Unexpected High Intragenomic Variation in Two of Three Major Pest Thrips Species Does Not Affect Ribosomal Internal Transcribed Spacer 2 (ITS2) Utility for Thrips Identification

The mitochondrial cytochrome oxidase I gene (mtCO1) and the ribosomal internal transcribed spacer 2 region (ITS2) are among the most widely used molecular markers for insect taxonomic characterization. Three economically important species of thrips, Scirtothrips dorsalis, Thrips palmi, and Frankliniella occidentalis were selected to examine the extent of intragenomic variation within these two marker regions in the family Thripidae, and determine if this variation would affect the utility of markers in thrips molecular diagnostics. For each species, intragenomic (within individual) variation and intergenomic (among individuals) variation was assessed by cloning and sequencing PCR-amplified copies. Intergenomic variation was generally higher than intragenomic variation except in cases where intergenomic variation was very low, as in mtCO1 from S. dorsalis and F. occidentalis. Intragenomic variation was detected in both markers in all three of the thrips species, however, 2–3 times more intragenomic variation was observed for ITS2 than mtCO1 in both S. dorsalis and T. palmi. Furthermore, levels of intragenomic variation were low for both of the genes in F. occidentalis. In all of the three thrips species, no sex-based clustering of haplotypes was observed in either marker. Unexpected high intragenomic variation in ITS2 for two of three thrips species did not interfere with thrips diagnostics. However, caution should be taken in applying ITS2 to certain studies of S. dorsalis and T. palmi when high levels of intragenomic variation could be problematic or confounding. In such studies, mtCO1 may be a preferable marker. Possible reasons for discrepancies in intragenomic variation among genomic regions are discussed

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

African ancestry of New World, Bemisia tabaci-whitefly species

Bemisia tabaci whitefly species are some of the world’s most devastating agricultural pests and plant-virus disease vectors. Elucidation of the phylogenetic relationships in the group is the basis for understanding their evolution, biogeography, gene-functions and development of novel control technologies. We report here the discovery of five new Sub-Saharan Africa (SSA) B. tabaci putative species, using the partial mitochondrial cytochrome oxidase 1 gene: SSA9, SSA10, SSA11, SSA12 and SSA13. Two of them, SSA10 and SSA11 clustered with the New World species and shared 84.8‒86.5% sequence identities. SSA10 and SSA11 provide new evidence for a close evolutionary link between the Old and New World species. Re-analysis of the evolutionary history of B. tabaci species group indicates that the new African species (SSA10 and SSA11) diverged from the New World clade c. 25 million years ago. The new putative species enable us to: (i) re-evaluate current models of B. tabaci evolution, (ii) recognise increased diversity within this cryptic species group and (iii) re-estimate divergence dates in evolutionary time

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

Stylet Morphometrics and Ultrastructure in Relation to Feeding Behavior and Pathogen Transmission by Nymphs and Adults of the Asian Citrus Psyllid Diaphorina citri, Vector of Citrus Huanglongbing Bacterium

The feeding behavior and stylet morphometrics were studied in nymphs and adults of the Asian citrus psyllid (ACP), Diaphorina citri (Hemiptera, Psyllidae), vector of Candidatus Liberibacter asiaticus (Las) associated with citrus huanglongbing (HLB) disease. The stylet length of first instar nymphs averaged 266 µm (83% of body length) whereas that of 5th instar nymphs was 615 µm (34% of body length). Younger ACP nymphs feed only on young citrus leaves on smaller veins or on the sides of the midrib, whereas adults can feed anywhere on the veins of young or old citrus leaves. Epifluorescence microscopy of cross sections in citrus leaves indicated that the thick-walled fibrous layer around the phloem is much more prominent in older than in younger leaves. Additionally, first instar nymphs can reach the phloem because the distance to the phloem is shorter from the sides of the midrib compared to that from the top, and is considerably shorter in younger than in older/mature leaves. Ultrastructural studies on ACP stylets show that the width of the maxillary food canal in first instar nymphs is wide enough for Las bacteria to go through during food ingestion (and Las acquisition). However, the width of the maxillary salivary canal in first instar nymphs may not be wide enough for Las bacteria to go through during salivation (and inoculation of Las bacterium) into host plants. This may explain previous studies indicating that older ACP nymphs and adults can transmit HLB bacterium whereas younger nymphs probably cannot

Stylet Morphometrics and Ultrastructure in Relation to Feeding Behavior and Pathogen Transmission by Nymphs and Adults of the Asian Citrus Psyllid Diaphorina citri, Vector of Citrus Huanglongbing Bacterium

The feeding behavior and stylet morphometrics were studied in nymphs and adults of the Asian citrus psyllid (ACP), Diaphorina citri (Hemiptera, Psyllidae), vector of Candidatus Liberibacter asiaticus (Las) associated with citrus huanglongbing (HLB) disease. The stylet length of first instar nymphs averaged 266 µm (83% of body length) whereas that of 5th instar nymphs was 615 µm (34% of body length). Younger ACP nymphs feed only on young citrus leaves on smaller veins or on the sides of the midrib, whereas adults can feed anywhere on the veins of young or old citrus leaves. Epifluorescence microscopy of cross sections in citrus leaves indicated that the thick-walled fibrous layer around the phloem is much more prominent in older than in younger leaves. Additionally, first instar nymphs can reach the phloem because the distance to the phloem is shorter from the sides of the midrib compared to that from the top, and is considerably shorter in younger than in older/mature leaves. Ultrastructural studies on ACP stylets show that the width of the maxillary food canal in first instar nymphs is wide enough for Las bacteria to go through during food ingestion (and Las acquisition). However, the width of the maxillary salivary canal in first instar nymphs may not be wide enough for Las bacteria to go through during salivation (and inoculation of Las bacterium) into host plants. This may explain previous studies indicating that older ACP nymphs and adults can transmit HLB bacterium whereas younger nymphs probably cannot