Second messengers mediating the effects of testis ecdysiotropin in testes of the gypsy moth,Lymantria dispar

Exposure of larval and pupal testes of Lymantria dispar to diacyl glycerol mimics, phorbol, 12-myristate, 13-acetate, and 11, 12 dibutyryl phorbol ester, induced or augmented synthesis of immunodetectable ecdysteroids. The non-esterified analog, 4α-phorbol, had little effect. H-7, a protein kinase C inhibitor, inhibited ecdysteroid synthesis. When testis ecdysiotropin and phorbol esters were administered together at the maximum effective dose of each, steroidogenesis was further enhanced. Therefore, diacyl glycerol may be a second messenger for testis ecdysiotropin in testes. In addition, testis ecdysiotropin induced a rapid rise and fall in cAMP titers in both larval and pupal testes. The cyclic AMP analog, dibutyryl cyclic AMP, induced ecdysteroid synthesis in larval testes, but had little steroidogenic effect in pupal testis sheaths. However, dibutyryl cyclic AMP inhibited the steroidogenic effect of testis ecdysiotropin in larval as well as pupal testes. Cyclic AMP may act to modulate the effects of testis ecdysiotropin in inducing ecdysteroid synthesis by testes of L. dispar. © 1993 Wiley-Liss, Inc.     

Identification and characterization of an ecdysiotropic peptide from brain extracts of the gypsy moth,Lymantria dispar

A peptide (Lymantria TE) was isolated from brains of the gypsy moth, Lymantria dispar, which stimulates synthesis of ecdysteroid in the testes of larval and pupal insects. This ecdysiotropic peptide was purified and its structure determined to be NH₂-IIe-Ser-Asp-Phe-Asp-Glu-Tyr-Glu-Pro-Leu-Asn-Asp-Ala-Asp-Asn-Asn-Glu-Val-Leu-Asp-Phe-OH using protein sequence analysis and electrospray mass spectrometry. The peptide was biphasic in activity, with maximal activity in the pupal testes at 10⁻¹³ M and 10⁻⁹ M, with a minimum at 10⁻¹⁰ M, and with maxima at 10⁻¹⁵ M and 10⁻¹⁰ M and minimum at 10⁻¹³ M for larval testes. Arch. Insect Biochem. Physiol. 34:175–189, 1997. © 1997 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Naturally occurring analogs of Lymantria testis ecdysiotropin,a gonadotropin isolated from brains of Lymantria dispar pupae1

Lymantria testis ecdysiotropin (LTE) was isolated from the most prominent peptide peak corresponding to an active fraction obtained by high pressure liquid chromatographic (HPLC) separation of a homogenate of 13,000 Lymantria dispar pupal brains. In this work we examined the other active fractions from this separation as well as a second HPLC separation of an additional 2,300 pupal brains. Bioassay of the ecdysteroidogenic effects of each peak on L. dispar testes allowed detection of 20 peptide peaks with testis ecdysiotropic activity in addition to LTE. Of these, ten peptides were purified and sequenced. All of them were comparable to LTE in molecular weight. The amino acid sequences of five of the peptides were similar enough to LTE to be considered to be members of an LTE family. However, the other five peptides had no significant homology with LTE or with each other. A BLAST database search indicated LTE family homology with portions of inhibitory peptides such as those inhibiting cytolysis. In contrast, non-LTE ecdysiotropic peptides, in which undetermined residues designated X were assumed to be cysteine, were strikingly homologous to portions of vertebrate and invertebrate zinc finger peptides and to vertebrate and invertebrate virus proteins. Arch. Insect Biochem. Physiol. 36:37–50, 1997.Published 1997 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Action cascade of an insect gonadotropin,testis ecdysiotropin,in male Lepidoptera

