The introduced solitary bee, Osmia cornifrons (Hymenoptera: Megachilidae), is an essential pollinator species in managed ecosystems. Naturalized populations of this bee are part of the approximately 50 wild species of bees that have replaced the honey bee for pollination of almond and apple orchards in USA (1, 2). Mason bees face numerous challenges, including habitat fragmentation, pathogens, and pesticides (2, 3). Among pesticides, fungicides can reduce energy gain, foraging (4) and fitness (5, 6). Although recent studies indicate mason bee fitness is directly affected by symbiotic and exobiotic microbes (7, 8) because bacteria and fungi can influence nutrition and immune response, the effects of exposure to fungicides on microbial diversity of mason bees are beginning to be explored.
Before and during flowering, apple orchards are typically sprayed with fungicides with varying modes of action (contact and systemic) to treat diseases such as apple scab, bitter rot, brown rot, and powdery mildew (9, 10). Historically, unlike insecticides, fungicides were assumed harmless to pollinators; hence, they were recommended to growers during flowering. Contact and ingestion exposure of these fungicides to honey bees are relatively well known since it is part of the pesticide registration process by the US-Environmental Protection Agency and many other countries' regulatory agencies (11–13). However, the effects of orchard pesticides on non-honey bees are less well known as they are not required to be registered in the US, there is a general lack of standardized testing protocols, and it is challenging to maintain colonies that provide bees for testing (14). Various species of managed Osmia in Europe and the US are increasingly being used to examine pesticide effects on wild bees, and recently, a standardized protocol was developed for O. cornifrons (15).
Osmia cornifrons is univoltine and has been commercially used in tree fruit crops to supplement or replace honey bees. These bees emerge between March and April, with protandrous males emerging three to four days ahead of females. After mating, females actively collect pollen and nectar for provisioning a series of brood cells within tubular nest cavities (natural or artificial) (16, 17). Eggs are laid on the pollen provisions within a cell; then, the female builds a mud partition before provisioning the next cell (18). The first larval instar is enclosed within the chorion, feeding on embryonic fluids. From the second to fifth instar, the larva feeds on pollen provisions, and once it reaches the fifth instar (prepupa), it defecates for the first time, forming a meconium. Once the pollen provision is completely consumed, the larva forms a cocoon, pupates, and becomes an adult in the same brood cell, usually by the end of summer (17, 19). The adult emerges from the cocoon and the tubular nest in the following spring (20). Adult survival is correlated with net energy gain (weight gain) based on the provisions consumed. Therefore, the nutritional quality of the pollen, as well as other factors such as weather or pesticide exposure, are determinants of survival and fitness (21).
Application of fungicides is the predominant method for disease control in tree fruit crops, and they are commonly applied during bloom. Fungicides are classified into two main categories: contact and systemic. Contact fungicides have preventive action by inhibiting fungi before infecting plant tissues. The plant assimilates systemic fungicides to varying degrees and can somewhat cure existing infections (22). Hence, pollinators are likely to be directly exposed to fungicides during bloom and may be further exposed as larvae from contaminated pollen and nectar. There is an increasing number of studies about the impacts of fungicides on pollinators. For example, some fungicides can reduce feeding and alter metabolism (23, 24). However, understanding how fungicides affect critical aspects of pollinator health, such as the microbiome, is crucial in developing strategies and policies to minimize the risks posed by their use.
Pre-bloom sprays of insecticides and fungicides with the ability to move within the plant vascular system to varying degrees from translaminar (e.g., able to move from the top surface of a leaf to the bottom surface as with some fungicides)(25) to the truly systemic neonicotinoid insecticides that can move from root applications up into the canopy have been previously shown to move into the nectar of apple bloom (26) where they can kill adult O. cornifrons (27). Some of these pesticides can also move into the pollen, affecting O. cornifrons larval development and cause delayed mortality (15). Other studies have shown some fungicides can dramatically change nesting behavior in a congener, O. lignaria (28). Furthermore, laboratory and field studies simulating pesticide (including fungicides) exposure scenarios demonstrated adverse effects on physiology, morphology, and survival in honey bees and some solitary bees (11). The impact of various fungicide sprays applied directly to open flowers at bloom, which would contaminate the pollen collected by adult O. cornifrons for larval development, has yet to be explored.
It is increasingly recognized that larval development is affected by the microbial community present in the pollen and digestive system. The bee microbiome influences parameters such as body mass (29), metabolism alterations, and susceptibility to pathogens. Prior research has investigated the effects of developmental stages, nutrients, and environment on solitary bee microbiome. These studies revealed similarities in structure and abundance of the microbiome of both larvae and pollen (30) and the most abundant bacteria genera, Pseudomonas and Delftia, in solitary bee species (31, 32). However, the impact of fungicides on the larval microbiome through direct oral exposure remains unexplored despite its relevance for strategies aimed at preserving bee health.
This study tested the effects of field-realistic doses of six commonly used contact and systemic fungicides via oral exposure to O. cornifrons larvae from contaminated provisions. We found contact and systemic fungicides reduced bee weight gain and increased mortality, with the most severe impact associated with mancozeb and penthipyrad. Then, we compared the microbial diversity of larvae fed with pollen provisions treated with mancozeb against those fed with control provisions. We discuss potential mechanisms underpinning the lethality and implications for integrated pest and pollinator management (IPPM) programs.