Cuprina Larvae Research Articles

The use of cell and larval assays to identify target genes for RNA interference-meditated control of the Australian sheep blowfly (Lucilia cuprina).

Flystrike, primarily caused by Lucilia cuprina, is a major health and welfare issue for sheep wool industries. Current chemical-based controls can have limited effectiveness due to the emergence of resistance in the parasite. RNA interference (RNAi), which uses double-stranded RNA (dsRNA) as a trigger molecule, has been successfully investigated for the development of innovative pest control strategies. Although RNAi offers great potential, the efficient identification, selection of target genes and delivery of dsRNA represent challenges to be overcome for the successful application of RNAi for control of L. cuprina. A primary L. cuprina (blowfly) embryo cell line (BFEC) was established and confirmed as being derived from L. cuprina eggs by PCR and amplicon sequencing. The BFECs were successfully transfected with plasmids and messenger RNA (mRNA) expressing fluorescent reporter proteins and dsRNA using lipid-based transfection reagents. The transfection of dsRNA into BEFC in this study suggested decreased mRNA levels of target gene expression, which suggested RNAi-mediated knockdown. Three of the dsRNAs identified in this study resulted in reductions of in target gene mRNA levels in BFEC and loss of biological fitness by L. cuprina larvae in a feeding bioassay. This study confirms that the novel BFEC cell line can be used to improve the efficacy of dsRNA-mediated screening to accelerate the identification of potential target genes in the development of RNAi mediated control approaches for L. cuprina. The research models established in this study are encouraging with respect to the use of RNAi as a blowfly control method, however further improvement and validation are required for field applicationsnot prefect, and could be ongoing developing. © 2024 The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Exploring Life History Choices: Using Temperature and Substrate Type as Interacting Factors for Blowfly Larval and Female Preferences.

Blowflies (Diptera: Calliphoridae) present a wide range of larval lifestyles, typically classified as obligate parasitism, facultative parasitism, and complete sapro-necrophagy. Several parasitic species, both obligate and facultative, are considered to be of sanitary and economic importance, as their larvae can cause myiasis (maggot infestation in live tissue). However, it is noteworthy that the adult female plays a decisive role as she chooses the oviposition site, and, therefore, largely determines the feeding habit and developmental conditions of the larvae. In this study, two protocols are proposed to test larval feeding preference and female oviposition site preference considering two interacting factors: meat substrate type and temperature. The setups presented here allowed to test Lucilia cuprina larvae and gravid females in a four-choice assay with two temperatures (33 ± 2 °C and 25 ± 2 °C) and two types of meat substrates (fresh meat supplemented with blood and 5-day-old rotten meat). Larvae or gravid females can choose to burrow or lay their eggs, respectively, in either of the following: rotten meat at 25 °C (simulating a necrophagous species condition), fresh meat supplemented with blood at 33 °C (simulating a parasitic species condition), and two controls, rotten meat at 33 °C, or fresh meat supplemented with blood at 25 °C. The preference is assessed by counting the number of larvae or eggs laid in each option for each replicate. Comparing the observed results to a random distribution allowed for the estimation of the statistical significance of the preference. The results indicated that L. cuprina larvae have a strong preference for the rotten substrate at 25 °C. Conversely, oviposition-site preference by females was more varied for the meat type. This methodology can be adapted to test the preference of other insect species of similar size. Other questions can also be explored by using alternative conditions.

Elucidation of lipopeptide biosurfactants responsible for the larvicidal activity of Bacillus velezensis PHP1601 towards Lucilia cuprina larvae

