The antibacterial potency of the medicinal maggot, Lucilia sericata (Meigen): Variation in laboratory evaluation

Research to quantify the potency of larval excretion/secretion from Lucilia sericata using liquid culture assays has produced contradictory results. In this study, viable counting was used to investigate the effectiveness of excretion/secretion against three marker bacterial species (Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli) and the effects of varying growing conditions in assays. Results demonstrate that factors such as number of larvae, species of bacteria and addition of nutrient influence its antibacterial potency. Therefore a standardised method should be employed for liquid culture assays when investigating the antibacterial activity of larval excretion/secretion from L. sericata.     

In vitro antibacterial activity and physicochemical properties of a crude methanol extract of the larvae of the blow fly Lucilia cuprina

The emergence of multidrug‐resistant bacterial strains has prompted the reintroduction of maggot therapy in the treatment of chronic, infected wounds. Many previous studies have demonstrated the potent antibacterial activity of larval excretions/secretions of the blowfly Lucilia sericata (Meigen) (Diptera:Calliphoridae) against bacteria. However, the antibacterial activity of its sibling species, Lucilia cuprina (Wiedemann) (Diptera:Calliphoridae) against a wide range of pathogenic bacteria has never been determined. The aim of this study was to develop a new procedure to produce whole body extract of larvae of L. cuprina via methanol extraction as well as to demonstrate the in vitro antibacterial activity of this extract against seven selected wound pathogens (Staphylococcus aureus, methicillin‐resistant S. aureus, S. epidermidis, Streptococcus pyogenes, Klebsiella pneumoniae, Pseudomonas aeruginosa and Escherichia coli). The turbidimetric assay demonstrated that L. cuprina larval extract was significantly potent against all bacteria tested (P < 0.001). Additionally, colony‐forming unit (CFU), agar well diffusion and minimum inhibitory concentration assays have confirmed the apparent potency of larval extract against P. aeruginosa. The reconstituted larval extract was highly robust and thermally stable. These observations substantiated the feasibility of the methanol extraction method in the production of larval extract.     

Antimicrobial compounds from the excretions of surgical maggots, Lucilia sericata (Meigen) (Diptera, Calliphoridae)

Maggots of Lucilia sericata are widely used in the therapy of infected wounds and skin ulcers. Antimicrobial materials released by the insects during their feeding period in order to suppress microbial competitors and potential pathogens play the key role both in the maggots’ survival in their natural habitats (animal corpses) and their therapeutic efficacy. Although the antimicrobial activity of the maggots’ excretion was demonstrated about a hundred years ago, little is known about the nature of its active compounds. We studied the structural characteristics and antimicrobial activities of the compounds released by L. sericata maggots into the environment. To isolate the compounds, excretion was collected from the culture of actively feeding last instar larvae, active compounds were purified using a combination of liquid chromatography and antibacterial growth inhibition assay and characterized by mass spectrometry. Two groups of antibacterial compounds were isolated from the excretion: polypeptides with molecular masses ranging from 6466 to 9025 Da and small molecules with molecular masses ranging from 130 to 700 Da. The polypeptides characterized by the masses of 8882 and 9025 Da and showing selective activity against Gram-negative bacteria correspond well to diptericins, antimicrobial peptides previously found in the hemolymph of Calliphoridae maggots and known to be part of immune response to bacterial pathogens. Other high-molecular compounds with masses 6466, 6633, 5772, and 8631 Da have no clear analogs among antimicrobial peptides present in the hemolymph. The nature of small molecules present in the excretion awaits further study. Thus, the diversity of antimicrobial compounds discovered in Lucilia excretion demonstrates a sophisticated strategy that helps the maggots to fight bacteria and other microorganisms settling their environment. The strategy combines secretion of a set of antibacterial peptides involved in insect immune response as well as molecules which function outside the host organism.     

