Immune Priming, Fat Reserves, Muscle Mass and Body Weight of the House Cricket is Affected by Diet Composition

Abstract

Some insect species are capable of producing an enhanced immune response after a first pathogenic encounter, a process called immune priming. However, whether and how such ability is driven by particular diet components (protein/carbohydrate) have not been explored. Such questions are sound given that, in general, immune response is dietary dependent. We have used adults of the house cricket Acheta domesticus L. (Orthoptera: Gryllidae) and exposed them to the bacteria Serratia marcescens. We first addressed whether survival rate after priming and nonpriming treatments is dietary dependent based on access/no access to proteins and carbohydrates. Second, we investigated how these dietary components affected fat reserves, muscle mass, and body weight, three key traits in insect fitness. Thus, we exposed adult house crickets to either a protein or a carbohydrate diet and measured the three traits. After being provided with protein, primed animals survived longer compared to the other diet treatments. Interestingly, this effect was also sex dependent with primed males having a higher survival than primed females when protein was supplemented. For the second experiment, protein-fed animals had more fat, muscle mass, and body weight than carbohydrate-fed animals. Although we are not aware of the immune component underlying immune priming, our results suggest that its energetic demand for its functioning and/or consequent survival requires a higher demand of protein with respect to carbohydrate. Thus, protein shortage can impair key survival-related traits related to immune and energetic condition. Further studies varying nutrient ratios should verify our results.

Full text

SYSTEMATICS, MORPHOLOGY AND PHYSIOLOGY
Immune Priming, Fat Reserves, Muscle Mass and Body Weight
of the House Cricket is Affected by Diet Composition
ACÓRDOBA-AGUILAR
1
,ANAVA-SÁNCHEZ
1
,DMGONZÁLEZ-TOKMAN
1,2
,RMUNGUÍA-STEYER
3
,
AE G
UTIÉRREZ-CABRERA
4
1
Depto de Ecología Evolutiva, Instituto de Ecología, Univ Nacional Autónoma de México, Ciudad Universitaria, Mexico, D.F., Mexico
2
CONACyT Research Fellow, Instituto de Ecología, Xalapa, Mexico
3
Unidad de Morfología y Función, Fac de Estudios Superiores Iztacala, Univ Nacional Autónoma de México, Tlalnepantla, Mexico
4
CONACyT Research Fellow, Centro de Investigación en Enfermedades Infecciosas, Instituto Nacional de Salud Pública, Cuernavaca, Morelos,
Mexico
AbstractKeywords
Carbohydrate, house cricket,
immunological priming, protein
Correspondence
A C órdoba-Aguilar,
Depto de Ecología
Evolutiva, Instituto de Ecología, Univ
Nacional Autónoma de México, Ciudad
Universitaria, Mexico, D.F., Mexico;
acordoba@iecologia.unam.mx
Edited by Guilherme D Rossi – UNESP
Received 12 May 2015 and accepted 15
March 2016
* Sociedade Entomológica do Brasil 2016
Some insect species are capable of producing an enhanced immune re-
sponse after a first pathogenic encounter, a process called immune prim-
ing. However, whether and how such ability is driven by particular diet
components (protein/carbohydrate) have not been explored. Such ques-
tions are sound given that, in general, immune response is dietary depen-
dent. We have used adults of the house cricket Acheta domesticus L.
(Orthoptera: Gryllidae) and exposed them to the bacteria Serratia
marcescens. We first addressed whether survival rate after priming and
nonpriming treatments is dietary dependent based on access/no access to
proteins and carbohydrates. Second, we investigated how these dietary
components affected fat reserves, muscle mass, and body weight, three
key traits in insect fitness. Thus, we exposed adult house crickets to either
a protein or a carbohydrate diet and measured the three traits. After being
provided with protein, primed animals survived longer compared to the
other diet treatments. Interestingly, this effect was also sex dependent
with primed males having a higher survival than primed females when
protein was supplemented. For the second experiment, protein-fed ani-
mals had more fat, muscle mass, and body weight than carbohydrate-fed
animals. Although we are not aware of the immune component underlying
immune priming, our results suggest that its energetic demand for its
functioning and/or consequent survival requires a higher demand of pro-
tein with respect to carbohydrate. Thus, protein shortage can impair key
survival-related traits related to immune and energetic condition. Further
studies varying nutrient ratios should verify our results.
