Die rolle der Carotinoide und des Gallenfarbstoffs bei der Farbmodifikation der Puppen von Pieris brassicae

The carotenoids and the bile pigment in larvae and pupae of Pieris brassicae were analysed. Their rôle in the morphological colour adaptation of the pupae was studied by quantitative measurements.The carotenoids are β-carotene, lutein mono-ester, free lutein, and zeaxanthin. Metabolized carotenoids were not found. There are no differences between pupae showing different grades of melanization in the quality of the carotenoids, or in the total amounts, or in the relative portions of each carotenoid fraction. However, the carotenoid content of the integument alone is twofold in the light pupae as compared to dark ones. The integumental carotenoids are deposited mainly in the epidermis. β-Carotene, lutein, and zeaxanthin are selectively absorbed by the larvae from the diet. β-Carotene and lutein ester are localized mainly in the fat body, whereas lutein is predominant in the haemolymph and in the integument.The pupal bile pigment is protobiliverdin-IXγ (pterobilin), which is also known to be the larval pigment. The bile pigment is synthesized mainly during the last larval instar up to the pharate pupal stage. In the pupae the bile pigment content is related to the melanization: pupae exposed to the same light conditions contain less bile pigment the more melanized they are (negative correlation). On the whole there is a strong enhancement by blue light of the bile pigment content besides the known stimulation of melanization (positive correlation). But within such a sample the negative correlation between the amounts of bile pigment and melanin is maintained.     

The role of carotenoids in the "golden glance" of danaid pupae (Insecta: Lepidoptera)

Silver replaces the golden areas of danaid pupae if the larvae are reared on a carotenoid-free diet. These pigments are therefore responsible for the yellow colouring of the "golden glance". Lutein is the dominant carotenoid present in pupal exuviae. In response to light cues, these pigments can be re-routed into deeper tissues, thus pupae of Euploea core become silver instead of gold if reared against a dull white background.     

Water and carbohydrate levels at different developmental stages and dynamics in hibernating pupae of Pieris melete (Lepidoptera: Pieridae)

For insight into the physiological indicators of diapause in Pieris melete, water and carbohydrate (glycogen and trehalose) levels were measured under both natural and laboratory conditions. The highest water content (3.71–3.79 mg/mg dry weight) was found in larvae and developing pupae, which was substantially higher than in diapausing pupae (2.59 mg/mg dry weight). Water content was almost stable during diapause, except for individuals approaching diapause termination (3.43–3.58 mg/mg dry weight). The total carbohydrate level was significantly higher in pre‐pupae (47.41 μg/mg) compared to larvae (22.80 μg/mg) and developing pupae (21.48 μg/mg). The highest level of trehalose was detected in winter diapausing pupae, and no trehalose was found in larvae or developing pupae. Levels of glycogen were highest in pre‐pupae and lowest in diapausing pupae. Levels of total carbohydrate decreased as diapause proceeded, and no significant changes were found in trehalose levels for diapausing pupae under natural conditions or treated for 60–90 days at 5°C. Pupae treated at 20°C for 60–90 days had significantly lower levels of trehalose than those treated for 30 days. Glycogen content was relatively stable at 5°C, but increased after treatment under natural conditions and 20°C for more than 60 days. These results suggest that the dynamics of water and carbohydrate levels are potential physiological diapause indicators, which show metabolic differences between trehalose and glycogen during diapause development.     

Pupal polymorphism in the butterfly Danaus chrysippus (L.): environmental, seasonal and genetic influences

