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Keywords:

  • Complex life cycle;
  • ecological immunology;
  • host resistance;
  • host shift;
  • immune priming;
  • metamorphosis;
  • ontogeny;
  • stage-structure

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

1. Insects lack the acquired immune system of vertebrates, but there is some evidence that insect immunity can be primed against an encountered pathogen to mitigate the intensity of future infections within a life stage.

2. Many invertebrates have multiple life-history stages separated by complete metamorphosis, but different life stages can often be infected by the same pathogens, and the potential loss of immune priming during metamorphosis could therefore have detrimental effects on the host. Evidence that invertebrate immune priming can persist through metamorphosis is still missing, and consequently it is unclear how host–parasite interactions change across different life-history stages in the context of infection history.

3. By experimentally manipulating the infection history of the flour beetle Tribolium confusum, we show that intestinal gregarine parasite infections during the larval stage reduced parasite load in adults, demonstrating that a host-controlled mechanism for parasite resistance can persist through complete metamorphosis in insects.

4. Infections reduced larval developmental rates and increased host mortality but only during the crucial metamorphic stage, indicating that parasites impact multiple life stages. In general, our results demonstrate that invertebrates can show surprisingly robust immune priming despite dramatic physiological changes and protect hosts across completely different life-history stages.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Host–pathogen interactions have important ecological and evolutionary consequences for natural populations and communities (Rolff & Siva-Jothy, 2003). The interactions between pathogens and their hosts depend on the ecology and physiology of the host. For example, the host's environment, specific immune system, energy resources, and even predation risk can all determine its infection risk by a specific pathogen (reviewed in Rolff & Siva-Jothy, 2003; Schmid-Hempel & Ebert, 2003; Joop & Rolff, 2004; Schmid-Hempel, 2005). Many organisms, however, experience considerable shifts in their ecology and physiology during development. The most striking shifts occur in species with metamorphosis (e.g. amphibians, holometabolous insects, marine invertebrates, parasites, and copepods), where individuals can experience substantial changes in their ecology and physiology, including alterations to the immune system (Russell & Dunn, 1996; Rollins-Smith, 1998). Despite these changes, different life stages are commonly infected by the same pathogens, and the loss of primed immunity during metamorphosis could be detrimental for the host. However, the influences of metamorphosis on host–parasite interactions across different life-history stages are poorly understood.

Priming of the immune system can reduce the risk of secondary infections and strongly have an impact on the interactions between hosts and their pathogens. Here we use the term ‘primed immunity’ to refer to the phenomenological observation of increased resistance to parasites upon secondary exposure, without implying that observed priming is necessarily pathogen specific. Emergent evidence suggests that an invertebrate's innate immune response can be primed to protect hosts from repeated infections and that this protection can be pathogen specific (Moret & Siva-Jothy, 2003; Sadd & Schmid-Hempel, 2006; e.g. Kurtz & Franz, 2003; Pham et al., 2007; Roth et al., 2009). Although these studies indicate that such immunity can last from several days to several weeks, they generally focus on a single life stage and there has been no evidence that this primed immunity can persist through metamorphosis (Meylaers et al., 2007; Pham & Schneider, 2007). Metamorphosis is a critical transition stage where a variety of hormones orchestrate the loss and reorganisation of tissues and many organs, including the immune system. In vertebrates with metamorphosis (i.e. amphibians), many immune functions are lost during metamorphosis, but some memory-B cells persist and can provide protection against secondary infections. Invertebrates, however, lack these cells, and their immune system differs in many other aspects from that of vertebrates (Rolff & Siva-Jothy, 2003). Some studies suggest that metamorphosing insects experience increased antibacterial immune protection in the midgut (Russell & Dunn, 1996) and restructuring of cellular components responsible for the production of constitutive immune molecules like phenoloxidase (Hoffmann, 1995), but the range of changes to the immune system during insect metamorphosis is not well defined. Consequently it remains unknown whether priming of the immune system protects invertebrate hosts from secondary infections for their whole life cycle, or simply for a single life stage. Given that the majority of insects go through metamorphosis, identifying the impact of metamorphosis on host–pathogen interactions is of key importance for understanding the ecological and evolutionary dynamics of host–pathogen systems.

