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Fungicides are often used during tree fruit flowering and can threaten insect pollinators. However, little is known about how non-bee pollinators (e.g., solitary bees, Osmia cornifrons) respond to contact and systemic fungicides commonly used on apples during flowering. This knowledge gap limits regulatory decisions determining safe concentrations and timing of fungicide spraying. We assessed the effects of two contact fungicides (captan and mancozeb) and four interlayer/phytosystem fungicides (ciprocycline, myclobutanil, pyrostrobin and trifloxystrobin). Effects on larval weight gain, survival, sex ratio and bacterial diversity. The evaluation was conducted using a chronic oral bioassay in which pollen was treated in three doses based on the currently recommended dose for field use (1X), half dose (0.5X) and low dose (0.1X). All doses of mancozeb and pyritisoline significantly reduced body weight and larval survival. We then sequenced the 16S gene to characterize the larval bacteriome of mancozeb, the fungicide responsible for the highest mortality. We found that bacterial diversity and abundance were significantly reduced in larvae fed on mancozeb-treated pollen. Our laboratory results indicate that spraying some of these fungicides during flowering is particularly harmful to the health of O. cornifrons. This information is relevant for future management decisions regarding the sustainable use of fruit tree protection products and serves as the basis for regulatory processes aimed at protecting pollinators.
The solitary mason bee Osmia cornifrons (Hymenoptera: Megachilidae) was introduced to the United States from Japan in the late 1970s and early 1980s, and the species has played an important pollinator role in managed ecosystems ever since. Naturalized populations of this bee are part of approximately 50 species of wild bees that complement the bees that pollinate almond and apple orchards in the United States2,3. Mason bees face many challenges, including habitat fragmentation, pathogens, and pesticides3,4. Among insecticides, fungicides reduce energy gain, foraging5 and body conditioning6,7. Although recent research suggests that the health of Mason bees is directly influenced by commensal and ectobactic microorganisms, 8,9 because bacteria and fungi can influence nutrition and immune responses, the effects of fungicide exposure on the microbial diversity of Mason bees are just beginning to be studied.
Fungicides of various effects (contact and systemic) are sprayed in orchards before and during flowering to treat diseases such as apple scab, bitter rot, brown rot and powdery mildew10,11. Fungicides are considered harmless to pollinators, so they are recommended to gardeners during the flowering period; The exposure and ingestion of these fungicides by bees is relatively well known, as it is part of the pesticide registration process by the US Environmental Protection Agency and many other national regulatory agencies12,13,14. However, the effects of fungicides on non-bees are less known because they are not required under marketing authorization agreements in the United States15. In addition, there are generally no standardized protocols for testing single bees16,17, and maintaining colonies that provide bees for testing is challenging18. Trials of different managed bees are increasingly being conducted in Europe and the USA to study the effects of pesticides on wild bees, and standardized protocols have recently been developed for O. cornifrons19.
Horned bees are monocytes and are commercially used in carp crops as a supplement or replacement for honey bees. These bees emerge between March and April, with the precocious males emerging three to four days before the females. After mating, the female actively collects pollen and nectar to provide a series of brood cells within the tubular nest cavity (natural or artificial)1,20. The eggs are laid on pollen inside the cells; the female then builds a clay wall before preparing the next cell. The first instar larvae are enclosed in the chorion and feed on embryonic fluids. From the second to the fifth instar (prepupa), the larvae feed on pollen22. Once the pollen supply is completely depleted, the larvae form cocoons, pupate and emerge as adults in the same brood chamber, usually in late summer20,23. Adults emerge the following spring. Adult survival is associated with net energy gain (weight gain) based on food intake. Thus, the nutritional quality of pollen, as well as other factors such as weather or exposure to pesticides, are determinants of survival and health24.
