Vinay Kalia
Division of Entomology, ICAR- Indian Agricultural Research Institute, New Delhi [email protected]
RNAi is a post transcriptional mechanism of silencing gene function by inserting short homologous sequence of messenger RNA (mRNA) to prevent translation of proteins. RNAi is a gene silencing mechanism triggered by double-stranded RNA (dsRNA). In the first step, the trigger dsRNA is processed by Dicer (species-specific RNase-III like enzymes) into short interfering RNA (siRNA). In the second step, siRNAs are loaded into the effector complex RNA-induced silencing complex (RISC) which is associated with an Argonaut protein (AGO).
The Argonaut protein uses the antisense/guide RNA to associate with target mRNA and then slicing of the target occurs.
An important aspect of the RNA interference pathway is the transport of RNAi information. In insects RNAi can be divided in cell-autonomous [RNA silencing effect takes place within cells where dsRNA is expressed or introduced] and non-cell-autonomous RNAi, the interfering effect takes place in tissues/cells different from the location of application or production of the dsRNA. There are two different kinds of non-cell-autonomous RNAi i.e., environmental RNAi [Signal is picked up by cells from immediate environment such as gut or haemocoel] and systemic RNAi response [when silencing signal spreads to neighbouring cells from epicentre of cells].
For the efficient application of RNAi in insect control, we have to focus on non-cell- autonomous RNAi. The insect will have to internalize the dsRNA of a target gene through feeding. In order to silence the target gene, this dsRNA must be taken up from the gut lumen into the gut cells demonstrating environmental RNAi. The midgut is designed to absorb nutrients from the gut lumen with its large absorption area created by the microvilli. These characteristics make the tissue very interesting as a potential dsRNA uptake location. If the target gene is expressed in a tissue outside of the gut, the silencing signal will also have to spread via cells and tissues, which is systemic RNAi.
Chapter-15
Page | 90 In this class we will evaluate the efficacy of synthesised dsRNA against Helicoerpa armigera by feeding bioassays
Principle: Bioassay refers to the procedure for determination of the relation between a physiologically active agent and the effect, which it produces in a living organism. According to Finney (1952), the term biological assay means the potency of any stimulus, physical, chemical or biological, physiological or psychological by means of the reactions which it produces in living matter. The principle of bioassay is to compare the response of treated insect to those of untreated insect under the same conditions. The response may be based upon mortality, growth inhibition, antifeedant activity etc. Bioassay is affected by experimental conditions therefore success of bioassay depends upon both biotic (type of test insect as well as stage, age, size and sex of insect) and abiotic factors (temperature, humidity, amount of illumination, amount and type of food).
Materials Required
1. ds RNA construct; 2. Negative control dsRNA construct; 3. neonates of H. armigera; 4.
Diet; 5. Microapplicator; 6. Plastic containers; 7. Tissue role; 8. Spatula; 9 nuclease free water and Pestle mortar
Method
1. Prepare different concentrations viz., 0.5, 1.0 and 5.0 µg of dsRNA/g of diet using stock solution suitably diluted to volume of 0.1 ml nuclease free water to mix uniformly in 10 gm of diet.
2. As given above do similarly for –ve control dsRNA construct. In control use nuclease free water only
3. After mixing, divide the diet in three parts and transferred to small plastic containers (5×2 cm). Each container serve as one replicate, with three replications per concentration.
4. Release ten neonates on the treated diet (3g diet) per replication and fed for four days after that they were separated and transferred individually on fresh untreated diet.
5. Record observations on mortality and phenotypic effect after every 24 h up to the adult emergence.
6. As given in step 1 to 4 repeat and kept the treated as well as control sample separately.
7. Collect samples for expression analysis from second fraction after every 24 hours till 96 hours from treatment and control.
8. Wash collected larvae with 70% alcohol followed by nuclease free water and kept in RNAse free tube in -80oC in RNAse later™ for further use expression analysis by Real Time PCR.
Page | 91 Datasheet for Bioassay
1. Name of dsRNA construct (gene) --- 2. No. of concentration/dose --- 3. List of concentrations --- 4. Control (nuclease free water) --- 5. Negative control (unrelated dsRNA) ---
6. Insect name ---
7. Stage of insect ---
8. No. of insect / concentration --- 9. Total No. of insects including control --- 10. Location of insect source if laboratory; --- 11. Method of Bioassay:
Diet incorporation/Diet overlay --- 12. Conditions of bioassay:
Temperature ---
Humidity ---
Light:Dark period --- 13. Duration of bioassay --- 14. Observation Record:
S.No. Conc. of Toxin Mortality/total No. of insects Total Mortality
24h 48h 72h 96h
1. Control 2.
3.
4.
5.
6.
7.
8.
9.
10.
15. Analyze the mortality data by probit analysis; Calculate LC50 and LT50 using MLP (Ross, 1977)
16. Observation on growth inhibition : Take weight of 10-15 larva: control and treatment References
Finney, D.J. (1952). Probit analysis. Cambridge University Press. 2nd Ed.
Ross G.E.S. (1977). Maximum likelihood programme. The numerical algorithms Group, Rothamsted Experiment Station, Harpenden, UK.
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