cDNA cloning and transcriptional regulation of the vitellogenin receptor from the imported fire ant, Solenopsis invicta Buren (Hymenoptera: Formicidae).

We describe the cloning of the first hymenopteran vitellogenin receptor (VgR) cDNA from the imported fire ant, Solenopsis invicta, an invasive pest. Using reverse transcription polymerase chain reaction and rapid amplification of cDNA ends, fragments encompassing the entire coding region of a putative VgR were cloned and sequenced. The complete 5764 bp cDNA encodes a 1782 residue protein with a predicted molecular mass of 201.3 kDa (=SiVgR). Northern blot analysis demonstrated that the 7.4 kb SiVgR transcript was present only in ovaries of reproductive females (virgin alates and queens). The temporal profile of transcriptional expression showed that SiVgR mRNA increased with age in virgin alate females and that this was up-regulated by methoprene, a juvenile hormone (JH) analogue. This suggests that the SiVgR gene is JH regulated.

A hexamerin protein, AgSP-1, is associated with diapause in the boll weevil(1).

The objective of this research was to identify a reliable biochemical indicator for diapause (dormancy) in the boll weevil, Anthonomus grandis. Hemolymph polypeptides from reproductive and diapausing weevils were compared using denaturing sodium dodecyl sulpfate (SDS)-polyacrylamide gel electrophoresis (PAGE). A 77-kDa protein, which proved to be a hexamerin (AgSP-1), strongly correlated with morphological diapause characters in both male and female adult weevils. N-terminal sequence analysis identified the first 25 amino acids of the mature protein and was used to develop an antibody to AgSP-1. Anti-AgSP-1 reacted only with hemolymph from diapausing weevils of both sexes but not with hemolymph from reproductive weevils. Also, the yolk protein, vitellogenin (VG), inversely correlated with AgSP-1. When hemolymph VG was high, AgSP-1 was absent or barely perceptible.Juvenile hormone regulates VG synthesis in most insect species. Juvenile hormone is reported to stimulate reproductive maturation in the boll weevil (Physiological Entomology 22 (1997) 261) and to be absent during diapause (Physiological Entomology 22 (1997a) 269). Therefore, the juvenile hormone (JH) mimic, methoprene, was used to assess the role of JH activity in the boll weevil for terminating diapause, stimulating reproductive maturation and possibly influencing AgSP-1 titers. Application of methoprene was not effective in activating reproductive development. Hemolymph from methoprene-treated, females contained VG and AgSP-1 titers that were similar to acetone-treated and untreated control weevils.Using a genomic DNA library and 3' RACE, two clones were isolated that yielded the complete sequence of AgSP-1 as well as a portion of the 5' untranslated region. Northern blot analysis confirmed the presence of a 2.5 kB transcript for AgSP-1 in the fat body of diapausing weevils. AgSP-1 was also present in the fat body of reproductive weevils, but to a lesser extent. No sex-related differences in gene expression were observed; diapausing weevils of both sexes showed similar levels of AgSP-1 expression. An inverse correlation was observed between the VG transcript and AgSP-1 mRNA. VG was highly expressed in the fat body of reproductive females and only slightly expressed in tissue from diapausing females. Our data suggests that AgSP-1 is a diapause-specific protein in adult weevils and that JH, alone, is not effective in terminating diapause.

Characterization of vitellogenin in the red imported fire ant, Solenopsis invicta (Hymenoptera: Apocrita: Formicidae).

Vitellin (VN) and vitellogenin (VG) profiles were analyzed in monogyne and polygyne colonies of the red imported fire ant, Solenopsis invicta. Non-denaturing and SDS-polyacrylamide gel electrophoresis (PAGE) analyses indicated that the native VN was likely 350 kDa and comprised of two subunits in the molecular size range of 170-185 kDa. SDS-PAGE of hemolymph showed that the relative mobilities and subunit patterns of VG and VN were similar. VG was present in the hemolymph of reproductive queens; alate, virgin queens; and workers, but not in males. Anti-VN, prepared from polygyne egg homogenates, reacted with egg homogenates and with hemolymph VG from reproductive, monogyne and polygyne queens and alate, virgin polygyne queens. Analysis of circulating VG and ovarian development in alate, virgin queens showed that low levels of VG appeared by five days following adult eclosion, but egg development was not observed until seven weeks. VG was evident in newly inseminated queens, and increased steadily for the first three weeks following dealation. VG levels declined slightly near eclosion of the first workers (= nanitics) and dropped sharply after nanitic emergence at five weeks following dealation. Oocyte maturation peaked at days 15-25 following dealation, but otherwise remained low but steady. These studies provide the basis for future investigations into endocrine regulations of vitellogenesis in S. invicta queens.

