Microfluorometric Method Research Articles

1. ニンヒドリン蛍光反応によるペプチド, アミノ酸, 1級アミン類の微量分析

1. ニンヒドリン蛍光反応によるペプチド, アミノ酸, 1級アミン類の微量分析

Abstracts: Reduction‐induced surface modification of human hair

pp. 1–12 A microfluorometric method has been developed to characterize lipid removal or ‘delipidation’ of the human hair cuticula during light exposure and chemical grooming processes such as oxidation (bleaching) and reduction. In the case of photochemical and chemical oxidation, lipid removal (‘delipidation’ of the F‐layer or lipid‐layer) from the outer‐layer of the exposed scale faces and generation of cysteic acid groups occurs. This ‘delipidation,’ which ultimately results in ‘acidification’ of the scale faces, leading to a change in surface chemistry from hydrophobic to hydrophilic, can be detected and quantified by microfluorometry by tagging, e.g. with the cationic fluorochrome Rhodamine B. In the case of reduction, similar tagging of the acid sites on the scale faces is possible, but this time, Rhodamine B reacts with the mixed disulfide containing a carboxyl group that will be ionized above a pH of about 4. In addition to this, we have shown by microfluorometric scanning that the negative charges generated in the cuticle surface can be used to bind low‐molecular‐weight quaternary conditioners. This process can be considered as ‘relipidation’ or ‘refatting of the scale faces. We have shown in earlier studies (1) that this entire process of oxidation‐induced ‘delipidation’ and subsequent ‘relipidation’ of the acidic scale faces with a cationic conditioning molecule can also be reliably quantified by X‐ray photoelectron spectroscopy (XPS). Furthermore, single‐fiber wettability scanning using the Wilhelmy technique, which is highly sensitive to any changes in surface chemistry, is well‐suited to detect and characterize treatment‐induced changes in the chemical nature of the hair surface from hydrophobic to hydrophilic.

Collecting lymphatic vessel permeability response to atrial natriuretic peptide

While the lymphatic vasculature is known to regulate fluid and solute homeostasis, the mechanisms by which it accomplishes this task are not clear. Previously we determined that the basal solute permeability (Ps, cm·s−1) of rat mesenteric collecting lymphatics does not differ from that of the venules. Whether collecting lymphatic Ps is dynamic, able to change in response to disease, remains unknown. Using a microfluorometric method we tested whether, like venules, collecting lymphatic Ps to rat albumin is increased by atrial natriuretic peptide (ANP), a vasoactive hormone secreted by the heart in response to stretch (e.g., congestive heart failure, microgravity, hypertension). Perfusion with 100 nM ANP in vivo resulted in a 2.5 fold (n=4) elevation in collecting lymphatic Ps, a change similar to that reported for venules. Coupled with our previous findings these data demonstrate a novel ability of collecting lymphatics to act as exchange vessels, modulating their Ps in response to vasoactive hormones. Collecting lymphatic vessels represent an additional site for vascular volume regulation, increasing the surface area available for shifting solute and fluid from the vascular to the interstitial compartment. Supported by NIH HL078816 & NASA NNJ05HF37G.

Activity of increased specific and non-specific esterases and glutathione transferases associated with resistance to permethrin in pediculus humanus capitis (phthiraptera: pediculidae) from Argentina

Enhanced metabolism by oxidative enzymes is a major cause of pyrethroid resistance in insects. In this work, we evaluated the role of specific and non-specific esterases in head louse populations from Buenos Aires with different levels of resistance to permethrin. As esterase activity is substrate-dependent, four different esters were used as unspecific substrates in order to obtain a better characterization of the possible role of these enzymes in the resistance phenomenon. The unspecific substrates were phenylthioacetate, 1- and 2-naphtyl-acetate, and p-nitrophenyl acetate. A 7-coumaryl permethrate was synthesized and used as a specific substrate to measure pyrethroid esterases by a very sensitive microfluorometric method. The results on pyrethroid esterase activity obtained with this substrate showed that these enzymes contribute to the detoxifying activity in resistant populations, although no correlation was found between pyrethroid esterase activity and resistance ratios. In this study, we established that the activity of esterase against specific and non-specific substrates is increased in pyrethroid-resistant populations of head lice from Buenos Aires. Also, dichlorodiphenyltrichloroethane (DDT) resistance values demonstrated that there is a DDT cross-resistance phenomenon in pyrethroid-resistant head louse populations and suggested that an alteration in the receptor of the nervous system (kdr gen) is a key factor of the resistance phenomena in these head louse populations.

