Journal of Apicultural Research

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Histomorphological description of the

reproductive system in mated honey bee queens

Ivanna V. Kozii, Sarah C. Wood, Roman V. Koziy & Elemir Simko

To cite this article: Ivanna V. Kozii, Sarah C. Wood, Roman V. Koziy & Elemir Simko (2022)
Histomorphological description of the reproductive system in mated honey bee queens,
Journal of Apicultural Research, 61:1, 114-126, DOI: 10.1080/00218839.2021.1900636

To link to this article: https://doi.org/10.1080/00218839.2021.1900636

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Journal of Apicultural Research, 2022
Vol. 61, No. 1, 114–126, https://doi.org/10.1080/00218839.2021.1900636

ORIGINAL RESEARCH ARTICLE

Histomorphological description of the reproductive system in mated honey
bee queens
Ivanna V. Kozii
, Sarah C. Wood , Roman V. Koziy and Elemir Simko

Department of Veterinary Pathology, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, Canada

(Received 6 December 2018; accepted 28 August 2020)

The accelerating decline of pollinator populations has become increasingly concerning in the last two decades. Honey
bees are economically important pollinators and a model species to evaluate pollinator health. The decline in queen
quality is one of the frequently reported causes associated with honey bee losses worldwide. Histopathology is an
essential tool used for diagnostics and research in mammalian species and may provide insight into the histomorphologi-
cal basis of a decline in queen quality. Thus, the purpose of this study was to summarize the previously described nor-
mal morphology of the entire reproductive tract of mated honey bee queens and to illustrate it by high quality
photomicrographs. Accordingly, reproductive tracts of one-year old mated honey bee queens were processed for hist-
ology, serially sectioned, stained with hematoxylin and eosin, and used for the capture of high quality histological photo-
micrographs of the entire reproductive tract. This study illustrates the microscopic morphology of the queen
reproductive tract which may facilitate further investigations of the cause and pathogenesis of the recent decline in
reproductive fitness of honey bees using histopathology.

Keywords: honey bee queen; Apis mellifera; histology; reproductive tract

Introduction or evaluation of host response to noxious stimuli. It is

Histology is an essential tool that facilitates accurate used extensively for diagnostics, research, and regulatory
evaluation of the microscopic tissue structures and allows purposes (risk and safety assessment) (Bernet et al.,
for a basic assessment of organ function. In vertebrate 1999; Brown et al., 2016). However, histopathology has
species, the study of microscopic morphologic alterations not been developed and utilized to the same extent in
(histopathology) is the basis for the diagnosis of disease insects as it has been in vertebrate species.

Corresponding author. Email: ivanna.kozii@usask.ca; elemir.simko@usask.ca
IK and ES designed experiment; IK, SW, RK, and ES participated in the design and interpretation of the data; IK performed
experiments and analysis; IK and ES wrote the manuscript. All authors participated in the revision of the manuscript, read and
approved the final manuscript.

