Daily temperature fluctuation interacts with the mean temperature to increase the toxicity of a pyrethroid insecticide in a moth.

Climate change complicates ecotoxicology studies because species responses to pesticides depend on temperature. Classically illustrated by the effect of constant laboratory temperatures, a recent review revealed that the toxicity of pesticides is also often increased by daily temperature fluctuations. Here, we investigated the combined effects of daily temperature fluctuation and mean temperature on the toxicity of two insecticides in the moth Spodoptera littoralis. Our study tested the toxicity of chlorpyrifos and deltamethrin on larvae of six experimental groups that crossed three treatments of daily temperature fluctuations (0, 5 or 10 °C) and two treatments of mean temperatures (25 or 33 °C). We showed that daily temperature fluctuation increased larval mortality induced by chlorpyrifos and deltamethrin. However, the response differed between the organophosphorus insecticide chlorpyrifos and the pyrethroid insecticide deltamethrin. The increase in chlorpyrifos toxicity by daily temperature fluctuation did not differ between mean temperatures of 25 and 33 °C. Remarkably, the increase in deltamethrin toxicity by daily temperature fluctuation was dependent on the crossed effects of the amplitude of daily fluctuation and mean temperature. This increase in deltamethrin toxicity occurred with a daily fluctuation of only 5 °C for larvae reared at 25 °C and a daily fluctuation of 10 °C in larvae reared at 33 °C. To confidently quantify the responses of insecticide toxicity to temperature, future ecotoxicology studies will have to evaluate the generality of the interaction between the effects of daily temperature fluctuation and mean temperature.

Fitness under high temperatures is overestimated when daily thermal fluctuation is ignored.

Experimental studies on the thermal biology of organisms have become crucial to investigate the impact of climate warming. However, most laboratory studies are carried out under constant temperatures and assume a negligible effect from daily fluctuating temperatures. We tested this assumption on multiple fitness traits of the moth Spodoptera littoralis, and a literature review on insects complements this study. Tests on S. littoralis focused on its optimal and maximal critical temperatures by comparing constant and daily fluctuating temperatures (±5 °C) at mean temperatures of 25, 29 and 33 °C. The nine fitness parameters investigated were influenced by mean temperature. The overall effect was a maximal multiplication rate at 29 °C and a marked decrease under the fluctuating regime at 33 °C. Effects of fluctuating temperatures differed between mean temperatures. Developmental and larval survival rates at 33 °C were lower under the fluctuating thermal regime than under a constant temperature. Our literature review also illustrates that ignoring daily fluctuations based on constant temperatures commonly leads to overestimate fitness traits at high temperatures. Overlooking the experimental bias associated with constant temperatures minimizes the expected impact of climate warming on fitness traits.

Diet impacts the reproductive system's maturation in the male moth Agrotis ipsilon (Noctuidae, Lepidoptera).

In male moth Agrotis ipsilon, sexual maturation occurs between the third and the fifth day of adult life and is characterized by the development of the reproductive organs such as testes and accessory sex glands. Since sexual maturation requires considerable energy investment, we hypothesized that diet would be an essential regulatory factor in this developmental process. Indeed, the links between the male diet and reproductive physiology have not been described as in females. To test the previous hypothesis, we offered male moths diets corresponding to different flower nectars found in nature, and measured morphological and functional changes in the testes and accessory sex glands. In comparison to a diet composed of sucrose only, males fed with a diet composed of diverse sugars, including glucose, supplemented with sodium led to an earlier increase in the length and the protein content of accessory sex glands, as well as a reduction of the testicular volume accompanied by an acceleration of the sperm bundle transfer from the testes to the duplex. These results show that these specific diets accelerate the maturation of the reproductive system in male moth Agrotis ipsilon.

Methoprene-tolerant and Krüppel homolog 1 are actors of juvenile hormone-signaling controlling the development of male sexual behavior in the moth Agrotis ipsilon.