Isoliquiritigenin, a flavonoid from licorice, relaxes guinea-pig tracheal smooth muscle in vitro and in vivo: Role of cGMP/PKG pathway

Licorice root is used to treat asthma as a component of Shaoyao-Gancao-tang, a traditional Chinese medicine formula. In this study, we investigated the tracheal relaxation effects of isoliquiritigenin, a flavonoid isolated from the roots of Glycyrrhiza glabra (a kind of Licorice), on guinea-pig tracheal smooth muscle in vitro and in vivo. The tension changes of isolated tracheal rings were isometrically recorded on a polygraph. The large-conductance Ca 2+-activated K + channels (BK Ca) were measured by inside-out patch-clamp techniques and intracellular Ca 2+ concentrations ([Ca 2+] i) were tested by microfluorometric method in guinea-pig tracheal smooth muscle cells (TSMCs). Isoliquiritigenin produced concentration-dependent relaxation in isolated guinea-pig tracheal rings precontracted with acetylcholine, KCl, and histamine. Pretreatments with charybdotoxin, ODQ and KT5823 attenuated the relaxation induced by isoliquiritigenin. Isoliquiritigenin significantly increased intracellular cGMP level in cultured TSMCs and inhibited the activity of phosphodiesterase (PDE) 5 in human platelets. Moreover, isoliquiritigenin increased by 9-fold the probability of BK Ca channel openings of TSMCs in inside-out patches and markedly reduced [Ca 2+] i rise induced by acetylcholine in TSMCs, pretreatment with KT5823 attenuated above two responses to isoliquiritigenin. In vivo experiment isoliquiritigenin significantly prolonged the latency time of histamine–acetylcholine aerosol-induced collapse and inhibited the increase of lung overflow induced by intravenously administered histamine dose-dependently. These data indicate that isoliquiritigenin relaxes guinea-pig trachea through a multiple of intracellular actions, including sGC activation, inhibition of PDEs, and associated activation of the cGMP/PKG signaling cascade, leading to the opening of BK Ca channels and [Ca 2+] i decrease through PKG-dependent mechanism and thus to tracheal relaxation.

The use of microfluorometric method for activity-guided isolation of antiplasmodial compound from plant extracts

In vitro antiplasmodial activity of methanolic extracts of 16 medicinal plants was evaluated by fluorometric assay using PicoGreen. The IC50s, as determined by parasite DNA concentration, ranged from <11 to >200 and <13 to >200 microg/ml for Plasmodium falciparum 3D7 and K1, respectively; and the most active extracts were those from Anogeissus leiocarpus and Terminalia avicennoides (<11-> or =14 microg/ml). Aqueous, butanolic, ethyl acetate, and methanolic fractions of these two extracts revealed butanolic fraction to have a relatively better activity (IC50, 10-12 microg/ml). Activity-guided chromatographic separation of the butanolic fraction on Sephadex LH-20 followed by nuclear magnetic resonance and correlation high-performance liquid chromatography revealed the presence of known hydrolysable tannins and some related compounds-castalagin, ellagic acid, flavogallonic acid, punicalagin, terchebulin, and two other fractions. The IC50s of all these compounds ranged between 8-21 microg/ml (8-40 microM) against both the strains. Toxicity assay with mouse fibroblasts showed all the extracts and isolated compounds to have IC50 > or = 1500 microg/ml, except for Momordica balsamina with <1500 microg/l. All the extracts and isolated compounds did not affect the integrity of human erythrocyte membrane at the observed IC50s. However, adverse effects manifest in a concentration-dependent fashion (from IC50 > or = 500 microg/ml).

Nesfatin-1: Distribution and Interaction with a G Protein-Coupled Receptor in the Rat Brain

