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Belgica antarctica (Diptera: Chironomidae), a brachypterous midge endemic to the maritime Antarctic, was first described in 1900. Over more than a century of study, a vast amount of information has been compiled on the species (3 750 000 Google search results as of January 10, 2021), encompassing its ecology and biology, life cycle and reproduction, polytene chromosomes, physiology, biochemistry and, increasingly, omics. In 2014, B. antarctica's genome was sequenced, further boosting research. Certain developmental stages can be cultured successfully in the laboratory. Taken together, this wealth of information allows the species to be viewed as a natural model organism for studies of adaptation and function in extreme environments. [ABSTRACT FROM AUTHOR]

Genome of antarctic endemic Belgica antarctica Jacobs has been sequenced. However, no set of inversion diagnostic markers has ever been assigned for the species. Using the classical method of polytene chromosome squash preparation, we found three heterozygous inversions located on the second (two heterozygous inversions) and third chromosomes (one heterozygous inversion) in the Belgica antarctica population of a cape of Wiencke Island, 500 m to SW from Port Lockroy. The chromosome set and chromosome variability did not differ from those described in the literature (Atchley and Davis 1979). Every salivary gland chromosome had its own markers by which it can be determined. However, we did not find a sex-linked inversion on chromosome III and heterozygous inversion on chromosome I, reported in earlier studies. For the first time, we observed a strong heterochromatin band in chromosome III at the telomere of one arm. Our data show not only the stability of the described inversions in the population but also the usefulness of the squash preparation technique in the studies of genetic variability of Belgica antarctica in present time. [ABSTRACT FROM AUTHOR]

The Rosenthal Islands lie along the western edge of the Antarctic Peninsula. They are largely inaccessible and the few research projects in the area have focused on seabird colonies, so nothing has been known about the arthropod fauna on these islands. We conducted a preliminary survey of the arthropods associated with large penguin colonies. We identified two species of Collembola (Cryptopygus antarcticus and Friesea grisea), four species of mites (Alaskozetes antarcticus, Hydrogamasellus racovitzai, Tectopenthalodes villosus and Rhagidia sp.) and one insect (Belgica antarctica). The mite A. antarcticus and the collembolan C. antarcticus were common in large aggregations at our collecting sites and were occasionally observed on the surface of penguin guano without vegetative cover. The insect, a chironomid midge, was less common and found only in vegetated areas.

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.

Changes in climate and environment can impact the sustainability of populations and biodiversity. Understanding population genetic diversity in the past and present can help us better predict species' responses to future environmental change. Antarctica has experienced drastic environmental change which threatens its biodiversity. In this study, we characterized the phylogeography and population genetic structure of Belgica antarctica , a wingless midge that is endemic to the western Antarctic Peninsula. This insect has adaptive features to withstand extremes in temperature, salinity, humidity, anoxia and pH. Belgica antarctica is widespread on widely dispersed islands of ice-free habitat, but questions remain regarding its genetic history, diversity and gene flow. We created nuclear-based, single nucleotide polymorphism (SNP) markers and genotyped 229 individuals from 11 populations to examine historical and current population genetic patterns. Our results support recent divergence among populations on different islands within the last 1 Mya. Furthermore, despite a lack of wings, B. antarctica exhibited frequent migration among islands, perhaps via ocean currents or phoresy with Antarctic vertebrates (e.g. seabirds). The close link between the evolutionary history of B. antarctica and the region's environment and ecology emphasize the importance of understanding its population dynamics to predict its persistence under environmental change. [ABSTRACT FROM AUTHOR]

Desiccation resistance of adult males and females of the midge, Belgica antarctica (Diptera: Chironomidae) was evaluated to determine how this short-lived stage maintains water balance in the dry Antarctic environment. Both sexes had slightly lower water content (≈60%) and a higher dehydration tolerance (>30% water loss) than most other insects. Water loss rates were high and increased rapidly at temperatures above 15°C, indicating that the adult midges are more hygric than many other polar terrestrial arthropods. Water gain was accomplished by free water uptake with minimal or no contribution from absorption of water vapor or metabolic water production. Parameters related to water balance did not differ among populations from different islands. Overall, the high water requirements of the adult midge appear to be a significant challenge and presumably dictate that the adult midges must emerge during the brief period when free water is readily available and seek protected microhabitats that facilitate water retention.

