Host range of Lepidelphax pistiae (Hemiptera: Delphacidae) and its potential impact on Pistia stratiotes L. (Araceae)

ABSTRACT Pistia stratiotes L. (Araceae) is a floating aquatic plant that has become invasive in Florida. It is primarily controlled with herbicides, but two biocontrol agents have previously been released to assist in management of this species. A new potential biocontrol agent from Argentina, Lepidelphax pistiae Remes Lenicov (Hemiptera: Delphacidae), has been evaluated comprehensively for specificity after initial host range studies done in its native range indicated that it is likely specific to P. stratiotes. Host range studies indicated that this insect is specific to P. stratiotes, with no survival or reproduction occurring on any of the 42 other plant species tested. Impact studies indicated that this insect can significantly damage P. stratiotes at medium and high population densities, which were comparable to those seen in its native range. Lepidelphax pistiae is sufficiently specific enough to warrant release and has a high probability of aiding management of P. stratiotes populations in Florida. GRAPHICAL ABSTRACT

Herbicide application is the preferred management strategy for keeping floating aquatic vegetation (mainly P. stratiotes and Pontederia [Eichhornia] crassipes [Martius] Solms. [Pontederiaceae]) at maintenance levels in Florida. Because this is a management strategy, herbicide application must be done regularly and in perpetuity to maintain population control, costing millions of dollars annually (Netherland, Getsinger, & Stubbs, 2005). In order to improve the suppression of this plant, a new potential biocontrol agent, Lepidelphax pistiae de Remes Lenicov (Hemiptera: Delphacidae) was collected from Argentina for evaluation.
Lepidelphax pistiae was described from the Paraná/Uruguay basin in Argentina and is the only species within the Lepidelphax genus (de Remes Lenicov & Walsh, 2013). It has been observed feeding and reproducing only on P. stratiotes both in the field and the laboratory (Cabrera Walsh, Maestro, Sosa, & Tipping, 2014;de Remes Lenicov & Walsh, 2013;de Remes Lenicov, Defea, Rusconi, & Cabrera Walsh, 2017). Lepidelphax pistiae by itself can significantly impact growth of P. stratiotes, reducing final biomass by approximately one third and resulting in ∼20% fewer rosettes compared to controls (Cabrera . Other studies on this species show that L. pistiae, together with Neohydronomous affinis, reduce P. stratiotes biomass and coverage in field settings (Cabrera Walsh & Maestro, 2016).
Previously, host range testing of L. pistiae was conducted at Fundación para el Estudio de Especies Invasivas (FUEDEI) in Hurlingham, Buenos Aires, Argentina and at the USDA-ARS Invasive Plant Research Laboratory (IPRL) in Ft. Lauderdale, FL, USA. This testing involved no-choice specificity tests on 25 species of Araceae and four additional wetland plant species with 100% adult mortality and no progeny produced on any plant other than P. stratiotes (Cabrera . Herein, L. pistiae was tested on an additional 37 plant species including common landscaping plants and economically important crops, along with additional members of the Araceae family, both native and exotic to Florida. The effects of L. pistiae were also tested to determine the damage this species could inflict directly on P. stratiotes.

Methods and materials
Host range and oviposition testing No-choice specificity tests were conducted on non-target plants (Table 1) in a quarantine greenhouse at IPRL under natural light conditions, 25-27°C, and 70-85% RH. Test plants were selected based on their relatedness to P. stratiotes, use of similar habitats, and economic importance. Testing was done using whole plants because L. pistiae feeds on the plant's fluids.
Damage from this insect is seen primarily as 'hopper burn' (chlorosis) on leaves and mean growth rate reduction (de Remes Lenicov & Walsh, 2013). Feeding from an individual insect is difficult to detect, so P 1 survival and F 1 nymph emergence were used as a sign of host plant suitability. Caging appropriate for each plant type and size was used and included sleeved cages and 40 L aquaria or 5 L plastic jars with screened lids. A randomised, complete block design with five replications of each test plant species was used throughout and each trial contained three to five plant species at a time, including a P. stratiotes control. Each test plant was enclosed separately and five (3 ♀ / 2 ♂) 1-2 week old L. pistiae adults were placed on each plant for seven days; they were then removed and the P 1 mortality was recorded. Test plants and P. stratiotes control plants were then monitored for appearance of F 1 adults. Monitoring was discontinued after F 1 adults were found on P. stratiotes control plants.
Insects were also tested for longevity without access to P. stratiotes or water to simulate what would occur if all of their host plant died off or they were accidentally transported away from their host plant. A randomised design with ten replicates of each treatment was used and the experiment was repeated three times with equal numbers of male and female L. pistiae. Each replicate consisted of a single L. pistiae placed in a 500 mL plastic container closed with a friction fitting, screened lid that contained either one P. stratiotes rosette in fertilised water (water soluble fertiliser [Peters Professional 24-8-16 Fertilizer, Everris, Geldermalsen, Netherlands] at a rate of 1 g / 5 L deionised water), one filter paper sheet moistened with DI water (water only treatment), or nothing (control). The filter paper was remoistened as necessary during the experiment and treatments were monitored twice daily for insect mortality until 100% mortality occurred in the water and control treatments.

