practical-use-rna-interference-oral-delivery-double-stranded-rna

Practical Use of RNA Interference: Oral Delivery of Double-stranded RNA in Liposome Carriers for Cockroaches

RNA interference (RNAi) has been widely applied for uncovering the biological functions of numerous genes, and has been envisaged as a pest control tool ...

National Taiwan University

Hypertrehalosemic hormone (HTH) is a neuropeptide within the adipokinetic hormone (AKH) family that induces a release of trehalose from fat body into hemolymph in a number of insects. In this study, we first showed that female adult German cockroach, Blattella germanica, displayed a cyclic fluctuation of hemolymph trehalose levels correlated to the maturation of oocytes in the reproductive cycle. After cloning the HTH cDNA from the German cockroach (Blage-HTH), expression studies indicated that Blage-HTH mRNA showed the cyclic changes during the first reproductive cycle, where peak values occurred in 8-day-old virgin female cockroaches, which were going to produce oothecae. The functions of Blage-HTH were studied using RNA interference (RNAi) to knockdown its expression. Adult virgin females of B. germanica injected with Blage-HTH dsRNA increased hemolymph trehalose levels in the late period of vitellogenesis more slowly than control. Furthermore, RNAi of Blage-HTH delayed oviposition time and some (10%) individuals did not produce the first ootheca until 15 days after eclosion, whereas the control group produced ootheca before 9 days in all cases.

RNA interference unveils functions of the hypertrehalosemic hormone on cyclic fluctuation of hemolymph trehalose and oviposition in the virgin female Blattella germanica.

Hypertrehalosemic hormone (HTH) is a peptide hormone that belongs to the adipokinetic hormone/red pigment concentrating hormone (AKH/RPCH) family, which exerts pleiotropic actions related to catabolic reaction and stress response. AKH peptides have been demonstrated to participate in stress response including oxidative stress in several insects. In order to study the signaling pathway of HTH involved in anti-oxidative stress, we have characterized a HIH receptor cDNA in Blattella germanica (Blage-HTHR) in structural and in functional terms using RNA interference (RNAi). Blage-HTHR is expressed in various female adult tissues (brain-CC-CA, ventral nerve cord, midgut, fat body, oviduct), but maximal expression is observed in the fat body. RNAi-mediated knockdown of Blage-HTHR expression results in a significantly lower level of hemolymph trehalose, even though HTH is exogenously administered. Paraquat elicits lethal oxidative stress in B. germanica, and co-injection of paraquat and HTH reduces this detrimental effect and extends the median survival time. Interestingly, the "rescue" effect of HTH on mortality caused by paraquat is diminished in specimens with depleted expression of Blage-HTH and Blage-HTHR. Finally, lipid peroxidation in the hemolymph increases 4 h after paraquat treatment, in comparison with control specimens or with HTH-treated specimens. However, lipid peroxidation induced by paraquat was not "rescued" by HTH in Blage-HTH and Blage-HTHR knockdown specimens. Our results demonstrate that HTH acts as a stress hormone mediating anti-oxidative protection in B. germanica, and that its receptor, Blage-HTHR, is essential for this action.

Functional Characterization of Hypertrehalosemic Hormone Receptor in Relation to Hemolymph Trehalose and to Oxidative Stress in the Cockroach Blattella germanica.

The daily susceptibility rhythm to permethrin and the expression level of the delta class glutathione S-transferase (BgGSTD1) gene were investigated in Blattella germanica. Male cockroaches were exposed to the same concentration of permethrin at different times in a light-dark cycle, and results showed that the highest resistance occurred at night. Furthermore, the circadian rhythmicity of permethrin susceptibility was demonstrated by the highest resistance at subjective night under constant darkness. The mRNA level of the BgGSTD1 gene in the fat body of B. germanica peaked early in the day or subjective day under light-dark or constant dark conditions, whereas enzyme activity of cytosolic GSTs did not reflect the rhythmic pattern as well as BgGSTD1 expression. RNA interference (RNAi) was employed to study the function of BgGSTD1 in the circadian rhythm of permethrin susceptibility in B. germanica. Both BgGSTD1 mRNA level and cytosolic GSTs activity were significantly decreased by dsGSTD1 injection. In addition, survival of B. germanica with silenced BgGSTD1 was significantly decreased at night but not in the day when the cockroaches were exposed to permethrin. Total cytosolic GSTs activity demonstrated that is not the only gene involved in the circadian regulation of the permethrin resistance, although it is one of the major regulators of permethrin resistance. 

Circadian regulation of permethrin susceptibility by glutathione S-transferase (BgGSTD1) in the German cockroach (Blattella germanica).

In the past years, the concept of RNAi application for insect pest control has been proposed, considering the disruption of vital genes. However, the efficiency of RNAi is variable between different insect groups, especially by oral delivery of dsRNA. The purpose of this study is to assess the possibilities of RNAi as a tool for pest control using oral delivery of the dsRNAs encapsulated by liposome in the German cockroach Blattella germanica, which is highly sensitive to RNAi by injection of dsRNAs.

Oral delivery of dsRNA lipoplexes to German cockroach protects dsRNA from degradation and induces RNAi response.

Acetic acid is a fermentation product of many microorganisms, including some that inhabit the food and guts of Drosophila. Here, we investigated the effect of dietary acetic acid on oviposition and larval performance of Drosophila. At all concentrations tested (0.34-3.4%), acetic acid promoted egg deposition by mated females in no-choice assays; and females preferred to oviposit on diet with acetic acid relative to acetic acid-free diet. However, acetic acid depressed larval performance, particularly extending the development time of both larvae colonized with the bacterium Acetobacter pomorum and axenic (microbe-free) larvae. The larvae may incur an energetic cost associated with dissipating the high acid load on acetic acid-supplemented diets. This effect was compounded by suppressed population growth of A. pomorum on the 3.4% acetic acid diet, such that the gnotobiotic Drosophila on this diet displayed traits characteristic of axenic Drosophila, specifically reduced developmental rate and elevated lipid content. It is concluded that acetic acid is deleterious to larval Drosophila, and hypothesized that acetic acid may function as a reliable cue for females to oviposit in substrates bearing microbial communities that promote larval nutrition.

Article	Year
RNA interference unveils functions of the hypertrehalosemic hormone on cyclic fluctuation of hemolymph trehalose and oviposition in the virgin female Blattella germanica. Journal of insect physiology\| PubMed ID: 21439292	2011
Functional Characterization of Hypertrehalosemic Hormone Receptor in Relation to Hemolymph Trehalose and to Oxidative Stress in the Cockroach Blattella germanica. Frontiers in endocrinology\| PubMed ID: 22649400	2011
Circadian regulation of permethrin susceptibility by glutathione S-transferase (BgGSTD1) in the German cockroach (Blattella germanica). Journal of insect physiology\| PubMed ID: 24819204	2014
Oral delivery of dsRNA lipoplexes to German cockroach protects dsRNA from degradation and induces RNAi response. Pest management science\| PubMed ID: 27470169	2017
Physiological responses of insects to microbial fermentation products: Insights from the interactions between Drosophila and acetic acid. Journal of insect physiology\| PubMed ID: 28522417	2018

Jia-Hsin Huang

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