Diapause in Univoltine and Semivoltine Life Cycles.

Although it is generally more adaptive for insects to produce additional generations than to have longer life cycles, some insects produce one or fewer generations per year (univoltine or semivoltine life cycles, respectively). Some insects with the potential to produce multiple generations per year produce a univoltine life cycle in response to environmental conditions. Obligatory univoltine insects have a single long diapause or multiple diapauses in different seasons. Semivoltine insects have multiple diapauses in different years, a prolonged diapause for more than a year, or diapause controlled by a circannual rhythm. Diapause in these insects greatly varies among species both in the physiological mechanism and in the evolutionary background, and there is no general rule defining it. In this review, we survey the physiological control of univoltine and semivoltine insects' diapause and discuss the adaptive significance of the long life cycles. Although constraints such as slow development are sometimes responsible for these life cycles, the benefits of these life cycles can be explained by bet-hedging in many cases. We also discuss the effect of climate warming on these life cycles as a future area of research.

Functional Analysis of a Juvenile Hormone Inducible Transcription Factor, Krüppel homolog 1, in the Bean Bug, Riptortus pedestris.

Juvenile hormone (JH) has two major functions in insects, i.e., suppression of metamorphosis in the larval or nymphal stage and promotion of reproduction in the adult stage. Krüppel homolog 1 (Kr-h1), a C2H2 zinc-finger type transcription factor, is reported to act downstream of the JH receptor complex. In the present study, the function of Kr-h1 was examined in adults and nymphs of Riptortus pedestris by RNA interference (RNAi). After injection of adults with dsRNA of Kr-h1, the expression level of Kr-h1 was significantly decreased in the abdomen. Kr-h1 dsRNA-injection resulted in a lower proportion of individuals with developed ovaries, but the difference was not statistically significant. The transcript levels of cyanoprotein-α and vitellogenin-1, which are JH-inducible genes encoding yolk proteins, were not affected in the abdomen by Kr-h1 knockdown. Kr-h1 dsRNA-injection was effective for suppression of Kr-h1 expression in nymphs. Some Kr-h1 dsRNA-injected fifth (final) instar nymphs had morphological defects in the wing bud. Moreover, they had several adult morphological features, including ocelli in the head, connexivum in the abdomen, coloring of the dorsal abdomen, and genitals. The nymphs possessing adult features did not emerge as adults during 1 month. These results demonstrated that Kr-h1 is necessary for maintaining nymphal characters in R. pedestris. The function of Kr-h1 in ovarian development remains unclear in R. pedestris.

Circadian Clock Genes Regulate Temperature-Dependent Diapause Induction in Silkworm Bombyx mori.

The bivoltine strain of the domestic silkworm, Bombyx mori, exhibits a facultative diapause phenotype that is determined by maternal environmental conditions during embryonic and larval development. Although a recent study implicated a circadian clock gene period (per) in circadian rhythms and photoperiod-induced diapause, the roles of other core feedback loop genes, including timeless (tim), Clock (Clk), cycle (cyc), and cryptochrome2 (cry2), have to be clarified yet. Therefore, the aim of this study was to elucidate the roles of circadian clock genes in temperature-dependent diapause induction. To achieve this, per, tim, Clk, cyc, and cry2 knockout (KO) mutants were generated, and the percentages of diapause and non-diapause eggs were determined. The results show that per, tim, Clk, cyc, and cry2 regulated temperature-induced diapause by acting upstream of cerebral γ-aminobutyric acid (GABA)ergic and diapause hormone signaling pathways. Moreover, the temporal expression of the clock genes in wild-type (wt) silkworms was significantly different from that of thermosensitive transient receptor potential ankyrin 1 (TRPA1) KO mutants during embryonic development. Overall, the findings of this study provide target genes for regulating temperature-dependent diapause induction in silkworms.

Involvement of the Clock Gene Period in the Photoperiodism of the Silkmoth Bombyx mori.

