Title: Female investment in terminal reproduction or somatic maintenance depends on infection dose

Immune responses are energetically costly to produce and often trade off with investment in reproduction and offspring provisioning. We predicted that hosts can interpret the magnitude of the infection threat from cues like initial bacterial load to adjust reproductive investment into offspring quantity and quality. To test this prediction, we exposed female flour beetles ( Tribolium castaneum ) to naïve or sterile media controls or one of three increasing doses of heat-18 killed Bacillus thuringiensis (Bt). To estimate offspring quantity, we measured the number of eggs and hatched larvae from each maternal treatment group. To estimate offspring quality, we measured egg protein content, development to pupation, pupal weight, and offspring survival 21 against Bt infection. Compared to naïve controls, low and intermediate bacterial doses resulted in lower female fecundity, suggesting a shift to a somatic maintenance strategy. Meanwhile, in the 23 highest-dose group, fecundity was negatively correlated with egg protein content, suggesting a trade-off between offspring quality and quantity that could mask terminal investment based on metrics of quantity alone. Our results underscore the need to account for the magnitude of environmental cue when quantifying plasticity and trade-offs among life history traits, and 27 provide new insight into the trans-generational effects of immune responses.


Introduction 2 9
Parasites and pathogens steal energetic resources from their hosts and damage host 3 0 tissues. The energy that hosts allocate to resist infections, repair damage, and maintain 3 1 homeostasis can come at the expense of other life history traits like reproduction. Sometimes, it terminal investment dilemma focuses on the dimension of offspring quantity, investment in 1 3 8 naïve (handled but not injected) and sterile saline-injected controls, we made two tenfold with the number of CFUs/mL in our priming doses and confirmed that the heat had killed all 1 4 4 cells, respectively. We randomly assigned all sexually mature females to one of the five priming 1 4 5 treatment groups.

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We carried out the priming as previously described, but without anaesthetizing the 1 4 7 beetles [26]. In short, we pricked beetles with an ultrafine needle between occiput and pronotum was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 25, 2022. ;https://doi.org/10.1101https://doi.org/10. /2022 Fecundity after Bt exposure 2 7 6 We counted eggs produced by each mating pair for each of the six oviposition periods. We 2 7 7 combined all counts for each pair into one value because the egg-laying rate did not vary 2 7 8 significantly over time between the treatment groups (Suppl. Figure 1, Suppl. Table 2). Egg 2 7 9 production was significantly reduced in the group that had received the low Bt dose compared to  Table 2). To exclude the 2 8 1 possibility that the reduced egg laying rate from females that died during the experiment was the 2 8 2 sole contributor to this finding, we ran the same analysis on the data set containing only egg Estimate=-0.242, p=0.002, Figure 2A, Suppl. Table 2). Females that received a low or  reduced by 7% and 6% respectively and did not differ significantly from those laid by the naïve 2 9 0 group (wounding control: Estimate=-0.108, p=0.143; high dose: Estimate=-0.047, p=0.55, Figure   2 9 1 2A, Suppl. Table 2).

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For oviposition periods 1, 3 and 5, we let the eggs develop and counted live larvae produced by each female two weeks later. We also combined these counts into one total value 2 9 4 per female for the analysis. Similar to the egg count, females that received a low or intermediate dose had significantly fewer larval offspring than the naïve group (low dose: 24% reduction, there was no significant difference between the naïve group and the wounding control (15% 2 9 8 . CC-BY-NC 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 25, 2022. ;https://doi.org/10.1101https://doi.org/10. /2022 fewer larvae, Estimate=-0.162, p=0.094) or the group that received the high dose of heat killed 2 9 9 bacteria (7% fewer larvae, Estimate=-0.017, p=0.871) ( Figure 2B, Suppl. Table 2). None of the 3 0 0 four treatment groups differed in their hatching rate (number of larvae/number of eggs) from the 3 0 1 naïve group (Suppl. Figure 3, Suppl. Table 2).  We evaluated the total protein content per egg for eggs laid during the second oviposition  Table 3). However, we did see evidence of interaction effects 3 1 5 between egg number and egg protein content. While egg number did not significantly predict 3 1 6 protein content in eggs from naïve (R 2 =0.09, p=0.14) or saline-exposed mothers (R 2 =0.03, 3 1 7 p=0.24), those from a mother receiving a low bacterial dose exhibited a positive correlation 3 1 8 on . CC-BY-NC 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 25, 2022. ;https://doi.org/10.1101https://doi.org/10. /2022 between the two metrics (R 2 =0.29, p=0.019), potentially revealing a quality gradient of 3 1 9 reproductive potential among the females ( Figure 3B). In the high dose group, on the other hand, there was a significant negative relationship between egg quantity and protein content (R 2 =0.23, 3 2 1 p=0.036), suggesting a forced trade-off in reproductive investment ( Figure 3B).
CC-BY-NC 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 25, 2022. ;https://doi.org/10.1101https://doi.org/10. /2022 Offspring developmental speed and size are reliable indicators of their quality 3 3 1 [17,18,40]. Earlier pupation means the individual spends less time in its most vulnerable state, 3 3 2 and larger size is associated with higher reproductive fitness. We measured developmental speed 3 3 3 by recording the proportion of individuals that had pupated sixteen days post oviposition. There  Table 5). Low (Estimate=-0.319, p=0.093) and high dose groups (Estimate=-   Table 5).

