Xiao-Ping Wang

Advances in the use of the RNA interference technique in Hemiptera

Abstract  RNA interference (RNAi) suppresses the expression of target genes by post‐transcriptional regulation. Because double‐stranded RNA (dsRNA) mediated gene silencing is a conserved mechanism in many eukaryotes, RNAi has become a valuable tool for unveiling gene function in many model insects. Recent research has also shown that RNAi can also be effective in the downregulation of target genes in Hemiptera. In this review, we discuss the use of the RNAi technique in gene functional analysis in hemipterans, highlighting the methods of dsRNA uptake by these insects and discuss the knock‐down efficiency of these techniques. Although the RNAi technique has disadvantages, our primary goal here is to determine whether it can be exploited further in the discovery of new gene functions, and as a pest control strategy, in some important Hemipteran pests.

Projecting Overwintering Regions of the Beet Armyworm, Spodoptera exigua in China using the CLIMEX Model

Abstract The beet armyworm, Spodoptera exigua Hübner (Lepidoptera: Noctuidae) is a serious agricultural pest worldwide. However, population sources of S. exigua in outbreak regions are still vague due to the lack of understanding the distribution of overwintering regions, especially in China. In the present study, the potential overwintering regions of S. exigua in China are projected using the method of Compare Location in the CLIMEX model in order to understand the population sources in outbreak regions and establish an accurate forecasting system. The results showed the southern and northern overwintering boundaries near the Tropic of Cancer (about 23.5 °N) and the Yangtze River valley (about 30 °N), respectively. Meanwhile, the projection was supported by the data of fieldwork in 14 countries/cities during winter from 2008–2010. In conclusion, results of this study indicated that the overwintering regions of S. exigua were accurately projected by the CLIMEX model.

Relationships between body weight of overwintering larvae and supercooling capacity; diapause intensity and post-diapause reproductive potential in Chilo suppressalis Walker.

The rice stem borer, Chilo suppressalis Walker, overwinters in China as a larva in facultative diapause. The instars and body weights of overwintering larvae vary widely. In this paper, the relationships between body weight and supercooling capacity, diapause intensity and post-diapause reproductive potential of overwintering larvae collected in late-stage rice field were examined. There was a significant positive correlation between body weight, instar, and head capsule width, thus the overwintering larvae were divided into five groups based on body weight (I, up to 35.0mg; II, 35.1-57.0mg; III, 57.1-79.0mg; IV, 79.1-101.0mg; and V, over 101.1mg) for further analysis. The body water content of the lighter group (I) was significantly higher than that of the heavier groups (IV-V). However, the mean supercooling point decreased with an increase of the mean larval body weight in five groups; mean supercooling point of group I was significantly lower than that of group V, except in January 2009. After transfer of overwintering larvae to 15, 20 and 25°C on different dates, smaller individuals pupated slightly faster than larger ones at the same temperature, suggesting that diapause was less intense in smaller overwintering larvae. On 19 March 2009 there was a strong positive correlation between larval body weight and the weight of 3 day-old pupae, and the number of eggs carried by 2 day-old adult females at 15, 20 and 25°C. The average number of eggs carried by 2 day-old adult females differed significantly among different groups. The average number of eggs carried by 2 day-old adult females in group V was significantly greater than those of other groups, and that of group I was significantly lower than those of other groups, suggesting that post-diapause reproductive potential was determined, to a certain extent, by body weight of the overwintering larvae.

A de novo transcriptome and valid reference genes for quantitative real-time PCR in Colaphellus bowringi.

Background The cabbage beetle Colaphellus bowringi Baly is a serious insect pest of crucifers and undergoes reproductive diapause in soil. An understanding of the molecular mechanisms of diapause regulation, insecticide resistance, and other physiological processes is helpful for developing new management strategies for this beetle. However, the lack of genomic information and valid reference genes limits knowledge on the molecular bases of these physiological processes in this species. Results Using Illumina sequencing, we obtained more than 57 million sequence reads derived from C. bowringi, which were assembled into 39,390 unique sequences. A Clusters of Orthologous Groups classification was obtained for 9,048 of these sequences, covering 25 categories, and 16,951 were assigned to 255 Kyoto Encyclopedia of Genes and Genomes pathways. Eleven candidate reference gene sequences from the transcriptome were then identified through reverse transcriptase polymerase chain reaction. Among these candidate genes, EF1α, ACT1, and RPL19 proved to be the most stable reference genes for different reverse transcriptase quantitative polymerase chain reaction experiments in C. bowringi. Conversely, aTUB and GAPDH were the least stable reference genes. Conclusion The abundant putative C. bowringi transcript sequences reported enrich the genomic resources of this beetle. Importantly, the larger number of gene sequences and valid reference genes provide a valuable platform for future gene expression studies, especially with regard to exploring the molecular mechanisms of different physiological processes in this species.

