Discussion
The results of our study show that both macronutrient intake and a
simulated infection threat influence the expression of life history
traits in decorated crickets, G. sigillatus . Specifically, both
diet and an immune challenge with heat-killed bacteria: 1) affected male
reproductive effort, quantified here as male calling effort and 2)
greatly altered male survival. The two diets used in the current study
were deliberately selected because they had been shown previously to
either maximize calling effort (P:C = 1:8, high carbohydrate diet) or to
maximize immune function, at least with respect to encapsulation ability
(P:C = 5:1, high protein diet) (Rapkin et al., 2018), thereby making it
more likely that the effects of our immune challenge would be contingent
on the diet on which males had been maintained. However, we found no or,
at best, weak evidence of an interaction between the diet on which males
were maintained and the level of the immune challenge that they
experienced in the expression of life-history traits. Moreover, although
our results aligned with the anticipated effects of these two diets on
male calling, surprisingly, they were in apparent contradiction of the
anticipated effect on male immunity. We elaborate on these differences
below and seek to identify potential mechanisms mediating observed
treatment effects and the factors potentially constraining an
interaction between diet and the magnitude of an immune challenge on
male life history strategy.
Males maintained on a high protein diet exhibited significantly higher
mortality than those held on the high carbohydrate diet. Nearly 30% (68
out of 249) of males maintained on the high protein diet died prior to
end to the experiment compared with less than 14% (33 out of 245) of
males held on the high carbohydrate diet. This is consistent with
numerous findings across taxa showing that lifespan is truncated in
animals fed a high protein, low carbohydrate diet (reviewed in Le
Couteur et al., 2016 and Simpson et al., 2017), including G.
sigillatus (J. Hunt, unpublished data). These recent discoveries, which
contrast with conventional Y resource allocation models (van Noordwijk
and de Jong, 1986; Zera and Harshman, 2001), have inspired the lethal
protein hypothesis (Lee et al., 2008; Fanson et al., 2009; Simpson and
Raubenheimer, 2009). Proposed mechanisms mediating the lethal protein
hypothesis include increased mitochondrial generation of radical oxygen
species (Sanz et al., 2004), changes in the relationship between
insulin/IGF-1 and amino acid signaling (e.g., TOR) pathways (Kapahi et
al., 2004), and damage to organs from nitrogenous waste products.
Although further studies are needed to conclusively determine whether
the diet-induced mortality in the present study is due to an excess of
protein or a deficit of carbohydrates, it is unlikely due to the latter
as males were always provided more food than they could eat, suggesting
that they were not carbohydrate limited. Males held on the high protein
diet consumed a greater amount of food than those held on the high
carbohydrate diet across the entire range of body sizes, which could
have been due to a greater nutritional demand for carbohydrates in the
former. Indeed, Rapkin et al. (2018) showed that when male G.
sigillatus are given a dietary choice, they regulate their intake of
protein and carbohydrate to a P:C ratio of 1:2.
As was the case with diet, infection cue dose had a significant effect
on male survival through the end of the two-day calling period.
Specifically, the highest infection cue dose resulted in higher male
mortality than any of the other infection cue levels. This suggests that
our simulated infection threat imposed a physiological cost on males,
and that at the most severe infection threat level, this cost was
manifest in increased mortality. This is consistent with a cost of using
the immune system (Sadd and Schmid-Hempel, 2009), which has been shown
to have consequences for survival in other insect taxa (Moret and
Schmid-Hempel, 2000; Armitage et al., 2003). This has also previously
been shown to be independent of nutritional condition in another cricket
species (Jacot et al., 2004).
Both diet and infection cue treatment had significant effects on male
reproductive effort, measured here as the time spent calling to attract
a mate (termed calling effort). Calling effort was significantly higher
in males held on the high carbohydrate diet than in those held on the
high protein diet, consistent with previous work (Rapkin et al., 2018).
We also found that infection cue, specifically at a low dose, impacts
male reproductive effort. If they called, males injected with a low
infection cue dose called more relative to naive controls. This
increased calling effort is consistent with a pattern of terminal
investment (Duffield et al., 2017), but, surprisingly, neither of the
two higher infection cue treatments differed from the naive control
group with respect to calling effort. However, at least in the case of
males injected with the high infection cue dose, males experienced at
least a two-fold higher mortality during the calling period compared
with males in the other infection cue treatments, and thus, males in
this group were, to some extent, self-selected. If the males that died
were those that were more inclined to terminally invest, whereas those
that survived prioritized maintenance over reproduction, then this would
attenuate any effect of infection cue treatment on calling effort at the
more severe infection threat levels.
