Evidence against the existence of acetylated Sudan Black B

Summary The nature of acetylated Sudan Black B (aSBB) has been investigated, and it has been found, by thin layer chromatography, that each fraction of aSBB has an R _f which is the same as that of a similar fraction of Sudan Black B (SBB). However, aSBB has been found to have fewer fractions, 9–12 than SBB, 14–16. The two major fractions from aSBB and SBB were examined, and a great similarity was found between the absorption spectra of the respective fractions of aSBB and SBB. The major fraction of aSBB was investigated by mass spectroscopy and found to have a similar molecular weight to that expected of SBB. This demonstrates that aSBB is not in fact acetylated, and that the components of aSBB are chemically no different from the corresponding components of SBB.     

Thin layer chrornatography and histochemistry of Sudan Black B

Summary Thin layer chromatography of commercial Sudan Black B on silica gel with chloroform-benzene (11) as the developing solvent reveals two blue main fractions with R_f values of 0.49 and 0.19, SBB-I and SBB-II respectively. Furthermore at least eighteen secondary fractions or impurities have been found. SBB-I and SBB-II were isolated and purified by preparative thin layer chromatography. Commercial Sudan Black B consists of about 20 p.c. SBB-I, 60 p.c. SBB-II and 20 p.c. secondary fractions.From spectrophotometrical and histochemical investigations it appeared that SBB-I stains lipids more pronounced than SBB-II; moreover SBB-I is more specific for neutral lipids than SBB-II, which fraction may also stain some proteins and acid mucopolysaccharides. Contrary to SBB-II the staining with SBB-I is fairly independent of pH. Finally, the colour of SBB-II changes under the influence of light and air, while SBB-I is much more stable.A physico-chemical study of the nature of SBB-I and SBB-II, including spectrophotometry, chromatography, infrared spectroscopy and chemical analysis revealed, that SBB-II is a basic dye, while SBB-I in spite of the structural resemblance behaves as a neutral one, dissolving therefore better in neutral lipids.As yet the chemical composition of SBB-I and SBB-II, and the relation to the scheme of synthesis of Sudan Black B has not been solved. The unspecificity of lipid staining by Sudan Black B is due to the basicity of SBB-II, and to the instability of this dye toward light and air. Moreover the some eighteen impurities may have some influence on the staining properties. The question of solubility or adsorption processes in the case of lipid staining by Sudan dyes is at least partially answered by the proposition of a dissolving fraction SBB-I and an adsorbed fraction SBB-II. The changing absorption spectra by the corresponding solvatochromic and metachromatic effects may give information about the nature of the lipids stained.     

Detecting senescence: a new method for an old pigment

Cellular senescence is a state of irreversible cell cycle arrest induced by different types of cellular stresses. The field of senescence has made significant advances in the understanding of many of the mechanisms governing this phenomenon; however, a universal biomarker that unambiguously distinguishes senescent from proliferating cells has not been found. In this issue of Aging Cell, Evangelou and colleagues developed a sensitive method for identification of senescent cells in different types of biological material based on the detection of lipofuscin using an analogue of Sudan Black B (SBB) histochemical dye coupled with biotin, which they named GL13. The authors propose that this method is more sensitive and versatile than using SBB alone. Lipofuscin, a nondegradable oxidation product of lipids, proteins and metals, is found in senescent cells. Detection of lipofuscin using GL13 staining may be a more feasible method than others currently used for identification of senescent cells both in cell culture and tissues.     

A simple adhesion assay for studying interactions between platelets and endothelial cells in vitro

Cell adhesion plays a key role during various physiological and pathological processes. Many studies have been performed to understand the interaction of platelets with endothelial cells (ECs) during the past decades. Modulation of their interaction has been shown to be therapeutically useful in thrombotic diseases. Some methods of labeling platelets such as counting and radiolabeling have been applied in the study of the platelets-ECs interaction, but these methods did not obtain full approval. A rapid, simple and sensitive assay for platelets-ECs interaction was developed in this paper. Platelets were labeled with Sudan Black B (SBB) before adding to confluent ECs monolayer. Non-adherent platelets were removed by washing with PBS. The adherent platelets were lysed with dimethylsulfoxide (DMSO) and the absorbance was recorded at 595 nm by spectrophotometer. A linear correlation was observed between the absorbance of SBB and the number of platelets. By employing the SBB method, the influence of heparin on platelets-ECs interactions was observed. Heparin (3–100 units/mL) obviously reduced platelets adhering to ECs in a concentration-dependent manner.     

