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Toxicity of spirotetramat to F. occidentalis eggsThe indoor toxicity of spirotetramat to 0-h-, 12-h-, and 24-h-old eggs of F. occidentalis using egg dipping method and leaf dipping method was shown in Table 1 after egg hatching was observed for 144 h. The results suggested that the LC50 value gradually decreased as the egg age increased. The two methods have the same trend. And in the leaf dipping method, according to the confidence limits analysis, 0-h-old eggs are significantly more sensitive to spirotretramat than 24-h-old eggs.Table 1 Toxicity of spirotetramat to F. occidentalis eggs.Full size tableEgg external shape observationsExternal morphology of normally developed isolated 0-h-old eggs of F. occidentalis in the control treatment (Fig. 1a) were compared with those treated with spirotetramat (Fig. 1b, c). After spirotetramat treatment, some eggs appeared darker, yellowish-brown, and with embryonic development abnormalities (Fig. 1b); some of the egg embryo cells treated with spirotetramat appeared atrophied, and there were obvious gaps between the outer and the inner egg embryo cells, compared with the control treatment (Fig. 1c). Some eggs ruptured at the top after the egg shell was treated with spirotetramat, and the internal egg embryo cells flowed out, thus failing to form a complete embryo (Fig. 1d); the full egg embryo cells formed a control treatment.Figure 1The effect of spirotetramat on external morphology of isolated 0-h-old eggs of F. occidentalis. (a) 0-h-old isolated eggs of normal developing thrips; (b) yellowish-brown, developmentally deformed eggs after spirotetramat treatment; (c) eggs with shrunken oocytes after spirotetramat treatment; (d) eggs with apical rupture of the eggshell after spirotetramat treatment.Full size imageThe effect of spirotetramat on external morphology of live F. occidentalis 0-h-old eggs (Fig. 2b, c) was compared with normal development in the control treatment (Fig. 2a). Similar to the effect on external morphology of isolated 0-h-old eggs of F. occidentalis, the eggs treated with spirotetramat also showed abnormal embryonic development (Fig. 2b), egg embryo cell atrophy (Fig. 2c) and the phenomenon of rupture of the egg shell and outflow of embryo cells.Figure 2The effect of spirotetramat on external morphology of living 0-h-old eggs of F. occidentalis. (a) External morphology of live 0-h-old eggs of normal developing thrips; (b) developmentally deformed eggs after spirotetramat treatment; (c) eggs with shrunken oocytes after spirotetramat treatment; (d) eggs with apical rupture of the eggshell after spirotetramat treatment.Full size imageCompared with the control (Fig. 3a), the isolated 24-h-old eggs treated with spirotetramat (Fig. 3b) did not show obvious external morphological differences. After spirotetramat treatment, the eggs were still white and plump and with no embryonic deformities, egg cell atrophy or egg shell rupture, and could still develop normally. There were clear red eye spots on the head end, and embryo movement was clearly seen under the super-depth microscope (Fig. 3b). Similarly, the live 24-h-old eggs in the control treatment (Fig. 4a) and those treated with spirotetramat (Fig. 4b) showed no obvious external morphological differences, and the eggs developed normally.Figure 3The effect of spirotetramat on external morphology of isolated 24-h-old eggs of F. occidentalis. (a) Normally developing 24-h-isolated eggs in the control group; (b) 24-h-old eggs after spirotetramat treatment.Full size imageFigure 4The effect of spirotetramat on external morphology living of 24-h-old eggs of F. occidentalis. (a) Normally developing 24-h-live eggs in the control group; (b) live 24-h-old eggs after spirotetramat treatment.Full size imageEffect of egg hatchingThe 0-h-old eggs of F. occidentalis treated with spirotetramat did not hatch normally, and the mortality rate was 100% (Fig. 5). Among them, 77 eggs eventually showed rupture of the egg shell, the internal egg embryo cells flowed out and they did not hatch; 23 eggs showed no changes in external morphology, but