[Pollinator] Cell death localization in situ in laboratory reared honey bee (Apis mellifera L
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Paper attached and embedded below. Important new information. Laurie
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Cell death localization in situ in laboratory reared honey bee (Apis
mellifera L.) larvae treated with pesticides
Ales Gregorc*, James D. Ellis
Honey Bee Research and Extension Laboratory, Department of Entomology and
Nematology, University of Florida, P.O. Box 110620, Bldg 970 Natural Area
Drive, Gainesville, FL, USA 32611
*Corresponding author email and present mail address:
_ales.gregorc at kis.si_ (mailto:ales.gregorc at kis.si) ; Agricultural Institute of Slovenia,
Hacquetova 17, SI-1000 Ljubljana, Slovenia, tel: +386-1- 28 05 150
Abstract
In this study, cell death detected by DNA fragmentation labeling and
phosphatidylserine (PS) localization was investigated in the honey bee (Apis
mellifera L.) midgut, salivary glands and ovaries after treating larvae with
different pesticides offered via an artificial diet. To do this, honey bee
larvae reared in an incubator were exposed to one of nine pesticides:
chlorpyrifos, imidacloprid, amitraz, fluvalinate, coumaphos, myclobutanil,
chlorothalonil, glyphosate and simazine. Following this, larvae were fixed and
prepared for immunohistologically detected cellular death using two TUNEL
techniques for DNA fragmentation labeling and Annexin V to detect the
localization of exposed PS specific in situ binding to apoptotic cells. Untreated
larvae experienced ~10% midgut apoptotic cell death under controlled
conditions. All applied pesticides triggered an increase in apoptosis in treated
compared to untreated larvae. The level of cell death in the midgut of
simazine-treated larvae was highest at 77% mortality and statistically similar
to the level of cell death for chlorpyrifos (65%), imidacloprid (61%),
myclobutanil (69%), and glyphosate (69%) treated larvae. Larvae exposed to
fluvalinate had the lowest midgut columnar apoptotic cell death (30%) of any
pesticide treated larvae. Indications of elevated apoptotic cell death in
salivary glands and ovaries after pesticides applications were detected.
Annexin V localization, indicative of apoptotic cell deletion, had an extensive
distribution in the midgut, salivary glands and ovaries of
pesticide-treated larvae. The data suggest that the tested pesticides induced apoptosis in
tissues of honey bee larvae at the tested concentrations. Cell death
localization as a tool for a monitoring the subclinical and sub-lethal effects of
external influences on honey bee larval tissues is discussed.
Keywords: Apis mellifera, immunohistology, cell death, TUNEL, insecticide,
herbicide, fungicide
Introduction
Globally, the environment around honey bee (Apis mellifera) colonies can
be contaminated with toxic chemicals from industrial, agricultural and
domestic activities. In many cases, these chemicals are pesticides which
encompass an array of compounds designed to repel or kill insects (insecticides),
plants (herbicides), fungi (fungicides) and other organisms considered
pests. Though honey bees are non-target organisms for most pesticide
applications, they nevertheless can be exposed to pesticides while collecting pollen
and nectar from flowers, collecting resins from various plants, drinking
water from rivers/lakes/ponds/etc., breathing, and during flight (if the
pesticides are airborne). These pesticides may be brought back inadvertently
to the colony where their levels are concentrated further in the waxy nest
infrastructure. In surveys of North American honey bee colonies conducted in
2007 and 2008, investigators found 121 different pesticides and
metabolites in wax, pollen, bees, and corresponding hive samples [1], thus
illustrating the need to understand how pesticides may affect individual honey bees
and the social colonies in which they reside.
Many of the pesticides to which honey bees are exposed have insecticidal
properties and may be harmful to bees. For example, pesticides are known to
lower the developmental rate of queen honey bees, increase the occurrence
of queen rejection, and lower queen weight [2-4], affect honey bee
cardiotoxicity [5], and affect forager bee mobility and communicative capacity [6],
all among other effects documented in the literature. In our effort, we
broaden the study of pesticide effects on honey bees by investigating
pesticide effects on cell death and localization in pesticide-treated, honey bee
larvae.
There are many reasons to look at pesticide effects in bee larvae tissues.
First, toxic effects of pesticides have been shown to manifest in
mammalian tissue and alter enzymatic levels, blood biochemistry and tissue
histology [7], thus providing evidence that toxins can affect tissues in
pesticide-exposed organisms. Second, histological changes in treated individuals
provide a rapid detection method for the effects of toxicants, especially
chronic irritants, in various tissues and organs [8]. Third, many of the
studies where the effects of pesticides on honey bees are discussed focus on
toxin effects on adult bees rather than immature ones, resulting in a lack of
information concerning the latter. Fourth, previous immunocytochemical
studies of cell death and the localization of heat-shock proteins in larval
honey bee tissues after acaricide application have fostered a
better-understanding the adverse effects acaricides may have on bees [9-11]. Finally,
there is an overall lack of histopathological studies on pesticide treated
animal tissues. For all of these reasons, we studied the effects of pesticides
on larval honey bees at the cellular level.
