Optimal dsRNA Concentration for RNA Interference in Asian Citrus Psyllid.

The Asian citrus psyllid (ACP) is a citrus pest and insect vector of "Candidatus Liberibacter asiaticus", the causal agent of citrus greening disease. Double-stranded RNA (dsRNA) biopesticides that trigger RNA interference (RNAi) offer an alternative to traditional insecticides. Standardized laboratory screening of dsRNA requires establishing the minimal effective concentration(s) that result in effective RNAi "penetrance" and trigger RNAi, resulting in one or more measurable phenotypes, herein, significant gene knockdown and the potential for mortality. In this study, knockdown was evaluated for a range of dsRNA concentrations of three ACP candidate genes, clathrin heavy chain (CHC), vacuolar ATPase subunit A (vATPase-A), and sucrose non-fermenting protein 7 (Snf7). Gene knockdown was quantified for ACP teneral adults and 3rd instar nymphs allowed a 48 h ingestion-access period (IAP) on 10, 50,100, 200, and 500 ng/µL dsRNA dissolved in 20% sucrose followed by a 5-day post-IAP on orange jasmine shoots. Significant gene knockdown (p < 0.05) in ACP third instar nymphs and adults ranged from 12-34% and 18-39%, 5 days post-IAP on dsRNA at 10-500 and 100-500 ng/µL, respectively. The threshold concentration beyond which no significant gene knockdown and adult mortality was observed post-48 h IAP and 10-day IAP, respectively, was determined as 200 ng/µL, a concentration indicative of optimal RNAi penetrance.

UV-LASER adjuvant-surfactant-facilitated delivery of mobile dsRNA to tomato plant vasculature and evidence of biological activity by gene knockdown in the potato psyllid.

BACKGROUND: Double-stranded RNA (dsRNA) biopesticides are of interest for the abatement of insect vectors of pathogenic bacteria such as 'Candidatus Liberibacter', which infects both its psyllid and plant hosts. Silencing of genes essential for psyllids, or for Liberibacter, is anticipated to lead to mortality or impeded bacterial multiplication. Foliar delivery is preferred for biopesticide application; however, the cuticle impedes dsRNA penetration into the vasculature. Here, conditions were established for wounding tomato leaves using ultraviolet light amplification by stimulated emissions of radiation (UV-LASER) to promote dsRNA penetration into leaves and vasculature. RESULTS: UV-LASER treatment with application of select adjuvants/surfactants resulted in vascular delivery of 100-, 300- and 600-bp dsRNAs that, in general, were correlated with size. The 100-bp dsRNA required no pretreatment, whereas 300- and 600-bp dsRNAs entered the vasculature after UV-LASER treatment only and UV-LASER adjuvant/surfactant treatment, respectively. Of six adjuvant/surfactants evaluated, plant-derived oil combined with an anionic organosilicon compound performed most optimally. Localization of dsRNAs in the tomato vasculature was documented using fluorometry and fluorescence confocal microscopy. The biological activity of in planta-delivered dsRNA (200-250 bp) was determined by feeding third-instar psyllids on tomato leaves post UV-LASER adjuvant/surfactant treatment, with or without psyllid cdc42- and gelsolin dsRNAs. Gene knockdown was quantified by quantitative, real-time polymerase chain reaction with reverse transcription (RT-qPCR) amplification. At 10 days post the ingestion-access period, knockdown of cdc42 and gelsolin expression was 61% and 56%, respectively, indicating that the dsRNAs delivered to the tomato vasculature were mobile and biologically active. CONCLUSION: Results indicated that UV-LASER adjuvant/surfactant treatments facilitated the delivery of mobile, biologically active dsRNA molecules to the plant vasculature. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

The Bemisia tabaci cryptic (sibling) species group - imperative for a taxonomic reassessment.

The taxonomy of the Bemisia tabaci cryptic species group remains a challenge due to the lack of morphological differentiation and porous species boundaries among its members. Additionally, it is unclear whether B. tabaci consists of several species in evolutionary stasis with limited morphological change or is the result of a recent adaptive radiation characterized by great ecological diversity but little morphological divergence. Here, a historical overview of the development of the nomenclature used to classify B. tabaci is provided covering changes after synonymizing several species in 1957 until recent insights gained from whole-genome sequencing data. The article discusses the limitations of using a 3.5% mtCOI threshold and argues that a 1% nuclear divergence cutoff better reflects ecological and biogeographic species boundaries. Finally, a plan of action is outlined for naming B. tabaci species using a Latin binomial system in accordance with the International Comission on Zoological Nomenclature (ICZN) regulations.