ß 2021 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group
This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License (http://creativecommons.org/
licenses/by-nc-nd/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited, and is not
altered, transformed, or built upon in any way.
 Histomorphological description of the reproductive system
Despite the growing public awareness of pollinators’
importance, the beginning of the 21st century has been
marked by compelling evidence of the decline in pollina-
tors and dependent plants (Burkle et al., 2013; Morse &
Calderone, 2000; Potts et al., 2010; Vanbergen &
Initiative, 2013). Honey bees are the most researched
pollinators; the decline in honey bee populations and
deterioration of their health has been linked to a num-
ber of causes, including pesticide exposure (S
anchez-
Bayo et al., 2016; Wood et al., 2018), climate (Beyer
et al., 2018), infectious and parasitic diseases (Grozinger
& Flenniken, 2019; Higes et al., 2008; Mart
ın-Hern
andez
et al., 2011), agricultural intensification (Ricketts et al.,
2008; Winfree et al., 2009), and loss of genetic diversity
(Forfert et al., 2017). Rigorous field and laboratory pro-
tocols have been established to study declines in honey
bees. However, the results are often controversial and
inconsistent between field and laboratory-based studies,
leaving room for improvement of standard honey bee
research techniques.
Queen failure and poor queen quality are consist-
ently among the top three reported causes of honey
bee colony losses (Amiri et al., 2017). The decreased
quality of queens has been associated with the use of
pesticides (Haarmann et al., 2002; Wu-Smart & Spivak,
2016), nosemosis (Alaux et al., 2011; Simeunovic et al.,
2014; Traver & Fell, 2012), varroosis, and viral infec-
tions (Gauthier et al., 2011); however, the cause and
pathogenesis remain unclear. Histopathological evalu-
ation of the honey bee reproductive tract in addition to
the current standard methods used for assessment of
reproductive fitness (e.g. queen morphometric meas-
ures, ovariole count, sperm evaluation, brood pattern)
may enhance our understanding of disease development,
treatment, and prevention.
Previously, normal macroscopic and microscopic
anatomy of the entire reproductive tract of mated
honey bee queens was described and illustrated by dia-
grams (Snodgrass, 1985), and certain segments of the
reproductive tract were well-characterized by electron
microscopy (Bili
nski & Jaglarz, 1999; Cruz-Landim &
Patr
ıcio, 2010; Gutzeit et al., 1993; Patr
ıcio & Cruz-
Landim, 2002, 2008) or light microscopy (Camargo &
Mello, 1970; Gutzeit et al., 1993; Stell, 2012). However,
neither of these individual studies encompasses the
entire reproductive tract in sufficient detail to provide a
comprehensive reference for histopathology. Therefore,
to facilitate the future histopathological evaluation of
abnormalities in the honey bee reproductive tract, a
comprehensive study with high quality photomicro-
graphs illustrating the normal reproductive tract of
mated honey bee queens is needed.
The purpose of this study was to i) perform serial
histological sections of the abdomen of mated honey
bee queens, ii) illustrate the entire reproductive tract of
a mated honey bee queen with high quality photomicro-
graphs, and iii) using these photomicrographs as a
115
reference, summarize previously published descriptions
of normal queen morphology.
Materials and methods
Honey bee queen origin and dissection
All queens in this study originated from healthy honey
bee colonies managed for honey production in a
research apiary located at the Goodale Research and
Teaching Farm, University of Saskatchewan
(52 01'50.6"N 106 32'26.6"W) during summer 2015.
The research apiary was surrounded by commercial
beekeeping operations, pastures, and agricultural crops,
including alfalfa, canola, and cereals treated with pesti-
cides according to standard agricultural practices. All
colonies received standard spring and fall treatments
of amitraz and oxytetracycline according to label
instructions. The research apiary was established pre-
dominantly from local stock, with a minor contribution
of New Zealand packaged bees that were cross-bred
and represent a genetic mixture most closely resem-
bling Apis mellifera carnica. All of the studied queens
were naturally mated during early summer 2015 and
collected in 10% formalin at the end of summer. To
avoid artifact introduced by sectioning through the
hard cuticle, the exoskeleton was removed from the
abdomen prior to sectioning. The 7th sternum was left
intact, as it is involved in forming part of the repro-
ductive tract.
Tissue processing
The reproductive tracts, together with associated
