Environ Sci Pollut Res DOI 10.1007/s11356-015-5923-3
ECOTOX, THE INRA'S NETWORK OF ECOTOXICOLOGISTS
Unexpected effects of sublethal doses of insecticide on the peripheral olfactory response and sexual behavior in a pest insect Lisa Lalouette 1 & Marie-Anne Pottier 1 & Marie-Anne Wycke 1 & Constance Boitard 1 & Françoise Bozzolan 1 & Annick Maria 1 & Elodie Demondion 2 & Thomas Chertemps 1 & Philippe Lucas 2 & David Renault 3 & Martine Maibeche 1 & David Siaussat 1
Received: 12 May 2015 / Accepted: 3 December 2015 # Springer-Verlag Berlin Heidelberg 2015
Abstract Pesticides have long been used as the main solution to limit agricultural pests, but their widespread use resulted in chronic or diffuse environmental pollutions, development of insect resistances, and biodiversity reduction. The effects of low residual doses of these chemical products on organisms that affect both targeted species (crop pests) but also beneficial insects became a major concern, particularly because low doses of pesticides can induce unexpected positive—also called hormetic—effects on insects, leading to surges in pest population growth at greater rate than what would have been observed without pesticide application. The present study aimed to examine the effects of sublethal doses of deltamethrin, one of the most used synthetic pyrethroids, known to present a residual activity and persistence in the environment, on the peripheral olfactory system and sexual behavior of a major pest insect, the cotton leafworm Spodoptera littoralis. We highlighted here a hormetic effect of sublethal dose of deltamethrin on the male responses to sex pheromone, without Responsible editor: Philippe Garrigues Electronic supplementary material The online version of this article (doi:10.1007/s11356-015-5923-3) contains supplementary material, which is available to authorized users. * David Siaussat
[email protected] 1
Sorbonne Université, UPMC—Paris 6, Institute of Ecology and Environnemental Sciences of Paris—Sensory Ecology Department, 7 Quai Saint Bernard, F-75005, Paris, France
2
Institute of Ecology and Environnemental Sciences of Paris— Sensory Ecology Department, INRA, Route de Saint-Cyr, F-78000, Versailles, France
3
Université de Rennes 1, UMR CNRS 6553 Ecobio, 263 Avenue du Gal Leclerc, CS 74205, 35042 Rennes, France
any modification of their response to host-plant odorants. We also identified several antennal actors potentially involved in this hormetic effect and in the antennal detoxification or antennal stress response of/to deltamethrin exposure. Keywords Crop pest . Sublethal pesticide doses . Detoxification . Olfaction . Behavior . Hormesis
Introduction The widespread use of pesticides that have long been applied as the main solution to limit agricultural pests is not without risk; environmental problems can be imposed, including chronic or diffuse environmental pollution and development of pest insect resistance and reduced biodiversity (Casida 2009). Even though the effects of moderate to large insecticide doses were definitively established as deleterious to biodiversity, low residual doses might have prominent effects on targeted species (crop pests) or beneficial insects (Maxim and van der Sluijs 2007). These environmentally relevant concentrations have not been thoroughly examined because the risk assessment is currently based only on the determination of acute toxicity as the critical toxicological value (OEPP/EPPO 2003). Yet, it has long been noted that following insecticide application, a surge in pest population growth is sometimes observed at a rate greater than would be observed without chemical application (review in Cutler 2013). Identification of this phenomenon which might be linked to an insecticide residual dose hormetic effect on various physiological reproductive processes appeared to be particularly important due to the potential implications in agriculture and pest management. Pyrethroids exhibit high bioefficacy on targeted organisms and decreased toxicity on non-targeted vertebrates (Bradbury
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and Coats 1989; Goulding et al. 2013). One of the most worldwide used synthetic pyrethroids is deltamethrin (WHO 1989). The molecule exerts toxic and paralyzing effects on the central and peripheral nervous systems of insects by interacting with the voltage-gated sodium channels (Haug and Hoffman 1990). As one of numerous sprayed pesticides (Soderlund 2004), deltamethrin can enter the atmosphere via drift or volatilization and can therefore be found in all environmental compartments, including the periphery or outside treated areas. Deltamethrin is also known for its high residual activity and persistence in the environment (De Jong et al. 2008); therefore, the diffuse environmental pollution caused by this pesticide can lead to direct or indirect insect body surface exposure to lower pesticide doses without any apparent mortality suffered in insect populations (Soderlund 2004). Consequently, sublethal deltamethrin doses disrupt various biological processes, such as longevity, oviposition, and crop pest reproduction (Cutler 2013; Lee et al. 1998; Yang and Du 2003), but deleterious effects are also observed in beneficial arthropods and parasitoids (Delpuech et al. 2012; Desneux et al. 2007). Most insects use olfactory cues to communicate with mating partners and localized trophic patches and oviposition sites (Roelofs and Comeau 1969); therefore, several studies have indeed focused on the impact of sublethal deltamethrin doses on olfaction using sex pheromone communication as a model. At the behavioral level, sublethal deltamethrin doses induced various perturbations in different insect species; for example, male sex pheromone responses were disrupted in various Trichogramma species (Delpuech et al. 2012). In the moth Ostrinia furnacalis, emerging males from deltamethrintreated larvae exhibited a reduced behavioral response to the sex pheromone, and consistent with this result, Spodoptera litura male moths were less likely to locate a sex pheromone source following deltamethrin treatment (Wei and Du 2004; Wei et al. 2004). Pheromone perception at the peripheral (i.e., antennal) level involves specific receptor excitation that elicits nerve impulses interpreted by the central nervous system (Kaissling 1996). Insecticides, by obstructing nerve impulse transmission, might interfere with pheromone perception. However, to our knowledge, only three studies have examined insecticide effects on insect peripheral olfactory systems (Zhou et al. 2005; Wang et al. 2011) and only one study evaluated deltamethrin (Lucas and Renou 1992). In this latter case, deltamethrin application at lethal doses induced disruption in olfactory receptor neuron (ORN) responses to the sex pheromone in males of two Mamestra species. The mechanistic nature of insecticide effects on the olfactory system at sublethal doses within the antennae remains poorly investigated. Several recent transcriptomic and proteomic studies—including in the cotton leafworm Spodoptera littoralis, a major crop pest—showed that insect antennae indeed possess numerous detoxification enzymes and genes involved in stress response (review in Siaussat et al. 2014).
