Biological Control 42 (2007) 129–138 www.elsevier.com/locate/ybcon

Preliminary evaluation of Megamelus scutellaris Berg (: ), a candidate for biological control of waterhyacinth

A.J. Sosa *, H.A. Cordo, J. Sacco

USDA, ARS, South American Biological Control Laboratory (SABCL), Bolivar 1559, (B1686EFA) Hurlingham, Argentina

Received 14 September 2006; accepted 16 April 2007 Available online 4 May 2007

Abstract

The Megamelus scutellaris (Hemiptera: Delphacidae) is a potentially valuable for biological control of waterhya- cinth (Eichhornia crassipes (Mart.) Solms.: Pontederiaceae), a serious aquatic weed in many tropical and subtropical countries. Field surveys done in South America revealed that this insect is sympatric with waterhyacinth throughout the native range of the plant, from Peru to as far south as Buenos Aires Province, Argentina. The host range of this insect was preliminarily evaluated, as was its ability to damage the plant, in order to determine whether further study was warranted. Feeding was compared, using a preference index, among leaf disks of test plants in two multiple-choice experiments: one with and one without members of the family Pontederiaceae. A similar no-choice experiment employed various species of Pontederiaceae along with rice and maize. A paired oviposition choice experiment was done by caging gravid females on E. crassipes and Pontederia cordata plants and then counting eggs deposited as well as oviposition scars. The ability of the insect to damage waterhyacinth was assessed in a greenhouse by inoculating a single waterhyacinth plant with none, 10, or 20 adult . The planthopper effect was measured by comparing plant biomass (dry weight) among treatments after 1 month. All of these studies suggested that M. scutellaris is host specific and has the ability to damage waterhyacinth. Some feeding and development occurred on other species in the family Pontederiaceae which may have been laboratory artifacts, but which mandates a cautious, further evaluation of this planthopper prior to its use as a classical biological control agent. Published by Elsevier Inc.

Keywords: Megamelus scutellaris; Eichhornia crassipes; Host specificity; Classical biological control

1. Introduction niae Warner and N. bruchi Hustache (Coleoptera: Curculi- onidae) (Julien et al., 1999; Julien, 2001). However, they The South American waterhyacinth, Eichhornia crassi- are not successful in all circumstances, so additional con- pes (Martius) Solms-Laubach (Pontederiaceae) has been trol measures are still required (Cordo, 1996; Julien, introduced throughout tropical and subtropical regions 2001). New biological control agents have been recom- of the world, and has become a serious aquatic weed. Sev- mended for further studies (Herna´ndez et al., 2004; Bickel eral methods to control this weed have been developed, but and Herna´ndez, 2004; Cordo, 1996, 1999), one of which is biological control provides the only sustainable control the planthopper Megamelus scutellaris Berg (Hemiptera: option for most of the countries invaded (Center, 1996; Delphacidae) (Sosa et al., 2004, 2005). Julien et al., 1999; Julien, 2001). Six South American bio- The host plant plays an important role in the develop- logical agents have been released to control waterhyacinth, ment of planthoppers, not only as a substrate for feeding, the most successful being two weevils: Neochetina eichhor- but also as a substrate for communication between individ- uals of different sexes, for mating, and for oviposition * Corresponding author. (Claridge, 1985; den Bieman, 1987). Host plants are known E-mail addresses: [email protected] (A.J. Sosa), hacordo@ for only seven species of Megamelus, and are all aquatic arnet.com.ar (H.A. Cordo), [email protected] (J. Sacco). plants, including three Pontederiaceae species (Au, 1941;

1049-9644/$ - see front matter Published by Elsevier Inc. doi:10.1016/j.biocontrol.2007.04.012 130 A.J. Sosa et al. / Biological Control 42 (2007) 129–138

