Beetle Tribolium Castaneum Research Articles

Animal personalities influence male mating behavior and reproductive fitness

Abstract The relationship between individual personality and sexual selection has the potential to provide insights into how variation in personality traits is maintained. However, whether and how individuals with different personality traits have different mating behaviors and the fitness consequences has remained largely unexplored. We tested the hypothesis that male individuals have different mating behavior depending on their personality traits, and that there are fitness effects of personality traits. To do so, we conducted trials to quantify personality traits and observed mating behavior under standardized conditions in the red flour beetle (Tribolium castaneum). We also collected the number of offspring produced by their female mates to assess the association between male personality and their reproductive fitness. We found that male beetles showed consistent inter-individual differences in walking distance, emergence time from shelter and death-feigning time, which likely corresponding to two personality traits of activity and boldness. Although larger males had higher mating success and produced more offspring, some personality traits were also strongly related to mating behavior and reproductive fitness. Males with longer walking distances tended to seek female mates more promptly. Other than the morphological trait of body size, the emergence time from shelter (boldness) also showed a positive relationship with the number of matings and offspring reproduced. Furthermore, we found that the interaction between death-feigning time and walking distance in males influenced the number of matings they obtain, with either shy but active or bold but inactive males achieving more matings. We therefore highlight that alongside morphological traits, certain personality traits can also play a role in affecting mating behaviors and even reproductive fitness in males.

Superior target genes and pathways for RNAi-mediated pest control revealed by genome-wide analysis in the beetle Tribolium castaneum.

An increasing human population, the emergence of resistances against pesticides and their potential impact on the environment call for the development of new eco-friendly pest control strategies. RNA interference (RNAi)-based pesticides have emerged as a new option with the first products entering the market. Essentially, double-stranded RNAs targeting essential genes of pests are either expressed in the plants or sprayed on their surface. Upon feeding, pests mount an RNAi response and die. However, it has remained unclear whether RNAi-based insecticides should target the same pathways as classic pesticides or whether the different mode-of-action would favor other processes. Moreover, there is no consensus on the best genes to be targeted. We performed a genome-wide screen in the red flour beetle to identify 905 RNAi target genes. Based on a validation screen and clustering, we identified the 192 most effective target genes in that species. The transfer to oral application in other beetle pests revealed a list of 34 superior target genes, which are an excellent starting point for application in other pests. Gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) analyses of our genome-wide dataset revealed that genes with high efficacy belonged mainly to basic cellular processes such as gene expression and protein homeostasis - processes not targeted by classic insecticides. Our work revealed the best target genes and target processes for RNAi-based pest control and we propose a procedure to transfer our short list of superior target genes to other pests. © 2024 The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

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.

Long-read genome assembly of the insect model organism Tribolium castaneum reveals spread of satellite DNA in gene-rich regions by recurrent burst events.

Eukaryotic genomes are replete with satellite DNAs (satDNAs), large stretches of tandemly repeated sequences that are mostly underrepresented in genome assemblies. Here we combined nanopore long-read sequencing with a reference-guided assembly approach to generate an improved, high-quality genome assembly, TcasONT, of the model beetle Tribolium castaneum Enriched by 45 Mb in repetitive regions, the new assembly comprises almost the entire genome sequence. We use the enhanced assembly to conduct global and in-depth analyses of abundant euchromatic satDNAs. Unexpectedly, we show the extensive spread of satDNAs in gene-rich regions, including long arrays. The sequence similarity relationships between satDNA monomers and arrays indicate a recent exchange of satDNA arrays between different chromosomes. We propose a scenario of their genome dynamics characterized by repeated bursts of satDNAs spreading through euchromatin, followed by a process of elongation and homogenization of arrays. We find that suppressed recombination on the X Chromosome has no significant effect on the spread of satDNAs but the X rather tolerates the amplification of satDNAs into longer arrays. Analyses of arrays' neighboring regions show a tendency of one satDNA to be associated with transposable-like elements. Using 2D electrophoresis followed by Southern blotting, we prove Cast satDNAs' presence in the fraction of extrachromosomal circular DNA (eccDNA). We point to two mechanisms that enable this satDNA spread to occur: transposition by transposable elements and insertion mediated by eccDNA. The presence of such a large proportion of satDNA in gene-rich regions inevitably gives rise to speculation about their possible influence on gene expression.

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.

Nutrition affects larval survival and the development of morphological traits in male and female flour beetles, but genital size and shape remains canalised.

