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Accepted: 27 March 2025
Published: 2 April 2025
Citation: Diedrick, W.A.; Kanga,
L.H.B.; Mallinger, R.; Pescador, M.;
Elsharkawy, I.; Zhang, Y. Molecular
Assessment of Genes Linked to
Honeybee Health Fed with Different
Diets in Nuclear Colonies. Insects
2025,16, 374. https://doi.org/
10.3390/insects16040374
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Article
Molecular Assessment of Genes Linked to Honeybee Health Fed
with Different Diets in Nuclear Colonies
Worrel A. Diedrick 1, Lambert H. B. Kanga 1,* , Rachel Mallinger 2, Manuel Pescador 1, Islam Elsharkawy 3and
Yanping Zhang 4
1Entomology Department, College of Agriculture and Food Sciences, Florida A&M University,
Tallahassee, FL 32307, USA; worrel1.diedrick@famu.edu (W.A.D.); manuel.pescador@gmail.com (M.P.)
2Department of Entomology and Nematology, University of Florida, Gainesville, FL 32608, USA;
rachel.mallinger@ufl.edu
3Center for Viticulture and Small Fruits Research, Tallahassee, FL 32307, USA; islam.elsharkawy@famu.edu
4Interdisciplinary Center for Biotechnology Research, University of Florida, Gainesville, FL 32610, USA;
yanp@ufl.edu
*Correspondence: lambert.kanga@famu.edu
Simple Summary: Agriculture increasingly depends on honeybees for crop pollination,
and the added value of crops pollinated by honeybees has been estimated at USD 20 billion;
it is, therefore, vital to sustaining a profitable agriculture industry. Unfortunately, the
number of colonies has continued to decline over the last few decades. Colony losses
have occurred concurrently with an increasing demand for the pollination of fiber, fruit,
vegetable, and nut crops. This decline is attributed to a combination of factors including
parasites, diseases, pesticides, and nutrition. In this study, using nuclear colonies (nucs), we
assessed the impact of different diets on the expression of genes linked to honeybee health.
These included immune function genes [Cactus, immune deficiency (IMD), Spaetzle)], genes
involved in nutrition, cellular defense, longevity, and behavior (Vitellogenin,Malvolio), a
gene involved in energy metabolism (Maltase), and a gene associated with locomotory
behavior (Single-minded). The diets included (a) commercial pollen patties and sugar
syrup, (b) monofloral (anise hyssop), and (c) polyfloral (marigold, anise hyssop, sweet
alyssum, and basil). At the end of a 2.7-month experimental period, we found that the diets
significantly affected honeybee health and triggered an up- and downregulation of these
genes, which correlated with the health and activities of the honeybee colonies. Overall,
we found that monofloral and polyfloral diets provided higher nutritional benefits, thus
enhancing honeybee health, compared with the pollen patty and sugar syrup. Unlike the
findings in several reports on honeybee nutrition, our data indicated that a single-species
diet (such as anise hyssop) is nutritionally adequate and better or comparable to polyfloral
diets. However, this result will depend on the nutritional value and bloom period of
individual plant species. Thus, we recommend that the landscape of any apiaries include
resource-rich and abundant flowering plants, like anise hyssop, throughout the season to
enhance honeybee health.
Abstract: Honeybees are of economic importance not only for honey production, but
also for crop pollination, which amounts to USD 20 billion per year in the United States.
However, the number of honeybee colonies has declined more than 40% during the last
few decades. Although this decline is attributed to a combination of factors (parasites,
diseases, pesticides, and nutrition), unlike other factors, the effect of nutrition on honeybee
health is not well documented. In this study, we assessed the differential expression of
seven genes linked to honeybee health under three different diets. These included immune
function genes [Cactus, immune deficiency (IMD), Spaetzle)], genes involved in nutrition,
Insects 2025,16, 374 https://doi.org/10.3390/insects16040374
Insects 2025,16, 374 2 of 20
cellular defense, longevity, and behavior (Vitellogenin,Malvolio), a gene involved in energy
metabolism (Maltase), and a gene associated with locomotory behavior (Single-minded).
The diets included (a) commercial pollen patties and sugar syrup, (b) monofloral (anise
hyssop), and (c) polyfloral (marigold, anise hyssop, sweet alyssum, and basil). Over
the 2.7-month experimental periods, adult bees in controls fed pollen patties and sugar
syrup showed upregulated Cactus (involved in Toll pathway) and IMD (signaling pathway
controls antibacterial defense) expression, while their counterparts fed monofloral and
polyfloral diets downregulated the expression of these genes. Unlike Cactus and IMD,
the gene expression profile of Spaetzle (involved in Toll pathway) did not differ across
treatments during the experimental period except that it was significantly downregulated
on day 63 and day 84 in bees fed polyfloral diets. The Vitellogenin gene indicated that
monofloral and polyfloral diets significantly upregulated this gene and enhanced lifespan,
foraging behavior, and immunity in adult bees fed with monofloral diets. The expression of
Malvolio (involved in sucrose responsiveness and foraging behavior) was upregulated when
food reserves (pollen and nectar) were limited in adult bees fed polyfloral diets. Adult bees
fed with monofloral diets significantly upregulated the expression of Maltase (involved
in energy metabolisms) compared to their counterparts in control diets to the end of the
experimental period. Single-Minded Homolog 2 (involved in locomotory behavior) was also
upregulated in adult bees fed pollen patties and sugar syrup compared to their counterparts
fed monofloral and polyfloral diets. Thus, the food source significantly affected honeybee
health and triggered an up- and downregulation of these genes, which correlated with
the health and activities of the honeybee colonies. Overall, we found that the companion
crops (monofloral and polyfloral) provided higher nutritional benefits to enhance honeybee
health than the pollen patty and sugar syrup used currently by beekeepers. Furthermore,
while it has been reported that bees require pollen from diverse sources to maintain a
healthy physiology and hive, our data on nuclear colonies indicated that a single-species
diet (such as anise hyssop) is nutritionally adequate and better or comparable to polyfloral
diets. To the best of our knowledge, this is the first report indicating better nutritional
benefits from monofloral diets (anise hyssop) over polyfloral diets for honeybee colonies
(nucs) in semi-large-scale experimental runs. Thus, we recommend that the landscape of
any apiary include highly nutritious food sources, such as anise hyssop, throughout the
season to enhance honeybee health.
Keywords: honeybee colonies; gene expression; immune response; monofloral and polyflo-
ral diets; pollen patty; sugar syrup; honeybee health; Varroa mite; companion crops;
landscape
1. Introduction
Food production and forest ecosystems are largely dependent on insect pollinators.
The implications of pollinator health (location, density, and activity) are important, with a di-
rect economic value worldwide greater than USD 175 billion [
1
–
3
]. Unfortunately, bees and
other insect pollinators have suffered a sharp population decline in recent decades caused
by parasites, pesticides, pathogens, land development, and habitat fragmentation [
4
–
6
]. The
detrimental effects of pesticides, parasites, and pathogens on honeybee health have been
well documented [
7
–
10
]. Honeybees have an innate immune system, which include cellular
and humoral responses against pathogens and parasites [
11
]. Cellular responses consist of
phagocytosis, encapsulation, and myelinization mechanism [
12
]. The humoral response
system includes pattern recognition receptors that interacts with pathogen-associated
Insects 2025,16, 374 3 of 20
molecules which stimulate different pathways (Toll, IMD, JNK, JAK/STAT) depending
on each type of pathogen to produce anti-microbial peptides (AMPs) [
13
]. The immune
system of individual bees plays a key role in colony health status [
13
,
14
]. For example,
using relish (IMD pathway) and defensin (Toll pathway) as immune markers is useful for
monitoring colony health when compared to using of deformed wing virus or mite load
for assessing colony health [
15
]. Honeybees are generalist pollinators, having relationships
with several plant species for their nutritional requirements, to ensure the normal growth
and development of bee colonies [
16
,
17
]. Floral species produce pollens which differ in
their nutritional contents; the pollen quality and availability of polyfloral pollen diets
influence pollen intake, which, in turn affect, honeybee health. Some immune functions,
particularly glucose oxidase (GOX) activity (an enzyme involved in the synthesis of anti-
septic hydrogen peroxide in honey), can be enhanced by polyfloral diets when compared
with monofloral diets [
18
]. Overall, the health of honeybee colonies is maintained not
by a monospecific pollen diet, but by a more diverse pollen diet which augments the
lifespan of honeybees [
18
]. Moreover, worker bees fed with a polyfloral pollen diet can
have increased immune functions against pathogenic invasions [
19
]. It has been shown that
honeybees fed with a diverse pollen diet had a higher level of glucose oxidase and brood
food compared with those fed with a monofloral diet with a higher protein content [
19
].
The pollen preferences of the foragers are determined by the requirements of the colony;
preferred pollen sources are influenced more by the composition of fatty and amino acids
of the pollen than by the total protein content [
16
,
17
]. Pollen and nectar are essential dietary
sources for honeybees [
20
]. Pollen contains proteins, amino acids, lipids, vitamins, and
minerals, all of which are vital for honeybee health, colony growth and development [
20
,
21
].
