Acquired resistance in sheep to infection with larvae of the blowfly, Lucilia cuprina

Acquired resistance in sheep to infection with larvae of the blowfly, Lucilia cuprina. International Journal for Parasitology16: 69–75. Resistance to blowfly larvae infections developed in sheep exposed to at least four consecutive infections. Half of the sheep treated showed significant levels of resistance, the others remained susceptible. This resistance took the form of a decreased yield of third instar larvae in comparison to controls and sheep which remained susceptible. In addition an increased sensitivity to larvae developed, as shown by the area of wound obtained per maggot recovered, by the early appearance in resistant sheep of exudate from the infection site and by skin reactions to larval products. Radioimmunoassays demonstrated high levels of serum antibody against larval excretory/secretory antigens, though the response did not peak until after four infections. Resistant animals showed somewhat lower antibody titres than susceptible sheep. Consecutive infections of only 50 larvae failed to induce resistance to larger challenge infections. It is suggested that consecutive infections of larger numbers of maggots induce a hypersensitivity response which may effect larval survival especially of first and second instar maggots.     

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.     

Eicosanoids mediate nodulation reactions to a mollicute bacterium in larvae of the blowfly, Chrysomya megacephala

Nodulation is the temporally and quantitatively most important cellular defense response to bacterial, fungal and some viral infections in insects. We tested the hypothesis that prostaglandins and other eicosanoids are responsible for mediating nodulation reactions to bacterial infection in larvae of the blowfly Chrysomya megacephala. Third-instar larvae treated with Ureaplasma urealyticum formed nodules in a challenge dose-dependent manner. Nodulation was evoked shortly after injection and reached a maximum of approximately 25 nodules/larva within 8 h. Larvae treated with the glucocorticoid, dexamethasone and the cyclooxygenase inhibitors, indomethacin and piroxicam were impaired in their ability to form nodules following U. urealyticum infection. The number of nodules decreased with increasing doses of piroxicam. Contrarily, treating larvae with the lipooxygenase inhibitor, esculetin, and the dual cyclooxygenase/lipooxygenase inhibitor, phenidone did not influence nodulation reactions to infection. Supplying dexamethasone-treated larvae with the eicosanoid precursor, arachidonic acid, reversed the inhibitory effect of dexamethasone on nodulation. We infer from these results that eicosanoids mediate nodulation reactions to infection of a bacterial species that lacks cell walls in larvae of the blowfly, C. megacephala.     

Influence of temperature on the survival and infectivity of Trichinella spiralis larvae in Sarcophaga argyrostoma (Diptera, Sarcophagidae) maggots

To investigate the role of fleshfly maggots as a paratenic host for Trichinella spiralis larvae, maggots of Sarcophaga argyrostoma (Muscidae, Sarcophagidae) kept at different temperatures (26, 22, 20, 16, 12, 8, and 4 C) were allowed to feed on T. spiralis-infected mouse meat. Trichinella larvae found in maggots kept at 8-26 C were able to cause infection when inoculated in mice. Infective larvae survived in maggots up to 5 days postinfection at 8 C and for shorter periods of time at higher temperatures. The survival time in maggots was negatively related to the temperature of maggot breeding. The results suggest that the role of S. argyrostoma in the dissemination of Trichinella larvae in nature is limited in comparison to the role played by mammals with scavenger and cannibalistic behavior.     

Blow fly Lucilia sericata nuclease digests DNA associated with wound slough/eschar and with Pseudomonas aeruginosa biofilm

In chronic wounds, it may be clinically important to remove extracellular bacterial and patient DNA as its presence may impede wound healing and promote bacterial survival in biofilm, in which extracellular DNA forms part of the biofilm architecture. As medicinal maggots, larvae of Lucilia sericata Meigen (Diptera: Calliphoridae) have been shown to efficiently debride wounds it became of interest to investigate their excretions/secretions (ES) for the presence of a deoxyribonuclease (DNAse) activity. Excretions/secretions products were shown to contain a DNAse, with magnesium, sodium and calcium metal ion dependency, and a native molecular mass following affinity purification of approximately 45 kDa. The affinity purified DNAse degraded genomic bacterial DNA per se, DNA from the slough/eschar of a venous leg ulcer, and extracellular bacterial DNA in biofilms pre‐formed from a clinical isolate of Pseudomonas aeruginosa. The latter finding highlights an important attribute of the DNAse, given the frequency of P. aeruginosa infection in non‐healing wounds and the fact that P. aeruginosa virulence factors can be toxic to maggots. Maggot DNAse is thus a competent enzyme derived from a rational source, with the potential to assist in clinical wound debridement by removing extracellular DNA from tissue and biofilm, and promoting tissue viability, while liberating proteinaceous slough/eschar for debridement by the suite of proteinases secreted by L. sericata.     

