Exposure to exogenous enkephalins disrupts reproductive development in the Eastern lubber grasshopper, Romalea microptera (Insecta: Orthoptera).

Enkephalins play a major role in reproductive physiology in crustaceans; however their role in reproductive development in insects is largely unknown. We investigated the effect of exposure to exogenous leucine-enkephalin (Leu-Enk), methionine-enkephalin (Met-Enk), and the opioid antagonist naloxone on gonad development in the Eastern lubber grasshopper, Romalea microptera. Injection of either Leu-Enk or naloxone alone significantly increased the testicular index and testicular follicular diameter in males, and the ovarian index, oocyte length, and oocyte diameter in females. In contrast, injection of Met-Enk inhibited all measures of reproductive development in both sexes. Surprisingly, co-injection of naloxone with either enkephalin enhanced the effect associated with administration of the enkephalin alone. This study clearly demonstrates the ability of enkephalins to disrupt insect sexual development and also suggests the existence of conserved enkephaline-dependent regulatory mechanisms in insects and crustaceans.

Molecular cloning and sequence of retinoid X receptor in the green crab Carcinus maenas: a possible role in female reproduction.

Retinoid X receptor (RXR) belongs to an ancient superfamily of nuclear hormone receptors, and plays an important role in reproduction of vertebrates. However, the reproductive role of RXR has not been clarified in crustaceans. In this investigation, we first report the cloning of two alternative splice variants of RXR cDNA from green crab ovarian RNA. RXR mRNA levels were quantified in different vitellogenic stages of the crab hepatopancreas (HP) and ovary. The expression of RXR mRNA relative to the arginine kinase mRNA was significantly increased in the HP of vitellogenic crabs in a stage-dependent manner. The relative levels of RXR mRNA in the ovary were significantly lower in vitellogenic stage III crabs than in crabs in the other three stages. These data indicate that the HP and ovary of the crab are capable of expressing RXR, which may regulate, in part, vitellogenesis in the crab. We also examined the effects of methyl farnesoate (MF) and RXR-dsRNA treatments on vitellogenin and RXR gene expression. Vitellogenin and RXR mRNA levels in HP and ovarian fragments incubated in MF were significantly (P<0.001) higher than in control tissue fragments prepared from the same animal. Treatment of crabs with RXR-dsRNA significantly (P<0.001) reduced mRNA levels for RXR and for vitellogenin as well as MF levels in hemolymph. These results indicate that, MF and RXR form a complex (MF-RXR) directly and together stimulate ovarian development in these green crabs. This interaction of RXR, MF, and ovary development axis is a novel finding and is the first report to the best of our knowledge.

Methyl farnesoate synthesis in the lobster mandibular organ: the roles of HMG-CoA reductase and farnesoic acid O-methyltransferase.

Eyestalk ablation (ESA) increases crustacean production of methyl farnesoate (MF), a juvenile hormone-like compound, but the biochemical steps involved are not completely understood. We measured the activity of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) and farnesoic acid O-methyl transferase (FAOMeT), an early step and the last step in MF synthesis. ESA elevated hemolymph levels of MF in male lobsters. Enzyme activity suggested that increased MF production on day one was due largely to elevated HMGR activity while changes in FAOMeT activity closely paralleled changes in MF levels on day 14. Transcript levels for HMGR and FAOMeT changed little on day one, but both increased substantially on day 14. We treated ESA males with a partially purified mandibular organ-inhibiting hormone (MOIH) and observed a significant decline in MF levels, FAOMeT activity, and FAOMeT-mRNA levels after 5h. However, no effect was observed on HMGR activity or its mRNA indicating that they must be regulated by a separate sinus gland peptide. We confirmed that lobster HMGR was not a phosphoprotein and was not regulated by reversible phosphorylation, an important mechanism for regulating other HMGRs. Nevertheless, molecular modeling indicated that the catalytic mechanisms of lobster and mammalian HMGR were similar.

Effects of eyestalk ablation on carbonic anhydrase activity in the euryhaline blue crab Callinectes sapidus: neuroendocrine control of enzyme expression.