Cloning of an aquaporin-like cDNA and in situ hybridization in adults of the mosquito Aedes aegypti (Diptera: Culicidae).

A cDNA encoding a putative water channel protein, aquaporin, was cloned from a cDNA library of Aedes aegypti Malpighian tubules. The cDNA encodes a 26.11 kDa protein similar to insect aquaporins from Haematobia irritans exigua (Diptera) and Cicadella viridis (Homoptera), and to mammalian aquaporin 4. Localization of the messenger RNA (mRNA) was performed by in situ hybridization of Malpighian tubules and analysed by fluorescence and confocal microscopy. The mRNA was localized in tracheolar cells associated with the Malpighian tubules. No signal was detected in the Malpighian tubule epithelium. The molecular mechanisms for water movement between tissues and tracheoles are not yet elucidated in insects. Our results suggest a model to explain fluid movements in tracheoles during insect respiration.

Primary structure of CHH/MIH/GIH-like peptides in sinus gland extracts from Penaeus vannamei.

Peptides belonging to the CHH/MIH/GIH-family of crustacean hormones were isolated from acetic acid extracts of sinus glands isolated from eyestalks of the shrimp, Penaeus vannamei. The peptides were isolated by chromatography and molecular weights determined by MALDI mass spectrometry. Peptides in the range of 7-9 kDa and containing three disulfide bridges were selected for amino acid sequence analysis. Three peptides with the requisite properties were present in sufficient amounts for sequence analysis. Two peptides had unique sequences similar to CHH/MIH/GIH peptides from other crustaceans. A third peptide seemed to be a truncated form of one of the previous sequences.

Juvenile hormone inhibition of gene expression for cytochrome P4504C1 in adult females of the cockroach, Blaberus discoidalis.

The regulation and pattern of gene expression for cytochrome P4504C1 was measured in the fat body of adult females of the cockroach, Blaberus discoidalis. The level of CYP4C1-mRNA was high at adult emergence but disappeared after 4 days of adult life. In starved females, CYP4C1-mRNA levels declined by day 4 but increased steadily thereafter; by 25 days, the levels were nearly twice those observed at eclosion. Both the rapid early disappearance of the transcript and the starvation-related increase failed to occur following decapitation. Allatectomy also prevented the disappearance of CYP4C1-mRNA at day 4, and treatment of decapitated females with methoprene (JHA) stimulated a 70% decrease in transcript within 24 h. Injection of synthetic Blaberus hypertrehalosemic hormone (HTH) increased CYP4C1-mRNA by six-fold in the fat body of both intact and decapitated females. CYP4C1-mRNA in the fat body of males did not respond to JHA treatment. The dynamics of CYP4C1-mRNA in the fat body of females could be explained based on an inhibition of CYP4C1 expression by JH that was overcome by HTH.

The effect of biogenic amines and their analogs on carbohydrate metabolism in the fat body of the cockroach Blaberus discoidalis.

Several biogenic amines and their analogs were examined for stimulatory effects on glycogen phosphorylase activity and trehalose biosynthesis in fat body of the cockroach, Blaberus discoidalis. Octopamine and synephrine were the most potent activators of fat body phosphorylase; 10 muM octopamine being nearly as effective as the hypertrehalosemic hormone (HTH). Epinephrine, norepinephrine, and tyramine produced intermediate effects, whereas dopamine, 5-hydroxytryptamine, and melatonin had no effect. The fat body octopamine receptors appeared to be pharmacologically related to vertebrate alpha-adrenergic receptors and belonged to the Octopamine1 class receptor. In contrast to previous reports, synephrine also induced both phosphorylase activation and hypertrehalosemia as effectively as octopamine. Demethylchlordimeform, a formamidine insecticide structurally similar to octopamine, also strongly activated fat body phosphorylase, possibly by interaction with the octopamine receptor.

Feeding effects on gene expression of the hypertrehalosemic hormone in the cockroach, Blaberus discoidalis.