Robust,universal biomarker assay to detect senescent cells in biological specimens

Cellular senescence contributes to organismal development, aging, and diverse pathologies, yet available assays to detect senescent cells remain unsatisfactory. Here, we designed and synthesized a lipophilic, biotin‐linked Sudan Black B (SBB) analogue suitable for sensitive and specific, antibody‐enhanced detection of lipofuscin‐containing senescent cells in any biological material. This new hybrid histo‐/immunochemical method is easy to perform, reliable, and universally applicable to assess senescence in biomedicine, from cancer research to gerontology.     

Multiacetylated forms of H4 are found in a putative transcriptionally competent chromatin fraction from trout testis.

We have examined the distribution of acetylated histones derived from various trout testis chromatin fractions of different composition. Our results indicate that a chromatin fraction, preferentially solubilized by micrococcal nuclease, containing the bulk of the HMG proteins and similar to a fraction released from intact trout nuclei and previously shown to be enriched in transcribed DNA sequences also possesses high levels of multiacetylated species of H4. Histones 2A, 2B and 3 are also acetylated in this particular chromatin fraction. Monoacetylated species of the 4 inner nucleosomal histones appear to be characteristic of the nucleohistone portion of trout testis chromatin.     

Chromatography and Biological Stains: II. Column Chromatographic Separation of the Colored Materials in Commercial Sudan III

Commercial Sudan III was separated into various fractions by the usual column chromatographic technics using Celite-silicic acid as the adsorbent and commercial petroleum ether (B) as the solvent. By analysis, using both spectrophotometric and paper strip chromatographic methods, it was possible to identify fractions from the commercial sample, including several fractions which were indubitably very highly purified Sudan III. In addition several other fractions containing mixtures of colored components have been isolated.     

Release of ribonucleoprotein during digestion of rat testis chromatin with deoxyribonuclease II (3.1.4.6)

The composition of rat testis chromatin proteins in fractions produced by limited DNase II digestion followed by differential precipitation with MgCl2 has been studied. Over 50% of the acid-soluble proteins in the soluble chromatin fraction appeared to be quite similar to proteins which are associated with ribonucleoprotein (RNP) particles in HeLa cells. Although the ratios of the testis RNP protein components differed from those of HeLa RNP particles, the three major polypeptides were most similar to the HeLa components designated A2, B2, and C1. The soluble chromatin fraction was also enriched in the high mobility group proteins HMG1 and HMG2.     

Sudan Black B: chemical structure and histochemistry of the blue main components.

Sudan Black B contains two blue main components, SSB-I and SSB-II. Their chemical structures were determinated by the aid of two-dimensional thin-layer chromatography, column chromatography, absorption, IR, mass, H1-NMR, and C13-NMR spectroscopy and were proved by alternate synthesis. SSB-I has been found to be 2,3-dihydro-2,2-dimethyl4-[(4-phenylazo-1-naphthalenyl)-azol]-1H-perimidine. For SSB-II was confirmed the known structure 2,3-dihydro-2,2-dimethyl-6-[(4-phenylazo-1-naphthalenyl)-azo]-1H-permidine. Relations of chemical structure of SSB-I and SSB-II to their staining properties are discussed.     

Monoclonal antibodies specific for an acetylated form of alpha-tubulin recognize the antigen in cilia and flagella from a variety of organisms

