Evaluating the efficacy of Oxyops vitosa in Florida

Explorations for potential agents in western Australia resulted in the collection of >450 arthropods associated with melaleuca. Among the most promising candidates, the melaleuca snout beetle (O. vitiosa) was the first agent selected for quarantine-based host specificity testing. These tests predicted that the weevil was sufficiently host specific for release and in 1997 O. vitiosa was introduced at 13 locations in south Florida. Nascent populations were established at 9 of the original 13 release sites.

Adults and larvae of the melaleuca snout beetle feed on young foliage produced on both saplings and older, mature trees. The larvae are the most damaging life stage, feeding on one side of a leaf through to the cuticle on the opposite side, which results in a window-like feeding scar. This damage may persist for months, ultimately resulting in leaf drop. Adult feeding is characterized by the narrow scars along the leaf surfaces.

Intuitively, the next stage of research involves assessing the impact of this feeding damage on Melaleuca performance, population dynamics and ecological interactions in the new habitat. More generally, how does foliar damage aid in the suppression of Melaleuca populations below an ecological threshold that results in the replacement of the target weed by more desirable vegetation? In the following sections we describe preliminary results that are aimed at answering these questions.

Geographic Distribution

It has been suggested that ecological impacts can be evaluated as a function of 1) the agent’s geographic distribution, 2) population densities and 3) suppressive effect per individual. A preliminary estimate of the first parameter, snout weevil’s distribution, can be determined from the point localities were the weevil was released or redistributed. This method assumes that no appreciable dispersal occurs after release (see below). To do this we simply estimated the weevil distribution by recording the location of each release with a real-time differential global positioning system.

The geographic distribution of O. vitiosa is presented in Figure 2. To date, the biological control agent has been redistributed to 135 locations in south Florida and now inhabits 10 counties including Broward, Dade, Charlotte, Collier, Glades, Lee, Martin, Monroe, Palm Beach and Sarasota (Table 1).The number of releases per county does not appear to correlate with area infested per county (Table 1). For instance, Dade county has the highest number of releases (81) yet has less melaleuca than many other counties. In contrast, Palm Beach Co. has the greatest area infested by the weed but has received only 5 releases. One explanation for this phenomenon may be related to direct funding of releases by specific counties.

The absence of weevils in certain melaleuca dominated regions is disturbing (Figure 2). To ensure regional impacts of the biological control agent are realized, a combined redistribution effort is being organized among USDA/ARS, Department of Environmental Protection, South Florida Water Management District and the Florida State Extension Service.

Table1. Releases of O. vitiosa in relation to Melaleuca infestations occurring in south Florida

County

No. of releases

% of total releases

% of total Melaleuca infestation

Broward

22.22

11.41

Charlotte

0.74

4.09

Collier

5.19

17.89

Dade

60.00

12.31

Glades

2.96

3.97

Lee

2.96

19.67

Martin

1.48

4.50

Palm Beach

3.70

25.89

Sarasota

0.74

0.27

As described above, the geographic distribution of a biological control agent is a key parameter when estimating impacts on a target weed. In early stages of a biological control program, calculation of geographic distribution is limited to initial release localities. As target weeds deteriorate, or otherwise become unsuitable, the agent is forced to disperse and the agent’s distribution increases. Therefore, evaluating the biological control agent’s rate of spread is integral for assessing its geographic distribution and quantifying impacts on weeds. Therefore, we wished to assess the rate of spread of the melaleuca weevil (O. vitiosa).