Beamer, 1955; O’Brien and Wilson, 1985; Wilson et al., ia montevidensis Chamisso et Schlechtedahl, Echinodorus 1994; Wilson and McPherson, 1981a; Nickel and Hilde- grandifolium (Chamisso et Schlechtedahl) Micelli (Alismat- brandt, 2003). In North America, Pontederia cordata aceae), Oplismenopsis najada (Haccktel et Arechavaleta) L. is the only known host of M. palaetus (Van Duzee) Parodi, Panicum elephantipes Nees (Poaceae), Enydra (Beamer, 1955; Wilson and McPherson, 1979), whereas in anagallis Gardner (Compositae), and Ludwigia sp. South America M. electrae Muir (Cruttwell, 1973) and (Onagraceae). M. scutellaris have been associated with waterhyacinth and have both been considered as biological control candi- 2.2. Host specificity tests dates. Studies on M. electrae were halted (Cruttwell, 1973), but studies on M. scutellaris, the most abundant species on Experiments were conducted at the South American waterhyacinth, have continued in Argentina since 1999 Biological Control Laboratory. Three series of experiments (Sosa et al., 2004, 2005). were carried out: (i) feeding multiple-choice tests, (ii) Determinations of host specificity of a potential agent no-choice test, and (iii) oviposition choice test. and its ability to damage the weed are crucial issues in a weed biological control program. The level of host specific- 2.2.1. Feeding multiple-tests ity predicts the risk that the agent will impart to nontarget Two tests were carried out to estimate adult feeding plant species (Heard, 1997) whereas prerelease evaluations preference of M. scutellaris under laboratory conditions. attempt to predict whether the candidate will be effective, Test plants were selected according to their phylogenetic thereby reducing the risk of indirect effects that might relatedness (Kelch and McClay, 2004) to waterhyacinth result from the persistence of large numbers of ineffective (Eckenwalder and Barrett, 1986; Graham et al., 2002), their agents (Balciunas, 2004). economic importance and because they share the same Since waterhyacinth continues to be an uncontrolled habitat. In the first test conducted in December 2001, 15 weed in several environments and new biocontrol agents aquatic plants from different families, except Pontederia- are required, the possible use of M. scutellaris for classical ceae, were tested: E. crassipes (target weed), Canna glauca, biological control of E. crassipes is being evaluated. Results Limnobium laevigatum espongia, Sagittaria montevidensis, of field host range, host specificity, and host damage stud- Echinodorus grandifolium, Thalia geniculata L. (Marantha- ies of this insect are presented herein. ceae), Oplismenopsis najada, Panicum elephantipes, Pistia stratiotes L. (Araceae), Colocasia esculenta (L.) Schott 2. Materials and methods (Araceae), Salvinia biloba Raddi emnd. De la Sota, Com- melina sp. (Commelinaceae); Enydra anagallis and Typha 2.1. Field survey latifolia L. In the second multiple-choice test, conducted in late The field host range of Megamelus scutellaris was February 2002, species exclusively in the Pontederiaceae recorded during surveys conducted at 158 sites (Fig. 1)in were tested: E. crassipes, E. azurea, P. cordata var. cordata, Brazil (southeastern Brazil April 2000; Pantanal August P. cordata var. lancifolia, P. rotundifolia, P. subovata,and 2002), Northern Argentina (December 1997, 1998, October H. reniformis. All test plants were obtained from seedlings 1999), Peru-upper Amazon basin (April 1999, November in the laboratory or by individual plants collected in the 2001), Uruguay (January 2002), and the Delta of the Par- field about 50 km apart. So, this way we assure genetic var- ana River in Argentina (at least five times a year from iability among plants, particularly in the control treatment 1999 to 2005). (adults and nymphs) were collected with only waterhyacinth. from plants, preserved in 70% ethyl alcohol and identified The same test design was used for both multiple-choice up to species in the laboratory. tests, although they were not run simultaneously. Six Plants examined were mostly members of Pontederia- adults, from the laboratory culture, chosen at random ceae: Eichhornia crassipes, E. azurea (Swartz) Kunth, Pont- and starved for 24 h, were placed in a petri dish (147 mm ederia cordata L. (both lancifolia and cordata varieties), diameter by 24.9 mm height) with circular pieces of leaves P. rotundifolia L., P. subovata (Seubert) Low, P. parviflora (20 mm diameter) from the tested plants. The leaf discs Alexander, and Heteranthera reniformis Ruı´z et Pavo´n. were randomly arranged in a circle. Two experiments were Additionally, other aquatic plants that are known host set up: the first included all test species except waterhya- plants of Megamelus spp. and plants sharing the same cinth (WH) and the second included waterhyacinth as habitat were checked: Typha sp. (Typhaceae), Carex sp., well (WH+). Two types of control were used: the first Cyperus sp. (Cyperaceae), Canna glauca L. (Cannaceae), (CI), included only waterhyacinth leaf discs, which pro- Hydrocleis nymphoides (Wilds.) (Limnocharitaceae), vided the standard for optimal insect performance under Nymphoides indica (L.) Kuntze (Menyanthaceae), Salvinia the experiment conditions. In the second control (CII), spp. (Salviniaceae), Limnobium laevigatum espongia no plants were provided. This provided a negative standard (Humboldt et Bonpland) Heine (Hydrocharitaceae), for insect survival in the absence of food. There were 10 Alternanthera philoxeroides (Martius) Grisebach, Alternan- replicates, each one consisting on one petri dish as thera aquatica (Parodi) Chodat (Amaranthaceae), Sagittar- described above, for each treatment and control. A.J. Sosa et al. / Biological Control 42 (2007) 129–138 131

80º 60º 40º 0º 0º

Brazil 10º Perú 10º

Bolivia

20º Paraguay 20º

30º 30º Uruguay Chile

Argentina km 0 200 400 40º 40º 80º 60º 40º

Fig. 1. Native range of Megamelus scutellaris. Grey circles indicate locations where the insect was recorded in our surveys.

To estimate the preference we used an index derived calculated as the number of insects alive at the end divided from Manly’s (1974), that is based on the proportion of by the number at the beginning. consumed items. Since the feeding process of M. scutellaris e1 does not leave any evidence on the plant (Sosa et al., per- b1 ¼ s ð3Þ sonal observation), we followed the assumption that a ðÞe1 þ e2 þþek planthopper resting on a leaf is feeding on it as suggested According to this index for treatments with s close to one, by Virla and Maragliano (1993). Therefore, we replaced b is close to one when a plant i is the most selected or the proportion of consumed items by the mean proportion i closed to zero when the plant i the most avoided, and b of insects on test plants on each petri dish every 6 h for i is close to 1/k when selection for any plant i is at random. 2–4 days until mortality reached more than 90% in control For treatments with s close to zero, any b should be close II (without plants): i to zero which means that any plant i in the trial is not suit- e1 able for feeding or is rejected. Selection in Control I (only b ¼ A1 ð1Þ 1 e1 þ e2 þþ ek waterhyacinth) is expected to be random and survivorship A1 A2 Ak close to one. Thus, preference for any waterhyacinth plant should be bEc =1/k; in this case, k is the number of water- b1 is the preference observed for a plant 1, e1/A1 is the mean frequency of insects on plant 1 (e1 insects on plant hyacinth discs exposed in the arena. 1, and A1 total number of insects), e2/A2, ek/Ak are the As a secondary indication of the insect feeding, the pres- mean frequency of insects on plant 2 and k, respectively, ence and abundance of honey dew was noted as the area covered in a categorical scale as follow: 0—absent (no evi- and Pk is the number of items of plants used per trial (k = i). dences of honey dew), 1—very low (one or few drops that covered less than 10% of the disc), 2—present (represented For each observation A1 = A2 = = Ak, then (1) should be replaced as the following: by few drops that covered >10% and <50% of the leaf disc), 3—abundant (represented by honey dew that covered e1 >50% and <75% of the leaf disc), and 4—very abundant b ¼ ð2Þ 1 ðe1 þ e2 þþekÞ (honey dew overflowing the leaf disc). Preference was statistically analyzed using Friedman It is expected that the number of insects alive at the end of analysis and the nonparametric multiple comparisons sug- the experiment should differ depending on the mortality gested by Conover (1999) were used to separate medians. during the trial. To correct this, a factor s was added, These analyses are more appropriate than standard which is the survivorship at the end of the experiment ANOVA techniques because, as noted by Roa (1992) and 132 A.J. Sosa et al. / Biological Control 42 (2007) 129–138

