Symbiotic streptomycetes in antennal glands of the South American digger wasp genus Trachypus (Hymenoptera,Crabronidae)

Several insect taxa have evolved symbioses with actinobacteria that protect the host or its nutritional resources against pathogens. Digger wasps of the genus Philanthus (‘beewolves’; Hymenoptera, Crabronidae) cultivate ‘Candidatus Streptomyces philanthi’ (Ca. S. philanthi) in specialized antennal glands and transfer them to subterranean brood cells, where the symbionts provide protection for the beewolf larva against pathogens by producing a cocktail of antibiotic substances. The present study investigates the occurrence of antennal symbionts in two species of the genus Trachypus, which is the closest relative to Philanthus. Cross sections of the antennae from females of both Trachypus denticollis and Trachypus boharti reveal bacteria‐containing gland reservoirs that are morphologically very similar to those found in Philanthus spp. Polymerase chain reaction‐based screens with specific primers, sequencing of the partial 16S rRNA gene, and fluorescence in situ hybridization confirm the presence of close relatives of ‘Ca. S. philanthi’ in T. denticollis and T. boharti. However, 16S rRNA sequence divergence between Trachypus spp. and Philanthus spp. symbionts is much higher for T. boharti than for T. denticollis, suggesting that horizontal transmission and/or de novo uptake of symbionts from the environment occurs occasionally. The results obtained indicate that the protective symbiosis with antennal actinobacteria is older and more widespread than previously recognized and occurs in at least two genera of digger wasps that comprise approximately 170 species.     

Food wrapping by females of the European Beewolf, Philanthus triangulum, retards water loss of larval provisions

Abstract.  Females of the European beewolf, Philanthus triangulum F. (Hymenoptera, Crabronidae), embalm the provisions of their larvae, paralyzed honeybees, with a secretion from a postpharyngeal gland prior to oviposition. This food wrapping is known to delay fungus infestation of the prey. In the present study, the hypothesis that the food wrapping has an additional function, namely the prevention of prey desiccation, is tested. Water loss of paralyzed but unembalmed honeybees and embalmed honeybees is measured and the composition and quantity of their cuticular hydrocarbons analyzed by coupled gas chromatography–mass spectrometry. Water loss is significantly lower in embalmed compared with unembalmed bees. This might have important advantages for the larvae under the warm and dry conditions that prevail in some brood cells. The embalming by beewolf females increases the total amount of hydrocarbons on the surface of the bees by almost ten-fold. Moreover, the proportion of unsaturated and short-chained hydrocarbons is significantly increased. Unsaturated and short-chain hydrocarbons are usually less effective against water loss, so the increased protection against water loss appears to be mediated mainly by the thickness of the hydrocarbon layer.     

Selective preying of the sphecid wasp Trachypus boharti on the meliponine bee Scaptotrigona postica: potential involvement of caste‐specific cuticular hydrocarbons

The specialist digger wasp Trachypus boharti Rubio‐Espina preys exclusively on males of the stingless bee Scaptotrigona postica Latreille 1807, although the hunting attacks involve both male and worker bees of S. postica and members of its own species. To understand the mechanism of prey selection, the cuticular hydrocarbon patterns of workers and males of S. postica are analyzed in detail, and the mandibular secretion of males is examined. The cuticular profiles of males and workers are distinctively different. The major group of cuticular compounds, heptacosene isomers, is twice as abundant in workers as in males. There is no clear distinction between worker and male mandibular secretions. Such a distinct and straightforward caste‐specific difference in cuticular hydrocarbons could function as a recognition cue by which T. boharti distinguishes between workers and males of S. postica.     

Structure, chemical composition and putative function of the postpharyngeal gland of the emerald cockroach wasp, Ampulex compressa (Hymenoptera, Ampulicidae)

The postpharyngeal gland (PPG) plays a major role in the social integration of ant colonies. It had been thought to be restricted to ants but was recently also described for a solitary wasp, the European beewolf (Philanthus triangulum). This finding posed the question whether the gland has evolved independently in the two taxa or has been inherited from a common ancestor and is hence homologous. The latter alternative would be supported if a PPG was found in more basal taxa. Therefore, we examined a species at the base of the Apoidea, the solitary ampulicid wasp Ampulex compressa, for the existence of a PPG. Both sexes of this species possess a cephalic gland that branches off the posterior part of the pharynx, is lined by a cuticular intima and surrounded by a monolayered epithelium with the epithelial cells bearing long hairs. Most of these morphological characteristics conform to those of the PPG of ants and beewolves. Chemical analysis of the gland content revealed that it contains mainly hydrocarbons and that there is a congruence of the pattern of hydrocarbons in the gland, on the cuticle, and in the hemolymph, as has also been reported for both ants and beewolves. Based on these morphological and chemical results we propose that the newly described cephalic gland is a PPG and discuss its possible function in A. compressa. The present study supports the view of a homologous origin of the PPG in the aculeate Hymenoptera.     

