Impact of Formulation and Suspension Properties on Redispersion of Aluminum-Adjuvanted Vaccines.

The adsorption of antigens to the surface of 2 commonly used insoluble adjuvants, aluminum phosphate and aluminum hydroxide, has been well characterized. In spite of the pharmaceutical benefits, alum-based vaccine formulations can present challenges in redispersion of the final product after storage. Inability to resuspend alum-based vaccines during administration results in inadequate dosing, thus rendering the product unusable. Here, the influence of formulation conditions on the resuspendability of aluminum adjuvant-containing vaccines was investigated. Particle size analysis by Micro-Flow Imaging (MFI™), zeta potential measurement, and sedimentation analysis by Turbiscan® were used to characterize suspension properties. Ionic strength, pH, and antigen concentration were found to significantly influence sedimentation behavior, particle size, and redispersion. Increasing ionic strength increased the sedimentation rate of adjuvants favoring resuspendability. The addition of bovine serum albumin to aluminum phosphate reduced resuspendability more significantly than the addition of lysozyme. Decreased resuspendability correlated with an increase in fine-to-large particle ratio and decrease in sedimentation rate. In summary, resuspendability of adjuvant drug product is favored by increased flocculation, decrease in fine-to-large particle ratio, and reduction in surface charge of antigen and adjuvant. A careful balance of these formulation conditions can therefore be an effective means to mitigate challenges of alum adjuvant redispersion.

Multidimensional approach to invasive species prevention.

Nonindigenous species (NIS) cause global biotic homogenization and extinctions, with commercial shipping being a leading vector for spread of aquatic NIS. To reduce transport of NIS by ships, regulations requiring ballast water exchange (BWE) have been implemented by numerous countries. BWE appears to effectively reduce risk for freshwater ports, but provides only moderate protection of marine ports. In the near future, ships may be required to undertake ballast water treatment (BWT) to meet numeric performance standards, and BWE may be phased out of use. However, there are concerns that BWT systems may not operate reliably in fresh or turbid water, or both. Consequently, it has been proposed that BWE could be used in combination with BWT to maximize the positive benefits of both management strategies for protection of freshwater ports. We compared the biological efficacy of "BWE plus BWT" against "BWT alone" at a ballast water treatment experimental test facility. Our comparative evaluation showed that even though BWT alone significantly reduced abundances of all tested organism groups except total heterotrophic bacteria, the BWE plus BWT strategy significantly reduced abundances for all groups and furthermore resulted in significantly lower abundances of most groups when compared to BWT alone. Our study clearly demonstrates potential benefits of combining BWE with BWT to reduce invasion risk of freshwater organisms transported in ships' ballast water, and it should be of interest to policy makers and environmental managers.

Climate change, breeding date and nestling diet: how temperature differentially affects seasonal changes in pied flycatcher diet depending on habitat variation.

1. Climate warming has led to shifts in the seasonal timing of species. These shifts can differ across trophic levels, and as a result, predator phenology can get out of synchrony with prey phenology. This can have major consequences for predators such as population declines owing to low reproductive success. However, such trophic interactions are likely to differ between habitats, resulting in differential susceptibility of populations to increases in spring temperatures. A mismatch between breeding phenology and food abundance might be mitigated by dietary changes, but few studies have investigated this phenomenon. Here, we present data on nestling diets of nine different populations of pied flycatchers Ficedula hypoleuca, across their breeding range. This species has been shown to adjust its breeding phenology to local climate change, but sometimes insufficiently relative to the phenology of their presumed major prey: Lepidoptera larvae. In spring, such larvae have a pronounced peak in oak habitats, but to a much lesser extent in coniferous and other deciduous habitats. 2. We found strong seasonal declines in the proportions of caterpillars in the diet only for oak habitats, and not for the other forest types. The seasonal decline in oak habitats was most strongly observed in warmer years, indicating that potential mismatches were stronger in warmer years. However, in coniferous and other habitats, no such effect of spring temperature was found. 3. Chicks reached somewhat higher weights in broods provided with higher proportions of caterpillars, supporting the notion that caterpillars are an important food source and that the temporal match with the caterpillar peak may represent an important component of reproductive success. 4. We suggest that pied flycatchers breeding in oak habitats have greater need to adjust timing of breeding to rising spring temperatures, because of the strong seasonality in their food. Such between-habitat differences can have important consequences for population dynamics and should be taken into account in studies on phenotypic plasticity and adaptation to climate change.

