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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.

A structure-function study was performed on the synthetic 21 residue neuropeptide, Lymantria testis ecdysiotropin (LTE), originally isolated from brains of Lymantria dispar pupae. The peptide induces ecdysteroid synthesis by testis sheaths of various lepidopteran species. LTE, as well as synthetic LTE 1-11, 11-21, and 11-15, stimulated synthesis in larval and pupal testes of Lymantria dispar at concentrations of 10(-9) to 10(-15) M; LTE 16-21 was weakly active, and an elongated LEU-LTE was inhibitory to synthesis at all but extremely low concentrations (10(-15) M). Since the sequence and polarity of residues in LTE 1-11, 11-15, and 11-21 are quite different, several parts of the molecule must activate receptors which initiate the cascade, resulting in ecdysiogenesis in Lepidopteran testes.

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.

14C-penfluron was used in the study of its metabolism in Musca domestica L. After injection of 298,000 dpm (11 μg)/female, the distribution of radioactivity was determined and percentages were calculated on the basis of the total injected amount. At 72 h posttreatment, the recovery was 95.2% in treated flies, 94.6% as unchanged penfluron, 0.57% as conjugates, and 2% as conjugates in the excreta. At one wk posttreatment, recovery was 87% in treated flies, 86.2% as unchanged penfluron, 0.82% as conjugates, and 5.6% as conjugates in the excreta. The conjugates consisted of 4 metabolites that upon acid hydrolysis yielded a single relatively nonpolar metabolite which was assumed to be a hydroxylated derivative of penfluron.

Summary Testes from young fifth instar Ostrinia nubilalis produced very small amounts of ecdysteroids while those from larvae that had purged their gut produced considerably more immunodetectable ecdysteroid in vitro. Larval testes that had fused produced 2.2 times more ecdysteroid than those that remained separate. It was the sheath of the testes rather than the contents that was physiologically active. Synthesis was questionable in testes from day-1 pupae and was not observed in testes from pharate pupae, from day-2 pupae or from pharate adults. Thus, synthesis only occurred at specific times in the life cycle. Ecdysteroid profiles for testes from wandering larvae whose testes had fused showed a net increase in all normally observed ecdysteroids, with the greatest increase being in 20-hydroxyecdysone. For testes from day-1 pupae, the nature of the ecdysteroid profile changed after 24 h of incubation, with some ecdysteroids showing increases and other decreases. There appear to be considerable difference...

Haemolymph from female houseflies at 4, 48 and 96 h after emergence contained approximately equivalent amounts of 20-hydroxyecdysone that varied from 2.9 to 4.1 pg/μl. Polar ecdysteroids decreased with the insect age from 12.9 pg/μl at 4 h to 5.1 pg/μl at 96 h. At 48 h the haemolymph contained ecdysone and 20,26-dihydroxyecdysone in addition to 20-hydroxyecdysone and the polar materials. When ovaries were culture in vitro , the culture medium contained ecdysone as the major ecdysteroid (70%) with lesser concentrations of 26-hydroxyecdysone, 20-hydroxyecdysone, and 20,26-dihydroxyecdysone. Haemolymph from ovariectomized flies contained 20-hydroxyecdysone, some polar materials, and very little ecdysone. Thus, it appears that the ovaries are the source of ecdysone. Since all samples contained 20-hydroxyecdysone, its source is not known. Removal of the corpus allatum-cardiacum complex decreased the amounts of all ecdysteroids in the haemolymph sample. Ecdysone and 20-hydroxyecdysone were both present in amounts of 1 pg/μl and 20,26-dihydroxyecdysone was barely detectable.

Hydrolysis of a polar ecdysteroid HPLC fraction by treatment with glucuronidase revealed the presence of at least six ecdysteroid conjugates in haemolymph from pupal and pharate adult European corn borers. Conjugates of ecdysone, an ecdysteroid more polar than ecdysone, 26-hydroxyecdysone, 20-hydroxyecdysone and two ecdysteroids more polar than 20-hydroxyecdysone were detected. Titres of conjugates were maximal on day 2, 3 or 4 post-pupation and fell to lower levels on day 5. This pattern is similar to that for the corresponding free haemolymph ecdysteroids.