abdominal organs, were processed according to the
standard mammalian procedure (Mepham, 1991) and
embedded in Paraplast Plus (Leica Biosystems,
Richmond, IL, USA) in 3 positions: cranial, lateral, and
ventral to obtain sections in 3 different planes, namely:
transverse, sagittal and dorso-ventral. Five mm serial
sections were collected every 100 mm for histological
evaluation. The slides were dried for 1 hour at 65  C
and stained using routine Harris’ hematoxylin and eosin
(H&E) staining protocol (Luna, 1968); briefly: xylene
2 min., xylene 2 min., 100% ethyl alcohol 2 min., 100%
ethyl alcohol 2 min., 90% ethyl alcohol 2 min., 70% ethyl
alcohol 2 min., running 40  C water 1 min., Surgipath
Harris’s hematoxylin 560 (Leica Biosystems, Richmond,
IL, USA) 5 min., running 40  C water 1 min., 0.3% acid
alcohol 15 sec., running 40  C water 1 min., Surgipath
blue buffer 8 (Leica Biosystems, Richmond, IL, USA)
1 min., running 40  C water 3 min., 80% ethyl alcohol
1 min., Surgipath alcoholic eosin 515 Y (1%) (Leica
Biosystems, Richmond, IL, USA) 30 sec., 90% ethyl alco-
hol 1 min., 90% ethyl alcohol 1 min., 100% ethyl alcohol
15 sec., 100% ethyl alcohol 15 sec., 100% ethyl alcohol
15 sec., xylene 2 min., xylene 1 min., xylene 1 min. The
slides were then cover slipped in a fume hood using
Micromount (Leica Biosystems, Richmond, IL, USA. We
116 I. V. Kozii et al.
Figure 1. Macroscopic image of the reproductive tract of for-
malin fixed honey bee queen: (A) Upper reproductive tract
including ovaries (1), calyx (2), lateral oviducts (3) (the right
oviduct is distended with eggs), median oviduct (4), and
spermatheca covered by tracheal network (5) (bar ¼ 1mm);
(B) Middle region of the reproductive tract including lateral
oviducts (3), median oviduct (4), spermatheca (5), spermathecal
glands (6), common duct (7), spermathecal duct (8), sperm
pump (9); (bar ¼ 0.5 mm).
deliberately chose hematoxylin and eosin staining proto-
col for tissue evaluation because it is the most widely
used stain in vertebrate histopathology for diagnostics,
research, and regulatory risk assessment purposes
(Fischer et al., 2008).
Photomicrographs
For macroscopic images, queens were dissected under
a stereomicroscope Olympus SZ61 and photographed
with Olympus DP71 microscope digital camera
(Olympus, Japan). Each specimen was photographed at
7-12 consecutive field depths. The digital images were
overlaid with Helicon Focus 6.7.1. (Helicon Soft Ltd.,
Ukraine) to obtain a full-depth, in-focus image.
Histological images were captured using an Olympus
BX51 microscope and Olympus DP71 microscope
digital camera using various magnifications (reference
bars indicated in figure legends).
Terminology and description of macroscopic and
microscopic anatomy
The terminology and description of macroscopic and
microscopic anatomy used in this manuscript are based
on multiple sources of previously published literature
referenced in the discussion section.
Results
In this study, we reproduced the histological morph-
ology of the entire reproductive system of mated honey
bee queens and illustrated it with high quality photomi-
crographs. The reproductive system of the honey bee
queen consists of paired ovaries (Figure 1A-1) con-
nected by calices (Figure 1A-2) to paired lateral ovi-
ducts (Figure 1A-3, 1B-3), which merge to form a
median oviduct (Figure 1A-4, 1B-4) connected dorsally
to the spermatheca (Figure 1A-5, 1B-5) and caudally to
the terminal portion of the reproductive tract (i.e. geni-
tal chamber and bursa copulatrix with lateral pouches)
(Figure 8). The spermatheca (Figure 1B-5) is connected
to the median oviduct (Figure 1B-4) via the spermathe-
cal duct (Figure 1B-8). Paired spermathecal glands
(Figure 1B-6) are connected to the spermatheca via the
common duct (Figure 1B-7). The sperm pump is shown
in Figure ure 1B-9.
Histological morphology of the reproductive system
of the mated honey bee queen
Ovaries
Each ovary (Figures 1A-1 and 2A–C) consists of densely
packed clusters of ovarioles (Figure 2A) which are thin
chains of eggs that widen posterior, representing sequen-
tial, linear maturation of the oocytes (Figure 2A-1) and
trophocytes (Figure 2A-2). Macroscopically, an ovary is
structurally reminiscent of a bunch of bananas, where
each banana represents a separate ovariole. Each ovariole
has four distinct regions which are: a short terminal fila-
ment, a germarium of intermediate length, and a long
vitellarium which ends with the ovariole pedicle, all
encased within an epithelial sheath (Figure 2B-4). The
terminal filament (Figure 2B-solid bar) is composed of
undifferentiated stem cells embedded between discoid
 Histomorphological description of the reproductive system 117

