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Ecological Parasitology and Immunology
Vol. 3 (2014), Article ID 235884, 7 pages
doi:10.4303/epi/235884
ASHDIN
publishing
Research Article
Comparing Natural Parasitism and Resistance with Proxies of Host
Immune Response in Lestid Damselflies
Laura Nagel,1 Julia J. Mlynarek,2 and Mark R. Forbes2
1 Department
of Biology, Queen’s University, Kingston, ON, Canada K7L 3N6
of Biology, Carleton University, Ottawa, ON, Canada K1S 5B6
Address correspondence to Laura Nagel, [email protected]
2 Department
Received 25 July 2014; Revised 26 September 2014; Accepted 15 October 2014
Copyright © 2014 Laura Nagel et al. This is an open access article distributed under the terms of the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract Commonly used proxies for measuring immune responses
in invertebrates include the amount of melanin deposited on nylon
inserts and assays of activity of the enzyme phenoloxidase (PO) in the
haemolymph. We used these proxies to estimate immunity in unparasitized individuals from four Lestid damselfly species from populations with different levels of water mite parasitism. Levels of parasitism and resistance by hosts were population level estimates from
published papers. These parasitism levels were not correlated positively with immune response measured by proxies in the current study.
The species with the strongest melanization response to the inserts
and the highest PO levels was the one that currently experienced no
mite parasitism. The species with the weakest response to the inserts
and the lowest PO levels had low current levels of parasitism. The
two species that are heavily parasitized had an intermediate response.
Natural resistance levels were also not correlated with the response
measured by proxies, but the species with a strong response had high
levels of resistance in the past. This finding is supported by earlier
work done with Lepidoptera in which the most well-defended species
currently experience no natural parasitism.
Keywords damselflies; water mites; immune response; phenoloxidase
(PO); melanization; parasitism
1. Introduction
Immune responses are costly for hosts [16, 45], and there
are many studies documenting evolutionary tradeoffs
between immune traits or their proxies and other important
reproductive traits and survival measures [4, 10, 12, 19,
35, 37]. Since measuring immune responses in animals is
an important component of many behavioral, ecological,
and evolutionary studies, researchers need to measure
appropriate proxies of what is assumed to be the natural
immune response. However, finding a method that gives the
most accurate estimate and is relatively easy to measure
has proved difficult for many organisms [22, 32], partly
because immune systems are characterized by both parasite
recognition and defense against parasites.
In invertebrates, a common physiological response to
parasitism is encapsulation of a foreign object by layers of
haemocytes. A prophenoloxidase enzymatic reaction kills
the parasite through the synthesis of melanin, which has
toxic effects [33, 37]. Because of this process, many studies measuring resistance in insects measure the amount of
melanin deposited on a synthetic object (e.g., a small segment of nylon monofilament or sephadex beads) as a proxy
of the amount of resistance that the host is capable of mounting to a natural parasite (e.g., [10, 14, 15, 17, 18, 27, 31]). This
induced response allows researchers to determine levels of
defense following a challenge, and low levels of melanization of the object are interpreted as weak innate immunity.
Other studies measure levels of the enzyme phenoloxidase (PO) in the haemolymph because this gives information
about the amount of enzyme available to catalyze the reaction that results in melanization [5, 22]. PO activity is a measure of constitutive levels of immune effectors that can be
used to mount a defense against a parasite. When a parasite
attacks a host, the host can respond immediately by forming toxins and pigments such as melanin. PO is produced
last in a chain of prophenoloxidase reactions (prophenoloxidase is activated via a serine protease cascade), so levels
of PO activity should indicate the magnitude of the immune
response that is at least possible in the absence of substrate
limitation [5, 33]. Measuring PO activity in invertebrates is
thought by some researchers to be an indicator of a host
animal’s ability to minimize fitness costs of parasitism. For
example, [36] demonstrates a link between increased PO
levels and increased melanin production. Variation in host
resistance could therefore be partially explained by variation in PO activity, although PO activity does not appear
to be important for some invertebrates in resistance against
parasites [6].
