This method can help answer key questions in the chronobiology and stress physiology field, such as what cues effect the timing of insect emergence. The main advantage of this technique is that it is automated, and can be used to measure emergence of large numbers of insects. The following parts are required for each channel being constructed.
One collector manifold, one end cap, six platform supports, four tube rack base plates, and four tube rack face plates. With three platform supports and a 33 by 30 centimeter piece of corrugated plastic, use hot glue to assemble two tube rack platforms per channel being constructed. Install electronics into the collector manifold by first soldering a 120 ohm resistor to the anode of both the infrared emitter and the infrared detector and a five-centimeter length of 22-gauge electrical wire to both cathodes.
Carefully insert the detector into one socket of the collector manifold, and the emitter into the second socket. Both components should fit snugly. Feed the detector wires through the cabling channel and pull both wires through the access hole.
Solder all four wires to an RJ45 ethernet jack using the back row of pins. Both anodes should be soldered to the leftmost pin, the cathode of the emitter to the rightmost pin, and the cathode of the detector to either of the center pins. Secure the RJ45 jack over the collector manifold access hole with hot glue, ensuring no bare wires are touching inside the manifold.
Next, begin construction of the central processor for the system by soldering female headers on the through holes labeled for Arduino nano, temp, clock, and SD module. Also, solder a female header on the unlabeled two-by-five through hole in the upper-left corner of the PCB board for the LCD screen. Snap in and solder six RJ45 jacks along the lower edge of the PCB board.
Solder six 470 kilo ohm pull-down resistors into the through hole sites, located just above the RJ45 jacks. Install the Arduino nano, DHT temperature and humidity sensor, clock, and SD module onto the PCB board. Finally, connect a ten-connector ribbon wire to the LCD screen connector of the PCB board.
Once the detection system has been assembled, load and place two racks immediately prior to running an experiment. First, ensure that all holes in the rack contain a 0.5 milliliter micro-centrifuge tube with the cap removed, and that the tubes fit snugly. Fill each tube with one insect brood cell, making sure the flat edge side is facing toward the opening.
Then add one air soft pellet, and finally, one metal BB.Then use quarter-inch nylon screws to affix the tube rack faceplate, with the rounded edge towards the bottom of the rack. Places the tube racks on the rack platform. Start with the openings facing upwards, and then gently rotate into place to ensure the metal BBs are not released.
Racks should be placed at the edge of the platform so that a metal BB can fall freely into the collector without bouncing against another portion of the structure. Insert an SD card into the adapter, and then start the central processor by plugging a micro-USB connector into the Arduino, and the other end into any appropriate USB adapter. The LCD screen will display numbers one through six when ready.
Drop a single metal BB into the ball collector of each channel, and watch for the corresponding count to appear on the screen, and for the correct time to display at the bottom of the screen. After all insects have emerged, power down the apparatus by unplugging the Arduino. Racks may be disassembled and cleaned for re-use.
During the emergence experiment, data is stored on the SD card in a comma-delimited file. Use the SD card to transfer data to the computer, and RStudio to auto-generate bubble plots of the data. Both event and temperature data are saved in the same file for data integrity.
Prior to analysis, import the comma-delimited file into a spreadsheet program. Columns I and J are the date and time of emergence for bees. Make them columns A and B by cutting and pasting columns A through E into a second spreadsheet, and save as a separate file of temperature data for further analysis.
This figure displays bee emergence under a four degree Celsius thermo-period after exposure to a cold stress during development. This figure shows the same data set as just seen, but with one of the six channels clogged with BBs, creating the large bubble on the graph as the lack of signal is repeatedly counted. Data from this channel can justifiably be removed from the analysis.
While attempting this procedure, it's important to remember to check all parts soldered to make sure the connections are working. After its development, this technique paved the way for researchers in the field of chronobiology and stress physiology to explore what cues effect the timing of emergence in solitary bees.
