Effects of Pyriproxyfen on Female Reproduction in the Common Cutworm, Spodoptera litura (F.) (Lepidoptera: Noctuidae)

Abstract

The common cutworm, Spodoptera litura, is a rapidly reproducing pest of numerous agricultural ecosystems worldwide. The use of pesticides remains the primary means for controlling S. litura, despite their negative ecological impact and potential threat to human health. The use of exogenous hormone analogs may represent an alternative to insecticides. Juvenile hormones (JHs) play an important role in the reproductive systems of female insects, but the effects of pyriproxyfen, a JH analog, on reproduction in S. litura were poorly understood. In this paper, we topically treated the newly emerged females with 20, 60, or 100 μg of pyriproxyfen to determine its effects on reproduction. Then, we examined the expression of vitellogenin (Vg) and three hormone receptors, USP, HR3, and EcR, using quantitative reverse transcription and real-time polymerase chain reaction (qRT-PCR), and found that pyriproxyfen up-regulated the expression of Vg, USP, and HR3, whereas the expression of EcR was unaffected. An analysis of fecundity showed that the peak oviposition day, lifespan, and oviposition period were progressively shortened as the pyriproxyfen dosage increased. We also found that pyriproxyfen decreased egg laying amount, whereas the number of mature eggs that remained in the ovarioles of dead females increased as the pyriproxyfen dosage increased. We examined oocytes using transmission electron microscopy and found that treatment with 100 μg of pyriproxyfen increased the metabolism by increasing the amount of rough endoplasmic reticulum and mitochondria in the primary oocytes. Our results suggest that the topical application of pyriproxyfen on newly emerged females can efficiently reduce reproduction in S. litura and may represent an alternative to the use of insecticides for controlling the agricultural pest.

Fig 1. Correlation between hormone levels and Vg mRNA expression levels in S. litura.

The level of Vg mRNA in female adults (A) and pupae (B). (C) Correlation analysis of hormone (JH III and 20E) and Vg expression levels. (D) The levels of JH III and 20E at different developmental stages. Total RNA was extracted from the fat body for qRT-PCR. The data represent at least three independent experiments with five female insects in each experiment, and are normalized relative to the level of β-actin mRNA. Vertical error bars indicate standard errors (**P ≤ 0.01; ***P ≤ 0.001).

Fig 2. Topical application of pyriproxyfen increased Vg, HR3, USP, and EcR gene expression levels in (A) adults and (B) pupae of S. litura.

Total RNA was extracted from the fat body at three time points. Data were collected from three independent experiments with five female insects in each experiment, and were normalized to the level of the β-actin mRNA. Vertical error bars indicate standard errors (*P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001).

Fig 3. Effects of pyriproxyfen on adult reproduction and longevity.

Newly emerged adult females were treated topically with pyriproxyfen, and survival (A), the number of eggs laid and the number of mature eggs in the ovarioles (B), the length of the oviposition period (C), and peak oviposition (D) were determined. Vertical error bars indicate standard errors, n = 10 to 30. (*P ≤ 0.05 and ***P ≤ 0.001).

Fig 4. Effects of pyriproxyfen treatment on the ultrastructure of follicle cells and oocytes in the ovary.

The transmission electron micrographs (TEM) images of the pyriproxyfen-treated and control females were compared. The first and second columns represent the TEM of the follicle cells and oocytes of control female S. litura; the third and fourth columns represent the TEM of the follicle cells and oocytes of pyriproxyfen-treated female S. litura. The fifth line represents the developed eggs. The oocytes (Oo) in the stage 5 of the controls were entirely surrounded by follicle cells (Fc) with numerous microvilli (Mv) (A), whereas fewer microvilli were observed in the ovaries of the pyriproxyfen-treated insects (a). FcN = the follicular epithelium cell nucleus. ON = oocyte nucleus. The endoplasmic reticulum (ER) can be observed in the control group (B), and yolk granules (YG) and glycogenosomes (GG) can be observed in pyriproxyfen-treated female S. litura (b). Follicle cells (Fc) and oocytes of control and pyriproxyfen-treated female in stage 6 (C, c), spaces appear between follicle cells. The pyriproxyfen treatment increased the amount of ER or rough ER (RER), the number of mitochondria (Mt), and the accumulation of lipids (L) in stage 6 and stage 7 (D, E and F; d, e and f). In stage 8 and stage 9 oocytes reduced numbers of YG and Mt were observed (G, H and I; g, h and i), and a comb-like dentate structure was observed in the mature oocytes (stage 9) of both groups (J and j). Scale Bars showed on down-left of each Figure.