Lockwood (1998), the consumptions within an arena may Additionally, the number of mature follicles and ovu- violate ANOVA independence assumptions. lated eggs that remained in the oviduct were counted in Finally, mortality was compared for all treatments; 10 randomly selected females from the initial field-collected ANOVA and honest significant differences (HSD) Tukey pool before the experiment and those remaining in the test were used to compare means; the data were trans- females at the end of the experiment (control and formed with angular transformation. treatment). Data were transformed using angular transfor- mation and they were analyzed using one-way ANOVA. 2.2.2. No-choice test Tukey HSD test for unequal samples was used to compare The ability of M. scutellaris to develop from egg to adult means. was evaluated in a no-choice test on: E. crassipes, E. azurea, P. cordata var cordata, P. cordata var lancifolia, 2.3. Impact study P. rotundifolia, H. reniformis, maize, and rice. Ten newly emerged nymphs were placed in a glass container with To evaluate the damage caused by M. scutellaris to leaves or pieces of leaves of each tested species and kept waterhyacinth, laboratory-cultivated plants (n = 18) i.e., in rearing chamber at 25 C and 90% RH. This was repli- ramets of similar sizes, were selected. Six of them were ran- cated 10 times. To evaluate the ability of the insect to domly separated and dried to a constant weight in an oven develop in each test plant, the proportion of nymphs that at 60 C to determine initial dry biomass. Those remaining reached the adult stage and their nymph developmental on the plant were separated in cages and randomly times were recorded and compared. ANOVA and Tukey assigned to three treatments per cage with four replicates: HSD test were used to compare means of data that had no insects (control), 10 adults (T1), and 20 adults (T2) been transformed using angular transformation. Addition- per cage with male–female ratio of 1:1. The replicate con- ally, the survivorship rate for each instar on each plant was sisted of a ramet. The damage produced by M. scutellaris recorded as the proportion of individuals of a particular was estimated by comparing the waterhyacinth dry bio- instar that reached the following instar. MANOVA and mass (green or the live aerial portion above the soil, roots Tukey HSD test were used to compare means. or the buried portion including stems and fibrous roots, dead material, and total) in the cages after 1 month. Addi- 2.2.3. Oviposition choice test tionally, the carbon–nitrogen rate was registered and com- A paired choice-test was conducted to evaluate the ovi- pared for all treatments. Results were analyzed with position preference of M. scutellaris, E. crassipes, and MANOVA followed by ANOVA and Tuckey-HSD to P. cordata. In a plastic container (40 cm length, 32 cm separate the means. Statistical analyses were performed width, 15 cm high) filled with water and placed in the gar- using Statistica 6.0 (StatSoft, 2001) software. den (mean temperature = 25 C), two pots were placed, one with E. crassipes (plant 1) and the other with P. cordata 3. Results (plant 2). One single petiole of each plant was chosen and both (petiole of plant 1 + petiole of plant 2) were confined 3.1. Field survey into a plastic cylinder (78 mm diameter, 139 mm length), closed at both ends with pieces of a fine-mesh gauze. Three The planthopper M. scutellaris was found throughout gravid brachypterous females, which had been collected at the range of the weed from 5S latitude (Iquitos, Peru) to Isla Talavera (340409800S; 584805900W), Buenos Aires 36S latitude (Buenos Aires, Argentina) (Fig. 1). Adults Province in January 2004 and starved for 24 h were placed and nymphs were abundant at every site and only recorded in the cylinder. In addition, a control treatment was on waterhyacinth. In addition, six other species of included, which consisted of randomly assigned pairs of Megamelus were found; four of which are undescribed spe- waterhyacinth plants (plants 1 and 2). Both treatment cies (unpublished data). Three species were collected on and control were replicated 10 times. After 6 days, the den- E. crassipes: M. scutellaris, M. electrae,andM. bellicus sity of eggs per plant (No. eggs/mm2) and density of ovipo- n.sp. Remes Lenicov and Sosa (Sosa et al., in press). sition scars (No. probes/mm2) were recorded. To estimate Megamelus electrae was also found on E. azurea,and the oviposition preference for a particular plant in both M. bellicus was found on several species of Pontederiaceae treatments, the following index D was developed: (E. crassipes, E. azurea, P. cordata, and P. rotundifolia). d1 d2 D1 ¼ 3.2. Host specificity tests d1 þ d2 where d1 is the egg density or oviposition-scar density 3.2.1. Feeding multiple-choice test observed in the plant 1, d2 is the egg density or oviposi- No preference was shown for any waterhyacinth plants tion-scar density observed in the plant 2. in control treatments of either tests (F = 6.958, df = 9, 63, A D close to 1 indicates a preference for plant 1, close to P = 0.00001; F = 16.536, df = 6, 54, P = 0.00001) (Figs. 2 zero indicates no preference (random choice), and close to and 3). The insect remained in the experimental arena 1 it indicates preference for plant 2. most of the time when waterhyacinth was not available. A.J. Sosa et al. / Biological Control 42 (2007) 129–138 133