ABSTRACT Lipopeptide biosurfactant compounds derived from cultures of Bacillus velezensis PHP1601 (Bacillales: Bacillaceae) show antagonism towards the larval stage of Lucilia cuprina (Diptera: Calliphoridae), a blowfly pest of agricultural significance. A study was undertaken to characterise and elucidate the lipopeptide biosurfactant compounds contributing to this effect. Lipopeptide extracts were obtained from cultures grown in Landy medium by acid precipitation and methanol extraction. Thin layer chromatography and UPLC ESI-TOF MS were used to partially purify and characterise the lipopeptides present in the extract. Lipopeptide fractions contained homologues of surfactin (C13–C17), fengycin (C14–C17) and iturin (C14–C17). Each lipopeptide fraction (20 µg g−1) displayed larvicidal activity against second-instar L. cuprina larvae, with a highly polar surfactin fraction (Rf: 0.90) being the most effective. The potency of surfactin was confirmed with bioassays incorporating a surfactin standard whereby a LT50 of 179.97 h and LC50 of 9.87 µg g−1 was determined. Interestingly, larvae cadavers recovered from the bioassays displayed significant physiological discolouration and stunting; this was attributed to the biosurfactant nature of the lipopeptide compounds. These findings corroborate the role of lipopeptide compounds, specifically surfactin, in the fly biocontrol mechanism of PHP1601 and constitute the first report of these compounds being insecticidal towards blowfly larvae.

Safety and efficacy of Lucilia cuprina maggots on treating an induced infected wound in Wistar rats

Infection is one of the main complications that hinder wound healing. Currently, antibiotic-resistant bacteria, such as Methicilin-resistant Staphylococcus aureus (MRSA), are a concern worldwide for both humans and animals. Maggot therapy is re-emerging as an alternative to intractable wounds and may be an option to the traditional antibiotic treatment. Although the species of choice is Lucilia sericata, reports of clinical use have led us to evaluate the efficacy and safety of using Lucilia cuprina larvae on induced infected wounds in Wistar rats. In short, 32 male Wistar rats were divided into 4 groups: Group I - saline solution treated; Group II - antibiotic-treated; Group III - treated with larval debridement, and Group IV - without wound and treatment. Skin wounds were induced in groups I, II and III. All treatments were performed once and held for 48 h. Clinical, microbiological, histopathological, hematological, and biochemical analyses were done. Significant wound area contraction was found (>95%) in group III on day 9 compared to day 15 in group II. Complete elimination (0.0 ± 0.0 CFU/mL) of bioburden was achieved after the second treatment (day 6) in both the II and III groups, compared to an increase in Group I (6.51 ± 0.37 CFU/mL). A cleaner wound was also observed in the histopathological evaluation of group III, with adequate collagen formation and re-epithelialization on day 15. Furthermore, larvae increased blood platelet levels after the first treatment. L. cuprina larvae have proven safe and effective in accelerating wound treatment and eliminating MRSA.

Reduction Chemistry of Natural Pyrethrins and Preliminary Insecticidal Activity of Reduced Pyrethrins

The natural extract pyrethrum is an insecticidal oil derived from Tanacetum cinerariifolium that is commonly used in domestic and agricultural pesticides. The major constituents of the extract are the Pyrethrins, six esters that provide pyrethrum with its insecticidal properties. These Pyrethrins readily degrade through several environmental means and as such, there can be significant Pyrethrin losses during processing and long-term storage of pyrethrum-based insecticides. This work attempts to alleviate the effect of these degradative processes through the pursuit of stabilised Pyrethrins by chemically removing oxidatively sensitive functionality. Several reduced Pyrethrin analogues were produced and a method to convert the more sensitive Pyrethrins present in the pyrethrum concentrate into their respective more stable jasmolin counterparts, as a mixture with the over-reduced tetrahydropyrethrins, was developed. All other reduction processes abolished insecticidal activity against Lucilia cuprina larvae, whereas some isomerised analogues showed comparable potency with the individual natural pyrethrin esters. This work has revealed new insights into the structure–activity relationships in this unique class of insecticide.

Insecticide activity of Curcuma longa (leaves) essential oil and its major compound α-phellandrene against Lucilia cuprina larvae (Diptera: Calliphoridae): Histological and ultrastructural biomarkers assessment

