Science of the Total Environment 830 (2022) 154747

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Science of the Total Environment

journal homepage: www.elsevier.com/locate/scitotenv

Parasitism by metacercariae modulates the morphological, organic and

mechanical responses of the shell of an intertidal bivalve to
environmental drivers
Oscar Varas a,f, José Pulgar f, Cristian Duarte f, Claudio García-Herrera g, Aldo Abarca-Ortega c,g, Cristian Grenier d,e,
Alejandro B. Rodríguez-Navarro e, Javier Zapata h, Nelson A. Lagos a,
⁎
M. Roberto García-Huidobro a,b, , Marcela Aldana a,b
a
Centro de Investigación e Innovación para el Cambio Climático (CiiCC), Facultad de Ciencias, Universidad Santo Tomás, Ejército 146, Santiago, Chile
b
Programa de Doctorado en Conservación y Gestión de la Biodiversidad, Facultad de Ciencias, Universidad Santo Tomás, Ejército 146, Santiago, Chile
c
Centro de Tecnología Biomédica, Universidad Politécnica de Madrid, 28223 Pozuelo de Alarcón, Madrid, Spain
d
Departamento de Estratigrafía y Paleontología, Universidad de Granada, Granada, Spain
e
Departamento de Mineralogía y Petrología, Universidad de Granada, Granada, Spain
f
Departamento de Ecología y Biodiversidad, Facultas de Ciencias de la Vida, Universidad Andres Bello, República 440, Santiago, Chile
g
Laboratorio de Biomecánica y Biomateriales, Departamento de Ingeniería Mecánica, Universidad de Santiago de Chile, Santiago, Chile
h
Departamento de Ecología, Pontificia Universidad Católica de Chile, Santiago, Chile

H I G H L I G H T S G R A P H I C A L A B S T R A C T

• The effect of parasitism depends on local

environmental conditions.
• Under some environmental conditions,
parasitized host can increase its perfor-
mance.
• Should considering parasitism when eval-
uating the effect of environment on organ-
isms

A R T I C L E I N F O A B S T R A C T

Article history: Environmental variation alters biological interactions and their ecological and evolutionary consequences. In coastal
Received 27 October 2021 systems, trematode parasites affect their hosts by disrupting their life-history traits. However, the effects of parasitism
Received in revised form 4 March 2022 could be variable and dependent on the prevailing environmental conditions where the host-parasite interaction oc-
Accepted 18 March 2022
curs. This study compared the effect of a trematode parasite in the family Renicolidae (metacercariae) on the body
Available online 23 March 2022
size and the shell organic and mechanical characteristics of the intertidal mussels Perumytilus purpuratus, inhabiting
Editor: Julian Blasco two environmentally contrasting localities in northern and central Chile (ca. 1600 km apart). Congruent with the en-
vironmental gradient along the Chilean coast, higher levels of temperature, salinity and pCO2, and a lower pH charac-
Keywords: terise the northern locality compared to that of central Chile. In the north, parasitised individuals showed lower body
Environmental variability size and shell resistance than non-parasitised individuals, while in central Chile, the opposite pattern was observed.
Bivalves Protein level in the organic matter of the shell was lower in the parasitised hosts than in the non-parasitised ones re-
Trematode metacercariae gardless of the locality. However, an increase in polysaccharide levels was observed in the parasitised individuals
Productivity/upwelling from central Chile. These results evidence that body size and shell properties of P. purpuratus vary between local

⁎ Corresponding author at: Centro de Investigación e Innovación para el Cambio Climático (CiiCC), Facultad de Ciencias, Universidad Santo Tomás, Ejército 146, Santiago, Chile.
E-mail address: mgarciahuidobro@santotomas.cl (M.R. García-Huidobro).

