Artifical light at night triggers slight transcriptomic effects on melatonin signaling but not synthesis in tadpoles of two anuran species.

The worldwide expansion of artificial light at night (ALAN) is acknowledged as a threat to biodiversity through alterations of the natural photoperiod triggering the disruption of physiological functions. In vertebrates, melatonin production during the dark phase can be decreased or suppressed by nocturnal light as shown in many taxa. But the effect of ALAN at low intensity mimicking light pollution in peri-urban area has never been investigated in amphibians. We filled this gap by studying the impact of low ALAN levels on the expression of genes related to melatonin synthesis and signaling in two anurans (agile frog, Rana dalmatina, and common toad, Bufo bufo). Circadian expression of genes encoding enzymes catalyzing melatonin synthesis (aralkylamine N-acetyltransferase, AANAT and acetylserotonin O-methyltransferase, ASMT) or melatonin receptors (Mel1a, Mel1b and Mel1c) was investigated using RT-qPCR after 23 days of nocturnal exposure to control (< 0.01 lx) or low ALAN (3 lx). We showed that the relative abundance of most transcripts was low in late afternoon and early evening (06 pm and 08 pm) and increased throughout the night in R. dalmatina. However, a clear and ample nocturnal pattern of target gene expression was not detected in control tadpoles of both species. Surprisingly, a low ALAN level had little influence on the relative expression of most melatonin-related genes. Only Mel1c expression in R. dalmatina and Mel1b expression in B. bufo were affected by ALAN. This target gene approach provides experimental evidence that melatonin signaling pathway was slightly affected by low ALAN level in anuran tadpoles.

Artificial light at night decreases the pupillary light response of dark-adapted toads to bright light.

Artificial light at night (ALAN) is expanding worldwide. Many physiological effects have been reported in animals, but we still know little about the consequences for the visual system. The pupil contributes to control incoming light onto the retina. Sudden increases in light intensity evokes the pupil light reflex (PLR). Intrinsically photosensitive retinal ganglion cells (ipRGC) affect PLR and melatonin expression, which largely regulate circadian rhythms and PLR itself. IpRCG receive inputs from various photoreptors with different peak sensitivities implying that PLR could be altered by a broad range of light sources. We predicted ALAN to enhance PLR. Contrary to our prediction, dark-adapted cane toads Rhinella marina, exposed to ALAN (5 lx) for 12 days, exhibited a lower PLR than controls and individuals exposed to 0.04 lx, even after 1 h in bright light. We cannot conclude whether ALAN induced a larger pupil size in dark-adapted toads or a slower initial contraction. Nevertheless, the response was triggered by a light source with an emission peak (590 nm) well above the sensitivity peak of melanopsin, the main photoreceptor involved in PLR. Therefore, ALAN alters the capacity of toads to regulate the incoming light in the eye at night, which may reduce the performance of visually guided behaviors, and increase mortality by predators or road kills at night. This first study emphasizes the need to focus on the effect of ALAN on the vision of nocturnal organisms to better understand how this sensory system is altered and anticipate the consequences for organisms.

Discrimination of conspecifics from heterospecifics in a hybrid zone: Behavioral and chemical cues in ants.

Species and nestmate recognition in social insects occurs mostly through cuticular hydrocarbons acting as chemical cues. These compounds generate a colony-specific odor profile depending on genetic and environmental factors. Species and nestmate recognition results in specific behavioral responses, regulating the level of aggression toward other individuals during an interaction. Although species discrimination and recognition cues have been poorly studied in the context of interspecific hybridization, such systems offer an opportunity to further investigate the influence of heritable and environmental factors on recognition. We explored the strength of discrimination in a hybrid zone between two ant species-Tetramorium immigrans and T. caespitum-by comparing cuticular hydrocarbon profiles and measuring intra- and interspecific worker aggression in both areas of sympatry and areas of allopatry among species. Species cuticular hydrocarbon profiles were well-differentiated and interspecific aggression was high, revealing highly discriminating species recognition cues. Hybrids' cuticular hydrocarbon profiles consisted of a mixture of the parental bouquets, but also exhibited hybrid-specific patterns. Behavioral assays showed that T. immigrans is as aggressive toward hybrids as toward heterospecifics. Finally, aggression between heterospecific workers was lower when interacting individuals came from areas of sympatry among species than from areas of allopatry. Taken as a whole, these findings paint a particularly complex picture of the recognition system in T. immigrans, T. caespitum, and their hybrids, and highlight that hybrid zones afford a still underexplored opportunity for investigating recognition mechanisms and discrimination between species.

