Spatial variation of pollen receipt and effects of heterospecific pollen on seed set in Salvia przewalskii.

Generalized pollinators visit multiple co-flowering plant species and may transfer heterospecific pollen grains. Recent studies have indicated that the effect of heterospecific pollen (HP) on reproduction success is variable and depends on the identity of donor and recipient species. However, few studies have documented variation in HP receipt and evaluated the reproductive effects of HP receipt across geographic locations under natural conditions. We investigated the spatial variation of pollen deposition across eight sites and how the pollen receipt related to the seed set of Salvia przewalskii, a subalpine perennial herb in Hengduan Mountain in southwest China. We found that stigmatic pollen loads substantially varied among sites for several metrics, including quantities of conspecific and heterospecific pollen, the proportion of HP, and species composition of HP donors. Five different plant families were the most common HP source at one or two sites, and the proportion of HP ranged from 3.4% to 51.3% across sites. The association of conspecific pollen with seed set was positive and variable among sites, whereas the association of HP receipt and seed set was negative and not significantly different among sites. Our results demonstrate variation in the quantity and fitness effect of pollen receipt across sites, which is a precondition for evolution of local adaptation. Further study of variation in patterns and effects of HP receipt for the same recipient species across natural communities would allow better understanding of the ecological and evolutionary consequences of HP receipt.

Evidence for asymmetrical hybridization despite pre- and post-pollination reproductive barriers between two Silene species.

Interspecific hybridization is widespread among plants; nevertheless, pre- and post-zygotic isolating mechanisms may maintain species integrity for interfertile species in sympatry despite some gene flow. Interspecific hybridization and potential isolating barriers were evaluated between co-flowering Silene asclepiadea and Silene yunnanensis in an alpine community in southwest China. We investigated morphological and molecular (nuclear microsatellites and chloroplast gene sequence) variation in sympatric populations of S. asclepiadea and S. yunnanensis. Additionally, we analyzed pollinator behaviour and compared reproductive success between the putative hybrids and their parental species. Both the molecular and morphological data indicate that there were putative natural hybrids in the field, with S. asclepiadae the ovule parent and S. yunnanensis the pollen parent. Bumblebees were the primary visitors to S. asclepiadae and putative hybrids, while butterflies were the primary visitors to S. yunnanensis Pollen production and viability were significantly lower in putative hybrids than the parental species. The direction of hybridization is quite asymmetric from S. yunnanensis to S. asclepiadea Protandry combined with later peak flowering of S. yunnanensis, and pollinator preference may have contributed to the asymmetric pattern of hybridization, but putative hybrids were rare. Our results thus suggest that despite gene flow, S. asclepiadea and S. yunnanensis can maintain species boundaries, perhaps as a result of floral isolation and low fecundity of the hybrids.

Induction of phagocytosis and intracellular signaling by an inhibitory channel catfish leukocyte immune-type receptor: evidence for immunoregulatory receptor functional plasticity in teleosts.

Immunoregulatory receptors are categorized as stimulatory or inhibitory based on their engagement of unique intracellular signaling networks. These proteins also display functional plasticity, which adds versatility to the control of innate immunity. Here we demonstrate that an inhibitory catfish leukocyte immune-type receptor (IpLITR) also displays stimulatory capabilities in a representative myeloid cell model. Previously, the receptor IpLITR 1.1b was shown to inhibit natural killer cell-mediated cytotoxicity. Here we expressed IpLITR 1.1b in rat basophilic leukemia-2H3 cells and monitored intracellular signaling and functional responses. Although IpLITR 1.1b did not stimulate cytokine secretion, activation of this receptor unexpectedly induced phagocytosis as well as extracellular signal-related kinase 1/2- and protein kinase B (Akt)-dependent signal transduction. This novel IpLITR 1.1b-mediated response was independent of an association with the FcRγ chain and was likely due to phosphotyrosine-dependent adaptors associating with prototypical signaling motifs within the distal region of its cytoplasmic tail. Furthermore, compared to a stimulatory IpLITR, IpLITR 1.1b displayed temporal differences in the induction of intracellular signaling, and IpLITR 1.1b-mediated phagocytosis had reduced sensitivity to EDTA and cytochalasin D. Overall, this is the first demonstration of functional plasticity for teleost LITRs, a process likely important for the fine-tuning of conserved innate defenses.

Channel catfish leukocyte immune-type receptor mediated inhibition of cellular cytotoxicity is facilitated by SHP-1-dependent and -independent mechanisms.