The pupae of the tropical butterfly Danaus chrysippus are either green or pink the switch being operated by a ‘greening’ hormone produced in the larval head. Both environmental and genetic cues are involved in controlling the endocrine mechanism. The environmental factors identified are of two distinct kinds: proximate factors influence pupal colour after the larva has selected its pupation site, whereas ultimate factors are effective at an earlier stage, either prompting choice of pupation site by the larva or priming pupation physiology in a particular direction. Genetic factors preadapt the larva to form a pupa which will be cryptic in the normal or average conditions, climatic or biogeographical, anticipated in its environment. The proximate factors demonstrated are background colour, darkness, light quality (wavelength) and humidity. There is some evidence that substrate texture may also be relevant. Ultimate factors are temperature, humidity and species of larval foodplant. Two closely linked gene loci which govern the phenotype of adult morphs and races either have a pleiotropic effect on pupa colour or are closely linked with other genes which do so. Moreover, the two loci interact epistatically with respect to their pupation effects. Factors producing predominantly green pupae are plant substrates, yellow background, darkness, yellow light, high humidity, high temperature, the b allele at the B locus when homozygous and, on non-plant substrates, the C allele at the C locus. High frequencies of pink pupae result on non-plant substrates, red backgrounds, in blue light, low humidity, low temperatures and in B- and cc genotypes. The C locus alleles, C and c, interact epistatically with the B alleles, their effect on choice of pupation site being determined by linkage phase. Of the two foodplants tested, Calotropis produced a high frequency of green pupae and Tylophora of pinks. The seasonal cycling of rainfall, temperature, availability or condition of foodplant, and gene frequencies are all correlated with oscillations in the frequencies of green and pink pupae. Though genotype influences pupa colour, all genotypes are capable of forming pupae of both colours. The variation can therefore be attributed to an environmental polyphenism superimposed upon a genetic polymorphism. The hormone producing green pupae emanates from the head during the prepupal period. Denied hormonal influence, the pupa is pink. Pupal colour is judged to be aposematic at close range and cryptic at distance.     

Regulatory mechanisms in phenotypic plasticity of diapause and nondiapause pupal colouration of the swallowtail butterfly Papilio machaon

Nondiapause pupae of Papilio machaon L. exhibit pupal colour diphenism comprising green–yellow and brown–white types. To understand the regulatory mechanism underlying the control of pupal colouration in P. machaon, the effect of environmental cues on diapause and nondiapause pupal colouration is investigated. When larvae reared under short‐day and long‐day conditions are allowed to pupate in sites with a smooth surface and a yellow background colour, all diapause pupae exhibit a brown–white type and 89.5% of nondiapause pupae exhibit a green–yellow type, respectively. With rough‐surface pupation sites, all diapause pupae exhibit brown–white and intermediate types, whereas a large proportion of nondiapause pupae exhibit brown–white and intermediate types, although some exhibit a green–yellow type. When extracts prepared from the head‐thoracic and thoracic‐abdominal regions of larval central nervous systems are injected into the ligated abdomens of P. machaon short‐day pharate pupae, all recipients exhibit a brown–white colouration. Furthermore, when each extract is injected into the ligated abdomen of Papilio xuthus L. short‐day pharate pupae with orange‐pupa‐inducing factor activity, recipients injected with the head‐thoracic extract exhibit the brown type, whereas those injected with the thoracic‐abdominal extract exhibit an orange colour. The results indicate that the response to the environmental cues of pupation site in P. machaon changes according to the photoperiodic conditions experienced during larval stages, and that at least two hormonal factors producing brown–white pupae are located in the larval central nervous system, with the secretion of these factors being regulated by the recognition of environmental cues in long‐day larvae.     

寄主植物对棉铃虫越冬蛹抗寒能力的影响

通过对滞育蛹过冷却点的测定 ,初步明确寄主对棉铃虫Helicoverpaarmigera的越冬抗寒性有影响 ,幼虫取食棉花时产生的滞育蛹的过冷却点低于取食玉米的滞育蛹的过冷却点 ,即前者的抗寒能力高于后者 ;但用Bt棉喂养后的棉铃虫滞育蛹抗寒能力下降     

Cold hardiness of the fly pupal parasitoid Nasonia vitripennis is enhanced by its host Sarcophaga crassipalpis

Supercooling points (SCPs) and low temperature survival were determined for diapausing and nondiapausing larvae of the ectoparasitoid Nasonia vitripennis. Neither nondiapausing nor diapausing larvae could survive tissue freezing. The SCP profiles were nearly identical for nondiapause-destined (-27 degrees C) and diapausing larvae (-25 degrees C), but these values were not indicative of the lower limits of tolerance in either type of larvae: larvae were killed by chilling at temperatures well above the SCP. Diapausing larvae could withstand low temperature exposures 3-8 times longer than their nondiapausing counterparts. Low temperature survival was enhanced in diapausing and nondiapausing larvae by their encasement within the puparium of the host flesh fly, SARCOPHAGA CRASSIPALPIS: the LT(50)s determined for nondiapausing and diapausing larvae enclosed by fly puparia were 2-3 times higher than values calculated for larvae removed from the puparia. Additional low temperature protection was gained through acquisition of host cryoprotectants during larval feeding: nondiapausing parasitoid larvae that fed on diapausing flesh fly pupae with high levels of glycerol were able to survive exposure to a subzero temperature 4-9 times longer than wasps reared on nondiapausing fly pupae that contained lower quantities of glycerol. Alanine may also contribute to the cold hardiness of N. vitripennis, as evidenced by the fact that larvae feeding on diapausing fly pupae both contained higher concentrations of alanine and exhibited greater cold hardiness. The results thus demonstrate that several critical features of cold hardiness in the wasp are derived from biochemical and physical attributes of the host.     

