Control of Diapause by Acidic pH and Ammonium Accumulation in the Hemolymph of Antarctic Copepods

Life-cycles of polar herbivorous copepods are characterised by seasonal/ontogenetic vertical migrations and diapause to survive periods of food shortage during the long winter season. However, the triggers of vertical migration and diapause are still far from being understood. In this study, we test the hypothesis that acidic pH and the accumulation of ammonium (NH₄ ⁺) in the hemolymph contribute to the control of diapause in certain Antarctic copepod species. In a recent study, it was already hypothesized that the replacement of heavy ions by ammonium is necessary for diapausing copepods to achieve neutral buoyancy at overwintering depth. The current article extends the hypothesis of ammonium-aided buoyancy by highlighting recent findings of low pH values in the hemolymph of diapausing copepods with elevated ammonium concentrations. Since ammonia (NH₃) is toxic to most organisms, a low hemolymph pH is required to maintain ammonium in the less toxic ionized form (NH₄ ⁺). Recognizing that low pH values are a relevant factor reducing metabolic rate in other marine invertebrates, the low pH values found in overwintering copepods might not only be a precondition for ammonium accumulation, but in addition, it may insure metabolic depression throughout diapause.     

Aspects of the study of the life cycles of Antarctic copepods

Different approaches to the study of life cycle strategies of Antarctic copepods are described in an attempt to shed new light on our present knowledge. To date, most studies were carried out on abundance, horizontal and vertical distribution and stage composition during different seasons and in various regions. Hence, the seasonal pictures had to be compiled from different years and sampling regions. The physiological method includes measurements on e.g. egg production, feeding, respiration and excretion rates, C:N and O:N ratios, lipid and protein contents. However, both physiological and biochemical data are still rare. Results of field observations are given in this paper for investigations conducted within the last 15 years in the eastern Weddell Sea, while data of physiological parameters are based on a broader geographical region. In the eastern Weddell Sea, eight copepod species account for about 95% of copepod abundance and for more than 80% of copepod biomass. Within the calanoids, the small species Microcalanus pygmaeus dominates by numbers with 66%, while the large species Calanoides acutus and Calanus propinquus comprise together 52% of the biomass. Species abundance is lowest in winter and highest in summer/autumn, however, seasonal changes in the abundance of M. pygmaeus are small and this species occurs in similar quantities throughout the year. All copepod species show a distinct seasonal vertical distribution pattern and they occur in upper water layers in summer, in contrast to the other seasons. However, the depth layers of maximum concentration differ between species. The ontogenetic vertical migration is most pronounced in C. acutus and relatively weak in C. propinquus. The age structure also shows seasonal differences with the youngest population observed in summer for C. acutus, C. propinquus, Ctenocalanus citer or autumn for Metridia gerlachei, whereas the M. pygmaeus population is oldest during summer. The youngest copepodite stage and the males are not always present in C. acutus and C. propinquus. In contrast, all developmental stages and both sexes occur throughout the year in M. gerlachei, M. pygmaeus and C. citer. Gonad maturation in the dominant calanoid species proceeds well before the onset of phytoplankton production in the eastern Weddell Sea. However, the highest portion of females with ripe gonads and hence highest egg production rates coincide with the productive period in spring and summer. In autumn, ovaries of the three larger species C. acutus, C. propinquus and M. gerlacheiare all spent. In contrast, the percentage of ripe females of the two smaller species, C. citer and M. pygmaeus, stays high in autumn. Egg production rates are highly variable within one region and species. Many copepods accumulate large depots of lipid, mainly wax esters. In contrast, five species (C. propinquus, C. simillimus, Euchirella rostromagna, Stephos longipes and Paralabidocera antarctica) almost exclusively synthesise triacylglycerols and not wax esters. The lipid content exhibits distinct seasonal patterns, and is highest in autumn. A seasonal difference is also obvious in metabolic activities with lowest rates during the dark season. The adaptation to the pronounced seasonality in the Southern Ocean differs greatly between copepod species, and most Antarctic copepods stay active during the dark season. Calanoides acutus seems to be the only true diapause species. Calculations of summer developmental rates and winter mortality rates of the large species C. acutus and C. propinquus suggest that both species have a 1-year life cycle with few females overwintering and probably spawning a second time. In contrast, a 2-year life cycle is more likely in R. gigas. However, life cycle durations of all species studied are still uncertain and regional differences are very probable.     