Channel catfish (Ictalurus punctatus) leukocyte immune-type receptors (IpLITRs) are immunoregulatory proteins belonging to the immunoglobulin superfamily that likely play an important role in the regulation of teleost immune cell effector responses. IpLITRs are expressed by myeloid and lymphoid subsets and based on their structural features can be classified as either putative stimulatory or inhibitory forms. We have recently demonstrated at the biochemical and functional levels that stimulatory IpLITR-types induced intracellular signaling cascades resulting in immune cell activation. Alternatively, we have shown that putative inhibitory IpLITRs may abrogate immune cell responses by recruiting teleost Src homology 2 (SH2) domain-containing cytoplasmic phosphatases (SHP) to their tyrosine-containing cytoplasmic tails. In the present study, we used vaccinia virus to express recombinant chimeric proteins encoding the extracellular and transmembrane regions of human KIR2DL3 fused with the cytoplasmic tails of two putative inhibitory IpLITRs (i.e. IpLITR1.2a and IpLITR1.1b) in mouse spleen-derived cytotoxic lymphocytes. This approach allowed us to study the specific effects of IpLITR-induced signaling on lymphocyte killing of B cell targets (e.g. 721.221 cells) using a standard chromium release assay. Our results suggest that both IpLITR1.2a and IpLITR1.1b are potent inhibitors of lymphocyte-mediated cellular cytotoxicity. Furthermore, using a catalytically inactive SHP-1 mutant in combination with site-directed mutagenesis and co-immunoprecipitations, we also demonstrate that the IpLITR1.2a-mediated functional inhibitory response is SHP-1-dependent. Alternatively, IpLITR1.1b-mediated inhibition of cellular cytotoxicity is facilitated by both SHP-1-dependent and independent mechanisms, possibly involving the C-terminal Src kinase (Csk). The involvement of this inhibitory kinase requires binding to a tyrosine residue encoded in the unique membrane proximal cytoplasmic tail region of IpLITR1.1b. Overall, this represents the first functional information for inhibitory IpLITR-types and reveals that catfish LITRs engage SHP-dependent and -independent inhibitory signaling pathways to abrogate lymphocyte-mediated killing.

Effects of floral restrictiveness and stigma size on heterospecific pollen receipt in a prairie community.

Plant species vary greatly in the degree to which floral morphology restricts access to the flower interior. Restrictiveness of flower corollas may influence heterospecific pollen receipt, but the impact of floral morphology on heterospecific pollen transfer has received little attention. We characterized patterns of pollinator visitation and quantities of conspecific and heterospecific pollen receipt for 29 species with a range of floral morphologies in a prairie community dominated by the introduced plant Euphorbia esula (leafy spurge) which has an unrestrictive morphology. Pollinator overlap was significantly greater between Euphorbia and other unrestrictive flowers than restrictive flowers. Compared to flowers with restrictive morphologies, unrestrictive flowers received significantly more Euphorbia pollen, more heterospecific pollen from other sources, and a greater diversity of pollen species, but not more conspecific pollen. However, stigmatic surface area was significantly larger for flowers with unrestrictive morphologies, and the density of Euphorbia and other heterospecific pollen per stigmatic area did not significantly differ between flower types. These findings suggest that the smaller stigma size in restrictive flowers partly accounts for their decreased heterospecific pollen receipt, but that restrictiveness also allows species to increase the purity of pollen loads they receive. Given that restrictive flowers receive fewer heterospecific pollen grains but at a higher density, the effect of restrictiveness on fecundity depends on whether absolute quantity or density of heterospecific pollen affects fecundity more. Our results also indicate that abundant neighbors are not necessarily important heterospecific pollen sources since Euphorbia pollen was rarely abundant on heterospecifics.

Examination of the stimulatory signaling potential of a channel catfish leukocyte immune-type receptor and associated adaptor.

Expressed by various subsets of myeloid and lymphoid immune cells, channel catfish (Ictalurus punctatus) leukocyte immune-type receptors (IpLITRs) are predicted to play a key role in the initiation and termination of teleost cellular effector responses. These type I transmembrane proteins belong to the immunoglobulin superfamily and display features of immunoregulatory receptors with inhibitory and/or stimulatory signaling potential. Expanding on our previous work, which demonstrated that putative stimulatory IpLITR-types associated with the catfish adaptor proteins IpFcRγ and FcRγ-L, this study focuses on the functional significance of this immune receptor-adaptor signaling complex. Specifically, we generated an epitope-tagged chimeric receptor construct by fusing the extracellular domain of IpLITR 2.6b with the transmembrane region and cytoplasmic tail of IpFcRγ-L. This chimera was stably expressed in a rat basophilic leukemia (RBL) cell line, RBL-2H3, and following cross-linking of the surface receptor with an anti-hemagglutinin monoclonal antibody or opsonized microspheres, the chimeric teleost receptor induced cellular degranulation and phagocytic responses, respectively. Site-directed mutagenesis of the immunoreceptor tyrosine-based activation motif encoded within the cytoplasmic tail of the chimera confirmed that these functional responses were dependent on the phosphorylated tyrosines within this motif. Using a combination of phospho-specific antibodies and pharmacological inhibitors, we also demonstrate that the IpLITR/IpFcRγ-L-induced degranulation response requires the activity of Src homology 2 domain containing protein tyrosine phosphatases, phosphatidylinositol 3-kinase, protein kinase C, and mitogen-activated protein kinases but appears independent of the c-Jun N-terminal kinase and p38 MAP kinase pathways. In addition to this first look at stimulatory IpLITR-mediated signaling and its influence on cellular effector responses, the advantage of generating RBL-2H3 cells stably expressing a functional IpLITR-adaptor chimera will be discussed.

