Online electrochemical system as an in vivo method to study dynamic changes of ascorbate in rat brain during 3-methylindole-induced olfactory dysfunction.

This study demonstrates the application of an online electrochemical system (OECS) as an in vivo method to investigate the dynamic change of microdialysate ascorbate in the olfactory bulb (OB) of rats during the acute period of olfactory dysfunction induced by intraperitoneal (i.p.) injection of 3-methylindole (3-MI). The OECS is developed by directly coupling an electrochemical detector to in vivo microdialysis for the direct monitoring of ascorbate. The system benefits from the good electrochemical activity of single-walled carbon nanotubes towards the oxidation of ascorbate and exhibits high selectivity, good stability, reproducibility and linearity for the measurement of ascorbate in the OB under physiological conditions. With this method, the basal level of microdialysate ascorbate in the OB is determined to be 48.64 ± 5.44 µM. The administration of 3-MI clearly increases the microdialysate ascorbate in the OB after 3-MI treatments and this increase is obviously alleviated by intravenous administration of ascorbate and glutathione (GSH) within 10 min after i.p. injection of 3-MI. These observations with the OECS suggest that ascorbate may be involved in chemical processes during the early stages of 3-MI-induced olfactory dysfunction. This study essentially validates the OECS as an in vivo method for effective measurement of ascorbate in the OB in rat brain and such a method will find interesting applications in investigating chemical process associated with ascorbate underlying olfactory dysfunction.

Inhibitory effect of skatole (3-methylindole) on enterohemorrhagic Escherichia coli O157:H7 ATCC 43894 biofilm formation mediated by elevated endogenous oxidative stress.

UNLABELLED: We investigated the effects of skatole (3-methylindole), which is one of the indole derivatives on the biofilm formation of EHEC O157:H7. Notably, skatole (100 µg ml⁻¹) significantly reduced EHEC O157:H7 ATCC 43894 biofilm formation by 52% in 96-well polystyrene plates under quiescent conditions, with no effect on planktonic cell growth. The skatole sample was maintained in stable conditions for 24 h without degradation or evaporation via EHEC O157:H7 ATCC 43894. Importantly, skatole negatively triggered the expression of catalase in EHEC strains, as well as altered EHEC surface morphology. Our finding indicated that suppressed catalase activity via skatole might have been responsible for elevated endogenous oxidative stress and increment in oxidative metabolites might have led to damaged cell surfaces and a reduction in biofilm formation of EHEC O157:H7 ATCC 43894. SIGNIFICANCE AND IMPACT OF THE STUDY: Our findings suggest that inefficient catalase activity of skatole-exposed enterohemorrhagic Escherichia coli (EHEC) O157:H7 ATCC 43894 may account for elevated endogenous oxidative stress, leading to damaged cell surfaces and reduction in biofilm formation. Our results also provide that skatole as a new candidate for bacterial signalling may be applied for inhibiting bacterial biofilms in food and feed industry.

Odorant receptor from the southern house mosquito narrowly tuned to the oviposition attractant skatole.

Oviposition attractants are environmental cues that allow Culex gravid female mosquitoes to locate suitable sites for egg-laying and, therefore, may be exploited for environmentally friendly strategies for controlling mosquito populations. Naturally occurring skatole has been identified as an oviposition attractant for the Southern House mosquito, Culex quinquefasciatus. Previously, we identified in Cx. quinquefasciatus female antennae an olfactory receptor neuron (ORN) highly sensitive to skatole and an odorant-binding protein involved in the detection of this semiochemical. Here, we describe the characterization of an odorant receptor (OR), CquiOR10, which is narrowly tuned to skatole when expressed in the Xenopus oocyte system. Odorant-induced response profiles generated by heterologously expressed CquiOR10 suggest that this OR is expressed in the mosquito ORN sensitive to skatole. However, geranylacetone, which stimulates the antennal ORN, was not detected by CquiOR10-expressing oocytes, thus raising interesting questions about reception of oviposition attractants in mosquitoes.

Culex mosquitoes (Diptera: Culicidae) egg laying in traps loaded with Bacillus thuringiensis variety israelensis and baited with skatole.

