BIOLOGICAL CONTROL 8, 65–73 (1997) ARTICLE NO. BC960491

Establishment of Hydrellia pakistanae (Diptera: ) for the Biological Control of the Submersed Aquatic Plant verticillata (Hydrocharitaceae) in the Southeastern United States

TED D. CENTER,* MICHAEL J. GRODOWITZ,† ALFRED F. C OFRANCESCO,† GREG JUBINSKY,‡ EDWARD SNODDY,§ AND JAN E. FREEDMAN† *USDA-ARS, Aquatic Plant Control Research Unit, 3205 College Avenue, Fort Lauderdale, Florida 33314; †USAE Waterways Experiment Station, 3909 Halls Ferry Road, Vicksburg, Mississippi; ‡Florida Department of Environmental Protection, Bureau of Aquatic Plant Control, 3917 Commonwealth Boulevard, Tallahassee, Florida 32399; §Tennessee Valley Authority, OSA 1B, Muscle Shoals, Alabama 35611

Received May 24, 1996; accepted October 8, 1996

Control of hydrilla is currently achieved primarily The Indian leaf-mining fly Hydrellia pakistanae De- with herbicides or by the release of exotic herbivorous onier was released in the United States during October fish (Sutton and Vandiver, 1986), but both methods are 1987 as a potential agent for the biological control of expensive for widespread use. Development of alterna- the submersed aquatic plant, Hydrilla verticillata (L.f.) tive control methods has been assigned high priority by Royle. Since then, nearly 3 million individuals have various federal and state agencies. One possible man- been released in four states at over 25 separate loca- agement alternative involves ‘‘inoculative’’ or ‘‘classi- tions. Hydrellia pakistanae is now permanently estab- cal’’ biological control, i.e., the introduction of host- lished at many locations in Florida as well as other specific plant-feeding obtained from within the portions of the southeastern United States. Despite native range of hydrilla. Searches for potential biologi- early failures, populations established from at least cal control agents began during the 1970s in India, 70% of the release attempts. Modifications of rearing Pakistan, and Africa and resumed during the 1980s in and release procedures that considered the biology of other areas of the world (Baloch and Sana-Ullah, 1974; the agent in relation to various habitat characteristics Baloch et al., 1980; Sankaran and Rao, 1972; Pember- facilitated successful establishment. 1997 Academic Press r ton, 1980; Center et al., 1990). The resultant surveys KEY WORDS: aquatic weeds; hydrilla control; biologi- revealed the presence of several biological control candi- cal control of weeds; Hydrellia pakistanae; Hydrilla dates including an undescribed Hydrellia species later verticillata; phytophagous insects; bioagent release named H. pakistanae Deonier (Deonier, 1978). techniques; Ephydridae; Hydrocharitaceae. Hydrellia pakistanae is a small, ephydrid fly whose larvae mine hydrilla leaves. Eggs are laid on floating leaves and the larvae either enter the leaves directly or INTRODUCTION descend into the water column before entering the plant. Females apparently oviposit indiscriminately, Hydrilla, Hydrilla verticillata (L.f.) Royle (Hydro- but the larvae are quite selective (Buckingham et al., charitaceae), is a submersed, leafy-stemmed vascular 1989; Wheeler and Center, 1996). Each mines hydrophyte that is widely distributed in the Old World. about 12 leaves during the course of its development It was introduced into the United States through the and then pupates within an air-filled puparium that is aquarium trade in the early 1950s (Schmitz et al., attached in a leaf axil (Baloch and Sana-Ullah, 1974; 1991). Currently, infestations of this invasive plant Buckingham et al., 1989). Total generation time is 18 to constitute the most severe aquatic plant problem in the 30 days (Baloch and Sana-Ullah, 1974). southern United States. Hydrilla infested 22,000 ha in Laboratory studies done in Pakistan (Baloch et al., Florida by 1988 and the state was spending about $7 1980) and later in a U.S. quarantine facility located in million annually in attempts to control about 6000 ha Gainesville, Florida (Buckingham et al., 1989), verified (Schmitz et al., 1991). During this same period it that H. pakistanae was host-specific to hydrilla. Accord- rapidly expanded its range and now occurs as far west ingly, Dr. Gary Buckingham petitioned the USDA Ani- as California (Sonder, 1979). mal and Plant Health Inspection Service—Plant Protec-

