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Impact of herbivory by Hydrellia pakistanae (Diptera: Ephydridae) on growth and photosynthetic potential of Hydrilla verticillata
Biological Control 24 (2002) 221–229 www.academicpress.com
Impact of herbivory by Hydrellia pakistanae (Diptera: Ephydridae) on growth and photosynthetic potential of Hydrilla verticillata Robert D. Doyle,a,* Michael Grodowitz,b R. Michael Smart,c and Chetta Owensd a University of North Texas, Denton, TX 76203-0559, USA U.S. Army Engineer Research and Development Center, Vicksburg, MS 39180, USA U.S. Army Engineer Research and Development Center, Lewisville Aquatic Ecosystem Research Facility, Lewisville, TX 75056, USA d ASI, Lewisville Aquatic Ecosystem Research Facility, Lewisville, TX 75056, USA b
c
Received 22 June 2001; accepted 24 January 2002
Abstract The impacts of varying levels of herbivory by Hydrellia pakistanae on the dioecious ecotype of Hydrilla verticillata were evaluated by conducting a 10-week growth experiment within mesocosm tanks. The observed leaf damage to H. verticillata stems was highly correlated with the total number of immature H. pakistanae in H. verticillata tissue at the time of harvest (P < 0:001, R2 > 0:80). Increasing levels of insect herbivory significantly impacted biomass and growth morphology of H. verticillata. Relative to control tanks, plants under intermediate or high levels of herbivory produced progressively less biomass. Insect herbivory also significantly impacted investment of energy in sexual and asexual reproduction. Plants under an intermediate or high level of herbivory produced fewer than 15% of the number of pistillate flowers produced by plants in control tanks. Furthermore, plants subject to high insect herbivory produced fewer and smaller tubers than control tanks. Finally, herbivory had a strong impact on the photosynthetic potential of stems. With 10–30% leaf damage, the maximum rate of light-saturated photosynthesis was reduced 30–40% relative to undamaged controls. Total daily photosynthetic production in these stems was estimated to balance, just barely, the daily respiratory needs of stems. Photosynthetic rate was reduced by about 60% in stems showing 70–90% leaf damage. This level of photosynthetic reduction would make continued survival of the plants unlikely since they would be unable to meet daily respiratory demands. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Hydrilla verticillata; Hydrellia pakistanae; Insect herbivory; Biocontrol; Photosynthesis; Aquatic weed; Invasive species; Aquatic plant management
1. Introduction Hydrilla verticillata (L.f.) Royle (hydrilla) is a major aquatic pest in North America (Pieterse, 1981). It is described as the perfect aquatic weed due to many phenological and physiological adaptations, which allow it to thrive in a variety of shallow aquatic environments (Langeland, 1996). The high biomass production and formation of a dense surface canopy are among the most undesirable characteristics of the species (Smart * Corresponding author. Present address: Department of Biology, Baylor University, P.O. Box 97388, Waco, TX 76798, USA. Fax: 254710-2969. E-mail address: [email protected] (R.D. Doyle).
and Doyle, 1995). These characteristics negatively impact the environment, interfere with management objectives, and may be responsible for the dominance of this species over native plants (Van et al., 1999). Control options for aquatic weeds include various mechanical, physical, chemical, and biological methods (Madsen, 1997). Classical biological control relies on use of host-specific insect herbivores or plant pathogens from the native range of an alien plant (Cofrancesco, 1998). This method has recently been described among the most desirable for the long-term control of nuisance alien plants (National Academy of Sciences, 1987). This method minimizes the chances of unexpected negative impacts seen in control efforts that utilized generalized vertebrate agents (DeLoach, 1991). However, biocontrol
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agents are unlikely to eradicate a species because the impacts of sustained insect herbivory are typically loss of plant vigor and loss of competitive advantages rather than direct plant mortality (Crawley, 1989; Okasnen, 1990). The combination of insect herbivory, in conjunction with establishment of competitive native species, may offer the best long-term solutions (Grodowitz et al., 2000a). Four host-specific insect biological control agents have been introduced into North America for the control of H. verticillata (Buckingham, 1994; Center et al., 1997). The leaf-mining fly Hydrellia pakistanae Deonier (Diptera: Ephydridae) was introduced in 1987 and has been the most effective. The host-specificity of H. pakistanae was confirmed in studies conducted in both Pakistan (Baloch et al., 1980) and in the United States (Buckingham and Okrah, 1993; Buckingham et al., 1989) before release. The life cycle of H. pakistanae is well known (Baloch and Sana-Ullah, 1974; Baloch et al., 1980; Buckingham et al., 1989; Wheeler and Center, 1996). Adult H. pakistanae oviposit on floating or emergent vegetation. After eclosion, the larvae actively search out H. verticillata. The larvae feed exclusively on H. verticillata leaves and damage 9–12 leaves during their three larval instars. The larvae form a puparium from the last larval cuticle and pupate attached to stems of H. verticillata. Total generation time varies between 18 and 30 days depending on temperature and nutritional content of the plant (Grodowitz, unpublished data; Wheeler and Center, 1996). More than three million individuals of H. pakistanae have been released at 25 sites in the United States (Florida, Georgia, Alabama, Louisiana, Texas, and California) and establishment has been confirmed at most sites. In some instances, H. pakistanae populations have been present for over six years (Center et al., 1997). Extensive geographic range-expansion is documented, with populations occurring throughout Florida, north to Muscle Shoals, Alabama, and west to Austin, Texas (Grodowitz et al., 1999; Grodowitz et al., 2000b). Numerous factors including nutritional quality of H. verticillata, life stage of release organisms, number of individuals released, predation, and parasitism appear to impact establishment (Center et al., 1997; Grodowitz, unpublished data; Wheeler and Center, 1996). Although larvae of H. pakistanae unquestionably damage H. verticillata leaves, studies quantifying the impacts of H. pakistanae on H. verticillata are few. In laboratory experiments, damage to canopy leaves of H. verticillata approached 100% in plants infested with 4000 larvae per m2 (Wheeler and Center, 2001). Grodowitz et al. (1997) reports estimates of 20% leaf damage under field conditions when levels reached 2320 larvae per kg of H. verticillata tissue.
This study quantifies the impacts of varying levels of herbivory by H. pakistanae on H. verticillata growth. Furthermore, the specific impact of herbivory by H. pakistanae on the photosynthetic potential of H. verticillata is evaluated for the first time.
