Introduction

Ecdysis is shedding of the old cuticle during the molting process in insects. Pupal ecdysis refers to the shedding of the last instar cuticle during the pupal molt. In Lepidoptera pupal ecdysis, the role of neuroendocrine hormones, specifically corazonin, ecdysis triggering hormones (ETH), pre-ecdysis triggering hormones (PETH), and eclosion hormone (EH), have been reasonably elucidated1,2,3. The PETH or ETH activates L3,4 neurons and elicits pre-ecdysis behavior through the release of kinins and diuretic hormone (DH). The ETH and/or EH activate different subsets of crustacean cardioactive peptide (CCAP) interneurons and elicit ecdysis and post-ecdysis behavior through the release of various peptides, including CCAP, myoinhibitory peptides (MIPS), and bursicon. Studies indicate that other factors, including excitatory and inhibitory neurotransmitters, could also play an important role in the regulation of CCAP neurons3,4. For example, inhibitory signals coming from cranial and thoracic ganglia can delay the release of neuropeptides from CCAP neurons during the pre-ecdysis stages1,2,3; these signals could be produced by the inhibitory neurotransmitter gamma-aminobutyric acid (GABA)4. In Drosophila, acetylcholine (ACh) and GABA modulate calcium signaling in CCAP neurons through their respective receptors5. In addition, somatic deletion of a nicotinic ACh receptor (nAChR) subunit in Drosophila CCAP neurons was sufficient to disrupt bursicon-driven wing expansion6. However, the roles of ACh, GABA, and other neurotransmitters in regulation of the insect ecdysis process have not been well elucidated.

We previously demonstrated that low doses of imidacloprid, clothianidin, and thiamethoxam, three neonicotinoid insecticides that mimic ACh and bind to nAChRs, disrupt pupal ecdysis in Lepidoptera7. This phenomenon, which we termed arrested ecdysis (AE), is the process in which an organism initiates but does not finish ecdysis. Pupal AE is characterized by delayed initiation of pupal ecdysis, incompletely shed larval cuticle (often with no breakage of the larval ecdysial line), and non-expansion of pupal appendages. As release of peptides from CCAP neurons are responsible for the successful and timely completion of ecdysis and successful expansion of pupal and adult appendages8,9,10, we hypothesized that neonicotinoids are interfering with the release of CCAP neuronal peptides, likely by preventing sufficient and timely disinhibition of CCAP neurons during ecdysis7. In other words, we hypothesized that the excitatory neurotransmitter ACh regulates the function of CCAP neurons, likely by binding to nAChRs on CCAP neurons or their upstream efferent inhibitory (likely GABA-releasing) neurons5.

As our hypothesis suggests that perturbation of ACh signaling would interfere with the activation and/or secretion of peptides from CCAP neurons leading to AE, we assessed two classes of insecticides that disrupt ACh signaling: neonicotinoid and organophosphate insecticides. Neonicotinoids bind to and activate nAChRs; however, these insecticides cannot be hydrolyzed by ACh esterase (AChE). Organophosphates irreversibly bind to AChE, which prevents the hydrolysis of ACh and results in constant activation of nAChRs, as well as muscarinic ACh receptors (mAChRs), another target receptor of ACh.

Neonicotinoids can be subdivided based on whether they contain a nitro (e.g., imidacloprid and nitenpyram) or a cyano functional group (e.g., acetamiprid and thiacloprid) as well as their receptor subunit interaction profiles11. Studies in Drosophila11,12 have found that different neonicotinoid insecticides bind to different subunits of nAChRs. Imidacloprid and thiacloprid bind to nAChR subunits α1, α2, β1, β2, acetamiprid binds to α1, β1, β2, and nitenpyram binds to either α1, β1, β2 or α1, α3, β1. In addition, sulfoxaflor, a non-neonicotinoid nAChR agonist, binds to either nAChR subunits β1 or α3, β1. Nicotine is believed to be a non-selective nAChR agonist in insects13. We assessed the potency of six nAChR agonists on eliciting AE to explore if different nAChR subunits may be associated with AE. Since toxicokinetic factors (absorption, metabolism, and excretion) can also influence potency in eliciting AE, we analyzed internal larval concentrations of three compounds with differing AE susceptibilities.

Since CCAP neurons in Drosophila also contain mAChRs5, we undertook studies with organophosphate insecticides to evaluate the role of mAChRs in pupal ecdysis. If mAChRs are involved, we would expect to see very high rates of AE following exposure to low doses of chlorpyrifos, a thionate organophosphate, and naled, an oxon organophosphate, as deactivation of AChE would result in continuous activation of both nAChRs and mAChRs. To further explore the potential role of mAChRs in pupal ecdysis and AE, we undertook experiments with pilocarpine, a mAChR agonist.

