Uptake and fate of specific antibody in feeding larvae of the sheep blowfly,Lucilia cuprina

Abstract. The quantity of specific antibody ingested by larvae of Lucilia cuprina and its fate after ingestion were studied in larvae grown on sheep and on an artificial diet. Larvae grown to late first or early second instar on sheep vaccinated with horse myoglobin contained 66% less specific antibody detected by enzyme linked immunosorbent assay than larvae grown to a similar stage on an artificial diet containing 75% serum from the same sheep. A similar result was obtained when larvae were grown to mid-third instar. Larvae grown on sheep to first or second instar contained approximately the same quantity of specific antibody per unit weight of larvae as those grown to third instar. Larvae grown on diet to third instar contained 22% less specific antibody per unit weight than those grown to first or second instar. In larvae grown on diet to late third instar, ingested diet retained 91 ± 12% of its original specific antibody activity in the crop, 50 ± 11% in the anterior midgut, 8 ± 2% in the posterior midgut and 13 ± 6% in the hindgut. The mean concentration of total immunoglobulin detectable in the haemolymph of individual third instar larvae grown on diet was 1.7 ± 2.8 ug/ml. Assays of specific antibody in the haemolymph of similarly reared larvae indicated that all or most of this immunoglobulin remained functional. The implications of the quantities and distribution of ingested functional antibody found in feeding larvae of L.cuprina are discussed in relation to the possibility of vaccinating sheep against these larvae and the selection of likely internal targets as sources of potential protective antigens.     

Tryptic and chymotryptic proteases released by larvae of the blowfly, Lucilia cuprina

Proteases released by larvae of the sheep blowfly have been suggested to have a primary role in wound formation and larval nutrition. Assays were carried out on two larval products to analyse the substrate specificity of these proteases, their abundance and approximate molecular weights. Tryptic and chymotryptic activities were found in both products though there were more chymotrypsin-like enzymes in products from 48 h cultures (CESP) than in product collected direct from 48 h larvae (LESP). Sodium dodecyi sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) gels incubated with azocasein showed plaques of major enzyme activity at molecular weights of 20,000 and 26,000 in LESP and at 20,000 in CESP. SDS-PAGE gels, when reacted with peptide substrates showed tryptic activity at 20,000 and 26,000 in LESP, whereas CESP showed only chymotryptic activity at 20,000 and higher molecular weights. The results suggest at least three enzymes, a trypsin and chymotrypsin in LESP, a chymotrypsin in CESP and a tryptic enzyme which is not stable to SDS-PAGE probably in both LESP and CESP. In addition, reactivity with elastase and plasmin substrates suggests the presence of enzymes with general effects on skin substrates and inflammatory pathways.     

Juvenile hormone synthesis by ring glands of the blowfly Lucilia cuprina

The major radiolabelled product released from ring gland and brain-ring gland complexes of third instar larval and pre-pupal stages of the sheep blowfly Lucilia cuprina upon incubation with L-[methyl-³H]methionine corresponded to one diastereomer of juvenile hormone III bisepoxide (JHB₃). Endocrine glands incubated with the juvenile hormone precursor 2E,6E-farnesoic acid released increased quantities of JHB₃, together with significant amounts of juvenile hormone III but not the isomeric methyl 2E-6,7-epoxyfarnesoate. Synthesis of JHB₃ was developmentally and neurally regulated. Ring glands and brain-ring gland complexes from third instar larvae released more JHB₃ than comparable preparations from pre-pupae, while isolated corpus allatum segments of the gland were more active than intact brain-gland complexes. These results reinforce the emerging status of JHB₃ as the characteristic juvenile hormone of dipteran insects. Arch. Insect Biochem. Physiol. 34:239–253, 1997. © 1997 Wiley-Liss, Inc.     