2. Materials and methods 2.1. Experimental design The impact of H. pakistanae on the growth and photosynthetic potential of dioecious H. verticillata was evaluated in a 10-week growth experiment conducted at the Lewisville Aquatic Ecosystem Research Facility (LAERF), Lewisville, Texas (33E040 4500 N, 96E570 3000 W). The experiment was conducted between early August and mid October, a time when H. verticillata is still growing vigorously and when the shortening day lengths stimulate tuber production. The experiment was conducted in eighteen 114-liter, white plastic tanks (45 cm diameter, 70 cm tall). The tanks were set inside a waterfilled raceway (45–55 cm deep) and beneath a translucent fiberglass canopy. Each tank housed six 1-liter pots (11.3 cm diameter, 0:01 m2 surface area per pot). The pots were filled with fine-textured, heat-sterilized sediment from the LAERF ponds. After sterilization, the sediment was amended with ammonium sulfate (0.04 g per liter dry sediment) and homogenized in a large motorized mortar mixer. Each pot was planted in late June 1999 with two H. verticillata sprigs (15-cm apical tips) collected from local research ponds. The H. verticillata was precultured under greenhouse conditions for 5 weeks to allow the sprigs to establish and begin active growth. Following the establishment period, the pots were moved into the 114-liter tanks on August 4, 1999. The tanks were filled with alum-treated pond water to a depth of 45 cm over the sediment surface of the pots. The alum treatment removes most dissolved phosphorus from the water and minimizes the growth of algae and periphyton within the experimental tanks. The water within each tank was continuously bubbled to replenish CO2 and to keep the water in the tanks mixed. Evaporative loss of water from the tanks (2–3 cm per week) was replaced with additional alum-treated pond water. H. pakistanae larvae were added to each of the 12 tanks on August 5, 1999. In an effort to achieve differing levels of H. pakistanae establishment, six tanks received approximately 125 second and third instar larvae, six additional tanks received 250 larvae, and the final six tanks (controls) received no H. pakistanae. Tanks containing H. pakistanae were tightly covered with 50 24 mesh antivirus insect screen (Green-tek, Edgerton, WI). A cloth sleeve sewn into each screen provided daily access to the tanks to feed adult flies. Tanks that did not re-
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ceive H. pakistanae flies were loosely covered with a screen so that light conditions within all tanks would be similar. Although the nutritional needs of adult H. pakistanae flies are poorly understood, sucrose and protein amendments (sucrose ¼ 7 g table sugar dissolved in 10 ml water; protein ¼ 7 g table sugar + 4 g yeast hydrolysate dissolved in 10 ml water) were provided daily as small liquid droplets in petri dishes that floated on small Styrofoam platforms within each tank. Water temperature was measured several times per week throughout the experiment. Photosynthetically active radiation (PAR) penetrating to the water surface within the tanks was measured at the beginning and the end of the experiment under cloudless conditions utilizing a spherical quantum sensor (Li-Cor LI-190SA, Lincoln, NE). 2.2. Harvest On October 14, 1999, the experiment was terminated and all plants were harvested. Prior to harvest, six apical stems (ca. 25 cm length) of H. verticillata stems were collected from each tank to quantify the abundance of H. pakistanae immatures in each tank. Additional stems to be used for determination of photosynthetic potential were also collected from several tanks believed to show a range of H. pakistanae establishment. Each pot in each tank was then harvested individually, and the basal stems at the sediment surface were cut, thereby separating the above- and belowground tissues. The number of basal stems in each pot was enumerated along with the number of lateral branches, number of axial turions, and number of pistillate flowers. The aboveground tissues were gently washed to remove accumulated sediments and epiphytes. Belowground tissues were washed over a 1-mm sieve to remove sediment and debris. Tubers (subterranean turions), if present, were counted and separated from the remainder of the belowground tissues. Plant tissue samples were bagged and oven-dried to constant weight at 60EC in a forced-draft drying oven. 2.3. Quantification of H. pakistanae The abundance of H. pakistanae immatures ðlarvae þ pupaeÞ was determined from the stems collected for this purpose. Each stem was individually measured in length and examined to determine the total number of larvae and pupae per stem and the proportion of leaves damaged by larval feeding. Stems were then dried to constant weight at 60 °C and larval and immature abundance expressed on a dry-weight basis and on the basis of per m of stem length. Dry weight biomass values for these stems were added to the biomass totals of the tanks from which the samples were collected.
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2.4. Analysis of photosynthetic potential The rate of dark respiration and the photosynthetic potential of H. verticillata stems exhibiting varying levels of H. pakistanae damage were determined in laboratory assays at the time of harvest in October 1999. Net photosynthesis and respiration was determined by oxygen change in clear BOD bottles incubated under various light conditions. Photosynthesis assays were conducted in freshly prepared culture solution (Smart and Barko, 1985). The initial oxygen concentration of the solution was lowered to about 30% air-saturation by bubbling the solution with a mixed gas containing 6% O2 , 0.035% CO2 , and balance N2 for 15 min. Reductions in initial oxygen content were made to minimize the potential of photorespiration caused by high dissolved oxygen concentrations as the assays progressed. The rate of oxygen consumption or evolution was measured for each stem at five light intensities ranging from complete darkness to 450 lE m2 s1 . Oxygen was measured by a self-stirring polarographic oxygen sensor attached to an oxygen meter (YSI 5010 and 5000, respectively, YSI, Yellow Springs, OH). Following exposure to all of the light levels, each stem was examined under a dissecting microscope (10–40 magnification) to determine the proportion of leaves damaged by H. pakistanae larval feeding. The stems were dried to constant weight at 60 °C. Rates of respiration or net photosynthesis were computed from the rate of oxygen change over time and normalized by stem dry weight. Gross photosynthesis was estimated by adding the rate of dark respiration to the net photosynthesis rate computed at each light level. Dry weight biomass values were added to the biomass totals of the tanks from which the samples were collected. 2.5. Statistical analysis Data from all pots in a tank were summed to provide totals for each tank for analysis. Each tank represented one replicate. Pots from within the same tank had similar total biomass, but were not analyzed separately because they were not statistically independent (i.e., they were pseudo-replicates). Four different statistical analyses were performed using Statgraphics Plus version 5.0 (Manugistics, Rockville, MD). (1) Linear regression analysis was used to examine relationships between the number of H. pakistanae immatures and observed leaf damage. (2) One-way analysis of variance (ANOVA) followed by means separation test (Tukey’s HSD) was used to determine differences among three damage-level groups for each of the H. verticillata parameters measured. Tanks were divided into three groups according to the degree of actual H. pakistanae impact as measured by average percent damage to canopy leaves at the time of harvest [0–5% leaf damage (N ¼ 10), 15–40%
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leaf damage (N ¼ 4), and 50–75% leaf damage (N ¼ 4)]. Due to the low number of experimental units in each category, a P value of 0.10 was accepted for statistical significance. (3) Nonlinear regression analysis was used to fit data from each individual H. verticillata stem analyzed for photosynthetic potential to the hyperbolic tangent function of Jassby and Platt (1976). This analysis provided best-fit data for Pmax (rate of gross photosynthesis under light-saturating conditions) and a (initial slope of photosynthesis vs. light curve at low light). (4) A t statistic was used to compare the a value of undamaged stems to that of H. pakistanae-impacted stems.
factor that makes establishment of H. pakistanae more difficult. Although H. pakistanae adults were never observed in control tanks, three of the loosely covered control tanks apparently become colonized by very low numbers of flies. At the time of harvest, three of the six control tanks had no H. pakistanae larvae and had no leaf damage. The remaining three control tanks had minimal numbers of immatures (<50=kg) and leaf damage in these tanks was always <2% of the leaves. Because the control tanks were only loosely covered with screens, any adult flies that escaped from adjacent tanks during the daily feeding could have colonized these control tanks.
3. Results and discussion
3.2. Impacts of H. pakistanae herbivory on H. verticillata growth
3.1. Environmental conditions and H. pakistanae establishment The combination of fiberglass roofing and insect screens over each tank removed about 55% of total incident light. Maximum daily PAR penetrating to the surface of the water averaged about 900 lE m2 s1 . The water temperature of the tanks ranged between 24 and 32 °C throughout the experiment. The degree of H. pakistanae establishment, measured as abundance of larvae and pupae at harvest, was highly variable among tanks and unrelated to the initial stocking density of H. pakistanae larvae. Of the four tanks showing greatest H. pakistanae impacts (average of 50–75% surface canopy leaves damaged) two were initially stocked with 250 larvae while the other two were initially stocked with only 125 H. pakistanae larvae. Four tanks initially stocked with H. pakistanae had little or no fly establishment (<500/kg) and showed virtually no leaf damage. The reason for the lack of establishment in some of the tanks initially inoculated with H. pakistanae is not completely known. However, we did observe that the parasitic wasp Trichopria columbiana Ashmead (Hymenoptera: Diapriidae) was present in some of the tanks. This wasp is present at the LAERF research station and may have been inadvertently introduced to some tanks during the experimental setup. The wasp is known to parasitize aquatic immatures of H. pakistanae in H. verticillata plant tissues and may have contributed to the lack of establishment in these tanks. Wheeler and Center (2001) report parasitism rates due to T. columbiana of 9% or greater for H. pakistanae samples collected from Florida lakes and suggest that such parasitism may contribute to the apparent cyclic nature of H. pakistanae abundance in field samples. Grodowitz (unpublished data) has observed T. columbiana parasitism rates as high as 90% for some field populations of H. pakistanae, and considers this a
Although the levels of H. pakistanae establishment observed did not correlate with the original infestation pattern, the impacts observed on H. verticillata were clearly related to the actual establishment pattern of the biocontrol agent. The degree of leaf damage per stem was highly correlated with density of immatures at harvest for most of the tanks (Fig. 1). High leaf damage (average of 50–75% surface canopy leaves damaged) was observed in tanks having immature levels greater than ca. 6000 immatures per kg (>15 immatures per m stem length) (Fig. 1). Stems with 2000–4000 immatures per kg (3–10 per m) had an intermediate average damage level of 15–40% of canopy leaves damaged. The 10 tanks with 0–5% average leaf damage were observed to have zero or very few larvae and pupae. Two tanks had high levels of leaf damage but relatively few larvae or pupae at the time sampled (Fig. 1, open symbols). Stems from these tanks had a large number of empty pupal cases (not quantified) and very high numbers of eggs (>130,000 eggs per kg). Apparently, these two tanks had just experienced an adult emergence event followed by oviposition by adult females. In the absence of herbivory, H. verticillata quickly grew to the surface of the tanks and during the 10-week growth period produced about 28 g total dry biomass per tank. Since each 45 cm diameter tank contained six 0:01 m2 pots, this total tank biomass corresponds to 176 g per m2 water surface area or 465 g per m2 sediment surface area. The degree of H. pakistanae herbivory, as measured by leaf damage at harvest, significantly affected biomass accumulation, tuber production, and the developmental pattern observed in the H. verticillata plants (Table 1). Total biomass in the tanks declined significantly as leaf herbivory increased (Fig. 2). At the highest level of herbivory, the biomass was reduced by about 30% relative to controls. Herbivory did not impact total number of basal stems but did produce more highly
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Fig. 1. Relationship between abundance of H. pakistanae immatures and observed damage to leaves of H. verticillata stems at the end of a 10-week growth period. Number of immatures are counted as the sum of larvae + pupae observed on 25-cm apical tips of H. verticillata stems. Data shown are values for individual stems harvested from each of the 18 tanks utilized in the experiment (six stems per tank). Open circles or squares indicate stems from two tanks with recent adult emergence.
