Effect of Rapid Cold Hardening on the Cold Tolerance of the Larvae of the Rice Stem Borer,Chilo suppressalis(Walker)

The effects of rapid cold hardening （RCH） on the cold tolerance of the last instar larvae of Chilo suppressalis （Walker） were evaluated for the first time. The discriminating temperature, induction, detection, duration and extent of RCH of the larvae in the laboratory were tested, and the supercooling points （SCPs） and the contents of water and lipid of the larvae after RCH treatment were determined, respectively. The results showed that the discriminating temperature of the larvae was about -21℃. Mean survival rates of the larvae which exposed to either 0 or 5℃ for 2 and 4 h before exposure to the discriminating temperature for 2 h were significantly higher than those of the control groups （P 〈 0.05）. Moreover, the highest survival rate appeared in the larvae after 0℃ for 4 h treatment. The protection against low temperature gained by RCH at 0℃ for 4 h was rapidly lost on return to 28℃. Mean survival rates of RCH larvae were significantly higher than those of non-acclimated （NACC） larvae and acclimation （ACC） larvae when they were exposed to the discriminating temperature for 2 or 4 h （P〈 0.05）. Moreover, the rates of NACC, ACC, RCH and ACC ＋ RCH larvae from 2 to 6 h to the discriminating temperature resulted in a significant decline. The values of SCPs and the contents of lipid of the larvae which exposed to either 0 or 5℃ for 2 and 4 h showed no significant difference at 0.05 level compared to those of the control groups. But the contents of water in the larvae were obviously decreased. Therefore, it could be concluded that RCH could enhance cold tolerance and affect partly physiological and biochemical components of the larvae of C. suppressalis, but the underlying mechanisms needs to be further explored.

Evaluation of Lethal Effects of Chlorantraniliprole on Chilo suppressalis and Its Larval Parasitoid, Cotesia chilonis

Chlorantraniliprole was introduced into China from 2008 as a novel insecticide to control the rice stem borer, Chilo suppressalis （Walker）. Cotesia chilonis （Matsumura） is the major parasitoid of C. suppressalis. We collected seven populations of C. suppressalis and two populations of C. chilonis in different locations in China in 2009 to investigate the lethal effects of chlorantraniliprole on them. The populations of C. suppressalis from different regions showed various LD50 values ranging from 2.00 to 18.70 ng per larva. Chlorantraniliprole has negligible acute contact toxicity （LC50500 mg L-1） to C. chilonis and its oral toxicity is also much lower than that of fipronil （2 800-fold difference in LC50）. The results indicated that chlorantraniliprole is a good alterative in rice integrated pest management （IPM） programs. The susceptibility data of C. suppressalis will be useful for monitoring resistance levels in future.

Application of vetiver grass Vetiveria zizanioides: Poaceae(L.) as a trap plant for rice stem borer Chilo suppressalis: Crambidae(Walker) in the paddy fields

Rice is an important staple food in China and it is at risk of attack by rice striped stem borer Chilo suppressalis, which occurs in most rice growing areas. In recent years, severe C. suppressalis outbreaks have been observed in China mainly due to changes in the rice cultivation systems, wide adoption of hybrid varieties and resistance to the dominant insecticides. Management relies primarily on chemical insecticides and resistance is an important contributing factor in these outbreaks. As a result, food safety of agricultural produce is reduced and the ecological and environmental integrities are threatened as well. Recently, environmentally friendly pest management measures, such as trap plants have been introduced for C. suppressalis management and this method can greatly reduce insecticide use. Our previous results indicated that the vetiver grass(Vetiveria zizanioides) is a dead-end trap plant that can effectively attract the adult females of C. suppressalis to lay eggs on it but where larvae are unable to complete their life cycle. This paper further explored the application of vetiver grass as a trap plant to manage C. suppressalis in the paddy fields. This environment-friendly tool can not only reduce C. suppressalis populations, it can also increase the diversity and abundance of natural enemies that can provide better environmental conditions for rice production.

Electrophysiological responses of the rice striped stem borer Chilo suppressalis to volatiles of the trap plant vetiver grass (Vetiveria zizanioides L.)

