Chihiro Katagiri

Radiation tolerance in the tardigrade Milnesium tardigradum

Purpose: Tardigrades are known to survive high doses of ionizing radiation. However, there have been no reports about radiation effects in tardigrades under culture conditions. In this study, we investigated tolerance of the tardigrade, Milnesium tardigradum, against gamma-rays and heavy ions by determining short-term or long-term survival, and reproductive ability after irradiation. Materials and methods: Hydrated and anhydrobiotic animals were exposed to gamma-rays (1000 – 7000 Gy) or heavy ions (1000 – 8000 Gy) to evaluate short-term survival at 2, 24 and 48 h post-irradiation. Long-term survival and reproduction were observed up to 31 days after irradiation with gamma-rays (1000 – 4000 Gy). Results: At 48 h after irradiation, median lethal doses were 5000 Gy (gamma-rays) and 6200 Gy (heavy ions) in hydrated animals, and 4400 Gy (gamma-rays) and 5200 Gy (heavy ions) in anhydrobiotic ones. Gamma-irradiation shortened average life span in a dose-dependent manner both in hydrated and anhydrobiotic groups. No irradiated animals laid eggs with one exception in which a hydrated animal irradiated with 2000 Gy of gamma-rays laid 3 eggs, and those eggs failed to hatch, whereas eggs produced by non-irradiated animals hatched successfully. Conclusion: M. tardigradum survives high doses of ionizing radiation in both hydrated and anhydrobiotic states, but irradiation with >1000 Gy makes them sterile.

Establishment of a Rearing System of the Extremotolerant Tardigrade Ramazzottius varieornatus: A New Model Animal for Astrobiology

Studies on the ability of multicellular organisms to tolerate specific environmental extremes are relatively rare compared to those of unicellular microorganisms in extreme environments. Tardigrades are extremotolerant animals that can enter an ametabolic dry state called anhydrobiosis and have high tolerance to a variety of extreme environmental conditions, particularly while in anhydrobiosis. Although tardigrades have been expected to be a potential model animal for astrobiological studies due to their excellent anhydrobiotic and extremotolerant abilities, few studies of tolerance with cultured tardigrades have been reported, possibly due to the absence of a model species that can be easily maintained under rearing conditions. We report the successful rearing of the herbivorous tardigrade, Ramazzottius varieornatus, by supplying the green alga Chlorella vulgaris as food. The life span was 35 +/- 16.4 d, deposited eggs required 5.7 +/- 1.1 d to hatch, and animals began to deposit eggs 9 d after hatching. The reared individuals of this species had an anhydrobiotic capacity throughout their life cycle in egg, juvenile, and adult stages. Furthermore, the reared adults in an anhydrobiotic state were tolerant of temperatures of 90 degrees C and -196 degrees C, and exposure to 99.8% acetonitrile or irradiation with 4000 Gy (4)He ions. Based on their life history traits and tolerance to extreme stresses, R. varieornatus may be a suitable model for astrobiological studies of multicellular organisms.

Sexual isolation and cuticular hydrocarbons in Drosophila elegans

In Drosophila elegans, partial sexual isolation has developed between the brown and black morphs, which are distributed allopatrically. The present study aims to understand how they discriminate between potential mates. Mating experiments show that the females of the two morphs differ in sexual signal(s) and the males discriminate using these differences. Body colouration is not used as a sexual cue in this species. Between the females of the two morphs, a large difference was observed in the percentages of 7-pentacosene and 9-pentacosene on the cuticle. Genetical analysis using recombinant inbred lines supported the possibility that the concentration of these pentacosenes plays a role in mate discrimination of these two morphs. However, males did not respond to killed females at all, suggesting that cuticular hydrocarbons of females are not the only cue for the induction of male courtship behaviour. It may be that unknown signals or substances are essential to induce male courtship and pentacosenes modulate the attractiveness of females, positively in the black morph and negatively in the brown morph. Drosophila elegans F1 offspring had intermediate characteristics in mate discrimination and hydrocarbon composition between the parental brown and black morph strains. The number of loci responsible for the differences in the concentration of pentacosenes and the male and female components in the mate recognition between these two morphs is suggested to be more than one.

