Effects of precocene II on female-specific hemolymph polypeptides in Oncopeltus fasciatus

Using polyacrylamide gel electrophoresis (PAGE) under denaturing conditions, two major polypeptides of 200,000 and 170,000 daltons were detected in the hemolymph of mature female Oncopeltus fasciatus, but they were not found in the hemolymph of males or newly emerged females. Those polypeptides constituted the two major bands of early vitellogenic oocytes; however, they were absent from the yolk of mature eggs. The slower-migrating band (200,000 daltons) appears to correspond to a vitellogenic protein already identified in O. fasciatus, whose synthesis has been suggested to be independent of juvenile hormone (JH). Treatment of newly emerged adult females with the corpus allatum cytotoxin precocene II prevented the appearance of the female-specific bands and induced an important accumulation of other proteins in the hemolymph. Yolk deposition was also inhibited in those animals. Topical application of JH to precocene-treated females restored the appearance of the 200,000 and 170,000 dalton polypeptides in the hemolymph. These results suggest that JH is required for the synthesis of female-specific polypeptides in O. fasciatus.     

Variations induced by juvenile hormone and precocene II in the metabolic rate of 3 species of insects

The effects of juvenile hormone and precocoene II on the metabolic rate of Blattela germanica L. (Blattaria, Blattellidae), Spilostethus pandurus Scop. (Heteroptera: Lygaeidae) and Oncopeltus fasciatus Dallas (Heteroptera: Lygaeidae) 5th instar nymphs and adults have been investigated. Direct volumetric method was used to determine the oxygen consumption rate. JH produced a stage and/or sex dependent increase in the metabolic rate according to the species considered. On the other hand, PII decreased oxygen consumption in Blattella germanica and S. pandurus fifth instar and males, whereas O. fasciatus metabolic rate was not affected by the treatment.     

Effect of ecdysterone on ethyl trimethyl dodecadienoate juvenile hormone action in Oncopeltus fasciatus

Ethyl trimethyl dodecadienoate exhibited a strong juvenile hormone action in large milkweed bug larvae (Oncopeltus fasciatus) whereas ecdysterone caused minimal juvenilizing effects. Topically applied ethyl trimethyl dodecadienoate was not synergized by simultaneously injected ecdysterone.     

Mating behaviour in Oncopeltus fasciatus (Dallas): effects of diet, photoperiod, juvenoids and precocene II

ABSTRACT. When adults of the large milkweed bug, Oncopeltus fasciatus (Dallas) were starved or fed non-host seeds, their mating activity during the 30 days following emergence was reduced by c. 50%. Topical applications of a juvenoid to adults fed non-host seeds usually increased mating activity, sometimes to near the level of milkweed-fed controls. An optimal juvenoid dose applied to adults reared and fed on sunflower seeds increased mating activity by only 9% to 28% from that of controls reared and fed on milkweed. Rearing and maintenance of the sexes on separate diets before pairing indicated that both the juvenoid-restored and non-restored milkweed stimulatory effects on mating probably acted exclusively on the males. Topical application of the anti-allatotropic agent precocene II to milkweed-fed males reduced mating activity by 75%. Simultaneous juvenoid treatment prevented most, but not all, of the precocene II effect. Juvenoid treatment completely prevented the depressed mating activity under short days. Rearing and maintenance of the sexes under different photo-periods before pairing under short days showed that photoperiod acted solely on the males. It is postulated that JH and one or more photosensitive and diet-sensitive factors in males regulate mating activity, JH being stimulatory but nonessential. A partial dietary control over mating may optimize male activity according to the population density and female reproductive activity.     

Metabolism of juvenile hormone in cultures of ovaries and fat body in the cockroachperiplaneta Americana

Summary The time course of juvenile hormone (JH) metabolism is examined in cultures ofPeriplaneta americana fat body and ovaries in medium containingManduca sexta carrier protein or cockroach hemolymph. In the absence ofM. sexta carrier protein or cockroach hemolymph, both tissues extensively catabolize exogenous [³H]JH in the medium. Addition of the carrier protein or hemolymph to the culture system prevents the hydrolysis of the hormone in the medium. Within the tissues JH is degraded whether or not carrier protein or hemolymph is present which suggests that the protective role of these molecules is exclusively extracellular. Incubation of [³H]JH with medium preconditioned with tissue results in destruction of the hormone. This suggests that the fat body secretes esterases into the medium. In contrast, the ovarioles hydrolyze the hormone by means of cell-associated enzyme. The relationship of these phenomena to insect development is discussed. This work supported by NSF Grant PCM 76-02229 and University of Kansas Biomedical Sciences Grant RR-07037.     

