low doses of ivermectin cause sensory and locomotor disorders in dung beetles - purified air

A veterinary drug that is commonly used to control ecto-
However, its use has adversely affected cohabiting insects, resulting in a decline in population numbers and loss of biodiversity.
There is currently no information about the direct effects of ivicin on the physiology and behavior of dung beetles.
Here, based on the electrical diagram of the antenna and the spontaneous muscle strength test, we show
Ivicin is a critical species of dung beetle in the Mediterranean ecosystem-the sensation of cicadas and the deadly disease caused by the motor system.
Our findings suggest that ivamex reduces the olfactory and motor abilities of dung beetles and prevents them from performing basic biological activities.
These effects were observed at lower concentrations than normally measured in treated livestock feces.
Considering the effect of Averin on both MSGgated and GABA-
We predict that the role of Ivex at physiological levels may affect many members of the fecal pat community.
The results suggest that the decline in the population of dung beetles may be related to the harmful effects of chemical contamination in feces.
Collect individuals from the Donna Biological Reserve (DBR-ICTS)Averin-
Free venues in Donna National Park (Huelva)
Summer in southern Spain (July 2013).
All of them are kept in plastic containers (60x40x40 cm)
At 20 °c until they arrive at the lab, they are maintained in the climate chamber at 29 °c. L: D)
80 ± 5 RH with a light time of 14: 10 (L : D).
These conditions simulate the best conditions on site.
In order to ensure the common physiological state of all individuals and thus allow the comparison of physiological and behavioral measures in treatment, we have chosen the following methods :(a)
Only mature specimens according to external age
Scoring method (e. g.
Erosion of Shin and clypeus)
Persons of approximately the same age are allowed to be identified; (b)
Mature individuals of the same weight (About 1. 2 g); (c)
The gender ratio was 1: 1 in each experiment.
This work is in compliance with Spanish legal requirements, including those related to protection and welfare.
In addition, beetle collections related to collection and field research were conducted at Donna National Park. Non-
Obtaining contaminated cattle feces from ivamex
Free cattle in Donna Bio Reserve.
Collect fresh feces the first two hours of the morning to avoid the colonisation of feces animals and minimize physical-
Chemical changes in feces.
Freeze the stored feces if not immediately used (−20u2009°C)or cold (3u2009°C)
Until it's used.
All feces collected (~20u2009kg)
Homogenize using an electric paint mixer.
According to the literature and the recommendation of the OECD, the concentration of Averin was selected.
Six concentrations of fresh manure, 1. 0, 3. 3, 10. 0, 33. 3, 100. 0, and 200.
Use 0 μg kg plus untreated controls.
Through the dissolution of Averin (Sigma-Aldrich Co. )
Absolute ethanol (Sigma-Aldrich Co. )
, Add six 2 ml equally divided samples of selected concentrations to 2 kilograms of fresh feces and mix them for 20 minutes through a kitchen machine mixer.
For untreated controls, absolute ethanol (2u2009ml)
Is applied to the same amount of feces.
The residual ethanol was removed by evaporation before the manure treatment was transferred to individual experimental units.
For antenna measurement (EAG)
Muscle strength and ataxia
Mortality test, each individual experimental unit consists of a plastic container of 15 × 10 × 7 cm, which is based on bentonite.
Stool treatment is provided in a volume of 4 ml to better quantify the amount of ivysin consumed by each person.
Remove unconsumed feces and measure them every three days (in ml)
, Add a new section of the corresponding manure treatment.
For EAG tests, each treated beetle was fed treated feces within 12 days prior to biometrics.
In the case of the foreleg muscle strength test, the time for ivamin poisoning before the bioassay was 12-18 days.
For the EAG and foreleg muscle strength tests, 10 repetitions per treatment, for ataxia-
Mortality tests were repeated 20 times per treatment.
The number and weight of the beetle (body mass)
Before each treatment.
In the case of the olfactory instrument test, the beetle is also stored in a plastic container (60x40x40 cm)
The substrate is moist and sterile vermiculite.
Prior to the test, the Beetle was supplied with feces within two weeks, replacing them every three days to ensure that the food supply was not a limiting factor.
For this biological assay, ten repetitions per treatment.
Recording antenna signals with EAG system (
Syntech in kirschzarten, Germany)
A universal single-ended probe (Type PRS-1)
Stimulus controller (CS-55)
Data acquisition interface board (Type IDAC-02)
, And stimulate the air controller (Type CS-01).
Remove the antenna and insert it into a small drop of the electrode gel (
Spectra 360, Fairfield Park Laboratory, New Jersey, USA)
And separately installed between the electrodes in the antenna bracket under the purified air flow (500u2009ml/min)(). A Syntech PC-
The EAG signal is amplified by signal processing.
