Linkage disequilibrium and natural selection for mimicry in the Batesian mimic Hypolimnas misippus (L.) (Lepidoptera: Nymphalidae) in the Afrotropics

On two occasions, on opposite sides of the African continent (Cape Coast, Ghana, and Dar es Salaam, Tanzania), high adult population densities in the polymorphic butterfly Hypolimnas misippus (a presumed mimic of Danaus chrysippus) were followed by linkage disequilibrium in combinations of fore‐ and hindwing colour patterns. On both occasions, disequilibrium was caused by significant changes in morph frequencies favouring rarer and more mimetic forms. Recaptures were too few for analysis at Dar, although the changes there took place within a single generation and must have been the result of differential survival. Recapture rate data and survival rate estimates at Cape Coast support the hypothesis that selective predation was responsible, as does the observation of synchronous linkage disequilibrium at Dar in the model D. chrysippus, indicating parasitic mimicry. There was clear selection for the perfection of mimicry for forewings at Dar and for hindwings at Cape Coast. Disequilibrium is also reported for two other sites, Legon (Ghana) and Boksburg (South Africa) and, in all four sites, it was associated with an increase in the most mimetic forms. New chemical evidence is presented to support the contention that D. chrysippus is a defended model. Although all the evidence leads to the conclusion that H. misippus is a Batesian mimic of D. chrysippus, many questions remain, particularly with regard to the identity of predators, the episodic nature of selective predation events, and their apparent lack of lasting and significant impact on overall gene frequencies. We conclude that H. misippus presents both challenges and opportunities for studies on mimicry, and we suggest that linkage disequilibrium can be a useful generic indicator for Gestalt predation on polymorphic prey. © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 100 , 180–194.     