Unexpected Positive Buoyancy in Deep Sea Sharks,Hexanchus griseus,and a Echinorhinus cookei

We do not expect non air-breathing aquatic animals to exhibit positive buoyancy. Sharks, for example, rely on oil-filled livers instead of gas-filled swim bladders to increase their buoyancy, but are nonetheless ubiquitously regarded as either negatively or neutrally buoyant. Deep-sea sharks have particularly large, oil-filled livers, and are believed to be neutrally buoyant in their natural habitat, but this has never been confirmed. To empirically determine the buoyancy status of two species of deep-sea sharks (bluntnose sixgill sharks, Hexanchus griseus, and a prickly shark, Echinorhinus cookei) in their natural habitat, we used accelerometer-magnetometer data loggers to measure their swimming performance. Both species of deep-sea sharks showed similar diel vertical migrations: they swam at depths of 200–300 m at night and deeper than 500 m during the day. Ambient water temperature was around 15°C at 200–300 m but below 7°C at depths greater than 500 m. During vertical movements, all deep-sea sharks showed higher swimming efforts during descent than ascent to maintain a given swimming speed, and were able to glide uphill for extended periods (several minutes), indicating that these deep-sea sharks are in fact positively buoyant in their natural habitats. This positive buoyancy may adaptive for stealthy hunting (i.e. upward gliding to surprise prey from underneath) or may facilitate evening upward migrations when muscle temperatures are coolest, and swimming most sluggish, after spending the day in deep, cold water. Positive buoyancy could potentially be widespread in fish conducting daily vertical migration in deep-sea habitats.     

The occurrence of Rhincalanus gigas,Calanoides acutus,and Calanus propinquus (Copepoda: Calanoida) in late May in the area of the Antarctic Peninsula

Summary The Antarctic copepod species Rhincalanus gigas, Calanoides acutus and Calanus propinquus were studied in the area of the Antarctic Peninsula in May 1986. Research was focussed on vertical distribution and stage composition of the populations. Rhincalanus gigas occurred in greatest densities in the upper layers of the water column, and copepodite stages CI and CII and nauplii dominated the population. Gut content analyses suggest that R. gigas was actively feeding. Copepodite stage CV dominated the Calanoides acutus population. At two deep basin stations (water depth>1000 m) the C. acutus population occurred below 500 m, whereas at shallower stations the majority was found above 300 m. Most specimens had empty guts. Calanus propinquus occurred in low densities, mainly in the upper water layers, and copepodite stage CV dominated. Most individuals of stage V had food in their guts. Our results suggest that C. acutus had ceased feeding and was overwintering in a resting stage (diapause), while C. propinquus and R. gigas were still active, the latter species having finished a late autumn spawning.     

Diapause of Cyclops vicinus (Uljanin) in Lake Søbygård: Indication of a Risk-spreading Strategy

Cyclops vicinus is reported to enter summer diapause triggered by day length in order to survive food scarcity and fish predation. Development ceases and the fourth or fifth copepodid stages persist in the sediment for several weeks. In Lake Søbygård, however, a small eutrophic lake in Denmark, C. vicinus is found in the sediment as well as in the open water throughout the year. We performed laboratory experiments to elucidate the life cycle strategy of this population. In order to find the factors that induce diapause we tested the influence of food composition and light regime on the proportion of copepods entering diapause. Further we examined the diapause pattern of the offspring from diapausing and non-diapausing parents to see whether the co-occurrence of diapausing and non-diapausing copepods results from genetic differentiation within the population. In all experiments some of the copepods developed directly into adults, while others remained at the fourth or fifth copepodid stage and displayed diapause features. The proportion of copepods that developed directly into adults was influenced by food supply. Fewer individuals entered diapause when they received a mixed diet of algae and ciliates or algae, ciliates and seston compared with a pure algal diet. The response to light was different than described in literature: about 30% of the copepods entered diapause under dark conditions, and the diapause frequency was not higher when copepods had been exposed to continuous light. The offspring from diapausing parents showed no higher tendency to enter diapause than the offspring of non-diapausing parents, indicating no genetic differentiation. We conclude that diapause in C. vicinus represents a risk-spreading strategy, modified by food. The simultaneous production of diapausing and non-diapausing offspring ensures survival under harsh conditions; the influence of the food supply on the proportion of individuals entering diapause may adjust the population to the actual environmental condition. This flexible life cycle strategy might contribute to the exclusive dominance of C. vicinus in Lake Søbygård.     

