Cochineal Research Articles

Molecular cloning, expression, and characterization of a major 38-kd cochineal allergen

Carmine is a natural red pigment obtained from dried gravid female cochineal insects (Dactylopius coccus or Coccus cacti). There have been several reports of allergies to carmine, but the major allergens responsible have not been identified. To identify the major allergenic proteins in cochineal. Immunoblots of purified cochineal extract were probed with sera from 3 patients with allergy. Partial amino acid sequences were determined for the proteins bound by IgE, and the corresponding cDNA, containing a complete coding region, was cloned by 5' and 3' rapid cDNA extension and PCR. The recombinant protein was expressed in yeast and subjected to immunoblotting. We identified a full-length cDNA encoding a protein, which we named CC38K, with 335 amino acids and a molecular mass calculated as 38 kd. This amino acid sequence included all the partial amino acid sequences obtained from the purified proteins identified by IgE from patients with allergy. Recombinant CC38K protein was recognized by patients' sera, indicating that this is a major allergen present in carmine. The CC38K sequence showed homology to phospholipases. We have, for the first time, identified the major allergen in cochineal extract. This protein may be a phospholipase or related enzyme, both of which are known to be allergens in other insects.

The biology ofDactylopius tomentosus(Hemiptera: Dactylopiidae)

Dactylopius tomentosus (Lamarck) (Hemiptera: Dactylopiidae) is a cochineal insect whose host range is restricted to Cylindropuntia species (Caryophyllales: Cactaceae). This insect has been utilized successfully for biological control of Cylindropuntia imbricata (Haw.) F.M. Knuth in Australia and South Africa. Despite this, its biology has not been studied previously, probably due to the widely held belief that the biology of all Dactylopius species is similar. This study investigated the life cycle and the morphological and reproductive characteristics of D. tomentosus. Results revealed some unique characteristics of D. tomentosus: (i) eggs undergo a much longer incubation period, an average of 17 days compared to <1 day in its congeners; (ii) eggs are laid singly but are retained as an egg mass secured in a mesh of waxy threads attached to the female; (iii) the developmental times of males and females are longer compared to other Dactylopius spp. due to a longer egg incubation period; (iv) D. tomentosus does not undergo parthenogenesis; (v) D. tomentosus is smaller in size than its congeners; and (vi) male mating capacity and reproductive potential were both high and variable between males. There was a significant, strong, positive relationship (r = 0.93) between female mass and fecundity, whereas the relationship between the number of females mated per male that became gravid and their fecundity was negative (r = -0.68). Besides contributing to our knowledge of this economically important species, the finding of unique characteristics of D. tomentosus biology underlines the need to study each species in this genus.

Carmine hypersensitivity masquerading as azithromycin hypersensitivity

Macrolide hypersensitivity is a rarely reported event. However, carmine dye has become increasingly important as a provocative agent. We present a case of a woman with documented carmine hypersensitivity, who reported anaphylaxis 90 minutes after ingestion of a generic azithromycin. Our investigations revealed that this was an allergy to the carmine dye in the tablet's coating rather than to the antibiotic. Seven extracts were prepared including carmine dye, crushed dried female cochineal insects, crushed tablets of Zithromax (Pfizer Inc.) and generic azithromycin (Teva Pharmaceuticals), and the crushed colored coatings from both tablets. These were suspended in preservative-free normal saline, and then applied as a skin-prick test and read at 30 minutes. The skin-prick skin test results were 4+ to histamine and carmine dye, but negative to cochineal insect extract, Pfizer crushed tablets, and negative control. The patient was 1+ to the Teva crushed tablet, but was 4+ to the Teva brand coating and negative to the Pfizer brand coating, which did not contain carmine. The patient subsequently ingested Pfizer Zithromax without any sequelae. To our knowledge, this is the first reported case of carmine anaphylaxis attributed to carmine-containing medication. Careful history and skin-prick testing to the appropriate agents allowed elucidation of the subtlety of the true offending agent without unnecessary avoidance of the medication class. Patients with a carmine hypersensitivity should actively check with their pharmacy or prescribing physician to verify their medications are free of this offending agent.

