The honeybee is the most important economic insect. It is not only the ideal pollinator of crops and wild plants that subserve the agricultural production, plant diversity and ecological balance but also provides nutritious bee products (Gallai et al., 2009; Champetier et al., 2015; Zheng et al., 2019). In recent years, the honeybee colony losses were reported and the dramatic reductions caused significant economic losses all over the world (Van der Zee et al., 2012; van Dooremalen et al., 2018; Calatayud-Vernich et al., 2019; Kablau et al., 2020). The continued decline of honeybee colonies brings a serious crisis to plant pollination and food production, because nearly 75% of the major crop species rely on pollinators (Clermont et al., 2015; Shi et al., 2020). The main reasons were diverse agrochemicals, parasites, viruses, adjustment of crop planting structure and distribution, especially the noticeable toxic effects of pesticides on honeybees, and also these factors were complex and interacting (van Dooremalen et al., 2018; Duan et al., 2020a; Shi et al., 2020b). During the foraging process, honeybees can directly contact the pesticides remaining on the surface of plants (Legard et al., 2001; Xiong et al., 2022). At the same time, the systemic pesticide residues could be absorbed by plants and remain in nectar and pollen, then were taken back to the colony by the foraging bee and led to the contamination of honey, pollen and comb which were consumed by other members in the colony (Mullin et al., 2010). As a metamorphosis development insect, the life cycle of the honeybee consists of four developmental phases, including egg, larva, pupa, and adult. Furthermore, these pesticide residues could harm the health of honeybees at different stages of individuals and colonies by direct or indirect exposure (Krupke et al., 2017). Consequently, the primary problem in modern agricultural production is how to balance between protecting crops efficiently against pests and diseases and maintaining healthy pollinator populations (Godfray and Garnett, 2014; Xiong et al., 2022).

As an important class of plant protection products, fungicides have already accounted for more than 35% of the global pesticide market and are widely used in the disease control of nectar and pollen plants, such as maize, rape, sunflower, and alfalfa, which account for approximately 11% of total global pesticide use (Liao et al., 2018; Zhang 2018; Zubrod et al., 2019). According to the data of the Fungicide Resistance Action Committee (FRAC), the action mechanisms of fungicides to plant pathogenic microorganisms were the negative effects on the nucleic acid and protein synthesis, respiration, signal transduction, cell division, and membrane structure and function of microorganisms (Fungicide Resistance Action Committee, 2021). Therefore, the bioassay results showed that most fungicides were lower acutely toxic to honeybees and other non-target insects (Liao et al., 2018; Simondelso et al., 2018; Xiong et al., 2022). Normally, the field-realistic concentrations or residue in the colonies were considered to be low toxicity to cause illness or death of honeybees (Pettis et al., 2013). Furthermore, the fungicides were considered to be safe for honeybees and the chronic toxicity was ignored (Tadei et al., 2019; Duan et al., 2020a; Xiong et al., 2022). Thus bees are more likely to encounter fungicides than insecticides because fungicides can even be sprayed when insect-attractive crops are in bloom (Favaro et al., 2019; Gierer et al., 2019; Rondeau and Raine 2022).

However, a large number of scientific investigations have reported the chronic toxicity of fungicides on the development, detoxification, and immune function, foraging, and homing ability, olfactory memory of bees, which cause serious damage to the individual and colony of honeybees (Zhu et al., 2014; Carneiro et al., 2019; Duan et al., 2020a; Dai et al., 2021; Fisher et al., 2021; Traynor et al., 2021). After exposure to chlorothalonil for 3 days, the mortality of A. mellifera 4-day-old larvae was significantly increased over two-fold compared to untreated larvae, and also the pairing of chlorothalonil and coumaphos or fluvalinate produced synergistic interactions on the mortality of larvae (Zhu et al., 2014). The low field concentration of dimertachlone, prochloraz and iprodione could induce the activities of catalase (CAT), carboxylesterases (CarE) and glutathione S-transferase (GSTs), but the high concentration inhibits their activity (Duan et al., 2020a). The Iprodione (2 mg/kg) was not lethal to newly emerged bees, but it can inhibit the synthesis of glutathione, leading to the generation of reactive oxygen species and the cells of treated bees had signs of apoptosis (Carneiro et al., 2019). The benomyl stress (5 g/kg) led to a total of 5,759 DEGs being upregulated in the worker bees of A. mellifera, and most of the DEGs were involved in the functions of immunity, detoxification, biological metabolism, and regulation, such as light conduction, MAPK, calcium ion pathway and other 12 pathways (Dai et al., 2021). DesJardins et al. (2021) found the compound fungicides Pristine^® showed significant sublethal effects on the learning performance of A. mellifera and lead the work type conversion of nurse bee to forage bee.