Lipid dynamics and feeding of dominant Antarctic calanoid copepods in the eastern Weddell Sea in December

Lipid content, fatty acid composition, and feeding activity of the dominant Antarctic copepods, Calanoides acutus, Calanus propinquus, and Metridia gerlachei, were studied at a quasi-permanent station in the eastern Weddell Sea in December 2003. During 3 weeks of the spring phytoplankton development, total lipid levels of females and copepodite stages V (CVs) of C. acutus were almost doubled. Meanwhile, only a slight increase in total lipid content occurred in M. gerlachei, and no clear trend was observed in lipids of C. propinquus females. The pronounced increase of lipids in C. acutus was due to an accumulation of wax esters. The proportion of wax esters in the lipids of M. gerlachei was clearly lower, while triacylglycerols played a more important role. In C. propinquus, triacylglycerols were the only neutral lipid class. There were no pronounced changes in the feeding activity of M. gerlachei, whereas the feeding activity of C. acutus had rapidly increased with the development of the phytoplankton bloom in December, which explains its rapid lipid accumulation. The combination of gut content and fatty acid trophic marker analyses showed that C. acutus was feeding predominantly on diatoms. The typical diatom fatty acid marker, 16:1(n-7), slightly decreased and the tracer for flagellates, 18:4(n-3), increased in females and CVs of C. acutus. This shift indicates the time, when the significance of flagellates started to increase. The three copepod species exhibited different patterns of lipid accumulation in relation to their trophic niches and different duration of their active phases. The investigations filled a crucial data gap in the seasonal lipid dynamics of dominant calanoid copepods in the Weddell Sea in December and support earlier hypotheses on their energetic adaptations and life cycle strategies.     

Feeding patterns of dominant Antarctic copepods: an interplay of diapause,selectivity, and availability of food

Gut contents and feeding activity of five dominant Antarctic copepods (Calanus propinquus, Calanoides acutus, Rhincalanus gigas, Metridia gerlachei and Microcalanus pygmaeus) were studied from samples collected during several cruises of the RV Polarstern to the eastern Weddell Sea. In summer, feeding activity, estimated as percentage of copepods with food in the guts, was high in all the species, and diatoms dominated all gut contents. In winter, C. acutus was trophically inactive, and C. propinquus and R. gigas considerably decreased their feeding activity, while a decrease in feeding of M. gerlachei and M. pygmaeus was less pronounced. Unidentified mass dominated gut contents in winter, supplemented by phytoplankton and protozoans. Prior to the spring bloom, feeding activity of C. acutus was low, with unidentified food predominating, while carnivory was important in actively feeding C. propinquus. Rhincalanus gigas tended to be more carnivorous than C. acutus, however with less feeding activity than C. propinquus. Seasonal changes in feeding patterns are discussed.     

