Blow Fly Larvae Research Articles

Flame impingement affects survival and development of Lucilia sericata (Diptera: Calliphoridae) on securely wrapped remains

Wrapping and burning are techniques to hide or destroy evidence on human remains. Both may influence the ability for forensically important arthropods, such as blow flies (Diptera: Calliphoridae), to colonize and develop to adulthood on remains. The combined effects of wrapping and burning a body on blow flies has not been examined in the literature, therefore this was examined using two experiments: (1) measuring the potential delays in oviposition due to wrapped remains and (2) the survival of blow fly larvae on wrapped bodies exposed to flame impingement. First, three domestic pig carcasses were wrapped in 100% polyester blankets and sealed at the end with duct tape, while three remained un-wrapped. After three days of exposure to natural colonization, blow flies delayed oviposition by at least 21 h on the wrapped remains. Second, six pig carcasses were colonized with 250 first instar Lucilia sericata (Meigen). Three of these carcasses were wrapped as described in the first experiment. Larvae were allowed three days of feeding and development, then the pigs were transferred to a rural, two-storey farmhouse, which was set ablaze. The local fire department suppressed the fire and removed the pigs. Compared to un-wrapped carcasses, wrapping increased survival of live larvae and recoverability of live and dead larvae. The blanket acted as an extra layer of protection, increasing maggot survival, however, live insect evidence collected and reared post-burn provided a less accurate minimum post-mortem interval estimate than maggots killed by the fire.

Insect rearing protocols in forensic entomology: Benefits from collective rearing of larvae in a carrion beetle Necrodes littoralis L. (Silphidae).

Forensic entomologists frequently use a developmental method to estimate a post-mortem interval (PMI). Such estimates are based usually on the blow fly larvae or puparia. Data on their development is obtained by rearing them in colonies. In the case of beetles, which can be also useful for PMI estimation, development data is frequently collected by rearing them individually. However, some carrion beetles are gregarious, for instance, Necrodes littoralis (Linnaeus, 1758) (Silphidae). We compared mortality, rate of development and body size of emerged adult beetles reared individually and in aggregations. Mortality was much higher for beetles reared individually, particularly at low temperatures. The rearing protocol affected the time of immature development and the size of adult insects. Individually reared specimens developed much longer at 16°C, whereas at 20°C and 26°C development times of individually reared beetles were slightly shorter. Significant differences in the body size were observed only at 16°C; beetles that developed in aggregations were larger at this temperature. These findings demonstrate that aggregating is particularly beneficial for larvae of N. littoralis at low temperatures, where it largely reduces mortality and facilitates growth. Moreover, these results indicate that in forensic entomology the protocol of individual rearing is unsuitable for gregarious beetles, as it produces reference developmental data of low quality.

Experimental manipulation of cavity temperature produces differential effects on parasite abundances in blue tit nests at two different latitudes.

Although different predictive models forecast that climate change will alter the distribution and incidence of parasitic diseases, few studies have investigated how microclimatic changes may affect host-parasite relationships. In this study, we experimentally increased the temperature inside nest boxes of the blue tit Cyanistes caeruleus during the nestling period at two different latitudes (central Spain and central Germany) to determine its effect on parasite abundance. The two localities have contrasting climate conditions: the southern one in Spain is warmer and drier than the northern one in Germany. Consistent with this, we observed that the parasitic fauna in nests at the two localities differs. The flea species Ceratophyllus gallinae was more abundant in the northern locality, while the blowfly species Protocalliphora azurea and biting midge species of the genus Culicoides were more abundant in the southern one, as were blood parasites. Moreover, dermanyssid mites and blackflies (Simuliidae) were observed only in the southern locality. The temperature inside nest boxes was increased using heat mats placed underneath the nest material during the nestling period (day 3 to day13 post-hatching). Compared with control nests, the average temperature in heated nests increased by 2.24 °C and 1.35 °C at night in Spain and Germany, respectively. Consequently, the average relative humidity in heated versus control nests decreased 4.93 and 0.82 units in Spain and Germany, respectively. The abundance of blowfly pupae in the heated nests was significantly lower than that of control nests at both localities. The abundance of larval fleas was also lower in the heated nests, but only at the Spanish locality. Infection by the blood parasites Haemoproteus/Plasmodium was higher in males attending the heated nests in Germany, and the control nests in Spain. Moreover, both male body mass and nestling wing length were negatively related to the abundance of larval fleas. In conclusion, our results indicate that increased temperature at the nestling stage may affect the fitness of blue tits by altering parasite prevalence rates.

