Cryptic microsporidian parasites differentially affect invasive and native Artemia spp.

International Journal for Parasitology 43 (2013) 795–803

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Cryptic microsporidian parasites differentially affect invasive and native Artemia spp. q Nicolas O. Rode ⇑, Eva J.P. Lievens, Adeline Segard, Elodie Flaven 1, Roula Jabbour-Zahab, Thomas Lenormand Centre d’Ecologie Fonctionnelle et Evolutive, UMR CNRS 5175, 1, 1919 route de Mende, 34293 Montpellier cedex 5, France

a r t i c l e

i n f o

Article history: Received 25 March 2013 Received in revised form 13 April 2013 Accepted 22 April 2013 Available online 11 July 2013 Keywords: Microsporidiosis Enemy release Biological invasions Artemia parthenogenetica Artemia franciscana Artemia salina Anostracospora rigaudi Enterocytospora artemiae

a b s t r a c t We investigated the host specificity of two cryptic microsporidian species (Anostracospora rigaudi and Enterocytospora artemiae) infecting invasive (Artemia franciscana) and native (Artemia parthenogenetica) hosts in sympatry. Anostracospora rigaudi was on average four times more prevalent in the native host, whereas E. artemiae was three times more prevalent in the invasive host. Infection with An. rigaudi strongly reduced female reproduction in both host species, whereas infection with E. artemiae had weaker effects on female reproduction. We contrasted microsporidian prevalence in native A. franciscana populations (New World) and in both invaded and non-invaded Artemia populations (Old World). At a community level, microsporidian prevalence was twice as high in native compared with invasive hosts, due to the contrasting host-specificity of An. rigaudi and E. artemiae. At a higher biogeographical level, microsporidian prevalence in A. franciscana did not differ between the invaded populations and the native populations used for the introduction. Although E. artemiae was the only species found both in New and Old World populations, no evidence of its co-introduction with the invasive host was found in our experimental and phylogeographic tests. These results suggest that the success of A. franciscana invasion is probably due to a lower susceptibility to virulent microsporidian parasites rather than to decreased microsporidian prevalence compared with A. parthenogenetica or to lower microsporidian virulence in introduced areas. Published by Elsevier Ltd. on behalf of Australian Society for Parasitology Inc.

1. Introduction Parasites have been shown to play a prominent role in determining the success and extent of biological invasions (Dunn, 2009). Studies investigating the importance of parasites in mediating invasion success usually rely on ‘community’ studies which contrast parasite prevalence in native and invasive species cooccurring within the same habitat, or on biogeographical studies which compare parasite prevalences in native versus introduced populations (Colautti and MacIsaac, 2004; Colautti et al., 2004). However, both approaches have rarely been conducted simultaneously using the same model system (Colautti et al., 2004; Liu and Stiling, 2006). In recent decades, New World brine shrimps, Artemia franciscana, have been repeatedly introduced to Old World salterns,

q Note: Nucleotide sequence data reported in this paper are available in the GenBank™ database under Accession Nos.: JX839889, JX839890, JX839891, JX915754, JX915755, JX915756, JX915757, JX915761. ⇑ Corresponding author. Tel.: +33 467 613 259; fax: +33 467 613 336. E-mail address: [email protected] (N.O. Rode). 1 Current address: Institut des Sciences de l’Evolution (UM2-CNRS), Université Montpellier 2, Montpellier, France.

which has led to a severe decline in native populations of Artemia parthenogenetica and Artemia salina (Amat et al., 2005, 2007; Mura et al., 2006; Scalone and Rabet, 2013). In southern France, dormant eggs, also called cysts, were intentionally and massively introduced from 1970 to 1983 from two American populations (San Francisco Bay and The Great Salt Lake, D. Facca, personal communication). Artemia are infected by several parasites, among which the microsporidia form an important group (Ovcharenko and Wita, 2005). Microsporidian parasites can have a strong impact on host fitness in natural populations (e.g. Stirnadel and Ebert, 1997; Otti and Schmid-Hempel, 2008; Ryan and Kohler, 2010). In addition, they are relatively host-specific compared with other parasites (Solter and Maddox, 1998; Shaw et al., 2000; Ebert, 2005; Saito and Bjornson, 2008). Hence, they are likely to affect the fitness of invasive and native hosts differentially and to play a role in the outcome of host competition. Surprisingly, only a handful of studies have investigated the potential role of microsporidia in host invasions (Slothouber Galbreath et al., 2004, 2010; Wattier et al., 2007; Yang et al., 2010). More generally, little is known about the importance of microsporidia in mediating invasion success. The goal of the current study is to examine the impact of two gut microsporidian parasites, Anostracospora rigaudi and

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N.O. Rode et al. / International Journal for Parasitology 43 (2013) 795–803