The spent medium from ten established cell lines was extracted and tested for ecdysteroids by radioimmunoassay. Of the seven lepidopteran lines tested, only IAL-TNDI and MRRL-CH showed evidence of ecdysteroid production. However, the results were erratic and difficult to evaluate and these lines were dropped from further consideration. However, of the three cockroach cell lines tested, one, UMBGE 4, produces ecdysteroid and consistently releases virtually all of it into the medium. The main ecdysteroid was identified as ecdysone and the increase was logarithmic during the first 11 days of the subculture, with a decrease from day 11 to day 14. UMBGE 4 is a vesicle cell line which also tested positive for chitin synthesis. When the pH of the medium was lowered from pH 7.4 to pH 6.3, both the chitin synthesis and the ecdysone synthesis dropped by roughly 50%.

The haemolymph ecdysteroids were examined in fifth-stage larvae of Nezara viridula, Podisus maculiventris and Dysdercus cingulatus (Hemiptera-Heteroptera) using high-pressure liquid chromatography to separate the ecdysteroids and a radioimmunoassay to detect the fractionated ecdysteroids. The length of the fifth stage ranged from 5 to 8 days, and a peak in ecdysteroid titre (1700–2650 ng/ml) occurred 2–3 days prior to ecdysis to the adult. An ecdysteroid matching the retention time of makisterone A (24-methyl-20-hydroxyecdysone) was clearly present in haemolymph taken at the time of peak titre in all 3 of these true bugs, whereas little, if any, ecdysone or 20-hydroxyecdysone was detected. These data, along with previously reported data for the milkweed bug Oncopeltus fasciatus, are persuasive evidence that makisterone A is the larval moulting hormone of a group of closely related Heteroptera called the Trichophora (Lygaeoida, Pentatomoidea, Pyrrhocoroidea and Coreoidea).

Testes or the isolated sheaths of testes from late-last-instar larvae of the tobacco budworm, Heliothis virescens, spontaneously secreted ecdysteroids in vitro. Exogenous 20-hydroxyecdysone, incubated with the testes or isolated testis sheaths, functioned in a local positive feedback system to increase production of the several ecdysteroids typical of H. virescens testes. The increase in ecdysteroid production by testis tissue was dependent on discrete ranges in exogenous ecdysteroid titer. Activation ratios of 2–5 resulted from exogenous 20-hydroxyecdysone titers of 0.5 to 15 pg/μl, while activation ratios of 10–30 resulted from exogenous titers of 15–100 pg/μl of 20-hydroxyecdysone. Activation ratios approached 200 with exogenous titers of 100–1,000 pg/μl of 20-hydroxyecdysone.

1. 1. Haemolymph ecdysteroid titers of nondiapause-bound European corn borers (LD 16: 8, 30°C) were low at the beginning of the fifth (last) instar, increased gradually between days 2 and 4 and peaked sharply (4400 pg/μl) just before pharate pupal formation. 2. 2. Time of day influenced prepupal ecdysteroid titers in that levels rose sharply between 14:00 and 24:00 hr AZT (arbitrary Zeitgeber time) in nondiapause-bound animals. 3. 3. Haemolymph ecdysteroid titers decreased in pharate and new pupae, peaked in 2-day pupae (26,000 pg/μl) just before pharate adult formation and then fell more gradually in 3–5-day pupae. 4. 4. In diapause-bound and diapausing 5th instars haemolymph ecdysteroid titers were relatively low, 11–25 pg/μl 5. 5. HPLC followed by RIA of 5th stage larval and pupal haemolymph revealed the presence of a polar peak, 2 small peaks following the polar peak, material that comigrated with 20-hydroxyecdysone and ecdysone, and in 2- and 3-day pupae, material that comigrated with 26-hydroxyecdysone. 6. 6. Before formation of the pharate pupa and pharate adult, ecdysone levels were relatively low as compared to 20-hydroxyecdysone levels.

Total ecdysteroid levels as well as concentrations of several individual ecdysteroids were determined for hemolymph and testes of fifth instars, pupae, and pharate adults of the European corn borer, Ostrinia nubilalis (Hubner). For total levels, the patterns of fluctuation in hemolymph and testes were similar, but the concentrations in testes were lower than those in hemolymph. In both hemolymph and testes there were two ecdysteroid peaks: the first just prior to the formation of the pharate pupa, the second just prior to the formation of the pharate adult. An examination of ecdysteroid profiles revealed some important differences. Ecdysone was either absent or present at extremely low levels in larval testes, whereas in hemolymph there was a premolt ecdysone peak. In pupal testes, ecdysone was present, but levels of 26-hydroxyecdysone were much lower than those in hemolymph. Thus, in regard to ecdysteroids, testes have the ability to control their own internal milieu.