To determine pesticide effects on the cellular tissues of larval honey
bees, we looked specifically at unintentional cell death (necrosis) and
programmed cell death (apoptosis)
[12]. Necrotic cell death is induced by external influences with evident
morphological changes: i.e. the chromatin condenses and clumps are formed at
the nuclear periphery [12]. Necrosis refers to the post mortem changes
that occur following the death of the cell [13]. Apoptosis on the other hand
presents a range of morphological symptoms including cell shrinkage and
chromatin margination, the latter of which is followed by DNA fragmentation
and the formation of apoptotic bodies [14]. Apoptosis originally was
defined as the physiological death of cells and tissues associated with
developmental remodeling [15] and can be induced by genetic [16] and non-genetic
[17] means.
We used multiple cell death assays to determine the effects of various
pesticides on honey bee larvae. The first method we used to determine the
progression of cell death in situ was the TUNEL (terminal deoxynucleotidyl
transferase–mediated dUTP nick end-labeling) method which assesses DNA
breakdown preceding the nuclear collapse of apoptotic nuclei [18] and consists
of the visualization of fragmented DNA in the nucleus [19]. Cell death
previously has been characterized using the terminal TUNEL technique method in
the honey bee midgut [10, 20] and larval salivary glands [11] where the
death of salivary gland tissues in honey bee larvae was detected [21]. We
decided to use two TUNEL methods in our experiment because others have provided
data which show that different TUNEL kits can indicate different levels of
cell death in target tissues [10]. For example, the in situ cell death
detection kit AP was unable to differentiate between apoptosis and necrosis in
different human tissues and detected both [22]. Furthermore, DNA
fragmentation and a TUNEL-positive reaction can occur after different kinds of cell
death using various kits. Regardless, immunocytochemical methods assaying
DNA fragmentation [24] are useful techniques for detecting impending
apoptosis due to larval exposure to pesticides while feeding [25].
The second method we employed to monitor cell death was via our use of
Annexin V to detect the localization of exposed phosphatidylserine (PS)
specific in vivo binding to apoptotic cells. In dying cells, PS is externalized
actively to the plasma membrane’s outer leaflet parallel to the
extracellular environment [26]. Most forms of cell death share the phenomenon of cell
surface expression of PS [27]. Externalization of PS is an early event in
the sequence of steps leading to cell death which starts well before
changes in the cell nuclei and plasma membrane integrity are compromised [28].
PS on the cell surface can be detected using Annexin V, a member of the
annexin protein family that binds in a calcium-dependent way to PS-containing
membranes [29]. The Annexin V affinity assay discriminates among living
cells, cells in the early phase of cell death and (secondary) necrotic cells
that have a compromised cell membrane [30].
In our study, induced cell death and PS localization was investigated in
honey bee midguts after treating larvae with one of nine different
pesticides offered via an artificial diet. The tested pesticides (with insecticide
class in parentheses) included 2 fungicides [myclobutanil (azole),
chlorothalonil (substituted benzene)], 2 herbicides [simazine (triazine), glyphosate
(phosphonoglycine)], and 5 insecticides/miticides [fluvalinate
(pyrethroid), imidacloprid (nicotinoid), coumaphos (organophosphate), chlorpyrifos
(organophosphate), amitraz (amidine)] and represent a range of
modes-of-actions and pesticide families. With the exception of glyphosate, all have been
found as residues in honey bee colonies [1]. Immunohistological methods
using both TUNEL assays and Annexin 5 were employed in order to reduce the
probability of extraneous artifacts [25], in an attempt to define the specific
modes of cell death, and for the broad quantification of cell death
observed in larval midguts. We hypothesized that increased apoptotic cell death
(determined using the TUNEL technique) occurs in pesticide treated larvae in
comparison to untreated larvae and that PS exposure on the plasma membrane
of apoptotic cells (determined using Annexin V) would be present in
pesticide treated larvae.