Ovariole Structure of the Cochineal Scale Insect,Dactylophis coccus

The ovaries of the adult cochineal scale insect, Dactylopius coccus Costa (Hemiptera: Coccoidea: Dactylopiidae) are made up of more than 400 short ovarioles of the telotrophic type. The ovarioles develop asynchronously. The ovarioles consist of a germarium with six or seven nurse cells, a vitellarium with an oocyte, and pedicel. A terminal filament is lacking. A maturing oocyte was attached to the trophic core by the trophic cord during previtellogenesis and most of vitellogenesis.

Towards the identification of characteristic minor components from textiles dyed with weld (Reseda luteola L.) and those dyed with Mexican cochineal (Dactylopius coccus Costa)

The identity of a minor flavonoid component observed in extracts of textile samples dyed with weld (Reseda luteola L.) is confirmed as chrysoeriol (3′-O-methylluteolin) by HPLC-PDA analysis. HPLC-PDA, HPLC-MS and NMR techniques have been used to show that the unknown dcII component found in extracts of textiles dyed with Mexican cochineal (Dactylopius coccus Costa) is most probably the 7-C-glycoside of flavokermesic acid. In addition, the unknown dcIV and dcVII components have been shown to be isomeric with carminic acid, most probably differing only in the stereochemistry of the sugar moiety.

Free radical generation during the activation of hemolymph prepared from the homopteran Dactylopius coccus

Superoxide anion (O(-) (2)) and nitric oxide (NO) generation in Dactylopius coccus hemolymph obtained by perfusion and activated with zymosan was studied. Activated hemolymph reduced 3-[4,5 dimethylthiazolil-2]-2,5-diphenyl tetrazolium bromide. This reduction was prevented by superoxide dismutase (SOD) indicating O(-) (2) generation. This activity was dependent on temperature, and hemolymph incubated at 75 degrees C lost its activity. Chromatocytes incubated with zymosan released their content and produced O(-) (2). Activated hemolymph also produced NO and this activity was prevented in the presence of NG-nitro-L-arginine methyl ester, suggesting that nitric oxide synthase (NOS) might be present in D. coccus hemolymph. The probable source of O(-) (2) in the D. coccus hemolymph is the anthraquinone oxidation, since commercial carminic dye produced O(-) (2) during its oxidation by Agaricus bisporus tyrosinase. Gram+ Micrococcus luteus exposed to activated hemolymph were killed in vitro, and addition of NG-nitro-L-arginine methyl ester and D-Mannitol (a hydroxyl radical scavenger) prevented their killing. The cytotoxic effect produced by the activated hemolymph was not observed with the Gram- bacteria Serratia marcescens. These results suggest that D. coccus activated hemolymph generates reactive oxygen intermediates (ROI) and reactive nitrogen intermediates (RNI) that may limit M. luteus growth.

Local identification and valuation of ecosystem goods and services from Opuntia scrublands of Ayacucho, Peru

Opuntia scrublands are important ecological–economic systems in the Andean area. They perform a major role protecting slopes against erosion, improving the soil properties and providing a variety of products employed in the human diet, and in animal feeding, as well as cochineal insects, a highly valued source of dyes. The collection of the insects has represented an important economic activity for the local communities since pre-Columbian times. Current Peruvian production represents between 85% and 90% of the global market, and is mainly based on collection of the insect in natural Opuntia scrublands located in the poorest Andean areas of Ayacucho. Although much is known about the financial benefits of cochineal for exporters and dye manufacturers, information about the value of standing Opuntia scrubs to collectors and the relative contribution of Opuntia scrubs to their household economies is scarce. Here we contribute to the estimation of the use value of Opuntia scrublands to local communities in Ayacucho by initially exploring the cultural domain of Opuntia in order to identify the ecosystem goods and services recognized by peasants, and later presenting empirical estimates of their importance to annual household income.

