Function and immuno-localization of aquaporins in the Antarctic midge Belgica antarctica

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.     

Sex ratio and adult behaviour of the Antarctic midge Belgica antarctica (Diptera, Chironomklae)

Abstract. 1. The sex ratio in populations of the Antarctic chironomid Belgica antarctica approximates 1:1 at eclosion.
2. Male predominance in surface populations of the short-lived adult persists throughout the summer. The mean ratio, based on data from surface aggregations (austral summer 1977–78) and from sticky traps (1978–79) is about 6:1, with a steady seasonal decline from 10–20:1 to 2–5:1.
3. Males live slightly longer than females but the difference does not account for observed ratios.
4. Samples of adult populations taken from subsurface sites have sex ratios nearer to equality than surface populations.
5. It is concluded that the male-dominated surface populations are equivalent to the male swarms typical of winged Chironomidae.
6. Capture of airborne adults shows that aerial transport as well as water-surface rafting may play a role in dispersal of adults.     

Mechanisms to reduce dehydration stress in larvae of the Antarctic midge, Belgica antarctica

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 (< or = 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.     

Mechanisms of freezing tolerance in an Antarctic midge, Belgica antarctica

ABSTRACT. The larvae and adults of Belgica antarctica were studied in an attempt to identify the mechanism of low temperature adaptation that enables this species to survive in the Antarctic. Larvae are freezing-tolerant during the austral summer and elaborate a complex of cryoprotectants including erythritol, glucose, sucrose and trehalose. Adults are freezing-susceptible and lack adequate quantities of cryoprotectants. Maintenance on artificial diets indicated that cryoprotectant profiles have food-source and temperature-dependent components. In addition, direct utilization of dietary cryoprotectants is suggested.     

A dipteran from south of the Antarctic Circle: Belgica antarctica (Chironomidae) with a description of its larva

Belgica antarctica Jacobs is recorded for the first time from two localities south of the Antarctic circle: Orford Cliff (66°55'S) on the Continental mainland and the Refuge Islands (68°21'S). The larva is described, and measurements of head capsule lengths indicate four instars. The larvae south of the Antarctic circle appear to be slightly smaller than those from within the main distributional range. Belgica antarctica is certainly the earth's southernmost free-living holometabolous insect, but the flea Glaciopsyllus antarcticus has been recorded 14 minutes of latitude farther south than the southernmost records of B. antarctica.     

The winged Antarctic midge Parochlus steinenii (Gerke) (Diptera: Chironomidae) in the South Shetland Islands

The morphological variation of adult Parochlus steinenii (Gerke) is described from measurements of two populations from the South Shetland Islands. Morphologically, the population from Ardley Island is significantly larger than the population from Livingston Island, and in both populations variation in forefemur length is generally greater than variation in either antennal or wing length. The final instar larva of P. steinenii is described in detail. A consideration of the species' distribution in three geographically isolated areas, as well as of the greater morphological variation in polar as opposed to temperate populations, indicates that a flexible life history strategy in the larval stage may be important for survival in extreme environments.     

Life history traits of adults and embryos of the Antarctic midge Belgica antarctica

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.     

The larva and habitat ofParakiefferiella nigra Brundin (Diptera: Chironomidae)

Collections of adults ofParakiefferiella nigra Brundin indicate that this chironomid occurs widely in arctic and subarctic zones. In addition, it has occasionally been collected in cool temperate and boreal forest lakes of both North America and Europe. Although widely distributed, the larva and its habitat have not previously been described. Identity of the larva ofP. nigra has been established by studying associated reared specimens. The distinctive larva, with reduced second lateral mental teeth, is stenotopic, and on the basis of modern collections appears to be most abundant in cold, oligotrophic lakes. More data is required to establish the range of thermal environments inhabited by the larva. Larval head capsule remains ofP. nigra are common in late-glacial sediments of southwestern British Columbia lakes and provide important evidence for oligotrophic conditions during late-glacial time.     

Metabolomics reveals unique and shared metabolic changes in response to heat shock, freezing and desiccation in the Antarctic midge, Belgica antarctica

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 (6 h 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 1 h at 30 °C elevated -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.     

Identification of instars and species in some larval Polypedilum (Polypedilum) (Diptera: Chironomidae)

Instars II and III of Polypedilum aviceps Townes, Polypedilum convictum (Walker), and Polypedilum illinoense (Malloch) can be identified to species by associating them with instar IV because key taxonomic characters remain relatively unchanged from instar to instar. Instars I cannot be identified to species or genus unless they are associated with older, identifiable larvae reared from the same egg masses. No single character evaluated on slide material can be used to clearly separate instars in all three species. Larvae of P. aviceps can be separated into instars based on any four of seven characters; P. convictum by either of two characters; and, P. illinoense by a combination of two characters. Changes in structures of instars II, III, and IV are described for all three species. Growth ratios for some structures are compared and discussed with regard to Dyar's Rule.     