Feeding effects on hypertrehalosemic hormone (HTH) transcript levels in corpora cardiaca (CC) of adult females of the cockroach, Blaberus discoidalis were measured using dot blot hybridization. HTH transcript levels were nearly doubled in CC from females withheld from food and water for ten days compared to CC from fed females. The increase in HTH-mRNA was a response to starvation, not dehydration, and reversed within 2 days after exposure to food. HTH-mRNA was elevated in CC from fed insects that had their recurrent nerve severed, but low fecal output by insects with severed nerves indicated that feeding and digestion were impaired. Thus, the increased HTH synthesis likely resulted from starvation rather than disruption of neural regulation. CC from starved females that were refed with either solutions or agar that contained glucose did not show down-regulation of HTH-mRNA. Likewise, injections of glucose or trehalose did not suppress HTH-mRNA levels in CC of starving insects. Down-regulation of the starvation-related increase in HTH-mRNA appears to be a response to consumption of a complex of nutrients and not to increased carbohydrates or mechanical aspects of feeding.

Hypertrehalosemic hormone in a cockroach: molecular cloning and expression.

Hypertrehalosemic hormone (HTH) is a neuropeptide in the adipokinetic hormone/red pigment-concentrating hormone (AKH/RPCH) family that stimulates the synthesis of trehalose, the main blood sugar of many insects. The preproHTH of the cockroach Blaberus discoidalis was cloned from the corpora cardiaca (CC), the endocrine source for HTH, and the deduced sequence and organization of preproHTH were compared with other AKH/RPCH precursors. PreproHTH mRNA was determined to be approximately 0.5 kb in length as predicted by DNA sequence analysis. Northern blot analysis of the CC, ventral nerve cord, brain and fat body detected HTH-mRNA only in the CC. Levels of the HTH transcript in the CC were determined according to age, gender and mating. The HTH message was most abundant in the CC during the first several days of adult life in both sexes, then declined by 50% and were stable. HTH-mRNA levels in the CC did not respond to mating.

Physiological effectors of hyperglycemic neurohormone biosynthesis in an insect.

Biosynthesis of hypertrehalosemic hormone (HTH) by isolated corpora cardiaca (CC) from Blaberus discoidalis cockroaches was measured for the 3-week period following adult ecdysis using an in vitro bioassay based on the incorporation of [3H]tryptophan. Synthetic rates for both males and females were highest following the molt and declined with age. Rates of HTH secretion and levels of stored HTH followed a similar pattern. We found no neural or endocrine brain effects or photophase influence on HTH synthesis. Synthesis was unaffected by treatment with 20-hydroxyecdysone or the juvenile hormone analog methoprene. In starved insects, HTH synthesis doubled and secretion increased 66% relative to fed controls.

Free amino acids in the hemolymph of the cockroach, Blaberus discoidalis.

The concentrations of free amino acids in the hemolymph of adult males of the cockroach Blaberus discoidalis were determined by automated amino acid analysis. Eighteen of the twenty standard amino acids as well as two nonstandard amino acids (hydroxyproline and ornithine) were found. Aspartic acid and cysteine were not present. Proline levels were up to 100-fold higher than those of any other amino acid. Total amino acid concentration was comparable to those reported for most other cockroaches. The reported amino acid profile formed the basis for an organ culture medium which sustained metabolic viability of isolated B. discoidalis neuroendocrine glands and brain-gland complexes for at least 24 hr.

Calcium-dependent action of hypertrehalosemic hormone on activation of glycogen phosphorylase in cockroach fat body.

Signaling mechanisms for Blaberus discoidalis hypertrehalosemic hormone (Bld-HrTH)-dependent glycogen phosphorylase activation were investigated in vitro using fat body of the tropical cockroach, B. discoidalis. Brief treatment of fat bodies with Bld-HrTH in the absence of extracellular Ca2+ showed a significant activation of phosphorylase. Although extracellular Ca2+ was required for a full activation of phosphorylase by Bld-HrTH, stimulation in the absence of extracellular Ca2+ suggested that intracellular Ca2+ was also involved in Bld-HrTH signal transduction. Thapsigargin and thimerosal mobilize Ca2+ from intracellular stores by different mechanisms, and both chemicals stimulated phosphorylase activities as effectively as a maximum dose of Bld-HrTH. Bld-HrTH likely induces intracellular Ca2+ release followed by extracellular Ca2+ entry across the plasma membrane. Inositol-1,4,5-trisphosphate (InsP3) levels were greatly increased by Bld HrTH in a time- and dose-dependent manner, suggesting that InsP3 might be a Ca(2+)-mobilizing intracellular second messenger for Bld-HrTH.

Calcium-dependent signal transduction by the hypertrehalosemic hormone in the cockroach fat body.

