Effects of adult temperature on gene expression in a butterfly: identifying pathways associated with thermal acclimation.

BACKGROUND: Phenotypic plasticity is a pervasive property of all organisms and considered to be of key importance for dealing with environmental variation. Plastic responses to temperature, which is one of the most important ecological factors, have received much attention over recent decades. A recurrent pattern of temperature-induced adaptive plasticity includes increased heat tolerance after exposure to warmer temperatures and increased cold tolerance after exposure to cooler temperatures. However, the mechanisms underlying these plastic responses are hitherto not well understood. Therefore, we here investigate effects of adult acclimation on gene expression in the tropical butterfly Bicyclus anynana, using an RNAseq approach. RESULTS: We show that several antioxidant markers (e.g. peroxidase, cytochrome P450) were up-regulated at a higher temperature compared with a lower adult temperature, which might play an important role in the acclamatory responses subsequently providing increased heat tolerance. Furthermore, several metabolic pathways were up-regulated at the higher temperature, likely reflecting increased metabolic rates. In contrast, we found no evidence for a decisive role of the heat shock response. CONCLUSIONS: Although the important role of antioxidant defence mechanisms in alleviating detrimental effects of oxidative stress is firmly established, we speculate that its potentially important role in mediating heat tolerance and survival under stress has been underestimated thus far and thus deserves more attention.

A reply to Nieberding and Holveck: beyond experimental design and proximate mechanisms - mate choice in the face of sexual conflict.

We summarise our work on male mating behaviour in the tropical butterfly Bicyclus anynana, responding to the commentary provided by Nieberding and Holveck. We acknowledge that our laboratory studies are not free of shortcomings and potential caveats, though we attempted to address or highlight these within each paper. The concerns raised seem to stem mainly from different notions with respect to the proximate basis of old male mating advantage, and specifically the relative importance of male behaviour versus pheromone blend. In our view, our experiments provided compelling evidence for a prominent role of male behaviour, while we were unable to obtain clear evidence for a major role of male sexual pheromones. In addition to the lack of evidence we argue that a preference of females for older males based on pheromone blend is unlikely, as pheromone titres do not seem to indicate male quality and, more importantly, females actually suffer a fitness cost when mating with older males. The latter suggests that old male mating advantage arises from sexual conflict rather than cooperation. We thus highlight the importance of considering both the proximate and the ultimate level for gaining an integrative understanding of complex behavioural patterns.

Old-male paternity advantage is a function of accumulating sperm and last-male precedence in a butterfly.

Old-male mating advantage has been convincingly demonstrated in Bicyclus anynana butterflies. This intriguing pattern may be explained by two alternative hypotheses: (i) an increased aggressiveness and persistence of older males during courtship, being caused by the older males' low residual reproductive value; and (ii) an active preference of females towards older males what reflects a good genes hypothesis. Against this background, we here investigate postcopulatory sexual selection by double-mating Bicyclus anynana females to older and younger males, thus allowing for sperm competition and cryptic mate choice, and by genotyping the resulting offspring. Virgin females were mated with a younger virgin (2-3 days old) and afterwards an older virgin male (12-13 days old) or vice versa. Older males had a higher paternity success than younger ones, but only when being the second (=last) mating partner, while paternity success was equal among older and younger males when older males were the first mating partner. Older males produced larger spermatophores with much higher numbers of fertile sperm than younger males. Thus, we found no evidence for cryptic female mate choice. Rather, the findings reported here seem to result from a combination of last-male precedence and the number of sperm transferred upon mating, both increasing paternity success.

Bladder Microbiome in the Context of Urological Disorders-Is There a Biomarker Potential for Interstitial Cystitis?