Host plant exodus and larval wandering behaviour in a butterfly: diapause generation larvae wander for longer periods than do non‐diapause generation larvae

1. Prior to pupation, lepidopteran larvae enter a wandering phase lasting up to 30 h before choosing a pupation site. Because stillness is important for concealment, this behaviour calls for an adaptive explanation. 2. The explanation most likely relates to the need to find a suitable pupation substrate, especially in terms of shelter from predation, and given that many predators and parasitoids use host plants as prey‐location cues, mortality probably decreases with distance from the host plant. Hence, remaining on the host includes a long‐term risk, while moving away from the host introduces an increased risk during locomotion. 3. Bivoltine species that overwinter in the pupal stage produce two kinds of pupae; non‐diapausing pupae from which adults emerge after 1–2 weeks, or diapausing pupae that overwinter with adults emerging after 8–10 months. 4. Given the hypothesis of distance‐from‐host‐plant‐related predation, this should select for phenotypic plasticity with larvae in the diapausing generation having a longer wandering phase than larvae under direct development, if there is a trade‐off between mortality during the wandering phase and accumulated mortality during winter. 5. Here this prediction is tested by studying the duration of the wandering period in larvae of the partially bivoltine swallowtail butterfly, Papilio machaon, under both developmental pathways. 6. The results are in agreement with the predictions and show that the larval wandering phase is approximately twice as long under diapause development. The authors suggest that the longer duration of the wandering phase in the diapause generation is a general phenomenon in Lepidoptera.     

Interactions of environmental factors influencing pupal coloration in swallowtail butterfly Papilio xuthus

The swallowtail butterfly Papilio xuthus Linné [Lepidoptera: Papilionidae] exhibits pupal protective color polyphenism. Interactions of various environmental factors on pupal coloration were analyzed in non-diapausing individuals. Under sufficient light (200lux), most pupating larvae became green pupae when the surface of the pupation site was smooth, while they became brown when the surface was rough. Tactile signals are the positive environmental factors causing induction of the brown pupal coloration. In dark boxes, the induction of the brown pupal coloration was easily induced even on a smooth surface, suggesting that light suppresses induction of brown coloration. Different colors of pupation sites did not affect pupal coloration under sufficient light. Environmental factors received during a critical period both before girdling and after girdling affected pupal coloration. When tactile signals received from rough surfaces reach threshold levels during pupation, brown pupal coloration is determined. Larvae reared under a daily periodicity of natural light formed a girdle at midnight, subsequently, the prepupae received strong daylight the following day. Under natural light most larvae produced brown pupae on rough surfaces and green pupae on smooth surfaces.     

The major carotenoid pigments of the grain aphid, Sitobion avenae (F.) (Hemiptera: Aphididae)

The economically important grain aphid, Sitobion avenae (F.) shows colour polymorphism, with brown and green forms predominating. Colour is determined both genetically and in response to environmental factors, including nutrition. The biological significance of the colour polymorphism is unknown, although seasonal changes occur in the frequency of colour morphs in the field, whilst the brown morph may have adaptive significance in terms of hymenopterous endoparasitism. The ground colour of aphids is produced by haemolymph pigments, aphins (glucosides) and carotenoids. The latter may be under the synthetic control of intracellular endosymbiotic bacteria. In this study, the major carotenoid pigments of a brown and a green clone of S. avenae were examined using thin layer chromatography (TLC) and high-performance liquid chromatography (HPLC), and their absorbance spectra recorded. Using TLC, the brown clone produced five bands of different R_f, ranging from yellow, to orange-pink to pink in colour. In contrast, the green clone gave only a single yellow band of higher R_f than any of the bands of brown aphids. Following separation of carotenoids by HPLC, brown aphids gave seven peaks and green aphids five. Comparison of absorbance maxima with known published values for carotenoids provides strong evidence for the identification of four of the carotenoid pigments from brown aphids (RB-4, 3,4-didehydrolycopene; RB-5, torulene; RB-6; lycopene; RB-7, γ-carotene) and one from green aphids (RG-2, α-carotene). The other carotenoids remain unidentified. The biosynthesis and possible biological relevance of the various pigments of S. avenae are briefly discussed.     