The effect of the receding ice edge on the condition of copepods in the northwestern Weddell Sea: results from biochemical assays

We compared six biochemical measures of nutritional condition: citrate synthase activity (CS), malate and lactate dehydrogenase activity (MDH and LDH), RNA:DNA ratio, and percent body protein and lipid. Adult females of five species of calanoid copepod (Calanoides acutus, Calanus propinquus, Metridia gerlachei, Rhincalanus gigas and Paraeuchaeta antarctica) were collected in the marginal ice zone of the northwestern Weddell Sea at the time of the annual phytoplankton bloom that occurs in association with the receding ice edge during austral spring. Three zones within the marginal ice zone were sampled: heavy-ice-cover pre-bloom, ice-edge bloom and low-ice-cover post-bloom. Lipid generally increased greatly from ice-covered to open water zones, and its importance in the life of polar copepods cannot be overstated. Increases in protein from ice-covered to open water were also observed, but were of less significance. Each species exhibited significant changes in at least one enzyme activity level. Citrate synthase activity in C. acutus, C. propinquus and R. gigas, all herbivores, increased between pre- and post-bloom stations. C. propinquus and M. gerlachei, which feed during winter, had large increases in LDH activity between pre- and post-bloom stations. Rhincalanus gigas and P. antarctica, the two largest species studied, showed variations in MDH activity, with peak enzyme activity occurring in post-bloom stations. RNA:DNA ratio did not change in any species. The effects of size, shipboard handling and freezer storage were easily corrected statistically, and did not alter any conclusions. The patterns observed in copepod nutrition at the Antarctic ice edge were consistent with existing models of life history for each species. The observations reported here, in conjunction with previously reported data, suggested that measurement of metabolic enzyme activity, especially in concert with lipid, enables estimation of nutritional condition in adult copepods. Additional studies comparing metabolic activity and ecology of common species should yield more information on the ecology of rarer species.     

Effects of the ice-edge bloom and season on the metabolism of copepods in the Weddell Sea,Antarctica

The metabolic responses of several species of Antarctic copepods to primary productivity and changes between seasons were investigated. To examine the influence of the spring ice-edge bloom on the metabolism of copepods, oxygen consumption rates were determined on specimens from three zones of widely different ice coverage and chlorophyll biomass: pack ice (pre-bloom), ice edge (bloom) and open water (post-bloom). Summer metabolic rates were compared with published winter rates. Field work was done in the Weddell Sea in the region of 60 °S, 36°W in late November and December 1993. Oxygen consumption rates were determined by placing individuals in syringe respirometers and monitoring the oxygen partial pressure for 10–20 hours. Higher metabolic rates were observed in the primarily herbivorous copepods, Calanoides acutus, Rhincalanus gigas and Calanus propinquus in regions of higher primary production: ice edge and open water. The carnivorous Paraeuchaeta antarctica showed a similar pattern. The omnivorous copepods Metridia gerlachei and Gaetanus tenuispinus showed no changes in metabolism between zones. Data on routine rates of copepods from the winter were available for C. propinquus and P. antarctica. In P. antarctica, rates were higher in the summer. Calanus propinquus showed a higher metabolic rate in the summer than in the winter, but the difference was not significant at the 0.05 level. It was concluded that copepods near the ice zone in the ice zone in the Antarctic rely on the spring ice-edge bloom for growth and completion of their life cycle.     

The zooplankton community in the vicinity of the ice edge,western Weddell Sea,March 1986

Summary The zooplankton community in the vicinity of the ice edge in the west central Weddell Sea was investigated in the late austral summer (March 1986). Sampling was done with two ships operating concurrently, one in the pack ice and the other in the adjcent open sea. Metazoan microzooplankton (<1 mm) was most abundant in the epipelagic zone. It consisted mostly of copepod nauplii and copepods of the genera Oithona, Oncaea, Ctenocalanus and Microcalanus. While species composition was similar in both areas, vertical patterns differed in that the microzooplankton had sparse populations in the upper 50 m under the ice. This may have been related to water temperature which in the upper 50 m under the ice was more than 1°C cooler than in the open sea. Zooplankton in the 1–20 mm size range was dominated by the calanoid copepods Metridia gerlachei, Calanus propinquus and Calanoides acutus which constituted half the biomass in the upper 1000 m. Their populations had highest densities in the upper 150 m, though much of the C. acutus population resided below 300 m. Metridia gerlachei and C. propinquus underwent diel vertical migrations in both areas whereas C. acutus did not migrate. Species diversity in the epipelagic zone was moderate and the fauna was characterized by species typical of the oceanic east wind drift. Diversity increased with depth and was due primarily to the appearance of circumpolar mesopelagic copepods in Weddell Warm Deep Water. Biomass of 1–20 mm zooplankton in the 0–1000 m zone was low (1.1–1.3 gDWm^-2) compared to other Southern Ocean areas investigated with comparable methods. It is suggested that this is related to Weddell circulation patterns and the resulting low annual primary production in the central Weddell Sea.     

