Lilioceris egena Air Potato Biocontrol Environmental Assessment

Field Release of the Beetle

Lilioceris egena (Coleoptera:

Chrysomelidae) for Classical

Biological Control of Air

Potato,

Dioscorea bulbifera

(Dioscoreaceae), in the

Continental United States

Environmental Assessment,

February 2021

United States

Department of

Agriculture

Marketing and

Regulatory

Programs

Field Release of the Beetle Lilioceris

egena (Coleoptera: Chrysomelidae)

for Classical Biological Control of Air

Potato, Dioscorea bulbifera

(Dioscoreaceae), in the Continental

United States

Environmental Assessment,

February 2021

Agency Contact:

Colin D. Stewart, Assistant Director

Pests, Pathogens, and Biocontrol Permits

Plant Protection and Quarantine

Animal and Plant Health Inspection Service

U.S. Department of Agriculture

4700 River Rd., Unit 133

Riverdale, MD 20737

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Contents

I. Purpose and Need for the Proposed Action .................................................................................................... 1

II. Alternatives ..................................................................................................................................................... 2

A. No Action ..................................................................................................................................................................... 3

B. Issue Permits for Environmental Release of Lilioceris egena. ..................................................................................... 3

III. Affected Environment .................................................................................................................................... 7

A. Taxonomy and Description of Air Potato ..................................................................................................................... 7

B. Areas Affected by Air Potato ........................................................................................................................................ 8

C. Plants Related to Air Potato and Their Distribution ................................................................................................... 11

IV. Environmental Consequences .................................................................................................................... 13

A. No Action ................................................................................................................................................................... 13

B. Issue Permits for Environmental Release of Lilioceris egena .................................................................................... 15

V. Other Issues ................................................................................................................................................. 19

VI. Agencies, Organizations, and Individuals Consulted ................................................................................... 20

VII. References ................................................................................................................................................. 21

Appendix 1. ...................................................................................................................................................................... 30

Appendix 2. ...................................................................................................................................................................... 74

1

I. Purpose and Need for the Proposed Action

The U.S. Department of Agriculture (USDA), Animal and Plant Health

Inspection Service (APHIS), Plant Protection and Quarantine (PPQ), Pests,

Pathogens, and Biocontrol Permits (PPBP) is proposing to issue permits for

release of the beetle Lilioceris egena (Weise) (Coleoptera: Chrysomelidae).

Lilioceris egena would be used for the classical biological control of air potato,

Dioscorea bulbifera L. (Dioscoreaceae), in the continental United States.

Classical biological control of weeds is a weed control method where natural

enemies from a foreign country are used to reduce exotic weeds that have

become established in the United States. Several different kinds of organisms

have been used as biological control agents of weeds: insects, mites, nematodes,

and plant pathogens. Efforts to study and release an organism for classical

biological control of weeds consist of the following steps (TAG, 2016):

1. Foreign exploration in the weed’s area of origin.

2. Host specificity studies.

3. Approval of the exotic agent by PPBP.

4. Release and establishment in areas of the United States invaded by the target

weed.

5. Post-release monitoring.

This environmental assessment

1

(EA) has been prepared, consistent with

USDA, APHIS' National Environmental Policy Act of 1969 (NEPA)

implementing procedures (Title 7 of the Code of Federal Regulations (CFR),

part 372). It examines the potential effects on the quality of the human

environment that may be associated with the release of L. egena to control

infestations of air potato within the continental United States. This EA considers

the potential effects of the proposed action and its alternatives, including no

action. Notice of this EA was made available in the Federal Register on January

8, 2021 for a 30-day public comment period. Fourteen comments were received

on the EA by the close of the comment period. All comments were in favor of

the proposed release of L. egena.

APHIS has the authority to regulate biological control organisms under the

Plant Protection Act of 2000 (Title IV of Pub. L. 106–224). Applicants who

wish to study and release biological control organisms into the United States

must receive PPQ Form 526 permits for such activities. The PPBP received a

permit application requesting environmental release of the beetle, L. egena,

from China, and the PPBP is proposing to issue permits for this action. Before

1

Regulations implementing the National Environmental Policy Act of 1969 (42

United States Code 4321 et seq.) provide that an environmental assessment “shall include brief discussions

of the need for the proposal, of alternatives as required by section 102(2)(E), of the environmental impacts of

the proposed action and alternatives, and a listing of agencies and persons consulted.” 40 CFR § 1508.9.

2

permits are issued, the PPBP must analyze the potential impacts of the release

of this agent into the contiguous United States.

The applicant’s purpose for releasing L. egena is to reduce the severity of

infestations of air potato in the contiguous United States. Air potato is a twining

vine (65 feet long or greater) that often forms single species stands in Florida

(Schmitz et al., 1997; Langeland and Craddock Burks, 1998; Gordon et al.,

1999) and constitutes one of the most aggressive weeds introduced into the state

(Hammer, 1998). Noted horticulturalist Henry Nehrling described his concern

about the plant’s invasiveness early in the 20th century, stating that “with the

exception of the kudzu vine, I have never seen a more aggressive and dangerous

vine in Florida” (Nehrling, 1933). Similar warnings were expressed in the

1970s, with recommendations to limit the planting of this ornamental species

(Long and Lakela, 1976; Morton, 1976; Ward, 1977). By the 1980s, air potato

was commonly growing in thickets, waste areas, and along hedges or fencerows

in south and central Florida (Bell and Taylor, 1982). By the end of the 20th

century, air potato was listed as a noxious weed by the Florida Department of

Agricultural and Consumer Services (FDACS) (FL-EPPC, 2003). Air potato is

considered the most serious type of environmental threat, described as a

Category I weed by the Florida Exotic Pest Plant Council (FL-EPPC, 2003) as

“invasive exotics that are altering native plant communities by displacing native

species, changing community structure or ecological functions, or hybridizing

with natives.” Presently, air potato is well established in Florida, and is

spreading into surrounding states (Raz, 2002) where it has the potential to

severely disrupt entire ecosystems (Hammer, 1998).

Existing options for management of air potato, such as chemical and mechanical

methods, provide only temporary solutions that require retreatment and are

harmful to non-target species associated with the weed. Lilioceris egena is also

expected to complement the activity of a previously released beetle, Lilioceris

cheni, for biological control of air potato. For these reasons, the applicant has a

need to release L. egena, a host-specific, biological control organism for the

control of air potato, into the environment.

II. Alternatives

This section will explain the two alternatives available to the PPBP—no action

and issuance of permits for environmental release of L. egena. Although the

PPBP’s alternatives are limited to a decision on whether to issue permits for

release of L. egena, other methods available for control of air potato are also

described. These control methods are not decisions to be made by the PPBP,

and their use is likely to continue whether or not permits are issued for

environmental release of L. egena, depending on the efficacy of L. egena to

control air potato. These are methods presently being used to control air potato

by public and private concerns.

3

A third alternative was considered, but will not be analyzed further. Under this

third alternative, the PPBP would have issued permits for the field release of L.

egena; however, the permits would contain special provisions or requirements

concerning release procedures or mitigating measures. No issues have been

raised that would indicate special provisions or requirements are necessary.

A. No Action

Under the no action alternative, PPBP would not issue permits for the field

release of L. egena for the biological control of air potato. The release of this

biological control agent would not take place. The following methods are

presently being used to control air potato; these methods will continue under the

“No Action” alternative and will likely continue even if permits are issued for

release of L. egena, depending on the efficacy of the organism to control air

potato.

Chemical control of air potato vines requires repeated basal applications of

herbicides (e.g., glyphosate, triclopyr), and these treatments need to be repeated

over a two or three year period (Mullahey and Brown, 1999).

Removal of aboveground plants and bulbils by hand is a method of reducing air

potato infestations (e.g., Duxbury et al., 2003). Hand removal was found to be

as effective at controlling air potato as a combination of herbicide and hand

pulling of air potato plants.

The Asian beetle Lilioceris cheni Gressitt and Kimoto was developed

(Pemberton and Witkus, 2010) and released (Center et al., 2013) as a biological

control agent of air potato. This beetle has been distributed throughout Florida

and is dispersing at a rate of 8.2 kilometers (km)/year, with a maximum

dispersal of 67 km from nearest release point (Overholt et al., 2016).

B. Issue Permits for Environmental Release of Lilioceris

egena.

Under this alternative, the PPBP would issue permits for the field release of the

beetle L. egena for the biological control of air potato. These permits would

contain no special provisions or requirements concerning release procedures or

mitigating measures.

Biological Control Agent Information

Lilioceris egena is assigned to the insect order Coleoptera, family

Chysomelidae, and subfamily Criocerinae. There are currently only two

Lilioceris species in North America: the invasive Lilioceris lilii (Scopoli), and

the biological control agent L. cheni (Center et al., 2013). A.S. Konstantinov,

1. Chemical

Control

2. Mechanical

Control

1. Taxonomy

and

Description

3. Biological

Control

4

USDA-Agricultural Research Service, Systematic Entomology Laboratory,

Beltsville, MD, determined the specimens collected by F.A. Dray and G.

Witkus from air potato in southern China during May 2011 to be Lilioceris

egena, a member of the L. impressa group (see Tishechkin et al., 2011).

Specimens collected in Yunnan Province, China in May 2011 were deposited by

A.S. Konstantinov at the National Museum of Natural History, Smithsonian

Institution, Washington, D.C.

Adult L. egena beetles are shiny black in color except for their red-reddish

brown wing covers (elytra). Like other species in the subfamily Criocerinae, L.

egena are elongate (1 centimeter (cm) long by 0.5 cm wide at the abdomen),

with a narrow thorax and an even more narrow head with bulging eyes.

The genus Lilioceris is a widespread group that Berti and Rapilly (1976)

proposed as originating in southern China/northern southeast Asia. The known

distribution of L. egena includes China (Anhui, Yunnan, Fujian, Sichuan, and

Hong Kong Provinces), India (Assam, Karnataka, and Uttarhand), Nepal

(Province 3), Laos (Vientianne Province), Vietnam (Tây Nihn and Ho Chi Minh

Provinces), and Singapore (Tishechkin et al., 2011). In addition, Warchalowski

(2011) reports L. egena as occurring in Hainan, China and Taiwan.

The average male L. egena beetle is only slightly longer-lived than the average

female (126.2 ± 9.69 days vs. 110.7 ± 7.70 days) (± standard error (s.e.)

hereafter); both have a maximum lifespan of 207 days. Females have a pre-egg

laying period of 9.5 ± 0.75 days, and produce eggs for 95.3 ± 9.14 days. They

deposit their eggs singly or in clutches of 2 to 14. Lilioceris egena eggs are

frequently deposited on the undersides of aerial tubers, known as bulbils, that

have fallen to the ground and split open, and there, the eggs are protected by the

soil. The eggs may also be laid immediately above the soil/bulbil interface.

Eggs are also deposited inside adult feeding holes in bulbils (Figure 1b). The

eggs are attached to the bulbil with a gluey greenish substance exuded by the

beetle that darkens over time. Eggs are occasionally found on nearby soil

(Figure 1a), although it is unclear whether these are placed there by the females

or represent eggs displaced when the bulbils are disturbed. Females only rarely

oviposit (lay eggs) on air potato leaves.

Eggs are initially creamy yellow (Figure 1a), but darken (greenish-gray) as the

larva inside develops (Figure 1b). The head capsule becomes visible through the

end of the opaque egg midway through development, causing that end to appear

to be darker than the rest of the egg (Figure 1b). Soon after, the developing

larval eyes become visible as two distinct maroon spots. Neonate larvae eclose

(hatch) from the eggs 4.8 days after oviposition.

2. Geographical

Range of L.

egena

3. Life History of

L. egena

5

Figure 1. Lilioceris egena eggs deposited (a) on soil at base of an air potato

bulbil, and (b) in an adult feeding hole on a bulbil. Eggs in (a) are less than 24

hours old, whereas eggs in (b) are about 48 hours old. [Photos: F.A. Dray Jr.]

Neonates (newly hatched larvae) are translucent white with black legs, head

capsules, and thoracic plates. As they age, the larvae become dark grey before

pupation. The larvae feed singly, whether scraping the undersides of the foliage

(Figure 2a) or mining within the bulbils (Figure 2b). The larvae prefer tender

newly emerged air potato leaves, but can also eat older toughened leaves. Early

instar larvae can consume air potato bulbils if there is an initial tear through

itsthe bulbil surface, but more mature larvae can eat intact bulbils without

difficulty.

Figure 2. Lilioceris egena (a) newly hatched (neonate) larva feeding on

underside of air potato leaf, and (b) 2nd instar larva beginning to tunnel into

bulbil inside adult feeding scar. Note neonate exuvia on edge of adult feeding

scar in (b). [Photos: F.A. Dray Jr.]

Although larvae can feed on foliage (Figure 2a), they prefer bulbils (Figure 2b),

and develop through four instars (immature developmental stages) after which

they exit the bulbils (Figure 3a). Neonates are unable to penetrate the periderm

(outer ‘skin’) of the bulbil, so they use adult feeding holes (Figure 2b) to access

6

the bulbil interior. Neonates have also been observed entering the bulbil via

breaks in the periderm formed when the bulbil begins to sprout. Later (2nd and

3rd) instar larvae can penetrate the periderm and have also been observed

cannibalizing younger instars. The peach-colored pre-pupae (4th instar; see

Figure 3c) exit the bulbil and crawl to the soil. Although some form naked

pupae (Figure 3b), the vast majority form a cocoon composed of a white

substance secreted from their mouths. Soil adheres to this material as it hardens

to a Styrofoam™-like texture thereby forming the puparium. A puparium is the

hardened last larval skin that encloses the pupa. Puparia can be affixed to the

undersides of the bulbil or can be free in the soil, and they often are found in

clusters of 2–8 individuals (Figure 3d). Development from neonate to adult

requires slightly less time on foliage compared to bulbils (27.4 ± 0.17 days for

foliage vs. 28.9 ± 0.23 days for bulbils). However, a much greater proportion

(62.9 vs. 44.3 percent) of larvae successfully complete development on bulbils.

Figure 3. Lilioceris egena (a) 2nd to 4th instar larvae exiting air potato bulbil,

(b) naked pupa, (c) pre-pupae, and (d) conjoined puparia. Note larval exuviae

above pupa in (b) and at back of puparium on bottom left in (d). [Photos: F.A.

Dray Jr.]

Egg production and adult emergence from pupae were greatly reduced during

December and January indicating the existence of a reproductive diapause (a

period of suspended development) that coincides both with cooler weather in

the more temperate regions of L. egena’s distribution and with the period when

air potato is typically leafless with vines that have died back. The aerial tubers

of air potato fall to the ground soon after the leaves fall, thereby becoming

available on the ground where pupation occurs.

7

III. Affected Environment

A. Taxonomy and Description of Air Potato

Class: Equisitopsida (Embryophytes)

Subclass: Magnoliidae (Angiosperms)

Superorder: Lilianae (monocots)

Order: Dioscoreales

Family: Dioscoreaceae (yams)

Genus: Dioscorea L.

Species: bulbifera L

Synonyms: Helmia bulbifera (L.) Kunth., Dioscorea sativa F.M. Bailey, D.

sativa Thumb., D. latifolia Benth., D. anthropophagorum A. Chev. (Wilkin

2001, Gövaerts et al., 2007); D. crispata Roxb., D. dicranandra Donn. Sm., D.

heterophylla Roxb., D. hoffa Cordem., D. pulchella Roxb., D. tamnifolia

Salisb., D. tenuiflora Salisb., Smilax decipens Spreng. (Wunderlin et al., 2017);

D. oppositifolia L., D. papilaris Blanco, D. tunga Hamilton (Coursey, 1967).

Common names: acom, air potato, air yam, ñame, ala-ala, hoi

Air potato is an herbaceous twining vine, growing 65 feet or more in length.

Leaves are broadly cordate (heart shaped) and alternately arranged on stems. A

distinguishing characteristic of air potato is that all leaf veins arise from the leaf

base. Flowers are inconspicuous (Figure 4a), arising from leaf axils in panicles

4 inches long. Flowers are rarely seen in Florida. The fruit-type produced by

female plants in the native range of air potato is a dry capsule which is pale

brown at maturity (Figure 4a-c) (Coursey, 1967; Hamon et al., 1995; Raz,

2002). Seeds are winged, elongate, and are slightly curved at the point of

attachment (Hamon et al., 1995; Raz, 2002) (Figure 4c). Seeds range in length

from 12–22 millimeters (mm) (Raz, 2002). In Florida, vegetative reproduction

is the primary mechanism of spread. This is through the formation of aerial

tubers, or bulbils, which are formed in leaf axils.

Aerial tubers (bulbils; Figure 4d) may be produced throughout the active

growing cycle of the plant, but tend to be more common later in the cycle when

stem and leaf development is complete (Coursey, 1967; Miller, 2010). Bulbils

are vegetative organs with a shape that is similar to a condensed stem (Coursey,

1967). One to four bulbils may be produced per leaf axil. Bulbils can reach 12

cm in length and have a potato-like appearance. New plants develop from

bulbils, and these bulbils serve as a means of dispersal.

Bulbils produced by air potato in Florida are primarily of two types (Figure 4e)

matching descriptions of Asian varieties of the plant (Martin, 1974). The

majority of bulbils are dark coffee-colored with a bumpy texture (Figure 4e,

right). Some plants, however, have been found to produce light tan or grey

bulbils with smoother skin (Figure 4e, left; Hammer, 1998; Overholt et al.,

8

2003). A third type of bulbil (Figure 4f) is very rare in Florida, but matches with

descriptions of edible African varieties (Martin, 1974), especially D. bulbifera

var. anthropophagorum (Matthews and Terauchi, 1994).

Figure 4. Reproductive structures of air potato: (a) herbarium specimen

showing flowers and fruits, (b) close-up of ripening fruits on the vine, (c) close-

up of dried fruits showing winged seed, (d) aerial tubers (bulbils) along stem at

leaf axils, (e) smooth tan and bumpy brown bulbil types common in Florida, and

(f) bulbil of edible African variety uncommon in Florida. [Photos: (a) Kew

Herbarium, (b) N. Sasidharan, Kerala Forest Research Institute, (c) Kew

Herbarium, (d) K. and F. Starr, Hawaii, (e) W. Overholt, University of Florida,

and (f) D. Goodman, TheSurvivalGardener.com.]

B. Areas Affected by Air Potato

Air potato is broadly pantropical, with two primary types - one African and one

Asian - within the species (Terauchi et al., 1991; Figure 5). Chevalier (1936)

believed that air potato was originally Asian (likely Chinese), transported to

East Africa via Arabian merchants, and then to West Africa via Portuguese

merchants. Although Chevalier (1936) may ultimately be correct about the

Chinese origin, his assertion about the derivation of the two types is

unsupported by the molecular data which suggests that they diverged about 10

million years ago during the Pliocene Epoch (Terauchi et al., 1991). Each type

is quite diverse, with Miège (1982) listing 11 varieties in West Africa, and

Yifeng et al. (2008) reporting four varieties in China. Both Terauchi et al.

1. Native Range

of Air Potato

9

(1991), and Zheng et al. (2006) proposed that Yunnan Province, China, was the

center of diversification for Asian varieties of this species. The native range of

air potato extends from Africa and Asia (including India) through Malaysia to

Australia and the Pacific Islands (Prain and Burkill, 1936; Coursey, 1967;

Wilson and Hamilton, 1988; Williams, 2012).

Figure 5. Worldwide distribution of Dioscorea bulbifera L. (air potato) (Dray,

2017).

The earliest U.S. record for air potato is William Bartram’s (1791) observation

during 1777 of this vine in a garden in Mobile, Alabama. The plants he observed

likely derived from Africa given that he described the bulbils as being kidney-

shaped, a description that fits the African but not the Asian varieties (Martin,

1974; Matthews and Terauchi, 1994; see also Figure 4f). There is no evidence

that these plants persisted into the present, however, and Croxton et al. (2011)

found that the predominant varieties of air potato in Florida did not match plants

tested from West Africa. The Florida types instead match plants from Asia and

Oceania, including Hawaii (Croxton et al., 2011). These findings match well

with USDA plant importation records showing that the earliest verifiable

introduction of air potato into Florida was from Hawaii (as D. sativa) in March

1899 (USDA, 1900). Hillebrand (1888) described the bulbils of this species as

“green globular bulbs” (a description that fits immature bulbils in Florida) and

noted that this species is not native to Hawaii, but was introduced with many

other species via Oceanian cultures. The common name “hoi” used in Hawaii

(Hillebrand, 1888; Kinsey, 2016) is also used for this plant in Sumatra.

Since its introduction to Florida, air potato has aggressively spread throughout

the state: from Baldwin County in the northwestern panhandle to Miami-Dade

County at the southern tip of the state. Collections from herbaria and reports

from state regional biologists listed 60 of 67 Florida counties infested with air

potato in 2017 (Wunderlin et al., 2017; EDDMaps 2017; Figure 6), up from the

29 counties reported by Wheeler (2007). This species is reportedly naturalized

2. Introduced

Range of Air

Potato

10

in Georgia, Alabama, Mississippi, Louisiana, Texas, and Hawaii (Nesom and

Brown, 1998; EDDMapS, 2017). Villaseñor and Espinosa-Garcia (2004) also

report air potato from 10 states in Mexico.

Figure 6. Distribution of Dioscorea bulbifera L. in the United States

(EDDMapS, 2017).

Based on the known range of air potato in the United States (Figure 6), the plant

can survive in areas with an average annual minimum temperature range of

-12.2 to -9.5°C (10 to 15°F) — zone 8b on the USDA Hardiness Zone Map.

Climatic data (minimum January temperature and annual rainfall) from

locations where air potato is known to occur in Florida were extrapolated

outside of Florida by Overholt et al. (2014) to estimate its potential distribution

in the United States (Figure 7). These data suggest that air potato may be able to

spread more extensively throughout the Gulf coast and along the Atlantic coast

as far north as Charleston, South Carolina.

Figure 7. Potential range of air potato in North America (from Overholt et al.,

2014).

11

In Florida, air potato is frequently found in tropical and subtropical hammocks

but may also invade disturbed uplands, scrub, sinkholes, alluvial flood plain

forests, urban lots (Schultz, 1993; Gann et al., 2001), pinelands (Langeland and

Craddock-Burks, 1998), and hedges or fencerows (Bell and Taylor, 1982).

