Introduction

Chemical pesticides remain the primary means of controlling many of the world’s most damaging arthropod crop pests (1, 2). However, pesticide applications can result in pest resurgence, increases in pest insect populations that exceed natural, untreated population sizes, following an initial reduction of the pest population (3–5). Two mechanisms have been implicated in pest resurgence - the loss of beneficial insects including natural enemies and pesticide-enhanced pest reproduction (3). In the case of the latter, several pesticides, such as the insecticides triazophos, deltamethrin and the fungicide jinggangmycin, have been reported to stimulate pest reproduction (6–8). Pesticide-enhanced pest reproduction has been linked to changes in physiology and biochemistry of pest organisms after exposure to pesticides (3, 4). However, the molecular mechanisms underlying enhanced reproduction associated with pest resurgence remain poorly resolved.

The brown planthopper (BPH), Nilaparvata lugens (Stål), is a notorious pest of rice crops throughout Asia causing annual losses of ∼300 million dollars across major rice producing countries (2, 3). BPH inhibits the growth of rice plants by feeding, and also transmits highly damaging plant viruses including rice grassy stunt virus and rice ragged stunt virus (9). Currently, chemical insecticides play an indispensable role in the control of BPH due to their efficiency, rapid effect, and low cost. However, due to the widespread and intensive use of chemical insecticides, BPH has developed resistance to the majority of compounds used for control (2, 10).

Emamectin benzoate (EB) and abamectin are avermectin pesticides, and act as allosteric modulators of insect glutamate gated chloride channels (GluCls), inhibiting muscle contractions that lead to the cessation of insect feeding and subsequent death (11). These insecticides exhibit particularly strong activity against Lepidoptera such as the rice leaffolder, Cnaphalocrocis medinalis Guénee, an important foliage-feeding insect which attacks rice during the vegetative stage (12). Both BPH and the rice leaffolder are migratory pests with overlapping migratory paths, however, their occurrence period in the field differs by approximately one month, with leaffolders appearing earlier than BPH. Therefore, the use of EB to control rice leaffolder has the potential to impact BPH arriving later, via exposure to sublethal concentrations of this compound. In this regard, we have observed that when farmers use EB and abamectin to control leaffolders on rice crops in China, BPH outbreaks frequently occur in the same field. While sublethal doses of certain pesticides have been shown to enhance fecundity in BPH, including the insecticides triazophos and deltamethrin (6, 7, 13, 14) and the fungicides carbendazim and jinggangmycin (8), whether avermectins trigger resurgence in BPH via insecticide-enhanced reproduction remains unclear.

Reproduction in insects is influenced by external factors such as light (15), temperature (16), humidity (17) and nutrition (18), and endogenous factors such as the juvenile hormone (JH) (19), ecdysone (20), insulin (21) and TOR (22) pathways (23, 24). Of these, JH has been particularly implicated in insecticide-induced enhanced fecundity, with triazophos and deltamethrin treatments leading to increased circulating JH III titers in BPH females (3). JH is synthesized and secreted by the corpora allata in insects (25), and can promote reproduction by regulating the synthesis and secretion of vitellogenin (vg) in the female fat body, and stimulating the absorption of vg by the developing oocyte (19). However, the regulation of JH is complex (19) and the key actors involved in JH-mediated pesticide-enhanced reproduction remains an open question. Previous finding reported that GluCl receptor is involved in JH biosynthesis in cockroach (26–28). However, we know little whether insecticide which target on the GluCl can stimulate reproduction in insect pest and how GluCl receptor regulates JH production is still unknown.

In this study, we used a diverse range of approaches to investigate the impact of sublethal doses of avermectins on BPH fecundity, and unravel the molecular mechanisms mediating enhanced reproduction following exposure to this insecticide class. We show that avermectin exposure results in profound changes in the expression of a key suite of genes that in combination regulate JH, resulting in increased JH titer in adult females, which promotes fecundity. Interestingly, we found that GluCl channel modulators can inhibit Ast/AstAR expression and then promotes JH increasing.

Results

GluCl allosteric modulators (emamectin benzoate and abamectin) stimulate fecundity of female N. lugens

To investigate whether GluCl modulators affect fecundity in BPH, we first determined the sub-lethal and median lethal doses of emamectin benzoate (EB) to 4^th instar nymphs, newly emerged males and females of BPH (Table S1). For this we employed two different bioassay methods, the rice seedling dip bioassay method and topical application bioassay method (2, 29), in order to assess both the systemic and contact toxicity of these insecticides (Table S1). We then systemically treated 4^th instar nymphs of BPH with the estimated LC₁₅ and LC₅₀ concentrations of EB and examined the fecundity of BPH after these individuals mated with treated or untreated individuals. We use the term “t” to represent individuals treated with EB and “ck” to indicate control individuals that were treated with insecticide diluent minus insecticide. After treatment with the LC₁₅ and LC₅₀ concentrations of EB, the number of eggs laid per female of BPH in ♀t × ♂t crosses increased by 1.48 and 1.40 times compared with control ♀ck × ♂ck crosses (Figure 1A); the number of eggs laid per female of BPH in ♀t × ♂ck crosses increased by 1.53 and 2.07 times compared with control crosses (Figure 1B); However, the number of eggs laid by per female of BPH in ♀ck × ♂t crosses did not increase significantly compared to control ♀ck × ♂ck crosses (Figure 1C).

Figure 1.