The Southern house mosquito, Culex quinquefasciatus, is an important human health pest as a vector of several pathogens, including agents of lymphatic filariasis and arboviruses like West Nile virus. We conducted preliminary experiments in Recife, Brazil, to explore applications of Culex oviposition attractants in combination with Bacillus thuringiensis variety israelensis (Bti) in an attract-and-kill approach. Simple, cost-effective oviposition traps, BR-OVT, loaded with Bti and baited with or without attractant, were deployed in 10 homes for 30 d in 2 consecutive yr. Significantly higher numbers of egg rafts were deposited in traps baited with skatole or infusion than the control water traps. In the first year, 2006, significantly higher numbers of eggs were deposited in infusion-baited traps, particularly in the first 15 d of the experiment, than in skatole traps, but in the following year no significant difference was observed between synthetic and natural attractants. The tests strongly demonstrate that skatole or infusion can be used to enhance the number of egg rafts deposited on Bti-treated oviposition traps.

An unsettling explanation for the failure of skatole-baited ovitraps to capture Culex mosquitoes.

Culex mosquitoes are primarily found in temperate and tropical regions worldwide where they play a crucial role as main vectors of filarial worms and arboviruses. In Recife, a northeast city in Brazil, high densities of Culex quinquefasciatus are often found in association with human populated areas. In marked contrast to another part of the city, field tests conducted in the neighborhood of Sítio dos Pintos showed that trapping of mosquitoes in skatole-baited ovitraps did not differ significantly from captures in control (water) traps. Thus, classical and molecular taxonomic approaches were used to analyze the Culex species circulating in Sítio dos Pintos. Results obtained from both approaches agreed on the cocirculation of Culex quinquefasciatus and Culex nigripalpus in three different areas of this neighborhood. What was initially considered as an unexpected failure of this lure turned out to be a more unsettling problem, that is, the first report in Recife of Culex nigripalpus, a vector of Venezuelan equine encephalitis virus and West Nile virus. Unplanned urbanization processes close to remnants of the Atlantic forest, such as observed in Sítio dos Pintos, may have contributed to the introduction of Cx. nigripalpus in urban areas.

Mechanism-based inactivation of lung-selective cytochrome P450 CYP2F enzymes.

3-Methylindole (3MI) is a pneumotoxin that requires P450-catalyzed metabolic activation (dehydrogenation), to an electrophilic methylene imine to elicit toxicity. Previous studies have shown that the human pulmonary cytochrome P450 enzyme, CYP2F1, and its goat analog, CYP2F3, catalyzed the dehydrogenation of 3MI. However, it was not known whether the dehydrogenation product could bind to active site nucleophilic residues to inactivate these enzymes. Therefore, the purpose of this study was to determine whether 3MI is a mechanism-based inhibitor of CYP2F3 and CYP2F1. The results showed that both enzymes were highly susceptible to 3MI-mediated suicide inactivation. The k(inact) and the K(I) for CYP2F3 were 0.09/min and 160 microM, respectively, and the approximate partition ratio was 220. Although CYP2F3 lost approximately 80% of its activity in 30 min, a concurrent loss of its reduced carbon monoxide complex was not observed, suggesting that the heme was not destroyed/modified during the inactivation. The exogenous nucleophile, glutathione, did not protect CYP2F1 from 3MI-mediated inactivation, suggesting that the reactive intermediate did not diffuse from the active site before inactivation events. Dialysis of 3MI-inactivated CYP2F3 did not restore activity, and alternate substrates protected CYP2F3. In addition, 3MI inhibited the 7-ethoxycoumarin deethylase activity of human CYP2F1 in a time- and concentration-dependent manner; the k(inact) and K(I) were 0.025/min and 49 microM, respectively. In conclusion, this study presents evidence that 3MI is a mechanism-based inhibitor of both CYP2F3 and CYP2F1, which are important enzymes in the bioactivation of pneumotoxicants such as 3MI or 1,1-dichloroethylene or carcinogens such as naphthalene, benzene, and styrene.

Regulation of CYP2A6 protein expression by skatole, indole, and testicular steroids in primary cultured pig hepatocytes.

