Edward P. Masler

Inhibitory effects of oostatic hormone on ovarian maturation and ecdysteroid production in diptera

Extracts from post-vitellogenic ovaries of Musca domestica, Aedes atropalpus and Drosphila melanogaster were examined for the ability to inhibit the vitellogenic phase of growth in newly emerged A. atropalpus and D. melanogaster adult females. All extracts were inhibitory in A. atropalpus. D. melanogaster females were less sensitive than A. atropalpus females, only showing inhibition at a two-fold greater concentration of M. domestica extract. The titre of oostatic hormone in A. atropalpus ovaries correlated with the decline in ecdysteroid synthesis by these ovaries. A. atropalpus ovaries containing post-vitellogenic follicles were shown to inhibit ovarian ecdysteroid synthesis in vitro by vitellogenic ovaries of both A. atropalpus and D. melanogaster. D. melanogaster ovaries were not capable of eliciting such an inhibition in vitro, at least with the numbers of ovaries utilized in our experiments.

Responses of Heterodera glycines and Meloidogyne incognita to exogenously applied neuromodulators.

Biogenic amines regulate important behaviours in nematodes and are associated with pharyngeal activity in plant-parasitic nematodes. A robust behavioural assay based upon nematode body movements was developed to expand the study of these and other neuroregulators in plant-parasitic nematodes. Dopamine, octopamine and serotonin each had significant but differing effects on the behaviour of soybean cyst nematode Heterodera glycines and root-knot nematode Meloidogyne incognita juveniles. Body movement frequency was increased twofold in H. glycines by 5 mM dopamine (P = 0.0001), but decreased by 50 mM dopamine in H. glycines (88%) and M. incognita (53%) (P < 0.0001). Movement frequency in both species was increased by 50-70% (P < 0.0001) by 50 mM octopamine, and 5 mM octopamine increased M. incognita movement frequency more than twofold (P < 0.0001). Movement frequency in each species was reduced by more than 90% by 5 mM serotonin (P < 0.0001). While amplitude of body movement in H. glycines was unaffected by any amine, it was significantly reduced in M. incognita by all amines (P < 0.0006). Stylet pulsing frequencies in either species were unaffected by dopamine or octopamine, but 5 mM serotonin stimulated pulsing in H. glycines by nearly 13-fold (P < 0.0001) and in M. incognita by more than 14-fold (P < 0.0001). The invertebrate neuropeptide FLRFamide (N-Phe-Leu-Arg-Phe) increased M. incognita body movement frequency 45% (P = 0.02) at 1 mM but did not affect stylet activity. Finally, H. glycines egg hatch was completely suppressed by 50 mM serotonin, and partially suppressed by 50 mM dopamine (75%; P < 0.0001) and 50 mM octopamine (55%; P < 0.0001).

Prothoracicotropic Hormone Stimulation of Ecdysone Synthesis by the Prothoracic Glands of the Gypsy Moth, Lymantria Dispar

Kopec (1922), using Lymantria, first demonstrated that a factor(s) from the head was necessary for insect metamorphosis. This factor was later termed prothoracicotropic hormone (PTTH) (Kobayashi et al., 1965). PTTH stimulates ecdysone synthesis by the paired prothoracic glands (PGs) in lepidopterans, and ecdysone is subsequently converted by other tissues to the active hormone, 20-hydroxyecdysone (see Gilbert et al., 1980). In vitro bioassays for PTTH have been established in both Bombyx mori (Nagasawa et al., 1984; Okuda et al., 1985) and Manduca sexta (Bollenbacher et al., 1979) and have been utilized to quantitate PTTH activity in various tissues and fractions (see Ishizaki and Suzuki, 1984; Bollenbacher and Granger, 1985).

