Watermelon is one of the major Cucurbitaceae crops and the recent availability of genome sequence greatly facilitates the fundamental researches on it. Quantitative real-time reverse transcriptase PCR (qRT–PCR) is the preferred method for gene expression analyses, and using validated reference genes for normalization is crucial to ensure the accuracy of this method. However, a systematic validation of reference genes has not been conducted on watermelon. In this study, transcripts of 15 candidate reference genes were quantified in watermelon using qRT–PCR, and the stability of these genes was compared using geNorm and NormFinder. geNorm identified ClTUA and ClACT, ClEF1α and ClACT, and ClCAC and ClTUA as the best pairs of reference genes in watermelon organs and tissues under normal growth conditions, abiotic stress, and biotic stress, respectively. NormFinder identified ClYLS8, ClUBCP, and ClCAC as the best single reference genes under the above experimental conditions, respectively. ClYLS8 and ClPP2A were identified as the best reference genes across all samples. Two to nine reference genes were required for more reliable normalization depending on the experimental conditions. The widely used watermelon reference gene 18SrRNA was less stable than the other reference genes under the experimental conditions. Catalase family genes were identified in watermelon genome, and used to validate the reliability of the identified reference genes. ClCAT1and ClCAT2 were induced and upregulated in the first 24 h, whereas ClCAT3 was downregulated in the leaves under low temperature stress. However, the expression levels of these genes were significantly overestimated and misinterpreted when 18SrRNA was used as a reference gene. These results provide a good starting point for reference gene selection in qRT–PCR analyses involving watermelon.

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The effect of B. subtilis on growth performance of broilers was evaluated and the relationship between growth and caecal microbiota was revealed. The results of this study help to promote application of probiotics in poultry industry.

Summary In the present study, Bacillus subtilis Ch9 was evaluated as a probiotic in grass carp, Ctenopharyngodon idella (Valenciennes, 1844). For 56 days the grass carp (50 ± 2.5 g) were given a feed containing B. subtilis Ch9 in three concentrations: 1.0 × 109 (T1), 3.0 × 109 (T2) and 5.0 × 109 (T3) CFU kg−1 feed in triplicate treatments. The control group (T0) was given feed without B. subtilis Ch9 for the same period. Determined were the specific growth rate (SGR), feed conversion ratio (FCR), and digestive enzyme activities in the intestine and hepatopancreas as well as the intestinal microflora. After 56 days, fish receiving the diets supplemented with B. subtilis Ch9 showed significantly higher SGR and lower FCR (P < 0.05) than those fed the control diet. There was no significant different in SGR and FCR among T1, T2 and T3 nor was the survival rate affected (P > 0.05) by the dietary treatments. From days 14 to 56 of the experiment, higher protease, amylase and lipase activities in the foregut, midgut hindgut and hepatopancreas were observed in T1, T2 and T3 (P < 0.05) compared with the control over a short‐term (14–28 days). Enzyme activity did not increase after long‐term feeding with B. subtilis Ch9 (56 days), but was still higher than that of control fish (P < 0.05). Fish fed the probiotic had an increase in trend of total aerobic and facultative anaerobic bacterial quantity (P > 0.05), but the ratio of Bacillus was significantly higher (P < 0.05) than in control fish. The total anaerobic bacterial quantity, Bifidobacterium and Lactobacillus were significantly higher (P < 0.05) in fish fed B. subtilis Ch9 compared with fish fed control feed. In conclusion, an optimum dose of B. subtilis Ch9 could modulate intestinal microflora, induce digestive enzyme activity and potentially promote the digestion and absorption of nutrients, as well as improve the growth performance of grass carp significantly.

Melon (Cucumis melo. L) is not only an economically important cucurbitaceous crop but also an attractive model for studying many biological characteristics. Screening appropriate reference genes is essential to reverse transcription quantitative real-time PCR (RT-qPCR), which is key to many studies involving gene expression analysis. In this study, 14 candidate reference genes were selected, and the variations in their expression in roots and leaves of plants subjected to biotic stress, abiotic stress, and plant growth regulator treatment were assessed by RT-qPCR. The stability of the expression of the selected genes was determined and ranked using geNorm and NormFinder. geNorm identified the two most stable genes for each set of conditions: CmADP and CmUBIep across all samples, CmUBIep and CmRPL in roots, CmRAN and CmACT in leaves, CmADP and CmRPL under abiotic stress conditions, CmTUA and CmACT under biotic stress conditions, and CmRAN and CmACT under plant growth regulator treatments. NormFinder determined CmRPL to be the best reference gene in roots and under biotic stress conditions and CmADP under the other experimental conditions. CmUBC2 and CmPP2A were not found to be suitable under many experimental conditions. The catalase family genes CmCAT1, CmCAT2, and CmCAT3 were identified in melon genome and used as target genes to validate the reliability of identified reference genes. The catalase family genes showed the most upregulation 3 days after inoculation with Fusarium wilt in roots, after which they were downregulated. Their levels of expression were significantly overestimated when the unsuitable reference gene was used for normalization. These results not only provide guidelines for the selection of reference genes for gene expression analyses in melons but may also provide valuable information for studying the functions of catalase family genes in stress responses.

