| Area-wide commercial cultivation of transgenic Bt (Bacillus thuringiensis) cotton has effectively controlled the cotton bollworm Helicoverpa armigera Hubner and resulted in a sharp reduction in the use of broad-spectrum insecticides in cotton production. Meanwhile, the green mirid bug Apolygus lucorum (Meyer-Diir) (Hemiptera:Miridae) increased its population size and became the dominant species on Bt cotton. Furthermore, this pest has spread to jujube tree Ziziphus jujuba Mill., grape tree Vitis vinifera L. and other crops in northern China, and caused great damage. A. lucorum overwinters with diapausing eggs, and diapause trait is an important indicator of ecological adaptation of this pest. Short day photoperiods induce diapause in A. lucorum. Chilling at low temperature of 2 or 4? and exposure to constant light (photoperiod of L24:D0) for more than 30 days terminated diapause effectively. However, it is not clear whether there are differences among different geographical populations of A. lucorum, and how environmental factors (temperature, photoperiod and water) affect the whole process of diapause, including diapause induction, diapause development and post-diapause development, and its molecular mechanism is unclear as well. In the present study, investigations were carried out in the field and laboratory to elucidate (1) the seasonal occurrence and damage of overwintering populations, (2) ecological effects of temperature, photoperiod and water on diapause regulation in different geographical populations and (3) possible molecular mechanism of diapause in A. lucorum.The main results are as follows:(1) Overwintering site of A. lucorum on winter jujube and grape trees and oviposition site of 1st generation adults on carrots were investigated in the field. The egg density of A. lucorum overwintering populations on winter buds of different grape varieties with two management practices was investigated, and population dynamics and host transfer of this species were monitored as well in this paper. The results showed that overwintering eggs of A. lucorum in Henan province were laid mainly in pruned wounds of winter jujube trees and winter buds of grape. The results also revealed that the 1st generation adults of A. lucorum prefer laying eggs in big faceplate leaves and flowers of carrots. The investigation also indicated that the percent winter buds with eggs and overwintering eggs per one hundred winter buds of extensively managed grapes were all significantly higher than that in intensively managed grapes. Grape varieties of 'Hutai 8','Red Globe' and 'Summer Black' were easily damaged by A. lucorum. The population dynamics of A. lucorum on grape and host transfer rule were revealed as well. The investigation also suggested that the dispersed hatching dates of overwintering eggs, not only because of rainfall, but also a result of synchronized development with host plants.(2) To investigate geographic variation in the critical photoperiod for diapause induction in A. lucorum, bugs were collected from nine localities with latitude varied from 30.47°N (Wuhan) to 39.32°N (Langfang), and reared successively under a warm long-day photoperiod of 16 hours light and 8 hours dark (L16:D8) at 26?. The 10th generation were used to investigate the critical photoperiod for each geographic population. The results indicated that the critical photoperiods (p50) for diapause induction in A. lucorum at 17,20,23,26 and 29? were prolonged with the increase of original latitude, but only at lower temperature of 17?, had a significant positive relationship with the original latitude (La), and could be described as pso=0.0454La +12.3927 (P= 0.0259), there were no significant positive relationships at other higher temperatures. We also found that the relationship between the exposure temperautres and the critical photoperiods were fitted with quadratic parabolic regressions (first prolonged and then shortened with the increase of temperature), rather than a simple linear relationship as reported before.(3) Huaiyang population collected in 2007 was used to study the combination effects of temperatures and photoperiods on diapause development and diapause termination in A. lucorum. The combinations of temperatures and photoperiods are as follows:(1) lower temperatures of 0,4,10? and L0:D24 photoperiod, (2) higher temperatures of 17,20,23? and L24:D0, L14:D10, L13:D11, L12:D12, L11:D13, L10:D14, L0:D24 photoperiods. The results revealed that each combination of temperature and photoperiod could terminate diapause effectively (except 0?). The average pre-hatching period of these eggs placed under 26?, L16:D8 photoperiod (66.17±12.56 d) was significantly shorter than those exposed to various combinations of temperatures and photoperiods (P< 0.05). Pre-hatching period prolonged following with the prolonged exposure time under combinations of temperatures and photoperiods, and had significantly linear relationship (Pp= at+b, a> 0). And the relationship among exposure time (x1), day length of photoperiod (x2) and temperature (x3) could be described as:Pp= 86.202+0.450x1-0.560x2-0.534x3 (R = 0.941, R2= 0.886, P= 0.000). This indicated that long day photoperiod and temperature had effects on diapause development and hatching, though pre-hatching period prolonged following with the prolonged exposure time. Thus, pre-hatching period was not unlimited prolonged, but had periodically period to hatch.