65 1049-9644/97 $25.00 Copyright r 1997 by Academic Press All rights of reproduction in any form reserved. 66 CENTER ET AL. tion and Quarantine (USDA-APHIS-PPQ) in June 1987 ther separately or in mixture (4 g yeast hydrolysate for permission to release this species. Permission was plus 7 g sucrose in 7 ml of water). Partially submersed obtained after review by the Technical Advisory Group hydrilla sprigs placed in shallow dishes within the on Biological Control of Weeds (Coulson, 1992) and this chambers provided the oviposition substrate. Egg- was first released on 29 October 1987 (Bucking- laden sprigs were transferred to new water-filled con- ham, 1988). In this paper, we document the results of tainers after 1 to 3 days to perpetuate the rearing this and later releases of H. pakistanae and the subse- process. quent establishment of self-perpetuating field popula- The second rearing method consisted of placing tions. This project represents the first attempt to use an freshly harvested hydrilla in large tanks. Either adults insect to control a weed in the submersed aquatic or hydrilla sprigs containing immatures were placed on environment by implementation of the ‘‘classical’’ ap- the floating hydrilla. populations were allowed to proach to biological control. increase in size within the tanks without additional handling. The hydrilla was then removed and trans- ferred to field sites. MATERIALS AND METHODS Releases. The basic release method involved the Insects. Buckingham et al. (1989) documented the placement of insects directly on hydrilla infestations collection of the original H. pakistanae stock from India either within open hydrilla beds or in protective cages and its receipt and disposition at the Gainesville, (Fig. 2). The cages were constructed of two frames made 2 Florida, quarantine facility during May to August 1985. of 2.5 cm (cross section), channeled aluminum extru- This initial stock was used for all host specificity sion rods. The frames were attached to one another testing and provided progeny for the initial U.S. re- with a piano hinge. The tops of the upper frames were leases made during 1987 to 1990. However, because of covered with fine-mesh screen (different materials with the risk associated with excessive inbreeding of the varying mesh sizes were used but 60% greenhouse original stock, fresh material was obtained from over- shade cloth usually sufficed). Floatation was provided seas. The Gainesville quarantine facility received a by filling the hollow aluminum extrusion rods with fresh consignment of field-collected H. pakistanae from polystyrene. The cages were placed at each site on the Kumbaleagodu, Bangalore, Karnataka State, India, on water surface with the screened side up. To prevent 25 August 1990 and one from Rawalpindi, Pakistan, on lateral movement, the cage was anchored by passing 28 August 1990. These insects were reared in quaran- poles through U-bolts attached to the outside of the tine for less than three generations prior to field release lower frame. The ends of the poles were forced into the to confirm identifications and to eliminate parasites. bottom soil. The hinged upper frame provided access After permission was granted for release of H. paki- into the cage for fly releases and for later assessments. stanae, a portion of the original quarantine colony was Numerous variations of the basic release technique used for the first release, a portion was retained in were employed. For example, individuals from both quarantine, and a portion was transferred to the U.S. laboratory colonies and from previously established Department of Agriculture, Agricultural Research Ser- field sites were released. Most of the individuals from vice (ARS), Aquatic Plant Control Research Unit in laboratory colonies were progeny of the original quaran- Fort Lauderdale, Florida. Insects reared at ARS were tine colony. However, some freshly acquired material later provided to the Waterways Experiment Station from overseas was released at selected sites. All life (WES) of the U.S. Army Corps of Engineers in Vicks- stages were released although the majority were eggs burg, Mississippi, the Tennessee ValleyAuthority (TVA) or larvae. Immature stages were transferred on or in Muscles Shoals, Alabama, and to the Florida Depart- within the plant tissue to minimize mortality. ment of Natural Resources (FDNR, now the Depart- The number of releases made and the total number of ment of Environmental Protection) Bureau of Aquatic individuals released varied among sites. In some cases, Plant Control in Tallahassee, Florida. predetermined quantities were released. In other in- Rearing. Two basic techniques were used to rear stances, releases were made repeatedly and were discon- H. pakistanae. The primary method was modified tinued only after evidence of establishment was ob- slightly from that described by Buckingham et al. tained. The brief growing season limited the numbers (1989). Hydrilla sprigs containing freshly oviposited released at more northern sites (i.e., Alabama). eggs were placed in 2- to 4-liter water-filled containers Assessment of establishment. Sites were checked for containing approximately 100 g of hydrilla. Adults were the presence of H. pakistanae by a variety of qualitative removed after emergence and transferred to oviposition sampling methods. These included hand collection of chambers. The oviposition chambers consisted of wooden adults from the water surface, microscopic examination boxes covered with either plastic or glass tops and fitted of randomly selected hydrilla sprigs for the presence of with cloth sleeves covering entry ports (Fig. 1). Adults immatures and associated damage, extraction of imma- were provided sugar water and yeast hydrolysate ei- tures from hydrilla samples via Berlese funnels, and RELEASE AND ESTABLISHMENT OF Hydrellia pakistanae 67

