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J. Parasitol., 95(1), 2009, pp.

䉷 American Society of Parasitologists 2009


Marta I. Sánchez*, Frédéric Thomas, Marie-Jeanne Perrot-Minnot†, David G. Biron‡, Justine Bertrand-Michel§, and
Dorothée Missé
Equipe ‘‘Evolution des Systèmes Symbiotiques,’’ GEMI, UMR CNRS/IRD 2724, IRD, 911 Avenue Agropolis, BP 64501, 34394 Montpellier
CEDEX 5, France. e-mail:

ABSTRACT: There are many impressive examples of host manipulation by parasites, but mechanisms underlying these ethological
changes, as well as their physiological consequences, are not well characterized. Here, we analyzed part of the cerebral proteome
of brine shrimp Artemia infected by manipulative cestodes, using for the first time the ProteinChip Surface-Enhanced Laser
Desorption Ionization and Time of Fly Mass Spectrometry (SELDI-TOF MS) system, which has been proposed as an excellent
way to analyze the host genome during the host–parasite interaction processes. We found 2 peptides downregulated in individuals
infected by the dilepidid, Anomotaenia tringae (4.5 kDa), and by the 2 hymenolepidids, Flamingolepis liguloides and Confluaria
podicipina (3.9 kDa), which are potential candidates for involvement with the manipulation process. The identification of 2 head
peptides (4.1 and 4.2 kDa) overexpressed in all the categories in brine shrimp living at the surface (both infected individuals and
uninfected controls) suggests its association with the different environmental conditions experienced at the water surface. In
parallel, brine shrimp infected by C. podicipina showed significant values of triglycerides, potentially augmenting their profit-
ability and attractiveness for the predaceous definitive host (grebes). We discuss our findings in relationship with current ideas
on the complexity of parasitically modified organisms.

During the last 3 decades, the study of phenotypic changes of the avian definitive hosts. The newly hatched oncosphere
in parasitized organisms has attracted the interest of many re- penetrates into the hemocoel, where it develops into a cysticer-
searchers (Adamo, 2002; Moore, 2002; Thomas et al., 2005). coid. In at least 3 cestode species (Flamingolepis liguloides
In many of the best-known examples, changes displayed by infecting flamingos, Confluaria podicipina infecting grebes, and
infected hosts appear advantageous for the parasites. For in- Anomotaenia tringae infecting shore birds), the presence of cys-
stance, parasite-induced alterations in host behavior can result ticercoids is associated with changes in host behavior and color
in the infected host becoming more vulnerable to predation by (Gabrion et al., 1982; Amat et al., 1991; Robert and Gabrion,
the parasite’s next host (Lafferty, 1999; Poulin, 2007). The al- 1991; Sánchez, Georgiev et al., 2006; Sanchez et al., 2007),
tered host phenotype may also cause the host to migrate toward making infected individuals more vulnerable to foraging birds.
particular microhabitats, where the parasite must be to complete Adult tapeworms develop and reproduce in the small intestine
its transmission cycle (Moore, 2002; Poulin, 2007). An impor- of bird final hosts, concluding their life cycle.
tant aspect of recent articles on parasite manipulations (see We first designed the present study to explore some of the
Thomas et al., 2005) is the recognition that these phenomena proximate mechanisms underlying behavioral modifications in
are probably more complex than traditionally viewed, i.e., ma- parasitized A. parthenogenetica. Although it is generally ad-
nipulated hosts being altered by a range of changes, in addition mitted that manipulative parasites can change host behavior by
to the most obvious ones, e.g., aberrant behavior. Along the directly and/or indirectly modulating the host’s central nervous
same idea, it seems that the mechanisms underlying ethological system, empirical demonstrations of such neurophysiological
changes following infection are in many cases sophisticated, interaction between manipulated hosts and parasites are very
some sort of chemical interference with host neurochemistry, scarce. Recently, proteomics has been shown to offer an ex-
endocrine function, and/or gene expression (Thomas et al., cellent way to analyze the host genome during the host–parasite
2005). interaction processes (Biron, Joly et al., 2005). Here, we used
The representatives of the cosmopolitan crustacean Artemia for the first time The ProteinChip Surface-Enhanced Laser De-
(Branchiopoda: Anostraca) are key species in extreme hyper- sorption Ionization and Time of Fly Mass Spectrometry (SEL-
saline environments (e.g., Sánchez, Green et al., 2006). In salt- DI-TOF MS) system (Cipergen娂, Freemont, California) (Vor-
pans of southwest Spain, Artemia parthenogenetica is inter- derwülbecke et al., 2005), to contrast the head proteome of
mediate host for at least 10 species of cyclophyllidean tape- uninfected individuals with brine shrimp infected by several
worms, the adults of which infect several species of water birds species of manipulative cestodes. Because of parasite-induced
(see Georgiev et al., 2005). Brine shrimp become infected by behavioral changes, we predict that there should be substantial
consuming cestode eggs released into the water with the feces differences in the protein profiles of infected and uninfected
individuals. A second aim of this work was to compare the lipid
contents of infected and uninfected A. parthenogenetica. In-
Received 6 December 2007; revised 9 April 2008, 26 May 2008; deed, for reasons that are still unclear, several recent articles
accepted 26 May 2008.
* Present address: CEFE, UMR 5175, CNRS, 1919 Route de Mende, suggest that aquatic crustaceans parasitized by manipulative
34293 Montpellier, France. helminths completing their life cycles in aquatic birds, i.e.,
† Equipe Ecologie Evolutive, UMR CNRS 5561 Biogeosciences, Uni- trematodes (Ponton et al., 2005), acanthocephalans (Plaistow et
versité de Bourgogne, 6 Boulevard Gabriel, 21000 Dijon, France. al., 2001), and cestodes (Amat et al., 1991), possess signifi-
‡ HPMCT Proteome Analysis, 223 St. Antoine, St. Elphège, Quebec,
Canada J0G 1J0.
cantly higher levels of lipids than uninfected conspecifics. Here,
§ INSERM, Institut Claude de Préval, IFR30, Plateau Technique de Lip- we attempted to further probe several components of the lipid
idomique, Toulouse F-31300, France. storage forms in infected and uninfected A. parthenogenetica.