Figure 2. Photomicrographs of the ovary segments: (A) Low magnification view of the ovarioles within the ovaries showing sequential
linear maturation of the oocytes (1), accompanied by trophocytes (2) (bar ¼ 1mm); (B) The most cranial portion of an ovariole is
the terminal filament (solid bar) composed of somatic and undifferentiated stem cells embedded between discoid cells (3) within an
epithelial sheath (4). Germarium (dashed bar) includes cystocyte rosettes (5) which develop into a single oocyte and accompanying
trophocytes (bar ¼ 20mm); (C) Cranial portion of vitellarium showing the distinct arrangement of oocytes (1) into a single row, sepa-
rated by nurse cell chambers (2). There is a distinct germinal vesicle within each oocyte (3) (bar ¼ 100 mm).

cells; the stem cells have large nuclei and poorly stained
cytoplasm (Figure 2B-3). The germarium (Figure 2B-
dashed bar) contains germline cells (cystocytes) clus-
tered into rosette-like structures (Figure 2B-5). In the
distal portion of the germarium, the oocytes (Figure
2C-1) become arranged into a single row, separated by
nurse cell chambers (Figure 2C-2), which constitutes
the transition to the vitellarium. Somatic cells are hap-
hazardly arranged in the proximal germarium but form
a distinct follicular epithelial lining around an oocyte
and its corresponding trophocytes in the vitellarium
(Figure 3A-2 and 5).
In the centro-caudal portion of the ovariole, the ovum
is almost completely covered by simple cuboidal somatic
epithelium with a central nucleus, densely stippled chro-
matin, and eosinophilic cytoplasm (Figure 3A-2), leaving
only a narrow opening on the anterior side, the trophic
stalk (Figure 3A-3) which allows access of nutrients from
the trophocytes (Figure 3A-4). There is a distinct germi-
nal vesicle within individual oocytes (Figure 2C-3). At the
late stages of vitellogenesis, the epithelium of the ovum is
blunted (cuboidal) (Figure 3B-8) and subsequently
becomes attenuated (Figure 3B-9).
The trophocytes, or nurse cells, are large cells with
dense, granular, eosinophilic cytoplasm and a large, pale,
polymorphic nucleus with finely to coarsely stippled
chromatin (Figure 3A-4). As they move posterior along
the ovariole, the trophocytes increase in size and even-
tually undergo degeneration, characterized by cytoplas-
mic eosinophilic granularity with pallor (rarefaction),
dispersal of the coarsely stippled nuclear chromatin, and
eventual effacement of the nuclear membrane and cellu-
lar borders (Figure 3A-7). Small median follicular cells,
previously described as intertrophocytic median follicu-
lar cells, are occasionally observed in between tropho-
cytes within the nurse chamber (Figure 3A-6).
Calyx
The calyx (Figures 1A-2, 4A-1, and 4B) is a funnel-like
structure on the caudal aspect of the ovaries that facili-
tates the transfer of eggs from ovarioles to the lateral ovi-
duct (Figures 1A-3 and 4A-2). The calyx is composed of
simple to the bi-layered, tall, columnar, densely packed
epithelium (Figure 4B-4) along an eosinophilic basement
membrane (Figure 4B-5). The epithelial cells have a cen-
trally located nucleus and an eosinophilic cytoplasm that
contains round, optically vacant vacuoles (Figure 4B-6)
that increase in size posterior and expand the cytoplasm.
118 I. V. Kozii et al.

