Red Flour Beetle Tribolium Castaneum Research Articles

Bioassays of Fumigant Toxicity and Repulsiveness of Haplophyllum tuberculatum Extracts

Abstract Food grains were severely damaged by stored goods insects. The red flour beetle Tribolium castaneum, the cigarette beetle Lasioderma serricorne and the rice weevil Sitophilus orysae are some of the most destructive pests which attack raw cereals throughout the world. In stored grain mill, fumigation with synthetic fumigants was the most economical tool for managing stored pests. Nevertheless, the application of chemical pesticides caused adverse effects on humans and environment. Chloroform, acetone, methanol and water extracts and essential oil of Haplophyllum tuberculatum aerial parts were tested for their fumigant and repellent effect against adults of the three stored products pests (T. castaneum, L. serricorne and S. oryzae). GC/MS analysis of the essential oil resulted in determination of 19 compounds. The major constituents were β-phellandrene (16.17%), trans-p-menth-2-ene-1-ol, (13.22%) and cis-p-menth-2-ene-1-ol (8.29%). Thus, it seemed that the essential oil had the highest fumigant activity against L. serricorne after 12 h of exposure compared to the two other insects with a LC50 equal to 38.59 µ.l-1 air. The best repellency was observed for the chloroform and methanol extracts against T. castaneum. The respective repellent doses RD50 values were = 0.03 and 0.04 mg.ml-1 after 24 h of exposure. However, after 24 h of exposure acetone extract seems to be more repellent with respective repellent dose values of = 0.14 mg.ml-1 (RD50) against L. serricorne. H. tuberculatum essential oil and extract proved to be effective in control pest infestations in grains and dry food products.

Isolation, Identification and Evaluation of the Effective Phyto-Compounds for The Management of Groundnut Beetles Infesting Stored Groundnut (Arachis Hypogaea L.)

The effectiveness of various botanicals was tested against the Major Pest of shelled groundnuts, the red flour beetle, Tribolium castaneum (Herbst) (Coleoptera: Tenbebrionidae), under artificial infestation, in Laboratory conditions for one year. Essential Oils isolated by Clevengers Apparatus , @1%v/w of Pongamia glabra (Karanja Oil), Azadirachta indica (Neem Oil), Brassica campestris (Mustard Oil) , Crown oil (Resin of Shorea robusta) , Dalbergia sisso (Sisso Oil), Oryza sativa (Rice bran oil), Helianthus annus (Sunflower Oil) , Ricinus communis (Castor Oil), Aegle marmelos (Bael Oil), Sesamum indicum (Til Oil), etc were tested for their bioefficacy against the Major Pest, of shelled groundnuts, red flour beetle Tribolium castaneum, (Herbst) Tenebrionidae, Coleoptera, under artificial infestation, in Laboratory conditions for one year. Out of all these plant products only, Crown oil (Resin of Shorea robusta) gave 100 % pod/Kernel protection for one year. The LD 50 values were found out to be - 0.5% v/w. Spectroscopic GC MS analysis of, Crown oil (Resin of Shorea robusta) are identified as 2-Ethyl-oxetane (RT 2.223), as the major product It may be due to the strong aromatic nature of these phyto-product. Hence these phyto-product may be used as fumigants or insect repellants against Tribolium castaneum and can be included in the package of practices to save stored groundnut in storage.

Knockdown of TcGluCl leads to the premature pupation of Tribolium castaneum larvae possibly by influencing the calcium-mediating hormone homeostasis

The glutamate-gated chloride channels (GluCls) are widely existed in the neural and nonneural tissues of invertebrate. In addition to play important roles in signal transduction, the GluCls also showed multiple physiological functions in insects such as participate in the juvenile hormone synthesis. In the present study, the potential roles of TcGluCl in growth and development of the red flour beetle Tribolium castaneum were explored. Knockdown of TcGluCl showed no effects on the survivability, weight growth, final pupation rate, eclosion and fecundity of T. castaneum, whereas resulted in the significant premature pupation of larvae. Inhibition of TcGluCl expression significantly changed the levels of juvenile hormone and ecdysone as well as the expressions of hormone biosynthetic genes. The increased ecdysone level and decreased juvenile hormone level were observed at the late stage of dsGluCl-treated larvae. Knockdown of TcGluCl significantly reduced the expressions of TcSTIM1 and TcOrai1, which were the primary proteins in store-operated calcium entry (SOCE) mediated Ca2+ influx mechanism. Whilst the L-glutamic acid treatment led to the increased TcOrai1 expression in T. castaneum. These findings suggested that knockdown of TcGluCl increased the ecdysone level and contributed to the premature pupation of larvae, which might be due to the reduced Ca2+ influx caused by the decreased expressions of TcSTIM1 and TcOrai1. These studies provide novel insights on the function of GluCls in insects.