Clustered O-Glycans of IgA1: DEFINING MACRO- AND MICROHETEROGENEITY BY USE OF ELECTRON CAPTURE/TRANSFER DISSOCIATION*

IgA nephropathy (IgAN) is the most common primary glomerulonephritis in the world. Aberrantly glycosylated IgA1, with galactose (Gal)-deficient hinge region (HR) O-glycans, plays a pivotal role in the pathogenesis of the disease. It is not known whether the glycosylation defect occurs randomly or preferentially at specific sites. We have described the utility of activated ion-electron capture dissociation (AI-ECD) mass spectrometric analysis of IgA1 O-glycosylation. However, locating and characterizing the entire range of O-glycan attachment sites are analytically challenging due to the clustered serine and threonine residues in the HR of IgA1 heavy chain. To address this problem, we analyzed all glycoforms of the HR glycopeptides of a Gal-deficient IgA1 myeloma protein, mimicking the aberrant IgA1 in patients with IgAN, by use of a combination of IgA-specific proteases + trypsin and AI-ECD Fourier transform ion cyclotron resonance (FT-ICR) tandem mass spectrometry (MS/MS). The IgA-specific proteases provided a variety of IgA1 HR fragments that allowed unambiguous localization of all O-glycosylation sites in the six most abundant glycoforms, including the sites deficient in Gal. Additionally, this protocol was adapted for on-line liquid chromatography (LC)-AI-ECD MS/MS and LC-electron transfer dissociation MS/MS analysis. Our results thus represent a new clinically relevant approach that requires ECD/electron transfer dissociation-type fragmentation to define the molecular events leading to pathogenesis of a chronic kidney disease. Furthermore, this work offers generally applicable principles for the analysis of clustered sites of O-glycosylation.Glycosylation is one of the most common post-translational modifications of proteins. It is estimated that over half of mammalian proteins are glycosylated. Patients with several autoimmune disorders, chronic inflammatory diseases, and some infectious diseases exhibit abnormal glycosylation of serum immunoglobulins and other glycoproteins (1–5). The biological functions of these modifications in health and disease have become a significant area of interest in biomedical research (6). A subset of these glycoproteins has clustered sites of O-glycosylation with serine- and threonine-rich stretches within the amino acid sequence. Mucins, such as membrane-associated MUC1, are perhaps the best known family of proteins that are heavily O-glycosylated. Their altered expression and aberrant glycosylation have made them potential targets as biomarkers for early detection of cancer (7). Immunoglobulin A1 (IgA1)1 contains both O- and N-glycans (Fig. 1). Aberrant O-glycosylation of IgA1 is involved in the pathogenesis of IgA nephropathy (IgAN) and the closely related Henoch-Schönlein purpura nephritis (1, 8). Interestingly, the aberrantly glycosylated molecules, IgA1 in IgAN and MUC1 in cancer, are recognized by the immune system as neoepitopes as evidenced by formation of specific antibodies (9–11). Mucin-like bacterial surface proteins exhibit similar properties: the molecules have clustered bacterial O-glycans that mediate cellular adhesion, and blocking antibodies target these glycan-containing epitopes (12).Open in a separate windowFig. 1.IgA1 structural elements. IgA1 has N-linked glycans (filled circles) and O-linked glycans (open circles). The O-glycosylated sites are in the HR between the first and second constant region domains of the heavy chains. The HR is a Pro-rich segment with nine possible sites of O-glycan attachment. Underlined serine and threonine residues are usually glycosylated (31). Arrows show cleavage sites of trypsin and IgA-specific proteases.An O-glycosylated protein from a single source contains a population of variably O-glycosylated isoforms that show a distinct distribution of microheterogeneity of the O-glycan chains in terms of number, sites of attachment, and composition. Characterizing these clustered sites and understanding how the distributions change under different biological conditions or disease states are an analytical challenge. Enzymatic or chemical release of O-glycans is not selective. The heterogeneity, composition, and quantitative aspects of different O-glycan chains can be assessed and quantified by gas chromatographic and/or mass spectrometric techniques. However, the site-specific information and context of location and composition of adjacent chains are lost. Carbohydrate-specific lectin analysis of O-glycoproteins can provide information on glycan composition and comparative differences between samples, such as those from healthy controls and patients with various disease states. We have successfully demonstrated this in the analysis of IgA1 O-glycans from patients with IgAN versus healthy controls and disease controls (13–15). This included proximal assessment of sites with galactose (Gal)-deficient O-glycans after digests with IgA-specific proteases (8). Several studies have demonstrated the value of mass spectrometry (MS) in identifying Gal-deficient IgA1 in patients with IgAN (16–21), including our work that demonstrated the first direct localization of native sites of O-glycan chains in the hinge region (HR) of IgA1 by use of electron capture dissociation (ECD) (20, 22). ECD and the more recently developed electron transfer dissociation (ETD) have been used to identify sites of O-glycosylation on a variety of proteins (23–26). This includes the analysis of sites of O-glycosylation by on-line LC-ECD/ETD MS/MS methods (23, 26, 27).IgAN is the most common primary glomerulonephritis worldwide (28) with about 20–40% of patients developing end stage renal failure. It is characterized by mesangial deposits of IgA1-containing immune complexes (28). The distinctive O-glycan chains of IgA1 molecules play a pivotal role in the pathogenesis of IgAN (1, 10, 14–16, 29, 30). IgA1 contains an HR between the first and second heavy chain constant region domains with a high content of Ser, Thr, and Pro. This segment usually has three to five O-glycan chains per HR (31) (see Fig. 1). Aberrantly glycosylated IgA1, deficient in Gal in some of the O-glycans in the HR, in serum is rare in healthy individuals but is present at elevated levels in IgAN patients (13, 15). This distinctive IgA1 is in circulating immune complexes (8, 10, 15) and in the glomerular deposits of IgAN patients (16, 29). The absence of Gal apparently leads to the exposure of neoepitopes, including terminal and sialylated N-acetylgalactosamine (GalNAc) residues (9, 10). These epitopes are recognized by naturally occurring anti-glycan IgG or IgA1 antibodies and, consequently, circulating immune complexes are formed (9, 10, 15) that can deposit in the glomerular mesangia. To identify the pathogenic forms of IgA1, a thorough analysis of O-glycan microheterogeneity, including identification of the attachment sites, will be required.In this work, we demonstrate the complete analysis of O-glycoform microheterogeneity and site localization of the glycoforms in a naturally Gal-deficient IgA1 (Ale) myeloma protein that mimics the nephritogenic IgA1 in patients with IgAN (8, 9). Reversed phase (RP) LC FT-ICR MS successfully identified 10 distinct IgA1 HR fragments representing >99% of total IgA1. AI-ECD of the six most abundant IgA1 HR glycoforms (>95% of total IgA1) was accomplished with three distinct IgA-specific protease + trypsin digestions, identifying sites of Gal deficiency across four distinct IgA1 O-glycoforms. Based on the success of the ECD fragmentation of these IgA1 HR fragments, we adapted the analysis for on-line LC-MS/MS methods for both ECD and ETD. The variety of IgA1 HR proteolytic fragments provides a practical set of guidelines for the ECD/ETD analysis of clustered sites of O-glycosylation on this and other proteins. These results also provide insight into the order of attachment of the O-glycans in the IgA1 HR.     

