Identification and evaluation of suitable reference genes for gene expression studies in the whitefly Bemisia tabaci (Asia I) by reverse transcription quantitative realtime PCR

This study presents a reliable method for performing reverse transcription quantitative realtime PCR (RT-qPCR) to measure gene expression in the whitefly Bemisia tabaci (Asia I) (Gennadius) (Hemiptera: Aleyrodidae), utilising suitable reference genes for data normalisation. We identified orthologs of commonly used reference genes (actin (ACT), cyclophilin 1 (CYP1), elongation factor 1α (EF1A), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), ribosomal protein L13a (RPL13A), and α-tubulin (TUB1A)), measured the levels of their transcripts by RT-qPCR during development and in response to thermal stress, and evaluated their suitability as endogenous controls using geNorm, BestKeeper, and NormFinder programs. Overall, TUB1A, RPL13A, and CYP1 were the most stable reference genes during B. tabaci development, and TUB1A, GAPDH, and RPL13A were the most stable reference genes in the context of thermal stress. An analysis of the effects of reference gene choice on the transcript profile of a developmentally-regulated gene encoding vitellogenin demonstrated the importance of selecting the correct endogenous controls for RT-qPCR studies. We propose the use of TUB1A, RPL13A, and CYP1 as endogenous controls for transcript profiling studies of B. tabaci development, whereas the combination of TUB1A, GAPDH, and RPL13A should be employed for studies into thermal stress. The data pre- sented here will assist future transcript profiling studies in whiteflies

Suitable Reference Gene Selection for Different Strains and Developmental Stages of the Carmine Spider Mite, Tetranychus cinnabarinus, using Quantitative Real-Time PCR

Reference genes are used as internal controls in gene expression studies, but their expression levels vary according to tissue types and experimental treatments. Quantitative real-time PCR (qPCR) is the most sensitive technique for transcript quantification provided that gene transcription patterns are normalized to an evaluated reference gene. In this study, the suitability of eight commonly used genes (β?-actin, 5.8SrRNA, α?-TUB, GAPDH, RPL13a, RPS18, TBP, SDHA) were cloned and investigated to find the most stable candidates for normalizing real-time PCR data generated from the four different strains (abamectin-resistant, fenpropathrin-resistant, omethoate-resistant, and susceptible strains) and different developmental stages (eggs, protonymphs, nymphs, and adults) of carmine spider mite, Tetranychus cinnabarinus (Boisduval) (Acarina: Tetranychidae). The stability of gene expression was assessed using two different analysis programs, geNorm and NormFinder. Using these analyses, RPS18 and 5.8SrRNA had the most stable expression regardless of the four different strains, whereas RPS18 and α?-TUB were expressed most stably in different developmental stages

Robust RT-qPCR Data Normalization: Validation and Selection of Internal Reference Genes during Post-Experimental Data Analysis

Reverse transcription and real-time PCR (RT-qPCR) has been widely used for rapid quantification of relative gene expression. To offset technical confounding variations, stably-expressed internal reference genes are measured simultaneously along with target genes for data normalization. Statistic methods have been developed for reference validation; however normalization of RT-qPCR data still remains arbitrary due to pre-experimental determination of particular reference genes. To establish a method for determination of the most stable normalizing factor (NF) across samples for robust data normalization, we measured the expression of 20 candidate reference genes and 7 target genes in 15 Drosophila head cDNA samples using RT-qPCR. The 20 reference genes exhibit sample-specific variation in their expression stability. Unexpectedly the NF variation across samples does not exhibit a continuous decrease with pairwise inclusion of more reference genes, suggesting that either too few or too many reference genes may detriment the robustness of data normalization. The optimal number of reference genes predicted by the minimal and most stable NF variation differs greatly from 1 to more than 10 based on particular sample sets. We also found that GstD1, InR and Hsp70 expression exhibits an age-dependent increase in fly heads; however their relative expression levels are significantly affected by NF using different numbers of reference genes. Due to highly dependent on actual data, RT-qPCR reference genes thus have to be validated and selected at post-experimental data analysis stage rather than by pre-experimental determination