Lucilia cuprina, known as the Australian blowfly, is of high medico-sanitary and veterinary importance due to its ability to induce myiasis. Synthetic products are the most frequent form of fly control, but their indiscriminate use has selected for resistant populations and accounted for high levels of residues in animal products. This study aimed to assess the effect of essential oil from leaves of Curcuma longa (CLLEO), and its major compound α-phellandrene against L. cuprina L3. An additional goal was to determine the morphological alterations in target organs/tissues through ultrastructural assessment (SEM) and light microscopy, as well as macroscopic damage to cuticle induced by CLLEO. Groups of 20 L3 were placed on filter paper impregnated with increasing concentrations of CLLEO (0.15 to 2.86 μL/cm2) and α-phellandrene (0.29 to 1.47 μL/cm2). Efficacy was determined by quantifying L3 mortality 6, 24 and 48 h after contact with CLLEO and by measuring the structural damage to L3. CLLEO and α-phellandrene inhibited adult emergence by 96.22 and 100%, respectively. Macroscopic cuticle damage, appeared as diffuse pigment and darkening of larval body, was caused by both extracts. The SEM revealed dryness on the cuticle surface, distortion of the sensorial structures and general degeneration in treated L3. Furthermore, alterations in target organs (digestive tract, fat body and brain) were noticed and shall be used as biomarkers in future attempts to elucidate the mechanism of action of these compounds. The vacuolar degeneration and pyknotic profiles observed in the brain tissue of treated larvae with both extracts and the decreased motility within <6 h after treatment leads us to suggest a neurotoxic activity of the products. This work demonstrates the potential use of CLLEO and α-phellandrene as bioinsecticides to be used against L. cuprina, representing an ecofriendly alternative for myiasis control in humans and animals.

Sterilization of blow fly eggs, Chrysomya megacephala and Lucilia cuprina, (Diptera: Calliphoridae) for maggot debridement therapy application.

Maggot debridement therapy (MDT) is an application of sterile laboratory-reared blow fly larvae to remove necrotic tissue and disinfect wounds for medical conditions. For effective application, the blow fly larvae used in the wound treatment are required to be in aseptic condition. Here, we report the results of a detailed assessment of bacteria and fungi isolated from the eggs of two blow fly species, Chrysomya megacephala (F.) and Lucilia cuprina (Wiedemann) before and after sterilization by disinfectants Chlorhex-C, povidone-iodine, and sodium hypochlorite. We also assess the survival ability of larvae and their sterility after the cleansing process. The results indicate that the isolated microorganisms from the control group of both the species consisted of 10 species of gram-positive bacteria, 21 species of gram-negative bacteria, and 4 species of yeast. As for sterility testing, the eggs and the larvae of C. megacephala were found to have been completely sterilized after being subjected to thioglycollate medium for 5days, leading to aseptic larvae. By contrast, some microorganisms from the bacterial culture were still detected in the L. cuprina larvae treated with Chlorhex-C and povidone-iodine. The survival ability of the larvae in both the species was not significantly different between the treated and the control groups. Due to its high disinfection efficacy in destroying microorganisms in both the blow fly eggs, sodium hypochlorite is recommended for preparing sterile larvae before using MDT.

Cloning, recombinant expression and inhibitor profiles of dihydrofolate reductase from the Australian sheep blow fly, Lucilia cuprina

While dihydrofolate reductase (DHFR) is an important drug target in mammals, bacteria and protozoa, no inhibitors of this enzyme have been developed as commercial insecticides. We therefore examined the potential of this enzyme as a drug target in an important ectoparasite of livestock, the Australian sheep blow fly, Lucilia cuprina (Diptera: Calliphoridae) (Wiedemann). The non-specific DHFR inhibitors aminopterin and methotrexate significantly inhibited the growth of L. cuprina larvae, with IC50 values at µg levels. Trimethoprim and pyrimethamine were 5-30-fold less active. Relative IC50 values for the inhibition of recombinant L. cuprina DHFR by various inhibitors were in accordance with their relative effects on larval growth. The active-site amino acid residues of L. cuprina DHFR differed by between 34% and 50% when compared with two mammalian species, as well as two bacteria and two protozoa. There were significant charge and size differences in specific residues between the blow fly and human DHFR enzymes, notably the L. cuprina Asn21, Lys31 and Lys63 residues. This study provides bioassay evidence to highlight the potential of blow fly DHFR as an insecticide target, and describes differences in active site residues between blow flies and other organisms which could be exploited in the design of blow fly control chemicals.

