Larval feeding behavior of the truncatus group of Thrypticus (Diptera: Dolichopodidae) that breed in the aerenchyma of Pontederiaceae

Comportamiento alimentario de las larvas del grupo truncatus de Thrypticus (Diptera: Dolichopodidae) que se crían en el aerénquima de Pontederiaceae

Hernández, M. Cristina

South American Biological Control Laboratory, USDA-ARS. Bolívar 1559, (B1686EFA) Hurlingham, Buenos Aires, Argentina; e-mail: crisher@speedy.com.ar

RESUMEN. Se da a conocer el comportamiento alimentario de las larvas de algunas especies del grupo truncatus de Thrypticus (Diptera: Dolichopodidae), que se desarrollan en plantas hospedadoras de Pontederiaceae. Los estadios inmaduros de T. circularis Bickel & Hernández se crían en los pecíolos globosos de Eichhornia crassipes (Martius) Solms-Laubach (Pontederiaceae), minando cerca de la epidermis y abriendo varios orificios al exterior. La larva de T. romus Bickel & Hernández crece en E. azurea (Sw.) Kunth, formando una mina curva, corta en comparación con las otras especies. La larva de T. formosensis Bickel & Hernández se desarrolla en Pontederia cordata L. (Pontederiaceae), excavando la mina entre la epidermis y la gran celda central de los pecíolos. Por último, la larva de T. taragui Bickel & Hernández se cría en tallos sumergidos de P. subovata (Seub. in Mart.) Lowden, formando una mina subepidérmica con ramificaciones hacia la estela central del tallo. No se pudieron asociar las minas correspondientes a las especies T. yanayacu, T. chanophalus y T. azuricola. No se encontraron predadores ni parásitos de larvas o pupas, se observaron casos de canibalismo entre larvas cuando el trazado de dos minas confluye. Las especies del grupo truncatus poseen un modo de alimentación sumamente específico, confinadas al aerénquima, se alimentan de la savia extraída de los orificios roídos en los haces vasculares de los pecíolos y tallos, posiblemente con levaduras simbiontes como suplemento para la nutrición. Numerosas colecciones en el campo y pruebas en el laboratorio, indican que estos insectos están asociados con plantas hospedadoras específicas dentro de la misma familia. Esta especialización sugiere una larga asociación insecto-hospedadora.

PALABRAS CLAVE. Grupo truncatus de Thrypticus; Eichhornia crassipes; E. azurea;Pontederia spp; Fitofagia.

ABSTRACT. Larval feeding behavior of the truncatus group of Thrypticus (Diptera: Dolichopodidae) that breed in Pontederiaceae species are presented. The larvae of T. circularis Bickel & Hernández develop in globous petioles of Eichhornia crassipes (Martius) Solms- Laubach (Pontederiaceae), digging a mine near the epidermis and forming several holes to the exterior. T. romus Bickel & Hernández develop in E. azurea (Sw.) Kunth petioles; the mine is curved and short compared with other Thrypticus species. T. formosensis Bickel & Hernández develops in Pontederia cordata L. (Pontederiaceae), and digs the mine between the epidermis and the big central cell of the petioles. T. taragui Bickel & Hernández breeds in submersed stems of P. subovata (Seub. in Mart.) Lowden, and forms a mine close to the epidermis with branches to the central vascular stele. The mines of T. yanayacu, T. chanophalus and T. azuricola could not be associated with the corresponding species. Neither predators nor parasites were found associated with the group, but some cases of cannibalism were observed when two mines were confluent. The truncatus group species have a very specific feeding habit, live completely enclosed within host plant tissues, and feed on the sap obtained from holes chewed in the vascular bundles of the petioles, possibly using yeast as supplementary nourishment. Extensive field collections and laboratory tests indicate that each species in the truncatus group is associated with a specific host plant in the Pontederiaceae. Such specialization suggests a long association between the members of this group and their respective host plants.

KEY WORDS. Truncatus group of Thrypticus; Eichhornia crassipes; E. azurea;Pontederia spp; Phytophagy.