Insecticides and fungicides applied before flowering are able to move within the plant vasculature to varying degrees, from translaminar (e.g., able to move from the upper surface of leaves to the lower surface, like some fungicides) 25 to truly systemic effects. , which can penetrate the crown from the roots, can enter the nectar of apple flowers26, where they can kill adult O. cornifrons27. Some pesticides also leach into pollen, affecting the development of maize larvae and causing their death19. Other studies have shown that some fungicides can significantly alter the nesting behavior of the related species O. lignaria28. In addition, laboratory and field studies simulating pesticide exposure scenarios (including fungicides) have shown that pesticides negatively affect the physiology 22 morphology 29 and survival of honey bees and some solitary bees. Various fungicidal sprays applied directly to open flowers during flowering may contaminate pollen collected by adults for larval development, the effects of which remain to be studied30.
It is increasingly recognized that larval development is influenced by pollen and microbial communities of the digestive system. The honeybee microbiome influences parameters such as body mass31, metabolic changes22 and susceptibility to pathogens32. Previous studies have examined the influence of developmental stage, nutrients, and environment on the microbiome of solitary bees. These studies revealed similarities in the structure and abundance of the larval and pollen microbiomes33, as well as the most common bacterial genera Pseudomonas and Delftia, among solitary bee species. However, although fungicides have been associated with strategies to protect bee health, the effects of fungicides on larval microbiota through direct oral exposure remain unexplored.
This study tested the effects of real-world doses of six commonly used fungicides registered for use on tree fruit in the United States, including contact and systemic fungicides administered orally to corn hornworm moth larvae from contaminated food. We found that contact and systemic fungicides reduced bee body weight gain and increased mortality, with the most severe effects associated with mancozeb and pyrithiopide. We then compared the microbial diversity of larvae fed on the mancozeb-treated pollen diet with those fed on the control diet. We discuss potential mechanisms underlying mortality and implications for integrated pest and pollinator management (IPPM)36 programs.
Adult O. cornifrons overwintering in cocoons were obtained from the Fruit Research Center, Biglerville, PA, and stored at −3 to 2°C (±0.3°C). Before the experiment (600 cocoons in total). In May 2022, 100 O. cornifrons cocoons were transferred daily into plastic cups (50 cocoons per cup, DI 5 cm × 15 cm long) and wipes were placed inside the cups to promote opening and provide a chewable substrate, reducing stress on the stony bees37 . Place two plastic cups containing cocoons in an insect cage (30 × 30 × 30 cm, BugDorm MegaView Science Co. Ltd., Taiwan) with 10 ml feeders containing 50% sucrose solution and store for four days to ensure closure and mating . 23°C, relative humidity 60%, photoperiod 10 l (low intensity): 14 days. 100 mated females and males were released every morning for six days (100 per day) into two artificial nests during peak apple flowering (trap nest: width 33.66 × height 30.48 × length 46.99 cm; Supplementary Figure 1 ). Placed at the Pennsylvania State Arboretum, near cherry (Prunus cerasus ‘Eubank’ Sweet Cherry Pie™), peach (Prunus persica ‘Contender’), Prunus persica ‘PF 27A’ Flamin Fury®), pear (Pyrus perifolia ‘Olympic’, Pyrus perifolia ‘ Shinko’, Pyrus perifolia ‘Shinseiki’), coronaria apple tree (Malus coronaria) and numerous varieties of apple trees (Malus coronaria, Malus), domestic apple tree ‘Co-op 30′ Enterprise™, Malus apple tree ‘Co-Op 31′ Winecrisp™, begonia ‘Freedom’, Begonia ‘Golden Delicious’, Begonia ‘Nova Spy’). Each blue plastic birdhouse fits on top of two wooden boxes. Each nest box contained 800 empty kraft paper tubes (spiral open, 0.8 cm ID × 15 cm L) (Jonesville Paper Tube Co., Michigan) inserted into opaque cellophane tubes (0.7 OD see Plastic plugs (T-1X plugs) provide nesting sites.