FIG. 1. A typical sleeve cage used as an egging chamber for Hydrellia pakistanae. Females oviposited on exposed leaves of partially submersed hydrilla sprigs. The resultant progeny were released at field sites. collection of emerging adults from hydrilla samples of the release attempts; i.e., H. pakistanae adults were held in containers for at least 30 days. We considered H. collected at least 4 months after releases were discontin- pakistanae to be established when we consistently ued (Tables 1–3). Hydrellia pakistanae has now been recovered adults, using any of the above sampling present at some of these sites for over 6 years. Popula- methods, from release sites for five generations (i.e., tions at many of these sites became large enough to about 4 months) after the last release. support redistributions of H. pakistanae to other loca- Voucher specimens were prepared and the identity of tions (Table 3). For example, over 4000 adults were the first specimens were verified by Dr. Richard L. collected at the Miami Canal in south Florida during a Deonier (Gainesville, FL), a taxonomic expert on the single week in June 1993 for incorporation into colonies genus Hydrellia. Voucher specimens were also depos- at TVA and WES. A large population of H. pakistanae ited in the Florida State Collection of existed at the Miami Canal even though H. pakistanae (Gainesville, FL). was never released there, the closest release site being over 15 km away. We also collected an estimated 2 RESULTS AND DISCUSSION million immatures from a pond in Muscle Shoals, Alabama, which were later moved to Lake Seminole, Hydrellia pakistanae is now clearly established at Georgia, and Lake Boeuf, Louisiana. These high num- several of the release sites in the southeastern United bers resulted from the release of only 50,000 individu- States, especially in southern Florida. This is indicated als in the area. Thriving H. pakistanae populations now by the fact that populations persisted from at least 70% also occur at many sites where they were not released. 68 CENTER ET AL.

FIG. 2. Hydrellia pakistanae release cages were constructed of hollow, channeled aluminum extrusion bars filled with polystyrene for floatation. U-bolts on the sides encircled poles anchored into the bottom soil to hold the cage at the surface while allowing it to rise or fall with changing water levels.

For example, during November 1991, H. pakistanae Leonore) and Rodman Reservoir did not result in adults were recovered near Naples, Florida, at a site at establishment. We therefore released larger numbers least 120 km from the nearest established release site. over shorter time intervals. The first successful estab- This rapid dispersal demonstrates the vitality of these lishment of H. pakistanae occurred nearly simulta- populations and supports the conclusion that H. paki- neously at the Palm Beach Airport and Hacienda stanae is permanently established. It is now difficult to Village pond sites (Table 2). This required an intensive find hydrilla beds in southern Florida that do not effort. For example, establishment of a persistent popu- support H. pakistanae populations. This represents the lation at the Hacienda Village site required nine succes- first successful establishment of an introduced insect sive releases made at weekly intervals that totaled biological control agent for the management of a weed nearly 30,000 individuals. This suggested that single growing in the submersed aquatic environment. releases and the small numbers previously released While these results substantiate our conclusion that inhibited establishment and dictated the strategy of H. pakistanae is well established in the southeastern frequently releasing large numbers over shorter time United States, early releases failed to produce persis- intervals (Table 2). To meet the associated demand for tent field populations (Table 1). Success came only after higher numbers of individuals, we increased rearing altering rearing, release, and establishment tech- capabilities at the Fort Lauderdale ARS laboratory and niques. Modifications included the rearing and subse- later at TVA facilities in Muscle Shoals, Alabama, and quent release of larger numbers of individuals, the use WES facilities in Vicksburg, Mississippi. These added of individuals not subjected to intensive, long-term facilities supported release efforts in Georgia, Alabama, laboratory rearing, as well as alterations to actual Louisiana, and Texas as well as supplemental efforts in release procedures and site selection based on recogni- Florida (Table 3). In addition to laboratory-reared tion of mitigating factors such as plant quality (Wheeler individuals, the releases were supplemented with field- and Center, 1996). These changes, taken together, collected adult flies to maximize the numbers released. apparently facilitated establishment of H. pakistanae. We eventually released nearly 3 million individuals The initial supply of insects for release originated from laboratory and field sources. from 2-year-old colonies maintained at the quarantine We also identified several problems that were likely facility. The first releases were necessarily small and impediments to establishment. These included possible infrequent because of the limited numbers of insects inbreeding and other adverse effects of laboratory available (Table 1). For example, the releases of fewer rearing. Selective breeding induced by laboratory- than 10,000 individuals at Lake Patrick (aka Lake rearing is known to reduce competitive ability in re- RELEASE AND ESTABLISHMENT OF Hydrellia pakistanae 69