Summary

Testis sheaths from late last instar larvae and mid-developing pupae of Heliothis virescens and Lymantria dispar synthesize ecdysteroid in vitro. Gonadal ecdysteroid can stimulate the production of growth factors from the sheaths which, in turn, promote the growth and development of the genital tract. Ongoing basal synthesis is controlled by positive feedback to exogenous ecdysteroid; titers of this hormone approaching those of molting last instar larvae and developing pupae effect maximum synthesis. These findings suggest that circulating titers of ecdysteroid hormone promote gonadal ecdysteroidogenesis, and thus coordinate the actions of the gonads with metamorphic events in the whole animal. Synthesis of ecdysteroid by testes is initiated, however, by a brain neuropeptide, testis ecdysiotropin (TE). TE is a 21 amino acid peptide of molecular weight 2472 Da. TE boosts basal steroid synthesis by pupal testis sheaths as well. It acts primarily via G_i protein and second messengers diacyl glycerol and low calcium influx, resulting in stimulation of phosphokinase C. G_s protein and its resultant messenger, cyclic AMP, also play roles in activation and inhibition of ecdysteroidogenesis. The interplay of controlling systems probably serves to fine tune a system essential to gonadal development and function.     

Effect of calcium ions on ecdysteroid secretion by testes of Heliothis virescens

• 1.1. Testes of Heliothis virescens synthesized ecdysteroid in media containing low titers of calcium; the optimum calcium titer for testis sheaths stimulated to synthesize ecdysteroid in vivo was ca 1 mM, while the optimum of testes stimulated in vitro with the peptide testis ecdysiotropin was ca 0.3 mM calcium.
• 2.2. Verapamil at concentrations lower than 10⁻³ M induced increases in ecdysteroid synthesis, indicating more efficient synthesis when calcium influx was inhibited.
• 3.3. Hemolymph of H. virescens was 7 mM in calcium, while whole testes were maintained at 1–2 μM calcium.

     

Characterisation of three Alphabaculovirus isolates from the gypsy moth,Lymantria dispar dispar (Lepidoptera: Erebidae), in Turkey

In this study, Lymantria dispar dispar larvae, collected from three different localities in Turkey, were examined for the presence of inclusion bodies under phase contrast and electron microscopes. Inclusion bodies from infected larvae were subjected to polymerase chain reaction using the conserved primers for polyhedrin (polh), late expression factor 8 (lef-8) and late expression factor 9 (lef-9) genes. Sequence analysis confirmed that larvae collected from the three different localities contained multiple nucleopolyhedrosis viruses (MNPVs). These isolates were designated LdMNPV-T1, LdMNPV-T2 and LdMNPV-T3. Phylogenetic analyses of these isolates were performed using target genes polh, lef-8 and lef-9. Restriction endonuclease analysis of the three geographic isolates with EcoRI and PstI enzymes demonstrated some differences existed among the isolates. According to the EcoRI profile, the mean estimated size for the complete genome of each isolate (LdMNPV-T1, LdMNPV-T2 and LdMNPV-T3) was calculated to be approximately 170, 153 and 170?kb, respectively. Insecticidal activities of each isolate were tested on L. d. dispar larvae using four different viral concentrations between 10³ and 10⁶?OBs/ml. Results showed that the mortalities for LdMNPV-T1, -T2 and -T3 ranged between 13–53%, 47–100% and 46–93%, respectively. The LC₅₀ and LC₉₅ values of LdMNPV-T2 were not significantly different from the respective corresponding values of the other two isolates. However, isolate LdMNPV-T2 killed larvae with a LC₅₀ value that was lower than the other two isolates. Our results suggested there are promising LdMNPV isolates in Turkey that can be used for microbial control of L. d. dispar larvae.     

An Investigation of the Endocrine-Disruptive Effects of Bisphenol A in Human and Rat Fetal Testes

