Diversity of honey stores and their impact on pathogenic bacteria of the honeybee,Apis mellifera

Honeybee colonies offer an excellent environment for microbial pathogen development. The highest virulent, colony killing, bacterial agents are Paenibacillus larvae causing American foulbrood (AFB), and European foulbrood (EFB) associated bacteria. Besides the innate immune defense, honeybees evolved behavioral defenses to combat infections. Foraging of antimicrobial plant compounds plays a key role for this “social immunity” behavior. Secondary plant metabolites in floral nectar are known for their antimicrobial effects. Yet, these compounds are highly plant specific, and the effects on bee health will depend on the floral origin of the honey produced. As worker bees not only feed themselves, but also the larvae and other colony members, honey is a prime candidate acting as self‐medication agent in honeybee colonies to prevent or decrease infections. Here, we test eight AFB and EFB bacterial strains and the growth inhibitory activity of three honey types. Using a high‐throughput cell growth assay, we show that all honeys have high growth inhibitory activity and the two monofloral honeys appeared to be strain specific. The specificity of the monofloral honeys and the strong antimicrobial potential of the polyfloral honey suggest that the diversity of honeys in the honey stores of a colony may be highly adaptive for its “social immunity” against the highly diverse suite of pathogens encountered in nature. This ecological diversity may therefore operate similar to the well‐known effects of host genetic variance in the arms race between host and parasite.     

The distribution of Paenibacillus larvae spores in adult bees and honey and larval mortality, following the addition of American foulbrood diseased brood or spore-contaminated honey in honey bee (Apis mellifera) colonies

Within colony transmission of Paenibacillus larvae spores was studied by giving spore-contaminated honey comb or comb containing 100 larvae killed by American foulbrood to five experimental colonies respectively. We registered the impact of the two treatments on P. larvae spore loads in adult bees and honey and on larval mortality by culturing for spores in samples of adult bees and honey, respectively, and by measuring larval survival. The results demonstrate a direct effect of treatment on spore levels in adult bees and honey as well as on larval mortality. Colonies treated with dead larvae showed immediate high spore levels in adult bee samples, while the colonies treated with contaminated honey showed a comparable spore load but the effect was delayed until the bees started to utilize the honey at the end of the flight season. During the winter there was a build up of spores in the adult bees, which may increase the risk for infection in spring. The results confirm that contaminated honey can act as an environmental reservoir of P. larvae spores and suggest that less spores may be needed in honey, compared to in diseased brood, to produce clinically diseased colonies. The spore load in adult bee samples was significantly related to larval mortality but the spore load of honey samples was not.     

Effects of Imidacloprid and Varroa destructor on survival and health of European honey bees,Apis mellifera

There has been growing concern over declines in populations of honey bees and other pollinators which are a vital part to our food security. It is imperative to identify factors responsible for accelerated declines in bee populations and develop solutions for reversing bee losses. While exact causes of colony losses remain elusive, risk factors thought to play key roles are ectoparasitic mites Varroa destructor and neonicotinoid pesticides. The present study aims to investigate effects of a neonicotinoid pesticide Imidacloprid and Varroa mites individually on survivorship, growth, physiology, virus dynamics and immunity of honey bee workers. Our study provides clear evidence that the exposure to sublethal doses of Imidacloprid could exert a significantly negative effect on health and survival of honey bees. We observed a significant reduction in the titer of vitellogenin (Vg), an egg yolk precursor that regulates the honey bees development and behavior and often are linked to energy homeostasis, in bees exposed to Imidacloprid. This result indicates that sublethal exposure to neonicotinoid could lead to increased energy usage in honey bees as detoxification is a energy‐consuming metabolic process and suggests that Vg could be a useful biomarker for measuring levels of energy stress and sublethal effects of pesticides on honey bees. Measurement of the quantitative effects of different levels of Varroa mite infestation on the replication dynamic of Deformed wing virus (DWV), an RNA virus associated with Varroa infestation, and expression level of immune genes yields unique insights into how honey bees respond to stressors under laboratory conditions.     