branched plants at the highest level of herbivory (Table 2). Apparently, herbivory on apical stem tips was sufficiently disruptive to free lateral buds from apical dominance and cause significantly more branching under high levels of herbivory. Van et al. (1998) also report significant suppression of H. verticillata grown in the presence of H. pakistanae in experimental tanks. In two experiments conducted in Florida, controls averaged about 50 g H. verticillata biomass per tray (500 g per m2 ) while plants infested with H. pakistanae produced only about 30% of that total. However, the experiments conducted by Van et al. (1998) utilized H. pakistanae stocking rates 3–5 times higher than those utilized in this experiment. Furthermore, while actual larval abundance was not quantified, they must have experienced much higher levels of in-
225
festation since the herbivory was sufficient to completely defoliate H. verticillata stems and prevent the formation of a surface canopy. There is little information in the literature concerning the level of H. pakistanae infestation needed to impact the development of H. verticillata. In what appears to be the only study reported to date, Wheeler and Center (2001) state that H. pakistanae larval densities of approximately 4000 larvae per m2 within small enclosures resulted in near complete defoliation of H. verticillata. They also report a H. verticillata canopy biomass (top 20 cm) of 25–30 g dry weight for the 0.159 m2 enclosures (37 43 cm) utilized in the experiment. From these data, we estimate that the larval density in the experiments conducted by Wheeler and Center (2001) to have been about 21,000–25,000 larvae per kg canopy biomass, a value >3-fold higher than any obtained in the present study. In the present experiment, an abundance of 15–18 immatures per m canopy stem length was sufficient to produce damage to about 60% of the leaves in the H. verticillata canopy. These results appear reasonable given the feeding rates reported by Buckingham et al. (1989) and the total number of leaves per m we measured. In this experiment, the apical tips of H. verticillata stems averaged about 350 leaves per m. If each larvae consumed 12 leaves during development (Buckingham et al., 1989), the higher level of H. pakistanae immature density found in our study (15–18 per m) should have damaged 50–60% of the leaves, a value remarkably similar to that observed (Fig. 1). These rates of leaf damage are likely only valid for the terminal portions of the stems since fly larvae are likely to be less abundant beneath the canopy surface. Wheeler and Center (1996) report preferential feeding of larvae on the terminal leaves of H. verticillata stems and relatively little impact to leaves removed from the stem tip. Herbivory also impacted the investment of energy to sexual reproduction (flower formation) and asexual formation of tubers (Table 2). The significant decline in number of pistillate flowers observed as herbivory increased may have little ecological significance given that seed production is likely of minor importance in this species relative to vegetative reproduction (Langeland, 1996) and that seed production and viability of monoecious H. verticillata are low compared to other weed species (Langeland and Smith, 1984). Of more obvious ecological importance is the reduction in tuber number and tuber mass (Fig. 2, Table 2). The prolific production of tubers within the sediment provides H. verticillata with an effective mechanism for regrowth following disturbances in the ecosystem (Netherland, 1999) and is one of the characteristics that makes this plant so difficult to control (Doyle and Smart, 2001a). Relative to controls, tuber numbers were reduced by
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Table 1 One-way ANOVA results for effect of herbivory treatment on the measured growth variables of H. verticillata Variable
Source of error
df
Total biomass
Herbivory
2
111.59
15
2.26
2
0.228
15
0.036
Error Tuber biomass
Herbivory Error
No. of tubers
Herbivory Error
No. of basal stems
Herbivory Error
No. of branches
Herbivory Error
No. of flowers
Herbivory Error
Mass per tuber
Herbivory Error
No. of axial turions
Herbivory Error
MS
2
76.63
15
24.12
2
516.3
15
992.6
2
11072
15
1296
2
292.3
15
80.1
2
940.0
15
279.0
2
13.01
15
15.27
F ratio
P
49.28
0.000
6.32
0.010
3.18
0.071
0.52
0.605
8.54
0.003
3.65
0.051
3.37
0.062
0.85
0.44
55% (Fig. 2) and individual tuber mass was reduced by 60% under the highest level of herbivory (Table 2). However, since the tubers were not yet fully formed at the time of harvest, it is unclear whether the trend towards smaller tubers would have held up after the tubers matured. If the trend in tuber size held true for mature tubers, this would constitute a significant finding since tuber size is a good indicator of propagule vigor (Doyle and Smart, 2001b; Netherland, 1999). Assuming this to be true, H. pakistanae herbivory appears to force H. verticillata plants to produce not only fewer, but also weaker tubers.
Fig. 2. Impact of varying levels of H. pakistanae herbivory on H. verticillata total biomass, tuber biomass, and tuber production. Data shown are means SE for groups established based on level of leaf damage (N ¼ number of tanks in a particular category).
Van et al. (1998) also demonstrated reductions in H. verticillata tuber number and biomass of individual tubers due to H. pakistanae herbivory. In their experiments, tuber number was reduced by 30% and the size of individual tubers was reduced by 24%. No impact to tuber numbers by fly herbivory was reported in Wheeler and Center (2001) at either high or low fertilization treatments, possibly because the experiments were conducted during the spring, a period when little tuber production is expected. Biomass was reduced in treat-
Table 2 Impact of H. pakistanae herbivory on various developmental parameters of H. verticillataa Variable
No. of basal stems No. of branches No. of flowers Mass per tuber (mg) No. of axial turions a
Leaf damage level (% of leaves damaged) Very low (0–5%, N ¼ 10)
Intermediate (15–40%, N ¼ 4)
High (50–75%, N ¼ 4)
187:6 12:0 96:6 14:0 a 12:7 3:6 a 43:1 5:7a 4:2 1:6
169:5 4:0 99:5 10:3 a 1:8 1:2 b 34:6 9:7 ab 2:8 0:6
188:0 12:2 181:8 7:0 b 0:8 0:8 b 17:5 3:6 b 1:3 0:6
Shown are means SE for groups of tanks at different levels of leaf damage. Number of tanks (N) falling into each group is indicated. A mean separation test (Tukey’s HSD) was applied to variables having a significant one-way ANOVA (P ¼ 0.10). Means in columns followed by different letters are significantly different at P ¼ 0:10.