The extreme seasonal shifts of day length in polar regions, ranging from constant light in the summer to constant darkness in the winter, pose an intriguing environment for probing activity rhythms and the functioning of circadian clocks. Here, we monitor locomotor activity during the summer on the Antarctic Peninsula and under laboratory conditions, as well as the accompanying patterns of clock gene expression in the Antarctic midge, the only insect endemic to Antarctica. Larvae and adults are most active during the warmest portion of the day, but at a constant temperature they remain continuously active regardless of the photoregime, and activity also persists in constant darkness. The canonical clock genes period , timeless , Clock , and vrille are expressed in the head but we detected no cycling of expression in either the field or under diverse photoregimes in the laboratory. The timekeeping function of the clock has possibly been lost, enabling the midge to opportunistically exploit the unpredictable availability of permissive thermal conditions for growth, development, and reproduction during the short summer in Antarctica. [ABSTRACT FROM AUTHOR]

Since biotic interactions within the simple terrestrial communities on the Antarctic Peninsula are limited compared with tropical and temperate regions, survival is largely dictated by the numerous abiotic challenges. Our research focuses on adaptations to environmental stresses experienced by the Antarctic midge ( Belgica antarctica Jacobs, 1900), the southernmost free-living insect. Midge larvae can survive freezing and anoxia year-round. Not only can frozen larvae undergo rapid cold-hardening (RCH) at temperatures as low as -12 °C, but RCH develops more rapidly in frozen compared with supercooled larvae. Whether larvae overwinter in a frozen state or cryoprotectively dehydrated may depend on hydration levels within their hibernacula. Larvae constitutively up-regulate genes encoding heat shock proteins, as well as the antioxidant enzymes superoxide dismutase and catalase. Larvae accumulate osmoprotectants in response to freezing, desiccation, and exposure to seawater; exposure to one of these osmotic stressors confers cross-tolerance to the others. Molecular responses to dehydration stress include extensive genome-wide changes that include differential expression of aquaporins among tissues, upregulation of pathways associated with autophagy, inhibition of apoptosis, and downregulation of metabolism and ATP production. [ABSTRACT FROM AUTHOR]

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The terrestrial midge Belgica antarctica relies extensively on dehydration to survive the low temperatures and desiccation stress that prevail in its Antarctic habitat. The loss of body water is thus a critical adaptive mechanism employed at the onset of winter to prevent injury from internal ice formation; a rapid mechanism for rehydration is equally essential when summer returns and the larva resumes the brief active phase of its life. This important role for water movement suggests a critical role for aquaporins (AQPs). Recent completion of the genome project on this species revealed the presence of AQPs in B. antarctica representing the DRIP, PRIP, BIB, RPIP, and LHIP families. Treatment with mercuric chloride to block AQPs also blocks water loss, thereby decreasing cell survival at low temperatures. Antibodies directed against mammalian or Drosophila AQPs suggest a wide tissue distribution of AQPs in the midge and changes in protein abundance in response to dehydration, rehydration, and freezing. Thus far, functional studies have been completed only for PRIP1. It appears to be a water-specific AQP, but expression levels are not altered by dehydration or rehydration. Functional assays remain to be completed for the additional AQPs. [ABSTRACT FROM AUTHOR]

Although larvae of the Antarctic midge, Belgica antarctica, live for more than 2 years, the adult and embryonic stages are brief and are less well known than the larvae. In this report, we provide additional details of these understudied life stages with laboratory observation on adult emergence, longevity, preoviposition period and embryonic development. Male adults emerged slightly earlier than females, and they also lived longer. More than a half (57 %) of the adults that emerged in the laboratory were males. Females produced only a single egg mass and died within a day after oviposition. Embryonic development required 16 days at 4 °C, and prior to hatching, the pharate larvae perform a distinct sequence of behaviors that include drinking and peristaltic movement. We also discuss points that need to be resolved for laboratory propagation of this species. [ABSTRACT FROM AUTHOR]

The terrestrial midge, Belgica antarctica, occupies a diverse range of microhabitats along the Antarctic Peninsula. Although overwintering larvae have the physiological potential to survive by freezing or cryoprotective dehydration, use of the latter strategy may be constrained by inoculative freezing within hibernacula. To investigate the influence of microhabitat type on larval overwintering, we selected four substrate types that differed markedly in their composition and hydric characteristics. Organic content of these substrates ranged from 14 to 89 %. High organic content was associated with higher values for saturation moisture content (up to 2.0 HO g dry mass) as well as elevated levels of field moisture content. Seasonal values of field moisture content remained near or above the saturation moisture value for each microhabitat type, and when larvae were cooled in substrates rehydrated to field-based levels, they were unable to avoid inoculation by environmental ice, regardless of substrate type. Consequently, our data suggest that wet hibernacula would force most larvae to overwinter in a frozen state. Yet, dehydrated larvae were collected in April during the seasonal transition to winter suggesting that spatial and temporal variations in precipitation and microhabitat conditions may expose larvae to dehydration and promote larval overwintering in a cryoprotectively dehydrated state. [ABSTRACT FROM AUTHOR]