0.8

0.7

0.6 a

0.5

0.4

0.3 Preference index 0.2 b 0.1 b b b b b b b b 0

Pistia Canna Typha Thalia Enhydra Salvinia Panicum Comelina Sagittaria Colocasia E.crassipes Limnobium Alternanthera Echinodorus Oplismenopsis

Fig. 2. Feeding preference of Megamelus scutellaris in the first multiple-choice test (without Pontederiaceae). Striped bars refer to the experiments that included waterhyacinth (WH+) and white bars to those without waterhyacinth (WH). Same type of bars with different letters indicate significant differences (Friedman, P < 0.05).

0.8

0.7 a

0.6

0.5

0.4

0.3 Preference Index 0.2 b b b 0.1 bbb

0

E.azurea E.crassipes P.cord.lanc. P.subovata P.rotundifolia H.reniformis P.cord.cord.

Fig. 3. Feeding preference of Megamelus scutellaris in the second multiple-choice test (with Pontederiaceae). Striped bars refer to the experiments that included waterhyacinth (WH+) and white bars to those without waterhyacinth (WH). Same type bars with different letters indicate significant differences (Friedman, P < 0.05). P.cord.lanc = Pontederia cordata var. lancifolia; P.cord.cord = P. cordata var. cordata.

Nevertheless, in the second test preference was also regis- (Fig. 5). In the control treatment without food, some tered on P. cordata, P. rotundifolia, and H. reniformis insects survived for 90 h. Furthermore, survival ranked in (F = 10.867, df = 5, 45, P = 0.00001), but with low index increasing order as follows: (1) no food (2 ± 2%), (2) plants values. No preference was shown for any clone of waterhy- without waterhyacinth (WH) (44 ± 5%), (3) plants plus acinth in control treatments of either test (F = 1.497, df = waterhyacinth (WH+) (83 ± 5%), and (4) control with only 13, 117, P = 0.130; F = 0.572, df = 9, 99, P = 0.817). Sur- waterhyacinth (97 ± 2%). vival differed in every treatment. In the first multiple-choice Honey dew exudate was abundant on waterhyacinth test (without waterhyacinth) 6 ± 4% of the insects survived, discs (in choice experiments where the plant was present). which was similar to that in the control treatment without In multiple-choice test without Pontederiaceae, honey food. On the other hand, 40 ± 29% of the insects were alive dew was recorded on waterhyacinth and only one drop after 30 h when discs of all tested plants were offered along was observed on L. laevigatum spongia in the choice treat- with one disc of waterhyacinth. Finally, survival was ments without waterhyacinth. In the second test, a few 95 ± 4 in the control treatments where only waterhyacinth drops of honey dew were present on P. cordata var lancifo- leaf discs were available (Fig. 4). Whereas in the second lia and var cordata, P. rotundifolia, P. subovata, and H. ren- multiple-choice test, the results were slightly different iformis (Table 1). 134 A.J. Sosa et al. / Biological Control 42 (2007) 129–138

1 Table 1 0.9 Honey dew recorded as categorical values on test plants in both multiple- a 0.8 choice tests 0.7 Multiple-choice I Multiple-choice II 0.6 Plants WH+ WH Plants WH+ WH 0.5 0.4 Eichhornia 4—Eichhornia 4— crassipes crassipes Survivorship 0.3 Canna maluca 00E. azurea 00 0.2 Limnobium 01Pontederia cordata 01 0.1 espongia cordata 0 Sagittaria 00P. cordata 11 22 24 26 28 30 Time (hours) montevidensis lancifolia Control 1 Control 2 WH - WH + Echinodorus 00P. subovata 01 grandiflorum Fig. 4. Mean survivorship of adults of Megamelus scutellaris in the first Thalia geniculata 00Heteranthera 01 multiple-choice test (no Pontederiaceae). Control 1 = with no food, empty reniformis rombe; Control 2 = only waterhyacinth fill in black; WH = without Oplismenospsis 00 waterhyacith, empty circle; WH+ = with waterhyacinth fill in black. najada Pistia stratiotes 00 Colocasia 00 1 esculenta 0.9 Salvinia herzogi 00 0.8 Comelina sp. 0 0 0.7 Panicum 00 0.6 elephantipes 0.5 Typha latifolia 00 0.4 a 4—very abundant; 3—abundant; 2—present; 1—very low; 0—absent. 0.3 Survivorship 0.2 0.1 the end of the experiment as compared to those from the 0 initial field collection. The number in E. crassipes–P. corda- 26 50 80 90 Time (hours) ta (1.8 ± 0.2 ‘‘eggs’’/female) treatments was similar to Control 1 Control 2 WH - WH + E. crassipes–E. crassipes (2.1 ± 0.1 ‘‘eggs’’/female) and less Fig. 5. Mean survivorship of adults of Megamelus scutellaris in the second than that observed for field-collected females before the multiple-choice test (Pontederiaceae). Control 1 = with no food, empty experiment (3.8 ± 0.2 eggs/female) (Fig. 2, ANOVA; rombe; Control 2 = only waterhyacinth fill in black; WH = without F2,23 = 14.40; P < 0.001) (Fig. 8). waterhyacith, empty circle; WH+ = with waterhyacinth fill in black.