http://dx.doi.org/10.1016/j.scitotenv.2022.154747
0048-9697/© 2022 Elsevier B.V. All rights reserved.
O. Varas et al.
populations and that they respond differently when confronting the parasitism impacts. Considering that the parasite
prevalence reaches around 50% in both populations, if parasitism is not included in the analysis, the true response of
the host species would be masked by the effects of the parasite, skewing our understanding of how environmental var-
iables will affect marine species. Considering parasitism and identifying its effects on host species faced with environ-
mental drivers is essential to understand and accurately predict the ecological consequences of climate change.
1. Introduction
Increasing atmospheric CO2 has simultaneously caused a decrease in
seawater pH (ocean acidification, OA) and a global increase in seawater
temperature (ocean warming, OW) (IPCC, 2014). Ocean acidification neg-
atively affects the shell formation of many marine coastal taxa, in which
lower calcification rates, a significant reduction in shell growth, altered
shell mineral composition, and high shell mineral disorganisation have
been observed (Kroeker et al., 2013a; Coleman et al., 2014; Fitzer et al.,
2016; Lagos et al., 2016). However, some marine calcifiers can maintain,
or even enhance, shell growth and strength under OA (Leung et al.,
2017a, 2017b, 2019a), deploying compensatory mechanisms to buffer its
impacts by modifying shell properties (Ries, 2011; Leung et al., 2017a).
These responses are driven by the energy budget determined by physiolog-
ical performance and, therefore, by marine organisms' temperature and
thermal tolerance (Pörtner, 2012; Leung et al., 2019b, 2020). In this con-
text, responses of marine calcifying organisms to climatic change depends
on the interplay between OA and OW, and due to the differential impact
on species, they are further subject to possible indirect effects resulting
from interactions with other species (Kroeker et al., 2013a, 2013b; Poore
et al., 2013; Duarte et al., 2016). These effects can even reverse the direct
negative impact of climate change, greatly complicating predictions based
on abiotic variables due to cascading effects through food webs (Kroeker
et al., 2013b; Ghedini and Connell, 2017).
The intertidal zone is one of the marine habitats most likely to be af-
fected by atmospheric and oceanic changes (Harley et al., 2006; Helmuth
et al., 2006; Poulin and Mouritsen, 2006). In this ecosystem, animals and
algae are subject to environmental challenges of aquatic and aerial climatic
regimes and therefore subjected to rapid changes in temperature and pH
(Helmuth and Hofmann, 2001; Hofmann et al., 2011). The impact of envi-
ronmental stressors (e.g., temperature, pH, salinity) on intertidal organisms
is well documented (see Kroeker et al., 2013a). For example, high temper-
ature and low pH led to a significant decrease in shell hardness in oyster
Crassostrea virginica and clam Mercenaria mercenaria (Ivanina et al., 2013).
Low pH leads to a decrease of carbonate ions in seawater, impacting the
ability to build shells and alter the shells mechanical properties (Pörtner
et al., 2005; Guinotte and Fabry, 2008; Doney et al., 2009; Ramajo et al.,
2013; Fitzer et al., 2014; Milano et al., 2016; Leung et al., 2017a).
Along with abiotic stressors, intertidal organisms are also subjected to
biotic stressors induced by parasitic infections. In this regard, parasitic
trematodes are common in the intertidal ecosystem, with some species in-
fecting up to 80–100% of the host population (Mouritsen and Poulin,
2002; Fredensborg et al., 2005) and affecting the physiology and behaviour
of individual hosts, the host population dynamics, and the structure and
biodiversity of communities (Mouritsen and Poulin, 2002, 2005; Wood
et al., 2007; Buck and Ripple, 2017). In addition, the mollusc-trematode as-
sociation is an ideal model to evaluate the interactive effects of OW and OA
(Poulin and Mouritsen, 2006) since molluscs with shell act as intermediate
hosts for most trematode species and, as calcifying organisms, face signifi-
cant physiological challenges caused by OA and OW (MacLeod and
Poulin, 2012; Gazeau et al., 2013; Kroeker et al., 2014a). Thus, understand-
ing how the environment-parasite interaction influences the performance
of calcifier hosts is necessary to improve understanding of the effect of cli-
mate change on host-parasite dynamics (MacLeod and Poulin, 2012;
Kroeker et al., 2014a; García-Huidobro et al., 2019, 2020).
Numerous studies have documented the role of parasites in structuring
communities by directly reducing the abundance of its host, with cascade
effect at the scale of populations, communities and ecosystems (Sousa,
2
Science of the Total Environment 830 (2022) 154747
1991; Mouritsen and Poulin, 2002). However, some parasites can induce
more subtle modifications of host phenotype by altering the physiology
or behaviour of key-stone host species, for example, ecosystem engineer
species (Thomas et al., 1998, 1999; Mouritsen and Poulin, 2010; Gopko
and Mikheev, 2019). Consequently, parasitism can also influence the func-
tional role of the infected host through indirect trait-mediated effects
(Mouritsen and Poulin, 2005; Lefèvre et al., 2009a; Mouritsen and Haun,
2008). Furthermore, parasites may modify the host phenotype in ways
that benefit this host under particular conditions while still exacting fitness
costs under other environmental conditions (“conditionally helpful para-
sites”; Fellous and Salvaudon, 2009).
In wild marine populations, the effect of parasitism varies considerably
with environmental factors (e.g., physical-chemical variables); however,
parasites and stressors, such as OA, affect molluscs in fundamentally similar
ways. While exposure to high CO2 seawater increases the physiological
costs of maintenance via homeostasis and calcification (Pörtner et al.,
2004; Sokolova et al., 2012; Parker et al., 2013; Pan et al., 2015), infection
increases metabolic demands as the host supplies energy to the parasite and
meets its energetic requirements (Bates et al., 2011; MacLeod, 2017). In
this sense, parasitism is considered an additional stressor increasing host
mortality rates exacerbated by abiotic conditions (synergistic or additive ef-
fects) (MacLeod and Poulin, 2012; MacLeod, 2017). However, parasites
often face the challenge of not overexploiting their host, as host death
means concurrent death of the parasite (Poulin et al., 1994; Maure et al.,
2016). Thus, parasites may adopt exploitation strategies adjusted to the
physiological state of the host (Thomas et al., 2002; Hechinger, 2010), re-
sulting in a better condition of hosts parasitised under stress than under
stress without infection (antagonistic effect; MacLeod and Poulin, 2016a;
Selbach et al., 2020).
Parasitic trematodes using bivalves as first intermediate host have a se-
vere impact on hosts as their offspring production is reduced or they be-
come castrated by reproducing trematode larvae (cercariae) located in
the host gonads (Lauckner, 1983; Sousa, 1983; Minchella, 1985). In con-
trast, trematodes that use bivalves as second intermediate hosts typically
occur in a stage called metacercaria, presumably inflicting less harm as
they do not reproduce inside the bivalve, but facilitate its transmission to
the definitive host through predation. Nonetheless, parasites in this second
intermediate host have also been reported to cause significant alterations.
Effects induced by the metacercaria presence in bivalves include: decreased
clearance rates and heart rates, reduced byssal thread production, reduced
tolerance of anoxia, impaired growth, reduced ability to burrow, calcareous
irregularities inside the shell and possibly enhanced mortality (Wegeberg
and Jensen, 2003; Thieltges, 2006; Stier et al., 2015; O'Connell-Milne
et al., 2016; Bakhmet et al., 2017, 2019; Vázquez et al., 2020). Thus, the ev-
idence suggests that parasites can have significant implications on the en-
ergy budget of the host (Agnew et al., 2000; Aldana et al., 2020).
Consequently, trematode infection on bivalves could be relevant in the pre-
cipitation of shell carbonates, an energetically expensive structure, because
of the synthesis of its organic components (Palmer, 1992). In addition, the
combination of environmental stressors and parasitic infection could syner-
gistically alter the performance of host species.
The mollusc Perumytilus purpuratus Lamarck, 1819 (Bivalvia: Mytilidae)
inhabits rocky intertidal coast in the southeast of the Pacific from Ecuador
(2°00′S 77°20′W), down the west coast of South America to Cape Horn, and
then up the Atlantic South, up to Lobería on the coast of Argentina (41°8′S
63°0′W) (Prado and Castilla, 2006). This species forms dense three-
