Development of two forensically important blowfly species (Chrysomya megacephala and Chrysomya rufifacies) (Diptera: Calliphoridae) at four temperatures in India

The development of the Oriental latrine fly, Chrysomya megacephala (Fabricius), and hairy maggot blowfly, C. rufifacies (Macquart) (Diptera: Calliphoridae), was studied at four different temperatures (22°C, 25°C, 29°C and 31°C) in order to draw correlations between larval age, body length and body dry weight. The mean larval body length increased steadily from a minimum of 1.4 mm for C. megacephala and 1.8 mm for C. rufifacies to a maximum of 17.4 mm for C. megacephala and 15.9 mm for C. rufifacies at different temperatures. Similarly, the mean dry weight increased steadily from a minimum of 0.0007 g for C. megacephala (second instar) and 0.0008 g for C. rufifacies (second instar) to a maximum of 0.0290 g for C. megacephala and 0.0270 g for C. rufifacies at different temperatures. Entomological evidence is often used to estimate the minimum postmortem interval (mPMI) and both of these species are important from a forensic point of view. Graphs of age of larvae vs. body length and age of larvae vs. dry body weight at different temperatures can be used to estimate the larval age of these two species.     

Effects of bait age, larval chemical cues and nutrient depletion on colonization by forensically important Calliphorid and Sarcophagid flies

Species colonization patterns on corpses and the frequency of carrion fly oviposition and larviposition are affected by decomposition stage and previous maggot colonization. This study investigated these effects on meat bait colonization by Victorian Diptera of forensic importance. Bait treatments were: 'aged' (aged for 4 days at 22 °C, allowing some decomposition); 'nutrient-depleted' [aged for 4 days at 22 °C with feeding Calliphora vicina (Robineau-Desvoidy) (Diptera: Calliphoridae) larvae]; 'extract' (fresh bait mixed with liquid formed by feeding C. vicina larvae), and 'fresh' (untreated control bait). Statistical analysis (α = 0.05) revealed that colonization frequency differed significantly among treatments (Welch's F(3,18.83) = 4.66, P < 0.05). Post hoc tests showed that fresh and extract baits were colonized extensively throughout the experiment with no significant difference, whereas the colonization of nutrient-depleted baits was significantly lower. This suggests that larval digestive enzymes, larval excreta and cuticular hydrocarbons have less effect on colonizing Diptera than the nutritional content of meat. The colonization of aged baits did not differ significantly from that of fresh, extract or nutrient-depleted baits. A further experiment testing 'very aged' (aged for 8 days at 28 °C), 'larvae-added' (fresh bait with C. vicina larvae added before placement) and 'fresh' (untreated control) baits revealed that very aged baits were colonized significantly less frequently than either fresh or larvae-added baits (Welch's F(2, 6.17) = 17.40, P < 0.05).     

The effect of larval temperature and photoperiod on the incidence of larval diapause in the blowfly, Calliphora vicina

ABSTRACT. The blowfly, Calliphora vicina Meigen (Diptera: Calliphoridae), displays a maternally induced larval diapause. Progeny of adults exposed to short days enter diapause if the larval temperature is 15C or below; exposing adults to long days, or larvae to temperatures above 15C, results in non-diapause. By keeping progeny from short-day parents at the parental temperature of 23.5C for various lengths of time before transfer to 11C, it could be shown that the diapause-averting effect of high temperature operates in the late wandering stage of larva, possibly at the moment of the decision for the brain-ring gland complex to release PTTH/ecdysone at the diapause/non-diapause stage.
Although photoperiodic induction in the blowfly is maternal, previous work had shown that the larvae were also slightly sensitive to photoperiod. From the present study it is evident that also the eggs are photo-sensitive.     

Low temperature episodes in development of blowflies: implications for postmortem interval estimation

Traditionally the calculation of accumulated degree days or hours (ADD or ADH) involves the concept of a minimum threshold temperature below which development ceases. Hence in fluctuating conditions, where temperatures drop below this threshold, there may be periods of time when development is taken to be zero. This has important implications when the calculation of postmortem interval (PMI) is based on the ADD or ADH of larval dipterans. Normal development of larvae of the blowflies Calliphora vicina Robineau-Desvoidy and C. vomitoria L. (Diptera: Calliphoridae) at 20 degrees C was interrupted by cold episodes. The expectation was that total development time would increase by the period at low (therefore no development) temperature but the total ADD or ADH should be the same as non-cold treated cohorts. The results, however, showed that total ADH for both species decreased linearly with increasing temperature with no evidence of any minimum threshold temperature effect. The increased ADH at low temperatures may be due to either continued but reduced development or a delay in development restarting after the cold episode. Use of ADH in PMI estimations has shortcomings particularly during the winter period where low temperatures are involved or where there are sudden summer cold spells during the development period. As blowfly development progresses from egg to pupa such errors will be compounded.     