Effects of Polyphosphate Additives on Campylobacter Survival in Processed Chicken Exudates

Campylobacter spp. are responsible for a large number of the bacterial food poisoning cases worldwide. Despite being sensitive to oxygen and nutritionally fastidious, Campylobacter spp. are able to survive in food processing environments and reach consumers in sufficient numbers to cause disease. To investigate Campylobacter persistence on processed chicken, exudates from chickens produced for consumer sale were collected and sterilized. Two types of exudates from chicken products were collected: enhanced, where a marinade was added to the chickens during processing, and nonenhanced, where no additives were added during processing. Exudates from enhanced chicken products examined in this study contained a mixture of polyphosphates. Exudate samples were inoculated with Campylobacter jejuni or Campylobacter coli strains and incubated under a range of environmental conditions, and viable bacteria present in the resultant cultures were enumerated. When incubated at 42°C in a microaerobic environment, exudates from enhanced chicken products resulted in increased survival of C. jejuni and C. coli compared with that in nonenhanced exudates in the range of <1 to >4 log CFU/ml. Under more relevant food storage conditions (4°C and normal atmosphere), the exudates from enhanced chicken products also demonstrated improved Campylobacter survival compared with that in nonenhanced exudates. Polyphosphates present in the enhanced exudates were determined to be largely responsible for the improved survival observed when the two types of exudates were compared. Therefore, polyphosphates used to enhance chicken quality aid in sustaining the numbers of Campylobacter bacteria, increasing the opportunity for disease via cross-contamination or improperly cooked poultry.Campylobacter species are the major causative agent of food-borne gastrointestinal bacterial infections in the developed world (6, 11, 21). Poultry products are a major source for the introduction of Campylobacter into the food supply (15, 16). Improperly cooked poultry and cross-contamination of other foods by raw poultry are common methods for transmission of Campylobacter to humans (5). However, Campylobacter spp. are nutritionally fastidious organisms that are sensitive to the oxygen levels present in a normal environment (O₂ = 20.9%) (21). Therefore, Campylobacter appears an unlikely candidate to persist within poultry processing and storage environments at levels sufficient to cause human disease. This conundrum directly leads to a question: what then are the elements that contribute to the ability of Campylobacter to survive through poultry processing and cold storage?To investigate this question, a food-relevant environment consisting of chicken weepage or exudate can be used to perform survival experiments on Campylobacter species. Strains of Campylobacter jejuni and Campylobacter coli were used for the survival studies since these two species are responsible for the vast majority of human cases of campylobacteriosis (20, 28). Chicken exudate is the fluid that seeps out from processed poultry carcasses and is often found to be contaminated with considerable numbers of Campylobacter bacteria. It is comprised of water, blood, fats, and other materials added to the poultry during processing. Sterilized poultry exudates make for a convenient experimental material that is also relevant to the conditions which Campylobacter will experience as a contaminant of processed poultry (2, 3). Two different types of chicken exudates were collected from commercial producers, one from chickens processed without additives (nonenhanced) and the other from chickens that were treated with a commercial marinade to increase the quality and appeal of the meat at market (enhanced). The commercial poultry marinades contain a significant amount of polyphosphate additives. Polyphosphates comprise a group of food additives that are utilized within poultry processing to enhance the moisture absorbance, color, and flavor and to reduce product shrinkage of poultry (24, 29-32). Polyphosphates have also been shown to have an antimicrobial effect on several different bacterial species (8, 10, 12). The goal of the research was to determine if polyphosphates contribute to the ability of Campylobacter to survive and persist through the supply chain, thus directly increasing the opportunity for Campylobacter-mediated food poisoning of consumers.     

