A comparison of host range and performance of congeneric leaf-mining flies, Hydrellia pakistanae (Diptera: Ephydridae) and Hydrellia sp., two candidate biological control agents for the South African biotype of Hydrilla verticillata (Hydrocharitaceae)

Biological Control 84 (2015) 44–52

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A comparison of host range and performance of congeneric leaf-mining flies, Hydrellia pakistanae (Diptera: Ephydridae) and Hydrellia sp., two candidate biological control agents for the South African biotype of Hydrilla verticillata (Hydrocharitaceae) A. Bownes ⇑ Agricultural Research Council – Plant Protection Research Institute (ARC-PPRI), Private Bag X6006, Hilton, 3245, South Africa School of Life Sciences, University of KwaZulu-Natal, Scottsville, 3209, South Africa

h i g h l i g h t s

g r a p h i c a l a b s t r a c t

 Two Hydrellia flies were assessed as

biocontrol agents for Hydrilla verticillata.  Host range, performance and leafmining were compared.  Host range and damage potential were similar but performance differed significantly.  Hydrellia sp. was selected for biocontrol of H. verticillata in South Africa.

a r t i c l e

i n f o

Article history: Received 10 November 2014 Accepted 19 February 2015 Available online 26 February 2015 Keywords: Hydrilla verticillata Hydrellia pakistanae Hydrellia sp. Host specificity Fitness Host plant genotype

a b s t r a c t A monoecious Malaysian/Indonesian biotype of the invasive aquatic weed, Hydrilla verticillata (L.f.) Royle (Hydrocharitaceae) recently invaded South Africa and has been targeted for biological control. Two ephydrid leaf-mining flies, originating on genetically distinct H. verticillata biotypes were considered as prospective agents. Hydrellia pakistanae Deonier (Diptera: Ephydridae) is a widely established biological control agent of a dioecious Indian/Pakistani H. verticillata biotype in North America, and an unnamed Hydrellia fly, Hydrellia sp. was collected in Singapore on a similar host plant biotype to the one invading South Africa. The suitability of the two fly species as biocontrol agents for H. verticillata in South Africa was assessed by comparing their fundamental host ranges as well as their performance and leaf-mining on the South African biotype of H. verticillata. Additionally, fitness parameters of H. pakistanae reared on South African H. verticillata were compared to the same parameters measured on the U.S. dioecious biotype. The two fly species showed minor differences in non-target host use in no-choice larval development trials. Hydrellia sp. had higher survival, longevity and fecundity and shorter egg to adult development times on South African H. verticillata compared to H. pakistanae. Further, leaf-mining by the two fly species was similar and H. pakistanae’s performance on South African H. verticillata was inferior in comparison to its performance on the U.S. dioecious H. verticillata biotype. These findings guided a decision to reject H. pakistanae as a biocontrol agent for H. verticillata in South Africa in favor of its congener, Hydrellia sp. Ó 2015 Elsevier Inc. All rights reserved.

⇑ Address: Agricultural Research Council – Plant Protection Research Institute (ARC-PPRI), Private Bag X6006, Hilton 3245, South Africa. Fax: +27 (0) 33 355 9423. E-mail address: [email protected] http://dx.doi.org/10.1016/j.biocontrol.2015.02.004 1049-9644/Ó 2015 Elsevier Inc. All rights reserved.

A. Bownes / Biological Control 84 (2015) 44–52

1. Introduction Hydrilla, Hydrilla verticillata (L.f.) Royle (Hydrocharitaceae) is a rooted, submerged aquatic macrophyte, native to the warmer regions of Asia and Australia and isolated parts of Europe and central Africa, that it is now cosmopolitan, occurring on all continents except Antarctica (Balciunas et al., 2002; Cook and Lüönd, 1982; Thomaz et al., 2009). H. verticillata thrives in a variety of freshwater habitats (Cook and Lüönd, 1982) and its growth habit of forming a dense canopy at the water surface (Haller and Sutton, 1975), coupled with its ecological adaptability, make it a highly aggressive competitor in aquatic environments (Langeland, 1996). H. verticillata is considered to be genetically diverse (Madeira et al., 1997, 2007; Verkleij, 1983), although the presence of three cryptic species within the Hydrilla genus was recently proposed by Benoit (2011). The plant can be monoecious or dioecious, diploid, triploid or tetraploid (Cook and Lüönd, 1982) and there is biotype variation in reproduction, growth form and morphology (Steward, 1993; Van, 1989). H. verticillata is regarded as the worst aquatic weed in the southeastern states of North America (Balciunas et al., 2002; Center et al., 1997) and it was recently discovered during 2006 in Pongolapoort Dam (27°240 0900 S 31°570 3100 E), a major tourism destination in northern KwaZulu-Natal (KZN), South Africa (Henderson, 2006). RAPD (Random Amplified Polymorphic DNA) analyses were used to determine the origins of the H. verticillata biotypes invading both North America (Madeira et al., 1997) and South Africa (Madeira et al., 2007). In North America, a female dioecious form was first discovered in Florida in the 1950s (Schmitz et al., 1991) and was later determined to originate in India (Madeira et al., 1997). A monoecious H. verticillata biotype invaded the northern U.S. states (Steward et al., 1984) in the 1970s and was found to be of Korean origin (Madeira et al., 1997). The South African biotype was found to be monoecious and identical to Malaysian and Indonesian H. verticillata (Madeira et al., 2007). A classical biological control programme against H. verticillata was initiated in Florida in 1981, with the evaluation of 28 insects and pathogens, following extensive surveys in the native range (Balciunas et al., 2002). Although at the time, the origin of the U.S. dioecious biotype was not yet confirmed, a leaf-mining fly, Hydrellia pakistanae Deonier (Diptera: Ephydridae), originating on dioecious H. verticillata from India and Pakistan (Baloch et al., 1980; Buckingham et al., 1989) was the first agent released in 1987 (Buckingham, 1988). H. pakistanae has been the most successful of the four biocontrol agents released in North America, based on its establishment, range expansion and damage potential (Grodowitz et al., 1999, 2003). With the extensive biological control research conducted on H. verticillata in North America and with a view to the invasive potential of this aquatic weed, South Africa developed a biological control programme soon after H. verticillata was discovered. The programme benefitted from the experiences of North America and two of the four agents released, H. pakistanae and a stem-mining weevil Bagous hydrillae O’Brien (Coleoptera: Curculionidae), were prioritized for possible introduction into South Africa. Although H. pakistanae was a promising candidate, the genetic diversity of H. verticillata (Madeira et al., 1997, 2007; Verkleij, 1983), its disparate geographical origins and the provenance of the South African H. verticillata biotype, caused concerns over potentially mismatched insect and host. Weed biological control agents are introduced into the invasive range of a target weed with the expectation that they will reach damaging densities, ultimately suppressing populations of the weed. The potential for insect biocontrol agents to build up suitable populations is dependent on many interacting factors, one of which may be host plant genotype