We established a knockout strain of a clock gene, period (per), by using TALEN in a bivoltine strain (Kosetsu) of Bombyx mori (Insecta, Lepidoptera), and examined the effect of per knockout on the circadian rhythm and photoperiodism. The generated per knockout allele was considered to be null, because a new stop codon was present in the insertion allele. The wild type (Kosetsu) showed clear circadian rhythms in eclosion and hatching, whereas the per knockout strain showed arrhythmic eclosion and hatching under constant darkness. In this strain, moreover, temporal expression changes of clock genes per and timeless were disrupted. The wild type showed a clear long-day response for induction of embryonic diapause: when larvae were reared under long-day and short-day conditions at 25°C, adults produced nondiapause and diapause eggs, respectively. However, the per knockout strain lost the sensitivity to photoperiod and laid nondiapause eggs under both conditions. We conclude that per plays an important role both in circadian rhythms and in photoperiodism of B. mori, indicating the involvement of the circadian clock consisting of per in the photoperiodism.

Juvenile hormone III skipped bisepoxide is widespread in true bugs (Hemiptera: Heteroptera).

Juvenile hormone (JH) plays important roles in almost every aspect of insect development and reproduction. JHs are a group of acyclic sesquiterpenoids, and their farnesol backbone has been chemically modified to generate a homologous series of hormones in some insect lineages. JH III (methyl farnesoate, 10,11-epoxide) is the most common JH in insects, but Lepidoptera (butterflies and moths) and 'higher' Diptera (suborder: Brachycera; flies) have developed their own unique JHs. Although JH was first proposed in the hemipteran suborder Heteroptera (true bugs), the chemical identity of the heteropteran JH was only recently determined. Furthermore, recent studies revealed the presence of a novel JH, JH III skipped bisepoxide (JHSB3), in some heteropterans, but its taxonomic distribution remains largely unknown. In the present study, we investigated JHSB3 production in 31 heteropteran species, covering almost all heteropteran lineages, through ultra-performance liquid chromatography coupled with tandem mass spectrometry. We found that all of the focal species produced JHSB3, indicating that JHSB3 is widespread in heteropteran bugs and the evolutionary occurrence of JHSB3 ascends to the common ancestor of Heteroptera.

The formation of a hatching line in the serosal cuticle confers multifaceted adaptive functions on the eggshell of a cicada.

Insect eggshells must meet various demands of developing embryos. These demands sometimes conflict with each other; therefore, there are tradeoffs between eggshell properties, such as robustness and permeability. To meet these conflicting demands, particular eggshell structures have evolved in diverse insect species. Here, we report a rare eggshell structure found in the eggshell of a cicada, Cryptotympana facialis. This species has a prolonged egg period with embryonic diapause and a trait of humidity-inducible hatching, which would impose severe demands on the eggshell. We found that in eggs of this species, unlike many other insect eggs, a dedicated cleavage site, known as a hatching line, was formed not in the chorion but in the serosal cuticle. The hatching line was composed of a fine furrow accompanied by ridges on both sides. This furrow-ridge structure formed in the terminal phase of embryogenesis through the partial degradation of an initially thick and nearly flat cuticle layer. We showed that the permeability of the eggshell was low in the diapause stage, when the cuticle was thick, and increased with degradation of the serosal cuticle. We also demonstrated that the force required to cleave the eggshell was reduced after the formation of the hatching line. These results suggest that the establishment of the hatching line on the serosal cuticle enables flexible modification of eggshell properties during embryogenesis, and we predict that it is an adaptation to maximize the protective role of the shell during the long egg period while reducing the barrier to emerging nymphs at the time of hatching.

Transcriptomic changes in the pea aphid, Acyrthosiphon pisum: Effects of the seasonal timer and photoperiod.

Many insect species use photoperiod as a cue for induction of seasonal responses, including seasonal polyphenism. Although most aphid species viviparously produce parthenogenetic and sexual morphs under long and short days, respectively, a seasonal timer suppresses the sexual morph production over several successive generations during a few months following hatching of a sexually produced diapause egg. To reveal the relative influences of photoperiod and the seasonal timer on the reproductive polyphenism at the gene expression level, we performed RNA sequencing-based transcriptome analyses in the pea aphid, Acyrthosiphon pisum (Hemiptera: Aphididae). Under short days, aphids with an expired seasonal timer showed a higher expression level in hundreds of genes than those with an operative seasonal timer. In contrast, aphids with an operative seasonal timer did not show upregulation in most of these genes. Functional annotations based on gene ontology showed that histone modifications and small non-coding RNA pathways were enriched in aphids with an expired seasonal timer under short-day conditions, suggesting that these epigenetic regulations on gene expression might be involved in a mechanism of maternal switching from the parthenogenetic to sexual morph production.

