Loss of functional cryptochrome 1 reduces robustness of 24-hour behavioral rhythms in monarch butterflies.

Light is one of the strongest cues for entrainment of circadian clocks. While some insect species rely only on visual input, others like Drosophila melanogaster use both the visual system and the deep-brain blue-light photoreceptor cryptochrome for entraining circadian rhythms. Here, we used the monarch butterfly Danaus plexippus (dp), which possesses a light-sensitive cryptochrome 1 (dpCry1), to test the conservation of mechanisms of clock entrainment. We showed that loss of functional dpCry1 reduced the amplitude and altered the phase of adult eclosion rhythms, and disrupted brain molecular circadian rhythms. Robust rhythms could be restored by entrainment to temperature cycles, indicating a likely functional core circadian clock in dpCry1 mutants. We also showed that rhythmic flight activity was less robust in dpCry1 mutants, and that visual impairment in dpNinaB1 mutants impacted flight suppression at night. Our data suggest that dpCRY1 is a major photoreceptor for light-entrainment of the monarch circadian clock.

Probing Transcriptional Crosstalk between Cryptochromes and Iron-sulfur Cluster Assembly 1 (MagR) in the Magnetoresponse of a Migratory Insect.

Many organisms can sense and respond to magnetic fields (MFs), with migratory species in particular utilizing geomagnetic field information for long-distance migration. Cryptochrome proteins (Crys) along with a highly conserved Iron-sulfur cluster assembly protein (i.e., MagR) have garnered significant attention for their involvement in magnetoresponse (including magnetoreception). However, in vivo investigations of potential transcriptional crosstalk between Crys and MagR genes have been limited. The brown planthopper, Nilaparvata lugens, is a major migratory pest insect and an emerging model for studying MF intensity-related magnetoresponse. Here, we explored in vivo transcriptional crosstalk between Crys (Cry1 and Cry2) and MagR in N. lugens. The expression of Crys and MagR were found to be sensitive to MF intensity changes as small as several micro-teslas. Knocking down MagR expression led to a significant downregulation of Cry1, but not Cry2. The knockdown of either Cry1 or Cry2 individually did not significantly affect MagR expression. However, their double knockdown resulted in significant upregulation of MagR. Our findings clearly indicate transcriptional crosstalk between MagR and Crys known to be involved in magnetoresponse. This work advances the understanding of magnetoresponse signaling and represents a key initial step towards elucidating the functional consequences of these novel in vivo interactions.

Adaptive migratory orientation of an invasive pest on a new continent.

Many species of insects undertake long-range, seasonally reversed migrations, displaying sophisticated orientation behaviors to optimize their migratory trajectories. However, when invasive insects arrive in new biogeographical regions, it is unclear if migrants retain (or how quickly they regain) ancestral migratory traits, such as seasonally preferred flight headings. Here we present behavioral evidence that an invasive migratory pest, the fall armyworm moth (Spodoptera frugiperda), a native of the Americas, exhibited locally adaptive migratory orientation less than three years after arriving on a new continent. Specimens collected from China showed flight orientations directed north-northwest in spring and southwest in autumn, and this would promote seasonal forward and return migrations in East Asia. We also show that the driver of the seasonal switch in orientation direction is photoperiod. Our results thus provide a clear example of an invasive insect that has rapidly exhibited adaptive migratory behaviors, either inherited or newly evolved, in a completely alien environment.

Reliable reference genes for gene expression analyses under the hypomagnetic field in a migratory insect.

Manipulating the hypomagnetic field (HMF), which is the absence or significant weakening (<5 µT) of the geomagnetic field (GMF), offers a unique tool to investigate magnetic field effects on organismal physiology, development, behavior and life history. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) has been utilized to study changes in gene expression associated with exposure to the HMF. However, selecting appropriate reference genes (RGs) with confirmed stable expression across environments for RT-qPCR is often underappreciated. Using three algorithms (BestKeeper, NormFinder, and GeNorm), we investigated the expression stability of eight candidate RGs when exposed to the HMF condition versus local GMF during developmental from juveniles to adults in the migratory insect pest, the brown planthopper Nilaparvata lugens. During the nymphal stage, RPL5 & α-TUB1, EF1-α & ARF1, RPL5 & AK, EF1-α & RPL5, and ARF1 & AK were suggested as the most stable RG sets in the 1st to 5th instars, respectively. For 1- to 3-day-old adults, AK & ARF1, AK & α-TUB1, AK & ARF1 and EF1-α & RPL5, AK & α-TUB1, AK & EF1-α were the optimal RG sets for macropterous and brachypterous females, respectively. ACT1 & RPL5, RPL5 & EF1-α, α-TUB1 & ACT1 and EF1-α & RPL5, ARF1 & ACT1, ACT1 & ARF1 were the optimal RG sets for macropterous and brachypterous males, respectively. These results will facilitate accurate gene expression analyses under the HMF in N. lugens. The verification approach illustrated in this study highlights the importance of identifying reliable RGs for future empirical studies of magnetobiology (including magnetoreception) that involve magnetic field intensity as a factor.