Convergent evolution of neuroendocrine control of phenotypic plasticity in pupal colour in butterflies

Phenotypic plasticity in pupal colour occurs in three families of butterflies (the Nymphalidae, Papilionidae and Pieridae), typically in species whose pupation sites vary unpredictably in colour. In all species studied to date, larvae ready for pupation respond to environmental cues associated with the colour of their pupation sites and moult into cryptic light (yellow–green) or dark (brown–black) pupae. In nymphalids and pierids, pupal colour is controlled by a neuroendocrine factor, pupal melanization-reducing factor (PMRF), the release of which inhibits the melanization of the pupal cuticle resulting in light pupae. In contrast, the neuroendocrine factor controlling pupal colour in papilionid butterflies results in the production of brown pupae. PMRF was extracted from the ventral nerve chains of the peacock butterfly Inachis io (Nymphalidae) and black swallowtail butterfly Papilio polyxenes (Papilionidae). When injected into pre-pupae, the extracts resulted in yellow pupae in I. io but brown pupae in P. polyxenes. These results suggest that the same neuroendocrine factor controls the plasticity in pupal colour, but that plasticity in pupal colour in these species has evolved independently (convergently).     

Juvenile hormone and diapause in the Mediterranean corn borer,Sesamia nonagrioides

Juvenile hormone content of Sesamia nonagrioides larvae reared under different environmental conditions that induce diapause was studied using a bioassay on newly-emerged Tribolium confusum pupae. Two analytical methods were also used to measure JHIII content. Extracts from larvae that developed under different conditions of photoperiod and temperature caused different effects in T. confusum pupae. This can be related to different diapause intensities. Extracts from diapausing larvae produced a higher juvenilizing effect than the expected, considering their JHIII titer calculated by the chromatographic analysis. This indicates that hormones other than JHIII must be present in extracts of diapausing larvae of S. nonagrioides. The analytical procedures confirm that diapausing larvae have a higher JHIII titer in the haemolymph than non-diapausing larvae. This shows that JH is involved in the maintenance of diapause in this species.     

Differences in pupal cold hardiness and larval food consumption between overwintering and non‐overwintering generations of the common yellow swallowtail,Papilio machaon (Lepidoptera: Papilionidae), from the Osaka population

To clarify differences in pupal cold hardiness and larval food consumption between overwintering and non‐overwintering generations of the common yellow swallowtail, Papilio machaon, we reared larvae from the Osaka population under photoperiods of 16 h light : 8 h dark (LD 16:8) (long day) or LD 12:12 (short day) at 20°C. We examined the relationship between food consumption and weight during the final larval stadium and pupae, and measured the pupal supercooling point (SCP). Although the ratio of assimilation to consumption did not differ significantly between photoperiods, the ratio of assimilation to pupal weight differed significantly between individuals reared under long and short days. All diapausing pupae were brown, whereas 56% of non‐diapausing pupae were green with the remainder brown. The mean pupal body length (L), dorsal width (W₁) and lateral width (W₂) were larger in non‐diapausing than in diapausing pupae, and the W₁/L and W₁/W₂ ratios differed significantly between non‐diapausing and diapausing pupae. SCP was approximately –20°C and did not differ among pupae 5, 15 and 30 days after pupation under long‐day conditions. However, under short‐day conditions, mean SCP gradually decreased, stabilizing at approximately –24 to –25°C by 30 days after pupation. After freezing, some diapausing pupae emerged as adults, whereas all non‐diapausing pupae died. Both egestion and assimilation were greater under long‐day conditions. The results revealed that pupae of this papilionid exhibit seasonal polyphenism in physiological and morphological traits. Energy from food appears to be expended on increasing cold hardiness in the overwintering generation and on reproduction in the non‐overwintering generation.     

