The effect of postmortem penetrative trauma on oviposition behavior of Calliphora vicina.

Flies in the family Calliphoridae are purported to demonstrate a strong attraction to and preferential colonization of wounds when present on human corpses or carrion. This well-circulated concept in Forensic Entomology is based on surprisingly few empirical studies that have examined the oviposition behavior of necrophagous Diptera toward wounds of vertebrate animals. In the present study, the oviposition behavior of Calliphora vicina toward piglets inflicted with postmortem sharp force trauma was examined during a 10-h test period under controlled laboratory conditions and in an outdoor urban environment. Three species of flies (C. vicina, C. livida and Cynomya cadaverina) deposited eggs on wounded and non-wound piglets during the field studies, although more than 80% of all eggs laid were by C. vicina regardless of wound status. For all species, oviposition occurred predominately on the head, including in eyes, ears, nose and mouth, and less frequently in other locations on the body. Eggs were never found in or near wounds. Similar oviposition patterns were displayed by C. vicina under laboratory conditions, with the exception for limited egg deposition in wounds under overcrowded conditions. In this latter scenario, egg deposition represented only 0.14% of the total reproductive output of adult flies. The findings do not support the contention that calliphorids preferentially oviposit in wounds resulting from sharp force penetrating trauma.

Differential responses of adult Calliphora vicina to dry bloodstains on porous versus non-porous surface materials.

Necrophagous flies are presumed to feed on wet and dried blood at crime scenes, but no empirical information exists detailing fly interactions with dried bloodstains. In the present study, the foraging behavior of adult Calliphora vicina was characterized during interactions with dried bloodstains formed on a variety of porous, and non-porous materials that are commonly encountered in a household. Continuous digital recording and image analysis were used to monitor fly interactions with dried bloodstains and to determine mechanisms of stain modification. Flies displayed differential responses to bloodstains based on the porosity and topography of the surface material. For instance, blood that was not tightly adhered to the materials was flaked or dislodged by fly activity and was not consumed by the flies. On other non-porous surfaces, most stains were consumed following moistening by regurgitation. Feeding activity on such bloodstains frequently yielded partial, perimeter and skeletal stains. In contrast, adult flies rarely mechanically altered dried blood on porous fabrics and there was no evidence of modification due to feeding. Feeding avoidance behavior was observed due to tactile inhibition with blood dried on rough, uneven surfaces like cotton and denim.

Retention of Human Body Fluids in Adults of Calliphora vicina (Diptera: Calliphoridae).

Foraging by Calliphora vicina Robineau-Desvoidy often leads to a period of bubbling behavior, followed by either deposition of the regurgitate onto surfaces or reuptake of the bubble. Eventually, the partially or undigested food is passed in the excreta forming fecal or defecatory stains on surfaces in which deposition occurs. This study examined the digestive artifacts (i.e., regurgitate and defecatory stains) formed following consumption of human blood and semen by adult flies in an attempt to determine the length of time the meal was retained in the crop. The morphological appearance of either type of stain appeared consistent with the color of blood or semen for 10-20 d after feeding. When tested with ABA Hematrace immunochromatographic strip assays, blood was detectable in at least 33% of fly artifacts 25 d after the initial consumption of blood. Similarly, semen was detected in nearly 34% of digestive artifacts 30 d after feeding on human semen when using ABA p30 cards. Human body fluids were also detected in fly artifacts when using RSID lateral flow assays, but a much lower percentage of artifacts tested positive for blood (4.9%) and semen (4.6%) 25-d postfeeding in comparison to ABA strip assays. The difference between the types of lateral flow assays appeared to be due to extraction efficiencies of the buffers used for isolation of blood or semen from the fly artifacts. The implications of these observations in reference to seasonal adaptations and to bloodstain pattern analysis at crime scenes are discussed.

Characterization of Insect Stains Deposited by Calliphora vicina (Diptera: Calliphoridae) on Shirt Fabrics.