Rearing bacteria and maggots concurrently: a protocol using Lucilia sericata (Diptera: Calliphoridae) as a model species

Maggot debridement therapy using live Lucilia sericata (Meigen) larvae is an efficient and cost-effective way to treat chronic wounds. The recent increase in studies to assess the antibacterial properties of L. sericata has created a need for a simple, low-cost, and comprehensible rearing and investigative method for researchers with little or no entomological experience. This paper describes and evaluates a reproducible protocol for sterilising and rearing blowfly larvae utilising two sterile artificial diets (blood–yeast agar and pre-prepared blood agar plates) that is suitable for directly investigating the effect of larvae on microbial growth. Using Lucilia sericata as a model, the results show that larval growth on the pre-prepared blood agar diet is detrimental to larval growth and survival, whereas larval growth and survival on the blood–yeast agar diet are comparable to those of larvae raised on porcine liver. This diet is proposed as a standard for blowfly and bacteria interaction studies investigating clinical microbial strains. Developmental data are provided for L. sericata larvae raised on both sterile and nonsterile diets so that researchers can determine the effect of treatment based on the length of time for larvae to reach the required life stage at 25 ± 2 °C. Information on larval ageing (instars at an average of 1, 2, 3 and 4 days), oviposition times (4–5 days after adult emergence) and adult longevity on the diets (102–116 days) is also given.     

Impacts of dietary amino acid composition and microbial presence on preference and performance of immature Lucilia sericata (Diptera: Calliphoridae)

1. As primary colonisers of ephemeral resources, the common green bottle fly, Lucilia sericata, must exercise efficient resource exploitation strategies to maximise fitness. Foraging incurs costs, so the efficient evaluation of resources is essential for individuals relying on fragmented patches whose dissimilarity may result in fitness consequences for offspring. Thus, selective pressures between carrion‐using insects and carrion‐associated microbes are expected to occur. 2. Microbes play a pivotal role in adult fly assessment of resources via production of volatile organic compounds (VOCs) resulting from the breakdown of essential amino acids. If one assumes that chemoreception of these VOCs provides public information regarding the corresponding essential amino acids, the resulting effects on maternal decision‐making could impact larval resource exploitation. 3. The essential amino acids phenylalanine, methionine, valine, and tryptophan, the corresponding VOCs of which are known fly attractants, were used to construct imbalanced diets with and without antimicrobial treatment to investigate the impact of suboptimal food sources on larval L. sericata. 4. The results of this study demonstrate that both amino acid composition and microbial presence influence behaviour, with larvae displaying a preference for untreated diets lacking methionine and a complete reversal in preference for antimicrobial‐treated diets. This interaction between diet and antimicrobial treatment indicates that the presence of microbes that produce essential amino acids may impact larval resource utilisation strategies. This investigation into larval performance on diets deficient in amino acids that provide important VOC cues to maternal flies provides a stepping stone for dissecting larval behavioural strategies on ephemeral resources when faced with limited choices.     

A new method for the production of sterile colonies of Lucilia sericata

Maggot debridement therapy (MDT) refers to the use of blowfly larvae to clean or debride an infected wound. Most commonly, larvae of Lucilia sericata (Meigen) (Diptera: Calliphoridae) are used, and are sterilized prior to use to ensure no further bacterial infections are introduced during treatment. Current methods sterilize eggs from laboratory‐reared blowfly colonies, after which sterile early second instar maggots can be provided to hospitals for use in treatment. Maggots not required for treatment are used for colony regeneration, in which sterility is not maintained. The ability to maintain sterility beyond this would allow further research into fly–bacteria interactions and the effects of different bacteria on the blowfly lifecycle. This study aimed to produce a colony of sterile adults, using current egg sterilization practice, but maintaining sterility through to pupation and emergence. The production of a sterile colony allows further research into the impact of bacteria on fly development and survival. Eggs were placed on a sterile food source within autoclaved plant tissue culture containers to allow growth under sterile conditions. Nutrient agar plating of sterilized and non‐sterilized eggs, larvae and adults (post‐emergence), as well as the pupation medium and feed source in nutrient broth confirmed the aerobic sterility of all samples involved. The lifecycle of L. sericata was successfully completed through pupation to emergence with no effects on lifespan or oviposition by the newly emerged, sterile adult colony.     

