1. Introduction
Pyridaben is an efficient and low-toxicity acaricide with strong contact action [
1]. It is an acaricide with a wide spectrum of action against all plant-feeding mites [
2], such as
Panonychus citri, from eggs to adult mites [
3]. However, with the extensive annual use of pyridaben, mite resistance to this acaricide is continuously increasing, necessitating the development of more efficient, safer, and more sustainable control technologies. Biological control, known for being environmentally friendly and not restricted by mite resistance, can be used in conjunction with acaricides to enhance control efficiency. For example, Iwassaki et al. [
4] achieved a significant reduction in the damage levels of
Tetranychus urticae by combining
Neoseiulus californicus with pyridaben. Similarly, Jin et al. [
5] reported effective control of
T. urticae by combining
Phytoseiulus persimilis with bifenthrin. Another study demonstrated that using two species of predatory mites in conjunction with etoxazole provided effective control of
T. urticae [
6]. Similarly, a study found that combining mineral oil emulsions with
Neoseiulus barkeri provided good synergistic control of
P. citri [
7]. In the application of acaricides and predatory mites for controlling pest mites, acaricides not only directly kill mites but also unavoidably affect the behavior and physiology of naturally occurring or subsequently released predatory mites. These sublethal effects can reduce the reproductive capacity of predatory mites, diminish their prey-searching ability, and decrease their population size, potentially leading to pest mite resurgence and hindering pest control efforts [
8,
9].
Research on the sublethal effects of low doses of pyridaben on pest mites has primarily focused on life history traits (development time, fecundity, and longevity) and population parameters. Generally, these sublethal effects are inhibitory, limiting population growth by reducing the lifespan and reproductive capacity of adult females, affecting egg hatching, and prolonging the development period of the F1 generation. However, there are instances where sublethal effects can trigger “overcompensation effects” or “corrective overcompensation effects” in pest mite populations, leading to a stimulatory effect [
10,
11]. As pyridaben is a broad-spectrum acaricide with secondary insecticidal properties, it can also harm predatory mites, as well as some insect predators of plant-feeding mites. Consequently, recent research has shifted focus to the impact of sublethal effects of pyridaben on behavior, i.e., predation of pest mites by their natural enemies. Another study observed significant declines in the predation ability and searching efficiency of
N. californicus after treatment with pyridaben at LC
10 and LC
30 concentrations [
12]. Further research discovered that treatment with pyridaben at the LC
30 concentration slightly reduced the predation of
N. barkeri on
T. urticae and significantly induced the activities of glutathione S-transferases (GSTs) and carboxylesterases (CarEs), while inhibiting multi-function oxidases (MFOs) [
13].
Few researchers have deeply analyzed the mechanisms by which the sublethal effects of pyridaben impact the predatory functions of predatory mites. Plants generate herbivore-induced plant volatiles (HIPVs) when damaged by pests, which have been widely studied in plant pest resistance strategies.
N. womersleyi may locate prey through HIPVs produced after
T. urticae feeding, as demonstrated in attraction experiments with common predatory mites, such as
Neoseiulus cucumeris and
P. persimilis [
14,
15,
16,
17,
18]. Due to the lack of eyes and antennae,
N. womersleyi uses its chelicerae and legs to continuously tap and perform olfactive functions to locate prey. The physiological functions of Niemann–Pick-type C2 (NPC2) proteins in vertebrates, such as cholesterol and lipid binding and transport, are well known [
19]. In contrast, studies on the functions of
NPC2 genes in insects and mites are limited. Some
NPC2 genes were highly expressed, specifically in the first pair of feet [
20,
21].
NPC2 genes may be involved in the collection and transport of HIPVs in predatory mites, thereby participating in chemical communication for locating hosts and effective foraging [
22,
23,
24,
25]. Hypotheses suggest that NPC2 proteins in insects may rely on the hemolymph pH in antennae to alter binding affinities for different odor molecules, although this remains unconfirmed. Alternatively, NPC2 proteins may undergo conformational changes induced by odor molecules to bind various odors [
26].
Currently, the relationship between NPC2 genes and HIPVs in predatory mites and their connection to predatory function and searching efficiency requires further in-depth research. This study focuses on N. womersleyi, a mite with strong control capabilities against various pest mites, and uses T. urticae, a severely damaging spider mite, as prey. By examining the sublethal effects of pyridaben on the NPC2 gene, this research aims to elucidate the mechanisms affecting the predation responses of natural enemies, providing theoretical support for resolving conflicts in the use of acaricides and predatory mites in practical production. This study has practical significance for the selection of safe acaricides, rational pesticide application, and the protection of natural enemies.