A fourth set of studies were undertaken to determine if AE in Lepidoptera could also occur during the pupal to adult molt (i.e., adult ecdysis). We had previously determined, in both corn earworms (Helicoverpa zea) and monarch butterflies (Danaus plexippus), that AE did not occur between larval-to-larval molts7,14,15. However, our preliminary studies indicate that with three beetle species [red flour beetle (Tribolium castaneum), mealworm (Tenebrio molitor), and Colorado potato beetle (Leptinotarsa decemlineata)], AE occurred during the pupal to adult molt. Beetle pupae exposed to low doses of neonicotinoids incompletely shed the pupal cuticle and failed to expand adult wings. We therefore undertook studies with Lepidoptera pupae to determine if ACh agonists could cause arrested adult ecdysis.

Finally, to further evaluate our hypothesis of CCAP neuronal involvement in AE, we undertook detailed observations of pupal ecdysis movements between control and neonicotinoid-treated larvae and compared them with published findings in CCAP neuron-knockout (CCAP-KO) Drosophila larvae8. We also evaluated the time taken for larvae to complete various ecdysis stages in final-instar larvae.

Studies described in the current paper were undertaken with two lepidopteran species: corn earworms and fall armyworms (Spodoptera frugiperda). Both species are polyphagous noctuid moths that have a similar life cycle and size16. We previously determined that they had very different susceptibilities to imidacloprid7. We further explore this difference in susceptibility by undertaking studies with a suite of nAChR agonists and organophosphates.

Results

For both corn earworms and fall armyworms, low rates of control mortality were observed across different life stages and routes of exposure (see Table 1). Given these low mortality rates (≤ 6%), the mortality in the treatment groups were not corrected. In acetone-treated corn earworms, no AE symptomology was observed and 100% of larvae that successfully pupated emerged as adults. With acetone-treated fall armyworms, four out of the five dead larvae exhibited AE symptomology, indicating background levels of AE in this species. Of the fall armyworm larvae that successfully pupated, 99% emerged as adults (104 adults from 105 pupae).

Table 1 Mortality in the control (acetone-treated) group for corn earworms and fall armyworms.
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Assessing the potency of nAChR agonists in eliciting pupal AE

In corn earworms, topical exposure to 20 µg/larva imidacloprid and nitenpyram resulted in 87 and 57% AE respectively, with no larval mortality (Fig. 1a and S1). Acetamiprid and thiacloprid were more potent, with 1 µg/larva causing 73 and 93% AE, respectively, while 20 µg/larva caused 100 and 90% AE, respectively. Fall armyworms were significantly less susceptible to neonicotinoids (p < 0.0001): topical exposure of 200 µg/larva imidacloprid, nitenpyram, acetamiprid, and thiacloprid caused 0, 20, 7, 13%, respectively, of combined mortality and AE (Fig. 1b and S1). Studies with 20 µg/larva sulfoxaflor caused no mortality or AE in both species (data not shown). At 200 µg/larva, sulfoxaflor caused 17% (fall armyworm) to 30% (corn earworm) combined mortality and AE. We also undertook screening studies with soybean looper (Chrysodeixis includens) larvae and found they responded similarly to corn earworm larvae; treatment with 20 µg imidacloprid, 20 µg nitenpyram, and 100 µg sulfoxaflor caused 93, 20, and 0% combined mortality and AE, respectively (Table S1).

Fig. 1
Fig. 1The alternative text for this image may have been generated using AI.Fig. 1The alternative text for this image may have been generated using AI.
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Stacked barplots depicting percent of larval mortality (red) and pupal AE (green) observed following topical exposure to nAChR agonists in (a) sixth instar corn earworms and (b) sixth instar fall armyworms. Corresponding Figure S1 includes error bars.

Following dietary exposure, 500 µg/g diet of imidacloprid, acetamiprid, and thiacloprid caused between 95 and 100% AE in corn earworms, with no larval mortality (Fig. 2a and S2). Thiacloprid was the most potent with 50 µg/g diet causing 97% AE. Nitenpyram was the least effective neonicotinoid, with 500 and 1000 µg/g diet causing 47 and 75% AE, respectively. A diet concentration of 1000 µg/g sulfoxaflor or nicotine caused 20 and 0% AE, respectively. In fall armyworms, dietary exposure to neonicotinoids caused no mortality or AE at 125 µg/g and caused 5 to 20% combined mortality and AE at 1000 µg/g (Fig. 2b and S2). Thus, fall armyworms are also significantly less susceptible to dietary neonicotinoid exposure as compared to corn earworms (p < 0.0001). Additionally, fall armyworms did not exhibit mortality or AE at dietary concentrations of 1000 µg/g sulfoxaflor or nicotine.

