Importation of L. oregmae

Adults of L. oregmae were shipped from Guam into the high security quarantine facility at the Division of Plant Industry Department of Agriculture and Consumer Services, in Gainesville, Florida under USDA-APHIS Permit 954945 on Aug 19, 1999. On Dec 6, 1999 a subculture was transferred to the Department of Entomology and Nematology quarantine facility at the University of Florida, Gainesville with a permit.

Mass Rearing of L. oregmae

Lipolexis oregmae was cultured in a synchronous tritrophic rearing system (Hill & Hoy 2003; Walker & Hoy 2003). Potted citrus plants were infested with mixed stages of brown citrus aphids and then exposed to parasitoids within plastic cylinders in the Division of Plant Industry quarantine facility or in mesh cages in the quarantine laboratory of the Department of Entomology and Nematology, University of Florida, Gainesville. Parasitoids emerging from the rearing cages were stored in plastic vials (6.5 × 2.5 cm) containing a fluted paper (2 × 5 cm) for refuge and a moistened honey-saturated absorbent paper strip as a nutrient source. Parasitoids were stored in groups of up to 50 individuals per vial and were either used to initiate cultures or shipped overnight to collaborators in styrofoam boxes cooled with frozen cold packs or taken directly to citrus groves within 1-3 d of emergence for release.

Release Protocol for L. oregmae

All releases were made in citrus groves with abundant flush and had at least 3 to 5 trees well infested with brown citrus aphids (>3,000 aphids each). The location of each release site was recorded with a handheld Geographic Positioning Systems receiver (Magellan GPS 310, Orbital Products, San Dimas, CA.) as well as on a Florida state map (Florida Atlas and Gazetteer 1997, Delorme, Maine). During releases, the cap on the vial containing parasitoids was unscrewed and the vial held upright under the brown citrus aphid colonies. Most release sites received single releases of 100 to 200 newly emerged L. oregmae adults, although some sites received multiple releases. Females were often observed stinging brown citrus aphids moments after their release. No releases were carried out in heavy rain or at locations where irrigation sprays would have washed the aphids off the foliage, or in groves where pesticides were recently applied.

Effect of Storing on Fecundity of Released L. oregmae Females

To confirm that we could store females for 1-3 d at 15°C without a loss in vigor when it was difficult to release parasitoids on the day of emergence, 10 females of L. oregmae were individually placed on a potted citrus plants infested with 250-300 aphids of mixed stages on the day of parasitoid emergence and left for 24 h. The plant was covered with a ventilated plexiglass cylinder and a moist cotton pad and honey-saturated paper strip within for water and nutrients, respectively. Female parasitoids were allowed to mate individually with 1-d-old males in gelatin capsules (size 00) and then exposed to 5 aphids for 5 min prior to introducing them to the aphid-infested plants. Any female that did not mate or did not oviposit into aphids was not used.

After female parasitoids were removed, the plants were held in the laboratory at 22-24°C, 55-65% RH and 16L:8D cycle until emergence of adult parasitoids 13-14 d later. Any replicate in which the female could not be located after 24 h was rejected. The experiment was repeated with 10 females that were stored in a growth chamber at 15°C under a 16L:8D cycle for 3 d and 10 females stored for 6 d. All emerging parasitoids from the 10 replicates of the 3 treatments were counted and One-way Analysis of Variance and Least Significant Difference (Statview ver 5.0) was used to compare the mean fecundity for each treatment.

Survey Sites and Sampling Protocol for 2001 and 2002

Survey sites during 2001 and 2002 were all former release sites or within a 4.8-km (3-mile) radius of former release sites. All flushes on all trees that could be examined within 45 min were inspected, and all flushes infested with brown citrus aphids were collected. Citrus flushes were placed between loosely folded paper towels in a ‘Ziploc’ plastic bag (Fisher Scientific) (22 × 14 cm) which was moderately inflated and stored in an insulated cooler (65 × 54 × 24 cm). The temperature (10-17°C) within the cooler was maintained by a single layer of ‘blue ice’ packets and separated from direct contact with the sample bags by 4 layers of heavy plastic sheeting. Once in the laboratory, bags were wiped inside to remove condensation and were fully inflated with a laboratory air pump, resealed, and stored on bench tops under a 16L:8D photoperiod.