Density dependence in blowfly populations: experimental evaluation of non‐parametric time‐series modelling

Two approaches for describing density dependence in demographic rates of stage‐structured populations are compared in this study. Time‐series data from laboratory blowfly populations (Lucilia sericata) have been analysed in a separate study, with a statistical modelling approach that incorporated density dependences as unspecified (non‐parametric) functions. In this study, we assessed density‐dependent structures by manipulating densities of larvae and adults in cohorts of blowflies and measuring the demographic rates. We here compare the density‐dependent structures revealed by the cohort experiments with those estimated by the non‐parametric model. This model estimates the demographic rates to have the following density‐dependent structures: (i) larval survival was non‐linearly density‐dependent (a ‘humped’ function), (ii) adult survival was density‐independent, and (iii) reproductive rate decreased with adult density. In the cohort experiments reported here, (i) juvenile survival exhibited a positive density dependence in low densities (facilitation), which became negative at higher densities (competition). Pupal and adult size decreased with initial larval density. (ii) Adult survival was reduced by high initial larval density, but it was independent of adult density. (iii) Reproductive rate was reduced by high initial larval density, and by high adult density in populations of large individuals (from low larval density). Hence, the results from these experiments support the non‐parametric model estimates regarding density‐dependent structures of demographic rates in the blowfly populations. The mean demographic rates, however, were apparently underestimated by the model. We conclude that non‐parametric modelling is a useful first approach for exploratory analysis of ecological time‐series data.     

Changes in dry weight, protein, and nucleic acid content during diapause and normal development of the blowfly, Lucilia sericata

Dry weight (D.W.), protein, RNA, and DNA have been determined in the blowfly, Lucilia sericata, throughout all stages of normal development in long and short photoperiod regimes and during larval diapause. During normal development protein/D.W. levels fluctuate markedly during the larval and puparial stages, increased levels being correlated with the synthesis of new cuticle, etc. prior to ecdysis and the histogenesis of adult tissues prior to emergence. Protein levels remain relatively high and constant during adult life to senescence. RNA/D.W. levels are highest in first instar larvae but decline rapidly during larval development until just before puparium formation. Sharp increases are found prior to pupation and then again prior to adult emergence. In the adult stage, the levels decline steadily throughout the life span. DNA/D.W. levels are very low in the egg but rach their highest levels in early first instar larvae. They then decline during larval development, with small increases being found prior to puparium formation and adult emergence. Adult levels remain relatively constant throughout the life span. The ratio has extremely high values in the egg, indicating the high degree of synthetic activity that takes place during embryogenesis. There is a steady decline in values during adult life to senescence in both sexes, suggesting that physiological ageing in L. sericata is accompanied by a decrease in protein synthesis potential.During larval diapause all parameters, with the exception of the ratio, are maintained at low and constant levels, reflecting the fact that diapause is a period when synthetic and mitotic activity are minimal. The great variations in levels, however, indicate that individuals within a group of larvae can terminate diapause spontaneously at 24°C and return to the normal processes of morphogenesis.     

A simple,heat‐sterilizable artificial diet excluding animal‐derived ingredients for adult blowfly,Lucilia sericata

Artificial diets have been developed for Lucilia sericata (Meigen) blowfly larvae; however, diets for adults have not yet been developed. An adult diet that excludes animal tissues and animal‐derived ingredients and promotes not only ovarian development, but also oviposition, would aid in basic research and maggot debridement therapy. We have successfully developed artificial diets that exclude animal tissues and animal‐derived ingredients for L. sericata adults. The outcomes of the diets were comparable with those of a beef liver diet in terms of oviposition, adult survival and number of offspring.     

