Multiple paternity in superfetatious live-bearing fishes.

Superfetation, the ability to carry several overlapping broods at different developmental stages, has evolved independently multiple times within the live-bearing fish family Poeciliidae. Even though superfetation is widespread among poeciliids, its evolutionary advantages remain unclear. Theory predicts that superfetation should increase polyandry by increasing the probability that temporally overlapping broods are fertilized by different fathers. Here, we test this key prediction in two poeciliid species that each carry two temporally overlapping broods: Poeciliopsis retropinna and P. turrubarensis. We collected 25 females per species from freshwater streams in South-Eastern Costa Rica and assessed multiple paternity by genotyping all their embryos (420 embryos for P. retropinna; 788 embryos for P. turrubarensis) using existing and newly developed microsatellite markers. We observed a high frequency of unique sires in the simultaneous, temporally overlapping broods in P. retropinna (in 56% of the pregnant females) and P. turrubarensis (79%). We found that the mean number of sires within females was higher than the number of sires within the separate broods (2.92 sires within mothers vs. 2.36 within separate broods in P. retropinna; and 3.40 vs 2.56 in P. turrubarensis). We further observed that there were significant differences in the proportion of offspring sired by each male in 42% of pregnant female P. retropinna and 65% of female P. turrubarensis; however, this significance applied to only 9% and 46% of the individual broods in P. retropinna and P. turrubarensis, respectively, suggesting that the unequal reproductive success of sires (i.e. reproductive skew) mostly originated from differences in paternal contribution between, rather than within broods. Together, these findings tentatively suggest that superfetation may promote polyandry and reproductive skew in live-bearing fishes.

Effects of lighting schedule during incubation of broiler chicken embryos on leg bone development at hatch and related physiological characteristics.

Providing a broiler chicken embryo with a lighting schedule during incubation may stimulate leg bone development. Bone development may be stimulated through melatonin, a hormone released in darkness that stimulates bone development, or increased activity in embryos exposed to a light-dark rhythm. Aim was to investigate lighting conditions during incubation and leg bone development in broiler embryos, and to reveal the involved mechanisms. Embryos were incubated under continuous cool white 500 lux LED light (24L), continuous darkness (24D), or 16h of light, followed by 8h of darkness (16L:8D) from the start of incubation until hatching. Embryonic bone development largely takes place through cartilage formation (of which collagen is an important component) and ossification. Expression of genes involved in cartilage formation (col1α2, col2α1, and col10α1) and ossification (spp1, sparc, bglap, and alpl) in the tibia on embryonic day (ED)13, ED17, and at hatching were measured through qPCR. Femur and tibia dimensions were determined at hatch. Plasma growth hormone and corticosterone and pineal melatonin concentrations were determined every 4h between ED18.75 and ED19.5. Embryonic heart rate was measured twice daily from ED12 till ED19 as a reflection of activity. No difference between lighting treatments on gene expression was found. 24D resulted in higher femur length and higher femur and tibia weight, width, and depth at hatch than 16L:8D. 24D furthermore resulted in higher femur length and width and tibia depth than 24L. Embryonic heart rate was higher for 24D and 16L:8D in both its light and dark period than for 24L, suggesting that 24L embryos may have been less active. Melatonin and growth hormone showed different release patterns between treatments, but the biological significance was hard to interpret. To conclude, 24D resulted in larger leg bones at hatch than light during incubation, but the underlying pathways were not clear from present data.

In vivo kinetics of cytokine expression during peritonitis in carp: evidence for innate and alternative macrophage polarization.

Despite the discovery of many cytokine genes in fish, knowledge on their functional homology is limited. To enlighten the biological function of inflammation-related mediators, we studied their kinetics of gene expression during peritonitis in carp. Zymosan-induced intraperitoneal influx of phagocytes reached a maximum at 24h. In peritoneal leukocytes (PTL) up-regulation of IL-1beta, TNF-alpha, CXCa, and chemokine receptor CXCR1 preceded this peak. Delayed up-regulation of these genes in the head kidney (HK) indicates emigration of antigen-presenting cells from peritoneum into HK and/or systemic spreading of inflammation. In turn, early increase in expression of anti-inflammatory genes in HK (6h) precede their up-regulation in the focus of inflammation. In PTL peaks of IL-10 and arginase 2 expression were recorded at 96 and 168h, respectively. These results give evidence that carp macrophages in vivo differentiate into a continuum of different activation states with innate and alternative activation representing the extremes.

Characterization of mutants with reduced seed dormancy at two novel rdo loci and a further characterization of rdo1 and rdo2 in Arabidopsis.

Seed dormancy and germination are complex traits that are controlled by many genes. Four mutants in Arabidopsis thaliana exhibiting a reduced dormancy phenotype, designated rdo1, rdo2, rdo3, and rdo4, have been characterized, both genetically and physiologically. Two of these mutants, rdo1 and rdo2, have been described before, the other two represent novel loci. The mutants mapped on chromosome 1 (rdo3), chromosome 2 (rdo2 and rdo4), and chromosome 3 (rdo1). None of these loci has been related to dormancy before. All four mutants show pleiotropic effects in the adult plant stage, which are different for each mutant. None of the mutants is deficient in ABA. Compared to Ler (wild-type), ABA sensitivity is not altered either, thereby excluding the possibility that ABA is involved in causing the reduced dormancy phenotype. The GA requirement was studied by using the GA biosynthesis inhibitor paclobutrazol, and genetically by generating double mutants with the GA-deficient mutant ga1-3. The results obtained by these two methods were comparable for all but one mutant: rdo1. In a GA-deficient background, rdo1, rdo2 and rdo3, all show sensitivity to GA between that of ga1-3 and ga1-3 aba1. However, when using paclobutrazol rdo1 exhibited the same sensitivity as rdo4 and wild-type. Analysis of double mutants among the rdo mutants revealed a very complex and inconsistent pattern.