The samples were checked daily at 900, 1200, and 1600 h for adult parasitoid emergence; paper towels within each bag were replaced if wet. Because most mummified brown citrus aphids containing L. oregmae form in or on the soil (Hill & Hoy 2003), freshly collected flush samples rarely contained mummies. Adult wasps were identified with the guidelines of Evans & Stange (1997). Samples were held for 8 to 10 d, by which time any brown citrus aphids containing eggs or larvae of either parasitoid species would become mummified.

Mortality Associated with Sampling Protocol

To determine whether the sampling protocol caused significant mortality of aphids or immature parasitoids, a laboratory assessment was conducted. Each of 10 third-instar brown citrus aphids was exposed to either L. oregmae or L. testaceipes in a petri dish arena according to methods described by Persad & Hoy (2003), and the parasitized aphids were returned to tender new leaves on potted citrus. To confirm parasitism, 3 d later a third of the exposed aphids were removed and dissected in 0.7% saline under a microscope to record the number of parasitoid eggs or larvae. One-third of the aphids were removed along with leaves and stored in bags as described above, and the remaining third were reared on the flushed citrus plants. If there was less than the expected 97 to 100% parasitism rate in any group (Persad & Hoy 2003), that trial was rejected. Because up to 75% mortality occurred before adult parasitoids emerged in both the plastic bags and the rearing treatments, a molecular test was developed (Persad et al. 2004) and used during 2003 to monitor field-collected foliage containing aphids for the presence of L. oregmae.

High-fidelity PCR Protocol for Monitoring L. oregmae and L. testaceipes in 2003

In 2003, field samples were split into 2 treatments; 1 consisted of pooled (potentially parasitized) aphids (70% of the sample obtained from a particular location), and the other consisted of individual parasitoid larvae dissected from the remaining 30% of the aphids. The assay conducted on pooled aphids (36 aphids or fewer) used the High-fidelity PCR protocol developed by Persad et al. (2004). For the pooled aphids, DNA was extracted from groups of up to 36 aphids (and potential parasitoids) in 0.5-ml thick-walled eppendorf tubes, each containing 50 μl of 5% Chelex (Bio-Rad, Hercules, CA) resin. The extraction took 1 h at 60°C and 5 min at 94°C (Edwards & Hoy 1993). To grind the aphids and parasitoids in Chelex, a pestle was made by slowly heating a standard pipette tip which was then inserted into an empty 0.5-mL eppendorf tube so that the tip assumed the shape of the base of the tube to form a close-fitting pestle, which was used once only. The remaining aphids (30%) were dissected in saline under a microscope and any parasitoid larva found was individually identified by High-fidelity PCR protocol because larvae of L. oregmae and L. testaceipes cannot be discriminated after the first instar.

One microliter of each Chelex preparation was used for High-fidelity PCR, which was performed in a 50-μL reaction volume containing 50 mM Tris, pH 9.2, 16 mM ammonium sulfate, 1.75 mM MgCl2, 350-μM each of dATP, dGTP, dCTP, dTTP, 800 pmol of primers, 1 unit Tgo DNA polymerase, and 5 units of Taq DNA polymerase (Roche Molecular Biochemicals). The PCR primers used code for the nuclear rRNA ITS-2 region and include an L. oregmae-specific forward primer (LO-ITSF 5’-GGCCAGTTGTCGAGTCC-3’) based on the complete ITS-2 sequence. DNA from any larva not identified as L. oregmae was retested with a species-specific primer to determine the presence of L. testaceipes, the only other primary parasitoid recovered during the surveys in 2001 and 2002. The L. testaceipes-specific forward primer (LT-ITSF 5’-CTAGCGATAAATGAATGTTC-3’) was designed after obtaining a partial ITS-2 sequence from L. testaceipes (Persad et al. 2004). PCR products were separated on a 2% agarose gel, stained with ethidium bromide, and photographed under UV light. The L. testaceipes-specific primers produced 520-bp bands, while those of L. oregmae produced 270-bp bands.