Interpreting the results of proxies can be difficult
because immune systems are so complex and exhibit
redundancy [2]. There are a few studies that have attempted
to elucidate the relationship between natural immune
responses and these induced and constitutive proxies in
2
invertebrates. Oliver and Fisher [25] reviewed the efficacy
of immunomarkers in bivalves and determined that the
presence of cytotoxic molecules does not reflect disease
resistance. Mucklow et al. [22] showed that estimating PO
activity is not a reliable measure of parasite resistance in the
crustacean Daphnia magna. In insects, some studies that
measured PO concluded that it is a good indicator of natural
immune response [7], but others have not [42]. Results have
also been mixed in studies using artificial inserts across a
broad range of insect species. Honkavaara et al. [15] found
that nylon insert encapsulation was negatively correlated
with numbers of water mites on a damselfly. Rantala and
Roff [27] demonstrated that the ability of the moth Epirrita
autumnata to encapsulate nylon inserts was correlated with
the ability to resist a fungal pathogen in a natural population.
However, Schwartz and Koella [34] found no correlation
between the encapsulation response to artificial objects and
the response to malaria parasites in mosquitoes. Mallon
et al. [21] showed that bumblebees have a weak immune
response to nylon monofilament despite having strong
natural resistance responses to trypanosome parasites.
Smilanich et al. [38] demonstrated that tropical caterpillar
species that experience low levels of natural parasitism
by parasitoids have much higher levels of encapsulation
response to artificial beads than those that are parasitized
more frequently. The authors suggest that this could be
due to past antagonistic coevolution, where species that
mounted immune responses which killed wasp parasitoids
in the past are now avoided by the wasps.
In studies that measure both PO activity and melanization, the two proxies are not always positively correlated.
Cotter et al. [8] showed that a lepidopteran species
selected for a dark phenotype had lower PO activity,
suggesting a tradeoff between melanin and PO activity in
cuticular melanization (as opposed to encapsulation related
melanization). In another lepidopteran, PO activity was high
but encapsulation responses were low after starvation experiments [42], suggesting that PO is affected by resource availability. In damselflies, the relationship between PO activity
and melanization response is sometimes inconsistent [14].
We examined the strength of encapsulation response and
PO activity and compared these with parasite prevalence and
resistance in our study system of two species of parasitic
mites and four species of Lestid damselfly. Some temperate
damselfly species and populations are heavily parasitized by
mites, while others avoid parasitism completely (possibly
due to emergence timing, which the mites need to match
closely in order to parasitize hosts). Some damselflies mount
immune responses to mites that kill the parasites [29, 43].
Nagel et al. [24] showed that natural but narrow ranges of
air temperature and host sex and size do not significantly
affect expression of resistance by damselflies against larval
mites in nature (but see [29]).
Ecological Parasitology and Immunology
This study follows [23], which examined the response of
two Lestid species to nylon inserts and compared those with
natural patterns of parasitism. They found that the damselfly
species that had high resistance in nature (Lestes forcipatus)
also deposited more melanin on inserts than a species with
lower natural levels of resistance (Lestes disjunctus). However, they also found that there was no correlation between
an individual’s natural resistance to mites and its response to
nylon filaments. The present study differs in that it includes
two other Lestid species and measures PO activity in addition to melanization of nylon inserts. We also did not measure these proxies on the same individuals that were parasitized naturally but rather used population level parasitism
from other studies for comparison. Our goal was to see if
these two proxies reflect parasitism and resistance patterns
in the six populations that we examined at two sites.
Based on our initial study [23], we expected that
damselfly populations with high levels of natural resistance
would melanize inserts more and also have higher phenoloxidase levels than those that do not resist mites. We
expected that damselfly populations that do not experience
high levels of parasitism naturally would have lower
immune responses. This prediction makes intuitive sense if
we assume that immune responses are costly to maintain
and may be lost or reduced if there is little selection from
parasitism in a population.