TABLE 1 Early Hydrellia pakistanae Releases Made from October 1987 to April 1989

Release series Quantity released

No. Eggs First Duration No. site and Late Infested Source Release State County Location date (days) releases visits neonates instarts Adults plantsa codeb methodc Resultd

Florida Broward Everglades 26-Feb-88 123 2 2 6,089 0 0 1 CONE Holiday Park Florida Broward North New River 23-Jan-89 — 1 1 0 0 0 1 CONE Canal Florida Glades Intersection US 28-Apr-89 — 2 1 609 0 0 2 CONE 27 and SR 78 Florida Glades Lake 18-Nov-88 109 7 7 10,351 0 0 2 CONE Okeechobee, Big Bear Beach Florida Glades Lake 11-Aug-88 — 1 1 0 0 0 1 CONE Okeechobee, Fisheating Bay Florida Glades Lake Hicpochee 17-Mar-88 — 1 1 0 0 0 1 CONE Florida Glades Lake Hicpochee 16-Mar-89 53 2 2 1,138 0 0 2 CONE Florida Marion Rodman Reser- 9-Nov-87 — 1 1 1,000 0 0 2 CONE voir Florida Osceola Lake Tohopeka- 1-Mar-89 — 7 1 5,055 18 110 2 CONE liga Florida Palm Beach Lake Osborne 23-May-88 — 1 1 3,870 0 0 2 CONE Florida Polk Lake Patrick 29-Oct-87 — 3 1 8,600 0 600 1 CONE (Lake Lenore) Florida Polk Sears Lake 2-Mar-89 42 2 2 3,266 0 0 2 CONE Totals 30 21 37,761 18 710

a Infested plants containing unknown quantities of larvae and/or eggs. b C, from laboratory cultures. c O, released in open hydrilla beds. d NE, no evidence of establishment. leased populations (Dame and Birkenmeyer, 1975; Bush this fresh stock, though, we had established several and Neck, 1976; Chambers et al., 1983; and Grodowitz field populations at other locations using insects from et al., 1992). As a case in point, observations of adult old cultures, so either approach can be effective. More H. pakistanae behavioral changes suggested that the research is needed on the effects of laboratory rearing original individuals had adapted to laboratory condi- on field establishment and the subsequent performance tions and were therefore poorly suited to natural of biological control agents. habitats. This was deduced from a progressive reduc- Another possible problem was associated with the tion of adult activity and reproduction. We therefore life stage released. Earliest releases of H. pakistanae imported new colonies from India and Pakistan. About were composed mainly of eggs (Table 1). Egg-laden 2000 immatures from these new colonies were released sprigs were transferred directly from oviposition cham- during November of 1990 in southern Florida at two bers to field sites. These infested sprigs were usually small catchment ponds in northern Broward County submersed among leafy stems in the upper layer of the (designated the Lakeview sites in Table 1). The Indian existing hydrilla beds. However, eggs are usually laid strain was released at the north pond (about 1700 at the water surface in protected sites on the plant immatures) and the Pakistani strain at the south pond tissue. With no establishment resulting from the exclu- (about 300 immatures). Two months later, H. paki- sive release of eggs, we suspected that poor survivor- stanae was not found at the north site but 15 individu- ship resulted from submerging the eggs too deeply. als were collected at the south site at the point of their Moreover, we noted that eggs were easily dislodged release. Hence, it appears that the Pakistani strain of from the hydrilla sprigs. This prompted us to release H. pakistanae colonized the south site despite the small larvae rather than eggs. We believed that second and numbers released. This suggests that insects released third instars would survive better because, in labora- immediately after importation directly in the field may tory experiments, we observed higher mortality rates indeed establish more readily. By the time we received associated with first instars. Eggs were obtained as 70 CENTER ET AL.

