To eat or get heat: Behavioral trade‐offs between thermoregulation and feeding in gregarious necrophagous larvae

The thermoregulation behavior of Lucilia sericata larvae (Diptera: Calliphoridae), a necrophagous species that feeds on vertebrate cadavers, was investigated. These larvae require high heat incomes to develop, and can elevate temperatures by forming large aggregates. We hypothesized that L. sericata larvae should continue to feed at temperatures up to 38 °C, which can be reached inside larval masses. Thermal regulation behavior such as movement between a hot food spot and colder areas was also postulated. The hypotheses were tested by tracking for 1 h the activity of single, starved third instar larvae in a Petri dish containing 1 food spot (FS) that was heated to a constant temperature of 25 °C, 34 °C or 38 °C with an ambient temperature of 25 °C. The influence of previous conspecific activity in the food on larval behavior was also tested. The crops of larvae were dissected to monitor food content in the digestive systems. Based on relative crop measurements, larvae fed at all food temperatures, but temperature strongly affected larval behavior and kinematics. The total time spent by larvae in FS and the duration of each stay decreased at high FS temperature. Previous activity of conspecifics in the food slightly increased the time spent by larvae in FS and also decreased the average distance to FS. Therefore, necrophagous L. sericata larvae likely thermoregulate during normal feeding activities by adjusting to local fluctuations in temperature, particularly inside maggot masses. By maintaining a steady internal body temperature, larvae likely reduce their development time.     

The effect of bacterially‐dense environments on the development and immune defences of the blowfly Lucilia sericata

Competitive interactions between insects and microbes and the associated cost of development in bacterially‐dense environments are investigated using the blowfly Lucilia sericata (Meigen) (Diptera: Calliphoridae) as a model. The effects of developing in a bacterially‐dense environment are measured by assessing the fitness consequences of competition using the pathogen Staphylococcus aureus. Fitness is quantified in terms of larval survival, puparial development and adult emergence.The influence of bacteria on larval immune defences is investigated using optical density to assess whether antibacterial potency of the larval excretion/secretion changes in response to the degree of contamination of the larval environment. The results obtained demonstrate that bacterial presence has no detrimental effect on survival of L. sericata from egg to adult eclosion, or on puparial size. Additionally, the level of microbial contamination of larvae has no effect on the antibacterial potency of the larval excretion/secretion. These findings confirm that larval antibacterial activity is not induced by the presence of environmental bacteria but is produced constitutively.     

Synomones in necrophagous larvae of the blow flies Lucilia sericata and Calliphora vomitoria

Chemical signals are widespread in insects, but those resulting in interspecific communication (i.e., synomones) remain understudied. Here, we analysed chemicals left on substrates by two species of blow fly larvae, Lucilia sericata (Meigen) and Calliphora vomitoria (Linneaus) (Diptera: Calliphoridae), which can aggregate together on carrion. Using solid-phase microextraction and dynamic headspace analysis, we identified six compounds common to both species: the decanoic, tetradecanoic, pentadecanoic, hexadecanoic and octadecanoic acids, and the 2-ethylhexyl salicylate. We then tested the behavioural effects of the decanoic and pentadecanoic acids using binary-choice experiments, along with the (Z)-9-tricosene, a pheromone found in many arthropods. The time spent by a larva and its average crawling speed were measured in two sides of an arena, where only one contained a compound at 0.25 or 25 μg/μl. No effect was observed when testing the decanoic acid. The pentadecanoic acid only reduced the speed of C. vomitoria larvae at 25 μg/μl. Finally, L. sericata larvae spent less time in the side containing the (Z)-9-tricosene at 0.25 μg/μl, whereas C. vomitoria spent more time and crawled faster in this side at 25 μg/μl. Although these results did not directly evidence synomones, they suggest that the (Z)-9-tricosene could regulate larval aggregations on carrion.     