Carbonic anhydrase (CA) activity in the gills of the euryhaline blue crab, Callinectes sapidus, was measured in response to acute low-salinity transfer and treatment with eyestalk ablation (ESA) in an attempt to elucidate potential regulatory mechanisms of salinity-mediated CA induction. ESA alone resulted in an approximate doubling of CA activity in the posterior, ion-transporting gills of crabs acclimated to 35 ppt. Transfer of intact crabs to 28 ppt, a salinity at which the blue crab is still an osmotic and ionic conformer, had no effect on CA activity, but treatment with ESA prior to transfer resulted in a 5-fold increase. Hemolymph osmolality was unaffected by ESA. There was a 7-fold induction of CA activity in posterior gills of intact crabs transferred from 35 to 15 ppt, and this was potentiated by about 100% by ESA. Hemolymph osmolality was slightly elevated in the ESA-treated crabs. CA activity in anterior gills did not increase in response to any treatment. Hemolymph concentrations of methyl farnesoate (MF) were measured for all experimental animals. MF concentrations were undetectable in all intact crabs, regardless of salinity. Treatment with ESA resulted in elevated levels of hemolymph MF, but these levels were still relatively low and unrelated to salinity. These results suggest that CA induction is under the control of a regulatory substance located in the eyestalk. This substance appears to be a CA repressor, keeping CA expression at low levels in the gills of crabs acclimated to high salinity. Exposure to low salinity, or treatment with ESA, removes the effects of this putative repressor and allows CA induction to occur.

The effect of seawater composition and osmolality on hemolymph levels of methyl farnesoate in the green crab Carcinus maenas.

Green crabs, Carcinus maenas, exposed to dilute seawater (e.g., 5 ppt salinity, approximately 150 mOsm/kg) have hemolymph levels of methyl farnesoate (MF) that are up to 10-fold higher than animals in isosmotic seawater (27 ppt, approximately 800 mOsm/kg). In this paper, we examine aspects of osmotic and ionic stress to identify factors involved in elevating MF levels. MF levels did not rise after exposure to concentrated seawater, so only hypoosmotic stress elevates MF. MF levels rose in animals exposed to dilute seawater containing mannitol to make it isosmotic, indicating that the hypoosmotic rise in MF is due to decreased ion concentrations. Individual ions were investigated by exposing crabs either to isosmotic seawater with low concentrations of an ion or to dilute seawater with high concentrations of an ion. Ca(2+) and Mg(2+) in combination affected MF levels. Finally, we found that the increase in MF levels was accelerated when hemolymph osmolality was precociously lowered by partially replacing hemolymph with deionized water prior to transferring animals to dilute seawater. Thus, the 6-8 h delay between exposing crabs to dilute sea water and observing an increase in MF appears to reflect the time needed for specific hemolymph ions to decrease below a threshold concentration.

Starvation affects vitellogenin production but not vitellogenin mRNA levels in the lubber grasshopper, Romalea microptera.

The interaction of juvenile hormone (JH) and nutrition was studied during the oviposition cycle of the Eastern Lubber grasshopper (Romalea microptera). Starvation of females early or in the middle of the cycle inhibited oocyte growth. Starvation for 4 days also reduced hemolymph levels of JH III and vitellogenesis (Vg) to 25% and 15%, respectively, of the levels in fed animals. Likewise, Vg production by fat body fragments incubated in vitro was reduced to 2% of the levels in fed animals and total protein synthesis was reduced to 25%, suggesting that starvation had a stronger effect on Vg synthesis than on protein synthesis. These effects were reversed when starved animals were fed again. However, fat body levels of Vg-mRNA were similar in fed and starved animals, indicating that starvation did not affect transcript levels. We tested whether the decline in JH levels mediated the other starvation effects by infusing animals with JH III or vehicle for 2 days at the onset of starvation. Infusion of JH elevated JH and Vg-mRNA levels 670% and 103%, respectively, above the levels in vehicle-infused animals. However, Vg production and hemolymph levels of Vg were similar to the levels in vehicle-infused animals. These data suggest that JH alone is insufficient to stimulate Vg production.

Allatotropin regulation of juvenile hormone synthesis by the corpora allata from the lubber grasshopper, Romalea microptera.

The in vitro synthesis of juvenile hormone (JH) by corpora allata (CA) from the lubber grasshopper (Romalea microptera) was stimulated by low concentrations of brain extract and this effect was reduced at higher concentrations, suggesting the presence of allatotropin (AT) and allatostatin (AST) factors in the brain. The AT activity of brain extracts caused a rapid and reversible stimulation and appeared to be a peptide(s). Reversed phase (C18) HPLC analysis of brain extracts disclosed two peaks of AT activity but no significant AST activity. Manse-AT, Schgr-NPF, and Locmi-FLRF had no effect on JH synthesis by lubber CA, indicating that the Rommi-AT factors are distinct from these peptides. High concentrations of Dippu-AST-7 and Grybi-AST-1 inhibited JH synthesis, implying that AST factors might be present in lubber grasshoppers. CA response to AT activity of brain extracts varied during the oviposition cycle ( approximately 35 days), with the maximum response occurring on days 16-18. AT activity of brain extracts also varied during the cycle, being highest on day 25. Our data suggest that the lubber CA is largely regulated by AT activity, and that JH synthesis reflects both CA response to AT activity and the level of AT activity in the brain.