In insects, juvenile hormone (JH) is critical for the orchestration of male reproductive maturation. For instance, in the male moth, Agrotis ipsilon, the behavioral response and the neuronal sensitivity within the primary olfactory centers, the antennal lobes (ALs), to the female-emitted sex pheromone increase with fertility during adulthood and the coordination between these events is governed by JH. However, the molecular basis of JH action in the development of sexual behavior remains largely unknown. Here, we show that the expression of the paralogous JH receptors, Methoprene-tolerant 1 and 2 (Met1, Met2) and of the JH-inducible transcription factor, Krüppel homolog 1 (Kr-h1) within ALs raised from the third day of adult life and this dynamic is correlated with increased behavioral responsiveness to sex pheromone. Met1-, Met2- and Kr-h1-depleted sexually mature males exhibited altered sex pheromone-guided orientation flight. Moreover, injection of JH-II into young males enhanced the behavioral response to sex pheromone with increased AL Met1, Met2 and Kr-h1 mRNA levels. By contrast, JH deficiency suppressed the behavioral response to sex pheromone coupled with reduced AL Met1, Met2 and Kr-h1 mRNA levels in allatectomized old males and these inhibitions were compensated by an injection of JH-II in operated males. Our results demonstrated that JH acts through Met-Kr-h1 signaling pathway operating in ALs, to promote the pheromone information processing and consequently the display of sexual behavior in synchronization with fertility to optimize male reproductive fitness. Thus, this study provides insights into the molecular mechanisms underlying the hormonal regulation of reproductive behavior in insects.

The impact of temperature on insecticide sensitivity depends on transgenerational effects.

The combined effects of insecticides and temperature are increasingly being studied because species are expected to change their responses to insecticides with climate warming. As recently highlighted, the impact of temperature on insecticide sensitivity might be influenced by the environment experienced by the previous generation. However, a pioneering study that showed this transgenerational effect in the mosquito Culex pipiens needs to be confirmed because two other studies did not show similar results. Here, we performed an experiment on the moth Spodoptera littoralis to test this hypothesis. We analysed reaction norms among experimental families to test transgenerational effects, i.e., the variation in the response of families to the combined effects of the insecticide chlorpyrifos and developmental temperature. Reaction norm analyses revealed that the responses of the families to chlorpyrifos and temperature differed for developmental time and larval survival, two key parameters in S. littoralis. Crucially, for larval survival, a family effect influenced the impact of temperature on chlorpyrifos sensitivity. This finding confirms the pioneering study on C. pipiens that showed transgenerational effects on the combined effects of insecticides and temperature. This result also highlights that transgenerational plasticity can be important to consider in ecotoxicology.

Combined influences of transgenerational effects, temperature and insecticide on the moth Spodoptera littoralis.

Climate warming is expected to impact the response of species to insecticides. Recent studies show that this interaction between insecticides and temperature can depend on other factors. Here, we tested for the influence of transgenerational effects on the Insecticide × Temperature interaction in the crop pest moth Spodoptera littoralis. Specifically, we analysed reaction norms among experimental clutches based on a split-plot design crossing the factors temperature, insecticide and clutch. The study was performed on 2280 larvae reared at four temperatures (23, 25, 27 and 29 °C), and their response to the insecticide deltamethrin (three concentrations and a control group) was tested. Temperature had a global influence with effects on larval survival, duration of development, pupal body mass, and significant reaction norms of the clutches for temperature variations of only 2 °C. In addition to the expected effect of deltamethrin on mortality, the insecticide slightly delayed the development of S. littoralis, and the effects on mortality and development differed among the clutches. Projection models integrating all the observed responses illustrated the additive effects of deltamethrin and temperature on the population multiplication rate. Variation in the response of the clutches showed that transgenerational effects influenced the impact of insecticide and temperature. Although no evidence indicated that the Insecticide × Temperature interaction depended on transgenerational effects, the studies on the dependence of the Insecticide × Temperature interaction on other factors continue to be crucial to confidently predict the combined effects of insecticides and climate warming.

Involvement of Methoprene-tolerant and Krüppel homolog 1 in juvenile hormone-signaling regulating the maturation of male accessory glands in the moth Agrotis ipsilon.

Male accessory glands (MAGs) produce seminal fluid proteins that are essential for the fertility and also influence the reproductive physiology and behavior of mated females. In many insect species, and especially in the moth Agrotis ipsilon, juvenile hormone (JH) promotes the maturation of the MAGs but the underlying molecular mechanisms in this hormonal regulation are not yet well identified. Here, we examined the role of the JH receptor, Methoprene-tolerant (Met) and the JH-inducible transcription factor, Krüppel homolog 1 (Kr-h1) in transmitting the JH signal that upregulates the growth and synthetic activity of the MAGs in A. ipsilon. We cloned two full length cDNAs encoding Met1 and Met2 which are co-expressed with Kr-h1 in the MAGs where their expression levels increase with age in parallel with the length and protein content of the MAGs. RNAi-mediated knockdown of either Met1, Met2, or Kr-h1 resulted in reduced MAG length and protein amount. Moreover, injection of JH-II into newly emerged adult males induced the transcription of Met1, Met2 and Kr-h1 associated to an increase in the length and protein content of the MAGs. By contrast, JH deficiency decreased Met1, Met2 and Kr-h1 mRNA levels as well as the length and protein reserves of the MAGs of allatectomized old males and these declines were partly compensated by a combined injection of JH-II in operated males. Taken together, our results highlighted an involvement of the JH-Met-Kr-h1 signaling pathway in the development and secretory activity of the MAGs in A. ipsilon.