Mate searching in the scale insect, Dactylopius coccus (Hemiptera: Coccoidea: Dactylopiidae)

Dactylopius coccus is a sessile scale insect living on cladodes of Opuntia ficus indica which has commercial importance as a source of carminic acid. We herein present behavioural evidence of the existence of a sex pheromone in D. coccus, based on olfactometric experiments using both biological sources of odours and collected chemical extracts.

Evaluation of Three Varieties of Opuntia Ficus-Indica (L.) Miller as Hosts of the Cochineal Insect Dactylopius Coccus Costa (Homoptera: Dactylopiidae) in a Semiarid Area of Northeastern Mexico

Three varieties of prickly pear Opuntia ficus-indica (Villanueva, Copena, and Jalpa varieties) were evaluated in their capacity to be host of the cochineal insect (Dactylopius coccus Costa) in two localities of northeastern Mexico. The experiment was carried out for two seasons, winter and summer, in 1998. Variables under evaluation were: fresh weight of grana, dry weight of grana, and number of insects. The Villanueva variety showed the highest production for the three variables and there were no differences between the other two varieties. Iturbide locality was superior to Linares for the three variables. Higher production (both dry and fresh weight of grana) was obtained during winter and there were no differences between seasons for number of insects. Figures obtained for the Villanueva variety are slightly higher than those reported in literature for the most productive cochineal areas in Mexico.

Land clearance and social capital in mountain agro–ecosystems: the case of Opuntia scrubland in Ayacucho, Peru

Opuntia scrublands are important ecological–economic systems in rural Andean areas. They provide goods for peasants' diet and animal feed, as well as cochineal insects, a highly valued source of dyes. Land clearance on these scrublands promotes changes in land use, from nonproductive wilderness to cochineal and fruit harvest areas, grazing lands, and fuel–wood supply zones. Here we empirically explore the role of social capital on land clearance, based on a case study of the Humanga region of Ayacucho, Peru. The literature on social capital is showing evidence of the positive effect of social capital on development actions and on environmental conservation in rural areas of developing countries such as participatory forest management. Here, the notion of social capital is captured by the idea that some features of social organization, such as networks, facilitate coordination and cooperation within the peasant communities that help in managing different economic activities, both on- and off-farm. The habilitation of the scrublands in the Andean mountain environment could be generating negative externalities as a consequence of altering the vegetation cover such as soil erosion. Because the benefits arising from land clearance activities are obtained by cochineal collecting households and the negative environmental effects are costs imposed upon noncochineal collectors, the social capital associated with scrubland habilitation could be perverse from an agro–environmental viewpoint and a revision of some government policies and of some NGOs actions for rural development through the promotion of various community networks in this deprived area could be needed.

Land clearance and social capital in mountain agro?ecosystems: the case of Opuntia scrubland in Ayacucho, Peru*1

Opuntia scrublands are important ecological–economic systems in rural Andean areas. They provide goods for peasants' diet and animal feed, as well as cochineal insects, a highly valued source of dyes. Land clearance on these scrublands promotes changes in land use, from nonproductive wilderness to cochineal and fruit harvest areas, grazing lands, and fuel–wood supply zones. Here we empirically explore the role of social capital on land clearance, based on a case study of the Humanga region of Ayacucho, Peru. The literature on social capital is showing evidence of the positive effect of social capital on development actions and on environmental conservation in rural areas of developing countries such as participatory forest management. Here, the notion of social capital is captured by the idea that some features of social organization, such as networks, facilitate coordination and cooperation within the peasant communities that help in managing different economic activities, both on- and off-farm. The habilitation of the scrublands in the Andean mountain environment could be generating negative externalities as a consequence of altering the vegetation cover such as soil erosion. Because the benefits arising from land clearance activities are obtained by cochineal collecting households and the negative environmental effects are costs imposed upon noncochineal collectors, the social capital associated with scrubland habilitation could be perverse from an agro–environmental viewpoint and a revision of some government policies and of some NGOs actions for rural development through the promotion of various community networks in this deprived area could be needed.

