Reproduction and Development of the Biocontrol AgentHydrellia pakistanae(Diptera: Ephydridae) on Monoecious Hydrilla

BIOLOGICAL CONTROL ARTICLE NO.

7, 275–280 (1996)

0094

Reproduction and Development of the Biocontrol Agent Hydrellia pakistanae (Diptera: Ephydridae) on Monoecious Hydrilla F. ALLEN DRAY JR.

AND

TED D. CENTER

University of Florida Fort Lauderdale Research and Education Center, 3205 College Avenue, Fort Lauderdale, Florida 33314; and United States Department of Agriculture, Agricultural Research Service, Aquatic Weed Control Laboratory, 3205 College Avenue, Fort Lauderdale, Florida 33314 Received August 2, 1995; accepted July 1, 1996

Previous investigations of the laboratory biology and host range of Hydrellia pakistanae, a biological control of Hydrilla verticillata (hydrilla), used the dioecious hydrilla biotype common to Florida, Texas, and California. A monoecious biotype that is now spreading throughout the mid-Atlantic states, California, and Washington was not investigated during these original studies. We therefore compared the dioecious and monoecious hydrilla biotypes as hosts for H. pakistanae. Female H. pakistanae accepted the two biotypes equally as ovipositional substrates. Overall developmental success differed little: 42% of eggs oviposited on monoecious hydrilla produced adults compared to 39% of eggs oviposited on dioecious hydrilla. Fly development required about 33 days on both biotypes (at 22 6 2°C), but larvae that completed development mined 1.6 times as many leaves on monoecious hydrilla as on dioecious plants. These data suggest that H. pakistanae would be a useful biocontrol agent of monoecious hydrilla, should this plant invade areas where it can grow as a perennial. r 1996 Academic Press, Inc. KEY WORDS: Hydrilla verticillata; Hydrellia pakistanae; classical biological control; biotypes; herbivory; monoecious; dioecious; aquatic weed; submerged macrophyte.

INTRODUCTION

Hydrellia pakistanae Deonier is a small ephydrid fly native to tropical and temperate regions of Asia (Deonier, 1978, 1993). The larvae mine in leaves of the freshwater macrophyte Hydrilla verticillata (L.f.) Royle, often causing extensive damage to the plant (Baloch and Sana-Ullah, 1974; Deonier, 1978; Buckingham et al., 1989). Field surveys and preliminary laboratory host-range tests conducted in Pakistan indicated that hydrilla was the sole host of this fly (Baloch and Sana-Ullah, 1974). As a result, Baloch et al. (1980) recommended H. pakistanae as a biocontrol agent for use against hydrilla. Further testing by Buckingham et

al. (1989) confirmed its host specificity, and in late 1987 H. pakistanae was approved for release in the United States. The first H. pakistanae released in the United States were progeny of insects originally collected in subtropical Bangalore, India (Buckingham et al., 1989). Later releases also included progeny of flies collected in Pakistan (Center, 1992). These releases occurred during 1987–1990 in Florida (Center, 1989, 1992; Center et al., 1991) where the subtropical climate and dense hydrilla monocultures seemed ideally suited for establishment of this biocontrol agent. By 1990, persistent H. pakistanae populations had established on several Florida waterways and the fly was rapidly dispersing throughout the state (Center et al., 1991; Center, 1992). The adventive range of H. verticillata in the United States extends well into the mid-Atlantic states and westward to Texas and California (Steward and Van, 1987). Comparisons of multi-isoenzymic phenotypes suggest that United States populations represent two distinct hydrilla biotypes, both of which differ from the 21 biotypes presently known from other regions of the world (Verkleij et al., 1983; Pieterse et al., 1984, 1985; Ryan, 1989, 1991). Plants found in the mid-Atlantic states are monoecious, representing a single multiisoenzymic phenotype (Verkleij et al., 1983; Steward et al., 1984; Verkleij and Pieterse, 1986; Ryan, 1991). In contrast, the exclusively dioecious female plants found in Florida, Texas, and California share a multi-isoenzymic phenotype that is quite distinct from the more northern biotype (Verkleij et al., 1983; Steward et al., 1984; Verkleij and Pieterse, 1986; Ryan, 1991). The ranges of the two biotypes overlap along the Virginia/North Carolina border (Ryan et al., 1995). Populations in other southern states are assumed to be dioecious, but have not been characterized through electrophoresis. Farmer and Adams (1989) suggested that high genetic diversity in plant species may indicate the presence of physiological races. Further, they speculated that these races could respond differently to biological