2. Methods
2.1. Study sites and relevant natural history
Larval water mites are phoretic on the final aquatic larval
instars of many odonates. Behavioral defenses against larval
mites include larval damselflies emerging at a time when
mites are not active and grooming [20]. If grooming by larval damselflies is unsuccessful, the larval mites transition
to newly emerged adults when the hosts eclose. The larval
mites then form a blind-ended feeding tube [39]. The effects
of engorging mites on hosts appear to be severe, as they can
affect perching and flight [13, 28], cause cellular and tissue
damage [1], reduce condition [30], and are associated with
reduced mating success [39].
We used Lestid damselflies from two study sites
located near the Queen’s University Biology Station in
eastern Ontario, Canada. Barb’s Marsh (44°31 27.54 N,
76°22 25.89 W) is a permanent marsh and Yzerinac’s Pond
(44°32 12.82 N, 76°22 58.29 W) is an ephemeral pond.
At Barb’s Marsh, Lestes disjunctus is very common, while
L. congener and L. rectangularis are rare. At Yzerinac’s
Pond, Lestes forcipatus is very common, while L. congener
and L. rectangularis are again rare. These patterns are
reflected in our sample sizes (Table 1).
Arrenurus planus is a generalist mite that parasitizes
several Lestid damselflies and Sympetrum dragonflies at
Yzerinac’s Pond and was the only mite species present at
Ecological Parasitology and Immunology
3
Table 1: Parasite prevalence (mean % parasitism) and damselfly resistance (mean % of damselflies with one or more dead
mites) estimated over a period of 5–10 years (see text for details and sample sizes). GI indicates mean melanization (grayscale
index out of 100 where 0 = pure black) and PO activity (N = sample sizes) for four damselfly species from two sites in
Ontario, Canada.
Damselfly species
Barb’s Marsh
L. disjunctus
L. rectangularis
L. congener
Yzerinac’s Pond
L. forcipatus
L. rectangularis
L. congener
1 46%
2 50%
Prevalence
Resistance
GI (N )
PO (N )
90
26
0
3
0
0
59.5 ± 2.4 SE(18)
62.2 ± 4.0 SE(3)
39.5 ± 1.6 SE(7)
0.02 ± 2.4e-3 SE(47)
NA
NA
82
15
01
66
0
02
55.6 ± 1.6 SE(18)
76.3 ± 1.6 SE(5)
44.3 ± 1.6 SE(10)
0.01 ± 2.1e-3 SE(20)
0.02 ± 2.8e-3 SE(19)
0.04 ± 5.2e-3 SE(14)
in 1998 [44].
in 1998 [44].
this site during the study. Arrenurus pollictus appears to
specialize on Lestes disjunctus at Barb’s Marsh [24] and is
the only mite species present at this site. Mites cannot move
to other hosts or form another feeding tube once feeding
begins. Host resistance to mites is through melanotic
encapsulation of mite feeding tubes, which usually occurs
within 24 hours of damselfly emergence [39]. Dead mites
remain attached to the host and are flattened, dark, and
always associated with a melanized feeding tube in these
populations [44]. During mate guarding and oviposition,
fully engorged mites drop off of their hosts (often leaving a
scar). Detachment from hosts appears to be synchronized, as
damselflies at each site are rarely found with both engorged
mites and scars. Larval mites then go through predatory
nymphal and adult stages in water.
The parasite prevalence and resistance data used for this
study includes data obtained from 1998–2012 using aerial
sweep nets at the same two sites used in this study [23,
24, 29, 43]. In those surveys, we recorded host sex, wing
length and number of live mites, dead mites, and scars left by
mites. We define prevalence as the percentage of individuals
sampled that were parasitized by one or more mites. Resistance is the percentage of parasitized individuals sampled
that had one or more dead mites on them. Each damselfly
was marked individually on one of the hind wings with a
permanent marker (Stanford Sharpie) to avoid including it
in further surveys. At Barb’s Marsh, mite prevalence has
been estimated from 2002–2012 in L. disjunctus and has varied from 75–100% (mean = 90 ± 0.01 SE; N = 2068) [29].