Nesfatin-1 is a recently identified satiety molecule detectable in neurons of the hypothalamus and nucleus of solitary tract (NTS). Immunohistochemical studies revealed nesfatin-1-immunoreactive (irNEF) cells in the Edinger-Westphal nucleus, dorsal motor nucleus of vagus, and caudal raphe nuclei of the rats, in addition to the hypothalamus and NTS reported in the initial study. Double-labeling immunohistochemistry showed that irNEF cells were vasopressin or oxytocin positive in the paraventricular and supraoptic nucleus; cocaine-amphetamine-regulated transcript or tyrosine hydroxylase positive in arcuate nucleus; cocaine-amphetamine-regulated transcript or melanin concentrating hormone positive in the lateral hypothalamus. In the brainstem, irNEF neurons were choline acetyltransferase positive in the Edinger-Westphal nucleus and dorsal motor nucleus of vagus; tyrosine hydroxylase positive in the NTS; and 5-hydroxytryptamine positive in the caudal raphe nucleus. The biological activity of rat nesfatin-1 (1-82) (100 nm) was assessed by the Ca(2+) microfluorometric method. Nesfatin-1 elevated intracellular Ca(2+) concentrations [Ca(2+)](i) in dissociated and cultured hypothalamic neurons. The response was prevented by pretreating the cells with pertussis toxin (100 nm) or Ca(2+)-free solution and by a combination of the L-type and P/Q-type calcium channel blocker verapamil (1 microm) and omega-conotoxin MVIIC (100 nm). The protein kinase A inhibitor KT 5720 (1 microm) suppressed nesfatin-1-induced rise in [Ca(2+)](i). The result shows that irNEF is distributed to several discrete nuclei in the brainstem, in addition to the hypothalamus and NTS reported earlier, and that the peptide interacts with a G protein-coupled receptor, leading to an increase of [Ca(2+)](i), which is linked to protein kinase A activation in cultured rat hypothalamic neurons.

Fluorescence measurements of serotonin-induced V-ATPase-dependent pH changes at the luminal surface in salivary glands of the blowflyCalliphora vicina

Secretion in blowfly salivary glands is induced by the neurohormone serotonin and powered by a vacuolar-type H(+)-ATPase (V-ATPase) located in the apical membrane of the secretory cells. We have established a microfluorometric method for analysing pH changes at the luminal surface of the secretory epithelial cells by using the fluorescent dye 5-N-hexadecanoyl-aminofluorescein (HAF). After injection of HAF into the lumen of the tubular salivary gland, the fatty acyl chain of the dye molecule partitions into the outer leaflet of the plasma membrane and its pH-sensitive fluorescent moiety is exposed at the cell surface. Confocal imaging has confirmed that HAF distributes over the entire apical membrane of the secretory cells and remains restricted to this membrane domain. Ratiometric analysis of HAF fluorescence demonstrates that serotonin leads to a reversible dose-dependent acidification at the luminal surface. Inhibition by concanamycin A confirms that the serotonin-induced acidification at the luminal surface is due to H(+) transport across the apical membrane via V-ATPase. Measurements with pH-sensitive microelectrodes corroborate a serotonin-induced luminal acidification and demonstrate that luminal pH decreases by about 0.4 pH units at saturating serotonin concentrations. We conclude that ratiometric measurements of HAF fluorescence provide an elegant method for monitoring V-ATPase-dependent H(+) transport in the blowfly salivary gland in vivo and for analysing the spatiotemporal pattern of pH changes at the luminal surface.

Change in surface chemistry of the cuticle of human hair by chemical and photochemical oxidation

This is a multifaceted study on the characterization and quantification of damage to the hair fiber surface caused by photochemical and cosmetic chemical oxidative processes. Unique techniques were used, including a microfluorometric method that had been adapted to characterize and quantify the delipidation and acidification of the human hair surface during light exposure and cosmetic chemical grooming processes such as bleaching. During photochemical and chemical oxidation, breakdown of the lipid domains (also called the F‐layer) of the outer b‐layer occurs on the exposed scale faces and cysteic acid groups are generated on the cuticle cell surface. The newly formed acid functionalities can be tagged with the cationic fluorochrome Rhodamine B, allowing not only quantification of the level of progressive acidification but also localization of the newly formed acid functionalities. On the other hand, the negative charges generated on the hair surface by oxidation can also bind low molecular weight quaternary cationic conditioning compounds such as cetyltrimethylammonium bromide. This process can be considered a relipidation by adsorption. We have shown that the entire process of delipidation/acid formation and subsequent relipidation by adsorption on the scale faces can be quantified by X‐ray photoelectron spectroscopy. Since X‐ray photoelectron spectroscopy analysis is highly sensitive and able to detect atomic species at the very surface of the hair fibers, receiving signals from an escape depth as shallow as 25 Å, it appeared ideal for the characterization of treatment‐induced changes in the hair surface. However, X‐ray photoelectron spectroscopy provides an average elemental composition of the hair surface including scale faces and scale edges. The microfluorometric technique, on the other hand, can distinguish progressive delipidation of the scale faces from changes occurring at the broken scale edges. This distinction was shown and characterized in detail by slow speed microfluorometric scanning of the hair surface. Chemical and photochemical oxidative processes at the hair surface result in certain collateral effects. Particularly changes in surface wettability and fiber friction are of significance to the cosmetic chemist because they affect the spreading and wicking of products in hair as well as the managability and the body of hair assemblies. Methods of characterizing these effects are discussed briefly.