Exposure of 4^th instar nymphs with the LC₁₅ and LC₅₀ concentrations of EB in contact bioassays also significantly stimulated fecundity. After treatment with the LC₁₅ and LC₅₀ concentrations of EB, the number of eggs laid by per female of BPH in ♀t × ♂t crosses increased by 1.18 and 1.26 times compared with the control (♀ck × ♂ck) (Figure 1D); The number of eggs laid per female of BPH in ♀t × ♂ck crosses increased by 1.27 and 1.56 times compared with the control crosses (Figure 1E); However, there was no significant difference in egg-laying number between ♀ck × ♂t crosses and controls (♀ck × ♂ck) (Figure 1F). These results reveal that EB can stimulate the fecundity of females following both the systemic and contact routes of exposure.

We next examined whether EB treatment of adult BPH also stimulates reproduction. Indeed, treating newly emerged adults with the LC₁₅ and LC₅₀ concentrations of EB significantly stimulated the number of eggs laid per female (Figure 1-figure supplement 1A). Furthermore, sub-lethal exposure of 4^th instar BPH nymphs to another GluCl allosteric modulator, abamectin (LC₁₅ and LC₅₀ concentrations) was also found to significantly enhance reproduction (Figure 1-figure supplement 1B).

To examine if EB also stimulates egg-laying in other insect species we conducted bioassays on the small brown planthopper, Laodelphax striatellus, the white backed planthopper, Sogatella furcifera and fruit flies Drosophila melanogaster. In contrast to our findings on BPH, we found that sub-lethal doses (LC₁₅ and LC₅₀) of EB inhibits fecundity of female L. striatellus, (Figure1-figure supplement 2A-C) and has no impact on the fecundity of S. furcifera, (Figure1-figure supplement 2D-F). In addition, we found that sublethal doses (LC₁₅ or LC₅₀) of EB also inhibit fecundity in D. melanogaster (Figure1-figure supplement 2G and H). These results indicate that the stimulation of reproduction by EB in BPH is species-specific and does not extend to even related insect species.

The impact of EB treatment on BPH reproductive fitness

To better understand the effects of EB on the reproductive fitness of BPH, the preoviposition period, emergence rate, female ratio, female longevity and female weight were evaluated following exposure using the systemic bioassay. The preoviposition period of females treated with the LC₅₀ of EB decreased significantly compared with the control (Figure 1-figure supplement 3A). In contrast no significant effects of EB on emergence rate and female ratio were observed (Figure 1-figure supplement 3B and C). Female survival analysis showed that exposure of 4^th instar nymphs to the LC₅₀ of EB has no impact on female longevity (Figure 1-figure supplement 3D). Interestingly, brachypterism (long-wing) female ratio and female weight were significantly increased after EB exposure (Figure 1-figure supplement 3E and F).

EB promotes ovarian development in BPH

To investigate the cause of increased female weight following EB exposure we examined if EB influences ovarian development in BPH. We dissected and compared the ovaries of females treated with the LC₅₀ of EB at 1, 3, 5 and 7 days after eclosion (DAE) with control females. At 3, 5 and 7 DAE, the number of detained eggs of BPH in the EB treated group were significantly higher than that of controls (Figure 2A and B). We also explored whether EB treatment could enhance or impair oogenesis in BPH. However, dissection of various developmental stages revealed that emamectin benzoate treatment has no significant effects on oogenesis (Figure 2C and D).

Figure 2.

EB exposure enhances the abundances of storage macromolecules and circulating sugars in BPH

Nutritional status is an important indicator of reproductive fitness. Thus, to investigate whether EB affects intermediary metabolism and energy storage of BPH, glycogen, triacylglyceride (TAG), total protein content, cholesterol and four circulating carbohydrates were quantified in 4^th instar BPH nymphs following exposure to the LC₅₀ of EB.

We found that EB exposure has no impact on glycogen levels (Figure 2-figure supplement 1A). The amounts of TAG in EB-treated BPH were 27% higher (p < 0.05) than those in controls, but only in BPH of the late fifth instar (5L) stage, with no significant differences observed in subsequent developmental stages (Figure 2-figure supplement 1B). The amount of total protein content in EB-treated BPH was higher than the control groups in the case of all developmental stages from 5L nymph to 7DAE (Figure 2-figure supplement 1C). EB exposure also increased cholesterol levels at 4 and 5 DAE (Figure 2-figure supplement 1D). Compared with the solvent control, EB treatment caused significant increases (p < 0.05) in the levels of sucrose, glucose, fructose, and trehalose (Figure 2-figure supplement 1E-H). Thus, collectively, these data provide evidence that EB exposure leads to energy mobilization and the metabolism of carbohydrates and lipids in BPH.

EB stimulates egg-laying that is mediated by the JH signaling pathway

Given the important role of juvenile hormone (JH) in vitellogenesis and egg development in insects (19, 25, 30–32), we asked whether EB-treatment could influence the titer of JH in BPH. As measured by ELISA, the juvenile hormone titer of BPH nymphs treated with the LC₅₀ concentration of EB was significantly lower than that of controls in systemic bioassays during the middle and late stages of the 4^th instar (Figure 3A). However, at 2, 3 and 4 DAE, the JH titer in the EB treated group was significantly higher than that of the control (Figure 3A). Interestingly, the titer of another important insect hormone, the steroid ecdysone, was not significantly different between EB-treated BPH and solvent-treated controls (Figure 3-figure supplement 1). To independently validate the results of ELISA assays, we employed HPLC-MS/MS to measure JH titer in BPH following EB exposure (30, 33, 34). The results showed that JH III titer significantly decreased after EB-treatment at the late 4^th instar nymph stage (Figure 3A and B), but significantly increased at the third day after eclosion (3 DAE) (Figure 3A and C). To further investigate the role of JH in EB- enhanced fecundity in BPH, we treated BPH with methoprene and pyriproxyfen, JH analogues or biologically active JH mimics respectively, to determine whether they can stimulate fecundity in BPH. Both compounds significantly increased egg-laying in BPH (Figure 3D). Takes together these results reveal that EB stimulates an increase in JH titer that induces fecundity in BPH. Since we found that EB could induce JH synthesis in the BPH, we asked whether EB could influence the expression of genes that are involved in JH synthesis or degradation. For this we treated 4^th instar nymphs of BPH with the LC₅₀ concentration of EB using systemic bioassays, and then collected early (5E), middle (5M) and late (5L) stage of 5^th instar nymph and 1-7 DAE female adults (DAE) for analysis. Quantitative PCR was then used to examine the expression of key genes previously implicated in the regulation of JH (35, 36).