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ments with higher fly densities and low fertilizer treatments (Wheeler and Center, 2001). 3.3. Impacts of herbivory on photosynthetic potential Herbivory by H. pakistanae substantially impacted the maximum photosynthetic potential of H. verticillata (Fig. 3). Six control (undamaged) and 10 damaged stems were assayed. Leaf damage due to insect herbivory ranged from 10% to 90%. Control stems had an average Pmax (light-saturated rate of gross photosynthesis) of 1.22 ( 0:05) mg O2 per g dry weight (gdw) per hour although the rate dropped sharply in damaged stems. Pmax was suppressed by 30–40% in stems exhibiting 10–30% leaf damage. In stems with 70–90% of leaves damaged, Pmax averaged only 40% of undamaged stems. Damaged stems appear not to be able to use low light levels as efficiently as undamaged stems. Although the a value, which measures the ability to utilize low levels of light, showed no consistent linear pattern with increased leaf damage, the value was significantly lower in H. pakistanae-impacted stems compared to control stems (t statistic, P < 0:01). Control stems had an average a value of 0.018 mg O2 / gdw/h per lE m2 s1 0:019 ðN ¼ 6Þ while damaged stems averaged only 0.012 mg O2 /gdw/h per lE m2 s1 0:001 ðN ¼ 10Þ. Dark respiration rate of H. pakistanae-impacted stems was not significantly different (P > 0:05) from that of undamaged control stems and averaged 0:38 0:05 and 0:43 0:05 mg O2 per gdw per hour, respectively. Simulating the net photosynthetic production of undamaged control and H. pakistanae-impacted stems through a 24-h period illustrates the potential impact of herbivory on the plant’s daily carbon balance. The fol-
227
lowing assumptions are made: (1) the stems are subjected to 8 h of light-saturating conditions (i.e., PAR >250 lE m2 s1 ), (2) the stems are subjected to 2 h each of light conditions where photosynthesis averages 75%, 50%, and 25% of Pmax , and (3) that rates of dark respiration are constant over 24 h. Under these conditions, the control (undamaged) stems would have a daily surplus of photosynthetic production available for growth equal to about 30% of gross production. In contrast, the H. pakistanae stems with 20% damaged leaves would just break even, with photosynthetic production barely balancing respiratory demands and no surplus remaining for growth. Stems with >20% damaged leaves would suffer a net daily deficit, with respiratory demands exceeding photosynthetic production. For example, with 50% leaf damage, the stems produced only 66% of the total daily respiratory needs; stems with 70–90% leaf damage produced only 55% of total daily respiratory needs. Obviously, under such conditions, the plant could not survive since they would consume more organic material via respiration than was being produced by photosynthesis. Although significant differences in H. verticillata biomass were observed in this experiment, the magnitude of the impact is likely underestimated due to the short-term nature of the experiment. At the end of 10 weeks of growth, the tanks with the highest herbivory had a standing-crop biomass that was lower than that of the controls by about 30%. Had the experiment continued, it is likely that the difference in plant biomass would have increased through time. The plants in the control tanks are likely to have increased in biomass, while the biomass of those subject to H. pakistanae herbivory, with lowered photosynthetic potential, increased at much slower rates. 3.4. Management implications
Fig. 3. Impact of H. pakistanae herbivory (0–90% leaf damage) on maximum photosynthetic potential (Pmax ) of H. verticillata stems. Each symbol represents a single data point (Pmax determined from single stem) except for the first point (0% leaf damage), which shows the mean and standard error of six stems.
Results of this study indicate that establishment of high levels of H. pakistanae (>6000 immatures per kg) should result in substantial H. verticillata leaf damage (>50%) and should impact the accumulation of biomass and the production of tubers by H. verticillata relatively quickly. Unfortunately, field populations to date have rarely achieved densities sufficient to produce such extensive damage to H. verticillata canopies. However, it is encouraging that much less damage (10–30%) to the canopy leaves appears to be sufficient to substantially reduce photosynthetic surplus and dramatically slow the rate of H. verticillata growth. This level of H. pakistanae establishment in the field may be possible. In a recent survey of six field sites where H. pakistanae larvae had been released in Texas, two sites showed H. pakistanae establishment of 1800 and 3100 immatures per kg with associated canopy leaf
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damage of 13% and 19%, respectively (Grodowitz, unpublished data). Our data indicate that these levels of H. pakistanae establishment may be sufficient to impact the long-term growth of H. verticillata. For example, at one of these sites, (i.e., Coleto Creek, Texas) significant declines in the status of the hydrilla have been observed even at only low to moderate fly levels (Grodowitz et al., 1999; Grodowitz, unpublished data).
Acknowledgments The experiments described and the resulting data presented herein, unless otherwise noted, were obtained from research conducted under the Aquatic Plant Control Research Program of the U.S. Army Engineer Research and Development Center, Waterways Experiment Station. Permission was granted by the Chief of Engineers to publish this information. The authors wish to thank Robin Bare and Christi Snell (University of North Texas) for their assistance in setting up, monitoring, and harvesting the experiment.
References Baloch, G.M., Sana-Ullah, 1974. Insects and other organisms associated with Hydrilla verticillata (L.f.) L.C. (Hydrocharitaceae) in Pakistan. In: Proceedings of the 3rd International Symposium of Biological Control of Weeds, Montpellier, France. Commonwealth Institue of Biological Control, Slough, UK, pp. 61–66, Misc. Publ. No. 8. Baloch, G.M., Sana-Ullah, Ghani, M.A., 1980. Some promising insects for the biological control of Hydrilla verticillata in Pakistan. Trop. Pest Manage. 26, 194–200. Buckingham, G.R., 1994. Biological control of aquatic weeds. In: Rosen, D., Bennett, F.D., Capinera, J.L. (Eds.), Pest Management in the Subtropics: Biological Control—A Florida Perspective. Intercept Ltd, Andover, Hampshire, UK, pp. 413–480. Buckingham, G.R., Okrah, E.A. 1993. Biological and host range studies with two species of Hydrellia (Diptera: Ephydridae) that feed on hydrilla. Technical Report A-93-7. U.S. Army Engineer Waterways Experiment Station. Vicksburg, MS. Buckingham, G.R., Okrah, E.A., Thomas, M.C., 1989. Laboratory host range test with Hydrellia pakistanae (Diptera: Ephydridae), an agent for biological control of Hydrilla verticillata (Hydrocharitaceae). Environ. Entomol. 18, 164–171. Center, T.D., Grodowitz, M.J., Cofrancesco, A.F., Jubinsky, G., Snoddy, E., Freeman, J.E., 1997. Establishment of Hydrellia pakistanae (Diptera: Ephydridae) for the biological control of the submersed plant Hydrilla verticillata (Hydrocharitaceae) in the southeastern United States. Biol. Control 8, 65–73. Crawley, M.J., 1989. Insect herbivores and plant population dynamics. Annu. Rev. Entomol. 34, 531–564. Cofrancesco, A.F., 1998. Overview and future direction of biological control technology. J. Aquat. Plant Manage. 36, 49–53.