Environmental control of pupal colour in swallowtail butterflies (Lepidoptera: Papilioninae): Battus philenor (L.) and Papilio polyxenes Fabr.

Abstract. 1. Some swallowtail butterflies produce both green and brown pupae. The phenotypes result from the joint action of genotype and environment and usually make the pupae cryptic in their habitats.
2. The major environmental cues influencing pupal colour in two swallowtail species were determined to be textural and optical.
3. Differences in the usage of these kinds of cues in the two species are thought to have evolved because of major differences in the pupation habitats. P.polyxenes , which usually pupates on slender stems amidst vegetation, responds more strongly to optical cues. B.philenor , which usually pupates on exposed surfaces of tree trunks and cliffs, responds more strongly to textural cues.
4. Differences in the overall tendency to produce brown pupae ('sensitivity': Hazel, 1977) are thought to be related to the frequency of brown pupation sites utilized by these two species: high average sensitivity in philenor , which often uses brown sites, and lower average sensitivity in polyxenes , which often uses green sites.     

Effect of light intensity on colour morph formation and performance of the grain aphid Sitobion avenae F. (Homoptera: Aphididae)

The grain aphid Sitobion avenae F., one of the major pest aphids of cereals in Central Europe, exhibits colour polymorphism, even within the same clones. Although there is evidence that green and brown morphs of S. avenae contain different carotenoids, the mechanisms determining the induction of colour morphs are unknown. The common understanding is that the formation of colour morphs is controlled by light and affected by genetic and environmental factors and by host plant species. So far, there is no unequivocal evidence that light intensity, photoperiod, or a mixture of several variables are involved in the induction of S. avenae colour formation, resulting in the induction of S. avenae colour formation and carotenoid synthesis.Here we determined the effect of light intensity on the colour formation and performance of ten clones of S. avenae with experiments that controlled for the effects of host plant and genetic factors. We found that some clones remained green under all test conditions. In other clones, colour morph formation was controlled by light. The synthesis of carotenoids correlated with changes in colour formation. Host plant did not affect colour formation in the ten clones studied. Although colour of the aphid clones did not affect their performance, high light intensity increased the fecundity and fresh weight of S. avenae clones, while low light intensity stimulated the production of alatae.     

Antioxidant defense in mitochondria during diapause and postdiapause development of European corn borer (Ostrinia nubilalis, Hubn.)

Antioxidant enzymes (CAT, catalase; GPx, selenium nondependent glutathione peroxidase; GST, glutathione-S-transferase; GR, glutathione reductase; DHAR, dehydroascorbate reductase) were determined in the mitochondria of diapausing and non-diapausing larvae and pupae of both diapausing and non-diapausing larvae of the European corn borer (Ostrinia nubilalis, Hubn., Lepidoptera: Pyralidae). CAT, GST, and DHAR activity in mitochondria of diapausing larvae were reduced compared to non-diapausing larvae. Pupae of diapaused-larvae possessed lower GST, but higher DHAR activities compared to pupae of non-diapaused individuals. Comparison between larvae and pupae revealed lower GPx activity in the mitochondria of pupae. CAT activity in the mitochondria of pupae was higher compared to diapausing larvae, but lower than in non-diapausing ones. Correlation and canonical discriminant analyses revealed different antioxidant enzyme compositions for a particular stage and developmental pattern. Our results show that antioxidant enzymes have a similar role in the regulation of energetics in mitochondria as that in diapause and metamorphosis.     

Brain glycogen during non-diapausing and diapausing development of Pieris brassicae larvae and pupae

Summary

Histological and cytological localization of glycogen was studied in the brain of Pieris brassicae larvae and pupae by histochemistry and electron microscopy. The major glycogen deposits were observed in glial cells located between the cortex and the neuropile and also in perineurial cells. The concentrations of brain glycogen were measured in the larvae and the pupae during non-diapausing and diapausing development. In addition, we demonstrated that starvation reduces the density of the brain glycogen deposits as well as the concentrations of glycogen.     