Few studies have been undertaken to assess the possible effects of bisphenol A (BPA) on the reproductive hormone balance in animals or humans with often contradictory results. We investigated possible direct endocrine disruption by BPA of the fetal testes of 2 rat strains (14.5–17.5 days post-coitum) and humans (8–12 gestational weeks) and under different culture conditions. BPA concentrations of 10^-8M and 10^-5M for 72h reduced testosterone production by the Sprague-Dawley fetal rat testes, while only 10^-5M suppressed it in the Wistar strain. The suppressive effects at 10^-5M were seen as early as 24h and 48h in both strains. BPA at 10^-7-10^-5M for 72h suppressed the levels of fetal rat Leydig cell insulin-like factor 3 (INSL3). BPA exposure at 10^-8M, 10^-7M, and 10^-5M for 72h inhibited testosterone production in fetal human testes. For the lowest doses, the effects observed occurred only when no gonadotrophin was added to the culture media and were associated with a poorly preserved testicular morphology. We concluded that (i) BPA can display anti-androgenic effects both in rat and human fetal testes; (ii) it is essential to ascertain that the divergent effects of endocrine disruptors between species in vitro do not result from the culture conditions used, and/or the rodent strain selected; (iii) the optimization of each in vitro assay for a given species should be a major objective rather than the search of an hypothetical trans-species consensual model-system, as the organization of the testis is intrinsically different between mammalian species; (iv) due to the uncertainty existing on the internal exposure of the human fetal testis to BPA, and the insufficient number of epidemiological studies on the endocrine disruptive effects of BPA, caution should be taken in the extrapolation of our present results to the human reproductive health after fetal exposure to BPA.     

Gonadal maturation is dependent on body size in the sea urchin,Echinometra tsumajiroi

Summary

This study examined gonadal maturation in the sea urchin, Echinometra tsumajiroi, in relationship to body size. In the breeding season, E. tsumajiroi were collected and divided into six groups based on the length of the major axis (LM) as a measure of body size: Group 1 (6–10 mm in LM), Group 2 (11–15 mm in LM), Group 3 (16–20 mm in LM), Group 4 (21–25 mm in LM), Group 5 (26–30 mm in LM), and Group 6 (> 31 mm in LM). Although gonads were present in individuals larger than 8 mm in LM, the germ cells were undifferentiated in Group 1. Gonadal sex differentiation was first seen in Group 2: a few young oocytes appeared at the periphery of the ovary, and both spermatogonia and spermatocytes were also observed at the periphery of the testis. As size increased from Group 3 to Group 4, more mature germ cells such as ripe ova or spermatozoa were observed in ovaries and testes. In Group 5, the ovaries and testes were filled with a large number of mature eggs and spermatozoa, respectively. Spawning could be induced in this group by the injection of 0.5 M KCl into the coelomic cavity. In contrast, gonads of some of the biggest individuals, belonging to Group 6, appeared to be spent due to spawning, because few eggs were present in the ovary and relict spermatozoa were present in the testis. These results suggest that reproductive activity in the sea urchin, E. tsumajiroi, depends on body size.     

Testis ecdysiotropin, an insect gonadotropin that induces synthesis of ecdysteroid.

Testes of lepidoptera synthesized ecdysteroid in a somewhat different temporal pattern than the prothoracic glands that release ecdysteroid to the hemolymph. Brain extracts from Heliothis virescens and Lymantria dispar induced testes to synthesize ecdysteroid, but did not affect prothoracic glands. The testis ecdysiotropin (LTE) was isolated from L. dispar pupal brains by a series of high-pressure chromatography steps. Its sequence was Ile-Ser-Asp-Phe-Asp-Glu-Tyr-Glu-Pro-Leu-Asn-Asp-Ala-Asp-Asn-Asn-Glu-Val-Leu-Asp-Phe-OH, of molecular mass 2,473 Daltons. The predominant signaling pathway for LTE was via G(i) protein, IP3, diacylglycerol and PKC; a modulating pathway, apparently mediated by an angiotensin II-like peptide, was controlled via G(s) protein, cAMP, and PKA. Testis ecdysteroid caused isolated testis sheaths to also synthesize a growth factor that induced development of the male genital tract. The growth factor appeared to be a glycoprotein similar to vertebrate alpha-1-glycoprotein. A polyclonal antibody to LTE indicated LTE-like peptide in L. dispar brain medial neurosecretory cells, the suboesophageal, and other ganglia, and also in its target organ, the testis sheath. LTE immunoreactivity was also seen in testis sheaths of Rhodnius prolixus. LTE-like immunoactivity was also detected in developing optic lobes, antennae, frontal ganglia, and elongating spermatids of developing L. dispar pupae. This may indicate that LTE has a role in development as well as stimulation of testis ecdysteroid synthesis. Published 2001 Wiley-Liss, Inc.     