Native and Non-Native Bemisia tabaci NAFME Haplotypes Can Be Implicated in Dispersal of Endemic and Introduced Begomoviruses in Oman.

Irrigated agriculture and global trade expansion have facilitated diversification and spread of begomoviruses (Geminiviridae), transmitted by the Bemisia tabaci (Gennadius) cryptic species. Oman is situated on major crossroads between Africa and South Asia, where endemic/native and introduced/exotic begomoviruses occur in agroecosystems. The B. tabaci 'B mitotype' belongs to the North Africa-Middle East (NAFME) cryptic species, comprising at least eight endemic haplotypes, of which haplotypes 6 and/or 8 are recognized invasives. Prevalence and associations among native and exotic begomoviruses and NAFME haplotypes in Oman were investigated. Nine begomoviral species were identified from B. tabaci infesting crop or wild plant species, with 67% and 33% representing native and exotic species, respectively. Haplotypes 2, 3, and 5 represented 31%, 3%, and 66% of the B. tabaci population, respectively. Logistic regression and correspondence analyses predicted 'strong'- and 'close' virus-vector associations involving haplotypes 5 and 2 and the exotic chili leaf curl virus (ChiLCV) and endemic tomato yellow leaf curl virus-OM, respectively. Patterns favor a hypothesis of relaxed virus-vector specificity between an endemic haplotype and the introduced ChiLCV, whereas the endemic co-evolved TYLCV-OM and haplotype 2 virus-vector relationship was reinforced. Thus, in Oman, at least one native haplotype can facilitate the spread of endemic and introduced begomoviruses.

RNA interference-mediated knockdown of genes involved in sugar transport and metabolism disrupts psyllid Bactericera cockerelli (Order: Hemiptera) gut physiology and results in high mortality.

Introduction: The causal agent of zebra chip of potato and vein-greening diseases of tomato is "Candidatus Liberibacter solanacearum" (CLso), a fastidious bacterium transmitted by the potato psyllid. In the absence of disease-resistant cultivars, disease management has relied on minimizing vector population size to reduce CLso transmission, which requires frequent insecticide applications. There is growing interest in the use of RNA interference (RNAi) technology to supplant traditional insecticides with biopesticides. This requires knowledge of genes essential for insect livelihood whose knockdown leads to significant mortality or other phenotypes. Such candidate genes can be evaluated by reverse genetics approaches to further corroborate predicted gene function. Methods: Here, five potato psyllid genes involved in sugar homeostasis in the potato psyllid gut, α-glucosidase1 (AGLU1), aquaporin2 (AQP2), facilitated trehalose transporter1 (TRET1), Trehalase1 (TRE1), and Trehalase2 (TRE2), were investigated as candidates for effective gene silencing. Potato psyllid dsRNAs were designed to optimize knockdown of gene targets. Third instar PoP nymphs were given a 48-hr ingestion-access period (IAP) on individual or groups of dsRNA in 20% sucrose. Mortality was recorded 0, 3, 5, 7, and 9 days post-IAP. Gene knockdown was analyzed 9 days post-IAP by quantitative real-time reverse-transcriptase polymerase chain reaction amplification. Results: The individual or stacked dsRNA combinations resulted in 20-60% and 20-40% knockdown, respectively, while subsequent psyllid mortality ranged from 20-40% to >60% for single and stacked dsRNA combinations, respectively. Reverse genetics analysis showed that simultaneous knockdown of the five selected candidate genes with predicted functions in pathways involved in sugar-homeostasis, metabolism, and -transport yielded the highest mortality, when compared with single or combinations of targets. Discussion: Results confirmed the functions afforded by psyllid gut genes responsible for osmotic homeostasis and sugar metabolism/transport are essential for livelihood, identifying them as potentially lucrative RNAi biopesticide targets and highlighted the translational relevance of targeting multiple nodes in a physiological pathway simultaneously.

Characterization of the Asian Citrus Psyllid-'Candidatus Liberibacter Asiaticus' Pathosystem in Saudi Arabia Reveals Two Predominant CLas Lineages and One Asian Citrus Psyllid Vector Haplotype.