The hypertrehalosemic hormones (HrTH) are insect peptides that stimulate the fat body to synthesize trehalose. The present research investigated the relative importance of extra- and intracellular Ca2+ for HrTH signal transduction in the fat body of the cockroach, Blaberus discoidalis. Although trehalose synthesis could be stimulated significantly by B. discoidalis HrTH (Bld-HrTH) in the absence of Ca2+, the presence of Ca2+ was necessary to attain a maximum hormonal response. Entry of extracellular Ca2+ mediated by ionophores (A23187, ionomycin) produced no significant hypertrehalosemic effects, whereas release of intracellular Ca2+ by thimerosal or thapsigargin significantly stimulated trehalose synthesis. The hypertrehalosemic effect by thimerosal occurred independently of extracellular Ca2+. Dantrolene inhibits intracellular Ca2+ release, and its presence partially suppressed Bld-HrTH-dependent hypertrehalosemia. In conclusion, the presence of either extracellular Ca2+ or intracellular Ca2+ enhanced the Bld-HrTH response, but agents that affected intracellular Ca2+ release produced the more conclusive effects.

In vitro hormonal regulation of glycogen phosphorylase activity in fat body of the tropical cockroach, Blaberus discoidalis.

Blaberus hypertrehalosemic hormone (Bld-HTH)-dependent glycogen phosphorylase activation was investigated using in vitro fat bodies from the cockroach, Blaberus discoidalis. Resting levels of active phosphorylase were decreased by the presence of trehalose and glucose. Phosphorylase activation was dose-responsive to Bld-HTH and increased ca. 3-fold over a range of 0.02 to 2 nM Bld-HTH. Maximum phosphorylase activation required only 5-min exposure to Bld-HTH; reversion to the inactive state began within 15 min after Bld-HTH removal and was completed by 60 min. Octopamine also activated phosphorylase but required 10(3)-fold higher concentrations than did Bld-HTH. Concentrations of Bld-HTH and octopamine that increased active phosphorylase did not elevate fat body cAMP levels, although a high concentration of octopamine increased tissue cAMP levels. cAMP did not increase phosphorylase activity, but Ca2+ was important for both Bld-HTH- and octopamine-dependent phosphorylase activation.

In vitro bioassay for hypertrehalosemic hormone-dependent trehalose biosynthesis by fat body from adult Blaberus discoidalis cockroaches.

An in vitro bioassay suitable for routine use to investigate hypertrehalosemic hormone (HTH)-dependent trehalose biosynthesis was developed for the cockroach fat body. Blaberus discoidalis fat bodies were isolated and divided so that eight matched pieces from a single tissue could be compared for multiple control and experimental treatments. Optimum incubation conditions and the properties of HTH-dependent trehalose synthesis were determined. Dose-response studies determined an EC50 of 0.044 nM. HTH for male fat body and 0.16 nM HTH for female tissue. HTH increased trehalose production by male fat body 3-fold compared to only a 67% maximum increase by the female tissue, and only the male tissue was used in subsequent studies. Fat body required only 5-min exposure to HTH for maximum trehalose production for 1 h. Trehalose synthesis was inhibited by > or = 15 mM trehalose in the incubation medium. The fat body showed a developmental increase in trehalose synthesis in vitro that was reflected by hemolymph trehalose in vivo. Basal and HTH-related trehalose synthesis were low between days 0 and 10, increased 3-fold by day 20, and were high thereafter. These studies have established baseline data for future investigations to identify the signal transduction mechanisms involved in HTH regulation of fat body metabolism.

Intracellular transduction of trehalose synthesis by hypertrehalosemic hormone in the fat body of the tropical cockroach, Blaberus discoidalis.

Fat body of adult male Blaberus discoidalis cockroaches exposed to B. discoidalis hypertrehalosemic hormone (HTH) in vitro showed a decline in tissue glycogen as carbohydrate increased in the medium. In vivo HTH injections increased hemolymph carbohydrate and fat body glycogen phosphorylase activity > 2-fold compared to controls. In vivo trehalose synthesis was unaffected by agents that enhance intracellular levels of cyclic nucleotides including: dibutyrl cAMP, dibutyryl cGMP, forskolin (adenylyl cyclase activator) and isobutyl methylxanthine (IBMX) or theophylline (cAMP phosphodiesterase inhibitors). DbcAMP+IBMX stimulated trehalose biosynthesis of fat body in vitro and had additive effects with a minimally stimulatory HTH concentration. However, adenylyl cyclase activity was unaffected by HTH either with isolated fat body or fat body membrane preparations. We conclude that cAMP is not a second messenger for HTH, but cAMP can stimulate trehalose production independent of HTH through actions on common regulatory events related to trehalose biosynthesis. Dibutyryl cGMP and phorbol esters and diacylglycerol (activators of protein kinase C) also failed to stimulate trehalose biosynthesis in vitro. Extracellular Ca2+ enhanced HTH-dependent phosphorylase activity, glycogenolysis and hypertrehalosemia and maintained basal levels of phosphorylase a at twice those observed in the absence of Ca2+. However, Ca2+ entry by Ca2+ ionophore (A23187) did not mimic HTH effects. Results of these studies demonstrated that extracellular Ca2+ is important for HTH-dependent trehalose biosynthesis but cAMP and cGMP are not involved.

