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- Effects of cypermethrin as a model chemical on life cycle and biochemical responses of the tropical stingless bee Meliponula bocandei Spinola, 1853
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- Environmental Advances 5 (2021) 100074
Contents lists available at ScienceDirect
Environmental Advances
journal homepage: www.sciencedirect.com/journal/environmental-advances
Effects of cypermethrin as a model chemical on life cycle and biochemical
responses of the tropical stingless bee Meliponula bocandei Spinola, 1853
Glory U. Chibee a, Olajumoke M. Ojelabi b, Hamzat O. Fajana c, d, Bolajoko A. Akinpelu b,
Temitope O. Kehinde a, Olufemi M. Awodiran a, Efere M. Obuotor b, Olugbenga J. Owojori a, *
a
Department of Zoology, Obafemi Awolowo University, Ile-Ife, Nigeria
b
Department of Biochemistry and Molecular Biology, Obafemi Awolowo University, Ile-Ife, Nigeria
c
Toxicology Centre, University of Saskatchewan, Saskatoon SK S7N 5B3, Canada
d
Department of Soil Science, University of Saskatchewan, Saskatoon SK S7N 5A8, Canada
A R T I C L E I N F O A B S T R A C T
Keywords: The tropical stingless bee, Meliponula bocandei Spinola, 1853 is an important pollinator in West Africa but there is
West Africa no study on the effect of pesticides on this species. This study assessed the effects of cypermethrin, a common
Stingless bees pyrethroid on survival, taste (sucrose sensitivity), and biochemical responses of M. bocandei. The biochemical
Pesticides
markers were superoxide dismutase [SOD], catalase [CAT], glutathione S-transferase (GST), reduced glutathione
Ecotoxicology
[GSH]), and acetylcholinesterase [AChE]) as well as glucose, trehalose and total protein concentrations. Test
temperature was optimized by acclimatizing adult worker bees collected from a pristine natural colony to
different temperature regimes in the laboratory and fed with sucrose solution. The optimized temperature (22◦ C)
for survival and sucrose consumption was adopted for the toxicity test. The 24–48 h oral lethal dose (LD50) and
24–96 h indirect contact lethal concentration (LC50) of cypermethrin on the bees was in the range of 0.66–0.76
µg/ml and 92.24–223.69 µg/ml respectively. Also, the overall PER response reduced below 50% in bees that
were orally exposed to high doses of cypermethrin. There was significant decrease in glucose, trehalose, total
protein and GSH concentrations in bees when compared with the control. Also, significant alterations were
observed in SOD, CAT, GST and AChE activity for both oral and contact exposure pathways. In conclusion,
cypermethrin modulated the activities of biomarkers of oxidative stress and altered the level of energy metab
olites in the bees.
Introduction pesticides applied on farmlands during foraging. Stingless bees are
referred to as true generalists as they forage on vast arrays of plants
To combat food shortage and meet the world’s rising human popu (Lima et al., 2016). They are non-target organisms of contact and oral
lation, the use of agrochemicals was introduced with the intention of exposure to pesticides through aerial and soil application (Lima et al.,
improving agricultural yield. Despite the apparent influence of pesti 2016). Moreover, despite these two main modes of exposure, larva and
cides and fertilizers on increasing crop yield, the associated hazard to other castes of the colony can be affected by the contaminants when
the environment and especially to non-target species is worrisome when contaminated pollen and nectars are relocated from contaminated areas
used indiscriminately. This, among other effects, has resulted in the to their hives (Pham-Delegue et al., 2002). This can have adverse effects
decline of pollinators whose ecosystem services enhance the provision of on the individual or colony and sublethal effects on bees’ behaviour,
food and other hive products, thereby affecting agricultural productivity development, longevity, and immune system (Thompson, 2003; Des
globally (Kwapong et al., 2010; Pashte and Patil, 2017). neux et al., 2007).
Bees are non-target arthropods exposed continuously to pesticides Pesticide use varies globally, but there has been a surge in pesticide
either by direct or indirect contact on farmlands (McArt et al., 2017). use in developing countries in recent times. In Nigeria, pesticide use
Some species of bees have a well-organized worker caste system, a social ranges between 12500-13000 tonnes yearly (Asogwa and Dongo, 2009)
structure that makes them susceptible to unintended exposure to and commonly used pesticides include pyrethroids, organophosphate,
* Corresponding author.