Transcriptome-wide deregulation of gene expression by artificial light at night in tadpoles of common toads.

Artificial light at night (ALAN) affects numerous physiological and behavioural mechanisms in various species by potentially disturbing circadian timekeeping systems and modifying melatonin levels. However, given the multiple direct and indirect effects of ALAN on organisms, large-scale transcriptomic approaches are essential to assess the global effect of ALAN on biological processes. Moreover, although studies have focused mainly on variations in gene expression during the night in the presence of ALAN, it is necessary to investigate the effect of ALAN on gene expression during the day. In this study, we combined de novo transcriptome sequencing and assembly, and a controlled laboratory experiment to evaluate the transcriptome-wide gene expression response using high-throughput (RNA-seq) in Bufo bufo tadpoles exposed to ecologically relevant light levels. Here, we demonstrated for the first time that ALAN affected gene expression at night (3.5% and 11% of differentially expressed genes when exposed to 0.1 and 5 lx compared to controls, respectively), but also during the day (11.2% of differentially expressed genes when exposed to 5 lx compared to controls) with a dose-dependent effect. ALAN globally induced a downregulation of genes (during the night, 58% and 62% of the genes were downregulated when exposed to 0.1 and 5 lx compared to controls, respectively, and during the day, 61.2% of the genes were downregulated when exposed to 5 lx compared to controls). ALAN effects were detected at very low levels of illuminance (0.1 lx) and affected mainly genes related to the innate immune system and, to a lesser extend to lipid metabolism. These results provide new insights into understanding the effects of ALAN on organism. ALAN impacted the expression of genes linked to a broad range of physiological pathways at very low levels of ALAN during night-time and during daytime, potentially resulting in reduced immune capacity under environmental immune challenges.

Ecdysteroids affect female reproductive status and outcome of contest over hosts in the parasitoid wasp Eupelmus vuilleti.

Ecdysteroids are a family of insect hormones that may play a role in modulating aggressive behavior in reproductive contexts. In Hymenoptera, the few studies investigating the link between ecdysteroid titers, reproduction and aggressiveness during contests concern solely eusocial species. Here, we explored whether ecdysteroid titers influenced female reproductive status as well as aggressiveness and resolution of conflict in a solitary ectoparasitoid, Eupelmus vuilleti (Hymenoptera: Eupelmidae). Eupelmus vuilleti females parasitize and feed upon juvenile stages of Callosobruchus maculatus (Coleoptera: Bruchidae). When two E. vuilleti females are simultaneously present on a patch, they tend to protect the host they exploit by displaying aggressive behaviors towards conspecific competitors. To our knowledge, nothing is known about the association between ecdysteroids and aggressiveness or the outcome of contests for host access in solitary insects. First, we quantified ecdysteroid titers that naturally circulate in females without fighting experience and after a contest over host access. Ecdysteroid titers measured after the contest did not correlate with female aggressiveness during the contest, but winner wasps had higher titers than both losers and females that did not fight. Then, we manipulated hormone titers via injection: ecdysone favored egg maturation (i.e., gonadotropic effect) within 24 h and increased almost immediately the females' probability of winning host access without affecting their aggressiveness. Our results represent an important step in understanding how hormones, such as ecdysteroids, mediate insect behavior during intraspecific competition.

Artificial light at night alters the sexual behaviour and fertilisation success of the common toad.

Artificial Light At Night (ALAN) is an emerging pollution, that dramatically keeps on increasing worldwide due to urbanisation and transport infrastructure development. In 2016, it nearly affected 23% of the Earth's surface. To date, all terrestrial and aquatic ecosystems have been affected. The disruption of natural light cycles due to ALAN is particularly expected for nocturnal species, which require dark periods to forage, move, and reproduce. Apart from chiropterans, amphibians contain the largest proportion of nocturnal species among vertebrates exhibiting an unfavourable conservation status in most parts of the world and living in ALAN polluted areas. Despite the growing number of studies on this subject, our knowledge on the direct influence of nocturnal lighting on amphibians is still scarce. To better understand the consequences of ALAN on the breeding component of amphibian fitness, we experimentally exposed male breeding common toads (Bufo bufo) to ecologically relevant light intensities of 0.01 (control), 0.1 or 5 lux for 12 days. At mating, exposed males took longer than controls to form an amplexus, i.e. to pair with a female, and broke amplexus before egg laying, while controls never did. These behavioural changes were associated with fitness alteration. The fertilisation rate of 5 lux-exposed males was reduced by 25%. Salivary testosterone, which is usually correlated with reproductive behaviours, was not altered by ALAN. Our study demonstrates that ALAN can affect the breeding behaviour of anuran species and reduce one component of their fitness. Given the growing importance of ALAN, more work is needed to understand its long-term consequences on the behaviour and physiology of individuals. It appears essential to identify deleterious effects for animal populations and propose appropriate management solutions in an increasingly brighter world.