Importation and Quarantine Evaluation

The application to release L. oregmae was reviewed by the Institute of Food and Agricultural Sciences of the University of Florida and the Division of Plant Industry of the Florida Department of Agriculture and Consumer Services. The identity of L. oregmae was confirmed by Peter Stary, Institute of Entomology, Academy of Sciences, Czech Republic. Specimens of L. oregmae were deposited at the Florida State Collection of Arthropods, Division of Plant Industry (DPI), Gainesville, as voucher specimens FBCL-2002-2. The parasitoid was released under DPI-Florida Department of Agriculture and Consumer Services permit number 208-00-026, dated 18 February 2000. A copy of the application to release is available at  http://ipm.ifas.ufl.edu/reports/L._scutellaris_5.31.00.pdf, where a description is given of the potential hosts of L. oregmae in Florida and its potential nontarget effects. The potential benefits of establishing L. oregmae in Florida’s citrus were considered to be greater than potential risks to native aphid species because there are no known threatened or endangered aphids in Florida (S. Halbert, personal communication).

Releases of L. oregmae

After approval of the application to release L. oregmae was obtained on 21 Jun 2000, releases were made in 15 counties throughout the approximately 348,031 ha of citrus in Florida (Table 1). Releases of approximately 20,200, 12,100, and 1,260 adults of L. oregmae were made in citrus groves in Florida during 2000, 2001, and 2002, respectively.

The 24-h test of fecundity of L. oregmae females conducted to confirm that the holding and shipping conditions were suitable was not different for females held for 1 or 3 d at 15°C. A mean (±SE) of 28.6 (±2.1) adult parasitoid progeny were produced if their mothers were held for 1 d, and a mean of 39.4 (2.9) progeny were produced if females were first held for 3 d at 15°C. However, significantly fewer progeny (22.6, SE = 2.1) were produced if the mothers were held for 6 d at 15°C (One-way ANOVA, F = 2.81, df = 29, P = 0.02). Because most parasitoids were shipped on the day of emergence and were released by collaborators the next day or releases were made by the authors within 3 d of adult parasitoid emergence, females should have been sufficiently fit that they could locate hosts and begin oviposition after release. Observations made by the authors of females immediately after release in the field indicated that females could find and attack hosts readily.

Recovery of L. oregmae from Citrus Groves in Florida in 2001 and 2002

Recoveries of L. oregmae at or near release sites occurred in 10 citrus groves in 4 counties during 2001 and in 4 groves in 3 counties in 2002 (Table 1). Between Mar and Oct, there are periods (typically 3 or 4) when citrus trees are putting out large amounts of new growth (flush) suitable for the development of large brown citrus aphid populations; during the rest of the growing season, aphid populations are often very low due to a lack of tender new flush. Although monitoring of citrus groves took place between Mar and Oct each year, the majority of brown citrus aphids were found between Jun and Aug. The percentage parasitism of brown citrus aphids by L. oregmae at these sites ranged from 0.7 to 3.3%. Because brown citrus aphids containing L. oregmae pupate in or on the soil, predation by ground foragers such as red imported fire ants, Solenopsis invicta Buren, may contribute to the low incidence of L. oregmae in these samples (Hill & Hoy 2003; Persad & Hoy 2004). For unknown reasons, the number of groves found to contain brown citrus aphids decreased from 58 sites out of 122 (48%) monitored in 2001 to 12 of 70 (17%) evaluated in 2002. This made evaluation of the establishment and dispersal of L. oregmae difficult, so we decided to evaluate other aphid species on additional host plants in 2003 to resolve whether L. oregmae was established.