A PCR-based method that permits specific detection of Paenibacillus larvae subsp. larvae, the cause of American Foulbrood of honey bees, at the subspecies level

AIMS: A reliable procedure for the identification of Paenibacillus larvae subsp. larvae, the causal agent of American Foulbrood disease of honey bees (Apis mellifera L.) based on the polymerase chain reaction (PCR) and subspecies - specific primers is described. METHODS AND RESULTS: By using ERIC-PCR, an amplicon of ca 970 bp was found among P. l. larvae strains but not in other closely related species. Based on the nucleotide sequence data of this amplicon, we designed the pair of oligonucleotides KAT 1 and KAT 2, which were assayed as primers in a PCR reaction. A PCR amplicon of the expected size ca 550 bp was only found in P. l. larvae strains. CONCLUSIONS: This PCR assay provides a specific detection for P. l. larvae. SIGNIFICANCE AND IMPACT OF THE STUDY: The developed PCR assay is highly specific because can differentiate Paenibacillus larvae subsp. larvae from the closely related Paenibacillus larvae subsp. pulvifaciens. The technique can be directly used to detect presence or absence of P. l. larvae spores in honey bee brood samples and contaminated honeys.     

Regulation of nectar collection in relation to honey storage levels by honey bees, Apis mellifera

Honey bees collect distinct nutrient sources in the form ofnectar (energy) and pollen (nitrogen). We investigated the effectof varying energy stores on nectar and pollen foraging. We foundno significant changes in nectar foraging in response to changesin honey storage levels within colonies. Individual foragersdid not vary activity rates or nectar load sizes in responseto changes in honey stores, and colonies did not increase nectarintake rates when honey stores within the hive were decreased.This result contrasts with pollen foraging behavior, which isextremely sensitive to colony state. Our data show that individualforaging decisions during nectar collection and colony regulationof nectar intake are distincdy different from pollen foraging.The behavior of honey bees illustrates that foraging strategy(and therefore foraging models) can incorporate multiple currencies,including both energy and protein intake.[Behav Ecol 7: 286–291(1996)]     

Heterologous expression of green fluorescent protein in Paenibacillus larvae, the causative agent of American Foulbrood of honey bees

Aims: We aimed at expressing heterologous proteins in Paenibacillus larvae, the causative agent of American Foulbrood of honey bees, as a prerequisite for future studies on the molecular pathogenesis of P. larvae infections. Methods and Results: For this purpose, we established a protocol for the transformation of the plasmid pAD43‐25 carrying a functional GFP gene sequence (gfpmut3a) into different P. larvae strains representing the two most relevant P. larvae genotypes ERIC I and ERIC II. We determined the optimal field strength for electroporation and the optimal regeneration time after transformation. Stable GFP expression could be detected in the mutants during their entire life cycles and even after sporulation and re‐germination. Conclusions: This method is suitable not only for the expression of GFP in P. larvae but also for the expression of heterologous proteins or GFP‐tagged proteins in P. larvae. Mutants can be used for infection assays because GFP expression remained stable after sporulation and re‐germination. Significance and Impact of the Study: This method provides the first true molecular tool for P. larvae and, therefore, is an immense advancement from what we had previously at our hands for the study of P. larvae pathogenesis.     

Chemical Composition,Antimicrobial Activity,and Mode of Action of Essential Oils against Paenibacillus larvae,Etiological Agent of American Foulbrood on Apis mellifera

This study aimed to characterize the chemical composition of Aloysia polystachia, Acantholippia seriphioides, Schinus molle, Solidago chilensis, Lippia turbinata, Minthostachys mollis, Buddleja globosa, and Baccharis latifolia essential oils (EOs), and to evaluate their antibacterial activities and their capacity to provoke membrane disruption in Paenibacillus larvae, the bacteria that causes the American Foulbrood (AFB) disease on honey bee larvae. The relationship between the composition of the EOs and these activities on P. larvae was also analyzed. Monoterpenes were the most abundant compounds in all EOs. All EOs showed antimicrobial activity against P. larvae and disrupted the cell wall and cytoplasmic membrane of P. larvae provoking the leakage of cytoplasmic constituents (with the exception of B. latifolia EO). While, the EOs’ antimicrobial activity was correlated most strongly to the content of pulegone, carvone, (Z)‐β‐ocimene, δ‐cadinene, camphene, terpinen‐4‐ol, elemol, β‐pinene, β‐elemene, γ‐cadinene, α‐terpineol, and bornyl acetate; the volatiles that better explained the membrane disruption were carvone, limonene, cis‐carvone oxide, pentadecane, trans‐carvyl acetate, trans‐carvone oxide, trans‐limonene oxide, artemisia ketone, trans‐carveol, thymol, and γ‐terpinene (positively correlated) and biciclogermacrene, δ‐2‐carene, verbenol, α‐pinene, and α‐thujene (negatively correlated). The studied EOs are proposed as natural alternative means of control for the AFB disease.     

Social control of air ventilation in colonies of honey bees,Apis mellifera

Fanning behaviour inside the nest of honey bees is an effective mechanism of ventilation. The following results are reported: (1) With only a single small entrance, the fanning is controlled so as to induce tidal ventilation of the nest as in a typical breathing pattern. (2) Periodic active fanning moves an air current out followed by a passive influx of air. (3) Fanning bees show negative phototaxis. (4) The colonial respiratory activity decreases at night following a pronounced day-night cycle.     