Widespread genetic linkage of mating signals and preferences in the Hawaiian cricket Laupala.

The evolution of novel sexual communication systems is integral to the process of speciation, as it discourages gene flow between incipient species. Physical linkage between genes underlying male-female communication (i.e. sexual signals and preferences for them) facilitates both rapid and coordinated divergence of sexual communication systems between populations and reduces recombination in the face of occasional hybridization between diverging populations. Despite these ramifications of the genetic architecture of sexual communication for sexual selection and speciation, few studies have examined this relationship empirically. Previous studies of the closely related Hawaiian crickets Laupala paranigra and Laupala kohalensis have indirectly suggested that many of the genes underlying the difference in pulse rate of male song are physically linked with genes underlying the difference in female preference for pulse rate. Using marker-assisted introgression, we moved 'slow pulse rate' alleles from L. paranigra at five known quantitative trait loci (QTL) underlying male pulse rate into the 'fast pulse rate' genetic background of L. kohalensis and assessed the effect of these loci on female preference. An astounding four out of five song QTL predicted the preferences of female fourth-generation backcrosses, providing direct evidence for the extensive genetic linkage of song and preference in one of the fastest diversifying genera currently known.

Evaluating efficacy of an environmental policy to prevent biological invasions.

Enactment of any environmental policy should be followed by an evaluation of its efficacy to ensure optimal utilization of limited resources, yet measuring the success of these policies can be a challenging task owing to a dearth of data and confounding factors. We examine the efficacy of ballast water policies enacted to prevent biological invasions in the Laurentian Great Lakes. We utilize four criteria to assess the efficacy of this environmental regulation: (1) Is the prescribed management action demonstrably effective? (2) Is the management action effective under operational conditions? (3) Can compliance be achieved on a broad scale? (4) Are desired changes observed in the environment? The four lines of evidence resulting from this analysis indicate that the Great Lakes ballast water management program provides robust, but not complete, protection against ship-mediated biological invasions. Our analysis also indicates that corresponding inspection and enforcement efforts should be undertaken to ensure that environmental policies translate into increased environmental protection. Similar programs could be implemented immediately around the world to protect the biodiversity of the many freshwater ecosystems which receive ballast water discharges by international vessels. This general framework can be extended to evaluate efficacy of other environmental policies.

Multiple genetic linkages between female preference and male signal in rapidly speciating Hawaiian crickets.

Diverging sexual communication systems can lead to the evolution of new species that no longer recognize each other as potential mates. The coevolution of male and female components of sexual communication is facilitated by physical linkage between genes underlying signals and preferences. By crossing two closely related Hawaiian crickets (Laupala kohalensis and Laupala paranigra) with vastly different song pulse rates and female preferences, and assessing segregation of songs and preferences among second generation backcrosses, we show a strong genetic correlation between song and preference variation. Furthermore, multiple, but not all, quantitative trait loci underlying song variation also predict female preferences. This physical linkage or pleiotropy may have facilitated the striking diversification of pulse rates observed among Laupala species in conjunction with one of the most rapid species radiations so far recorded.

Life-history divergence facilitates regional coexistence of competing Ficedula flycatchers.

