Diagnosis of American foulbrood in honey bees: a synthesis and proposed analytical protocols.

Worldwide, American foulbrood (AFB) is the most devastating bacterial disease of the honey bee (Apis mellifera). Because the distinction between AFB and powdery scale disease is no longer considered valid, the pathogenic agent has recently been reclassified as one species Paenibacillus larvae, eliminating the subspecies designations Paenibacillus larvae subsp. larvae and Paenibacillus larvae subsp. pulvifaciens. The creamy or dark brown, glue-like larval remains of infected larvae continue to provide the most obvious clinical symptom of AFB, although it is not conclusive. Several sensitive and selective culture media are available for isolation of this spore-forming bacterium, with the type of samples that may be utilized for detection of the organism being further expanded. PCR methods for identification and genotyping of the pathogen have now been extensively developed. Nevertheless, biochemical profiling, bacteriophage sensitivity, immunotechniques and microscopy of suspect bacterial strains are entirely adequate for routine identification purposes.

Prevalence and transmission of honeybee viruses.

Transmission mechanisms of six honeybee viruses, including acute bee paralysis virus (ABPV), black queen cell virus (BQCV), chronic bee paralysis virus (CBPV), deformed wing virus (DWV), Kashmir bee virus (KBV), and sacbrood bee virus (SBV), in honey bee colonies were investigated by reverse transcription-PCR (RT-PCR) methods. The virus status of individual queens was evaluated by examining the presence of viruses in the queens' feces and tissues, including hemolymph, gut, ovaries, spermatheca, head, and eviscerated body. Except for head tissue, all five tissues as well as queen feces were found to be positive for virus infections. When queens in bee colonies were identified as positive for BQCV, DWV, CBPV, KBV, and SBV, the same viruses were detected in their offspring, including eggs, larvae, and adult workers. On the other hand, when queens were found positive for only two viruses, BQCV and DWV, only these two viruses were detected in their offspring. The presence of viruses in the tissue of ovaries and the detection of the same viruses in queens' eggs and young larvae suggest vertical transmission of viruses from queens to offspring. To our knowledge, this is the first evidence of vertical transmission of viruses in honeybee colonies.

Quantitative real-time reverse transcription-PCR analysis of deformed wing virus infection in the honeybee (Apis mellifera L.).

Deformed wing virus (DWV) can cause wing deformity and premature death in adult honeybees, although like many other bee viruses, DWV generally persists as a latent infection with no apparent symptoms. Using reverse transcription (RT)-PCR and Southern hybridization, we detected DWV in all life stages of honeybees, including adults with and without deformed wings. We also found DWV in the parasitic mite Varroa destructor, suggesting that this mite may be involved in the transmission of DWV. However, the detection of the virus in life stages not normally associated with mite parasitism (i.e., eggs and larvae) suggests that there are other modes of transmission. The levels of DWV in different life stages of bees were investigated by using TaqMan real-time quantitative RT-PCR. The amounts of virus varied significantly in these different stages, and the highest levels occurred in pupae and in adult worker bees with deformed wings. The variability in virus titer may reflect the different abilities of bees to resist DWV infection and replication. The epidemiology of DWV is discussed, and factors such as mite infestation, malnutrition, and climate are also considered.

Larval Apis mellifera L. (Hymenoptera: Apidae) mortality after topical application of antibiotics and dusts.

