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Ecología en Bolivia

Print version ISSN 1605-2528On-line version ISSN 2075-5023

Ecología en Bolivia vol.39 no.2 La Paz Oct. 2004

 

Artículo

 

Ectoparasites (Crustacea: Branchiura) of Pseudoplatystoma fasciatum (surubí) and P. tigrinum (chuncuina) in Bolivian whitewater floodplains

 

Ectoparásitos (Crustacea: Branchiura) de Pseudoplatystoma fasciatum (surubí) y P. tigrinum (chuncuina) en planicies de aguas blancas en Bolivia

 

 

M. Mamani1, C. Hamel1,2 & P.A. Van Damme1,3,4

1Unidad de Limnología y Recursos Acuáticos (ULRA), Universidad Mayor de San Simón, Cochabamba, Bolivia
2Present address: Asociación Armonía, Santa Cruz, Bolivia
3Asociation FaunAgua, Bolivia, email: Paul.vandamme@bo.net
4Laboratory of Aquatic Ecology, Katholieke Universiteit Leuven, Belgium.

 

 


Abstract

The ectoparasites of Pseudoplatystoma fasciatum (surubí) and P. tigrinum (chuncuina) from Río Ichilo, from oxbow lakes in the Río Ichilo floodplain and from Río Beni were studied. Sampling was carried out between August 1996 and February 1997 and in August and September 1999. Overall, the ectoparasites from the skin, the branchial cavity, the fins and the gills of 163 P. fasciatum and of 42 P. tigrinum were collected. All fish were found to be infested with ectoparasites. Seven species of Branchiura were recorded, all belonging to the family Argulidae: Argulus juparanaensis, A. pestifer, A. elongatus, A. nattereri, Dolops discoidalis, D. carvalhoi and Dipteropeltis hirundo. The data of 1996-1997 show that P. fasciatum and P. tigrinum harbour the same parasite species, D. hirundo being the only species that was not recorded on the latter, and the infestation levels on both hosts were similar. In general, A. juparanaensis, A. pestifer and D. carvalhoi were the most common species on both fish species. There was a marked difference between the parasite community of P. fasciatum in the Río Ichilo (dominated by Argulus species) and oxbow lakes (dominated by Dolops carvalhoi). For this fish species, the similarity between ectoparasite communities from different rivers (Ichilo and Beni) was higher than the similarity between ectoparasite communities from different habitats (river channel and oxbow lakes in the Ichilo basin). The relevance of these results for the ecology of the fish host species is discussed.

keywords: Argulus, dolops, branchiura, ectoparasites, Bolivia, Pseudoplatystoma.


Resumen

Los ectoparásitos de Pseudoplatystoma fasciatum (surubí) y P. tigrinum (chuncuina) en el Río Ichilo, en lagunas de várzea del Río Ichilo y en el Río Beni fueron estudiados. El muestreo se llevó a cabo entre agosto de 1996 y febrero de 1997 y en los meses de agosto y septiembre de 1999. Las muestras fueron tomadas de la piel, cavidad branquial, agallas y aletas de 163 individuos de P. fasciatum y 42 de P. tigrinum. Todos los peces se encontraron infestados con ectoparásitos. Se encontraron siete especies de Branchiura (ectoparásitos) que corresponden a la familia Argulidae: Argulus juparanaensis, A. pestifer, A. elongatus, A. nattereri, Dolops discoidalis, D. carvalhoi y Dipteropeltis hirundo. Los datos de 1996-1997 mostraron que P. fasciatum y P. tigrinum tienen las mismas especies, con excepción de D. hirundo la cual no fue encontrada en la última. A. juparanaensis, A. pestifer y D. carvalhoi fueron las especies más comunes. Sin embargo, hubo una marcada diferencia entre las comunidades de Branchiura de ríos y de lagunas de varzea en P. fasciatum. Se observó que la similitud entre comunidades de ectoparásitos originando de diferentes ríos (Ichilo y Beni) fue mayor que la similitud entre comunidades de río y comunidades de laguna de várzea. Se discute el significado de los resultados para la ecología de los huéspedes.

Palabras clave: Argulus, dolops, branchiura, ectoparásitos, Bolivia, Pseudoplatystoma.