However, a knowledge gap still exists regarding signaling pathways activated in the insect olfactory organ by exposure to pesticides and particularly at environmentally relevant doses. In this ecotoxicological context, it is important to study the global effects of sublethal pesticide doses on insect vital physiological functions, including olfaction, which could have profound effects on life cycle. The objectives of the present study were to examine the effects of sublethal deltamethrin doses on the peripheral olfactory system and sexual behavior of a terrestrial insect using a physiological approach by direct antennal exposure. The model used here is the cotton leafworm S. littoralis, a major crop pest known to possess systemic detoxification mechanisms for deltamethrin (Riskallah et al. 2006; Miles and Lysandrou 2007). The sex pheromone system of this noctuid moth has been well characterized, and a variety of antennal genes involved in detoxification and stress response has been identified (reviewed in Siaussat et al. 2014), making this species a viable model to investigate the effects of deltamethrin on insect olfaction. Furthermore, because sublethal pesticide doses might induce cryptic effects at various levels, we used a multidisciplinary strategy by combining molecular and biochemical Bomics^ methods with electrophysiological and behavioral approaches. Our results highlighted the toxicity of sublethal insecticide doses and revealed a hormetic effect on the responses to the sex pheromone in males exposed to sublethal deltamethrin doses, both at the behavioral and electrophysiological levels. We also identified variations in the expression of various antennal proteins and genes, such as odorant-binding proteins (OBP), or xenobiotic degrading enzymes, which could explain this hormetic effect at the peripheral olfactory level.
Methods Insect rearing, treatment, and tissue collection S. littoralis larvae were reared on a semi-artificial diet (Hinks and Byers 1976) at 24 °C, 60–70 % relative humidity, and a 16:8 light/dark cycle until emergence. Individuals were sexed as pupae. Male adult antennae were removed, dissected, frozen in liquid nitrogen, and stored at −80 °C until used in transcriptomics and proteomics. LD50 and NOAEL of deltamethrin-exposed males Adult males were maintained in plastic boxes (length 15 cm, width 5 cm) and supplied with water-dissolved sugar (20 g/L). Two-day-old males (body mass 0.11 ± 0.02 g) were exposed to a range of increasing doses of deltamethrin (45423, SIGMA PESTANAL, France) diluted in hexane, either by a 1-μL drop topical application on each antenna (N = 30 for each
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condition). Control insects were treated using hexane for each experimental procedure. Twenty-four hours after deltamethrin application, percent mortality was determined. Individual behavioral experiments All the experiments were conducted 24 h following the treatments, i.e., deltamethrin application on antennae either at a LD50 1/10 dose (referred to as LD501/10) or at a LD50 1/100 dose (referred to as LD501/100) or control treatments. S. littoralis males were placed individually in a plastic petri dish (Ø = 20 cm) before onset of the scotophase. Individuals were maintained in the dark and acclimatized until the experiment was initiated in mid-scotophase. Analyses were performed under red light conditions (T = 22 ± 1 °C, RH = 70 ± 10 %) and recorded over a 4-min period with QuickCam® Pro 9000. Pheromonal stimulation was performed under a constant stream of air (0.6 m/min) supplied with a Pasteur pipette, with the thinner end inserted through the side of the petri dish. The Pasteur pipette contained a filter paper loaded with either 10 μl of the pheromonal component (Z9,E11–14Ac diluted at 10 ng/μl in hexane) or with 10 μl of hexane for the control batch. The filter paper was replaced every 15 min. The latencies of stereotyped behavioral items were individually screened as follows: from the activation step, which included antennal erect (AE), wing fanning (WF), extrusion of genitalia (EG), and movement towards the pheromonal stimulation (M). Insects were considered non-activated when not demonstrating the stereotypic behavioral sequence described above. Competitive behavioral experiments Experiments were conducted 24 h following the treatments, either with deltamethrin applications at LD501/10 or at LD501/100, LD30 or control treatments. Treated and control males were placed together in a same plastic square box (12 × 12 × 2 cm) before onset of the scotophase. They were maintained in the dark and acclimatized until the experiment was initiated in mid-scotophase by introduction of a 2-day-old female in the box. Analyses were performed under red light conditions (T = 22 ± 1 °C, RH = 70 ± 10 %) and recorded over a 15-min period. The sexual behavior of males competing for the same female was then analyzed and the respective success rates of males were calculated. Since the female mates with only one male during a test, a mated male gets a score of B1^ and an unmated male a score of B0^. The tests during which both males did not exhibit any sexual behavior were not included in this calculation. Electroantennography recordings Electroantennography (EAG) recordings were performed as described in Bigot et al. (2012) in mid-scotophase, 24 h after
deltamethrin application on antennae at LD501/10 or control treatments. S. littoralis female main sex pheromonal component, (Z9,E11)-tetradecadienyl acetate (Z9,E11-14:Ac) (Tamaki and Yushima 1974), was diluted in hexane (>98 % purity, CAS 110-54-3). Males were exposed to 10 and 100 ng of Z9,E11-14:Ac. Plant volatile compounds (PVs) from several S. littoralis host plants, including (Z)-3-hexenyl acetate (Z3-6:OAc), linalool, α-humulene, and 2-phenylethanol (2P), were diluted in white mineral oil (Sigma; CAS 8042-47-5). Among the PVs, we focused on (Z)-3-hexenyl acetate, a substance naturally released from damaged bean and known to attract numerous insect pest species, with a dose response assay of 1, 10, and 100 μg, whereas one dose of 100 μg was used for the other PVs. Stimulus cartridges contained nonsaturating doses of pheromone or PVs, which consisted of Pasteur pipettes containing a filter paper (8 × 12 mm) loaded with 10 μl of the stimulus chemicals at the appropriate concentration. Stimulus cartridges were changed between each insect. After offline low-pass filtering (50 Hz, Gaussian filter), the following four parameters were measured: (i) amplitude in millivolts; (ii) half the depolarization time in seconds (1/2 time); (iii) amplitude in millivolts (2/3 repolarization); and (iv) 2/3 repolarization time in seconds (2/3 time repolarization).