3.2.2. No-choice test 3.3. Impact studies Megamelus scutellaris completed nymphal development on waterhyacinth, P. cordata var. lancifolia and P. rotundi- These results revealed the impact of M. scutellaris on folia. Most of the nymphs reached adult stage only on waterhyacinth (Table 4). The leaves of plants initially waterhyacinth (ANOVA; F = 19.57; df = 3.35; P < 0.001) exposed to 20 adults (T2) was reduced relative to the control (Fig. 6) where survivorship rate per instar was higher from (MANOVA, Wilks-Lambda = 0.011537, df = 26.92172, the fouth instar (MANOVA; F = 5.4608; df = 20; P < 0.0001; ANOVA, F3,13 = 83.2307, P < 0.0001). 133.6149; P < 0.001) (Table 2). Furthermore, the develop- mental time on waterhyacinth was shorter than on any of 4. Discussion the Pontederia species (ANOVA; F = 6.4708; df = 2, 18; P < 0.001) (Fig. 7). Host specificity is fundamental to the selection of an agent in weed biological control programs. Thus, it is nec- 3.2.3. Oviposition choice test essary to predict the host range while considering motiva- The density of eggs and oviposition marks were higher tion, learning, previous experiment, and success of the in waterhyacinth than pickerel weed in the trial that development of the agents (Singer, 2004; Sheppard et al., included the latter (Table 3). The preference indices were 2005). Based on our field observations and laboratory test- close to 1 indicating a strong preference for waterhyacinth ing, the delphacid M. scutellaris showed a high degree of petioles (F = 101.9; df = 1, 9; P = 0.0001) (F = 77.4; feeding and ovipositional specificity towards E. crassipes. df = 1, 9; P = 0.001), whereas in the control treatment Megamelus scutellaris chose waterhyacinth as food host the index was close to 0 which indicates the random choice in both multiple-choice experiments, even when adults were of insects for waterhyacinth petioles. highly motivated causing the test to become more conser- After the experiment, females had almost half of the pre- vative (less rejection expected). In the first test, the high liminary number of follicles maturing in the ovarioles at value of the preference index, the presence of honey dew A.J. Sosa et al. / Biological Control 42 (2007) 129–138 135

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Proportion of nymphs that reached adult stage 0.2

0.1

0 E.crassipes P.c. P. P.c. cordata E. azurea H.reniformis Maize Rice lancifolia rotundifolia

Fig. 6. No-choice test. Performance of M. scutellaris estimated through the proportion of nymphs that reach the adult stage on E. crassipes compared to other hosts. Bars (mean value ± SE) with different letters indicate significant differences (ANOVA, P < 0.05).

Table 2 Survivorship rates of nymph instars of M. scutellaris in different plants in no-choice test Instar I Instar II Instar III Instar IV Instar V Total E. crassipes 0.81 ± 0.05a 0.90 ± 0.04a 0.94 ± 0.05a 0.98 ± 0.02a 0.83 ± 0.08a 0.55 ± 0.26a E. azurea 0.20 ± 0.07 0 0 0 0 0 P. cordata var. cordata 0.46 ± 0.10a 0.14 ± 0.08c 0.10 ± 0.11c 0 0 0 P. cordata var. lancifolia 0.79 ± 0.05a 0.70 ± 0.09a 0.52 ± 0.14b 0.45 ± 0.15b 0.27 ± 0.13b 0.11 ± 0.11b P. rotundifolia 0.63 ± 0.08a 0.69 ± 0.09a 0.70 ± 0.09a 0.48 ± 0.14b 0.34 ± 0.14b 0.1 ± 0.1b H. reniformis 0.60 ± 0.10a 0.52 ± 0.11b 0.11 ± 0.06c 0 0 0 Zea mays 0.15 ± 0.06 0 0 0 0 0 Oryza sativa 0.13 ± 0.07 0 0 0 0 0 F value 2.315 15.8678 13.7619 18.5678 11.834 19.57 P value 0.931305 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 Survivorship values (means ± SE) with same letters within each column indicates no significant differences (ANOVA, P > 005). only on waterhyacinth discs, and the high survivorship rate by inserting their stylets and then reject the plant after on waterhyacinth discs and WH+ treatments supports the phloem probing (Cook and Denno, 1994). The index values hypothesis that selection was due to feeding. In the second recorded for the alternative hosts in Pontederiaceae (P.cor- experiment, although the preference for E. crassipes was data lancifolia, P. rotundifolia, and H. reniformis) could higher, P. cordata, P. rotundifolia, and H. reniformis were have been explained by this process. This aspect should secondarily selected. Also, the occurrence of honey dew be seriously considered because it is uncertain if M. scutel- and the survivorship data support this secondary prefer- laris is a vector or a potential vector of plant diseases as is ence which was independent of the availability of known for other delphacids (e.g., Delphacoes kucsheli Fen- waterhyacinth. nah transmits a virus disease to maize, which is not a suit- The preference index obtained form multiple-choice able host) (Brentasi, 2003). tests was derived from observations of the insects resting The different survivorship patterns found in both multi- on plant discs; so if the acceptance of a host plant requires ple-choice experiments could be explained by insect and/or a certain duration (from visual selection to phloem plant phenological state. The first experiment was done probing), it is probable that M. scutellaris, like other at the beginning of summer, when all individuals were monophagous delphacids, could try nonpreferred plants brachypterous (nondispersible form), and the second 136 A.J. Sosa et al. / Biological Control 42 (2007) 129–138

35

30 b b

25

a 20

15 Duration (days)

10

5

0 E.crassipes P.c.lancifolia P.rotundifolia P.c.cordata E.azurea H.reniformis Maize Rice

Fig. 7. No-choice test. Duration of the whole immature stage of M. scutellaris on E. crassipes compared to other test plants. Bars (mean value ± SE) with different letters indicate significant differences (ANOVA, P < 0.05).

