Gasterosteus aculeatus Linnaeus, 1758, a new host fish for the endangered Margaritifera auricularia (Spengler, 1793) (Unionoida: Margaritiferidae)

The giant freshwater pearl mussel Margaritifera auricularia (Spengler, 1793) is considered the most imperilled bivalve species in Europe (Araujo & Ramos, 2001; Prié, 2010). Although likely widespread in most western European rivers at the beginning of the twentieth century, its decline in both abundance and distribution range has been estimated to be over 90% (Prié, 2010) and it is now nearly extinct. Only a few populations remain in Spain (Ebro Basin) and France (Loire, Charente, Garonne and Adour basins) (Araujo & Ramos, 2000a; Cochet, 2001; Prié et al., 2018; Soler et al., 2018).

Various causes have likely contributed to the extreme decline of M. auricularia populations, including pollution, climate change, commercial exploitation, host fish loss due to exotic fishes and dam construction (Araujo & Álvarez-Cobelas, 2016; Prié et al., 2018). Freshwater mussels have a temporary but obligatory parasitic stage in which the larvae (glochidia) attach to the external surface or gill filaments of their vertebrate hosts (mainly fishes). The glochidia of most freshwater mussels develop successfully into juveniles only on a limited number of host fish species (Jansen, Bauer & Zahner-Meike, 2001; Barnhart, Haag & Roston, 2008; Strayer, 2008). Therefore, mussel survival depends not only on habitat conservation but also on the availability of host fishes. Among 21 fish species tested, complete metamorphosis of M. auricularia glochidia into juveniles, which takes place after 4–6 weeks in the gills, has only been shown to occur in four sturgeon species (Acipenser sturio, A. naccarii, A. ruthenus and A. baerii) and the river blenny (Salaria fluviatilis) (Araujo & Ramos, 1998, 2000b; Araujo, Bragado & Ramos, 2001; Altaba & López, 2001; Araujo, Quirós & Ramos, 2003; López & Altaba, 2005; López et al., 2007; K. Nakamura, E. Elbaile, M.A. Muñoz-Yanguas, C. Catalá & C. Salinas, personal communication).

Scarce but recent recruitment of M. auricularia has been observed in several French localities (Prié et al., 2018); however, none of the known host fishes co-occur with these M. auricularia populations. The European sturgeon A. sturio is the only native Acipenser species that occurs in France. However, this species was extirpated from most European rivers during the twentieth century and is now almost extinct. Only one remaining reproductive population is known in the Garonne River (France). The river blenny is a Mediterranean species, and its distribution range does not coincide with that of the French populations of M. auricularia. Therefore, these populations appear to use an unknown host fish species and its determination is the objective of this note.

Given that host fish species for M. auricularia and for the majority of Margaritiferidae species have a close relationship with the marine environment, we hypothesized a similar relationship for this unknown host fish species. Sturgeons are anadromous fishes and the river blenny, although restricted to freshwater, can tolerate high salinity levels (Plaut, 1998) and is the only freshwater species of an otherwise entirely marine family. Furthermore, it has been observed that margaritiferid mussels frequently use anadromous host fishes (Curole, Foltz & Brown, 2004; Araujo et al., 2017): Margaritifera margaritifera, M. laevis, M. falcata, M. dahurica and M. middendorffi only use salmonids (Murphy, 1942; Karna & Milleman, 1978; Kobayashi & Kondo, 2005; Kondo & Kobayashi, 2005; Klishko & Bogan, 2013). However, the recent discovery of exclusively freshwater host fishes of the family Hiodontidae for M. monodonta (Sietman et al., 2017) and Esocidae for M. marrianae and M. hembeli (Fobian et al., 2017; P. Johnson 2018, personal communication) calls into question this hypothesis, although the salinity tolerances of these fishes are unknown.