2. Materials and Methods
2.1. Larval rearing, treatment and sampling
Experiments were conducted at the University of Florida Honey Bee Research
and Extension Laboratory, Department of Entomology and Nematology,
Gainesville, FL. Queens in three production honey bee colonies housed in 10-frame
Langstroth-style equipment were confined to a section of newly-drawn comb
using a metal queen excluder cage (~10 × 10 × 3 cm) at time t = -12 h. The
caged queen and frame were returned to the center of the brood nest where
worker bees could access and tend the queen. After 24 h of queen confinement,
t = 12 h [ 31, 32], we removed the queen from the cage and replaced the
cage on the comb as before but this time for 108 h (from t = 0) to allow the
eggs to hatch and larvae to reach an appropriate age for grafting. During
this time, worker bees were able to access the comb to feed the developing
larvae. At 108 h, we removed the test frames (now containing 36 ± 12 h old
larvae) from the colonies and took them to the laboratory.
At the laboratory, the larvae were grafted to sterile, 96-well tissue
culture plates (well volume = 0.32 mL, Fisher Scientific, Pittsburgh, PA, USA).
Prior to grafting the larvae into plates, we pipetted 20 µL of larval diet
into the bottom of each cell. The diet had a pH that ranged from 4.0-4.5
and consisted of 50% royal jelly (Glory Bee Foods, Eugene, OR, USA), 6%
D-glucose (Fischer Chemical, Fair Lawn, NJ, USA), 6% D-fructose (Fischer
Chemical, Fair Lawn, NJ), 37% double distilled water, and 1% yeast extract (Bacto
™, Sparks, MD, USA) by volume [32]. Prior to adding the diet to each
cell, we pre-warmed it to 35oC in an incubator (Percival Scientific Inc, Perry,
IA, USA).
Each subsequent day, we transferred larvae to a clean culture plate
provisioned with fresh diet. The amount of artificial diet provided to each larva
depended on the larva’s age. We fed larvae 20 µL of diet at hours 108 and
132, 30 µL on hour 156, 40 µL on hour 180, and 50 µL on hour 204 [33, 34].
At 204 h post oviposition (larvae are 132 ± 12 h old), we transferred the
larvae to a 48-well plate (Becton Dickinson Labware, Franklin Lakes, NJ,
USA, wells were 13 × 17 mm) because the growing larvae were too large to
handle delicately in a 96-well plate. Throughout the study, trays containing
larvae were incubated in the dark at 35oC and ~96% RH [31].
To test the effects of pesticides on developing larvae, specific pesticide
concentrations were mixed with the larval diet daily for 4 days beginning
the second day larvae were in the laboratory (132 h = 60 h old larvae).
Nine treatment groups of larvae were established in all, each group being
composed of 12 treated larvae. Each group of test larvae was treated with 1 of
the following pesticide doses: 1.6 ppm chlorpyrifos, 400 ppm imidacloprid,
400 ppm amitraz, 200 ppm fluvalinate, 100 ppm coumaphos, 400 ppm
myclobutanil, 400 ppm chlorothalonil, 400 ppm glyphosate, and 400 ppm simazine. The
respective pesticide doses are at or below LC50 values known for honey bee
larvae (unpublished data). Originally, we wanted to standardize the dose
delivered across all pesticides at 400 ppm to bracket the upper residue limit
that any of these pesticides have been found in honey bee colonies [1].
However, chlorpyrifos has a low LC50 value and fluvalinate/coumaphos LC50
values do not fit standard toxicity curves (unpublished data). As such, these
three pesticides were administered at different doses than were the other
pesticides. All applied pesticides were obtained from Chem Service, West
Chester, PA, USA.
Prior to administration to the larval diet, each pesticide was diluted
individually in an acetone solvent. The diet/pesticide combinations were
prepared and stored in 1.5 ml snap-top plastic vials (Fisher Scientific,
Pittsburgh, PA, USA). We included two control groups in the study: larvae feeding
on diet containing acetone and larvae feeding on an untreated diet. All
larvae were sampled on day 6 (h = 228), 24 hours after the application of the
last pesticide treatment. Sampled larvae were fixed in 10% formalin for 24
h, dehydrated in a series of alcohols and xylene, and finally embedded in
paraffin wax as described by Gregorc and Bowen [9]. Sections of 5 μm were
cut on a 2030 Rechert/Young Microtome (Cambridge Instrument GmbH., Germany),
floated on distilled water at 40°C, collected on cleaned slides, and kept
in an drying oven at 60°C for ~4 h. Slides then were stored at room
temperature until later analyses.
2.2. Immunohistology
The paraffin wax was removed from the tissue sections in three washes of
xylene and three washes of absolute alcohol. Sections then were rinsed in
Phosphate Buffer Solution (PBS, 0.01 M, pH 7.1) and prepared for staining.
2.3. DeadEnd colorimetric TUNEL system
The DeadEnd system (Promega, Madison, WI, USA) labels fragmented DNA of
apoptotic cells in situ using the TUNEL assay. After applying proteinase K,
the larval sections were incubated with the TdT reaction mixture and then
with a horseradish peroxidase-labeled streptavidin solution.