4. Discussion
This study explored the effects of LC
30 and LC
50 concentrations of pyridaben on the predatory function and searching efficiency of
N. womersleyi. Under LC
30 treatment, an increase was observed in both the predatory capacity (
a/
Th) of
N. womersleyi on
T. urticae larvae and the maximum daily predation rate (1/
Th) on various stages of
T. urticae. It is speculated that the reduction in handling time (
Th) for
N. womersleyi when preying on
T. urticae may lead to less thorough consumption, thereby increasing the number of preys consumed. Compared to previous studies on the predation efficiency of
N. pseudolongispinosus on
T. urticae [
36], our results indicated that
N. womersleyi exhibited a Holling Type II functional response to different stages of
T. urticae. Additionally, it was found that
N. pseudolongispinosus had a higher searching efficiency for
T. urticae eggs and larvae compared to nymphs, which aligns with our findings. Research on the predatory functional response of
A. pseudolongispinosus to
Tetranychus cinnabarinus [
37] found that the functional responses to three different stages of
T. cinnabarinus also followed Holling Type II. Under similar control treatment densities, the predatory capacity,
a/
Th, and maximum daily predation rate,
1/
Th, of
A. pseudolongispinosus for
T. cinnabarinus eggs, larvae, and nymphs were lower than those observed in our study for
N. womersleyi preying on
T. urticae eggs, larvae, and nymphs. Another study on the effects of sublethal doses of abamectin on the predatory function of
P. persimilis on
T. urticae [
38] revealed a decrease in the instantaneous attack rate (
a), an increase in the handling time (
Th), a reduction in the predatory capacity (
a/
Th), and a decrease in the maximum daily predation rate (1/
Th). This ultimately reduced the control efficiency of
P. persimilis on the prey. Our study similarly concluded that LC
30 and LC
50 concentrations of pyridaben negatively affected the predation efficiency and searching ability of
N. womersleyi.
The study successfully cloned a NPC2 protein gene,
NwNPC2a, from
N. womersleyi for the first time. Under LC
30 and LC
50 treatments of pyridaben, the relative expression levels of the
NwNPC2a gene were found to decrease compared to the control group. As the concentration of pyridaben increased, the relative expression levels of the genes further decreased, indicating that this gene might be involved in the olfactory-related predatory behavior of
N. womersleyi. The identification of NPC2 proteins from the antennae of the Japanese carpenter ant demonstrated their ability to deliver various hydrophobic signaling substances to chemosensory neurons, marking the first study on the role of NPC2 proteins in olfactory communication [
39]. According to Li et al. [
21], three full-length
NPC2 genes (
NbNPC2-1, NbNPC2-2, and
NbNPC2-3) were cloned based on the transcriptome data of
N. barkeri, suggesting that NPC2 proteins might be involved in the chemical communication of male mites.
Previous research on the factors affecting RNAi efficiency showed that RNAi efficiency might vary with insect species, dsRNA molecule length, target gene, and other experimental factors [
40]. Due to the small size and fast crawling speed of
N. womersleyi, methods such as microinjection are highly challenging. In RNAi studies on the
Metaseiulus occidentalis and
N. barkeri, the feeding method was used to deliver dsRNA into predatory mites. Using the feeding method, RNAi treatment of
NbarNPC2a and
NbarNPC2b genes in
N. barkeri resulted in expression reductions of 59% and 44%, respectively, achieving high levels of gene silencing [
32,
41]. In this study, the feeding method was used for interference treatment, and the relative expression level of the
NwNPC2a gene was significantly reduced compared to the control (
p < 0.001). This indicates that the feeding method effectively interfered with the gene expression in
N. womersleyi, significantly silencing the target
NPC2 gene. After interference treatment,
N. womersleyi females showed significant reductions in the attack coefficient (
a), handling time (
Th), predatory capacity (
a/
Th), and maximum daily predation rate (
1/
Th) when preying on
T. urticae. The searching efficiency (
S) was also lower than the control and slightly decreased with the increasing prey density. This suggests that NPC2 proteins play an important role in the predation of
T. urticae by
N. womersleyi. The internal molecular mechanism by which pyridaben affects the predation ability of
N. womersleyi may be through the inhibition of
NwNPC2a expression. Similar findings were reported in RNAi studies, where the daily predation rates of
N. barkeri significantly decreased under 16 °C and 24 °C conditions after interference with
NbarNPC2a and
NbarNPC2b genes [
41]. Additionally, RNAi treatment in
P. persimilis resulted in reduced sensitivity to plant odors, suggesting the involvement of the
PpNPC2a gene in the recognition of plant volatiles [
25]. In the two-spotted spider mite, silencing of a highly expressed
NPC2 transcript (
NP1) by 48–59% led to reduced feeding and reproduction but did not affect the host-seeking ability. Conversely, downregulation of the
NP5 transcript by more than 50% significantly impaired the host-seeking ability, indicating that
NP1 might be involved in short-range host recognition signals, while
NP5 might be involved in long-range host recognition signals [
20,
42].
N. womersleyi is a widely distributed predatory mite with effective control over various harmful agricultural spider mites. Exploring the effects of pyridaben on N. womersleyi and its impact on predatory behavior mechanisms provides theoretical guidance for the field application of pyridaben. In arthropods, NPC2 proteins primarily function as olfactory-binding proteins. Predatory mites may rely on NPC2 proteins to collect HIPVs and other odor molecules to locate and pursue their prey. Further in-depth research is needed to elucidate the relationship between NPC2 proteins and HIPVs within predatory mites and their connection to predatory functions and searching efficiency.