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1049-9644/96 $18.00 Copyright r 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

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controls. Plant biochemistry mediates adaptive processes thought to drive herbivorous insects toward host specificity (Hanson, 1983; Strong et al., 1984). Thus, biochemical differences between physiological races could dramatically alter the performance of biological control insects. Prerelease investigations of H. pakistanae’s host range included over 50 plant species in 27 families (Buckingham et al., 1989), but did not include the monoecious United States hydrilla biotype. We therefore decided to compare oviposition, larval development, pupation, and adult emergence of H. pakistanae on the monoecious and dioecious hydrilla biotypes. MATERIALS AND METHODS

The hydrilla used in these experiments was grown in outdoor tanks (1.7-m2 area, 0.6 m deep) at the Fort Lauderdale Research and Education Center (FLREC). Constant water flow from a pond at the FLREC provided complete volume (about 1000 liters) exchange every 12 h. Two plant biotypes were used, the dioecious female hydrilla found in Florida and the monoecious form infesting the Potomac River near Washington, DC. The biotypes were cultivated from subterranean turions (also called tubers) and were kept in separate tanks to prevent cross-contamination. The tubers were provided by Dr. Kerry Steward from cultures maintained at the USDA, ARS Aquatic Weed Control Laboratory (AWCL) in Fort Lauderdale, Florida. These were sprouted in a plant growth chamber and transplanted to pans containing a mixture of 95% sandy loam and 5% cow manure. Each pan received 10 sprouted tubers, and 12 pans were placed in each tank. Hydrilla sprigs used in our experiments were harvested only after the plant material formed a thick canopy over the water surface. Environmental conditions in the two tanks remained identical throughout the study. Hydrilla sprigs of each biotype were collected randomly from their respective tanks. These sprigs were lyophilized, ground, and analyzed for chemical and proximate constituents following the procedures of Allen et al. (1974). Proximate constituents included percentages of dry weight, ash, lipids (nonvolatile), and crude fiber. Soluble protein was extracted with 0.10 N NaOH and estimated as ribulose 1,5-diphosphate carboxylase (RUBISCO) using Bio-Rad Protein Assay reagent (Bio-Rad Laboratories, Richmond, CA) (Jones et al., 1989). Nitrogen (N), phosphorus (P), potassium (K), magnesium (Mg), calcium (Ca), iron (Fe), manganese (Mn), and zinc (Zn) were extracted with a sulfuric acid–hydrogen peroxide digest and quantified using spectrophotometry (Center and Van, 1989). These analyses were intended only to characterize the plant tissues; hence, the results were not statistically compared between biotypes.