Lipid composition of the fat body and haemolymph and its relation to lipid release in Oncopeltus fasciatus

Lipid composition of the fat body and haemolymph of male milkweed bug, Oncopeltus fasciatus, was determined. Triglycerides were the predominant lipids of the fat body while diglycerides accounted for the major lipid in the haemolymph. Sterols, sterol esters, and non-esterified fatty acids were present in both fat body and haemolymph besides triglycerides and diglycerides. Only traces of monoglycerides were detected.Gas chromatographic analysis of the fatty acids revealed a difference in the fatty acid composition between fat body and haemolymph glycerides and sterol esters. Oleate and linoleate were the predominant unsaturated fatty acids in both fat body and haemolymph lipids and in the milkweed seeds as well.When fat body was labelled in vivo and in vitro with ¹⁴C-palmitate, the fatty acid was incorporated largely into the triglycerides. When the prelabelled fat body was incubated with a medium containing haemolymph the fat body released lipids mainly as diglycerides. Some radioactivity was observed in the triglycerides and non-esterified fatty acids also.Electrophoretic analysis of the incubation medium containing the haemolymph revealed that the released lipids were bound to three haemolymph lipoprotein bands. Lipid mobilization, release, and transport in Oncopeltus are discussed in relation to studies on other insects.     

Feeding, nutrient flow, and digestive enzyme release in the giant milkweed bug, Oncopeltus fasciatus

Abstract.  The giant milkweed bug, Oncopeltus fasciatus , is an intermittent feeder, spending most of its time in compact groups away from the food, with feeding bouts lasting 30–60 min. They macerate a small area of the seed with their stylets, secreting large amounts of saliva (which suspends and emulsifies the seed tissue), and quickly suck the emulsion into the gut. Therefore, little digestive activity occurs in the seed tissue. Freshly ingested food in the first gut region (V1) consists of 50% water, 46% lipid, 5% protein, and 1% soluble carbohydrates. The junction of the midgut and hindgut lacks a lumen in the larvae stages, and a 3 μL large oil drop (> 85% triacylglycerides) accumulates in the third gut region (V3). Approximately 24 h after the imaginal moult, this large volume of oil and faeces is defecated, and thereafter an oily faeces is defecated several times daily. It is postulated that the delayed egestion in larvae protects them against suffocation due to self-soiling with oily faeces. Serine proteases are absent in the gut and, instead, a cystein proteinase, inhibited by E64, is present (gut pH 6.0). High lipase activity was found throughout the gut, but only limited amylase activities. Basal levels of all three enzymes occurred throughout the gut, but at lower levels in starved bugs. Salivary gland extracts contain a cysteine proteinase and a lipase, but no amylase.     

Effects of juvenile hormone I and precocene I & II on Microplitis rufiventris when administered via its host, Spodoptera littoralis

We investigated the effect of juvenile hormoneI (JHI) and precocene I & II (PI & PII) onthe endo-developmental stages, reproductiveperformance and longevity of Microplitisrufiventris Kok. after topical treatment ofhost larvae of Spodoptera littoralis(Boisd.). Host larvae were treated with each ofthe test compounds by a single dose 2–4 h afterparasitization or 1 day prior to parasitoidemergence, or by repeated daily applications 2–4 h after parasitization to parasitoid emergence.JHI-treatments increased parasitoiddevelopmental time, induced developmentalabnormalities and inhibition of parasitoidegression. Interestingly, one of the causal factorsthat inhibited the parasitoid egression was themoulting of some parasitoid larvae into asupernumerary instar. Applications of PI or PIIto parasitized S. littoralis larvae didnot affect parasitoid developmental time, butsome developmental abnormalities were noted.Melanin formation in host's blood was greaterin PI-treatments than in PII-treatments.PII-treatments arrested development of somefirst instar parasitoid larvae typical of JHIexcess. Adult female wasps that emerged fromJHI-treated hosts lived for significantlylonger periods than control females, but didnot produce more offspring. The longevity andreproductive capacity of females whichdeveloped in hosts treated by precocenes werereduced compared with the females emerged fromcontrol hosts. Even the small number ofadult wasps from PII-treated hosts which livedfor the same period as control wasps, showed asignificant reduction in their reproductiveperformance. Repeated daily applications of thetest compounds reduced wasp fecundity andlongevity to a significantly greater degreethan the single applications, except for a lowdose of JHI (1 µg). The study demonstratesthat growth disrupters, such as JHs andprecocenes have a delayed developmental effect onM. rufiventris and points out the need tobe aware of these effects in implementingcontrol strategies of S. littoralis innatural habitats.     