Juvenile hormone facilitates the antagonism between adult reproduction and diapause through the methoprene-tolerant gene in the female Colaphellus bowringi.

In insects, the process whereby juvenile hormone (JH) regulates short-day (SD)-induced reproductive diapause has been previously investigated. However, we still do not understand the mechanism by which JH regulates long-day (LD)-induced reproductive diapause. In this study, we use a cabbage beetle, Colaphellus bowringi, which is a serious pest of cruciferous vegetables in Asia capable of entering reproductive diapause under LD conditions, as a model to test whether JH regulates female reproductive diapause similar to the mechanism of SD-induced diapause. Our results showed that the JH analog (JHA) methoprene significantly induced ovarian development but inhibited lipid accumulation of diapause-destined adults. Meanwhile, the transcripts of the vitellogenin (Vg) genes were upregulated, whereas the expression of the fat synthesis and stress tolerance genes were downregulated. RNA interference of the JH candidate receptor gene methoprene-tolerant (Met) blocked JH-induced ovarian development and Vg transcription, suggesting a positive regulatory function for JH-Met signaling in reproduction. Furthermore, under reproduction-inducing conditions, Met depletion promoted a diapause-like phenotype, including arrested ovarian development and increased lipid storage, and stimulated the expression of diapause-related genes involved in lipid synthesis and stress tolerance, suggesting JH-Met signaling plays an important role in the inhibition of diapause. Accordingly, our data indicate that JH acts through Met to facilitate development of the reproductive system by upregulating Vg expression while inhibiting diapause by suppressing lipid synthesis and stress tolerance in the cabbage beetle. Combined with previous studies in SD-induced reproductive diapause, we conclude that JH may regulate female reproductive diapause using a conserved Met-dependent pathway, regardless of the length of the photoperiod inducing diapause in insects.

Analysis of pupal head proteome and its alteration in diapausing pupae of Helicoverpa armigera

The proteomic approach has proven to be an useful tool for understanding insect diapause processes. Using 2D gel electrophoresis and matrix assisted laser/desorption ionization (MALDI) time of flight (TOF), we identified 24 proteins in the head of Helicoverpa armigera pupae with diverse functional characteristics, including cytoskeleton proteins, heat-shock proteins, insect development regulation factors, ATPases, proteins regulating signal pathway and enzymes involved in metabolism, etc. A proteomic comparison between nondiapausing and diapausing pupae revealed three proteins that were present only in nondiapausing pupae, and six proteins represented >or=2.0-fold or <or=0.5-fold changes. The differentially expressed proteins, including heat-shock protein 90, chitin deacetylase, alpha-tubulin and transitional endoplasmic reticulum ATPase, etc. were reported for the first time in H. armigera. Identification of these proteins will enable us to further characterize the regulated functions of diapause in this important species.

Effect of soil moisture on overwintering pupae in Spodoptera exigua (Lepidoptera: Noctuidae)

Spodoptera exigua Hübner (Lepidoptera: Noctuidae), can overwinter as pupae at depths of 0- to 5-cm beneath the ground. In order to understand the effect of soil moisture on the overwintering success of this species, we tested the supercooling point and survival of pupae, and the growth rate of prepupae that were exposed to different temperature and soil moisture in the laboratory. Results showed that supercooling points, body water contents, and survivals after the different pre-treatments were not significantly affected by the soil moisture. The developmental progress of prepupae, survival of prepupae and pupae were negatively correlated with soil moisture as well as the delay of exposure time, which supports the hypothesis that soil moisture acts as a developmental modulator. We presumed that the delay of pupation and lower prepupae survival under higher soil moisture was due to lack of a complete pupal chamber for protection at low temperatures. Low pupal survival was likely attributed to lack of oxygen in the soil, especially under the condition of higher soil moisture. We suggest that using strategies of irrigation and soil tillage during winter may decrease the overwintering population of S. exigua from the perspective of integrated pest management.