During the austral summer, larvae of the terrestrial midge Belgica antarctica (Diptera: Chironomidae) experience highly variable and often unpredictable thermal conditions. In addition to remaining freeze tolerant year-round, larvae are capable of swiftly increasing their cold tolerance through the rapid cold-hardening (RCH) response. The present study compared the induction of RCH in frozen versus supercooled larvae. At the same induction temperature, RCH occurred more rapidly and conferred a greater level of cryoprotection in frozen versus supercooled larvae. Furthermore, RCH in frozen larvae could be induced at temperatures as low as -12°C, which is the lowest temperature reported to induce RCH. Remarkably, as little as 15 min at -5°C significantly enhanced larval cold tolerance. Not only is protection from RCH acquired swiftly, but it is also quickly lost after thawing for 2 h at 2°C. Because the primary difference between frozen and supercooled larvae is cellular dehydration caused by freeze concentration of body fluids, we also compared the effects of acclimation in dehydrated versus frozen larvae. Because slow dehydration without chilling significantly increased larval survival to a subsequent cold exposure, we hypothesize that cellular dehydration caused by freeze concentration promotes the rapid acquisition of cold tolerance in frozen larvae. [ABSTRACT FROM AUTHOR]

This study describes the ecology and distribution of the only two native Antarctic insects, the chironomid species Parochlus steinenii and Belgica antarctica, both found on Byers Peninsula (Livingston Island, South Shetland Islands). Parochlus steinenii inhabits lakes of the central plateau of Byers Peninsula associated with aquatic mosses on the bottom of lakes and in some streams of the South Beach area. Some streams have stable populations which are able to complete their life cycle while other streams have temporary, unstable populations. Belgica antarctica also inhabits streams running through mosses located in the South Beach area. Our data indicate that this species has a limited dispersal capability which is positively light activated for both adults and pupae. Both Antarctic midge species coexists on Byers Peninsula and share some stretches of streams. Isotopic studies show a non-selective feeding regime for both species with mixed carbon sources associated with both biofilm/microbial mats and mosses. [ABSTRACT FROM PUBLISHER]

The Antarctic midge, Belgica antarctica, experiences sub-zero temperatures and desiccating conditions for much of the year, and in response to these environmental insults, larvae undergo rapid shifts in metabolism, mobilizing carbohydrate energy reserves to promote synthesis of low-molecular-mass osmoprotectants. In this study, we measured the expression of 11 metabolic genes in response to thermal and dehydration stress. During both heat and cold stress, we observed upregulation of phosphoenolpyruvate carboxykinase ( pepck) and glycogen phosphorylase ( gp) to support rapid glucose mobilization. In contrast, there was a general downregulation of pathways related to polyol, trehalose, and proline synthesis during both high- and low-temperature stress. Pepck was likewise upregulated in response to different types of dehydration stress; however, for many of the other genes, expression patterns depended on the nature of dehydration stress. Following fast dehydration, expression patterns were similar to those observed during thermal stress, i.e., upregulation of gp accompanied by downregulation of trehalose and proline synthetic genes. In contrast, gradual, prolonged dehydration (both at a constant temperature and in conjunction with chilling) promoted marked upregulation of genes responsible for trehalose and proline synthesis. On the whole, our data agree with known metabolic adaptations to stress in B. antarctica, although a few discrepancies between gene expression patterns and downstream metabolite contents point to fluxes that are not controlled at the level of transcription. [ABSTRACT FROM AUTHOR]

Abstract: During the 1960s, the midge, Eretmoptera murphyi, was transferred from sub-Antarctic South Georgia (55oS 37oW) where it is endemic to a single location on maritime Antarctic Signy Island (60oS 45oW). Its distribution has since expanded considerably, suggesting that it is pre-adapted to the more severe conditions further south. To test one aspect of the level of its pre-adaptation, the rapid cold hardening (RCH) response in this species was investigated. When juvenile (L1–L2) and mature (L3–L4) larvae of E. murphyi were directly exposed to progressively lower temperatures for 8h, they exhibited Discriminating Temperatures (DTemp, temperature at which there is 10–20% survival of exposed individuals) of −11.5 and −12.5°C, respectively. The mean SCP was above −7.5°C in both larval groups, confirming the finding of previous studies that this species is freeze-tolerant. Following gradual cooling (0.2°Cmin−1), survival was significantly greater at the DTemp in both larval groups. The response was strong, lowering the lower lethal temperature (LLT) by up to 6.5°C and maintaining survival above 80% for at least 22h at the DTemp. RCH was also exhibited during the cooling phase of an ecologically relevant thermoperiodic cycle (+4°C to −3°C). Mechanistically, the response did not affect freezing, with no alteration in the supercooling point (SCP) found following gradual cooling, and was not induced while the organism was in a frozen state. These results are discussed in light of E. murphyi’s pre-adaptation to conditions on Signy Island and its potential to colonize regions further south in the maritime Antarctic. [Copyright &y& Elsevier]