E-mail address: gowojori@oauife.edu.ng (O.J. Owojori).
https://doi.org/10.1016/j.envadv.2021.100074
Received 17 May 2021; Received in revised form 28 May 2021; Accepted 2 June 2021
Available online 6 June 2021
2666-7657/© 2021 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
- G.U. Chibee et al. Environmental Advances 5 (2021) 100074
carbamates, neonicotinoids, organochlorines, insect growth regulators Experimental procedures
and chlorinated cyclodienes (Asogwa and Dongo, 2009; Natala and
Ochoje, 2009; Akinwande et al., 2013). In most parts of the world, there The study was in two phases. The first phase involved the optimi
has been a shift towards more recent pesticide classes such as neon zation of temperature for the survival and sucrose consumption of
icotinoids and synthetic pyrethroids (FAO, 2010), although some M. bocandei in the laboratory. The second phase assessed cypermethrin’s
neonicotinoids (clothianidin, imidacloprid and thiamethoxam) now toxicity on the stingless bees at the optimized temperature for survival
have restricted use in Europe (Regulation (EU) No 485/2013). The and sucrose consumption.
synthetic pyrethroid, cypermethrin, is a commonly used broad-spectrum
neurotoxic insecticide (USEPA, 2006). It is used in Nigeria as an insec Optimization of test conditions
ticide targeted for pests on cocoa, vegetables, and other food products
(Bateman, 2008). There are indications that cypermethrin affects the Thirty (n = 30) adult worker stingless bees were sorted into a plastic
survival, reproduction, and learning potential of honeybees (Taylor cage designed to minimize handling stress on the bees. The stingless bees
et al., 1987, Desneux et al., 2007, Decoutye et al., 2005). While there is a were then acclimated to different temperature regimes (18, 20, 22, 25
lot of literature on pesticides’ effect on honey bees, only limited data and 28 ◦ C) in the climate chamber (Memmert GmbH, Germany) for 14
exist for stingless bees. Most of these data are from the neotropical re days, and the test for each of the temperature regimes was run in trip
gions of the world like Brazil and the southern part of Australia (Lima licates. The survival and sucrose (50% w/v) consumption of the stingless
et al., 2016). bees in response to the different temperature regimes were assessed. The
Meliponula bocandei is a stingless bee species common in tropical amount of sucrose consumed was determined using a 2 ml syringe of
regions (Fabre Anguilet et al., 2015). The tropical stingless bee has been known weight (Abou-Shaara et al., 2012). The weight of sucrose
studied extensively in Africa, focusing on their biology and ecology, but consumed was then calculated by subtracting the syringe’s initial weight
little information exists on the hazards posed by pesticides on them. with sucrose solution from the final weight.
Moreover, the effect of pesticides on bees and non-pest insects has been
poorly studied in Africa (Akinwande et al., 2013). Although test pro
tocols for honey bees (Apis mellifera) exist (Medrzycki et al., 2013), Toxicity of Cypermethrin on M. bocandei
toxicity test protocols for the toxic assessment of contaminants on
stingless bees are not currently available. To correctly assess the harmful Test chemical
effect of pesticides on tropical arthropods in the laboratory, test condi The insecticide selected for this study is a synthetic pyrethroid, 10%
tions need to be optimized (Owojori et al., 2019). EC with cypermethrin as the active ingredient (100 g/L a.i). The
Temperature is a critical factor affecting insect development, chemical used was manufactured in India by Meghnami Organics
behaviour, and growth because insects need to regulate their internal Limited.
body temperature with ambient temperature
(Abou-Shara et al., 2012). Temperature tolerance is one condition to Experimental conditions
be ascertained for a toxicity test to be valid under laboratory conditions The experimental procedure used in this study were in accordance
(Medrzycki et al., 2013). Since stingless bees (M. bocandei) are social with OECD guideline (1998) for honey bees. Ten (N = 10) adult worker
insects, acclimating them to the laboratory environment is crucial for an bees were placed in plastic cages inside a climate chamber at a tem
ecotoxicity study. Moreover, in an insect’s body, essential macromole perature of 22 ◦ C and relative humidity of 60%. For each test exposure, a
cules such as carbohydrates, lipids and proteins are closely linked with control experiment was set up, and all tests were done in triplicate (n =
critical metabolic processes. The susceptibility of insects to pesticides 3).
may reflect these macromolecules’ alteration (Piri et al., 2014; Xu et al.,
2016). Mode of exposure
This study aimed to assess the toxicity of the synthetic pyrethroid
cypermethrin on the workers of tropical stingless bee, M. bocandei. The Indirect contact test. Worker bees were exposed to different cypermeth
specific objectives were to provide information on the optimal temper rin concentrations (400 mg/L, 200 mg/L, 100 mg/L, 50 mg/L and 25
ature for rearing M. bocandei in the laboratory and the lethal and sub mg/L) prepared from the stock solution (100 g/L of cypermethrin).
lethal effects of cypermethrin on the insect. Endpoints selected included These concentrations were chosen based on a range-finding test (Data
survival, sucrose consumption and biochemical responses. not shown). The dosing was carried out by drenching uncontaminated
filter papers with cypermethrin at the different concentrations, while for
Materials and methods the control test, the filter paper was drenched with distilled water. Both
the control and treated filter papers were air-dried for 5 min before bees
Collection of M. bocandei were allowed to walk on the filter paper in a cage exposure for four
hours, after which the filter papers were removed. This was done to
Adult workers of the stingless bees, M. bocandei were collected from mimic field-realistic indirect contact exposure. During this experiment,
two healthy natural colonies in the botanical garden of the Obafemi the bees were fed with 50% (w/v) of sucrose solution and housed in a
Awolowo University, Ile Ife, Nigeria. Due to increasing threats and climate chamber at the optimum temperature (i.e., 22 ◦ C) obtained from
apparent conservation pressures, bees were collected from two colonies the optimization test. The mortality of the bees was monitored from 24
to limit potential colony loss that often results from frequent visits to the to 96 h (4 days) post-exposure.