Juvenile hormone III skipped bisepoxide, not its stereoisomers, as a juvenile hormone of the bean bug Riptortus pedestris.

Juvenile hormone (JH) plays a pivotal role in many aspects of insect physiology. Although its presence was first reported in a blood-sucking bug belonging to the suborder Heteroptera (true bugs), JH species in the group has long been controversial. Although some recent studies proposed a putative JH molecular species in several Heteropteran species, it is not conclusive because physicochemical analyses were insufficient in most cases. Here, we studied this issue with an ultraperformance liquid chromatography-tandem mass spectrometer (UPLC-MS/MS) equipped with C18 and chiral columns in the bean bug Riptortus pedestris (Heteroptera, Alydidae), in which the JH species has long been controversial. Although a recent study describes JHSB3 as the major JH of this species, that finding was not conclusive because its chirality has not been clarified. In the present study, we detected methyl (2R,3S,10R)-2,3;10,11-bisepoxyfarnesoate, commonly named juvenile hormone III skipped bisepoxide (JHSB3), in the culture media of the corpora cardiaca-corpus allatum (CC-CA) complex and in the hemolymph of this species by a chiral ultraperformance liquid chromatography- tandem mass spectrometer (UPLC-MS/MS). Other JHSB3 stereoisomers were not detected. Topical application of JHSB3 effectively averted diapause. These results indicate that JHSB3 is the major JH of R. pedestris. The present study further revealed that JHSB3 and its (2R,3S,10S) isomer are more potent than (2S,3R,10R) and (2S,3R,10S) isomers, which suggests that there is a significance to the configuration of the 2,3-epoxide moiety in JH action. We further found a supplemental significance to the configuration of the 10-position.

Involvement of the Clock Gene period in the Circadian Rhythm of the Silkmoth Bombyx mori.

In Lepidoptera, the roles of period ( per) and the negative feedback involving this gene in circadian rhythm are controversial. In the present study, we established a per knockout strain using TALEN in Bombyx mori, and compared eclosion and hatching rhythms between the per-knockout and wild-type strains to examine whether per is actually involved in these rhythms. The generated per knockout allele was considered null, because it encoded an extensively truncated form of PERIOD (198 aa due to a 64-bp deletion in exon 7, in contrast to 1113 aa in the wild-type protein). In this per knockout strain, circadian rhythms in eclosion and hatching were disrupted. Under LD cycles, however, a steep peak existed at 1 h after lights-on in both eclosion and hatching, and was considered to be produced by a masking effect-a direct response to light. In the per-knockout strain, temporal expression changes of per and timeless ( tim) were also lost. The expression levels of tim were continuously high, probably due to the loss of negative feedback by per and tim. In contrast, the expression levels of per were much lower in the per knockout strain than in the wild type at every time point. From these results, we concluded that per is indispensable for circadian rhythms, and we suggest that the negative feedback loop of the circadian rhythm involving per functions for the production of behavioral rhythms in B. mori.

Egg-Cracking Vibration as a Cue for Stink Bug Siblings to Synchronize Hatching.

Egg clutches of many animals hatch synchronously due to parental control [1, 2] or environmental stimulation [3, 4]. In contrast, in some animals, embryos actively synchronize their hatching timing with their siblings to facilitate adaptive behavior in sibling groups, such as mass migration [5, 6]. These embryos require synchronization cues that are detectable from eggs and indicative of when the siblings hatch, such as pre-hatching vocalizations in birds and crocodiles [7, 8]. Previous studies, using methods including artificial presentation of non-specific mechanical stimuli, demonstrated that vibrations or other mechanical forces caused by sibling movements are cues used by some turtles and insects [9-13]. However, there is no evidence about which movements of tiny embryos or hatchlings, among multiple possibilities, can generate mechanical cues actually detectable through eggs. Here, we show that embryos of the brown marmorated stink bug, Halyomorpha halys, synchronize hatching by responding to single pulsed vibrations generated when siblings crack open their eggshells. An egg-cracking vibration seems to be transmitted to distant eggs within a clutch while still maintaining its function as a cue, thus leading to the highly synchronized hatching pattern previously reported [14]. In this species, it is possible that embryos attempt to hatch with short lags after earlier-hatched siblings to avoid egg cannibalism by them [14]. The present study illustrates the diversity of social-information use by animal embryos for success in the sibling group.