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.     

Temperature-related development of the parasitoid wasp Nasonia vitripennis as forensic indicator

Development times of the forensically significant parasitic wasp Nasonia vitripennis (Walker) (Hymenoptera: Pteromalidae) from oviposition to pupation, and from oviposition to adult emergence, were studied in the laboratory at temperatures of 15-35 degrees C using host pupae of the blowfly Protophormia terraenovae (Robineau-Desvoidy) (Diptera: Calliphoridae). Total developmental time of N. vitripennis from oviposition to adult emergence (mean+/-SD) was 43.5+/-2.4, 22.5+/-1.1, 14.8+/-1.7 and 11.3+/-0.9 days when reared at 15, 20, 25 and 30 degrees C, respectively. At 35 degrees C, N. vitripennis did not develop successfully. The rate of total immature development (1/days) increased with temperature. From linear regression of development rates, it was determined that the minimum threshold (tL) for total immature development was 9.8 degrees C (approximately 10 degrees C). Above this threshold, the overall thermal constant (K) for N. vitripennis was found to be 224.3+/-1.7 degree-days.     

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.     

Blowfly succession from possum (Trichosurus vulpecula) carrion in a sheep-farming zone

The significance of brushtail possum, Trichosurus vulpecula Kerr (Diprotodontia: Phalangeridae) carcasses to the succession and production of Diptera species and its relevance to fly strike management in Tasmania, Australia was examined. Calliphora stygia (Fabricius), Lucilia sericata (Meigen) and Calliphora vicina Robineau-Desvoidy (Diptera: Calliphoridae) were found to be the most abundant and Lucilia cuprina (Wiedemann) (Diptera: Calliphoridae) always the least abundant (< 1%) of the putative primary fly invading species to emerge. Carcasses that were left for up to 15 days in the field before being exposed to flies for 2 days also acted as breeding sites for large numbers of all primary fly species, with the exception of L. cuprina. Ordination analysis revealed no relationship between possum carcasses according to their length of exposure but did show significant negative associations between the number of putative secondary invaders (Chrysomya rufifacies (Macquart) (Diptera: Calliphoridae), Chrysomya varipes (Macquart) (Diptera: Calliphoridae) and putative tertiary flies (Hydrotaea rostrata Robineau-Desvoidy (Diptera: Muscidae)) to the number of C. vicina or C. stygia to emerge. There was enormous variability in the numbers of secondary/tertiary fly species to emerge from carcasses (0-11 450) that negatively correlated with the proportion of all flies to emerge that were primary, and with the mean size of adult L. sericata. Although carcass temperatures, especially those with a large larval population, were elevated, this did not appear to result in significant pre-adult fly mortality. The most important primary fly strike species L. cuprina was only found in insignificant numbers, whereas three other members of the fly strike fauna C. stygia, L. sericata and Ch. rufifacies did use possum carrion as an important breeding resource, but left implications for fly strike management inconclusive.     

Detection of food source by PCR analysis of the gut contents of Aldrichina grahami （Aldrich） （Diptera： Calliphoridae） during post-feeding period

The last meal of sarcophagous maggots may be useful in identifying the species on whose flesh they have fed （the＂host＂species）. The DNA profile of the host species may indeed be detectable in the＂last meal＂. In this paper, mitochondrial DNA analysis of gut contents was used to identify the prior host of post-feeding larvae of Aldrichina grahami （Aldrich） （Diptera： Calliphoridae）. A modified logistic equation was fitted to estimate the probability of identifying the host under five different constant temperatures （16, 20, 24, 28 and 32 ℃）. Our results shows that the detected time ranged from a maximum of 24 h at 32℃ to 42 h at 16℃ and a minimum of 12 h at 32~C to 30 h at 16℃. Furthermore, the host detection time was also calculated to give the maximal time after larval hatching from the egg. These results indicate that, in criminal cases where the maggots stray from the corpse, the last meal of the larvae should not be overlooked as potentially critical evidence.     