Fig. 2
Fig. 2The alternative text for this image may have been generated using AI.Fig. 2The alternative text for this image may have been generated using AI.
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Stacked barplots depicting percent of larval mortality (red) and pupal AE (green) observed following dietary exposure to nAChR agonists in (a) sixth instar corn earworms and (b) sixth instar fall armyworms. Corresponding Figure S2 includes error bars.

Across insecticide groups, 96–100% of corn earworm and fall armyworm larvae that successfully pupated following topical exposure to nAChR agonists emerged as adults. Following dietary exposure, 96–100% of corn earworm larvae and 89–97% of fall armyworm larvae that successfully pupated emerged as adults (Table S2).

Assessing toxicokinetic differences of nAChR agonists in eliciting pupal AE

Toxicokinetic studies were undertaken with nominal stock solution concentrations of 20 µg/µL imidacloprid, nitenpyram, and sulfoxaflor, which corresponded to measured stock solution concentrations of 22.4, 15.9, and 17.3 µg/µL, respectively. At 0 h, the mean internal concentrations of corn earworm and fall armyworm larvae treated with 22.4 µg imidacloprid were 38.4 ± 11 and 38.5 ± 8 µg/g, respectively (Fig. 3). At 6 h, the internal concentrations dropped to 25.8 ± 14 (p = 0.2) and 17.5 ± 10 (p = 0.02) µg/g, respectively. At 24 h, both corn earworms and fall armyworms had significantly lower concentrations compared to 0 h (p < 0.001) and, at pupation, only 2 and 1 samples, respectively, had detectable/quantifiable concentrations. When corn earworm and fall armyworm were provided 15.9 µg/larva nitenpyram, the mean internal concentrations at 0 h were 19.2 ± 4.5 and 26 ± 6.4 µg/g, respectively. For both species, the mean internal concentrations significantly dropped at 6 h and 24 h (p < 0.001), with all samples having non-detectable/non-quantifiable concentrations at pupation. When provided 86.5 µg sulfoxaflor/larva (or 5 µL of 17.3 µg/µL), corn earworms and fall armyworms had mean internal concentrations of 93.2 ± 34 and 61.4 ± 14 µg/g, respectively, at 0 h. For both species, the mean internal concentrations significantly dropped at 6 h (p = 0.02 and p = 0.002, respectively) and 24 h (p < 0.001), with most/all samples having non-detectable/non-quantifiable concentrations at pupation (Fig. 3).

Fig. 3
Fig. 3The alternative text for this image may have been generated using AI.
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Mean internal concentrations (± standard deviation) of imidacloprid, nitenpyram, and sulfoxaflor in corn earworm (A, B, C) and fall armyworm (D, E, F) final instars. Larvae were collected at the following time points after topical treatment with 20 µg imidacloprid, 20 µg nitenpyram, or 100 µg sulfoxaflor: 0 h, 4 h, 24 h, and after arrested ecdysis or pupation (AE/P). Different lower-case letters above bars denote significant differences (p < 0.05) between control and treated larvae using Dunnett’s test for multiple comparisons.

Assessing the potency of organophosphates in eliciting pupal AE

In corn earworms, topical exposure to 0.2, 2, 20, and 200 µg chlorpyrifos/larva resulted in 0, 30, 63, and 95% combined mortality and AE (percentage of AE ranged from 0 to 33%), respectively (Fig. 4a and S3). Fall armyworms were more susceptible than corn earworms when exposed to 2 and 20 µg/larva chlorpyrifos (p = 0.0002 and p = 0.002, respectively) and these doses caused 80 and 100% combined mortality and AE, with 21 and 30% AE, respectively (Fig. 4b and S3). Similar to chlorpyrifos, naled was more toxic to fall armyworms but only at a dose of 20 µg/larva (p < 0.0001). While 20 µg naled/larva caused 10% combined mortality and AE in corn earworms, it caused 95% combined mortality and AE in fall armyworms. Corn earworms exposed to 200 µg naled/larva had 85% combined mortality and AE (47% AE).

Fig. 4
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Stacked barplots depicting percent of larval mortality (red) and pupal AE (green) observed following topical exposure to organophosphates in (a) sixth instar corn earworms and (b) sixth instar fall armyworms. Corresponding Figure S3 includes error bars.

Results of dietary exposure to chlorpyrifos and naled in corn earworms are shown in Fig. 5 and S4. Increasing concentrations of chlorpyrifos and naled had increased rates of non-AE mortality. Fall armyworms were again more susceptible to the compounds; for example, when treated with 25 µg/g chlorpyrifos and naled, they had significantly more mortality/AE (83 to 95%) compared to corn earworms exposed to 50 µg/g of the same compounds (35 to 60%; p = 0.0003 and p = 0.008, respectively).