Diaminobenzidine (DAB) substrate was applied onto the tissue sections to develop a brown
reaction product. The sections were counterstained with Mayer’s
hematoxylin. Negative control labeling was achieved by substituting the
deoxynucleotidyl transferase (TdT) enzyme with PBS.
2.4. In situ cell death detection kit, AP (ISCDDK)
Dewaxed and rehydrated tissue sections were incubated with proteinase K
(20 μg/mL in 10 mM Tris/HCl, pH 7.4). Labeling was conducted by covering the
tissue section with a TUNEL reaction mixture composed of terminal
deoxynucleotidyl transferase (TdT) from calf thymus. TdT enzymes with fluorescein
were detected using “converter-AP” consisting of anti-fluorescein
antibodies from sheep, conjugated with alkaline phosphatase. The substrate solution
was obtained using a Vector® Red Alkaline Phosphatase Substrate Kit (Vector
Laboratories, Burlingame, CA, USA). Sections were incubated with the
substrate (AP) and washed in tap water for 5 min. Counterstaining was
accomplished by transferring the sections into Mayer’s hematoxylin and then rinsing
the sections under running tap water. As a negative control, we labeled a
subgroup with terminal transferase, rather than TUNEL reaction mixture.
2.5. Quantification of cell type and apoptosis
TUNEL labeled tissue slides were used for quantification of cell type and
apoptosis as determined using Dead End and ISDDK kits. For each treated
group of larvae, approximately 300 total cells from at least three larvae on
different slides were counted in random fields within the tissue. The
results were expressed as the proportion of cells counted that gave positive
staining. To confirm reproducibility, 25% of the slides were chosen randomly
and scored twice. The proportion of cells that gave positive staining was
analyzed by treatment (9 pesticides and 2 controls) with a one way ANOVA for
both staining techniques (Dead End and ISDDK). Furthermore, we used a two
way ANOVA to test the effects of technique, overall treatment and the
interaction of treatment × technique on the proportion of cells with positive
staining. Prior to all analyses, the proportion data were transformed with an
asin √x transformation. The untransformed means are reported in the
manuscript. Where necessary, we used Student’s T-tests to compare means, accepting
differences at P ≤ 0.05.
2.6. Immunohistochemical localization of PS
Dewaxed and rehydrated tissue sections were placed in PBS (0.01 M, pH 7.1)
and incubated with a primary antibody solution. Rabbit antibodies
polyclonal to Annexin V were obtained from Abcam (Abcam Inc., Cambridge, MA,
USA). Antibodies were used at a concentration of 2 µg/ml in PBS with 1% bovine
serum albumin. After incubating the primary antibodies overnight at 4°C,
the sections were covered with biotinylated universal secondary antibodies
for 30 min. Alkaline phosphatase reagent also was applied for 30 min. Both
reagents were obtained in the Vecastain Universal ABC-AP kit (Vector
Laboratories, Burlingame, CA, USA). The substrate solution was obtained using the
Vector® Red Alkaline Phosphatase Substrate Kit (Vector Laboratories,
Burlingame, CA, USA). Sections were incubated with the substrate (AP) and
counterstaining was accomplished by transferring sections into Mayer’s
hematoxylin. As a control, no primary antibody was applied to the tissue sections.
Sections were mounted in Faramount aqueous mounting medium (Dako,
Carpinteria, CA, USA). All slides were examined with a Leica light microscope (Leica
Microsystems, Germany) at 400× magnification.
3. Results
3.1. DeadEnd colorimetric TUNEL system
The brown reaction product obtained from the Promega DeadEnd kit indicated
DAB-positive, impending apoptotic cell death in all test larvae. Pesticide
specific levels of apoptosis detected in the midgut tissue are shown in
Table 1. The DAB reaction product was detected in the midguts of all
pesticide-treated larvae in larger percentages than in control larvae fed either a
diet containing acetone or pure diet (Table 1). In all DAB-positive cells,
the brown reaction product was localized to the nuclei. The largest
percentages of DAB-positive cells in the midgut epithelium were observed in
larvae exposed to simazine, glyphosate, myclobutanil and amitraz (>60%, Table
1). There were some incongruities between the two TUNEL techniques used to
estimate cell mortality, but these usually were orders of magnitude
differences in the data because the trends detected by both TUNEL techniques were
similar (Table 1). In general, pesticides that resulted in high levels of
apoptosis as detected by the ISDDK technique resulted in the same as detected
by the DeadEnd technique (Table 1). Notably, fluvalinate, on average,
resulted in the lowest level of apoptosis of any tested pesticide (Table 1).