dimensional matrices that provide biogenic microhabitats for several inter-
tidal species (Prado and Castilla, 2006) and hosts an encysted metacercaria
O. Varas et al.
of the Renicolidae family acting as the second intermediate host
(Montenegro et al., 2012; Vázquez et al., 2020). Several metacercariae
can occur in the palps, gills, and visceral mass of bivalves that negatively af-
fect the mussel growth (Lauckner, 1983; Thieltges, 2006; Thieltges and
Rick, 2006). Despite the extensive and abundant coverage of P. purpuratus
in the intertidal zone and the high prevalence of this parasite species
(59.83% in northern Chile; Montenegro et al., 2012), there are no studies
that address the concomitant effect of environmental drivers
(e.g., temperature and pH) and parasitism on this bivalve species. In the
present study, we perform a field assessment including two local popula-
tions experiencing contrasting regimes in the physical-chemical parameters
of seawater. We hypothesise that parasitised P. purpuratus mussels,
inhabiting a locality with higher temperature and lower pH (northern
Chile) will present a lower body size, organic and mechanical properties
of the shell than non-parasitised individuals from the same locality, and
than those individuals (parasitised and non-parasitised) that inhabit a local-
ity with lower temperature and higher pH (central Chile). Additionally, we
measured the cyst size of the metacercariae and compared between loca-
tions.
2. Materials and methods
2.1. Study locality, biological and environmental sample collection
The Chilean coast (with 4270 km long, between latitudes 17° and 56°S),
presents a gradual decrease in sea surface temperature (SST) from north to
south, and a corresponding environmental gradient in the intensity of
coastal upwelling (Strub et al., 1998; Lagos et al., 2005, 2008). The coastal
upwelling interact with the geographical variations in the air-sea CO2 fluxes
along the Chilean coast, leading to high pCO2 conditions and low levels of
the mineral saturation state (Ω) in seawater (Torres et al., 2011; Mayol
et al., 2012) that can induce significant impact especially on calcifying or-
ganisms (Feely et al., 2008). The contribution of freshwater to the coast
due to an increase in river discharge from the arid region of northern
Chile to the rainy areas of the south (Dávila et al., 2002; Ramajo et al.,
2013, 2015), promotes high temperature and salinity in the northern
coastal area, generally decreasing towards the south (Ramajo et al., 2013,
2015; García-Huidobro et al., 2020). The northern region also presents
more variable pH and Ω values compared to the central-southern regions
of Chile (Torres et al., 2011; Mayol et al., 2012; Ramajo et al., 2013, 2015).
We selected two regions of the Chilean coast, separated by ca. 1600 km,
that experience contrasting patterns in temperature, pH and salinity: north
(Antofagasta, 23°46′S) and central (Valparaíso, 32°43′S) regions. In each lo-
cality, between December and March (2018–2019), we collected 981
adults (mature >10 mm of shell length) mussels of Perumytilus purpuratus
from the mid-intertidal zone (n = 633 from north and n = 348 from cen-
tral locality). All specimens were deposited in plastic bags, labelled, and
transported to the laboratory, where they were frozen (−20 °C) until
their dissection to evaluate the parasite burden and shell morphology (see
below).
At each locality, CTDO casts (Ocean Seven 304plus Logger, © Idronaut)
were performed in shallow waters (∼2 m in depth) in front of each rocky
platform to characterise variation in Sea Surface Temperature (SST) and sa-
linity (PSU). Simultaneously, three water samples were collected and proc-
essed for pHNBS using a Metrohm pH-meter (model pH mobile 888),
connected to a combined electrode (double juncture), calibrated using
Metrohm buffers (pH = 4.0, 7.0 and 9.0) at 25 °C in a temperature-
controlled water bath (Torres et al., 2011). At the same time, discrete sam-
ples of 500 ml of seawater were collected for Total Alkalinity (AT) analyses,
poisoned in situ using mercuric chloride (0.2 cm3 of a 50% saturated solu-
tion), and maintained in dark conditions and ambient temperature until
analysis in the laboratory. Measurements of AT were done using potentio-
metric titration, with the Metrohm Titrando 888 (Haraldsson et al.,
1997). Temperature, salinity, pH and AT data were used to calculate the car-
bonate system parameters (pCO2, aragonite saturation [Ωar]). Analyses
were performed using CO2SYS software in MS Excel (Pierrot et al., 2006)
3
Science of the Total Environment 830 (2022) 154747
set with Mehrbach solubility constants (Mehrbach et al., 1973), refitted
by Dickson and Millero (1987).
2.2. Parasitism magnitude and cyst body sizes
The mussel individuals were dissected to determine the condition of
parasitism by Renicolidae metacercariae by examining the surface mussels'
soft tissue (mantle, digestive gland and foot) under a stereomicroscope. All
encysted metacercariae found were extracted, counted, and stored in 95%
alcohol. All cysts were morphologically identical and were not considered
hosts with infections and co-infections by other parasite species. The mag-
nitude of parasitism in P. purpuratus of each locality was estimated by the
prevalence (percentage of infected host individuals) and the parasitic inten-
sity (mean number of parasites per infected host) (Bush et al., 1997).
From 27 parasitised hosts from both localities (north = 14, central =
13) were obtained among 5 to 11 encysted metacercaria by host were ran-
domly selected and transferred to a glass slide to determine the diameter
(μm) and thickness cyst (μm) under a microscope (Fig. 1S in supplementary
material) (De Mountaudouin et al., 2009).
2.3. Body size of Perumytilus purpuratus
For all P. purpuratus collected, parasitised and non-parasitised, the shell
length (l), width (w), and height (h) were measured with a digital calliper
(±0.1 mm) (Fig. 2SA in supplementary material), while total weight, soft
body wet weight, and shell weight were measured with an analytical bal-
ance (Mettler Toledo ±0.0001 g). From the length measurements, the vol-
ume of each mussel was approximated to an ellipsoid volume: [(4π / 3) ×
(l) × (w) × (h)] (Calvo-Ugarteburu and McQuaid, 1998). The shell thick-
ness was estimated using the relationship between shell weight and shell
surface area (Briones et al., 2014). Briefly, the shells surface incorporated
the possible effect of the valve curvature (using shell depth or width as a
proxy variable) on the surface estimation and was based on the formula
of shell surface proposed by Reimer and Tedengren (1996): [l (h2 +
w2)0.5 π / 2], where l, h, and w were the maximum shell length, height,
and width, respectively. This formula describes a figure that resembles an
ellipsoid and should give estimates relatively close to the true shell surface
area (Reimer and Tedengren, 1996). We estimated the adjusted shell thick-
ness using the relationship between shell weight and shell surface area
(Raubenheimer and Cook, 1990; Guiñez, 1996; Lord and Whitlatch,
2012, 2013). An analysis of covariance (ANCOVA) was used to compare
means for shell weight corrected for shell surface (see Statistical analysis).
2.4. Shell periostracum organic composition
These analyses are not introduced in the chronological order in which
they were performed but in their appearance in the Results section. After
evaluating the mussel's size variables and inspecting for parasites, we se-
lected 66 left valves (parasitized =17 and non-parasitized = 18 from
northern population; and parasitized = 19 and non-parasitized = 12
from central population). Biomechanical analyses were carried out first,
followed by the analysis of the organic composition of the periostracum
shell edge (Fig. 2SB–C in supplementary material).
The composition of organic matter at the periostracum edge of the
growth shell was measured by Attenuated Total Reflection-Fourier Trans-
form Infrared spectroscopy (ATR-FTIR) (e.g., Ramajo et al., 2016a; Checa
et al., 2019; García-Huidobro et al., 2020). For ATR-FTIR analysis, after
the mechanical analyses of shells (see below), a small piece was collected
from the shell edge. Samples were measured by pressing the outer shell sur-
face (periostracum) on the ATR diamond crystal and recording the IR spec-
tra at a 2 cm−1 resolution and 100 scans using Fourier Transform Infrared
(FTIR) spectrometer (model 6200, JASCO Analytical Instrument). The rela-
tive amount of main chemical components (i.e., polysaccharides, proteins,
water, and carbonates) were estimated from the absorption peak areas asso-
ciated with the characteristic molecular group of each component
(e.g., COC: sugars/polysaccharides), amine: proteins, OH: water, CO
O. Varas et al. Science of the Total Environment 830 (2022) 154747