MATERIALS AND METHODS software (Ciphergen Biosystems). A range of 2–30-kDa molecular

weights (SELDI TOF-MS is especially sensitive at this low-molecular-
Sampling mass range) was analyzed for differences in peptide profile. Spectra
Artemia parthenogenetica were collected with a 0.1-mm-mesh net were mass aligned, baseline-subtracted with the use of a smoothing
from an industrial saltpan in the Odiel Marshes (Huelva, southwest feature, and then normalized with the use of total ion current normali-
Spain, 37⬚17⬘N, 06⬚55⬘W) during August 2005 for the proteomic anal- zation before statistical analysis was performed. The total ion current
ysis and June 2006 for the lipid analysis. Infected individuals displaying method assumes that, on average, the total number of proteins being
aberrant behavior were sampled from the water surface, whereas unin- expressed is constant across the samples being normalized. The process
fected ones were collected from the bottom (a direct consequence of takes the total ion current used for all the spots, averages the intensity,
the infection is the spatial segregation of infected and uninfected indi- and adjusts the intensity scales for all the spots to display data on the
viduals in the water column). Given the important ecological differences same scale. The m/z of each peak to be quantified was determined
between these 2 microhabitats (food, light, oxygen, disturbance, tem- according to externally calibrated standards (Ciphergen Biosystems).
perature, predation risk, etc.), and knowing that not only infection sta- Univariate analyses, Mann–Whitney nonparametric U-tests, were used
tus, but also environmental conditions, may have a significant influence to compare the mean intensity values of each recognized peak at the
on protein expression, we created a control group by placing uninfected molecular mass range of 2–30 kDa. A significance threshold was set at
brine shrimp from the bottom in the same ecological conditions as in- P ⬍ 0.05.
fected ones from the surface (as a control for environmental conditions).
We placed a sample of uninfected brine shrimp in a metallic cage (50-
Lipid analysis
cm diameter) located at the surface (1 cm) at the collecting site for 24
hr. For the host head proteome analysis, we used A. parthenogenetica We measured free cholesterol, diglycerides (including phospholipids,
as host species and 3 cestode species, i.e., F. liguloides, C. podicipina, DG ⫹ PL), cholesterol esters (Cho Est), and triglycerides (TG). The
and A. tringae, known to use brine shrimp as intermediate hosts. For measurements of neutral lipid molecular species were performed by
the SELDI-TOF experiment, we used 5 categories of individuals: (1) gas–liquid chromatography. Brine shrimp (10 individuals per group)
uninfected A. parthenogenetica from the bottom (NP); (2) uninfected were individually crushed with the use of an Ultra Turax in 1 ml of
A. parthenogenetica from the bottom placed at the surface as controls methanol/5 mM EGTA (2:1 v/v). Aliquots (50 ␮l) were evaporated, the
(NPC); (3) A. parthenogenetica infected by F. liguloides (AP FL); (4) dry pellets were dissolved in 0.2 ml of NaOH (0.1 M) overnight, and
A. parthenogenetica infected with C. podicipina (AP CP); and (5) A. proteins were measured with the Bio-Rad assay (Bio-Rad Laboratories,
parthenogenetica infected by A. tringae (AP AT). For the lipid analysis, Hercules, California). Lipids corresponding to 0.5 ml of the homoge-
we were unable to find enough individuals infected by A. tringae so nized body were extracted according to Bligh and Dyer (1959) in chlo-
that only 3 categories were considered, i.e., uninfected individuals, in- roform/methanol/water (2.5:2.5:2.1, v/v/v), in the presence of the inter-
fected by F. liguloides, and infected by C. podicipina.
nal standards: 6 ␮g of stigmasterol, 2 ␮g of 1,3-dimyristine, 2 ␮g of
In the laboratory, immediately following the collection, individuals
cholesteryl heptadecanoate, and 3 ␮g of glyceryl triheptadecanoate. The
were carefully checked for the presence of cysticercoids, dried on ab-
chloroform phase was filtered over glass wool, evaporated to dryness,
sorbent paper, and individually stored in a 1.5-ml Eppendorf tube at
and dissolved in 20 ␮l of ethyl acetate. One microliter of the lipid
⫺80 C until the analyses were undertaken. Only individuals infected