Recovery of L. oregmae within 5 Aphid Species in or near Citrus Groves in Florida in 2003 Detected by High-fidelity PCR

High-fidelity PCR assays detected the presence of L. oregmae in 5 species of aphids (Aphis spiraecola, A. gossypii, A. craccivora, T. citricida, and T. aurantii) collected from citrus foliage (c), vegetables (v), or weeds (w) in 4 of the 8 counties surveyed in Florida during 2003 (Table 2). The percentage parasitism of these aphids by L. oregmae ranged from 2.0 to 12.9% when parasitism was estimated from larvae dissected out of their aphid hosts (Table 2). Of the 7 aphid species collected in the survey, only Myzus persicae (v) and Uroleucon pseudambrosiae (w) did not contain immature L. oregmae.

Lipolexis oregmae was found in both the individual and group analyses of aphids collected from Alachua, Indian River, or Lake counties (Table 2). In Hendry county, assays on individually dissected parasitoid larvae were negative for L. oregmae, but pooled aphid samples from that county yielded two groups positive for L. oregmae. The absence of L. oregmae larvae in the dissected aphids suggests that parasitism was very low in this site, but the positive PCR results from the pooled samples from the same location indicate establishment at low levels by L. oregmae. Sensitivity analysis has shown the High-fidelity PCR assay can detect a single parasitized aphid when combined with up to 499 unparasitized aphids (Persad et al. 2004).

An advantage of the High-fidelity PCR assay is that it is sensitive and allows monitoring of large numbers of pooled aphids for the presence of L. oregmae (as well as of L. testaceipes). Furthermore, the PCR assay eliminates losses from mortality of immature parasitoids while they are held in plastic bags until adult parasitoids emerge. The High-fidelity PCR assay takes less time than rearing the parasitoids in bags because L. oregmae can be detected within 1 d after the sample is collected. The disadvantage of conducting the PCR assay on pooled aphids is that it provides only qualitative data on the presence of L. oregmae and it is expensive. However, the High-fidelity PCR assay allows us to evaluate larger numbers of aphids and sites when monitoring for establishment and distribution.

Lysiphlebus testaceipes and Hyperparasitoids

Lysiphlebus testaceipes was collected in all brown citrus aphid samples in which L. oregmae was found during the 3 field seasons. This suggests that L. testaceipes may be a better parasitoid of the brown citrus aphid than previously thought. For example, of 158 parasitoid larvae dissected from 5 aphid species during 2003 and assayed by High-fidelity PCR, 140 (89%) were L. testaceipes (Table 2). Several authors (Michaud 1998; Michaud & Browning 1999; Yokomi & Tang 1996) have rated L. testaceipes as ineffective because few adults emerge from brown citrus aphid mummies located on foliage due to the phenomenon called incomplete parasitization. However, Persad & Hoy (2003) discovered that a large proportion of brown citrus aphids parasitized by L. testaceipes mummify off the citrus foliage and a high proportion of these mummies can produce adults. We conclude that L. testaceipes is an effective parasitoid of the brown citrus aphid in citrus groves in Florida.

Two species of hyperparasitoids were recovered from the field-collected foliage held in plastic bags in 2001 and 2002. These comprised approximately 1.1 and 2.1% of all emerged parasitoids, respectively. They were identified as Pachyneuron aphidis Bouche and the ‘Alloxysta megourae complex’. Because the bags contained adults of L. testaceipes, as well as L. oregmae, we could not resolve which parasitoid produced these hyperparasitoids. To resolve whether these hyperparasitoids could develop on L. oregmae, we placed potted citrus trees containing brown citrus aphids parasitized by L. oregmae in the citrus grove located next to the Department of Entomology and Nematology building in Gainesville during Jun 2002; both hyperparasitoids were recovered at low rates (1-2%), confirming that these two hyperparasitoids will attack L. oregmae.

The results of surveys conducted in 15 counties containing approximately 348,031 ha of citrus between 2001 and 2003 show that L. oregmae was recovered from multiple sites some distance from the original release sites. Because L. oregmae was not released after 2002, yet was found in multiple sites during 2003, L. oregmae appears to have successfully persisted, overwintered, and dispersed from the original release sites in Florida citrus groves.