Proteomics analysis Experiments were conducted 24 h following deltamethrin application at LD501/10 or control treatments. Three biological replicates were prepared for each condition (control and LD501/10) and analyzed using a proteomics approach as described in Bigot et al. (2012). An equal amount of antennal protein extract from paired samples was labeled with three different CyDye DIGE (Cy2, Cy3, or Cy5). The samples were simultaneously separated on a single 2D gel, using isoelectric focusing in the first dimension and SDS polyacrylamide gel electrophoresis in the second dimension. The gel was scanned using a Typhoon image scanner to reveal each CyDye signal and overlay images using ImageQuant software (GE Healthcare, Life Sciences, USA). A comparative analysis of all spots of interest was performed using DeCyder Bin-gel^ or Bcross-gel^ analysis software to establish protein expression ratios between deltamethrin-treated and control antennae. Differences in protein quantities were considered significant when ratio values were ≤−1.5 or ≥1.5, as suggested by Contreras et al. (2013). Protein spots presenting this range of ratio values were then automatically chosen from the 2D gel with the Ettan Spot Picker and identified using MALDI-TOF or MALDI-TOF/TOF procedure (Applied Biosystems Proteomics Analyzer, GE Healthcare, Life Sciences, USA) and primary sequence databases (NCBInr and SwissProt) using GPS Explorer software equipped with the MASCOT
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search engine (GE Healthcare, Life Sciences, USA) (See Table S1 for details). RNA isolation and cDNA synthesis Total RNAs (5 μg) were extracted with TRIzol reagent (Invitrogen, Carlsbad, CA, USA), treated with DNase I (Ambion, USA) in accordance with the manufacturer’s instructions, and quantified by spectrophotometry at 260 nm (BioPhotometer, Eppendorf, Hamburg, Germany). Singlestranded cDNAs were synthesized from total RNAs (5 μg) with SuperScript II Reverse Transcriptase (Gibco BRL, Invitrogen) as described in Bigot et al. (2012), and based on the manufacturer’s protocol. For each experimental condition, four to seven biological replicates were performed. PCR and qPCR All PCRs and real-time quantitative PCRs were conducted as described in Bigot et al. (2012). PCR amplification products were separated on 1 % agarose gels and visualized by SYBR safe fluorescence. The cycle threshold values (Ct values) were determined for the reference and the candidate genes with LightCycler ® 480 software (Roche, France). The average Ct value of each triplicate reaction was used to normalize the candidate gene expression level to the geometric mean of the reference gene’s level in QGene (Simon 2003). Seven different genes (Ac tin , R pl 13 , R pl 8, GAPDH, tub ul i n, AT Pase , Ubiquitin) were tested as putative reference genes. Specific qPCR primers for housekeeping genes or targeted genes were designed using eprimer3 software (http://emboss.bioinformatics.nl/cgi-bin/emboss/eprimer3) , and optimal primer annealing temperatures were optimized using qPCR tests. After BestKeeper analysis (Pfaffl et al. 2004), Rpl13 was selected as the reference gene due to its consistent expression under all experimental conditions (see Table S2 for additional information). Normalized expression was determined relative to the control expression. We studied expression variation of the following 32 antennal genes: six P450s (CYP6AE49, CYP6AE47, CYP9A52, CYP4L12, CYP6AE48, CYP6AE50; Pottier et al. 2012), antennal cytochrome P450 reductase, also called CPR (Pottier et al. 2012), seven GSTs (four epsilon forms SlitGSTe1, SlitGSTe2, S l i t G S Te 3 , a n d S l i t G S Te 4 a n d t w o d e l t a f o r m s SlitGSTd1, SlitGSTd2, and SlitGSTd3), eight CXEs (SlitCXE3, SlitCXE5, SlitCXE7, SlitCXE11, SlitCXE15, SlitCXE21, SlitCXE27, SlitCXE28) (Durand et al. 2010a, b), four heat shock proteins (HSPs) (SlitHsp20.7, SlitHsp60, SlitHsp70, SlitHsp90), and four OBPs (SlitGOBP2, SlitPBP1,SlitPBP2, SlitPBP2). GST and HSP gene identification and different subfamily
classification was achieved through a bioinformatics analysis performing nucleic acid sequence alignments of selected EST database genes (Legeai et al. 2011) and sequence databases using NCBI BLAST (http://blast.ncbi. nlm.nih.gov/) and CLUSTAL W (BioEdit Sequence Alignment Editor; Hall 1999). Statistical analyses Lethal doses for 50 % of the population was computed using Probit analyses with an α risk of 0.05. Analysis of variance (ANOVA) with subsequent post hoc comparisons was performed using Tukey’s test for molecular and electrophysiological data. For individual behavioral experiments, a timedependent Cox regression was used (also known as proportional hazard survival analysis, see Dacher and Smith 2008). The Cox regression compares across treatment batches the probability to observe a positive response during the given time period of 210 s or the Bsurvival rate^ in the considered time-period (see Dacher and Smith 2008 for more details). For each behavior, we summed every 30 s for Bresponses^ or Bno responses^ until the end of the record, and the Cox regression analysis was performed for each behavioral observation (AS, WG, EG, and M). A χ2 test was also performed to compare the success rate between deltamethrin-treated males and controls in the competitive behavior experiments. These statistical analyses were conducted using Minitab™ 13 Statistical Software (Minitab Inc., State College, PA, USA) and STATISTICA (StatSoft Enterprise, USA) and computed with R 2.4.
