The pattern of ecdysone levels during development in the desert locust, Schistocerca gregaria

A simple chemical assay, sensitive to picogram amounts (10^?12 g) of ecdysones in crude biological materials is described. By a simple, rapid purification procedure followed by conversion to their trimethylsilyl ethers, ecdysones are determined quantitatively by gas chromatography with an electron capture detector. The method has been applied to the determination of ecdysones in the fourth and fifth instars of the desert locust. 20-Hydroxyecdysone is always present in higher concentration than ecdysone and the former rises to a sharp maximum 1 to 2 days before ecdysis.     

Thin-layer chromatographic in situ analysis of insect ecdysones via fluorescence-quenching

The possibility of quantitating insect ecdysones $\text{in situ}$ on thin-layer Chromatographic plates was examined. Two approaches were evaluated: 1) the induction of ecdysone fluorescence by sulfurio acid treatments and 2) the fluorescence-quenching of fluorescent thin-layer Chromatographic plates by ecdysones. The fluorescence-quenching method was found to be most suitable and had a linear response range from 0.5 to 3 μg for α-ecdysone and 20-hydroxyecdysone. Fluorescence-quenching and high pressure liquid Chromatographic analyses obtained, from extracts of α-ecdysone 20-hydroxylase incubations gave similar results. New data concerning the acid-induced fluorescence of ecdysones showed α-ecdysone to be twice as fluorescent as 20-hydroxyecdysone.     

Behavioral responses of male lobsters to ecdysone metabolites

A molting hormone metabolite of crustecdysone 4,4‐dimethylbutyrolactone, 4, and six structurally related compounds were tested for their capacity to elicit behavioral responses, especially sexual responses, in the male lobster (Homarus americanus). Of these compounds, 6, 7, 8, and especially 9 provoked alert responses. Moreover, compounds 7 and 9 startled the lobsters, which responded with defensive and perhaps aggressive postures. No feeding or sexual behavior was observed. These results do not support the hypothesis that metabolites of molting hormones act as sex pheromones in the lobster. A comparison of chemical structure with behavioral response between the most active compound 9 and the inactive compound 10 is made.     

Comprehensive Study of the Phenolics and Saponins from Helleborus niger L. Leaves and Stems by Liquid Chromatography/Tandem Mass Spectrometry

The aerial parts of the medicinal plant Helleborus niger L. comprise a substantial number of constituents with only few of them identified so far. To expand the knowledge of its secondary metabolite profile, extracts from H. niger leaves and stems were investigated by liquid chromatography/tandem mass spectrometry (LC/MSⁿ). Specific identification strategies using LC/MS are established and discussed in detail. The leaves turned out to contain acylated and non‐acylated quercetin and kaempferol oligoglycosides, protoanemonin and its precursor ranunculin, β‐ecdysone, and a variety of steroidal saponins, mainly in the furostanol form. The sapogenins were elucidated as of sarsasapogenyl, diosgenyl, and macranthogenyl structures, and confirmed by comparison with the respective reference compounds. The secondary metabolite profiles were almost identical in both plant parts except that the stems lacked kaempferol derivatives and some saponins. The ranunculin derivatives and β‐ecdysone were found in both plant parts. Correlations between the location of the compound groups and the plant's defense strategies are proposed. Additionally, the role of the detected secondary metabolites as protective substances against exogenic stress and as a defense against herbivores is discussed.     

Independence of Sexual and Anti-Predator Perceptual Functions in an Acoustic Moth: Implications for the Receiver Bias Mechanism in Signal Evolution

The receiver bias model for the evolution of mating signals proposes that selection favors male displays that happen to stimulate a general, ancestral perception in females such that receptivity and successful courtship increase. If these male signals do arise, however, the female perception will operate in two contexts, the original, typically non‐sexual, one and courtship. We may then ask whether these two functions represent the same or distinct traits, which may be under separate neural and genetic control. We studied this question in Achroia grisella, a pyralid moth species in which males attract females, with an ultrasonic mating song. Hearing in pyralid moths is widespread and originated in an anti‐predator context – the perception and avoidance of echolocating bats – and it is inferred that the male song found in A. grisella, and in several other pyralid species, arose subsequently via a receiver bias mechanism: Females perceiving male‐produced ultrasound and responding with anti‐predator behavior normally exhibited in the presence of bat echolocations may have inadvertently increased the likelihood of successful courtship. We measured hearing responses in both sexual and anti‐predator contexts in inbred lines developed from an A. grisella population. Significant inter‐line variance was observed for sensitivity thresholds for female response to male song and for both female and male responses to synthetic bat echolocation signals. Female responses to male song and to synthetic echolocations differed markedly in sensitivity, and the median sexual and anti‐predator responses in the various lines were not correlated. However, a higher level of similarity occurs between the female and male anti‐predator responses. Thus, genetic variance for sexual and anti‐predator responses appears to exist, and the two responses may represent independent traits. These results imply that when male signals originate via a receiver bias mechanism, female perception does not necessarily remain fixed in its ancestral state but has the potential for continued modification.     

