Sex‐specific graphs: Relating group‐specific topology to demographic and landscape data

Sex‐specific genetic structure is a commonly observed pattern among vertebrate species. Facing differential selective pressures, individuals may adopt sex‐specific life history traits that ultimately shape genetic variation among populations. Although differential dispersal dynamics are commonly detected in the literature, few studies have used genetic structure to investigate sex‐specific functional connectivity. The recent use of graph theoretic approaches in landscape genetics has demonstrated network capacities to describe complex system behaviours where network topology represents genetic interaction among subunits. Here, we partition the overall genetic structure into sex‐specific graphs, revealing different male and female dispersal dynamics of a fisher (Pekania [Martes] pennanti) metapopulation in southern Ontario. Our analyses based on network topologies supported the hypothesis of male‐biased dispersal. Furthermore, we demonstrated that the effect of the landscape, identified at the population level, could be partitioned among sex‐specific strata. We found that female connectivity was negatively correlated with snow depth, whereas connectivity among males was not. Our findings underscore the potential of conducting sex‐specific analysis by identifying landscape elements or configuration that differentially promotes or impedes functional connectivity between sexes, revealing processes that may otherwise remain cryptic. We propose that the sex‐specific graph approach would be applicable to other vagile species where differential sex‐specific processes are expected to occur.     

Species‐specific and temporal scale‐dependent responses of birds to drought

Global climate change is increasing the frequency and intensity of weather extremes, including severe droughts in many regions. Drought can impact organisms by inhibiting reproduction, reducing survival and abundance, and forcing range shifts. For birds, considering temporal scale by averaging drought‐related variables over different time lengths (i.e., temporal grains) captures different hydrologic attributes which may uniquely influence food supplies, vegetation greenness/structure, and other factors affecting populations. However, studies examining drought impacts on birds often assess a single temporal grain without considering that different species have different life histories that likely determine the temporal grain of their drought response. Furthermore, while drought is known to influence bird abundance and drive between‐year range shifts, less understood is whether it causes within‐range changes in species distributions. Our objectives were to (a) determine which temporal grain of drought (if any) is most related to bird presence/absence and whether this response is species specific; and (b) assess whether drought alters bird distributions by quantifying probability of local colonization and extinction as a function of drought intensity. We used North American Breeding Bird Survey data collected over 16 years, generalized linear mixed models, and dynamic occupancy models to meet these objectives. Different bird species responded to drought at different temporal grains, with most showing the strongest signal at annual or near‐annual grains. For all drought‐responsive species, increased drought intensity at any temporal grain always correlated with decreased occupancy. Additionally, colonization/extinction analyses indicated that one species, the dickcissel (Spiza americana), is more likely to colonize novel areas within the southern/core portion of its range during drought. Considering drought at different temporal grains, along with hydrologic attributes captured by each grain, may better reveal mechanisms behind drought impacts on birds and other organisms, and therefore improve understanding of how global climate change impacts species and the landscapes they inhabit.     

Female‐ and male‐specific signals of quality in the barn owl

Most bird studies of female signalling have been confined to species in which females display a male‐ornament in a vestigial form. However, a great deal of benefit may be gained from considering phenotypic traits that are specific to females. This is because (1) sex‐specific traits may signal sex‐specific qualities and (2) females may develop a male‐ornament not because they are selected to do so, but because fathers transmit to daughters the underlying genes for its expression (genetic correlation between the sexes). We investigated these two propositions in the barn owl Tyto alba, a species in which male plumage is lighter in colour and less marked with black spots than that of females. Firstly, we present published evidence that female plumage spottiness reflects parasite resistance ability. We also show that male plumage coloration is correlated with reproductive success, male feeding rate and heart mass. Secondly, cross‐fostering experiments demonstrate that plumage coloration and spottiness are genetically correlated between the sexes. This implies that if a given trait value is selected in one sex, the other sex will indirectly evolve towards a similar value. This prediction is supported by the observation that female plumage coloration and spottiness resembled that of males, in comparisons at the level of Tyto alba alba populations, Tyto alba subspecies and Tyto species. Our results therefore support the hypothesis that sex‐specific traits signal sex‐specific qualities and that a gene for a sex‐specific trait can be expressed in the other sex as the consequence of a genetic correlation between the sexes.     

