James Y. Bradfield

Ovarian protein synthesis in the South American white shrimp, Penaeus vannamei, during the reproductive cycle

Summary Ovarian development was induced by eyestalk ablation in the South American white shrimp, Penaeus vannamei. During the gonadotrophic cycle, ovarian protein increased from virtually undetectable levels in undeveloped ovaries to >400 mg in mature ovaries. Total protein was separated by SDS-PAGE and high molecular weight polypeptides of ~175–200 kDa were abundant in vitellogenic ovaries but absent in previtellogenic ovaries. These polypeptides are probably the yolk polypeptides. We determined in vitro protein synthesis rates for the ovary and hepatopancreas during ovarian development. Tissues from animals at the onset of vitellogenesis incorporated radiolabelled amino acids at the most active rate. Using autoradiography, we demonstrated that the ovary synthesized many polypeptides, including the major, putative yolk polypeptides. We did not detect synthesis of these polypeptides by the hepatopancreas.

Juvenile hormone acid: Evidence for a hormonal function in induction of vitellogenin in larvae of Manduca sexta†

In the tobacco hornworm (Manduca sexta), vitellogenin (Vg), the major yolk protein precursor, and its mRNA are first detectable in the prepupal stage; and production of both can be enhanced by methoprene, a juvenile hormone (JH) analog. Competence to respond to methoprene is acquired after ecdysteroid-initiated commitment for metamorphosis. Here we show that acquisition of competence requires prior exposure to JH-II acid in addition to ecdysteroid. Application of 20-hydroxyecdysone or RH5992, an ecdysteroid analog, to isolated abdomens from feeding larvae (precommitment) results in exposure of the dorsal vessel (EDV), a sign of metamorphic commitment--but such abdomens do not make Vg in response to methoprene. However, injection of JH-II acid along with 20-hydroxyecdysone into isolated abdomens causes Vg production in response to methoprene. Methoprene acid similarly induces competence to respond to methoprene. Northern blot analysis confirmed that Vg transcripts are present in fat body only if isolated abdomens were pretreated with both ecdysteroid, and JH-II acid or methoprene acid. The latter two can induce competence even in precocious prepupae resulting from removal of the corpora allata (the glands that produce JH) from early penultimate larvae. JH-III acid and related metabolites such as farnesol, farnesoic acid, and methyl farnesoate do not induce competence. Hitherto, JH acids have been regarded as precursors or catabolites of JHs. Here we show for the first time that JH acid has a hormonal function that cannot be performed by JH itself.

Aromatic hexamerin subunit from adult female cockroaches (Blaberus discoidalis): Molecular cloning, suppression by juvenile hormone, and evolutionary perspectives

Abstract In an effort to identify several polypeptides that are strongly suppressed by juvenile hormone (JH) in fat body of adult female Blaberus discoidalis cockroaches, we have cloned a cDNA representing a polypeptide member of the hexamerin family of arthropod serum proteins. The deduced primary translation product consists of a 17-residue signal peptide and a mature protein of 716 residues (86.1kDa) composed of 21% tyrosine plus phenylalanine. The latter property leads us to assign the name arylphorin to the cockroach protein. In pairwise comparisons Blaberus arylphorin (BAr) has 30–39% positional identity with other insect hexamerins and with cheliceratan and crustacean hemocyanins. In multiple alignment with 21 arthropod hexamerins there are 18 invariant residues. We present the profile of BAr mRNA as correlated to the reproductive cycle, where BAr accumulates dramatically between cycles of JH-dependent vitellogenesis.

Physiological actions by hypertrehalosemic hormone and adipokinetic peptides in adult Blaberus discoidalis cockroaches

Abstract Members of the adipokinetic hormone/red pigment-concentrating hormone (AKH/RPCH) family characteristically cause metabolite mobilization by the insect fat body. The present study identified several additional physiological actions in adult Blaberus discoidalis cockroaches that were influenced by synthetic Blaberus hypertrehalosemic hormone (HTH) and other AKH/RPCH family peptides. HTH elevated blood carbohydrate by 4-fold and cytochrome heme a + b synthesis of fat body mitochondria by 3-fold. Both carbohydrate and heme synthesis were dose-responsive to HTH. Carbohydrate synthesis was 10 times more sensitive to HTH than heme synthesis. Heme synthesis was also stimulated by Periplaneta cardioacceleratory hormones (CAH)-I and -II and RPCH but not by AKH-I or -II, at the doses tested. HTH showed strong cardioexcitatory activity. Long-term treatment of decapitated female B. discoidalis with juvenile hormone analog (JHA = methoprene) stimulated by 2.6-fold the rate of synthesis of secreted fat body proteins. HTH enhanced the JHA-dependent export protein synthesis by 42% above that observed with JHA alone. SDS-PAGE demonstrated that JHA determined the nature of the newly synthesized polypeptides; HTH enhanced their synthesis rate. Neither AKH-I nor HTH affected protein synthesis when added directly to isolated fat body. These results demonstrate that peptides of the AKH/RPCH family have multiple physiological actions related to fat body energy metabolism.