Prevalence in the other two Lestid species at this site has
been estimated from 2008–2012 and has remained low in L.
rectangularis (mean = 26 ± 0.06 SE; N = 108) and 0% in
L. congener (N = 79) (L. Nagel, unpublished data). Only
L. disjunctus resists mites at this site, but at low levels [24].
These data are summarized in Table 1.
At Yzerinac’s Pond, mite prevalence was measured in
1998 and then from 2008–2012. It has varied from 66–85%
in the L. forcipatus population (mean = 82 ± 0.01 SE; N =
400) and 15% in L. rectangularis (mean = 15 ± 0.02 SE;
N = 34) [43]. Yourth et al. [44] reported parasitism levels
of 46% in L. congener in 1998, but since 2008, we have not
seen any parasitized individuals at this site (N = 181 from
2008–2012). Resistance levels in the L. forcipatus population have remained steady at around 66%. In 1998, 50% of
infested L. congener resisted one or more mites. Resistance
has not been observed in L. rectangularis.
2.2. Melanization of inserts
Damselflies were captured using aerial sweep nets in
June and July 2012. In order to mimic the location where
Arrenurid mites attach and feed in these hosts, we inserted
nylon monofilaments into the underside of the thorax
of unparasitized adult female and male damselflies. The
timing of insertion of nylon monofilaments does not reflect
the exact time of natural parasitism because parasitism
occurs during host emergence and the adults that we used
were a few days old. However, newly emerged damselflies
always died in preliminary tests after insertion of the nylon,
probably because the cuticle was still hardening and too
much tissue damage occurred.
We intended to use only males in the study in order
to control for possible effects of sex but were unable to
capture enough males of L. disjunctus and L. forcipatus.
Therefore, we included some females in those samples (all
other samples were of males only). At Barb’s Marsh, we
collected Lestes disjunctus (N = 18, 6), L. rectangularis
(N = 3), and L. congener (N = 7). At Yzerinac’s Pond,
we used L. congener (N = 10), L. forcipatus (N = 18, 8),
and L. rectangularis (N = 5). Damselflies were brought to
a nearby lab within 2 h of capture and housed individually
in an opaque, plastic 100 mL drinking cup with a mesh
top, and a wood dowel (to perch on). Cups were placed
in a large plastic tub containing wet towel paper in order
to avoid insect desiccation. Temperatures varied naturally
between 20 °C and 23 °C (photoperiod 16:8 L: D). We
used fine forceps to insert one sterile 2 mm length of
4
nylon monofilament (diameter 0.20 mm; rubbed with fine
sandpaper) into the thorax of the damselfly above the lateral
stripe. This location is close to the venter of the thorax
where most mites are located on their hosts [23, 24]. The
length of the right forewing between nodus and tip was
measured with digital calipers (±0.01 mm). Insects were
left for 24 h [24, 37], then killed by decapitation, and stored
in an Eppendorf tube in 80% ethanol alcohol.
Implants were removed from the insect with fine forceps
and photographed under a Leica dissecting microscope at
3.2× with a Leica DMRB digital camera using Northern
Eclipse 8.0 software. Three different pictures were taken
of the nylon insert. The amount of melanin on inserts in
each image was quantified using ImageJ software (U. S.
National Institutes of Health, Bethesda, Maryland, USA,
http://rsb.info.nih.gov/ij/) to obtain the mean gray value
(±SD) of the part of the nylon that was inside the damselfly
thorax. We refer to this measure of melanin on a nylon insert
as the “grayscale index” (GI). In this measure, pure black is
given a value of 0 and pure white is assigned 255 (i.e., there
are 255 possible gray states that are scored in each image).
A mean GI value of the three images was used. The GI
score obtained from ImageJ was changed to a value out of
100 for statistical analyses and in Table 1. This value is not
the proportion of gray on an insert; a lower GI value (e.g.,
37.2) means that more melanin was present than a higher
GI value (e.g., 62.3) in the images of the nylon inserts.