Monitoring and selection of resistance to pyrethroids in the Australian sheep blowfly, Lucilia cuprina

Field and laboratory populations of the Australian sheep blowfly, Lucilia cuprina (Wiedemann) (Calliphoridae), were surveyed by bioassay for possible resistance to the synthetic pyrethroids, a group of insecticides under development for blowfly control. A normal distribution of LC50 values was found using deltamethrin, the test pyrethroid, with no indication of specific resistance despite widespread use of deltamethrin on sheep to control the sheep body louse, Damalinia ovis (Schrank) (Trichodectidae). There was no cross-resistance to deltamethrin from existing organophosphate (OP) resistance nor from previous use of DDT. Selection with deltamethrin on a combined field strain, CSF85, increased the LC50 gradually over the first twenty generations until it stabilized at approximately 25x that of the unselected CSF85. This laboratory-induced resistance extended to other pyrethroids, cypermethrin (16x), cyhalothrin (25x) and cycloprothrin (10x), and increased the existing resistance of CFS85 to the OP diazinon (11x) and the carbamate, butacarb (83x).     

Circadian control of spontaneous flight activity in the blowfly, Lucilia cuprina

ABSTRACT. Under laboratory light: dark cycles, the flight activity of adult Lucilia cuprina (Wied.) was low during darkness and uniformity high during light. This pattern persisted as a rhythm both in constant darkness and in constant light of intensity up to 1lx, with a period of approximately 22 h in each. Light pulses of 15 min at l00lx applied to the free-running rhythm in constant darkness generated phase shifts of up to 60°, 12-h light pulses of the same intensity generated maximal (180°) phase shifts. The phase response curves had shapes similar to those of a number of other insect rhythms. When exposed to light periods (70 lx) of greater than 12 h followed by constant darkness, the rhythm reinitiated at the light-dark transition from a constant phase equivalent to that at the time of the light-dark transition in the LD 12:12 cycle.     

The responses of Lucilia cuprina to odours from sheep, offal and bacterial cultures

Abstract. The responses of gravid female Lucilia cuprina to odours from sheep urine, faeces and gut mucus, and to odours from liver/sodium sulphide mixtures was tested using a bioassay which measured the movement and probing response of walking flies. The same bioassay was used to test the response to odours from cultures of bacteria isolated from liver/sodium sulphide and liver/water mixtures. A significant movement towards odours from faeces, gut mucus and urine was observed. Odours from cultures of the bacteria Proteus mirabilis, Dermatophilus congolensis and Serratia marcescens also elicited significant movement. A probing response was elicited by odours from gut mucus, fresh urine, liver/sodium sulphide mixtures and cultures of P.mirabilis, D.congolensis and gram-positive species. Odours from cultures of Pseudomonas aeruginosa, Entero-bacter aerogenes and Citrobacterfreundii did not elicit significant movement or probing. The movement and probing responses are discussed with reference to the possible uses of the substances tested as a bait for attracting L.cuprina.     

Initial characterisation of the sheep immune response to infections of Lucilia cuprina

Assays for total serum antibody, histamine sensitivity and the presence of reaginic antibodies were carried out on sheep repeatedly infected with first stage larvae of Lucilia cuprina. Effects of the sheep response on the larvae were monitored at the final infection and compared with control animals by the recovery of larvae and measurement of the wound caused by the larvae. Overall larval survival was not significantly different in the pre-infected group though wound sizes were smaller. Histamine sensitivity appeared to correlate with wound size only in the control group. Thus recent infection experience may lead to immune responses which override non-specific inflammatory events and cause smaller wound sizes. A sub-group of the pre-infected sheep had lower larval survival and smaller wound sizes than the other animals and this correlated with increased levels of reaginic antibody and lower total antibody levels. The results suggest a genetic basis for resistance to fly strike and the possible involvement of reaginic antibody in protective responses.     

Synthesis and deposition of chitin in larvae of the Australian sheep blowfly,Lucilia cuprina

