The paratransgenic potential of Lactobacillus kunkeei in the honey bee Apis mellifera.

The honey bee (Apis mellifera) is a domestic insect of high value to human societies, as a crop pollinator in agriculture and a model animal in scientific research. The honey bee, however, has experienced massive mortality worldwide due to the phenomenon Colony Collapse Disorder (CCD), resulting in alarming prospects for crop failure in Europe and the USA. The reasons for CCD are complex and much debated, but several honey bee pathogens are believed to be involved. Paratransgenesis is a Trojan horse strategy, where endogenous microorganisms are used to express effector molecules that antagonise pathogen development. For use in honey bees, paratransgenesis must rely on a set of criteria that the candidate paratransgenic microorganism must fulfil in order to obtain a successful outcome: (1) the candidate must be genetically modifiable to express effector molecules; (2) the modified organism should have no adverse effects on honey bee health upon reintroduction; and (3) it must survive together with other non-pathogenic bee-associated microorganisms. Lactic acid bacteria (LAB) are common gut bacteria in vertebrates and invertebrates, and some have naturally beneficial properties in their host. In the present work we aimed to find a potential paratransgenic candidate within this bacterial group for use in honey bees. Among isolated LAB associated with bee gut microbiota, we found the fructophilic Lactobacillus kunkeei to be the most predominant species during foraging seasons. Four genetically different strains of L. kunkeei were selected for further assessment. We demonstrated (1) that L. kunkeei is transformable; (2) that the transformed cells had no obvious adverse effect on honey bee survival; and (3) that transformed cells survived well in the gut environment of bees upon reintroduction. Our study demonstrates that L. kunkeei fulfils the three criteria for paratransgenesis and can be a suitable candidate for further research on this strategy in honey bees.

Mitochondrial DNA integrity changes with age but does not correlate with learning performance in honey bees.

The honey bee is a well-established model organism to study aging, learning and memory. Here, we used young and old forager honey bees to investigate whether age-related learning capacity correlates with mitochondrial function. The bees were selected for age and learning performance and mitochondrial function was evaluated by measuring mtDNA integrity, mtDNA copy number and mitochondrial gene expression. Quite unexpectedly, mtDNA from young bees showed more damage than mtDNA from older bees, but neither mtDNA integrity, nor mtDNA copy number nor mitochondrial gene expression correlated with learning performance. Although not statistically significant (p=0.07) the level of L-rRNA increased with age in good learners whereas it decreased in poor learners. Our results show that learning performance in honey bee does not correlate with absolute mitochondrial parameters like mtDNA damage, copy number or expression of mitochondrial genes, but may be associated with the ability to regulate mitochondrial activity.

Size-related variation in protein abundance in the brain and abdominal tissue of bumble bee workers.

Female bumble bee workers of the same species often show a profound body size variation that is linked to a division of labour. Large individuals are more likely to forage whereas small individuals are more likely to perform in-nest activities. A higher sensory sensitivity, stronger circadian rhythms as well as better learning and memory performances appear to better equip large individuals for outdoor activities compared to their smaller siblings. The molecular mechanisms underlying worker functional polymorphism remain unclear. Proteins are major determinants of an individual's morphology and behaviour. We hypothesized that the abundance of proteins such as metabolic enzymes as well as proteins involved in neuronal functions would differ with body size and provide insights into the mechanisms underlying size-dependent physiological specialization in bumble bee workers. We conducted protein quantification measurements based on liquid chromatography coupled with tandem mass spectrometry on tissue samples derived from small and large Bombus impatiens and Bombus terrestris workers. Proteins found to differ significantly in abundance between small and large workers belong to the categories of structure, energy metabolism and stress response. These findings provide the first proteomic insight into mechanisms associated with size-based division of labour in social insects.

Larval and nurse worker control of developmental plasticity and the evolution of honey bee queen-worker dimorphism.

Social evolution in honey bees has produced strong queen-worker dimorphism for plastic traits that depend on larval nutrition. The honey bee developmental programme includes both larval components that determine plastic growth responses to larval nutrition and nurse components that regulate larval nutrition. We studied how these two components contribute to variation in worker and queen body size and ovary size for two pairs of honey bee lineages that show similar differences in worker body-ovary size allometry but have diverged over different evolutionary timescales. Our results indicate that the lineages have diverged for both nurse and larval developmental components, that rapid changes in worker body-ovary size allometry may disrupt queen development and that queen-worker dimorphism arises mainly from discrete nurse-provided nutritional environments, not from a developmental switch that converts variable nutritional environments into discrete phenotypes. Both larval and nurse components have likely contributed to the evolution of queen-worker dimorphism.

Genetic architecture of ovary size and asymmetry in European honeybee workers.

