Guiding RNAi Design Through Characterization of Endogenous Small RNA Pathways.

RNA interference (RNAi) is a common eukaryotic gene regulation process driven by small RNA effectors. Mechanisms that govern regulatory small noncoding RNA behavior have been extensively described in only a handful of organisms, which suggests that the most effective RNAi approach in many organisms, such as insect pests, remains to be determined. Taking advantage of advances in high-throughput sequencing, characterization of small RNA molecules can be achieved through bioinformatic approaches without the need for genetic experiments. This chapter describes pipelines for characterizing three main classes of small RNAs (microRNAs, small-interfering RNAs, and piwi-associated RNAs) using computationally determined small RNA biogenesis signatures. Obtaining information regarding the abundance of different small RNA classes through these pipelines will lead to a better-informed RNAi strategy, thereby identifying the most efficacious approach for RNAi.

Double-Strand RNA (dsRNA) Delivery Methods in Insects: Diaphorina citri.

RNAi is a gene-silencing mechanism conserved in the vast majority of eukaryotes. It is widely used to study gene function in animals due to the ease of eliciting gene knockdown. Beyond research applications, RNAi technology based on exogenous dsRNA is a promising candidate for next generation insect pest control. An advantage of using RNAi is that design of dsRNA essentially requires only the sequence of the target gene. The greatest challenge, however, is dsRNA delivery for large-scale insect control. Delivery methods that have widely been used are oral, injection, or via soaking. Unfortunately, each insect presents its own challenges owing to the differences in the presence of dsRNA degrading enzymes, cellular uptake efficiency, expression of core RNAi machinery, the nature of the target gene, the concentration and persistence of the dsRNA, as well as the particular way of feeding of each insect, which together cause variations in the efficiency of RNAi. In this chapter, a protocol for the synthetic production of dsRNA is described along with three methods for delivery that have been successful in one of the more problematic insects, Diaphorina citri.

The Chorion Proteome of Diaphorina citri, the Vector of Huanglongbing Disease in Citrus.

Nowadays, the Asian citrus psyllid, Diaphorina citri (Kuwayama) (Hemiptera: Liviidae) is considered the most devastating pest of citrus because it transmits "Candidatus Liberibacter asiaticus", the putative causal agent of huanglongbing (HLB) or citrus greening. Controlling the vector is the main strategy used to mitigate HLB. Targeting D. citri at the very early stages of its development may offer an effective control strategy. Identifying chorion proteins will contribute to a better understanding of embryo development and egg hatching and thus could lead to valuable targets to better control psyllid populations. Herein, we analyze the chorion proteins of D. citri. Mass spectrometry-based bottom-up/shotgun proteomics and databases were queried to achieve protein identification. Fifty-one proteins were identified in D. citri chorion. The D. citri chorion proteins were divided into eight categories according to their biological or molecular function: i-enzymes (25%); ii-binding proteins (10%); iii-structural proteins (8%); iv-homeostasis-related proteins, mostly vitellogenins (8%); v-proteins related to gene expression (6%); vi-immune system proteins (6%); vii-other proteins (16%); and viii-uncharacterized proteins (21%). The composition of the chorion proteome suggested that the hatching rate could be reduced by silencing chorion-related genes. The proteomic analysis of D. citri chorion tissue allowed us to identify its proteins, providing promising new targets for D. citri control through RNA interference technology.

Silencing of Aquaporin Homologue Accumulates Uric Acid and Decreases the Lifespan of the Asian Citrus Psyllid, Diaphorina citri (Hemiptera: Liviidae).

The Asian citrus psyllid, Diaphorina citri Kuwayama is devastating the citrus industry worldwide. It transmits 'Candidatus Liberibacter asiaticus', the pathogen of Huanglongbing in citrus. RNA interference is an excellent tool for functional genomics and for screening target genes for pest control. Herein, we silenced the aquaporin (AQP) gene (DcAQP) homologue in D. citri to study its functionality and whether it could be a good target for a control strategy. AQP is an integral membrane channel protein that aids in the rapid flux of water and other small solutes that move across the lipid membrane. In Hemiptera, it is well established that AQP plays important roles in adjusting to physiological challenges including (1) regulating osmotic stress between the gut lumen and hemolymph after imbibing large quantities of a low nitrogen, sugar-rich liquid diet; (2) avoiding or preventing dehydration and desiccation; and (3) surviving at elevated temperatures. The dsRNA-DcAQP was applied twice to nymphs of the 4th and 5th instars through a soaking technique. Silencing AQP caused a significant increase in nymph mortality. Emerged adults showed malformations and a shorter lifespan. Silencing DcAQP provoked alterations in some metabolites and increased the uric acid content in emerged adults. DcAQP could be a useful target to control D. citri.