Impact testing
Two experiments were conducted to evaluate the impact of L. pistiae on P. stratiotes: 1) a two-treatment randomised block design with five blocks and, 2) a four-treatment randomised block design with six blocks. For the two-treatment experiment (repeated four times between February 2017 and March 2018), a low density insect treatment (2 ♀ / 1 ♂ L. pistiae) was compared to a no-insect control treatment. For the four-treatment experiment (conducted May through June 2018), three insect densities of L. pistiae (low -2 ♀ / 1 ♂, medium -10 ♀ / 5 ♂, and high -20 ♀ / 10 ♂) were compared to a no-insect control treatment. Densities were chosen based on previous experiments done in Argentina, where 30 L. pistiae were able to negatively impact caged areas of P. stratiotes (Cabrera . Cabrera  also noted that L. pistiae is highly mobile in the field, so the medium and low density treatments in this current experiment were meant to mimic other observed field densities.
For each sample, one P. stratiotes rosette was placed in an 11 L plastic tub with a screen lid. Initial fresh weight biomass of each P. stratiotes was recorded, as well as the number of leaves within each tub.
Plants were fertilised with the same water-soluble fertiliser and rate used as before, and Aquashade (Arch Chemicals, Inc., Germantown, Wisconsin) was added at the label rate to reduce algal growth. For the insect treatments, 1-2 week old brachypterous adult L. pistiae were placed in each insect treatment tub for 1 week and then removed and mortality recorded. Any F 1 nymphs that emerged were permitted to develop into adults, which were counted and removed over the course of 1-2 weeks. Final fresh weight biomass of each P. stratiotes was recorded, as well as the final number of rosettes and leaves within each tub.

Statistical analysis
Analyses were performed using R (version 3.3.2; R Core Team, 2016). For the two-treatment impact experiments, treatments and blocks were compared using ANOVAs to determine differences in initial fresh weight and number of leaves and two sample t-tests were used to compare changes in fresh weight, number of leaves, and number of rosettes. For the four-treatment impact experiment, treatments and blocks were compared using ANOVA. Data were then evaluated post-hoc using Tukey's tests to determine differences among treatments. A significance level of α < 0.05 was used for all statistical tests.

Results and discussion
Host range and oviposition Adult L. pistiae did not survive on, nor did any nymphs emerge from, any plants except for P. stratiotes, where up to 204 nymphs emerged from each plant (Mean (± SE) number of nymphs = 84.8 ± 9.26; average P 1 survival = 0.63 ± 0.047) ( Table 1). Mean survival of adults was 1.38 ± 0.10 days in the water only treatment and 0.63 ± 0.04 days in the control treatment. Average survival of L. pistiae under the same ambient conditions in the P. stratiotes treatment was 70 ± 8.5% over three days, similar to what was seen in the host range no-choice tests. The no-choice experiments show that in the event that this insect is separated from its host plant, the adults are not able to survive more than a few days without feeding. The specificity of this insect and its inability to survive off of its host plant for any significant period indicate that it would be a safe biological control agent for P. stratiotes.

Impact
There was no significant difference in initial fresh weight or initial number of leaves between treatments or blocks in the two-treatment impact experiment. At low density (2 ♀ / 1 ♂) L. pistiae did not reduce P. stratiotes growth or vegetative reproduction. There was a change in fresh weight (df = 3, F = 26.65, p < 0.001) and a difference in total F 1 L. pistiae (df = 3, F = 7.483, p = 0.00167) among treatments in the four-treatment experiment. Further, Tukey's tests indicated that the control and low density treatments had greater increases in fresh weight compared to the medium and high density treatments (p < 0.001, Figure 1). The impact experiments found lower insect densities did Figure 1. Effect of L. pistiae density on fresh weight (g) of P. stratiotes. Mean (± SE) change in the biomass of P. stratiotes when exposed to a range of densities of L. pistiae. *Letters indicate significant differences determined by Tukey's test at p < 0.05. not affect the growth rate of P. stratiotes while moderate to higher densities did. The high density treatment resulted in the complete destruction of the experimental plant population, thereby causing the insects to starve and the final total F 1 count to be zero for most of the samples. This explains why the medium density treatment had significantly more total F 1 L. pistiae than either of the other insect treatments or the control (p < 0.05, Figure 2).
In a previous lab study, L. pistiae reached similarly high densities to those in this study . In field studies in the native range of L. pistiae, caged plots containing P. stratiotes that were exposed to 30 L. pistiae adults (comparable to the high density impact treatment in this study) produced lower biomass and plants with smaller diameters than those in control enclosures . Lepidelphax pistiae also produced high population densities and dispersed across a 7,000 m 2 lake within 40 days of release (Cabrera Walsh & Maestro, 2016). In this study, L. pistiae exposures at medium density resulted in a ∼36% increase in biomass, compared to a > 200% increase on average in the control treatment. These results suggest that, if released, Figure 2. Effect of L. pistiae density on the number of F 1 L. pistiae adults. Mean (± SE) number of F 1 adults produced under different initial densities of L. pistiae. The high density treatment only had one replication with live plant material at the end of the experiment. *Letters indicate significant differences determined by Tukey's test at p < 0.05. this insect may be able to reach useful densities in the field and cause significant damage to P. stratiotes.
Lepidelphax pistiae has been shown to be highly specific to P. stratiotes and can be damaging at medium and higher densities, indicating that it would be a suitable biological control agent for this species. However, the question of whether P. stratiotes is native to Florida must be considered and requires further research (Evans, 2013).

Disclosure statement
No potential conflict of interest was reported by the authors.