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Asterisks indicate significant differences with *=p<.05 and ***=p<.001; n.s.=not significant. . CC-BY-NC 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 25, 2022. ;https://doi.org/10.1101https://doi.org/10. /2022 For each mating pair, we randomly chose two offspring pupae and weighed them. Pupae  We septically infected offspring larvae from oviposition period 1, 3 and 5 with a 66%-91% among the oviposition periods (Suppl. Table 6). However, there were no significant 3 5 5 differences in survival between any of the four maternal treatment groups and the naïve control 3 5 6 (Suppl. Table 7). Theory predicts that host resource allocation to physiological processes like survival and reproduction should evolve to be sensitive to the magnitude of threats in the environment 3 6 0 [14,25,41]. In the case of allocation trade-offs between immunity and reproduction, we predicted results suggest that female responses to the inactivated pathogens were indeed dose dependent. suggesting the adoption of a somatic maintenance strategy that prioritized survival and immune 3 6 7 investment over reproduction. Females exposed to the highest bacterial dose did not exhibit the 3 6 8 same reduction in fecundity but did reveal a trade-off between offspring quantity and quality, as was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 25, 2022. ; https://doi.org/10.1101/2022.07.25.501224 doi: bioRxiv preprint despite low resources, partially consistent with a terminal investment strategy. To the best of our 3 7 1 knowledge, this study is the first to investigate the impact of exposure dose on the sensitivity of 3 7 2 maternal investment into offspring quantity and quality, and sheds new light on the evolutionary 3 7 3 drivers of plasticity in resource allocation to survival and reproduction. reproductive period of the parent [15,16,42]. In a study of pea aphids infected with an ultimately 3 7 7 lethal bacterium, for example, the lowest initial dose spurred a decrease in reproductive output.

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Aphid fecundity recovered as dose subsequently increased, suggesting that a shift to somatic 3 7 9 maintenance at low doses was replaced by terminal investment as infection-induced mortality 3 8 0 became more certain [15]. Aphid reproductive investment never became significantly greater 3 8 1 than the fecundity of controls, however, suggesting that microbe-induced pathology could be 3 8 2 inducing side-effects like damage to reproductive tissues that obscure the true magnitude of  As a proxy for host fitness, fecundity is often the most straightforward trait to measure 3 9 0 and interpret. However, fitness is maximized only if those offspring are also viable and 3 9 1 competitive in their environment. Therefore, increased investment in offspring quality that 3 9 2 comes at the expense of current or later fecundity and survival could also fall under the umbrella 3 9 3 . CC-BY-NC 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 25, 2022. ; https://doi.org/10.1101/2022.07.25.501224 doi: bioRxiv preprint of terminal investment because it temporarily boosts the production of viable offspring [44,45].