Vetiver grass （Vetiveria zizanioides L.） was previously found to effectively attract female adults of Chilo suppressalis （Walker）, an important pest of rice. To determine the volatile compounds involved in this attraction, electroantennography （EAG） responses to seven synthetic volatiles released from vetiver plants were examined. Our results indicated that the responses of C. suppressalis adult antennae to the different compounds varied widely. The compounds elicited strong EAG responses in female antennae were subsequently selected for further EAG response tests, namely, caryophyllene, β-ocimene, linalool and a-pinene. EAG responses to a combination of these four compounds did not differ significantly from the individual compounds. However, pair combination tests indicated that 0.01μg μL-1linalool and 50 μg μL-1 α-pinene, 50 μgμL-1 caryophyllene and 0.01μg μL-1 linalool, 0.01 μg μL-1 13-ocimene and 0.01 μg μL-1 linalool, and 0.01μg μL-1 β-ocimene and 50 μg μL-1 caryophyllene elicited significantly greater EAG responses in 3-day female moths compared to the 1-day female. These compound combinations and the corresponding ratios are probably playing an important role in attracting female adults of C. suppressalis to the vetiver grass.

Preliminary Study on Resistance of the Rice Stem Borer (Chilo Suppressalis) to Fipronil

By means of topical application, fipronil resistance was surveyed in the rice stem borer, Chilo suppressalis, from 12 diferent areas in east China from 2001 to 2004. The rice stem borers in most regions of Jiangsu and Anhui were still susceptible to fipronil. But in Wuxi, Jiangsu Province and Cixi, Zhejiang Province, their sensitivity became decreased （resistance ratios were 3.1 and 3.6, respectively）, and the medium level of resistance （resistance ratio was 21.2） was found in Cangnan, Zhejiang Province. So, it was still at the early stage for fipronil resistance development in this pest. Synergism experiments showed that piperonyl butoxide（PBO） had a little effect on both susceptible and resistant borers （synergism ratios were 1.1-1.2）. Though triphenyl phosphate （TPP） and diethyl meleate （DEM） had no effect on the susceptible borers, they had significant synergism on fipronil in the resistant population to fipronil （synergism ratios were 1.8 and 1.6, respectively）, indicating esterase and glutathion S-transferase may be involved in the resistance mechanism. Bioassay with currently used insecticides indicated that triazophos （because of high resistance）, trichlorphon and acephate had very low toxicity to resistant borers. But diazinon, pyridaphenthion, decamethrin and avermeatin showed high toxicity and had no cross resistance to fipronil, which could be considered as substitute insecticides in the resistance managment.

Causes of Recurrence of Rice Chilo suppressalis( Walker) in Longyou County and Prevention and Control Measures

In recent years,the occurrence of rice Chilo suppressalis( Walker) in Longyou County of Zhejiang Province has increased year by year and has become aggravated. This poses a great threat to the safety of rice production. In order to curb the recurrence momentum of rice C. suppressalis,according to the dynamic monitoring and field system survey of rice C. suppressalis in Longyou County since 2015,the causes of the annual increase of rice C. suppressalis in recent years were analyzed,and the recurrence area of C. suppressalis was introduced,and finally some prevention and control measures were put forward.

Molecular cloning and expression patterns of two small heat shock proteins from Chilo suppressalis(Walker)

Small heat shock protei ns(sHSPs)are a very complex protei n superfamily that increase in sect temperature tolera nee.In order to deeply understand the function and role of sHSPs in Chilo suppressalis(Walker),this study isolated and identified two CsHSP genes lacking introns from C.suppressalis,Cshsp23.9 and Cshsp27.3.The cDNA full-length of Cshsp23.9 and Cshsp27.3 were 909 and 1036 bp encoding 220 and 242 amino acids,respectively.Alignment with homologs and phylogenetic analysis indicated Cshsp23.9 and Cshsp27.3 were two new types of Cshsps in C.suppressalis.Real-time quantitative PCR(qPCR)revealed that Cshsp23.9 had the highest relative expression in hindgut compared with other tissues(head,epidermis,foregut,midgut,fat body,Malpighian tubules,and hemocytes),while Cs/?sp27.3 expressed the highest in fat body con tent.Whe n exposed to thermal stress from-11 to 43°C for 2 h,two genes showed differe nt expression patterns.Cshsp23.9 did not respond to low temperature,but could be up-regulated by high temperature and the highest expression temperature was at 36°C.Cshsp27.3 could only be induced by mild temperature,with the highest expression at 15 and 30°C.In conclusion,Cshsp23.9 and Cshsp27.3 existed in different tissues/organs of C.suppressalis,and played different important roles in C.suppressalis to resist temperature stress and regulate physiological activities.