Since the development of modern cultivation and sequencing techniques, the human microbiome has increasingly become the focus of scientific attention. Even in the bladder, long considered to be a sterile niche, a highly variable and complex microbial colonization has now been demonstrated. Especially in the context of diseases such as interstitial cystitis, whose etiopathogenesis is largely unknown, and whose diagnosis is based on a process of exclusion of confusable diseases, science hopes to gain far-reaching insights for etiology and diagnosis, including the identification of potential biomarkers. While for functional disorders such as urge urinary incontinence and overactive bladder syndrome, initial associations have been demonstrated between reduced microbial diversity and increased symptomatology, as well as shifts in the abundance of specific microorganisms such as Lactobacillus or Proteus, studies in interstitial cystitis show conflicting results and have failed to identify a putative organism or urotype that clearly distinguishes the urinary microbiome of patients with IC/BPS from that of healthy controls. At the present time, therefore, the new insights into the bladder microbiome and its potential influence on urologic disease cannot yet be used in the context of elucidating possible etiopathogenetic causes, as well as in the use of a biomarker for diagnostic or prognostic purposes. Further studies should focus primarily on uniform procedures and detection methods to achieve better comparability of results and increase the likelihood of detecting hidden patterns.

Assay conditions in laboratory experiments: is the use of constant rather than fluctuating temperatures justified when investigating temperature-induced plasticity?

Temperature is an important selective agent in nature. Consequently, temperature-induced plasticity which may help buffering detrimental effects of temperature variation has received considerable attention over recent decades. Laboratory studies have almost exclusively used constant temperatures, while in nature, temperature typically shows pronounced daily fluctuations. Using a factorial design with constant versus fluctuating temperatures and a higher versus a lower mean temperature, we here investigate in the butterfly Lycaena tityrus whether the use of constant temperatures is justified. Fluctuating compared to constant temperatures caused shorter development times, increased heat but decreased cold stress resistance, decreased heat-shock protein expression, and increased immunocompetence. Thus, overall, fluctuating temperatures were more beneficial to the butterflies compared to constant ones. However, despite substantial variation across temperature regimes, the ranking of trait values among treatments remained largely unaffected (e.g. lower constant as well as fluctuating temperatures caused increased pupal mass). Thus, we tentatively conclude that there is no general reason for concern about using constant temperatures in studies investigating phenotypic plasticity, which seem to comprise a fair proxy. However, substantial differences in mean values as well as interactive effects suggest that one needs to be cautious. We further demonstrate negative effects of high temperatures on butterfly immune function, which seem to result from a trade-off between the latter and the heat shock response.

No trade-off between growth rate and temperature stress resistance in four insect species.

Although fast growth seems to be generally favored by natural selection, growth rates are rarely maximized in nature. Consequently, fast growth is predicted to carry costs resulting in intrinsic trade-offs. Disentangling such trade-offs is of great ecological importance in order to fully understand the prospects and limitations of growth rate variation. A recent study provided evidence for a hitherto unknown cost of fast growth, namely reduced cold stress resistance. Such relationships could be especially important under climate change. Against this background we here investigate the relationships between individual larval growth rate and adult heat as well as cold stress resistance, using eleven data sets from four different insect species (three butterfly species: Bicyclus anynana, Lycaena tityrus, Pieris napi; one Dipteran species: Protophormia terraenovae). Despite using different species (and partly different populations within species) and an array of experimental manipulations (e.g. different temperatures, photoperiods, feeding regimes, inbreeding levels), we were not able to provide any consistent evidence for trade-offs between fast growth and temperature stress resistance in these four insect species.

Why get big in the cold? Towards a solution to a life-history puzzle.

The temperature-size rule (TSR), which states that body size increases at lower developmental temperatures, appears to be a near-universal law for ectotherms. Although recent studies seem to suggest that the TSR might be adaptive, the underlying developmental mechanisms are thus far largely unknown. Here, we investigate temperature effects on life-history traits, behaviour and physiology in the copper butterfly Lycaena tityrus in order to disentangle the mechanistic basis for the above rule. In L. tityrus the larger body size produced at a lower temperature was proximately due to a greater increase in mass, which was caused by both behavioural and physiological mechanisms: a much-increased food intake and a higher efficiency in converting ingested food into body matter. These mechanisms, combined with temperature-induced changes at the cellular level, may provide general explanations for the TSR. Body fat and protein content increased in butterflies reared at the higher temperature, indicating favourable growth conditions. As predicted from protandry theory, males showed reduced development times, caused by higher growth rates compared to females. The latter was itself related to a higher daily food consumption, while the total food consumption (due to the females' longer developmental period) and assimilation was higher in females and may underly the sexual body size dimorphism.