Gene expression studies in organisms are often conducted using reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR), and the accuracy of RT-qPCR results relies on the stability of reference genes. We examined ten candidate reference genes in Sclerodermus guani, a parasitoid wasp that is a natural enemy of long-horned beetle pests in forestry, including ACT, EF1α, Hsc70, Hsp70, SRSF7, α-tubulin, RPL7A, 18S, 28S, and SOD1, regarding variable biotic and abiotic factors such as body part, life stage, hormone, diet, and temperature. Data were analysed using four dedicated algorithms (ΔCt, BestKeeper, NormFinder, and geNorm) and one comparative tool (RefFinder). Our results showed that the most stable reference genes were RPL7A and EF1α regarding the body part, SRSF7 and Hsc70 regarding the diet, RPL7A and α-tubulin regarding the hormone, SRSF7 and RPL7A regarding the life stage, and SRSF7 and α-tubulin regarding temperature. To ascertain the applicability of specific reference genes, the expression level of the target gene (ACPase) was estimated regarding the body part using the most stable reference genes, RPL7A and EF1α, and the least stable one, SOD1. The highest expression level of ACPase was observed in the abdomen, and the validity of RPL7A and EF1α was confirmed. This study provides, for the first time, an extensive list of reliable reference genes for molecular biology studies in S. guani.

The silkworm Bombyx mori (Lepidoptera: Bombycidae) is a lepidopteran model insect of great economic importance. The parasitoid Exorista sorbillans (Diptera, Tachinidae) is the major pest of B. mori and also a promising candidate for biological control. However, the molecular interactions between hosts and dipteran parasitoids have only partially been studied. Gene expression analysis by reverse-transcription quantitative real-time polymerase chain reaction (RT-qPCR) is indispensable to characterise their interactions. Accurate normalisation of RT-qPCR-based gene expression requires the use of reference genes that are constantly expressed irrespective of experimental conditions. In this study, the expression stability of 13 traditionally used reference genes was estimated by five statistical algorithms (ΔCt, geNorm, Normfinder, BestKeeper, and RefFinder) to determine the best reference genes for gene expression studies in different tissues of B. mori under E. sorbillans parasitism. Specifically, TATA-box-binding protein was the best reference gene in epidermis and testis, while elongation factor 1α was the most stable gene in prothoracic gland and midgut. Elongation factor 1γ, ribosomal protein L3, actin A1, ribosomal protein L40, glyceraldehyde-3-phosphate dehydrogenase and eukaryotic translation initiation factor 4A were the most suitable genes in head, silk gland, fat body, haemolymph, Malpighian tubule and ovary, respectively. Our study offers a set of suitable reference genes for gene expression normalisation in B. mori under the parasitic stress of E. sorbillans, which will benefit the in-depth exploration of host-dipteran parasitoid interactions, and also provide insights for further improvements of B. mori resistance against parasitoids and biocontrol efficacy of dipteran parasitoids.

Exploration of novel strategies for early pregnancy diagnosis is pivotal in enhancing the reproductive potential and monetary gains from dairy herds. In buffalo, the trophectoderm cells of the elongating conceptus secrete interferon-tau that stimulates the transcription of various genes in peripheral blood mononuclear cells (PBMC) during the peri-implantation period. We explored the differential expression of classical (ISG15) and novel (LGALS3BP and CD9) early pregnancy markers in PBMC of buffaloes during various stages of pregnancy. Natural heat was detected in buffaloes by assessing the vaginal fluid, and artificial insemination (AI) was done. Whole blood was collected from the jugular vein in EDTA-containing vacutainers for PBMC isolation before AI (0-day) and 20, 25 and 40 d post-AI. On day 40, transrectal ultrasonography examination was performed to confirm pregnancy. The inseminated non-pregnant animals served as control. Total RNA was extracted using the TRIzol method. The temporal abundance of ISG15, LGALS3BP and CD9 genes in PBMC was compared between pregnant and non-pregnant groups (n = 9 per group) using real time-qPCR. Results showed transcripts of ISG15 and LGALS3BP were more abundant at 20 d in the pregnant group compared to the 0 d and 20 d values of the non-pregnant group. However, due to variability in expression, threshold (Ct) cycle of RT-qPCR alone could not distinguish pregnant and non-pregnant animals. In conclusion, ISG15 and LGALS3BP transcripts abundance in PBMCs are potential candidate biomarkers for early prediction of buffalo pregnancy 20-days post-AI, but further work is required to allow the development of a reliable new methodology.