45

(Goolsby et al., 2006; Grevstad et al., 2013; Lym and Carlson, 2002; Lym et al., 1996; Manrique et al., 2008). Indeed, the H. verticillata biocontrol programme in North America has shown that biotype matching is a crucial factor, due to the plant’s wide geographical distribution (Cook and Lüönd, 1982) and genotypic (Madeira et al., 1997, 2007) and phenotypic variation (Van, 1989; Steward, 1993). H. pakistanae, although able to develop equally well on the monoecious U.S. biotype of H. verticillata in the laboratory (Dray and Center, 1996), failed to establish widely or to persist on this biotype in the field (Grodowitz et al., 2013). Furthermore, another leaf-mining Hydrellia fly, Hydrellia balciunasi Bock of Australian origin has had limited establishment on H. verticillata in North America, despite widespread, intensive releases. One of the attributing factors was a mismatch of the agent and the target weed due to genetic differences between Australian H. verticillata and the two biotypes in North America (Grodowitz et al., 1997); another contributing factor is winter die-back of the monoecious biotype which leaves little suitable habitat for H. pakistanae to overwinter (Harms and Grodowitz, 2011). Several other studies have demonstrated the importance of considering host plant genotype when selecting candidate biological control agents (Goolsby et al., 2006; Grevstad et al., 2013; Lym and Carlson, 2002; Manrique et al., 2008). As a consequence, another leaf-mining fly, Hydrellia sp. from Singapore, originating on a similar biotype to South African H. verticillata, was considered as a prospective agent. Although biotype matching is an increasingly important factor considered in weed biological control programmes, a suitably narrow host range remains the fundamental basis for selecting or rejecting prospective agents and the most important factor in minimizing potential risks. Hence the more specific of the two fly species should be the preferred agent before biotype matching is considered. The objective of this study was to determine which of the two fly species is more suitable for biological control of H. verticillata in South Africa. Agent suitability was assessed by direct comparisons of the host ranges of the two fly species as well as their performance (survival, fecundity, longevity and egg viability) and leaf mining on the South African biotype of H. verticillata. Additionally, this paper examines the performance of H. pakistanae on the South African biotype of H. verticillata in comparison to the U.S. dioecious form (Buckingham et al., 1989), a genetically similar biotype to H. pakistanae’s native host plant biotype in India. These findings guided a decision to reject H. pakistanae in favor of Hydrellia sp. for biological control of H. verticillata in South Africa. 2. Materials and methods 2.1. Study species H. verticillata is the only species in the Hydrilla genus which is in the frog’s bit family, Hydrocharitaceae. The Hydrocharitaceae includes 17 aquatic plant genera (Les et al., 2006), five of which are native to southern Africa and three of which are exotic, including Hydrilla. The ephydrid fly genus, Hydrellia is cosmopolitan, with over 120 named species (Deonier, 1971). H. pakistanae and Hydrellia sp. are closely related species in the H. pakistanae species complex. Hydrellia sp. is in the process of being described by dipteran taxonomist, Dr. John Deeming, who has designated the fly as a sibling species of H. balciunasi, to which it is very similar. There are a few minor differences between H. balciunasi and Hydrellia sp. however the fused surstyli in male Hydrellia sp. are unique in the H. pakistanae species group (Deeming, unpubl.). Voucher specimens of Hydrellia sp. have been deposited at SANC (South African National Collection of Insects, Pretoria) with the database accession number DIPT04104.

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A. Bownes / Biological Control 84 (2015) 44–52

H. pakistanae and Hydrellia sp. are morphologically similar (small, dark gray with bright green eyes), with similar life histories. Adult flies oviposit on exposed plant material and the larvae mine the submerged leaves of H. verticillata, passing through 3 instars during their development. The cuticle of the final larval instar forms the puparia and pupae develop at the leaf axils, resembling leaf buds. Adult flies float to the water surface in an air bubble and are able to fly within a few minutes of emerging (Buckingham et al., 1991). At a constant temperature of 27 °C, the egg to adult life cycle of both fly species is approximately 23 days, measured on U.S. dioecious H. verticillata for H. pakistanae (Buckingham and Okrah, 1993) and South African monoecious H. verticillata for Hydrellia sp. (Bownes, unpubl.). The host range of H. pakistanae was investigated by Baloch et al. (1980) in the native range and Buckingham et al. (1989) in North America and it was found to be suitably host specific. Host range studies on Hydrellia sp. in South Africa indicated the fly has a strong preference for its host plant, H. verticillata and limited potential to establish permanent populations on native plant species (Bownes, 2014) however, a release permit is not yet issued. H. pakistanae was imported into a quarantine facility of the Agricultural Research Council – Plant Protection Research Institute (ARC-PPRI) in Pretoria, South Africa in November 2007. The laboratory culture was initiated with adults that emerged from infested dioecious H. verticillata, imported from a mass rearing facility, the U.S. Army Engineers Research and Development Center in Vicksburg, Mississippi. The Hydrellia sp. starter culture was imported from Singapore in March 2008. Larvae developing within leaves of monoecious H. verticillata were collected from a stream flowing from a reservoir in Bishan Park, Singapore (01°210 9500 N 103°500 1900 E) (M. Purcell, pers. comm.). Both fly cultures were transferred to another ARC-PPRI quarantine facility at Cedara (Provincial Department of Agriculture and Environmental Affairs headquarters) near Pietermaritzburg, KZN in July 2008. Cultures of the two fly species were maintained in separate facilities to prevent accidental mixing, however all trials comparing their host range and performance on South African H. verticillata were conducted simultaneously in the same facility, under the same environmental conditions. All tests were conducted with H. verticillata that was either field collected from Pongolapoort Dam and held in outdoor pools or was cultivated at the research facility in 80 L plastic tubs in pond sediment fertilized with ammonium nitrate (NH4NO3) and potassium dihydrogen orthophosphate (KH2PO4). Field plants were used where possible, but when in poor condition, stock plants were used as an alternative. For every trial, test plants or H. verticillata of the same origin (i.e. either freshly collected from the field or cultivated in the same tub) were used to ensure, as far as possible, consistency in tissue nutrient content and other plant characteristics that may influence insect performance and response.