Color Quality of Pigments in Cochineals (Dactylopius coccusCosta). Geographical Origin Characterization Using Multivariate Statistical Analysis

The commercial value of a cochineal (Dactylopius coccus Costa) sample is associated with its color quality. Because the cochineal is a legal food colorant, its color quality is generally understood as its pigment content. Simply put, the higher this content, the more valuable the sample is to the market. In an effort to devise a way to measure the color quality of a cochineal, the present study evaluates different parameters of color measurement such as chromatic attributes (L*, and a*), percentage of carminic acid, tint determination, and chromatographic profile of pigments. Tint determination did not achieve this objective because this parameter does not correlate with carminic acid content. On the other hand, carminic acid showed a highly significant correlation (r = - 0.922, p = 0.000) with L* values determined from powdered cochineal samples. The combination of the information from the spectrophotometric determination of carminic acid with that of the pigment profile acquired by liquid chromatography (LC) and the composition of the red and yellow pigment groups, also acquired by LC, enables greater accuracy in judging the quality of the final sample. As a result of this study, it was possible to achieve the separation of cochineal samples according to geographical origin using two statistical techniques: cluster analysis and principal component analysis.

Detection of colour adulteration in cochineals by spectrophotometric determination of yellow and red pigment groups

A rapid method was developed for the determination of yellow and red pigment groups in cochineals, a natural food colourant. The method was based on UV–visible spectrophotometric measurement at two characteristic wavelengths. The wavelengths 420 and 500 nm were selected as the most appropriate for simultaneous quantification of these pigment groups from a cochineal extract in methanol:water (65:35, v:v). Determination of the mean absorption coefficients for the yellow pigment group, at 420 nm, and the red pigment group, at 500 nm, yielded equations that serve to calculate the concentration of the two pigment groups (C420 and C500, respectively) and the ratio between them (C420/C500). Because the ratio between pigment groups can be considered constant (comprised between 0.93 and 1.10), this parameter can be used for quality control. Fluctuations outside the set limits may indicate that the colour of the cochineal has been enhanced by colourants foreign to the raw material. A study of adulterations showed the capacity of the proposed method to detect these adulterations. There were no significant differences when comparing the determination of the pigment groups by the proposed spectrophotometric method with the results obtained by liquid chromatography.

Carminic acid dye from the homopteran Dactylopius coccus hemolymph is consumed during treatment with different microbial elicitors.

The activation of Dactylopius coccus (Costa) hemolymph with microbial polysaccharide molecules was studied. Hemolymph incubated in the presence of laminarin, zymosan, and N-acetyl glucosamine produced a dark fibrillar precipitated, and the red pigment (carminic acid) was consumed (measured spectrophotometrically at 495 nm). Lipopolysaccharide (LPS) did not induce any response. The reaction was inhibited with millimolar concentrations of serine and cysteine protease inhibitors, EGTA and phenyl thiourea. It was also diminished by prostaglandin synthesis inhibitors: dexamethasone, acetylsalicylic acid, and indomethacin. However, Mg2+ chelator EDTA did not inhibit hemolymph activation. Hemolymph proteins were depleted from soluble phase during treatment with laminarin, but a group of around 34 kDa remained unmodified. These results showed that D. coccus hemolymph is activated by microbial elicitors, its activation depends on eicosanoids, and suggest participation of a prophenoloxidase (PPO)-like activation system that could consume carminic acid. We are currently dissecting the molecular factors involved in D. coccus hemolymph activation to determine homologies and differences with other arthropods immune response pathways.