A maternal effect influences sensitivity to chlorpyrifos pesticide in the pest moth Spodoptera littoralis.

Transgenerational effects on sensitivity to pesticides are poorly studied. This study investigated the transgenerational influences of maternal body mass in the major pest moth Spodoptera littoralis, with a focus on sensitivity to chlorpyrifos pesticide. In 147 clutches of a laboratory strain of S. littoralis, we compared larval mortality between control larvae and larvae treated with chlorpyrifos. Because of the classic positive relationships between offspring size and maternal size and between offspring size and offspring quality, sensitivity to chlorpyrifos was predicted to be lower in larvae of larger mothers. Surprisingly, we found the opposite result, with higher pesticide toxicity in larvae of larger mothers. This result is partly explained by the lack of a relationship between larval mass and larval sensitivity to chlorpyrifos. This means that another offspring characteristic linked to maternal size should have affected larval sensitivity to chlorpyrifos. More generally, knowledge of the effects of the traits and ecological environments of mothers on offspring sensitivity to pesticides remains limited. Ecotoxicologists should pay more attention to such maternal effects on sensitivity to pesticides, both in pests and non-target species.

Responsiveness to Sugar Solutions in the Moth Agrotis ipsilon: Parameters Affecting Proboscis Extension.

Adult moths need energy and nutrients for reproducing and obtain them mainly by consuming flower nectar (a solution of sugars and other compounds). Gustatory perception gives them information on the plants they feed on. Feeding and food perception are integrated in the proboscis extension response, which occurs when their antennae touch a sugar solution. We took advantage of this reflex to explore moth sugar responsiveness depending on different parameters (i.e., sex, age, satiety, site of presentation, and composition of the solution). We observed that starvation but not age induced higher response rates to sucrose. Presentation of sucrose solutions in a randomized order confirmed that repeated sugar stimulations did not affect the response rate; however, animals were sometimes sensitized to water, indicating sucrose presentation might induce non-associative plasticity. Leg stimulation was much less efficient than antennal stimulation to elicit a response. Quinine prevented and terminated sucrose-elicited proboscis extension. Males but not females responded slightly more to sucrose than to fructose. Animals of either sex rarely reacted to glucose, but curiously, mixtures in which half sucrose or fructose were replaced by glucose elicited the same response rate than sucrose or fructose alone. Fructose synergized the response when mixed with sucrose in male but not female moths. This is consistent with the fact that nectars consumed by moths in nature are mixtures of these three sugars, which suggests an adaptation to nectar perception.

Exposure to Conspecific and Heterospecific Sex-Pheromones Modulates Gustatory Habituation in the Moth Agrotis ipsilon.

In several insects, sex-pheromones are essential for reproduction and reproductive isolation. Pheromones generally elicit stereotyped behaviors. In moths, these are attraction to conspecific sex-pheromone sources and deterrence for heterospecific sex-pheromone. Contrasting with these innate behaviors, some results in social insects point toward effects of non-sex-pheromones on perception and learning. We report the effects of sex-pheromone pre-exposure on gustatory perception and habituation (a non-associative learning) in male Agrotis ipsilon moths, a non-social insect. We also studied the effect of Z5-decenyl acetate (Z5), a compound of the sex-pheromone of the related species Agrotis segetum. We hypothesized that conspecific sex-pheromone and Z5 would have opposite effects. Pre-exposure to either the conspecific sex-pheromone or Z5 lasted 15 min and was done either immediately or 24 h before the experiments, using their solvent alone (hexane) as control. In a sucrose responsiveness assay, pre-exposure to the conspecific sex-pheromone had no effect on the dose-response curve at either delays. By contrast, Z5 slightly improved sucrose responsiveness 15 min but not 24 h after pre-exposure. Interestingly, the conspecific sex-pheromone and Z5 had time-dependent effects on gustatory habituation: pre-exposing the moths with Z5 hindered learning after immediate but not 24-h pre-exposure, whereas pre-exposure to the conspecific sex-pheromone hindered learning at 24-h but not immediate pre-exposure. They did not have opposite effects. This is the first time a sex-pheromone is reported to affect learning in a non-social insect. The difference in modulation between conspecific sex-pheromone and Z5 suggests that con- and hetero-specific sex-pheromones act on plasticity through different cerebral pathways.