Freshwater mussel host species can be inferred from natural infestations of glochidia on fishes, but it is preferable to combine this evidence with artificial laboratory infections to determine host suitability (e.g., Zale & Neves, 1982; Araujo, Gómez & Machordom, 2005; Taeubert et al., 2012) or by retrieving fully metamorphosed juveniles from naturally infected fish (Sietman et al., 2017).

Fish communities in the vicinity of major M. auricularia populations from the Charente, Vienne and Creuse rivers were assessed for natural infestation by electrofishing boat in April 2016, coinciding with their recently determined glochidial release period (Soler et al., 2018). A total of 613 specimens belonging to 23 fish species were analysed. Although the presence of three-spined stickleback (Gasterosteus aculeatus) is known in nearby small tributaries of the Vienne and Creuse rivers, it was not found in the stretches of these two rivers examined in this study. However, in the Charente River, we found M. auricularia glochidia attached to the gills of 2 of the 29 G. aculeatus specimens examined. This observation represents the first reported natural infestation by M. auricularia glochidia in the wild. No glochidia of other species of freshwater mussels were found in the inspected fish.

To test the susceptibility of G. aculeatus as a host to M. auricularia, 20 stickleback specimens were captured by electrofishing in a small stream tributary of the Vienne River near Chinon (France) on 28 March 2017. This stream was not colonized by M. auricularia. The animals were transferred to the laboratory in aerated containers. Thirty gravid M. auricularia specimens were collected from the Charente River on 26 March 2017 and maintained until mature glochidia emerged. Mussels and fish were kept in separate aquaria filled with aerated water from their respective rivers (without sediment) and at an average water temperature of 12.6 °C and 18 °C, respectively.

For induced infestation, glochidia were removed from the exhalant apertures of the mussels using a pipette and placed in a plastic bucket filled with aerated water (2,100 glochidia per l in 3.5 l) and the 20 fish. After 15 min, fish were transferred to a cylindroconical tank (250 l) equipped with biological and mechanical filters. They were kept in aerated river water at an average temperature of 19.3 °C and fed red mosquito larvae. Due to their small size, fish had to be sacrificed to verify glochidia encystment in the gills. Fish were anaesthetized then sacrificed at 1, 5, 10, 18 and 26 days after infestation.

Encysted glochidia were found in the gills of all inspected specimens, showing a gradual loss as metamorphosis progressed (Table 1). An outbreak of the fish parasite Ichthyophthirius sp. occurred 26 days after infestation. Though the fish were treated with malachite green, all died a day later (27 days after infestation). Nevertheless, on this day, eight fully transformed live juveniles were recovered from the gills of two of the fishes (Fig. 1A), thus indicating that the three-spined stickleback is a suitable host for M. auricularia glochidia.

Table 1.

Evolution of infestation intensity by Margaritifera auricularia glochidia during the encystment period on Gasterosteus aculeatus and Acipenser baerii.

Species	Days after infestation	Number of fish	Cysts/fish	Glochidia loss
G. aculeatus	1	2	29
	5	1	18	38%
	10	1	17	41%
	18	1	12	59%
	26	1	4	86%
A. baerii	6	1	2675
	28–43	1	2446	9%
	29–44	1	2517	6%
	35–50	1	2735	2%
	36–51	1	2685	0%

Figure 1.

Juvenile of Margaritifera auricularia at the end of metamorphosis in the gill of Gasterosteus aculeatus (light microscopy) (A) and Acipenser baerii (scanning electron microscopy) (B). Scale bars: A = 200 μm; B = 1 mm.

Gasterosteus aculeatus (Actinopterygii: Gasterosteidae) is a small fish (6 cm in length) with a large circumarctic and temperate distribution. Three-spined sticklebacks are known to inhabit brackish water, and even sea water, and anadromous populations are relatively frequent. Their ecological and behavioural characteristics may render them especially vulnerable to infestation by glochidia, since they are benthic feeders and may prey on glochidia (Dartnall & Walkey, 1979). It has been reported as a host for many unionid mussel species distributed throughout Europe, North America and Asia (Table 2) and, according to Lopes-Lima et al. (2017), is the host fish most commonly used by European freshwater mussels. Due to its ability to facilitate the metamorphosis of glochidia of mussels in a variety of genera and tribes, G. aculeatus could be considered a ‘universal host’, similar to other host species of the families Fundulidae and Poeciliidae (Haag, 2012).

