Continuous mowing differentially affects floral defenses in the noxious and invasive weed Solanum elaeagnifolium in its native range.

In weeds, disturbance has been found to affect life history traits and mediate trophic interactions. In urban landscapes, mowing is an important disturbance, and we previously showed that continuous mowing leads to enhanced fitness and defense traits in Solanum elaeagnifolium, Silverleaf Nightshade (SLN). However, most studies have been focused on foliar defenses, ignoring floral defenses. In this study we examined whether continuous mowing affected floral defenses in SLN using mowed and unmowed populations in South Texas, their native range. We found flowers of mowed SLN plants larger but lighter than unmowed plants. Additionally, flowers on plants that were mowed frequently were both heavier and larger. Mowed plants had higher spine density and consequently unmowed flowers had higher herbivore damage. Additionally, early instar Manduca sexta fed on mowed flower-based artificial diets showed no difference in mass than the control and unmowed; however, later instars caterpillars on unmowed diets gained significantly more mass than the mowed treatment and control. Mowed plants had higher spine density which may shed light on why unmowed flowers experienced higher herbivore damage. We found caterpillars fed on high mowing frequency diets were heavier than those on low mowing frequency diets. Collectively, we show that mowing compromises floral traits and enhances plant defenses against herbivores and should be accounted for in management.

A conserved asparagine residue stabilizes iron binding in Manduca sexta transferrin-1.

Transferrin 1 (Tsf1) is an insect-specific iron-binding protein that is abundant in hemolymph and other extracellular fluids. It binds iron tightly at neutral pH and releases iron under acidic conditions. Tsf1 influences the distribution of iron in the body and protects against infection. Elucidating the mechanisms by which Tsf1 achieves these functions will require an understanding of how Tsf1 binds and releases iron. Previously, crystallized Tsf1 from Manduca sexta was shown to have a novel type of iron coordination that involves four iron-binding ligands: two tyrosine residues (Tyr90 and Tyr204), a buried carbonate anion, and a solvent-exposed carbonate anion. The solvent-exposed carbonate anion was bound by a single amino acid residue, a highly conserved asparagine at position 121 (Asn121); thus, we predicted that Asn121 would be essential for high-affinity iron binding. To test this hypothesis, we analyzed the iron-binding and -release properties of five forms of recombinant Tsf1: wild-type, a Y90F/Y204F double mutant (negative control), and three Asn121 mutants (N121A, N121D and N121S). Each of the Asn121 mutants exhibited altered spectral properties, confirming that Asn121 contributes to iron coordination. The N121D and N121S mutations resulted in slightly lower affinity for iron, especially at acidic pH, while iron binding and release by the N121A mutant was indistinguishable from that of the wild-type protein. The surprisingly minor consequences of mutating Asn121, despite its high degree of conservation in diverse insect species, suggest that Asn121 may play a role that is essential in vivo but non-essential for high affinity iron binding in vitro.

Identification of immunity-related genes distinctly regulated by Manduca sexta Spӓtzle-1/2 and Escherichia coli peptidoglycan.

The immune system of Manduca sexta has been well studied to understand molecular mechanisms of insect antimicrobial responses. While evidence supports the existence of major immune signaling pathways in this species, it is unclear how induced production of defense proteins is specifically regulated by the Toll and Imd pathways. Our previous studies suggested that diaminopimelic acid-type peptidoglycans (DAP-PG) from Gram-negative and some Gram-positive bacteria, more than Lys-type peptidoglycans (Lys-PG) from other Gram-positive bacteria, triggers both pathways through membrane-bound receptors orthologous to Drosophila Toll and PGRP-LC. In this study, we produced M. sexta proSpätzle-1 and proSpätzle-2 in Sf9 cells, identified their processing enzymes, and used prophenoloxidase activating protease-3 to activate the cytokine precursors. After Spätzle-1 and -2 were isolated from the reaction mixtures, we separately injected the purified cytokines into larval hemocoel to induce gene transcription in fat body through the Toll pathway solely. On the other hand, we treated a M. sexta cell line with E. coli DAP-PG to only induce the Imd pathway and target gene expression. RNA-Seq analysis of the fat body and cultured cells collected at 0, 6, and 24 h after treatment indicated that expression of diapausin-4, -10, -12, -13, cecropin-2, -4, -5, attacin-5, -11, and lebocin D is up-regulated predominantly via Toll signaling, whereas transcription of cecropin-6, gloverin, lysozyme-1, and gallerimycin-2 is mostly induced by DAP-PG via Imd signaling. Other antimicrobial peptides are expressed in response to both pathways. Transcripts of most Toll-specific genes (e.g., lebocin D) peaked at 6 h, contrasting the gradual increase and plateauing of drosomycin mRNA level at 24-48 h in Drosophila. We also used T (oll)-I (md) ratios to estimate relative contributions of the two pathways to transcriptional regulation of other components of the immune system. The differences in pathway specificity and time course of transcriptional regulation call for further investigations in M. sexta and other insects.

