R. Michael Roe

The juvenile hormones: historical facts and speculations on future research directions

In all of endocrinology there is no more wondrous name for a hormone than the insect juvenile hormone (JH). Could V.B. Wigglesworth have predicted some six decades ago that his term “juvenile hormone” would offer promise of immortal youth to the aged, the expectation of a bloom of dollars to agrochemical concerns, and the hope of solutions to basic problems by developmental biologists and entomologists. The aged have been disappointed and the high expectations of commercial firms have not been met, but hope remains that JH can be used as a probe to ultimately solve basic questions in development. It has been more than two centuries since Lyonet (1762) described granulated vessels in the thorax of lepidopteran larvae that proved to be the prothoracic glands. By contrast, the corpora allata were not mentioned in the literature until Mu ̈ller (1828) described organs in the cockroach that he called pharyngeal bodies and which he thought innervated the dorsal vessel and esophagus. During the remainder of the 19th century, the corpora allata were described as sympathetic ganglia or other components of the nervous system, as indicated by the various descriptive terms given them, e.g. accessory ganglia, tracheal ganglia, lateral ganglion, lateral head ganglion, appendage of the pharyngeal ganglion, etc. In 1899, Heymons dubbed these organs the corpora allata and correctly described their embryological origin, but also believed that they were a pair of sympathetic ganglia concerned with the innervation of the digestive system.

Molecular traces of alternative social organization in a termite genome

Although eusociality evolved independently within several orders of insects, research into the molecular underpinnings of the transition towards social complexity has been confined primarily to Hymenoptera (for example, ants and bees). Here we sequence the genome and stage-specific transcriptomes of the dampwood termite Zootermopsis nevadensis (Blattodea) and compare them with similar data for eusocial Hymenoptera, to better identify commonalities and differences in achieving this significant transition. We show an expansion of genes related to male fertility, with upregulated gene expression in male reproductive individuals reflecting the profound differences in mating biology relative to the Hymenoptera. For several chemoreceptor families, we show divergent numbers of genes, which may correspond to the more claustral lifestyle of these termites. We also show similarities in the number and expression of genes related to caste determination mechanisms. Finally, patterns of DNA methylation and alternative splicing support a hypothesized epigenetic regulation of caste differentiation.

A partition assay for the simultaneous determination of insect juvenile hormone esterase and epoxide hydrolase activity

A partition assay was developed to measure insect juvenile hormone (JH) I and III metabolism in biological samples containing both JH esterase and JH epoxide hydrolase activity. The assay utilizes commercially available radiochain 3H-labeled JH as substrate and the selective JH esterase inhibitor 3-octylthio-1,1,1-trifluoro-2-propanone. JH partitions into an isooctane phase and the metabolites JH acid, JH diol, and JH diol-acid into aqueous methanol after incubation of JH substrate with inhibited and uninhibited sample. The assay provides a time- and cost-efficient alternative to the currently available thin-layer chromatography method for the measurement of JH esterase and epoxide hydrolase activity.

The movement of proteins across the insect and tick digestive system.

The movement of intact proteins across the digestive system was shown in a number of different blood-feeding and non-blood-feeding insects in the orders Blattaria, Coleoptera, Diptera, Hemiptera, Lepidoptera, Orthoptera, Neuroptera and Siphonaptera, as well as in two tick families Ixodidae and Argasidae. Protein movement was observed for both normal dietary and xenobiotic proteins, which suggest that the mechanism for transfer is not substrate specific. The number of studies on the mechanism of movement is limited. The research so far suggests that movement can occur by either a transcellular or an intercellular pathway in the ventriculus with most of the research describing the former. Transfer is by continuous diffusion with no evidence of pinocytosis or vesicular transport common in mammalian systems. Proteins can move across the digestive system without modification of their primary or multimeric structure and with retention of their functional characteristics. Accumulation in the hemolymph is the result of the protein degradation rate in the gut and hemolymph and transfer rate across the digestive system and can be highly variable depending on species. Research on the development of delivery systems to enhance protein movement across the insect digestive system is in its infancy. The approaches so far considered with some success include the use of lipophilic-polyethylene glycol (PEG) polymers, the development of fusion proteins with lectins, reduced gut protease activity and the development of amphiphilic peptidic analogs. Additional research on understanding the basic mechanisms of protein delivery across the insect digestive system, the importance of structure activity in this transfer and the development of technology to improve movement across the gut could be highly significant to the future of protein and nucleic acid-based insecticide development as well as traditional chemical insecticidal technologies.