Dam, Boschhoek Golf Club (29°200 59.600 S 30°050 48.300 E); Lagarosiphon verticillifolius Obermeyer, seasonal pan, Ndumo Game Reserve (26°5200 53.400 S 32°150 03.300 E); Lagarosiphon cordofanus Obermeyer, Driekoppies Dam (25°460 04.700 S 31°280 04.500 E); Najas marina L., Mzinene River (27°520 17.100 S 32°210 85.500 E); Lagarosiphon muscoides Harvey, forestry dam, Cedara (29°330 29.500 S 30°150 17.300 E); Egeria densa Planchon, Midmar Dam (29°310 20.500 S 30°120 59.200 E). All tests were conducted from January to May in 2012 and 2013. Plants were washed well in reverse osmosis water prior to use to eliminate predatory insects. Plants collected in Pongolapoort Dam were routinely treated with DipelÒ prior to use because of the presence of a leaf-cutting moth, Parapoynx diminutalis Snellen (Lepidoptera: Crambidae) that attacks hydrilla (Bownes, 2010) and other aquatic plant species (Bownes, unpubl.) in the dam. The Bt is specific to lepidopteran larvae and does not impact negatively on Hydrellia sp., however plants were left for a week after treatment and washed well before use. An indigenous Hydrellia fly, Hydrellia lagarosiphon Deeming was present on L. major, however because of its larger size compared to Hydrellia sp. and its low occurrence on L. major at the time plants were collected, its presence was not seen to be a potential confounding factor. Enough plant material was provided to ensure that food availability did not limit larval survival. Test plant species were selected on the basis of phylogenetic relatedness to H. verticillata (phylogeny of the southern African Alismatales provided by botanist, R. Glen, SANBI) as well as their susceptibility to Hydrellia sp. in the trials conducted to evaluate its safety for release in South Africa (Bownes, 2014). H. verticillata was treated as a test plant and not as a control and a total of 11 plant species (Table 1) were tested, 10 of which are in the Hydrocharitaceae. Eggs for the trials were obtained by exposing sprigs or portions of the test plants to approximately 100 adult flies per fly species in separate isolation boxes (43  31  55 cm). Both fly species demonstrate indiscriminate oviposition (Bownes, 2014; Buckingham et al., 1989), however eggs for the trial with N. marina had to be transferred on minute pieces of H. verticillata due to limited oviposition by females. The plants were held in Petri dishes with reverse osmosis water and with some plant material exposed for oviposition. These were left in the oviposition chambers for 24 h, at which time the eggs were collected on tiny plant fragments with fine forceps, under a Zeiss microscope at 6 magnification. Ten eggs per replicate were transferred to 500 ml transparent plastic containers containing reverse osmosis water and the same test plant on which they were oviposited. The jars were enclosed with white netting held in place by an elastic band and were held in a quarantine glasshouse with a 22/28 °C min/max and a 14:10 L:D photoperiod for larval development. There were 10 replicates per test plant for each fly species. The number of larvae to complete development (i.e. to survive to adulthood) and the number of days to eclosion were recorded.

2.2. Comparison of host range No-choice larval development trials were used to compare the host ranges of H. pakistanae and Hydrellia sp. Tests were initiated with plants collected from rivers, seasonal pans or impoundments in South Africa. In most cases, the plants were used within a few days of field collecting, however two species, Lagarosiphon ilicifolius Obermeyer (Hydrocharitaceae) and Vallisneria spiralis L. (Hydrocharitaceae) were first cultivated at ARC-PPRI’s research facilities at Cedara to obtain enough plant material for testing. Another two test plant species, Najas horrida Brown ex Magnus (Hydrocharitaceae) and Potamogeton crispus L. (Potamogetonaceae) were collected from Pongolapoort Dam (including H. verticillata). The remaining six test species were collected at the following localities: Lagarosiphon major Ridley (Moss), Ndiza

2.3. Survival and development To compare Hydrellia sp. and H. pakistanae survival and speed of development, one-day old eggs of each fly species were obtained, as above. Ten eggs per replicate (n = 15/spp.) were transferred to 700 ml transparent plastic containers with reverse osmosis water and 12 cm apical sprigs of H. verticillata. Enough plant material was provided in the jars to ensure that food availability did not limit larval survival and development. The jars were enclosed with netting and an elastic band and were held in a quarantine glasshouse with a 14:10 h L:D photoperiod and a min/max of 22/ 28 °C. The number of adults to emerge and the number of days for egg to adult development were recorded.

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A. Bownes / Biological Control 84 (2015) 44–52 Table 1 Summary of no-choice larval development trials investigating the host ranges of Hydrellia pakistanae and Hydrellia sp.

1 a *

Family

Genus

Species and species authority

No. eggs tested/per spp.

% H. pakistanae adults1 (±SE)a

% Hydrellia sp. adults1 (±SE)a

H. pakistanae mean development time (days) (±SE)a

Hydrellia sp. mean development time (days) (±SE)a

Hydrocharitaceae

Hydrilla

100

87a (±3.35)

70b (±5.37)

25.45a (±0.12)

25.85a (±0.26)

Hydrocharitaceae Hydrocharitaceae Hydrocharitaceae Hydrocharitaceae Hydrocharitaceae Hydrocharitaceae Hydrocharitaceae Hydrocharitaceae Hydrocharitaceae Potamogetonaceae

Lagarosiphon Lagarosiphon Lagarosiphon Lagarosiphon Lagarosiphon Najas Najas Vallisneria Egeria Potamogeton

verticillata (L.f.) Royle (monoecious Malaysian/ Indonesian biotype) major Ridley (Moss) muscoides Harvey ilicifolius Obermeyer verticillifolius Obermeyer cordofanus Caspary horrida A. Brown ex Magnus marina L. spiralis L. densa Planchon crispus L.

100 100 100 100 100 100 100 100 100 100

11a 31a 42a 0 0 0 0 0 0 33a

6a (±3.14) 25a (±4.53) 40a (±3.89) 0 0 0 0 0 0 3b (±8.17)

27.14a (±0.66)

27.9a (±0.79)

(±2.67) (±6.05) (±8.43)

(±1.53)

*

*

28.13a (±0.28) n/a n/a n/a n/a n/a n/a 29a (±0.33)

27.03a (±0.56) n/a n/a n/a n/a n/a n/a 29.3a (±0.88)

(Total number of adults/total No. of eggs)  100. Means followed by the same letter within a row (for the same parameter) are not statistically different according to a one-way ANOVA (P < 0.05). Not recorded.