The DAB reaction product was observed in columnar midgut epithelial cells
in simazine treated larvae (Fig. 1A) and in nearly all of the regenerative
cells in chlorpyrifos treated larvae (Fig. 1B). Furthermore, there were
midgut regions in amitraz treated larvae with both DAB-positive columnar and
regenerative epithelial cells (Fig. 1C), and regions in imidacloprid treated
larvae with columnar DAB-positive and regenerative negative cells (Fig.
1D). In larvae with high proportions of DAB-positive cells, the positive
cells were localized in compartmental areas of the midgut, but tissues also
were observed containing only solitary DAB-positive cells (chlorothalonil
treated larvae, Fig. 1E). In untreated larvae, ~10% of the midgut epithelial
cells were DAB-positive (Fig. 1F).
The DAB reaction product also was localized in the salivary glands and the
ovaries of treated larvae. Salivary gland tissue expressed high levels of
DAB-positive cells in larvae exposed to amitraz (Fig. 1G). Similar levels
also were found in salivary glands in simazine, imidacloprid, glyphosate,
myclobutanil or fluvalinate treated larvae. In the ovarian tissue, high
levels of DAB-positive nurse cells were found in imidacloprid-treated larvae
(Fig. 1H). In ovaries of larvae treated with the remaining pesticides, the DAB
reaction product was found in similar amounts as in ovaries of untreated
larvae. At normal tissue turnover, up to 20% of nurse cells were
DAB-positive (Fig. 1I). Negative control sections showed no presence of the DAB
reaction product, and endogenous peroxidase also was quenched successfully (Fig.
1J).
3.2. In situ cell death detection kit, AP (ISCDDK)
Twenty-four hours after honey bee larvae were exposed to the last of four
pesticide treatments, the red azo-dye reaction product was found in
increased levels of the midgut columnar-cell nuclei and also in the midgut
regenerative-epithelial cells. In chlorpyrifos-treated larvae, the level of
positive-reaction product in the columnar midgut cells (Fig. 2A) had risen to
~74% (Table 1). In simazine, myclobutanil, imidacloprid, chlorpyrifos,
chlorothalonil and glyphosate-treated larvae, the level of positive columnar
epithelial cells with red azo-dye reaction product was ≥65% (Table 1). Simazine
induced localization of red azo-dye reaction product to the columnar and
regenerative cells (Fig. 2B). In coumaphos-treated larvae, the reaction
product was found in ~48% of all columnar and regenerative epithelial cells
(Fig. 2C). The reaction product in the salivary glands was found in
myclobutanil-treated larvae, where a majority of cells were positive (Fig. 2D). In
untreated larvae, low amounts of reaction products were observed, though
sporadic cells were positive (Fig. 2E). The red azo-dye product in the ovarian
tissue of all treated and untreated control larvae ranged from 5 to 10 %
(Fig. 2F).
3.3. Immunohistochemical localization of PS
In the pesticide-treated larvae, the red azo-dye reaction product detected
by Annexin V, which characteristically localizes PS, was found to be
present abundantly in the midgut epithelium, salivary glands and ovaries. Thus,
it was possible to delineate the PS boundary at the apical columnar cell
membrane in the brush border and at the basal cell cytoplasm bound to basal
membrane by immunostaining of Annexin V. The Red azo-dye reaction product
was localized and bound to the apical brush border in chlorpyrifos- treated
larvae (Fig. 3A). Annexin V staining spread throughout the midgut epithelium
cells noticeably, where immunostaining was diffuse and the entire cell
cytoplasm of glyphosate-treated tissue was stained (Fig. 3B). In the
glyphosate-treated larvae, Annexin V was abundant and PS was localized in the basal
and apical cell cytoplasm (Fig. 3C). Staining of the cytoplasm in a group
of columnar cells at the basal area was uneven and spotty and bound to the
basal membrane in simazine-treated larvae (Fig. 3D). Red azo-dye was present
abundantly in salivary glands of mycobutanil (Fig. 3E) and ovaries of
glyphosate-treated larvae (Fig. 3F). Staining was less intensive in salivary
gland cells in untreated larvae (Fig. 3G). In untreated larvae, Annexin V
was present in some sections of the midgut epithelium and immunostainning was
bound to the apical and basal cell membrane (Fig. 3H) while the cytoplasm
of the midgut cells was not stained. Results indicate that Annexin V binds
to cells of the midgut, salivary glands and ovaries of all pesticide
treated larvae abundantly while in untreated larvae Annexin V binding was not as
evident. In both groups of control larvae, the general morphology of the
epithelium was unchanged.
