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appetitive floral odours prevent aggression in honeybees
by：Bestway
2020-02-19
Bees actively defend against intruders and release an effective alarm information element to recruit young birds for defensive missions.
Although these olfactory cues are linked to the biology of bees, the effect of floral flowers on this behavior has never been studied.
Here, we use a new analytical approach to study social and olfactory cues that drive bee defense behavior.
We show that social interaction is necessary in order to reveal the recruitment function and specific floral fragrance of alarm informationlinalool and 2-phenylethanol—
There is an amazing ability to stop hiring through alarm information.
This effect is not due to the smell of flowers that mask the sex hormones, but is related to their appetite values.
In addition to their potential applications, these findings provide new insights into how bees make decisions to engage in defense and how conflict information affects the process.
In all experiments, bees were collected from several unrelated bee populations, except for field trials ()
University of Queensland St. Lucia campus (
Brisbane, Queensland, Australia)
, April 2013 to October 2014, excluding winter months-June to August.
During the experiment, all colonies were free to feed and received routine beekeeping tests and honey collection.
The same number of bees from four different groups took part in the competition.
On sunny days, bees are caught in two rounds (
About 0930 and 1100 h per colony)
To ensure that any colony is disturbed in a manner that does not exceed once every 48 hours.
This delay makes the hive completely stable between disturbances, and in fact, no increase in aggression has been observed over time.
Selection of BEE groups participating in group defense (
Guards and soldiers)
The bee waved a large black feather in front of the entrance to the hive and collected it for a few seconds.
Once the feather is covered by about 30-40 attack bees, it is quickly put into a sealed plastic bag and in a refrigerator at 20 °c.
Check the status of the bees after 5 minutes, then check every 1-2 minutes until they are not moving (on average 8.
25 minutes in the fridge).
Choose to restore the fastest Bee to put in a 50 ml syringe alone or in pairs (Terumo)
Contains wet tissue and three drops of sugar (
50% sugar water, volume/volume).
The tip of the syringe is cut off and replaced by a plastic sliding door fixed with a paper clip.
In pairs, the chest of a bee is marked as a red dot (enamel paint)
And the other one is not marked.
Similarly, half of the single bees are marked in the same way, while the other half is still not marked to control the possible effects of the enamel paint.
The data shows that there is no difference in the attack behavior of marked bees and unmarked bees (=1. 575, df=2, =0. 455).
Once this step is done, all bees can recover for 10 and 80 minutes before the test-
Investigate Aggressive Behavior
If one or two bees show signs of poor recovery when they put into the cloth scene --up (
Difficult to reverse, clumsy and/or walk slowly)
The whole trial was excluded from further analysis.
All materials used to hold bees are washed, washed and dried with detergent after each use.
A large number of different scents were tested in the main attack experiments.
Since it is technically impossible to test all of these scents at the same time, the scents are divided into four groups, each of which includes aba and solvent TEC as reference points.
Since there is no statistical difference between the four groups of references (see results)
Data is gathered together.
As a result, 128 pairs of bees were built and 32 pairs of bees were built.
Experiments that tested the role of social interaction included 32 pairs or individuals in each group.
This sample size was selected based on the pilot experiment.
Experiments using full SAP included 48 pairs of bees under each odor condition.
The sample size of this experiment was increased to obtain the statistical capability required to detect this smaller effect.
No bees were tested more than once and not released (
They were killed).
A live test was conducted at the beekeeping farm at Paul Sabatier University (
Toulouse, France)
In summer 2015
August-September).
Three colonies of the same sub-species ()
Participated in the experiment.
During the course of the experiment, they were all free to feed, received Varroa treatment, and received routine beekeeping tests.
On a sunny morning, colonies are tested every 24 hours.
All smells are pure chemicals. 98–99. 9% purity)from Sigma-
In the refrigerator (−20u2009° C).
Prior to each set of experiments, a batch of fresh odor agent dilution was prepared using TEC as a solvent and kept throughout the experiment.
