Insect herbivores associated with Ludwigia species, Oligospermum section, in their Argentine distribution.

The South American water primroses, Ludwigia grandiflora (Michx.) Greuter & Burdet, L. grandiflora subsp. hexapetala (Hook. & Arn.) G.L. Nesom & Kartesz, Ludwigia peploides (Kunth) P.H. Raven, and L. p. subsp. montevidensis (Spreng.) P.H. Raven (Onagraceae, Section Oligospermum), have become invasive in several watersheds of the United States and Europe. Surveys were carried out in center-east of Argentina to find insect species that might serve as biological control agents for L. g. subsp. hexapetala in California and elsewhere. Stems (0.5-0.6 m) of Ludwigia species, Sect. Oligospermum, were collected in 41 sites and analyzed in the laboratory; immature insects were reared to adults. The plant species found in the area were L. grandiflora (2 sites), L. g. subsp. hexapetala (33 sites), and L. p. subsp. montevidensis (4 sites). There was a variety of insect guilds feeding on L. g. subsp. hexapetala, including six species with stem-borer larvae, one species with fruit-feeding larvae, four species with defoliating larvae, two species with defoliating larvae on young leaves and axil meristems, one species of cell content feeder, and three species of sap feeders. Nine of these species also have defoliating adults. Biological information on most of them is provided. Of these insect herbivores, only two species were also found on L. grandiflora, and one on L. peploides. Several of the species found on L. g. hexapetala, such as the cell-content feeder Liothrips ludwigi (Thysanoptera), the stem-borers Merocnemus binotatus (Boheman) and Tyloderma spp. (Coleoptera), are promising candidates for biocontrol agents.

The South American water primroses species, Ludwigia grandiflora (Michx.) Greuter & Burdet , L. grandiflora subsp. hexapetala (Hook. & Arn.) G.L. Nesom & Kartesz , Ludwigia peploides (Kunth) P.H. Raven, and L. p. subsp. montevidensis (Spreng.) P.H. Raven (Onagraceae, Section Oligospermum), have become invasive in several watersheds of the United States and Europe ( Okada et al. 2009 , Haury et al. 2011 , Nehring and Kolthoff 2011 ).

It is widely accepted that the genus Ludwigia may have originated in South America because the less derived members and the main number of species in the genus are predominantly South American ( Raven and Axelrod 1974 , Skvarla et al. 1975 , Wagner and Hoch 2005 , Wagner et al. 2007 ). The species in the Oligospermum section of Ludwigia are perennial forbs with creeping stems that root freely at the nodes, growing in mud or shallow waters ( Fig. 1 ). They can have floating stems and erect aerial stems, connected by running stems on or under the ground ( Munz 1942 ). This group of species is characterized by having five sepals, twice as many stamens as sepals; pollen shed singly; capsule thick walled, irregularly and tardily dehiscent, terete; seeds uniseriate in each locule, firmly embedded in woody coherent endocarp ( Raven 1963 ).

Fig. 1.

L. g. subsp. hexapetala . (a) Flowers. (b) Typical presence at road side ditch in Buenos Aires province.

These species are polyploids that reproduce both sexually and vegetatively competing aggressively with native species in the invaded fresh water bodies. Attempts to chemical and/or mechanical control of L. grandiflora subsp. hexapetala in California did not give long-term results because remaining stems and seeds restored the previous situation in 4 years. Neither glyphosate nor triclopyr provided systemic control at the rates used ( Meisler 2009 ).

The use of biological control agents may produce a new equilibrium in the system giving the native plant species an opportunity to compete.

Past surveys for natural enemies of Ludwigia spp. in Argentina resulted in the discovery of two unidentified species of Tyloderma sp., Auleutes bosqui Hustache, and Onychylis sp. (all Coleoptera: Curculionidae); and Lysathia flavipes (Boheman) (Coleoptera: Chrysomelidae) ( Cordo and DeLoach 1982a , b ). The plants surveyed were identified at that time as L . peploides and Ludwigia uruguayensis ; but later Zardini et al. (1991) divided uruguayensis into the species grandiflora and hexapetala , based on their chromosome numbers. More recently, hexapetala was defined as a subspecies of grandiflora ( Nesom and Kartesz 2000 ). In host range studies, it is essential to have accurate knowledge on the host plant taxonomy, otherwise host preference tests of potential biocontrol agents may give erroneous or contradictory results.