Fig. 5
Fig. 5The alternative text for this image may have been generated using AI.
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Stacked barplots depicting percent of larval mortality (red) and pupal AE (green) observed following dietary exposure to organophosphates in (a) sixth instar corn earworms and (b) sixth instar fall armyworms. Corresponding Figure S4 includes error bars.

Across insecticide groups, 93–97% of corn earworm and fall armyworm larvae that successfully pupated following topical exposure to organophosphates emerged as adults. Following dietary exposure, 93–100% of corn earworm larvae and 73–100% of fall armyworm larvae that successfully pupated emerged as adults (Table S2).

In corn earworms, topical studies with 20 µg pilocarpine/larva resulted in 15% larval mortality with no AE (see Table S3). In fall armyworms (n = 20), the same dose caused 10% larval mortality and 5% AE. In both species, all the larvae that successfully pupated emerged as adults.

Assessing the potency of nAChR agonists and organophosphates in eliciting adult AE

None of the treated pupae exhibited arrested adult ecdysis. Following topical exposure to 200 µg of nAChR agonists, 0 to 20% of corn earworm pupae did not emerge as adults. In fall armyworms, this ranged from 0 to 15% (p = 0.1; Figure S5). Topical exposure to 20 and 200 µg of organophosphate insecticides caused significantly more pupal mortality in fall armyworms (65–100%) compared to corn earworms (0–55%; p < 0.0001).

Evaluating corn earworm pupation events in control and AE larvae

All 17 acetone-treated corn earworm larvae successfully pupated while all acetamiprid-treated larvae had AE. Out of 20 imidacloprid-treated larvae, 13 had AE and were analyzed. Similar to what we had observed previously7, AE larvae had delayed initiation of pupal ecdysis, with acetamiprid-treated larvae initiating ecdysis several days after acetone-treated larvae (data not shown). Five acetamiprid-treated (56%) and 11 imidacloprid-treated (85%) larvae initiated abdominal peristalsis; the remaining tanned without underdoing peristalsis and subsequent pupation events. Fewer neonicotinoid-treated larvae proceeded to the next stage as pupation progressed, with no acetamiprid-treated larvae initiating shedding of the fifth tracheal segment and only one imidacloprid-treated larvae successfully freeing its head (Fig. 6). Additionally, acetamiprid- and imidacloprid-treated larvae showed significantly slower progression of ecdysial events following peristalsis. Compared to controls, acetamiprid-treated larvae took 11 to 23 times longer to initiate shedding of various tracheal segments (p values calculated when two or more larvae were present range from 0.01 to 0.002) and larvae dosed with imidacloprid took 2 to 3 times longer to initiate shedding of various tracheal segments and break the ecdysial line, although only 2 out of 13 progressed to that stage (p values range from 0.07 to 0.0009) (see Fig. 7 and Table S4).

Fig. 6
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Percentage of control (17 treated with acetone) and arrested ecdysis (13 treated with 20–40 µg imidacloprid and 9 treated with 20 µg acetamiprid) larvae initiating various pupation events (see Table 2 for details).

Fig. 7
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Mean (± standard deviation) time taken for control (acetone-treated) and arrested ecdysis (imidacloprid- and acetamiprid-treated) larvae to initiate various pupation events following start of abdominal peristalsis (see Table 2 for details).

Table 2 Denotation and definition of various pupation events observed.
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We also undertook a more qualitative observation of acetone- and neonicotinoid-treated larvae undergoing pre-ecdysis and ecdysis movements. Acetone-treated larvae progressed steadily through pre-ecdysis and ecdysis with strong consistent motions. At the end of pre-ecdysis movements they started rolling with writhing body movements, followed by posterior to anterior peristalsis of abdominal segments to initiate the shedding of the larval cuticle and tracheal lining. Neonicotinoid-treated larvae progressed more slowly through pre-ecdysis. Larvae that did not reach ecdysis performed mild flexing and wriggling while larvae that progressed to ecdysis exhibited slower and less robust pre-ecdysis behaviors than acetone-treated larvae. As neonicotinoid-treated larvae spent longer time in pre-ecdysis, they often achieved a similar number of repetitions of each behavior, just at a slower rate and less rapidly. Finally, we observed two of the four imidacloprid-treated larvae perform reverse peristalsis of abdominal segments, i.e., peristalsis that progressed from the anterior to posterior.