Despite the fact that necrophagous flies are known to alter bloodstains and create unique artifacts, no research has occurred to date that has examined the characteristics of insect stains on textiles or fabrics. This study represents the first effort to characterize artifacts produced by adult Calliphora vicina Robineau-Desvoidy deposited on a range of shirt fabrics that varied in type, color, orientation, and yarn tension. In general, artifact morphology on any type of fabric was distorted in comparison to those observed on smooth and/or nonporous surfaces in previous studies. Consequently, distinction of artifact type could only be made broadly as digestive artifacts and transfer patterns, in which the latter was predominantly detected as tarsal tracks. None of the artifacts displayed satellite stains typical of human bloodstains found on textiles. Wicking was evident on all fabrics but was most pronounced with dri wick and jersey knit polyester in comparison to cotton knit. Digestive artifacts on any colored fabric, but especially with green and yellow shirt samples, resembled the reported color, size, and morphology of bloodstains generated in laboratory studies on a range of fabrics. Unique digestive artifacts were also detected as small, black, and nearly spherical. These defecatory stains did not appear to wet or wick into any of the fabrics. Digestive artifacts and tarsal tracks differentially interacted with front face stitch loops of clothing fabrics to yield distinct stain patterns. The implications of these observations in reference to bloodstain pattern analysis at crime scenes are discussed.

Detection of fly artifacts from four species of necrophagous flies on household materials using immunoassays.

An immunoassay was previously developed as a technique to improve methods for detection and analysis of fly artifacts found at crime scenes. The dot blot assay utilized a polyclonal antiserum (anti-md3) based on a unique digestive cathepsin D found in cyclorrhaphous Diptera. In this study, artifacts produced by adults of Calliphora vicina, Cynomya cadaverina, Sarcophaga bullata, and Protophormia terraenovae were examined using the immunoassay to determine if insect-derived stains could be distinguished from a range of human body fluid stains. A lift technique was developed which permitted transfer of fly artifacts from test materials to filter paper for dot blot analyses. All species readily deposited artifacts on all test household materials regardless of diet consumed. Despite differences in texture and porosity of the household materials, artifacts of all species transferred to the filter paper. With all fly species, anti-md3 serum bound to artifacts produced after feeding on semen, blood, feces, urine, and saliva. By contrast, anti-md3 serum did not react with any of the human fluids tested, nor with any of the lifts from household materials not exposed to flies. There was no evidence of false positives with any of the fly species tested, regardless of diet consumed. There was also no indication of false negatives with any of the dot blot assays. These observations suggest that immunoassays using anti-md3 serum performed on a simple lift of suspected fly artifacts can be used effectively as a confirmatory assay to distinguish fly regurgitate and fecal stains from human body fluids.

Immunoassay detection of fly artifacts produced by several species of necrophagous flies following feeding on human blood.

Foraging behavior of necrophagous flies commonly leads to distortion of human bloodstains and production of artifacts that confound reconstruction efforts at crime scenes. Currently there is no reliable method for detection of fly-derived stains or distinction of the artifacts from human bloodstains. To overcome these deficiencies, a confirmatory test was developed based on immunological detection of cathepsin D found in digestive fluids of Musca domestica and Protophormia terraenovae. Anti-serum (anti-md3 serum) was generated toward a 17-amino acid synthetic peptide based upon predicted antigenic amino acid sequences for the propeptide and mature enzyme of cathepsin D proteinase from larvae of M. domestica. The serum was used to test the hypothesis that digestive artifacts produced by an array of necrophagous flies associated with human decomposition could be detected with the immunoassay. Anti-md3 serum was able to bind artifacts from 27 species of flies representing 9 families. The antiserum reacted with both regurgitate and defecatory stains, but not transfer patterns. Stains from 4 fly species displayed no reactivity with anti-serum in dot blot assays. Anti-md3 serum did not bind to either human or bovine blood stains on filter paper. However, when both types of blood were spiked with synthetic md3 peptide the antiserum was able to bind. Dot blot assays displayed positive reactions with stains produced from larvae and teneral adults of Sarcophaga bullata, and with artifacts as old as 7-years after deposition. These observations indicate that the immunoassay permits distinction of artifacts from a wide range of species from human bloodstains, from multiple development stages, and from artifacts that remain at crime scenes for many months to years after deposition. Further work is needed to determine whether the detection of fly artifacts using the antiserum is suitable for non-laboratory conditions.