Distribution of six major copepod species around South Georgia in early summer

Summary An intensive net sampling survey was conducted around the island of South Georgia during November/December 1981. The distribution and copepodite stage structure of the dominant copepods Calanoides acutus, Calanus simillimus, C. propinquus, Rhincalanus gigas, Metridia lucens and Metridia gerlachei were compared. The herbivorous species had completed their spring vertical migration and their summer generations were developing during the survey. At every station, Calanoides acutus was noticeably more advanced than Rhincalanus gigas in its reproductive cycle. The species were also more advanced in their development in the SE oceanic part of the survey area than in the NW. However, copepod development rates are rapid at this time of year, and this apparent regional difference may be due mainly to temporal variation during the four weeks of the survey. A truly regional variation in timing of reproduction was found when development was compared between shelf and oceanic waters. Spawning of Rhincalanus gigas and particularly of Calanoides acutus was later over the shelf. The age structure of the epi-mesopelagic metridinids also differed between shelf and oceanic waters, but no age differences were found for either Calanus simillimus or C. propinquus, both of which live and spawn higher in the water column than the other species. The presence of the South Georgia shelf also limited the overall abundance of the two deeper living metridinids. However, the length of time between spawning (and production of large numbers of early copepodids) and sampling had the largest influence on observed species abundance within the survey area.     

Population differences in the timing of diapause: a test of hypotheses

Summary The reproductive phenology of the freshwater copepod Diaptomus sanguineus differs markedly between populations residing in two Rhode Island ponds. In a permanent pond the population switches abruptly from making subitaneous (immediately hatching) eggs to diapausing eggs at the end of March each year. In contrast, a temporary pond population switches egg types in May, returns to production of subitaneous eggs in June, and concludes the reproductive season by making diapausing eggs in July. An ESS model suggests that the pattern of diapause expected of a copepod population is a function of annual variation in the onset of harsh conditions (catastrophe date). When variation is relatively low, the superior strategy is for diapause to begin a constant period before the mean catastrophe date. When variation is high, females should make first subitaneous eggs and then diapausing eggs irrespective of the expected catastrophe date. With discrete generations, such a population would alternate between egg types. In the permanent pond, variation of catastrophe date the spring onset of planktivory by sunfish is low, whereas in the temporary pond variation of the catastrophe (pond drying) is high. The model predicts well the phenology of the two copepod populations.In the research reported here, we tested the hypothesis that copepods from the permanent pond, which switch to diapause at the same time every year, are cued by the environment to begin diapause (i.e. by photoperiod, temperature, or both), whereas those from the temporary pond make both egg types regardless of environmental conditions. In opposition to our hypothesis, experimental results indicate that diapause in both populations is cued by the environment. The distinct reproductive phenologies documented in the two populations apparently result from the copepods responding to different environmental cues, rather than one being responsive to the environment while the other is not.     

The Insect Growth Regulator Pyriproxyfen Terminates Egg Diapause in the Asian Tiger Mosquito,Aedes albopictus