Regional coexistence of ecologically similar species is facilitated when fluctuations in environmental conditions favor different species at different times or places. However, why species with similar ecology should vary in their response to environmental change is unclear. In this study, we explore the role of a life-history divergence in causing changes in relative fitness across environmental conditions experienced by populations of two closely related Ficedula flycatchers on the Baltic island of Oland, Sweden. We compared patterns of nestling survival between Pied (Ficedula hypoleuca) and Collared (F. albicollis) Flycatchers in relation to two factors known to influence the environment experienced by nestlings: natural variation in their parents' onset of breeding and artificial manipulation of the brood size. Possible differences in the location of the nests (i.e., microhabitat differences) or in habitat use (i.e., feeding patterns) by the adult birds were controlled for by partial cross-fostering of young between the two species. We found that nestling mortality was relatively higher among Collared Flycatchers and that this difference increased with later breeding. Mass gain, which predicted survival probability, of nestling Collared Flycatchers did not respond to the seasonal decline in environmental conditions when they were raised in nests with reduced brood size (i.e., where sibling competition was experimentally reduced). This latter result suggests that the smaller clutch size of Collared Flycatchers reflects an adaptive adjustment to their offspring's higher sensitivity to environmental change. We discuss the possibility that the divergence in life-history traits between the two species represents adaptation to different environments experienced during their recent evolutionary history. We conclude that the survival of nestling Collared Flycatchers is more sensitive to harsh environment and that this is likely to limit where and when the more aggressive Collared Flycatchers are able to displace Pied Flycatchers. Our results provide support for models of species coexistence that emphasize the importance of spatial or temporal heterogeneity in relative fitness or life-history divergence. More precisely, our results demonstrate that variation in life-history adaptations may result in changes in relative fitness of species across environments despite their use of similar resources.

Postzygotic isolation over multiple generations of hybrid descendents in a natural hybrid zone: how well do single-generation estimates reflect reproductive isolation?

Understanding speciation depends on an accurate assessment of the reproductive barriers separating newly diverged populations. In several taxonomic groups, prezygotic barriers, especially preferences for conspecific mates, are thought to play the dominant role in speciation. However, the importance of postzygotic barriers (i.e., low fitness of hybrid offspring) may be widely underestimated. In this study, we examined how well the widely used proxy of postzygotic isolation (reproductive output of F(1) hybrids) reflects the long-term fitness consequences of hybridization between two closely related species of birds. Using 40 species-specific single nucleotide polymorphism (SNP) markers, we genotyped a mixed population of collared and pied flycatchers (Ficedula albicollis and F. hypoleuca) to identify grand- and great grand-offspring from interspecific crosses to derive an accurate, multigeneration estimate of postzygotic isolation. Two independent estimates of fitness show that hybridization results in 2.4% and 2.7% of the number of descendents typical of conspecific pairing. This postzygotic isolation was considerably stronger than estimates based on F(1) hybrids. Our results demonstrate that, in nature, combined selection against hybrids and backcrossed individuals may result in almost complete postzygotic isolation between two comparatively young species. If these findings are general, postzygotic barriers separating hybridizing populations may be much stronger than previously thought.

Natural and sexual selection against hybrid flycatchers.

While sexual selection is generally assumed to quickly cause or strengthen prezygotic barriers between sister species, its role in causing postzygotic isolation, through the unattractiveness of intermediate hybrids, is less often examined. Combining 24 years of pedigree data and recently developed species-specific molecular markers from collared (Ficedula albicollis) and pied (Ficedula hypoleuca) flycatchers and their hybrids, we were able to quantify all key components of fitness. To disentangle the relative role of natural and sexual selection acting on F1 hybrid flycatchers, we estimated various fitness components, which when combined represent the total lifetime reproductive success of F1 hybrids, and then compared the different fitness components of F1 hybrids to that of collared flycatchers. Female hybrid flycatchers are sterile, with natural selection being the selective force involved, but male hybrids mainly experienced a reduction in fitness through sexual selection (decreased pairing success and increased rate of being cuckolded). To disentangle the role of sexual selection against male hybrids from a possible effect of genetic incompatibility (on the rate of being cuckolded), we compared male hybrids with pure-bred males expressing intermediate plumage characters. Given that sexual selection against male hybrids is a result of their intermediate plumage, we expect these two groups of males to have a similar fitness reduction. Alternatively, hybrids have reduced fitness owing to genetic incompatibility, in which case their fitness should be lower than that of the intermediate pure-bred males. We conclude that sexual selection against male hybrids accounts for approximately 75% of the reduction in their fitness. We discuss how natural and sexual selection against hybrids may have different implications for speciation and conclude that reinforcement of reproductive barriers may be more likely when there is sexual selection against hybrids.