Effect of density and species preferences on collective choices: an experimental study on maggot aggregation behaviours.

Collective decisions have been extensively studied in arthropods, but they remain poorly understood in heterospecific groups. This study was designed to (1) assess the collective behaviours of blow fly larvae (Diptera: Calliphoridae) in groups varying in density and species composition, and (2) relate them to the costs and benefits of aggregating on fresh or decomposed food. First, experiments testing conspecific groups of Lucilia sericata and Calliphora vicina larvae, two species feeding at the same time on fresh carcasses, demonstrated decreases in growth and survival on rotten beef liver compared with fresh liver. However, mixing species together reduced this adverse impact of decomposition by increasing the mass of emerged adults. Second, larval groups were observed in binary choice tests between fresh and rotten liver (i.e. optimal and sub-optimal food sources). The results showed that larvae interacted with each other and that these interactions influenced their food preferences. We observed that (1) larvae were able to collectively choose the optimal food, (2) their choice accuracy increased with larval density and (3) the presence of another species induced a reversal in larval preference towards rotten food. These results highlight the ubiquity of collective decision properties in gregarious insects. They also reveal an unexpected effect of interspecific association, suggesting the colonization of new resources through a developmental niche construction.

Evolutionary profile of the family Calliphoridae, with notes on the origin of myiasis.

The family Calliphoridae is a group of heterogenous calyptrate flies with a worldwide distribution including species of ecological, veterinary, medical, and forensic importance. Notorious for their parasitic habits, the larvae of many blowflies are characterised – like some other dipteran larvae – by their ability to develop in animal flesh. When parasitism affects a living host, it is termed “myiasis”. This has led the Calliphoridae to be considered as a pivotal family in its relationship with a man. Nevertheless, even after more than 50 years of research, the phylogenetic relationships among calliphorid subfamilies together with the evolutionary origin of myiasis remain unclear. In order to elucidate these problems, we constructed three phylogenetic trees by using nucleotide sequence data from cytochrome oxidase subunit one (COI), representing a mitochondrial conservative gene, and nuclear 28S subunit of ribosomal RNA gene (28S rRNA) in order to interpret the evolutionary profile of myiasis in the family Calliphoridae. The sequenced data represented species associated with ectoparasitic life-styles, either saprophagy or facultative and obligate parasitism. A total number of 50 accessions were collected for 28S rRNA, 56 for COI, and 38 for combined sequences phylogeny. Molecular Evolutionary Genetics Analysis (MEGA) software was used to align 2197 nucleotide positions of 28S rRNA and 1500 nucleotide positions of COI with a gap opening penalties and gap extension penalties equalling 20 and 0.1 respectively. The results reveal the non-monophyly of the family Calliphoridae despite the stable monophyletic status of the Chrysomyinae, Luciliinae, and Auchmeromyiinae. Also, our findings recommend ranking the Toxotarsinae as a separate family. Furthermore, comparative analysis of the phylogenetic trees shows that the habit of obligatory myiasis originated independently more than five times. This strengthens our hypothesis that the origin of eating fresh meat is a case of convergent evolution that has taken place after speciation events millions of years ago. Finally, estimating the divergence dates between lineages from molecular sequences provides a better chance of understanding their evolutionary biology.

Colonisation of finfish substrate inhabited by black soldier fly larvae by blow flies, bacteria, and fungi

Black soldier fly,Hermetia illucens (L.) (Diptera: Stratiomyidae) can be used for recycling a wide variety of organic wastes. However, these wastes are also suitable for colonisation by pest fly species, including the blow flyLucilia sericata (Meigen) (Diptera: Calliphoridae), and by pathogenic microorganisms. We tested effects of the presence of black soldier fly larvae (BSFL) on recruitment and retention of blow fly larvae, as well as on composition and dynamics of microbial communities in finfish-based substrates. Substrates inhabited by BSFL contained approximately one-tenth of theL. sericata pupae found in the control substrate even when BSFL were removed prior to the introduction of gravidL. sericata females. Furthermore, almost three-quarters ofL. sericata released into substrates previously colonised by BSFL emigrated within 24 h, while no such emigration was detected for the control substrates. Unlike previous studies, bacterial community was heavily dominated by Gram-positive species. Overall bacterial abundance on BSFL substrates declined approximately two-fold compared to the control substrate, while fungal abundance increased. However, surviving microbial communities were still robust and diverse, and continued to be dominated by Gram-positive bacteria. On the other hand, Gram-negativeShigella sonnei, which is a common cause of diarrhoea in humans, was more than 200 times less abundant in BSFL substrates compared to the control throughout the experiment. Repellent and antibiotic properties observed in this study are advantageous for using BSFL in remediation of finfish wastes. However, additional treatment of remaining residue may still be necessary to eliminate all biological contamination.