Rapid colour changes in Euglena sanguinea (Euglenophyceae) caused by internal lipid globule migration

The accumulation of red pigments, frequently carotenoids, under chronic stress is a response observed in diverse kinds of eukaryotic photoautotrophs. It is thought that red pigments protect the chlorophyll located underneath by a light-shielding mechanism. However, the synthesis or degradation of carotenoids is a slow process and this response is usually only observed when the stress is maintained over long periods of time. In contrast, rapid colour changes have been reported in the euglenophyte Euglena sanguinea. Here we study the ecophysiological process behind this phenomenon through chlorophyll fluorescence, and pigment, colour and ultrastructural analyses. Reddening in E. sanguinea was due to the presence of a large amount of free and esterified astaxanthin (representing 80% of the carotenoid pool). The process was highly dynamic, shifting from green to red in 8 min (and vice-versa in 20 min). This change was not due to de novo carotenogenesis, but to the relocation of cytoplasmic lipid globules where astaxanthin accumulates. Thus, red globules were observed to migrate from the centre of the cell to peripheral locations when exposed to high light. Globule migration seems to be so efficient that other classical photoprotective mechanisms are not operative in this species. Despite the presence and operation of the diadino-diatoxanthin cycle, non-photochemical quenching was almost undetectable. Since E. sanguinea forms extensive floating colonies, reddening can be observed at a much greater scale than at the cellular level and the mechanism described here is one of the fastest and most dramatic colour changes attributable to photosynthetic organisms at cell and landscape level. In conclusion E. sanguinea shows an extremely dynamic and efficient photoprotective mechanism, based more on organelle migration than on carotenoid biosynthesis, which prevents excess light absorption by chlorophylls reducing the need for other protective processes related to energy dissipation.     

Effects of transgenic Bt cotton on overwintering characteristics and survival of Helicoverpa armigera

The effects of transgenic Bt cotton on the overwintering generation of the cotton bollworm, Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae), are unknown. We hypothesized that a Bt cotton diet may adversely affect fitness of this generation and examined fresh weight, lipids, glycogens, low-molecular-weight sugars and SCPs (supercooling points) of pupae, as well as survival of larvae, diapausing pupae and adult emergence in comparison with controls. Field and laboratory experiments showed that larvae fed on Bt cotton had a decreased pupation rate, and fewer entered diapause and emerged as adults compared with larvae fed non-Bt cotton. Furthermore, larvae fed Bt cotton had reduced pupal weight, glycogen content and trehalose levels both in diapausing and in non-diapausing pupae, and only diapausing pupae had an increased SCP compared to controls. The SCPs of diapausing pupae reared on Bt cotton were significantly higher than those reared on non-Bt cotton. The trehalose levels of diapausing pupae reared on Bt cotton were significantly lower than those of larvae reared on non-Bt cotton. Thus, these results suggest that a Bt cotton diet weakens the preparedness of cotton bollworm for overwintering and reduces survival of the overwintering generation, which will in turn reduce the density of the first generation in the following year. Effects of transgenic Bt cotton on the overwintering generation of cotton bollworm appear to have significantly contributed to the suppression of cotton bollworm observed throughout northern China in the past decade.     

Environmental factors affecting black/white coloration of the silken girdle in the swallowtail butterfly, Atrophaneura alcinous (Lepidoptera: Papilionidae)

The silken girdles of pupae of the swallowtail butterfly Atrophaneura alcinous show black and white color diphenism. Field observations revealed that all pupae observed on non-food plants and the leaves and stems of the larval food plant Aristolochia debilis were classified as a silken girdle of a black type, while a large portion of pupae pupating on the twigs and trunks of cherry trees in close proximity to A. debilis were classified as a silken girdle of a black type. Additionally, all pupae observed on the surfaces of artificial objects in areas where there are no surrounding plants or trees were classified as a silken girdle of a white type. We demonstrated the effect of day length and the texture, light, plant odor and humidity of pupation sites on the coloration of the silken girdle in A. alcinous. Regardless of long-day or short-day day length conditions, light conditions of constant light or dark, or the presence of a plant odor of A. debilis as environmental cues, all larvae placed at over 80% relative humidity (R.H.) developed into pupae with a silken girdle of a black type. However, all larvae developed into pupae with a silken girdle of a white type when R.H. was below 75%. Furthermore, when pupae with a silken girdle of a white type were transferred to conditions of 90% R.H. within 24 hr of pupation, the white color of the silken girdle changed into a black type within 24 hr of the transfer. The present data suggest that the induction of a black coloration of the silken girdle in A. alcinous requires a R.H. of approximately 80% or more as an environmental factor.