Laboratory colonies maintained at the FLREC provided insects for these experiments. These colonies derived from quarantine stock provided by Dr. Gary Buckingham (USDA, ARS Biological Control Laboratory, Gainesville, FL) and represented progeny of flies collected in Bangalore, India. Fly colonies were maintained in 3.8-liter (1 gallon) glass jars following procedures developed by Buckingham et al. (1989). Oviposition trials. Ovipositional preference tests were conducted in glass-topped, wooden cages (i.e., sleeve cages). Holes in the fronts of these cages were fitted with muslin sleeves to facilitate reaching into the cages while they remained closed, thereby preventing escape of adult flies. Young hydrilla sprigs were measured volumetrically (each sprig displaced 5 ml of water) to ensure that we presented the flies with equal amounts of plant material. We then placed the sprigs in petri dishes (10 cm diameter) and added water so that half of each sprig was exposed. Two petri dishes, one containing dioecious hydrilla and the other monoecious hydrilla, were placed in a sleeve cage into which adult flies were then released. A minimum of 10 females was included in each test (the absolute number varied depending upon fly availability), and the flies were allowed to oviposit for 24 to 72 h. The petri dishes were then removed from the sleeve cage and the eggs on each biotype were counted. The adult flies were provided a yeast hydrolysate–sugar solution (Buckingham et al., 1989) as a food source prior to and during the ovipositional tests. The trials were conducted under fluorescent lighting at 44.7 µE/ m2/s; ambient temperature during the trials was 22 6 2°C. Differences in the numbers of eggs oviposited on each biotype during 51 trials (conducted during April 1–August 17, 1989) were evaluated using a paired comparisons t test. Colony success trials. We inoculated hydrilla cultures with eggs to examine H. pakistanae’s ability to establish colonies on each biotype. Each culture in these colony success trials was composed of a 3.8-liter jar covered by nylon organdy cloth and filled with pond water containing one of the hydrilla biotypes. Twentysix trials were conducted during April 1–July 28, 1989, consisting of two culture jars (one of each hydrilla biotype) per trial. Jars received equal amounts of plant material (measured volumetrically: initially 150 ml displacement, later 100 ml). Eggs were obtained by allowing females to oviposit on hydrilla in sleeve cages as described above. The egg-infested plant material was transferred to paired culture jars (one jar of each biotype), taking care not to cross-contaminate the biotypes. Each pair of jars was maintained under artificial light (44.7 µE/m2/s) for 28 days during which the number of adults emerging from each jar was recorded. Ambient temperatures ranged from 23 to 35°C over the course of the study. Colony success was

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H. pakistanae DEVELOPMENT ON MONOECIOUS HYDRILLA

evaluated in terms of the number of adults that emerged as a proportion of the eggs with which the culture was inoculated. The data were analyzed using a paired comparisons t test. Developmental trials. We also investigated the effect of host plant biotype on development and survival of immature H. pakistanae. Single whorls of hydrilla leaves, each with only 1 egg, were isolated in small (35 mm diameter, 25 mm deep) cups covered with ventilated caps. Dates for eclosion, larval molts, pupation, and emergence were recorded for 200 eggs, 100 on each biotype. The number of leaves mined during each stadium and the sex ratio of emerging adults were also noted. Mined whorls were replaced with fresh whorls as necessary. The experiment was conducted under fluorescent lighting at 44.7 µE/m2/s; ambient temperature during the experiment was 22 6 2°C. Differences in survival were analyzed using a G test for goodness of fit with Williams’ correction applied to the result (Sokal and Rohlf, 1981). Developmental times were analyzed using an unpaired t test. RESULTS AND DISCUSSION

Oviposition trials. H. pakistanae females preferred neither biotype as an ovipositional substrate (6268 eggs on monoecious hydrilla vs 7020 eggs on dioecious hydrilla; paired comparison t 5 1.65, P . 0.10). This is not surprising. Buckingham et al. (1989) and Buckingham and Okrah (1993) reported that H. pakistanae females oviposited on several aquatic macrophytes (e.g., Elodea canadensis Rich. in Michx.) in nearly equal proportion to hydrilla during host screening trials. Also, Baloch et al. (1980) reported that H. pakistanae females oviposit indiscriminately on a broad spectrum of plants in Pakistan. The adaptive significance of this behavior becomes apparent when one considers that hydrilla populations do not always extend from the hydrosoil to the water surface. Such populations represent an ephemeral ovipositional substrate that could often be unavailable to fastidious females. Buckingham and Okrah (1993) demonstrated that H. pakistanae neonates are able to locate and infest hydrilla up to 1 m below the water surface, however. Consequently, females that oviposit on alternative substrates intermixed with hydrilla can successfully produce progeny under circumstances where more discriminating females, those accepting only hydrilla, cannot. Less discriminating females thus have an adaptive advantage over their more fastidious counterparts. Colony success trials. H. pakistanae productivity was highly variable during the colony success trials, with survival from the egg stage through adult emergence ranging from 0 to 87%. This variability is not unexpected as Buckingham and Okrah (1993) reported