Metabolic Profiling of Hybrids Generated from Pummelo and Citrus latipes in Relation to Their Attraction to Diaphorina citri, the Vector of Huanglongbing.

The citrus industry at present is severely affected by huanglongbing disease (HLB). HLB is caused by the supposed bacterial pathogen "Candidatus Liberibacter asiaticus" and is transmitted by the insect vector, the Asian citrus psyllid, Diaphorina citri Kuwayama. Developing new citrus hybrids to improve HLB management is much needed. In this study, we investigated the metabolomic profiles of three new hybrids produced from the cross of C2-5-12 Pummelo (Citrus maxima (L.) Osbeck) × pollen from Citrus latipes. The hybrids were selected based on leaf morphology and seedling vigor. The selected hybrids exhibited compact and upright tree architecture as seen in C. latipes. Hybrids were verified by simple sequence repeat markers, and were subjected to metabolomic analysis using gas chromatography-mass spectrometry. The volatile organic compounds (VOCs) and polar metabolites profiling also showed that the new hybrids were different from their parents. Interestingly, the levels of stored VOCs in hybrid II were higher than those observed in its parents and other hybrids. The level of most VOCs released by hybrid II was also higher than that released from its parents. Additionally, the preference assay showed that hybrid II was more attractive to D. citri than its parents and other hybrids. The leaf morphology, compact and upright architecture of hybrid II, and its attraction to D. citri suggest that it could be used as a windbreak and trap tree for D. citri (double duty), once its tolerance to HLB disease is confirmed. Our results showed that metabolomic analysis could be successfully used to understand the biochemical mechanisms controlling the interaction of D. citri with its host plants.

Tracing Penicillin Movement in Citrus Plants Using Fluorescence-Labeled Penicillin.

Huánglóngbìng (HLB), citrus greening, is one of the most destructive diseases of citrus plants worldwide. In North America, HLB is caused by the phloem-limited bacterium Candidatus Liberibacter asiaticus and is transmitted by the Asian citrus psyllid, Diaphorina citri. No cure exists at present, and the use of antibiotics for the control of HLB has gained interest due to the significant losses to the citrus industry. Because of unsatisfactory results when using foliar applications of antibiotics, concerns were raised regarding the uptake and translocation of these materials within trees. We, therefore, investigated a method that allows us to study the movement of antibiotic materials in citrus plants. Herein, we utilized a fluorescence-labeled penicillin, BOCILLINTM FL-Penicillin (FL-penicillin), to study the uptake and translocation of penicillin in citrus plants. FL-penicillin was applied by puncture to the stem of young citrus seedlings and was traced by using fluorescence microscopy. After application, we detected FL-penicillin in the leaves and in the stem xylem and phloem tissues above and below the application site in both intact and partially bark-girdled citrus seedlings, indicating that it is easily taken up and transported through the plant vascular system. In addition, we detected FL-penicillin in the gut of D. citri, which were allowed to feed on the treated plants, suggesting translocation of this molecule into the vascular tissue. We propose that the use of fluorescent-labeled molecules could be an effective tool for understanding the uptake and translocation of antibiotics and other macromolecules in plants and insects.

Silencing of sucrose hydrolase causes nymph mortality and disturbs adult osmotic homeostasis in Diaphorina citri (Hemiptera: Liviidae).

