Fungal ectoparasites increase winter mortality of ladybird hosts despite limited effects on their immune system.

Winter represents a challenging period for insects inhabiting temperate regions. A plethora of studies have investigated how environmental conditions such as temperature affect insect overwintering success. However, only a few studies have focused on biotic factors and the mechanisms affecting the overwintering performance of insects. Here, we investigated the effects of the parasitic fungus Hesperomyces virescens on the overwintering performance and immune system functioning of the invasive ladybird Harmonia axyridis. Winter survival was significantly lower for infected than for uninfected ladybirds. Body mass loss during overwintering tends to be higher for infected individuals compared to uninfected ones and for larger ladybirds. In addition, parasitic infection reduced post-winter longevity without food in male but not female ladybirds. Total haemocyte and protein concentration as well as antimicrobial activity against Escherichia coli significantly decreased during ladybird overwintering. However, haemolymph parameters were only poorly affected by Hesperomyces infection, with the exception of antimicrobial activity against E. coli that tended to be higher in infected ladybirds. Interestingly, none of the pre-winter haemolymph parameters were good predictors of ladybird winter survival. Overall, our results indicate that energy exhaustion unrelated to immune system challenge is the most probable explanation for increased overwintering mortality in infected ladybirds.

Increased pupal temperature has reversible effects on thermal performance and irreversible effects on immune system and fecundity in adult ladybirds.

The environmental conditions an organism encounters during development vary in their lasting impact on adult phenotypes. In the context of ongoing climate change, it is particularly relevant to understand how high developmental temperatures can impact adult traits, and whether these effects persist or diminish during adulthood. Here, we assessed the effects of pupal temperature (17 °C - normal temperature, 26 °C - increased temperature, or 35 °C - heat wave) on adult Harmonia axyridis thermal stress tolerance, immune function, starvation resistance, and fecundity. The temperature during pupation significantly affected all investigated traits in fresh adults. Heat acclimation decreased adult haemocyte concentration, cold tolerance, and total egg production, and had a positive effect on heat tolerance and starvation resistance. The negative effects of heat acclimation on cold tolerance diminished after seven days. In contrast, heat acclimation had a lasting positive effect on adult heat tolerance. Our results provide a broad assessment of the effects of developmental thermal acclimation on H. axyridis adult phenotypes. The relative plasticity of several adult traits after thermal acclimation may be consequential for the future geographic distribution and local performance of various insect species.

Changes in haemolymph parameters and insect ability to respond to immune challenge during overwintering.

Overwintering is a challenging period in the life of temperate insects. A limited energy budget characteristic of this period can result in reduced investment in immune system. Here, we investigated selected physiological and immunological parameters in laboratory-reared and field-collected harlequin ladybirds (Harmonia axyridis). For laboratory-reared beetles, we focused on the effects of winter temperature regime (cold, average, or warm winter) on total haemocyte concentration aiming to investigate potential effects of ongoing climate change on immune system in overwintering insects. We recorded strong reduction in haemocyte concentration during winter; however, there were only limited effects of winter temperature regime on changes in haemocyte concentration in the course of overwintering. For field-collected beetles, we measured additional parameters, specifically: total protein concentration, antimicrobial activity against Escherichia coli, and haemocyte concentration before and after overwintering. The field experiment did not investigate effects of winter temperature, but focused on changes in inducibility of insect immune system during overwintering, that is, measured parameters were compared between naïve beetles and those challenged by Escherichia coli. Haemocyte concentration decreased during overwintering, but only in individuals challenged by Escherichia coli. Prior to overwintering, the challenged beetles had a significantly higher haemocyte concentration compared to naïve beetles, whereas no difference was observed after overwintering. A similar pattern was observed also for antimicrobial activity against Escherichia coli as challenged beetles outperformed naïve beetles before overwintering, but not after winter. In both sexes, total protein concentration increased in the course of overwintering, but females had a significantly higher total protein concentration in their hemolymph compared to males. In general, our results revealed that insect's ability to respond to an immune challenge is significantly reduced in the course of overwintering.

Effects of the winter temperature regime on survival, body mass loss and post-winter starvation resistance in laboratory-reared and field-collected ladybirds.

Ongoing climate change results in increasing temperatures throughout the seasons. The effects of climate change on insect performance are less studied during the winter season than during the growing season. Here, we investigated the effects of various winter temperature regimes (warm, normal and cold) on the winter performance of the invasive ladybird Harmonia axyridis (Coleoptera: Coccinellidae). Winter survival, body mass loss and post-winter starvation resistance were measured for a laboratory-reared population as well as three populations collected from the field prior to overwintering. The warm winter regime increased the survival rate and body mass loss and reduced post-winter starvation resistance compared to those of the ladybirds in the cold winter regime. The effects of the temperature regime were qualitatively similar for the laboratory-reared and field-collected beetles; however, there were significant quantitative differences in all measured overwintering parameters between the laboratory-reared and field-collected populations. The winter survival of the laboratory-reared beetles was much lower than that of the field-collected beetles. The laboratory-reared beetles also lost a larger proportion of their body mass and had reduced post-winter starvation resistance. Winter survival was similar between the females and males, but compared to the males, the females lost a smaller proportion of their body mass and had better post-winter starvation resistance. The pre-overwintering body mass positively affected winter survival and post-winter starvation resistance in both the laboratory-reared and field-collected ladybirds. The significant differences between the laboratory-reared and field-collected individuals indicate that quantitative conclusions derived from studies investigating solely laboratory-reared individuals cannot be directly extrapolated to field situations.

Physiological costs of chemical defence: repeated reflex bleeding weakens the immune system and postpones reproduction in a ladybird beetle.

In insects, external chemical defences, such as reflex bleeding, have been proved to be an efficient strategy against various predators. At the same time, significant costs of reflex bleeding can be expected because bled haemolymph is lost and all valuable components included have to be renewed. Interestingly, this issue has rarely been investigated for adult insects. In this study, we examined the immune and fitness costs of repeated reflex bleeding in adults of the invasive ladybird Harmonia axyridis, investigating several haemolymph parameters. Reflex bleeding induced twice a week for three weeks resulted in a significant reduction in haemocyte concentration, total protein content, and antimicrobial activity against Micrococcus luteus, and a marginally non-significant decrease in antimicrobial activity against Escherichia coli. Repeated reflex bleeding did not result in significant body mass reduction. Interestingly, resource limitation in the form of complete food absence did not significantly interact with reflex bleeding, even though starvation itself had a strong negative effect on all haemolymph parameters investigated and individual body mass. Daily reflex bleeding did not result in decreased fecundity of young ladybirds during the first 30 days of their adult life, but the start of ladybird reproduction was delayed by about two days. Moreover, ladybirds bleeding larger amounts of haemolymph started their reproduction significantly later. Overall, our results indicate that repeated reflex bleeding weakens a ladybird's immune system and can increase their susceptibility to pathogens, but a ladybird's reproductive potential remains almost unaffected, even by very intensive reflex bleeding.