Evidence also suggests that air potato aggressively exploits forest canopies

damaged by hurricanes, thereby impeding the reestablishment of native species

(Horvitz et al., 1998; Gordon et al., 1999).

C. Plants Related to Air Potato and Their Distribution

The family Dioscoreaceae currently includes 644 species in four genera:

Dioscorea, Stenomeris, Tacca, and Trichopus (Gövaerts et al., 2007; Viruel et

al., 2016). The largest genus in the family, Dioscorea, contains approximately

600 species (Raz, 2002), most of which grow in the subtropics or tropics, with

only a few species growing in temperate regions (Al-Shehbaz and Schubert,

1989; Raz, 2002). The Dioscorea genus is grouped into subgeneric sections

(Uline, 1897; Kunth, 1924). Traditionally, air potato has been placed in section

Opsophyton along with a few other tropical Old World (Africa, Asia and

Europe) species (Kunth, 1924; Huber, 1998). The two native North American

(north of Mexico) species, D. floridana (Florida yam) and D. villosa (wild

yam), are assigned to the section Macropoda (Kunth, 1924; Raz, 2002). Raz

(2016) suggests that these two U.S. Dioscorea species are separated in

relatedness from air potato.

The native West Indian Dioscoreaceae are represented by 28 species of

Dioscorea, 19 of which are found in Rajania, a subgenus recently merged into

the genus Dioscorea (Raz, 2016). The center of origin for this group appears to

be Cuba (Kunth, 1924; Raz, 2016).

Mexico harbors 73 species of Dioscorea, including the introduced D. bulbifera

(air potato) and D. alata. None of the native species in Mexico are in the section

Opsophyton to which air potato belongs, and so the Mexican species are also

less closely related to air potato.

The remaining three genera in the family Dioscoreaceae represent the type

genera for their former respective families. Stenomeris contains two species

found only in Southeast Asia. Similarly, Trichopus contains one Asian species

and one species native to Madagascar. However, the genus Tacca with 13

tropical species contains some New World (North, Central, and South America

and nearby islands) representatives.

The family Dioscoreaceae is placed in the order Dioscoreales, a small order that

has had its member families change considerably with DNA analysis (Judd et

al., 2002). The order Dioscoreales was more recently redefined (Caddick et al.,

2002a, 2002b; Judd et al., 2002; Gövaerts et al., 2007) to contain, in addition to

3. Habitats

Where Air

Potato is

Found in North

America

1. Native and

Non-Native

Relatives

12

the family Dioscoreaceae, the family Nartheciaceae and the tiny mycoparastic

herbs in the family Burmanniaceae.

The family Burmanniaceae has 14 genera, three of which (Burmannia, Apteria,

and Thismia) are represented in the plants of North American (Gövaerts et al.,

2007). The genus Burmannia has three species in North America (B. biflora, B.

capitata, and B. flava), all of which occur in Florida (Wunderlin et al., 2017).

The genus Apteria has a single species which also occurs in Florida (A. aphylla)

(Wunderlin et al., 2017), whereas Thismia’s single species is limited to a small

area in northern Illinois (Gövaerts et al., 2007). The family Nartheciaceae

contains five genera: three in North America (Narthecium, Lophiola, and

Aletris), one limited to Japan and Korea (Metanarthecium), and one in northern

South America (Nietneria) (Fuse et al., 2012). The genus Aletris has five

species in North America (A. aurea, A. bracteata, A. farinosa, A. lutea, and A.

obovata), all of which occur in Florida (Gövaerts et al., 2007; Wunderlin et al.,

2017). The genus Lophiola has a single species (L. aurea) found from Nova

Scotia south to Florida (Wunderlin et al., 2017). The genus Narthecium has

seven species total, with two found in North America: one (N. californicum)

from Oregon and California, and one (N. americanum) scattered from New

Jersey to North Carolina (Gövaerts et al., 2007).

The subtropical order Pandanales is a sister group to the Dioscoreales in the

most current analyses (Hertweck et al., 2015). It contains five families (APG

IV, 2016): Cyclanthaceae which is found from Mesoamerica through South

America, and in the West Indies; Pandanaceae which occurs in the Old World

tropics and subtropics; Stemonaceae which is largely Asian and Australian, but

contains a single North American species - the Florida (and southeastern U.S.)

native Croomia pauciflora (Wunderlin et al., 2017); Triuridaceae which is

scattered across the Old and New World tropics and has no North American

representatives; and Velloziaceae which occurs in Africa, Asia, and South

America.

The genus Dioscorea contains the true yams, several of which are important

food crops in tropical and subtropical countries worldwide (Martin, 1974;

Coursey, 1981; Prance and Nesbitt, 2005; Wheeler et al., 2007; FAO, 2017).

None of the cultivated Dioscorea species are grown commercially in the

continental United States (Wheeler et al., 2007; FAO, 2017), but a few are

important commodities in the New World tropics. Dioscorea alata has

historically been grown in the region and is still grown in the Bahamas (Correll

and Correll, 1982), Cuba (Leon and Alain, 1974), Hispaniola (Liogier, 2000),

Jamaica (Adams, 1972), Puerto Rico (Liogier and Martorell, 1982), and the

Virgin Islands (USDA-NRCS, 2002). Dioscorea cayenensis (reported as D.

occidentalis in Leon and Alain, 1974) is cultivated in Cuba, Jamaica, and Puerto

Rico (Liogier and Martorell, 1982).

2. Economically

and

Environment-

ally Important

Relatives

13

IV. Environmental Consequences

A. No Action

a. Animals

Air potato often dominates habitats that it invades. It negatively impacts wildlife

dependent on native vegetation for forage, nesting, and cover.

b. Native Plants

Air potato poses a threat to natural areas because of its ability to trellis over and

out-compete native vegetation for limited resources, especially sunlight (Figure

8; Schmitz, 1994; Langeland and Craddock Burks, 1998; Gordon et al., 1999).

Wunderlin et al. (2017) lists over 75 species in Florida that have a vining habit.

Given that air potato shares this growth form and invades a variety of plant

communities, it seems likely that some of these native vines (e.g., Dioscorea

floridana) are at risk of replacement by air potato.

Figure 8. Dioscorea bulbifera trellising over native palms, pines, hardwoods,

and understory vegetation in Snyder Park, Broward County, Florida, during June

2012. [Photo: T. Center, U.S. Dept. Agriculture]

c. Human Health

No reports of detrimental health effects from air potato are known, although

aerial bulbils of the main variety in Florida are reportedly poisonous (Martin,

1974).

d. Social and Recreational Uses

Children reportedly play with the aerial bulbils which, because of their size and

weight, make them appealing to carry and throw – similar to snowballs in more

temperate climatic regions (Pemberton, 2009). This and the unusual shapes of

some bulbils promote their collection (Pemberton, 2009). However, this play

and collection can result in the spread of bulbils and the vine.

1. Impact of Air

Potato

14

e. Beneficial Uses

Air potato was introduced into Florida both as an ornamental and as a potential

crop plant (Fairchild, 1938; Nehrling, 1944), but was never really a commercial

success in either arena. Dioscorea alata is a popular ethnic West Indian food

plant. Yet despite this crop’s presence in over 400 gardens during the early 20th

century (Young, 1923), commercial production failed to persist and home

cultivation of this species in Florida today is uncommon. No purposeful

cultivation of air potato or any other Dioscorea is known in Florida today,

although one local survivalist does recommend wild D. alata (Good, 2016).

The continued use of chemical, mechanical, and biological controls at current

levels would be a result if the “no action” alternative is chosen. These

environmental consequences may occur even with the implementation of the

biological control alternative, depending on the efficacy of L. egena to reduce

air potato populations in the contiguous United States.

a. Chemical Control

Herbicidal control with glyphosate applications in heavily infested areas (e.g.,

Fern Forest, Broward County, Florida) that included other invasive weeds such

as Brazilian peppertree and bishop wood, cost $1,750/hectare (ha)/year. In this

example, complete control was not achieved as re-sprouts continued despite

three herbicide treatments over nearly two years. It has been estimated that five

years or more of herbicidal treatments and monitoring would be required to

achieve control. The herbicidal control method has additional costs as non-

target species, especially native species, suffer damage from non-selective

products.

b. Mechanical Control

Mechanical control is labor-intensive and provides only a temporary solution

that requires constant retreatment as plants continue to sprout up from bulbils.

Plants in isolated locations are difficult to access. Manual removal of air potato

plants is harmful to the native plant species that is being climbed by the vine.

c. Biological Control

Foliar damage caused by L. cheni is credited with reducing abundance and

overall biomass of air potato bulbils (Overholt et al., 2016). Despite the

observed reductions attributable to L. cheni, bulbil production remains a serious

concern because air potato is not known to reproduce sexually in North

America, so the primary means of multiplying and spreading in Florida is via

bulbils.

2. Impact

from Use of

Other

Control

Methods

15

B. Issue Permits for Environmental Release of Lilioceris

egena

Host specificity of L. egena to air potato has been demonstrated through

scientific literature, field observations, and host range testing. If the candidate

biological control agent only attacks one or a few plant species closely related to

the target weed, it is considered to be very host-specific. Host specificity is an

essential trait for a biological control organism proposed for environmental

release.

a. Scientific Literature

Tishechkin et al. (2011) reports L. egena adults feeding on Dioscorea subcalva,

a plant known only from Yunnan, Guangxi, Guizhou, and Chongqing Provinces

in central China.

b. Field Observations

During collecting trips in China and Nepal, the permit applicant/researcher

observed adult L. egena feeding only on D. bulbifera (air potato) (see also

Center et al., 2013).

c. Host Range Testing

Quarantine host range testing was conducted to determine the specificity of L.

egena for air potato and to determine if plants in the continental United States

could be at risk of attack by L. egena.

(1) Site of Quarantine Studies in the United States

Quarantine host specificity studies were conducted at the Invasive Plant

Research Laboratory, USDA Agricultural Research Service, 3225 College

Avenue, Fort Lauderdale, FL 33314.

(2) Test Plant List

Test plant lists are developed by researchers for determining the host specificity

of biological control agents of weeds in North America. Test plant lists are

usually developed on the basis of phylogenetic relationships between the target

weed and other plant species (Wapshere, 1974). It is generally assumed that

plant species more closely related to the target weed species are at greater risk

of attack than more distantly related species.

The host specificity test strategy as described by Wapshere (1974) is “a

centrifugal phylogenetic testing method which involves exposing to the

organism a sequence of plants from those most closely related to the weed

1. Impact of L.

egena on

Nontarget

Plants

16

species, progressing to successively more and more distantly related plants until

the host range has been adequately circumscribed.” Researchers do not pursue

release of biological control agents that do not demonstrate high host specificity

to the target weed.

For selecting the test plants for L. egena, the primary strategy followed the

centrifugal phylogenetic method (Wapshere 1974) modified, in part, per Briese

and Walker (2008). Host range of L. egena was determined through testing of

82 plant species in 46 families and 25 orders: 15 species within the

Dioscoreaceae and 67 species outside of the Dioscoreaceae. Test plants within

the Dioscoreaceae were chosen to represent the major taxonomic sections of the

family with representatives in Florida and the West Indies, and species of

economic importance. Additionally, the ornamental species Tacca chantrieri

was tested, which is a member of the only other genus in the family

Dioscoreaceae that occurs in Florida. Also, representative species were included

whose tubers/corms/expanded rhizomes are economically important crops. This

was important given that L. egena has proven to be an air potato storage organ

(bulbil) specialist.

Representatives of the Burmanniaceae, sister family to Dioscoreaceae (Judd et.

al., 2002; Merckx et al., 2006; 2010), were considered for testing – including

representatives of the three Burmannia species and the single Apteria species

that occurs in Florida (Wunderlin et al., 2017). However, these plants are tiny,

short-lived, fungal parasites with thread-like flower stalks that rise only a few

inches above the ground and thus were considered to be wholely inadequate to

support any L. egena feeding and development.

Members of Nartheciaceae, the only other family in the order Dioscoreales,

were also considered for testing – including the five species of Aletris and the

single Lophiola species that occur in Florida but no sources of these species

could be found. However, Lophiola and Aletris primarily inhabit locations with

saturated soils, such as bogs, swamps, and moors (Fuse et al., 2012; Wunderlin

et al., 2017) that would be unsuitable habitat for successful L. egena pupation.

The most closely related order to the Dioscoreales is the Pandanales (APG IV,

2016), represented in testing by Pandanus tectorius. Test plants outside the

Dioscoreales/Pandanales group represented orders within superorder Lilianae

with Florida natives, as well as families and orders within and outside the

Lilianae that are economically important species and/or have

tubers/corms/expanded rhizomes.

(3) Discussion of Host Specificity Testing

See appendix 1 for a complete description of host specificity test design and

results.

The results of host range testing indicate that the beetle Lilioceris egena is

17

highly specialized on its target host, Dioscorea bulbifera (air potato).

Oviposition occurred only on this plant, and females tended to hold eggs while

on air potato foliage only to lay eggs as soon as being placed upon bulbils.

Thus, the ovipositional specificity occurs at the organ level within a single

species, and not just the species as a whole. Neonates failed to develop on any

plant species aside from air potato and developed better on air potato

bulbils/tubers than on leaves. Further, in a preliminary choice test in which

neonates were placed in arenas with leaves and bulbil slices, the larvae always

moved onto the bulbils, even if placed directly on the leaves first. The reverse,

larvae abandoning bulbils for leaves, never occurred. Finally, the data from

2nd/3rd instar larval trials suggest that late instar larvae from air potato leaves

or bulbils may occasionally migrate to, and complete development on, a few

Dioscorea congeners (D. alata, D. cordata, D. trifida) in areas of Florida and

the Caribbean where these congeners are intermixed and the larvae cannot

locate their preferred host. The exteme rarity of this occurance in the no choice

trials (3 of 456 2nd/3rd instar larvae on Dioscoreaceae; less than 1 percent),

which forced the larvae to stay on the non-target host, indicates that the

likelihood of this occurring in nature is very low. However, even should this

occur, the failure of adults to oviposit and neonates to develop on non-target

plants assures that persistent populations could not develop on these non-targets.

Release of L. egena is expected to directly impact air potato reproduction.

Successful establishment of L. egena on air potato will complement effects

already being realized by the release in 2011 of L. cheni (Center et al., 2013).

Where L. cheni is already slowing growth and reducing air potato’s dominance

in invaded plant communities, production of vegetative propagules (i.e., bulbils)

continues though at a reduced level (Overholt et al., 2016). Adult L. egena will

contribute to foliar damage. More importantly, however, L. egena’s strong

preference to lay eggs on air potato bulbils should lead to large numbers of

bulbils being damaged by this beetle, thereby reducing their ability to sprout

(Pemberton and Witkus, 2010). Vegetative propagation is the means by which

this vine expands its geographic range, so damage to bulbils will likely restrict

further spread of this invasive weed in Florida and the southern United States.

Reduction of air potato by L. egena could be beneficial to animals because it

would reduce the potential of air potato to dominate animal habitats. Air potato

negatively impacts wildlife dependent on native vegetation for forage, nesting,

and cover.

Direct impact of the beetle will be restricted to the target weed, air potato. Adult

feeding on native U.S., Caribbean, and Mesoamerican Dioscorea species found

in close proximity to air potato is possible should heavy beetle infestations

develop. Such damage would be short lived and only cosmetic; however, L.

egena’s close connection to its host precludes oviposition and neonate

development upon non-hosts, as demonstrated in host range testing.

Because air potato is uncommon in agricultural areas, except occasionally along

3. Animals

.4. Native Plants

2. Impact of L.

egena on Air

Potato

18

fence lines, the primary benefits derived from release of L. egena will occur in

natural areas. The beneficial indirect impact on native plants, will be substantial

if L. egena results in reduction in air potato vine densities. Trellising of vines

that smother trees and shrubs will be reduced, thereby fostering a more diverse

canopy and mid-story plants. Population reductions of air potato will also, in

conjunction with defoliation by L. cheni, promote light penetration to the forest

floor thereby stimulating understory plant growth.

Some varieties of air potato are cultivated for consumption in Asia and Africa,

but this yam is not an important food crop anywhere in the New World (Asiedu

and Sartie, 2010; FAO, 2017). Similarly, although dried bulbils/tubers are used

in traditional medicines in Asia and Africa, air potato is not known to be used

similarly in the United States or Caribbean. Many varieties are considerably

bitter and can cause vomiting and diarrhea (Kawasaki et al., 1968; Martin,

1974; Telek et al., 1974; Webster et al., 1984; Bhandari and Kawabata, 2005),

so control of the plant will reduce potential for human illness arising from

consumption. Thus, control of this weed will not negatively affect human

health, and may in fact have some small positive human health benefit.

No human health effects are known to be associated with L. egena or any other

Lilioceris species.

Lilioceris egena would reduce the quantity of bulbils for children to play with,

collect, and throw.

Once a biological control agent such as L. egena is released into the

environment and becomes established, there is a slight possibility that it could

move from the target plant (air potato) to attack nontarget plants. Host shifts by

introduced weed biological control agents to unrelated plants are rare

(Pemberton, 2000). Native species that are closely related to the target species

are the most likely to be attacked (Louda et al., 2003). If other plant species

were to be attacked by L. egena, the resulting effects could be environmental

impacts that may not be easily reversed. Biological control agents such as L.

egena generally spread without intervention by man. In principle, therefore,

release of this biological control agent at even one site must be considered

equivalent to release over the entire area in which potential hosts occur, and in

which the climate is suitable for reproduction and survival. However, significant

non-target impacts on plant populations from previous releases of weed

biological control agents are unusual (Suckling and Sforza, 2014).

In addition, this agent may not be successful in reducing air potato populations

in the contiguous United States. Worldwide, biological weed control programs

have had an overall success rate of 33 percent; success rates have been

considerably higher for programs in individual countries (Culliney, 2005).

Actual impacts on air potato by L. egena will not be known until after release

occurs and post-release monitoring has been conducted (see Appendix 2 for

release protocol and post-release monitoring plan). It is expected that L. egena

6. Beneficial Uses

.5. Human Health

7. Uncertainties

R

egarding the

Environ-

mental

Release of L.

egena

19

will damage bulbils, likely restricting further spread of air potato in Florida and

the southern United States.

“Cumulative impacts are defined as the impact on the environment which

results from the incremental impact of the action when added to other past,

present and reasonably foreseeable future actions regardless of what agencies or

person undertakes such other actions” (40 CFR 1508.7).

Other private and public concerns work to control air potato in invaded areas

using available chemical, mechanical, and biological control methods. Release

of L. egena is not expected to have any negative cumulative impacts in the

continental United States because of its host specificity to air potato. Effective

biological control of air potato will have beneficial effects for Federal, State,

local, and private weed management programs, and may result in a long-term,

non-damaging method to assist in the control of air potato.

Section 7 of the Endangered Species Act (ESA) and ESA’s implementing

regulations require Federal agencies to ensure that their actions are not likely to

jeopardize the continued existence of federally listed threatened and endangered

species or result in the destruction or adverse modification of critical habitat.

In the continental United States, there are no plants that are federally listed or

proposed for listing in the family Dioscoreaceae, the same family as the target

weed. However, in host range testing, minor feeding occurred on foliage or

storage organs of some plants in the families Poaceae, Amaranthaceae,

Apiaceae, Liliaceae, Convolvulaceae, Fabaceae, and Brassicaceae that contain

federally listed and candidate plant species. Most of these plant species would

not overlap with the projected distribution of air potato. Based on the host

specificity of L. egena reported in testing, field observations, and in the

scientific literature, APHIS has determined that environmental release of L.

egena may affect, but is not likely to adversely affect listed plants in the

families Poaceae, Amaranthaceae, Apiaceae, Liliaceae, Convolvulaceae,

Fabaceae, and Brassicaceae. APHIS has also determined that the release of L.

egena may affect beneficially the Stock Island tree snail, Orthalicus reses.

A biological assessment was prepared and submitted to the U.S. Fish and

Wildlife Service (FWS) and is part of the administrative record for this EA

(prepared by T.A. Willard, May 31, 2018, and revised November 6, 2018).

APHIS requested concurrence from the FWS on these determinations, and

received a concurrence letter dated February 21, 2019.

V. Other Issues

Consistent with Executive Order (EO) 12898, “Federal Actions to Address

Environmental Justice in Minority Populations and Low-income Populations,”

APHIS considered the potential for disproportionately high and adverse human

health or environmental effects on any minority populations and low-income

populations. There are no adverse environmental or human health effects from

9

. Endangered

Species Act

8. Cumulative

Impacts

20

the field release of L. egena and will not have disproportionate adverse effects

to any minority or low-income populations.

Consistent with EO 13045, “Protection of Children from Environmental Health

Risks and Safety Risks,” APHIS considered the potential for disproportionately

high and adverse environmental health and safety risks to children. No

circumstances that would trigger the need for special environmental reviews are

involved in implementing the preferred alternative. Therefore, it is expected that

no disproportionate effects on children are anticipated as a consequence of the

field release of L. egena.

EO 13175, “Consultation and Coordination with Indian Tribal Governments,”

was issued to ensure that there would be “meaningful consultation and

collaboration with tribal officials in the development of Federal policies that

have tribal implications….”

APHIS is consulting and collaborating with Indian tribal officials to ensure that

they are well-informed and represented in policy and program decisions that

may impact their agricultural interests in accordance with EO 13175.

VI. Agencies, Organizations, and Individuals

Consulted

The Technical Advisory Group for the Biological Control Agents of Weeds

(TAG) recommended the release of L. egena on March 30, 2018. The TAG

members that reviewed the release petition (17-01) (Dray, 2017) included

USDA representatives from the National Institute of Food and Agriculture, and

Agricultural Research Service; U.S. Department of Interior’s U.S. Geological

Survey, Bureau of Land Management and U.S. Fish and Wildlife Service; U.S.

Army Corps of Engineers; and representatives from California Department of

Food and Agriculture (National Plant Board), Mexico Secretariat of Agriculture,

Livestock, Rural Development, and Fisheries, and Agriculture and Agri-Food

Canada.