colonies (Kerr et al., 2001). Bees were collected using a modified
bologna trap that was attached to a wooden cage of dimension 29.5 cm Chronic oral exposure
× 27.5 cm × 40.5 cm. The botanical garden is pristine, with no history of
agrochemical usage. Adult worker bees (foragers) are most vulnerable to Worker bees were continuously fed for 10 days with 50% w/v su
pesticide exposure in the wild (Alburaki et al., 2017). The bees were crose solution dosed with 0.088 mg/L, 0.176 mg/L, 0.352 mg/L and
transported to the laboratory, acclimated for a few hours at room tem 0.528 mg/L of cypermethrin. The definitive test concentrations were
perature, fed sucrose solution (50% w/v), and transferred into a climate selected after a range-finding test via oral exposure of the bees to
chamber at 22 ◦ C. cypermethrin (0.01–1 mg/L) for 10 days (Data not shown). The median
lethal dose (LD50) of cypermethrin was then estimated for ten days of
exposure. During this exposure, feeders were changed daily for both the
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- G.U. Chibee et al. Environmental Advances 5 (2021) 100074
control and experimental cages. prepared under the same conditions, in the absence of the enzyme; the
trehalose amount was corrected for the glucose content in these samples.
Gustatory responsiveness (Proboscis extension reflex-PER)
Protein concentration
Out of a total of 140 bees surviving at the end of the ten days chronic
oral exposure, only 112 responded when probed with water and these The gut homogenate (20 µl) was added to 200 µl of alkaline copper
were the ones used in the gustatory responsiveness test. The test was reagent, which was freshly prepared by mixing 2% (w/v) Na2CO3 in 0.1
done following McCabe et al. (2007) by touching the antenna of the bees M NaOH, 0.5% (w/v) CuSO4.5H2O and 1% Na-K tartrate.4H2O in a
selected with sucrose solution in increasing order of concentrations (0.5, volumetric ratio of 98:1:1 v/v/v. The mixture was vortexed and allowed
1, 3, 5, 10, 30, and 50% w/v) while the control received only distilled to stand for 10 min, followed by the addition of 20 µl of Folin-Ciocalteau
water. This was done to evaluate the effect of the pesticide on gustatory colour reagent. The resulting reaction mixture was vortexed and allowed
responsiveness at different sucrose concentrations. The PER was esti to stand at room temperature in the dark for an hour. Absorbance was
mated as the number of bees that responded when the antenna was read at 660 nm against a reagent blank (same as the test sample except
elicited with the varying sugar concentrations. Before this procedure, 20 µl distilled water instead of the homogenate. The protein concen
bees were starved for four hours to exclude effects of thirst or habitua tration of the test samples was extrapolated from a standard curve ob
tion. Also, water was offered before every tested sugar solution. A full tained using Bovine Serum Albumin (BSA) (Lowry et al., 1951).
extension of the proboscis after touching the antenna was considered a
positive response to the stimulus, and bees not responding to water or Estimation of biomarkers of oxidative stress
sugar concentration were not considered for PER estimation.
Reduced Glutathione [GSH]
Biochemical analyses The gut homogenate (1 ml) was added to 0.5 ml of Ellman’s reagent
(10 mM), and 2 ml of phosphate buffer (0.2 M, pH 8.0) was added. The
The biochemical parameters were selected to reflect the biomarker of yellow colour that developed was read at 412 nm with a blank (0.5 ml of
oxidative stress and energy metabolism in the bees. Superoxide dis Ellman’s reagent and 3 ml phosphate buffer). Glutathione was used as
mutase (SOD), Catalase (CAT), Glutathione peroxidase (GPx) were standard. The GSH level was extrapolated from the glutathione standard
measured to assess sublethal doses of cypermethrin on oxidative stress; calibration curve, and the amount of GSH was expressed in mg/weight
Glutathione S-transferase on detoxification of the pesticide, acetylcho of homogenate, according to Beutler (1963).
line esterase on possible impairment of neurological response. Glucose,
trehalose as well as total protein were measured as indicators of energy Superoxide Dismutase [SOD] activity
metabolism. About 20 µl of the gut homogenate was added to 30 mM EDTA in 75
mM Tris-HCl buffer (pH = 8.2) and 30 µl of 2 mM of pyrogallol, and then
Preparation of M. bocandei Homogenate properly mixed. Then the absorbance was measured in a uv/vis spec
trophotometer at 420 nm for 3 min. One unit of enzyme activity rep
A total of 140 adult worker bees (from parallel exposure) were used resents 50% inhibition of the rate of auto-oxidation of pyrogallol as
for the biochemical tests which was carried out after the PER test since determined by change in absorbance/min at 420 nm. The activity of
the bees used for the PER would not be suitable for the biochemical test. SOD was expressed as units/mg protein (Marklund and Marklund,
They were anesthetized after the indirect contact and oral exposure to 1974).