Table 3 Oviposition choice test of Megamelus scutellaris using Eichhornia crassipes and Pontederia cordataa Test treatment Control treatment E. crassipes P. cordata E. crassipes E. crassipes Egg density (·103 eggs/mm2) 19.19 ± 7.75 0.46 ± 0.04 6.38 ± 2.02 7.97 ± 2.05 Oviposition marks (·103 marks/mm2) 10.58 ± 4.35 0.03 ± 0.01 3.76 ± 1.20 4.48 ± 1.04 DE 0.96 ± 0.02b 0.06 ± 0.11c DO 0.96 ± 0.03b 0.04 ± 0.12c a Means ± SEMs are shown. b Test conducted with three gravid females of M. scutellaris isolated in plastic cylinders containing two petioles of two live plants (Test: plant 1 = E. crassipes; plant 2 = P. cordata; control: plant 1 = plant 2 = E. crassipes). DE preference index for eggs, DO preference index for oviposition. c Means within each column with the same letters are not significantly different (ANOVA, P > 0.05).

4.5

4 a

3.5

3

2.5 b 2 b

1.5 No. eggs in ovarioles 1

0.5

0 Before EC-PC EC-EC

Fig. 8. Eggs maturing in the ovarioles of M. scutellaris females, before and after the oviposition choice test (treatment EC–PC [E. crassipes–P. cordata] and in treatment EC–EC [E. crasssipes–E. crassipes]). Bars (mean value ± SE) with different letters indicate significant differences (ANOVA, P < 0.05). experiment was carried out at the end of summer, whereas tions. However, there are no records of differences in the a 3:1 brachypterous/macropterous proportion. The dis- mortality of winged forms in delphacids (Denno and Rod- persal form is able to resist long periods of adverse condi- erick, 1990; Matsumura, 1996, 1997). A.J. Sosa et al. / Biological Control 42 (2007) 129–138 137

Table 4 Impact of M. scutellaris on E. crassipes (mean ± SE) Treatment Leaves Roots Dead material Total Initial Biomass (g) 2.2 ± 0.7a, n = 6 1.4 ± 0.5a, n = 6 0.13 ± 0.01a, n = 6 3.7 ± 1.2a, n =6 Control (no insects) Biomass (g) 17.3 ± 0.8c, n = 4 18.5 ± 3.1b, n = 4 11.8 ± 1.4b, n = 4 47.5 ± 3.7b, n =4 T1 Biomass (g) 14.3 ± 0.7bc 13.5 ± 1.1b 10.8 ± 0.6b 39.0 ± 2.0b Beginning: 10 adults n =4 n =4 n =4 n =4 End: 15.8 ± 4.1 nymphs; 2.6 ± 1.1 adults T2 Biomass (g) 13.9 ± 1.1b 15.6 ± 1.2b 12.7 ± 0.6b 42.2 ± 2.7b Beginning: 20 adults n =3 n =3 n =3 n =3 End: 16.7 ± 3.6 nymphs; 1.8 ± 1.2 adults Means followed by same letters within each column are not significantly different, MANOVA/ANOVA, P > 0.05.

The no-choice test implicated two additional potential tory planthoppers mate after dispersal and colonization of hosts of M. scutellaris: P. cordata var. lancifolia and new habitat. Monophagy may therefore be essential for P. rotundifolia. However, during field explorations, in areas mate location particularly in the absence of pheromone where these two species coexisted with waterhyacinth, communication (Cook and Denno, 1994). M. scutellaris was only found on E. crassipes. Therefore, Prerelease efficacy assessments are estimated through the limited acceptance of P. cordata var. lancifolia and impact studies on the target weed at plant individual level. P. rotundifolia might be an artifact of the testing method. In this sense, the planthopper M. scutellaris reduced leaf Similar results have been recorded from other species as biomass (photosynthetic portion) by about 20% relative potential agents and in one case in delphacids. The poten- to the control in 1 month. However, it is remarkable that tial host range of several Ribautodelphax species (Delphaci- there was no difference between insect densities in the dae) is wider than their actual host ranges (monophagous two treatments. Perhaps, if the experiment had lasted in most cases) (den Bieman, 1987). However, experiments longer, the effects might have been clearer. The results of that use cut foliage, principally with leaf discs (Jones and the host specificity test (feeding and ovipositing), impact Coleman, 1988) are extremely conservative, particularly studies, and field data suggest that M. scutellaris is a for sap-feeding insects (Palmer, 1999), so it is advisable monophagous and damaging insect on waterhyacinth and to use whole plants in choice tests in future studies. hence a potential candidate for biological control. The motivation for ovipositing was not affected in both treatments, or it was affected in the same way. Females pre- Acknowledgments ferred to oviposit on waterhyacinth rather than P. cordata petioles in spite of being in close proximity to each other. We thank A. Marino de Remes Lenicov (Facultad de In control treatment the density of eggs was similar for Ciencias Naturales y Museo, La Plata, Argentina) for help- each waterhyacinth petiole and was almost the double for ing us in identifications and for invaluable comments and the only one waterhyacinth petiole in the test treatment suggestions, M.C. Herna´ndez (SABCL) for comments (E. crassipes–P. cordata). and suggestions, J. Dorado, E. Martinez Mullo, M. Szu- Since waterhyacinth was introduced to the USA about druk, M. V. Cardo, and M. Telesnicki (SABCL) for field 125 years ago, there are no records of the North American and laboratory assistance. We also thank to the editor Megamelus species (M. palaetus) from P. cordata expand- and the anonymous reviewers for their invaluable com- ing its host range to use E. crassipes. This provides a good ments and suggestions that improved the original example of absence of natural host range extension to a manuscript. closely related species. One species of this genus, M. davisi Van Duzee is considered a pest in Hawaii on water lily References Nuphar advena (Aiton), an introduced ornamental plant (Au, 1941). However, this is not an example of host-shift- Asche, M.A., 1997. A review of the systematics of Hawaiian planthoppers ing as this planthopper was introduced from the mainland (Hemiptera: Fulgoroidea). Pac. Sci. 51, 366–376. USA with its original host plant (Pemberton, 1947; Zim- Au, S.H., 1941. Megamelus davisi infesting water lily in Hawaii. J. Econ. merman, 1948; Beardsley, 1990; Asche, 1997). Entomol. 34, 415. Balciunas, J.K., 2004. Are mono-specific agents necessarily safe? The need For species like M. scutellaris that mate on its host for pre-release assessment of probable impact of candidates of plant, monophagy may facilitate mate-finding by increas- biocontrol agents, with some examples. In: Cullen, J.M., Briese, ing encounter rates among conspecifics. Not only do plant- D.T., Kriticos, D.J., Londsale, W.M., Morin, L., Scott, J.K. (Eds.), hoppers mate on their hosts, but the mate location is Proceedings of the XI International Symposium on Biological Control dependent on acoustic signals transmitted through the of Weeds. CSIRO Entomology, Canberra, Australia, pp. 252–257. Beamer, R.H., 1955. A revision of the genus Megamelus in America North plant substrate (Claridge, 1985), which could probably of Mexico. J. Kans. Entomol. Soc. 28, 29–46. function in M. scutellaris as in other delphacids. Further- Beardsley, J.W., 1990. Notes on immigrant Delphacid planthoppers in more, Denno and Roderick (1990) pointed out that migra- Hawaii (Homoptera: Fulgoroidea). Proc. Entomol. Soc. 30, 121–129. 138 A.J. Sosa et al. / Biological Control 42 (2007) 129–138

