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

Characterization of the RNA Interference Response in the Asian Citrus Psyllid

The Asian citrus psyllid (ACP), Diaphorina citri, is a major pest of citrus since it is the only known vector of ‘Candidatus Liberibactor’ species, the bacterium associated with citrus greening disease.  Since control of the psyllid is the only effective defense so far against citrus greening, and heavy reliance upon pesticides is not sustainable, an RNA interference (RNAi) strategy for ACP control was investigated. RNA interference is an innate immune response triggered by the cellular uptake of double stranded RNA (dsRNA) and studies were conducted to determine if ACP could be killed by oral uptake of dsRNA that target essential ACP transcripts. An artificial diet system was designed that facilitated the ingestion of dsRNA complementary to genes involved in digestion. Using this system, ACP mortality was observed as a result of oral uptake of dsRNAs targeting an apparent essential ACP gene and this mortality was shown to be dose responsive, reaching a maximum of 37% at high dsRNA concentrations.  There was also what appeared to be sequence independent ACP toxicity of large doses of dsRNA, concentrations above 48 ng/uL in the diet.  However, this sequence independent mortality was not as high as that observed for the targeted ACP gene, never rising above 17%.   These results provide support for the concept that RNAi could be adapted for use as a control strategy for the ACP

Huanglongbing Resistance and Tolerance in Citrus

Huanglongbing (HLB) is severely impacting Florida citrus. Productivity declines in many HLB-affected genotypes, often with greatly thinned canopies. Fruit size and quality are often adversely affected as the disease advances. HLB was assessed in diverse cultivars in commercial groves with high HLB-incidence. ‘Temple’ had the lowest HLB symptoms and Liberibacter (Las) titer, while ‘Murcott’ and ‘Minneola’ had the highest. The USDA Ft. Pierce, FL farm is managed to reveal genotype responses to HLB. Some current cultivars and hybrid seedlings demonstrate resistance/tolerance, at least to strain(s) of Las present. C. trifoliata is the best documented citrus resistance source with Las titers suppressed even when C. trifoliata is grafted onto severely-infected rootstocks. Some cultivars and hybrids have abundant foliage symptoms, but full canopies and seemingly normal fruit set and size. In 3-years of data from a replicated trial of ‘Triumph’(T), ‘Jackson’(J), ‘Flame’(F), and ‘Marsh’(M), HLB symptoms were severe in all trees and Liberibacter titers were similar. However, F&M were almost completely defoliated in some years while T&J had full canopies. Cumulative fruit/tree was greater for T&J (255&220) than for F&M (29&66). T&J fruit met commercial standards and had normal size but F&M fruit were unacceptable with many small and misshapen. Evidence mounts that useful resistance/tolerance to HLB is present in cultivated citrus and this is a focus of the USDA citrus breeding program

Huanglongbing Resistance and Tolerance in Citrus

Huanglongbing (HLB) is severely impacting Florida citrus. Productivity declines in many HLB-affected genotypes, often with greatly thinned canopies. Fruit size and quality are often adversely affected as the disease advances. HLB was assessed in diverse cultivars in commercial groves with high HLB-incidence. ‘Temple’ had the lowest HLB symptoms and Liberibacter (Las) titer, while ‘Murcott’ and ‘Minneola’ had the highest. The USDA Ft. Pierce, FL farm is managed to reveal genotype responses to HLB. Some current cultivars and hybrid seedlings demonstrate resistance/tolerance, at least to strain(s) of Las present. C. trifoliata is the best documented citrus resistance source with Las titers suppressed even when C. trifoliata is grafted onto severely-infected rootstocks. Some cultivars and hybrids have abundant foliage symptoms, but full canopies and seemingly normal fruit set and size. In 3-years of data from a replicated trial of ‘Triumph’(T), ‘Jackson’(J), ‘Flame’(F), and ‘Marsh’(M), HLB symptoms were severe in all trees and Liberibacter titers were similar. However, F&M were almost completely defoliated in some years while T&J had full canopies. Cumulative fruit/tree was greater for T&J (255&220) than for F&M (29&66). T&J fruit met commercial standards and had normal size but F&M fruit were unacceptable with many small and misshapen. Evidence mounts that useful resistance/tolerance to HLB is present in cultivated citrus and this is a focus of the USDA citrus breeding program