cypermethrin and dissected with the head excised separately. The
weight of the gut and head were obtained and homogenized for 20 Catalase [CAT] activity
minutes at 4 ◦ C in 5 ml of 10% (w/v) extraction solution (40 mM The gut homogenate, 50 μl was added to 450 μl phosphate buffer
phosphate buffer pH 7.4 containing 10 mM NaCl) (Carvalho, 2010). The (0.1 M, pH = 7.0) and 500 μl of 20 mM H2O2 in a 1 ml cuvette. The
bee samples from the control group were prepared in the same manner. absorbance was taken at 240 nm at an interval of 15 s for 1 min using a
The supernatants were carefully collected into vials and stored at -20 ◦ C spectrophotometer. The molar extinction coefficient of H2O2 (0.0436
for further biochemical analysis. M− 1 cm− 1) was used to determine the catalase activity which is
expressed as units per milligram of protein (Aebi, 1984).
Estimation of energy metabolites
Energy metabolites (glucose, trehalose, and protein) were quantified Glutathione S-transferase [GST] activity
in the bees’ gut homogenate because of the difficulty in getting hae The reaction mixture, 1 ml containing 0.1 M phosphate buffer at pH
molymph from the bees due to their small size. of 6.5, 1 mM GSH, and 1 mM 1-chloro-2,4-dinitrobenzene (CDNB) was
added to the homogenate and vortexed gently. The absorbance of the
Glucose concentration mixture was measured at 340 nm at 15 s intervals for 3 min. Distilled
water was used as a blank (Habig et al., 1974).
Glucose concentration in the gut homogenate (in phosphate buffer of
pH = 7.0) was estimated using the Randox glucose kit (Randox Labo Acetylcholinesterase [AChE] activity
ratories Ltd, Antrim, UK), and the absorbance was measured at 546 nm A mixture of 240 µl of phosphate buffer (0.1 M at pH=8.0) and 20 µl
using glucose solution as standard at a baseline concentration of 5.44 of the bees’ head homogenate was incubated in a dry bath incubator for
mmol/l. 30 min at 37 ◦ C, after which 20 µl of 25 mM acetylthiocholine iodide
(ATChl) and 20 µl of 10 mM 5,5′ -dithiobis-2-nitrobenzoic acid (DTNB)
Trehalose concentration were added to initiate the reaction. Then, the change in absorbance at
30 s intervals was measured for 4 min at an absorbance of 412 nm
Trehalose concentration was determined according to the method of (Ellman et al. 1961).
Santos et al. (2008). Aliquots (50 µl) of the gut homogenate were incu
bated with trehalase (0.1 U) (EC 3.2.1.28; Sigma Chemical Co., St. Louis, Data analyses
MO) in 200 ul phosphate buffer (40 mM, pH 5.5) for 4 h at 40 ◦ C. After Survival data was expressed as a percentage (n = 3 replicates) prior
incubation, the glucose generated from trehalose was determined as to use for dose-response analysis. The median lethal concentrations of
described above in the estimation of glucose content. The blank was cypermethrin for contact (LC50) and oral exposure (LD50), were
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- G.U. Chibee et al. Environmental Advances 5 (2021) 100074
calculated in R using the package MASS (Ripley et al., 2013). The LD50 Sublethal effect of cypermethrin on the stingless bees
of cypermethrin (in µg/ml) for oral exposure was converted to a.i/bee by
multiplying LD50 by the average volume of sucrose consumed per bee, i. Behavioural response
e., 0.022 ml. Proboscis extension reflex (PER) was expressed as a per An olfactory behavioural response, Proboscis Extension Reflex
centage response to sucrose relative to the control response. A one-way (PER), was conducted on the stingless bees that survived the chronic oral
analysis of variance (ANOVA) was used to determine significant differ exposure to cypermethrin. For the bees fed with increasing sucrose
ence at 5% probability level for (i) the effect of temperature on sucrose concentration without any pesticides, the PER increased as the amount
consumption of the bees and (ii) the effect of varying sublethal con of sucrose given increased (50% response in 0.5% and peaking at 86%
centrations of cypermethrin on certain energy metabolites and enzy response in 50% w/v sucrose). The PER of the bees to 0.5%, 1% w/v of
matic activities of the bees. Tukey post-hoc test was used for pairwise sucrose relative to the control response for each group, followed a dose-
comparison of treatment with the control. response pattern with the highest effect on PER (40% and 37% relative
to control for 1 and 0.5% respectively) recorded for bees exposed to the
Results highest concentrations (Fig. 3). This pattern was less evident at higher
sucrose concentration, suggesting a probable sucrose oversaturation
Influence of temperature on the survival and sucrose consumption of masking the pesticide effect (Fig. 3).