Prominent immunogenicity of monosialosyl galactosylgloboside, carrying a stage-specific embryonic antigen-4 (SSEA-4) epitope in the ACHN human renal tubular cell line-a simple method for producing monoclonal antibodies against detergent-insoluble microdomains/raft.

The binding of Shiga toxin (Stx) to Gb3Cer† in detergent-insoluble microdomains (DIM)/raft of the ACHN human renal tubular cell line causes the temporal activation of the Src-family kinase Yes [1]. As a strategy for examining signaling mechanisms in DIM/raft, monoclonal antibodies (MAbs) are reliable tools for characterizing the constituent molecules in these microdomains. Thus, we employed DIM/raft suspensions of ACHN cells as an immunogen to develop MAbs. Simply subcutaneous injections of ACHN DIM/raft could elevate the serum titer after several boosts. The first screening was performed using dot-blot immunostaining with culture supernatants on a polyvinylidene difluoride (PVDF) membrane, on which DIM/raft or their chloroform/methanol (C/M) (2:1, v/v) extracts were dot-blotted. The next screening was performed by flowcytometric analysis of ACHN cells treated with or without a permeabilizing reagent. Many of the clones (21/31 clones=68%) thus obtained were also found to recognize to lipid fractions of the DIM/raft. Strikingly, all of the 21 clones that reacted to the lipid fraction were found to recognize monosialosyl galactosylgloboside (MSGG) or GL7, which carries the SSEA-4 epitope. Using DIM/raft as immunogens may enable us to easily obtain MAbs for glycolipids.

Causal involvement of mammalian‐type cryptochrome in the circadian cuticle deposition rhythm in the bean bug Riptortus pedestris

Mammalian‐type CRYPTOCHROME (CRY‐m) is considered to be a core repressive component of the circadian clock in various insect species. However, this role is based only on the molecular function of CRY‐m in cultured cells and it therefore remains unknown whether CRY‐m is indispensable for governing physiological rhythms at the organismal level. In the present study, we show that RNA interference (RNAi) targeting of cry‐m in the bean bug Riptortus pedestris disrupts the circadian clock governing the cuticle deposition rhythm and results in the generation of a single cuticle layer. Furthermore, period expression was induced in cry‐m RNAi insects. These results verified that CRY‐m functions as a negative regulator in the circadian clock that generates physiological rhythm at the organismal level.

Heat coma as an indicator of resistance to environmental stress and its relationship to ocean dynamics in the sea skaters, Halobates （Heteroptera： Gerridae）

Abstract  The tolerance to temperature increase was tested for Halobates individuals collected during two cruises in the western tropical Pacific Ocean (MR‐06‐05‐Leg 3, December 21, 2006–January 12, 2007, 0°N‐8°N; KH‐06‐02‐Leg 5, August 18–31, 2006, 12°N–17°N). High temperature coma experiments were conducted on adults and 5th instar larvae. On average, H. sericeus (distributed in the wide latitude zone of 5°N–40°N), H. germanus (distributed in the moderate latitude zone of 0°N–35°N) and H. micans (distributed mainly in the lower latitudes around the equator) were on average paralyzed at 35.6°C (SD: 0.89), 32.9°C (SD: 2.17) and 31.6°C (SD: 2.60), respectively (P= 0.035). According to the current dynamics during the cruise, the colony of H. sericeus at one station (5°N 137°E) may have been transferred from the northern area of 14°N by three currents (North Equatorial Current, Mindanao Current and North Equatorial Counter Current) to the area of 5°N 138°E. Extremely high heat resistance was shown by the adults of H. germanus in the sea area around the equator. Dynamic current and air movements in this area around the equator, that is a “warm seawater pool”, could be hypothesized to be related to the high resistance to heat shown in this study.