Unidirectional en masse larval dispersal of blow flies (Diptera: Calliphoridae)

Decomposition of carrion is a complex ecological process wherein nutrients and energy are recycled in the environment. After feeding but prior to pupation, blow fly larvae disperse from decomposing carrion, presumably to escape unfavorable microsite conditions or perhaps predation. By leaving the immediate area, larvae are effectively transporting carrion-derived nutrients throughout the ecosystem. This dispersal is generally characterized as individual-based and moving randomly from the carcass in every direction, however, non-random, en masse maggot dispersal has been documented at least twice. Here, we report another incidence of en masse blow fly larval dispersal traveling due east in a small column. At approximately 2 m, the column stopped its unidirectional path and dispersed in a radial, 360° pattern. A subsample of 5 larvae was collected, returned to the lab, and identified as Lucilia coeruleiviridis, a common blow fly often involved in carrion decomposition in nature. These observations contribute to an increasing body of evidence that migrating fly larvae do not always disperse randomly, which may have important implications for the dispersal of carrion-derived nutrients as well as interactions with blow fly predators.

Expanding our view of Bartonella and its hosts: Bartonella in nest ectoparasites and their migratory avian hosts

BackgroundBartonella is a genus of Gram-negative facultative intracellular Alphaproteobacteria of public health importance. Although they are known to mainly infect mammalian hosts with some blood-feeding arthropods having been confirmed as vectors, there is some evidence of Bartonella association with non-mammalian hosts including birds.MethodsHere we used high-throughput sequencing of 16S rRNA and Sanger sequencing of the citrate synthase (gltA) genes to test for the presence of Bartonellaceae in the blood of three migratory cavity nesting bird species, purple martins (Progne subis), tree swallows (Tachycineta bicolor) and eastern bluebirds (Sialia sialis) and their most prevalent and abundant nest ectoparasites, Dermanyssus prognephilus (mite), Ceratophyllus idius (flea) and Protocalliphora sialia (bird blow fly larva). We constructed maximum likelihood phylogenetic trees to verify the placement of the resulting sequences in the Bartonellaceae.ResultsWe found evidence of Bartonella in all three bird species and all three arthropod species tested. We report multiple instances of identical Bartonella sequences in both birds and parasites, leading to the likely hypothesis that these ectoparasites are potential vectors of Bartonella. Our phylogenetic analysis suggests that ‘avian Bartonella’ may form its own sub-clade within the genus Bartonella.ConclusionsTo the best of our knowledge, we provide the first confirmation of overlapping Bartonella strains among bird hosts and various species of nest-associated ectoparasites from the same system, suggesting a possible Bartonella host–vector relationship between these arthropods and a non-mammalian host. Our study adds to the growing appreciation of the Bartonellaceae as a phylogenetically diverse group with a wide range of hosts.

A Survey on Inhibitory Effect of Whole-Body Extraction and Secretions of Lucilia sericata's Larvae on Leishmania major In vitro.

Background:Leishmaniasis is a skin disease caused by Leishmania parasite. Despite being self-limiting, must be treated. Available drugs have side effects and drug resistance has also been seen.Materials and Methods:Maggot debridement therapy (MDT) is using sterile fly larvae (maggots) of blow flies (Lucilia sericata) for the treatment of different types of tissue wounds. Larvae have excreted and secreted substances that have been proved to have antimicrobial effects, in addition to the some other specifications.Results:In this study, the anti-leishmanial effects of extracts and secretions of sterile second- and third-instar larvae of L. sericata on the growth of Leishmania major promastigotes and amastigotes in the J774 macrophages have been evaluated in vitro.Conclusion:The results showed that extracts and secretions had almost the same leishmaniocidal effect on promastigotes and intracellular amastigotes without cytotoxic effect on macrophages.

