Cuticular Lipid Topology on Insect Body Surfaces Studied by Synchrotron Radiation FTIR ATR Microspectroscopy.

The cuticular lipid covering the integument of insects is exposed to the environment and involved in a variety of functions offered by insect body surfaces, ranging from protection against the environment, such as the control of water transpiration, the reduction of abrasive damage, and the prevention of pathogen intrusion, to the communication between insects from intraspecific to interspecific interactions. In comparison with the importance of their physiological functions, there is remarkably little information on the structure and physical property of cuticular lipids on insect body surfaces. The lipid layer on the outer exoskeleton is very thin, estimated on the order of 0.01-1 µm or less, and this has led to a lack of practical methodologies for detailed structural analyses. To fill this devoid, we have exploited the characteristics of Fourier transform infrared (FTIR) attenuated total reflection (ATR) spectroscopy, which allows us to conduct a chemical analysis on insect body surfaces and also to investigate depth-dependent structural changes. We have applied a combination of FTIR ATR microspectroscopy with IR radiation provided by a synchrotron facility to obtain in situ two-dimensional (2D) information of the cuticular lipid layer on the surface of the integument. The 2D FTIR spectra measured on the two-spotted cricket and the American cockroach show that the IR bands due to the cuticular lipid, such as CH2 symmetric and antisymmetric stretch, νa(CH2) and νs(CH2), change in intensity significantly, depending on the location of measurements. As if to keep pace with this, the bands of the amide group for the underlying cuticular layer also change in intensity significantly, although the changes are in the opposite direction; as the lipid bands increase in intensity, the amide band decreases, and vice versa. The ATR spectral analysis, which takes into account the characteristics of the evanescent wave, points out that the lipid layer would vary tens of times in the range of 0.01-1 µm significantly. The νa(CH2) and νs(CH2) bands show frequency shifts, which correlate to some extent with their intensity changes, suggesting that the drastic uneven distribution of the cuticular lipid would be related to the solid-liquid phase separation and also the coarsening of the solid phase domains. The formation of such topological features, significant heterogeneity in the lipid layer thickness, and solid-liquid phase ratios would be accompanied by the partitioning of lipid components according to molecular structures and physicochemical properties. Considering that each lipid component in insect body surface lipids is involved in various physiological roles, the segregation of lipid components during the formation of such heterogeneous structures is thought to have a significant impact on the functionality of the insect body surface.

First In Situ X-ray Scattering Measurements of Insect Body Surface Lipids: American Cockroach.

In situ X-ray scattering measurements of insect body surface lipids were successfully attempted by using a synchrotron X-ray source. The temperature-dependent structural changes of the cuticular hydrocarbons covering the forewing of an American cockroach were able to be followed, which showed that the majority of the hydrocarbons were in a liquid state even far below the critical temperature of water transpiration through the body surface. The results clearly demonstrated that synchrotron radiation X-ray scattering has the potential to obtain the detailed information about the intact lipid structure and physical properties on insect body surfaces.

ATR FTIR Spectroscopic Study on Insect Body Surface Lipids Rich in Methylene-Interrupted Diene.

To protect themselves, insects cover their bodies with what is called cuticular lipid. The cuticular lipid of an American cockroach has a unique lipid content; the most abundant is a cis-alkadiene, cis, cis-6,9-heptacosadiene, amounting to about 70%, which is followed by a branched alkane 3-methylpentacosane. In order to clarify the structural features of the unique lipid composition below the critical temperature, the cuticular lipid was studied by Fourier transform infrared (FTIR) spectroscopy in combination with an attenuated total reflection (ATR) sampling technique. The infrared spectra measured on an extracted lipid sample at 20 °C suggested that the lipid keeps an appreciable level of conformational and lateral packing regularity, in spite of a high cis-unsaturated lipid content, and also a highly disordered condition around the methyl terminals and cis-olefin groups. The CH2 scissoring and the CH2 rocking regions showed the characteristics of the O⊥ subcell. The same characteristics were observed also by in situ measurements on a forewing of the American cockroach. Combining the spectral features of these bands and the physicochemical properties of each component, it can be inferred that saturated lipids form highly ordered domains within the liquid containing the cis, cis-diene as the main component. For comparison, the cuticular lipid of a male cricket, which consisted of many different hydrocarbons, including 15% of unsaturated hydrocarbons, showed a lower regularity both in the conformation and in the lateral packing of hydrocarbon chains. These results imply that not only the degree of cis-unsaturation but also the chemical structure diversity of hydrocarbons are the important factors to determine the physicochemical properties of cuticular lipid.