To understand the dynamics of natural host–parasite interactions and the role of the host immune system, whole-organism experimentation with natural host-parasite systems provides important guidance for mechanistic approaches (Little et al., 2005). Here, we experimentally investigate whether infection history alters host–pathogen interactions across life stages in invertebrates using an insect (Tenebrionidae: Tribolium confusum)–parasite (Gregarinidae: Gregarina minuta) system. In particular, we tested whether infection in the larval stage altered the infection risk of adults and estimated the associated fitness costs of the parasite infection. Results indicate that infection history reduced parasite loads in the adult stage, suggesting that some form of immune priming can persist across metamorphosis in invertebrates.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The life cycle of T. confusum beetles and their gut parasites makes them particularly suited to examine host–pathogen interactions across metamorphosis because the gut parasites are naturally shed during complete metamorphosis together with the beetle's gut lining (Fig. 1) (Detwiler & Janovy, 2008; A. M. Thomas, pers. obs.). This naturally produces uninfected adults and offers a convenient way to manipulate the infection in the adult and larval stage independently, without the use of artificial treatments or the risk of overlapping parasite cohorts. In addition, T. confusum should greatly benefit from immune priming because it lives in dense colonies and is prone to re-infection by the same parasite (Janovy et al., 2007). Previous studies on the closely related Tribolium castaneum also suggest that Tribolium can show highly specific immune priming (Roth et al., 2009) and alter their development in response to different bacterial pathogens (Roth & Kurtz, 2008).

image

Figure 1. Life cycle of the flour beetle host and its intestinal gregarine parasite. Flour beetle larvae and adults can ingest parasite oocysts (parasitic stage) from the flour, which mature into trophozoites (parasitic stage) in the host gut epithelia. The trophozoites mate and the hosts pass the resulting gametocysts with their faecal matter. Gametocysts then dehisce into oocysts to be ingested by a new host. Parasites are naturally shed from the host during metamorphosis (pupal stage) resulting in uninfected adult hosts. This allowed us to independently manipulate infections in larval and adult hosts to test whether infections during the larval stage altered infection risk in the adult stage.

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Flour beetles and their parasites were randomly obtained from a large (1500 + individuals) infected T. confusum stock colony maintained at Rice University according to standard protocols. All experimental individuals were kept at 28 ± 2°C and 35 ± 3% humidity. Although parasites descended from a single flour beetle stock colony, the extent of their genetic diversity is currently unknown. We placed randomly selected older larvae from infected colonies into individual wells in 60-well small ice cube trays. The infected larvae excreted gametocysts for 1 day and were subsequently removed. Resulting gametocysts in each well were then covered in 2 ml of wheat flour for 3 days to allow production of oocysts. For adult infections we additionally homogenized infectious material by removing the flour from experimental larval wells upon larval pupation, and mixing the flour together to create a large infected flour stock that was distributed evenly into new wells.

We gathered 80 eggs from an infected T. confusum colony and allowed them to hatch. To avoid differences in hatching dates we only used 70 larvae that successfully hatched within 2 days of each other and were similar in size. We distributed the young larvae randomly across 30 well-trays with infected and 40 well-trays with uninfected whole wheat flour. At day 12 post-hatching, we examined six larvae from the infected treatment to get an estimate for infection prevalence and intensity, and five larvae from the uninfected treatment to confirm uninfected status. We transferred new pupae from both treatments to infected wells on the same tray, marked the date of pupation and eclosion, and inspected individuals for infection intensity and prevalence at day 12 post-eclosion, for an average of 32 days (uninfected treatment) and 39 days (infected treatment) after the first larval exposure to oocysts. To determine infection intensity, we surgically removed the gut, treated it with two drops of 5% iodine solution to stain the trophozoites, and counted trophozoites at 100 × magnification (Detwiler & Janovy, 2008). Midguts that were damaged by the dissection process were excluded from the parasite count and discarded. Dissections confirmed that the infection treatment was highly successful: in infected treatments 100% of the larvae were infected with an average of 150 trophozoites 12 days post-hatching (n = 6), whereas in uninfected treatments 0% of the larvae were infected (n = 5). This is consistent with pilot studies where more larvae were dissected. To confirm that individuals shed infection during the pupal stage to emerge as uninfected adults, we placed a subset (n = 6) of pupae that were infected as larvae in uninfected flour. At day 12 post-eclosion, all adults were free of parasites, which is consistent with results from previous work (Detwiler & Janovy, 2008). To estimate the fitness cost of parasites on their host, we monitored the developmental time and survival rates of larvae and survival rates during the metamorphic stage (i.e. after pupation and before 3 days into adulthood).

