Phenology and life history of Belgica antarctica, an Antarctic midge (Diptera: Chironomidae)

ABSTRACT.

• 1 An account of the life-history with emphasis on phenology and number of instars is presented for Belgica antarctica Jacobs, the southernmost holometabolous insect.
• 2 Contrary to earlier reports, Belgica has four instars, in common with most other chironomids. Mean head capsule lengths varied between different populations but no overlap was found between discrete size classes of successive instars.
• 3 Belgica overwinters in all four instars.
• 4 Relative frequency of different instars from samples taken through the season indicates that a 2-year life-span is the commonest pattern.
• 5 Emergence of adults is relatively synchronous. Belgica exhibits protandry, which may be established at the time of pupation.

     

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.     

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.     

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.     

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.     

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.     

Wet hibernacula promote inoculative freezing and limit the potential for cryoprotective dehydration in the Antarctic midge,Belgica antarctica

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 H₂O g^?1 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.     

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.     

Onset of seasonal metabolic depression in the Antarctic midge Belgica antarctica appears to be independent of environmental cues

Seasonal progression is tracked in most animals by changes in daylength, thus allowing reliable synchrony with abundant food and favourable developmental conditions. In polar regions, daylength varies extensively, fluctuating at the highest latitudes from persistent light to persistent dark. The Antarctic midge Belgica antarctica has a narrow seasonal window in which to feed and develop, and previous work shows that this insect, despite having the elements of a circadian clock, remains continuously active when temperatures are permissive. The present study aims to clarify seasonal tracking in B. antarctica during the austral summer by monitoring oxygen consumption rates in a field population and in experimental groups exposed to shortened daylength, dehydration and chilling. Remarkably, during March, coordinated decreases in oxygen consumption are observed, ranging from 18% to 42%, in all treatment groups, indicating an anticipatory response to seasonal change regardless of the environmental cues. These results suggest that B. antarctica relies on an intrinsic mechanism to program metabolic depression at the onset of the long austral winter.     

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.     

High resistance to oxidative damage in the Antarctic midge Belgica antarctica, and developmentally linked expression of genes encoding superoxide dismutase, catalase and heat shock proteins

Intense ultraviolet radiation, coupled with frequent bouts of freezing-thawing and anoxia, have the potential to generate high levels of oxidative stress in Antarctic organisms. In this study, we examined mechanisms used by the Antarctic midge, Belgica antarctica, to counter oxidative stress. We cloned genes encoding two key antioxidant enzymes, superoxide dismutase (SOD) and catalase (Cat), and showed that SOD mRNA was expressed continuously and at very high levels in larvae, but not in adults, while Cat mRNA was expressed in both larvae and adults but at a somewhat reduced level. SOD mRNA was expressed at even higher levels in larvae that were exposed to direct sunlight. Catalase, a small heat shock protein, Hsp70 and Hsp90 mRNAs were also strongly upregulated in response to sunlight. Total antioxidant capacity of the adults was higher than that of the larvae, but levels in both stages of the midge were much higher than observed in a freeze-tolerant, temperate zone insect, the gall fly Eurosta solidaginis. Assays to measure oxidative damage (lipid peroxidation TBARS and carbonyl proteins) demonstrated that the Antarctic midge is highly resistant to oxidative stress.     

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.     

Sex ratio in relation to season and host plant quality in a monogenous stem-galling midge (Diptera: Cecidomyiidae)

Abstract.  1. The sex determination mechanism in gall midges is little understood, although it is known that the females of several species primarily or exclusively produce unisexual broods throughout their lifetime.
2. The gall midge Izeniola obesula Dorchin is a multivoltine species, inducing multi-chambered stem galls on the salt-marsh plant Suaeda monoica . Each gall contains 5–70 individuals, all being the progeny of a single female. Sampling of more than 450 galls, from which adult midges were reared, suggested that I .  obesula exhibits strict monogeny, resulting in galls that contain either all female or all male progeny.
3. Characterisation of the growth pattern of young S .  monoica shoots revealed that shoots in apical positions grew more rapidly than shoots in more basal positions. Galls that were induced on such shoots were larger and yielded more adult midges.
4. No difference in the site of gall induction was found between male and female galls, with galls of either sex being mainly induced on more rapidly growing shoots. It is concluded that I .  obesula females cannot control the sex of their progeny, and that both female-producers and male-producers strive to maximise their reproductive success by choosing the faster-growing shoots for gall induction.
5. Female galls were larger and more abundant than male galls at almost all times. The sex ratio among galls fluctuated throughout the year, ranging from 4:1 in spring to 1:1 in winter. The skewed sex ratio among galls possibly results from greater mortality rates among male galls than among female galls, due to either primary or secondary factors. Alternatively, it is possible that the number or fitness of male-producers in the population is reduced relative to female-producers.     