INTRODUCTION

The aquatic plant Eichhornia crassipes (Martius) Solms-Laubach (Pontederiaceae), commonly called waterhyacinth, was released as ornamental in several countries in the late XIX century. It is currently a mayor invasive weed throughout the tropics and other regions of the world. It grows very rapidly, occupying any open water body, covering rivers and lakes, clogging drainage or irrigation canals. Several methods have been used to control its populations, but to little avail. Consequently, the search for natural enemies for biological control commenced in the 1960s (Center, 1994; Cordo, 1999; Julien, 2001). During one of the first surveys for insects associated with waterhyacinth in Guyana, Surinam and Brazil, a mining fly larva of Thrypticus Gerstäcker was found tunneling the basal part of the petioles (Bennett & Zwolfer, 1968). Many years later, taxonomic studies on the Thrypticus associated with Pontederiaceae established that there was a group of nine new species in South America (Bickel & Hernández, 2004). Five of these species, Thrypticus truncatus Bickel & Hernández (B&H), T. sagittatus B&H, T. yanayacu B&H, T. chanophalus B&H and T. circularis B&H, develop in Eichhornia crassipes petioles. Two species, T. romus B&H and T. azuricola B&H, breed in Eichhornia azurea (Sw) Kunth. Thrypticus formosensis B&H develops in Pontederia cordata L., and T. taragui B&H breeds in submersed stems of P. subovata (Seub. in Mart.) Lowden. These nine Thrypticus species each have a diagnostic oviscapt and male genitalia that allows them to be clearly separated from congeneric species, and together they form a monophyletic taxon, the truncatus group.This group was aboundant on E. crassipes and therefore became the subject of taxonomic and ecological study (Cordo et al. 2000; Bickel & Hernández, 2004).
The Pontederiaceae is a small monocotyledonous family composed of six to nine genera and 35 to 40 species, most of which are native to South America (Eckenwalder & Barrett, 1986; Barrett & Graham, 1997). All but one of the Eichhornia species are Neotropical, and all the Pontederia species are New World (Barrett, 1993). Eichhornia and Pontederia are two major genera in the family, and there is phylogenetic evidence that Eichhornia is the ancestor of Pontederia.
The petiole anatomy of the leaves of all species in the Pontederiaceae shows many characters typical of aquatic plants, such as the aerenchyma. This is a specialized tissue with large cells filled with air that help the plant to float and exchange gases. In a cross section of the petiole they have a thin layer of epidermal cells and a central aerenchyma with vascular bundles dispersed throughout (Castellanos, 1959; Gonzalez, 2002; Mahmood et al., 2005).
Although there are 94 known species of Thrypticus (Grichanov, 2007), their biology is poorly known (Dyte, 1959; Stiling & Strong, 1983; Dyte, 1993); e.g., the mine of T. fraterculus (Wheeler) is described as a small blotch or a blister in the leaves of Scirpus acutus (Muhl.) (Cyperaceae) (Green 1954). All the known Thrypticus larvae are miners in aquatic or semi-aquatic monocots, and this is the only genus with phytophagous habits in the Dolichopodidae family (Dyte, 1993).
Both Thrypticus truncatus and T. sagittatus burrow and form mines across the basal part of a petiole. The larva goes through the aerenchyma finding the vascular bundles and chewing a small hole in each one, without crossing through them. They chew through the lateral septa of the aerenchyma until they find other vascular bundles. In both species, the completed mine has an opening at each end. The larvae feed on sap from the holes in the bundles, going forward and backward along the mine. The trace of the larval mines is irregular and it may or may not be branched, but the mines lack any distinctive pattern diagnostic for either of the two species. The larva never comes out of its own mine, and it is incapable of feeding outside of it, and it is unable to dig a new mine if it is removed and transferred to another part of the aerenchyma. Close to one of the outside orifices of the mine, the full grown larva will build a chamber where pupation occurs. The larva cuts an operculum in the epidermis, and afterwards seals the interior of the chamber with a translucent film, where it rests until pupation with the body folded in «U».
Thrypticus truncatus and T. sagittatus have been studied as biocontrol agents for waterhyacinth. All the information gathered in that study is presented in another paper in preparation at this moment (Hernández, unpubl.).
The objective of this work is to present the larval feeding habits of six species of truncatus group of Thrypticus which are associated with Eichhornia crassipes, E. azurea, Pontederia cordata and P. subovata.