Table 1
Study localities and summarised carbonate systems parameters (Mean ± SE) mea-
sured in situ during the sampling period. Letters represent significant differences be-
tween localities (P < 0.05).
Environmental variables Localities

Northern (23°S) Central (33°S)

Temperature (°C) 19.8 ± 0.53a 16.8 ± 0.84b

Salinity (PSU) 34.05 ± 0.26a 33.30 ± 0.16b
pHNBS 8.02 ± 0.02a 8.17 ± 0.03b
AT (μmol kg−1) 2226.4 ± 25a 2232 ± 17a
pCO2 (μatm) 575 ± 36a 364 ± 17b
ΩAR 2.52 ± 0.5a 3.40 ± 0.33b

carbonates). Calculated peak areas of the absorbance spectrum follow pro-

cedures described by Rodríguez-Navarro et al. (2013).

2.5. Shell biomechanical properties

A universal testing machine Instron (Model 3342, Norwood, MA) was
used to measure the load, time, and mechanical work required to fracture
the valves (similar to Burnett and Belk, 2018). The valves were compressed
by Instron at a rate of 0.1 mm/min, and the resulting compression and load
on the valve were recorded (Fig. 2SB in supplementary material). The com-
pression (mm) and load (N) at which the valve fractured were recorded by a
computer. The area beneath the curve in the graph of the compression and
load on the valve gave the mechanical work required to fracture the shell
(Fig. 2SC in supplementary material).
2.6. Statistical analysis
Natural variation in environmental parameters was compared between
study localities (north vs central) using a one-way analysis of variance
(ANOVA). The parasite prevalence between localities was compared
using a contingency table with Chi2 statistics. The parasite intensity, diam-
eter and thickness cyst were compared using analyses of co-variance
(ANCOVA), with the host shell lengths used as a covariate. To avoid
pseudo-replication, the diameter and thickness were averaged for host
from each locality. We performed two-way ANOVA to test for differences
in the shell length of P. purpuratus between parasitised conditions and local-
ities. ANCOVAs were used to compare total weight, soft tissue weight, shell
weight and volume, between parasitised conditions and localities, with the
host shell lengths used as covariates. In the case of shell thickness, ANCOVA
analysis incorporated the shell weight as a dependent variable and shell
surface as covariate (García-Huidobro et al., 2019). Previous to these anal-
yses, all variables were log10 transformed. We show the Least Square Means
(LSM), the predicted response of parasitised and non-parasitised, at the
mean value of the covariate for each morphological variable. The corre-
sponding bivariate regression analyses and the estimation of their parame-
ters are presented as supplementary material (Table 1 in supplementary
material). The periostracum organic composition (percentage of polysac-
charides, proteins, water, and CO3) and mechanical work required to frac-
ture the shell were compared between parasitised conditions and
localities by two-way ANOVA. In all cases, Tukey posthoc analysis was
Fig. 1. Shell length of parasitised and non-parasitised P. purpuratus from north and
central localities. Asterisks and letters indicate significant differences between
parasite conditions and localities (P < 0.05). Error bars indicate ±1 standard errors.
performed to determine significant differences between groups (Zar,
1996). All analyses were performed using Minitab 14® ver 13.3.2.
3. Results
3.1. Environmental variability at study localities
Both localities exhibited significant contrasting environmental condi-
tions in SST, PSU, pH, partial pressure of CO2 (pCO2) and Ω aragonite satu-
ration state (Table 1). In general, the northern location showed a higher
SST, salinity and pCO2 than the central Chile location, while the pH and
the saturation states of Ω aragonite were lower in the northern location
than in the central Chile location.
3.2. Parasitism magnitude and cyst body sizes
The prevalence was similar between localities (North = 48.6% and
Central = 51.7%. Chi2 = 0.84; P = 0.359). In the same way, the cyst's in-
tensity, diameter, and thickness were similar between localities (Table 2).
3.3. Body size of P. purpuratus
Parasitised mussels showed greater shell length than non-parasitised
mussels (F(1, 977) = 38.7; P < 0.001. Fig. 1), and they were longer in the
north than in the central locality (F(1, 977) = 273.7; P < 0.001. Fig. 1).
Soft weight was higher in the northern than central locality, with no differ-
ences between parasitism conditions (Fig. 2B, Table 3). Comparing mor-
phological variables between mussels with different parasite conditions at
the mean shell length (i.e., LSM value of the covariate in ANCOVA), the
total weight, shell weight, volume and thickness were on average 8%
lower in parasitised than non-parasitised individuals from the north