11–70% of H. pakistanae eggs produced adults in their studies with dioecious hydrilla. Twenty-one of the 26 colonies initiated on monoecious hydrilla produced adults (compared to 25 of 26 on dioecious hydrilla), so the monoecious biotype was clearly an acceptable host to this fly. However, colonies maintained on dioecious hydrilla were more successful than those on monoecious hydrilla (n 5 26 pairs, paired comparison t 5 4.10, P , 0.001). Only 844 adults emerged from cultures containing monoecious hydrilla. This represents just 20% of the 4273 eggs with which these cultures were inoculated. In contrast, 1337 (35%) of the 3777 eggs oviposited on dioecious hydrilla produced adults. Developmental trials. Egg to adult survival on monoecious hydrilla was greater during the developmental trials (42%; Table 1) than during the colony success trials. Reasons for the discrepancy are unclear. Perhaps the intensive care that flies received during the developmental studies enhanced their chances for survival. Alternatively, jars may have been overstocked with plant material during colony success trials. Recent observations suggest this could have rendered the cultures suboptimal for fly development. Even so, survival on monoecious hydrilla in both studies was within the range obtained by Buckingham and Okrah (1993). Increased mortality during the colony success trials, relative to the developmental studies, may have occurred during the early stages in H. pakistanae’s life cycle. Table 1 shows that 16% of the eggs used to initiate the developmental studies failed to eclose and 38% of the neonates died before molting. Overall, nearly 80% of the mortality observed during the developmental trials occurred during these early stages. This high level of mortality by the mid-larval stage is a common phenomenon among insects (Price, 1975). Egg mortality during the developmental trials was inexplicably higher (Gadj 5 4.63, P , 0.05) on dioecious

TABLE 1 Survival of H. pakistanae when Cultured on Monoecious vs Dioecious Hydrilla Biotypes a Dioecious hydrilla

Egg First instar Second instar Third instar Pupa

Monoecious hydrilla

Survival (%)

n

Survival (%)

n

79.0 57.0 97.8 97.7 90.7

100 79 45 44 43

90.0 66.7 95.0 80.7 93.5

100 90 60 57 46

Gadj 5 4.63, P , 0.05 Gadj 5 1.67, P . 0.10 Gadj 5 0.51, P . 0.10 Gadj 5 7.84, P , 0.01 Gadj 5 0.01, P . 0.90

a Survival represents the number of individuals that successfully completed a stadium as a proportion of the number that entered that stadium. Data were compared using a G test for goodness of fit with Williams’ correction applied to the result (Sokal and Rohlf, 1981).

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hydrilla than on monoecious hydrilla (Table 1). This was compensated for by a substantially greater pupation success on the dioecious biotype, however. About 98% of the third-instar larvae feeding on dioecious hydrilla pupated, compared to only 81% on monoecious hydrilla (Gadj 5 7.87, P , 0.01). Survival of first and second instars did not differ among biotypes (Gadj 5 1.66 and 0.51, respectively, P . 0.10) (see also Table 1). Overall H. pakistanae developed equally well on the dioecious and monoecious hydrilla biotypes (Gadj 5 0.18, P . 0.50) during these trials. Successful development required about 33 days on either biotype (Table 2). Larvae fed dioecious hydrilla mined 12 leaves prior to pupation (Table 2). This was identical to results obtained by Buckingham and Okrah (1993) and Baloch and Sana-Ullah (1974). These values are lower than the 19 leaves mined by larvae fed monoecious hydrilla (t 5 6.91, P , 0.0001), however. The disparity was most dramatic among third-instar larvae (Table 2), which may account for the lower pupation success for individuals on the monoecious biotype. Leaves from monoecious hydrilla were generally longer, more slender, and had a more delicate appearance than those from dioecious hydrilla, suggesting that leaf size may have been a factor in the differential feeding. We did not quantify these differences. Verkleij et al. (1983), however, were unable to distinguish between the two biotypes (called Washington, DC and Miami, Florida, in their study) in terms of average leaf area (28.6 vs 28.5 mm2, respectively) or length/width ratios (6.6 vs 5.1, respectively). The higher proportion of leaves mined by larvae fed monoecious hydrilla is also suggestive of compensatory feeding (Slansky, 1993) and could thus be construed as indicating that nutritional quality was deficient in these tissues. Preliminary analysis of plant nutritional