Both nest boxes faced east and were covered with green plastic garden fencing (Everbilt model #889250EB12, opening size 5 × 5 cm, 0.95 m × 100 m) to prevent rodent and bird access and placed on the soil surface next to the nest box soil boxes. Nest box (Supplementary Figure 1a). Corn borer eggs were collected daily by collecting 30 tubes from nests and transporting them to the laboratory. Using scissors, make a cut at the end of the tube, then disassemble the spiral tube to expose the brood cells. Individual eggs and their pollen were removed using a curved spatula (Microslide tool kit, BioQuip Products Inc., California). The eggs were incubated on damp filter paper and placed in a Petri dish for 2 hours before being used in our experiments (Supplementary Figure 1b-d).
In the laboratory, we evaluated the oral toxicity of six fungicides applied before and during apple blossom at three concentrations (0.1X, 0.5X, and 1X, where 1X is the mark applied per 100 gallons of water/acre. High field dose = concentration in the field). , Table 1). Each concentration was repeated 16 times (n = 16). Two contact fungicides (Table S1: mancozeb 2696.14 ppm and captan 2875.88 ppm) and four systemic fungicides (Table S1: pyrithiostrobin 250.14 ppm; trifloxystrobin 110.06 ppm; myclobutanil azole 75 .12 ppm; cyprodinil 280.845 ppm) toxicity to fruits, vegetables and ornamental crops. We homogenized the pollen using a grinder, transferred 0.20 g to a well (24-well Falcon Plate), and added and mixed 1 μL of fungicide solution to form pyramidal pollen with 1 mm deep wells into which the eggs were placed. Place using a mini spatula (Supplementary Figure 1c,d). Falcon plates were stored at room temperature (25°C) and 70% relative humidity. We compared them with control larvae fed a homogeneous pollen diet treated with pure water. We recorded mortality and measured larval weight every other day until the larvae reached prepupal age using an analytical balance (Fisher Scientific, accuracy = 0.0001 g). Finally, sex ratio was assessed by opening the cocoon after 2.5 months.
DNA was extracted from whole O. cornifrons larvae (n = 3 per treatment condition, mancozeb-treated and untreated pollen) and we performed microbial diversity analyzes on these samples, especially because in mancozeb the highest mortality was observed in larvae. receiving MnZn. DNA was amplified, purified using the DNAZymoBIOMICS®-96 MagBead DNA kit (Zymo Research, Irvine, CA), and sequenced (600 cycles) on an Illumina® MiSeq™ using the v3 kit. Targeted sequencing of bacterial 16S ribosomal RNA genes was performed using the Quick-16S™ NGS Library Prep Kit (Zymo Research, Irvine, CA) using primers targeting the V3-V4 region of the 16S rRNA gene. Additionally, 18S sequencing was performed using 10% PhiX inclusion, and amplification was performed using the primer pair 18S001 and NS4.
Import and process paired reads39 using the QIIME2 pipeline (v2022.11.1). These reads were trimmed and merged, and chimeric sequences were removed using the DADA2 plugin in QIIME2 (qiime dada2 noise pairing)40. The 16S and 18S class assignments were performed using the object classifier plugin Classify-sklearn and the pre-trained artifact silva-138-99-nb-classifier.
All experimental data were checked for normality (Shapiro-Wilks) and homogeneity of variances (Levene’s test). Because the data set did not meet the assumptions of parametric analysis and the transformation failed to standardize the residuals, we performed a nonparametric two-way ANOVA (Kruskal-Wallis) with two factors [time (three-phase 2, 5, and 8 day time points) and fungicide] to evaluate the effect of treatment on larval fresh weight, then post hoc nonparametric pairwise comparisons were performed using the Wilcoxon test. We used a generalized linear model (GLM) with a Poisson distribution to compare the effects of fungicides on survival across three fungicide concentrations41,42. For differential abundance analysis, the number of amplicon sequence variants (ASVs) was collapsed at the genus level. Comparisons of differential abundance between groups using 16S (genus level) and 18S relative abundance were performed using a generalized additive model for position, scale, and shape (GAMLSS) with beta zero-inflated (BEZI) family distributions, which were modeled on a macro . in Microbiome R43 (v1.1). 1). Remove mitochondrial and chloroplast species before differential analysis. Because of the different taxonomic levels of 18S, only the lowest level of each taxon was used for differential analyses. All statistical analyzes were performed using R (v. 3.4.3., CRAN project) (Team 2013).