Population Genetic Analysis of the Rice Stem Borer, Chilo suppressalis, in the South China

Genetic variation and patterns of genetic differentiation of the rice stem borer, Chilo suppressalis （Lepidoptera： Pyralidae）, from the South China were analyzed using 6 microsatellite markers and two partial mtDNA （cox1 and cox2） regions. All of the 6 microsatellite loci were polymorphic in the studied seven populations. The allelic richness per population ranged between 5.67 and 14.00, and average H E and H O values were 0.6246-0.8329 and 0.2634-0.6061, respectively. As the mitochondrial genome is a single genetic locus, we only present results for the concatenated data set （cox1 plus cox2 gene sequences, 513 bp）. The concatenated data showed high level of genetic diversity and there are 23 variable polymorphic sites among the 513 sites in concatenated data. Nearly all of （20 of 21） pairwise F ST comparisons among populations showed genetic differentiation with moderate to high pairwise F ST values based on microsatellite markers. However, for the mtDNA data, most of the seven populations did not show significant differentiation with other populations. The differences of population differentiation obtained with the two different genetic markers could be mainly attributed to the different mutation rates of microsatellite and mtDNA. There was not genetic structure existed in these studied populations based on microsatellite loci and mtDNA data. The analysis based on network, mismatch distribution, Tajima＇s D and F S indicated that the studied populations were from the recent same ancestor or the same refuge and followed by a sudden demographic expansion condition.

Suppression of Rice Stem Borer, Chilo suppressalis by Mass Trapping Using Synthetic Sex Pheromone in Paddy Field

Suppressing effects of mass trapping using synthetic sex pheromone with main active ingredients of Z-11-hexadecenal, Z-13-octadecenal and Z-9-hexadecenal on the rice stem borer, Chilo suppressalis were investigated during the flight periods of the overwintering and 1st generation in the paddy area of 60 ha in 1999. Population density of C. suppressalis in the pheromone-treated fields was lower than that in control. The egg masses decreased by 74.39% and population size of adult males of the 1st generation decreased by 61.64% in the treated fields as compared to control. Meanwhile, the damage by C. suppressalis larvae was significantly lower in the treated fields than control (t-test, P=0.05). Percentages of brownish leaf sheath, dead heart, and white head in the treated fields were decreased by 70.90%, 57.01% and 44.30%, respectively in contrast to control. The present study demonstrated that mass trapping to C. suppressalis using synthetic sex pheromone shows great potential as an alternative measure in an environment-friendly pest management and at the same challenging the insecticide use that has some environment impacts.

Effects of High-Quality Aromatic Rice Varieties on the Fitness of the Striped Stem Borer, Chilo suppressalis (Walker) in Central China

The striped stem borer （SSB）, Chilo suppressalis （Walker） （Lepidoptera： Pyralidae）, is a major pest of rice in China. Variation in host-plant quality may affect the body size of herbivorous insects, which in turn, can determine their life-history parameters such as survival, fecundity and fitness. In this study, we tested the effects of high-quality aromatic rice varieties on the fitness of SSB in China. Results showed that 1st instar larvae had higher penetrating rates and survival rates on the high-quality aromatic rice varieties （EX-1 and WX-988） than that on the non-aromatic rice varieties （EZ-5 and LYP-9）. In addition, shorter developmental periods, greater female pupal weights and higher of other life-history parameters （hatching rate, pupation rate, eclosion rate and increase index） were found on the high-quality aromatic rice varieties, although only female pupal weight showed a significant difference between the two varieties. The highest dead heart rate was found on the aromatic rice variety of EX-1. These results indicate that SSB sustains a lower fitness cost when consuming the high-quality aromatic rice varieties （EX-1 and WX-988） than on the non-aromatic rice varieties （EZ-5 and LYP-9） in Central China.