Growth and survival of blowfly Lucilia sericata larvae under simulated wound conditions: implications for maggot debridement therapy

Maggot debridement therapy has become a well‐established method of wound debridement. Despite its success, little information is available about the optimum duration of the treatment cycle and larval growth in wounds. This study examines the development of Lucilia sericata (Diptera: Calliphoridae) larvae under two containment conditions (bagged and free range) under simulated wound conditions and assesses the impact of transport and further storage of larvae on their survival and growth. There was no significant difference in size between bagged and free‐range larvae over the 72‐h experimental period. Larvae grew fastest 8–24 h after inoculation and completed their growth at 40–48 h. Mortality rates were similar (0.12–0.23% per hour) in both containment conditions and did not differ significantly (P = 0.3212). Survival of free‐range larvae was on average 16% lower than survival of bagged larvae. Refrigeration of larvae upon simulated delivery for > 1 day reduced their survival to < 50% and caused a reduction in growth of up to 30% at 12 h, but not at 48 h, of incubation. Therefore, it is recommended that free‐range larvae are left in the wound for a maximum of 40–48 h, and bagged larvae for 48–72 h. Larvae should be used within 24 h of delivery to avoid high mortality caused by prolonged refrigeration.     

Stocking rate and organic waste type affect development of three Chrysomya species and Lucilia sericata (Diptera: Calliphoridae): Implications for bioconversion

Fly larvae can be used effectively to reduce various organic waste types and produce value-added products, including protein as an ingredient in livestock feeds and oil for biodiesel production. However, fly development on different waste types may cause differences in growth rate and the body composition, which can further be influenced by fly species and their stocking rate. This study explored the impact of different waste types (kitchen waste, abattoir waste and swine manure) and larval stocking rate on growth and body composition of four blowfly species, Chrysomya chloropyga (Wiedemann), Chrysomya megacephala (Fabricius), Chrysomya putoria (Wiedemann) and Lucilia sericata (Meigen). First-instar larvae (20, 50 or 100), less than 3 hr old, were placed on 100 g of each waste type. Pre-pupal mass at commencement of post-feeding larval dispersal, time to onset of dispersal, survival and nutrient reserves were determined for each species, stocking rate and waste type. Our results revealed that larvae fed kitchen and abattoir waste had significantly higher dry mass, crude protein and lipid content compared with those fed swine manure. Higher survival rate was observed with increasing larval stocking rate. We provide important information to guide the mass production of high-quality nutrient-rich larvae and recommend C. putoria, which is versatile and effective on a range of waste products, as well as high in protein and lipids. The implications for waste management are discussed.     

Antimicrobial components of haemolymph and exosecretion of larvae <Emphasis Type="Italic">Lucilia sericata</Emphasis> (Meigen) (Diptera,Calliphoridae)

The present study deals with molecular nature and peculiarities of the functioning of two main protective systems of larvae Lucilia sericata—the antimicrobial compounds of haemolymph and exosecretion released by feeding larvae into environment. In the haemolymph of larvae undergone to bacterial infestation, the chromato-masspectrometry methods identified a set of inducible antibacterial peptides including defensins (3844, 4062, and 4117 Da), P-peptide (3043 Da), and four new polypeptides (3235, 3702, 3746, and 3768 Da). The exosecretion of Lucilia sericata maggots contains the peptides analogous or identical to the haemolymph antimicrobial peptides (diptericins: 8882 Da and 9025 Da), high molecular compounds of the peptide nature (6466 Da, 6633 Da, 5772 Da, 8631 Da, etc.) differing from the known haemolymph components, and the low molecular compounds (130–700 Da). The spectrum of exosecretion bactericidal activity includes the representatives of various groups of bacteria including pathogen the most actual from the medical point of view-the methicillin-resistant Staphylococcus aureus that does not have anti-staphylococcal activity in contrast to haemolymph. The exosecretion components suppressing growth and development of this staphylococcus represent the substances of low molecular mass (from 160 to 1020 Da). The performed studies characterize the strategies used by “surgical maggots” for protection from pathogens and for suppression of microbial competitors, and allow better understanding of molecular mechanisms of larval therapy of purulent infectious diseases. These studies in perspective can serve the basis for creation of the principally new drugs for struggle with usual and antibiotics-resistant bacterial infections.     