data do not support this interpretation, however. Although leaves from the monoecious biotype contained more water, seemingly making the nutrient content more dilute, they also contained 20% more lipids and 15% more nitrogen (% dry weight basis) than the dioecious plants (Table 3). Consequently, monoecious and dioecious plants provided about the same nutrition (% fresh weight basis). An alternative explanation might be that smaller quantities of available tissue were ingested per leaf for monoecious vs. dioecious plants. Unfortunately, this was beyond the scope of our investigation. However, the nutritional ecology of H. pakistanae in relation to the various hydrilla biotypes is a subject that warrants further attention. From a biological control standpoint, the more leaves mined per larva the better. Thus, our data suggest that similar densities of H. pakistanae may be more efficacious on the monoecious hydrilla biotype than on the dioecious biotype. Of course, differences in climate might, by limiting the period during which damage to the plant can accumulate, offset any advantage this feeding disparity provides. This, too, invites further investigation. In summary, H. pakistanae should readily oviposit on field populations of the monoecious hydrilla biotype found in some United States waterways. Absence of hydrilla at the water surface should not interfere with population establishment because females oviposit on many aquatic and semiaquatic plant species (Baloch and Sana-Ullah, 1974; Buckingham et al., 1989; Buckingham and Okrah, 1993). Neonates abandon nonhydrilla ovipositional substrates and drop through the water column to locate submersed hydrilla (Buckingham and Okrah, 1993). Despite high mortality (due perhaps to host-searching behavior) during early sta-

TABLE 2 Comparison of Larval H. pakistanae Development and Feeding when Cultured on Monoecious vs Dioecious Hydrilla Biotypes Dioecious hydrilla

Developmental period, d Egg First instar Second instar Third instar Pupa Total Leaves mined, no. First instar Second instar Third instar Total

Monoecious hydrilla

Mean (S.D.)

Range

n

Mean (S.D.)

Range

n

3.9 (0.25) 5.4 (1.01) 4.4 (1.28) 9.1 (2.38) 10.8 (1.31) 33.1 (4.54)

3–4 3–7 2–7 6–15 9–14 27–42

79 45 44 43 38 a 38 a

4.0 (0.11) 5.6 (0.81) 4.3 (0.93) 8.6 (2.04) 11.3 (1.28) 33.4 (2.18)

3–4 4–8 2–7 6–18 10–15 29–39

90 60 57 46 42 42

t 5 1.84, P 5 0.0681 t 5 1.13, P 5 0.2618 t 5 0.84, P 5 0.4001 t 5 1.13, P 5 0.2623 t 5 1.69, P 5 0.0958 t 5 0.40, P 5 0.6903

1.0 (0.0) 1.7 (0.95) 9.2 (4.74) 12.0 (4.95)

1 1–5 3–19 5–22

45 44 43 39

1.1 (0.32) 2.8 (1.02) 15.3 (4.52) 19.1 (4.82)

1–2 1–6 5–25 9–29

60 57 46 42

t 5 2.39, P 5 0.0188 t 5 5.16, P , 0.0001 t 5 6.15, P , 0.0001 t 5 6.91, P , 0.0001

a The date of emergence was uncertain for one adult; thus, pupal duration for this individual could not be included in the analyses. However, all other data for this individual were included in the study.