Cloning,Tissue Distribution,and Transmembrane Orientation of the Olfactory Co-Receptor Orco from Two Important Lepidopteran Rice Pests,the Leaffolder(Cnaphalocrocis medinalis) and the Striped Stem Borer(Chilo suppressalis)

In insects,the sense of smell is mainly mediated by olfactory receptors（Ors）.Olfactory co-receptor（Orco）,which is coexpressed with the Ors in almost all olfactory receptor neurons（ORNs）,is demonstrated to be an essential component in the insect olfactory system.It can be potential target for developing novel olfactory-disruption strategy to control insect pests.In this study,two full-length cDNA sequences encoding Orcos（CmedOrco and ChsupOrco） were cloned from two Lepidopteran rice pests,the rice leaffolder,Cnaphalocrocis medinalis and the rice striped stem borer,Chilo suppressalis.The amino acid sequences of CmedOrco and ChsupOrco showed high similarity to the previously identified Orcos from other insect species. Bioinformatic prediction and cellular immunofluorescence indicated that CmedOrco and ChsupOrco were both seven-transmembrane proteins with intracellular N-termini and extracellular C-termini.mRNA expression levels of the two Orcos were much higher in male and female antennae than those in non-olfactory tissues,and the ChsupOrco transcripts reached a peak level in adults compared to other life stages.Our results provide a foundation from which it will be possible to elucidate the roles of Orco in moth olfaction and for the development of environment-friendly management strategies of these two rice insect pests.

Characterization of T-complex polypeptide 1 (TCP-1) from the Chilo suppressalis HSP60 family and its expression in response to temperature stress

Many proteins require assistance from molecular chaperones at various stages to attain correctly folded states and functional conformations during protein synthesis. In this study, the gene encoding T-complex polypeptide 1（TCP-1）, which belongs to the heat shock protein 60（HSP60） family, was isolated and characterized from the rice stem borer, Chilo suppressalis, by RACE and q PCR, respectively. The full-length c DNA of Tcp-1 was 2 144 bp and encoded a 1 635-bp ORF; the deduced translational product contained 545 amino acids with 5′-and 3′-UTRs and an isoelectric point of 5.29. Cluster analysis confirmed that the deduced amino acid sequence shared high identity（60–99%） with TCP-1 from other insects. To investigate Tcp-1 expression in response to abiotic stress, q PCR was used to analyze expression levels of Tcp-1 m RNA in C. suppressalis larvae exposed to temperatures ranging from –11 to 43°C. With respect to heat shock, Tcp-1 expression was higher than the control after a 2-h exposure to 30 and 36°C and declined at 39 and 43°C. Difference in Tcp-1 expression was observed at temperatures ranging from –11 to 27°C. q PCR analyses revealed that Tcp-1 expression was the highest in hindgut tissue as compared to heads, epidermis, fat body, foregut, midgut, and malpighian tubules. Our results indicated that Tcp-1 expression was differentially expressed in C. suppressalis tissues, and was impacted by temperature stress.

The insecticide resistance in stripped stem borer,Chilo suppressalis(Walker)

The stripped stem borer (SSB),Chilo suppressalis (Walker) is one ofthe major insect pests of rice in Chi-na. Chemical control has been acommon practice in SSB managementsince 1950s. Insecticides used in-cluded BHC before 1983; organophos-

Identification of Bt-transgenic Rice Plants for Resistance to Stripe Stem Borer (Chilo suppressalis) and Genetic Analysis of the Transgenes

Resistance to herbicide Basta was used to identify Bt-transgenic rice plants and the progenies of crosses between the Bt-transgenic rice and the rice varieties cultivated in the Huang Huai area of China. The results demonstrated that the Basta-positive rice plants were highly resistant to stripe stem borer (Chilo sup-pressalis) both in the laboratory and field tests. Both cryIA (b) and bar genes were expressed and co-inherited in both self ing and crossing progenies. Mendelian segregation of the marker gene bar was observed in F2 and BC1 progenies. The results implicates that it is possible to transfer cryIA (b) gene into other cultivated varieties through crosses and back crosses.

