Spatial Genetic Heterogeneity in Populations of a Newly Invasive Whitefly in China Revealed by a Nation-Wide Field Survey

Background

Even though introductions of exotic species provide ready-made experiments of rapid evolution, few studies have examined the genetic structure of an exotic species shortly after its initial introduction and subsequent spread. To determine the genetic structure of its populations during the initial introduction, we investigated the invasive sweet potato whitefly (Bemisia tabaci Q, commonly known as B. tabaci biotype Q) in China, which was introduced in approximately 2003. A total of 619 B. tabaci Q individuals in 20 provinces throughout China were collected and analyzed using five microsatellite loci.

Results

The introduced populations of B. tabaci Q in China represent eight genetic clusters with different geographic distributions. The populations in Yunnan Province, where B. tabaci Q was first detected, are genetically different from the other populations in China.

Conclusion

The introduced populations of B. tabaci Q in China have high spatial genetic heterogeneity. Additional research is required to determine whether the heterogeneity results from multiple introductions, rapid evolution following one or few introductions, or some combination of multiple introductions and rapid evolution. The heterogeneity, however, is inconsistent with a single introduction at Yunnan Province, where B. tabaci Q was first detected, followed by spread.     

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.     

Plant Virus Differentially Alters the Plant's Defense Response to Its Closely Related Vectors

Background

The whitefly, Bemisia tabaci (Hemiptera: Aleyrodidae), is one of the most widely distributed agricultural pests. In recent years, B. tabaci Q has invaded China, and Q has displaced B in many areas now. In a number of regions of the world, invasion by B and/or Q has been followed by outbreaks of tomato yellow leaf curl virus (TYLCV). Our previous study showed TYLCV directly and indirectly modified the feeding behavior of B. tabaci in favor of Q rather than B.

Methodology/Principal Findings

In this study, we quantified the salicylic acid (SA) titers and relative gene expression of SA in tomato leaves that were infested with viruliferous or non-viruliferous B and Q. We also measured the impacts of exogenous SA on the performance of B and Q, including the effects on ovary development. SA titer was always higher in leaves that were infested with viruliferous B than with viruliferous Q, whereas the SA titer did not differ between leaves infested with non-viruliferous B and Q. The relative gene expression of SA signaling was increased by feeding of viruliferous B but was not increased by feeding of viruliferous Q. The life history traits of B and Q were adversely affected on SA-treated plants. On SA-treated plants, both B and Q had lower fecundity, shorter longevity, longer developmental time and lower survival rate than on untreated plants. Compared with whiteflies feeding on control plants, those feeding on SA-treated plants had fewer oocytes and slower ovary development. On SA-treated plants, viruliferous B had fewer oocytes than viruliferous Q.

Conclusions/Significance

These results indicate that TYLCV tends to induce SA-regulated plant defense against B but SA-regulated plant defense against Q was reduced. In other words, Q may have a mutualistic relationship with TYLCV that results in the reduction of the plant''s defense response.     

An Invasive Whitefly Feeding on a Virus-Infected Plant Increased Its Egg Production and Realized Fecundity

Background

Plant-pathogenic begomoviruses have a complex association with their insect vectors. The interactions of begomoviruses and reproduction of their vectors are poorly understood. Bemisia tabaci is known to transmit many begomoviruses, and the spread of B. tabaci, especially the B and Q ‘biotypes’, has been accompanied by the epidemics of begomoviruses. One of these identified disease-causing agents was Tomato yellow leaf curl China virus (TYLCCNV).