Testes of the gypsy moth, Lymantria dispar, secrete ecdysteroid in vitro during the silk-spinning pharate pupa stage and in mid to late adult development during the pupal stage. Low titers (3–6 pg/μl) of exogenous 20-hydroxyecdysone are required to elicit endogenous ecdysteroid production. Testis ecdysiotropin (TE) was detected in acid methanolic extracts of brains of immobile pharate pupae and mid-development pupae. It was possible to activate early last-instar testes of both L. dispar and Heliothis virescens to synthesize ecdysteroid by exposing them to TE prepared from pupae of either species. Although testes of L. dispar contained no detectable ecdysone, they were able to convert (3H) ecdysone to 20-hydroxyecdysone and a highly polar fraction. The active ecdysteroid product of L. dispar testes is probably 20-hydroxyecdysone.

The ovaries of adult Drosophila melanogaster were shown to produce and secrete ecdysteroids in vitro by an ecdysteroid radioimmunoassay. The major secretory products were determined by HPLC/RIA analysis to be ecdysone, 20-hydroxyecdysone, and a highly polar fraction. Based on competitive binding studies, the secreted ecdysteroids contain 2–3 times more 20-hydroxyecdysone than ecdysone.

European corn borer testes were cultured in vitro to determine the effects of physiological doses of ecdysteroids on apyrene spermiogenesis. Ecdysone and 20-hydroxyecdysone stimulated spermiogenesis in larval testes excised 4–5 days after ecdysis to the last instar. The stimulatory effect on the testes was direct. Both the degree of larval maturation and the concentration of ecdysteroid in the incubation medium affected the amount of stimulation. Promotional capability of 20-hydroxyecdysone was greatest in wandering larvae that had not yet completed gut purge. Spermatocysts from larval testes taken 3 days after ecdysis were refractory to stimulation. Determination of 20-hydroxyecdysone levels in larval, pharate pupal and day-1 pupal haemolymph confirmed that the observed stimulation occurred at physiological doses.

A procedure suitable for sterilizing both sexes of Anthonomus grandis grandis Boheman on a large scale was developed. First, the weevils were fumigated with bisazir ( P,P -bis(1-aziridinyl)- N -methylphosphinothioic amide, AI3-61585) at atmospheric pressure for 90 min, and 30 min later, they were dipped for 5 sec into a 1.35% solution of penfluron (2,6-difluoro- N -[[[4-(trifluoromethyl) phenyl]amino]carbonyl]benzamide, AI3-63223) in acetone. In either sex, the sterility exceeded 99%, and 59.3% of the treated males survived 10 days posttreatment. A new laboratory-size fumigation chamber with a rotating bisazir-treated paddle was designed and scaled up to a field-size chamber suitable for treating up to 200,000 weevils at a time. Monitoring of the sterilizing treatment consisted of measuring the concentration of bisazir vapors during the fumigation and analyzing the treated weevils for residues of bisazir and penfluron. One day posttreatment, no residues of bisazir or of its aziridinyl metabolite could be detected in the treated weevils, but penfluron residues decreased only slowly from 39 to 8.8 μg/weevil within 7 days. Oviposition of untreated females mated to treated males was decreased; also, treated males were less effective than untreated ones in transferring sperm. On the other hand, when various ratios of treated and untreated males were combined with untreated females, the treated males appeared more competitive because they suppressed reproduction more effectively than their proportional number would warrant. The development of the new sterilizing procedure now makes it possible to test the performance of chemosterilized weevils in large field tests.

The major ecdysteroids of last-stage Oncopeltus fasciatus larvae were determined by HPLC/RIA analysis. An ecdysteroid other than ecdysone or 20-hydroxyecdysone, probably makisterone A, was the major ecdysteroid in hemolymph and in whole body extracts of 4- to 5-day-old larvae, when hemolymph ecdysteroid titers were at their peak. Little, if any, ecdysone or 20-hydroxyecdysone was detected in the hemolymph. Bioassay of the relative activities of ecdysone, 20-hydroxyecdysone, and makisterone A in last-stage larvae revealed that makisterone A was ten times more active than 20-hydroxyecdysone, and that ecdysone was inactive at doses as high as 1 mg/ml, its limit of solubility in 10% ethanol. Thus, it is suggested that makisterone A is the molting hormone in Oncopeltus. The implications of this discovery are discussed.