did not hatch after continuous observation for 144 h, and showed no developmental phenomena such as embryo movement under a super-depth microscope, which was regarded as egg death. In the control treatment, 96 eggs hatched normally, and only six eggs did not rupture but did not hatch normally and were considered dead.Figure 5Effect of spirotetramat on hatching rate of F. occidentalis 0-h-old eggs.Full size imageThere was no significant difference between the 24-h-old eggs of F. occidentalis treated with spirotetramat and the control treatment. After spirotetramat treatment, 93 eggs hatched normally, and the shells of seven eggs were not ruptured (Fig. 6). Any eggs not hatched after 144 h of continuous observation were considered dead. In the control treatment, 95 eggs hatched normally and five eggs did not rupture but did not hatch normally, and so were considered dead.Figure 6The effect of spirotetramat on hatching rate of F. occidentalis 24-h-old eggs.Full size imageSEM observationsThe F. occidentalis eggs in the control treatment were kidney-shaped, with regular egg morphology, smooth surfaces and no folds or protrusions (Fig. 7a). At 24 h after spirotetramat treatment, part of the egg shells treated with spirotetramat had fallen off the chorion, and the embryonic material was exposed (Fig. 7b). The surface of the egg shell was uneven and severely wrinkled (Fig. 7c). The pores of some eggs treated with spirotetramat were sunken down and shrunken (Fig. 7d). Spirotetramat treatment of 0-h-old eggs affect clearly egg shells, resulting in shrinkage of egg shells, ovarian depression and egg malformations, and destroyed the egg shell structure. Thus, normal embryonic development was affected, and disrupted normal hatching.Figure 7The effect of spirotetramat on the surface of egg shells of F. occidentalis 0-h-old eggs. (a) 0-h-old eggs in the control treatment; (b) eggs shells were shed 24 h after treatment with spirotetramat; (c) the surface of the egg shell was uneven and severely wrinkled; (d) the pores of some eggs were sunken down and shrunken after treatment with spirotetramat.Full size imageThe shells of eggs treated with spirotetramat (Fig. 8b) showed no significant difference compared with controls (Fig. 8a). The eggs of the two groups of F. occidentalis were regular in shape, with smooth surfaces and without folds or protrusions. Thus, development of 24-h-old eggs showed some resistance to spirotetramat. Spirotetramat did not destroy the egg shell surface structure of 24-h-old eggs, indicating a high resistance to spirotetramat.Figure 8The effect of spirotetramat on the egg shell surface of F. occidentalis 24-h-old eggs. (a) 24-h-old eggs in the control treatment; (b) 24-h-old eggs in the spirotetramat treatment.Full size imageTEM observationsThe TEM observations showed that the egg structure of the control treatment was complete, the protoplasm and yolk were clearly observed inside the egg and the yolk was packed in the void of the protoplasm network (Fig. 9a). The egg shell structure was clear, and the outer and inner egg shell were clearly observed, as was the yolk membrane and the dense layer structure (Fig. 9c). Eggs treated with spirotetramat were flocculent, and no clear internal material was observed. The protoplasm and yolk structure were blurred, and flocculation in the protoplasm appeared to agglomerate and form blocks (Fig. 9b). The egg shell structure was unclear, and no clear outer egg shell, inner egg shell, yolk membrane and lamellar structures were observed. The egg shell was also filled with many flocs (Fig. 9d).Figure 9The effect of spirotetramat on the structure of F. occidentalis 0-h-old eggs. (a) and (b) 0-h-old eggs in the control treatment; (c) and (d) 0-h-old eggs in the spirotetramat treatment.Full size imageEffect on embryonic developmentThe initial eggs of the control group were kidney-shaped, white and full of vitellin (Fig. 10a). After 12 h of development, the eggs were larger and of oval shape (Fig. 10b). After 24 h of development, the egg had increased in volume, a partially transparent region appeared in the embryo and the embryo