Swim-training changes the spatio-temporal dynamics of skeletogenesis in zebrafish larvae (Danio rerio).

Fish larvae experience many environmental challenges during development such as variation in water velocity, food availability and predation. The rapid development of structures involved in feeding, respiration and swimming increases the chance of survival. It has been hypothesized that mechanical loading induced by muscle forces plays a role in prioritizing the development of these structures. Mechanical loading by muscle forces has been shown to affect larval and embryonic bone development in vertebrates, but these investigations were limited to the appendicular skeleton. To explore the role of mechanical load during chondrogenesis and osteogenesis of the cranial, axial and appendicular skeleton, we subjected zebrafish larvae to swim-training, which increases physical exercise levels and presumably also mechanical loads, from 5 until 14 days post fertilization. Here we show that an increased swimming activity accelerated growth, chondrogenesis and osteogenesis during larval development in zebrafish. Interestingly, swim-training accelerated both perichondral and intramembranous ossification. Furthermore, swim-training prioritized the formation of cartilage and bone structures in the head and tail region as well as the formation of elements in the anal and dorsal fins. This suggests that an increased swimming activity prioritized the development of structures which play an important role in swimming and thereby increasing the chance of survival in an environment where water velocity increases. Our study is the first to show that already during early zebrafish larval development, skeletal tissue in the cranial, axial and appendicular skeleton is competent to respond to swim-training due to increased water velocities. It demonstrates that changes in water flow conditions can result into significant spatio-temporal changes in skeletogenesis.

Colonization of the Arabidopsis rhizosphere by fluorescent Pseudomonas spp. activates a root-specific, ethylene-responsive PR-5 gene in the vascular bundle.

Plants of which the roots are colonized by selected strains of non-pathogenic, fluorescent Pseudomonas spp. develop an enhanced defensive capacity against a broad spectrum of foliar pathogens. In Arabidopsis thaliana, this rhizobacteria-induced systemic resistance (ISR) functions independently of salicylic acid but requires responsiveness to jasmonic acid and ethylene. In contrast to pathogen-induced systemic acquired resistance (SAR), ISR is not associated with systemic changes in the expression of genes encoding pathogenesis-related (PR) proteins. To identify genes that are specifically expressed in response to colonization of the roots by ISR-inducing Pseudomonas fluorescens WCS417r bacteria, we screened a collection of Arabidopsis enhancer trap and gene trap lines containing a transposable element of the Ac/Ds system and the GUS reporter gene. We identified an enhancer trap line (WET121) that specifically showed GUS activity in the root vascular bundle upon colonization of the roots by WCS417r. Fluorescent Pseudomonas spp. strains P. fluorescens WCS374r and P. putida WCS358r triggered a similar expression pattern, whereas ISR-non-inducing Escherichia coli bacteria did not. Exogenous application of the ethylene precursor 1-aminocyclopropane-1-carboxylate (ACC) mimicked the rhizobacteria-induced GUS expression pattern in the root vascular bundle, whereas methyl jasmonic acid and salicylic acid did not, indicating that the Ds element in WET121 is inserted in the vicinity of an ethylene-responsive gene. Analysis of the expression of the genes in the close vicinity of the Ds element revealed AtTLP1 as the gene responsible for the in cis activation of the GUS reporter gene in the root vascular bundle. AtTLP1 encodes a thaumatin-like protein that belongs to the PR-5 family of PR proteins, some of which possess antimicrobial properties. AtTLP1 knockout mutant plants showed normal levels of WCS417r-mediated ISR against the bacterial leaf pathogen Pseudomonas syringae pv. tomato DC3000, suggesting that expression of AtTLP1 in the roots is not required for systemic expression of ISR in the leaves. Together, these results indicate that induction of AtTLP1 is a local response of Arabidopsis roots to colonization by non-pathogenic fluorescent Pseudomonas spp. and is unlikely to play a role in systemic resistance.

Regulation of Arabidopsis thaliana Em genes: role of ABI5.

In order to identify new factors involved in Em (a class I Late Embryogenesis Abundant protein) gene expression, Arabidopsis mutants with an altered expression of an Em promoter GUS fusion construct and a modified accumulation of Em transcripts and proteins were isolated. Germination tests on ABA showed that the most affected mutant had a weak abi phenotype. Complementation tests further revealed this mutant to be a new abi5 allele, consequently named abi5-5. In addition to reducing the final level of Em transcripts in the dry seed, the abi5-5 mutation causes a delay in the accumulation of AtEm1 during seed development. An additional characteristic of the abi5-5 mutant, is the ability of its seeds to germinate at high concentrations of salt and mannitol. The abi5-5 mutation was characterized at the molecular level and was shown to result from a two base pair deletion in the coding sequence of the ABI 5 gene. The wild type and mutant recombinant proteins were produced in E. coli and were assayed for DNA-binding activity on their target promoters by electrophoretic mobility shift assay (EMSA). The ABI5 recombinant protein binds the ABRE sequence in the AtEm6 promoter as shown by Dnase footprinting. Among the ABRE-type sequences selected on both Em promoters, the G-box type AGACACGTGGCATGT element of the AtEm6 promoter shows the strongest binding by EMSA quantification.