DeLoach, C.J., 1991. Past successes and current prospects in biological control of weeds in the United States and Canada. Natural Areas J. 11, 129–142. Doyle, R.D., Smart, R.M., 2001a. Effects of drawdown and dessication on tubers of Hydrilla verticillata, an exotic aquatic weed. Weed Sci. 49, 135–140. Doyle, R.D., Smart, R.M., 2001b. Impacts of water column turbidity on the survival and growth of Vallisneria americana winterbuds and seedlings. Lake Reservoir Manage. 17, 17–28. Grodowitz, M.J., Center, T.D., Cofrancesco, A.F., Freedman, J.E., 1997. Release and establishment of Hydrellia balciunasi (Diptera: Ephydridae) for the biological control of the submersed aquatic plant Hydrilla verticillata (Hydrocharitaceae) in the United States. Biol. Control 9, 15–23. Grodowitz, M.J., Freedman, J.E., Cofrancesco, A.F., and Center, T.D. 1999. Status of Hydrellia spp. (Diptera: Ephydridae) release sites in Texas as of December 1998. Miscellaneous Paper A-99-1, U.S. Army Engineer Research and Development Center, Vicksburg, MS. Grodowitz, M.J., Doyle, R.D., Smart, R.M. 2000a. Potential use of insect biocontrol agents for reducing the competitive ability of Hydrilla verticillata. ERCD/EL SR-00-1, U.S. Army Engineer Research and Development Center, Vicksburg, MS. Grodowitz, M.J., Freedman, J.E., Jones, H., Jeffers, L., Lopez, C., Nibling, F., 2000b. Status of waterhyacinth/hydrilla infestations and associated biological control agents in lower Rio Grande valley cooperating irrigation districts. ERDC/EL SR-00-11, U.S. Army Engineer Research and Development Center, Vicksburg, MS. Jassby, A.D., Platt, T., 1976. Mathematical formulation of the relationship between photosynthesis and light for phytoplankton. Limnol. Oceanogr. 21, 540–547. Langeland, K.A., 1996. Hydrilla verticillata (L.f.) Royle (Hydrocharataceae), A The perfect aquatic weed. Castanea 61, 293– 304. Langeland, K.A., Smith, C.B., 1984. Hydrilla produces viable seed in North Carolina Lakes. Aquatics 6, 20–22. Madsen, J.D., 1997. Methods for management of nonindigenous aquatic plants. In: Luken, J.O., Thieret, J.W. (Eds.), Assessment and Management of Plant Invasions. Springer, New York, pp. 145–171. National Academy of Sciences, 1987. Research briefings 1987: report of the research briefing panel on biological control in managed ecosystems. National Academy Press, Washington, DC. Netherland, M.D., 1999. Management impacts on the quiescence and sprouting of subterranean turions of dioecious hydrilla [Hydrilla verticillata (L.f.) Royle]. Ph.D. Dissertation, University of Florida, Gainesville, FL. Okasnen, L., 1990. Predation, herbivory, and plant strategies along gradients of primary productivity. In: Grace, J.B., Tilman, D. (Eds.), Perspectives on Plant Competition. Academic Press, New York, pp. 445–474. Pieterse, A.H., 1981. Hydrilla verticillata-a review. Abstr. Trop. Agric. 7, 9–34. Smart, R.M., Barko, J.W., 1985. Laboratory culture of submersed freshwater macrophytes on natural sediments. Aquat. Bot. 21, 251– 263. Smart, R.M., Doyle, R.D., 1995. Ecological theory and management of submersed aquatic plant communities. Information Exchange Bulletin A-95-3, APCRP, U.S. Army Engineer Waterways Experiment Station, Vicksburg, MS. Van, T.K., Wheeler, G.S., Center, T.D., 1998. Competitive interactions between Hydrilla (Hydrilla verticillata) and Vallisneria (Val-
R.D. Doyle et al. / Biological Control 24 (2002) 221–229 lisneria americana) as influenced by insect herbivory. Biol. Control, 185–192. Van, T.K., Wheeler, G.S., Center, T.D., 1999. Competition between Hydrilla verticillata and Vallisneria americana as influenced by soil fertility. Aquatic Bot. 62, 225–233. Wheeler, G.S., Center, T.D., 1996. The influence of hydrilla leaf quality on larval growth and development for the biological control
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agent Hydrellia pakistanae (Diptera: Ephydridae). Biol. Control 7, 1–9. Wheeler, G.S., Center, T.D., 2001. Impact of the biological control agent Hydrellia pakistanae (Diptera: Ephydridae) on the submersed aquatic weed Hydrilla verticillata (Hydrocharitaceae). Biol. Control 21, 168–181.
Impact of herbivory by Hydrellia pakistanae (Diptera: Ephydridae) on growth and photosynthetic potential of Hydrilla verticillata Robert D. Doyle,a,* Michael Grodowitz,b R. Michael Smart,c and Chetta Owensd a University of North Texas, Denton, TX 76203-0559, USA U.S. Army Engineer Research and Development Center, Vicksburg, MS 39180, USA U.S. Army Engineer Research and Development Center, Lewisville Aquatic Ecosystem Research Facility, Lewisville, TX 75056, USA d ASI, Lewisville Aquatic Ecosystem Research Facility, Lewisville, TX 75056, USA b
c
Received 22 June 2001; accepted 24 January 2002
Abstract The impacts of varying levels of herbivory by Hydrellia pakistanae on the dioecious ecotype of Hydrilla verticillata were evaluated by conducting a 10-week growth experiment within mesocosm tanks. The observed leaf damage to H. verticillata stems was highly correlated with the total number of immature H. pakistanae in H. verticillata tissue at the time of harvest (P < 0:001, R2 > 0:80). Increasing levels of insect herbivory significantly impacted biomass and growth morphology of H. verticillata. Relative to control tanks, plants under intermediate or high levels of herbivory produced progressively less biomass. Insect herbivory also significantly impacted investment of energy in sexual and asexual reproduction. Plants under an intermediate or high level of herbivory produced fewer than 15% of the number of pistillate flowers produced by plants in control tanks. Furthermore, plants subject to high insect herbivory produced fewer and smaller tubers than control tanks. Finally, herbivory had a strong impact on the photosynthetic potential of stems. With 10–30% leaf damage, the maximum rate of light-saturated photosynthesis was reduced 30–40% relative to undamaged controls. Total daily photosynthetic production in these stems was estimated to balance, just barely, the daily respiratory needs of stems. Photosynthetic rate was reduced by about 60% in stems showing 70–90% leaf damage. This level of photosynthetic reduction would make continued survival of the plants unlikely since they would be unable to meet daily respiratory demands. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Hydrilla verticillata; Hydrellia pakistanae; Insect herbivory; Biocontrol; Photosynthesis; Aquatic weed; Invasive species; Aquatic plant management
1. Introduction Hydrilla verticillata (L.f.) Royle (hydrilla) is a major aquatic pest in North America (Pieterse, 1981). It is described as the perfect aquatic weed due to many phenological and physiological adaptations, which allow it to thrive in a variety of shallow aquatic environments (Langeland, 1996). The high biomass production and formation of a dense surface canopy are among the most undesirable characteristics of the species (Smart * Corresponding author. Present address: Department of Biology, Baylor University, P.O. Box 97388, Waco, TX 76798, USA. Fax: 254710-2969. E-mail address: [email protected] (R.D. Doyle).
and Doyle, 1995). These characteristics negatively impact the environment, interfere with management objectives, and may be responsible for the dominance of this species over native plants (Van et al., 1999). Control options for aquatic weeds include various mechanical, physical, chemical, and biological methods (Madsen, 1997). Classical biological control relies on use of host-specific insect herbivores or plant pathogens from the native range of an alien plant (Cofrancesco, 1998). This method has recently been described among the most desirable for the long-term control of nuisance alien plants (National Academy of Sciences, 1987). This method minimizes the chances of unexpected negative impacts seen in control efforts that utilized generalized vertebrate agents (DeLoach, 1991). However, biocontrol
1049-9644/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S 1 0 4 9 - 9 6 4 4 ( 0 2 ) 0 0 0 2 4 - 5
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agents are unlikely to eradicate a species because the impacts of sustained insect herbivory are typically loss of plant vigor and loss of competitive advantages rather than direct plant mortality (Crawley, 1989; Okasnen, 1990). The combination of insect herbivory, in conjunction with establishment of competitive native species, may offer the best long-term solutions (Grodowitz et al., 2000a). Four host-specific insect biological control agents have been introduced into North America for the control of H. verticillata (Buckingham, 1994; Center et al., 1997). The leaf-mining fly Hydrellia pakistanae Deonier (Diptera: Ephydridae) was introduced in 1987 and has been the most effective. The host-specificity of H. pakistanae was confirmed in studies conducted in both Pakistan (Baloch et al., 1980) and in the United States (Buckingham and Okrah, 1993; Buckingham et al., 1989) before release. The life cycle of H. pakistanae is well known (Baloch and Sana-Ullah, 1974; Baloch et al., 1980; Buckingham et al., 1989; Wheeler and Center, 1996). Adult H. pakistanae oviposit on floating or emergent vegetation. After eclosion, the larvae actively search out H. verticillata. The larvae feed exclusively on H. verticillata leaves and damage 9–12 leaves during their three larval instars. The larvae form a puparium from the last larval cuticle and pupate attached to stems of H. verticillata. Total generation time varies between 18 and 30 days depending on temperature and nutritional content of the plant (Grodowitz, unpublished data; Wheeler and Center, 1996). More than three million individuals of H. pakistanae have been released at 25 sites in the United States (Florida, Georgia, Alabama, Louisiana, Texas, and California) and establishment has been confirmed at most sites. In some instances, H. pakistanae populations have been present for over six years (Center et al., 1997). Extensive geographic range-expansion is documented, with populations occurring throughout Florida, north to Muscle Shoals, Alabama, and west to Austin, Texas (Grodowitz et al., 1999; Grodowitz et al., 2000b). Numerous factors including nutritional quality of H. verticillata, life stage of release organisms, number of individuals released, predation, and parasitism appear to impact establishment (Center et al., 1997; Grodowitz, unpublished data; Wheeler and Center, 1996). Although larvae of H. pakistanae unquestionably damage H. verticillata leaves, studies quantifying the impacts of H. pakistanae on H. verticillata are few. In laboratory experiments, damage to canopy leaves of H. verticillata approached 100% in plants infested with 4000 larvae per m2 (Wheeler and Center, 2001). Grodowitz et al. (1997) reports estimates of 20% leaf damage under field conditions when levels reached 2320 larvae per kg of H. verticillata tissue.