These smells are delivered at room temperature (25u2009° C)
In the refrigerator (4u2009° C)
When not in use.
All the scents used and their concentration are shown.
Concentration of 0. 075% (vol/vol)
For all plant odors, the plant was selected for reference with the concentration of Pr-
Derived odors used in our work (0. 03% Z-3-hexen-1-ol, 0. 03% E-2-hexenal and 0. 015% α-pinene in TEC).
This concentration has been shown to reduce cortical steroids, glucose, and redox reactions caused by psychological stress in rats and is therefore used as a starting point for our study.
For on-site testing, in response to the large amount of air that must deliver the smell, the concentration of the smell increased to 1%.
The attack of bees was evaluated in the circular arena (
4 cm in diameter, in height)
Made of transparent plastic.
The sliding door on the side allows the introduction of bees from the syringe.
The various scents used are blown into the arena through three entry points (4u2009mm ID)
Regular intervals and intermediate heights along the wall.
About 40 holes are drilled regularly on the arena cover (1u2009mm ID)
To avoid the accumulation of smells in the arena. A 1-
Cm holes are also opened in the middle of the arena floor to allow the stepping motor shaft to pass through (
Aviosys DYO AK27PCB).
The stepping motor is connected to a DC power unit set to 9 v and 0. 25u2009A.
Before each trial, wipe the arena clean using a 70% ethanol solution and put a wet filter paper on the floor to keep the humidity.
Place the dummy horizontally at the top of the stepping motor shaft with blue stickiness.
Four dummies were made, each of which was made by a 3-ml syringe (
Cylinder 5-6 cm in length, 1 cm in diameter)
Rectangular patch of black suede lint (4. 5 × 7u2009cm)
Extend at one end with soft black feathers.
The use of the four dummies is always balanced under different conditions.
The leather patch is secured with four pieces of yellow tape and is replaced whenever stung.
Rinse the stinging leather patch with clear water before using it again and dry it outside for at least 24 hours;
Feathers are also cleaned with 70% ethanol.
In order to increase the jitter of the movement, the stepping motor is used at the lowest speed: therefore, the dummy rotates horizontally in the middle of the arena floor, and the black feathers gently flick through both sides.
The size and shape of the dummy allows the bee to move freely along the side and lid of the box without touching the box.
The purpose of the black feathers is to disturb them without causing bee pain.
In fact, this feather is only touching bees, and it is not enough to change the path of walking bees.
Medical Air (
Bank of China, North Ryder, Australia)
From 680-
L can and feed customization-
Design the olfactory stimulation controller.
This olfactory instrument provides a constant clean air flow for 1 minute.
PTFEPTFE)Teflon tubing (3u2009mm ID)
Causing this air to flow to 15-
Ml Falcon tube with filter paper with smell placed inside.
On both sides of the Falcon, three more Teflon tubes are attached, which terminate at the other end to the truncated pipet tip.
The resulting device can easily insert and exit the three odor entry points of the arena ().
In order to avoid contamination, eight of these devices have been manufactured, each for the delivery of an odor agent (
Or a combination of smells)
In the course of the experiment.
Between the two experiments, they were thoroughly cleaned with 70% ethanol and dried for at least 24 hours and then used for another set of scents.
In each trial, two sheets of filter paper were placed in a Falcon tube dedicated to odor delivery.
Depending on the smell combination tested, they are either blank (
No odor control)
, One is soaked with a 10 μ l odor agent, and the other is soaked with a 10 μ l solvent (odourant alone)
Or one soaked with a scent agent of 10 μ l, and the other with an alarm element of 10 μ l (
(Inclusive).
For example, for TEC control, both documents have been soaked by TEC and are only for testing Lim, the combination is Lim TEC, for testing the interaction between Lim and alarm information, A piece of filter paper carries the aba and another Lim.
To ensure the homogeneity of the data, the presentation of different odors is balanced in terms of colonies and the time of day.