In Saudi Arabia (SA), the citrus greening disease is caused by 'Candidatus Liberibacter asiaticus' (CLas) transmitted by the Asian citrus psyllid (ACP) Diaphorina citri. The origin and route(s) of the ACP-CLas pathosystem invasion in SA have not been studied. Adult ACP were collected from citrus trees in SA and differentiated by analysis of the mitochondrial cytochrome oxidase I (mtCOI) and nuclear copper transporting protein (atox1) genes. A phylogenetic analysis of the Wolbachia spp. surface protein (wsp) gene was used to identify the ACP-associated Wolbachia spp. A phylogenetic analysis of the atox1 and mtCOI gene sequences revealed one predominant ACP haplotype most closely related to the Indian subcontinent founder populations. The detection and identification of CLas in citrus trees were carried out by polymerase chain reaction (PCR) amplification and sequencing of the 16S rDNA gene. The CLas-integrated prophage genomes were sequenced, annotated, and used to differentiate CLas populations. The ML and ASTRAL trees reconstructed with prophages type 1 and 2 genome sequences, separately and concatenated, resolved two major lineages, CLas-1 and -2. The CLas-1 clade, reported here for the first time, consisted of isolates from SA isolates and Pakistan. The CLas-2 sequences formed two groups, CLas-2-1 and -2-2, previously the 'Asiatic' and 'Floridian' strains, respectively. Members of CLas-2-1 originated from Southeast Asia, the USA, and other worldwide locations, while CLas-2-2 was identified only in Florida. This study provides the first snapshot into the status of the ACP-CLas pathosystem in SA. In addition, the results provide new insights into the pathosystem coevolution and global invasion histories of two ACP-CLas lineages with a predicted center of origin in South and Southeast Asia, respectively.

Earlier than expected introductions of the Bemisia tabaci B mitotype in Brazil reveal an unprecedented, rapid invasion history.

During 1991, in Brazil, the presence of the exotic Bemisia tabaci B mitotype was reported in São Paulo state. However, the duration from the time of initial introduction to population upsurges is not known. To investigate whether the 1991 B mitotype outbreaks in Brazil originated in São Paulo or from migrating populations from neighboring introduction sites, country-wide field samples of B. tabaci archived from 1989-2005 collections were subjected to analysis of mitochondrial cytochrome oxidase I (mtCOI) and nuclear RNA-binding protein 15 (RP-15) sequences. The results of mtCOI sequence analysis identified all B. tabaci as the NAFME 8 haplotype of the B mitotype. Phylogenetic analyses of RP-15 sequences revealed that the B mitotype was likely a hybrid between a B type parent related to a haplotype Ethiopian endemism (NAFME 1-3), and an unidentified parent from the North Africa-Middle East (NAF-ME) region. Results provide the first evidence that this widely invasive B mitotype has evolved from a previously undocumented hybridization event. Samples from Rio de Janeiro (1989) and Ceará state (1990), respectively, are the earliest known B mitotype records in Brazil. A simulated migration for the 1989 introduction predicted a dispersal rate of 200-500 km/year, indicating that the population was unlikely to have reached Ceará by 1990. Results implicated two independent introductions of the B mitotype in Brazil in 1989 and 1990, that together were predicted to have contributed to the complete invasion of Brazil in only 30 generations.

Knockdown of ecdysteroid synthesis genes results in impaired molting and high mortality in Bactericera cockerelli (Hemiptera: Triozidae).

BACKGROUND: RNA-mediated interference (RNAi) has become a promising biopesticide technology with which to direct sequence-specific gene knockdown of key targets in the potato psyllid (PoP) Bactericera cockerelli, resulting in significant mortality. In this study, three strategically selected target genes, ATF4, C7 and D24, essential for the biosynthesis and regulation of ecdysteroids, were evaluated for knockdown and mortality using oral delivery of individual, paired and all three double-stranded RNAs (dsRNAs), in five replicated experiments. Knockdown was determined as the fold-change in gene expression using a quantitative polymerase chain reaction. RESULTS: Knockdown of the D24 target, at 39%-45%, resulted in 51% PoP mortality by 10 days post-ingestion (dpi) of dsRNA. Knockdown of C7, at 38%-61%, resulted in 53% mortality by 10 dpi, whereas dsD24 ingestion resulted in 65% mortality by 10 dpi when dsD24 and dsC7 were co-delivered. Three phenotypes, INCOMEC, PREMEC and SWOLLEN, were observed at a frequency of 4%-12%, and are consistent with incomplete ecdysis in immature and/or adult PoP. Adult PoP exhibiting INCOMEC survived for several days but were unable to mate or fly, whereas SWOLLEN and PREMEC were lethal to the immature instars. Knockdown of ATF4 did not result in the mortality or malformations in immature and adult PoP. CONCLUSIONS: Compared with knockdown of individual D24 and C7 targets, significantly greater RNAi penetrance was achieved following delivery of combined dsRNAs. The highest knockdown that resulted in incomplete ecdysis and/or mortality was obtained for targets with predicted involvement in the same or interacting pathway(s). Knockdown of ATF4 was apparently "rescued" by uncharacterized compensatory gene(s) or effects. © 2022 Society of Chemical Industry.