Methodology/Principal Findings

In this study, we compared the egg production and realized fecundity of two ‘biotypes’ or putative species of the whitefly B. tabaci, including the alien invasive B and the indigenous ZHJ1 from Zhejiang, China, feeding on either healthy or TYLCCNV-infected tobacco plants. The ovary of the whitefly was composed of 12–22 telotrophic ovarioles. According to the morphology of the oocytes and level of yolk content, oocytes in ovarioles were divided into four developmental phases (I-IV). Significantly higher proportion of immature oocytes (phase II, III) and mature oocytes (phase IV) was observed in ovary of females that fed on TYLCCNV-infected tobacco compared to that on healthy plants. Moreover, there was significant increase of eggs laid of B whitefly that fed on TYLCCNV-infected tobacco plants during the early developmental stages. In contrast, the proportion of oocytes of different developmental phases and eggs laid had no significant differences between ZHJ1 whiteflies feeding on TYLCCNV-infected and non-infected host plants.

Conclusions/Significance

The invasive B whitefly benefits from feeding on a begomovirus-infected plant through increased egg production and realized fecundity.     

Identification of Conserved and Novel microRNAs from Liriodendron chinense Floral Tissues

Background

Liriodendron chinense (L. chinense) is an endangered basal angiosperm plant in China because of its low reproductive efficiency. Recently, miRNAs have obtained great attention because they can play important roles. Through high throughput sequencing technique, large amount of miRNAs were identified from different plant species. But there were few studies about the miRNAs in the basal angiosperms especially in the sexual reproduction process.

Results

Deep sequencing technology was applied to discover miRNAs in L. chinense flowers at different stages. After bioinformatic analysis, 496 putative conserved miRNAs representing 97 families and 2 novel miRNAs were found. Among them, one is previously regarded as gymnosperm specific. Their expressions were further validated by Real-time PCR for 13 selected miRNAs. Putative targeting genes were predicted and categorized with gene ontology (GO) analysis. About ten percents of the targets are involved in the reproduction process. Further expressional analysis showed that many of these miRNAs were highly related to the reproductive growth.

Conclusions

This is the first comprehensive identification of conserved and novel miRNAs in L. chinense. The data presented here might not only help to fill the gap of miRNA registered about basal angiosperm plants but also contribute to understanding the evolution of miRNAs. The differential expression of some of the miRNAs and the prediction of their target genes are also helpful in understanding the regulation of L. chinense sexual reproduction.     

Parallel analysis of RNA ends enhances global investigation of microRNAs and target RNAs of Brachypodium distachyon

Background

The wild grass Brachypodium distachyon has emerged as a model system for temperate grasses and biofuel plants. However, the global analysis of miRNAs, molecules known to be key for eukaryotic gene regulation, has been limited in B. distachyon to studies examining a few samples or that rely on computational predictions. Similarly an in-depth global analysis of miRNA-mediated target cleavage using parallel analysis of RNA ends (PARE) data is lacking in B. distachyon.

Results

B. distachyon small RNAs were cloned and deeply sequenced from 17 libraries that represent different tissues and stresses. Using a computational pipeline, we identified 116 miRNAs including not only conserved miRNAs that have not been reported in B. distachyon, but also non-conserved miRNAs that were not found in other plants. To investigate miRNA-mediated cleavage function, four PARE libraries were constructed from key tissues and sequenced to a total depth of approximately 70 million sequences. The roughly 5 million distinct genome-matched sequences that resulted represent an extensive dataset for analyzing small RNA-guided cleavage events. Analysis of the PARE and miRNA data provided experimental evidence for miRNA-mediated cleavage of 264 sites in predicted miRNA targets. In addition, PARE analysis revealed that differentially expressed miRNAs in the same family guide specific target RNA cleavage in a correspondingly tissue-preferential manner.

Conclusions

B. distachyon miRNAs and target RNAs were experimentally identified and analyzed. Knowledge gained from this study should provide insights into the roles of miRNAs and the regulation of their targets in B. distachyon and related plants.     

An extensive field survey combined with a phylogenetic analysis reveals rapid and widespread invasion of two alien whiteflies in China

Background

To understand the processes of invasions by alien insects is a pre-requisite for improving management. The whitefly Bemisia tabaci is a cryptic species complex that contains some of the most invasive pests worldwide. However, extensive field data to show the geographic distribution of the members of this species complex as well as the invasion by some of its members are scarce.