All the trials were carried out under the HD camera above the arena.
Each test was performed as follows: First, turn on the camera and the stepping motor of the moving dummy.
Then insert the tip of the syringe containing bees into the arena.
The olfactory instrument always opens before the arena introduces bees, while the doors of the arena have been opened, but the door of the syringe has not yet been opened.
As a result, the bees quickly received a scent before facing the dummy, thus mimicking the continuous steps of group defense that usually occur in nature.
The syringe door is then opened and, if necessary, the bee gently pushes to the arena with a piston.
The odourant airflow remained in operation throughout the test (3u2009min).
In the trial, the direction of rotation of the dummy was manual and randomly changed multiple times.
The sting reaction of the bee was visually graded and defined as the bee holding at least 3 m/s in the dummy, and the abdominal tip pressing on the dummy in a typical sting position;
The vast majority (90. 2%)
The recorded attack further confirmed the presence of the stinger device still embedded in fake leather. Another 5.
7% of aggressive bees only stay on their feathers, which is believed to be the reason why their sting has not been pulled away.
Finally, the remaining 4.
The attack score of 1% corresponds to the Bee who chooses to bite the dummy, or (
In rare cases)
Apparently, the bees try to sting the dummy, although the reason for not being able to retrieve the stinger is unknown.
Less than 1% of the trials are considered marginal (
For example, when a fidgety Bee comes into contact with the dummy many times but does not show any other criteria)
Excluded.
In each trial, if at least one bee attacks the dummy, the offensive response score is 1 point, and if all bees remain calm, it is 0.
Before the start of the experiment, the size of the landing board was standardized (5. 5 × 53u2009cm)
For all colonies, by placing two vertical wooden walls, an open box is created around the entrance to the Hive (10u2009cm high)
Transparent plastic roof at each end and at the top of the landing board ().
At the beginning of each test, the box was closed by adding the front door, thus creating a stable atmosphere of about 2.
5 liters at the entrance of the hive, you can smell the smell.
To do this, insert two 15 ml filter paper containing 10 μ l odor into the hole in the side wall ().
Four small holes are drilled at the bottom and the lid is modified so that the tube can be easily attached to the output of the aquarium air pump (
Rena 300, provides a total airflow of about 3. 3u2009lu2009min).
To avoid contamination, a pair of tubes were made for each smell tested.
To measure aggression at the group level, the black leather patch (4. 5 × 7u2009cm)
On a wooden pole in front of the entrance of the hive, 1-2 cuccm from the landing board and shake through a small motor (
XL-Lego PowerMotor).
The square mark on the ground ensures that the flag is in the same position within a few days.
The test started by closing the front door of the box and opening the air pump to pass on the smell, while the flag remained intact.
The order of smell presentation is random.
After 2 minutes of exposing the smell, open the flag motor and remove the door, thus allowing the bee to face the moving flag (
The smell continues; ).
This step lasted another minute (
So it takes a total of 3 minutes for the whole test)
After that, the motor stopped working and the flag was quickly sealed in a plastic box so that no additional bees would enter.
Then calculate the number of thorns embedded in the leather and use it as a measure of aggression at the colony level.
The flag was discarded after being stung and during the trial all materials were cleaned with 70% ethanol.
All trials were recorded with a camera placed above the landing plate.
Each of the three colonies was tested six times, each smell (=18 per group)
Data normalization for each group (see below)
To illustrate different international
The colony was aggressive before it was merged.
In the morning, the Falcon tube captured an equal number of bees from four colonies at the entrance of the hive.
They were then subjected to cold anesthesia in the refrigerator within 5 minutes and were tied to the suppression tube used for each adjustment.
Feed them with a drop of sugar (50% vol/vol)
Before being placed in a dark incubator (
26 °c, humidity 85%)for 3u2009h.
This is the standard procedure for homogenizing the satiety of bees and adapting them to the suppression tube.