The impact of the crayfish Orconectes virilis on aquatic macrophytes

SUMMARY. 1. The impact of crayfish on the biomass, density and shoot morphology of four submersed plant species was examined under semi-natural conditions. Male or female crayfish ( Orconectes virilis ) were held for 5 weeks at biomasses of 0, 5, 10 or 18 g m⁻² (live weight) in twelve plastic pools (4.67 m², surface area) containing Potamogeton richardsonii, Myriophyllum exalbescens, Nuphar variegatum and Sparganium eurycarpum .
2. Crayfish significantly affected biomass, density and/or shoot morphology of all four macrophyte species. Differences in the effect of crayfish on macrophyte growth were related to plant species, crayfish sex and activity, and the abundance of alternative foods.
3. The effect of female crayfish on macrophyte growth was generally stimulatory. Myriophyllum and Potamogeton biomass, Potamogeton density and Myriophyllum length increased in the presence of female crayfish, possibly due to the reduction in herbivorous snails as a result of crayfish predation. In contrast, plant growth decreased in the presence of male crayfish: Myriophyllum, Nuphar and Potamogeton biomass, Myriophyllum and Sparganium density, and Sparganium and Poiamogeton length were reduced at male crayfish biomasses between 5 and 18 g m⁻².
4. These results indicate that even relatively low densities of crayfish can greatly affect the growth of submersed aquatic plants. Because of their ability to modify aquatic macrophyte, macroinvertebrate and, ultimately, fish communities, the introduction of crayfish into lakes where they do not occur could have a major effect on the structure and composition of the littoral zone.     

A simplified high-throughput method for pyrethroid knock-down resistance (kdr) detection in Anopheles gambiae

Background

A single base pair mutation in the sodium channel confers knock-down resistance to pyrethroids in many insect species. Its occurrence in Anopheles mosquitoes may have important implications for malaria vector control especially considering the current trend for large scale pyrethroid-treated bednet programmes. Screening Anopheles gambiae populations for the kdr mutation has become one of the mainstays of programmes that monitor the development of insecticide resistance. The screening is commonly performed using a multiplex Polymerase Chain Reaction (PCR) which, since it is reliant on a single nucleotide polymorphism, can be unreliable. Here we present a reliable and potentially high throughput method for screening An. gambiae for the kdr mutation.

Methods

A Hot Ligation Oligonucleotide Assay (HOLA) was developed to detect both the East and West African kdr alleles in the homozygous and heterozygous states, and was optimized for use in low-tech developing world laboratories. Results from the HOLA were compared to results from the multiplex PCR for field and laboratory mosquito specimens to provide verification of the robustness and sensitivity of the technique.

Results and Discussion

The HOLA assay, developed for detection of the kdr mutation, gives a bright blue colouration for a positive result whilst negative reactions remain colourless. The results are apparent within a few minutes of adding the final substrate and can be scored by eye. Heterozygotes are scored when a sample gives a positive reaction to the susceptible probe and the kdr probe. The technique uses only basic laboratory equipment and skills and can be carried out by anyone familiar with the Enzyme-linked immunosorbent assay (ELISA) technique. A comparison to the multiplex PCR method showed that the HOLA assay was more reliable, and scoring of the plates was less ambiguous.

Conclusion

The method is capable of detecting both the East and West African kdr alleles in the homozygous and heterozygous states from fresh or dried material using several DNA extraction methods. It is more reliable than the traditional PCR method and may be more sensitive for the detection of heterozygotes. It is inexpensive, simple and relatively safe making it suitable for use in resource-poor countries.