The ecdysteroid and juvenile hormone titre of 4th and 5th-instar larvae of Trichoplusia ni was determined by radioimmunoassay and the black mutant bioassay. Application of 50 nmol juvenile hormone II accelerated by several hours the delayed peak of ecdysteroids occurring in neck-ligated larvae. Inject of 20 nmol of 20-hydroxyecdysone initially accelerated prepupal development and later blocked the process. Application of 100 nmol O-ethyl-S-phenol-phosphoroamidothiolate (EPPAT) to post-wandering larvae inhibited the juvenile hormone esterase activity occurring at the time of normal juvenile hormone decline, and indirectly maintained an elevated ecdysteroid titre at the time when the controls moulted. Most of these EPPAT-treated prepupae were delayed in normal prepupal development and/or failed to normally ecdyse. Thus, the interaction of juvenile hormone, its esterase and ecdysteroids is an important aspect of prepupal development in T. ni.

The metabolism of 14C-penfluron (2,6-difluoro- N -[[[4-(trifluoromethyl)phenyl]amino]14C-carbonyl]benzamide) was studied in Anthonomus grandis grandis Boheman. Recoveries of radioactivity of 99 and 94.36% were obtained after 4 days and 1 week, respectively, from weevils that had been injected with 124,970 dpm (4.61 μg/weevil). On the basis of the total injected amount, 97.74% of the label recovered from the extract of weevils 4 days posttreatment were unchanged penfluron and 0.16% was conjugates. Similarly at 1 week posttreatment, 90.56% were unchanged penfluron and 0.2% was conjugates. Less than 1% of additional radioactivity was recovered from weevil residues in both experiments. Four days posttreatment, recovery from excreta extract was 1.02%; 0.33% was unchanged penfluron; 0.06% was 2,6-difluorobenzamide; and 0.63% was conjugates. Similarly, at 1 week posttreatment the recovery from excreta extract was 3.41%; 1.33% were unchanged penfluron; 0.20% was 2,6-difluorobenzamide; and 1.87% were conjugates. The conjugates consisted of 2 metabolites which upon acid hydrolysis yielded a single relatively nonpolar metabolite that was assumed to be a hydroxylated derivative of penfluron.

Radioimmunoassay (RIA) of whole body extracts of Drosophila melanogaster males and females demonstrates that at eclosion all individuals contain high levels of ecdysteroid. Highly polar ecdysteroids (presumably metabolites) in the meconium represent approximately half of the total ecdysteroid RIA-activity present at this time and are subsequently eliminated. Ecdysteroids remaining after the elimination of the meconium are also highly polar as shown by reverse-phase high pressure liquid chromatography (RP-HPLC). The amount of ecdysteroid RIA-activity found in whole body extracts declines in both sexes until 18 h post-eclosion when levels begin to increase in the female and drop to undetectable levels in the male. In the female the ovaries are the major source of ecdysteroid. The increase in whole body ecdysteroid in the female coincides with the initiation of ovarian ecdysteroid production and accumulation. Topical application of methoprene, a juvenile hormone (JH) analog, stimulates ovarian ecdysteroid synthesis in apterous-ts 78j (ap ts 78j ) , a temperature-sensitive juvenile hormone-deficient mutant, corroborating previous results suggesting a role of juvenile hormone in ovarian ecdysteroid production. Stage 8–9 follicles, whose development is juvenile hormone dependent, are shown to be the most active in ecdysteroid production. The regulatory potential of these stages is discussed.