2.3. Phenoloxidase content
Unparasitized adult female and male damselflies were captured a few days after emergence using aerial sweep nets in
June and July 2012. Again, we intended to use only males
in the study but were unable to capture enough males of L.
disjunctus and L. forcipatus. Therefore, we included some
females in those samples (all other samples were of males
only). At Barb’s Marsh, we collected Lestes disjunctus (N =
47, 16). PO activity was not measured in other species at
this site due to logistical problems. At Yzerinac’s Pond, we
used L. congener (N = 14), L. forcipatus (N = 20, 6), and
L. rectangularis (N = 19).
Damselflies were stored in separate vials in an ice-filled
cooler in the field until being moved to a fridge at 3 °C two
hours later. Wing length, from tip to base, was measured
using electronic calipers and the wing was then placed in
liquid nitrogen for 10 seconds and placed in −80 °C freezer.
The protocol used followed that of [40]. The damselflies
were dissected and the head, pronotum, wings, legs, and
abdomen were removed and stored in 95% ethanol. The
thorax was placed in an Eppendorf tube and dipped in liquid
nitrogen for 10 s. It was then crushed with a hand-held pistil.
300 µL of cooled cacodylate buffer (0.01 M C2 H6 AsNaO2 0.005 M CaCl2 , pH = 7.0) was added to each Eppendorf
tube. The cell walls were removed by centrifugation at 4 °C
Ecological Parasitology and Immunology
at a speed of 26916 g for 10 min. 100 µL of the extract
was placed into a well of the 96-well microplate along
with 35 µL PBS buffer, 5 µL chemo. It was allowed to
react for 5 min. Finally 60 µL of L-DOPA (10 mmol/L in
cacodylate buffer) was added. This was always done in pairs
so that each damselfly extract was placed in two wells. The
microplate was placed in a spectrophotometer (FLUOstar
OPTIMA microplate reader, BMG Latch). The reaction
proceeded for 30 min at 30 °C using an excitation filter of
wavelength 485. There were 20 cycles that each ran for 89 s.
Between the readings the microplate was shaken for 10 s
prior to the beginning of a new cycle. The readings were
done at the beginning of each cycle. Phenoloxidase activity
was measured as the slope of the reaction curve during the
linear phase.
2.4. Thoracic protein content
Protein content in the samples was always scaled towards
a standard curve with known concentrations. Therefore 6
dilutions of the protein BSA were prepared (0, 25, 50, 100,
150, 200, 250 µL/mL BSA). The dilution series was placed
in two rows of the microplate. The remaining extract, from
the PO content (5 µL), was placed in wells of a new 96-well
microplate with 155 µL Mili-Q and 40 µL Bradford reagent.
The microplate was placed in the spectrophotometer on an
endpoint reading using an absorption filter wavelength of
595 nm. The reaction was run at 30 °C for 6 min with continuous shaking.
2.5. Statistical analyses
Logarithmic transformations of wing length and grayscale
index (GI) were done to satisfy assumptions of normality.
Analysis of variance (ANOVA) was used to test for the
effect of sex, wing length, and species on LogGI and PO at
each site (since the two sites had different species of mites
and hosts). We tested for effects of sex in all the samples
where females were used and there were no differences
between males and females. Phenoloxidase levels were
regressed against protein content to control for size of the
species. The residuals from the regression of PO vs. protein
content were the response variable and damselfly species
was the explanatory variable in an analysis of variance
(ANOVA) in R (version 2.14.2: R Development Core
Team, 2012). Following the ANOVA, a post hoc Tukey test
determined which species was significantly different in PO
activity levels.
3. Results
3.1. Melanization of inserts
Melanization response or grayscale index (GI) was variable
within a species (Table 1). In the damselfly species from
Barb’s Marsh, species explained significant variation in
the ANOVA (F (1, 28) = 12.5, P = .0002), but sex and
Ecological Parasitology and Immunology
wing length or any interaction between these terms was
not significant (sex: F (1, 28) = 0.08, P = .77, wing length
F (1, 28) = 0.02, P = .86). L. congener had significantly
different LogGI scores compared to L. disjunctus and L.
rectangularis, meaning that L. congener melanized nylon
filaments more than the other two species.