The regulation of PER is a classical regulatory analysis in which the controlled bee learns to associate the smell with the appetite reward of the sucrose solution.
When the tentacles of a hungry, driven Bee are touched by a solution of sucrose, the animal reflexively stretches its proboscis and reaches out its hand to absorb sucrose (PER).
If the smell is present immediately before the sucrose solution (Forward Pairing)
, Will form an association that enables odourant to release PER in the next Test.
In our experiment, bees were exposed to a scent (CS)
For 6 u2009 s, then the presentation of the sucrose solution (Volume 50%/volume, United States of America)for 3u2009s.
CS and US overlap during the 3 u2009 s.
Bees were tested four times for 13 minutes. Forty-
Five minutes after the last regulation trial, the bee\'s response to three or four scents was randomly tested without any sugar reward.
There\'s a time of 13 minutes.
Test interval between tests.
Three sets of experiments were conducted.
In the first group, bees received hormone regulation, regulated with hormones, hormones and hormones, or regulated with hormones, hormones and hormones.
In the second group, bees are trained with a mixture (
IAA, phenylalanine, IAA Pr)
Test the plant smell separately with the same mixture (PhE or Pr)and β-c (novel odour).
Finally, in the third group of experiments, bees were trained in plant odors (PhE or Pr)
With the same plant smell, the corresponding mixture (
IAA, phenylalanine, IAA Pr), IAA and β-c.
The six test groups included 53, 54, 56, 53 and 56 bees, respectively.
These sample sizes are within the standard range used to ensure statistical capacity during each experimental analysis.
Two bee populations were tested in this experiment.
As mentioned above, defensive bees are captured directly from colonies.
To test if some of the scents are inherently delicious, we made the scents-
The innocent bee places an upper-cap hatchling box in the dark Hatchery (34u2009°C)
Collect new bees every day.
20-year-old
Then, match the bees in the same incubator for 10 days in a mesh cage.
They got water and a tasteless solution of sugar (50% vol/vol)
, Except the night before the test sugar solution was removed to increase their power.
Fresh food and water are served daily.
On the morning of the test, all bees were given cold anesthesia, placed in the suppression tube used for each test, and fed a drop of sugar water before the test, then place it for 4 hours in a dark incubator at 26 °c.
A total of 101 childish bees and 110 aggressive bees participated in the experiment.
Each bee was randomly arranged at intervals of 13 minutes to test six scents.
It is important that there is no training before the test and no reward is given during the test.
At the end of the test process, PER was triggered by touching the tentacles of bees with sugar water, and a small number of bees that did not respond to this stimulus were excluded from the analysis. We used -
To analyze the test of the data generated by the experiment and investigate the role of social interaction, since observation is independent, all expected cell counts are> 10.
To calculate the theoretical data, we believe that the frequency of active trials of a single bee under a given condition represents the probability of a bee moving from this population to sting under these conditions.
Then, using the classical law of probability of two independent events, the probability of obtaining positive trials from two such bees is calculated.
So, or more generally, the expected results are obtained by multiplying this probability by the sample size.
Analyze all other attack data using GLM set
Use the logical link function suitable for binomial data.
In the field test data set, two outliers must be removed for each group.
They all correspond to extreme radical trials, during which they collect two to nine times more sting than usual.
Deleting them does not change the overall pattern of the observed response, but allows the dataset to satisfy the normal assumption (
Shapiro-Wilk test)
Analysis of variance using repeated measurements is necessary.
By subtracting the group average from each data point and dividing it by the group s, the data for each group is also standardized. d. (standard score).
This is done to homogenize the data because the baseline attack levels are different for each group (from 1. 76 to 17.
For the most aggressive groups, there are an average of 6 responding bees).
Correct the comparison in pairs with the Bonferroni program.
Tested with cronran test the potential difference between the percentage of bees showing each reaction when different scents are presented, as it is suitable for repeated measurements with binary reactions.
If this test is significant, an analysis is performed using multiple McNemar tests and a notability threshold adjusted through Bonferroni correction.
