Michael Barrett

faculty_img Academic Title: Emeritus

Campus: St. George

CSB Appointment: Full

Primary Undergraduate Department:
Cell & Systems Biology

Graduate Programs:
Cell & Systems Biology

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Mailing Address
Department of Cell & Systems Biology
University of Toronto
25 Harbord St.
Toronto, ON M5S 3G5
Canada

 

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Office phone: 416-978-3488
Office: RW 403A
Lab: RW 403
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Email: fm.barrett@utoronto.ca
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Serotonin at 10−6M concentration induces an average 12-fold increase in cyclic AMP levels in dorsal abdominal epidermis of fifth-instar larval Rhodnius prolixus. The epidermal serotonin receptor is inhibited by a number of mammalian 5-HT2 receptor antagonists but only α-methyl 5-HT among the 5-HT2 receptor agonists tested caused any increase in cyclic AMP levels. Isolated anterior midgut from adult male Rhodnius also shows dose-dependent cyclic AMP elevation in response to serotonin (threshold approx. 10−7M, maximum 5 × 10−6M) with receptor characteristics similar to the hypothesized 5-HT2-like serotonin receptor in larval epidermis. HPLC analysis showed a rapid rise in serotonin concentration in the haemolymph of adult male Rhodnius during feeding (peak 91 nM at 3 min after the onset of gorging) similar to, although less pronounced than, that previously reported for fifth-instar larvae. These findings are consistent with the hypothesis that serotonin released into the haemolymph and onto target tissues during feeding plays an important natural role in the integration of epidermal cell responses, fluid transport across the anterior midgut, diuresis, and other feeding-related functions in Rhodnius.
The concentration of serotonin in the haemolymph of fifth-instar larvae of Rhodnius prolixus increase quickly after the onset of feeding. In insects fed on either blood or artificial diet the haemolymph serotonin concentration increases from approx. 7 nM in unfed insects to over 100 nM by 5 min after the onset of feeding whereafter it decreases again to low levels at 20 min. If insects are interrupted after 1 or 2 min of feeding the haemolymph serotonin concentration declines rather than following the predictable pattern of increase achieved when feeding is allowed to continue. One source of serotonin appears to be neurohaemal areas lying upon the abdominal nerves, since the intensity of serotonin-like immunoreactivity is greatly reduced in these areas following 15 min of feeding.We conclude that serotonin is a neurohormone in Rhodnius and that feeding is the natural stimulus responsible for its release and subsequent elevated concentration in the haemolymph.

Immunohistochemistry and high-performance liquid chromatography coupled with electrochemical detection has been used to demonstrate the presence of serotonin within the nerve supply to the epidermis of 5th-instar and adult Rhodnius prolixus. Serotonin is released from dorsal integument in vivo in response to feeding, and in vitro in response to high-potassium saline. Thus, the intensity of serotonin-like immunoreactive staining of the processes on the dorsal integument is much greater in unfed insects than in those examined 15 min after the onset of feeding. In addition the release of serotonin from dorsal integument into the perfusion medium is elevated in high-potassium saline in the presence of calcium. Serotonin-like immunoreactive processes are also found associated with salivary glands and digestive tract, as well as with skeletal muscle. The results support the notion that serotonin plays a key role in the integration of feeding activity and is probably the plasticising factor in Rhodnius.
The distribution of proctolin throughout the central nervous system and visceral structures of Rhodnius prolixus has been analysed using reversed-phase high-performance liquid chromatography (RP-HPLC) together with a sensitive bioassay. Immunohistochemistry has also been used to identify somata and terminals reactive with a proctolin antiserum.The central nervous system of adult females contained the equivalent of about 378 fmol proctolin distributed unequally throughout all of the ganglia. The highest amount was found in the mesothoracic ganglionic mass, the lowest in the brain. Proctolin was found to be associated with the reproductive and digestive systems, with substantial amounts present in the oviduct and high concentrations per mg tissue proteins in ovaries, oviduct and hindgut.The number and diameter of proctolin-immunoreactive neurones present in each ganglion correlates well with the content of proctolin quantified following RP-HPLC. Over 200 proctolin-staining neurones were distributed over dorsal and ventral aspects of the various ganglia. In addition terminals showing proctolin-like immunoreactivity were observed on the oviducts and hindgut.
The ultrastructural localization of two types of biochemically characterized phenol oxidase activity is described in the larva of the sheep blowfly, Lucilia cuprina. Cuticular tyrosinase activity (enzyme A) is seen in epicuticular filaments and procuticle. Procuticle activity can be detected only after a presumed process of activation takes place in damaged cuticle. By using either the dopamine reaction or inducing melanization by hot-water treatment, tyrosinase is readily shown in haemopoietic tissue which, in L. cuprina, occurs subdermally as well as being associated with the dorsal vessel. The adaptation of the diaminobenzidine technique, used to stain laccase in electrophoretic gels, to ultrastructural cytochemistry has made it possible to demonstrate enzymic activity probably due to laccase (enzyme B). The laccase activity is present in the inner epicuticle of late wandering third instar larvae (about to pupariate) but is not present in the epicuticle of younger larvae.
A tyrosinase, enzyme A (EC 1.10.3.1, o-diphenol: O2 oxidoreductase), and a laccase, enzyme B (EC 1.10.3.2, p-diphenol: O2 oxidoreductase), have been partially purified and characterized from larval cuticle of the sheep blowfly, Lucilia cuprina. Enzyme A is active toward a range of o-diphenols but not p-diphenols, is strongly inhibited by thiourea and phenylthiourea, has a pH optimum between 6.5 and 7.0, and yields a single, 60,000 molecular weight subunit following SDS gel electrophoresis. Enzyme B is active toward both o-diphenols and p-diphenols, is only slightly inhibited by phenylthiourea, has a pH optimum near 4.5, is highly thermostable, and has an apparent molecular weight of 90,000. Enzyme A appears to be activated from an inactive proenzyme in the cuticle and to be present throughout the wandering phase of the final larval instar, declining at pupariation. Enzyme B is present in active form, increases greatly in the cuticle just at the time of pupariation, and then decreases as sclerotization occurs. Antibodies against enzyme A have been raised in sheep and rabbits, and against enzyme B in rabbits, but diets containing antiphenoloxidase antibodies did not affect development or mortality of fly larvae.