This EA was prepared by personnel at APHIS and ARS. The addresses of

participating APHIS units, cooperators, and consultants follow.

U.S. Department of Agriculture

Animal and Plant Health Inspection Service

Policy and Program Development

Environmental and Risk Analysis Services

4700 River Road, Unit 149

Riverdale, MD 20737

U.S. Department of Agriculture

Animal and Plant Health Inspection Service

21

Plant Protection and Quarantine

Pests, Pathogens, and Biocontrol Permits

4700 River Road, Unit 133

Riverdale, MD 20737

U.S. Department of Agriculture

Agricultural Research Service

Invasive Plant Research Laboratory,

3225 College Ave.

Ft. Lauderdale, FL 33314

U.S. Fish and Wildlife Service

Branch of Environmental Review

5275 Leesburg Pike, MS:ES

Falls Church, VA 22041

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30

Appendix 1. U.S. host-specificity testing methods and results (Dray, 2017).

Experimental Design

All 82 species of plants were included in foliage feeding and oviposition trials, and 33 of these

were included in storage organ feeding and oviposition trials (see Table 1-1).

Source of plants

The D. bulbifera bulbils, or plants grown from bulbils, used in this study were collected either at

Tree Tops Park, Davie FL (26.070434°N, -80.270151°W) or at Easterlin Park, Oakland Park,

Florida (26.170197°N, -80.160279°W). Colonies of adult beetles were maintained on bulbils

collected at these same sites. Some test plants remaining from the L. cheni host range studies

(Pemberton and Witkus, 2010) and otherwise unobtainable (e.g., Dioscorea altissima) were

incorporated into this present project but may not have been included in all trial types due to

limited numbers (e.g., Ipomoea pandurata). Other plants were obtained from a variety of

commercial sources; some as live plants, others as seed. A few species (e.g., Smilax laurifolia)

were hand dug at field sites and repotted for cultivation. Foliage for trials of large trees was

collected from species that are growing live on the USDA campus (e.g., Ficus aurea) or at

nearby parks (e.g., Salix caroliniana). All field collected material was done so with permission

of the appropriate land owners and with appropriate state and local permits.

31

Table 1-1. Plant species included in the host range trials with Lilioceris egena.

Order

Family

1

Genus (Section

2

) species Authority

3

Common names

Distribution/Status/Comments

4

Category 1 - Genetic Type of the Target Weed Found in North America

Dioscoreales

Dioscoreaceae

* Dioscorea (Opsophyton) bulbifera L.

air potato

OWT; FL, GA, LA, Carib., M.Am.

Category 2 (a) - Species in the Same Genus as the Target Weed: North American (excluding Mexico)

Dioscoreales

Dioscoreaceae

* Dioscorea (Macropoda) floridana Bartlett

Florida yam

FL, GA, SC

Dioscoreaceae

* Dioscorea (Macropoda) villosa L.

fourleaf yam, wild yam

FL north to Canada, west to Texas; /hort. (OH,

Canada)

Category 2 (b) - Species in the Same Genus as the Target Weed: West Indian/Mesoamerican/South American

Dioscoreales

Dioscoreaceae

Dioscorea (Chondrocarpa) altissima Lam.

dunguey

Brazil; Puerto Rico

Dioscoreaceae

Dioscorea (Rajania) cordata (L.) Raz

himber

Puerto Rico, Cuba, Jamaica

Dioscoreaceae

* Dioscorea (Dematostemon) pilosiuscula Bertero ex

Spreng.

bulbous yam, air yam, dungeuy

Carib., M.Am., trop. S.Am.

Dioscoreaceae

* Dioscorea (Lynchnostemon) polygonoides Humb. and

Bonpl. ex Willd.

Jamaican bitter yam, mata gallina,

M.Am., trop. S.Am.

Dioscoreaceae

* Dioscorea (Macrogynodium) trifida L.f.

yampi, cush-cush, mapuey, inhame,

tabena, sacha papa

M.Am., trop. S.Am. /crop

Category 2 (c) - Species in the Same Genus as the Target Weed: Weedy and/or Exotic in U.S.

Dioscoreales

Dioscoreaceae

* Dioscorea (Enantiophyllum) alata L.

purple yam, water yam, white yam,

winged yam, name blanco

trop. Asia; SE US, Puerto Rico, Virgin Islands /crop

(not US)

Dioscoreaceae

* Dioscorea (Enantiophyllum) cayenensis Lam.

(combines subspp)

yellow yam, Lagos yam, name amarillo

west and central Africa; Carib., M.Am., S.Am. /crop

Dioscoreaceae

* Dioscorea (Combilium) esculenta (Lour.) Burkill

lesser yam, Asiatic yam, gan shu

trop. and subtrop. Asia; Carib. /crop

Dioscoreaceae

* Dioscorea (Enantiophyllum) oppositifolia L.

nagaimo, Chinese yam

southern India; (where this species is listed as in U.S.,

the plants are actually D. polystachya)

Dioscoreaceae

* Dioscorea (Enantiophyllum) polystachya Turcz.

Chinese yam, shan yao, cinnamon vine

temp. Asia; FL north to MA, west to AR (often listed

under its synonym, D. batatas); /hort. (as D. batatas)

Dioscoreaceae

* Dioscorea (Opsophyton) sansibarensis Pax

Zanzibar yam

trop. Africa; FL (but possibly eradicated)

Category 3 - Species in Other Genera in the Same Family (Dioscoreaceae) as the Target Weed

Dioscoreales

Dioscoreaceae

* Tacca chantrieri André

batflower, devil flower

trop. Asia; /FL hort.

Category 4 - Threatened and Endangered Species in the Same Family (Dioscoreaceae) as the Target Weed

Dioscoreales

Dioscoreaceae

There are no U.S. or Florida listed species in this family

Category 5 - Species in Other Families in the Same Order (Dioscoreales) with Similarities to the Target Weed

Dioscoreales

Burmanniaceae

Species in this family were unobtainable

32

Order

Family

1

Genus (Section

2

) species Authority

3

Common names

Distribution/Status/Comments

4

Nartheciaceae

Species in this family were unobtainable

Category 6 (a) - Species in Closely Related Orders to the Target Weed

Pandanales

Pandanaceae

Pandanus tectorius Park. ex Du Roi

variegated dwarf pandanus

Malesia, AU, Pacific Islands; /hort (often sold as P.

baptistii)

Category 6 (b) - Species in the Same SuperOrder (Lilianae) as the Target Weed

Alismatales

Alismataceae

Sagittaria latifolia Willd.

arrowhead, wapato

N.Am.

Araceae

Alocasia cucullata (Lour.) Schott

dwarf elephant ear

trop. Asia; /crop

Araceae

Caladium bicolor (Aiton) Vent.

angel wings, heart of Jesus, elephant ear

M.Am., S.Am.; trop. India, trop. Africa /hort.

Araceae

* Colocasia esculenta (L.) Schott

taro

Malaysia, AU, PNG; India, Egypt, Africa, Carib., M.Am.,

S.Am., SE US;

Araceae

Symplocarpus foetidus Salisb.

skunk or swamp cabbage

eastern US to Canada

Araceae

* Xanthosoma sagittifolium (L.) Schott

arrowleaf elephant ear, nampi, malanga

NWT; /crop

Araceae

Zantedeschia aethiopica (l.) Spreng.

calla lily, arum lily

southern Africa; AU

Arecales

Arecaceae

Sabal palmetto (Walt.) Lodd.

cabbage palm

SE US, Bahamas, Cuba; /hort.

Asperagales

Amaryllidaceae

Crinum americanum L.

bog lily, string lily

SE US, Mexico, Cuba, Jamaica; Puerto Rico

Amaryllidaceae

Zephyranthes minuta (Kunth) D. Dietr.

pink rain lily

M.Am.; Carib., SE US, HI; /hort. [Z. atamasco (L.)

Herb., Z. simpsonii Chapm., and Z. treatiae S.Watson

are all FL state threatened]

Commelinales

Commelinaceae

Tradescantia pallida (Rose) D.R. Hunt

wandering jew, purple heart

Mexico; /hort.

Pontedariaceae

Pontederia cordata L.

pickerelweed

SE US

Poales

Cyperaceae

Cladium mariscus subsp. jamaicense (Crantz) Kük.

sawgrass

M.Am., S.Am. SE US, Carib.; trop. Africa, New Guinea,

HI

Juncaceae

Juncus effusus L.

soft rush, corkscrew rush

EU, Asia, Africa, N.Am., S.Am.; AU, Madagascar,

Pacific Is.

Musaceae

Musa acuminata Colla

wild banana

SE Asia; /crop

Poaceae

Saccharum officinarum L.

sugar

SE Asia; /crop

Poaceae

Zea mays L.

corn

Mexico; /crop

Zingiberales

Cannaceae

Canna glauca L.

canna lily

Carib., M.Am., S.Am., TX, LA; OWT

Cannaceae

Canna cultivar americanallisvar. variegata

variegated canna lily, Bengal tiger lily

uncertain, possibly India; worldwide trop. and

subtrop. /hort.

Costaceae

Costus woodsonii Maas

red button ginger, lipstick plant

M.Am; /hort.

Heliconiaceae

Heliconia bihai (L.) L.

lobster claw, macawflower

northern S.Am., Carib.; /hort.

Marantaceae

* Maranta arundinacea L.

arrowroot, maranta, araru

M.Am., S.Am., Carib.; FL, OWT /crop

Marantaceae

Thalia geniculata L.

arrowroot, fireflag, alligatorflag

M.Am, S.Am., SE US, Carib., Africa

Zingiberaceae

* Curcuma longa L.

turmeric

trop. and subtrop. Asia; /crop

33

Order

Family

1

Genus (Section

2

) species Authority

3

Common names

Distribution/Status/Comments

4

Zingiberaceae

* Hedychium coronarium J. Koenig

white ginger lily

trop. Asia; Carib., S.Am., SE US, HI /hort.

Zingiberaceae

* Zingiber officinale Roscoe

ginger

uncertain, possibly India; worldwide /crop

Category 6 (c) - Species outside the Superorder (Lilianae) containing the Target Weed

Apiales

Apiaceae

Apium graveolens L.

celery

uncertain; worldwide /crop

Apiaceae

* Daucus carota L.

carrot

EU, SW Asia; worldwide /crop

Araliaceae

* Panax ginseng C.A. Mey.

Asian ginseng

temp. Asia; /crop

Asterales

Asteraceae

* Arctium lappa L.

burdock, gobo, lappa

temp. EU, temp. Asia; worldwide /crop

Brassicales

Brassicaceae

* Brassica rapa L.

turnip

temp. Asia; worldwide /crop

Brassicaceae

* Raphanus sativus L.

radish

possibly SE Asia; worldwide /crop

Caryophyllales

Amaranthaceae

* Beta vulgaris L.

beet

southern coastal EU, N Africa, W Asia; worldwide

/crop

Fabales

Fabaceae

Glycine max (L.) Merr.

soybean

E Asia, AU; worldwide /crop

Fabaceae

Mimosa pudica L.

sensitive plant, touch-me-not

M.Am., S.Am., Carib.; pantropical

Fabaceae

* Pachyrhizus erosus (L.) Urb.

jicama, Mexican yam, Mexican turnip

Mexico; trop. Asia /crop

Gentianales

Rubiaceae

Guettarda scabra (L.) Vent.

rough velvetseed, wild guave

Carib., FL, N S.Am.;

Laurales

Calycanthaceae

Calycanthus floridus L.

Carolina allspice, sweet shrub

E US [FL state endangered]; China

Magnoliales

Annonaceae

Annona glabra L.

pond apple

FL, Carib., M.Am., S.Am.; AU, Sri Lanka

Malphigiales

Chrysobalanaceae

Chrysobalanus icaco L.

cocoplum

NWT, FL, trop. Africa; /hort.

Euphorbiaceae

* Manihot esculenta Crantz

cassava, yuca, manioc, tapioca-root

Brazil; OWT /crop

Piperales

Aristolochiaceae

Aristolochia tomentosa Sims

Dutchman's pipe vine

FL [state endangered] north to MA, west to MO;

/hort.

Solanales

Convolvulaceae

* Ipomoea batatas (L.) Lam.

sweet potato

NWT; worldwide trop. and warm temp. /crop /hort.

[I. microdactyla Griseb. and I. tenuissima Choisy are

both FL state endangered]

Convolvulaceae

Ipomoea pandurata (L.) G.F.W.Mey.

man-of-the-earth, wild sweet potato

SE US;

Category 7 (a) - Species on Which the Proposed Agent Has Been Recorded

Dioscoreales

Dioscoreaceae

Dioscorea subclava Prain and Burkill is the only species aside from the the target from which L.

egena has been reported

China; was unobtainable for the study

Category 7 (b) - Species (or surrogates) on Which Congeners of the Proposed Agent Have Been Recorded

7

within Lilianae

Asperagales

Amaryllidaceae

* Allium cepa L.

onion

uncertain, probably Asia; /crop

Amaryllidaceae

* Allium sativum L.

garlic

uncertain, possibly central Asia; /crop

Asparagaceae

Asparagus densiflorus (Kunth) Jessop

Sprenger's asparagus fern

S. Africa; AU, Carib., FL, CA, HI

Asparagaceae

Asparagus officinalis L.

asparagus

N. Africa, EU, Asia; worldwide /crop

34

Order

Family

1

Genus (Section

2

) species Authority

3

Common names

Distribution/Status/Comments

4

Asphodelaceae

Aloe vera (L.) Burm.f.

aloe

unknown, possibly N Africa; worldwide /hort

Iridaceae

Sisyrinchium angustifolium Mill.

narrow-leaf blue-eyed grass

FL to Canada west to TX and MN

Orchidaceae

Bletilla striata (Thunb,) Rchb.f.

hyacinth orchid

Japan, Korea, China, Myanmar; FL /hort.

Dioscoreales

Dioscoreaceae

* Dioscorea alata L.

purple yam, water yam, white yam,

winged yam, name blanco

trop. Asia; SE US, Puerto Rico, Virgin Islands /crop

(not US)

Liliales

Liliaceae

* Lilium michauxii Poir

wild lily

FL north to VA, west to TX [FL state endangered, as

are L. iridollae M.K.Henry and L. superbum L.; L.

catesbaei Walter is FL state threatened]

Liliaceae

* Tricyrtis lasiocarpa Matsum.

toad lily

Taiwan; /hort.

Smilacaceae

Smilax laurifolia L.

greenbrier

FL north to NJ and west to Arkansas, Cuba, Bahamas

Pandanales

Pandanaceae

Pandanus tectorius Park. ex Du Roi

variegated dwarf pandanus

Malesia, AU, Pacific Islands; /hort (often sold as P.

baptistii)

Poales

Poaceae

Triticum aestivum L.

wheat

worldwide

non-Lilianae

Apiales

Araliaceae

Schefflera actinophylla (Endl.) Harms

umbrella tree, octopus tree

AU, New Guinea, Java; FL, HI, Carib. /hort.

Asterales

Campanulaceae

Lobelia cardinalis L.

cardinal flower

N.Am. [FL state threatened], M.Am., Columbia; /hort.

[L. boykinii Torr. and A.Gray ex A.DC.is FL state

endangered]

Caryophyllales

Polygonaceae

Persicaria glabra (Willd.) M. Gomez

swamp smartweed

N.Am., elsewhere uncertain

Fabales

Fabaceae

Senna (Cassia) ligustrina (L.) H.S. Irwin and Barneby

privet cassia

OWT; NWT [S. mexicana (Jacq.) H.S.Irwin and

Barneby var. chapmanii is FL state threatened]

Fagales

Betulaceae

Corylus americana Marshall

American hazelnut

eastern and central N.Am.

Fagaceae

Quercus virginiana Miller

southern live oak

SE US; [Q. arkansana Sarg. is FL state threatened]

Gentianales

Apocynaceae

Asclepias tuberosa L.

butterfly milkweed

N.Am.

Lamiales

Verbenaceae

Callicarpa americana L.

American beautyberry

Carib., SE US

Malpighiales

Salicaceae

Salix caroliniana Michx.

coastal plain willow

Carib., SE US, M.Am. [S. eriocephala Michx. and S.

floridana Chapm. are both FL state endangered]

Rosales

Moraceae

Ficus aurea Nutt.

strangler fig

FL, Carib., M.Am.

Solanales

Solanaceae

* Solanum tuberosum L.

Irish potato

Peru; worldwide /crop

out of Magnoliidae (Angiosperms)

Cycadales

Cycadaceae

Cycas revoluta Thunb.

Sago palm

Japan; worldwide /hort.

1

Species preceded by an * were incorporated in storage organ trials as well as foliage trials.

2

Raz (2016) was followed in using the Dioscorea sections erected by Kunth (1924) despite some deviations in the latter from currently accepted phylogenies.

3

Florida native species in bold.

4

Distribution of species are indicated as follows: OWT=Old World tropics/subtropics, NWT=New World tropics/subtropics, N.Am.=North America, M.Am.=Mesoamerica, S.Am.=South America,

Carib.=Caribbean, EU=Europe, AU=Australia, Africa, Asia. U.S. state designations follow the standard two digit postal code (e.g., Florida=FL). Regions where a species is native are in regular Calibri font,

whereas areas of naturalization/invasion are in Cambria italics. Horticultural species denoted as /hort. Agricultural species denoted as /crop.

35

Number of replicates

Each trial was usually replicated a minimum of five times, generally using five individual plants.

In some cases, fewer than five individuals were available. In such cases multiple leaves were

selected from one individual at random. Similarly, storage organs were divided into sections

when necessary to achieve the desired five replicates. Dioscorea altissma represents an example

of a few cases wherein the only specimens were left over from earlier L. cheni host specificity

research, but developed a disease that prevented completion of all L. egena trials and

replacement of the specimens was not possible. Data for such species are presented herein, but

will appear incomplete.

Trial methodology

a. Multi-choice (choice minus control) adult storage organ feeding trials

Preliminary adult feeding trials were conducted on the storage organs of 25 species in 13

families (including Dioscoreaceae) and 10 orders (including Dioscoreales), using a multiple

choice minus control (D. bulbifera) scenario (Table 1-1). These species were selected primarily

to represent commercially available crop species that might be encountered by L. egena adults in

Florida or the Caribbean. Each trial was composed of 9–10 plastic petri dishes (25 cm diameter x

10 cm deep), with each dish containing storage organs from three or four test plant species. A

positive control of a separate container containing a D. bulbifera bulbil was run together with

each trial. The storage organs were placed on moist filter paper and the dishes were sealed with

Parafilm® to inhibit desiccation of the test materials. Storage organs that were too large for the

containers were sectioned, with the cut ends sealed with Parafilm

®

. This assured that all beetles

had to penetrate the storage organs’ epidermal layers to feed, as they would in nature. Five

beetles of uniform age were placed in each test arena and monitored for seven days, after which

the storage organs were examined for the presence of eggs and scored for feeding damage on a

qualitative scale of 0 (no feeding), 1 (a few scrapes), 2 (many scrapes and/or a few notches), 3

(many notches and/or holes), and 4 (burrowing inside bulb/tuber/etc.). These tests were

replicated a minimum of five times using fresh storage organs each time. Data were analyzed

using non-parametric multiple comparison tests available in SigmaPlot

©

12.3 (Systat 2011).

b. No choice adult storage organ feeding/ovipositional trials

Adult no choice storage organ trials were conducted on 33 plant species in 15 families and 11

orders (see Table 1-2). Storage organs were placed on moist filter paper in test arenas composed

of either a plastic soup (11 cm diameter, 7 cm deep) or food storage (16 x 11 x 7 cm) container

ventilated by cutting a rectangular hole in the lid which was then covered with 290 μ mesh

screening. Three beetles (two females, one male) were placed in each test arena and monitored

30 days or until there were no live beetles in any test arena containing a non-target host,

whichever was longer. Storage organs were replaced as needed (if they began to desiccate or

decay). These storage organs were then scored for presence/absence of eggs and assessed for the

amount of tissue consumed by all of the beetles in that trial. The latter was accomplished by

36

developing a novel volumetric technique (Dray, in prep.) that required injecting water from a 0.3

mL syringe into the feeding scars/tunnels and then tallying the total across scars, tunnels, and

storage organs.

Table 1-2. Species included in preliminary multi-choice adult feeding trials. Most (though not

all, e.g. I. pandurata, T. chantrieri) are crop plants cultivated for their storage organs.

Order

Family

Genus (Section) species Authority

1

Alismatales

Araceae

Colocasia esculenta (L.) Schott

Apiales

Apiaceae

Apium graveolens L.

Apiaceae

Daucus carota L.

Asperagales

Amaryllidaceae

Allium cepa L.

Amaryllidaceae

Allium sativum L.

Asterales

Asteraceae

Arctium lappa L.

Brassicales

Brassicaceae

Brassica rapa L.

Brassicaceae

Raphanus sativus L.

Caryophyllales

Amaranthaceae

Beta vulgaris L.

Fabaceae

Pachyrhizus erosus (L.) Urb.

Dioscoreales

Dioscoreaceae

Dioscorea (Opsophyton) bulbifera L.

Dioscoreaceae

Dioscorea (Macropoda) floridana Bartlett

Dioscoreaceae

Dioscorea (Macropoda) villosa L.

Dioscoreaceae

Dioscorea (Enantiophyllum) alata L.

Dioscoreaceae

Dioscorea (Enantiophyllum) cayenensis Lam.

(combines subspp cayenensis and rotundata)

Dioscoreaceae

Dioscorea (Enantiophyllum) oppositifolia L.

Dioscoreaceae

Dioscorea (Opsophyton) sansibarensis Pax

Dioscoreaceae

Tacca chantrieri André

Malphigiales

Euphorbiaceae

Manihot esculenta Crantz

Solanales

Convolvulaceae

Ipomoea batatas (L.) Lam.

Convolvulaceae

Ipomoea pandurata (L.) G.F.W.Mey.

Solanaceae

Solanum tuberosum L.

Zingiberales

Marantaceae

Maranta arundinacea L.

Zingiberaceae

Curcuma longa L.