Table 2
Summary of analyses of parasite intensity, and cyst body sizes (diameter and thickness) (mean ± standard error), controlled by the host body size between localities
(ANCOVA). bP = bNP are slope homogeneity analyses. Bold indicated significant differences.
Variable Hypothesis ANCOVA
bP = bNP
source of variation df F P-value Mean ± standard error

Intensity F(1, 484) = 0.13 Shell length 1, 485 12.27 <0.01 North = 7.37 ± 1.13
P = 0.712 Locality 1, 485 1.09 0.295 Central = 9.43 ± 1.4
Cyst diameter (μm) F(1,23) = 3.21 Shell length 1, 24 1.36 0.253 North = 251.96 ± 5.41
P = 0.086 Locality 1, 24 0.02 0.876 Central = 253.16 ± 5.21
Cyst thickness (μm) F(1, 23) < 0.001 Shell length 1, 24 0.39 0.533 North = 27.42 ± 1.42
P = 0.996 Locality 1, 24 0.7 0.409 Central = 25.74 ± 1.37

4
O. Varas et al. Science of the Total Environment 830 (2022) 154747

Fig. 2. Least square means (LSM), the predicted value of the response variable at the mean value of the covariate (shell length and shell surface in E) in ANCOVA. Responses
are (A) total weight, (B) soft tissue weight, (C) shell weight, (D) volume, (E) shell thickness of parasitised and non-parasitised P. purpuratus from north and central localities.
Asterisks and letters indicate significant differences between parasite conditions and localities, respectively (P < 0.05). Error bars indicate ±1 standard errors.

locality. In contrast, in the central locality, these morphological variables
were on average 2% higher in parasitised than non-parasitised individuals
(Fig. 2A, C, D, E, Table 3).
3.4. Shell periostracum organic composition
The IR spectra showed that all samples of P. purpuratus have organic
matter in shell periostracum, but there were significant differences in the
periostracum composition depending on parasitism condition and study lo-
cality (Fig. 3). The relative amount of proteins and water were on aver-
age 19% and 12% respectively lower in parasitised than non-
parasitised individuals in both study localities, and also were lower in
the north than central locality (Fig. 4B, C, Table 4). The relative amount
of polysaccharides was 35% higher in parasitised than non-parasitised
individuals in the central locality, and on the north locality not be ob-
served differences in the relative amount of polysaccharides between
parasitism condition (Fig. 4A, Table 4. Finally, the relative amount of
carbonates was higher in the north than in the central locality
(Fig. 4D, Table 4).
3.5. Mechanical properties of the shell
The mechanical work to fracture shell valve in the north locality was
27% lower in parasitised than non-parasitised individuals, while in the cen-
tral locality was 28% higher in parasitised than non-parasitised individuals
(F(1, 62) = 5.81; P = 0.018. Fig. 5).
4. Discussion
The similarity in the parasite magnitude between localities (prevalence
and intensity) suggests that the host taxa that participate in the life cycle of
the Renicolidae are present in both localities (Aldana et al., 2014). Further-
more, the parasite body size (cyst diameter and thickness) was also similar
between localities, suggesting that environmental differences are not a re-
striction for the recruitment and growth of the parasite in the host. Our re-
sults about the mussel's body size initially suggested that individuals from
the northern population were larger than the central Chilean population
(Figs. 1 and 2B). However, after considering the parasitism condition, the
body size difference described between localities was only observed in

5
O. Varas et al. Science of the Total Environment 830 (2022) 154747

Table 3
Summary of analyses of total, soft, and shell weight, volume and shell thickness of P. purpuratus, controlled by the host body size between localities and parasitism condition
(ANCOVA). bP = bNP are slope homogeneity analyses. Bold indicated significant differences.
Variable Hypothesis ANCOVA
bP = bNP
Source of variation df F P-value

Total weight (g) F(1, 973) = 0.02 Locality (L) 1, 976 27.04 <0.001
P = 0.884 Parasitism condition (Pc) 1, 976 6.81 0.009
L × Pc 1, 976 25.21 <0.001
Soft tissue weight (g) F(1, 972) = 2.28 Locality (L) 1, 975 17.59 <0.001
P = 0.115 Parasitism condition (Pc) 1, 975 0.3 0.579
L × Pc 1, 975 0.3 0.581
Shell weight (g) F(1, 972) = 0.1 Locality (L) 1, 975 17.2 <0.001
P = 0.748 Parasitism condition (Pc) 1, 975 9.35 0.002
L × Pc 1, 975 27.79 0.001
Volume (mm3) F(1, 973) = 0.54 Locality (L) 1, 976 55.85 <0.001
P = 0.460 Parasitism condition (Pc) 1, 976 8.3 <0.001
L × Pc 1, 976 19.91 <0.001
Shell thickness F(1, 972) = 0.11 Locality (L) 1, 975 1.14 0.285
P = 0.737 Parasitism condition (Pc) 1, 975 5.24 0.022
L × Pc 1, 975 14.39 <0.001