Is the postpharyngeal gland of a solitary digger wasp homologous to ants? Evidence from chemistry and physiology

The postpharyngeal gland (PPG) was thought to be restricted to ants where it serves a crucial function in the generation of the colony odour. Recently, head glands that closely resemble the PPG of ants were discovered in females of a solitary digger wasp, the European beewolf. The function of this gland necessarily differs from ants: beewolf females apply the secretion of their PPG onto the bodies of paralysed honeybees that serve as larval provisions in order to delay fungus growth. Since ants and digger wasps are not closely related, the occurrence of this gland in these two taxa might either be due to convergent evolution or it is a homologous organ inherited from a common ancestor. Here we test the hypothesis that the PPGs of both taxa are homologous by comparing characteristics of chemical composition and physiology of the PPG of beewolves and ants. Based on reported characteristics of the PPG content of ants, we tested three predictions that were all met. First, the PPG of beewolves contained mainly long-chain hydrocarbons and very few compounds with functional groups. Second, the composition of hydrocarbons in the beewolf PPG was similar to that of the hemolymph. Taking the structure of the gland epithelium and the huge requirements of beewolf females for gland secretion into account this result suggests that the content of the PPG is also sequestered from the hemolymph in beewolves. Third, the chemical composition of the PPG and the cuticle was similar in beewolves since cuticular hydrocarbons derive either from the hemolymph or the PPG. Taking the considerable morphological similarities into account, our results support the hypothesis of a homologous origin of the PPG in beewolves and ants.     

Phenylpropanoid glycoside analogues: enzymatic synthesis, antioxidant activity and theoretical study of their free radical scavenger mechanism

Phenylpropanoid glycosides (PPGs) are natural compounds present in several medicinal plants that have high antioxidant power and diverse biological activities. Because of their low content in plants (less than 5% w/w), several chemical synthetic routes to produce PPGs have been developed, but their synthesis is a time consuming process and the achieved yields are often low. In this study, an alternative and efficient two-step biosynthetic route to obtain natural PPG analogues is reported for the first time. Two galactosides were initially synthesized from vanillyl alcohol and homovanillyl alcohol by a transgalactosylation reaction catalyzed by Kluyveromyces lactis β-galactosidase in saturated lactose solutions with a 30%–35% yield. To synthesize PPGs, the galactoconjugates were esterified with saturated and unsaturated hydroxycinnamic acid derivatives using Candida antarctica Lipase B (CaL-B) as a biocatalyst with 40%–60% yields. The scavenging ability of the phenolic raw materials, intermediates and PPGs was evaluated by the 2,2-diphenyl-1-picrylhydrazyl radical (DPPH•) method. It was found that the biosynthesized PPGs had higher scavenging abilities when compared to ascorbic acid, the reference compound, while their antioxidant activities were found similar to that of natural PPGs. Moreover, density functional theory (DFT) calculations were used to determine that the PPGs antioxidant mechanism proceeds through a sequential proton loss single electron transfer (SPLET). The enzymatic process reported in this study is an efficient and versatile route to obtain PPGs from different phenylpropanoid acids, sugars and phenolic alcohols.     

Release from prey preservation behavior via prey switch allowed diversification of cuticular hydrocarbon profiles in digger wasps

The cuticle of insects is covered by a layer of hydrocarbons (CHC), whose original function is the protection from desiccation and pathogens. However, in most insects CHC profiles are species specific. While this variability among species was largely linked to communication and recognition functions, additional selective forces may shape insect CHC profiles. Here, we show that in Philanthinae digger wasps (Crabronidae) the CHC profile coevolved with a peculiar brood‐care strategy. In particular, we found that the behavior to embalm prey stored in the nest with hydrocarbons is adaptive to protect larval food from fungi in those species hunting for Hymenoptera. The prey embalming secretion is identical in composition to the alkene‐dominated CHC profile in these species, suggesting that their profile is adaptively conserved for this purpose. In contrast, prey embalming is not required in those species that switched to Coleoptera as prey. Released from this chemical brood‐care strategy, Coleoptera‐hunting species considerably diversified their CHC profiles. Differential needs to successfully protect prey types used as larval food have thus driven the diversification of CHCs profiles of female Philanthinae wasps. To the best of our knowledge, this is the first evidence of a direct link between selection pressure for food preservation and CHC diversity.     