Plasticity in juvenile hormone in male burying beetles during breeding: physiological consequences of the loss of a mate.

Burying beetles, Nicrophorus orbicollis, have facultative biparental care. They bury and prepare small vertebrate carcasses that provide food for their young. Here we establish the juvenile hormone (JH) profiles of paired females, paired males and single males and investigate some of the environmental and social factors that may affect these profiles. Before larvae hatch JH profiles of paired males and females were similar. However, after larvae hatch and during brood care, JH titers of females were very high and those of single males were significantly higher than those of paired males. We tested the hypothesis that higher JH was a response to the need for increased parental care by manipulating brood size. Although JH titers of single males caring for small versus large broods were not significantly different, when comparing JH titers and larval growth (a measure of parental effort), a significant positive correlation emerged. In contrast, we found that food quality had no effect on JH levels suggesting that increased feeding by males and females after carcass discovery cannot explain the elevation of JH. The regulation of JH in male burying beetles appears thus to be dependent on the presence of a mate and on critical stimuli from young.

Purification and characterization of a mandibular organ protein from the American lobster, Homarus americanus: a putative farnesoic acid O-methyltransferase.

Methyl farnesoate (MF) appears to have important roles in the development, morphogenesis, and reproduction of crustaceans. To better understand the regulation of MF synthesis, we studied farnesoic acid O-methyltransferase (FAOMeT, the final enzyme in the MF biosynthetic pathway) in the American lobster (Homarus americanus). FAOMeT purified from mandibular organ (MO) homogenates had a MW of approximately 38,000. The sequences of trypsin fragments of purified FAOMeT were used to design PCR primers to amplify a cDNA fragment, which was used to isolate a full-length cDNA containing a single open reading frame (ORF) of 828 bp encoding a protein of 276 amino acids. The deduced amino acid sequence of this putative FAOMeT protein contained two copies of a conserved approximately 135 amino acid domain we term the CF (CPAMD8/FAOMeT) domain; single copies of this domain also occur in the human CPAMD8 protein (a member of the alpha-2 macroglobulin family) and an uncharacterized Drosophila protein. The recombinant protein had no FAOMeT activity. However, its addition to MO homogenates from eyestalk ablated (ESA) lobsters increased enzyme activity by up to 75%, suggesting that FAOMeT may require an additional factor or modification (e.g., phosphorylation) for its activation. The mRNA for the putative FAOMeT was primarily found in the proximal region of the MO, the predominant site of MF synthesis. FAOMeT transcripts were found in muscle tissue from ESA animals, but not in green gland, hepatopancreas, or in muscle tissue from intact animals. FAOMeT mRNA was also detected in embryos and larval stages. This is the first comprehensive report of this protein in the lobster, and is an important step in elucidating the functions of MF in these animals.

Increased juvenile hormone levels after long-duration flight in the grasshopper, Melanoplus sanguinipes.

Although, in many insects, migration imposes a cost in terms of timing or amount of reproduction, in the migratory grasshopper Melanoplus sanguinipes performance of long-duration flight to voluntary cessation or exhaustion accelerates the onset of first reproduction and enhances reproductive success over the entire lifetime of the insect. Since juvenile hormone (JH) is involved in the control of reproduction in most species, we examined JH titer after long flight using a chiral selective radioimmunoassay. JH levels increased on days 5 and 8 in animals flown to exhaustion on day 4 but not in 1-h or non-flier controls. No difference was seen in the diel pattern of JH titer, but hemolymph samples were taken between 5 and 7 h after lights on. Treatment of grasshoppers with JH-III mimicked the effect of long-duration flight in the induction of early reproduction. The increased JH titer induced by performance of long-duration flight is thus at least one component of flight-enhanced reproduction. To test the possibility that post-flight JH titer increases are caused by adipokinetic hormone (AKH) released during long flights, a series of injections of physiological doses of Lom-AKH I were given to unflown animals to simulate AKH release during long flight. This treatment had no effect on JH titers. Thus, although AKH is released during flight and controls lipid mobilization, it is not the factor responsible for increased JH titers after long-duration flight.

The lobster mandibular organ produces soluble and membrane-bound forms of 3-hydroxy-3-methylglutaryl-CoA reductase.