Seven monoclonal antibodies raised against tubulin from the axonemes of sea urchin sperm flagella recognize an acetylated form of alpha-tubulin present in the axoneme of a variety of organisms. The antigen was not detected among soluble, cytoplasmic alpha-tubulin isoforms from a variety of cells. The specificity of the antibodies was determined by in vitro acetylation of sea urchin and Chlamydomonas cytoplasmic tubulins in crude extracts. Of all the acetylated polypeptides in the extracts, only alpha-tubulin became antigenic. Among Chlamydomonas tubulin isoforms, the antibodies recognize only the axonemal alpha-tubulin isoform acetylated in vivo on the epsilon-amino group of lysine(s) (L'Hernault, S.W., and J.L. Rosenbaum, 1985, Biochemistry, 24:473-478). The antibodies do not recognize unmodified axonemal alpha-tubulin, unassembled alpha-tubulin present in a flagellar matrix-plus-membrane fraction, or soluble, cytoplasmic alpha-tubulin from Chlamydomonas cell bodies. The antigen was found in protein fractions that contained axonemal microtubules from a variety of sources, including cilia from sea urchin blastulae and Tetrahymena, sperm and testis from Drosophila, and human sperm. In contrast, the antigen was not detected in preparations of soluble, cytoplasmic tubulin, which would not have contained tubulin from stable microtubule arrays such as centrioles, from unfertilized sea urchin eggs, Drosophila embryos, and HeLa cells. Although the acetylated alpha-tubulin recognized by the antibodies is present in axonemes from a variety of sources and may be necessary for axoneme formation, it is not found exclusively in any one subset of morphologically distinct axonemal microtubules. The antigen was found in similar proportions in fractions from sea urchin sperm axonemes enriched for central pair or outer doublet B or outer doublet A microtubules. Therefore the acetylation of alpha-tubulin does not provide the mechanism that specifies the structure of any one class of axonemal microtubules. Preliminary evidence indicates that acetylated alpha-tubulin is not restricted to the axoneme. The antibodies described in this report may allow us to deduce the role of tubulin acetylation in the structure and function of microtubules in vivo.     

Sudan Black B: Chemical structure and histochemistry of the blue main components

Summary Sudan Black B contains two blue main components, SSB-I and SSB-II. Their chemical structures were determinated by the aid of twodimensional thin-layer chromatography, column chromatography, absorption, IR, mass, H¹-NMR, and C¹³-NMR spectroscopy and were proved by alternate synthesis. SSB-I has been found to be 2,3-dihydro-2,2-dimethyl-4-[(4-phenylazo-l-naphthalenyl)-azo]-lH-perimidine. For SSB-II was confirmed the known structure 2,3-dihydro-2,2-dimethyl-6-[(4-phenylazo-l-naphthalenyl)-azo]-lH-perimidine. Relations of chemical structure of SSB-I and SSB-II to their staining properties are discussed.Professor Dr. G. Holle on the occasion on his 65th birthday     

Role of pyruvate and S-adenosylmethioine in activating the pyruvate formate-lyase of Escherichia coli

The pyruvate formate-lyase activity of extracts of Escherichia coli is stimulated and the dilution effect is abolished by the addition of pyruvate to the extract. The activity can be purified fourfold from pyruvate-supplemented extracts by isoelectric precipitation under anaerobic conditions. The activity of extracts not supplemented with pyruvate has been separated into two fractions by treatment with protamine sulfate-fraction PS, the soluble portion, and fraction N, an extract of the precipitate formed upon the addition of protamine sulfate. After treatment of these fractions with charcoal, pyruvate formate-lyase activity is stimulated by the addition of S-adenosylmethionine. When sodium pyruvate is added to the crude extract before the fractionation, fraction PS has full enzymatic activity and is not stimulated by fraction N or by S-adenosylmethionine. Incubation of the inactive fractions with pyruvate and S-adenosylmethionine in the absence of other substrates similarly results in a highly active preparation, not subject to the "dilution effect" obtained when the fractions are added separately to the assay. These observations suggest that the component in the protamine supernatant fraction is activated by the other fraction and that S-adenosylmethionine and pyruvate are required for the activation reaction. The activating factor present in the protamine precipitate fraction may be further purified by heating for 10 min at 100 C under H(2) atmosphere. The yield of this factor from crude extract is not affected by activation of the pyruvate formate-lyase of the extract, indicating that the factor acts catalytically. The requirement for pyruvate is only partially satisfied by alpha-ketobutyrate and not at all by other alpha-keto acids, acetyl phosphate, or adenosine triphosphate. The rate of activation is maximal at 0.01 m sodium pyruvate and 3 x 10(-4)mS-adenosylmethionine; it is linearly dependent on the amount of activating factor added. The rate of activation is the same when the activation reaction is initiated by addition of any of the four required components, indicating that no slow step of activation can be carried out by any three of the components. A similar pyruvate formate-lyase system was found in extracts of the methionine/B(12) autotroph 113-3, grown with methionine supplement, indicating that vitamin B(12) derivatives do not participate in the system.     