Enterocytospora artemiae, infecting native and invasive Artemia hosts in sympatry (Rode et al., 2013). These two cryptic parasites are both difficult to detect (spore size 1 lm) and difficult to distinguish from other small gut microorganisms (e.g. bacteria, yeast, unicellular algae), which makes them difficult to identify without molecular methods (Rode et al., 2013). Using field, phylogeographic and experimental approaches, we (i) investigated microsporidian host-specificity by comparing prevalence in the invasive A. franciscana and in the native A. parthenogenetica, (ii) quantified the impact of microsporidian parasites on the reproduction of sympatric females, (iii) compared microsporidian prevalence at the community and biogeographical scales and (iv) tested for the potential introduction of a microsporidian parasite with the invasive host. 2. Materials and methods 2.1. Anostracospora rigaudi and E. artemiae prevalence, phenotypic effects and effect on brooding probability of sympatric females from Aigues-Mortes, France Juvenile and adult Artemia were sampled with a plankton net in three shallow salterns (depth <30 cm) in Aigues-Mortes (France) in May 2011. Two samples were taken from Site 1 and Site 2, whereas one sample was taken from Site 3 (Table 1). Samples were kept in 10 L tanks (salinity 50 g/L, Thalasea, Camargue-Pêche, Grau du Roi, France) and fed ad libitum with an algal solution (Dunaliella tertiolecta). Individuals were randomly selected from each sample and their total length was measured to the nearest 0.05 mm. Sex, species, the number of infecting cestodes (mostly Flamingolepis liguloides) and the reproductive status of females (empty brood pouch versus presence of ovules/embryos) were observed using a binocular microscope. Artemia parthenogenetica females with atrophied or absent brood pouch due to F. liguloides infection were considered to be mature and non-reproducing when they measured more than 0.7 mm (which was 0.1 mm longer than the smallest reproducing female). The sample size of A. parthenogenetica was increased in order to obtain a higher number of A. parthenogenetica females not infected with F. liguloides, and thus increase our statistical power to independently disentangle the effects of single infections versus co-infections by several parasites on female brooding probability. The final sample size was 243 A. franciscana and 987 A. parthenogenetica individuals. Upon observation, individuals were killed and placed in ethanol in 96-well plates and PCR was performed using species-specific microsporidian primers (Msp1p2f/ Msp1p3r and Msp2p1f/Msp2p3r, see Rode et al., 2013 for the complete protocol). Microsporidian infection was judged based on the presence of bands on electrophoretic gels. We investigated the differential prevalence of An. rigaudi and E. artemiae in A. franciscana and A. parthenogenetica from Aigues-Mortes using v2 tests for independence (‘stats’ package in R 2.14.2). Analysis of female brooding probability was performed using a generalised linear model with a Bernouilli error distribution (‘stats’ package in R 2.14.2). Models included host species, presence of An. rigaudi, presence of E. artemiae, presence of F. liguloides, sample identity (five in total), length, the square of length, the interaction between the three parasite species together with each interaction between length and each of the other effects tested. In addition, we tested for differential phenotypic effects on invasive and native species by including an interaction between host species and the presence of An. rigaudi or E. artemiae. The effects of length (as a proxy for age) on the probabilities of infection by An. rigaudi and E. artemiae were analysed similarly. Models included host species, length and their interaction. All model selections were based on the corrected Akaike’s information criterion (AICc), which was computed independently for the

different models (Hurvich and Tsai, 1989). We discuss the effects of the best models (i.e. all models whose AICc differs by less than two from the lowest AICc; Burnham and Anderson, 2002).

2.2. Microsporidian prevalence, diversity and polymorphism in New and Old World populations Average microsporidian prevalence is usually low among Artemia populations, but generally high within infected populations (Codreanu, 1957; Martinez et al., 1992), a pattern commonly found in microsporidian parasites infecting other crustacean hosts (e.g. Ebert et al., 2001; Terry et al., 2004). To accurately estimate microsporidian prevalence despite this large between-population variation, we sampled a few individuals at a large number of sites. In order to investigate differential microsporidian prevalence between A. franciscana-invaded and native populations, we examined the presence of microsporidian parasites in 27 invaded and eight native salterns (n = 437 and n = 70, respectively; Tables 1 and 2). To test for parasite spillback in invaded populations and for E. artemiae introduction along with A. franciscana (see below and Section 2.3), microsporidian parasite prevalence was investigated in A. parthenogenetica and A. salina from eight invaded and nine non-invaded salterns from northern Africa, Europe and central Asia (n = 1040 and 67, respectively, Tables 1 and 2). All individuals were killed and preserved in 96% ethanol. DNA from each individual was screened for microsporidian infection using V1f/530r primers and An. rigaudi and E. artemiae species-specific primer sets (A. salina DNA quality was checked with the COI_FolF/COI_Fol-R primers; see Muñoz et al., 2008; Rode et al., 2013 for complete protocols). Importantly, such universal primers might fail to detect microsporidian species that have accumulated mutations in one of the primer sites. However, we did not expect any bias in the geographical distribution of these potentially undetected species, so our results should not be biased. Sample sizes were lower in non-French samples, resulting in a less accurate estimation of microsporidian prevalence within each population (Tables 1 and 2). We used v2 tests that account for the actual number of individuals sampled to compare microsporidian prevalence either between A. franciscana native and invaded populations or between A. parthenogenetica populations invaded by A. franciscana and non-invaded A. parthenogenetica populations. Hence, our results should be robust despite the low size of some population samples. To investigate the phylogeography of microsporidian parasites, we sequenced eight and 12 positive V1f/530r-PCR products from Old and New World populations, respectively, on an ABI prism 3130xl genetic analyser (Applied Biosystems, Foster City, California, USA) with an ABI Prism Big Dye Terminator cycle sequencing kit. Sequences were aligned using the ClustalW algorithm in BioEdit v7.0.9 (Hall, 2001, North Carolina State University, Raleigh, North Carolina, USA). A BLAST database search in GenBank was used to identify and select the closest matches together with already characterised clade-specific microsporidian sequences (Supplementary Table S1, Vossbrinck and Debrunner-Vossbrinck, 2005; Wang et al., 2005). Phylogenetic analyses were performed only on those portions of the sequences that could be unambiguously aligned. The selection of the best model for the base frequencies and substitution rates was based on AICc calculated using jModelTest 0.1, (TPM3 model with a gamma variation rate among sites; Posada and Crandall, 1998; Posada, 2008). Maximum likelihood analyses were carried out using Phyml v3.0 (Guindon et al., 2010) and robustness of nodes was assessed with 100 bootstrap replications. Representative sequences of newly characterised lineages (Microsporidium sp. 3, Microsporidium sp. 4) have been deposited in GenBank (Accession Nos.: JX839890, JX839891).