Exposure to mancozeb, pyrithiostrobin, and trifloxystrobin significantly reduced body weight gain in O. cornifrons (Fig. 1). These effects were consistently observed for all three doses assessed (Fig. 1a–c). Cyclostrobin and myclobutanil did not significantly reduce the weight of larvae.
Average fresh weight of stem borer larvae measured at three time points under four dietary treatments (homogeneous pollen feed + fungicide: control, 0.1X, 0.5X and 1X doses). (a) Low dose (0.1X): first time point (day 1): χ2: 30.99, DF = 6; P < 0.0001, second time point (day 5): 22.83, DF = 0.0009; third time; point (day 8): χ2: 28.39, DF = 6; (b) half dose (0.5X): first time point (day 1): χ2: 35.67, DF = 6; P < 0.0001, second time point (day one). ): χ2: 15.98, DF = 6; P = 0.0090; third time point (day 8) χ2: 16.47, DF = 6; (c) Site or full dose (1X): first time point (day 1) χ2: 20.64, P = 6; P = 0.0326, second time point (day 5): χ2: 22.83, DF = 6; P = 0.0009; third time point (day 8): χ2: 28.39, DF = 6; nonparametric analysis of variance. Bars represent mean ± SE of pairwise comparisons (α = 0.05) (n = 16) *P ≤ 0.05, **P ≤ 0.001, ***P ≤ 0.0001.
At the lowest dose (0.1X), larval body weight was reduced by 60% with trifloxystrobin, 49% with mancozeb, 48% with myclobutanil, and 46% with pyrithistrobin (Fig. 1a). When exposed to half the field dose (0.5X), the body weight of mancozeb larvae was reduced by 86%, pyrithiostrobin by 52% and trifloxystrobin by 50% (Fig. 1b). A full field dose (1X) of mancozeb reduced larval weight by 82%, pyrithiostrobin by 70%, and trifloxystrobin, myclobutanil and sangard by approximately 30% (Fig. 1c).
Mortality was highest among larvae fed mancozeb-treated pollen, followed by pyrithiostrobin and trifloxystrobin. Mortality increased with increasing doses of mancozeb and pyritisoline (Fig. 2; Table 2). However, corn borer mortality increased only slightly as trifloxystrobin concentrations increased; cyprodinil and captan did not significantly increase mortality compared with control treatments.
Mortality of borer fly larvae was compared after ingestion of pollen individually treated with six different fungicides. Mancozeb and pentopyramide were more sensitive to oral exposure to corn maggots (GLM: χ = 29.45, DF = 20, P = 0.0059) (line, slope = 0.29, P < 0.001; slope = 0.24, P <0.00)).
On average, across all treatments, 39.05% of patients were female and 60.95% were male. Among the control treatments, the proportion of women was 40% in both the low-dose (0.1X) and half-dose (0.5X) studies, and 30% in the field-dose (1X) studies. At 0.1X dose, among pollen-fed larvae treated with mancozeb and myclobutanil, 33.33% of adults were female, 22% of adults were female, 44% of adult larvae were female, 44% of adult larvae were female. female, 41% of adult larvae were females, and controls were 31% (Fig. 3a). At 0.5 times the dose, 33% of adult worms in the mancozeb and pyrithiostrobin group were female, 36% in the trifloxystrobin group, 41% in the myclobutanil group, and 46% in the cyprostrobin group. This figure was 53% in the group. in the captan group and 38% in the control group (Fig. 3b). At 1X dose, 30% of the mancozeb group were women, 36% of the pyrithiostrobin group, 44% of the trifloxystrobin group, 38% of the myclobutanil group, 50% of the control group were women – 38.5% (Fig. 3c).