The role of associative learning process on the response of fledgling great tits (Parus major) to mobbing calls.

When they detect a predator, many species emit anti-predator vocalizations. In some cases, they emit mobbing calls, which are associated with the caller approaching and harassing the predator while attracting others to join it. Surprisingly, although mobbing has been widely reported in adults of numerous species, there has been no test of the role of learning in mobbing call recognition, especially during ontogeny. Here, we exposed wild great tit (Parus major) nestlings to playbacks of an unthreatening novel sound either associated with conspecific mobbing calls (experimental treatment) or with another unthreatening novel sound (control treatment). We then tested them as nestlings and fledglings to see how they respond to the novel sound compared to conspecific mobbing calls. Results revealed that fledglings in the experimental treatment behaved similarly to conspecific mobbing calls and the novel sound associated with conspecific mobbing calls. Because mobbing efficiency is often linked to interspecific communication, associative learning should be used by heterospecifics as mobbing calls recognition mechanism. Regardless of treatment during the nestling phase, fledglings always were sensitive to the playback of conspecific mobbing calls. However, fledglings from the control group were more likely to approach the loudspeaker than those from the experimental group when mobbing calls were played suggesting that overexposure during the nestling phase altered mobbing learning. Overall, these results suggest that learning could play a role in the recognition of calls, like heterospecific mobbing calls, when paired with conspecific mobbing, and that mobbing is perceived as a threatening stimulus from a very young age.

Artificial light at night disturbs the activity and energy allocation of the common toad during the breeding period.

The presence of artificial light at night (ALAN) is currently a global phenomenon. By altering the photoperiod, ALAN may directly affect the physiology and behaviour of many organisms, such as the timing of daily rhythms, hormonal regulation, food intake, metabolism, migration and reproduction. Surprisingly while it is known that ALAN exposure strongly influences health of humans and laboratory animals, studies on wildlife remain scarce. Amphibians are one of the most nocturnal groups of vertebrates and exhibit an unfavourable conservation status in most parts of the world. In order to gain insight into the consequences of ALAN, we experimentally exposed 36 adult breeding male common toads, Bufo bufo, to a light intensity of 0.1, 5 or 20 lux for 20 days, to investigate the activity using infrared cameras and the whole-body oxygen consumption by respirometry, as well as body mass and food intake. ALAN reduced toad activity over 24 h by 56% at 5 lux and by 73% at 20 lux. It did not affect the total energy expenditure but altered energy allocation. Indeed, standard energy expenditure increased by 28% at 5 lux and by 58% at 20 lux, while activity energy expenditure decreased by 18% at 5 lux and 38% at 20 lux. Finally, body mass and food intake were not affected. This study suggests that ALAN plays a large role in the activity and energy metabolism of common toads, which may have a long-term negative effect on the fitness of common toad populations. Generalizing these results to other taxa is crucial for conservation of biodiversity in an increasingly light world.

Variability of surface and underwater nocturnal spectral irradiance with the presence of clouds in urban and peri-urban wetlands.

Artificial light at night (ALAN) is an increasing phenomenon worldwide. It causes a wealth of biological and ecological effects that may eventually affect populations and ecosystems. Despite the growing concern about ALAN, little is known about the light levels species are exposed to at night, especially for wetlands and underwater habitats. We determined nocturnal irradiance in urban and peri-urban wetlands above and under water, and assessed the effect of cloud cover on the variability of ALAN across the urban gradient. Even in aquatic habitats, cloud cover could increase irradiance beyond values observed during clear full moon nights. We report a negative relationship between baseline irradiance and the increase in irradiance during overcast nights. According to this result and previous studies, we propose that the change in the variation regime of ALAN between the urban center and rural land at its periphery is a usual feature. We discuss the ecological and evolutionary implications of this spatial variation in the urban and peri-urban environment.

Doping for sex: Bad for mitochondrial performances? Case of testosterone supplemented Hyla arborea during the courtship period.