In Vitro Evaluation of Native <i>Bacillus thuringiensis</i> (Berliner) Isolates against Different Insects

Evaluation of native strains of Bacillus thuringiensis (Berliner) isolates against different orders of insects and characterization of responsible Cry genes was carried out. Among the native isolates, BGC-1 showed the least LC50 value of 5.24 μg/ ml and was comparable to the reference strain HD1 (2.89 μg/ ml) against tobacco caterpillar Spodoptera litura (Fabricius). The isolate RCM-1 recorded least LC50 value of 4.69 μg/ ml against the housefly Musca domestica Linnaeus. One isolate viz., GHB-1 was found to be potential against both the larvae of S. litura and M. domestica. The isolate RCM-2 registered the least LC50 value of 8.21 μg/ ml against grubs of red flour beetle Tribolium castaneum (Herbst). Several isolates had more than one Cry gene in them. Among them, 12 isolates (63.15%) were found to contain Cry3 genes, 11 isolates harbour Cry4 genes, five isolates had Cry1, Cry2 and Cry11 genes each and four isolates contain Cry7 genes each. Native isolate, GHB-1 had Cry1, Cry3, Cry4 and Cry11 genes.

Carboxylesterase α-EST5 is required for the red flour beetle to metabolize mugwort essential oil

The red flour beetle (Tribolium castaneum) is a global agricultural grain storage pest that seriously jeopardizes the food safety of wheat, corn, and other grains. Carboxylesterases belong to a family of multifunctional enzymes that can participate in the metabolism of toxic substances ingested by T. castaneum, conferring drug resistance. In this study we have cloned from T. castaneum a gene encoding carboxylesterase, α-EST5, which is expressed mainly in T. castaneum late larvae and late adults. Tissue expression pattern analysis showed that α-EST5 was mainly expressed in the larval gut and adult head. Moreover, α-EST5 was able to be induced to be expressed by mugwort essential oils and significantly increased the enzymatic activity of carboxylesterase in T. castaneum after mugwort essential oil treatment. The sensitivity of T. castaneum to mugwort essential oils was significantly increased after knockdown the expression of α-EST5 using RNAi (RNA interference) technology. Through our study, we demonstrated that the carboxylesterase α-EST5 plays an important role in the detoxification of mugwort essential oils by T. castaneum. This study results provide a reference for understanding the detoxification mechanism of T. castaneum to external toxic substances.

Toxicity and Repellent Effect of Three Plant Based Essential Oils against the Red Flour Beetle <i>Tribolium castaneum</i> (Herbst)

In this study jasmine (Jasminum officinale), lemon grass (Cymbopogon citratus), and sandalwood (Santalum album) essential oils were investigated for their efficacy against the red flour beetle Tribolium castaneum. Sandalwood oil consistently showed the highest contact toxicity, with LD50 values decreasing over time. Jasmine and lemon grass oils ranked lower but still exhibited toxicity. The order of contact toxicity was sandalwood > jasmine > lemon grass at each exposure period. Fumigation treatments revealed sandalwood’s initial superiority, though jasmine surpassed it at 48 hr. The order of fumigation toxicity shifted to jasmine > sandalwood > lemon grass at this extended exposure time. Repellency tests showed dose and time-dependent responses for both larval and adult stages, with occasional fluctuations. Sandalwood’s significance differed for adult beetles between time intervals, and jasmine exhibited insignificance in its efficacy for larvae at certain intervals. This study provides insights into the potential of these essential oils as alternatives for pest control.

Beyond adult models: Tribolium castaneum larval timekeeping reveals unexpected robustness and insights into circadian clock.