Two species of chironomid midges are currently described in the genus Belgica Jacobs, 1900. Belgica antarctica Jacobs, 1900 is endemic to parts of the maritime Antarctic, and Belgica albipes (Séguy, 1965) is endemic to Îles Crozet, a sub-Antarctic archipelago in the southern Indian Ocean. The relationships between these species, and their closest known relative ( Eretmoptera murphyi Schaeffer, 1914, endemic to sub-Antarctic South Georgia), were examined by sequencing DNA fragments for domains 1 and 3-5 of 28S ribosomal DNA and the mitochondrial gene cytochrome c oxidase 1 ( cox1). The resulting molecular relationships between the three species were unclear, although their position within the subfamily Orthocladiinae of the Chironomidae, as generated by classical taxonomy, was confirmed. Our data reinforce earlier doubts, based on classical morphological approaches, that the generic placement of E. murphyi may be incorrect. Further analyses may indeed confirm that the species represents a third member of the genus Belgica. Genetic distance analysis, limited to the barcode region of cox1, indicated high differentiation between the two populations of B. albipes sampled (one obtained from the type location), suggesting the likely presence of cryptic species within this taxon, and that the taxonomic status of this species should be revised. Analysis of cox1 sequences in B. antarctica highlighted a strong genetic structure between populations obtained from 12 locations along the Antarctic Peninsula and the South Shetland Islands archipelago, with a number of distinctive mtDNA lineages inhabiting geographically distinct areas. In particular, we found four different haplogroups constituting geographically close but genetically distinct populations, a pattern likely to have been encouraged by the brachyptery of the members of this genus. We suggest that the different genetic patterns shown by each haplogroup have probably been determined by historical dispersal and colonization events during the Pleistocene, and are consistent with their survival in refuges in situ during successive glacial maxima over this period. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, 106, 258-274. [ABSTRACT FROM AUTHOR]

Abstract: Larvae of the Antarctic midge, Belgica antarctica, routinely face periods of limited water availability in their natural environments on the Antarctic Peninsula. As a result, B. antarctica is one of the most dehydration-tolerant insects studied, surviving up to 70% loss of its body water. While previous studies have characterized the physiological effects of a single bout of dehydration, in nature larvae are likely to experience multiple bouts of dehydration throughout their lifetime. Thus, we examined the physiological consequences of repeated dehydration and compared results to larvae exposed to a single, prolonged period of dehydration. For the repeated dehydration experiment, larvae were exposed to 1–5 cycles of 24h dehydration at 75% RH followed by 24h rehydration. Each bout of dehydration resulted in 30–40% loss of body water, with a concomitant 2- to 3-fold increase in body fluid osmolality. While nearly 100% of larvae survived a single bout of dehydration, <65% of larvae survived five such cycles. Larvae subjected to multiple bouts of dehydration also experienced severe depletion of carbohydrate energy reserves; glycogen and trehalose content decreased with each successive cycle, with larvae losing 89% and 48% of their glycogen and trehalose, respectively, after five cycles of dehydration/rehydration. Larvae exposed to prolonged dehydration (99% RH for 10d) had 26% less water, 43% less glycogen, and 27% less lipid content than controls, but did not experience any mortality. Thus, both repeated and prolonged dehydration results in substantial energetic costs that are likely to negatively impact fitness. [Copyright &y& Elsevier]

Abstract: Aquaporins (AQPs) are water channel proteins facilitating movement of water across the cell membrane. Recent insect studies clearly demonstrate that AQPs are indispensable for cellular water management under normal conditions as well as under stress conditions including dehydration and cold. In the present study we cloned an AQP cDNA from the Antarctic midge Belgica antarctica (Diptera, Chironomidae) and investigated water transport activity of the AQP protein and transcriptional regulation of the gene in response to dehydration and rehydration. The nucleotide sequence and deduced amino acid sequence of the cDNA showed high similarity to AQPs in other insects and also showed characteristic features of orthodox AQPs. Phylogenetic analysis revealed that Belgica AQP is a homolog of dehydration-inducible AQP of another chironomid, Polypedilum vanderplanki. A swelling assay using a Xenopus oocyte expression system verified that Belgica AQP is capable of transporting water, but not glycerol or urea. The AQP mRNA was detected in various organs under non-stressed conditions, suggesting that this AQP plays a fundamental role in cell physiology. In contrast to our expectation, AQP transcriptional expression was not affected by either dehydration or rehydration. [Copyright &y& Elsevier]

Abstract: Aquaporin (AQP) water channel proteins play key roles in water movement across cell membranes. Extending previous reports of cryoprotective functions in insects, this study examines roles of AQPs in response to dehydration, rehydration, and freezing, and their distribution in specific tissues of the Antarctic midge, Belgica antarctica (Diptera, Chironomidae). When AQPs were blocked using mercuric chloride, tissue dehydration tolerance increased in response to hypertonic challenge, and susceptibility to overhydration decreased in a hypotonic solution. Blocking AQPs decreased the ability of tissues from the midgut and Malpighian tubules to tolerate freezing, but only minimal changes were noted in cellular viability of the fat body. Immuno-localization revealed that a DRIP-like protein (a Drosophila aquaporin), AQP2- and AQP3 (aquaglyceroporin)-like proteins were present in most larval tissues. DRIP- and AQP2-like proteins were also present in the gut of adult midges, but AQP4-like protein was not detectable in any tissues we examined. Western blotting indicated that larval AQP2-like protein levels were increased in response to dehydration, rehydration and freezing, whereas, in adults DRIP-, AQP2-, and AQP3-like proteins were elevated by dehydration. These results imply a vital role for aquaporin/aquaglyceroporins in water relations and freezing tolerance in B. antarctica. [Copyright &y& Elsevier]