Sexual selection has been widely explored from numerous perspectives, including behavior, ecology, and to a lesser extent, energetics. Hormones, and specifically androgens such as testosterone, are known to trigger sexual behaviors. Their effects are therefore of interest during the breeding period. Our work investigates the effect of testosterone on the relationship between cellular bioenergetics and contractile properties of two skeletal muscles involved in sexual selection in tree frogs. Calling and locomotor abilities are considered evidence of good condition in Hyla males, and thus server as proxies for male quality and attractiveness. Therefore, how these behaviors are powered efficiently remains of both physiological and behavioral interest. Most previous research, however, has focused primarily on biomechanics, contractile properties or mitochondrial enzyme activities. Some have tried to establish a relationship between those parameters but to our knowledge, there is no study examining muscle fiber bioenergetics in Hyla arborea. Using chronic testosterone supplementation and through an integrative study combining fiber bioenergetics and contractile properties, we compared sexually dimorphic trunk muscles directly linked to chronic sound production to a hindlimb muscle (i.e. gastrocnemius) that is particularly adapted for explosive movement. As expected, trunk muscle bioenergetics were more affected by testosterone than gastrocnemius muscle. Our study also underlines contrasted energetic capacities between muscles, in line with contractile properties of these two different muscle phenotypes. The discrepancy of both substrate utilization and contractile properties is consistent with the specific role of each muscle and our results are elucidating another integrative example of a muscle force-endurance trade-off.

Immune challenges and visual signalling in tree frogs.

In animals, mate-choice is often based on sexual signals that carry information and help the receiver make the best choice to improve the receiver's fitness. Orange visual sexual signals have been hypothesised to carry immune information because they are often due to carotenoid pigments which are also involved in immunity response. Although many studies have focused on the direct relationships between coloration and immunocompetence, few studies have simultaneously studied immunocompetent response and coloration variation after an immune challenge. We tested this hypothesis on starved and ad libitum-fed males of the European tree frog Hyla arborea. Our results show that male coloration is not a reliable indicator of its immune response capacity in this species. However, after an immune challenge induced by a PHA (Phaseolus vulgaris phytohaemagglutinin) injection, starved males presented a significant coloration loss and this alteration was related to the immune response intensity. Taken together, these results suggest that the brighter (lighter) coloration may be used as a cue by female to exclude males with a recent immune challenge, due to diseases or parasites for example.

Does testosterone affect foraging behavior in male frogs?

During the breeding season, males often produce costly and extravagant displays or physical ornaments to attract females. Numerous studies have established that testosterone could directly influence the expression of certain sexual signals. However, few of these studies have focused on the indirect role that testosterone could play in modulating prey detection and visual performance to improve the foraging ability of males and hence their acquisition of nutritional resource. In the present study, we experimentally modified the testosterone levels of European tree frog males (Hyla arborea), staying in the natural range previously measured in the field, and we investigated the effect of testosterone on the foraging ability of individuals. Foraging capacities were measured on males placed in an arena with a virtual cricket moving on a computer screen. Our results demonstrated a significant effect of testosterone on the hunting behavior of H. arborea. We observed that testosterone reduced the orientation latency to virtual prey for supplemented males compared to controls. In addition, testosterone significantly increased the attack promptness of male frogs. Finally, our experiment did not demonstrate any impact of testosterone on male attack success.

Effects of traffic noise on tree frog stress levels, immunity, and color signaling.

During the last decade, many studies have focused on the detrimental effects of noise pollution on acoustic communication. Surprisingly, although it is known that noise exposure strongly influences health in humans, studies on wildlife remain scarce. In order to gain insight into the consequences of traffic noise exposure, we experimentally manipulated traffic noise exposure as well as the endocrine status of animals to investigate physiological and phenotypic consequences of noise pollution in an anuran species. We showed that noise exposure increased stress hormone level and induced an immunosuppressive effect. In addition, both traffic noise exposure and stress hormone application negatively impacted H. arborea vocal sac coloration. Moreover, our results suggest profound changes in sexual selection processes because the best quality males with initial attractive vocal sac coloration were the most impacted by noise. Hence, our study suggests that the recent increases in anthropogenic noise worldwide might affect a broader range of animal species than previously thought, because of alteration of visual signals and immunity. Generalizing these results to other taxa is crucial for the conservation of biodiversity in an increasingly noisy world.