Beekeepers apply various dusts to honey bee, Apis mellifera L., colonies to dislodge parasitic mites and control bacterial brood diseases. Anecdotal reports by beekeepers indicate that the antibiotic oxytetracycline (OTC) can be toxic when applied in powdered sugar to cells containing immature bee brood, but it was not known whether the purported toxicity is caused by the antibiotic or the sugar carrier. Additionally, the toxicity of various dusts, proposed for parasitic mite control, is poorly known. In the current studies, we tested OTC and two other antibiotics (tylosin and lincomycin, candidate compounds for use in honey bee colonies) in a powdered sugar carrier for larval toxicity. We also tested for larval toxicity, several dusts that have been proposed for mite control. OTC caused significant brood mortality of approximately 80% at the concentrations used in the hive (200 mg in 20 g sugar). In contrast, tylosin and lincomycin at the 200 mg dose were both similar to untreated controls, and only five times that concentration (1000 mg) caused significant brood mortality of approximately 65%. The addition of dusts, wheat flour, talc, and a commercially available protein supplement, BeePro, resulted in mortality levels between 65 and 80%, similar to that seen with OTC. The common antibiotic carrier, powered confectioners sugar, was nontoxic. The use of 100 unsealed brood cells was demonstrated to be a reliable means of assessing potential adverse affects of dry compounds on larval honey bees. Two new candidate antibiotics for use in honey bee colonies were less toxic to larval bees than the currently labeled antibiotic, OTC.

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.

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.

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.

Neutral sterols of sawflies (Symphyta): their relationship to other Hymenoptera.

To investigate sterol utilization in sawflies, the neutral sterols of four species of sawflies were determined by gas-liquid chromatography and mass spectrometry, and compared to the respective dietary plant material. Cholesterol was the predominant (55-76%) sterol in all species and stages of sawflies examined. Host plants, however, contained primarily sitosterol (50-88%), along with other 24-alkylsterols and only 0.5-5.9% cholesterol, indicating that the sawflies examined are capable of dealkylating the C28 and C29 phytosterols in their diet to cholesterol. Comparative sterol metabolism in Hymenoptera is discussed.

Degradation of pheromone biosynthesis-activating neuropeptide (PBAN) by hemolymph enzymes of the tobacco hornworm, Manduca sexta, and the corn earworm, Helicoverpa zea.

The tritium-labeled bis-norleucine analog of Helicoverpa zea pheromone biosynthesis-activating neuropeptide ([3H]NLPBAN) was incubated in vitro with hemolymph from Manduca sexta or H. zea adult females. The incubations resulted in the formation of several tritium-labeled degradation products. At a [3H]NLPBAN concentration of 0.9 microM the degradation proceeded at a very slow but physiologically plausible rate (2-10 fmol/min/microliters hemolymph). The primary [3H]NLPBAN degradation reaction in M. sexta hemolymph was not inhibited by 20 microM leupeptin, 0.1 mM amastatin, 1 mM EDTA, 1 mM EGTA, 1 mM 1,10-phenanthroline, or 2 mM 4-(2-aminoethyl)benzenesulfonyl fluoride; but secondary reactions may have been affected, as some of the inhibitors changed the radio-HPLC profile of the degradation products. It is concluded that hemolymph of M. sexta and H. zea contains peptidase(s) capable of inactivating circulating PBAN.

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.

Ecdysteroids in Axenically Propagated Caenorhabditis elegans and Culture Medium.

Ecdysteroids (insect molting hormones) from Caenorhabditis elegans were chromatographically purified and quantified by radioimmunoassay. Nematodes from semidefined medium contained the immunoreactive equivalent of 460 pg ecdysone per gram dry weight. Culture medium, however, contained the immunoreactive equivalent of 68 times the quantity within the nematodes. In a defined medium lacking immunoreactivity, C. elegans contained 520 pg ecdysone equivalents per gram dry weight but reproduced slowly. Reproduction of C. elegans in defined medium was enhanced by formulation in agar. Propagation of C. elegans in either agar-based or aqueous defined medium supplemented with [(1)C]cholesterol of high specific activity failed to result in production of radiolabeled free ecdysteroids or polar or apolar ecdysteroid conjugates. Failure to demonstrate ecdysteroid biosynthesis in C. elegans raises questions about the ecdysteroids identified previously in nematodes being products of endogenous biosynthesis, a necessary condition for these compounds to be nematode hormones.

Sterol Composition and Ecdysteroid Content of Eggs of the Root-knot Nematodes Meloidogyne incognita and M. arenaria.

