Marleen Brunain

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.

Insights into the venom composition of the ectoparasitoid wasp Nasonia vitripennis from bioinformatic and proteomic studies.

With the Nasonia vitripennis genome sequences available, we attempted to determine the proteins present in venom by two different approaches. First, we searched for the transcripts of venom proteins by a bioinformatic approach using amino acid sequences of known hymenopteran venom proteins. Second, we performed proteomic analyses of crude N. vitripennis venom removed from the venom reservoir, implementing both an off‐line two‐dimensional liquid chromatography matrix‐assisted laser desorption/ ionization time‐of‐flight (2D‐LC‐MALDI‐TOF) mass spectrometry (MS) and a two‐dimensional liquid chromatography electrospray ionization Founer transform ion cyclotron resonance (2D‐LC‐ESI‐FT‐ICR) MS setup. This combination of bioinformatic and proteomic studies resulted in an extraordinary richness of identified venom constituents. Moreover, half of the 79 identified proteins were not yet associated with insect venoms: 16 proteins showed similarity only to known proteins from other tissues or secretions, and an additional 23 did not show similarity to any known protein. Serine proteases and their inhibitors were the most represented. Fifteen nonsecretory proteins were also identified by proteomic means and probably represent so‐called ‘venom trace elements’. The present study contributes greatly to the understanding of the biological diversity of the venom of parasitoid wasps at the molecular level.

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.

Polar tube protein gene diversity among Nosema ceranae strains derived from a Greek honey bee health study.

Honey bee samples from 54 apiaries originating from 37 geographic locations of Greece were screened for Nosema apis and Nosema ceranae. Furthermore 15 samples coming from 12 geographic locations were screened also for Paenibacilluslarvae and Melissococcus plutonius and seven honey bee virus species, for the first time on a nation-wide level. There was a tendency in finding proportionally higher spore counts in samples from apiaries that suffered important colony losses. P. larvae bacteria were identified in two samples and each of the tested bee viruses could be detected in at least one of the examined samples, with IAPV, CBPV and SBV being the least abundant and BQCV and DWV being the most abundant. In the study we focused on polymorphism of a N. ceranae gene encoding a polar tube protein (PTP) as similar genes were proven to be highly polymorphic in the microsporidian parasites Encephalitozoon cuniculi and Encephalitozoon hellem. The polymorphism observed in the PTP gene sequences from a single sample (bee hive) was unexpected and can thus be considered to be a major obstacle for genotyping.

Extending the honey bee venome with the antimicrobial peptide apidaecin and a protein resembling wasp antigen 5

Honey bee venom is a complex mixture of toxic proteins and peptides. In the present study we tried to extend our knowledge of the venom composition using two different approaches. First, worker venom was analysed by liquid chromatography‐mass spectrometry and this revealed the antimicrobial peptide apidaecin for the first time in such samples. Its expression in the venom gland was confirmed by reverse transcription PCR and by a peptidomic analysis of the venom apparatus tissue. Second, genome mining revealed a list of proteins with resemblance to known insect allergens or venom toxins, one of which showed homology to proteins of the antigen 5 (Ag5)/Sol i 3 cluster. It was demonstrated that the honey bee Ag5‐like gene is expressed by venom gland tissue of winter bees but not of summer bees. Besides this seasonal variation, it shows an interesting spatial expression pattern with additional production in the hypopharyngeal glands, the brains and the midgut. Finally, our immunoblot study revealed that both synthetic apidaecin and the Ag5‐like recombinant from bacteria evoke no humoral activity in beekeepers. Also, no IgG4‐based cross‐reactivity was detected between the honey bee Ag5‐like protein and its yellow jacket paralogue Ves v 5.

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.

Bee pathogens found in Bombus atratus from Colombia: A case study.