Differential Effects of Bisphenol A and Diethylstilbestrol on Human,Rat and Mouse Fetal Leydig Cell Function

Endocrine disruptors (ED) have been incriminated in the current increase of male reproductive alterations. Bisphenol A (BPA) is a widely used weak estrogenic environmental ED and it is debated whether BPA concentrations within the average internal exposure are toxic. In the present study we investigated the effects of 10⁻¹² to 10⁻⁵ M BPA concentrations on fetal Leydig cell function, as fetal life is a critical period of sensitivity to ED effects on male reproductive function. To this aim, fetal testes from human at 6.5–10.5 gestational weeks (GW) or from rat and mouse at a comparable critical period of development (14.5 days post-coitum (dpc) for rat and 12.5 dpc for mouse) were explanted and cultured using our validated organotypic culture system in the presence or absence of BPA for 1–3 days. BPA concentrations as low as 10⁻⁸ M reduced testosterone secretion by human testes from day 1 of culture onwards, but not by mouse and rat testes where concentrations equal to 10⁻⁵ M BPA were required. Similarly, 10⁻⁸ M BPA reduced INSL3 mRNA levels only in human cultured testes. On the contrary, 10⁻⁵ and 10⁻⁶ M diethylstilbestrol (DES), a classical estrogenic compound, affected testosterone secretion only in rat and mouse testis cultures, but not in human testis cultures. Lastly, contrarily to the DES effect, the negative effect of BPA on testosterone produced by the mouse fetal testis was maintained after invalidation of estrogen receptor α (ERα). In conclusion, these results evidenced i) a deleterious effect of BPA on fetal Leydig cells function in human for concentrations from 10⁻⁸ M upwards, ii) species-specific differences raising concerns about extrapolation of data from rodent studies to human risk assessment, iii) a specific signaling pathway for BPA which differs from the DES one and which does not involve ERα.     

In vitro maintenance and sperm development in the testis of the house cricket (Acheta domesticus)

Summary

Whole testes of Acheta domesticus were maintained in vitro for up to 48 h. Development of sperm could not be induced in the penultimate stage testis irrespective of hormone influence. A partial stimulation of spermatogenesis in the ultimate stage testis was achieved using 10^?6 M 20-hydroxyecdysone but completion of spermiogenesis was not seen.     

Susceptibility of gypsy moth larvae toLymantria spp. nuclear-and cytoplasmic-polyhedrosis viruses

SevenLymantria spp. viruses from widely separated sources were used in a screening test for virulence. Laboratory data show thatLymantria dispar L. cytoplasmic-polyhedrosis virus of Japan, and the nuclear-polyhedrosis virus of Connecticut (U.S.A.) are more virulent than other five viruses when tested on the larvae of the gypsy moth. The nuclear-and cytoplasmic-polyhedrosis viruses ofLymantria fumida Btl. were also infectious forL. dispar larvae.

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Résumé On a comparé au laboratoire la virulence de 7 souches différentes de virus des polyédroses nucléaire et cytoplasmique isolées deLymantria spp. Les essais effectués par contamination de la nourriture montrent que les chenilles deLymantria dispar L. sont sensibles à tous les virus employés. Les données permettent d’établir que la souche américaine de virus de la polyédrose nucléaire et la souche japonaise de virus cytoplasmique deL. dispar sont les plus virulentes.

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Presented at the 1st meeting of the “Working Group onLymantria dispar, O. I. L. B.”, Tempio Pausania, Italy, October 16–17, 1969.     

