This method can provide important information related to the pupation ecology of tea leaf caterpillars, Ectropis grisescens, such as their pupation behaviors and the pupae survivorship in response to different environmental factors. The main advantage of this technique, it allows you to use a simple method such as measuring the pupation preference and emergence success of Ectropis grisescens. To begin, cut fresh shoots from tea plants.
Then fill a 250 milliliter triangular flask with tap water and place the shoots into the flask. Transfer three to four flasks to a plastic basin. Next, release 1, 000 to 2, 000 larvae from a laboratory colony of E.grisescens onto the leaves of the tea shoots.
Maintain the larvae in controlled laboratory conditions. Obtain the mature, last-instar larvae that fall from the leaves of the shoots and actively wander along the bottom of the basin. Sterilize soil and sand in an 80 degree Celsius oven dryer for three days.
Then dry both the soil and sand at 50 degrees Celsius for several weeks. The substrate samples are sufficiently dried when the weight does not change over time. Use a wooden pestle and mortar to grind the dried soil.
Then sift the sand and the ground soil through a three millimeter sieve. Add the required amount of distilled water to the sealed bags containing the substrates and mix thoroughly. Next, use PVC sheets to divide polypropylene containers into six equal chambers.
Fix the PVC sheets and seal the cracks with hot glue. Next, fill the six chambers with the same type of substrate with different moisture contents. Use small pieces of tape to paste four to six pieces of fresh leaves to the inner surface of the lid of the polypropylene container.
Next, release 30 mature last-instar larvae onto the leaves pasted to the lid of the chambered container. Carefully turn the lid over and cover the container. Maintain the bioassay arena in an environmental chamber with a 14 to 10 light/dark photoperiod at 26 degrees Celsius.
On day five, count the number of pupae on the surface of the substrate in each chamber. Dismantle the bioassay and count the number of pupae. First, use PVC sheets to divide the polypropylene containers into four chambers.
Then, fix and seal the PVC sheets with hot glue. Fill the chambers of the container with four types of substrate with the same moisture content and randomly assigned orders. Then, use tape to paste fresh tea leaves to the lid before turning it over onto the container.
Next, transfer larvae to the leaves and record and analyze the data as outlined in the text protocol. After preparing the 12 substrate moisture content treatments, add the substrate to plastic containers. Then release 15 mature last-instar larvae into the substrate of each arena.
Maintain each of the bioassay arenas in an environmental chamber with a 14 to 10 light/dark photoperiod at 26 degrees Celsius. On day three, count the number of pupae and any dead larvae on the surface of the substrate of each arena. Finally, record the number of emerging adults each day until no new adults emerge for 15 days.
In this protocol, moisture and substrate choice bioassays were used to study the pupation preference of E.grisescens. The moisture choice bioassays showed that more individuals pupated on or within the 5%and 35%moisture sand compared to the 80%moisture sand. However, significantly more individuals preferred to pupate on or within the soil with intermediate moisture content.
Substrate choice bioassays revealed that sand was most preferred by E.grisescens under 20%moisture conditions, and under 80%moisture conditions. However, no pupation preference was found among the four substrates under 50%moisture conditions. After development, this technique paved the way for researchers in the field of entomology to explore the pupation ecology of many soil-pupating insects.
