Sepsis and Hemocyte Loss in Honey Bees (Apis mellifera) Infected with Serratia marcescens Strain Sicaria.

Global loss of honey bee colonies is threatening the human food supply. Diverse pathogens reduce honey bee hardiness needed to sustain colonies, especially in winter. We isolated a free-living Gram negative bacillus from hemolymph of worker honey bees (Apis mellifera) found separated from winter clusters. In some hives, greater than 90% of the dying bees detached from the winter cluster were found to contain this bacterium in their hemolymph. Throughout the year, the same organism was rarely found in bees engaged in normal hive activities, but was detected in about half of Varroa destructor mites obtained from colonies that housed the septic bees. Flow cytometry of hemolymph from septic bees showed a significant reduction of plasmatocytes and other types of hemocytes. Interpretation of the16S rRNA sequence of the bacterium indicated that it belongs to the Serratia genus of Gram-negative Gammaproteobacteria, which has not previously been implicated as a pathogen of adult honey bees. Complete genome sequence analysis of the bacterium supported its classification as a novel strain of Serratia marcescens, which was designated as S. marcescens strain sicaria (Ss1). When compared with other strains of S. marcescens, Ss1 demonstrated several phenotypic and genetic differences, including 65 genes not previously found in other Serratia genomes. Some of the unique genes we identified in Ss1 were related to those from bacterial insect pathogens and commensals. Recovery of this organism extends a complex pathosphere of agents which may contribute to failure of honey bee colonies.

Honey bee hemocyte profiling by flow cytometry.

Multiple stress factors in honey bees are causing loss of bee colonies worldwide. Several infectious agents of bees are believed to contribute to this problem. The mechanisms of honey bee immunity are not completely understood, in part due to limited information about the types and abundances of hemocytes that help bees resist disease. Our study utilized flow cytometry and microscopy to examine populations of hemolymph particulates in honey bees. We found bee hemolymph includes permeabilized cells, plasmatocytes, and acellular objects that resemble microparticles, listed in order of increasing abundance. The permeabilized cells and plasmatocytes showed unexpected differences with respect to properties of the plasma membrane and labeling with annexin V. Both permeabilized cells and plasmatocytes failed to show measurable mitochondrial membrane potential by flow cytometry using the JC-1 probe. Our results suggest hemolymph particulate populations are dynamic, revealing significant differences when comparing individual hive members, and when comparing colonies exposed to diverse conditions. Shifts in hemocyte populations in bees likely represent changing conditions or metabolic differences of colony members. A better understanding of hemocyte profiles may provide insight into physiological responses of honey bees to stress factors, some of which may be related to colony failure.

Reduced nicotinamide adenine dinucleotide phosphate oxidase-independent resistance to Aspergillus fumigatus in alveolar macrophages.

The fungal pathogen Aspergillus fumigatus is responsible for increasing numbers of fatal infections in immune-compromised humans. Alveolar macrophages (AM) are important in the innate defense against aspergillosis, but little is known about their molecular responses to fungal conidia in vivo. We examined transcriptional changes and superoxide release by AM from C57BL/6 and gp91(phox)(-/-) mice in response to conidia. Following introduction of conidia into the lung, microarray analysis of AM showed the transcripts most strongly up-regulated in vivo to encode chemokines and additional genes that play a critical role in neutrophil and monocyte recruitment, indicating that activation of phagocytes represents a critical early response of AM to fungal conidia. Of the 73 AM genes showing > or = 2-fold changes, 8 were also increased in gp91(phox)(-/-) mice by conidia and in C57BL/6 mice by polystyrene beads, suggesting a common innate response to particulate matter. Ingenuity analysis of the microarray data from C57BL/6 mice revealed immune cell signaling and gene expression as primary mechanisms of this response. Despite the well-established importance of phagocyte NADPH oxidase in resisting aspergillosis, we found no evidence of this mechanism in AM following introduction of conidia into the mouse lung using transcriptional, luminometry, or NBT staining analysis. In support of these findings, we observed that AM from C57BL/6 and gp91(phox)(-/-) mice inhibit conidial germination equally in vitro. Our results indicate that early transcription in mouse AM exposed to conidia in vivo targets neutrophil recruitment, and that NADPH oxidase-independent mechanisms in AM contribute to inhibition of conidial germination.