 

 

Introduction

Pseudoplatystoma fasciatum Linnaeus 1840 (surubí) and P. tigrinum Valenciennes 1840 (chuncuina), belonging to the family Pimelodidae (Siluriformes), are widespread fish species in Latin America. They colonized almost all possible habitats in the Amazon: they are common both in the lower and the upper Amazon and reach the headwaters of black and white water rivers (Rodríguez 1992). They can be found in river channels, in inundated forests and in floodplain lakes (Barthem & Goulding 1997). In the Mamoré river basin, they migrate upstream between July and October to spawn in the headwaters of the Ichilo river around January (Muñoz & Van Damme 1998). The growth and migration patterns of these fish species in the Bolivian Amazon was recently studied by Loubens and Panfili (2000).

Pseudoplatystoma species are carnivores that exert a strong pressure on lower food chain links (Barthem & Goulding 1997; Agudelo Córdoba et al. 2000) and probably play a key role in aquatic habitats of the Amazon basin. They undertake complex lateral migrations between rivers, lakes and river floodplains as well as longitudinal movements along river channels (Barthem &Goulding 1997). The latter authors argue that more detailed studies are needed to understand how environmental factors affect the distribution and migration patterns of these pimelodid fish species.

Disease caused by parasite infestation is one of the environmental factors that may affect pimelodid fish biology and regulate fish abundance. According to various authors, ectoparasites have the potential to exert a strong effect on the behaviour and biology of their fish hosts (Scott & Dobson 1989, Dobson 1988). In Bolivia, the most notorious ectoparasites of Amazon fish species belong to the order Branchiura.

Notwithstanding their wide distribution, the branchiuran parasites of fish species in the Bolivian Amazon are poorly known. Most of the previous research was carried out in Brasil and Paraguay by Thatcher (1990). In Bolivia, Hugghins (1970) described the Branchiura of six host species collected in the Mamoré river. Thatcher (1990) reported 8 Branchiuran species on P. fasciatum in the Brazilian Amazon and concluded that this is the fish host species with the highest number of recorded Branchiura species so far.

In the present study, we explore the Branchiuran parasite community on two pimelodid fish species (P. tigrinum and P. fasciatum) from the Bolivian Amazon. Besides the elaboration of a parasite species list, we focus on the distribution patterns of the parasite species in the host populations and on the habitat characteristics that may affect parasite occurrence.

 

Study area


The Ichilo and Beni rivers form part of the Madeira river basin. The río Ichilo represents the border between the departments of Cochabamba and Santa Cruz, situated in the center of Bolivia. After its confluence with the Río Grande, its name changes to Mamoré (Fig. 1). The Río Beni originates in the department of La Paz and flows in northeast direction before it joins the Río Mamoré in Guayaramerín (Fig.1).

Both rivers have high discharge rates and have extensive white-water floodplains, characterized by inundated forest and oxbow lakes (Navarro & Maldonado 2003). Both rivers are important for commercial fisheries, with Puerto Villarroel (Río Ichilo) and Rurrenabaque (Río Beni) being the most important fishery ports.

 

Methods

The results presented in this study were obtained during two fish sampling campaigns. During the first sampling campaign, between August 1996 and February 1997, 82 P. fasciatum and 42 P. tigrinum were collected in the Río Ichilo basin (in the river channel and oxbow lakes). During the second sampling campaign, in August and September 1999, 75 P. fasciatum were collected in the Río Ichilo, in oxbow lakes of the Ichilo basin, and in the Río Beni. Fishes were captured using gill nets and immediately after capture samples of ectoparasites were taken with pincets from the skin, the gill cavity, the gills and the fins. The parasites were fixed and conserved in alcohol 85 %. After parasite collection, the total length of the hosts was measured with an ichthyometer. Fish weight was measured with a balance of 50 kg capacity and a sensitivity of ± 0.5 kg.

In the laboratory, the ectoparasites were separated, counted and determined to species level with a binocular. Identification guides from Lemos de Castro (1986) and Thatcher (1990) were used for this purpose.

For each parasite species, the following parameters were calculated (according to Margolis et al. 1981):

The Dispersion index (DI) was calculated as variance / abundance (Elliot 1977).

