Insect prothoracicotropic hormone: evidence for two molecular forms.

In an insect, the tobacco hornworm Manduca sexta, the cerebral neuropeptide prothoracicotropic hormone (PTTH), the primary effector of postembryonic development, exists as two molecular forms. These two PTTH's elicit characteristic in vitro dose responses of activation of prothoracic glands from different developmental stages, an indication that during development the glands change in their sensitivity to the neurohormones. Both PTTH's are active in a specific in situ bioassay. Since they may be released in situ at stage-specific times to evoke distinctly different developmental responses, the PTTH neuroendocrine axis appears to be an effective system for determining the functions of molecular forms of a neurohormone in the regulation of growth and development.

Regeneration of the neurohemal terminals for identified cerebral neurosecretory cells in an insect.

The axons of specific neurosecretory cells, L-NSC III, in the brain of the tobacco hornworm, Manduca sexta, were transected during larval-pupal development to study the effects of this type of lesion on these peptidergic neurons and to begin to identify factors that may regulate their regeneration and growth. The two somata of these bilaterally paired neurons produce the prothoracicotropic hormone and are located in the pars intercerebralis. Their axons exit from the contralateral brain lobe via a retrocerebral nerve and pass through the corpus cardiacum before terminating at the glandular corpus allatum. At the corpus allatum, the L-NSC III axons arborize to form the terminal neurohemal organ for prothoracicotropic hormone release. The retrocerebral nerve was severed either in vitro followed by brain transplantation or in situ; in either protocol, the distal axon segments and corpus allatum were removed. The ability of the injured L-NSC III axons to regenerate was assessed immunocytologically by using a monoclonal antibody against the prothoracicotropic hormone. In both treatments, the proximal axon stumps exhibited regenerative growth as early as 1 day after axotomy, and, by the third day, neurites had extended. By the fifth day, the regenerating axons had branched to form terminal varicosities similar to those of a normal neurohemal organ. The regenerated neurohemal structure appeared to be functional, because larvae that had been bilaterally axotomized were able to metamorphose to pupae, a process requiring temporally precise periods of prothoracicotropic hormone release. In addition to the regeneration of the terminal axon structures, several other responses to axotomy and retrocerebral organ excision occurred. These included an apparent accumulation of prothoracicotropic hormone in the axons and regenerating neurohemal-like structure, sprouting of ectopic neurites from the axotomized somata, and a change in shape of the cell bodies from spherical to avoid.

Houseflies: not simple mechanical vectors of enterohemorrhagic Escherichia coli O157:H7.

An epidemic of enterohemorrhagic colitis caused by Escherichia coli O157:H7 (EHEC-O157) occurred in a nursery school in a rural area of Japan in September 1996. The EHEC-O157 were isolated both from patients and houseflies collected at the school. The flies were suspected to be mechanical vectors of the pathogen. Feeding experiments of EHEC-O157 to houseflies showed that the ingested bacteria were harbored in the intestine of flies and continued to be excreted at least for 3 days after feeding. Scanning electron microscopy showed that a large number of EHEC-O157 adhered to the surface of the housefly mouthparts and actively proliferated in the minute spaces of the labellum. Food masses containing EHEC-O157 in the fly intestine were completely surrounded by a peritrophic membrane during digestion and discharged rapidly. The persistence of bacteria in the intestine and feces is mainly a result of proliferation in the mouthparts and accumulation in the crop. Our results strongly suggest that houseflies are not simple mechanical vectors of EHEC. The epidemiologic potential of houseflies to disseminate EHEC-O157 may be greater than initially suspected.

Epidemiological potential of excretion and regurgitation by Musca domestica (Diptera: Muscidae) in the dissemination of Escherichia coli O157: H7 to food.