Fatty acids of membrane phospholipids in Drosophila melanogaster lines showing rapid and slow recovery from chill coma.

We investigated the fatty acid compositions of phospholipids in Drosophila melanogaster lines showing rapid (CR), intermediate (CTL), or slow (CS) recovery from chill coma, which were established by artificial selection or by free recombination without selection. Compared to CTL, CS showed a low composition of dienoic acids and a small number of double bonds in the fatty acids. The ratio of unsaturated fatty acids and saturated fatty acids (UFAs/SFAs) was significantly lower in CS than in CTL. CR had higher monoenoic acid composition and lower dienoic acid composition than CTL. In addition, the amount of SFAs was lower and therefore the UFAs/SFAs ratio considerably higher in CR than in CTL. These changes in phospholipid fatty acids probably contributed to losing and maintaining the homeoviscosity of the cellular membranes in CS and CR, respectively, at low temperature and therefore produced their distinct phenotypes in recovery from chill coma.

Cuticular lipids and odors induce sex-specific behaviors in the male cricket Gryllus bimaculatus

Male crickets display sex-specific (e.g., mating and agonistic) behaviors towards conspecific individuals. One of the key signals for these behaviors is the chemical substance on the cricket body surface. In the present study, we analyzed female and male cuticular substances in behavioral assays. Antennal contact stimulation using female forewings elicited a mating behavior in males, while that using male forewings elicited an agonistic behavior in males. Thin-layer-chromatographic and other techniques analysis showed that saturated cuticular lipids were present in both female and male cuticles and that unsaturated lipids were present only in the male cuticle. Filter papers soaked with saturated or unsaturated cuticular lipids were applied to antennae of male crickets. Males showed mating behavior in response to stimulation with saturated lipids from both females and males but showed avoidance behavior in response to stimulation with male unsaturated lipids. Because cuticular lipids did not induce agonistic behavior in males, we collected odors from male crickets and found that these odors induced agonistic behavior in males. Therefore, we concluded that the key signals for mating, avoidance and agonistic behaviors of male crickets are comprised of at least three different components, saturated and unsaturated cuticular lipids and male odors, respectively.

Localization of trehalase in the haemolymph of the American cockroach, Periplaneta americana

Abstract Trehalase activity in cockroach haemolymph was determined by different methods using 14 C-labelled trehalose and gas chromatography. It was found that no such activity was detected in the serum, whereas significant activity was found in the haemocytes.

Tolerance of Anhydrobiotic Eggs of the Tardigrade Ramazzottius varieornatus to Extreme Environments

Tardigrades are tiny (less than 1 mm in length) invertebrate animals that have the potential to survive travel to other planets because of their tolerance to extreme environmental conditions by means of a dry ametabolic state called anhydrobiosis. While the tolerance of adult tardigrades to extreme environments has been reported, there are few reports on the tolerance of their eggs. We examined the ability of hydrated and anhydrobiotic eggs of the tardigrade Ramazzottius varieornatus to hatch after exposure to ionizing irradiation (helium ions), extremely low and high temperatures, and high vacuum. We previously reported that there was a similar pattern of tolerance against ionizing radiation between hydrated and anhydrobiotic adults. In contrast, anhydrobiotic eggs (50% lethal dose; 1690 Gy) were substantially more radioresistant than hydrated ones (50% lethal dose; 509 Gy). Anhydrobiotic eggs also have a broader temperature resistance compared with hydrated ones. Over 70% of the anhydrobiotic eggs treated at either -196°C or +50°C hatched successfully, but all the hydrated eggs failed to hatch. After exposure to high-vacuum conditions (5.3×10(-4) Pa to 6.2×10(-5) Pa), the hatchability of the anhydrobiotic eggs was comparable to that of untreated control eggs.