Crop-emptying rate and nectar resource allocation in a nectivorous pollinator.

In nectivorous pollinators, timing and pattern of allocation of consumed nectar affects fitness traits and foraging behavior. Differences in male and female behaviors can influence these allocation strategies. These physiological patterns are not well studied in Lepidoptera, despite them being important pollinators. In this study we investigate crop-emptying rate and nectar allocation in Manduca sexta (Sphingidae), and how sex and flight influence these physiological patterns. After a single feeding event, moths were dissected at fixed time intervals to measure crop volume and analyze sugar allocation to flight muscle and fat body. Then we compared sedentary and flown moths to test how activity may alter these patterns. Sedentary males and females emptied their crops six hours after a feeding event. Both males and females preferentially allocated these consumed sugars to fat body over flight muscle. Moths began to allocate to the fat body during crop-emptying and retained these nutrients long-term (four and a half days after a feeding event). Males allocated consumed sugar to flight muscles sooner and retained these allocated nutrients in the flight muscle longer than did females. Flight initiated increased crop-emptying in females, but had no effect on males. Flight did not significantly affect allocation to flight muscle or fat body in either sex. This study showed that there are inherent differences in male and female nectar sugar allocation strategies, but that male and female differences in crop-emptying rate are context dependent on flight activity. These differences in physiology may be linked to distinct ways males and females maximize their own fitness.

Olfaction in the Anthropocene: NO3 negatively affects floral scent and nocturnal pollination.

There is growing concern about sensory pollutants affecting ecological communities. Anthropogenically enhanced oxidants [ozone (O3) and nitrate radicals (NO3)] rapidly degrade floral scents, potentially reducing pollinator attraction to flowers. However, the physiological and behavioral impacts on pollinators and plant fitness are unknown. Using a nocturnal flower-moth system, we found that atmospherically relevant concentrations of NO3 eliminate flower visitation by moths, and the reaction of NO3 with a subset of monoterpenes is what reduces the scent's attractiveness. Global atmospheric models of floral scent oxidation reveal that pollinators in certain urban areas may have a reduced ability to perceive and navigate to flowers. These results illustrate the impact of anthropogenic pollutants on an animal's olfactory ability and indicate that such pollutants may be critical regulators of global pollination.

Serine protease homolog pairs CLIPA4-A6, A4-A7Δ, and A4-A12 act as cofactors for proteolytic activation of prophenoloxidase-2 and -7 in Anopheles gambiae.

Phenoloxidase (PO) catalyzed melanization and other insect immune responses are mediated by serine proteases (SPs) and their noncatalytic homologs (SPHs). Many of these SP-like proteins have a regulatory clip domain and are called CLIPs. In most insects studied so far, PO precursors are activated by a PAP (i.e., PPO activating protease) and its cofactor of clip-domain SPHs. Although melanotic encapsulation is a well-known refractory mechanism of mosquitoes against malaria parasites, it is unclear if a cofactor is required for PPO activation. In Anopheles gambiae, CLIPA4 is 1:1 orthologous to Manduca sexta SPH2; CLIPs A5-7, A12-14, A26, A31, A32, E6, and E7 are 11:4 orthologous to M. sexta SPH1a, 1b, 4, and 101, SPH2 partners in the cofactors. Here we produced proCLIPs A4, A6, A7Δ, A12, and activated them with CLIPB9 or M. sexta PAP3. A. gambiae PPO2 and PPO7 were expressed in Escherichia coli for use as PAP substrates. CLIPB9 was mutated to CLIPB9Xa by including a Factor Xa cleavage site. CLIPA7Δ was a deletion mutant with a low complexity region removed. After PAP3 or CLIPB9Xa processing, CLIPA4 formed a high Mr complex with CLIPA6, A7Δ or A12, which assisted PPO2 and PPO7 activation. High levels of specific PO activity (55-85 U/µg for PO2 and 1131-1630 U/µg for PO7) were detected in vitro, indicating that cofactor-assisted PPO activation also occurs in this species. The cleavage sites and mechanisms for complex formation and cofactor function are like those reported in M. sexta and Drosophila melanogaster. In conclusion, these data suggest that the three (and perhaps more) SPHI-II pairs may form cofactors for CLIPB9-mediated activation of PPOs for melanotic encapsulation in A. gambiae.