Excretions/Secretions from Bacteria-Pretreated Maggot Are More Effective against Pseudomonas aeruginosa Biofilms

Background

Sterile larvae—maggots of the green bottle blowfly Lucilia sericata are employed as a treatment tool for various types of chronic wounds. Previous studies reported that excretions/secretions (ES) of the sterile larvae could prevent and remove the biofilms of various species of bacteria. In the present study we assessed the effect of ES from the larvae pretreated with Pseudomonas aeruginosa on the bacteria biofilms.

Methods and Findings

We investigated the effects of ES from the maggot pretreated with P. aeruginosa on the biofilms using microtitre plate assays and on bactericidal effect using the colony-forming unit (CFU) assay. The results showed that only 30 µg of the ES from the pretreated maggots could prevent and degrade the biofilm of P. aeruginosa. However, the CFU count of P. aeruginosa was not decrease when compared to the ES from non pretreated maggots in this study condition. It is suggested that the ES from the pretreated maggot was more effective against biofilm of P. aeruginosa than sterile maggot ES.

Conclusions

Our results showed that the maggot ES, especially the bacteria-pretreated larva ES may provide a new insight into the treatment tool of the bacterial biofilms.     

Lucilia sericata Chymotrypsin Disrupts Protein Adhesin-Mediated Staphylococcal Biofilm Formation

Staphylococcus aureus and Staphylococcus epidermidis biofilms cause chronic infections due to their ability to form biofilms. The excretions/secretions of Lucilia sericata larvae (maggots) have effective activity for debridement and disruption of bacterial biofilms. In this paper, we demonstrate how chymotrypsin derived from maggot excretions/secretions disrupts protein-dependent bacterial biofilm formation mechanisms.     

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.     

Aminopeptidases as potential targets for the control of the Australian sheep blowfly, Lucilia cuprina.

A series of experiments were carried out to investigate the role of proteinase enzymes in the growth of larvae of the sheep blowfly, Lucilia cuprina. First, instar larvae were incubated on an artificial growth media in the presence of various concentrations of inhibitors of all the major proteinase classes. Inhibitors of serine proteinases and aminopeptidases were found to cause significant growth inhibition and in some cases death of the larvae within 24 h, suggesting that these enzymes were the major classes involved in protein digestion in the gut of the insect. A second group of experiments analysed the effects of two inhibitors from the same or different proteinase classes in the growth media. Synergistic inhibition of larval growth was observed with the incorporation of inhibitors of serine proteinases and aminopeptidases. The results suggest that these classes of proteinases are both central to protein digestion in this insect, probably in the gut, and that the inhibition of both types of activity leads to an almost complete blockade of digestion. Testing in vivo gave similar results with infections on sheep skin inhibited by either serine proteinase or aminopeptidase enzyme inhibitors and the combination of both stopped the infection process. The role of aminopeptidases in larval metabolism and as potential targets for blowfly control agents is examined.     

韭菜根蛆的发生危害及综合防治技术研究——公益性行业(农业)科研专项“作物根蛆类害虫综合防治技术研究与示范”进展

针对我国根蛆类害虫严重危害葱蒜类蔬菜作物,特别在韭菜上问题突出的现状,项目开展了根蛆的生物学和生态学、种群规律、单项防治技术及综合防治技术的研究与示范工作。明确了我国大部分地区韭菜上的根蛆主要是韭菜迟眼蕈蚊Bradysiaodoriphaga Yang et Zhang,在甘肃和辽宁地区的优势种为异迟眼蕈蚊B.difformis Frey。采用黄板诱集成虫结合挖根查幼虫的方法,明确了华北、东北、西北和华东地区根蛆在田间的发生规律。根蛆不耐高温耐低温,春、秋和冬季发生严重,在夏季有转主危害的趋势,冬季以老熟幼虫在0~5 cm土层中越冬。蕈蚊成虫对黑色和棕色趋性最强,研发出特异性的黑色粘虫板。覆盖50~60目防虫网对根蛆的防效最佳,且对韭菜生长有促进作用。建立了蕈蚊成虫的优势捕食性天敌——瘦弱秽蝇Coenosia attenuata的饲养技术和田间应用技术,筛选出了对蕈蚊幼虫高致病力的昆虫病原线虫和Bt菌株,田间防效达60%以上。研发出保护地灌溉臭氧水、减量施药和喷淋施药等轻简化的根蛆防治技术10项。根据不同地区韭菜的种植习惯和根蛆的发生特点,在辽宁、河北、山东、甘肃等地建立了区域性的根蛆类害虫综合防治技术,建立核心示范区10个,进行技术培训5 000人次,取得了一定的经济、社会和生态效益。     