Extracts of four plant species used traditionally to treat myiasis influence pupation rate, pupal mass and adult blowfly emergence of Lucilia cuprina and Chrysomya marginalis (Diptera: Calliphoridae)

Ethnopharmacological relevanceAloe zebrina, Clausena anisata, Erythrina lysistemon and Spirostachys africana are used traditionally in southern Africa to combat and/or treat myiasis, the infestation of the skin of mammals by larvae of a variety of fly species belonging to the arthropod order Diptera.The objective of the study was to establish the in vitro effect of extracts of these plant species on blowfly larvae to ascertain the potential use of these extracts and validate the traditional use. Materials and methodsChrysomya marginalis and Lucilia cuprina larvae cultured in the laboratory under controlled conditions were exposed to meat treated with acetone and aqueous extracts of four different plant species. In addition to solvent controls, the effects of the plant extracts were tested at concentrations of 10, 25, 50, 75, 100 and 150mg/ml. Larval behaviour, larval development and emergence of adult flies were assessed after exposing the larvae to the treated meat. Ivermectin was used as a positive control while acetone and water served as solvent controls. Cytotoxicity of the extracts was determined using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide] assay against C3A human liver cells, bovine dermis cells and Vero African green monkey kidney cells. ResultsAn increase in the concentration of the acetone plant extracts resulted in a decrease in the ingestion of meat by larvae, pupal mass and adult emergence rates. The R2 values for the correlation between dose and pupal mass was 0.92, 0.74 and 0.65 for A. zebrina, C. anisata, and E. lysistemon respectively. For C. anisata and S. africana extracts the increase in the concentration was also associated with the emerging adult flies being smaller. C. anisata was the most toxic plant extract, with an average IC50 of 39μg/ml, while E. lysistemon was the least toxic. Vero cells were the most susceptible cell type, with an average IC50 of less than 100μg/ml, while the human liver cells were the most resistant to the effect of the plant extracts. ConclusionsThe results suggest that the plants may contain compounds that interfere with larval feeding and the neuroendocrine control mechanisms in the blowfly. It appears that some of these plant species have the potential to deliver a product that can be used to control myiasis.

The Influence of Temperature on the Behavior of Burrowing in Larvae of the Blowflies,Chrysomya albicepsandLucilia cuprina, Under Controlled Conditions

Blowflies use discrete, ephemeral substrates for larval development. After exhaustion of the food supply, larvae will disperse in search of sites to burrow and pupate or will seek other sources of food in a process known as post-feeding larval dispersal. In this study, the effect of temperature was investigated as it is one of the most important aspects of the environmental variables in this process. 800 larvae of the blowflies Chrysomya albiceps (Wiedemann 1819) and Lucilia cuprina (Wiedemann 1830) (Diptera: Calliphoridae) were incubated in tubes covered with vermiculite at 15, 20, 25 and 30°C. For each pupa, the body weight, sex and depth of burrowing were determined. Statistical tests were used to examine the relationship of depth of burrowing and body weight to the temperature at which burrowing occurred. Depth of burrowing was affected differently by temperature for both of the species studied; L. cuprina larvae burrowed deeper at lower and higher temperatures while C. albiceps larvae burrowed less at extreme temperatures. Additionally, temperature had a significant effect on the body weight of L. cuprina larvae as body weight decreased as temperature increased, whereas for C. albiceps, pupal weight increased up to 25°C and then decreased abruptly at a higher temperatures. The maximum body weight was also differently affected in the two species; in L. cuprina, the maximal weight was at 15°C and for C. albiceps weight was maximal at 20°C.