Discovery and Partial Characterization of Prothoracicotropic Hormones of the Gypsy Moth, Lymantria Dispar

The brain neuropeptide prothoracicotropic hormone (PTTH) is an ecdysiotropin that drives post-embryonic growth and development through the stimulation of ecdysteroid production by the prothoracic glands (PG) (Gilbert et al., 1980). Research to isolate PTTH has utilized primarily two species, Bombyx mori (Ishizaki and Suzuki, 1984) and Manduca sexta (Bollenbacher and Granger, 1985). Characterization and isolation studies have used both in vivo (Bombyx, Ishizaki et al, 1983, Nagasawa et al, 1984a; Manduca, Kingan, 1981) and in vitro (Bombyx, Nagasawa et al, 1984a; Okuda et al, 1985; Manduca, Bollenbacher et al, 1979) bioassays. Recent work on Bombyx and Manduca PTTH demonstrates that biological activity is associated with two molecular-weight ranges of ca. 20–28 kD and ca. 4–7 kD (Ishizaki et al., 1983, Bollenbacher et al., 1984). A family of 4 kD PTTH’s has been isolated from Bombyx (Nagasawa et al., 1984b).

Vitellogenin synthesis in female larvae of the gypsy moth, Lymantria dispar (L.): suppression by juvenile hormone

Abstract 1. 1. Fat body from feeding-phase, last instar gypsy moth females incorporates l -[35S]methionine in vitro into two vitellogenins with the same molecular masses (165 and 180 kDa) as the apo-vitellogenins found in teh hemolymph and the apo-vitellins in teh eggs. 2. 2. Both apo-vitellogenins are observed in the medium of fat body cultures, but only the 180 kDa apo-vitellogenin is observed in extracts of cultured tissue. 3. 3. Synthesis and accumulation of the apo-vitellogenins are suppressed in a dose-dependent manner by topical treatment with the juvenile hormone analog, methoprene, prior to day 4. 4. 4. This suppression suggests that a declining juvenile hormone titre is involved in the initiation of vitellogenin synthesis.

Comparison of FaRP Immunoreactivity in Free‐living Nematodes and in the Plant‐parasitic Nematode Heterodera glycines

Abstract: The family of FMRFamide‐related peptides (FaRPs) is widely distributed among invertebrates, where the peptides serve as neuromodulators. Published reports indicate that numerous FaRP sequences exist in free‐living and animal parasitic nematodes. Using a FMRFamide ELISA, FaRP immu‐noreactivity was detected in extracts of the soybean cyst nematode, Heterodera glycines, in both sexes and at all developmental stages. HPLC‐ELISA results revealed a number of immunoreactive components in H. glycines preparations, and a comparison with extracts of the free‐living nematodes Caenorhabditis el‐egans and Panagrellus redivivus showed significant qualitative differences in FaRP immunoreactivity between the plant parasite and the two free‐living nematodes. Total and specific immunoreactivities varied during H. glycines development, with the highest specific activity in juveniles and males, and the highest total activity in mature females. Total female immunoreactivity was located primarily within the mature eggs. A significant portion, however, was as‐sociated with the female body, perhaps with egg laying.

Aminopeptidase-like activities in Caenorhabditis elegans and the soybean cyst nematode, Heterodera glycines

Aminopeptidase-like activities in crude whole body extracts of the free-living nematode Caenorhabditis elegans and the plant parasitic soybean cyst nematode Heterodera glycines were examined. General characteristics including pH optima, heat lability, and inactivation of enzyme by organic solvent were the same for the two species. All developmental stages of H. glycines exhibited activity. In older females, activity was present primarily in the eggs. Affinity for the substrate L-alanine-4-nitroanilide was the same regardless of the stage examined, and was similar for the two species (m for C. elegans and m for H. glycines). Nearly all (>95%) of C. elegans aminopeptidase-like activity was present in the soluble fraction of the extract, while H. glycines activity was distributed between the soluble and membrane fractions. Specific activities of the soluble enzymes were highest in C. elegans and H. glycines juveniles. The C. elegans enzyme was susceptible to a number of aminopeptidase inhibitors, particularly to amastatin and leuhistin, each of which inhibited aminopeptidase-like activity more than 90% at 90 microm. In H. glycines, aminopeptidase-like activity was inhibited 39% by amastatin at 900 microm. The apparent molecular weight of the soluble C. elegans enzyme is 70-80 kDa. Some activity in H. glycines is present in the 70-80 kDa range, but most activity (80-90%) is associated with a very high molecular weight (>240 kDa) component.