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Fitness costs

Parasites conferred a clear fitness cost for the host. In general, infections increased larval development time by 38% (average larval stage, uninfected = 14.9 ± 2.3 days; infected = 22.6 ± 7.8 days, unequal variance Welch's t-test, t = 4.7, d.f. = 19, P = 0.0002, Fig. 2B). Infected larvae died more often (n = 18, 11% mortality rate) than uninfected larvae (n = 34, 0% mortality rate) during metamorphosis [one-tailed randomization test (Edgington, 1995), P = 0.02]. Since the sample size was low, we tested how robust this pattern was by combining this data with the mortality data from a pilot experiment that used identical procedures (A. Thomas, unpublished). The effect remained highly significant, with the larger data set showing similar mortality patterns (infected: n = 50, 20% mortality rate; uninfected: n = 48, 0% mortality rate, randomization test, P = 0.004). No mortality occurred during the larval stage.

image

Figure 2. (A) Mean (± 1 SD) parasite infection loads of adults that were uninfected or infected during the larval stage. Individuals that were heavily infected as larvae had significantly (P < 0.001) fewer parasites when re-infected after complete metamorphosis, demonstrating the priming effect that larval infection can have on adult infections. (B) Infected larvae exhibited significant delayed development into the next life stage (dark) as compared with uninfected larvae (light). Furthermore, infections during the larval stage significantly increased host mortality relative to their uninfected cohorts (P = 0.02) but only during the metamorphic stage, indicating the costs of infections for the hosts.

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Primed parasite resistance

If immune priming against the parasites persists across metamorphosis, we would expect that infection in the larval stage reduces infections in the subsequent adult stage. As predicted, we found that infections in adults were clearly dependent on the infection history of individuals as larvae (Fig. 2A). On average, adults that were infected as larvae had a 59% lower parasite load (4.25 ± 2.28 trophozoites, n = 16), than adults that were uninfected as larvae (10.4 trophozoites ± 3.12 SD, n = 26, Mann–Whitney test, Z = 4.7, P < 0.001).

It is possible that the higher mortality rate in previously infected larvae may select for more resistant individuals that might support a lower infection load during adulthood as well. Meanwhile, uninfected larvae would not go through this selection process, which could result in a higher average infection load as adults, simply as a result of selective mortality. To rule out this possibility, we excluded 11% of the uninfected group adults with the highest infection intensity from the analysis, to mimic the 11% mortality rate of the infected group. Accounting for this selective mortality did not alter the results. The previously uninfected group still had a significantly higher infection intensity than the infected group (Mann–Whitney test, Z = 4.5, P < 0.001) suggesting that selective mortality alone cannot explain the apparent immune priming effect.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Our results indicate that infection by a parasite during the larval stage can reduce the infection load in adults. To the best of our knowledge this is the first demonstration that infection history can have persistent effects across metamorphosis in insects, and provides novel insights into host–pathogen interactions across different life-history stages in hosts with metamorphosis. Infections can weaken the immune system and make it more prone to secondary infections (Rolff & Siva-Jothy, 2003). However, this is not consistent with the lower infection load of previously infected, as opposed to uninfected, adults. It is also unlikely that the parasites themselves are responsible for the reduced infection load as all parasites are shed during metamorphosis before secondary infections occur. Similarly, results suggest that differences in the host energy resources are not likely responsible for potential differences in infection loads. Adults had more time (12 days) to restore any nutritional or immune deficiencies than needed by similar species (Siva-Jothy & Thompson, 2002) and qualitative comparisons upon dissection indicated similar levels of haemolymph and fat bodies in adults of both treatments.

It could also be argued that the observed difference in parasite loads between adult treatment groups was due to the selective mortality. In treatments where larvae were infected, only the fittest individuals may have survived the infections, whereas the control individuals were not subject to this selective mortality process. Assuming that surviving the infection as larvae also correlates with higher adult resistance to parasite infections, the difference in mortality among treatments could have led to the observed differences in parasite loads across treatments. However, even when correcting our analysis for the potential confounding effects that could arise from differences in mortality across treatments, the previously uninfected group still had an average infection load that was significantly greater than that of previously infected individuals. This indicates that selective mortality alone cannot be used to explain the observed difference in parasite loads between treatments. Instead, this suggests that previously infected individuals are undergoing a physiological priming process to mitigate the intensity of future infections.

