Mark F. Feldlaufer

Comparison of juvenile hormone and ecdysteroid haemolymph titres in adult worker and queen honey bees (Apis mellifera)

Abstract Measurements of both juvenile hormone and ecdysteroid haemolymph titres were made from the same individuals to explore the possibility that there is an interaction between these hormones in the regulation of adult honey bee behaviour and physiology. Queens, egg-laying workers, and workers engaged in brood care (nurses) had low titres of juvenile hormone whereas foragers had significantly higher titres, as in previous studies. In contrast, ecdysteroid titres were undetectably low in both nurses and foragers, higher in laying workers, and higher still in laying queens. Measurements of juvenile hormone titres are consistent with previous findings demonstrating that this hormone regulates worker age polyethism but does not play a typical role in reproduction. Comparison of juvenile hormone and edcysteroid titres suggests that ecdysteroids are not involved in the regulation of age polyethism but may play a role in the regulation of reproduction in honey bees.

Makisterone A: The major ecdysteroid from the pupa of the honey bee, Apis mellifera

Abstract Makisterone A , a 28-carbon moulting hormone, has been identified as the major free pupal ecdysteroid in the honey bee, Apis mellifera. The pupal ecdysteroid was isolated and identified by normal and reversed-phase high performance liquid chromatography in conjunction with a radioimmune assay. The compound was further characterized physico-chemically by both mass spectrometry and nuclear magnetic resonance spectroscopy. No C27 ecdysteroids (i.e. 20-hydroxyecdysone or ecdysone) were detected at this stage of development. This is the first isolation and identification of a 28-carbon ecdysteroid in an insect species from the order Hymenoptera. Utilization of dietary sterols by honey bees is also discussed.

Responsiveness of honey bee (Apis mellifera L.) corpora allata to allatoregulatory peptides from four insect species

Five neuropeptides with known allatotropic or allatostatic activity in other insect species were examined for their effects on honey bee corpora allata. Using an in vitro radiochemical assay, we assessed the ability of these peptides to affect the biosynthesis of juvenile hormone III and its immediate precursor methyl farnesoate, as well as their effects on the conversion of methyl farnesoate into juvenile hormone. None of the allatostatins tested affected JH biosynthesis during the last larval instar of honey bee workers. Manduca sexta allatotropin, however, stimulated JH biosynthesis in a stage-specific and dose-dependent manner. Analysis of intraglandular contents of juvenile hormone and its precursor revealed that the allatotropin significantly increased JH precursor but did not overcome the stage-specific block in the terminal step of JH biosynthesis that is typical for early fifth-instar worker larvae. Studies also indicated that the allatotropic effect was reversible at the level of methyl farnesoate production.

Neutral sterol metabolism in insects

Since they are unable to biosynthesize sterols, many phytophagous and omnivorous insects satisfy their cholesterol requirement by side chain dealkylation of the C-24 alkyl group of dietary C28 and C29 phytosterols. However, not all insects that can dealkylate the phytosterol side chain produce cholesterol. In addition, certain insects,e.g., some Hymenoptera, Hemiptera, and Diptera, are unable to dealkylate the sterol side chain. Although C27 ecdysteroids (molting hormones), which are biosynthesized from cholesterol, are the major ecdysteroids in most insects, many of those species that are unable to dealkylate phytosterols utilize campesterol as a precursor for the C28 ecdysteroid makisterone A. The considerable diversity of steroid utilization between certain insect species makes it difficult to generalize about insect steroid biochemistry. The ability to disrupt certain unique aspects of steroid utilization and metabolism in insects might be exploited for developing new insect control technology.

Performance of honey bee (Apis mellifera) queens reared in beeswax cells impregnated with coumaphos

SUMMARY The sublethal effects of coumaphos on developing queens were investigated. Young honey bee larvae were transferred into beeswax cups containing known concentrations (0 to 1000 mg/kg) of coumaphos. The cups with larvae were placed in queenless colonies for rearing and 10 days later acceptance was determined by recording mature queen cells. The queen cells were placed in small mating colonies for 21 days, when the queens were collected after being rated for commercial acceptability. The queens were then either introduced to production colonies and monitored for six months or dissected to determine mating success. All but one queen failed to develop at 1000 mg/kg of coumaphos, and greater than 50% of cells were rejected at 100 mg/kg coumaphos. The queens that were reared were lighter in weight as pre-emergence pupae. The effects on other characters related to queen performance are discussed. The presence of coumaphos in queen rearing cells can reduce the number of queens still functioning in colonies at six months by as much as 75%.

Relationship of the neutral sterols and ecdysteroids of the parasitic mite, Varroa jacobsoni to those of the honey bee, Apis mellifera.