Figure 3.

Farnesoic acid O-methyltransferase (FAMeT) (37), is known as an important enzyme in the JH biosynthetic pathway, catalyzing methylation of farnesoic acid (FA) to methyl farnesoate (MF) (36). We found that this gene was significantly upregulated in 5M instar nymph to 4 DAE after EB- treatment (1.0-fold to 3.0-fold) (Figure 3E). Another gene, juvenile hormone acid methyltransferase (JHAMT) (36, 38), which is involved in the biosynthesis of JH, was also upregulated in EB-treated BPH at 1 to 5 DAE than controls (1.5-fold to 3.0-fold) (Figure 3F). Methoprene-tolerant (Met), belongs the basic helix–loop–helix Per/Arnt/Sim (bHLH-PAS) family of transcription factors and is the intracellular (nuclear) receptor of JH (39, 40). The levels of met mRNA increased in EB-treated BPH at the 5M and 5L instar nymph and 1 to 5 DAE stages compared to controls (1.7-fold to 2.9-fold) (Figure 3G). Krüppel homolog 1 (Kr-h1), a transcriptional target of JH signaling, is reported to be sensitive to levels of JH and its expression levels are directly correlated with JH titers (35, 41, 42). We found that Kr-h1 was significantly upregulated in the adults of EB-treated BPH at the 5M, 5L nymph and 4 to 5 DAE stages (4.7-fold to 27.2-fold) (Figure 3H). Similarly, the expression levels of vitellogenin (Vg), a key downstream component of JH signaling triggering ovarian development in insects including BPH (43), was markedly increased in females at 2–5 DAE by EB (1.7-fold to 5.5-fold) (Figure 3I). Juvenile hormone esterase (JHE) is the primary JH-specific degradation enzyme that plays a key role in regulating JH titers (44). Interestingly, we observed a significant upregulation of JHE mRNA levels in the early and middle 5^th instar nymph stage followed by downregulation in 5L instar nymphs of EB-treated BPH (1.3-fold to 2.6-fold) (Figure 3J). In combination these results reveal that EB has profound impacts on the expression of key genes involved in the synthesis of JH or downstream signaling pathway genes that might promote egg development.

To further understand whether these JH pathway-related genes were involved in egg-laying behavior in BPH, we performed RNAi experiments to downregulate the expression of JHAMT, Met and Kr-h1 (Figure 3-figure supplement 2A-C). We found that silencing of these three genes downregulated the titer of JH in BPH providing functional evidence of their role in the regulation of JH (Figure 3-figure supplement 2D). Furthermore, silencing of Kr-h1 inhibited FAMeT and Vg gene expression while increasing JHE gene expression (Figure 3-figure supplement 2E-H). Importantly, silencing of JHAMT, Met and Kr-h1 gene in female BPH was also found to suppress egg-laying (Figure 3K-M). However, this phenotype was rescued by EB treatment, which restored egg-laying to normal levels in BPH injected with JHAMT, Met and Kr-h1 dsRNA (Figure 3K-M). Together these results provide a mechanistic understanding of how EB enhances fecundity in BPH by modulating the expression of key genes involved in JH synthesis (Figure 3N).

EB induces JH biosynthesis through the peptidergic AstA/AstAR signaling pathway

The timing and level of JH biosynthesis can be precisely regulated by the neuropeptides, stimulatory allatotropins (ATs) and inhibitory allatostatins (Asts), in many insects (45–52). Insects can, in a species-specific manner, produce one type of AT and three types of Asts: FGL-amide Ast (AstA) (46, 53); myoinhibitory peptide (MIP or AstB) and PISCF Ast (AstC) (50, 54). In some species, there also exists two paralogue genes of AstCs which are named AstCC and AstCCC (55, 56). Interestingly, the allatostatic activity of these three types of Ast peptides varies between insect species so that in each species only one type of Ast (for example AstA) controls JH production (50, 51, 54, 57).

Analysis of the BPH neural transcriptome sequence data has revealed the presence of one AT, four types of Asts and four corresponding receptors, allatotropin receptor (A16, ATR), AstA receptor (A2, AstAR), AstB (MIP) receptor (A10, AstBR or MIPR) and AstC receptor (A1, AstCR) (58). We cloned the five neuropeptide genes (AT, AstA, AstB/MIP, AstCC and AstCCC) and confirmed the sequence obtained from transcriptome data (Figure 4-figure supplement 1) (58). Interestingly, we found that AstC is missing in the genome of BPH and only AstCC and AstCCC are present (Figure 4-figure supplement 1). Next, we also cloned their corresponding receptors (55) including ATR (A16), AstAR (A2), AstBR (A10) and AstCR (A1) which might be activated by AstCC and/or AstCCC (42, 55, 59). Sequence alignments and phylogenetic analysis are shown in Figure 4-figure supplement 2.