In this study, we examined the effects of repeated cold exposure (RCE) on the survival, energy content and stress protein expression of larvae of the Antarctic midge, Belgica antarctica (Diptera: Chironomidae). Additionally, we compared results between larvae that were frozen at -5°C in the presence of water during ACE and those that were supercooled at -5°C in a dry environment. Although >95% of larvae survived a single 12h bout of freezing at -5°C, after five cycles of ACE survival of frozen larvae dropped below 70%. Meanwhile, the survival of control and supercooled larvae was unchanged, remaining around 90% for the duration of the study. At the tissue level, frozen larvae had higher rates of cell mortality in the midgut than control and supercooled larvae. Furthermore, larvae that were frozen during ACE experienced a dramatic reduction in energy reserves; after five cycles, frozen larvae had 25% less lipid, 30% less glycogen and nearly 40% less trehalose than supercooled larvae. Finally, larvae that were frozen during ACE had higher expression of hsp7O than those that were supercooled, indicating a higher degree of protein damage in the frozen group. Results were similar between larvae that had accumulated 60h of freezing at -5°C over five cycles of ACE and those that were frozen continuously for 60h, suggesting that the total time spent frozen determines the physiological response. Our results suggest that it is preferable, both from a survival and energetic standpoint, for larvae to seek dry microhabitats where they can avoid inoculative freezing and remain unfrozen during ACE. [ABSTRACT FROM AUTHOR]

During the late 1960s, larvae of the flightless midge Eretmoptera murphyi Schaeffer were accidentally transferred from the sub-Antarctic island of South Georgia to Signy Island in the maritime Antarctic. Higher insects are rare in the Antarctic and the introduction and establishment of a new species is an unusual event. The fly has overcome the two major barriers to colonization of the Antarctic by new species: the geographical isolation of the region and its severe climate. Larvae of the flightless midge overwinter in the surface layers of soil on Signy Island where the temperature may fall to below −10 °C, compared with as little as −1.5 °C on South Georgia. This suggests the possession of a level of pre-adaption to colder conditions. Summer-collected larvae have a supercooling point (SCP or whole body freezing point) of approximately −5.0 °C but survive experimental exposure to −13 °C, giving them a level of freeze tolerance. After acclimation at −4 °C for 4 days, the SCP changes little but the temperature at which 50% of the population would die decreases to lower than −19 °C. Larvae are also resistant to dehydration. Under experimental conditions of 88% relative humidity at 5 °C, larvae lose water linearly (0.42% h−1) over the first 30 h but resist further water loss once their water content decreases to approximately 1.4 g g−1 dry weight. All larvae survive these conditions for the duration of the experiment (55 h). Eretmoptera murphyi is well adapted to survive on Signy Island, and these studies suggest that it has the ability to survive at more extreme locations at higher latitudes if it were to be inadvertently transferred to a suitable habitat. [ABSTRACT FROM AUTHOR]

Abstract: On the Antarctica continent the wingless midge, Belgica antarctica (Diptera, Chironomidae) occurs further south than any other insect. The digestive tract of the larval stage of Belgica that inhabits this extreme environment and feeds in detritus of penguin rookeries has been described for the first time. Ingested food passes through a foregut lumen and into a stomodeal valve representing an intussusception of the foregut into the midgut. A sharp discontinuity in microvillar length occurs at an interface separating relatively long microvilli of the stomodeal midgut region, the site where peritrophic membrane originates, from the midgut epithelium lying posterior to this stomodeal region. Although shapes of cells along the length of this non-stomodeal midgut epithelium are similar, the lengths of their microvilli increase over two orders of magnitude from anterior midgut to posterior midgut. Infoldings of the basal membranes also account for a greatly expanded interface between midgut cells and the hemocoel. The epithelial cells of the hindgut seem to be specialized for exchange of water with their environment, with the anterior two-thirds of the hindgut showing highly convoluted luminal membranes and the posterior third having a highly convoluted basal surface. The lumen of the middle third of the hindgut has a dense population of resident bacteria. Regenerative cells are scattered throughout the larval midgut epithelium. These presumably represent stem cells for the adult midgut, while a ring of cells, marked by a discontinuity in nuclear size at the midgut-hindgut interface, presumably represents stem cells for the adult hindgut. [Copyright &y& Elsevier]