Pheromonotropic activity in the gypsy moth Lymantria dispar: evidence for a neuropeptide

The brain-suboesophageal ganglion complex of the gypsy moth, Lymantria dispar, contains pheromonotropic activity detectable using a Helicoverpa zea in vivo bioassay for pheromone-biosynthesis-activating neuropeptide. Pheromonotropic activity was detected as early as the third larval instar and was present throughout development and through day 6 post-eclosion. Activity in the adult is presumably associated with pheromone production, while it is speculated that larval activity may be related to melanization. Adult pheromonotropic activity is associated with a peptide of approximately 3.500 kDa. It is heat labile and only partially stable when incubated at 35°C or exposed to freeze-thawing. Isolation of L. dispar pheromonotropic factor should facilitate the elucidation of the mechanism of pheromone production in this insect pest.Abbreviations ED ₅₀ dose at which one-half maximal response is observal - eq equivalent - MRCH melanization and reddish colorization hormone - MW molecular weight - PBAN pheromone biosynthesis activating neuropeptide - SOG suboesophageal ganglion - TFA trifluoroacetic acid - Z11-16: Ald (Z)-11-hexadecenal     

Effect of potassium phenosan on structure of plasma membranes of mice liver cells in vitro

The effect of synthetic anti-oxidant potassium phenosan (PP, potassium salt of β-(4-hydroxy-3,5-ditretbutil-phenyl)-propionic acid) on the structural state of the surface (8 Å) and deep (20–22 Å) lipid regions of plasma membranes of mice liver cells was studied by spin probes method in vitro in a wide range of concentrations (10^?5–10^?21 M). Two stable free radicals, 5- and 16-doxyl-stearic acids (C₅ and C₁₆), were used as spin probes. The nonlinear polymodal dose-effect dependences were obtained for parameters that characterize the microviscosity of the lipid bilayer (τ_c) in the site of localization of the probe C₁₆, and the order parameter (S), which characterizes the stiffness of the surface layers of lipids in the site of localization of the probe C₅. Statistically a reliable increase was observed for parameter τ_c after addition of PP at concentrations 10^?5–10^?7 M and 10^?18–10^?19 M, and for parameter S after addition of PP at concentrations 10^?6–10^?7 M and 10^?13–10^?15 M. Peaks on both dose-effect curves were separated by the intervals of concentrations where PP had no effect on the studied physico-chemical characteristics of biomembranes. For PP concentrations which caused maximal changes in τ_c and S, we investigated thermal dependence of these parameters and determined the thermally induced structural transitions. Comparing with control, ultra-low doses of PP (10^?13–10^?15 M) and (10^?18–10^?19 M) caused an appearance of additional thermally induced structural transition in the surface and deep regions of plasma membrane lipids. The possible role of the interaction of PP molecules with specific binding sites on plasma membranes and formation of nanoparticles of PP in very dilute aqueous solutions are discussed.     

A comparative study of three closely related cytoplasmic polyhedrosis viruses

A cytoplasmic polyhedrosis virus (CPV) from the pine caterpillar, Dendrolimus spectabilis, was compared with Japanese isolates of closely related viruses from the silkworm, Bombyx mori, and gypsy moth, Lymantria dispar. The sizes of the viral RNA genome segments were almost identical, although the CPVs from D. spectabilis and L. dispar could be distinguished from the silkworm virus by a small size difference (0.03 × 10⁶ daltons) in one segment. The same viruses were also distinguishable by RNA homology differences of 25–50% measured by the reannealing of ³H-labeled single-stranded viral messenger RNA (synthesized in vitro) to heat-denatured viral double-stranded RNA. Antigenic differences were also detected by gel immunodiffusion tests. CPVs of D. spectabilis and L. dispar were indistinguishable by these criteria.     

Activity of lymantriid NPVS from the People's Republic of China against North AmericanLymantria Dispar

Six different geographical sources ofLymantria dispar (L.) andLymantria mathura (L.) in the People's Republic of China were sampled for nucleopolyhedrosis virus. Comparative bioassays of theL. dispar sources showed the U.S. Hamden standard to be more effective than the PRC nucleopolyhedrosis viruses. TheL. mathura NPV equalled the effectiveness of the Hamden standard even after 3 passages in lab rearedL. dispar larvae. This is the 1st report ofL. mathura NPV effect on a U.S. strain ofL. dispar.