Although diet and infection cue treatment independently influenced male
calling effort, we found no evidence of an interaction between these two
factors. The absence of such an interaction contrasts with an early
study demonstrating an interaction between male age and infection cue
dose, in which older male G. sigillatus increased their calling
effort in response to the same graded increase in infection threat used
here, whereas younger males did not (Duffield et al., 2018). Why do
males retain this level of plasticity in calling effort with respect to
age, but not diet? One possibility is that age overrides any influence
of diet on the propensity to shift life history strategy, given that it
has been previously shown that males at the age used in this study
exhibit terminal investment, whereas younger males do not (Duffield et
al., 2018). A standard lab diet was provided in this previous study, but
it is possible that terminal investment in younger males could be
elicited by certain combinations of diet and infection cue dose, in line
with the dynamic terminal investment threshold model (Duffield et al.,
2017). An alternative is that males were terminally investing in other
important components of reproductive effort that we did not measure in
the present study. For example, the spermatophore transferred by males
at copulation includes a large gelatinous mass, the spermatophylax, that
the female consumes after mating as a nuptial food gift, which is
critical to male fertilization success (Sakaluk, 1984; Sakaluk, 1986;
Sakaluk and Eggert, 1996; Eggert et al., 2003). Duffield et al. (2015)
showed that immune-challenged males terminally invest by altering the
free amino acid profile of the spermatophylax, enhancing its gustatory
appeal to females (see also Gershman et al., 2012). Moreover, male
chemical cues, in the form of cuticular hydrocarbons (CHCs), greatly
influence whether a female mounts a male, a necessary antecedent to
copulation (Weddle et al., 2013; Capodeanu-Nägler et al., 2014). The CHC
profile of a male, and by extension, his attractiveness, can be
significantly influenced by his nutritional environment (Weddle et al.
2012). Both CHC expression (Rapkin et al., 2016) and the amino acid
composition of the spermatophylax (Rapkin et al., 2017a) are optimized
at a P:C ratio of 1:1.3 and 1:1.5, respectively, ratios far removed from
the two diets offered in the present study.
Despite our initial expectation, we found no evidence that males held on
the high protein diet exhibit enhanced immune function. Specifically,
there was no positive effect of diet on the number of circulating
hemocytes, presence of microaggregations, or antibacterial activity of
the hemolymph. We did, however, find a significant interaction between
diet and infection cue dose on the incidence of circulating
microaggregations of hemocytes, suggesting that both factors could
affect this immune response. This contrasts with the results of a
previous study showing that among 24 diets differing in P∶C ratio and
distributed along six nutritional rails, a P:C = 5:1 ratio optimizes
immune function as assessed using an encapsulation assay (Rapkin et al.,
2018). In the present study, however, we did not measure the
encapsulation ability of males, and the difference between the two
studies may be a function of the different components of immunity that
were measured. Different facets of immunity can be triggered by
different types of threats or regulated independently, resulting in
positive, negative, or no associations between immune components (Adamo,
2004; Forsman et al., 2008). Indeed, although Rapkin et al. (2018)
demonstrated a significant effect of macronutrient intake on male
encapsulation ability, they found no effect of diet on the activity of
phenoloxidase, an important enzyme in the melanization cascade.
Ultimately, we are cautious to draw any firm conclusions about the
influence of diet and infection cue on immune parameters, due to the
interesting but confounding differential effects of these factors on
survival prior to the assaying of immunity.
In conclusion, both macronutrient intake and a simulated infection
threat independently influenced the survival and reproductive effort of
male G. sigillatus . There was evidence for terminal investment,
as males increased calling effort at the low infection cue dose, but
interpretation of responses at the higher threat levels was hampered by
the differential mortality of males across diet and infection cue
treatments, the latter demonstrating a cost of immune activation for
survival. There was, however, no evidence of an interaction between diet
and infection cue dose in their influence on calling effort, suggesting
that the threshold for terminal investment was not contingent on diet,
in contrast to earlier work documenting a shifting terminal investment
threshold contingent on male age (Duffield et al., 2018). The absence of
a dynamic terminal investment threshold may have been due to the
previously documented influence of age masking other intrinsic and
extrinsic factors, or, alternatively, males prioritizing investment in
other components of reproductive effort. Regardless, how reproductive
effort changes in accordance with the various intrinsic and extrinsic
factors that alter an individual’s residual reproductive value remains a
fertile area of inquiry (Duffield et al., 2017), but any generalizations
must await additional comparative studies that preferably incorporate
multiple facets of male reproductive effort.