CYP2A6 is one of the enzymes involved in the hepatic metabolism of a naturally produced compound, skatole, in the pig. Low CYP2A6 activity has been linked to excessive accumulation of skatole in pig adipose tissue and development of the phenomenon "boar taint." CYP2A6 activity varies between male and female animals, suggesting the involvement of sex hormones in regulation of the enzyme activity and/or expression. The present study investigated whether pig hepatic CYP2A6 protein expression is regulated by the testicular steroids testosterone, androstenone, or estrone sulfate using primary cultured hepatocytes as a model system. The study has also examined whether CYP2A6 expression can be modulated by the boar taint compounds skatole and indole. The research has established that androstenone inhibits CYP2A6 protein expression at the concentration of 1, 10, and 100 nM by 55, 37, and 44%, respectively. In contrast to androstenone, skatole and indole (final concentrations, 1, 10, and 100 nM) had a stimulatory effect on CYP2A6 expression. The effect of indole was more pronounced than that of skatole (maximum induction by 145 and 70%, respectively). Estrone sulfate and testosterone did not have a significant effect on CYP2A6 protein level. This is, as far as we know, the first communication to report the regulation of pig hepatic CYP2A6 expression by steroids and boar taint compounds. The hormonal modulation of CYP2A6 expression might contribute to gender-related differences in pig hepatic CYP2A6 activity and skatole accumulation in pig adipose tissue.

Growth-suppressing and algicidal properties of an extract from Arundo donax, an invasive riparian plant, against Prymnesium parvum, an invasive harmful alga.

This study examined the ability of acidic and neutral/alkaline fractions of a methanolic extract from giant reed (Arundo donax) and of two of its constituents, gramine and skatole, to inhibit growth of the ichthyotoxic golden alga (Prymnesium parvum) in batch culture. For this study, growth suppression was defined as inhibition of maximum cell density, algicidal activity as early occurrence of negative growth, and algistatic activity as lack of net growth. The acidic fraction did not affect algal growth. The neutral/alkaline fraction showed growth-suppressing and algicidal activities but no signs of algistatic activity - namely, cells in cultures surviving a partial-algicidal exposure concentration (causing transient negative growth) were later able to initiate positive growth but at higher concentrations, algicidal activity was full and irreversible. Gramine suppressed growth more effectively than skatole and at the highest concentration tested, gramine also showed partial-algicidal and algistatic activity. While the partial-algicidal activities of the neutral/alkaline fraction and of gramine were short-lived (≤6days) and thus may share similar mechanisms, algistatic activity was unique to gramine and persisted for >3 weeks. Given gramine's reported concentration in the neutral/alkaline fraction, its corresponding level of algicidal activity is much lower than the fraction's suggesting the latter contains additional potent algicides. Inhibition of maximum cell density by all test compounds was associated with reductions in exponential growth rate, and in the case of the neutral/alkaline fraction and gramine also reductions in early (pre-exponential) growth. These results indicate that giant reed is a potential source of natural products to control golden alga blooms. Giant reed is an invasive species in North America, thus also providing incentive for research into strategies to couple management efforts for both species.

Characteristic Scent from the Tahitian Liverwort, Cyathodium foetidissimum.

The volatile components of the Tahitian liverwort Cyathodium foetidissimum was analyzed using headspace solid phase micro-extraction (SPME) and GC-MS. Three volatile components, 4-methoxystyrene (24.4%), 3,4-dimethoxystyrene (28.7%), and skatole (15.9%) were identified as the major components from the fresh C. foetidissimum, along with several aliphatic aldehydes, n-octanal, n-nonanal, and n-decanal. However, (E)-2-nonenal recognized as aged malodor was not identified. In GC-O analysis, 2-aminoacetophenone was detected as one of the minor components with a strong aging note. In fact, C. foetidissimum showed the characteristic aging odor reminiscent the damp smell from old chest of drawers, or the civet like note with very strong feces and urine odor. The mixture consisted of 4-methoxystyrene, 3,4-dimethoxystyrene, and skatole in the detected ratio showed the sedative effect on CNV (contingent negative variation) measurement.

[Exploratory activity of mice of different genetic strains after olfaction disruption by 3-methylindole (skatole)].

The behavior of mice of two inbred strains (C57BL/6J and CBA) and their F1 hybrids was evaluated in the open field test after intraperitoneal administration of 3-methylindole (skatole) disrupting epithelium of the main olfactory system. High motor and exploratory activities and emotional sensitivity was observed in intact C57BL/6J mice compared to CBA mice and their hybrids. Anosmia induced by intraperitoneal administration of skatole changed the behavior of C57BL/6J and CBA mice. The direction of the observed changes in the orientation and exploratory behavior of anosmic animals was different. Anosmia decreased motor and exploratory activities in C57BL/6J mice and increase them in CBA mice. Intact hybrid mice demonstrated the predominance of the CBA genotype in the orientation and exploratory activity in the test used. Anosmia in hybrid animals had no significant effect on the orientation and exploratory behavior.