non-parasitised individuals, since the parasitised ones showed similar
values between the northern and central localities (total weight, shell
weight and volume; Fig. 2A, C, D) or presented a significantly higher
value in central Chile (shell thickness; Fig. 2E). These results suggest that
contrasting environmental scenarios in northern and central Chile in
terms of SST, PSU, pH, pCO2 and Ω aragonite saturation state, may drive
geographic variability of the P. purpuratus shell (see similar examples in
Ramajo et al., 2013, 2015; García-Huidobro et al., 2019, 2020), but the
changing trend depends on the parasitism condition by Renicolidae
metacercariae. Therefore, this parasite species can significantly alter the
response of the P. purpuratus host and confuse the results of the ocean
acidification evaluation when the infection has not been considered
(MacLeod, 2017).
4.1. Parasite effect on the P. purpuratus body size
Trematode metacercariae are among the most common parasites of bi-
valves (Lauckner, 1983) and, in many cases, they are harmful to this host
group as second intermediate hosts, significantly reducing the body size
or shell growth rate of bivalves (Thieltges, 2006; Stier et al., 2015;
O’Connell-Milne et al., 2016; Bakhmet et al., 2017, 2019). It has been sug-
gested that this effect would be associated with the high energetic demands
of the metacercariae growing within the host (Stier et al., 2015; O’Connell-
Milne et al., 2016; Magalhães et al., 2018). Consistent with this approach,
in the northern locality, the parasitised individuals showed smaller body
size than the non-parasitised individuals (total weight, and weight, volume
and thickness of the shell. Fig. 2), highlighting the high energy demand

Fig. 3. ATR-FTIR spectra of the outer shell edge surface of parasitised and non-parasitised P. purpuratus from north and central localities. The spectra are offset along the Y-
axis. Main IR bands are highlighted in the graph.

6
O. Varas et al.
Fig. 4. Normalised intensity of main absorption bands of ATR-FTIR spectra (polysaccharides, proteins, water, and carbonates) of the outer shell edge surface of parasitised and
non-parasitised P. purpuratuss from north and central localities. Asterisks and letters indicate significant differences between parasite conditions and localities, respectively (P
< 0.05). Error bars indicate ±1 standard errors.
imposed by the parasite growth and/or tissue repair post-infection
(Thieltges, 2006) in a locality with a higher temperature and lower pH com-
pared to the central locality. These results support the premise that both
parasitic infection and ocean acidification increase the metabolic demands
of the host (MacLeod and Poulin, 2015a, 2016b). Northern Chile has been
described as an area of wide variations in the physical-chemical parameters
of seawater (Torres et al., 2011; Mayol et al., 2012; Ramajo et al., 2013,
2015), which can impose a high energy cost for the animals that inhabit
these areas, altering the energy available to deal with infectious diseases.
Furthermore, parasites exposed to acidified waters, as well as non-
parasitic organisms, are challenged by physiological stressors such as in-
creased metabolic costs of ionoregulation and calcification (MacLeod,
2017), which would cause the survival reduction of parasites (MacLeod
and Poulin, 2015b; Guilloteau et al., 2016). Considering that below a spe-
cific pH value, the internal fluids of host organisms may become acidified,
as the ionoregulatory ability of the host organism is overwhelmed
(Reipschläger and Pörtner, 1996), then, beyond that point, the endopara-
sites are directly exposed to an acidified environment (extracellular fluid)
(MacLeod, 2017). Under these conditions, endoparasites would increase
their metabolic energy consumption and cause a corresponding increase
in the energy requirements of the host. The host organisms would then
have to adapt to a greater overall energy budget or, if this were not possible,
experience a gradual decline in energy stores as demand outpaces supply.
On the other hand, several authors found that filtration rates and oxy-
gen consumption of bivalves are reduced as a result of parasite infection
(Calvo-Ugarteburu and McQuaid, 1998; Stier et al., 2015; Magalhães
et al., 2020), either due to direct impact resulting from mechanical distur-
bance of the cysts or indirect impact resulting from negative effects on
host overall condition. Metabolic suppression or a reduced resting meta-
bolic rate could lead to an overall increase of the glycogen content in in-
fected hosts (Oppliger et al., 1996). In fact, some studies have reported
that metacercariae infection tended to induce a reduction in host oxygen
7
Science of the Total Environment 830 (2022) 154747
consumption, resulting in a glycogen and lipid reserves accumulation
(Sánchez et al., 2016; Magalhães et al., 2018, 2020). Thus, parasite infec-
tion can lead to an energy imbalance in which the host generates more en-
ergy reserves than required by the parasite, resulting in energy
accumulation (Sánchez et al., 2016; Vivas et al., 2018). In this context, al-
though parasitic infection is usually detrimental, it can be beneficial to
the host in some situations, such as in the central locality where parasitised
P. purpuratus individuals appear to have an energetic advantage compared
with uninfected conspecifics, since not only the total weight of the
parasitised mussels was higher, but also the volume, weight and thickness
of the shell (Fig. 2A, C, D, E).
The increased body size of parasitised individuals in the central locality
suggests a direct parasite effect on host energy budgets. Increased growth of
the first trematode-infected intermediate hosts has been related to hosting
castration that would leave a large reserve of energy to redirect into somatic
growth (Sousa, 1983; Minchella, 1985; García-Huidobro et al., 2019). How-
ever, trematode does not reproduce on the second intermediate host and
thus are not as destructive to tissues as asexual larvae in the first intermedi-
ate host (Mouritsen and Poulin, 2002). It has been suggested that the effect
of the parasite interacts with the availability of food, host condition, and en-
vironmental factors, influencing the severity of the energetic exploitation of
the parasite and consequently the energy budget of the host (Wegeberg and
Jensen, 2003; Thieltges, 2006; Fellous and Salvaudon, 2009; Wolinska and
King, 2009; Vale et al., 2011). In this sense, the environmental productivity
associated with coastal upwelling systems along with the South American
Pacific coast (Feely et al., 2008; Cao et al., 2011; Vargas et al., 2016)
could affect the interaction performance between P. purpuratus and the
Renicolidae metacercariae that it harbours, as has already been evidenced
in other host-parasite systems (García-Huidobro et al., 2019; Aldana
et al., 2020).
The two localities evaluated in this study have been described as an up-
welling focus (Thiel et al., 2007); however, the amplitude and intensity of
O. Varas et al. Science of the Total Environment 830 (2022) 154747