Functional annotation of insecta transcriptomes: A cautionary tale from Lepidoptera.

Functional annotation is a critical step in the analysis of genomic data, as it provides insight into the function of individual genes and the pathways in which they participate. Currently, there is no consensus on the best computational approach for assigning functional annotation. This study compares three functional annotation methods (BLAST, eggNOG-Mapper, and InterProScan) in their ability to assign Gene Ontology terms in two species of Insecta with differing levels of annotation, Bombyx mori and Manduca sexta. The methods were compared for their annotation coverage, number of term assignments, term agreement and non-overlapping terms. Here we show that there are large discrepancies in gene ontology term assignment among the three computational methods, which could lead to confounding interpretations of data and non-comparable results. This study provide insight into the strengths and weaknesses of each computational method and highlight the need for more standardized methods of functional annotation.

OXIDOSQUALENE CYCLASE 1 and 2 influence triterpene biosynthesis and defense in Nicotiana attenuata.

Triterpenes are a class of bioactive compounds with diverse biological functions, playing pivotal roles in plant defense against biotic stressors. Oxidosqualene cyclases (OSCs) serve as gatekeepers in the biosynthesis of triterpenes. In this study, we utilized a Nicotiana benthamiana heterologous expression system to characterize NaOSC1 from Nicotiana attenuata as a multifunctional enzyme capable of synthesizing lupeol, dammarenediol II, 3-alpha,20-lupanediol, and 7 other triterpene scaffolds. We also demonstrated that NaOSC2 is, in contrast, a selective enzyme, producing only the ß-amyrin scaffold. Through virus-induced gene silencing and in vitro toxicity assays, we elucidated the roles of NaOSC1 and NaOSC2 in the defense of N. attenuata against Manduca sexta larvae. Metabolomic and feature-based molecular network analyses of leaves with silenced NaOSC1 and NaOSC2 unveiled 3 potential triterpene glycoside metabolite clusters. Interestingly, features identified as triterpenes within these clusters displayed a significant negative correlation with larval mass. Our study highlights the pivotal roles of NaOSC1 and NaOSC2 from N. attenuata in the initial steps of triterpene biosynthesis, subsequently influencing defense against M. sexta through the modulation of downstream triterpene glycoside compounds.

Retrospective radiological analysis to establish anatomical variation in angulation between medial canthus and the middle turbinate, for optimal positioning of Lester Jones Lacrimal bypass tubes in treatment of Epiphora.

PURPOSE: To determine the optimum angle for placement of Lester Jones lacrimal bypass tube using fixed radiological markers on CT scan head with axial and coronal cuts, as well as analysing the anatomical variation and range of angulation between individuals within our local population. METHODS: A retrospective radiological study conducted on a randomly selected sample of 384 adult patients in a UK Teaching Hospital. The angle between the medial canthus and the middle turbinate was measured on CT scans of the head using fixed radiological anatomical landmarks and analysed using the IMPAX software. Patients with orbital or nasal fractures, as well as those with history of surgical procedures involving the facial bones, were excluded. The accuracy of our measurements was validated using three dimensional (3D) CT head reconstruction technology. RESULTS: Analysis of the results showed a range of angulation between 28-45 degrees, with a mean angle of 36.99 ± 4.78 SD. There was no significant correlation found when comparing the different age groups using the One Way ANOVA test. Furthermore, a non-significant correlation was found between males and females when their mean angles were compared using the independent t-test. CONCLUSION: Our study showed that the ideal angle for insertion of Lester Jones tube would be between 30-45 degrees, with a mean of 37 degrees. No significant correlation was found between the age of the patient and the ideal angle of insertion of Lester Jones tube. Moreover, no significant difference was found in the angle measurements between males and females.

Critical re-examination of known purported fossil Bombycoidea (Lepidoptera).

