Immune‐related proteins induced in the hemolymph after aseptic and septic injury differ in honey bee worker larvae and adults

Abstract

We have employed the proteomic approach in combination with mass spectrometry to study the immune response of honey bee workers at different developmental stages. Analysis of the hemolymph proteins of noninfected, mock-infected and immune-challenged individuals by polyacrylamide gel electrophoresis showed differences in the protein profiles. We present evidence that in vitro reared honey bee larvae respond with a prominent humoral reaction to aseptic and septic injury as documented by the transient synthesis of the three antimicrobial peptides (AMPs) hymenoptaecin, defensin1, and abaecin. In contrast, young adult worker bees react with a broader spectrum of immune reactions that include the activation of prophenoloxidase and humoral immune responses. At least seven proteins appeared consistently in the hemolymph of immune-challenged bees, three of which are identical to the AMPs induced also in larvae. The other four, i.e., phenoloxidase (PO), peptidoglycan recognition protein-S2, carboxylesterase (CE), and an Apis-specific protein not assigned to any function (HP30), are induced specifically in adult bees and, with the exception of PO, are not expressed after aseptic injury. Structural features of CE and HP30, such as classical leucine zipper motifs, together with their strong simultaneous induction upon challenge with bacteria suggest an important role of the two novel bee-specific immune proteins in response to microbial infections.

Honey bee drones maintain humoral immune competence throughout all life stages in the absence of vitellogenin production

“…The E. coli strain was cultivated in nutrient broth (NB medium), whereas M. flavus was grown in lysogeny broth (LB medium) as described previously (Randolt et al, 2008). All ingredients were purchased from Becton Dickinson (Heidelberg, Germany).…”

“…Aseptic and septic wounding of drone larvae was performed in a manner similar to that established for worker larvae (Randolt et al, 2008). For injection, we used disposable calibrated (1-5l) glass capillaries (Servoprax, Hartenstein, Würzburg, Germany) with fine tips that were generated by a P-2000 laser based micropipette puller (Sutter Instrument, Novato, CA, USA).…”

“…In addition, newly emerged adults -but not larvae -reacted with the upregulation of several large proteins, including a member of the carboxylesterase family (CE 1), peptidoglycan recognition protein-S2 (PGRP-S2) and a protein with a molecular mass of 30kDa (IRp30) of unknown function (Randolt et al, 2008).…”

Detailed proteome mapping of newly emerged honeybee worker hemolymph and comparison with the red-eye pupal stage

“…FABPs are also known to be linked to inflammatory pathways (Makowski and Hotamisligil 2004) and are thought to be connected to the insulin/ insulin-like signaling network, which is conserved among animals and is central to growth and development (Wolschin et al 2011). FABPs were found to be continuously highly expressed in honeybee worker larvae, but showed decreased expression in adults (Randolt et al 2008), suggesting age-dependent decreases in the worker hemolymph. Here, we found FABPs to be of low abundance in the emerged bee hemolymph compared to pupa.…”

Expression of larval jelly antimicrobial peptide defensin1 in Apis mellifera colonies

“…Colonies exhibit different degrees of resistance against AFB (Rothenbuhler & Thompson 1956;Bambrick 1964;Hansen & Brødsgaard 1999) which might be associated with the efficacy of individual and social/collective defense traits of larvae and adult bees (Cremer et al 2007;Wilson-Rich et al 2009;Evans & Spivak 2010). Several defense mechanisms act-Defensin1 expression in honeybee colonies 201 ing against AFB in colonies have been described: (1) filtering of spores from contaminated food in stomach of adult bees (Sturtevant & Revell 1953), (2) the inhibitory effect of other microorganisms present in the gut of larvae (Rinderer et al 1974;Forsgren et al 2010), (3) the action of cellular and humoral immune reactions in the hemolymph of larvae (Wilson-Rich et al 2008) including age-and/or pathogen-dependent synthesis of antimicrobial substances and peptides in larvae (Wedenig et al 2003;Evans 2004;Evans & Pettis 2005;Evans et al 2006;Randolt et al 2008), (4) and ability of bees with heightened olfactory sensitivity to detect and remove infected larvae from a hive before pathogens reach infectious stage -hygienic behavior (Woodrow and Holst 1942;Spivak & Gilliam 1998a, b;Masterman et al 2001).…”