Table 4 on shell formation in bivalves (Dauphin and Cuif, 2018), but the organic
Statistical analyses for intensity of absorption bands of ATR-FTIR spectra of the shell component, occluded in biologically assembled crystals as part of the shell's
edge periostracum of P. purpuratus between localities and parasitism condition. Bold protective coating (periostracum), is not fully understood (Chen et al.,
indicated significant differences. 2008; Meyers et al., 2008; Schmahl et al., 2012). The periostracum of
Variable Source of variation df F P-value P. purpuratus is a proteinous layer rich in sulfated polysaccharides
Polysaccharides (%) Locality (L) 1, 54 22.19 <0.001 (Ramajo et al., 2016b), and it has been reported that it varies in response
Parasitism condition (Pc) 1, 54 3.82 0.055 to the food availability, pH and salinity of seawater (Ramajo et al., 2016a,
L × Pc 1, 54 11.91 0.001 2016b). Recently, the periostracum composition has been associated with
Proteins (%) Locality (L) 1, 54 18.59 <0.001
the parasitism influence (García-Huidobro et al., 2020), which is also evi-
Parasitism condition (Pc) 1, 54 8.03 0.006
L × Pc 1, 54 0.18 0.666 dent in the differences observed in proteins and water between parasitised
Water (%) Locality (L) 1, 54 18.5 <0.001 and non-parasitised P. purpuratus individuals (Fig. 4). The secretion of a
Parasitism condition (Pc) 1, 54 3.99 0.05 thinner periostracum involving lower levels of organic components would
L × Pc 1, 54 0.05 0.812 be consistent with the diversion of resources by the parasite, which has
CO3 (%) Locality (L) 1, 54 28.22 <0.001
been reported in other study models (e.g., Fissurella-Proctoeces sp., García-
Parasitism condition (Pc) 1, 54 2.66 0.108
L × Pc 1, 54 0.37 0.541 Huidobro et al., 2020). Surprisingly, however, the level of polysaccharides
was higher in the parasitised individuals from central Chile than in the
other three groups. Since energy is provided by food, these results highlight
this phenomenon along the Chilean coast increase from north to south, with the potential contribution of increased resource abundance in the upwell-
a positive effect on the productivity of benthic resources (Anguita et al., ing centre at the central zone (Aldana et al., 2017, 2020), important for
2020). Increased productivity could attenuate the resource conflict be- the host when it comes to compensating for high pCO2 conditions and par-
tween host and parasite by decreasing the host exploitation rate and para- asitism costs. In central Chile, parasitised mussels show a better perfor-
site pathogenicity, and hence the negative parasite effects on host fitness mance in terms of body and shell condition and the periostracum organic
(Bedhomme et al., 2004; Hall et al., 2009; Vale et al., 2011). Thus, the bet- composition, suggesting that they would have a higher energy budget
ter performance in terms of body size (total weight, weight, volume and than non-parasitised ones from the northern and central localities.
thickness of the shell) of parasitized than non-parasitized central zone
hosts, suggests reduced metabolic stress in infected individuals inhabiting 4.3. Parasite effect on mechanical properties of the shell of P. purpuratus.
this productive site. Analogous to this host-parasite system (second inter-
mediate host mollusc – metacercaria), Aldana et al. (2020) showed that In general, the pattern of the results described is transferred to the me-
in central Chile, the intertidal mollusc Fissurella crassa parasitised by chanical work necessary to fracture the shell of P. purpuratus. In the north-
Proctoeces humboldti metacercariae presented greater body size and gonadal ern population, parasitised individuals need less mechanical work to
biomass, associated with lower oxygen consumption. Apparently, in areas fracture the shell than non-parasitised individuals, while the opposite pat-
with high availability of food resources, host molluscs can prosper, and par- tern was observed in the central population. Proteins and polysaccharides
asitic metacercariae grow, with little impact on host health (Aldana et al., provide flexibility and tensile strength while the mineral provides a high
2020). elastic modulus, compressive strength, hardness and resistance to abrasion;
therefore, the organic matrix is also related to the mechanical properties of
4.2. Parasite effect on shell periostracum organic composition of P. purpuratus the shell (Chen et al., 2008; Meyers et al., 2008; Schmahl et al., 2012). In
our study, parasitised mussels presented a lesser amount of proteins in
Because shell formation is a biologically regulated process that requires both localities, but was observed an increase in the amount of polysaccha-
metabolic energy (Palmer, 1992), the energy budget of organisms is ex- rides of this organic coating in the parasitised individuals of the central lo-
pected to govern shell building. Some studies support this approach show- cality, thus rendering shell carbonate more resistant.
ing a positive relationship between energy budget and shell growth (Leung To date, only two publications have investigated the combined effects of
et al., 2020; Ramajo et al., 2020), which may also explain why some marine acidified seawater and parasitic infections on the calcifying capacity or met-
calcifiers are able to produce thicker shells under ocean acidification abolic status of molluscs (MacLeod and Poulin, 2015a; MacLeod and
(Kroeker et al., 2014b; Leung et al., 2019a). Much information is available Poulin, 2016a). Our results in the northern locality agree with the additive
or synergistic effects of the stressors reported in these studies. However, an
opposite pattern emerges in the central locality suggesting that climatic
drivers and the metacercaria Renicolidae act antagonistically on
P. purpuratus. Thus, in this locality, the parasite would reduce the negative
impact of OA, potentially mediated by environmental productivity. Al-
though parasites are largely defined by the harm they cause to their
hosts, it has been described that in some situations they can be beneficial
by providing new functions or modifying phenotypic traits that increase
host performance (Weinersmith and Early, 2016, see example in Aldana
et al., 2020). Parasites with this effect are known as “conditionally useful”
(Fellous and Salvaudon, 2009) because they benefit the host under particu-
lar environmental conditions (e.g., central Chile), while imposing fitness
costs under other environmental conditions (e.g., northern Chile).
On the other hand, due to the absence of laboratory-controlled experi-
ments of our study, it is possible to propose as an alternative explanation
to the results obtained, that in the central locality naturally larger or
better-conditioned mussels are more susceptible to infection. In contrast,
in the northern locality weaker or smaller P. purpuratus are preferred
Fig. 5. Mechanical work to fracture the left valve of parasitised and non-parasitised hosts for the parasite. This interpretation implies a state-dependent infec-
P. purpuratus from north and central localities. Asterisks and letters indicate tion dynamics, where the parasite would adjust the host's infection or ex-
significant differences between parasite conditions (P < 0.05). Error bars indicate ploitation strategy depending on its environment (immediate or host, and
±1 standard errors. external or ecosystem where interaction occurs) (Thomas et al., 2002). A