Automated measurement of site‐specific N‐glycosylation occupancy with SWATH‐MS

Asparagine‐linked glycosylation is a common post‐translational modification of proteins catalyzed by oligosaccharyltransferase that is important in regulating many aspects of protein function. Analysis of protein glycosylation, including glycoproteomic measurement of the site‐specific extent of glycosylation, remains challenging. Here, we developed methods combining enzymatic deglycosylation and protease digestion with SWATH‐MS to enable automated measurement of site‐specific occupancy at many glycosylation sites. Deglycosylation with peptide‐endoglycosidase H, leaving a remnant N‐acetylglucosamine on asparagines previously carrying high‐mannose glycans, followed by trypsin digestion allowed robust automated measurement of occupancy at many sites. Combining deglycosylation with the more general peptide‐N‐glycosidase F enzyme with AspN protease digest allowed robust automated differentiation of nonglycosylated and deglycosylated forms of a given glycosylation site. Ratiometric analysis of deglycosylated peptides and the total intensities of all peptides from the corresponding proteins allowed relative quantification of site‐specific glycosylation occupancy between yeast strains with various isoforms of oligosaccharyltransferase. This approach also allowed robust measurement of glycosylation sites in human salivary glycoproteins. This method for automated relative quantification of site‐specific glycosylation occupancy will be a useful tool for research with model systems and clinical samples.     

Hunger‐dependent and sex‐specific antipredator behaviour of larvae of a size‐dimorphic mosquito

1. Modification of behaviours in the presence of predators or predation cues is widespread among animals. The costs of a behavioural change in the presence of predators or predation cues depend on fitness effects of lost feeding opportunities and, especially when organisms are sexually dimorphic in size or timing of maturation, these costs are expected to differ between the sexes. 2. Larval Aedes triseriatus (Say) (Diptera: Culicidae) were used to test the hypothesis that behavioural responses of the sexes to predation cues have been selected differently due to different energy demands. 3. Even in the absence of water‐borne predation cues, hungry females (the larger sex) spent more time browsing than did males, indicating a difference in energy needs. 4. In the presence of predation cues, well‐fed larvae of both sexes reduced their activity more than did hungry larvae, and males shifted away from high‐risk behaviours to a greater degree than did females, providing the first evidence of sex‐specific antipredator behaviour in foraging mosquito larvae. 5. Because sexual size dimorphism is common across taxa, and energetic demands are probably correlated with size dimorphism, this research demonstrates the importance of investigating sex‐specific behaviour and behavioural responses to enemies, and cautions against generalising results between sexes.     

Maternal pathogen exposure causes diet‐ and pathogen‐specific transgenerational costs

Transgenerational effects, whereby the environment experienced by a parent leads to an altered offspring phenotype, have now been described in a variety of taxa. In invertebrates, much of the research on these effects has concentrated on the role of parental exposure to pathogens or immune elicitors in determining offspring immune investment or disease resistance. To date, however, studies of transgenerational effects in invertebrates have generally been restricted to single infections or immune elicitors in ideal laboratory environments. Animals in field situations will commonly experience sub‐optimal environments and co‐infection by multiple species of parasites and pathogens, leading to increased relative costs of immune investment and changing fitness benefits from offspring responses to the parental environment. Here we investigate a more ecologically realistic scenario involving both multiple infections and resource limitation, using the Indian meal moth Plodia interpunctella as a model host, challenged with the entomopathogenic bacterium Bacillus thuringiensis and fungus Beauveria bassiana. Mothers were exposed to low doses of one or both pathogens, or a control. Offspring from each family were reared on either good‐ or poor‐quality food and then exposed to one or both pathogens. Maternal exposure to pathogens led to reduced pathogen resistance in offspring, depending on the combination of maternal and offspring pathogen‐specific infections and resource limitation in the offspring generation. Much research to date has focussed on trans‐generational immune priming, in which parental exposure to pathogens or immune elicitors leads to upregulated immune reactivity in their offspring. The lack of any such effects in our system suggests that the production of less resistant offspring following parental exposure to pathogens might be an important alternative, driven by costs of resistance rather than adaptive benefits.     