Principal Components Analysis (PCA) was used to represent fish individuals in the plane formed by the two first principal components. Factor loadings were calculated using the program STATISTICA. Subsequently, the correlation between factor scores and fish length was calculated (Pla 1986, Ludwig & Reynolds 1988).

 

Results

Seven Branchiura species were recorded, four of which belong to the genus Argulus (A. elongatus Heller 1857, A. pestifer Ringuelet 1948, A. nattereri Heller 1857, A. juparanaensis Lemos de Castro 1950), two belong to the genus Dolops (D. discoidalis Bouvier 1899, D. carvalhoi Lemos de Castro 1949) and one belongs to the genus Dipteropeltis (D. hirundo Calman 1912).

The prevalence, abundance and intensity of infestation of the 7 species of Branchiura recorded from Pseudoplatystoma fasciatum and P. tigrinum from the Río Ichilo basin in 19961997 are presented in Table 1. Argulus pestifer, A. juparanaensis and Dolops carvalhoi were the most abundant parasite species in both hosts. The infection intensities in both species were similar, Dipteropeltis hirundo being the only branchiuran species that was not recorded from P. tigrinum.

The infestation intensities, prevalences and abundances of parasites on P. fasciatum from the Río Ichilo, oxbow lakes in the Ichilo basin and the Río Beni in 1999 are presented in Table 2. In general, fish from oxbow lakes were more heavily infected (18 parasites/fish) than river fish (Río Ichilo: 4.5 parasites/fish; Río Beni: 3.1 parasites/fish). However, there were two Branchiuran species which were never recorded in oxbow lakes: Argulus juparanaensis and A. nattereri.

In the Ichilo, the most abundant species was A. juparanaensis, whereas it was Dipteropeltis hirundo in the Beni. In oxbow lakes of the Río Ichilo basin, D. carvalhoi (prevalence 79%) was the most common species, followed by D. discoidales (prevalence 68 %), two species which were never recorded in rivers.

Figure 2A shows the plane formed by the two first principal components of a PCA analysis of parasites abundances on P. fasciatum from the 1999 sampling campaign. There was a remarkable superposition of the fish populations of rivers Ichilo and Beni, whereas the lake fish populations were clearly separated from the riverine populations along the first axis. In figure 2B, the loadings of the Branchiuran species, relative to the two first principal components, are shown. The length of the fish was correlated significantly with the second principal component (Pearson Correlation, p < 0.05).

Table 3 and Figure 3 show that the Dispersion Index for most parasite species is generally significantly larger than 1, illustrating the aggregation of the parasites on a few host individuals. Some species (for example D. hirundo) have Dispersion Indices close to one and show truncated dispersion patterns.

In Table 4, all the recorded host species for the branchiuran parasites species that were recorded during the present study are listed. It can be seen that three of the Argulus species (A. pestifer, A. elongatus, A. nattereri) have the highest host specificity, whereas Dolops species in general seem to be less specific.

 

Discussion

Pseudoplatystoma fasciatum and P. tigrinum are very similar species that speciated about two million years ago (Coronel et al. 2004). In Table 3, it is shown that the branchiuran parasite communities of these two species have a similar composition, making it highly probable that the speciation within the Branchiura has occurred a lot earlier than within host species. Possibly, speciation within the Branchiura has occurred on the ancestor of P. tigrinum and P. fasciatum, and these host species inherited the same parasite species. However, we need studies on the phylogeny of branchiuran parasite species to test these hypotheses.

In general, in the case of fish parasites with a relatively high to very high host specificity (such as monogean ectoparasites, for example), related host species have a higher probability to possess a similar parasite fauna than unrelated host species (Boeger & Kritsky 1997, Poulin 2002). However, in the particular case of branchiurans, which are known by their frequent host switching and rather low host specificity (Thatcher 1991), there are other influencing factors: for this group, the similarity of parasite fauna may be more related to (dis)similarity in host behaviour than to genetic similarity. Indeed, if we look at host checklists for branchiuran parasites (Table 4), both genetical and ecological factors may be involved to various degrees.