Distinction of Fly Artifacts from Human Blood using Immunodetection.

Insect stains produced by necrophagous flies are indistinguishable morphologically from human bloodstains. At present, no diagnostic tests exist to overcome this deficiency. As the first step toward developing a chemical test to recognize fly artifacts, polyclonal antisera were generated in rats against three distinct antigenic sequences of fly cathepsin D-like proteinase, an enzyme that is structurally distinct in cyclorrhaphous Diptera from other animals. The resulting rat antisera bound to artifacts produced by Protophormia terraenovae and synthetic peptides used to generate the polyclonal antisera, but not with any type of mammalian blood tested in immunoassays. Among the three antisera, anti-md3 serum displayed the highest reactivity for fly stains, demonstrated cross-reactivity for all synthetic peptides representing antigenic sequences of the mature fly enzyme, and bound artifacts originating from the fly digestive tract. Further work is needed to determine whether the antisera are suitable for non-laboratory conditions.

Morphological Features of Regurgitate and Defecatory Stains Deposited by Five Species of Necrophagous Flies are Influenced by Adult Diets and Body Size.

The morphological characteristics of artifacts from five species of necrophagous flies were examined following feeding on several types of diets. Four types of insect stains were produced by each species: regurgitate, defecatory, translocation, and tarsal tracks. Regurgitate was the most frequent type deposited (70.9 ± 2.4%), followed by defecatory (19.8 ± 4.0%), tarsal tracks (8.6 ± 1.2%), and translocation (0.7 ± 0.1%). Artifact shapes, sizes, and color were highly variable and species and diet specific. Calliphora vicina and Sarcophaga bullata consistently deposited the largest artifacts after feeding, whereas Chrysomya rufifacies and Ch. megacephala produced more tarsal tracks than the other species examined. Artifacts with tails were infrequently observed (4.1 ± 0.6% of all stains) but occurred as either defecatory or regurgitate stains. The widely variable morphologies of all types of fly artifacts underscores the view that insect stains cannot be distinguished from human bloodstains based on morphology alone.

First Record of the Larval Endoparasitoid Tachinaephagus zealandicus (Hymenoptera: Encyrtidae) From Fly Hosts Developing on Burnt Remains and in Pennsylvania.

Adults of the gregarious larval endoparasitoid Tachinaephagus zealandicus Ashmead (Hymenoptera: Encyrtidae) were collected from two species of carrion-inhabiting flies, Phormia regina Meigen (Diptera: Calliphoridae) and Hydrotaea aenescens Wiedemann (Muscidae), associated with the burnt remains of a domesticated pig, Sus scrofa L. (Artiodactyla: Suidae), during late summer in south central Pennsylvania. This represents the first reported occurrence of the wasp in the state and only the second in the mid-Atlantic region. Collection from P. regina is a new host record for T. zealandicus and also the first for this parasitoid being attracted to flies on burnt remains.

Spatial characterization of proteolytic enzyme activity in the foregut region of the adult necrophagous fly, Protophormia terraenovae.