For this study we randomly selected 4 release sites to estimate the rate spread of O. vitiosa from the respective release date to May 2000 (Table 1). In general, weevil populations at study sites had not coalesced with those of other release sites and M. quinquenervia was widely, although sometimes patchily, distributed in all 4 cardinal directions. The point of release for each site was recorded with the GPS system as described earlier. The distance O. vitiosa dispersed from each release point was measured by noting the most distant individual or signs of weevil damage along transects radiating in the 4 cardinal directions (N, S, E, W) with the GPS unit. Foliar damage by all stages of O. vitiosa is diagnostic and discloses the presence of the otherwise cryptic adults at very low population densities. In all cases, melaleuca trees were searched along transects for a minimum of 0.75 km beyond the last observed weevil or sign.From dispersal distances measured along each transect we calculated the rate of spread for each site as:

where R is the rate of spread for an individual site, d is the distance traveled by O. vitiosa, and N, S, E, W represent transects in the 4 cardinal directions and t is time (years) since release (adapted from Andow et al. 1993).We also estimated the percent of trees attacked by O. vitiosa at 0.25 km intervals along each transect by randomly selecting 100 trees at each sample interval and assessing the presence or absence of damage.

To elucidate parameters that may influence the rate of spread, various characteristics of each transect were noted, including cardinal direction, weed fragmentation, hydroperiod, years since release, predominate wind direction, maximum and mean wind speed, and number of individuals released. Melaleuca fragmentation along each transect was evaluated according to 3 categories: dense stands with breaks <10m (0 fragmentation), moderate fragmentation with breaks of 10-50 m, and widely fragmented stands separated by more than 50 m. Hydroperiod was classified as: Dry = never inundated; short = inundated < 6 months; moderate = 6-9 months. Wind data were gathered at 1 h intervals from individual weather monitoring stations located <40 km from each study site. Wind direction was categorized into 8 cardinal directions (N, NE, E, SE, S, SW, W, NW). Only wind data from 1997-2000 were used in this study. Stepwise regression was then used to distinguish which parameter significantly influenced the linear distance traveled by O. vitiosa along each transect.

When averaging among all directions and sites, O. vitiosa spread from release points at a rate of 0.99 (±0.28) km/yr, ranging from 0.10 to 2.78 km/yr. This preliminary rate of spread estimate is minimal when compared to that of other introduced weevils. For instance, the average rate of spread of the boll weevil (Anthonomus grandis grandis) was estimated to be 95.3 km/yr with a range of 64 to 193 km/yr. The disparity among these rates of spread may be related to differences in the amount of time used to acquire the estimate. For instance, we calculated the dispersal rates of O. vitiosa from data collected 2-3 years after introduction versus data that averaged spread for the boll weevil over ca. 20 yrs of invasion. When calculating estimates from larger temporal intervals, slow initial rates of spread may be masked by acceleration of an invasion front as it increases over time. One advantage of this apparently slow rate of initial dispersal may be the concentration of herbivory, resulting in high levels of localized plant damage.

Among all parameters measured, melaleuca fragmentation, the number of weevils released and time since release significantly influenced the rate of spread of O. vitiosa (F= 19.2, df = 1,14, P=<0.0001). Mean rate of spread is positively correlated with each of the 3 fragmentation levels: high= 2.04, medium= 1.07 and low= 0.30 km/yr. The most probable explanation for this relationship is that weevils increase linear dispersal to locate and attack widely separated melaleuca stands. Assuming that a high rate of spread is desired, increasing the number O. vitiosa individuals released at future sites will increase localized damage and expedite the movement of weevils through out the melaleuca infested region.

Indirect effects of herbivory by O. vitiosa negatively impacts the reproductive performance of M. quinquenervia.

To test this hypothesis we are currently evaluating: 1. What impact does the cumulative herbivory (3-4 yrs) have on flower production, 2. Does defoliation during a single growing season alter flower production, 3.Is flower production further reduced by an increase in herbivory frequency, 4. Does seed fill and viability differ among damaged and non-damaged trees?

Figure5. Oxyops vitiosa herbivory; damaged (left) and non-damaged (right) Melaleuca branches.