extract was analyzed by gas–liquid chromatography on a FOCUS Ther-
with 1 cestode species were employed in the study. Using a partheno-
mo Electron system (Waltham, Massachusetts) with the use of a Zebron-
genetic species (all the individuals were females) limits possible host
sex-specific effects. For the SELDI-TOF analysis, only the heads, after 1 Phenomenex (Phenomenex, Torrance, California) with fused silica
being carefully removed from the bodies, were preserved on ice. The capillary columns (5 m ⫻ 0.32–mm i.d., 0.50-mm film thickness) (Bar-
bodies were then placed in temporary glycerol mounts and cysticercoids rans et al., 1994). Oven temperature was programmed from 200 to 350
were examined with the use of stereo or ordinary light microscopy to C at a rate of 5 C per min and the carrier gas was hydrogen (0.5 bar).
identify the cestode species. If the identification of the cysticercoids The injector and the detector were at 315 and 345 C, respectively.
was not possible at this stage, whole infected brine shrimp or isolated All statistical analyses were performed following Sokal and Rohlf
cysticercoids were prepared as permanent mounts in Berlese’s medium (1981), Siegel and Castellan (1988), and Zar (1999). When data devi-
to facilitate observations on the morphology of rostellar hooks. The ated from normality and/or did not fit a normal distribution after trans-
same protocol was followed to verify the absence of cysticercoids for formation, we used nonparametric statistics. Post hoc comparisons were
individuals collected from the bottom and those experimentally placed performed according to Zar (1999) with a significance level of P ⬍
in the surface. 0.05. P values were Bonferroni corrected to avoid Type I errors.
Throughout the article, values given are mean ⫾ SE. Results were con-
Proteomic analysis (SELDI-TOF MS) sidered significant at the 5% level.
The ProteinChip SELDI system was used as previously described
(Missé et al., 2007). We used protein chip arrays, i.e., solid-phase chro- RESULTS
matographic surface chips, to ionize proteins from host heads and then
analyze them. Preactivated differential binding surfaces permitted mul-
tidimensional chromatography. Three types of protein chip surfaces, i.e., Proteomic analysis
hydrophobic (H50), normal phase (NP20), and immobilized metal af-
finity capture (IMAC 30), were used to perform preliminary analysis to SELDI-TOF analysis of A. parthenogenetica head extracts
search for the most suitable binding surface for protein retention. IMAC showed specific peptide profiles for each category in the range
30 ProteinChip Arrays (Ciphergen Biosystems, Fremont, California) of 2–5 kDa (Fig. 1). This representative peptide profile also
previously activated with 50 mmol/L NiSO4 (according to manufactur-
er’s instructions) were used to perform the final analysis.
revealed several differentially expressed peptide peaks. The
In total, the heads of 52 uninfected, 42 uninfected controls, and 56 presence of a soluble head peptide with a molecular mass of
infected brine shrimp were examined. Infected A. parthenogenetica in- 4.5 kDa was underexpressed in the heads of A. tringae–infected
cluded 21 by A. tringae, 11 by F. liguloides, and 24 by C. podicipina. individuals (AP AT) compared with controls (NPC and NP), as
Brine shrimp heads were individually bound to activated chip spots and
incubated for 24 hr at 4 C. After incubation, unbounded proteins were
well as in the other 2 infected groups (AP CP and AP FL; P
removed by 3 successive washes of phosphate-buffered saline. Chip- ⬍ 0.001). SELDI analysis also showed an absence of a 3.9-
captured proteins were air dried at room temperature for 15 min, fol- kDa head peptide in A. tringae- and F. liguloides–infected in-
lowed by 2 additions (1 ␮l each) of a saturated solution of ␣-cyano-4- dividuals compared with controls and C. podicipina–infected
OH-cinnamic acid (CHCA) matrix. The ionized and desorbed proteins
were detected and their molecular masses displayed on a proteogram.
individuals. Two head peptides of 4.1 and 4.2 kDa were over-
Protein peaks were analyzed by SELDI-TOF MS analysis with the Pro- expressed in the majority of infected individuals, as well as in
tein-Chip Biology System II software (PBS II) and Ciphergen Peaks uninfected controls placed at the surface (P ⬍ 0.05).