Pyraclostrobin is a high-efficiency, low-toxic, and broad-spectrum systemic strobilurin fungicide which was registered and widely used to prevent and control diseases caused by fungi on various nectar and pollen plants (Bartlett et al., 2002). The bactericidal mechanism of pyraclostrobin was to inhibit cell respiration in fungi and the acute oral and contact toxicity of pyraclostrobin to the worker bee of A. mellifera was low toxicity (LC₅₀ > 100 μg (a.i.)/bee) (Earley et al., 2012; Tan et al., 2021). Pyraclostrobin was chronic toxicity to honeybees and could directly inhibit the mitochondrial function in vitro (Campbell et al., 2016; Nicodemo et al., 2020). The field-relevant doses of pyraclostrobin decreased the height of secretory cells and volume of mandibular glands with 6 days continuous exposure and influence the behavior of newly emerged workers and young workers (Zaluski et al., 2017; Tadei et al., 2019). Meanwhile, pyraclostrobin was widely residual in the pollen of treated crops and in honeybee colonies which may influence the health of honeybees (Yoder et al., 2013; David et al., 2015).

However, the effect of pyraclostrobin on the development and physiology of larvae and pupae of A. mellifera is rarely known. To investigate the influence of field-realistic concentrations of pyraclostrobin on larvae and pupae, the survival and developmental state of A. mellifera worker bees from larvae to adult stage in each treatment were documented daily. Further, the effect of pyraclostrobin on the development-related genes ecdysone receptor (Ecr) and ultraspiracle protein (Usp), nutrient metabolism-related genes insulin-like peptides 1 (ILP 1), insulin-like peptides 2 (ILP 2), Hexamerin 70b (Hex 70b), Hexamerin 110 (Hex 110), Vitellogenin (Vg) and immune-related genes Apidaecin, Abaecin, Hymenoptaecin, Defensin1 in larvae and pupae were examined, respectively. This study will provide new evidence of pyraclostrobin exposure on honeybee larvae and pupae development, and also provide the theoretical basis for the pollination safety and the management of pesticides.

So far, pyraclostrobin has been on the market for over 20 years and was registered and used for fungi diseases control of various plants in different countries (Luo et al., 2022). With widely and irrational use, the accumulation and pollution of pyraclostrobin in the soil, water and other ecosystems represent high potential risks to the environment and organisms. As an indicator of environmental pollution, honeybees also directly or indirectly contact with this pollutant which led to harmful effects on individuals and colonies (Yoder et al., 2013; David et al., 2015; Zaluski et al., 2017; Tadei et al., 2019; Xiong et al., 2022).

Despite the low acute oral and contact toxicity of pyraclostrobin to honeybees (Tan et al., 2021), long-time exposure to pyraclostrobin could lead to irreversible adverse effects on honeybees. The results showed pyraclostrobin (100 and 83.3 mg/L) were chronic toxicity effects on the survival and development of A. mellifera. da Costa Domingues et al. (2020) found the forager workers of Melipona scutellaris exposed to pyraclostrobin showed a reduced survival rate. Compared with control, both two pyraclostrobin concentrations exposure significantly reduced the survival rate of A. mellifera larvae with a significant concentration effect (p < 0.05; Table 2). Meanwhile, the significantly low capped rate of larvae and emergence rate of pupae after pyraclostrobin exposure causes unsuccessful metamorphic development from larvae to pupae with a high mortality rate. During the process of pupation and emergence, honeybees consume the energy which they have previously stored to synthesize new substances. Owing to the low concentration of pyraclostrobin could inhibit the mitochondrial respiratory of A. mellifera (Nicodemo et al., 2020), which means the fungicide exposure-treated A. mellifera larvae and pupae need to consume more material to complete metamorphosis. Thus significantly decreased the weight of pupae and newly emerged bees (Table 3), suggesting pyraclostrobin could affect the normal growth and metabolism of A. mellifera larvae and pupae, especially for pupae with a low emergence rate and weight, though they did not feed during pupal stage. The quantity and quality of brood (larvae and pupae) are critical to the population size of the colony (Duan et al., 2020b; Xiong et al., 2022) and these findings indicate pyraclostrobin can cause serious damage to bee colonies by suppressing the survival and development of individuals.

During the larval-pupal transition of A. mellifera, the development rhythm of metamorphosis was regulated by juvenile hormones (JH) and molting hormone (20-hydroxyecdysone, 20E) (Liu et al., 2014). The 20E, ecdysteroid receptor (Ecr) and ultraspiracle protein (Usp) constitute the ligand-receptor complex (20E-Ecr-Usp) and then activate the metamorphosis process (Riddiford et al., 2000). Therefore, the Ecr and Usp were considered to be key genes responsible for the transduction of the JH/20E signals during metamorphosis development (Barchuk et al., 2008; Duan et al., 2021). In the present study, the RT-PCR results showed the expression levels of Ecr and Usp in larvae and pupae were altered with pyraclostrobin exposure. The expression level of Ecr and Usp in pupae were significantly upregulated after pyraclostrobin exposure (100 and 83.3 mg/L). Furthermore, the low emergence rate and weight of pupae also confirmed that pyraclostrobin could disturb the normal development process leading to a high mortality rate of A. mellifera pupae.