H. pakistanae DEVELOPMENT ON MONOECIOUS HYDRILLA

TABLE 3 Tissue Analysis of Monoecious and Dioecious Hydrilla Biotypes Cultivated under Identical Conditions

Fresh weight (fw) (g) Dry weight (dw) (g) Dry weight (% fw) Ash (% dw) Lipids (% dw) Fiber (% dw) Total nitrogen (% dw) Crude protein (N 3 6.25) (% dw) Ketohexoses (% dw) Total reducing sugars (% dw) Phosphorus (% dw) Potassium (% dw) Calcium (% dw) Magnesium (% dw) Iron (ppm dw) Manganese (ppm dw) Zinc (ppm dw)

Dioecious

Monoecious

104.02 4.69 4.51 21.40 4.00 34.08 3.87 24.19 2.70 5.56 0.47 5.50 4.07 0.43 815.5 142.5 98.0

147.71 5.04 3.41 25.20 4.80 10.72 4.45 27.81 3.22 4.92 0.65 4.61 4.23 0.34 1084.0 177.0 94.0

dia, enough individuals should complete development to ensure persistence of fly populations throughout the warmer months. This would be particularly true in regions where the monoecious hydrilla biotype performs as a perennial (Sutton et al., 1992). Recent recoveries of H. pakistanae from Beijing, China (Deonier, 1993) show that this species is well adapted to temperate as well as tropical climates. Further, an H. pakistanae population has persisted at Muscle Shoals, Alabama, since 1992, thereby demonstrating the fly’s ability to overwinter in temperate regions of the United States (Grodowitz et al., 1994). Overwintering as larvae or pupae would appear to be difficult at sites where hydrilla performs as an annual. However, some other ephydrids (e.g., Parydra breviceps Loew and Parydra quadrituberculata Loew) overwinter primarily as adults (Deonier and Regensburg, 1978; Bischof and Deonier, 1985) which would overcome the obstacle posed to immature stages by the absence of hydrilla. Also, the fact that populations persist as far north as Beijing (40° N; equal to Philadelphia, Pennsylvania), where hydrilla is also likely to perform as an annual, suggests that H. pakistanae has developed an adaptive strategy capable of overcoming this limitation. Finally, higher levels of leaf damage on the monoecious hydrilla biotype relative to the dioecious biotype, suggest that this fly could be an effective control agent of the monoecious biotype in regions of the United States where the fly is able to persist. ACKNOWLEDGMENTS We are grateful to B. Maharajh and R. Moriones (University of Florida, Institute of Food and Agricultural Sciences), who enthusias-

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tically carried out many of the technical aspects of this study. Several colleagues also deserve special thanks: Dr. R. Hammerschlag (U.S. Department of the Interior, National Park Service) provided the impetus for initiating this work, Dr. K. K. Steward (U.S. Department of Agriculture, Agricultural Research Service) generously contributed tubers and helped us develop cultures of the two hydrilla biotypes, and Dr. G. R. Buckingham (U.S. Department of Agriculture, Agricultural Research Service) graciously advised us concerning Hydrellia culture techniques. Constructive comments by Drs. G. S. Wheeler (U.S. Department of Agriculture, Agricultural Research Service), G. R. Buckingham, M. J. Grodowitz (U.S. Army Engineers, Waterways Experiment Station), and anonymous reviewers helped improve the manuscript and are truly appreciated. This study is published as Florida Agricultural Experiment Station Journal Series R-05137. It was conducted through Cooperative Agreement 58-43YK8-0005 between the United States Department of Agriculture, Agricultural Research Service, and the University of Florida, Institute of Food and Agricultural Sciences. It was funded through Cooperative Agreement 43YK-8-2911 between the United States Department of Agriculture, Agricultural Research Service, and the United States Department of the Interior, National Park Service.

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