We critically re-examine 17 records of fossils currently assigned to the lepidopteran superfamily Bombycoidea, which includes the silk moths, emperor moths and hawk moths. These records include subfossils, compression and impression fossils, permineralizations and ichnofossils. We assess whether observable morphological features warrant their confident assignment to the superfamily. None of the examined fossils displays characters that allow unequivocal identification as Sphingidae, but three fossils and a subfossil (Mioclanis shanwangiana Zhang, Sun and Zhang, 1994, two fossil larvae, and a proboscis in asphaltum) have combinations of diagnostic features that support placement in the family. The identification of a fossil pupa as Bunaeini (Saturniidae) is well supported. The other fossils that we evaluate lack definitive bombycoid and, in several cases, even lepidopteran characters. Some of these dubious fossils have been used as calibration points in earlier studies casting doubt on the resulting age estimates. All fossil specimens reliably assigned to Bombycoidea are relatively young, the earliest fossil evidence of the superfamily dating to the middle Miocene.

Degumming and characterization of Bombyx mori and non-mulberry silks from Saturniidae silkworms.

In this study, cocoons and degummed silk samples of Bombyx mori and twenty Saturniidae species of the genera Actias, Attacus, Argema, Antheraea, Caligula, Callosamia, Cricula, Epiphora, Hyalophora, Loepa, Samia and Saturnia are studied to gain an insight into their morphology, chemical composition and physical structure. For this purpose, silk samples are characterized by optical microscopy and FTIR spectroscopy in attenuated total reflection mode (ATR-FTIR spectroscopy). Furthermore, degummed silk samples are analyzed for their amino acid (AA) composition by GC-FID. In the course of method development, various degumming methods are tested using alkalis, citric acid, enzymes and detergents. A mixture of 0.1% sodium carbonate and 2.5% ethylenediamine proves to be an effective agent for degumming Saturniidae and B. mori cocoons. After hydrolysis of the fibroin filaments with 6 N hydrochloric acid and derivatization with propyl chloroformate, fifteen AAs are identified and qualified. This method shows a satisfactory overall analytical performance with an average recovery rate of 95% at the medium concentration level. The chemical composition of the different silks was considered comparatively. Within a genus, the analyses usually show a high degree of similarity in AA composition and the resulting structural indices, whereas differences are found between genera.

Differential expression of immunity-related genes in larval Manduca sexta tissues in response to gut and systemic infection.

Introduction: The midgut epithelium functions as tissue for nutrient uptake as well as physical barrier against pathogens. Additionally, it responds to pathogen contact by production and release of various factors including antimicrobial peptides, similar to the systemic innate immune response. However, if such a response is restricted to a local stimulus or if it appears in response to a systemic infection, too is a rather underexplored topic in insect immunity. We addressed the role of the midgut and the role of systemic immune tissues in the defense against gut-borne and systemic infections, respectively. Methods: Manduca sexta larvae were challenged with DAP-type peptidoglycan bacteria - Bacillus thuringiensis for local gut infection and Escherichia coli for systemic stimulation. We compared the immune response to both infection models by measuring mRNA levels of four selected immunity-related genes in midgut, fat body, hematopoietic organs (HOs), and hemocytes, and determined hemolymph antimicrobial activity. Hemocytes and HOs were tested for presence and distribution of lysozyme mRNA and protein. Results: The midgut and circulating hemocytes exhibited a significantly increased level of lysozyme mRNA in response to gut infection but did not significantly alter expression in response to a systemic infection. Conversely, fat body and HOs responded to both infection models by altered mRNA levels of at least one gene monitored. Most, but not all hemocytes and HO cells contain lysozyme mRNA and protein. Discussion: These data suggest that the gut recruits immune-related tissues in response to gut infection whereas systemic infections do not induce a response in the midgut. The experimental approach implies a skewed cross-talk: An intestinal infection triggers immune activity in systemic immune organs, while a systemic infection does not elicit any or only a restricted immune response in the midgut. The HOs, which form and release hemocytes in larval M. sexta, i) synthesize lysozyme, and ii) respond to immune challenges by increased immune gene expression. These findings strongly suggest that they not only provide phagocytes for the cellular immune response but also synthesize humoral immune components.

Wettability and morphology of proboscises interweave with hawkmoth evolutionary history.