stingless bees
Biochemical response of stingless bees to cypermethrin
The highest average survival of bees (>80%) was observed at 22 ◦ C
from day 3 to 14 of acclimation of the bees to laboratory conditions at Indirect contact acute toxicity. The biochemical responses of stingless
60% relative humidity (Fig. S1 supplementary information). The sur bees to concentrations that were in the range of the 96 h acute toxicity,
vival at 20 ◦ C was the next highest survival but decreased below 80% LC50 = 92.24 (66.59–127.78) µg/mL of cypermethrin via indirect con
(cut-off for optimum survival) from day 3 of acclimation. Therefore, tact was assessed for energy metabolites, oxidative stress biomarkers,
22 ◦ C appeared to be the optimum temperature for the survival of and acetylcholinesterase (AChE) activities (Fig.s 4 and 5). Cypermethrin
M. bocandei in the laboratory. significantly (Tukey test: p < 0.05) reduced the concentration of glucose,
Sucrose consumption by the stingless bees was significantly higher at trehalose, and total protein after a 96 h exposure via indirect contact
22, 25, and 28 ◦ C compared to 18 and 20 ◦ C at 3 days (Tukey test: F(4,15) (Fig. 4). However, an increase in reduced glutathione [GSH] concen
= 15.37, p < 0.001) (Fig. 1a). At day 14 of acclimation to laboratory tration was observed, but the increase was not significant (Fig. 4d).
condition, only bees from 20 and 22 ◦ C survived; however, sucrose Cypermethrin had no significant effect on the activity of superoxide
consumption was significantly higher at 22 ◦ C (paired t-test: p = 0.036) dismutase (SOD) of bees that were exposed to cypermethrin via indirect
(Fig. 1b). contact for 96 h (Fig. 5a) but significantly (p < 0.001) induced the ac
tivity of catalase enzyme (CAT) in the stingless bees at the highest
Acute and chronic toxicity of cypermethrin on stingless bee survival at exposure concentration, 113.53 µg/ml (Fig. 5b). The activity of
22 ◦ C glutathione-S-transferase (GST) in the bees significantly increased at the
intermediate dose of cypermethrin (Fig. 5c). Also, there was a significant
The stingless bees, M. bocandei was exposed to cypermethrin at the dose-dependent increase in the activity of acetylcholinesterase (AChE)
optimum temperature, 22 ◦ C. Cypermethrin via indirect contact had of the stingless bees, thus indicating an enzymatic induction in response
acute toxicity on the stingless bees at 24, 48, 72 and 96 h of exposure to cypermethrin (Fig. 5d).
(Fig. 2). However, the highest acute toxicity on the bees was at 96 h with
an acute median lethal concentration (LC50) value of 92.24
Oral chronic toxicity
(66.59–127.78) µg/ml (Fig. 2d). The predicted chronic LD50 of cyper
methrin on the bees was not much different at 24 h, and 240 h and the
Cypermethrin via oral exposure significantly reduced the concen
latter values were 0.97 (0.26–3.65) µg/ml of 50% w/v sucrose, equiv
tration of glucose, trehalose, and total protein of the stingless bees,
alent to 0.021 µg a.i/bee (Fig. S2 of supplementary information).
especially at the highest dose, 0.53 µg/ml in sucrose after 240 h (Fig. 6a-
c). There was a significant decrease in the concentration of GSH at in
termediate doses of cypermethrin, but GSH at the highest dose was not
significantly different from control (Fig. 6d).
Fig. 1. Sucrose consumption of stingless bee, Meliponula bocandei at (a) 3 days (b) 14 days of acclimation to laboratory conditions at 60% RH. Bars with different
letters are significantly different at p < 0.05.
Note: Sucrose consumption at 14 days was only estimated for 20 and 22 ◦ C because the bees did not survive at other temperature regimes.
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Fig. 2. Dose-response for (a) 24 h (b) 48 h (c) 72 h and (d) 96 h acute toxicity of cypermethrin on the survival of the stingless bees, Meliponula bocandei via an indirect
contact exposure at an optimum temperature of 22 ◦ C in the laboratory.
Fig. 3. Proboscis Extension Reflex (PER) of cypermethrin-dosed stingless bees, M. bocandei to different concentrations of sucrose.