Effectiveness and potential toxicological impact of the PERACLEAN Ocean ballast water treatment technology.

The efficacy and the potential toxicological impact of a proposed ballast water treatment (PERACLEAN Ocean) using peracetic acid (PAA) as active substances to control species introduction was assessed in both fresh- and salt water experiments at very cold water temperatures (1-2 degrees C). Levels of PAA gradually declined over the 5-day experiments, while levels of hydrogen peroxide remained relatively stable. The rate of decay of both the PAA and hydrogen peroxide in water was accelerated in the presence of sediments. Water quality properties varied significantly with treatment level with a maximum reduction of pH by 2.0 units and a concomitant 20-fold increase in dissolved organic carbon levels. Living biomass of organisms in treated water was reduced by 99% after 2 days. Results from six toxicological tests revealed very steep dose-response curves of the treatment. The toxic response of treated waters was higher in fresh water than in salt water. The PERACLEAN Ocean treatment may represent an effective technology to treat ballast waters under a wide range of temperature and salinity conditions. The discharge of treated fresh water may however pose some toxicological risk to fresh water receiving environments and to cold waters in particular.

Sex chromosome-linked species recognition and evolution of reproductive isolation in flycatchers.

Interbreeding between species (hybridization) typically produces unfit offspring. Reduced hybridization should therefore be favored by natural selection. However, this is difficult to accomplish because hybridization also sets the stage for genetic recombination to dissociate species-specific traits from the preferences for them. Here we show that this association is maintained by physical linkage (on the same chromosome) in two hybridizing Ficedula flycatchers. By analyzing the mating patterns of female hybrids and cross-fostered offspring, we demonstrate that species recognition is inherited on the Z chromosome, which is also the known location of species-specific male plumage traits and genes causing low hybrid fitness. Limited recombination on the Z chromosome maintains associations of Z-linked genes despite hybridization, suggesting that the sex chromosomes may be a hotspot for adaptive speciation.

An introduction to Na(18)F bone scintigraphy: basic principles, advanced imaging concepts, and case examples.

Na(18)F, an early bone scintigraphy agent, is poised to reenter mainstream clinical imaging with the present generations of stand-alone PET and PET/CT hybrid scanners. (18)F PET scans promise improved imaging quality for both benign and malignant bone disease, with significantly improved sensitivity and specificity over conventional planar and SPECT bone scans. In this article, basic acquisition information will be presented along with examples of studies related to oncology, sports medicine, and general orthopedics. The use of image fusion of PET bone scans with CT and MRI will be demonstrated. The objectives of this article are to provide the reader with an understanding of the history of early bone scintigraphy in relation to Na(18)F scanning, a familiarity with basic imaging techniques for PET bone scanning, an appreciation of the extent of disease processes that can be imaged with PET bone scanning, an appreciation for the added value of multimodality image fusion with bone disease, and a recognition of the potential role PET bone scanning may play in clinical imaging.

Species divergence in offspring begging intensity: difference in need or manipulation of parents?

