Real-time PCR, qPCR, Primer design

The aim of this study was to assess physical and chemical quality of raw cow's milk produced and marketed in Shashemene town, Ethiopia. A total of 48 samples of raw cow's milk were collected in the morning. All of the samples were collected using proportional random sampling method. The means for temperature, pH, specific gravity, titratable acidity, total solids, fat, solids-not-fat, protein, ash and lactose contents of milk samples were 22.83 ± 1.22°C, 6.32 ± 0.07, 1.030 ± 0.000, 0.194 ± 0.006%, 12.87 ± 0.11%, 4.28 ± 0.05%, 8.59 ± 0.07%, 3.43 ± 0.00%, 0.74 ± 0.00% and 4.43 ± 0.06%, respectively. Significant differences (P<0.05) were found for the values of temperature, pH, titratable acidity, total solids, fat, protein, ash and lactose contents between the sources of milk samples (dairy cooperative milk collection centers, hotels, small shops and small scale milk producers). Therefore, it was concluded that the chemical composition was adequate as compared to the standard level.

Pathogen prevalence within blacklegged ticks (Ixodes scapularis Say, 1821) tends to vary across sites and geographic regions, but the underlying causes of this variation are not well understood. Efforts to understand the ecology of Lyme disease have led to the proposition that sites with higher host diversity will result in lower disease risk due to an increase in the abundance of inefficient reservoir species relative to the abundance of species that are highly competent reservoirs. Although the Lyme disease transmission cycle is often cited as a model for this “dilution effect hypothesis”, little empirical evidence exists to support that claim. Here we tested the dilution effect hypothesis for two pathogens transmitted by the blacklegged tick along an urban-to-rural gradient in the northeastern United States using landscape fragmentation as a proxy for host biodiversity. Percent impervious surface and habitat fragment size around each site were determined to assess the effect of landscape fragmentation on nymphal blacklegged tick infection with Borrelia burgdorferi and Anaplasma phagocytophilum. Our results do not support the dilution effect hypothesis for either pathogen and are in agreement with the few studies to date that have tested this idea using either a landscape proxy or direct measures of host biodiversity.

Real-time quantitative polymerase chain reaction (q-PCR) is an advanced molecular method for determining the amount of nucleic acid in both gene expression analysis and routine Deoxyribo Nucleic Acid (DNA) measurement. An accurate measurement method is essential given that the labelling threshold for genetically modified organisms (GMO) residues in food and feed products is 5% in Japan and 0,9% in the European Union. Determination of GMO components, quantification of exact amount and determination of trace amounts in food matrices are possible in q-PCR. Various q-PCR chemicals are used for this purpose. These; intercalation dyes, primary based, chemicals and probe based chemicals. With the increasing number of GMO products in the grocery stores, the number of analyses performed per sample and thus the cost of analysis increase. For this purpose, in GMO studies, improved detection methods are needed to determine the presence of GMOs in order to perform fast and economically feasible scans. In this study, q-PCR chemistries were compared in terms of cost, efficiency and applicability.

Karnal bunt (KB) in wheat caused by Tilletia indica Mitra has been placed as quarantine pest in more than 70 countries. Despite its economic importance, little information about the molecular components of disease resistance is known. To elucidate the molecular mechanisms involved in compatible and incompatible interactions of wheat genotypes to Tilletia indica Mitra, real time PCR based expression profiling of pathogenesis related (PR)-proteins (PR1, PR5 and PDF1.2) in resistant (HD 29) and susceptible (WL 711) genotype at different point times was carried out. All the three genes regulating accumulation of PR-proteins were always expressed at higher levels in resistant genotype relative to susceptible one. The average amount of individual PR protein induced in resistance cultivar was: PDF1.2 (16.33 folds), PR1 (15.93 folds) and PR5 (5.74 folds) relative to control. In case of susceptible cultivar, the expression of PDF1.2 (18.77 folds) was higher than PR1 (4.13 folds) and PR5 (3.12 folds) at 5 dpi and in next two days sharp decrease in the accumulation of PDF1.2 transcript (3.05 folds) was recorded.  The time of up-regulation of these genes differ in both genotypes, suggesting that the differences between these wheat genotypes in susceptibility and resistance are a matter of timing and magnitude of the expression of PR proteins.