Because brown citrus aphids collected from Polk County during Oct 2005 produced adults of L. oregmae, this parasitoid has persisted in Florida since 2002. Interestingly, L. oregmae was discovered during 2004 to be well established on brown citrus aphids in Jamaica, where it was fortuitously introduced from an unknown source (Hoy et al. unpublished data). It is likely that L. oregmae was introduced into Jamaica from Florida because Florida is the only known location where it has been released and established in the Western Hemisphere. Because L. oregmae parasitizes several aphid hosts, it could have been introduced on aphid species and host plants other than citrus.

During the 2001 through 2003 surveys in Florida, L. testaceipes was found consistently and was the only other primary parasitoid of brown citrus aphid. Its abundance raised the question as to whether L. testaceipes would interfere with the establishment of, or abundance of, L. oregmae. However, interspecific competition studies indicated that neither L. oregmae nor L. testaceipes were intrinsically superior to the other when reared on the brown citrus aphid in the laboratory (Persad & Hoy 2003).

Brown citrus aphids parasitized by L. oregmae do not mummify on the foliage, as is common with many other parasitized aphids. These mummies can be preyed on by the red imported fire ant, Solenopsis invicta Buren, and the Asian cockroach, Blattella asahinai Mizukubo (Hill & Hoy 2003; Persad & Hoy 2004). Furthermore, red imported fire ants forage in citrus trees and will selectively remove brown citrus aphids parasitized by L. oregmae (Persad & Hoy 2004). Because red imported fire ants are very abundant in citrus groves throughout Florida, their predation on parasitized aphids or mummies may be reducing the effectiveness of L. oregmae as a natural enemy of the brown citrus aphid in Florida.

Our data indicate that sampling methods designed to evaluate parasitism of brown citrus aphids by either L. oregmae and L. testaceipes should take into account the fact that the majority of parasitized brown citrus aphids move off the foliage to mummify. As a result, mortality estimates based on the few mummies retained on citrus foliage will underestimate mortality. Our data also indicate that L. oregmae and L. testaceipes are coexisting in citrus groves in Florida.

The use of alternative aphid host species on citrus, vegetables, and weeds was expected, and the survey conducted during 2003 confirms that L. oregmae is using these alternative hosts in Florida. Lipolexis oregmae is oligophagous (Stary 1988; Stary & Ghosh 1983; Stary & Zeleny 1983; Ahmad & Singh 1996) and all aphid species that we found parasitized are considered pests of economic importance. The use of alternative aphid species may prove advantageous for the efficacy of L. oregmae in citrus in Florida because it could allow L. oregmae populations to be sustained at higher densities when brown citrus aphids are scarce in groves. Because Singh & Hoy (unpublished) recently found that L. oregmae could parasitize milkweed aphids, Aphis nerii Boyer de Fonscolombe, on scarlet milkweed (Asclepias curassivica L.) in Florida, the host range of L. oregmae needs further analysis. Brown citrus aphid populations are maintained at very low densities in citrus groves unless abundant new growth is present. As a result, brown citrus aphid populations (and any parasitoids attacking them) in groves probably are limited to intervals when major flush cycles occur because the host range of brown citrus aphids is restricted to Citrus species and some Rutaceae such as lime berry (Triphasia trifolia), box-orange (Severinia buxifolia (Poir.)), and orange jasmine (Murraya paniculata (L.)), which are used as landscape hedges in south Florida (Tsai 1998).

Quantification of the impact of L. oregmae and L. testaceipes, in combination with the role of endemic predators and pathogens, on brown citrus aphid populations in Florida remains to be determined because L. oregmae was still in the process of colonizing suitable aphid species on citrus, vegetables, ornamentals, and weeds during this study. Thus, the impact of L. oregmae on brown citrus aphid populations, and on other aphid species on other host plants in Florida remains to be resolved.