Influence of development and prothoracicotropic hormone on the ecdysteroids produced in vitro by the prothoracic glands of female gypsy moth (Lymantria dispar) pupae and pharate adults

Abstract Fluctuations in hemolymph ecdysteroid titer are part of a complex mechanism that regulates pupal-adult development. The amount of ecdysteroid produced in vitro by prothoracic glands from female Lymantria dispar (L.) (Lepidoptera: Lymantriidae) pupae and pharate adults, as well as the competency of these glands to respond to a prothoracicotropic hormone (PTTH) stimulus in vitro , each correspond temporally with hemolymph ecdysteroid titers. Based on studies of gland kinetics and dose-responses to brain extract using prothoracic glands from different female pupal and pharate adult ages, an in vitro bioassay for the quantification of PTTH activity was developed using glands from day 2 females incubated without stimulus for 1 h followed by a 3 h incubation with stimulus. Only extracts of brains and corpora allata from pupae and pharate adults possess a PTTH factor. This factor is heat stable and can be separated on high performance size exclusion chromatography into two molecular sizes of 13.75 and 3.2 kDa. Ecdysone and 3-dehydroecdysone are produced in vitro by prothoracic glands from all ages of female L. dispar pupae and pharate adults tested. The amount of ecdysone produced by these glands exceeds that of 3-dehydroecdysone production after 4 h of incubation.

In vitro proteolysis of nematode FMRFamide-like peptides (FLPs) by preparations from a free-living nematode (Panagrellus redivivus) and two plant-parasitic nematodes (Heterodera glycines and Meloidogyne incognita).

Proteolytic activities in extracts from three nematodes, the plant parasites Heterodera glycines and Meloidogyne incognita, and the free-living Panagrellus redivivus, were surveyed for substrate preferences using a battery of seven FRET-modified peptide substrates, all derived from members of the large FMRF-amide like peptide (FLP) family in nematodes. Overall protease activity in P. redivivus was four- to fivefold greater than in either of the parasites, a result that might reflect developmental differences. Digestion of the M. incognita FLP KHEFVRFa (substrate Abz-KHEFVRF-Y(3-NO2)a) by M. incognita extract was sevenfold greater than with H. glycines extract and twofold greater than P. redivivus, suggesting species-specific preferences. Additional species differences were revealed upon screening 12 different protease inhibitors. Two substrates were used in the screen, Abz-KHEFVRF-Y(3-NO2)a and Abz-KPSFVRF-Y(3-NO2)a), which was digested equally by all three species. The effects of various inhibitor, substrate and extract source combinations on substrate digestion suggest that M. incognita differs significantly from P. redivivus and H. glycines in its complement of cysteine proteases, particularly cathepsin L-type protease.

Responses of Heterodera glycines and Meloidogyne incognita to exogenously applied biogenic amines

Hatching and head movement behaviours of second-stage juvenile (J2) of two agriculturally important plant-parasitic nematodes were affected by the in vitro application of biogenic amines. The behavioural responses of Heterodera glycines and Meloidogyne incognita to treatments of serotonin, octopamine and dopamine were qualitatively similar, but significant quantitative differences between the species were revealed. The frequency of J2 head movement was decreased by as little as 250 μM serotonin in H. glycines and 500 μM serotonin in M. incognita, with effective doses (ED50) of 0.73 mM for H. glycines and 1.72 mM for M. incognita. Octopamine had the opposite effect of serotonin, increasing J2 head movement frequency at thresholds of 2 mM in H. glycines and 1 mM in M. incognita. Octopamine ED50 values were 32.35 mM and 1.91 mM, respectively. Dopamine had no effect on head movement in either species up to concentrations of 20 mM. Serotonin inhibited hatch in both species but was more potent against H. glycines (90% inhibition at 1 mM) than M. incognita (40% inhibition at 5 mM). Octopamine reduced hatch equally in both species with over 95% inhibition at 80 mM. Dopamine had no effect on hatch in M. incognita but did inhibit H. glycines hatch over 60% at 40 mM. The value of detailed quantitative analyses of plant-parasitic nematode responses to biogenic amines for studies on nematode control is discussed.