Skatole (3-Methylindole) Is a Partial Aryl Hydrocarbon Receptor Agonist and Induces CYP1A1/2 and CYP1B1 Expression in Primary Human Hepatocytes.

Skatole (3-methylindole) is a product of bacterial fermentation of tryptophan in the intestine. A significant amount of skatole can also be inhaled during cigarette smoking. Skatole is a pulmonary toxin that induces the expression of aryl hydrocarbon receptor (AhR) regulated genes, such as cytochrome P450 1A1 (CYP1A1), in human bronchial cells. The liver has a high metabolic capacity for skatole and is the first organ encountered by the absorbed skatole; however, the effect of skatole in the liver is unknown. Therefore, we investigated the impact of skatole on hepatic AhR activity and AhR-regulated gene expression. Using reporter gene assays, we showed that skatole activates AhR and that this is accompanied by an increase of CYP1A1, CYP1A2 and CYP1B1 expression in HepG2-C3 and primary human hepatocytes. Specific AhR antagonists and siRNA-mediated AhR silencing demonstrated that skatole-induced CYP1A1 expression is dependent on AhR activation. The effect of skatole was reduced by blocking intrinsic cytochrome P450 activity and indole-3-carbinole, a known skatole metabolite, was a more potent inducer than skatole. Finally, skatole could reduce TCDD-induced CYP1A1 expression, suggesting that skatole is a partial AhR agonist. In conclusion, our findings suggest that skatole and its metabolites affect liver homeostasis by modulating the AhR pathway.

3-Methylindole alters both olfactory and trigeminal nasal mucosal potentials in rats.

Data from human studies imply that vanillin is an olfactory stimulant, whereas CO2 activates intranasal trigeminal nociceptors. We examined the effects of the olfactotoxin 3-methylindole (3-MI) on nasal mucosal potentials evoked by vanillin and CO2 in rats. A single i.p. administration of 300 mg/kg 3-MI altered both olfactory and trigeminal mucosal responses. Relative to amplitude values determined in non-3-MI-injected rats, the response to vanillin was reduced to 6%, 7%, and 43%, and the response to CO2, recorded in the same rats, decreased to 25%, 38%, and 51% at 4, 8 and 16 days post-3-MI, respectively. The results suggest that 3-MI affects both olfactory and trigeminal elements within the nasal mucosa.

Cytochrome P450IIE1 (CYP2E1) is induced by skatole and this induction is blocked by androstenone in isolated pig hepatocytes.

Skatole, a derivative of tryptophan, is produced in the hind-gut of pigs and is metabolised via hepatic cytochrome P4502E1 (CYP2E1). Excessive accumulation of skatole together with androstenone, a metabolite of testosterone, in adipose tissue in some pigs is a major cause of 'boar taint' and is associated with defective expression of CYP2E1. This phenomenon is not understood because factors regulating CYP2E1 expression in pig liver have not yet been characterised. Therefore effects of skatole and androstenone on CYP2E1 expression were studied using isolated pig hepatocytes as a model system. Skatole induced CYP2E1 protein expression to the same degree as did acetone, a known CYP2E1 inducer. Induction by skatole was maximum between 20 and 28 h and a half-maximum effect was obtained at a skatole concentration of 0.2 mM. Induction of CYP2E1 by skatole was protein-synthesis dependent. Androstenone antagonised the effect of skatole on CYP2E1 expression but did not affect the CYP2E1 protein level when added alone. These results suggest that defective expression of CYP2E1 in some pigs is due to excessive concentrations of androstenone which prevent CYP2E1 induction by its substrate skatole. As a result, skatole metabolism is reduced and skatole is accumulated in adipose tissue.

The effect of 3-methylindole on the uptake and incorporation of 14C-choline into phospholipids in lung tissue slices.