Genetic resistance to growth of Lucilia cuprina larvae in Merino sheep

Cutaneous myiasis (fly strike), caused by Lucilia cuprina, is a major ectoparasitic infection of sheep. Previous research has identified contributions of body conformation, wool characteristics and resistance to bacterial dermatoses to resistance to fly strike. This paper investigates whether genetic variation occurs amongst sheep in growth of larvae on skin. Mixed sex Merino sheep in 27 half-sibling groups were challenged with freshly hatched Lucilia cuprina larvae, and survival and growth of larvae were measured after 50 h. Growth but not survival of larvae had moderate heritability (0.29 ± 0.22), comparable to that seen for resistance to nematode parasites in sheep. Phenotypic correlations between resistance to larval growth and wool traits, skin wheal response to intradermal injection of larval excretory secretory products and resistance to internal parasites were negligible; however, there was a significant negative phenotypic correlation with fleece rot score after exposure of sheep to simulated rain at a time independent to that of measurement of larval growth. Larval growth was negatively correlated with peripheral blood eosinophil numbers measured either before or after larval challenge. In addition, larval growth in vitro on serum collected from challenged sheep was moderately associated with larval growth in vivo. A search for quantitative trait loci (QTL) for larval survival and growth was conducted in data from 94 half-sibling progeny of a Merino × Romney sire backcrossed to Merino ewes. Potential QTL for larval growth were identified on chromosome 11 and for larval survival on chromosome 18, although phenotyping greater numbers of sheep and a higher marker density on these chromosomes is necessary to confirm the result. We conclude that this study has identified a novel level of resistance of Merino sheep to growth of L. cuprina larvae that may be mediated in part through actions of anti-larval factors in serum and eosinophils. Further studies are required to establish the impact of growth retardation on the severity of systemic responses of sheep to fly strike and on the biology of adult flies.

In vitro larvicidal efficacy of flystrike dressings against the Australian sheep blowfly

The abilities of 14 registered flystrike products to prevent development of Australian sheep blowfly (Lucilia cuprina) larvae were measured by laboratory bioassay. Larvae were exposed to the products for periods ranging from 5 to 180 s. Most products were quite effective against susceptible-strain larvae but less effective in field strains due to organophosphate resistance. Some products killed fewer than 20% of field larvae, even after 180 s exposure. However, field strain and susceptible-strain L. cuprina were equally likely to succumb after thorough exposure to a powder formulation of diazinon or to products containing ivermectin or cyromazine. Most products had no obvious effects on larvae until 2–8 h after treatment. Exceptions were non-aqueous, ready to use formulations such as Kleendok with diazinon, Defiance S and Coopers Flystrike Powder. In the cases of Kleendok with diazinon and Defiance S it is likely that formulation components other than the active ingredient were responsible for the fast action. Of the aqueous organophosphate formulation products Supona, containing chlorfenvinphos, was the fastest acting. Greater proportions of susceptible larvae responded over time but among field larvae that responded initially, there were some that recovered within 24 h. Increasing concern over insecticide residues in wool following jetting (topical treatment) of whole sheep, has placed greater reliance on dressing products to control flystrike. With so few effective products available there is a ready market for fast-acting, efficacious products.

Inhibition of growth of Lucilia cuprina larvae using serum from sheep vaccinated with first-instar larval antigens

Whole first-instar Lucilia cuprina larvae were homogenised and sequentially extracted with a series of buffers of progressively more severe solubilising power. The final extract, using a buffer containing 6 M-urea, was fractionated by preparative isoelectric focussing. At each step in this process, protein fractions were tested in sheep vaccination trials for their ability to induce immune responses affecting the growth of L. cuprina larvae which fed on the sera from vaccinated sheep. One isoelectric focussing fraction (pH 5.9–6.7) containing a number of larval proteins induced an immune response which inhibited the growth of larvae by a mean of 84±7% in an in vitro feeding bioassay. The recovery of larvae after feeding on sera from sheep vaccinated with this fraction was significantly reduced by 35±13%. This antilarval effect was shown to be mediated by ingested ovine antibodies. Immunofluorescence and immunogold localisations showed that the immune response was directed at proteins from the larval peritrophic membrane, larval cuticle and, to lesser extent, basement membranes and microvilli of digestive epithelial cells. Electron microscopic examination of larvae feeding on sera from sheep vaccinated with this fraction showed that the normally semi-permeable peritrophic membrane was blocked on the luminal side by an electron-lucent layer of undefined composition. It is postulated that this layer prevents nutrients from moving from the gut to the underlying digestive epithelial cells, thereby starving the larvae. The sera from sheep vaccinated with another isoelectric focussing fraction (pH 3.4–5.5) reduced the mean larval weight by 56±13% without significant effects on larval survival.