Cryptochrome 1 mediates light-dependent inclination magnetosensing in monarch butterflies.

Many animals use the Earth's geomagnetic field for orientation and navigation. Yet, the molecular and cellular underpinnings of the magnetic sense remain largely unknown. A biophysical model proposed that magnetoreception can be achieved through quantum effects of magnetically-sensitive radical pairs formed by the photoexcitation of cryptochrome (CRY) proteins. Studies in Drosophila are the only ones to date to have provided compelling evidence for the ultraviolet (UV)-A/blue light-sensitive type 1 CRY (CRY1) involvement in animal magnetoreception, and surprisingly extended this discovery to the light-insensitive mammalian-like type 2 CRYs (CRY2s) of both monarchs and humans. Here, we show that monarchs respond to a reversal of the inclination of the Earth's magnetic field in an UV-A/blue light and CRY1, but not CRY2, dependent manner. We further demonstrate that both antennae and eyes, which express CRY1, are magnetosensory organs. Our work argues that only light-sensitive CRYs function in animal light-dependent inclination-based magnetic sensing.

Geomagnetic field absence reduces adult body weight of a migratory insect by disrupting feeding behavior and appetite regulation.

The geomagnetic field (GMF) is well documented for its essential role as a cue used in animal orientation or navigation. Recent evidence indicates that the absence of GMF (mimicked by the near-zero magnetic field, NZMF) can trigger stress-like responses such as reduced body weight, as we have previously shown in the brown planthopper, Nilaparvata lugens. In this study, we found that consistent with the significantly decreased body weight of newly emerged female (-14.67%) and male (-13.17%) adult N. lugens, the duration of the phloem ingestion feeding waveform was significantly reduced by 32.02% in 5th instar nymphs reared under the NZMF versus GMF. Interestingly, 5th instar nymphs that exhibited reduced feeding had significantly higher glucose levels (+16.98% and +20.05%; 24 h and 48 h after molting), which are associated with food aversion, and expression patterns of their appetite-related neuropeptide genes (neuropeptide F, down-regulated overall; short neuropeptide F, down-regulated overall; adipokinetic hormone, up-regulated overall; and adipokinetic hormone receptor, down-regulated overall) were also altered under the absence of GMF in a manner consistent with diminishing appetite. Moreover, the expressions of the potential magnetosensor cryptochromes (Crys) were found significantly altered under the absence of GMF, indicating the likely upstream signaling of the Cry-mediated magnetoreception mechanisms. These findings support the hypothesis that strong changes in GMF intensity can reduce adult body weight through affecting insect feeding behavior and underlying regulatory processes including appetite regulation. Our results highlight that GMF could be necessary for the maintenance of energy homeostasis in insects.

Change in geomagnetic field intensity alters migration-associated traits in a migratory insect.

Geomagnetic field (GMF) intensity can be used by some animals to determine their position during migration. However, its role, if any, in mediating other migration-related phenotypes remains largely unknown. Here, we simulated variation in GMF intensity between two locations along the migration route of a nocturnal insect migrant, the brown planthopper Nilaparvata lugens, that varied by approximately 5 µT in field intensity. After one generation of exposure, we tested for changes in key morphological, behavioural and physiological traits related to migratory performance, including wing dimorphism, flight capacity and positive phototaxis. Our results showed that all three morphological and behavioural phenotypes responded to a small difference in magnetic field intensity. Consistent magnetic responses in the expression of the phototaxis-related Drosophila-like cryptochrome 1 (Cry1) gene and levels of two primary energy substrates used during flight, triglyceride and trehalose, were also found. Our findings indicate changes in GMF intensity can alter the expression of phenotypes critical for insect migration and highlight the unique role of magnetoreception as a trait that may help migratory insects express potentially beneficial phenotypes in geographically variable environments.