Results Lethal dose determination The deltamethrin dose that yielded 50 % mortality (LD50) in 2-day-old males following antennal application was 7.6 ng ± 2.3 (Fig. 1). This value of LD50 following antennal application was quite consistent with those previously reported for other insect species after deltamethrin application on other body parts or oral route administration: 12 ng per insect in Blattella germanica (Lee et al. 1998), 0.5 to 125.1 ng in Musca domestica (Sukontason et al. 2005), 24.4 to 132.7 ng in Chrysomya megacephala (Sukontason et al. 2005), 620 ng in Apis mellifera (Decourtye et al. 2005; Dai et al. 2010), and 60 ng in Cimex lectularius (Zhu et al. 2012). We set the no observable adverse effect level (NOAEL)— which is conventionally described as the amount of pesticide that has any effect on the organism—as 0.15 ng. Indeed, the mortality obtained for this dose was equivalent to the mortality of the control insects. For the subsequent experiments, two deltamethrin sublethal doses corresponding to 1/10 and 1/
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100 of the LD50 (referred to as LD501/10 and LD501/100) and one lethal dose (i.e., LD30) were thus selected and used for antennal applications (Fig. 1).
30.9 ± 0.04%; chi-square = 14.44000 df = 1 p < 0.000145, Fig. 2g). Effects of the LD501/10 sublethal dose of deltamethrin on male antennal electrophysiological responses
Effects of sublethal and lethal doses of deltamethrin on male sexual behavior The global effects of sublethal deltamethrin doses were measured by studying the sexual behavior of males after insecticide treatment. Four behavior idioms characteristic of male courtship, namely AE, WF, EG, and oriented movement towards the pheromone source (M), were recorded. The initiation times of all idioms were significantly reduced in males treated with deltamethrin at LD501/10 whereas no difference between control and LD501/100-treated males was observed (Fig. 2a–d). Males treated with deltamethrin at LD501/10 were thus much more responsive compared to control and LD501/100-treated males. A faster courtship behaviour could putatively lead to a higher reproductive success in treated males. We then tested if males treated with this LD501/10 sublethal dose were more successful for mating than control males when in a competition threesome with a single female. Males exposed to the LD501/10 dilution showed indeed a higher rate of reproductive success compared to control males (i.e., 60.5 ± 0.04% vs 39.5 ± 0.04 %; chi-square = 4.326400 df = 1 p < 0.037526, Fig. 2e). Moreover, as expected, in the same conditions, the LD501/100treated males have similar reproductive success than the control ones (49.2 ± 0.03% vs 50.8±0.03%; chi-square = 0.0400000 df = 1 p < 0.0841481, Fig. 2f) whereas the LD30treated males showed a lower success (i.e., 69.1 ± 0.04 % vs 100
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Dose (ng per insect) Fig. 1 Antennal toxicology of deltamethrin in S. littoralis males (circles). The 50 % lethal dose (LD50) was estimated 24 h following antennal exposure of 2-day-old S. littoralis males to deltamethrin serial dilutions (N = 30 for each dilution) using a probit analysis. No observed effect level (NOEC) was set by taking into consideration the mortality observed in the control insects without any pesticide treatment (squares). LD30 (5.05 ng), LD501/10 (0.76 ng), and LD501/100 (0.076 ng) were used for the following behavioral experiments
Since behavioral effects were only observed in males exposed to LD501/10 of deltamethrin, we focused on this dose to test its effect on odorant reception. We analyzed if this treatment at sublethal dose could modify the detection of both the sex pheromone main component (Z9,E11-14:Ac) (Fig. 3) and four host-plant odorants (Fig. 4) by performing EAGs. Deltamethrin at LD501/10 significantly reduced the 2/3 repolarization time (10 ng H2 = 9.17, p < 0.05; 100 ng H2 = 18.14, p < 0.001; Fig. 3d), regardless of the pheromone dose; whereas the 1/2 depolarization time, the maximum depolarization amplitude, and the 2/3 repolarization amplitude time were not affected (Fig. 3a–c). The intensity of the antennal response was thus not affected, but the dynamics of the repolarization was modified. The EAG profiles recorded from the same deltamethrin-exposed males in response to four PVs showed no significant difference compared to controls, under all parameters (Fig. 4), suggesting a specific effect of the treatment on pheromone detection. Effects of the LD501/10 sublethal dose of deltamethrin on antennal gene expression and proteome We performed a proteomic approach to identify antennal proteins subjected to quantitative variation following a 24-h LD501/10 treatment. Variation in protein expression ratios between the control and the LD501/10-treated insects was significant for 52 proteins. They were subsequently identified using MASCOTT (Table S1) and then classed into eight functional groups according to gene ontology (Fig. 5). The most abundant regulated proteins in treated antennae were primarily involved in energy metabolism (tricarboxylic acid cycle—TCA cycle, glycolysis, ATP synthesis, and fatty acid degradation). In addition, seven classes involved in cuticular structure, cytoskeletal organization, and microtubule movement showed decreased expression, as well as most proteins with fat lipid transport functions. Three detoxification enzymes were differentially regulated, with increased levels of glutathione S-transferases (GST), carboxylesterase 5 (SlCXE5), and the redox partner of the cytochrome P450 enzyme (CPR) (2.80-, 1.5-, and 1.5-fold changes, respectively), whereas UGT levels decreased. In addition, prophenoloxidase, known to control pathogen melanization and damaged tissues in invertebrates, was reduced (Cerenius and Söderhäll 2004). Variability in protein profiles involved in protein folding processes was also detected, including a decrease in two HSPs from the HSP70 family and an increase in cyclophilin A, also known as peptidylprolyl isomerase A. Finally, two proteins related to