Manipulation of host ecdysteroid hormone levels facilitates infection by the fungal insect pathogen,Metarhizium rileyi

Entomopathogenic fungi such as Metarhizium rileyi and Beauveria bassiana are widely used insect biological control agents. Little, however, is known concerning genetic or enzymatic factors that differentiate the mechanisms employed by these two fungal pathogens to infect target hosts. Infection by either of these organisms is known to increase levels of the growth and molting hormone, ecdysone, which also regulates the expression of a number of innate immune pathways. M. rileyi, but not B. bassiana, has apparently evolved an ecdysteroid-22-oxidase (MrE22O) that inactivate ecdysone. We show that deletion of MrE22O impaired virulence compared with the wild-type strain, with an increase in ecdysone titer seen in hosts that was coupled to an increase in the expression of antimicrobial genes. An M. rileyi strain engineered to overexpress MrE22O (MrE22O^OE), as well as trans-expression in B. bassiana (Bb::MrE220^OE) resulted, in strains displaying enhanced virulence and dampening of host immune responses compared with their respective wild-type parental strains. These results indicate that ecdysone plays an important role in mediating responses to fungal infection and that some insect pathogenic fungi have evolved mechanisms for targeting this hormone as a means for facilitating infection.     

The molting hormones from the embryonated egg of the tobacco hornworm, Manduca sexta (L.)

26-Hydroxyecdysone is the predominant molting hormone in 24- to 44-hour-old embryonated tobacco hornworm eggs, accounting for approximately 80% of the ecdysones present at this stage of development. This molting hormone was previously shown to be the major ecdysone present in 48- to 64-hour-old embryonated eggs of this insect. During both of these periods of embryonic development in the hornworm 20-hydroxyecdysone is a minor component, in contrast to its presence as the major ecdysone in the hornworm during certain stages of post-embryonic development.     

Non‐Conceptive Sexual Behavior in Spiders: A Form of Play Associated with Body Condition,Personality Type,and Male Intrasexual Selection

In the socially polymorphic spider Anelosimus studiosus, males mature early in the reproductive season and recruit to the webs of juvenile females and guard them until they mature. During the period before females mature, males and females engage in repeated bouts of non‐conceptive (play) sexual behavior, where the pair courts and engages in mock copulation; both males and females gain performance‐enhancing experience via these encounters. In this study, we examined the factors that underlie individual variation in the tendency to engage in non‐conceptive mating and determine whether it impacts male–male competition for females. We found that docile females, being less resistant to mating in general, are more likely to accept male courtship and non‐conceptive copulation as juveniles. Personality type influenced the exhibition of non‐conceptive sexual behavior in males as well. High body condition males of the aggressive phenotype were more likely to engage in non‐conceptive sexual behavior than males with lower body condition. Body condition did not influence docile males’ propensity to engage in non‐conceptive sexual behavior, but female size did. Docile males engaged in more non‐conceptive sexual displays with larger females. Engaging in non‐conceptive sexual displays negatively impacted male performance in staged male–male contests for access to females. This cost was greatest for males of the aggressive phenotype, which are otherwise favored in male–male contests. Our findings indicate expression of non‐conceptive sexual displays is linked to personality and results in reproductive performance trade‐offs for male A. studiosus.     

Sex‐biased gene expression during the adult stage of the Indian meal moth Plodia interpunctella

Sexual dimorphisms are primary regulated by sex‐biased gene expression. In the present study, using real‐time polymerase chain reaction, we determined the expression profiles of nine genes associated with development, metabolism, stress, and defense throughout adulthood of the Indian meal moth Plodia interpunctella, a global pest of stored food products. Most genes were differentially expressed in a sex‐biased manner during the adult lifespan of the moth. Expression of the heat shock protein genes hsp25 and hsp90 and the antioxidant gene thioredoxin peroxidase (Tpx) was highly female biased, whereas the expression of a gene related to host development (ecdysone receptor [EcR]) and two genes associated with immunity (β‐glycan recognition protein [βgrp] and prophenoloxidase [ProPO]) was male biased. In contrast, the expression of hsp70, glucose‐regulated protein 78 (grp78) and ultraspiracle (USP) was not sex biased. The results of the present study provide important insights into the role of sex‐biased genes in the physiology and behavior of P. interpunctella.     