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N.O. Rode et al. / International Journal for Parasitology 43 (2013) 795–803 Table 1 Prevalence of microsporidian infection in A. franciscana invaded salterns from France and Israel. Sampling site

Host species

Microsporidia sp.

Prevalence in Ap (%)

Prevalence in Af (%)

n Sampled (Ap; Af)

Latitude– longitude

Sampling date

Invaded populations France Aigues-Mortes (Site 1)a

Ap; Af

Ar

60.1

12.7

336; 55

25 May 2011

Aigues-Mortes (Site 1)a

Ap; Af

Ea

17

52.7

336; 55

Aigues-Mortes (Site 2)a

Ap; Af

Ar

62.4

14.7

404; 95

Aigues-Mortes (Site 2)

a

Ap; Af

Ea

14.11

41.1

404; 95

Aigues-Mortes (Site 3)

a

Ap; Af

Ar

65.2

15.1

247; 93

Aigues-Mortes (Site 3)a

Ap; Af

Ea

15.8

45.2

247; 93

N43°310 2500 – E4°100 3700 N43°310 2500 – E4°100 3700 N43°310 2600 – E4°100 3500 N43°310 2600 – E4°100 3500 N43°300 5100 – E4°100 4200 N43°300 5100 – E4°100 4200 N47°240 55’’– W2°250 2’’ N46°590 56’’– W2°16’36’’ N46°590 56’’– W2°16’36’’ N46°590 56’’– W2°16’36’’ N43°210 48’’– E4°420 45’’ N43°210 48’’– E4°420 45’’ N43°210 48’’– E4°420 45’’ N43°200 3800 – E3°340 1000 N43°200 4000 – E3°340 1300 N43°220 5600 – E3°370 3100 N43°220 5100 – E3°370 2300 N43°220 5000 – E3°370 2300 N43°280 1300 – E5°90 3100 N43°280 4800 – E5°80 2600 N43°250 50’’– E4°570 00’’ N43°250 49’’– E4°570 01’’ N43°30 5000 – E6°80 4300 N43°40 3600 –E6°– 80 3400 N 43°40 3000 – E6°80 4000 N 43°40 2900 – E6°80 4000 N 43°70 2500 – E6°120 3300 N 43°70 2200 – E6°120 4200 N43°50 1300 – E3°50 400

Saline la Haye; Mès basin; Ansséracb

Af

–

–

–

0; 16

b

Af

–

–

–

0; 16

Petite Loire; Noirmoutierb

Af

–

–

–

0; 16

Petite Loire; Noirmoutierb

Af

–

–

–

0; 8

Salin-de-Giraud salterns; Salin-de-Giraud (Saint Genest)c Salin-de-Giraud salterns; Salin-de-Giraud (Saint Genest)c Salin-de-Giraud salterns; Salin-de-Giraud (Saint Genest)c Thau castellas; Marseilland

Ap

Ar

42.9

–

7; 0

Ap; Af

Ar

88.2

100

17; 3

Ap; Af

Ea

0

33

17; 3

Af

–

–

–

0; 8

Thau castellas; Marseillan (Site 1)

Af

–

–

–

0; 15

Sète-Villeroy (Thau Listel); Sèted

Af

–

–

–

0; 8

Ap

–

–

–

15; 0

Af

–

–

–

0; 15

Af

–

–

–

0; 8

Berre salterns; Berre l’Etang (Site 1)b

Ap

–

–

–

10; 0

Fos-sur-mer (Fos I)b

Af

–

–

–

0; 7

Af

–

–

–

0; 6

Pesquiers salterns; Hyèrese

Af

–

–

–

2; 6

Pesquiers salterns; Hyères (Site 1)e

Af

–

–

–

0; 10

Pesquiers salterns; Hyères (Site 2)e

Af

–

–

–

0; 10

e

Pesquiers salterns; Hyères (Site 3)