Introduction
Several studies have shown that invertebrates can respond
more effectively after a first encounter with a pathogen, a pro-
cess otherwise known as immun e priming (e.g., Cisarovsky et al
2012,Dauksteet al 2012 but see González-Tokman et al 2010,
Reber & Chapuisat 2012). Unlike vertebrates, this memory-like
ability in invertebrates is based on germline-encoded molecules
and epigenetic processes which allows recognizing pathogens
effectively (e.g., Watson et al 2005,Donget al 2006,Gómez-
Díaz et al 2012, Mukherjee et al 2015).
Despite the fact that there is interindividual variation in
immune priming (Daukste et al 2012), few studies have ex-
plored why this is the case. It has been proposed that priming
Neotrop Entomol
DOI 10.1007/s13744-016-0391-0

ability can be affected by individual condition (González-
Tokman et al 2010) and age (Daukste et al 2012), and one
underlying explanation for this is that priming is costly. For
example, it was recently shown that females of Anopheles
albimanus Wiedemann (Diptera: Culicidae) pay a reproductive
cost via immune priming when infected with the parasite
Plasmodium berghei (Contreras-Garduño et al 2013). The au-
thors found a lower egg-hatching success in primed females
than in control females (Contreras-Garduño et al 2013). One
environmental source explaining priming costs is diet.
Moreover, assuming that dietary resources are generally limit-
ed and shared among several traits, one should expect trade-
offs between the immune system and other life history traits
(Sheldon & Verhulst 1996). Therefore, only individuals in good
nutritional condition are able to generate effective immune
responses. To our knowledge, this hypothesis has not been
tested in terms of immune priming in invertebrates. A related
issue, however, is that several have studied the effect of pa-
rental diet on offspring diet and immune reactivity (e.g., Alaux
et al 2010, Cotter et al 2011, Triggs & Knell 2012).
Not all dietary components impact immune ability similar-
ly (Lee et al 2006,Poveyet al 2009,Pontonet al 2013). One
case is that of protein and carbohydrate components which
are key during parasite defense (Lee et al 2006,Poveyet al
2009, 2014,Cotteret al 2011), but their impact on immune
components varies: proteins promote phenoloxidase activi-
ty, encapsulation response, and antimicrobial activity (Lee
et al 2006 , Povey et al 2009, Roth et al 2010, González-
Santoyo & Córdoba-Aguilar 2012), while carbohydrates in-
crease lysozyme-like antibacterial activity (Srygley & Lorch
2013). In terms of immune priming, there are at least two
gaps in relation to the effect of protein and carbohydrate
dietary components: (1) how these components indepen-
dently affect immune priming and (2) the role of these com-
ponents on other nonimmunological traits that also play a
role in insect fitness, to have a more integrated framework of
the action of diet on trade-offs between immunological and
nonimmunological traits. Three such nonimmunological
traits that are energetically costly and thus likely to trade-
off with immune priming are adult fat reserves, muscle mass,
and body weight. Our understanding of the use of fat re-
serves and muscle mass indicates that insect fitness depends
on these traits for a plethora of activities. Some examples are
male-male aggressive contests (e.g., Lailvaux & Irschick
2006), predator avoidance (e.g., MacLeod et al 2007), migra-
tion (McWilliams et al 2004), hibernation (e.g., Humphries
et al 2003), egg production (e.g., Jervis
et al 20
05), and
immune ability (e.g., Jimén ez-Cortés & Córdo ba-Agui lar
2013), among others.