Enhancement of the geomagnetic field reduces the phototaxis of rice brown planthopper Nilaparvata lugens associated with frataxin down-regulation.

The geomagnetic field (GMF) is an environmental cue that provides directional information for animals. The intensity of GMF is varied over space and time. Variations in the GMF intensity affect the navigation of animals and their physiology. In this study, the phototaxis of the migratory insect rice planthopper Nilaparvata lugens (N. lugens) and frataxin in N. lugens (Nl-fh), which is a mitochondrial protein required for cellular iron homeostasis and iron-sulfur cluster assembly, were investigated by using different intensities of magnetic field. From the results, individuals of N. lugens showed decreased phototaxis when reared and tested in a behavioral arena under a strong magnetic field. Besides the reduction in performance, an accompanying effect of the strong magnetic field condition was a reduced level of Nl-fh-messenger RNA, and a Nl-fh knockdown indeed impaired the phototactic behavior in a tested sample of insects. This leads to the conclusion that the expression of frataxin is dependent on the strength of the surrounding magnetic field and that functional frataxin facilitates phototactic behavior in N. lugens.

Molecular characterization, spatial-temporal expression and magnetic response patterns of iron-sulfur cluster assembly1 (IscA1) in the rice planthopper, Nilaparvata lugens.

The mechanisms of magnetoreception have been proposed as the magnetite-based, the chemical radical-pair and biocompass model, in which magnetite particles, the cryptochrome (Cry) or iron-sulfur cluster assembly 1 (IscA1) may be involved. However, little is known about the association among the molecules. Here we investigated the molecular characterization and the mRNA expression of IscA1 in different developmental stages, tissues and magnetic fields in the migratory brown planthopper (BPH), Nilaparvata lugens. NlIscA1 contains an open reading frame of 390 bp, encoding amino acids of 129, with the predicted molecular weight of 14.0 kDa and the isoelectric point of 9.10. Well-conserved Fe-S cluster binding sites were observed in the predicted protein. Phylogenetic analysis demonstrated NlIscA1 to be clustered into the insect's IscA1. NlIscA1 showed up-regulated mRNA expression during the period of migration. The mRNA expression of NlIscA1 could be detected in all the three tissues of head, thorax and abdomen, with the highest expression level in the abdomen. For the macropterous migratory Nilaparvata lugens, mRNA expression of NlIscA1 and N. lugens cryptochrome1 (Nlcry1) were up-regulated under the magnetic fields of 5 Gauss and 10 Gauss in strength (vs. local geomagnetic field), while N. lugens cryptochrome2 (Nlcry2) remained stable. For the brachyterous non-migratory Nilaparvata lugens, no significant changes were found in mRNA expression of NlIscA1, Nlcry1 and Nlcry2 among different magnetic fields. These findings preliminarily reveal that the expression of NlIscA1 and Nlcry1 exhibited coordinated responses to the magnetic field. It suggests some potential associations among the putative magneto-sensitive molecules of cryptochrome and iron-sulfur cluster assembly.

Effects of seed mixture sowing with resistant and susceptible rice on population dynamics of target planthoppers and non-target stemborers and leaffolders.

BACKGROUND: The widespread planting of insect-resistant crops has caused a dramatic shift in agricultural landscapes, thus raising concerns about the potential impact on both target and non-target pests worldwide. In this study, we examined the potential effects of six seed mixture ratios of insect-resistance dominance [100% (R100), 95% (S05R95), 90% (S10R90), 80% (S20R80), 60% (S40R60), and 0% (S100)] on target and non-target pests in a 2-year field trial in southern China. RESULTS: The occurrence of the target pests Nilaparvata lugens and Sogatella furcifera decreased with an increase in the ratio of resistant rice, and mixture ratios with ≥90% resistant rice significantly increased the pest suppression efficiency, with the lowest occurrences of the non-target pests Sesamia inferens, Chilo suppressalis and Cnaphalocrocis medinalis for S100 and S10R90 seed mixture ratios. Furthermore, there were no significant differences in the 1000-grain dry weight and grain yield between R100 and other treatments with ≥80% resistant seeds in the mixture (S20R80, S10R90 and S05R95). CONCLUSION: S10R90 produced a good yield and provided the most effective control of both target and non-target pests, with the potential to significantly reduce the application of chemical pesticides for integrated pest management in paddy fields. It is further presumed that the strategy of seed mixture with resistant and susceptible rice would be advantageous for rice yield stability. © 2018 Society of Chemical Industry.

