Physiological Entomology (2017), DOI: 10.1111/phen.12186 The more, the merrier? Obligate symbiont density changes over time under controlled environmental conditions, yet holds no clear fitness consequences JASMINE F. PARKINSON 1 , BRUNO GOBIN 2 and WILLIAM O. H. HUGHES 1 1 Department of Evolution, Behaviour and Environment, School of Life Sciences, University of Sussex, Falmer, U.K. and 2 PCS-Ornamental Plant Research, Destelbergen, Belgium Abstract. Symbiotic bacteria are highly diverse, play an important role in ecology and evolution, and are also of applied relevance because many pest insects rely on them for their success. However, the dynamics and regulation of symbiotic bacteria within hosts is complex and still poorly understood outside of a few model systems. One of the most intriguing symbiotic relationships is the obligate, tripartite nutritional mutu- alism in sap-feeding, economically-destructive mealybugs (Hemiptera: Sternorrhyncha: Pseudococcidae), which involves γ-proteobacteria hosted within β-proteobacteria hosted within the mealybugs. The present study examines whether there is population variation in symbiont density (i.e. infection intensity, or titre) in the citrus mealybug Planococcus citri (Risso) and how this impacts host life-history. Symbiont density is found to differ significantly between populations when reared under controlled environmental condi- tions, indicating that the density of symbiont infections is influenced by host or symbiont genotype. However, symbiont density changes in populations over multiple generations, indicating that symbiont densities are dynamic. Surprisingly, given that the symbionts are essential nutritional mutualists, the density of the symbionts does not correlate sig- nificantly with either host fecundity or development. Higher levels of symbionts have no clear benefit to hosts and therefore appear to be superfluous, at least under constant, optimized environmental conditions. Excessive symbiont density may be an evolution- ary artefact from a period of inefficient vertical transmission when the balance of conflict between host and symbiont was still being established. Key words. Endosymbionts, fecundity, Hemiptera, mealybug, Planococcus citri, Proteobacteria, Pseudococcidae, qPCR reproduction, size. Introduction Symbiotic bacteria are now understood to be highly diverse and influential players in eukaryotic ecology and evolution (Saffo, 1992; Moran, 2001; Douglas, 2009). They are fundamental to many aspects of life, having given rise to mitochondria and chloroplasts, as well as numerous other pivotal evolutionary steps, such as nitrogen fixation and bioluminescence (Giobel, 1926; Yasaki, 1928; Schwartz & Dayhoff, 1978; Peix et al., Correspondence: Jasmine Parkinson, Department of Evolution, Behaviour and Environment, School of Life Sciences, University of Sussex, John Maynard Smith Building, North South Road, Falmer, Brighton BN1 9RH, U.K. Tel.: +44 7540687532; e-mail: jasmine.parkinson@gmail.com 2015). The impacts that symbionts have on their hosts can range from mutualistic, to commensal, to parasitic. However, rather than residing in strict categories, the relationships between hosts and symbionts exist on a dynamic spectrum, and may often be context-specific (Swain, 2012; Gerardo, 2015). Each member in a symbiotic relationship will ultimately evolve to maximize its own fitness rather than that of its partner, so conflict between host and symbiont may often occur, even in mutualistic associations (Bennett & Moran, 2015). The maintenance of beneficial symbionts will still incur some cost to the host, and the host should reduce symbiont density (i.e. infection intensity) when it is in excess, whereas the symbiont should seek to optimize its density to maximize the likelihood of its own transmission to new hosts (Falkowski et al., 1993; © 2017 The Royal Entomological Society 1