We previously reported that enterohemorrhagic Escherichia coli O157: H7 (EHEC) proliferates in the mouthparts of the house fly Musca domestica vicina Macquart and are excreted for at least 3 d after ingestion. However, the role of the crop and excretory behavior of the house fly in contamination of human food with EHEC is not known. In the current experiments, EHEC persisted in the crop of house flies for at least 4 d. The number of EHEC in an excreted droplet was approximately 10(4) 1 h after bacterial feeding, > 1.8 x 10(5) 3 h after feeding, and then drastically decreased after 24 h. Excretion is one of the major mechanisms for decreasing number of EHEC in the crop and gut of the house fly. The frequency of excretion by females with developing eggs in their ovary was clearly higher (6.5 min per drop) than for males or females with mature eggs. Minute eosin-sign around a container filled with eosin-supplemented trypticase soy broth might be derived from frequent contact by house fly contaminated mouthparts. These results show that frequent excretion potentially enhances the dissemination of EHEC to foods, particularly during the first 24 h after ingestion of the bacteria.

Electrical properties of the cerebral prothoracicotropic hormone cells in diapausing and non-diapausing pupae of the tobacco hornworm, Manduca sexta.

Prothoracicotropic hormone (PTTH) is an insect brain neuropeptide that is a primary factor regulating an insect development. Curtailment of its release is thought to be responsible for the pupal diapause of tobacco hornworm, Manduca sexta. The cell synthesizing and secreting the PTTH has been identified as a pair of neurosecretory cells in the pars lateralis on each brain hemisphere. Using intracellular recording techniques, we have demonstrated electrical properties of the PTTH cells in different physiological status, i.e., diapausing and developing pupae. In diapausing pupae, they showed threshold value increasing and input resistance decreasing with the progress of diapausing state, indicating that they were getting unexcitable. Spontaneous action potentials and excitatory postsynaptic potentials (EPSPs) were rarely observed in deeply diapausing state. Non-diapausing PTTH cells were almost silent except day-2, showing rather constant values of electrical properties. On day-2, a significant proportion of the cells had spontaneous action potentials, showing less negative membrane potential values than inactive cells. Exclusively inhibitory postsynaptic potentials (IPSPs) were observed in significant numbers of the cells during the period from day-2 to day-5. On the basis of the results obtained, we proposed a working hypothesis that electrical activities of the PTTH cell may be primarily regulated by its membrane properties which are further modulated by the synaptic mediation.

The parasitoid Apanteles kariyai inhibits pupation of its host, Pseudaletia separata, via disruption of prothoracicotropic hormone release.

When the parasitoid Apanteles kariyai laid eggs into host Pseudaletia separata larvae, before prothoracicotropic hormone (PTTH) was released in the last instar preparatory to metamorphosis, the host did not pupate and the larvae of the wasps emerged. The ecdysteroid titer of unparasitized intact larvae increased up to 1 microgram/ml 1 day before pupation, whereas the titer of parasitized larvae was maintained at a low level without the surge. Isolated prothoracic glands from intact larvae synthesized much more ecdysone than those of parasitized larvae both in vivo and in vitro. Administration of exogenous PTTH caused the activation of the prothoracic glands seen during parasitization. Injection of 20-hydroxyecdysone (20-HE) into the parasitized larvae caused by host's pupation, but did not affect the development of the wasp larvae. However, the sensitivity of the integument to 20-HE was lower in parasitized than in unparasitized larvae. Injection of a mixture of adult wasp calyx and venom fluids into last instar unparasitized larvae delayed their pupation, suggesting that calyx and venom fluids are factors contributing to disturbance of the normal function of brain-prothoracic gland system. These results show that parasitization inhibits secretion and/or synthesis of PTTH and also delays the larval-pupal commitment of the integument by keeping the ecdysteroid level low.

Effect of culture medium on the in vitro secretion activity of prothoracic glands from Pseudaletia separata.