Circadian rhythms are self-sustained endogenous oscillations that are found in all living organisms. In insects, circadian rhythms control a wide variety of behavioral and physiological processes, including feeding, locomotion, mating, and metabolism. While the role of circadian rhythms in adult insects is well-understood, it is largely unexplored in larvae. This study investigates the potential for larval synchronized activity in the red flour beetle (Tribolium castaneum), a species exhibiting solitary and aggregation phases. We hypothesized that, similar to adults, larvae would exhibit a daily activity pattern governed by an endogenous circadian clock. We further predicted that the transition between the solitary and gregarious phases extends to unique temporal activity patterns. Our results revealed unique timekeeper gene expression in larvae, leading to a distinct daily rhythm characterized by nocturnal activity. Cues indicating on potential cannibalism did not change daily activity peak. However, the absence of these cues significantly reduced the proportion of rhythmic larvae and led to higher variation in peak activity, highlighting the crucial role of social interactions in shaping their rhythmicity. This study sheds light on the evolution and function of larval synchronization in group-living insects, offering novel insights into this complex behavior.

Effects of nutrition on recovery, mortality, and mobility of adult Tribolium castaneum after exposure to long-lasting insecticide-incorporated netting.

Long-lasting insecticide-incorporated netting (LLIN) has been used to deliver contact insecticides as an integrated pest management tool for stored product insect pests in food facilities. Although the presence of food is known to improve insect recovery after exposure, it is not clear whether food nutritional quality plays a role. Here, the red flour beetle Tribolium castaneum adults were exposed to two commercially available LLINs, Carifend (active ingredient α-cypermethrin) and D-Terrence (deltamethrin), then transferred to Petri dishes with foods with varying nutritional quality (e.g., 0-100% ratios of flour to non-nutritive cellulose). We investigated the effects of nutrition, LLIN type, and exposure time on post-exposure recovery, mortality, and mobility. After exposure for 2-168 h, the immediate mortality of T. castaneum adults ranged from 0.5% to 91.0% with Carifend and 0% to 75.3% with D-Terrence. Adult recovery and delayed mortality were significantly affected by nutritional quality, LLIN type, exposure time, and recovery time. For both LLINs, adult recovery increased over time, with a trend for higher recovery and lower mortality with increasing nutritional quality and decreasing exposure time. In addition, adult mobility decreased multiple-fold after Carifend or D-Terrence exposure for 30, 60 or 90 min compared to 10 min. This study shows nutrition significantly modulates the efficacy of LLIN against T. castaneum, and thus strengthens the rationale for implementing stringent sanitation protocols for food facility managers. © 2024 Society of Chemical Industry. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

Deciphering a Beetle Clock: Individual and Sex-Dependent Variation in Daily Activity Patterns.

Circadian clocks are inherent to most organisms, including cryptozoic animals that seldom encounter direct light, and regulate their daily activity cycles. A conserved suite of clock genes underpins these rhythms. In this study, we explore the circadian behaviors of the red flour beetle Tribolium castaneum, a significant pest impacting stored grain globally. We report on how daily light and temperature cues synchronize distinct activity patterns in these beetles, characterized by reduced morning activity and increased evening activity, anticipating the respective environmental transitions. Although less robust, rhythmicity in locomotor activity is maintained in constant dark and constant light conditions. Notably, we observed more robust rhythmic behaviors in males than females with individual variation exceeding those previously reported for other insect species. RNA interference targeting the Clock gene weakened locomotor activity rhythms. Our findings demonstrate the existence of a circadian clock and of clock-controlled behaviors in T. castaneum. Furthermore, they highlight substantial individual differences in circadian activity, laying the groundwork for future research on the relevance of individual variation in circadian rhythms in an ecological and evolutionary context.

Impact of Different Food Sources on the Developmental Biology and Host Preferences of the Red Flour Beetle Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae)

An experiment conducted at the seed center laboratory at Tamil Nadu Agricultural University during June 2022 evaluated the host preferences and developmental biology of the red flour beetle (Tribolium castaneum) across three food sources: broken rice with rice flour, broken wheat with wheat flour, and tapioca flour. Beetle development was observed in ventilated containers under controlled conditions (28-30°C, 65% relative humidity) with 25 beetles per container. Significant differences in beetle survival and development. Wheat flour was the most favourable host, with 304 live adults, followed by broken wheat (145 live adults. Rice flour supported 20 adults and 11 larvae, indicating moderate suitability. Whole rice grains and tapioca flour were the least effective, with only dead adults and few larvae. Despite host quality variations, the developmental stages (egg incubation: 4-7 days, larval: 65-70 days, pupal: 6-9 days) were consistent across food sources. Wheat flour is the most supportive environment for T. castaneum development.