The molecular basis of complex traits is increasingly understood but a remaining challenge is to identify their co-regulation and inter-dependence. Pollen hoarding (pln) in honeybees is a complex trait associated with a well-characterized suite of linked behavioral and physiological traits. In European honeybee stocks bidirectionally selected for pln, worker (sterile helper) ovary size is pleiotropically affected by quantitative trait loci that were initially identified for their effect on foraging behavior. To gain a better understanding of the genetic architecture of worker ovary size in this model system, we analyzed a series of crosses between the selected strains. The crossing results were heterogeneous and suggested non-additive effects. Three significant and three suggestive quantitative trait loci of relatively large effect sizes were found in two reciprocal backcrosses. These loci are not located in genome regions of known effects on foraging behavior but contain several interesting candidate genes that may specifically affect worker-ovary size. Thus, the genetic architecture of this life history syndrome may be comprised of pleiotropic, central regulators that influence several linked traits and other genetic factors that may be downstream and trait specific.

Structural and proteomic analyses reveal regional brain differences during honeybee aging.

Among insects, learning is particularly well studied in the fruit fly Drosophila melanogaster and the honeybee Apis mellifera. A senescence-dependent decline in classic pavlovian conditioning is demonstrated for both species. To understand how aging affects learning, genetic approaches used with Drosophila can benefit from complementary studies in Apis. Specifically, honeybees have a larger brain size allowing for compartment-specific approaches, and a unique life-history plasticity. They usually perform within-nest tasks early in life (nest bees) and later they collect food (foragers). Senescence of learning performance is a function of the bees' foraging duration but underlying causes are poorly understood. As cognitive aging is commonly associated with structural and biochemical changes in the brain, we hypothesized that brain areas implicated in learning change in synaptic and biochemical composition with increased foraging duration. First, we used synapse-specific immunohistochemistry and proteomics to screen for alterations in the calyx region of the mushroom body, a key structure for memory formation. Using proteomics, we next profiled the central brain, which comprises all higher-order integration centers. We show that, with increased foraging duration, levels of kinases, synaptic- and neuronal growth-related proteins decline in the central brain while the calyx region remains intact both in structure and biochemistry. We suggest that proteome-level changes within major anatomical sites of memory formation other than the calyx region could be central to learning dysfunction. These include large compartments of the central brain, such as the mushroom body's output regions and the antennal lobes. Our data provide novel information toward heterogeneity in the aging insect brain, and demonstrate advantages of the honeybee for invertebrate neurogerontological research.

Brood pheromone suppresses physiology of extreme longevity in honeybees (Apis mellifera).

Honeybee (Apis mellifera) society is characterized by a helper caste of essentially sterile female bees called workers. Workers show striking changes in lifespan that correlate with changes in colony demography. When rearing sibling sisters (brood), workers survive for 3-6 weeks. When brood rearing declines, worker lifespan is 20 weeks or longer. Insects can survive unfavorable periods on endogenous stores of protein and lipid. The glyco-lipoprotein vitellogenin extends worker bee lifespan by functioning in free radical defense, immunity and behavioral control. Workers use vitellogenin in brood food synthesis, and the metabolic cost of brood rearing (nurse load) may consume vitellogenin stores and reduce worker longevity. Yet, in addition to consuming resources, brood secretes a primer pheromone that affects worker physiology and behavior. Odors and odor perception can influence invertebrate longevity but it is unknown whether brood pheromone modulates vitellogenin stores and survival. We address this question with a 2-factorial experiment where 12 colonies are exposed to combinations of absence vs presence of brood and brood pheromone. Over an age-course of 24 days, we monitor the amount of vitellogenin stored in workers' fat body (adipose tissue). Thereafter, we track colony survival for 200 days. We demonstrate that brood rearing reduces worker vitellogenin stores and colony long-term survival. Yet also, we establish that the effects can result solely from exposure to brood pheromone. These findings indicate that molecular systems of extreme lifespan regulation are integrated with the sensory system of honeybees to respond to variation in a primer pheromone secreted from larvae.

Ageing in a eusocial insect: molecular and physiological characteristics of life span plasticity in the honey bee.

Commonly held views assume that ageing, or senescence, represents an inevitable, passive, and random decline in function that is strongly linked to chronological age. In recent years, genetic intervention of life span regulating pathways, for example, in Drosophila as well as case studies in non-classical animal models, have provided compelling evidence to challenge these views.Rather than comprehensively revisiting studies on the established genetic model systems of ageing, we here focus on an alternative model organism with a wild type (unselected genotype) characterized by a unique diversity in longevity - the honey bee.Honey bee (Apis mellifera) life span varies from a few weeks to more than 2 years. This plasticity is largely controlled by environmental factors. Thereby, although individuals are closely related genetically, distinct life histories can emerge as a function of social environmental change.Another remarkable feature of the honey bee is the occurrence of reverted behavioural ontogeny in the worker (female helper) caste. This behavioural peculiarity is associated with alterations in somatic maintenance functions that are indicative of reverted senescence. Thus, although intraspecific variation in organismal life span is not uncommon, the honey bee holds great promise for gaining insights into regulatory pathways that can shape the time-course of ageing by delaying, halting or even reversing processes of senescence. These aspects provide the setting of our review.We will highlight comparative findings from Drosophila melanogaster and Caenorhabditis elegans in particular, and focus on knowledge spanning from molecular- to behavioural-senescence to elucidate how the honey bee can contribute to novel insights into regulatory mechanisms that underlie plasticity and robustness or irreversibility in ageing.