Effect of Gnaphalium conoideum HBK on guinea pig airway smooth muscle: role of L-type Ca 2+ channels

Plants from the Gnaphalium genus have been used in the Mexican traditional medicine for digestive and respiratory complaints. In the present study, the effect of methanolic extract from Gnaphalium conoideum HBK on the responses to contractile agonists was assessed in guinea pig tracheas, and the possible role of L-type Ca 2+ channels was explored in tracheal guinea pig isolated myocytes. Cumulative concentration–response curves to carbachol or histamine, as well as contractile responses to 60 mM KCl were evaluated with or without 30 min preincubation with 20 or 100 μg ml −1 Gnaphalium conoideum. Likewise, intracellular Ca 2+ concentrations were measured by microfluorometric method (fura-2 AM) in isolated tracheal myocytes with or without preincubation with 0.1, 0.31 or 1 μg ml −1 Gnaphalium conoideum. We found that methanolic extract from Gnaphalium conoideum significantly diminished the contractile responses to histamine, but not to carbachol or KCl. In isolated myocytes, Gnaphalium conoideum significantly reduced the intracellular Ca 2+ rise induced by 60 mM KCl. Because histamine contractile responses are largely dependent on extracellular Ca 2+, and KCl responses are mainly mediated through L-type Ca 2+ channels, our results suggested that methanolic extract from Gnaphalium conoideum might be acting as a partial blocker of these Ca 2+ channels.

A microfluorometric method for quantifying RNA and DNA in terrestrial insects.

Evidence is accumulating for a mechanistic linkage between body phosphorus content and growth and reproduction of individual organisms, due in part to variation in allocation of resources to ribosomal RNA. Testing this connection requires reliable methods of quantifying the nucleic acid content of individual organisms. Although methods for quantifying nucleic acids are available for a wide array of organisms, adaptation of such methods for study of insects has been neglected. Sensitive stains and high throughput fluorometric measurements are now available that substantially improve past methodologies. Here we present methods for the extraction and quantification of insect RNA and DNA based on the use of N-lauroylsarcosine and sonication for extraction, the nucleases RNase and DNase, and the use of microplate fluorescent assays to quantify nucleic acids as percent of body weight in insects. We illustrate the method using Drosophila and curculionid weevils.

A microfluorometric method for quantifying RNA and DNA in terrestrial insects.

Evidence is accumulating for a mechanistic linkage between body phosphorus content and growth and reproduction of individual organisms, due in part to variation in allocation of resources to ribosomal RNA. Testing this connection requires reliable methods of quantifying the nucleic acid content of individual organisms. Although methods for quantifying nucleic acids are available for a wide array of organisms, adaptation of such methods for study of insects has been neglected. Sensitive stains and high throughput fluorometric measurements are now available that substantially improve past methodologies. Here we present methods for the extraction and quantification of insect RNA and DNA based on the use of N-lauroylsarcosine and sonication for extraction, the nucleases RNase and DNase, and the use of microplate fluorescent assays to quantify nucleic acids as percent of body weight in insects. We illustrate the method using Drosophila and curculionid weevils.

Evaluation of β-amyloid peptide 25–35 on calcium homeostasis in cultured rat dorsal root ganglion neurons