Stable reference genes for the measurement of transcript abundance during larval caste development in the honeybee

Many genes are differentially regulated by caste development in the honeybee. Identifying and understanding these differences is key to discovering the mechanisms underlying this process. To identify these gene expression differences requires robust methods to measure transcript abundance. RT-qPCR is currently the gold standard to measure gene expression, but requires stable reference genes to compare gene expression changes. Such reference genes have not been established for honeybee caste development. Here, we identify and test potential reference genes that have stable expression throughout larval development between the two female castes. In this study, 15 candidate reference genes were examined to identify the most stable reference genes. Three algorithms (GeNorm, Bestkeeper and NormFinder) were used to rank the candidate reference genes based on their stability between the castes throughout larval development. Of these genes Ndufa8 (the orthologue of a component of complex one of the mitochondrial electron transport chain) and Pros54 (orthologous to a component of the 26S proteasome) were identified as being the most stable. When these two genes were used to normalise expression of two target genes (previously found to be differentially expressed between queen and worker larvae by microarray analysis) they were able to more accurately detect differential expression than two previously used reference genes (awd and RpL12). The identification of these novel reference genes will be of benefit to future studies of caste development in the honeybee

Differential Protein Expression in Honeybee (Apis mellifera L.) Larvae: Underlying Caste Differentiation

Honeybee (Apis mellifera) exhibits divisions in both morphology and reproduction. The queen is larger in size and fully developed sexually, while the worker bees are smaller in size and nearly infertile. To better understand the specific time and underlying molecular mechanisms of caste differentiation, the proteomic profiles of larvae intended to grow into queen and worker castes were compared at 72 and 120 hours using two dimensional electrophoresis (2-DE), network, enrichment and quantitative PCR analysis. There were significant differences in protein expression between the two larvae castes at 72 and 120 hours, suggesting the queen and the worker larvae have already decided their fate before 72 hours. Specifically, at 72 hours, queen intended larvae over-expressed transketolase, aldehyde reductase, and enolase proteins which are involved in carbohydrate metabolism and energy production, imaginal disc growth factor 4 which is a developmental related protein, long-chain-fatty-acid CoA ligase and proteasome subunit alpha type 5 which metabolize fatty and amino acids, while worker intended larvae over-expressed ATP synthase beta subunit, aldehyde dehydrogenase, thioredoxin peroxidase 1 and peroxiredoxin 2540, lethal (2) 37 and 14-3-3 protein epsilon, fatty acid binding protein, and translational controlled tumor protein. This differential protein expression between the two caste intended larvae was more pronounced at 120 hours, with particular significant differences in proteins associated with carbohydrate metabolism and energy production. Functional enrichment analysis suggests that carbohydrate metabolism and energy production and anti-oxidation proteins play major roles in the formation of caste divergence. The constructed network and validated gene expression identified target proteins for further functional study. This new finding is in contrast to the existing notion that 72 hour old larvae has bipotential and can develop into either queen or worker based on epigenetics and can help us to gain new insight into the time of departure as well as caste trajectory influencing elements at the molecular level

Validation of reference genes for expression analysis in the salivary gland and the intestine of Rhodnius prolixus (Hemiptera, Reduviidae) under different experimental conditions by quantitative real-time PCR