At the Yzerinac’s Pond site, species also explained significant variation in the model, with sex and wing length and
any interaction between these terms not being significant
factors (F (1, 33) = 13.7, P < .0001); (sex: F (1, 33) = 1.9,
P = .17, wing length F (1, 33) = 2.3, P = .14). Again, L.
congener had significantly different LogGI scores compared
to L. forcipatus and L. rectangularis.
3.2. Phenoloxidase content
There was a significant difference among species at
Yzerinac’s Pond in phenoloxidase levels (F (3, 96) = 14.48,
P < .0001). Post hoc Tukey’s HSD tests showed that L.
congener had significantly higher phenoloxidase than the
other species. All other comparisons were not significant.
The phenoloxidase levels were highest in L. congener,
followed by L. disjunctus and L. forcipatus (Table 1).
4. Discussion
Measuring different components of the immune system
should improve the accuracy of estimates of an animal’s
ability to mount an immune response. In our study, both
PO activity and melanization of nylon monofilament
inserts gave similar results in four species of damselflies.
This contrasts some studies with insects that have found
conflicting results using these two proxies [8, 14, 42].
However, there were minor exceptions to this pattern that
suggest that the two proxies are not always positively
correlated. For example, L. disjunctus melanized the inserts
slightly less than L. forcipatus but had higher PO activity
levels. The PO activity levels in L. rectangularis were
slightly higher than in L. forcipatus, but L. rectangularis
deposited the least amount of melanin on the inserts of
all the four species. Potential problems with our protocol
for measuring PO are that we activated the proPO with
chymotrypsin. Other studies may not have used activated
proPO but instead measured the activity of PO. In addition,
we crushed the thorax of each damselfly for the PO assay, so
the sample presumably contained PO from the cuticle, gut,
and haemolymph, increasing our estimates of PO activity.
High PO activity and strong melanotic encapsulation
response to objects may reflect high immunocompetence to
natural parasites. However, we found that species with
a strong immune response to the inserts were from
populations with low parasite prevalence, which seems
counterintuitive. L. congener had the highest PO activity
levels and melanized the inserts more than the other species,
yet we have not seen parasitized individuals at either site
5
for at least five years. If we assume that immune responses
are costly to maintain and may be lost or reduced if there
is little selection from parasitism in a population, then
this result is unexpected. One explanation is either that
natural selection does not act on immune response or that
not enough time has passed since the reduction in mite
parasitism occurred in the host population. There is also the
obvious explanation that maintaining high immune response
may be adaptive if the species encounters other parasites
(the only other macroparasites that we have found in these
Lestidae are gregarines, which are found at very low levels
in L. congener).
Immune responses in nature are affected by many
factors, including parasite recognition, costs of parasitism,
host condition, environmental stress, and predation pressure,
which will differ among host-parasite associations. In
general, immune activity should be an adaptive response
to parasitism because variation in immune response can be
due to genetic effects in some insects [33]. Most researchers
assume that selection pressure from parasites causes the
evolution of increased immune responses in hosts. However,
coevolution between hosts and parasites can mean that
results from proxies will not necessarily reflect current
conditions, but rather past patterns or associations.
Smilanich et al. [38] found that lepidopteran larvae from
species that were not parasitized had higher immune
responses than those that were. They suggested that
parasitoids may avoid a host species that is capable of
killing them. In our system, Lestes congener may enjoy its
current parasite free status because mites avoid them due to
past selection pressure from this host. The emergence period
of L. congener has not been monitored regularly at these
sites, and it is possible that the species has shifted to a later
emergence period. This could have resulted in the avoidance
of parasitism that we have seen in the last few years. Natural
selection will have acted on mites to be ready to search for
hosts during the short window of time when the host insects
emerge from the water. Time spent searching for hosts
after the mites hatch can affect their ability to successfully
parasitize hosts [20, 29]. Lestes congener is one of the latest
Lestidae to emerge in this region [41], so the fact that it was
parasitized at all in the past may have been a chance occurrence or might relate to the site being more of an ephemeral
pond in the past (which selects for advanced emergence
timing in all host species and could mean that L. congener
overlapped its emergence with larval activity of A. planus).