A serine protease-associated lectin in the cytolytic system of blowfly (Chrysomya megacephala) larvae: Evidence and characterization.

Cytolytic activity against invading microorganisms is one of the innate forms of immunity in invertebrates. A serine protease-associated sialic acid-specific cytolytic lectin was purified using glutaraldehyde-fixed ox erythrocytes from the larval extract of blowfly (Chrysomya megacephala). The purified lectin lysed vertebrate erythrocytes with effective haemolysis of ox red blood cells (RBCs) in an isotonic medium. The degree of haemolytic (HL) activity of the purified cytolytic lectin depended on its concentration, pH, temperature, and calcium ions. It was sensitive to ethylenediaminetetraacetic acid. The native molecular mass of the C-type lectin was 260 ± 26 kDa, comprising four different polypeptide subunits of 75 kDa (pI ~8), 69 kDa (pI ~7.0), 61 kDa (pI ~5.3), and 55 kDa (pI ~4.6). The association between the C-type lectin and serine protease was confirmed by MALDI-TOF-MS analysis that revealed its homology in the same spectral peak as well as the proteases and phenylmethylsulphonyl fluoride inhibition of HL activity. Haemolysis inhibition by N-acetylneuraminic acid and other sugars revealed the properties of the lectin. The purified lectin distorted the integrity of ox RBCs and Paenalcaligenes hermetiae. This in vitro study documents the presence of a cytolytic system in blowfly (C. megacephala) larvae for the clearance of invading microbial pathogens in their feeding niche.

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

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

Microbial communities of salmon resource subsidies and associated necrophagous consumers during decomposition: Potential of cross-ecosystem microbial dispersal

Ephemeral resource subsidies affect ecosystems at both short- and long-term temporal scales. One such resource is carrion, which varies in size and nutrient composition. Salmon of the Pacific Northwest have an anadromous life cycle resulting in immatures feeding and developing in vast oceanic expanses and adults returning to freshwater streams for spawning and eventual death, leaving large numbers of carcasses to decompose. Nutrients from these carcasses act as subsidies for both aquatic and terrestrial communities. However, what is less understood is the fate of microbial communities of the salmon upon death and then decomposition in watershed ecosystems. We evaluated the microbiomes of salmon carcasses and how they changed in conjunction with associated necrophagous larval blow fly consumers (Diptera: Calliphoridae). We determined microbes were shared among the carcass resource, internal communities of blow fly larvae, and excretions and secretions from blow fly larval aggregates. Larval feeding also represented a possible filtering effect on some microbial taxa from the collective community due to the observed decrease in microbial richness and diversity during larval growth and development. These results suggest some carcass resource microbes are not detected in the primary insect consumers during feeding, assimilation, and egestion. Ultimately, taxa remaining with larvae can potentially be dispersed into an ecosystem by adult flies. Our findings support a more general hypothesis of the potential of microbial transfer among ecosystems via salmon necrobiome food webs. Additional controlled field studies are needed to test and identify the ubiquity and mechanism of these potential bottom-up trophic dynamics.

Proteomics and Lipidomics of Black Soldier Fly (Diptera: Stratiomyidae) and Blow Fly (Diptera: Calliphoridae) Larvae.

Farming insects has recently emerged as a new source of protein and lipid production. To date, research has mostly focused on food applications of insects. Focusing on nonfood potential of oil and proteins of insects, high-throughput studies of insect lipids and proteins are needed. We performed proteomics and lipidomics investigation on black soldier fly (Hermitia illucens) and blow fly (Lucilia sericata) larvae to investigate new potential and applications. We used mass spectrometry for proteomics and lipidomics analysis of control and treated larvae. Treatment was performed by incubation with a biological decomposer. We provide the list of all fatty acids with their concentration in control and treated larvae. This result showed high levels of lauric acid in black soldier fly, which could even increase after biological decomposition. Proteomics analysis showed the presence of proteins like collagen of cosmetic interest, and proteins with antimicrobial properties such as phenoloxidases and enzymatic activities, such as amylase and trypsin. Insects harbor high potential for nonfood usage as additives, antimicrobial effects, and even pharmaceuticals and cosmetics. These data open avenues for future research in pharmacological and cosmetic approaches to find new molecules of interests.