All or nothing: Survival, reproduction and oxidative balance in Spotted Wing Drosophila (Drosophila suzukii) in response to cold.

Winter severity and overwintering capacity are key ecological factors in successful invasions, especially in ectotherms. The integration of physiological approaches into the study of invasion processes is emerging and promising. Physiological information describes the mechanisms underlying observed survival and reproductive capacities, and it can be used to predict an organism's response to environmental perturbations such as cold temperatures. We investigated the effects of various cold treatments on life history and physiological traits of an invasive pest species, Drosophila suzukii, such as survival, fertility and oxidative balance. This species, a native of temperate Asian areas, is known to survive where cold temperatures are particularly harsh and has been recently introduced into Europe and North America. We found that cold treatments had a strong impact on adult survival but no effect on female's fertility. Although only minor changes were observed after cold treatment on studied physiological traits, a strong sex-based difference was observed in both survival and physiological markers (antioxidant defences and oxidative markers). Females exhibited higher survival, reduced oxidative defences, less damage to nucleic acids, and more damage to lipids. These results suggest that D. suzukii relies on a pathway other than oxidative balance to resist cold injury. Altogether, our results provide information concerning the mechanisms of successful invasion by D. suzukii. These findings may assist in the development of population models that predict the current and future geographic ranges of this species.

Impact of cadmium on the ecdysteroids production in Gammarus fossarum.

Gammarus fossarum is an important test organism which is currently used as a bio-indicator as well as in ecotoxicological tests. Nevertheless, data on ecdysteroids in endocrine toxicity test are not yet available for these species, despite its crucial role in molting and reproduction. In the present paper, ecdysteroids concentrations were studied during the molt cycle (in females) and embryonic development in G. fossarum (Crustacea, Amphipoda) in order to propose an ecdysteroids toxicity test. Ecdysteroids levels in G. fossarum showed a single peak during premolt at stage Dl-D2. In embryos, ecdysteroids levels progressively increased over stages 3 and 4, with peak levels at stage 4. A Cadmium toxicity test was proposed to examine if the molting and embryogenesis disturbances previously observed after cadmium exposure (Geffard et al. 2010) could be attributed to changes in ecdysteroids titers. Exposure to the different cadmium concentrations (3; 9; 300; 900 µg/l) increased ecdysteroids secretion by Y-organs in vitro, but it had no significant effect on exposed embryos (in vivo). Based on previous findings, we are led to conclude that the molting impairments in cadmium-exposed females of G. fossarum is connected to the changes in ecdysteroids concentrations.

The energetic costs of case construction in the caddisfly Limnephilus rhombicus: direct impacts on larvae and delayed impacts on adults.

Caddisflies, whose aquatic larvae build a portable case with silk, are a suitable model organism to test the impacts of resource allocation trade-off during development and examine the evolution of life-history strategies. In the caddisfly Limnephilus rhombicus, adult feeding is minimal. Therefore, the whole resources are acquired during the larval phase and must be allocated to case construction, growth and reproduction. In this study, the larval energetic reserves of L. rhombicus were manipulated by forcing larvae to rebuild their cases in the final larval stage. This allowed us to measure the physiological cost of construction. First, we recorded oxygen consumption during case reconstruction. Second, we measured the sugar, protein and lipid contents of larvae forced to rebuild their case and of larvae required only to re-enter on their case. Larvae had their sugar, protein and lipid content measured after the rebuilding event and 72 h later. The same analyses were carried out with adults immediately after emergence. We found that larvae forced to rebuild a case consumed 1.5 times more oxygen than control larvae. This energy expenditure generated a cost that was estimated to be a loss of larval protein of approximately 35%. Insects were unable to compensate for this loss of proteins during the end of the larval stage, and their metamorphosis to adults was also impacted. Therefore, we suggest that loss of larval protein is linked to silk production and may alter fitness.

Reproduction in a variable environment: How does Eupelmus vuilleti, a parasitoid wasp, adjust oogenesis to host availability?