TABLE 2 Releases of Hydrellia pakistanae Made from November 1989 to March 1992

Release series Quantity released

No. Eggs First Duration No. site and Late Infested Source Release State County Location date (days) releases visits neonates instars Adults plantsa codeb methodc Resultd

Alabama Jackson Comer 29-Aug-90 49 12 12 0 29,512 0 2 CCNE Bridge Alabama Jackson Chisenhall Jul-91 ? ? ? 0 25,000 0 2 CCNE Alabama Lauderdale Muscle 1-Aug-91 ? ? ? 0 12,633 0 2 CCE? Shoals Pond Facility Alabama Marshall Murphy Hill 1-Sep-90 — 1 1 0 1,245 0 2 CCE? Alabama Marshall Murphy Hill Aug-91 ? ? ? 0 9,670 0 2 CCE Florida Brevard Stick Marsh 12-Nov-91 49 4 4 1,969 0 5,500 2 F,C O E Florida Broward Lake View, 5-Nov-90 9 3 2 0 1,300 0 1 C O,C E North Pond Florida Broward Lake View, 14-Nov-90 — 2 1 0 200 0 1 CCE South Pond Florida Glades Lake 6-Nov-90 — 1 1 0 0 1,030 2 CONE Okeechobee, Fisheating Bay Florida Jefferson Wacissa 31-Oct-90 7 9 2 10,430 3,514 3,834 1 FR,C O TE River Florida Jefferson Wacissa 22-Feb-91 52 4 4 46,390 0 0 1 FR,C O TE River Florida Lake Lake Robin 20-Dec-90 — 1 1 0 0 1,200 2 FONE Hood Florida Okeechobee Lake 21-Nov-90 121 13 10 147,984 0 3,000 1 C,F O,C E Okeechobee, Eagle Bay Florida Osceola Lake Toho- 13-Dec-90 7 2 2 0 0 3,300 2 FONE pekaliga Florida Osceola Lake Toho- 12-Feb-92 — 1 1 0 0 0 1 CONE pekaliga Florida Polk Cattle Farm 10-Feb-92 81 3 3 0 0 0 1 CCNE Lake Florida Broward Hacienda 8-Jan-90 44 10 9 0 28,831 0 2 CCE Village Florida Glades Lake Hicpo- 2-Mar-90 189 33 17 0 31,904 0 2 CCE chee Florida Palm Beach Palm Beach 3-Nov-89 70 11 11 5,826 11,610 0 2 CCE Airport Georgia Decatur Lake Semi- 26-Jun-90 112 21 8 20,110 16,916 3,244 2 F, FR,C C NE nole Georgia Decatur Lake Semi- 2-May-91 106 23 18 96,305 51,281 300 2 F, FR CTE nole Georgia Decatur Lake Semi- 17-Mar-92 177 21 16 1,804 93,620 0 2,000,000 F C E? nole Totals 175 123 235,141 132,946 22,668

Note. Predominant method used shown in boldface. a Infested plants containing unknown quantities of larvae and/or eggs. b C, from laboratory cultures; F, field collected (usually adults); FR, field collected, then partially reared (mostly eggs of field-collected adults). c O, released in open hydrilla beds; C, released in cage. d E, permanently established; E?, weakly established; TE, temporarily established; NE, no evidence of establishment. RELEASE AND ESTABLISHMENT OF Hydrellia pakistanae 71

TABLE 3 Redistributions of Hydrellia pakistanae from Field-Established Colonies (August 1991 to August 1995)

Release series Quantity released

No. Eggs County/ First Duration No. site and Late Infested Source Release State parish Location date (days) releases visits neonates instars Adults plantsa codeb methodc Resultd

