This protocol can be used to predict and mitigate the adverse effects of pesticides on solitary bees. The main advantage of this technique is simulating larval feeding on residual pesticide in provisions. Visual demonstration of this method is critical because the biological characteristics of Osmia excavata are unique compared with social bees, and little research has been conducted on O.excavata, especially the larva.
To prepare the feeding tube, punch a 0.3 millimeter diameter hole into the lid of a two milliliter centrifuge tube using an electric winding iron. To prepare the pesticide, dissolve the technical grade pesticide in acetone to acquire stock solutions of 10 milligrams per milliliter, then perform gradient dilutions of the solution to more than five concentrations. Next, acquire plastic bee tubes containing provisions and newly hatched Osmia excavata larva from a mass rearing program.
Using a soft brush, gently separate the larva from the provisions, select female larvae based on provision size and cell position within the nest, then place the uniform size provisions with the selected female larva in 60 millimeter diameter Petri dishes and set them aside for use. Before proceeding to the pesticide treatment, weigh the provisions. To treat the provisions with pesticides, first, measure the volume of pesticide solution.
Then soak the selected evenly sized provisions in the diluted pesticide solutions for 10 seconds. After treating the provisions with the pesticide, measure the volume of the pesticide solutions again. After air drying the provisions on a sterile work table, add them to the prepared centrifuge tubes with holes.
Next, weigh 60 female larvae and using a soft brush, transfer the larvae to the surface of the naturally dried provisions, adding one larva per tube. Rear the larvae in a growth chamber in the dark at 25 plus or minus two degrees Celsius with 65 to 75%relative humidity. For the acute lethal toxicity test, measure the mortality of the larva after being placed onto the treated and the control provisions for 48 hours.
Next, weigh the provisions remaining after 48 hours of insect rearing to determine the amount of provision consumed by each larva, then using this formula, calculate the dose of pesticide at each concentration consumed by each larva according to the percentage of provision eaten and the pesticide content in each provision. For the sublethal toxicity test, observe the O.excavata daily during cocooning under black light lamps to measure the larval development duration. Then weigh the larvae after 14 days of treatments to determine the larval weight gain.
Next, weigh the provisions remaining after 14 days of feeding to calculate the consumption and the efficiency of conversion of ingested food. And examine the number of eclosion by sniping the cocoons using a small scissor when the control bees emerge into adults. The acute lethal toxicity test showed that the LD50 value of the chlorpyrifos for the O.excavata larvae was 0.001 micrograms per bee.
In the sublethal toxicity test, as the dose increased, the index values of larval weight gain, consumption, and efficiency of conversion of ingested food decreased for treatments, with the lowest values relative to the control observed in the 0.013 micrograms chlorpyrifos per bee. Conversely, the most extended larval developmental duration was observed in 0.016 micrograms chlorpyrifos per bee, compared to the control treatment. Evaluation of the impact of chlorpyrifos on eclosion rate revealed a significant negative linear relationship between ingested dosages of chlorpyrifos and the eclosion rate of O.excavata.
The eclosion rate was considerably lower when the ingested dosages exceeded 0.02 micrograms per bee than those in the control treatment. In this protocol, screening for provisions of uniform size is critical to minimize the test error. This technique is helpful in exploring the effects of pesticides on the adult flight and fecundity of solitary bees, thus solving problems of low recovery rate and population decline.