Af

–

–

–

0; 10

Vieux salins; Hyères (Site 1)b

Af

–

–

–

0; 10

Ap; Af

–

–

–

1; 9

Ap; Af

Ea

100

33.3

1; 6

Israel Ein Evrona salterns; Eilat (Site 63)f

Af

–

–

–

0; 1

Ein Evrona salterns; Eilat (Site 65)f

Petite Loire; Noirmoutier

d

Sète-Villeroy (Thau Listel); Sète (Site 1)d d

Sète-Villeroy (Thau Listel); Sète (Site 2) Berre salterns; Berre l’Etang

Fos-sur-mer (Fos II)

b

b

Vieux salins; Hyères (Site 2)b Saint Martin salterns; Gruissan

c

Af

Ea

–

100

0; 3

f

Af

Ea

–

100

0; 1

f

Af

–

–

–

0; 1

Eilat salterns; Eilat (Site 201)f

Af

Ea

–

100

0; 1

Eilat salterns; Eilat (Site 104) Eilat salterns; Eilat (Site 200)

N29°370 4600 – E34°590 5600 N29°370 2400 – E34°590 5400 N29°340 900 – E34°580 500 N29°330 4400 – E34°580 000 N29°330 1900 – E34°580 1900

25 May 2011

25 May 2011

15 November 2011 9 May 2011 17 May 2011 21 October 2011 20 May 2011 19 October 2011 19 October 2011 14 May 2002 31 March 2012 14 May 2002 31 March 2012 31 March 2012 15 May 2002 16 April 2012 15 May 2002 15 May 2002 15 May 2002 16 April 2012 16 April 2012 16 April 2012 16 April 2012 16 April 2012 28 December 2011 2008 2008 2008 2008 2008

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N.O. Rode et al. / International Journal for Parasitology 43 (2013) 795–803

Ap, Artemia parthenogenetica; Af, Artemia franciscana; Ar, Anostracospora rigaudi; Ea, Enterocytospora artemiae. a Introduction with San Francisco Bay cysts in September 1970 and from 1974 to 1979. Introduction with Great Salt Lake cysts in 1979 (D. Facca, personal communication). b Unknown introduction date. c No human introduction, introduction most likely through waterbirds (D. Facca, personal communication). d Introduction in July 1970 (D. Facca, personal communication). e Introduction before 1970 (D. Facca, personal communication). f Introduction in 1979 (Zmora and Popper, 1985).

Table 2 Prevalence of microsporidian infections in non-invaded Old World populations and in original New World populations. Sampling site

Host Species

Microsporidia sp.

Prevalence (%)

n Sampled

Latitude–longitude

Sampling date

Ap

–

–

3

Unknown

Unknown

– – – –

8

N39°120 21’’–E9°90 24’’

Non-invaded Old World populations Germany Province of Thüringen, Germany (Unknown) Italy Vasche retrolitorali, Molentargius Park, Cagliari Marroco Oualidia

As

– – – –

Spain Odiel (E13) Odiel (E14)

Ap Ap

– –

Ukraine Ukraine (Unknown) Ukraine (Unknown)

Ap Ap

Uzbekistan Cape Aktumsuk, Utsyurt, Karakalpakstan, Uzbekistan Tashkent, Uzbekistan

As

0 00

0

12 October 2011 00

15

N32°50 6 –W8°54 11

– –

13 13

N37°150 1700 –W6°590 1800 N37°150 1700 –W6°590 2200

26 July 2011 26 July 2011

Ar Ar

25 100

4 4

Unknown Unknown

1998 1998

Ap Ap

– –

– –

4 3

N44°350 5700 –E58°170 5600 Unknown

2002 2000

USA Great Salt Lake, Utah (Spiral Jetty)

Af

Ea

18.8

16

1 October 2011

Great Salt Lake, Utah (Spiral Jetty)

Af

Msp4

6.3

16

Mono Lake, CA

Af

Ea

31.8

22

Alviso Pond, SFB, California (A22)

Af

–

–

4

Alviso Pond, SFB, California (A23)

Af

–

–

4

Eden Landing Pond, SFB, California (B5C)

Af

–

–

4

Eden Landing Pond, SFB, California (B8X)

Af

Msp3

37.5

8

Eden Landing Pond, SFB, California (B12)

Af

–

–

4

Ravenswood Pond, SFB, California (R1)

Af

–

–

8

N41°260 1600 – W112°400 03’’ N41°260 1600 – W112°400 03’’ N37°590 27’’– W119°080 04’’ N37°290 03’’– W121°570 40’’ N37°280 31’’– W121°570 39’’ N37°340 21’’– W122°050 56’’ N37°360 15’’– W122°070 05’’ N37°360 56’’– W122°070 26’’ N37°290 56’’– W122°080 35’’

20 March 2012

New World populations

1 October 2011 14 October 2010 25 March 2008 25 March 2008 26 March 2008 20 March 2008 20 March 2008 18 March 2008

SFB: San Francisco Bay. Ap, Artemia parthenogenetica; Af, Artemia franciscana; Ar, Anostracospora rigaudi; Ea, Enterocytospora artemiae; Msp3, Microsporidium sp. 3; Msp4, Microsporidium sp. 4.