Alabama Lauderdale Muscle Shoals Aug-91 ? ? ? 0 12,633 0 2 CCE? Pond Facility Alabama Lauderdale Muscle Shoals 4-Aug-92 17 5 5 0 33,259 0 2 CCE Pond Facility Alabama Lauderdale Muscle Shoals 18-Aug-93 71 49 14 52,075 51,179 0 2 CCE Pond Facility Alabama Lauderdale Muscle Shoals 27-Sep-94 — 1 1 0 528 0 2 CCE Pond Facility Alabama Lauderdale Muscle Shoals 27-Apr-95 126 5 2 8,277 6,188 0 2 CCE Pond Facility Alabama Jackson Sublet Ferry 30-Jun-93 90 18 18 0 43,372 0 2 CCE Alabama Jackson Sublet Ferry 7-Jul-94 126 22 22 0 101,671 0 2 CCE Californiae Imperial New Spruce 5-Apr-94 — 1 1 0 3,000 0 2 FR O TE Canal Californiae Imperial Sumac Main 5-Apr-94 — 1 1 0 3,100 0 2 FR O TE Canal Californiae Imperial Thistle Main 5-Apr-94 — 1 1 0 3,125 0 2 FR O TE Canal Florida Marion Blue Run 10-Sep-92 28 3 2 0 0 1,100 1 F, FR O E Florida Hillsborough Hillsborough 8-Apr-94 26 3 2 0 0 14,481 2 FOE River Florida Polk Lake Fannie 19-Aug-94 77 4 3 17,100 0 2,700 2 FR,F C NE Florida Hernando Weeki Wachee 30-Aug-94 — 1 1 4,000 0 0 2 FR O E River Florida Broward Orange Brook 6-Sep-94 49 3 3 16,500 0 2,410 2 F, FR O, C SE Golf Course Florida Palm Beach West Jupiter 27-Mar-95 43 10 10 0 0 66,500 2 FR C E Louisiana LaFourche Lake Bouef 15-Aug-91 117 14 14 0 71,051 0 1 CCNE Louisiana LaFourche Lake Bouef 3-Sep-92 — 2 1 0 7,160 0 2 CCNE Louisiana LaFourche Lake Bouef 1-Jan-93 25 4 3 0 8,128 0 250,000 F O NE Louisiana LaFourche Lake Bouef 20-Oct-93 — 1 1 0 3,870 0 2 CONE Texas Goliad Coleto Creek 2-June-94 27 8 4 0 19,584 0 2 C, FR OE Reservoir Texas Live Oak Choke Canyon 20-Jan-93 — 1 1 0 3,693 0 2 CCNE Texas Live Oak Choke Canyon 23-Oct-93 89 9 8 53,178 35,753 0 2 C, FR OTE Texas Live Oak Choke Canyon 26-Sep-94 179 19 9 0 76,693 0 2 CONE Totals 185 127 151,130 484,023 87,191