Detection and partial characterisation of two antibacterial factors from the excretions/secretions of the medicinal maggot Lucilia sericata and their activity against methicillin-resistant Staphylococcus aureus (MRSA)

Maggot therapy is a simple and highly successful method for cleansing infected and necrotic wounds. The use of maggots has become increasingly important in the treatment of non-healing wounds, particularly those infected with the multidrug-resistant pathogen, methicillin-resistant Staphylococcus aureus (MRSA). The increasing challenge concerning the treatment of MRSA infections and the recent finding of vancomycin-resistant strains of MRSA have elicited the search for novel antibacterial compounds and, in particular, investigations into the potent antibacterial mechanism(s) behind maggot therapy. In this study, we report that excretions/secretions (ES) from the blowfly, Lucilia sericata, exhibit potent, thermally stable, protease resistant antibacterial activity against MRSA in vitro. We describe the initial characterisation of two antibacterial factors from native ES of L. sericata. A small, <500 Da factor with significant antibacterial activity against MRSA was partially isolated using ultrafiltration techniques. The potent activity of this factor was comparable to that of native excretions/secretions. A larger, 0.5-3-kDa factor with significant activity against S. aureus was also partially characterised.     

家蝇和大头金蝇在麦麸和猪瘦肉上的产卵选择和发育差异

在中国许多地区, 大头金蝇Chrysomya megacephala F.已侵入过去由家蝇Musca domestica L.占绝对优势的垃圾生态位, 逐渐成为城市蝇类的优势种. 为了解单独和混合饲养时食物种类对家蝇和大头金蝇幼虫生长发育的影响, 在室内观察了野外大头金蝇和家蝇F1代在湿麦麸、猪瘦肉以及两者混合物上的产卵选择和生活史. 结果显示: (1)大头金蝇嗜在含有猪瘦肉的基质上产卵, 而家蝇嗜在含有麦麸的基质上产卵;(2)初孵家蝇和大头金蝇幼虫都能在含有瘦肉的食物上发育至成虫. 在麦麸上, 初孵家蝇幼虫能发育至成虫, 而初孵和2龄大头金蝇幼虫在进入下一龄期前全部死亡, 但少数3龄大头金蝇幼虫能发育至成虫;(3)等量初孵家蝇与大头金蝇在含有猪瘦肉的食物上共同生长时, 与家蝇相比, 大头金蝇的发育历期较短、存活率较高. 与家蝇在麦麸上共同生长时, 与在麦麸上独立生长的同龄大头金蝇相比, 大头金蝇的发育历期较短、存活率较高. 这些结果表明, 共生时家蝇可促进大头金蝇对植物质营养的利用, 这也许是大头金蝇能成功侵入家蝇占绝对优势的垃圾生态位的一个重要原因.     

Consumption and utilization of a soyflour-wheat germ diet by larvae of the tobacco budworm parasitized by the tachinidEucelatoria sp

The number ( \(\bar X\) =2.4) ofEucelatoria sp. maggots that completed development in 4th- or 5th-instar larvae of the tobacco budworm (TBW),Heliothis virescens (F.), was significantly greater (P<0.05) than the number ( \(\bar X\) =1.2) that completed development in 3rd-instar larvae. Maggot development time decreased with increasing number of maggots per host larva. It also decreased with advancing larval instars. The range was 6.9±1.1 days in early 3rd-instar TBW larvae and 5.0±0.8 days in early 5th-instar TBW larvae. Unparasitized 3rd- or 4th-instar TBW larvae consumed significantly more food than did similar aged larvae parasitized byEucelatoria sp., but larvae parasitized during the early 5th-instar consumed more food than did similar aged unparasitized larvae. Consumption by 4th- or 5th-instar larvae increased significantly as maggot densities increased from 1 to 3 per host larva, but decreased at a density of 4 or more maggots per host larva. Although body weight gain and consumption were both significantly reduced 48 and 120 h after parasitization of late 3rd-instar larvae (6 days old), the approximate digestibility (AD) value was significantly greater for parasitized than for unparasitized larvae. Unparasitized larvae were more efficient in converting digested food to body substance (ECD) than parasitized larvae, but the efficiency in conversion of ingested food to body substance (ECI) was similar for both parasitized and unparasitized larvae.     

