Mass-Rearing and Molecular Studies in Tortricidae Pest Insects

We present fundamental, but highly in demand masses for mass rearing, observations and molecular studies using serious pest Tortrix masses, to study their biology. So far, the structure of the insect eggs have prevented further analysis. Notably, we enabled the study of thin, soft and fragile eggs covered by maternal secretion.

There is simple techniques expected to be applicable for further research on tortrix, trying other insects and other taxa. Begin by placing a female and two males in a 120 milliliter plastic cup with a piece of paraffin paper to establish a Matra line. Slice approximately 60 grams of artificial diets using a Grater for mass rearing.

Place the egg mass filled with matured embryos on the sliced artificial diet in a plastic container. Place paraffin papers on the egg mass with the sliced diets. Place 15 males and 10 females in a plastic box for mating at 25 degrees Celsius.

Collect eggs every five to seven days and repeat mass rearing steps for each generation. Soak the egg mass in 1000 microliters of 1.2%sodium hypochlorite aqueous solution for 10 minutes or into 1000 microliters of five molar potassium hydroxide aqueous solution for 30 minutes to separate the eggs. Wash the separated eggs in 1000 microliters of PBSt.

Soak eggs in a weight by volume mixture of 500 microliters of 100%heptane and 500 microliters of 4%Paraformaldehyde PBSt solution. Mix for 10 minutes at 1500 rotations per minute using a vortex mixer. Soak the eggs into a mixture of 500 microliters of 100%heptane and 500 microliters of 100%methanol.

Mix for 10 minutes at 1500 rotations per minute. Wash the eggs twice with 1000 microliters of 100%methanol and store them at four degrees Celsius in 100%methanol. Soak the eggs sequentially in 99%70%and 50%ethanol and PBSt for five minutes each for hydrophilization.

Wash the eggs with PBSt. Immerse the eggs in one microgram per milliliter DAPI solution for five minutes, and then wash the eggs with PBS twice. Soak the eggs in 20 microliters of 1.25%lactic, acetic or CN solution, to visualize heterochromatin until the nuclei exhibit a brilliant red color.

Transfer the stained eggs using a pipette to a glass slide, then enclosed them with an anti-fade reagent and a cover glass. Extract Ferrate, Amonamagnetama and Adoxophyes honmai larvae from the egg masses using forceps. Dissect the larvae on the glass slide.

Fix the tissues with a mixture of one to three 99.7%ascetic acid in 100%methanol for five minutes. Stain those with 1.25%weight by weight lactic, ascetic, or CN solution until the nuclei are stained. Determine the sex of each specimen by observing the presence of heterochromatin for females or absence for males under a microscope.

To extract DNA and RNA from sex determined Ferrate larvae, pool 12 sex determined male or female ferrate larvae, and add either cell lysis buffer or RNA extraction reagents into one 1.5 milliliter tube. The fixation, Permeabilization and staining steps enable the visualization of nuclei with DAPI solution. Lactic, ascetic or CN staining using the dissected Ferrate larvae revealed that females exhibit heterochromatin as a dot, but males lack the heterochromatin.

The modified protocols using a spin column improved the quality of the RNA producing 900 to 1500 nanograms of product with a 260 to 200 ratio of 1.9 to 2.1, and a 260 to 280 ratio of 1.9 to 2.3. The RNA concentration ratio for the modified protocol was below 1.2, meeting the quality requirements for next generation sequencing. The intactness of the RNA extracted from sex determined Ferrate larvae using the modified protocol was confirmed using microchip electrophoresis.

The prepared libraries were confirmed to yield high Q 30 score reads. Our message contributes to fundamental insect studies and pest tools. Moreover, our protocols have the potential to be used to assess the effective chemical pesticides or inter cell microbes during embryogenesis.