 

A phenoloxidase has been extracted, purified, and characterized from cuticle of last-instar larvae of the red-humped oakworm, Symmerista cannicosta. It is a typical tyrosinase (EC 1.10.3.1., o-diphenol:O2 oxidoreductase), active toward o-diphenols but not p-diphenols, inhibited by thiourea and phenylthiourea, with a pH optimum between 6.0 and 7.2. In these respects it resembles enzyme A of C. vicina, one of the few species from which this presumed wound healing enzyme has been purified and characterized. Hydrolysis of either exuviae or intact cuticle from last instar larvae yielded a number of ketocatechols of which the most abundant, 2-hydroxy, 3′,4′-dihydroxyacetophenone, represented 2.9% of the dry weight of head capsule exuviae, 0.3% of exuviae from the remainder of the body, and 4.6% of the dry weight of head capsule cuticle from previously frozen intact larvae. Differences in the type and amount of ketocatechol recovered from these cuticles are described.
The extent of diuresis following ingestion of different artificial diets has been used to investigate, in vivo, the mechanism of fluid absorption from the anterior midgut in Rhodnius prolixus. Urine output in the first 3 hr after feeding declined as sodium concentration in the diet was reduced (Na+ + K+ maintained at 150 mM). The diminished diuresis following low-sodium diets has been explained not as a failure of Malpighian tubule function, since Rhodnius tubules will function in a variety of ionic media, but as a failure of fluid absorption from the crop. The process requires dietary sodium and a Na+-dependent pump in the crop is postulated as the driving force for this movement of water into the haemolymph immediately following a meal.
Three different phenoloxidases have been purified from the cuticle of mature larvae of the blowfly, Calliphora vicina. Enzyme A is a typical tyrosinase (EC 1.10.3.1., : O2 oxidoreductase). It oxidizes both monophenols and , is inactive towards , and is readily inhibited by thiourea and phenylthiourea. Enzymes B and C are laccases (EC 1.10.3.2., : O2 oxidoreductase), that will oxidize both o- and but are inactive towards monophenols. The molecular weight of enzyme B was estimated to be 90,000. The pH-optima for enzymes A and C are close to neutrality, whereas enzyme B has its pH-optimum at 4.5.It is suggested that enzyme A is involved in wound healing, and that enzyme B plays a role in sclerotization of the puparium.
Changes in coital experience, birth control usage, and sex education between 1968, 1971, 1974, and 1978 were studied in unmarried Canadian university students. The percentage of coitally experienced females increased from 32% in 1968 to 58% in 1978 and of males from 40% to 62%. There was no significant change between 1974 and 1978 for either sex, nor were there sex differences in coital experience in either year. Coitally experienced females were more likely to have had intercourse in the 3 months preceding both 1974 and 1978 surveys, but otherwise there were no sex differences in coital frequency, lifetime number of coital partners, or age at first intercourse. Religion was a more reliable indicator of coital experience in 1968 than in 1978. Males were more likely than females to report postpubertal sexual contacts which they classified as homosexual (16.5% vs. 7%) whereas 12-13% of both sexes reported no sexual contacts of any kind with another person. Contraceptive risk-taking was high at first intercourse and among those having infrequent coitus. However, both this group and more coitally active students were less likely to take such risks in 1978 than in 1968, almost entirely because of increased pill use. Coitally experienced vs. inexperienced students differed in their ranking of the major influences on their knowledge and attitudes about sex. PIP Changes in coital experience, birth control usage, and sex education between 1968, 1971, 1974, and 1978 were studied in unmarried Canadian University students. The % of sexually experienced females increased from 32% in 1968 to 58% in 1978 and of males from 40% to 62%. There was no significant change between 1974 and 1978 for either sex, nor were there sex differences in sexual experience in either year. Sexually experienced females were more likely to have had intercourse in the 3 months preceding both 1974 and 1978 surveys, but otherwise there were no sex differences in coital frequency, lifetime number of sexual partners, or age at 1st intercourse. Religion was a more reliable indicator of coital experience in 1968 that in 1978. Males were more likely than females to report postpubertal sexual contacts which they classified as homosexual (16.5% vs. 7%) whereas 12-13% of both sexes reported no sexual contacts of any kind with another person. Contraceptive risk taking was high at 1st intercourse and among those having infrequent coitus. However, both this group and more coitally active students were less likely to take such risks in 1978 than in 1968, almost entirely because of increased pill use. Coitally experienced vs. inexperienced students differed in their ranking of the major influences on their knowledge and attitudes about sex.