Glyceraldehyde-derived advanced glycation end-products having pyrrolopyridinium-based crosslinks

Reducing sugars and reactive aldehydes, such as glyceraldehyde, non-enzymatically react with amino or guanidino groups of proteins to form advanced glycation end-products (AGEs) by the Maillard reaction that involves Schiff base formation followed by Amadori rearrangement. AGEs are found relatively in abundance in the human eye and to accumulate at a higher rate in diseases that impair vision such as cataract, diabetic retinopathy or age-related macular degeneration. We identified two novel AGEs of pyrrolopyridinium lysine dimer derived from glyceraldehyde, PPG1 and PPG2, in the Maillard reaction of N^α-acetyl-l-lysine with glyceraldehyde under physiological conditions. Having fluorophores similar to that of vesperlysine A, which was isolated from the human lens, PPGs were found to act as photosensitizers producing singlet oxygen in response to blue light irradiation. Moreover, PPG2 interacts with receptor for AGE (RAGE) in vitro with a higher binding affinity than GLAP, a well-known ligand of the receptor. We also proposed a pathway to form PPGs and discussed how they would be formed in vitro. As glyceraldehyde-derived AGEs have been studied extensively in connection with various hyperglycemia-related diseases, further studies will be required to find PPGs in vivo such as in the lens or other tissues.     

Ontogeny of hydrocarbon profiles in the ant Aphaenogaster senilis and effects of social isolation

In ants, cuticular hydrocarbons are used for nestmate recognition; they are stored in the postpharyngeal gland and shared among the individuals. Newly emerged ants have a very small quantity of hydrocarbons. We studied the ontogeny of the hydrocarbon profile in Aphaenogaster senilis. The total quantities of both cuticular and postpharyngeal gland (PPG) hydrocarbons increased with age from 0 to 20 days after emergence and then stabilised. These quantities are correlated with the development of the ovary. Under individual social isolation, cuticular hydrocarbons increased as normal, but the total quantity of PPG hydrocarbons never increased from the initial low level. This effect of social isolation on the PPG hydrocarbon level indicates the importance of hydrocarbon transfer between nestmates through the PPG and lends support to the gestalt model of nestmate recognition. To cite this article: K. Ichinose, A. Lenoir, C. R. Biologies 332 (2009).     

Comparative analysis of dufour's gland secretions of two carpenter bees (xylocopinae: anthophoridae) with different nesting habits

• 1.1. The chemistry and function of Dufour's gland secretions of two carpenter bees were studied.
• 2.2. Dufour's gland of Proxylocopa olivieri, a ground nesting bee, produces long chain hydrocarbons that are utilized to line its brood cells and are mixed with its bee bread.
• 3.3. Dufour's gland secretion of Xylocopa sulcatipes, on the other hand, is dominated by ethyl eicosanoate and ethyl docosanoate accompanied by the corresponding methyl esters and high boiling hydrocarbons.
• 4.4. This wood nesting bee apparently does not use Dufour's gland secretion to line its nest.
• 5.5. The relationship between the respective chemistry and function of Dufour's gland secretion and the nesting ecology of the two bees is discussed.

     

Nestmate recognition signals of the leaf-cutting ant Atta laevigata

Behavioral tests with field colonies of Atta laevigata were performed in order to identify the source of the odors used in nestmate recognition. We tested the postpharyngeal (PPG) and mandibular glands (MG) as putative organs producing chemical signals for nestmate recognition. Chemical analyses of PPG were also undertaken. With a series of bioassays, we confirmed that nestmate recognition is based on cephalic odors and that these odors come mainly from the mandibular gland secretion. We show chemical evidence that odors from MG are dispersed all over the cuticle. Although odors from PPG elicited colony-specific behavioral responses, the types of behaviors they elicited differed from those of nestmate recognition of whole ants or MG extracts. PPG secretion was characterized by long-chain alkanes and methyl branched alkanes of low volatility, whereas MG contained volatile ketones and alcohols.     

