The Antarctic midge, Belgica antarctica, is a wingless, non-biting midge endemic to Antarctica. Larval development requires at least 2 years, but adults live only 2 weeks. The nonfeeding adults mate in swarms and females die shortly after oviposition. Eggs are suspended in a gel of unknown composition that is expressed from the female accessory gland. This project characterizes molecular mechanisms underlying reproduction in this midge by examining differential gene expression in whole males, females, and larvae, as well as in male and female accessory glands. Functional studies were used to assess the role of the gel encasing the eggs, as well as the impact of stress on reproductive biology. RNA-seq analyses revealed sex- and development-specific gene sets along with those associated with the accessory glands. Proteomic analyses were used to define the composition of the egg-containing gel, which is generated during multiple developmental stages and derived from both the accessory gland and other female organs. Functional studies indicate the gel provides a larval food source as well as a buffer for thermal and dehydration stress. All of these function are critical to juvenile survival. Larval dehydration stress directly reduces production of storage proteins and key accessory gland components, a feature that impacts adult reproductive success. Modeling reveals that bouts of dehydration may have a significant impact on population growth. This work lays a foundation for further examination of reproduction in midges and provides new information related to general reproduction in dipterans. A key aspect of this work is that reproduction and stress dynamics, currently understudied in polar organisms, are likely to prove critical in determining how climate change will alter their survivability.
Predation by phagocytic predators is a major source of bacterial mortality. The first steps in protozoan predation are recognition and consumption of their bacterial prey. However, the precise mechanisms governing prey recognition and phagocytosis by protists, and the identities of the molecular and cellular factors involved in these processes are, as yet, ill-characterized. Here, we show that that the ability of the phagocytic bacterivorous amoebae, Acanthamoeba castellanii, to recognize and internalize Escherichia coli, a bacterial prey, varies with LPS structure and composition. The presence of an O-antigen carbohydrate is not required for uptake of E. coli by A. castellanii. However, O1-antigen types, not O157 O-antigen types, inhibit recognition and uptake of bacteria by amoeba. This finding implies that O-antigen may function as an antipredator defence molecule. Recognition and uptake of E. coli by A. castellanii is mediated by the interaction of mannose-binding protein located on amoebae's surface with LPS carbohydrate. Phagocytic mammalian cells also use mannose-binding lectins to recognize and/or mediate phagocytosis of E. coli. Nonetheless, A. castellanii's mannose binding protein apparently displays no sequence similarity with any known metazoan mannose binding protein. Hence, the similarity in bacterial recognition mechanisms of amoebae and mammalian phagocytes may be a result of convergent evolution.
Freeze-tolerance, or the ability to survive internal ice formation, is relatively rare among insects. Larvae of the Antarctic midge Belgica antarctica are freeze-tolerant year-round, but in dry environments, the larvae can remain supercooled (i.e., unfrozen) at subzero temperatures. In previous work with summer-acclimatized larvae, we showed that freezing is considerably more stressful than remaining supercooled. Here, these findings are extended by comparing survival, tissue damage, energetic costs, and stress gene expression in larvae that have undergone an artificial winter acclimation regime and are either frozen or supercooled at −5 °C. In contrast to summer larvae, winter larvae survive at −5 °C equally well for up to 14 days, whether frozen or supercooled, and there is no tissue damage at these conditions. In subsequent experiments, we measured energy stores and stress gene expression following cold exposure at −5 °C for either 24 h or 14 days, with and without a 12 h recovery period. We observed slight energetic costs to freezing, as frozen larvae tended to have lower glycogen stores across all groups. In addition, the abundance of two heat shock protein transcripts, hsp60 and hsp90, tended to be higher in frozen larvae, indicating higher levels of protein damage following freezing. Together, these results indicate a slight cost to being frozen relative to remaining supercooled, which may have implications for the selection of hibernacula and responses to climate change.
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