JIA-2019-11

2580 CUI Dong-nan et al. Journal of Integrative Agriculture 2019, 18(11): 2579–2588 Depending on the species, insect diapause can occur in embryos, larvae, pupae or adults (Tatar and Yin 2001). The migratory locust, Locusta migratoria L., a facultative diapause insect, enters diapause in the egg stage (Uvarov 1966, 1977). The factors that influence diapause in the migratory locust are temperature, photoperiod and maternal effect (Tanaka 1992; Tanaka 1994; Tu et al . 2014). The effects of temperature and photoperiod on diapause have been widely reported (Sawchyn and Church 1973; Kobayashi and Numata 1975; Yi et al. 2007; Saunders 2010) including studies on the regulatory mechanisms of temperature and photoperiod (Williams and Adkisson 1964; Saunders 2014; Meuti et al. 2015). Maternal control of diapause in a few insects and mites has been reported (Mousseau and Fox 1998). Generally speaking, females experiencing deteriorating environmental conditions are more likely to produce diapause offspring (Mousseau and Fox 1998). In particular, maternal photoperiodic response on offspring diapause has been reported. In Trichogramma , the percentage of diapause prepupae was significantly dependent on the photoperiodic conditions of the preimaginal development of the maternal generation (Vaghina et al. 2013). Maternal photoperiodic response on diapause incidence of the parasitoid has also been reported in Cotesia plutellae (Guo et al. 2007). The migratory locust is an economically important species of insect because it threatens agricultural production. Maternal photoperiodic response affects embryo diapause in L . migratoria L. For example, in the laboratory, female migratory locusts produced non-diapause eggs when exposed to long photoperiods (16 h L:8 h D) and diapause eggs when exposed to short photoperiods (10 h L:14 h D or 12 h L:12 h D) (Tanaka 1994). In order to explain the regulatory mechanism of diapause in migratory locust, the previous studies analyzed the transcriptome and proteomics of the locust eggs at different embryonic states. The results showed that “juvenile hormone biosynthesis, insulin and PPAR signaling pathways” may play a critical role on diapause regulation in migratory locust (Tu et al. 2015; Hao 2017). How the female adults of migratory locust transfer the environmental information to offspring and induce egg diapause is still unclear. To reveal the molecular regulatory mechanisms influenced by maternal photoperiod on locust diapause, we performed a label-free protocol for a comparative analysis of proteomes between diapause and non-diapause eggs isolated from the ovaries of females exposed to long or short photoperiods. The present study can improve our understanding of the genetic and molecular mechanisms underlying diapause in an agriculturally important insect pest. 2. Materials and methods 2.1. Insect materials Eggs of migratory locust were collected from the field at Tianjin, China (38°49´N, 117°18´E). Eggs were dug out from the soil, gently sieved and placed into a re-sealable bag before placing into an insulated container to keep cool. Eggs were then transferred to the Langfang Field Station of the Institute of Plant Protection, Chinese Academy of Agricultural Sciences. 2.2. Insect growth conditions The eggs were placed in vermiculite with 20–30% water content and then hatched at 30°C and 60% relative humidity in an artificial climate box (PRX-250B-30, Haishu Saifu Experimental Instrument Factory, Ningbo, China). The emerged nymphs were transferred to wooden rearing chambers (60 cm×50 cm×60 cm), and fed with wheat seedlings. Second instars were exposed to either long-day or short-day conditions. We used two artificial climate boxes to mimic the natural daily photoperiod cycles for the long and short days. The photoperiodic regime used for non- diapause locusts in the experiment was 16 h L:8 h D. The temperature was 27°C and relative humidity was 60%. To induce diapause, locusts were reared at 10 h L:14 h D, 27°C and 60% relative humidity (Tanaka 1994; Wang et al. 2014). We recorded temperature data per hour for each 24-h day for each artificial climate box using a HOBO U23 Pro v2 Temperature/Relative Humidity Data Logger (Onset, USA). 2.3. Production of non-diapause and diapause eggs We reared adults under long and short photoperiods to obtain non-diapause and diapause egg-producing females, respectively. Females were dissected two weeks after reaching adulthood and eggs from the ovaries were collected. The ovaries of 15-day-old adult females were typically at the 4th developmental stage with an approximate size of about 15 mm (L) and 10 mm (W) (Guo et al. 1991). Dissection was performed on ice, and the extracted eggs were immediately put into liquid nitrogen and stored at –80°C for further analysis. Three independent biological replicates were prepared for each photoperiod treatment. Every replicate involved 60 eggs dissected from three female locusts. 2.4. Diapause rate The remnant locusts were reared at 27°C in both long or short photoperiods until they laid eggs. After oviposition, we