1. Please state project objectives and what work was done this quarter to address them:
Worth note, with the unprecedented heat and the duration of it this summer, lebbeck mealybug activity in groves was far less than anticipated. We know that the population generally slows during the hotter month(s) of the year, as we have seen in previous years, however this year the impacts were seen for a much longer period of time.
1. Project objectives and what was done this quarter to address them:
(1a) Develop monitoring tools to time management actions
Due to unprecedented heat this summer, lebbeck mealybug populations were low in the late summer when odorant lure were ready for field deployment. Despite this, ongoing observations from a study on a different pest organism indicated the lebbeck mealybug persists throughout much of the year in CUPS. Odors found to be promising for lebbeck mealybug attraction in the laboratory were placed in CUPS houses with ongoing infestations in late September 2023 as populations are beginning to recover from the heat. We anticipate having data from this in our next report.
(1a-1) Movement
Hibiscus mealybugs have a high propensity for dispersal. They can be dispersed passively and actively. Passively, they are spread from one grove to another on clothing of farm workers and on farm equipment. Understanding the seasonal distribution and dispersal behavior of this pest would be important in developing effective monitoring and management strategies for improved control of this pest. Therefore, we conducted this study to describe the seasonal distribution of hibiscus mealybug within citrus trees and its migration patterns on the plants.
Method
We deployed corrugated cardboard band traps (CCBTs) with one end sealed with molten parafilm-wax to prevent escape of mealybugs that enter through the unsealed end. We deployed 3 pairs of these on three main branches and trunk of five infested citrus trees such that the sealed ends of each pair were adjacent to each other, such that we could trap mealybugs migrating downward from the canopy and mealybugs into the canopy from the trunks (mealybugs ascending and descending (Fig. 1). Canopy traps were collected monthly and number of mealybugs in defined developmental groups (eggs, crawlers, immatures, and adult females) were counted under a microscope in the laboratory. The study was conducted from August 2021 to July 2022 (sample processing and data analysis just completed).
Results
Strata Distribution
Mealybugs were collected from citrus canopies and trunks throughout the sampling period, except in the months of September, November, and December 2021, and February and March 2022, when there was no mealybug captured on the trunks of sampled citrus trees. The result of statistical analysis showed that the proportion of mealybug collected from citrus canopy was significantly greater (.2 = 7.95, d.f. = 1, p = 0.005) than those collected from the tree trunk throughout the sampling period, except in the months of January, April, and July 2022 (Fig. 2).
We recorded mealybug movement activities throughout the year, but greater activities were recorded in the months of April, July, and August 2022. The result of statical analysis showed that migration pattern of this mealybug differed significantly across the sampling period (.2 = 27.40, d.f.= 8, p < 0.001). We observed distinct patterns in the movement of the mealybug within the citrus trees sampled. Significantly greater number of mobile mealybugs were captured ascending into the citrus trees in April 2022, which coincides with the periods of fruit setting of citrus trees in Florida (Olabiyi et al. Unpublished). Conversely, a significantly greater number of mealybugs were captured descending from the citrus trees in July 2022, which is probably to move away from the developing fruits at the terminal ends of the branched into the canopy away from the heat from increased sunlight intensity in the summer months of June - August 2022 (Fig. 3).
(1c) Evaluate promising materials in open grove settings
Insecticide field evaluations were planned for July 2023, however our laboratory colony was similarly impacted by the heat as we do not have temperature controls in the space in which we are maintaining them.