Percentage of female and male borers after larval stage fungicide exposure. (a) Low dose (0.1X). (b) Half dose (0.5X). (c) Field dose or full dose (1X).
16S sequence analysis showed that the bacterial group differed between larvae fed with mancozeb-treated pollen and larvae fed with untreated pollen (Fig. 4a). The microbial index of untreated larvae fed on pollen was higher than that of larvae fed on mancozeb-treated pollen (Fig. 4b). Although the observed difference in richness between groups was not statistically significant, it was significantly lower than that observed for larvae feeding on untreated pollen (Fig. 4c). Relative abundance showed that the microbiota of larvae fed on control pollen was more diverse than that of larvae fed on mancozeb-treated larvae (Fig. 5a). Descriptive analysis revealed the presence of 28 genera in control and mancozeb-treated samples (Fig. 5b). c Analysis using 18S sequencing revealed no significant differences (Supplementary Figure 2).
SAV profiles based on 16S sequences were compared with Shannon richness and observed richness at the phylum level. (a) Principal coordinate analysis (PCoA) based on overall microbial community structure in untreated pollen-fed or control (blue) and mancozeb-fed larvae (orange). Each data point represents a separate sample. PCoA was calculated using the Bray-Curtis distance of the multivariate t distribution. Ovals represent the 80% confidence level. (b) Boxplot, raw Shannon wealth data (points) and c. Observable wealth. Boxplots show boxes for median line, interquartile range (IQR), and 1.5 × IQR (n = 3).
Composition of microbial communities of larvae fed on mancozeb-treated and untreated pollen. (a) Relative abundance of microbial genera reads in larvae. (b) Heat map of identified microbial communities. Delftia (odds ratio (OR) = 0.67, P = 0.0030) and Pseudomonas (OR = 0.3, P = 0.0074), Microbacterium (OR = 0.75, P = 0.0617) (OR = 1.5, P = 0.0060); Heat map rows are clustered using correlation distance and average connectivity.
Our results show that oral exposure to contact (mancozeb) and systemic (pyrostrobin and trifloxystrobin) fungicides, widely applied during flowering, significantly reduced weight gain and increased mortality of maize larvae. In addition, mancozeb significantly reduced the diversity and richness of the microbiome during the prepupal stage. Myclobutanil, another systemic fungicide, significantly reduced larval body weight gain at all three doses. This effect was evident at the second (day 5) and third (day 8) time points. In contrast, cyprodinil and captan did not significantly reduce weight gain or survival compared with the control group. To our knowledge, this work is the first to determine the effects of field rates of different fungicides used to protect corn crops through direct pollen exposure.
All fungicide treatments significantly reduced body weight gain compared to control treatments. Mancozeb had the greatest effect on larval body weight gain with an average reduction of 51%, followed by pyrithiostrobin. However, other studies have not reported adverse effects of field doses of fungicides on larval stages44. Although dithiocarbamate biocides have been shown to have low acute toxicity45, ethylene bisdithiocarbamates (EBDCS) such as mancozeb can degrade to urea ethylene sulfide. Given its mutagenic effects in other animals, this degradation product may be responsible for the observed effects46,47. Previous studies have shown that the formation of ethylene thiourea is influenced by factors such as elevated temperature48, humidity levels49 and length of product storage50. Proper storage conditions for biocides can mitigate these side effects. In addition, the European Food Safety Authority has expressed concern about the toxicity of pyrithiopide, which has been shown to be carcinogenic to the digestive systems of other animals51.