Synthetic Co-Attractants of the Aggregation Pheromone of the Date Palm Root Borer Oryctes agamemnon.

Laboratory and field investigations to identify and evaluate plant co-attractants of the aggregation pheromone of the date palm pest Oryctes agamemnon are reported. Volatiles emitted by freshly cut palm core and palm core with feeding males, were collected, analyzed by gas chromatography coupled to mass spectrometry and evaluated in olfactometers alone or combined with synthetic pheromone. A collection of palm odor without male effluvia was attractive alone and enhanced attraction to synthetic pheromone in an olfactometer similar to that to a collection of palm odor emitted with feeding males and containing natural pheromone. Behavioral responses to collections of palm volatiles were correlated to the amount of volatiles material in them. Enhancement of the attractiveness of the pheromone was not correlated to chemicals specific to beetle feeding. The chemicals common to the active collections extracts were benzoate esters, mostly ethyl benzoate, anisole derivatives and sesquiterpenes. Blends of the most abundant components of the extracts were evaluated for enhancement of the attractiveness of pheromone (1 µg) in olfactometers at 1 or 10 µg doses. The mixtures were further evaluated by field trapping in Tunisia at 3-10 mg/day using reference (6 mg/day) or experimental pheromone formulations. A mixture of ethyl benzoate, 4-methylanisole and farnesol (1:1:1 w/w at 6.5 mg/day) enhanced captures in pheromone baited traps in 2014 and 2015 and this mixture was as active as the natural palm bait. The practical prospect of the result for the management for O. agamemnon, and other palm beetles is discussed.

Identification of the Aggregation Pheromone of the Date Palm Root Borer Oryctes agamemnon.

Laboratory and field investigations aimed to characterize the chemical communication system of the date palm pest Oryctes agamemnon. Live males or extracts of male effluvia attracted conspecifics in an olfactometer, whereas female effluvia attracted only males. Volatile emissions from adults feeding on sugarcane were sampled and analysed by gas chromatography (GC) and GC/mass spectrometry (GC/MS). Males emitted a blend of 1) ethyl 4-methyloctanoate, 2) 4-methyloctanoic acid, 3) 4-methyloctanyl acetate, and 4) 4-methyloctanol in variable ratio. Single sensillum recordings demonstrated that compounds 1, 2, and 3 are detected by specific olfactory receptor neurons. Olfactometric experiments showed that compounds 1 and 3 attract both sexes of O. agamemnon, but females are more attracted by compound 1 and males by compound 3. Compound 2 was more attractive for females, especially virgin ones. Field experiments confirmed that compound 1 and compound 2 attracted O. agamemnon of both sexes and showed synergy with palm odors. No clear activity of compound 3 was observed. A mix of compounds 1 and 2 with date palm core odor was significantly the most attractive, and captured more females than males. The male aggregation pheromone of O. agamemnon appears therefore to be based on a mixture in contrast to previously identified Oryctes pheromones. Our results provide the basis for developing mass trapping to control this pest.

Divergence in delta(13)C of dark respired CO(2) and bulk organic matter occurs during the transition between heterotrophy and autotrophy in Phaseolus vulgaris plants.

Substantial evidence has been published in recent years demonstrating that postphotosynthetic fractionations occur in plants, leading to (13)C-enrichment in heterotrophic (as compared with autotrophic) organs. However, less is known about the mechanism responsible for changes in these responses during plant development. The isotopic signature of both organic matter and respired CO(2) for different organs of French bean (Phaseolus vulgaris) was investigated during early ontogeny, in order to identify the developmental stage at which isotopic changes occur. Isotopic analyses of metabolites and mass balance calculations helped to constrain the metabolic processes involved. At the plant scale, apparent respiratory fractionation was constantly positive in the heterotrophic phase (c. 1 per thousand) and turned negative with autotrophy acquisition (down to -3.08 per thousand). Initially very close to that of the dry seed (-26.83 +/- 0.69 per thousand), isotopic signatures of organic matter and respired CO(2) diverged (in opposite directions) in leaves and roots after onset of photosynthesis. Respired CO(2) reached values up to -20 per thousand in leaves and became (13)C-depleted down to -29 per thousand in roots. It was concluded that isotopic differences between organs occurred subsequent to metabolic changes in the seedling during the transition from heterotrophy to autotrophy. They were especially related to respiration and respiratory fractionation.