Figure 4.

Quantitative PCR was then used to examine if EB treatment could influence the expression of the genes encoding these neuropeptides and their receptors. Treating BPH with the LC₅₀ concentration of EB significantly increased the expression of AT, ATR and AstCCC while resulting in the downregulation of AstA, AstB/mip, AstCC, AstAR and AstBR/mipr at the adult stage (Figure 4A and B, Figure 4-figure supplement 3A-G). Among these, AstA and AstAR were the most downregulated genes after EB treatment (Figure 4A and B, Figure 4-figure supplement 3H) and thus the AstA/AstAR signaling system was selected for subsequent functional analysis. Silencing of AstAR in female BPH using RNAi (Figure 4C), resulted in an increased number of eggs laid per female compared with dsgfp-injected controls (Figure 4D). Interestingly, silencing AstAR also resulted in the upregulation of FAMeT, JHAMT, Met and Kr-h1 which are involved in the JH biosynthesis/signaling (Figure 4E-H). However, JHE was not influenced by AstAR silencing (Figure 4I). We therefore investigated whether silencing the AstAR gene could influence JH titer in BPH. A significantly increased JH titer was observed in AstAR silenced BPH compared with controls (Figure 4J). Thus, our data strongly suggest that AstA is a key inhibitor of JH production in BPH.

Finally, we investigated whether injection of mature Ast and AT peptides could influence the number of eggs laid and JH titer in BPH. We synthesized one AT, six AstAs (AstA1 to AstA6), one AstCC and one AstCCC peptide according to their determined sequences (Figure 4-figure supplement 1). Indeed, we found that injection of AstA1 and AstA6 reduced the number of eggs laid per female in 48 h (Figure 4K). We also observed that AstA1 injection after 16h and 48h decreased the JH titer significantly (Figure 4L), and AT injection increased the JH titer after 2h but levels returned to normal 4h after injection (Figure 4M). Collectively, these results provide compelling evidence that EB induces reproduction in BPH through the AstA/AstAR and JH signaling pathways (Figure 4N) and further supports the role of AstA and AT in regulation of JH titer.

EB-enhanced fecundity in BPH is dependent on its molecular target protein the GluCl

EB and abamectin, are allosteric modulators, which target glutamate-gated chloride channels (GluCls) (60–62). Hence, we examined whether EB-stimulated fecundity in BPH is influenced by its molecular target GluCl. The full length GluCl coding sequence from BPH was cloned and sequenced (Figure 5-figure supplement 1) and the impact of EB on GluCl gene expression examined using quantitative PCR. Treatment of BPH with the LC₅₀ concentration of EB significantly downregulated GluCl gene expression at the 5E and 5M nymph stages while upregulating GluCl gene expression at 2 DAE and 5 DAE in the adult stage (Figure 5A). To examine the role of GluCl gene in BPH fecundity, RNAi was used to knockdown expression of this gene in female BPH (Figure 5B). A significant decrease in the number of eggs laid by per female was observed in dsGluCl-injected insects compared with dsgfp-injected insects (Figure 5C). However, treatment with EB was found to rescue the decreased egg-laying phenotype induced by dsGluCl injection (Figure 5C). To investigate the mechanism by which GluCl expression modulates fecundity we examined if silencing GluCl influences JH titer and JH-related gene expression. Indeed, we observed that RNAi knockdown of GluCl leads to a decrease in JH titer (Figure 5D) and down-regulation of genes including JHAMT which is responsible for JH synthesis, and the JH signaling downstream genes Met and Kr-h1 (Figure 5E-G). In contrast, expression of FAMeT and JHE were not changed in the GluCl silencing insects (Figure 5H and I). We also examined whether silencing GluCl impacts the AstA/AstAR signaling pathway. Silencing GluCl was found to have no impact on the expression of AT, AstA, AstB, AstCC, AstAR, and AstBR. However, the expression of AstCCC and AstCR was significantly upregulated in dsGluCl-injected insects (Figure 5-figure supplement 2A-H). These results suggest that EB activates GluCl which induces JH biosynthesis and release, which in turn stimulates reproduction in BPH (Figure 5J).

Figure 5.

Discussion

Pesticide-induced resurgence of pest insects is a serious problem in rice and several other cropping systems (3). However, the mechanisms underpinning pesticide-enhanced reproduction in insects remain poorly understood. Here we reveal that a suite of molecular actors underlie this trait that, in combination, mediate profound physiological changes in the reproductive fitness of female BPH. Our data provide fundamental insights into the molecular mechanisms by which xenobiotics modify insect reproduction and have applied implications for the control of a highly damaging crop pest. We discuss these topics below.

Sublethal doses of GluCl modulators, EB and abamectin, stimulates fecundity in BPH

We show that in both contact and systemic assays EB and abamectin stimulate reproduction in BPH. Thus, insecticide-enhanced reproduction is likely a key factor in the BPH resurgence observed when farmers use EB and abamectin to control leaffolders in rice crops in China (29). Although this is the first report of sublethal doses of avermectins enhancing insect fecundity, our findings are consistent with previous studies which have shown that certain insecticides, herbicides and fungicides stimulate BPH reproduction (3, 7, 14, 63–68). Intriguingly, we show that EB only induces fecundity in female adults and is specific to BPH, with EB exposure failing to enhance reproduction in two related species, the small brown planthopper, L. striatellus and the white backed planthopper, S. furcifera, or the model insect D. melanogaster. Thus, the mechanisms underpinning this trait appear to act exclusively on female BPH and may be specific to this species.