Bacterial microbiota assemblage in Aedes albopictus mosquitoes and its impacts on larval development

Interactions between bacterial microbiota and mosquitoes play an important role in mosquitoes’ capacity to transmit pathogens. However, microbiota assemblages within mosquitoes and the impact of microbiota in environments on mosquito development and survival remain unclear. This study examined microbiota assemblages and the effects of aquatic environment microbiota on the larval development of the Aedes albopictus mosquito, an important dengue virus vector. Life table studies have found that reducing bacterial load in natural aquatic habitats through water filtering and treatment with antibiotics significantly reduced the larva‐to‐adult emergence rate. This finding was consistent in two types of larval habitats examined—discarded tires and flowerpots, suggesting that bacteria play a crucial role in larval development. Pyrosequencing of the bacterial 16S rRNA gene was used to determine the diversity of bacterial communities in larval habitats and the resulting numbers of mosquitoes under both laboratory and field conditions. The microbiota profiling identified common shared bacteria among samples from different years; further studies are needed to determine whether these bacteria represent a core microbiota. The highest microbiota diversity was found in aquatic habitats, followed by mosquito larvae, and the lowest in adult mosquitoes. Mosquito larvae ingested their bacterial microbiota and nutrients from aquatic habitats of high microbiota diversity. Taken together, the results support the observation that Ae. albopictus larvae are able to utilize diverse bacteria from aquatic habitats and that live bacteria from aquatic habitats play an important role in larval mosquito development and survival. These findings provide new insights into bacteria's role in mosquito larval ecology.     

Looking into the puparium: Micro‐CT visualization of the internal morphological changes during metamorphosis of the blow fly,Calliphora vicina,with the first quantitative analysis of organ development in cyclorrhaphous dipterans

Metamorphosis of cyclorrhaphous flies takes place inside a barrel‐like puparium, formed by the shrinking, hardening and darkening of the third‐instar larval cuticle. The opacity of this structure hampers the visualization of the morphological changes occurring inside and therefore a full understanding of the metamorphosis process. Here, we use micro‐computed tomography (micro‐CT) to describe the internal morphological changes that occur during metamorphosis of the blow fly, Calliphora vicina Robineau‐Desvoidy 1830 (Diptera: Calliphoridae) at a greater temporal resolution than anything hitherto published. The morphological changes were documented at 10% intervals of the total intra‐puparial period, and down to 2.5% intervals during the first 20% interval, when the most dramatic morphological changes occur. Moreover, the development of an internal gas bubble, which plays an essential role during early metamorphosis, was further investigated with X‐ray images and micro‐CT virtual sections. The origin of this gas bubble has been largely unknown, but micro‐CT virtual sections show that it is connected to one of the main tracheal trunks. Micro‐CT virtual sections also provided enough resolution for determining the completion of the larval‐pupal and pupal‐adult apolyses, thus enabling an accurate timing of the different intra‐puparial life stages. The prepupal, pupal, and pharate adult stages last for 7.5%, 22.5%, and 70% of the total intra‐puparial development, respectively. Furthermore, we provide for the first time quantitative data on the development of two organ systems of the blow fly: the alimentary canal and the indirect flight muscles. There is a significant and negative correlation between the volume of the indirect flight muscles and the pre‐helicoidal region of the midgut during metamorphosis. The latter occupies a large portion of the thorax during the pupal stage but narrows progressively as the indirect flight muscles increase in volume during the development of the pharate adult.     

Quickie well done: no evidence of physiological costs in the development race of Lucilia sericata necrophagous larvae

The present study focuses on trade-offs between the development rates and their life-history traits of feeding larvae. Indeed, quick growth is considered to be vital for necrophagous insects such as larvae because they are part of a rapidly changing ecosystem and at the mercy of many predators. However, excessively quick growth can have a negative effect on other life-history traits (e.g. survivorship and body size). The blowfly Lucilia sericata (Diptera: Calliphoridae) is used in the present study because this species is frequently found on carcasses in Central Europe and is a well-known experimental model in insect physiology and ecology. Individuals are reared from first instars to adults at two different constant temperatures (i.e. 15 and 28 °C) and under three different conditions: 100 Lucilia sericata (i.e. small monospecific condition), 250 L. sericata (i.e. large monospecific) or 125 L. sericata + 125 Calliphora vicina (i.e. heterospecific). The development time, pupal and larval survival rate and pupal size are determined individually under each condition. Regarding size and development time, substantial variation is observed between the different growth conditions and within a larval group under the same conditions. However, no trade-offs between development rate and size or survival are detected. These results demonstrate that, under the range of developmental conditions tested in the present study, the quick development of L. sericata larvae does not affect their size or mortality. This developmental plasticity may explain the evolutionary success of this species, which is present in several ecosystems worldwide and dominates the fresh-carrion ecosystem.     