Pseudoplatystoma fasciatum and P. tigrinum, are infestated with a remarkably high number of branchiuran parasites species (see also Malta 1984), whereas other fish species of the same family, such as Brachyplatystoma sp., do not have branchiuran parasite species (Table 3) (Thatcher 1990, Van Damme unpublished data). This difference is probably related with the biology of the host species. The presence of a high number of Branchiura on P. fasciatum and P. tigrinum may be a consequence of their life style, both species displaying a characteristic “sit-and-wait” behavior, staying at any time in close contact with the sediment of rivers or oxbow lakes (Barthem & Goulding 1997). As a consequence of this life style, they are probably very vulnerable to infestation with the infective (larval) stages of Branchiuran parasites. This probably also explains why species with amore pelagic life style, such as Brachyplatystoma spp. do not harbour branchiuran parasites. The pelagic environment is probably less suitable for Branchiuran species as a result of high current velocities.

Moreover, it is probable that host species that occupy a variety of different habitats are infected with more parasite species. This might be one of the reasons why Pseudoplatystoma species, which can be found in all possible habitats of the floodplain (river, inundated forest, oxbow lakes, streams) (Barthem & Goulding 1997), have relatively more parasite species. An alternative explication may be that host species found in different habitats are made up of different populations, which may have favored parasitic speciation, and a consequent increase in parasite species richness.

Apart from the abovementioned factors, host density, which in the case of Pseudoplatystoma spp. is thought to be different in lakes and rivers, might also influence the success of parasite transmission. However, there are not enough data to validate this assumption.

Consequently, we suggest that pimelodid fish species that occupy a variety of different habitats and/or that display a sit-and-wait “benthic” behaviour have more parasite species. There seems to be a gradient Pseudoplatystoma-Phractocephalus-Brachyplatystoma. However, the same is not necessarily true for characids, where for example a species such as Salminus maxillosus, a pelagic species “por excelencia”, does have a quite high number of Branchiuran parasite species (Thatcher 1990). Besides the mentioned ecological factors, genetical factores may also play an important role.

The P. fasciatum of oxbow lakes of the Río Ichilo River basin had a higher load of Branchiuran parasites than riverine fishes. However, species diversity in the two habitats is equal. Dolops species seem to be more common in oxbow lakes (D. discoidales and D. carvalhoi exclusively occurring in lakes during the 1999 campaign), whereas the genus Argulus has both riverine species (A. juparanensis and A. nattereri) and species with a preference towards lakes (A. pestifer and A. elongatus). The latter may explain why Malta (1982a) hardly found A. juparanensis in Brazilian oxbow lakes.

The observation that parasite community structure on P. fasciatum in lakes and rivers is different may suggest that fish host populations are (genetically) different. However, it is known that P. fasciatum spawn in the river channels (Barthem & Goulding 1997, Muñoz & Van Damme 1998), and lake fish probably move to the river channel during the spawning season. The previous makes it more plausible that the Branchiuran parasites lose hold when the host moves from one to another habitat, for example because they can not resist high current velocities. If this is so, Branchiura would be bad indicators of the origin or migration patterns of their hosts.

Thatcher (1990) found that A. pestifer infesting Pseudoplatystoma species in the Brazilian Amazon was found in high numbers during the low water season (novemberdecember), whereas all the other species were more common in June and July, when the water level was higher. Malta (1983) observed similar seasonal patterns for A. pestifer in a Brazilian oxbow lake. The abundance of Dolops carvalhoi was also related to water levels, maximum peaks of infestation occurring during high water (Malta & Varella 1983). The same seasonal pattern was found for Dolops discoidales by Malta (1982b). This type of seasonality may also have affected parasite abundance during the present study.

The species-rich Branchiuran parasite community on P. fasciatum offers an excellent opportunity to study host-parasite interactions, parasite isolation effects, and gene fluxes between parasite populations. Moreover, we recommend studying the impact of these abundant parasites on their fish host populations. Branchiuran parasites (as most other parasites) generally show an overdispersed distribution in their host populations (Anderson & Gordon 1982). This phenomenon, confirmed during the present study (Fig. 3; Table 3), suggests that some of these parasites may be controlling host population size. The observation that some parasite species seem to show truncated distributions (D. hirundo in Table 3 and Figure 3), suggesting host mortality at high parasite densities, should be investigated in more detail.