Pyraclostrobin could cause the energy deficiency of A. mellifera, and more nutrition materials need to be metabolized for normal development (Nicodemo et al., 2020). Moreover, extra nutrient consumption may cause the weight loss of pupae and newly emerged bees. The present results suggested that the weight loss might come from either some nutrient metabolic pathway disturbance or the decreased hexamerins for building the pupae tissues, which may already be disrupted by pyraclostrobin. There are two insulin-like peptides (ILPs) in honeybees that have profound effects on invertebrate metabolism, nutrient storage and fertility (de Azeved and Hartfelder, 2008; Duan et al., 2021). The ILP1 gene potentially functions in lipid and protein metabolism while ILP2 is a more general indicator of nutritional status (Ihle et al., 2014). Compared with control, the high concentration of pyraclostrobin (100 m/L) inhibits ILP1 expression in both larvae and pupae. However, the expression of ILP2 in 83.3 mg/L treatment exhibited upregulation. The abnormal expression phenomenon of ILP1 and ILP2 in honeybees would lead to nutritional and metabolic disorders (Wang et al., 2013). The hexamerins were synthesized in the fat body during the larval growth phase and used for pupal development and adult differentiation (Burmester and Scheller, 1999). The subunits of Hex 110 were highly abundant in A. mellifera larval hemolymph and the gene activity obeys a nutritional control (Bitondi et al., 2006). The expression of Hex100 was significantly downregulated indicating malnutrition and developmental abnormalities of larvae and pupae after pyraclostrobin exposure. Meanwhile, the larvae could use Hex 70b to compensate for the lack of proteins (Cunha et al., 2005). And the expression of Hex 70b was significantly induced in pupae by pyraclostrobin (Figure 2). Vitellogenin (Vg) is an egg-yolk precursor in insect reproduction and multiple roles of Vgs, such as immunity, life span, and antioxidation in non-reproduction were also uncovered (Havukainen et al., 2013; Salmela et al., 2015; Salmela and Sundström, 2017). Pyraclostrobin exposure could result in oxidative stress in zebrafish embryos (Li et al., 2018; Kumar et al., 2020). In the present study, Vg has significantly upregulated expression in larvae and pupae which acts as a ‘defender’ against infection and reactive oxygen species for a prolonged life span (p < 0.05) (Havukainen et al., 2013; Salmela et al., 2015).

Honey bee innate immunity provides immediate responses against invading pathogens, especially antimicrobial peptides (AMPs) in cell-free humoral immunity (Danihlík et al., 2015; Duan et al., 2021; Xiong et al., 2022). Four families of AMPs (i.e., apidaecins, abaecin, hymenoptaecin and defensins) with a variety of antimicrobial activities have been described in the honey bee and their expressions were regulated by two intracellular signaling pathways Toll and Imd/JNK (Evans et al., 2006; Danihlík et al., 2015). It should be noted that that pyraclostrobin had negative effects on the immunity of bee larvae and pupae, leading to a low survival rate (Figure 3). At the larvae stage, the expression of Apidaecin and Abaecin were significantly downregulated (p < 0.05) indicating that exposure to pyraclostrobin makes the honeybee would be more sensitive to pathogens, which may be the main reason for the increased Nosema ceranae infection rates in adult bees (Pettis et al., 2013). While the Defensin1 and Hymenoptaecin genes exhibited upregulation in two pyraclostrobin treatments suggesting that larvae can coordinate different immune genes in response to the effects of fungicides on their immunity (Shi et al., 2020). However, at the pupae stage, the pyraclostrobin exposure could induce the expression of Apidaecin and Hymenoptaecin, and inhibit the expression of Defensin1 which means pupae had different defense strategies for stress to immunity than larvae. Furthermore, combined with the results of immune-genes expression with different exposure concentrations, these four immune genes have different response mechanisms to two pyraclostrobin treatment concentrations. Considering the regulation of four immune genes by Toll and Imd/JNK metabolic pathways (Evans et al., 2006), the influence of pyraclostrobin on two Toll and Imd/JNK metabolic pathways also needs to be further evaluated and attention.

In the current study, two field-recommended concentrations pyraclostrobin (100 and 83.3 mg/L) showed significant adverse effects on the development of honey bee, resulting in a significantly lower survival rate, capped rate, emergence rate and body weight. Meanwhile, with long-term pyraclostrobin exposure, the expression levels of development-, nutrient- and immune-related genes in both larvae and pupae were also abnormally altered, indicating that pyraclostrobin could impair the development, nutrient metabolism and immunity of larvae and pupae. These findings demonstrate that the low acute toxic fungicide pyraclostrobin has deleterious effects on A. mellifera larvae and pupae with continuous exposure. Thus, it is necessary to re-evaluate the safety and potential risks of the fungicides to honey bee, bumble bee and solitary bee in the future. And the health welfare of pollinators should be emphasized in integrated pest management.