Effects of temperature and other environmental factors on the post‐diapause development of walnut husk fly Rhagoletis completa (Diptera: Tephritidae)

To more effectively manage walnut husk fly Rhagoletis completa (Diptera: Tephritidae), in California walnut orchards, it is important to understand the factors that affect the timing of adult emergence. In the present study, we examine the effects of incubation temperature, pre‐chill and chill durations, latitude, cultivar and size on the post‐diapause development of R. completa puparia. The lower developmental threshold, upper developmental threshold and optimal temperature for puparial development are estimated to be 4, 34 and 26.6 °C, respectively. The thermal requirement for adult emergence after 120 days of chilling is estimated to be 2024 degree days. Percentage adult emergence declines at both higher and lower incubation temperatures. Chill duration at 5 °C for diapausing puparia has a nonlinear negative effect on the thermal requirement but no effect on percentage emergence. Insufficient chilling leads to poor synchronization of adult emergence. Greater pre‐chill duration at room temperature increases the thermal requirement and slightly decreased percentage emergence. Latitude has a negative effect on the thermal requirement. Puparia from northern California black walnut (Juglans hindsii) have a slightly greater thermal requirement than puparia from cultivated walnut (Julgans regia). There is no significant difference in puparial fresh weight or mean thermal requirement between males and females, although the positive correlation between thermal requirement and puparial fresh weight is stronger for females than males. The effects of temperature and other environmental factors on the post‐diapause development of R. completa are discussed in relation to observations from other Rhagoletis species.     

Behavioural response of Lucilia sericata to a decaying body infested by necrophagous insects

Numerous insect species are necrophagous, with Dipterans and Coleopterans being the most abundant on a corpse. Whether the presence of necrophageous species on a corpse affects the attraction of adult blowflies to the corpse is sparsely studied. We test the hypothesis that Lucilia sericata can discriminate the odour of a noncolonized cadaver from the odour of a cadaver on which conspecific and/or heterospecific necrophagous insects are feeding. The volatile organic compounds are collected from decomposing rats under four modalities: (i) in the absence of insects; (ii) in the presence of L. sericata adults; (iii) in the presence of Dermestes frischii adults; (iv) and in the presence of both D. frischii adults and L. sericata adults. During a multiple‐choice bioassay, blowflies are exposed to the four odour samples, and are shown to prefer the odour of a corpse where conspecific larvae are present. We also expect blowflies to avoid cadavers on which predators where present, although L. sericata are not repulsed by the odour of a cadaver colonized by hide beetles. We then compare the average quantities of all 61 volatile molecules identified, finding that the presence of necrophagous insects impacts some of them. However, none of the volatile organic compounds previously reported in the literature as being attractive for L. sericata adults are impacted by the presence of necrophagous insects. The results of the present study suggest that blowfly larvae modify the volatilome of a corpse, enabling adults to discriminate a colonized from a noncolonized corpse.     

Expression of lucifensin in Lucilia sericata medicinal maggots in infected environments

Lucifensin, a novel larval defensin, is one of the antibacterial agents of medicinal maggots involved in maggot therapy. The goal of this study was to examine lucifensin expression in various larval tissues during Lucilia sericata development and in maggots exposed to a variety of infectious environments in vitro. In situ hybridisation revealed lucifensin expression in the salivary glands of all larval stages. Expression was occasionally detected in a few cells of the fat body and in the grease coupler of the salivary glands. Expression of lucifensin in the salivary glands was initiated 5–6 h after hatching from the egg. Maximum expression was reached about 24 h after hatching, remained strong during the second and third instars and declined at the end of the third instar, before the wandering stage. Expression of lucifensin was also investigated in maggots after oral ingestion of certain pathogens regularly found in infected chronic wounds. No differences were detected in the salivary glands after stimulation by wound bacterial isolates. However, lucifensin expression was strongly stimulated in the fat body by the presence of Staphylococcus aureus and Pseudomonas aeruginosa. Our data suggest that certain infectious environments increase lucifensin expression only in the fat body, whereas its production and antimicrobial activity in excretion/secretion products are not affected.     