The correlation between the two data sets was tested using Pearson test.
Although these olfactory cues are linked to the biology of bees, the effect of floral flowers on this behavior has never been studied.
Here, we use a new analytical approach to study social and olfactory cues that drive bee defense behavior.
We show that social interaction is necessary in order to reveal the recruitment function and specific floral fragrance of alarm informationlinalool and 2-phenylethanol—
There is an amazing ability to stop hiring through alarm information.
This effect is not due to the smell of flowers that mask the sex hormones, but is related to their appetite values.
In addition to their potential applications, these findings provide new insights into how bees make decisions to engage in defense and how conflict information affects the process.
In all experiments, bees were collected from several unrelated bee populations, except for field trials ()
University of Queensland St. Lucia campus (
Brisbane, Queensland, Australia)
, April 2013 to October 2014, excluding winter months-June to August.
During the experiment, all colonies were free to feed and received routine beekeeping tests and honey collection.
The same number of bees from four different groups took part in the competition.
On sunny days, bees are caught in two rounds (
About 0930 and 1100 h per colony)
To ensure that any colony is disturbed in a manner that does not exceed once every 48 hours.
This delay makes the hive completely stable between disturbances, and in fact, no increase in aggression has been observed over time.
Selection of BEE groups participating in group defense (
Guards and soldiers)
The bee waved a large black feather in front of the entrance to the hive and collected it for a few seconds.
Once the feather is covered by about 30-40 attack bees, it is quickly put into a sealed plastic bag and in a refrigerator at 20 °c.
Check the status of the bees after 5 minutes, then check every 1-2 minutes until they are not moving (on average 8.
25 minutes in the fridge).
Choose to restore the fastest Bee to put in a 50 ml syringe alone or in pairs (Terumo)
Contains wet tissue and three drops of sugar (
50% sugar water, volume/volume).
The tip of the syringe is cut off and replaced by a plastic sliding door fixed with a paper clip.
In pairs, the chest of a bee is marked as a red dot (enamel paint)
And the other one is not marked.
Similarly, half of the single bees are marked in the same way, while the other half is still not marked to control the possible effects of the enamel paint.
The data shows that there is no difference in the attack behavior of marked bees and unmarked bees (=1. 575, df=2, =0. 455).
Once this step is done, all bees can recover for 10 and 80 minutes before the test-
Investigate Aggressive Behavior
If one or two bees show signs of poor recovery when they put into the cloth scene --up (
Difficult to reverse, clumsy and/or walk slowly)
The whole trial was excluded from further analysis.
All materials used to hold bees are washed, washed and dried with detergent after each use.
A large number of different scents were tested in the main attack experiments.
Since it is technically impossible to test all of these scents at the same time, the scents are divided into four groups, each of which includes aba and solvent TEC as reference points.
Since there is no statistical difference between the four groups of references (see results)
Data is gathered together.
As a result, 128 pairs of bees were built and 32 pairs of bees were built.
Experiments that tested the role of social interaction included 32 pairs or individuals in each group.
This sample size was selected based on the pilot experiment.
Experiments using full SAP included 48 pairs of bees under each odor condition.
The sample size of this experiment was increased to obtain the statistical capability required to detect this smaller effect.
No bees were tested more than once and not released (
They were killed).
A live test was conducted at the beekeeping farm at Paul Sabatier University (
Toulouse, France)
In summer 2015
August-September).
Three colonies of the same sub-species ()
Participated in the experiment.
During the course of the experiment, they were all free to feed, received Varroa treatment, and received routine beekeeping tests.
On a sunny morning, colonies are tested every 24 hours.
All smells are pure chemicals. 98–99. 9% purity)from Sigma-
In the refrigerator (−20u2009° C).
Prior to each set of experiments, a batch of fresh odor agent dilution was prepared using TEC as a solvent and kept throughout the experiment.
These smells are delivered at room temperature (25u2009° C)
In the refrigerator (4u2009° C)
When not in use.