Efficiency of Alkaloid and Phenolic Extracts of Seeds and Leaves Sesbania Plant on Adults Red Flour Beetle Tribolium castaneum Herbst (Coleoptera : Tenebrionidae)

Efficiency of Alkaloid and Phenolic Extracts of Seeds and Leaves Sesbania Plant on Adults Red Flour Beetle Tribolium castaneum Herbst (Coleoptera : Tenebrionidae)

Characterization of double-stranded RNA and its silencing efficiency for insects using hybrid deep-learning framework.

RNA interference (RNAi) technology is widely used in the biological prevention and control of terrestrial insects. One of the main factors with the application of RNAi in insects is the difference in RNAi efficiency, which may vary not only in different insects, but also in different genes of the same insect, and even in different double-stranded RNAs (dsRNAs) of the same gene. This work focuses on the last question and establishes a bioinformatics software that can help researchers screen for the most efficient dsRNA targeting target genes. Among insects, the red flour beetle (Tribolium castaneum) is known to be one of the most sensitive to RNAi. From iBeetle-Base, we extracted 12027 efficient dsRNA sequences with a lethality rate of ≥20% or with experimentation-induced phenotypic changes and processed these data to correspond to specific silence efficiency. Based on the first complied novel benchmark dataset, we specifically designed a deep neural network to identify and characterize efficient dsRNA for RNAi in insects. The dna2vec word embedding model was trained to extract distributed feature representations, and three powerful modules, namely convolutional neural network, bidirectional long short-term memory network, and self-attention mechanism, were integrated to form our predictor model to characterize the extracted dsRNAs and their silencing efficiencies for T. castaneum. Our model dsRNAPredictor showed reliable performance in multiple independent tests based on different species, including both T. castaneum and Aedes aegypti. This indicates that dsRNAPredictor can facilitate prescreening for designing high-efficiency dsRNA targeting target genes of insects in advance.

Temporal control of RNAi reveals both robust and labile feedback loops in the segmentation clock of the red flour beetle

Animals from all major clades have evolved a segmented trunk, reflected in the human spine or the insect segments. These units emerge during embryogenesis from a posterior segment addition zone (SAZ), where repetitive gene activity is regulated by a mechanism described by the clock and wavefront/speed gradient model. In the red flour beetle Tribolium castaneum, RNA interference (RNAi) has been used to continuously knock down the function of primary pair-rule genes (pPRGs), caudal or Wnt pathway components, which has led to the complete breakdown of segmentation. However, it has remained untested, if this breakdown was reversible by bringing the missing gene function back to the system. To fill this gap, we established a transgenic system in T. castaneum, which allows blocking an ongoing RNAi effect with temporal control by expressing a viral inhibitor of RNAi via heat shock. We show that the T. castaneum segmentation machinery was able to reestablish after RNAi targeting the pPRGs Tc-eve, Tc-odd, and Tc-runt was blocked. However, we observed no rescue after blocking RNAi targeting Wnt pathway components. We conclude that the insect segmentation system contains both robust feedback loops that can reestablish and labile feedback loops that break down irreversibly. This combination may reconcile conflicting needs of the system: Labile systems controlling initiation and maintenance of the SAZ ensure that only one SAZ is formed. Robust feedback loops confer developmental robustness toward external disturbances.

Insecticidal activity on Tribolium castaneum and Phenacoccus solenopsis and characterization of active phytonanoparticles

Phytonanoparticles can be used as an alternative method for pest control in agriculture. The objective of this study was to evaluate the insecticidal activity of mono, bi and trimetallic phytonanoparticles (PNPs) synthesized from cachanilla (Pluchea sericea Nutt. Coville) and epazote (Chenopodium ambrosioides L.) against the red flour beetle (Tribolium castaneum) and the cotton mealybug (Phenacoccus solenopsis). Overall, T. castaneum showed greater sensitivity to PNPs than P. solenopsis. The trimetallic PNPs, Zn/Cu/Mn of P. sericea showed significantly higher effect against T. castaneum than the crude aqueous extract of P. sericea. No other PNPs showed more effect than their respective aqueous plant extract. Physicochemical characterization of the most active PNPs showed that the Zn/Cu/Mn NP of P. sericea had a strong zeta potential of +200 mV, hydrodynamic size of 16 nm, and contains zinc (6.73%), copper (0.13%), and manganese (4.69%). This study suggests that the trimetallic Zn/Cu/Mn PNPs of P. sericea have high insecticidal effect on T. castaneum, but low toxicity on P. solenopsis. Additional research on the mode of action of PNPs on insect targets is required.