Observations on myiasis by the calliphorids, Bufolucilia silvarum and Bufolucilia elongata, in wood frogs, Rana sylvatica, from southeastern Wisconsin

Larvae of certain species of blowflies (Calliphoridae) can cause myiasis in frogs and toads, but there are few reports from North American amphibians. Of these, most are from toads (bufonids). In this study, we observe primary myiasis in a population of juvenile wood frogs, Rana sylvatica, collected on 22-23 August 2003, from southeastern Wisconsin and compare our observations with previous studies on myiasis from toads. Two (5%) of 39 frogs were infected by the blow fly Bufolucilia silvarum, with an intensity of 28 and 31, whereas 1 (2.5%) of 39 frogs was infected by the blow fly Bufolucilia elongata with an intensity of 14. We found that (1) B. silvarum lay eggs on healthy wood frogs, (2) eggs hatch, with first-instar maggots penetrating under the skin, (3) maggots develop to mature third instars within 13-16 hr of egg hatching, (4) maggots kill the host within 7-47 hr of egg hatching, and (5) maggots consume the entire frog carcass reducing it to bones within 42-59 hr of egg hatching. Our observations on the time of death and how quickly carcasses of wood frogs were consumed by these maggots compared with previous studies on toads suggest that finding infected juvenile wood frogs may be uncommon. Therefore, myiasis by these flies on wood frogs and other small terrestrial anurans may be a phenomenon that is much more common than is currently observed. This is the first report of B. silvarum and B. elongata causing myiasis in wood frogs.     

Delaying insect access alters community composition on small carrion: a quantitative approach

We present a study of interactions in the highly competitive insect communities inhabiting the carrion of small mammals. Via manipulation in a fully quantitative design, we delayed community development by excluding insect colonization in mouse and rat carcasses for 3 days, to study the role of early competitively dominant colonizers [burying beetles (Nicrophorus spp.; Coleoptera: Silphidae) and blowfly larvae (Diptera: Calliphoridae)] in the course of heterotrophic succession on small cadavers. Earlier studies demonstrated that in the case of large mammalian carrion, exclusion of insects’ access to the carcass in the early stages of decomposition altered the successional trajectory and species assemblages. However, the effect of such manipulation in easy monopolizable small vertebrate carrion remained unknown. Our results demonstrate that delaying insect access to carrion significantly lowered blowfly larvae abundances, while it simultaneously had no effect on colonization and carrion burial by burying beetles. Higher abundances of blowfly larvae seem to deter necrophagous beetles, whereas they are not harmful to the larvae of flesh flies, at least in larger rat carcasses. Predatory beetle species preferred the lower abundances of blowfly larvae, presumably due to better accessibility of their prey. Our results therefore suggest that in the course of the entire season, larvae of blowflies are the dominant competitors in small carcasses, and significantly affect the assembly of other insect groups, whereas burying beetles may exhibit a more temporal pattern of dominance.     

自然感染方法诱导蝇蛆抗菌活性研究

研究通过自然感染方法诱导的丝光绿蝇蛆虫的抗菌活性.用大肠埃希菌感染无菌蛆虫,用灭菌的PBS作为对照.提取血淋巴,以金黄色葡萄球菌、绿脓杆菌作指示菌,采用平板法作抑菌试验,检验蛆虫血淋巴的抗菌活性.结果发现:感染的蛆虫比无菌蛆虫有较好的抗菌能力;大肠埃希菌预处理12 h后能够诱导蛆虫的抗菌活性,在24h达到峰值然后下降;血淋巴对金黄色葡萄球菌的抗菌活性高于绿脓杆菌.研究表明,通过自然感染的方法可以诱导丝光绿蝇幼虫抗菌活性.     

Myiasis by screw worm Cochliomyia hominivorax (Coquerel) (Diptera: Calliphoridae) in a wild maned wolf Chrysocyon brachyurus (Mammalia: Canidae), in Brasília, Brazil

In April 2009, a wild maned wolf, Chrysocyon brachyurus, was captured in an area of cerrado in Brasília, DF, Brazil, with screw worm maggots in external wounds. Fifty larvae were bred in the laboratory and eight adults of Cochliomyia hominivorax (Coquerel) emerged 10 days after pupation. This is the first report of a myiasis by C. hominivorax in a free-living maned wolf in Brazil.     