Antennal and behavioral responses of female Maruca vitrata to the floral volatiles of Vigna unguiculata and Lablab purpureus

In many herbivorous insects, the attraction to suitable host plants is in part mediated by similar olfactory cues from different host plants. This study investigated the electrophysiological and behavioral responses of female Maruca vitrata Fabricius (Lepidoptera: Crambidae) to floral volatiles from two host plants, Vigna unguiculata (L.) Walpers and Lablab purpureus (L.) Sweet (both Fabaceae). Qualitative and quantitative differences were found between the odor profiles of two host plants by gas chromatography‐mass spectrometry (GC‐MS). Nine electroantennography‐active compounds were detected in the headspace collections of the two host plants by means of coupled GC‐electroantennographic detection. In a wind tunnel, female behaviors (upwind flight and source approach) were not affected by stimuli or their interaction with host plants, but the behaviors were influenced by host plants. A mixture of the nine bioactive compounds, including limonene, 1,3‐diethylbenzene, benzaldehyde, acetophenone, 4‐ethylbenzaldehyde, 1‐(4‐ethylphenyl)‐ethanone, 2‐methyl‐3‐phenylpropanal, 1H‐indol‐4‐ol, and 1,1′‐(1,4‐phenylene)bis‐ethanone mimicking L. purpureus (in a ratio of 0.4:9.7:2.4:5.7:78.1:4.8:100:0.6:30.3) attracted significantly more females approaching the sources compared with the mixture of compounds mimicking V. unguiculata (in a ratio of 0.3:1.4:1.6:3.7:50.8:3.2:100:4.2:16.7). Further bioassays revealed that a subtractive 4‐component blend [limonene, 1,3‐diethylbenzene, 4‐ethylbenzaldehyde, and 1‐(4‐ethylphenyl)‐ethanone in a ratio of 0.4:9.7:78.1:4.8] was essential for maximal attraction. Our study suggested that female M. vitrata might use similar odors from V. unguiculata and L. purpureus to locate suitable hosts and that an artificial lure, containing the major floral volatiles released by the two host plants, might be useful in exploring efficiency monitoring and/or control strategies of this moth in the field.

The emergence and mating rhythms of the legume pod borer, Maruca vitrata (Fabricius, 1787) (Lepidoptera: Pyralidae)

The legume pod borer, Maruca vitrata (Fabricius, 1787) is a serious pantropical insect pest of grain legumes. The emergence and mating rhythms of the legume pod borer adults were investigated at 29±1°C and 75–80% r.h. under 14L:10D. The results showed that emergence activity of M .vitrata occurred throughout the day, 86% of females and 73% of males emerged at night. The highest emergence frequency occurred at 2300–2400 h and 0200–0300 h for females and males, respectively. The highest mating frequency occurred between 2300 and 2400 h for 3-day-old moths. Single mating peaks were observed for 1-, 6-and 7-day-old moths and double mating peaks were observed for 2- to 5-day-old moths. The percentage of pairs mating at the first peak was higher than at the second peak. More time was spent in mating during the first half of scotophase than the second one. The mating behaviour of M. vitrata was age-dependent. Young and old pairs spent more time in mating than middle-aged pairs. Advanced onset of time and peak for mating were observed directly by older moths. The emergence activities of both sexes were almost synchronous. Emergence peaks were almost synchronous with the two peaks of mating at different time of day.

Differences in the pre‐diapause and pre‐oviposition accumulation of critical nutrients in adult females of the beetle Colaphellus bowringi

In many insect species, the differentiation of development between diapause and reproduction first becomes obvious during the diapause preparation (pre‐diapause) and pre‐oviposition phases. However, the differentiation of nutrient accumulation between these two phases remains unclear. We compared the weights of pre‐diapause and reproductive adult female Colaphellus bowringi Baly (Coleoptera: Chrysomelidae), and measured their triacylglycerol (TAG), protein, and carbohydrate content from emergence until they had fed for 4 days post‐eclosion. We also compared the ovarian development and accumulation of lipid droplets between pre‐diapause and reproductive adult females in order to determine whether we could visually detect differences in nutrient allocation. The weights of both pre‐diapause and reproductive females increased with duration of feeding. The fresh weight and water content of pre‐diapause females was significantly lower than that of reproductive females after feeding for 3 days post‐eclosion. Pre‐diapause females channeled their reserves into TAG in the fat body, whereas reproductive females converted nutrients into proteins and carbohydrates for egg development. These results quantify differences in nutrient accumulation between pre‐diapause and reproductive adult female C. bowringi, and provide clues for understanding the molecular mechanisms underlying differences in the allocation of nutrients between diapause and reproduction in insects.