A new suite of reporter vectors and a novel landing site survey system to study cis-regulatory elements in diverse insect species

Comparative analyses between traditional model organisms, such as the fruit fly Drosophila melanogaster, and more recent model organisms, such as the red flour beetle Tribolium castaneum, have provided a wealth of insight into conserved and diverged aspects of gene regulation. While the study of trans-regulatory components is relatively straightforward, the study of cis-regulatory elements (CREs, or enhancers) remains challenging outside of Drosophila. A central component of this challenge has been finding a core promoter suitable for enhancer-reporter assays in diverse insect species. Previously, we demonstrated that a Drosophila Synthetic Core Promoter (DSCP) functions in a cross-species manner in Drosophila and Tribolium. Given the over 300 million years of divergence between the Diptera and Coleoptera, we reasoned that DSCP-based reporter constructs will be useful when studying cis-regulation in a variety of insect models across the holometabola and possibly beyond. To this end, we sought to create a suite of new DSCP-based reporter vectors, leveraging dual compatibility with piggyBac and PhiC31-integration, the 3xP3 universal eye marker, GATEWAY cloning, different colors of reporters and markers, as well as Gal4-UAS binary expression. While all constructs functioned properly with a Tc-nub enhancer in Drosophila, complications arose with tissue-specific Gal4-UAS binary expression in Tribolium. Nevertheless, the functionality of these constructs across multiple holometabolous orders suggests a high potential compatibility with a variety of other insects. In addition, we present the piggyLANDR (piggyBac-LoxP AttP Neutralizable Destination Reporter) platform for the establishment of proper PhiC31 landing sites free from position effects. As a proof-of-principle, we demonstrated the workflow for piggyLANDR in Drosophila. The potential utility of these tools ranges from molecular biology research to pest and disease-vector management, and will help advance the study of gene regulation beyond traditional insect models.

Diatomaceous earth as insecticide: physiological and morphological evidence of its underlying mechanism.

Wheat grain containers or silos can be perfect habitats for insects, which generate large economic losses to grain production. Natural alternatives to synthetic insecticides have grown in popularity because of health, economic and ecological issues. Diatomaceous earth is a natural compound that has an insecticide effect by enhancing an insect's dehydration with no toxicity on mammals including humans. The aim of this study is to confirm the effect of diatomaceous earth as an insecticide for the wheat grain pest, the red flour beetle Tribolium castaneum (Coleoptera: Tenebrionidae) and demonstrate its underlying mechanisms as an insecticide by open-flow respirometry and scanning electron microscopy. Survival bioassays of T. castaneum revealed a dose-dependent insecticide effect of diatomaceous earth. Gravimetric measurements showed that 2 days exposure to diatomaceous earth produces a significant increase of mass loss. Open-flow respirometry measurements showed an increase of total water emission rate on insects due to an increase of both, respiratory and cuticular water loss. Our study revealed that diatomaceous earth produces an increase of insect's cuticle permeability, which is responsible for elevated cuticular water loss. Scanning electron microscopy images provided visual evidence of the lipid absorbent properties of diatomaceous earth particles, and showed a tendency for higher, although not significant, damaged area of the cuticle's surface from diatomaceous earth treated insects compared to control ones. With state-of-the art techniques like open-flow respirometry and scanning electron microscopy, we demonstrated the underlying mechanism of diatomaceous earth as an insecticide and provided new cues for understanding the properties of the cuticle and its ecological importance. © 2024 Society of Chemical Industry.

Structural Characterization and Functional Analysis of Mevalonate Kinase from Tribolium castaneum (Red Flour Beetle).