Wastewater treatment plants (WWTPs) are an effective barrier in the protection of human and environment health around the world, although WWTPs also are suggested to be selectors and-or reservoirs of antibiotic resistance genes (ARGs) before entering the environment. The dogma about WWTPs as “ARG selectors” presumes that biotreatment compartments (e.g., activated sludge; AS) are single densely populated ecosystems with elevated horizontal gene transfer. However, recent work has suggested WWTP biotreatment compartments may be different than previously believed relative to antibiotic resistance (AR) fate, and other process factors, such as bacterial separation and specific waste sources, may be key to ARGs released to the environment. Here we combined 16S rRNA metagenomic sequencing and high-throughput qPCR to characterise microbial communities and ARGs across a wastewater network in Spain that includes both community (i.e., non-clinical urban) and hospital sources. Contrary to expectations, ARGs found in downstream receiving waters were not dominated by AS biosolids (RAS), but more resembled raw wastewater sources. In fact, ARGs and microbial communities in liquid-phase WWTP effluents and RAS were significantly different (Bray–Curtis dissimilarity index = 0.66 ± 0.11), with a consequential fraction of influent ARGs and organisms passing directly through the WWTP with limited association with RAS. Instead, ARGs and organisms in the RAS may be more defined by biosolids separation and biophysical traits, such as flocculation, rather than ARG carriage. This explains why RAS has significantly lower ARG richness (47 ± 4 ARGs) than liquid-phase effluents (104 ± 5 ARGs), and downstream water column (135 ± 4 ARGs) and river sediments (120 ± 5 ARGs) (Tukey's test, p < 0.001). These data suggest RAS and liquid-phase WWTP effluents may reflect two parallel ecosystems with potentially limited ARG exchange. As such, ARG mitigation in WWTPs should more focus on removing bacterial hosts from the liquid phase, AR source reduction, and possibly disinfection to reduce ARG releases to the environment.

Agriculture accounts for ~ 70% of all water use and the world population is increasing annually; soon more people will need to be fed, while also using less water. The use of plant-associated bacteria (PAB) is an eco-friendly alternative that can increase crop water use efficiency. This work aimed to study the effect of some PAB on increasing soybean tolerance to drought stress, the mechanisms of the drought tolerance process, and the effect of the PAB on promoting plant growth and on the biocontrol of Sclerotinia sclerotiorum. PAB were isolated from soybean rhizosphere and S. sclerotiorum sclerotia. The strains identified as UFGS1 (Bacillus subtilis), UFGS2 (Bacillus thuringiensis), UFGRB2 and UFGRB3 (Bacillus cereus) were selected on their ability to grow in media with reduced water activity. Soybean plants were inoculated with the PAB and evaluated for growth promotion, physiological and molecular parameters, after drought stress. Under drought stress, UFGS2 and UFGRB2 sustained potential quantum efficiency of PSII (Fv/Fm), while a decrease was found in the control plants. Moreover, UFGS2 and UFGRB3 maintained the photosynthetic rates in non-stressed conditions compared to the control. UFGS2-treated plants showed a higher stomatal conductance and higher transpiration than the control, after drought stress. Some PAB-treated plants also had other beneficial phenotypes, such as increases in fresh and dried biomass relative to the control. Differential gene expression analysis of genes involved in plant stress pathways shows changes in expression in PAB-treated plants. Results from this study suggest that PAB can mitigate drought stress in soybean and may improve water efficiency under certain conditions.