We know by previous studies that adult crickets Acheta
domesticus L. (Orthoptera: Gryllidae) have an enhanced
phagocytic activity after a previous encounter with a patho-
genic bacteria, Serratia marcescens (Nava-Sánchez et al
2015). Notice that this previous study, however, did not take
into account the effect of diet (fish food and water were
provided) on immune ability. In the present study, we inves-
tigated the effects of protein and carbohydrate components
on immune priming and nonimmunological traits. We reared
male and female crickets under different dietary qualities—
protein and carbohydrate based—and measured survival af-
ter repeated encounters with the same bacteria. In a second
experiment, we measured adult fat reserves, muscle mass,
and body weight after a period of 15 days under these dietary
conditions. We do not have specific predictions in regard to
how proteins/carbohydrates will affect immune ability dur-
ing priming. This is partly because the mechanism of how
insect immune priming works is not clear and so there are
no straightforward connections of how proteins and/or car-
bohydrates will impact it. Nevertheless, for fat reserves, mus-
cle mass, and body weight, predictions can be put forward
based on previous studies in insects: while carbohydrate will
increase fat reserves, protein diets will positively affect mus-
cle mass (e.g., Chapman 2012, Roeder & Behmer 2014).
However, a balanced or slightly protein-based diet will posi-
tively affect body weight (e.g., Roeder & Behmer 2014).
Material and Methods
Acheta domesticus, the house cricket, has been widely used
as a study model in the field of ecological immunology (e.g.,
Adamo 1999, Ryder & Siva-Jothy 2000). For example, it is
known that there are trade-offs between immune response
and reproduction in both sexes (Bascuñan-García et al 2010).
Animals were derived from the vivarium of the Facultad de
Estudios Superiores Iztacala (Universidad Nacional
Autónoma de México). Eggs were kept in a humid substrate
(peat moss, commercial substrate) at 27 ±2°C with natural
photoperiod day/night. After hatching, crickets were kept in
a glass box (20 cm × 40 cm × 25 cm) covered with a glass
cover. No more than 15 crickets were placed inside each glass
box. Fish food was supplied on a cotton wool ball placed at
the bottom of each container. This diet was provided until
the experiment started which was 30 days before reaching
adulthood. The cotton was changed for a fresh one every
day. When the crickets reach the adult stage, males and
females were separated and left individually for the rest of
the experiment. This was done to avoid cannibalism given
the experimental diet restrictions.
Experiment 1—diet effects on priming
For assessing diet effects, crickets were reared on diets
of different compositions. A first gro up of 68 individuals
(34 males and 34 females) w ere fed on a protein diet
and a second group of 66 individ uals (33 males and 33
Córdoba-Aguilar et al

females) were fed on a carbohydrate diet (for the prep-
aration of both diets, see below). The two groups were
maintained on th ese diets for an approximate time of
45 days for each group: 30 days before and 15 days after
reaching ad ultho od. We chose to keep o ur animals un-
der this diet for this 15-day time window, to solve the
logistic compromise of measuring survival after chal-
lenge, which t ook an extra 15 days. Since adults usually
survive for 60 – 90 days in controlled conditions, the first
15 days (under our dietary conditi on) plus the 15 d ays of
survival a ssessment provide enough time to assess their
survival. After this period , e ach group was divided into a
priming group (n =35 protein diet; n = 32 carbohydrate
diet) and a contro l group (n = 33 protein diet; n =34 car-
bohydrate diet). The primed group was injected with
3 μ L Grace medium solution containing a LD
10
(2.5 × 10
5
cfu/μL) of S. marcescens bacteria, while the
control group was injected with 3 μLofGracemedium.
After 7 days, the four group s were i njected with 3 μLof
S. marcescens in culture media at a dose LD
75
(9 × 10
5
cfu/μL). These LD
10
and LD
75
doses were deter-
mined f rom a previous stu dy using the same h ost and
pathogen (Nava-Sán chez et al 2015 ). We also used this
previous study for the 7-day period, as this is the time
where priming using S. marcescens develops (Nava-
Sánchez et al 2015). After the seco nd injection, we mea-
sured survival every day for a w eek.