Objective 1. To assess the impacts of cumulative herbivory on floral development among insect damaged and undamaged trees, we have randomly selected 3 of the original 9 O. vitiosa release sites (see above). These localities represent a range in the weed’s geographic distribution, hydroperiods and invaded soil types. These 3 release sites have also been paired with nearby melaleuca stands that have not been exposed to O. vitiosa damage and served as “controls” for statistical comparisons. To quantify the incidence of flowering among the 6 replicated melaleuca stands, we evaluated floral history and development for ca. 200 randomly selected trees per site within a range of tree sizes (1 cm-6 cm diameter at breast height (dbh)). This was done by noting the dbh, the historical production of flowers (as described by the number of serotinous capsules on the tree), development of the current seasons floral production and intensity of herbivory for each tree along a randomly selected transect.

To date, we have collected the first sample and are currently processing the second. Preliminary data from the first assessment indicates that flower production, not surprisingly, is related to tree size (Figure 7). In undamaged trees, for instance, flowers production occurs among 25% of the trees measuring 1 cm dbh and increases to approximately 75% when trees are 6 cm. In contrast, a significant difference in floral development exists among those trees that are damaged by the snout weevil and undamaged trees (Figure 7). Flower production for damaged trees is nearly non-existent among small saplings and peaks at approximately 25% for larger trees with a dbh of 6 cm. This finding suggests that damage from the snout weevil, O. vitiosa, significantly reduces and retards Melaleuca flower production over a range of plant sizes. Analysis of the second sampling interval will aid in elucidating additional impacts of the weevil on plant phenology.

Figure7.Flower production among melaleuca trees damaged by the biological control agent O. vitiosa and undamaged “controls”. Herbivory by the weevil significantly reduced or delayed flowering within the range of tree sizes (dbh) studied herein (ANCOVA P<0.0001).

Objectives 2, 3 & 4. The previous studies evaluate the impact of O. vitiosa on a range of melaleuca plant sizes (dbh). Unfortunately, in those studies we are unable to ascertain the impacts of attack frequency on the study trees. For instance, what impact does the removal of new foliar tissue have on the current years flower production (objective 2) and does additional defoliation during the same growing season cause a further reduction (objective 3)? Does seed fill and viability differ among damaged and non-damaged trees (objective 4)? To answer these questions a stand of melaleuca trees growing along a road cut was selected for experimental manipulations.In contrast to the earlier studies, we sought to use similar sized trees that met the following criteria: trees of similar dbh, of similar height, reproductively mature, and possessing similar densities of seed capsule clusters. Prior to initiation of the test each tree was evaluated for the number of capsules per cluster, capsule cluster length and number of branch tips.Sixty trees meeting the above criteria were identified, providing 10 replicate trees to be randomly assigned each of six treatments:

1.Single defoliation event:A lightweight nylon screen cage was placed individually over the canopy of each of ten treatment trees. Approximately 200 third and fourth O. vitiosa larvae were placed into each cage and both larvae and cages were removed after all new foliar tissue has been consumed (ca. 1 wk).

2.Two defoliation events: In addition to those listed above, ten trees were caged and inoculated with O. vitiosa larvae as describe in treatment 1. In response to herbivory, damaged melaleuca trees produced adventitious growth to replace the consumed foliage. At this time we re-caged the trees and inoculate the selected trees with 200 larvae as before.

3.Mechanical defoliation: All leaves within the canopy were removed on the same date as the weevil inoculation (treatment 1). Removal of leaves was done by stripping petioles from branches. This treatment is designed to provide the highest level of damage and to estimate (although grossly) the impact of additional arthropods attacking mature leaves.

5.Cage effect 1: Trees were caged in conjunction with those in treatment 1 but no larvae were added. This treatment quantifies the effect of the cage on reproduction.

6.Cage effect 2: Trees caged as in treatment 5 and repeated again in conjunction with the second defoliation in treatment 2.

Trees from each treatment were evaluated biweekly. Specific data gathered for this study included phenological development (i.e. induced meristematic growth, flowering episodes) among treatments, impacts on reproduction (flower number, seed capsule abortion, capsule cluster size, densities of capsules and clusters, seed fill and viability) and plant growth (tree height, number of branches, dbh). Currently, seeds from randomly selected capsules produced among treatments are being evaluated for viability and germinability using the 2,3,5, -triphenol tetrazolium chloride (TTC) technique.