Quality control of a medicinal larval (Lucilia sericata) debridement device based on released gelatinase activity

Lucilia sericata Meigen (Diptera: Calliphoridae) larvae are manufactured worldwide for the treatment of chronic wounds. Published research has confirmed that the primary clinical effect of the product, debridement (the degradation of non‐viable wound tissue), is accomplished by a range of enzymes released by larvae during feeding. The quality assessment of larval activity is currently achieved during production using meat‐based assays, which monitor insect growth and/or the reduction in substrate mass. To support this, the present authors developed a complementary radial diffusion enzymatic assay to produce a visual and measureable indication of the activity of larval alimentary products (LAP) collected under standardized conditions, against a gelatin substrate. Using basic laboratory equipment and reagents, the assay is rapid and suited to high throughput. Assay reproducibility is high (standard deviation: 0.06–0.27; coefficient of variation: 0.75–4.31%) and the LAP collection procedure does not adversely affect larval survival (mortality: < 2%). Because it is both cost‐ and time‐effective, this method is suited to both academic and industrial use and supports good manufacturing and laboratory practice as a quality control assay.     

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.     

A reverse temperature‐size rule associated with a negative relationship between larval development time and pupal weight in a tropical population of Ostrinia furnacalis

Empirical studies demonstrate a negative relationship between body size and temperature [i.e. the temperature‐size rule (TSR)] and a positive relationship between body size and development time (a trade‐off). However, many exceptions are also reported. The present study tests the two relationships in a tropical population of the Asian corn borer Ostrinia furnacalis under a wide range of temperatures from 18 to 31 °C. The results show that larval time is decreased significantly and pupal weight is increased significantly with an increasing rearing temperature, exhibiting the reverse‐TSR. Growth rate is positively correlated with temperature. The relationships between body size and development time among individuals show a negative slope (i.e. pupal weight tends to decrease with increases in development time). Females are significantly larger than males at all temperatures, showing a female‐biased sex size dimorphism. It is also found that development time and pupal weight vary greatly among individuals, particularly at lower temperatures. The results of the present study demonstrate a reverse‐TSR associated with a negative relationship between larval development time and pupal weight in a tropical population of O. furnacalis.     

Influence of parasitism and soil compaction on pupation of the green bottle fly,Lucilia sericata

The influence of parasitoids and soil compaction on pupation behavior of blow flies was examined in a host–parasitoid system involving Lucilia sericata (Meigen) (Diptera: Calliphoridae) and Nasonia vitripennis (Walker) (Hymenoptera: Pteromalidae). Larvae of L. sericata were introduced to containers with soil of different compaction levels, with or without parasitoids. Although females of N. vitripennis did not significantly affect the pupation depth of L. sericata, they increased the rate of pupal development by 15.0–23.7 h at 28.4 ± 1.2 °C, and increased the clumping of puparia. Pupation depth of L. sericata was negatively related to soil compaction; mean depth of pupation was 4.4 cm in uncompacted soil and 0.5 cm in high‐compaction soil. In high‐compaction soil, pupal development increased by 10.5–18.8 h at 25.2 ± 0.3 °C, and puparia were clumped. These results provide a framework for locating puparia in forensic investigations and releasing appropriate parasitoids for biological control of blow flies.     

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.     

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.     

How do food quality and larval crowding affect performance of the autumnal moth,Epirrita autumnata?