MATERIALS AND METHODS

The specimens were collected by H. A. Cordo, A. J. Sosa and the author from 1995 to 2004, during trips in the provinces of Buenos Aires, Entre Ríos, Corrientes, Chaco and Formosa, on the Paraguay-Paraná Rivers basins. H. A. Cordo also surveyed on E. crassipes in the upper Amazon River, near Iquitos in Perú (6-22 Nov. 2001), and near Rio de Janeiro, Brazil (5-25 Apr. 2000) (Cordo, unpubl.). These trips were part of the explorations for the natural enemies of waterhyacinth performed at the South American Biological Control Laboratory (SABCL), in Hurlingham, Buenos Aires, Argentina.
Petioles and stems of Eichhornia crassipes, E. azurea, Pontederia cordata and P. subovata with mines of Thrypticus sp. were gathered. They were kept at room temperature in opaque bottles with a translucent vial in the cap. The newly emerged flies were collected from the vials once a day, and preserved in 80 % ethanol in the SABCL collection. The species identification was carried out by the author, based on the male genitalia characters. To discover the mine's characteristics and the larval feeding habits, sections of the petioles were cut and the mines were traced using a stereomicroscope.

RESULTS

More than 500 adults of Thrypticus truncatus, T. sagittatus, T. circularis, T. yanayacu and T. chanophalus were obtained from Eichhornia crassipes, about 150 adults of T. romus and 6 adults of T. azuricola from E. azurea, 120 adults of T. formosensis from Pontederia cordata, and 50 adults of T. taragui from P. subovata. Each Thrypticus species was obtained invariably from the same host species, this being evidence of fidelity to host plant in natural conditions.
The length of the larvae depends on the age and the nutrition received, and therefore the dimensions of the specimens obtained in the field are the result of several factors. In general, the larvae of these species are 2-3 mm long.
The species of Thrypticus studied in this paper develop inside the aerenchyma of their host plants. The structure of this aerenchyma is similar between species (Fig. 1), consisting, from the epidermis inwards, of a thin, compact layer of parenchymatous cells with small vascular bundles right under the epidermis; below this the petiole is composed by air-filled cells surrounding the vascular bundles distributed regularly within the petiole. These vascular bundles are bigger than those found under the epidermis. Particular variations of this basic morphology can be found among the species.

Figs. 1-9. Thrypticus mines in their host plants. 1, Aerenchyma of Eichhornia crassipes. ac, air-filled cell;,di, diaphragm; ep, epidermis; sp, septa; vb, vascular bundle. 2, Thrypticus truncatus in E. crassipes. 3, T. sagittatus in E. crassipes. 4, globous petioles of E. crassipes with T. circularis mines. 5, detail of T. circularis mines, orifices to exterior. 6, T. romus in E. azurea. 7, T. formosensis in Pontederia cordata. 8, sub-aquatic rhizome of P. subovata with T. taragui. 9, T. taragui in P. subovata stem. Arrows in Figs. 2, 4, 6 and 8, signal the mines. Scale bars = 1 cm.

The mines are made by a series of adjacent air cells connected by the holes made in the septa by the larvae. The size of the holes is always equal to the diameter of the larval body. There are indications that suggest that when the larva is burrowing the mine, it may choose the direction of the mine according to the size of the aerial cell in front of its head. The larva rests its body in the septa of the aerenchyma, therefore if the size of the airfilled cell immediately ahead is longer than the length of the first part of the body, it changes direction and moves towards a more closely positioned septum. Larvae of all the observed species have similar feeding habits, obtaining their nourishment from the sap of the feeding holes scraped in the vascular bundles.

