Huipeng Pan

Selection of Reference Genes for RT-qPCR Analysis in Coccinella septempunctata to Assess Un-intended Effects of RNAi Transgenic Plants.

The development of genetically engineered plants that employ RNA interference (RNAi) to suppress invertebrate pests opens up new avenues for insect control. While this biotechnology shows tremendous promise, the potential for both non-target and off-target impacts, which likely manifest via altered mRNA expression in the exposed organisms, remains a major concern. One powerful tool for the analysis of these un-intended effects is reverse transcriptase-quantitative polymerase chain reaction, a technique for quantifying gene expression using a suite of reference genes for normalization. The seven-spotted ladybeetle Coccinella septempunctata, a commonly used predator in both classical and augmentative biological controls, is a model surrogate species used in the environmental risk assessment (ERA) of plant incorporated protectants (PIPs). Here, we assessed the suitability of eight reference gene candidates for the normalization and analysis of C. septempunctata v-ATPase A gene expression under both biotic and abiotic conditions. Five computational tools with distinct algorisms, geNorm, Normfinder, BestKeeper, the ΔCt method, and RefFinder, were used to evaluate the stability of these candidates. As a result, unique sets of reference genes were recommended, respectively, for experiments involving different developmental stages, tissues, and ingested dsRNAs. By providing a foundation for standardized RT-qPCR analysis in C. septempunctata, our work improves the accuracy and replicability of the ERA of PIPs involving RNAi transgenic plants.

Further Spread of and Domination by Bemisia tabaci (Hemiptera: Aleyrodidae) Biotype Q on Field Crops in China

ABSTRACT The sweetpotato whitefly, Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae), causes severe crop losses to many crops. The worst of these losses are often associated with the invasion and establishment of biotypes B and Q of this pest. Previous research in 2007 showed that biotype Q occurred with other biotypes in most field populations in China. To determine the current status of the biotype composition in the field, an extensive survey covering mainly eastern parts of China was conducted in 2009. Using polymerase chain reaction primers specific for the mitochondrial cytochrome oxidase I of biotypes B and Q and gene sequencing, we determined the biotypes composition in 61 whitefly populations and their distribution across 19 provinces in China. Our research revealed that only biotypes B and Q have been found in the field in 2009 in China. Among them, biotype Q was dominant in 44 locations (100.0%) and biotype B was dominant in 17 locations (100.0%). The current survey indicates that biotype Q has rapidly displaced biotype B in most locations in China.

Reference gene selection for qRT-PCR analysis in the sweetpotato whitefly, Bemisia tabaci (Hemiptera: Aleyrodidae).

Background Accurate evaluation of gene expression requires normalization relative to the expression of reliable reference genes. Expression levels of “classical” reference genes can differ, however, across experimental conditions. Although quantitative real-time PCR (qRT-PCR) has been used extensively to decipher gene function in the sweetpotato whitefly Bemisia tabaci, a world-wide pest in many agricultural systems, the stability of its reference genes has rarely been validated. Results In this study, 15 candidate reference genes from B. tabaci were evaluated using two Excel-based algorithms geNorm and Normfinder under a diverse set of biotic and abiotic conditions. At least two reference genes were selected to normalize gene expressions in B. tabaci under experimental conditions. Specifically, for biotic conditions including host plant, acquisition of a plant virus, developmental stage, tissue (body region of the adult), and whitefly biotype, ribosomal protein L29 was the most stable reference gene. In contrast, the expression of elongation factor 1 alpha, peptidylprolyl isomerase A, NADH dehydrogenase, succinate dehydrogenase complex subunit A and heat shock protein 40 were consistently stable across various abiotic conditions including photoperiod, temperature, and insecticide susceptibility. Conclusion Our finding is the first step toward establishing a standardized quantitative real-time PCR procedure following the MIQE (Minimum Information for publication of Quantitative real time PCR Experiments) guideline in an agriculturally important insect pest, and provides a solid foundation for future RNA interference based functional study in B. tabaci.

Rapid Spread of Tomato Yellow Leaf Curl Virus in China Is Aided Differentially by Two Invasive Whiteflies