The number and health of honeybees in the landscapes can be increased through habitat
restoration and other land management practices [
21
]. Little is known about factors related
to nutritional fitness and the composition of the landscape on honeybee health. Previous
studies suggested that strategies should include a mixture of plant species (companion
crops) to provide continual blooms and complementary nutritional profiles throughout
the growing season for use by insect pollinators. Indeed, reduced floral resource diversity
can lead to suboptimal bee nutrition, resulting in a weakened immune system and poor
overall bee health [
22
,
23
]. However, in some cases, highly nutritious plants with a long
bloom period may be adequate for generalist pollinators, like honeybees [24].
Diet is an important factor in the development of individuals and the entire colony.
Alaux et al. [
19
] reported that an improperly balanced diet may weaken the immune system
and increase the susceptibility of workers to diseases. Bry´s et al. [
25
] reported that a
honeybee diet based on multiflower pollen is more desirable than a monoflower diet but
must be properly balanced. In addition, while pollen from a single species has specific
benefits, bees may require pollen from diverse sources to maintain their individual health
and a healthy hive.
The objectives of this study were to assess the impact of nutrition on honeybee health.
These include (a) the strength and growth of the honeybee colony, and (b) the response of
genes linked to bee health when provided with different diets. We compared the differential
expression of seven genes, including immune function genes (Cactus, immune deficiency,
Spaetzle), genes involved in nutrition, longevity, and behavior (Vitellogenin,Malvolio), a gene
involved in energy metabolism (Maltase), and a gene associated with locomotory behavior
(Single-minded) linked to honeybee health in adult bees when fed with the following three
diets: a control diet (commercial pollen and sugar patties), a monofloral diet (anise hyssop),
and a polyfloral diet (companion crops). To the best of our knowledge, this is the first study
of its kind using nuclear (nuc) honeybee colonies. The findings should provide useful
insights into the development of the best beekeeping practices.
Insects 2025,16, 374 4 of 20
2. Materials and Methods
2.1. Honeybee Colonies and Treatments
Twenty-one colonies with uniform honeybee (Apis mellifera) populations were estab-
lished in our apiary at the Research Experimental Farm in Quincy, FL. The experiments
were conducted in 15 flight cages (Figure 1) (each 6 m
×
3 m
×
1.8 m, or 32.4 m
2
), with 5
flight cages per treatment and 1 nuclear (nuc) colony per flight cage, totaling 15 colonies.
The honeybees were provided with three diets (treatments). Treatment 1 (control) received
commercial pollen patties (Dadant & Sons, Inc., Hamilton, IL, USA) and sugar syrup. Treat-
ment 2 (monofloral) received anise hyssop (Agastache foeniculum). Treatment 3 (polyfloral)
was provided with a companion crop mix [anise hyssop (A. foeniculum), marigold (Calendula
officinalis), sweet alyssum (Lobularia maritima) and basil (Ocimum basilicum)]. The companion
crops were evenly spaced in the flight cages and covered 10% of the total area to serve as a
food source (pollen, nectar) for the bees [
26
]. A water source was also provided to the bees
(Figure 1). The companion plants were chosen because of their high nutritional benefits
(nectar and pollen), long blooming period, attractiveness to honeybees, and easiness to
grow [
27
–
30
]. Honeybees also select plants to forage based on their colony’s needs [
31
].
The food sources (companion plants) made up 10% of the total area [
26
] (Figures 1and 2).
The experiments ran from 4 October to 27 December 2023.
Insects 2025, 16, x FOR PEER REVIEW 4 of 21
findings should provide useful insights into the development of the best beekeeping prac-
tices.
2. Materials and Methods
2.1. Honeybee Colonies and Treatments
Twenty-one colonies with uniform honeybee (Apis mellifera) populations were estab-
lished in our apiary at the Research Experimental Farm in Quincy, FL. The experiments
were conducted in 15 flight cages (Figure 1) (each 6 m × 3 m × 1.8 m, or 32.4 m2), with 5
flight cages per treatment and 1 nuclear (nuc) colony per flight cage, totaling 15 colonies.
The honeybees were provided with three diets (treatments). Treatment 1 (control) re-
ceived commercial pollen paies (Dadant & Sons, Inc., Hamilton, IL, USA) and sugar
syrup. Treatment 2 (monofloral) received anise hyssop (Agastache foeniculum). Treatment
3 (polyfloral) was provided with a companion crop mix [anise hyssop (A. foeniculum),
marigold (Calendula officinalis), sweet alyssum (Lobularia maritima) and basil (Ocimum ba-
silicum)]. The companion crops were evenly spaced in the flight cages and covered 10% of
the total area to serve as a food source (pollen, nectar) for the bees [26]. A water source
was also provided to the bees (Figure 1). The companion plants were chosen because of
their high nutritional benefits (nectar and pollen), long blooming period, aractiveness to
honeybees, and easiness to grow [27–30]. Honeybees also select plants to forage based on
their colony’s needs [31]. The food sources (companion plants) made up 10% of the total
area [26] (Figures 1 and 2). The experiments ran from 4 October to 27 December 2023.
Figure 1. Flight cage with nuclear honeybee colony, companion plants, and water source.
Figure 1. Flight cage with nuclear honeybee colony, companion plants, and water source.
Insects 2025, 16, x FOR PEER REVIEW 5 of 21
Figure 2. Companion plants (sweet alyssum, anise hyssop, marigold, and basil) inside the flight
cage.
2.2. Assessment of Varroa Mite Infestations and Honeybee Colony Strength
Mite infestations of adult bees in the nucs were assessed at the start of the experi-
ments and at 84 days post-treatment using the alcohol wash method as described by
Delaplane et al. [32]. The strength of the bee colonies was evaluated by visually inspecting
both sides of each frame in the hive to estimate the area covered with adult bees, brood,
pollen, and honey. The estimates were made in tenths of frame equivalents (for a standard
232 mm-deep Langstroth frame [33,34]. The assessments of colony strength were con-
ducted at the beginning and at the end of the 84-day experimental period.
2.3. Honeybee Samples
Samples of 9 adult female worker bees were collected from each of the 5 treatments
every 21 days. Adult bees were randomly collected from the frames and placed in plastic
crush-proof containers. Samples were held on liquid nitrogen before being stored in a −80
°C freezer. Samples were later processed using reverse transcription droplet digital PCR
(RT-ddPCR).
2.4. RNA Extraction and Reverse Transcription
Adult honeybees were ground in liquid nitrogen. Ribonucleic acid (RNA) was iso-
lated from nine individual worker bees using the RNeasy Mini Kit (Qiagen, Hilden, Ger-
many, catlog #74104) according to the manufacturer’s protocol. RNA concentration was
quantified using the Qubit® 2.0 Fluorometer method (Invitrogen, Waltham, MA, USA)
and RNA quality was assessed using the Agilent TapeStation 2200 (Agilent Technologies,
Inc., Santa Clara, CA, USA). Then, 500 ng of total RNA was used for reverse transcription
using SuperScript™ IV VILO™ Master Mix (Thermo Fisher, Waltham, MA, USA, Cat. No:
11766050) according to the user guide.
2.5. Primer Design
The potential impact on the function of genes linked to honeybee health were as-
sessed from a subset of known genes (Table 1) from the literature [35–37]. These include
genes involved in nutrition and cellular defense [Cactus, immune deficiency (IMD),
Spaele (Spz), Malvolio (Mvl), and Vitellogenin (Vg)] and genes involved in energy and be-
havior (Maltase and Single-minded homolog 2). Primers were designed using information
from the National Center for Biotechnology Information, (hp://www.ncbi.nlm.nih.gov,
NCBI) and Bee Base (hp://hymenopteragenome.org). Primer conditions were optimized
Figure 2. Companion plants (sweet alyssum, anise hyssop, marigold, and basil) inside the flight cage.
Insects 2025,16, 374 5 of 20
2.2. Assessment of Varroa Mite Infestations and Honeybee Colony Strength
Mite infestations of adult bees in the nucs were assessed at the start of the experiments
and at 84 days post-treatment using the alcohol wash method as described by Delaplane
et al. [
32
]. The strength of the bee colonies was evaluated by visually inspecting both
sides of each frame in the hive to estimate the area covered with adult bees, brood, pollen,
and honey. The estimates were made in tenths of frame equivalents (for a standard 232
mm-deep Langstroth frame [
33
,
34
]. The assessments of colony strength were conducted at
the beginning and at the end of the 84-day experimental period.
2.3. Honeybee Samples
Samples of 9 adult female worker bees were collected from each of the 5 treatments
every 21 days. Adult bees were randomly collected from the frames and placed in plastic
crush-proof containers. Samples were held on liquid nitrogen before being stored in a
−80 ◦C
freezer. Samples were later processed using reverse transcription droplet digital
PCR (RT-ddPCR).
2.4. RNA Extraction and Reverse Transcription
Adult honeybees were ground in liquid nitrogen. Ribonucleic acid (RNA) was isolated
from nine individual worker bees using the RNeasy Mini Kit (Qiagen, Hilden, Germany,
catlog #74104) according to the manufacturer’s protocol. RNA concentration was quantified
using the Qubit
®
2.0 Fluorometer method (Invitrogen, Waltham, MA, USA) and RNA qual-
ity was assessed using the Agilent TapeStation 2200 (Agilent Technologies, Inc., Santa Clara,
CA, USA). Then, 500 ng of total RNA was used for reverse transcription using SuperScript™
IV VILO™ Master Mix (Thermo Fisher, Waltham, MA, USA, Cat. No: 11766050) according
to the user guide.
