Fluorescein-dextran sequestration in the reproductive tract of the migratory grasshopper Melanoplus sanguinipes (Orthoptera, Acridiidae).

The fluid dynamics of the reproductive system of the migratory grasshopper, Melanoplus sanguinipes F. (Orthoptera: Acrididae) was examined by the introduction of fluorescein-dextran (FD) into the hemocoel and observing its tissue specific sequestration. Male grasshoppers were observed to sequester FD first in the apical end of each sperm tube. FD then moved into the vasa deferentia and ejaculatory duct. This suggests that materials, that transit the hemolymph could be a component of the spermatophore in M. sanguinipes. Female grasshoppers were observed to sequester hemolymph FD into vitellogenic oocytes and to sometimes reuptake the FD during the resorption of oocytes in the formation of yellow bodies or corpora lutea. Female M. sanguinipes who performed long duration flight sequestered FD in their oocytes to a greater degree than controls as determined by fluorescence intensity data collected as the mean gray value of the flourescein emission channel. Transfer of hemolymph FD from males to females was observed at the pores along the margin of the operculum of eggs in the female common oviduct following mating. FD has the potential to be an effective tracker of male reproductive secretions and as a tool for the observation of insect reproductive tract development.

Comparison of radioimmunoassay and liquid chromatography tandem mass spectrometry for determination of juvenile hormone titers.

This paper compares the results of juvenile hormone (JH) titer determinations in two insect species, Melanoplus sanguinipes, a migratory grasshopper, and Acyrthosiphon pisum, the pea aphid, using a chiral-specific JH radioimmunoassay (RIA) and liquid chromatography tandem mass spectrometry (LC-MS/MS), after extraction of JH with either hexane or isooctane-methanol. We compared results of JH titer determinations done on extracts of M. sanguinipes hemolymph taken from animals flown to exhaustion in tethered flight tests or unflown controls and from whole body extracts of A. pisum raised at two different temperatures. In each case the two different treatments experienced by the experimental animals were expected to result in widely differing JH titers. Methoprene and precocene II were used as internal standards. Samples were split and titers determined simultaneously with both the LC-MS/MS and RIA procedures. Unambiguous detection of JH III by LC-MS/MS was done by identification of its specific parent ion and its mass fingerprint (m/z 289, 267, 249, 235, 217, and 189). We conclude that isooctane-methanol-extracted JH samples can be accurately analyzed by LC-MS/MS, but not by RIA without further separation of JH from contaminating lipids. Hexane extracted JH samples from hemolymph can be analyzed accurately by both RIA and LC-MS/MS. However, the RIA results from whole body extracts of aphids reared at two different temperatures were initially obscured with excess lipids even when hexane was the extraction solvent. Thus samples were further purified by Waters Sep-Pak C18 column, but contaminating phospholipids continued to cause problems with the RIA assay. The detection limit of JH III standard for RIA was 13.75+/-2.39 pg whereas that for LC-M/MS was 8.25+/-1.44 pg in our experimental conditions.

JH III production, titers and degradation in relation to reproduction in male and female Anthonomus grandis.

Juvenile hormone (JH) is necessary for the production of vitellogenin (Vg) in the boll weevil, Anthonomus grandis. Occurrence of Vg in this species is typically restricted to reproductively competent females, and is not detected in untreated males. However, the JH analog, methoprene stimulates Vg production in intact males and in the isolated abdomens of both male and female boll weevils (where in each case no Vg is detected without treatment), suggesting that males are competent to produce Vg but are normally not stimulated to do so. Preliminary work indicating that male boll weevil corpora allata (CA) produced little or no JH in vitro suggested that failure of males to produce Vg might be due to very low JH levels compared to females. This study re-examines the question of JH in male boll weevils by determining in vitro production of JH III by male CA during the first 10 days after adult emergence, determining hemolymph JH esterase activity during this same time period and hemolymph JH III titers in adults of both sexes. We also re-examine the ability of isolated male abdomens to produce Vg in response to hormonal stimulation, analyzing the effect of a wide range of methoprene and JH III dosages. Results indicate that male A. grandis have circulating JH titers and JH production similar to females. JH esterase activity is slightly but significantly higher in males than females. Vg production by isolated abdomens of both sexes after stimulation with methoprene or JH III was confirmed. Dose response studies indicated that high levels of methoprene were less effective than intermediate doses in stimulating Vg synthesis in both sexes. We conclude that the sexually dimorphic effect of JH on Vg synthesis is not due to differences in JH production or differences in JH titer between the sexes.