This study quantifies the impacts of varying levels of herbivory by H. pakistanae on H. verticillata growth. Furthermore, the specific impact of herbivory by H. pakistanae on the photosynthetic potential of H. verticillata is evaluated for the first time.
2. Materials and methods 2.1. Experimental design The impact of H. pakistanae on the growth and photosynthetic potential of dioecious H. verticillata was evaluated in a 10-week growth experiment conducted at the Lewisville Aquatic Ecosystem Research Facility (LAERF), Lewisville, Texas (33E040 4500 N, 96E570 3000 W). The experiment was conducted between early August and mid October, a time when H. verticillata is still growing vigorously and when the shortening day lengths stimulate tuber production. The experiment was conducted in eighteen 114-liter, white plastic tanks (45 cm diameter, 70 cm tall). The tanks were set inside a waterfilled raceway (45–55 cm deep) and beneath a translucent fiberglass canopy. Each tank housed six 1-liter pots (11.3 cm diameter, 0:01 m2 surface area per pot). The pots were filled with fine-textured, heat-sterilized sediment from the LAERF ponds. After sterilization, the sediment was amended with ammonium sulfate (0.04 g per liter dry sediment) and homogenized in a large motorized mortar mixer. Each pot was planted in late June 1999 with two H. verticillata sprigs (15-cm apical tips) collected from local research ponds. The H. verticillata was precultured under greenhouse conditions for 5 weeks to allow the sprigs to establish and begin active growth. Following the establishment period, the pots were moved into the 114-liter tanks on August 4, 1999. The tanks were filled with alum-treated pond water to a depth of 45 cm over the sediment surface of the pots. The alum treatment removes most dissolved phosphorus from the water and minimizes the growth of algae and periphyton within the experimental tanks. The water within each tank was continuously bubbled to replenish CO2 and to keep the water in the tanks mixed. Evaporative loss of water from the tanks (2–3 cm per week) was replaced with additional alum-treated pond water. H. pakistanae larvae were added to each of the 12 tanks on August 5, 1999. In an effort to achieve differing levels of H. pakistanae establishment, six tanks received approximately 125 second and third instar larvae, six additional tanks received 250 larvae, and the final six tanks (controls) received no H. pakistanae. Tanks containing H. pakistanae were tightly covered with 50 24 mesh antivirus insect screen (Green-tek, Edgerton, WI). A cloth sleeve sewn into each screen provided daily access to the tanks to feed adult flies. Tanks that did not re-
R.D. Doyle et al. / Biological Control 24 (2002) 221–229
ceive H. pakistanae flies were loosely covered with a screen so that light conditions within all tanks would be similar. Although the nutritional needs of adult H. pakistanae flies are poorly understood, sucrose and protein amendments (sucrose ¼ 7 g table sugar dissolved in 10 ml water; protein ¼ 7 g table sugar + 4 g yeast hydrolysate dissolved in 10 ml water) were provided daily as small liquid droplets in petri dishes that floated on small Styrofoam platforms within each tank. Water temperature was measured several times per week throughout the experiment. Photosynthetically active radiation (PAR) penetrating to the water surface within the tanks was measured at the beginning and the end of the experiment under cloudless conditions utilizing a spherical quantum sensor (Li-Cor LI-190SA, Lincoln, NE). 2.2. Harvest On October 14, 1999, the experiment was terminated and all plants were harvested. Prior to harvest, six apical stems (ca. 25 cm length) of H. verticillata stems were collected from each tank to quantify the abundance of H. pakistanae immatures in each tank. Additional stems to be used for determination of photosynthetic potential were also collected from several tanks believed to show a range of H. pakistanae establishment. Each pot in each tank was then harvested individually, and the basal stems at the sediment surface were cut, thereby separating the above- and belowground tissues. The number of basal stems in each pot was enumerated along with the number of lateral branches, number of axial turions, and number of pistillate flowers. The aboveground tissues were gently washed to remove accumulated sediments and epiphytes. Belowground tissues were washed over a 1-mm sieve to remove sediment and debris. Tubers (subterranean turions), if present, were counted and separated from the remainder of the belowground tissues. Plant tissue samples were bagged and oven-dried to constant weight at 60EC in a forced-draft drying oven. 2.3. Quantification of H. pakistanae The abundance of H. pakistanae immatures ðlarvae þ pupaeÞ was determined from the stems collected for this purpose. Each stem was individually measured in length and examined to determine the total number of larvae and pupae per stem and the proportion of leaves damaged by larval feeding. Stems were then dried to constant weight at 60 °C and larval and immature abundance expressed on a dry-weight basis and on the basis of per m of stem length. Dry weight biomass values for these stems were added to the biomass totals of the tanks from which the samples were collected.
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2.4. Analysis of photosynthetic potential The rate of dark respiration and the photosynthetic potential of H. verticillata stems exhibiting varying levels of H. pakistanae damage were determined in laboratory assays at the time of harvest in October 1999. Net photosynthesis and respiration was determined by oxygen change in clear BOD bottles incubated under various light conditions. Photosynthesis assays were conducted in freshly prepared culture solution (Smart and Barko, 1985). The initial oxygen concentration of the solution was lowered to about 30% air-saturation by bubbling the solution with a mixed gas containing 6% O2 , 0.035% CO2 , and balance N2 for 15 min. Reductions in initial oxygen content were made to minimize the potential of photorespiration caused by high dissolved oxygen concentrations as the assays progressed. The rate of oxygen consumption or evolution was measured for each stem at five light intensities ranging from complete darkness to 450 lE m2 s1 . Oxygen was measured by a self-stirring polarographic oxygen sensor attached to an oxygen meter (YSI 5010 and 5000, respectively, YSI, Yellow Springs, OH). Following exposure to all of the light levels, each stem was examined under a dissecting microscope (10–40 magnification) to determine the proportion of leaves damaged by H. pakistanae larval feeding. The stems were dried to constant weight at 60 °C. Rates of respiration or net photosynthesis were computed from the rate of oxygen change over time and normalized by stem dry weight. Gross photosynthesis was estimated by adding the rate of dark respiration to the net photosynthesis rate computed at each light level. Dry weight biomass values were added to the biomass totals of the tanks from which the samples were collected. 2.5. Statistical analysis Data from all pots in a tank were summed to provide totals for each tank for analysis. Each tank represented one replicate. Pots from within the same tank had similar total biomass, but were not analyzed separately because they were not statistically independent (i.e., they were pseudo-replicates). Four different statistical analyses were performed using Statgraphics Plus version 5.0 (Manugistics, Rockville, MD). (1) Linear regression analysis was used to examine relationships between the number of H. pakistanae immatures and observed leaf damage. (2) One-way analysis of variance (ANOVA) followed by means separation test (Tukey’s HSD) was used to determine differences among three damage-level groups for each of the H. verticillata parameters measured. Tanks were divided into three groups according to the degree of actual H. pakistanae impact as measured by average percent damage to canopy leaves at the time of harvest [0–5% leaf damage (N ¼ 10), 15–40%
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leaf damage (N ¼ 4), and 50–75% leaf damage (N ¼ 4)]. Due to the low number of experimental units in each category, a P value of 0.10 was accepted for statistical significance. (3) Nonlinear regression analysis was used to fit data from each individual H. verticillata stem analyzed for photosynthetic potential to the hyperbolic tangent function of Jassby and Platt (1976). This analysis provided best-fit data for Pmax (rate of gross photosynthesis under light-saturating conditions) and a (initial slope of photosynthesis vs. light curve at low light). (4) A t statistic was used to compare the a value of undamaged stems to that of H. pakistanae-impacted stems.
factor that makes establishment of H. pakistanae more difficult. Although H. pakistanae adults were never observed in control tanks, three of the loosely covered control tanks apparently become colonized by very low numbers of flies. At the time of harvest, three of the six control tanks had no H. pakistanae larvae and had no leaf damage. The remaining three control tanks had minimal numbers of immatures (<50=kg) and leaf damage in these tanks was always <2% of the leaves. Because the control tanks were only loosely covered with screens, any adult flies that escaped from adjacent tanks during the daily feeding could have colonized these control tanks.