2.4. Fecundity, longevity and egg viability This trial followed a similar methodology to Buckingham et al. (1989) when investigating the performance of H. pakistanae on a test plant species versus H. verticillata. One-day old H. pakistanae and Hydrellia sp. eggs were collected from H. verticillata sprigs in the culture rearing boxes (75  55  55 cm) approximately 24 h after fresh plants had been exposed to the adult flies. In order to ensure that virgin females were used in the trial, the eggs were placed individually in 35 ml glass vials with reverse osmosis water and a 8 cm apical sprig of H. verticillata. The vials were left in a quarantine glasshouse for larval development under the same conditions specified above. At eclosion, adults of each fly species were paired (n = 10 per fly species) and placed in 500 ml plastic containers containing 4–5 apical sprigs of H. verticillata and reverse osmosis water. A yeast and sugar solution (Freedman et al., 2001) was provided as food for the adults. The containers were covered with white netting, held in place with an open-holed lid and were kept in a Labcon growth chamber set at 27 °C with a 16:8 L:D photoperiod. The H. verticillata, water and artificial food supplement were replaced every two to three days. The containers were checked daily for adult mortality and to collect and count the eggs. Up to 15 eggs per replicate were collected each day to observe egg hatch. The eggs were collected on minute pieces of H. verticillata and were placed in 35 ml transparent plastic containers with a moist cotton pad to prevent desiccation. The egg containers with lids were held in the same growth chamber as the adults and were checked daily to record egg hatch. The trial ran until the last adult of each fly species died. 2.5. Impact The per capita impact of male and female H. pakistanae and Hydrellia sp. was compared by investigating leaf mining during immature development. Eggs for the trial were obtained by exposing sprigs of H. verticillata in Petri dishes with reverse osmosis water to 50 newly eclosed adult H. pakistanae and Hydrellia sp. in separate isolation boxes (43  31  55 cm) for approximately 24 h. Eggs were collected as above. Forty eggs per fly species were randomly selected for the trial and larvae were reared individually in 35 ml glass vials and a 8 cm apical sprig of H. verticillata and reverse osmosis water. The vials were held for larval development in a Labcon growth chamber set at a constant temperature of 27 °C,

with a 16:8 L:D photoperiod. The vials were capped with a gauze lid and were checked daily to top up with water as necessary. The sex of the emerging adults, and the number of leaves mined during their immature development were recorded. Partially mined leaves were included in the count record as one damaged leaf, although larvae typically mined all or nearly all of a leaf before entering a new one. 2.6. Performance of H. pakistanae on S.A. H. verticillata To enable comparison, H. pakistanae’s performance parameters were investigated under the same environmental conditions assigned by Buckingham et al. (1989), when investigating its performance on the U.S. dioecious biotype (in comparison to a test plant). Trials were conducted in a Labcon growth chamber set at 27 °C with a 16:8 h L:D photoperiod. Lighting was provided by both incandescent and fluorescent globes. Eggs for the trial were obtained as above. Egg to adult survival of H. pakistanae on S.A. H. verticillata was investigated by transferring one-day old eggs to 500 ml plastic containers with reverse osmosis water and apical sprigs of H. verticillata that was cultivated in the laboratory. Enough plant material was provided in the jars to ensure that resource-limitation was not a confounding factor. The number of adults and the number of days to eclosion were recorded. This trial was replicated 7 times with 10 H. pakistanae eggs per replicate. Mean percent egg to adult survival and the mean number of days to emergence of 50% of the adults were compared to these parameters measured for H. pakistanae developing on the U.S. dioecious H. verticillata biotype (Buckingham et al., 1989). The methodology used to investigate fecundity, adult longevity and egg viability of H. pakistanae was based on that used by Buckingham et al. (1989), except adult flies for the trial were reared individually in 35 ml glass vials containing a 8 cm apical sprig of South African H. verticillata. The vials (n = 40) contained reverse osmosis water, were capped with a gauze lid and were held in a quarantine glasshouse with a 22/28 °C min/max and a 14:10 h L:D photoperiod. At eclosion, pairs (n = 9) of adults were placed in 500 ml plastic containers containing H. verticillata in reverse osmosis water and a yeast/sugar solution (Freedman et al., 2001) as food. Droplets of the artificial food supplement were smeared on the inside of the jar and were reapplied, as necessary. The containers were covered with white netting held in place with an openholed lid and were placed in a growth chamber with the same

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temperature and light regime specified above. Containers were checked daily for adult mortality and eggs were collected and counted every one to two days. Up to 15 eggs per replicate were kept at each sampling interval to observe hatching. The eggs were collected on pieces of H. verticillata and stored in 35 ml transparent plastic containers with a cotton pad, sprayed with reverse osmosis water. The trial ran until the last adult died. These data were compared to the same parameters measured for H. pakistanae reared on U.S. dioecious H. verticillata (Buckingham et al., 1989). 2.7. Statistical analysis All the data analyzed statistically conformed to the assumptions of normality and homogeneity of variances and were compared using Analysis of Variance (ANOVA). Survival and duration of immature development of H. pakistanae and Hydrellia sp. in the host range trials were compared (independently) by one-way ANOVA. Survival, egg to adult development time, adult longevity, lifetime fecundity of females and egg viability were compared between H. pakistanae and Hydrellia sp. reared on S.A. H. verticillata with a one-way ANOVA. A two-way ANOVA with interaction was used to compare the numbers of leaves mined by male and female H. pakistanae and Hydrellia sp. Tukey’s HSD test for unequal sample sizes was used to separate the means (Zar, 1999). All statistics analyses were conducted in Statistica ver. 12 (Statsoft, 2013). 3. Results 3.1. Comparison of host range Both H. pakistanae and Hydrellia sp. larvae were able to develop to adults on five of the 11 plants species tested, including H. verticillata. Larval survival rates were similar on L. muscoides, L. major and L. ilicifolius, however, P. crispus, the only species outside of the Hydrocharitaceae that was tested, supported higher larval survival rates (F1;18 = 13.02; P = 0.002) of H. pakistanae (Table 1). On the host plant, H. verticillata, larval survival of H. pakistanae was significantly lower (F1;18 = 7.21; P = 0.015) than that of Hydrellia sp. Eighty-seven percent of the Hydrellia sp. eggs tested developed to adults compared to 70% of H. pakistanae eggs. Mean larval development times of the two fly species were similar on all the plant species tested, including H. verticillata (Table 1). 3.2. Survival and development Egg to adult survival was significantly different (F1;28 = 5.60; P = 0.025) between the two fly species. An average of 79% of the Hydrellia sp. eggs tested successfully completed development in comparison to an average of 65% of the H. pakistanae eggs tested (Fig. 1). Likewise, speed of development differed (F1;28 = 104.41; P < 0.0001). The mean development time to 50% eclosion varied from 24 days for Hydrellia sp. to 29 days for H. pakistanae. Hence immature development took, on average, five days longer for H. pakistanae compared to Hydrellia sp. (Fig. 1). 3.3. Fecundity, longevity and egg viability Adult longevity of H. pakistanae and Hydrellia sp. was significantly different in both males (F1;18 6.56; P = 0.020) and females (F1;18 = 6.70; P = 0.019) (Table 2). Male Hydrellia sp. lived for an average of 23.4 days compared to 18.4 days for H. pakistanae and female Hydrellia sp. lived for 19.8 days compared to only 14.9 days for H. pakistanae females. This equates to a difference of an average of five days for both sexes. Egg production by females differed significantly (F1;18 = 10.48; P = 0.005) between the two fly species

