DNA Extraction and Comparison between Old and Fresh Necrophilic Fly Samples.

A total of five samples of Chrysomya megacephala samples - three fresh samples, one sample stored in alcohol for 2 years, and one sample stored in dry sealed storage for 2 years protected from light only - were selected to investigate whether a blood DNA extraction kit could extract DNA from necrophilous flies and to determine whether alcohol could prolong the preservation of necrophilous flies' DNA. First, the blood DNA extraction kit was used to extract DNA from their thorax tissues. Then, the DNA purity and concentration were examined using a microplate reader and a fluorometer. Finally, PCR amplification and electrophoresis of the extracted DNA were done with necrophilic fly-specific primers located in the mitochondrial CO I gene sequence. The results showed that the DNA purity of all samples was greater than 2.0. The DNA concentration was observed to be of the following order: fresh samples > alcohol-preserved old samples > untreated, old samples. All samples had specific electrophoretic bands after PCR amplification. In conclusion, a blood DNA extraction kit can be used to extract DNA from necrophilic flies successfully, and the DNA concentration of fresh fly samples is greater than that of old fly samples. The flies can be stored in alcohol for a long time.

Therapeutic Targeting of Nrf2 Signaling by Maggot Extracts Ameliorates Inflammation-Associated Intestinal Fibrosis in Chronic DSS-Induced Colitis.

Intestinal fibrosis is induced by excessive myofibroblast proliferation and collagen deposition, which has been regarded as a general pathological feature in inflammatory bowel disease (IBD). Therefore, identifying clinical markers and targets to treat and prevent intestinal fibrosis is urgently needed. The traditional Chinese medicine maggot, commonly known as "wu gu chong", has been shown to reduce oxidative stress and alleviate inflammation in chronic colitis. This study investigated the mechanisms underlying the effects of maggot extract (ME) on inflammation-associated intestinal fibrosis in TGF-ß1-stimulated human intestinal fibroblasts (CCD-18Co cells) and dextran sodium sulphate (DSS)-induced chronic colitis murine model. To assess the severity of inflammation and fibrosis, histological and macroscopic evaluation were carried out. The results showed that ME was a significant inhibitor of body weight loss and colon length shortening in mice with chronic colitis. In addition, ME suppressed the intestinal fibrosis by downregulating TGF-ß1/SMADs pathway via upregulation of Nrf2 expression at both protein and mRNA levels. ME markedly increased the expression of Nrf2, thus resulting in a higher level of HO-1. After treatment with Nrf2 inhibitor (ML385) or siRNA-Nrf2 for deactivating Nrf2 pathway, the protective effects of ME were abolished both in vitro and in vivo. Moreover, the histopathological results for the major organs of DSS mice treated with ME showed no signs of clinically important abnormalities. Treatment with ME had no effect on the viability of CCD-18Co cells, suggesting its low in vitro cytotoxicity. Furthermore, ME could mediate intestine health by keeping the balance of the gut microbes through the enhancement of beneficial microbes and suppression of pathogenic microbes. In conclusion, this is the first ever report demonstrating that ME ameliorates inflammation-associated intestinal fibrosis by suppressing TGF-ß1/SMAD pathway via upregulation of Nrf2 expression. Our findings highlight the potential of Nrf2 as an effective therapeutic target for alleviating intestinal fibrosis.

Larval excretion/secretion of dipters of Lucilia cuprina species induces death in promastigote and amastigote forms of Leishmania amazonensis.

Leishmaniasis is a neglected tropical disease that affects millions of people around the world. Larval excretion/secretion (ES) of the larvae of flies of the Calliphoridae family has microbicidal activity against Gram-positive and Gram-negative bacteria, in addition to some species of Leishmania. Our study aimed at assessing the in vitro efficacy of Lucilia cuprina larval ES against the promastigote and amastigote forms of Leishmania amazonensis, elucidating possible microbicidal mechanisms and routes of death involved. Larval ES was able to inhibit the viability of L. amazonensis at all concentrations, induce morphological and ultrastructural changes in the parasite, retraction of the cell body, roughness of the cytoplasmic membrane, leakage of intracellular content, ROS production increase, induction of membrane depolarization and mitochondrial swelling, the formation of cytoplasmic lipid droplets and phosphatidylserine exposure, thus indicating the possibility of apoptosis-like death. To verify the efficacy of larval ES on amastigote forms, we performed a phagocytic assay, measurement of total ROS and NO. Treatment using larval ES reduced the percentage of infection and the number of amastigotes per macrophage of lineage J774A.1 at all concentrations, increasing the production of ROS and TNF-α, thus indicating possible pro-inflammatory immunomodulation and oxidative damage. Therefore, treatment using larval ES is effective at inducing the death of promastigotes and amastigotes of L. amazonensis even at low concentrations.

Volatile compounds reveal age: a study of volatile organic compounds released by Chrysomya rufifacies immatures.