Discussion

In fall armyworms, background levels of AE symptomology were observed in acetone-treated larvae and also untreated colony larvae. While only 5% of acetone-treated larvae did not pupate, this included 4% AE larvae (i.e., 4 out of 5 non-pupated acetone-treated larvae had AE). In addition, only low levels of AE were observed in fall armyworm larvae that had topical and dietary exposure to neonicotinoids (0 to 17% of treated larvae). Our toxicokinetic analysis indicates that fall armyworms and corn earworms have similar rates of neonicotinoid metabolism/excretion in larvae. It is possible that interspecies variations of AE responses could be due to variations in sequences of nAChR subunit binding domains or differences in regulation of pupal ecdysis. Neither of these hypotheses can be currently tested as we lack adequate information on nAChR binding domains and pupal ecdysis regulation in corn earworms and fall armyworms.

Fall armyworms that had topical or dietary exposures to organophosphates had relatively low levels of AE (2 to 32% of larvae that did not pupate had AE), particularly when compared to acetone AE levels (80% of larvae that did not pupate had AE). Interestingly, fall armyworms were significantly more susceptible than corn earworms to organophosphates. This difference between the two species could be due to several factors, including binding potential to AChE and organophosphate metabolism/excretion rates. This difference in susceptibility was also observed in other instars; for example, second instar fall armyworms were ten times more susceptible to chlorpyrifos (LD50 = 0.03 µg/larva17) than third instar corn earworms (LD50 = 0.4 µg/larva18). Additionally, our topical pupal studies also indicate that fall armyworms have greater susceptibility to organophosphates.

In corn earworms, AE symptomology was caused by insecticidal disruption of pupal ecdysis as none of the controls (acetone-treated or untreated colony larvae) exhibited AE. Additionally, larval mortality was rare in neonicotinoid-treated larvae; nearly 100% of non-pupation incidences (following both topical and dietary exposures) were due to AE. Acetamiprid and thiacloprid were more potent at inducing AE compared to imidacloprid and nitenpyram. While both sulfoxaflor and nicotine are also agonists of nAChRs, they caused significantly less AE. Toxicokinetic analysis of sulfoxaflor, which showed a similar rate of larval degradation/excretion as imidacloprid and nitenpyram at 24 h post-exposure, indicates that the differences in susceptibilities could be due to toxicodynamic factors. Specifically, differences in nAChR subunit binding could contribute to differences in susceptibility; in Drosophila, all four neonicotinoids bind to α1, β1, and potentially β211,12. Sulfoxaflor does not bind to α1 or β2 and nicotine is believed to be a non-selective nAChR agonist in insects13. While lepidopteran nAChR subunits are not well annotated, our findings suggest that in Lepidoptera, amino acid sequences corresponding to α1, β1, and β2 subunit receptors in Drosophila could largely mediate AE. This finding is similar to Christesen et al.6. , where they found that CCAP neurons mediate adult wing expansion through the β1 nAChR subunit in Drosophila.

With topical and dietary exposures to organophosphate insecticides, which should increase muscarinic and nicotinic ACh signaling through inhibition of AChE, the rate of AE was significantly lower than what was observed with neonicotinoids. Additionally, pilocarpine, a mAChR agonist, caused no AE. These data suggest that AE is primarily mediated through nAChRs.

In preliminary studies, we found that beetle pupae of three species (red flour beetle, mealworm beetle, and Colorado potato beetle) topically treated with low doses of neonicotinoids exhibited arrested adult ecdysis, i.e., the adult could not completely emerge from the pupal cuticle. The incompletely emerged adults also had unexpanded and stunted wings. In red flour beetle and Drosophila, CCAP neurons were shown to be responsible for successful adult ecdysis and adult wing expansion6,8,9. Thus, we wanted to determine if arrested adult ecdysis would also occur in Lepidoptera. Our findings indicate this is not the case, as we did not observe AE symptomology in the transition from pupal to adult stage following topical exposures to neonicotinoids and organophosphates (also see15 which presents similar results for monarch butterfly pupae). This indicates that different neurotransmitters, peptides, or subunit sequences could be responsible for the differences in AE susceptibilities between lepidopteran larvae and pupae and lepidopteran and coleopteran pupae. There could also be interspecies differences in toxicokinetics; for e.g., differences in absorption through lepidopteran and coleopteran pupal cases or differing metabolic rates.