Antibacterial properties of larval secretions of the blowfly, Lucilia sericata

The antibacterial properties of secretions aseptically collected from larvae of the greenbottle fly Lucilia sericata (Meigen) (Diptera: Calliphoridae) were examined. These investigations revealed the presence of small (<1 kDa) antibacterial factor(s) within the larval secretions, active against a range of bacteria. These include the Gram-positive Staphylococcus aureus, both methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-sensitive Staphylococcus aureus (MSSA), Streptococcus pyogenes and to a lesser extent the Gram-negative Pseudomonas aeruginosa. These secretions were shown to be highly stable as a freeze-dried preparation and, considering the activity against organisms typically associated with clinical infection, may be a source of novel antibiotic-like compounds that may be used for infection control and in the fight against MRSA.     

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.     

Description of the Photoperiodic Control of Larval Burrowing in the Blowfly Lucilia Cuprina: a Novel Index for Photoperiodic Research

The photoperiodic control of larval burrowing depth in the Australian sheep blowfly Lucilia cuprina (Wiedemann) (Diptera: Calliphoridae) was investigated by measuring burrowing depth under controlled laboratory photo-periods. The results demonstrated that larvae exposed to long photoperiods (LD 18:6) burrowed to deeper depths than those in shorter photoperiods (LD 12:12), and that this behavior was induced during the third instar stage. The ecological significance of this behavior is discussed, as are the ways in which daylength is measured and depth assessed. The use of burrowing depth could prove to be a novel index of a photoperiodic response and provide a far simpler approach to the study of photoperiodism in certain insect species. (Chronobiology International, 14(3), 247–252, 1997)     

Development changes of cuticular hydrocarbons in Chrysomya rufifacies larvae: potential for determining larval age

Age determination is the basis of determining the postmortem interval using necrophagous fly larvae. To explore the potential of using cuticular hydrocarbons for determining the ages of fly larvae, changes of cuticular hydrocarbons in developing larvae of Chrysomya rufifacies (Macquart) (Diptera: Calliphoridae) were investigated using gas chromatography with flame-ionization detection and gas chromatography-mass spectrometry. This study showed that the larvae produced cuticular hydrocarbons typical of insects. Most of the hydrocarbons identified were alkanes with the carbon chain length of 21-31, plus six kinds of alkenes. The hydrocarbon composition of the larvae correlated with age. The statistical results showed that simple peak ratios of n-C29 divided by another eight selected peaks increased significantly with age; their relationships with age could be modelled using exponential or power functions with R(2) close to or > 0.80. These results suggest that cuticular hydrocarbon composition is a useful indicator for determining the age of larval C. rufifacies, especially for post-feeding larvae, which are difficult to differentiate by morphology.     

Toxicity and behavioural effects of diet-borne alkaloids on larvae of the black blowfly,Phormia regina

Larvae of the black blowfly, Phormia regina (Meigen) (Diptera: Calliphoridae) were exposed for 24 h to artificial diets that contained one of the following alkaloids: arecoline, caffeine, nicotine, quinine, sparteine or strychnine at either 1000 or 100 p.p.m. Each of the alkaloids caused reduced weight gain, relative to a control population in a no-choice bioassay and, with the exception of quinine, all alkaloids caused reduced larval weights in a choice bioassay. Larvae were unable to move away from diets containing arecoline (1000 and 100 p.p.m) and congregated away from diets containing 1000 p.p.m. quinine. Arecoline (1000 p.p.m) and both concentrations of nicotine caused significant mortality of larvae. Over a longer period (120 h), 10 and 1 p.p.m. nicotine resulted in significant numbers of larvae congregating away from a treated diet. Ten p.p.m. nicotine caused reduced weight gain over 120 h, although larvae provided with a choice were less affected. Exposure of larvae to dried residues of nicotine for 2 h did not affect subsequent development.     

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.     