In a previous study [Li, Wagner, Friesen and Borst (2003) Gen. Comp. Endocrinol. 134, 147-155], we showed that the MO (mandibular organ) of the lobster Homarus americanus has high levels of HMGR (3-hydroxy-3-methylglutaryl-CoA reductase) and that most (approx. 75%) of the enzyme activity is soluble. In the present study, we report the biochemical and molecular characteristics of this enzyme. HMGR had two forms in the MO: a more abundant soluble form (66 kDa) and a less abundant membrane-bound form (72 kDa). Two cDNAs for HMGR were isolated from the MO. A 2.6-kb cDNA encoded HMGR1, a 599-amino-acid protein (63 kDa), and a 3.2-kb cDNA encoded HMGR2, a 655-amino-acid protein (69 kDa). These two cDNAs had identical 3'-ends and appeared to be products of a single gene. The deduced amino acid sequences of these two proteins revealed a high degree of similarity to other class I HMGRs. Hydropathy plots indicated that the N-terminus of HMGR1 lacked a transmembrane region and HMGR2 had a single transmembrane segment. Recombinant HMGR1 expressed in Sf9 insect cells was soluble and had kinetic characteristics similar to native HMGR from the MO. Treatment with phosphatase did not affect HMGR activity, consistent with the observation that neither HMGR1 nor HMGR2 has a serine at position 490 or 546, the position of a conserved phosphorylation site found in class I HMGR from higher eukaryotes. Other lobster tissues (i.e. midgut, brain and muscles) had low HMGR activities and mRNA levels. MO with higher HMGR activities had higher HMGR mRNA levels, implying that HMGR is regulated, in part, at the transcription level.

Juvenile hormone synthesis, metabolism, and resulting haemolymph titre in Heliothis virescens larvae parasitized by Toxoneuron nigriceps.

Last instar larvae of the tobacco budworm, Heliothis virescens F., fail to pupate and have little 20-hydroxyecdysone when parasitized by Toxoneuron nigriceps (Viereck). In this paper, we extend these observations to juvenile hormone (JH) to determine if parasitism by this wasp affects other endocrine systems. To this end, we compared the production of JH by corpora cardiaca-corpora allata complexes (CC-CA), the metabolism of JH by haemolymph enzymes, and the haemolymph titre of JH in parasitized and non-parasitized control larvae of H. virescens during the last larval instar. CC-CA from parasitized and control larvae had similar peaks of JH synthesis on day 1 of the fifth instar, with JH II accounting for more than 90% of total JH in both groups. On subsequent days, JH synthesis dropped to undetectable levels more quickly in non-parasitized controls than in parasitized larvae. JH metabolism by haemolymph of parasitized and control animals increased from low levels on day 1 of the fifth instar to high levels on days 2 and 3 of the instar. JH metabolism was significantly higher in control larvae than in parasitized larvae. After day 3, JH metabolism decreased in both groups, but was significantly higher in parasitized larvae. The major metabolite of JH in both groups was JH acid, though traces of JH diol and JH acid diol were also detected. The haemolymph titre of JH in both groups peaked on day 1 of the fifth instar and, similar to the synthesis of JH by CC-CA, decreased more rapidly in control larvae. As a result, non-parasitized animals had significantly lower JH titres on day 2. The higher JH titres observed in parasitized larvae during the early fifth instar may contribute to their developmental arrest. The possible role of these JH alterations in the host developmental and metabolic redirection is discussed and a more comprehensive physiological model accounting for host-parasitoid interactions is proposed.

3-hydroxy-3-methylglutaryl-coenzyme A reductase in the lobster mandibular organ: regulation by the eyestalk.

The mandibular organ (MO) of the lobster, Homarus americanus, produces the isoprenoid methyl farnesoate (MF), a compound related to insect juvenile hormone (JH). To better understand the synthesis and regulation of MF, we studied 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase (HMGR), the rate-limiting enzyme in isoprenoid biosynthesis. Lobster HMGR had a Km of 11.4 microM for HMG-CoA, a Km of 14.8 microM for NADPH, and was at least 2000-fold more selective for this cofactor than for NADH. Lovastatin and mevalonic acid inhibited HMGR, with KI values of 1.3 nM and 25.3 microM, respectively, whereas MF, farnesoic acid, cholesterol, 20-hydroxyecdysone, and progesterone had no effect. Approximately 75% of the HMGR activity in lobster MO was soluble. Similar levels of HMGR activity were observed in all regions of the MO. Eyestalk removal increased MF synthesis and the activity of farnesoic acid O-methyltransferase (FAOMeT, the final step in MF synthesis) in the MO by 10.7- and 5.7-fold, respectively, and caused a 3.1-fold increase of HMGR activity. Injection of the eyestalk ablated lobsters with an extract of two sinus glands (SG), a neuroendocrine organ in the eyestalk, decreased MF synthesis, FAOMeT activity and HMGR activity to 3, 8, and 20%, respectively, of the levels observed in saline-treated animals. The regulation of crustacean HMGR by the SG suggests that the lobster MO is a useful model system for investigating the cellular regulation of HMGR activity.