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We measured several potential indicators of offspring quality, including egg protein content, 3 9 5 development rate, pupal weight, and survival against infection, all of which are positively 3 9 6 associated with quality. Our results suggested that while individuals exposed to sham or low 3 9 7 doses might produce larvae that develop faster (potentially to escape a hostile environment) [28], 3 9 8 there were no other consistent trends of offspring quality variation across exposure dose. When considering the interaction of fecundity and egg protein content, however, we did discover that 4 0 0 the relationship between offspring quality and quantity shifts across dose. Mothers exposed to  [29], we expected offspring from exposed mothers in this study to exhibit increased survival 4 1 2 after live Bt infection relative to those born to unexposed mothers [22,27,48]. Alternately, if 4 1 3 exposure forced a decrease in maternal provisioning and offspring quality, we would expect to 4 1 4 see offspring from exposed mothers become more susceptible to infection [49]. However, we 4 1 5 observed no consistent phenotype that would indicate either a priming response or a more 4 1 6 . CC-BY-NC 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made natural or laboratory populations [50,51]. It is also possible that any net phenotypes were 4 1 9 cancelled out by the opposing forces of priming, which could increase with dose [52], and poor 4 2 0 offspring provisioning, which could get more severe at higher doses. Future work would benefit 4 2 1 from using a beetle strain with a strong priming phenotype to try to disentangle these potential  the mortality rates in our study were similar across heat-killed doses, meaning that we do not 4 3 3 expect the observed fecundity and provisioning differences among these groups to be attributable  . CC-BY-NC 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 25, 2022. ; https://doi.org/10.1101/2022.07.25.501224 doi: bioRxiv preprint imminent to maximize fitness. If the risk of death is less severe, the host might invest in somatic   All data is included in the supplemental materials. The authors declare no conflict of interest. pilot work that informed the experimental design. This work was supported in part by Alfred P.   . CC-BY-NC 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made . T  a  t  e  A  T  ,  G  r  a  h  a  m  A  L  .  2  0  1  5  T  r  a  n  s  -g  e  n  e  r  a  t  i  o  n  a  l  p  r  i  m  i  n  g  o  f  r  e  s  i  s  t  a  n  c  e  i  n  w  i  l  d  f  l  o  u  r  b  e  e  t  l  e  s  r  e  f  l  e  c  t  s  5  3  3  t  h  e  p  r  i  m  e  d  p  h  e  n  o  t  y  p  e  s  o  f  l  a  b  o  r  a  t  o  r  y  p  o  p  u  l  a  t  i  o  n  s  a  n  d  i  s  i  n  h  i  b  i  t  e  d  b  y  c  o  -i  n  f  e  c  t  i  o  n  w  i  t  h  a  c  o  m  m  o  n  5  3  4  p  a  r  a  s  i  t  e  .  F  u  n  c  t  i  o  n  a  l  E  c  o  l  o  g  y  2  9  ,  1  0  5  9  -1  0  6  9  .  (  d  o  i  :  1  0  .  1  1  1  1  /  1  3  6  5  -2  4  3  5  .  1  2  4  1  1  )  5  3  5   3  0  .  J  e  n  t  D  ,  P  e  r  r  y  A  ,  C  r  i  t  c  h  l  o  w  J  ,  T  a  t  e  A  T  .  2  0  1  9  N  a  t  u  r  a  l  v  a  r  i  a  t  i  o  n  i  n  t  h  e  c  o  n  t  r  i  b  u  t  i  o  n  o  f  m  i  c  r  o  b  i  a  l  5  3  . CC-BY-NC 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made  4  3  .  P  i  k  e  V  L  ,  F  o  r  d  S  A  ,  K  i  n  g  K  C  ,  R  a  f  a  l  u  k  -M  o  h  r  C  .  2  0  1  9  F  e  c  u  n  d  i  t  y  c  o  m  p  e  n  s  a  t  i  o  n  i  s  d  e  p  e  n  d  e  n  t  o  n  t  h  e  5  6  8  g  e  n  e  r  a  l  i  z  e  d  s  t  r  e  s  s  r  e  s  p  o  n  s  e  i  n  a  n  e  m  a  t  o  d  e  h  o  s  t  .  E  c  o  l  o  g  y  a  n  d  E  v  o  l  u  t  i  o  n  9  ,  1  1  9  5  7  -1  1  9  6  1  .  5  6  9  (  d  o  i  :  1  0  .  1  0  0  2  /  E  C  E  3  .  5  7  0  4  )  5  7  0   4  4  .  D  a  v  i  s  A  A  ,  V  a  n  n  a  t  t  a  J  T  ,  G  u  t  i  e  r  r  e  z  S  O  ,  M  i  n  c  h  e  l  l  a  D  J  .  2  0  2  1  L  a  c  k  o  f  t  r  a  d  e  -o  f  f  s  i  n  h  o  s  t  o  f  f  s  p  r  i  n  g  5  7  1 p was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint (which this version posted July 25, 2022. ;https://doi.org/10.1101https://doi.org/10. /2022