Thrypticus species by host plant

Eichhornia crassipes. The two most common species in the Paraná-Paraguay Rivers are Thrypticus truncatus and T. sagittatus. The mines of these species can have branches of different length and shape (Figs. 2 and 3). In addition, the species T. yanayacu and T. chanophalus emerged from samples from Perú, but unfortunately the mines were not identified. Thrypticus circularis larvae were obtained from globular petioles only (Fig. 4). Inside the globular petioles the aerenchyma has very large cells, and perhaps this is why the mines of this species are dug near the epidermis, where the cells are smaller, and have several openings to the exterior (Fig. 5). No larvae of any Thrypticus species were found in the E. crassipes stems, possibly because it has compact tissues.
Eichhornia azurea: In the E. azurea petioles, the cells of the aerenchyma are slightly bigger toward the center than near the epidermis. The Thrypticus romus mines are curved, without ramifications, with two orifices to the exterior, and comparatively shorter than the mines from other Thrypticus species (Fig. 6). The Thrypticus azuricola mines have not been identified.
Pontederia cordata: Frequently, the petioles in this plant have a big central cell along the axis; therefore, the Thrypticus formosensis mines are located between the epidermis and the central lacuna wall. The mines have some short ramifications, and may have more than two orifices to the exterior (Fig. 7).
Pontederia subovata: The Thrypticus taragui mines are located in the submerged stems or in the rhizomes of Pontederia subovata (Fig. 8), but not in the petioles as in the other species of the group. The stems have a homogeneous aerenchyma with the vascular bundles concentrated in the central stele and near the epidermis. Therefore, the main part of the mine is located near the epidermis and has ramifications to the central stele (Fig.9). The mine has more than two orifices to the exterior.
All the larvae in the truncatus group are sap feeders but the different species make a different number of feeding holes. The shortest mines, and consequently the lowest number of vascular bundles visited, corresponded to Thrypticus romus. The longest were formed by T. circularis and T. taragui, probably because both species burrow the mines in the portion of the aerenchyma with smaller vascular bundles. In addition, it is probable that their nutrition is supplemented by symbiotic yeasts, given that yeasts were isolated from the larvae of T. truncatus and T. sagittatus (Hernández, et al. 2007).
The mortality factors observed were microbial diseases or drowning by mine flooding. Also, the larvae can attack each other if they are in contact, e.g. when one mine accidentally intercepts another. Despite the large number of larvae reared, neither larval nor pupal predators nor parasitoids were found.

DISCUSSION

The truncatus group species share morphological and bionomic characters. They are all associated to the Eichhornia and Pontederia genera, and use the microenvironment inside the aerenchyma to develop. This habitat provides them with nutrition and protection from predators and parasites. Such specialized way of life associated with two genera of plants, belonging to the same family, can reflect an ancient insect-host relationship. This assumption is supported by the fact that 7 species in the truncatus group were found on the genus Eichhornia, ancestor of Pontederia, on which only two species were found.
Thrypticus truncatus, T. sagittatus, T. circularis, T. yanayacu and T. chanophalus, all of them associated with Eichhornia crassipes, are sympatric in several places (Bickel & Hernández, 2004); they use the same environment, the aerenchyma, and seem to have the same niche, as sap feeders developing enclosed in the aerenchyma.
Thrypticus circularis has differentiated utilizing only globular petioles, but the other species live in the same part of the elongated petioles and seem to have similar behavior. More detailed research is necessary to understand if they really have overlapped niches, and if they experience inter-specific competition.

ACKNOWLEDGEMENTS

My gratitude to Dr. Axel O. Bachmann for his professional example and unconditional help in different times during my career. I also want to thank Hugo Cordo and Alejandro Sosa for the survey's trip; to Guillermo Willie Cabrera, Joaquín Sacco and anonymous reviewers for reading the manuscript and providing valuable suggestions.