Figure 3. Photomicrographs of the follicles: (A) The oocyte with accompanying trophocytes form a follicle (black outline). The
oocyte (1) is surrounded by a layer of single cuboidal epithelium (2). Trophic stalk (3) connects the oocyte with the trophocytes (4).
The trophocyte chamber is surrounded by a thin layer of attenuated epithelial cells (5). Intertrophocitic median follicular cells (6) are
located between trophocytes. The size of trophocytes increases with oocyte maturation and they eventually undergo degeneration
(7) (bar ¼ 50mm); (B) Blunted (8) and attenuated (9) follicular epithelium surrounding oocytes (1) in the middle and caudal sections
of vitellarium (bar ¼ 20mm).

Lateral oviducts multiple invaginations (Figure 5B-7). The epithelium of

The lateral oviducts (Figures 1A-3, 1B-3, and 4A-2)
extend bilaterally from the calyx and unite ventrally
toward the spermatheca (Figure 1A-5), to form the
median oviduct (Figure 1A-4 and 1B-4). The oviducts
are lined by cuboidal epithelium (Figure 4D-9) on an
eosinophilic basement membrane (Figure 4D-5) with a
thin, outer layer of longitudinal muscle (Figure 4D-10).
The epithelial cells have eosinophilic cytoplasm with a
well-defined, apical to central nucleus. The luminal side
of the epithelial lining is covered by a thin, chitinous,
strongly basophilic intima that has numerous cteniform
(comb-like) spines pointing caudally (Figure 4D-11). The
lining epithelium is attenuated when the oviduct lumen
is distended by an egg (Figure 4C-8).
Median oviduct and valve fold
The median oviduct (Figure 1A-4, 1B-4, and 5A-1) is a
short, muscular tubule that joins both lateral oviducts. It
is posterior defined by the valve fold (Figure 5B circled),
which also constitutes the transition point of the
median oviduct into the genital chamber. On the ventral
surface, the median oviduct has two lateral folds (Figure
5A-4) connected to sphincter muscles (Figure 5A-3). It
is lined by epithelium similar to that found in the lateral
oviducts, but instead of cteniform spines, it is covered
by a thin, pale, amphophilic intima (Figure 5B-8) that
thickens posterior along the genital tract (Figure 8B-5).
The valve fold is a deep transverse epithelio-muscular
inward projection on the ventral aspect of the posterior
median oviduct (Figure 5B circled). The stalk of the
valve fold is composed of loosely arranged muscle
(Figure 5B-9) and is covered by a single layer of
cuboidal to columnar epithelium (Figure 5B-6) with
the valve fold is covered by a thin, amphophilic intima,
similar to the median oviduct (Figure 5B-8).
Spermatheca, spermathecal duct and glands
The spermatheca (Figure 1B-5 and 6A-1) contains
densely packed spermatozoa (Figure 6A-2) in the mated
queen and is connected to the adjacent median oviduct
via the spermathecal duct (Figure 1B-8 and 6A-4). The
spermatheca is lined by simple cuboidal epithelium
(Figure 6B-5) covered by a thin cuticle (Figure 6B-7).
The spermatheca is surrounded by the well-developed
tracheal network (Figure 6B-8) which is intimately asso-
ciated with the basement membrane (Figure 6B-6) of
the spermathecal epithelium (Figure 6B-5) to facilitate
gas exchange. The orifice connecting the spermatheca
with the spermathecal duct (Figure 7A-2) is lined by tall
columnar, slender epithelial cells with basal nuclei
(Figure 7A-1 and 7B-4). The lumen of the duct is cov-
ered by a thick layer of basophilic cuticle (Figure 7B-5).
At the base of the duct, where it is still in contact with
spermatheca, there is a set of circular and longitudinally
arranged muscles called the sperm pump (Figure 7B-6).
Two long, convoluted, symmetrical spermathecal
glands (Figures 1B-6 and 6A-3) are connected to the
spermatheca via the common duct (Figure 1B-7). The
spermathecal glands are lined by two epithelial layers –
an external glandular layer (Figure 6C-9) and an internal
intima (Figure 6C-12). The external layer is composed
of the tall columnar glandular epithelium (Figure 6C-9)
that has large, basal, densely basophilic nuclei and foamy
cytoplasm with at least one large, distinct, pale eosino-
philic, apical intracytoplasmic vacuole (Figure 6C-10).
The external epithelium is surrounded by a thin, baso-
philic basement membrane formed by attenuated
 Histomorphological description of the reproductive system
Figure 4. Photomicrographs of the cranial tubular region of the
reproductive tract: (A) Low magnification view of the calyx (1)
transition into lateral oviducts (2) with centrally located ventriculus