Zingiberaceae

Zingiber officinale Roscoe

No choice adult foliage feeding/oviposition trials

Adult no choice foliage trials were conducted on 82 plant species in 46 families and 25 orders

(Table 1-1). Trials were conducted in test arenas (Figure 1-1a,b) composed of plexiglass sleeves

(8 cm diameter x 15 cm long) ventilated with four 5 cm holes drilled in the sides and covered

with 290 μ mesh insect screening. The sleeves were fitted over leaves of live plants, with a soft

foam bung inserted into the bottom of the sleeve and fitted around the plant stem. Another bung

sealed the top after the insects were inserted into the sleeve (Figure 1-1b). Test arenas were held

37

in place on bamboo stakes using rubber bands. Three beetles (two females, one male) were

placed in each test arena and monitored 30 days or until there were no live beetles in any test

arena containing a non-target host, whichever was longer.

Figure 1-1. Plexiglass sleeve cages used for adult L. egena foliage feeding/oviposition trials; (a)

test array, (b) individual test arena. [Photos: F.A. Dray Jr.]

Test arenas were moved onto fresh leaf material as needed. All leaves presented to the beetles

were scored for presence/absence of eggs. Damaged leaves were subsequently pressed and dried,

then scanned on a flatbed scanner. The resultant image was imported into the ImageJ software

package (version 1.46r; Schneider et al., 2012) where the image was converted to 8-bit format.

The paintbrush tool was used to draw lines filling in any gaps in the leaf perimeter. The image

was then converted into binary (black and white) and the Analyze Particles function was applied

twice. In the first analysis, the “include holes” option was selected, thereby providing a

measurement of the entire area of the leaf prior to insect feeding. In the second analysis, the

“include holes” option was deselected so that feeding damage was excluded from the overall

area. By subtracting the second quantity from the first, a measure of the leaf material (mm

2

)

consumed by all of the beetles in a given trial could be obtained.

No choice neonate storage organ feeding/developmental trials

Neonate storage organ feeding/development trials were conducted on the same 33 plant species

(see Table 1-2) as the adult storage organ trials. Naïve neonates were obtained by collecting L.

egena eggs from the colonies and placing them on moist filter paper in small (9 cm diameter)

petri dishes. The dishes were sealed with Parafilm

®

(to prevent desiccation and larval escape)

and monitored for eclosion. Neonate mandibles are unable to penetrate the skin (epidermal +

38

peridermal layers) of D. bulbifera bulbils, so these larvae accessed the flesh (medulla) via adult

feeding scars or breaks in the skin caused by eruption of the root radicle or cotyledon. Thus, to

maximize the possibility that the neonates might feed on a proffered test plant, a portion of the

skin from each storage organ was excised prior to their being placed on moist filter paper in the

test arenas. These latter were composed of either a small (118 mL) Gladware

®

mini-round

storage bowl ventilated with holes punched in the lid (Figure 1-2), a small plastic soup container

(11 cm diameter x 7 cm high) or a plastic storage container (11 x 7 x 16 cm). To conduct the

trials, freshly eclosed (<24 hour) neonates were transferred (using a fine 00 gauge paint brush)

onto the bare patches of the storage organs and each trial was examined daily for larval

mortality. Trials continued for 35 days, or until all larvae were dead and the positive controls (D.

bulbifera) had produced adult beetles (whichever was longer). At the end of each trial, all

puparia were dissected to check for the possibility that an adult beetle had formed but failed to

emerge (pharate adult). As in the other storage organ trials, L. egena feeding was evaluated using

the volumetric method previously described in the adult no choice storage organ trial section.

Figure 1-2. Example of an L. egena no choice neonate larval storage organ

feeding/developmental trial.

No choice neonate foliage feeding/developmental trials

Neonate foliage feeding/development trials were conducted on the same 82 plant species (see

Table 1-1) as the adult foliage trials. Naïve neonates were obtained for these trials in a similar

manner as above. Freshly eclosed (<24 h) neonates were then transferred as before onto leaf

material from the test plants. Generally, whole leaves were placed in the test arenas, but in rare

cases it was only possible to use a section of larger leaves. The leaves were placed on moist filter

paper in medium (15 cm diameter) petri dishes sealed with Parafilm® to prevent desiccation and

39

larval escape. Larval mortality was monitored daily, and the filter paper was moistened and

leaves replaced as needed. Trials continued for 35 days, or until all larvae were dead and the

positive controls (D. bulbifera) had produced adult beetles (whichever was longer). At the end of

each trial, all puparia were dissected to check for pharate adults. As in other foliage trials,

feeding damage was assessed quantitatively using the leaf scan method previously described in

the adult foliage trial section.

No choice 2

nd

/3

rd

instar larval storage organ feeding/development trials

These larval storage organ feeding/development trials were conducted on the same 33 plant

species (see Table 1-2) as the adult storage organ trials. Larvae for these trials were obtained in

two different ways. Initially, ages of test larvae were estimated by size, but for most studies,

neonates were instead collected within 24 hours of eclosion and placed on thin (~1 cm) slices of

D. bulbifera bulbil (5–10 neonates per slice) on moist filter paper in clear plastic salad containers

(19 x 19 x 7 cm). The neonates were allowed to feed and develop for 4–5 days until they were

2

nd

or early 3

rd

instars.

Unlike the insects used in the previously described trials, these larvae were necessarily not naïve.

Also, unlike the neonates, these larger larvae had mandibles capable of penetrating the bulbil’s

periderm (skin) and thus were transferred onto whole storage organs or sections of storage

organs the ends of which were sealed by thermoplastic adhesive from a hot glue gun

(preliminary tests showed no larval mortality resulted from the presence of the adhesive once it

had cooled). This forced the larvae to access the flesh of the storage organs only after penetrating

the periderm, to mimic conditions that a wandering larva might encounter in nature. Trials

continued for 35 days or until all larvae were dead and the positive controls (D. bulbifera) had

produced adult beetles (whichever was longer). At the end of each trial, all puparia were

dissected to check for pharate adults. As in the other storage organ trials, L. egena feeding was

evaluated quantitatively using the volumetric method developed.

No choice 2

nd

/3

rd

instar larval foliage feeding/development trials

These 2

nd

/3

rd

instar foliage feeding/development trials were conducted on the same 82 plant

species (see Table 1-1) as the adult foliage trials. Larvae for these trials were obtained by

collecting neonates as described above, placing them on thin (~ 1 cm) slices of D. bulbifera

bulbil in clear plastic salad containers (19 x 19 x 7 cm), and allowing them to feed and develop

for 4–5 days to the 2

nd

or early 3

rd

instar. As in the 2

nd

/3

rd

instar storage organ trials, these larvae

were necessarily not naïve. The larvae were then transferred onto leaf material from the test

plants. Generally, whole leaves were placed in the test arenas, but in rare cases it was only

possible to use a section of a leaf. The leaves were placed on moist filter paper in medium-sized

(15 cm diameter) petri dishes sealed with Parafilm® to prevent desiccation and larval escape

(Figure 1-3). Larval mortality was monitored daily, and the filter paper was moistened and leaves

replaced as needed. Trials continued for 35 days, or until all larvae were dead and the positive

controls (D. bulbifera) had produced adult beetles (whichever was longer). At the end of each

trial, all puparia were dissected to check for pharate adults. As in other foliage trials, feeding

40

damage was assessed quantitatively using the leaf scan method described previously.

Figure 1-3. Example of an L. egena no choice 2nd/3rd instar larval foliage

feeding/developmental trial. Note the extensive feeding on D. bulbifera.

Two choice adult oviposition and development tests – adults on whole plants

During the adult foliage trials, feeding on the Caribbean endemic D. (Rajania) cordata exceeded

10 percent of the leaf material presented to the beetles, the only plant besides D. bulbifera for

which this was true. Despite failure of adults to live beyond 25 days on D. cordata, and the

absence of oviposition on this plant, a side-by-side whole plant choice trial was conducted using

these two species. To do so potted 1 m tall vines of each species were placed into three large (1 x

1 x 2 m) pop-up cages. Additionally, a pot with bulbils lying on the soil was placed into each

cage. Each cage was inoculated with 5 pairs (one female, one male) of L. egena, and the trial ran

for 7 days. The trial was scored for proportion of leaves of each species suffering feeding

damage, the location of oviposition, and the locations of the adults at the conclusion of the trial.

Ovipositional preference trials

During preliminary foliage feeding trials, females only rarely produced or deposited eggs on D.

bulbifera foliage. This prevented having a positive control for the ovipositional portion of the

adult foliage trials. Thus, female discrimination of ovipositional substrates was assessed.

41

In the first test, females were removed from D. bulbifera leaves at the ends of a subset of the

foliage feeding trials and moved them onto D. bulbifera bulbils. Any eggs produced were

subsequently evaluated for viability by monitoring for eclosion of neonate larvae.

In a second experiment, female performance (feeding and oviposition) was compared on tubers

versus bulbils, and whether the females would burrow through a substrate (soil or vermiculite) to

locate, feed upon, and oviposit on tubers was also assessed. This experiment was conducted in

three separate trials with slightly different conditions. The first trial used naïve adults (two

females + one male) with a vermiculite substrate, the second trial used naïve adults with a

heavier gardening soil as a substrate, and the third used fecund females (already ovipositing)

with gardening soil as a substrate. Gardening soil was used in the latter two trials to better mimic

conditions that the beetles would encounter in the field. Each trial included the following

treatments: (a) a bulbil placed on the substrate surface as a positive control, (b) a tuber placed on

the substrate surface, (c) a tuber buried 5 cm below the substrate surface, (d) a tuber buried at 10

cm below the surface, (e) a sprouting tuber buried at 10 cm below the soil surface, but with its

stem emerging above the surface, and (f) a tuber buried at 25 cm below the surface. These depths

were selected because tubers in the field are typically recovered 10–25 cm below the surface.

Further, observations in China (FAD) suggest that these beetles and their congener, L. cheni,

seek out very young sprouting vines during early spring and so L. egena may use this to locate

underground food resources.

Positive Controls

D. bulbifera was included as a positive control in all host range trials.

Rationale for Study Design and Execution

The Asian beetle L. egena was first collected in China during May 2011 on an expedition that

was focused on collecting the air potato leaf beetle L. cheni (Center et al., 2013). Efforts to

maintain these insects during the expedition, and subsequently in the quarantine laboratory,

revealed that these two beetles differed in their feeding predilections – whereas L. cheni was

primarily a leaf feeder, adult L. egena fed both on foliage and on bulbils (Prain and Burkill,

1936; Martin, 1974). The latter are the primary means by which this invasive vine propagates in

the United States. Thus, the decision was made to test L. egena both on foliage and on

representative plant storage organs – especially those cultivated as food crops.

Unlike its congener L. cheni, which oviposits on leaves but also indiscriminately (Pemberton and

Witkus, 2010), female L. egena prefer to deposit eggs in or on D. bulbifera bulbils and only

occasionally oviposit on the undersides of the containers holding these bulbils. Thus,

ovipositional trials were incorporated in adult feeding trials for this beetle. Preliminary

reproductive studies showed a pre–ovipositional period of 7–10 days, so adult feeding trials with

beetles younger than 7 days old were initiated – thereby assuring that potential egg production

would be influenced by the proffered host plant. Further, to avoid possible effects of host plant

imprinting, these adults were collected from pupation cages from which all food materials were

42

removed prior to adult emergence, thereby assuring naïve adults were used. In a preliminary

trial, freshly emerged naïve adults died after 22 days with no food or water. Therefore, adult

trials were conducted for a minimum of 30 days. Males and females were differentiated based on

gross morphological features (e.g., size and shape of abdomen), but this was an inexact science

(~70 percent accuracy). The error was usually misidentifying a large male as female, so two

putative females and one putative male were placed into each test arena to increase the likelihood

of getting at least one complete mating pair.

Neonate and later instar larvae often abandon unsuitable host materials to search for more

acceptable food. Thus, the potential exists for these stages to encounter and potentially feed upon

novel food items. Therefore, in addition to the adult feeding trials, host trials both with neonates

and with late 2

nd

/early 3

rd

instar larvae were conducted. Each was tested on foliage and on

storage organs for the same reasons cited above for adult feeding trials.

Given that two distinct biogeographical populations were under colonization in quarantine, both

biotypes were incorporated into the host range trials. The Chinese insects were tested against the

entire suite of 82 plant species (Table 1-1). However, to avoid unnecessary duplication of effort

(and associated time delays), the Nepalese insects were only tested against those species

considered potentially most susceptible (the Dioscoreaceae). Substantive differences between the

two biotypes in terms of responses to the Dioscoreaceae would have triggered an expanded list

of host plants offered to the Nepalese biotype. This followed the protocols employed when

releasing Chinese L. cheni after host trials had been conducted exclusively on Nepalese insects

(see Center et al., 2013). Fortunately, the two L. egena biotypes performed similarly on the

Dioscoreaceae, so further tests outside of this family with the Nepalese biotype were

unnecessary. The Chinese and Nepalese data are reported separately within each of the tables,

otherwise discussions and graphs are generally inclusive of the combined datasets.

Host Range Testing Results

Multi-choice (choice minus control) adult storage organ feeding trials

These multi-choice trials provided preliminary evidence that L. egena is host specific, given that

the beetles ignored these initial test plant (predominantly crop) species (Table 1-3). The beetles

produced minor surface “scrapes” on three species of Dioscorea (Table 1-3) compared with

extensive tunneling inside D. bulbifera (both bulbils and tubers). Also, females oviposited only

on D. bulbifera (Table 1-3).

An abundance of zero feeding scores caused us to collapse the data into the following groups for

statistical analysis: (1) D. bulbifera, (2) native eastern U.S. Dioscoreaceae (D. floridana + D.

villosa), (3) Dioscoreaceae crops (D. cayenensis/rotundata + D. alata), (4) other Dioscoreaceae

(D. oppositifolia + D. sansibarensis, + T. chantrieri), and (5) non-Dioscoreaceae (all other

species in Table 1-4). These data still demonstrated a non-normal distribution and had unequal

variances, so they were analyzed using a Kruskal-Wallis One-way ANOVA on Ranks, with

Dunn’s Multiple Comparison test applied post-hoc. Results showed that the groups had highly

43

divergent feeding scores (H=70.25, P < 0.001) - with D. bulbifera differing from each of the

other 4 groups, but none of the rest differing among each other (Table 1-4).

Table 1-3. Summary results from multi-choice (choice minus control) adult storage organ

feeding trials. The target weed (D. bulbifera) is highlighted in tan. See Table 1-2 for additional

information (e.g., common names) on these species.

Order

Family

Genus (Section) species Authority

Replicates

Avg.

feeding

score

1

Oviposition

Alismatales

Araceae

Colocasia esculenta (L.) Schott

5

0

no

Apiales

Apiaceae

Apium graveolens L.

4

0

no

Apiaceae

Daucus carota L.

6

0

no

Asperagales

Amaryllidaceae

Allium cepa L.

4

0

no

Amaryllidaceae

Allium sativum L.

4

0

no

Asterales

Asteraceae

Arctium lappa L.

4

0

no

Brassicales

Brassicaceae

Brassica rapa L.

4

0

no

Brassicaceae

Raphanus sativus L. ("cherry belle" and "daikon")

8

0.3

no

Caryophyllales

Amaranthaceae

Beta vulgaris L.

4

0

no

Fabaceae

Pachyrhizus erosus (L.) Urb.

5

0

no

Dioscoreales

Dioscoreaceae

Dioscorea (Opsophyton) bulbifera L.

10

3.5

yes

Dioscoreaceae

Dioscorea (Opsophyton) sansibarensis Pax

5

0

no

Dioscoreaceae

Dioscorea (Macropoda) floridana Bartlett

5

0.2

no

Dioscoreaceae

Dioscorea (Macropoda) villosa L.

5

0

no

Dioscoreaceae

Dioscorea (Enantiophyllum) alata L.

4

0

no

Dioscoreaceae

Dioscorea (Enantiophyllum) cayenensis Lam.

(combines subspp cayenensis and rotundata)

15

0.3

no

Dioscoreaceae

Dioscorea (Enantiophyllum) oppositifolia L.

4

0.3

no

Dioscoreaceae

Tacca chantrieri André

4

0

no

Malphigiales

Euphorbiaceae

Manihot esculenta Crantz

5

0

no

Solanales

Convolvulaceae

Ipomoea batatas (L.) Lam.

13

0

no

Convolvulaceae

Ipomoea pandurata (L.) G.F.W.Mey.

4

0

no

Solanaceae

Solanum tuberosum L.

5

0

no

Zingiberales

Marantaceae

Maranta arundinacea L.

4

0

no

Zingiberaceae

Curcuma longa L.

4

0

no

Zingiberaceae

Zingiber officinale Roscoe

4

0

no

1

Qualitative feeding scored as: 0 - no feeding, 1 - a few scars, 2 - many scars and/or a few notches, 3 - many notches and/or holes,

and 4 - burrowing inside bulb/tuber/etc.

44

Table 1-4. Comparison of the five groups of plant species (see text) subjected to multi-choice

(choice minus control) adult L. egena feeding trials. Numbers in the upper quadrat (peach boxes)

represent the Dunn’s Multiple Comparison Q values for the various comparisons, those in the

lower quadrat (green boxes) represent the associated significance values.

Groups

(1)

(2)

(3)

(4)

(5)

(1) D. bulbifera

2.970

3.621

3.956

4.769

(2) D. floridana + D. villosa

P<0.05

0.192

0.588

0.794

(3) D. cayenensis/rotundata + D. alata

P<0.05

NS

0.485

0.769

(4) other Dioscoreaceae

P<0.05

NS

0.128

(5) non-Dioscoreaceae

P<0.05

NS

No choice adult storage organ feeding/ovipositional trials

Naïve female L. egena produced eggs and oviposited only on storage organs of the target plant,

D. bulbifera (Table 1-5) during the storage organ feeding/oviposition trials. In reproductive trials

conducted separately from the feeding trials, Chinese females produced an average 8.4 ± 0.28

eggs/day, and a total of 931.0 ± 103.49 eggs/lifetime, on D. bulbifera bulbils.

Adult L. egena fed readily on D. bulbifera bulbils, with about 82 percent of Chinese and 92

percent of Nepalese biotype beetles surviving the 30-day trials. Overall, beetles fed non-host

storage organs lived only about a third as long as those fed D. bulbifera bulbils (Table 1-5;

Figure 1-4). However, a total of seven individuals (1 percent of the beetles placed on non-host

plants) from a total of four different trials survived longer on three Dioscorea congeners (Table

1-5. None of these plants are native to Florida, but one (D. trifida) is native to Mesoamerica

(Table 1-1). In each of these seven cases, the beetles on the D. bulbifera control out-lived the

beetles on the congeners (Table 1-5). Trials were always terminated a few days after death of the

last adult on non-host plants because adults on D. bulbifera can live 6 months or longer (one

adult from an unrelated experiment has lived 373 days), so it was not necessary to maintain the

adults from each trial until they died. The longest-lived non-control beetle (one on D.

polystachya) consumed substantially less storage organ tissue than did the controls on D.

bulbifera (Table 1-5), and none of these adults produced any eggs (Table 1-5).

Chinese and Nepalese beetles consumed the similar amounts of D. bulbifera bulbil tissue (2.845

± 0.3698 vs 1.737 ± 0.4081 mL; Mann-Whitney U = 113.5, P = 0.104). Adult beetles fed native

U.S. Dioscoreaceae (category 2a plants) and Dioscoreaceae native to the Caribbean and/or

Mesoamerica (category 2b plants) consumed less than 1 percent of the storage organ tissue as

those fed D. bulbifera bulbils (Table 1-5, Figure 1-5). Adults fed the remaining Dioscorea

species (category 2c) consumed more tissue than those on other non-target species, but this still

equaled only 6.2 percent of D. bulbifera bulbil tissue consumption in the controls (Table 1-5,

Figure 1-5). There was virtually no feeding on storage organs from plants outside the

Dioscoreaceae (Table 1-5, Figure 1-5).

45

Table 1-5. Results of the adult L. egena no choice bulbil feeding/oviposition trials. Uncolored

rows represent host trials utilizing Chinese beetles, whereas gray colored rows represent host

trials utilizing Nepalese beetles. The latter were only conducted on members of the order

Dioscoreales. Tan colored rows represent summaries by TAG Category. See Table 1-1 for

additional information regarding the test plant species.

TAG

Category

Order

Family

Species

1

Total

reps

Longevity (d)

(avg ± s.e.)

Females

produced

eggs

(Y/N)

Storage organ

tissue consumed

(mL)

(avg ± s.e.)

2

1

Dioscoreales

Dioscoreaceae

Dioscorea bulbifera

39

>30

±

--

Y

2.845

±

0.3698

9

>30

±

--

Y

1.737

±

0.4081

Category 1 summary

48

>30

±

--

Y

2.291

±

0.3148

2a

Dioscoreales

Dioscoreaceae

Dioscorea floridana

6

10.3

±

0.92

N

0.003

±

0.0011

5

10.8

±

0.67

N

0.020

±

0.0095

Dioscoreaceae

Dioscorea villosa

6

10.8

±

1.66

N

0.001

±

0.0008

5

11.3

±

1.24

N

0.000

±

0.0000

Category 2a summary

22

10.8

±

0.57

N

0.006

±

0.0027

2b

Dioscoreales

Dioscoreaceae

Dioscorea pilosiuscula

5

9.3

±

1.11

N

0.005

±

0.0027

5

9.3

±

1.13

N

0.001

±

0.0010

Dioscoreaceae

Dioscorea polygonoides

3

5

6.1

±

0.44

N

0.000

±

0.0000

0

--

±

--

N

--

±

--

Dioscoreaceae

Dioscorea trifida

3

5

24.2

±

9.95

N

0.108

±

0.0474

0

--

±

--

N

--

±

--

Category 2b summary

20

11.2

±

2.82

N

0.019

±

0.0152

2c

Dioscoreales

Dioscoreaceae

Dioscorea alata

8

12.1

±

3.60

N

0.108

±

0.1081

5

9.3

±

1.63

N

0.043

±

0.0320

Dioscoreaceae

Dioscorea cayenensis

3

6

10.1

±

1.89

N

0.053

±

0.0375

0

--

N

--

Dioscoreaceae

Dioscorea esculenta

5

11.7

±

1.44

N

0.034

±

0.0189

5

8.8

±

1.36

N

0.000

±

0.0000

Dioscoreaceae

Dioscorea oppositifolia

5

31.6

±

14.30

N

0.864

±

0.4155

5

10.9

±

0.76

N

0.186

±

0.1050

Dioscoreaceae

Dioscorea polystachya

7

28.7

±

11.94

N

0.288

±

0.2273

5

17.3

±

3.19

N

0.065

±

0.0215

Dioscoreaceae

Dioscorea sansibarensis

5

9.4

±

0.59

N

0.000

±

0.0000

5

7.5

±

0.44

N

0.000

±

0.0000

Category 2c summary

61

14.0

±

2.03

N

0.143

±

0.0518

3

Dioscoreaceae

Tacca chantrieri

3

1

9.7

±

--

N

0.000

±

0.0000

5

9.6

±

1.02

N

0.000

±

0.0000

Category 3 summary

6

9.6

±

0.83

N

0.000

±

0.0000

4

Dioscoreales

No native N. American Dioscoreaceae are threatened or endangered (Neither U.S. nor Florida)

5

Dioscoreales

No species available for testing, see text.