Hovering hawkmoths expend significant energy while feeding, which should select for greater feeding efficiency. Although increased feeding efficiency has been implicitly assumed, it has never been assessed. We hypothesized that hawkmoths have proboscises specialized for gathering nectar passively. Using contact angle and capillary pressure to evaluate capillary action of the proboscis, we conducted a comparative analysis of wetting and absorption properties for 13 species of hawkmoths. We showed that all 13 species have a hydrophilic proboscis. In contradistinction, the proboscises of all other tested lepidopteran species have a wetting dichotomy with only the distal ∼10% hydrophilic. Longer proboscises are more wettable, suggesting that species of hawkmoths with long proboscises are more efficient at acquiring nectar by the proboscis surface than are species with shorter proboscises. All hawkmoth species also show strong capillary pressure, which, together with the feeding behaviors we observed, ensures that nectar will be delivered to the food canal efficiently. The patterns we found suggest that different subfamilies of hawkmoths use different feeding strategies. Our comparative approach reveals that hawkmoths are unique among Lepidoptera and highlights the importance of considering the physical characteristics of the proboscis to understand the evolution and diversification of hawkmoths.

Standard- and large-sized eggs of Trichuris trichiura in the feces of schoolchildren in the Yangon Region, Myanmar: Morphological and molecular analyses.

Standard- and large-sized eggs of Trichuris trichiura were found in the feces of schoolchildren in Yangon, Myanmar during epidemiological surveys and mass deworming with albendazole in 2017-2019. The standard-sized eggs were identified as those of T. trichiura, but it was necessary to exclude the possibility of the large-sized eggs belonging to Trichuris vulpis, a dog whipworm. We conducted morphological and molecular studies to determine the species of the 2 types of Trichuris eggs. Individual eggs of both sizes were isolated from Kato-Katz fecal smears (n=20) and mechanically destroyed using a 23G injection needle. Nuclear DNA was extracted, and the 18S rRNA region was sequenced in 15 standard-sized eggs and 15 large-sized eggs. The average size of standard-sized eggs (T. trichiura) was 55.2×26.1 µm (range: 51.7-57.6×21.3-28.0 µm; n=97), whereas the size of large-sized eggs was 69.3×32.0 µm (range: 65.1-76.4×30.1-34.5 µm; n=20), slightly smaller than the known size of T. vulpis. Regarding standard-sized eggs, the 18S rRNA nucleotide sequences exhibited 100% homology with T. trichiura deposited in GenBank and 88.6-90.5% homology with T. vulpis. Regarding large-sized eggs, the nucleotide sequences showed 99.8-100% homology with T. trichiura in GenBank and 89.6-90.7% homology with T. vulpis. Both standard- and large-sized eggs of Trichuris spp. found in Myanmar schoolchildren during 2017-2019 were morphologically and molecularly confirmed to belong to T. trichiura. The conversion of eggs from smaller to large sizes might be due to anthelmintic treatments with albendazole.

Manduca sexta caterpillars parasitized by the wasp Cotesia congregata stop chewing despite an intact motor system.

The parasitic wasp Cotesia congregata suppresses feeding in its host, the caterpillar Manduca sexta, during specific periods of wasp development. We examined both feeding behaviour and the neurophysiology of the mandibular closer muscle in parasitized and unparasitized control M. sexta to determine how the wasp may accomplish this. To test whether the wasps activated a pre-existing host mechanism for feeding cessation, we examined the microstructure of feeding behaviour in caterpillars that stopped feeding due to illness-induced anorexia or an impending moult. These microstructures were compared with that shown by parasitized caterpillars. While there were overall differences between parasitized and unparasitized caterpillars, the groups showed similar progression in feeding microstructure as feeding ended, suggesting a common pattern for terminating a meal. Parasitized caterpillars also consumed less leaf area in 100 bites than control caterpillars at around the same time their feeding microstructure changed. The decline in food consumption was accompanied by fewer spikes per burst and shorter burst durations in chewing muscle electromyograms. Similar extracellular results were obtained from the motorneuron of the mandibular closer muscle. However, chewing was dramatically re-activated in non-feeding parasitized caterpillars if the connectives posterior to the suboesophageal ganglion were severed. The same result was observed in unparasitized caterpillars given the same treatment. Our results suggest that the reduced feeding in parasitized caterpillars is not due to damage to the central pattern generator (CPG) for chewing, motor nerves or chewing muscles, but is more likely to be due to a suppression of chewing CPG activity by ascending or descending inputs.

3D reconstruction of larval and adult brain neuropils of two giant silk moth species: Hyalophora cecropia and Antheraea pernyi.