Biology and genetics of a laboratory strain of the tobacco budworm, Heliothis virescens (Lepidoptera: Noctuidae), highly resistant to spinosad

Abstract Tobacco budworm larvae, Heliothis virescens (F.), were collected from the field in North Carolina in 1996 and 1997 and established as a laboratory (parental) strain. When a subset of these insects was selected by the topical application of technical spinosad (a mixture of spinosyns A and D) every generation for 13 generations, they became highly resistant to the insecticide. The resistance ratio for topically applied spinosad based on differences in the LD50 between the parental (susceptible) and the resistant (generation 19) strain was 669-fold when fourth stadium larvae were treated. The susceptible strain LD50 18 d after treatment was 0.11 μg of active ingredient per larva while the LD50 for generation (G) 19 of the resistant strain was 73.55 μg per larva. Reciprocal single pair matings between the resistant and the parental strain and backcrosses of F 1 (R×S) females with resistant males indicated that a non-sex linked, (partially) recessive single gene was responsible for spinosad resistance. The F1 larvae were only slightly (5.3–5.6-fold) resistant compared to the parental strain. The stability of resistance was tested by removing spinosad selection for five generations. In the absence of immigration of susceptible budworms into the population and insecticide treatments, the LD50 decreased only 1.4-fold. The only differences noted in the biology of the parental and resistant strain was that the resistant males developed slower as larvae and emerged as adults later than the susceptible males and had a slightly smaller 1 d old pupal wet weight. However, when 80% highly resistant and 20% parental moths of both sexes were allowed to mate freely, the majority of the offspring (84.6%) were susceptible to spinosad. This suggests a reduced reproductive competitiveness for the resistant strain.

Changes in biosynthesis and degradation of juvenile hormone during breeding by burying beetles: a reproductive or social role?

Burying beetles, Nicrophorus orbicollis, depend on the location of an unpredictable resource, a small vertebrate carcass, for reproduction. When they discover a carcass, they undergo a correlated rapid rise in titers of juvenile hormone (JH) in the hemolymph and ovarian development. This study investigates the regulation of the changes in JH during breeding in both male and female burying beetles and the role of JH in ovarian development. JH biosynthesis by the corpora allata (CA), measured in vitro, increased in females within an hour of their discovery of a carcass and increased later in males. After returning to low rates as oviposition began, JH biosynthesis rose again 3 days later in females but not in males. Neither the ovaries nor testes synthesized JH. There was a concomitant fall in JH esterase activity within 12 h of discovery of the carcass in both males and females. Although the rise in JH titers and biosynthesis and the fall in JH esterase is correlated with ovarian development, application of methoprene or JH III in the absence of a carcass did not result in vitellogenin uptake by the oocytes. Therefore, we conclude that, in spite of the rapid rise in JH before oviposition, it is not sufficient to regulate vitellogenin synthesis and/or its uptake by the ovaries. We suggest that its role has been preempted to organize social behavior and coordinate parental behavior between mates.

Haemolymph juvenile hormone esterase during the life cycle of the tobacco hornworm, Manduca sexta (L.)

Abstract Juvenile hormone (JH) esterase activity was found in the plasma of larvae, pupae and adults of wild-type tobacco hornworms, Manduca sexta . There was a single peak of plasma JH esterase activity approx. 28 h prior to ecdysis in each instar from the second through the fourth instar and a peak of activity prior to both wandering and pupation in the fifth (last) instar. JH esterase activity was high in newly formed male and female pupae but declined to minimal levels by day 1 of the pupal stage. For the remainder of the pupal period, activity was at background levels. JH esterase activity increased again in newly emerged, virgin male and female adults but declined and remained at a low level 1 day after emergence through death. Gel filtration analysis of larval, pupal and adult plasma resolved a single peak of JH esterase activity with an apparent molecular weight of 66,000. However, isoelectric focusing revealed three forms with isoelectric points of 5.5, 5.8 and 6.1. These isoelectric forms were also found in black and white mutants of last instar M. sexta and in purified JH esterase from wild-type larvae. The plasma JH esterase activity metabolized JH I 2–3 times faster than JH III and was sensitive to inhibition by octylthio-1,1,1-trifluoro-2-propanone and insensitive to O , O -diisopropyl phosphorofluoridate. Gel filtration, isoelectric focusing, substrate specificity and developmental studies suggest that the same JH esterases are found in the plasma of larvae, pupae and adults and appear to be different from general (α-NA) esterase.

Developmental profile, isolation, and biochemical characterization of a novel lipoglycoheme-carrier protein from the American dog tick, Dermacentor variabilis (Acari: Ixodidae) and observations on a similar protein in the soft tick, Ornithodoros parkeri (Acari: Argasidae)