Removal of ammonium and phosphates from wastewater resulting from the process of cochineal extraction using MgO-containing by-product

The wastewater produced by the cochineal extract process to obtain the carminic acid colouring pigment (carmin red E120) has high concentrations of phosphates and ammonium. It is known that both ions can be precipitated with magnesium in the form of struvite, MgNH 4PO 4, or ammonium magnesium phosphate (MAP) compounds. In this study, the use of an alternative MgO-containing by-product is investigated. The optimal pH, reaction time and solid/liquid ratio have been studied. It has been found that the low-grade MgO needed is greater than the stoichiometric value for the full removal of ammonium and phosphate as MAP compounds. Although the low-grade MgO (LG-MgO) reacts slower than pure MgO, it has considerable economic advantages. A batch process has been proposed for the removal of ammonium and phosphates from wastewater obtained in cochineal extracts processing, previously to biological treatment to diminish the COD.

Asma y alergia por el colorante carmín

Cochineal carmine, or simply carmine (E120), is a red colouring that is obtained from the dried bodies of the female insect Dactylopius Coccus Costa (the cochineal insect). We have evaluated the prevalence of sensitization and asthma caused by carmine in a factory using natural colouring, following the diagnosis of two workers with occupational asthma. The accumulated incidence of sensitization and occupational asthma due to carmine in this factory are 48.1% and 18.5% respectively, figures that make the introduction of preventive measures obligatory. Occupational asthma caused by inhaling carmine should be considered as a further example of the capacity of certain protein particles of arthropods (in this case cochineal insects) to act as aeroallergens. Carmine should be added to the list of agents capable of producing occupational asthma, whose mechanism, according to our studies, would be immunological mediated by IgE antibodies in the face of diverse allergens of high molecular weight, which can vary from patient to patient. Nonetheless, given the existence of different components in carmine, it cannot be ruled out that substances of low molecular weight, such as carminic acid, might act as haptenes. Besides, since we are dealing with a colouring that is widely used as a food additive, as a pharmaceutical excipient and in the composition of numerous cosmetics, it is not surprising that allergic reactions can appear both through ingestion and through direct cutaneous contact. We find ourselves facing a new example of an allergen that can act through both inhalation and digestion, giving rise to an allergolical syndrome that can show itself clinically with expressions of both respiratory allergy and alimentary allergy.

Biological control of cactus weeds: implications of hybridization between control agent biotypes

Summary Results of recent research on Dactylopius opuntiae, a biological control agent for cactus weeds (Opuntia spp.) in South Africa and elsewhere, challenge the maxim that genetic diversity of agents necessarily enhances the chances of success in biological weed control. Two biotypes of D. opuntiae, each specific to a different Opuntia species, interbred freely, at least under insectary conditions. We therefore carried out cross‐breeding experiments to determine the viability and host‐preferences of progeny produced by these crosses. Unlike their parents, F1 hybrids were not species‐specific, developing equally well on either of the parental hosts, Opuntia ficus‐indica and Opuntia stricta. The situation was more complex in F2 back‐crosses between hybrids and in crosses between parent strains and hybrids because male cochineal insects contributed only maternally inherited genes to their progeny, due to their unusual haploid‐diploid (lecanoid) mechanism of sex determination. Some F2 combinations produced cohorts of progeny that were either entirely true‐bred (i.e. host‐specific) or entirely hybrid (i.e. not host‐specific) genotypes, while other combinations produced groups of siblings with some individuals (theoretically half) that were true‐bred genotypes and the balance were hybrid genotypes. The lack of host‐specificity of hybrids should enhance overall biological control of the target species directly, because hybrids attack both host‐plants, and indirectly, because hybrid nymphs have greater chances of finding a suitable host‐during passive dispersal. However, this advantage will be negated when F2 crosses produce host‐specific nymphs on host‐plants that are incompatible for their survival. These findings show that only pure strains of D. opuntiae should be released in monocultures of the target weeds. More generally, they caution that the possible consequences of mixing genotypes of a biological control agent species should be investigated before different provenances are amalgamated to enhance genetic diversity.