Free and esterified sterols of eggs of the root-knot nematodes Meloidogyne incognita races 2 and 3 and M. arenaria race 1 were isolated and identified by gas-liquid chromatography-mass spectrometry. The major sterols of eggs of each race were 24-ethylcholesterol (33.4-38.8% of total sterol), 24-ethylcholestanol (18.3-25.3%), 24-methylcholesterol (8.6-11.7%), 24-methylcholestanol (7.7-12.5%), and cholesterol (4.6-11.6%). Consequently, the major metabolic transformation performed by Meloidogyne females or eggs upon host sterols appeared to be saturation of the sterol nucleus. The free and esterified sterols of the same race did not differ appreciably, except for a slight enrichment of the steryl esters in cholesterol. Although the sterol composition of Meloidogyne eggs differed from that of other life stages of other genera of plant-parasitic nematodes, the three Meloidogyne races could not be distinguished from each other by their egg sterols. Ecdysteroids, compounds with hormonal function in insects, were not detected by radioimmunoassay in the Meloidogyne eggs either as free ecdysteroids or as polar conjugates.

The fate of radiolabeled steroids in ovaries and eggs of the tobacco hornworm,Manduca sexta.

To determine the precursors and the fate of 26-hydroxyecdysone in eggs, the fate of labeled putative ecdysteroid precursors was examined in the tobacco hornworm,Manduca sexta. Following injection of [(14)C]cholesterol, 22,25[(14)C]dideoxyecdysome or [(3)H]ecdysone into female pupae (day 16), only [(14)C]cholesterol was incorporated and metabolized. It was converted to labeled nonecdysteroid and ecdysteroid conjugates, of which the latter in ovaries and 48- to 64-hr-old eggs is mainly 26-hydroxyecdysone 26-phosphate (>85% in ovaries). Quantitation of the ecdysteroid conjugate by reversed-phase high performance liquid chromatography (RP-HPLC) showed that the levels of 26-hydroxyecdysone 26-phosphate were 31 µg/g of ovaries from 4-day-old females and 25 µg/g and 17 µg/g of 48- to 64-hr-old and 72- to 88-hr-old eggs, respectively. The RP-HPLC of the conjugate fraction of 48- to 64-hr-old eggs showed an additional peak of radioactive material eluting about three min before the 26-hydroxyecdysone 26-phosphate. The quantity of this material increased in the 72-to 88-hr-old eggs, though it was not detected in the analyses of the conjugate fraction from ovaries. Additional peaks of radioactive material eluting before the 26-hydroxyecdysone 26-phosphate peak were observed in the chromatogram of the conjugates of 72- to 88-hr-old eggs. These radioactive materials need to be identified to determine the ultimate fate of ecdysteroids in the developing embryos of the tobacco hornworm. No radioactive free ecdysteroids were detected in either egg age group.

Selective sterol transfer in the honey bee: Its significance and relationship to other hymenoptera.

The honey bee,Apis mellifera, is one of only a few species of phytophagous insects known to be unable to convert C-24 alkyl phytosterols to cholesterol. Regardless of the dietary sterols available to worker bees, the major tissue sterol of brood reared by the workers is always 24-methylenecholesterol, followed by sitosterol and isofucosterol. Normally, little or no cholesterol is present in honey bee sterols. The maintenance of high levels of certain sterols is accomplished through a selective transfer of sterols from the endogenous sterol pools of the workers to the developing larvae through the brood food material secreted from the hypopharyngeal and mandibular glands and/or the honey stomach of the workers. The selective uptake and transfer of radiolabeled C27, C28 and C29 sterols have been studied to correlate these aspects of sterol utilization with the discovery of an unusual molting hormone (ecdysteroid) in honey bee pupae as the major ecdysteroid of this stage of development. The phylogenetic implications of this selective transfer phenomenon in the honey bee and comparison with sterol metabolism in certain other hymenopteran species emphasize the diversity of steroid biochemistry in insects.