Cloned cDNA and Antibody for an Ovarian Cortical Granule Polypeptide of the Shrimp Penaeus vannamei

A cloned cDNA was generated to a transcript for a major ovarian polypeptide (200 kDa) of the South American white shrimp, Penaeus vannamei. The cloned cDNA hybridized to a single transcript in ovaries but not to RNA from the hepatopancreas or muscle. For immunodetection and quantitation, a monospecific polyclonal antibody was raised against the cDNA translation product expressed in bacteria. The antibody was used to show that the 200 kDa ovarian polypeptide accumulated in cortical granules during ovarian development to comprise ∼11% of the total ovarian protein and disappeared during early embryonic development. These studies begin to explain a gene-product relationship essential for reproduction in P. vannamei.

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.

Neuroendocrine and juvenile hormone effects on fat body protein synthesis during the reproductive cycle in female Blaberus discoidalis cockroaches

Ovarian protein content and fat body protein synthesis were measured during the first gonotrophic cycle in virgin female Blaberus discoidalis cockroaches. Protein synthesis was measured for in vitro fat bodies from animals treated with combinations of a juvenile hormone analog (JHA = methoprene) and corpora cardiaca (CC) extracts. Ovarian protein content began to increase on Day 5 of adult life and reached its maximum at Days 20-22. Synthesis of proteins secreted by the in vitro fat body increased by 12-fold between emergence and its maximum on Day 18, then declined to nearly its original level by Day 33. Synthesis of nonsecreted, fat body proteins increased by 4-fold between emergence and a maximum on Day 21, then declined. CC extracts and JHA were administered to decapitated females to determine their regulative effects on fat body protein synthesis. The synthesis by the fat body of nonsecreted proteins was increased by both JHA and CC extracts. In contrast, synthesis of secreted proteins increased only in the presence of JHA. CC extracts, alone, had no effects on the synthesis of secreted proteins, but administration of CC extracts in combination with JHA increased the synthesis of the secreted proteins by 55% above that observed with JHA alone. SDS-PAGE of proteins secreted into the medium by the in vitro fat body demonstrated that JHA stimulated the synthesis of specific polypeptides, whereas CC extracts did not affect the synthesis of specific-secreted polypeptides. These results suggest that JH regulates specific protein synthesis by the fat body of B. discoidalis; neurohormones elevate the general capacity of the fat body for protein synthesis and amplify the specific effects of JH.

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.

NEUROHORMONE SIGNAL TRANSDUCTION FOR DUAL REGULATION OF METABOLISM AND GENE EXPRESSION IN INSECTS : HYPERTREHALOSEMIC HORMONE AS A MODEL

The hypertrehalosemic hormone of the cockroach, Blaberus discoidalis (Bld-HTH), stimulates trehalose biosynthesis by the fat body and is a member of the adipokinetic hormone/red pigment-concentrating hormone family of arthropods. Bld-HTH also stimulates expression of the gene for cytochrome P4504C1 (CYP4C1) in the fat body. The present studies were undertaken to determine if simultaneous stimulation of trehalose biosynthesis and CYP4C1 expression by Bld-HTH occurs in response to independent signal transduction cascades or if they are sequential events of a common cascade. The Bld-HTH signal transduction cascade does not involve cyclic nucleotides but appears to employ inositol 1,4,5-triphosphate (IP3) to release intracellular Ca2+ for activation of phosphorylase which increases trehalose biosynthesis and CYP4C1 expression. Trehalose-6-phosphate synthase activity was also stimulated by Bld-HTH. Both phosphorylase activation and CYP4C1 expression appear regulated by a common, IP3/Ca2+-based signal transduction cascade.

Contrasting modulations of gene expression by a juvenile hormone analog

Decapitation altered the pattern of in vitro protein synthesis by the fat body of adult female Blaberus discoidalis cockroaches. Several polypeptides showed marked diminution, whereas two polypeptides showed markedly increased synthesis. The polypeptide profile was restored to normal in decapitated cockroaches by treatment with the juvenile hormone analog (JHA) methoprene. Cloned cDNAs that were isolated represented two fat body transcripts: one transcript was strongly stimulated and the other strongly suppressed by JHA. JHA acted on fat bodies in vitro to modulate the levels of the two transcripts. These studies indicate a system where juvenile hormone may provoke vividly contrasting gene expressions in the same tissue at the same time.