Comparative analysis of septic injury-inducible genes in phylogenetically distant model organisms of regeneration and stem cell research, the planarian Schmidtea mediterranea and the cnidarian Hydra vulgaris

Background

Host-parasite interactions are among the most important biotic relationships. Host species should evolve mechanisms to detect their enemies and employ appropriate counterstrategies. Parasites, in turn, should evolve mechanisms to evade detection and thus maximize their success. Females of the European beewolf (Philanthus triangulum, Hymenoptera, Crabronidae) hunt exclusively honeybee workers as food for their progeny. The brood cells containing the paralyzed bees are severely threatened by a highly specialized cuckoo wasp (Hedychrum rutilans, Hymenoptera, Chrysididae). Female cuckoo wasps enter beewolf nests to oviposit on paralyzed bees that are temporarily couched in the nest burrow. The cuckoo wasp larva kills the beewolf larva and feeds on it and the bees. Here, we investigated whether H. rutilans evades detection by its host. Since chemical senses are most important in the dark nest, we hypothesized that the cuckoo wasp might employ chemical camouflage.

Results

Field observations suggest that cuckoo wasps are attacked by beewolves in front of their nest, most probably after being recognized visually. In contrast, beewolves seem not to detect signs of the presence of these parasitoids neither when these had visited the nest nor when directly encountered in the dark nest burrow. In a recognition bioassay in observation cages, beewolf females responded significantly less frequently to filter paper discs treated with a cuticular extract from H. rutilans females, than to filter paper discs treated with an extract from another cuckoo wasp species (Chrysis viridula). The behavior to paper discs treated with a cuticular extract from H. rutilans females did not differ significantly from the behavior towards filter paper discs treated with the solvent only. We hypothesized that cuckoo wasps either mimic the chemistry of their beewolf host or their host's prey. We tested this hypothesis using GC-MS analyses of the cuticles of male and female beewolves, cuckoo wasps, and honeybee workers. Cuticle extracts of Hedychrum nobile (Hymenoptera: Chrysididae) and Cerceris arenaria (Hymenoptera: Crabronidae) were used as outgroups. There was little congruence with regard to cuticular compounds between H. rutilans females and honeybees as well as females of C. arenaria and H. nobile. However, there was a considerable similarity between beewolf females and H. rutilans females. Beewolf females show a striking dimorphism regarding their cuticular hydrocarbons with one morph having (Z)-9-C25:1 and the other morph having (Z)-9-C27:1 as the major component. H. rutilans females were more similar to the morph having (Z)-9-C27:1 as the main component.

Conclusion

We conclude that H. rutilans females closely mimic the composition of cuticular compounds of their host species P. triangulum. The occurrence of isomeric forms of certain compounds on the cuticles of the cuckoo wasps but their absence on beewolf females suggests that cuckoo wasps synthesize the cuticular compounds rather than sequester them from their host. Thus, the behavioral data and the chemical analysis provide evidence that a specialized cuckoo wasp exhibits chemical mimicry of the odor of its host. This probably allows the cuckoo wasp to enter the nest with a reduced risk of being detected by olfaction and without leaving traitorous chemical traces.     

Repair effect of phenylpropanoid glycosides on thymine radical anion induced by pulse radiolysis

Repair effects on thymine radical anion by six phenylpropanoid glycosides (PPGs), isolated from Pedicularis species, were studied using pulse radiolysis method. The thymine radical anion was produced by the reaction of hydrated electron with thymine. PPGs were added into the thymine solution saturated with N(2). Kinetic analysis showed that transient absorption spectrum of thymine radical anion formed at first, and then after several microseconds of pulse radiolysis changed to that of PPG radical anion. The evidence indicated that thymine radical anion was repaired through one-electron transfer between the radical anion and PPG. Electrophilic phenyl-substituted unsaturated carboxylic group containing in PPGs' structure was able to capture electron from thymine radical anion before it undergo reversible protonation. The reaction rate constants of electron transfer from thymine radical anion to PPGs were within 1.16-2.29 x 10(9) dm(3) mol(-1) s(-1).     