The objective of this study was to investigate the insect species associated with L. g. subsp. hexapetala , L . grandiflora , and L. peploides in their native range to evaluate, in sequential steps, their potential as biocontrol agents for these weeds. A second goal was to obtain basic biological information on each insect species. References to L . grandiflora will henceforth include all subspecies in the species, except L. g. subsp. hexapetala.

Materials and Methods

Surveys

Our insect collections were made on Ludwigia species within the Oligospermum section, in central and eastern Argentina ( Fig. 2 ) (roughly between 25° 55′ S–38° 39′ S; and 56° 38′ W–61° 38′ W) where the main subtropical and temperate wetlands are located. Surveys were carried out from 2006 to 2009, mostly from spring to autumn, and some sites were visited more than once. Plants were found mainly along road ditches, streams, and shallow water wetlands ( Fig. 1 b). Live and pressed samples were transported to the laboratory for identification. Plants were identified by the first author using morphological characters ( Munz 1942 , Raven 1963 , Zardini et al. 1991 , Dietrich 2005 ) and chromosome counts obtained following the cytological techniques of Singh (2003) and Zardini et al. (1990) .

Fig. 2.

(a) Surveyed area in Argentina. (b) Sampled sites; filled circles, L. grandiflora ; gray circles, L. g . hexapetala ; asterisk, L. p. subsp. montevidensis.

Insects

Some herbivore arthropods were collected on the plants directly with aspirators, but the main collection method was rearing immature stages present on the plants to ensure that the association with the host was not casual; aerial stems were collected in plastic bags and separated in the laboratory according to damage type and incubated individually to identify the immature stages and adults of each natural enemy.

The percentage of stems used by each herbivorous species in seven different populations of L. g. hexapetala was calculated from random samples of 10 stems (0.5–0.6 m) gathered in each site and inspected in the laboratory. General observations were focused on the diversity of insect herbivores, the diversity of host plants of each insect species, the type of injury produced to the plant, and the morphology of the oviposition scars left by the different species of borers.

A basic no-choice feeding tests were carried out on Archips sp. (Lepidoptera: Tortricidae). An apical portion of stem was offered to 20 newly emerged larvae per test plant. The stem was inserted in a water pick and enclosed in a plastic container with wet tissue paper on the bottom and held at room temperature. Three replications per test plant were performed, and L.g. hexapatala was used as control. The test plants used were Ludwigia elegans, L. grandiflora, Ludwigia leptocarpa, Myriophyllum aquaticum, and Alternanthera phylloxeroides.

Results

L . g. hexapetala was found in 33 of 41 sites sampled. L. grandiflora was found in two sites in the north, and L. p. montevidensis in four sites in the southern region ( Fig. 2 ). In total, 19 insect species were obtained from L. g. hexapetala , which belonged to 6 guilds: stem borers, fruit feeders, defoliators, defoliators of apical or axillary leaflets, cell-content feeders, and sap feeders ( Table 1 ).

Table 1.

Guilds and insects species breeding on Ludwigia g. subsp. hexapetala in Argentina