Pre-harvest sprouting (PHS) induced by the absence of seed dormancy causes a severe reduction in crop yield and flour quality. In this study, we isolated and characterized TaABI4, an ABA-responsive transcription factor that participates in regulating seed germination in wheat. Sequence analysis revealed that TaABI4 has three homologues, located on chromosomes 1A/1B/1D. TaABI4 contains a conserved AP2 domain, and AP2-associated, LRP and potential PEST motifs. Putative cis-acting regulatory elements (CE1-like box, W-box, ABRE elements and RY elements) were identified in the TaABI4 promoter region that showed high conservation in 17 wheat cultivars and wheat-related species. Expression profiling of TaABI4 indicated that it is a seed-specific gene accumulating during the middle stages of seed development. Transcript accumulation of TaABI4 in wheat cultivar Chuanmai 32 (CM32, PHS susceptible) was 5.07-fold and 1.39-fold higher than that in synthetic hexaploidy wheat SHW-L1 (PHS resistant) at 15 and 20 DPA, respectively. Six expression quantitative trait loci (eQTL) of TaABI4 on chromosomes 2A, 2D, 3B and 4A were characterized based on the accumulated transcripts of TaABI4 in SHW-L1 and CM32-derived recombinant inbred lines. These QTLs explained 10.7 to 46.1% of the trait variation with 4.53–10.59 of LOD scores, which contain genes that may affect the expression of TaABI4.

Apis mellifera is infected by more than 24 virus species worldwide, mainly positive-sense, single-stranded RNA viruses of the Dicistroviridae and Iflaviridae families. Among the viruses that infect honey bees, Deformed wing virus is the most prevalent and is present as three master variants DWV-A, B, and C. Given that the ectoparasitic mite Varroa destructor vectors these virus variants, recombination events between them are expected, and variants and their recombinants can co-exist in mites and honeybees at the same time. In this study, we detect, through RT-qPCR, the presence of DWV-A and B in the same samples of adult bees from colonies of Argentina. Total RNA was extracted from pools of ten adult bees from 45 apiaries distributed across the main beekeeping Provinces of Argentina (Buenos Aires, Santa Fe, Córdoba, Santiago del Estero, Río Negro, and Mendoza); then RT-qPCR reactions were performed to detect DWV-A and B, with specific primer pairs. After the amplifications, PCR products (204 and 660 bp amplicons for DWV-B, and ~250 bp for DWV-A) were purified and sequenced to verify that they corresponded to reported sequences, analyzing them using the Blast software. Of the 45 samples analyzed by RT-qPCR, over 90% were infected with DWV-A and 47% were also positive for DWV-B, where it was found in high prevalence specifically in colonies of A. mellifera of the Buenos Aires Province. Future studies will determine the impact of this type of the virus and its ability to recombine with the other DWV types in the apiaries of our country.

Tramadol is a centrally acting analgesic drug, used for the management of moderate to severe pain in a variety of diseases. The long-term use of tramadol can induce endocrinopathy. This study aimed to evaluate the effect of tramadol dependence on the adrenal cortex and the effect of its withdrawal. Thirty adult male rats were divided into three experimental groups: the control group, the tramadol-dependent group that received increasing therapeutic doses of tramadol orally for 1 month, and the recovery group that received tramadol in a dose and duration similar to the previous group followed by a withdrawal period for another month. Specimens from the adrenal cortex were processed for histological, immunohistochemical, enzyme assay, and quantitative real-time PCR (RT-qPCR) studies. Tramadol induced a significant increase in malondialdehyde level and a significant decrease in the levels of glutathione peroxidase and superoxide dismutase. A significant decrease in the levels of adrenocorticotrophic hormones, aldosterone, cortisol, corticosterone, and dehydroepiandrosterone sulfate was also detected. Severe histopathological changes in the adrenal cortex were demonstrated in the form of disturbed architecture, swollen cells, and shrunken cells with pyknotic nuclei. Inflammatory cellular infiltration and variable-sized homogenized areas were also detected. A significant increase in P53 and Bax immunoreaction was detected and confirmed by RT-qPCR. The ultrastructural examination showed irregular, shrunken adrenocorticocytes with dense nuclei. Dilated smooth endoplasmic reticulum, mitochondria with disrupted cristae, and numerous coalesced lipid droplets were also demonstrated. All these changes started to return to normal after the withdrawal of tramadol. Thus, it was confirmed that the long-term use of tramadol can induce severe adrenal changes with subsequent insufficiency.