ble I). Differences between uninfected individuals and those

infected with C. podicipina were significant for TG C49. There
was no significant effect of parasite load on the lipid content
(Spearman rank test, P ⬎ 0.107 for C. podicipina and P ⬎ 0.08
for F. liguloides).

Proteomic analysis
Studying processes that explain parasite strategies is critical
to understanding the evolution of host–parasite interactions. Al-
teration of the central nervous system is commonly used by
parasites to alter host behavior (Holmes and Zohar, 1990;
Thompson and Kavaliers, 1994; Adamo, 2002; Helluy and
Thomas, 2003; Beckage and Gelman, 2004; Biron, Marché et
al., 2005), although the mechanisms responsible are not well
Proteomics constitute a powerful tool to investigate the mo-
lecular cross talk established during the manipulative processes
(Biron, Moura et al., 2005; Thomas et al., 2005). A major ap-
proach for protein biomarker discovery is differential protein
expression profiling, in which protein expression levels are
compared across samples. Here we used for the first time SEL-
DI-TOF MS to compare the head proteomes of uninfected or-
ganisms and those infected by a manipulative parasite. Al-
though this approach permitted a rapid discovery of differen-
tially expressed proteins in manipulated individuals, difficulty
in identifying proteins of interest is a major limitation of this
technology. Despite this problem, a SELDI-TOF approach per-
mitted rapid and reproducible protein profiling directly from
crude samples, and allowed us to bind a range of proteins and
peptides to different molecular surfaces without the requirement
for antibody production.
FIGURE 1. Surface-enhanced laser adsorption/ionization time-of- In the present study, SELDI-TOF analysis detected 2 peptides
flight (SELDI-TOF) MS analysis of head proteome of Artemia showing whose expression is altered in individuals infected by the di-
differentially expressed protein peaks. NPC (nonparasitized control
placed at the surface), NP (nonparasitized), AP AT (infected by An- lepidid, A. tringae (downregulated 4.5-kDa peptide), and by the
omotaenia tringae), AP CP (infected by Confluaria podicipina), AP FL 2 hymenolepidids F. liguloides and C. podicipina (downregu-
(infected by Flamingolepis liguloides). Arrows indicate peptidic bio- lated 3.9-kDa peptide). Because parasitized individuals also
marker. typically displayed the altered behavior, these peptides are po-
tential candidates for involvement with the manipulation pro-
cess. However, to confirm this hypothesis, further work is re-
Lipid analysis
quired, particularly in identification of these molecules and their
It was not possible to select individuals of the same size for biological functions.
infected and uninfected categories, because some species, e.g., Another interesting finding is the identification of 2 head pep-
F. liguloides (Amat et al., 1991), induce gigantism. Thus, the tides (4.1 and 4.2 kDa) overexpressed in all the categories in
mean body size was slightly different between uninfected brine shrimp living at the surface (both infected individuals and