Maritime midge radiations in the Pacific Ocean (Diptera: Chironomidae)

Maritime chironomid midges (Diptera) are diverse, yet these ‘pearls of the ocean’ are little known. Emphasizing Pacific Ocean taxa, we used six genetic markers (18S, 28S, CAD1, CAD4, FolCOI and COI) and fossil calibrations to produce Bayesian time-calibrated phylogenies to date eight independent marine transitions in three subfamilies. Deep nodes involve subfamily Telmatogetoninae (originating mid-Cretaceous, 101–128, 114 Ma), with sister genera Telmatogeton Schiner and Thalassomya Schiner splitting later in the Cretaceous (56–82, 69 Ma). Two transitions in Orthocladiinae involve Clunio Haliday and Pseudosmittia Edwards, dating from the upper Cretaceous, both with Eocene crown groups. In subfamily Chironominae, transitions to marine occur in two tribes. Four transitions occur within the otherwise nonmarine crown groups Kiefferulus Goetghebuer, Dicrotendipes Kieffer, Polypedilum Kieffer and Ainuyusurika Sasa & Shirasaka. Two separate robust clades in tribe Tanytarsini involve: (1) a minor radiation within Paratanytarsus dated to the mid-Eocene around 43 Ma; and (2) an unexpected but fully supported diversification in Pontomyia Edwards plus Yaetanytarsus Sasa dated to around 47 Ma, with separation of Pontomyia from Yaetanytarsus around 40 Ma. Crown Pontomyia, represented by three species, was estimated to have diverged around 19 Ma, whereas the crown radiation of Yaetanytarsus, with 12 sampled species, dates to the mid-Eocene. In a comprehensive global review we concisely document new synonymies and new combinations revealed by the study. The evolutionary timing estimate provides insights into the frequency of marine transitions and diversifications in the Chironomidae in association with dynamic oceanic changes during the Oligocene and Miocene.     

Energetic consequences of repeated and prolonged dehydration in the Antarctic midge, Belgica antarctica

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 24 h dehydration at 75% RH followed by 24 h 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.     

Regulation of foraging trips and costs of incubation shifts in the Antarctic petrel (Thalassoica antarctica)

In species where incubation is shared by both parents, the mate'sability to fast on the nest may constrain the time availablefor foraging. The decision to return to the nest should thereforebe a compromise between an animal's own foraging success andits mate's ability to fast on the nest. To examine how the bodyconditions of incubating Antarctic petrels, Thalassoica antarctica,influence both the length of foraging trips and incubation shifts,we experimentally handicapped females by increasing their flightcosts during a foraging trip by adding lead weights to theirlegs. Handicapped females spent more time at sea and had lowerbody conditions at arrival to the colony than controls, and,moreover, females in poor body condition at arrival to the colonyspent generally more time at sea than those with higher bodycondition. The prolonged time period spent at sea by handicappedfemales was associated with higher desertion rates than amongcontrols. The time the incubating mates fasted increased withtheir body condition at arrival to the colony, suggesting thata high body condition of the incubating bird may reduce theprobability of nest desertion. Accordingly, our results suggestthat the time spent foraging is adjusted to the body conditionsof both the foraging and incubating mate.     

Limnophyes (Diptera: Chironomidae) from northwestern Russia

The nominal family group taxa of the Ephemeroptera are listed along with their authors, dates of publication and bibliographic references.     

Afrotropical montane midges (Diptera, Chironomidae, Diamesa)

The tribe Diamesini is a characteristic element of the chironomid fauna of the Holarctic alpine and montane areas. It is represented in the E African mountains by three species: Diamesa kenyae Freeman, Diamesa ruwenzoriensis Freeman and Diamesa freemani sp. nov. The adults of the three species, the pupa and larva of D. freemani , and the larva of D. kenyae , are all described in detail. Several morphological features indicate that the Afrotropical Diamesa are derived from a relatively plesiomorphic lineage of the genus. Other characters indicate possible sister-species occurring in the Alps, the Caucasus mountains and the Himalaya. Three lines of evidence suggest a northern origin of the Afrotropical Diamesa: the relatively young age of the E African mountain peaks, the proposed phylogeny of the genus, and the present distribution of the tribe Diamesini.     

Qiniella,a new orthoclad genus from China (Diptera: Chironomidae)

Quiniella lii gen. nov., sp. nov. from China is describedas male imago. The genus share bare eyes, wing and squama and extended costa with most members of the Parakiefferiella group sensu S\sgmaelig;ther (1983), and the absence of a scutaltubercle, hump or microtrichial tuft and strongly developedvirga with Krenosmittia Thienemann & Krüger, but isamong other distinguished by a unique trifid gonostylus. This revised version was published online in July 2006 with corrections to the Cover Date.     

Rapid cold-hardening in larvae of the Antarctic midge Belgica antarctica: cellular cold-sensing and a role for calcium

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 degrees C significantly increased organismal freezing tolerance at both -15 degrees C and -20 degrees C. Likewise, RCH enhanced cell survival of fat body, Malpighian tubules, and midgut tissue of larvae frozen at -20 degrees 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 (MTT)-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 degrees C and those frozen at -5 degrees 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.     

A New Hairy-Winged Pseudorthocladius (Diptera: Chironomidae) from Luxemburg

Pseudorthocladius cristagus sp. n. is described as male imago. It differs from all other members of the genus Pseudorthocladius v. d. Wulp except P. pilosipennis Brundin by having setae on the wing membrane. It can be separated from P. pilosipennis by having a gonostylus with a prominent crista dorsalis and an outer corner or heel. The species was collected with emergence traps from a helocrene spring in northern Luxemburg.