Brown planthopper Nilaparvata lugens was concentrated at the rear of the typhoon Soudelor in Eastern China in August 2015.

Sometimes, extreme weather is vital for the population survival of migratory insects by causing sudden population collapse or outbreak. Several studies have shown that rice planthopper migration was significantly influenced by typhoons in eastern Asia. Most typhoons occur in the summer, especially in August. In August, brown planthopper Nilaparvata lugens (Stål) migrates northward or southward depending on wind direction, and thus typhoons can potentially influence its migration process and population distribution. However, this has not yet been studied. This paper reported a case study on the effects of Typhoon Soudelor on the summer migration of N. lugens in eastern China in 2015. The migration pathways of N. lugens were reconstructed for the period under the influence of a typhoon by calculating the trajectories and migration events in eight counties of the Yangtze River Valley region with ancillary information. Trajectory modelling showed that most migrants took short distance migrations (less than 200 km) under the influence of the Typhoon Soudelor. Numerous N. lugens migrants were concentrated and deposited at the rear of the typhoon during the last 5 days of Typhoon Soudelor on August 9-13 due to horizontal convergence, and this led to an outbreak population. These results indicated that the N. lugens population was redistributed by the typhoon in the summer and that the population dynamics at the rear of a typhoon should be kept under close surveillance. This study provided insight into migratory organisms adapting to atmospheric features.

Impacts of elevated CO2 on exogenous Bacillus thuringiensis toxins and transgene expression in transgenic rice under different levels of nitrogen.

Recent studies have highlighted great challenges of transgene silencing for transgenic plants facing climate change. In order to understand the impacts of elevated CO2 on exogenous Bacillus thuringiensis (Bt) toxins and transgene expression in transgenic rice under different levels of N-fertilizer supply, we investigated the biomass, exogenous Bt toxins, Bt-transgene expression and methylation status in Bt rice exposed to two levels of CO2 concentrations and nitrogen (N) supply (1/8, 1/4, 1/2, 1 and 2 N). It is elucidated that the increased levels of global atmospheric CO2 concentration will trigger up-regulation of Bt toxin expression in transgenic rice, especially with appropriate increase of N fertilizer supply, while, to some extent, the exogenous Bt-transgene expression is reduced at sub-N levels (1/4 and 1/2N), even though the total protein of plant tissues is reduced and the plant growth is restricted. The unpredictable and stochastic occurrence of transgene silencing and epigenetic alternations remains unresolved for most transgenic plants. It is expected that N fertilization supply may promote the expression of transgenic Bt toxin in transgenic Bt rice, particularly under elevated CO2.

Behavioral evidence for a magnetic sense in the oriental armyworm, Mythimna separata.

Progress has been made in understanding the mechanisms underlying directional navigation in migratory insects, yet the magnetic compass involved has not been fully elucidated. Here we developed a flight simulation system to study the flight directionality of the migratory armyworm Mythimna separata in response to magnetic fields. Armyworm moths were exposed to either a 500 nT extreme weak magnetic field, 1.8 T strong magnetic field, or a deflecting magnetic field and subjected to tethered flight trials indoors in the dark. The moths were disoriented in the extreme weak magnetic field, with flight vectors that were more dispersed (variance=0.60) than in the geomagnetic field (variance=0.32). After exposure to a 1.8 T strong magnetic field, the mean flight vectors were shifted by about 105° in comparison with those in the geomagnetic field. In the deflecting magnetic field, the flight directions varied with the direction of the magnetic field, and also pointed to the same direction of the magnetic field. In the south-north magnetic field and the east-west field, the flight angles were determined to be 98.9° and 166.3°, respectively, and formed the included angles of 12.66° or 6.19° to the corresponding magnetic direction. The armyworm moths responded to the change of the intensity and direction of magnetic fields. Such results provide initial indications of the moth reliance on a magnetic compass. The findings support the hypothesis of a magnetic sense used for flight orientation in the armyworm Mythimna separata.

Evidence for the presence of biogenic magnetic particles in the nocturnal migratory brown planthopper, Nilaparvata lugens.