<p>Abstract</p> <p>Background</p> <p><it>Rhodnius prolixus </it>is a blood-feeding insect that can transmit <it>Trypanosoma cruzi </it>and <it>Trypanosoma rangeli </it>to vertebrate hosts. Recently, genomic resources for invertebrate vectors of human pathogens have increased significantly, and <it>R. prolixus </it>has been one of the main species studied among the triatomines. However, the paucity of information on many of the fundamental molecular aspects of this species limits the use of the available genomic information. The present study aimed to facilitate gene expression studies by identifying the most suitable reference genes for the normalization of mRNA expression data from qPCR.</p> <p>Results</p> <p>The expression stability of five candidate reference genes (<it>18S </it>rRNA, <it>GAPDH</it>, β-actin, α-tubulin and ribosomal protein <it>L26</it>) was evaluated by qPCR in two tissues (salivary gland and intestine) and under different physiological conditions: before and after blood feeding and after infection with <it>T. cruzi </it>or <it>T. rangeli</it>. The results were analyzed with three software programs: geNorm, NormFinder and BestKeeper. All of the evaluated candidate genes proved to be acceptable as reference genes, but some were found to be more appropriate depending on the experimental conditions. <it>18S</it>, <it>GAPDH </it>and α-tubulin showed acceptable stability for studies in all of the tissues and experimental conditions evaluated. β-actin, one of the most widely used reference genes, was confirmed to be one of the most suitable reference genes in studies with salivary glands, but it had the lowest expression stability in the intestine after insect blood feeding. <it>L26 </it>was identified as the poorest reference gene in the studies performed.</p> <p>Conclusions</p> <p>The expression stability of the genes varies in different tissue samples and under different experimental conditions. The results provided by three statistical packages emphasize the suitability of all five of the tested reference genes in both the crop and the salivary glands with a few exceptions. The results emphasise the importance of validating reference genes for qRT-PCR analysis in <it>R. prolixus </it>studies.</p

Evaluation of endogenous references for gene expression profiling in different tissues of the oriental fruit fly Bactrocera dorsalis (Diptera: Tephritidae)

<p>Abstract</p> <p>Background</p> <p>Quantitative real-time reverse transcriptase PCR (RT-qPCR) has been widely used for quantification of mRNA as a way to determine key genes involved in different biological processes. For accurate gene quantification analysis, normalization of RT-qPCR data is absolutely essential. To date, normalization is most frequently achieved by the use of internal controls, often referred to as reference genes. However, several studies have shown that the reference genes used for the quantification of mRNA expression can be affected by the experimental set-up or cell type resulting in variation of the expression level of these key genes. Therefore, the evaluation of reference genes is critical for gene expression profiling, which is often neglected in gene expression studies of insects. For this purpose, ten candidate reference genes were investigated in three different tissues (midgut, Malpighian tubules, and fat body) of the oriental fruit fly, <it>Bactrocera dorsalis </it>(Hendel).</p> <p>Results</p> <p>Two different programs, <it>geNorm </it>and <it>Normfinder</it>, were used to analyze the data. According to <it>geNorm</it>, α-TUB + ACT5 are the most appropriate reference genes for gene expression profiling across the three different tissues in the female flies, while ACT3 + α-TUB are considered as the best for males. Furthermore, we evaluated the stability of the candidate reference genes to determine the sexual differences in the same tissue. In the midgut and Malpighian tubules, ACT2 + α-TUB are the best choice for both males and females. However, α-TUB + ACT1 are the best pair for fat body. Meanwhile, the results calculated by <it>Normfinder </it>are quite the same as the results with <it>geNorm</it>; α-TUB is always one of the most stable genes in each sample validated by the two programs.</p> <p>Conclusions</p> <p>In this study, we validated the suitable reference genes for gene expression profiling in different tissues of <it>B. dorsalis. </it>Moreover, appropriate reference genes were selected out for gene expression profiling of the same tissues taking the sexual differences into consideration. This work not only formed a solid basis for future gene expression study in <it>B. dorsalis</it>, but also will serve as a resource to screen reference genes for gene expression studies in any other insects.</p

Diversity in Protein Glycosylation among Insect Species

status: publishe

Down-Regulation of Honey Bee IRS Gene Biases Behavior toward Food Rich in Protein