Guild	Species	Observations
Stem borers	Merocnemus binotatus (Coleoptera: Curculionidae)	E in oval scar with fiber in one edge. La. digs in the medulla. Could be associated with apical damage. Total span life around 45 days.
	Tyloderma affine (Coleoptera: Curculionidae)	E in oval scar with fibers in the middle and La. digs initially one round mine, sub-epidermal, after that it goes to the vascular medulla.
	Tyloderma longisquameum (Coleoptera: Curculionidae)	E laid in axilar bud and La. digs in the medulla. P last 7–8 days.
	Tyloderma elongatus (Coleoptera: Curculionidae)	Without information
	Tyloderma sp1. (Coleoptera: Curculionidae)	E in irregular slit, enlarged in one end. La. digs in the medulla. P last 7–8 days.
	Tyloderma sp2,( aeneotinctum group) (Coleoptera: Curculionidae)	E in oval scar with fibers in the middle. La. digs in the medulla. P last 8–10 days. A remains inside the mine several days, after that cuts a circular exit.
	Microlepidoptera, (Lepidoptera)	La. dig helicoidal mines under the epidermis in the stems.
Fuit feeder	Tyloderma nigromaculatum (Coleoptera: Curculionidae)	Egg at sepal base, the whole development goes inside the fruit but several seeds remain undamaged.
Defoliators	Eumorpha fasciata (Lepidoptera: Sphyngidae).	Young La. green, changing to reddish in middle age, and full grown La. with transverse black, red and white stripes. Specificity: Onagraceae and Vitaceae.
	Archips sp. (Lepidoptera: Tortricidae)	Leaf tier. Eggs in long stripe mass on the leaves. Larvae black head and greenish body, very agile. Specificity: low. In no-choice, full development on L. elegans , L. grandiflora , L. leptocarpa , My. aquaticum , A. phylloxeroides.
	Noctuidae (Lepidoptera)	Defoliator. Black larvae. Not frequent.
	Lysathia sp. (Coleoptera: Chrysomelidae)	La. and A feed on leaves. Specificity: All stadia were found on L. g . hexapetala , L. leptocarpa , L. bonariensis.
	Galerucella obliterata (Coleoptera: Chrysomelidae)	A and La. were found defoliating whole plants, and the yellow eggs were found in clutches of 10–20 eggs on the leaves and stems.
	Lagideus badoae (Hymenoptera: Pergidae).	La. feed on the leaves.
Defoliators of apical or axillar leaflet	Auleutes bosqui (Coleoptera. Curculionidae)	La. bore through the buds killing them. A, defoliator.
	Sphenarches ? sp., (Lepidoptera:Pterophoridae)	La. feed on young leaves and use them as protection. La. and P with dorsal hairs similar to the hairs in the plants.
Cell-content feeder	Liothrips ludwigi (Thysanoptera: Phlaeothripidae)	Eggs are laid on the surface of the leaves. All nymphs and A feed on the young leaves, apical and axilar. They produce dead cell spots and even the death of the buds. It is a new species under description and evaluation as candidate for biocontrol.
Sap feeders	Delphacidae sp.1, (Hemiptera)	Dark spot in base of tegmina. Biology not available.
	Delphacidae sp.2, (Hemiptera)	The eggs are inserted in the stems and it produce the bend of the stems. White nymphs. Biology not available.
	Delphacidae sp.3, (Hemiptera)	Yellow nymphs and adults dark brown with a transverse white line in the edge of the tegmina, brachypterous. Biology not available.

A, adult; E, eggs; La., larva; P, pupa.

Stem Borers

Merocnemus binotatus (Boheman) and five species of Tyloderma spp. Say (Curculionidae: Cryptorhynchinae: Cryptorhynchini) were found in this guild. The six species are stem borers as larvae, and use the medulla as food and shelter. Mines were practically indistinguishable among species, but the oviposition scars were slightly different (described further on), which allowed identification of the species of larva present within the stem. Pupation occurred in the mine, and the newly emerged adult often stayed inside several days until it eventually cut a round exit hole. Cannibalism was observed between larvae, and eggs were parasitized frequently. These weevil species were found in sites north of 35° S, and were generally more abundant in the subtropical latitudes ( Figs. 3 and 4 ).

Fig. 3.

Sampled sites and Tyloderma species presence. (a) T. affine. (b) T. longisquameum. (c) Tyloderma sp.1. (d) Tyloderma sp.2, aeneotinctum group. (e) T. elongatum . (f) T. nigromaculatum . Filled circles, presence; open circles, absence.

Fig. 4.

(a) M. binotatus . (b) A. bosqui . Filled circles, presence on L. g . subsp. hexapetala ; open circles, absence.

The female of M. binotatus (Coleoptera: Curculionidae) ( Fig. 5 ) laid a single egg in a small hole chewed in the epidermis of the stems and covered it with torn epidermis fibers. Newly emerged larvae would chew into the stem and develop to adults in it. This species overwintered as larvae in the stem mine. The period from egg to adult in the laboratory was 45 ± 3 days (mean ± SD; n  = 5), suggesting this species may have three to four generations per year. It was found mining in L.g. hexapetala exclusively ( Fig. 4 a).