Methodology/Principal Findings

We used field surveys and published data to assess the current diversity and distribution of B. tabaci cryptic species in China and relate the indigenous members to other Asian and Australian members of the complex. The survey covered the 16 provinces where indigenous B. tabaci occur and extends this with published data for the whole of China. We used molecular markers to identify cryptic species. The evolutionary relationships between the different Asian B. tabaci were reconstructed using Bayesian methods. We show that whereas in the past the exotic invader Middle East-Asia Minor 1 was predominant across China, another newer invader Mediterranean is now the dominant species in the Yangtze River Valley and eastern coastal areas, and Middle East-Asia Minor 1 is now predominant only in the south and south eastern coastal areas. Based on mtCO1 we identified four new cryptic species, and in total we have recorded 13 indigenous and two invasive species from China. Diversity was highest in the southern and southeastern provinces and declined to north and west. Only the two invasive species were found in the northern part of the country where they occur primarily in protected cropping. By 2009, indigenous species were mainly found in remote mountainous areas and were mostly absent from extensive agricultural areas.

Conclusions/Significance

Invasions by some members of the whitefly B. tabaci species complex can be rapid and widespread, and indigenous species closely related to the invaders are replaced.     

Deep Sequencing and Microarray Hybridization Identify Conserved and Species-Specific MicroRNAs during Somatic Embryogenesis in Hybrid Yellow Poplar

Background

To date, several studies have indicated a major role for microRNAs (miRNAs) in regulating plant development, but miRNA-mediated regulation of the developing somatic embryo is poorly understood, especially during early stages of somatic embryogenesis in hardwood plants. In this study, Solexa sequencing and miRNA microfluidic chips were used to discover conserved and species-specific miRNAs during somatic embryogenesis of hybrid yellow poplar (Liriodendron tulipifera×L. chinense).

Methodology/Principal Findings

A total of 17,214,153 reads representing 7,421,623 distinct sequences were obtained from a short RNA library generated from small RNAs extracted from all stages of somatic embryos. Through a combination of deep sequencing and bioinformatic analyses, we discovered 83 sequences with perfect matches to known miRNAs from 33 conserved miRNA families and 273 species-specific candidate miRNAs. MicroRNA microarray results demonstrated that many conserved and species-specific miRNAs were expressed in hybrid yellow poplar embryos. In addition, the microarray also detected another 149 potential miRNAs, belonging to 29 conserved families, which were not discovered by deep sequencing analysis. The biological processes and molecular functions of the targets of these miRNAs were predicted by carrying out BLAST search against Arabidopsis thaliana GenBank sequences and then analyzing the results with Gene Ontology.

Conclusions

Solexa sequencing and microarray hybridization were used to discover 232 candidate conserved miRNAs from 61 miRNA families and 273 candidate species-specific miRNAs in hybrid yellow poplar. In these predicted miRNAs, 64 conserved miRNAs and 177 species-specific miRNAs were detected by both sequencing and microarray hybridization. Our results suggest that miRNAs have wide-ranging characteristics and important roles during all stages of somatic embryogenesis in this economically important species.     

Identification and Comparative Analysis of the Tegillarca granosa Haemocytes MicroRNA Transcriptome in Response to Cd Using a Deep Sequencing Approach

Background

MicroRNAs (miRNAs) are endogenous non-coding small RNAs (sRNAs) that can base pair with their target mRNAs, which represses their translation or induces their degradation in various biological processes. To identify miRNAs regulated by heavy metal stress, we constructed two sRNA libraries for the blood clam Tegillarca granosa: one for organisms exposed to toxic levels of cadmium (Cd) and one for a control group.