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Fig. 2 Behavioral responses of S. littoralis males exposed to sublethal deltamethrin doses. a–d Twenty-four hours following antennal application of deltamethrin at LD501/10 and at LD501/100 in 2-day-old males, the latencies of stereotypical behaviors in male S. littoralis after pheromonal stimulation were individually screened from the activation step, which included antennal erect, wing fanning extrusion of genitalia, and movement towards the pheromonal stimulation. The black line represents control insects (N = 25), the dashed curved line indicates
LD50 -treated insects (N = 22), and the dotted line indicates LD501/100-treated insects (N = 24). e–g Twenty-four hours following antennal application of deltamethrin, behavioral experiments were carried out using competitive assays (two males versus one female). e LD30-treated males versus control males (N = 55), f LD501/100-treated males versus control males (N = 55), and g LD501/10-treated males versus control males (N = 53). Asterisk indicates a significant difference between control and the labeled conditions (χ2 test)
G proteins and three OBPs, including two pheromone-binding proteins (PBP1 and PBP2) putatively involved in odorant signal processing, were also regulated in deltamethrin-treated antennae. To complete the proteomic approach, we analyzed the transcription levels of several antennal genes known for their involvement in pyrethroid detoxification and odorant transport (OBPs). Six cytochrome P450s (P450s or CYPs) and CPR (Pottier et al. 2012), seven GSTs (Pottier et al., pers.com.), and eight carboxylesterases (CXEs) (Durand et al. 2010a, b) were selected based on sequence similarities with genes involved in pyrethroid metabolism in closely related species (Zhou et al. 2010; Farnsworth et al. 2010; Ketterman et al. 2011; Brun-Barale et al. 2010). The transcription levels of several genes encoding HSPs involved in cellular stress responses (reviewed in Siaussat et al. 2014) were also measured. The transcript levels of one P450 (Slcyp6ae50), two GSTs (Slgste1, Slgste2), and one esterase (Slcxe7) exhibited significant increases in treated males (7-, 1.6-, 3-, and 4.8-fold, respectively) (Fig. 6), whereas another esterase (Slcxe21)
showed a 2-fold decrease. Transcript levels of the four OBP genes, including PBPs, were not modified.
Discussion Our results reveal the various effects of a sublethal exposure of deltamethrin on the olfaction of an insect, from peripheral reception to behavioral responses to sex pheromones (Fig. 7). Our molecular data support previous hypotheses deduced from the analysis of antennal transcriptome and proteome of various insect species (Siaussat et al. 2014), which proposed that antennae have multiple functions in addition to odorant reception, including physiological plasticity under environmental stress, such as under pesticide exposure. Application of different substances directly on the antennae had been employed in various contexts, such as for studying olfaction disruption by pheromone inhibitors (Munoz et al. 2011) or antennal flight posture disruption by motoneuron-
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A
Fig. 3 Electroantennography (EAG) responses in males of S. littoralis to sex pheromone. EAG recordings were performed 24 h after antennal exposure of 2day-old males to LD501/10. The following four parameters were measured: a amplitude in millivolts; b half the depolarization time in seconds (1/2 time); c amplitude in millivolts (2/3 repolarization); and d 2/3 repolarization time in seconds (2/3 time repolarization). N = 24 for control insects and N = 34 for 1/10 treated males. Asterisks indicate significant differences among means (p < 0.05) between control (black bars) and treated (grey bars) individuals
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Fig. 4 Electroantennography (EAG) responses in S. littoralis males to plant odors. EAG recordings were performed 24 h after a 2-day-old male antennal exposure to LD501/10. One dose of each plant odor stimulus was applied, and the following four parameters were measured: a amplitude in
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millivolts; b half the depolarization time in seconds (1/2 time); c amplitude in millivolts (2/3 repolarization); and d 2/3 repolarization time in seconds (2/3 time repolarization). Results found that plant volatile compounds stimulated a total of 17 ± 3 control or treated males
Environ Sci Pollut Res hypothecal protein hypothecal protein KGM
pupal cucle protein precursor cucular protein hypothecal 30 precursor cucular protein hypothecal 30 precursor hemocyte aggregaon inhibitor protein precursor heat shock protein 70 heat shock cognate protein 70 histone H2B-like protein cyclophilin A histone H2B-like protein histone H2A-like protein 2
gi|187423907
-2.10
gi|357631640
-3.20
gi|290560816
-1.80
gi|357625069
gi|219671577 -2.00 gi|112982828 -4.30 gi|357625089 gi|298111994 gi|357625089 gi|237648978
gi|270006324 gi|357627147
-2.70 -2.30
vacuolar ATP synthase catalyc subunit A enolase citrate synthase isocitrate dehydrogenase isocitrate dehydrogenase dihydrolipoamide dehydrogenase glyceraldehyde-3-phosphate dehydrogenase ATP synthase fay acid beta-oxidaon complex subunit beta succinate dehydrogenase vacuolar ATP synthase subunit E
Unknown funcon (5%)
-1.60
Structural proteins of the cucle (9%)
gi|148298878
1.80
gi|270298186 gi|357612136 gi|114051866 gi|357622716 gi|357631306
1.50 1.60 -1.60 1.50 -3.70
gi|319998854
-1.90
gi|114052278
-3.00
gi|283993139
-4.30
gi|357613438 gi|114052088
-1.50 -2.40
Intermediary metabolism (26%)
-1.50 1.70 -1.50 Regulaon of 1.80 gene expression.