Summer storms along the Antarctic Peninsula can cause microhabitats of the terrestrial midge Belgica antarctica to become periodically inundated with seawater from tidal spray. As microhabitats dry, larvae may be exposed to increasing concentrations of seawater. Alternatively, as a result of melting snow or following rain, larvae may be immersed in freshwater for extended periods. The present study assessed the tolerance and physiological response of B. antarctica larvae to salinity exposure, and examined the effect of seawater acclimation on their subsequent tolerance of freezing, dehydration and heat shock. Midge larvae tolerated extended exposure to hyperosmotic seawater; nearly 50% of larvae survived a 10-day exposure to 1000 mOsm kg-1 seawater and -25% of larvae survived 6 days in 2000 mOsm kg-1 seawater. Exposure to seawater drastically reduced larval body water content and increased hemolymph osmolality. By contrast, immersion in freshwater did not affect water content or hemolymph osmolality. Hyperosmotic seawater exposure, and the accompanying osmotic dehydration, resulted in a significant correlation between the rate of oxygen consumption and larval water content and induced the de novo synthesis and accumulation of several organic osmolytes. A 3-day exposure of larvae to hyperosmotic seawater increased freezing tolerance relative to freshwater-acclimated larvae. Even after rehydration, the freezing survival of larvae acclimated to seawater was greater than freshwater-acclimated larvae. Additionally, seawater exposure increased the subsequent tolerance of larvae to dehydration. Our results further illustrate the similarities between these related, yet distinct, forms of osmotic stress and add to the suite of physiological responses used by larvae to enhance survival in the harsh and unpredictable Antarctic environment. [ABSTRACT FROM AUTHOR]

We investigated molecular responses elicited by three types of dehydration (fast, slow and cryoprotective), rehydration and overhydration in larvae of the Antarctic midge, Belgica antarctica. The larvae spend most the year encased in ice but during the austral summer are vulnerable to summer storms, osmotic stress from ocean spray and drying conditions due to wind and intense sunlight. Using suppressive subtractive hybridization (SSH), we obtained clones that were potentially responsive to dehydration and then used northern blots to evaluate the gene’s responsiveness to different dehydration rates and hydration states. Among the genes most responsive to changes in the hydration state were those encoding heat shock proteins ( smHsp, Hsp70, Hsp90), antioxidants ( superoxide dismutase, catalase), detoxification ( metallothionein, cytochrome p450), genes involved in altering cell membranes ( fatty acid desaturase, phospholipase A2 activating protein, fatty acyl CoA desaturase) and the cytoskeleton ( actin, muscle-specific actin), and several additional genes including a zinc-finger protein, pacifastin and VATPase. Among the three types of dehydration evaluated, fast dehydration elicited the strongest response (more genes, higher expression), followed by cryoprotective dehydration and slow dehydration. During rehydration most, but not all, genes that were expressed during dehydration continued to be expressed; fatty acid desaturase was the only gene to be uniquely upregulated in response to rehydration. All genes examined, except VATPase, were upregulated in response to overhydration. The midge larvae are thus responding quickly to water loss and gain by expressing genes that encode proteins contributing to maintenance of proper protein function, protection and overall cell homeostasis during times of osmotic flux, a challenge that is particularly acute in this Antarctic environment. [ABSTRACT FROM AUTHOR]

Abstract: Larvae of the Antarctic midge, Belgica antarctica (Diptera: Chironomidae), are frequently exposed to dehydrating conditions on the Antarctic Peninsula. In this study, we examined how rates and levels of dehydration alter heat and cold tolerance and how these relate to levels of trehalose within the insect. When dehydrated, larvae tolerated cold and heat stress more effectively, although resistance to cold was more pronounced than heat resistance. Slow dehydration was more effective than rapid dehydration in increasing temperature tolerance. Severe dehydration (50% reduction in water content) caused a much greater increase in temperature tolerance than did mild dehydration (e.g. 10% water loss). Larvae severely dehydrated at a slow rate (98% RH) were more temperature tolerant than those dehydrated quickly (0 or 75% RH). These results indicate that the slower dehydration rate allows the larvae to more effectively respond to reduced water levels and that physiological adjustments to desiccation provide cross tolerance to cold and heat. Levels of trehalose increased during dehydration and are likely a major factor increasing subsequent cold and heat resistance. This hypothesis was also supported by experimental results showing that injection of trehalose enhanced resistance to temperature stress and dehydration. We conclude that changes in temperature tolerance in B. antarctica are linked to the rate and severity of dehydration and that trehalose elevation is a probable mechanism enhancing this form of cross tolerance. [Copyright &y& Elsevier]