Study on food consumption by blow fly maggots and their percent adult emergence under laboratory conditions

Abstract Blow flies plays an important role in decomposition process and consume the maximum carrion. To study the food consumption by blow fly larvae and percent adult emergence, the present research works was carried out at Entomology research laboratory, The University of Agriculture, Peshawar during the year 2015. In 1st experiment, two rabbits were killed by intra cardiac injection of 10cc magnesium sulphate per rabbit and one each was placed in sunny and shaded areas. In 2nd Experiment, one rabbit was killed by intra cardiac injection of magnesium sulphate while one rabbit was slaughtered with a sharp knife. Blowfly maggots were collected from the rabbit carrion with the help of forceps and were brought to the laboratory for rearing on cow meat at 25 ± 2 oC and RH 65 ± 10% to get the adult blowfly. Meat consumption was calculated by exposing 30 grams meat slice to 10 actively feeding larvae. The meat consumption per larvae was recorded highest (1.95 gram) in larvae collected from slaughtered rabbit and lowest (1.91 gram) in larva collected from the rabbit killed by injection. Furthermore, results revealed that the meat consumption per larva and percent adult emergence appeared to be similar in both sunny and shaded areas while the rabbit killed by injection yielded higher percent adult emergence (88.97%) in comparison to slaughtered rabbit with (83.39%) emergence. Keywords: Blow flies; Carrion flies; Food consumption; Forensic entomology; Percent adult emergence http://dx.doi.org/10.19045/bspab.2019.80021

Technical note: A rapid, non-invasive method for measuring live or preserved insect specimens using digital image analysis

The measurement of insects is an important component of many entomological applications, including forensic evidence, where larvae size is used as a proxy for developmental stage, and hence time since colonization/death. Current methods for measuring insects are confounded by varying preservation techniques, biased and non-standardized measurements, and often a lack of sample size given practical constraints. Towards enhanced accuracy and precision in measuring live insects to help avoid these variables, and that allows for different measurements to be analyzed, we developed a non-invasive, digital method using widely available free analytical software to measure live blow fly larvae. Using crime scene photographic equipment currently standard in investigation protocols, we measured the live length of 282 Phormia regina larvae. Repeated measurements of maggots, for all instars, were performed for several orientations and images. Most accurate measurements were obtained when maggots were oriented in their natural full extension. Killed specimens resulted in greater length measurements (Mean 1.79 ± 1.11 mm) when compared to live length. Herein, we report a technically simple, fast, and accurate measurement technique adapted for field and lab-based measurements, as well as, a simple linear equation for conversion of live length to standard killed length measurements. We propose this method be utilized for the standardization of forensic entomological evidence collection and development model creation.

The effects of bacterial endotoxin (LPS) on cardiac function in a medicinal blow fly (Phaenicia sericata) and a fruit fly (Drosophila melanogaster)

The bacterial endotoxins, lipopolysaccharides (LPS), are known to have direct effects on mammalian heart cells; thus, LPS is likely to have some effects in other cardiac models. Drosophila melanogaster was used since it serves as a model for cardiac physiology. Larvae of blow flies (Phaenicia sericata) commonly used as therapy for debriding dead tissue, are exposed to high levels of bacterial endotoxins, but their mechanisms of LPS resistance are not entirely understood. Comparative effects of LPS on heart rate (HR) were examined for both Drosophila and blowfly larvae. Acute 10-min direct exposure of in situ heart tubes with saline containing 1, 100, and 500 μg/ml LPS from two common bacterial stains (Pseudomonas aeruginosa and Serratia marcescens) revealed a dose-dependent effect. The effects differed between the two fly models. Larval hearts of Drosophila stopped rapidly in low Ca2+ containing saline, but the hearts of blow flies appear unaffected for >30 min. S. marcescens increased HR initially in Drosophila followed by a reduction for low and high doses, but no change was observed in larvae of blow flies. Whereas P. aeruginosa at a high dose decreased HR in larvae of Drosophila but increased HR in larvae of blow flies. The goal of this study is to better the understanding in the direct action of LPS on HR. Knowing the acute and direct actions of LPS exposure on HR in different species of larvae may aid in understanding the underlying mechanisms in other animals during septicemia.

Two species of Caiusa Surcouf (Diptera: Calliphoridae) new to India, with data on larval behaviour and morphology.