nucleic acid and protein metabolism (14%) Oxidave enzymes and defense mechanisms CPR gi|38679387 1.50 (12%) antennal esterase CXE gi|257480037 1.50
Olfacon: Signaling pathway, Odorant binding protein, etc… (12%)
heterotrimeric guanine nucleodegi|357626837 -2.00 binding protein β subunit GCYDA_DROME 1.80 Soluble guanylate cyclase general odorant-binding protein II gi|157312500 -1.80 pheromone-binding protein 1 gi|148533559 -2.40 pheromone-binding protein 2
prophenol oxidase gi|56694122 -2.80 Glutathion-S-transferase gi|357617691 2.80 UDP-glycosyltransferase gi|363896200 -5.30
Transport (12%)
Cytoskeletal organizaon and microtubule movement (9%)
gi|365919036 -2.00
Apolipophorin Apolipophorin-3 Apolipophorins cellular renoic acid binding protein fay acid-binding protein 3
APL3_GALME gi|68564994 APLP_MANSE gi|357631570 gi|171740911
-2.10 -3.60 -3.00 2.60 -6.50
Chain A. Moesin past-1 Chain A. Moesin myosin heavy chain
gi|122920502 gi|357624424 gi|357613203 gi|170029182
-2.10 -2.80 -5.60 -2.90
Fig. 5 Functional categories of proteins displaying a change in the deltamethrin/control ratio. Proteins of controls and LD501/10-treated males were analyzed by 2D-DIGE methods, identified by MALDI-TOF or MALDI-TOF/TOF procedures, and classified into eight functional categories. Protein percentage based on the number of given protein
over the total number of proteins displaying a significant difference in protein quantity with ratio values ≤−1.5 or ≥1.5 was indicated for each category. The results of protein identification are given in Table S1 (name of the identified protein, accession number, and the protein ratio of deltamethrin-treated males compared to control males)
targeting products (Gebhardt 2004). However, a direct application of deltamethrin on antennae was rarely used before. Lucas and Renou (1992) have tested the effect of deltamethrin antennal application on the EAG responses of two noctuids, Mamestra suasa and M. brassicae, but without reproducible results. Interestingly, we demonstrated here a toxicity of deltamethrin after antennal route in S. littoralis males. This toxicity might be associated with the multiporous structure of the cuticular olfactory hairs, potentially facilitating insecticide penetration within the insect body. Despite the presence of septate junctions, which isolated receptor-lymph, subcuticular, and hemolymph spaces (Keil and Steinbrecht 1987), deltamethrin seemed to cross these barriers, reaching the central nervous system, as demonstrated with xenobiotics penetration in the olfactory mucosa of mammals (Minn et al. 2005). Additional studies should examine the possible penetration of xenobiotics from sensilla to ORNs and then to the insect brain and whole body.
At the behavioral level, our results highlighted an improved mating success in males exposed to the LD501/10 sublethal dose and its reduction with the lethal LD30 dose. We can suppose that this higher mating rate for LD501/10-treated males is a result of their faster courtship. Such a biphasic response following a pollutant exposure with beneficial effects at low doses and adverse effect at high doses is characteristic of a hormetic effect, as defined in various taxa (Calabrese and Baldwin 2002; Cedergreen et al. 2006; Calabrese and Mattson 2011). In the context of the phytosanitary treatments, targeted insects can be exposed to lower or sublethal doses because many biotic (e.g., plant uptake, microbial, and plant degradation) and abiotic (e.g., wind, volatilization, chemical degradation, and dripping) processes can spatially and temporally change the pesticide dose an insect is exposed to in the field. Thus, hormesis appeared to be one of the potential causes underlying pest resurgence and outbreaks notably because the increase of the reproduction
Environ Sci Pollut Res Fig. 6 Effect of sublethal doses of deltamethrin on S. littoralis antennal mRNA levels of a cytochrome P450 (CYP),b glutathione-S-transferase (GST), c carboxylesterase enzymes (CXE), d heat shock proteins (HSP), and e odorant or pheromonal binding protein (OBP and PBP). Twenty-four hours after a 2-day-old male antennal exposure to LD501/10, mRNA levels of several cyp, gst, cxe, and hsp genes were analyzed by qPCR using the Rpl13 gene as a housekeeping gene (N = 4 to 7 biological replicates). Bars with asterisks indicate significant differences (*p < 0.05)
A
*
8 6 4 2 0
Control Control
DL501/10 1/10 LD50 CYP6AE49
CYP6AE49
B
Control Control
DL50 1/10 1/10 LD50
Control Control
CYP6AE47
DL50 1/10 1/10 LD50
Control Control
CYP9A52
CYP6AE47
DL50 1/10 1/10 LD50 CYP4L12
CYP9A52
CYP4L12
2
Control Control
DL50 1/10 1/10 LD50 CYP6AE48
CYP6AE48
*
Control Control
DL50 1/10 1/10 LD50 CYP6AE50
CYP6AE50
Control Control
DL50 1/10 1/10 LD50 CPR
CPR
*
1 0
DL50 1/10 1/10 LD50
Control Control
GSTd1
C
5 4 3 2 1 0
GSTd1
DL50 1/10 1/10 LD50
Control Control
GSTd2
DL50 1/10 1/10 LD50
Control Control
GSTe1
GSTd3
GSTd2
GSTd3
Control Control
DL50 1/10 1/10 LD50 GSTe2
GSTe1
GSTe2
DL501/10 1/10 LD50
Control Control
GSTe3
Control Control
DL501/10 1/10 LD50 GSTe4
GSTe3
GSTe4
* * Control Control
DL50 1/10 1/10 LD50 CXE3
CXE3
D
DL50 1/10 1/10 LD50
Control Control
Control Control
DL50 1/10 1/10 LD50
Control Control
CXE5
DL50 1/10 1/10 LD50 CXE7
CXE5
CXE7
Control Control
DL50 1/10 1/10 LD50 CXE11
Control Control
DL501/10 1/10 LD50 CXE15
CXE11
CXE15
Control Control