Bickel, D.J., Herna´ndez, M.C., 2004. Neotropical Thrypticus (Diptera: International Symposium on Biological Control of Weeds. CSIRO Dolichopodidae) reared from water hyacinth, Eichhornia crassipes, and Enthomology, Canberra, Australia, pp. 287–296. other Pontederiaceae. Ann. Entomol. Soc. Am. 97, 437–449. Lockwood III, J.R., 1998. On the statistical analysis of multiple-choice Brentasi, M.E., 2003. Estudio de la interaccio´n planta-insecto. Compor- feeding preference experiments. Oecologia 116, 475–481. tamiento alimentario del vector del ‘‘Mal de Rı´o Cuarto del maı´z’’, Manly, B.F.J., 1974. A model for certain type of selection experiments. Delphacodes kuscheli Fennah (Insecta—Hemiptera—Fulgoromor- Biometrics 30, 281–294. pha—Delphacidae). Tesis Doctoral. Facultad de Ciencias Naturales Matsumura, M., 1996. Genetic analysis of a threshold trait: density- y Museo, Universidad de La Plata. dependent wing dimorphism in Sogatella furcifera (Horva´th) (Hemip- Center, T., 1996. Biological control of waterhyacinth in the United States. tera: Delphacidae), the whitebacked planthopper. Heredity 76, 229– In: Charudattan, R., Labrada R., Center, T.D., Kelly-Begazo, C. 237. (Eds.), Strategies for Water Hyacinth Control. Report of a Panel of Matsumura, M., 1997. Correlated responses of life history traits, wing Experts Meetings. Fort Lauderdale, FL, pp. 165–173. length, and flight propensity to wing-form selection in the whitebacked Claridge, M.F., 1985. Acoustic signal in the Homoptera: behaviour, planthopper, Sogatella furcifera (Horva´th) (Hemiptera: Delphacidae). and evolution. Annu. Rev. Entomol. 30, 297–317. Appl. Entomol. Zool. 32, 437–445. Conover, W.J., 1999. Practical Nonparametric Statistics. Wiley, New Nickel, H., Hildebrandt, J., 2003. Auchenorrhyncha communities as York. indicators of disturbance in grasslands (Insecta, Hemiptera)—a case Cook, A.G., Denno, R.F., 1994. Planthopper/plant interactions: feeding study from the Elbe flood plains (northern Germany). Agric. Ecosys. behavior, plant nutrition, plant defense, and host plant specialization. Environ. 98, 183–199. In: Denno, F.D., Perfect, T.J. (Eds.), Planthoppers. Their Ecology and O’Brien, L.B., Wilson, S.W., 1985. Planthoppers systematics and external Management. Chapman and Hall, New York, pp. 114–139. morphology. In: Nault, L.R., Rodrı´guez, J.G. (Eds.), The Leafhoppers Cordo, H.A., 1996. Recommendations for finding and prioritizing new and Planthoppers. Wiley, New York, pp. 61–102. agents for biocontrol of water hyacinth. In: Charudattan, R., Labrada Palmer, W.A., 1999. The use of cut foliage instead of whole plants for host R., Center, T.D., Kelly-Begazo, C. (Eds.), Strategies for Water specificity testing of weed biocontrol insects—is this acceptable Hyacinth Control. Report of a Panel of Experts Meetings, Fort practice? In: Whithers, T.M., Barton Brown, L., Stanley J. (Eds.), Lauderdale, FL, pp. 181–185. Host Specificity Testing in Australia: Towards Improved Assays for Cordo, H.A. 1999. New agents for biological control of water hyacinth. Biological Control. CRC for Tropical Pest Management, Brisbane, In: Hill, M., Julien, M., Center, T. (Eds.), Proceeding of the First Australia, pp. 20–29. IOBC Global Working Group Meeting for the Biological Control of Pemberton, C.E., 1947. Some insect pests of the mainland of the United Water Hyacinth, Harare, Zimbabwe, pp. 68–74. States occurring also in Hawaii. Hawaiian Planthopper Rec. 51, 85–87. Cruttwell, R.E., 1973. Preliminary Investigations on Some Insects Causing Roa, R., 1992. Design and analysis of multiple-choice feeding-preference Minor Damage to Water Hyacinth, Eichhornia crassipes. Report West experiments. Oecologia 89, 509–515. Indian Station, CIBC, Trinidad. Sheppard, A.W., van Klinken, R.D., Heard, T.A., 2005. Scientific den Bieman, C.F.M., 1987. Host plant relations in the planthopper genus advances in the analysis of direct risks of weed biological control Ribautodelphax (Homoptera, Delphacidae). Ecol. Entomol. 