There was a significant inhibition of SOD activity of bees that were Discussion and Conclusion
exposed to cypermethrin via oral contact for 240 h (Fig. 7a) but an in
duction in the activity of catalase enzyme (CAT) at the highest exposure Optimization and sucrose consumption
dose, 0.53 µg/ml (Fig. 7b). The activity of glutathione-S-transferase
(GST) in the bees significantly reduced at intermediate doses of cyper This study has provided baseline data for using the stingless bees
methrin (Fig. 7c). There was a significant inhibition in acetylcholines M. bocandei in ecotoxicity assessment in the tropics. The optimum
terase activity (AChE) in the stingless bees (Fig. 7d). temperature for the survival of M. bocandei during acclimation to lab
oratory conditions was 22 ◦ C. The temperature selected for assessing
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Fig. 4. Toxicity of cypermethrin on the concentration of (a) glucose (b) trehalose (c) total protein, and (d) reduced glutathione (GSH) of the stingless bees, Meliponula
bocandei after a 96 h exposure to cypermethrin. *p < 0.05, **p < 0.01 significance, (Error bars are standard error).
optimal temperature (20-28 ◦ C) in our study is not extreme. In fact, warmer nectar, and their sucrose consumption increased with an in
similar studies in Africa on same species indicated a temperature of crease in temperature (20–35 ◦ C) irrespective of sucrose concentration.
26 ◦ C and 70% relative humidity was used to maintain M. bocandei in the In addition, the ambient temperature was found to influence the sting
laboratory (Bobadoye et al., 2016). Stingless bees need a higher tem less bee, T. carborania’s preference for warm nectar, and the preference
perature at flight than when resting in the hives (Kajobe and Echa increased with an increase in temperature (Norgate et al., 2010). In our
zarreta, 2005). Therefore, the 22 ◦ C, which is lower than the ambient air study, the optimum acclimation temperature, i.e., 22 ◦ C, supported the
temperature in the study area, is probably the bees’ resting temperature survival and sucrose consumption of M. bocandei in the laboratory and is
during acclimation in the laboratory and their hives. The optimum therefore recommended for future tests.
temperature found for M. bocandei in this study is similar to other
stingless bees found elsewhere in Africa. Kajobe and Echazarreta (2005) Acute toxicity
showed that the African stingless bees, Meliponula ferruguinea and
M. nebulatathe were found to have high adult mortality at a low tem The 24 and 96-h acute toxicity of cypermethrin on M. bocandei (LC50
perature range of − 10 to 2 ◦ C; however, mortality dropped and of 223.69 and 92.24 µg/ml) via indirect contact is now reported for the
remained stable at 5 to 36 ◦ C. Also, pupae mortality of the tropical first time. Although there is no data for cypermethrin’s toxic effect on
stingless bees (Melipona colimana, M. beecheii, and Scaptotrigona hellwe other similar species, data available for deltamethrin on
geri) was minimal at 25 ◦ C (Macías-Macías et al., 2011). Although M. quadrifasciata via indirect contact showed a lower LC50 value of 5.6
experimental bees were limited to two colonies, due to increasing (4.4–6.9) µg/ml for 24 h (Del Sarto et al., 2014). In both cases, these
threats and conservation pressures, the findings here provide timely values were expectedly higher than those obtained for acute oral
information on the potential of M. bocandei for ecotoxicity assessment toxicity. Since the exposure was via indirect contact, the bees were only
and behavioral response of the bees to pesticides. passively exposed without topical contact. However, the indirect contact
Sucrose consumption is a relevant sublethal endpoint for assessing exposure route mimics possible exposure in the field after hours of
the effect of pesticides and other chemicals on bees (Decourtye et al. spraying flowers with pesticides; thus, the bees are accidentally exposed
2005). Sucrose consumption by M. bocandei within 72 h seemed to be to the pesticide residues on the surface of flowers. Cypermethrin takes
favoured by test temperature between 22–28 ◦ C. However, because of an average of 1 to 3 days to dissipate from vegetation (Chai et al., 2009).
high bee mortality at the higher tested temperatures of 25 and 28 ◦ C at Therefore, there is a window of exposure of bees to cypermethrin via
14 days, 22 ◦ C, which is the optimum temperature for survival, seemed indirect contact, which could cause acute toxicity, with a predicted LC50
to be the optimal temperature for sucrose consumption in our study. Our of 92.24 µg/ml within the dissipation time.
result is similar to the findings of Nicolson et al. (2013). They reported The acute oral toxicity with 24 h LD50 of 0.017 (0.013–0.021) µg a.i/
that the honeybee, Apis mellifera scutellata has a higher preference for bee found in this study is similar or lower than those reported for similar
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Fig. 5. Toxicity of cypermethrin on the activities of the enzymes (a) superoxide dismutase [SOD] (b) catalase [CAT] (c) glutathione S-transferase [GST] and (d)
acetylcholinesterase (AChE) of the stingless bees, Meliponula bocandei after a 96 h exposure to cypermethrin. *p < 0.05, **p < 0.01 significance.