The spatial distribution of proteolytic enzymes in the adult foregut of Protophormia terraenovae was studied in the context of protein digestion and regurgitation. Based on substrate specificity, pH optima, and use of specific protease inhibitors, all adults tested displayed enzyme activity in the foregut consistent with pepsin, trypsin and chymotrypsin. Chymotrypsin-like and trypsin-like enzyme activity were detected in all gut fluids and tissues tested, with chymotrypsin displaying the highest activity in saliva and salivary gland tissue, whereas maximal trypsin activity was evident in the crop. Pepsin-like activity was only evident in crop fluids and tissues. The activity of all three enzymes was low or undetectable (pepsin) in the fluids and tissue homogenates derived from the esophagus and cardia of any of the adults assayed. Fed adult females displayed higher enzyme activities than fed males, and the activity of all three enzymes were much more prevalent in fed adults than starved. The pH optimum of the trypsin-like enzyme was between pH 7.0 and 8.0; chymotrypsin was near pH 8.0; and maximal pepsin-like activity occurred between pH 1.0 and 2.0. Regurgitate from fed adult females displayed enzyme activity consistent with the proteolytic enzymes detected in crop gut fluids. Enzymes in regurgitate were not derived from food sources based on assays of bovine liver samples. These latter observations suggest that adult flies release fluids from foregut when encountering dry foods, potentially as a means to initiate extra-oral digestion.

Water balance characteristics of pupae developing in different size maggot masses from six species of forensically important flies.

The impact of maggot mass size on body water content, net transpiration rate, and dehydration tolerance of fly pupae was examined in six species of necrophagous flies. Species that spent more time on food as larvae, produced pupae with high body water contents. Dehydration tolerance limits of pupae were modest, matching the moisture-rich conditions of decaying carrion for larvae. Protophormia terraenovae pupariates on food as it dries, and this was reflected by pupae having the highest body water content and lowest net transpiration rate. Megaselia scalaris featured the lowest body water content and highest dehydration tolerance, implying that this species is arid-suited, which matches its ability to feed and colonize on post-decay carrion. Lucilia illustris was the most sensitive to larval overcrowding, resulting in a dramatic decrease in pupal size, early dispersal from food, fed less and had fast net transpiration rates. By contrast, Lucilia sericata was the most resistant, by showing no pupal size decrease and no change in net transpiration rate. Other species were between these extremes, requiring larger maggot mass sizes to produce the effect of decreasing pupal size and increasing net transpiration rate. We conclude: (1) the pupa's response to overcrowding and water balance profile are species-specific, varying according to pupal size and net transpiration rate as independent characteristics; (2) water balance profile of the pupae reflects the behavior and microhabitat of the larva; and (3) danger of lethal desiccation to smaller-sized pupae is circumvented by a faster developmental rate rather than enhanced water conservation.

Oviposition restraint and developmental alterations in the ectoparasitic wasp, Nasonia vitripennis, when utilizing puparia resulting from different size maggot masses of Lucilia illustris, Protophormia terraenovae, and Sarcophaga bullata.

Adult females of the ectoparasitoid Nasonia vitripennis (Walker) are capable of distinguishing between hosts of different quality, and then correspondingly adjust clutch sizes and sex ratios of the offspring. In this study, we examined whether the size of the maggot mass, and presumably the developmental temperature, influenced the suitability of the resulting fly pupal and pharate adult stages as hosts for N. vitripennis. Three sizes of maggot masses (100; 500; and 1,000 individuals per mass) were selected for use to generate hosts based on previous studies characterizing developmental and heat shock response differences for the flies. For all host species tested (Lucilia illustris, Protophormia terraenovae, and Sarcophaga bullata), the rate of parasitism by N. vitripennis decreased with increasing maggot mass size. When successful parasitism did occur, parasitoid development increased in duration, clutch sizes decreased, mortality from egg hatch to adult emergence elevated, male biased sex ratios were produced, and adult wasp body sizes were truncated with increasing fly larval density. These wasp life history features are consistent with reductions in host quality. Host quality reductions corresponded to production of heat shock proteins 23, 60, and 70. Heat shock protein synthesis appeared to occur at the expense of normal protein production because total hemolymph protein concentrations decreased with increased larval density in maggot masses. These observations argue that use of N. vitripennis in criminal investigations to estimate periods of insect activity or a minimum post mortem interval must take into account the maggot mass history of the hosts used by the wasp.