Ozone treatment ameliorates oil sands process water toxicity to the mammalian immune system.

We evaluated whether ozonation ameliorated the effects of the organic fraction of oil sands process water (OSPW) on immune functions of mice. Ozonation of OSPW eliminated the capacity of its organic fraction to affect various mouse bone marrow-derived macrophage (BMDM) functions in vitro. These included the production of nitric oxide and the expression of inducible nitric oxide synthase, the production of reactive oxygen intermediates and the expression of NADPH oxidase subunits, phagocytosis, and the expression of pro-inflammatory cytokine genes. Ozone treatment also eliminated the ability of OSPW organic fraction to down-regulate the expression of various pro-inflammatory cytokine and chemokine genes in the liver of mice, one week after oral exposure. We conclude that ozone treatment may be a valuable process for the remediation of large volumes of OSPW.

X linkage of AP3A, a homolog of the Y-linked MADS-box gene AP3Y in Silene latifolia and S. dioica.

BACKGROUND: The duplication of autosomal genes onto the Y chromosome may be an important element in the evolution of sexual dimorphism. A previous cytological study reported on a putative example of such a duplication event in a dioecious tribe of Silene (Caryophyllaceae): it was inferred that the Y-linked MADS-box gene AP3Y originated from a duplication of the reportedly autosomal ortholog AP3A. However, a recent study, also using cytological methods, indicated that AP3A is X-linked in Silene latifolia. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we hybridized S. latifolia and S. dioica to investigate whether the pattern of X linkage is consistent among distinct populations, occurs in both species, and is robust to genetic methods. We found inheritance patterns indicative of X linkage of AP3A in widely distributed populations of both species. CONCLUSIONS/SIGNIFICANCE: X linkage of AP3A and Y linkage of AP3Y in both species indicates that the genes' ancestral progenitor resided on the autosomes that gave rise to the sex chromosomes and that neither gene has moved between chromosomes since species divergence. Consequently, our results do not support the contention that inter-chromosomal gene transfer occurred in the evolution of SlAP3Y from SlAP3A.

Teleost IgSF immunoregulatory receptors.

In all animals innate immunity is the first line of immune defense from invading pathogens. The prototypical innate cellular responses such as phagocytosis, degranulation, and cellular cytotoxicity are elicited by leukocytes in a diverse range of animals including fish, amphibians, birds and mammals reinforcing the importance of such primordial defense mechanisms. In mammals, these responses are intricately controlled and coordinated at the cellular level by distinct subsets of immunoregulatory receptors. Many of these surface proteins belong to the immunoglobulin superfamily and in mammals elaborate immunoregulatory receptor networks play a major role in the control of infectious diseases. Recent examination of teleost immunity has begun to further illustrate the complexities of these receptor networks in lower vertebrates. However, little is known about the mechanisms that control how immunoregulatory receptors influence cellular decision making in ectothermic vertebrates. This review focuses on several families of recently discovered immunoglobulin superfamily members in fish that share structural, phylogenetic and in some cases functional relationships with mammalian immunoregulatory receptors. Further characterization of these teleost innate immune receptor families will provide detailed information regarding the conservation and importance of innate immune defense strategies throughout vertebrate evolution.

Asymmetrical conspecific seed-siring advantage between Silene latifolia and S. dioica.