(3) (bar ¼ 500mm); (B) Calyx is formed by an irregularly thickened
wall, lined by simple to bi-layered, slender, tall, columnar, densely-
packed epithelium (4) on an eosinophilic basement membrane (5)
and contains apical, optically vacant vacuoles (6) (bar ¼ 50mm); (C)
Cross section of a distended lateral oviduct with eggs (8): distention
is facilitated by longitudinal folds (7) lined by attenuated epithelium
(bar ¼ 100mm); (D) Longitudinal section of a lateral oviduct lined
by cuboidal epithelium (9) on an eosinophilic basement membrane
(5) with an underlying, single layer of longitudinal muscle cells (10).
The epithelium is covered by a chitinous, strongly basophilic intima
that has numerous cteniform spines (11) (bar ¼ 20mm).
119
epithelial cells (Figure 6C-11). The internal intima is
composed of much smaller cells (Figure 6C-12) with ill-
defined cell borders; small, round, basal nuclei; and mul-
tiple, optically vacant, linear, intracytoplasmic canaliculi
directed toward the glandular lumen (Figure 6C-13).
The glandular lumen is covered by amorphous ampho-
philic material (Figure 6C-14).
Genital chamber, bursa copulatrix, and bursal pouches
The median oviduct continues posterior to the valve
fold to become the genital chamber (Figure 8A-1) which
is limited posterior by a cleft-like widening called the
bursa copulatrix (Figure 8A-2) with bilateral connections
to two bursal pouches (Figure 8A-3). The genital cham-
ber, bursa copulatrix, and bursal pouches are lined by a
single layer of epithelium (Figure 8B-4) covered by a
cuticle (Figure 8B-5) which has a similar histological
structure to the median oviduct (Figure 5A). The most
caudal portion of the reproductive tract is formed dor-
sally by the ventral part of the sting and ventrally by the
7th sternum.
Discussion
In this study, we reproduced the histomorphology of
the entire reproductive system of mated honey bee
queens and illustrated it by high quality photomicro-
graphs. These photomicrographs augment and enhance
previous diagrams and descriptions of macroscopic and
microscopic queen reproductive anatomy illustrated by
Snodgrass (1985) and Camargo and Mello (1970); they
described and illustrated by diagrams the reproductive
system of the honey bee queen in the order from cra-
nial to caudal consisting of paired ovaries, paired lateral
oviducts, a median oviduct with a valve fold, a genital
chamber (vagina), a bursa copulatrix with lateral
pouches, the sperm recepticulum: the spermatheca with
its glands and a spermathecal duct.
Ovaries
The ovaries form a paired organ that takes up about
2/3 of the abdominal cavity in the honey bee queen and
are located latero-ventral to the upper digestive tract
(Camargo & Mello, 1970; Snodgrass, 1985). The ovaries
are composed of ovarioles divided into four regions (i.e.
terminal filament, germarium, vitellarium, and ovariole
pedicle) based on morphological differentiation (Patr
ıcio
& Cruz-Landim, 2002, 2008). The stem cells (terminal
filament) differentiate into germinal cells forming ros-
ette-like clusters (germarium); subsequently, each ros-
ette produces a single oocyte and accompanying
trophocytes which together comprise a follicle (vitella-
rium) (Martins et al., 2011; Patr
ıcio & Cruz-Landim,
2002, 2008; Snodgrass, 1985; Tanaka & Hartfelder,
2004), as illustrated in the present study (Figure 2A–C).
120 I. V. Kozii et al.
Figure 5. Photomicrographs of (A) the median oviduct (1) which is lined by simple cuboidal epithelium (2) and is externally sur-
rounded by a well-developed outer sphincter muscle (3) that forms two lateral folds (4) of the median oviduct by protruding inwards;
egg (5); (bar ¼ 100mm); (B) The valve fold (circled) is a deep, transverse, epithelio-muscular inward projection, lined by simple
cuboidal epithelium (6) that forms multiple invaginations (7), covered by a thin intima (8). The core of the stalk is composed of loosely
arranged musculature (9) (bar ¼ 100mm).
The ovary of the honey bee queen is polytrophic
(multiple trophocytes per follicle) and meroistic (pro-
duce nutritive trophocytes and ova). Within each fol-
licle, the trophocytes (nurse cells) (Figure 3A-4) are
attached by intercellular bridges and connected to the
oocyte by the trophic stalk (a narrow opening) (Figure
3A-3) which allows the transport of nutrients, RNA,
and ribonucleoproteins from the trophocytes to the
ovum during maturation (Figure 3A-1) as described in
previous studies (Berger & da Cruz-Landim, 2009;
nski & Jaglarz, 1999; Cruz-Landim & Patr
ıcio, 2010;
Bili