Isolation, identification, and characterization of histones from plasmodia of the true slime mold Physarum polycephalum using extraction with guanidine hydrochloride

Histones from plasmodia of the true slime mold Physarum polycephalum have been prepared free of slime by an approach to histone isolation that uses extraction of nuclei with 40% guanidine hydrochloride and chromatography of the extract on Bio-Rex 70. This procedure followed by chromatography or electrophoresis has been used to obtain pure fractions of histones from Physarum microplasmodia. Physarum microplasmodia have five major histone fractions, and we show by amino acid analysis, apparent molecular weight on three gel systems containing sodium dodecyl sulfate, mobility on gels containing Triton X-100, and other characterizations that these fractions are analogous to mammalian histones H1, H2A, H2B, H3, and H4. Significant differences between Physarum and mammalian histones are noted, with histone H1 showing by far the greatest variation. Histones H1 and H4 from Physarum microplasmodia have similar, but not identical, products of partial chymotryptic digestion compared with those of calf thymus histones H1 and H4. Labeling experiments, in vivo, showed that histone H1 is the major phosphorylated histone and approximately 15 separate phosphopeptides are present in a tryptic digest of Physarum histone H1. The core histones from Physarum, histones H2A, H2B, H3, and H4, are rapidly acetylated; histone H4 shows five subfractions, analogous to the five subfractions of mammalian histone H4 (containing zero to four acetyllysine residues per molecule); histone H3 has a more complex pattern that we interpret as zero to four acetyllysine residues on each of two sequence variants of histone H3; histones H2A and H2B show less heterogeneity. Overall, the data show that Physarum microplasmodia have a set of histones that is closely analogous to mammalian histones.     

Characterization and metabolic fate of two very-low-density lipoprotein subfractions separated by heparin-sepharose chromatography

Human very-low-density lipoproteins (VLDL) have been separated into two discrete subfractions by heparin-Sepharose chromatography. The retained fraction relative to the unretained fraction is characterized by an increased cholesterol ester/triacylglycerol ratio and an increased ratio of apolipoprotein E relative to apolipoprotein C. We have subfractionated VLDL from type IV hyperlipoproteinemic subjects and characterized these subfractions with respect to (i) composition and (ii) the metabolic fate of apolipoprotein B of each subfraction. The unretained fraction accounted for an average of 42% of total VLDL in type IV subjects. A similar distribution was obtained with VLDL from Type III subjects; however, only 25% of normal VLDL is in the unretained fraction. The apolipoprotein E/apolipoprotein C ratio was 2-8-fold higher in the retained fraction. The distribution of apolipoprotein E isomorphs and the individual C apolipoproteins were similar in each fraction. Retained and unretained fractions were labelled with 125I and/or 131I and injected simultaneously into miniature pigs. Apolipoprotein B of retained fractions was catabolized at a greater rate (fractional catabolic rate = 0.98 h-1 vs. 0.54 h-1, n = 7, P less than 0.05) compared to unretained fractions. These results are consistent with the concept that reduced content of C apolipoproteins in VLDL is correlated with enhanced uptake by perfused rat livers. Apolipoprotein B from retained fractions was converted to intermediate-density lipoproteins (IDL) at a greater rate, and apolipoprotein B from both fractions were converted to low-density lipoproteins (LDL). Although the unretained fraction may be the precursor of the retained fraction, the possibility exists that each fraction is largely synthesized and catabolized independently.     

Identification of aldosterone metabolites formed by A6 cells

The A6 cell line of the toad kidney is well known to form an Na+ transporting tight epithelium in culture and is often used as an experimental model for Na+ transport systems. Although it has been shown that A6 cells can convert aldosterone to polar metabolites, these metabolites have not been identified. Therefore, in this study, we tried to identify the metabolites of aldosterone formed by A6 cells in culture. A6 cells at confluence were incubated with serum-free culture media containing [3H]aldosterone. When radioactive compounds in incubation media were separated by reversed phase high-pressure liquid chromatography (HPLC), four fractions (fractions A-D) were obtained. Fraction A, a mixture of two components, comprised the majority of metabolites formed. The more polar material (fraction A-1) and the less polar material (fraction A-2) of fraction A contained 47-71 and 9-19% of total radioactivity, respectively. When incubated in cell-free media, fraction A-2 was found to be unstable and partially converted to fraction A-1. Fraction B, 0.7-1.5% of total radioactivity, and fraction C, 8-21% of total radioactivity, cochromatographed with iso-aldosterone and D-aldosterone, respectively. Fraction D, 4-8% of total radioactivity, was a mixture of two components, which cochromatographed with 3 beta,5 beta-tetrahydroaldosterone and 5 alpha-dihydroaldosterone, respectively. In order to identify fraction A-2 material, large-scale cultures were performed and fraction A-2 was separated and purified by reversed phase HPLC. The purified material was analyzed by fast atom bombardment mass spectrometry and nuclear magnetic resonance spectroscopy. These two procedures unambiguously revealed that this material was 6 beta-hydroxyaldosterone. These results demonstrate that aldosterone can be converted to at least four metabolites by the incubation with A6 cells, and that major metabolites are polar compounds, a portion of which is 6 beta-hydroxyaldosterone.     