Our video analyses of AE larvae show most had initiated peristalsis (73%) and tracheal shedding (68%) but only a few could successfully shed all tracheal segments (14%). Fewer larvae could break their ecdysial line (9%) and free their head (5%). These data indicate AE larvae can often initiate ecdysis but not complete it. As CCAP neuronal peptides are needed to complete, but not initiate, ecdysis8,9,10, this suggests that neonicotinoids are unlikely to disrupt signaling events upstream of the CCAP neurons. Additionally, a couple of neonicotinoid-treated larvae exhibited anterior to posterior peristalsis (a behavior not observed in controls); this behavior is similar to that observed in CCAP neuron-KO Drosophila larvae8. As acetamiprid-treated larvae took longer to undergo tracheal shedding and fewer larvae progressed through pupation, our data also demonstrate that, in Lepidoptera, neonicotinoids containing cyano-functional groups (e.g., acetamiprid) are more potent than neonicotinoids containing nitro-functional groups (e.g., imidacloprid).

Our results suggest that many lepidopteran larvae, with the exception of fall armyworms and European corn borers (Ostrinia nubilalis; see7), exhibit AE when exposed to relatively low doses of neonicotinoids. Screening bioassays with soybean loopers show they behave similarly to corn earworms; none of the acetone-treated and untreated larvae in the colony exhibited AE while topical exposure to neonicotinoids caused AE. Previously we had determined that neonicotinoid-induced AE was also prevalent in other final-instar lepidopteran larvae, including monarch butterflies, wax moths (Galleria mellonella), and painted ladies (Vanessa cardui)7. Topical exposure of 20 µg imidacloprid to sixth-instar true armyworms (Mythimna unipuncta) also produced high rates of AE (70% AE, with no mortality/AE in control larvae).

Based on data gathered to date, we propose that ACh (through nAChRs) regulates the release of peptides from CCAP neurons during lepidopteran pupal ecdysis. Specifically, we hypothesize that ACh regulates CCAP interneurons directly or through upstream inhibitory GABAergic neurons that contain nAChRs. We had previously proposed detailed mechanisms to describe ACh’s role in lepidopteran pupal ecdysis, and how exposure to low doses of neonicotinoids could lead to arrested ecdysis7. Specifically, we had proposed that neonicotinoids could (a) desensitize nAChRs on CCAP neurons at low concentrations19,20, thereby reducing their excitability and release of neuropeptides or (b) activate nAChRs on upstream GABA neurons resulting in continual inhibition of CCAP neurons7. Additional mechanistic research is needed to test our proposed hypothesis for ACh involvement in regulation of CCAP neurons.

While the present paper has increased our understanding of the potential role of ACh in insect pupal ecdysis, the possible role of GABA needs further study. Preliminary studies undertaken in our lab with the GABA antagonist, fipronil, and type II pyrethroids, beta-cyfluthrin and cypermethrin, caused relatively high rates of AE in corn earworms, suggesting GABA involvement in the regulation of pupal ecdysis. While the primary mode of action of pyrethroid insecticides concerns modifying the activity of voltage-gated sodium channels, studies in the malaria mosquito, Anopheles gambiae, and rats have shown a secondary mode of action involving inhibition of GABA signaling with type II pyrethroids21,22, which are more potent than Type I pyrethroids in inhibiting GABA-induced Cl- influx22. Future studies are needed to assess potential GABA involvement, in combination with ACh signaling, in the regulation of lepidopteran pupal ecdysis.

Methods

Insects and chemicals

Corn earworm. fall armyworm, and soybean looper eggs were purchased from Benzon Research, Carlisle, PA. Upon hatching, the larvae were plated individually into 1-oz plastic cups containing Stonefly Heliothis diet (Ward’s Sciences, West Henrietta, NY) provided ad libitum. The larvae were reared in an incubator maintained at 26.7ºC and 16:8 light: dark cycle. The average weights and surface areas (measured using methods described in14) of two-day-old sixth-instar corn earworms, fall armyworms, and soybean loopers were 0.355 g and 4.55 cm2, 0.266 g and 3.59 cm2, and 0.26 g and 4.27 cm2 (n = 15 each), respectively.

Analytical grade insecticides (IUPAC name; CAS number; percentage purity) were purchased from Sigma-Aldrich, St. Louis, MO. This included imidacloprid (N-[1-[(6-chloropyridin-3-yl)methyl]-4,5-dihydroimidazol-2-yl]nitramide; 138261-41-3; 100%), nitenpyram ((E)-1-N’-[(6-chloropyridin-3-yl)methyl]-1-N’-ethyl-1-N-methyl-2-nitroethene-1,1-diamine; 150824-47-8; 99.7%), acetamiprid (N-[(6-chloropyridin-3-yl)methyl]-N’-cyano-N-methylethanimidamide; 160430-64-8; 99.9%), thiacloprid ([3-[(6-chloropyridin-3-yl)methyl]-1,3-thiazolidin-2-ylidene]cyanamide; 111988-49-9; 99.8%), chlorpyrifos (diethoxy-sulfanylidene-(3,5,6-trichloropyridin-2-yl)oxy-λ5-phosphane; 2921-88-2; 98.5%), and naled ((1,2-dibromo-2,2-dichloroethyl) dimethyl phosphate; 300-76-5; 95.2%). Sulfoxaflor ([methyl-oxo-[1-[6-(trifluoromethyl)pyridin-3-yl]ethyl]-λ6-sulfanylidene]cyanamide; 946578-00-3; 95.6%) was provided by Corteva AgriScience (Indianapolis, IN). Certified ACS reagent grade acetone was purchased from Fisher Scientific (Hampton, NH) and used for stock solution preparations and serial dilutions. Definitive doses and concentrations in the topical and dietary studies were determined using data from Krishnan et al. 20217 and by undertaking preliminary range-finding studies (see Table S3).