 

A number of ketocatechols have been isolated and identified from acid hydrolysates of larval and pupal exuviae of the spruce budworm, Choristoneura fumiferana. The most abundant of these, 2-hydroxy-3′,4′-dihydroxyacetophenone, represented 5.8% of the dry weight of pupal exuviae, 4.6% of larval head capsules, and 0.3% of the remaining larval exuvium. This compound is thought to be the primary breakdown product arising from crosslinks between cuticular proteins and the aliphatic sidechain of a phenolic tanning compound. The yields therefore reflect the extent of β-sclerotization, one of two mechanisms proposed for the hardening of insect cuticle. Budworm is similar to other species with respect to the types of hydrolysis products recovered although three previously undescribed compounds were detected.
Acid hydrolysis of exuviae from the cicada, Tibicen pruinosa, gave rise to to a number of previously identified ketocatechols, the most abundant of which was 2-hydroxy-3′,4′-dihydroxyacetophenone. When different parts of the exuvium were tested, the clear, colourless compound eye exocuticle proved to be the richest source of neutral ketocatechol, yielding 11.79% by weight. In contrast, the brown foreleg femur which appeared hard and modified for digging, gave only 5.62%. It is hypothesized that these differences reflect the existence of two different mechanisms of sclerotization in cicada. β-sclerotization is presumed to be the predominant or even exclusive means of hardening compound eye exocuticle, whereas both this mechanism and quinone tanning are thought to contribute to sclerotization of foreleg femur. The unexpectedly high degree of cross-linking of eye cuticle is considered a possible adaptation to the insect’s underground larval existence.
1.1. Twenty-nine free amino acids and amino acid derivatives were detected in the haemolymph of Calpodes ethlius sampled at different times during the fifth instar.2.2. The changes in total free amino acid concentration throughout the fifth instar showed no obvious trends and no major differences between males and females were observed.3.3. Glycine undergoes a large and sustained concentration increase approx 48 hr after the onset of wax secretion, a change that is paralleled by increased concentration of a ninhydrin-positive compound tentatively identified as α-amino adipic acid.4.4. Histidine, serine, lysine and glutamine are the other predominant amino acids in Calpodes haemolymph, although tyrosine is present in high concentrations in the first few hr after ecdysis into the fifth instar.5.5. The possibility that changes in the concentration of specific free amino acids can be related to specific developmental events is discussed.
1.1. The total concentration of free amino acids in the haemolymph of adult male Rhodnius prolixus sampled at different intervals over the first 40 days following a blood meal ranged from 397·9 mg/100 ml to 683·3 mg/100 ml.2.2. Totals for females ranged from 424·6 mg/100 ml to 777·8 mg/100 ml with proline being the predominant amino acid in both sexes contributing between 38·4 and 59·2% by weight of the total free amino acid detected.3.3. Elevated post-feeding concentrations of valine, tyrosine and serine were observed in both males and females until about 20 days after the blood meal when the levels of these amino acids dropped below the concentrations found in unfed insects.4.4. Methionine concentrations were generally higher in females than males both before and after feeding and substantially elevated concentrations observed in females at 5 and 7 days after feeding were not detected in males.