A 174-kilodalton ATPase/dATPase polypeptide and a glycoprotein of apparently identical molecular weight are common but distinct components of higher eukaryotic nuclear structural protein subfractions

A 174-kilodalton polypeptide of the Drosophila nuclear matrix-pore complex-lamina fraction has been identified as an ATPase/dATPase on the basis of direct UV photoaffinity labeling studies; a polypeptide with similar properties has been found in nuclear envelope fractions prepared from several vertebrate sources (Berrios, M., Blobel, G., and Fisher, P. A. (1983) J. Biol. Chem. 258, 4548-4555). This ATPase/dATPase polypeptide co-migrates on sodium dodecyl sulfate (SDS)-polyacrylamide gels with a glycoprotein also found in all of these fractions. In rat liver, this glycoprotein has been localized to the nuclear pore complex by means of immunoelectron microscopy (Gerace, L., Ottaviano, Y., and Kondor-Koch, C. (1982) J. Cell Biol. 95, 826-837). Following SDS denaturation and reduction/alkylation, chromatography of the Drosophila nuclear matrix-pore complex-lamina fraction on hydroxylapatite columns run in the presence of SDS results in the separation of two quantitatively major 174-kilodalton polypeptides. The peak of glycoprotein elution from the SDS-hydroxylapatite column correlates exactly with that of the early eluting 174-kilodalton species while the photolabeled ATPase/dATPase polypeptide co-chromatographs with the late eluting one. Identical results have been obtained with the rat liver nuclear envelope fraction. The chromatographically separated 174-kilodalton species from both organisms have been further distinguished through the use of polypeptide-specific antisera; finally, the glycoprotein purified from Drosophila embryos is fully sensitive to limited degradation by endoglycosidase H whereas the ATPase/dATPase polypeptide is completely resistant. We have thus established, using material obtained from two widely divergent higher eukaryotes, that the 174-kilodalton ATPase/dATPase is a quantitatively major nuclear matrix-pore complex-lamina component distinct from the nuclear pore complex glycoprotein of apparently identical molecular weight.     

Comparison of the crystallin mRNA populations from rat, calf and duck lens. Evidence for a longer alpha A2-mRNA and two distinct alpha B2-mRNAs in the birds

Total cytoplasmic poly(A)-containing RNA from rat, calf and duck lens was fractionated by electrophoresis in methylmercury hydroxide-containing agarose gels. RNA electrophoresed in parallel lanes was either transferred onto nitrocellulose and hybridized with total cDNA synthesized on the initial mRNA or was recovered from individual gel fractions for in vitro translation in a reticulocyte cell-free system. This allowed the identification and size-characterization of individual mRNA species encoding alpha-, beta-, gamma- and delta-crystallin polypeptides. The 14 S mRNA fraction of rat lens comprises two alpha A2-mRNAs of approximately 1250 and 1350 nucleotides and the alpha AIns-mRNA with a size similar to that of the largest alpha A2-mRNA. The calf lens 14 S mRNA fraction harbors a heterogeneous population of alpha A2-mRNA. In the same fraction another mRNA encoding a polypeptide, designated X, has been found sharing no homology with alpha A sequences. The duck lens alpha A2-mRNA appears to be 400-450 bases longer than the rat and calf lens alpha A2-mRNAs. Furthermore, in contrast to the single alpha B2-mRNA in rat and calf lens, two alpha B2-mRNAs have been identified in duck lens, one, the major species, similar in size to the alpha B2-mRNA in rat and calf lens (800 bases), and the other species 700 nucleotides longer. The large size differences among the alpha A2- and alpha B2-mRNAs most likely reside in their 3'-untranslated sequences.     