In many insects, the rapid cold-hardening (RCH) response significantly enhances cold tolerance in minutes to hours. Larvae of the Antarctic midge, Belgica antarctica, exhibit a novel form of RCH, by which they increase their freezing tolerance. In this study, we examined whether cold-sensing and RCH in B. antarctica occur in vitro and whether calcium is required to generate RCH. As demonstrated previously, 1 h at -5°C significantly increased organismal freezing tolerance at both -15°C and -20°C. Likewise, RCH enhanced cell survival of fat body, Malpighian tubules, and midgut tissue of larvae frozen at -20°C. Furthermore, isolated tissues retained the capacity for RCH in vitro, as demonstrated with both a dye exclusion assay and a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (M]IT)-based viability assay, thus indicating that cold- sensing and RCH in B. antarctica occur at the cellular level. Interestingly, there was no difference in survival between tissues that were supercooled at -5°C and those frozen at -5°C, suggesting that temperature mediates the RCH response independent of the freezing of body fluids. Finally, we demonstrated that calcium is required for RCH to occur. Removing calcium from the incubating solution slightly decreased cell survival after RCH treatments, while blocking calcium with the intracellular chelator BAPTA-AM significantly reduced survival in the RCH treatments. The calmodulin inhibitor N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7) also significantly reduced cell survival in the RCH treatments, thus supporting a role for calcium in RCH. This is the first report implicating calcium as an important second messenger in the RCH response. [ABSTRACT FROM AUTHOR]

Abstract: The midge, Belgica antarctica Jacobs, is subjected to numerous environmental stressors during its 2-year life cycle on the Antarctic Peninsula, and in response it has evolved a suite of behavioral, physiological, and life-cycle modifications to counter these stressors, but thus far only a limited number of biochemical adaptations have been identified. In this study, we use a metabolomics approach to obtain a broad overview of changes in energy metabolism, amino acids, and polyols in response to three of the midge''s major stresses: heat, freezing, and desiccation. Using GC-MS analysis, a total of 75 compounds were identified. Desiccation (50% water loss) elicited the greatest physiological response (as determined by principal components analysis) when compared to untreated controls, with many elevated metabolites from pathways of central carbohydrate metabolism and a decrease in free amino acids. When larvae were frozen (6h at −10°C), alanine and aspartate increased as well as urea. Freezing also increased three polyols (glycerol, mannitol, erythritol), while desiccation increased only two polyols (glycerol, erythritol). Heating the midges for 1h at 30°C elevated α-ketoglutarate and putrescine while suppressing glycerol, glucose, and serine levels. Freezing and desiccation elicited elevation of four shared metabolites, whereas no shared metabolites were elevated by heat. All three treatments resulted in a reduction in serine, potentially identifying this amino acid as a marker for stress in this species. A number of metabolic changes, especially those in the sugar and polyol pools, are adaptations that have potential to enhance survival during both cold and desiccation. [Copyright &y& Elsevier]

During winter, larvae of the Antarctic midge, Belgica antarctica (Diptera, Chironomidae), must endure 7-8 months of continuous subzero temperatures, encasement in a matrix of soil and ice, and severely desiccating conditions. This environment, along with the fact that larvae possess a high rate of water loss and are extremely tolerant of desiccation, may promote the use of cryoprotective dehydration as a strategy for winter survival. This study investigates the capacity of larvae to resist inoculative freezing and undergo cryoprotective dehydration at subzero temperatures. Slow cooling to -3°C in an environment at equilibrium with the vapor pressure of ice reduced larval water content by ~40% and depressed the body fluid melting point more than threefold to -2.6°C. This melting point depression was the result of the concentration of existing solutes (i.e. loss of body water) and the de novo synthesis of osmolytes. By day 14 of the subzero exposure, larval survival was still >95%, suggesting larvae have the capacity to undergo cryoprotective dehydration. However, under natural conditions the use of cryoprotective dehydration may be constrained by inoculative freezing as result of the insect's intimate contact with environmental ice. During slow cooling within a substrate of frozen soil, the ability of larvae to resist inoculative freezing and undergo cryoprotective dehydration was dependent upon the moisture content of the soil. As detected by a reduction of larval water content, the percentage of larvae that resisted inoculative freezing increased with decreasing soil moisture. These results suggest that larvae of the Antarctic midge have the capacity to resist inoculative freezing at relatively low soil moisture contents and likely undergo cryoprotective dehydration when exposed to subzero temperatures during the polar winter. [ABSTRACT FROM AUTHOR]