2.5. Primer Design
The potential impact on the function of genes linked to honeybee health were assessed
from a subset of known genes (Table 1) from the literature [
35
–
37
]. These include genes
involved in nutrition and cellular defense [Cactus, immune deficiency (IMD), Spaetzle (Spz),
Malvolio (Mvl), and Vitellogenin (Vg)] and genes involved in energy and behavior (Maltase
and Single-minded homolog 2). Primers were designed using information from the National
Center for Biotechnology Information, (http://www.ncbi.nlm.nih.gov, NCBI) and Bee Base
(http://hymenopteragenome.org). Primer conditions were optimized by determining the
optimal annealing temperature (Ta) and primer concentration [
38
]. Before real-time PCR
(RT-PCR), each primer was validated by specific PCR amplifications and 1.5% agarose gel
electrophoresis in TBE buffer with ethidium bromide staining.
Table 1. Primers used for quantitative PCR of honeybee immune-linked genes for nutrition.
Gene NCBI Forward Primer
(5′to 3′)
Reverse Primer
(5′to 3′)Function
Cactus FJ546095.1 ACATAGTTCGG
GCCACACTG
AGGTGCGGTTGCA
GTATTCA Immune response [35]
Immune deficiency
(IMD)NM_001163717.2 ACCCGCCAAAT
GCCAATAGA
AGTCGATGGTGGTA
ATGGTACT
Antimicrobial
defense [37]
Spaetzle XM_026444398.1 GTGTCAGTGGC
GGTGACTAA
GCACGTGTTGATG
TATCCGC
Member of the
TOLL-immune signaling
pathway against
bacteria and fungi [36]
Insects 2025,16, 374 6 of 20
Table 1. Cont.
Gene NCBI Forward Primer
(5′to 3′)
Reverse Primer
(5′to 3′)Function
Vitellogenin (Vg) NM_001011578.1 AACGCTTTTACTG
TTCGCGG
TATGCACGTCCGAC
AGATCG
Immune function and
longevity [36]
Maltase XM_006564751.3
CGAAAGCAGCAAC
GAATGGG
ACAGGTTTATCGC
TGTTACCGA Energy metabolism [36]
Malvolio (mvl) XM_006563052.3 TCCCCGCCAAGAT
CACATTT
ACCACACCAAGTCT
TGCACT
Involved in sucrose
responsiveness [36]
Single-minded
homolog 2 XM_016914389.2
TGCGATCGGGAGA
AAGTGTC
TTTCGCCTCCAACT
ACCGAC
Locomotor behavior [
36
]
2.6. Droplet Digital Polymerase Chain Reaction (ddPCR)
Droplet digital PCR amplification was performed with nine individual adult bees
collected from each of the five treatments. The oligonucleotide amplification primers are
listed in Table 1. The gene expressions of Cactus, immune deficiency, Malvolio,Maltase,
Single-Minded, Spaetzle, and Vitellogenin were tested using a BioRad QX200 AutoDG Droplet
Digital PCR System (Bio-Rad, Hercules, CA, USA). Briefly, each of the 20
µ
L reactions
contained 10 uL QX200 EvaGreen Supermix (Bio-Rad, Hercules, CA, USA), 250 nM gene-
specific primers, and 1
µ
L of the cDNA sample with 6.67 ng of total RNA input. The 20
µ
L
reaction was mixed with 20
µ
L of droplet generation oil and partitioned in ~20,000 droplets
after droplet generation. Then, the nanoliter-sized droplets were PCR amplified in a thermal
cycler with enzyme activation at 95
◦
C for 10 min followed by a two-step cycling protocol
for 40 cycles (94
◦
C for 30 s; 60
◦
C for 1 min); after enzyme deactivation at 98
◦
C for
10 min, the final step was to hold at 4
◦
C indefinitely. A ramp rate of 2
◦
C/s and a lid
temperature of 105
◦
C were used. Finally, the fluorescence intensity of individual droplets
was measured with the QX200 Droplet Reader (Bio-Rad). The data analysis was performed
with Quantasoft droplet reader software (v. 1.4) (Bio-Rad). Positive and negative droplet
populations were detected automatically. The absolute transcript expression was reported
in copies/ng of total RNA input. The RNA extraction, RT, and ddPCR were performed
at the University of Florida’s ICBR Gene Expression Core (https://biotech.ufl.edu/gene-
expression-genotyping/ (accessed on 6 March 2023), RRID:SCR_019145).
2.7. Statistical Analysis
Data on colony development and mite infestations were subjected to analysis of
covariance using pre-treatment data as the covariate [
39
]. The data on adult bee populations,
brood, pollen, and honey were transformed to log (x + 1) to satisfy the assumptions
of normality before analysis. Means between treatments were separated using Tukey’s
studentized range test. Data on gene expression (transcript abundances) for bee samples
from the different treatments were subjected to repeated measures analysis [
39
]. Treatments
were modeled as fixed effects; date and date-by-treatment interactions were modeled as
random effects. The data on gene expression were transformed to log (x + 1) to satisfy the
assumptions of normality before analysis. A significant level of alpha = 0.05 was used for
all statistical tests.
3. Results
3.1. Honeybee Colony Growth and Development
The numbers of mites per adult bees were similar in all treatment groups at the
start of the experiment on day 0 (Table 2). Mite infestations per adult bees did not vary
Insects 2025,16, 374 7 of 20
significantly between treatment groups (F = 0.19; df = 2, 3; p= 0.8356) at the end of the
84-day experimental period. The average number of bees in the colonies did not differ
between the treatments at the beginning of the experiment (day 0). At the end of the
experimental run, bee populations were significantly reduced as compared to the initial
size of the populations for all treatment groups (F = 8.77; df = 5, 13; p= 0.0008) except for the
monofloral diet group (Table 2). The honey reserves were significantly different between
treatment groups (F = 8.77; df = 5, 13; p= 0.0008) and significantly higher in adult bees fed
with pollen patties and sugar syrup as compared to their counterparts fed with monofloral
and polyfloral diets (Table 2). The experiments were conducted in the fall season, and the
colonies (nucs) had no brood production nor pollen reserves (only traces) at the end of the
84-day experimental period. Our observations during the experimental run indicated that
the pollen that was collected by adult bees was not stored but was instead probably used
for the daily maintenance of the colonies.
Table 2. Mite infestations and colony development of adult bees fed with control (pollen patties and
sugar syrup), monofloral (anise hyssop), and polyfloral (companion crops). Means in rows are not
significantly different if followed by the same upper-case letter (p> 0.05). Means in columns are not
significantly different if followed by the same lower-case letter (p< 0.05; Tukey test).
Treatments
1
Number of Mites per Hundred
Adult Bees (Mean ±SE)
2Colony Strength Parameters (Mean ±SE)
Bees Honey
Before After Before After Before After
Control 0.11 ±0.01 aA 0.94 ±0.61 aA
5.20
±
0.52 aA
0.72 ±0.67 aB
2.28
±
0.30 aA 1.67
±
1.07 aA
Monofloral 0.35 ±0.14 aA 0.41 ±0.19 aA
5.04
±
1.00 aA 2.92
±
0.82 aA 3.08
±
0.32 aA
0.32 ±0.19 aB
Polyfloral 0.01 ±0.00 aA 0.20 ±0.12 aA
5.54
±
0.27 aA
1.12 ±0.73 aB
2.14
±
0.27 aA
0.01 ±0.00 aB
1
The initial numbers of mites per hundred adult bees (mite infestations) were not significantly different among
treatments. 2Estimates were made in tenths of standard Langstroth frames [33,34].
3.2. Expression of Genes Linked to Honeybee Health Fed with Different Diets
3.2.1. Cactus Gene Expression
Expression levels (transcript abundances) of Cactus, a gene involved in the immune
response in the Toll pathway [
40
,
41
] were similar in all treatment groups in flight cages
at the start of the experiments on day 0 (F = 0.05; df = 2, 3; p= 0.9476) and on day 21
(F = 0.47; df = 2, 3; p= 0.6658) (Figure 3). However, on day 42, gene expression varied
significantly between control groups fed with pollen patties and sugar syrup and their
counterparts fed with the monofloral (anise hyssop) and polyfloral (companion crops) diets
(F = 9.46; df = 2, 6; p= 0.0140). Gene expression levels of Cactus on day 42 were similar
(
t = −0.36;
df = 3; p= 0.7445) in adult bees fed with monofloral and polyfloral diets. On day
63, differential gene expression levels of adult bees were significantly different between the
control groups and both monofloral and polyfloral groups (F = 10.89; df = 2, 9; p= 0.0040)
but not between the monofloral and polyfloral diets (t =
−
0.28; df = 3; p= 0.7958). At the
end of the 84-day experimental period, gene expression levels varied significantly between
treatments (
F = 15.19;
df = 2, 12; p= 0.0005). The time effect (a measure of within-treatment
variability over time) was not significant (F = 1.79; df = 4,12; p= 0.1949) nor were the
time-by-treatment interactions (F = 2.04; df = 8,12; p= 0.1280). However, gene expression
levels of Cactus did not differ (t =
−
0.30; df = 3; p= 0.7832) between the monofloral and
polyfloral diets on day 84. Overall, adult honeybees in the control groups fed with pollen
patties and sugar syrup upregulated Cactus gene expression compared to their counterparts
fed with monofloral (anise hyssop) and polyfloral (companion crops) diets (Figure 1).