A New and Simple Method for Collecting Eggs of the Striped Stemborer,Chilo suppressalis

The method for collecting eggs of the striped stem borer, Chilo suppressalis using plastic bags was studied in comparison with using caged rice plants. There was no significant difference in fecundity of C. suppressalis at 279 eggs/moth and in percentage of hatched eggs at 95% between in plastic bags and on rice plants. More egg masses were collected on plastic bags than on rice plants, whereas more smaller egg masses (less than 100 eggs per mass) in plastic bags than on rice plants. The advantages in collecting eggs of C. suppressalis and other insects by using plastic bags were also discussed.

Preliminary Study on Shelf Life of Artificial Feed of Chilo suppressalis

In order to preliminarily explore the preservation effect of ordinary refrigerator( 4℃) on artificial diet of Chilo suppressalis,the effects of artificial feed preserved at refrigerator for different time on percentage of pupation and pupa weight of C. suppressalis were investigated to understand the impact of long-term storage of artificial feed on the growth and development of C. suppressalis. The results showed that the percentage of pupation of C. suppressalis fed with artificial diet that had been stored in refrigerator for 40 d( T40) was highest( 67. 1%),followed by CK( 1 d,63. 5%),and the percentage of pupation of C. suppressalis fed with artificial diet that had been stored in refrigerator for10 d( T10) was 61. 9%. No significant difference was found in percentage of pupation among the treatment groups( P > 0. 05). The female pupa weight of the short-term preservation treatment group( 71. 9 mg) was significantly larger than that of the long-term preservation treatment group( 66. 1 mg)( P < 0. 05),and was insignificantly different from that of CK( P > 0. 05). The male pupa weight of the short-term preservation treatment group( 51. 2 mg) was significantly larger than that of CK( 47. 7 mg)( P < 0. 05),and the male pupa weight of the long-term preservation treatment group was slightly larger than that of CK( P > 0. 05). In conclusion,the artificial diet of C. suppressalis can be stored for at least 40 d in refrigerator( 4℃).

Molecular and functional analysis of chitin synthase genes in Chilo suppressalis(Lepidoptera:Crambidae)

The rice stem borer,Chilo suppressalis,has developed a high level of resistance to many of the compounds currently used for control.There is therefore an urgent need to develop novel control methods for C.suppressalis.Insect chitin synthases(CHS)have attracted interest as a potential target for insect pest management.However,to date,CHS have not been characterized in C.suppressalis.Two CHS genes(CsCHSI and CsCHS2)were identified and cloned from C.suppressalis.Two transcript variants were identified for CsCHS1,CsCHSla and CsCHS1b.Spatiotemporal expression profiling showed that both transcripts of CsCHS1 are most highly expressed on the last day of each larval instar stage and show the highest expression levels in the integument.In contrast,CsCHS2 is predominantly expressed during the larval feeding stages and shows the highest expression levels in the midgut.Knockdown of CsCHSI by RNA interference significantly inhibited the molting and pupation of C.suppressalis,and knockdown of CsCHS2 significantly affected growth during the larval stage,but had no significant effect on the pupation.Moreover,knockout of CsCHS1 by CRISPR/Cas9 genome editing severely lowered the hatching rate,larval survivorship,pupation rate,and eclosion rate,but only larval survivorship at the GO generation was lowered after the knockout of CsCHS2.These results demonstrate that CsCHS1 and CsCHS2 play vital roles in the growth and development of C.suppressalis,and so have potential as insecticidal targets for the control of this highly damaging pest.