The Asian tiger mosquito, Aedes albopictus, is a highly invasive mosquito species that transmits chikungunya and dengue. This species overwinters as diapausing eggs in temperate climates. Early diapause termination may be a beneficial strategy for winter mosquito control; however, a mechanism to terminate the diapause process using chemicals is not known. We tested the hypothesis that a hormonal imbalance caused by the administration of juvenile hormone analog would terminate egg diapause in A. albopictus. We tested the insect growth regulator pyriproxyfen on all developmental stages to identify a susceptible stage for diapause termination. We found that pyriproxyfen treatment of mosquito eggs terminated embryonic diapause. The highest rates of diapause termination were recorded in newly deposited (78.9%) and fully embryonated (74.7%) eggs at 0.1 and 1 ppm, respectively. Hatching was completed earlier in newly deposited eggs (25–30 days) compared to fully embryonated eggs (71–80 days). The combined mortality from premature diapause termination and ovicidal activity was 98.2% in newly deposited and >98.9% in fully embryonated eggs at 1 ppm. The control diapause eggs did not hatch under diapausing conditions. Pyriproxyfen exposure to larvae, pupae and adults did not prevent the females from ovipositing diapausing eggs. There was no effect of pyriproxyfen on diapausing egg embryonic developmental time. We also observed mortality in diapausing eggs laid by females exposed to pyriproxyfen immediately after blood feeding. There was no mortality in eggs laid by females that survived larval and pupal exposures. In conclusion, diapausing eggs were the more susceptible to pyriproxyfen diapause termination compared to other life stages. This is the first report of diapause termination in A. albopictus with a juvenile hormone analog. We believe our findings will be useful in developing a new control strategy against overwintering mosquito populations.     

How are the vertical migrations of copepods controlled?

Using Calanus finmarchicus (Gunnerus) as a model organism, a hypothesis is suggested to explain the diel and seasonal vertical migrations of herbivorous copepods in boreal and polar waters. The hypothesis is based on the following assumptions. Hungry copepods are assumed to react to food smell by increased swimming. High lipid content is assumed to turn the copepods upside down. Light avoidance is assumed to operate solely while the copepods are satiated. The following three major peaks in downflux of phytoplankton remains are assumed to reach 1000 m depth or more: pre- and post-spring bloom peaks and the autumn increase. A minor “afternoon peak” in short-range downflux of phytoplankton is also assumed to exist. The assumptions are used to explain the following main traits in copepod migrations. The afternoon increase in downflux of phytoplankton material induces upward swimming of hungry copepods. If satiated, light avoidance brings them down again at dawn. The late stages of many species of copepods accumulate large amounts of lipids and if the above assumptions are valid, they will be turned upside down and swim down if activated. During midsummer, the downflux does not reach deep water and the copepods are assumed to spend some time in midwater until they moult. Copepods moulting from stage V into female adults use up to half of their lipids to produce eggs, which are more anteriorly located. This is assumed to turn their bodies back into an upright position and the copepods are assumed to swim up to the surface again when they smell sinking phytoplankton remains. Fat copepods are assumed to follow the downflux of phytoplankton material down to diapause depths, especially at the end of the spring bloom and in autumn. It is assumed that enough lipids are used up during the diapause to turn the copepods into head-up position again. The smell of fast-sinking fecal pellets containing prebloom phytoplankton is assumed to bring the copepods up from diapause again in late winter. The probable implications for the survival of cod larvae are discussed.     

Population differences in the timing of diapause: adaptation in a spatially heterogeneous environment

Summary Populations of the planktonic copepod, Diaptomus sanguineus, live in permanent and temporary freshwater ponds in Rhode Island. All ponds in which they occur become uninhabitable at some time during the year, but the nature and timing of the harsh period varies both spatially and temporally. Females produce discrete clutches either of subitaneous eggs which hatch immediately or of diapausing eggs which hatch the following season. The two egg types show distinct chorion morphologies under transmission electron microscopy. In permanent ponds the copepods start making diapausing eggs in March, one month before rising water temperatures induce planktivorous sunfish to become active. In temporary ponds diapausing eggs are produced, in a complex pattern from May to July, before the water disappears in late summer or early fall.We investigated the spatial scale at which D. sanguineus is adapted to this complex environment. In a reciprocal transfer experiment between temporary and permanent bodies of water, female copepods placed in new ponds made subies of water, female copepods placed in new ponds made subitaneous and diapausing eggs in the same sequence as control females retained in their home ponds. The copepod populations enter diapause at times appropriate for the local habitat conditions they experience, but inappropriate for other, nearby ponds. Transplanted females were unable to sense a change in pond type or to adjust egg production accordingly. We conclude that D. sanguineus populations are adapted to the specific conditions of isolated ponds rather than to a broader geographical region containing several pond types.     