Seasonality of salt foraging in honey bees (Apis mellifera)

1. Honey bees (Apis mellifera) prefer foraging at compound‐rich, ‘dirty’, water sources over clean water sources. As a honey bee's main floral diet only contains trace amounts of micronutrients – likely not enough to sustain an entire colony – it was hypothesised that honey bees forage in dirty water for physiologically essential minerals that their floral diet, and thus the colony, may lack. 2. While there are many studies regarding macronutrient requirements of honey bees, few investigate micronutrient needs. For this study, from 2013 to 2015, a series of preference assays were conducted in both summer and autumn. 3. During all field seasons, honey bees exhibited a strong preference for sodium in comparison to deionised water. There was, however, a notable switch in preferences for other minerals between seasons. 4. Calcium, magnesium, and potassium – three minerals most commonly found in pollen – were preferred in autumn when pollen was scarce, but were avoided in summer when pollen was abundant. Thus, as floral resources change in distribution and abundance, honey bees similarly change their water‐foraging preferences. 5. Our data suggest that, although they are generalists with relatively few gustatory receptor genes, honey bee foragers are fine‐tuned to search for micronutrients. This ability likely helps the foragers in their search for a balanced diet for the colony as a whole.     

Calorimetric investigations of the different castes of honey bees,Apis mellifera carnica

Summary Honey bees of different age and castes were investigated calorimetrically at 20, 25 and 30 °C. Experiments were completed by endoscopic observation of the insects in the visible and the near infrared range and by acoustical monitoring and subsequent frequency analysis of various locomotor activities. Direct calorimetric results of this paper are compared with data of indirect calorimetry from the literature using a respiratory quotient of 1.00 and 21.13 J consumed. Agreements between both methods are generally good. The results show that weight-specific heat production rates increase with age of worker bees by a factor of 5.6 at 30 °C, 3.7 at 25 °C and 40.0 at 20 °C. In groups of foragers the heat production decreases with growing group size to around 6% of the value for an isolated bee. The presence of a fertile queen or of brood reduces the heat output of a small worker group significantly. Adult drones exhibit a much higher metabolic rate (up to 19.7-fold at 20 °C) than juveniles with strong fluctuations in the power-time curves. Fertile queens show a less pronounced heat production rate than virgin queens (54% at 30 °C, 87% at 25 °C and 77% at 20 °C). Calorimetric unrest is much higher for young than for adult queens. Heat production is very low in both uncapped and capped brood and less than 30% of that of a newly emerged worker. In most cases temperature showed a significant influence on the metabolic level, although its sign was not homogeneous between the castes or even within them. Locomotor activities are easily recorded by the acoustic frequency spectrum (0–7.5 kHz) and in good agreement with endoscopic observations and calorimetric traces.Abbreviations RQ respiratory quotient - ww wet weight This paper is part of the PhD thesis of L.F.     

Comparative susceptibility and immune responses of Asian and European honey bees to the American foulbrood pathogen,Paenibacillus larvae

American foulbrood (AFB) disease is caused by Paenibacillus larvae. Currently, this pathogen is widespread in the European honey bee— Apis mellifera. However, little is known about infectivity and pathogenicity of P. lan'ae in the Asiatic cavity-nesting honey bees, Apis cerana. Moreover, comparative knowledge of P. larvae infectivity and pathogenicity between both honey bee species is scarce. In this study, we examined susceptibility, larval mortality, survival rate and expression of genes encoding antimicrobial peptides (AMPs) including defensin, apidaecin, abaecin, and hymenoptaecin in A. mellifera and A. cerana when infected with P. larvae. Our results showed similar effects of P. larvae on the survival rate and patterns of AMP gene expression in both honey bee species when bee larvae are infected with spores at the median lethal concentration (LC5 0 ) for A. mellifera. All AMPs of infected bee larvae showed significant upregulation compared with noninfected bee larvae in both honey bee species. However, larvae of A. cerana were more susceptible than A. mellifera when the same larval ages and spore concentration of P. larvae were used. It also appears that A. cerana showed higher levels of AMP expression than A. mellifera. This research provides the first evidence of survival rate, LC50 and immune response profiles of Asian honey bees, A. cerana, when infected by P. larvae in comparison with the European honey bee, A. mellifera.     