The prothoracic glands (PGs) taken from the last instar of the common armyworm, Pseudaletia separata, were cultured in various media for the purpose of finding a suitable medium for relatively long-term culture of PGs. Among the tested culture media, MGM-450 medium without serum was the best to maintain PG cells viable for relatively long periods, and to continue to secrete ecdysteroids. Secretion of ecdysteroid by the PG in vitro became marked when the PG was taken from last instar larvae older than 2 days after the last molt. PGs cultured in any of the media secreted ecdysteroid only within the first 2 h after placing them in culture, however, in the MGM-450 medium, the PGs secreted ecdysteroid even after 5 days of culture.

In vitro activation of insect prothoracic glands by the prothoracicotropic hormone.

An in vitro assay for the prothoracicotropic hormone has been developed that utilizes an ecdysone radioimmunoassay to quantify the increase in the rate of ecdysone synthesis elicited by the neurohormonal activation of the prothoracic glands. The rapidity, reproducibility, and accuracy of the assay were maximized by using one member of a gland pair as the control and the other as the test gland. This was possible because the basal rates of ecdysone synthesis by the members of a gland pair were equivalent. Activation was demonstrated to be dose dependent and specific, with prothoracicotropic hormone activity present only in homogenates of brain. The in vitro activation of the prothoracic glands was verified with the Manduca bioassay for the prothoracicotropic hormone in which the morphological responses to the hormone were correlated with increased in vivo ecdysone titers. These results provide unequivocal evidence that the activation of the prothoracic glands by the prothoracicotropic hormone is direct and suggest that activation represents an increase in a basal rate of ecdysone synthesis.

Cellular localization of the insect prothoracicotropic hormone: In vitro assay of a single neurosecretory cell.

The distribution of prothoracicotropic hormone in the pupal brain of Manduca sexta has been determined by an in vitro assay for prothoracic gland activation. Prothoracicotropic activity was observed in both the brain and retrocerebral complex, but predominantly in the dorosolateral regions of the protocerebrum. Of the two groups of neurosecretory cells present in this area of the brain, only the two lateral type III neurosecretory cells exhibited significant prothoracicotropic hormone activity. Further analysis revealed that the neurohormone was localized in only one of the two type III cells, suggesting that a single neurosecretory cell in each hemisphere is the source of the hormone at the stage examined (day 0). Prothoracicotropic hormone activity was detected in both the corpora allata and the corpora cardiaca, but the corpora allata contained 2 to 9 times the activity of the corpora cardiaca, depending on developmental stage. The significantly higher level of activity in the corpora allata suggests that they may be the neurohemal organs through which the prothoracicotropic hormone of Manduca is released.

Polymorphism and linkage analysis of the prothoracicotropic hormone gene in the silkmoth, Bombyx mori.

We looked for polymorphism of the prothoracicotropic hormone gene locus (Ptth) among inbred strains of the silkmoth, Bombyx mori, by in vitro DNA amplification (polymerase chain reaction), and found three alleles, PtthA, PtthB and PtthC. The PtthA allele contained a third intron consisting of 680 bp and a fourth intron of 350 bp. PtthB contained the same size third intron but a longer fourth intron of 490 bp, while PtthC had a longer third intron of 1080 bp and a shorter fourth intron of 350 bp. In 29 strains which we examined, 9 strains had PtthA, 8 strains had PtthB, and 2 strains had PtthC. The other 10 strains had heterogeneous genotypes with the same 3 alleles. Linkage analyses using these polymorphisms showed that Ptth was linked to the or gene on chromosome 22, and was independent of other chromosome markers. To determine the map position, we obtained an F1 hybrid between the w41 strain (PtthC+or+mw) and the w22 strain (PtthB or mw), and backcrossed the F1 hybrid to females of the w22 strain. From the segregation of or, mw and Ptth in 861 progeny, recombination values were calculated as 18.5% between or and mw, 23.9% between mw and Ptth, and 7.3% between Ptth and or. Because or and mw have already been mapped at positions 22-8.9 cM and 22-25.2 cM, respectively, we mapped Ptth at 22-2.5 cM. The Ptth locus is independent of any loci which are known to control growth and molting.