Outbreak densities of autumnal moth, Epirrita autumnata (Borkhausen) (Lepidoptera: Geometridae), lead to high larval crowding. Phenotypic responses of E. autumnata to larval crowding and to food quality were studied by measuring growth and consumption as well as pupal weight and fecundity. Crowding may trigger increased consumption and faster development to avoid impending food shortage on good quality food. This is suggested by the result that on a good‐quality diet, the growth of crowded larvae was better than that of solitary larvae, though they did not consume more food than solitary larvae. Crowded larvae also completed the last instar earlier than solitary larvae. The fecundity of crowded autumnal moths was not lower than the fecundity of solitarily grown autumnal moths. This may provide conditions for extra rapid population build‐up of E. autumnata. During the population increase phase the crowding effect may facilitate larval performance; however, at peak density the crowding starts to have negative effects on the performance of larvae. On a poor‐quality diet, the performance of crowded and solitary larvae did not differ. The growth of larvae was better on a good‐quality diet than on a poor‐quality diet, due to higher efficiency in food utilization. Larvae feeding on low‐quality diet did not prolong their development time, but pupated at smaller size; this resulted in lower fecundity. A decrease in food quality can be seen as a cue of oncoming food shortage and resource depletion; it may be advantageous to pupate at a smaller size and ensure survival till reproduction, rather than risk prolonging development to achieve larger size and higher fecundity.     

Geographic origin affects larval competitive ability in European populations of the blow fly, Lucilia sericata

The blow fly, Lucilia sericata (Meigen) (Diptera: Calliphoridae), is found throughout Europe. In northern areas, in addition to breeding in carrion, it can also commonly act as primary facultative ectoparasite of sheep, resulting in animal suffering and production losses. However, in the south of Europe, L. sericata is not usually implicated in myiasis and is found almost entirely in carrion. The causes of this behavioural difference are unclear. To begin to address the possible reasons for this difference, the present study examined the effects of competition on the survival and development of L. sericata populations derived from southern Spain, England, and a hybrid derived from a cross between Spanish females and UK males. Larvae were reared at a range of different initial densities and the mortality, development rate, and size of the resultant adults were measured. Mortality increased significantly with density, but the Spanish population had a significantly higher mortality rate than the UK population at all densities. Similarly, adult size declined with increased larval density, but individuals from the Spanish population were consistently larger than those from the UK population at all densities. The mortality and size of the hybrids was consistently intermediate between the Spanish and UK populations. No consistent effects on development rate were observed. This study demonstrates that insects from northwestern and southern European population have qualitatively different life history traits and competitive abilities and may contribute towards explaining the different behaviour of populations of this species in different part of its distribution range.     

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.     

Extra molting,cannibalism and pupal diapause under unfavorable growth conditions in Atrophaneura alcinous (Lepidoptera: Papilionidae)

Atrophaneura alcinous adopt multiple strategies such as extra molting, cannibalism and pupal diapause under unfavorable growth conditions. The conditions under which these strategies are adopted have been separately verified, but their relationship has often been overlooked. We examined which strategy A. alcinous adopted and the relative advantages of strategies using four experimental groups under different food quantity and individual density conditions. Our results indicated that A. alcinous often extra‐molted, prolonged the larval period, and larval and pupal weights were lighter under food shortage. Cannibalism and disease often occurred under the high density and food shortage condition. We also showed that individuals cannibalized during the prepupal or pupal period were less likely to extra molt before they were killed. Extra molting tended to occur more frequently in females than in males under a shortage of food. In addition, we showed that, when food was insufficient, A. alcinous might initiate pupal diapause under low densities, but not under high densities. These results suggest that A. alcinous prolongs the larval period by engaging in extra molting during times of unfavorable food conditions. This strategy might decrease the risk of cannibalization because surrounding larvae who could potentially cannibalize others pupate before larvae who prolong the larval period pupate. Our results also suggest that diapausing pupae under a shortage of food can only survive when there are few surrounding conspecifics, due to a lower cannibalization risk. In conclusion, there is a complex interaction between extra molting, cannibalism and pupal diapause strategies.     