6a

Pandanales

The only U.S. species [Croomia pauciflora (Nutt.) Torr. (Stemonaceae)] within this order has no storage organs

6b

Alismatales

Araceae

Colocasia esculenta

6

7.2

±

0.65

N

0.000

±

0.0000

Araceae

Xanthosoma sagittifolium

5

7.5

±

1.63

N

0.000

±

0.0000

Zingiberales

Marantaceae

Maranta arundinacea

7

10.6

±

1.29

N

0.000

±

0.0000

Zingiberaceae

Curcuma longa

5

10.8

±

1.85

N

0.000

±

0.0000

Zingiberaceae

Hedychium coronarium

5

7.5

±

0.67

N

0.000

±

0.0000

46

TAG

Category

Order

Family

Species

1

Total

reps

Longevity (d)

(avg ± s.e.)

Females

produced

eggs

(Y/N)

Storage organ

tissue consumed

(mL)

(avg ± s.e.)

2

Zingiberaceae

Zingiber officinale

6

10.3

±

1.41

N

0.000

±

0.0000

Category 6b (+7b) Lilianae summary

57

9.6

±

0.41

N

0.000

±

0.0000

6c

Apiales

Apiaceae

Daucus carota

6

9.4

±

0.96

N

0.001

±

0.0008

Araliaceae

Panax ginseng

5

6.4

±

0.69

N

0.000

±

0.0000

Asterales

Asteraceae

Arctium lappa

6

6.5

±

0.75

N

0.000

±

0.0000

Brassicales

Brassicaceae

Brassica rapa

5

6.7

±

0.62

N

0.000

±

0.0000

Brassicaceae

Raphanus sativus

6

8.0

±

0.98

N

0.000

±

0.0000

Caryophyllales

Amaranthaceae

Beta vulgaris

6

7.9

±

0.62

N

0.000

±

0.0000

Fabaceae

Pachyrhizus erosus

5

6.9

±

1.05

N

0.000

±

0.0000

Malpighiales

Euphorbiaceae

Manihot esculenta

5

9.3

±

1.27

N

0.000

±

0.0000

Solanales

Convolvulaceae

Ipomoea batatas

5

7.5

±

0.47

N

0.000

±

0.0000

Category 6c (+ 7c) non-Lilianae summary

55

7.8

±

0.30

N

0.000

±

0.0001

7a

Monocot

Dioscoreaceae

Dioscorea subclava

Known only from China, not available for testing.

7b

Asparagales

Amaryllidaceae

Allium cepa

7

9.5

±

0.78

N

0.000

±

0.0000

Allium sativum

6

7.3

±

0.69

N

0.000

±

0.0000

Dioscoreales

Dioscoreaceae

Dioscorea alata

13

10.7

±

2.27

N

0.105

±

0.0681

Liliales

Liliaceae

Lilium michauxii

5

13.6

±

1.93

N

0.000

±

0.0000

Liliaceae

Tricyrtus lasiocarpa

5

11.9

±

1.49

N

0.000

±

0.0000

Category 7b Lilianae summary

36

10.9

±

0.94

N

0.044

±

0.0262

7c

Solanales

Solanaceae

Solanum tuberosum

6

9.3

±

1.12

N

0.000

±

0.0000

Category 7 combined summary

42

10.9

±

0.67

N

0.044

±

0.0190

1

Species from Category 7 also qualify as belonging in Category 6 (with the exception of D. alata, which is omitted) and so are included in the

appropriate Category 6 summaries.

2

Obtaining potential oviposition required having multiple individuals (2 females, 1 male) in each trial replicate. Thus, the leaf tissue consumed

represents the combined feeding activity of the three adult beetles in each replicate.

3

These plants died out part way through the trials, and additional plants could not be obtained to complete the studies.

47

N/A

Adult longevity on storage organs (d)

0 5 10 15 20 25 30 35

7b. other Lilioceris hosts

7a. reported L. egena hosts

6c. non-Lilianae

6b. other Lilianae

6a. Pandanales (sister order)

5. other Dioscoreales

4. T&E Dioscoreales

3. other Dioscoreaceae

2c. Dioscorea (US weeds)

2b. Dioscorea (WI/CA natives)

2a. Dioscorea (US natives)

1. D. bulbifera

Adult L. egena can live

22 d w/o food or water

Adult L. egena can live

>6 mo. on D. bulbifera

N/A

Figure 1-4. Adult L. egena longevity (mean ± s.e.) during storage organ feeding trials, presented

as averages across TAG categories.

Volume of tissue consumed (mL) by adults

0.0 0.5 1.0 1.5 2.0 2.5 3.0

7b. other Lilioceris hosts

7a. reported L. egena hosts

6c. non-Lilianae

6b. other Lilianae

6a. Pandanales (sister order)

5. other Dioscoreales

4. T&E Dioscoreales

3. other Dioscoreaceae

2c. Dioscorea (US weeds)

2b. Dioscorea (WI/CA natives)

2a. Dioscorea (US natives)

1. D. bulbifera

N/A

Figure 1-5. Adult L. egena tissue consumption (mean ± s.e.) during storage organ feeding trials,

presented as averages across TAG categories.

No choice adult foliage feeding/oviposition trials

Female L. egena produced eggs on live D. bulbifera leaves in only five (three Chinese biotype

and two Nepalese biotype) of the 81 (6.2 percent) successful adult feeding trials, and never

produced eggs on other test plant species (Table 1-6). A total of 77 viable eggs were produced on

D. bulbifera foliage in four of these five trials for an average of 0.03 eggs/female/day during the

30 days. In contrast, females in reproductive studies produced 251.5 ± 34.68 eggs/day over their

first 30 days on bulbils. This suggests that the eggs deposited on foliage are an aberration.

48

Adult L. egena fed readily on D. bulbifera foliage, with about 75 percent of each biotype

surviving the 30-day trials and consuming an average 14,839.9 ± 993.23 mm

2

of leaf tissue per

trial (Table 1-6, Figures 1-6 and 1-7). In contrast, Chinese beetles survived an average of 23.2 ±

1.43 days on wheat (Triticum aestivum) and 22.7 ± 3.33 days on yucca (Manihot esculenta), but

did not feed (Table 1-6). The closest congener in terms of beetle longevity was the U.S. native D.

villosa, on which adults survived an average 18.7 ± 1.48 or 14.1 ± 3.29 days (Chinese vs.

Nepalese beetles, respectively) - roughly 15 percent of the average lifespan of a beetle on D.

bulbifera (Table 1-6). Beetles lived an average 15.7 ± 1.16 and 7.9 ± 1.33 days (Chinese vs.

Nepalese beetles, respectively) on the other U.S. native, D. floridana (Table 1-6). In both of

these cases, the beetles fed very little (Table 1-6). The closest host in terms of consumption was

the West Indian endemic D. cordata on which L. egena consumed 1,196.2 ± 630.83 and 2,715.8

± 1504.51 mm

2

(Chinese vs. Nepalese beetles, respectively) of available leaf tissue - roughly 8

and 18 percent, respectively, of consumption on D. bulbifera (Table 1-6).

On average, adult beetles consumed 95.5 ± 52.66 mm

2

of leaf tissue while surviving 14.1 ± 2.58

days on native U.S. Dioscoreaceae (category 2a plants) (Table 1-6, Figures 1-6 and 1-7). Adult

beetles offered Dioscoreaceae native to the Caribbean and/or Mesoamerica (category 2b plants)

consumed 546.0 ± 192.33 mm

2

, but survived a shorter duration (9.2 ± 0.91 days) than on the

U.S. natives (Table 1-6, Figures 1-6 and 1-7). Adults survived less than two weeks on other test

plant species, including other Dioscoreaceae (categories 2c and 3), and consumed less than 1

percent of the leaf tissue that was consumed by L.egena on D. bulbifera (Table 1-6, Figures 1-6

and 1-7).

Failure of adult L. egena to oviposit upon, survive upon, or consume significant quantities of leaf

tissue from plant species other than D. bulbifera supports the contention that this beetle has close

host fidelity to air potato.

49

Table 1-6. Results of the adult L. egena no choice foliage feeding/oviposition trials. Uncolored

rows represent host trials utilizing Chinese beetles, whereas gray colored rows represent host

trials utilizing Nepalese beetles. The latter were only conducted on members of the order

Dioscoreales. Tan colored rows represent summaries by TAG Category. See Table 1-1 for

additional information regarding the test plant species.

TAG

Category

Order

Family

Species

1

Total

reps

Longevity (d)

(mean ± s.e.)

Females

produced

eggs (Y/N)

Leaf tissue

consumed (mm

2

)

(mean ± s.e.)

2

1

Dioscoreales

Dioscoreaceae

Dioscorea bulbifera

68

>30

±

--

Y

14981.9

±

1082.3

13

>30

±

--

N

14800.7

±

2601.7

Category 1 summary

81

>30

±

--

Y

14839.9

±

993.2

2a

Dioscoreales

Dioscoreaceae

Dioscorea floridana

5

15.7

±

1.16

N

55.3

±

21.1

5

7.9

±

1.33

N

191.0

±

54.3

Dioscoreaceae

Dioscorea villosa

5

18.7

±

1.48

N

6.9

±

4.5

5

14.1

±

3.29

N

128.7

±

69.6

Category 2a summary

20

14.1

±

2.58

N

95.5

±

52.7

2b

Dioscoreales

Dioscoreaceae

Dioscorea altissima

3

4

8.9

±

2.03

N

318.7

±

141.9

0

--

N

--

Dioscoreaceae

Dioscorea cordata

5

9.7

±

3.80

N

1196.2

±

630.8

4

13.5

±

4.72

N

2715.8

±

1504.5

Dioscoreaceae

Dioscorea pilosiuscula

5

15.7

±

3.11

N

4.8

±

3.5

5

6.3

±

0.65

N

0.0

±

0.0

Dioscoreaceae

Dioscorea polygonoides

5

4.6

±

0.99

N

3.6

±

2.2

5

10.5

±

2.49

N

26.6

±

15.1

Dioscoreaceae

Dioscorea trifida

5

7.1

±

1.16

N

355.4

±

346.5

5

7.5

±

1.18

N

513.9

±

311.4

Category 2b summary

43

9.2

±

0.91

N

546.0

±

192.3

2c

Dioscoreales

Dioscoreaceae

Dioscorea alata

5

10.9

±

1.01

N

4.0

±

3.4

5

6.9

±

1.94

N

0.0

±

0.0

Dioscoreaceae

Dioscorea cayenensis

10

10.1

±

1.34

N

54.7

±

39.2

6

17.4

±

9.74

N

175.9

±

91.3

Dioscoreaceae

Dioscorea esculenta

5

6.7

±

1.28

N

0.5

±

0.4

5

6.9

±

0.95

N

0.0

±

0.0

Dioscoreaceae

Dioscorea oppositifolia

3

5

12.5

±

1.84

N

4.7

±

2.9

0

--

N

--

Dioscoreaceae

Dioscorea polystachya

5

6.9

±

0.70

N

24.5

±

7.8

5

5.3

±

0.58

N

3.7

±

3.7

Dioscoreaceae

Dioscorea sansibarensis

5

6.5

±

1.34

N

6.7

±

6.7

5

6.7

±

0.45

N

6.4

±

4.2

Category 2c summary

56

9.0

±

1.05

N

32.0

±

12.3

3

Dioscoreales

Dioscoreaceae

Tacca chantrieri

5

9.4

±

1.35

N

13.8

±

13.8

4

10.8

±

2.85

N

8.4

±

8.4

Category 3 summary

9

9.8

±

1.38

N

9.9

±

8.1

4

Dioscoreales

No native N. American Dioscoreaceae are threatened or endangered (Neither U.S. nor Florida)

5

Dioscoreales

No species available for testing, see text.

6a

Pandanales

Pandanaceae

Pandanus tectorius

5

7.0

±

0.61

N

0.0

±

0.0

6b

Alismatales

Alismataceae

Sagittaria latifolia

5

12.3

±

1.93

N

0.0

±

0.0

Araceae

Alocasia cuculata

5

5.7

±

1.85

N

0.0

±

0.0

50

TAG

Category

Order

Family

Species

1

Total

reps

Longevity (d)

(mean ± s.e.)

Females

produced

eggs (Y/N)

Leaf tissue

consumed (mm

2

)

(mean ± s.e.)

2

Araceae

Caladium bicolor

5

8.1

±

1.49

N

0.0

±

0.0

Araceae

Colocasia esculenta

7

9.5

±

1.31

N

0.0

±

0.0

Araceae

Symplocarpus foetidus

5

6.5

±

0.43

N

0.0

±

0.0

Araceae

Xanthosoma sagittifolium

5

11.2

±

0.78

N

0.0

±

0.0

Araceae

Zantedeschia aethiopica

5

8.1

±

0.70

N

0.0

±

0.0

Arecales

Arecaceae

Sabal palmetto

5

4.1

±

0.07

N

0.0

±

0.0

Asperagales

Amaryllidaceae

Crinum americanum

5

17.7

±

8.30

N

0.0

±

0.0

Amaryllidaceae

Zephyranthes grandiflora

5

7.7

±

0.78

N

0.0

±

0.0

Commelinales

Commelinaceae

Tradescantia pallida

5

8.4

±

1.00

N

0.2

±

0.1

Pontedariaceae

Pontederia cordata

5

8.7

±

1.02

N

0.0

±

0.0

Poales

Cyperaceae

Cladium jamaicense

5

7.7

±

0.67

N

0.0

±

0.0

Juncaceae

Juncus effusus

5

14.3

±

7.28

N

0.0

±

0.0

Musaceae

Musa acuminata

5

7.7

±

1.19

N

0.0

±

0.0

Poaceae

Saccharum officinarum

5

7.3

±

1.27

N

1.3

±

1.3

Poaceae

Zea mays

5

4.7

±

0.52

N

0.0

±

0.0

Zingiberales

Cannaceae

Canna glauca

5

13.1

±

4.12

N

0.0

±

0.0

Cannaceae

Canna americanallis

5

15.4

±

6.61

N

1.8

±

1.8

Costaceae

Costus woodsonii

5

15.6

±

9.63

N

2.2

±

2.2

Heliconiaceae

Heliconia caribaea

5

13.1

±

1.34

N

0.0

±

0.4

Marantaceae

Maranta arundinacea

5

5.7

±

1.81

N

0.0

±

0.0

Marantaceae

Thalia geniculata

5

6.0

±

1.37

N

0.1

±

0.1

Zingiberaceae

Curcuma longa

5

8.7

±

1.21

N

0.0

±

0.0

Zingiberaceae

Hedychium coronarium

5

7.5

±

0.79

N

0.0

±

0.0

Zingiberaceae

Zingiber officinale

5

6.5

±

0.56

N

0.0

±

0.0

Category 6b (+7b) Lilianae summary

192

9.6

±

0.56

N

0.2

±

0.1

6c

Apiales

Apiaceae

Apium graveolens

5

6.7

±

1.06

N

0.0

±

0.0

Apiaceae

Daucus carota

5

6.9

±

0.76

N

0.0

±

0.0

Araliaceae

Panax ginseng

5

14.9

±

1.96

N

1.9

±

1.7

Asterales

Asteraceae

Arctium lappa

5

12.1

±

1.74

N

0.0

±

0.0

Brassicales

Brassicaceae

Brassica rapa

5

6.1

±

0.97

N

0.0

±

0.0

Brassicaceae

Raphanus sativus

6

7.6

±

1.39

N

3.9

±

3.8

Caryophyllales

Amaranthaceae

Beta vulgaris

6

7.6

±

1.10

N

0.9

±

0.9

Fabales

Fabaceae

Glycine max

5

7.2

±

0.20

N

0.0

±

0.0

Fabaceae

Mimosa pudica

5

13.0

±

5.36

N

0.0

±

0.0

Fabaceae

Pachyrhizus erosus

5

7.4

±

0.85

N

0.0

±

0.0

Gentianales

Rubiaceae

Guettarda scabra

5

5.8

±

0.50

N

0.0

±

0.0

Laurales

Calycanthaceae

Calycanthus floridus

5

4.8

±

0.74

N

0.0

±

0.0

Magnoliales

Annonaceae

Annona glabra

5

6.5

±

0.83

N

0.0

±

0.0

Malphigiales

Chrysobalanaceae

Chrysobalanus icaco

6

8.9

±

1.06

N

0.0

±

0.0

Euphorbiaceae

Manihot esculenta

5

22.7

±

3.33

N

0.0

±

0.0

Piperales

Aristolochiaceae

Aristolochia tomentosa

5

5.7

±

1.81

N

0.0

±

0.0

Solanales

Convolvulaceae

Ipomoea batatas

5

5.8

±

0.57

N

0.0

±

0.0

Category 6c (+ 7c) non-Lilianae summary

149

9.0

±

0.54

N

0.3

±

0.2

7a

Monocot

Dioscoreaceae

Dioscorea subclava

Known only from China, not available for testing.

7b

Asperagales

Amaryllidaceae

Allium cepa

5

7.5

±

0.92

N

0.0

±

0.0

Amaryllidaceae

Allium sativum

5

6.9

±

0.53

N

0.0

±

0.0

Asparagaceae

Asparagus densiflorus

5

4.9

±

1.14

N

0.0

±

0.0

51

TAG

Category

Order

Family

Species

1

Total

reps

Longevity (d)

(mean ± s.e.)

Females

produced

eggs (Y/N)

Leaf tissue

consumed (mm

2

)

(mean ± s.e.)

2

Asparagaceae

Asparagus officinalis

5

3.7

±

0.24

N

0.0

±

0.0

Asphodelaceae

Aloe vera

5

10.5

±

0.89

N

0.0

±

0.0

Iridaceae

Sisyrinchium angustifolium

5

9.3

±

1.16

N

0.0

±

0.0

Orchidaceae

Bletilla striata

5

10.5

±

1.33

N

0.0

±

0.0

Dioscoreales

Dioscoreaceae

Dioscorea alata

10

8.9

±

1.22

N

4.0

±

1.7

Liliales

Liliaceae

Lilium michauxii

5

5.3

±

0.21

N

0.0

±

0.0

Liliaceae

Tricyrtus lasiocarpa

5

15.1

±

2.93

N

1.4

±

1.4

Smilacaceae

Smilax laurifolia

5

5.4

±

0.77

N

0.0

±

0.0

Pandanales

Pandanaceae

Pandanus tectorius

5

7.0

±

0.61

N

0.0

±

0.0

Poales

Poaceae

Triticum aestivum

5

23.2

±

1.43

N

0.0

±

0.0

Category 7b Lilianae summary

70

8.1

±

0.67

N

0.2

±

0.3

7c

Apiales

Araliaceae

Schefflera actinophylla

5

9.0

±

0.82

N

0.0

±

0.0

Asterales

Campanulaceae

Lobelia cardinalis

5

11.1

±

0.69

N

0.0

±

0.0

Caryophyllales

Polygonaceae

Persicaria glabra

5

6.0

±

0.61

N

0.0

±

0.0

Fabales

Fabaceae

Senna ligustrina

5

6.4

±

0.40

N

0.0

±

0.0

Fagales

Betulaceae

Corylus americana

5

8.0

±

0.55

N

0.0

±

0.0

Fagaceae

Quercus virginiana

5

15.1

±

1.58

N

0.0

±

0.0

Gentianales

Apocynaceae

Asclepias tuberosa

5

10.1

±

0.75

N

0.0

±

0.0

Lamiales

Verbenaceae

Callicarpa americana

5

7.9

±

0.81

N

0.0

±

0.1

Malpighiales

Salicaceae

Salix caroliniana

5

11.1

±

1.07

N

0.0

±

0.0

Rosales

Moraceae

Ficus aurea

5

8.7

±

0.54

N

0.0

±

0.0

Solanales

Solanaceae

Solanum tuberosum

6

10.5

±

3.27

N

0.1

±

0.0

Cycadales

Cycadaceae

Cycas revoluta

5

8.1

±

1.26

N

0.0

±

0.0

Category 7c non-Lilianae summary

61

9.3

±

0.48

N

0.0

±

0.0

Category 7 combined summary

131

8.5

±

0.42

N

0.1

±

0.1

1

Species from Category 7 also qualify as belonging in Category 6 (with the exception of D. alata, which is omitted) and so are included in the

appropriate Category 6 summaries.

2

Obtaining potential oviposition required having multiple individuals (2 females, 1 male) in each trial replicate. Thus, the leaf tissue consumed

represents the combined feeding activity of the three adult beetles in each replicate.

3

These plants died out part way through the trials, and additional plants could not be obtained to complete studies.

52

Adult L. egena feeding

trials stopped at 30 d

Adult L. egena live 111 (F) - 126 (M) d

Adult longevity on foliage (d)

0 5 10 15 20 25 30 35

7c. Lilioceris hosts (non-Lilianae)

7b. Lilioceris hosts (Lilianae)

7a. reported L. egena hosts

6c. non-Lilianae

6b. other Lilianae

6a. Pandanales (sister order)

5. other Dioscoreales

4. T&E Dioscoreales

3. other Dioscoreaceae

2c. Dioscorea (US weeds)

2b. Dioscorea (WI/CA natives)

2a. Dioscorea (US natives)

1. D. bulbifera

Adult L. egena feeding

trials stopped at 30 d

N/A

Adult L. egena live 111 (F) - 126 (M) d

Figure 1-6. Adult L. egena longevity (mean ± s.e.) during foliage feeding trials, presented as

averages across TAG categories.