Phylogenetic analysis of Starmerella apis in honey bees (Apis mellifera)

Honey bees are among the most effective pollinators that promote plant reproduction. Bees are highly active in the pollen collection season, which can lead to the transmission of selected pathogens between colonies. The clade Starmerella comprises yeasts that are isolated mainly from bees and their environment. When visiting plants, bees can come into contact with Starmerella spp. The aim of this study was to determine the prevalence and phylogenetic position of S. apis in bee colonies. Bee colonies were collected from nine apiaries in three regions. Ten colonies were sampled randomly from each apiary, and pooled samples were collected from the central part of the hive in each colony. A total of 90 (100%) bee colonies from nine apiaries were examined. Starmerella apis was detected in 31 (34.44%) samples, but related species were not identified. The 18S rRNA amplicon sequences of S. apis were compatible with the GenBank sequences of Starmerella spp. from India, Japan, Syria, Thailand, and the USA. The amplicon sequences of S. apis were also 99.06% homologous with the sequences deposited in GenBank under accession numbers JX515988 and NG067631 .This is the first study to perform a phylogenetic analysis of S. apis in Polish honey bees.     

The ontogeny of immunity in the honey bee, Apis mellifera L. following an immune challenge

The honey bee, Apis mellifera, is an ideal system for investigating ontogenetic changes in the immune system, because it combines holometabolous development within a eusocial caste system. As adults, male and female bees are subject to differing selective pressures: worker bees (females) exhibit temporal polyethism, while the male drones invest in mating. They are further influenced by changes in the threat of pathogen infection at different life stages. We investigated the immune response of workers and drones at all developmental phases, from larvae through to late stage adults, assaying both a constitutive (phenoloxidase, PO activity) and induced (antimicrobial peptide, AMP) immune response. We found that larval bees have low levels of PO activity. Adult workers produced stronger immune responses than drones, and a greater plasticity in immune investment. Immune challenge resulted in lower levels of PO activity in adult workers, which may be due to the rapid utilisation and a subsequent failure to replenish the constitutive phenoloxidase. Both adult workers and drones responded to an immune challenge by producing higher titres of AMPs, suggesting that the cost of this response prohibits its constant maintenance. Both castes showed signs of senescence in immune investment in the AMP response. Different sexes and life stages therefore alter their immune system management based on the combined factors of disease risk and life history.     

Kin recognition of worker brood by worker honey bees,Apis mellifera L.

Experimental colonies of honey bees consisting of two patrilines were observed as they reared worker brood. Seven behavior patterns that relate to brood care were recorded. Worker bees biased the care they provided to eggs and larvae destined to become workers on the basis of brood patrilines. Both patrilineal and antipatrilineal preferences in various behavioral patterns were observed. There was variation among colonies that may have been the result of the frequencies of brood of each patriline and the total amount of brood available to be reared. In addition, there were some differences between workers of the two patrilines in the way that they cared for the two patrilines of brood.     

Halocyntin and papillosin,two new antimicrobial peptides isolated from hemocytes of the solitary tunicate,Halocynthia papillosa

We report here the screening of five marine invertebrate species from two taxa (tunicates and echinoderms) for the presence of cationic antimicrobial peptides (AMP) in defence cells (hemocytes). Antimicrobial activities were detected only in the two tunicates Microcosmus sabatieri and Halocynthia papillosa. In addition, we report the isolation and characterization of two novel peptides from H. papillosa hemocytes. These molecules display antibacterial activity against Gram‐positive and Gram‐negative bacteria. Complete peptide characterization was obtained by a combination of Edman degradation and mass spectrometry. The mature molecules, named halocyntin and papillosin, comprise 26 and 34 amino acid residues, respectively. Their primary structure display no significant similarities with previously described AMP. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