(10.45 ⫾ 0.33, mean ⫾ SE) and infected individuals (11.4 ⫾ uninfected controls). This suggests that some peptide expres-
0.33 for F. liguloides; 11.9 ⫾ 0.34 for C. podicipina) (ANOVA, sions are more the consequence than the cause of the behavioral
F ⫽ 4.96, P ⫽ 0.015; post hoc comparisons, Student’s t-test, P change, i.e., different environmental conditions experienced at
⬍ 0.047, for uninfected–F. liguloides and uninfected–C. podi- the water surface. This of course also underlines the need in
cipina). To avoid size-confounding effects, we performed all proteomic investigations for considering control categories.
the analyses on the residuals of the relationship between lipid Further identification of these peptides would be necessary to
content (ln) and brine shrimp size (ln). Details of lipid content understand their role. At the moment, we can speculate about
for the different categories are presented in Table I. Values for their relation in the synthesis of all-trans-canthaxanthins, as in-
diglycerides were in general significantly higher in uninfected fected photophilic individuals (displaying bright red coloration)
A. parthenogenetica than in infected ones. Conversely, for tri- are more exposed to UV radiation, which stimulates synthesis
glycerides and Cho Est content, the general tendency was to of photoprotective carotenoids. The next step to improve our
exhibit an increase from individuals infected with F. liguloides understanding of the molecular mechanisms implied in the
(with minimum values), followed by uninfected controls, to modification of host behavior in this system will be the iden-
those infected with C. podicipina (with the highest values; Ta- tification (isolation, purification, and characterization) of the

TABLE I. Artemia parthenogenetica lipid content (nmol/mg, mean ⫾ SE) for individuals infected with Confluaria podicipina (cp), Flamingolepis
liguloides (fl), and for uninfected individuals (ni). Measures of diglycerides (DG) include phospholipids (PL).

Post hoc comparisons

ANOVA (Bonferroni corrected)
Composes Uninfected C. podicipina F. liguloides F P ⬍0.05 ⬍0.01

Cholesterol 27.96 ⫾ 3.89 22.99 ⫾ 2.45 28.19 ⫾ 4.71

DG 16-16 0.00 ⫾ 0.00 0.00 ⫾ 0.00 0.88 ⫾ 1.29
DG 16-18 21.96 ⫾ 6.40 8.82 ⫾ 2.30 10.82 ⫾ 2.34 10.25 * ni ⬎ cp, fl
DG 18-18 22.79 ⫾ 7.66 9.90 ⫾ 3.49 14.20 ⫾ 4.90
DG 18-20 0.00 ⫾ 0.00 0.00 ⫾ 0.00 0.78 ⫾ 1.68
DG ⫹ PL 44.75 ⫾ 13.60 18.72 ⫾ 5.27 26.67 ⫾ 7.99 ni ⬎ cp
C18 0.55 ⫾ 0.49 0.06 ⫾ 0.19 0.04 ⫾ 0.08
C20:4 2.96 ⫾ 1.94 6.35 ⫾ 2.97 2.33 ⫾ 2.52
Total cholesterol esters 3.65 ⫾ 1.48 6.41 ⫾ 2.86 2.37 ⫾ 2.48 7.86 † cp ⬎ fl
Triglycerides (TG) C49 0.50 ⫾ 0.82 3.89 ⫾ 1.92 2.10 ⫾ 2.29 cp ⬎ ni
C51 12.79 ⫾ 6.82 17.99 ⫾ 8.18 8.21 ⫾ 9.00
C53 27.14 ⫾ 12.50 38.35 ⫾ 18.74 15.89 ⫾ 18.06 7.65 † cp ⬎ fl
C55 27.25 ⫾ 13.87 42.39 ⫾ 25.40 16.14 ⫾ 19.91 8.19 † cp ⬎ fl
C57 16.15 ⫾ 8.67 27.86 ⫾ 17.93 9.56 ⫾ 11.46 8.87 † cp ⬎ fl
Total TG 83.84 ⫾ 39.80 130.49 ⫾ 68.58 51.90 ⫾ 60.01 7.88 † cp ⬎ fl
Pic 1 (27,85⬘) 13.42 ⫾ 6.50 19.33 ⫾ 11.15 7.43 ⫾ 8.02 cp ⬎ fl
Pic 2 (29,27⬘) 4.60 ⫾ 2.56 14.85 ⫾ 14.34 3.25 ⫾ 3.69 cp ⬎ fl
Pic 3 (30,59⬘) 3.01 ⫾ 1.59 5.08 ⫾ 3.37 1.64 ⫾ 1.86 cp ⬎ fl