Seasonal Change in Distribution and Heat Coma Temperature of Oceanic Skaters, Halobates (Insecta, Heteroptera: Gerridae).

A series of studies were conducted during two cruises between Tokyo and Honolulu in September 2010 and from February to March 2012. The aims of the studies were to (1) compare the distribution of three species of Halobates oceanic skaters, H. germanus, H. micans, and H. sericeus, with respect to their temperature limits; (2) identify the lower temperature limit of H. sericeus, the species that displays the widest distribution range (40°N⁻35°S) latitude; and (3) test the hypothesis that H. sericeus can change their temperature tolerance to adapt to seasonal changes in sea surface temperatures. The heat coma temperature (HCT) was measured during the two cruises and the values were compared between the two populations of H. sericeus. The species collected in September 2010 were H. germanus, H. micans, and H. sericeus. H. sericeus was dominant, occupying more than 90% of the collecting sites. H. germanus and H. micans were collected in the northern and western part of the cruise track (29⁻34°N, 141⁻151°E), and not in the southern and eastern part. The population density of these two species was 9000⁻150,000/km² in the first cruise, which took place in summer. On the other hand, H. sericeus was collected throughout the cruise track during that cruise. The population density of H. sericeus was relatively high, at 4000⁻310,000/km², in the southern and eastern part of the cruise track (19⁻29°N, 152°E⁻165°W). In February and March 2012, only H. sericeus was collected at a density of 17,000⁻80,000/km² and only in the eastern and southern part, at 25°â»28°N, 169°E⁻178°W. No Halobates oceanic skaters were found in the western or northern part (30°N and further north, 159°E and further west) during that cruise. The lower limit for the inhabitation of sea surface temperatures appeared to be 27.8 °C or slightly lower for H. germanus and H. micans, but was 22.1 °C or slightly lower for H. sericeus. H. sericeus specimens, mostly adults, that had been collected during the two cruises were used in heat coma experiments. Summer specimens showed significantly higher heat coma temperatures (HCTs) than the winter specimens. This difference in HCTs may be the result of relatively long term temperature acclimation in the summer or winter for the adults that inhabit the temperate and subtropical areas along the cruise tracks between Tokyo and Honolulu in the Pacific Ocean. This temperature plasticity of H. sericeus may be related to the wider latitude area inhabited by this species (main range: 40°N⁻25°S).

The First Finding of Six Instars of Larvae in Heteroptera and the Negative Correlation between Precipitation and Number of Individuals Collected in Sea Skaters of Halobates (Heteroptera: Gerridae).

This study, conducted during a scientific cruise, MR15-04, aims, first, to examine species and larval/adult components of Halobates (Heteroptera: Gerridae) inhabiting the tropical Indian Ocean of 4°00' S-7°00' S, 101°00' E-103°00' E and, second, to examine the correlative relationship between precipitation just before collection and the number of sea skaters collected in November and December 2015. Near Sumatra (50 km south-west), larvae and adults of four species of Halobates (Halobates germanes White, 1883; Halobates micans Eschscholtz, 1822; Halobates princeps White, 1883; undescribed species: Halobates sp.) were collected. Adults of an undescribed species had about a 5 mm long body in a gourd-like shape. One male adult specimen of H. princeps was collected. Body length, body width, and head width was measured in all specimens of Halobates. Six larval stages were detected in all three species of sea skaters as the first finding for Heteropteran insects. There was a negative correlation between amount of precipitation for 19 h before collection and the number of Halobates individuals collected by the neuston net. Death or (positive or passive) sinking by sea skaters could be due to occasional rain fall on the sea surface.

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.

Apolipophorin-III expression and low density lipophorin formation during embryonic development of the silkworm, Bombyx mori.