Insects 2025,16, 374 8 of 20
Insects 2025, 16, x FOR PEER REVIEW 8 of 21
3.2. Expression of Genes Linked to Honeybee Health Fed with Different Diets
3.2.1. Cactus Gene Expression.
Expression levels (transcript abundances) of Cactus, a gene involved in the immune
response in the Toll pathway [40,41] were similar in all treatment groups in flight cages at
the start of the experiments on day 0 (F = 0.05; df = 2, 3; p = 0.9476) and on day 21 (F = 0.47;
df = 2, 3; p = 0.6658) (Figure 3). However, on day 42, gene expression varied significantly
between control groups fed with pollen paies and sugar syrup and their counterparts
fed with the monofloral (anise hyssop) and polyfloral (companion crops) diets (F = 9.46;
df = 2, 6; p = 0.0140). Gene expression levels of Cactus on day 42 were similar (t = −0.36; df
= 3; p = 0.7445) in adult bees fed with monofloral and polyfloral diets. On day 63, differen-
tial gene expression levels of adult bees were significantly different between the control
groups and both monofloral and polyfloral groups (F = 10.89; df = 2, 9; p = 0.0040) but not
between the monofloral and polyfloral diets (t = −0.28; df = 3; p = 0.7958). At the end of the
84-day experimental period, gene expression levels varied significantly between treat-
ments (F = 15.19; df = 2, 12; p = 0.0005). The time effect (a measure of within-treatment
variability over time) was not significant (F = 1.79; df = 4,12; p = 0.1949) nor were the time-
by-treatment interactions (F = 2.04; df = 8,12; p = 0.1280). However, gene expression levels
of Cactus did not differ (t = −0.30; df = 3; p = 0.7832) between the monofloral and polyfloral
diets on day 84. Overall, adult honeybees in the control groups fed with pollen paies and
sugar syrup upregulated Cactus gene expression compared to their counterparts fed with
monofloral (anise hyssop) and polyfloral (companion crops) diets (Figure 1).
Figure 3. Paerns of the gene expression profile of Cactus in adult bees fed with control (pollen and
sugar syrup), monofloral (anise hyssop), and polyfloral (companion crops) diets during the 84-day
experimental period. Means between lines within each sampling date are not significantly different
if followed by the same lower-case leer (p > 0.05; Tukey test).
3.2.2. Immune Deficiency Gene Expression.
The gene expression levels of immune deficiency (IMD) (an immune defense path-
way against bacteria and fungi) [42] were different (F = 20.27; df = 2, 3; p = 0.0181) between
treatments at the start of the experiments on day 0 (Figure 4). Gene expression continued
to vary significantly between treatments on day 21 (F = 188.15; df = 2, 3; p = 0.0007). Thus,
adult bees fed with pollen paies and sugar syrup displayed significantly higher
Days After Treatments
Day 0 Day 21 Day 42 Day 63 Day 84
Gene Expression (copies/uL)
180
200
220
240
260
280
300
320
Control
Polyfloral
Monofloral
a
a
a
a
a
a
a
b
b
a
a
b
b
b
b
Figure 3. Patterns of the gene expression profile of Cactus in adult bees fed with control (pollen and
sugar syrup), monofloral (anise hyssop), and polyfloral (companion crops) diets during the 84-day
experimental period. Means between lines within each sampling date are not significantly different if
followed by the same lower-case letter (p> 0.05; Tukey test).
3.2.2. Immune Deficiency Gene Expression
The gene expression levels of immune deficiency (IMD) (an immune defense pathway
against bacteria and fungi) [
42
] were different (F = 20.27; df = 2, 3; p= 0.0181) between
treatments at the start of the experiments on day 0 (Figure 4). Gene expression continued
to vary significantly between treatments on day 21 (F = 188.15; df = 2, 3; p= 0.0007). Thus,
adult bees fed with pollen patties and sugar syrup displayed significantly higher expression
of the IMD gene than their counterparts fed with monofloral (anise hyssop) diets (t = 19.33;
df = 3; p= 0.0003) and polyfloral diets (t = 11.07; df = 3; p= 0.0016). Also, adult bees fed
with monofloral diets had a significantly lower expression of IMD gene compared to their
counterparts fed with polyfloral diets (t =
−
8.26; df = 3; p= 0.0037). Differential gene
expression levels of adult bees were significantly different between the control groups
and both monofloral and polyfloral groups on day 42 (F = 88.57; df = 2, 3; p= 0.0021) and
day 63 (F = 51.90; df = 2, 3; p= 0.0047). The patterns of the IMD gene profile for days
42 and 63 were similar to that of day 21. At the end of the 84-day experimental period,
gene expression levels of IMD gene varied significantly between treatments (F = 65.98;
df = 2, 3; p= 0.0033) as in previous sampling dates. The time effect was not significant
(
F = 1.96;
df = 4,12; p= 0.1649) but the time-by treatment interactions were significant
(
F = 3.10; df = 8, 12; p= 0.0382
). Adult bees fed the control diets displayed a significantly
upregulated expression of IMD gene compared to those fed with monofloral diets (t = 11.40;
df = 3; p= 0.0014) and those fed with polyfloral diets (t = 6.93; df = 3; p= 0.0062). Adult
bees fed with monofloral diets had a significantly lower expression of IMD gene compared
to their counterparts fed with polyfloral diets (t = −4.47; df = 3; p=−0.0209).
3.2.3. Spaetzle Gene Expression
The expression levels of the gene Spaetzle (Spz), a member of the Toll immune signaling
pathway against fungi and bacteria [
36
,
42
], were similar (F = 2.0; df = 2, 3; p= 0.2805) in
all treatment groups at the start of the experiments on day 0, on day 21 (F = 2.55; df = 2,
3;
p= 0.2257
), and on day 42 (F = 4.20; df = 2, 3; p= 0.1348) (Figure 5). However, gene
expression varied significantly between treatments (F = 10.71; df = 2, 3; p= 0.0242) on
day 63. The Spaetzle expression levels were similar between the control and monofloral
Insects 2025,16, 374 9 of 20
diets (
t = 0.46;
df = 3; p= 0.6751), but not polyfloral diets (t = 4.22; df = 3; p= 0.0344). At
the end of the experiments on day 84, the expression of Spz was significantly different
(
F = 8.61;
df = 2, 3; p= 0.0501) between treatments. The time effect was not significant
(
F = 2.66; df = 4, 12; p= 0.0849
) nor were the time-by-treatment interactions (F = 1.85; df = 8,
12;
p= 0.1633
). Adult honeybees in control groups fed with pollen patties and sugar syrup
and in monofloral (anise hyssop) groups showed significantly upregulated gene expression
compared to their counterparts fed with polyfloral diets (t = 4.00; df = 3; p= 0.0281), and
(t = 2.97; df = 3; p= 0.0510), respectively, at the end of the 84-day experimental period.
Insects 2025, 16, x FOR PEER REVIEW 9 of 21
expression of the IMD gene than their counterparts fed with monofloral (anise hyssop)
diets (t = 19.33; df = 3; p = 0.0003) and polyfloral diets (t = 11.07; df = 3; p = 0.0016). Also,
adult bees fed with monofloral diets had a significantly lower expression of IMD gene
compared to their counterparts fed with polyfloral diets (t = −8.26; df = 3; p = 0.0037). Dif-
ferential gene expression levels of adult bees were significantly different between the con-
trol groups and both monofloral and polyfloral groups on day 42 (F = 88.57; df = 2, 3; p =
0.0021) and day 63 (F = 51.90; df = 2, 3; p = 0.0047). The paerns of the IMD gene profile for
da ys 42 a nd 63 w ere simil ar to t hat of day 21. At the en d of the 84 -day e xpe rimen tal pe rio d,
gene expression levels of IMD gene varied significantly between treatments (F = 65.98; df
= 2, 3; p = 0.0033) as in previous sampling dates. The time effect was not significant (F =
1.96; df = 4,12; p = 0.1649) but the time-by treatment interactions were significant (F = 3.10;
df = 8, 12; p = 0.0382). Adult bees fed the control diets displayed a significantly upregulated
expression of IMD gene compared to those fed with monofloral diets (t = 11.40; df = 3; p =
0.0014) and those fed with polyfloral diets (t = 6.93; df = 3; p = 0.0062). Adult bees fed with
monofloral diets had a significantly lower expression of IMD gene compared to their
counterparts fed with polyfloral diets (t = −4.47; df = 3; p = −0.0209).
Figure 4. Paerns of the gene expression profile of immune deficiency in adult bees fed with control.
(pollen and sugar syrup), monofloral (anise hyssop), and polyfloral (companion crops) diets during
the 84-day experimental period. Means between lines within each sampling date are not signifi-
cantly different if followed by the same lower-case leer (p > 0.05; Tukey test).