Targeting juvenile hormone metabolic genes in the Asian citrus psyllid (Diaphorina citri Kuwayama) as a strategy to reduce the spread of citrus greening disease

Diaphorina citri Kuwayama, the Asian citrus psyllid (ACP), is a devastating citrus pest due to its transmission of a phloem-limited bacterial pathogen, Candidatus Liberibacter asiaticus, that causes citrus greening. Psyllid control is a major part of effective greening disease management, and our research targets perturbation of insect juvenile hormone metabolism as a new psyllid control strategy.  Previous studies have shown that application of a juvenile hormone (JH) analogue, pyriproxyfen, is known to produce ovicidal/nymphicidal effects, morphological abnormalities, and reduced fecundity in ACP adults. These observations prompted us to identify JH biosynthetic and degradative pathways as targets for biologically-based control strategies, including RNA interference, as alternatives to heavy reliance on broad-spectrum pesticides. First, candidate genes/cDNAs encoding the JH metabolic enzymes, juvenile hormone acid methyl transferase (JHAMT) and juvenile hormone esterase, were identified through computational analysis of the D. citri genome. Second, JHAMT cDNA was cloned, expressed in E. coli and a functional protein was purified. This JHAMT had a high affinity for substrates leading to JHI and JHIII synthesis, making it plausible that both juvenoids are present in the ACP. Results are discussed with respect to mechanism(s) of juvenile hormone biosynthesis/catabolism in the ACP and targeting this process as an interdiction point for a bio-rational ACP control strategy

Targeting juvenile hormone metabolic genes in the Asian citrus psyllid (Diaphorina citri Kuwayama) as a strategy to reduce the spread of citrus greening disease

Diaphorina citri Kuwayama, the Asian citrus psyllid (ACP), is a devastating citrus pest due to its transmission of a phloem-limited bacterial pathogen, Candidatus Liberibacter asiaticus, that causes citrus greening. Psyllid control is a major part of effective greening disease management, and our research targets perturbation of insect juvenile hormone metabolism as a new psyllid control strategy.  Previous studies have shown that application of a juvenile hormone (JH) analogue, pyriproxyfen, is known to produce ovicidal/nymphicidal effects, morphological abnormalities, and reduced fecundity in ACP adults. These observations prompted us to identify JH biosynthetic and degradative pathways as targets for biologically-based control strategies, including RNA interference, as alternatives to heavy reliance on broad-spectrum pesticides. First, candidate genes/cDNAs encoding the JH metabolic enzymes, juvenile hormone acid methyl transferase (JHAMT) and juvenile hormone esterase, were identified through computational analysis of the D. citri genome. Second, JHAMT cDNA was cloned, expressed in E. coli and a functional protein was purified. This JHAMT had a high affinity for substrates leading to JHI and JHIII synthesis, making it plausible that both juvenoids are present in the ACP. Results are discussed with respect to mechanism(s) of juvenile hormone biosynthesis/catabolism in the ACP and targeting this process as an interdiction point for a bio-rational ACP control strategy

Whole Genome PCR Sequencing Strategy for ‘Candidatus’ Liberibacter asiaticus: Analyzing Sequence Diversity Among U.S. Isolates

The inability to culture the ‘Candidatus’ Liberibacter asiaticus (CLas) bacterium has greatly hindered research on the etiology of the citrus disease Huanglongbing which is associated with this bacterium.  This is especially true with respect to possible links between strain/isolate diversity and disease symptom variations and development.  Past genetic marker research indicates that there is considerable CLas isolate diversity even within Florida (a location that has only recognized the presence of the disease since 2005); however, no effort has been made to correlate this diversity with symptom differences.  To advance our understanding of CLas geographic spread and strain/isolate diversity, we have developed a whole-genome PCR amplification strategy that can be used in conjunction with next generation genome sequencing to rapidly obtain near whole genome sequence for specific isolates.  This method was used to generate genome sequence data (~93% of the total genome with an average ~300x coverage) from numerous isolates within Florida that are known to be different with respect to previously characterized genetic markers, and also to compare genomic sequence with isolates that may induce different symptoms within citrus.  Results will be presented that show the diversity among Florida CLas isolates and will also be contextualized within the diversity observed sequence variation among global isolates