2.3. Test for the possible introduction of E. artemiae spores with A. franciscana cysts As E. artemiae was found in both Old World and New World samples (see Section 3.3) and since horizontal transmission following spore desiccation has been reported in some microsporidian species (Vizoso et al., 2005), we investigated the co-introduction of E. artemiae parasites together with A. franciscana cysts (direct transplantation of infected individuals from American to European or Israeli populations is very unlikely, D. Facca and O. Zmora, personal communication). We investigated E. artemiae polymorphism in populations where A. franciscana is native and invasive and we sequenced a larger fragment of the small subunit gene using nested PCRs. We first amplified a fragment with the V1F/1537R primers (Vossbrinck et al., 1993) in E. artemiae-infected individuals from native (two from The Great Salt Lake, Utah, USA and two from Mono Lake, California, USA) or invasive populations (two from

Eilat, Israel). We subsequently amplified nested fragments with the V1F/HG4R primers (Gatehouse and Malone, 1998) and the 734F/1537R primers (Rode et al., 2013). To assess E. artemiae polymorphism, the reference French sequence (JX915760) was aligned with the six new sequences using the ClustalW algorithm in BioEdit v7.0.9 (Hall, 2001). The new E. artemiae sequences have been deposited in GenBank with the Accession Nos.: JX839889, JX915761 (The Great Salt Lake, Utah, USA), JX915754, JX915755 (Mono Lake, California, USA), JX915756 and JX915757 (Ein Evrona, Eilat, Israel). Second, we used cysts collected in different years from the two American populations which were used for the introduction of A. franciscana in Aigues-Mortes (San Francisco Bay, 1979, 1989, 2005 and The Great Salt Lake 1977, 1989, 2005) and used cysts from two Aigues-Mortes populations with high E. artemiae prevalence as positive controls (Site 9, 2010, N43°320 2500 –E4°130 2600 , Pont l’Abbé, 2011, N43°320 4000 –E4°90 17). We tested for the

N.O. Rode et al. / International Journal for Parasitology 43 (2013) 795–803

presence of microsporidia on the cyst surface by grinding 0.1 g (104 embryos) of intact cysts from each sample, using bleachdecapsulated cysts as negative controls (bleach is known to remove microsporidian spores from the egg surface; Goertz and Hoch, 2008). DNA was extracted using a HotShot method (Sigma, Saint Louis, Missouri, USA) and PCR tested for E. artemiae DNA. Since DNA detection of E. artemiae is facilitated in early infection stages compared with spores (Hatakeyama and Hayasaka, 2002; Rode et al., 2013), we also hatched 0.1 g of intact and bleachdecapsulated cysts from San Francisco Bay (2005), The Great Salt Lake (2005) and Aigues-Mortes (2011). After 1 month, we tested 15 adults per treatment/population combination using PCR, as described previously.

3. Results 3.1. Anostracospora rigaudi and E. artemiae prevalence and phenotypic effects on sympatric hosts from Aigues-Mortes The prevalence of An. rigaudi in Aigues-Mortes was approximately 15% in A. franciscana and 60% in A. parthenogenetica (v2ð1Þ = 179.6, P < 105, Fig. 1). Hence, the native host species appears to be more susceptible to An. rigaudi infection. In contrast, E. artemiae prevalence was approximately 45% and 15% in A. franciscana and A. parthenogenetica, respectively (v2ð1Þ = 102.8, P < 105, Fig. 1). Hence, the invasive host species appears more susceptible to E. artemiae infection. 3.2. Effect of parasites on female brooding probability and effect of length on parasitic infection probability Both An. rigaudi and F. liguloides infections had a clear negative impact on female brooding probability (Fig. 2, Table 3). In contrast, although E. artemiae also had a negative impact on female brooding probability, this effect was much smaller and was not present in all

1.0

*** Microsporidian Prevalence in A. franciscana and A. parthenogenetica

0.8 ***

0.6

0.4

0.2

799

of the best models (Fig. 2, DAICc = 1.17, Table 3). Furthermore, all best models included host species, length and sample (DAICc < 2, Table 3). Indeed, A. parthenogenetica females were on average more likely be reproducing than A. franciscana females at the time of sampling (Fig. 2A versus B), and large females were more likely to be reproducing than small ones (Fig. 2). Importantly, none of the best models included an interaction between female species and either An. rigaudi or E. artemiae infection (DAICc > 2, Table 3), indicating than the decrease in brooding probability did not differ between A. franciscana and A. parthenogenetica infected females. In addition, the positive interaction between female length and An. rigaudi presence found in several of the best models suggested a 20% decrease in brooding probability in small infected compared with non-infected females (Fig. 2, Table 3), whereas no difference was observed in large females (3% increase in Fig. 2). Again, this decrease in small females did not differ between host species (DAICc > 2, Table 3). Finally, the negative interaction found between An. rigaudi and F. liguloides infections in some of the best models suggested a greater reproductive cost of the presence of both parasites in co-infected A. parthenogenetica females (Fig. 2A, Table 3). Length had a positive effect on the infection probability by either An. rigaudi or E. artemiae (Fig. 3, Tables 4 and 5). Models with different length effects for A. franciscana and A. parthenogenetica females had low supports (DAICc > 1.7, Tables 4 and 5). 3.3. Microsporidian prevalence, diversity and polymorphism in New and Old World populations Two new sets of divergent microsporidian sequences (hereafter Msp3, Msp4) were found in American populations (Fig. 4). These species clustered in a clade of microsporidian parasites infecting mostly the gut tissues of their insect and crustacean hosts (Vossbrinck and Debrunner-Vossbrinck, 2005; Rode et al., 2013; Supplementary Fig. S1). At the biogeographical level, the average microsporidian prevalence in A. franciscana tended to be higher in introduced than in native ranges, but the difference was not significant (v2ð1Þ = 2.8, P = 0.09, Fig. 3). In contrast, at the community level, average microsporidian prevalence was twice as high in A. parthenogenetica as in A. franciscana from French and Israeli invaded populations (65% versus 30%, v2ð1Þ = 157.8, P < 105, Fig. 3). These results held true qualitatively when analyses were restricted to samples with at least one A. parthenogenetica and A. franciscana individual (67% versus 47%, v2ð1Þ = 37.4, P < 105). Hence, differential susceptibility between A. franciscana and A. parthenogenetica could contribute to the invasive success of A. franciscana. Furthermore, microsporidian prevalence in A. parthenogenetica in invaded populations was much greater than in non-invaded populations (65% versus 11%, v2ð1Þ = 53.0, P < 105, Fig. 4). No sequence polymorphism of the ribosomal small subunit (340 bp) was found within An. rigaudi, E. artemiae and Msp3 fragments, for which different samples were available. Each sequence is thus likely to represent only one parasite species with a wide (An. rigaudi and E. artemiae) or narrow (Msp3) population range. 3.4. Test for E. artemiae spore introduction with A. franciscana cysts