when reared on South African H. verticillata. Hydrellia sp. females laid 75.2 eggs during their lifetime, compared to an average of 50.4 for H. pakistanae. Egg viability was high (94–96%) and did not differ between H. pakistanae and Hydrellia sp. (Table 2). 3.4. Impact (leaf mining) A total of 29 (72.5%) Hydrellia sp. larvae completed development and of these, 12 (41.4%) were males and 17 (58.6%) were females. A total of 22 (55%) H. pakistanae developed to adults, 8 (36.4%) of which were males and 14 (63.6%) were females. In both cases, there was a female bias. Hydrellia sp. males mined, on average, 8.4 leaves during their immature development compared to 10.1 for H. pakistanae males. Female Hydrellia sp. mined 13.2 leaves as opposed to 13.8 for H. pakistanae (Fig. 2). There were no significant differences in the numbers of leaves mined by male or female Hydrellia sp. and H. pakistanae or a significant interaction. However, with the fly species data combined, females mined more leaves than males (F1;47 = 48.10; P < 0.0001). 3.5. Performance of H. pakistanae on S.A. H. verticillata In general, H. pakistanae performed better on U.S. dioecious H. verticillata compared to South African H. verticillata, with the exception that individuals reared on the South African biotype lived longer than those reared on the U.S. dioecious biotype (Table 3). Larval survival on the U.S. dioecious biotype was 1.4-fold higher (Buckingham et al., 1989) than on the South African biotype. Mean larval development time to emergence of 50% of the adults was extended by 18 days on average when larvae were reared on the South African monoecious biotype in comparison to the U.S. dioecious biotype (Buckingham et al., 1989). Further, reproductive success of H. pakistanae females reared on U.S. dioecious H. verticillata was greater than on South African H. verticillata. Females, on average, produced 51.6 more eggs during their lifetime and their eggs were 1.4 times more viable when reared on U.S. dioecious H. verticillata (Buckingham et al., 1989) (Table 3). 4. Discussion The laboratory host ranges of H. pakistanae and Hydrellia sp. were found to be almost identical, indicating that the two species have similar response cues to physiologically suitable host plants. Although closely related herbivorous insects often demonstrate similar patterns of host plant use (Ehrlich and Raven, 1964), host races/biotypes of herbivorous insects can differ in their ability to survive and develop on plants not normally used as hosts, including different host-plant genotypes (Grevstad et al., 2013). H. pakistanae and Hydrellia sp. were able to complete their life cycle on exactly the same range of host plants and were equally successful on all the non-target species with the exception of P. crispus, where higher numbers of H. pakistanae adults were produced. Although preliminary host range testing of H. pakistanae was conducted in South Africa, Hydrellia sp. was subjected to intensive host range testing because of its origins on a more suitable biotype of H. verticillata. Comparing these results (Bownes, 2014) with those of Buckingham et al. (1989) for H. pakistanae, Hydrellia sp. would initially appear to have the broader host range. H. pakistanae developed to adults on three test plant species compared to five for Hydrellia sp. and in much lower proportions (1–7% compared to 4–39%). On closer inspection, the results support the findings presented here that H. pakistanae and Hydrellia sp. have similar fundamental host ranges. Both Hydrellia sp. (Bownes, 2014) and H. pakistanae (Buckingham et al., 1989) were able to develop on Najas species native to the relevant recipient region and both

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Fig. 1. Mean (±SE) percent survival and mean egg to adult development times of Hydrellia pakistanae and Hydrellia sp. reared on the South African biotype of Hydrilla verticillata. Means compared by one-way ANOVA; different letters indicate statistically significant differences (P < 0.05).

Table 2 Summary of performance parameters of Hydrellia pakistanae and Hydrellia sp. reared on the South African biotype of their host plant, Hydrilla verticillata. Performance parameter Adult longevity ± SE (n) No. of eggs/female ± SE (n) Egg viability ± SE (n)

Hydrellia sp. a

$$ 19.8 ± 1.58 (10) ## 23.4a ± 1.67 (10) 75.2a ± 6.75 (10) 94.06a ± 1.97 (10)

Hydrellia pakistanae $$ 14.9b ± 1.04 (10) ## 18.4b ± 1.01 (10) 50.4b ± 3.63 (10) 95.76a ± 1.38 (10)

a Means followed by the same letter within a row are not significantly different according to a one-way ANOVA (P < 0.05).

developed on P. crispus. Representatives of the African endemic plant genus, Lagarosiphon (Hydrocharitaceae), which was equally susceptible to both fly species were, for obvious reasons, not tested in the laboratory host range trials conducted with H. pakistanae in North America. Particularly intriguing is the response of both flies to P. crispus, which may be indicative of biochemical similarities to H. verticillata. Interestingly, H. balciunasi, also released for biological control of H. verticillata in North America, developed on P. crispus in the laboratory host range trials (Buckingham et al., 1991). Representatives of the Potamogetonaceae were selected for testing due to their similarities in ecology and habitat requirements to H. verticillata but typically test plant lists for screening are devised according to phylogenetic relationships (Briese and Walker, 2002; Wapshere, 1974). Phylogenetic theory would not have predicted the consistent use of P. crispus by these closely related flies, which begs the question of whether the phylogenetic approach alone adequately assesses risk involved with biocontrol agent introductions. Wheeler (2005) and Wheeler et al. (2014) demonstrated that secondary plant chemistry can be an important determinant of

host plant use by specialist insects, and argued that screening protocols that strictly follow phylogenetic relationships may exclude unrelated plant taxa that may be at risk of attack. Despite this, Pemberton’s (2000) review shows that the risk of non-target host use by introduced biocontrol agents can be reliably predicted using phylogeny. Although P. crispus is readily accepted in the laboratory under no-choice conditions, collections of this plant in the native range in India did not yield H. pakistanae (Baloch et al., 1980). This suggests the positive results for H. pakistanae on P. crispus and quite possibly Hydrellia sp. are most likely laboratory artifacts. Further, H. pakistanae failed to persist on P. crispus after the seventh generation in Buckingham et al.’s (1989) laboratory trials and individuals with reduced fitness were produced on P. crispus in comparison to the host plant (Buckingham et al., 1989). The population dynamics of insect biocontrol agents in the recipient region can depend on their adaptation to the environment and a number of bottom-up factors, most notably host plant quality (Price, 2000). However, host plant genotype is increasingly recognized as a bottom-up factor of considerable importance, particularly in weed biological control programmes targeting genetically diverse weeds (Grevstad et al., 2013; Lym and Carlson, 2002; Lym et al., 1996; Manrique et al., 2008; Zachariades et al., 2011). This, coupled with evidence of severe limitations in the H. verticillata biological control programme in North America due to biotype mismatches (Grodowitz et al., 1997; Grevstad et al., 2013), strongly suggested that Hydrellia sp. should be the preferred candidate agent of the two fly species. However, the results presented here provide the most convincing support for this selection. McClay and Balciunas (2005) note that the range and abundance of insect biological control agents are primarily functions of their life history characteristics, which suggests agent efficacy at the population level should be greater for an insect with higher survival and reproductive success, all other

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Fig. 2. Mean (±SE) number of Hydrilla verticillata leaves mined by male and female Hydrellia pakistanae and Hydrellia sp. Means compared by two-way ANOVA; different letters indicate statistically significant differences according to Tukey’s HSD test for unequal sample sizes (P < 0.05).