The neutral sterols of the parasitic mite Varroa jacobsoni were compared with Apis mellifera carnica drone pupae. Analysis by GLC-mass spectrometry indicated mite sterols were reflective of the sterol composition of the drones; 24-methylenecholesterol was the major sterol in both species, with lesser amounts of sitosterol and isofucosterol. Cholesterol accounted for less than 1% of the total sterols. Ecdysteroid analyses indicated drones contained primarily makisterone A. In addition to makisterone A, mites contained ecdysone and 20-hydroxyecdysone, which accounted for over 66% of the ecdysteroid detected. These results indicate that while V. jacobsoni are apparently unable to convert dietary sterols to cholesterol, they are able to produce significant amount of C(27) ecdysteroids in a low cholesterol environment.

Terminal steps in JH biosynthesis in the honey bee (Apis mellifera L. ): developmental changes in sensitivity to JH precursor and allatotropin.

Juvenile hormone (JH) is considered the prime endogenous signal for the induction of queen development in honey bees (Apis mellifera L.). At the beginning of the last (5th) larval stadium, worker corpora allata synthesize less JH than queen corpora allata as a consequence of a limited production of JH precursors and a caste- and stage-specific block of the terminal step in JH biosynthesis. As previously shown, the Manduca sexta allatotropin stimulates JH biosynthesis in honey bee corpora allata in a dose-dependent and reversible manner, but can not overcome the stage-specific block in the terminal step of JH biosynthesis that is typical for worker early 5th instars. In experiments with M. sexta allatotropin and with the JH precursor farnesoic acid, we found characteristic stage-specific differences in their effects on JH biosynthesis. From the end of the spinning stage on, corpora allata could be stimulated by farnesoic acid to a much higher extent than in earlier developmental stages, suggesting a sudden increase in epoxidase activity. Manduca sexta allatotropin, however, stimulated corpora allata activity until the end of the spinning stage, at which time the corpora allata become suddenly insensitive. These data suggest that in worker larvae, important changes in the regulation of the terminal enzymatic steps in JH biosynthesis occur at the transition from the spinning stage to the prepupal stage. However, the analysis of in vitro activities of the involved enzymes, O-methyltransferase and methyl farnesoate epoxidase, remained inconclusive.

4-Oxo-Aldehydes From the Dorsal Abdominal Glands of the Bed Bug (Hemiptera: Cimicidae)

ABSTRACT Analyses of the dorsal abdominal glands of fourth- and fifth-instar nymphs of the bed bud, Cimex lectularius L. (Hemiptera: Cimicidae), indicated the predominant constituents were (E)-2-hexenal and (E)-2-octenal, with lesser amounts of 4-oxo-(E)-2-hexenal and 4-oxo-(E)-2-octenal. The latter two compounds have not been reported previously as occurring in bed bugs. There were no differences in the chemical composition of the dorsal abdominal glands excised from exuviae left behind by either male or female adults, nor from glands excised from fourth-instar exuviae. Because the two oxo-aldehydes made up at least 16% of the gland contents, further study of the functional role of these chemicals seem advisable.

Ecdysteroid production in Drosophila melanogaster reared on defined diets.

Larvae of Drosophila melanogaster were reared aseptically on defined diets containing either cholesterol, campesterol or sitosterol as the only dietary sterol. Sterol analyses of pupae revealed that insects reared on campesterol and sitosterol diets contained 3.3 and 8.1% cholesterol, indicative of an ability to accumulate this sterol. Ecdysone and 20-hydroxyecdysone were the predominant ecdysteroids in insects from all diet studies, though makisterone A was detected in pupae reared on campesterol and sitosterol.

The influence of RNA integrity on the detection of honey bee viruses: molecular assessment of different sample storage methods

Summary RNA quality has been considered to be one of the most critical components for the overall success of RNA-based assays. To ensure accuracy of virus diagnosis by the RT-PCR method, it is important to identify an optimal sample storage method that stabilizes RNA and protects RNA from the activities of RNase in intact samples. We conducted studies to evaluate the effects of seven different storage conditions on the integrity of RNA and the influence of RNA integrity on the detection of virus infections in honey bees. RNA was isolated from samples processed under one of six storage conditions: 1) bees stored at 4°C; 2) bees stored at–20°C; 3) bees stored at–80°C; 4) sliced bees immersed in RNAlater at 4°C; 5) crushed bee immersed in RNAlater at 4°C; 6) intact bees immersed in RNAlater at 4°C, or 7) bees immersed in 70% ethanol at room temperature. The results indicated that bee samples stored at–80°C,–20°C, cut in slices and then immersed in RNAlater at 4°C, and crushed into a paste and then immersed in RNAlater at 4°C provided successful RNA stabilization, suggesting any one of these four storage methods is the method of choice for storing bee samples intended for virus analysis. RNA extracted from bee samples stored at 4°C or whole bees immersed in RNAlater at 4°C was partially degraded one week post treatment, suggesting that a temperature of 4°C could not prevent RNA from activities of RNase completely and that the size of tissue is critical for successful stabilization of samples immersed in RNAlater. 70% ethanol caused quick and strong degradation of RNA and therefore bee samples that are stored in 70% ethanol are not the recommended starting material for virus analysis. The information obtained from this study is relevant to other researchers and to apiary inspectors involved in epidemiological surveillance of bee viral infections.