In this study, a quantitative real-time PCR (qPCR) assay targeting the second internal transcribed spacer (ITS2) of the nuclear-encoded ribosomal RNA gene (rDNA) was developed for Alexandrium tamiyavanichii, a harmful tropical marine dinoflagellate. This species is of concern because it produces toxins that cause paralytic shellfish poisoning (PSP). The qPCR assay employed hydrolysis probe technology and showed high specificity, with a detection limit of 102 gene copies (less than one cell equivalent). Using this assay, the spatial distribution of A. tamiyavanichii was assessed, for the first time, in the southeastern South China Sea and the Sulu Sea. Plankton samples were collected from 71 stations during a scientific cruise from the Research Vessel Sonne as part of the joint EU project on Stratosphere ozone: Halogens in a Varying Atmosphere (SHIVA), conducted in November 2011. The highest cell densities were detected offshore of Kuching, southern Borneo (150 cells l−1) and exceeded the threshold level of 20–40 cells l−1 where the bioaccumulation of PSP toxins by shellfish is of concern. The distribution of A. tamiyavanichii was patchy horizontally with the highest cell concentrations found mainly offshore of southern Borneo, and a heterogeneous vertical distribution was observed above the pycnocline. The A. tamiyavanichii qPCR assay proved its applicability, specificity and sensitivity, and provides an alternative implementation tool for harmful microalgae monitoring programs.

Mining activities pollute the environment with by-products that cause unpredictable impacts in surrounding areas. Cartagena-La Unión mine (Southeastern-Spain) was active for >2500years. Despite its closure in 1991, high concentrations of metals and waste residues remain in this area. A previous study using nematodes suggested that high lead content diminished soil biodiversity. However, the effects of mine pollution on specific ecosystem services remain unknown. Entomopathogenic nematodes (EPN) play a major role in the biocontrol of insect pests. Because EPNs are widespread throughout the world, we speculated that EPNs would be present in the mined areas, but at increased incidence with distance from the pollution focus. We predicted that the natural enemies of nematodes would follow a similar spatial pattern. We used qPCR techniques to measure abundance of five EPN species, five nematophagous fungi species, two bacterial ectoparasites of EPNs and one group of free-living nematodes that compete for the insect-cadaver. The study comprised 193 soil samples taken from mining sites, natural areas and agricultural fields. The highest concentrations of iron and zinc were detected in the mined area as was previously described for lead, cadmium and nickel. Molecular tools detected very low numbers of EPNs in samples found to be negative by insect-baiting, demonstrating the importance of the approach. EPNs were detected at low numbers in 13% of the localities, without relationship to heavy-metal concentrations. Only Acrobeloides-group nematodes were inversely related to the pollution gradient. Factors associated with agricultural areas explained 98.35% of the biotic variability, including EPN association with agricultural areas. Our study suggests that EPNs have adapted to polluted habitats that might support arthropod hosts. By contrast, the relationship between abundance of Acrobeloides-group and heavy-metal levels, revealed these taxa as especially well suited bio-indicators of soil mining pollution.

Methanogens, a key contributor in global carbon cycling, methane emission, and alternative energy production, generate methane gas via anaerobic digestion of organic matter. The methane emission potential depends upon methanogenic diversity and activity. Since they are anaerobes and difficult to isolate and culture, their diversity present in the landfill sites of Delhi and marshlands of Southern Assam, India, was analyzed using molecular techniques like 16S rDNA sequencing, DGGE, and qPCR. The sequencing results indicated the presence of methanogens belonging to the seventh order and also the order Methanomicrobiales in the Ghazipur and Bhalsawa landfill sites of Delhi. Sequences, related to the phyla Crenarchaeota (thermophilic) and Thaumarchaeota (mesophilic), were detected from marshland sites of Southern Assam, India. Jaccard analysis of DGGE gel using Gel2K showed three main clusters depending on the number and similarity of band patterns. The copy number analysis of hydrogenotrophic methanogens using qPCR indicates higher abundance in landfill sites of Delhi as compared to the marshlands of Southern Assam. The knowledge about " methanogenic archaea composition " and " abundance " in the contrasting ecosystems like " landfill " and " marshland " may reorient our understanding of the Archaea inhabitants. This study could shed light on the relationship between methane-dynamics and the global warming process.