4. Discussion
Honey bee larvae reared in an incubator and treated with one of nine
pesticides undergo subclinical, cellular changes that can be detected using
immunohistochemical methods. ISCDDK showed comparable levels of apoptosis with
that shown using the DeadEnd kit. Both TUNEL kits indicated induction of
DNA strand breaks after pesticide treatments and differences in apoptosis
levels in the tissue sections. There were variations in the distribution of
apoptosis, which was uneven and inconsistent. ISCDDK was found to demonstrate
DNA-fragmentation after both apoptotic and necrotic cell death [22, 23].
There were differences in apoptosis appearance in the midgut and apoptotic
cells were observed randomly in the epithelium of pesticide-treated
larvae. Normal apoptotic cell death level in the epithelium observed in both
groups of control larvae (untreated diet and acetone treated diet) was ~10%.
Observed elevated death rates in the midgut columnar cells and in ovarian
or salivary gland cells of pesticide treated larvae may be triggered by an
apoptotic pathway after pesticide application. All applied pesticides
induced significant apoptotic cell death in the larvae midgut as demonstrated
through the use of both TUNEL kits. Necrosis, which usually is caused by a
lethal accident or disease opposed to a programmed process, can be detected by
TUNEL as found in Orita et al. [33] and in previous experiments where
larvae were water-treated [10].
Interestingly, fluvalinate resulted in the lowest levels of observed cell
death of any pesticide treated larvae. Fluvalinate has been used in the
U.S. for over two decades to control Varroa destructor Anderson and Trueman,
the varroa mite. Our data suggest that honey bee larvae may have developed
some level of resistance to fluvalinate exposure. Equally interesting is
that the herbicides glyphosate and simazine and fungicides myclobutanil and
chlorothalonil induced elevated apoptotic cell death in an insect. Though
unclear how this may affect honey bees at the individual organism or colony
level, the data suggest that herbicides and fungicides cannot be presumed
innocuous to bees. Regardless, the level of stress-induced apoptosis related
to pesticide treatment in bee larvae in our experiment was comparable to
that experienced by two invasive bivalves exposed to a molluscicide [35].
In our experiment, the tested insecticides, fungicides and herbicides
induced elevated level of apoptosis in the larval midgut. In previous
experiments, lower concentrations of coumaphos applied to adult worker bees did not
trigger increased levels of apoptosis in hypopharyngeal glands compared to
that in untreated bees [20]. In contrast, honey bee larvae treated with
acaricides experienced apoptosis and stress-induced, necrotic cell deletion
[10], indicating that these different types of cell death can occur
simultaneously after exposure to pesticides [32].
Follicular maturation during oogenesis involves necrosis along with
apoptosis [36] and investigators have shown that necrosis potentially can
accompany apoptosis during normal development as shown in experiments with mouse
cell embryos [37]. Thus, necrotic and apoptotic cell death often occur
simultaneously during many pathological processes, as seen in the present
study, and during normal processes such as tissue renewal, embryogenesis, and
immune response.
In our study, we confirmed elevated levels of apoptosis in larvae treated
with pesticides. The epithelial cell nuclei remained morphologically
unchanged but became TUNEL-positive, indicating that the DNA was fragmented but
not different from neighboring cell nuclei otherwise. It is possible that
the induced larval cell apoptosis trigged by pesticide treatment in our study
may have been a reversible process in the midgut tissue, one from which
the affected larvae could recover. On the other hand, the appearance of
apoptosis may precede further tissue deletion, the development of necrosis in
the midgut cells, or cell death altogether. The TUNEL method thus is a useful
diagnostic tool to monitor subclinical changes in honey bee larvae induced
by external influences.
The apoptosis of regenerative cells observed in the basal area of the
epithelium of pesticide treated larvae may function to maintain the proper
ratio of cells in the midgut, i.e. large numbers of regenerative cells may die
to compensate for the inadequate number of epithelial cells. This apoptotic
mechanism has been suggested for Drosophila cell mechanisms which cause
dying germline and follicle cells in Drosophila ovaries [38]. Other
investigators observed that the percentage of epithelial cells labeled with
digoxigenin using the ISCDDK increased to 70% in 3-day-old larvae when to the
larvae were treated with formic acid [39]. The high cell death levels detected
using ISCDDK likely indicated accidental cell death leading to necrosis,
triggered by necrotic injury [39]. Further studies should be performed to
establish whether higher pesticide concentrations can decrease apoptosis and
increase necrosis in honey bee larvae and how this may affect clinical
symptoms or larvae mortality.