3-Methylindole (3MI) is the causative agent in the development of acute bovine pulmonary edema. Microscopic studies revealed a structural disruption in the lamellar bodies of type II cells, indicating an abnormal metabolism of phospholipid in the lung of 3MI treated animals. In the present study, lung slices from 4 goats were used to investigate the changes in phosphatidylcholine metabolism induced by 3MI. Eighteen slices were cut from each healthy lung and divided into control and 3MI groups. After a 4-hr pretreatment with 3MI (.19 or .57 mM) or carrier, the level of incorporation of 14C-choline into phosphatidylcholine, sphingomyelin and their water soluble intermediates was studied. The uptake of 14C-choline and its incorporation into phosphatidylcholine and sphingomyelin was depressed by 3MI treatment. In the water soluble fractions, the radioactivity increased in free choline and CDP-choline, while it decreased in P-choline. This suggests that choline kinase and the P-choline transferases have become relatively more rate limiting and may play a role in the depressed de novo synthesis of phosphatidylcholine induced by 3MI.

Effect of serotonergic blockade on calf pulmonary function after the intravenous administration of 3-methylindole.

This study was designed to investigate whether 3-methylindole (3-Mi), a tryptamine analogue, could cause pulmonary injury in calves other than by cytotoxicity. Injection of 3-Mi resulted in a marked increase of respiratory rate, decrease of tidal volume and increase in minute ventilation. Pulmonary mechanics values were also profoundly affected, lung dynamic compliance being reduced to approximately one-third of its baseline value and total pulmonary resistance being increased two-fold. Arterial oxygen partial pressure was dramatically reduced. Successive challenges with 3-Mi after physiological saline pretreatment resulted in quantitatively identical alterations of pulmonary function values. Conversely, all these ventilatory, mechanical and gas exchange changes were abolished by pretreatment with serotonergic antagonists. It was concluded that intravenous administration of 3-Mi to healthy calves induced immediate and reversible bronchoconstriction which affected both central and peripheral airways. Because the effect was abolished by pretreatment with antiserotonin drugs, it is suggested that 3-Mi acts either directly by stimulating serotonergic receptors or indirectly through the release of serotonin from platelets. Current concepts of the physiopathological cascade underlying the toxicity of 3-Mi should, therefore, be re-evaluated in the light of this pharmacological mechanism.

Effect of energy or protein supplements containing monensin on ruminal 3-methylindole formation in pastured cattle.

The metabolism of 3-methylindole (3MI), a ruminal degradation product of L-tryptophan, results in acute bovine pulmonary edema and emphysema. The effect of feeding an energy or protein supplement containing monensin on ruminal 3MI formation in pastured beef cattle was investigated. A luxuriant pasture of orchard grass was established in a field that was seeded 1 year before the start of the grazing period. This 4-ha pasture was cut, fertilized, divided into 2 equal plots, and then irrigated during a 22-day growth period. All cows were fed a restricted quantity of low-quality alfalfa hay for 33 days before the grazing period. Two experiments were conducted, using 38 cows (30 of the cows were used in experiment I and all 38 cows were used in experiment II). Cows in each experiment were randomly allotted to 2 groups. One group was designated in each experiment as the control group. The control group for experiment I was fed an energy supplement. The control group for experiment II was fed a protein supplement. The 2nd group in each experiment was given the same supplement as the respective control group with 200 mg of monensin added/! kg of feed. Supplements were fed on days - 1, 0, 1, 2, 3, 4, 5, 6, and 7 of each experimental period. Supplements were fed twice daily to provide 1 kg of supplement/cow. Cows were given access to orchard grass pasture on day 0 of each experiment. Ruminal fluid was collected daily for analysis of 3MI, indole, and volatile fatty acids. Ruminal fluid pH was recorded immediately after collection. Ruminal pH of all cows decreased from 7.3 to 6.2 during the first few days of grazing the orchard grass. Ruminal pH then gradually increased toward neutrality by experimental day 10. Significantly (P < 0.01) higher molar percentages of pro-pionate and lower (P < 0.01) molar percentages of acetate and butyrate were observed in the 2 groups fed the supplements with added monensin. These changes in propionate and acetate remained different (P < 0.01) from those of the controls for 10 days (or 3 days after the last monensin feeding). Compared with pregrazing ruminal concentrations of 3MI, the 3MI values were elevated (P < 0.01) by day 1 in all groups, except in the monensin-treated cows of experiment I. In experiment I, 3MI concentrations were highest on experimental days 5 and 10 in control and monensin-treated cows, respectively. In experiment II, 3MI concentrations peaked on day 4 for the control cows and day 6 for the monensin-treated cows. Monensin supplementation reduced (P < 0.05) 3MI formation on days 1 through 5 in experiment I and on days 1 through 3 in experiment II. Formation of 3MI was increased in ruminal fluid of all cows after an abrupt change to the pasture forage, but the rate of 3MI production was slower, and a lower peak concentration of 3MI was reached in cows fed monensin than was observed in the controls. These results indicate that monensin administration in either an energy or protein supplement effectively reduced ruminal 3MI formation in pasture-fed cattle.