Oogenesis of the parasitoid wasp Eupelmus vuilleti is known to be dependent on host availability. However, examination of ovarian dynamics by microscopy showed that oogenesis and vitellogenesis are initiated before female eclosion and proceed 1-2 days after, independent of host presence. Oogenesis continued beyond the 2nd day only in the presence of hosts, otherwise it was replaced by egg resorption. It is thus possible to distinguish between host-independent and host-dependent periods of oogenesis. In the presence of host, each ovariole (three per ovary) contained generally three oocytes: a fully mature oocyte, a nearly mature one and an immature one. However, host deprived-females resorbed their most mature and their smallest oocytes, but kept one almost mature oocyte per ovariole. Comparison of zero, short and long host deprivation periods showed that females always had the ability to quickly lay eggs to exploit any new host. However, increased deprivation led to a reduction in the number and the viability of eggs. Enzymo-immunological measurements of ecdysteroids were made in whole females, in dissected ovaries and in newly laid eggs. Our results indicated that ecdysteroids play a major role as circulating hormones involved in the regulation of oogenesis.

Host-induced ecdysteroids in the stop-and-go oogenesis in a synovigenic parasitoid wasp.

Eupelmus vuilleti (Hymenoptera; Eupelmidae) is a solitary ectoparasitoid producing yolk-rich eggs. The female oviposits mainly on the fourth larval instar of Callosobruchus maculatus (Coleoptera; Bruchidae), which develop within pods and seeds of Vigna unguiculata (Fabacae). Parasitoid females are synovigenic, i.e., they are born with immature eggs and need to feed from the host to sustain egg production during their entire lifetime. However, eggs are rapidly resorbed in unfavourable conditions and an efficient stop-and-go mechanism controls oogenesis in such animals. In this study, the possible involvement of ecdysteroids in the regulation of parasitoid oogenesis is examined. In a first step, the identity and titre of ecdysteroids in reproductively active and inactive female parasitoids were investigated by high performance liquid chromatography followed by enzyme immuno-assay (EIA/HPLC). A larger secretion of ecdysone was found in female during their reproductive period compared with inactive females. In a second step, both the secretion of ecdysteroids into the medium of in vitro incubated ovaries and the ecdysteroid content of females reared with or without host were measured (EIA). The presence of the host, which represents both the oviposition site and the nutritional source, induced an active biosynthesis of ecdysone. This synthesis started at a slow rate after host introduction and reached a maximum after 48 h. When hosts were available, this synthesis was cyclic and continuous during the entire female lifetime. These results showed that host presence triggered ovarian synthesis of ecdysteroids, which are involved in a stop-and-go regulation of egg production linked to host availability.

Importance of sterols acquired through host feeding in synovigenic parasitoid oogenesis.

Eupelmus vuilleti (Hymenoptera; Eupelmidae) is a host feeding ectoparasitoid of fourth-instar larvae or pupae of Callosobruchus maculatus (Coleoptera; Bruchidae) infecting Vigna unguiculata seed and pods (Fabacae). Parasitoid females are synovigenic, i.e. they are born with immature eggs and need to feed from the host in order to sustain egg production. In this study, the role of sterols obtained through host feeding in parasitoid oogenesis are examined. Quantitative and qualitative analyses of the sterol contents in each partner of the tritrophic interaction show that a parasitoid female's larval sterol contents is sufficient to produce only 30% of the total number of eggs laid throughout a female's life cycle. In a second step, by manipulating the composition of the sterols hemolymph in the host, it is shown that cholesterol obtained through adult nutrition plays a crucial role in the eggs viability but does not affect the egg production quantitatively. This result has important implications for understanding both the nutrient allocation strategy in this species and the impact of cholesterol in parasitoid reproduction.

Increased sulfur precursors and volatiles production by the leek Allium porrum in response to specialist insect attack.

To defend themselves against herbivory, plants use a variety of direct and indirect strategies involving induced increases in secondary substances. Species of the Allium genus (Alliaceae), such as the leek Allium porrum (L.), produce nonprotein sulfur amino acids derived from cysteine, i.e., alk(en)yl-cysteine sulfoxides that are precursors of volatile thiosulfinates and disulfides. These defend most species including the specialist leek moth, Acrolepiopsis assectella. We determined by measuring the increase in the sulfur precursor propyl-cysteine sulfoxide (PCSO) if production of this precursor is induced in response to moth attack and mechanical wounding. The concentration of PCSO was determined by HPLC in 2- or 6-mo-old leeks after attacks of various intensity either by the specialist leek moth or by a generalist moth, Agrotis ipsilon. Injury-induced release of sulfur volatiles was measured by GC/MS after the attacks. Results showed an increase in the production of sulfur compounds in both the precursor and volatile form, occurring only in association with intensive attacks by leek moths. The increase in sulfur precursors also led to an increase in the release of sulfur volatiles. This induced response may provide an effective defense strategy against the plant's main natural enemy, both directly and indirectly by attracting entomophagous insects.