Background Tomato yellow leaf curl virus (TYLCV) was introduced into China in 2006, approximately 10 years after the introduction of an invasive whitefly, Bemisia tabaci (Genn.) B biotype. Even so the distribution and prevalence of TYLCV remained limited, and the economic damage was minimal. Following the introduction of Q biotype into China in 2003, the prevalence and spread of TYLCV started to accelerate. This has lead to the hypothesis that the two biotypes might not be equally competent vectors of TYLCV. Methodology/Principal Findings The infection frequency of TYLCV in the field-collected B. tabaci populations was investigated, the acquisition and transmission capability of TYLCV by B and Q biotypes were compared under the laboratory conditions. Analysis of B. tabaci populations from 55 field sites revealed the existence of 12 B and 43 Q biotypes across 18 provinces in China. The acquisition and transmission experiments showed that both B and Q biotypes can acquire and transmit the virus, however, Q biotype demonstrated superior acquisition and transmission capability than its B counterparts. Specifically, Q biotype acquired significantly more viral DNA than the B biotype, and reached the maximum viral load in a substantially shorter period of time. Although TYLCV was shown to be transmitted horizontally by both biotypes, Q biotype exhibited significantly higher viral transmission frequency than B biotype. Vertical transmission result, on the other hand, indicated that TYLCV DNA can be detected in eggs and nymphs, but not in pupae and adults of the first generation progeny. Conclusions/Significance These combined results suggested that the epidemiology of TYLCV was aided differentially by the two invasive whiteflies (B and Q biotypes) through horizontal but not vertical transmission of the virus. This is consistent with the concomitant eruption of TYLCV in tomato fields following the recent rapid invasion of Q biotype whitefly in China.

Multiple Forms of Vector Manipulation by a Plant-Infecting Virus: Bemisia tabaci and Tomato Yellow Leaf Curl Virus

ABSTRACT For many insect-vectored plant viruses, the relationship between feeding behavior and vector competence may prove integral to an understanding of the epidemiology of the resulting plant disease. While plant-infecting viruses are well known to change host plant physiology in a way that makes them more attractive to vectors, viral manipulation of the vectors themselves has only recently been reported. Previous research suggested that the rapid spread of Tomato yellow leaf curl virus (TYLCV) throughout China has been facilitated by its primary vector, the whitefly Bemisia tabaci. We conducted two experiments testing the impact of TYLCV infection of the host plant (tomato) and vector (B. tabaci biotypes B and Q) on whitefly feeding behavior. Whiteflies of biotypes B and Q both appeared to find TYLCV-infected plants more attractive, probing them more quickly and having a greater number of feeding bouts; this did not, however, alter the total time spent feeding. Viruliferous whiteflies fed more readily than uninfected whiteflies and spent more time salivating into sieve tube elements. Because vector salivation is essential for viral transmission, this virally mediated alteration of behavior should provide TYLCV a direct fitness benefit. This is the first report of such manipulation by a nonpropagative virus that belongs to an exclusively plant-infecting family of viruses (Geminiviridae). In the context of previous research showing that feeding on TYLCV-infected plants harms biotype B but helps biotype Q, the fact that both biotypes were equally affected by TYLCV also suggests that the virus may alter the biotype B-biotype Q competitive interaction in favor of biotype Q.

Factors Affecting Population Dynamics of Maternally Transmitted Endosymbionts in Bemisia tabaci

While every individual of Bemisia tabaci (Hemiptera: Aleyrodidae) harbors the primary symbiont (P-symbiont) Portiera, the infection frequencies of the six secondary symbionts (S-symbionts) including Hamiltonella, Arsenophonus, Cardinium, Wolbachia, Rickettsia and Fritschea vary greatly among different populations. To characterize the factors influencing the infection dynamics of the six S-symbionts in B. tabaci, gene-specific PCR were conducted to screen for the presence of the P-symbiont Portiera and the six S-symbionts in 61 (17 B and 44 Q biotypes) field populations collected from different plant species and locations in China. All individuals of the 61 populations hosted the P-symbiont Portiera, but none of them harbored Arsenophonus and Fritschea. The presence and infection rates of Hamiltonella, Cardinium, Rickettsia, Wolbachia and their co-infections Rickettsia + Hamiltonella (RH), Rickettsia + Cardinium (RC), Hamiltonella + Cardinium (HC) and Rickettsia + Hamiltonella + Cardinium (RHC) varied significantly among the 61 field populations; and the observed variations can be explained by biotypes, sexes, host plants and geographical locations of these field populations. Taken together, at least three factors including biotype, host plant and geographical location affect the infection dynamics of S-symbionts in B. tabaci.

Pyrosequencing the Bemisia tabaci transcriptome reveals a highly diverse bacterial community and a robust system for insecticide resistance.

Background Bemisia tabaci (Gennadius) is a phloem-feeding insect poised to become one of the major insect pests in open field and greenhouse production systems throughout the world. The high level of resistance to insecticides is a main factor that hinders continued use of insecticides for suppression of B. tabaci. Despite its prevalence, little is known about B. tabaci at the genome level. To fill this gap, an invasive B. tabaci B biotype was subjected to pyrosequencing-based transcriptome analysis to identify genes and gene networks putatively involved in various physiological and toxicological processes. Methodology and Principal Findings Using Roche 454 pyrosequencing, 857,205 reads containing approximately 340 megabases were obtained from the B. tabaci transcriptome. De novo assembly generated 178,669 unigenes including 30,980 from insects, 17,881 from bacteria, and 129,808 from the nohit. A total of 50,835 (28.45%) unigenes showed similarity to the non-redundant database in GenBank with a cut-off E-value of 10–5. Among them, 40,611 unigenes were assigned to one or more GO terms and 6,917 unigenes were assigned to 288 known pathways. De novo metatranscriptome analysis revealed highly diverse bacterial symbionts in B. tabaci, and demonstrated the host-symbiont cooperation in amino acid production. In-depth transcriptome analysis indentified putative molecular markers, and genes potentially involved in insecticide resistance and nutrient digestion. The utility of this transcriptome was validated by a thiamethoxam resistance study, in which annotated cytochrome P450 genes were significantly overexpressed in the resistant B. tabaci in comparison to its susceptible counterparts. Conclusions This transcriptome/metatranscriptome analysis sheds light on the molecular understanding of symbiosis and insecticide resistance in an agriculturally important phloem-feeding insect pest, and lays the foundation for future functional genomics research of the B. tabaci complex. Moreover, current pyrosequencing effort greatly enriched the existing whitefly EST database, and makes RNAseq a viable option for future genomic analysis.