We examined the expression of apolipophorin-III (apoLp-III) during embryonic development of the silkworm Bombyx mori. ApoLp-III mRNA was first expressed 24h after oviposition, which corresponds to the time of germ band formation. The amount of apoLp-III in the eggs increased from day 2, peaked on day 4, and then gradually decreased until hatching (on day 9.5). ApoLp-III was apparently synthesized during early embryogenesis, as radioactive amino acids were incorporated into newly synthesized apoLp-III in three-day-old eggs. Moreover, radioactive apoLp-III was found only in the embryo and not in the extraembryonic tissue. KBr density gradient ultracentrifugation of egg homogenates showed that apoLp-III was associated with low-density lipophorin (LDLp). These results suggest that LDLp is required for the delivery of lipids for organogenesis during embryogenesis.

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.

Direct observation of fat crystallization in a living fly by X-ray diffraction: fat crystallization does not cause the fly's instantaneous death, but ice formation does.

A cool-temperate fly, Drosophila triauraria, stores fat, triacylglycerol (TAG), primarily in the fat storage organ, the fat body, and then diapauses to pass the winter in imago stage. TAG crystallization and ice formation taking place in a living fly by lowering temperatures were studied, in order to clarify the relationship between crystallizations and the fly's death at lower temperatures. X-ray diffraction, a direct non-invasive method, was used to detect the liquid-to-crystal transformations of TAG and water. During cooling, TAG crystallization preceded ice formation. It was also found that ice formation causes the fly to die instantaneously whereas the TAG crystallization does not.

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.

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.

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.

A simple method to discriminate diapause from non-diapause pupae in large and small white butterflies, Pieris brassicae and P. rapae crucivora.

Differences in colour and shape have been used to discriminate diapause pupae from non-diapause pupae in butterflies. In the present study, we describe a simple discrimination method based on differences in the specific gravity of diapause and non-diapause pupae of large and small white butterflies, Pieris brassicae and Pieris rapae crucivora. When put into water, diapause pupae sink to the bottom (specific gravity is above 1.0), whereas non-diapause pupae float on the surface (specific gravity is below 1.0). Nuclear magnetic resonance microimaging revealed that this difference in specific gravity is due to a difference in the volume of an internal cavity located between the thorax and the abdomen in the pupae. The cavity appears quite early in development. We also examined the cavity in pupae of the unrelated swallowtail butterfly, Papilio xuthus, and found a similar cavity. However, in this species, the cavity is very small and, as a consequence, non-diapause as well as diapause pupae sink in water.

The basis for colorless hemolymph and cocoons in the Y-gene recessive Bombyx mori mutants: a defect in the cellular uptake of carotenoids.

Bombyx mori is an excellent model for the study of carotenoid-binding proteins (CBP). In previous papers, we identified and molecularly characterized a CBP from the Y-gene dominant mutants. In the present study, we attempted to correlate and establish lipid metabolism and distribution in these mutants. When [3H]-triolein was fed to the mutants, typical patterns of uptake of labeled fatty acids from midgut to hemolymph and subsequent delivery to fat body and silk glands were obtained in all mutants. Further analysis of lipid and carotenoid profiles revealed that the yellow coloration in the hemolymph associated with lipophorin is not attributed to a difference in lipophorin concentrations among the mutants, nor to its lipid composition, but rather to its carotenoid content. Lipophorin of the Y+I mutant exhibited the highest concentration of total carotenoids of 55.8 microg/mg lipophorin compared to 3.1 microg/mg in the +Y+I mutant, 1.2 microg/mg in the YI mutant and 0.5 microg/mg in the +YI mutant. Characteristic retention time in HPLC of the different classes of carotenoids of lipophorin identified the presence of lutein as the major chromophore (62-77%), followed by beta-carotenes (22-38%). Although lutein and beta-carotene content of mutants' lipophorin differed significantly, the ratio of lutein to beta-carotene of 3:1 was not different among mutants. Similarly, lipid compositions of mutant silk glands were not significantly different, but carotenoid contents were. The significantly high concentration of lutein in the Y+I mutant silk gland represented more than 160-fold increase compared to +Y+I mutant (p<0.001). In this report, we conclude that lipid metabolism in the mutants is not defected and that the molecular basis for colorless hemolymph and cocoons is a defect in the cellular uptake of lutein associated with the Y-gene recessive mutants.

Epicuticular wax of large and small white butterflies, Pieris brassicae and P. rapae crucivora: qualitative and quantitative comparison between diapause and non-diapause pupae.