Fig. 5.

M. binotatus . (a) Adult dorsal. (b) Lateral. (c) Oviposition scar with one egg under fibers, arrow. (d) Stem showing the mine and characteristic frass mixed with long fibers rolled, arrow.

Tyloderma affine Wibmer, ( Figs. 3 a and 6 ) has a wide distribution in South America, and it was previously collected on L. peploides and Oenothera mollisima (L.) (Onagraceae) ( Wibmer 1989 ). L. g . hexapetala is thus added to the list of host plants. Females laid one egg in the middle of an oval scar, which was covered with torn epidermis fibers. Larvae dug initially a superficial, round, subepidermal mine, after which they tunneled in the vascular medulla.

Fig. 6.

T. affine. (a) Adult. (b) Oviposition scar. (c) Fibers moved to show the egg laid in a shallow hole dug in the epidermis. (d) Oviposition scar and the subepidermal mine dug by the larva, arrow.

Tyloderma longisquameum Wibmer, ( Figs. 3 b and 7 ) was quoted by Wibmer (1989) as known only from eastern Argentina but without host plant data. This is the first host plant association reported, and so far, it has only been collected on L. g. hexapetala. Females laid their eggs in the axil of the leaves, and the larvae mined in the same place.

Fig. 7.

T. longisquameum . (a) Adult. (b) Pupa inside the stem, arrow. (c) Egg scar in the axil, arrow.

Tyloderma elongatum Wibmer ( Figs. 3 e and 8 ) was obtained from three localities in the provinces of Buenos Aires, Entre Ríos, and Corrientes, but biological information could not be obtained. Wibmer (1989) cites it as collected on L. peploides , Polygonum sp. (Polygonaceae), and Azolla filiculoides Lam. (Salviniaceae), from eastern Argentina, Uruguay, Paraguay, and southern Brazil.

Fig. 8.

Tyloderma elongatum .

Tyloderma sp.1 ( Figs. 3 c and 9 ) laid its eggs in accidental scars in the stem, apparently not producing lesions of its own. Some of the larvae did not enter the stem directly under the egg chorion, as the other species did, but dug a mine displaced a few millimeters from it.

Fig. 9.

Tyloderma sp.1. (a) Adult. (b) Oviposition scar located under the petiole axil, arrow.

Tyloderma sp.2 ( aeneotinctum group according to Wibmer 1989 ) ( Figs. 3 d and 10 ) laid its eggs in oval scars with fibers piled in the middle. The larva dug in the medulla. Pupal stage lasted 8–10 days. Adults remained inside the mine several days and later cut a circular exit hole.

Fig. 10.

Tyloderma sp. ( aeneotinctum group of Wibmer 1989 , p. 52). (a and b) Adults. (c) oviposition scar with one egg under fibers, arrow.

Fruit Feeders

Tyloderma nigromaculatum Hustache ( Figs. 3 f and 11 ) showed a clear niche differentiation from the stem borer guild, as it laid its eggs at the base of sepals, not covered by fibers nor inserted in the tissue. The larvae fed on the seeds and more than one egg could be found in one fruit. Wibmer (1989) reports that it collected on L. p. subsp. montevidensis and Ludwigia repens .

Fig. 11.

T. nigromaculatum . (a) Adult. (b and c) Eggs on the fruits at sepal bases, arrows.

Defoliators

Larvae of Eumorpha fasciata (Sulzer) (Lepidoptera: Sphingidae) ( Fig. 12 ) were often found feeding on L. g . hexapetala . The cryptic larvae changed the general body color and the pattern of lines and spots in each instar, resembling the green of the leaves in the youngest, reaching an intense red in the fourth, similar to the red stems of this Ludwigia species. The multicolored mature larva pupates in a subterranean burrow. The host range of this species includes several genera in the Onagraceae and Vitaceae.

Fig. 12.

E. fasciata . (a) Male adult. (b) Young instar. (c) Third instar. (d) Fourth instar, red color similar to Ludwigia ’s stems. (e) fifth instar. Scale: 1 cm.