Table 2.

Reported mussel species using Gasterosteus aculeatus as host fish.

Mussel species	Region	Type of infestation	Reference
Anodonta beringiana	NE Asia and NW America	NI	Saenko, Shedko & Kholin (2001)
		NI	Cope (1959)
Anodonta kennerlyi	NW America	NI + AI	Martel & Lauzon-Guay (2005)
Anodonta nuttalliana	NW America	AI	Maine, Arango & O’Brien (2016)
Margaritifera falcata	NW America	NI	Karna & Millemann (1978)
Pyganodon cataracta	NE America	NI	Lambert & Martel (2012)
		NI	Beaudet (2006)
		NI	Threlfall (1986)
Anodonta implicata or Pyganodon cataracta	NE America	NI	Wiles (1974)
Elliptio complanata	NE America	NI	Kneeland & Rhymer (2008)
Anodonta anatina	Europe	AI	Teutsch (1997)
Anodonta cygnea	Europe	AI	Teutsch (1997)
		NI	Dartnall & Wakey (1979)
Pseudoanodonta complanata	Europe	AI	Hüby (1988)
Unio pictorum	Europe	AI	McIvor (2004)
Unio tumidus	Europe	AI	Fleischauer-Rossing (1990)
Unio crassus	Europe	NI	Engel (1990)
		AI	Taeubert et al. (2012)
Margaritifera auricularia	Europe	AI	This study

Abbreviations: NI, natural infestation; AI, artificial infestation.

Although further research is required to establish the relative suitability of G. aculeatus, the high glochidial mortality found during the encystment period suggests that this host would not be ideal for M. auricularia glochidia. This observation is consistent with the low glochidial survival rates usually found in universal hosts (Teutsch, 1997; Haag, 2012; Taeubert et al., 2012).

Araujo et al. (2002) reported high survival rates of M. auricularia in the gills of the exotic species A. baerii, the most common host fish used in conservation programs. We re-inspected the preserved fish gills used in that experiment (Araujo et al., 2002) (Fig. 1B) and determined that glochidial mortality during metamorphosis ranged between 0% and 9% (Table 1). Margaritifera auricularia transformation on the small Spanish native fish Salaria fluviatilis was 318 juveniles per fish (Araujo et al. 2003), suggesting low mortality during metamorphosis in this host.

Overall our results suggest that, in France, G. aculeatus may serve as a native host for M. auricularia, similar to S. fluviatilis in Spain. Given the inadvisability of introducing exotic species into the French rivers supporting M. auricularia populations, and the difficulty of reintroducing the endangered A. sturio, G. aculeatus could be used to augment M. auricularia populations by releasing infested fish in river sections where both species occur.

Due to the small size of G. aculeauts, its absence in the Vienne River and the low glochidial survival rate observed, we think that another fish species could also be responsible for the maintenance of the French populations of M. auricularia. Thus, further research should be devoted in order to find other potential host species for this endangered freshwater mussel.

ACKNOWLEDGEMENTS

This work was supported by the LIFE+ project ‘LIFE13 BIO/FR/001162 Conservation of the Giant Pearl Mussel in Europe’. Thanks to the Charente-Maritime and Indre-Loire Prefectures for the collection permits. We also thank CETU ELMIS, BIOTOPE, FDAAPPMA 37 and 16, LOGRAMI and ONEMA for their contribution to the electrofishing surveys. Thanks to Yann Guerez, and Marjolaine Sicot for the capture and maintenance of the three-spined sticklebacks for experimental purposes and to Nina Richard, Philippe Jugé and Laure Morisseau for their contribution to the project. Thanks also to two anonymous referees and P.D. Johnson for their comments.