Caiusa Surcouf (Diptera: Calliphoridae) is an Old World genus of blow flies, the larvae of which feed on egg masses in the foam nests of various species of rhacophorid tree frogs. Here, we provide the first records for India (West Bengal, Eastern India) of Caiusa coomani Séguy, 1948 and C. karrakerae Rognes, 2015, together with new information on the behaviour and morphology of their larvae. Active surface swimming to disperse from infested nests is documented in blow fly larvae for the first time, as is the presence of a large internal air sac presumably acting as a floating aid. Chiromantis simus (Annandale, 1915) (Anura: Rhacophoridae) egg masses are first recorded as a feeding substrate of Caiusa larvae.

Collective exodigestion favours blow fly colonization and development on fresh carcasses

Necrophagous flies breeding on carcasses face high selection pressures and therefore provide interesting opportunities to study social adaptations. We postulated that gregariousness in necrophagous blow fly larvae is an adaptive response to the environmental constraints of fresh carcasses. Cooperation is indeed believed to be key to the global success of social species. To test this idea, the development of Lucilia sericata (Diptera: Calliphoridae) larvae growing on low- or high-digestibility food substrate (control or trypsin-added ground beef muscle, respectively) at different larval densities was monitored. Results showed that larvae developed faster and had decreased mortality at high than low larval density. Furthermore, aggregation had no deleterious effect on the morphological characteristics (e.g. size) of postfeeding larvae and adult flies. We concluded that increased density positively affected population fitness, which is a conclusion consistent with the predictions of the Allee effect. Compared with those fed on regular food, larvae fed on high-digestibility food had reduced mortality and faster development on average. From these results, we postulated that collective exodigestion might be an adaptive response allowing blow flies to colonize fresh carcasses before the arrival of other insects and the multiplication of microbes. This hypothesis is consistent with the idea that cooperation may enable species to expand their niches.

The movement of fly (Diptera) larvae within a feeding aggregation

AbstractDipteran larvae from a number of families feed in aggregations. Rotation of blow fly (Diptera: Calliphoridae) larvae within an aggregation has been reported anecdotally many times. However, there is a lack of quantitative data on such larval movement, which is necessary to better understand the advantage of this gregarious behaviour. A recent development in tagging methods provided an opportunity to address this gap in knowledge. In 15 aggregations of 500 Lucilia sericata (Meigen) (Diptera: Calliphoridae) larvae, the location of four-tagged individuals was recorded at 10-minute intervals. All larvae were seen to rotate, alternating between the periphery and within. There was much variation in the relative proportions that larvae were seen in these two locations among aggregations (χ2=78.4, df=58, P=0.038), perhaps as a result of differences in mass shape and, therefore, surface area: volume ratio. There were also differences between larvae within aggregations (χ2=25.6, df=14, P=0.029), which may give rise to differences in development rate, perhaps as a result of intraspecific competition. Further work would be required to verify this competition, and to establish whether the limited resource is temperature, food, oxygen, or some other requirement.

Dating Pupae of the Blow Fly Calliphora vicina Robineau-Desvoidy 1830 (Diptera: Calliphoridae) for Post Mortem Interval-Estimation: Validation of Molecular Age Markers.

Determining the age of juvenile blow flies is one of the key tasks of forensic entomology when providing evidence for the minimum post mortem interval. While the age determination of blow fly larvae is well established using morphological parameters, the current study focuses on molecular methods for estimating the age of blow flies during the metamorphosis in the pupal stage, which lasts about half the total juvenile development. It has already been demonstrated in several studies that the intraspecific variance in expression of so far used genes in blow flies is often too high to assign a certain expression level to a distinct age, leading to an inaccurate prediction. To overcome this problem, we previously identified new markers, which show a very sharp age dependent expression course during pupal development of the forensically-important blow fly Calliphora vicina Robineau–Desvoidy 1830 (Diptera: Calliphoridae) by analyzing massive parallel sequencing (MPS) generated transcriptome data. We initially designed and validated two quantitative polymerase chain reaction (qPCR) assays for each of 15 defined pupal ages representing a daily progress during the total pupal development if grown at 17 °C. We also investigated whether the performance of these assays is affected by the ambient temperature, when rearing pupae of C. vicina at three different constant temperatures—namely 17 °C, 20 °C and 25 °C. A temperature dependency of the performance could not be observed, except for one marker. Hence, for each of the defined development landmarks, we can present gene expression profiles of one to two markers defining the mentioned progress in development.