LITERATURE CITED

1. BARRETT, S. C. H. 1993. The evolutionary biology of tristyly. In: Futuyama, D. & J. Antonovics (Eds.), Oxford Surveys in Evolutionary Biology, Vol.9: 283-326. Oxford University Press, Oxford, UK.
2. BARRETT, S. C. H. & S. W. GRAHAM. 1997. Adaptive radiation in the aquatic plant family Pontederiaceae: insights from phylogenetic analysis. In: Givnish, T. I. & K. Sytsma, Molecular evolution and adaptive radiation, Cambridge University Press, pp. 225- 258.
3. BENNETT, F. D. & H. ZWOLFER. 1968. Exploration for natural enemies of the waterhyacinth in northern South America and Trinidad. Hyacinth Cont. J. 7: 44-52.
4. BICKEL, D. J. & M. C. HERNÁNDEZ. 2004. Neotropical Thrypticus (Diptera: Dolichopodidae) reared from Water Hyacinth, Eichhornia crassipes, and other Pontederiaceae. Ann. Entomol. Soc. Am. 97(3): 437- 449.
5. CASTELLANOS, A. 1959. Las Pontederiaceae de Brasil. Arch. Jard. Bot. Rio de Janeiro 16: 149- 218, pl. 1-18.
6. CENTER, T. D. 1994. Biological control of weeds: Waterhyacinth and waterlettuce: 481-521. In: Rosen, D., F. D. Bennett & J. L. Capinera. (eds.). Pest Management in the Subtropics: Biological Control - A Florida Perspective. Intercept Publishing Company, Andover, United Kingdom.
7. CORDO, H. A. 1999. New agents for biological control of water hyacinth: 68-74. In Hill, M. P., M. H. Julien & T. D. Center (eds.). Proceedings of the First Global Working Group Meeting for the Biological and Integrated Control of Water Hyacinth. November 16-19, 1998, Harare, Zimbabwe. Plant Protection Research Institute, Pretoria, South Africa.
8. CORDO, H. A., A. J. SOSA, & M. C. HERNÁNDEZ. 2000. The petiole mining fly Thrypticus sp. (Diptera: Dolichopodidae), a new agent for the biological control of waterhyacinth (Eichhornia crassipes). In: N. R. Spencer (ed.), Proceedings of the X International Symposium on Biological Control of Weeds, 4-14 July 1999, Montana State University, Bozeman, USA, pp. 315-323.
9. DYTE, C. E. 1959. Some interesting habitats of larval Dolichopodidae (Diptera). Entom. Mon. Mag. 95: 139-143.
10. DYTE, C. E. 1993. The occurrence of Thrypticus smaragdinus Gerst. (Dipt. Dolichopodidae) in Britain, with remarks on plant hosts in the genus. Entomologist 112: 81-84.
11. ECKENWALDER, J. E. & S. C. H. BARRETT.1986. Phylogenetic Systematics of Pontederiaceae. Systematic Bot. 11(3): 373-391.
12. GONZALEZ, A. M. 2002. Anatomía del vástago en especies selectas de plantas vasculares hidrófilas. In: Arbo, M. M. & S. G. Tressens (Eds.). Flora del Iberá, EUDENE, Corrientes, Argentina, Cap.9.
13. GREEN, C. T. 1954. Larva and pupa of Thrypticus fraterculus (Wheeler) with new original notes on the habits of the family Dolichopodidae (Diptera). Entom. News 65: 89-92.
14. GRICHANOV, I. Y. 2007. A check list of species of the family Dolichopodidae (Diptera) of the World, arranged by alphabetic list of generic names. Dolichopodidae Homepage. http://www.fortunecity.com/greenfield/porton/875/Genera 3.com.
15. HERNÁNDEZ, M. C. Unpublished. Estudio de especies de Thrypticus asociadas con Eichhornia crassipes y otras Pontederiáceas en América del Sur. Tesis doctoral. 2007. Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, 130 pp.
16. HERNÁNDEZ, M. C., M. B. PILDAIN, M. V. NOVAS, J. SACCO, & S. E. LOPEZ. 2007. Mycobiota associated with larval mines of Thrypticus truncatus and T. sagittatus (Diptera, Dolichopodidae) on Waterhyacinth, Eichhornia crassipes, in Argentina. Biol. Control 41: 321-326.
17. JULIEN, M. 2001. Biological control of water hyacinth with arthropods: a review to 2000: 8- 20. In: Julien, M., M. Hill, T. Center, and Ding, J. (Eds.), Proceedings of the Second Meeting of the Global Working Group for the Biological and Integrated; Control of Water Hyacinth, Beijing, China, 9-12 December 2000. Australian Centre for International Agricultural Research, Canberra, Australia.
18. MAHMOOD, Q., P. ZHENG, M. R. SIDDIQI, E. ISLAM, M. R. AZIM & Y. HAYAT. 2005. Anatomical studies on water hyacinth (Eichhornia crassipes (Mart.) Solms) under the influence of textile wastewater. J. of Zheijiang Univ. Science 6B 10:991-998.
19. STILING, P. S. & D. R. STRONG. 1983. Weak competition among Spartina stem borers, by means of murder. Ecology 6(4): 770-778.         [ Links ]         [ Links ]         [ Links ]         [ Links ]         [ Links ]         [ Links ]         [ Links ]         [ Links ]         [ Links ]         [ Links ]         [ Links ]         [ Links ]         [ Links ]         [ Links ]         [ Links ]         [ Links ]         [ Links ]         [ Links ]         [ Links ]

Recibido: 11-05-2007;
aceptado: 4-09-2007