Given the prevalence of pathogens and parasites, an individual’s ability to overcome potential infections through immunity is a critical defensive strategy. Immunity serves as a general mechanism for lowering host fitness costs associated with these interactions. The encapsulation-melanization immune response (hereafter melanotic encapsulation), for example, is a component of the insect immune system. It occurs when a foreign object that is too large to be phagocytized penetrates the body. Cellular and humoral processes cause hemocyte aggregation and melanin deposition around the object, often resulting in a dark capsule (Siva-Jothy et al., 2005). This is how arthropods neutralize endoparasitoid wasps and flies (Salt, 1970, Kraaijeveld et al., 2001), as well as virus-infected cells (Washburn et al., 1996). Inserting a nylon monofilament implant into the body cavity and measuring the implant’s melanization score is the easiest and probably the most informative way to assess the strength of immunocompetence in insects (Moreno-García et al., 2013). It has been shown that the ability to melanize a nylon monofilament better is strongly related to a lower likelihood of being successfully parasitized (Doums & Schmid-Hempel, 2000) as well as a higher ability to encapsulate real pathogens, parasitoids, and parasites (Rantala and Roff, 2007, Smilanich et al., 2009, Nagel et al., 2011).

The melanotic encapsulation, as well as other mechanisms related to immune function, are costly (Sheldon and Verhulst, 1996, Rigby et al., 2002), and usually, individuals cannot maintain them at high levels throughout their lives. The relationship between immune function and age (either positive or negative) reported in many taxa has often been attributed to immunosenescence (Müller et al., 2013, Verges and Nehring, 2016). However, immune function can be context-dependent instead of inevitably subjected to aging (Silva et al., 2013, Reavey et al., 2015). The exposure risk hypothesis (Schmid-Hempel, 1998; Bocher et al., 2007) proposes that individuals engaging in riskier behaviors in terms of infection, such as allogrooming in honeybees, are expected to invest more in personal immune function (Cini et al., 2020).

In social hymenopterans, worker behavior is biased toward specific tasks based on age (Wilson, 1971, Jeanne, 1986). Typically, in social wasps, a young worker (one-week-old) stays inside the nest interacting with immature brood and acting as a nurse, inspecting cells. When it is older (two-week-old), it can move to the nest surface to defend the colony as a guard. Finally, when older (three-week-old), it begins to leave the nest to forage for food and nest-building materials, acting as a forager (O'Donnell, 2001). This age polyethism provides an interesting framework to study modulators of immune function. A link between age polyethism and immune function has often been reported in some social bees and ants. Specifically, immune function of young workers performing tasks inside the nest differs from older workers performing tasks outside the nest, and the direction of this difference varies across taxa and the specific component of the immune system measured (Doums & Schmid-Hempel, 2000; Doums et al., 2002, Bocher et al., 2007, Wilson-Rich et al., 2008, Moret and Schmid-Hempel, 2009, Armitage and Boomsma, 2010, Laughton et al., 2011, Helft et al., 2012, Cappa et al., 2020). However, despite the potential to critically support or refute the immunosenescence and the exposure risk hypotheses, the relative effects of age and behavior on the strength of immune response has rarely been tested in social hymenopterans. To our knowledge, the best evidence came from honeybees, where well-controlled experiments demonstrated that worker immune function changes with age after controlling for behavior (Roberts and Hughes, 2014, Lourenço et al., 2019) and task after controlling for age (Amdam et al., 2005, Cini et al., 2020).

Epiponini wasps (Hymenoptera:Vespidae) are similar to honeybees in that they are both highly eusocial, with large colonies containing hundreds of individuals, and workers exhibit age polyethism (da Silva et al., 2021, Noll et al., 2021). These neotropical wasps provide an opportunity to study whether and how age and behavior influence worker immune function in a clade where sociality has evolved independently of bees (Hughes et al., 2008). Polybia paulista workers are highly susceptible to parasites such as gregarines (Kudô 2021). Gametocytes of these protozoans are confined within the hemocoel of the wasp and can only be released into the environment from dead or dismembered bodies of infected wasps, where they can be transmitted horizontally (Bouwma et al., 2005). Because wasps usually die outside the nest (Bouwma et al., 2005), gregarine infection may be primarily associated with foraging rather than inside nest activities. One could also expect that foraging increases worker exposure risk to other pathogens (viral and fungal) that might be a risk for the whole colony (Cremer et al., 2007). Therefore, the ability to neutralize foreign bodies (e.g. melanotic encapsulation) might be an important component of worker fitness.

We test whether behavior and age modulate immune function. Specifically, we compared the strength of melanotic encapsulation in workers displaying age polyethism or experimentally prevented from behavioral specialization. Under the exposure risk hypothesis, we expect melanotic encapsulation to be associated with worker tasks and to be especially high in foragers. Instead, under the immunosenescence hypothesis, we expect melanotic encapsulation to be associated with worker age.