Cardiac‐specific overexpression of metallothionein attenuates L‐NAME‐induced myocardial contractile anomalies and apoptosis

Hypertension contributes to the high cardiac morbidity and mortality. Although oxidative stress plays an essential role in hypertensive heart diseases, the mechanism remains elusive. Transgenic mice with cardiac overexpression of metallothionein, a heavy metal‐binding scavenger, were challenged with N^G‐nitro‐L‐arginine methyl ester (L‐NAME) for 14 days prior to measurement of myocardial contractile and intracellular Ca²⁺ anomalies as well as cell signalling mechanisms using Western blot and immunofluorescence analysis. L‐NAME challenge elicited hypertension, macrophage infiltration, oxidative stress, inflammation and cardiac dysfunction manifested as increased proinflammatory macrophage marker F4/80, interleukin‐1β (IL‐1β), intracellular production, LV end systolic and diastolic diameters as well as depressed fractional shortening. L‐NAME treatment reduced mitochondrial membrane potential (MMP), impaired cardiomyocyte contractile and intracellular Ca²⁺ properties as evidenced by suppressed peak shortening, maximal velocity of shortening/relengthening, rise in intracellular Ca²⁺, along with elevated baseline and peak intracellular Ca²⁺. These unfavourable mechanical changes and decreased MMP (except blood pressure and macrophage infiltration) were alleviated by overexpression of metallothionein. Furthermore, the apoptosis markers including BAD, Bax, Caspase 9, Caspase 12 and cleaved Caspase 3 were up‐regulated while the anti‐apoptotic marker Bcl‐2 was decreased by L‐NAME treatment. Metallothionein transgene reversed L‐NAME‐induced changes in Bax, Bcl‐2, BAD phosphorylation, Caspase 9, Caspase 12 and cleaved Caspase 3. Our results suggest that metallothionein protects against L‐NAME‐induced myocardial contractile anomalies in part through inhibition of apoptosis.     

Serum‐specific stimulation of proliferation and mineralization of fish bone‐derived cells

Teleost fish have recently been implemented as suitable model organisms to study vertebrate development, in particular skeletogenesis. In vitro cell systems derived from fish bone have been successfully established, although their development has been hampered by the limited availability of fish serum to supplement culture medium. Commercially available sera are mostly of mammalian origin and thus not necessarily adequate to fish cell growth. The main objective of this work was to compare proliferative and mineralogenic potential of bovine and fish sera using fish bone‐derived cell lines VSa13 and VSa16. Fish serum was shown to (i) strongly stimulate cell proliferation in an apparent dose‐dependent and cell type‐specific manner, (ii) induce morphological changes, and (iii) enhance extracellular matrix mineralization of bone cells, although cytotoxic for fish osteoblast‐like cells at the concentration tested. To better understand mechanisms underlying mineralogenic effect of fish serum in fish chondrocytes, expression of several mineralization‐related genes was evaluated by qPCR. Regulation of matrix Gla protein (MGP) and bone morphogenetic protein 2 (BMP2) gene expression was modified upon culture with fish serum in a way compatible with an early onset and an increase in mineralization. In conclusion, fish serum was shown to be more adequate to proliferation and differentiation/mineralization of fish bone‐derived cells.     