(17.9 % of leaf area available)

Area of tissue consumed (mm

2

)

0

2000 4000

6000 8000 10000

12000 14000 16000

7c. Lilioceris hosts (non-Lilianae)

7b. Lilioceris hosts (Lilianae)

7a. reported L. egena hosts

6c. non-Lilianae

6b. other Lilianae

6a. Pandanales (sister order)

5. other Dioscoreales

4. T&E Dioscoreales

3. other Dioscoreaceae

2c. Dioscorea (US weeds)

2b. Dioscorea (WI/CA natives)

2a. Dioscorea (US natives)

1. D. bulbifera

(17.9 % of leaf area available)

N/A

Figure 1-7. Adult L. egena leaf tissue consumption (mean ± s.e.) during foliage feeding trials,

presented as averages across TAG categories.

No choice neonate storage organ feeding/developmental trials

Neonates placed on non-target storage organs usually quickly abandoned the proffered host after

minor taste-testing as evidenced by the positions of dead neonates which were usually scattered

throughout the testing arenas but seldom found on the storage organs. In contrast, neonates

placed on D. bulbifera generally moved very little prior to initiating feeding and instead began

53

burrowing into the D. bulbifera bulbil (or tuber), forming extensive tunnels where they fed until

exiting for pupation. Neonates developing on D. bulbifera consumed an average of 1.81 ± 0.22

mL of plant tissue, whereas the maximum consumed on any other species was 0.02 ± 0.02 mL on

P. erosus (jicama) which constituted 1 percent of the average damage on D bulbifera. Most test

plants remained undamaged (Table 1-7, Figure 1-8).

Whereas 62.9 percent of the L. egena neonates on D. bulbifera storage organs developed into

adults, none of the neonates completed development on any non-target storage organs (Table 1-

7, Figure 1-9). Average developmental time for L. egena neonates on D. bulbifera was 29.0 ±

0.39 days, whereas most neonates on non-targets survived less than 3 days (Table 1-7, Figure 1-

9). The longest lived neonates other than those on D. bulbifera were two that survived 8 days on

P. erosus (jicama) and one that survived 7 days on D. polystachya (cinnamon vine), but all three

failed to molt and become 2

nd

instar larvae.

These results suggest that storage organs of the non-target species lack the cues necessary to

stimulate neonate feeding, and are nutritionally inadequate to permit neonates to develop.

54

Table 1-7. Outcomes of Lilioceris egena no choice neonate storage organ feeding/developmental

trials. Uncolored rows represent host trials utilizing Chinese beetles, whereas gray colored rows

represent host trials utilizing Nepalese beetles. The latter were only conducted on members of

the order Dioscoreales. Tan colored rows represent summaries by TAG Category.

TAG

Category

Order

Family

Species

Total

reps

Longevity (d)

(mean ± s.e.)

F

1

adults

produced

(mean ± s.e.)

Storage organ

tissue consumed

(mL)

(mean ± s.e.)

2

1

Dioscoreales

Dioscoreaceae

Dioscorea bulbifera

35

29.0

±

0.40

3.2

±

0.1

1.810

±

0.2167

12

29.4

±

1.01

3.5

±

0.3

1.866

±

0.2644

Category 1 summary

47

29.2

±

0.39

3.3

±

0.1

1.838

±

0.1710

2a

Dioscoreales

Dioscoreaceae

Dioscorea floridana

4

1.3

±

0.13

0.0

±

0.0

0.004

±

0.0013

5

1.2

±

0.12

0.0

±

0.0

0.000

±

0.0000

Dioscoreaceae

Dioscorea villosa

5

1.4

±

0.10

0.0

±

0.0

0.000

±

0.0000

5

1.0

±

0.00

0.0

±

0.0

0.000

±

0.0000

Category 2a summary

19

1.2

±

0.06

0.0

±

0.0

0.001

±

0.0004

2b

Dioscoreales

Dioscoreaceae

Dioscorea pilosiuscula

5

1.4

±

0.27

0.0

±

0.0

0.000

±

0.0000

5

1.0

±

0.04

0.0

±

0.0

0.000

±

0.0000

Dioscoreaceae

Dioscorea polygonoides

5

2.7

±

0.27

0.0

±

0.0

0.004

±

0.0023

5

1.0

±

0.00

0.0

±

0.0

0.000

±

0.0000

Dioscoreaceae

Dioscorea trifida

5

2.3

±

0.74

0.0

±

0.0

0.004

±

0.0010

5

2.9

±

0.26

0.0

±

0.0

0.024

±

0.0128

Category 2b summary

30

1.9

±

0.20

0.0

±

0.0

0.005

±

0.0025

2c

Dioscoreales

Dioscoreaceae

Dioscorea alata

5

1.4

±

0.18

0.0

±

0.0

0.001

±

0.0010

5

2.3

±

0.68

0.0

±

0.2

0.003

±

0.0020

Dioscoreaceae

Dioscorea cayenensis

3

4

1.5

±

0.22

0.0

±

0.0

0.002

±

0.0012

0

--

Dioscoreaceae

Dioscorea esculenta

5

1.0

±

0.00

0.0

±

0.0

0.000

±

0.0000

5

3.3

±

0.46

0.0

±

0.0

0.000

±

0.0000

Dioscoreaceae

Dioscorea oppositifolia

4

1.2

±

0.12

0.0

±

0.0

0.002

±

0.0012

5

1.1

±

0.12

0.0

±

0.0

0.000

±

0.0000

Dioscoreaceae

Dioscorea polystachya

5

3.1

±

0.23

0.0

±

0.0

0.001

±

0.0000

5

1.3

±

0.20

0.0

±

0.0

0.001

±

0.0010

Dioscoreaceae

Dioscorea sansibarensis

6

1.0

±

0.03

0.0

±

0.0

0.000

±

0.0002

5

1.0

±

0.00

0.0

±

0.0

0.000

±

0.0000

Category 2c summary

54

1.7

±

0.14

0.0

±

0.0

0.001

±

0.0003

3

Dioscoreaceae

Tacca chantrieri

5

1.2

±

0.20

0.0

±

0.0

0.000

±

0.0000

5

3.4

±

0.24

0.0

±

0.0

0.000

±

0.0000

Category 3 summary

10

2.3

±

0.40

0.0

±

0.0

0.000

±

0.0000

4

Dioscoreales

No native N. American Dioscoreaceae are threatened or endangered (Neither U.S. nor Florida)

5

Dioscoreales

No species available for testing, see text.

6a

Pandanales

The only U.S. species [Croomia pauciflora (Nutt.) Torr. (Stemonaceae)] within this order has no storage organs

6b

Alismatales

Araceae

Colocasia esculenta

5

1.0

±

0.04

0.0

±

0.0

0.000

±

0.0000

Araceae

Xanthosoma

sagittifolium

6

1.7

±

0.19

0.0

±

0.0

0.001

±

0.0008

Zingiberales

Marantaceae

Maranta arundinacea

5

1.0

±

0.00

0.0

±

0.0

0.000

±

0.0000

Zingiberaceae

Curcuma longa

5

1.0

±

0.00

0.0

±

0.0

0.000

±

0.0000

Zingiberaceae

Hedychium coronarium

5

1.0

±

0.00

0.0

±

0.0

0.000

±

0.0000

55

Zingiberaceae

Zingiber officinale

5

1.4

±

0.21

0.0

±

0.0

0.000

±

0.0000

Category 6b (+7b) Lilianae summary

51

1.3

±

0.07

0.0

±

0.0

0.000

±

0.0001

6c

Apiales

Apiaceae

Daucus carota

5

2.1

±

0.05

0.0

±

0.0

0.000

±

0.0002

Araliaceae

Panax ginseng

5

1.0

±

0.00

0.0

±

0.0

0.000

±

0.0000

Asterales

Asteraceae

Arctium lappa

5

2.0

±

0.31

0.0

±

0.0

0.000

±

0.0000

Brassicales

Brassicaceae

Brassica rapa

5

1.6

±

0.17

0.0

±

0.0

0.000

±

0.0000

Brassicaceae

Raphanus sativus

4

1.4

±

0.18

0.0

±

0.0

0.000

±

0.0000

Caryophyllales

Amaranthaceae

Beta vulgaris

5

1.5

±

0.14

0.0

±

0.0

0.000

±

0.0002

Fabaceae

Pachyrhizus erosus

5

1.6

±

0.56

0.0

±

0.0

0.022

±

0.0220

Malpighiales

Euphorbiaceae

Manihot esculenta

5

1.0

±

0.00

0.0

±

0.0

0.000

±

0.0000

Solanales

Convolvulaceae

Ipomoea batatas

5

1.8

±

0.35

0.0

±

0.0

0.000

±

0.0000

Category 6c (+ 7c) non-Lilianae summary

49

1.5

±

0.09

0.0

±

0.0

0.002

±

0.0021

7a

Monocot

Dioscoreaceae

Dioscorea subclava

Known only from China, not available for testing.

7b

Asparagales

Amaryllidaceae

Allium cepa

5

2.0

±

0.25

0.0

±

0.0

0.000

±

0.0000

Allium sativum

5

1.1

±

0.08

0.0

±

0.0

0.000

±

0.0000

Dioscoreales

Dioscoreaceae

Dioscorea alata

5

1.4

±

0.18

0.0

±

0.0

0.001

±

0.0010

Liliales

Liliaceae

Lilium michauxii

5

1.0

±

0.00

0.0

±

0.0

0.000

±

0.0000

Liliaceae

Tricyrtus lasiocarpa

5

1.9

±

0.21

0.0

±

0.0

0.000

±

0.0002

Category 7b Lilianae summary

25

1.5

±

0.11

0.0

±

0.0

0.000

±

0.0002

7c

Solanales

Solanaceae

Solanum tuberosum

5

1.5

±

0.23

0.0

±

0.0

0.000

±

0.0000

Category 7 combined summary

30

1.5

±

0.10

0.0

±

0.0

0.000

±

0.0002

1

Species from Category 7 also qualify as belonging in Category 6 (with the exception of D. alata, which is omitted) and so are included in the

appropriate Category 6 summaries.

2

Each trial replicate contained three 2nd/3rd instar larvae. Although survivorship could be tracked individually, tissue consumption could not.

Thus, the leaf tissue consumed represents the combined feeding activity of the three larvae in each replicate.

3

These plants died out part way through the trials, and additional plants could not be obtained to complete the studies.

56

1 mL = 1000 mm

3

Storage organ tissue consumption (mL) by neonates

0.0 0.5 1.0 1.5 2.0 2.5

7b. other Lilioceris hosts

7a. reported L. egena hosts

6c. non-Lilianae

6b. other Lilianae

6a. Pandanales (sister order)

5. other Dioscoreales

4. T&E Dioscoreales

3. other Dioscoreaceae

2c. Dioscorea (US weeds)

2b. Dioscorea (WI/CA natives)

2a. Dioscorea (US natives)

1. D. bulbifera

N/A

Figure 1-8. Consumption (mean ± s.e.) of storage organ tissues by Lilioceris egena neonates,

averaged across TAG categories.

Neonate longevity on storage organs (d)

0 5 10 15 20 25 30 35

7b. other Lilioceris hosts

7a. reported L. egena hosts

6c. non-Lilianae

6b. other Lilianae

6a. Pandanales (sister order)

5. other Dioscoreales

4. T&E Dioscoreales

3. other Dioscoreaceae

2c. Dioscorea (US weeds)

2b. Dioscorea (WI/CA natives)

2a. Dioscorea (US natives)

1. D. bulbifera

62.9% of neonates became adults

}

No neonates became adults

N/A

Figure 1-9. Survivorship (mean ± s.e.) of neonate Lilioceris egena on storage organs of test

plants, averaged across TAG categories.

57

No choice neonate foliage feeding/developmental trials

Neonates placed on foliage other than D. bulbifera leaves exhibited the same wandering behavior

as those placed on storage organs. Some neonates scraped a small bit of tissue from the food

resource prior to abandoning it, usually within minutes. As before, dead neonates were seldom

found on the test leaves, but instead were elsewhere in the testing arenas.

In contrast, neonates placed on D. bulbifera generally wandered a bit initially, presumably

seeking a storage organ, but then settled into feeding on the leaf (Table 1-8). Neonates

developing on D. bulbifera consumed an average of 9,082.6 ± 554.9 mm

2

of leaf tissue. In

contrast, the maximum consumed on any other species was 56.7 ± 41.0 mm

2

(on D. alata), and

most test plants remained undamaged (Table 1-8, Figure 1-10).

An average of 47.8 percent of the L. egena neonates developed into adults on D. bulbifera

leaves (as compared to the 62.9 percent on bulbils) while no neonates completed development on

any non-target foliage (Table 1-8, Figure 1-11). Average developmental time for L. egena

neonates on D. bulbifera was 28.0 ± 0.4 days, whereas neonates on any other plant species

generally survived less than 3 days (Table 1-8, Figure 1-11). The longest lived neonates, other

than those on D. bulbifera, were one on D. cordata that survived 10 days and two that survived 6

days – one on D. cordata and one on D. alata, but none of these molted to become 2

nd

instar

larvae.

Thus, the neonate foliage feeding/development trials further supports the contention that L.egena

is an air potato specialist, and the lower survivorship on foliage versus bulbils indicates this

beetle is primarily adapted to the storage organs of its host.

Table 1-8. Outcomes of Lilioceris egena no choice neonate foliage feeding/developmental trials.

Uncolored rows represent host trials utilizing Chinese beetles, whereas gray colored rows

represent host trials utilizing Nepalese beetles. The latter were only conducted on members of

the order Dioscoreales. Tan colored rows represent summaries by TAG Category.

TAG

Category

Order

Family

Species

1

Total

reps

Longevity (d)

(mean ± s.e.)

F

1

adults

produced

(mean ± s.e.)

Leaf tissue

consumed (mm

2

)

(mean ± s.e.)

2

1

Dioscoreales

Dioscoreaceae

Dioscorea bulbifera

78

28.5

±

0.4

2.3

±

0.2

8828.0

±

594.2

9

27.4

±

0.5

2.7

±

0.4

9337.2

±

1577.3

Category 1 summary

87

28.0

±

0.4

2.5

±

0.2

9082.6

±

554.9

2a

Dioscoreales

Dioscoreaceae

Dioscorea floridana

6

1.8

±

0.3

0.0

±

0.0

0.4

±

0.1

6

1.3

±

0.2

0.0

±

0.0

±

0.0

Dioscoreaceae

Dioscorea villosa

5

1.6

±

0.2

0.0

±

0.0

±

0.0

6

1.6

±

0.2

0.0

±

0.0

±

0.0

Category 2a summary

23

1.6

±

0.1

0.0

±

0.0

0.1

±

0.0

2b

Dioscoreales

Dioscoreaceae

Dioscorea altissima

3

2

1.4

±

0.4

0.0

±

0.0

0.4

±

0.4

58

TAG

Category

Order

Family

Species

1

Total

reps

Longevity (d)

(mean ± s.e.)

F

1

adults

produced

(mean ± s.e.)

Leaf tissue

consumed (mm

2

)

(mean ± s.e.)

2

0

--

Dioscoreaceae

Dioscorea cordata

6

3.1

±

0.6

0.0

±

0.1

4.0

±

3.5

6

1.7

±

0.2

0.0

±

0.0

0.4

±

0.3

Dioscoreaceae

Dioscorea pilosiuscula

7

1.8

±

0.4

0.0

±

0.0

±

0.0

6

1.3

±

0.2

0.0

±

0.0

±

0.0

Dioscoreaceae

Dioscorea polygonoides

7

2.3

±

0.5

0.0

±

0.0

±

0.0

6

1.5

±

0.2

0.0

±

0.0

±

0.0

Dioscoreaceae

Dioscorea trifida

5

1.8

±

0.3

0.0

±

0.2

0.1

±

0.1

5

1.5

±

0.2

0.0

±

0.0

±

0.0

Category 2b summary

50

1.8

±

0.1

0.0

±

0.0

0.5

±

0.4

2c

Dioscoreales

Dioscoreaceae

Dioscorea alata

5

2.5

±

0.7

0.0

±

0.0

56.7

±

41.0

7

1.7

±

0.5

0.0

±

0.0

±

0.0

Dioscoreaceae

Dioscorea cayenensis

8

1.8

±

0.2

0.0

±

0.0

±

0.0

8

1.4

±

0.1

0.0

±

0.0

±

0.0

Dioscoreaceae

Dioscorea esculenta

7

1.9

±

0.2

0.0

±

0.0

±

0.0

6

1.5

±

0.2

0.0

±

0.0

±

0.0

Dioscoreaceae

Dioscorea oppositifolia

3

5

1.4

±

0.2

0.0

±

0.0

±

0.0

0

--

Dioscoreaceae

Dioscorea polystachya

6

1.9

±

0.3

0.0

±

0.0

±

0.0

5

1.5

±

0.3

0.0

±

0.0

±

0.0

Dioscoreaceae

Dioscorea sansibarensis

5

0.9

±

0.1

0.0

±

0.0

±

0.0

5

1.4

±

0.3

0.0

±

0.0

±

0.0

Category 2c summary

67

1.6

±

0.1

0.0

±

0.0

4.1

±

3.3

3

Dioscoreales

Dioscoreaceae

Tacca chantrieri

5

1.7

±

0.4

0.0

±

0.0

0.2

±

0.2

7

1.3

±

0.1

0.0

±

0.0

±

0.0

Category 3 summary

12

1.5

±

0.2

0.0

±

0.0

0.1

±

0.1

4

Dioscoreales

No native N. American Dioscoreaceae are threatened or endangered (Neither U.S. nor Florida)

5

Dioscoreales

No species available for testing, see text.

6a

Pandanales

Pandanaceae

Pandanus tectorius

5

1.4

±

0.1

0.0

±

0.0

±

0.0

6b

Alismatales

Alismataceae

Sagittaria latifolia

5

1.5

±

0.1

0.0

±

0.0

±

0.0

Araceae

Alocasia cuculata

5

1.1

±

0.0

±

0.0

±

0.0

Araceae

Caladium bicolor

5

1.7

±

0.2

0.0

±

0.0

±

0.0

Araceae

Colocasia esculenta

8

1.5

±

0.2

0.0

±

0.0

±

0.0

Araceae

Symplocarpus foetidus

5

1.2

±

0.2

0.0

±

0.0

±

0.0

Araceae

Xanthosoma sagittifolium

5

1.5

±

0.1

0.0

±

0.0

±

0.0

Araceae

Zantedeschia aethiopica

5

1.8

±

0.2

0.0

±

0.0

±

0.0

Arecales

Arecaceae

Sabal palmetto

5

1.4

±

0.2

0.0

±

0.0

±

0.0

Asperagales

Amaryllidaceae

Crinum americanum

8

1.9

±

0.4

0.0

±

0.0

±

0.0

Amaryllidaceae

Zephyranthes grandiflora

5

1.5

±

0.4

0.0

±

0.0

±

0.0

59

TAG

Category

Order

Family

Species

1

Total

reps

Longevity (d)

(mean ± s.e.)

F

1

adults

produced

(mean ± s.e.)

Leaf tissue

consumed (mm

2

)

(mean ± s.e.)

2

Commelinales

Commelinaceae

Tradescantia pallida

5

1.2

±

0.1

0.0

±

0.0

±

0.0

Pontedariaceae

Pontederia cordata

5

1.6

±

0.2

0.0

±

0.0

±

0.0

Poales

Cyperaceae

Cladium jamaicense

5

1.4

±

0.1

0.0

±

0.0

±

0.0

Juncaceae

Juncus effusus

5

1.0

±

0.1

0.0

±

0.0

±

0.0

Musaceae

Musa acuminata

5

4.3

±

0.1

0.0

±

0.0

±

0.0

Poaceae

Saccharum officinarum

9

1.3

±

0.1

0.0

±

0.0

±

0.0

Poaceae

Zea mays

7

1.8

±

0.2

0.0

±

0.0

±

0.0

Zingiberales

Cannaceae

Canna glauca

5

0.8

±

0.1

0.0

±

0.0

±

0.0

Cannaceae

Canna americanallis

5

1.0

±

0.3

0.0

±

0.0

±

0.0

Costaceae

Costus woodsonii

5

0.9

±

0.2

0.0

±

0.0

±

0.0

Heliconiaceae

Heliconia caribaea

5

1.8

±

0.2

0.0

±

0.0

±

0.0

Marantaceae

Maranta arundinacea

5

1.1

±

0.1

0.0

±

0.0

±

0.0

Marantaceae

Thalia geniculata

6

1.2

±

0.2

0.0

±

0.0

±

0.0

Zingiberaceae

Curcuma longa

5

1.2

±

0.0

±

0.0

±

0.0

Zingiberaceae

Hedychium coronarium

10

1.2

±

0.1

0.0

±

0.0

±

0.0

Zingiberaceae

Zingiber officinale

5

1.5

±

0.1

0.0

±

0.0

±

0.0

Category 6b (+7b) Lilianae summary

215

1.4

±

0.0

±

0.0

±

0.0

6c

Apiales

Apiaceae

Apium graveolens

5

1.9

±

0.4

0.0

±

0.0

±

0.0

Apiaceae

Daucus carota

5

1.6

±

0.3

0.0

±

0.0

±

0.0

Araliaceae

Panax ginseng

3

1.3

±

0.3

0.0

±

0.0

±

0.0

Asterales

Asteraceae

Arctium lappa

5

1.3

±

0.1

0.0

±

0.0

±

0.0

Brassicales

Brassicaceae

Brassica rapa

5

1.7

±

0.3

0.0

±

0.0

±

0.0

Brassicaceae

Raphanus sativus

5

1.4

±

0.2

0.0

±

0.0

±

0.0

Caryophyllales

Amaranthaceae

Beta vulgaris

6

1.4

±

0.2

0.0

±

0.0

±

0.0

Fabales

Fabaceae

Glycine max

5

1.4

±

0.2

0.0

±

0.0

±

0.0

Fabaceae

Mimosa pudica

5

1.0

±

0.1

0.0

±

0.0

±

0.0

Fabaceae

Pachyrhizus erosus

5

1.5

±

0.2

0.0

±

0.0

±

0.0

Gentianales

Rubiaceae

Guettarda scabra

5

1.3

±

0.1

0.0

±

0.0

±

0.0

Laurales

Calycanthaceae

Calycanthus floridus

5

1.8

±

0.2

0.0

±

0.0

±

0.0

Magnoliales

Annonaceae

Annona glabra

5

2.3

±

0.1

0.0

±

0.0

±

0.0

Malphigiales

Chrysobalanaceae

Chrysobalanus icaco

5

1.4

±

0.4

0.0

±

0.0

±

0.0

Euphorbiaceae

Manihot esculenta

5

1.8

±

0.1

0.0

±

0.0

±

0.0

Piperales

Aristolochiaceae

Aristolochia tomentosa

5

1.5

±

0.2

0.0

±

0.0

±

0.0

Solanales

Convolvulaceae

Ipomoea batatas

5

1.6

±

0.1

0.0

±

0.0

±

0.0

Category 6c (+ 7c) non-Lilianae summary

147

1.5

±

0.0

±

0.0

±

0.0

7a

Monocot

Dioscoreaceae

Dioscorea subclava

Known only from China, not available for testing.