Females of the European beewolf preserve their honeybee prey against competing fungi

1. Females of the European beewolf Philanthus triangulum (Hymenoptera, Sphecidae) provision brood cells with paralysed honeybees as larval food. Because brood cells are located in warm, humid locations there is a high risk of microbial decomposition of the provisions. Low incidence of fungus infestation (Aspergillus sp.) in nests in the field suggested the presence of an anti‐fungal adaptation. 2. To test whether the paralysis caused the protection from fungus infestation, the timing of fungus growth on bees that were freeze‐killed, paralysed but not provisioned, and provisioned regularly by beewolf females was determined. Fungus growth was first detected on freeze‐killed bees, followed by paralysed but not provisioned bees. By contrast, fungus growth on provisioned bees was delayed greatly or even absent. Thus, paralysis alone is much less efficient in delaying fungus growth than is regular provisioning. 3. Observations of beewolves in their nests revealed that females lick the body surface of their prey very thoroughly during the period of excavation of the brood cell. 4. To separate the effect of a possible anti‐fungal property of the brood cell and the licking of the bees, a second experiment was conducted. Timing of fungus growth on paralysed bees did not differ between artificial and original brood cells. By contrast, fungus growth on bees that had been provisioned by a female but were transferred to artificial brood cells was delayed significantly. Thus, the treatment of the bees by the female wasp but not the brood cell caused the delay in fungus growth. 5. Beewolf females most probably apply anti‐fungal chemicals to the cuticle of their prey. This is the first demonstration of the mechanism involved in the preservation of provisions in a hunting wasp. Some kind of preservation of prey as a component of parental care is probably widespread among hunting wasps and might have been a prerequisite for the evolution of mass provisioning.     

Leishmania major: effects of proteophosphoglycan on reactive oxygen species, IL-12, IFN-gamma and IL-10 production in healthy individuals

Protozoan parasites of the genus Leishmania secrete a range of proteophosphoglycans (PPG) known to be important for successful colonization of Leishmania in the sandfly and for virulence in the mammalian host. PPGs are a large family of extensively glycosylated proteins with some unusual and unique features. In this study we purified PPG from culture supernatant of Leishmania major metacyclic promastigotes. In discontinuous SDS-PAGE, PPG could not enter the resolving gel but after mild acid hydrolysis several bands resolved. Agarose gel electrophoresis and immunoblot analysis using monoclonal antibody (WIC 79.3) indicated that the PPG preparation consisted of heterogeneous molecules. Compositional analysis showed that the PPG preparation contained 67% glycan, 28% protein and 5% phosphate. Additionally, the effect of PPG on reactive oxygen species (ROS) production and induction of IL-10, IL-12 and IFN-γ secretion by human peripheral blood mononuclear cells (PBMCs) isolated from healthy individuals was investigated. The water-soluble secreted form of PPG at a concentration of 1 μg glycan/ml seems to be a potent inducer of ROS and IL-10 and to a lesser extent of IFN-γ and IL-12. Cytokines and ROS production was decreased in a dose-dependent manner as the concentration of PPG was increased to 100 μg glycan/ml.     

The dufour gland substances of the workers of Formica fusca and Formica lemani (hymenoptera: formicidae)

• 1.1. The Dufour gland secretions of Formica fusca consist mainly of saturated straight and branched chain hydrocarbons (C₉–C₁₉), one unsaturated hydrocarbon (C₁₃) and two sesquiterpenoids, farnesene and homofarnesene.
• 2.2. In F. lemani, the Dufour gland contains branched, saturated and unsaturated hydrocarbons (C₉–C₁₉) and two farnesenes.
• 3.3. The two species were distinguished chiefly by the presence of a relatively large proportion of farnesene in F. fusca, with very little homofarnesene and by contrast, little farnesene but much more homofarnesene in F. lemani.
• 4.4. The contents of the Dufour gland can be used as a chemotaxonomic clue to distinguish between the species.

     

Influence of Resource Availability on the Foraging Strategies of the Triangle Butterflyfish Chaetodon triangulum in the Maldives