Asymmetric competition between larvae of the blowflies Calliphora vicina and Lucilia sericata in carrion

1. The relative abundance of the blowflies Calliphora vicina (R.-D.) and Lucilia sericata (Meigen) in carrion was considered in relation to inter- and intraspecific larval competition and the distribution of adults between habitat types. 2. In mixed and pure laboratory cultures of L. sericata and C. vicina the mortality of both species increased and adult size declined as the initial larval number was increased. However, for L. sericata at initial numbers greater than ten larvae/g of liver, the effects of competition on adult size and mortality were greater in the mixed cultures than in the pure cultures. In contrast, for C. vicina at numbers greater than ten larvae/g of liver, the effects of competition on adult size and mortality were greater in the pure cultures than in the mixed cultures. 3. The laboratory data suggest therefore that for L. sericata the effects of interspecific competition with C. vicina on size and mortality were greater than the effects of intraspecific competition in a pure culture at the same initial number. Notably, however, the intensity of interspecific competition was not sufficiently asymmetric to allow C. vicina to exclude L. sericata even at the highest numbers examined. 4. In the field, higher numbers of adult L. sericata emerged from the carcasses of laboratory mice placed in open pasture than in woodland or hedgerow sites. In contrast, higher numbers of C. vicina emerged from carcasses placed in woodland and hedgerow sites. 5. Trapping showed that in the field adult L. sericata were relatively more abundant in open pasture than in woodland and hedgerow sites, while C. vicina were more abundant in woodland and hedgerow sites than in open pasture. 6. It is concluded that the low numbers of L. sericata that emerge from carrion relative to the numbers of C. vicina may, in part, be the result of asymmetric interspecific competition, but that the uneven distribution of adults of the two species among habitat types also plays a major role in shaping the blowfly community in carrion.     

Complex competitive interactions in four species of Lucilia blowflies

Abstract.

• 1 The effects of intraspecific and interspecific larval competition on larval survival, adult size, adult longevity and fecundity were quantified in four species of coexisting Lucilia blowflies: illustris, silvarum, sericata and caesar.
• 2 There was a general negative effect of increasing density on larval and adult survival, size and fecundity. Additionally, complex species-specific and frequency-dependent responses were identified, which were not expected in these biologically and morphologically closely similar species.
• 3 Lucilia illustris, the numerically dominant species in the natural community, was a superior competitor to L.silvarum at intermediate densities but an inferior competitor at high density. Such nonlinear responses may be related to differences in the life histories and larval behaviour of the species (bigger eggs and more contest-type outcome of competition in L.silvarum).
• 4 We parameterized a model of interspecific competition on a subdivided resource in an attempt to reconcile the conflicting results on larval competitive abilities and the abundances of the species in the field. Using laboratory and field-estimated parameter values the model predicted coexistence of L.illustris and L.silvarum and the observed numerical dominance of the former species. The average densities of flies in the field are limited to relatively low levels, apparently preventing L.silvarum (the superior competitor at high density) from dominating and excluding L. illustris.

     

In Vitro Rearing of the Parasitoid Exorista larvarum (L.) (Diptera: Tachinidae) on Meat Homogenate-Based Diets

The tachinid Exorista larvarum (L.), a polyphagous gregarious larval endoparasitoid of Lepidoptera, was reared from egg to fecund adult on media containing commercial meat homogenates for babies as the main ingredient. Four media, each containing a diverse homogenate supplemented with extract of Galleria mellonella L. pupae, were tested first. Despite the difference in nutrient content, the kind of homogenate did not significantly affect the adult yields (30.2 to 40.7%) or puparial weights. Two other diets free of host materials (I and II) were then tested. Both were based on Gerber veal homogenate combined with different amounts of yeast extract and chicken egg yolk and were supplemented with wheat germ (I) or saccharose (II). Adult yields (28.7 to 32.7%) and puparial weights did not differ significantly between the two diets. Fly longevity and fecundity of the females obtained on diet I were comparable to those of the females emerged from puparia formed in G. mellonella larvae. Male and female puparial weights were, however, higher and development times longer on the diet than in the host.     