The Chelonid herpesvirus 5 (ChHV5) has been consistently associated with fibropapil-lomatosis (FP), a transmissible neoplastic disease of marine turtles. Whether ChHV5 plays a causal role remains debated, partly because while FP tumours have been clearly documented to contain high concentrations of ChHV5 DNA, recent PCR-based studies have demonstrated that large proportions of asymptomatic marine turtles are also carriers of ChHV5. We used a real-time PCR assay to quantify the levels of ChHV5 Glycoprotein B (gB) DNA in both tumour and non-tumour skin tissues, from clinically affected and healthy turtles drawn from distant ocean basins across four species. In agreement with previous studies, higher ratios of viral to host DNA were consistently observed in tumour versus non-tumour tissues in turtles with FP. Unexpectedly however, the levels of ChHV5 gB DNA in clinically healthy turtles were significantly higher than in non-tumour tissues from FP positive turtles. Thus, a large proportion of clinically healthy sea turtle populations worldwide across species carry ChHV5 gB DNA presumably through persistent latent infections. ChHV5 appears to be ubiquitous regardless of the animals' clinical conditions. Hence, these results support the theory that ChHV5 is a near ubiquitous virus with latency characteristics requiring co-factors, possibly environmental or immune related, to induce FP.

Sweet corn, silage and grain crops have been facing the threat of severe corn disease for many years, spreading to various parts of Israel. Over the past few years, we conducted a series of field experiments, with the assistance of Netafim Israel, growers' associations and farmers from the Galilee region. In 2017-2018, this research led to a historic breakthrough: for the first time, 60 years since its discovery, an economically feasible solution was found for late wilt disease of corn in Israel. The successful treatment protocol is based on changing the cultivation method, changing the traditional irrigation method used in most corn growing areas in Israel, and the sophisticated integration of pesticide mixtures in a schedule adapted to key points in the development of the disease. This method can now be widely applied in commercial fields for the protection of sensitive corn varieties.

Late wilt disease in corn is caused by the fungus Harpophora maydis, which is distributed in the soil and seeds, penetrates the plant and blocks the water supply to its upper parts. The disease, considered to be the most severe corn disease in our region, was first discovered in Egypt in 1960, where it still causes serious damage. The disease is now reported in 11 countries. In Israel, it has existed for 40 years in the Upper Galilee, especially in the Hula Valley, and in the past decade, has spread to the south of the country. In heavily contaminated fields and in sensitive maize cultivars, the pathogen can cause 100% infection and total yield loss. The disease is characterized by rapid wilt of corn, which usually occurs two to three weeks before harvesting. In the past, attempts had been made to control the pathogen using different methods, including agricultural (balanced soil fertility and flood fallowing), chemical, biological, physical (solar heating) and plant compounds. Despite the potential of some of these methods, the only means applied in Israel to date to restrict the disease is the use of resistant corn varieties. However, in Egypt and Spain, virulent pathogen strains that threaten resistance maize cultivars have been found.

The research team led by Dr. Ofir Degani from the Migal - Galilee Research Institute and Tel-Hai College, funded and supported by Netafim Israel, the Consultation Service of the Israel Ministry of Agriculture and Rural Development, and growers' associations has recently led to a breakthrough of historical significance. For the first time, 60 years since the discovery of late wilt disease, an efficient and economically feasible solution has been found that can be used on a large scale to protect sensitive corn varieties in commercial fields.