Synthesis and Biological Activity of Allosamidin and Allosamidin Analogues

The chitinase inhibitor/insect ecdysis inhibitor allosamidin and eight allosamidin analogues have been synthesised from simple carbohydrate starting materials. Allosamidin was assayed against Tineola bisselliella (Hummel) larvae and all nine compounds were examined for their effects on the development of larvae of Lucilia cuprina (Wiedemann). High larval mortality compared to controls resulted when T. bisselliella and L. cuprina larva were exposed to allosamidin. The (1→3) linked regioisomer, the dimeric analogue and the gluco-configurated dimeric analogue of allosamidin all showed high activity against L. cuprina larvae. The regioisomer, the (1→3) linked isomer and its dimeric analogue, as well as the monomer allosamizoline and its regioisomer, were inactive. These new in-vivo results are consistent with known in-vitro insect chitinase inhibition data, in that greatest larval mortality was exhibited by the best inhibitors.

Activity of extracts and compounds from New Zealand gymnosperms against larvae of Lucilia cuprina (Diptera: Calliphoridae)

A modified serum‐based bioassay was used to screen plant extracts and assess compound activity against newly hatched larvae of the Australian sheep blowfly Lucilia cuprina. Larval instar mortality and physical appearance were recorded after exposure for 24 h to the treatments. Extracts from New Zealand gymnosperms shown to have activity against L. cuprina larvae were: Podocarpus totara; P. acuteafolius; Dacrycarpus dacrydioides; Halocarpus bidwillii; H. biformis; H. kirkii; Lagarostrobos colensoi; Lepidothamnus intermedius; L. laxifolius; Phyllocladus tricho‐manoides; P. glaucus, and Agathis australis. The phytoecdysones 20‐hydroxyecdysone, 5,20‐dihydroxyecdysone, and ponasterone A inhibited larval moulting. Dyshomoerythrine, dacrysterone, and nagilactone C were insecticidal, with the latter also retarding growth at sublethal rates.

Expression of angiotensin-converting enzyme-related carboxydipeptidases in the larvae of four species of fly

HieACE, a soluble 70 kDa protein related to the angiotensin-converting enzyme (ACE) has recently been identified, characterized and cloned from the adult buffalo fly ( Haematobia irritans exigua). HieACE is enzymatically similar to the mammalian ACEs and its predicted amino acid sequence has 42% identity with the mammalian testicular ACEs. In adult H.i. exigua, HieACE expression is restricted to the compound ganglion and posterior midgut, and the maturing male reproductive system. Western blot analysis was used to investigate the expression of HieACE and its homologues in the larvae of H.i. exigua, Drosophila melanogaster, the sheep blowfly ( Lucilia cuprina), the Old World screwworm fly ( Chrysomya bezziana) and a secondary strike fly, Chrysomya rufifacies. Dipteran ACE homologues of 65–70 kDa were detected in all the larval instars investigated. Most of the immunoreactive proteins were concentrated in the soluble fraction. The first and second larval instars of L. cuprina and C. bezziana appeared to express two ACE homologues. These larvae were also found to secrete (or excrete) the ACE homologue in larval cultures. The presence of ACE-like enzymes in these larvae was confirmed by the measurement of carboxydipeptidase activity that was inhibited by the specific ACE inhibitor, captopril. The tissue distributions of the ACE homologues in the third instar larvae of H.i. exigua and L. cuprina were examined. As in adult H.i. exigua, HieACE was detected in the larval ganglion, but in contrast to the restricted distribution in the adult stage midgut, HieACE was found throughout the digestive system, and in the salivary glands of H.i. exigua larvae. The expression pattern in the gut of L. cuprina larvae was similar despite the differences in diet and habitat. The most striking difference from the adult stage H.i. exigua was the expression of HieACE and its L. cuprina homologues in the hindgut and Malpighian tubules of these larvae. These results suggest that the role(s) played by the dipteran ACE-like enzymes differ between the adult and larval stages.

The major excretory/secretory protease from Lucilia cuprina larvae is also a gut digestive protease

The larvae of the fly Lucilia cuprina excrete or secrete a chymotrypsin (LCTb) onto the skin of sheep to facilitate the establishment of the larval infestation. A combination of immunoblotting and RT-PCR approaches has established that this protease is also a gut digestive protease. LCTb is synthesized primarily in the cardia, a small highly specialized organ located at the anterior end of the midgut and by midgut cells. There is also some expression by the hindgut but no expression by salivary glands. Excretion of LCTb with waste products or regurgitation of the gut contents of the larvae may explain how this protease is transferred from the larval gut onto ovine skin. LCTb is first expressed in eggs and constitutively expressed throughout each larval instar, but is not expressed in pupae or adult flies. It is concluded that LCTb could be involved in the establishment of larvae on sheep skin as well as acting as a general gut digestive enzyme.