Effects of juvenile hormone I, precocene I and precocene II on the progeny of Microplitis rufiventris Kok. female when administered via its host, Spodoptera littoralis (Boisd.)

Reproductive biologies of Microplitis rufiventris Kok. females resulting from topically treated Spodoptera littoralis (Boisd.) larvae with constant effective doses of juvenile hormone I (JHI, 1 μg), precocene I (PI, 25 μg) or PII (25 μg) were investigated. Although the female wasps were treated during their presence as eggs or larvae in their hosts, the complete effects of the test compound were not apparent until the wasps had become adults. On the bases of the obtained results, the reproductive inhibition activity caused by the test compounds comprises of two categories: (1) reduction in progeny production, and (2) induction of significant proportion of imperfect ‘non‐functional’ parasitoid progeny. Whereas, the adverse effect of JHI is only restricted to the second category, the adverse effects of PI or PII fall into both categories. Thus, workers should be aware of the delayed effects of new generations of pesticides which may occur in later stages of the non‐target insects.     

Moulting hormone,juvenile hormone and the ultrastructure of the fat body of adult Sarcophaga bullata (Diptera)

Summary In the ovoviviparous fly, Sarcophaga bullata, vitellogenesis is cyclic; a process reflected in ultrastructural changes in the fat body cells and oenocytes. At eclosion the larval fat body has not yet completely disappeared. During vitellogenesis the fat body cells are specialized for intensive protein synthesis showing a very extensive RER and numerous invaginations of the plasma membrane. These features disappear when the eggs descend into the oviducts to complete embryogenesis. The predominant feature of the oenocytes is their very prominent SER. The fat body cells of the males are never as specialized for protein synthesis as those of the females. Feeding of ecdysterone to males for 3 or more days induces a rather extensive subcellular apparatus for protein synthesis, i.e., invaginations of the plasma membrane and an extensive RER. Juvenile hormone is completely ineffective in this respect. Both ecdysterone and juvenile hormone have pronounced but different effects on the oenocytes of males.     

Comparative studies on pharmacokinetics and metabolism of the anti-juvenile hormone precocene II

The cuticular penetration and pharmacokinetics of the anti-juvenile hormone precocene II were determined in a sensitive species (Oncopeltus fasciatus) and an insensitive species (Heliothis zea). Precocene was sequestered by the fat body and slowly metabolized in Oncopeltus, but rapidly metabolized and excreted in Heliothis. Studies in vitro using inhibitors for cytochrome P-450 and for cyt P-450-NADPH-reductase, confirmed the anticipated detoxification of precocene by a mixed-function oxidase via the 3,4-epoxide. Use of the inhibitors in vivo had no influence on the metabolism of precocene.     

Insecticidal effect of Canavalia ensiformis major urease on nymphs of the milkweed bug Oncopeltus fasciatus and characterization of digestive peptidases

Jackbean (Canavalia ensiformis) ureases are entomotoxic upon the release of internal peptides by insect’s digestive enzymes. Here we studied the digestive peptidases of Oncopeltus fasciatus (milkweed bug) and its susceptibility to jackbean urease (JBU). O. fasciatus nymphs fed urease showed a mortality rate higher than 80% after two weeks. Homogenates of midguts dissected from fourth instars were used to perform proteolytic activity assays. The homogenates hydrolyzed JBU in vitro, yielding a fragment similar in size to known entomotoxic peptides. The major proteolytic activity at pH 4.0 upon protein substrates was blocked by specific inhibitors of aspartic and cysteine peptidases, but not significantly affected by inhibitors of metallopeptidases or serine peptidases. The optimal activity upon N-Cbz-Phe-Arg-MCA was at pH 5.0, with complete blockage by E-64 in all pH tested. Optimal activity upon Abz-AIAFFSRQ-EDDnp (a substrate for aspartic peptidases) was detected at pH 5.0, with partial inhibition by Pepstatin A in the pH range 2-8. Fluorogenic substrates corresponding to the N- and C-terminal regions flanking a known entomotoxic peptide within urease sequence were also tested. While the midgut homogenate did not hydrolyze the N-terminal peptide, it cleaved the C-terminal peptide maximally at pH 4.0-5.0, and this activity was inhibited by E-64 (10 ??M). The midgut homogenate was submitted to ion-exchange chromatography followed by gel filtration. A 22 kDa active fraction was obtained, resolved in SDS-PAGE (12%), the corresponding band was in-gel digested by trypsin, the peptides were analyzed by mass spectrometry, retrieving a cathepsin L protein. The purified cathepsin L was shown to have at least two possible cleavage sites within the urease sequence, and might be able to release a known insecticidal peptide in a single or cascade event. The results suggest that susceptibility of O. fasciatus nymphs to jackbean urease is, like in other insect models, due mostly to limited proteolysis of ingested protein and subsequent release of entomotoxic peptide(s) by cathepsin-like digestive enzymes.     