Obligate coral feeders such as many members of the Chaetodontidae family (also known as butterflyfish) often show strong preferences for particular coral species. This is thought to have evolved through natural selection as an energy-maximising strategy. Although some species remain as highly specialised feeders throughout their lifetime, many corallivores show a degree of dietary versatility when food abundance is limited; a strategy described by the optimal foraging theory. This study aimed to examine if, within-reef differences in the feeding regime and territory size of the Triangle Butterflyfish Chaetodon triangulum occurred, as a function of resource availability. Results showed that the dietary specialisation of C. triangulum was significant in both areas of low and high coral cover (χL2² = 2.52 x 102, P<0.001 and χL2² = 3.78 x 102, P<0.001 respectively). Resource selection functions (RSFs), calculated for the two main sites of contrasting coral assemblage, showed that in the resource-rich environments, only two Genera (Acropora and Pocillopora) were preferentially selected for, with the majority of other corals being actively ‘avoided’. Conversely, in territories of lower coral coverage, C. triangulum was being less selective in its prey choice and consuming corals in a more even distribution with respect to their availability. Interestingly, coral cover appeared to show no significant effect on feeding rate, however it was a primary determinant of territory size. The findings of the study agree with the predictions of the optimal foraging theory, in that where food supply is scarce, dietary specialisation is minimised and territory size increased. This results in maximising energy intake. This study represents the first scientific evidence that C. triangulum is an obligate corallivore and, as with many other butterflyfish, is therefore dependent on healthy scleractinian corals for survival.     

A pollinators' eye view of a shelter mimicry system

Background and Aims

‘Human-red’ flowers are traditionally considered to be rather unpopular with bees, yet some allogamous species in the section Oncocyclus (genus Iris, Iridaceae) have evolved specialized interactions with their pollinators, a narrow taxonomic range of male solitary bees. The dark-red, tubular flowers of these irises are nectarless but provide protective shelters (i.e. a non-nutritive form of reward) primarily to male solitary bees (Apidae, Eucerini) that pollinate the flowers while looking for a shelter. An earlier study on orchids suggested that species pollinated predominantly by male solitary bees produce significantly larger amounts and larger numbers of different n-alkenes (unsaturated cuticular hydrocarbons). Whether or not this also applies to the Oncocyclus irises and whether pollinators are attracted by specific colours or scents of these flowers is unknown.

Methods

Using Iris atropurpurea, recording of pollinator preferences for shelters with different spatial parameters was combined with analyses of floral colours (by spectrophotometry) and scents (by gas chromatography–mass spectrometry) to test the hypotheses that (a) pollinators significantly prefer floral tunnels facing the rising sun (floral heat-reward hypothesis), and that (b) flowers pollinated predominantly by male solitary bees produce significantly larger amounts and larger numbers of unsaturated cuticular hydrocarbons (n-alkenes) in their floral scent (preadaptation to sexual-deception hypothesis).

Key Results

Male bees do not significantly prefer shelters facing the rising sun or with the presence of high absolute/relative amounts and numbers of n-alkenes in the floral scent.

Conclusions

The results suggest that the flowers of I. atropurpurea probably evolved by pollinator-mediated selection acting primarily on floral colours to mimic large achromatic (‘bee-black’) protective shelters used preferentially by male solitary bees, and that pollinator visits are presumably not the result of an odour-based sexual stimulation or motivated by an increased morning floral heat reward in tunnels facing the rising sun.     

Reduced aggregation and improved specificity of G-rich oligodeoxyribonucleotides containing pyrazolo[3,4-d]pyrimidine guanine bases

Guanine (G)-rich oligodeoxyribonucleotides (ODNs) can form undesired complexes by self association through non-Watson–Crick interactions. These aggregates can compromise performance of DNA probes and make genetic analysis unpredictable. We found that the 8-aza-7-deazaguanine (PPG), a pyrazolo[3,4-d]pyrimidine analog, reduces guanine self association of G-rich ODNs. In the PPG heterocycle, the N-7 and C-8 atoms of G are interposed. This leaves the ring system with an electron density similar to G, but prevents Hoogsteen-bonding associated with N-7. ODNs containing multiple PPG bases were easily prepared using a dimethylformamidine-protected phosphoramidite reagent. Substitution of PPG for G in ODNs allowed formation of more stable DNA duplexes. When one or more PPGs were substituted for G in ODNs containing four or more consecutive Gs, G aggregation was eliminated. Substitution of PPG for G also improved discrimination of G/A, G/G and G/T mismatches in Watson–Crick hybrids. Use of PPG in fluorogenic minor groove binder probes was also explored. PPG prevented aggregation in MGB probes (MGB^TM is a trademark of Epoch Biosciences) and allowed use of G-rich sequences. An increased signal was observed in 5′-PPG probes due to reduced quenching of fluorescein by PPG. In summary, substitution of PPG for G enhances affinity, specificity, sensitivity and predictability of G-rich DNA probes.