A comparison of inflammatory exudates released from myiasis wounds on sheep bred for resistance or susceptibility to Lucilia cuprina

Sheep bred for resistance (R) or susceptibility (S) to fleece rot and myiasis (blowfly strike) were experimentally infected with L. cuprina larvae. Exudates released from the wound site were collected during the infection at 6, 12, 18 and 24 h. The exudates were separated using two-dimensional gel electrophoresis, and proteins were silver stained and identified by immunoblotting with specific antibody and by their isoelectric points and molecular weights. Comparisons of exudate composition were made over time and between R and S sheep. Between 6 and 12 h post larval implantation the exudate was rich in IgG and fibrinogen, which is before extensive tissue damage and suggests that the exudate is not simply tissue haemorrhage but the result of an inflammatory response by the sheep to L. cuprina. The exudate grew in complexity between 12 and 18 h and contained a maximum of 74 distinct peptide spots by 24 h. Exudate from wounds on resistant sheep contained many more peptides in the first 12 h of infection, suggesting a more rapid inflammatory response. The source of proteins from the exudate remains speculative; it appears to be composed of many acute-phase proteins, large amounts of immunoglobulin G and proportionally low levels of serum albumin. Exudate composition is likely to be influenced by the local synthesis of acute-phase proteins and perhaps immunoglobulins, selective transport to the infection site and also enzymic degradation by L. cuprina larval enzymes. The more rapid exudation of acute-phase and serum proteins at infection sites on R sheep may allow the inhibition of the establishment of fleece rot bacteria or L. cuprina larvae under natural challenge.

Effect of parenteral antioxidants on adrenal pathobiology and leukocytes in hyperammonaemic toxaemia.

Infestation of sheep by L. cuprina larvae produces extensive skin wounds, severe dermatitis, hyperammonaemia and stress with adrenal necrosis and haemmorhage. In infested sheep, intramuscular (im) injections of Dl-Alpha tocopherol induced wool shedding and Desferrioxamine im prevented declines in white blood cells (WBC). In further trials, daily im injections of sodium ascorbate with Dl-alpha tocopherol, desferrioxamine and oral butylated-hydroxyanisole prevented adrenal damage and induced adrenocortical hypertrophy of the zona fasciculata. The treatment boosted the levels of mature and juvenile neutrophils, and blood glucose. Increases in toxic ammonia levels were correlated with increased toxic and band neutrophils, and globulin levels in treated sheep and toxic neutrophils in non-treated sheep. Decreases in serum zinc were correlated with declining lymphocytes and globulin levels. The results suggested that antioxidants protect and enhance adrenal activation in hyperammonaemic toxaemia. The changes in WBC, globulins and glucose were consistent with protected adrenocortical activation.

Larvicidal activity of lectins on Lucilia cuprina: mechanism of action

AbstractLarvae of the blowfly Lucilia cuprina (Wied.) (Diptera: Calliphoridae) were grown in vitro on a serum‐free medium in the presence of a number of lectins. Lectins with specificities for β‐(1,4)‐N‐acetylglucosamine (wheat germ lectin) and α‐D‐mannopyranosyl and (α‐D‐glucopyranosyl residues (lentil lectin and Con A) caused strong concentration‐dependent inhibition of the growth of the larvae and substantial mortality. Wheat germ lectin had the strongest effects, showing 50% inhibition of larval growth at a concentration of 2 μM and 100% mortality at 25 μM. Other lectins with different sugar specificities had much less effect. The mechanism of the larvicidal action(s) of wheat germ lectin, lentil lectin and Con A was investigated. There were at least three effects of these lectins on L. cuprina larvae. First, these lectins bound to and reduced the permeability of the peritrophic membrane of the larvae. Second, they reduced ingestion of diet medium by larvae. Third, the lectins also bound to the apical membranes of larval gut epithelial cells although there were no obvious signs of damage to these cells. It is concluded that the combination of these effects probably results in the starvation of the larvae. The implications of these results in terms of possible control strategies for L. cuprina are discussed.