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.

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.

EGF-like ligands mediate progesterone's anti-apoptotic action on macaque granulosa cells.

A local autocrine/paracrine role for progesterone is an absolute requirement for corpus luteum formation in primates. Despite this, the mechanism(s) remain obscure, although existing data suggest an anti-apoptotic action to be central. There are a limited number of progestin-regulated gene targets identified in the luteinizing primate follicle, suggesting that a small number of important genes may mediate progesterone action. Possible gene targets could be the epidermal growth factor (EGF) family members amphiregulin (AREG) and epiregulin (EREG). Using macaques undergoing controlled ovarian stimulation cycles, we show that the phosphorylation of EGF receptor (EGFR), ERK 1/2, and AKT increases 6 h after an ovulatory human chorionic gonadotropin (hCG) stimulus and remains activate through 24 h. Immunoreactive EREG and AREG ligands in the follicular fluid both increased in a time frame commensurate with EGFR phosphorylation. The mRNA expression of AREG and EREG in nonluteinized granulosa cells (NLGC) was induced in culture with hCG, an effect blocked by progesterone receptor (PGR) antagonists. Overexpression of PGR B in NLGC and treatment with a nonmetabolizable progestin did not increase either gene, indicating both progesterone and luteinizing hormone/CG are necessary. Addition of EGF and EGF-like ligands did not promote steroidogenesis in vitro by granulosa cells in the presence of gonadotropin, but were able to partially reverse RU486-induced cell death. These data suggest that progesterone promotes the expression of AREG and EREG, which in turn maintain viability of luteinizing granulosa cells, representing one possible mechanism whereby progesterone promotes corpus luteum formation in the primate.

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.

Dynamics of intra-follicular glucose during luteinization of macaque ovarian follicles.

Glucose is important to the maturation of the oocyte and development of the embryo, while hyperglycemia results in profound reproductive and developmental consequences. However, the normal physiology of glucose in the ovary remains poorly understood. The goal of this study was to determine intra-follicular glucose dynamics during the periovulatory interval in non-human primates undergoing controlled ovarian stimulation protocols. Follicular fluid and mural granulosa cells were isolated before or up to 24h after an ovulatory hCG bolus, and the human granulosa-lutein cell line hGL5 was used. Intra-follicular glucose increased 3h after hCG, and remained at that level until 12h when levels decline back to pre-hCG concentrations. Pyruvate and lactate concentrations in the follicle were not strongly altered by hCG. Mural granulosa cell expression of hexokinase 1 and 2, and glucose-6-phosphate dehydrogenase mRNA decreased following hCG, while glycogen phosphorylase (liver form) increased following hCG. Glucose uptake by hGL5 cells was delayed until 24h following stimulation. In summary, intra-follicular glucose increases following an ovulatory stimulus and mural granulosa cells do not appear able to utilize it, sparing the glucose for the cumulus-oocyte complex.

Deficiency of scavenger receptor class B type I negatively affects progesterone secretion in human granulosa cells.

Our goal was to examine the effect of deficiency of the lipoprotein receptor, scavenger receptor class B type I (SR-BI), on progesterone secretion in human granulosa cells (HGL5). Scrambled or SR-BI small interfering RNA [knockdown (KD)] cells were exposed to dimethylsulfoxide [DMSO, vehicle for forskolin (Fo)], Fo, serum, high-density lipoprotein, low-density lipoprotein (LDL), or Fo plus lipoproteins or serum for 24 h. Progesterone secretion was lower in all of the SR-BI KD cells regardless of treatment. We examined progesterone secretion in SR-BI KD, LDL receptor KD, and double KD cells incubated with DMSO, Fo, LDL, or Fo + LDL for 6-24 h. As compared with scrambled cells, progesterone secretion was lower in SR-BI and double KD cells regardless of treatment; whereas progesterone secretion was only lower in LDL receptor KD cells incubated with LDL and Fo + LDL. We measured phosphorylation of hormone-sensitive lipase (pHSL) expression, intracellular total cholesterol (TC) mass, and progesterone secretion in scrambled and SR-BI KD cells incubated with DMSO or Fo for 2-24 h. The expression of pHSL was similar between the cells and conditions. The mean change in TC mass and progesterone secretion was lower in SR-BI KD cells exposed to DMSO and Fo. Incubating SR-BI KD cells with 22-hydroxy cholesterol did not overcome the reduction in progesterone secretion. At different time points, RNA expression of steroidogenic acute regulatory protein, side-chain cleavage, and 3ß-hydroxysteroid dehydrogenase was significantly lower in SR-BI KD cells incubated with Fo. In conclusion, SR-BI protein deficiency, in part, might explain progesterone deficiency in some infertile women.

