Larval Exposure to Parasitic Varroa destructor Mites Triggers Specific Immune Responses in Different Honey Bee Castes and Species.

Innate immune systems are key defenses of animals and particularly important in species that lack the sophisticated adaptive immune systems as found in vertebrates. Here, we were interested to quantify variation in innate immune responses of insects in hosts that differ in their parasite susceptibility. To do this, we studied immune responses in honey bees, which can host a remarkable number of different parasites, which are major contributors of declining bee health and colony losses. The most significant parasite of honey bees is the mite Varroa destructor, which has infested the majority of global honey bee populations, and its control remains a major challenge for beekeepers. However, a number of nonmanaged honey bees seem able to control Varroa infections, for example, the Eastern honey bee Apis ceranacerana or the African honey bee Apis mellifera scutellata. These bees therefore make interesting study subjects to identify underlaying resistance traits, for example, by comparing them to more susceptible bee genotypes such as Western honey bees (A. melliferaligustica). We conducted a series of interlinked experiments and started with behavioral assays to compare the attractiveness of bee larvae to mites using different honey bee genotypes and castes. We found that 6-day-old larvae are always most attractive to mites, independently of genotype or castes. In a next step, we compared volatile profiles of the most attractive larvae to test whether they could be used by mites for host selection. We found that the abundance of volatile compounds differed between larval ages, but we also found significant differences between genotypes and castes. To further study the expected underlaying physiological differences between potentially resistant and susceptible host larvae, we compared the larval hemolymph proteomes of the three honey bee genotypes and two castes in response to mite exposure. We identified consistent upregulation of immune and stress-related genes in Varroa-exposed larvae, which differed between genotypes and castes. Tolerant honey bee castes and genotypes were characterized by stronger or more distinct immune esponses. In summary, we provide first insights into the complex involvement of the innate immune system of tolerant honey bees against mite infestations, which could be used for future breeding purposes.

Proteomic Analysis Reveals the Molecular Underpinnings of Mandibular Gland Development and Lipid Metabolism in Two Lines of Honeybees (Apis mellifera ligustica).

The mandibular glands (MGs) of honeybee workers are vital for the secretion of lipids, for both larval nutrition and pheromones. However, knowledge of how the proteome controls MG development and functionality at the different physiological stages of worker bees is still lacking. We characterized and compared the proteome across different ages of MGs in Italian bees (ITBs) and Royal Jelly (RJ) bees (RJBs), the latter being a line bred for increasing RJ yield, originating from the ITB. All 2000 proteins that were shared by differently aged MGs in both bee lines (>4000 proteins identified in all) were strongly enriched in metabolizing protein, nucleic acid, small molecule, and lipid functional groups. The fact that these shared proteins are enriched in similar groups in both lines suggests that they are essential for basic cellular maintenance and MG functions. However, great differences were found when comparing the proteome across different MG phases in each line. In newly emerged bees (NEBs), the unique and highly abundant proteins were enriched in protein synthesis, cytoskeleton, and development related functional groups, suggesting their importance to initialize young MG development. In nurse bees (NBs), specific and highly abundant proteins were mainly enriched in substance transport and lipid synthesis, indicating their priority may be in priming high secretory activity in lipid synthesis as larval nutrition. The unique and highly abundant proteins in forager bees (FBs) were enriched in lipid metabolism, small molecule, and carbohydrate metabolism. This indicates their emphasis on 2-heptanone synthesis as an alarm pheromone to enhance colony defense or scent marker for foraging efficiency. Furthermore, a wide range of different biological processes was observed between ITBs and RJBs at different MG ages. Both bee stocks may adapt different proteome programs to drive gland development and functionality. The RJB nurse bee has reshaped its proteome by enhancing the rate of lipid synthesis and minimizing degradation to increase 10-hydroxy-2-decenoic acid synthesis, a major component of RJ, to maintain the desired proportion of lipids in increased RJ production. This study contributes a novel understanding of MG development and lipid metabolism, and a potential starting point for lipid or pheromone biochemists as well as developmental geneticists.

Proteome Analysis of the Hemolymph, Mushroom Body, and Antenna Provides Novel Insight into Honeybee Resistance against Varroa Infestation.

Varroa destructor has been identified as a major culprit responsible for the losses of millions of honeybee colonies. Varroa sensitive hygiene (VSH) is a suite of behaviors from adult bees to suppress mite reproduction by uncapping and/or removing mite infested pupae from a sealed brood. Despite the efforts to elucidate the molecular underpinnings of VSH, they remain largely unknown. We investigated the proteome of mushroom bodies (MBs) and antennae of adult bees with and without VSH from a stock selected for VSH based on their response to artificially Varroa-infected brood cells by near-infrared camera observation. The pupal hemolymph proteome was also compared between the VSH-line and the line that was not selected for VSH. The identified 8609 proteins in the hemolymph, MBs, and antennae represent the most depth coverage of the honeybee proteome (>55%) to date. In the hemolymph, the VSH-line adapts a unique strategy to boost the social immunity and drive pupal organogenesis by enhancing energy metabolism and protein biosynthesis. In MBs, the up-regulated proteins implicated in neuronal sensitivity suggest their roles to promote the execution of VSH by activation of synaptic vesicles and calcium channel activities. In antennae, the highly expressed proteins associated with sensitivity of olfactory senses and signal transmissions signify their roles by inputting a strong signal to the MBs for initiating VSH. These observations illustrate that the enhanced social immunities and olfactory and neuronal sensitivity play key roles in the combat against Varroa infestation. The identified candidate markers may be useful for accelerating marker-associated selection for VSH to aid in resistance to a parasite responsible for decline in honeybee health.

Biodegradation of neonicotinoid insecticide, imidacloprid by restriction enzyme mediated integration (REMI) generated Trichoderma mutants.

REMI (restriction enzyme-mediated integration) technique was employed to construct Trichoderma atroviride strain T23 mutants with degrading capability of neonicotinoid insecticide, imidacloprid. The plasmid pBluescript II KS-hph used for integration in REMI mutants was confirmed by PCR and Southern hybridization. Among 153 transformants, 57% of them have showed higher neonicotinoid insecticide, imidacloprid, degradation ability than the wild strain T23 (p<0.01). More specifically, seven single-copied T. atroviride T23 transformants have confirmed a 30% higher degradation rate than their parent isolate. Among all transformed mutants, a 95% imidacloprid degradation rate was identified as the highest. This study, thus, provided an effective approach for improving neonicotinoid insecticide-degrading capability using REMI transformed T. atroviride mutants.