Testes from late last stage larvae of the tobacco budworm, Heliothis virescens, were incubated with [3H]ecdysone and [3H]cholesterol. [3H]Ecdysone was converted to six other major ecdysteroids, identified by cochromatography in reverse-phase high-pressure liquid chromatography (RPHPLC); four of them were verified by normal-phase HPLC. A highly polar fraction, moderately polar ecdysteroids (20,26-dihydroxyecdysone, 3-epi-20-hydroxyecdysone, and 20-hydroxyecdysone) and low-polarity ecdysteroids, including 2-deoxyecdysone, were detected after incubation with [3H]ecdysone. Compounds that reacted positively to antibodies to progesterone and testosterone were detected in the low-polarity fractions. Testes were incubated in fractions corresponding to each of the major ecdysteroid peaks derived from [3H]ecdysone metabolism. Although most of the radioactive ecdysteroid fractions were further metabolized to high- and low-polarity endpoints, 88% of the [3H]20-hydroxyecdysone peak apparently remained unmetabolized. 20-Hydroxyecdysone may be the primary ecdysteroid product of testes of H. virescens. [3H]Cholesterol was not metabolized to any appreciable extent.

Developing ovarian follicles of the silkmoth Hyalophora cecropia accumulate large amounts of ecdysteroids during oogenesis. As measured by an ecdysteroid radioimmunoassay (RIA), this accumulation begins near the end of vitellogenesis, just prior to nurse cell collapse, and continues through the beginning of chorion formation. Analysis of ovarian ecdysteroids by a combination of high-performance liquid chromatography and RIA demonstrates that the major proportion of these are present in a highly polar form, most likely as conjugates; ecdysone and 20-hydroxyecdysone were present as well, in much lower proportions. Light microscopic autoradiographs of photoactivated follicles after in vivo incubation with [3H]ecdysone indicate that within the oocyte ecdysteroids are associated with the yolk sphere membranes.

Oostatic hormone, prepared from mature female house flies, Musca domestica, was examined for its effect on processes mediated by egg development neurosecretory hormone (EDNH). When injected into newly emerged adult female mosquitoes, Aedes atropalpus, oostatic extracts inhibited vitellogenic growth of the ovaries. Comparison of oostatic activities in heads, thoraces, and abdomens of house flies showed that the major portion was present in the abdomens, apparently associated with the ovaries. In contrast to untreated vitellogenic females, no ecdysone or 20-hydroxyecdysone could be detected in female mosquitoes injected with the oostatic extract. Vitellogenic growth and ecdysteroid levels were suppressed in decapitated females injected with a mixture of extracts containing EDNH and oostatic activities. Since the normal vitellogenic and steroidogenic responses to EDNH can be suppressed by simultaneous injection of EDNH and oostatic material, it is suggested that oostatic hormone acts at a level subsequent to EDNH release. However, we cannot rule out at this time that EDNH release, itself, is also affected.

In male Musca domestica L., N2,N2,N4,N4-tetramethylmelamine (TMM) was metabolized to N2,N2,N4trimethylmelamine and N2,N4-dimethylmelamine. The absence of the other isomeric trimethyl- and dimethylmelamines among the metabolites indicated that the dimethylamino group was demethylated in preference to the methylamino group. Since more than 80% of the originally injected TMM was recovered as metabolites containing the s-triazine ring, the main metabolic pathway was a demethylation and did not involve ring cleavage.

A closed fumigation system with circulating atmosphere saturated with vapors of tepa, thiotepa (tris (l-aziridinyl) phosphine sulfide), hempa, thiohempa (hexa-methylphosphorothioictriamide), bis (l-aziridinyl)- N methyl-phosphinothioic amide, or bis (l-azilidinyl)- N -methylphosphinothioic amide was used to stetilize male Musca domestica L. Chemical and physical characteristics of the chemosterilant affected the fumigation period required for 50 or 95% sterilization (ST50 and ST95), but the vapor pressure of a sterilant was not directly proportional to its effectiveness. On the other hand, an increase in ambient temperature or in the treatment period invariably increased sterility. The uptake of hempa or thiohempa by treated flies was similar to the injection dose needed for the same degree of sterility, and each compound was metabolized to a different extent during the fumigation period. The closed fumigation system is safe to the operator, and the procedure is rapid and economical both in use of sterilant and in handling of insects. For mass treatments, the equipment could be easily scaled up.