Diet preparation. We prepared the diet based on the meth-
odology used by Simpson et al (2006), which has been used
in other studies (Srygley & Lorch 2011, 2013). These artificial
diets were created considering the optimal growth of locusts
Schistocerca gregaria (Forskål) and Locusta migratoria
(Linnaeus) which feed mainly on plants but also bran, sugar
cubes, wood, paper, and wax (Dadd 1960). According to this
methodology, the protein diet consisted of 42% of a 3:1:1
mixture of casein, peptone, and albumin, while the carbohy-
drate diet consisted of 42% of a 1:1 mixture of sucrose and
dextrin. The remainder of the two diets comprised 54% cel-
lulose, which is a key factor in insect growth, 2% cholesterol,
and 2% linoleic acid. The reasons for using these components
are the following: Although cellulose is frequently used as a
bulking agent in insect diets (Lee et al 2006) and is indigest-
ible by most insects, it is an important factor for maximum
growth in crickets (Neville & Luckey 1962,Lynet al 2011). On
the other hand, both cholesterol and linoleic acid satisfy the
lipid requi rements in orthop terans (S. gregaria and
L. migratoria) in the adult stage (Dadd 1960). We did not
want to add Wesson components typical of insect diets (min-
erals and vitamins) as, given their role in insect development
(Nation 2016), they may also have unpredicted effects on
immune ability that may have affected our results.
Experiment 2—diet effects on muscle mass and fat load
We used 61 animals that were divided into two groups. The
first group was fed with the protein diet (n =28; 14 males and
14 females), while the second was fed with the carbohydrate
diet (n = 33; 16 males and 17 females). This feeding protocol
was provided 30 days before and 15 day s after reaching
adulthood, the time at which we measured fat load, muscle
mass, and body weight.
Quantification of fat reserves, muscle mass, and body weight.
Crickets were left without food for 3 days to purge their guts
after which they were sacrificed in 90% ethanol. After this,
we placed the body in a desiccator (for 48 h) and obtained
their dry weight to the nearest 0.001 g. Initial dry weight
includes basically the exoskeleton, fat, and muscle. We then
placed samples in chloroform/methanol for 24 h and added
salt to extract the fat (for a full description of methodological
details, see Barnes & Blackstock 1973). Samples were then
redesiccated and reweighed. We quantified fat content as
the difference between the initial weight and the second
weight (Contreras Garduño et al 2008). After fat extraction,
we submerged the samples in 0.8 M potassium hydroxide for
48 h to extract the muscle (Plaistow & Siva-Jothy 1996). The
fact that samples were not boiled, however, avoided glyco-
gen extraction (Carroll et al 1956
). The weight of the body
wa
s measured previous to and after the extraction, and the
difference was interpreted as total muscle mass. For body
weight, we used that recording of dry body weight before
chloroform/methanol-based fat extraction explained above.
To account for the effect of body size in fat, muscle, and body
weight quantification, we measured the length of the left
femur with a digital micrometer (precision ±0.01 mm; for
the use of leg length as an indicator of body size, see
Simmons 1986). After checking that our measurement error
was low (see below), femur length was measured by the
same person three times, so that we used an average for
subsequent analyses.
Statistical analysis
For experiment 1, we assessed survival of adult individuals
using treatment (priming, no priming), diet group (protein,
carbohydrate), and gender as predictor variables of individu-
al survival. As the hazard rate was variable, we used para-
metric models with a Weibull distribution . We estimated
Kaplan-Meier survival curves for each group according to
treatment and diet.
For experiment 2, we tested for differences in fat load,
muscle mass, and body weight using three general linear
models. In the global (most parametrized) models, we includ-
ed diet and femur length as predictor variables as well as
their second and third order interactions. From these global
A Protein-Based Diet Enhances Immune Priming in the House Cricket

models, we fitted simpler models with fewer interactions
and predictor variables. We then performed model selection
and ranked those models using AIC values that showed the
best compromise between fit and parsimony of each partic-
ular model (Johnson & Omland 2004). Additionally, we per-
formed sequential (type I) analyses of deviance in the most
supported models. Previous to this, we made sure that our
measurement error was small enough by using random ef-
fects analysis of variance (Bailey & Byrnes 1990, Yezerinac
et al 1992). This analysis pro duced a low error measure-
ment = 3.10% . Fat load data was not normally distributed
(Shapiro-Wilk test) and was adjusted by using their log
10
-
transformed values. All statistical analyses were carried out
in software R v. 3.02 (R Core Development Team 2013).