This solution, published in December 2018 in the leading scientific journal PloS ONE, saves about 40% of irrigation costs and combines antifungal mixtures with different action mechanisms to prevent resistance development. The new application was tested in field trials in 2017-2018 and included the injection of irrigation material at intervals of 15, 30 and 45 days from sowing of the substances Difenoconazole + Azoxystrobin (commercial name Ortiva-Top, manufactured by Syngenta, Basel, Switzerland, supplied by Adama Makhteshim, Airport City, Israel) or other fungicide mixtures. Savings in the deployment of irrigation lines was ensured by using a drip line for two adjacent rows (a row spacing of 50 cm instead of 96 cm), a row space that maintains the effective concentration of the fungicide in the soil. Recently developed real-time PCR-based molecular detection showed that following the treatment of Ortiva-Top, the amount of pathogen DNA in the host tissue decreased to near zero levels. The successful treatments using Ortiva-Top resulted in a 100% decrease in dehydration symptoms and a 100% increase in crop yield, together with an approximately 40% increase in crop quality (the yield classified as A class that had a cob weight exceeding 250 g). These results were also obtained in an alternative protocol – the replacement of pesticides based on Ortiva-Top in the first application and the application of pesticides with a different mechanism of action in the second and third applications. A remote sensing evaluation of the efficacy of the treatments using a quadcopter equipped with a thermal camera carried out by Asaf Chen’s research team (Migal - Galilee Research Institute) supported the results and proved its effectiveness as a research tool for diagnosing infected fields.

These results are the fruits of a decade of research (since 2008) in which a fundamental understanding of the pathogen H. maydis and the generation and development of late wilt disease in Israel was established. During this period, new research methods such as sensitive molecular tests (PCR and qPCR) were developed to diagnose fungal violence and detect the pathogen inside the host plant tissue. In addition, a link was found to plant hormones that regulates the development of the pathogen. Complex field trials that were carried out annually over a full growing season (about 80 days) since 2009 led to this important discovery. However, the work on late wilt disease does not end here. In the past two years, Dr. Degani's research team identified the presence of H. maydis in secondary hosts, such as cotton, watermelon and green foxtail (Setaria viridis), which could assist in the survival of the pathogen, developed an environmentally friendly biological control method to control the pathogen, and established a new bio-assay for examining soils suspected of being infected with the pathogen. These new findings, which are currently in the process of being prepared for scientific publication, are encouraging the continuation and enhancement of the research.

Late wilt, a disease severely affecting maize fields throughout Israel, is characterized by relatively rapid wilting of maize plants before tasseling and until shortly before maturity. The disease's causal agent is the fungus Harpophora maydis, a soil-borne and seed-borne pathogen , which is currently controlled using reduced sensitivity maize cultivars. In a former study, we showed that Azoxystrobin (AS) injected into a drip irrigation line assigned for each row can suppress H. maydis in the field and that AS seed coating can provide an additional layer of protection. In the present study, we examine a more cost-effective protective treatment using this fungicide with Difenoconazole mixture (AS+DC), or Fluazinam, or Fluo-pyram and Trifloxystrobin mixture, or Prothioconazole and Tebuconazole mixture in combined treatment of seed coating and a drip irrigation line for two coupling rows. A recently developed Real-Time PCR method revealed that protecting the plants using AS+DC seed coating alone managed to delay pathogen DNA spread in the maize tissues, in the early stages of the growth season (up to the age of 50 days from sowing), but was less effective in protecting the crops later. AS+DC seed coating combined with drip irrigation using AS+DC was the most successful treatment, and in the double-row cultivation, it reduced fungal DNA in the host tissues to near zero levels. This treatment minimized the development of wilt symptoms by 41% and recovered cob yield by a factor of 1.6 (to the level common in healthy fields). Moreover, the yield classified as A class (cob weight of more than 250 g) increased from 58% to 75% in this treatment. This successful treatment against H. maydis in Israel can now be applied in vast areas to protect sensitive maize cultivars against maize late wilt disease.