A tetracycline‐inducible and skeletal muscle‐specific Cre recombinase transgenic mouse

We have generated a transgenic mouse that expresses Cre recombinase only in skeletal muscle and only following tetracycline treatment. This spatiotemporal specificity is achieved using two transgenes. The first transgene uses the human skeletal actin (HSA) promoter to drive expression of the reverse tetracycline‐controlled transactivator (rtTA). The second transgene uses a tetracycline responsive promoter to drive the expression of Cre recombinase. We monitored transgene expression in these mice by crossing them with ROSA26 loxP‐LacZ reporter mice, which express β‐galactosidase when activated by Cre. We find that the expression of this transgene is only detectable within skeletal muscle and that Cre expression in the absence of tetracycline is negligible. Cre is readily induced in this model with tetracycline analogs at a range of embryonic and postnatal ages and in a pattern consistent with other HSA transgenic mice. This mouse improves upon existing transgenic mice in which skeletal muscle Cre is expressed throughout development by allowing Cre expression to begin at later developmental stages. This temporal control of transgene expression has several applications, including overcoming embryonic or perinatal lethality due to transgene expression. This mouse is especially suited for studies of steroid hormone action, as it uses tetracycline, rather than tamoxifen, to activate Cre expression. In summary, we find that this transgenic induction system is suitable for studies of gene function in the context of hormonal regulation of skeletal muscle or interactions between muscle and motoneurons in mice. © 2009 Wiley Periodicals, Inc. Develop Neurobiol, 2009     

Tissue‐specific and light‐dependent regulation of phytochrome gene expression in rice

Phytochromes are red‐ and far red light photoreceptors in higher plants. Rice (Oryza sativa L.) has three phytochromes (phyA, phyB and phyC), which play distinct as well as cooperative roles in light perception. To gain a better understanding of individual phytochrome functions in rice, expression patterns of three phytochrome genes were characterized using promoter‐GUS fusion constructs. The phytochrome genes PHYA and PHYB showed distinct patterns of tissue‐ and developmental stage‐specific expression in rice. The PHYA promoter‐GUS was expressed in all leaf tissues in etiolated seedlings, while its expression was restricted to vascular bundles in expanded leaves of light‐grown seedlings. These observations suggest that light represses the expression of the PHYA gene in all cells except vascular bundle cells in rice seedlings. Red light was effective, but far red light was ineffective in gene repression, and red light‐induced repression was not observed in phyB mutants. These results indicate that phyB is involved in light‐dependent and tissue‐specific repression of the PHYA gene in rice.     

Patch exploitation strategies of parasitoids under indirect intra‐ and inter‐specific competition

1. Insect parasitoids are expected to evolve behavioural strategies to exploit resources in competitive environments optimally. Indirect competition between parasitoids is particularly common because exploited host patches remain available in the environment for other foraging individuals. 2. The effects of indirect competition on the behaviour of two closely related generalist egg parasitoids were investigated: Trichogramma pintoi Voegelé and Trichogramma minutum Riley (Hymenoptera: Trichogrammatidae). Patch residence time, a patch‐leaving mechanism, and progeny sex allocation of females foraging were analysed: (i) alone, (ii) in patches partially parasitised by conspecifics, and (iii) in patches partially parasitised by heterospecifics. 3. Each species responded differently to indirect competition. Trichogramma pintoi females shortened their patch residence times, but they did not adjust their progeny sex ratios. In contrast, T. minutum females did not modify their patch residence times, but they did increase their progeny sex ratios in response to competition. Both Trichogramma species used host rejection, either by antenna rejection or by ovipositor rejection, as a patch‐leaving mechanism. 4. In agreement with a companion study of direct competition using the same model species, the present results indicate that even amongst closely related species, responses to competition can vary considerably.     