Results
Experiment 1—diet effects on immune priming
Survival after a second bacterial challenge was dependent on
treatment (deviance = 34.80, df =1, p < 0.001 ), diet (d evi-
ance=42.08, df =1, p < 0.001), and the interaction
diet × treatment (deviance = 15.79, df =1, p <0.001; Fig 1).
According to this interaction, survival was higher in primed
male and female reared under the protein diet compared to
the other groups (Fig 1).Thedifferenceinmediansurvivaltime
observed between samples was approximately 2 days (Fig 2).
Survival was not different for the other groups. Survival was
also dependent on gender (deviance = 9.62, df =1, p <0.001),
with males surviving longer than females (Fig 2).
Experiment 2—diet effects on muscle mass, fat load,
and body weight
Muscle mass. The most supported model regarding muscle
mass considered size (F =44.1; df =1, 57; p <0.001), sex
(F = 23.7; df = 1, 57; p < 0.001), and diet (F = 11.4; df = 1, 57;
p = 0.001). These results implied that muscle content was
higher in (a) larger individuals, (b) females than in males,
and (c) protein-fed individuals compared to carbohydrate-
fed animals (Fig 3).
Fat content. The most supported model included size, diet,
and their interaction as predictor variables. These results
implied that larger individuals had more fat content
(F = 13.26; df =1, 57; p <0.001) and that protein-fed individ-
uals had a higher fat load than carbohydrate-fed animals
(F = 16.07; df =1,57;p <0.001;Fig3). The interaction between
size and diet was not significant (F =1.95;df =1,57;p =0.168).
Body weight. The most supported model included size, diet,
weight, and the interaction between size and sex. These results
meant that larger individuals weighed more than smaller ani-
mals (F = 83.68; df
=1, 56; p <
0.0001), that males were heavier
than females (F = 26.88; df =1, 56; p< 0.001), and that protein-
Fig 1 Survival curves according to sex, treatment, and diet interaction in Acheta domesticus crickets previously exposed to pathogenic bacteria,
Serratia marcescens: a female and b male.
Córdoba-Aguilar et al

fed individuals weighted more than carbohydrate-fed animals
(F =10.29; df =1, 56; p = 0.002). The interacti on between size
and sex was not significant (F =2.80; df =1, 56; p =0.099).
Discussion
As expected by the results of a previous study (Nava-Sánchez
et al 2015), we have corroborated that male and female
A. domesticus crickets that experienced a first encounter
with the pathogenic bacterium S. marcescens gained protec-
tion against a second infection with the same pathogen later
in life. However, in both males and females, the protective
effect of the first encounter took place only when crickets
were fed with protein and not when fed on carbohydrates
alone during all adult life. This result confirms our prediction
that, just as other components of insect immune response
(Schmid-Hempel 2005), priming response is a costly trait that
only individuals in good physiological condition (based on
good nutritional state) can afford. Alternatively, it may also
be that carbohydrate-fed individuals may have also devel-
oped a priming response but that the large amount of re-
sources demanded to survive after being primed cannot be
satisfied. This issue can be partly resolved once the mecha-
nism that underlies priming responses is clarified to then
assess how costly immune priming is. On the other hand, it
would have been desirable to verify that bacteria were in-
deed present in the infected groups to confirm that it was
the infection that explained survival differences. We also
showed that a protein diet improved individual muscle mass,
fat load, and body weight compared to a carbohydrate diet.
Except for muscle mass, these results were not sex depen-
dent. Such protein effect is against previous knowledge that
indicated that carbohydrates are converted into storage
lipids (Arrese & Soulages 2010) and that protein may slightly
contribute to body weight (Roeder & Behmer 2014). The
general salient interpretation of our two experiments is that,
unlike carbohydrates, proteins not only favor immune prim-
ing but also physiological traits (muscle, fat reserves, and
body weight) closely linked to fitness (Lee et al 2006,
Povey et al 2009). However, to confirm this, one needs to
vary nutrient ratios (e.g., Behmer et al 2001) and expect, for
example, that protein levels positively and gradually affect
immune priming and physiological state.