Recent studies have demonstrated that immune activation can persist from several days up to 3 weeks, and can be pathogen specific in some invertebrates, including Tribolium (Moret & Siva-Jothy, 2003; Sadd & Schmid-Hempel, 2006; e.g. Kurtz & Franz, 2003; Pham et al., 2007; Roth et al., 2009). These studies provided important first evidence that invertebrate immune responses can be highly specific and persist for extended periods of time, but they typically focused on priming within a life stage. Many invertebrates, however, go through metamorphosis, which results in dramatic loss and reorganization of tissues and many organs, including the immune system (Russell & Dunn, 1996). Consequently, it remained uncertain whether immune priming only protects individual life stages or whether it can protect invertebrates during their whole life cycle (Pham & Schneider, 2007). Our results demonstrate that immune priming can persist across metamorphosis in invertebrates and can protect invertebrates across very different life stages despite substantial physiological changes. Given that the majority of invertebrates go through metamorphosis, the importance of this insight into the long-term disease resistance capabilities of invertebrates cannot be overstated. It complements recent evidence suggesting that the offspring of infected invertebrate females are born with a more robust constitutive immune response (Moret & Schmid-Hempel, 2001), and that some mechanisms of maternally transferred immunity are pathogen specific (Little et al., 2003; Sadd et al., 2005; Moret, 2006; Sadd & Schmid-Hempel, 2007). Together with studies that demonstrate trans-generational immune priming, our study suggests that immune priming can provide protection to secondary infections across the whole life cycle of invertebrate hosts and their offspring.

Demonstrating that parasite resistance can persist across metamorphosis to provide long-term protection across very different life stages advances our understanding of the ecology and evolution of host–pathogen interactions and could explain patterns observed in natural populations. Current empirical data suggests that hosts with metamorphosis often exhibit large differences in infection prevalence of the same infectious disease across life stages. For example, in different Tenebrio species, adults show typically much lower infection prevalence of intestinal parasites than larvae, and in some species parasites only infect the adults but not the larvae (Clopton et al., 1992; Clopton & Janovy, 1993). Similar differences between stages are found in odonates (e.g. Siva-Jothy & Plaistow, 1999; Hecker et al., 2002). However, it is unclear what causes the differences in disease prevalence across stages. Although changes in the host's ecology over ontogeny (e.g. changes in abiotic conditions of the habitat) may explain some of the differences in parasite prevalence (Yan & Stevens, 1995), such clear environmental shifts are often absent in other systems. Our results suggest that even without environmental shifts, host–parasite interactions can differ over ontogeny because infection in the larval stage indirectly alters the risk of secondary infections in the adult stage.

We found that parasites conferred a clear fitness cost for the host by delaying the host's developmental time and increasing its mortality rate. Surprisingly, the mortality occurred only after the parasite was naturally shed from the host during the early pupal stage. Although the mortality rates were low, similar mortality patterns were observed in other studies (A. M. Thomas, unpublished; Kang and Rudolf, unpublished), suggesting that this might be a general pattern in T. confusum. The unusual timing of the mortality may give another clue to the identity of host–parasite interactions in this system. All larvae entered the pupal stage with a normal morphology, but previously infected larvae either failed to eclose or eclosed with such grossly aberrant structural contortions, that they did not survive more than 3 days as an adult. Parasites can substantially drain the resources of the hosts (Schmid-Hempel, 2005) and it is probable that this mortality effect represents extreme malnutrition imposed upon the host by the parasite that manifests only during the energy-intensive physiological remodelling process during the pupal stage. Infection during the larval stage clearly resulted in developmental changes during metamorphosis which reduced the risk of infection in the adult stage. Thus, this mortality could also be a cost, in part, of physiological changes associated with immune system priming. It is also possible that the mortality effect is due to an overaggressive immune-mediated pathology. Irrespective of the causes, the parasite-mediated indirect cost of metamorphosis provides an important novel finding and warrants further study to elucidate the underlying mechanisms.

Our results demonstrate that immune priming can persist across metamorphosis and can protect hosts across very different life stages. While our study did not focus on the detailed molecular mechanisms that underlie the observed increase in resistance, demonstrating such an immune function and its cost in a whole-organisms experiment is a first crucial step towards understanding how host–pathogen interactions change during the whole life cycle of invertebrate hosts. It would be interesting to look for specificity in the insect immune response to gregarine protozoa, as the vast majority of specificity experiments have focused on the response to different strains or species of bacteria. Plans are underway to characterise the genetic diversity of the parasites, and to create strains that will be useful for future studies on the immunological consequences of gregarine infections in flour beetles. Finally, further research on the molecular mechanisms that allow immune priming to persist across metamorphosis will be an important next step towards understanding the invertebrate immune system.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

We are grateful to J. Janovy and R. Beeman for advice on maintenance and manipulation of the host–parasite system and to A. E. Dunham and two anonymous reviewers for critical comments on earlier versions of the manuscript.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References