Age determination of insects collected from vertebrate remains is an essential step in estimating time since colonization as related to the post-mortem interval. Long-established methods for making such estimates rely on determining age related to stage of development at the time of collection in relation to conditions experienced. However, such estimates are based on the completion of a stage of development. Methods allowing for more precise estimates of age (i.e., within a stage of development) are sorely needed. This study examined the potential of volatile organic compounds emitted by blow fly, Ch. rufifacies (Macquart), immatures to determine stage of development, which could potentially be used to estimate the age. Volatile organic compounds (VOCs) from the larval and pupal stages of Ch. rufifacies were collected by headspace solid-phase micro-extraction followed by gas chromatography-mass spectrometry (GC-MS). Analyses indicated 37 compounds shift quantitatively, as well as qualitatively, as the larvae and pupae age. Furthermore, compounds, such as 2-ethyl-1-hexanol, phenol, butanoic acid, hexadecanoic acid, octadecanoic acid, 2-methyl propanamide, and 2-methyl butanoic acid, serve as indicator compounds of specific stages within Ch. rufifacies development. This information could be important to determine if these compounds can be used in the field to predict the presence of certain developmental stages, in order to determine the potential of using volatile markers to estimate time of colonization.

Cuticular hydrocarbons as a tool for determining the age of Chrysomya rufifacies (Diptera: Calliphoridae) larvae.

Calliphoridae are one of the most important insect groups encountered as evidence collected from a crime scene. Age determination of the immature stages of these necrophagous flies is an important step toward estimating the time of colonization and inferring a minimum postmortem interval (PMImin ) in most instances. To determine if the cuticular hydrocarbons could be used to establish whether the development stages yield characteristics profiles, allowing for age estimation, hydrocarbons were extracted from 1st and 2nd, as well as feeding and post-feeding 3rd instar Chrysomya rufifacies, the hairy maggot blow fly. Extracted hydrocarbons were analyzed using gas chromatography coupled to mass spectrometry with the aim to investigate the changes in chemical profiles of each larval stage. A total of 23 compounds were identified with most of them being alkanes (65%) with carbon chain lengths of 9-33 carbons, alkenes (18%), and methyl-branched alkanes (17%). All the hydrocarbons except pentadecane (C15), hexadecane (C16), and nonacosane (C29) showed significant differences in their expression throughout larval development. For 1st instars, nonane was the most abundant (17% of the total hydrocarbons content) compound. Accounting for 11% and 10% of the cuticular hydrocarbons, tricosane and pentacosane, respectively, were the notable hydrocarbons in 2nd instars. For post-feeding 3rd instars, hentriacontane and tritriacontane were present with relative abundances 18% and 15%, respectively. On average, there was a shift from low to high molecular weight hydrocarbons as the larvae aged. These results indicate the change in hydrocarbons makeup as larvae age and could potentially be used to determine the age of immature C. rufifacies and hence aid in PMImin estimations. However, future research is needed to validate these results under natural conditions in the field.

Detection of Pb, Ba, and Sb in Cadaveric Maggots and Pupae by ICP-MS.

The concentrations of lead (Pb), barium (Ba), and antimony (Sb), characteristic of GSR, were determined in soil sediments and immature (larvae) of cadaveric flies of the family Calliphoridae, by inductively coupled plasma mass spectrometry (ICP-MS). This research refers to a case study from two real crime scenes in which the corpses were in an advanced state of decomposition. In case 1, the victim had holes similar to gunshot wounds, and in case 2, there was no evidence of perforations in the corpse. Soil sediment collection was performed at three different points of the terrain, at a minimum distance of 10 m from the corpse, for cases 1 and 2. In relation to the collection of immatures, larvae were collected in regions of the mouth, nose, and orifices similar to the entry of firearms projectile into the body, for case 1, and collection of larvae and pupae, located on the body and underneath it, for case 2. It was possible to detect and quantify the three elements of interest (Pb, Ba, and Sb) by ICP-MS in both sediment and cadaveric larvae. Concentrations of 4.44, 8.74, and 0.08 µg/g were obtained for Pb, Ba, and Sb, respectively, in the soil for case 1. For the case 2, the concentrations in Pb, Ba, and Sb were from 16.34 to 26.02 µg/g; from 32.64 to 57.97 µg/g and from 0.042 to 0.30 µg/g, respectively. In the larvae, Pb, Ba, and Sb were quantified in cases 1 and 2 with a concentration of 6.28 and 1.78 µg/g for Pb, 1.49 and 2.94 µg/g for Ba, 0.50 µg/g and

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.

Characterization of the Volatiles' Profiles of the Eggs of Forensically Relevant Lucilia sericata and Phormia regina (Diptera: Calliphoridae) Blow Flies by SPME-Facilitated GC-MS.

The attraction of necrophagous insects, particularly blow flies, to corpses and carrion is of ecological, economic, and agricultural importance, although the mechanisms by which it occurs are not well understood. Much of the published research on blow fly attractants has focused on volatiles emitted from carrion surrogates, but little attention has been given to the possibility that blow fly eggs themselves may emit chemical cues that are responsible for conspecific and heterospecific insect attraction. In this study, the headspace volatiles emitted from eggs representing two aggregated oviposition events that were collected 1 mo apart from two species of the Calliphoridae family (Order: Diptera), Lucilia sericata (Meigen), and Phormia regina (Meigen) were analyzed via solid-phase microextraction-facilitated GC-MS. The volatiles' profiles were found to be consistent between samples representing the same species, but unique between the two species. Over 100 molecules covering a wide range of compound classes that included alcohols, aldehydes, esters, amines, ketones, and organosulfur compounds were identified. The profile of volatiles emitted from the L. sericata eggs contained several alkanes and aldehydes, whereas salient features of the P. regina headspace included numerous esters and ketones. Between the two species, 42 compounds were shared, several of which were carboxylic acids. Little overlap between the range of compounds detected and those reported to be emitted from decomposing remains was observed.