Conflicts over the delivery and sharing of food among family members are expected to lead to evolution of exaggerated offspring begging for food. Coevolution between offspring begging intensity and parent response depends on the genetic architecture of the traits involved. Given a genetic correlation between offspring begging intensity and parental response, there may be fast and arbitrary divergence in these behaviours between populations. However, there is limited knowledge about the genetic basis of offspring solicitation and parental response and whether these traits are genetically correlated. In this study, we performed a partial cross-fostering experiment of young between pied and collared flycatchers (Ficedula hypoleuca and Ficedula albicollis) and recorded the behaviour of individual offspring and their (foster)parents. We found that nestling collared flycatchers reached a higher phenotypic quality, estimated both as mass at fledging and as intensity of their T-lymphocyte-mediated immune response when raised by heterospecific foster parents. However, although collared flycatchers begged relatively more intensively, we found no evidence of corresponding higher resistance (i.e. lower feeding rate) of adult collared flycatchers than of adult pied flycatchers. Thus, the difference in offspring begging intensity between the two species seems not to be a result of a difference in escalation of the parent-offspring conflict. Instead, the species' divergence in exaggeration of offspring begging intensity 'honestly' matches a difference between the species in offspring need. This interpretation is strengthened by the fact that the difference in begging intensity between the two species increased as the season progressed, coinciding with the higher sensitivity of nestling collared flycatchers to the seasonal decline in food availability. Thus, the behavioural differentiation appears to be a direct consequence of a life-history differentiation (offspring growth patterns).

Cross-fostering reveals seasonal changes in the relative fitness of two competing species of flycatchers.

Spatial and temporal heterogeneity in relative fitness of competing species is a key factor affecting the structure of communities. However, it is not intuitive why species that are ecologically similar should differ in their response to environmental changes. Here we show that two sympatric flycatchers differ in reproductive strategy and in sensitivity to harsh environment. The fitness of collared flycatchers (Ficedula albicollis), which are dominant in interference competition, is more sensitive than the fitness of pied flycatchers (Ficedula hypoleuca) to the seasonal decline in environmental conditions. In order to control for the possibility that this pattern arises solely from differences in microhabitat use (i.e. a local niche differentiation), we performed a partial cross-fostering experiment of young between the two species (i.e. resulting in nests containing young of both species). Our results show that the growth of nestling pied flycatchers is less influenced by the seasonal decline in environmental conditions. We suggest that a life-history trade-off between interference competitive ability and robustness to harsh environment promotes a regional coexistence of the two species.

Hybridization cost of delayed maturation of secondary sexual traits in the collared flycatcher.

In many species, individuals do not attain their full adult coloration until one or several years after reaching sexual maturity, and this signaling of juvenile status is thought to enable young individuals to avoid aggression from older, dominant conspecifics. We propose that hybridization may be one of several costs and benefits associated with such delayed maturation. We tested this idea in a hybrid zone of collared (Ficedula albicollis) and pied (F. hypoleuca) flycatchers on the Baltic islands of Oland and Gotland. One-year-old (subadult) male collared flycatchers differed from older birds in many plumage traits, and approached male pied flycatchers in phenotype. On both islands, subadult male collared flycatchers hybridized at a higher rate than adults. Mate-choice experiments in aviaries suggest that this difference is at least partly due to female pied flycatchers having a preference for subadults when constrained to choose a heterospecific mate. Because novel morphologies are often derived from changes in ontology, juvenile forms may resemble adults of closely related taxa. When such juveniles are reproductively mature, their phenotypic similarity to the adults of closely related species may increase their risk of hybridization.

Testosterone is correlated with courtship but not aggression in the tropical buff-banded rail, Gallirallus philippensis.

Past studies into the roles of testosterone in birds have focused on species that occur in temperate regions. In such species, plasma testosterone levels are high during the establishment of territories in the spring and are associated with increased aggression. In contrast to most temperate species, tropical birds frequently defend territories year-round, during which time territoriality often occurs in a nonsexual context. The few studies that have been carried out on tropical birds show lower levels of circulating testosterone than occur in their temperate counterparts. In some year-round territorial tropical species, testosterone and aggression are dissociated, while in other species testosterone still plays a role in regulating aggression. This study examined the relationship between aggression and plasma testosterone levels in a year-round territorial, subtropical population of the buff-banded rail with characteristics typical of tropical species. Peak testosterone levels were substantially lower than those found in temperate species. Males displayed a seasonal peak in plasma testosterone level when their partners were most likely to be fertile. At other times, testosterone levels were mostly undetectable, despite year-round territoriality. We found that T levels increased with courtship behavior but showed no relationship with aggression, supporting the hypothesis that dissociation between testosterone and territoriality may be widespread among tropical avian taxa.