The anatomy, histology, release site, and function of Brindley’s glands were studied in adults of the bloodsucking bug, Rhodnius prolixus (Stål). The 2 glands, present in both males and females, are located dorsally extending into the lateral part of the 2nd abdominal segment. Brindley’s gland secretion is released by fed or unfed males and females in response to various types of handling or mechanical stimulation and can apparently be synthesized over an extended period of time. The presumed major volatile constituent of the scent is isobutyric acid. Introduction of pure isobutyric acid vapor into an olfactometer containing adult insects caused rapid arousal and disruption of an equilibrium aggregation whereas no such effect was observed on a comparable equilibrated aggregation of 5th-stage insects. Furthermore, 0.5 μl of pure isobutyric acid applied to the thorax of 5th-stage R. prolixus caused rapid paralysis whereas twice this amount applied to either head, thorax, or abdomen of adults had no effect. The implications of these findings in relation to a possible defensive function for the Brindley gland secretion are discussed.
Fifth instar Rhodnius prolixus given a single topical application of synthetic juvenile hormone (JH) on one of the first 12 days after feeding showed a sigmoid relationship between day of treatment and duration of the moulting cycle. A similar relationship was observed between day of treatment and mean ‘juvenile score’ in subsequently emerging insects. The linear relationship between mean ‘juvenile score’ and duration of the moulting cycle suggests that the increased time from feeding to ecdysis for late-treated insects is a result of the morphogenetic effects of the JH treatment. Some of the partially juvenilized insects obtained by treatment after day 5 produced an odour characteristically detected only from adult insects. The possible significance of this observation is discussed.
1.1. The total concentration of seventeen free amino acids in the haemolymph of fifth instar Rhodnius prolixus ranged from 372·4 mg/100 ml 5 days after a blood meal to 624·6 mg/100 ml 18 days after a blood meal.2.2. Proline was the predominant amino acid in the haemolymph at all stages of the fifth instar contributing between 31·8 and 57·3 per cent of the total mM concentration.3.3. Tyrosine concentration increased more than tenfold during the moulting cycle, presumably in preparation for cuticular sclerotization although changes in other amino acids were less pronounced and did not suggest any obvious connection with specifically identifiable developmental events.4.4. The concentrations of proline, leucine, phenylalanine and histidine differ considerably from the only other published quantitative values for R. prolixus haemolymph.5.5. Analyses done on the parent colony from which the present study population was taken showed no such differences.
Glycine-1-14C, glycine-2-14C, and sodium formate-14C were used to investigate the origin of the carbon atoms of uric acid in Rhodnius prolixus. As in other uricotelic organisms, the carboxyl carbon of glycine is incorporated into position 4 of uric acid, the alpha carbon of glycine goes to position 5, and the carbon of sodium formate is incorporated into positions 2 and 8.The carboxyl carbon of glycine is converted to CO2 approximately twenty-four times more rapidly than is the alpha carbon, and much of this CO2 is incorporated into carbon 6 of uric acid. The alpha carbon of glycine is used in the synthesis of formate.The use of isotopically labelled precursors to determine the origin of all five carbon atoms of uric acid has not previously been reported for any species of insect.
The concentration of uric acid in the haemolymph of fifth-instar larvae of Rhodnius prolixus ranged from 9·5 to 17·0 mg/100 ml. In insects unfed for from 32 to 66 days it varied from 9·9 to 15·0 mg/100 ml; insects unfed for the longest periods of time had the highest concentrations. Feeding induced concentration increases of as much as 5·3 mg/100 ml in 24 hr, suggesting a digestive and metabolic activation. The total amount of uric acid in the haemolymph reached a maximum of from 4·1 to 4·5 μg, in the period between the fourth and seventh day after feeding. A 2·9 mg/100 ml increase in uric acid concentration on the third day after moulting was correlated with the degeneration of the moulting