Comparison of three transgenic Bt rice lines for insecticidal protein expression and resistance against a target pest, Chilo suppressalis （Lepidoptera： Crambidae）

Two transgenic rice lines （T2A-1 and T1C-19b） expressing cry2A and crylC genes, respectively, were developed in China, targeting lepidopteran pests including Chilo suppressalis （Walker） （Lepidoptera： Crambidae）. The seasonal expression of Cry proteins in different tissues of the rice lines and their resistance to C. suppressalis were assessed in comparison to a Bt rice line expressing a crylAb/Ac fusion gene, Huahui 1, which has been granted a biosafety certificate. In general, levels of Cry proteins were T2A- 1 〉 Huahui I 〉 T1C-19b among rice lines, and leaf 〉 stern 〉 root among rice tissues. The expression patterns of Cry protein in the rice line plants were similar： higher level at early stages than at later stages with an exception that high Cry 1C level in T 1 C- 19b stems at the maturing stage. The bioassay results revealed that the three transgenic rice lines exhibited significantly high resistance against C. suppressalis larvae throughout the rice growing season. According to Cry protein levels in rice tissues, the raw and corrected mortalities of C. suppressalis caused by each Bt rice line were the highest in the seedling and declined through the j ointing stage with an exception for T1 C-19b providing an excellent performance at the maturing stage. By comparison, T1C-19b exhibited more stable and greater resistance to C. suppressalis larvae than T2A-1, being close to Huahui 1. The results suggest crylC is an ideal Bt gene for plant transformation for lepidopteran pest control, and T1C-19b is a promising Bt rice line for commercial use for tolerating lepidopteran rice pests.

Molecular characterization and expression profiles of nicotinic acetylcholine receptors in the rice striped stem borer, Chilo suppressalis （Lepidoptera： Crambidae）

Nicotinic acetylcholine receptors （nAChRs） are members of the cys-loop ligand- gated ion channel （cysLGIC） superfamily, mediating fast synaptic cholinergic transmission in the central nervous system in insects. Insect nAChRs are the molecular targets of economically important insecticides, such as neonicotinoids and spinosad. Identification and characterization of the nAChR gene family in the rice striped stem borer, Chilo suppressa[is, could provide beneficial information about this important receptor gene family and contribute to the investigation of the molecular modes of insecticide action and resistance for current and future chemical control strategies. We searched our C. suppressalis transcriptome database using Bombyx mori nAChR sequences in local BLAST searches and obtained the putative nAChR subunit complementary DNAs （cDNAs） via reverse transcription polymerase chain reaction （RT-PCR） and rapid amplification of cDNA ends methods. Similar to B. mori, C. suppressalis possesses 12 nAChR subunits, including nine c^-type and three/%type subunits. Quantitative RT-PCR analysis revealed the expression profiles of the nAChR subunits in various tissues, including the brain, subesophageal ganglion, thoracic ganglion, abdominal ganglion, hemocytes, fat body, foregut, midgut, hindgut and Malpighian tubules. Developmental expression analyses showed clear differential expression of nAChR subunits throughout the C. suppressalis life cycle. The identification of nAChR subunits in this study will provide a foundation for investigating the diverse roles played by nAChRs in C. suppressalis and for exploring specific target sites for chemicals that control agricultural pests while sparing beneficial species

Gene cloning and expression of aminopeptidase N and cadherin from midgut of the rice stem borer, Chilo suppressalis

The rice stem borer, Chilo suppressalis Walker is one of the most important insect pests on rice in Asia, north Africa and southern Europe. Transgenic Bt rice has been developed in the laboratory with good resistance to this pest and other Lepidopteran insects, which will provide a possible alternative tool for this pest control. The full-length cDNAs encoding an aminopeptidase N （CsAPN） and a cadherin （CsCad） were cloned from C. suppressalis. CsAPN showed common features of, and high identities to, other insect APNs in its deduced amino acid sequence. Although a full-length cDNA encoding cadherin-like protein has been reported in GenBank, the newly isolated cadherin here （CsCad） showed some differences in its amino acid sequence, especially at the 7th cadherin repeat region （CR7）, which indicated the newly isolated CsCad might be another allele. CsAPN and CsCad were successfully expressed in insect Tn cells, and the blot analysis showed these two proteins could bind Bt toxin CrylAb. The results will provide valuable information for the studies of toxin mode of action and the possible toxin resistance mechanisms in this pest.