Assessing the potency of nAChR agonists in eliciting pupal AE

Typically, two-day-old sixth-instar larvae (which were within two to three days of pupal ecdysis) were used. The larvae were randomly assigned to treatments. The following nAChR agonists were assessed: imidacloprid, nitenpyram, acetamiprid, thiacloprid, sulfoxaflor, and nicotine.

For topical studies, the highest stock solution concentration prepared was 20 µg/µL, as this concentration was approaching the solubility limit of most insecticides. Serial dilutions were made to obtain lower concentrations (down to 0.001 µg/µL). To obtain larval doses ≤ 20 µg, one µL of the insecticide-acetone solution (using a calibrated 0.1 to 2.5 µL pipette) was placed on the dorsal prothorax. To administer higher doses, up to 200 µg/larva, correspondingly higher volumes of the 20 µg/µL was placed on the dorsal prothorax. Control larvae were provided 10 µL acetone, which was the highest volume applied in the treatments. Ten larvae were employed for each insecticide concentration and the studies were repeated in duplicate or triplicate (where each replicate was obtained from a different generation), depending on the consistency of the results across runs. Following treatments, daily observations of mortality, AE, and adult eclosion were recorded.

For dietary studies, insecticide diets were prepared by incorporating insecticide-acetone solution in the water: vinegar mixture of the Stonefly Heliothis diet. The liquid mixture was then mixed thoroughly with the Stonefly powder. Control diets only contained acetone; the highest volume of acetone that did not cause mortality in corn earworms and fall armyworms was 1000 µL per 20 g diet. As our highest insecticide stock solution concentration was 20 µg/µL, the highest insecticide diet concentration we could obtain was 1000 µg/g. Approximately 2 g of diet was provided to each larva. Ten larvae were employed for each insecticide concentration and the studies were repeated in duplicate or triplicate. Following treatments, daily observations on mortality, AE, and adult eclosion were recorded.

Assessing toxicokinetic differences of nAChR agonists in eliciting pupal AE

To assess toxicokinetic differences of nAChR agonists that elicit pupal AE responses, individual sixth instar corn earworms and fall armyworms were topically treated with 20 µg imidacloprid (elicits high levels of AE), 20 µg nitenpyram (intermediate levels of AE), or 100 µg sulfoxaflor (low levels of AE). The larvae were collected at the following time points after application: 0 h, 6 h, 24 h, and after pupation or AE. For each timepoint, five samples were collected. Each sample consisted of two larvae, individually treated but combined for analysis (to obtain the mass necessary for successful extraction and detection of the insecticides). The larvae were frozen in plastic vials immediately following collection and stored at -80 °C.

To extract the insecticides, larvae samples were first diced into small pieces using scissors and placed in 50 mL plastic conical tube. A 0.4 g portion of the partially homogenized larvae was extracted into acetonitrile. Approximately 1 mL of the extract was transferred to a dispersive solid phase extraction (dSPE) tube containing 150 mg MgSO4, 50 mg PSA, and 50 mg C18. Extracts were diluted with 50:50 acetonitrile: water, and internal standards were added prior to LC-MS analysis. An injection volume of 2 µL was used for all samples.

The samples were injected into a Vanquish Flex LC pump interfaced with a TSQ Altis triple quadrupole mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA). All insecticides were analyzed in positive electrospray ionization mode. The MS ionization source conditions were as follows: spray voltage 3700 V, sheath gas 30 (Arb), auxiliary gas 6 (Arb), sweep gas 1 (Arb), ion transfer tube temperature 325 °C, and vaporizer temperature 350 °C. MS acquisition was performed in selected reaction monitoring (SRM) mode with argon used as the collision gas. The cycle time of the method was 0.4 s, and the chromatographic peak width was 6 s. The resolution of Q1 and Q3 were 0.7 FWHM. Data analysis was performed using Xcalibur 4.5 software (Thermo Fisher Scientific, San Jose, CA).

