Fine-scale variation in microhabitat conditions influences physiology and metabolism in an Antarctic insect.

Microhabitats with distinct biotic and abiotic properties exist within landscapes, and this microhabitat variation can have dramatic impacts on the phenology and physiology of the organisms occupying them. The Antarctic midge Belgica antarctica inhabits diverse microhabitats along the Western Antarctic Peninsula that vary in macrophyte composition, hygric qualities, nutrient input, and thermal patterns. Here, we compare seasonal physiological changes in five populations of B. antarctica living in close proximity but in different microhabitats in the vicinity of Palmer Station, Antarctica. Thermal regimes among our sample locations differed in both mean temperature and thermal stability. Between the warmest and coldest sites, seasonal mean temperatures differed by 2.6˚C and degree day accumulations above freezing differed by a factor of 1.7. Larval metabolic and growth rates varied among the sites, and adult emergence occurred at different times. Distinct microhabitats also corresponded with differences in body composition, as lipid and carbohydrate content of larvae differed across sites. Further, seasonal changes in carbohydrate and protein content were dependent on site, indicating fine-scale variation in the biochemical composition of larvae as they prepare for winter. Together, these results demonstrate that variation in microhabitat properties influences the ontogeny, phenology, physiology, and biochemical makeup of midge populations living in close proximity. These results have implications for predicting responses of Antarctic ecosystems to environmental change.

Multi-level analysis of reproduction in an Antarctic midge identifies female and male accessory gland products that are altered by larval stress and impact progeny viability.

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.

Changes in Energy Reserves and Gene Expression Elicited by Freezing and Supercooling in the Antarctic Midge, Belgica antarctica.

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.

Two isoforms of Pepck in Sarcophaga bullata and their distinct expression profiles through development, diapause, and in response to stresses of cold and starvation.

Pepck is a metabolic enzyme that participates in gluconeogenesis through the conversion of oxaloacetate into phosphoenol pyruvate. Numerous transcriptomic studies have identified Pepck as a potential key player during diapause and various stresses responses. Here, we describe expression patterns of both cytosolic and mitochondrial isoforms of Pepck throughout development, during diapause, and in response to starvation and cold shock in the flesh fly, Sarcophaga bullata. We cloned full-length transcripts for both Pepck isoforms and observed that expression of both genes varied throughout development. Diapausing pupae have the highest relative expression of both isoforms, suggesting participation in the anticipatory production of sugars and sugar alcohols that occurs during this overwintering stage. In response to acute stress, the cytosolic isoform was upregulated whereas the mitochondrial variant remained unchanged. Cytosolic Pepck was strongly upregulated after 2 h recovery from cold shock and returned to baseline levels within 8 h. In response to 24 h of starvation, the cytosolic isoform was similarly upregulated and returned to control levels after 24 h of recovery. Acute stress is known to incur a metabolic cost, and Pepck could be a key player in this response. Although it remains unclear why there is such a dramatic divergence in the expression of the two isoforms, the distinction suggests specific roles for the two isoforms that depend on the developmental status of the fly and the stress conditions to which it is exposed.

Determinants that govern the recognition and uptake of Escherichia coli O157 : H7 by Acanthamoeba castellanii.

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.