Frans J. Jacobs

Reference Gene Selection for Insect Expression Studies Using Quantitative Real-Time PCR: The Head of the Honeybee, Apis mellifera, After a Bacterial Challenge

Abstract In this study an important and often neglected aspect of gene expression studies in insects, the validation of appropriate reference genes with stable expression levels between sample groups, is addressed. Although in this paper the reference gene selection for the honeybee, Apis mellifera L. (Hymenoptera: Apidae) head was tested in the context of bacterial challenge with Escherichia coli, this work can serve as a resource to help select and screen insect reference genes for gene expression studies in any tissue and under any experimental manipulation. Since it is recommended to use multiple reference genes for accurate normalization, we analyzed the expression of eleven candidate reference genes in the honeybee head, for their potential use in the analysis of differential gene expression following bacterial challenge. Three software programs, BestKeeper, Normfinder and geNorm, were used to assess candidate reference genes. GeNorm recommended the use of four reference genes. Both geNorm and Normfinder identified the genes GAPDH, RPS18, actin and RPL13a as the most stable ones, only differing in their ranking order. BestKeeper identified RPS18 as being the reference gene with the least overall variation, but also actin and GAPDH were found to be the second and third most stable expressed gene. By a combination of three software programs the genes actin, RPS18 and GAPDH were found suitable reference genes in the honeybee head in the context of bacterial infection.

Proteomic analysis of the honey bee worker venom gland focusing on the mechanisms of protection against tissue damage.

Honey bee workers use venom for the defence of the colony and themselves when they are exposed to dangers and predators. It is produced by a long thin, convoluted, and bifurcated gland, and consists of several toxic proteins and peptides. The present study was undertaken in order to identify the mechanisms that protect the venom gland secretory cells against these harmful components. Samples of whole venom glands, including the interconnected reservoirs, were separated by two-dimensional gel electrophoresis and the most abundant protein spots were subjected to mass spectrometric identification using MALDI TOF/TOF-MS and LC MS/MS. This proteomic study revealed four antioxidant enzymes: CuZn superoxide dismutase (SOD1), glutathione-S-transferase sigma 1 isoform A (GSTS1), peroxiredoxin 2540 (PXR2540) and thioredoxin peroxidase 1 isoform A (TPX1). Although glutathione-S-transferase (GST) has also been associated with xenobiotic detoxification, the protein we found belongs to the GST Sigma class which is known to protect against oxidative stress only. Moreover, we could demonstrate that the GST and SOD activity of the venom gland was low and moderate, respectively, when compared to other tissues from the adult honey bee. Several proteins involved in other forms of stress were likewise found but it remains uncertain what their function is in the venom gland. In addition to major royal jelly protein 9 (MRJP9), already found in a previous proteomic study, we identified MRJP8 as second member of the MRJP protein family to be associated with the venom gland. Transcripts of both MRJPs were amplified and sequenced. Two endocuticular structural proteins were abundantly present in the 2D-gel and most probably represent a structural component of the epicuticular lining that protects the secretory cells from the toxins they produce.

Molecular cloning and expression of icarapin, a novel IgE-binding bee venom protein

The 1045 bp full‐length cDNA sequence of a new bee venom component was obtained by rapid amplification of cDNA ends. The 672 bp coding sequence corresponds to a protein with a signal peptide and multiple carbohydrate binding sites, and it was named icarapin. It has the new consensus sequence N‐[TS]‐T‐S‐[TV]‐x‐K‐[VI](2)‐[DN]‐G‐H‐x‐V‐x‐I‐N‐[ED]‐T‐x‐Y‐x‐[DHK]‐x(2,6)‐ [STA]‐[VLFI]‐x‐[KR]‐V‐R‐[VLI]‐[IV]‐[DN]‐V‐x‐P. At least two transcript variants were found. Recombinant icarapin was tested for recognition by IgE antibodies and gave a positive dot blot with sera from 4 out of 5 bee venom allergic patients, all beekeepers. Indirect immunofluorescent staining localized the protein in the cuticular lining of the venom duct.

Honey bee colony losses in Belgium during the 2008–9 winter

(2010). Honey bee colony losses in Belgium during the 2008–9 winter. Journal of Apicultural Research: Vol. 49, No. 4, pp. 337-339.

Two novel proteins expressed by the venom glands of Apis mellifera and Nasonia vitripennis share an ancient C1q-like domain.