Northward expansion of the bivoltine life cycle of the cricket over the last four decades.

Recent climate warming has affected some life-history traits of insects, including voltinism and body size. The magnitude of changes in these traits may differ latitudinally within a species because of the differing lengths of season available for growth. The present study aims to estimate the change in voltinism of the lawn ground cricket, Polionemobius mikado (Shiraki) (Orthoptera: Trigonidiidae), over the last four decades by comparing the body size between adults collected from a wide range of latitudes in Japan in recent years (2015-2017) and those collected four decades ago (1969-1976). The body size of adults collected in recent years showed a latitudinal saw-tooth cline, in the same way as body size did four decades ago, and the cline shifted northward over the last four decades: In 2015-2017, the body size decreased slightly with increasing latitude from 31°N to 36°N, and then increased to 40°N, and again decreased from 40°N to 44°N. Comparison of the body size between recent years and four decades ago revealed that the body size has decreased significantly at the middle latitudes (36-40°N), suggesting that the proportion of smaller bivoltine individuals there has increased over the last four decades. The sum of effective temperatures for postdiapause embryonic development at around 36°N in recent years was comparable to that at 31-35°N four decades ago, at which P. mikado populations were bivoltine. Taken together, these findings suggested that the latitudinal range suitable for the bivoltine life cycle of P. mikado has expanded northward over the last four decades because of climate warming. This is the first report that shows that a decrease in body size can be caused by climate warming via an increase in voltinism.

Effects of pars intercerebralis removal on circatidal rhythm in the mangrove cricket, Apteronemobius asahinai.

The circatidal rhythm is an endogenous rhythm corresponding to the tidal cycles, and its neural mechanism remains unknown. The mangrove cricket, Apteronemobius asahinai, possesses both circatidal and circadian clocks, and simultaneously exhibits circatidal and circadian rhythms in its locomotor activity. In a previous study, we showed that surgical removal of the optic lobes, the principal circadian clock locus in crickets, disrupted their circadian rhythm, but not their circatidal rhythm. In this study, we focused on the pars intercerebralis (PI) because surgical removal of the PI disrupts the circadian rhythm and causes arrhythmic activity in some cricket species. After surgical removal of the PI, the proportion of crickets displaying circatidal rhythm decreased, and more than half of the crickets exhibited arrhythmic activity. Surgical removal of the regions around the PI also caused a similar effect on locomotor activity. Our results indicate that the PI and/or its surrounding regions are important not only for circadian but also for circatidal rhythm. This suggests the presence of a neural or hormonal pathway in the PI and/or its surrounding regions that is common to the circatidal and circadian rhythms.

Transgenerational seasonal timer for suppression of sexual morph production in the pea aphid, Acyrthosiphon pisum.

Many aphid species switch reproductive modes seasonally, with the sexual generations appearing in autumn. Sexual generations are induced by short days. It has been reported that the appearance of sexual morphs is suppressed by a transgenerational factor (a seasonal timer) over several generations after hatching from overwintered eggs. The present study examined whether the seasonal timer measures the number of days from hatching or the number of generations from hatching using the pea aphid, Acyrthosiphon pisum Harris (Homoptera: Aphididae). Effects of temperature and photoperiod on the seasonal timer were also examined by successive rearing. The ability to produce sexual morphs was strongly suppressed in stem mothers (the foundress generation), and gradually recovered over successive generations produced during a few months. The duration for which the seasonal timer could function depended on the number of days from hatching and temperature, but not on photoperiod or the number of generations from hatching. We thus showed in a single study that the seasonal timer of the pea aphid has all the physiological characteristics shown in separate studies in different aphid species.

Common features in diverse insect clocks.