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Résumé Six populations deLymantria dispar L. et desLymantria mathura L. provenant de différentes régions de République populaire de Chine furent examinées pour la présence de virus de la nucléopolyhédrose. Des essais comparatifs sur ces populations deL. dispar ont démontré que l'étalon de virus du “U.S. Hamden” est plus actif que ceux de la Chine. Le NPV deL. mathura, après passages successifs dans des larves en élevage en laboratoire fut également actif par rapport à l'étalon “Hamden” après le 3ème passage. Ceci est le premier rapport concernant l'effet du NPV deL. mathura contre une population deL. dispar d'origine américaine.

     

Molecular characterization and functional analysis of cyp11a and cyp11b in black rockfish (Sebastes schlegelii)

The cyp11 includes cyp11a and cyp11b in most mammals and teleosts, encoded cholesterol side chain lyase and 11β-hydroxylase, respectively. It is essential in steroid hormone synthesis. However, studies on the regulation of cyp11 are limited, especially in teleosts. In this study, the molecular characterization and function of cyp11a and cyp11b of black rockfish was investigated. Both of them showed high homology with other teleost counterparts by phylogenetic analysis. The expression of cyp11a and cyp11b exhibited a clear sexually dimorphic pattern, with a higher expression level in testis than that of in ovaries. During the different developmental stages (40 dpf, 80 dpf, 190 dpf, 360 dpf, 720 dpf), the expression of cyp11a was earlier than cyp11b. In situ hybridization results showed that cyp11a and cyp11b were mainly expressed in oogonia and oocytes of the ovary. They were located in spermatogonia and interstitial compartment in the 1.5-year-old gonads, and spermatocytesgonia and the peritubular myoid cell of the testis in the 2.5-year-old gonads. To explore the distinct roles of cyp11a and cyp11b in gonads, oestrogen and androgens were used to stimulate the primary testicular and ovarian cells. The expressions of cyp11a and cyp11b were tested under different dose of 17α-methyltestosterone (17α-MT) and 17β-estradiol (E2). The results showed cyp11a was significantly increased at 10⁻⁶ mol ml^–1 of 17α-MT and 10⁻⁸ mol ml^–1 of E2 in ovary and 10⁻¹⁰ mol ml^–1 of 17α-MT and E2 in testis, while cyp11b was significantly decreased after 17α-MT and E2 treatment. These results indicated that cyp11a and cyp11b were likely to have different functions, and also implied they might play an important roles in the differentiation of gonads and the synthesis of steroids in black rockfish.     

Cold hardiness of Lymantria monacha and L. dispar (Lepidoptera: Erebidae) eggs to extreme winter temperatures: implications for predicting climate change impacts

1. It has been predicted that temperature increases of 3.6–5.8 °C would shift the northern distribution limit of Lymantria monacha (Linnaeus) and Lymantria dispar (Linnaeus) by 500–700 km, but these predictions ignore the effects of minimum winter temperatures. It was hypothesised that winter cold can limit range expansion due to high egg mortality in cold temperatures. 2. The present study determined the supercooling points of overwintering eggs of these forest pests, and compared these with recent minimum winter temperatures in the areas of origin of three populations. Eggs from one L. monacha and one L. dispar population from the species' core distribution area in Germany were included, as well as L. monacha eggs from Finland, near the northern border of the species' distribution. 3. The median supercooling points of both species were more than 10 °C lower than the median minimum winter temperatures of their areas of origin, and the median supercooling points of Finnish and German L. monacha eggs did not differ significantly. The median supercooling point of German L. monacha eggs differed from that of German L. dispar eggs. 4. Previous literature on the topic is referenced, and translations of the old German and Russian sources are given. Based on these results, it is argued that the frequent claim that L. monacha eggs can survive cold down to ?40 °C is unsupported, with a value near ?30 °C being a more likely limit. 5. Winter cold alone can limit the predicted range shifts of these species to 200–300 km under 3.6–5.8 °C increase scenarios, which is less than half the value of earlier estimates.