A novel lipoglycoheme-carrier protein (CP) in the American dog tick, Dermacentor variabilis (Say) has been purified and characterized. CP was purified by native-PAGE from partially fed virgin females. CP has a density of 1.25 g/ml with a molecular weight of 200 K by native-PAGE and 340 K by gel filtration chromatography. CP is comprised of two majour subunits, 98 K and 92 K in molecular weight by SDS-PAGE. Separate amino acid composition of the two subunits indicated high contents of As(x), Gl(x) and leucine. However, the N-terminal amino acid sequence of the two subunits was only 13% identical. The lower molecular weight subunit showed 61% identity to artemocyanin (biliprotein) in fairy shrimps, 46% identity to minor vitellogenin in chickens and 13% identity to vitellin of the black-legged tick. No similarity match was found for the other subunit. CP is a lipoglycoheme-protein as indicated by selective staining of native-PAGE gel for lipids, carbohydrates and heme. Lipid analysis by thin layer chromatography revealed the presence of cholesterol, phospholipids, monoacylglycerides, triacylglycerides and free fatty acids. Heme associated with purified CP demonstrated a lambda(max) of 397.5 nm while the lambda(max) of crude hemolymph plasma was 402.5 nm. The presence of CP in whole body homogenates of eggs, unfed and fed larvae and fed nymphs as well as in the plasma of unfed and fed adults including vitellogenic females was demonstrated by native-PAGE. Although a protein of analogous size was not found in the soft tick, Ornithodoros parkeri Cooley, a high molecular weight protein (500 K) is the predominant plasma protein in both unfed and fed male and female adults of that species as determined by native-PAGE. Also, CP appears to function as a biliprotein which sequesters heme.

Absence of insect juvenile hormones in the American dog tick, Dermacentor variabilis (Say) (Acari:Ixodidae), and in Ornithodoros parkeri Cooley (Acari:Argasidae)

Synganglia, salivary gland, midgut, ovary, fat body and muscle alone and in combination from the ixodid tick, Dermacentor variabilis (Say), or the argasid tick, Ornithodoros parkeri Cooley, were incubated in vitro in separate experiments with L-[methyl-(3)H]methionine and farnesoic acid or with [1-(14)C]acetate. Life stages examined in D. variabilis were 3 and 72 h old (after ecdysis) unfed nymphs, partially fed nymphs (18 and 72 h after attachment to the host), fully engorged nymphs (2 d after detachment from host), 3 and 72 h old (after eclosion) unfed females, partially fed unmated females (12-168 h after attachment to host) and mated replete females (2 d after detachment from the host). Those from O. parkeri were third and fourth stadium nymphs and female O. parkeri, 1-2 d after detachment. Corpora allata from Diploptera punctata, Periplaneta americana and Gromphadorina portentosa were used as positive controls in these experiments. No farnesol, methyl farnesoate, JH I, JH II, JH III, or JHIII bisepoxide was detected by radio HPLC from any tick analysis while JH III, methyl farnesoate, and farnesol were detected in the positive controls. To examine further for the presence of a tick, insect-juvenilizing agent, Galleria pupal-cuticle bioassays were conducted on lipid extracts from 10 and 15 d old eggs, unfed larvae (1-5 d after ecdysis), unfed nymphs (1-7 d after ecdysis), and partially fed, unmated female adults (completed slow feeding phase) of D. variabilis. Whole body extracts of fourth stadium D. punctata and JH III standard were used as positive controls. No juvenilizing activity in any of the tick extracts could be detected. Electron impact, gas chromatography-mass spectrometry of hemolymph extracts from fed, virgin (forcibly detached 7 d after attachment) and mated, replete (allowed to drop naturally) D. variabilis and fully engorged (1-2 d after detachment) O. parkeri females also failed to identify the common insect juvenile hormones. The same procedures were successful in the identification of JH III in hemolymph of fourth stadium D. punctata. Last stadium nymphal (female) O. parkeri implanted with synganglia from second nymphal instars underwent normal eclosion to the adult. The above studies in toto suggest that D. variabilis and O. parkeri do not have the ability to make the common insect juvenile hormones, and these juvenile hormones do not regulate tick metamorphosis or reproduction as hypothesized in the literature.

Juvenile hormone metabolism during the fourth stadium and pupal stage of the southern house mosquito, Culex quinquefasciatus Say

Abstract Juvenile hormone (JH) esterase activity did not change significantly during the fourth stadium in Culex quinquefasciatus , but JH epoxide hydrolase activity peaked in 36-h-old fourth instars and pharate pupae at levels 5.7- and 3.0-times that of JH esterase, respectively. JH epoxide hydrolase activity was greater than the JH esterase activity throughout larval development and in 0-h-old pupae. Both activities declined and were equivalent in pupae more than 8-h-old. 1-Naphthyl acetate (NA) esterase activity remained constant during the fourth stadium and peaked in pupae 16–24-h-old. Different isoelectric forms of JH esterase and epoxide hydrolase were found in fourth instars and pharate pupae. Subcellular distribution studies indicated that the majority of JH esterase and JH epoxide hydrolase were membrane bound. Inhibition of larval JH esterase with O,O-diisopropyl phosphorofluoridate revealed an I 50 3000-times that of 1-NA esterase but both enzymes had approximately equivalent I 50 values in pupae that were more than 8-h-old. Methoprene treatment of fourth instars significantly extended the duration of the fourth stadium, but did not extend the duration of the pupal stage. Methoprene also eliminated the normal peaks in JH epoxide hydrolase activity found during the last stadium, increased the 1-NA esterase activity, but had no effect on JH esterase activity. Methoprene treatment of fourth instars had no effect on the pupal JH epoxide hydrolase and JH esterase activity but depressed the overall 1-NA esterase activity eliminating the pupal 1-NA esterase peak.