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.

Relationship of adipokinetic hormone I and II to migratory propensity in the grasshopper, Melanoplus sanguinipes.

This report examines three aspects of adipokinetic hormone (AKH) involvement in migratory flight behavior in the grasshopper, Melanoplus sanguinipes. The titer of hemolymph AKH I during long-duration tethered flight was examined using radioimmunoassay (RIA) after narrow bore RP-HPLC. The hemolymph fraction containing AKH I was assayed using commercially available anti-Tyr1-AKH I serum. Titer determinations of hemolymph AKH were done at rest and after various periods of flight. The amount of AKH I released from the corpora cardiaca during flight was estimated. When resting levels of AKH I and II in corpora cardiaca (CC) of migrants and non-migrants were examined with HPLC, no significant differences in AKH levels were detected between non-migrants, animals that had flown for 1 h to identify them as migrants, and animals that had flown to exhaustion (i.e., voluntary cessation). CC levels of both AKH I and II were less in this species than in locusts. When the lipid mobilization in response to AKH I and II was compared in migrants (animals that had self-identified as migrants in a 1-h tethered flight test) and non-migrants (animals that would not perform a 1-h flight in a tethered flight test), the adipokinetic response to AKH I was greater in migrants than in non-migrants, possibly indicating differences in level of sensitivity or number of receptors in the target tissues. AKH II had little effect on hemolymph lipid levels in either flight group, and may not play a significant role in lipid mobilization in this species.

Discovery and characterization of Melanoplus sanguinipes AKH II by combined HPLC and mass spectrometry methods.

Reversed-phase high-performance liquid chromatography (RP-HPLC) allows for fractionation of extract from the corpora cardiaca of insects for examination of bioactivity or immunoreactivity and/or subsequent characterization of the peptides. Using RP-HPLC, we have previously identified an adipokinetic hormone (AKH) from the corpora cardiaca of the migratory grasshopper Melanoplus sanguinipes. This hydrophobic decapeptide has the same primary structure as locust (Locusta migratoria; Lom) AKH I. Initially, only one HPLC peak with the same retention time as Lom-AKH I was detected; however, we found a second putative AKH within this fraction by using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and micro HPLC tandem electrospray ionization mass spectrometry. Armed with this information, we were able to improve the resolution of the native AKHs by micro HPLC, which allowed us to characterize the peptide. Our results show the power of coupling HPLC and mass spectrometric methods to resolve and identify very similar hydrophobic peptides at the lower picomole level.

DIFFERENT PATHWAYS IN ARTHROPOD POSTEMBRYONIC DEVELOPMENT.

To investigate the consequences of canalization and plasticity in arthropod developmental pathways, we developed a model that predicts eight possible combinations among three larval developmental parameters. From the descriptions of insect and spider postembryonic development, it is apparent that not all aspects of juvenile development are plastic and that species differ in which traits are plastic. Most strikingly, only four of the possible eight combinations of canalized and plastic parameters have been found in nature. Using this model, we show that the identity of the canalized developmental parameters and the degree of genetic variation in the value at which a given parameter is fixed have important implications for the ecology and evolution of complex life cycles.

Sexually differentiated flight responses of the Mexican bean beetle to larval and adult nutrition.

Flight of male and female Mexican bean beetle adults was examined in laboratory tests. The experimental design made it possible to examine flight behavior not only with respect to different types of hosts (young vs senescent common bean foliage) but also with respect to effects due to their utilization during particular stages of beetle development. The median flight time of males was significantly affected by the adult host, but not by the juvenile host; whereas, the median flight time of females tended to be more affected by the juvenile than by the adult host. These different effects of hosts on the flight times of males and females resulted in sexual dimorphism in flight when the sexes were fed senescent foliage as adults. Although age significantly affected the flight time of both males and females, the reproductive status of females did not affect their flight times. The significance of these results are discussed with respect to the influence of the nutritional complexity of habitats on life history strategies and population dynamics.