3.2.3. Spaele Gene Expression.
The expression levels of the gene Spaele (Spz), a member of the Toll immune signal-
ing pathway against fungi and bacteria [36,42], were similar (F = 2.0; df = 2, 3; p = 0.2805)
in all treatment groups at the start of the experiments on day 0, on day 21 (F = 2.55; df = 2,
3; p = 0.2257), and on day 42 (F = 4.20; df = 2, 3; p = 0.1348) (Figure 5). However, gene
expression varied significantly between treatments (F = 10.71; df = 2, 3; p = 0.0242) on day
63. The Spaele expression levels were similar between the control and monofloral diets (t
= 0.46; df = 3; p = 0.6751), but not polyfloral diets (t = 4.22; df = 3; p = 0.0344). At the end of
the experiments on day 84, the expression of Spz was significantly different (F = 8.61; df =
2, 3; p = 0.0501) between treatments. The time effect was not significant (F = 2.66; df = 4, 12;
Days After Treatments
Day 0 Day 2 1 Day 42 Day 63 Day 8 4
Gene Expression (copies/uL)
0
50
100
150
200
250
300
350
400
Control
Monofloral
Polyfloral
a
ab
b
aaaa
cccc
bbbb
Figure 4. Patterns of the gene expression profile of immune deficiency in adult bees fed with control.
(pollen and sugar syrup), monofloral (anise hyssop), and polyfloral (companion crops) diets during
the 84-day experimental period. Means between lines within each sampling date are not significantly
different if followed by the same lower-case letter (p> 0.05; Tukey test).
Insects 2025, 16, x FOR PEER REVIEW 10 of 21
p = 0.0849) nor were the time-by-treatment interactions (F = 1.85; df = 8, 12; p = 0.1633).
Adult honeybees in control groups fed with pollen paies and sugar syrup and in mon-
ofloral (anise hyssop) groups showed significantly upregulated gene expression com-
pared to their counterparts fed with polyfloral diets (t = 4.00; df = 3; p = 0.0281), and (t =
2.97; df = 3; p = 0.0510), respectively, at the end of the 84-day experimental period.
Figure 5. Paerns of the gene expression profile of Spaele in adult bees fed with control (pollen and
sugar syrup), monofloral (anise hyssop), and polyfloral (companion crops) diets during the 84-day
experimental period. Means between lines within each sampling date are not significantly different
if followed by the same lower-case leer (p > 0.05; Tukey test).
3.2.4. Vitellogenin Gene Expression.
The expression levels of vitellogenin (Vg), a gene associated with immune function
and longevity [36], were similar (F = 0.78; df = 2, 3; p = 0.5319) in all treatment groups at
the start of the experiments on day 0 (Figure 6). However, differential gene expression
levels of adult bees were significantly different between control groups and both monoflo-
ral and polyfloral groups on day 21 (F = 14.83; df = 2, 3; p = 0.0048), day 42 (F = 14.83; df =
2, 3; p = 0.0048), and day 63 (F = 8.71; df = 2, 3; p = 0.0503). Expression levels of Vg were
similar between adult bees fed with monofloral and polyfloral diets on day 21 (t = 0.76; df
= 3; p = 0.5035), day 42 (t = 0.73; df = 3; p = 0.5181), and day 63 (t = 0.56; df = 3; p = 0.6175).
At the end of the 84-day experimental period, the gene expression levels of Vg varied
significantly between treatments (F = 12.35; df = 2, 3; p = 0.0356) and followed the same
paern as on days 21, 42, and 63, as the expression levels of Vg were similar in adult bees
fed with monofloral and polyfloral diets (t = 0.90; df = 3; p = 0.4329). The time effect was
significant (F = 2.39; df = 4, 12; p = 0.0510), as were the time-by treatment interactions (F =
3.81; df = 8, 12; p = 0.0188). Adult honeybees in the control groups fed with pollen paies
and sugar syrup had significantly reduced Vg gene expression compared to their coun-
terparts fed with monofloral (anise hyssop) and polyfloral diets, and the expression levels
of the gene varied during the experimental run.
Days After Treatments
Day 0 Day 21 Day 42 Day 63 Day 84
Gene Expression (copies/uL)
25
30
35
40
45
50
Control
Monofloral
Polyfloral
a
aa
aa
a
a
aaa
aaa
bb
Figure 5. Patterns of the gene expression profile of Spaetzle in adult bees fed with control (pollen and
sugar syrup), monofloral (anise hyssop), and polyfloral (companion crops) diets during the 84-day
experimental period. Means between lines within each sampling date are not significantly different if
followed by the same lower-case letter (p> 0.05; Tukey test).
Insects 2025,16, 374 10 of 20
3.2.4. Vitellogenin Gene Expression
The expression levels of vitellogenin (Vg), a gene associated with immune function and
longevity [
36
], were similar (F = 0.78; df = 2, 3; p= 0.5319) in all treatment groups at the
start of the experiments on day 0 (Figure 6). However, differential gene expression levels of
adult bees were significantly different between control groups and both monofloral and
polyfloral groups on day 21 (F = 14.83; df = 2, 3; p= 0.0048), day 42 (F = 14.83; df = 2, 3;
p= 0.0048
), and day 63 (F = 8.71; df = 2, 3; p= 0.0503). Expression levels of Vg were similar
between adult bees fed with monofloral and polyfloral diets on day 21 (t = 0.76; df = 3;
p= 0.5035
), day 42 (t = 0.73; df = 3; p= 0.5181), and day 63 (t = 0.56; df = 3;
p= 0.6175
).
At the end of the 84-day experimental period, the gene expression levels of Vg varied
significantly between treatments (F = 12.35; df = 2, 3; p= 0.0356) and followed the same
pattern as on days 21, 42, and 63, as the expression levels of Vg were similar in adult bees
fed with monofloral and polyfloral diets (t = 0.90; df = 3; p= 0.4329). The time effect was
significant (F = 2.39; df = 4, 12; p= 0.0510), as were the time-by treatment interactions
(F = 3.81; df = 8, 12; p= 0.0188). Adult honeybees in the control groups fed with pollen
patties and sugar syrup had significantly reduced Vg gene expression compared to their
counterparts fed with monofloral (anise hyssop) and polyfloral diets, and the expression
levels of the gene varied during the experimental run.
Insects 2025, 16, x FOR PEER REVIEW 11 of 21
Figure 6. Paerns of the gene expression profile of Vitellogenin in adult bees fed with control (pollen
and sugar syrup), monofloral (anise hyssop), and polyfloral (companion crops) diets during the 84-
day experimental period. Means between lines within each sampling date are not significantly dif-
ferent if followed by the same lower-case leer (p > 0.05; Tukey test).
3.2.5. Malvolio Gene Expression.
The expression level of Malvolio (mvl), a gene involved in sucrose responsiveness
[36,43], was similar at the start of the field trials (F = 2.99; df = 2, 3; p = 0.1933) (Figure 7).
Gene expression of mvl did not differ significantly between treatments on day 21 (F = 2.41;
df = 2, 3; p = 0.2378), day 42 (F = 1.22; df = 2, 3; p = 0.4096), and day 63 (F = 0.87; df = 2, 3; p
= 0.5022). However, the expression levels of mvl were significantly different between the
treatments at the end of the experimental run on day 84 (F = 3.77; df = 2, 3; p = 0.0503). The
time effect was not significant (F = 0.92; df = 4, 12; p = 0.4851), nor were the time-by-treat-
ment interactions (F = 1.07; df = 8, 12; p = 0.4439). Adult honeybees fed with polyfloral diets
showed upregulated mvl expression compared to their counterparts in the control groups
fed with pollen paies and sugar syrup (t = 4.05; df = 3; p = 0.0482). Adult bees fed with
monofloral (anise hyssop) diets had a similar expression of the mvl gene to that of the
control groups and polyfloral groups. (t = −4.0; df = 3; p = 0.4647).
Days After Treatments
Day 0 Day 21 Day 42 Day 63 Day 84
Gene Expression (copies/uL)
400
600
800
1000
1200
1400
1600
Monofloral
Polyfloral
Control
a
aa
a
a
a
b
ab
b
a
bb
b
b
Figure 6. Patterns of the gene expression profile of Vitellogenin in adult bees fed with control (pollen
and sugar syrup), monofloral (anise hyssop), and polyfloral (companion crops) diets during the
84-day experimental period. Means between lines within each sampling date are not significantly
different if followed by the same lower-case letter (p> 0.05; Tukey test).
3.2.5. Malvolio Gene Expression
The expression level of Malvolio (mvl), a gene involved in sucrose
responsiveness [36,43],
was similar at the start of the field trials (F = 2.99; df = 2, 3; p= 0.1933) (Figure 7). Gene
expression of mvl did not differ significantly between treatments on day 21 (
F = 2.41;
df = 2,
3; p= 0.2378), day 42 (F = 1.22; df = 2, 3; p= 0.4096), and day 63 (F = 0.87; df = 2, 3; p= 0.5022).
However, the expression levels of mvl were significantly different between the treatments
at the end of the experimental run on day 84 (F = 3.77; df = 2, 3; p= 0.0503). The time
Insects 2025,16, 374 11 of 20
effect was not significant (F = 0.92; df = 4, 12; p= 0.4851), nor were the time-by-treatment
interactions (F = 1.07; df = 8, 12; p= 0.4439). Adult honeybees fed with polyfloral diets
showed upregulated mvl expression compared to their counterparts in the control groups
fed with pollen patties and sugar syrup (t = 4.05; df = 3; p= 0.0482). Adult bees fed with
monofloral (anise hyssop) diets had a similar expression of the mvl gene to that of the
control groups and polyfloral groups. (t = −4.0; df = 3; p= 0.4647).
Insects 2025, 16, x FOR PEER REVIEW 12 of 21
Figure 7. Paerns of the gene expression profile of Malvolio in adult bees fed with control (pollen
and sugar syrup), monofloral (anise hyssop), and polyfloral (companion crops) diets during the 84-
day experimental period. Means between lines within each sampling date are not significantly dif-
ferent if followed by the same lower-case leer (p > 0.05; Tukey test).