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.

Expression of the insulin-like growth factor and insulin systems in the luteinizing macaque ovarian follicle.

OBJECTIVE: To determine intrafollicular hormone levels and characterize the mRNA expression of the insulin-like growth factor (IGF) receptors, IGF binding proteins (IGFBP), and pregnancy-associated plasma protein-A (PAPP-A) in granulosa cells before and after an ovulatory hCG stimulus. DESIGN: Experimental animal study. SETTING: Academic medical center. ANIMAL(S): Adult rhesus macaques. INTERVENTION(S): Animals received exogenous FSH to promote the development of multiple preovulatory follicles. Follicles were aspirated before (0 hours) or 3, 6, 12, or 24 hours after an ovulatory hCG bolus. MAIN OUTCOME MEASURE(S): IGF1, IGF2, and insulin levels in follicular fluid were determined by radioimmunoassay. Messenger RNA (mRNA) levels in granulosa cells were determined by real-time reverse transcriptase-polymerase chain reaction. IGFBPs and PAPP-A in follicular fluid were determined by Western blot analysis and enzyme-linked immunosorbent assay. RESULT(S): IGF1, IGF2, and insulin in follicular fluid did not change during luteinization. IGF1R, IGFBP1, and IGFBP2 mRNAs were unchanged by hCG. IGF2R, IGFBP3, -5, and -6 and PAPP-A mRNA levels increased after hCG administration, while insulin receptor and IGFBP4 mRNA levels decreased after hCG administration. IGFBP3 and -6 and PAPP-A protein increased after hCG administration. CONCLUSION(S): Dynamic changes in the expression of the IGFBPs and PAPP-A suggest tight regulation of IGF action during ovulation and corpus luteum formation.

Dynamics of Myc/Max/Mad expression during luteinization of primate granulosa cells in vitro: association with periovulatory proliferation.

Granulosa cell luteinization involves the attenuation of gonadotropin-induced proliferation. Although recent evidence indicates that primate granulosa cells stop dividing within 12 h of an ovulatory stimulus, early events in cell cycle arrest remain unknown. In the current study an in vitro model of primate granulosa cell luteinization is established that allows assessment of early events in terminal differentiation. A luteinizing dose of human chorionic gonadotropin (hCG) results in a secondary rise in proliferation before cell cycle arrest that is paralleled by a transient increase in the expression of c-Myc. In contrast, the c-Myc antagonists Mad1, Mad4, and Mxi1 are transiently repressed by hCG. Max, the common dimerization partner for Myc and Mad, is similarly repressed by hCG, suggesting that changes in the expression of this gene may further regulate the activity of Myc and Mad. To determine whether other cell cycle regulatory families are involved in luteinization, the expression of p53 and the wild-type p53-inducible phosphatase (wip1) was examined. Similar to Mad and Max, p53 and wip1 are transiently repressed by hCG, suggesting that the p53 and Mad pathways have either parallel or cooperative roles in luteinization. Thus, luteinization of primate granulosa cells is preceded by a burst of proliferation that is regulated by changes in the relative levels of c-Myc, Max, and Mad as well as p53.