Hemolymph ecdysteroids do not affect vitellogenesis in the lubber grasshopper.

The role of hemolymph ecdysteroids in the reproduction of non-dipteran insects is unclear. We examine the role(s) of hemolymph ecdysteroids during egg production in the lubber grasshopper, Romalea microptera. In all individuals, hemolymph ecdysteroids rose to a sharp peak with similar maxima and then fell to undetectable levels. The time from the adult molt to the maximum ecdysteroid titer (E(max) titer) varied in response to food availability, whereas the time from E(max) titer to oviposition was unrelated to food availability. Because both the timing of egg production and the timing of E(max) responded similarly to environmental changes, ecdysteroids may be involved in egg production. We hypothesized that this role is the stimulation of vitellogenesis. Ovariectomized females had vitellogenin but no ecdysteroids, so ecdysteroids are not necessary for vitellogenin production. In addition, treatment of females with ecdysteroids altered neither Vg titers nor ovarian growth. Ovarian ecdysteriods increased at the same age in development as hemolymph ecdysteroids. In contrast to hemolymph ecdysteroids, ovarian ecdysteroids persisted until oviposition. Despite this, [(3)H]ecdysone injected into the hemolymph was detected later only at very low levels in the ovary, suggesting that hemolymph ecdysteroids are not sequestered by the ovary. In summary, our studies indicate that hemolymph ecdysteroids in adult females of the lubber grasshopper are associated with the timing of egg production, but they neither regulate vitellogenesis nor act as a source of ecdysteroids for the ovary.

Plasticity and canalization in the control of reproduction in the lubber grasshopper.

The ability to change reproductive tactics during adult development in response to environmental variation is predicted to enhance fitness. Many organisms show phenotypic plasticity early in non-embryonic development, but later exhibit phases of developmental inflexibility (=canalization). Therefore, we studied reproduction-related hormones and proteins and their relationships to plasticity in the Eastern lubber grasshopper. Diet-switching experiments demonstrated plasticity early in the egg production cycle, but a switch to canalization late in the cycle. We measured developmental titers of 4 hemolymph compounds from single individuals from adult molt until first oviposition. These 4 compounds were the egg-yolk precursor protein vitellogenin, juvenile hormone (the central regulator of insect reproduction), major hemolymph proteins, and ecdysteroids (the arthropod molting hormone that ultimately is stored in the egg). Using diet manipulations, we investigated how these developmental titers relate to the switch from plastic to canalized egg production. All 4 hemolymph compounds reached their peak levels during the canalized phase, about 12 day before oviposition. Diet switches after these peak levels did not affect the timing to oviposition. Therefore, these peak titers were physiological events that occurred after the individual committed to laying. We compared these patterns in reproduction to the development toward adult molt, another major life-history event in insects. We observed an extended canalized phase before the adult molt. This canalized phase always included a peak of ecdysteroids. The similar patterns in the physiology of these life-history events suggested that common limitations may exist in major developmental processes of insects that are directed by hormones.

Characterization and quantification of yolk proteins in the lobster, Homarus americanus.

Yolk protein (vitellin, Vn) and its precursor (vitellogenin, Vg) were isolated and characterized in the ovary and hemolymph, respectively, of the adult female lobster, Homarus americanus. Vn had a molecular mass of 360 kDa when analyzed by gel filtration. When analyzed by SDS-PAGE, Vn had six bands (110, 105, 94, 90, 81, and 78 kDa). An anti-Vn antiserum was developed using purified Vn, and the antiserum was used to detect Vn and Vg by ELISA and western blot techniques. ELISA analysis of hemolymph proteins separated by gel filtration indicated that Vg was similar in mass to Vn (360 kDa). However, western blots of hemolymph proteins separated by SDS-PAGE indicated that Vg contained a pair of protein subunits, 194 kDa and 179 kDa. Furthermore, the elution profiles of Vn and Vg from anion exchange chromatography indicated that Vg had a more negative charge. Thus, Vg appears to be processed after its uptake by the ovary to form Vn. Vg was undetectable in hemolymph from adult males by either ELISA or by western blot analysis. However, hemolymph levels of Vg in adult females increased 40-fold during the reproductive cycle, rising from 18 microg/mL in ovarian stage II to 789 microg/mL at stage V. This increase correlates well with oocyte growth during the cycle. Hence, this method may be useful for studying the regulation of lobster vitellogenesis.