Pesticides may stimulate insect reproduction through a variety of physiological and molecular mechanisms. Our data reveal that exposure to sub-lethal concentrations of EB results in profound changes to female BPH fitness, leading to increases in female weight, total protein content, cholesterol content, sugar content and egg production and decreases in duration of the preoviposition period. Some of these findings exhibit parallels with previous studies, which demonstrated that treating third-instar BPH nymphs with either deltamethrin, triazophos, or imidacloprid led to increased soluble sugar levels in the corresponding adults (69). Such metabolites provide the energy that drives BPH reproduction and resurgence (3). Thus, together with prior work, our results suggest that pesticides associated with resurgence stimulate nutritional intake in BPH to fuel enhanced energy-intensive reproduction.

The JH signaling pathway plays an essential role in EB-induced fecundity in BPH

JH is a pleiotropic hormone which plays important roles in development and reproduction in insects (19, 70). Circulating JH titers are regulated by factors that control JH production in the corpora allata including biosynthetic enzymes and catabolic enzymes that regulate JH levels. Our results show that EB increases circulating JH III titers in BPH females over 2–4 days after eclosion (DAE) and promotes ovarian development. Previous studies have reported that triazophos and deltamethrin treatments also lead to increased circulating JH III titers in BPH females over 1–3 days post emergence. Similarly, jinggangmycin treatments were found to lead to increased JH titers (by approximately 45–50%) in BPH females over two days post emergence (71). Thus our findings, in combination with these previous studies, demonstrate that insecticide treatments can have dramatic effects on the regulation of key insect hormones involved in pest reproduction which can in turn drive pesticide resurgence.

Although increased JH titers following pesticide exposure have been correlated with reduced levels of active JH esterase during the first three days PE (6), the type and number of mechanisms mediating the observed increase in hormone titer has remained an open question. Our data reveal that elevated JH titer in EB-exposed BPH is associated with the upregulation of genes that encode biosynthetic enzymes for JH (JHAMT) and downstream signaling genes that can induce vg gene expression (met and kr-h1). Using RNAi we provide functional evidence of the role of these genes in the regulation of JH III and fecundity of female BPH, and demonstrate that EB can restore the reduction in egg production resulting from the knockdown of JHAMT, met and kr-h1 expression.

JHMAT is an enzyme that catalyzes the conversion of inactive precursors of JH to active JH in the final stages of JH biosynthesis (36, 38). Interestingly, while it has not been previously implicated in pesticide resurgence, treatment of the stored product pest Sitotroga cerealella with diallyl trisulfide, an insecticidal compound in garlic essential oil, was found to increase JHAMT mRNA levels (72). Because JHMAT is the key rate-limiting enzyme in regulation of JH titer our results suggest that its enhanced expression is a key molecular mechanism of pesticide resurgence in BPH.

Met is a ligand-activated member of the basic helix–loop–helix Per/Arnt/Sim (bHLH-PAS) transcription factors and is the intracellular receptor for JH (39, 40). Kr-h1 is a zinc finger protein that acts downstream of Met and is expressed in response to JH signaling. Although the genes encoding these proteins have not been previously linked to pesticide resurgence, our finding that they are upregulated following EB exposure, and demonstration of their role of in promoting fecundity, is consistent with previous studies. Specifically, treatment of BPH with JH III or the insecticidal analogues methoprene or pyriproxifen was found to induce the expression of Kr-h1 (73). Furthermore, knockdown of Met and Kr-h1 in BPH brachypterous females was found to result in delayed ovariole development and this was significantly more pronounced than the response observed in BPH treated separately with dsNlMet or dsNlKr-h1 (74). This finding provides evidence of a possible interaction between Met and Kr-h1 and, in combination with our data, suggests that Met and Kr-h1 may act in concert to mediate EB-enhanced fecundity.

EB-induced fecundity in BPH is dependent on the allatostatin signaling pathway

In addition to regulatory proteins that promote JH production, insects have peptides that inhibit JH biosynthesis. These include the allatostatins: FGLamides (FGLa; AstA), the W(X)6Wamides (AstB), and the PISCFs (AstC) (50, 51, 57, 58, 75, 76). Interestingly, our results showed that EB exposure results in the marked downregulation of the expression of the genes encoding allatostatin AstA and its receptor AstAR. We provide clear evidence of the functional impact of this on JH synthesis and BPH fecundity by: i) demonstrating that RNAi knockdown of AstAR expression results in increased JH titer and enhanced female egg production, and, ii) showing that injection of female BPH with synthetic AStA peptide reduces JH titer and decreases egg production. Thus, our data provide unequivocal evidence that AstA is a key inhibitor of JH production in BPH. This finding is consistent with previous work which has shown that FGLa/ASTs inhibit JH biosynthesis in cockroaches, and termites (46, 77). To our knowledge, our study is the first report of insecticides inhibiting the expression of the neuropeptide receptor, AstAR, and linking this to increases in JH titer and enhanced reproduction in insects.

Interestingly knockdown of AstAR resulted in significant increases in the expression of genes involved in JH synthesis/signaling including FAMeT and JHAMT Met and Kr-h1. Related to this finding, previous work has shown that knockdown of the AstA receptor gene, Dar-2, in D. melanogaster results in changes in the expression of genes encoding Drosophila insulin-like peptides (DILPs) and adipokinetic hormone (AKH) signaling proteins (78). Together with our findings, this demonstrates that AstA receptors may modulate the expression of numerous downstream genes involves in metabolism, energy store and reproduction. In the case of pesticide resurgence our results imply significant cross-talk in the expression of genes that inhibit JH production and those that promote it.