0.0 A. rigaudi

E. artemiae

A. franciscana

A. rigaudi

E. artemiae

A. parthenogenetica

Fig. 1. Observed prevalence of Anostracospora rigaudi and Enterocytospora artemiae parasites in the invasive Artemia franciscana and native Artemia parthenogenetica hosts. Error bars: S.D. across samples. ⁄⁄⁄P < 0.001. n = 243 for A. franciscana and 987 for A. parthenogenetica.

A very low polymorphism was observed when comparing the seven E. artemiae sequences (length 876 bp) from France, Israel and the USA. Indeed, the sequences comprised only seven polymorphic sites (four substitutions, two deletions, one insertion). Interestingly, one substitution (C to T) was shared among invaded populations from France and Israel, but was absent from the two American populations. All of the other variable sites were sam-

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B

1.0

Fitted Brooding Probability for Af females

Fitted Brooding Probability for Ap females

A

0.8

0.6

0.4

0.2

0.0

1.0

0.8

0.6

0.4

No infection A. rigaudi infection

0.2

E. artemiae infection F. liguloides infection A. rigaudi and F. liguloides infection

0.0 0.6

0.7

0.8

0.9

1.0

0.6

Female Length (cm)

0.7

0.8

0.9

1.0

Female Length (cm)

Fig. 2. Fitted effects of Anostracospora rigaudi, Enterocytospora artemiae and Flamingolepis liguloides infections on Artemia parthenogenetica (Ap) (A) and Artemia franciscana (Af) (B) female brooding probability. Fitted values were computed with the best model including the effect of E. artemiae infection (DAICc = 1.17, Table 3).

Table 3 Effect of parasite infection on female brooding probability. K

logLik

AIC

AICc

DAICc

wi

Parameters

11 9 12 10 9 7 5 13 11 11 6 11 6 9 12 13 14 14 15 9

224.56 226.90 224.12 226.51 227.64 229.70 231.85 223.80 225.88 226.00 231.14 226.36 231.51 228.60 225.60 225.53 225.10 225.53 225.03 628.59

471.11 471.81 472.24 473.01 473.28 473.40 473.70 473.60 473.76 474.00 474.28 474.71 475.02 475.20 475.21 477.07 478.20 479.07 480.07 1275.17

471.38 471.99 472.55 473.23 473.46 473.51 473.76 473.96 474.02 474.26 474.36 474.97 475.10 475.38 475.52 477.43 478.62 479.49 480.55 1275.35

0.00 0.61 1.17 1.85 2.08 2.14 2.38 2.59 2.64 2.89 2.99 3.60 3.72 4.00 4.14 6.05 7.24 8.11 9.17 803.98

0.18 0.13 0.10 0.07 0.06 0.06 0.05 0.05 0.05 0.04 0.04 0.03 0.03 0.02 0.02 0.01 0.00 0.00 0.00 0.00

Length*Ar + Fl*Ar + species + sample Length + Fl + Ar + species + sample Ea + length*Ar + Fl*Ar + species + sample Length + Fl + species + Ar + Ea + sample Length + Fl + Ea + species + sample Length*Ar + Fl*Ar + species Length + Fl + species + Ar Ea + length*Ar + Fl*Ar + species + length*Ar*species + sample Ea + length*Ar + Fl + species + sample Ea + length + Fl*Ar + species + sample Length*Ar + Fl + species Length2 + length + Fl + Ar + Ea + species + sample Length + Fl*Ar + species Length + Fl + Ar + Ea + sample Length2 + length + Fl + length*Ar + Ea + species + Ar + Ea + sample Length2 + length + Fl + length*Ar + length*Ea + species + Ar + Ea + sample Length2 + length + Fl + length*Ar + length*Ea + species*Ar + Ea + sample Length2 + length + Fl + length*Ar + length*Ea + Ar + species*Ea + sample Length2 + length + Fl + length*Ar + length*Ea + species*Ar + species*Ea + sample Length + Ar + Ea + species + sample