Impacts of oxygen deficiency on embryo life-history traits of migratory locust Locusta migratoria from low and high altitudes

Hypoxia challenges aerobic organisms in numerous environments, and hypoxic conditions may become more severe under future climate-change scenarios. The impact of hypoxia on the development of terrestrial insect embryos is not well understood. Here, to address this gap, embryonic life-history traits of migratory locust Locusta migratoria from low-altitude and high-altitude regions were compared under 2 oxygen levels: normoxia (i.e., 21 kPa oxygen partial pressure and mild hypoxia (i.e., 10 kPa oxygen partial pressure). Our results demonstrated that, whether reared under normoxia or mild hypoxia, L. migratoria from high-altitude populations had longer developmental times, reduced weight, and lower mean relative growth rate as compared with those from low-altitude populations. When transferred from normoxia to mild hypoxia, nearly all the tested life-history traits presented significant negative changes in the low-altitude populations, but not in the high-altitude populations. The factor ‘strain’ alone explained 18.26%–54.59% of the total variation for traits, suggesting that the phenotypic differences between L. migratoria populations from the 2 altitudes could be driven by genetic variation. Significant genetic correlations were found between life-history traits, and most of these showed differentiation between the 2 altitudinal gradients. G-matrix comparisons showed significant structural differences between L. migratoria from the 2 regions, as well as several negative covariances (i.e., trade-offs) between traits in the low-altitude populations. Overall, our study provides clear evidence that evolutionary divergence of embryonic traits between L. migratoria populations from different altitudes has occurred.     

A Jonah-like chymotrypsin from the therapeutic maggot Lucilia sericata plays a role in wound debridement and coagulation

Lucilia sericata larvae are used in maggot debridement therapy, a traditional wound healing approach that has recently been approved for the treatment of chronic wounds. Maggot excretion products (MEP) contain many different proteases that promote disinfection, debridement and the acceleration of wound healing, e.g. by activating the host contact phase/intrinsic pathway of coagulation. In order to characterise relevant procoagulant proteases, we analysed MEP and identified a chymotrypsin-like serine protease with similarities to Jonah proteases from Drosophila melanogaster and a chymotrypsin from Lucilia cuprina. A recombinant form of the L. sericata Jonah chymotrypsin was produced in Escherichia coli. The activated enzyme (Jonah_m) had a pH optimum of 8.0 and a temperature optimum of 37 °C, based on the cleavage of the chromogenic peptide s-7388 and casein. Jonah_m reduced the clotting time of human plasma even in the absence of the endogenous protease kallikrein, factor XI or factor XII and digested the extracellular matrix proteins fibronectin, laminin and collagen IV, suggesting a potential mechanism of wound debridement. Based on these characteristics, the novel L. sericata chymotrypsin-like serine protease appears to be an ideal candidate for the development of topical drugs for wound healing applications.     

Effect of bisabolangelone on development ofCydia pomonella larvae and oviposition of the females

The effects of bisabolangelone on the development of codling moth larvae,Cydia pomonella L., and on ovipositional behaviour of the females were studied under laboratory conditions. Entry of the neonate larvae into apples and their development on a semi-synthetic medium were completely inhibited when the larvae were exposed respectively to 10 μg a.i./cm² and 20 μg a.i./ml of this compound. While concentrations of 1.25 to 5.0 μg a.i./ml bisabolangelone in the medium did not significantly affect larval development, exposure of the larvae to a higher rate (10 μg a.i./ml) resulted in 80% mortality during the first week. Nevertheless, the larvae which survived the treatments underwent further development until emergence of the adults. No significant changes in duration of larval or pupal periods were recorded. Oviposition of the females in plastic beakers, whose inner surfaces were partially painted with two concentrations of bisabolangelone (1.25 to 5.0 μg a.i./cm²), was significantly reduced and the eggs were mainly laid on the parts painted with the ethanol solvent alone. When the inner surface of the cups was completely treated (i.e. top, bottom, and side) with similar concentrations of bisabolangelone, a dramatic reduction in oviposition occurred and the eggs were mostly laid on the bottom of the beakers. While this compound did not significantly influence egg hatch, it inhibited the normal upward movement of the adults in the cups and migration of the newly hatched larvae through perforations of the lids.