Mevalonate kinase (MevK) is an important enzyme in the mevalonate pathway that catalyzes the phosphorylation of mevalonate into phosphomevalonate and is involved in juvenile hormone biosynthesis. Herein, we present a structure model of MevK from the red flour beetle Tribolium castaneum (TcMevK), which adopts a compact α/β conformation that can be divided into two parts: an N-terminal domain and a C-terminal domain. A narrow, deep cavity accommodating the substrate and cofactor was observed at the junction between the two domains of TcMevK. Computational simulation combined with site-directed mutagenesis and biochemical analyses allowed us to define the binding mode of TcMevK to cofactors and substrates. Moreover, TcMevK showed optimal enzyme activity at pH 8.0 and an optimal temperature of 40 °C for mevalonate as the substrate. The expression profiles and RNA interference of TcMevK indicated its critical role in controlling juvenile hormone biosynthesis, as well as its participation in the production of other terpenoids in T. castaneum. These findings improve our understanding of the structural and biochemical features of insect Mevk and provide a structural basis for the design of MevK inhibitors.

Toxicity of Two Plant Essential Oils as Eco-friendly Fumigants against the Red Flour Beetle Tribolium castaneum

Background: This study aims to extract essential oils from Artemisia herba alba and Eucalyptus camaldolensis plants and evaluate their effectiveness as eco-friendly natural fumigants against larvae and adults of red flour beetle Tribolium castaneum. Methods: The current study was conducted under laboratory conditions for the period from 2023-4 to 2023-8 in the Plant Protection Laboratory in the Diyala Agriculture Directorate, Iraq. Different concentrations of 2, 4, 6, 8 and 10 µL-1 air for exposure period of 24, 48 and 72 hours. Result: Larval mortality rates reached 100% at concentrations of 8 and 10 µL L-1 air for Eucalyptus and Artemisia, respectively. The mortality rate increases with increasing concentration in adults, where the highest killing rate was recorded at 8.10 µL L-1 air, reaching 90% and 100% for Artemisia, while it reached 96.6% and 100% for Eucalyptus, respectively. Mortality rates were high throughout the duration of the first exposure and these rates increased as the exposure period increased to 48 hours and 72 hours, with the next highest killing rates for concentrations of 6, 8 and 10 µL L-1 air for Artemisia and Eucalyptus oils. There was no significant difference with pesticide treatment. This indicates the possibility of using essential oils in the applied field to manage pest populations in store products.

Influences of compound age and identity in the effectiveness of insect quinone secretions against the fungus Beauveria bassiana.

Chemical defences against parasites and pathogens can be seen in a wide range of animal taxa, including insect pests such as the red flour beetle Tribolium castaneum. Antimicrobial quinone-based secretions can be used by these beetles to defend against various parasites, particularly the fungal entomopathogen Beauveria bassiana. While quinone secretions can inhibit B. bassiana growth, it is unknown how long they remain effective or how individual secretion compounds contribute to growth inhibition. Here, we tested each individual component of the quinone secretions (methyl-1,4-benzoquinone, ethyl-1,4-benzoquinone, and 1-pentadecene), as well as two mixed solutions that represent the composition range found in natural T. castaneum secretions, after aging for 0, 24, or 72h. The two quinone compounds equally contributed to B. bassiana inhibition, but their efficacy was significantly reduced after 24h, with no growth inhibition after 72h. This indicates that quinones protect insects against B. bassiana for only a limited time, perhaps requiring constant secretion into the environment to effectively defend against this fungal threat. Future investigations may consider the extent to which quinone secretions are effective against other parasites, as well as how their ability to cause parasite damage changes with compound age.

Transcriptomic comparison between populations selected for higher and lower mobility in the red flour beetle Tribolium castaneum

Movement is an important behavior observed in a wide range of taxa. Previous studies have examined genes controlling movement using wing polymorphic insects and genes controlling wing size. However, few studies have investigated genes controlling movement activity rather than morphological traits. In the present study, we conducted RNA sequencing using populations with higher (WL) and lower (WS) mobility established by artificial selection in the red flour beetle Tribolium castaneum and compared gene expression levels between selected populations with two replicate lines. As a result, we found significant differences between the selected populations in 677 genes expressed in one replicate line and 1198 genes expressed in another replicate line, of which 311 genes were common to the two replicate lines. Furthermore, quantitative PCR focusing on 6 of these genes revealed that neuropeptide F receptor gene (NpF) was significantly more highly expressed in the WL population than in the WS population, which was common to the two replicate lines. We discuss differences in genes controlling movement between walking activity and wing polymorphism.