Preliminary data demonstrate that, when undamaged by the biological control agent, the study trees produced 10 cumulative inflorescences per plant per year (Figure 8). In contrast, when new, developing leaves on Melaleuca trees were defoliated once per year flower production was reduced to 1.5 inflorecences per plant. In response to the removal of the new leaves, the melaleuca trees reinitiated bud and leaf development. When these new leaves were also defoliated by the weevil cumulative flower production was reduced to 1.0 inflorescence per plant. By mechanically removing all leaves (old and new) once per year flower production was eliminated (Figure 8). When comparing these results statistically, there are no differences between the insect and mechanical treatments but stark differences exist among the defoliation treatments and the undamaged trees. These preliminary results suggest that removal (defoliation) of new Melaleuca leaves once per year by the weevil can reduce flower and subsequent seed production by 90% in the size class studied herein. We are currently collecting the data for the seed viability and germinability study, which will be presented in next years report.

Classical weed biological control involves the reuniting of an invasive plant with co-evolved natural enemies from its native range. To ensure the introduced natural enemy does not impact other native or economically important plants, intense host specificity testing is done under quarantine conditions prior to release. Before release of the melaleuca biological control agent Oxyops vitiosa, for example, tests to evaluate oviposition, feeding and development of the agent when held with closely related native and horticultural plants were performed. Findings from these tests included limited oviposition, adult feeding and larval feeding on various native myrtaceous plants and some ornamentals. In no cases were O. vitiosa larvae able to complete development on any of the native species tested so the weevil was cleared for release by federal regulatory agencies.

This method of estimating the host range of biological control agents has been criticized because arthropods may behave differently when held in controlled environments versus natural settings. Rarely have the predictions of host specificity been evaluated after release of the biological control agent into the new environment. Therefore the goal of this research is to quantify the quarantine-based predictions for non-target impacts by O. vitiosa (and other future agents) on native plants. Specific objectives include: 1) Survey O. vitiosa release sites for incidence of non-target damage on the native plants identified in quarantine as sub-optimal hosts. 2) Quantify the utilization of native, potential non-target plants in a common locality where O. vitiosa weevils have overexploited the surrounding melaleuca plants.

Although some of the non-target plants are quite prevalent at the current release sites, particularly Myrica cerifera, all plants are not equally represented in the surveys. To ensure adequate representation of the plants potentially impacted by O. vitiosa, we have developed a field plot by planting the species of interest amongst the melaleuca dominated system. Species used in this study include: Calyptranthes pallens (Spicewood, Florida Threatened), Eugenia rhombea (Florida Endangered), Eugenia axillaris (White Stopper, native), Eugenia foetida (Spanish-Stopper, native), Myrcianthes simpsonii (Simpson’s Stopper, Florida Threatened), Callistemon viminalis (Weeping bottlebrush, exotic, ornamental), Callistemon rigidis (bottlebrush, exotic, ornamental), Myrica cerifera (Wax Myrtle, native). Five replicate plants of each selected species were planted among existing melaleuca trees in a randomized block design. Incidence of O. vitiosa (adults, eggs or larvae) on plants, area of the leaf damaged by feeding, and impact of feeding on the plant species is monitored monthly.

Data collected from this study have not been analyzed but preliminary indications suggest that feeding damage from both adults and larval weevil stages negatively impact the two Callistemon species (Figure 9). This result is not surprising when one considers how closely related these species are to the target plant and the fact that similar results were obtained in quarantine based tests. While monitoring the Myrica plants, we have observed various herbivores attacking the foliage, including the exotic Diaprepes abbreviates. Only an occasional O. vitiosa weevil has been seen on Myrica plants and very little feeding damage has been observed. Our preliminary findings suggest that quarantine based host specificity predictions are consistent with field-based, post release tests.