The parasitoid Gonatopus bartletti reduces presence of plant-pathogenic Spiroplasma kunkelii within the leafhopper vector Dalbulus maidis

Homopteran vectors (e.g., leafhoppers) of plant pathogens are vessels for reproduction of cell wall‐free bacteria. These vectors also serve as hosts for larval parasitoid dipterans, hymenopterans, and strepsipterans. However, no study has explored the relationship among these wall‐free bacteria and parasitoid larvae within the insect host. We studied the corn stunt spiroplasma (CSS), Spiroplasma kunkelii Whitcomb (Mycoplasmatales: Spiroplasmataceae), a bacterium that originated from secondary symbionts that cause corn stunt disease in maize, Zea mays L., and its reproduction in the haemolymph of the corn leafhopper, Dalbulus maidis (Delong and Wolcott) (Homoptera: Cicadellidae). We also studied the dryinid parasitoid Gonatopus bartletti Olmi (Hymenoptera: Dryinidae), the larva of which feeds in the corn leafhopper haemolymph. Our results showed that when CSS and the wasp coexisted in D. maidis, the development of the parasitoid was not affected by S. kunkelii. Parasitoid development was successfully completed when leafhoppers acquired S. kunkelii before or after parasitism and when CSS had median (10 days) and long (20 days) incubation periods in the leafhopper before parasitization. The presence of S. kunkelii did not affect parasitoid development to the adult stage. However, polymerase chain reaction showed that the presence (survival) of S. kunkelii in the leafhopper was negatively affected by the parasitoid larva. Fewer leafhoppers had CSS before and after parasitization compared with leafhoppers that only acquired the CSS. This negative effect helps to explain the high parasitism rate by G. bartletti in D. maidis and the low presence of S. kunkelii in the corn leafhopper when CSS and the wasp parasitoid overlap throughout their geographic distribution. The parasitoid larva may negatively affect S. kunkelii by (1) producing antibacterial peptides that are toxic to CSS; (2) producing teratocytes that take nutrients from the host for larval development, but these nutrients are required by CSS; (3) affecting, indirectly, CSS through other symbiotic microorganisms; and (4) producing proteins with antibacterial activity that are present in the venom of the wasp parasitoid.     

Antibacterial Potential of Garlic-Derived Allicin and Its Cancellation by Sulfhydryl Compounds

Allicin (allyl 2-propenethiosulfinate), an antibacterial principle of garlic, has drawn much attention, since it has potent antimicrobial activity against a range of microorganisms, including methicillin-resistant Staphylococcus aureus. There have been many reports on the antibacterial properties of allicin, but no quantitative comparison of antibacterial activities between freshly prepared garlic extract and clinically useful antibiotics has been performed. To verify the substantial antibacterial effect of aqueous garlic extract, we compared it with those of allicin and several clinically useful antibiotics using two representative bacteria commonly found in the human environment, Gram-positive S. aureus and Gram-negative Escherichia coli. The garlic extract had more potent anti-staphylococcal activity than an equal amount of allicin. In terms of antibiotic potency against Gram-positive and Gram-negative bacteria, authentic allicin had roughly 1–2% of the potency of streptomycin (vs. S. aureus), 8% of that of vancomycin (vs. S. aureus), and only 0.2% of that of colistin (vs. E. coli). The antibacterial activity of allicin was completely abolished by cysteine, glutathione and coenzyme A, but not by non-SH-compounds. The oxygen in the structure (–S(=O)–S–) of allicin therefore functions to liberate the S-allyl moiety, which might be an offensive tool against bacteria.