Other reasons for the strong immune response shown
by Lestes congener may have little to do with adaptive
responses to parasitism. Although there is no reason to
expect taxonomic relatedness to be correlated with immune
response, we note that L. congener is not closely related to
the other three Lestid species [9]. L. congener is a very dark
damselfly, appearing almost black from above. Dark color
6
and melanization rely on the same biochemical processes,
so darker insects may be more resistant to parasites [3, 11].
Yourth et al. [44] recorded natural resistance levels of
50% for L. congener and 70% for L. forcipatus, yet measuring the immune response of L. congener by PO activity
or melanization of an insert would suggest to a researcher
that L. congener has a much stronger immune response to
parasitism. However, PO activity and melanization of nylon
inserts were not measured in 1998 when these natural resistance estimates were taken. The results from L. congener
are only one example of how it is sometimes unclear what
these proxies are actually measuring. In another comparison, L. forcipatus and L. disjunctus have similar PO activity
levels and responses to nylon inserts, as well as parasitism
prevalence levels (about 85% of the population). However,
L. forcipatus resists mites at very high levels (70%), while
in L. disjunctus it is usually below 5%.
Nagel et al. [23] examined the response of individuals
from two Lestid species to nylon inserts and compared
those with natural parasitism on the same individual. They
found that there was no correlation between damselflies that
resist mites naturally and the same individual’s response
to nylon filaments. That study would have benefited from
having melanotic encapsulation estimates and PO measures
from (age-controlled) mite-infected damselflies from all
the species used in the present study (which uses only
unparasitized insects). The measurements of grayscale
index (GI) obtained in this study were almost the same
as those from [23], suggesting that these measures are
repeatable.
However, both [23] and the present study suggest that
caution should be taken when using proxies. If the goal in
using a proxy such as nylon monofilaments is to determine
which species or individual has higher immunocompetence
in nature, our study suggests that this proxy is not an
accurate reflection of natural parasitism patterns. Levels of
current mite parasitism were not correlated positively with
immune response measured by either proxy. Measuring
immune response using these proxies therefore does not
reflect mite prevalence in these populations but may be a
good estimate of past or current resistance levels in one
species (L. congener).
Nylon filaments should be good surrogates for actual
mite parasitism (mimicking mite feeding tubes and being
placed in the same location on the host thorax that mites
attach to). The choice of a proxy is crucial to the question
being explored, so researchers need to consider what is
being measured and how appropriate the proxy is. For
example, if the goal of a study is to estimate the likelihood
of a host responding to a parasite that is assumed to be
costly, using a parasite that has coevolved with the host may
not be appropriate. If the two organisms are at a point in
their evolution where the host is well adapted to coping with
Ecological Parasitology and Immunology
the parasite, the immune response measured may not reflect
the expected costs. If we expect a host to show high innate
immunity, using nylon implants may be more appropriate
because parasite recognition will not be a confounding
factor in the study design. If we expect a host to show
greater specific immunity, using real parasites may be more
appropriate.
This study is correlational, and we acknowledge that
many factors affect investment in immunity (e.g., age and
tolerance) [26]. Our results do not question the roles that PO
activity or melanization plays in natural immune response,
but they do suggest that measuring immune response
using these proxies does not always reflect current parasite
pressures in a population.
Acknowledgments The authors thank André Morrill for assistance in
the field and many insightful discussions and Arne Iserbyt for aid with
the PO component. The Natural Sciences and Engineering Research
Council of Canada funded L. Nagel and M. R. Forbes.
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