CN-770000-18120-TA 19304.pdf

Multi-Year Project Request

Date: 24 April 2018

Project Title: Quarantine Testing to Develop Biological Control of Old World Climbing Fern, Lygodium microphyllum, in Florida

Contact Information:

USDA ARS Invasive Plant Research Laboratory (IPRL): Ellen C. Lake (PI), 3225 College Avenue, Fort Lauderdale, FL 33314, email: ellen.lake@ars.usda.gov, Phone: (954) 475-6547, Fax: 954-476-9169

Need for Research and Summary of Recent Progress:

Lygodium microphyllum, Old World climbing fern, continues to expand its range in Florida. The two established biological control agents Neomusotima conspurcatalis, the brown lygodium moth, and the lygodium mite, Floracarus perrepae, are being mass reared and released and are dispersing and establishing new populations across the landscape. However, additional biological control agents would benefit efforts to control this invasive weed.

Hurricane Irma significantly slowed progress on quarantine research projects in the fall of 2017. This was primarily due to the need to cull colonies to life stages that could survive several days with limited care, i.e. eggs and pupae, without stress increasing the risk of disease in the colonies. It then took time post-hurricane to rebuild the colonies to a larger size to continue with testing. Despite these challenges, in FY2017 progress was made with the quarantine testing of several candidate biological control agents. The original colony of Lygomusotima stria was merged with a colony established from L. stria collected in Singapore in 2016. This improved the genetic diversity and health of the combined colony, which will be used for the remaining testing. We successfully reared Siamusotima disrupta stemborer larvae from overseas shipments to adults but limited numbers and asynchronous emergence prevented colony establishment. The size of the colony of the sawfly Neostrombocerus albicomus was substantially increased to facilitate multi-generational testing on L. microphyllum and three congeners, these tests will begin as soon as adults emerge in late April or early May 2018. To date, no-choice tests with Callopistria sp. have been conducted with 25 and all larvae died as first instars on non- Lygodium species. Callopistria sp. have completed development on several Lygodium species, but preliminary data suggests that L. micophyllum and L. japonicum are the most suitable hosts.

Multi-Year Project Objectives and Methods:

Objective 1: Colonize new collections of Lygodium stem borers to begin host-range testing and determine suitability for biological control.

2017 Accomplishments:

We succeeded in rearing larvae received from shipments received from ABCL collections in Hong Kong in June and August of 2017 to adults. However, only one F1 adult emerged from mattings of the adults reared from the June shipment. Emergence of parental adults was lower and asynchronous from the August larval shipment. This very limited overlap in emergence of males and females severely limited any chance of successful reproduction. The new cage design continues to provide favorable rearing conditions.

2018 Plans:

We anticipate shipments of S. disrupta from Hong Kong in May/ June and August of 2018. Plant preparation is underway to ensure that the optimal size and quality plants for stem borers are available for the spring shipments. We will continue to dissect larvae out of the shipped L. microphyllum stems and transfer them onto whole plants in the large acrylic cages, which provided favorable abiotic conditions for the stem borers. If numbers permit, we will also work to learn more about the life history of S. disrupta, including development time and adult longevity and fecundity, which will help us to better plan our rearing efforts and rotation of plants and insects in rearing cages to prevent overcrowding. This will be accomplished by caging one male and one female on an individual plant. Numbers permitting, we also plan to pursue host-range testing with the 2018 collections, focusing on the Lygodium congeners.

Objective 2: Complete multi-generational host-specificity testing with Lygodium congeners for the sawfly Neostrombocerus albicomus.

2017 Accomplishments:

Further improvements were made to the rearing methods for the sawfly, including changing the pupation substrate to sand, which facilitates better monitoring of pupating individuals than the previous media used. The size of the sawfly colony was substantially increased over several generations to produce the number of adults needed to simultaneously initiate multi-generational testing with multiple replicates of the four Lygodium species on which the sawfly completed development during no-choice testing. The sawfly individuals needed to start these tests are currently pupating, we anticipate adult emergence in late April or early May 2018. Plants have been grown and are ready for these tests.

2018 Plans:

Once adults emerge from pupation they will be used to inoculate whole plants of L.

microphyllum, L. oligostachyum, L. volubile, and L. japonicum in small pop up cages. Methods for this testing have been revised to incorporate the improvements we have made in rearing this insect, including both changes to handling of the insect and use of the same oviposition cages used for colony maintenance. In accordance with this successful rearing protocol, 25 adults will be added to each replicate cage to produce the first generation: 15 mated females, 5 unmated females, and 5 unmated males. We will run these tests for several generations to determine if the sawfly can develop sustainable populations on any of the congeners besides L. microphyllum.