DL501/10 1/10 LD50 CX21
8
Control Control
DL501/10 1/10 LD50
Control Control
CX27
CXE21
CXE27
DL501/10 1/10 LD50 CX28
CXE28
*
6 4 2 0
CT Control
LD50101/10 HSP90
HSP90
LD50101/10
CT Control
CT Control
HSP70
LD50101/10 HSP60
HSP70
HSP60
CT Control
LD50101/10 HSP40
CT Control
LD50101/10 HSP21.4
HSP40
HSP21.4
CT Control
LD50101/10 HSP20.7
HSP20.7
E 1.5 1 0.5 0
C Control
101/10 LD50 GOBP2
GOBP2
10 1/10 LD50
C Control
C Control
CT Control
1/10 1_10 LD50 PBP3
PBP2
abilities in insects exposed to lower or sublethal doses is usually described as a way to bypass the pollutant stress, favoring the development of resistance or adaptation (Guedes et al. 2010). However, hormesis remains poorly studied in entomology and acarology (Hardin et al. 1995; Morse 1998; Cohen Fig. 7 Proposed effect’s model of sublethal deltamethrin doses in Spodoptera littoralis. Deltamethrin entrance induces modulation of several antennal actors involved in detoxification process, global stress responses, and olfactory process. Deltamethrin (or its catabolites) induces then a faster ORN response and a faster behavioral response to sex pheromone in males
101/10 LD50 PBP2
PBP1
PBP1
PBP3
2006; Guedes and Cutler 2014). Some hormetic effects of insecticide treatments on population growth and female reproductive physiology were reported in various insect species, including S. littoralis (reviewed in Cutler 2013). However, positive effects of sublethal doses on male sexual behavior Expression modulaons of several antennal actors…
Deltamethrin OBP or detoxificaon enzymes
Pore Dendrite Auxiliary cells
Detoxicaon process (CYP6ae50, GSTe1, GSTe2) Global stress responses (HSP20.7, cyclophilin A…) Olfactory processes (OBP/PBP, CXE7…)
(tormogen, trichogen and thecogen cells)
Cucle
Antennal nerve
Epithelium OR N
Hemolymph Deltamethrin or its catabolites ?
Brain
Axon
Antennal nerve Faster response of ORN involved in pheromone detecon
Gut Hemolymph
Faster behavioral responses of males to pheromone smulaon Malpighian tubules
Environ Sci Pollut Res
or on peripheral response to a sex pheromone were rarely highlighted. This is the case in the Oriental fruit moths, in which the pheromone-mediated flight behavior of males is stimulated after topical application of chlordimeform at sublethal concentrations (Linn and Roelofs 1984, 1985). But most of the time, a decrease in the behavioral responses to the sex pheromone, or a loss in the olfactory discrimination, is observed in species exposed to sublethal doses of pesticides (Delpuech et al. 2001, 2005, 2012; Wei and Du 2004; Wei et al. 2004). The positive behavioral effects induced here in S. littoralis males by the deltamethrin at LD501/10 sublethal dose were associated with a modified ORN response to pheromonal stimulation, with a faster signal termination. Up to date, the scarce literature that examined peripheral insecticide disruption mainly reported negative effect of sublethal doses on ORN responses (review in Tricoire-Leignel et al. 2012). In honeybees, high doses of tetramethrin and permethrin strongly increased ORN repolarization time by prolonging sodium channel opening (Kadala et al. 2011), delaying signal termination. In O. furnacalis males exposed to sublethal doses of malathion, reduction in ORN depolarization was observed in response to the pheromone, a lower intensity of response which was correlated with a decreased pheromone-induced behavior (Zhou et al. 2005). Moreover, unlike other previous studies that have not studied the responses to plant odors, our results in S. littoralis showed that this alteration of antennal response appeared to be specific to sex pheromone detection, suggesting a more focused effect of the treatment on pheromone processing pathways. At the molecular level, various antennal transcript/protein amounts putatively involved in cell functioning, stress response, cell defense and/or detoxification, and olfactory processes were modified by exposure to deltamethrin at sublethal doses. Results were notable for several genes due to the absence of a correlation between transcript levels and protein variations, which can be partly explained by a different dynamic in protein turnover. Most of proteins involved in energy metabolism were down-regulated, suggesting toxic effects of deltamethrin on antennal cells. However, several of them were up-regulated, congruent with the recruitment of metabolic pathways involved in stress response and compensation for survivability, as observed in insects and shrimps following pesticide exposure (Reddy and Rao 1988). Evidence for disruption in energy metabolism was indicated by downregulation of proteins involved in lipid transport and lipoprotein metabolism, which are considered as environmental and chemical stress exposure biomarkers (Choi et al. 2001; Rizwan-ul-Haq et al. 2011). Although it is difficult to determine whether these changes could result in a gain or loss of energy, they are often considered as viable pesticide exposure biomarkers, even at sublethal doses (Yuk et al. 2012; De Coen et al. 2001). Several HSPs were up- or down-regulated,