Plant piercing sucking insects mainly feed on phloem sap containing a high amount of sucrose. To enhance the absorption of sucrose from the midgut, sucrose hydrolase digests sucrose into glucose and fructose. In this study, a sucrose hydrolase homolog (DcSuh) was identified and targeted in Diaphorina citri, the vector of huanglongbing (HLB), by RNA interference (RNAi). In silico analysis revealed the presence of an Aamy domain in the DcSUH protein, which is characteristic of the glycoside hydrolase family 13 (GH13). Phylogenetic analysis showed DcSuh was closely related to the sucrose hydrolase of other Hemiptera members. The highest gene expression levels of DcSuh was found in the 4th and 5th instar nymphs. dsRNA-mediated RNAi of DcSuh was achieved through topical feeding. Our results showed that application of 0.2 µL of 500 ng µL-1 (100 ng) dsRNA-DcSuh was sufficient to repress the expression of the targeted gene and cause nymph mortality and reduce adult lifespan. The reduction in gene expression, mortality, and lifespan was dose-dependent. In agreement with the gene expression results, treatment with dsRNA-DcSuh significantly reduced sucrose hydrolase activity in treated nymphs and emerged adults from treated nymphs. Interestingly, some emerged adults from treated nymphs showed a swollen abdomen phenotype, indicating that these insects were under osmotic stress. Although the percentage of swollen abdomens was low, their incidence was significantly correlated with the concentration of applied dsRNA-DcSuh. Metabolomic analyses using GC-MS showed an accumulation of sucrose and a reduction in fructose, glucose and trehalose in treated nymphs, confirming the inhibition of sucrose hydrolase activity. Additionally, most of the secondary metabolites were reduced in the treated nymphs, indicating a reduction in the biological activities in D. citri and that they are under stress. Our findings indicate that sucrose hydrolase might be a potential target for effective RNAi control of D. citri.

Ausencia de efecto piramidal sobre cardiomiocitos aislados de rata / No pyramidal effect on cardiomyocytes isolated from rats

Se valoró que los promotores de la existencia de la energía piramidal sugieren que pirámides no ferrosas de determinadas proporciones y con cierta orientación, son capaces de concentrar energía que proporciona efectos beneficiosos a los objetos situados en su interior. No obstante, científicos cubanos han argumentado la imposibilidad de la existencia de esta energía. Se propuso una acción bactericida de la energía piramidal relacionada con cambios inducidos en el agua, lo cual ayudaría a preservar materias orgánicas. El objetivo fue estudiar la supervivencia de cardiomiocitos ventriculares de rata, conservados dentro de una pirámide no ferrosa. Los resultados mostraron que no existió diferencia estadísticamente significativa en la supervivencia de cardiomiocitos conservados dentro de la pirámide, fuera de esta o dentro de la pirámide mal orientada. Estos datos indicaron que las pirámides no ejercen acción directa ni sobre los cardiomiocitos, ni sobre el agua o los componentes de la solución de conservación.

Promoters of the so-called Pyramidal Energy suggest that non-ferrous pyramids of certain proportions and orientation could concentrate energy, thus conferring beneficial effects on the objects inside them. However, Cuban scientists have argued against the existence of this energy. Bactericidal action by pyramidal energy has been proposed in relation to water-induced changes, which could help to preserve organic matter. The aim was to study the survival of rat cardiomyocytes preserved inside a non-ferrous pyramid. The results showed no statistically significant difference in survival between preserved cardiomyocytes either inside or outside the pyramid or inside a wronly positioned pyramid. They indicated that pyramids exert no direct action either on cardiomyocytes or on water and the elements of the preserving solution.

Ausencia de efecto piramidal sobre cardiomiocitos aislados de rata / No pyramidal effect on cardiomyocytes isolated from rats

Se valoró que los promotores de la existencia de la energía piramidal sugieren que pirámides no ferrosas de determinadas proporciones y con cierta orientación, son capaces de concentrar energía que proporciona efectos beneficiosos a los objetos situados en su interior. No obstante, científicos cubanos han argumentado la imposibilidad de la existencia de esta energía. Se propuso una acción bactericida de la energía piramidal relacionada con cambios inducidos en el agua, lo cual ayudaría a preservar materias orgánicas. El objetivo fue estudiar la supervivencia de cardiomiocitos ventriculares de rata, conservados dentro de una pirámide no ferrosa. Los resultados mostraron que no existió diferencia estadísticamente significativa en la supervivencia de cardiomiocitos conservados dentro de la pirámide, fuera de esta o dentro de la pirámide mal orientada. Estos datos indicaron que las pirámides no ejercen acción directa ni sobre los cardiomiocitos, ni sobre el agua o los componentes de la solución de conservación(AU)

Promoters of the so-called Pyramidal Energy suggest that non-ferrous pyramids of certain proportions and orientation could concentrate energy, thus conferring beneficial effects on the objects inside them. However, Cuban scientists have argued against the existence of this energy. Bactericidal action by pyramidal energy has been proposed in relation to water-induced changes, which could help to preserve organic matter. The aim was to study the survival of rat cardiomyocytes preserved inside a non-ferrous pyramid. The results showed no statistically significant difference in survival between preserved cardiomyocytes either inside or outside the pyramid or inside a wronly positioned pyramid. They indicated that pyramids exert no direct action either on cardiomyocytes or on water and the elements of the preserving solution(AU)