A periplasm in Bacillus subtilis.

The possibility of there being a periplasm in Bacillus subtilis, in the distinct cell compartment bounded by the cytoplasmic membrane and the thick cell wall, has been investigated quantitatively and qualitatively. Cytoplasmic, membrane, and protoplast supernatant fractions were obtained from protoplasts which were prepared isotonically from cells grown under phosphate limitation. The contents of the protoplast supernatant fraction represent an operational definition of the periplasm. In addition, this cell fraction includes cell wall-bound proteins, exoproteins in transit, and contaminating cytoplasmic proteins arising through leakage from, or lysis of a fraction of, protoplasts. The latter, measured by assay of enzyme markers and by radiolabeled RNA and protein, was found to represent 7.6% of total cell protein, yielding a mean of 9.8% +/- 4.8% for B. subtilis 168 protein considered periplasmic. Qualitatively, after subjection of all cell fractions to polyacrylamide gel electrophoresis, RNase and DNase, zymographs revealed that (i) each cell fraction had a unique profile of nucleases and (ii) multiple species and a major fraction of both nucleases were concentrated in the periplasm. We conclude that the operationally defined periplasmic fraction corresponds closely, both quantitatively and qualitatively, to the contents of the periplasm of Escherichia coli. We discuss evidence that the maintenance of the components of this surface compartment in B. subtilis is compatible with the thick negatively charged cell wall acting as an external permeability barrier.     

Quenching autofluorescence in the alimentary canal tissues of Bactericera cockerelli (Hemiptera: Triozidae) for immunofluorescence labeling

Immunofluorescence has been widely used to localize microbes or specific molecules in insect tissues or cells. However, significant autofluorescence is frequently observed in tissues which can interfere with the fluorescent identification of target antigens, leading to inaccurate or even false positive fluorescent labeling. The alimentary canal of the potato psyllid, Bactericera cockerelli ?ulc, exhibits intense autofluorescence, hindering the application of immunolocalization for the detection and localization of the economically important pathogen transmitted by this insect, “Candidatus Liberibacter solanacearum” (Lso). In the present study, we tested the use of irradiation, hydrogen peroxide (H₂O₂) and Sudan black B (SBB) treatments to reduce the autofluorescence in the B. cockerelli alimentary canal tissues. Furthermore, we assessed the compatibility of the above‐mentioned treatments with Lso immunolocalization and actin staining using phalloidin. Our results showed that the autofluorescence in the alimentary canal was reduced by irradiation, H₂O₂, or SBB treatments. The compatibility assays indicated that irradiation and H₂O₂ treatment both greatly reduced the fluorescent signal associated with Lso and actin. However, the SBB incubation preserved those target signals, while efficiently eliminating autofluorescence in the psyllid alimentary canal. Therefore, herein we propose a robust method for reducing the autofluorescence in the B. cockerelli alimentary canal with SBB treatment, which may improve the use of immunofluorescence labeling in this organism. This method may also have a wide range of uses by reducing the autofluorescence in other arthropod species.     

Oil-soluble dyes for marking Spodoptera frugiperda (Lepidoptera: Noctuidae)

Although various biological aspects of Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae) have been examined, adult movement and dispersal of this insect pest is not well understood. Release-recapture techniques by using marked insects is a useful approach for dispersal studies; however, the marking technique should not significantly affect insect biology or behavior. Therefore, the effect of different concentrations of oil-soluble dyes (Solvent Blue 35 [C.I. 61554], Sudan Red 7B [C.I. 26050], Sudan Black B [26150], Sudan Orange G [C.I. 11920], and Sudan I 103624 [C.I. 12055]) on development, mortality, and fecundity of S. frugiperda was evaluated. Dyes were added to artificial diet used to feed larvae. Larval and pupal development and mortality, adult longevity, and female fecundity were evaluated. High concentrations (400 and 600 ppm) of all dyes led to longer larval and pupal stages. Adult life span and number of eggs were not affected by the dyes. Sudan Red 7B marked both adults and eggs very well. Solvent Blue 35 marked both adults and eggs, but the blue-marked eggs could not be distinguished from some bluish eggs laid by nonlabeled females. Adults and eggs were not adequately marked by the Sudan Black B, Sudan Orange G, and Sudan I 103624 (a yellow dye).