Table 3 Summary of Hydrellia pakistanae performance parameters when reared on the monoecious Hydrilla verticillata biotype from South Africa in comparison to the U.S. dioecious H. verticillata biotype. Trials conducted under the same environmental conditions.

* a b

Hydrilla verticillata biotype

% Adults ± SD (n)

Days to 50% adult emergence ± SD (n)

Longevity in days ± SD (n) ## $$

Eggs/$ ± SD (n)

% Egg hatch ± SD

S.A. monoecious Hydrilla verticillata * U.S. dioecious Hydrilla verticillata

42.9 ± 6.8 (7) 59.3 ± 7.6 (3)

38.3 ± 0.9 (7) 19.7 ± 0.6 (3)

16.8 ± 3.7 (9) 11.4 ± 3.3 (5)

26.4 ± 6.3 (9) 78.0 ± 19.0 (5)

61.3a ± 39.9 85.6b ± 4.3

18.3 ± 4.2 (9) 10.3 ± 2.3 (5)

Buckingham et al. (1989). Samples of up to 15 eggs selected from the total number of eggs collected at each sampling interval for each replicate. Means of all samples in the replicate. Three samples of five eggs deposited each day in each replicate. Means of all samples in the replicate.

factors being equal. H. pakistanae and Hydrellia sp. differed significantly in their performance on the S.A. biotype of H. verticillata. Yet, when studied separately on their respectively more suitable host plant genotypes, but under the same environmental conditions (Buckingham et al., 1989; unpubl.), the life history parameters of the two fly species are similar. For example, egg to adult development averaged 23 days for both flies, and lifetime fecundity ranged from 78 for H. pakistanae (Buckingham et al., 1989) to 102 for Hydrellia sp. (Bownes, unpubl.), although an average of 82.9 eggs per female has also been recorded for Hydrellia sp., under the same environmental conditions (Bownes, 2014). These similarities give a strong indication that host plant genotype was indeed responsible for the observed differences in the functional responses of the two fly species. Furthermore, by all measures, with the exception of adult longevity, the South African monoecious biotype of H. verticillata is an inferior host for H. pakistanae in comparison to the U.S. dioecious biotype, a close relative of the fly’s native host plant genotype (although it should be noted that this interpretation does have limitations since other factors such as plant nutritional quality or fly strains may have differed and could possibly have influenced the performance of H. pakistanae, despite having used similar environmental rearing conditions). Host plant genotype was not predicted to limit H. pakistanae in North America (Dray and Center, 1996), however, the fly failed to

establish widely on a genetically distinct biotype (i.e. U.S. monoecious H. verticillata) to its native host plant but conversely, established well on the U.S. dioecious form (Grodowitz et al., 1999, 2003). This clearly demonstrates the importance of old associations in weed biological control, where specialist insects that have co-evolved with a particular genotype of the targeted weed are usually preferable. Although these results only show the potential effects of host plant genotype on the short term dynamics of H. pakistanae and Hydrellia sp., Underwood and Rausher (2000) showed that differences in the performance parameters of herbivorous insects according to host plant genotypes have the potential to influence long-term population dynamics of such herbivores. Although the specific plant quality traits responsible for the inferior performance of H. pakistanae on South African H. verticillata in comparison to Hydrellia sp., as well as in comparison to the U.S. dioecious biotype are unknown, biochemical differences between H. pakistanae’s native host plant genotype and South African H. verticillata are a possible explanation. H. verticillata displays high variability in isoenzyme patterns between different populations (Verkleij, 1983), which may influence the performance of specialized phytophagous insects. Farmer and Adams (1989) suggest that high genetic diversity within plant species may be indicative of different physiological races, which may have different responses to management interventions, including biological

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control. Alternatively, Benoit’s (2011) proposal of three separate Hydrilla lineages could explain the superior performance of the flies on the host plant genotypes on which they originated. It is also noted that other, unknown factors such as differences in the nutritional requirements of the two fly species or unobvious differences in colony maintenance conditions may have influenced their life histories. Larval mining by H. pakistanae (and Hydrellia sp.) during their immature development reduces H. verticillata’s leaf surface area for photosynthesis which causes an associated reduction in plant growth and competitive ability (Doyle et al., 2007; Van et al., 1998). Doyle et al. (2002) showed that reductions in H. verticillata tuber production and biomass accumulation were directly proportional to the amount of leaf material damaged by H. pakistanae larval mining (Doyle et al., 2002). In the present study, H. pakistanae and Hydrellia sp. used similar amounts of plant tissue during their larval development, suggesting their per capita impact on H. verticillata will be similar. The flies also demonstrated similar patterns of larval mining. Early instar larvae usually mined leaflets in the crown and mature larvae tended to mine larger leaves below the crown before pupating on the stem. The difference in the number of leaves mined by males and females of both species was expected due to sexual dimorphism, with females, in both cases, being larger than the males. The reason for the strong female bias is unknown, particularly since the laboratory cultures of both species exhibited a sex ratio of almost 1:1 (Buckingham et al., 1989; Bownes, unpubl.). Bownes et al. (2013) found plant tissue nutrient levels to have significant effects on sex ratios of a leaf-feeding grasshopper, Cornops aquaticum Brüner (Orthoptera: Acrididae), being female-biased when plant nitrogen levels were high and male-biased when plant nitrogen levels were low. This is a plausible explanation since the H. verticillata used in this trial was cultivated in pond sediment fertilized with ammonium nitrate (NH4NO3). In the more recent history of weed biological control, the science has been criticized for a lack of predictability in terms of agent efficacy (McEvoy and Coombs, 1999), which encouraged practitioners to be more selective of prospective agents (McClay and Balciunas, 2005; Sheppard, 2003). Although host specificity is the primary consideration and both an ethical and regulatory requirement, potential efficacy and climate- and biotype-compatibility are important secondary motivating factors in the selection of agents. With the aim to be more selective and to avoid potential negative competition effects between two closely-related insects occupying the same niche (Grodowitz et al., 1997), only one of the two fly species was deemed to be suitable for biological control of H. verticillata in South Africa. The similar host ranges of H. pakistanae and Hydrellia sp. eliminated host specificity as the crucial determinant hence biotype matching, potential efficacy and host plant genotype-suitability guided the decision to reject H. pakistanae in favor of Hydrellia sp., which is considered to have a narrow host range (Bownes, 2014). Indeed, if Hydrellia sp. fails to establish or contribute significantly to the suppression of H. verticillata in South Africa, H. pakistanae may be reconsidered. Acknowledgments The KwaZulu-Natal Department of Agriculture and Environmental Affairs – Invasive Alien Species Programme (KZN DAEA – IASP) and the Department of Environmental Affairs – Natural Resource Management Programmes (DEA-NRMP) provided research funding. Mr. Matthew Purcell (CSIRO Australia) and Dr. Michael Grodowitz (U.S. Army Corps of Engineers) are gratefully acknowledged for providing starter cultures of the flies and ARCPPRI support staff, particularly Ms. Lynnet Khumalo and Mr. Njabulo Mngomezulu, for technical assistance. I am extremely grateful to Dr. John Deeming (National Museum of Wales) for his