(2c) Determine what insecticide chemistries inhibit feeding
We are using EPG techniques (Electropenetrography) to better understand the feeding behavior of the second instar of the hibiscus mealybug, N. viridis. By using a four-channel AC-DC monitor that generates waveforms. To do this, we first need to create a waveform library (i.e. records of waveforms at different settings- this describes the interactions leading to ingestion). This first step will allow us to better identify and characterize the feeding behavior of this insect and compare it to previous studies done on other mealybugs and scales. Comparing our waveforms with previous studies helps us in the interpretation of their biological meaning (pathway phase, xylem ingestion, phloem salivation
). In addition, determining the electrical origin of the waveforms also helps in understanding their biological meaning. Waveforms have two major components: the electromotive force, [emf] component, and Resistance, [R] component. R is the physical resistance to the applied electrical signal conveyed by ionized fluids moving through the stylets. Emf is biopotential generated from depolarization events (plant cell membrane breakage). Hence, to get the maximum information waveforms should have a 50:50 R:emf balance that is to say Ra=Ri. This means we need to choose the input resistance (Ri) setting that matches the inherent resistance of the mealybug (calculated to be at 1012 Ra) (Figure 1). For these reasons, we are trying to build a waveform library that tests three Ri which are bracketing the Ra of N. viridis (1012):
- 109 Ri with substrate voltage at 250mV (some R component)
- 1010 Ri with substrate voltage at 100mV
- 1013 Ri with substrate voltage at 0mV (pure emf signal)
- We are also trying a voltage setting at 1013 Ri with substrate voltage at 50mV (addition of voltage to add some R component).
The waveform's appearance will be compared according to the settings, and we will choose the best monitor setting for future studies.
Our final goal is to test the effect of insecticides (i.e. neonicotinoids drench) to see how they interfere with the feeding behavior of second instars mealybug (i.e. do insecticides prevent phloem salivation, phloem ingestion or xylem ingestion?)
We compared our results with 4 species of mealybugs and one species of scale:
- The longtailed mealybug, Pseudococcus longispinus
- The solenopsis mealybug, Phenacoccus solenopsis
- The citrus mealybug, Planococcus citri
- The cassava mealybugs, Phenacoccus manihoti
- The crapemyrtle bark scale, Acanthococcus lagerstroemiae
In our preliminary recordings, we observed two main issues:
- Noise in our recordings prevents us from distinguishing E1 (phloem salivation) from E2 (phloem ingestion)
- Most of the recordings are spent in the pathway phase (test probing) and little time is spent on phloem or xylem ingestion (<15%). Yet, mealybugs are phloem feeders and should spend more time feeding on the sap elements (xylem and or phloem).
We will discuss progress made on these points in the following report.
- Noise problem
A voltage regulator (Furman®, Model: P-1800 AR) was bought to fix the noise issue. We are currently using two EPG monitors. The voltage regulator was installed on one of the EPG monitors and based on our last recordings, we observed cleaner signals with significantly less noise. Another voltage regulator was purchased and will be installed on the second monitor.
- Behavior problem
We hypothesize that the reason why N. viridis spends so little time doing phloem ingestion/salivation is due to our wiring method. The gold wire could be too thick (25µm diameter) for the size of the mealybug and prevent it from moving freely and thus expressing its natural feeding behavior. We ordered some platinum Wollaston wire which is smaller and more flexible (2.5µm diameter, Sigmund Cohn Inc.) and should allow better movement of the mealybug. The wire will take up from 12 to 15 weeks to be delivered (July-August 2023). We will compare recordings with both wires and see if there is a change in probing behavior.
All the studies cited were done with adult mealybugs or scales. The percentage time spent in sieve elements varies from 26-61%. Since we are using N. virdis nymph (2nd instar) we are expecting them to feed longer on the phloem and xylem than adults (need to feed more to complete growth). So far, the time spent in phloem and xylem varies greatly among recordings (see waveform library, from 8% to 64%). We hope that using the platinum Wollaston wire will fix this issue and that mealybugs will feed longer on the sieve element (>50%).
2. Please state what work is anticipated for next quarter:
We will continue working on the odorant lures as long as populations remain active in CUPS or open groves throughout the fall. We will also continue working through the challenges with EPG to deliver the wave form library necessary to evaluate chemical feeding inhibition and/or death. This information will help determine rates necessary for managing the mealybug inside of IPCs.
3. Please state budget status (underspend or overspend, and why):
We are on track financially and may have a small amount of funds remaining at the culmination of this funding period.
4. Please show all potential commercialization products resulting from this research, and the status of each:
If the lures under evaluation are indeed able to detect mealybug populations before they achieve damaging levels, then we will have a product that could be commercialized.