3.2.6. Maltase Gene Expression.
The expression levels of the gene Maltase, a gene associated with energy metabolism
[36], were similar (F = 2.73; df = 2, 3; p = 0.2110) in all treatment groups at the start of the
experiments on day 0 (Figure 8). However, the differential gene expression levels of Malt-
ase in adult bees were significantly different between the control, monofloral, and polyflo-
ral groups on day 21 (F = 15.38; df = 2, 3; p = 0.0265), on day 42 (F = 14.74; df = 2, 3; p =
0.0281), and on day 63 (F = 9.44; df = 2, 3; p = 0.0501). Expression levels of Maltase were
significantly lower in adult bees fed with pollen and sugar syrup than those of their coun-
terparts fed with monofloral diets on day 21 (t = −5.00; df = 3; p = 0.0154), on day 42 (t =
−3.88; df = 3; p = 0.0303). and day 63 (t = −3.70; df = 3; p = 0.0343). Adult bees in control
groups had a similar expression of Maltase gene as their counterparts in polyfloral groups
on day 21 (t = −2.34; df = 3; p = 0.1015), day 42 (t = −1.97; df = 3; p = 0.1436) and on day 63 (t
= −2.07; df = 3; p = 0.1303). Similarly, the expression of Maltase did not differ between adult
bees fed with monofloral and polyfloral diets on day 21 (t = 2.67; df = 3; p = 0.0759), on day
42 (t = 1.91; df = 3; p = 0.1522), and on day 63 (t = 1.62; df = 3; p = 0.2016). At the end of the
84-day experimental period, expression levels of Maltase varied significantly between
treatments (F = 12.31; df = 2, 3; p = 0.0358). The time effect was significant (F = 84.35; df =
4,12; p = 0.0001) as were the time-by treatment interactions (F = 4.36; df = 8, 12; p = 0.0113).
Adult honeybees in the control groups fed with pollen paies and sugar syrup had sig-
nificantly lower expression levels of Maltase compared to their counterparts fed with mon-
ofloral (t = −4.74; df = 3; p = 0.0178) and polyfloral diets (t = −365; df = 3; p = 0.0355) diets.
There were no significant differences (t = 1.09; df = 3; p = 0.3566) in gene expression levels
between adult bees fed with monofloral and polyfloral diets. Overall, expression levels of
Maltase varied over time during the 84-days of the field trials.
Days After Treatment
Day 0 Day 21 Day 42 Day 63 Day 84
Gene Expression (Copies/uL)
2
3
4
5
Polyfloral
Monofloral
Control
a
a
a
a
aaa
ab
aaa
b
a
aa
Figure 7. Patterns of the gene expression profile of Malvolio in adult bees fed with control (pollen and
sugar syrup), monofloral (anise hyssop), and polyfloral (companion crops) diets during the 84-day
experimental period. Means between lines within each sampling date are not significantly different if
followed by the same lower-case letter (p> 0.05; Tukey test).
3.2.6. Maltase Gene Expression
The expression levels of the gene Maltase, a gene associated with energy metabolism [
36
],
were similar (F = 2.73; df = 2, 3; p= 0.2110) in all treatment groups at the start of the
experiments on day 0 (Figure 8). However, the differential gene expression levels of Maltase
in adult bees were significantly different between the control, monofloral, and polyfloral
groups on day 21 (F = 15.38; df = 2, 3; p= 0.0265), on day 42 (F = 14.74; df = 2, 3; p= 0.0281),
and on day 63 (F = 9.44; df = 2, 3; p= 0.0501). Expression levels of Maltase were significantly
lower in adult bees fed with pollen and sugar syrup than those of their counterparts fed
with monofloral diets on day 21 (t =
−
5.00; df = 3; p= 0.0154), on day 42 (t =
−
3.88;
df = 3;
p= 0.0303). and day 63 (t =
−
3.70; df = 3; p= 0.0343). Adult bees in control groups had
a similar expression of Maltase gene as their counterparts in polyfloral groups on day 21
(
t = −2.34;
df = 3; p= 0.1015), day 42 (t =
−
1.97; df = 3; p= 0.1436) and on day 63 (
t = −2.07;
df = 3; p= 0.1303). Similarly, the expression of Maltase did not differ between adult bees
fed with monofloral and polyfloral diets on day 21 (t = 2.67; df = 3; p= 0.0759), on day
42
(t = 1.91;
df = 3; p= 0.1522), and on day 63 (t = 1.62; df = 3; p= 0.2016). At the end of
the 84-day experimental period, expression levels of Maltase varied significantly between
treatments (F = 12.31; df = 2, 3; p= 0.0358). The time effect was significant (
F = 84.35;
df = 4,12; p= 0.0001) as were the time-by treatment interactions (F = 4.36; df = 8, 12;
p= 0.0113
). Adult honeybees in the control groups fed with pollen patties and sugar syrup
had significantly lower expression levels of Maltase compared to their counterparts fed with
monofloral (t =
−
4.74; df = 3; p= 0.0178) and polyfloral diets (t =
−
365; df = 3;
p= 0.0355
)
diets. There were no significant differences (t = 1.09; df = 3; p= 0.3566) in gene expression
Insects 2025,16, 374 12 of 20
levels between adult bees fed with monofloral and polyfloral diets. Overall, expression
levels of Maltase varied over time during the 84-days of the field trials.
Insects 2025, 16, x FOR PEER REVIEW 13 of 21
Figure 8. Paerns of the gene expression profile of Maltase in adult bees fed with control (pollen and
sugar syrup), monofloral (anise hyssop), and polyfloral (companion crops) diets during the 84-day
experimental period. Means between lines within each sampling date are not significantly different
if followed by the same lower-case leer (p > 0.05; Tukey test).
3.2.7. Single-Minded Homolog 2 Gene
The expression levels of the gene single-minded Homolog 2 (Smh2), a gene involved in
locomotory behavior [36] were similar (F = 0.23; df = 2, 3; p = 0.8091) in all treatment groups
at the start of the experiments on day 0 (Figure 9). However, the gene expression levels of
adult bees were significantly different between control groups and both monofloral and
polyfloral groups on day 21 (F = 41.25; df = 2, 3; p = 0.0066) and on day 42 (F = 55.85; df =
2, 3; p = 0.0001). There were no significant differences between monofloral and polyfloral
diets on day 21 (t = −0.07; df = 3; p = 0.9467) and day 42 (t = 0.82; df = 3; p = 0.4722). On day
63 of the field trials, gene expression levels of single-minded were significantly different (F
= 41.25; df = 2, 3; p = 0.0066) between the control diets and monofloral diets (t = −2.37; df =
3; p = 0.0507), but they were similar to that of polyfloral diets (t = 1.39; df = 3; p = 0.2601).
At the end of the 84-day experimental period, expression levels of single-minded varied
significantly between treatments (F = 4.76; df = 2, 3; p = 0.0300). The time effect was signif-
icant (F = 57.40; df = 4, 12; p = 0.0001), as were the time-by treatment interactions (F = 3.74;
df = 8, 12; p = 0.0200). Adult honeybees in the control groups fed with pollen paies and
sugar syrup had similar gene expression levels compared to their counterparts fed with
polyfloral diets (t = 1.21; df = 3; p = 0.3142). Similarly, there were no differences (t = −1.29;
df = 3; p = 0.2890) in gene expression between adult bees fed with monofloral and polyflo-
ral diets. Overall, the differential gene expression of Sim2 varied over time during the 84-
day experimental period.
Days After Treatments
Day 0 Day 21 Day 42 Day 63 Day 84
Gene Expression (copies/uL)
0
5
10
15
20
25
30
35
Monofloral
Polyfloral
Control
a
a
a
a
a
aa
ab
ab
ab
b
b
b
b
b
Figure 8. Patterns of the gene expression profile of Maltase in adult bees fed with control (pollen and
sugar syrup), monofloral (anise hyssop), and polyfloral (companion crops) diets during the 84-day
experimental period. Means between lines within each sampling date are not significantly different if
followed by the same lower-case letter (p> 0.05; Tukey test).
3.2.7. Single-Minded Homolog 2 Gene
The expression levels of the gene single-minded Homolog 2 (Smh2), a gene involved in
locomotory behavior [
36
] were similar (F = 0.23; df = 2, 3; p= 0.8091) in all treatment groups
at the start of the experiments on day 0 (Figure 9). However, the gene expression levels of
adult bees were significantly different between control groups and both monofloral and
polyfloral groups on day 21 (F = 41.25; df = 2, 3; p= 0.0066) and on day 42 (F = 55.85;
df = 2, 3;
p= 0.0001). There were no significant differences between monofloral and polyfloral diets
on day 21 (t =
−
0.07; df = 3; p= 0.9467) and day 42 (t = 0.82; df = 3; p= 0.4722). On day 63 of
the field trials, gene expression levels of single-minded were significantly different (
F = 41.25;
df = 2, 3; p= 0.0066) between the control diets and monofloral diets (t =
−
2.37;
df = 3;
p= 0.0507), but they were similar to that of polyfloral diets (t = 1.39; df = 3;
p= 0.2601
).