Optimizing conditions for the extraction of pigments in cochineals (Dactylopius coccus Costa) using response surface methodology.

A simple method was developed for the extraction and determination of color pigments in cochineals (Dactylopius coccus Costa). The procedure was based on the solvent extraction of pigments in insect samples using methanol:water (65:35, v:v) as extractant. Two-level factorial design was used in order to optimize the solvent extraction parameters: temperature, time, methanol concentration in the extractant mixture, and the number of extractions. The results suggest that the number of extractions is statistically the most significant factor. The separation and determination of the pigments was carried out by high-performance liquid chromatography with UV-visible detection. Because the absorption spectra of different pigments are different in the visible region, it is convenient to use a diode array detector to obtain chromatographic profiles that allow for the characterization of the extracted pigments.

Use of Live Fences of Nopal (Opuntia) and Associated Crops to Rehabilitate and Protect Sloping Land in Loja, Ecuador

A live fences project in Ecuador sought to associate the idea of environmental recovery, characterized by an agro-ecological focus, with a perspective on social and economic development. Cultivation of the Opuntia cactus and the cochineal insect (Dactylopus coccus), environmentally and culturally adapted to the region, permitted the recovery of several degraded areas and generated income for rural dwellers, especially during periods of drought. Among the most important project impacts were: recovery of traditional knowledge, cultural values, ancestral skills, and inveterate attachment to communal properties; determination of the ecotones of the cactus for production of cochineal, fruits, forage, and live barriers; recovery of areas eroded by overexploitation and inadequate management; increased sensitivity among political leaders regarding the problems of desertification and the need to support a second phase of the project; decision-making by community-based organizations; and commitment of the community to the activities of the project, based on agreements between authorities, academia, and the community. In 1999, the project was honored with the “Saving the Drylands” Award given by the United Nations Environment Programme (UNEP) at Recife, Brazil, during the Third Conference of Parties (COP 3) of the Convention to Combat Desertification (CCD).

Preventive effects of anthraquinone food pigments on the DNA damage induced by carcinogens in Drosophila

We have previously demonstrated the inhibitory effect of chlorophyllin, a green food additive, on the genotoxicities of various carcinogens in Drosophila. Recently, we reported that purpurin, a component of a red food additive produced from madder root ( Rubia tinctorium), inhibits the bacterial mutagenicity of heterocyclic amines. In the present study, we examined antigenotoxic activities of various pigments that are either constituents of food or food additives, using Drosophila in vivo DNA repair assay. Third instar larvae of Drosophila were fed a mutagen with or without pigment. The resulting adult flies were monitored for their male (repair deficient)/female (repair proficient) ratios, which reflect the DNA damage. We tested a total of 20 pigments, which are mainly of plant origins, including flavonoids, carotenoids, anthocyanins, anthraquinones and β-diketone (curcumin)-derivatives, against the genotoxicities of eight carcinogens; IQ, MeIQx, AFB1, NDMA, 2-AAF, DMBA, 4NQO, and MNU. Four anthraquinone pigments (alizarin, purpurin, lac color, and cochineal extract) showed significant antigenotoxic activities. Alizarin and purpurin suppressed the DNA damage induced by IQ, MeIQx, AFB1, NDMA, 2-AAF, DMBA, and MNU. Lac color and cochineal extract showed inhibition against IQ, MeIQx, AFB1, 2-AAF and DMBA. In these inhibitions, suppression of metabolic enzymes may be involved. Since purpurin and alizarin suppressed the activity of MNU, a direct alkylating agent, there may also be a mechanism distinct from enzyme inhibitions in these anthraquinone-mediated suppressions of DNA damage.