吡虫啉胁迫对意大利蜜蜂哺育蜂免疫解毒相关基因表达及酶活力的影响

蜜蜂作为世界上最重要的授粉性昆虫,在采集过程中易接触到杀虫剂,前人研究表明新烟碱类杀虫剂吡虫啉（imidaclorprid）影响意大利蜜蜂Apis mellifera ligustica（简称“意蜂”）的存活和舞蹈、采集等行为。本研究旨在探究亚致死剂量吡虫啉胁迫对意大利蜜蜂哺育蜂（8日龄成年工蜂）免疫解毒相关基因表达、免疫解毒酶系活力及存活率的影响。结果显示哺育蜂连续取食3 d和9 d含0.1 ng/μL吡虫啉的蔗糖液后,其存活率与对照组（饲喂含等量丙酮的蔗糖溶液）无显著差异;连续饲喂11 d含0.1 ng/μL吡虫啉的50%蔗糖溶液后,其存活率与对照组有显著差异。荧光定量PCR检测及双抗体一步夹心法酶联免疫吸附试验结果显示哺育蜂取食吡虫啉3 d后,蜜蜂体内免疫基因多酚氧化酶基因（PPOA3,GB43738）,Abaecin类抗菌肽基因（ABA,GB18323）,葡萄糖脱氢酶基因（GLD, GB43007）和解毒基因细胞色素P450基因（CYP450 6a2,GB49876）,细胞色素B561基因（CYB561 2-like,GB40148）,葡萄糖醛酸转移酶（UDP-glucuronosyltransferase,GB52179）的表达及蜂体内体内细胞色素P450酶（cytochrome P450,CYP450）含量均有上调趋势,超氧化物岐化酶（superoxide dismutase,SOD）和过氧化氢酶（catalase,CAT）均有显著下调趋势;哺育蜂取食吡虫啉9 d后,PPOA3,ABA,GLD,CYP450 6a2,CYB561 2-like,UDP-glucuronosyltransferase的表达及蜂体内体内细胞色素P450酶含量均有下调趋势,多酚氧化酶（polyphenol oxidase,PPO）,超氧化物歧化酶和过氧化氢酶酶活力均有显著下调趋势。本研究在分子水平上提供了亚致死剂量吡虫啉是通过扰乱蜜蜂正常的免疫系统进而影响蜜蜂行为的证据,以期为维护蜜蜂健康提供一定的理论依据。     

In situ localization of heat-shock and histone proteins in honey-bee (Apis mellifera l.) larvae infected with Paenibacillus larvae

The immunohistochemical localization of the heat shock proteins (Hsp70 and Hsp90) and histone protein in healthy and Paenibacillus larvae infected honeybee (Apis mellifera L.) larvae has been studied. Hsp70 was found in the nuclei and the cytoplasm of infected midgut, salivary gland cells and haemocytes, but not in uninfected larvae. Hsp90 was localized in both infected and uninfected cells. Exposed histone proteins were localized in the nuclei of dying uninfected cells undergoing programmed cell death. The distribution of histone protein in uninfected cells of midgut, salivary gland, and other tissues was nuclear and indicative of normal programmed cell death at levels between 1 and 5%.After applying histone protein antibodies to P. larvae infected honeybee larvae, the DAB based reaction product was located in the nuclei or immediate surroundings of all larval cells. The Hsp70, Hsp90 and histone protein distribution patterns are discussed in relation to the morphological, cytochemical and immunocytochemical characteristics of programmed cell death and pathological necrosis. Results produced by methyl green-pyronin staining confirm an elevation of RNA levels in normal programmed cell death and a reduced staining for RNA in necrotic infected cells.     

Within- and across-colony effects of hyperpolyandry on immune function and body condition in honey bees (Apis mellifera)

Honey bees (Apis mellifera) have become a model system for studies on the influence of genetic diversity on disease. Honey bee queens mate with a remarkably high number of males-up to 29 in the current study-from which they produce a colony of genetically diverse daughter workers. Recent evidence suggests a significant benefit of intracolony genetic diversity on disease resistance. Here, we explored the relationship between the level of genetic diversity and multiple physiological mechanisms of cellular and humoral immune defense (encapsulation response and phenoloxidase activity). We also investigated an effect of genetic diversity on a measure of body condition (fat body mass). While we predicted that mean colony phenoloxidase activity, encapsulation response, and fat body mass would show a positive relationship with increased intracolonial genetic diversity, we found no significant relationship between genetic diversity and these immune measures, and found no consistent effect on body condition. These results suggest that high genetic diversity as a result of extreme polyandry may have little bearing on the physiological mechanisms of immune function at naturally occurring mating levels in honey bees.     

Diagnosis of American foulbrood in honey bees: a synthesis and proposed analytical protocols

Worldwide, American foulbrood (AFB) is the most devastating bacterial disease of the honey bee (Apis mellifera). Because the distinction between AFB and powdery scale disease is no longer considered valid, the pathogenic agent has recently been reclassified as one species Paenibacillus larvae, eliminating the subspecies designations Paenibacillus larvae subsp. larvae and Paenibacillus larvae subsp. pulvifaciens. The creamy or dark brown, glue-like larval remains of infected larvae continue to provide the most obvious clinical symptom of AFB, although it is not conclusive. Several sensitive and selective culture media are available for isolation of this spore-forming bacterium, with the type of samples that may be utilized for detection of the organism being further expanded. PCR methods for identification and genotyping of the pathogen have now been extensively developed. Nevertheless, biochemical profiling, bacteriophage sensitivity, immunotechniques and microscopy of suspect bacterial strains are entirely adequate for routine identification purposes.