7b

Asperagales

Amaryllidaceae

Allium cepa

6

1.0

±

0.0

±

0.0

±

0.0

Amaryllidaceae

Allium sativum

6

1.2

±

0.2

0.0

±

0.0

±

0.0

Asparagaceae

Asparagus densiflorus

5

1.0

±

0.0

±

0.0

±

0.0

60

TAG

Category

Order

Family

Species

1

Total

reps

Longevity (d)

(mean ± s.e.)

F

1

adults

produced

(mean ± s.e.)

Leaf tissue

consumed (mm

2

)

(mean ± s.e.)

2

Asparagaceae

Asparagus officinalis

5

1.3

±

0.1

0.0

±

0.0

±

0.0

Asphodelaceae

Aloe vera

5

1.0

±

0.0

±

0.0

±

0.0

Iridaceae

Sisyrinchium angustifolium

5

1.5

±

0.1

0.0

±

0.0

±

0.0

Orchidaceae

Bletilla striata

5

1.0

±

0.0

±

0.0

±

0.0

Dioscoreales

Dioscoreaceae

Dioscorea alata

12

2.1

±

0.4

0.0

±

0.0

28.4

±

18.0

Liliales

Liliaceae

Lilium michauxii

5

2.0

±

0.0

±

0.0

±

0.0

Liliaceae

Tricyrtus lasiocarpa

10

1.1

±

0.1

0.0

±

0.0

±

0.0

Smilacaceae

Smilax laurifolia

5

1.0

±

0.0

±

0.0

±

0.0

Pandanales

Pandanaceae

Pandanus tectorius

5

1.4

±

0.1

0.0

±

0.0

±

0.0

Poales

Poaceae

Triticum aestivum

5

1.2

±

0.1

0.0

±

0.0

±

0.0

Category 7b Lilianae summary

79

1.5

±

0.1

0.0

±

0.0

±

0.0

7c

Apiales

Araliaceae

Schefflera actinophylla

5

1.5

±

0.2

0.0

±

0.0

±

0.0

Asterales

Campanulaceae

Lobelia cardinalis

5

1.2

±

0.1

0.0

±

0.0

±

0.0

Caryophyllales

Polygonaceae

Persicaria glabra

5

1.2

±

0.1

0.0

±

0.0

±

0.0

Fabales

Fabaceae

Senna ligustrina

5

1.3

±

0.1

0.0

±

0.0

±

0.0

Fagales

Betulaceae

Corylus americana

5

1.4

±

0.1

0.0

±

0.0

±

0.0

Fagaceae

Quercus virginiana

5

2.0

±

0.0

±

0.0

±

0.0

Gentianales

Apocynaceae

Asclepias tuberosa

5

1.3

±

0.1

0.0

±

0.0

±

0.0

Lamiales

Verbenaceae

Callicarpa americana

8

1.5

±

0.2

0.0

±

0.0

±

0.0

Malpighiales

Salicaceae

Salix caroliniana

5

1.0

±

0.0

±

0.0

±

0.0

Rosales

Moraceae

Ficus aurea

5

1.4

±

0.1

0.0

±

0.0

±

0.0

Solanales

Solanaceae

Solanum tuberosum

5

1.1

±

0.1

0.0

±

0.0

±

0.0

Cycadales

Cycadaceae

Cycas revoluta

5

2.0

±

0.0

±

0.0

±

0.0

Category 7c non-Lilianae summary

63

1.4

±

0.1

0.0

±

0.0

±

0.0

Category 7 combined summary

142

1.5

±

0.0

±

0.0

±

0.0

1

Species from Category 7 also qualify as belonging in Category 6 (with the exception of D. alata, which is omitted) and so are included in the

appropriate Category 6 summaries.

2

Each trial replicate contained three 2nd/3rd instar larvae. Although survivorship could be tracked individually, tissue consumption could not. Thus,

the leaf tissue consumed represents the combined feeding activity of the three larvae in each replicate.

3

These plants died out part way through the trials, and additional plants could not be obtained to complete the studies.

61

Neonate longevity on foliage (d)

0 5 10 15 20 25 30

7c. Lilioceris hosts (non-Lilianae)

7b. Lilioceris hosts (Lilianae)

7a. reported L. egena hosts

6c. non-Lilianae

6b. other Lilianae

6a. Pandanales (sister order)

5. other Dioscoreales

4. T&E Dioscoreales

3. other Dioscoreaceae

2c. Dioscorea (US weeds)

2b. Dioscorea (WI/CA natives)

2a. Dioscorea (US natives)

1. D. bulbifera

}

46.7% of neonates became adults

No neonates became adults

N/A

Figure 1-10. Neonate Lilioceris egena developmental period/longevity (mean ± s.e.) on foliage,

averaged across TAG categories.

Volume of tissue consumed (mm

2

)

0 2000 4000 6000 8000 10000 12000

7c. Lilioceris hosts (non-Lilianae)

7b. Lilioceris hosts (Lilianae)

7a. reported L. egena hosts

6c. non-Lilianae

6b. other Lilianae

6a. Pandanales (sister order)

5. other Dioscoreales

4. T&E Dioscoreales

3. other Dioscoreaceae

2c. Dioscorea (US weeds)

2b. Dioscorea (WI/CA natives)

2a. Dioscorea (US natives)

1. D. bulbifera

(36.4 % of leaf area available)

N/A

Figure 1-11. Neonate Lilioceris egena leaf tissue consumption (mean ± s.e.), averaged across

TAG categories.

No choice 2

nd

/3

rd

instar storage organ feeding/development trials

Roughly two-thirds (67.9 percent) of 2

nd

/3

rd

instars developed into adults when fed D. bulbifera

storage organs, requiring an average of 25.2 ± 0.57 days to complete development. An additional

5.4 percent of the larvae pupated, but failed to produce adults. In contrast, larvae fed non-target

hosts generally survived a week or less, and only one (0.01 percent of all 2

nd

/3

rd

instars included

in these trials) developed into an adult (Table 1-9). This single individual was a Nepalese 3

rd

instar exposed to bulbils of D. alata (an Asian native, cultivated in the Caribbean and

62

Mesoamerica, and invasive in Florida). The larvae in these trials were not naïve, and had been

feeding and developing in D. bulbifera bulbils prior to being moved onto the storage organs of

test plants. Such a scenario makes it likely this lone 3

rd

instar had acquired sufficient fat reserves

to survive and complete development without feeding upon the test plant. All-in-all, though, no

other 2

nd

/3

rd

instars pupated or completed development on plants other than D. bulbifera (Table

1-9, Figure 1-12).

Aside from D. bulbifera, three trials produced substantial damage (Table 1-9, Figure 1-13).

Larvae in two of the trials on tuberous jicama (Pachyrhizus erosus) roots consumed 4.65 and

4.80 mL (per trial, respectively) of tissue, roughly three times the amount of tissue consumed in

successful D. bulbifera trials (Table 1-9). These jicama-fed larvae (three total) died after molting

from 2

nd

to 3

rd

instar. The large consumption coupled with failure to pupate or complete

development indicates that the jicama roots are nutritionally inadequate for L. egena, but may

lack feeding deterrants. Similarly, two larvae in a single D. oppositifolia trial molted into third

instars, but then failed to develop further despite consuming a toal of 2.750 mL of tissue.

The data from these no choice 2

nd

/3

rd

instars storage organ feeding/development trials suggests

the possibility that late instars from eggs deposited upon D. bulbifera bulbils could occassionally

migrate to, and complete development on D. alata bulbils in areas of Florida and the Caribbean

where the two species are intermixed. However, the failure of adults to oviposit and failure of

neonates to develop on D. alata suggests that persistent populations would not develop on this

plant.

Table 1-9. Outcomes of Lilioceris egena no choice 2

nd

/3

rd

instar larval storage organ

feeding/developmental trials. Uncolored rows represent host trials utilizing Chinese beetles,

whereas gray colored rows represent host trials utilizing Nepalese beetles. The latter were only

conducted on members of the order Dioscoreales. Tan colored rows represent summaries by

TAG Category.

TAG

Category

Order Family Species

1

Total

reps

Longevity (d)

(avg ± s.e.)

F

1

adults

produced

(avg ± s.e.)

Storage organ tissue

consumed (mL)

(avg ± s.e.)

2

1

Dioscoreales

Dioscoreaceae

Dioscorea bulbifera

43

25.8

±

0.72

2.0

±

0.1

1.615

±

0.1105

11

24.7

±

0.38

1.9

±

0.3

1.775

±

0.2478

Category 1 summary

54

25.2

±

0.57

2.0

±

0.1

1.695

±

0.1015

2a

Dioscoreales

Dioscoreaceae

Dioscorea floridana

5

2.9

±

0.66

0.0

±

0.0

0.018

±

0.0143

5

3.1

±

0.23

0.0

±

0.0

0.000

±

0.0000

Dioscoreaceae

Dioscorea villosa

5

3.3

±

0.32

0.0

±

0.0

0.010

±

0.0100

5

2.7

±

0.36

0.0

±

0.0

0.000

±

0.0000

Category 2a summary

20.0

3.0

±

0.18

0.0

±

0.0

0.004

±

0.0028

2b

Dioscoreales

Dioscoreaceae

Dioscorea pilosiuscula

5

2.0

±

0.38

0.0

±

0.0

0.022

±

0.0058

5

4.5

±

0.45

0.0

±

0.0

0.028

±

0.0166

Dioscoreaceae

Dioscorea polygonoides

5

5.8

±

1.11

0.0

±

0.0

0.105

±

0.0371

5

3.5

±

0.17

0.0

±

0.0

0.049

±

0.0175

Dioscoreaceae

Dioscorea trifida

5

3.7

±

0.43

0.0

±

0.0

0.137

±

0.1136

5

3.4

±

0.99

0.0

±

0.0

0.002

±

0.0012

63

TAG

Category

Order Family Species

1

Total

reps

Longevity (d)

(avg ± s.e.)

F

1

adults

produced

(avg ± s.e.)

Storage organ tissue

consumed (mL)

(avg ± s.e.)

2

Category 2b summary

30.0

3.8

±

0.32

0.0

±

0.0

0.050

±

0.0195

2c

Dioscoreales

Dioscoreaceae

Dioscorea alata

6

5.0

±

0.37

0.0

±

0.0

0.138

±

0.0225

5

3.1

±

1.82

0.2

±

0.2

0.138

±

0.1256

Dioscoreaceae

Dioscorea cayenensis

3

5

1.9

±

0.20

0.0

±

0.0

0.005

±

0.0050

0

--

Dioscoreaceae

Dioscorea esculenta

5

2.7

±

0.33

0.0

±

0.0

0.003

±

0.0030

5

3.6

±

0.75

0.0

±

0.0

0.014

±

0.0068

Dioscoreaceae

Dioscorea oppositifolia

5

4.8

±

1.09

0.0

±

0.0

0.834

±

0.5479

5

5.9

±

0.27

0.0

±

0.0

1.651

±

0.2433

Dioscoreaceae

Dioscorea polystachya

5

4.6

±

0.54

0.0

±

0.0

0.261

±

0.1217

5

5.1

±

0.69

0.0

±

0.0

0.000

±

0.0000

Dioscoreaceae

Dioscorea sansibarensis

11

1.5

±

0.27

0.0

±

0.0

0.066

±

0.0267

5

1.9

±

0.08

0.0

±

0.0

0.097

±

0.0238

Category 2c summary

62.0

3.6

±

0.27

0.0

±

0.0

0.285

±

0.0749

3

Dioscoreaceae

Tacca chantrieri

3

5

4.3

±

0.42

0.0

±

0.0

0.014

±

0.0083

0

--

Category 3 summary

5

4.3

±

0.42

0.0

±

0.0

0.014

±

0.0083

4

Dioscoreales

No native N. American Dioscoreaceae are threatened or endangered (Neither U.S. nor Florida)

5

Dioscoreales

No species available for testing, see text.

6a

Pandanales

The only U.S. species [Croomia pauciflora (Nutt.) Torr. (Stemonaceae)] within this order has no storage organs

6b

Alismatales

Araceae

Colocasia esculenta

5

2.5

±

0.20

0.0

±

0.0

0.000

±

0.0002

Araceae

Xanthosoma sagittifolium

5

5.3

±

1.02

0.0

±

0.0

0.011

±

0.0077

Zingiberales

Marantaceae

Maranta arundinacea

5

3.9

±

0.94

0.0

±

0.0

0.000

±

0.0000

Zingiberaceae

Curcuma longa

5

2.7

±

0.07

0.0

±

0.0

0.000

±

0.0000

Zingiberaceae

Hedychium coronarium

5

1.3

±

1.28

0.0

±

0.0

0.000

±

0.0002

Zingiberaceae

Zingiber officinale

5

2.1

±

0.25

0.0

±

0.0

0.003

±

0.0030

Category 6b (+7b) Lilianae summary

49.0

3.0

±

0.21

0.0

±

0.0

0.003

±

0.0011

6c

Apiales

Apiaceae

Daucus carota

5

4.1

±

0.66

0.0

±

0.0

0.014

±

0.0070

Araliaceae

Panax ginseng

5

2.5

±

0.29

0.0

±

0.0

0.000

±

0.0000

Asterales

Asteraceae

Arctium lappa

5

3.6

±

0.80

0.0

±

0.0

0.000

±

0.0000

Brassicales

Brassicaceae

Brassica rapa

10

3.1

±

0.49

0.0

±

0.0

0.004

±

0.0026

Brassicaceae

Raphanus sativus

5

3.5

±

0.50

0.0

±

0.0

0.204

±

0.1231

Caryophyllales

Amaranthaceae

Beta vulgaris

5

5.6

±

0.39

0.0

±

0.0

0.017

±

0.0118

Fabaceae

Pachyrhizus erosus

5

7.0

±

1.66

0.0

±

0.0

1.893

±

1.1564

Malpighiales

Euphorbiaceae

Manihot esculenta

5

4.0

±

0.38

0.0

±

0.0

0.000

±

0.0000

Solanales

Convolvulaceae

Ipomoea batatas

5

2.6

±

0.32

0.0

±

0.0

0.000

±

0.0000

Category 6c (+ 7c) non-Lilianae summary

55.0

3.8

±

0.27

0.0

±

0.0

0.191

±

0.1194

7a

Monocot

Dioscoreaceae

Dioscorea subclava

Known only from China, not available for testing.

7b

Asparagales

Amaryllidaceae

Allium cepa

5

3.1

±

0.95

0.0

±

0.0

0.012

±

0.0097

Allium sativum

4

3.3

±

0.16

0.0

±

0.0

0.000

±

0.0000

Dioscoreales

Dioscoreaceae

Dioscorea alata

6

5.0

±

0.37

0.0

±

0.0

0.138

±

0.0225

Liliales

Liliaceae

Lilium michauxii

5

3.9

±

0.48

0.0

±

0.0

0.000

±

0.0000

Liliaceae

Tricyrtus lasiocarpa

5

2.0

±

0.00

0.0

±

0.0

0.000

±

0.0000

Category 7b Lilianae summary

25

3.5

±

0.30

0.0

±

0.0

0.035

±

0.0129

7c

Solanales

Solanaceae

Solanum tuberosum

5

3.3

±

0.33

0.0

±

0.0

0.000

±

0.0000

Category 7 combined summary

30.0

3.5

±

0.25

0.0

±

0.0

0.030

±

0.0110

64

TAG

Category

Order Family Species

1

Total

reps

Longevity (d)

(avg ± s.e.)

F

1

adults

produced

(avg ± s.e.)

Storage organ tissue

consumed (mL)

(avg ± s.e.)

2

1

Species from Category 7 also qualify as belonging in Category 6 (with the exception of D. alata, which is omitted) and so are included in the

appropriate Category 6 summaries.

2

Each trial replicate contained three 2nd/3rd instar larvae. Although survivorship could be tracked individually, tissue consumption could not.

Thus, the leaf tissue consumed represents the combined feeding activity of the three larvae in each replicate.

3

These plants died out part way through the trials, and additional plants could not be obtained to complete the studies.

N/A

0 5 10 15 20 25 30

7b. other Lilioceris hosts

7a. reported L. egena hosts

6c. non-Lilianae

6b. other Lilianae

6a. Pandanales (sister order)

5. other Dioscoreales

4. T&E Dioscoreales

3. other Dioscoreaceae

2c. Dioscorea (US weeds)

2b. Dioscorea (WI/CA natives)

2a. Dioscorea (US natives)

1. D. bulbifera

No larve became adults

69.0% of larvae became adults

}

Late 2

nd

/early 3

rd

instar larval longevity

on storage organs (d)

No larve became adults

One larva (of 186 in the category; 0.5%) became adults

Figure 1-12. Developmental period/longevity (mean ± s.e.) of 2nd/3rd instar Lilioceris egena

larvae on plant storage organs, averaged across TAG categories.

65

Volume of tissue consumed (mL)

by late 2

nd

/early 3

rd

instar larvae

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0

7b. other Lilioceris hosts

7a. reported L. egena hosts

6c. non-Lilianae

6b. other Lilianae

6a. Pandanales (sister order)

5. other Dioscoreales

4. T&E Dioscoreales

3. other Dioscoreaceae

2c. Dioscorea (US weeds)

2b. Dioscorea (WI/CA natives)

2a. Dioscorea (US natives)

1. D. bulbifera

Figure 1-13. Consumption of plant storage organ tissues (mean ± s.e.) by 2

nd

/3

rd

instar Lilioceris

egena larvae, averaged across TAG categories.

No choice 2

nd

/3

rd

instar larval foliage feeding/development trials

Roughly two-thirds of 2

nd

/3

rd

instar L. egena larvae produced adults when fed D. bulbifera

foliage, requiring 21.6 ± 0.3 days to complete development (Table 1-10, Figure 1-14). Given that

these larvae had developed 4–5 days from eclosion, this developmental period is consistent with

the neonate developmental data (Table 1-8).

Two adults were produced on plants aside from D. bulbifera in these trials, though neither was

on a species found in the United States. A single adult was produced on Dioscorea cordata (a

West Indian endemic), and another on Dioscorea trifida (Mesoamerica to northern South

America) (both category 2b plants). Each adult represents 2.8 percent of the larvae offered these

plants. Once again, the ability of a 2

nd

/3

rd

instar L. egena larva to occasionally finish

development on another species in the genus Dioscorea is unsurprising, given the larvae in these

trials were not naïve but had been feeding and developing in D. bulbifera bulbils prior to being

moved onto the foliage of test plants. Such a scenario makes it likely that an occasional 3

rd

instar

larva would be produced with sufficient fat reserves to complete development even without

feeding upon the test plant.

In general, 2

nd

/3

rd

instar L. egena larvae died within a week of being placed on test plant foliage

(Table 1-10, Figure 1-14). Similar to the neonate larvae, the 2

nd

/3

rd

instar larvae frequently

abandoned the proffered host plant to seek a more suitable food resource. The extended lifespan,

as compared to neonates, may have been a result of fat reserves built up while developing on a

D. bulbifera bulbil slice prior to being transferred onto the test plant. This conclusion is

supported by the fact that average L. egena 2

nd

/3

rd

instar larval consumption of non-target

66

Dioscorea species (categories 2a, 2b, 2c) was very low (1–15 percent) when compared to the

amount of D. bulbifera leaf material consumed, thus making it highly unlikely these older larvae

would have survived for 7 days without the “jump start” that the production technique provided.

Results from this set of trials suggests that there is a small chance that late 3

rd

instar L. egena

larvae which initiate development on D. bulbifera bulbils or leaves could possibly transfer and

complete development on the leaves of a couple of Dioscorea congeners. However, given the

extreme ovipositional preference of female L. egena for D. bulbifera storage organs, and the

inability of neonates to develop on anything other than D. bulbifera, it is unlikely that a

persistent population could develop on any other Dioscorea species (Dray, 2017).

67

Table 1-10. Outcomes of Lilioceris egena no choice 2

nd

/3

rd

instar larval foliage

feeding/developmental trials. Uncolored rows represent host trials utilizing Chinese beetles,

whereas gray colored rows represent host trials utilizing Nepalese beetles. The latter were only

conducted on members of the order Dioscoreales. Tan colored rows represent summaries by

TAG Category.

TAG

Category

Order

Family

Species

1

Total

reps

Longevity (d)

(avg ± s.e.)

F

1

adults

produced

(avg ± s.e.)

Leaf tissue

consumed (mm

2

)

(avg ± s.e.)

