Study on the process of egg maturation and classification of ovary development in codling moths (Cydia pomonella L.)

Abstract:【Objective】The codling moth is a kind of quarantine pests of fruit trees in both China and the world because of its severe damage and strong fecundity.The egg-laying amount in nature is much higher than that in laboratory level, reaching more than 300 eggs.Thus, it is of great significance to study the process of egg maturation and ovary development with the codling moths.The aim of this study is to observe the process of egg maturation in Varian tubules and the developmental characteristics of ovaries in different stages after the emergence of female adults, which will provide a basis for the integrated management of the codling moths.【Methods】The larvae of codling moth were originally collected from an abandoned apple orchard, Wuwei city nearby, Gansu province and maintained under standard laboratory conditions at (25±1) ℃ with a relative humidity of 75%±5%, and a 16/8 light/dark cycle.The larvae of codling moths were reared up to the adult stage in spawning boxes by providing 10%honey water.When the process of eggs maturation was studied, the female adults emerging after 24-48h were dissected in the chilled insect ringer solution and observed under a stereoscopic binocular microscope for anatomical observations.The ovaries were fixed in Bouin’s fixative for 12 h and washed in water and then dehydrated gradually with the ethyl alcohol.After dehydration, tissues were cleared in nbutyl alcohol, infiltrated and embedded in paraffin wax at 60 ℃.Tissues were sectioned at 5μm after the clotting of the tissue block.Sections were stained with Haematoxylene-Eosin.The slices were observed and photographed under a binocular EX20 fluorescence microscope.In order to examine the developmental process of ovaries, a female adult and a male adult on the same day of age were collected into a disposable plastic cup (6-8 cm in diameter) .The plastic cups were sealed by using a fresh-keeping bag with the absorbent cotton with 10%honey water.The fixed (30 min) insect body was dissected day by day in a Petri dish with Ring's physiological saline under the Motic K series anatomy microscope.The morphological structures of the ovaries were observed under the Zeiss Stereo Discovery V12stereoscopic microscope, photographed with a Axio Cam MRc 5 and the pictures were processed with Adobe Photoshop【.Results】The process of egg maturation of codling moths was divided into five stages according to the size of the eggs, the morphological changes of the follicular and trophoblast cells, and the yolk deposition, which were named the previtellogenesis stage, the early stage of vitellogenesis, the middle stage of vitellogenesis, the late stage of vitellogenesis and maturation stage, respectively.A few cells on the top and a large number of cells on the lower took place in the egg at the previtellogenesis.Eggs were nearly round at the early stage of vitellogenesis and surrounded by 1-2 layers of cells except for follicular cells that were arranged loosely.There were different sizes of droplets in whole eggs after they grew into the oval shape at the middle stage of vitellogenesis.The shapes of follicular cells in epithelial tissue were spherical to oval.The trophoblastic cells gradually decreased.In the late stage of yolk occurrence, the egg volume became larger, and the yolk drops dispersed in the whole cytoplasm at the late stage of vitellogenesis.At this stage, the follicle membrane was composed of dense squamous epithelial cells, and the trophoblastic cells almost disappeared.The maturation stage was the last period of the development of the egg.Eggshell formed when the egg increased to a certain size but there was no trophoblast at this stage.The ovarian development of codling moths was divided into five phases according to the shapes of ovary, yolk deposition and appearance or disappearance of an egg chamber, which were named the initial phase (StageⅠ) , yolk deposition phase (StageⅡ) , egg maturation phase (StageⅢ) , peak phase of oviposition (StageⅣ) and terminal phase of oviposition (StageⅤ) , respectively.StageⅠ:The tube of the ovary was very small with no egg chamber forming within the growth area of the ovarian tube and was transparent.Fat body was spherical and milky white.There were no eggs in ovarian tube handle, lateral fallopian tube and middle fallopian tube.StageⅡ:The ovarian tube thickened and grew.The yolk sediments were filled with the egg chamber, and there was an obvious overflowing margin between the egg chambers.The adipocytes became oblong with milky white color.StageⅢ:The ovarian tube became thicker and the boundaries between the egg chambers were not obvious.The fat body cells were long filamentous and the eggs arranged closely in the lateral fallopian and middle fallopian tubes.StageⅣ:The boundary between the egg chambers disappeared.The fat body was significantly reduced and almost depleted.The eggs in the lateral fallopian tube and the middle oviduct were high and easy to fall off.StageⅤ:There was a boundary between the egg chambers.The remaining mature eggs containing about 3-4 grains existed in the growing area of the ovarian tube.The ovarian tube base was orange yellow and the fat body was depleted.【Conclusion】In this study, we observed the morphological change of the egg and ovarian development stages of the codling moth, which could provide a scientific reference for forecasting and integrated management on this pest.