The GluCl plays an essential role in EB-induced fecundity in BPH

EB and abamectin are allosteric modulators of GluCls (60–62). Our data revealed that EB exposure modifies expression of the GluCl in BPH, and knockdown of GluCl expression resulted in a reduction in both JH levels and egg production. Interestingly, the GluCl has been reported to inhibit the biosynthesis of JH in the cockroach, Diploptera punctata (28). Recent work has also reported that modulation of glutamatergic signals may contribute to the photoperiodic control of reproduction in bean bug, Riptortus pedestris (79). Interestingly, work on D. punctata has revealed that application of the GluCl channel agonist ivermectin, which like EB belongs to the avermectin family, caused a decline in JH synthesis in corpus allatum glands (28). While the inhibitory effect of ivermectin observed in this previous study differs from the activating effect of EB we observed in our study, it is consistent with our finding of a role for GluCl channel in the regulation of JH regulation. Interestingly, we found that knockdown of GluCl gene expression results in the down-regulation of JHAMT, Met and Kr-h1, further revealing significant convergent relationships between genes underpinning pesticide resurgence.

Conclusion

Our study has revealed a diverse suite of genes that act in combination to enhance JH titer and thus fecundity following BPH exposure to EB. A schematic of how these factors promote ovarian development in the adult stage of N. lugens through the JH signaling pathway is provided in Figure 6. Our findings provide the foundation for further work to understand exactly how these genes interact and the mechanisms by which their expression is activated or repressed by EB. Furthermore, our findings provide fundamental insights into the molecular response in insects to xenobiotic stress and illustrate that pesticides can have unexpected and negative impacts on pest populations. In this regard our findings also have applied implications for the control of a highly damaging crop pest. Previous studies have reported that avermectins such as abamectin are toxic to the wolf spider Pardosa pseudoannulata, which is the main predator of BPH in rice crops (80, 81). Thus, these insecticides both stimulate reproduction in BPH while killing their natural enemies providing a ‘perfect storm’ for damaging BPH outbreaks. Based on these findings, to avoid BPH resurgence, we suggest that the GluCls, EB and abamectin, should not be (or rarely be) applied to rice plants at growth stages when BPHs are present. On a more optimistic note, our findings have identified numerous genes that play key roles in BPH reproduction and thus represent promising targets for the development of novel controls against this important pest.

Figure 6.

Materials and methods

Insects

BPH was initially collected from Wanan, JiangXi Province in 2020, reared on ‘Taichung Native 1’ (TN1) rice seedlings in the laboratory without exposure to any insecticides. The strain was maintained in a climatic chamber at 27 ± 1°C, with relative humidity of 70 ± 10% and a light: dark = 16 h: 8 h photoperiod.

Chemicals

Emamectin benzoate (95.2%) was obtained from Hebei Weiyuan Co., Ltd. (Hebei, China). Abamectin (96.8%) was obtained from Hebei Weiyuan Co., Ltd. (Hebei, China). Pyriproxyfen (98%) was obtained from Shanghai Shengnong Co., Ltd. (Shanghai, China). Methoprene (S)-(+) (mx7457-100mg) was purchased from Shanghai MaoKang Biotechnology Co., Ltd., (Shanghai, China). Juvenile hormone standard sample (J912305-10mg) was purchased from Shanghai Macklin Biotechnology Co., Ltd., (Shanghai, China).

Bioassay

Different life stages of insects were used to perform bioassay to investigate the effects of insecticide on nymphs and adults. To test whether treatment of the nymph stage of insects would promote reproduction in female, we used 4^th instar nymphs of BPH or 3^rd instar nymph of Laodelphax striatellus and Sogatella furcifera to perform bioassays. To test whether treatment of the adult stage of insects would promote reproduction in female, we used newly emerged male and female BPH.

Systemic route

The rice-seeding dipping bioassay method was used to evaluate the susceptibility of BPH, L. striatellus and S. furcifera to EB. Technical-grade insecticides were dissolved in acetone as stock solution then diluted in a series of six concentrations with water containing 0.1% Triton. Selected rice seedlings at the 6-8 cm growth stage were dipped in insecticide solutions for 30 s and then air-dried at room temperature. The roots of the rice seedlings were wrapped with cotton strips and placed seedlings placed in a plastic cup 5 cm in diameter. Fifteen insects were introduced into each plastic cup for each replicate. The top of the cup was then selaed with gauze to prevent escape. All experiments comprised at least three biological replicates. Control rice seedings were treated with 0.1% Triton X-100 water solution only. All treatments were maintained under standard conditions of 27 ± 1 °C and 70– 80% relative humidity with a 16 h light/8 h dark photoperiod. Mortality was assessed after 4 d for N. lugens or 2 d for L. striatellus and S. furcifera after treatment with insecticides. The insects were considered dead if they were unable to move after a gentle prodding with a fine brush.

For Drosophila larvae bioassay, we adopted a method described previously in our lab with minor modifications (82). Briefly, twenty third instar larvae were placed in fly vials containing fly food (based on corn powder, brown sugar, yeast and agar) supplemented with EB of different concentrations. Four concentrations (LC₁₀, LC₃₀ and LC₅₀) were tested together with a negative (no insecticide) control. For Drosophila adult bioassays, we selected virgin females three days after eclosion. Several concentrations were overlaid onto fly food in standard Drosophila vials and allowed to dry overnight at room temperature. 15 adult flies (three days after eclosion) were then added to each vial and mortality assessed after 2 d. Four replicates were carried out for each concentration. Control mortality was assessed using vials containing food with solvent minus insecticide.