Snodgrass, 1985; Tanaka et al., 2006). After transferring
their cytoplasmic contents to the developing ovum, the
trophocytes undergo programmed cell death (apoptosis)
(Figure 3A-7) as found in earlier studies (Mpakou et al.,
2006; Patr
ıcio & Cruz-Landim, 2008). Thus, the oocytes
in the proximal vitellarium are small, followed by an
organized cluster of trophocytes (Figure 3A circle),
whereas the oocytes in the distal vitellarium are greater
in size, followed by apoptotic/degenerating trophocytes
(Figure 3A-7), as described by Snodgrass, 1985.
Ramamurty (1977) suggested that degenerated tropho-
cytes are phagocytosed by the intertrophocytic median
follicular cells (Figure 3A-6).
During the late stages of vitellogenesis, the epithe-
lium surrounding the ovum progressively attenuates and
produces chorion and vitelline membrane (Figure 3B-9).
At the end of the vitellogenesis, the trophic stalk
(Figure 3A-3), forms an elevated, sieve-like micropyle
(pore) in the ovum, through which the sperm enters
during fertilization in the median oviduct (Landim &
Yabuki, 1995; Nelson, 1915). When a mature ovum
progresses from an ovariole to the calyx during ovula-
tion, the ovum epithelium collapses and forms a corpus
luteum (Patr
ıcio & Cruz-Landim, 2002), which later
undergoes degeneration and resorption (Ramamurty,
1977). The corpus luteum is an unorganized mass of
cells that obstructs the ovariole pedicle, thus temporar-
ily preventing subsequent ovulation by the same ovar-
iole (Patr
ıcio & Cruz-Landim, 2008). A mature ovum
can be ovulated approximately every 3-5 hours from
each ovariole (Ramamurty, 1977).
Calyx and lateral oviducts
After ovulation, the egg passes from the calyx (Figure
4B) into a lateral oviduct (Figure 4C) which has multiple
accordion-like, longitudinal folds which allow expansion
of the oviduct lumen for the passing egg (Laidlaw, 1944;
Mackensen & Tucker, 1970) as illustrated in Figures 4C-
7 and 8. One of the distinctive features of the lateral
oviduct is the presence of cteniform spines in the epi-
thelial intima (Figure 4D-11) which have been described
in earlier studies (Berger-Twelbeck & Dorn, 1994;
Gerber et al., 1978; Laidlaw, 1944). These spines are
more numerous and pronounced in the anterior por-
tion of the oviducts adjacent to the calyx and are absent
in the median oviduct. The spines are believed to have
a role in the unidirectional progression of the egg from
the lateral to the median oviduct by preventing its cra-
nial movement during muscle contraction (Gerber
et al., 1978).
Median oviduct and the valve fold
Fertilization of an egg takes place in the median oviduct.
It is believed that the valve fold (Figure 5B-9), together
with the median oviduct sphincter (Figure 5A-3), slow
down the egg when it passes through the median ovi-
duct and push the micropylar end of the egg against the
opening to the spermathecal duct to facilitate fertiliza-
tion (Colin et al., 1999). The valve fold is also thought
 Histomorphological description of the reproductive system
Figure 6. Photomicrographs of the spermatheca with its
glands and ducts: (A) Low magnification image of the
spermatheca (1) containing sperm (2); spermathecal glands
(3) and spermathecal duct (4) (bar ¼ 200mm); (B) The
spermatheca is lined by simple cuboidal epithelium (5) on a
thin, anucleate, basophilic basement membrane (6) with an
overlying thin, basophilic cuticle (7). The spermatheca is
surrounded by tracheal network (8). The fecundated
spermatheca is filled with tightly packed spermatozoa (2)
(bar ¼ 20mm); (C) The spermathecal glands are lined by an
external glandular epithelial layer (9), formed by tall, colum-
nar epithelium that has large, basal nuclei and foamy cyto-
plasm with at least one, distinct, apical, intracytoplasmic
vacuole (10), on a basophilic membrane formed by attenu-
ated epithelium (11). The inner intima (12) is formed
by smaller, tall columnar cells with basal nuclei and multiple
intracytoplasmic canaliculi (13). The glandular lumen is
covered by amorphous amphophilic material (14) (bar
¼ 20mm).
121
to regulate sperm outflow from the spermathecal duct
during fertilization (Camargo & Mello, 1970).
Spermatheca, spermathecal duct and glands
The spermatheca (Figure 1B-5 and 6A-1), spermathecal
duct (Figure 1B-8 and 6A-4), and the spermathecal
glands (Figures 1B-6 and 6A-3) are accessory organs of
the reproductive system of the honey bee queen
studied in closer detail by Kapil (1962). The sperma-
theca is located craniodorsally over the median oviduct
at the level of the 5th sternum. It is a round, 1.2-1.3 mm
in diameter, sperm recepticulum (Tarpy et al., 2011).
The spermathecal glands (Figure 1B-6) secrete proteins
into the spermatheca via the common duct (Figure 1B-
7) to replace the proteinaceous fluid lost from the
spermatheca with the sperm during fertilization (Klenk
et al., 2004). At the base of the spermathecal duct, the
muscular sperm pump (Figure 7B-6) provides a constant
sperm volume necessary for the fertilization of a single
egg (Baer et al., 2016; Bresslau, 1905).
Genital chamber, bursa copulatrix, and
bursal pouches
The posterior portion of the genital tract comprises the
genital chamber, bursa copulatrix, and bursal pouches.
Several papers refer to the genital chamber as the
“vagina” (Kapil, 1962; Laidlaw, 1944; Snodgrass, 1985).
However, Camargo and Mello (1970) contend that this
term is not appropriate as the genital chamber does not
receive the drone’s endophallus during mating, except
on rare occasions, and therefore this term should not
be borrowed from mammalian anatomy. During copula-
tion, the chitinous plates of the drone endophallus
attach to the bursal pouches (Camargo & Mello, 1970;
Woyke, 2011) and sperm is ejaculated into the lateral
oviducts where it then travels backward via the sperma-
thecal duct to be stored in the spermatheca within the
next several hours. The valve fold is thought to regulate
the backflow of sperm into the spermatheca by prevent-
ing its retrograde flow into the posterior genital cham-
ber (Laidlaw, 1944). However, since sperm consistently
escape backward into the bursa copulatrix after mating,
the efficacy of regulation of sperm flow by the valve
fold has been questioned by Camargo and Mello (1970).
Use of light and electron microscopy in honey
bee research
Histological evaluation of microscopic tissue samples is
a universal gold standard technique used for studying
normal tissue structures, detection and diagnosis of dis-
ease, and toxicologic risk assessment in vertebrate spe-
cies (Crissman et al., 2004; Fiette & Slaoui, 2011).
Historically, some of the first histological studies were
used to understand and describe insect respiration in
silkworms by Marcello Malpighi (1628–1694) who is
122 I. V. Kozii et al.
Figure 7. Spermathecal duct: (A) The orifice connecting the spermatheca and the spermathecal duct lined by high columnar, slender
epithelial cells (1) covered by basophilic cuticle (2); sperm (3) (bar ¼ 50mm); (B) The spermathecal duct is lined by tall columnar epi-