An in‐depth proteomic study of previously unidentified two‐dimensional polyacrylamide gel electrophoresis spots of honey bee (Apis mellifera, Hymenoptera) venom revealed a new protein with a C1q conserved domain (C1q‐VP). BlastP searching revealed a strong identity with only two proteins from other insect species: the jewel wasp, Nasonia vitripennis (Hymenoptera), and the green pea aphid, Acyrthosiphon pisum (Hemiptera). In higher organisms, C1q is the first subcomponent of the classical complement pathway and constitutes a major link between innate and acquired immunity. Expression of C1q‐VP in a variety of tissues of honey bee workers and drones was demonstrated. In addition, a wide spatial and temporal pattern of expression was observed in N. vitripennis. We suggest that C1q‐VP represents a new member of the emerging group of venom trace elements. Using degenerate primers the corresponding gene was found to be highly conserved in eight hymenopteran species, including species of the Aculeata and the Parasitica groups (suborder Apocrita) and even the suborder Symphyta. A preliminary test using recombinant proteins failed to demonstrate Am_C1q‐VP‐specific immunoglobulin E recognition by serum from patients with a documented severe bee venom allergy.

The possible role of Varroa destructor in the spreading of American foulbrood among apiaries

The aim of this investigation was to establish whether Varroa destructor can play a role in the transmission of Paenibacillus larvae larvae spores from infected to healthy bee colonies. Mites, collected from an Apis mellifera carnica colony heavily infected with American foulbrood and treated with Apistan®, were suspended in distilled water and treated in three different ways: homogenizing, shaking and stirring, or sonication. The resulting fluid samples were transferred onto selective agar medium. All culture plates showed colonies that could be identified as P. l. larvae. In view of the numbers of spores they can carry, it is concluded that mites may transmit American foulbrood from infected to healthy bee colonies.

Comparison of Two Commercial Kits for Biochemical Characterization of Paenibacillus Larvae Larvae in the Diagnosis of AFB

colony for 26 days to remove varroa. At this time the strips were removed, and the queen was caged for nine days. There was only capped brood in the colony at the end of this period. A single patch of 400–500 eggs (aged about 18 h) on a brood comb was placed in the colony, and the 73 tagged mites were added to the colony. The queen remained caged until the end of the test. The frame that had contained the eggs was removed after another 15 days. At this time, the worker bee pupae had tan coloured bodies. Only 40 of the original 73 mites were recovered, and 37 of those mites had laid eggs. They averaged 3.450 ± 0.75 progeny, which is not different from the 3–4 progeny expected from unmarked mites found on host pupae at the same age (Ifantidis, 1983). The families produced by marked mites appeared normal (most had adult males, protonymphs, deutonymphs, and adult daughters). No attempt was made to locate tagged mites on the adult bees at the end of the test.

The buzz about bees and poverty alleviation : identifying drivers and barriers of beekeeping in sub-Saharan Africa

The potential of beekeeping to mitigate the exposure of rural sub-Sahara African farmers to economic stochasticity has been widely promoted by an array of development agencies. Robust outcome indicators of the success of beekeeping to improve household well-being are unfortunately lacking. This study aimed to identify the key drivers and barriers of beekeeping adoption at the household level, and quantified the associated income contribution in three agro-ecological zones in Uganda. Beekeepers were generally the most economically disadvantaged people in the study areas and tended to adopt beekeeping following contact with non-government organisations and access to training. Whilst incomes were not statistically lower than their non-beekeeping counterparts; their mean household well-being scores were significantly lower than non-beekeeping households. The inability of beekeeping to significantly improve well-being status can in part be attributed to a lack of both training in bee husbandry and protective equipment provision such as suits, gloves and smokers. These are critical tools for beekeepers as they provide the necessary confidence to manage honey bees. Rather than focussing solely on the socio-economic conditions of farmers to effectively adopt beekeeping, future research should also attempt to evaluate the effectiveness of development agencies’ provision to the beekeeping sector.

Characterization of Native Honey Bee Subspecies in Republic of Benin Using Morphometric and Genetic Tools

Abstract Morphometric characteristics combined with genetic markers are powerful tools used for determining honey bee subspecies. Bees samples collected from 94 established apiaries distributed throughout all of the Republic of Benin were morphometricaly characterized using seven parameters and the COI-COII regions of mitochondrial DNA were sequenced. Based on the morphometric data the native honey bees could be divided into three distinct ecotypes - the Benino-dry-tropical-ecotype in the north, the Benino-Sudanian-ecotype in the central part and the Benino-Sudano-Guinean-ecotype in the south. The DNA COI-COII regions sequence analyses confirmed that the honey bee population of the Republic of Benin belongs to different mitotypes but do not correspond with the determined ecotypes. We could determine three new haplotypes which missed the P0 segment but the Q region was duplicated or triplicated. Phylogenetic analyses clustered them together in the A evolutionary lineage. In conclusion, morphometric and genetic analysis of the native West African honey bees indicated that each of the different mitotypes was able to adapt to the different ecological conditions in the country by morphometric adjustments.