The chemosterilant hemel is metabolized by male Musca domestica L. to lower methylmelamines and to other, possibly acyclic, products. The methylmelamines isolated from treated flies and from their excreta were identified as: pentamethylmelamine, N2, N2, N4, N6 -tetramethylmelamine, N2, N4, N6 -trimethylmelamine, and N2, N2, N4 -trimethylmela. mine. Mono- and dimethylmelamines were not detected and unchanged hemel was found only in the flies. In 24 hours, the treated flies converted about 11% of the methyl carbons of hemel to respiratory CO2. Hemel uniformly labeled with C14 in the methyl groups was synthesized and used in the initial experiments, but the identity of the methylmelamine metabolites was confirmed by isolating them and comparing them with synthetic reference compounds. Also, the sterilizing activity of all trio, tetra, and pentamethylmelamines was determined. The most active methyl melamine, N2, N2, N4, N4 -tetramethylmelamine, was not found in the treated flies or in their excreta.

Screening 852 compounds as additives to the diet of Musca domestica L. yielded 60 active sterilants, 33 of them aziridines. Other categories of known chemosterilants,, i.e., phosphor amides, melamines, N -mustards, boron compounds, ureas, and triazoles, were also represented among the active compounds.

Tepa uniformly labeled with C14 with a specific activity of 12 mc/mmole was used to study the metabolism and mechanism of sterilization in male house flies, Musca fomestica L. A wet-combustinon method was developed for C14-labeled compounds which gave quantitative recovery and reproducible results. The injection of C14-labeled tepa was adjusted to about 1 µg per male fly. Total recovery of radioactivity from treated flies and their excreta was almost quantitative; radioactivity in respiratory CO2 was only 0.75% of the injected dose during the first 12 hours. When male flies were allowed normal activities after an injection of about 1 µg of C14-labeled tepa, 50% of the dose was retained in the fly 5 hours after treatment. Radioactivity was present in treated flies as tepa or aziridinyl metabolites; radioactive metabolites in the excreta did not contain aziridinyl groups. Radiometric and colorimetric determinations indicated that treated flies retained about 9 and 5%, respectively, of the injected dose over prolonged periods. Radioactivity was transferred to female flies by copulation with treated males. Because this radioactivity was found within the female body and not on its surface, we concluded that the transfer was by copulation rather than by body contact. However, no detectable radioactivity was found in the sperm or the seminal fluid present in the spermathecae of inseminated females. An electron microscopic scrutiny of house fly sperm failed to reveal any structural changes brought about by the chemosterilant.

When hempa was uniformly labeled with C14 and its metabolism in male house flies, Musca domestica L., was studied, total recovery of radioactivity from treated flies and their excreta was about 90%. The recovery of radioactivity from respiratory CO2 was only 1.25% of the injected 5.71 μg of C14-hempa/fly during the first 24 hours. Radiometric and colorimetric determinations indicated that 2.5-3 hours after treatment, treated flies retained 50% of the dose and that at the end of 24 hours they retained about 2-3% of the dose. The treated flies were 50% sterile 4 hours 45 minutes after treatment and completely sterile about 7 hours after treatment. The only major metabolite of hempa found in treated flies and in their excreta was pentamethylphosphoric triamide. Unchanged hempa and its metabolite were separated from the extracts of treated flies and their excreta by thin-layer chromatography, gas-liquid chromatography, and radiochromatography. For identification, the 2 compounds were isolated by thin-layer chromatography, and their structure was confirmed by infrared spectroscopy.

Testes of last-instar larvae of the tobacco budworm release five times more ecdysteroid into incubation medium (judged by radioimmunoassay) in 2.5 hours than is found in testis homogenates. Incubation of testicular components indicates that the testis sheath may be the site of ecdysteroid synthesis. Fractionation of hemalymph, testis homogenate, and incubation medium by high-performance liquid chromatography produces a distinct ecdysteroid pattern in each case. Thus, released testis ecdysteroids are probably converted to other forms for use, sequestration, or general circulation. Their functions are unknown.

When sterilizing doses SD50 and SD95 of 10 substituted bis (1-aziridinyl) phosphine oxides and 10 substituted bis (1-aziridinyl) phosphine sulfides were determined in male Musca domestica L., the alkylamino-substituted compounds were the most effective sterilants, followed by the alkylthio-, alkoxy-, and alkyl-substituted analogues. Within each homologous series, activity decreased with increasing size of the alkyl group. Only minor differences in activity were found between analogous phosphine oxides and sulfides.