All the scents used and their concentration are shown.
Concentration of 0. 075% (vol/vol)
For all plant odors, the plant was selected for reference with the concentration of Pr-
Derived odors used in our work (0. 03% Z-3-hexen-1-ol, 0. 03% E-2-hexenal and 0. 015% α-pinene in TEC).
This concentration has been shown to reduce cortical steroids, glucose, and redox reactions caused by psychological stress in rats and is therefore used as a starting point for our study.
For on-site testing, in response to the large amount of air that must deliver the smell, the concentration of the smell increased to 1%.
The attack of bees was evaluated in the circular arena (
4 cm in diameter, in height)
Made of transparent plastic.
The sliding door on the side allows the introduction of bees from the syringe.
The various scents used are blown into the arena through three entry points (4u2009mm ID)
Regular intervals and intermediate heights along the wall.
About 40 holes are drilled regularly on the arena cover (1u2009mm ID)
To avoid the accumulation of smells in the arena. A 1-
Cm holes are also opened in the middle of the arena floor to allow the stepping motor shaft to pass through (
Aviosys DYO AK27PCB).
The stepping motor is connected to a DC power unit set to 9 v and 0. 25u2009A.
Before each trial, wipe the arena clean using a 70% ethanol solution and put a wet filter paper on the floor to keep the humidity.
Place the dummy horizontally at the top of the stepping motor shaft with blue stickiness.
Four dummies were made, each of which was made by a 3-ml syringe (
Cylinder 5-6 cm in length, 1 cm in diameter)
Rectangular patch of black suede lint (4. 5 × 7u2009cm)
Extend at one end with soft black feathers.
The use of the four dummies is always balanced under different conditions.
The leather patch is secured with four pieces of yellow tape and is replaced whenever stung.
Rinse the stinging leather patch with clear water before using it again and dry it outside for at least 24 hours;
Feathers are also cleaned with 70% ethanol.
In order to increase the jitter of the movement, the stepping motor is used at the lowest speed: therefore, the dummy rotates horizontally in the middle of the arena floor, and the black feathers gently flick through both sides.
The size and shape of the dummy allows the bee to move freely along the side and lid of the box without touching the box.
The purpose of the black feathers is to disturb them without causing bee pain.
In fact, this feather is only touching bees, and it is not enough to change the path of walking bees.
Medical Air (
Bank of China, North Ryder, Australia)
From 680-
L can and feed customization-
Design the olfactory stimulation controller.
This olfactory instrument provides a constant clean air flow for 1 minute.
PTFEPTFE)Teflon tubing (3u2009mm ID)
Causing this air to flow to 15-
Ml Falcon tube with filter paper with smell placed inside.
On both sides of the Falcon, three more Teflon tubes are attached, which terminate at the other end to the truncated pipet tip.
The resulting device can easily insert and exit the three odor entry points of the arena ().
In order to avoid contamination, eight of these devices have been manufactured, each for the delivery of an odor agent (
Or a combination of smells)
In the course of the experiment.
Between the two experiments, they were thoroughly cleaned with 70% ethanol and dried for at least 24 hours and then used for another set of scents.
In each trial, two sheets of filter paper were placed in a Falcon tube dedicated to odor delivery.
Depending on the smell combination tested, they are either blank (
No odor control)
, One is soaked with a 10 μ l odor agent, and the other is soaked with a 10 μ l solvent (odourant alone)
Or one soaked with a scent agent of 10 μ l, and the other with an alarm element of 10 μ l (
(Inclusive).
For example, for TEC control, both documents have been soaked by TEC and are only for testing Lim, the combination is Lim TEC, for testing the interaction between Lim and alarm information, A piece of filter paper carries the aba and another Lim.
To ensure the homogeneity of the data, the presentation of different odors is balanced in terms of colonies and the time of day.
All the trials were carried out under the HD camera above the arena.