Biogenic magnetic particles have been detected in some migratory insects, which implies the basis of magnetoreception mechanism for orientation and navigation. Here, the biogenic magnetic particles in the migratory brown planthopper (BPH), Nilaparvata lugens were qualitatively measured by SQUID magnetometry, and their characteristics were further determined by Prussian Blue staining, electron microscopy and energy dispersive x-ray spectroscopy. The results indicate that there were remarkable magnetic materials in the abdomens and not in the head or thorax of the 3(rd)-5(th) instar nymphs, and in macropterous and brachypterous female and male adults of BPH. The size of magnetic particles was shown to be between 50-450 nm with a shape factor estimate of between 0.8-1.0 for all the tested BPHs. Moreover, the amount of magnetic particles was associated with the developmental stage (the 3(rd)-5(th) instar), wing form (macropterous vs. brachypterous) and sex. The macropterous female adults had the largest amount of magnetic particles. Although the existence of magnetic particles in the abdomens of BPH provides sound basis for the assumption of magnetic orientation, further behavioral studies and complementary physical characterization experiments should be conducted to determine whether the orientation behavior of BPH is associated with the magnetic particles detected in this study.

Molecular characterization, tissue and developmental expression profiles of cryptochrome genes in wing dimorphic brown planthoppers, Nilaparvata lugens.

Cryptochromes (CRYs) are blue and UV light photoreceptors, known to play key roles in circadian rhythms and in the light-dependent magnetosensitivity of insects. Two novel cryptochrome genes were cloned from the brown planthopper, and were given the designations of Nlcry1 and Nlcry2, with the accession numbers KM108578 and KM108579 in GenBank. The complementary DNA sequences of Nlcry1 and Nlcry2 are 1935 bp and 2463 bp in length, and they contain an open reading frame of 1629 bp and 1872 bp, encoding amino acids of 542 and 623, with a predicted molecular weight of 62.53 kDa and 70.60 kDa, respectively. Well-conserved motifs such as DNA-photolyase and FAD-binding-7 domains were observed in Nlcry1 and Nlcry2. Phylogenetic analysis demonstrated the proteins of Nlcry1 and Nlcry2 to be clustered into the insect's cryptochrome 1 and cryptochrome 2, respectively. Quantitative polymerase chain reaction showed that the daily oscillations of messenger RNA (mRNA) expression in the head of the brown planthopper were mild for Nlcry1, and modest for Nlcry2. Throughout all developmental stages, Nlcry1 and Nlcry2 exhibited extreme fluctuations and distinctive expression profiles. Cryptochrome mRNA expression peaked immediately after adult emergence and then decreased subsequently. The tissue expression profiles of newly emerged brown planthopper adults showed higher expression levels of CRYs in the head than in the thorax or abdomen, as well as significantly higher levels of CRYs in the heads of the macropterous strain than in the heads of the brachypterous strain. Taken together, the results of our study suggest that the two cryptochrome genes characterized in the brown planthopper might be associated with developmental physiology and migration.

Rice stripe virus counters reduced fecundity in its insect vector by modifying insect physiology, primary endosymbionts and feeding behavior.

Virus-vector relationships can be complex and diverse as a result of long-term coevolution. Understanding these interactions is crucial for disease and vector management. Rice stripe virus (RSV) is known to be transovarially transmitted within its vector, Laodelphax striatellus, and causes serious rice stripe disease. In RSV-infected L. striatellus, we found contrasting changes in vector fecundity, physiology, primary endosymbionts (i.e. yeast-like symbionts, YLS) and feeding behavior that can interact to affect the spread of RSV. RSV-infected L. striatellus exhibited a significant decrease in fecundity that could lead a reduction of viruliferous individuals in populations. As a potential response to this loss, RSV infection also significantly shortened nymphal stage duration, which can strengthen RSV vertical circulation in L. striatellus populations and promote RSV spreading by adult migration and dispersal. Down-regulated JHAMT and up-regulated CYP307A1 in the juvenile hormone and ecdysteroid pathways, respectively, were linked to accelerated development. RSV-infected adults were also found to have higher body weight in conjunction with increased YLS abundance. Furthermore, prolonged host plant phloem exposure to salivation by RSV-infected adults should further enhance RSV horizontal transmission. Our study highlights potential strategies of RSV in enhancing its transmission, and provides new insights into the complexity of virus-vector interactions.

Cryptochromes and Hormone Signal Transduction under Near-Zero Magnetic Fields: New Clues to Magnetic Field Effects in a Rice Planthopper.