We present a detailed analysis of the brain anatomy of two saturniid species, the cecropia silk moth, Hyalophora cecropia, and the Chinese oak silk moth, Antheraea pernyi, including 3D reconstructions of the major brain neuropils in the larva and in male and female adults. The 3D reconstructions, prepared from high-resolution optical sections, showed that the corresponding neuropils of these saturniid species are virtually identical. Similarities between the two species include a pronounced sexual dimorphism in the adults in the form of a male-specific assembly of markedly enlarged glomeruli forming the so-called macroglomerular complex. From the reports published to date, it can be concluded that the neuropil architecture of saturniids resembles that of other nocturnal moths, including the sibling family Sphingidae. In addition, compared with previous anatomical data on diurnal lepidopteran species, significant differences were observed in the two saturniid species, which include the thickness of the Y-tract of the mushroom body, the size of the main neuropils of the optic lobes, and the sexual dimorphisms of the antennal lobes.

An information theoretic method to resolve millisecond-scale spike timing precision in a comprehensive motor program.

Sensory inputs in nervous systems are often encoded at the millisecond scale in a precise spike timing code. There is now growing evidence in behaviors ranging from slow breathing to rapid flight for the prevalence of precise timing encoding in motor systems. Despite this, we largely do not know at what scale timing matters in these circuits due to the difficulty of recording a complete set of spike-resolved motor signals and assessing spike timing precision for encoding continuous motor signals. We also do not know if the precision scale varies depending on the functional role of different motor units. We introduce a method to estimate spike timing precision in motor circuits using continuous MI estimation at increasing levels of added uniform noise. This method can assess spike timing precision at fine scales for encoding rich motor output variation. We demonstrate the advantages of this approach compared to a previously established discrete information theoretic method of assessing spike timing precision. We use this method to analyze the precision in a nearly complete, spike resolved recording of the 10 primary wing muscles control flight in an agile hawk moth, Manduca sexta. Tethered moths visually tracked a robotic flower producing a range of turning (yaw) torques. We know that all 10 muscles in this motor program encode the majority of information about yaw torque in spike timings, but we do not know whether individual muscles encode motor information at different levels of precision. We demonstrate that the scale of temporal precision in all motor units in this insect flight circuit is at the sub-millisecond or millisecond-scale, with variation in precision scale present between muscle types. This method can be applied broadly to estimate spike timing precision in sensory and motor circuits in both invertebrates and vertebrates.

A salivary GMC oxidoreductase of Manduca sexta re-arranges the green leaf volatile profile of its host plant.

Green leaf volatiles (GLVs) are short-chain oxylipins that are emitted from plants in response to stress. Previous studies have shown that oral secretions (OS) of the tobacco hornworm Manduca sexta, introduced into plant wounds during feeding, catalyze the re-arrangement of GLVs from Z-3- to E-2-isomers. This change in the volatile signal however is bittersweet for the insect as it can be used by their natural enemies, as a prey location cue. Here we show that (3Z):(2E)-hexenal isomerase (Hi-1) in M. sexta's OS catalyzes the conversion of the GLV Z-3-hexenal to E-2-hexenal. Hi-1 mutants that were raised on a GLV-free diet showed developmental disorders, indicating that Hi-1 also metabolizes other substrates important for the insect's development. Phylogenetic analysis placed Hi-1 within the GMCß-subfamily and showed that Hi-1 homologs from other lepidopterans could catalyze similar reactions. Our results indicate that Hi-1 not only modulates the plant's GLV-bouquet but also functions in insect development.

Muscle in the caterpillar Manduca sexta responds to an immune challenge, but at a cost, suggesting a physiological trade-off.

Although skeletal muscle is a specialized tissue that provides the motor for movement, it also participates in other functions, including the immune response. However, little is known about the effects of this multitasking on muscle. We show that muscle loses some of its capacity while it is participating in the immune response. Caterpillars (Manduca sexta) were exposed to an immune challenge, predator stress or a combination of immune challenge and predator stress. The expression of immune genes (toll-1, domeless, cactus, tube and attacin) increased in body wall muscle after exposure to an immune challenge. Muscle also showed a reduction in the amount of the energy storage molecule glycogen. During an immune challenge, the force of the defensive strike, an important anti-predator behaviour in M. sexta, was reduced. Caterpillars were also less able to defend themselves against a common enemy, the wasp Cotesia congregata, suggesting that the effect on muscle is biologically significant. Our results support the concept of an integrated defence system in which life-threatening events activate organism-wide responses. We suggest that increased mortality from predation is a non-immunological cost of infection in M. sexta. Our study also suggests that one reason non-immunological costs of infection exist is because of the participation of diverse organs, such as muscle, in immunity.