Note. Predominant method used shown in boldface. a Infested plants containing unknown quantities of larvae and/or eggs. Numbers, when given, are estimates derived from subsamples. b C, from laboratory cultures; F, field collected (usually adults); FR, field collected, then partially reared (mostly eggs of field-collected adults). c O, released in open hydrilla beds; C, released in cage. d E, permanently established; E?, weakly established; TE, temporarily established; NE, no establishment; SE, supplemental release at established site. e California releases made by (and data courtesy of) Drs. C. Turner, C. Godfrey, and L. Anderson from insects collected in Florida. before, but the egg-laden sprigs were held for as long as larly by damselflies, might have further reduced the 14 days prior to release. reproductive population. Additionally, we observed coots We also suspected other problems. The few adults and moorhens selectively eating the infested hydrilla that emerged might have been dispersing too quickly, sprigs placed at the sites, thereby reducing or eliminat- thereby reducing mating encounters. Predation, particu- ing the released individuals. This prompted us to 72 CENTER ET AL. institute the use of shallow, floating cages to protect the addition, one female H. pakistanae was recovered in plant material harboring the larvae as well as the Lake Seminole at Fireman’s Cut during July 1993, 1 emerging adults (Fig. 2). year after the last release at that specific area and 8 The cages served several functions. They did not months after the last release (i.e., December 1992) confine the insects completely, but merely detained anywhere in the reservoir. Hence, the H. pakistanae them, thereby increasing the likelihood of mating en- population at Lake Seminole successfully overwin- counters among emerging adults. The cages also clearly tered, although the resultant establishment seemed demarcated the point of release, thus facilitating recov- tenuous. ery of successive generations of flies to verify establish- Variation in plant quality was probably the most ment. The cages were shallow (with onlya2cminterior important factor limiting establishment at some sites. space above the water surface), limiting maneuverabil- For example, Wheeler and Center (1996) found that ity by flying predators within the cages and presumably leaf texture and tissue nitrogen concentrations affect decreasing predation and improving adult survival. larval survival, duration of larval stadia, and ultimate The cages also protected the plant material from larger adult size of H. pakistanae. They reared H. pakistanae herbivores such as coots and moorhens. The first suc- on field-collected plants from a variety of conditions. cessful establishment attempts made at the Hacienda Coarse plants with low tissue-nitrogen concentrations Village and Palm Beach Airport sites involved the use were less suitable than soft plants with higher nitrogen of these cages (Table 2). levels. Hence, establishment of populations on low All modifications of the original rearing and release quality hydrilla might be impossible, or could require procedures apparently contributed to the ultimate es- more inoculum to overcome low survival in the found- tablishment of H. pakistanae. All of these corrective ing population. measures were instituted simultaneously, so it is not In conclusion, permanently established H. paki- possible to isolate any one change that seemed more stanae populations now occur from northern Alabama significant than the others. However, the data indi- to the southern tip of Florida and further releases are cated that establishment occurred only after all of these continuing throughout the region. Meanwhile, addi- corrective measures were implemented. Most of the tional sites are being colonized by naturally dispersing populations. This success was apparently based on early open releases using mainly small numbers of eggs modifications of initial rearing and release procedures. failed to establish (e.g., Everglades Holiday Park) but Modifications made were based on our increased under- later caged releases using large numbers of late instar standing of both the insect biological control agent and larvae were quickly successful (e.g., Hacienda Village). its associated habitats. While success has been exten- This underscores the importance of understanding the sive, it has not been universal. Research is needed into biology of the biocontrol agent as well as the biotic and the causes of failed attempts (Wheeler and Center, abiotic conditions associated with recipient habitats 1996), but it is clear that success can be attained even when designing release protocols. when initial attempts fail. Harris (1988) noted that Hydrellia pakistanae populations are now well estab- about a third of all insects released as weed biological lished across a broad geographical range. Populations control agents fail to establish but that half of those at some sites have repeatedly failed to establish, or that do establish consume appreciable amounts of the persisted at very low levels, despite massive releases targeted weed. A higher percentage of projects might be (Tables 2 and 3). These include sites in Georgia (i.e., successful if more persistent effort was directed toward Lake Seminole), Florida (i.e., the Wacissa River), and ensuring agent establishment. Louisiana (i.e., Lake Boeuf). For example, we estimated that over 2 million insects were released throughout Lake Seminole but only very weak establishment re- ACKNOWLEDGMENTS sulted in one area (the Fireman’s Cut). While the The data presented herein, unless otherwise noted, were obtained causes of these failures are unknown, they are most from research funded by the U.S. Army Corps of Engineers Water- likely associated with site and