Our data indicate that Annexin V has a widespread distribution in
pesticide treated larvae being found in the midgut epithelium, salivary glands and
ovaries. Microscopic analyses on cellular localization of Annexin V would
help to obtain information on its function. Intracellular and extracellular
localizations of Annexin V have been reported in human cardiac muscle and
vary based on changes in disease states [40]. It has also been reported that
in the ischemic rat heart, Annexin V leaked from cardiac cells into the
extracellular space and that the cardiac cell membrane was stained intensely
by the anti-Annexin V antibody [41]. In our study, the varied localization
of Annexin V suggests that this protein is related closely to apoptotic
cell death in tissues of bee larvae exposed to pesticides. These findings may
contribute to a better understanding of potential cell injury during and
after pesticide exposure, especially due to the possibility of
false-negatives produced using TUNEL kits [28]. Moreover, cell death detection and
quantification can be more accurate and potential artifacts can be reduced by
using more than one assay [25].
Collectively, our data indicate that the nine test pesticides can induce
apoptosis in tissues of honey bee larvae reared in an incubator. The data
also suggest that the pesticide doses we tested were tolerable to larvae
because apoptosis likely was initiated as a protective mechanism in the midgut,
salivary glands or ovaries, though further expansion into necrosis, tissue
deletion and larval death is a potential development of these events.
Future studies will be necessary to explore the effects and modes of action of
different doses of these pesticides on larvae at the cellular and tissue
levels. The quantification of cell death could be used to monitor the
subclinical and sub-lethal effects of applied pesticides on larval tissue. Honey
bee larvae reared in vitro could be used in the future as models for
studying the effects of chemicals on living tissues at the cellular level.
Acknowledgements
We would like to thank Jeanette Klopchin and Michelle Kelley (University
of Florida Honey Bee Research and Extension Laboratory) for their
technical assistance with this project. We also thank Catherine Zettel Nalen (UF
HBREL) for editing an earlier draft of the manuscript and Michael Scharf (UF
Department of Entomology and Nematology) for assistance with pesticide
dosing. We would like to thank Prof. Dr. Elaine C. M. Silva-Zacarin
(Universidade Federal de Sa˜o Carlos (UFSCar), Campus Sorocaba, Brazil) for reading
the manuscript and useful suggestions. This work was supported by the
National Honey Board and the Florida Department of Agriculture and Consumer
Services through the work of the Honey Bee Technical Council.
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Table 1. Mortality in the midgut columnar cells determined using two
TUNEL kits, DeadEnd and ISCDDK. Data are mean ± s.e. proportion cells
positively stained, N out of at least 300 cells counted from a minimum of 3 larvae.
When both treatments were analyzed together, neither technique (DeadEnd or
ISCDDK - F = 1.1; df = 1,123; P = 0.29) nor the interaction between
technique and treatment (F = 1.6; df = 10,123; P = 0.13) affected the proportion
of cells positively stained. Data in columns followed by the same letter
are not different at P < 0.05. Students T tests were used to compare means.
Type of pesticide
Treatment ↓
Dead End technique
ISDDK technique
Both techniques analyzed together
Insecticide
Chlorpyrifos
0.56 ± 0.08, 6abc
0.74 ± 0.04, 6a
0.65 ± 0.05, 12abc
Imidacloprid
0.51 ± 0.12, 6bcd
0.76 ± 0.02, 4a
0.61 ± 0.08, 10abc
Amitraz*
0.64 ± 0.07, 5abc
0.37 ± 0.03, 4c
0.52 ± 0.06, 9c
Fluvalinate*
0.29 ± 0.04, 7d
0.32 ± 0.02, 3c
0.30 ± 0.03, 10d
Coumaphos*
0.58 ± 0.12, 8abc
0.48 ± 0.03, 5bc
0.54 ± 0.07, 13c
Fungicide
Myclobutanil
0.62 ± 0.11, 5abc
0.78 ± 0.06, 4a
0.69 ± 0.07, 9ab
Chlorothalonil
0.49 ± 0.08, 7cd
0.66 ± 0.06, 4ab
0.55 ± 0.06, 11bc
Herbicide
Glyphosate
0.74 ± 0.05, 5ab
0.65 ± 0.15, 5a
0.69 ± 0.08, 10ab
Simazine
0.76 ± 0.06, 7a
0.77 ± 0.06, 7a
0.77 ± 0.04, 14a
Control
Diet with acetone
0.10 ± 0.01, 8e
0.11 ± 0.03, 5d
0.11 ± 0.01, 13e
Diet only
0.10 ± 0.02, 8e
0.09 ± 0.02, 5d
0.10 ± 0.02, 13e
ANOVA→
F = 10.6; df = 10,71; P < 0.01
F = 16.3; df = 10,51; P < 0.01
Treatment effect: F = 22.2; df = 10,123; P < 0.01
*Used in honey bee colonies to control varroa mites
Figure 1. Staining of formalin-fixed, paraffin-embedded larvae on which
the DeadEnd colorimetric apoptosis detection system (Promega, Madison, WI,
USA) was used. Larvae were 6 d old, 24 h after the last of four consecutive
daily pesticide treatments and prepared for immunohistology. Peroxidase
conjugated anti-digoxigenin secondary antibody and DAB as a substrate were
used to obtain a specific brown reaction product. The DAB reaction product
localized to the nuclei of the columnar midgut epithelial cells is indicated