* P ⬍ 0.01 after Bonferroni correction.

† P ⬍ 0.05 after Bonferroni correction.

candidate peptides potentially linked to the manipulative pro- to be a good predictor of copepod survival (Franz and Kurtz,
cess, followed by functional analysis. 2002), so that manipulation of energetic storages may also func-
tion as a parasite strategy to increase the longevity of infected
Effect of infection on lipid levels individuals and, therefore, their probability of encountering a
final host. The greater survival of infected crustaceans reported
Contrary to the findings of Amat et al. (1991) showing higher by Amat et al. (1991) supports this hypothesis.
levels of total lipid reserves in F. liguloides–infected brine Artemia parthenogenetica is a keystone species in hypersa-
shrimp, we found an opposite result. Thus, there were decreased line habitats and is a major food resource not only for grebes,
levels of all the specific lipids analyzed. However, brine shrimp but also for a wide range of water birds (Cooper et al., 1984;
infected by C. podicipina (adult parasite of Black-necked grebe Britton et al., 1986; Verkuil et al., 2003; Sánchez et al., 2005),
Podiceps nigricollis) showed a tendency (significant for TG especially during migration, when it is well established that
C49) to increased levels of triglycerides. By increasing the en- birds accumulate fat reserves by increasing food intake and by
ergetic level of their intermediate host, C. podicipina potentially choosing high-energy diets (Blem, 1990; Stiles, 1993; Bairlein
augment their profitability and attractiveness for the grebe final and Gwinner, 1994; Biebach, 1996; McWilliams and Karasov,
host. Fatty acids in wild bird food are mostly in the triglyceride 2004). Because the cestodes studied here appear to enhance not
form (Zurovchak et al., 1999), with elevated plasma of triglyc- only the accessibility to predation, but, in the case of species
eride levels in birds being indicative of fat deposition (Robin- such a C. podicipina, also their nutritional value (specific tri-
son, 1970; Ramenofsky, 1990). glycerides), ecologists and conservationists should devote fur-
Black-necked grebes forage primarily on brine shrimp, a ther attention to exploring the relationships between parasites
highly digestible and energy-rich food for them (Caudell and and the trophic potential of habitats.
Conover, 2006). The Odiel marshes (in southwestern Spain) are
1 of the major stopover sites for grebes when they molt. This
period, in which grebes become flightless for up to several
months, is a very energy-demanding experience; there are dra- The first author was supported by a postdoctoral grant from the Min-
isterio de Ciencia y Tecnologı́a (Spain). Consejerı́a de Medio Ambiente,
matic increases in body fat stores, leading to a doubling of their
Junta de Andalucı́a, and Aragonesas Industrias y Energı́a S.A. provided
body mass (Jehl, 1997). Observations of grebes picking brine permission to work in the saltworks. We are grateful to A. J. Green,
shrimp from the surface (infected and segregated individuals) anonymous referees, the Associate Editor Mike Sukhdeo, and the Editor
instead of the most common way to feed, i.e., while diving (M. Gerald W. Esch for helpful comments on the manuscript.
I. Sánchez, pers. obs.), support the idea that infected A. par-
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