had transparent top follicles (Fig. 10c). After 36 h of development, some yolk granules disappeared and eggs became smooth and translucent (Fig. 10d). After 48 h of development, the insect outline was visible within the egg, a pair of antennae were visible on the head and a red eye point was clearly observed on the head during the blastokinesis phenomenon (Fig. 10e). After 60 h of development, embryo color deepened, the eye point was clearer and the head, femur, tibia and tarsus were clear (Fig. 10f). After 72 h of development, the egg shell began to break at the head, the tail constantly jittered, internal fluid flowed and the larva hatched from the top of the egg (Fig. 10g).Figure 10The embryonic development process of control 0-h-old eggs of F. occidentalis. (a) Control initial eggs; (b) eggs after 12 h of development; (c) eggs after 24 h of development; (d) eggs after 36 h of development; (e) eggs after 48 h of development; (f) eggs after 60 h of development; (g) eggs hatching as larvae after 72 h of development.Full size imageEggs of F. occidentalis were initially white, kidney-shaped and full of vitellin (Fig. 11a). Following treatment with spirotetramat, after 12 h of development, the eggs became large and oval, and the embryo was a pale brown color (Fig. 11b). After 24 h of development, color of the egg deepened to dark brown. There was a gap between the egg and the egg shell, and a small amount of spillage appeared at the end of the egg (Fig. 11c). After 36 h of development, the egg shell ruptured, material flowed out of the egg and embryo development did not proceed (Fig. 11d).Figure 11Effects of spirotetramat on development of 0-h-old eggs of F. occidentalis. (a) Frankliniella occidentalis initial eggs; (b) eggs developing 12 h after spirotetramat treatment; (c) eggs developing 24 h after spirotetramat treatment; (d) eggs developing 36 h after spirotetramat treatment.Full size imageIn the control treatment, the egg volume increased at 24 h, the embryo had a partially transparent area and there was a transparent follicle on the top of the embryo (Fig. 12a). After 12 h of development, some of the yolk particles disappeared and the egg body was smooth and translucent (Fig. 12b). After 24 h of development, the body outline, a pair of antennae and red eye spots were visible, and there was obvious embryo movement (Fig. 12c). At 36 h of development, the head, leg segments, tibia and tarsus were apparent (Fig. 12d). After 48 h of development, the embryo moved violently, internal body fluid flowed and the larva was ready to hatch (Fig. 12e). After 60 h of development, the larva emerged from its shell (Fig. 12f).Figure 1224-h-old eggs embryo development of F. occidentalis in control treatment. (a) Control 24-h-old eggs; (b) eggs after 12 h of development; (c) eggs after 24 h of development; (d) eggs after 36 h of development; (e) eggs after 48 h of development; (f) eggs hatching as larvae after 60 h of development.Full size imageThe 24 h old eggs of F. occidentalis showed enlarged volume, and there were transparent follicles on the top of the embryo (Fig. 13a). After 24 h eggs were treated with spirotetramat, they developed for 12–36 h, and the developmental status was the same as that of the control. The embryos developed normally, and there was no egg body discoloration or egg shell rupture (Fig. 13b–d). After 48 h of development, hairy scales appeared on the surface of the egg shell, and the egg body turned yellowish-brown in color, but the egg shell was not broken and no internal material overflow was seen (Fig. 13e). After 60 h of development, larvae hatched normally (Fig. 13f).Figure 13The effect of spirotetramat on embryonic development of F. occidentalis 24-h-old eggs. (a) Frankliniella occidentalis 24-h-old eggs; (b) eggs developing 12 h after spirotetramat treatment; (c) eggs developing 24 h after spirotetramat treatment; (d) eggs developing 36 h after spirotetramat treatment; (e) eggs developing 48 h after spirotetramat treatment; (f) eggs hatching as larvae after 60 h of development.Full size image More

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