Larry L. Keeley

Characterization of the Molt Stages in Penaeus vannamei: Setogenesis and Hemolymph Levels of Total Protein, Ecdysteroids, and Glucose

The molting cycle of Penaeus vannamei ju veniles was characterized by distinct and predictable changes in the setae of pleopods. The molt pattern was diecdysic with a relatively short intermolt period (40%) and a long proecdysial period (>53%). The levels of both total protein and ecdysteroids increased in the hemo lymph during proecdysis, whereas the level of hemo lymph glucose was low at metecdysis and proecdysis and maximal during anecdysis. As revealed by SDS-PAGE, the relative concentrations of two polypeptides (32 kD; 175 kD) changed during the molting cycle.

Ecdysteroid-titre reduction and developmental arrest of last-instar Heliothis virescens larvae by calyx fluid from the parasitoid Campoletis sonorensis

Abstract Fifth-instar Heliothis virescens larvae did not pupate after injections of Campoletis sonorensis calyx fluid in or before the burrow-digging stage of development. Arrested development occurred in 40% of larvae injected at the cell-formation stage. Further experiments showed that the particles in calyx fluid were responsible for developmental arrest. Arrested development due to calyx fluid could be reversed by injecting 10 μg of either ecdysone or 20-hydroxyecdysone, although a second injection of 20-hydroxyecdysone was needed for some larvae 3 days after the first treatment. Ecdysteroid production ceased for up to 10 days in 5th-instar H. virescens after calyx-fluid injection. After 10 days, some experimental larvae began to produce ecdysteroids again but remained developmentally arrested. The head, thorax, or abdomen of larvae were isolated by ligations and calyx fluid injected into the isolated body region. After 24 h, ligatures were released and the larvae observed for developmental arrest. Only injections into the isolated thorax stopped development. This, along with ecdysteroid data, indicated that C. sonorensis calyx fluid may directly affect the prothoracic glands of 5th-instar H. virescens.

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.

Insect hypertrehalosemic hormone: Isolation and primary structure from Blaberusdiscoidalis cockroaches

A new neurohormone was isolated and structurally characterized that increased hemolymph carbohydrate (trehalose) levels in the cockroach, Blaberus discoidalis. The hormone was isolated in high yield by a rapid HPLC procedure. The sequence, pGlu-Val-Asn-Phe-Ser-Pro-Gly-Trp-Gly-Thr-NH2, was suggested from gas-phase Edman degradation of a peptide fragment of the natural peptide after deblocking with pyroglutamate aminopeptidase. The structure was confirmed by synthesis of the suggested sequence. The synthetic peptide had identical chromatographic and biological properties as the natural peptide.

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.

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.

Characterization of insect fat body mitochondria isolated by a rapid procedure.

Abstract 1. 1. A rapid method is described for isolating mitochondria from the fat body tissue of the cockroach Blaberus discoidalis. 2. 2. Oxidative activity was comparable to that of conventional procedures with a recovery of 50 per cent of the initial total respiratory activity. With succinate, ADP:O = 1.6 and respiratory control ratios were between 3 and 4. 3. 3. Electron microscopy showed isolated fat body mitochondria to be about 1.0 μm in dia. with a condensed ultrastructure. Only slight amounts of membraneous contamination were found in pellets, and the symbiotic micro-organisms found in fat body mycetocytes were rare.

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.

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

SummarySeveral 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”.

Endocrine regulations of ovarian maturation in the cockroach Blaberus discoidalis

The first gonotrophic cycle of oocyte maturation was defined for virgin Blaberus discoidalis cockroaches. A high correlation was found between growth of the basal oocytes and increased ovarian protein content. Allatectomy blocked ovarian protein formation, whereas JH-III administration compensated for the loss of the corpora allata. Experiments involving neuro-endocrine surgery and hormone replacement therapy indicated that JH, alone, could stimulate ovarian protein formation; however, the addition of neuroendocrine extracts enhanced the JH effects. The data suggest that both JH and neurohormones stimulate aspects of ovarian protein formation in this insect.