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work on the description of the Singapore Hydrellia. KZN Wildlife and Boschoek Golf Club are thanked for facilitating collection of some test plants. References Balciunas, J.K., Grodowitz, M.J., Cofrancesco, A.F., Shearer, J.F., 2002. Hydrilla. In: van Driesche, R., Blossey, B., Hoddle, M., Lyon, S., Reardon, R. (Eds.), Biological Control of Invasive Plants in the Eastern United States. USDA Forest Service, Morgantown, pp. 91–114. 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. Benoit, L.K., 2011. Cryptic speciation, genetic diversity and herbicide resistance in the invasive aquatic plant Hydrilla verticillata. Doctoral Dissertations, Paper AAI3492149, University of Connecticut. Bownes, A., 2010. Asian aquatic plant moth, accidentally introduced earlier, contributes to control of an emerging aquatic weed, Hydrilla verticillata, in South Africa. AJAS 35, 307–311. Bownes, A., 2014. Suitability of a leaf-mining fly, Hydrellia sp., for biological control of the invasive aquatic weed, Hydrilla verticillata in South Africa. BioControl 59, 771–780. Bownes, A., Hill, M.P., Byrne, M.J., 2013. Nutrient-mediated effects on Cornops aquaticum Brüner (Orthoptera: Acrididae), a potential biological control agent of water hyacinth, Eichhornia crassipes (Mart.) Solms (Pontederiaceae). Biol. Control 67, 548–554. Briese, D.T., Walker, A., 2002. A new perspective on the selection of test plants for evaluating the host specificity of weed biological control agents: the case of Deuterocampta quadrijuga, a potential insect control agent of Heliotropium amplexicaule. Biol. Control 25, 273–287. Buckingham, G.R., 1988. Reunion in Florida: hydrilla, a weevil and a fly. Aquatics 10, 19–25. Buckingham, G.R., Okrah, E.A., Thomas, M.C., 1989. Laboratory host range tests with Hydrellia pakistanae (Diptera: Ephydridae), an agent for biological control of Hydrilla verticillata (Hydrocharitaceae). Environ. Entomol. 18, 164–171. Buckingham, G.R., Okrah, E.A., Christian-Meier, M., 1991. Laboratory biology and hot range of Hydrellia balciunasi (Diptera: Ephydridae). Entomophaga 36, 575– 586. Buckingham, G.R., Okrah, E.A., 1993. Biology and host range studies with two species of Hydrellia (Diptera: Ephydridae) that feed on hydrilla. Technical Report A-93-7. U.S. Army Engineer Research and Development Center, Vicksburg. Center, T.D., Grodowitz, M.J., Cofrancesco, A.F., Jubinsky, G., Snoddy, E., Freedman, J.E., 1997. Establishment of Hydrellia pakistanae (Diptera: Ephydridae) for the biological control of the submersed aquatic plant Hydrilla verticillata (Hydrocharitaceae) in the southeastern United States. Biol. Control 8, 65–73. Cook, C.D.K., Lüönd, R., 1982. A revision of the genus Hydrilla (Hydrocharitaceae). Aquat. Bot. 13, 485–504. Deonier, D.L., 1971. A systematic and ecological study of Nearctic Hydrellia (Diptera: Ephydridae). Smithson. Contrib. Zool. 68, 1–147. Doyle, R.D., Grodowitz, M.J., Smart, R.M., Owens, C., 2002. Impact of herbivory by Hydrellia pakistanae Deonier (Diptera: Ephydridae) on growth and photosynthetic potential of Hydrilla verticillata. Biol. Control 24, 221–229. Doyle, R.D., Grodowitz, M.J., Smart, R.M., Owens, C., 2007. Separate and interactive effects of competition and herbivory on the growth, expansion and tuber formation of Hydrilla verticillata. Biol. Control 41, 327–338. Dray, F.A., Center, T.D., 1996. Reproduction and development of the biocontrol agent Hydrellia pakistanae (Diptera: Ephydridae) on monoecious hydrilla. Biol. Control 7, 275–280. Ehrlich, P.R., Raven, P.H., 1964. Butterflies and plants: a study in coevolution. Evolution 18, 586–608. Farmer, A.M., Adams, M.S., 1989. A consideration of the problems of scale in the study of the ecology of aquatic macrophytes. Aquat. Bot. 33, 177–189. Freedman, J.E., Grodowitz, M.J., Cofrancesco, A.E., Bare, R., 2001. Mass rearing Hydrellia pakistanae Deonier, a biological control agent of Hydrilla verticillata (L.f.) Royle, for release and establishment. Report ERDC/EL TR-01-24. US Army Engineer Research and Development Center, Vicksburg. Goolsby, J.A., De Barro, P.J., Makinson, J.R., Pemberton, R.W., Hartley, D.M., Frohlich, D.R., 2006. Matching the origin of an invasive weed for selection of a herbivore haplotype for a biological control program. Mol. Ecol. 15, 287–297. Grevstad, F., Shaw, R., Bourchier, R., Sanguankeo, P., Cortat, G., Reardon, R.C., 2013. Efficacy and host specificity compared between two populations of the psyllid Aphalara itadori, candidates for biological control of invasive knotweeds in North America. Biol. Control 65, 53–62. 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., 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. Grodowitz, M.J., Cofrancesco, A.F., Stewart, R.M., Madsen, J., Morgan, D., 2003. Possible impact on Lake Seminole Hydrilla by the introduced leaf-mining fly,