This review describes common features among diverse biological clocks in insects, including circadian, circatidal, circalunar/circasemilunar, and circannual clocks. These clocks control various behaviors, physiological functions, and developmental events, enabling adaptation to periodic environmental changes. Circadian clocks also function in time-compensation for celestial navigation and in the measurement of day or night length for photoperiodism. Phase response curves for such clocks reported thus far exhibit close similarities; specifically, the circannual clock in Anthrenus verbasci shows striking similarity to circadian clocks in its phase response. It is suggested that diverse biological clocks share physiological properties in their phase responses irrespective of period length. Molecular and physiological mechanisms are best understood for the optic-lobe and mid-brain circadian clocks, although there is no direct evidence that these clocks are involved in rhythmic phenomena other than circadian rhythms in daily events. Circadian clocks have also been localized in peripheral tissues, and research on their role in various rhythmic phenomena has been started. Although clock genes have been identified as controllers of circadian rhythms in daily events, some of these genes have also been shown to be involved in photoperiodism and possibly in time-compensated celestial navigation. In contrast, there is no experimental evidence indicating that any known clock gene is involved in biological clocks other than circadian clocks.

Urban soil compaction reduces cicada diversity.

INTRODUCTION: Urbanization converts animal habitats into globally homogeneous environments. Consequently, urban communities have low diversity and are often dominated by a few species. However, proximate environmental factor(s) causing community degradation have rarely been identified among diverse and co-varying urban parameters. RESULTS: The present study addresses the recent loss of cicada diversity in Osaka, Japan, where cicada communities are overwhelmed by a single species, Cryptotympana facialis. A field survey across an urban-forest gradient revealed that the trend of decreasing cicada diversity toward the urban core was mostly associated with the soil hardness among the environmental variables examined. Simultaneously, the proportion of C. facialis increased with soil hardness, although this effect was partially mitigated in forest patches. Newly hatched nymphs of C. facialis exhibited greater burrowing ability than that in other native species. CONCLUSIONS: These findings identify soil compaction due to urbanization as a possible cause of cicada diversity loss, as it impedes the passage of nymphs to underground nests. This impact of urban soil compaction may influence ecosystem functioning of soil-dwelling arthropods and their trophically associated animals.

Silencing the circadian clock gene Clock using RNAi reveals dissociation of the circatidal clock from the circadian clock in the mangrove cricket.

Whether a clock that generates a circatidal rhythm shares the same elements as the circadian clock is not fully understood. The mangrove cricket, Apteronemobius asahinai, shows simultaneously two endogenous rhythms in its locomotor activity; the circatidal rhythm generates active and inactive phases, and the circadian rhythm modifies activity levels by suppressing the activity during subjective day. In the present study, we silenced Clock (Clk), a master gene of the circadian clock, in A. asahinai using RNAi to investigate the link between the circatidal and circadian clocks. The abundance of Clk mRNA in the crickets injected with double-stranded RNA of Clk (dsClk) was reduced to a half of that in control crickets. dsClk injection also reduced mRNA abundance of another circadian clock gene period (per) and weakened diel oscillation in per mRNA expression. Examination of the locomotor rhythms under constant conditions revealed that the circadian modification was disrupted after silencing Clk expression, but the circatidal rhythm remained unaffected. There were no significant changes in the free-running period of the circatidal rhythm between the controls and the crickets injected with dsClk. Our results reveal that Clk is essential for the circadian clock, but is not required for the circatidal clock. From these results we propose that the circatidal rhythm of A. asahinai is driven by a clock, the molecular components of which are distinct from that of the circadian clock.

The circatidal rhythm persists without the optic lobe in the mangrove cricket Apteronemobius asahinai.

Whether the circatidal rhythm is generated by a machinery common to the circadian clock is one of the important and interesting questions in chronobiology. The mangrove cricket Apteronemobius asahinai shows a circatidal rhythm generating active and inactive phases and a circadian rhythm modifying the circatidal rhythm by inhibiting activity during the subjective day simultaneously. In the previous study, RNA interference of the circadian clock gene period disrupted the circadian rhythm but not the circatidal rhythm, suggesting a difference in molecular mechanisms between the circatidal and circadian rhythms. In the present study, to compare the neural mechanisms of these 2 rhythms, we observed locomotor activity in the mangrove cricket after surgical removal of the optic lobe, which has been shown to be the locus of the circadian clock in other crickets. We also noted the pigment-dispersing factor immunoreactive neurons (PDF-IRNs) in the optic lobe, because PDF is a key output molecule in the circadian clock system in some insects. The results showed that the circadian modulation was disrupted after the removal of the optic lobes but that the circatidal rhythm was maintained with no remarkable changes in its free-running period. Even in crickets in which some PDF-immunoreactive somata remained after removal of the optic lobe, the circadian rhythm was completely disrupted. The remnants of PDF-IRNs were not correlated to the occurrence and free-running period of the circatidal rhythm. These results indicate that the principal circatidal clock is located in a region(s) different from the optic lobe, whereas the circadian clock is located in the optic lobe, as in other crickets, and PDF-IRNs are not important for circatidal rhythm. Therefore, it is suggested that the circatidal rhythm of A. asahinai is driven by a neural basis different from that driving the circadian rhythm.