Chitin synthesis in third-instar Lucilia cuprina larvae cultured at 23 °C was investigated using in vivo and in vitro systems, the latter with whole and with homogenized integuments. Synthesis was at a maximum between 24 and 48h after ecdysis from the second instar. Chitin was deposited in layers, and labeled GlcNAc was rapidly cleared from the hemolymph. In in vitro homogenate systems, the rapid conversion of UDP-([¹⁴C]GlcN)Ac to ([¹⁴C]GlcN)Ac and its 1-phosphate derivative contributed to the low incorporation of this precursor into chitin. The extent of the conversion was reduced by the addition of KCN or phenylthiourea. In in vivo and in vitro tissue systems the level of incorporation of ([¹⁴C]ClcN)Ac was higher than that of UDP-([¹⁴C]GlcN)Ac. However, in in vitro homogenate systems there was no difference unless UTP was added when the level of incorporation of only ([¹⁴C]GlcN)Ac was increased (by a factor of 9). Incorporation of UDP-([¹⁴C]GlcN)Ac, but not that of ([¹⁴C]GlcN)Ac, was decreased when larvae were deprived of food. Soluble oligosaccharides were detected in in vitro homogenate systems. They were formed during chitin synthesis and may represent newly initiated chitin chains. A reappraisal of current ideas on chitin synthesis in insects is needed.     

Resistance to two organophosphorus insecticides in New Zealand populations of the Australian sheep blowfly, Lucilia cuprina

Abstract. A treated-surface test was used in a bioassay to detect insecticide resistance to the organophosphorus compounds diazinon and propetamphos, in larvae of field strains of Lucilia cuprina in New Zealand during 1990/91 and 1991/92 (December-April). Mean LC₅₀s for both diazinon and propetamphos showed a significant increase between seasons although this is not necessarily indicative of a trend. Mean LC₅₀s for diazinon increased from 1.16 to 1.60 mg/1 (P< 0.001) and for propetamphos from 0.49 to 0.59 mg/1 (P< 0.005) in pooled populations. Diazinon and propetamphos resistance was significantly correlated in both seasons, suggesting side-resistance between the two insecticides.     

Responsiveness to light of the circadian clock controlling eclosion in the blowfly, Lucilia cuprina

ABSTRACT. Eclosion in Lucilia cuprina (Wiedemann) occurs near dawn. The rhythm of eclosion persists in both darkness and constant light of high intensity (490μW cm^-2) with a period close to 24h. The sensitivity to light of the circadian clock controlling eclosion varies greatly according to the stage of the life cycle. During larval life the free running rhythm in darkness can be phase shifted by light pulses of 100μW cm^-2 intensity, with the transition from a Type 1 phase response curve to a Type 0, occurring with pulses of between 1 and 8h. Extending the last light period of LD to 24 h followed by constant darkness resets the phase of the rhythm by 12h, a transition from constant light to constant darkness initiates rhythmicity in flies made arrhythmic by being reared from eggs collected from adults maintained in constant light. After pupariation, the rhythm is relatively insensitive to light. Rhythmicity is sometimes induced by a transition from constant light to constant darkness, but the phase of the rhythm is not shifted by extending the last light period of LD before entering constant darkness. Repeated LD cycles applied after pupariation initiate and entrain the rhythm.     

The patterning of compensatory sugar feeding in the Australian sheep blowfly

Abstract. The pattern of feeding is described for males of the Australian sheep blowfly, Lucilia cuprina , with ad libitum access to either 0.1 M or 1.0 M glucose solution. Flies given the 0.1 M solution ingested nearly 3 times the volume taken by flies given the 1.0 M solution by eating meals of, on average, twice the size about 1.5 times as frequently. Flies were usually relatively inactive following a meal, with the extent of this post-prandial quiescence being a function both of meal size and concentration of sugar. Quiescence lasted only about 20% of the average intermeal interval, however, and there was no correlation between meal size and time to the next meal. The crop emptied more slowly when it contained 1.0 M rather than 0.1 M glucose solution and the crop was, on average, fuller at the beginning of a meal on the higher concentration. The volume of solution imbibed during a meal was positively correlated with time since the end of the preceding meal. The average crop volume at the end of a meal was similar in flies feeding on 0.1 M and 1.0 M solutions. The results are considered in relation to published information on control of feeding and compensation in the blowfly Phormia regina.     

The genetic bases of high-level resistance to diflubenzuron and low-level resistance to cyromazine in a field strain of the Australian sheep blowfly, Lucilia cuprina (Wiedemann) (Diptera: Calliphoridae)

Abstract  Several genes on chromosomes IV and VI have a significant influence on high-level resistance to diflubenzuron in a strain of the Australian sheep blowfly from Tara, Queensland. Low-level resistance to cyromazine in the same strain is due to genes on these chromosomes with a gene (gene complex) in the sv marker region of chromosome IV being particularly important. For both insecticides, genetic background influences resistance status. If the results of the Tara strain prove typical for those of other populations, resistance to diflubenzuron in the Australian sheep blowfly has potentially significant consequences for woolgrowers.     

