Final Report for GS02-019
The findings of earlier research suggest that Microtheca ochroloma undergo a facultative diapause during the summer and re-colonize field plots from alternate host plants nearby. The cause of aestival induction is unknown, although temperature and day length are likely cues. Pinpointing the cues and timing of the beetle’s aestival cycle will help farmers stagger their plantings to avoid beetle outbreaks. Determining how beetles colonize a field; by walking and/or flying and from which location or direction will suggest additional control methods.
Yellowmargined leaf beetles are oligophagous, and are highly selective in their food choices within a small group of suitable host plants. Like other specialized crucifer feeders, they must be able to distinguish between host and non-host plants. Understanding beetle responses to host plant volatiles and/or conspecific synomones will help farmers to develop cultural controls for this pest.
1.To demonstrate how temperature and day length influence the dormancy of Microtheca ochroloma.
2. To determine if proximity to old feeding sites determines the location of new beetle outbreaks.
3. To determine what chemical cues (plant-insect and insect-insect) adult beetles use to locate host plants.
A HOBO weather station (Onset Computer Corporation, Bourne, MA) was deployed in the experimental field in Tallahassee FL (30.47 N, -84.28 W) to record temperature, precipitation and wind data. Napa cabbage, Brassica rapa pekinensis, and mizuna, Brassica rapa japonica (both from Johnny’s Seeds, Albion ME) were seeded on 4 October 2004 and greenhouse grown using Scott’s Metro Mix 200. Plants were fertilized once a week using Miracle-Gro 10-10-10. Host plants were transplanted on 4 November 2004, in a single east-west orientated 30 m row in alternating blocks of 7.5 m each. This field had been planted in tomatoes for the spring 2004 field season and had been fallow since then.
Adult beetles used in for this objective were from a laboratory colony maintained at 25 C and 14:10 L:D photoperiod and fed napa cabbage. A total of 250 mixed age and sex beetles were marked and released. Beetles were marked by placing them in a plastic 9 dram aspirator vial (BioQuip Products, Rancho Dominguez, CA) with fluorescent powder (BioQuip Products) and gently shaking them until the beetles were coated in powder.
Mizuna was seeded on 1 September 2003 and two mizuna plants per pot were transplanted to gallon pots (Cassco) on 17 November. On 18 November, a 83 ft by 109 ft rectangle was measured out in the field and four gallon pots were placed together on each corner of the rectangle. Clockwise from the southeast corner, the corners were numbered from one to four and assigned a color. The fifth color was assigned to the point at the center of the rectangle. No pots were placed in the middle of the rectangle. Plants were checked daily for beetles, which were removed and brought to the lab. Only two adults and 10 larvae were found. On 8 December, fifty adult beetles were marked and released at each of the four corner plots. Beetles were gently tapped onto and around the four plants at each corner plot. At the center of the rectangle, beetles were tapped onto the grass. After 72 hours, plants were visually sampled for marked beetles.
Adult beetles used in for this objective were from a laboratory colony maintained at 25 C and 14:10 L:D photoperiod and fed napa cabbage. Late instar larvae were separated into individual Petri dishes and provided a leaf disk for feeding. After emergence, adult beetles were sexed, then starved for at least 24 hours and run through the olfactometer.
The olfactometer (Analytical Research Systems, Inc. Gainesville, FL) consists of a glass y-tube (90 degree, 24mm/25mm joints) connected with Teflon tubing (0.25” od) connected to glass volatile collection chambers (6”dia x 12” l ). Compressed air (Airgas, Inc., Atlanta, GA) flowed through a carbon filter and into the chambers.
Mizuna was seeded in the greenhouse and then transplanted (2 plants per pot) into 4 inch pots (Cassco, Montgomery, AL). For experiments requiring non-host plants, we seeded Florida 47 (Seedway, Elizabethtown, PA ) and transplanted one tomato and one mizuna plant together into a single 4” pot.
There were five different choice tests. Table 1 lists the treatment and control for each test. For test 3, I cut the 16 largest leaves off of the treatment plants. For test 4, I starved four third instars for 24 hours and placed them (2 per plant) on the treatment plants. For test 5, I starved 20 adult beetles (10 female and 10 male) for 24 hours and placed them on the treatment plants one hour before beginning the test
The procedure for each choice replicate was as follows: air was turned on and pressure regulators set at 10psi (tank) and 0.5 lpm (chamber inlet). A pot was placed into each chamber and air was allowed to run for at least 10 minutes. In some cases, the air was allowed to run longer, as in the feeding damage trials (see above). A beetle was placed into the insect inlet at the bottom of the y-tube and its movements were recorded with Observer software. The stem of the y-tube was marked with a solid black line below where the tube branches to the right and left chambers. Beetles that did not cross this line were recorded as “no response”. For beetles that did cross the line, the time each beetle spent in the left or right branch of the tube was recorded.
After six minutes the beetle was removed from the apparatus. After each observation all glass which came into contact with the insect was washed and rinsed with Liqui-nox and allowed to dry. After half the beetles for any given replicate had run through the olfactometer, we switched the treatments, moving the right chamber to the left side and vice versa.
Adult beetles and eggs were initially found on 17 December. Beetles did not show a preference for one host plant over the other. Beetles were found in both the east and west ends of the experimental row. The western edge is closer to site of the 2003 marking trials while the eastern edge is closer a residential neighborhood. Field populations are still relatively low and there is little beetle damage evident on the host plants. No beetles have been located in nearby patches of wild radish.
After 72 hours, very few beetles were found on host plants. Only 20 adult beetles were found on the host plants and of those only half had any obvious mark. A single beetle released in the center of the experimental rectangle (released into the grass) was found in on a host plant. Recapture rates for marked adult beetles were very low for all experimental plots (1-6 % recapture). There is little evidence that beetles moved between experimental plots; i.e. all but one of the marked beetles were found in the plots were they were released.
In general there was little difference between the time that beetles spend moving toward treatments versus control for tests 2,3 and 5. In these test, beetles made no choice 86 %, 84.9 % and 76 % of the time, respectively. Beetles never moved towards the empty pots in test 1 and moved towards undamaged host plants about 25 % of the time. There is some evidence that adult yellowmargined leaf beetles respond to host plant-larvae complex. In olfactometer choice tests, beetles spent almost twice as much time moving toward host plants with feeding larvae as they did moving towards an undamaged host plant (Figure 1).
Areas needing additional study
Weather data will continue to be collected in order to correlate the appearance of beetles with temperature and day length information. Data over several seasons must be collected in order to firmly establish the aestival patterns of M. ochroloma.
Field exclusion cages will be set up this spring to determine how long the beetles will remain active under increasing temperature and day length conditions.
A larger scale mark recapture study is planned for this spring.
More replicates of the olfactometer trials will establish which of the choice combinations is most attractive to adult beetles.