Chromatographic separation was done using an HypersilGold column (50 × 3 mm, 1.9 μm Thermo Fisher Scientific). The column compartment temperature was 30 °C. Mobile Phase A was water + 5mM ammonium formate + 0.1% formic acid and Mobile Phase B was methanol + 5mM ammonium formate + 0.1% formic acid. The gradient conditions were as follows: start at 10% B, linear ramp to 100% B at 4 min, hold at 100% B for 1 min, drop to 10% B in 0.01 min, and hold at 10% B for 2 min. The flow rate was 0.3 mL/minute, and the total run time of the method was 7 min.

A calibration curve was prepared in control larvae with a range from 0.25 to 31.25 µg/g for imidacloprid and nitenpyram and 1.25–156.25 µg/g for sulfoxaflor. For fall armyworms, quality control (QC) samples were prepared in triplicate at 3.75 µg/g (imidacloprid and nitenpyram) and 18.75 µg/g (sulfoxaflor) and analyzed with study samples. For corn earworms, five replicates of QC samples at 3.75 µg/g (imidacloprid and nitenpyram), 18.75 µg/g (sulfoxaflor), 18.75 µg/g (imidacloprid and nitenpyram) and 93.75 µg/g (sulfoxaflor) were prepared and analyzed with the study samples. All QC samples from both larval species had a calculated concentration within 15% of the nominal value for all insecticides. Five replicates of the limit of quantification (LOQ; 0.25 µg/g for imidacloprid and nitenpyram and 1.25 µg/g for sulfoxaflor) were prepared for corn earworms and analyzed along with the samples. Three replicates of the LOQ (0.25 µg/g for imidacloprid and nitenpyram and 1.25 µg/g for sulfoxaflor) were prepared for fall armyworms and analyzed along with the samples. Bias and precision were calculated at the LOQ for both species.

Assessing the potency of organophosphates in eliciting pupal AE

Topical and dietary studies were undertaken with chlorpyrifos and naled on sixth-instar larvae using the same techniques described in the previous section (Assessing the potency of nAChR agonists in eliciting pupal AE). The highest stock solution concentration prepared for both insecticides was 20 µg/µL in acetone. Preliminary topical studies were also undertaken with pilocarpine; however, its maximum solubility in acetone was ~ 2 µg/µL. We applied 10 µL of the stock solution in both species to achieve a dose of 20 µg pilocarpine/larva.

Assessing the potency of nAChR agonists and organophosphates in eliciting adult AE

Topical studies were undertaken with pupae that were within two to four days of adult ecdysis/emergence. We employed the same stock solutions that were prepared for larval studies; all 8 compounds were assessed. A microliter pipette was used to spread one or ten µL of the insecticide-acetone solution (or acetone) on the pupal spiracles to ensure absorption. Twenty larvae were treated per concentration and observations on adult ecdysis, eclosion success, and appendage expansion were noted.

Evaluating corn earworm pupation events in control and AE larvae

Early to mid-sixth instar corn earworm larvae were provided 20–40 µg imidacloprid (n = 20), 20 µg acetamiprid (n = 9), or acetone (n = 17). When larvae were within a day of pupal ecdysis (i.e., when larval eye pigments had migrated away from the stemmata; see Figure S6), a video camera (Panasonic HC-V180 high definition) was used to record their movements until pupation. The camera was held 8–12 inches above the larvae. The light source was a NOEVSBIG soldering light kept 12–14 inches above the larvae. It was bright white and did not produce heat. We evaluated the time taken (in minutes) for the control and treated larvae to complete various ecdysis stages (see Table 2 for stages and definitions). Additionally, we visually assessed the pre-ecdysis and ecdysis movements of 12 larvae (four each from control, imidacloprid, and acetamiprid) in detail.

Statistical analyses

All statistical analyses were done in RStudio 2023.12.1 (R version 4.3.2). All routes of exposure, life stages, and insecticides were analyzed independently. Mortality and AE data across species and insecticide doses/concentrations were analyzed using a binomial or a quasibinomial (to account for overdispersion) generalized linear model with two-way ANOVA. Whenever treatment effects were significant at the p = 0.05 level, Tukey’s test was employed to undertake pairwise dose/concentration comparisons. To assess internal insecticide concentrations in larvae over time in control- and neonicotinoid-treated larvae, a gaussian generalized linear model was employed. When treatment effects were significant at the p = 0.05 level, Dunnett’s test was employed to undertake comparisons against the control group. One-way ANOVA, followed by the non-parametric post-hoc Mann-Whitney Wilcoxon test, was used to analyze differences in the number of minutes it took larvae in acetone-, imidacloprid-, and acetamiprid-treated larvae to complete various pupation events.