Accumulation of β-amyloid (Aβ) protein in brain is an important characteristic for the etiology of Alzheimer’s disease. Of all the possible processes generating the neurotoxic effects by Aβ, disruption of intracellular Ca 2+ homeostasis is the primary event. In this process, various intracellular Ca 2+ regulatory mechanisms are reported to be involved. Using patch-clamp techniques, both low and high voltage activated Ca 2+ channel currents were recorded in the cultured dorsal root ganglion (DRG) neurons. Application of Aβ protein fragment, Aβ 25–35 (2 μM), for 30 s increased the amplitude in both currents. The Aβ-triggered facilitation effect of Ca 2+ channel was found in all the depolarized potentials tested, as shown in the current–voltage relationship. Furthermore, after applying single cell Ca 2+ microfluorometric method, it was found that Aβ 25–35 alone could trigger elevations of intracellular Ca 2+ concentration ([Ca 2+] i) level in 90% of the cells tested. The elevation diminished completely by cumulatively adding CdCl 2, NiCl 2, thapsigargin (TG), FCCP and Zn 2+ in the normal bath solution. Combining pharmacological approaches, we found that voltage-dependent Ca 2+ channels, Ca 2+ stores and a putative Zn 2+-sensitive extracellular Ca 2+ entry, respectively, makes 61.0, 25.1, and 13.9% contribution to the [Ca 2+] i increase caused by Aβ. When tested in a Ca 2+-free buffer, mitochondria was found to contribute 41.3% of Aβ produced [Ca 2+] i elevation and the remaining 58.7% was attributed to endoplasmic reticulum (ER) release.

Response of the Photosynthetic Apparatus of the Diatom Thallassiosira weisflogii to High Irradiance Light

The response of the photosynthetic apparatus to high irradiance illumination (440–2200 W/m2) was studied in the diatom Thallassiosira weisflogii by fluorescence methods. Changes in the photosynthetic apparatus were monitored by measuring characteristics of chlorophyll fluorescence F0, Fm, Fv /Fm, and qN for several hours after illumination of the alga with high-intensity light. Incubation of the alga with 2 mM DTT, an inhibitor of de-epoxidase of carotenoids in the diadinoxanthin cycle, led to a decrease in the nonphotochemical quenching of chlorophyll fluorescence and a drop in the Fv /Fm ratio, a characteristic that reflects the quantum efficiency of the functioning of the photosynthetic apparatus. Light-induced absorption changes associated with transformations of carotenoids of diadinoxanthin cycle were recorded in vivo in algal suspensions in the absence and in the presence of DTT. Using the microfluorometric method, we measured cell distribution over the efficiency of the primary processes of photosynthesis (Fv /Fm) after illumination. We found cells with a high tolerance of their photosynthetic apparatus to photooxidative damage. The relatively high tolerance of a portion of the cell population to high-light illumination can be related to light-induced transformation of carotenoids and to the functioning of other protective systems of the photosynthetic apparatus in diatoms.

A Direct Microfluorometric Method for Measuring Subpicomole Amounts of Nicotinamide Adenine Dinucleotide Phosphate, Glucose, and Glycogen

A Direct Microfluorometric Method for Measuring Subpicomole Amounts of Nicotinamide Adenine Dinucleotide Phosphate, Glucose, and Glycogen

Microfluorometric analyses of glycogen in freshly dissected, single skeletal muscle fibres of the cane toad using a mechanically skinned fibre preparation.

The main objective of this study was to analyse glycogen in single muscle fibres, using a recently developed microfluorometric method which detects subpicomol amounts of NADPH, glucose and glycogen (as glucosyl units) (detection limit 0.16-0.17 pmol in a 25 nl sample) without fluorochrome amplification. The fibres were freshly dissected from the twitch region of the iliofibularis muscle of the cane toad (Bufo marinus), and were mechanically skinned under paraffin oil to gain access to the intracellular compartments. The results show that (1) glycogen concentrations in toad skeletal muscle fibres range between 25.8 and 369 mmol glucosyl units/litre fibre volume; (2) there is a large variation in glycogen content between individual fibres from the iliofibularis muscle of one animal; (3) there are seasonal differences in the glycogen content of toad single muscle fibres; (4) the total amount of glycogen in single muscle fibres of the toad does not decrease significantly when storing the tissue, under paraffin oil, at 20-25 degree C for up to 6 h or at 4 degree C for up to 24 h; and (5) 15-26% of fibre glycogen can be washed in an aqueous solution at pH 5-7, within 5 min, while 74-85% of fibre glycogen remains associated with the washed skinned fibre, even after 40 min exposure of the skinned fibre preparation to the aqueous environment. The retention of most glycogen in the fibre preparation after mechanical removal of the plasma membrane and extensive washing indicates that in toad skeletal muscle fibres the largest proportion of glycogen is tightly bound to intracellular structures. The results also show that the skinned muscle fibre preparation is well suited for microfluorometric glycogen determination, since low molecular weight non-glycogen contributors to the fluorescence signal can be removed from the myoplasmic space prior to the glycogen hydrolysis step.