Induction of antibacterial activity in larvae of the blowfly Lucilia sericata by an infected environment

Maggot debridement therapy (MDT) is a method for the treatment of intractable, infected and necrotic wounds. In MDT, sterile larvae of Lucilia sericata Meigen (Diptera: Calliphoridae) are applied to infected wounds, where they exert antibacterial effects. Once the larvae are placed in the wound, they are no longer germ-free. This study analysed the influence of infected environments on larval antibacterial activities. Sterile larvae were mixed in a test tube containing a bacterial suspension of Staphylococcus aureus or Pseudomonas aeruginosa, transferred to liver puree agar, and incubated at 25 °C for set periods. To collect the larval extracts, the incubated larvae were transferred to a test tube containing phosphate buffered saline (PBS), cut into multiple pieces with scissors, and centrifuged. The supernatant was used to test antibacterial activities. The results showed that infected larvae had better antibacterial capacities than sterile larvae. Antibacterial activities were induced by pretreatment with a single bacterial species, S. aureus or P. aeruginosa, within 24 h and 12 h, respectively, and disappeared after 36 h. The activities were effective against S. aureus, but not against P. aeruginosa. This natural infection model is very similar to the clinical wound context in MDT and will be a powerful tool with which to study the antibacterial activities of L. sericata larvae in MDT.     

Larviposition in the ovoviviparous blowfly Calliphora dubia

This study examined larviposition in Calliphora dubia Macquart (Diptera: Calliphoridae), an ovoviviparous blowfly of considerable forensic importance in Australia. Females in the field carried 22–83 live larvae, exhibiting a strong linear relationship between female size and the number of live larvae carried. Females took just over 1 min (mean 67.7 ± 7.7 s, n = 54) to larviposit live larvae on or near fresh liver in the laboratory. Females laid larvae at a mean rate of 1.2 ± 0.1 larvae/s, with the fastest rate being 3.4 larvae/s. Most females (70%) laid live larvae only, but 14% laid larvae and eggs at the same time and 16% laid eggs only (none of the eggs laid were viable). Females laying only live larvae laid a mean of 53.7 ± 2.3 larvae, whereas those laying only eggs laid a mean of 48.6 ± 2.8 eggs on each occasion. None of the eggs laid were viable. Most females (86%) laid all their larvae in a single spot, even if they engaged in several bouts of laying live larvae. Nearly one‐third of females did not lay all the live larvae in their ovisacs, but retained half of their complement of developed larvae. Females may be opting to spread their larvae across several carcasses in order to increase their survival and not to overcrowd small, ephemeral carcasses. The fact that a blowfly can lay either eggs or live larvae has enormous implications for the accurate determination of the post‐mortem interval (PMI) as the presence of larvae derived from eggs laid on the body add 6–18 h to the PMI. This paper represents the first report of the ability of female calliphorids to resorb some of their own live larvae.     

Maggot excretions/secretions inhibit multiple neutrophil pro-inflammatory responses

There is renewed interest in the use of maggots (Lucilia sericata) to aid in healing of chronic wounds. In such wounds neutrophils precipitate tissue damage rather than contribute to healing. As the molecules responsible for the beneficial actions of maggots are contained in their excretions/secretions (ES), we assessed the effects of ES on functional activities of human neutrophils. ES dose-dependently inhibited elastase release and H(2)O(2) production by fMLP-activated neutrophils; maximal inhibition was seen with 5-50 microg of ES/ml. In contrast, ES did not affect phagocytosis and intracellular killing of Candida albicans by neutrophils. Furthermore, 0.5 microg of ES/ml already inhibited neutrophil migration towards fMLP. ES dose-dependently reduced the fMLP-stimulated expression of CD11b/CD18 by neutrophils, suggesting that ES modulate neutrophil adhesion to endothelial cells. ES did not affect the fMLP-induced rise in [Ca(2+)](i) in neutrophils, indicating that ES act down-stream of phospholipase C-mediated activation of protein kinase C. In agreement, ES inhibited PMA-activated neutrophil functional activities. ES induced a rise in intracellular cAMP concentration in neutrophils and pharmacological activators of cAMP-dependent mechanisms mimicked their inhibitory effects on neutrophils. The beneficial effects of maggots on chronic wounds may be explained in part by inhibition of multiple pro-inflammatory responses of activated neutrophils by ES.