Quantitative aspects of the effect of mating on readiness to lay in the Australian sheep blowfly,Lucilia cuprina

Virgin females of Lucilia cuprinararely lay eggs, whereas mated females do so readily. This effect of mating is due entirely to increased readiness to lay, and not to any effect on ovarian development. An investigation was made of how readiness to lay was affected by matings which differed in terms of the male's chemical and mechanical contribution. Individual males were mated, during 1 day, to a succession of females whose readiness to lay was determined 1 or 8 days after mating. On both days, the proportion of females laying was inversely related to the number of females with which the male had previously mated. A high proportion of females that had mated with previously unmated or oncemated males laid at both 1 and 8 days after mating. However, this proportion tended to decline between day 1 and day 8 in females that had mated with males with two or more previous matings, and this effect was most evident in females mated with males that had previously mated with four or more females. When matings were manually terminated as soon as coupling had occurred, the proportion laying remained as low as in virgins. This proportion progressively increased as mating duration increased from 2 to 6 min. The proportion that laid after mating terminated at 6 or 8 min was as high as that for females from full-term matings (mean duration, 12.5 min). The results are generally similar to those obtained in parallel experiments on the effect of mating on sexual receptivity in this species and, therefore, indicate that the physiological bases for the two effects of mating might be the same.     

Regulation of Juvenile Hormone synthesis in the blowfly Lucilla cuprina

Abstract. Juvenile Hormone III bisepoxide synthesis by ring gland complexes from third-instar larvae of the blowfly Lucilia cuprina Weidemann (Diptera, Calliphoridae) was measured using a radiochemical assay in vitro. Hormone synthesis is regulated by three distinct mechanisms during development of the final larval instar up to the time of pupariation. The first type of regulation is detected as a rapid decline in hormone release coinciding with the final phase of commitment to pupariation. The second is a neurally mediated inhibition by the brain that acts at all stages of development in third-instar larvae. A protease-sensitive factor from brains of third-instar larvae causes dose-dependent reversible inhibition of Juvenile Hormone III bisepoxide synthesis. The third regulatory signal is a neural inhibition, observed in brain-ring gland complexes of prepupal stages. The first two levels of regulation appear to act early in the synthetic pathway for Juvenile Hormone (JH), whereas the third acts on the final steps of bisepoxide synthesis.     

Pre-adult mortality in the blowfly Lucilia sericata

Populations of the blowfly, Lucilia sericata (Meigen) (Diptera: Calliphoridae), have a considerable potential for rapid increase; the lifetime reproductive output of each adult female has been estimated to be between 130 and 172 eggs. Nevertheless, in the field, absolute population densities of this species are relatively low. To account for this difference, the levels of mortality affecting the eggs, feeding and wandering larvae and pupae of L. sericata were assessed in the field and laboratory. Percentage egg hatch was dependent on relative humidity with no egg eclosion at humidities below 50%; there was no significant effect of temperature on egg hatch. On infested sheep, the mean mortality of feeding larvae was 53%, but this ranged widely from 0% to 96%. There was no effect of atmospheric temperature or humidity on the mortality of feeding larvae in vivo. In the laboratory, only 10% of wandering larvae pupariated at 10 degrees C. At above 20 degrees C pupariation was consistently almost 100%. Percentage emergence increased from 0% at 10 degrees C to about 80% between 20 and 30 degrees C. The upper lethal temperature for pupae was approached at 35 degrees C. Analysis of the predation of pupae in the field revealed a weak, but significant curvilinear relationship between temperature and proportionate mortality and a median mortality of 0.49% per 24 h exposure (interquartile range = 5.2%). There was no evidence of density dependence in pupal predation. Overall, it is estimated that pre-adult mortality accounts for losses of approximately 97% of each generation, but this figure is subject to considerable variation depending on factors such as climate, time of year and host susceptibility.