Necrotic enteritis (NE) caused by Clostridium perfringens is one of the most detrimental infectious diseases in poultry. This study examined the effect of blends of essential oils (BEOs) (25% thymol and 25% carvacrol) on NE and bacterial dynamics and functions in chicks challenged with C. perfringens. Chicks were assigned to a Control diet and BEOs diet (Control diet + 120 mg/kg BEOs), were challenged with C. perfringens from days 14 to 20 and were killed on day 21 for assessment. Supplementation with BEOs decreased the mortality, alleviated gut lesions, and decreased the virulence factors of pathogenic bacteria (VF 0073-ClpE, VF0124-LPS, and VF0350-BSH). Lack of supplementation also changed the nutrient and immunological dynamics of host microbiota in responding to C. perfringens infection. Adding BEOs changed the host ileum microbial population by increasing the numbers of Lactobacillus crispatus and Lactobacillus agilis, and decreasing Lactobacillus salivarius and Lactobacillus johnsonii. The functional roles of these changing host bacterial populations coupled with the putative reduced pathogenicity of C. perfringens by BEOs contributed to the reduction in gut lesions and mortality in infected chickens. It suggests that dietary supplementation with BEOs could significantly reduce the impact of NE caused by C. perfringens on broilers.

A 2×2 factorial experiment was conducted to study the effect of dietary calcium and non-phytate phosphorus (nPP) imbalance on calbindin and NaPi-IIb mRNA levels in the small intestine and tibia parameters of broiler chicks. One hundred and forty four 1-d-old Arbor Acres male broiler chicks were divided into four treatments consisted of six replicates with six chicks each. The two dietary calcium levels were 1.10% and 0.60%, and two dietary nPP levels were 0.50% and 0.27%. Results showed that a high Ca/nPP ratio diet (4.07:1) significantly depressed feed intake and weight gain of broilers (p<0.05), but a lower Ca:nPP ratio (1.2:1) had no influence (p>0.05). Low-Ca with low-P diet resulted in low tibia minerals and tibia breaking strength of broilers, and all the tibia parameters were further decreased when the dietary ratio of Ca to P was relative higher. Low dietary Ca or P up-regulated the calbindin and NaPi-IIb mRNA expression levels. Low Ca with normal P diet up-regulated duodenal calbindin mRNA expression level to the greatest extent. Low P with a normal Ca diet significantly enhanced NaPi-IIb mRNA expression level to the highest extent. These results suggest that the calbindin and NaPi-IIb mRNA expression were enhanced by the imbalance between dietary Ca and nPP, and their expression were not only influenced by Ca or nPP level, but also the ratio of Ca:nPP.

BackgroundCytokines are important mediators and regulators of host responses against foreign antigen, with their main function to orchestrate the functional activities of the cells of the immune system. However little is known about the role of cytokines in pathogenesis and immune responses caused by infectious bursa disease virus (IBDV). The aim of this study was to examine the transcripts of cell-mediated immune response-related cytokine genes in the bursal tissues of chickens infected with IBDVs of varying virulence to gain an understanding of pathological changes and mechanisms of immunosuppression caused by IBDV infection and the immune responses evoked.ResultsReal-time quantitative PCR analysis revealed that the expression levels of both Th1 [interferon (IFN)-γ, interleukins (IL)-2 and IL-12p40] and Th2 (IL-4, IL-5, IL-13 and IL-10) cytokines were significantly up-regulated following challenge with the H strain (vvIBDV) and up to 2- and 30-fold, respectively (P < 0.05). Following infection with the Ts strain (cell-adapted virus) these cytokine transcripts were up-regulated at 5 days post-infection (dpi), 2- and 13-fold respectively (P < 0.05), while the expression levels of IL-2 and IL-4 were not significantly different (P > 0.05). A higher degree of cytokine expression was induced by the H strain compared with the Ts strain.ConclusionThe results indicate that the expression of cell-mediated immune-related cytokine genes is strongly induced by IBDV, especially by the vvIBDV, H strain and reveal that these cytokines could play a crucial role in driving cellular immune responses during the acute phase of IBDV infection, and the cellular immune responses caused by IBDV of varying virulence are through different signaling pathways.