Abstract: The Antarctic midge, Belgica antarctica, is exposed to frequent periods of dehydration during its prolonged larval development in the cold and dry Antarctic environment. In this study, we determined the water requirements of the larvae and the mechanisms it exploits to reduce the stress of drying. Larvae lost water at an exceptionally high rate (>10%/h) and tolerated losing a high portion (>70%) of their water content. Larvae were unable to absorb water from subsaturated water vapor (⩽0.98 a v) to replenish their water stores, thus this midge relies exclusively on the intake of liquid water to increase its pool of body water and maintain water balance. To reduce dehydration stress, the midge employed a variety of mechanisms. Behaviorally, the larvae suppressed water loss by clustering. In response to slow dehydration, glycerol concentration increased 2-fold and trehalose concentration increased 3-fold, responses that are known to decrease the rate of water loss and increase dehydration tolerance. No changes in the mass of cuticular lipids occurred in response to desiccation, but the observed shift to longer hydrocarbons likely contributes to reduced water loss as the larvae dehydrate. As the larvae dehydrated, their oxygen consumption rate dropped, resulting in a reduction of water loss by respiration. Lastly, one bout of slow dehydration also enhanced the larva''s ability to survive subsequent dehydration, suggesting that the larvae have the capacity for drought acclimation. Thus, these hydrophilic midge larvae prevent dehydration by multiple mechanisms that collectively reduce the water loss rate and increase dehydration tolerance. [Copyright &y& Elsevier]

Antarctica's terrestrial environment is a challenge to which very few animals have adapted. The largest, free-living animal to inhabit the continent year-round is a flightless midge, Belgica antarctica. Larval midges survive the lengthy austral winter encased in ice, and when the ice melts in summer, the larvae complete their 2-yr life cycle, and the wingless adults form mating aggregations while subjected to surprisingly high substrate temperatures. Here we report a dichotomy in survival strategies exploited by this insect at different stages of its life cycle. Larvae constitutively up-regulate their heat shock proteins (small hsp, hsp70, and hsp90) and maintain a high inherent tolerance to temperature stress. High or low temperature exposure does not further up-regulate these genes nor does it further enhance thermotolerance. Such ‘preemptive’ synthesis of hsps is sufficient to prevent irreversible protein aggregation in response to a variety of common environmental stresses. Conversely, adults exhibit no constitutive up-regulation of their hsps and have a lower intrinsic tolerance to high temperatures, but their hsps can be thermally activated, resulting in enhanced thermotolerance. Thus, the midge larvae, but not the adults, have adopted the unusual strategy of expressing hsps continuously, possibly to facilitate proper protein folding in a cold habitat that is more thermally stable than that of the adults but a habitat subjected frequently to freeze-thaw episodes and bouts of pH, anoxic, and osmotic stress. [ABSTRACT FROM AUTHOR]

The chironomid midges Belgica antarctica, Eretmoptera murphyi (subfamily Orthocladiinae) and Parochlus steinenii (subfamily Podonominae), are the only Diptera species currently found in Antarctica. The relationships between these species and a range of further taxa of Chironomidae were examined by sequencing domains 1 and 3–5 of 28S ribosomal RNA. The resulting molecular relationships between B. antarctica and E. murphyi, within Orthocladiinae, were highly supported by validation analyses, confirming their position within Chironomidae, as generated by classical taxonomy. Within Podonominae, P. steinenii from the Maritime Antarctic was more closely related to material from sub-Antarctic South Georgia than to material from Patagonia. Taking advantage of the availability of a molecular substitution rate calculated for this gene in Diptera, a dating of divergence between our study taxa was tentatively established. The divergence dates obtained were 49 million years (Myr), between B. antarctica and E. murphyi, and 68.5 Myr between these species and the closest Orthocladiinae taxon tested from Patagonia, suggesting that B. antarctica and E. murphyi were representatives of an ancient lineage. As both are endemic to their respective tectonic microplates, their contemporary distribution is, therefore, likely to have been shaped by vicariance rather than dispersal. [ABSTRACT FROM AUTHOR]

Rapid cold-hardening (RCH) is well known to increase the tolerance of chilling or cold shock in a diverse array of invertebrate systems at both organismal and cellular levels. Here, we report a novel role for RCH by showing that RCH also increases freezing tolerance in an Antarctic midge, Belgica antarctica (Diptera, Chironomidae). The RCH response of B. antarctica was investigated under two distinct physiological states: summer acclimatized and cold acclimated. Summer-acclimatized larvae were less cold tolerant, as indicated by low survival following exposure to -10°C for 24 h; by contrast, nearly all cold- acclimated larvae survived -10°C, and a significant number could survive -15°C. Cold-acclimated larvae had higher supercooling points than summer larvae. To evaluate the RCH response in summer-acclimatized midges, larvae and adults, maintained at 4°C, were transferred to -5°C for 1 h prior to exposures to -10, -15 or -20°C. RCH significantly increased survival of summer-acclimatized larvae frozen at -10°C for 1 h compared with larvae receiving no cold-hardening treatment, but adults, which live for only a week or so in the austral summer, lacked the capacity for RCH. In cold-acclimated larvae, RCH significantly increased freeze tolerance to both -15 and -20°C. Similarly, RCH significantly increased cellular survival of fat body, Malpighian tubules and gut tissue from cold-acclimated larvae frozen at -20°C for 24 h. These results indicate that RCH not only protects against non-freezing injury but also increases freeze tolerance. [ABSTRACT FROM AUTHOR]