Archips sp. (Tortricidae: Lepidoptera) ( Fig. 13 ), females laid strips of hundreds of eggs on the upper surface of the leaves (32 ± 2.8 eggs per mm 2 ). The larva lives protected by the rolled leaves of the branch tips. Pupation occurs in the same place within a silk cocoon. This species was found developing only on L. g . hexapetala in the field. However, in no-choice feeding tests, the larvae reached full development on L. elegans, L. grandiflora, L. leptocarpa, My . aquaticum, and Al. phylloxeroides. Because of its apparently wide host range, Archips sp. was discarded for further studies.

Fig. 13.

Archips sp. (a and b) Adults and pupal exuvium. (c) Adult, lateral view. (d) Clutch of eggs. (e) stripe of eggs and newly emerged larva. (f) cocoon and secondary fungal infection of the leaf. (g) larva.

Lysathia sp. (Coleoptera: Chrysomelidae) ( Fig. 14 ) laid bright yellow eggs on the leaves in irregular clutches. Larvae and adults fed on the apical leaves and were very damaging. Specimens of this genus were collected in the field feeding on L. grandiflora , L.g. hexapetala , L. elegans , L. leptocarpa , and L. bonariensis . However, species identification is still pending because the genus is under revision and could be a complex of species.

Fig. 14.

Lysathia sp. (a) Adult. (b) Larva. (c) Clutch of eggs. (d) Damage on Ludwigia g . subsp. hexapetala .

Galerucella obliterata Olivier (Coleoptera: Chrysomelidae) was collected on L.g. hexapetala and L. grandiflora in the provinces of Formosa and Buenos Aires. Several specimens collected in the 1970s in the province of Santa Fe mention it on L. repens , which is a synonym of L. peploides. Adults and larvae were found defoliating whole plants and the yellow eggs were found in clutches of 10–20 eggs on the leaves and stems.

Lagideus badoae Smith (Hymenoptera: Pergidae) is a sawfly that produces heavy defoliation. It had been reported feeding on L. peploides and Fuchsia sp. (Onagraceae) in Argentina and Uruguay ( Smith and Bado 2004 ), so no further testing was attempted.

Defoliators of Apical or Axillary Meristems

A. bosqui (Hustache) (Coleoptera: Curculionidae) ( Figs. 4 b and 15 ) has tip-feeding larvae and defoliating adults. Larval feeding activity may produce the death of the apical and axillary meristems. They move downward to pupate forming a spherical cocoon. It was found developing on L. grandiflora , L. g . hexapetala , L. peploides , L. elegans, and L. leptocarpa.

Fig. 15.

A. bosqui . (a) Adult. (b) Larva feeding on young leaf tip.

Sphenarches sp., (Lepidoptera: Pterophoridae) ( Fig. 16 ) larvae fed on the young apical leaves, producing moderate damage in the field. It was present on L. g. hexapetala only in two sites, one in Lobos, Buenos Aires province and the other in Goya, Corrientes province.

Fig. 16.

Sphenarches sp. (a) Adult. (b) Larva. (c) Pupa.

Cell-Content Feeders

Liothrips ludwigi Zamar (Thysanoptera: Phlaeothripidae) ( Fig. 17 ) is a new species recently described as consequence of this research ( Zamar et al. 2013 ). It feeds and breeds in the apical buds of L. grandiflora and L. g . hexapetala all year round. Adults are black and larvae intense red; both share the same feeding-refuge sites in young leaves, moving to a new one when the leaves deteriorate. Ludwigia plants show typical round necrotic spots, around 0.3–2 mm across, in the feeding sites. Symptoms include curly or deformed leaves, death of apical buds and, if the attack persists, a general deterioration of the plant.

Fig. 17.

Li. ludwigi (Phlaeothripidae). (a) Adults and nymph. (b) nymphs in a characteristic position in a young leaf. (c) Ludwigia leaves damaged by Liothrips.

Sap Feeders

Two species of yet unidentified Delphacidae (Hemiptera) were found in low densities on L. g. hexapetala ( Fig. 18 a–c). Sp.1 was found in Santa Fe province (S 28.71577–W 59.43899), and sp.2 was found in the Paraná delta (Otamendi S 34.06339–W 58.82211). The insertion of the eggs of sp.1 produces corkscrew distortion of the stems ( Fig. 18 b). Neither species could be colonized for specificity testing.