The brain neuropeptide prothoracicotropic hormone (PTTH) is an ecdysiotropin that drives post-embryonic growth and development through the stimulation of ecdysteroid production by the prothoracic glands (PG) (Gilbert et al., 1980). Research to isolate PTTH has utilized primarily two species, Bombyx mori (Ishizaki and Suzuki, 1984) and Manduca sexta (Bollenbacher and Granger, 1985). Characterization and isolation studies have used both in vivo (Bombyx, Ishizaki et al, 1983, Nagasawa et al, 1984a; Manduca, Kingan, 1981) and in vitro (Bombyx, Nagasawa et al, 1984a; Okuda et al, 1985; Manduca, Bollenbacher et al, 1979) bioassays. Recent work on Bombyx and Manduca PTTH demonstrates that biological activity is associated with two molecular-weight ranges of ca. 20–28 kD and ca. 4–7 kD (Ishizaki et al., 1983, Bollenbacher et al., 1984). A family of 4 kD PTTH’s has been isolated from Bombyx (Nagasawa et al., 1984b).

The effects of juvenile hormone (JH) and egg development neurosecretory hormone (EDNH) on ovarian ecdysteroid production during vitellogenesis in the autogenous mosquito, Aedes atropalpus , were investigated using in vitro techniques coupled with radioimmunoassay (RIA) and high-pressure liquid chromatography (HPLC). Normal females were characterized by quantitative, qualitative, and temporal patterns of in vitro ovarian ecdysteroid production. Females decapitated at emergence showed little ovarian ecdysteroid production and did not undergo vitellogenesis. A 500-ng dose of JH-I applied topically to decapitated females restored normal patterns of ecdysteroid production. In both normal and experimental females, ecdysone constituted the major portion of the ecdysteroids secreted by the ovaries in vitro . However, significant amounts of other RIA-active materials were detected, one of which was probably 20-OH-ecdysone. Fat body incubations indicated that these tissues produce little RIA-active material during the peak of vitellogenesis. During that period, the ovaries were the major source of ecdysteroid. Various doses of JH-I, applied to adbomens isolated at emergence, enhanced ovarian responsiveness to subsequent applications of head extracts containing EDNH both in vivo and in vitro . A 500-ng dose of JH-I, applied topically to blood-fed, decapitated Aedes aegypti , stimulated a significant increase in in vitro ovarian ecdysteroid production. Similarities between these data and those demonstrating prothoracicotropic effects of JH in the Lepidoptera are discussed.

Brain neuropeptides induce target glands to secrete ecdysteroid in two well-characterized insect systems: the prothoracicotropic hormone (PTTH) activates prothoracic glands to produce ecdysone, and the egg development neurosecretory hormone (EDNH) activates ovaries to produce ecdysteroid (reviewed by Wigglesworth, 1985). Bollenbacher et al. (1979) studied the PTTH system isolated in vitro by incubating brain extracts containing PTTH with prothoracic glands, detecting the resultant ecdysteroid by radioimmunoassay (RIA) (Borst and O’Connor 1972). Similarly, Hagedorn et al. (1979) elicited immunoreactive ecdysone production directly from mosquito ovaries by incubating them with head extracts containing EDNH. Testes of Heliothis virescens produce ecdysteroid in vitro at specific periods during development (Loeb et al. 1984). Incubation with a peptide-like factor from male brains can initiate ecdysteroid synthesis by testes in vitro.

Makisterone A, a 28-carbon (C-24 alkyl) hexahydroxy steroid, has been identified by mass spectrometry as the major ecdysteroid in last-stage larvae of the large milkweed bug,Oncopeltus fasciatus, a phytophagous hemipteran. Similarly, it is a major molting hormone in 2 phytophagous and 1 predacious species of Hemiptera belonging to the group, Pentatomomorpha. It is not, however, a major ecdysteroid in another group of Hemiptera, the Cimicomorpha, where 1 predacious and 2 hematophagous species contain ecdysone and 20-hydroxyecdysone as their major molting hormones.

Chemosterilizing activity of 41 bis(1-aziridinyl)phosphine oxides and sulfides was determined in male Musca domestica L. by the circulatory fumigation technique. In homologous series, the methyl and ethyl compounds were more effective than the higher alkyl or aryl compounds; and in analogous pairs, the phosphine sulfides were more active than the oxides. Two monoaziridines also were highly active. However, no direct relationship between the effectiveness of compounds injected or used as fumigants was evident.