environmental condi- ways Experiment Station, Aquatic Plant Control Research Program; tions. Lack of establishment or poor establishment Florida Department of Natural Resources, Bureau of Aquatic Plant cannot be attributed to the release methodology or the Control; and the USDA, Agricultural Research Service. Permission quality of the insects released because successes were was granted by the Chief of Engineers to publish this information. We gratefully acknowledge the persistent efforts put forth by Mr. W. C. attained with even less effort at many other locations Durden, Mr. F. A. Dray, Mr. Boudanath Maharajh, and Ms. D. using the same techniques and insect stock. Also, the Niehaus to accomplish the objectives of this project. We are also colder winters and associated senescence of hydrilla at grateful to the many student assistants who assisted with field work northern sites could not account for the lack of establish- and rearing of the insects. We are especially indebted to Dr. Gary Buckingham and Ms. Christine Bennett who graciously provided ment because verifiable establishment has occurred at stock cultures of H. pakistanae, processed foreign consignments of the northernmost sites in northeastern Alabama (i.e., insects through quarantine, and provided invaluable advice and Muscle Shoals and Guntersville, Tables 2 and 3). In suggestions. This project would have been much more difficult RELEASE AND ESTABLISHMENT OF Hydrellia pakistanae 73 without their valuable insight. We also thank Dr. Charley Turner, Dr. shop, June 10–12, 1991, Vienna, VA’’ (R. Charudattan and H. W. Cris Godfrey, and Dr. Lars Anderson, who provided information on Browning, Eds.), pp. 53–60. Inst. Food Agric. Sci., Univ. Florida, the release of Hydrellia pakistanae in California. Gainesville. Dame, D. A., and Birkenmeyer, D. R. 1975. The dispersal and REFERENCES survival of laboratory-bred and native Glossina moristans moris- tans Westw. (Diptera: Glossinidae) in the field. Bull. Entomol. Res. 65, 453–457. Baloch, G. M., and Sana-Ullah 1974. Insects and other organisms associated with Hydrilla verticillata (L.f.) L.C. (Hydrocharitaceae) Deonier, R. L. 1978. New species of Hydrellia reared from aquatic in Pakistan. In ‘‘Proceedings of the III International Symposium on macrophytes in Pakistan (Diptera: Ephydridae). Entomol. Scand. Biological Control of Weeds, 10–14 September 1973, Montpellier, 9, 188–197. France’’ (A.J. Wapshere, Ed.), Misc. Publ. 8, pp. 61–66. Common- Grodowitz, M. J., Lloyd, E. P., and Mckibben, G. H. 1992. Comparison wealth Institute of Biological Control, U.K. of feeding and olfactory behaviors between laboratory-reared and Baloch, G. M., Sana-Ullah, and Ghani, M. A. 1980. Some promising overwintered native boll weevils (Coleoptera: Curculionidae). J. insects for the biological control of Hydrilla verticillata in Pakistan. Econ. Entomol. 85, 2201–2210. Trop. Pest Manage. 26, 194–200. Harris, P. 1988. Environmental impact of weed-control insects. Buckingham, G. R. 1988. Reunion in Florida: Hydrilla, a weevil, and BioScience 38, 542–548. afly.Aquatics 10, 19–25. Pemberton, R. W. 1980. ‘‘Exploration for natural enemies of Hydrilla Buckingham, G. R., Okrah, E. A., and Thomas, M. C. 1989. Labora- verticillata in Eastern Africa,’’ U.S. Army Eng., Waterways Exp. tory host range tests with Hydrellia pakistanae (Diptera: Ephydri- Sta., Misc. Paper A-80-1. dae), an agent for biological control of Hydrilla verticillata (Hydro- Sankaran, T., and Rao, V. P. 1972. An annotated list of insects charitaceae). Environ. Entomol. 18, 164–71. attacking some terrestrial and aquatic weeds in India, with records Bush, G. L., and Neck, R. W. 1976. Ecological genetics of the of some parasites of the phytophagous insects. Commonw. Inst. screw-worm fly, Cochliomyia hominovorax (Diptera: Calliphoridae) Biol. Control Tech. Bull. 15, 131–157. and its bearing on the quality control of mass-reared insects. Schmitz, D. C., Nelson, B. V., Nall, L. E., and Schardt, J. D. 1991. Environ. Entomol. 5, 821–826. Exotic aquatic plants in Florida: A historical perspective in review Center, T. D., Cofrancesco, A. F., and Balciunas, J. K. 1990. Biological of the present aquatic plant regulation program. In ‘‘Proceedings of control of aquatic and wetland weeds in the southeastern United a Symposium on Exotic Pest Plants, Miami, Florida, 2–4 Nov. 1988’’ States. In ‘‘Proceedings of the VII International Symposium on (T. D. Center, R. F. Doren, R. L. Hofstetter, R. L. Myers, and L. D. Biological Control of Weeds, 6–11 March 1988, Rome, Italy’’ (E. S. Whiteaker, Eds.), pp. 303–323. U.S. Dept. Interior, Tech. Rep. Delfosse Ed.), pp. 239–262. Istituto Sperimentale per la Patologia NPS/NREVER/NRTR-91/06, Natl. Park Serv., Washington, DC. Vegetale (Ministero dell’Agricoltura e delle Foreste), Rome, Italy. Sonder, L. W. 1979. Hydrilla infestations in California. Proc. Calif. Chambers, D. L., Calkins, C. O., Boller, E. F., Ito, Y., and Cunning- Weed Conf. Sacramento, CA. 1979, 122–125. ham, R. T. 1983. Measuring, monitoring, and improving the quality Sutton, D. L., and Vandiver, V. V. 1986. ‘‘Grass carp. A Fish for of mass-reared Mediterranean fruit flies, Ceratitus capitata (Wied.). Biological Management of Hydrilla and Other Aquatic Weeds in 2. Field tests for confirming and extending laboratory results. Z. Florida,’’ Fla. Agr. Exp. Sta. Bull. 867. Univ. Florida, Gainesville. Angew. Entomol. 95, 285–303. Wheeler, G. S., and Center, T. D. 1996. The influence of hydrilla leaf Coulson, J. R. 1992. The TAG: Development, functions, procedures, quality on larval growth and development of the biological control and problems. In ‘‘Regulations and Guidelines: Critical Issues in agent Hydrellia pakistanae (Diptera: Ephydridae). Biol. Control 7, Biological Control. Proceedings of a USDA/CSRS National Work- 1–9.