Fig. 18.

Sap feeders Delphacidae. (a) Sp1. Nymph I. (b) Sp.1 produces distorted stems. (c) Sp.2 nymph and brachypterous adult.

In addition to this guild, two species of unidentified sap feeding Fulgoroidea were collected in the provinces of Santa Fe and Buenos Aires.

Herbivore Diversity

The species diversity per site was dissimilar ( Table 2 ). Liothrips sp. was the most frequently found in the sites surveyed and the most frequent species per stem sample. Stem borers were the best represented guild with four species of Tyloderma and one species of Merocnemus feeding in the core of the stems. In most sites, however, more than one guild (usually 3 or 4) was represented. Even the species that were not very frequent seem to be widely dispersed, as they have been found in sites far apart.

Table 2.

Percentage of insect species rearing from L. g. subsp. hexapetala in seven sites of Argentina

		Sites
		SF-GUS	CH-SAL	CO-VCU	CO-ITU	CO-GOY	CO-BV1	CO-CAR
Stem miners	M. binotatus	30	—	10	—	—	10	—
	T. affine	—	20	—	—	—	—	—
	T. longiscuameum	—	20	—	—	50	—	30
	Tyloderma sp.1	10	—	20	—	—	—	—
	Tyloderma sp.2	10	90	—	30	—	—	—
Fruit feeder	T. nigromaculatum	—	10	—		10	—	10
Defoliators	E. fasciata	10	—	—	—	—	—	—
	Archips sp.	70	—	—	—	—	—	—
	Lysathia sp.	—	—	—	10	—	—	—
Defoliators apice and axil	A. bosqui	30	20	—	10	—	50	—
Cell content	Li. ludwigi	100	30	40	60	70	20	—

SF-GUS, Santa Fe province S29 35 33.6.W59 46 19.3; CH-SAL, Chaco prov. S27 33 23.6 W59 08 49.7; CO-VCU, Corrientes prov. S27 28 20.5 W57 18 06.3; CO-ITU, Corrientes prov. S27 37 08.7 W56 43 13.4; CO-GOY, Corrientes prov. S29 10 27.7 W59 14 45.7; CO-BV1, Corrientes prov. S28 54 34.5 W59 05 27.1; CO-CAR, Corrientes prov. S29 53 12.9 W59 27 50.5.

A few species of herbivores were collected once, or very rarely, including one Curculionidae (Coleoptera), possibly Onychylis sp.; one species of microlepidopteran with larvae that bore under the epidermis of the stems; and one Noctuidae (Lepidoptera), possibly a generalist, with black larvae that produced heavy defoliation.

Discussion

The goal of this study was directed to identify the herbivorous insects associated with the aerial parts of all L. grandiflora and L. peploides subspecies, in the native range of these aggressive weeds. The species of Ludwigia grouped in the Oligospermum section are a complex of polyploids that produce hybrids ( Raven and Tai 1979 , Zardini et al. 1990 ). As a consequence, the morphology of the plants is highly variable and species are very difficult to identify. This constitutes an extra difficulty for the field host range studies, which was addressed here by means of chromosome counts of the plants sampled, to be sure of the identity of the host plants.

In the central-east region, the most abundant taxon was L. g. hexapetala , which was also the taxon with the highest number of herbivore species. It was found that there were different guilds utilizing all the important aerial parts of the plant. Visible damage was easily observed in the field when the thrips on apical and axil meristems were present. Leaves would grow deformed as a result of feeding and even death of the bud could occur when thrips density was high.

Less obvious were the effects produced by the stems borers, in spite of them being capable of eating the medulla in its entirety. This apparent lack of impact on plant survival is probably due to the ability of this plant to produce roots and leaves in each node, constituting virtually independent plants. However, considering the role of pith in nutrient storage ( Carr and Jaffe 1995 ), the action of these herbivores could conceivably affect plant growth, if not survival.

The thrips species, Liothrips ludwigi , and some of the stem borers species, M. binotatus and Tyloderma spp., appear to be promising candidates for biocontrol agents. These species were selected for further studies on their specificity, bioecology, and damage on L.g. hexapetala .