3. Results and discussion
3.2. Impacts of H. pakistanae herbivory on H. verticillata growth
3.1. Environmental conditions and H. pakistanae establishment The combination of fiberglass roofing and insect screens over each tank removed about 55% of total incident light. Maximum daily PAR penetrating to the surface of the water averaged about 900 lE m2 s1 . The water temperature of the tanks ranged between 24 and 32 °C throughout the experiment. The degree of H. pakistanae establishment, measured as abundance of larvae and pupae at harvest, was highly variable among tanks and unrelated to the initial stocking density of H. pakistanae larvae. Of the four tanks showing greatest H. pakistanae impacts (average of 50–75% surface canopy leaves damaged) two were initially stocked with 250 larvae while the other two were initially stocked with only 125 H. pakistanae larvae. Four tanks initially stocked with H. pakistanae had little or no fly establishment (<500/kg) and showed virtually no leaf damage. The reason for the lack of establishment in some of the tanks initially inoculated with H. pakistanae is not completely known. However, we did observe that the parasitic wasp Trichopria columbiana Ashmead (Hymenoptera: Diapriidae) was present in some of the tanks. This wasp is present at the LAERF research station and may have been inadvertently introduced to some tanks during the experimental setup. The wasp is known to parasitize aquatic immatures of H. pakistanae in H. verticillata plant tissues and may have contributed to the lack of establishment in these tanks. Wheeler and Center (2001) report parasitism rates due to T. columbiana of 9% or greater for H. pakistanae samples collected from Florida lakes and suggest that such parasitism may contribute to the apparent cyclic nature of H. pakistanae abundance in field samples. Grodowitz (unpublished data) has observed T. columbiana parasitism rates as high as 90% for some field populations of H. pakistanae, and considers this a
Although the levels of H. pakistanae establishment observed did not correlate with the original infestation pattern, the impacts observed on H. verticillata were clearly related to the actual establishment pattern of the biocontrol agent. The degree of leaf damage per stem was highly correlated with density of immatures at harvest for most of the tanks (Fig. 1). High leaf damage (average of 50–75% surface canopy leaves damaged) was observed in tanks having immature levels greater than ca. 6000 immatures per kg (>15 immatures per m stem length) (Fig. 1). Stems with 2000–4000 immatures per kg (3–10 per m) had an intermediate average damage level of 15–40% of canopy leaves damaged. The 10 tanks with 0–5% average leaf damage were observed to have zero or very few larvae and pupae. Two tanks had high levels of leaf damage but relatively few larvae or pupae at the time sampled (Fig. 1, open symbols). Stems from these tanks had a large number of empty pupal cases (not quantified) and very high numbers of eggs (>130,000 eggs per kg). Apparently, these two tanks had just experienced an adult emergence event followed by oviposition by adult females. In the absence of herbivory, H. verticillata quickly grew to the surface of the tanks and during the 10-week growth period produced about 28 g total dry biomass per tank. Since each 45 cm diameter tank contained six 0:01 m2 pots, this total tank biomass corresponds to 176 g per m2 water surface area or 465 g per m2 sediment surface area. The degree of H. pakistanae herbivory, as measured by leaf damage at harvest, significantly affected biomass accumulation, tuber production, and the developmental pattern observed in the H. verticillata plants (Table 1). Total biomass in the tanks declined significantly as leaf herbivory increased (Fig. 2). At the highest level of herbivory, the biomass was reduced by about 30% relative to controls. Herbivory did not impact total number of basal stems but did produce more highly
R.D. Doyle et al. / Biological Control 24 (2002) 221–229
Fig. 1. Relationship between abundance of H. pakistanae immatures and observed damage to leaves of H. verticillata stems at the end of a 10-week growth period. Number of immatures are counted as the sum of larvae + pupae observed on 25-cm apical tips of H. verticillata stems. Data shown are values for individual stems harvested from each of the 18 tanks utilized in the experiment (six stems per tank). Open circles or squares indicate stems from two tanks with recent adult emergence.
branched plants at the highest level of herbivory (Table 2). Apparently, herbivory on apical stem tips was sufficiently disruptive to free lateral buds from apical dominance and cause significantly more branching under high levels of herbivory. Van et al. (1998) also report significant suppression of H. verticillata grown in the presence of H. pakistanae in experimental tanks. In two experiments conducted in Florida, controls averaged about 50 g H. verticillata biomass per tray (500 g per m2 ) while plants infested with H. pakistanae produced only about 30% of that total. However, the experiments conducted by Van et al. (1998) utilized H. pakistanae stocking rates 3–5 times higher than those utilized in this experiment. Furthermore, while actual larval abundance was not quantified, they must have experienced much higher levels of in-
225
festation since the herbivory was sufficient to completely defoliate H. verticillata stems and prevent the formation of a surface canopy. There is little information in the literature concerning the level of H. pakistanae infestation needed to impact the development of H. verticillata. In what appears to be the only study reported to date, Wheeler and Center (2001) state that H. pakistanae larval densities of approximately 4000 larvae per m2 within small enclosures resulted in near complete defoliation of H. verticillata. They also report a H. verticillata canopy biomass (top 20 cm) of 25–30 g dry weight for the 0.159 m2 enclosures (37 43 cm) utilized in the experiment. From these data, we estimate that the larval density in the experiments conducted by Wheeler and Center (2001) to have been about 21,000–25,000 larvae per kg canopy biomass, a value >3-fold higher than any obtained in the present study. In the present experiment, an abundance of 15–18 immatures per m canopy stem length was sufficient to produce damage to about 60% of the leaves in the H. verticillata canopy. These results appear reasonable given the feeding rates reported by Buckingham et al. (1989) and the total number of leaves per m we measured. In this experiment, the apical tips of H. verticillata stems averaged about 350 leaves per m. If each larvae consumed 12 leaves during development (Buckingham et al., 1989), the higher level of H. pakistanae immature density found in our study (15–18 per m) should have damaged 50–60% of the leaves, a value remarkably similar to that observed (Fig. 1). These rates of leaf damage are likely only valid for the terminal portions of the stems since fly larvae are likely to be less abundant beneath the canopy surface. Wheeler and Center (1996) report preferential feeding of larvae on the terminal leaves of H. verticillata stems and relatively little impact to leaves removed from the stem tip. Herbivory also impacted the investment of energy to sexual reproduction (flower formation) and asexual formation of tubers (Table 2). The significant decline in number of pistillate flowers observed as herbivory increased may have little ecological significance given that seed production is likely of minor importance in this species relative to vegetative reproduction (Langeland, 1996) and that seed production and viability of monoecious H. verticillata are low compared to other weed species (Langeland and Smith, 1984). Of more obvious ecological importance is the reduction in tuber number and tuber mass (Fig. 2, Table 2). The prolific production of tubers within the sediment provides H. verticillata with an effective mechanism for regrowth following disturbances in the ecosystem (Netherland, 1999) and is one of the characteristics that makes this plant so difficult to control (Doyle and Smart, 2001a). Relative to controls, tuber numbers were reduced by
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Table 1 One-way ANOVA results for effect of herbivory treatment on the measured growth variables of H. verticillata Variable
Source of error
df
Total biomass
Herbivory
2
111.59
15
2.26
2
0.228
15
0.036
Error Tuber biomass
Herbivory Error
No. of tubers
Herbivory Error
No. of basal stems
Herbivory Error
No. of branches
Herbivory Error
No. of flowers
Herbivory Error
Mass per tuber
Herbivory Error
No. of axial turions
Herbivory Error
MS
2
76.63
15
24.12
2
516.3
15
992.6
2
11072
15
1296
2
292.3
15
80.1
2
940.0
15
279.0
2
13.01
15
15.27
F ratio
P
49.28
0.000
6.32
0.010
3.18
0.071
0.52
0.605
8.54
0.003
3.65
0.051
3.37
0.062
0.85
0.44
55% (Fig. 2) and individual tuber mass was reduced by 60% under the highest level of herbivory (Table 2). However, since the tubers were not yet fully formed at the time of harvest, it is unclear whether the trend towards smaller tubers would have held up after the tubers matured. If the trend in tuber size held true for mature tubers, this would constitute a significant finding since tuber size is a good indicator of propagule vigor (Doyle and Smart, 2001b; Netherland, 1999). Assuming this to be true, H. pakistanae herbivory appears to force H. verticillata plants to produce not only fewer, but also weaker tubers.