Venom proteins from endoparasitoid wasps and their role in host-parasite interactions.

Endoparasitoids introduce a variety of factors into their host during oviposition to ensure successful parasitism. These include ovarian and venom fluids that may be accompanied by viruses and virus-like particles. An overwhelming number of venom components are enzymes with similarities to insect metabolic enzymes, suggesting their recruitment for expression in venom glands with modified functions. Other components include protease inhibitors, paralytic factors, and constituents that facilitate/enhance entry and expression of genes from symbiotic viruses or virus-like particles. In addition, the venom gland may itself support replication/production of some viruses or virus-like entities. Overlapping functions and structural similarities of some venom, ovarian, and virus-encoded proteins suggest coevolution of molecules recruited by endoparasitoids to maintain their fitness relative to their host.

Pathological and ultrastructural changes in cultured cells induced by venom from the ectoparasitic wasp Nasonia vitripennis (Walker) (Hymenoptera: Pteromalidae).

The ectoparasitic wasp Nasonia vitripennis produces a proteinaceous venom that induces death in fly hosts by non-paralytic mechanisms. Previous in vitro assays have suggested that the primary cause of cell and tissue death is oncosis, a non-programmed cell death (PCD) pathway characterized by cellular swelling and lysis. However, ultrastructural analyses of BTI-TN-5B1 cells exposed to LC(99) doses of wasp venom revealed cellular changes more consistent with apoptosis and/or non-apoptotic PCD than oncosis or necrosis: By 3h after incubation with venom, susceptible cells displayed indentations in the nuclear membranes, large nucleoli, and extensive vacuolization throughout the cytoplasm. In the vast majority of venom treated cells, annexin V bound to the plasma membrane surface within 15 min after treatment, a characteristic consistent with translocation of phosphatidylserine to the cell surface during the early stages of apoptosis. Likewise, mitochondrial transmembrane potential was depressed in cells within 15 min in venom-treated cells, an event that occurred in the absence of mitochondrial swelling or rupturing of cristae. Active caspase 3 was detected by fluorescent labeling in nearly all venom treated cells 3h after exposure to venom, and in turn, the potent caspase 3 inhibitor Z-VAD-FMK attenuated the morphological changes elicited by wasp venom and afforded protection to BTI-TN-5B1-4 cells.

Changes in development and heat shock protein expression in two species of flies (Sarcophaga bullata [Diptera: Sarcophagidae] and Protophormia terraenovae [Diptera: Calliphoridae]) reared in different sized maggot masses.

Development of two species of necrophagous flies, Sarcophaga bullata Parker (Sarcophagidae) and Protophormia terraenovae (Robineau-Desvoidy) (Calliphoridae), was examined in different size maggot masses generated under laboratory conditions. Larvae from both species induced elevated mass temperatures dependent on the number of individuals per mass. The relationship was more evident for S. bullata, as larvae generated higher temperatures in every size maggot mass than P. terraenovae. Several development events were altered with increasing maggot mass size of flesh flies, and to a lesser extent blow flies, which corresponded with elevated temperatures. Duration of development of all feeding larval stages decreased with increased size of maggot mass. However, the length of development during puparial stages actually increased for these same flies. Puparial weights also declined with maggot mass size, as did the ability to eclose. The altered fly development was attributed to the induction of heat stress conditions, which was evident by the expression of heat shock proteins (23, 60, 70, and 90) in larval brains of both fly types.

Venom proteins of the parasitoid wasp Nasonia vitripennis: recent discovery of an untapped pharmacopee.

Adult females of Nasonia vitripennis inject a venomous mixture into its host flies prior to oviposition. Recently, the entire genome of this ectoparasitoid wasp was sequenced, enabling the identification of 79 venom proteins. The next challenge will be to unravel their specific functions, but based on homolog studies, some predictions already can be made. Parasitization has an enormous impact on hosts physiology of which five major effects are discussed in this review: the impact on immune responses, induction of developmental arrest, increases in lipid levels, apoptosis and nutrient releases. The value of deciphering this venom is also discussed.