Bombus atratus bumblebees from Colombia that were caught in the wild and from breeding programs were screened for a broad set of bee pathogens. We discovered for the first time Lake Sinai Virus and confirmed the infection by other common viruses. The prevalence of Apicystis bombi, Crithidia bombi and Nosema ceranae was remarkably high. According to other studies the former two could have been co-introduced in South America with exotic bumble bees as Bombus terrestris or Bombus ruderatus. Given the fact that none of these species occur in Colombia, our data puts a new light on the spread of these pathogens over the South American continent.

Cranial architecture of tube-snouted gasterosteiformes (Syngnathus rostellatus and Hippocampus capensis).

The long snout of pipefishes and seahorses (Syngnathidae, Gasterosteiformes) is formed as an elongation of the ethmoid region. This is in contrast to many other teleosts with elongate snouts (e.g., butterflyfishes) in which the snout is formed as an extension of the jaws. Syngnathid fishes perform very fast suction feeding, accomplished by powerful neurocranial elevation and hyoid retraction. Clearly, suction through a long and narrow tube and its hydrodynamic implications can be expected to require certain adaptations in the cranium, especially in musculoskeletal elements of the feeding apparatus. Not much is known about which skeletal elements actually support the snout and what the effect of elongation is on related structures. Here, we give a detailed morphological description of the cartilaginous and bony feeding apparatus in both juvenile and adult Syngnathus rostellatus and Hippocampus capensis. Our results are compared with previous morphological studies of a generalized teleost, Gasterosteus aculeatus. We found that the ethmoid region is elongated early during development, with the ethmoid plate, the hyosymplectic, and the basihyal cartilage being extended in the chondrocranium. In the juveniles of both species almost all bones are forming, although only as a very thin layer. The elongation of the vomeral, mesethmoid, quadrate, metapterygoid, symplectic, and preopercular bones is already present. Probably, because of the long and specialized parental care which releases advanced developmental stages from the brooding pouch, morphology of the feeding apparatus of juveniles is already very similar to that of the adults. We describe morphological features related to snout elongation that may be considered adaptations for suction feeding; e.g. the peculiar shape of the interhyal bone and its saddle‐shaped articulation with the posterior ceratohyal bone might aid in explosive hyoid retraction by reducing the risk of hyoid dislocation. J. Morphol., 2010.

Nosema neumanni n. sp. (Microsporidia, Nosematidae), a new microsporidian parasite of honeybees, Apis mellifera in Uganda

The microsporidium Nosema neumanni n. sp., a new parasite of the honeybee Apis mellifera is described based on its ultra-structural and molecular characteristics. Structures resembling microsporidian spores were found by microscopic examination of honeybees from Uganda. Molecular confirmation failed when PCR primers specific for Nosema apis and Nosema ceranae were used, but was successful with primers covering the whole family of Nosematidae. We performed transmission electron microscopy and found typical microsporidian spores which were smaller (length: 2.36±0.14μm and width: 1.78±0.06μm; n=6) and had fewer polar filament coils (10-12) when compared to those of known species infecting honeybees. The entire 16S SSU rRNA region was amplified, cloned and sequenced and was found to be unique with the highest resemblance (97% identity) to N. apis. The incidence of N. neumanni n. sp. in Ugandan honeybees was found to be much higher than of the two other Nosema species.

Implementation of quality control and biosafety measurements in the diagnosis of honey bee diseases

Summary A quality control system is of paramount importance to boost the confidence of clients of a bee disease diagnostic laboratory, namely beekeepers and the responsible authorities, and can guarantee that tests are performed with the same accuracy in different bee laboratories. ISO 17025 is an International Standard that specifies the general requirements for the competence to carry out tests and/or calibrations. A quality control system can be recognized by accreditation, but there are important drawbacks that often drive a laboratory to focus on a limited number of highlights of the standard only. The legal framework for biosafety measurements can differ between the countries, but it is advisable (sometimes obligatory) to take measurements to protect the laboratory worker exposed to biological agents and to prevent the spread of important bee pathogens from the diagnostic lab.