thelium with basal nuclei (4), covered by a thick layer of basophilic cuticle (5). At the base of the duct, there is a set of circular and
longitudinal muscles called the sperm pump (6) (bar ¼ 50mm).
now known as the “father of microscopic anatomy”
(DiDio, 1995; West, 2013). However, unlike research in
vertebrate species, insect studies today rarely use histo-
logical tissue evaluation as a research tool.
Nevertheless, in recent years histological and ultra
structural morphologic alterations have been success-
fully used to study honey bee diseases and toxicities.
For example, Koziy et al. (2019) investigated the histo-
logical appearance of brood-food glands in nurse bees
affected with deformed wing virus – a nearly ubiquitous
honey bee disease worldwide. In this study, both the
hypopharyngeal and mandibular glands of affected bees
were markedly hypoplastic and contained a greater
number of apoptotic cells (Koziy et al., 2019). These
findings contribute to further understanding of
deformed wing virus pathogenesis. Similarly, Zaluski
et al. (2017) described histopathology of morphologic
changes in mandibular (reduced height of secretory
cells) and hypopharyngeal (reduced acini size) glands of
worker bees exposed to pyraclostrobin and fipronil.
The authors concluded that these changes to the glan-
dular morphology may negatively affect overall colony
development, maintenance, and survival (Zaluski et al.,
2017). Histological examination of the brain (de
Almeida Rossi et al., 2013; Tom
e et al., 2012) and the
hepato-nephrotic system (Domingues et al., 2017) of
honey bees has also enhanced the current body of lit-
erature on the pathogenesis and effect of certain pesti-
cides on honey bee health revealing an array of
degenerative sublethal changes in response to insecti-
cide treatment. Using histological tissue evaluation has
enabled the above researchers to detect and describe
minute changes in bees in response to studied stimuli.
Their discoveries now help us better understand the
pathogenesis of disease and some toxicity.
Light microscopic and ultrastructural evaluation of
ovaries but no other structures of the honey bee queen
reproductive tract, were used in several studies. These
studies focus on understanding both physiologic and
pathologic processes in the ovary in response to various
stimuli. For instance, ultrastructural evaluation of the
ovary showed that delayed mating increases apoptotic
and necrotic death of the ovarian germ line and somatic
interstitial cells (Berger & da Cruz-Landim, 2009).
Histology was used for more precise and reproducible
estimation of ovariole numbers in honey bee queens
(Jackson et al., 2011), which has since become one of
the standard methods in honey bee research (Carreck
et al., 2013). Additionally, histology was used in studies
by Fyg in 1964, who described the rare direct infections
of the queen reproductive system with yeast-like micro-
organisms (H-melanosis) and bacteria (B-melanosis) and
characterized vitellarium degeneration in queens as an
indirect effects of Nosema spp infection. These studies
further emphasize the importance and benefit of thor-
ough histopathological examination of the honey bee
queen reproductive system.
The importance and novelty of the current study is
that it provides high quality, detailed photomicrographs
with a thorough sequential description of the hematoxy-
lin and eosin stained histological structures of the entire
reproductive tract of mated honey bee queen. We
deliberately chose hematoxylin and eosin staining proto-
col for tissue evaluation because it is the most widely
used stain in vertebrate histopathology for diagnostics,
research, and regulatory risk assessment purposes and
it allows detailed evaluation of microscopic changes in
cells and tissues (Fischer et al., 2008).
By synthesizing descriptions and diagrams provided in
earlier studies, this manuscript summarizes in detail the
normal structure, nomenclature, and function of the
 Histomorphological description of the reproductive system
Figure 8. Lower reproductive tract: (A) The genital chamber (1) is a continuation of the median oviduct posterior to the valve fold;
through a cleft-like opening, it transitions into the bursa copulatrix (2) with bilateral bursal pouches (3) (bar ¼ 200mm); (B) Enlarged
squared area in A: genital chamber (1), bursa copulatrix (2), and bursal pouch (3) lined by cuboidal epithelium (4) with prominent
cuticle (5) (bar ¼ 50mm).
microscopic structures of the queen’s reproductive
tract and provides a visual example of each structure.
Histological evaluation of reproductive histopathology is
an essential step in toxicological risk assessment in ver-
tebrate species, especially so in pharmaceutical drug
development (Halpern et al., 2016) as well as in therio-
genology research. Accordingly, the comprehensive
characterization and illustration of normal light micro-
scopic morphology of the entire reproductive tract of
the honey bee queen performed in the current study is
anticipated to serve as a reference for the future histo-
pathological investigations of reproductive problems
affecting honey bee queens.
Conclusions
The current study summarized the normal histological
structure and functions of the entire honey bee queen
reproductive tract and illustrated it with high-quality
photomicrographs. It is anticipated that this study will
facilitate further investigations of the pathogenesis of
the recent decline in reproductive fitness of honey bees
using histopathology.
Acknowledgements
We thank Claire Janse van Rensburg, Dr. Igor Moshynskyy,
Dr. Sophie Derveau, Dr. Maud Ferrari, and Dr. Ahmad Al-
Dissi for their contribution and constructive feedback. We
also thank Madeleine Friesen and Phil Dillman for field and
histology assistance, respectively.
Authors contribution
IK and ES designed experiment; IK, SW, RK, and ES partici-
pated in the design and interpretation of the data; IK per-
formed experiments and analysis; IK and ES wrote the
manuscript. All authors participated in the revision of the
manuscript, read and approved the final manuscript.
123
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This work was supported by the Western Grains Research
Foundation (ADF 20150125, 2016); Saskatchewan Canola
Development Commission (ADF 20150125, 2016);
Saskatchewan Agricultural Development Fund (20160157,
2017); MITACS Accelerate (Canada, IT09712); Project Apis m.
(Costco, Canada); North American Pollinator Protection
Campaign (2015), Canadian bee research fund (2015),
Saskatchewan Beekeeping Development Commission (2015),
and WCVM Wildlife Health Research Fund (2016). The per-
sonal support of IK was provided by the University of
Saskatchewan Graduate Scholarship, WCVM Graduate
enhancement fund, Saskatchewan Innovation & Opportunity
Scholarship, MITACS Accelerate, and the PEO International
Peace Scholarship.
ORCID
Ivanna V. Kozii http://orcid.org/0000-0002-6802-2762
Sarah C. Wood http://orcid.org/0000-0001-8613-156X
Roman V. Koziy http://orcid.org/0000-0003-1009-7814
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