The life cycle of Cyclops vicinus in Lake Søbygård: new aspects derived from sediment analyses

Cyclops vicinus is the only copepod species in the pelagic zone of Lake Søbygård and can be found there all year round. We studied the population dynamics of this copepod over a one-year period. In contrast to earlier studies we included the copepods in the pelagic zone as well as the copepods resting in the sediment. Cyclops vicinuswas found not only in the open water, but also in the sediment throughout the year. From the fluctuations of the abundances in both habitats we suggest that the life cycle is more complex than known so far: different diapause pattern appear within the population and the induction of diapause is different than in other populations. We assume that these features contribute to the predominance of C. vicinusin Lake Søbygård.     

Finding copepod footprints: a protocol for molecular identification of diapausing eggs in lake sediments

Even though calanoid copepods produce diapausing eggs that stay alive in lake sediments, these eggs have rarely been used paleolimnologically, as they lack diagnostic morphological features. In this study, we developed a method to identify copepod diapausing eggs in Japan as a clue toward reconstructing past plankton populations. We first determined a 28S ribosomal DNA (rDNA) (i.e., nc28S) regional sequence library (240 bp) of various calanoid copepod species using ethanol-fixed plankton samples collected from across the Japanese archipelago. Then we applied the UltraSHOT method to extract DNA from an individual diapausing egg. Finally, the nc28S region of diapausing eggs collected from various lakes was sequenced and compared with the regional library for species identification. In total, 21 haplotypes of the nc28S region were recovered from planktonic samples of 11 Japanese freshwater calanoid copepod species. Despite the short length of this region, no identical haplotypes were shared among the species analyzed, including the Acanthodiaptomus pacificus complex treated as a species. Even different lineages of A. pacificus could be separated. These results indicate that the nc28S region can be used as a barcode in Japan. A total of 112 diapausing eggs collected from various lakes and ponds was processed, and the nc28S region of each was successfully sequenced. All of these egg sequences matched one or the other of the nc28S haplotypes in the regional library mentioned above. The set of protocols we applied (i.e., preparing a comprehensive regional sequence library and sequencing egg DNA) is thus useful for involving copepod diapausing eggs in paleolimnological studies in lakes. The nc28S region treated in this study has a strong potential to uncover the paleodiversity of copepods, at least in Japan.     

The environmental regulation of adult diapause in Drosophila littoralis

Drosophila littoralis overwinters in the adult stage in a reproductive diapause. During the warm season there are one or two generations in Finland. The diapause appears to be a prolongation of the post-eclosion immaturity of young females. The termination of diapause is controlled by a combination of adequate temperature and sufficiently long photophases. The diapausing status of females is ascertained by inspecting the developmental stage of their ovaries. In laboratory experiments the maturity of ovaries is not closely correlated with the receptivity of females.     

Effects of acclimation and diapause on the thermal tolerance of the two-spotted spider mite Tetranychus urticae

The two-spotted spider mite, Tetranychus urticae, is a worldwide pest species that overwinters as diapausing females. Cold hardening is presumed to start during diapause development to ensure the successful overwintering of this species. To address this hypothesis, we compared cold tolerance between non-diapausing and diapausing females. We measured supercooling point (SCP) and survival to acute cold stress by exposing the mites at a range of sub-zero temperatures (from −4 to −28 °C for 2 h). The mean SCPs of non-diapausing and diapausing females were −19.6±0.5 and −24.7±0.3 °C respectively, and freezing killed the mites. Diapausing females were significantly more cold tolerant than non-diapausing ones, with LT₅₀ of −19.7 and −13.3 °C, respectively. Further, we also examined the effects of cold acclimation (10 d at 0 or 5 °C) in non-diapausing and diapausing females. Our findings indicated that diapause decreased SCP significantly, while cold acclimation had no effect on the SCP except for non-diapausing females that were acclimated at 5 °C. Acclimation at 5 °C enhanced survival to acute cold stress in diapausing and non-diapausing females, with LT₅₀ of −22.0 and −17.1 °C, respectively. Altogether, our results indicate that T. urticae is a chill tolerant species, and that diapause and cold acclimation elevate cold hardiness in this species.