Results

Sequencing of the two libraries and subsequent analysis revealed 215 conserved and 39 new miRNAs. Most of the new miRNAs in T. granosa were up- or down-regulated in response to Cd exposure. There were significant differences in expression between the Cd and control groups for 16 miRNAs. Of these, five miRNAs were significantly up-regulated and 11 were significantly down-regulated in the Cd stress library. Potential targets were predicted for the 16 differential miRNAs in pre-miRNAs identified according to sequence homology. Some of the predicted miRNA targets are associated with regulation of the response to stress induced by heavy metals. Five differentially expressed miRNAs (Tgr-nmiR-8, Tgr-nmiR-21, Tgr-miR-2a, Tgr-miR-10a-5p, and Tgr-miR-184b) were validated by qRT-PCR.

Conclusion

Our study is the first large-scale identification of miRNAs in T. granosa haemocytes. Our findings suggest that some miRNAs and their target genes and pathways may play critical roles in the responses of this species to environmental heavy metal stresses.     

Inactivation of Wolbachia Reveals Its Biological Roles in Whitefly Host

Background

The whitefly Bemisia tabaci is cryptic species complex composed of numerous species. Individual species from the complex harbor a diversity of bacterial endosymbionts including Wolbachia. However, while Wolbachia is known to have a number of different roles, its role in B. tabaci is unclear. Here, the antibiotic rifampicin is used to selectively eliminate Wolbachia from B. tabaci so as to enable its roles in whitefly development and reproduction to be explored. The indirect effects of Wolbachia elimination on the biology of Encarsia bimaculata, a dominant parasitoid of B. tabaci in South China, were also investigated.

Methodology/Principal Finding

qRT-PCR and FISH were used to show that after 48 h exposure to 1.0 mg/ml rifampicin, Wolbachia was completely inactivated from B. tabaci Mediterranean (MED) without any significant impact on either the primary symbiont, Portiera aleyrodidarum or any of the other secondary endosymbionts present. For B. tabaci MED, Wolbachia was shown to be associated with decreased juvenile development time, increased likelihood that nymphs completed development, increased adult life span and increased percentage of female progeny. Inactivation was associated with a significant decrease in the body size of the 4^th instar which leads us to speculate as to whether Wolbachia may have a nutrient supplementation role. The reduction in nymph body size has consequences for its parasitoid, E. bimaculata. The elimination of Wolbachia lead to a marked increase in the proportion of parasitoid eggs that completed their development, but the reduced size of the whitefly host was also associated with a significant reduction in the size of the emerging parasitoid adult and this was in turn associated with a marked reduction in adult parasitoid longevity.

Conclusions/Significance

Wolbachia increases the fitness of the whitefly host and provides some protection against parasitization. These observations add to our understanding of the roles played by bacterial endosymbionts.     

Acylsucrose-producing tomato plants forces Bemisia tabaci to shift its preferred settling and feeding site

Background

The whitefly Bemisia tabaci (Genn.) causes dramatic damage to plants by transmitting yield-limiting virus diseases. Previous studies proved that the tomato breeding line ABL 14-8 was resistant to B. tabaci, the vector of tomato yellow leaf curl disease (TYLCD). This resistance is based on the presence of type IV glandular trichomes and acylsucrose production. These trichomes deter settling and probing of B. tabaci in ABL 14-8, which reduces primary and secondary spread of TYLCD.

Methodology/Principal Findings

Whitefly settlement preference was evaluated on the adaxial and abaxial leaf surfaces of nearly-isogenic tomato lines with and without B. tabaci-resistance traits, ‘ABL 14-8 and Moneymaker’ respectively, under non-choice and free-choice conditions. In addition, the Electrical Penetration Graph technique was used to study probing and feeding activities of B. tabaci on the adaxial and abaxial leaf surfaces of the same genotypes. B. tabaci preferred to settle on the abaxial than on the adaxial surface of ‘Moneymaker’ leaves, whereas no such preference was observed on ABL 14-8 tomato plants at the ten-leaf growth stage. Furthermore, B. tabaci preferred to feed on the abaxial than on the adaxial leaf surface of ‘Moneymarker’ susceptible tomato plants as shown by a higher number of sustained phloem feeding ingestion events and a shorter time to reach the phloem. However, B. tabaci standard probing and feeding behavior patterns were altered in ABL 14-8 plants and whiteflies were unable to feed from the phloem and spent more time in non-probing activities when exposed to the abaxial leaf surface.