consistent with reports in Drosophila exposed to organochlorine (Sharma et al. 2012). Interestingly, a network of detoxifying enzymes was also modulated by deltamethrin exposure at the antennal level. In several insect species, elevated activities and/or expression of these genes in the midgut or fat body have been associated with resistance to pyrethroids (Enayati and MotevalliHaghi 2007; Ahmad et al. 2007), including deltamethrin (Jagadeshwaran and Vijayan 2009; Vontas et al. 2002). Evidence for the role of carboxylesterases in pyrethroid metabolism in various species is well supported, including in spodopteran pests (Farnsworth et al. 2010; Coppin et al. 2012); therefore, some of these enzymes might also be involved in deltamethrin detoxification within antennae. Interestingly, several genes/proteins involved more precisely in odorant processing were also modulated. This is the case for one CXE, SlCXE7, involved in pheromone catabolism and thus characterized as a pheromone/odorant-degrading enzyme (PDE/ODE) in S. littoralis (Durand et al. 2011). Its regulation by insecticide exposure might impact the dynamic of pheromone signal termination, as shown in Drosophila after ODE/ PDE in vivo inhibition (Chertemps et al. 2012; 2015). Moreover, a decrease in the amount of three OBP proteins was also observed. Although OBP roles remain controversial, the proteins reportedly trigger olfactory reception by transporting odorant/pheromone molecules within the sensillar lymph to the olfactory receptors (review in Fan et al. 2011). They could also play a role in signal inactivation or detoxification (Boudjelal et al. 1996; Bianchi et al. 2012). In mammals, OBPs have been shown to protect cells, including ORNs, against various oxidative, cytotoxic, and carcinogenic compounds by scavenging toxic molecules (Pevsner et al. 1990; Grolli et al. 2006; Mitchell et al. 2008) or by presenting toxins to the intracellular detoxification or inactivation machinery (Bianchi et al. 2012). Strotmann and Breer (2011) emphasized rapid internalization of OBP/odorant complexes into lysosomes, suggesting the degradation of OBP/ odorant complex, a mechanism that might also allow the elimination of toxic molecules. Therefore, the OBP/PBP protein decrease observed here 24 h after LD501/10 exposure might be the consequence of their turnover, associated with pesticide detoxification and/or pheromone inactivation. Some changes in protein/transcript levels could be probably under the control of signaling pathways involved in the stress and hormetic response. For example, the increase of GST and the disruption of intermediary metabolism enzymes involved in the NADPH homeostasis can be typically due to activation of the nuclear factor-erythroid 2-related factor 2 (Nrf2)/Keap1pathway (Johnson et al. 2008). This pathway modulates the expression of numerous antioxidant and cytoprotective genes in response to toxicity, oxidative stress, tissue inflammation, and neurodegeneration (Johnson et al., 2008; Marini et al. 2008; Son et al. 2008, Ma, 2013; Surh, 2011). This pathway is also involved in the neurohormetic
Environ Sci Pollut Res
effects of various phytochemicals (Mattson and Cheng 2007; Murugaiyah and Mattson, 2015). It contributes to oxidative stress tolerance and lifespan as well as the activation of detoxification genes and apparition of insecticide resistance (Sykiotis and Bohmann 2008; Misra et al. 2011, 2013). Likewise, we can assume the involvement of HSP expression-controlling pathways, such as the heat shock factor (HSF) or the FOXO pathways (Mattson and Cheng 2007; Murugaiyah and Mattson, 2015), which were known to play a major role in response to low hormetic stress in various tissues (Tower 2011, 2015; Boncristiani et al. 2012; DiasSantagata et al. 2007; Salgueiro et al. 2014). There is also more and more evidence of the strong interaction of these previous pathways with many other pathways such as the NF-κB pathway, for example (Luna-López et al. 2013; Chirumbolo 2012). In our study, variations of many molecular actors could thus be due to the activation or to the synergistic effects of these signaling pathway networks. To conclude, our study highlighted the effects of sublethal doses of deltamethrin on the peripheral olfactory system and the sexual behavior of a pest insect, revealing in particular a hormetic effect on sexual behavior. At the molecular level, we observed the modulation of detoxification mechanisms, pesticide stress responses, and the modulation of some actors involved in the olfactory processes. The hormetic effects induced by sublethal doses of deltamethrin in experimental conditions, if observed in the field, could potentially contribute to the adaptation of this pest insect to pesticides, with as possible consequence an acceleration of population growth. In a context of reducing pesticide use or in an ecotoxicological context with the presence of pesticide residues, our study emphasizes the need to assess the effects of sublethal doses and to elucidate the mechanisms involved in exposed organisms. Acknowledgments We thank Dr. Matthieu Dacher for his assistance in the statistical analyses, Dr. Emmanuelle Jacquin-Joly for access to the S. littoralis EST database, and Dr. Didier Rochat for helpful commentaries regarding this study. Lisa Lalouette was supported by a DIM ASTREA (Région Ile de France) post-doctoral fellowship. Marie-Anne Pottier and all experiments were supported by an BEMERGENCE^ grant from the University Pierre and Marie Curie (UPMC-Paris 6) and PHEROTOX grant from ANR BIOADAPT.
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