K, number of parameters; logLik, loglikelihood; AIC, Akaike Information Criterion; AICc, AIC corrected for small sample size; DAICc, AICc difference with the best model; wi, AICc weights; Parameters, variables included in the model; Fl, Ar, Ea, infection by either Flamingolepis liguloides, Anostracospora rigaudi or Enterocytospora artemiae; length, female length; species, female host species; sample, sample identity. * Denotes an interaction between two variables.

ple-specific. Hence, E. artemiae differentiation between Old World and New World strains appeared very low. No microsporidian DNA was detected in the raw and decapsulated cysts from the two American populations (San Francisco Bay, Great Salt Lake) used for A. franciscana introduction in Aigues-Mortes. We also failed to detect any infection in the positive controls (cysts from Aigues-Mortes populations with high E. artemiae prevalence). In addition, no microsporidian infection was detected in any of the adults hatched from raw and decapsulated cysts from the same populations. Hence, we did not find any evidence that E. artemiae could have been co-introduced during the intentional introduction of A. franciscana cysts in Aigues-Mortes and Israel. However, since these introductions implied massive amounts of cysts, our experiment may not be entirely appropriate to detect very rare E. artemiae introduction events that may have occurred.

4. Discussion Parasites can favour biological invasion, whenever they disproportionately affect the fitness of native compared with invasive hosts (Dunn, 2009). We investigated the role of microsporidian parasites in mediating the competition between invasive and native Artemia hosts. At the community scale, the native A. parthenogenetica was on average twice as susceptible to microsporidian infection as the invasive A. franciscana. Investigating microsporidian infection at the species level allowed us to unravel the causes of these discrepancies. Indeed, A. parthenogenetica hosts appear four times as susceptible to the virulent An. rigaudi compared with A. franciscana hosts, whereas A. parthenogenetica hosts are only onethird as susceptible to the less virulent E. artemiae compared with A. franciscana hosts (Figs. 1 and 2). The latter result appears quite robust, since it did not account for the larger size (and the likely

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B

1.0

Probability of Infection by E. artemiae

Probability of Infection by A. rigaudi

A

0.8

0.6

0.4

0.2

0.0

1.0

0.8

0.6

0.4

0.2

0.0

0.6

0.7

0.8

0.9

1.0

Female Length (cm)

0.6

0.7

0.8

0.9

1.0

Female Length (cm)

Fig. 3. Fitted effect of female length on Anostracospora rigaudi (A) or Enterocytospora artemiae (B) infection probability for Artemia parthenogenetica (solid line) and Artemia franciscana (broken line) females. Observed infection probabilities are represented for the different classes (60.7 cm, 60.8 cm and 61.28 cm) of A. parthenogenetica (solid circles) and A. franciscana (open circles) female length.

Table 4 Effect of length on Anostracospora rigaudi infection probability. K

logLik

AIC

AICc

DAICc

wi

Parameters

3 4 2 2

656.79 656.66 658.81 678.82

1319.57 1321.32 1321.62 1361.64

1319.59 1321.36 1321.63 1361.65

0.00 1.76 2.03 42.06

0.56 0.23 0.20 0.00

Length + species Length*species Species Length

K, number of parameters; logLik, loglikelihood; AIC, Akaike Information Criterion; AICc, AIC corrected for small sample size; DAICc, AICc difference with the best model; wi, AICc weights; Parameters, variables included in the model; length, female length; species, female host species. * Denotes an interaction between two variables.

Table 5 Effect of length on Enterocytospora artemiae infection probability. K

logLik

AIC

AICc

DAICc

wi

Parameters

3 4 2 2

455.13 454.99 460.90 471.77

916.26 917.97 925.80 947.54

916.29 918.01 925.81 947.56

0.00 1.72 9.52 31.27

0.70 0.30 0.01 0.00

Length + species Length * species Species Length

K, number of parameters; logLik, loglikelihood; AIC, Akaike Information Criterion; AICc, AIC corrected for small sample size; DAICc, AICc difference with the best model; wi, AICc weights; Parameters, variables included in the model; length, female length; species, female host species. * Denotes an interaction between two variables.

higher rate of spore ingestion) of A. parthenogenetica compared with A. franciscana (Triantaphyllidis et al., 1995). Hence, microsporidian parasites impose greater demographic costs on A. parthenogenetica compared with A. franciscana, in addition to the castrating effect of F. liguloides on A. parthenogenetica which is highlighted in this and other studies (Fig. 2, Amat et al., 1991; Varó et al., 2000; Sánchez et al., 2012). Interestingly, the detrimental effects of An. rigaudi tended to be restricted to small females (Fig. 2). This decrease could be due to old (and consequently large) females becoming parasitised while already carrying eggs, to large females being more able to cope with parasitic infection or to the better survival of females that can tolerate the deleterious effects of An. rigaudi and reproduce. We found a positive effect of female length on infection probability (Fig. 3). The higher ingestion rate (Reeve, 1963) and the longer exposition to parasites of old (and large)