We compared the quantity and quality of the epicuticular wax of diapause and non-diapause pupae in two closely related Pieris species, P. brassicae and P. rapae crucivora. Main components of their epicuticular wax were identified as hydrocarbons. In P. brassicae, more than 95% of hydrocarbons were saturated regardless of whether the pupae were in diapause or not. In P. rapae crucivora, 93% of hydrocarbons were saturated in non-diapause pupae whereas in diapause pupae 41% were saturated and 59% unsaturated. From measurements of body surface area by nuclear magnetic resonance microimaging, we calculated the average thickness of the wax layer. The thickness in diapause and non-diapause pupae of P. brassicae was 800 and 160 nm, respectively. In P. rapae crucivora, the thickness was 195 nm in diapause and 11 nm in non-diapause. This is the first report to clarify the compositional difference in epicuticular wax between diapause and non-diapause pupae.

Two-dimensional diffraction study of ice crystallisation in polymer gels.

Ice crystallisation in crosslinked dextran (Sephadex) gels was studied by the method of two-dimensional X-ray diffraction (XRD) in combination with the simultaneous measurement of differential scanning calorimetry (DSC). With a Sephadex G25 gel where an exotherm due to ice crystallisation is observed in the DSC rewarming trace, it was indicated by the XRD pattern that small ice crystals less than approximately 10 microns in diameter are readily formed during freezing, and that the endothermic trend prior to the exotherm is not due to the glass transition but due to the melting of the small ice crystals. Moreover, the diffraction pattern observed with frozen Sephadex gels depended on the density of crosslink indicating that ice crystals of different size and dimension are formed in the gels.

Mate discrimination and cuticular hydrocarbons in Drosophila elegans and D. gunungcola.

In Drosophila elegans, two morphs are known, the brown-morph occurring from southern China to Indonesia and the black-morph occurring in the Ryukyu Islands, Japan, and Taiwan, and brown-morph populations at high altitudes in Indonesia at least sympatrically occur with a sibling species D. gunungcola. Sexual isolation has developed between the two morphs of D. elegans to some extent; females of the black-morph have a higher concentration of pentacosenes on cuticle than those of the brown-morph, and males of these morphs discriminate between the females based on this difference. In this study, it was examined whether sympatry and allopatry with D. gunungcola have resulted in the differentiation of mate recognition system in D. elegans. No significant difference was observed in the degree of mate discrimination between a sympatric pair of D. elegans and D. gunungcola and their allopatric pairs. Thus, no support was obtained for the above notion. Males of the brown- and black-morphs of D. elegans discriminate between females of own morphs and D. gunungcola. However, brown-morph males did not discriminate between females of the black-morph and D. gunungcola, and also black-morph males did not discriminate between females of the brown-morph and D. gunungcola. This may be attributed to that D. gunungcola females retained an intermediate level of pentacosenes between brown- and black-morph females.

Purification and characterization of biliverdin-binding vitellogenin from the hemolymph of the common cutworm, Spodoptera litura.

Biliverdin-binding vitellogenin (Vg) was purified from adult female hemolymph of the common cutworm, Spodoptera litura, by using gel filtration and ion exchange chromatographies. The molecular mass of the protein was 490 kDa and it was composed of two 188-kDa subunits. Three internal amino acid sequences obtained by digestion of the protein with lysylendopeptidase showed high similarity to those of Bombyx mori Vg, supporting the purified blue protein to be vitellogenin. latroscan analyses demonstrated the presence of biliverdin in Vg that occupied 2.4% of total lipid components. Among the lipids of Vg (9.5 micrograms total lipids per 100 micrograms protein), diacylglycerol was the most predominant, followed by phospholipid, hydrocarbons, and then triacylglycerol, while in biliverdin-binding proteins (BPs) purified from larval hemolymph (3.1 micrograms total lipids per 100 micrograms protein), phospholipid was the most abundant lipid followed by diacylglycerol; hydrocarbons and triacylglycerol were minor components. Vg was first detected in the hemolymph of female pupae one day before eclosion, but injection of 5 micrograms of methoprene into a 3-day-old pupa induced Vg in the hemolymph 4 days earlier than in the control. Methoprene also induced a faster decline in BP-A and BP-B titers in the hemolymph with a corresponding increase of the Vg titer. These results suggest that juvenile hormone (JH) induces not only vitellogenesis but also the uptake of these proteins by stimulating the metamorphosis of fat body during the pupal stage.