Involvement of the brain region containing pigment-dispersing factor-immunoreactive neurons in the photoperiodic response of the bean bug, Riptortus pedestris.

The concept of insect photoperiodism based on a circadian clock has been supported by many studies demonstrating that the behavioural circadian rhythm and the photoperiodic response are driven by the same circadian clock genes. However, the neuronal mechanism of the circadian clock underlying photoperiodism is poorly understood. To examine whether circadian rhythm and photoperiodism share a neuronal mechanism, we focused on the neurons that express neuropeptide pigment-dispersing factor (PDF) in the bean bug, Riptortus pedestris. PDF has been identified as an important regulator of the insect circadian rhythm and is expressed in circadian clock neurons of various insect species. In R. pedestris, PDF immunoreactivity was detected in some clusters of cells and their fibres in the optic lobe and the protocerebrum. cDNA encoding a PDF precursor protein was highly conserved between R. pedestris and many other insects. Differences between day and night were not observed in the immunolabelling intensity in cell bodies of PDF-immunoreactive neurons and pdf mRNA expression levels in the head. Surgical removal of the region containing PDF-immunoreactive cell bodies at the medulla disrupted the photoperiodic regulation of diapause. However, gene suppression of pdf by RNA interference did not affect the photoperiodic response. These results suggest that the region containing PDF-immunoreactive somata is important for the photoperiodic response in R. pedestris, but pdf mRNA expression is probably not required for the response.

Both the anterior and posterior eyes function as photoreceptors for photoperiodic termination of diapause in the two-spotted spider mite.

Photoreceptors involved in photoperiodism in insects and mites can be either the retinal photoreceptors in the visual system or nonvisual extraretinal photoreceptors. Mites with no eyes have a clear photoperiodic response, suggesting the involvement of extraretinal photoreceptors in mite photoperiodism. In mites equipped with eyes, however, it is not known whether the retinal or extraretinal photoreceptors are involved in photoperiodism. The two-spotted spider mite Tetranychus urticae possesses two pairs of eyes. Adult females of this species terminate diapause in response to long days. To investigate whether the eyes function as photoperiodic photoreceptors in T. urticae, their eyes were ablated using a laser ablation system. Mites with their eyes intact terminated diapause under long days after low temperature exposure, whereas they remained in diapause under short days. Under constant darkness, they did not terminate diapause. When all eyes were removed, the mites remained in diapause even when they were maintained under long days. In contrast, the mites showed clear photoperiodic response when only the anterior or posterior eyes were removed. These results indicate that both the anterior and posterior eyes function as photoreceptors in photoperiodic termination of diapause in T. urticae.

Desiccation and heat tolerance of eggs of the Asian tadpole shrimp, Triops granarius.

Triops granarius (Lucas) is a freshwater crustacean that is distributed from South Africa to the Eurasian continent. This species lives in temporary water pools and is exposed to desiccation and extreme temperatures after the water dries up in its habitat. To withstand this severe environment, Triops eggs enter anhydrobiosis when dehydrated. To clarify the physiological characteristics of T. granarius anhydrobiosis, we examined hatching rates after rehydration of eggs that were dehydrated at several humidity levels for 10 or 100 days. Lower humidity produced higher hatching rates when dehydration was continued for 100 days. These results suggest that drying at low humidity is required for long-term anhydrobiosis of T. granarius eggs. The eggs survived desiccation when dehydrated at the blastula, gastrula, and early organogenesis stages. The most dehydration-tolerant stage was early organogenesis. Non-dehydrated eggs hatched after temperature treatments of up to 50°C for 1 h, but did not hatch after exposure to 60°C for 1 h in air and under water. Similar results were obtained for dehydrated eggs exposed to high temperatures under water. In contrast, dehydrated eggs hatched after 1 h at 80°C in air but did not after 1 h at 90°C in air. Our results show that Triops eggs exhibit tolerance for desiccation and high temperature in a dried state, once they have entered anhydrobiosis.