females are likely explanations for this pattern. This higher parasite prevalence in large females also suggests that parasitic effects on female survival are absent or low (as shown in related intestinal microsporidian species; Decaestecker et al., 2003). Importantly, none of the phenotypic effects detected differed between A. parthenogenetica and A. franciscana females. In particular, the detrimental effect of microsporidian infection did not differ between hosts. Hence, host-specific transmission rather than host-specific virulence is likely to mediate the competitive advantage of A. franciscana over A. parthenogenetica. At a biogeographical scale, average microsporidian prevalence was much higher in A. parthenogentica populations invaded by A. franciscana compared with A. parthenogenetica non-invaded populations (65% versus 11%). These differences could be due to genetic differentiation between populations of hosts and/or parasites. An alternative hypothesis would be that the coexistence of both hosts leads to an increase in parasite prevalence (de Castro and Bolker, 2005). For instance, A. parthenogenetica disappears almost completely during winter in Aigues-Mortes (most likely due to low temperatures; Sánchez et al., 2012). The invasive A. franciscana represents a reservoir host for An. rigaudi that can survive winter in infected A. franciscana individuals. Anostracospora rigaudi could thus reach higher prevalence in new A. parthenogenetica generations hatching from cysts in spring. Given its higher susceptibility to An. rigaudi, we predict A. parthenogenetica should suffer higher demographic costs from such spillback. Empirical and theoretical studies have shown that temporal or spatial variations in susceptible hosts can greatly affect parasite prevalence in host populations (Altizer et al., 2006; Condeso and Meentemeyer, 2007; Kelly et al., 2009; O’Brien et al., 2011). Furthermore, we suspected a co-introduction of E. artemiae with A. franciscana in France and Israel. Indeed, E. artemiae was the only microsporidian species found in both New and Old World populations. In addition, E. artemiae was consistently more prevalent in A. franciscana than in A. parthenogenetica when in sympatry. In favour of this hypothesis, we found a very low spatial structure in E. artemiae compared with microsporidian phylogeographic studies based on the same marker (Krebes et al., 2010; Wilkinson et al., 2011; Li et al., 2012). We found only one single nucleotide polymorphism (SNP) differentiating Old World from New World isolates. Given the relatively small size of our sample, this SNP could actually be present in American populations. Our

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No Infection A. rigaudi

11% 20% Af (Native Range)

E. artemiae

30% Af (Invasive range)

Ap (Invasive range of Af)

Ap (Non-introduced range of Af)

65%

A. rigaudi / E. artemiae Msp3 Msp4

Fig. 4. Comparison of microsporidian prevalence between New World native Artemia franciscana (Af) populations, Old World A. franciscana and Artemia parthenogenetica (Ap) invaded populations and Old World A. parthenogenetica populations where A. franciscana was not introduced. Percentages represent average prevalence when pooling the different microsporidian species. Microsporidian prevalence in Artemia salina was zero and is not represented.

experimental tests in the laboratory, mimicking the introduction of undecapsulated cysts in Aigues-Mortes (F. René, D. Facca, personal communication) failed to demonstrate any transmission of E. artemiae to nauplii hatched from raw cysts. Hence, these results suggest that the genetic exchange between Old World and New World populations (e.g. through birds; Higes et al., 2008; Valera et al., 2011) predated the invasion of A. franciscana. More extensive sampling of native A. franciscana populations (San Francisco Bay, Great Salt Lake) combined with large-scale genotyping would allow us to accurately trace the historical gene flow between American and Old World populations of E. artemiae. Overall, A. franciscana invasion success is likely due to a lower susceptibility to (virulent) microsporidian parasites compared with its A. parthenogenetica congener rather than to decreased microsporidian prevalence or lower parasite virulence in introduced areas. Microsporidian prevalence in A. franciscana native and invaded ranges did not differ, but the invader was four times less likely to be infected by the virulent An. rigaudi compared with native A. parthenogenetica. Artemia franciscana consequently pays lower demographic costs in invaded areas where An. rigaudi is present. This is further evidence that parasites play an important role in altering the outcome of competition between invasive and native Artemia spp. (Sánchez et al., 2012). More specifically, we show here that widespread parasites, which can easily remain undetected, can nevertheless have strong and differential fitness effects. As A. parthenogenetica and A. franciscana have coexisted for more than 40 years in some invaded populations, the ecological factors counter-balancing the detrimental fitness effects of their different parasites remain to be determined. Acknowledgements The authors are grateful to C. Vivares, P. Agnew, Y. Michalakis, T. Rigaud and M.-P. Dubois for their advice and help regarding the molecular investigation of microsporidiosis. We are indebted to M. Sanchez, C. Mathieu, G. Van Stappen, JP. Rullmann, R. Jellison, JY. Takekawa, AG. Saez, J. Butler, S. Bonnet-Questiau, I. Gallois-Morin, A. Atzeni, G. Martin, P-A. Crochet for providing some of the samples. We also wish to thank T. Pugliano, JL. Kammradt, T. Gout and F. Gout who assisted in sampling and R. Tkavc for pointing out the presence of a microsporidium in Eilat and providing individual samples. We thank O. Zmora, D. Facca, F. René, B. Menu, A. Muller-Feuga, P. Serene and L. Euzet for their information regarding the introduction of A. franciscana in Southern France and Israel. We also thank two anonymous reviewers who provided useful comments. Financial support was provided by the European

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