(4) Diapausing eggs of nine populations were exposed to low temperature of 4? chilling for 60 d to terminate diapause, then were placed under different temperatures ranging 13 to 26? and photoperiod of L16:D8 to study the post-diapause threshold temperature and effective accumulative temperature in A. lucorum. The results showed that the post-diapause threshold temperatures of three different populations were lower than others, e.g. Wuhan, Nanyang and Yancheng, was -3.29,-1.33 and 1.78?, respectively, and post-diapause effective accumulative temperature of these three populations was 1428.57,1111.11 and 1111.11 day degrees, respectively, higher than others. We found that the relationship between post-diapause threshold temperature and effective accumulative temperature was fitted with a negative linear model:DD=-72.9ST0+1119.13 (R2= 0.88, P= 0.00018). And the relationships between post-diapause threshold temperatures (T0) or effective accumulative temperatures (DD) and original latitude (L) were fitted with quadratic parabolic regressions, could be described as:T0=-0.21L2+15.47L-279.51 (R2= 0.771, P= 0.012) and DD= 15.77L2-1164.75L+22312.75 (R2= 0.879, P= 0.0018), respectively. So post-diapause threshold temperatures were first increased and then decreased with the increase of original latitude, while effective accumulative temperatures were first decreased and then increased, and the turning point of both was between 36 and 37°N. There was geographic variation in the post-diapause threshold temperature and effective accumulative temperature.(5) The effects of water on diapause development of the whole diapause process ('diapause-post-diapause' development), and diapause termination and post-diapause development of two distinct sub diapause stage were studied in this paper using laboratory induced diapause eggs. The results indicated that water was crucial for diapause development of A. lucorum and diapausing eggs could not develop without water. However, excessive water was detrimental to diapause development, as indicated by decreased diapause termination rate and elongated developmental duration, compared with the intermittent dry conditions. Variety of water treatments were applied to diapausing eggs while the diapause was terminated naturally under warm long day conditions (WLD:26?,16 hours light,8 hours darkness, abbreviated as L16:D8). The pre-hatching period (Pp) exponentially increased with increasing time to watering or number of dry days (T1), and the relationship could be described as Pp= 72.53 exp (0.0037 T1). The development duration of post-diapause eggs Dp (defined as the duration from transferring to WLD conditions after chilling up to the hatching of nymphs) increased logistically with increased time to watering T2 (defined as the period from transferring to WLD conditions after chilling up to the watering day, or number of dry days experienced before eggs were watered during the post-diapause development duration), i.e., the nonlinear relationship could be described as:Dp= 19.12+(31.08-19.12)/(1+exp ((8.90-T2)/0.56)). Therefore, water is not only a trigger factor, but also an important factor regulating the process of diapause.(6) De novo transcriptome assembly and gene expression analysis of non-diapause (NDA), diapause induction (DA) and diapause reversal (RNDA) adults in A. lucorum affected by photoperiod was performed using the platform of Illumina Hiseq 2000. There were 16,027 differentially expressed genes (DEGs) up-regulated and 7,395 DEGs down-regulated in NDA-vs-DA of All-Unigene,10,279 DEGs up-regulated and 9,811 DEGs down-regulated in NDA-vs-RNDA of All-Unigene, 10,439 DEGs up-regulated and 18,274 DEGs down-regulated in DA-vs-RNDA of All-Unigene. The results also showed that circadian genes of Per, Dbt, dCLK and Tim were up or down-regulated in different levels, especially gene Per (unigene-29686_All) and gene Tim (unigene31119_All) was up-regulated in NDA-vs-DA and down-regulated in DA-vs-RNDA of All-Unigene, but was not up or down-regulated in NDA-vs-RNDA of All-Unigene. Circadian genes Per and Tim may be related to diapause regulation in A. lucorum affected by photoperiod, but need further validation studies in the future. |
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