Food choice and eating behavior affect health and longevity. Large-scale research efforts aim to understand the molecular and social/behavioral mechanisms of energy homeostasis, body weight, and food intake. Honey bees (Apis mellifera) could provide a model for these studies since individuals vary in food-related behavior and social factors can be controlled. Here, we examine a potential role of peripheral insulin receptor substrate (IRS) expression in honey bee foraging behavior. IRS is central to cellular nutrient sensing through transduction of insulin/insulin-like signals (IIS). By reducing peripheral IRS gene expression and IRS protein amount with the use of RNA interference (RNAi), we demonstrate that IRS influences foraging choice in two standard strains selected for different food-hoarding behavior. Compared with controls, IRS knockdowns bias their foraging effort toward protein (pollen) rather than toward carbohydrate (nectar) sources. Through control experiments, we establish that IRS does not influence the bees' sucrose sensory response, a modality that is generally associated with food-related behavior and specifically correlated with the foraging preference of honey bees. These results reveal a new affector pathway of honey bee social foraging, and suggest that IRS expressed in peripheral tissue can modulate an insect's foraging choice between protein and carbohydrate sources

Assessment and validation of a suite of reverse transcription-quantitative PCR reference genes for analyses of density-dependent behavioural plasticity in the Australian plague locust

<p>Abstract</p> <p>Background</p> <p>The Australian plague locust, <it>Chortoicetes terminifera</it>, is among the most promising species to unravel the suites of genes underling the density-dependent shift from shy and cryptic solitarious behaviour to the highly active and aggregating gregarious behaviour that is characteristic of locusts. This is because it lacks many of the major phenotypic changes in colour and morphology that accompany phase change in other locust species. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is the most sensitive method available for determining changes in gene expression. However, to accurately monitor the expression of target genes, it is essential to select an appropriate normalization strategy to control for non-specific variation between samples. Here we identify eight potential reference genes and examine their expression stability at different rearing density treatments in neural tissue of the Australian plague locust.</p> <p>Results</p> <p>Taking advantage of the new orthologous DNA sequences available in locusts, we developed primers for genes encoding 18SrRNA, ribosomal protein L32 (RpL32), armadillo (Arm), actin 5C (Actin), succinate dehydrogenase (SDHa), glyceraldehyde-3P-dehydrogenase (GAPDH), elongation factor 1 alpha (EF1a) and annexin IX (AnnIX). The relative transcription levels of these eight genes were then analyzed in three treatment groups differing in rearing density (isolated, short- and long-term crowded), each made up of five pools of four neural tissue samples from 5^thinstar nymphs. SDHa and GAPDH, which are both involved in metabolic pathways, were identified as the least stable in expression levels, challenging their usefulness in normalization. Based on calculations performed with the geNorm and NormFinder programs, the best combination of two genes for normalization of gene expression data following crowding in the Australian plague locust was EF1a and Arm. We applied their use to studying a target gene that encodes a Ca²⁺binding glycoprotein, <it>SPARC</it>, which was previously found to be up-regulated in brains of gregarious desert locusts, <it>Schistocerca gregaria</it>. Interestingly, expression of this gene did not vary with rearing density in the same way in brains of the two locust species. Unlike <it>S. gregaria</it>, there was no effect of any crowding treatment in the Australian plague locust.</p> <p>Conclusion</p> <p>Arm and EF1a is the most stably expressed combination of two reference genes of the eight examined for reliable normalization of RT-qPCR assays studying density-dependent behavioural change in the Australian plague locust. Such normalization allowed us to show that <it>C. terminifera </it>crowding did not change the neuronal expression of the <it>SPARC </it>gene, a gregarious phase-specific gene identified in brains of the desert locust, <it>S. gregaria</it>. Such comparative results on density-dependent gene regulation provide insights into the evolution of gregarious behaviour and mass migration of locusts. The eight identified genes we evaluated are also candidates as normalization genes for use in experiments involving other Oedipodinae species, but the rank order of gene stability must necessarily be determined on a case-by-case basis.</p