Each test was performed as follows: First, turn on the camera and the stepping motor of the moving dummy.
Then insert the tip of the syringe containing bees into the arena.
The olfactory instrument always opens before the arena introduces bees, while the doors of the arena have been opened, but the door of the syringe has not yet been opened.
As a result, the bees quickly received a scent before facing the dummy, thus mimicking the continuous steps of group defense that usually occur in nature.
The syringe door is then opened and, if necessary, the bee gently pushes to the arena with a piston.
The odourant airflow remained in operation throughout the test (3u2009min).
In the trial, the direction of rotation of the dummy was manual and randomly changed multiple times.
The sting reaction of the bee was visually graded and defined as the bee holding at least 3 m/s in the dummy, and the abdominal tip pressing on the dummy in a typical sting position;
The vast majority (90. 2%)
The recorded attack further confirmed the presence of the stinger device still embedded in fake leather. Another 5.
7% of aggressive bees only stay on their feathers, which is believed to be the reason why their sting has not been pulled away.
Finally, the remaining 4.
The attack score of 1% corresponds to the Bee who chooses to bite the dummy, or (
In rare cases)
Apparently, the bees try to sting the dummy, although the reason for not being able to retrieve the stinger is unknown.
Less than 1% of the trials are considered marginal (
For example, when a fidgety Bee comes into contact with the dummy many times but does not show any other criteria)
Excluded.
In each trial, if at least one bee attacks the dummy, the offensive response score is 1 point, and if all bees remain calm, it is 0.
Before the start of the experiment, the size of the landing board was standardized (5. 5 × 53u2009cm)
For all colonies, by placing two vertical wooden walls, an open box is created around the entrance to the Hive (10u2009cm high)
Transparent plastic roof at each end and at the top of the landing board ().
At the beginning of each test, the box was closed by adding the front door, thus creating a stable atmosphere of about 2.
5 liters at the entrance of the hive, you can smell the smell.
To do this, insert two 15 ml filter paper containing 10 μ l odor into the hole in the side wall ().
Four small holes are drilled at the bottom and the lid is modified so that the tube can be easily attached to the output of the aquarium air pump (
Rena 300, provides a total airflow of about 3. 3u2009lu2009min).
To avoid contamination, a pair of tubes were made for each smell tested.
To measure aggression at the group level, the black leather patch (4. 5 × 7u2009cm)
On a wooden pole in front of the entrance of the hive, 1-2 cuccm from the landing board and shake through a small motor (
XL-Lego PowerMotor).
The square mark on the ground ensures that the flag is in the same position within a few days.
The test started by closing the front door of the box and opening the air pump to pass on the smell, while the flag remained intact.
The order of smell presentation is random.
After 2 minutes of exposing the smell, open the flag motor and remove the door, thus allowing the bee to face the moving flag (
The smell continues; ).
This step lasted another minute (
So it takes a total of 3 minutes for the whole test)
After that, the motor stopped working and the flag was quickly sealed in a plastic box so that no additional bees would enter.
Then calculate the number of thorns embedded in the leather and use it as a measure of aggression at the colony level.
The flag was discarded after being stung and during the trial all materials were cleaned with 70% ethanol.
All trials were recorded with a camera placed above the landing plate.
Each of the three colonies was tested six times, each smell (=18 per group)
Data normalization for each group (see below)
To illustrate different international
The colony was aggressive before it was merged.
In the morning, the Falcon tube captured an equal number of bees from four colonies at the entrance of the hive.
They were then subjected to cold anesthesia in the refrigerator within 5 minutes and were tied to the suppression tube used for each adjustment.
Feed them with a drop of sugar (50% vol/vol)
Before being placed in a dark incubator (
26 °c, humidity 85%)for 3u2009h.
This is the standard procedure for homogenizing the satiety of bees and adapting them to the suppression tube.
The regulation of PER is a classical regulatory analysis in which the controlled bee learns to associate the smell with the appetite reward of the sucrose solution.