A microassay for the detection of low levels of cytochrome P450 O-deethylation activities with alkoxyresorufin substrates.

A microfluorometric method for the detection of low levels of cytochrome P450 was developed to increase the sensitivity of the assay, since a low level of CYP450 associated enzymatic activities was expected in human placenta tissues and small samples of placenta (approximately 10 g) could be easily collected, stored and processed. The dual fluorescence assay of Kennedy et al. [1], which was developed to simultaneously quantitate microsomal proteins and ethoxyresorufin-O-deethylase (EROD) activity was adapted for 96 wells microtiter plates. Placental microsomes samples were analyzed. For samples obtained from non-smoking mothers from the general southern Quebec population, results ranged from less than 1-3.3 pmol/mg protein.min. Samples collected from smoking mothers showed activity levels ranging from 30-69 pmol/mg protein.min. These results showed the suitability of the microassay for measuring low level of CYP450 activity in tissues such as placenta.

EFFECT OF LOCATION ON VITAMIN C CONTENT OF SELECTED BELL PEPPER CULTIVARS

Vitamin C (VC) levels (mg/l00 g FW) were determined in 10 varieties of colored bell pepper grown under different field conditions. VC was determined by the microfluorometric method. `Orobelle' (169 mg), `King Arthur' (143 mg), `Valencia' (141 mg), and `Chocolate Bell' (134 mg) had significantly higher VC levels than `Dove' (109 mg), `Ivory' (106 mg), `Blue Jay' (93 mg), `Canary' (90 mg), and `Black Bird' (65 mg). The largest variability (53 mg) in VC levels were observed for varieties that had the highest VC content. Mean VC levels were 143a, 143a, 141a, 136a, 108ab, 93bc, and 63c for the yellow, red, orange, brown, white, purple, and black colors, respectively. Since the Recommended Daily Allowance (RDA) for VC is 60 mg per day, these results suggested that a 100-g serving of fresh bell pepper or less would supply 100% RDA of VC. Therefore, after selecting a color, growers still have the freedom to grow a variety that performs well in their area to produce peppers of high VC contents.

The role of membrane-bound porphyrin-type compound as endogenous sensitizer in photodynamic damage to yeast plasma membranes

The effect of visible light (400–600 nm) on Candida guilliermondii and Saccharomyces cerevisiae was studied, and irradiation fluences killing the yeast cells without exogenous sensitizers were determined. The lethal effects are strongly oxygen-dependent, suggesting the involvement of photodynamic reactions mediated by endogenous sensitizer(s). Repair-deficient strains of S. cerevisiae show the same photosensitivity as the wild-type strain indicating that visible light does not photosensitize repairable DNA lesions. As was demonstrated using the microfluorometric method with the fluorochrome primulin, photodestruction of plasma membrane permeability barriers is important for yeast cell lethality. Visible light at cell-killing fluences induces lipid peroxidation in plasma membrane ghosts isolated from C. guilliermondii. Data obtained suggest the important role of singlet oxygen photogenerated by endogenous sensitizer(s) in initiating oxidative reactions. A spectrofluorometric analysis of the plasma membrane ghosts revealed one compound fluorescent in the visible spectral region at 683 nm. Its fluorescence excitation and absorption spectra have structures typical for porphyrins. The plasma membrane-bound porphyrin-like compound is different in some fluorescence properties from mitochrondrial porphyrins. Estimation of its amount gives a value of 0.1 nmol porphyrin per milligram of protein of the plasma membrane ghosts. This porphyrin-type compound is considered to be the most probable candidate for the role of the sensitizer in photodynamic damage to yeast plasma membrane and cell inactivation by visible light.

A Microfluorometric Method for Measuring Ethoxycoumarin-O-deethylase Activity on Individual Drosophila melanogaster Abdomens: Interest for Screening Resistance in Insect Populations

We developed a method for measuring ethoxycoumarin deethylase (ECOD) activity using a single Drosophila abdomen. The activities obtained were well correlated with the classic method from microsomes (r = 0.902). This new method, performed in microtitration plates, was at least six times more sensitive compared to the conventional cuvet fluorometric one. Moreover, it was possible among a large number of insects to differentiate those with low or high ECOD activities. This improved procedure has been checked upon crosses between resistant strain (with high ECOD activity) and susceptible strain (with low ECOD activity). The results demonstrate the possible separation of resistant phenotypes and emphasize the importance of this approach in assessing the spreading of insecticide resistance in natural populations of insects.