The caloric content and macronutrient ratio of diet consumed is a major source of phenotypic variation in most animal populations. While these nutritional effects have been well-documented for a variety of life-history and morphological traits, the effects of nutrition on male genitals are poorly understood but genitals are thought to be more canalised than general morphology and hence less susceptible to variation in nutrition. Even less is known about the effects of nutrition on female genital form, which to our knowledge, have never been investigated. Here we tested for effects of juvenile dietary macronutrients (protein and carbohydrate) on larval survival, adult morphology, including genital size and shape in male and female flour beetles (Tribolium castaneum). We found there was nutritionally induced plasticity in larval survival and morphology, although the latter effect was variable, with body size being most responsive to dietary macronutrients and genital size and shape being least responsive. Functionally equivalent morphological traits in the sexes responded similarly to nutrition. Previously, we showed that the genitalia of male and female T. castaneum are subject to strong stabilising sexual selection, and our current findings suggest that developmental mechanisms reduce the nutritional sensitivity of male and female genitals, possibly to ensure matching during mating.

Experimental evolution reveals that males evolving within warmer thermal regimes improve reproductive performance under heatwave conditions in a model insect.

Climate change is increasing mean temperatures, and intensifying heatwaves. Natural populations may respond to stress through shorter-term acclimation via plasticity and/or longer-term inter-generational evolution. However, if the pace and/or extent of thermal change is too great, local extinctions occur; one potential cause in ectotherms is identified to be the heat-liability of male reproductive biology. Recent data from several species, including the beetle Tribolium castaneum, confirmed that male reproductive biology is vulnerable to heatwaves, which may constrain populations. However, such reproductive-damage may be overestimated, if there is potential to adapt to elevated mean temperatures associated with climate change via evolution and/or acclimation. Here, we tested this to evaluate whether pre-exposures could improve heatwave tolerance (adaptation or acclimation), by experimentally evolving Tribolium castaneum populations to divergent thermal regimes (30 °C vs. 38 °C). Findings across assays revealed that relative to 30 °C-regime males, males from the 38 °C regime, maintained constantly at 8 °C warmer for 25 generations, displayed an increase; (i) in post heatwave (42 °C) reproductive fitness by 55%, (ii) survival by 33%, and (iii) 32% larger testes volumes. Unexpectedly, in the acclimation assay, warm-adapted males' post-heatwave survival and reproduction were best if they experienced cool developmental acclimation beforehand, suggesting a cost to adapting to 38 °C. These results help progress knowledge of the potential for survival and reproduction to adapt to climate change; trait specific adaptation to divergent thermal regimes can occur over relatively few generations, but this capacity depended on the interaction of evolutionary and thermal acclimatory processes.

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.

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.

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.

Relationship between walking movement and reproductive traits in the red flour beetle Tribolium castaneum

AbstractMany animal species have movement abilities. Behavior is important for evolutionary ecology because animal movement leads to dispersal, migration, search for food and mates, and escaping from enemies. However, individual differences in movement activity are found within a population. This phenomenon can be affected by various factors, one of which is suggesting that higher moving activity has fitness cost, whereas lower moving activity has benefits. Animal movement may also affect reproduction (e.g., resource allocation tradeoff between movement and reproduction as well as intra‐ and intersexual selection). Although many previous studies have investigated the relationship between movement and reproduction, less attention has been paid to walking movement. In this study, previous studies that investigated the relationship between movement and reproduction were reviewed using the flour beetle Tribolium castaneum, a model insect in behavioral ecology and genetics. Several previous studies suggest that beetle walking is strongly associated with male and female reproductive traits. In recent years, empirical studies on the correlation between walking and other traits have increased, particularly in T. castaneum. Although this species can fly and walk, the movement is often discussed without discriminating between flight and walk. Differences in modes of movement may affect the correlation between movement and other traits; thus, discussing each mode of movement separately is necessary.

Tissue-Level Integration Overrides Gradations of Differentiating Cell Identity in Beetle Extraembryonic Tissue.

During animal embryogenesis, one of the earliest specification events distinguishes extraembryonic (EE) from embryonic tissue fates: the serosa in the case of the insects. While it is well established that the homeodomain transcription factor Zen1 is the critical determinant of the serosa, the subsequent realization of this tissue's identity has not been investigated. Here, we examine serosal differentiation in the beetle Tribolium castaneum based on the quantification of morphological and morphogenetic features, comparing embryos from a Tc-zen1 RNAi dilution series, where complete knockdown results in amnion-only EE tissue identity. We assess features including cell density, tissue boundary morphology, and nuclear size as dynamic readouts for progressive tissue maturation. While some features exhibit an all-or-nothing outcome, other key features show dose-dependent phenotypic responses with trait-specific thresholds. Collectively, these findings provide nuance beyond the known status of Tc-Zen1 as a selector gene for serosal tissue patterning. Overall, our approach illustrates how the analysis of tissue maturation dynamics from live imaging extends but also challenges interpretations based on gene expression data, refining our understanding of tissue identity and when it is achieved.

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.