Phylogeographic and SNPs Analyses of Bemisia tabaci B Mitotype Populations Reveal Only Two of Eight Haplotypes Are Invasive.

The Bemisia tabaci cryptic species contains 39 known mitotypes of which the B and Q are best recognized for having established outside their extant endemic range. In the 1980s, previously uncharacterized haplotype(s) of the B mitotype rapidly established in tropical and subtropical locales distant from their presumed center of origin, leading to displacement of several native mitotypes and extreme damage to crops and other vegetation particularly in irrigated agroecosystems. To trace the natural and evolutionary history of the invasive B haplotypes, a phylo-biogeographic study was undertaken. Patterns of single nucleotide polymorphisms (SNPs) and signatures potentially indicative of geographic isolation were investigated using a globally representative mitochondrial cytochrome oxidase I gene (mtCOI) sequence database. Eight haplotype groups within the North Africa-Middle East (NAFME) region were differentiated, NAFME 1-8. The NAFME 1-3 haplotypes were members of the same population that is associated with warm desert climate niches of the Arabian Peninsula and east coastal Africa-Ethiopia. The NAFME 4 and 5 haplotypes are endemic to warm and cold semi-arid niches delimited by the Irano-Turanian floristic region, itself harboring extensive biodiversity. Haplotypes 6 and 7 co-occurred in the Middle East along eastern Mediterranean Sea landmasses, while NAFME 8 was found to be endemic to Cyprus, Turkey, and desert micro-niches throughout Egypt and Israel. Contrary to claims that collectively, the B mitotype is invasive, NAFME 6 and 8 are the only haplotypes to have established in geographical locations outside of their zone of endemism.

Genetic variability, community structure, and horizontal transfer of endosymbionts among three Asia II-Bemisia tabaci mitotypes in Pakistan.

Endosymbionts associated with the whitefly Bemisia tabaci cryptic species are known to contribute to host fitness and environmental adaptation. The genetic diversity and population complexity were investigated for endosymbiont communities of B. tabaci occupying different micro-environments in Pakistan. Mitotypes of B. tabaci were identified by comparative sequence analysis of the mitochondria cytochrome oxidase I (mtCOI) gene sequence. Whitefly mitotypes belonged to the Asia II-1, -5, and -7 mitotypes of the Asia II major clade. The whitefly-endosymbiont communities were characterized based on 16S ribosomal RNA operational taxonomic unit (OTU) assignments, resulting in 43 OTUs. Most of the OTUs occurred in the Asia II-1 and II-7 mitotypes (r 2 = .9, p < .005), while the Asia II-5 microbiome was less complex. The microbiome OTU groups were mitotype-specific, clustering with a basis in phylogeographical distribution and the corresponding ecological niche of their whitefly host, suggesting mitotype-microbiome co-adaptation. The primary endosymbiont Portiera was represented by a single, highly homologous OTU (0%-0.67% divergence). Two of six Arsenophonus OTUs were uniquely associated with Asia II-5 and -7, and one occurred exclusively in Asia II-1, two only in Asia II-5, and one in both Asia II-1 and -7. Four other secondary endosymbionts, Cardinium, Hemipteriphilus, Rickettsia, and Wolbachia OTUs, were found at ≤29% frequencies. The most prevalent Arsenophonus OTU was found in all three Asia II mitotypes (55% frequency), whereas the same strain of Cardinium and Wolbachia was found in both Asia II-1 and -5, and a single Hemipteriphilus OTU occurred in Asia II-1 and -7. This pattern is indicative of horizontal transfer, suggestive of a proximity between mitotypes sufficient for gene flow at overlapping mitotype ecological niches.