 

Acknowledgements

We thank Simon Claus, Jorge Coronel and Huascar Muñoz for assistance during field sampling. This study was realized in the framework of the project PROLIMCO (Own Initiative, Flemish University Council) and the Inter-university Cooperation (I.U.C.) between the Flemish University Council and the San Simón University of Cochabamba. Two anonymous referees are acknowledged for comments on an earlier version of the manuscript.

 

References

Agudelo Córdoba, E., Salinas Coy, Y., Sáncez Páez, C.L., Muñoz-Sosa, D.L., Alonso González, J.C., Arteaga Díaz, M.E., Rodríguez Prieto, O.J., Anzola Potes, N.R., Acosta Muñoz, L.E., Núñez Avellaneda, M. & Valdés Carrillo, H. 2000. Bagres de la Amazonía Colombiana: un recurso sin fronteras. SINCHI, Bogotá. 253 p.

        [ Links ]

Anderson, R.M. & Gordon, D.M. 1982. Processes influencing the distribution of parasite numbers within host populations with special emphasis on parasite-induced host mortalities. Parasitology 85: 373-398.

        [ Links ]

Barthem, R. & Goulding, M. 1997. The catfish connection. Ecology, migration and conservation of Amazon predators. Ed. Columbia University, Nueva York, USA. 144 p.

        [ Links ]

Bouvier, E.L. 1899. Les crustaces parasites du genre Dolops Audouin. Bulletin de la Societe Philomathique de Paris. 8(10)23:53-81.

Boeger, W.A. & Kritsky, D.C. 1977. Coevolution of the Monogenoidea (Platyhelminthes) based on a revised hypothesis of parasite phylogeny. International Journal for Parasitology 27: 1495-1511.

        [ Links ]

Calman, W.T. 1912. On Dipteropeltis, a new genus of the crustacean Order Branchiura. Proceedings of the Zoological Society of London. 763-766, pl.84.

        [ Links ]

Coronel, J.S., Maes, G.E., Claus, S., Van Damme, P.A., Volckaert, A.M. &P.A. Van Damme 2004. Differential population history in the migratory catfishes Brachyplatystoma flavicans and Pseudoplatystoma fasciatum (Pimelodidae) from the Bolivian Amazon assessed with nuclear and mitochondrial DNA markers. Journal of Fish Biology 65: 859-868.

Dobson, A.P. 1988. The population biology of parasite-induced changes in host behavior. The Quarterly Review of Biology 63(2): 139-159.

        [ Links ]

Elliot, J.M. 1977. Some methods for the statistical analysis of samples of benthic invertebrates. Freshwater Biological Association, Scientific Publication 25. 156 p.

        [ Links ]

Heller, C. 1857. Beitraege zur Kenntniss der Siphonostomen. Sitzungsberichten der Kaiserlichen Akademie der Wissenschaften in Wien, Mathematische-naturwissenschaftliche Klasse 25(1): 89-108, pls. I-III.

        [ Links ]

Hugghins, E.J. 1970. Argulids (Crustacea: Branchiura) from Ecuador and Bolivia. Journal of Parasitology. 56(5):1003.

        [ Links ]

Lemos de Castro, A. 1949. Contribución al conocimiento de crustáceos Argulideos de Brasil (Branchiura, Argulidae), con descripción de una nueva especie. Mus. Nac. Río de Janeiro, Zool. 93: 1-7.

        [ Links ]

Lemos de Castro, A. 1986. Branchiura. Manual de Identificacao de Invertebrados Limnicos do Brasil. Ministerio da Ciencia e Tecnologia, Conselho Nacional de Ciencia e Tecnologia, Coordenacao Editorial, Brasilia D.F. 30:1-23.

        [ Links ]

Loubens, G. & Panfili, J. 2000. Biologie de Pseudoplatystoma fasciatum et P. tigrinum (Teleostei : Pimelodidae) dans le bassin du Mamoré (Amazonie Bolivienne). Ichthyol. Explor. Freshwaters 11: 13-34.

        [ Links ]

Ludwig, J.A. & Reynolds, J.F. 1988. Statistical ecology: A primer on methods and computing. A Wiley-Interscience Publication, Nueva York. 327 p.