The signal is further analyzed using EAG 2000 software (
Syntech in kirschzarten, Germany).
Stimulation test was carried out by applying puffs of humidified air (200u2009ml/min)
Use a stimulus controller to flow through a pasteurized transfer tube containing a small piece of filter paper (Whatman no. 1)strip (1u2009cm)
One of the test compounds that flow in a stainless steel conveying pipe (1u2009cm diameter)
The socket is located about 1 cm from the antenna.
In each experiment, the standard reference compound hexane (
Sigma-HPLC gradeAldrich Co. )
And then test the odor agent by injection.
Highly specific sensitivity based on insect olfactory sensory neurons (OSNs)
Several compounds related to protein decomposition
Organic materials including ammonia and Amine, ammonia standard (
KGaA 25% v/v aqueous solution of damstadt Merck, Germany)
And trimethamine (
Sigma-45 % v/v aqueous solutionAldrich Co. )
Was chosen to test the smell.
Puffs of the test compound apply at least 10 times on each antenna every 1 minute.
Copying with different individuals (
= 201710 per treatment).
The beetle was first fixed with a polyfluoride bandage (PTFE)
Around the chest and abdomen.
To avoid unwanted movement, after cutting off one of the front legs, the Beetle was placed in the base Chamber of Sylgard, piercing the four fluorine around the body using an insect pin.
The remaining front leg of the beetle is attached to the force sensor by a surgical thread tied between the femur and the Shin (
Knot bonded with cyano acrylic adhesive)().
When the beetle is positioned correctly, the femur is vertical, and its spindle is perpendicular to the force sensor (
Fort WPI Inc. 250Stevenage, UK).
The force sensor is mounted in a micro-manipulator that allows the legs to stretch to just tighten.
Spontaneous muscle strength (SMF)
Measure, record each equal length contraction within 5 minutes.
At this time, the legs are stretched to a position that causes the maximum contraction. LabChart v. 7 software (
ADInstruments Inc.
Dunedin, New Zealand)
Run in PowerLab 8/35 (
ADInstruments Inc.
Dunedin, New Zealand)
For data collection.
Maximum self-Force ('Peak' variable; in g)
And the area points of self-force in time ("Region" variable; in g ms)
Using Microcal Origin (
Microsoft software
United States, MA, Northampton).
Copying using different individuals (
= 201710 per treatment). Four four-
Use arm olfactometers to test the possible effects of Averin on the smell detection and motor ability of feces.
Olfactometers consists of a central arena based on a sterile dry vermiculite stone, four 5-
Cm diameter hole connecting the pipe (arms)
The end is equipped with a plastic container with test samples ().
Verd u developed and described these olfactmeters in detail.
In this study, plastic containers were designed to allow beetle entry and exit that responded to test resources.
In each olfactory instrument, 20 grams of fresh cow dung were provided to two plastic containers, respectively, and two empty containers were used as controls.
In each trial, food sources were randomly placed in the olfactory instrument.
The temperature of the biometrics chamber is maintained at 27-28 °c. Each test (
= 201710 per treatment)
We counted the time of the beetle (
20 Beatles per trial)
From the arena to the container, measure the time it takes to get two 25% (t25%; 5 individuals)and 50% (t50%; 10 individuals).
For each of the above experimental devices, we provide 4 ml of the feces (
Control or treated feces).
Every three days, we conducted two observations to test possible negative symptoms associated with ivamin intake:)
B) walking coordination
Reflection avoidance of landscape
The flower stalk joint of the antenna.
When both observations were negative, we concluded that the beetle was healthy.
In general, both observations are made in chronological order, so if they are partially paralyzed (ataxia)
We observed on the legs and or tentacles, we recorded the date of observation and described the symptoms.
In this trial, 20 repetitions per treatment (=u200920).
Design and analyze data by variance analysis using a general linear model (GLM)
If necessary, follow the normal distribution after the logarithmic transformation of the dependent variable.
Use of Kolmogorov-check the normal conditionSmirnov test (Pu2009>u20090. 05 in all cases).
Although the selection of beetles is to reduce the variability of individual weight, we suspect that some differences in body mass between individuals may lead to random variability in physiological measurements.
Therefore, in order to significantly reduce the error variance, we included weight as covariates in the analysis. Multiple post-
A special comparison was made between the treatment group and the control group using Dunnet's test (
EAG test: mean value of treatment mean of control;
Olfactory instrument test: processing average value> controlling average value;
Ataxia and mortality test: Mean of treatment> mean of control).
The software Statistics v8. 0 (
StatSoft Inc. , Tulsa, Oakland, United States of America)
For all statistical analysis.