The swarming behaviour of the Scottish biting midge, Culicoides impunctatus (Diptera: Ceratopogonidae)

Abstract.

• 1 Swarms of Culicoides impunctatus males were examined in the field in Scotland. Most swarms were close to midge emergence/breeding grounds over a variety of vegetation, some of which clearly acted as swarm markers. Low light (0–1000 lux) and still/humid conditions favoured swarming.
• 2 Swarm size ranged between less than 10 and more than one thousand midges. The modal size was 200 individuals. The smallest swarms were usually columnar in shape and the larger swarms ovoid.
• 3 Midges behaved individually within swarms, moving in an elliptical manner characteristic of other dipterans in swarms.
• 4 Swarms were classified as either ‘persistent’ or ‘transient’ in terms of their shape, size and stability. Wind was the most influential factor in disrupting swarms.
• 5 Swarms were confirmed as mating sites for C.impunctatus.

     

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.     

Ultrastructure of the retrocerebral complex in the adult midge,chironomus riparius MG (Diptera : Chironomidae)

The organisation of the retrocerebral complex of adult Chironomus riparius Mg. (Diptera : Chironomidae) has previously been compared with that of the larva. The primary objective of the present work was to examine the ultrastructure of the components of the complex in neoimaginal male C. riparius to enable their further comparison with similar structures in other dipterans and determine whether this information supports the contention that the midge has a relatively primitive neuroendocrine system.The hypocerebral ganglion has a complex innervation, is composed of non- neurosecretory neurons and glial cells and, with the adjacent aortic wall, is the major neurohaemal site of the complex. The corpora cardiaca are not intimately associated with the ganglion. They are not apparently neurohaemal but are composed of cells containing granules with a bimodal size distribution. The corpora allata are of the pseudolymphoid type and are devoid of extracellular stromal channels. Peritracheal tissue persists into the neoimaginal midge but is apparently inactive and undergoing degeneration.The post-cerebral glands have been included in all previous accounts of the retrocerebral complex in chironomids but the present work confirms the view that they are not endocrine in nature despite their close proximity to the corpora cardiaca. Their ultrastructural appearance suggests that they may be functionally related to pericardial cells in other insects.It is concluded that the retrocerebral complex is indeed rather primitive both in its organisation and in the structure of its components.     

Sex ratio, emergence and infection rates of Heterotylenchus-exposed Musca autumnalis (Diptera: Muscidae)

Experimental infections were produced with the host-specific nematode Heterotylenchus autumnalis, which sterilizes its host, the face fly (Musca autumnalis). The high infection rate achieved (means = 82.5%) was similar for male and female flies. The pupal emergence rate was significantly lower for fly larvae exposed to the nematodes than for unexposed controls. Heterotylenchus-exposed flies had a sex ratio skewed in favor of males. This indicates that in addition to sterilization, H. autumnalis has other deleterious effects on its host. Heterotylenchus autumnalis is therefore a candidate for use as a potential biological control agent of the face fly.     

Complete mitochondrial genome of the Antarctic amphipod Gondogeneia antarctica (Crustacea, amphipod)

The complete sequence of the mitochondrial genome of the Antarctic amphipod Gondogeneia antarctica was determined to be 18,424 bp in length, and to contain 13 protein-coding genes (PCGs), 22 tRNA genes, and large (rrnL) and small (rrnS) rRNA genes. Its total A+T content is 70.1%. The G. antarctica mitogenome is the largest known among those of crustaceans, due to the existence of two relatively large intergenic non-coding sequences. The PCG arrangement of G. antarctica is identical to that of the ancestral pancrustacean ground pattern, although the tRNA arrangement differs somewhat. The complete mitogenome sequences of 68 species of pancrustacea have been added to the NCBI database, only 4 of which represent complete mitogenome sequences from amphipods. This is the first report of a mitogenome sequence of an Antarctic amphipod, and provides insights into the evolution of crustacean mitochondrial genomes, particularly in amphipods.