Oral administration of mancozeb, pyrithiostrobin, and trifloxystrobin increases mortality of corn borer larvae. In contrast, myclobutanil, ciprocycline and captan had no effect on mortality. These results differ from those of Ladurner et al.52, who showed that captan significantly reduced the survival of adult O. lignaria and Apis mellifera L. (Hymenoptera, Apisidae). In addition, fungicides such as captan and boscalid have been found to cause larval mortality52,53,54 or alter feeding behavior55. These changes, in turn, can affect the nutritional quality of the pollen and ultimately the energy gain of the larval stage. Mortality observed in the control group was consistent with other studies 56,57.
The male-favoring sex ratio observed in our work may be explained by factors such as insufficient mating and poor weather conditions during flowering, as previously suggested for O. cornuta by Vicens and Bosch. Although females and males in our study had four days to mate (a period generally considered sufficient for successful mating), we deliberately reduced light intensity to minimize stress. However, this modification may unintentionally interfere with the mating process61. In addition, bees experience several days of adverse weather, including rain and low temperatures (<5°C), which can also negatively impact mating success4,23.
Although our study focused on the entire larval microbiome, our results provide insight into potential relationships among bacterial communities that may be critical to bee nutrition and fungicide exposure. For example, larvae fed mancozeb-treated pollen had significantly reduced microbial community structure and abundance compared to larvae fed untreated pollen. In larvae consuming untreated pollen, the bacterial groups Proteobacteria and Actinobacteria were dominant and were predominantly aerobic or facultatively aerobic. Delft bacteria, usually associated with solitary bee species, are known to have antibiotic activity, indicating a potential protective role against pathogens. Another bacterial species, Pseudomonas, was abundant in larvae fed untreated pollen, but was significantly reduced in mancozeb-treated larvae. Our results support previous studies identifying Pseudomonas as one of the most abundant genera in O. bicornis35 and other solitary wasps34. Although experimental evidence for the role of Pseudomonas in the health of O. cornifrons has not been studied, this bacterium has been shown to promote the synthesis of protective toxins in the beetle Paederus fuscipes and promote arginine metabolism in vitro 35, 65. These observations suggest a potential role in viral and bacterial defense during development time of O. cornifrons larvae. Microbacterium is another genus identified in our study that is reported to be present in high numbers in black soldier fly larvae under starvation conditions66. In O. cornifrons larvae, microbacteria may contribute to the balance and resilience of the gut microbiome under stress conditions. Additionally, Rhodococcus is found in O. cornifrons larvae and is known for its detoxification abilities67. This genus is also found in the gut of A. florea, but in very low abundance68. Our results demonstrate the presence of multiple genetic variations across numerous microbial taxa that can alter metabolic processes in larvae. However, a better understanding of the functional diversity of O. cornifrons is needed.
In summary, the results indicate that mancozeb, pyrithiostrobin, and trifloxystrobin reduced body weight gain and increased mortality of corn borer larvae. Although there is growing concern about the effects of fungicides on pollinators, there is a need to better understand the effects of residual metabolites of these compounds. These results can be incorporated into recommendations for integrated pollinator management programs that help farmers avoid the use of certain fungicides before and during fruit tree flowering by selecting fungicides and varying the timing of application, or by encouraging the use of less harmful alternatives 36. This information is important for developing recommendations. on pesticide use, such as adjusting existing spray programs and changing spray timing when selecting fungicides or promoting the use of less hazardous alternatives. Further research is needed into the adverse effects of fungicides on sex ratio, feeding behavior, gut microbiome, and the molecular mechanisms underlying corn borer weight loss and mortality.
Source data 1, 2 and 3 in Figures 1 and 2 have been deposited in the figshare data repository DOI: https://doi.org/10.6084/m9.figshare.24996245 and https://doi.org/10.6084/m9. figshare.24996233. The sequences analyzed in the current study (Figs. 4, 5) are available in the NCBI SRA repository under accession number PRJNA1023565.
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Post time: May-14-2024