Pharmacological observations on body fluids from the milkweed bug,Oncopeltus fasciatus (Dallas) (Heteroptera: Lygaeidae)

A report is given on the pharmacological activity of body fluids from milkweed bugs maintained on a diet of sunflower seeds, together with observations on the toxin accumulating system of the adult insect.     

Effects of metabolic level on the body size scaling of metabolic rate in birds and mammals

Metabolic rate is traditionally assumed to scale with body mass to the 3/4-power, but significant deviations from the '3/4-power law' have been observed for several different taxa of animals and plants, and for different physiological states. The recently proposed 'metabolic-level boundaries hypothesis' represents one of the attempts to explain this variation. It predicts that the power (log-log slope) of metabolic scaling relationships should vary between 2/3 and 1, in a systematic way with metabolic level. Here, this hypothesis is tested using data from birds and mammals. As predicted, in both of these independently evolved endothermic taxa, the scaling slope approaches 1 at the lowest and highest metabolic levels (as observed during torpor and strenuous exercise, respectively), whereas it is near 2/3 at intermediate resting and cold-induced metabolic levels. Remarkably, both taxa show similar, approximately U-shaped relationships between the scaling slope and the metabolic (activity) level. These predictable patterns strongly support the view that variation of the scaling slope is not merely noise obscuring the signal of a universal scaling law, but rather is the result of multiple physical constraints whose relative influence depends on the metabolic state of the organisms being analysed.     

Hepatotoxicity of the anti-juvenile hormone precocene II and the generation of dihydrodiol metabolites

Precocene II (6,7-dimethoxy-2,2-dimethylchromene), the most active anti-juvenile hormone isolated from Ageratum houstonianum, has been shown to be hepatotoxic in male Sprague-Dawley rats. A single 300-mg/kg dose of precocene II administered via i.p. injection caused extensive necrosis of parenchymal cells in the hepatic centrolobular areas. Liver functions were markedly affected as shown by the significant increases of glutamic-oxalacetic transaminase and glutamic-pyruvic transaminase in the serum. By means of reversed-phase high pressure liquid chromatography (HPLC), [³H]precocene II was found to be rapidly metabolized in vitro by rat liver microsomes in an NADPH-generating system. Approximately 5% (3.4 nmol/mg protein) of the radioactivity from the [³H]precocene II substrate was covalently bound to the macromolecular pellet at the end of a 15-min incubation period when phenobarbital (PB)-induced microsomes were used. Results obtained from experiments using different incubation systems indicated the involvement of the cytochrome P-450-dependent monooxygenases in the metabolism of precocene II and the concurrent covalent binding. The most predominent metabolite was isolated and accounted for >90% of the radioactivity associated with the ethylacetate-extractable metabolites. Further analysis by mass spectrometry and proton nuclear magnetic resonance (NMR) spectroscopy identified it as a 37 : 63 stereoisomeric mixture of the cis and trans 3,4-dihydrodiols of precocene II. A highly reactive (3,4-epoxy-6,7-dimethyl-2,2-dimethylchromane (precocene-3,4-epoxide) was thus suggested as a crucial metabolic intermediate which may be responsible for the histopathological changes seen in rat liver.     

Chemical structure, juvenile hormone activity and persistence within the insect of juvenile hormone mimics for Rhodnius prolixus.

41 chemicals were tested for juvenile hormone (JH) activity for Rhodnius prolizus by two methods of application. Several different classes of chemical were represented, viz. long chain terpenoid compounds, aryl terpenoid compounds, aryl terpenoid amines and aryl terpenoid ethers.All classes of chemical contained members which showed at least moderate activity. The most active compound was N-(2,5-dichlorophenyl)-3,7-dimethyl-2,6-octadienylamine; 0.0024 μg induced the production of an insect which was halfway between a normal adult and a complete supernumerary larva. Since several compounds with widely differing chemical groupings showed high JH activity, it is considered that overall molecular size and shape are important considerations in determining JH activity, although they do not by themselves provide a complete explanation. Some chemicals tested are specific in their JH activity to relatively few insects. JH mimic specificity is discussed and it is considered that molecular shape can explain some aspects of this phenomenon.The persistence within the insect of the JH mimics varied greatly. In general, compounds with a methyl ester group at one end and an epoxide group at the other had least persistence. The absence of one or more of these features tends to induce greater persistence. Within most classes of chemical, the members with highest JH activity tended to have least persistence and it is considered that the major factor affecting persistence with the insect is the rate of enzymic degradation of the JH mimic.