Although there are considerable reports of magnetic field effects (MFE) on organisms, very little is known so far about the MFE-related signal transduction pathways. Here we establish a manipulative near-zero magnetic field (NZMF) to investigate the potential signal transduction pathways involved in MFE. We show that exposure of migratory white-backed planthopper, Sogatella furcifera, to the NZMF results in delayed egg and nymphal development, increased frequency of brachypterous females, and reduced longevity of macropterous female adults. To understand the changes in gene expression underlying these phenotypes, we examined the temporal patterns of gene expression of (i) CRY1 and CRY2 as putative magnetosensors, (ii) JHAMT, FAMeT and JHEH in the juvenile hormone pathway, (iii) CYP307A1 in the ecdysone pathway, and (iv) reproduction-related Vitellogenin (Vg). The significantly altered gene expression of CRY1 and CRY2 under the NZMF suggest their developmental stage-specific patterns and potential upstream location in magnetic response. Gene expression patterns of JHAMT, JHEH and CYP307A1 were consistent with the NZMF-triggered delay in nymphal development, higher proportion of brachypterous female adults, and the shortened longevity of macropterous female adults, which show feasible links between hormone signal transduction and phenotypic MFE. By conducting manipulative NZMF experiments, our study suggests an important role of the geomagnetic field (GMF) in modulating development and physiology of insects, provides new insights into the complexity of MFE-magnetosensitivity interactions, and represents an initial but crucial step forward in understanding the molecular basis of cryptochromes and hormone signal transduction involved in MFE.

Bio-effects of near-zero magnetic fields on the growth, development and reproduction of small brown planthopper, Laodelphax striatellus and brown planthopper, Nilaparvata lugens.

Magnetic fields markedly affect the growth and development of many species of organisms potentially due to cryptochrome and endogenous presence of magnetic materials. Sensitivity to magnetic fields can also be involved in geomagnetic orientation by some long-distance migratory insects. In this study, near-zero magnetic fields (NZMF) in relation to normal geomagnetic fields (GMF) were setup using the Hypomagnetic Field Space System (HMFs) to investigate the effects of magnetic fields on the growth, development and reproduction of two species of migratory planthopper, the small brown planthopper (abbr. SBPH), Laodelphax striatellus, and the brown planthopper (abbr. BPH), Nilaparvata lugens. Exposure of both L. striatellus and N. lugens to NZMF delayed egg and nymphal developmental durations and decreased adult weight and female fecundity. The 1st-5th instars of SBPH and BPH showed different responses to NZMF. The 4th instar was significantly affected by NZMF, especially for BPH males, in which NZMF exposure reduced the difference in development duration between females and males. Compared with GMF, the vitellogenin transcript levels of newly molted female adults and the number of eggs per female were significantly reduced in both planthopper species, indicating a negative effect on fertility under NZMF. Our findings provided experimental evidence that NZMF negatively affected the growth and development of SBPH and BPH, with particularly strong effects on reproduction.

Single and fused transgenic Bacillus thuringiensis rice alter the species-specific responses of non-target planthoppers to elevated carbon dioxide and temperature.

BACKGROUND: The approval of transgenic Bacillus thuringiensis (Bt) rice by China was momentous for biotech crops, although it has yet to be approved for commercial production. Non-target pest problems in rice paddies, such as the three ecologically similar species of planthoppers Nilaparvata lugens, Laodelphax striatellus and Sogatella furcifera, could become increasingly serious under global climate change. Fused (Cry1Ab/Cry1Ac) and single (Cry1Ab) transgenic Bt rice were evaluated for effects on species-specific responses of planthoppers to elevated carbon dioxide (CO2) and temperature. RESULTS: Transgenic Bt rice lines significantly modified species-specific responses of the planthoppers to elevated CO2 and temperature. High temperature appears to favour outbreaks of S. furcifera relative to N. lugens and L. striatellus when feeding upon fused transgenic Bt rice, especially at elevated CO2 . Elevated CO2 at high temperature appears to be a factor reducing S. furcifera occurrence when feeding upon single transgenic Bt rice. CONCLUSION: Different types of transgenic Bt rice alter the species-specific responses of non-target planthoppers to elevated CO2 and temperature. Compared with their non-transgenic parental lines, the single transgenic Bt rice shows better performance in controlling the non-target planthopper S. furcifera by comparison with the fused transgenic Bt rice under elevated CO2 and temperature. It is suggested that multitypes of transgenic Bt rice be used in the field simultaneously in order to take advantage of high transgenic diversity for optimal performance against all pests in paddy fields.