We will complete the multi-generational testing and conduct additional replicates as needed in 2018.

No-choice host-range testing will be conducted on additional species to ensure we have covered all testing requirements recommended by the Technical Advisory Group for the Biological Control of Weeds (TAG). After completion of the multi-generational testing we will return to the life history studies of the sawfly – adult longevity, fecundity, sex ratio of offspring, and temperature dependent development.

Objective 3: Complete the suite of host-specificity trials on Lygomusotima stria in addition to the temperature tolerance studies.

2017 Accomplishments:

Following genetic analyses that confirmed that we were working with the same species, the old L. stria colony was merged with the new colony collected in 2016 from a site in Singapore very close to the collection site for the original colony. This process took more than two months but ensures that the remaining testing will be conducted with a robust colony that maximized the available genetic material.

We continue to analyze the cold tolerance and chill coma data, which will be included in the TAG petition. These data indicate that due to cool temperatures L. stria is unlikely to establish populations in northern Florida or beyond.

Results of chill coma determination the effect of reducing temperature on the immobilization of early, middle, and late instar larvae of Lygomusotima stria.

Lower thermal limits (LT50) values for cold tolerance tests for Lygomusotima stria early, middle, and late instar larvae, and pupae (D). Regression lines drawn with SAS NLIN using the y = B1 + B2 x (temperature) + B3 x (temperature 2).

2018 Plans:

We will collaborate with a niche modeler to incorporate our cold tolerance testing results into climate models to predict the potential range of L. stria and will conduct any additional cold tolerance tests as needed. We will obtain additional test plants to continue conducting no-choice testing, including adding additional species to fulfill TAG recommendations. To maintain consistency with earlier methods, these tests will consist of ten larvae per dish, five replicates per plant species. We will conduct additional replicates of multi-generational testing of L. stria on L.

microphyllum, L. japonicum, and L. palmatum. After this testing is complete, a petition will be prepared for TAG review.

Objective 4 (new in fall 2016): Initiate host-range testing with Callopistria sp. (Lepidoptera:

Noctuidae)

2017 Accomplishments:

A Callopistria species (Lepidoptera: Noctuidae) was collected in Hong Kong in 2016.

This species has been colonized in the IPRL quarantine and rearing methods have been established. Specimens of male and female adults, and larvae from each instar were prepared and submitted to Paul Goldstein, a noctuid expert at the USDA ARS Systematic Entomology Laboratory for identification. More was learned about the biology of this species by determining the duration of the larval instars. Knowing the duration of the larval instars on the target weed is particularly important for comparison to the response of the insect to non-target plants during no-choice testing.

Duration (days) of Callopistria sp. larval instars

1st 2nd 3rd 4th 5th

Mean ±

SEM

3.82 ± 0.06 2.72 ± 0.11 2.49 ± 0.08 2.83 ± 0.06 additional data needed N 49 44 40 35

Full or partial replicates of no-choice testing have been completed on 25 species. This testing was conducted with naïve Callopistria sp. larvae that were less than 24 hours old at the start of the test. All larvae died as first instars on all non-lygodium test plant species. Additional no-choice test replicates were conducted with L. microphyllum and its congeners. Although more testing is needed, based on decreased development time and higher percent survival to adults L.

microphyllum and L. japonicum are more suitable hosts than the other lygodium species.

Pinned male and female Callopistria sp. (Lepidoptera: Noctuidae) adults from colony started from the Hong Kong collections that were submitted to the USDA ARS Systematic Entomology Laboratory for identification.

2018 Plans:

We will continue to conduct host-range testing with Callopistria sp. Emphasis will be on increasing the number of replicates of Lygodium congeners while also running non-Lygodium species from the test plant list. For the Lygodium congeners we will continue to test one larva per petri dish so we can obtain the pupal weight and development time for each individual. This will contribute to our understanding of which species are optimal versus suboptimal hosts. No-choice tests with non-Lygodium species will consist of 10 larvae per dish, five dishes per species.

During each test, all required plant material for an individual dish will be sourced from the same plant. We hope to obtain more specimens of Callopistria sp. from collections by the Australian Biological Control Laboratory to increase the genetic diversity of the colony.

Deliverables:

A mid-point status report and a fiscal-year final report will be provided.

Budget: The requested budget of $55,550 includes support for one full-time technician allocated to USDA-ARS-IPRL in Fort Lauderdale. This technician will maintain quarantine colonies of candidate agents and conduct host-range testing. This proposed budget is leveraged with USDA- ARS funds that cover the salary of one USDA-ARS scientist, a technician, greenhouse and quarantine space, and vehicles. These matching contributions are made at no charge to FFWCC.