Short indicator RNA sequences (,100 bp) persist after autoclaving and are recovered intact by molecular
amplification. Primers targeting longer sequences are most likely to produce false positives due to
amplification errors easily verified by melting curves analyses. If short indicator RNAsequences are used for
virus identification and quantification then post autoclave RNA degradation methodology should be
employed, which may include further autoclaving

Fluoxetine (FLX) is a pharmaceutical used to treat affective disorders in humans, but as environmental contaminant also affects inadvertently exposed fish in urban watersheds. In humans and fish, acute FLX treatment and exposure are linked to endocrine disruption, including effects on the reproductive and stress axes. Using the zebrafish model, we build on the recent finding that developmental FLX exposure reduced cortisol production across generations, to determine possible parental and/or life-stage-dependent (age and/or breeding experience) contributions to this phenotype. Specifically, we combined control and developmentally FLX-exposed animals of both sexes (F 0) into four distinct breeding groups mated at 5 and 9 months, and measured offspring (F 1) basal cortisol at 12 dpf. Basal cortisol was lower in F 1 descended from developmentally FLX-exposed F 0 females bred at 5, but not 9 months, revealing a maternal, life-stage dependent effect. To investigate potential molecular contributions to this phenotype, we profiled maternally deposited transcripts involved in endocrine stress axis development and regulation, epigenetic (de novo DNA methyltransferases) and post-transcriptional (miRNA pathway components and specific miRNAs) regulation of gene expression in unfertilized eggs. Maternal FLX exposure resulted in decreased transcript abundance of glucocorticoid receptor, dnmt3 paralogues and miRNA pathway components in eggs collected at 5 months, and increased transcript abundance of miRNA pathway components at 9 months. Specific miRNAs predicted to target stress axis transcripts decreased (miR-740) or increased (miR-26, miR-30d, miR-92a, miR-103) in eggs collected from FLX females at 5 months. Increased abundance of miRNA-30d and miRNA-92a persisted in eggs collected from FLX females at 9 months. Clustering and principal component analyses of egg transcript profiles separated eggs collected from FLX-females at 5 months from other groups, suggesting that oocyte molecular signatures, and miRNAs in particular, may serve as predictive tools for the offspring phenotype of reduced basal cortisol in response to maternal FLX exposure.

East African cichlid fishes represent a model to tackle adaptive changes and their connection to rapid speciation and ecological distinction. In comparison to bony craniofacial tissues, adaptive morphogenesis of soft tissues has been rarely addressed, particularly at the molecular level. The nuchal hump in cichlids fishes is one such soft-tissue and exaggerated trait that is hypothesized to play an innovative role in the adaptive radiation of cichlids fishes. It has also evolved in parallel across lakes in East Africa and Central America. Using gene expression profiling, we identified and validated a set of genes involved in nuchal hump formation in the Lake Malawi dolphin cichlid, Cyrtocara moorii. in particular, we found genes differentially expressed in the nuchal hump, which are involved in controlling cell proliferation (btg3, fosl1a and pdgfrb), cell growth (dlk1), craniofacial morphogenesis (dlx5a, mycn and tcf12), as well as regulators of growth-related signals (dpt, pappa and socs2). This is the first study to identify the set of genes associated with nuchal hump formation in cichlids. Given that the hump is a trait that evolved repeatedly in several African and American cichlid lineages, it would be interesting to see if the molecular pathways and genes triggering hump formation follow a common genetic track or if the trait evolved in parallel, with distinct mechanisms, in other cichlid adaptive radiations and even in other teleost fishes. Given the striking adaptive morphological diversity of craniofacial structures in teleost fish, it comes with no surprise that these differences in naturally occurring systems have garnered considerable attention in studies of developmental and molecular biology, beyond models like zebrafish 1,2. Myriad molecular players and interconnected signalling pathways, which participate in development and morphogenesis of craniofacial musculoskeletal structures, are described in teleost fish 3,4. However, when it comes to craniofacial soft tissues, little is known about the morphogenic molecular factors and underlying signals. In cichlids for instance, researchers have only recently attempted to investigate the potential molecular mechanisms forming soft-tissue traits such as enlarged lips and nose flaps 5-9. Such extreme and novel phenotypes are commonly referred to as exaggerated traits and are thought to have contributed to the evolutionary success of the cichlid adaptive radiation. Another exaggerated craniofacial phenotype that is observed in different groups of teleost, including cichlid fishes, is the overgrowth and protrusion of soft and possibly underlying hard tissues on the forehead, called the nuchal hump, also known as forehead swelling 10-15. In the Central American cichlid species, Amphilophus citrinel-lus, which is well-known for its large nuchal hump (in both sexes), the hump tissue seems to develop in response to hormonal stimulus and contain modified nuchal hypodermis and high amount of fat stored in the same tissue 10. Similar nuchal humps have evolved in parallel in African cichlids, which shared a common ancestor with the South American cichlids ~100 MYA 16,17. Both species of the Lake Tanganyika endemic tribe Cyphotilapiini (Cyphotilapia gibberosa, Cyphotilapia frontosa and Ctenochromis benthicola) have nuchal humps, as well as the species Cyrtocara moorii from the hyperdiverse radiation of the tribe haplochromini in Lake Malawi 17. The nuchal hump in Cyphotilapia gibberosa is also formed by hypertrophy of hypodermis and excessive fat storage of in this tissue 18. The exact function of the nuchal hump in fish is still a subject of debate; however, a few plausible hypotheses have been proposed. For instance, its potential role in sex recognition (in dimorphic species), species recognition, mechanical advantage in a fight, improved hydrodynamics and anti-predation role 11,13,18 .