Interruption of proecdysis by autotomy of partially regenerated limbs in the land crab, Gecarcinus lateralis

In the land crab, Gecarcinus lateralis, autotomy of partially regenerated limbs before a critical stage in the premolt period results in (1) a very rapid decrease in the serum ecdysone titer, (2) a delay in the growth of partial regenerates remaining on the animal, (3) a delay in the deposition of gastroliths, and (4) a delay in cytological changes in the epidermis. Serum ecdysone titers remain low while new limb regenerates form at the sites of those removed. Ecdysone titers rise when these secondary regenerates complete basal regeneration. Premolt events, which had ceased at the time of autotomy of the partial regenerates, resume their normal patterns of development when ecdysone titers reach the level present in the serum at the time of this interruption. We propose that the effect of autotomy before a critical period is to reinitiate a normal proecdysis. The same pattern of events occurs following autotomy of partial regenerates of crabs without eyestalks, suggesting that the decrease of serum ecdysones is brought about by some mechanism other than changes in the titer of the molt inhibitory hormone.     

Ecdysteroids as a component of the genital sex pheromone in two species of hard ticks,Dermacentor variabilis andDermacentor andersoni (Acari: Ixodidae)

Neutering of part-fed females eliminated copulatory behavior inDermacentor variabilis (Say) andD. andersoni Stiles males. Extracts from the anterior reproductive tracts of part-fed (7 days) females partly restored the male copulatory behavior in conspecific neutered females. Similar extracts from unfed females did not restore the behavior, suggesting that the pheromone was produced during feeding. Perception of the genital sex pheromone by sensillae on the male cheliceral digits was confirmed by electrophysiological techniques.Males ofD. variabilis andD. andersoni responsed positively to authentic ecdysone and 20-hydroxyccdysone (20HE) in neutered female bioassays. Responses to sterols were significantly lower than to ecdysteroids. Electrophysiological assays suggest a sensitivity of males to high doses of ecdysteroids. The strongest responses were to 20HE in both species. Ecdysteroids, specifically ecdysone and 20HE were shown to be present in the anterior reproductive tracts in excess of amounts that could be explained by mere hemolymph contamination. Ecdysteroids were also found in washings of the vaginal lumen of these two species.Dermacentor andersoni females contained larger amounts of ecdysteroids thanD. variabilis females. 20-hydroxyecdysone and possibly ecdysone appear to be components of the genital sex pheromone ofD. variabilis andD. andersoni. Species recognition may be facilitated by these components, but the complete mechanism is not yet fully understood.Supported by grant AI10, 986 from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, U.S. Public Health Service, Department of Health and Human Services, Bethesda, Maryland.     

Identification and laboratory behavioral test of the alarm pheromone of Acanthocoris sordidus Thunberg (Hemiptera: Coreidae)

The winter cherry bug, Acanthocoris sordidus Thunberg (Hemiptera: Coreidae), widely spread in South Korea, Japan and Taiwan, is becoming an economic pest in these regions. Its elevated pest status in pepper plant, Capsicum annuum, is a trigger for eco‐friendly management tactics. In order to use its pheromone for pest management, the cuticular components of adult A. sordidus were identified and a behavioral bioassay on the functions of the components was conducted. From the whole‐body extracts of male and female A. sordidus, 13 compounds (hexanal, 1‐hexanol, 2‐hexanol, 3‐hexanol, hexanoic acid, hexyl acetate, heptyl acetate, octyl acetate, tetradecane, 2‐butyl‐2‐octenal, hexyl hexanoate and (2α,4α,6β)‐2,4,6‐tripentyl‐1,3,5‐trioxane (hexanal trimer, cis‐ and trans‐forms) were identified. In addition to these, two unidentified compounds, a male‐specific and a male‐rich compound, were also found. Hexanal provoked clear escaping behavior. 1‐Hexanol, hexyl acetate and hexanoic acid evoked weaker behavior than hexanal. We concluded that hexanal worked as an alarm pheromone and could be used as a pushing signal for a control program for A. sordidus.     

The apparent requirement of two hormones, alpha- and beta-ecdysone, for molting induction in insects

Puff formations at loci I-18-C and IV-2-B of the salivary gland chromosomes are early indications of a beginning molting process in Chironomus tentans larvae. The effectiveness of the two ecdysone analogs, α- and β-ecdysone, in inducing these puffs was compared. Incubation of salivary glands in vitro with β-ecdysone causes only puff IV-2-B to appear; incubation with α-ecdysone stimulates initially puffing at only I-18-C. After an injection of α-ecdysone, puffing at I-18-C begins within less than 15 min, whereas puffing at IV-2-B is delayed for more than 30 min. Following an injection of β-ecdysone, puffing at IV-2-B begins within less than 15 min, whereas puffing at I-18-C is delayed. Injected ³H-α-ecdysone is converted to β-ecdysone and a polar compound. Injected ³H-β-ecdysone is converted to a compound less polar than α-ecdysone and a polar metabolite which stimulates puffing at I-18-C, like α-ecdysone. It is suggested that the two ecdysones have different targets in the cell, that they can be rapidly converted to compounds with the activity of the other analog, and that the induction of a complete molt requires the action of both hormones.