When the tentacles of a hungry, driven Bee are touched by a solution of sucrose, the animal reflexively stretches its proboscis and reaches out its hand to absorb sucrose (PER).
If the smell is present immediately before the sucrose solution (Forward Pairing)
, Will form an association that enables odourant to release PER in the next Test.
In our experiment, bees were exposed to a scent (CS)
For 6 u2009 s, then the presentation of the sucrose solution (Volume 50%/volume, United States of America)for 3u2009s.
CS and US overlap during the 3 u2009 s.
Bees were tested four times for 13 minutes. Forty-
Five minutes after the last regulation trial, the bee\'s response to three or four scents was randomly tested without any sugar reward.
There\'s a time of 13 minutes.
Test interval between tests.
Three sets of experiments were conducted.
In the first group, bees received hormone regulation, regulated with hormones, hormones and hormones, or regulated with hormones, hormones and hormones.
In the second group, bees are trained with a mixture (
IAA, phenylalanine, IAA Pr)
Test the plant smell separately with the same mixture (PhE or Pr)and β-c (novel odour).
Finally, in the third group of experiments, bees were trained in plant odors (PhE or Pr)
With the same plant smell, the corresponding mixture (
IAA, phenylalanine, IAA Pr), IAA and β-c.
The six test groups included 53, 54, 56, 53 and 56 bees, respectively.
These sample sizes are within the standard range used to ensure statistical capacity during each experimental analysis.
Two bee populations were tested in this experiment.
As mentioned above, defensive bees are captured directly from colonies.
To test if some of the scents are inherently delicious, we made the scents-
The innocent bee places an upper-cap hatchling box in the dark Hatchery (34u2009°C)
Collect new bees every day.
20-year-old
Then, match the bees in the same incubator for 10 days in a mesh cage.
They got water and a tasteless solution of sugar (50% vol/vol)
, Except the night before the test sugar solution was removed to increase their power.
Fresh food and water are served daily.
On the morning of the test, all bees were given cold anesthesia, placed in the suppression tube used for each test, and fed a drop of sugar water before the test, then place it for 4 hours in a dark incubator at 26 °c.
A total of 101 childish bees and 110 aggressive bees participated in the experiment.
Each bee was randomly arranged at intervals of 13 minutes to test six scents.
It is important that there is no training before the test and no reward is given during the test.
At the end of the test process, PER was triggered by touching the tentacles of bees with sugar water, and a small number of bees that did not respond to this stimulus were excluded from the analysis. We used -
To analyze the test of the data generated by the experiment and investigate the role of social interaction, since observation is independent, all expected cell counts are> 10.
To calculate the theoretical data, we believe that the frequency of active trials of a single bee under a given condition represents the probability of a bee moving from this population to sting under these conditions.
Then, using the classical law of probability of two independent events, the probability of obtaining positive trials from two such bees is calculated.
So, or more generally, the expected results are obtained by multiplying this probability by the sample size.
Analyze all other attack data using GLM set
Use the logical link function suitable for binomial data.
In the field test data set, two outliers must be removed for each group.
They all correspond to extreme radical trials, during which they collect two to nine times more sting than usual.
Deleting them does not change the overall pattern of the observed response, but allows the dataset to satisfy the normal assumption (
Shapiro-Wilk test)
Analysis of variance using repeated measurements is necessary.
By subtracting the group average from each data point and dividing it by the group s, the data for each group is also standardized. d. (standard score).
This is done to homogenize the data because the baseline attack levels are different for each group (from 1. 76 to 17.
For the most aggressive groups, there are an average of 6 responding bees).
Correct the comparison in pairs with the Bonferroni program.
Tested with cronran test the potential difference between the percentage of bees showing each reaction when different scents are presented, as it is suitable for repeated measurements with binary reactions.
If this test is significant, an analysis is performed using multiple McNemar tests and a notability threshold adjusted through Bonferroni correction.
The correlation between the two data sets was tested using Pearson test.
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