Comparative analyses of gene regulation inform about the molecular basis of phenotypic trait evolution. Here, we address a fin shape phenotype that evolved multiple times independently across teleost fish, including several species within the family Cichlidae. In a previous study, we proposed a gene regulatory network (GRN) involved in the formation and regeneration of conspicuous filamentous elongations adorning the unpaired fins of the Neolamprologus brichardi. Here, we tested the members of this network in the blockhead cichlid, Steatocranus casuarius, which displays conspicuously elongated dorsal and moderately elongated anal fins. Our study provided evidence for differences in the anatomy of fin elongation and suggested gene regulatory divergence between the two cichlid species. Only a subset of the 20 genes tested in S. casuarius showed the qPCR expression patterns predicted from the GRN identified in N. brichardi, and several of the gene-by-gene expression correlations differed between the two cichlid species. In comparison to N. brichardi, gene expression patterns in S. casuarius were in better (but not full) agreement with gene regulatory interactions inferred in zebrafish. Within S. casuarius, the dorsoventral asymmetry in ornament expression was accompanied by differences in gene expression patterns, including potential regulatory differentiation, between the anal and dorsal fin. Gene regulatory networks (GRNs) describe the regulatory relationships among genes involved in the development and maintenance of phenotypes. A large body of work concentrated on selected developmental models, for instance sea urchins 1 , and synergistically constructed extensive GRNs including hundreds of transcription factors and signaling proteins. Additionally, comparative studies of gene regulation across different levels of biological organization and diversification (organs, individuals, populations, species) address the divergence and conservation of regulatory networks and their roles in evolutionary processes 2-4. For instance, parallel phenotypic variation across populations or species may be controlled by parallel regulatory changes, as demonstrated in the adaptive evolution of stickleback populations 5,6 and for leaf shape complexity in Solanum 7. In contrast, distinct changes in gene expression patterns were shown to underlie similar pigmentation patterns across Drosophila species 8. In fish, the molecular mechanisms controlling fin growth and regeneration have received particular attention not least due to their relevance for biomedical research 9. Most studies focussed on the caudal fin of the zebrafish Danio rerio, which exhibits a particularly well developed regeneration capacity 9. An additional reason to study the caudal fin of fishes in a comparative approach is to gain understanding about the evolution of body shape, for instance regarding the transition from the dorsoventrally asymmetrical (heterocercal) caudal fin of acip-enseriformes, gars and bowfin to the outer symmetry displayed by the homocercal caudal fins of teleost fish 10. Furthermore, within teleosts, variation in the outer shape and anatomy of caudal fins provided opportunities to identify genes and molecular mechanisms involved in fin shape formation 11-13. The examination of dorsoventral (a)symmetry in fish fin morphology can be extended to the dorsal and anal fins, e.g. by comparing the molecular correlates of ornamental filaments 14 or colour patterns 15 between these fins. In a previous study, we investigated the molecular mechanisms involved with the formation of elongated fin filaments, which adorn the dorsal, caudal and anal fins of the cichlid fish Neolamprologus brichardi 14,16. The extent of the outgrowth is similar across the

