Effect of Cannabidiol on the Modulation of Proteolytic Activity on the Honey Bee Workers’ Cuticle

Source / Quelle: https://www.mdpi.com/1420-3049/31/14/2498

Abstract

The aim of the study was to determine the effect of cannabidiol extract (CBD) on the activity of the proteolytic system and the concentrations of total proteins on the cuticular surface of worker bees. Marked one-day-old workers were introduced into established colonies, which were divided into three groups (two hives each): CSy (CBD in sugar syrup), CSt (CBD-soaked strip), and C (control, pure syrup). Every seven days, the bioactive cuticular layer was washed off from the marked bees for biochemical analysis. Total protein concentrations increased with worker age in C, CSy, and CSt, and were highest from day 7 in group C. Protease activities increased until day 21/28 of worker life in all groups. From day 21 for acidic proteases, day 28 for neutral proteases, and day 14 for alkaline proteases, the activities were highest in CSy. The activities of neutral and alkaline protease inhibitors decreased with bee age and were always higher in CSy and CSt than in C, regardless of worker age. CBD favorably modulates the proteolytic system on the cuticle of honeybees.

Keywords:

 proteases; protease inhibitors; immunoproteins; body surface; cannabidiol; hemp extract; Apis mellifera

1. Introduction

Honeybees, as key pollinators, currently face numerous factors that negatively impact their quality of life. Despite the large number of apiaries in many regions, maintaining colonies in good condition, meaning an adequate number of healthy individuals with a biologically appropriate lifespan, has become a challenge [1]. These problems had “cradle” from the declining immunity of these pollinators, as noted by many scientists. The effectiveness of immune processes depends on the ability of bees to adapt to rapidly changing environmental conditions influenced by climate and anthropogenic factors (industrialization, chemicalization). Bees in this state are unable to respond appropriately to emerging natural pathogens and parasites (e.g., Vairimorpha/Nosema spp., Varroa destructor, Paenibacillus larvae, etc.), which limits their defenses and shortens their lifespan [2]. In recent years, the scientific community has focused on solutions that support and stimulate these processes using biostimulants. Natural supplements, designed to be safe not only for the colony but also for its products, are becoming increasingly popular. Often, these substances have already been positively tested in other animal groups and humans. This reduces the risk of future consumption of bee products containing residues from the decomposition of biostimulants with antioxidant properties, such as piperine, curcumin, caffeine, coenzyme Q10, or substances for so-called “superfoods” like resveratrol or even “pickles” [3,4,5,6,7,8]. In our previous work we have shown that an example of such a substance is the hemp-derived CBD [9]. Its popularity and number of potential health-promoting applications are growing year by year. Demand for these characteristics and properties of the preparation are evident in ongoing attempts to optimize the production of these preparations [10,11,12,13]. To date, CBD has been used primarily for regenerative purposes, supporting the treatment of conditions (e.g., cancer), and rheumatological and neurological conditions (Alzheimer’s, multiple sclerosis, anxiety, sleep disorders) [14]. Recent research also demonstrates new potential for CBD. Dehner et al. (2025) reported significant immune modulation capabilities through changes in morphology (a decrease in leukocyte count in both sexes and changes in mean hemoglobin concentration per red blood cell, mean red blood cell volume, and percentage of neutrophils and monocytes) and alanine transaminase activities, depending on sex in the rats [12]. We also confirmed these properties in honeybees after supplementation in apiary conditions [15,16]. Interestingly, the study also noted the sequence of extract accumulation, indicating the highest levels in the brain, then the kidneys and finally the skin [12]. Other studies on cytokine storm-induced hyperinflammation have shown that CBD reduces pro-inflammatory cytokines (in human PBMC), demonstrating a reduction in the pro-inflammatory cytokines TNFα and IL-1β and a concomitant increase in the anti-inflammatory cytokine IL-10 in in vivo mouse studies [11].

Research on this extract is mostly conducted on vertebrates, where the presence of CB1/CB2 receptors has been described. In invertebrates, the actual process by which these compounds act on their bodies is not yet well understood. Specific cannabinoid receptors have not yet been observed in insects. Studies conducted on one of the most popular insect model organisms, Drosophila melanogaster, mainly suggested that these flies may have a preference for consuming CBD-laced food, and in other studies, this extract may affect their circadian rhythm and lifespan, and modulate motor functions [17,18]. An interesting example of CBD’s effects in a more detailed context was conducted on another group of invertebrates—Caenorhabditis elegans. A 2024 study described that C. elegans possesses systems useful for modeling the effects of cannabinoids, including the NPR-19 receptor, considered an ortholog functionally related to CB1, and TRP channels; CBD also reduced the response to oxidative stress induced by a high-fat diet [19]. However, there is still no clear information regarding the specific classical receptors, as in the case of vertebrates. Nevertheless, it is worth emphasizing that some patterns in studies on vertebrates and invertebrates published by other scientists, as well as our previous studies on CBD extract, have similar conclusions regarding the effects of its use.

As with immune processes in vertebrates, many patterns and processes are reflected in the body of honeybees. One of the first immune mechanisms in bees is the proteolytic system. The proteolytic system consists of proteolytic enzymes (e.g., serine protease, aspartate protease, metalloproteases) and protease inhibitors. Proteolytic enzymes are designed to inhibit and degrade proteins of pathogenic organisms by cleaving at specific bond sites in these compounds. Protease inhibitors, on the other hand, protect bee proteins (including the aforementioned immune proteins, e.g., those with antimicrobial activities) by inactivating pathogen proteases. Proteases and inhibitors also play a crucial role in modulating other cellular processes, such as those related to melanization, phagocyte activation, and lysozyme and peroxidase activities [20,21,22,23]. Although these systems and the synthesis of their components take place in the fat body of bees and are then distributed through the hemolymph, proteolytic activities have also been observed on the insect’s body surface, on the cuticle, where it constitutes the first bioactive immune barrier, supporting physiological and anatomical barriers [22,24,25]. This first-line defense structure is supported internally by supplements such as CBD extract. In previously published studies, we demonstrated the positive effect of the extract on the proteolytic system and other systems, such as the antioxidant system, as well as an increase in concentrations of total proteins in bee hemolymph under cage experiment conditions (in vitro) and in apiary conditions (in vivo) [9,16]. The proteolytic enzymes and proteins produced in the fat body are transported outside the bee’s body through glands located between the tergites. In this way, these enzymes create an immune layer on the outside of the body. Effective synthesis of proteolytic system compounds is not always synonymous with their high activities and action, also on the cuticle. Effective synthesis of proteolytic compounds does not always equate to their high activities and action, including on the cuticle. Hence, biostimulants are sought that will support these mechanisms. Since anatomical and physiological barriers (i.e., the cuticle, digestive system, and respiratory system) constitute a “defense wall” in the bee’s body against pathogen penetration, they should be strengthened. We hypothesize that since CBD increases the activities of proteolytic compounds in the bee’s hemolymph, it may have a similar effect as part of the protective cuticle biofilm [26]. Therefore, the aim of our study was to determine the activity of the proteolytic system and the concentrations of total proteins in the cuticle of honeybee workers, as the outermost protective barrier shaped by external and internal processes.

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