At the end of the 84-day experimental period, expression levels of single-minded varied
significantly between treatments (F = 4.76; df = 2, 3; p= 0.0300). The time effect was
significant (F = 57.40; df = 4, 12; p= 0.0001), as were the time-by treatment interactions
(
F = 3.74;
df = 8, 12; p= 0.0200). Adult honeybees in the control groups fed with pollen
patties and sugar syrup had similar gene expression levels compared to their counterparts
fed with polyfloral diets (t = 1.21; df = 3; p= 0.3142). Similarly, there were no differences
(
t = −1.29;
df = 3; p= 0.2890) in gene expression between adult bees fed with monofloral
and polyfloral diets. Overall, the differential gene expression of Sim2 varied over time
during the 84-day experimental period.
Insects 2025,16, 374 13 of 20
Insects 2025, 16, x FOR PEER REVIEW 14 of 21
Figure 9. Paerns of the gene expression profile of Single-minded homolog 2 in adult bees fed with
control (pollen and sugar syrup), monofloral (anise hyssop), and polyfloral (companion crops) diets
during the 84-day experimental period. Means between lines within each sampling date are not
significantly different if followed by the same lower-case leer (p > 0.05; Tukey test).
4. Discussion
Our studies included immune function genes (immune deficiency, Cactus, and
Spaele), genes involved in nutrition, cellular defense, and longevity (Vitellogenin and Mal-
volio), a gene involved in energy metabolism (Maltase), and a gene associated with loco-
motory behavior (Single-minded homolog 2). We found that the dietary treatments did not
significantly affect the strength of the colonies in terms of mite infestations, but the mon-
ofloral diet group had larger colonies by the end of the experiment. Additionally, there
were differences in gene expression across diets, and particularly between the monofloral
and control diet. Overall, the monofloral treatment appeared to be the best diet in com-
parison to polyfloral and pollen and sugar paies, probably due to specific nutritional
effects of anise hyssop (a companion crop).
4.1. Cactus Gene Expression
Adult bees fed pollen paies and sugar syrup showed upregulated Cactus gene ex-
pression during the 84-day experimental period while their counterparts fed with mon-
ofloral and polyfloral diets showed downregulated gene expression. The Cactus gene is
involved in the Toll pathways, providing inflammatory responses that include the recog-
nition of pathogens and the expression of antimicrobial peptides (AMPs) [42–44]. Yang
and Cox-Foster [4] reported that infestations of Varroa mite contributed to stress and
weakened honeybee colonies by directly affecting some genes encoding antimicrobial and
immune responses. Our data indicated that Varroa mite infestation levels of adult bees
were higher in the control groups (0.94 ± 0.61%) but did not differ significantly from those
in the monofloral (0.41 ± 0.19%) and polyfloral groups (0.20 ± 0.12%). The mite load in our
study was significantly lower than the recommended threshold (3–6 mites per 100 bees)
[45] for chemical treatments. That the expression of the Cactus gene did not display the
same trends as the Varroa mite load might suggest an absence of a strong association
Days After Treatments
Day 0 Day 21 Day 42 Day 63 Day 84
Gene Expression (copies/uL)
14
16
18
20
22
24
26
28
30
32
34
Control
Polyfloral
Monofloral
a
a
aaa
a
bb
b
b
b
b
ab
ab
a
Figure 9. Patterns of the gene expression profile of Single-minded homolog 2 in adult bees fed with
control (pollen and sugar syrup), monofloral (anise hyssop), and polyfloral (companion crops) diets
during the 84-day experimental period. Means between lines within each sampling date are not
significantly different if followed by the same lower-case letter (p> 0.05; Tukey test).
4. Discussion
Our studies included immune function genes (immune deficiency, Cactus, and Spaet-
zle), genes involved in nutrition, cellular defense, and longevity (Vitellogenin and Malvolio),
a gene involved in energy metabolism (Maltase), and a gene associated with locomotory
behavior (Single-minded homolog 2). We found that the dietary treatments did not signifi-
cantly affect the strength of the colonies in terms of mite infestations, but the monofloral
diet group had larger colonies by the end of the experiment. Additionally, there were
differences in gene expression across diets, and particularly between the monofloral and
control diet. Overall, the monofloral treatment appeared to be the best diet in comparison
to polyfloral and pollen and sugar patties, probably due to specific nutritional effects of
anise hyssop (a companion crop).
4.1. Cactus Gene Expression
Adult bees fed pollen patties and sugar syrup showed upregulated Cactus gene expres-
sion during the 84-day experimental period while their counterparts fed with monofloral
and polyfloral diets showed downregulated gene expression. The Cactus gene is involved
in the Toll pathways, providing inflammatory responses that include the recognition of
pathogens and the expression of antimicrobial peptides (AMPs) [
42
–
44
]. Yang and Cox-
Foster [
4
] reported that infestations of Varroa mite contributed to stress and weakened
honeybee colonies by directly affecting some genes encoding antimicrobial and immune
responses. Our data indicated that Varroa mite infestation levels of adult bees were higher
in the control groups (0.94
±
0.61%) but did not differ significantly from those in the
monofloral (0.41
±
0.19%) and polyfloral groups (0.20
±
0.12%). The mite load in our study
was significantly lower than the recommended threshold (3–6 mites per 100 bees) [
45
] for
chemical treatments. That the expression of the Cactus gene did not display the same trends
as the Varroa mite load might suggest an absence of a strong association between mite load
and the expression of the Cactus gene. The expression of Cactus was similar between the
Insects 2025,16, 374 14 of 20
control, monofloral, and polyfloral diets up to 21 days after the experiments were initiated.
Overall, the upregulation of Cactus in adult bees in the control groups from day 42 to the
end of the experiments without any significant infestation levels of Varroa mites suggested
that other factors may trigger the upregulation of the Cactus gene [
46
]. Adult bees in flight
cages fed monofloral and polyfloral diets generated similar patterns of expression of this
humoral defense gene during our 84-day experimental period. Whether the slightly higher
Varroa mite load in control groups adversely affected the expression of Cactus gene is yet
to be fully investigated. In a study by Barroso-Arévalo et al. [
15
] to assess whether the
expression levels of four immune system genes (dorsal, defensin, domeless, and relish)
could serve as biomarkers of colony mortality (0.3 to 28.85% mite infestation rates), they
noted a decreased expression of dorsal and defensin (Toll pathway) associated with high
deformed wing virus and Varroa mite loads. Marušˇcáková et al. [
47
] reported that when
bees were infested with Escherichia coli, the up-regulation of Cactus led to the termination of
AMP’s production. Hence, Cactus is a negative regulator of the Toll pathways.
4.2. Immune Deficiency
Expression levels of immune deficiency (IMD) gene in adult bees fed with pollen
patties and sugar syrup in flight cage treatments were significantly upregulated throughout
the 84-day experimental period compared with their counterparts fed with monofloral
diets and polyfloral diets. The IMD signaling pathway controls antibacterial defense [
48
,
49
].
Tanji and Ip [
50
] and Hoffmann [
51
] indicated that insect antibacterial immunity relies
heavily on the Toll and IMD pathways of the innate immune systems. Our data indicated
that relatively low mite loads of 0.94
±
61%, 0.20
±
0.12%, and 0.41
±
0.19% in the control,
polyfloral, and monofloral groups, respectively, did not significantly change the expression
of IMD genes within each diet from the beginning to the end of the experimental run.
Although the control groups have the highest infestations of Varroa mite [(0.94
±
0.61%)
mite per 100 bees)], the rates were not within the recommended threshold (3–6 mites
per 100 bees) [
45
] for chemical treatments. As in the Toll pathway, other factors (fungi,
protozoan diseases, beekeeping practices, etc.) may trigger the upregulation of IMD gene
expression. Our data also indicated that expression levels of IMD genes in adult bees fed
with monofloral diets were significantly lower during the 2.7-month experimental run. In a
study by Barosso-Arevalo et al. [
15
] on immune system genes in honeybees, he indicated
that the expression of relish (IMD pathway) was significantly higher in honeybee colonies
with high deformed wing virus and Varroa mite loads than those with low loads. Yang and
Cox-Foster [
4
] reported that parasitic mite infestation together with deformed wing virus
downregulated the expression of genes encoding antimicrobial peptides. However, the
levels of Varroa mite infestations in our study might not have provided detectable effects
of the expression profile of genes in the IMD pathway.
4.3. Spaetzle
We found that the expression of the Spaetzle gene in adult bees in controls, monofloral,
and polyfloral groups was similar except on day 63 and on day 84. On these two sampling
dates, adult bees fed with polyfloral diets significantly downregulated the expression of the
Spaetzle gene. Varroa mite infestation levels of adult bees were higher in the control groups
(0.94
±
0.61%) but did not differ significantly from the monofloral groups (0.41
±
0.19%)
and polyfloral groups (0.20
±
0.12%). In addition, this level of mite infestations was lower
than the recommended threshold (3–6 mites per 100 bees) [
45
] for chemical treatments.