BACKGROUND AND AIMS: Silene dioica and S. latifolia experience only limited introgression despite overlapping flowering phenologies, geographical distributions, and some pollinator sharing. Conspecific pollen precedence and other reproductive barriers operating between pollination and seed germination may limit hybridization. This study investigates whether barriers at this stage contribute to reproductive isolation between these species and, if so, which mechanisms are responsible. METHODS: Pollen-tube lengths for pollen of both species in styles of both species were compared. Additionally, both species were pollinated with majority S. latifolia and majority S. dioica pollen mixes; then seed set, seed germination rates and hybridity of the resulting seedlings were determined using species-specific molecular markers. KEY RESULTS: The longest pollen tubes were significantly longer for conspecific than heterospecific pollen in both species, indicating conspecific pollen precedence. Seed set but not seed germination was lower for flowers pollinated with pure heterospecific versus pure conspecific pollen. Mixed-species pollinations resulted in disproportionately high representation of nonhybrid offspring for pollinations of S. latifolia but not S. dioica flowers. CONCLUSIONS: The finding of conspecific pollen precedence for pollen-tube growth but not seed siring in S. dioica flowers may be explained by variation in pollen-tube growth rates, either at different locations in the style or between leading and trailing pollen tubes. Additionally, this study finds a barrier to hybridization operating between pollination and seed germination against S. dioica but not S. latifolia pollen. The results are consistent with the underlying cause of this barrier being attrition of S. dioica pollen tubes or reduced success of heterospecifically fertilized ovules, rather than time-variant mechanisms. Post-pollination, pre-germination barriers to hybridization thus play a partial role in limiting introgression between these species.

Stimulatory catfish leukocyte immune-type receptors (IpLITRs) demonstrate a unique ability to associate with adaptor signaling proteins and participate in the formation of homo- and heterodimers.

Channel catfish leukocyte immune-type receptors (IpLITRs) are immunoglobulin superfamily (IgSF) members believed to play a role in the control and coordination of cellular immune responses. Some IpLITR-types encode a transmembrane (TM) region containing a single positive charged lysine (K) residue, which is a key feature of stimulatory immune receptors that associate with immunoreceptor tyrosine-based activation motif (ITAM)-containing adaptor proteins. In this study we focused on identifying the signaling adaptor molecules recruited by putative stimulatory IpLITRs as a first step towards elucidating their ability to regulate catfish immune cell effector functions. Using HEK 293T cells co-transfected with epitope-tagged catfish proteins, we demonstrate that IpLITRs associated with the IpFcRgamma, IpFcRgamma-L, and IpCD3zeta-L adaptors, which all encode a negative charged aspartic acid (D) residue within their TM regions. Association of IpLITRs with IpFcRgamma and IpFcRgamma-L also enhanced cell surface expression of the receptor, which was not observed after co-transfections with IpCD3zeta-L, IpDAP12, or IpDAP10. Mutating the lysine residue (at amino acid position 199) within the TM region of IpLITR 2.6b to alanine (A(199)) did not prevent the association with IpFcRgamma-L and only slightly reduced receptor expression levels on the cell surface. Surprisingly, this mutation also facilitated IpLITR 2.6b association with IpDAP12 that correlated with an enhanced expression of the receptor. Conversely, an aspartic acid (D(30)) to A(30) switch within the IpFcRgamma-L TM region completely abrogated its assembly with the receptor and inhibited the IpFcRgamma-L induced surface expression of IpLITR 2.6b. In addition, co-transfections and immunoprecipitation of single (i.e. N-terminal HA) and double (i.e. N-terminal HA and C-terminal 3xFLAG) epitope-tagged stimulatory IpLITR-types revealed that these immune receptors formed non-covalent homo- and heterodimers through interaction(s) likely mediated by their extracellular immunoglobulin (Ig)-like domains. Combined with their unique association with adaptor proteins, dimerization may have profound effects on IpLITR-mediated regulation of teleost immune responses by influencing their signaling potential and/or ligand-binding properties.

Cell surface expression of channel catfish leukocyte immune-type receptors (IpLITRs) and recruitment of both Src homology 2 domain-containing protein tyrosine phosphatase (SHP)-1 and SHP-2.

Channel catfish leukocyte immune-type receptors (IpLITRs) are immunoglobulin superfamily (IgSF) members believed to play a role in the control and coordination of cellular immune responses in teleost. Putative stimulatory and inhibitory IpLITRs are co-expressed by different types of catfish immune cells (e.g. NK cells, T cells, B cells, and macrophages) but their signaling potential has not been determined. Following cationic polymer-mediated transfections into human cell lines we examined the surface expression, tyrosine phosphorylation, and phosphatase recruitment potential of two types of putative inhibitory IpLITRs using 'chimeric' expression constructs and an epitope-tagged 'native' IpLITR. We also cloned and expressed the teleost Src homology 2 domain-containing protein tyrosine phosphatases (SHP)-1 and SHP-2 and examined their expression in adult tissues and developing zebrafish embryos. Co-immunoprecipitation experiments support the inhibitory signaling potential of distinct IpLITR-types that bound both SHP-1 and SHP-2 following the phosphorylation of tyrosine residues within their cytoplasmic tail (CYT) regions. Phosphatase recruitment by IpLITRs represents an important first step in understanding their influence on immune cell effector functions and suggests that certain inhibitory signaling pathways are conserved among vertebrates.