetric displacement or incapacitation of first-male ejaculates. It is nevertheless clear that mating-order effects can be pronounced during the type of non-copulatory mating examined here, which is widespread in marine invertebrates.

Physiological and behavioral traits of sexually mature boars were compared between episodes of copulation and sexual frustration in order to determine reliable indicators of the differences in emotional states. Ten boars, approximately 6 mo of age, were trained to mount a stationary artificial sow (ArtSow) and to ejaculate when digital pressure was applied to the extended penis. This method of semen collection is the typical procedure of the industry. All 10 boars used in this study were fully trained to this procedure before the onset of the study. Each boar was subjected to trials in which one of the following two treatments was applied. In the control (CTRL) treatment, boars were treated the same as during their training (i.e., allowed to complete ejaculation). In the frustration (FRUS) treatment, boars were allowed to mount the ArtSow, but because no manual pressure was applied to the extended penis, ejaculation never occurred. Blood was collected via indwelling catheters before onset of the trial, during exposure to the ArtSow, and after returning to their home pen. Concentrations of testosterone, cortisol, and beta-endorphin were quantified. Behavior of the boars was recorded during exposure to the ArtSow and for 30 min after return to their home pen. Relative to preexposure levels, serum cortisol increased (P<.05) during CTRL exposure and after exposure to both treatments (CTRL; P<.04 and FRUS; P<.06). Serum testosterone did not change during and after either treatment. Serum concentrations of beta-endorphin did not change during or after CTRL trials, but serum beta-endorphin was greater (P<.05) during FRUS than during CTRL trials. Behavioral analysis revealed that boars spent less time lying down and more time moving about their home pen (P<.05) after a FRUS than after a CTRL trial. In summary, serum cortisol did not allow us to distinguish between the excitement of copulation and the negative affect associated with sexual frustration, whereas increases in serum beta-endorphin and motor activity seemed to be indicators of the negative emotional state of sexual frustration in trained boars.

Canada goose droppings, collected in parks to which the public had access, were screened for a range of bacteria that could be pathogenic in man. Droppings of Canada geese, and other waterfowl, did contain such bacteria, including some that are well-known causes of illness in man. These bacteria, plus a species of Salmonella that was experimentally inoculated into droppings, were shown to survive and multiply in the droppings for up to one month after their deposition by geese. Canada geese ranged further from water than other waterfowl species and thus distributed their droppings over a larger area of park grassland. This more widespread distribution of their droppings leads Canada geese to pose a greater potential health risk than other waterfowl studied here, but variations in human responses to challenge with bacteria, and variations in human and waterfowl behaviour in public parks, renders quantification of this risk impossible.