        [ Links ]

Malta, J.C.O. 1982a. Os argulídeos (Crustacea: Branchiura) da Amazonia Brasileira, 2. Aspectos da Ecologia de Dolops geayi Bouvier, 1897 e Argulus juparanaensis Castro, 1950. Acta Amazonica 12 (4): 701-706.

Malta, J.C.O. 1982b. Os argulídeos (Crustácea: Branchiura) da Amazonia Brasileira. Aspectos da ecología de Dolops discoidalis Bouvier, 1899 e Dolops bidentataBouvier, 1899. Acta Amazonica 12(3): 521-528.

        [ Links ]

Malta, J.C.O. 1983. Os argulídeos (crustacea: branchiura) da Amazonia brasileira. 4. Aspectos da ecologia de Argulus multicolor Stekhoven, 1937 e Argulus pestiferRinguelet, 1948. Acta Amazonica 13: 489-496.

Malta, J.C.O. 1983 & A. Varella 1983. Os argulídeos (crustacea: branchiura) da Amazonia Brasileira. Aspectos da ecología de Dolops striata Bouvier, 1899 e Dolops carvalhoi Castro, 1949. Acta Amazonica 13: 299-306.

Malta, J.C.O. 1984. Os peixes de um lago de várzea da Amazonia Central (Lago Janauacá, rio Solimoes) e suas relacoes com os crustaceos ectoparasitas (Branchiura: Argulidae). Acta Amazonica 14 (3/4): 355-372.

        [ Links ]

Margolis, L., Esch, G.W., Colmes, J.C., Curie, A.M. & Schad, G.A. 1981. The use of ecological terms in parasitology. J. Parasitol. 68: 133.

        [ Links ]

Muñoz, H. & Van Damme, P.A. 1998. Parámetros de reproducción de 4 especies de peces comerciales (Pseudoplatystoma fasciatum, P. tigrinum, Colossoma macropomum y Piaractus brachypomus) en la cuenca del río Ichilo (Cochabamba, Bolivia). Revista Boliviana de Ecología y Conservación Ambiental. 4: 39-54.

        [ Links ]

Navarro, G. & Maldonado, M. 2002. Geografía ecológica de Bolivia: Vegetación y ambientes acuáticos. Fundación Simon I. Patiño, Cochabamba. 719 p.

        [ Links ]

Nobre Carvalho, L., Del-Claro K. & R. Massato Takemoto 2003. Host-parasite interaction between branchiurans (Crustacea: Argulidae) and piranhas (Osteichthyes: Serrasalminae) in the Pantanal wetland of Brazil. Environmental Biology of Fishes 67 (3): 289-296.

Peralta, L., Solano, A., Carvalho, J.R., Matos, E. & Serra-Freire, N.M. 1998. Ocorrencia de Argulus juparanaensis Lemos de Castro, 1950 (Branchiura, Argulidae) em arraia de fogo Potamotrygon motoro (Müller & Henlz, 1841) (Rajifores: Potamotrygonidae) no Igarapé do Slito. Entomología y Vectores. 5 (1): 49-54.

Pla, L.E. 1986. Análisis multivariado: Método de componentes principales. Monografía Nº 27. Serie de Matemática. OEA. Washington. 95 p.

        [ Links ]

Poulin, R. 2002. The evolution of monogenean diversity. International Journal for Parasitology 32: 245-254.

        [ Links ]

Ringuelet, R. 1948. Argulídos del Museo de la Plata. Revista Mus. La Plata 5 (33): 81

        [ Links ]

Rodríguez, C. 1992. Bagres, malleros y cuerderos en el bajo río Caquetá. Ed. Tropenbos. Bogotá. 152 p.

        [ Links ]

Scott, M.E. & A. Dobson 1989. The role of parasites in regulating host abundance. Parasitology Today 5: 176-183.

Thatcher, V.E. 1990. Limnología et ecologia Regionalis Sistematis Fluminis Amazonas. Eds. Melonc y Kunk, W. Brasil. 11: 263-266.

        [ Links ]

Thatcher, V.E. 1991. Amazon fish parasites. Amazoniana 11 (3/4): 263-572.

        [ Links ]

 

Artículo recibido en: Enero de 2004

Manejado por: Robert Wallace

Aceptada en: Octubre de 2004.

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