A stable reference gene is a key prerequisite for accurate assessment of gene expression. At present, the real-time reverse transcriptase quantitative polymerase chain reaction has been widely used in the analysis of gene expression in a variety of organisms. Neoseiulus barkeri Hughes (Acari: Phytoseiidae) is a major predator of mites on many important economically crops. Until now, however, there are no reports evaluating the stability of reference genes in this species. In view of this, we used GeNorm, NormFinder, BestKeeper, and RefFinder software tools to evaluate the expression stability of 11 candidate reference genes in developmental stages and under various abiotic stresses. According to our results, β-ACT and Hsp40 were the top two stable reference genes in developmental stages. The Hsp60 and Hsp90 were the most stable reference genes in various acaricides stress. For alterations in temperature, Hsp40 and α-TUB were the most suitable reference genes. About UV stress, EF1α and α-TUB were the best choice, and for the different prey stress, β-ACT and α-TUB were best suited. In normal conditions, the β-ACT and α-TUB were the two of the highest stable reference genes to respond to all kinds of stresses. The current study provided a valuable foundation for the further analysis of gene expression in N. barkeri.

Ribonucleic acid interference (RNAi) is a sequence-specific gene silencing mechanism induced by double-stranded RNA (dsRNA). Recently, RNAi has gained popularity as a reverse genetics tool owing to its tremendous potential in insect pest management, which includes Helicoverpa armigera. However, its efficiency is mainly governed by dsRNA concentration, frequency of application, target gene, etc. Therefore, to obtain a robust RNAi response in H. armigera, we evaluated various concentrations of dsRNA and its frequency of applications delivered through diet in silencing a midgut gene, chymotrypsin and a non-midgut gene, juvenile hormone acid methyl transferase (jhamt) of H. armigera. The extent of target gene silencing was determined by employing reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR). Our study revealed four significant findings: (i) single application of dsRNA elicited a delayed and transient silencing, while multiple applications resulted in early and persistent silencing of the above genes; (ii) silencing of the non-midgut gene (jhamt) through diet delivered dsRNA revealed prevalence of systemic silencing probably due to communication of silencing signals in this pest; (iii) the extent of silencing of chymotrypsin was positively correlated with dsRNA concentration and was negatively correlated with jhamt; (iv) interestingly, over-expression (15–18 folds) of an upstream gene, farnesyl diphosphate synthase (fpps), in juvenile hormone (JH) biosynthetic pathway at higher concentrations of jhamt dsRNA was the plausible reason for lesser silencing of jhamt. This study provides an insight into RNAi response of target genes, which is essential for RNAi design and implementation as a pest management strategy.

The reliability of reverse transcription quantitative real-time PCR (RT-qPCR) depends on normalising the mRNA abundance using carefully selected, stable reference genes. Our aim was to propose sets of reference genes for normalisation in bovine or caprine adipose tissue (AT), mammary gland, liver and muscle. All of these tissues contribute to nutrient partitioning and metabolism and, thus, to the profitability of ruminant productions (i.e. carcasses, meat and milk). In this study, eight commonly used reference genes that belong to different functional classes (CLN3, EIF3K, MRPL39, PPIA, RPLP0, TBP, TOP2B and UXT) were analysed using the geNorm procedure to determine the most stable reference genes in bovine and/or caprine tissues. Abundances and rankings of reference genes varied between tissues, species and the combination of tissues and/or species. Therefore, we proposed 29 sets of reference genes that differed depending on the tissue and/or species. As examples of the 29 sets, EIF3K, TOP2B and UXT were proposed as the most stable reference genes in bovine AT; UXT, EIF3K and RPLP0 were the most stable reference genes in bovine and caprine AT. The optimal number of reference genes for data normalisation was 3 for 27 of the proposed 29 sets. In two of the 29 sets, four to five reference genes were necessary for data normalisation when the number of studied tissues was increased. For example, UXT, EIF3K, TBP, TOP2B and CLN3 were required for data normalisation in bovine mammary gland, AT, muscle and liver. We have evaluated some of our proposed sets of reference genes for the normalisation of CD36 gene expression. Normalisation using the three most stable reference genes has revealed downregulation of CD36 gene expression in bovine mammary gland by a concentrate-based diet that is supplemented with sunflower oil and upregulation of CD36 gene expression in caprine liver by including a rapidly degradable starch in the diet. The dietary regulation of the gene expression of CD36 has been erased by normalisation with the least stable reference genes, which may result in misinterpretation of CD36 gene regulation. To conclude, our results provide valuable reference gene sets for other studies that aim to measure tissue and/or species-specific mRNA abundance in ruminants.