Viable Triploid Honey Bees (Apis mellifera capensis) Are Reliably Produced in the Progeny of CO2 Narcotised Queens.

The haplodiploid system of sex determination of Hymenoptera acts as an exaptation for species to evolve novel forms of asexual reproduction including thelytoky (clonal offspring of the mother). During normal reproduction in Hymenoptera, three of the four products of meiosis that are present in newly-laid eggs are lost as polar bodies, while the remaining pronucleus either develops as a haploid male or fuses with a sperm nucleus to produce a diploid zygote. In contrast, in thelytokous reproduction, which is uncommon but taxonomically widespread, two of the four products of meiosis fuse, as if one acted as a sperm. Queenless workers of Apis mellifera capensis, a subspecies of honey bee from South Africa, routinely reproduce thelytokously. Unmated A. m. capensis queens can also be induced to lay thelytokously by narcosis with carbon dioxide, but mated queens are never thelytokous. We artificially inseminated A. m. capensis queens using CO2 narcosis. Up to 1/3 of offspring workers carried two maternal alleles and an allele of one father whereas no three-allele progeny were seen in control queens of the arrhenotokous (unfertilized eggs result in males) subspecies A. m. scutellata Flow cytometry of three-allele individuals revealed that they were triploid and arose from the fertilization of a thelytokous fusion nucleus. We then reared six queens from a narcotized A. m. capensis queen and determined the ploidy of the offspring queens based on microsatellites. One of the five daughters was triploid. Following artificial insemination, this queen produced unfertilized thelytokous diploid eggs at high frequency, and unfertilized triploid eggs at much lower frequency. If fertilized, thelytokous diploid eggs were non-viable, even though triploidy in itself does not impede normal development. In contrast, when the rarer triploid eggs were fertilized, a proportion developed into viable tetraploids. Our study highlights the extraordinary developmental flexibility of haplo-diploid systems.

The secreted lipoprotein, MPT83, of Mycobacterium tuberculosis is recognized during human tuberculosis and stimulates protective immunity in mice.

The long-term control of tuberculosis (TB) will require the development of more effective anti-TB vaccines, as the only licensed vaccine, Mycobacterium bovis bacille Calmette-Guérin (BCG), has limited protective efficacy against infectious pulmonary TB. Subunit vaccines have an improved safety profile over live, attenuated vaccines, such as BCG, and may be used in immuno-compromised individuals. MPT83 (Rv2873) is a secreted mycobacterial lipoprotein expressed on the surface of Mycobacterium tuberculosis. In this study, we examined whether recombinant MPT83 is recognized during human and murine M. tuberculosis infection. We assessed the immunogenicity and protective efficacy of MPT83 as a protein vaccine, with monophosphyl lipid A (MPLA) in dimethyl-dioctadecyl ammonium bromide (DDA) as adjuvant, or as a DNA vaccine in C57BL/6 mice and mapped the T cell epitopes with peptide scanning. We demonstrated that rMPT83 was recognised by strong proliferative and Interferon (IFN)-γ-secreting T cell responses in peripheral blood mononuclear cells (PBMC) from patients with active TB, but not from healthy, tuberculin skin test-negative control subjects. MPT83 also stimulated strong IFN-γ T cell responses during experimental murine M. tuberculosis infection. Immunization with either rMPT83 in MPLA/DDA or DNA-MPT83 stimulated antigen-specific T cell responses, and we identified MPT83(127-135) (PTNAAFDKL) as the dominant H-2(b)-restricted CD8(+) T cell epitope within MPT83. Further, immunization of C57BL/6 mice with rMPT83/MPLA/DDA or DNA-MPT83 stimulated significant levels of protection in the lungs and spleens against aerosol challenge with M. tuberculosis. Interestingly, immunization with rMPT83 in MPLA/DDA primed for stronger IFN-γ T cell responses to the whole protein following challenge, while DNA-MPT83 primed for stronger CD8(+) T cell responses to MPT83(127-135). Therefore MPT83 is a protective T cell antigen commonly recognized during human M. tuberculosis infection and should be considered for inclusion in future TB subunit vaccines.