Hypertrehalosemic hormone effects on transcriptional activity in the fat body of the cockroach, Blaberus discoidalis.

Gene expression was examined in the fat body of adult male Blaberus discoidalis cockroaches in response to the hypertrehalosemic hormone (HTH). HTH regulates a natural peak of heme synthesis in the fat body at day 4 of adult life. Inhibition of transcriptional activity by alpha-amanitin suppressed both the natural increase in heme biosynthesis at day 4, and the increase that was induced in decapitated animals by HTH administration. In contrast, the regimen of alpha-amanitin treatments that suppressed HTH-responsive heme biosynthesis had no effect on the ability of HTH to increase hemolymph carbohydrate. HTH administration to decapitated animals produced a 25% increase in total fat body RNA and a 3-fold increase in [3H]uridine incorporation. In vitro translation of fat body RNA showed that HTH increased the synthesis of polypeptides at 24, 60 and 77 kDa. The results suggest that HTH affects fat body heme synthesis through gene expression, and evidence was obtained for three polypeptides that increase in the fat body in response to HTH.

Cytochrome P450 family 4 in a cockroach: molecular cloning and regulation by regulation by hypertrehalosemic hormone.

Hypertrehalosemic hormone (a carbohydrate-mobilizing neuroendocrine decapeptide) and starvation markedly increased levels of a cockroach (Blaberus discoidalis) fat body cytochrome P450 message. The gene represented by the cloned P450 cDNA has been named CYP4C1 (cytochrome P450 family 4, subfamily C, gene 1), a newly identified member of the ubiquitous cytochrome P450 monooxygenase gene superfamily. Blaberus CYP4C1 (511 amino acids, Mr = 58,485) has a hydrophobic NH2 terminus and a sequence near the COOH terminus that is homologous to the cysteine-containing heme-binding region definitive of cytochromes P450. The cockroach sequence is 32-36% identical to mammalian family 4A and 4B enzymes. It contains a 13-residue sequence characteristic of family 4 but not other P450s. This study suggests that CYP4C1 is hormonally regulated in association with energy substrate mobilization and supports the idea that family 4 is an old and widespread gene family.

Structure-activity relationships on hyperglycemia by representatives of the adipokinetic/hyperglycemic hormone family in Blaberus discoidalis cockroaches.

Several members of the adipokinetic/hyperglycemic neurohormone family from several different invertebrate species have been prepared by solid-phase peptide synthesis and assayed by a modified in vivo hyperglycemic bioassay in Blaberus discoidalis cockroaches. The hypertrehalosemic hormone (HrTH) is the endogenous hypertrehalosemic factor for B. discoidalis and was the most potent peptide in the assay. The more divergent the sequence of a family member from Blaberus HrTH, the less potent was the bioanalog. Manduca adipokinetic hormone is the most divergent peptide of the family and was totally inactive in the bioassay. Locusta adipokinetic hormone I had reduced maximum activity in the assay, which suggests that Ser5 is an important residue for the transduction of the hyperglycemic response. The direct relation between bioanalog similarity to Blaberus HrTH sequence and potency suggests that the hormone and target cell receptor for HrTH have evolved to maintain an "optimal fit".

Adipokinetic hormone gene sequence from Manduca sexta.

A gene sequence encoding the neuropeptide adipokinetic hormone (AKH) was isolated from a genomic library of the tobacco hornworm, Manduca sexta. 2.5 x 10(5) recombinant plaques were screened with a 29-mer oligonucleotide substituted with deoxyinosine at six ambiguous codon positions. One clone was obtained from which we deduced an intronless gene encoding the AKH precursor (prepro-AKH). From NH2 to COOH terminus, prepro-AKH consists of a signal peptide, a single AKH peptide block followed by a Gly-Lys-Arg processing site, and a 34-residue sequence that appears unrelated to known peptides. The cloned AKH gene sequence hybridized to one transcript from the adult corpus cardiacum/corpus allatum complex. The Manduca genome appears to contain one copy of the AKH gene. These studies are a basis for explaining synthesis of a representative from a widespread, physiologically important family of arthropod neuropeptides.