12, 163– agents to nontarget plants. Biol. Control 35, 215–226. 172. Singer, M.C., 2004. Oviposition preference: its definition, measurements Denno, R.F., Roderick, G.K., 1990. Population biology of planthoppers. and correlates, and its use in assessing risk of host shifts. In: Cullen, Annu. Rev. Entomol. 35, 489–520. J.M., Briese, D.T., Kriticos, D.J., Londsale, W.M., Morin, L., Scott, Eckenwalder, J.E., Barrett, S.C.H., 1986. Phylogenetic systematics of J.K. (Eds.), Proceedings of the XI International Symposium on Pontederiaceae. Syst. Bot. 11, 373–391. Biological Control of Weeds. CSIRO Entomology, Canberra, Austra- Graham, S.W., Olmstead, R.G., Barret, S.C.H., 2002. Rooting phyloge- lia, pp. 235–244. netic trees with distant outgroups. A case study from Commelinoid Sosa, A.J., Marino de Remes Lenicov, A.M., Mariani, R., Cordo, H.A., Monocots. Mol. Biol. Evol. 19, 1769–1781. 2004. Redescription of Megamelus scutellaris Berg (Hemiptera: Delp- Heard, T., 1997. Host range testing of insects, pp. 77–82. In: Julien M., hacidae), a candidate for biological control of water hyacinth. Ann. White (Eds.), Biological Control of Weeds: Theory and Practical Entomol. Soc. Am. 97, 271–275. Application. ACIAR Monograph No. 49, 192 pp. Sosa, A.J., Marino de Remes Lenicov, A.M., Mariani, R., Cordo, H.A., Herna´ndez, M.C, Cordo, H.A., Hill, M., 2004. Studies in Argentina on 2005. Life history of Megamelus scutellaris with description of two species of Thrypticus (Diptera) as agents for the biological control immature Stages (Hemiptera: Delphacidae). Ann. Entomol. Soc. of water hyacinth. In: Cullen, J.M., Briese, D.T., Kriticos, D.J., Am. 98, 66–72. Lonsdale, W.M., Morin, L., Scott, J.K. (Eds.), Proceedings of the XI Sosa, A.J., Marino de Remes Lenicov, A.M., Mariani, R., in press. Species International Symposium on Biological Control of Weeds. CSIRO of Megamelus Fieber (Hemiptera: Delphacidae) associated with Enthomology, Canberra, Australia, pp. 117–120. Pontederiaceae in South America. Ann. Entomol. Soc. Am. Jones, C.G., Coleman, S., 1988. Leaf disc size and insect feeding StatSoft, 2001. STATISTICA for Windows. StatSoft, Tulsa, Oklahoma. preference: implications for assays and studies on induction of plant Virla, E.G., Maragliano, R.E., 1993. Preferencias alimentarias y sitios de defense. Entomol. Exp. Appl. 47, 167–172. oviposicio´ndeDelphacodes haywardi (Muir) en diferentes hue´spedes, Julien, M.H., Griffiths, M.W., Wright, A.D., 1999. Biological control of en condiciones de laboratorio (Homoptera: Delphacidae). Rev. Soc. water hyacinth. The weevils Neochetina bruchi and Neochetina Entomol. Arg. 52, 101–106. eichhorniae: biologies, host ranges, and rearing, releasing and mono- Wilson, S.W., McPherson, J.E., 1979. The first record of Megamelus toring techniques for biological control of Eichhornia crassipes. palaetus in Ilinois (Homoptera: Fulgoroidea: Delphacidae). Great ACIAR Monograph No. 60, 87 p. Lakes Entomol. 12, 27. Julien, M.H., 2001. Biological control of water hyacinth with : Wilson, S.W., McPherson, J.E., 1981a. Ontogeny of the tibial spur in a review to 2000. Biological and integrated control of water hyacinth Megamelus davisi (Homoptera: Delphacidae) and its bearing on Eichhornia crassipes. In: Julien, M.H., Hill, M.P., Center, T.D., delphacid classification. Great Lakes Entomol. 14 (1), 49–50. Jianqing, D. (Eds.), Proceedings of the Second Meeting of the Global Wilson, S.W., Mitter, C., Denno, R.F., Wilson, M.R., 1994. Evolutionary Working Group for the Biological and Integrated Control of Water patterns of host plant used by delphacid planthopper and their Hyacinth, pp. 8–19. relatives. In: Denno, R.F., Perfect, T.J. (Eds.), Planthoppers-Their Kelch, D.G., McClay, A., 2004. Putting the phylogeny into the centrifugal Ecology and Management. Chapman & Hall, New York, pp. 7–113. phylogenetic method. In: Cullen, J.M., Briese, D.T., Kriticos, D.J., Zimmerman, E.C., 1948. Homoptera: Auchenorrhyncha. Insects Hawaii Lonsdale, W.M., Morin, L., Scott, J.K. (Eds.), Proceedings of the XI 4, 248.