Alaux et al. [
7
] reported that Spaetzle transcripts were increased in honeybees fed polyfloral
pollen diets but decreased with the presence of Varroa mite. In our studies, Varroa mite load
was similar between the three types of diets, suggesting that the expression of this gene
Insects 2025,16, 374 15 of 20
was reduced, probably due to other pathways or factors (biological or environmental stres-
sors). In our studies, Spaetzle expression levels in adult bees in the control diets increased
significantly 21 days post-treatment, unlike in other diets, and the levels remained similar
to that of monofloral diets up to the end of the 84-day experimental period. The Spaetzle
gene is a member of the Toll immune signaling pathway against fungi and bacteria [
43
] that
results in the production of antimicrobial peptides [
52
]. Overall, the expression profiles of
the immune function genes (Cactus, immune deficiency, and Spaetzle) were affected by the
dietary treatments (pollen and sugar patties, monofloral, and polyfloral), and adult bees
fed with control diets upregulated these genes compared to their counterparts fed with
monofloral and polyfloral diets.
4.4. Vitellogenin
Adult bees fed with monofloral and polyfloral diets displayed significantly higher ex-
pression of the Vitellogenin (Vg) gene compared with their counterparts in control groups fed
with pollen patties and sugar syrup during the 2.7-month experimental period. Our results
indicated that pollen and nectar from monofloral diets and polyfloral diets upregulated
(2.7-fold) the expression of Vg. Similarly, Alaux et al. [
7
] and Chaimanee et al. [
53
] reported
that expression of Vg was increased in adult bees fed with polyfloral diets. Vitellogenin is a
major reproductive protein in insects and a proposed endocrine factor in honeybees; it has
antioxidant functions that protect bees from oxidative stress and enhance longevity [
54
,
55
]
affect foraging behavior, division of labor [
30
], and immunity [
42
]. Our data on colony
development indicated that a significantly higher population of bees remained (longer
life span) in the monofloral diet (2.92
±
0.0.82) groups compared to their counterparts in
the control diet fed with pollen patties and sugar syrup (0.72
±
0.67) (Table 2). Reduced
expression of Vg in control bees may be due to immune senescence (deterioration) in
foragers due to a behavioral stimulus and/or the absence of adequate nutrients [56,57].
4.5. Malvolio
The expression of the Malvolio (Mvl) gene was similar between adult bees fed with
control (pollen and sugar syrup), monofloral, and polyfloral diets up to day 63 post-
treatment. Our data indicated that feeding adult bees with the three types of pollen and
nectar did not affect Mvl expression 63 days after the experiments were initiated. However,
adult bees fed polyforal diets displayed higher expression of Mvl compared to the control
diets at the end of the 2.7-month experiment. The Malvolio gene is associated with sucrose
responsiveness [
58
] and the division of labor in honeybees. Ben-Shahr et al. [
59
] and Zayed
and Robinson [
60
] reported that foraging workers that are responsive to sucrose have
higher levels of Mvl in the brain than nurse bees, and that upregulation of Mvl contributed
to the transition from nursing to foraging behavior. In our study, before day 63, the Mvl
expression profile was similar in adult bees fed with all three diets but peaked on day 84 in
adult bees fed with polyfloral diets. This may suggest that by the end of the experiment
those bees fed with polyfloral diets performed significantly more outdoor feeding and
foraging activities compared to their counterparts in the control groups but not compared to
bees in the monofloral group. Because our data indicated low levels of food reserves (honey)
in adult bees fed with polyfloral diets, the upregulation of Malvolio may suggest that the
bees were increasing foraging in response to low food reserves (0.01
±
0.00) (Table 2). Our
data may follow findings on the gene expression profile of Seeley [
61
] and Page et al. [
62
]
that indicated that expression was low when bees were focused on in-hive tasks (at the start
of the experiments), but expression peaked when these bees performed outdoor activities
(foraging) [63] and then decreased again when outdoor activities reduced.
Insects 2025,16, 374 16 of 20
4.6. Maltase
Adult bees fed with monofloral diets significantly upregulated the expression of
Maltase compared to their counterparts fed with pollen patties and sugar syrup 21 days
after the experiments were initiated. The upregulation of Maltase in these bees continued
to the end of the 84-day experimental period. However, the differential gene expression
of Maltase in adult bees fed with monofloral diets was similar to that of their counterparts
fed with polyfloral diets throughout the experimental run. Furthermore, adult bees in
control groups displayed similar Maltase expressions with their counterparts in polyfloral
groups except at the end of the experiments on day 84, and, thus, differences were primarily
between the control and monofloral diet groups. Our data indicated that adult bees fed
with monofloral diets have relatively higher foraging behavior and may collect more pollen
and nectar/honey for their colonies than their counterparts fed with pollen patties and
sugar syrup. Our personal observations revealed that although control diets received the
highest number of visits (95 per 15 min, compared to 8 visits for the same amount of time
on monofloral diets), the average visit duration was longer (about 6 s) and the reward
(pollen/nectar) was larger (pollen basket) than that of the other two diets. We also found
that the pollen was used immediately for consumption and not for storage. Maltase is
involved in energy metabolism as it converts maltose into glucose [
64
]. Glucose is the main
energetic substrate for bee tissues, and it is stored in a form of glycogen that is used when
energy demands are high [65–67].
4.7. Single-Minded Homologue 2
Gene expression levels of Single-minded homolog 2 (Sim 2) were significantly higher
21-day post treatments in adult bees fed with control diets (pollen and sugar syrup) com-
pared to their counterparts fed with monofloral (anise hyssop) diets. Adult bees fed with
monofloral or polyfloral diets displayed similar levels of gene expression during the 84-day
experimental period. The Single-minded homolog 2 (Sim 2) gene encodes a transcription
factor that is a master regulator of neurogenesis and plays a critical role in the development
of the neurons, glia, and other nonneuronal cells that lie along the midline of the embryonic
CNS [
68
,
69
]. The gene is also involved in locomotory behavior [
70
] which supports our
personal observation that adult bees housed in flight cages fed with control diets were
hyperactive [as a result, the mortality was much higher (population declined by 86.2%),
Table 2)]. However, Pielage et al. [
70
] indicated that Sim 2 was important not only to
control locomotion, but also to correctly specify the formation of the central complex and
to correctly pattern the genital discs, as well as the anal pad anlage.
5. Conclusions
The availability of food (nutrients) is an important factor for the growth and survival
of an organism; thus, malnutrition can impair immune function and consequently affect
animal health, resistance to diseases, and survival [
7
]. In the case of honeybees, pollen
and nectar are essential dietary sources and are vital for individual honeybee health and
colony growth and development [
7
,
20
]. We found that the companion crops (monofloral
and polyfloral) provided higher nutritional benefits to enhance honeybee health than the
pollen patties and sugar syrup currently used by beekeepers. Furthermore, it has been
reported that while single-species pollen has specific benefits, bees require pollen from
diverse sources to maintain a healthy physiology and colony [
25
]. Our data on nuclear
colonies indicated that a single-species diet (such as anise hyssop) is nutritionally adequate,
as it is even much better than or comparable to polyfloral diets. The expression profiles
of the selected genes in this study were significantly affected by diets (pollen and sugar
patties, monofloral, and polyfloral) at the end of the experimental period. The potential
Insects 2025,16, 374 17 of 20
relationships between diet and gene expression varied among these genes and was the
highest with Vitellogenin and may translate into the behavior of adult bees. Based on the
gene expression profile, the monofloral (anise hyssop) diets provided quality nutrients
better than or comparable to those of polyfloral diets that were reported to be preferable to
pollinators [
7
]. Additionally, we found that the companion crops provided higher nutri-
tional benefits to enhance honeybee health than the pollen patties and sugar syrup currently
used by beekeepers. It is critically important to provide honeybees with sustainable and
nutritious food sources (plants, such as hyssop) throughout the season to enhance honeybee
health. These companion crops could be integrated into the fabric of any apiary, as not only
will they provide additional food sources (pollen and nectar) during the honey flow, but
they also serve as good quality food resources during the off season (the absence of honey
flow). These perennial crops (companion plants) could cover about 10% of the apiary and
be planted as perimeter cropping, intercropping, mosaic cropping, and/or random packets,
as well as part of the landscape of the apiary. Anise hyssop (perennial plant) could be one
of the best choices as it provides nutritional supplements to enhance honeybee health.
Author Contributions: Conceptualization, L.H.B.K., W.A.D., R.M., I.E. and M.P.; methodology, L.H.B.K.,
W.A.D., R.M. and M.P.; validation, I.E. and Y.Z.; formal analysis, I.E., R.M., M.P., Y.Z. and L.H.B.K.;
investigation, W.A.D. and L.H.B.K.; resources, L.H.B.K.; data curation, I.E. and Y.Z.; writing—original
draft preparation, L.H.B.K., W.A.D., R.M. and M.P.; writing—review and editing, R.M., M.P., L.H.B.K.,
W.A.D., I.E. and Y.Z.; supervision, L.H.B.K., R.M., I.E. and M.P.; project administration, L.H.B.K.,
R.M. and M.P.; funding acquisition, L.H.B.K. All authors contributed to the manuscript’s discussion,
reading, and approval. All authors have read and agreed to the published version of
the manuscript.
Funding: This study was supported by USDA-ARS-CMAVE FAIN number 58-6036-2-010 and USDA-
APHIS FAIN number AP23PPQSQT00C014.
Data Availability Statement: The original contributions presented in this study are included in the
article. Further inquiries can be directed to the corresponding author.
Acknowledgments: We are grateful to Marta Wayne, Blair Siegfried, James Nation, Janice Peters,
Jesusa Legaspi, Alexander Gaffke, Neil Miller, and Alejandro Bolques for providing scientific input,
technical support, and useful discussions and reviews of the manuscript.
Conflicts of Interest: The authors declare no conflicts of interest.
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