Background. Reference genes serve an important role as an endogenous control/s-tandard for data normalisation in gene expression studies. Although reference genes have recently been suggested for marsupials, independent analysis of reference genes on different immune tissues is yet to be tested. Therefore, an assessment of reference genes is needed for the selection of stable, expressed genes across different marsupial tissues. Methods. The study was conducted on red-tailed phascogales (Phascogale calura) using five juvenile and five adult males. The stability of five reference genes (glyceraldehyde-3-phosphate dehydrogenase, GAPDH ; β-actin, ACTB; 18S rRNA, 18S; 28S rRNA, 28S; and ribosomal protein L13A, RPL13A) was investigated using SYBR Green and analysed with the geNorm application available in qBase PLUS software. Results. Gene stability for juvenile and adult tissue samples combined show that GAPDH was most stable in liver and lung tissue, and 18S in small intestine and spleen. While all reference genes were suitable for small intestine and spleen tissues, all reference genes except 28S were stable for lung and only 18S and 28S were stable for liver tissue. Separating the two age groups, we found that two different reference genes were considered stable in juveniles (ACTB and GAPDH) and adults (18S and 28S), and RPL13A was not stable for juvenile small intestine tissue. Except for 28S, all reference genes were stable in juvenile and adult lungs, and all five reference genes were stable in spleen tissue. Discussion. Based on expression stability, ACTB and GAPDH are suitable for all tissues when studying the expression of marsupials in two age groups, except for adult liver tissues. The expression stability between juvenile and adult liver tissue was most unstable, as the stable reference genes for juveniles and adults were different. Juvenile and adult lung, small intestine and spleen share similar stable reference genes, except for small intestine tissues where all reference genes were stable in adults but RPL13A was not suitable in juveniles.

Bisphenol A (BPA) is an estrogenic contaminant linked to metabolic disruption. Developmental BPA exposure is of particular concern, as organizational effects may irreversibly disrupt metabolism at later life-stages. While BPA exposures in adult fish elicit metabolic perturbations similar to effects described in rodents, the metabolic effects of developmental BPA exposure in juvenile fish remain largely unknown. Following embryonic zebrafish exposure to BPA (0.1, 1 and 4 mg/L) and EE2 (10 ng/L) from 2 to 5 dpf, we assessed the metabolic phenotype in larvae (4–6 dpf) and juveniles (43–49 dpf) which had been divided into regular-fed and overfed groups at 29 dpf. Developmental BPA exposure in larvae dose-dependently reduced food-intake and locomotion and increased energy expenditure. Juveniles (29 dpf) exhibited a transient increase in body weight after developmental BPA exposure and persistent diet-dependent locomotion changes (43–49 dpf). At the molecular level, glucose and lipid metabolism-related transcript abundance clearly separated BPA exposed fish from controls and EE2 exposed fish at the larval stage, in juveniles on a regular diet and, to a lesser extent, in overfed juveniles. In general, the metabolic endpoints affected by BPA exposure were not mimicked by EE2 treatment. We conclude that developmental BPA exposure elicits acute metabolic effects in zebrafish larvae and fewer transient and persistent effects in juveniles and that these metabolic effects are largely independent of BPA’s estrogenicity.