Conclusions/Significance

The distorted behavior of B. tabaci on ABL 14-8 protects tomato plants from the transmission of phloem-restricted viruses such as Tomato yellow leaf curl virus (TYLCV), and forces whiteflies to feed on the adaxial side of leaves where they feed less efficiently and become more vulnerable to natural enemies.     

Identification and Characterization of Novel MicroRNAs from Schistosoma japonicum

Background

Schistosomiasis japonica remains a major public health problem in China. Its pathogen, Schistosoma japonicum has a complex life cycle and a unique repertoire of genes expressed at different life cycle stages. Exploring schistosome gene regulation will yield the best prospects for new drug targets and vaccine candidates. MicroRNAs (miRNAs) are a highly conserved class of noncoding RNA that control many biological processes by sequence-specific inhibition of gene expression. Although a large number of miRNAs have been identified from plants to mammals, it remains no experimental proof whether schistosome exist miRNAs.

Methodology and Results

We have identified novel miRNAs from Schistosoma japonicum by cloning and sequencing a small (18–26 nt) RNA cDNA library from the adult worms. Five novel miRNAs were identified from 227 cloned RNA sequences and verified by Northern blot. Alignments of the miRNAs with corresponding family members indicated that four of them belong to a metazoan miRNA family: let-7, miR-71, bantam and miR-125. The fifth potentially new (non conserved) miRNA appears to belong to a previously undescribed family in the genus Schistosome. The novel miRNAs were designated as sja-let-7, sja-miR-71, sja-bantam, sja-miR-125 and sja-miR-new1, respectively. Expression of sja-let-7, sja-miR-71 and sja-bantam were analyzed in six stages of the life cycle, i.e. egg, miracidium, sporocyst, cercaria, schistosomulum, and adult worm, by a modified stem-loop reverse transcribed polymerase chain reaction (RT-PCR) method developed in our laboratory. The expression patterns of these miRNAs were highly stage-specific. In particular, sja-miR-71 and sja-bantam expression reach their peaks in the cercaria stage and then drop quickly to the nadirs in the schistosomulum stage, following penetration of cercaria into a mammalian host.

Conclusions

Authentic miRNAs were identified for the first time in S. japonicum, including a new schistosome family member. The different expression patterns of the novel miRNAs over the life stages of S. japonicum suggest that they may mediate important roles in Schistosome growth and development.     

Mirna expression profiles identify drivers in colorectal and pancreatic cancers

Background and Aim

Altered expression of microRNAs (miRNAs) hallmarks many cancer types. The study of the associations of miRNA expression profile and cancer phenotype could help identify the links between deregulation of miRNA expression and oncogenic pathways.

Methods

Expression profiling of 866 human miRNAs in 19 colorectal and 17 pancreatic cancers and in matched adjacent normal tissues was investigated. Classical paired t-test and random forest analyses were applied to identify miRNAs associated with tissue-specific tumors. Network analysis based on a computational approach to mine associations between cancer types and miRNAs was performed.

Results

The merge between the two statistical methods used to intersect the miRNAs differentially expressed in colon and pancreatic cancers allowed the identification of cancer-specific miRNA alterations. By miRNA-network analysis, tissue-specific patterns of miRNA deregulation were traced: the driving miRNAs were miR-195, miR-1280, miR-140-3p and miR-1246 in colorectal tumors, and miR-103, miR-23a and miR-15b in pancreatic cancers.

Conclusion

MiRNA expression profiles may identify cancer-specific signatures and potentially useful biomarkers for the diagnosis of tissue specific cancers. miRNA-network analysis help identify altered miRNA regulatory networks that could play a role in tumor pathogenesis.