Senescence of the immune defences and reproductive trade-offs in females of the mealworm beetle, Tenebrio molitor.

In the theory of ageing, it has been assumed that ageing is associated with a decline in somatic defences, including the immune system, as a consequence of a trade-off with reproduction. While overall immunity suffers from age-related deterioration (immune senescence), the different components of the immune response appear to age differently. It is also likely that investment among the many arms of the immune system and reproduction with age is finely adjusted to the organisms' reproductive strategy. We investigated this possibility in females of Tenebrio molitor, a species of long-lived insect with reproductive strategies similar to those of long-lived mammals. We specifically tested the effects of immunological challenges imposed early or late in adult life on immune pathway activation as well as fertility early and late in life. We found complex patterns of changes in immune defences with age and age-specific immune challenges with contrasted relationships with female reproduction. While cellular and enzymatic defences showed signs of ageing, they did not trade-off with reproduction. By contrast, the induced antibacterial immune response was found to be unaffected by age and to be highly connected to female fecundity. These findings suggest that these immunological pathways have different functions with regard to female ageing in this insect species.

Age-specific fecundity under pathogenic threat in an insect: Terminal investment versus reproductive restraint.

The terminal investment hypothesis predicts that as an organism's prospects for survival decrease, through age or when exposed to a pathogenic infection, it will invest more in reproduction, which should trade-off against somatic maintenance (including immunity) and therefore future survival. Attempts to test this hypothesis have produced mixed results, which, in addition, mainly rely on the assessment of changes in reproductive effort and often overlooking its impact on somatic defences and survival. Alternatively, animals may restrain current reproduction to sustain somatic protection, increasing the chance of surviving for additional reproductive opportunities. We tested both of these hypotheses in females of the yellow mealworm beetle, Tenebrio molitor, an iteroparous insect with reproductive tactics similar to that of long-lived organisms. To achieve this, we mimicked pathogenic bacterial infections early or late in the life of breeding females by injecting them with a suspension of inactivated Bacillus cereus, a known natural pathogen of T. molitor, and measured female age-specific fecundity, survival, body mass and immunity. Inconsistent with a terminal investment, females given either an early or late-life immune challenge did not exhibit reduced survival or enhance their reproductive output. Female fecundity declined with age and was reduced by the early but not the late immune challenge. Both early and late-life fecundity correlated positively with life expectancy. Finally, young and old females exhibited similar antibacterial immune responses, suggesting that they both restrained reproduction to sustain immunity. Our results clearly demonstrate that age-specific reproduction of T. molitor females under pathogenic threat is inconsistent with a terminal investment. In contrast, our results instead suggest that females used a reproductive restraint strategy to sustain immunity and therefore subsequent reproductive opportunities. However, as infections were mimicked only, the fitness benefit of this reproductive restraint could not be shown.

Late-life reproduction in an insect: Terminal investment, reproductive restraint or senescence.

The terminal investment, reproductive restraint or senescence theories may explain individual late-life patterns of reproduction. The terminal investment hypothesis predicts that individuals increase reproductive allocation late in life as prospects for future survival decrease. The other two hypotheses predict reduced reproduction late in life, but for different reasons. Under the Reproductive Restraint hypothesis, individuals restrain their reproductive effort to sustain future survival and gain more time for reproducing, whereas under the Senescence process, reproduction is constrained because of somatic deterioration. While these hypotheses imply that reproduction is costly, they should have contrasted implications in terms of survival after late reproduction and somatic maintenance. Testing these hypotheses requires proper consideration of the effects of age-dependent reproductive effort on post-reproduction survival and age-related somatic functions. We experimentally tested these three hypotheses in females of the mealworm beetle, Tenebrio molitor, an iteroparous and income breeder insect. We manipulated their age-specific allocation into reproduction and observed the effects of this manipulation on their late-life fecundity, post-reproduction survival and immunocompetence as a measurement of somatic protection. We found that females exhibit age-related decline in fecundity and that this reproductive senescence is accelerated by a cost of early reproduction. The cost of reproduction had no significant effect on female longevity and their ability to survive a bacterial infection, despite that some immune cells were depleted by reproduction. We found that female post-infection survival deteriorated with age, which could be partly explained by a decline in some immune parameters. Importantly, females did not increase their reproductive effort late in life at the expense of their late-life post-reproduction survival. Late-life reproduction in T. molitor females is senescing and not consistent with a terminal investment strategy. Rather, our results suggest that females allocate resources according to a priority scheme favouring longevity at the expense of reproduction, which is in line with the reproductive restraint hypothesis. Such a priority scheme also shows that a relatively short-lived insect can evolve life-history strategies hitherto known only in long-lived animals. This puts in perspective the role of longevity in the evolution of life-history strategies.

Deciphering the molecular mechanisms of mother-to-egg immune protection in the mealworm beetle Tenebrio molitor.

In a number of species, individuals exposed to pathogens can mount an immune response and transmit this immunological experience to their offspring, thereby protecting them against persistent threats. Such vertical transfer of immunity, named trans-generational immune priming (TGIP), has been described in both vertebrates and invertebrates. Although increasingly studied during the last decade, the mechanisms underlying TGIP in invertebrates are still elusive, especially those protecting the earliest offspring life stage, i.e. the embryo developing in the egg. In the present study, we combined different proteomic and transcriptomic approaches to determine whether mothers transfer a "signal" (such as fragments of infecting bacteria), mRNA and/or protein/peptide effectors to protect their eggs against two natural bacterial pathogens, namely the Gram-positive Bacillus thuringiensis and the Gram-negative Serratia entomophila. By taking the mealworm beetle Tenebrio molitor as a biological model, our results suggest that eggs are mainly protected by an active direct transfer of a restricted number of immune proteins and of antimicrobial peptides. In contrast, the present data do not support the involvement of mRNA transfer while the transmission of a "signal", if it happens, is marginal and only occurs within 24h after maternal exposure to bacteria. This work exemplifies how combining global approaches helps to disentangle the different scenarios of a complex trait, providing a comprehensive characterization of TGIP mechanisms in T. molitor. It also paves the way for future alike studies focusing on TGIP in a wide range of invertebrates and vertebrates to identify additional candidates that could be specific to TGIP and to investigate whether the TGIP mechanisms found herein are specific or common to all insect species.