species and pyrethroids. For example, 24 h acute oral toxicity of delta hypoglycemia) in the treated stingless bees could be attributed to
methrin to M. quadrifasciata was 0.082 (0.065–0.097) µg/bee whereas 5 glucose being channeled to the hemolymph to compensate for energy
h LD50 of 0.014 (0.011–0.016) µg/bee of deltamethrin was reported for a crisis due to the toxicity of the pesticide (Surendra, 2002). This also
neotropical stingless bee, Partamona helleri (Tome et al., 2015). Cyper suggests glucose, a significant source of carbohydrate reserve, as prob
methrin and permethrin have 24 h acute LD50 values of 0.070 and 0.072 ably the first energy source to be deployed by the bees for the pesticide
µg a.i/bee respectively on the stingless bees, Trigona spinipes (Macieira detoxification process (Moolman et al. 2007).
and Hebling-Beraldo, 1989). However, PEC of 0.3 mg/kg (based on a The depletion of trehalose suggests breaking down of trehalose to
single application) was reported as the predicted environmental con generate more glucose for metabolic processes needed for adaptive
centration for tropical soil (Jegede et al., 2017). Suppose the PEC in soil response (Ali et al., 2014). Change in trehalose concentration has been
could be as low as 0.3 mg/kg. In that case, our data for 24 h oral toxicity reported as one of the strategies by which insects under stressful con
could suggest that M. bocandei may be relatively very sensitive to py ditions meet energy demand (Kalita et al., 2016). Our study has rela
rethroids in the environment compared to similar species and may be a tively shown that sublethal exposure of the stingless bees to
suitable bioindicator of pesticide pollution in the region. cypermethrin impaired glucose and trehalose synthesis, which might
likely disrupt the bees’ energy metabolism. However, further studies via
PER/Behavioral response expression of mRNA transcript abundance for important energy meta
bolism enzymes in the glycolytic pathway would elucidate how cyper
In this study, cypermethrin impaired the olfactory response indi methrin induces changes in energy metabolism in the stingless bees.
cated by the proboscis extension reflex (PER) of the stingless bees up to The significant decrease in total protein concentration of
63% at the highest cypermethrin concentration. This is contrary to cypermethrin-treated M. bocandei via oral and indirect contact exposure
Decourtye et al. (2005), who reported reduced sucrose sensitivity of is quite an indication of compensatory physiological mechanisms of the
49% and 39%, respectively for fipronil and endosulfan when Apis mel bees to provide intermediates tricarboxylic acid (TCA) cycle during
lifera were offered but found no effect for cypermethrin even at the insecticidal stress conditions, as observed by Nath et al. (1997) in silk
highest concentration tested. Only a few studies are available for com worm, Bombyx mori L after exposure to organophosphate pesticides.
parison, so it is unclear if these differences can be attributed to differ These changes in energy metabolites have also been reported to
ences in species alone. interfere with critical physiological activities such as thermoregulation
(Tosi et al., 2016), locomotion (Tosi and Nieh, 2017) and flight (Tosi
Biochemical response to cypermethrin toxicity et al., 2017) of bees, especially in honey bees.
The depletion of GSH in the treated M. bocandei during chronic oral
In this study, the treatment of M. bocandei with cypermethrin via oral and contact exposure at intermediate cypermethrin concentrations
or indirect contact at sublethal doses triggered a reduction in glucose could be attributed to the detoxification process by GSH metabolism
and trehalase concentration. The decrease in glucose concentration (i.e., enzymes such as glutathione S-transferase (GST). GSH level decreases
7
- G.U. Chibee et al. Environmental Advances 5 (2021) 100074
Fig. 6. Toxicity of cypermethrin on the concentration of (a) glucose (b) trehalose (c) total protein, and (d) reduced glutathione (GSH) of the stingless bees, Meliponula
bocandei after a 240 h chronic exposure to cypermethrin in 50% w/v of sucrose solution. *p < 0.05, **p < 0.01 significance.
when xenobiotics are removed through direct conjugation via GST or 2008; Badiou-Beneteau et al., 2012; Arora et al., 2017). The significant
glutathione (Farag et al., 2010). Reduced glutathione (GSH) is a critical inhibition in AChE activity of M. bocandei during oral exposure to sub
determinant of tissue susceptibility to oxidative damage, thus, the first lethal concentrations of cypermethrin (Fig. 7d) shows that cypermethrin
line of defence against free radicals. More GSH generated after 96 h was binds with the enzyme to prevent the breakdown of acetylcholine. The
probably to eliminate the high concentration of free radicals produced potent inhibitors of AChE are organophosphate and carbamate pesti
due to more prolonged exposure to the pesticide. cides, but synthetic pyrethroids such as cypermethrin have also been
The inhibition of GST activity might be as a result of the interaction reported to inhibit the activity of AChE in the brain of rats (Rao and Rao,
of the pesticides with cell metabolism (Otitoju and Onwurah, 2007) or 1995). Contrary to oral exposure, we observed a significant increase in
direct binding of pesticides with GST (Mecdad et al., 2011). GST con AChE for bees via contact exposure to cypermethrin (Fig. 6d). Other
tributes to cellular protection against oxidative damage (Hayes et al., studies have also reported elevated AChE activity in honeybees exposed
2005). to deltamethrin, a synthetic pyrethroid (Badiou and Belzunces, 2008)
The significant reduction in the SOD activity of the stingless bees that and a mixture of four organophosphate pesticides (Al Naggar et al.,
are orally exposed to sublethal concentrations of cypermethrin may be 2015).