Different components of insect innate immune response
are costly traits because their expression can be genetically
and physiologically linked to other immunological or life his-
tory traits (Sheldon & Verhu lst 1996, Cotter et al 2003,
Schmid-Hempel 2005). As a consequence, immune response
is highly sensitive to food quality (Lee et al 2006,Poveyet al
Fig 2 Expected median survival time according to sex, treatment, and diet in Acheta domesticus crickets. Error bars represent 95% confidence intervals.
Fig 3 Fat load and muscle mass (g) in relation to a protein and
carbohydrate diet treatments. Bars represent standard errors. ***
indicates significant differences at p < 0.001.
A Protein-Based Diet Enhances Immune Priming in the House Cricket

2009,Srygleyet al 2009,Pontonet al 2011, Triggs & Knell
2011). Our results that priming response against bacteria is
sensitive to protein availability imply that protein-deprived
insects may become immune depressed (notice, again, that
we are not aware of the immune parameter being affected).
Moreover, the fact that low protein negatively affected fat
reserves and muscle mass implies that these two traits are
very costly. This cost may in turn affect immune priming
although this cannot be assessed directly by our second ex-
periment. Given that one mechanism by which insect prim-
ing takes place is mediated by phagocytes (Pham et al 2007,
Roth & Kurtz 2009), which are protein-rich hemocytes
(Lemaitre & Hoffman 2007), one explanation for our exper-
iment is that protein limitation led to a reduction in hemo-
cyte number and, consequently, phagocytic activity. Future
studies should evaluate experimentally if phagocytosis is re-
duced upon repeated infections under low protein availabil-
ity and if the constant higher survival found in males with
respect to females (as previously observed by Nowosielski &
Patton 1965) is caused by sexual differences in phagocytic
activity. Related to this, Adamo et al (2001) found that fe-
males were more immunocompetent than males when both
sexes reached maturity and were previously infected with
S. marcescens. With respect to our results, since Adamo
et al (2001) did not measure priming, one explanation for
this discrepancy is that priming may be more energetically
demanding for females than males, a situation that should be
specifically looked for in future studies.
That protein input is key for an enhanced priming response
provides evidence that immune priming is condition depen-
dent. It also opens new questions in insect physiology and
evolutionary biology, including, for example, the evolution of
adaptive foraging and transgenerational immune ability. For
the case of adaptive foraging, since compensator y protein in-
take shown by infected individuals has consequences on im-
mediate immune responses (Lee et al 2006,Pontonet al 2011),
one question is whether insects augment their protein con-
sumption if encounter ing the same pathogen is likely. For the
case of transgenerational immune ability, given that parental
immunity can be transmitted across generations by both the
mother and the father (Little et al 2003,Rothet al 2010,Zanchi
et al 2011, also called as cryptic parental care, Jokela 2010),
transgenerational immune priming could be sensitive to paren-
tal condition.
Finally, while mathematical modeling predicts that the ben-
efits of immune priming depend on host lifespan and patho-
gen virulence (Miller et al 2007,Bestet al 2012), our results
show that priming response also depends on individual nutri-
tional condition. Thus, suboptimal nutrition has to be consid-
ered in future studies of factors affecting priming ability.
Acknowledgments This paper is a partial fulfillment of the require-
ment for the doctoral degree of A. Nava-Sánchez at the graduate
program Doctorado en Ciencias Biomédicas of the Universidad
Nacional A utónoma de México. Thanks to L. Cordero Méndez
(Facultad de Estudios Superiores Iztacala) who provided the crickets,
R. I. Martínez-Becerril for logistic support, and M. A. Moreno-García
and M. Tapia R. for key assistance in the laboratory.
Compliance with Ethical Standards
Conflict of Interest The authors declare that they have no conflicts of
interest.
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A Protein-Based Diet Enhances Immune Priming in the House Cricket