Cold susceptibility and disinfestation of Bactrocera invadens (Diptera: Tephritidae) in oranges

To develop a cold disinfestation treatment for the fruit fly Bactrocera invadens Drew, Tsuruta & White (Diptera: Tephritidae) that is rapidly spreading across Africa, research was conducted in Nairobi, Kenya, using flies from a laboratory culture and 'Valencia' orange (Citrus sinensis L. Osbeck) as the host. The developmental rate of B. invadens in Valencia oranges was determined at 28 degrees C, and the third instar was found to be the least susceptible of the egg and larval life stages to cold treatment at 1.1 degrees C in oranges. When 22,449 B. invadens third instars were exposed in oranges to a cold treatment with an approximate midpoint of 1.1 +/- 0.5 degrees C, the results suggested that a period of 16 d would be worthwhile verifying on a larger scale in oranges. Results from the first replicate of 16,617 larvae showed no survivors, but the second replicate of 23,536 larvae had three survivors. Because a longer cold treatment based on a mean temperature of 1.1 degrees C would create logistical difficulties for some export markets, further replicates were conducted at an approximate midpoint of 0.5 degrees C and at mean hourly maximum of 0.9 +/- 0.5 degrees C, for 16 d. After three replicates, in which 65,752 B. invadens third instars in total were treated with no survivors, the Japanese requirement of 99.99% mortality at the 95% confidence level was surpassed. The following treatment protocol for B. invadens larvae in oranges can therefore be recommended: fruit pulp to be maintained at temperatures of 0.9 degrees C or lower for 16 consecutive days.     

Effect of body length and temperature on the crawling speed of Protophormia terraenovae larvae (Robineau-Desvoidy) (Diptera Calliphoridae)

This work investigates variation in the crawling speed of Protophormia terraenovae larvae (Robineau-Desvoidy) (Diptera Calliphoridae) as a function of body length and ambient temperature. A video-tracking system was used to follow the movement of larvae in an experimental arena, and to estimate their average crawling speed. Recordings were carried out at various temperatures using larvae of different developmental stages. As temperature increased, the larvae crawled at a faster speed. Furthermore, speed increased as a function of body length. We present a general allometric relationship to express the average crawling speed of larvae as a combined function of temperature and body length, expressed in the following equation: speed (cm/min)=5.45 x log[length (mm)]+0.66 x temperature (degrees C)-12.8. These results are particularly useful in forensic entomology for estimating the time that elapsed following the larva's departure from the corpse. This information can provide more accuracy for post-mortem interval estimations and a better comprehension of larvae behaviour and displacement.     

Comparison of the predation rate of freshwater cyclopoid copepod species on larvae of the mosquito Culex pipiens

The predation rates of six copepod species: Acanthocyclops robustus G.O. Sars, Eucyclops neumani Pesta, Macrocyclops albidus Jurine, Mesocyclops longisetus Thibaud, Metacyclops grandis Kiefer and Metacyclops mendocinus Wierzejski (Copepoda: Cyclopidae) on mosquito larvae, Culex pipiens Linnaeus (Diptera: Culicidae) were assessed. Experiments consisted of 24-h incubations of one copepod and 10 larvae without extra food at 16 degrees C and 26 degrees C. Nine replicates were considered for each species and temperature. Predation rates (larvae per copepod per day) were: M. mendocinus (1.8), M. grandis (3.1), E. neumani (3.8), A. robustus (3.8), Ma. albidus (6.1) and Me. longisetus (7.0). There was a significant effect of both species and temperature on predation: all species experienced higher predation at 26 degrees C than at 16 degrees C, except for A. robustus whose predation rate was similar at both temperatures. These observations are consistent with previous results that point to Macrocyclops and Mesocyclops genera as important larval predators and suggest the need for field trials to evaluate the response of Ma. albidus and Me. longisetus under natural conditions in Uruguay.     

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 impacts of larval density and protease inhibition on feeding in medicinal larvae of the greenbottle fly Lucilia sericata

Larval therapy, the therapeutic use of blowfly larvae to treat chronic wounds, is primarily used in debridement. There are, however, gaps in current knowledge of the optimal clinical application of the therapy and mechanisms of action in the debridement process. Using an artificial assay, two studies were undertaken to investigate these aspects of larval debridement by Lucilia sericata Meigen (Diptera: Calliphoridae); the first studied the effects of the density of larvae on tissue digestion and larval mass, and the second considered the effects on the same parameters of incorporating protease inhibitors into the feeding substrate. The total mass of tissue digested increased with larval density until saturation was observed at 5.0–7.5 larvae/cm². This range was considered optimal as lower doses resulted in the removal of less tissue and higher doses offered no additional tissue removal and appeared to exacerbate competition for feeding. In the second study, increased protease inhibitor concentration led to significant decreases in tissue digestion and larval mass, suggesting that serine proteases, particularly trypsin, may play major roles in larval digestion. Such information is important in elucidating the main constituents that make up larval digestive products and may be significant in the development of new therapies.