attributed to the production of excess reactive oxygen species (ROS) and
intense SOD reutilization (Min and Ju-Chan, 2008). Pesticides such as Conclusion
synthetic pyrethroids cause ROS-mediated oxidative damage in bees and
other insects and trigger an imbalance in anti-oxidant levels (Chakra This study reports the lethal concentrations (LC50 – contact exposure
barti et al., 2015). A significant rise in the activities of anti-oxidants, and LD50 – oral exposure) of cypermethrin for M. bocandei for the first
such as SOD, are often initial signs of adaptive responses to oxidative time. Furthermore, the results showed that while sublethal effects were
stress (Oruc and Usta, 2007). slightly noticeable from behavioural responses, underlying biochemical
The significant induction of catalase (CAT) activity in the stingless responses were significantly impaired even at low concentrations of the
bees at the highest cypermethrin concentration for both oral and contact pesticide. Although conservation pressures limited the use of more
exposure in our study may be because of the conversion of ROS elements colonies for the current study, this study has provided toxicity data for
and peroxide radicals to water. Similar results were reported for the cypermethrin as a model chemical and baseline for other ecotoxico
bees, Apis dorsata and A. cerana from a high-intensity cropping area with logical studies on other pollinators in the region. This provides timely
elevated pesticide loads (Chakrabarti et al., 2015). information to guide policy on the protection of this important social bee
Acetylcholinesterase (AChE) is an important enzyme involved in the taxon.
hydrolyses of the neurotransmitter acetylcholine when insects are
exposed to organophosphates and carbamates (Badiou and Belzunces,
8
- G.U. Chibee et al. Environmental Advances 5 (2021) 100074
Fig. 7. Toxicity of cypermethrin on the activities of the enzymes (a) superoxide dismutase [SOD] (b) catalase [CAT] (c) glutathione S-transferase [GST] and (d)
acetylcholinesterase (AChE) of the stingless bees, Meliponula bocandei after a 240 h exposure to cypermethrin in 50% w/v of sucrose solution. *p < 0.05, **p < 0.01
significance.
Declaration of Competing Interest Naggar, Al, Y., Wiseman, S., Sun, J., Cutler, C., G., Aboul-Soud, M., Naiem, E., Mona, M.,
Seif, A., Giesy, J.P., 2015. Effects of environmentally-relevant mixtures of four
common organophosphorus insecticides on the honey bee (Apis mellifera L.).
The authors declare that they have no known competing financial J. Insect Physiol. 82, 85–91.
interests or personal relationships that could have appeared to influence Ali, S.N., Ali, S.S., Shakoori, A.R., 2014. Biochemical response of malathion-resistant and
the work reported in this paper. susceptible adults of Rhyzopertha dominica to the sublethal doses of deltamethrin.
Pak. J. Zool. 46 (2), 853–861.
Arora, S., Balotra, S., Pandey, G., Kumar, A., 2017. Binary combinations of
Acknowledgments organophosphorus and synthetic pyrethroids are more potent acetylcholinesterase
inhibitors than organophosphorus and carbamate mixtures: An in vitro assessment.
Toxicol. Lett. 268, 8–16.
This research is supported by the Nigerian Tertiary Education Trust Asogwa, E.U., Dongo, L.N., 2009. Problems associated with pesticide usage and
Fund (TETFUND) grant (TETF/DR&D/CE/NRF/STI/14) that was jointly application in Nigerian cocoa production: a review. Afr. J. Agric. Res. 4 (8),
awarded to B.A.A; T.O.K; O.M.A; E.M.O and the Alexander von Hum 675–683.
Badiou, A., Belzunces, L.P., 2008. Acetylcholinestaerse a pertinent biomarker to detect
boldt (AvH) Foundation, Bonn, Germany Research Grant (GA-Nr 19048) exposure of pyrethroids? A study case with deltamethrin. Chem. Biol. Interact. 175,
that was awarded to O.J.O. 406–409.
Badiou-B´ en´eteau, A., Carvalho, S.M., Brunet, J.L., Carvalho, G.A., Bulet´e, A., Giroud, B.,
Belzunces, L.P., 2012. Development of biomarkers of exposure to xenobiotics in the
Supplementary materials honey bee Apis mellifera: application to the systemic insecticide thiamethoxam.
Ecotoxicol. Environ. Saf. 82, 22–31.
Supplementary material associated with this article can be found, in Bateman, R., 2008. Pesticide use in Cocoa. A Guide for Training, Administrative and
Research Staff, 1st edition. ICCO/IPARC, London/Ascot, UK, p. 56.
the online version, at doi:10.1016/j.envadv.2021.100074.
Beutler, E., 1963. Improved method for the determination of blood glutathione. J. Lab.
Clin. Med. 61, 882–888.
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