by black arrows and the DAB reaction product localized to the regenerative
cells is indicated by a black arrow head. When the DAB reaction product is
absent, either the arrow (columnar midgut epithelial cells) or arrow head
(regenerative cells) is white. Panel 1A shows the midgut, of a
simazine-treated larva. The DAB reaction product is localized to the nuclei of the
most of the epithelial columnar and regenerative cells. Panel B shows
chlorpyrifos treated larva with the DAB reaction product localized to the
regenerative epithelial cells but not the columnar ones. Panel C shows an amitraz
treated larva where the DAB reaction product was localized in columnar and
regenerative epithelial cells. Panel D shows sections of the midgut of an
imidacloprid treated larva with the DAB reaction product localized in the
nuclei of columnar cells but not in regenerative cells. Panel E shows a
chlorothalonil treated larva. The DAB reaction product was found sporadically
in the migut columnar epithelial cells. Panel F shows an untreated larva
where only ~10 % DAB positive midgut epithelial cells were found. Panel G
shows an amitraz treated larva where DAB positive and negative salivary glands
cells were seen. Panel H shows an imidacloprid treated larva with
indicative DAB staining in ovariole nurse cells. Panel I shows a control larva
where the DAB reaction was distributed sporadically in ovariole nurse cells.
Panel J shows a control section of an imidacloprid treated larva where
endogenous peroxidase was quenched successfully and enzyme incubation was
omitted. No DAB reaction product was found. Magnification of all panels: 400×.
Figure 2. Sections of formalin-fixed, paraffin-embedded, 6 d old larvae,
24 h after the last of four consecutive daily pesticide treatments. Cell
death was detected using the TUNEL technique ISCDDK (Roche). TdT-mediated
dUTP for DNA labeling was employed, followed by the application of
anti-fluorescein alkaline phosphatase conjugated antibody, using fast red for
visualization, and counterstaining with haematoxylin. Dense red azo dye staining
localized to the nuclei of the midgut epithelial cells, in the salivary gland
cells, or in ovary nurse cells indicative of impending cell death is
indicated by a black arrow. Reaction product localization to the regenerative
cells is indicated by a black arrow head. Where the reaction product is
absent, either the arrow (midgut epithelial cells) or arrow head (regenerative
cells) is white. Panel A shows a chlorpyrifos-treated larva with red azo
dye staining localized to the midgut columnar epithelial-cell nuclei but not
the regenerative epithelial cells. Panel B shows midgut epithelium in a
simazine-treated larva. The Red azo-dye reaction product localized to the
columnar and regenerative cells. Panel C shows a midgut epithelium section of
a coumaphos-treated larva. The Red azo-dye reaction product localized to
the columnar and regenerative epithelial cells. Panel D shows salivary
gland tissue of a myclobutanil-treated larva where the majority of cells were
alkaline phosphatase positive. Panel E shows untreated, control larvae
with red azo-dye reaction product to sporadic salivary gland cells. Panel F
shows the red azo-dye product sporadically in nurse cells in ovaries of a
simazine-treated larva. Magnification of all panels: 400×.
Figure 3. Immunohistochemical localization of AnnexinV cells of
formalin-fixed, paraffin-embedded 6 d old larvae, 24 h after the last of four
consecutive daily pesticide treatments.
Panel A shows the red azo-dye reaction product bound to the apical brush
border (black arrow) in a chlorpyrifos-treated larva. The reaction product
was not localized in the midgut cells (white arrow head). Panel B shows
cells from a glyphosate-treated larva and indicates Annexin V staining
throughout the remaining midgut epithelium cytoplasm (black arrow). Panel C
shows a midgut section of a glyphosate-treated larva with red azo-dye
staining indicating PS localized to the basal and apical cell cytoplasm (black
arrow head). Panel D indicates that an alkaline phosphatase reaction product
in a simazine-treated larva was localized to the cytoplasm of the columnar
cells at the basal area (black arrow head). Panel E shows red azo-dye
localized in the salivary gland tissue of a myclobutanil-treated larva (black
arrow head). Panel F shows a glyphosate-treated larva in which red azo-dye
was localized to ovarian nurse cells (black arrow head). Panel G shows no
intensive staining in the salivary gland tissue of a larva consuming
untreated food. Panel H shows cells from an untreated, control larva with red
azo-dye reaction product to some sections of the midgut epithelium bound to
the apical and basal cell membrane (black arrow head). The cell cytoplasm
of the midgut cells was not stained. Magnification of all panels: 400×.
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