2

1

Dioscoreales

Dioscoreaceae

Dioscorea bulbifera

66

23.0

±

0.3

2.1

±

0.1

7255.6

±

410.4

11

20.2

±

0.0

1.8

±

0.3

7698.4

±

1598.6

Category 1 summary

77

21.6

±

0.3

1.9

±

0.1

7479.3

±

420.8

2a

Dioscoreales

Dioscoreaceae

Dioscorea floridana

7

3.8

±

0.5

0.0

±

0.0

33.8

±

9.7

5

3.9

±

0.6

0.0

±

0.0

155.9

±

118.1

Dioscoreaceae

Dioscorea villosa

7

4.4

±

0.6

0.0

±

0.0

106.2

±

70.9

5

4.4

±

0.7

0.0

±

0.0

288.3

±

115.1

Category 2a summary

24

4.0

±

0.3

0.0

±

0.0

150.1

±

28.7

2b

Dioscoreales

Dioscoreaceae

Dioscorea altissima

3

1

3.0

±

0.0

±

0.0

89.3

±

0.0

0

--

Dioscoreaceae

Dioscorea cordata

7

5.3

±

1.1

0.1

±

0.1

209.3

±

141.1

5

5.1

±

0.6

0.0

±

0.0

1051.5

±

289.9

Dioscoreaceae

Dioscorea pilosiuscula

6

3.2

±

0.5

0.0

±

0.0

±

0.0

5

4.3

±

0.3

0.0

±

0.0

1.1

±

1.1

Dioscoreaceae

Dioscorea polygonoides

6

5.3

±

0.6

0.0

±

0.0

225.0

±

126.7

5

5.0

±

0.4

0.0

±

0.0

1051.4

±

611.1

Dioscoreaceae

Dioscorea trifida

5

4.5

±

1.3

0.2

±

0.2

945.0

±

663.9

7

4.4

±

0.8

0.0

±

0.0

1029.7

±

544.2

Category 2b summary

47

4.6

±

0.3

0.0

±

0.0

549.4

±

102.7

2c

Dioscoreales

Dioscoreaceae

Dioscorea alata

6

3.7

±

0.4

0.0

±

0.0

371.5

±

325.5

5

4.8

±

0.5

0.0

±

0.0

13.4

±

13.4

Dioscoreaceae

Dioscorea cayenensis

9

3.4

±

0.3

0.0

±

0.0

19.1

±

15.5

7

4.4

±

0.3

0.0

±

0.0

27.6

±

21.1

Dioscoreaceae

Dioscorea esculenta

5

3.9

±

0.5

0.0

±

0.0

±

0.0

7

4.5

±

0.4

0.0

±

0.0

7.4

±

7.2

Dioscoreaceae

Dioscorea oppositifolia

3

5

4.0

±

0.7

0.0

±

0.0

±

0.0

0

--

Dioscoreaceae

Dioscorea polystachya

5

5.0

±

0.8

0.0

±

0.0

282.2

±

215.4

7

4.7

±

0.3

0.0

±

0.0

465.8

±

247.8

Dioscoreaceae

Dioscorea sansibarensis

5

1.9

±

0.1

0.0

±

0.0

14.7

±

9.8

7

2.0

±

0.2

0.0

±

0.0

46.6

±

26.0

Category 2c summary

68

3.8

±

0.2

0.0

±

0.0

115.9

±

45.8

3

Dioscoreales

Dioscoreaceae

Tacca chantrieri

6

2.9

±

0.5

0.0

±

0.0

7.7

±

7.7

68

TAG

Category

Order

Family

Species

1

Total

reps

Longevity (d)

(avg ± s.e.)

F

1

adults

produced

(avg ± s.e.)

Leaf tissue

consumed (mm

2

)

(avg ± s.e.)

2

7

2.3

±

0.4

0.0

±

0.0

7.0

±

3.6

Category 3 summary

13

2.6

±

0.3

0.0

±

0.0

7.3

±

5.0

4

Dioscoreales

No native N. American Dioscoreaceae are threatened or endangered (Neither U.S. nor Florida)

5

Dioscoreales

No species available for testing, see text.

6a

Pandanales

Pandanaceae

Pandanus tectorius

5

1.4

±

0.1

0.0

±

0.0

±

0.0

6b

Alismatales

Alismataceae

Sagittaria latifolia

5

2.8

±

0.2

0.0

±

0.0

±

0.0

Araceae

Alocasia cuculata

6

6.1

±

0.7

0.0

±

0.0

±

0.0

Araceae

Caladium bicolor

6

5.3

±

0.6

0.0

±

0.0

±

0.0

Araceae

Colocasia esculenta

5

3.0

±

0.3

0.0

±

0.0

±

0.0

Araceae

Symplocarpus foetidus

6

4.3

±

0.7

0.0

±

0.0

±

0.0

Araceae

Xanthosoma sagittifolium

5

3.8

±

0.3

0.0

±

0.0

±

0.0

Araceae

Zantedeschia aethiopica

5

4.9

±

0.5

0.0

±

0.0

±

0.0

Arecales

Arecaceae

Sabal palmetto

5

4.7

±

0.6

0.0

±

0.0

±

0.0

Asperagales

Amaryllidaceae

Crinum americanum

5

3.4

±

1.0

0.0

±

0.0

±

0.0

Amaryllidaceae

Zephyranthes grandiflora

5

2.9

±

0.9

0.0

±

0.0

±

0.0

Commelinales

Commelinaceae

Tradescantia pallida

5

3.4

±

0.7

0.0

±

0.0

0.1

±

0.1

Pontedariaceae

Pontederia cordata

5

4.0

±

0.3

0.0

±

0.0

±

0.0

Poales

Cyperaceae

Cladium jamaicense

5

4.7

±

0.1

0.0

±

0.0

±

0.0

Juncaceae

Juncus effusus

5

3.1

±

0.4

0.0

±

0.0

±

0.0

Musaceae

Musa acuminata

5

2.9

±

0.1

0.0

±

0.0

±

0.0

Poaceae

Saccharum officinarum

5

4.9

±

0.6

0.0

±

0.0

±

0.0

Poaceae

Zea mays

5

4.6

±

0.4

0.0

±

0.0

±

0.0

Zingiberales

Cannaceae

Canna glauca

5

4.2

±

0.6

0.0

±

0.0

1.3

±

0.6

Cannaceae

Canna americanallis

5

3.8

±

0.5

0.0

±

0.0

±

0.0

Costaceae

Costus woodsonii

5

2.6

±

0.7

0.0

±

0.0

±

0.0

Heliconiaceae

Heliconia caribaea

5

5.8

±

0.6

0.0

±

0.0

0.3

±

0.2

Marantaceae

Maranta arundinacea

5

3.7

±

0.3

0.0

±

0.0

±

0.0

Marantaceae

Thalia geniculata

5

6.6

±

0.4

0.0

±

0.0

0.9

±

0.9

Zingiberaceae

Curcuma longa

10

4.4

±

0.4

0.0

±

0.0

±

0.0

Zingiberaceae

Hedychium coronarium

5

4.5

±

0.6

0.0

±

0.0

±

0.0

Zingiberaceae

Zingiber officinale

5

5.5

±

0.3

0.0

±

0.0

±

0.0

Category 6b (+7b) Lilianae summary

210

4.0

±

0.1

0.0

±

0.0

0.1

±

0.0

6c

Apiales

Apiaceae

Apium graveolens

7

4.1

±

0.7

0.0

±

0.0

±

0.0

Apiaceae

Daucus carota

7

4.9

±

0.5

0.0

±

0.0

±

0.0

Araliaceae

Panax ginseng

5

3.7

±

0.3

0.0

±

0.0

±

0.0

Asterales

Asteraceae

Arctium lappa

5

1.9

±

0.1

0.0

±

0.0

±

0.0

Brassicales

Brassicaceae

Brassica rapa

7

5.1

±

0.7

0.0

±

0.0

±

0.0

Brassicaceae

Raphanus sativus

5

3.5

±

0.9

0.0

±

0.0

±

0.0

Caryophyllales

Amaranthaceae

Beta vulgaris

6

4.9

±

0.7

0.0

±

0.0

±

0.0

69

TAG

Category

Order

Family

Species

1

Total

reps

Longevity (d)

(avg ± s.e.)

F

1

adults

produced

(avg ± s.e.)

Leaf tissue

consumed (mm

2

)

(avg ± s.e.)

2

Fabales

Fabaceae

Glycine max

5

4.3

±

0.6

0.0

±

0.0

±

0.0

Fabaceae

Mimosa pudica

5

3.7

±

0.3

0.0

±

0.0

±

0.0

Fabaceae

Pachyrhizus erosus

7

4.8

±

0.7

0.0

±

0.0

±

0.0

Gentianales

Rubiaceae

Guettarda scabra

5

4.3

±

0.5

0.0

±

0.0

±

0.0

Laurales

Calycanthaceae

Calycanthus floridus

5

2.1

±

0.1

0.0

±

0.0

±

0.0

Magnoliales

Annonaceae

Annona glabra

5

2.3

±

0.2

0.0

±

0.0

±

0.0

Malphigiales

Chrysobalanaceae

Chrysobalanus icaco

5

3.2

±

0.7

0.0

±

0.0

±

0.0

Euphorbiaceae

Manihot esculenta

5

3.9

±

0.2

0.0

±

0.0

±

0.0

Piperales

Aristolochiaceae

Aristolochia tomentosa

5

2.1

±

0.1

0.0

±

0.0

±

0.0

Solanales

Convolvulaceae

Ipomoea batatas

6

4.1

±

0.6

0.0

±

0.0

5.4

±

5.4

Category 6c (+ 7c) non-Lilianae summary

152

4.1

±

0.1

0.0

±

0.0

0.2

±

0.2

7a

Monocot

Dioscoreaceae

Dioscorea subclava

Known only from China, not available for testing.

7b

Asperagales

Amaryllidaceae

Allium cepa

5

3.6

±

0.8

0.0

±

0.0

±

0.0

Amaryllidaceae

Allium sativum

6

3.8

±

0.5

0.0

±

0.0

0.8

±

0.8

Asparagaceae

Asparagus densiflorus

5

4.7

±

0.8

0.0

±

0.0

±

0.0

Asparagaceae

Asparagus officinalis

5

2.9

±

0.3

0.0

±

0.0

0.1

±

0.0

Asphodelaceae

Aloe vera

5

2.7

±

0.3

0.0

±

0.0

±

0.0

Iridaceae

Sisyrinchium angustifolium

5

4.1

±

0.4

0.0

±

0.0

±

0.0

Orchidaceae

Bletilla striata

5

3.0

±

0.2

0.0

±

0.0

±

0.0

Dioscoreales

Dioscoreaceae

Dioscorea alata

11

4.2

±

0.4

0.0

±

0.0

371.5

±

229.2

Liliales

Liliaceae

Lilium michauxii

5

3.3

±

0.3

0.0

±

0.0

±

0.0

Liliaceae

Tricyrtus lasiocarpa

5

4.1

±

0.1

0.0

±

0.0

±

0.0

Smilacaceae

Smilax laurifolia

5

2.7

±

0.2

0.0

±

0.0

±

0.0

Pandanales

Pandanaceae

Pandanus tectorius

5

3.4

±

0.7

0.0

±

0.0

±

0.0

Poales

Poaceae

Triticum aestivum

5

3.6

±

0.3

0.0

±

0.0

±

0.0

Category 7b Lilianae summary

72

4.2

±

0.1

0.0

±

0.0

6.73

±

0.9

7c

Apiales

Araliaceae

Schefflera actinophylla

5

5.5

±

0.5

0.0

±

0.0

±

0.0

Asterales

Campanulaceae

Lobelia cardinalis

5

3.7

±

0.4

0.0

±

0.0

±

0.0

Caryophyllales

Polygonaceae

Persicaria glabra

5

4.5

±

0.4

0.0

±

0.0

±

0.0

Fabales

Fabaceae

Senna ligustrina

5

3.3

±

0.2

0.0

±

0.0

±

0.0

Fagales

Betulaceae

Corylus americana

5

5.9

±

0.5

0.0

±

0.0

±

0.0

Fagaceae

Quercus virginiana

5

5.7

±

0.9

0.0

±

0.0

±

0.0

Gentianales

Apocynaceae

Asclepias tuberosa

5

2.9

±

0.2

0.0

±

0.0

±

0.0

Lamiales

Verbenaceae

Callicarpa americana

5

4.5

±

1.1

0.0

±

0.0

±

0.0

Malpighiales

Salicaceae

Salix caroliniana

5

3.7

±

0.4

0.0

±

0.0

±

0.0

Rosales

Moraceae

Ficus aurea

5

4.6

±

0.4

0.0

±

0.0

±

0.0

Solanales

Solanaceae

Solanum tuberosum

4

2.9

±

0.4

0.0

±

0.0

±

0.0

Cycadales

Cycadaceae

Cycas revoluta

5

5.3

±

0.5

0.0

±

0.0

±

0.0

Category 7c non-Lilianae summary

57

4.5

±

0.2

0.0

±

0.0

±

0.0

70

TAG

Category

Order

Family

Species

1

Total

reps

Longevity (d)

(avg ± s.e.)

F

1

adults

produced

(avg ± s.e.)

Leaf tissue

consumed (mm

2

)

(avg ± s.e.)

2

Category 7 combined summary

129

4.4

±

0.1

0.0

±

0.0

6.7

±

0.4

1

Species from Category 7 also qualify as belonging in Category 6 (with the exception of D. alata, which is omitted) and so are included in the

appropriate Category 6 summaries.

2

Each trial replicate contained three 2nd/3rd instar larvae. Although survivorship could be tracked individually, tissue consumption could not. Thus,

the leaf tissue consumed represents the combined feeding activity of the three larvae in each replicate.

3

These plants died out part way through the trials, and additional plants could not be obtained to complete the studies.

0 5 10 15 20 25

7c. Lilioceris hosts (non-Lilianae)

7b. Lilioceris hosts (Lilianae)

7a. reported L. egena hosts

6c. non-Lilianae

6b. other Lilianae

6a. Pandanales (sister order)

5. other Dioscoreales

4. T&E Dioscoreales

3. other Dioscoreaceae

2c. Dioscorea (US weeds)

2b. Dioscorea (WI/CA natives)

2a. Dioscorea (US natives)

1. D. bulbifera

Late 2

nd

/early 3

rd

instar larval longevity

on foliage (d)

No larvae became adults

67.9% of larvae became adults

}

No larvae became adults

Two larvae became adults

N/A

Figure 1-14. Developmental period/longevity (mean ± s.e.) of 2

nd

/3

rd

instar Lilioceris egena

larvae on foliage, averaged across TAG categories.

Two choice adult oviposition and development tests – adults on whole plants.

In this choice test, L. egena preferred D. bulbifera versus D. cordata in each of the three

parameters tested. The beetles damaged an average of 83.6 percent of the D. bulbifera leaves

available to them, as compared to 11.4 percent of the D. cordata leaves available. Two-thirds of

the D. bulbifera bulbils in the cages were damaged, and females only oviposited on these bulbils.

Finally, 60 percent of the adult beetles that were recovered were found on D. bulbifera leaves,

with another 20 percent on the bulbils. The remaining 20 percent of the adults were recovered

from D. coradata leaves. These data suggest that should L. egena migrate to regions of the

Caribbean where D. bulbifera and D. cordata co-occur in close proximity, the beetle would

likely cause some cosmetic damage to D. cordata. However, the absence of oviposition on D.

cordata (both in this trial and in the adult no choice foliage trials, Table 1-6), 100 percent

71

neonate mortality on D. cordata, and this species’ absence of above ground storage organs

means that persistent populations would not establish on this plant.

Ovipositional preference trials

Females rarely oviposited on live D. bulbifera leaves during the feeding trials (6.2 percent of

trials), whereas they oviposited on D. bulbifera bulbils in 90.3 percent of the storage organ

feeding trials. Also, oviposition occurred in 45 of 49 cases (91.8 percent) in which the females

were immediately moved from D. bulbifera leaves onto D. bulbifera bulbils at the conclusion of

foliage feeding trials, usually within 24–48 hours. This confirms that the females used in these

trials were capable of producing eggs, but did not oviposit until placed on their preferred

ovipositional host. From these data, the researchers conclude that female L. egena are so closely

adapted to their host plant, D. bulbifera, that acceptable ovipositional substrates are restricted to

the air potato vine’s storage organs (bulbils and tubers).

A follow-up question was whether L. egena females would demonstrate any difference in

ovipositional success between subterranean tubers and aerial bulbils (Figure 1-15a). In this series

of experiments, L. egena adults fed equally as well on tubers (Figure 1-15b) as bulbils when at

the surface, but did not burrow to locate tubers underground (Figure 1-15c). Likewise, the

females demonstrated no ovipositional preference between the two storage organs (Figure 1-

15d), but failed to burrow to locate tubers underground. Finally, when placed on exposed patches

(4.6 cm

2

) of tubers and bulbils that were otherwise buried in the soil, neonates developed equally

well on either storage organ (Figure 1-15e). Thus, although tubers are an acceptable food and

developmental substrate for these beetles, it is likely that the primary impact will be on bulbils

because L. egena requires tubers to be exposed before the beetles will attack them.

72

Figure 1-15. Comparison of adult Lilioceris egena performance on Dioscorea bulbifera tubers

vs bulbils (a) showed that adult feeding damage in tubers (b) did not differ from that in bulbils

(c). Similarly, neither was preferred as an ovipositional substrate (d), and neonates developed

equally well on both (e).

Researcher’s conclusions (Dray, 2017)

The results of these host range trials provide strong evidence that the beetle, Lilioceris egena, is

specialized on its target host, Dioscorea bulbifera (air potato). Oviposition occurred only on this

plant, and females tended to hold eggs while on air potato foliage only to initiate oviposition as

soon as being placed upon bulbils (and presumedly, exposed subterranean tubers). Thus, the

ovipositional specificity occurs at the organ level within a single species, and not just the species

as a whole. Neonates failed to develop on any plant species aside from D. bulbifera and

developed better on D. bulbifera bulbils/tubers than on leaves. Further, in a preliminary choice

test (not otherwise reported herein) in which neonates were placed in arenas with leaves and

73

bulbil slices, the larvae always moved onto the bulbils, even if placed directly on the leaves first.

The reverse, larvae abandoning bulbils for leaves, never occurred. Finally, the data from 2

nd

/3

rd

instar larval trials suggest that late instar larvae from D. bulbifera leaves or bulbils might on

occasionally migrate to, and complete development on, a few Dioscorea congeners (D. alata, D.

cordata, D. trifida) in areas of Florida and the Caribbean where these congeners are intermixed

and the larvae cannot locate their preferred host. The exteme rarity of this occurance in the no

choice trials (3 of 456 2

nd

/3

rd

instar larvae on Dioscoreaceae; <1 percent), which forced the

larvae to stay on the non-target host, indicates that the likelihood of this occurring in nature is

very low. However, even should this occur, the aforementioned failure of adults to oviposit and

neonates to develop on non-target plants assures that persistent populations could not develop on

these non-target plant species. Accordingly, L. egena appears to be a specific insect.

74

Appendix 2. Release Protocol and Post-Release Monitoring Plan for Lilioceris egena (Dray,

2017).

Release Protocol

Releases will be made of individuals descended from the Chinese and Nepalese colonies used in

the host range trials reported herein. These colonies have already been cleared of diseases,

natural enemies, and the possibility of cryptic species.

The initial insects for release will be reared in the USDA-ARS Fort Lauderdale quarantine

facility (APHIS Facility #106).

Initial releases will be composed of adults because they are long-lived, readily collected, easily

transported, and less likely than immature stages to be targeted by generalist predators. Later

releases may include larvae/egg-infested bulbils, however. Releases will be made in long-term

research plots where D. bulbifera bulbils are abundant and where impacts by the beetle can be

readily measured. These data will be compared to similar control plots where no releases will be

made. Releases will be made in southern, central, and northern Florida. A recent study with the

already released congener L. cheni (Lake et al., 2018), found 85 percent successful establishment

with releases of 100 individuals. Thus, 100 L. egena will initially be released per site and

adjusted as observations dictate.

The insects will be released as soon after PPBP provides a permit as practical. The insects enter a

reproductive diapause phase during late November through mid-February, so releases would not

occur during that season.

Post-Release Monitoring

The permittee and colleagues at the USDA Invasive Plant Research Lab will conduct monitoring

with cooperation from local land managers. APHIS, the permitting agency, does not have any

involvement in post-release monitoring.

None of the insects that feed on D. bulbifera in Florida damage the bulbils, which is L. egena’s

primary food resource. Thus, presence or absence of bulbil damage (rather than leaf damage)

will serve as the principle indicator for establishment, as will presence or absence of the insects.

Of course, L. egena’s congener, L. cheni, was released against D. bulbifera in Florida during

2011 (Center et al., 2013) and has been spread throughout state (Overholt et al., 2016).

Therefore, distinguishing between the two during field surveys will be important. Larvae of the

two species are largely indistinguishable in appearance. However, immature L. egena will

generally be found inside the bulbils, whereas immature L. cheni are found on the foliage and are

not known to burrow into bulbils. The adults of the two species are similar in appearance: shiny

black in color except for red to reddish brown elytra, having elongate bodies (1 cm long by 0.5

cm wide at the abdomen), with narrow thoraxes and even more narrow heads with bulging eyes.

The wing coloration makes them relatively easy to spot in the field, but offers little aid in parsing

75

between the two species. However, they can be distinguished by the presence or absence of setae

on the metasternal plate. This characteristic may be too subtle for the casual observer, but with a

modicum of training even citizen-scientists should be able to detect the differences.

Treatment plots where Lilioceris will be released and control plots where it will not be released

have been established in southern, central, and northern Florida. Given that L. cheni is already

producing impacts in terms of reduced vine densities and bulbil production, monitoring will

focus on proportions of damaged bulbils as a primary measure of L. egena impact. A subset of

damaged and undamaged bulbils from these research plots will be returned to the lab where they

will be planted and allowed to germinate. Germination success, and vine and propagule biomass

produced by germinating bulbils, will be compared between damaged (or possibly across

multiple damage levels) and undamaged bulbils. These data can then be extrapolated back to the

field sites using the field bulbil damage data. Adult beetles will be collected and the proportions

of L. egena to L. cheni will be calculated. Also, subsets of damaged bulbils will be returned to

the lab and dissected to provide estimates of larval abundance. Realized (i.e., ecological) host

range will be evaluated by infesting D. bulbifera and other Dioscoreaceae at a controlled field

site with L. egena in a manner simiar to Lake et al. (2015) and monitoring for dispersal from and

damage to non-hosts including the Florida natives Dioscorea floridana and D. villosa.