Contact route

For topical bioassays working insecticide solutions were prepared in acetone. 4^th instar nymphs or newly emerged males/females were anesthetized with carbon dioxide for 5 s, and then 0.04 μl/insect test solution applied topically to the dorsal plates with a capillary micro-applicator (29). Insects were then placed in an artificial climate incubator with a temperature of 27±1°C, a photoperiod of 16:8 h (L:D), and a humidity of 70%±10%. Mortality was determined 2 d after treatment. Data with over 20% mortality in the control treatment were discarded, and the assay was repeated at least three times.

Fecundity assays

Fourth instar nymphs of BPH and 3^rd instar nymph of L. striatellus and S. furcifera were treated with the LC₁₅ and LC₅₀ of EB, and then transferred to fresh rice seedlings. After eclosion, the adults were used in the following experiment. Newly emerged treated adults and untreated adults were paired to produce four groups: untreated males and untreated females (♂ck×♀ck; ck indicates untreated); untreated males and treated females (♂ck×♀t; t indicates insecticide treated); treated males and untreated females (♂t×♀ck); treated males and treated females (♂t×♀t). Each group comprised at least 10 mating pairs. All pairs were transferred to glass cylinders (diameter 5 cm and height 20 cm) containing rice plants (25 days old almost 20 cm high) as a food source for eleven days. The number of eggs and nymphs in plants were counted by a microscope.

For Drosophila egg-laying assay, we adopted our previous method (83). Briefly, insecticide-treated virgin females were paired with untreated males for three days and then the mated females transferred into the Drosophila ovipositing apparatus. The number of eggs were counted after 16 hours.

Fitness analysis

The fitness of EB-treated BPH were analyzed using methods reported previously (10). We selected two groups, (♂ck×♀ck) and (♂ck×♀t), to study the effects of the LC₅₀ concentration of EB on BPH fitness. In the case of systemic exposure, 4^th instar nymphs of BPH were treated with the LC₅₀ of EB for 4 days and then transferred to tubes containing untreated rice plants for individual rearing. The rice plants were replaced every three days with untreated plants. The emergence ratio, female ratio, preoviposition period, female longevity, brachypterism female ratio and female weight were calculated.

Examination of ovarian development

Adult females from ♂ck×♀ck control and ♂ck×♀t group on 1, 3, 5, 7 DAE were dissected for observe the ovarian development. The detained eggs in ovary were photographed and recorded. Each group has at least fifteen replicates.

To examination whether EB treated impairs egg maturation, we dissected untreated or EB-treated ovaries and fixed them in 4% paraformaldehyde (PFA) in phosphate buffered saline (PBS) for 30 min at room temperature. After four washes of 10 min (4 × 10 min) in PAT3 (PBS with 0.5% Triton X-100 and 0.5% bovine serum albumin), the ovaries were then incubated with DAPI (4’,6-diamidino-2-phenylindole, 100 nM) and Actin-stain 670 Fluorescent Phalloidin (200 nM). Imaging was performed using Zeiss LSM980 confocal laser microscope.

Measurements of glycogen, triglyceride, total protein content, cholesterol and four sugars

The content of glycogen, triglyceride, cholesterol and total protein was determined by spectrophotometry at 620 nm, 510 nm, 510 nm and 562 nm respectively using the glycogen assay kit (A043-1-1), triglyceride reagent kit (A110-1-1), cholesterol assay kit (A111-1-1) and total protein assay kit (A045-2-2) obtained from Nanjing Jiancheng Bioengineering Institute following to the manufacturer’s instructions. The determined results were normalized to the protein content in the sample, which was determined using BCA Protein Assay Reagent Kit (Thermo Scientific, Waltham, USA). Each sample contained tissue extracts from five adult female BPH, with three biological replicates per sample.

To assess the content of four sugars (sucrose, glucose, fructose and trehalose) in the extract of BPH tissue, the same extraction method was used as above. Sugar content was quantified using the colorimetric method by scanning at 290 nm, 505 nm, 285 nm and 620 nm respectively using the sucrose reagent kit (A099-1-1), glucose reagent kit (F006-1-1), fructose reagent kit (A085-1-1) and trehalose reagent kit (A150-1-1) obtained from Nanjing Jiancheng Bioengineering Institute based on the manufacturer’s instructions. Each sample contained tissue extracts from five adult female N. lugens, with three biological replicates per sample.

Determination of Juvenile hormone III and ecdysone titers of BPH by ELISA

The titer of Juvenile hormone III in BPH was measured using the Juvenile hormone ELISA Kit (Lot Number: HLE92086, Shanghai HaLing Biotechnology Co., Ltd., Shanghai, China) which employs the dual-antibody sandwich ELISA method. The titer of ecdysone in BPH were measured using the ecdysone ELISA Kit (Lot Number: ZK14705, Shanghai ZhenKe Biotechnology Co., Ltd., Shanghai, China). At least three biological replicates were employed for each treatment.