Genome sequencing of the sweetpotato whitefly Bemisia tabaci MED/Q

Abstract The sweetpotato whitefly Bemisia tabaci is a highly destructive agricultural and ornamental crop pest. It damages host plants through both phloem feeding and vectoring plant pathogens. Introductions of B. tabaci are difficult to quarantine and eradicate because of its high reproductive rates, broad host plant range, and insecticide resistance. A total of 791 Gb of raw DNA sequence from whole genome shotgun sequencing, and 13 BAC pooling libraries were generated by Illumina sequencing using different combinations of mate-pair and pair-end libraries. Assembly gave a final genome with a scaffold N50 of 437 kb, and a total length of 658 Mb. Annotation of repetitive elements and coding regions resulted in 265.0 Mb TEs (40.3%) and 20 786 protein-coding genes with putative gene family expansions, respectively. Phylogenetic analysis based on orthologs across 14 arthropod taxa suggested that MED/Q is clustered into a hemipteran clade containing A. pisum and is a sister lineage to a clade containing both R. prolixus and N. lugens. Genome completeness, as estimated using the CEGMA and Benchmarking Universal Single-Copy Orthologs pipelines, reached 96% and 79%. These MED/Q genomic resources lay a foundation for future ‘pan-genomic’ comparisons of invasive vs. noninvasive, invasive vs. invasive, and native vs. exotic Bemisia, which, in return, will open up new avenues of investigation into whitefly biology, evolution, and management.

A Comprehensive Selection of Reference Genes for RT-qPCR Analysis in a Predatory Lady Beetle, Hippodamia convergens (Coleoptera: Coccinellidae).

Reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) is a reliable, rapid, and reproducible technique for measuring and evaluating changes in gene expression. To facilitate gene expression studies and obtain more accurate RT-qPCR data, normalization relative to stable reference genes is required. In this study, expression profiles of seven candidate reference genes, including β-actin (Actin), elongation factor 1 α (EF1A), glyceralde hyde-3-phosphate dehydro-genase (GAPDH), cyclophilins A (CypA), vacuolar-type H+-ATPase (ATPase), 28S ribosomal RNA (28S), and 18S ribosomal RNA (18S) from Hippodamia convergens were investigated. H. convergens is an abundant predatory species in the New World, and has been widely used as a biological control agent against sap-sucking insect pests, primarily aphids. A total of four analytical methods, geNorm, Normfinder, BestKeeper, and the ΔCt method, were employed to evaluate the performance of these seven genes as endogenous controls under diverse experimental conditions. Additionally, RefFinder, a comprehensive evaluation platform integrating the four above mentioned algorithms, ranked the overall stability of these candidate genes. A suite of reference genes were specifically recommended for each experimental condition. Among them, 28S, EF1A, and CypA were the best reference genes across different development stages; GAPDH, 28S, and CypA were most stable in different tissues. GAPDH and CypA were most stable in female and male adults and photoperiod conditions, 28S and EF1A were most stable under a range of temperatures, Actin and CypA were most stable under dietary RNAi condition. This work establishes a standardized RT-qPCR analysis in H. convergens. Additionally, this study lays a foundation for functional genomics research in H. convergens and sheds light on the ecological risk assessment of RNAi-based biopesticides on this non-target biological control agent.

Differential effects of an exotic plant virus on its two closely related vectors

Concurrent spread of Tomato yellow leaf curl virus (TYLCV) with invasion of Bemisia tabaci Q rather than B in China suggests a more mutualistic relationship between TYLCV and Q than B. To assess this hypothesis, we quantified the impacts of TYLCV on the performance and competitiveness of B and Q in the laboratory. The results showed that relative to their non-infected counterparts feeding on cotton (a non-host for TYLCV), infected B exhibited significant reductions in life-history traits, whereas infected Q only showed marginal reductions. While Q performed better on TYLCV-infected tomato plants than on uninfected ones, the reverse was observed in B. Q displacement by B took one more generation on TYLCV-infected tomato plants than on healthy ones. These results demonstrate that TYLCV was indirectly mutualistic to Q but directly and indirectly parasitic to B.