Fig. 2. Impact of varying levels of H. pakistanae herbivory on H. verticillata total biomass, tuber biomass, and tuber production. Data shown are means SE for groups established based on level of leaf damage (N ¼ number of tanks in a particular category).
Van et al. (1998) also demonstrated reductions in H. verticillata tuber number and biomass of individual tubers due to H. pakistanae herbivory. In their experiments, tuber number was reduced by 30% and the size of individual tubers was reduced by 24%. No impact to tuber numbers by fly herbivory was reported in Wheeler and Center (2001) at either high or low fertilization treatments, possibly because the experiments were conducted during the spring, a period when little tuber production is expected. Biomass was reduced in treat-
Table 2 Impact of H. pakistanae herbivory on various developmental parameters of H. verticillataa Variable
No. of basal stems No. of branches No. of flowers Mass per tuber (mg) No. of axial turions a
Leaf damage level (% of leaves damaged) Very low (0–5%, N ¼ 10)
Intermediate (15–40%, N ¼ 4)
High (50–75%, N ¼ 4)
187:6 12:0 96:6 14:0 a 12:7 3:6 a 43:1 5:7a 4:2 1:6
169:5 4:0 99:5 10:3 a 1:8 1:2 b 34:6 9:7 ab 2:8 0:6
188:0 12:2 181:8 7:0 b 0:8 0:8 b 17:5 3:6 b 1:3 0:6
Shown are means SE for groups of tanks at different levels of leaf damage. Number of tanks (N) falling into each group is indicated. A mean separation test (Tukey’s HSD) was applied to variables having a significant one-way ANOVA (P ¼ 0.10). Means in columns followed by different letters are significantly different at P ¼ 0:10.
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ments with higher fly densities and low fertilizer treatments (Wheeler and Center, 2001). 3.3. Impacts of herbivory on photosynthetic potential Herbivory by H. pakistanae substantially impacted the maximum photosynthetic potential of H. verticillata (Fig. 3). Six control (undamaged) and 10 damaged stems were assayed. Leaf damage due to insect herbivory ranged from 10% to 90%. Control stems had an average Pmax (light-saturated rate of gross photosynthesis) of 1.22 ( 0:05) mg O2 per g dry weight (gdw) per hour although the rate dropped sharply in damaged stems. Pmax was suppressed by 30–40% in stems exhibiting 10–30% leaf damage. In stems with 70–90% of leaves damaged, Pmax averaged only 40% of undamaged stems. Damaged stems appear not to be able to use low light levels as efficiently as undamaged stems. Although the a value, which measures the ability to utilize low levels of light, showed no consistent linear pattern with increased leaf damage, the value was significantly lower in H. pakistanae-impacted stems compared to control stems (t statistic, P < 0:01). Control stems had an average a value of 0.018 mg O2 / gdw/h per lE m2 s1 0:019 ðN ¼ 6Þ while damaged stems averaged only 0.012 mg O2 /gdw/h per lE m2 s1 0:001 ðN ¼ 10Þ. Dark respiration rate of H. pakistanae-impacted stems was not significantly different (P > 0:05) from that of undamaged control stems and averaged 0:38 0:05 and 0:43 0:05 mg O2 per gdw per hour, respectively. Simulating the net photosynthetic production of undamaged control and H. pakistanae-impacted stems through a 24-h period illustrates the potential impact of herbivory on the plant’s daily carbon balance. The fol-
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lowing assumptions are made: (1) the stems are subjected to 8 h of light-saturating conditions (i.e., PAR >250 lE m2 s1 ), (2) the stems are subjected to 2 h each of light conditions where photosynthesis averages 75%, 50%, and 25% of Pmax , and (3) that rates of dark respiration are constant over 24 h. Under these conditions, the control (undamaged) stems would have a daily surplus of photosynthetic production available for growth equal to about 30% of gross production. In contrast, the H. pakistanae stems with 20% damaged leaves would just break even, with photosynthetic production barely balancing respiratory demands and no surplus remaining for growth. Stems with >20% damaged leaves would suffer a net daily deficit, with respiratory demands exceeding photosynthetic production. For example, with 50% leaf damage, the stems produced only 66% of the total daily respiratory needs; stems with 70–90% leaf damage produced only 55% of total daily respiratory needs. Obviously, under such conditions, the plant could not survive since they would consume more organic material via respiration than was being produced by photosynthesis. Although significant differences in H. verticillata biomass were observed in this experiment, the magnitude of the impact is likely underestimated due to the short-term nature of the experiment. At the end of 10 weeks of growth, the tanks with the highest herbivory had a standing-crop biomass that was lower than that of the controls by about 30%. Had the experiment continued, it is likely that the difference in plant biomass would have increased through time. The plants in the control tanks are likely to have increased in biomass, while the biomass of those subject to H. pakistanae herbivory, with lowered photosynthetic potential, increased at much slower rates. 3.4. Management implications
Fig. 3. Impact of H. pakistanae herbivory (0–90% leaf damage) on maximum photosynthetic potential (Pmax ) of H. verticillata stems. Each symbol represents a single data point (Pmax determined from single stem) except for the first point (0% leaf damage), which shows the mean and standard error of six stems.
Results of this study indicate that establishment of high levels of H. pakistanae (>6000 immatures per kg) should result in substantial H. verticillata leaf damage (>50%) and should impact the accumulation of biomass and the production of tubers by H. verticillata relatively quickly. Unfortunately, field populations to date have rarely achieved densities sufficient to produce such extensive damage to H. verticillata canopies. However, it is encouraging that much less damage (10–30%) to the canopy leaves appears to be sufficient to substantially reduce photosynthetic surplus and dramatically slow the rate of H. verticillata growth. This level of H. pakistanae establishment in the field may be possible. In a recent survey of six field sites where H. pakistanae larvae had been released in Texas, two sites showed H. pakistanae establishment of 1800 and 3100 immatures per kg with associated canopy leaf
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damage of 13% and 19%, respectively (Grodowitz, unpublished data). Our data indicate that these levels of H. pakistanae establishment may be sufficient to impact the long-term growth of H. verticillata. For example, at one of these sites, (i.e., Coleto Creek, Texas) significant declines in the status of the hydrilla have been observed even at only low to moderate fly levels (Grodowitz et al., 1999; Grodowitz, unpublished data).
Acknowledgments The experiments described and the resulting data presented herein, unless otherwise noted, were obtained from research conducted under the Aquatic Plant Control Research Program of the U.S. Army Engineer Research and Development Center, Waterways Experiment Station. Permission was granted by the Chief of Engineers to publish this information. The authors wish to thank Robin Bare and Christi Snell (University of North Texas) for their assistance in setting up, monitoring, and harvesting the experiment.
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