8
O. Varas et al.
parasite's ability to respond in a state-dependent manner can maximize its
lifetime reproductive success. Thus, in the northern locality, small and
less resistant hosts would optimize the parasite transmission to the next
host in its life cycle, while in the center, a higher filtration power of larger
mussels would favor parasitism and a better condition to be predated. Un-
doubtedly, the effect of environmental variables on the dynamics of para-
sitic infection is a topic that deserves study (Labaude et al., 2015;
Gleichsner et al., 2016; Lagrue et al., 2017).
In response to parasitism, many hosts display alterations in their behav-
iour and/or resource allocation-dependent traits, to alleviate parasites-
induced fitness costs, and benefit their survival and reproduction (Lefèvre
et al., 2008; Fellous and Salvaudon, 2009; Lefèvre et al., 2009b). For exam-
ple, parasitised hosts have increased energy requirements and forage more
to compensate for the negative effect of infection (Lefèvre et al., 2008,
2009b). This increased feeding activity could be of great benefit to calcify-
ing species, such as P. purpuratus, where a relatively large fraction of their
energy budget is allocated to shell production (Thomsen et al., 2013). In
this context, the parasitism effect on the filtration/feeding rate of
P. purpuratus would be advantageous in upwelling areas where the decrease
in pH and the saturation level of carbonate minerals negatively affect the
mussel feeding rate (Vargas et al., 2015) and calcification of many marine
organisms (Fabry et al., 2008; Gazeau et al., 2013; Kroeker et al., 2013a;
Lagos et al., 2016). In addition, the nutrient enrichment associated with
the upwelling center in the central zone could provide higher quality food
(Aldana et al., 2017, 2020). The quality together with a higher consump-
tion of food induced by the parasite would explain the better performance
of parasitized P. purpuratus observed in the present study (in terms of body
size, organic composition of the periostracum, and mechanical properties of
the shell). In fact, previous studies suggest that it is food supply, and not
pCO2, that is the main factor driving shell formation in mussel species
(Melzner et al., 2011; Thomsen et al., 2013). Currently we are still not cer-
tain that this physiological response underlies the observed patterns; how-
ever, our results are consistent with the reported background in this regard.
Our findings highlight the need to include parasitism in the physiologi-
cal response of bivalves to climatic stressors (MacLeod and Poulin, 2016a;
MacLeod, 2017; García-Huidobro et al., 2019, 2020). Environmental
physical–chemical parameters (i.e., SST, PSU, pH, pCO2 and Ω aragonite
saturation state), may lead to heavier and more resistant P. purpuratus shells
in the north than in the center (non-parasitized in Fig. 2A, B, E, Fig. 5).
However, parasitism and/or its interaction with the environment can
change this trend, cancelling out the differences or reversing the pattern
(parasitized in Fig. 2A, B, E, Fig. 5). Considering that the parasite preva-
lence reaches around 50% in both populations (north and center), if parasit-
ism is not included in the analysis, the true response of the host species
(i.e., the response of uninfected individuals) would be masked by the para-
site effects, skewing our understanding of how climate change will affect
marine species.
Finally, altering the physiology or behaviour of key-stone host species
(Thomas et al., 1998, 1999; Mouritsen and Poulin, 2010; Gopko and
Mikheev, 2019), the parasitism can influence the functional role of infected
hosts with a cascade effect on the populations, communities and ecosystems
(Mouritsen and Poulin, 2002, 2005; Lefèvre et al., 2009a; Mouritsen and
Haun, 2008). Bivalve species are important not only economically but
also as key determinants of biodiversity (Gutiérrez et al., 2003).
Perumytilus purpuratus is an ecosystem bioengineer that provides habitat
and other services for a rich diversity of organisms (Prado and Castilla,
2006). Consequently, considering parasitism and identifying its effects on
host species faced with environmental drivers is essential to understand
and accurately predict the ecological consequences of climate change.
CRediT authorship contribution statement
Oscar Varas: Conceptualization, Methodology, Investigation.
José Pulgar: Writing – Original Draft, Resources.
Cristian Duarte: Writing – Original Draft, Resources.
Claudio García: Resources, Methodology.
9
Science of the Total Environment 830 (2022) 154747
Aldo Abarca: Writing – Original Draft, Methodology.
Cristian Grenier: Methodology.
Alejandro B. Rodríguez: Methodology.
Javier Zapata: Methodology.
Nelson A. Lagos: Conceptualization, Resources, Writing – Original
Draft.
M. Robeto Garrcía-Huidobro: Conceptualization, Methodology, For-
mal analysis, Investigation, Resources, Writing – Review and Editing, Su-
pervision, Visualization, Project administration, Funding acquisition.
Marcela Aldana: Conceptualization, Writing – Review and Editing.
Declaration of competing interest
The authors declare that they have no known competing financial inter-
ests or personal relationships that could have appeared to influence the
work reported in this paper.
Acknowledgements
This study was funded by FONDECYT 3170522, 11220593 and ANID
PAI77190031 to RGH; PIA ANID ANILLOS ACT172037 and PCI ANID
REDES170106 to RGH, MA, NAL and ARN. JP and MA acknowledge the
support from FONDECYT 1200813, 1220866 and INACH RT09-18; AA-O
and CG to ANID PFCHA/DOCTORADOS BECAS CHILE CHILE/2019-
CEL0001151 and DICYT from Universidad de Santiago de Chile; CGR to
National Program of Granada Juvenile en I + D + í from European Social
Fund, Ministerio de Ciencias, Formación y Universidades, Junta de
Andalucia and the University of Granada and the support of FPI fellowship
(grant PRE2018-085419). CD acknowledges the support from FONDECYT
1200794. NAL acknowledge ANID-Millennium Science Initiative
Program-ICN2019_015 (SECOS). We thank three anonymous referees for
useful comments on an earlier draft.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.scitotenv.2022.154747.
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