Cardiomyocyte‐specific role of miR‐24 in promoting cell survival

Cardiomyocyte cell death is a major contributing factor to various cardiovascular diseases and is therefore an important target for the design of therapeutic strategies. More recently, stem cell therapies, such as transplantation of embryonic or induced pluripotent stem (iPS) cell‐derived cardiomyocytes, have emerged as a promising alternative therapeutic avenue to treating cardiovascular diseases. Nevertheless, survival of these introduced cells is a serious issue that must be solved before clinical application. We and others have identified a small non‐coding RNA, microRNA‐24 (miR‐24), as a pro‐survival molecule that inhibits the apoptosis of cardiomyocytes. However, these earlier studies delivered mimics or inhibitors of miR‐24 via viral transduction or chemical transfection, where the observed protective role of miR‐24 in cardiomyocytes might have partially resulted from its effect on non‐cardiomyocyte cells. To elucidate the cardiomyocyte‐specific effects of miR‐24 when overexpressed, we developed a genetic model by generating a transgenic mouse line, where miR‐24 expression is driven by the cardiac‐specific Myh6 promoter. The Myh6‐miR‐24 transgenic mice did not exhibit apparent difference from their wild‐type littermates under normal physiological conditions. However, when the mice were subject to myocardial infarction (MI), the transgenic mice exhibited decreased cardiomyocyte apoptosis, improved cardiac function and reduced scar size post‐MI compared to their wild‐type littermates. Interestingly, the protective effects observed in our transgenic mice were smaller than those from earlier reported approaches as well as our parallelly performed non‐genetic approach, raising the possibility that non‐genetic approaches of introducing miR‐24 might have been mediated via other cell types than cardiomyocytes, leading to a more dramatic phenotype. In conclusion, our study for the first time directly tests the cardiomyocyte‐specific role of miR‐24 in the adult heart, and may provide insight to strategy design when considering miRNA‐based therapies for cardiovascular diseases.     

Rapid detection and discrimination of fabaviruses by flow‐through hybridisation with genus‐ and species‐specific riboprobes

Viruses cause significant damage in agricultural crops worldwide. Disease management requires sensitive and specific tools for virus detection and identification. Also, detection techniques need to be rapid to keep pace with the continuous emergence of new viral diseases. The genus Fabavirus is composed of five viruses infecting many economically important crops worldwide. This research describes the development of a procedure based on flow‐through hybridisation (FTH), which is faster than and as sensitive as conventional hybridisation for virus detection in tissue‐prints from infected plants. Six digoxigenin‐labelled RNA probes were synthesised with two levels of specificity: (a) five specific for each viral species within this genus, and (b) a genus‐specific probe that hybridises with a nucleotide sequence signature only found in the 5′‐untranslated region of the genus Fabavirus, which is the first of this type reported for plant viruses. The new procedure developed is useful for rapid detection and discrimination of the five fabaviruses identified so far and opens the possibility of discovering new species of this genus.     

Isotype‐specific glycosylation analysis of mouse IgG by LC‐MS

With mice being the top model organism in immunology and with Fc glycosylation being increasingly recognized as important modulator of antibody function, the time has come to take a look at the glycosylation of mouse IgG isotypes. Tryptic glycopeptides of mouse IgG1, IgG2, and IgG3 differ in mass and so these three isoforms can be easily discriminated by MS. Commercial IgG contained a rare IgG1 variant but no IgG3, which, however, was found in sera of C57BL/6 and BALB/c mice. These strains deviated with regard to IgG2a and IgG2b alleles. The Ig2a B allele was not observed in any of the four samples investigated. All a/c isotypes contain the same glycopeptide sequence, which deviates from that of IgG2b by containing Leu instead of Ile. The Leu/Ile glycopeptide variants were separated by RP chromatography and the order of elution was determined. The major glycoforms on all isotypes were fucosylated with no and one galactose (GnGnF and GnAF) followed by fully galactosylated AAF and smaller amounts of mono‐ and disialylated N‐glycans. In the commercial serum pool, the relative ratios of glycans differed between isotypes. Sialic acid exclusively occurred as N‐glycolylneuraminic acid. Fucosylation was essentially complete. No bisected and no α1,3‐galactosylated glycans were found.