The mode of action of venom from the endoparasitic wasp Pimpla hypochondriaca (hymenoptera: ichneumonidae) involves Ca+2-dependent cell death pathways.

The endoparasitoid Pimpla hypochondriaca injects venom during oviposition to condition its lepidopteran hosts. Venom is a complex mixture of proteins and polypeptides, many of which have been identified as enzymes, including phenoloxidase, endopeptidase, aminopeptidase, hydrolase, and angiotensin-converting enzyme. Constituents of the venom have been shown to possess cytolytic and paralytic activity, but the modes of action of factor(s) responsible for exerting such effects have not been deciphered. In this study, we examined the mode of action of isolated venom using cultured cells (BTI-TN-5B1-4). A series of blockage and inhibition assays were performed using a potent inhibitor (phenylthiourea, PTU) of venom phenoloxidase, and anti-calreticulin antibodies. Monolayers exposed to venom alone were highly susceptible with more than 84.6+/-2.3% dead within 15 min. Susceptible cells displayed a retraction of cytoplasmic extensions, rounding, and swelling prior to lysis in more than half (55.7+/-1.7%) of the dying cells. Within 15 min of exposure to venom, cells displayed qualitative increases in [Ca(+2)](i) as evidenced by staining with the calcium-sensitive probe fluo-4 AM, and mitochondrial membrane potential (DeltaPsi(m)) was undetectable by 5 min post-treatment with venom. These venom-mediated changes occurred regardless of whether an external source of calcium was present, or whether venom was pre-treated with PTU. In contrast, venom toxicity was attenuated by treatment with anti-calreticulin antibodies. Not only did fewer cells die when exposed to antibody-treated venom but also cell swelling diminished and no increases in intracellular calcium were detected. A possible mode of action for the venom is discussed.

Characterization of phenoloxidase activity in venom from the ectoparasitoid Nasonia vitripennis (Walker) (Hymenoptera: Pteromalidae).

Crude venom isolated from the ectoparasitic wasp Nasonia vitripennis was found to possess phenoloxidase (PO) activity. Enzyme activity was detected by using a modified dot blot analysis approach in which venom samples were applied to nylon membranes and incubated with either L-DOPA or dopamine. Dot formation was most intense with dopamine as the substrate and no activators appeared to be necessary to evoke a melanization reaction. No melanization occurred when venom was incubated in Schneider's insect medium containing 10% fetal bovine serum or when using tyrosine as a substrate, but melanization did occur when larval or pupal plasma from the fly host, Sarcophaga bullata, was exposed to tyrosine. Only fly larval plasma induced an enzyme reaction with the Schneider's insect medium. The PO inhibitor phenylthiourea (PTU) and serine protease inhibitor phenylmethylsulfonylfluoride (PMSF) abolished PO activity in venom and host plasma samples, but glutathione (reduced) only inhibited venom PO. Elicitors of PO activity (sodium dodecyl sulfate and trypsin) had no or a modest effect (increase) on the ability of venom, or larval and pupal plasma to trigger melanization reactions. SDS-PAGE separation of crude venom followed by in-gel staining using L-DOPA as a substrate revealed two venom proteins with PO activity with estimated molecular weights of 68 and 160 kDa. In vitro assays using BTI-TN-5B1-4 cells were performed to determine the importance of venom PO in triggering cellular changes and evoking cell death. When cell monolayers were pre-treated with 10 mM PTU or PMSF prior to venom exposure, the cells were protected from the effects of venom intoxication as evidenced by no observable cellular morphological changes and over 90% cell viability by 24 h after venom treatment. Simultaneous addition of inhibitors with venom or lower concentrations of PMSF were less effective in affording protection. These observations collectively argue that wasp venom PO is unique from that of the fly hosts, and that the venom enzyme is critical in the intoxication pathway leading to cell death.