Determination of Juvenile hormone III titer in BPH using HPLC-MS/MS

The whole bodies of 5 individuals BPH were mixed with 1 ml of n-hexane, followed by centrifugation at 10,000×g for 10 min, the upper hexane layer was then dried with nitrogen, dissolved in methanol and sonicated for 10 min, after centrifugation at 10000×g for 10 min, the supernatant was collected through the organic filter membrane of 0.22 μm into 2 ml vials for JH Ⅲ determination. JH Ⅲ standard sample (J912305-10 mg) purchased from (Shanghai McLean Biochemical Technology Co. Ltd), dissolved in methyl alcohol as stock solution 10,000 mg/L was diluted in a series of six concentration gradients to serve as a reference. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was then carried out using UPLC Xevo TQ-S Micro (Waters technology), quantitative method according to the external standard, the chromatographic column was EC-C18 (4.6 mm×150 mm, 2.7 μm), column temperature was 30°C, injection volume was 20 μl, elution flow rate was 0.3 ml/min, and the mobile phase was acetonitrile:formic acid water (90:10), detection wavelength was 218 nm, the peak height was used for quantification.

Cloning, sequence and phylogenetic analysis

The NCBI database and BLAST program were used to carry out sequence alignment and analysis. Open Reading Frames (ORFs) were predicted with EditSeq. Primers were designed using the primer design tool in NCBI. Total RNA Extraction was extracted from 30 adults BPH using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. cDNA was synthesized using the Biotech M-MLV reverse transcription kit. Full-length gene sequences were amplified by PCR using cDNA as template and Rapid Taq Master Mix (Vazyme Biotech, Cat# P222-02). The PCR product was purified on a 1% agarose gel, cloned into pClone007 Simple Vector Kit (Tsingke Biotech, Cat# TSV-007S), and then sequenced using the 3730 XL DNA analyzer (Applied Biosystems, Carlsbad, CA, USA). Table S2 contains a list of the primers used in this study.

The exon and intron architectures of AT, AstA, AstB, AstCC and AstACCC were predicted based on the alignments of putative ORFs against their corresponding genomic sequences. Sequence similarity/annotations and orthologous gene searches were performed using BLAST programs available in NCBI. Multiple alignments of the complete amino acid sequences were performed with Clustal Omega (http://www.ebi.ac.uk/Tools/msa/clustalo). Phylogeny was conducted using the maximum likelihood technique to create phylogenetic trees and these were bootstrapped with 1000 replications were used using the MEGA 6 software (84).

RNA interference

Double-stranded RNA (dsRNA) of gfp (green fluorescent protein), JHAMT (juvenile hormone acid O-methyltransferase,), Met (methoprene-tolerant), Kr-h1 (krüppel homolog 1), AstAR (allatostatin-A receptor) and GluCl (glutamate-gated chloride channel) was prepared using Ambion’s MEGAscript T7 kit instructions following the manufacturer’s instructions. The primer sequences for double-stranded RNA synthesis are listed in Table S2. Newly emerged females were injected with 40 nl (5,000 ng/μl) of double-stranded RNA of gfp (dsgfp) or double-stranded RNA of the target genes in the conjunctive part between prothorax and mesothorax of insects. In the RNAi experiments, BPH were then treated with the LC₅₀ of EB 24h after dsRNA injection and the whole body sampled for qRT-PCR analysis.

Quantitative RT-PCR

Fourth instar nymphs of BPH were treated with EB after which total RNA was extracted from 5^th instar nymphs and 1-7 day post-eclosion females of N. lugens using the methods detailed above. The HiScript® II RT SuperMix for qPCR (+gDNA wiper) kit from Vazyme, Nanjing, China, was used to create first-strand cDNA. Primer3 (http://bioinfo.ut.ee/primer3/) was used to design real-time quantitative PCR (qPCR) primers listed in Table S2. mRNA levels of candidate genes were detected by qPCR using the UltraSYBR Mixture (with ROX) Kit (CWBIO, Beijing, China). Each reaction contained 2 μL of cDNA template (500 ng), 1 μL each forward and reverse qPCR primer (10 μM), 10 μL of UltraSYBR mixture buffer, and 6 μL of RNase-free water. Q-PCR was run on an ABI 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA) under the following conditions: 5 min at 95°C, followed by 40 cycles of 95°C for 10 s and 60°C for 30 s. Three independent biological replicates and four technical replicates were used in each qPCR experiment. The housekeeping genes 18S ribosomal RNA of BPH were selected to normalize the expression of candidate genes. The 2^−ΔΔCt method (Ct represents the cycle threshold) was used to measure relative expression levels (85). Three biological replicates were used for statistical comparison between samples. Table S2 contains a list of the primers used in this study.

Statistics

PoloPlus v2.0 (LeOra Sofware 2008) was used to calculate the lethal concentration (LC₅₀) and 95% fiducial limits (95% F.L.). GraphPad Prism 8.0 software (GraphPad Software Inc., San Diego, USA) was used to generate graphs and perform statistical analysis of data. Data presented in this study were first verified for normal distribution using the D’Agostino–Pearson normality test. One-way analysis of variance (ANOVA) with Duncan’s multiple range test was used to test differences among multiple groups of normally distributed groups of data. Student’s t test was used to test the differences between two groups. If not normally distributed, Mann–Whitney test was used for pairwise comparisons, and Kruskal–Wallis test was used for comparisons among multiple groups, followed by Dunn’s multiple comparisons. All data are presented as mean ± s.e.m. The sample sizes and statistical tests used for each experiment are stated in the figures or figure legends.

Acknowledgements

This research was funded by the National Key R&D Program of China to SFW (2022YFD1700200) (https://service.most.gov.cn/) and National Natural Science Foundation of China to SFW (32022011) (https://www.nsfc.gov.cn/).

Funding

This work was supported by the National Natural Science Foundation of China to SFW (No.32022011 & 31772205) (https://isisn.nsfc.gov.cn/egrantindex/funcindex/prjsearch-list?locale=zh_CN).

The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.