52

A. Bownes / Biological Control 84 (2015) 44–52

Hydrellia pakistanae. Report ERDC/TN APCRP-BC-15. U.S. Army Engineer Research and Development Center, Vicksburg. Grodowitz, M.J., Nachtrieb, J.G., Harms, N.E., Freedman, J.E., 2013. Suitability of using introduced Hydrellia spp. for management of monoecious Hydrilla verticillata. In: Wu, Y., Johnson, T., Sing, S., Raghu, S., Wheeler, G., Pratt, P., Warner, K., Center, T., Goolsby, J., Reardon, R. (Eds.), Proceedings of the XIII International Symposium on Biological Control of Weeds. FHTET, Hawaii, p. 189. Haller, W.T., Sutton, D.L., 1975. Community structure and competition between Hydrilla and Vallisneria. Hyacinth Contr. J. 13, 48–50. Harms, N.E., Grodowitz, M.J., 2011. Overwintering biology of Hydrellia pakistanae, a biological control agent of hydrilla. J. Aquat. Plant Manage. 49, 114–117. Henderson, L., 2006. Emerging weeds survey uncovers hydrilla in KwaZulu-Natal. PPRI News 67, 13. Langeland, K.A., 1996. Hydrilla verticillata (L.F.) Royle (Hydrocharitaceae), ‘‘The Perfect Aquatic Weed’’. Castanea 61, 293–304. Les, D.H., Moody, M.L., Soros, C.L., 2006. A reappraisal of phylogenetic relationships in the monocotyledon family Hydrocharitaceae (Alismatidae). Aliso 22, 211–230. Lym, R.G., Carlson, R.B., 2002. Effect of leafy spurge (Euphorbia esula) genotype on feeding damage and reproduction of Aphthona spp.: implications for weed biological control. Biol. Control 23, 127–133. Lym, R.G., Nissen, S.J., Rowe, M.L., Lee, D.J., Masters, R.A., 1996. Leafy spurge (Euphorbia esula L.) genotype affects gall midge (Spurgia esulae L.) establishment. Weed Sci. 44, 629–633. Madeira, P.T., Van, T.K., Steward, K.K., Schnell, R.J., 1997. Random amplified polymorphic DNA analysis of the phonetic relationships among world-wide accessions of Hydrilla verticillata. Aquat. Bot. 59, 217–236. Madeira, P.T., Coetzee, J.A., Center, T.D., White, E.E., Tipping, P.W., 2007. The origin of Hydrilla verticillata recently discovered at a South African dam. Aquat. Bot. 87, 176–180. Manrique, V., Cuda, J.P., Overholt, W.A., Williams, D.A., Wheeler, G.S., 2008. Effect of host–plant genotypes on the preference of three candidate biological control agents of Schinus terebinthifolius in Florida. Biol. Control 47, 167–171. McClay, A.S., Balciunas, J.K., 2005. The role of pre-release efficacy assessment in selecting classical biological control agents for weeds – applying the Anna Karenina principle. Biol. Control 35, 197–207. McEvoy, P., Coombs, E.M., 1999. Biological control of plant invaders: regional patterns, field experiments, and structured population models. Ecol. Appl. 9, 387–401. Pemberton, R.W., 2000. Predictable risk to native plants in weed biological control. Oecologia 125, 489–494. Price, P.W., 2000. Host plant resource quality, insect herbivores and biocontrol. In: Spencer, N.R. (Ed.), Proceedings of the X International Symposium on Biological Control of Weeds. Montana State University, Montana, pp. 583–590.

Schmitz, D.C., Nelson, B.V., Nall, L.E., Schardt, J.D., 1991. Exotic plants in Florida: a historical perspective and review of present aquatic plant regulation program. In: Center, T.D., Doren, R.F., Hofstetter, R.L., Myers, R.L. (Eds.), Proceedings of a Symposium on Exotic pest plants. United States Department of the Interior, National Park Service, Washington D.C., pp. 303–336. Sheppard, A.W., 2003. Prioritizing agents based on predicted efficacy. In: Jacob, H.S., Briese, D.T. (Eds.), Improving the Selection, Testing and Evaluation of Weed Biological Control Agents. CRC for Australian Weed Management, Glen Osmond, pp. 11–21. Statsoft Inc., 2013. Statistica 12.0 Data Analysis Software System. Statsoft Inc., Tulsa, Oklahoma. Steward, K.K., 1993. Comparative studies of monoecious and dioecious hydrilla (Hydrilla verticillata) biotypes. Aquat. Bot. 46, 169–183. Steward, K.K., Van, T.K., Carter, V., Pieterse, A.H., 1984. Hydrilla invades Washington, D.C. and the Potomac. Am. J. Bot. 71, 162–163. Thomaz, S.M., Carvalho, P., Padial, A.A., Kobayashi, J.T., 2009. Temporal and spatial patterns of aquatic macrophyte diversity in the Upper Paraná River floodplain. Braz. J. Biol. 69, 617–625. Underwood, N., Rausher, M.D., 2000. The effects of host plant genotype on herbivore population dynamics. Ecology 81, 1565–1576. Van, T.K., 1989. Differential responses to photoperiods in monoecious and dioecious Hydrilla verticillata. Weed Sci. 37, 552–556. Van, T.K., Wheeler, G.S., Center, T.D., 1998. Competitive interactions between Hydrilla (Hydrilla verticillata) and Vallisneria (Vallisneria americana) as influenced by insect herbivory. Biol. Control 11, 185–192. Verkleij, J.A.C., 1983. A comparative study of the morphology and isoenzyme patterns of H. verticillata (L.f.) Royle. Aquat. Bot. 17, 43–59. Wapshere, A.J., 1974. A strategy for evaluating the safety of organisms for biological weed control. Ann. Appl. Biol. 77, 201–211. Wheeler, G.S., 2005. Maintenance of a narrow host range by Oxyops vitiosa; a biological control agent of Melaleuca quinquenervia. Biochem. Syst. Ecol. 33, 383–965. Wheeler, G.S., Chawner, M., Williams, D.A., 2014. Predicting the host range of Nystalea ebalea: secondary plant chemistry and host selection by a surrogate biological control agent of Schinus terebinthifolia. Biol. Control 73, 39–49. Zachariades, C., Strathie, L.W., Retief, E., Dube, N., 2011. Progress towards the biological control of Chromolaena odorata (L.) R.M. King and H. Rob. (Asteraceae) in South Africa. Afr. Entomol. 19, 282–302. Zar, J.H., 1999. Biostatistical Analysis, fourth ed. Prentice Hall, Upper Saddle River, NJ.