Pathophysiologic studies of calves given 3-methylindole intraruminally.

Intraruminal administration of 0.25 g of 3-methylindole (3MI; skatole/kg of body weight) to seven young calves generally caused mild respiratory signs and lesions, accompanied by only slight changes in cardiopulmonary function. Moderate depression, trembling, and irregular respiratory rate were observed between postadministration hours (PAH) 6 and 12. By PAH 24 at this dosage, abnormal clinical signs were not present. Statistically significant (P less than or equal to 0.05) changes observed in blood gas data from the seven calves were a decrease in aortic oxygen tension at PAH 12, increases in free-flowing venous oxygen tension in the intervals between PAH 6 and 12 and between PAH 6 and 24, and an increase in occluded venous oxygen tension at PAH 24. All calves had increases (although generally not statistically significant) in heart rate, cardiac output, cardiac index, stroke volume, and stroke index after 3MI administration. Mean aortic and pulmonary arterial pressure changes were generally small and variable. At necropsy, the lungs of the calves did not collapse when the thorax was opened. Patchy areas of consolidation (0.5 cm in diameter) were scattered throughout the parenchyma. Pulmonary edema or emphysema was not observed grossly. Microscopically, the alveolar septae were irregularly thickened because of edema, infiltration by polymorphonuclear and mononuclear cells, and vascular congestion. Interstitial lesions were patchy in distribution and severity and corresponded to the areas of consolidation observed grossly. Alveolar epithelial hypertrophy and hyperplasia were present, and an occasional focus of alevoli contained fluid of edema. Degeneration of individual hepatocytes was observed in scattered areas of the liver, especially in the periportal areas. It was concluded that differences in 3MI dosage response may exist between young calves and adult cattle in which calves are more resistant to the pulmonary cytotoxicity of 3MI.

Comparisons of reactivity of pulmonary vascular and airway smooth muscle of sheep and calf to tryptamine analogues, histamine, and antigen.

Serotonin (5-hydroxytryptamine) contracted the pulmonary artery, trachea, and bronchus in both cattle and sheep. Serotonin contracted the calf pulmonary vein, but it relaxed the pulmonary vein of sheep. Histamine constricted the 2 pulmonary blood vessels and trachea in calves and sheep. In contrast, histamine relaxed the ovine bronchus, but not the bronchus of the calf. Antigen (equine serum) constricted pulmonary veins from sensitized calves, but relaxed pulmonary veins from similarly sensitized sheep. Apparently, sheep are naturally more resistant than calves to both histamine and serotonin which contribute to the pulmonary inflammatory reaction.

Identification of oviposition attractants for Culex quinquefasciatus from fermented Bermuda grass infusions.

Compounds which attract and stimulate oviposition by gravid Culex quinquefasciatus were isolated and identified from a fermented Bermuda grass infusion. The neutral portion of the ether extract of the aqueous infusion contained the stimulatory materials. Fractionation by liquid chromatography yielded an active fraction containing phenol, 4-methylphenol, 4-ethylphenol, indole and 3-methylindole. A blend of the 5 compounds strongly stimulated oviposition, as did blends of any 4 of them. Bioassays with individual compounds showed that only 3-methylindole consistently induced oviposition, in concentrations spanning 5 orders of magnitude.

Interaction of the Culex quinquefasciatus egg raft pheromone with a natural chemical associated with oviposition sites.

In laboratory bioassays, gravid Culex quinquefasciatus mosquitoes were strongly attracted and or stimulated to oviposit by a habitat-derived chemical cue, 3-methylindole, at several concentrations ranging from 0.01 to 1 microgram/liter in water. At concentrations above 10 micrograms/liter, 3-methylindole became repellent or deterrent. Responses to the known egg raft pheromone, 6-acetoxy-5-hexadecanolide, were much weaker and were relatively constant above a threshold dosage of about 0.1 microgram. Responses to blends of a fixed amount of the pheromone with variable doses of 3-methylindole were shown to be additive rather than synergistic.